Drugs and Pharmaceuticals

Current R & D Highlights

(RNA and Drug Discovery)








·      RNA and Drug Discovery                             1


·      RNA Therapeutics: Silencing the Culprit         7


News & Views                                            14


R&D Highlights                                           

·      MicroRNAs in Diseases and Drug

     Response                                                         19

·      Alternative Splicing and Disease                    22

RDH Abstracts

R& D Technology                                   48


New Leads                                                 56



·      RNA Interference: The Story of

     Gene Silencing in Plants and Humans             61

Bio Abstracts

Patents                                                         67


CSIR Publications                                    77










RNA and Drug Discovery

Sheela Tandon & VK Vohra

S&T Knowledge Resource Centre, CDRI, Lucknow-226001


       Ribonucleic acid (RNA) a macromolecule present in all the living cells, transporter of genetic information from DNA to protein that determines the structure and function of the cell, catalyzes chemical reactions and can alter the expression of proteins which may lead to various diseases.

       Living cells store their hereditary information in the form of double-stranded deoxyribonucleic acid (DNA) molecules. The DNA in genomes does not direct protein synthesis itself but instead uses RNA as intermediary molecules when the particular cell needs a specific protein. Nucleotide sequence of the appropriate portion of the immensely long DNA molecule in a chromosome is first copied into m-RNA a process called transcription. The copies of m-RNA segments of the DNA are used directly as templates to direct the synthesis of the protein  in a process called translation.

       The flow of genetic information in cells is therefore from DNA to RNA to protein. All cells from bacteria to humans, express their genetic information in this way – a principal so fundamental that it is termed as the “Central Dogma” of molecular biology. The process of regulation of gene expression is – that how cells “know” to make the right proteins at the right time in right amounts is the major focus of current research in molecular biology.

       Despite the universality of the “Central Dogma”, there are important variations in the way information flows from DNA to protein. Principal among these is that RNA transcripts (pre m-RNA) in eukaryotic cells are subject to a series of processing steps in the nucleus before the formation of mature m-RNA, which serves as template for protein synthesis.

       The protein coding sequences in the eukaryotic genes are typically interrupted by non-coding intervening sequences, discovered in 1977. This feature of eukaryotic genes came as a surprise to scientists who had been until that time, familiar only with bacterial genes, typically consisting of continuous stretches of coding DNA that is directly transcribed into m-RNA. In humans and other complex metazoans, the vast majority of protein-coding genes contain many segments (introns) that are part of the primary transcript (pre m-RNA) but are not included in mature m-RNA. The removal of introns and joining together of the sequences (exons) included in the final mature m-RNA is accomplished by pre-m-RNA splicing.

       The identification of exons and the execution of splicing reaction is mediated by the spliceosomes, a molecular complex composed of five snRNP (small nuclear RNA proteins), and a range of non-snRNP associated protein factors.

       Alternative splicing is a process by which the exons of the pre-m-RNA transcripts produced by transcription of a gene are reconnected in multiple ways. The resulting m-RNAs may be translated into different protein isoforms; thus, a single gene may code for multiple proteins. Alternative splicing occurs as a normal phenomenon in eukaryotes, where it greatly increases the diversity of proteins that can be encoded by the genome. In humans, over 80% of genes are alternatively spliced. There are numerous modes of alternative splicing observed, of which the most common is exon skipping. In this mode, a particular exon may be included in m-RNAs under some conditions or in particular tissues, and omitted from the m-RNA in others.

       The splicing of  pre-m-RNA  to m-RNA is a critical step in the expression of the majority of mammalian genes. Spliceosome, catalyzes the excision of intervening intron sequences and joining of the exon sequences. A typical human and mouse gene contains eight to ten exons, which can be joined in different arrangements by alternative splicing (AS). Recent computational studies have estimated that one- to two-thirds of human and mouse genes contain at least one alternative exon. It is widely assumed that AS is a key step in the generation of proteomic diversity in more complex organisms. AS can increase the coding capacity of the genome without increasing the number of genes.

       Alternative splicing is known to play numerous critical roles in regulatory pathways in metazoans, including those controlling cell growth, cell death, differentiation and development, and its mis-regulation has been implicated in many life-threatening human diseases. Many human gene mutations affect the splicing pattern of that gene. For example, a mutation in the sequence at an intron/exon junction that is recognized by the spliceosomes can cause this junction to be ignored. This causes splicing to occur to the next exon in line, leaving out the exon next to the mutation. This exon skipping usually results in an m-RNA that codes for a non-functional protein. Exon skipping and other errors in splicing are seen in many human genetic diseases (Table 1).

       Mutations that disrupt any of the components of RNPs, either RNA or proteins or the factors required for their assembly can be deleterious to cells and cause disease. To identify physiologically and diseases-relevant AS events and to determine where and when these occur, what their specific roles are, and how they are regulated is a priority research area.

       In this post-genomic era of biological sciences, it is more imperative than ever to utilize human DNA sequencing data in the process of drug design, which starts with target identification and validation. For decades, the pharmaceutical industry has been designing small molecules, peptides, and antibodies to inhibit clinically-relevant, human protein targets, many of which were identified and validated in the pre-genomic era. However, for a multitude of reasons, many clinically-relevant, human proteins are not druggable. Drug researchers continue to search for novel therapeutic modalities that can inhibit with greater potency, efficacy, and can be developed in less time and more cost-effectively. The most recent mission has been to target non-protein biomolecules—the most common of which is RNA—with inhibitory nucleic acids. However, this attempt is not a new one. The use of antisense nucleic acids to inhibit protein translation from complementary, clinically-relevant RNA in human cells has been in existence for many years. Other therapeutic modalities in this category include aptamers, ribozymes, and RNAi (a small inhibitory RNA molecule, or siRNA).

       There are a number of scientific and economical reasons for this trend shift in target identification. RNA offers a unique way to get at many drug targets that are currently un-druggable, but are very well validated. Some of therapeutic approaches that use or target RNAs are –


·        Antisense RNA

·        RNA interference

·        Small Molecules

·        RNA Aptamers

·        micro-RNAs


Antisense RNA

       Antisense therapy is a form of treatment for genetic disorders or infections. When the genetic sequence of a particular gene is known to be causative of a particular disease, it is possible to synthesize a strand of nucleic acid (DNA, RNA or a chemical analogue) that will bind to the messenger RNA (m-RNA) produced by that gene and inactivate it, effectively turning that gene "off". This is because m-RNA has to be single stranded for it to be translated. Alternatively, the strand might be targeted to bind a splicing site on pre-m-RNA and modify the exon content of an m-RNA.  


Table 1: The Affects of Alternative Splicing on Disease





Translationally Silent

Acute intermittent porphyria

Porphobilinogen deaminase



R28R(CG, 3)

Breast and ovarian cancer



E139K (G→T,18)


Carbohydrate-deficient glycoprotein type 1a



E139K (G→A,5)


Cerbotendinous xanthomatosis



E60X (G→T,3);

G112G (G→T, 2)

Cystic fibrosis



R75X (C→T,3);

R553X (C→T,11);




Ehlers-danlos syndrome type V1

Lysyl hydroxylase


Y511X (C→A,14);



Fanconi anemia



Q356X (C→T,8)



Frontotemporal dementia (FTDP-17)


S305N(G→A, 10)

N297K (T→G,10)



L284L (T→C10)

S305S (T→C10)

Hemophilia A

Factor VIII


E1978X (G→T,19)



HPRT deficiency

Hypoxanthine phosphoribosyl transferase



A161E (C→A,6)








Leigh’s encephalomyelopathy

Pyuvate dehydrogenase E1α




Marfan syndrome





Metachromatic leukodystrophy(juvenile form)

Arylsulfatase A

T4091 (C→T,8)



Neurofibromatosis  type 1







OCT deficiency

Ornithine carbamoyltransferase




Porphyria cutanea tarda

Uroporphyrinogen decarboxylase



E314E(G → A,9)

Sandhoff disease


P404L(C → T,11)



Severe combined immunodeficiency

Adenosine deaminase

R142Q(G → A,5)

R142X(C → T,5)


Spinal muscle atrophy



W102X(G →A,3)


Spinal muscle atrophy





Tyrosinemia type1

Fumaryl acetoacetate hydrolase




(Trends in Genetics, Vol.18, No. 4, April 2002, p.186)

       This synthesized nucleic acid is termed an "anti-sense" oligonucleotide because its base sequence is complementary to the gene's messenger RNA (m-RNA), which is called the "sense" sequence.

       Antisense drugs are being researched to treat cancers (including lung cancer, colorectal carcinoma, pancreatic carcinoma, malignant glioma and malignant melanoma), diabetes, ALS, Duchenne muscular dystrophy and diseases such as asthma and arthritis with an inflammatory component. Most potential therapies have not yet produced significant clinical results, though one antisense drug, fomivirsen, has been approved by the US FDA as a treatment for Cytomegalovirus retinitis.

RNA Interference

       The capacity to selectively eliminate an m-RNA of a disease causing allele or to prevent translation of a deleterious protein by



RNAi (RNA Interference) presents a wide range of targets for therapeutic modulation. RNAi relies on the base pairing interaction of 21-23 nucleotide RNAs, a size sufficient to uniquely target an m-RNA or even a specific splice variant, and provides a versatile and potent tool. RNAi-based strategies are applicable to all diseases in which decreasing expression of an RNA, whether from a mutant allele or an aberrantly expressed m-RNA, would have therapeutic effects. Great progress has been made toward translating the expertise of RNAi from an extensively used experimental tool to an effective and safe treatment. The main challenges again are optimal delivery to the appropriate tissues and cells, avoiding the cellular antiviral response to double-stranded RNA, and achieving the optimal balance of high potency without off-target effects.



Table2: Selected RNA-based Therapies in Development











High cholesterol

Phase II


ISIS 113715


Phase II

OncoGenex, Isis



Phase II

Eli Lilly, Isis

LY 2181308


Phase II

AVI BioPharma



Phase II




Phase I/II



Hepatitis C

Phase II




Phase I

Lorus Therapeutics


Renal cell carcinoma

Phase II







Acute coronary syndrome,percutaneous coronary intervention

Phase I


AS 1411

Renal cancer, acute myeloid leukaemia

Phase II

Small-interfering RNA




Opko Health

Bevasiranib (C and 5)


Phase III


AGN 211745 (Sirna-027)


Phase II

Silence Therapeutics, Quark Biotech, Pfizer

RTP 801i


Phase I



RSV infections

Phase II

RSV=respiratory syncytal virus;   AMD= age related macular degeneration

(Nature Reviews: Drug Discovery, Vol.6, Nov., 2007,p. 863)

RNA as Target for Small Molecules

       Alternative splicing is an attractive target for pharmacological intervention with small molecules. AS splicing of most of the introns is strongly dependent an serine-argenine rich (SR) proteins and hnRNP proteins. Small molecules that affect their activities or their relative amounts in the nucleus can profoundly modify splicing.

RNA Aptamers

       Aptamers are nucleic acids or species that have been engineered through repeated rounds of in vitro selection SELEX(systematic evolution of ligands by exponential enrichment) to bind to a specific target molecule. RNA aptamers have been shown to bind to proteins and perturb their function with a very high specificity and affinity making their potential high for use as therapeutic drugs and research tools. A system has been designed for in vitro selection (SELEX) of an RNA aptamer to maximize its binding capacity for a specified protein, which then is applied to a novel expression system that uses specific genetic constructs, designs and promoters along with transgenic techniques to produce either mono-or multivalent aptamers used as conditional alleles in vivo.


micro-RNA (mi-RNA)

       mi-RNAs are believed to regulate the expression of approximately 30% of all human genes. Thus, in contrast to antisense and RNAi, which target single genes, targeting mi-RNAs has the potential of addressing whole disease pathways.

       The normal function of the cell depends on accurate expression of various protein-coding and non-coding RNAs. These RNAs participate in transcription and translation. The RNPs are the functional forms of the corresponding RNAs and their normal activity depends on both the specific composition and the precise arrangement of their protein constituents. As there are numerous RNAs and a very large number of RNA-binding proteins, the biogenesis of RNPs must be orchestrated with great fidelity. Disrupted functions of RNAs and RNPs are the cause of numerous maladies.

       Reversal of defective protein or restoration of normal protein production can be achieved more efficaciously by eliminating or redirecting the splicing of pre-m-RNA.

       RNA-based strategies offer a series of novel therapeutic applications including altered processing of the target pre-m-RNA transcript, reprogramming of genetic defects through m-RNA repair, and the targeted silencing of allele-or isoforms-specific gene transcripts.














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RNATherapeutics: Silencing the Culprit

Ranjeet Kumar and Vinod Bhakuni

Division of Molecular and Structural Biology,

Central Drug Research Insttute, Lucknow-226001, India


RNA Biology

File:RNA chemical structure.GIF       The biology of RNA is quite intriguing. A contemporary discussion and thinking on the plethora of diversity they show in terms of structure, function and complexity demands understanding of the molecule in a blue sky perspective. The multiplicity of their forms from being coding, non-coding and their proven role in therapeutics makes the small molecule pivotal thus opening a new world of ribomics or ribonucleomics. The short review is a humble attempt to put the known scenario in right perspective; it does not endeavor to put copious information already circulating in the webosphere.



       Ribonucleic acid (RNA) is a biomolecule which consists of ordered repeats of nucleotide units. The backbone cocktail is a mixture of nitrogenous base, ribose sugar and phosphate moiety. RNA is quite similar to DNA but differs on the parameters of being single stranded, harboring a ribose sugar and thymine being replaced by uracil. DNA undergoes transcription to yield RNA the process mediated by RNA polymerases which is further translated to yield the building blocks of life-proteins. A fine orchestra of RNA and protein assembly is involved to produce life’s key molecular machine, ribosomes the site for protein synthesis, which is nature’s marvelous ergonomic design. Diversity exists in forms, structure and function of RNA and their crucial role in fine tuning and regulating intricate gene expression and being the genome material of viruses has evoked great interest globally to unveil these molecules. Thus RNA biology takes centre stage of today’s therapeutic scenario.

Types and Diversity of RNAs (their origin and implications)

       The diversity of RNA is mind boggling with pleotropicity in their functional assignments.      The family is mainly represented by coding and non coding RNAs. The transcriptomal process involves a consortium

Correspondence to Vinod Bhakuni, Division of Molecular and Structural Biology, CDRI, Lucknow    bhakuniv@rediffmail.com

of four major RNAs the hnRNA (hetronuclear) being processed further to yield m-RNA by polyadenylation at the 3’ tail and capping at 5’ end this forms the coding RNAs. All other RNA falls under the broad umbrella of non-coding RNA. The rRNA (ribosomal) and proteins combine together to form macromolecular machines called ribosomes which serve as site for protein synthesis. The tRNAs are the carrier and forwarding agents which read the code (codon –anticodon matching) and bring about one aminoacid from the cellular pool to be attached each time in the elongating chain of polypeptides. The cell harbours twenty different types of tRNAs which are appropriately recruited to form diverse protein molecules.

       The non coding RNAs form an even more interesting group of small RNAs. The members have quite interesting functional attributes. The siRNA christened as small interfering RNA or silencing RNA is a class of 20-25 nucleotides, double stranded RNA molecules which are mainly involved in RNAi (RNA interference) pathway. The molecule is involved in PTGS (Post Transcriptional Gene Silencing), antiviral mechanism and chroma in remodelling and dynamics. The enigma is now being investigated in greater details for discovery of the phenomenon by David Baulcombe’s in plants and Thomas Tuschl in mammalian cells which has opened up new vistas in biomedical research and drug devlopment and paved a new branch of interference biology.

       Micro RNA (mi RNA) is another member belonging to gene regulatory small RNAs. These 21-23 nucleotide species are synthesised and processed from single stranded RNA precursors and show partial complementarity to m-RNA target molecule. These intresting molecules have multiple function with their role in cellular growth, apoptosis, neuronal remodelling, their enhanced presence may lead to Fragile X mental retardation. They have been further attributed to cancer. These undergo various processing to form a dismantling machine that finally chew up m-RNA molecules thus regulating and sometime disorienting normal gene expression. Small nucleolar RNA (sno RNA) or sn RNA (small nuclear RNA) are mainly responsible for bringing out biochemical modification of such as methylation and pseudouridylation of rRNA, tRNA and other small nuclear RNA.

       There are some other types of RNAs such as telomerase RNA which provides RNA template that is acted upon by telomerase, a reverse transcriptase to synthesize DNA at the chromosomal ends as it gets shortened up in each replication cycle. The telomerase has role in aging and cancer and also in cri-du chat syndrome. RNase MRP RNA is a subunit component of mitochondrial RNA processing (MRP) enzyme complex. This enzyme is involved in multiple cellular RNA processing and is associated with cartilage-hair hypoplasia (CHH), a recessively inherited devlopmental disorder. Ribonuclease P (RNase P) is another type of catalytic RNA (Ribozymes) discovered by Sidney Altman (Nobel Prize in Chemistry 1989)-- it cleaves extra precursor RNA from tRNA molecules[1]. It’s further attributed to efficietly control transcription of small non coding RNA genes. Vault RNA (vRNA) is vault ribonucleoprotein complex constituted of major vault protein (MVP) and two minor vault proteins (VPARP and TEP1)  with small untranslated RNA molecules-- these are mainly attributed to drug resistance[2]. The YRNA are part of ribonucleoprotein particle (Ro RNP) first identified by Learner et.al as target of autoimmune antibodies in systemic lupus erythmatosus (SLE). Its main function is to put a quality check on maturing 5S rRNA and is proved to be required for DNA replication [3]. Piwi-interacting RNA (pi RNA) is the largest class of small RNA molecules expressed in animal cells[4]. They form riboprotein complex on interacting with piwi protein. They are crucial in transcriptional gene silencing of retrotransposons mainly in germ line cells. They are very unique from other family by virtue of their complexity, no sequence conservation and being 26-31 nucleotides in length.

       The RNA viruses or retroviruses have RNA as their genetic material. There biology opens an entirely new and exciting field in virology and pathogenesis that is RNA toxicity.

       Thus existence of a galaxy of RNA, sharing the common backbone structure but markedly differing in the functional arena controlling process of gene regulation, protein synthesis silencing genes, regulating chromatin dynamics and acting as a quality control machinery, all add to sea of avenues for harnessing their therapeutic potential and thus opening an exciting oddyssey for candid evaluation of the molecule in fathomic details.


















RNA and Disease (Mutations in exons/introns/RNPs)

       The transcriptome comprises of coding as well as significant regions of non coding RNA. The non coding sequences were earlier thought to be junk and no functional attribute was associated with them but with the advancement in trascriptomics they were known to influence  and fine tune gene regulation. Expansion of the microsatellite repeats in the non coding regions resulted in the synthesis of pathogenic RNA’s now thought to be the culprit behind some dominantly inherited neurological disorders[5]. The gain of function effects by these non coding regions can be attributed to different pathological consequences. Expression of the toxic RNA is associated with formation of nuclear inclusions and late-onset degenerative changes in brain, heart or skeletal muscle. Myotonic dystrophy is caused by one such phenomenon where regulation of alternative splicing gets compromised due to sequestering of RNA binding proteins by toxic RNAs.

       The finest example of toxic role of RNA is polyglutamine disease. In this case dangerous molecular shapes, that resulted by formation of hairpins in case of long CAG repeats were efficiently examined by altering the sequence CAACAG so that no more hairpin could be formed but the sanctity of protein under question and the blueprint of it remain unpertuerbed. It was found that this very alteration drastically reduces neurodegeneration by scrambling the RNA  structure mitigated toxicity. This common theme of triple repeat expansion diseases is also prevalent in fragile x syndrome and myotonic dystrophy.

       Myotonic Dystrophy (DM) in which either a CTG or CCTG expansion, located within noncoding regions of separate genes, results in strikingly similar effects. The role for an RNA gain-of-function has been firmly established as a major pathogenic event in DM. There is now substantial evidence that other diseases caused by noncoding expansions involve an RNA gain-of-function mechanism [6]. These diseases include fragile X tremor ataxia syndrome (FXTAS), spinocerebellar ataxia type 8 (SCA8), SCA10, SCA12, and Huntington's disease like 2 (HDL2)[7]. Recent progress in DM has provided a paradigm for understanding pathogenic mechanisms of RNA mediated disorders.

       Fragile X Syndrome (FXS) is caused by expansion of trinucleotide gene sequence CGG on X chromosome. This resulted in inability to express FMR-1 protein which is crucial for neuronal development. It’s an inherited mental impairment[8].

       The diseases which are based on toxic RNA are goverened by common mechanism of gain of function effect. An insight into the disorders that are being governed by toxic RNA becomes crucial due to absence and nearly no concrete therapeutic regimen against them.


Representative list of some common RNA mitigated diseases

Spinal muscular atrophy                                     (SMA)                                                   SMN2 splicing

Dyskeratosis congenita                                      (X-linked)                                              DKC1                                                                                                                                                                                                     telomerase/translation

Prostate cancer                                                                     SNHG5                                                   ribosome biogenesis

Myotonic dystrophy, type 1 (DM1)                                 DMPK (RNA gain of function)          protein kinase

Myotonic dystrophy, type 2 (DM2)                                 ZNF9 (RNA gain of function)            RNA binding

Spinocerebellar ataxia 8 (SCA8)                         ATXN8/ATXN8OS                                                                                                             (RNA gain of function)                                                                unknown/noncoding RNA                

Fragile X-associated tremor ataxia                    FMR1(RNA gain of function)                                                                            syndrome                                                                                                              translation/m-RNA localization

Fragile X syndrome                                             FMR1                                                     translation/m-RNA                                                                                                                                                                              localization

Retinitis pigmentosa                                            PRPF31                                                  splicing

Retinitis pigmentosa                                            PRPF8                                                    splicing

Retinitis pigmentosa                                            HPRP3                                                    splicing

Retinitis pigmentosa                                            PAP1                                                      splicing

Autism                                                                   7q22-q33 locus breakpoint                 noncoding RNA

Beckwith-Wiedemann syndrome                      (BWS) H19                                           noncoding RNA

Cancer                                                                    SFRS1                                                    splicing, translation, export

Cancer                                                                    RBM5                                                     splicing

Cancer                                                                    miR-17-92 cluster                  RNA interference

Cancer                                                                    miR-372, miR-373                    RNA interference


New Approaches of RNA (Therapeutics/ siRNA)

       The Human Genome Project delineated about 34,000 genes that code directly for functional proteins. Rest of the genome has been labeled as “junk” because of no obvious function[9]. Recently, RNA biology received global attention with a paradigm shift that the junk genome produces around half a million varieties of RNA which must be having regulatory roles rather than an evolutionary burden. This has open new vistas in research with discussions centered on staggering variety of RNA types produced from this “junk” and the huge potential implications that the finding promises. Specific genes associated with diseases processes can be targeted using RNA interference (RNAi)[10]. This innovative approach has great therapeutic implications, thus this very idea of harnessing the process as a therapeutic product will herald a new dimension in gene therapy and nucleic acid based therapeutics.

       The technology has been a boon since the first report came in 2001 regarding RNA mediated silencing of respiratory syncytial virus (RSV)[11]. The technology has undergone several studies and promises attractive alternative in case of hepatitis B and C virus (HBV and HCV, respectively) including dengue virus (DENV), Japanese encephalitis virus (JEV), yellow fever virus (YFV) and West Nile virus (WNV), recent report regarding HIV-1 reveals that targeting the chemokine receptor CCR5 host protein that act as coreceptor for the virus but whose mutation is compatible with normal life can be used for attenuation.

       Apart from viral diseases, protozoans that particularly cause havoc have also been shown to get attenuated and silenced. In Trypanosoma brucei dsRNA could induce sequence-specific m-RNA degradation [12].The study in case of plasmodium revealed that the mechanisms of RNAi like silencing do exist in plasmodium. But still dilemma exista as unlike T.brucei, P. falciparum has no relevant homolog to Dicer, Piwi, PAZ or other genes involved in the RNAi pathways [13,14]. The studies also become quite pivotal due to widespread resistence against currently available antimalarials.

       Mycobacterium tuberculosis the world’s most successful pathogen which does evade almost all available chemotherapy by its multidrug resistence has shown promising results in feasibility of utilizing antisense technology. One group has shown that when phosphorothioate-modified antisense oligodeoxyribonucleotides were used against the m-RNA of glutamine synthetase associated with Mycobacterium pathogenicity and formation of a poly-L-glutamate/glutamine cell wall structure, it reduces the expression and activity thereby having profound impact on bacterial replication[15]. Another recent study reports inhibition of mycobacterial growth by inhibition of the lysosomal enzyme beta-exosaminidase, which is a peptidoglycan hydrolase that facilitates mycobacteria-induced secretion of lysosomes at the macrophage plasma membrane [16].

       Thymine production is controlled by DHFR (Dihydro Folate Reductase) which is very important for rapidly dividing cells. Inhibiting DHFR will prevent the growth of neoplastic cancerous cells from ordinary cells that do get transformed in to cancerous cells as in prostate cancer (Dr Alexandre Akoulitchev, University of Oxford). Thus an RNA swith could efficently turn off cancer[17].

       Thus, a birds eye view of the landscape that the technology promises are virtually interesting because it provides an upper hand to silence toxic genes by using an endogenous mechanism that is inherently present in most of the organisms. Be it viral diseases or protozoan or dreaded diseases like tuberculosis and cancer all these can be visualised to be manipulated by this RNA based therapeutics.


RNA and Future

       This small molecule has been the pool of panacea for treatment of malignant diseases and rescue from other wide variety of old and emerging diseases. The current generation of targeted therapy, however, is not amicable to many new therapeutic targets and increasing drug resistance among patients which add to the burgeoning severity. The new school of thoughts now clearly put-forth the importance of genome based safe therapeutics including the RNA technology. Antisense armamentorium when amalgamated with the advance nanoscience and drug delievery strategy is surely bound to fulfill the long cherished dream of biochemists to device “magic bullets” for treatment of whole spectrum of diseases including cancer and AIDS. Recent reports suggest that RNA itself can be ergonomically prototyped to dock many therapeutic molecules simultaneously and target it to particular cell type. The fact that microRNA (miRNA) are evolutionarily conserved, suggests that miRNA therapeutics may have fewer side effects as compared to the artificial siRNAs. At present the miRNA/RNA-i therapeutics field is in its juvenile stage but a wave of optimism exists in the science fraternity to end up the gestation and prepare a firm platform for the birth of RNA therapeutics.



[1]     Guerrier-Takada C, Marsh T, Pace N, Altman S The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme, Cell 35 (1983) 849–857.

[2]     V.A. Kickhoefer, Y. Liu, L.B. Kong, B.E. Snow, P.L. Stewart, L. Harrington, L.H. Rome, The Telomerase/vault-associated protein TEP1 is required for vault RNA stability and its association with the vault particle, The Journal of cell biology 152 (2001) 157-164.

[3]     A. Belisova, K. Semrad, O. Mayer, G. Kocian, E. Waigmann, R. Schroeder, G. Steiner, RNA chaperone activity of protein components of human Ro RNPs, RNA (New York, N.Y 11 (2005) 1084-1094.

 [4]    A.G. Seto, R.E. Kingston, N.C. Lau, The coming

of age for Piwi proteins, Molecular cell 26 (2007) 603-609.

[5]     L.P. Ranum, T.A. Cooper, RNA-Mediated

Neuromuscular Disorders, Annual review of neuroscience  (2006).

[6]     G. Tiscornia, M.S. Mahadevan, Myotonic dystrophy: the role of the CUG triplet repeats in splicing of a novel DMPK exon and altered cytoplasmic DMPK m-RNA isoform ratios, Molecular cell 5 (2000) 959-967.

[7]     T.A. Cooper, L. Wan, G. Dreyfuss, RNA and disease, Cell 136 (2009) 777-793.

[8]     S.J. Moore, L. Strain, G.F. Cole, Z.





Miedzybrodzka, K.F. Kelly, J.C. Dean, Fragile X syndrome with FMR1 and FMR2 deletion, Journal of medical genetics 36 (1999) 565-566.

[9]     W.W. Gibbs, The unseen genome: gems among the junk, Scientific American 289 (2003) 26-33.

[10]   N. Agrawal, P.V. Dasaradhi, A. Mohmmed, P. Malhotra, R.K. Bhatnagar, S.K. Mukherjee, RNA interference: biology, mechanism, and applications, Microbiol Mol Biol Rev 67 (2003) 657-685.

[11]   V. Bitko, S. Barik, Phenotypic silencing of cytoplasmic genes using sequence-specific double-stranded short interfering RNA and its application in the reverse genetics of wild type negative-strand RNA viruses, BMC microbiology 1 (2001) 34.

[12]   H. Ngo, C. Tschudi, K. Gull, E. Ullu, Double-stranded RNA induces m-RNA degradation in Trypanosoma brucei, Proceedings of the National Academy of Sciences of the United States of America 95 (1998) 14687-14692.

[13]   L. Aravind, L.M. Iyer, T.E. Wellems, L.H. Miller, Plasmodium biology: genomic gleanings, Cell 115 (2003) 771-785.

[14]   E. Ullu, C. Tschudi, T. Chakraborty, RNA interference in protozoan parasites, Cellular microbiology 6 (2004) 509-519.

[15]   G. Harth, M.A. Horwitz, D. Tabatadze, P.C. Zamecnik, Targeting the Mycobacterium tuberculosis 30/32-kDa mycolyl transferase complex as a therapeutic strategy against tuberculosis: Proof of principle by using antisense technology, Proceedings of the National Academy of Sciences of the United States of America 99 (2002) 15614-15619.

[16]   I.C. Koo, Y.M. Ohol, P. Wu, J.H. Morisaki, J.S. Cox, E.J. Brown, Role for lysosomal enzyme beta-hexosaminidase in the control of mycobacteria infection, Proceedings of the National Academy of Sciences of the United States of America 105 (2008) 710-715.

[17]   I. Martianov, A. Ramadass, A. Serra Barros, N. Chow, A. Akoulitchev, Repression of the human dihydrofolate reductase gene by a non-coding interfering transcript, Nature 445 (2007) 666-670.















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Genesis R&D Taps International Partner for Gene Silencing

       Genesis Research and Development Corp., the Auckland-based biotechnology company, is planning to tap an international venture capital group to partner on a new subsidiary to undertake its gene silencing project.

       The company expects to finalise the new investment within the next few weeks, and will retain a majority holding in the subsidiary, chief executive Stephen Hall told BusinessWire. The company told shareholders yesterday it has an “urgent” need for cash, with funds on hand currently standing at NZ$ 300,000. Genesis will offer a share purchase plan to raise funds while it attempts to sell some assets and recoup debt.

       Genesis applied to patent its gene silencing technology, which uses RNA interference to target the growth and drug resistance of cancer cells. The company confirmed it would focus on the technology and seek a partner to continue its development of the RNAi mechanism.


Minicells' Breakdown Cancer Resistance

       Australian researchers have developed a new technique that could prevent resistance in cancer cells. The researchers say the breakthrough could lead to cheaper cancer treatments with fewer side effects.

       The new therapy used minicells to deliver cancer therapy drugs to resistant tumours in mice. The minicells were made from bacteria and contained pieces of genetic material known as short interference RNA (siRNA), which knockout or 'silence' the drug-resistant genes of tumours. Several days later, researchers injected another dose of minicells filled with chemotherapy drugs, which the tumour was previously resistant to.

       Molecular biologist and joint director of the biotechnology company Engeneic Dr Himanshu Brahmbhatt says cancer cells have an inbuilt mechanism to develop drug resistance over time. "[Drug resistance] is one of our biggest killers in terms of cancer therapy," he says. Brahmbhatt says by silencing the genes of the drug-resistant tumour cell, the cancers become sensitive to the chemotherapy again.

       Previously, researchers believed siRNA was unable to pass through cell membranes due to their size. But Brahmbhatt says their research has shown this isn't necessarily the case. "Bacterial membranes might be quite different because they have protein channels [in their membrane] through which siRNA's can enter [the minicell]." As well as being packed with gene silencing siRNA, the outside of the minicell membrane is coated with antibodies. Brahmbhatt says these antibodies to lock onto [antigen] receptors on the tumour cells. "The cancer cell then swallows the entire minicell."

       Once the minicell is inside the cancer cell, its breaksdown and the siRNA or the drug floods the interior of the tumour cell, says Brahmbhatt. "That's why we haven't seen any toxic side effects because this is intra-cellular delivery."

       The study shows that the combined minicell therapy can inhibit the growth of drug-resistant tumour xenographs, artificially manufactured tumours, for up to four months. Brahmbhatt says their studies have now moved beyond using mice models to dogs suffering relapsed cancers. "We've treated these dogs with sequential treatment, siRNAs followed by drugs, and we are getting the same sort of results in these animals with real cancers." Medical oncologist Stephen Clarke, of the University of Sydney says this research is proof that siRNA can be delivered to living creatures and produce the desired effect. He says the researchers should be applauded for their novel approach.

       But Clarke says this type of therapy is a long way off clinical use and more research needs to be done to ensure complications don't arise in humans. Clarke says the foreign antibodies on the outside of the minicells could create an immune response and negate their effect. Even if the antibodies don't activate the immune system, they may not be able to attach to all tumour cells, he says. "Human tumours are so molecularly diverse, it's possible that if not all the tumour are expressing the protein [antigen] you're targeting, you may have a patchy effect," says Clarke.


Genome-wide Map Shows Precisely Where microRNAs Do Their Work

       MicroRNAs are the newest kid on the genetic block. By regulating the unzipping of genetic information, these tiny molecules have set the scientific world alight with such wide-ranging applications as onions that can’t make you cry and therapeutic potential for new treatments for viral infections, cancer and degenerative diseases. But the question remains: How do they work?

       Robert B. Darnell, head of the Laboratory of Molecular Neuro-oncology, and his team at Rockefeller University provide a long-awaited key clue to answering that question. By using a technique that molecularly cements proteins to RNAs, the team has decoded a map of microRNA-messenger RNA interactions in the brain, an advance that holds promise for biology and human disease, for example by silencing trouble-making genes linked to disease.

       MicroRNAs rewrote the rules of gene expression in 2001 when they were found to bind to messenger RNA and shut down protein production, a process called RNA interference. By 2006, when the Nobel Prize in medicine was given for the discovery of RNA interference, scientists around the globe had even narrowed down microRNAs’ primary site of action to somewhere around the end of the RNA transcript. What scientists couldn’t nail down was the exact string of nucleotides to which the microRNAs bind along a messenger RNA transcript. “To understand exactly how microRNAs work, you want to know their precise targets,” says Darnell, who is a Howard Hughes Medical Institute investigator and Robert and Harriet Heilbrunn Professor at Rockefeller. “You want a map that tells you which messenger RNAs each microRNA targets and exactly where they are binding.”

       The problem was that on any given messenger RNA, there are many sites to which a single microRNA can theoretically bind, and there are hundreds of microRNAs in every cell. Prior techniques — primarily relying on computer predictions — weren’t very good at sorting through the morass of predictions to identify the real sites, explains Darnell. The trick to getting such a map was to freeze a snapshot of microRNAs directly bound to messenger RNA in living cells. Working specifically in mouse brain tissue, that’s what Darnell and his team did using a technique the lab developed called high throughput sequence-crosslinking immunoprecipitation, or HITS-CLIP.

       In order to shut down a gene before it is translated, microRNAs must be guided to their target messenger RNAs via a protein called Argonaute. The Argonaute-microRNA-messenger RNA complex now forms a sandwich structure where the microRNA is compressed in the middle. By using their technique to fuse Argonaute to these two RNAs, the team was then able to identify the bound microRNA and its precise target sites across all messenger RNAs expressed in the mouse brain.


Undruggable Cancer Genes May Not Be Invincible

       Nearly a third of all cancers have mutations in the gene KRAS, yet there are no drugs to combat these changes. And KRAS is not unusual—other common cancer genes are also considered "undruggable." But as two new studies report, cancer cells driven by these genes may be vulnerable to another kind of attack. A technology called RNA interference can identify "normal" genes in tumor cells that are required for the survival of these cells, and one of these genes may turn out to be an Achilles' heel.

       Two independent groups led by researchers at Harvard Medical School used this strategy to search for potential vulnerabilities in cancer cells with KRAS mutations. Both groups found proteins that were essential for the viability of the cells, including some protein kinases. These are promising targets because they can be inhibited by drugs. Imatinib (Gleevec) is one example.

       The researchers used short bits of RNA to target and silence individual genes in cells. While clinical trials will be needed to learn whether patients benefit from this approach, the findings are supported by smaller studies, including one that revealed potential drug targets for diffuse large B-cell lymphoma.

       "What these screens do is give us potential leads for new cancer drugs," said Dr. Stephen Elledge, one of the lead investigators. "There must be whole networks of non-oncogenes out there that tumors depend on," he noted, but the genes are difficult to find because they do not have any mutations or alterations.

       His team identified a number of genes related to mitosis, including one called PLK1, that are potential therapeutic targets. In tumor cells, KRAS mutations altered the fidelity of mitosis in a way that made the cells die when these genes were inhibited. Thus, cells with KRAS mutations may be vulnerable to antimitotic drugs that target these genes, the researchers said.

(National Cancer InstituteJune 16, 2009 • Volume 6 / Number 12)

Carbohydrate Acts as Tumor Suppressor

       Scientists at Burnham Institute for Medical Research have discovered that specialized complex sugar molecules (glycans) that anchor cells into place act as tumor suppressors in breast and prostate cancers. These glycans play a critical role in cell adhesion in normal cells, and their decrease or loss leads to increased cell migration by invasive cancer cells and metastasis. An increase in expression of the enzyme that produces these glycans, β3GnT1, resulted in a significant reduction in tumor activity.

       The specialized glycans are capable of binding to laminin and are attached to the α-DG cell surface protein. This binding facilitates adhesion between epithelial and basement membrane cells and prevents cells from migrating. The team of scientists, led by Professor Minoru Fukuda, demonstrated that β3GnT1 controls the synthesis of laminin-binding glycans in concert with the genes LARGE/LARGE2. Down-regulation of β3GnT1 reduces the number of glycans, leading to greater movement by invasive cancer cells. However, when the researchers forced aggressive cancer cells to express β3GnT1, the laminin-binding glycans were restored and tumor formation decreased.

       "These results indicate that certain carbohydrates on normal cells and enzymes that synthesize those glycans, such as β3GnT1, function as tumor suppressors," said Dr. Fukuda." Upregulation of β3GnT1 may become a novel way to treat cancer."



MicroRNAs Help Control HIV Life Cycle

       Scientists at Burnham Institute for Medical Research (Burnham) have discovered that specific microRNAs (non-coding RNAs that interfere with gene expression) reduce HIV replication and infectivity in human T-cells. In particular, miR29 plays a key role in controlling the HIV life cycle. The study suggests that HIV may have co-opted this cellular defense mechanism to help the virus hide from the immune system and antiviral drugs.

       Tariq Rana, director of the Program for RNA Biology at Burnham, and colleagues, found that the microRNA-miR29 suppresses translation of the HIV-1 genome by transporting the HIV m-RNA to processing-bodies (P-bodies), where they are stored or destroyed. This results in a reduction of viral replication and infectivity. The study also showed that inhibition of miR29 enhances viral replication and infectivity. The scientists further demonstrated that strains of HIV-1 with mutations in the region of the genome that interact with miR29 are not inhibited by miR29.


New Clues to Cholesterol Control

       Scientists have uncovered 20 "cholesterol control" genes that could help point to important new risk factors for heart disease. The researchers looked for genes with similar patterns of behaviour to those already known to be involved in cholesterol regulation.

       They then tested the activity of the 100 most promising candidates with a scientific technique called RNA interference (RNAi). The technique uses tiny bits of the genetic molecule RNA to block the protein-making "instructions" issued by genes. In this way, the function of genes can be assessed by effectively switching them off. The strategy identified 20 genes described as "immediately relevant" for maintaining cellular levels of cholesterol.

       Some them are thought to influence levels of low-density lipoprotein, or "bad" cholesterol, in the blood, a major heart disease risk factor. Study leader Dr Heiko Runz, from the University of Heidelberg in Germany, said: "High cholesterol in the blood is considered to be responsible for excess cardiovascular morbidity (illness) and mortality."Blood cholesterol levels are controlled by cholesterol in cells. Therefore, some of the genes identified by us as regulators of cellular cholesterol in future studies might turn out to be disease genes that contribute to hypercholesterolaemia (high cholesterol) in some cases."

Oncolytic Adenovirus Mediated Survivin Knockdown by RNA Interference Suppresses Human Colorectal Carcinoma Growth in vitro and in vivo.

       Colorectal cancer is a one of the most common alimentary malignancies. Survivin has been proved by many studies to be an ideal target for cancer gene therapy because of its strong anti-apoptotic effect. The reduction of Survivin expression by means of chemically synthesized small interfering RNA or small hairpin RNA expressed from plasmid and resulted growth inhibition of cancer cells had been proved by many studies including ours, but the transfection efficiency was not encouraging. So for the first time we constructed the Survivin shRNA into an oncolytic adenovirus, tested its effects on colorectal cancer cell lines and nude mice xenograft model. In this study, researchers constructed an oncolytic adenovirus with a Survivin targeted small hairpin RNA and a reporter gene (ZD55-Sur-EGFP). The expression of Survivin m-RNA and protein were analyzed by RT-PCR and western blot. The cell growth and apoptosis were tested by in vitro cytopathic assay, MTT assay and flow cytometry respectively. The effect of the constructed virus on xenograft model was evaluated by tumor volume and western blot analysis. RESULTS: ZD55-Sur-EGFP replicated in cancer cells specifically, reduced the expression of Survivin m-RNA and protein expression effectively (P < 0.0001), induced cancer cell apoptosis and inhibited SW480 cell growth both in vitro and in vivo significantly. We conclude Survivin RNA interference combining with oncolytic adenovirus virotherapy could be a promising treatment for colorectal cancer.

(J.of Experimental & Clinical Research 2009, 28; 81)





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MicroRNAs in Diseases and Drug Response



       MicroRNAs (miRNAs) are a class of endogenous non-coding RNAs of 19–24 nucleotides in length that play an important role in the negative regulation of gene expression by base-pairing to complementary sites on the target m-RNAs, causing a block of translation or degradation of the target m-RNA  The biogenesis of microRNAs is a complex and coordinated process in which different groups of enzymes and associated proteins, located in the nucleus and in the cytoplasm, carry out the multistep maturation of these tiny RNAs .

       The most important characterization of the function of microRNAs is the identification of m-RNA targets. Because the animal miRNAs have a 5′end restricted complementarity to the m-RNA target (only 5–8 nucleotides perfectly complementary), the RNA sequence named ‘seed region’, the miRNAs are predicted to regulate a large number of animal genes. Different algorithms have been developed to predict the animal miRNA targets; they are based on different criteria, resulting from the analysis of the in vivo demonstrated targets: (i) perfect or nearly perfect base-paring at the seed region and thermodynamically stability of the duplex miRNA–m-RNA; (ii) phylogenetic conservation of the seed region; (iii) multiple target sites in a single target by the same or different miRNAs; (iv) absence of strong secondary structures at the miRNA-binding site on the target. Several computational procedures are available to predict microRNA targets.

       In some studies, key target m-RNAs have been identified, but still relatively little is known of the functional role of miRNAs in mammalian species. It is known, however, that the miR-let7 family may play a role in oncogenesis via RAS oncogene m-RNAs. Enforced expression of the miR-17-92 cluster from chromosome 13q32-33, in conjunction with c-myc, accelerates tumor development in a mouse B cell lymphoma model .

       Functional studies indicated that miR-221 and 222 inhibit normal erythropoiesis and erythroleukemic cell growth at least partly via Kit receptor downmodulation, and their ectopic overexpression directly results in p27Kip1 downregulation in aggressive prostate Although miRNAs operate in a similar fashion as short interfering RNAs (siRNAs) , they typically target several transcripts instead of one specific gene , regulating about 30% of the protein coding genes of the human genome. MiR-21 has not only a role in tumorigenesis through regulation of the tumor suppressor genes tropomyosin 1 (TPM1) and PTEN, but is also involved in invasion and tumor metastasis by targeting programmed cell death 4 (PDCD4) and maspin.

       Most genes that are targeted by miRNAs appear to have multiple, co-clustered binding sites for multiple miRNAs in their m-RNA, and miRNA genes are thus likely to act combinatorially on target genes.

       Furthermore, miRNAs are differentially expressed in developmental stages, cell types, and tissues, and recent data suggest their role not only in physiological processes but also in many human diseases, including cancer, raising hope for the development of new drugs that targeting microRNAs and their gene networks could sensitize cells to therapy with greater efficacy.


MicroRNAs and Diseases  

       Currently the biological functions of miRNAs are actively being sought. Some studies have notably uncovered roles for miRNAs in cellular processes including apoptosis, proliferation, stress resistance, metabolism, defense against pathogenic infections, neural development and importantly, tumorigenesis .

Table 1.

Examples of microRNAs, which have been shown to be modulated in many pathological diseases, are indicated with their putative m-RNA targets


miRNAs Involved

Putative m-RNA(s) targets
















Liver dysfunction



Hepatic viral infection


HCV (Heptitis C virus)
















Sustained cardiac hyperthropathy


RhoA, Nelf-A/WHSC2



Kcnj2, GJA1



       To date, most efforts have been directed toward the study of alteration of miRNA expression in many diseases. Recent evidence suggests that miRNAs may be a contributing factor in neurodegeneration. Furthermore, studies in mammals and in invertebrates have suggested that miRNAs are involved in neuroprotection, fragile X syndrome , and schizophrenia. Together, these observations indicate that neurodegenerative diseases might result from the alteration of different cellular pathways, and miRNAs may have a role.

miRNAs and Cancer

       Although studies linking miRNA dysfunctions to human diseases are in their infancy, a great deal of data already exist, establishing an important role for miRNAs in the pathogenesis of cancer. The first evidence that miRNAs are involved in cancer comes from the finding that miR-15 and miR-16 are downregulated or deleted in most patients with chronic lymphocytic leukemia. This discovery has projected miRNAs to the center stage of molecular oncology. Several groups, in the past few years, have studied miRNA expression in cancer patients and found that miRNAs are differentially expressed not only in normal and tumor tissues  but are also differentially expressed in primary tumors and metastatic tissue . These differences are tumor specific and in some cases are associated with prognosis. Evidence indicates that some miRNAs can function either as oncogenes or tumor suppressors .

       The let-7 family contains miRNAs that have been shown to regulate the RAS family of oncogenes through post-transcriptional repression. Studies have indicated that a miRNA by itself could induce a neoplastic disease. Genome-wide analysis of miRNA expression in different steps of gastric carcinogenesis was performed. Deregulation of E2F1 activity and resistance to TGF-β are hallmarks of gastric cancer. miR-106b-25 cluster upregulation impairs the TGF-β tumor suppressor pathway, interfering with the expression of p21Waf1/Cip1 and Bim. These results suggest that the miR-106b-25 cluster plays a key role in gastric cancer interfering with proteins involved both in cell cycle and apoptosis .

       Researchers indicate that miR-126 and miR-335, whose expression is lost in human breast cancer cells, modulate metastatic potential.

MicroRNAs and Drug Response

       MiRNAs are attractive drug targets since they regulate expression of many proteins in the cell and are differentially expressed in malignant versus normal cells . Taking into account that according to recent computer predictions, each miRNA could have more than hundred of target m-RNAs in the cell, this multi-target regulation might be responsible for adverse or non-target effects in a future microRNA mediated-therapy.


       Since their discovery, 15 years ago, miRNAs have been recognized as mediators of transcription regulation. This review discusses their role in many diseases and an emerging, novel function of microRNA: their role in drug resistance.

       The molecular genetic basis of sensitivity and resistance to cancer therapeutics is complex, involving multiple processes such as drug transport, drug metabolism, DNA repair, and apoptosis. It is proposed that expression of more than one third of human genes are under microRNA control; this explains their wide action in many diseases, including cancer. Therefore, many genes that are drug targets may be regulated by miRNAs. A miRNA that targets tumor suppressor and/or pro-apoptotic proteins acts as an oncogene, giving rise to drugs resistance by inhibiting apoptosis and enhancing cell cycle. Conversely, a miRNA that targets an oncogene and/or anti-apoptotic proteins acts as a tumor suppressor, leading to drugs sensitivity by enhancing apoptosis and/or by blocking cell cycle. Here, a general model has been provided by which tumor cells could respond to drug treatments, but, obviously, other mechanisms such as gene amplification, deletion, or translocation can be involved in tumor development and drug response .

       Therefore, when combined with gene expression profiles and other biological data, microRNAs expression profile may provide crucial information for an understanding of cancer chemosensitivity and chemoresistance with very important implication in disease treatment and prevention.

(Based on the article written by Michela Garofalo et al and published in Current Opinion in Pharmacology (2008)8,661-67)





Alternative Splicing and Disease

1. General Principles of Alternative Splicing

1.1. Alternative pre-m-RNA splicing regulates the function of the majority of protein-coding genes

       An average human protein-coding gene contains a mean of 8.8 exons with a mean size of 145nt. The mean intron length is 3365nt and the 5′ and 3′ UTR are 770 and 300 nt, respectively; as a result, this “standard” gene spans about 27 kbp. After pre-m-RNA processing the average m-RNA exported into the cytosol consists of 1340 nt coding sequence, 1070 nt untranslated regions and a poly (A) tail. This shows that more than 90% of the pre-m-RNA is removed as introns, and only about 10% of the average pre-m-RNA is joined as exonic sequences by pre-m-RNA splicing. Almost all protein-coding genes contain introns that are removed in the nucleus by RNA splicing during pre-m-RNA processing. Exon usage is often alternative, i.e. the cell decides whether to remove a part of the pre-m-RNA as an intron or include this part in the mature m-RNA as an alternative exon. Alternative pre-m-RNA processing is a key regulator of gene expression as it generates numerous transcripts from a single protein-coding gene, which largely increases the use of genetic information. The process is more widely used than previously thought and was recently estimated to affect more than 88% of human protein-coding genes. An estimated 75% of alternative exons encode protein parts and their alternate use allows to generate multiple proteins from a single gene, which increases the coding potential of the genome. Mapping of alternatively spliced regions on known protein structures suggest that most alternative exons are in coiled or loop regions that are located on the surface . Alternative splicing generates protein isoforms with different biological properties that differ in protein:protein interaction, subcellular localization, or catalytic ability. More than a quarter of alternative exons introduce premature stop codons in their m-RNAs. This can result either in the formation of truncated proteins or in the degradation of the m-RNA in nonsense-mediated decay. Recent array analyses indicate that although frequently found, alternative exons with premature stop codons are present only in low abundance, which question their role as a general shut-off mechanism for protein production.

1.2. Changes in alternative splicing can be the cause or consequence of human diseases

       There are only a few reports of mutations in core elements of the splicing machinery that result in human diseases. For example, autosomal dominant forms of retinitis pigmentosa is caused by mutation in the splicing factors PRPF31/U4-61k . It is possible that defects in the general splicing machinery are generally not compatible with life, whereas changes in alternative splicing can be tolerated by an organism, although these changes might manifest in a disease. As alternative splicing affects numerous genes, it is not surprising that changes in alternative splicing are frequently associated with human diseases. It is often not clear whether a change in alternative splicing causes a disease or is an indicator for an underlying defect. A better mechanistical understanding of splice site selection has helped in distinguishing these effects. The first demonstration that exon sequences can have an effect on splice site selection was published 20 years ago . Ten years later, the first review about the impact of exonic mutations on splice site selection postulated that silent mutations can interfere with exon usage and explained how these mutations that do not change the predicted encoded protein can cause a human disease . Since then, a better understanding of alternative splice site selection contributed to a better understanding of human diseases and vice versa. The number of diseases reported to be associated with changes in alterative splicing increased dramatically and has been frequently reviewed , including in the form of a book. To facilitate the access to this fast growing area for colleagues in other fields, a brief summary disease-relevant aspects of splice site selection, are discussed with well-established examples of alternative splicing changes that lead to human disease and point out links between the diseases and aberrant splice site selection.

1.3. Alternative exons are regulated by combinatorial control through transient formation of recognition complexes

       Since splice sites follow only loose consensus sequences, the key questions in alternative splicing regulation are: How are splice sites recognized in the vast genomic sequence background, and how are they differentially regulated? The mechanism of alternative splice site recognition has been extensively reviewed. Exon recognition is regulated by the interaction of proteins and ribonuclear proteins (trans-factors), with sequence elements on the pre-m-RNA (cis-factors), which is summarized below.

1.4. Alternative exons are generated during evolution and their usage can be changed by point mutations located outside the splice sites

       Alternative exons can be generated by three mechanisms: (i) exon shuffling, where an existing exon is duplicated within the same gene and is then alternatively spliced, (ii) exonisation of mobile genetic elements, such as Alu elements  and (iii) a transformation of formally constitutive exons into alternative ones. Since the approximately one million human Alu elements are primate-specific elements that account for 10% of the human genome, their exonisation provides a large reservoir to generate new alternative exons. Numerous studies showed that synonymous mutations in coding regions can influence splice site selection. There is now also emerging evidence that intronic mutations and single-nucleotide polymorphism can alter exon usage. It is thus likely that alternative exon usage is an evolutionary ‘substrate’ that is subject to a large number of mutations. Due to the complexity of the splicing regulation, the effects of mutations are difficult to predict, but become obvious when they lead to human diseases.

       Each of the regulatory principles listed here can be altered to cause a human disease, which is schematically summarized in .

2. Examples of Diseases Caused by Alternative Splicing

       Exons associated with diseases mentioned in the text are listed. Since there are no unique accession numbers for exons, the NCBI accession number is listed. The mutations having an effect on splicing are listed under features and changed nucleotides are underlined. These nucleotides are underlined in the sequence. Capital letters are exons, small letters are introns.

2.1. Diseases caused by point mutations in regulatory sequences

       2.1.1. Spinal muscular atrophy as an example of a recessive disease caused by a point mutation in an exonic regulatory element

       Spinal muscular atrophy (SMA) describes several different diseases that are characterized by degeneration of alpha motoneurons in the brainstem and spinal cord. Autosomal recessive SMA associated with chromosome 5 is molecularly the best understood. It is characterized by progressive paralysis caused by the loss of alpha-motor neurons in the spinal cord. The incidence is 1:6000 to 1:10,000 for live births and the carrier frequency is 1 in 40 . SMA is the second most common autosomal recessive disorder.

       Since children suffering from cystic fibrosis now largely survive childhood, it is the most frequent genetic cause of infantile death. SMA is caused by the loss of the SMN1 gene that encodes the SMN protein, which regulates snRNP assembly. It is not clear how the loss of SMN protein causes the disease and leads to a specific death of motoneurons. Mouse studies revealed that the loss of SMN protein causes cell-type specific changes in snRNAs and a generally reduced snRNP assembly capacity. Numerous pre-m-RNA splicing events are deregulated in all tissues analyzed. Some of the changes observed reflect a shift in known alternative splicing patterns. However, the majority of the deregulated splicing events are aberrant m-RNAs, which are normally not produced. These findings suggest that (i) the selective death of motoneurons could be caused by the cumulative effect of aberrantly splicing m-RNAs and (ii) that changes in cells surrounding the motoneurons cause their death .

       Genetic studies identified six families with eight female members that were asymptomatic for SMA, although they inherited the same SMN1 and SMN2 alleles as their affected siblings . Plastin 3, an actin binding protein was identified as a modifier. Overexpression of plastin 3 in SMN knock-out mice partially rescued the short neuronal axon length causes by the absence of SMA protein. These findings argue that that the death of motoneurons could be caused by a mechanism different from a change in splicing. Although it is not understood how the loss of SMA protein causes the disease, it is clear that restoration of SMA protein production would be a therapeutic approach.

       Humans possess a gene, SMN2, that is almost identical to SMN1. SMN2 was generated through a recent duplication. Although both genes are almost identical in sequence, due to a translationally silent C→T change at position 6 in exon 7, they have different splicing patterns and exon 7 is predominantly excluded in SMN2. This exon-skipping event generates a truncated, less stable and probably non-functional protein. Therefore, SMN2 cannot compensate the loss of SMN1. At least one copy of SMN2 is retained in humans with SMA, as lack of both SMN2 and SMN1 is embryonically lethal. Mice have only one SMN gene where exon 7 is constitutively spliced. A homozygous knock out of this gene is lethal.

       To study the splicing regulation of the human gene in mice, transgenic animals that contain the human gene were developed . Although, in SMA patients the SMN protein is almost completely absent from all cells, for unknown reasons, alpha motoneurons are most severely affected and die, which causes the muscular atrophy. The disease can manifest in four phenotypes (type I to IV) that differ in onset and severity. The phenotypes correlate roughly with the number of SMN2 copies in the genome, most likely because more SMN2 copies produce more SMN protein. Since stimulation of SMN2 exon 7 usage would increase SMN protein levels and potentially cure the disease, work has concentrated on understanding the regulation of exon 7. Typical for the combinatorial control of exon regulation, multiple factors determine the regulation, including a suboptimal polypyrimidine tract, a central tra2-beta1-dependent enhancer  and the sequence around the C→T change at position 6. Recent large scale mutagenesis studies indicate that a composite regulatory exonic element termed EXINCT (extended inhibitory context) is responsible for the regulation of exon 7 inclusion . The exon skipping event is caused by the C→T change at position 6 and currently two models are proposed for its mechanism. In one model, the base exchange destroys the exonic enhancer that normally binds to SF2/ASF and in the other model, the mutation creates an hnRNPA1 binding site that acts as a silencer and . Both models can explain the predominant skipping of exon 7. Inclusion of exon 7 depends on a central tra2-beta1 enhancer sequence . Tra2-beta1 is an SR-related protein. Its activity is regulated by dephosphorylation mediated by protein phosphatase 1 and, not surprisingly, exon 7 usage depends on cellular PP1 activity .

SMN illustrates several common features of diseases caused by missplicing. Evolutionary changes in the genome, here the recent dublication of genes that facilitate their recombination, can manifest in splicing changes. Alternative exons are regulated by numerous factors and sequence elements and a single mutation can disturb the balance necessary for normal exon recognition. Finally, splicing factors are regulated by reversible phosphorylation controlled by cellular signaling pathways.

       2.1.2. Tauopathies as an example for a disease caused by a change in the ratio of protein isoforms generated by alternative splicing

       Tauopathies describe several diseases of the central nervous system that show intracellular accumulations of abnormal filaments that contain the microtubule associated protein tau. The tau protein is encoded by a single gene (MAPT, (microtubule associated protein tau) located on chromosome 17. The gene undergoes extensive alternative splicing and eight of the sixteen exons are alternatively spliced. In humans, these splicing events are spatially and temporally regulated. For example, exons 2, 3 and 10 are adult specific and show differences in splicing in various brain regions. The tau protein binds to microtubules via microtubule repeat regions. One of these microtubule binding regions is encoded by the alternatively used exon 10. Exon 10 inclusion creates a protein with four microtubule repeats (4R), whereas exon 10 skipping creates an isoform with three repeats (3R). This splicing event is species-specific in the adult. In humans, exon 10 is alternatively spliced in the adult, whereas in mice the exon is constitutively used. In both species, the exon usage is regulated during development.

       Genetic studies identified rare dominant mutations in the tau gene that caused frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), where currently 42 mutations are listed. The majority of the mutations affect the splicing regulation of exon 10 that encodes part of one microtubule binding site. The mutations in tau exon 10 helped dissect its regulatory elements. The exon shows an alternating arrangement of four enhancer and three silencer regions. A mutation that falls into a silencer or enhancer regions either promotes or decreases exon usage, respectively. Mutations in exon 10 alter its normal fraction of inclusion and changes of pre-m-RNA encoding 3R and 4R repeat tau isoforms were found associated with FTDP-17. These data clearly suggested that the splicing mutations cause the neuropathology by changing the ratio between the 3R and 4R isoforms. One mechanistically well understood mutation is N279K . This mutation is caused by changing a TAAGAAG into GAAGAAG. The GAAGAAG sequence forms the core of a tra2-beta1 binding site. Similar to the situation in exon 7 of SMN2, this mutated version contains two partially overlapping versions of the GAAG binding site. Biochemical studies showed that the mutation increases affinity to tra2-beta1 in vitro  and cotransfections experiments showed that tra2-beta1 promoted exon 10 inclusion in reporter gene constructs . In vitro studies showed that the asparagine to lysine exchange in the mutation does not alter the binding between tau and tubulin, the tau aggregation or microtubule assembly. These data suggested that mainly the change in the ratio of expressed isoforms is responsible for the disease. Testing this hypothesis in mouse models was difficult, as the mouse tau gene constitutively expresses the 4R isoform in the adult. Therefore, a minigene of human tau, containing the promoter and all exons flanked by shortened intronic regions was expressed in mice. These constructs show alternative splicing resembling the human situation and express human tau protein containing either 3 or 4 microtubule binding domains. When the mutation that promotes exon 10 inclusion (N279K) was introduced into exon 10 of this construct, pre-m-RNA splicing was shifted as expected towards exon 10 inclusion. Interestingly, the mice showed similar pathophysiology as humans with the same mutation and also showed behavioral changes . These data suggest that a change in the ratio between 3R and 4R tau isoforms is an important underlying cause for FTDP-17.

       2.1.3. Hutchinson–Gilford progeria syndrome as an example for a disease caused by an intronic mutation that activates a cryptic splice site

       Hutchinson–Gilford progeria syndrome (HGPS) is a rare genetic disorder phenotypically characterized by many features of premature aging. It is clinically characterized by postnatal growth retardation, midface hypoplasia, micrognathia, premature atherosclerosis, absence of subcutaneous fat, alopecia and generalized osteodysplasia. At birth, the appearance of patients is generally normal, but by one year of age patients show severe growth retardation, balding and sclerodermatous skin changes. Patients live a median of 13.4 years and die of heart attacks or congestive heart failure. Mutations causing HGPS have been identified in the nuclear lamin A/C (LMNA) gene. Lamin proteins are distributed throughout the nucleoplasm and are involved in numerous functions, including DNA replication, transcription, chromatin organization, nuclear positioning and shape, as well as the assembly/disassembly of the nucleus during cell division. Out of 14 mutations affecting lamin A/C, three have been reported to specifically alter lamin A splicing. The changes in splicing lead to the production of truncated protein products (p.G608G, p.T623S and IVS11+1G>A). Most of the typical Hutchinson–Gilford progeria cases are due to a recurrent, de novo point mutation in LMNA exon 11: c.1824C>T . This mutation occurs in a probable exon splicing enhancer. As a result, a cryptic splice site is activated in transcripts generated from the mutated allele, which is located 5 nucleotides upstream of the mutation. The use of the cryptic splice site leads to the production of a truncated Lamin A protein lacking the last 150 base pairs of exon 11. The truncated protein is called “progerin” and acts in a dominant fashion to generate the HGPS phenotype.

       This example shows how a mutation can cause activation of a nearby otherwise ‘hidden’, cryptic splice site.

       2.1.4. LDL receptor splicing variants caused by a single nucleotide polymorphism are a sex-specific factor for hypercholesterolemia

       Hypercholesterolemia is a major risk factor for arteriosclerosis. Low-density lipoproteins are removed from the bloodstream by the LDL receptor (LDLR). Mutations in the LDLR are a primary cause for hypercholesterolemia. Recently, a single nucleotide polymorphism was identified in exon 12 of the LDLR that promotes skipping of this exon. The SNP was found to promote exon 12 skipping in the liver of pre-menopausal women. However, the SNP had no effect on men and post-menopausal women. The SNP and the splicing pattern are associated with a higher level of cholesterol in pre-menopausal women, but not in men. Exon 12 skipping generates a truncated form of the receptor that lacks the transmembrane domain necessary for membrane binding and internalization. It is possible that the protein generated by exon 12 skipping prevents, in a dominant negative form, the uptake of LDL. This model explains the interesting finding that exon 12 skipping caused by this SNP is associated with cholesterol levels. The reason for the sex-dependency of the SNP is unclear, but is possible that high estrogen levels influence transcription level of the gene or its alternative splicing. Apo lipoprotein E (ApoE) is a ligand for the LDLR receptor and the apoE allele status is a major risk factor for Alzheimer's disease. It was therefore investigated whether the SNP in exon 12 of the LDLR associates with Alzheimer's disease. It was found that the SNP associates with an increased chance to develop Alzheimer's disease in males, but not females .

       This example nicely illustrates that a SNP can influence alternative splicing, which in turn predisposes to a disease. Reflecting the combinatorial control of alternative exon regulation, the result of a mutation depends on other factors, in this case the sex and age.

       2.1.5. Familial dysautonomia as an example for a disease caused by a mutation in the 5′ splice site

       About 10% of roughly 80,000 mutations reported in the human gene mutation database affect splice sites . Well-studied diseases caused by changes in splice site selection include thalassemias  and Familial dysautonomia (FD). FD, (also Riley–Day syndrome, hereditary sensory and autonomic neuropathy type III) is a recessive disease that is caused by loss of function of the i-kappa-B kinase complex associated protein (IKBKAP). In the Ashkenazi Jewish population, the incidence is 1/3600 in live birth (carrier frequency 1:30) . Affected children show abnormal development of the nervous system that is associated with demyelination in various regions. This leads to a large clinical spectrum that includes vomiting crises, unsteady gait, and decreased perception of pain. In more than 99.5% of FD patients the 5′ splice site of exon 20 is mutated T→C in position 6 of intron 20. This point mutation interrupts base pairing with U1snRNA. U1 snRNA interacts both with the last three nucleotides of the exon and the first six nucleotides of the downstream intron. The majority of 5′ splice sites show complementarity to seven base pairs of U1 snRNA. This means there are usually three mismatches between the 5′ splice site and U1 snRNA. Bioinformatic analyses indicate that these mismatches are not randomly distributed. They either weaken the exonic portion of the 5′ splice site, which is then compensated by strong binding to the intronic portion, or a weak intronic portion is compensated by a strong exonic portion. In exon 20 of the IKBKAP gene, the exonic part of the splice site is weak, due to an A at position − 1. The T→C mutation weakens the intronic part of the 5′ splice site, which causes exon skipping . Exon 20 usage is susceptible to a weak 5′ splice site, as the exon has a weak 3′ splice site that has an A at the − 3 position, and contains several exonic silencer elements . Array analysis indicates that IKBKAP promotes expression of genes involved in oligodendrocyte and myelin formation, which could explain the demyelination phenotype caused by the loss of IKBKAP .

       The example of FD illustrates the complexity of mutations in splice sites that have to be carefully analyzed within the context of other regulatory elements. It further shows that a missplicing event of a key regulatory gene can have profound impact by affecting other genes, and finally indicates that splicing is influenced by small substances.

       2.1.6. Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency illustrates how multiple mutations affect exon usage leading to a human disease

       Medium-chain acyl-CoA dehydrogenase (MCAD) is a mitochondrial enzyme that participates in the degradation of medium chain length fatty acids. Deficiencies of this enzyme are the most frequently diagnosed defect of mitochondrial beta-oxidation. The patients show metabolic crisis, characterized by hypoglycemia, lethargy and seizures, when first exposed to viral infections or challenged by fasting. About 20% of the infants die. MCAD deficiency results in accumulation of medium-chain acylcarnitines in the urine, which can be analyzed by mass-spectrometry. A large newborn screen showed an incidence from 1:15,000 in the US population. The major reason for the deficiency is a K304E missense mutation leading to a less active protein . The newborn screening project identified a 362C→T missense mutation in exon 5 of the MCAD gene that causes exon skipping and subsequent degradation of the m-RNA by nonsense-mediated decay . This mutation disrupts a splicing enhancer that is highly similar to the SF2/ASF enhancer in the SMN2 exon 7. Cotransfection experiments demonstrate that an increase of the SF2/ASF concentration promotes inclusion of the mutated exon, suggesting that the mutation weakens an SF2/ASF-dependent enhancer. Interestingly, a synonymous mutation 351A→C was identified 11 nucleotides upstream in the same exon. This mutation affects an hnRNP A1 dependent silencer. Since the exon is constitutively used in the absence of the 362C→T SF2/ASF enhancer mutation, it has no effect on splicing. However, in the presence of the 362C→T enhancer mutation, it promotes exon inclusion, which antagonized the exon-skipping effect of the 362C→T SF2/ASF enhancer mutation.

       This example illustrates the fine-tuned balance of positive and negative acting factors that exists in splicing regulation. It also shows that seemingly irrelevant mutations can have an effect on splicing when they are combined with other mutations. Finally, the similarities between the regulation of MCAD exon 5 and SMN2 exon 7 suggest that there are degenerate ‘building blocks’ or ‘regulatory modules’ in the splicing code.

       2.1.7. Frontotemporal lobar dementias are caused by the loss of the splicing factor TDP43

       TDP43 (TAR DNA binding protein 43 kDa) was originally identified as a transcriptional repressor that associates with the transcriptional activator DNA region (TAR) in HIV  and was later also found associated with the spermatid-specific gene SP-10 promoter , reviewed in . TDP43 is a member of the heterogeneous nuclear ribonucleoproteins (hnRNP) family of proteins. The protein was later identified as a factor that binds to 12 UG repeats that cause aberrant skipping of exon 9 of the CFTR gene, leading to cystic fibrosis . It contains two RNA binding domains. The first RNA binding domain is necessary and sufficient for binding to RNA that contains at least four UG repeats. In addition, TDP43 shows binding to single stranded TG DNA repeats in vitro . TDP43 is a nuclear protein and it was therefore completely surprising when it was detected in ubiquitin-positive, tau and alpha synuclein-negative cytosolic inclusions that are the characteristic feature of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS, Lou Gehring's disease) . During the disease, TDP43 is cleaved by caspase-3 and the cleavage fragments accumulate in the cytosol, where they form aggregates. The caspase-3 reaction is inhibited by progranulin, which explains why mutations reducing the progranulin expression cause FTLD . It is not clear whether the disease is caused by a loss of nuclear function of TDP43 or by a possible cytotoxic accumulation. However, siRNA mediated knock down of TDP43 in HeLa cells followed by RNA microarray analysis demonstrated a strong upregulation of cyclin-dependent kinase 6 (cdk6). The human Cdk6 gene contains numerous intronic TG repeats and interestingly a change in cdk6 expression was not observed for the chicken gene that lacks these repeats. These data strongly suggest that TDP43 represses cdk6 expression by sequestering its RNA by binding to UG repeats. Loss of TDP43 expression leads to abnormal cdk6 activity, resulting in pRB phosphorylation, genomic instability and apoptosis, which could explain the cell death in FTLD . Mutations of the gene encoding TDP43 are found in families with amyotrophic lateral sclerosis, as well as in sporadic cases, indicating a direct link between TDP43 and amyotrophic lateral sclerosis .

       This example illustrates that splicing factors like TDP43 operate in a regulatory network. A loss of their function can have drastic, but indirect effects. HnRNPs typically perform several different functions, which can obscure possible disease mechanisms. Finally, the interaction between UG repeats of the cdk6 gene and TDP43 illustrate how an hnRNP can be recruited to a new function during evolution.

       2.1.8. Diseases associated with repeat elements

       Short sequence repeats can be detected in numerous exons, where they serve to increase the recognition by a certain splicing factor , reviewed in . A change in length of simple repeat sequences can therefore change the splicing pattern of a gene. For example, the endothelial NO synthase gene contains an intronic polymorphic CA-repeat region and the number of repeats correlate with the risk of coronary artery disease and hyperhomo-cysteinemie in a sex-specific fashion . SELEX experiments and functional studies showed that CA repeats bind to hnRNP L and the action of hnRNP L depends on the CA repeat length. Microarray studies showed that intronic CA-rich repeats can influence alternative splicing decisions, strongly suggesting that other intronic CA-repeat polymorphisms could cause human disease . Another disease-relevant repeat is the UG repeat that can cause aberrant splicing of exon 9 of the CFTR gene, leading to cystic fibrosis . This repeat binds to TDP43, which is discussed below. Myotonic dystrophy (DM) is the most common form of muscular dystrophy in adults. The disease is caused by extensions of two repeats: a CUG repeat in the 3′ region of the DMPK gene leads to DM1 and extension of a CCUG repeat in an intron of the ZNF9gene leads to DM2 . The common CUG element binds to at least two groups of RNA binding proteins, muscleblind-like1 and CUG binding protein. Extension of the repeats leads a sequestration of muscleblind protein in nuclear foci. This sequestration of muscleblind-like 1 protein reduces its cellular concentration and changes alternative splicing events that depend on this protein. Surprisingly, the extended CUG repeats lead to an increase of CUG binding protein concentration. This increase is due to a stabilization of CUG-binding protein. CUG RNA repeats induce via an unknown mechanism protein kinase C, which phosphorylates the CUG protein, leading to a more stable protein .

       Alu elements are the largest group of repetitive elements in the human genome. They represent about 10% of the total genome sequence. Alu elements contain potential splice sites and can evolve into exons . It has been estimated that up to 5% of human alternative exons could be derived from Alu sequences . It is therefore not surprising that mutations in existing Alu elements can cause their abnormal inclusion in m-RNA, which leads to human diseases, such as the Alport syndrome , congenital cataracts facial dysmorphism neuropathy syndrome , and mucopolysaccharidosis type VII .

       These examples illustrate how repeats can change the balance of alternative splicing regulation, even when they are located in introns or in seemingly unrelated m-RNAs. The exonisation of Alu elements shows how new repetitive elements can be used by evolution to acquire new functions, and diseases caused by improper Alu-element exonisations can be viewed as failed evolutionary experiments.

3. Numerous Changes in Alternative Splicing are Found in Cancer

       Numerous reports have shown that alternative splicing patterns are changed in cancer. The expression of alternative or even tumour-specific splice variants significantly affects many cellular events critical for cancer biology such as cell proliferation, motility, and drug response . It is still unclear, however, to what extent alternative splicing functionally contributes to the initiation and progression of cancers. Most altered splicing patterns could be largely symptomatic and attributed to a generalized lack of fidelity of the splicing apparatus in cancer cells. The existence of particular splice variants in cancer could merely be a consequence of the malignant phenotype without contributing to the cancer phenotype. Here we will describe general changes of the splicing machinery in cancer and discuss specific examples illustrating the action on tumour suppressor genes.

4. Treatment Options for Missplicing Events

       The examples discussed here clearly show that a misregulation of alternative splicing plays a large role in numerous human diseases. The identification of molecules capable of correcting and or inhibiting pathological splicing events is therefore an important issue for future therapeutic approaches.

4.1. Antisense strategies

       The major challenge in treating splicing disorders is to specifically target one splicing event in a certain pre-m-RNA. Since there are several thousand other pre-m-RNAs in the cell, the selectivity of splice site intervention is a major problem. One way to address this problem is the use of oligonucleotides that will specifically bind to one sequence.

       Special chemistries were devised to prevent RNAseH-mediated cleavage of the RNA and to lower toxicity). A major drawback of the therapeutic use of oligonucleotides is their delivery and uptake in the cells. This problem has been addressed by coupling of oligonucleotides to arginine-rich cell penetrating peptides.

       Antisense oligonucleotides have been tested for beta thalassemias, Duchenne muscular dystrophy cystic fibrosis cyclophilin transcripts, Hutchinson Gilford Progeria Syndrome (HGPS) as well as block HIV replication and alter tau pre-m-RNAs.

       Antisense oligonucleotides can also be used to block splicing enhancers or silencers, which can be found in introns or exons quite far away from the splice sites . Inhibition of splicing silencers (ESSs or ISSs) is of particular interest since it provides a way to activate otherwise repressed exons. This was achieved for the α exon of fibroblast growth factor receptor-1 (FGFR1) transcripts  where an antisense morpholino oligonucleotide that blocks silencer elements in glioblastoma cells in culture promotes the inclusion of the α exon. The antisense approach was further developed in ESSENSE (exon-specific splicing enhancement by small chimeric effectors). ESSENSE uses bifunctional reagents that contain a peptide effector domain and an antisense-targeting domain. The effector domains of these protein-nucleic acids were arginine-serine (RS) repeats that mimic the effect of SR proteins . A second incarnation of bifunctional oligomers uses a 2′-Ome-modified binding domain and an effector domain, which is composed of RNA that contains binding sites for known splicing trans-acting factors. The recruited factors mediate activation or silencing of the targeted exon. An example of this type of bifunctional oligonucleotide acts as an ESE and promotes inclusion of the SMN2 exon 7 fibroblasts from SMA patients leading to partial restoration of the SMN function.

4.2. Substances that change alternative splicing

       The use of RNA-binding molecules as antibiotics, such as gentamicin, chloramphenicol, and tetracycline illustrates that drugs can be targeted against RNA and/or RNA binding proteins. High-throughput screens and testing of substances in model systems have now identified more than 30 substances that change splice site selection. The substances fall into several categories, including HDAC inhibitors, kinase and phosphatase inhibitors, as well as cAMP antagonist and agonists. The currently known substances are reviewed in and updated on the web.

       The usefulness of substances that change splice site selection is evident from potential HIV therapies that could be combined with other antiviral strategies. Using an in vitro splicing assay, a chemical screen was performed that identified an indole derivative (IDC16) that interferes with exonic splicing enhancer activity of the SR protein splicing factor SF2/ASF. IDC16 suppresses the production of key viral proteins and inhibits replication of macrophage- and T cell-tropic laboratory strains, clinical isolates, and strains with high-level resistance to inhibitors of viral protease and reverse transcriptase. Thus, human splicing factors represent novel and promising drug targets for the development of antiretroviral therapies, particularly for the inhibition of multidrug-resistant viruses.

(Based on the article written by Jamal Tazi et al.  and published in Biochimica et Biophysica Acta (BBA) –Molecular Basis of Disease Vol. 1792, Issue 1, January 2009, Pages 14-26)








       Mechanisms of RNA-mediated disease.

       Jason R. O'Rourke et al.

       J. of Biol. Chemistry 284(12), 7419

(March 2009)

       The human genome contains thousands of micro satellites, which are short (2-6 bp) polymorphic repetitive sequences, that can expand or contract due to mistakes in DNA replication, repair and recombination. Microsatellite expansions are particularly pathogenic and associated with a number of hereditary disorders. Repeat expansions in protein coding regions result in many of these diseases, including Huntington's disease, spinal bulbar muscular atrophy (Kennedy's disease) and at least six types of spinocerebellar ataxia. More unexpected is that a number of dominantly inherited disorders result from microsatellite expansions in noncoding regions. These diseases include myotonic dystrophy (DM), fragile X-associated






tremor/ataxia syndrome (FXTAS), spinocerebellar ataxia types 8, 10 and 12 (SCA8, 10, 12) and Huntington's disease-like 2. Interestingly, some of these expansions may also be located in a coding region. In this review, focus is on mechanisms which have been proposed to explain RNA-mediated pathogenesis in DM, FXTAS and SCA8, and mention how mutations in other types of ncRNAs might result in deleterious RNA gain-of-function effects.

       Ras promotes growth by alternative splicing-mediated inactivation of the KLF6 tumor suppressor in hepatocellular carcinoma.

       Yea, Steven et al.

       Gastroenterology, 134 (5), 1521 (May 2008)

       Hepatocellular carcinoma (HCC) is the fifth most prevalent cancer worldwide and the third most lethal. Dysregulation of alternative splicing underlies a number of human diseases, yet its contribution to liver cancer has not been explored fully. The Krüppel-like factor 6 (KLF6) gene is a zinc finger transcription factor that inhibits cellular growth in part by transcriptional activation of p21. KLF6 function is abrogated in human cancers owing to increased alternative splicing that yields a dominant-negative isoform, KLF6 splice variant 1 (SV1), which antagonizes full-length KLF6-mediated growth suppression. The molecular basis for stimulation of KLF6 splicing is unknown. In human HCC samples and cell lines, we functionally link oncogenic Ras signaling to increased alternative splicing of KLF6 through signaling by phosphatidylinositol-3 kinase and Akt, mediated by the splice regulatory protein ASF/SF2. In 67 human HCCs, there is a significant correlation between activated Ras signaling and increased KLF6 alternative splicing. In cultured cells, Ras signaling increases the expression of KLF6 SV1, relative to full-length KLF6, thereby enhancing proliferation. Abrogation of oncogenic Ras signaling by small interfering RNA (siRNA) or a farnesyl-transferase inhibitor decreases KLF6 SV1 and suppresses growth. Growth inhibition by farnesyl-transferase inhibitor in transformed cell lines is overcome by ectopic expression of KLF6 SV1. Down-regulation of the splice factor ASF/SF2 by siRNA increases KLF6 SV1 messenger RNA levels. KLF6 alternative splicing is not coupled to its transcriptional regulation. The findings expand the role of Ras in human HCC by identifying a novel mechanism of tumor-suppressor inactivation through increased alternative splicing mediated by an oncogenic signaling cascade.

       microRNA-146b inhibits glioma cell migration and invasion by targeting MMPs.

       Xia, Hongping et al.

       Brain Research, 1269, 158 (May 7, 2009)

       MicroRNAs (miRNAs) are a class of endogenous, small non-protein coding single-stranded RNA molecules, which are crucial post-transcriptional regulators of gene expression. Previous studies have shown that miRNAs participate in a wide range of biological functions and play important roles in various human diseases including glioma. However, the role of miRNAs in mediating glioblastoma cell migration and invasion has not been elucidated. Using miRNA microarray, miR-146b was identified as one of the miRNAs that is significantly dysregulated in human glioblastoma tissue. Authors showed that miR-146b overexpression by transfection with the precursor miR-146b, or knock-down by Locked Nucleic Acid (LNA)-modified anti-miR-146b, has no effect on the growth of human glioblastoma U373 cells. However, precursor miR-146b transfection significantly reduced the migration and invasion of U373 cells, while LNA-anti-miR-146b transfection generated the opposite result. Furthermore, it wasdiscovered that a matrix metalloproteinase gene, MMP16, is one of the downstream targets of miR-146b. Taken together, these findings suggest that miR-146b is involved in glioma cell migration and invasion by targeting MMPs, and implicate miR-146b as a metastasis-inhibiting miRNA in glioma.

       Signatures of purifying and local positive selection in human miRNAs

       The American Journal of Human Genetics, 84(3), 316 (Mar., 13, 2009)

       MicroRNAs (miRNAs) are noncoding RNAs involved in post-transcriptional gene repression, and their role in diverse physiological processes is increasingly recognized. Yet, few efforts have been devoted to evolutionary studies of human miRNAs. Knowledge about the way in which natural selection has targeted miRNAs should provide insight into their functional relevance as well as their mechanisms of action. Here authors used miRNAs as a model system for investigating the influence of natural selection on gene regulation by characterizing the full spectrum of naturally occurring sequence variation of 117 human miRNAs from different populations worldwide. Authors found that purifying selection has globally constrained the diversity of miRNA-containing regions and has strongly targeted the mature miRNA. This observation emphasizes that mutations in these molecules are likely to be deleterious, and therefore they can have severe phenotypic consequences on human health. More importantly, authors obtained evidence of population-specific events of positive selection acting on a number of miRNA-containing regions. Notably, this analysis revealed that positive selection has targeted a "small-RNA-rich island" on chromosome 14, harboring both miRNAs and small nucleolar RNAs, in Europeans and East Asians. These observations support the notion that the tuning of gene expression contributes to the processes by which populations adapt to specific environments. These findings will fuel future investigations exploring how genetic and functional variation of miRNAs under selection affects the repression of their m-RNA targets, increasing our understanding of the role of gene regulation in population adaptation and human disease.

       miR-155 gene: A typical multifunctional microRNA.

       Faraoni, Isabella et al.

       Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease

       In the last years small RNA molecules, i.e. microRNA (miRNA) encoded by miR genes, have been found to play a crucial role in regulating gene expression of a considerable part of plant's and animal's genome. Here, the essential information on biogenesis of miRNAs and recent evidence on their important role in human diseases has been reported. Emphasis has been given to miR-155, since this molecule represents a typical multifunctional miRNA. Recent data indicate that miR-155 has distinct expression profiles and plays a crucial role in various physiological and pathological processes such as haematopoietic lineage differentiation, immunity, inflammation, cancer, and cardiovascular diseases. Moreover, miR-155 has been found to be implicated in viral infections, particularly in those caused by DNA viruses. The experimental evidence indicating that miR-155 is over expressed in a variety of malignant tumors allows us to include this miRNA in the list of genes of paramount importance in cancer diagnosis and prognosis. Exogenous molecular control in vivo of miR-155 expression could open up new ways to restrain malignant growth and viral infections, or to attenuate the progression of cardiovascular diseases.               (ScienceDirect)

       Drug delivery of siRNA therapeutics: potentials and limits of nanosystems

       Reischl, Daniela et al.

       Nanomedicine: Nanotechnology, Biology and Medicine, 5(1), 8 (Mar., 2009)

       Gene therapy is a promising tool for the treatment of human diseases that cannot be cured by rational therapies. The major limitation for the use of small interfering RNA (siRNA), both in vitro and in vivo, is the inability of naked siRNA to passively diffuse through cellular membranes due to the strong anionic charge of the phosphate backbone and consequent electrostatic repulsion from the anionic cell membrane surface. Therefore, the primary success of siRNA applications depends on suitable vectors to deliver therapeutic genes. Cellular entrance is further limited by the size of the applied siRNA molecule. Multiple delivery pathways, both viral and nonviral, have been developed to bypass these problems and have been successfully used to gain access to the intracellular environment in vitro and in vivo, and to induce RNA interference (RNAi). The review focuses on different pathways for siRNA delivery and summarizes recent progress made in the use of vector-based siRNA technology.

       RNA-binding proteins in human genetic disease

       Lukong, Kiven E. et al.

       Trends in Genetics, 24(8), 416 (Aug., 2008)

       RNA-binding proteins (RBPs) are key components in RNA metabolism, regulating the temporal, spatial and functional dynamics of RNAs. Altering the expression of RBPs has profound implications for cellular physiology, affecting RNA processes from pre-m-RNA splicing to protein translation. Recent genetic and proteomic data and evidence from animal models reveal that RBPs are involved in many human diseases ranging from neurologic disorders to cancer. Here the emerging evidence has been reviewed showing the involvement of RBPs in many disease networks and concluding that defects in RNA metabolism caused by aberrations in RBPs might underlie a broader spectrum of complex human disorders.

       The enigmatic world of m-RNA-like ncRNAs: Their role in human evolution and in human diseases.

       Széll, Márta et al.

       Seminars in Cancer Biology, 18(2), 141 (Apr., 2008)

       Accumulating data on non-protein-coding transcripts suggest that besides the regulatory machinery driven by proteins, another yet enigmatic regulatory network of RNA molecules operates and has considerable impact on cell functions. Moreover, deregulation of these non-coding RNAs (ncRNAs) has been documented in several human diseases suggesting that they may significantly contribute to their pathogenesis. This review summarizes the present knowledge on the role of the so-called m-RNA-like ncRNAs in the complexity of multicellular organisms. Some examples to show how these m-RNA-like non-coding RNAs have been discovered, how their cellular functions and role in the pathogenesis of human diseases have been revealed are discussed.

       New modes of translational control in development, behavior, and disease.

       Sonenberg, Nahum et al.

       Molecular Cell, 28(5), 721 (Dec., 14., 2007)

       Over the last 10 years, the field of translational control has been enriched by atomic resolution structures of ribosomal complexes and factors in different functional states, and increased in sophistication by wedding genetics, reconstituted systems, and structural biology to elucidate basic reactions and m-RNA-specific control mechanisms. New regulatory principles have emerged, including repression by micro-RNAs (miRNAs) and m-RNA sequestration in cytoplasmic granules, and the field has extended its reach into development, brain function, and human disease. Some of the exciting developments of the last decade from the perspectives of authors’ own approaches and expertise; accordingly, many highly noteworthy achievements are highlighted in the review.

       A-to-I RNA editing: A new regulatory mechanism of global gene expression.

Amariglio, Ninette et al.

       Blood Cells, Molecules, and Diseases, 39(2), 151 (2007)

       The complexity of higher organisms is based on the number of different gene products available for structural, regulatory and enzymatic regulatory functions and the precise and coordinated control of gene expression. A-to-I RNA editing emerges as a global post-transcriptional modification that affects thousands of transcripts. RNA editing is becoming a central modification acting on double-stranded RNA and is therefore intimately associated with other basic processes such as RNA interference, microRNA and interferon response that also depend on double-stranded RNA. Recent research highlights the multiple roles of this mechanism in the transcriptome diversification and in the special and temporal gene expression control. Alterations in A-to-I RNA editing have now been linked to various human diseases. The increased understanding of the editing machinery and identification of the multiple targets are expected to improve our understanding of the roles of this mechanism in normal development and homeostasis and its derangement in disease states. Basic and translational research is expected to increase our understanding of this intriguing mechanism which is endowed with diagnostic and therapeutic implications.

       MicroRNA and esophageal carcinoma

       He, Xiaoting et al.

       Journal of Nanjing Medical University, 21(4), 201 (July 2007)

       An abundant class of non-coding small RNA molecules, 21-25 nucleotide in length, are widely found in animals and plants and named microRNA(miRNA). MiRNAs are highly evolutionarily conserved, expressing in specific tissue and timing, and negatively regulate the gene expressions at the posttranscriptional level, and subsequently control crucial physiological processes such as metabolism, amplification, differentiation, development and apoptosis. Therefore, miRNAs could provide an access to many human diseases in theory. Recent evidence demonstrates that miRNAs play an important role in the initiation and progression of human cancer, mainly by interrupting the cell cycle at the cellular level and by interacting with signaling. The expression profiling of miRNAs can be used as a tool of diagnosis, staging, prognosis and biotherapy of some tumors, as has already been proven to have superiority to m-RNA, in the categorization of tumors. This review focuses on the genesis, mechanism of action of miRNA and its relationship to tumors, detection methods and its potential effect on the diagnosis, staging, and biotherapy in esophageal carcinoma.

       An RNAi screening platform to identify secretion machinery in mammalian cells.

       Simpson, Jeremy C. et al.

       Journal of Biotechnology, 129(2), 352 (Apr., 30, 2007)

       Integrative approaches to study protein function in a cellular context are a vital aspect of understanding human disease. Genome sequencing projects provide the basic catalogue of information with which to unravel gene function, but more systematic applications of this resource are now necessary. Here, authors describe and test a platform with which it is possible to rapidly use RNA interference in cultured mammalian cells to probe for proteins involved in constitutive protein secretion. Synthetic small interfering RNA molecules are arrayed in chambered slides, then incubated with cells and an assay for secretion performed. Automated microscopy is used to acquire images from the experiments, and automated single-cell analysis rapidly provides reliable quantitative data. In test arrays of 92 siRNA spots targeting 37 prospective membrane traffic proteins, our approach identifies 7 of these as being important for the correct delivery of a secretion marker to the cell surface. Correlating these findings with other screens and bioinformatic information makes these candidates highly likely to be novel membrane traffic machinery components.

       Therapeutic RNA interference for neurodegenerative diseases: From promise to progress

       Gonzalez-Alegre, Pedro et al;.

       Pharmacology & Therapeutics, 114(1), 34 (Apr., 2007)

       RNA interference (RNAi) has emerged as a powerful tool to manipulate gene expression in the laboratory. Due to its remarkable discriminating properties, individual genes, or even alleles can be targeted with exquisite specificity in cultured cells or living animals. Among its many potential biomedical applications, silencing of disease-linked genes stands out as a promising therapeutic strategy for many incurable disorders. Neurodegenerative diseases represent one of the more attractive targets for the development of therapeutic RNAi. In this group of diseases, the progressive loss of neurons leads to the gradual appearance of disabling neurological symptoms and premature death. Currently, available therapies aim to improve the symptoms but not to halt the process of neurodegeneration. The increasing prevalence and economic burden of some of these diseases, such as Alzheimer's disease (AD) or Parkinson's disease (PD), has boosted the efforts invested in the development of interventions, such as RNAi, aimed at altering their natural course. This review will summarize where we stand in the therapeutic application of RNAi for neurodegenerative diseases. The basic principles of RNAi will be reviewed, focusing on features important for its therapeutic manipulation. Subsequently, a stepwise strategy for the development of therapeutic RNAi will be presented. Finally, the different preclinical trials of therapeutic RNAi completed in disease models will be summarized, stressing the experimental questions that need to be addressed before planning application in human disease.

       DrugScoreRNAknowledge-based scoring function to predict RNA−ligand inter-actions.

       Patrick Pfeffer  et al.

       J. Chem. Inf. Model., 2007, 47 (5), pp 1868–1876

       There is growing interest in RNA as a drug target due to its widespread involvement in biological processes. To exploit the power of structure-based drug-design approaches, novel scoring and docking tools need to be developed that can efficiently and reliably predict binding modes and binding affinities of RNA ligands. Authors  report for the first time the development of a knowledge-based scoring function to predict RNA−ligand interactions (DrugScoreRNA). Based on the formalism of the DrugScore approach, distance-dependent pair potentials are derived from 670 crystallographically determined nucleic acid−ligand and -protein complexes. These potentials display quantitative differences compared to those of DrugScore (derived from protein−ligand complexes) and DrugScoreCSD (derived from small-molecule crystal data). When used as an objective function for docking 31 RNA−ligand complexes, DrugScoreRNA generates “good” binding geometries (rmsd (root mean-square deviation) < 2 Å) in 42% of all cases on the first scoring rank. This is an improvement of 44% to 120% when compared to DrugScore, DrugScoreCSD, and an RNA-adapted AutoDock scoring function. Encouragingly, good docking results are also obtained for a subset of 20 NMR structures not contained in the knowledge-base to derive the potentials. This clearly demonstrates the robustness of the potentials. Binding free energy landscapes generated by DrugScoreRNA show a pronounced funnel shape in almost 3/4 of all cases, indicating the reduced steepness of the knowledge-based potentials. Docking with DrugScoreRNA can thus be expected to converge fast to the global minimum. Finally, binding affinities were predicted for 15 RNA−ligand complexes with DrugScoreRNA. A fair correlation between experimental and computed values is found (RS = 0.61), which suffices to distinguish weak from strong binders, as is required in virtual screening applications.

       Bacterial RNase P RNA is a drug target for aminoglycoside−arginine conjugates.

       Alexander Berchanski  et al.

       Bioconjugate Chem., 2008, 19 (9), pp 1896–1906

       The ribonuclease P (RNase P) holoenzymes are RNPs composed of RNase P RNA (PRNA) and a variable number of P protein subunits. Primary differences in structure and function between bacterial and eukaryotic RNase P and its indispensability for cell viability make the bacterial enzyme an attractive drug target. On the basis of previous studies by authors, aminoglycoside−arginine conjugates (AACs) bind to HIV-1 TAR and Rev responsive element (RRE) RNAs significantly more efficiently than neomycin B. Their specific inhibition of bacterial rRNA as well as the findings that the hexa-arginine neomycin derivative (NeoR6) is 500-fold more potent than neomycin B in inhibiting bacterial RNase P, led authors to explore the structure-function relationships of AACs in comparison to a new set of aminoglycoside-polyarginine conjugates (APACs). Authors here present predicted binding modes of AACs and APACs to PRNA. Authors used a multistep docking approach comprising rigid docking full scans and final refinement of the obtained complexes. Thesedocking results suggest three possible mechanisms of RNase P inhibition by AACs and APACs: competition with the P protein and pre-tRNA on binding to P1−P4 multihelix junction and to J19/4 region (probably including displacement of Mg2+ ions from the P4 helix) of PRNA; competition with Mg2+ ions near the P15 loop; and competition with the P protein and/or pre-tRNA near the P15 helix and interfering with interactions between the P protein and pre-tRNA at this region. The APACs revealed about 10-fold lower intermolecular energy than AACs, indicating stronger interactions of APACs than AACs with PRNA.

       Nonviral methods for siRNA delivery.

       Kun Gao  et al.

       Mol. Pharmaceutics, Article ASAP

       RNA interference (RNAi) as a mechanism to selectively degrade m-RNA (m-RNA) expression has emerged as a potential novel approach for drug target validation and the study of functional genomics. Small interfering RNAs (siRNA) therapeutics has developed rapidly and already there are clinical trials ongoing or planned. Although other challenges remain, delivery strategies for siRNA become the main hurdle that must be resolved prior to the full-scale clinical development of siRNA therapeutics. This review provides an overview of the current delivery strategies for synthetic siRNA, focusing on the targeted, self-assembled nanoparticles which show potential to become a useful and efficient tool in cancer therapy.

       Potent non-nucleoside inhibitors of the measles virus RNA-dependent RNA poly-merase complex.

       Aiming Sun  et al.

       J. Med. Chem., 2008, 51 (13), pp 3731–3741

       Measles virus (MV) is one of the most infectious pathogens known. In spite of the existence of a vaccine, approximately 350000 deaths/year result from MV or associated complications. Antimeasles compounds could conceivably diminish these statistics and provide a therapy that complements vaccine treatment. Authors recently described a high-throughput screening hit compound 1 (16677) against MV-infected cells with the capacity to eliminate viral reproduction at 250 nM by inhibiting the action of the virus’s RNA-dependent RNA polymerase complex (RdRp). The compound, 1-methyl-3-(trifluoromethyl)-N-[4-sulfonylphenyl]-1H-pyrazole-5-carboxamide, 1 carries a critical CF3 moiety on the 1, 2-pyrazole ring. Elaborating on the preliminary structure-activity (SAR) study, the present work presents the synthesis and SAR of a much broader range of low nanomolar nonpeptidic MV inhibitors and speculates on the role of the CF3 functionality.

       Selection of aptamers for molecular recognition and characterization of cancer cells.

       Zhiwen Tang  et al.

       Anal. Chem., 2007, 79 (13), pp 4900–4907

       In this paper, authors describe a new way to generate molecular probes for specific recognition of cancer cells. Molecular medicine will require a large number of probes for molecular recognition and characterization of a variety of diseased cells. Aptamers, single-stranded DNA/RNA probes, are poised to become a chemist's antibody and have the potential to serve as molecular probes for a variety of biomedical applications. By applying newly developed cell-SELEX (cell-based systematic evolution of ligands by exponential enrichment) against whole living cells, panels of aptamers have been evolved from an initial DNA library to characterize target cells at the molecular level. Ramos cells, a B-cell lymphoma cell line, were used as target cells for the generation of effective molecular probes. By taking advantages of the repetitive and broad enrichment strategy, the selected aptamers could bind to target cells and other closely related cell lines in variant patterns with an equilibrium dissociation constant (Kd) in the nanomolar range. Some aptamers could also specifically recognize the target lymphoma cells mixed with normal human bone marrow aspirates. The cell-based SELEX is simple, fast, and robust. The strategies used here will be highly useful for aptamer selection against complex target samples in order to generate a large number of aptamers in a variety of biomedical and biotechnological applications, paving the way for molecular diagnosis, therapy, and biomarker discovery.

       Design and creation of new nanomaterials for therapeutic RNAi.

       Huricha Baigude  et al.

       ACS Chem. Biol., 2007, 2 (4), pp 237–241

       RNA interference is an evolutionarily conserved gene-silencing phenomenon that shows great promise for developing new therapies. However, the development of small interfering RNA (siRNA)-based therapies needs to overcome two barriers and be able to (i) identify chemically stable and effective siRNA sequences and (ii) efficiently silence target genes with siRNA doses that will be clinically feasible in humans. Here, authors report the design and creation of interfering nanoparticles (iNOPs) as new systemic gene-silencing agents. iNOPs have two subunits: (i) a well-defined functionalized lipid nanoparticle as a delivery agent and (ii) a chemically modified siRNA for sustained silencing in vivo. When iNOPs containing only 1−5 mg kg–1 siRNA were injected into mice, an endogenous gene for apolipoprotein B (apoB) was silenced in liver, plasma levels of apoB decreased, and total plasma cholesterol was lowered. iNOP treatment was nontoxic and did not induce an immune response. The results show that these iNOPs can silence disease-related endogenous genes in clinically acceptable and therapeutically affordable doses.

       Structural characterization and biological evaluation of small interfering RNAs containing cyclohexenyl nucleosides.

       Koen Nauwelaerts  et al.

       J. Am. Chem. Soc., 2007, 129 (30), pp 9340–9348

       CeNA is an oligonucleotide where the (deoxy)ribose sugars have been replaced by cyclohexenyl moieties. Authors have determined the NMR structure of a CeNA:RNA duplex and have modeled this duplex in the crystal structure of a PIWI protein. An N puckering of the ribose nucleosides, a 2H3 conformation of the cyclohexenyl nucleosides, and an A-like helix conformation of the backbone, which deviates from the standard A-type helix by a larger twist and a smaller slide, are observed. The model of the CeNA:RNA duplex bound to the PIWI protein does not show major differences in the interaction of the guide CeNA with the protein when compared with dsRNA, suggesting that CeNA modified oligonucleotides might be useful as siRNAs. Incorporation of one or two CeNA units in the sense or antisense strands of dsRNA led to similar or enhanced activity compared to unmodified siRNAs. This was tested by targeting inhibition of expression of the MDR1 gene with accompanying changes in P-glycoprotein expression, drug transport, and drug resistance.

       Comparison of the RNase H cleavage kinetics and blood serum stability of the North-conformationally constrained and 2‘-alkoxy modified oligonucleotides.

       Dmytro Honcharenko  et al.

       Biochemistry, 2007, 46 (19), pp 5635–5646

       The RNase H cleavage potential of the RNA strand basepaired with the complementary antisense oligonucleotides (AONs) containing NorthEast conformationally constrained 1‘,2‘-methylene-bridged (azetidine-T and oxetane-T) nucleosides, North-constrained 2‘,4‘-ethylene-bridged (aza-ENA-T) nucleoside, and 2‘-alkoxy modified nucleosides (2‘-O-Me-T and 2‘-O-MOE-T modifications) have been evaluated and compared under identical conditions. When compared to the native AON, the aza-ENA-T modified AON/RNA hybrid duplexes showed an increase of melting temperature (ΔTm = 2.5−4 °C per modification), depending on the positions of the modified residues. The azetidine-T modified AONs showed a drop of 4−5.5 °C per modification with respect to the native AON/RNA hybrid, whereas the isosequential oxetane-T modified counterpart, showed a drop of 5−6 °C per modification. The 2‘-O-Me-T and 2‘-O-MOE-T modifications, on the other hand, showed an increase of Tm by 0.5 °C per modification in their AON/RNA hybrids. All of the partially modified AON/RNA hybrid duplexes were found to be good substrates for the RNase H mediated cleavage. The Km and Vmax values obtained from the RNA concentration-dependent kinetics of RNase H promoted cleavage reaction for all AON/RNA duplexes with identical modification site were compared with those of the reference native AON/RNA hybrid duplex. The catalytic activities (Kcat) of RNase H were found to be greater (1.4−2.6-fold) for all modified AON/RNA hybrids compared to those for the native AON/RNA duplex. However, the RNase H binding affinity (1/Km) showed a decrease (1.7−8.3-fold) for all modified AON/RNA hybrids. This resulted in less effective (1.1−3.2-fold) enzyme activity (Kcat/Km) for all modified AON/RNA duplexes with respect to the native counterpart. A stretch of five to seven nucleotides in the RNA strand (from the site of modifications in the complementary modified AON strand) was found to be resistant to RNase H digestion (giving a footprint) in the modified AON/RNA duplex. Thus, (i) the AON modification with azetidine-T created a resistant region of five to six nucleotides, (ii) modification with 2‘-O-Me-T created a resistant stretch of six nucleotides, (iii) modification with aza-ENA-T created a resistant region of five to seven nucleotide residues, whereas (iv) modification with 2‘-O-MOE-T created a resistant stretch of seven nucleotide residues. This shows the variable effect of the microstructure perturbation in the modified AON/RNA heteroduplex depending upon the chemical nature as well as the site of modifications in the AON strand. On the other hand, the enhanced blood serum as well as the 3‘-exonuclease stability (using snake venom phosphodiesterase, SVPDE) showed the effect of the tight conformational constraint in the AON with aza-ENA-T modifications in that the 3‘-exonuclease preferentially hydrolyzed the 3‘-phosphodiester bond one nucleotide away (n + 1) from the modification site (n) compared to all other modified AONs, which were 3‘-exonuclease cleaved at the 3‘-phosphodiester of the modification site (n). The aza-ENA-T modification in the AONs made the 5‘-residual oligonucleotides (including the n + 1 nucleotide) highly resistant in the blood serum (remaining after 48 h) compared to the native AON (fully degraded in 2 h). On the other hand, the 5‘-residual oligonucleotides (including the n nucleotide) in azetidine-T, 2‘-O-Me-T, and 2‘-O-MOE-T modified AONs were more stable compared to that of the native counterpart but more easily degradable than that of aza-ENA-T containing AONs.

       MicroRNAs and the advent of vertebrate morphological complexity

       Alysha M. Heimberg 

       PNAS February 26, 2008 vol. 105 no. 8 2946-2950

       The causal basis of vertebrate complexity has been sought in genome duplication events (GDEs) that occurred during the emergence of vertebrates, but evidence beyond coincidence is wanting. MicroRNAs (miRNAs) have recently been identified as a viable causal factor in increasing organismal complexity through the action of these ~22-nt noncoding RNAs in regulating gene expression. Because miRNAs are continuously being added to animalian genomes, and, once integrated into a gene regulatory network, are strongly conserved in primary sequence and rarely secondarily lost, their evolutionary history can be accurately reconstructed. Here, using a combination of Northern analyses and genomic searches, It is shown that 41 miRNA families evolved at the base of Vertebrata, as they are found and/or detected in lamprey, but not in either ascidians or amphioxus (or any other nonchordate taxon). When placed into temporal context, the rate of miRNA acquisition and the extent of phenotypic evolution are anomalously high early in vertebrate history, far outstripping any other episode in chordate evolution. The genomic position of miRNA paralogues in humans, together with gene trees incorporating lamprey orthologues, indicates that although GDEs can account for an increase in the diversity of miRNA family members, which occurred before the last common ancestor of all living vertebrates, GDEs cannot account for the origin of these novel families themselves. It is hypothesized that lying behind the origin of vertebrate complexity is the dramatic expansion of the noncoding RNA inventory including miRNAs, rather than an increase in the protein-encoding inventory caused by GDEs.

       MicroRNAs: novel biomarkers for human cancer

       Claudine L. Bartelsl 

       Clinical Chemistry 55: 623-631,2009.

       MicroRNAs (miRNAs), small RNA molecules of approximately 22 nucleotides, have been shown to be up- or downregulated in specific cell types and disease states. These molecules have become recognized as one of the major regulatory gatekeepers of coding genes in the human genome.

       miRNAs are produced in a tissue-specific manner, and changes in miRNA within a tissue type can be correlated with disease status are reviewed. miRNAs appear to regulate m-RNA translation and degradation via mechanisms that are dependent on the degree of complementarity between the miRNA and m-RNA molecules. miRNAs can be detected via several methods, such as microarrays, bead-based arrays, and quantitative real-time PCR. The tissue concentrations of specific miRNAs have been associated with tumor invasiveness, metastatic potential, and other clinical characteristics for several types of cancers, including chronic lymphocytic leukemia, and breast, colorectal, hepatic, lung, pancreatic, and prostate cancers.

       By targeting and controlling the expression of m-RNA, miRNAs can control highly complex signal-transduction pathways and other biological pathways. The biologic roles of miRNAs in cancer suggest a correlation with prognosis and therapeutic outcome. Further investigation of these roles may lead to new approaches for the categorization, diagnosis, and treatment of human cancers.

       RNA interference and tumor gene therapy.

       Ju, Ji-yu et al.

       Shipin Yu Yaopin (2009), 11(1), 54-57. Publisher: Shipin Yu Yaopin Bianjibu,

       RNA interference is a mechanism for controlling normal gene expression, which has been employed as a potential technol. for a wide range of disorders such as tumors, infectious diseases and metabolic disorders.  This paper reviews the discovery, mechanism of RNA interference and its application in tumor diagnosis and prevention, as well as some challenges.

       Nonviral vector-mediated RNA interference: Its gene silencing characteristics and important factors to achieve RNAi-based gene therapy.

       Takahashi, Yuki et al.

       Advanced Drug Delivery Reviews (2009), 61(9),  760-766.

       RNA interference (RNAi) is a potent and specific gene silencing event in which small interfering RNA (siRNA) degrades target m-RNA.  Therefore, RNAi is of potential use as a therapeutic approach for the treatment of a variety of diseases in which aberrant expression of m-RNA causes a problem.  RNAi can be achieved by delivering siRNA or vectors that transcribe siRNA or short-hairpin RNA (shRNA). The aim of this review is to examine the potential of nonviral vector-mediated RNAi technology in treating diseases. The characteristics of plasmid DNA expressing shRNA were compared with those of siRNA, focusing on the duration of gene silencing, delivery to target cells and target specificity. Recent progresses in prolonging the RNAi effect, improving the delivery to target cells and increasing the specificity of RNAi in vivo are also reviewed.

       Small silencing RNAs: State-of-the-art.

       Grimm, Dirke et al.

       Advanced Drug Delivery Reviews  (2009), 61(9), 672-703.

       Over just a single decade, there is rapid maturation of the field of RNA interference - the sequence-specific gene silencing mediated by small double-stranded RNAs - directly from its infancy into adulthood. With exciting data currently emerging from first clin. trials, it is now more likely than ever that RNAi drugs will soon provide another potent class of agents in our battle against infectious and genetic diseases. Accelerating this process and adding to RNAi's promise is our steadily expanding arsenal of innovative RNAi-based experimental tools and clinically applicable technologies. This presents a selection of relevant recent advances in RNAi therapeutics, from novel asymetry or bi-functional siRNA designs, deliberate use of small RNAs to regulate nuclear transcription, engineering of potent adeno-associated viral vectors for shRNA expression, exploitation of endogenous miRNAs to control transgene expression or vector tropism, to elegant attempts to inhibit cellular miRNAs involved in human disease.  The review includes cautionary notes on the potential risks inherent to in vivo RNAi applications, before discussing the latest surprising findings on circulating miRNAs in human body fluids, and concluding with an outlook into the possible future of RNAi as an increasingly powerful biomedical tool.

       MicroRNA expression and its implications for the diagnosis and therapeutic strategies of breast cancer.

       Shi, Ming et al.

       Cancer Treatment Reviews (2009), 35(4), 328-334.

       Alterations in microRNA (miRNA) expression have been associated with tumor suppression or tumorigenesis, metastasis and poor prognosis in human breast cancer.  Deregulation of miRNAs is emerging as a major aspect of cancer etiology because their capacity to direct the translation and stability of targeted transcripts can dramatically influence cellular physiology. Some miRNAs were considered to be associated with molecular subtypes of breast cancer and correlated with specific breast cancer clinicopathology factors, such as HER2, estrogen and progesterone receptor level, tumor stage, vascular invasion, or proliferation index. The expression level of miRNAs as suppressors, which can inhibit the expression of tumor promoting genes, is frequently down-regulated in breast cancer. Multiple lines of evidence also demonstrated the involvement of specific miRNAs as oncogenes in breast tumorigenesis. Some miRNAs have been considered to have potential clinical applications as a novel biomarker for breast cancer diagnosis and prognostic factor. Exploitation of the therapeutic potential of RNA interference will be an important task and achieved through the further understanding of the mechanisms of gene regulation by miRNAs.

       A novel mechanism is involved in cationic lipid-mediated functional siRNA delivery.

       Lu, James J. et al.

       Molecular Pharmaceutics (2009), 6(3), 763-771.

       A key challenge for therapeutic application of RNA interference is to efficiently deliver synthetic small interfering RNAs (siRNAs) into target cells that will lead to the knockdown of the target transcript (functional siRNA delivery). To facilitate rational development of nonviral carriers, authors have investigated by imaging, pharmacology and genetic approaches the mechanisms by which a cationic lipid carrier mediates siRNA delivery into mammalian cells. Authors show that approximately 95% of siRNA lipoplexes enter the cells through endocytosis and persist in endolysosomes for a prolonged period of time. However, inhibition of clathrin-, caveolin-, or lipid-raft-mediated endocytosis or macropinocytosis fails to inhibit the knockdown of the target transcript.  In contrast, depletion of cholesterol from the plasma membrane has little effect on the cellular uptake of siRNA lipoplexes, but it abolishes the target transcript knockdown. Furthermore, functional siRNA delivery occurs within a few hours and is gradually inhibited by lowering temperature.  These results demonstrate that although endocytosis is responsible for the majority of cellular uptake of siRNA lipoplexes, a minor pathway, probably mediated by fusion between siRNA lipoplexes and the plasma membrane, is responsible for the functional siRNA delivery. The findings suggest possible directions for improving functional siRNA delivery by cationic lipids.

       RNA oxidation in Alzheimer disease and related neurodegenerative disorders.       Nunomura, Akihiko et al.

       Acta Neuropathologica (2009), 118(1), 151-166. Publisher: Springer

       RNA oxidation and its biological effects are less well studied compared to DNA oxidation. However, RNA may be more susceptible to oxidative insults than DNA, for RNA is largely single-stranded and its bases are not protected by hydrogen bonding and less protected by specific proteins. Also, cellular RNA locates in the vicinity of mitochondria, the primary source of reactive oxygen species. Oxidative modification can occur not only in protein-coding RNAs, but also in non-coding RNAs that have been recently revealed to contribute towards the complexity of the mammalian brain. Damage to coding and non-coding RNAs will cause errors in proteins and disturbances in the regulation of gene expression. While less lethal than mutations in the genome and not inheritable, such sublethal damage to cells might be associated with underlying mechanisms of degeneration, especially age-associated neurodegeneration that is commonly found in the elderly population. Indeed, oxidative RNA damage has been described recently in most of the common neurodegenerative disorders including Alzheimer disease, Parkinson disease, dementia with Lewy bodies and amyotrophic lateral sclerosis. Of particular interest, the accumulating evidence obtained from studies on either human samples or experimental models coincidentally suggests that oxidative RNA damage is a feature in vulnerable neurons at early-stage of these neurodegenerative disorders, indicating that RNA oxidation actively contributes to the onset or the development of the disorders. Further investigations aimed at understanding of the processing mechanisms related to oxidative RNA damage and its consequences may provide significant insights into the pathogenesis of neurodegenerative disorders and lead to better therapeutic strategies.

       Riboactivators: transcription activation by noncoding RNA.

       Ansari, Aseem Z et al.

       Critical Reviews in Biochemistry and Molecular Biology (2009), 44(1), 50-61.

       The paradigm of gene regulation was forever changed by the discovery that short RNA duplexes could directly regulate gene expression.  Most regulatory roles attributed to noncoding RNA were often repressive.  Recent observations are beginning to reveal that duplex RNA molecule can stimulate gene transcription.  These RNA activators employ a wide array of mechanisms to up-regulate transcription of target genes, including functioning as DNA-tethered activation domains, as coactivators and modulators of general transcriptional machinery, and as regulators of other noncoding transcripts.  The discoveries over the past few years defy "Moore's law" in the breath-taking rapidity with which new roles for noncoding RNA in gene expression are being revealed.  As gene regulatory networks are reconstructed to accommodate the influence of noncoding RNAs, their importance in maintenance of cellular health will become increasingly apparent.  In fact, a new generation of therapeutic agents will focus on modulating the function of noncoding RNA.

       Antiangiogenesis of RNA interference on silencing VEGF and its receptor in colorectal cancer.

       Zhang, Hong-bin et al.

       Guoji Zhongliuxue Zazhi (2009), 36(2), 141-144.

       Researches find that VEGF and its receptor play an important role on angiogenesis of colorectal cancer, blocking the expression of VEGF and its receptor by RNA interference shows good prospects in anti-angiogenesis therapy of colorectal cancer.

       MicroRNAs in diabetes: tiny players in big disease.

       Pandey, Amit K. et al.

       Cellular Physiology and Biochemistry (2009), 23(46), 221-232.

       MicroRNAs (miRNAs) are a novel group of universally present small non-coding RNAs that have been implicated in wide ranging physiological processes and thereby are crit. in the manifestation of diverse diseases. Since their discovery as developmental regulators in C.elegans, they have come a long way and are currently associated with normal and diverse pathophysiol states including Parkinson's syndrome, cardiac hypertrophy, viral infection, diabetes and several types of cancer. Of special significance is their involvement in diabetes, an area in which several emerging reports point to the fact that these small RNA species could be special and critical in this complex disease and they or their specific inhibitors may be exploited as targets for therapeutic intervention. The stable nature of these miRNAs over m-RNAs is an added advantage of them being projected for the same. This review focuses on and discusses the current diabetic epidemic and the potential role(s) of these miRNAs in various physiological processes that lead to the diabetic phenotype with an objective of better understanding the emerging mechanisms of these small molecules in the development and progression of diabetes and its complications.

       Short interfering RNA-mediated gene silencing; towards successful application in human patients.

       Siomi, Mikiko C et al.

       Advanced Drug Delivery Reviews  (2009),  61(9), 668-671

       Gene silencing mechanisms that are mediated by small RNAs of 20-30 nucleotides(nt) are collectively called RNA silencing.  The representative mechanism is RNA interference (RNAi), in which   21-nt small RNAs (short interfering RNAs or siRNAs) efficiently trigger cleavage of target gene transcripts. As a result, proteins are no longer made from the targeted m-RNAs.  RNAi is tremendously specific and efficient with regard to recognizing target gene and disrupting their expression. The triggering molecules double-stranded (ds) RNAs, are quite stable in vivo and are basically indistinguishable from natural endogenous RNAs; thus, RNAi intrinsically has great potential for therapeutic use. In fact, RNAi-based clinical trials for treating human diseases are currently ongoing worldwide. However, optimization of the protocols still requires substantial investigation; indeed, relevant studies have been undertaken internationally by many researchers.  Given the growing anticipation of RNAi as a disease therapy, the RNAi mechanism and machinery from a biochemical perspective might provide a way to advance its successful application in the human body.

       Recent advances in the application of RNA interference techniques.

       Li, Shu-fang et al.

       Zhongguo Shiyan Dongwu Xuebao (2009), 17(1), 76-79.

       RNA interference (RNAi) has made considerable development in the biological research area during the ten years since it was discovered. It has been demonstrated its huge potential initially. This article summarizes the mediating methods of SiRNA and the newest application progress of RNAi in basic research, disease model building and medicinal research.

       Transcriptional and post-transcriptional impact of toxic RNA in myotonic dystrophy.

       Osborne, Robert J. et al.

       Human Molecular Genetics (2009), 18(8), 1471-1481.

       Myotonic dystrophy type 1 (DM1) is an RNA dominant disease in which mutant transcripts containing an expanded CUG repeat (CUGexp) cause muscle dysfunction by interfering with biogenesis of other m-RNAs.  The toxic effects of mutant RNA are mediated partly through sequestration of splicing regulator Muscleblind-like 1 (Mbnl1), a protein that binds to CUGexp RNA. A gene that is prominently affected encodes chloride channel 1 (Clcn1), resulting in hyperexcitability of muscle (myotonia).  To identify DM1-affected genes and study mechanisms for dysregulation, global m-RNA profiling in transgenic mice was performed that express CUGexp RNA, when compared with Mbnl1 knockout and Clcn1 null mice.  It was found that the majority of changes induced by CUGexp RNA in skeletal muscle can be explained by reduced activity of Mbnl1, including many changes that are secondary to myotonia. The pathway most affected comprises genes involved in calcium signaling and homeostasis.  Some effects of CUGexp RNA on gene expression are caused by abnormal alternative splicing or downregulation of Mbnl1-interacting m-RNAs.  However, several of the most highly dysregulated genes showed altered transcription, as indicated by parallel changes of the corresponding pre-m-RNAs.  These results support the idea that trans-dominant effects of CUGexp RNA on gene expression in this transgenic model may occur at the level of transcription, RNA processing and m-RNA decay, and are mediated mainly but not entirely through sequestration of Mbnl1.

       Disease phenocode analysis identifies SNP-guided microRNA maps (MirMaps) associated with human "master" disease genes.

       Glinsky, Gennadi V.

       Cell Cycle (2008), 7(23), 3680-3694. 

       Recently authors reported the results of a genome-wide disease phenocode analysis interrogating the relationships between structural features and gene expression patterns of disease-linked SNPs, microRNAs and m-RNAs of protein-coding genes in assocn. to phenotypes of 15 common human disorders.  One of the main implications of this analysis is that transcriptionally co-regulated SNP sequence-bearing RNAs are more likely to exert a cumulative effect in trans on phenotypes. In this work, the validity of a disease phenocode concept within a genomic context of distinct continuously spaced sets of disease-linked SNPs and m-RNAs of relevant protein-coding genes is verified. Authors report a sequence homological profiling of two sets of disease-linked SNPs which are located within continuous genomic regions associated with individual protein-coding genetic loci (NLRP1 and STAT4) and are likely to exhibit common profiles of transcriptional activity. Most of microRNAs (15 of 19; 79%) homologous to the NLPRP1- associated disease-linked SNPs have potential protein-coding m-RNA targets among the principal components of the nuclear import pathway (NIP) and/or inflammasome pathways, including KPNA1, NLRP1 and NLRP3 genes.  Authors demonstrate that cumulative targeting effects of microRNAs on m-RNAs within distinct allelic contexts of disease-linked SNPs are in agreement with microarray analysis-defined gene expression phenotypes associated with human genotypes of Crohn's disease (CD) and rheumatoid arthritis (RA) populations. Microarray experiments and disease phenocode analysis identify ten-gene expression signatures which seem to reflect the activated status of disease-linked SNPs/ microRNAs/ m-RNAs axis in peripheral blood mononuclear cells (PBMC) of 66% CD patients and 80% RA patients. Comparisons of ten-gene signature expression profiles and NLRP3/NLRP1 m-RNA expression ratios in PBMC of individual CD and RA patients and control subjects indicate that measurements of these markers may be useful for diagnostic applications.

       These findings demonstrate that NLPRP1- and STAT4- associated disease-linked SNPs have common sequence-defined features which are recapitulating the essential phenotype-affecting features of genome-wide disease-linked SNPs, suggesting that NLRP1 (NALP1) and STAT4 genetic loci may constitute "master" disease genes. It is concluded that both genome-wide SNP variations and SNP polymorphisms associated with "master" disease genes may cause similar genetically-defined malfunctions of the NIP and inflammasome/innate immunity pathways which are likely to contribute to pathogenesis of multiple common human disorders.

       Short hairpin RNA targeting NP m-RNA inhibiting Newcastle disease virus production and other viral structural m-RNA transcription.

       Yue, Hua; Deng, Shu  et al.

       Virus Genes (2009), 38(1), 143-148. 

       Newcastle disease virus (NDV), formally recognized as avian paramyxovirus 1 (APMV-1), is the etiologiocal agent of Newcastle disease (ND), an affliction which can cause severe losses in the poultry industry.  Better understanding of the molecular basis of viral structural genes involved with production. should contribute significantly toward the development of improved prophylactic and therapeutic reagents to control the infection. Here authors show that a short hairpin RNA (shRNA) eukaryotic expression vector targeting nucleocapsid (NP) gene of NDV can potently inhibit NDV production in both primary cells and embryonated chicken eggs. Moreover, shRNA specific for NP abolished the accumulation of not only the corresponding m-RNA but also P, HN, F, M gene m-RNA.  The findings reveal that newly synthesized NP m-RNA is essential for NDV transcription and replication, and provide a basis for the development of shRNAs as a prophylaxis and therapy for NDV infection in poultry.

       Dynamic combinatorial selection of molecules capable of inhibiting the (CUG) repeat RNA-MBNL1 interaction in vitro: discovery of lead compounds targeting myotonic dystrophy (DM1).

       Gareiss, Peter C. et al.

       Journal of the American Chemical Society  (2008), 130(48), 16254-16261

       Myotonic dystrophy type 1 (DM1), the most common form of muscular dystrophy in adults, is an RNA-mediated disease. Dramatically expanded (CUG) repeats accumulate in nuclei and sequester RNA-binding proteins such as the splicing regulator MBNL1. Authors have employed resin-bound dynamic combinatorial chemistry (RBDCC) to identify the first examples of compounds able to inhibit MBNL1 binding to (CUG) repeat RNA. Screening an RBDCL with a theoretical diversity of 11 325 members yielded several molecules with significant selectivity for binding to (CUG) repeat RNA over other sequences. These compounds were also able to inhibit the interaction of GGG-(CUG)109-GGG RNA with MBNL1 in vitro, with Ki values in the low micromolar range.

       The presence of rRNA sequences in polyadenylated RNA and its potential functions.

       Kong, Qiongman  et al.

       Biotechnology Journal  (2008), 3(8), 1041-1046

       Accumulating evidence has shown that various lengths of rRNA sequences are widely present in polyadenylated RNA cotaining transcripts (PART). PART are highly abundant and widely expressed in various tissues. It appears that there may be two types of PART. One type, type I, contains the rRNA segments (from .approximately 10 nucleotides up to several hundred nucleotides) located within the transcripts. It has been demonstrated that short rRNA sequences within type I PART may function as cis-regulatory elements that regulate translational efficiency. The other type, type II, contains large portions or almost entire sequences of rRNA with a cap at the 5' end and poly(A) at 3' end. Recent work has shown that some type II PART have functional significance for some neurodegenerative disease processes and may play an important role in the pathogenesis of diseases. Further investigation in this area is critical to understanding the basic biol. of PART and the potential role of PART in diseases.

       Neurodegeneration: RNA turns number one suspect in polyglutamine diseases.

       Birman, Serge

       Current Biology (2008), 18(15), R659-R661

       Polyglutamine expansion diseases are triggered by the accumulation of toxic proteins. A new study reports that RNA molecules containing long CAG repeats can also be toxic to neurons and may play a significant role in pathogenesis.

       RNA interference - potential therapeutic applications for the gastroenterologist.

       Pellish, R. S. et al.

       Alimentary Pharmacology and Therapeutics  (2008), 27(9), 715-723.

       A new technique of gene regulation, termed RNA interference, has emerged recently. RNA interference utilizes short double-stranded RNA to inhibit selectively gene expression of complementary RNA nucleotide sequences after transcription, but prior to translation. Gastrointestinal and hepatic disorders may be particularly amenable to therapeutic RNA interference intervention because of the relative ease of delivery of drugs to the gastrointestinal tract and liver. To examine the published literature for potential clinical uses of RNA interference in gastroenterology and speculate on future therapies for luminal disease.: Reports were identified using PubMed and the search term "RNA interference", focusing on therapeutic uses related to gastrointestinal and liver disease. Cellular and animal models demonstrate the potential application of short-interfering RNA-based therapies for viral hepatitis and inflammatory bowel disease. With validation of specific targets and better in vivo delivery of short-interfering RNA, RNA interference may represent a new frontier for molecular-targeted therapy in gastrointestinal and hepatological Short-interfering RNA provides a novel and specific means to inhibit gene expression. Translation to the clinical arena will require further definition of side-effects, off-target effects and delivery systems. Ultimately, mucosally applied or endoscopically delivered short-interfering RNA could be one of the earliest clinical uses of short-interfering RNA therapy.

       Small interfering RNA therapy in cancer: mechanism, potential targets, and clinical applications.

       Huang, Chuan  et al.

       Expert Opinion on Therapeutic Targets  (2008),  12(5), 637-645

       Small interfering RNA (siRNA) has become a powerful tool in knocking down or silencing gene expression in most cells. siRNA-based therapy has shown great promise for many diseases such as cancer. Major targets for siRNA therapy include oncogenes and genes that are involved in angiogenesis, metastasis, survival, antiapoptosis and resistance to chemotherapy. This review briefly summarizes current advances in siRNA therapy and clinical applications in cancers, especially in pancreatic cancer.

       This review article covers several aspects of siRNA therapy in cancer, which include the types of siRNA, the delivery systems for siRNA, and the major targets for siRNA therapy.  Specific attention is given to siRNA in pancreatic cancer, which is the main research focusof the authors. siRNA can be introduced into the cells by using either chemically synthesized siRNA oligo-nucleotides (oligos), or vector-based siRNA (shRNA), which allows long lasting and more stable gene silencing. Nanoparticles and liposomes are commonly used carriers, delivering the siRNA with better transfection efficiency and protecting it from degradation. In combination with standard chemotherapy, siRNA therapy can also reduce the chemoresistance of certain cancers, demonstrating the potential of siRNA therapy for treating many malignant diseases.










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       Nonviral methods for siRNA delivery.

       Kun Gaoand Leaf Huang

       Mol. Pharmaceutics

       RNA interference (RNAi) as a mechanism to selectively degrade m-RNA (m-RNA) expression has emerged as a potential novel approach for drug target validation and the study of functional genomics. Small interfering RNAs (siRNA) therapeutics has developed rapidly and already there are clinical trials ongoing or planned. Although other challenges remain, delivery strategies for siRNA become the main hurdle that must be resolved prior to the full-scale clinical development of siRNA therapeutics. This review provides an overview of the current delivery strategies for synthetic siRNA, focusing on the targeted, self-assembled nanoparticles which show potential to become a useful and efficient tool in cancer therapy.                                       (ScienceDirect)

       Targeted quantum dot conjugates for siRNA delivery

       Austin M. Derfus  et al.

       Bioconjugate Chem., 2007, 18 (5), pp 1391–1396

       Treatment of human diseases such as cancer generally involves the sequential use of diagnostic tools and therapeutic modalities. Multifunctional platforms combining therapeutic and diagnostic imaging functions in a single vehicle promise to change this paradigm. In particular, nanoparticle-based multifunctional platforms offer the potential to improve the pharmacokinetics of drug formulations, while providing attachment sites for diagnostic imaging and disease targeting features. Authors have applied these principles to the delivery of small interfering RNA (siRNA) therapeutics, where systemic delivery is hampered by rapid excretion and nontargeted tissue distribution. Using a PEGlyated quantum dot (QD) core as a scaffold, siRNA and tumor-homing peptides (F3) were conjugated to functional groups on the particle's surface. Authors found that the homing peptide was required for targeted internalization by tumor cells, and that siRNA cargo could be coattached without affecting the function of the peptide. Using an EGFP model system, the role of conjugation chemistry was investigated, with siRNA attached to the particle by disulfide cross-linkers showing greater silencing efficiency than when attached by a nonreducible thioether linkage. Since each particle contains a limited number of attachment sites, it is further explored the tradeoff between number of F3 peptides and the number of siRNA per particle, leading to an optimized formulation. Delivery of these F3/siRNA−QDs to EGFP-transfected HeLa cells and release from their endosomal entrapment led to significant knockdown of EGFP signal. By designing the siRNA sequence against a therapeutic target (e.g., oncogene) instead of EGFP, this technology may be ultimately adapted to simultaneously treat and image metastatic cancer.

       Controlling HBV replication in vivo by intravenous administration of triggered PEgylated siRNA-nanoparticles.

       Sergio Carmona  et al.

       Mol. Pharmaceutics, Article ASAP

       Harnessing RNA interference (RNAi) to inhibit hepatitis B virus (HBV) gene expression has promising application to therapy. Here authors describe a new hepatotropic nontoxic lipid-based vector system that is used to deliver chemically unmodified small interfering RNA (siRNA) sequences to the liver. Anti HBV formulations were generated by condensation of siRNA (A component) with cationic liposomes (B component) to form AB core particles. These core particles incorporate an aminoxy cholesteryl lipid for convenient surface postcoupling of polyethylene glycol (PEG; C component, stealth/biocompatibility polymer) to give triggered PEGylated siRNA-nanoparticles (also known as siRNA-ABC nanoparticles) with uniform small sizes of 80−100 nm in diameter. The oxime linkage that results from PEG coupling is pH sensitive and was included to facilitate acidic pH-triggered release of nucleic acids from endosomes. Nanoparticle-mediated siRNA delivery results in HBV replication knockdown in cell culture and in murine hydrodynamic injection models in vivo. Furthermore repeated systemic administration of triggered PEGylated siRNA-nanoparticles to HBV transgenic mice results in the suppression of markers of HBV replication by up to 3-fold relative to controls over a 28 day period. This compares favorably to silencing effects seen during lamivudine treatment. Collectively these observations indicate that our PEGylated siRNA-nanoparticles may have valuable applications in RNAi-based HBV therapy.

       Cellular siRNA delivery mediated by a cell-permeant RNA-binding protein and photoinduced RNA interference.

       Tamaki Endoh  et al.

       Bioconjugate Chem., 2008, 19 (5), pp 1017–1024

       HIV-1 TAT peptide, which is a cell-penetrating peptide (CPP), was fused to the U1A RNA-binding domain (TatU1A) to generate a sequence-specific siRNA delivery system for mammalian cells. The siRNA contained a short 5′-extension that is specifically recognized by the U1A RNA-binding domain (U1AsiRNA). Specific binding of TatU1A to the U1AsiRNA was confirmed using a gel mobility shift assay. The U1AsiRNA was internalized by cells only when it was preincubated with TatU1A before addition to the cells. Although most of the internalized siRNA seemed to be entrapped in endocytic compartments, efficient redistribution of the entrapped siRNAs was achieved by photostimulation of a fluorophore attached to TatU1A. Once in the cytoplasm, the siRNA induced RNAi-mediated gene silencing. Authors refer to this delivery strategy as CLIP-RNAi. CLIP-RNAi is a promising strategy for RNAi experiments and for pinpoint RNAi therapy.

       Inhalable siRNA: potential as a therapeutic agent in the lungs.

       Niamh Durcan  et al.

       Mol. Pharmaceutics, 2008, 5 (4), pp 559–566

       RNA interference (RNAi) is gaining increasing popularity both as a molecular biology tool and as a potential therapeutic agent. RNAi is a naturally occurring gene regulatory mechanism, which has a number of advantages over other gene/antisense therapies including specificity of inhibition, potency, the small size of the molecules and the diminished risk of toxic effects, e.g., immune responses. Targeted, local delivery of RNAi to the lungs via inhalation offers a unique opportunity to treat a range of previously untreatable or poorly controlled respiratory conditions. In this timely review look at the potential applications of RNAi in the lungs for the treatment of a range of diseases including inflammatory and immune conditions, cystic fibrosis, infectious disease and cancer. In 2006 Alnylam initiated the first phase 1 clinical study of an inhaled siRNA for the treatment of respiratory syncytial virus. If its potential as a therapeutic is to be realized, then safe and efficient means of targeted delivery of small interfering RNA (siRNA) to the lungs must be developed. Therefore in this review authors also present the latest developments in siRNA delivery to airway cells in vitro and the work to date on in vivo delivery of siRNA to the lungs for the treatment of a range of diseases.

       siRNA inhibition of telomerase enhances the anti-cancer effect of doxorubicin in breast cancer cells.

       Dong, Xuejun et al.

       BMC Cancer (2009), 9

       Doxorubicin is an effective breast cancer drug but is hampered by a severe, dose-dependent toxicity. Concomitant adminis-tration of doxorubicin and another cancer drug may be able to sensitize tumor cells to the cytotoxicity of doxorubicin and lowers the therapeutic dosage. In this study, the combined effect of low-dose doxorubicin and siRNA inhibition of telomerase as examined on breast cancer cells. Authors found that when used individually, both treatments were rapid and potent apoptosis inducers; and when the two treatments were combined, an enhanced and sustained apoptosis induction in breast cancer cells was observed.  siRNA targeting the m-RNA of the protein component of telomerase, the telomerase reverse transcriptase (hTERT), was transfected into two breast cancer cell lines. The siRNA inhibition was confirmed by RT-PCR and western blot on hTERT m-RNA and protein levels, respectively, and by measuring the activity level of telomerase using the TRAP assay.  The effect of the hTERT siRNA on the tumorigenicity of the breast cancer cells was also studied in vivo by injection of the siRNA-transfected breast cancer cells into nude mice.  The effects on cell viability, apoptosis and senescence of cells treated with hTERT siRNA, doxorubicin, and the combined treatment of doxorubicin and hTERT siRNA, were examd. in vitro by MTT assay, FACS and SA-b-galactosidase staining. The hTERT siRNA effectively knocked down the m-RNA and protein levels of hTERT, and reduced the telomerase activity to 30% of the untreated control.  In vivo, the tumors induced by the hTERT siRNA-transfected cells were of reduced sizes, indicating that the hTERT siRNA also reduced the tumorigenic potential of the breast cancer cells.  The siRNA treatment reduced cell viability by 50% in breast cancer cells within two days after transfection, while 0.5µM doxorubicin treatment had a comparable effect but with a slower kinetics.

       The combination of hTERT siRNA and 0.5µM doxorubicin killed twice as many cancer cells, showing a cumulative effect of the two treatments. The study demonstrated the potential of telomerase inhibition as an effective treatment for breast cancer.  When used in conjunction to doxorubicin, it could potentiate the cytotoxic effect of the drug to breast cancer cells.

       Acetylation of PAMAM dendrimers for cellular delivery of siRNA.

       Waite, Carolyn L. et al.

       BMC Biotechnology (2009), 9

       The advancement of gene silencing via RNA interference is limited by the lack of effective short interfering RNA (siRNA) delivery vectors. Rational design of polymeric carriers has been complicated by the fact that most chemical modifications affect multiple aspects of the delivery process. In this work, the extent of primary amine acetylation of generation 5 poly (amidoamine) (PAMAM) dendrimers was studied as a modification for the delivery of siRNA to U87 malignant glioma cells. PAMAM dendrimers were reacted with acetic anhydride to obtain controlled extents of primary amine acetylation. Acetylated dendrimers were complexed with siRNA, and physical properties of the complexes were studied. Dendrimers with up to 60% of primary amines acetylated formed apprroximately 200 nm complexes with siRNA. Increasing amine acetylation resulted in reduced polymer cytotoxicity to U87 cells, as well as enhanced dissocciation of dendrimer/siRNA complexes.  Acetylation of dendrimers reduced the cellular delivery of siRNA which correlated with a reduction in the buffering capacity of dendrimers upon amine acetylation. Confocal microscopy confirmed that escape from endosomes is a major barrier to siRNA delivery in this system. Primary amine acetylation of PAMAM dendrimers reduced their cytotoxicity to U87 cells, and promoted the release of siRNA from dendrimer/siRNA complexes.  A modest fraction (approx. 20%) of primary amines of PAMAM can be modified while maintaining the siRNA delivery efficiency of unmodified PAMAM, but higher degrees of amine neutralization reduced the gene silencing efficiency of PAMAM/siRNA delivery vectors.

       Application of RNA interference in the gene therapy of ovarian cancer.

       Zhou, Qing-feng et al.

       Shangqiu Shifan Xueyuan Xuebao  (2009), 25(3),  100-102

       RNA interference (RNAi) is a post - transcription gene silence which is derived by double strand RNA (dsRNA) specifically.  As an effective measure of gene silence, RNAi technology shows a promising prospect in the treatment of tumor.  Therefore, authors summarize the application of the RNAi technology in the treatment of ovarian cancer's gene therapy.

       Prospects of RNAi and microRNA-based therapies for hepatitis C.

       Pan, Qiuwei et al.

       Expert Opinion on Biological Therapy (2009), 9(6), 713-724.

       RNA interference (RNAi) represents a promising new approach to combat viral infections, and recent developments in the field of gene therapy have increased the feasibility of clinical applications to explore the utility of RNAi for the treatment of the ultimately life-threatening liver disease caused by hepatitis C virus (HCV), which affects approx. 170 million people worldwide.  The review summarises the current developments in liver-directed gene delivery and the potential application of RNAi for the treatment of HCV. In addition, the involvement of microRNAs (miRNA) in HCV infection and the potential therapeutic implications are emphasized. RNAi technologies have fuelled rapid progress in the basic understanding of HCV biological and revealed numerous new viral and host-cell factors as potential targets for therapy. Together with the improvement of gene delivery technology and the discovery of the critical role of miRNA in HCV infection, RNAi and miRNA-based antiviral strategies hold great promise for the future.

       Naked siLNA-mediated gene silencing of lung bronchoepithelium EGFP expression after intravenous administration.

       Glud, Sys Zoffmann et al.

       Oligonucleotides (2009), 19(2), 163-168.

       The use of systemic siRNA therapeutics for RNA interference-mediated silencing of disease genes is limited by serum instability and inadequate biodistribution. Authors have previously reported on the EGFP gene silencing effect of chitosan/siRNA nanoparticles in the bronchoepithelium of mice lungs following intranasal delivery and improved serum stability and reduced off-targeting effects in vitro by incorporation of locked nucleic acid (LNA). In this study, authors examine the pulmonary gene silencing effect of siLNAs targeting enhanced-green-fluorescent-protein (EGFP) in lung bronchoepithelium upon i.v. delivery of naked siLNAs and upon intranasal delivery of either naked siLNA or chitosan/siLNA nanoparticles.  It is shown that naked siLNA administered i.v. efficiently reduces the EGFP protein expression. A similar effect is obtained with intranasal delivery of chitosan nanoparticles containing siLNA whereas intranasally instilled naked siLNA did not cause a knockdown.

       RNA interference technology to improve recombinant protein production in Chinese hamster ovary cells.

       Wu, Suh-Chin et al.

       Biotechnology Advances (2009), 27(4), 417-422.

       RNA interference (RNAi) technology has become a novel tool for silencing gene expression in cells or organisms, and has also been used to develop new therapeutics for certain diseases. This review describes its other application of using RNAi technology to increase cellular productivity and the quality of recombinant proteins that are produced in Chinese hamster ovary (CHO) cells, the most important mammalian cell line used in producing licensed biopharmaceuticals in these days. The approaches reported include the silencing of apoptosis-associated gene expression, protein glycosylation-associated gene expression, lactate dehydrogenase involved in cellular metabolism, and dihydrofolate reductase used for gene amplification. All of these works belong to the single component approach therefore depends strongly on the identification of the down-regulation of the critical target gene which can markedly influence the cellular functions associated with recombinant protein expression in CHO cells. Future RNAi approaches can be extended to silence multiple targets involved in different cellular pathways for changing the global gene regulation in cells, as well as the targets related to microRNA molecules for cellular self regulation.

       Vero cells as a model to study the effects of adenoviral gene delivery vectors on the RNAi system in context of viral infection.

       Matskevich, Alexey A. et al.

       Journal of Innate Immunity (2009), 1(4), 389-394.

       Technology based on RNA interference (RNAi) is a promising source for new antiviral therapies. Although the application of RNAi has been studied extensively, significant problems with using RNAi remain.  Very few studies have specifically assessed model systems for testing the effects of viruses or gene delivery vectors on the RNAi system.  Since viruses have developed efficient strategies to circumvent the interferon (IFN) response, an IFN-deficient model system should be considered. Here authors show that Vero cells, which lack IFN-α and IFN-b genes, knockdown of Dicer, a key RNAi component, led to accelerated death of cells infected with other evolutionary distinct viruses: influenza A virus, vesicular stomatitis virus and poliovirus. Authors also demonstrate that transduction of Vero cells with adenoviral vector with subsequent infection with influenza A virus also resulted in increased mortality of infected cells.  These effects were much weaker in IFN-producing A549 and Hela cell lines.  Thus, the Vero cell line could serve as an interesting model for studying the effects of gene delivery vectors on the RNAi system in the context of virus-related disorders.

       Lentiviral delivery of short hairpin RNAs.

       Manjunath, N. et al.

       Advanced Drug Delivery Reviews  (2009), 61(9), 732-745.

       In less than a decade after discovery, RNA interference-mediated gene silencing is already being tested as potential therapy in clinical trials for a no. of diseases. Lentiviral vectors provide a means to express short hairpin RNA (shRNA) to induce stable and long-term gene silencing in both dividing and non-dividing cells and thus, are being intensively investigated for this purpose.  However, induction of long-term shRNA expression can also cause toxicities by inducing off-target effects and interference with the endogenous micro-RNA (miRNA) pathway that regulates cellular gene expression. Recently, several advances have been made in the shRNA vector design to mimic cellular miRNA processing and to express multiplex siRNAs in a tightly regulated and reversible manner to overcome toxicities.  In this review authors describe some of these advances, focusing on the progress made in the development of lentiviral shRNA delivery strategies to combat viral infections.

       Delivery of RNA interference therapeutics using polycation-based nanoparticles.

       Howard, Kenneth Alan.

       Advanced Drug Delivery Reviews (2009), 61(9), 710-720

       RNAi-based therapies are dependent on extracellular and intracellular delivery of RNA molecules for enabling target interaction.  Polycation-based nanoparticles (or polyplexes) formed by self-assembly with RNA can be used to modulate pharmacokinetics and intracellular trafficking to improve the therapeutic efficacy of RNAi-based therapeutics. This review describes the application of polyplexes for extracellular and intracellular delivery of synthetic RNA molecules. Focus is given to routes of administration and silencing effects in animal disease models. The inclusion of functional components into the nanoparticle for controlling cellular trafficking and RNA release is discussed. This work highlights the versatile nature of polycation-based nanoparticles to fulfil the delivery requirements for RNA molecules with flexibility in design to evolve alongside an expanding repertoire of RNAi-based drugs.

       Cellular siRNA delivery using cell-penetrating peptides modified for endosomal escape.

       Endoh, Tamaki et al.

       Advanced Drug Delivery Reviews  (2009), 61(9), 704-709

       RNAi-mediated silencing of specific genes is a promising strategy for gene therapy.  To utilize RNAi for therapy, an efficient and safe method for delivery of RNA into the cell cytosol is necessary. The plasma membrane is the primary, and most difficult, barrier for RNA to cross, because negative charged RNA is strongly repulsed by the negative charged membrane.  A variety of cationic polymers can be used as RNA carriers by interacting with RNA and covering its negative charges to form a cell-penetrating complex. Among the emerging candidates for RNA carriers are cationic cell-penetrating peptides (CPPs), which can cross the plasma membrane and internalize into cells together with RNA.  This review focuses on CPP-based RNA delivery strategies. In using CPP-based RNA delivery, most of the RNA internalized by the cell is entrapped in endosomes. Strategies for endosomal escape of RNAs are also reviewed.

       Hepatic delivery of RNA interference activators for therapeutic application.

       Arbuthnot, Patrick et al.

       Current Gene Therapy (2009), 9(2), 91-103

       Globally, hepatic diseases are an important cause of mortality and morbidity. Harnessing RNA interference (RNAi) to silence pathology-causing genes specifically offers exciting possibilities for improvement of treatment. Nevertheless achieving efficient and safe delivery of RNAi activators remains an important objective before this gene silencing approach realizes its full therapeutic potential. Several viral and non viral vectors (NVVs) are being developed for hepatotropic delivery of synthetic and expressed RNAi activators. Each has advantages and disadvantages that are suited to particular disease conditions.  Amongst the viral vectors, recombinant adeno- associated viruses and PEG-modified helper dependent adenoviruses show promise for situations that require intermediate to long term expression of RNAi activators.  Recombinant lentiviruses have not been used extensively as hepatotropic RNAi vectors, but are likely to find application where lasting therapeutic silencing is required.  NVVs are a particularly important class of vector and are effective for delivery of synthetic RNAi activators to the liver. Preclin. investigations using RNAi-mediated gene silencing to counter persistent hepatitis B virus, hepatitis C virus, hepatocellular carcinoma, hypercholesterolemia and cirrhosis are discussed in this review.  Although obstacles remain, vigorous research has given impetus to the field and RNAi-based treatment of liver diseases is likely to become a reality in the near future.

       Reversed-phase ion-pair liquid chromatography analysis and purification of small interfering RNA.

       McCarthy, Sean M. et al.

       Analytical Biochemistry  (2009), 390(2), 181-188.

       Small interfering RNA (siRNA)-induced gene silencing shows great promise in genomic research and therapeutic applications. siRNA duplexes are typically assembled from complementary synthetic oligonucleotides.  High-purity single-stranded species are required for in vivo applications. Methods for separation, characterization, and purification of short RNA strands have been developed based on reversed-phase ion-pair liquid chromatography. The purification strategies were developed for both single-stranded and duplex RNA species. The method of duplex purification uses on-column annealing of complementary RNA strands, followed by separation of the target duplex from truncated duplexes and single-stranded RNA forms.  The proposed method significantly reduces the purification time of synthetic siRNA.

       Biodegradable dextran nanogels for RNA interference: focusing on endosomal escape and intracellular siRNA delivery.

       Raemdonck, Koen et al.

       Advanced Functional Materials (2009), 19(9), 1406-1415.

       The successful therapeutic application of small interfering RNA (siRNA) largely relies on the development of safe and effective delivery systems that are able to guide the siRNA therapeutics to the cytoplasm of the target cell.  In this report, biodegradable cationic dextran nanogels are engineered by inverse emulsion photopolymerisation and their potential as siRNA carriers is evaluated.  The nanogels are able to entrap siRNA with a high loading capacity, based on electrostatic interaction. Confocal microscopy and flow cytometry anal. reveal that large amts. of siRNA-loaded nanogels can be internalized by HuH-7 human hepatoma cells without significant cytotoxicity.  Following their cellular uptake, it is found that the nanogels are mainly trafficked towards the endolysosomes. The influence of two different strategies to enhance endosomal escape on the extent of gene silencing is investigated. It is found that both the application of photochemical internalization (PCI) and the use of an influenza-derived fusogenic peptide (diINF-7) can significantly improve the silencing efficiency of siRNA-loaded nanogels. Furthermore, it is shown that an efficient gene silencing requires the degradation of the nanogels. As the degradation kinetics of the nanogels can easily be tailored, these particles show potential for intracellular controlled release of short interfering RNA.

       Proliferation of breast cancer cells inhibited by small interfering RNA directed against KDR gene in vitro and in vivo.

       Zhang, Xiaojing  et al.

       Xibao Yu Fenzi Mianyixue Zazhi (2008), 24(1), 58-61

       In vitro, small interfering RNA (siRNA) was transfected into MCF-7 cells to induce RNA interference (RNAi) by using cationic liposome Lipofectamine 2000TM. The changes of kinase insert domain-containing receptor (KDR) m-RNA and protein expressions in both siRNA treatment group and control group were measured by MTT assay and RT-PCR.  In vivo, the siRNA was transfected into transplanted tumor in nude mice by using cationic polymer nanoparticle in vivo jetPEITM.  Tumor growth was observed. The m-RNA and protein expressions of KDR were measured by RT-PCR and immunohistochem. staining. Experiments in vitro showed that siRNA directed against KDR effectively inhibited the proliferation of MCF-7 cells and downregulated KDR m-RNA expression. In vivo, the growth of tumor was visibly suppressed. Furthermore, RT-PCR and immunohistochemical results indicated that KDR m-RNA and protein expressions were reduced in excised tumors. RNAi mediated by chemically modified siRNA markedly decreased KDR gene expression and inhibited cellular proliferation. It may have the potential as a therapeutic method to treat human cancer.

       Trans-splicing into highly abundant albumin transcripts for production of therapeutic proteins in vivo.

       Wang, Jun  et al.

       Molecular Therapy (2009), 17(2), 343-351.

       Spliceosome-mediated RNA trans-splicing has emerged as an exciting mode of RNA therapy.  Here we describe a novel trans-splicing strategy, which targets highly abundant pre-m-RNAs, to produce therapeutic proteins in vivo.  First, a pre-trans-splicing molecule (PTM) was used that mediated trans-splicing of human apolipoprotein A-I (hapoA-I) into the highly abundant mouse albumin exon 1. Hydrodynamic tail vein injection of the hapoA-I PTM plasmid in mice followed by analysis of the chimeric transcripts and protein, confirmed accurate and efficient trans-splicing into albumin pre-m-RNA and production of hapoA-I protein.  The versatility of this approach was demonstrated by producing functional human papillomavirus type-16 E7 (HPV16-E7) single-chain antibody in C57BL/6 mice and functional factor VIII (FVIII) and phenotypic correction in hemophilia A mice.  Altogether, these studies demonstrate that trans-splicing to highly abundant albumin transcripts can be used as a general platform to produce therapeutic proteins in vivo.














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       Therapeutic siRNAs for dominant genetic skin disorders including pachyonychia congenital.

       Leachman, Sancy A. et al.

       Journal of Dermatological Science, 51(3), 151 (Sept., 2008)

       The field of science and medicine has experienced a flood of data and technology associated with the human genome project. Over 10,000 human diseases have been genetically defined, but little progress has been made with respect to the clinical application of this knowledge. A notable exception to this exists for pachyonychia congenita (PC), a rare, dominant-negative keratin disorder. Utilizing the technological by-products of the human genome project, such as RNA interference (RNAi) and quantitative RT-PCR (qRT-PCR), physicians and scientists have collaborated to create a candidate siRNA therapeutic that selectively inhibits a mutant allele of KRT6A, the most commonly affected PC keratin. In vitro investigation of this siRNA demonstrates potent inhibition of the mutant allele and reversal of the cellular aggregation phenotype. In parallel, an allele-specific quantitative real-time RT-PCR assay has been developed and validated on patient callus samples in preparation for clinical trials. If clinical efficacy is ultimately demonstrated, this "first-in-skin" siRNA may herald a paradigm shift in the treatment of dominant-negative genetic disorders.

       Discovery of ligands for a novel target, the human telomerase RNA, based on flexible-target virtual screening and NMR.

       Irene Gmez Pinto  et al.

       J. Med. Chem., 2008, 51 (22), pp 7205–7215

       The human ribonucleoprotein telomerase is a validated anticancer drug target, and hTR-P2b is a part of the human telomerase RNA (hTR) essential for its activity. Interesting ligands that bind hTR-P2b were identified by iteratively using a tandem structure-based approach: docking of potential ligands from small databases to hTR-P2b via the program MORDOR, which permits flexibility in both ligand and target, with subsequent NMR screening of high-ranking compounds. A high percentage of the compounds tested experimentally were found via NMR to bind to the U-rich region of hTR-P2b; most have MW < 500 Da and are from different compound classes, and several possess a charge of 0 or +1. Of the 48 ligands identified, 24 exhibit a decided preference to bind hTR-P2b RNA rather than A-site rRNA and 10 do not bind A-site rRNA at all. Binding affinity was measured by monitoring RNA imino proton resonances for some of the compounds that showed hTR binding preference.

       RNA aptamers selected against the GluR2 glutamate receptor channel.

       Zhen Huang  et al.

       Biochemistry, 2007, 46 (44), pp 12648–12655

       The excessive activation of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors, a subtype of glutamate ion channels, has been implicated in various neurological diseases such as cerebral ischemeia and amyotrophic lateral sclerosis. Inhibitors of AMPA receptors are drug candidates for potential treatment of these diseases. Using the systematic evolution of ligands by exponential enrichment (SELEX), authors have selected a group of RNA aptamers against the recombinant GluR2Qflip AMPA receptor transiently expressed in HEK-293 (human embryonic kidney) cells. One of the aptamers, AN58, is shown to competitively inhibit the receptor. The nanomolar affinity of AN58 rivals that of NBQX (6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione), one of the best competitive inhibitors. Like NBQX, AN58 has the highest affinity for GluR2, the selection target, among all AMPA receptor subunits. However, AN58 has a higher selectivity for the GluR4 AMPA receptor subunit and remains potent even at pH = 6.8 (i.e., a clinically relevant acidic pH), as compared with NBQX. Furthermore, this RNA molecule possesses stable physical properties. Therefore, AN58 serves as a unique lead compound for developing water-soluble inhibitors with a nanomolar affinity for GluR2 AMPA receptors.

       Discovery of (R)-6-cyclopentyl-6-(2-(2,6-diethylpyridin-4-yl)ethyl)-3-((5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)methyl)-4-hydroxy-5,6-dihydropyran-2-one (PF-00868554) as a potent and orally available hepatitis C virus polymerase inhibitor.

       Hui Li, John Tatlock  et al.

       J. Med. Chem., 2009, 52 (5), pp 1255–1258

       The HCV RNA-dependent RNA polymerase has emerged as one of the key targets for novel anti-HCV therapy development. Herein, authors report the optimization of the dihydropyrone series inhibitors to improve compound aqueous solubility and reduce CYP2D6 inhibition, which led to the discovery of compound 24 (PF-00868554). Compound 24 is a potent and selective HCV polymerase inhibitor with a favorable pharmacokinetic profile and has recently entered a phase II clinical evaluation in patients with genotype 1 HCV.

       Comparing in vitro and in vivo activity of 2′-O-[2-(methylamino)-2-oxoethyl]- and 2′-O-methoxyethyl-modified antisense oligonucleotides.

       Thazha P. Prakash  et al.

       J. Med. Chem., 2008, 51 (9), pp 2766–2776

       A number of 2′-O-modified antisense oligonucleotides have been reported for their potential use in oligonucleotide-based therapeutics. To date, most of the in vivo data has been generated for 2′-O-MOE (2′-O-methoxyethyl)- and 2′-O-Me (2′-O-methyl)-modified ASOs (antisense oligonucleotides). The synthesis and biological activity of another 2′-O-modification, namely 2′-O-[2-(methylamino)-2-oxoethyl] (2′-O-NMA) is reported. This modification resulted in an increase in the affinity of antisense oligonucleotides to complementary RNA similar to 2′-O-MOE-modified ASOs as compared to first-generation antisense oligodeoxynucleotides. The ASO modified with 2′-O-NMA reduced expression of PTEN m-RNA in vitro and in vivo in a dose-dependent manner similar to 2′-O-MOE modified ASO. Importantly, toxicity parameters such as AST, ALT, organ weights, and body weights were found to be normal similar to 2′-O-MOE ASO-treated animal models. The data generated in these experiments suggest that 2′-O-NMA is a useful modification for potential application in both antisense and other oligonucleotide-based drug discovery efforts.

       Design, synthesis, and properties of 2‘,4‘-BNANC:  A bridged nucleic acid analogue.

       S. M. Abdur Rahman et al.

       J. Am. Chem. Soc., 2008, 130 (14), pp 4886–4896

       The novel bridged nucleic-acid analogue 2‘,4‘-BNANC (2‘-O,4‘-C-aminomethylene bridged nucleic acid), containing a six-membered bridged structure with an N−O linkage, was designed and synthesized efficiently, demonstrating a one-pot intramolecular NC bond-forming key reaction to construct a perhydro-1,2-oxazine ring (11 and 12). Three monomers of 2‘,4‘-BNANC (2‘,4‘-BNANC[NH], [NMe], and [NBn]) were synthesized and incorporated into oligonucleotides, and their properties were investigated and compared with those of 2‘,4‘-BNA (LNA)-modified oligonucleotides. Compared to 2‘,4‘-BNA (LNA)-modified oligonucleotides, 2‘,4‘-BNANC congeners were found to possess:  (i) equal or higher binding affinity against an RNA complement with excellent single-mismatch discriminating power, (ii) much better RNA selective binding, (iii) stronger and more sequence selective triplex-forming characters, and (iv) immensely higher nuclease resistance, even higher than the Sp-phosphorthioate analogue. 2‘,4‘-BNANC-modified oligonucleotides with these excellent profiles show great promise for applications in antisense and antigene technologies.

       Docking to RNA via root-mean-square-deviation-driven energy minimization with flexible ligands and flexible targets.

       Christophe Guilbert  et al.

       J. Chem. Inf. Model., 2008, 48 (6), pp 1257–1268

       Structure-based drug design is now well-established for proteins as a key first step in the lengthy process of developing new drugs. In many ways, RNA may be a better target to treat disease than a protein because it is upstream in the translation pathway, so inhibiting a single m-RNA molecule could prevent the production of thousands of protein gene products. Virtual screening is often the starting point for structure-based drug design. However, computational docking of a small molecule to RNA seems to be more challenging than that to protein due to the higher intrinsic flexibility and highly charged structure of RNA. Previous attempts at docking to RNA showed the need for a new approach. Authors present here a novel algorithm using molecular simulation techniques to account for both nucleic acid and ligand flexibility. In this approach, with both the ligand and the receptor permitted some flexibility, they can bind one another via an induced fit, as the flexible ligand probes the surface of the receptor. A possible ligand can explore a low-energy path at the surface of the receptor by carrying out energy minimization with root-mean-square-distance constraints. Our procedure was tested on 57 RNA complexes (33 crystal and 24 NMR structures); this is the largest data set to date to reproduce experimental RNA binding poses. With the procedure, the lowest-energy conformations reproduced the experimental binding poses within an atomic root-mean-square deviation of 2.5 Å for 74% of tested complexes.

       Search for novel aminoglycosides by combining fragment-based virtual screening and 3D-QSAR scoring.

       Piotr Setny  et al.

       J. Chem. Inf. Model., 2009, 49 (2), pp 390–400

       Aminoglycosides are antibiotics targeting the 16S RNA A site of the bacterial ribosome. There have been many efforts directed toward design of their synthetic derivatives, however with only few successes. As RNA binders, aminoglycosides are also a difficult target for computational drug design, since most of the existing methods were developed for protein ligands. An approach that allows for evading the problems related to still poorly developed RNA docking and scoring algorithms is discussed. It is aimed at identification of new molecular scaffolds potentially binding to the A site. The considered molecules are based on the neamine core, which is common for all aminoglycosides and provides specificity toward the binding site, linked with diverse molecular fragments via its O5 or O6 oxygen atom. Suitable fragments are selected with the use of 3D searches of molecular fragments library against two distinct pharmacophores designed on the basis of available structural data for aminoglycoside-RNA complexes. The compounds resulting from fragments assembly with neamine are then scored with a 3D-QSAR model developed using the biological data for known aminoglycoside derivatives. Twenty-one new potential ligands are obtained, four of which have predicted activities comparable to less potent aminoglycoside antibiotics.

       Nerve injection of viral vectors efficiently transfers transgenes into motor neurons and delivers RNAi therapy against ALS.

       Wu, Rui et al.

       Antioxidants & Redox Signaling (2009), 11(7), 1523-1534.

       RNA interference (RNAi) mediates sequence-specific gene silencing, which can be harnessed to silencing disease-causing genes for therapy.  Particularly suitable diseases are those caused by dominant, gain-of-function type of gene mutations.  In these diseases, the mutant gene generates a mutant protein or RNA product, which possesses toxic properties that harm cells.  By silencing the mutant gene, the toxicity can be lessened because the amount of the toxic product is lowered in cells.  In this report, authors tested RNAi therapy in a mouse model for amyotrophic lateral sclerosis (ALS), which causes motor neuron degeneration, paralysis, and death. Authorsused a transgenic model that overexpresses mutant Cu, Zn superoxide dismutase (SOD1G93A), which causes ALS by a gained toxic property. RNAi using recombinant adenovirus (RAd) and adeno-associated virus serotype 2 (AAV2) were delivered and authors compared the efficiency of RNAi delivery between injecting the viral vectors into muscle and into nerve, and found that nerve injetion is more efficient in delivering RNAi to motor neurons. Based on this data, authors conducted therapeutic trials in the mouse model and found that nerve injection of RAd, but not AAV2, at the disease onset had a modest therapeutic efficacy. These results highlight the potential and the challenges in delivering RNAi therapy by gene therepy. 

       A novel program to design siRNAs simultaneously effective to highly variable virus genomes.

       Lee, Hui Sun  et al.

       Biochemical and Biophysical Research Communications  (2009), 384(4), 431-435. 

       A major concern of antiviral therapy using small interfering RNAs (siRNAs) targeting RNA viral genome is high sequence diversity and mutation rate due to genetic instability.  To overcome this problem, it is indispensable to design siRNAs targeting highly conserved regions. Authors thus designed CAPSID (Convenient Application Program for siRNA Design), a novel bioinformatics program to identify siRNAs targeting highly conserved regions within RNA viral genomes.  From a set of input RNAs of diverse sequences, CAPSID rapidly searches conserved patterns and suggests highly potent siRNA candidates in a hierarchical manner.  To validate the usefulness of this novel program, authors investigated the antiviral potency of universal siRNA for various Human enterovirus B (HEB) serotypes.  Assessment of antiviral efficacy using Hela cells, clearly demonstrates that HEB-specific siRNAs exhibit protective effects against all HEBs examd.  These findings strongly indicate that CAPSID can be applied to select universal antiviral siRNAs against highly divergent viral genomes.

       Prospects for the use of a synthon method for synthesis of modified oligonucleotides in solution and on a solid substrate.

       Bukowiecka-Matusiak, Malgorzata et al.

       Wiadomosci Chemiczne (2009), 63(1-2), 63-83.

       Studies on properties and function of nucleic acids constitute the most fascinating cognitive area in biol., chem. and medicine.  Dynamic development of the required techniques, primarily NMR (NMR), or crystalisation techniques, allowed to obtain a detailed information about structural diversity of complicated biological compounds for example peptides and nucleic acids. The replacement of one of the nonbonding oxygens of internucleotide bond by sulfur, selenium, Me or other functionalized alkyl groups creates a stereogenic center at the modified phosphorus atom. This arises a question about availability of stereoregular, P-defined analogs of DNA and RNA. Short synthetic oligonucleotides are indispensable tools in biomolicular and structural studies. They also have potential as therapeutics for manipulation of genes expression in a sequence specific manner.  The block synthesis assuming incorporation of P-chiral, diastereomerically pure dimeric building blocks is attractive, reliable and patent for automated approach to the synthesis of "chimeric oligonucleotides", both in soln. and on solid support. The attention of researches turned toward chimeric constructs of containing in successive internucleotide positions, phosphates and methane-phosphonates. Reynolds et al. found that for therapeutic applications, only chimeric oligonucleotides  with incorporated Rp-dinucleoside methanephosphonates had acceptable binding affinity towards complementary template of DNA and RNA.  Isosequential chimeric oligomers, constructed either from diastereomeric mixtures of dinucleoside methanephosphonates, or from those with Sp-configuration, form less stable duplexes with the same complementary RNA templates.  The preparation of the aforementioned chimeras utilized a "dimeric building blocks" approach. The corresponding dinucleoside (3',5')-methanephosphonates were separated into diastereomers by chromatography methods.

       After removal of the 3'-O-protecting group, the required Rp-isomers were activated at the 3'-O-position, and used as such for condensation via the phosphoramidite method .  Attempts towards their P-epimerization and recycling have failed. Such situation was notwithstanding the requirement of a cost-effective synthesis of new potential therapeutics. Therefore, Stec et al., made efforts in the design of a cost-effective synthesis of Rp-dinucleoside (3',5')-methanephosphonates 17.

       RNA interference compositions for conferring tolerance to viral disease in social insects, such as honeybees.

       Paldi, Nitzan et al.

       PCT Int. Appl. (2009), 66pp. CODEN: PIXXD2  WO  2009060429  A2  20090514  2009:580509

       The invention provides compotions and methods for reducing susceptibility to infectious disease in bees using RNA interference technology and more particularly, prevention and treatment of viral infections in honeybees such as Israel acute paralysis virus (IAPV) by feeding of pathogen-specific dsRNA. Further, multiple pathogen-specific dsRNAs are disclosed.













RNAInterference: The Story of

Gene Silencing in Plants and Humans


1. Introduction

       RNA interference (RNAi) is one of the most exciting discoveries of the past decade in the field of genomics. It is a phenomenon in which double stranded RNA (dsRNA) is the initiating factor in post-transcriptional gene silencing. It is a process in which the introduction of a double stranded RNA (dsRNA) into cells causes the specific degradation of m-RNA containing the same sequence. RNAi is rapidly becoming an important method for analyzing gene functions in eukaryotes. It is a post-transcriptional process triggered by the introduction of double stranded RNA (dsRNA), which leads to gene silencing in a sequence-specific manner. RNAi has been reported to occur naturally in organisms such as nematodes, trypanosmes, plants and fungi.

       The first evidence that dsRNA could achieve efficient gene silencing through RNAi came from studies on the nematode, Caenorhabditis elegans. Further analyses in the fruit fly Drosophila melanogaster indicated that RNAi is a two-step mechanism. First, long dsRNAs are cleaved by an enzyme known as DICER in 21–23 nucleotides fragments, called as small interfering RNAs (siRNAs). Then, siRNAs are recruited to RNA Induced Silencing Complex (RISC) which in turn mediates the cleavage of the target m-RNA. In this review article, the history of RNAi, its mechanism and applications in plants and human is briefly discussed.

2. History of RNA Interference

       The discovery of RNAi phenomenon came accidently when  injected the anti-sense strand to block expression of the par-1 gene in the nematode C. elegans. The expression was disrupted, however, upon performing their controls they found that the sense strand also reduced the expression of that gene. Even earlier biologists had unknowingly witnessed the process of RNA interference when performing experiments on petunias and found that when they introduced a pigment-producing gene under the control of a promoter into the flowers they did not get expected results. Instead of getting the expected deep purple color, the flowers were variegated or they were completely white.

       So what was the reason of these unusual results? It was in 1998 when Fire and Mello first injected double stranded RNA into C. elegans and were rewarded with a much more efficient gene silencing effect. Now the mystery was un-revealed and it was found that the initiator of this post-transcriptional gene silencing (PTGS) was dsRNA, but how it happens, was still a question.

3. The Mechanism of RNA Interference

       The double stranded RNA can be introduced into the cell in a number of ways. In simple organisms such as C. elegans and Paramecium, the dsRNA can be delivered by feeding the organisms with bacteria engineered to express the dsRNA of choice. In other cells the dsRNA may be injected directly. The process of RNAi is triggered by double stranded RNA precursors which are processed into siRNA in the presence of ATPs. Once the dsRNA is in the cell, it is the target for an enzyme named DICER. This enzyme is a dsRNA specific endonuclease that cuts it into smaller fragments, specifically into 21–23 nucleotides.

       These siRNAs are then incorporated to RNA-induced Silencing Complex (RISC) which contains several proteins besides siRNAs. Some well known proteins are AGO2, FMRP and P100. Now RISC is activated which is ATP dependent process and unwinds the double stranded siRNAs. It binds to the targeted m-RNA using the siRNA as a guide to find the target sequence and an endoribonuclease cleaves the m-RNA which is then degraded by exoribonucleases resulting in a loss of expression of the gene. Some of the double stranded siRNAs may be used as primers by an RNA-dependent RNA polymerase resulting in the formation of another long strand of dsRNA that can continue through the RNAi pathway. This may enhance the efficiency of the gene silencing by dsRNA.

       Since the RNAi pathway was first discovered in cells as a natural process, the question has arisen as to what its purpose is in the cell. Two answers came from scientists (1) to inhibit transposon mobilization  and (2) to act as an antiviral mechanism in plants. It has also been discovered that a disruption in the genes required for RNAi to take place often leads to developmental defects in the organism. This observation has suggested that the process of RNA interference is involved in at least one developmental pathway.

4. Silencing Hepatitis C Virus (HCV)

       Hepatitis C Virus (HCV) genome is a single-stranded RNA that functions as both a messenger RNA and a replication template, making it an attractive target for the study of RNA interference. RNA interference represents a promising new approach to tackling this problem. Previous results from identified a small (60-nucleotides) RNA from the yeast Saccharomyces cerevisiae that inhibited hepatitis C virus (HCV) 5′-untranslated region (5′UTR). RNA interference offers further hope to silence troublesome genes. The power of small RNAs to shut down specific gene activities has now been brought to bear on an animal model of hepatitis. Mice infused with a siRNA against a cell death receptor recovered their liver function after experimentally induced injury.

       Biologists have agreed that the best strategy would be to aim siRNA directly at hepatitis B or C viruses. Evidences suggest that, in petri dishes, siRNA can stop hepatitis C virus from replicating. RNAi targeting HCV IRES shows a strong inhibitive effect on the expression of the reporter gene controlled by this sequence, suggesting that RNAi-based anti-HCV strategy may represent a potential approach in the therapy of HCV infection.

       Synthetic small interfering RNAs could suppress transgene expression in adult mice and small-hairpin RNAs transcribed in vivo from DNA templates. Some scientists also showed the therapeutic potential of this technique by demonstrating effective targeting of a sequence from hepatitis C virus by RNA interference in vivo. The utility of siRNA as a therapy against HCV infection will depend on the development of efficient delivery systems that induce long-lasting RNAi activity. HCV is an attractive target for its localization in the liver, an organ that can be readily targeted by nucleic acid molecules and viral vectors.

       As therapeutic agents, siRNAs have remarkable properties. Their actions appear to be short-lived in mammals. They are sequence specific, natural and cellular products and may, therefore, not produce toxic metabolites. However, delivering siRNAs to the appropriate cells is a major challenge. Better delivery methods—such as formulation of siRNAs with compounds that promote transit across cell membranes are clearly required before siRNAs can be used in therapy, especially to suppress gene expression in tissues other than in the liver.

5. Silencing Human Immuno-deficiency Virus (HIV)

       RNA interference represents an exciting new technology that may have therapeutic applications in treating Human immuno-deficiency virus (HIV). Previous reports have shown that siRNA directed against the HIV genome can effectively inhibit virus production in cell-culture systems and RNAi activity directed toward the major HIV receptor protein, CD4, led to decreased entry of HIV into cells. siRNAs against cellular co-receptors CXCR4 and CCR5 had shown that down regulation of these surface molecules could prevent HIV-1 entry and confer viral resistance . By targeting several regions of the HIV-1 genome, showed siRNA mediated viral genome degradation and down regulation of viral gene expression. Further they proved that RNAi worked even when the viral genome was contained within the nucleoprotein complex. They also showed that intracellular siRNAs worked well, providing possible ways for delivering gene-therapy agents against HIV. To assess the effects of RNAi on HIV-1 infection, targeted both cellular and viral RNAs.

6. RNA Interference in Cancer Therapeutics

       The emergence of RNA interference (RNAi) as a mechanism to suppress gene expression has revolutionized genetics in mammalian cells and has begun to facilitate decoding gene functions on a genome scale . To develop siRNA for cancer therapy, several researchers have investigated siRNAs in animal models. To obtain efficient and long-lived gene silencing using RNAi, several groups have incorporated the siRNA expression cassettes into a variety of viral vectors.

       Although systemic siRNA delivery imposes several requirements and greater hurdles than local siRNA delivery. Diseases like cancer are considered as systemic diseases, including metastatic distribution of microdisseminated cells, and thus require systemic treatment with siRNA. In the near future the systemic delivery of siRNA will be required, possibly using a tissue-specific or cell-specific gene promoter vector or specific antibody-conjugated carriers, thus reducing applied dose of siRNA and resulting in decreased side effects. For specific targeting, angiogenesis and metastasis can be exploited for the differences between cancerous cells and normal cells, which include uncontrolled proliferation, insensitivity to negative growth regulation and antigrowth signals.

7. Advantages of RNA Interference

       One specific advantage of RNAi over other methods previously employed is that dsRNA activates a normal cellular process leading to a highly specific RNA degradation and perhaps more importantly, a cell-to-cell spreading of this gene silencing effect in several RNAi models. It is also a relatively quick method enhancing the genetic analysis of traditional model organisms and has provided a means of performing reverse genetics experiments on organisms lacking any established genetic tools. RNAi activity plays a role in host-cell protection from viruses and transposons in plants and insects. From a practical perspective, RNAi can therefore be used to target gene expression and has been proved to be a very powerful technique to knock down specific genes to evaluate their physiological roles in plants and humans.

8. Current and Potential Uses of RNAi

       RNAi technology has been described as “not only an extremely powerful instrument for functional genomic analyses, but also as a potentially useful method to develop highly specific dsRNA based gene silencing therapeutics”. Since RNA interference results in a cell or organism that is lacking in a particular transcript, biologists have a fairly reliable and quick method for creating mutants and performing “gene knockout” and “loss of phenotype” studies. There are many examples of experiments in which RNAi is used to assess the functions of particular proteins and to aid in the discovery of what enzymes and proteins are involved in certain metabolic pathways as in the Gibberellin/Abscisic acid-signalling pathway. Another type of experiment that is currently being done involves attempts to induce RNA interference by bi-directional transcription of a particular gene of interest or by flanking the gene to be silenced by two convergent promoters.

       So far we have seen that RNA interference can be an extremely valuable genetic tool when studying plants, insects and small invertebrates such as nematodes. But biologists are always looking ahead to how new discoveries can benefit human beings, and so this has led to the study of whether or not RNAi also takes place in mammalian cells. Could RNAi potentially be used as a form of gene therapy? Two problems were initially encountered when long strands of dsRNA were introduced into mammalian cells. Both these obstacles were due to the antiviral response of the cell against the foreign dsRNA. One of the problems is the activation of a protein kinase PKR, which stalls translation by phosphorylating an initiation factor, eIF2a. The other problem to the dsRNA is the activation of RNase L, an enzyme that degrades the m-RNA of the cell in a manner non-specific to the inducing dsRNA.

9. Future Directions

       The field of RNAi is moving at an impressive pace and generating exciting results that are clearly associated with RNA interference, transgene silencing and transposon mobilization . Possible links to X-chromosome inactivation, imprinting and interferon response have also been suggested, but not yet firmly established. RNAi also has a considerable economic potential, especially in agriculture. A better understanding of PTGS should allow a more efficient response to viral infection and the development of transgene/host associations that can override silencing to allow the expression of interested proteins. In the next 10 years, RNAi will probably be regarded as one of the major breakthroughs of the 21st century. In relation to RNA interference in mammals, it is important to note that in contrast to the sequence-specific RNAi effect observed in mouse embryos, this new study has shown that incubation of an m-RNA with rabbit reticulocyte lysates and dsRNA induces non-specific m-RNA degradation, one possible reason for this difference could be the interferon response present in rabbit reticulocyte lysate is not functional in early mouse embryos.

       Although antiviral RNAi technology has not yet been optimized, the phenomenon appears to be both general and effective. In 1988, the concept of "intracellular immunization" was proposed, whereby one could express within cells inhibitory molecules (usually proteins) that could protect these cells from specific viral infections in the future. The promise of intracellular immunization now appears to be closer to reality through the use of small RNAs rather than proteins. The potential of using RNAi activity for the treatment of viral diseases and cancer has aroused a great deal of interests in the scientific community. Many laboratories have reported the use of RNAi activity in cultured cell infected with HIV, human papillomavirus, and polio or containing a variety of cancer genes. The clinical applications of RNAi are just around the corner.

(Based on the article written by Mahmood-ur-Rahman et al., and published in Biotechnology Advances 26 (2008) 202-209)



       Genomic imprinting mechanisms in mammals

       Ideraabdullah, Folami Y. et al.

       Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 647(1-2), 77 (Dec., 1 2008)

       Genomic imprinting is a form of epigenetic gene regulation that results in expression from a single allele in a parent-of-origin-dependent manner. This form of monoallelic expression affects a small but growing number of genes and is essential to normal mammalian development. Despite extensive studies and some major breakthroughs regarding this intriguing phenomenon, we have not yet fully characterized the underlying molecular mechanisms of genomic imprinting. This is in part due to the complexity of the system in that the epigenetic markings required for proper imprinting must be established in the germline, maintained throughout development, and then erased before being re-established in the next generation's germline. Furthermore, imprinted gene expression is often tissue or stage-specific. It has also become clear that while imprinted loci across the genome seem to rely consistently on epigenetic markings of DNA methylation and/or histone modifications to discern parental alleles, the regulatory activities underlying these markings vary among loci. Here, we discuss different modes of imprinting regulation in mammals and how perturbations of these systems result in human disease. We focus on the mechanism of genomic imprinting mediated by insulators as is present at the H19/Igf2 locus, and by non-coding RNA present at the Igf2r and Kcnq1 loci. In addition to imprinting mechanisms at autosomal loci, what is known about imprinted X-chromosome inactivation and how it compares to autosomal imprinting is also discussed. Overall, this review summarizes many years of imprinting research, while pointing out exciting new discoveries that further elucidate the mechanism of genomic imprinting, and speculating on areas that require further investigation.

       Micro-RNAs as markers of the functional state of a dendritic cell.

       Skjoede Jensen, Simon et al.

       PCT Int. Appl. (2009), 41pp.

       The invention discloses the use of specified micro-RNAs as markers of the functional state of a dendritic cell.  In one aspect, the invention discloses a method for producing a quality-controlled therapeutic composition comprising dendritic cells. In another aspect, the invention discloses a method for in vitro screening of immunomodulatory compounds.

       Constructing and sequence analyzing of transcriptional small hairpin RNA recombinant plasmid targeting bcl-2 gene.

       Zhang, Yunuo et al.

       Chongqing Yixue (2009), 38(4), 434-435

       The objective of this study was to construct the recombinant plasmids expressing connective gene bcl-2 short hairpin RNA (shRNA) by Pgenesil-1 plasmid vector for the further searching new gene therapy method of the tumors.  Two DNA sequences containing small hairpin structure were designed and synthesized.  The recombinant plasmid was transformed into DH5 strain.  Then the recombinant plasmid identified by restriction enzyme was used for sequence analysis. The recombinant plasmid targeting bcl-2 gene was constructed and the aim sequence was obtained.

       Optimized basic conditions are essential for successful siRNA transfection into primary endothelial cells.

       Nolte, Andrea et al.

       Oligonucleotides (2009), 19(2), 141-150.

       RNA interference (RNAi) is a powerful technique in basic research and has a high potential for therapeutic applications. To realize its clinical applicability, introduction of short double-stranded RNA (dsRNA) has to be carried out under physiological conditions. This study evaluates two cationic liposomal transfection reagents on the efficiency of successful silencing of primary human endothelial cells. Transfection efficiency was investigated under different conditions, for example different media during transfection, duration of transfection, siRNA concentration and the use of serum and antibiotics. Viability after transfection was examined by CASY and MTT assay. Interferon response was examined by real-time PCR. First it was revealed that transfection carried out in the presence of serum and antibiotics caused good knockdown results only by the use of the novel lipid cationic transfection reagent. Both lipid cations had slightly the same transfection efficiency over the range of 10-150 nM siRNA concentration. Examination of interferon response showed increasing OAS1 and STAT1 expression, but not as high as if the transfections were carried out with synthetic polyinosinic-polycytidylic acid double-stranded RNA (poly[IC]). The optimized combination of basic conditions for transfection significantly enhanced the efficiency of the siRNA-mediated knockdown, without causing toxicity or stimulation of the interferon pathway.











      RNA interference mediated inhibition of cyclic nucleotide type 4 phosphodiesterase (PDE4B) gene expression using short interfering nucleic acid (siNA)

      Strapps Walter et al.

      Sirna Therapeutics, Inc.US

      WO 2008/137775, 13.11.2008

       The invention relates to compounds, compositions, and methods for the study, diagnosis, and treatment of traits, diseases and conditions that respond to the modulation of cyclic nucleotide type 4 phosphodiesterase (PDE4B) gene expression and/or activity, including PDE4B1, PDE4B2, and PDE4B3 gene expression and/or activity. The invention is also directed to compounds, compositions, and methods relating to traits, diseases and conditions that respond to the modulation of expression and/or activity of genes involved in cyclic nucleotide type 4 phosphodiesterase (PDE4B) gene expression pathways or other cellular processes that mediate the maintenance or development of such traits, diseases and conditions.

       RNA interference mediated inhibition of cyclic nucleotide type 4 phospho-diesterase (PDE4B) gene expression using short interfering nucleic acid (sina).

      Strapps Walter et al.

       Sirna Therapeutics, Inc. US

       WO 2008/137751 13.11.2008     C12N 15/11

       The invention relates to compounds, compositions, and methods for the study, diagnosis, and treatment of traits, diseases and conditions that respond to the modulation of cyclic nucleotide type 4 phosphodiesterase (PDE4B) gene expression and/or activity, including PDE4B1, PDE4B2, and PDE4B3 gene expression and/or activity.

      Nucleic acids hybridiazable to micro RNA and precursors thereof.

      Jayasena Sumedha D et al.

      Amgen Inc. US

      WO 2008/131191 30.10.2008

       Methods and compositions relating to nucleic acids targeting certain miRNA molecules are disclosed. The nucleic acids are useful in methods of increasing nuclear concentration of FKHR protein, decreasing cell viability, and treating cancer.

       Isolated nucleic acid molecules corresponding to micro RNA 145 (miRNA-145) and their use in treating colon cancer

       Shi Bin et al.

       Merck & Co., Inc. US

       WO 2008/127587 23.10.2008

       Provided herein are isolated nucleic acid molecule corresponding to miRNA145 that are useful in treating colon cancer. The disclosed miRNA145 nucleic acids specifically bind the 3' UTR within endogenous IRS-I such as to suppress or inhibit colon cell proliferation.

       Zinc-finger nuclease and RNA interference mediated inactivation of viral genomes

       Mccaffrey Anton P et al.

       University of IOWA Research Foundation

       WO 2008/119000  02.10.2008, ,

       The invention provides methods for targeted inactivation of viral genomes. In one embodiment, zinc-finger proteins in which DNA binding sites are altered such that they recognize and bind different, desired DNA sequences contained in hepatitis B virus (HBV) and that include nuclease domains are used for inactivation. Other embodiments for targeted inactivation of viral genomes use small nucleic acid molecules, such as short micro-RNA molecules or short hairpin RNA molecules capable of mediating RNA interference (RNAi) against the hepatitis B virus.

       Micro RNA profiles associated with endometrial cancer development and response to cisplatin and doxorubicin chemotherapy

       Lancaster Johnathan et al.

       University of South Florida , US

       WO 2008/109519 12.09.2008  

       A method predicting of cancer chemoresponse of the population of cancer cells to the one or more chemotherapeutic agents. Our ability to treat patients with advanced stage and recurrent endometrial cancer is hampered by an incomplete understanding of the molecular basis of disease development and response to therapy. A novel class of gene products called microRNA (miRNA) has recently been implicated in the etiology of several different human cancers. Altered levels of expression of specific miRNAs may contribute to cancer development in a variety of cancers such as endometrial cancer and may also influence response to cytotoxic chemotherapy or other cancer treatments. Evidence is provided that differential expression of miRNAs contributes

       The micrornaome.

       Cummins Jordan et al.

       The Johns Hopkins University, US

       WO 2008/103135 28.08.2008 ,

       MicroRNAs (miRNAs) are a class of small noncoding RNAs that have important regulatory roles in multicellular organisms.

       Multi-targeting short interfering RNAs

       Rossi John et al.

       Hope City ,US

       WO 2008/094516 07.08.2008,

       The present invention relates to novel short interfering RNA (siRNA) molecules that are multi-targeted. More specifically, the present invention relates to siRNA molecules that target two or more sequences. In one embodiment, multi-targeting siRNA molecules are designed to incorporate features of siRNA molecules and features of micro-RNA (miRNA) molecules. In another embodiment, multi-targeting siRNA molecules are designed so that each strand is directed to separate targets.

       Methods, compositions, and kits for detection ofmicro RNA

       Sorge Joseph et al.

       Stratagene California US

       WO 2008/092016 31.07.2008,

       The invention provides methods, nucleic acids, compositions, and kits for detecting microRNA (miRNA) in samples. The methods comprise designing m-RNA-specific primers, adding a polyA tail to the miRNA, and using reverse transcription and amplification to detect the miRNA. The nucleic acids, compositions, and kits typically comprise some or all of the components necessary to practice the methods of the invention.

       Chemically modified oligonucleotides for use in modulating micro RNA and uses thereof.

       Stoffel Markus et al.

       Alnylam Pharmaceuticals Inc., US

       WO 2008/091703 31.07.2008,

       This invention relates to chemically modified oligonuceotides useful for modulating expression of microRNAs and pre-microRNAs. More particularly, the invention relates to single stranded chemically modified oligonuceotides for inhibiting microRNA and pre-microRNA expression and to methods of making and using the modified oligonucleotides. Also included in the invention are compositions and methods for silencing microRNAs in the central nervous system.

       Method of estimating secondary structure in RNA and program and apparatus therefore.

       Nakamura Shingo, Japan 

       Takeda Pharmaceutical Company Limited

       WO 2008/072713 19.06.2008,

       Supposing a frame (F) having a definite short length (L2) on a transcript, the frame (F) is shifted finely stepwise at constant intervals (t). Thus, the structures in the individual micro sections are successively analyzed and the first probability in a specific secondary structure at a specific position in each micro section is determined. Next, one or more specific positions clarified in the individual micro sections are correspondingly located on the original transcript and the degree of the overlap between the individual specific positions is referred to as the second probability. By paying attention to these two probabilities, it can be estimated at an elevated reliability whether or not a desired secondary structure occurs in practice.

       Micro RNA targeting ETS1.

       Peschle Cesare

       Istituto Superiore Di Sanita, Italy

       WO 2008/068047 12.06.2008

       Micro RNA capable of interacting with the 3'untranslated region of Ets-1 protein m-RNA is useful in treating Ets-1-dependent tumours, and inhibitors therefor are useful in treating suppressed megakaryopoiesis in cancer patients or abnormal megakaryopoiesis. Micro RNA and inhibitors therefore are also useful in in vivo and ex vivo expansion of megakaryocytes and platelets.

       Micro RNA targeting AML1.

       Peschle Cesare

       Istituto Superiore Di Sanità,  Italy [IT]

       WO 2008/068046 12.06.2008

       MicroRNA in the miR-17-5p/106a/106b clusters capable of interacting with the 3-untranslated region of AML1 protein micro-RNA is useful in stimulating haematopoietic blast proliferation, and inhibiting differentiation into monocytes and dendritic cells. Micro-RNA and inhibitors thereof are also useful in the inhibitory and stimulatory control of in vivo and ex vivo production of monocytes and dendritic cells, as wells as in treating tumours related to enhanced microRNA level.

       Novel methods for quantification of microRNAs and small interfering RNAs.

       Mouritzen Peter

       Exiqon A/S, Denmark

       WO 2008/040355 10.04.2008,

       The invention relates to ribonucleic acids, probes and methods for detection, quantification as well as monitoring the expression of mature microRNAs and small interfering RNAs (siRNAs). The invention furthermore relates to methods for monitoring the expression of other non-coding RNAs, m-RNA splice variants, as well as detecting and quantifying RNA editing, allelic variants of single transcripts, mutations, deletions, or duplications of particular exons in transcripts, e.g., alterations associated with human disease such as cancer. The invention furthermore relates to methods for detection, quantification as well as monitoring the expression of deoxy nucleic acids.

       RNA interference mediated inhibition of histone deacetylase (HDAC) gene expression using short interfering nucleic acid (siNA)

       Jadhav Vasant et al.

       Sirna Therapeutics, Inc., US

       WO 2008/030239 13.03.2008

       The invention relates to compounds, compositions, and methods for the study, diagnosis, and treatment of traits, diseases and conditions that respond to the modulation of histone deacetylase (HDAC) gene expression and/or activity. The invention is also directed to compounds, compositions, and methods relating to traits, diseases and conditions that respond to the modulation of expression and/or activity of genes involved in HDAC gene expression pathways or other cellular processes that mediate the maintenance or development of such traits, diseases and conditions. Specifically, the invention relates to double stranded nucleic acid molecules including small nucleic acid molecules, such as short interfering nucleic acid (siNA).

       Methods for in vivo identification of endogenous m-RNA targets of microRNAS

       Keene Jack et al.

       Duke University, US

       WO 2008/024499 28.02.2008,

       A method of generating a gene expression profile of noncoding regulatory RNA (ncRNA; e.g. a microRNA) in a cell in vivo, is carried out by: (a) partitioning from a cell at least one m-RNA-protein (RNP) complex, the RNP complex comprising: (i) an RNA binding protein (RNABP) or RNA associated protein, (ii) at least one m-RNA bound to or associated with said protein, and (iii) at least one ncRNA bound to or associated with said protein, and then (b) identifying at least one ncRNA in-at least one RNP complex, thereby to produce a gene expression profile comprising the identity of an ncRNA in an RNP complex.

       Identification of a microRNA that activates expression of beta-myosin heavy chain.

       Olson Eric et al.

       Board of Regents of The University of Texas System, US

       WO 2008/016924 07.02.2008

       The invention relates to the identification of a microRNA, miR-208, that induces the expression of β-myosin heavy chain (β-MHC) and represses fast skeletal muscle contractile protein genes. Inhibition of this function is proposed as a treatment for cardiac fibrosis, hypertrophy and/or heart failure, and augmentation of this function can be used to repress slow fiber genes and activate fast fiber genes in the treatment of musculoskeletal disorders.

       RNA interference mediated inhibition of sterol regulatory element-binding protein 1 (SREBP1) gene expression using short interfering nucleic acid (SINA).

       Mcswiggen James et al.

       Sirna Therapeutics Inc., US

       WO 2008/011467 24.01.2008,

       The present invention relates to compounds, compositions, and methods for the study, diagnosis, and treatment of traits, diseases and conditions that respond to the modulation of Sterol Regulatory Element-Binding Protein 1 (SREBP1) gene expression and/or activity. The present invention is also directed to compounds, compositions, and methods relating to traits, diseases and conditions that respond to the modulation of expression and/or activity of genes involved in Sterol Regulatory Element-Binding Protein 1 (SREBP1) gene expression pathways or other cellular processes that mediate the maintenance or development of such traits, diseases and conditions. Specifically, the invention relates to double stranded nucleic acid molecules including s...

       RNA interference mediated inhibition of proprotein convertase subtilisin kexin 9 (PCSK9) gene expression using short interfering nucleic acid (SINA).

       Mcswiggen James et al.

       Sirna Therapeutics Inc. [US]

       WO 2008/011431 24.01.2008  C12N 15/11  PCT/US2007/073723

       The present invention relates to compounds, compositions, and methods for the study, diagnosis, and treatment of traits, diseases and conditions that respond to the modulation of Proprotein Convertase Subtilisin Kexin 9 (PCSK9) gene expression and/or activity. The present invention is also directed to compounds, compositions, and methods relating to traits, diseases and conditions that respond to the modulation of expression and/or activity of genes involved in Proprotein Convertase Subtilisin Kexin 9 (PCSK9) gene expression pathways or other cellular processes that mediate the maintenance or development of such traits, diseases and conditions.

       MicroRNA based methods and compositions for the diagnosis and treatment of colon cancer-related diseases

       Croce Carlo M et al.

       Ohio State University Research Foundation, US

       WO 2008/008430 17.01.2008  

       The invention provides novel methods and compositions for the diagnosis and treatment of colon cancers. The invention also provides methods of identifying inhibitors of tumorigenesis.

       RNA interference mediated inhibition of 11 beta-hydroxysteroid dehydrogenase-1 (11 beta-HSD-1) gene expression using short interfering nucleic acid (SINA)

       Mcswiggen James et al.

       Sirna Therapeutics, Inc.,US

       WO 2007/147143 21.12.2007  

       The invention relates to compounds, compositions, and methods for the study, diagnosis, and treatment of traits, diseases and conditions that respond to the modulation of 11 beta-hydroxysteroid dehydrogenase-1 (11 beta-HSD-1) gene expression and/or activity. The present invention is also directed to compounds, compositions, and methods relating to traits, diseases and conditions that respond to the modulation of expression and/or activity of genes involved in 11 beta-hydroxysteroid dehydrogenase-1 (11 beta-HSD-1) gene expression pathways or other cellular processes that mediate the maintenance or development of such traits, diseases and conditions.

       Detection of nucleic acids.

       Allawi Hatim T et al.

       Third Wave Technologies, Inc., US

       WO 2007/143097 13.12.2007

       The invention relates to compositions and methods for the detection and characterization of small nucleic acid molecules (e.g., RNA (e.g., small RNAs such as micro RNAs (miRNAs) and small interfering RNAs (siRNAs)) and other short nucleic acid molecules). More particularly, the present invention relates to methods for the detection and quantification of RNA expression. The present invention further provides for the detection of miRNA and siRNA variants.

       Compositions comprising a micro RNA and methods of their use in regulating cardiac remodeling.

       University of Texas,US

       WO 2009/0588 18 07.05.2009,

       The present invention relates to the identification of a microRNA, miR-21, that alters energy metabolism in cardiomyocytes and thus contributes to cardiac remodeling. Inhibition of this function is proposed as a treatment for cardiac hypertrophy, heart failure, and/or myocardial infarction.

       Methods and compositions for detection and enrichment of target small RNAS.

       Mcreynolds Lawrence et al.

       New England Biolabs, Inc. [US]

       WO 2009/058814 07.05.2009, G01N 33/53, PCT/US2008/081520

       Methods and compositions are provided for detecting small target RNAs where the target RNA may be single-stranded or double-stranded and may be contained in a mixture of RNAs of different types and sizes. The methods and compositions utilize a pl9 fusion protein that is capable of binding double-stranded RNA in a size-specific but sequence-independent manner and is further capable of binding to a matrix such as beads or plastic microwell plates. By labeling the pl9 fusion protein or the target RNA in a polynucleotide duplex either directly or indirectly, low levels of target RNA including microRNAs can be detected from cells. This can be applied to diagnosis of pathological conditions.

       Increasing erythropoietin using nucleic acids hybridizable to microRNA and precursors thereof.

       Jayasena Sumedha D et al.

       Amgen Inc., US

       WO 2009/045469 09.04.2009.

       Methods and compositions relating to nucleic acids targeting certain miRNA molecules are disclosed. The nucleic acids are useful, for example, in methods of increasing the expression and/or secretion of EPO and treating various disease states including anemia, hemophilia, and/or sickle cell disease.

       Nucleic acid capable of regulating the proliferation of cell.

       Nakano Haruo, et al.

       Kyowa Hakko Kirin Co., Ltd., US

       WO 2009/044899  09.04.2009.

       Disclosed are: an agent for inhibiting or promoting the proliferation of a cell; a diagnostic or therapeutic agent for a disease associated with the abnormal proliferation of a cell; an apoptosis inducer; an agent for inhibiting or promoting the expression of a target gene of a nucleic acid, such as microRNA; and a method for inhibiting or promoting the proliferation of a cell.

       Composition inhibiting the expression of target gene.

       Shinohara Fumikazu et al.

       Kyowa Hakko Kirin Co., Ltd.,Japan

       WO 2009/044895  09.04.2009.

       To provide a composition or the like having an effect of inhibiting the expression of a target gene and containing a microRNA or its derivative. Namely, it is intended to provide a composition or the like containing a liposome having a microRNA, an artificial microRNA, etc. encapsulated therein, which comprises a complex particle comprising a lead particle and the microRNA, artificial microRNA, etc. as described above together with a bimolecular lipid membrane coating the complex particle, wherein the component constituting the bimolecular lipid membrane is soluble in a specific polar organic solvent and the component constituting the bimolecular lipid membrane and the complex particle are dispersible in a liquid containing the polar organic solvent.

       Composition of asymmetric RNA duplex as microRNA mimetic or inhibitor.

       Li Chiang Jia et al.

       Boston Biomedical, Inc., US

       WO 2009/029690 05.03.2009..

       The invention provides double-stranded RNA molecules that are asymmetrical in strand length. The RNA molecule of the invention, the asymmetric RNA duplex, has one or two overhangs at the end. In one aspect, these novel RNA duplex molecules serve as effective mimetics of miRNA. In another aspect, they are designed to function as effective inhibitors of miRNA. Accordingly, the RNA molecules of the present invention can be used to modulate miRNA pathway activities, with tremendous implications for research, drug discovery and development, and treatment of human diseases.

       MicroRNAs for inhibiting viral replication.

       David Michael

       University of California, US

       WO 2009/029681 05.03.2009.

       The invention relates to reducing accumulation of viral genomes in a target cell. In particular the present invention provides compositions and methods for combating viral infection through RNA interference. Specifically the present invention provides cellular microRNA mimics for treating virus-infected subjects.

       Method for categorizing samples containing spermatozoa by molecular profiling.

       Simmet Christian et al.

       Minitüb Abfüll- Und Labortechnik Gmbh & Co. KG

       WO 2009/027096  05.03.2009.

       The invention relates to methods for categorizing samples containing spermatozoa by obtaining a RNA profile in said sample by hybridization and/or sequencing techniques, wherein the RNA is preferably selected from messenger RNA (m-RNA), noncoding RNA (ncRNA) and micro RNA (miRNA). The present invention relates further to the use of RNA profiles and/or translation product profiles as selection criterions, such as fertility and breeding selection, of the sample donor. The present invention allows for distinguishing male and female spermatozoa of a sample and subsequently separating male and female spermatozoa. With the methods of the invention categorized samples as well as male and female spermatozoa can be obtained.

       Methods of modulating mesenchymal stem cell differentiation.

       Federov Yuriy et al.

       Dharmacon, Inc., US

       WO 2009/023525 19.02.2009.

       The disclosure includes compositions and methods for modulating the differentiation of cells having osteogenic differentiation potential (such as mesenchymal stem cells (MSCs)) towards the osteogenic fate, and for obtaining diagnostic and prognostic information relating to diseases and disorders characterized by defects in osteogenic differentiation. The compositions include miRNAs, rm'RNA mimics, miRNA inhibitors, and siRNAs.

       A microRNA family that modulates fibrosis and uses thereof.

       Olson Eric et al.

       University of Texas, US

       WO 2009/018493, 05.02.2009.

       The invention relates to the identification of a microRNA family, designated miR-29a-c, that is a key regulator of fibrosis in cardiac tissue. The inventors show that members of the miR-29 family are down-regulated in the heart tissue in response to stress, and are up-regulated in heart tissue of mice that are resistant to both stress and fibrosis. Also provided are methods of modulating expression and activity of the miR-29 family of miRNAs as a treatment for fibrotic disease, including cardiac hypertrophy, skeletal muscle fibrosis other fibrosis related diseases and collagen loss-related disease.

       Alzheimer's disease-specific microRNA microarray and related methods.

       Wang Eugenia et al.

       University of Louisville Research Foundation, Inc.

       WO 2009/009457 15.01.2009.  

       The presently-disclosed subject matter provides methods of diagnosis and/or prognosis of Alzheimer's disease in subjects by measuring amounts of one or more micro-RNAs correlated with Alzheimer's disease present in a biological sample, including blood for example, from a subject.

       Mixed micelles including amphipathic conjugates of RNA agents, and uses thereof.

       Trochilin Valadmis et al.

       Northeastern University, Boston, US

       WO 2009/009025, 15.01.2009.

       Disclosed are improved pharmaceutical formulations for the delivery of RNA interference agents, such as antisense RNA, micro-RNA and siRNA. The formulations employ mixed micelles including amphipathic conjugates of the iRNA agents and amphipathic micelle- forming molecules with extended hydrophilic chains. Also disclosed are methods of using the pharmaceutical formulations to increase delivery of an iRNA agent to an intracellular target, and to decrease extracellular nuclease degradation of an iRNA agent in the formulations.

       Methods for cloning small RNA species.

       Devor Eric J.

       Integrated Dna Technologies, Inc., US

       WO 2009/006446 08.01.2009.

       This invention pertains to methods for cloning microRNA (miRNA) and other small ribonucleic acid (RNA) species from relevant cell sources.

       A method of typing a sample comprising colorectal cancer cells.

       Simon  Iris

       Agendia B.V., Amesterdam, Netherland

       WO 2009/002175 31.12.2008.

       The invention relates to a method of typing colorectal cancer cells by determining the RNA levels of a set of signature genes. Said typing can be use for predicting a risk for recurrence of said colorectal cancer. The invention further relates to a set of genes that can be used for normalizing the RNA levels of said set of signature genes., and to micro-array comprising said set of signture genes. In particular, the typing allows the distinction of stage II and III cancers.

       MicroRNAS modulating immunity and inflammation.

       Mohapatra Shyam S et al.

       University of South Florida, US

       WO 2008/147974 04.12.2008.

       MicroRNAs are shown to be up- and/or down-regulated in inflammation and immune cells using a mouse model of asthma and regulatory T cells as source of RNA, respectively. Modulating the expression of these microRNAs can be effective in redirecting inflammation and immunity and hence, may be beneficial as biomarkers or as therapeutic agents against diverse human immunologic and inflammatory diseases.

       Micro RNA scaffolds and non-naturally occurring micro RNA.

       Kelley Melissa et al.

       Dharmacon, Inc., US

       WO 2008/147839  04.12.2008.

       The disclosure provides a non-naturally occurring miRNA having a stem-loop structure comprising a scaffold derived from a first endogenous miRNA (e.g., miR-196a-2 or miR-204), a mature strand derived from a second endogenous miRNA, and a star strand sequence that is at least partially complementary to the mature strand sequence. The present disclosure also provides a non-naturally occurring miRNA having a stem-loop structure comprising a scaffold derived from an endogenous miRNA (e.g., miR-196a-2 or miR-204), a mature strand designed t be at least partially complementary to a target RNA, and a star strand sequence that is at least partially complementary to the mature strand sequence.  The methods and compositions of the disclosure may be used to mediate gene silencing via the RNAi pathway.

       Lipid nanoparticle based compositions and methods for the delivery of biologically active molecules.

       Chen Tongqian et al.

       Sirna Therapeutics, Inc., US

       WO 2008/147438  04.12.2008.

       The invention relates to novel cationic lipids, transfection agents, microparticles, nanoparticles, and short interfering nucleic acid (siNA) molecules. The invention also features compositions, and methods of use for the study, diagnosis, and treatment of traits, diseases and conditions that respond to the modulation of gene expression and/or activity in a subject or organism. Specifically, the invention relates to novel cationic lipids, microparticles, nanoparticles and transfection agents that effectively transfect or deliver biologically active molecules, such as antibodies (e.g., monoclonal, chimeric, humanized etc.), cholesterol, hormones, antivirals, peptides, proteins, chemotherapeutics, small molecules and vitamins.

       A primary microRNA expression cassette.

       Patrick Arbuthnot et al.

       University of The Witwatersrand, Johannesburg, South Aferica

       WO 2008/146251 04.12.2008.

       This invention relates to inhibition of hepatitis gene expression. More specifically, the invention relates to a method of using RNA sequences to inhibit Hepatitis B and C Virus replication. Expression cassettes that include DNA sequences derived from endogenous micro RNAs (miRs) are used in the method and are transcribed by Pol Il promoters, and then processed to generate sequences that are specific to target hepatitis virus sequences (RNAi effecter sequences). The RNAi effecter sequences can target the selected hepatitis virus sequences resulting in gene silencing or transcriptional inhibition of the hepatitis virus gene. The expression cassettes may be delivered in vitro or in vivo to host cells. A pharmaceutical composition containing t...

       Preparation of hydrophobic substance-modified nucleic acid aptamers with pleiotrophin binding activities for diagnostic and therapeutic use.

       Miyakawa, Shin et al.

       Ribomic Inc., Japan

       WO 2009063998 , 22.05.2009

       Nucleic acid aptamers having midkine -binding activities are provided.  The targeted midkines are more specifically pleiotrophins.  The nucleotide sequences of ten pleiotrophin-binding RNA aptamers are disclosed.  THe pleiotrophin-binding RNA aptamers are modified with hydrophobic substances such as cholesterol using polyethyleneglycol mols. as linkers.  The pleiotrophin-binding aptamers are further modified with functional moieties such indicators, enzymes or delivery carriers.  The modified pleiotrophin-binding aptamers are dissolved in neutral salt solution at concentration 0.1 .approximately 10 mg/mL and are immobilized on nanoparticles. The modified pleiotrophin-binding aptamers are used in the diagnostic assays based on pleiotrophin binding-assays. The modified pleiotrophin-binding aptamers are also therapeutically applied as the chemotaxis inhibitors or regulatory T-cell proliferation-promoting agents for prevention or treatment of autoimmune diseases.

       Micro-RNAs as markers of the functional state of a dendritic cell for use in screening of immunomodulatory compositions.

       Skjoede Jensen, Simon et al.

       Dendrit Biotech AS, DK

       WO 2009062515  22.05.2009

       The invention discloses the use of specified micro-RNAs as markers of the functional state of a dendritic cell.  In one aspect, the invention discloses a method for producing a quality-controlled therapeutic composition comprising dendritic cells. In another aspect, the invention discloses a method for in vitro screening of immunomodulatory compounds.

       Methods for epigenetic modification of somatic cell phenotype, fate or differentiation status by RNA transfer for use in transplantation therapy.

       Beyhan, Zeki et al.

       PCT Int. Appl. (2009), 40pp. CODEN: PIXXD2  WO 2009062157 A1 20090514

       The invention relates to methods for altering the fate or differentiation status of somatic cells by RNA transfer. These methods can be used to transdifferentiate or dedifferentiate somatic cells of one phenotype or lineage into pluripotent cells or into somatic cells of a different lineage or phenotype. The present invention may be used to provide a reproducible and renewable source of immunocompatible human cells/tissues for transplantation based therapies. The inventive method will allow the generation of cells that are fully compatible with the patient. For example, the subject invention may obtain a simple skin biopsy and isolate primary fibroblasts (or any other cell that is easy to obtain e.g. white blood cells, keratinocytes, etc.), expand them in vitro and later transdifferentiate or dedifferentiate them into desired cell populations by RNA transfection.  These methods may be used to produce pluripotent cells from somatic cells by the transfer of RNA from pluripotent cells as well as the production of different somatic cells by the introduction of RNA from somatic cells such as hepatocytes, beta-cells in order to transdifferentiate one type of somatic cell into another somatic cell type. By using epigenetic modifications, the present invention can dedifferentiate or transdifferentiate cells of a recipient, e.g., an individual in need of cell or gene therapy. This invention solves the problem of immunorejection as cells from one patient can be transformed into a different type of cell there by allowing for the production or creation of specific types of cells needed for the treatment of a particular disease the patient may be suffering from e.g., pancreatic islet cells for the treatment of diabetes or hepatocytes for the treatment of liver disease. Also, this invention provides for the formation of donor compatible pluripotent cells, e.g., stem cells thereby allowing for the derivation of different somatic cell phenotypes therefrom.  In addition, while the cells produced according to the invention are especially desired for cell therapy they may also be used for study of mechanisms involved in cell differentiation and disease progression. In particular embodiments, it provides methods for transdifferentiation of human primary fibroblasts into human hepatocytes, dedifferentiation of human fibroblasts into pluripotent cells, transdedifferentiation of white blood cells into beta cells, transdifferentiation of human keratinocytes into neural cells, dedifferentiation of primary keratinocytes into immunocompatible pluripotent cells and transdifferentiation of human primary fibroblasts into cardiac cells. It also provides methods for dedifferentiation of fibroblasts by transfection with RNA from pluripotent cells (oocyte, blCM, hNTera cells). Protein expression from transfected viral vectors and m-RNA in human fibroblasts.














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PapersPublished On RNA and Drug Discovery

Council of Scientific and Industrial Research, India

During the period from 1995 to 2009

1.    Differential Biophysical Behavior of Human Telomeric RNA and DNA Quadruplex.          

Arora A, Maiti S : Institute of Genomics and Integrative Biology, New Delhi.

J. of Phys. Chem. B (July 2009) – Ahead of Print.


2.    Micro RNAs in Diabetes : Tiny Players in Big Diseases.

Pandey Amit K, Agarwal Priyanka, Kaul Kirandeep, Datta Malabika : Institute of Genomics and Integrative Biology, New Delhi.

Cellular Physiology & Biochemistry (2009) ; 23 (4-6), 221-232.


3.    Antagomirzymes : Oligonucleotide Enzymes that Specifically Silence MicroRNA Function.

Jadhav V M, Scaria V, Maiti S : Institute of Genomics and Integrative Biology, New Delhi.

Angew Chem. Int. Ed. Eugl. (2009) ; 48 (14) : 2557-60.


4.    dbSMR : A Novel Resource of Genome – Wide SNPs Affecting MicroRNA Mediated


Hariharan M, Acaria V, Brahmachari S K : Institute of Genomics and Integrative Biology, New Delhi.

BMC Bioinformatics (April 2009) ; 10 : 108.


5.    Human Cellular MicroRNA has-miR-29a Interferes with Viral nef Protein Expression and

HIV-1 Replication.

Jasmine K Ahluwalia, Sohrab Zafar Khan, Kartik Soni, Pratima Rawat, Ankit Gupta, Manoj Hariharan, Vinod Scaria, Mukesh Lalwani, bina Pillai, Bebashish Mitra, Brahmachari S K.

Institute of Genomics and Integrative Biology, New Delhi.

National Centre for Cell Science (NCCS), University of Pune Campus, Pune.

Retrovirology (Dec. 2008) ; 5 : 117.


6.    The Guanine Nucleotide Exchange Factor, C3G Regulates Differentiation and Survival of

Human Nuroblastoma Cells.

Radha V, Rajanna A, Gupta R K, Dayma K, Raman T : Centre for Cellular and Molecular Biology, Hyderabad.

J. of Neurochem. (Dec. 2008) ; 107 (5) : 1424-35.


7.    Thermodynamic, Counterion and Hydration Effects for the Incorporation of Locked Nucleic

Acid   (LNA) Nucleotides in Duplex.

Kaur H, Arora A, Wengel J, Maiti S : Institute of Genomics and Integrative Biology, New Delhi.

Nucleic Acids Symp. Ser. – Oxf. (2008) ; (52) : 425.

8.    Inhibition of bfl-1/A1 by siRNA Inhibits Mycobacterial Growth in THP-1 Cells by

Enhancing Phagosomal Acidification.

Dhiman R, Kathania M, Raje M, Majumdar S : Institute of Microbial Technology, Chandigarh.

Biochim Biophys Acta (April 2008) ; 1780 (4) : 733-42.


9.    Thermodynamics of DNA-RNA Heteroduplex Formation : Effects of Locked Nucleic Acid

Nucleotides Incorporated into the DNA Strand.

Kaur H, Wengel J, Maiti S. : Institute of Genomics and Integrative Biology, New Delhi.

Biochemistry (Jan. 2008) ; 47 (4) : 1218-27.


10.  Host Virus Genome Interactions, Macro roles for MicroRNAs.

V Scaria, M Hariharan, B Pillai, S Maiti, S K Brahmachari : Institute of Genomics and Integrative Biology, New Delhi.

Cellular Microbiology (2007) ; Vol. 9 (12) : 2784-94.


11.  Perspectives on Chemistry and therapeutic Applications of Locked Nucleic Acid (LNA).

Kaur H, Babu B R, Maiti S : Institute of Genomics and Integrative Biology, New Delhi.

Chem. Rev. (2007) ; 107 (11) : 4672-97.


12.  Role of Locked Nucleic Acid Modified Complementary Strand in Quadruplex/Watson-Crick

Duplex Equilibrium.

Kumar N, Maiti S. : Institute of Genomics and Integrative Biology, New Delhi.

J. of Phys. Chem. B (Oct. 2007) ; 111 (42) : 12328-37.


13.  Serine/Threonine Kinase Dependent Transcription from the polyhedron Promoter of SpltNPV-1.

Mishra G, Gautam H K, Das R H : Institute of Genomics and Integrative Biology, New Delhi.

Biochem Biophys Res. Commun. (July 2007) ; 358 (3) : 942-47.


14.  MicroRNA : An Emerging Therapeutic.

Scaria V, Hariharan M, , Brahmachari S K, Maiti S, Pillai B : Institute of Genomics and Integrative Biology, New Delhi.

Chem. Med. Chem. (June 2007) ; 2 (6) : 789-92.


15.  Differential Expression of NF-Kappa B in Mycobacteria Infected THP-1 Affects Apoptosis.

Dhiman R, Raje M, Majumdar S : Institute of Microbial Technology, Chandigarh.

Biochim Biophys Acta (April 2007) ; 1770 (4) : 649-58.


16.  Quadifinder : server for Identification and Analysis of Quadruplex-forming Motifs in  Nucleotide Sequences.

Scaria V, Hariharan M, Maiti S : Institute of Genomics and Integrative Biology, New Delhi.

Nucleic Acids Res. (July 2006) ; 34 (Web Server Issue) – W683.


17.  Thermodynamic, Counterion and Hydration effects for the Incorporation of Locked Nucleic

Acid Nucleotides into DNA Duplexes.

Kaur H, Arora A, Wengel J, Maiti S : Institute of Genomics and Integrative Biology, New Delhi.

Biochemistry (June 2006) ; 45 (23) : 7347-55.


18.  Water soluble Nanoparticles from PEG-Based Cationic Hyperbranched Polymer and RNA that  Protect RNA from Enzymatic Degradation.

Khan J A, Kainthan R K, Ganguli M, Kizhakkedathu J N, Singh Y, Maiti S : Institute of Genomics and Integrative Biology, New Delhi.

Biomacromolecules (May 2006) ; 7 (5) : 1386-88.


19.  Targets for human Encoded MicroRNAs in HIV Genes.

M Hariharan, V Scaria, B Pillai, S K Brahmachari : : Institute of Genomics and Integrative Biology, New Delhi.

Biochemical and Biophysical Research Communications (Dec. 2005) ;Vol. 337(4):1214-18.


20.  Beta2 Adrenergic Receptor Polymorphisms and Asthma in the North Indian Population.

Bhatnagar P, Gupta S, Guleria R, Kukreti R : Institute of Genomics and Integrative Biology, New Delhi.

Pharmacogenomics (Oct. 2005) ; 6 (7) : 713-19.


21.  Beta2 Adrenergic Receptor Polymorphisms and Response to Salbutamol among Indian


Kukreti R, Bhatnagar P, Rao C, Gupta S, Madan B, Das C, Guleria R, Ahavale A U, Brahmachari S K, ghosh B : Institute of Genomics and Integrative Biology, New Delhi.

Pharmacogenomics (June 2005) ; 6 (4) : 399-410.


22.  Evolution and Distribution of RNA Polymerase II Regulatory Sites from RNA Polymerase III 

Dependant Mobile Alu Elements.

Ravi Shankar, Deepak Grover, S K Brahmachari, Maitali Mukerjee : Institute of Genomics and Integrative Biology, New Delhi.

BMC Evolutionary Biology (Oct. 2004) ; 4 : 37.


23.  Progressive Myoclonus Epilepsy [EPM1] Repeat d(CCCCGCCCCGCG)n Forms Folded 

Hairpin Structure at Physiological pH.

Pataskar S S, Dash D, Brahmachari S K :

Molecular Biophysics Unit, Indian Institute of science, Bangalore. Institute of Medical Science, Banaras Hindu University, Varanasi. Institute of Genomics and Integrative Biology, New Delhi.

J. of Biomol. Struct. Dyn. (Oct. 2001) ; 19 (2) : 293-305.


24.  Intramolecular i-Motif Structure at Acidic pH for progressive Myoclonus Epilepsy (EPM1)

repeat d(CCCCGCCCCGCG)n.

Pataskar S S, Dash D, Brahmachari S K :

Molecular Biophysics Unit, Indian Institute of Science, Bangalore. Institute of Medical Science, Banaras Hindu University, Varanasi. Institute of Genomics and Integrative Biology, New Delhi.

J. of Biomol. Struct. Dyn. (Oct. 2001) ; 19 (2) : 307-13.


25.  Piperine Impairs Cytochrome P4501A1 Activity by Direct Interaction with the Enzyme and not  by Down Regulation of CYP1A1 Gene Expression in the Rat Hepatoma 5L Cell Line.

Reen R K, Roesch S F, Kiefer F, Wiebel F J, Singh J : Indian Institute of Integrative Medicine, Jammu Tawi.

Biochem Biophys Res. Commun. (Jan. 1996) ; 218 (2) : 562-9.

Compiled by : Wamiq F. Rahman