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RNAi: towards paradigm therapeutic shift
ONE may reasonably think of RNA as one of the most primitive biological macromolecules on earth. Whether there was an ‘RNA world’ some four billion years ago or not, modern biology has ushered us into the world of RNA interference (RNAi), thriving within our own cells.
RNAi is an astonishing biological phenomenon which employs short interfering RNAs (siRNAs) complexed with proteins for RNA-based, gene-specific silencing and chromatin remodeling. Advanced methods for efficient siRNA delivery to mammalian cells augmented with computational siRNA design hold great promise for a paradigm shift in therapeutic approaches targeting HCV, HIV and cancer.
RNA is a biomolecule of diverse functionality. Besides its role in the encryption of DNA-derived genetic information leading to protein synthesis as messenger RNA (mRNA), it performs an array of structural, enzymatic and regulatory functions in the cell. RNAi is one of its regulatory functions, discovered recently. Eukaryotic cells have been found to have several genes encoding for double-stranded RNA (dsRNA) precursors, which are processed to short dsRNAs known as micro RNAs (miRNAs).
These miRNAs, ranging in length from 21 to 26 nucleotides, recognize and ultimately lead to the cleavage or translational repression of complementary single-stranded RNAs, such as mRNAs or viral genomic/antigenomic RNAs. The dsRNAs other than miRNA precursors are processed, short interfering RNA (siRNA) with same functions as miRNAs.
RNAi was originally identified as an antiviral defense mechanism. Later, it became important for its role in the regulation of cellular transcriptional programmes through gene-specific silencing and cellular DNA modification. RNAi can be triggered in mammalian cells by synthetic genes encoded by vectors that miRNAs’ mimics.
These are short, hairpin RNAs (shRNAs) ranging from 19 to 29 nucleotides in length, with various degrees of structural similarity to natural miRNAs. DNA vectors carrying shRNA-encoding segments can be delivered to cells by standard and stable transient transfection methods, using viruses ranging from retroviruses to adenoviruses.
The dsRNA molecules may arise in the cell from a replicating virus, from hybridization of overlapping mRNA transcripts of endogenous or vector-borne genes or finally from synthetic dsRNAs.
These dsRNA molecules are then processed into short RNA duplexes by an enzyme, containing RNaseIII and dsRNA binding domains, known as Dicer. The double-stranded siRNAs are passed to RNA-induced silencing complexes (RISC) — core RNAi devices performing crucial defence and regulation functions.
Several proteins are involved in RISC assembly. In human beings, RISCs are formed by the interaction of Argonaute proteins (Ago 2), eukaryotic initiation factor (eIF2C2), FMR protein, RNA helicases, etc. Once assembled, RISC uses the anti-sense strand (complementary to target mRNA) of siRNA to bind to and degrade the corresponding mRNA, resulting in gene-specific silencing.
RNAi and HIV
Currently, most anti-HIV infection treatments are based on combination therapies (“cocktails” of reverse transcriptase and protease inhibitors) like HAART. Despite the apparent success of new anti-retroviral drugs, there are challenges like drug-resistant viral variants and toxicities of combination drugs now in use. These drawbacks have allowed for new anti-retroviral therapeutics to come, with RNAi being the leading candidate.
Lifecycle and gene expression pattern of HIV is well-understood and the targets, well-identified. Synthetic siRNAs and expressed shRNAs have been used to target several HIV-encoded RNAs in cell lines and in primary haematopoietic cells. Given the high mutation rate and escape of variants from being targeted, RNAi-mediated downregulation of the cellular cofactors required for HIV infection is an attractive alternative. Cellular co-factors such as NF-kB, HIV receptor CD4, and co-receptors CXCR4 and CCR5 have been successfully downregulated by RNAi, resulting in the inhibition of HIV replication in numerous human cell lines and in primary cells including T lymphocytes and macrophages.
Macrophage-tropic CCR5 co-receptor holds particular promise as a target because it is not essential for normal immune function, and individuals homozygous for a 32-bp deletion in this gene are resistant to HIV infection, whereas individuals who are heterozygous for this deletion show delayed progression to AIDS.
The drawback is that non-infected cells will inevitably be targeted as well, leading to toxicities that are similar to those observed with the current anti-retroviral drugs. For efficient anti-viral RNAi strategies, viral targets with highly conserved sequences are very promising
The delivery of siRNAs or shRNAs to HIV-infected cells is also a challenge. For an anti-HIV therapy to be successful, it must cope with problems like short half-life of synthetic siRNAs and immunogenecity of shRNA encoding vectors.
The preferred method, so far proposed, is to isolate T-cells from patients; these T-cells are then transduced, expanded and re-infused into the same patients. In a continuing clinical trial, T-lymphocytes from HIV-infected individuals are transduced ex vivo with a lentiviral vector that encodes an anti-HIV, anti-sense RNA.
The cells are subsequently expanded and re-infused into patients. This approach would also be applicable to vector-borne siRNAs. The feasibility of this approach has been worked out and a human clinical trial for HIV treatment is expected to take place in a couple of years.
Grappling with HCV
HCV, a virus that now infects an estimated 3 per cent of the world’s population, is a major cause of chronic liver disease (hepatitis) leading to liver cirrhosis and hepatocellular carcinoma. The HCV genome is a sense (+)RNA molecule with a single-open reading frame encoding a polyprotein that is processed post-translationally to produce at least 10 proteins.
The only therapy currently available is a combination of interferon and ribavirin, but response to this therapy is often poor, particularly with certain HCV sub-types. Therefore, hepatitis caused by HCV and HBV is an important target for potential RNAi therapy.
A significant knockdown of the HBV core antigen in liver hepatocytes could be achieved by shRNA, providing important proof for future anti-viral applications of RNAi in hepatitis.
More advanced studies have been carried out for RNAi therapies against subgenomic and full-length HCV replicons that replicate and express HCV proteins in stable, transfected human hepatoma-derived cells.
As with HIV therapeutics, delivery of siRNAs or shRNA vectors is the main challenge for successful treatment of HCV. The method of delivery used in several in vivo studies — hydrodynamic intravenous injection — is not feasible for the treatment of human hepatitis. In mice, genetic material can be introduced into hepatocytes using catheters or even localized hydrodynamic procedures. However, it is yet to be determined whether such procedures can be used to deliver siRNAs in larger mammals.
Switching off oncogenes
Oncogenic cellular pathways lead to the uncontrolled proliferation of cells giving rise to tumours and associated cancerous disorders. Many studies have used siRNAs as an experimental tool to dissect these pathways
The first systemically delivered anti-sense oligonucleotide (an alternative and closely related strategy for gene silencing) for cancer treatment, which targets the anti-apoptotic gene BCL2, has shown promise in clinical trials for metastatic melanoma when used in combination with conventional chemotherapeutics.
The fact that RNAi is a potential anti-cancer therapeutic agent stems from the breakthrough in which an oncogene encoding Bcr-Abl cancer protein p210, characteristic of chronic myelogenous leukaemia (CML), was successfully targeted using RNAi. Bcr-Abl p210 has been selectively downregulated by both synthetic siRNAs and lentiviral vector-transduced shRNAs in cell lines. Importantly, the downregulation is selective only for p210 oncoprotein and its mRNA.
The potential for using RNAi to treat metastatic cancers depends on finding good cellular targets. Systemically delivered synthetic siRNAs are susceptible to degradation by serum nucleases. Therefore a highly efficient mechanism for delivery of siRNA to relevant cells is necessary for successful treatment of metastatic cancer. Delivery-related research has led to backbone modifications in serum-nuclease-resistant siRNAs. Liposome encapsulated-siRNAs have also been found particularly efficient in their cellular uptake.
Haematologic malignancies are often treated by bone-marrow transplantation. Therefore, a possible therapeutic application would be to transduce haematopoietic stem cells with vectors harbouring a gene that targets the mRNA-encoding, oncogenic p210 protein, thereby protecting patients from relapse caused by proliferation of latent leukaemic stem cells. Delivery is again the major issue; 100 per cent transduction of stem cells re-infused in a bone-marrow transplant setting will be required to make this therapeutically effective. Improvements in viral vector transduction would be potential areas to make this possible.
Computational siRNA design
siRNA design sequence is crucial for effective gene silencing. Rational design strategies for effective siRNAs are being developed based on an understanding of RNAi biochemistry and of naturally occurring microRNA (miRNA) function. Several research groups have proposed basic empirical guidelines for designing effective siRNAs that can be applied to the selection of potential target sequences. For a highly effective RNAi design, siRNA should be specific to the gene of interest and have approximately 20-50 per cent GC content. Moreover, siRNA should have A/U at the 5˘ end of antisense strand and G/C at the 5˘ end of sense strand as it favours it’s incorporation into RISC and offers significantly higher success rates. This principle was therefore implemented as one of the key algorithm determinants for maximizing silencing efficacy.
Several online design tools are available to assist in identifying potential siRNA targets. The basic functioning of these tools relies on an automated search for sequences satisfying siRNA design recommendations and BLAST-based search capability to ensure that selected sequences are specific to the gene of interest.
The main difficulty in designing siRNA sequences is the “off-target” silencing effects of siRNA. Base pairing between the antisense siRNA strand and non-targeted mRNA sequences affects silencing. Recent studies have shown that even a single mismatch in the centre of siRNA can eliminate silencing. While designing a highly effective siRNA, its homology to other sequences in the genome must be minimized.
The minimization of off-target silencing effects call for the selection of siRNA sequences that are guaranteed to have some mismatches to all unrelated sequences. The design process also includes a specificity check whereby both siRNA strands are subjected to customized homology searches to minimize risks of generating off-target effects and putative siRNA target sequences are screened against the most updated single-nucleotide polymorphism (SNP) databases to avoid variability in siRNA efficacy.
Most existing websites for designing siRNA sequences, such as siRNA Target Finder at the Ambion website, siDESIGN Center at Dharmacon, siRNA Target Finder at GenScript, and Gene specific siRNA Selector use BLAST to search for off-target candidates. Certain limitations are associated with BLAST.
For instance it may overlook off-target candidates. siDirect is another web-based online software system for computing highly effective small interfering RNA (siRNA) sequences with maximum target-specificity for mammalian RNA interference (RNAi). It also takes into account the overlooking of BLAST tool for off target sequences. Publicly available siRNA design programs have so far shown a 50-60 per cent success rate in generating siRNAs that can yield over 70 per cent silencing of target mRNA levels.
High through-put online computational tools that accept arbitrary sequences as input and return siRNA candidates with maximal target-specificity and take comprehensive account of off-target sequences provide a wide scope of applications in mammalian RNAi, including systematic functional genomics.
The writer
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