RNAi AND ITS APPLICATION IN HUMAN DISEASE THERAPY

PREPARED BY:

ROANNE ARNEDO

SEVERINE LANGON

ABSTRACT

RNA interference (RNAi) is a system within living cells that takes part in controlling which genes are active and how active they are. Two types of small RNA molecules- microRNA (miRNA) and small interfering RNA (siRNA) – are central to RNA interference. RNAs are the direct products of genes, and these small RNAs can bind to other specific RNAs (mRNA) and either increase or decrease their activity, for example by preventing messenger RNA from producing a protein. RNA interference has an important role in defending cells against parasitic genes –viruses and transposons – but also in directing development as well as gene expression in general.

The RNAi pathway is found in many eukaryotes including animals and is initiated by the enzyme Dicer, which cleaves long double-stranded RNA (dsRNA) molecules into short fragments of ~20 nucleotides. The siRNA will be unwound into two ssRNA, namely the passenger strand and the guide strand. The passenger strand will be degraded, and the guide strand is incorporated into the RNA-induced silencing complex (RISC). The most well-studied outcome is post-transcriptional gene silencing, which occurs when the guide strand base pairs with a complementary sequence of a messenger RNA molecule and induces cleavage by Argonaute, the catalytic component of the RISC complex. This process is known to spread systemically throughout the organism despite initially limited molar concentrations of siRNA.

WHAT IS RNAi?It is a recently discovered process in cells that stops the action of specific genes by destroying mRNA and thus preventing translation of the gene product.The diagram summarizes the process of RNAi;

• Dicer (enzyme) chops double-stranded RNA (dsRNA) into small piecescalled short interfering RNA (siRNA)

• siRNA combines with protein subunits to form an RNA-induced silencingcomplex (RISC)

• The siRNA within RISC unzips, exposing anticodons and thus "activating" theRISC

• Activated RISC binds to target mRNA (with complementary codons)

• RISC causes target mRNA to break apart, preventing translation of the geneproduct

CELLULAR MECHANISMDouble strand RNA (dsRNA)

RNAi is an RNA-dependent gene silencing process that is controlled by the RNA-induced silencing complex (RISC) and is initiated by short double-stranded RNA molecules in a cell's cytoplasm, where they interact with the catalytic RISC component argonaute.

Endogenous dsRNA initiates RNAi by activating the ribonuclease protein Dicer, which binds and cleaves double-stranded RNAs (dsRNAs) to produce double-stranded fragments of 20–25 base pairs with a 2 nucleotide overhang at 3' end.

Exogenous dsRNA is detected and bound by an effector protein, known as RDE-4 in C. elegans and R2D2 in Drosophila, that stimulates dicer activity.

MicroRNA

• (miRNAs) are short ribonucleic acid (RNA) molecules, on average only 22 nucleotides long and are found in all eukaryotic cells, except fungi and marine plants.

• miRNAs are post-transcriptional regulators that bind to complementary

sequences on target messenger RNA transcripts (mRNAs), usually resulting in translational repression and gene silencing.

Small interfering RNA (SiRNA)

• is a class of double-stranded RNA molecules, 20-25 nucleotides in length, that play a variety of roles in biology.

• The most notable role of siRNA is its involvement in the RNA interference (RNAi) pathway, where it interferes with the expression of a specific gene. In addition to its role in the RNAi pathway, siRNA also acts in RNAi-related pathways, e.g., as an antiviral mechanism or in shaping the chromatin structure of a genome; the complexity of these pathways is only now being elucidated.

RNAi in Anatomy and PhysiologyHere are a few (of many) reasons that including RNAi in an A&P course is useful:

RNAi plays a role in defending our cells against viruses by stopping viral genetic code from being translated in host cells 

RNAi likely plays a role in the normal regulation of gene activity in a cell by preventing translation of the gene product(s)

RNAi is increasingly used as method for "knocking out" a particular gene's effects in research animals in order to study the gene's functions

RNAi is being used to treat genetic disease. . . an application that will likely expand greatly over the next few decades

RNAi has been shown to be a mechanism of disease

Technological applicationsGene knockdown

The RNA interference pathway is often exploited in experimental biology to study the function of genes in cell culture and in vivo in model organisms. Double-stranded RNA is synthesized with a sequence complementary to a gene of interest and introduced into a cell or organism, where it is recognized as exogenous genetic material and activates the RNAi pathway. Using this mechanism, researchers can cause a drastic decrease in the expression of a targeted gene. Studying the effects of this decrease can show the physiological role of the gene product. Since RNAi may not totally abolish expression of the gene, this technique is sometimes referred as a "knockdown", to distinguish it from "knockout" procedures in which expression of a gene is entirely eliminated.

Functional genomics

Most functional genomics applications of RNAi in animals have used C. elegans and Drosophila, as these are the common model organisms in which RNAi is most effective. C. elegans is particularly useful for RNAi research for two reasons: firstly, the effects of the gene silencing are generally heritable, and secondly because delivery of the dsRNA is extremely simple. Through a mechanism whose details are poorly understood, bacteria such as E. coli that carry the desired dsRNA can be fed to the worms and will transfer their RNA payload to the worm via the intestinal tract. This "delivery by feeding" is just as effective at inducing gene silencing as more costly and time-consuming delivery methods, such as soaking the worms in dsRNA solution and injecting dsRNA into the gonads. Although delivery is more difficult in most other organisms, efforts are also underway to undertake large-scale genomic screening applications in cell culture with mammalian cells

Drosophila melanogaster – side view

A normal adult Drosophila fly, a common model organism used in RNAi experiments.

Medicine

It may be possible to exploit RNA interference in therapy. Although it is difficult to introduce long dsRNA strands into mammalian cells due to the interferon response, the use of short interfering RNA mimics has been more successful. Among the first applications to reach clinical trials were in the treatment of macular degeneration and respiratory syncytial virus, RNAi has also been shown to be effective in the reversal of induced liver failure in mouse models.Other proposed clinical uses center on antiviral therapies, including topical microbicide treatments that use RNAi to treat infection (at Harvard University Medical School; in mice, so far) by herpes simplex virus type 2 and the inhibition of viral gene expression in cancerous cells, knockdown of host receptors and coreceptors for HIV, the silencing of hepatitis A and hepatitis B genes, silencing of influenza gene expression, and inhibition of measles viral replication. Potential treatments for neurodegenerative diseases have also been proposed, with particular attention being paid to the polyglutamine diseases such as Huntington's disease.

An adult C. elegans worm

An adult C. elegans worm, grown under RNAi suppression of a nuclear hormone receptor involved in desaturase regulation. These worms have abnormal fatty acid metabolism but are viable and fertile.

Clinical Application

With unprecedented speed, RNA interference (RNAi) has advanced from its basic discovery in lower organisms to becoming a powerful genetic tool and perhaps our single most promising bio therapeutic for a wide array of diseases. Numerous studies document RNAi efficacy in laboratory animals, and the first clinical trials are underway and thus far suggest that RNAi is safe to use in humans. Yet substantial hurdles have also surfaced and must be surmounted before therapeutic RNAi applications can become a standard therapy. Here we review the most critical roadblocks and concerns for clinical RNAi transition, delivery, and safety. We highlight emerging solutions and concurrently discuss novel therapeutic RNAi-based concepts. The current rapid advances create realistic optimism that the establishment of RNAi as a new and potent clinical modality in humans is near.

Theoretically, it is sufficient to exogenously introduce, or alternatively, to intracellularly express, a short double-stranded RNA (dsRNA) with perfect complementarity to a target mRNA. Not surprisingly, the outstanding potency, simplicity, and specificity of this evolutionarily conserved gene silencing mechanism has fuelled a flurry of efforts to develop novel classes of bio therapeutics based on RNAi. Over the past 5 years, a plethora of in vitro and in vivo proof-of-concept studies have showed that practically every human disease with a gain-of-function genetic lesion can become a target for therapeutic RNAi.

Mechanism of RNAidsRNA is cleaved at specific sites by Dicer to form siRNA. siRNA can also be produced either in vitro, after which it can be conjugated to other molecules for efficient delivery into the target cells, or within cells, via DNA-based vectors encoding shRNA. siRNA binds to RISC, the action of which exposes the antisense strand of siRNA and allows it to recognize mRNA with a complementary sequence. Upon mRNA binding to RISC, the mRNA is cleaved and degraded, resulting in the posttranslational silencing of gene expression.

Toward an ideal vector for in vivo RNAi

As the RNAi field has rapidly matured since the early reports, it has soon become clear that there will be no universal vector for therapeutic applications in humans. In this respect, the situation with RNAi triggers is no different from previous experiences with other nucleic acid–based biodrugs, in which in vivo delivery posed a major challenge, especially to tissues deep within the body. In fact, there are only a few target tissues of interest that are easily accessible, including the human eye and the respiratory tract. In these two cases, local delivery of siRNAs in saline formulations can result in efficient knockdown of exo- or endogenous targets, i.e., VEGF and its receptor in the eye, or respiratory syncytial virus in the lung. Consequently, the first clinical phase I trials evaluating the safety of RNAi in humans are focusing on these particular combinations of tissues and targets.

• Recent progress with siRNA delivery.

Unfortunately, application of “naked” siRNA is not an option for organs or cells deep within the body that are only accessible in a clinically acceptable fashion through systemic RNAi delivery. In these cases, the siRNA not only needs to be protected from serum nucleases, but moreover, efficient and ideally specific delivery to the target tissue becomes more challenging. Therefore, a multitude of academic and industrial groups have recently zoomed in on the pivotal issues of siRNA stability, efficacy, and delivery. In fact, the short persistence of siRNA-mediated gene silencing, due to intracellular siRNA degradation, remains another potential hurdle to their broad clinical application, especially for chronic diseases that require persistent RNAi. Nonetheless, there are numerous examples of human diseases in which even short-term suppression of the underlying genes can result in therapeutic benefit and in which siRNAs remain an utmost viable and potent reagent.

Gene delivery vehicles for short hairpin RNA expression.

Despite these promising findings and similarly encouraging results with other siRNA delivery systems and cholesterol-siRNA-lipoprotein complexes, it is possible that treatment of many human disorders will exceed the capacities of the current siRNA generation. Crucial examples are viral infections and malignancies, both diseases that not only typically affect tissues that are difficult to access with siRNAs but that also require persistent suppression of the underlying gene(s). For the treatment of these challenging classes of diseases, a juxtaposition of RNAi methodology with gene therapy technology appears to be a most promising strategy.

Advantages:

• the majority of all available viral vectors have already been assessed clinically in phase I safety trials, with a number also in phase II/III efficacy studies.

• a thoughtful choice of viral vector type for a given target will provide the highest possible efficacy and specificity, to a degree beyond that currently achievable with siRNA

RNAi-based therapeutic strategies

Combinatorial RNAi.

This is especially true for RNA viruses, such as hepatitis C virus (HCV), which can incorporate 10–3 mutations per viral nucleotide per year, or HIV, which is one of the fastest evolving of all organisms. Unfortunately, in these cases, the superior specificity of RNAi can turn into a disadvantage, as a single nucleotide mismatch between siRNA/shRNA and (viral) target mRNA can abrogate recognition and thus silencing. A clever solution to the problem of viral variability is to target their genomes with a cocktail of siRNAs or with vectors expressing multiple shRNAs. Typically these RNAi triggers will simultaneously target different sites within the viral genome and, ideally, sequences that are highly conserved amongst all members of the viral family; the idea is that together these will effectively thwart the formation of escape mutants. Importantly, coRNAi strategies also hold promise for the treatment of other human diseases, including cancer.

miRNAs as therapeutic targets

According to current belief, a main function of the RNAi pathway in mammals and humans is the generation and processing of endogenously encoded miRNAs. Through binding to, and inhibition of, mRNAs with a partially complementary sequence (typically in their 3′UTR), miRNAs can post transcriptionally modulate the expression of probably thousands of genes. This likely occurs mostly through translational inhibition rather than mRNA degradation, although there are clearly overlapping processes, leaving the exact mechanism(s) not well understood. This makes miRNAs important regulators of multiple and diverse, benign, or malignant cellular processes such as differentiation, transformation, or viral infection. Not surprisingly, miRNAs therefore offer a vast potential for therapeutic intervention, and several different strategies to study and exploit these molecules have already been explored .

Segregation of transgene expression.

Notably, the above described study by Ebert et al. was not the first to actively exploit binding of endogenous miRNAs to a vector-expressed fusion gene, although the application was unique. In fact, in 2006, Brown et al. had already described their own strategy in which they inserted a binding site for a miRNA into the 3′UTR of the coding sequence for a strong neoantigen, GFP. This specific miRNA is selectively expressed in cells of the blood lineage. Here the idea was to systemically deliver the vector to the liver while selectively extinguishing expression from inadvertently transduced blood cells. Indeed, following lentiviral delivery to immunocompetent mice, GFP was only expressed in non-blood cells, particularly in hepatocytes and endothelial cells, but not in hematopoietic lineages. As a result, as GFP expression was absent in APCs, the mice did not mount an anti-GFP immune response, and vector expression was maintained. In a very recent follow-up study, the same principle was used to prevent off-target expression of the recombinant clotting human factor IX gene in hematopoietic cells. Indeed, immunocompetent haemophilia B mice treated with the hFIX–mir-142-3p fusion vector and evidence for phenotypic correction that persisted for over 9 months. Previously, segregation of transgene expression was mainly achieved through transcriptional control, i.e., the use of liver-specific promoters.

BiotechnologyRNA interference has been used for applications in biotechnology, particularly in the engineering of food plants that produce lower levels of natural plant toxins. Such techniques take advantage of the stable and heritable RNAi phenotype in plant stocks. For example, cotton seeds are rich in dietary protein but naturally contain the toxic terpenoid product gossypol, making them unsuitable for human consumption. RNAi has been used to produce cotton stocks whose seeds contain reduced levels of delta-cadinene synthase, a key enzyme in gossypol production, without affecting the enzyme's production in other parts of the plant, where gossypol is important in preventing damage from plant pests. Similar efforts have been directed toward the reduction of the cyanogenic natural product linamarin in cassava plants.

Although no plant products that use RNAi-based genetic engineering have yet passed the experimental stage, development efforts have successfully reduced the levels of allergens in tomato plants and decreased the precursors of likely carcinogens in tobacco plants. Other plant traits that have been engineered in the laboratory include the production of non-narcotic natural products by the opium poppy, resistance to common plant viruses, and fortification of plants such as tomatoes with dietary antioxidants Previous commercial products, including the Flavr Savr tomato and two cultivars of ringspot-resistant papaya, were originally developed using antisense technology but likely exploited the RNAi pathway.