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Maxwell Emerson Kruse 11/21/12 Bio 497A Term Paper

Viral Mediated RNA Interference: A Promising Therapeutic Approach for the Suppression of Dominantly Inherited Neurodegenerative Disease

Introduction

RNA Interference (RNAi) is a naturally occurring and evolutionarily conserved cellular mechanism which functions to regulate post-transcriptional gene expression, and also serves as an intrinsic defense mechanism against parasitic genomic material. RNAi allows for highly specific degradation of target RNAs, and therefore allows for highly specific gene silencing. Since the discovery of fundamental RNAi mechanism in 1998, it has been a focal point of biological research; RNAi has been utilized in a number of biological applications and has exhibited potential to become a highly effective mode of gene therapy both in vitro and in vivo. (Davidson and McCray, 2011)

Although it was observed in the early 1990s that RNA had the potential to inhibit gene expression, it was not until later that the mechanism of RNA interference was understood. It was initially believed that small, single stranded, antisense RNA fragments hybridized to endogenous mRNA and somehow this interfered with the transcripts expression. (Craig et al., 2010) In 1998, however, Mello and Fire, et al. published a Nobel Prize winning paper which elucidated that the most effective RNAi trigger was neither sense nor antisense RNA strands, but double stranded RNA constructs. (Fire et al., 1998) It was found that double stranded RNA (dsRNA) was more effective in silencing the targeted genes than either single sense or antisense RNA strands. In the 14 years since this discovery, many more mechanistic details underlying RNAi have been discovered; this process has become the most popular mode for probing gene function in eukaryotes, and is also emerging as therapeutic treatment for many previously incurable diseases. (Craig, 2010)

RNAi provides a versatile mechanism with the potential to allow researchers to target and silence virtually any gene. By triggering a cells natural RNAi machinery, exogenously applied RNA species can be engineered to catalyze the endogenous degradation of desired mRNA products, thus preventing the transcripts expression. Although RNAi has not yet been utilized successfully in a clinical setting, many studies and clinical trials have produced results that suggest RNAi

is a promising mode for therapeutic treatment of many diseases including: retinal degeneration, several neurodegenerative diseases, viral infections, respiratory infections, dysfunction metabolic processes, and cancer.

This review will highlight the basic mechanisms of RNA interference and will discuss two studies in which viral vectors were used to deliver small inhibitory RNA constructs that functioned to silence the expression of mutant genes responsible neurodegenerative disorders. Specifically, short hairpin RNAs (shRNAs) were utilized to trigger RNA interference that effectively rescued these mice from neurodegenerative phenotypes that mimicked symptoms associated with spinocerebellar ataxia and Amyotrophic lateral sclerosis.

Mechanism of RNA Interference

RNA interference is initiated in the dsRNA molecules present in the cytoplasm. These duplexed RNA molecules are recognized and bound by a multidomain RNase III endonuclease known as dicer that cleaves these dsRNAs into 2030 nucleotide fragments. (Davidson and McCray, 2011) Dicer catalyzes this cleavage by making staggered cuts which create overhangs of about 2-3 nucleotides, resulting in exposed 3-hydroxyl and 5-phosphate termini. (Neema, et al., 2003) These small dsRNA molecules are then bound by an Agronaute protein which is a core catalytic component of the RNA-Induced Silencing Complex (RISC). Once these short duplexes are bound, RISC separates the two homologous RNA strands; the sense strand, known as the passenger strand, is released from RISC and degraded, while the antisense strand, known as the guide strand is retained. The guide strand then directs RISC to the mRNA target and base pairs with this transcript via the guide strands seed, which is a sequence of about 7 bases that is complimentary to the target mRNA. (Davidson and McCray, 2011) Once the target mRNA is bound by RISC, the Argonaute protein catalyzes the cleavage of the mRNA strand. The Argonaute proteine is an RNase H- like endonuclease that cleaves the mRNA target at a single phosphodiester bond that lies between the tenth and eleventh base pairs of the guide strand. (Elif and Zamore, 2011) By catalyzing the degredation of mRNA transcripts, the expression of these transcripts is inhibited and the RISC has thus affected the silencing associated with RNA interference.

MicroRNAs (miRNAs) and small-interfering RNAs (siRNAs) are the two main categories of small dsRNA molecules that mediate the post-transcriptional gene silencing associated with RNAi. Both miRNA and siRNA are generated via the general mechanism described above. These two types of small RNA molecules mediate RNA interference in slightly different ways, but the overlying processes through which they modulate gene expression are highly similar.

Micro RNAs and Small Interfering RNAs

Both miRNA and siRNA mediate RNAi. Although the function of these molecules is essentially the same when exogenously applied in gene therapy and in genetic studies, the primary differences between miRNA and siRNA arise from their innate functions within the cell. Naturally, siRNAs function to protect the cells genome from parasitic genetic material, often associated with viral infection, and also affects the silencing of endogenous sequences such as retrotranspons and other repeate sequences. (Naqvi et al., 2009) While miRNAs are transcribed from specific genes, siRNAs arise when long dsRNA molecules are produced in the cytoplasm. These long dsRNA molecules are then cleaved into multiple fragments by Dicer, as described above, to produce many mature siRNAs that range from 20-24 nucleotides in length. These mature siRNAs are then able to associate with RISC and mediate RNAi. (Craig et al. 2010)

In contrast, miRNAs are produced by endogenous miRNA encoding genes. miRNAs function to regulate the translational efficiencies of various mRNA transcripts and also to regulate the longevity of various transcripts via modulating mRNA degradation. (Craig et al., 2010) Mature miRNAs are produced through the processing of the endogenous transcripts of specific genes within the cells genome. First, primary micro RNAs (pri-miRNAs), are transcribed from an endogenous gene by RNA polymerase II to form a long single stranded transcript. (Davidson and McCray, 2011) These pri-miRNAs are then cleaved by an RNAse III endonuclease, known as Drosha in animals, to form pre-miRNAs that range from about 60-70 nucleotides in length and subsequently fold into imperfect hairpin structures. (Yi et al., 2003) Much like Dicer, Drosha cleaves the pri-miRNA in a staggered fashion leaving 2-3 nucleotide on both 3 and 5 ends of the pre-miRNA. The 3 end of these pre-miRNAs associate with Exportin-5 which facilitates the transport of the pre-miRNA to the cytoplasm. After transport to the cytoplasm, pre-miRNAs are processed to mature miRNAs by Dicer, and are able to associate with RISC and thus to mediate post-transcriptional gene-silencing. (Yi et al., 2003)

Exogenous Initiation of RNA Interference

While RNA interference has evolved as an intrinsic mechanism through which a cell can regulate gene expression and protect itself from parasitic genomes, it has also been manipulated and utilized by researchers to probe for gene function and to silence malfunctioning gene products in disease therapies. Both siRNAs and miRNAs can be engineered and applied exogenously to trigger RNAi and to silence essentially any sequence with in the genome. Several strategies for delivering RNAi triggers exogenously have been explored, each utilizing a variety of different vectors and inhibitory RNA constructs. Small interfering RNA molecules can be engineered to mimic primary miRNAs, pre-miRNAs, and also immature or mature siRNAs. Each of these various constructs enter the RNAi biogenesis pathway at different points, but all serve as effective RNAi triggers. (Davidson and McCray, 2011)

Small Interfering RNAs are generally engineered to be transfected directly into the cells cytoplasm. They can be engineered as 25-27 nucleotide duplexes that will be cleaved into mature siRNAs by Dicer, and can also be transfected

as mature 21-22 nucleotide fragments ready to affect RNAi. (Davidson and McCray, 2011) Micro RNA constructs, however are generally engineered as short hairpin RNAs (shRNAs) that mimic pre-miRNAs or pri-mRNAs. These shRNAs are then processed and subsequently function as miRNAs in mediating RNAi. (Davidson and McCray, 2011)

Delivery of Inhibitory RNAs via Viral Vectors: Adeno-associated Viruses and Lentiviruses

Although a variety of vectors including bacterial vector species, nanoparticle assemblies, and stable nucleic acid lipid particles (SNALPs) have been utilized to deliver small interfering RNAs, viral vectors have proven to be highly effective in the delivery of RNAi triggers, and were utilized in the studies described in this review for the treatment of spinocerebellar ataxia and ALS phenotypes in mouse models. In the treatment of neurological disorders, the blood brain barrier limits the application of therapeutics to the central nervous system. Thus, direct injection of interfering RNA species to the diseased cells provides the most efficient recovery from the mutant phenotype. (Davidson and McCray, 2011)

Viral platforms that deliver shRNAs, mimicking miRNAs, are ideal for the treatment of chronic neurological disorders such as spinocerebellar ataxia and ALS because they provide prolonged expression of the RNAi trigger, while siRNAs have a much shorter life cycle and would require repeated re-dosing in the treatment of chronic disorders. Viral vectors deliver the shRNA constructs to the target cell and then utilize the hosts intrinsic transcriptional and RNAi machinery to elicit gene silencing. Viral vectors are also ideal in that the type of virus used and the cellular tropisms of the virus can be manipulated to transfect a variety of cell types with varying specificities. (Raoul et al., 2006) Several viral vectors can be used to deliver interfering RNAs such as lentiviruses, adenoviruses, adeno-associated viruses (AAVs), and herpesviruses. These viral vectors confer different advantages and disadvantages in their modes of delivery, and they differ in how their tissue tropisms will direct their distribution and in how their genomes will transfect the host cell. The choice of which viral vector is used to deliver the shRNA generally revolves around the packaging capacity of the virus, the integrative or non-integrative nature of the virus, and the cellular tropisms of their exterior. (Davidson and McCray, 2011) The viral vectors utilized in the papers discussed here were adeno-associated viruses (AAVs) and Lentiviruses, both of which show potential for the therapeutic treatment of neurodegenerative diseases connected with gain of function mutations. (Raoul et al., 2006)

Adeno-associated Viral Vectors

Adeno-associated viruses (AAVs) have small single stranded DNA genomes and have a packaging capacity of about 4.5 kb. These vectors are ideal for gene therapy because they are not known to cause disease in humans, and they remain episomal after the target cells are transfected, meaning that they do not integrate into the hosts genetic

material; the viral DNA remains free floating in the cell. The fact that they remain episomal is advantageous because this eliminates the possibility of insertional mutagenesis that may occur with vectors that integrate in to the host genome. (Raoul et al., 2006) Although their small genome size limits the packaging capacity of AAVs to 4.5kb, this is beneficial in that the entire viral genome is typically replaced by the engineered shRNA. Therefore, viral genomic material that could possibly insight an immune response by the host cell (which would prevent successful transduction of the shRNA ) is removed, and an immune response is avoided. (Davidson and McCray, 2011) Thus, AAVs are considered to be only mildly immunogenic. Although the fact that these vectors remain episomal prevents insertional mutagenesis, this also limits their transduction to cells that are slow to divide or to quiescent cells, because the episomal DNA will be lost after repeated division. The neurons and glia involved with neurodegenerative disorders, however, are non-dividing and slow to divide respectively so the episomal DNA will be expressed for prolonged periods of time. Ultimately, in the treatment of neurological disorders, AAVs are ideal vectors for delivering shRNAs because they have low immunogenicity, provide long-term expression in quiescent cells, and because their capsids can be engineered to target cells with a high specificity. (Raoul et al., 2006)

Lentiviral Vectors

Lentiviruses (LVs) are also idea viral vectors for transfecting cells with shRNAs. Lentiviruses are retroviruses that are unique in that they can infect both dividing and non-dividing cells. These vectors have a packaging capacity of about 13.5 kb, much larger than that of AAVs, and are also capable of eliciting prolonged expression of shRNAs. Although Lentiviral vectors generally exhibit a low immunogenicity, these retroviruses integrate into the hosts genome and therefore pose the risk of inducing insertional mutagenesis. (Davidson and McCray, 2011) Although these vectors have been used successfully to deliver RNAi triggers, their RNA genomes are often cleaved by RNA processing enzymes within the cytoplasm if they contain hairpin structures. This, along with the risk of insertional mutagenesis makes Lentiviruses less viable candidates for clinical use when compared to AAVs. (Raoul et al., 2006)

RNA Interference as a Therapy for Dominantly Inherited Neurological Disorders

There are several neurological disorders associated with dominant, gain of function mutations that pose chronic disruption to the processes of the central nervous system (CNS). Treatment of diseases affecting the CNS is difficult due to the blood brain barrier which inhibits the delivery of most therapeutics to the brain; thus, most of these disorders such as Huntingtons Disease, ALS, Parkinsons, Spinocerebellar Ataxia, and Alzheimers are currently incurable. Current technology that utilizes RNAi, however, shows potential for directing the therapeutic rescue of diseased cells from their mutant phenotypes.

Some of these neurological disorders, such as Huntingtons, spinocerebellar ataxias, and ALS are caused solely by the inheritance of gain of function mutations which result in malfunctioning gene products responsible for the pathology of the disease phenotypes. RNA interference provides the opportunity to surpass the issues of drug delivery associated with the blood brain barrier, and can allow for direct delivery of interfering RNA species to the diseased cells. Thus, through directly inhibiting the mutant genes causing the disease phenotypes, RNAi has been shown to successfully suppress the pathology of several neurological disorders associated with gain of function mechanism both in animal models and in clinical trials. (Baudreau and Davidson, 2010) The use of viral vectors to deliver long lasting and highly specific gene silencing in vivo has proven to be one of the most successful approaches in treating dominantly inherited neurodegenerative disorders. (Raoul et al., 2006)

Research Article #1: RNAi suppresses polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia

Polygultamine (polyQ) repeat diseases are dominantly inherited neurodegenerative disorders that result from mutations which cause an expansion of a trinucleotide repeat in a gene, and subsequently result in an expanded polyglutamine tract in the protein product of that gene. (Raoul et al., 2006) This polyQ expansion is a toxic gain of function mutation which causes the affected proteins to form intranuclear aggregates; these aggregations are thought to be the source of the neurodegeneration and disease phenotypes associated with diseases such as Huntingtons and spinocerebellar ataxias. (Matilla-Duenas et al., 2009) This study, conducted by Haibin, et al. utilized AAVs and shRNAs to affect RNAi in a mouse model of spinocerebellar ataxia type 1, and was the first to demonstrate that RNAi could be utilized in vivo as a therapeutic treatment for neurodegenerative disease. (Haibin et al., 2004) Spinocerebellar ataxia type 1 (SCA1) is a late onset disorder caused by a polyQ expansion of about 44-82 glutamines in the protein ataxin-1. This toxic gain of function mutation results in progressive cerebellar ataxia, cognitive impairment, degeneration of cerebellar tissue, and by the loss of neurons and Pukinje cells. (Matilla-Duenas et al., 2009) Therefore, the direct inhibition of mutant ataxin-1 allele expression should effectively prevent the formation of the intranuclear aggregates responsible for the pathology of SCA1. Haibin, et al. hypothesized that the introduction of viral vectors expressing short hairpin (shRNAs) directed against the transgenic mutated human ataxin-1 gene would reduce pathology and ataxia in a mouse SCA1 model. (Haibin et al., 2004) By delivering AAVs expressing shRNAs via intracerebellar injection to SCA1 transfected mice, Haibin, et al. were able to effectively prevent the onset of ataxia, improve the cerebellar morphology, and reduce the accumulation of mutant ataxin-1 aggregates in Purkinje cells. (Haibin et al., 2004)

Selecting target sequences in human ataxin-1 for Silencing with shRNA vectors

Developing shRNAs to target the mutant ataxin-1 allele

Before applying the shRNA constructs in vivo, a number of short hairpin RNA sequences were tested for their efficacy in silencing human ataxin-1. To accomplish this, HEK 293 cell cultures were cotransfected with FLAG-tagged ataxin-1 plasmids and plasmids expressing the shRNA candidates. Eleven shRNA sequences were tested under the expression of both pol III and pol II promoters. Below is an image of the 2.4-kb ataxin-1 cDNA and the locations of the sequences targeted by each shRNA candidate. (Haibin et al., 2004)

Figure 1a: Cartoon of the ataxin-1 cDNA and the regions tested for silencing (Haibin et al., 2004)

Through quantitative RT-PCR and western blot analyses, it was found that the shRNA constructs that targeted regions immediately flanking the CAG trinucleotide repeat were most effective in reducing RNA levels. In these experiments, shLacZ was utilized as a negative control to ensure that ataxin-1 silencing was specific to the shRNAs complementary to the target sequences. Actin levels were also assessed as another control to ensure that the shRNA constructs were effecting specific inhibition of ataxin-1 expression. As seen in Fig 1b below, the shSCA1.F10 and shSCA1.F11 constructs were most effective in reducing the mutant ataxin-1 RNA levels; these constructs reduced ataxin-1 levels by 80% and the ataxin-1 protein levels by about 60%. It was also found that the silencing induced by these shRNAs increased with increased molar ratio of shRNA to target ataxin-1 mRNA, as seen in Figure 1C. (Haibin et al., 2004)

Figure 1b: A western blot assessing ataxin-1 expression in transfected cells (Haibin et al., 2004)

Figure 1c: shRNA silencing of ataxin-1 is dose dependent (Haibin et al., 2004)

Determining if shSCA1.F10 and shSCA1.F11 are effective in neuronal cells

After the shSCA1.F10 and shSCA1.F11 vectors were selected to silence the mutant ataxin-1 allele, their efficacies were tested in PC6-3 neuronal cells. These neuronal cells were transfected with AAV vectors expressing shSCA1.F10, shSCA1.F11, or shLacZ control vectors, and the efficacies of each of these constructs were tested under the control of both pol II H1 promoters and pol III modified CMV (mCMV) promoters. Western blotting was used to measure the degree to which each of these constructs silenced ataxin-1. The western blot shown in Figure 1d, shows that both shSCA1.F10 and shSCA1.F11 constructs were indeed effective in silencing the mutant allele in neuronal cells. It was alos apparent that the shSCA1.F11 under the control of the mCMV proved to effect a higher degree of silencing than the same construct under the control of the H1 promoter. The mCMV pol III promoter was therefore selected to control the expression of these constructs for the remainder of the study. (Haibin et al., 2004)

Figure 1d: This western blot compares the silencing efficacies of the shRNA constructs in neuronal cells. The silencing of each construct was assessed under the control of both H1 and mCMV promoters. (Haibin et al., 2004)

Haibin, et al. note that previous studies suggest that the export of shRNA transcripts under the control of pol III promoters by exportin-5 to the cytoplasm may be inefficient. These studies indicate that nuclear export of the shRNAs can be made more efficient through the replacement of the loop structure of the engineered hairpins with loops expressed by endogenous miRNAs. Therefore, to improve the efficiency of ataxin-1 silencing, Haibin et al. modified the shSCA1 constructs to contain endogenous miRNA loops. These new constructs (denoted shSCA1.F10mi and shSCA1.F11mi) proved effective in enhancing the silencing of human ataxin-1, as can be seen in the western blot in Figure 1e below. Ultimately, these shSCA1.F10mi and shSCA1.F11mi constructs were selected for use in the silencing of SCA1 in vivo. (Haibin et al., 2004)

Figure 1e: This western blot compares the silencing efficiencies of the modified shSCA1mi constructs containing the endogenous miRNA loop against the shSCA1 constructs without the miRNA loops. (Haibin et al., 2004)

shSCA1 transduces Purkinje Cells

Once the shSCA1.F10mi and shSCA1.F11mi shRNAs were selected for use in vivo, recombinant AAV vectors expressing these two constructs were generated. hrGFP (humanized renilla reniformis green fluorescent protein) was utilized as a reporter in these vectors to allow for the detection of the transduced cells in vivo. To determine if these AAVs transduced cerebellar Pukinje cells, wild type mice were injected both with saline and the AAV shSCA1 vectors. These mice were sacrificed after 3 weeks, and the hrGFP expression was evaluated with fluorescent imaging. The mice injected with the AAV ShSCA1 vectors were shown to express hrGFP, indicating that the Purkinje cells were effectively transfected, while the saline injected mice exhibited no hrGFP expression. (Haibin et al., 2004)

To determine if these transduced cells actually expressed functional mature siRNA transcripts, mice were injected with the AAV shSCA1.F10mi and shLacZ vectors and harvested after 10 days. A northern blot was used to analyze the expression of these shRNA constructs in vivo. Figure 2C below indicates that both shRNA constructs were processed by the RNAi machinery, and that mature processed siRNA was present in the harvested cerebella. (Haibin et al., 2004)

Figure 2C: This northern blot indicates that processed shRNA transcripts were present in the harvested cerebella after 10d. The arrow head marks the location where the unprocessed transcript would be present, while the arrow marks the position of the processed siRNA. (Haibin, et al.)

shSCA1 induced RNAi improves motor coordination is SCA1 mice

The SCA1 transgenic mice used in this experiment express the mutant human ataxin-1 allele and thus exhibit progressive ataxia, atrophy of the cerebellar tissue, and also loss of Pukinje cells, essentially paralleling the disease characteristics seen in human spinocerebellar ataxia. The AAV shSCA1 vectors were therefore tested for their ability to suppress these three disease phenotypes in the mouse model. (Haibin et al., 2004) A rotorod test, in which the mice were tested for their ability to maintain balance on a rotating cylinder (Jones and Roberts, 1969), was utilized to assess the motor function of the mice at various time points, and thus to measure the rate of ataxia in the SCA1 mice. Both wild type and SCA1 mice were first assessed for a baseline rotarod performance, and were then injected with AAVs expressing shSCA1F10mi, shLacZ (a negative control), or saline (a negative control) at 7 weeks of age. The rotarod performance for each of these mice was assessed on a biweekly basis

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until the mice were sacrificed at 21 weeks of age. The results of these tests showed that the SCA1 mice transfected with the AAVshSCA1.F10 displayed significantly improved motor coordination, when compared to the mice injected with shLacZ and saline negative controls. This can be seen in Figure 2D below which clearly shows that the SCA1/shSCAq.F10 mice displayed a much stronger latency to fall. It was also noted that the wild type mice transfected with these vectors showed no apparent change in motor coordination over the course of 21 weeks, thus indicating that the expression of the shRNA constructs did appear to be toxic in wilt type Purkinje cells. These experiments therefore show that the AAV vectors expressing shSCA1.F10 were successful in suppressing the onset of ataxia, and were effective in improving motor coordination in SCA1 mice. (Haibin et al., 2004)

Figure 2D: This Figure compares the motor coordination of SCA1 and wild type mice injected with saline, shLacZ, and shSCA1.F10. The wild type mice preformed the best throughout the 21 week time period and did not display any signs of ataxia. The SCA1/shSCA1.F10 mice show reduced onset of ataxia, while the SCA1/saline and SCA1/shLacZ mice show progressive ataxia throughout the 21 week time period. (Haibin, et al.)

shSA1 induced RNAi prevents cerebellar atrophy and degradation of Pukinje cells

To test if the observed improvement in motor coordination was paralleled with improvements in the cerebellar neuropathology, the cerebellar lobes of these mice were analyzed after sacrifice to determine if the shSCA1 transduced SCA1 mice exhibited reduced neurodegeneration when compared to SCA1 mice injected with shLacZ and wild type mice. Cerebellar tissue was isolated and then stained with calbindin and analyzed with immunofluorescence imaging. The calbindin stain was used to determine the width of the molecular layer; a more intense staining indicated that there was little thinning of the cerebellar tissue and thus that significant neurodegeneration did not occur. The reduction of neuropathology in Purkinje cells was assessed by juxtaposing images of regions of transduced cells (expressing hrGFP) and untransduced cells (not expressing hrGFP), and then superimposing these images on the images of calbindin staining as seen in Figure 3a below:

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Figure 3a: SCA1 mice were injected with shSCA1.F10mi expressing AAVs and shLacZ. Wild type mice were also injected with AAV shSCA1.F10mi. After 9 weeks these mice were sacrificed and their cerebellar neuropathology was assessed. Each section of cerebellar tissue here was been stained with calbindin. Immunofluorescence images displaying hrGFP expression (top), calbinidin staining (middle), and merged images are shown. (Haibin et al., 2004)

Figure 3a shows that the transduced regions (regions expressing GFP) of the SCA1 mice transduced with shSCA1.F10mi AAVs display a more intense calbindin staining than the un-transduced regions (where GFP was not expressed), thus indicating that these shRNAs reduced the thinning of cerebellar tissue in the transduced regions. The SCA1 mice injected with shLacZ, however, displayed reduced calbindin staining in both transduced and un-transduced regions. Quantification of molecular layer widths in the wild-type mice and the SCA1 transfected mice confirmed that the shRNA1.F10mi RNAs reduced thinning of the cerebellar tissue, and also indicated that the wild-type mice were unaffected when injected with these AAVs. This can be seen in Figure 4a below. (Haibin et al., 2004) It could therefore be concluded that the shSCA1.F10mi RNAs were responsible for reducing the neurodegeneration associated with SCA1 and that these shRNAs were not toxic to wild-type mice. (Haibin et al., 2004)

Implications of the Study

Ultimately, the results of this study indicate that Haibin, et al. were successful in utilizing RNAi to suppress the neurodegeneration seen in spinocerebellar ataxia type 1. By utilizing AAVs to deliver shRNAs to SCA1 transgenic mice, the progression of ataxia in these mice was inhibited as was the atrophy of cerebellar tissue generally seen with the SCA1 phenotype. RNAi was therefore effective in silencing the mutant human ataxin-1 allele. These results suggest that effecting RNA interference through AAV mediated delivery of shRNAs may also be an effective therapy for treating human spinocerebellar ataxia and other human polyglutamine-induced disorders.

Silencing mutant SOD1 using RNAi protects against neurodegeneration and extends survival in an ALS model

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that causes the progressive degeneration of motor neurons in the brain stem, spinal cord, and cerebral cortex. The loss of motor neurons in many

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cases of ALS is the result of a gain of function mutation in the gene encoding superoxide dismutase (SOD1). Progression of the disease is rapid, and the neurodegeneration results in muscle atrophy, paralysis, and ultimately in death. (Raoul et al., 2006) Transgenic mice engineered to express mutant human SOD1, exhibit similar pathological features to human ALS victims such as motor impairment and rapid disease onset. (Ralph et al., 2005) In this study, Ralph, et al. utilized intramuscular injection of a lentiviral vector expressing shRNAs that specifically targeted the human SOD1 gene to affect RNAi of this mutant gene in vivo. Injection of this viral vector to SOD1 transgenic mice was shown to reduce the expression of mutant SOD1, reduced the atrophy of motor neurons, resulted in delayed onset of ALS symptoms, and also extended the survival rate of these SOD1 mice by over 80% of their typical life span. These data are the first to show a substantial extension of survival in an animal model of a fatal, dominantly inherited neurodegenerative condition using RNAi , (Ralph et al., 2005) and also displayed a remarkably high therapeutic efficacy of RNAi in treating this neurological disorder. (Ralph et al., 2005)

Development of shRNAs and Lentiviral Vectors targeting SOD1 in motor neurons

The disease symptoms associated with this mouse model of ALS result from a dominant mutation of SOD1 in the motor neurons of the brain and spinal cord. A lentiviral vector derived from the equine infectious anemia virus (EIAV) was selected to deliver the shRNAs targeting SOD1 to affect the RNAi mediated silencing of this mutant protein. These EIAV vectors are known to be highly efficient in targeting motor neurons within the brain and spinal cord following intramuscular injection, and would therefore be effective in delivering the therapeutic shRNAs to the diseased cell populations. (Ralph et al., 2005) Candidate target sites for the RNAi induced silencing of SOD1 were identified and analyzed with an online algorithm (Dharmacon) and BLAST searching of the GeneBank database. The researchers chose to engineer shRNA constructs complementary to the two target sites found in human SOD1 that displayed the least sequence homology to the mouse sod1 protein. This would ensure that the shRNAs would specifically target the mutant SOD1 gene, and would not interfere with the natural cellular processes mediated by the mouse sod1. (Ralph et al., 2005) EIAV vectors were engineered to express shRNAs targeting each of these selected target sequences. Both of these constructs were transfected under the control of the same Pol III H1 ribonuclease promoter. These two vectors were denoted EIAV-SOD1HP1 and EIAV-SOD1HP2. A vector expressing shRNA targeting the firefly luciferase gene (EIAVLucHP) and a vector containing empty cassette (EIAV-Emp) were also engineered as negative controls. (Ralph et al., 2005)

Selecting an EIAV-SOD1 Vector through in Vitro applications

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To determine the efficacy of these EIAV-SOD1 vectors in silencing mutant SOD1 in vitro, human kidney HEK293T cells were transfected with one of the two EIAV-SOD1 vectors, the EIAV-LucHP control vector, or the EIAV-Emp control vector. Seven days after transduction, western blotting and quantitative RT-PCR were used to determine the SOD1 silencing efficiencies of each vector. Neither control vector showed a reduction in SOD1 expression, while both EIAVSOD1 vectors exhibited reduction of both SOD1 mRNA and protein expression. Transduction of EIAV-SOD1H1, however, conferred a greater reduction of SOD1 expression than EIAV-SOD1H2. (Ralph et al., 2005) The western blot also analyzed luciferace and beta-actin mRNA levels present with in the cell culture. The expression of each of these proteins was unaffected by the EIAV-SOD1 vectors; cells co-transfected with the EIAC-LucHP vectors and a vector expressing the luciferase reporter, however, showed reduced luciferase expression, thus confirming that the EIAC-LucHP control also delivered functional shRNAs. These results indicate that the shRNA delivered by EIAVSOD1HP1 was effective in specifically reducing SOD1 expression levels, and also that the observed silencing was not the result of general shRNA expression. The EIAV-SOD1HP1 vector was therefore chosen for further investigation. (Ralph et al., 2005)

Determining the efficacy of EIAV-SOD1HP1 induced RNAi in neuronal Cells (in vitro)

Once the EIAV-SOD1HP1 vector was determined to selectively target and silence SOD1 in HEK293T cells, it was determined if this silencing could be applied in neuronal cells. First, to determine if these vectors could effectively transfect neuronal cells, neurons derived from mouse SOD1 embryos were transduced with the EIAV vectors. The EIAVSOD1HP1 vector was engineered to contain a LacZ reporter gene which codes for -galactosidases. Immunofluorescence imaging revealed that 95% stained positively for -galactosidase production, thus indicating that these vectors were capable of high levels of transduction. (Ralph et al., 2005) These SOD1 embryonic neurons were analyzed at days 4 and 7 after transduction through western blotting and quantitative RT-PCR to determine SOD1 mRNA and protein levels. The EIAV-SOD1HP1 traduced neurons exhibited a 50% reduction in SOD1 protein levels at day 4, and 70% reduction at day 7, thus indicating that silencing of the SOD1 gene was highly effective. It was also observed that mouse sod1 expression was not effected when wild-type mice were transduced with the EIAV-SOD1HP1 vector. (Ralph et al., 2005) It was therefore concluded that the EIAV-SOD1HP1 vector exhibited high levels of transduction in neuronal cells and was highly effective in silencing SOD1 expression. The silencing elicited by the EIAV-SOD1 vector was also highly specific in that it did not interfere with mouse sod1 expression. This later detail is important for in vivo applications because it ensured that the induced RNAi would not inhibit natural cellular processes through non-specific inhibition. (Ralph et al., 2005)

The application of EIAV Vectors in Vivo

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The EIAV-SOD1 vectors were modified for in vivo application to SOD1 transgenic mice, and rabies-Gpseudotyped EIAV vectors were generated. Rabies virus G-protein pseudotyping of lentiviral vectors confers a cellular tropism that allows for axonal retrograde transport of the vector to lumbar spinal cord. (Mazarakis et al., 2001) This pseudotyping was carried out to direct the lentiviral vector specifically to the motor neurons susceptible to degradation by mutant SOD1. (Ralph et al., 2005) The EIAV vectors were delivered to the hind limbs of SOD1 mice via intramuscular injection. Two weeks after injection the spinal cord sections from SOD1 mice were analyzed with Immunofluorescence imaging to determine the transduction efficiency of the EIAV-SOD1HP1 vector in vivo. The expression of the LacZ reporter gene was present in >50% of spinal neurons in the target vetral horn of lumbar spinal cord, thus indicating that the EIAV-SOD1 vector also exhibited a high transduction efficiency in vivo. Western blot and quantitative PCR analyses of these cells showed a significant decrease in SOD1 mRNA levels and 40% of SOD1 protein levels in the target cells of the spinal cord. (Ralph et al., 2005) It was therefore concluded that the EIAV-SOD1HP1 vector was delivered to SOD1 mice with a high specificity for the target cells of the lumbar spinal cord, and was also highly effective in triggering RNAi induced suppression of mutant SOD1 expression in vivo. (Ralph et al., 2005)

EIAV-SOD1HP1 vectors Delay the onset of ALS-like symptoms and extend life expectancy in SOD1 Mice

SOD1 transgenic mice exhibit a rapid onset of ALS-like symptoms including an overall reduction in mobility, loss of hind limb function, muscular atrophy, and behavioral changes such as a reduction in grooming. To determine if RNAi induced silencing of mutant SOD1 expression would cure these disease symptoms, the SOD1 mice were injected with either the EIAV-SOD1HP1 vector, EIAV-EMP vector, or were not injected at all at 7 days of age. Intramuscular injections of each vector were delivered to areas essential to the survival of the SOD1 mice. The muscle groups injected included the hindlimb (critical for mobility), the tongue (critical for feeding), and the diaphragm (critical for respiration). (Ralph et al., 2005) To determine the effect the EIAV vectors had on disease onset, the motor function of these injected mice was assessed each day via the rotarod test. Disease onset was characterized by a significant reduction in motor function associated with the end stages of the disease. The SOD1 mice injected with the EIAV-Emp control vector and the uninjected mice exhibited an average disease onset of about 94.3 days of age. After disease symptoms were first observed in these control mice, the disease symptoms were observed to progress rapidly until death. The SOD1 mice injected with the EIAV-SOD1HP1 vector, however, did not display disease symptoms until about 202.1 days of age, and the progression of the disease symptoms in these mice was much less severe. These mice did show reduced mobility, but never exhibited the sever hindlimb dysfunction seen in the control mice. The EIAV-SOD1HP1 vector therefore produced

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a 115% delay in the age of disease onset when compared to the uninjected animals, and was highly effective in preventing ALS-like symptoms. (Ralph et al., 2005) The EIAV-SOD1HP1 vector also proved effective in extending the life span of the SOD1 mice. The end stage of the disease was characterized by the point at which the mice were incapable of righting themselves within a 30 second time period of being placed on their sides; it was at this point the mice were sacrificed. The control mice survived for an average of 128.8 days, while the survival rate of the mice injected with the EIAV-SOD1HP1 vector was prolonged significantly to an average of 227.8 days. (Ralph et al., 2005)

The EIAV-SOD1HP1 vector improves the Neuropathology of SOD1 Mice

To determine if the suppression of disease phenotypes elicited by EIAV-SOD1HP1 were the result of improved neuropathology, a histological analysis of the brain and spinal cord was conducted. Sections of the lumbar spinal cord were from EIAV-SOD1HP1 transfected mice, EMP-transfected mice, and un-transduced mice were stained for facial nuceli with a cresyl violet stain. Cell counts elucidated that the control mice exhibited extensive motor neuron degeneration and vacuole formation characteristic of ALS neuropathology. The EIAV-SOD1HP1 transduced animals, however, showed a significant increase in surviving neurons. It was also observed that in the EIAV-SOD1HP1 transduced mice, that the cells expressing -galactosidase at the end stage of disease (which indicates that these cells were transduced with the shRNA) did not display the neurodegenration seen in degenerating cells of the same tissue. This finding thus indicates that the EIAV vectors delivered long term expression of the shRNA species, because SOD1 silencing persisted in these cells even to the end stages of the disease. (Ralph et al., 2005) It could therefore be concluded that the shRNAs expressed by the EIAV-SOD1HP1 vector were effective in silencing mutant SOD1 expression for a prolonged time period, and were thus effective in preventing the neurodegenration characterisitic of ALS. (Ralph et al., 2005)

Implications of the Study

This study, conducted by Ralph, et al. displayed that RNAi can be a highly effective approach for therapeutic treatment of dominantly inherited neurodegenerative disorders. The EIAV vectors provided high transduction efficiencies, targeted diseased tissues with a high level of specificity, and also afforded prolonged expressed of shRNAs targeting the mutant SOD1 allele. This EIAV-mediated silencing remarkably prevented the onset of ALS symptoms by over 100%, and increased the survival of these transgenic mice by over 77% of their usual life expectancy. If perfected, this viral system of RNAi delivery holds promising potential for clinical treatment of ALS and other neurodegenerative disorders. However, despite the notable success of these EIAV vectors in treating ALS-like symptoms this SOD1 mouse

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model, it is likely that ALS pathology in humans involves several other malfunctioning proteins and cellular mechanisms. It will be interesting to see how this viral platform for delivery of RNAi is refined and applied in a clinical setting.

References

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