[show abstract][hide abstract] ABSTRACT: Gene therapy has historically focused on delivering protein-coding genes to target cells or tissues using a variety of vectors. In recent years, the field has expanded to include gene-silencing strategies involving delivery of noncoding inhibitory RNAs, such as short hairpin RNAs or microRNAs (miRNAs). Often called RNA interference (RNAi) triggers, these small inhibitory RNAs are difficult or impossible to visualize in living cells or tissues. To circumvent this detection problem and ensure efficient delivery in preclinical studies, vectors can be engineered to coexpress a fluorescent reporter gene to serve as a marker of transduction. In this study, we set out to optimize adeno-associated viral (AAV) vectors capable of delivering engineered miRNAs and green fluorescent protein (GFP) reporter genes to skeletal muscle. Although the more broadly utilized enhanced GFP (eGFP) gene derived from the jellyfish, Aequorea victoria was a conventional choice, we were concerned about some previous studies suggesting this protein was myotoxic. We thus opted to test vectors carrying the humanized Renilla reniformis-derived GFP (hrGFP) gene, which has not seen as extensive usage as eGFP but was purported to be a safer and less cytotoxic alternative. Employing AAV6 vector dosages typically used in preclinical gene transfer studies (3×10(10) -1 × 10(11) particles), we found that hrGFP caused dose-dependent myopathy when delivered to wild-type (wt) mouse muscle, whereas identical titers of AAV6 carrying eGFP were relatively benign. Dose de-escalation at or below 8 × 10(9) AAV particles effectively reduced or eliminated hrGFP-associated myotoxicity, but also had dampening effects on green fluorescence and miRNA-mediated gene silencing in whole muscles. We conclude that hrGFP is impractical for use as a transduction marker in preclinical, AAV-based RNA interference therapy studies where adult mouse muscle is the target organ. Moreover, our data support that eGFP is superior to hrGFP as a reporter gene in mouse muscle. These results may impact the design of future preclinical gene therapy studies targeting muscles and non-muscle tissues alike.Molecular Therapy - Nucleic Acids (2013) 2, e86; doi:10.1038/mtna.2013.16; published online 16 April 2013.
[show abstract][hide abstract] ABSTRACT: Limb Girdle Muscular Dystrophy (LGMD) refers to a group of 25 genetic diseases linked by common clinical features, including wasting of muscles supporting the pelvic and shoulder girdles. Cardiac involvement may also occur. Like other muscular dystrophies, LGMDs are currently incurable, but prospective gene replacement therapies targeting recessive forms have shown promise in pre-clinical and clinical studies. In contrast, little attention has been paid to developing gene therapy approaches for dominant forms of LGMD, which would likely benefit from disease gene silencing. Despite the lack of focus to date on developing gene therapies for dominant LGMDs, the field is not starting at square one, since translational studies on recessive LGMDs provided a framework that can be applied to treating dominant forms of the disease. In this manuscript, we discuss the prospects of treating dominantly inherited forms of LGMD with gene silencing approaches.
Current Gene Therapy 08/2012; 12(4):307-14. · 5.32 Impact Factor
[show abstract][hide abstract] ABSTRACT: No treatment exists for facioscapulohumeral muscular dystrophy (FSHD), one of the most common inherited muscle diseases. Although FSHD can be debilitating, little effort has been made to develop targeted therapies. This lack of focus on targeted FSHD therapy perpetuated because the genes and pathways involved in the disorder were not understood. Now, more than 2 decades after efforts to decipher the root cause of FSHD began, this barrier to translation is finally lowering. Specifically, several recent studies support an FSHD pathogenesis model involving overexpression of the myopathic DUX4 gene. DUX4 inhibition has therefore emerged as a promising therapeutic strategy for FSHD. In this study, we tested a preclinical RNA interference (RNAi)-based DUX4 gene silencing approach as a prospective treatment for FSHD. We found that adeno-associated viral (AAV) vector-delivered therapeutic microRNAs corrected DUX4-associated myopathy in mouse muscle. These results provide proof-of-principle for RNAi therapy of FSHD through DUX4 inhibition.
[show abstract][hide abstract] ABSTRACT: RNA interference (RNAi) is a conserved gene silencing mechanism mediated by small inhibitory microRNAs (miRNAs).Promoter-driven miRNA expression vectors have emerged as important tools for delivering natural or artificially designed miRNAs to eukaryotic cells and organisms. Such systems can be used to query the normal or pathogenic functions of natural miRNAs or messenger RNAs, or to therapeutically silence disease genes.
As with any molecular cloning procedure, building miRNA-based expression constructs requires a time investment and some molecular biology skills. To improve efficiency and accelerate the construction process, we developed a method to rapidly generate miRNA expression vectors using recombinases instead of more traditional cut-and-paste molecular cloning techniques. In addition to streamlining the construction process, our cloning strategy provides vectors with added versatility. In our system, miRNAs can be constitutively expressed from the U6 promoter, or inducibly expressed by Cre recombinase. We also engineered a built-in mechanism to destroy the vector with Flp recombinase, if desired. Finally, to further simplify the construction process, we developed a software package that automates the prediction and design of optimal miRNA sequences using our system.
We designed and tested a modular system to rapidly clone miRNA expression cassettes. Our strategy reduces the hands-on time required to successfully generate effective constructs, and can be implemented in labs with minimal molecular cloning expertise. This versatile system provides options that permit constitutive or inducible miRNA expression, depending upon the needs of the end user. As such, it has utility for basic or translational applications.
[show abstract][hide abstract] ABSTRACT: Muscular dystrophies, and other diseases of muscle, arise from recessive and dominant gene mutations. Gene replacement strategies may be beneficial for the former, while gene silencing approaches may provide treatment for the latter. In the last two decades, muscle-directed gene therapies were primarily focused on treating recessive disorders. This disparity at least partly arose because feasible mechanisms to silence dominant disease genes lagged behind gene replacement strategies. With the discovery of RNA interference (RNAi) and its subsequent development as a promising new gene silencing tool, the landscape has changed. In this study, our objective was to demonstrate proof-of-principle for RNAi therapy of a dominant myopathy in vivo. We tested the potential of adeno-associated viral (AAV)-delivered therapeutic microRNAs, targeting the human Facioscapulohumeral muscular dystrophy (FSHD) region gene 1 (FRG1), to correct myopathic features in mice expressing toxic levels of human FRG1 (FRG1(-high) mice). We found that FRG1 gene silencing improved muscle mass, strength, and histopathological abnormalities associated with muscular dystrophy in FRG1(-high) mice, thereby demonstrating therapeutic promise for treatment of dominantly inherited myopathies using RNAi. This approach potentially applies to as many as 29 different gene mutations responsible for myopathies inherited as dominant disorders.
[show abstract][hide abstract] ABSTRACT: MicroRNAs (miRNAs) have emerged as important modulators of eukaryotic gene expression through a process called RNA interference
(RNAi). Over the last several years, a large amount of work has focused on understanding how miRNAs are expressed and processed
to a biologically functional form. This knowledge has enabled the development of RNAi as a molecular tool for investigating
basic biological questions or as a therapeutic technique. Artificial miRNA shuttle vectors can be engineered to mimic natural
miRNAs and subsequently used to suppress any gene of interest. Here, we describe a simple method to build and functionally
validate artificial miRNA shuttles.
Key wordsRNAi–MicroRNA–miRNA, siRNA–Inhibitory RNA–Gene silencing–Gene therapy
[show abstract][hide abstract] ABSTRACT: Facioscapulohumeral muscular dystrophy (FSHD) is associated with D4Z4 repeat contraction on human chromosome 4q35. This genetic lesion does not result in complete loss or mutation of any gene. Consequently, the pathogenic mechanisms underlying FSHD have been difficult to discern. In leading FSHD pathogenesis models, D4Z4 contractions are proposed to cause epigenetic changes, which ultimately increase expression of genes with myopathic potential. Although no gene has been conclusively linked to FSHD development, recent evidence supports a role for the D4Z4-encoded DUX4 gene in FSHD. In this study, our objective was to test the in vivo myopathic potential of DUX4.
We delivered DUX4 to zebrafish and mouse muscle by transposon-mediated transgenesis and adeno-associated viral vectors, respectively.
Overexpression of DUX4, which encodes a transcription factor, caused abnormalities associated with muscular dystrophy in zebrafish and mice. This toxicity required DNA binding, because a DUX4 DNA binding domain mutant produced no abnormalities. Importantly, we found the myopathic effects of DUX4 were p53 dependent, as p53 inhibition mitigated DUX4 toxicity in vitro, and muscles from p53 null mice were resistant to DUX4-induced damage.
Our work demonstrates the myopathic potential of DUX4 in animal muscle. Considering previous studies showed DUX4 was elevated in FSHD patient muscles, our data support the hypothesis that DUX4 overexpression contributes to FSHD development. Moreover, we provide a p53-dependent mechanism for DUX4 toxicity that is consistent with previous studies showing p53 pathway activation in FSHD muscles. Our work justifies further investigation of DUX4 and the p53 pathway in FSHD pathogenesis.
Annals of Neurology 03/2011; 69(3):540-52. · 11.19 Impact Factor
[show abstract][hide abstract] ABSTRACT: Protein phosphatase 2A (PP2A) is one of the most abundantly expressed serine/threonine protein phosphatases. A large body of evidence suggests that PP2A is a tumor suppressor and plays critical roles in regulating apoptosis. PP2A is a heterotrimeric protein complex. Its substrate specificity, localization, and activity are regulated by regulatory subunits of PP2A. A recent study has demonstrated that single nucleotide polymorphism in B56ε (PPP2R5E), a B56 family regulatory subunit of PP2A, is associated with human soft tissue sarcoma. This raises the possibility that B56ε is involved in tumorigenesis and plays important roles in regulating apoptosis. However, this hypothesis has not been tested experimentally. Our previous studies revealed that B56ε regulates a number of developmental signaling pathways during early embryonic patterning. Here we report novel functions of B56ε in regulating apoptosis. We provide evidence that B56ε has both anti- and pro-apoptotic functions. B56ε suppresses p53-independent apoptosis during neural development, but triggers p53-dependent apoptosis. Mechanistically, B56ε regulates the p53-dependent apoptotic pathway solely through controlling the stability of p53 protein. In addition to its function in regulating apoptosis, we show that B56ε undergoes proteolytic cleavage. The cleavage of B56ε is mediated by caspase-3 and occurs on the carboxyl side of an evolutionarily conserved N-terminal "DKXD" motif. These results demonstrate that B56ε, a substrate of caspase-3, is an essential regulator of apoptosis. So far, we have identified an alternative translation isoform and a caspase cleavage product of B56ε. The significance of post-transcriptional regulation of B56ε is discussed.
Journal of Biological Chemistry 11/2010; 285(45):34493-502. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Protein phosphatase 2A (PP2A) is one of the most abundantly expressed serine/threonine protein phosphatases. A large body of evidence suggests that PP2A is a tumor suppressor and plays critical roles in regulating apoptosis. PP2A is a heterotrimeric protein complex. Its substrate specificity, localization, and activity are regulated by regulatory subunits
of PP2A. A recent study has demonstrated that single nucleotide polymorphism in B56ϵ (PPP2R5E), a B56 family regulatory subunit of PP2A, is associated with human soft tissue sarcoma. This raises the possibility that B56ϵ is involved in tumorigenesis and plays important roles in regulating apoptosis. However, this hypothesis has not been tested
experimentally. Our previous studies revealed that B56ϵ regulates a number of developmental signaling pathways during early embryonic patterning. Here we report novel functions
of B56ϵ in regulating apoptosis. We provide evidence that B56ϵ has both anti- and pro-apoptotic functions. B56ϵ suppresses p53-independent apoptosis during neural development, but triggers p53-dependent apoptosis. Mechanistically, B56ϵ regulates the p53-dependent apoptotic pathway solely through controlling the stability of p53 protein. In addition to its function in regulating apoptosis, we show that B56ϵ undergoes proteolytic cleavage. The cleavage of B56ϵ is mediated by caspase-3 and occurs on the carboxyl side of an evolutionarily conserved N-terminal “DKXD” motif. These results demonstrate that B56ϵ, a substrate of caspase-3, is an essential regulator of apoptosis. So far, we have identified an alternative translation isoform and a caspase cleavage
product of B56ϵ. The significance of post-transcriptional regulation of B56ϵ is discussed.
Journal of Biological Chemistry 11/2010; 285(45):34493-34502. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Huntington disease is an incurable, dominant neurodegenerative disorder caused by polyglutamine repeat expansion in the huntingtin protein. Reducing mutant huntingtin expression may offer a treatment for Huntington disease. RNA interference has emerged as a powerful method to silence dominant disease genes. As such, it is being developed as a prospective Huntington disease therapy. Here I discuss the current progress and important remaining challenges of RNA interference therapy for Huntington disease.
Archives of neurology 09/2009; 66(8):933-8. · 6.31 Impact Factor
[show abstract][hide abstract] ABSTRACT: The transcription factor REST silences neuronal gene expression in non-neuronal cells. In neurons, the protein is sequestered in the cytoplasm in part through binding to huntingtin. Polyglutamine expansions in huntingtin, which causes Huntington's disease (HD), abrogates REST-huntingtin binding. Consequently, REST translocates to the nucleus, occupies RE1 repressor sequences and decreases neuronal gene expression. In this work, we found that levels of several microRNAs (miRNAs) with upstream RE1 sites are decreased in HD patient cortices relative to healthy controls. Interestingly, one of these, the bifunctional brain enriched miR-9/miR-9*, targets two components of the REST complex: miR-9 targets REST and miR-9* targets CoREST. These data provide evidence for a double negative feedback loop between the REST silencing complex and the miRNAs it regulates.
Journal of Neuroscience 01/2009; 28(53):14341-6. · 6.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: Huntington's disease (HD) is a fatal, dominant neurodegenerative disease caused by a polyglutamine repeat expansion in exon 1 of the HD gene, which encodes the huntingtin protein. We and others have shown that RNAi is a candidate therapy for HD because expression of inhibitory RNAs targeting mutant human HD transgenes improved neuropathology and behavioral deficits in HD mouse models. Here, we developed shRNAs targeting conserved sequences in human HD and mouse HD homolog (HDh) mRNAs to initiate preclinical testing in a knockin mouse model of HD. We screened 35 shRNAs in vitro and subsequently narrowed our focus to three candidates for in vivo testing. Unexpectedly, two active shRNAs induced significant neurotoxicity in mouse striatum, although HDh mRNA expression was reduced to similar levels by all three. Additionally, a control shRNA containing mismatches also induced toxicity, although it did not reduce HDh mRNA expression. Interestingly, the toxic shRNAs generated higher antisense RNA levels, compared with the nontoxic shRNA. These results demonstrate that the robust levels of antisense RNAs emerging from shRNA expression systems can be problematic in the mouse brain. Importantly, when sequences that were toxic in the context of shRNAs were placed into artificial microRNA (miRNA) expression systems, molecular and neuropathological readouts of neurotoxicity were significantly attenuated without compromising mouse HDh silencing efficacy. Thus, miRNA-based approaches may provide more appropriate biological tools for expressing inhibitory RNAs in the brain, the implications of which are crucial to the development of RNAi for both basic biological and therapeutic applications.
Proceedings of the National Academy of Sciences 05/2008; 105(15):5868-73. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: The ability to manipulate RNAi in cultured mammalian cells has provided scientists with a very powerful tool to influence gene expression. Neurons represent a cell type that initially displayed resistance to transduction by siRNAs or shRNA, when attempting to silence expression of endogenous genes. However, the development of lentiviral systems with that goal has facilitated the exogenous manipulation of RNAi in these postmitotic cells. Lentiviral-mediated RNAi experiments in cultured mammalian neurons can be designed to address a wide variety of biological questions or to test potential therapeutic hairpins before moving to treatment trials in vivo. We provide a practical approach to accomplish siRNA-mediated silencing of the disease-linked protein torsinA in primary neuronal cultures through the generation of lentiviral vectors expressing shRNAs.
Methods in molecular biology (Clifton, N.J.) 02/2008; 442:95-112.
[show abstract][hide abstract] ABSTRACT: Clathrin-coated vesicles (CCVs) are responsible for the endocytosis of multiple cargo, including synaptic vesicle membranes. We now describe a new CCV protein, termed connecdenn, that contains an N-terminal DENN (differentially expressed in neoplastic versus normal cells) domain, a poorly characterized protein module found in multiple proteins of unrelated function and a C-terminal peptide motif domain harboring three distinct motifs for binding the alpha-ear of the clathrin adaptor protein 2 (AP-2). Connecdenn coimmunoprecipitates and partially colocalizes with AP-2, and nuclear magnetic resonance and peptide competition studies reveal that all three alpha-ear-binding motifs contribute to AP-2 interactions. In addition, connecdenn contains multiple Src homology 3 (SH3) domain-binding motifs and coimmunoprecipitates with the synaptic SH3 domain proteins intersectin and endophilin A1. Interestingly, connecdenn is enriched on neuronal CCVs and is present in the presynaptic compartment of neurons. Moreover, connecdenn has a uniquely stable association with CCV membranes because it resists extraction with Tris and high-salt buffers, unlike most other CCV proteins, but it is not detected on purified synaptic vesicles. Together, these observations suggest that connecdenn functions on the endocytic limb of the synaptic vesicle cycle. Accordingly, disruption of connecdenn interactions with its binding partners through overexpression of the C-terminal peptide motif domain or knock down of connecdenn through lentiviral delivery of small hairpin RNA both lead to defects in synaptic vesicle endocytosis in cultured hippocampal neurons. Thus, we identified connecdenn as a component of the endocytic machinery functioning in synaptic vesicle endocytosis, providing the first evidence of a role for a DENN domain-containing protein in endocytosis.
Journal of Neuroscience 01/2007; 26(51):13202-12. · 6.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: RNA interference (RNAi) occurs naturally in plant and animal cells as a means for modulating gene expression. This process has been experimentally manipulated to achieve targeted gene silencing in cells, tissues, and animals, using a variety of vector systems. Here, we tested the hypothesis that vectors based on feline immunodeficiency virus (FIV) could be used for coexpression of reporter constructs and RNAi expression cassettes. We found, unexpectedly, in our initial constructs that placement of RNAi expression cassettes downstream from a polymerase II (pol II)-expressed reporter gene inhibited reporter expression but not vector titer. Through a series of intermediate vector constructs, we found that placement of the RNAi expression cassette relative to the Rev response element and the pol II expression cassette was critical for efficient RNAi and reporter gene expression. These results suggested that steric factors, including RNA structure and recruitment of competing transcriptional machinery, may affect gene expression from FIV vectors. In a second series of studies, we show that target sequence silencing can be achieved in cells transduced by FIV vectors coexpressing reporter genes and 3' untranslated region resident microRNAs. The optimized FIV-based RNAi expression vectors will find broad use given the extensive tropism of pseudotyped FIV vectors for many cell types in vitro and in vivo.
Journal of Virology 11/2006; 80(19):9371-80. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Molecular Therapy (2006) 13, S274|[ndash]|S275; doi: 10.1016/j.ymthe.2006.08.790
711. Allele-Specific Silencing of Mutant Huntingtin for Huntington's Disease Therapy
Alex Mas-Monteys1, Scott Q. Harper1, Brian L. Gilmore1, Patrick D. Staber1, Chris Schaffer2, Barry Polisky2, Chandra Vargeese2 and Beverly L. Davidson11Internal Medicine, University of Iowa, Iowa City, IA2Sirna Therapeutics, Boulder, CO
[show abstract][hide abstract] ABSTRACT: Molecular Therapy (2006) 13, S141|[ndash]|S141; doi: 10.1016/j.ymthe.2006.08.430
371. Optimization of Feline Immunodeficiency Viral Vectors for RNA Interference
Scott Q. Harper1, Patrick D. Staber1, Christine Rowley1, Sarah Fineberg1, Dalyz Ochoa1, Colleen Stein1 and Beverly L. Davidson11Internal Medicine, University of Iowa, Iowa City, IA
[show abstract][hide abstract] ABSTRACT: Huntington's disease (HD) is a dominant neurodegenerative disorder caused by an expansion in the polyglutamine (polyQ) tract of the huntingtin (htt) protein. PolyQ expansion in htt induces cortical and striatal neuron cell loss, and the formation of httcontaining aggregates within brain cells. HD patients have progressive psychiatric, cognitive and motor dysfunction and premature death. Early work in mouse models demonstrated that reduction of mutant protein after the onset of disease phenotypes could improve motor dysfunction and reduce htt-aggregate burden. Thus, reduction of mutant htt in patient brain may improve the disease.In recent work we showed that reduction of mutant htt in a mouse model of HD, using a viral vector expressing short hairpin RNAs (shRNAs), protected the animal from the onset of behavioral and neuropathological hallmarks of the disease. Here, we tested if delivery of synthetic siRNAs directly to the brain by nonviral methods could be similarly effective. This approach has many advantages, most important of which is that if problems arise, the therapy can be stopped. Major hurdles for nonviral delivery, however, include formulating the inhibitory nucleic acids for delivery to brain, and stabilizing the siRNAs for improved longevity in vivo. We tested chemically modified siRNAs specific for human and mouse huntingtin, encapsulated in various lipid nanoparticles (LNP). The prepared LNP formulations were screened for their ability to silence full-length htt in vitro, followed by testing in vivo. Using Alzet® osmotic pumps, siRNAs encapsulated in LNPs were infused into the lateral ventrical or striatum for 7 or 14 days, respectively, at concentrations ranging from 0.1 to 1 μg/μl (total dose ranging from 8.4 to 84 μg). We noted an impressive 80% reduction in htt mRNA levels by QPCR compared to scrambled control sequences, or naïve brain. This level of reduction is similar to that achieved with adenoassociated virus vector expressed shRNAs, and was accomplished at doses 18-fold lower than those previously reported as required for target gene reduction in adult rodent brain using other formulations. In summary, we show for the first time, reduction of target htt in vivo in adult mouse striatum, a major site of neurodegeneration in HD. Ongoing studies will determine the safety and feasibility of the synthetic siRNA approach for HD therapy.Collaborative effort between Sirna Therapeutics and the University of Iowa.