Artificial miRNAs mitigate shRNA-mediated toxicity in the brain: implications for the therapeutic development of RNAi.

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.

Long-term suppression of experimental arthritis following intramuscular administration of a pseudotyped AAV2/1-TNFR:Fc Vector.

We previously reported that administration of an adeno-associated virus 2 (AAV2) vector encoding a rat tumor necrosis factor (TNF) receptor-immunoglobulin Fc (TNFR:Fc) fusion gene to rats with streptococcal cell wall-induced arthritis resulted in suppression of joint inflammation and cartilage and bone destruction, as well as expression of joint proinflammatory cytokines. In this study, we used an alternate rat model of arthritis to compare the serum levels and duration of TNFR:Fc protein expression following intramuscular administration of pseudotyped AAV-TNFR:Fc vectors based on serotypes 1, 2, and 5. All three pseudotyped AAV-TNFR:Fc vectors led to sustained expression of serum TNFR:Fc protein for at least one year. Serum TNFR:Fc protein levels in rats administered intramuscularly with AAV2/1-TNFR:Fc vector were up to 100- and 10-fold higher than in rats administered the AAV2-TNFR:Fc or AAV2/5-TNFR:Fc vectors, respectively. A single intramuscular administration of AAV2/1-TNFR:Fc vector at vector doses ranging from 10(10) to 10(12) DNase-resistant particles (DRP) per animal, resulted in complete and long-term suppression of recurrent joint inflammation for at least 150 days. Our results establish a proof of concept for administration of an AAV2/1-TNFR:Fc vector to the muscle to achieve long-term, sustained and therapeutically relevant levels of TNFR:Fc protein to treat chronic systemic inflammatory joint diseases.

Persistent liver expression of murine apoA-l using vectors based on adeno-associated viral vectors serotypes 5 and 1.

Plasma levels of high-density lipoprotein-cholesterol (HDL-C) and apolipoprotein A-l (apoA-l) are inversely related to risk for coronary heart disease. Overexpression of apoA-l inhibits atherosclerosis in animal models. A method of stably expressing apoA-l using somatic gene transfer would be of interest. Pseudotyped adeno-associated virus (AAV) vectors comprised of inverted terminal repeats from AAV serotype 2 have been used for liver-directed gene transfers. We hypothesized that liver-directed gene transfer of apoA-l using vectors based on AAV serotypes 1 and 5 would result in higher-level, prolonged expression of apoA-l and increased HDL-C. To test this hypothesis we injected apoA-l-/- mice via the tail vein with either AAV2, AAV1 or AAV5 vectors encoding the murine apoA-l cDNA driven by the liver-specific thyroxine binding globulin promoter. Plasma levels of murine apoA-l and HDL-C were highest in mice injected with the AAV1-based vector and lowest in mice injected with the AAV2-based vector. Expression of apoA-l was stable up to 1 year after vector injection. These results indicate that AAV5 and AAV1 are more effective vectors for achieving higher levels of stable transgene expression of apoA-l after liver-directed gene transfer than AAV2. Furthermore, AAV1-based vectors generate higher apoA-l levels than AAV5-based vectors. It is possible that the levels of expression achieved using these vectors will be therapeutic in preventing atherosclerosis.

Secretion of a TNFR:Fc fusion protein following pulmonary administration of pseudotyped adeno-associated virus vectors.

This study evaluated and compared delivery of the tumor necrosis factor alpha receptor (TNFR)-immunoglobulin G1 (IgG1) Fc fusion (TNFR:Fc) gene to the lung by single and repeat administrations of multiple pseudotyped adeno-associated virus (AAV) vectors as a means for achieving systemic distribution of the soluble TNFR:Fc protein. A single endotracheal administration of AAV[2/5]cytomegalovirus (CMV)-TNFR:Fc vector (containing the AAV2 inverted terminal repeats and AAV5 capsid) to the rat lung resulted in long-term, high levels of serum TNFR:Fc protein that gradually declined over a period of 8 months. Endotracheal delivery of AAV[2/1]CMV-TNFR:Fc resulted in serum TNFR:Fc protein levels that were detectable for at least 4 months but were 10-fold lower than that of the AAV[2/5] vector. In contrast, secretion of the TNFR:Fc protein following pulmonary delivery of AAV[2/2]CMV-TNFR:Fc vector was very inefficient, and the protein was detected in the blood only when an airway epithelial cell-specific promoter, CC10, was substituted for the CMV enhancer/promoter to control transgene expression. In the context of AAV[2/5], the CC10 promoter was as efficient as CMV enhancer/promoter in generating similar levels of systemic TNFR:Fc protein, suggesting that this protein is secreted primarily from the airway epithelium. In mice, comparable long-term secretion of TNFR:Fc protein was demonstrated after AAV[2/2] and AAV[2/5] delivery, although the kinetics of transduction appeared to be different. All pseudotyped AAV vectors elicited serum anti-AAV capsid-neutralizing antibody responses, but these did not prevent lung transduction and efficient secretion of TNFR:Fc protein to the circulation following readministration with AAV[2/5]. These results highlight the potential utility of AAV vectors containing serotype 5 capsid to deliver and redeliver genes of secreted proteins to the lung to achieve long-term systemic protein expression.

Recombinant adeno-associated virus preferentially transduces human, compared to mouse, synovium: implications for arthritis therapy.

Despite a number of published reports, including from our own laboratory, suggesting that adeno-associated virus (AAV) transduces mouse synovium, a careful analysis demonstrated transduction predominantly of the subsynovial muscle tissue, while the synovial lining is poorly transduced. To investigate the potential of AAV to transduce human synovium, three human rheumatoid arthritis (RA) and two murine collagen-induced arthritis (CIA) synovial cell lines were infected with recombinant AAV (rAAV) vectors encoding either mouse IL-10 or IL-4. Low-level transgene expression was observed. However, either Gamma-irradiation or the addition of a low-titer E1-, E3-deleted recombinant adenovirus resulted in up to a 100-fold increase in transgene product in the human, but not the mouse, cell lines. RA synovial tissues implanted subcutaneously in severe combined immunodeficiency (SCID) mice, which were subsequently infected with rAAV, showed marked increases in transgene expression when co-infected with adenovirus. To our knowledge, this is the first study to show that intact human synovial tissues can be transduced by rAAV, and it suggests that murine arthritis may not be an optimal model to study rAAV as a gene transfer vector. Further studies to elucidate the mechanisms limiting gene transduction in human synovium may allow optimization of this vector for the treatment of arthritis.

Intraarticular gene transfer of TNFR:Fc suppresses experimental arthritis with reduced systemic distribution of the gene product.

Tumor necrosis factor alpha (TNFalpha) plays a pivotal role in the pathogenesis of rheumatoid arthritis (RA). Blockage of TNFalpha actions by systemic administration of TNF antagonists has recently been shown to ameliorate joint symptoms in RA patients. In the present study, a streptococcal cell wall (SCW)-induced rat arthritis model was used to evaluate the effect of different gene transfer routes of a TNF antagonist on the development and severity of arthritis. Successful delivery of a plasmid DNA encoding a rat TNF receptor-immunoglobulin Fc (TNFR:Fc) fusion gene prompted the subsequent administration of a recombinant adeno-associated virus (rAAV) vector encoding the antagonist, either locally (intraarticular) or systemically (intramuscular). The TNFR:Fc gene, delivered by either route, resulted in profound suppression of the arthritis as reflected in decreased inflammatory cell infiltration, pannus formation, cartilage and bone destruction, and mRNA expression of joint proinflammatory cytokines. Increased bioactive serum TNFR levels were detected as a result of rAAV-ratTNFR:Fc administration, concomitant with a decrease in circulating TNFalpha. Administration of the rAAV-ratTNFR:Fc vector to one joint also suppressed arthritis in the contralateral joint. Importantly, intraarticular administration resulted in significantly lower systemic distribution of the gene product. Hence, the use of rAAV as the delivery vector for TNFR:Fc effectively suppressed SCW-induced arthritis and may provide an approach for local delivery of antiarthritic therapy.