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.gene therapy ͉ Huntington's disease ͉ RNAi ͉ AAV

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.

SUMMARYPeriodontal disease is a chronic inflammatory condition induced by tooth-associated microbial biofilms that induce a host immune response. Therapeutic control of progressive tissue destruction in high-risk patients is a significant challenge in therapy. Soluble protein delivery of antagonists to tumor necrosis factor alpha (TNF-α) inhibits alveolar bone resorption due to periodontitis. However, protein therapy raises several concerns, such as recurrence of disease activity after treatment cessation and repeated dosing regimens. In this study, we used pseudotyped adeno-associated virus vector based on serotype 1 (AAV2/1) to deliver the TNF receptor-immunoglobulin Fc (TNFR:Fc) fusion gene to rats subjected to experimental Porphyromonas gingivalis (Pg)-lipopolysaccharide (LPS)-mediated bone loss. Animals received Pg-LPS delivered to the gingivae thrice weekly for 8 weeks, vehicle alone, Pg-LPS and intramuscular delivery of pseudotyped AAV2/1-TNFR:Fc vector (1×1011 DNase I-resistant particles) or AAV2/1-TNFR:Fc vector delivered to naïve animals. AAV2/1-TNFR:Fc therapy led to sustained therapeutic levels of serum TNFR protein and protected against Pg-LPS-mediated loss of bone volume and density. Furthermore, AAV2/1-TNFR:Fc administration reduced local levels of multiple pro-inflammatory cytokines and osteoclast-like cells at the periodontal lesions. These findings suggest that delivery of AAV2/1-TNFR:Fc may be a viable approach to modulate periodontal disease progression.

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