Search

The article presents the proceedings of the ASGCT Standardized Pathways Conference that was held in Silver Spring, Maryland, on February 20, 2014. It is mentioned that the premise of the conference was that the preclinical data and clinical outcomes from development and conduct of gene therapy clinical trials can be used to establish standardized pathways through the preclinical regulatory process.

Huntington's disease (HD) is a fatal neurodegenerative disease caused by mutant huntingtin (htt) protein, and there are currently no effective treatments. Recently, we and others demonstrated that silencing mutant htt via RNA interference (RNAi) provides therapeutic benefit in HD mice. We have since found that silencing wild-type htt in adult mouse striatum is tolerated for at least 4 months. However, given the role of htt in various cellular processes, it remains unknown whether nonallele-specific silencing of both wild-type and mutant htt is a viable therapeutic strategy for HD. Here, we tested whether cosilencing wild-type and mutant htt provides therapeutic benefit and is tolerable in HD mice. After treatment, HD mice showed significant reductions in wild-type and mutant htt, and demonstrated improved motor coordination and survival. We performed transcriptional profiling to evaluate the effects of reducing wild-type htt in adult mouse striatum. We identified gene expression changes that are concordant with previously described roles for htt in various cellular processes. Also, several abnormally expressed transcripts associated with early-stage HD were differentially expressed in our studies, but intriguingly, those involved in neuronal function changed in opposing directions. Together, these encouraging and surprising findings support further testing of nonallele-specific RNAi therapeutics for HD.Molecular Therapy (2009) 17 6, 1053–1063 doi:10.1038/mt.2009.17 [ABSTRACT FROM AUTHOR]

The successful application of gene therapy for the treatment of genetic diseases such as Fabry is reliant on the development of vectors that are safe and that facilitate sustained expression of therapeutic levels of the transgene product. Here, we report that intravenous administration of a recombinant AAV2 vector encoding human α-galactosidase A under the transcriptional control of a liver-restricted enhancer/promoter (AAV2/DC190-αgal) generated significantly higher levels of expression in BALB/c and Fabry mice than could be realized using the ubiquitous CMV promoter (AAV2/CMVHI-αgal). Moreover, AAV2/DC190-αgal-mediated hepatic expression of α-galactosidase A was sustained for 12 months in BALB/c mice and was associated with a significantly reduced immune response to the expressed enzyme. Subsequent challenge of the AAV2/DC190-αgal-treated animals with recombinant human α-galactosidase A at 6 months failed to elicit the production of anti-α-galactosidase A antibodies, suggesting the induction of immune tolerance in these animals. The levels of expression attained with AAV2/DC190-αgal in the Fabry mice were sufficient to reduce the abnormal accumulation of globotriaosylceramide in the liver, spleen, and heart to basal levels and in the kidney by approximately 40% at 8 weeks. Together, these results demonstrate that AAV2-mediated gene transfer that limits the expression of α-galactosidase A to the liver may be a viable strategy for treating Fabry disease. [Copyright &y& Elsevier]

We and others have demonstrated that RNA interference (RNAi) to silence human huntingtin transgenes in mouse models of Huntington's disease (HD) prevented inclusion formation and attenuated behavioral deficits (Harper et al., 2005 and Rodriguez- Lebron et al., 2005). In these previous studies, silencing was specific to the disease allele in transgenic mouse models. While allele specific silencing for HD would be ideal, only one known polymorphism for HD has been identified and is prevalent in only 40% of disease alleles in a specific population of patients. Therefore, the feasibility of non-allele specific silencing should be tested, as it would be universally applicable to all HD patients. To that end, we are testing RNAi in the CAG140 knock-in (KI) mouse model. In this study, we examined the anatomical, biochemical and behavioral effects of three hairpin constructs expressed from adeno-associated viral vectors (AAV), to the striatum. Each AAV-shHD construct was pre-screened in wildtype mice to evaluate vector distribution, cell type transduced, and the knock down of huntingtin mRNA. Additionally, we examined the potential toxicity associated with targeting both huntingtin alleles. Wildtype mice received unilateral injections (5μl) into the right striatum of AAV2/1shHD2.4-hrGFP) (n=5), AAV2/1shHD8.2- hrGFP (n=5), AAV2/1shHD30.1-hrGFP (n=5) or AAV1-hrGFP (n=5). All mice were sacrificed two weeks post-injection. For each viral vector construct, numerous GFP positive cells were observed throughout the rostral/caudal extent of the striatum, as well as in the globus pallidus and substantia nigra pars reticulata, regions of the basal ganglia which receive striatal projection. In all cases, GFP positive cells co-localized with the neuronal marker, NeuN, but not with astrocytic or oligodendrocytic markers, with no overt signs of toxicity. QPCR analyses demonstrated decreased huntingtin mRNA expression 2 wks after injection of AAVs expressing shHD2.4-GFP (44%), shHD8.2-GFP (36%) or shHD30.1-GFP (62%) compared to the un-injected hemisphere. No decrease in huntingtin mRNA was observed in mice injected with AAV-GFP. Following pre- screening in wildtype mice, hairpin constructs (with mismatch controls) were injected bilaterally into the CAG140 KI striata at week 5 of life (n=15 for each group). CAG140 KI mice exhibit HD behaviors by 5 months of age, but we assessed all groups every two weeks for changes in body weight, hind limb clasping and behavior on the accelerating rotarod, a measure of motor coordination, to assess a possible negative impact of reducing expression of both mutant and normal alleles. Importantly, at week 13 of life (to date) all mice show normal weight gain and rotarod behavior, and have not exhibited hind limb clasping. Thus, introduction of RNAi that reduces both huntingtin alleles in a KI model of HD is well tolerated acutely. Long term studies are underway to assess further any negative impact of non-allele specific silencing, and test if RNA inhibition of mutant huntingtin is efficacious.Molecular Therapy (2006) 13, S159–S159; doi: 10.1016/j.ymthe.2006.08.478 [ABSTRACT FROM AUTHOR]