414. Non-Allele Specific RNA Interference in the CAG140 Knock-In Mouse Model of Huntington's Disease (2006)

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. 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.