AAV5-miHTT gene therapy demonstrates suppression of mutant huntingtin aggregation and neuronal dysfunction in a rat model of Huntington’s disease

Abstract

Huntington’s disease (HD) is a fatal progressive neurodegenerative disorder caused by a mutation in the huntingtin (HTT) gene. To date, there is no treatment to halt or reverse the course of HD. Lowering of either total or only the mutant HTT expression is expected to have therapeutic benefit. This can be achieved by engineered micro (mi)RNAs targeting HTT transcripts and delivered by an adeno-associated viral (AAV) vector. We have previously showed a miHTT construct to induce total HTT knock-down in Hu128/21 HD mice, while miSNP50T and miSNP67T constructs induced allele-selective HTT knock-down in vitro. In the current preclinical study, the mechanistic efficacy and gene specificity of these selected constructs delivered by an AAV serotype 5 (AAV5) vector was addressed using an acute HD rat model. Our data demonstrated suppression of mutant HTT messenger RNA, which almost completely prevented mutant HTT aggregate formation, and ultimately resulted in suppression of DARPP-32-associated neuronal dysfunction. The AAV5-miHTT construct was found to be the most efficient, although AAV5-miSNP50T demonstrated the anticipated mutant HTT allele selectivity and no passenger strand expression. Ultimately, AAV5-delivered-miRNA-mediated HTT lowering did not cause activation of microglia or astrocytes suggesting no immune response to the AAV5 vector or therapeutic precursor sequences. These preclinical results suggest that using gene therapy to knock-down HTT may provide important therapeutic benefit for HD patients and raised no safety concerns, which supports our ongoing efforts for the development of an RNA interference-based gene therapy product for HD.

Gene Therapy for Neurodegenerative Diseases

“…In addition, nonhuman primate studies have demonstrated that reductions in normal Htt of up to 45% may be well tolerated [60,61]. An engineered microRNA targeting human huntingtin, delivered via AAV5 with a transgene encoding an engineered miRNA against HTT mRNA (AAV5-miHTT), has been developed [62][63][64]. Widespread distribution of the transgene has been observed, as would be expected from the properties of AAV5 [5], and as would be desired for this disorder.…”

Gene therapy conversion of striatal astrocytes into GABAergic neurons in mouse models of Huntington’s disease

“…Many of the symptoms observed in HD patients are highly related to the loss of MSNs in the striatum 6 and can be mimicked in rodent 7 , pig 8 , and non-human primate 9 models. Previous studies have achieved some success in HD animal models through cell transplantation [10][11][12] or using antisense oligonucleotides 13,14 and CRISPR/Cas9 gene editing technology 15 to reduce the mHtt level. Tetrabenazine has been used to treat chorea in HD and other movement disorders but with significant side effects 16 .…”

Translation of MicroRNA-Based Huntingtin-Lowering Therapies from Preclinical Studies to the Clinic

“…70 Several studies reported inhibition of mutant HTT aggregate formation, and this reduced neuronal dysfunction in HD rats. 38,71,72 In a nonhuman primate study, AAV1-miRNA targeting rhesus HTT was MRI-guided stereotactically injected in the right and left putamen, which induced 45% HTT reduction in the mid and caudal putamen, without inducing neuronal degeneration, astrogliosis, or an immune response until termination of the study at 6 weeks. 37 Similarly, injections of AAV2-shRNA targeting rhesus HTT induced welltolerated 30% HTT reduction in the injected putamen measured at 6 months post injections.…”

“…15 Those models are suitable for evaluating suppression of neurodegeneration, as indeed was demonstrated for different miRNAs and shRNAs that lower HTT. 71,72 The second HD rat model is transgenic, with a human HTT cDNA fragment containing 51 CAGs, whereas the expression is under control of the rat Htt promoter. 92 These rats manifest with adult-onset neurodegeneration and motor, cognitive, and behavioral deficits.…”

“…et al, unpublished data). 39,64,72,104,144 Measurements of On-Target Lowering Efficacy miRNA-based lowering strategies are designed to lower HTT mRNA levels and thereby reduce the overall mutant HTT protein. Already in early preclinical development, it is essential to establish the on-target activity of therapeutics by measuring their effects on HTT mRNA and protein levels.…”