Huntington disease (HD) is a fatal, neurodegenerative genetic disorder with aggregation of mutant Huntingtin protein (mutHTT) in the brain as a key pathological mechanism. There are currently no... Show moreHuntington disease (HD) is a fatal, neurodegenerative genetic disorder with aggregation of mutant Huntingtin protein (mutHTT) in the brain as a key pathological mechanism. There are currently no disease modifying therapies for HD; however, HTT-lowering therapies hold promise. Recombinant adeno-associated virus serotype 5 expressing a microRNA that targets HTT mRNA (AAV5-miHTT) is in development for the treatment of HD with promising results in rodent and minipig HD models. To support a clinical trial, toxicity studies were performed in non-human primates (NHP, Macaca fascicularis) and Sprague-Dawley rats to evaluate the safety of AAV5-miHTT, the neurosurgical administration procedure, vector delivery and expression of the miHTT transgene during a 6-month observation period. For accurate delivery of AAV5-miHTT to the striatum, real-time magnetic resonance imaging (MRI) with convection-enhanced delivery (CED) was used in NHP. Catheters were successfully implanted in 24 NHP, without neurological symptoms, and resulted in tracer signal in the target areas. Widespread vector DNA and miHTT transgene distribution in the brain was found, particularly in areas associated with HD pathology. Intrastriatal administration of AAV5-miHTT was well tolerated with no clinically relevant changes in either species. These studies demonstrate the excellent safety profile of AAV5-miHTT, the reproducibility and tolerability of intrastriatal administration, and the delivery of AAV5-miHTT to the brain, which support the transition of AAV5-miHTT into clinical studies. Show less
Huntington disease (HD) is a fatal neurodegenerative genetic disorder, thought to reflect a toxic gain of function in huntingtin (Htt) protein. Adeno-associated viral vector serotype 5 (AAV5)-... Show moreHuntington disease (HD) is a fatal neurodegenerative genetic disorder, thought to reflect a toxic gain of function in huntingtin (Htt) protein. Adeno-associated viral vector serotype 5 (AAV5)- microRNA targeting huntingtin (miHTT) is a HD gene-therapy candidate that efficiently lowers HTT using RNAi. This study analyzed the efficacy and potential for offtarget effects with AAV5-miHTT in neuronal and astrocyte cell cultures differentiated from induced pluripotent stem cells (iPSCs) from two individuals with HD (HD71 and HD180). One-time AAV5-miHTT treatment significantly reduced human HTT mRNA by 57% and Htt protein by 68% in neurons. Small RNA sequencing showed that mature miHTT was processed correctly without off-target passenger strand. No cellular microRNAs were dysregulated, indicating that endogenous RNAi machinery was unaffected by miHTT overexpression. qPCR validation of in silico-predicted off-target transcripts, next-generation sequencing, and pathway analysis confirmed absence of dysregulated genes due to sequence homology or seed-sequence activity of miHTT. Minor effects on gene expression were observed in both AAV5-miHTT and AAV5-GFP-treated samples, suggesting that they were due to viral transduction rather than miHTT. This study confirms the efficacy of AAV5-miHTT in HD patient iPSC-derived neuronal cultures and lack of off-target effects in gene expression and regulation in neuronal cells and astrocytes. Show less
Spinocerebellar ataxia type 3 (SCA3) or Machado-Joseph disease (MJD) is a progressiveneurodegenerative disorder caused by a CAG expansion in the ATXN3 gene. The expanded CAGrepeat is translated... Show moreSpinocerebellar ataxia type 3 (SCA3) or Machado-Joseph disease (MJD) is a progressiveneurodegenerative disorder caused by a CAG expansion in the ATXN3 gene. The expanded CAGrepeat is translated into a prolonged polyglutamine repeat in the ataxin-3 protein and accumulateswithin inclusions, acquiring toxic properties, which results in degeneration of the cerebellum and brainstem.In the current study, a non-allele specific ATXN3 silencing approach was investigated using artificialmicroRNAs engineered to target various regions of the ATXN3 gene (miATXN3). The miATXN3candidates were screened in vitro based on their silencing efficacy on a luciferase reporter co-expressing ATXN3. The three best miATXN3 candidates were further tested for target engagement andpotential off-target activity in induced-pluripotent stem cells (iPSC) differentiated into frontal brain-like neurons and in a SCA3 knock-in mouse model. Besides a strong reduction of ATXN3 mRNA andprotein, small RNA sequencing revealed efficient guide strand processing without passenger strandsbeing produced. We used different methods to predict alteration of off-target genes upon AAV5-miATXN3 treatment and found no evidence for unwanted effects. Furthermore, we demonstrated in alarge animal model, the minipig, that intrathecal delivery of AAV5 can transduce the main areasaffected in SCA3 patients. These results proved a strong basis to move forward to investigatedistribution, efficacy and safety of AAV5-miATXN3 in large animals. Show less
