Deimmunization for gene therapy: host matching of synthetic zinc finger constructs enables long term mutant Huntingtin repression in mice
BackgroundSynthetic zinc finger (ZF) proteins can be targeted to desired DNA sequences and are useful tools for gene therapy. We recently developed a ZF transcription repressor (ZF KOX1) able to bind to expanded DNA CAG repeats in the huntingtin (HTT) gene, which are found in Huntington's disease (HD) This ZF acutely repressed mutant HTT expression in a mouse model of HD and delayed neurological symptoms (clasping) for up to 3 weeks. In the present work, we sought to develop a long term single injection gene therapy approach in the brain.MethodSince non self proteins can elicit immune and inflammatory responses, we designed a host matched analogue of ZF KOX1 (called mZF KRAB) to treat mice more safely in combination with rAAV vector delivery. We also tested a neuron specific enolase promoter (pNSE) which has been reported as enabling long term transgene expression, to see whether HTT repression could be observed for up to 6 months after AAV injection in the brain.ResultsAfter rAAV vector delivery, we found that non self proteins induce significant inflammatory responses in the brain, in agreement with previous studies. Specifically, microglial cells were activated at 4 and 6 weeks after treatment with non host matched ZF KOX1 or GFP, respectively, and this was accompanied by a moderate neuronal loss. In contrast, the host matched mZF KRAB did not provoke these effects. Nonetheless, we found that using a pCAG promoter (CMV early enhancer element and the chicken b actin promoter) led to a strong reduction in ZF expression by 6 weeks after injection. We therefore tested a new non viral promoter to see whether the host adapted ZF expression could be sustained for a longer time. Vectorising mZF KRAB with a promoter enhancer from neuron specific enolase (Eno2, rat) resulted in up to 77 repression of mutant HTT in whole brain, 3 weeks after bilateral intraventricular injection of 1010 virions. Importantly, repressions of 48 and 23 were still detected after 12 and 24 weeks, respectively, indicating that longer term effects are possible.ConclusionHost adapted ZF AAV constructs displayed a reduced toxicity and a non viral pNSE promoter improved long term ZF protein expression and target gene repression. The optimized constructs presented here have potential for treating HD.
Molecular Neurodegeneration