Abstract
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 β-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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-016-0128-x) contains supplementary material, which is available to authorized users.
Highlights
Synthetic zinc finger (ZF) proteins can be targeted to desired DNA sequences and are useful tools for gene therapy
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
We showed that intracerebral administration of ZF-KOX1, in recombinant adeno-associated vectors, was able to acutely reduce the load of mutant huntingtin RNA, and HTT protein, in the striata of R6/2 mice
Summary
Synthetic 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. Site-specific nuclease technology is highly scaleable and, with the advent of vectorisable RNAprogrammable nucleases such as CRISPR/Cas lentiviruses [8], a revolution in genome editing is underway. Despite this progress, it is becoming clear that the host immune system is a major barrier to successful long term therapies. Even zinc finger nucleases use bacterial nuclease domains (FokI [9]) and it is unclear how they would be tolerated in vivo
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