Background: Synthetic zinc finger (ZF) proteins can be targeted to desired DNA sequences and are useful tools for gene\ntherapy. We recently developed a ZF transcription repressor (ZF-KOX1) able to bind to expanded DNA CAG-repeats in the\nhuntingtin (HTT) gene, which are found in Huntingtonâ��s disease (HD). This ZF acutely repressed mutant HTT expression in\na mouse model of HD and delayed neurological symptoms (clasping) for up to 3 weeks. In the present work, we sought\nto develop a long-term single-injection gene therapy approach in the brain.\nMethod: Since non-self proteins can elicit immune and inflammatory responses, we designed a host-matched analogue\nof ZF-KOX1 (called mZF-KRAB), to treat mice more safely in combination with rAAV vector delivery. We also tested a\nneuron-specific enolase promoter (pNSE), which has been reported as enabling long-term transgene expression, to see\nwhether HTT repression could be observed for up to 6 months after AAV injection in the brain.\nResults: After rAAV vector delivery, we found that non-self proteins induce significant inflammatory responses in the\nbrain, in agreement with previous studies. Specifically, microglial cells were activated at 4 and 6 weeks after treatment\nwith non-host-matched ZF-KOX1 or GFP, respectively, and this was accompanied by a moderate neuronal loss. In\ncontrast, the host-matched mZF-KRAB did not provoke these effects. Nonetheless, we found that using a pCAG promoter\n(CMV early enhancer element and the chicken �²-actin promoter) led to a strong reduction in ZF expression by 6 weeks\nafter injection. We therefore tested a new non-viral promoter to see whether the host-adapted ZF expression could be\nsustained for a longer time. Vectorising mZF-KRAB with a promoter-enhancer from neuron-specific enolase (Eno2, rat)\nresulted in up to 77 % repression of mutant HTT in whole brain, 3 weeks after bilateral intraventricular injection of 1010\nvirions. Importantly, repressions of 48 % and 23 % were still detected after 12 and 24 weeks, respectively, indicating that\nlonger term effects are possible.\nConclusion: Host-adapted ZF-AAV constructs displayed a reduced toxicity and a non-viral pNSE promoter improved\nlong-term ZF protein expression and target gene repression. The optimized constructs presented here have potential for\ntreating HD.
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