Background: 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 (ZFKOX1)
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.
Method: Since 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.
Results: After 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 hostmatched
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.
Conclusion: Host-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.