Deimmunization for gene therapy: host matching of synthetic zinc finger constructs enables long-term mutant Huntingtin repression in mice.
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. In the present work, we sought to develop a long-term single-injection gene therapy approach in the brain.



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.



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 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 10(10) virions. Importantly, repressions of 48 % and 23 % were still detected after 12 and 24 weeks, respectively, indicating that longer term effects are possible.



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.
Department of Life Sciences, Imperial College London, London, SW7 2AZ, UK.|Current address: Predepartmental Unit of Medicine, Faculty of Health Sciences, University Jaume I, Av. de Vicent Sos Baynat, s/n 12071, Castelló de la Plana, Spain.
Carmen
C
Agustín-Pavón
Department of Life Sciences, Imperial College London, London, SW7 2AZ, UK.
Michal
M
Mielcarek
Cell and Developmental Biology Program, Centre for Genomic Regulation (CRG), Dr. Aiguader 88, 08003, Barcelona, Spain.|Universitat Pompeu Fabra (UPF), Barcelona, Spain.
Mireia
M
Garriga-Canut
Department of Life Sciences, Imperial College London, London, SW7 2AZ, UK. m.isalan@imperial.ac.uk.
Mark
M
Isalan
D000818:Animals; D001921:Brain / Q000378:metabolism; D004195:Disease Models, Animal; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics; D000071058:Huntingtin Protein / Q000235:genetics / Q000378:metabolism; D006816:Huntington Disease / Q000235:genetics / Q000378:metabolism* / Q000628:therapy; D051379:Mice; D009474:Neurons / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D016335:Zinc Fingers
D000071058:Huntingtin Protein
Gene therapy; Host optimization; Huntington’s disease; Immune response; Monogenetic disease; Neurodegenerative disorder; Synthetic transcription factors; rAAV