Analysis of striatal zinc finger protein (ZFP) repression of mutant Huntingtin (HTT) in the zQ175DN knock-in mouse model of Huntington's disease (bulk RNA-seq)

We assessed the relative efficacy of selectively lowering mHTT in the striatum at different intervention times in a human HTT exon 1 knock-in mouse model of Huntington's disease, zQ175DN KI, using a virally delivered zinc finger protein transcriptional repressor directed against the expanded CAG repeat of the mutant HTT gene (Zeitler et al. Nat Med. 2019 Jul;25(7):1131-1142).Simultaneous bilateral striatal stereotactic injections were performed in zQ175DN heterozygous (zQ175) or wild type (WT) littermates at either 2 or 6 months of age. The zQ175 knock-in (zQ175 KI) mouse has the mouse Htt exon 1 replaced by the human HTT exon 1 sequence with a about 190 CAG repeat tract (JAX ID: B6.129S1-Htttm1.1Mfc/190ChdiJ, CHDI ID: CHDI-80003019). One striatal hemisphere of each mouse was injected with purified, endotoxin free AAV2/2+1 virus expressing C-terminally HA-tagged mutant Huntingtin (mHTT) zinc finger repressor protein (ZFP30645, AAV-SWB, HA), under control of the human synapsin (hsyn) promotor (CHDI-90003731, nZFP). This ZFP targets expanded CAG repeats in the mutant Htt DNA, resulting in robust and selective abrogation of mHTT production (>80%) in striatal neurons. The other striatal hemisphere of each mouse was injected with either a control (inactive) AAV2/2+1 virus expressing C-terminally HA-tagged mutant huntingtin ZFP repressor protein lacking the DNA binding domain, under the control of hsyn (CHDI-90003732; DBD) or with an alternative AAV2/2+1 control virus containing a non-protein encoding, non-mRNA encoding plasmid with a stuffer sequence between the inverted terminal repeats (transcription blocker (TB) / stop-EGFP nucleotide sequence (EstopB) / transcription blocker reverse complement (TBrc) and bovine growth hormone poly A (bGHpA)); (CHDI-90003959; Estop). Mice injected at 2 months were maintained for a further 4 or 6 months prior to humane euthanasia and focal striatal tissue (separate left and right hemisphere) collection at 6 months (2M_6M treatment group) or 8 months (2M_8M treatment group) of age respectively. Mice injected at 6 months were maintained for a further 2, 4 or 6 months prior to humane euthanasia and striatal tissue (separate left and right hemisphere) collection at 8 months (6M_8M treatment group) 10 months (6M_10M treatment group) or 12 months (6M_12M treatment group) of age respectively. Tissues were maintained at -80oC until processing. This culminated in 30 sample groups (5 treatment paradigms x 3 viral treatments per paradigm, applied to 2 genotypes (WT and zQ175)). Each of the 30 sample groups consisted of 7-8 striatal samples from an equally balanced proportion of injected male and female mice. Striatal samples were subjected to RNA isolation and sequencing (Illumina, stranded mRNAseq: 40-50M reads, Paired End, 100-150bp).