Allele-Selective Suppression of Mutant Huntingtin in Primary Human Blood Cells

Abstract

Post-transcriptional gene silencing is a promising therapy for the monogenic, autosomal dominant, Huntington’s disease (HD). However, wild-type huntingtin (HTT) has important cellular functions, so the ideal strategy would selectively lower mutant HTT while sparing wild-type. HD patients were genotyped for heterozygosity at three SNP sites, before phasing each SNP allele to wild-type or mutant HTT. Primary ex vivo myeloid cells were isolated from heterozygous patients and transfected with SNP-targeted siRNA, using glucan particles taken up by phagocytosis. Highly selective mRNA knockdown was achieved when targeting each allele of rs362331 in exon 50 of the HTT transcript; this selectivity was also present on protein studies. However, similar selectivity was not observed when targeting rs362273 or rs362307. Furthermore, HD myeloid cells are hyper-reactive compared to control. Allele-selective suppression of either wild-type or mutant HTT produced a significant, equivalent reduction in the cytokine response of HD myeloid cells to LPS, suggesting that wild-type HTT has a novel immune function. We demonstrate a sequential therapeutic process comprising genotyping and mutant HTT-linkage of SNPs, followed by personalised allele-selective suppression in a small patient cohort. We further show that allele-selectivity in ex vivo patient cells is highly SNP-dependent, with implications for clinical trial target selection.

Gene suppression approaches to neurodegeneration

“…Three such SNPs have been identified that could be used to treat a majority of patients in Europe and the USA [ 88 ]. A recent study has shown the feasibility of allele selective suppression in HD patient primary ex-vivo myeloid cells when using siRNA to target one of these potential SNPs, although the other SNPs tested did not achieve any allele selectivity [ 89 ]. This was thought to be due to the nucleotide sequence and tertiary structure of the transcript surrounding the base mismatch, and therefore a lower proportion of HD patients have targetable SNPs using this siRNA approach than originally thought.…”

Wild-type huntingtin regulates human macrophage function

“…with some indication of a similar effect in the control cells from healthy individuals 46,49 . Given the roles wtHTT might play in cytoskeleton remodelling, intracellular trafficking and transcription, it is plausible that a yet undescribed role for wtHTT could exist in cells of the innate immune system.…”

“…Previous data indicated the possibility of a role for wtHTT in cytokine release from monocytes and macrophages, whether they carry the Huntington's disease mutation or not 46,49 . In the present study, the cytokine profiles ex vivo of a large number of macrophage samples from normal, healthy individuals treated with either anti-HTT or scrambled siRNA GeRP-treated were obtained.…”

“…There is ample evidence, for example, showing HTT has the capacity to affect transcription, either by direct interaction with transcription factors 28 , or with chromatin modifying proteins 52 . Alternatively, HTT may interact with upstream intracellular signalling pathways, the effect of which is to alter transcription 46,49 . By any of or all these means, wtHTT could alter the expression of genes encoding pro-inflammatory cytokines.…”

“…Subjects with potential inflammatory or infective conditions were excluded from the study, as were subjects on immunomodulatory medications. Primary human macrophage cell cultures were derived as per previously published methods 31,46,48,49 . Briefly, whole blood samples were collected using BD Vacutainer Cell Preparation Tubes containing polyester gel and a density gradient liquid that allows for isolation of PBMCs through a single centrifugation step at 1000×g for 30 min, with the brake turned to "slow" for deceleration.…”