Stem cell-derived clade F AAVs mediate high-efficiency homologous recombination-based genome editing

Abstract

The precise correction of genetic mutations at the nucleotide level is an attractive permanent therapeutic strategy for human disease. However, despite significant progress, challenges to efficient and accurate genome editing persist. Here, we report a genome editing platform based upon a class of hematopoietic stem cell (HSC)-derived clade F adeno-associated virus (AAV), which does not require prior nuclease-mediated DNA breaks and functions exclusively through BRCA2-dependent homologous recombination. Genome editing is guided by complementary homology arms and is highly accurate and seamless, with no evidence of on-target mutations, including insertion/deletions or inclusion of AAV inverted terminal repeats. Efficient genome editing was demonstrated at different loci within the human genome, including a safe harbor locus, AAVS1, and the therapeutically relevant IL2RG gene, and at the murine Rosa26 locus. HSC-derived AAV vector (AAVHSC)-mediated genome editing was robust in primary human cells, including CD34 cells, adult liver, hepatic endothelial cells, and myocytes. Importantly, high-efficiency gene editing was achieved in vivo upon a single i.v. injection of AAVHSC editing vectors in mice. Thus, clade F AAV-mediated genome editing represents a promising, highly efficient, precise, single-component approach that enables the development of therapeutic in vivo genome editing for the treatment of a multitude of human gene-based diseases.

Homologous recombination-based genome editing by clade F AAVs is inefficient in the absence of a targeted DNA break

“…3A). 40 Since this construct lacks an internal promoter, GFP expression should only be observed following insertion into the host genome downstream of a promoter and within an intron and, as expected, episomal expression was not detected at day 2 in any of the samples transduced with AAV only (Fig 3B). At day 14, GFP expression ( Fig.…”

“…This MOI is comparable to that employed by Smith et al in HSPCs. 40 By day 10, GFP expression from all of the cells receiving AAV vectors alone had fallen to below 0.3% GFP + (Fig. 5B-C).…”

“…Smith et al reported that AAV genomes packaged into clade F capsids could direct high levels of homologous recombination-based genome editing in the absence of a catalyzing DSB. 40 The authors further suggested that the concentration of AAV genomic DNA within the nucleus may have played a role in this process, since they reported significantly better transduction and nuclear entry rates in human CD34 + HSPCs when using clade F AAVs compared to other serotypes, including AAV6, and that the efficiency of transduction correlated with the editing frequencies obtained. These results were somewhat surprising, given that we and others have previously reported that AAV6 vectors more efficiently transduce HSPCs than the prototype clade F vector, AAV9.…”

“…41 As a control, we also included AAV6 vectors. The AAVS1 homology donor genome we designed mimicked the approach used by Smith et al, 40 where a promoter-less GFP reporter was placed downstream of a splice acceptor cassette, so that site-specific integration into the PPP1R12C intron could result in GFP expression. In contrast, the CCR5 homology construct contained an internal PGK promoter, expected to drive GFP expression from both episomal and integrated forms of the vector genome.…”

“…At the same time, editing technologies that do not require DSBs are also being developed, such as those that exploit base editing [36][37][38] and potentially transposon integration. 39 Recently, Smith et al 40 reported that the use of AAV vectors generated with capsids from clade F viruses could mediate highly efficient HDR in the absence of targeted DNA breaks.…”