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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.

Adeno-associated virus (AAV) vectors are proving to be remarkably successful for in vivo gene delivery. Based upon reports of abundant AAV in the human marrow, we tested CD34(+) hematopoietic stem cells for the presence of natural AAV. Here, we report for the first time, the presence of novel AAV variants in healthy CD34(+) human peripheral blood stem cells. The majority of healthy peripheral blood stem cell donors were found to harbor AAV in their CD34(+) cells. Every AAV isolated from CD34(+) cells mapped to AAV Clade F. Gene transfer vectors derived from these novel AAVs efficiently underwent entry and postentry processing in human cord blood stem cells and supported stable gene transfer into long-term, in vivo engrafting human HSCs significantly better than other serotypes. AAVHSC-transduced human CD34(+) cells engrafted in vivo and gave rise to differentiated transgene-expressing progeny. Importantly, gene-marked CD34(+) stem cells persisted long term in xenograft recipients, indicating transduction of primitive progenitors. Notably, correlation of structure with function permitted identification of potential capsid components important for HSC transduction. Thus, AAVHSCs represent a new class of genetic vectors for the manipulation of HSC genomes.

Inverted terminal repeat (ITR) integrity is critical for the replication, packaging, and transduction of recombinant adeno-associated virus (rAAV), a promising gene therapy vector. Because AAV ITRs possess 70% GC content and are palindromic, they are notoriously difficult to sequence. The purpose of this work was to develop a reliable ITR sequencing method. The ITRs of two molecular clones of AAV2, pTZAAV and pAV2, were (1) sequenced directly from plasmid DNA in the presence of denaturant (direct sequencing method, DSM) or (2) first amplified in a reaction in which 7-deaza-dGTP was substituted for dGTP and the resultant amplification product sequenced (amplification sequencing method, ASM). The DSM and ASM techniques yielded clear chromatograms, read through the ITR hairpin, and revealed hitherto unreported mutations in each ITR. pTZAAV and pAV2 possess identical mutations at the upstream MscI site of the 5′ ITR (T>G, nt 2) and the downstream MscI site of the 3′ ITR (del. nt 4672–4679). The chromatograms for pAV2 also revealed that the ITRs of this construct were arranged in a FLOP/FLOP orientation. In addition, the DSM was successfully employed to recover ITR–chromosomal junction sequences from a variety of rAAV-transduced tissue types. Both the DSM and ASM can be employed to sequence through the AAV ITR hairpin, and both techniques reliably detect mutations in the ITR. Because the DSM and ASM offer a way to verify ITR integrity, they constitute powerful tools for the process development of rAAV gene therapy.

Zinc-finger nucleases (ZFN) are powerful genome editing tools that may be used to prevent expression of specified genes, correct mutations, or insert transgenes at safe harbor loci. ZFNs create double-stranded breaks at specified locations in the genome which may subsequently be repaired via the non-homologous end joining pathway to interrupt expression of a gene. Targeted integration of genes at pre-determined sites in the genome can be achieved through homology-directed repair of ZFN-mediated targeted double-strand breaks using a co-delivered donor construct. We recently isolated and characterized a panel of novel AAVs (AAVHSC) derived from CD34+ human hematopoietic stem cells (HSC) from healthy donors. We have previously shown that vectors derived from AAVHSCs have the ability to efficiently transduce CD34+ cells. Here we tested the hypothesis that delivery of ZFNs and donor DNA by AAVHSC vectors to CD34+ human cells results in the creation of double stranded site-specific breaks and subsequent efficient targeted transgene integration. A promoterless GFP gene was targeted to Intron 1 of the PPP1R12C gene in the AAVS1 locus such that expression would be driven by the endogenous PPP1R12C promoter following site specific transgene integration. ZFNs specific for PPP1R12C Intron 1 and donor DNA were packaged in AAVHSCs and used to transduce either K562 cells or primary human cytokine-primed peripheral blood CD34+ cells (PBSC). Flow-cytometric analysis at different time points post-transduction revealed specific GFP expression in ZFN + Donor-transduced cells but not in cells transduced with ZFN alone. The multiplicities of infection, ratio of ZFN:donor, and timing of transduction were optimized in K562 cells. Simultaneous transduction of AAVHSC encoding both ZFN and donor DNA at multiplicities of infection of 50,000 and 150,000 respectively was found to be optimal and resulted in GFP expression in up to 50% of transduced cells. Optimized conditions were confirmed in primary CD34+ PBSC from different donors. Targeted integration into AAVS1 was consistently found to be more efficient in CD34+ PBSC as compared with K562 cells, with GFP expression ranging from 10 to 65% of cells. Integrated GFP was expressed long term, with approximately 20% GFP positive cells persisting at 35 days post-transduction in vitro in the absence of selection. Targeted integration into the AAVS1 locus was independently confirmed by amplification using transgene-specific and chromosome-specific primers. Sequencing of the amplified vector chromosome junctions confirmed site-specific integration into AAVS1. No unexpected modifications were found at the junction of the AAVS1 chromosomal sequence and donor homology arms. These results indicate that AAVHSCs function as efficient delivery tools for genome editing in therapeutically relevant cells such as primary PBSC.

Background aims Although recombinant adeno-associated virus serotype 2 (AAV2) vectors have gained attention because of their safety and efficacy in numerous phase I/II clinical trials, their transduction efficiency in hematopoietic stem cells (HSCs) has been reported to be low. Only a few additional AAV serotype vectors have been evaluated, and comparative analyses of their transduction efficiency in HSCs from different species have not been performed. Methods We evaluated the transduction efficiency of all available AAV serotype vectors (AAV1 through AAV10) in primary mouse, cynomolgus monkey and human HSCs. The transduction efficiency of the optimized AAV vectors was also evaluated in human HSCs in a murine xenograft model in vivo. Results We observed that although there are only six amino acid differences between AAV1 and AAV6, AAV1, but not AAV6, transduced mouse HSCs well, whereas AAV6, but not AAV1, transduced human HSCs well. None of the 10 serotypes transduced cynomolgus monkey HSCs in vitro . We also evaluated the transduction efficiency of AAV6 vectors containing mutations in surface-exposed tyrosine residues. We observed that tyrosine (Y) to phenylalanine (F) point mutations in residues 445, 705 and 731 led to a significant increase in transgene expression in human HSCs in vitro and in a mouse xenograft model in vivo . Conclusions These studies suggest that the tyrosine-mutant AAV6 serotype vectors are the most promising vectors for transducing human HSCs and that it is possible to increase further the transduction efficiency of these vectors for their potential use in HSC-based gene therapy in humans.