(c) Real-time RT-PCR was performed on wt-CLEC and puro-CLEC to confirm transcriptome data of selected neighboring genes located within 1Mb of the AAVS1 integration site (left) and interacting partners ofPPP1R12C(right). GAPDH-normalized transcript levels (determined by themethod) in puro-CLEC are shown relative to wt-CLEC. mixed molecular features to enhance the accuracy and activity of ZFN-mediated transgenesis. Our data showed a low frequency of ZFN-associated indels, no detectable 3-Methyl-2-oxovaleric acid off-target transgene integrations or chromosomal rearrangements. ZFN-modified CLECs had very few dysregulated transcripts and no evidence of activated oncogenic pathways. 3-Methyl-2-oxovaleric acid We also showed AAVS1 ZFN activity and durable FVIII transgene secretion in primary human dermal fibroblasts, bone marrow- and adipose tissue-derived stromal cells. Our study suggests that, with close attention to Rabbit Polyclonal to HTR2B the molecular design of genome-modifying constructs, AAVS1 ZFN-mediated FVIII integration in several primary human cell types may be safe and efficacious. The bedrock of hemophilia A treatment is factor VIII (FVIII) protein replacement to restore hemostatic capacity to a level sufficient to enable normal blood coagulation during activities of daily living. Regular prophylaxis with plasma-free recombinant FVIII products is the treatment of choice as it greatly reduces the frequency of acute bleeding episodes, chronic musculoskeletal disability, and improves health-related quality of life. 1, 2Of the current global population of about 140, 000 people with hemophilia A, 75% receive little or no FVIII replacement. 3Even when FVIII products are affordable, regular prophylaxis is associated with frequent breakthrough bleeding, 4while the need for frequent intravenous access limits acceptance, especially among children for whom effective early intervention is especially important. 5 The high cost of FVIII replacement products for more than half the world’s population of hemophilia A patients motivates attempts to develop alternative therapies. In vivogene therapy using viral vectors is appealing for FVIII deficiency. Although it has not yet achieved the same success as gene therapy for hemophilia B, 6improvements in FVIII transgene expression and packaging in AAV vectors appear promising, as are approaches to minimize immune responses to AAV vectors. An alternative strategy is nonviral delivery of a FVIII transgene into autologous cellsex vivo. Proof of concept was demonstrated in a clinical trial of autologous dermal fibroblasts transfectedex vivowith a plasmid that delivered a B domain-deleted FVIII transgene. 7Since then, several programmable nucleases with the potential to modify genomes with high precision have emerged and can be delivered by nonviral vectors. Among these, zinc finger nuclease (ZFN) technology is currently most advanced towards possible clinical applications. A phase-1 clinical trial of ZFN-mediatedCCR5inactivation in autologous T cells reported no adverse event attributable to ZFN. 8Nonetheless, there is heightened awareness of potential oncogenic complications because clinical trials of transgene integration mediated by gammaretroviral vectors were marred by treatment-induced leukemias and myelodysplasia. 9, 10, 11The biosafety 3-Methyl-2-oxovaleric acid of all genome-modifying techniques is therefore crucial for clinical approval. Off-target genome modifications in ZFN-treated cells have not been comprehensively evaluated. They have been identified by screening bioinformatically predicted off-target sites, 12, 13in vitrocleavage of biased libraries14or sequencing the integration sites of integrase-defective lentiviral vectors. 15These studies reported frequencies of off-target events ranging from 1 to 6%. A machine-learning classifier16has partially resolved the issue of largely nonoverlapping off-target sites generated by different methods but there remain nontrivial method-dependent discrepancies in off-target site identifications. 14, 15No 3-Methyl-2-oxovaleric acid single technique for interrogating the genome suffices to reveal off-target modifications comprehensively; neither are there consensus standards for assessing biosafetyex vivo. 17, 18, 19We have adopted a suite of techniques comprising genomic analysis of bioinformatically predicted off-target sites, whole-genome sequencing (WGS), integration junction and donor vector copy number analysis, and RNA-seq transcriptome profiling to holistically evaluate the biosafety of AAVS1 ZFN-mediated FVIII transgene integration in primary human cells. We assembled codon-optimized AAVS1 ZFN monomers expressed from a single plasmid for stoichiometric expression of each monomer in transfected cells. This study aimed to address potential genotoxicity using AAVS1 ZFN designed for accurate and efficient on-target cleavage activity. In brief, monomers were mutated to suppress homodimerization, 20and additional mutations were introduced to restore nuclease activity which is greatly reduced in obligate heterodimericFokI catalytic domains. 21, 22The fully modified ZFN, named Enhanced Sharkey, could be expected to have substantially lower off-target activity (18. 9% homodimer versus 1 . 7% heterodimer), 20while mutations introduced to restore nuclease activity neither decrease nor increase cleavage specificity of the intended targets. 21, 22Thus, Enhanced Sharkey AAVS1 ZFN is novel in incorporating several molecular features that ought to favor accurate and efficient integration. We show that Enhanced Sharkey AAVS1 ZFN integrated a FVIII transgene and induced durable FVIII secretion by primary human umbilical cord-lining epithelial cells (CLECs) with negligible off-target effects. == Results == == Evaluation of different AAVS1 ZFN constructs == We tested three AAVS1 ZFN constructs: 3-Methyl-2-oxovaleric acid obligate heterodimer20; Sharkey21and Enhanced Sharkey22to quantify integration of pZDonor (50 bp) in K562 cells.