Multidimensional Genome-wide Analyses Show Accurate FVIII Integration by ZFN in Primary Human Cells

Costly coagulation factor VIII (FVIII) replacement therapy is a barrier to optimal clinical management of hemophilia A. Therapy using FVIII-secreting autologous primary cells is potentially efficacious and more affordable. Zinc finger nucleases (ZFN) mediate transgene integration into the AAVS1 locus but comprehensive evaluation of off-target genome effects is currently lacking. In light of serious adverse effects in clinical trials which employed genome-integrating viral vectors, this study evaluated potential genotoxicity of ZFN-mediated transgenesis using different techniques. We employed deep sequencing of predicted off-target sites, copy number analysis, whole-genome sequencing, and RNA-seq in primary human umbilical cord-lining epithelial cells (CLECs) with AAVS1 ZFN-mediated FVIII transgene integration. We combined molecular features to enhance the accuracy and activity of ZFN-mediated transgenesis. Our data showed a low frequency of ZFN-associated indels, no detectable off-target transgene integrations or chromosomal rearrangements. ZFN-modified CLECs had very few dysregulated transcripts and no evidence of activated oncogenic pathways. 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 the molecular design of genome-modifying constructs, AAVS1 ZFN-mediated FVIII integration in several primary human cell types may be safe and efficacious. Costly coagulation factor VIII (FVIII) replacement therapy is a barrier to optimal clinical management of hemophilia A. Therapy using FVIII-secreting autologous primary cells is potentially efficacious and more affordable. Zinc finger nucleases (ZFN) mediate transgene integration into the AAVS1 locus but comprehensive evaluation of off-target genome effects is currently lacking. In light of serious adverse effects in clinical trials which employed genome-integrating viral vectors, this study evaluated potential genotoxicity of ZFN-mediated transgenesis using different techniques. We employed deep sequencing of predicted off-target sites, copy number analysis, whole-genome sequencing, and RNA-seq in primary human umbilical cord-lining epithelial cells (CLECs) with AAVS1 ZFN-mediated FVIII transgene integration. We combined molecular features to enhance the accuracy and activity of ZFN-mediated transgenesis. Our data showed a low frequency of ZFN-associated indels, no detectable off-target transgene integrations or chromosomal rearrangements. ZFN-modified CLECs had very few dysregulated transcripts and no evidence of activated oncogenic pathways. 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 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.1Manco-Johnson MJ Abshire TC Shapiro AD Riske B Hacker MR Kilcoyne R et al.Prophylaxis versus episodic treatment to prevent joint disease in boys with severe hemophilia.N Engl J Med. 2007; 357: 535-544Crossref PubMed Scopus (1463) Google Scholar,2Collins P Faradji A Morfini M Enriquez MM Schwartz L Efficacy and safety of secondary prophylactic vs. on-demand sucrose-formulated recombinant factor VIII treatment in adults with severe hemophilia A: results from a 13-month crossover study.J Thromb Haemost. 2010; 8: 83-89Crossref PubMed Scopus (120) Google Scholar Of the current global population of about 140,000 people with hemophilia A, 75% receive little or no FVIII replacement.3World Federation of Hemophilia Report on the Annual Global Survey 2012, 2013 World Federation of Hemophilia Montreal, Canada.Google Scholar Even when FVIII products are affordable, regular prophylaxis is associated with frequent breakthrough bleeding,4Collins PW Blanchette VS Fischer K Björkman S Oh M Fritsch S rAHF-PFM Study Group et al.Break-through bleeding in relation to predicted factor VIII levels in patients receiving prophylactic treatment for severe hemophilia A.J Thromb Haemost. 2009; 7: 413-420Crossref PubMed Scopus (262) Google Scholar while the need for frequent intravenous access limits acceptance, especially among children for whom effective early intervention is especially important.5Santagostino E Mancuso ME Barriers to primary prophylaxis in haemophilic children: the issue of the venous access.Blood Transfus. 2008; 6:Suppl 2: s12-s16PubMed Google Scholar 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 vivo gene therapy using viral vectors is appealing for FVIII deficiency. Although it has not yet achieved the same success as gene therapy for hemophilia B,6Nathwani AC Reiss UM Tuddenham EG Rosales C Chowdary P McIntosh J et al.Long-term safety and efficacy of factor IX gene therapy in hemophilia B.N Engl J Med. 2014; 371: 1994-2004Crossref PubMed Scopus (879) Google Scholar improvements 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 cells ex vivo. Proof of concept was demonstrated in a clinical trial of autologous dermal fibroblasts transfected ex vivo with a plasmid that delivered a B domain-deleted FVIII transgene.7Roth DA Tawa Jr, NE O'Brien JM Treco DA Selden RF Factor VIII Transkaryotic Therapy Study Group Nonviral transfer of the gene encoding coagulation factor VIII in patients with severe hemophilia A.N Engl J Med. 2001; 344: 1735-1742Crossref PubMed Scopus (313) Google Scholar Since 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-mediated CCR5 inactivation in autologous T cells reported no adverse event attributable to ZFN.8Tebas P Stein D Tang WW Frank I Wang SQ Lee G et al.Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV.N Engl J Med. 2014; 370: 901-910Crossref PubMed Scopus (1008) Google Scholar Nonetheless, there is heightened awareness of potential oncogenic complications because clinical trials of transgene integration mediated by gammaretroviral vectors by and S A Wang J A E et in patients gene therapy of 2008; PubMed Scopus Google S S A B A et and with to gene therapy for chronic Med. 2010; PubMed Scopus Google I D EG M et of vectors with safety features for cell gene 8: PubMed Scopus Google Scholar The of genome-modifying is for clinical genome in cells have not have by predicted off-target D C M et of and in human and using 2009; PubMed Scopus Google Wang J et of in T cells by genome editing using 2008; PubMed Scopus Google Scholar in of off-target of nucleases by in 8: PubMed Scopus Google Scholar or sequencing the integration of R A A P C et of nuclease PubMed Scopus Google Scholar reported of off-target from to A M et of zinc finger nuclease of off-target PubMed Scopus Google Scholar has the issue of off-target by different but there in off-target off-target of nucleases by in 8: PubMed Scopus Google R A A P C et of nuclease PubMed Scopus Google Scholar for the genome to off-target are there for ex A G and off-target genome PubMed Scopus Google the of and 2014; PubMed Scopus Google T Lee J and off-target activities of programmable nucleases PubMed Scopus Google Scholar We have a of of predicted off-target sites, whole-genome sequencing integration and copy number analysis, and RNA-seq to the of AAVS1 ZFN-mediated FVIII transgene integration in primary human cells. We AAVS1 ZFN from a plasmid for expression of in transfected cells. study to potential genotoxicity using AAVS1 ZFN for and In to Wang J Lee I et nuclease for genome 2007; PubMed Scopus Google Scholar and to restore nuclease activity which is greatly in J T of and for zinc finger 2010; PubMed Scopus Google Wang J et activity with 8: PubMed Scopus Google Scholar The be to have off-target activity versus Wang J Lee I et nuclease for genome 2007; PubMed Scopus Google Scholar while to restore nuclease activity of the J T of and for zinc finger 2010; PubMed Scopus Google Wang J et activity with 8: PubMed Scopus Google Scholar AAVS1 ZFN is in several molecular features that to and integration. We that AAVS1 ZFN a FVIII transgene and durable FVIII secretion by primary human umbilical cord-lining epithelial cells (CLECs) with off-target We AAVS1 ZFN Wang J Lee I et nuclease for genome 2007; PubMed Scopus Google J T of and for zinc finger 2010; PubMed Scopus Google Scholar and Wang J et activity with 8: PubMed Scopus Google Scholar to integration of in cells plasmid The was as reported and to of that ZFN as and in of the to enhance activity in the was a to the in nuclease and in which was reported to activity while We of and the of by global and protein and of transfected gene 2010; 7: PubMed Scopus (120) Google Scholar and of integration and by ZFN was in of integration of and into the AAVS1 locus of cells was demonstrated by and integration of transfected with the on by integration integration for of integration integration of into of with in the of not evidence of transgene integration by integration and The delivered a B domain-deleted FVIII J integration in factor VIII expression in primary human cells 2014; PubMed Scopus Google integration of different integration of cells with for and with or AAVS1 ZFN and of transfected cells. the integration and on from cells treatment with or and treatment with and AAVS1 ZFN evidence of integration of a of the AAVS1 of AAVS1 ZFN-mediated integration of of the and integration on of cells with and ZFN with or of and are in AAVS1 ZFN are in a of the AAVS1 cells. of AAVS1 ZFN-mediated integration of of the and integration on of cells with FVIII or with ZFN by of and are in AAVS1 ZFN are in a of the AAVS1 cells. in the that for showed levels of ZFN expression with AAVS1 ZFN ZFN protein expression was also in CLECs to to CLECs which to ZFN activity was by the by integration and of results showed ZFN activity when and AAVS1 ZFN delivered as a to ZFN delivered as using a plasmid that delivered AAVS1 ZFN and integration and showed no integration in CLECs with of AAVS1 ZFN and to that by cell and The results showed that of AAVS1 ZFN with the of FVIII transgene was and because integration was the accuracy of genome in CLECs using gene vectors, AAVS1 and D C M et of and in human and using 2009; PubMed Scopus Google Scholar which delivered of CLECs demonstrated integration of and using which the integration and of integration of the of the was for a the and showed no or rearrangements. of integration and in the same gene integration and of CLECs with the FVIII transgene and AAVS1 ZFN showed integration of the in of by AAVS1 and sequencing of the FVIII transgene CLECs with the transgene population FVIII levels than CLECs versus off-target transgene integrations by of integration versus and off-target showed no in copy to of with P and P off-target integrations showed expression in to with transgene integration deep sequencing of the predicted most off-target D C M et of and in human and using 2009; PubMed Scopus Google Scholar showed and low frequency and in in to of was by AAVS1 with a a the AAVS1 of to sequencing the with a and levels a of deep sequencing of in predicted most AAVS1 ZFN off-target of of of of of of of predicted off-target for AAVS1 D C M et of and in human and using 2009; PubMed Scopus Google evaluated by deep sequencing of from from CLECs and of the chromosomal types of and are in are in genome to in a predicted off-target for AAVS1 D C M et of and in human and using 2009; PubMed Scopus Google evaluated by deep sequencing of from from CLECs and of the chromosomal types of and are in are in genome to D L et 2: and copy number in by PubMed Scopus Google Scholar of data to but in predicted off-target G C P genomes for zinc finger nuclease and off-target PubMed Scopus Google Scholar We to be off-target A Zinc finger and to the in vivo off-target activity of zinc finger PubMed Scopus Google Scholar of with in off-target had the combined number of and and also of than by to a of the AAVS1 locus genome to The ZFN was and of this of showed a A of this of showed a T or a C The off-target to in and potential AAVS1 The from from and from was a because data showed of different The was a of while the was a of a different not be to are because of and during by by Our data that off-target by of ZFN are chromosomal genome copy and was to chromosomal that the as it was to ZFN treatment had in employed a different on genome copy number in the predicted to have genome copy to the copy number of locus in of to the same locus in CLECs copy number locus was as the of of and was because different to of The of locus was to the of locus in the same locus of not be The of and as a The of to genome copy of not with a frequency of on the of chromosomal by and the of genome copy number by it was that ZFN treatment had chromosomal rearrangements. 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