Transduction of modified factor VIII gene improves lentiviral gene therapy efficacy for hemophilia A

Abstract

Hemophilia A (HA) is a bleeding disorder caused by deficiency of the coagulation factor VIII (F8). F8 replacement is standard of care, whereas gene therapy (F8 gene) for HA is an attractive investigational approach. However, the large size of the F8 gene and the immunogenicity of the product present challenges in development of the F8 gene therapy. To resolve these problems, we synthesized a shortened F8 gene (F8-BDD) and cloned it into a lentiviral vector (LV). The F8-BDD produced mainly short cleaved inactive products in LV-transduced cells. To improve F8 functionality, we designed two novel F8-BDD genes, one with an insertion of eight specific N-glycosylation sites (F8-N8) and another which restored all N-glycosylation sites (F8-299) in the B domain. Although the overall protein expression was reduced, high coagulation activity (>100-fold) was detected in the supernatants of LV-F8-N8- and LV-F8-299-transduced cells. Protein analysis of F8 and the procoagulation cofactor, von Willebrand Factor, showed enhanced interaction after restoration of B domain glycosylation using F8-299. HA mouse hematopoietic stem cell transplantation studies illustrated that the bleeding phenotype was corrected after LV-F8-N8 or -299 gene transfer into the hematopoietic stem cells. Importantly, the F8-299 modification markedly reduced immunogenicity of the F8 protein in these HA mice. In conclusion, the modified F8-299 gene could be efficiently packaged into LV and, although with reduced expression, produced highly stable and functional F8 protein that corrected the bleeding phenotype without inhibitory immunogenicity. We anticipate that these results will be beneficial in the development of gene therapies against HA. Hemophilia A (HA) is a bleeding disorder caused by deficiency of the coagulation factor VIII (F8). F8 replacement is standard of care, whereas gene therapy (F8 gene) for HA is an attractive investigational approach. However, the large size of the F8 gene and the immunogenicity of the product present challenges in development of the F8 gene therapy. To resolve these problems, we synthesized a shortened F8 gene (F8-BDD) and cloned it into a lentiviral vector (LV). The F8-BDD produced mainly short cleaved inactive products in LV-transduced cells. To improve F8 functionality, we designed two novel F8-BDD genes, one with an insertion of eight specific N-glycosylation sites (F8-N8) and another which restored all N-glycosylation sites (F8-299) in the B domain. Although the overall protein expression was reduced, high coagulation activity (>100-fold) was detected in the supernatants of LV-F8-N8- and LV-F8-299-transduced cells. Protein analysis of F8 and the procoagulation cofactor, von Willebrand Factor, showed enhanced interaction after restoration of B domain glycosylation using F8-299. HA mouse hematopoietic stem cell transplantation studies illustrated that the bleeding phenotype was corrected after LV-F8-N8 or -299 gene transfer into the hematopoietic stem cells. Importantly, the F8-299 modification markedly reduced immunogenicity of the F8 protein in these HA mice. In conclusion, the modified F8-299 gene could be efficiently packaged into LV and, although with reduced expression, produced highly stable and functional F8 protein that corrected the bleeding phenotype without inhibitory immunogenicity. We anticipate that these results will be beneficial in the development of gene therapies against HA. Hemophilia A (HA) is an X-linked monogenic coagulation disorder resulting from the genetic deficiency of the factor VIII (F8) gene in the intrinsic coagulation cascade (1Soucie J.M. Evatt B. J Ac Kson D. Occurrence of hemophilia in the United States.Am. J. Hematol. 2010; 59: 288-294Crossref Scopus (270) Google Scholar). The current treatment of HA is based on protein replacement therapy (PRT) through plasma-derived coagulation factors or recombinant proteins. The limitations of PRT include short half-life, high cost, and life-time requirement of the treatment. Thus, gene therapy has become highly promising for HA (2Croteau S.E. Wang M. Wheeler A.P. 2021 clinical trials update: Innovations in hemophilia therapy.Am. J. Hematol. 2021; 96: 128-144Crossref PubMed Scopus (18) Google Scholar, 3Guo X.L. Chung T.H. Qin Y. Zheng J. Zheng H. Sheng L. Wynn T. Chang L.-J. Hemophilia gene therapy: New development from bench to bed side.Curr. Gene Ther. 2019; 19: 264-273Crossref PubMed Scopus (8) Google Scholar, 4Rangarajan S. Walsh L. Lester W. Perry D. Madan B. Laffan M. Yu H. Vettermann C. Pierce G.F. Wong W.Y. Pasi K.J. AAV5-Factor VIII gene transfer in severe hemophilia A.N. Engl. J. Med. 2017; 377: 2519-2530Crossref PubMed Scopus (381) Google Scholar, 5Pasi K.J. Rangarajan S. Mitchell N. Lester W. Symington E. Madan B. Laffan M. Russell C.B. Li M. Pierce G.F. Wong W.Y. Multiyear follow-up of AAV5-hFVIII-SQ gene therapy for hemophilia A.N. Engl. J. Med. 2020; 382: 29-40Crossref PubMed Scopus (165) Google Scholar). The analysis of the human F8 gene revealed an obvious domain structure for the protein, represented as A1-A2-B-A3-C1-C2 (6Mannucci P.M. Tuddenham E.G.D. The hemophilias - from royal genes to gene therapy.N. Engl. J. Med. 2001; 344: 1773-1779Crossref PubMed Scopus (755) Google Scholar). The B domain is encoded by unusually large exons with highly conserved content of asparagine (N)-linked oligosaccharides (7Kumar H.P. Hague C. Haley T. Starr C.M. Besman M.J. Lundblad R.L. D B. Elucidation of N-linked oligosaccharide structures of recombinant human factor VIII using fluorophore-assisted carbohydrate electrophoresis.Biotechnol. Appl. Biochem. 2011; 24: 207-216Google Scholar). Miao et al. (8Miao H.Z. Sirachainan N. Palmer L. Kucab P. Cunningham M.A. Kaufman R.J. Pipe S.W. Bioengineering of coagulation factor VIII for improved secretion.Blood. 2004; 103: 3412-3419Crossref PubMed Scopus (154) Google Scholar) illustrated that a partial B-domain deletion of F8 (F8-BDD), leaving an N-terminal 226-aa-stretch containing six putative asparagine-linked glycosylation sites intact, was able to increase in vitro secretion of F8 by 10-fold. Thus, lowering F8-BDD retention in the endoplasmic reticulum (ER) whereas increasing transport to the Golgi is a rational approach to achieving increased secretion (8Miao H.Z. Sirachainan N. Palmer L. Kucab P. Cunningham M.A. Kaufman R.J. Pipe S.W. Bioengineering of coagulation factor VIII for improved secretion.Blood. 2004; 103: 3412-3419Crossref PubMed Scopus (154) Google Scholar, 9Cerullo V. Seiler M.P. Mane V. Cela R. Clarke C. Kaufman R.J. Pipe S.W. Lee B. Correction of murine hemophilia A and immunological differences of factor VIII variants delivered by helper-dependent adenoviral vectors.Mol. Ther. 2007; 15: 2080-2087Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Lentivirus is a class of retroviruses that can infect both dividing and nondividing cells (10Pan J. Dinh T.T. Rajaraman A. Lee M. Butcher E.C. Patterns of expression of factor VIII and von Willebrand factor by endothelial cell subsets in vivo.Blood. 2016; 128: 104Crossref PubMed Scopus (46) Google Scholar, 11Chang L.-J. Gay E.E. The molecular genetics of lentiviral vectors - current and future perspectives.Curr. Gene Ther. 2001; 1: 237-251Crossref PubMed Scopus (43) Google Scholar). Shi et al. (12Shi Q. Kuether E.L. Chen Y. Schroeder J.A. Montgomery R.R. Platelet gene therapy corrects the hemophilic phenotype in immunocompromised hemophilia A mice transplanted with genetically manipulated human cord blood stem cells.Blood. 2014; 123: 395-403Crossref PubMed Scopus (35) Google Scholar) reported that the transduction of hematopoietic stem cells (HSCs) using lentiviral vector (LV) expressing F8 could produce a therapeutic level of F8 in HA mice without antibody formation, illustrating that LV can be a valuable tool in F8 gene therapy applications. Here, we applied an advanced LV carrying a universal promoter-driving F8 transgene expression. To improve F8 secretion and function, we modified the glycosylation sites in the B domain based on a codon-optimized F8-BDD construct. The detailed analyses of F8 protein processing, cofactor interactions, secretion and function of these newly engineered F8-BDD constructs, and ex vivo blood clotting activities and in vivo immunogenicity in F8 knockout mice were presented. To increase expression, the nucleotide sequences of the full-length F8 (flF8) and F8-BDD constructs were selectively codon-optimized and chemically synthesized. All the constructs were cloned into a pEGWI-LV backbone under control of the ubiquitous EF1α promoter. We used quantitative PCR (qPCR) method to determine vector titer based on vector genomes in cells. The illustrated that was packaged into of the size To F8 expression, we cells and the cell and under to as illustrated in was from the cells to determine vector and the were against in and cells was and the transgene expression was with based on and The showed that the F8-BDD increased of as with the The F8 protein expression was using an F8 and The results showed that the expression of F8-BDD in was that of The detected full-length and products in the and to and products in the from the cells with the to were detected in the cells. The F8 coagulation function was based on using The was with from LV-transduced The results showed that therapeutic level of coagulation activities were of clotting activities based on the coagulation which illustrated that showed showed and the control showed clotting activity The F8-BDD produced of F8 in cells and clotting function, that clotting However, for the clotting function based on an of F8 protein as by the clotting of of protein was that of that the activities F8-BDD The analysis that the B domain that could F8 processing, and function The of F8 is to and In the functional F8 as a of a and a E.C. P. Kaufman R.J. A large to of human factor VIII is for in vitro S. A. PubMed Scopus Google Scholar, L. J. M. T. of recombinant human factor Full Text PDF PubMed Google Scholar). The F8-BDD is and is by to produce which is from von Willebrand as a of a and a from and a from L. J. M. T. of recombinant human factor Full Text PDF PubMed Google Scholar, R.J. A. processing, and secretion of recombinant human factor VIII in Full Text PDF PubMed Google Scholar, S. Walsh of human von Willebrand PubMed Scopus Google Scholar, of factor VIII in by the von Willebrand PubMed Scopus Google Scholar). the domain is F8 is Importantly, the B domain modification of the of F8 secretion and function, we designed two novel F8 genes with increased glycosylation the (F8-N8) by eight glycosylation sites and the (F8-299) by all of the glycosylation sites in the B domain. We and -299 and vector and vector titer by The modification the LV whereas the F8-299 increased the as with F8-BDD and To F8 and function, we a human endothelial cell the of of or and and F8 expression. The results showed that all transduction and of expression analysis of F8 in the of the cells revealed markedly increased F8 to produce the inactive protein in both the cells and the F8-BDD the and F8-299 cells which of the to products In the we detected increased F8 products from both and F8-299 cells and as with the F8 protein in the F8-BDD cells showed of F8 expression in the of F8-BDD cells the and F8-299 cells However, coagulation analyses based on and increased activities from both and F8-299 with the F8 activities detected in the F8-299 cells and that of F8-BDD and To N-glycosylation in we the from and F8-299 cells using glycosylation and which and molecular The results showed size in and F8-299 after the treatment for the F8-BDD protein To the of N-glycosylation in F8 function, we cells with which the in the of N-linked oligosaccharides in cells. In all LV-transduced we F8 activities after the in the and F8-299 cells the of glycosylation restoration in functional F8 secretion We the of glycosylation on the of the F8 The results showed that the F8 activities by after which is with the reported of F8 protein F8-299 F8 activities all with activities after increased of the F8-299 To the B domain with procoagulation we the of the cells with and The of enhanced F8 and produced protein which was with an increase in F8 activities as illustrated by functional the F8 activity increased in the F8-BDD and in the F8-299 S. We that was F8 by in the F8-299 S. The cofactor was with and using antibody to we detected of in the of and in the F8-BDD by the as human F8 and with We the of F8 with through using F8 antibody to protein and antibody to the The results illustrated a of increased of F8 with in the and F8-299 as with the F8-BDD In vitro improved coagulation activities of the modified F8 proteins. We these vectors in an mouse The were from of the HA mice and with or F8-299 for as illustrated in of the HA mice through analysis detected in the transduction The expression in the was using a after transplantation We detected for the F8-299 and and for the F8-BDD and and high the F8 activity in the F8-299 increased from to and stable for in the which was that of the F8-BDD and analysis of in blood cells using antibody showed and of F8 cells in and F8-BDD To the bleeding the HA mice were to bleeding by after and the results showed markedly bleeding in and F8-299 transplanted mice as with the HA mice The of in the blood is in with and increased bleeding to reduced Thus, we in the blood and the level of in the F8-299 the F8-BDD and mice The bleeding of the F8-299 was to the mice in and the bleeding the F8-BDD and results that the clotting function was restored in the F8-299 HA mice. HA treatment is a in F8 therapy. To the after LV gene we by in the and of the transplanted HA mice. We detected to LV-transduced cells in the and and LV was detected in the and In LV was in the blood after and was detected in F8-299 F8-BDD and in and F8-299 HA mice. A and in the and blood from the transplanted HA mice. The F8-299 showed the LV in all of the of in cell in and by The cells were from the and of and F8-299 mice and with as as cell for and and after analysis of titer using from transplanted HA mice. The was and the titer was based on a modified of by in the transplanted HA mice. The of transplanted mice was after transplantation and to determine In and the as by B-domain endothelial factor eight specific N-glycosylation hemophilia human lentiviral murine hematopoietic stem vector analyses of in hematopoietic of the F8-299 mice illustrated that cells and were detected in the and results were for the F8-BDD and mice to cells were detected in and and in the antibody in the after transplantation was based on to as The results showed to or of in the F8-299 was detected in the F8-BDD and D and The current treatment for hemophilia and life-time A as gene therapy is highly In of F8 or delivered by gene therapy can bleeding in vector hemophilia gene therapy has reported results in a of and in clinical the of product coagulation factors for hemophilia gene D. Full Text Full Text PDF Scopus Google Scholar). However, as antibody to the high immunogenicity of the protein, expression and and the high of vectors present hemophilia gene therapy has in and clinical Tuddenham E.G.D. C. P. J. M. E. N. D. A. J. Rangarajan S. D. M. et and of factor gene therapy in hemophilia Engl. J. Med. 2014; PubMed Scopus Google Scholar, A. A. D. R. H. of for the of the coagulation factor D. Full Text Full Text PDF Scopus Google Scholar). A large of studies and clinical that LV stable and gene expression with immunogenicity. of the of the gene can be into To the size of F8 gene without function has mainly to as the F8-BDD Wang Kaufman R.J. and in vivo functional of factor PubMed Google Scholar, Kaufman R.J. The of N-linked glycosylation and protein with the secretion of PubMed Scopus Google Scholar). The of LV is by the size of the and the specific nucleotide We that the size increased LV the titer of was that of the However, although F8-BDD is the F8 gene therapy under the of the protein the coagulation function of F8-BDD is as as that of which was in and that the B domain of F8 an in F8 The F8 coagulation function and cofactor interactions, which has The F8 expression and secretion both and protein transport from to the Golgi The Golgi transport with protein, which of oligosaccharide To transport of N-glycosylation modification in the B domain has (8Miao H.Z. Sirachainan N. Palmer L. Kucab P. Cunningham M.A. Kaufman R.J. Pipe S.W. Bioengineering of coagulation factor VIII for improved secretion.Blood. 2004; 103: 3412-3419Crossref PubMed Scopus (154) Google Scholar, B. J. S. C. L. C. D. cell on the and function of recombinant D. Full Text Full Text PDF Scopus Google Scholar, T. J. Tuddenham E.G.D. C. of human factor VIII to 2011; PubMed Scopus Google we a sites in and a restoration of N-glycosylation in F8-299. We a detailed analysis of F8 protein after B domain is to that from F8 the of in as the the of the of F8 to in it was that F8-299 increased the modification of F8 protein, which the of protein and in an increase in the secretion of an The results showed that the of F8 protein secretion and coagulation In the coagulation as a cofactor of factor and to the coagulation cascade of factor VIII and of cofactor 2004; PubMed Scopus Google Scholar, D. of the of factor by protein in the of protein and factor Full Text Full Text PDF PubMed Scopus (35) Google Scholar, J. J. R. M. R. analysis to PubMed Scopus Google Scholar, V. C. Y. N. H. C. factor factor VIII cofactor 2010; PubMed Scopus Google Scholar). The is one of the of the another cofactor, F8 F8 and synthesized and in in cell as and and can be in to a as of (10Pan J. Dinh T.T. Rajaraman A. Lee M. Butcher E.C. Patterns of expression of factor VIII and von Willebrand factor by endothelial cell subsets in vivo.Blood. 2016; 128: 104Crossref PubMed Scopus (46) Google Scholar). has that F8 is with from in that the function of F8 is through cofactor and highly to the modified Although the and in endothelial and hematopoietic the of F8 T. R. N. E. M. endothelial cells factor 2014; PubMed Scopus Google Scholar, D. coagulation factor VIII is synthesized in endothelial cells.Blood. 2014; 123: PubMed Scopus Google Scholar, Shi Q. H. Montgomery R.R. A knockout mouse endothelial cells as the and of factor 2014; 123: PubMed Scopus Google Scholar, S. R. E. D. V. S. A. expression by in hemophilic 2019; PubMed Scopus Google Scholar). F8 in and cell as the and reported L. S. J. of cells in with hemophilia A. A of Google Scholar, M.A. in bleeding Engl. J. Med. PubMed Scopus Google Scholar, of the on factor VIII Med. Google Scholar, Y. B. 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A. of endothelial cells and endothelial from PubMed Scopus Google Scholar). for HA gene can and the therapeutic into the that gene transfer or to the therapeutic which LV gene therapy an attractive gene therapy for HA. of HA gene vectors into using to expression and antibody could improve the and and the without ex vivo cell D. Miao of lentiviral vectors factor VIII expression in corrects murine hemophilia Ther. Full Text Full Text PDF PubMed Scopus Google Scholar, Hemophilia A gene therapy of factor J. 2016; PubMed Scopus Google Scholar, Li C. Chen J. J. Li L. W. M. Miao therapeutic of in vivo gene therapy in hemophilia A 2020; Google Scholar). of F8 knockout mice revealed that the F8-299 a therapeutic the F8-BDD and the gene transfer We that the F8 gene expression was enhanced through the of a universal and the increase in F8-BDD expression in activity by a inhibitory The analyses of F8 glycosylation and cofactor the of a of the F8 protein in clotting future human trials to the therapeutic of these novel F8 In conclusion, the F8-299 the F8 transgene with high LV a reduced protein level an increased and of the F8 which in of the genetic it a in HA gene therapy The codon-optimized gene was by the into the vector J.M. P. of putative in factor 2001; PubMed Scopus Google Scholar). The F8-BDD was by the using codon-optimized the and C.B. P. level expression of recombinant coagulation factor Full Text Full Text PDF PubMed Scopus Google Scholar). The two modified F8-BDD sequences were chemically and cloned into the The the containing eight putative glycosylation and the F8-299 all of the B which were were using the LV as L.-J. V. T. Y. J. and analyses of a recombinant human vector Ther. PubMed Scopus Google Scholar, T. Y. Chang L.-J. lentiviral vectors with and PubMed Scopus Google Scholar). and F8-299 were cloned into vector the human EF1α promoter. cloned cells were for vector The were and as J. Chang L.-J. of cell by lentiviral transduction of human 2004; 1: PubMed Scopus (35) Google Scholar, L.-J. vector transduction of cells for novel 2010; PubMed Scopus Google Scholar). 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