{"doi":"10.1016/j.omtm.2020.10.013","title":"Minimal Essential Human Factor VIII Alterations Enhance Secretion and Gene Therapy Efficiency","abstract":"One important limitation for achieving therapeutic expression of human factor VIII (FVIII) in hemophilia A gene therapy is inefficient secretion of the FVIII protein. Substitution of five amino acids in the A1 domain of human FVIII with the corresponding porcine FVIII residues generated a secretion-enhanced human FVIII variant termed B-domain-deleted (BDD)-FVIII-X5 that resulted in 8-fold higher FVIII activity levels in the supernatant of an in vitro cell-based assay system than seen with unmodified human BDD-FVIII. Analysis of purified recombinant BDD-FVIII-X5 and BDD-FVIII revealed similar specific activities for both proteins, indicating that the effect of the X5 alteration is confined to increased FVIII secretion. Intravenous delivery in FVIII-deficient mice of liver-targeted adeno-associated virus (AAV) vectors designed to express BDD-FVIII-X5 or BDD-FVIII achieved substantially higher plasma FVIII activity levels for BDD-FVIII-X5, even when highly efficient codon-optimized F8 nucleotide sequences were employed. A comprehensive immunogenicity assessment using in vitro stimulation assays and various in vivo preclinical models of hemophilia A demonstrated that the BDD-FVIII-X5 variant does not exhibit an increased immunogenicity risk compared to BDD-FVIII. In conclusion, BDD-FVIII-X5 is an effective FVIII variant molecule that can be further developed for use in gene- and protein-based therapeutics for patients with hemophilia A. One important limitation for achieving therapeutic expression of human factor VIII (FVIII) in hemophilia A gene therapy is inefficient secretion of the FVIII protein. Substitution of five amino acids in the A1 domain of human FVIII with the corresponding porcine FVIII residues generated a secretion-enhanced human FVIII variant termed B-domain-deleted (BDD)-FVIII-X5 that resulted in 8-fold higher FVIII activity levels in the supernatant of an in vitro cell-based assay system than seen with unmodified human BDD-FVIII. Analysis of purified recombinant BDD-FVIII-X5 and BDD-FVIII revealed similar specific activities for both proteins, indicating that the effect of the X5 alteration is confined to increased FVIII secretion. Intravenous delivery in FVIII-deficient mice of liver-targeted adeno-associated virus (AAV) vectors designed to express BDD-FVIII-X5 or BDD-FVIII achieved substantially higher plasma FVIII activity levels for BDD-FVIII-X5, even when highly efficient codon-optimized F8 nucleotide sequences were employed. A comprehensive immunogenicity assessment using in vitro stimulation assays and various in vivo preclinical models of hemophilia A demonstrated that the BDD-FVIII-X5 variant does not exhibit an increased immunogenicity risk compared to BDD-FVIII. In conclusion, BDD-FVIII-X5 is an effective FVIII variant molecule that can be further developed for use in gene- and protein-based therapeutics for patients with hemophilia A.","journal":"Molecular Therapy — Methods & Clinical Development","year":2020,"id":70705,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":19,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9512,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2020-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":273984,"name":"Biao Dong","orcid":"0000-0002-5991-1867","position":1,"is_corresponding":false},{"id":375296,"name":"Franziska Horling","orcid":null,"position":2,"is_corresponding":false},{"id":374030,"name":"Jenni Firrman","orcid":"0000-0002-9586-5204","position":3,"is_corresponding":false},{"id":374031,"name":"Johannes Lengler","orcid":"0000-0003-0004-7629","position":4,"is_corresponding":false},{"id":374032,"name":"Matthias Klugmann","orcid":"0000-0003-0574-612X","position":5,"is_corresponding":false},{"id":374033,"name":"Maurus de la Rosa","orcid":"0000-0002-6744-025X","position":6,"is_corresponding":false},{"id":374034,"name":"Wenman Wu","orcid":"0000-0002-4297-6904","position":7,"is_corresponding":false},{"id":301233,"name":"Qizhao Wang","orcid":"0000-0003-4466-7811","position":8,"is_corresponding":false},{"id":374035,"name":"Hongying Wei","orcid":"0000-0002-5714-879X","position":9,"is_corresponding":false},{"id":374036,"name":"Andrea R. Moore","orcid":"0000-0002-1236-1554","position":10,"is_corresponding":false},{"id":375297,"name":"S A Roberts","orcid":null,"position":11,"is_corresponding":false},{"id":374037,"name":"Carmen J. Booth","orcid":"0000-0002-2153-738X","position":12,"is_corresponding":false},{"id":375298,"name":"Werner Hoellriegl","orcid":null,"position":13,"is_corresponding":false},{"id":374038,"name":"Dong Li","orcid":"0000-0001-5510-9503","position":14,"is_corresponding":false},{"id":24983,"name":"Barbara A. Konkle","orcid":"0000-0002-3959-8797","position":15,"is_corresponding":false},{"id":374039,"name":"Carol H. Miao","orcid":"0000-0001-6520-2373","position":16,"is_corresponding":false},{"id":374040,"name":"Birgit M. 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