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Bioengineered coagulation factor VIII enables long-term correction of murine hemophilia A following liver-directed adeno-associated viral vector delivery.

ABSTRACT: Clinical data support the feasibility and safety of adeno-associated viral (AAV) vectors in gene therapy applications. Despite several clinical trials of AAV-based gene transfer for hemophilia B, a unique set of obstacles impede the development of a similar approach for hemophilia A. These include (i) the size of the factor VIII (fVIII) transgene, (ii) humoral immune responses to fVIII, (iii) inefficient biosynthesis of human fVIII, and (iv) AAV vector immunity. Through bioengineering approaches, a novel fVIII molecule, designated ET3, was developed and shown to improve biosynthetic efficiency 10- to 100-fold. In this study, the utility of ET3 was assessed in the context of liver-directed, AAV-mediated gene transfer into hemophilia A mice. Due to the large size of the expression cassette, AAV-ET3 genomes packaged into viral particles as partial genome fragments. Despite this potential limitation, a single peripheral vein administration of AAV-ET3 into immune-competent hemophilia A mice resulted in correction of the fVIII deficiency at lower vector doses than previously reported for similarly oversized AAV-fVIII vectors. Therefore, ET3 appears to improve vector potency and mitigate at least one of the critical barriers to AAV-based clinical gene therapy for hemophilia A.

SUBMITTER: Brown HC

PROVIDER: S-EPMC4362354 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Bioengineered coagulation factor VIII enables long-term correction of murine hemophilia A following liver-directed adeno-associated viral vector delivery.

Brown Harrison C HC Wright J Fraser JF Zhou Shangzhen S Lytle Allison M AM Shields Jordan E JE Spencer H Trent HT Doering Christopher B CB

Molecular therapy. Methods & clinical development 20140806

Clinical data support the feasibility and safety of adeno-associated viral (AAV) vectors in gene therapy applications. Despite several clinical trials of AAV-based gene transfer for hemophilia B, a unique set of obstacles impede the development of a similar approach for hemophilia A. These include (i) the size of the factor VIII (fVIII) transgene, (ii) humoral immune responses to fVIII, (iii) inefficient biosynthesis of human fVIII, and (iv) AAV vector immunity. Through bioengineering approaches ...[more]

PMID: 26015976

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Project description:Genetically modified, autologous hematopoietic stem and progenitor cells (HSPCs) represent a new class of genetic medicine. Following this therapeutic paradigm, we are developing a product candidate, designated CD68-ET3-LV CD34+, for the treatment of the severe bleeding disorder, hemophilia A. The product consists of autologous CD34+ cells transduced with a human immunodeficiency virus 1-based, monocyte lineage-restricted, self-inactivating lentiviral vector (LV), termed CD68-ET3-LV, encoding a bioengineered coagulation factor VIII (fVIII) transgene, termed ET3, designed for enhanced expression. This vector was shown capable of high-titer manufacture under clinical scale and Good Manufacturing Practice. Biochemical and immunogenicity testing of recombinant ET3, as well as safety and efficacy testing of CD68-ET3-LV HSPCs, were utilized to demonstrate overall safety and efficacy in murine models. In the first model, administration of CD68-ET3-LV-transduced stem-cell antigen-1+ cells to hemophilia A mice resulted in sustained plasma fVIII production and hemostatic correction without signs of toxicity. Patient-derived, autologous mobilized peripheral blood (mPB) CD34+ cells are the clinical target cells for ex vivo transduction using CD68-ET3-LV, and the resulting genetically modified cells represent the investigational drug candidate. In the second model, CD68-ET3-LV gene transfer into mPB CD34+ cells isolated from normal human donors was utilized to obtain in vitro and in vivo pharmacology, pharmacokinetic, and toxicology assessment. CD68-ET3-LV demonstrated reproducible and efficient gene transfer into mPB CD34+ cells, with vector copy numbers in the range of 1 copy per diploid genome equivalent without affecting clonogenic potential. Differentiation of human CD34+ cells into monocytes was associated with increased fVIII production, supporting the designed function of the CD68 promoter. To assess in vivo pharmacodynamics, CD68-ET3-LV CD34+ cell product was administered to immunodeficient mice. Treated mice displayed sustained plasma fVIII levels and no signs of product related toxicity. Collectively, the findings of the current study support the preclinical safety and efficacy of CD68-ET3-LV CD34+.

Dataset Information

Bioengineered coagulation factor VIII enables long-term correction of murine hemophilia A following liver-directed adeno-associated viral vector delivery.

Publications

Bioengineered coagulation factor VIII enables long-term correction of murine hemophilia A following liver-directed adeno-associated viral vector delivery.

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