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CD46 Null Packaging Cell Line Improves Measles Lentiviral Vector Production and Gene Delivery to Hematopoietic Stem and Progenitor Cells.

ABSTRACT: Lentiviral vectors (LVs) pseudotyped with the measles virus hemagglutinin (H) and fusion (F) glycoproteins have been reported to more efficiently transduce hematopoietic stem and progenitor cells (HSPCs) compared with vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped LVs. However, a limit to H/F LV use is the low titer of produced vector. Here we show that measles receptor (CD46) expression on H/F transfected HEK293T vector-producing cells caused adjacent cell membrane fusion, resulting in multinucleate syncytia formation and death prior to peak vector production, leading to contaminating cell membranes that co-purified with LV. H/F LVs produced in CD46 null HEK293T cells, generated by CRISPR/Cas9-mediated knockout of CD46, produced 2-fold higher titer vector compared with LVs produced in CD46+ HEK293T cells. This resulted in approximately 2- to 3-fold higher transduction of HSPCs while significantly reducing target cell cytotoxicity caused by producer cell contaminates. Improved H/F LV entry into HSPCs and distinct entry mechanisms compared with VSV-G LV were also observed by confocal microscopy. Given that vector production is a major source of cost and variability in clinical trials of gene therapy, we propose that the use of CD46 null packaging cells may help to address these challenges.

SUBMITTER: Ozog S

PROVIDER: S-EPMC6310745 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Publications

CD46 Null Packaging Cell Line Improves Measles Lentiviral Vector Production and Gene Delivery to Hematopoietic Stem and Progenitor Cells.

Ozog Stosh S Chen Craig X CX Simpson Elizabeth E Garijo Olivia O Timberlake Nina D ND Minder Petra P Verhoeyen Els E Torbett Bruce E BE

Molecular therapy. Methods & clinical development 20181124

Lentiviral vectors (LVs) pseudotyped with the measles virus hemagglutinin (H) and fusion (F) glycoproteins have been reported to more efficiently transduce hematopoietic stem and progenitor cells (HSPCs) compared with vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped LVs. However, a limit to H/F LV use is the low titer of produced vector. Here we show that measles receptor (CD46) expression on H/F transfected HEK293T vector-producing cells caused adjacent cell membrane fusion, resultin ...[more]

PMID: 30603655

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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

CD46 Null Packaging Cell Line Improves Measles Lentiviral Vector Production and Gene Delivery to Hematopoietic Stem and Progenitor Cells.

Publications

CD46 Null Packaging Cell Line Improves Measles Lentiviral Vector Production and Gene Delivery to Hematopoietic Stem and Progenitor Cells.

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