Efficient Human Hematopoietic Cell Transduction Using RD114- and GALV-Pseudotyped Retroviral Vectors Produced in Suspension and Serum-Free Media

Abstract

Retroviral vectors derived from the Moloney murine leukemia virus have been used in successful and promising gene therapy clinical trials. However, platforms for their large-scale production must be further developed. As a proof of principle, we reported the generation of a packaging cell line that produces amphotropic retroviral vectors in suspension and serum-free medium (SFM). In the present study, we have constructed and characterized two retroviral packaging cell lines designed for gene transfer in hematopoietic cells. These cell lines grow in suspension and SFM, and produce high-titer RD114- and gibbon ape leukemia virus (GALV)-pseudotyped vectors for a 3-month culture period. Viral particles released are as robust during repeated freeze-thaw cycles and on thermal inactivation at 37 degrees C as their counterparts produced in cells cultured adherently with serum. We also show that RD114- and GALV-pseudotyped vectors produced in suspension and SFM efficiently transduce human lymphocytes and hematopoietic stem cells. As these retroviral packaging cell lines distinctively maintain high vector titers while growing in suspension and SFM, we conclude that these cell lines are uniquely suitable for large-scale clinical-grade vector production for late-phase clinical trials involving gene transfer into hematopoietic cells.

Translating the combination of gene therapy and tissue engineering for treating recessive dystrophic epidermolysis bullosa

“…These cells are safer and release recombinant retroviruses with higher titers compared with former versions (Cosset et al, 1995;Sheridan et al, 2000). The GFP viruses used in this study were produced with a set of 293-based retrovirus packaging cell lines, allowing for the production of vectors with titers above 10 7 ivp/mL (Ghani et al, 2007;Ghani et al, 2009). The transduction ability of GFP vectors pseudotyped with the Ampho, Baev, Galv and RD114 envelopes was assessed.…”

“…HT-1080 cells, 293T cells, the retrovirus parental packaging cell lines 293Vec-Ampho, 293Vec-Baev, www.ecmjournal.org 293Vec-Galv, 293Vec-RD114 (Ghani et al, 2007;Ghani et al, 2009) Human fibroblasts (18-and 37-year-old donors) and keratinocytes (26-and 37-year-old donors) were obtained from reductive breast surgery of healthy adult women after informed consent was given. Fibroblasts and keratinocytes were isolated by the two-step thermolysine and trypsin method, as previously described Lavoie et al, 2013).…”

“…keratinocytes with vectors produced from 293Vec packaging cell lines in presence of the EF-C peptide Retroviral vectors pseudotyped with the RD114 and Galv envelopes are highly efficient to transduce cells from haematopoietic origin (Ghani et al, 2009;Kelly et al, 2000;Kiem et al, 1997). Our first objective was to discriminate which envelope among Ampho, Baev, Galv and RD114 was the most efficient for the transduction of human fibroblasts and keratinocytes.…”

“…The use of high-titer retroviral packaging cell lines (293Vec cells), constructed in our laboratory, could circumvent the titer issue. They release viral particles pseudotyped with the amphotropic (Ampho), the baboon endogenous virus (Baev), the gibbon ape leukaemia virus (Galv) and the feline endogenous retrovirus (RD114) envelopes (Ghani et al, 2007;Ghani et al, 2009). In the present study, the potential of GFP and COL7A1 vectors, produced from these packaging cell lines, for the transduction of human keratinocytes and fibroblasts was investigated.…”

Masked Chimeric Antigen Receptor for Tumor-Specific Activation

“…The cDNA of GALV was PCR-amplified from the genomic DNA of PG13 cell lines. 48 The primers used for the cloning are forward primer (EcoRI), 5 0 -tat GAA TTC gcc acc atg gta ttg ctg cct ggg tcc-3 0 and reverse primer (EcoRI), 5 0 -gcg GAA TTC tta aag gtt acc ttc gtt ctc tag ggc-3 0 . The resulting PCR fragment was then cloned into the pHCMV plasmid backbone from Addgene via the EcoRI site.…”