An all-feline retroviral packaging system for transduction of human cells

Abstract

Abstract The subgroup C feline leukemia virus (FeLV-C) receptor FLVCR is a widely expressed 12-transmembrane domain transporter that exports cytoplasmic heme and is a promising target for retrovirus-mediated gene delivery. Previous studies demonstrated that FeLV-C pseudotype vectors were more efficient at targeting human hematopoietic stem cells than those pseudotyped with gibbon ape leukemia virus (GALV), and thus we developed an all FeLV-C-based packaging system, termed CatPac. CatPac is helper-virus free and can produce higher titer vectors than existing gammaretroviral packaging systems, including systems mixing Moloney murine leukemia virus (MoMLV) Gag-Pol and FeLV-C Env proteins. The vectors can be readily concentrated (>30-fold), refrozen (three to five times), and held on ice (>2 days) with little loss of titer. Furthermore, we demonstrate that CatPac pseudotype vectors efficiently target early CD34(+)CD38(-) stem/progenitor cells, monocytic and erythroid progenitors, activated T cells, mature macrophages, and cancer cell lines, suggesting utility for human cell and cell line transduction and possibly gene therapy.

FIG. 1.

CatPac pseudotype vector stability. Fresh conditioned medium from CatPac6 cells (solid circles) and CatPac7 cells (solid squares) transfected with pMGIN were held at 37°C, 23°C, or 4°C as indicated, and then rapidly frozen and stored at −80°C. Vector titers (GFP⁺ TU/ml) were measured, using FEA cells as targets. Linear regression (dashed) lines were calculated with DeltaGraph 5.6. Half-life was calculated from the linear regression formula and is indicated for each regression line. Data are presented as means ± SEM of triplicate samples, representative of three similar experiments.

FIG. 2.

CD34⁺ progenitor cell transduction. CD34⁺ cells were transduced with Mock, CatPac6, GALV, or amphotropic pseudotype MGIN vector and then cultured for an additional 2 or 4 days as indicated. Cells were collected and stained for CD34 and CD38 and analyzed by flow cytometry for the frequency of GFP⁺ cells in the CD34⁺ subset (solid columns), the CD34⁺CD38^– subset (cross-hatched columns), or the CD34⁺CD38⁺ subset (open columns). The percentage of GFP⁺ cells in each subset was normalized to the percentage of GFP⁺CD34⁺ cells obtained with the amphotropic pseudotype vectors within each experiment. Averages ± SD of two independent experiments are presented; the CD34⁺ subset analysis is representative of four additional experiments.