Characterization of an alternative packaging system derived from the cat RD114 retrovirus for gene delivery

Retroviral vectors derived from the Moloney murine leukemia virus (MLV) are widely used in gene therapy. Pseudotyping of these vectors with the cat RD114 retrovirus envelope increases their potential for delivering genes into human hematopoietic cells. In the present study, we have further investigated the potential of the RD114 retrovirus in gene therapy. We describe and characterize an alternative retroviral packaging system derived from the RD114 retrovirus.

RD114-derived recombinant retroviruses were produced transiently by transfection of 293T cells, and viral titers were assessed on TE671 cells by measuring the percentage of infected green fluorescent protein (GFP) positive cells by fluorescence-activated cell sorter (FACS) analysis. Purified human hematopoietic cells (lymphocytes and CD34(+) cells) were activated and transduced on retronectin-coated plates. Two days later, the percentage of GFP positive cells was evaluated by FACS analysis.

We demonstrate that RD114 viral particles could package MLV transfer vectors, and that, in addition to its natural envelope, RD114 cores could be efficiently pseudotyped by the Gibbon ape leukemia, the MLV-amphotropic and the vesicular stomatitis virus G protein envelopes. Furthermore, we found that RD114 viral particles were highly efficient to transduce human lymphocytes and CD34(+) cells.

This is the first demonstration that replication-defective RD114 viral particles can be generated and used for efficient gene delivery into human hematopoietic cells. We conclude that RD114-derived vectors could be useful in the field of gene therapy.