Engraftment of NOD/SCID mice with human CD34(+) cells transduced by concentrated oncoretroviral vector particles pseudotyped with the feline endogenous retrovirus (RD114) envelope protein

Abstract

Oncoretrovirus vectors pseudotyped with the feline endogenous retrovirus (RD114) envelope protein produced by the FLYRD18 packaging cell line have previously been shown to transduce human hematopoietic progenitor cells with a greater efficiency than similar amphotropic envelope-pseudotyped vectors. In this report, we describe the production and efficient concentration of RD114-pseudotyped murine leukemia virus (MLV)-based vectors. Following a single round of centrifugation, vector supernatants were concentrated approximately 200-fold with a 50 to 70% yield. Concentrated vector stocks transduced prestimulated human CD34(+) (hCD34(+)) cells with approximately 69% efficiency (n = 7, standard deviation = 4.4%) using a single addition of vector at a low multiplicity of infection (MOI = 5). Introduction of transduced hCD34(+) cells into irradiated NOD/SCID recipients resulted in multilineage engraftment with long-term transgene expression. These data demonstrate that RD114-pseudotyped MLV-based vectors can be efficiently concentrated to high titers and that hCD34(+) cells transduced with concentrated vector stocks retain in vivo repopulating potential. These results highlight the potential of RD114-pseudotyped oncoretrovirus vectors for future clinical implementation in hematopoietic stem cell gene transfer.

FIG. 1

Production and concentration of feline endogenous virus (RD114) envelope protein-pseudotyped MLV vector particles. (A) FLYRD18/MSCV-EGFP cells were plated in triplicate, supernatants were harvested, and a titer (in i.u. per milliliter) was determined for unconcentrated supernatant collected every 24 h (circles), 48 h (diamonds), or 72 h (crosses). (B) For vector concentration, supernatants from FLYRD18/MSCV-EGFP cells were collected every 24 h, concentrated by centrifugation, and stored at −70°C. The titer (in i.u. per milliliter) of each preparation of vector was determined. Titers of unconcentrated supernatants (circles) and concentrated supernatants (triangles) were determined on HeLa cells by flow cytometry for EGFP expression.

FIG. 2

Transduction of hCD34⁺ cells by concentrated RD114-pseudotyped MLV vectors. hCD34⁺ cells were isolated from umbilical cord blood and transduced (MOI = 5) following 48 h of cytokine prestimulation. Flow-cytometric analysis was performed 48 h after transduction. The y axis represents the percentage of the total cell population. The x axis indicates the seven preparations of vector that were serially collected from a single set of plates and concentrated prior to addition to cells. Following transduction, cells were analyzed for hCD34 and EGFP expression by flow cytometry.

FIG. 3

Bone marrow engraftment of NOD/SCID recipients following transplantation of hCD34⁺ cells transduced with concentrated RD114-pseudotyped MLV vectors. Mononuclear cells were isolated from bone marrow 15 weeks posttransplant from animals receiving mock-transduced (A) or vector-transduced (B) cells. Isolated cells were stained with anti-hCD45 antibody conjugated to allophycocyanin (APC), and flow-cytometric analysis was performed. The upper left panels represent total live cells. The upper right and lower panels are gated for hCD45⁺ cells from the same sample. The y axis represents forward scatter (FSC), and the x axis represents staining for human cells (anti-hCD45–APC) (upper left panels), EGFP expression (upper right panels), anti-hCD34–PE (progenitor cells), anti-hCD19–PE (B cells), or hCD33–PE (myeloid cells) (lower panels). Values indicate the percentage of cells in each region compared to that of an unmanipulated mouse (that did not receive human cells) (upper left panel) or a mouse that received mock-transduced human cells (upper right panel).

FIG. 4

In vivo transgene expression following hematopoietic reconstitution. The results of flow-cytometric analyses of gated hCD45⁺ cells from one animal that received mock-transduced cells (upper panels) and one that received transduced cells (lower panels) are presented. The values in the upper panels indicate the percentage of total hCD45⁺ cells expressing the designated surface marker. The values in the lower panels indicate the percentage of cells expressing the designated surface marker that are EGFP⁺. Bone marrow mononuclear cells were isolated and stained with anti-hCD45–APC (human cells), anti-hCD34–PE (progenitor cells), anti-hCD19–PE (B cells), or hCD33–PE (myeloid cells). The y axis represents lineage-specific staining, and the x axis represents EGFP expression.