Efficient Gene Transfer to Hematopoietic Repopulating Cells Using Concentrated RD114-Pseudotype Vectors Produced by Human Packaging Cells
Authors: Tobias Neff, Laura J Peterson, Julia C Morris, Jesse Thompson, Xiaobing Zhang, Peter A Horn, Bobbie M Thomasson, Hans-Peter Kiem
Journal: Molecular Therapy (2004)

The use of alternative viral envelope proteins to pseudotype oncoretroviral vectors has contributed substantially to recent improvements in retroviral gene transfer to hematopoietic stem cells. The envelope protein of endogenous feline leukemia virus (RD114) has emerged as a particularly attractive candidate in this context. Initial studies of RD114-pseudotype vectors produced by FLYRD18 packaging cells have shown promise, but it has also been demonstrated that FLYRD18 produces soluble factors that interfere with stem cell maintenance. We hypothesized that novel production methods for RD114-pseudotype vector preparations could overcome this limitation and lead to consistent high-level gene transfer in a canine model of stem cell gene therapy. We have developed a new retroviral packaging cell line based on human 293T cells (Phoenix-RD114). Virally conditioned medium derived from this cell line was volume reduced by centrifugation and resuspension in 1% of the original volume. Here we show that vectors produced by this method led to highly efficient transduction of canine hematopoietic repopulating cells. These results suggest that Phoenix-RD114-derived vectors may allow for more consistent transduction of hematopoietic repopulating cells than FLYRD18-derived vectors. An important obstacle to achieving clinically relevant levels of gene transfer to stem cells has been the low expression of the amphotropic viral receptor on the most immature hematopoietic cell populations [1.Orlic D. Girard L.J. Jordan C.T. Anderson S.M. Cline A.P. Bodine D.M. The level of mRNA encoding the amphotropic retrovirus receptor in mouse and human hematopoietic stem cells is low and correlates with the efficiency of retrovirus transduction.Proc. Natl. Acad. Sci. USA. 1996; 93: 11097-11102Crossref PubMed Scopus (202) Google Scholar]. This has prompted the development of alternative envelope proteins for pseudotyping. The receptor for the feline endogenous retrovirus RD114 is a neutral amino acid transporter that has been shown to be highly expressed on a number of human tissues, including CD34+CD38− hematopoietic cells [2.Brenner S. et al.Concentrated RD114-pseudotyped MFGS-gp91phox vector achieves high levels of functional correction of the chronic granulomatous disease oxidase defect in NOD/SCID/{beta}2m −/− repopulating mobilized human peripheral blood CD34+ cells.Blood. 2003; Google Scholar]. High expression of the receptor for RD114 in these very primitive hematopoietic cells provides a good rationale for the use of the RD114 pseudotype in the context of clinical stem cell gene transfer. Consistent with this rationale, RD114-pseudotype vectors have been shown to transduce efficiently canine hematopoietic repopulating cells [3.Goerner M. et al.Sustained multilineage gene persistence and expression in dogs transplanted with CD34(+) marrow cells transduced by RD114-pseudotype oncoretrovirus vectors.Blood. 2001; 98: 2065-2070Crossref PubMed Scopus (68) Google Scholar] and human NOD/SCID repopulating cells [2.Brenner S. et al.Concentrated RD114-pseudotyped MFGS-gp91phox vector achieves high levels of functional correction of the chronic granulomatous disease oxidase defect in NOD/SCID/{beta}2m −/− repopulating mobilized human peripheral blood CD34+ cells.Blood. 2003; Google Scholar, 4.Kelly P.F. Vandergriff J. Nathwani A. Nienhuis A.W. Vanin E.F. Highly efficient gene transfer into cord blood nonobese diabetic/severe combined immunodeticiency repopulating cells by oncoretroviral vector particles pseudotyped with the feline endogenous retrovirus (RD114) envelope protein.Blood. 2000; 96: 1206-1214PubMed Google Scholar, 5.Kelly P.F. Carrington J. Nathwani A. Vanin E.F. RD114-pseudotyped oncoretroviral vectors. Biological and physical properties.Ann. N.Y. Acad. Sci. 2001; 938 (discussion 276–267): 262-276Crossref PubMed Scopus (33) Google Scholar, 6.Gatlin J. Melkus M.W. Padgett A. Kelly P.F. Garcia J.V. Engraftment of NOD/SCID mice with human CD34(+) cells transduced by concentrated oncoretroviral vector particles pseudotyped with the feline endogenous retrovirus (RD114) envelope protein.J. Virol. 2001; 75: 9995-9999Crossref PubMed Scopus (47) Google Scholar]. Their stability is a particularly attractive feature of RD114-pseudotype vector particles, allowing for volume reduction by centrifugation [2.Brenner S. et al.Concentrated RD114-pseudotyped MFGS-gp91phox vector achieves high levels of functional correction of the chronic granulomatous disease oxidase defect in NOD/SCID/{beta}2m −/− repopulating mobilized human peripheral blood CD34+ cells.Blood. 2003; Google Scholar, 6.Gatlin J. Melkus M.W. Padgett A. Kelly P.F. Garcia J.V. Engraftment of NOD/SCID mice with human CD34(+) cells transduced by concentrated oncoretroviral vector particles pseudotyped with the feline endogenous retrovirus (RD114) envelope protein.J. Virol. 2001; 75: 9995-9999Crossref PubMed Scopus (47) Google Scholar]. Centrifugation-mediated concentration of vector particles offers two important advantages. First, higher vector titers achieved by centrifugation and resuspension in a smaller volume could potentially lead to higher titers, saturating available receptors on stem cells, thereby overcoming limitations imposed on transduction by low receptor expression [1.Orlic D. Girard L.J. Jordan C.T. Anderson S.M. Cline A.P. Bodine D.M. The level of mRNA encoding the amphotropic retrovirus receptor in mouse and human hematopoietic stem cells is low and correlates with the efficiency of retrovirus transduction.Proc. Natl. Acad. Sci. USA. 1996; 93: 11097-11102Crossref PubMed Scopus (202) Google Scholar, 7.Kurre P. Morris J. Miller A.D. Kiem H.P. Envelope fusion protein binding studies in an inducible model of retrovirus receptor expression and in CD34(+) cells emphasize limited transduction at low receptor levels.Gene Ther. 2001; 8: 593-599Crossref PubMed Scopus (20) Google Scholar, 8.Kurre P. Kiem H.P. Morris J. Heyward S. Battini J.L. Miller A.D. Efficient transduction by an amphotropic retrovirus vector is dependent on high-level expression of the cell surface virus receptor.J. Virol. 1999; 73: 495-500PubMed Google Scholar]. Second, the concentration and resuspension significantly deplete potentially unwanted (e.g., FBS) as well as toxic by-products present in the virally conditioned supernatant harvested from producer cell lines. In this context it is important to note that the original RD114-producer cell line, FLYRD18, was reported to have a differentiating activity on repopulating hematopoietic cells [4.Kelly P.F. Vandergriff J. Nathwani A. Nienhuis A.W. Vanin E.F. Highly efficient gene transfer into cord blood nonobese diabetic/severe combined immunodeticiency repopulating cells by oncoretroviral vector particles pseudotyped with the feline endogenous retrovirus (RD114) envelope protein.Blood. 2000; 96: 1206-1214PubMed Google Scholar]. Production of transduction-inhibiting factors has been described for NIH3T3-derived producer clones [9.Le Doux J.M. Morgan J.R. Snow R.G. Yarmush M.L. Proteoglycans secreted by packaging cell lines inhibit retrovirus infection.J. Virol. 1996; 70: 6468-6473Crossref PubMed Google Scholar], but not for producer cell lines based on human 293 cells. Recently, we were able to demonstrate an advantage of packaging cell lines derived from human 293T cells (Phoenix-GALV) compared to murine NIH3T3-derived PG13 packaging cells. This combination has led to gene transfer levels of up to 25% in a nonhuman primate model of stem cell gene therapy [10.Horn P.A. Topp M.S. Morris J.C. Riddell S.R. Kiem H.P. Highly efficient gene transfer into baboon marrow repopulating cells using GALV-pseudotype oncoretroviral vectors produced by human packaging cells.Blood. 2002; 100: 3960-3967Crossref PubMed Scopus (84) Google Scholar]. Based on the encouraging results obtained with Phoenix-GALV-producer cells, we established a 293T-based RD114-pseudotype packaging cell line (Phoenix-RD114) in analogy to Phoenix-GALV cells [10.Horn P.A. Topp M.S. Morris J.C. Riddell S.R. Kiem H.P. Highly efficient gene transfer into baboon marrow repopulating cells using GALV-pseudotype oncoretroviral vectors produced by human packaging cells.Blood. 2002; 100: 3960-3967Crossref PubMed Scopus (84) Google Scholar]. We generated a helper-virus-free Phoenix-RD114 producer clone packaging an MGMT(P140K)- and GFP-containing MSCV-based vector (MIEG3Pl40K). Vector-conditioned medium harvested from this clone was volume-reduced by centrifugation (7277g, 4°C for 24 h). Titers were approximately 1.5 × 107/ml after centrifugation and resuspension in 1% of the original volume. Vector recovery after centrifugation and resuspension was typically 60%, with a range of 37–80%. We exposed the CD34+ cells twice on 2 consecutive days to a 1:10 dilution of concentrated vector for 4 h (effective titer 1.5 × 106/ml). In vitro gene transfer was comparable to results obtained with unconcentrated high-titer FLYRD18 - derived vector of approximately 10-fold higher titer (0.9–1.5 × 107) [3.Goerner M. et al.Sustained multilineage gene persistence and expression in dogs transplanted with CD34(+) marrow cells transduced by RD114-pseudotype oncoretrovirus vectors.Blood. 2001; 98: 2065-2070Crossref PubMed Scopus (68) Google Scholar]. However, marking during the early engraftment period was substantially higher using the Phoenix-RD114-derived vector compared to marking in the animals used to evaluated FLYRD18-derived vectors [3.Goerner M. et al.Sustained multilineage gene persistence and expression in dogs transplanted with CD34(+) marrow cells transduced by RD114-pseudotype oncoretrovirus vectors.Blood. 2001; 98: 2065-2070Crossref PubMed Scopus (68) Google Scholar]. Early after transplantation, we observed marking in excess of 30% in all three animals tested (Figs. 1a–1c) . As regularly observed after oncoretroviral gene transfer to hematopoietic stem cells, marking levels decreased somewhat in all three dogs and stabilized around day 90 after transplantation at levels of 3 to 19% in granulocytes. Using antibody staining with subset-specific antibodies, we were able to demonstrate marking in granulocytes, monocytes, T cells, and B cells (Fig. 1d). Gene marking was also observed in red blood cells and platelets (Fig. 1e), albeit at somewhat lower levels. Importantly, gene transfer at 3 months in our experience is a reliable predictor of long-term gene marking in dogs. To evaluate gene transfer into human hematopoietic cells with our novel Phoenix-RD114-producer clone, we transduced G-CSF-mobilized peripheral blood CD34+ cells from three different donors using a protocol similar to the one described for the dog studies. The mean gene transfer in three independent experiments was 90% (standard deviation 3%), as determined by flow cytometry, and 72% (standard deviation 5%) in CFU-C as determined by scoring under a fluorescence microscope, demonstrating highly efficient gene transfer. Combining the use of Phoenix-RD114 packaging cells with centrifugation-mediated concentration led to consistent, highly efficient in vivo gene marking in the dog. In fact, our vector production strategy yielded higher and more consistent in vivo gene marking than previous canine stem cell gene therapy studies, including those studies describing gene transfer with FLYRD18-based RD114-pseudotype vectors [3.Goerner M. et al.Sustained multilineage gene persistence and expression in dogs transplanted with CD34(+) marrow cells transduced by RD114-pseudotype oncoretrovirus vectors.Blood. 2001; 98: 2065-2070Crossref PubMed Scopus (68) Google Scholar], other pseudotypes [11.Kiem H.P. et al.Improved gene transfer into canine hematopoietic repopulating cells using CD34-enriched marrow cells in combination with a gibbon ape leukemia virus-pseudotype retroviral vector.Gene Ther. 1999; 6: 966-972Crossref PubMed Scopus (39) Google Scholar, 12.Goemer M. Bruno B. McSweeney P.A. Buron G. Storb R. Kiem H.P. The use of granulocyte colony-stimulating factor during retroviral transduction on fibronectin fragment CH-296 enhances gene transfer into hematopoietic repopulating cells in dogs.Blood. 1999; 94: 2287-2292Google Scholar], or other vector systems such as lentiviral vectors (manuscript submitted). Of note, the effective titer used in this study (1–2 × 106/ml) was lower than in our previous study using FLYRD18-derived vector, in which higher titers of 5 × 107 were necessary to achieve gene transfer to canine stem cells. This suggests that further concentration of Phoenix-RD114-derived vector to higher titers could potentially lead to even better in vivo marking. In conclusion, our data show that vectors produced by Phoenix-RD114 allow for efficient transduction of hematopoietic repopulating cells and should be considered in clinical gene therapy applications.