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Lentiviral vectors are established as efficient and convenient vehicles for gene transfer. They are almost always pseudotyped with the envelope glycoprotein of vesicular stomatitis virus (VSV-G) due to the high titers that can be achieved, their stability, and broad tropism. We generated a novel cocal vesiculovirus envelope glycoprotein plasmid and compared the properties of lentiviral vectors pseudotyped with cocal, VSV-G, and a modified feline endogenous retrovirus envelope glycoprotein (RD114/TR). Cocal-pseudotyped lentiviral vectors can be produced at titers as high as with VSV-G, have a broad tropism, and are stable, allowing for efficient concentration by centrifugation. Additionally, cocal vectors are more resistant to inactivation by human serum than VSV-G-pseudotyped vectors, and efficiently transduce human CD34(+) nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse-repopulating cells (SRCs), and long-term primate hematopoietic repopulating cells. These studies establish the potential of cocal-pseudotyped lentiviral vectors for a variety of scientific and therapeutic gene transfer applications, including in vivo gene delivery and hematopoietic stem cell (HSC) gene therapy.

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Overexpression of methylguanine methyltransferase P140K (MGMTP140K) has been successfully used for in vivo selection and chemoprotection in mouse and large animal studies, and has promise for autologous and allogeneic gene therapy. We examined the long-term safety of MGMTP140K selection in a clinically relevant dog model. Based on the association of provirus integration and protooncogene activation leading to leukemia in the X-linked immunodeficiency trial, we focused our analysis on the distribution of retrovirus integration sites (RIS) relative to proto-oncogene transcription start sites (TSS). We analyzed RIS near protooncogene TSS before (n ‫؍‬ 157) and after (n ‫؍‬ 129) chemotherapy in dogs that received MGMTP140K gene-modified cells and identified no overall increase of RIS near proto-oncogene TSS after chemotherapy. We also wanted to determine whether in vivo selected cells retained fundamental characteristics of hematopoietic stem cells. To that end, we performed secondary transplantation of IntroductionHematopoietic stem cell (HSC) gene therapy has advanced to the point at which engraftment of gene-modified cells in large animal models has reached clinically therapeutic levels (Ͼ 10%-15%) for a variety of diseases (for review, see Neff et al 1 ). In addition, several significant clinical gene therapy trials have clearly demonstrated the potential of retrovirus gene-modified cells as an effective cure for the genetic diseases of X-linked severe combined immunodeficiency (SCID-X1), 2 adenosine deaminase-deficient severe combined immunodeficiency, 3 and X-linked chronic granulomatous disease (X-CGD). 4 These large animal and clinical studies are aided by either the use of ablative radiation or an inherent growth/survival advantage of the gene-modified cells after transplantation. For many other single-gene genetic diseases (ie, ␤-thalassemias and pyruvate kinase deficiency), acquired diseases (ie, glioblastoma and acute myeloid leukemia), or infectious diseases (ie, AIDS) in which retrovirus gene therapy is appropriate, a potential limiting curative factor is gene-marking levels below therapeutic thresholds due to the inability to use ablative conditioning and no substantial growth advantage/ in vivo selection of gene-modified cells (for reviews, see Neff et al 1 and Trobridge et al 5 ). This requires alternate in vivo selection strategies after transplantation to increase the percentage of gene-modified cells to therapeutic levels. To realize the full clinical potential of retrovirus gene therapy, safe in vivo selection strategies using drug resistance gene therapy will be critical to propel these and other promising gene transfer applications.The basic premise of drug resistance gene therapy is to stably deliver a transgene that confers resistance to a cytotoxic drug via several different mechanisms (for reviews, see Neff et al, 1 Trobridge et al, 5 and Sorrentino 6 ). Several selectable markers have been extensively evaluated because of their ability to protect cells from cytotoxic agents, including d...

BackgroundHematopoietic stem cell (HSC) gene therapy has cured immunodeficiencies including X-linked severe combined immunodeficiency (SCID-X1) and adenine deaminase deficiency (ADA). For these immunodeficiencies corrected cells have a selective advantage in vivo, and low numbers of gene-modified cells are sufficient to provide therapeutic benefit. Strategies to efficiently transduce and/or expand long-term repopulating cells in vivo are needed for treatment of diseases that require higher levels of corrected cells, such as hemoglobinopathies. Here we expanded corrected stem cells in vivo in a canine model of a severe erythroid disease, pyruvate kinase deficiency.Methodology/Principal FindingsWe used a foamy virus (FV) vector expressing the P140K mutant of methylguanine methyltransferase (MGMTP140K) for in vivo expansion of corrected hematopoietic repopulating cells. FV vectors are attractive gene transfer vectors for hematopoietic stem cell gene therapy since they efficiently transduce repopulating cells and may be safer than more commonly used gammaretroviral vectors. Following transplantation with HSCs transduced ex vivo using a tri-cistronic FV vector that expressed EGFP, R-type pyruvate kinase, and MGMTP140K, we were able to increase marking from approximately 3.5% to 33% in myeloid long-term repopulating cells resulting in a functional cure.Conclusions/SignificanceHere we describe in one affected dog a functional cure for a severe erythroid disease using stem cell selection in vivo. In addition to providing a potential cure for patients with pyruvate kinase deficiency, in vivo selection using foamy vectors with MGMTP140K has broad potential for several hematopoietic diseases including hemoglobinopathies.