The potential for leukemia caused by retroviral vector integration has become a significant concern for hematopoietic stem cell gene therapy. We analyzed the distribution of vector integrants in pigtailed macaque and baboon repopulating cells for the two most commonly used retroviral vector systems, human immunodeficiency virus (HIV)-based lentiviral vectors and murine leukemia virus (MLV)-based gammaretroviral vectors, to help define their relative genotoxicity. All animals had polyclonal engraftment with no apparent adverse effects from transplantation with gene-modified cells. In all, 380 MLV and 235 HIV unique vector integration sites were analyzed and had distinct distribution patterns in relation to genes and CpG islands as observed in previous in vitro studies. Both vector types were found more frequently in and near proto-oncogenes in repopulating cells than in a random dataset. Analysis of functional classes of genes with integrants within 100 kilobases (kb) of their transcription start sites showed an over-representation of genes involved in growth or survival near both lentiviral and gammaretroviral integrants. Microarray analysis showed that both gammaretroviral and lentiviral vectors were found close to genes with high expression levels in primitive cells enriched for hematopoietic stem cells. These data help define the relative risk of insertional mutagenesis with MLV-, HIV-, and simian immunodeficiency virus (SIV)-based vectors in a highly relevant primate model.

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Foot-and-mouth disease virus (FMDV) produces one of the most infectious of all livestock diseases, causing extensive economic loss in areas of breakout. Like other viral pathogens, FMDV recruits proteins encoded by host cell genes to accomplish the entry, replication, and release of infectious viral particles. To identify such host-encoded proteins, we employed an antisense RNA strategy and a lentivirus-based library containing approximately 40,000 human expressed sequence tags (ESTs) to randomly inactivate chromosomal genes in a bovine kidney cell line (LF-BK) that is highly susceptible to FMDV infection and then isolated clones that survived multiple rounds of exposure to the virus. Here, we report the identification of ESTs whose expression in antisense orientation limited host cell killing by FMDV and restricted viral propagation. The role of one such EST, that of ectonucleoside triphosphate diphosphohydrolase 6 (NTPDase6; also known as CD39L2), a membrane-associated ectonucleoside triphosphate diphosphohydrolase that previously was not suspected of involvement in the propagation of viral pathogens and which we now show is required for normal synthesis of FMDV RNA and proteins, is described in this report.

In vivo selection strategies that convey a survival advantage to genetically modified cells carrying mutant forms of methylguanine methyltransferase (MGMT-P140K) have the potential to improve autologous and allogeneic stem cell gene therapy and transplantation. For some applications such as genetic diseases or anti-HIV strategies, in vivo selection may be required to increase initially low levels of gene-modified cells while for malignant diseases hematopoietic stem cell (HSC) chemo-protection may be necessary during chemotherapy dose escalation. Thus we have explored the use of gammaretrovirally expressed MGMT(P140K) mutant in three baboons. Animals received CD34−enriched cells transduced with a GALV-pseudotyped retroviral vector expressing a bicistronic message containing P140K and GFP. Two of the animals were part of a competitive repopulation assay in which one half of the cells were gene-modified with a GALV-pseudotyped vector expressing only YFP. After stable engraftment all three baboons were treated with various regimens of O6-benzylguanine (O6BG) and temozolomide (TMZ) or BCNU. Following treatment with O6BG/TMZ the selection was transient for ‘protected’ cells gene-modified with MGMT(P140K)-GFP, and the expected negative selection of ‘unprotected’ gene-modified cells (YFP transgene alone) was subtle. Conversely, positive selection of MGMT(P140K)-GFP gene-modified cells and negative selection of YFP gene-modified cells was dramatic and sustained following treatment with only a single dose of O6BG/BCNU. The increase in gene-marking (up to ~85%) is stable following selection out to 22 months. Importantly, selection of hematopoietic cells was polyclonal and no evidence of insertional mutagenesis has been detected. Aside from transient elevated liver enzymes following O6BG/BCNU treatment no additional extra-hematopoietic toxicity has been observed. We suspect that the delivery/absorption of TMZ in non-human primates is a contributing factor to transient selection because in animals with low levels (<1%) of MGMT(P140K) gene-modified cells no pronounced or sustained drop in white blood cell or platelet counts was observed following O6BG/TMZ. This is the case even up to TMZ dose levels of 700 mg/m2 that is above dose limiting toxicity in humans. In summary, MGMT selection is efficient and well tolerated in monkeys and we believe that these large animal studies closely reflect a clinical setting and will help to further improve clinical HSC gene therapy. Figure 1. Efficient in vivo selection and chemo-protection in non-human primates. (A) Representative gene-marking data in a baboon following chemotherapy treatment with either O6BG (120 mg/m2) and TMZ (600–1400 mg/m2) (solid arrows) or O6BG (120 mg/m2) and BCNU (40 mg/m2) (dashed arrows). The data is plotted as FACS+ MGMT-GFP granulocytes (closed circles) and FACS+ YFP granulocytes (open circles). (B) Absolute neutrophil counts following initial conditioning and subsequent chemotherapy treatment with O6BG/TMZ (solid arrows) or O6BG/BCNU (dashed arrow). Figure 1. Efficient in vivo selection and chemo-protection in non-human primates. (A) Representative gene-marking data in a baboon following chemotherapy treatment with either O6BG (120 mg/m2) and TMZ (600–1400 mg/m2) (solid arrows) or O6BG (120 mg/m2) and BCNU (40 mg/m2) (dashed arrows). The data is plotted as FACS+ MGMT-GFP granulocytes (closed circles) and FACS+ YFP granulocytes (open circles). (B) Absolute neutrophil counts following initial conditioning and subsequent chemotherapy treatment with O6BG/TMZ (solid arrows) or O6BG/BCNU (dashed arrow).

AIDS remains a significant health problem worldwide despite the advent of highly active antiretroviral therapy (HAART). Although substantial efforts have been made to develop a vaccine there is still no cure and alternative strategies are needed to treat HIV infection and to control its spread. Our goal is to evaluate lenti and foamy retroviral vectors that inhibit HIV replication by RNAi in a non-human primate SHIV model to develop a hematopoietic stem cell (HSC) gene therapy for AIDS. SHIV is a chimeric virus comprised of an SIV genome that contains the tat, rev and env genes of HIV and infects both T lymphocytes and macrophages. Infection of non-human primates with SHIV results in significant decreases in CD4+ T cells as early as 4 weeks post infection, and is currently the best large animal model available to test gene therapy strategies for AIDS. However inefficient gene delivery to hematopoietic stem cells has limited progress for AIDS gene therapy. We have developed both lenti and foamy retroviral vectors that contain methylguanine-DNA-methyltransferase (MGMT) expression cassettes to allow for in vivo selection, and have transduced macaque (M. nemestrina) long term repopulating cells with both vector systems. Following transplantation we observed rapid engraftment and levels of gene marking in the peripheral blood that should allow us to in vivo select both lenti and foamy-marked hematopoietic repopulating cells. In one animal transplanted with a lentiviral vector we obtained marking at 265 days post-transplant of over 30% in peripheral blood granulocytes and 20% in peripheral blood lymphocytes prior to in vivo selection. Anti-SHIV/HIV transgene cassettes targeting tat and rev that allow for potent inhibition of SHIV and HIV replication in vitro have been incorporated into both lenti and foamy vectors and we have transduced macaque long term repopulating cells with lenti vectors containing an anti-HIV cassette. We are currently developing protocols for efficient in vivo selection and future studies will investigate the ability of macaque hematopoietic repopulating cells transduced with lenti and foamy MGMT anti-HIV vectors to inhibit SHIV infection ex vivo and in vivo.