Identification of cellular genes affecting the infectivity of foot-and-mouth disease virus.

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.

Efficient transduction of pigtailed macaque hematopoietic repopulating cells with HIV-based lentiviral vectors.

Lentiviral vectors are attractive for hematopoietic stem cell (HSC) gene therapy because they do not require mitosis for nuclear entry, they efficiently transduce hematopoietic repopulating cells, and self-inactivating (SIN) designs can be produced at high titer. Experiments to evaluate HIV-derived lentiviral vectors in nonhuman primates prior to clinical trials have been hampered by low transduction frequencies due in part to host restriction by TRIM5alpha. We have established conditions for efficient transduction of pigtailed macaque (Macaca nemestrina) long-term repopulating cells using VSV-G-pseudotyped HIV-based lentiviral vectors. Stable, long-term, high-level gene marking was observed in 3 macaques using relatively low MOIs (5-10) in a 48-hour ex vivo transduction protocol. All animals studied had rapid neutrophil engraftment with a median of 10.3 days to a count greater than 0.5 x 10(9)/L (500/microL). Expression was detected in all lineages, with long-term marking levels in granulocytes at approximately 20% to 30%, and in lymphocytes at approximately 12% to 23%. All animals had polyclonal engraftment as determined by analysis of vector integration sites. These data suggest that lentiviral vectors should be highly effective for HSC gene therapy, particularly for diseases in which maintaining the engraftment potential of stem cells using short-term ex vivo transduction protocols is critical.

Comparison of HIV-derived lentiviral and MLV-based gammaretroviral vector integration sites in primate repopulating cells.

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.