Methotrexate (MTX) dose-escalation studies were conducted in C57BL/6 mice to determine the chemoprotective effect of transplantation using bone marrow transduced with lentivirus vectors expressing a drug-resistant variant of murine dihydrofolate reductase (DHFR). Methotrexate-resistant dihydrofolate reductase [tyrosine-22 (Tyr22)DHFR] and enhanced green fluorescent protein (GFP) coding sequences were inserted into self-inactivating lentiviral vectors as part of a genetic fusion or within the context of a bicistronic expression cassette. MTXtreated animals that received Tyr22DHFR-transduced marrow recovered to normal hematocrit levels by 3 weeks post-transplant and exhibited significant GFP marking in myeloid and lymphoid lineage-derived peripheral blood mononuclear cells (PBMCs). In contrast, MTX-treated animals transplanted with control GFP-transduced marrow exhibited extremely reduced hematocrits with severe marrow hypoplasia and did not survive MTX dose escalation. To minimize cell manipulation, we treated unfractionated marrow in an overnight exposure. Transduction at a multiplicity of infection of 10 resulted in up to 11% vectormodified PBMCs in primary recipients and successful repopulation of secondary recipients with vector-marked cells. Experimental cohorts exhibited sustained proviral expression with stable GFP fluorescence intensity. These results demonstrate the effectiveness of lentivirus vectors for chemoprotection in a well developed animal model, with the potential for further preclinical development toward human application.
Artemis is a hairpin-opening endonuclease involved in nonhomologous end-joining and V(D)J recombination. Deficiency of Artemis results in radiation-sensitive severe combined immunodeficiency (SCID) characterized by complete absence of T and B cells due to an arrest at the receptor recombination stage. We have generated several lentiviral vectors for transduction of the Artemis sequence, intending to complement the deficient phenotype. We found that transduction by a lentiviral vector in which Artemis is regulated by a strong EF-1a promoter resulted in a dose-dependent loss of cell viability due to perturbed cell cycle distribution, increased DNA damage, and increased apoptotic cell frequency. This toxic response was not observed in cultures exposed to identical amounts of control vector. Loss of cell viability was also observed in cells transfected with an Artemis expression construct, indicating that toxicity is independent of lentiviral transduction. Reduced toxicity was observed when cells were transduced with a moderate-strength phosphoglycerate kinase promoter to regulate Artemis expression. These results present a novel challenge in the establishment of conditions that support Artemis expression at levels that are nontoxic yet sufficient to correct the T À B À phenotype, crucial for preclinical studies and clinical application of Artemis gene transfer in the treatment of human SCID-A.
Proliferative enteritis is an enteric disease that affects a variety of animals. The causative agent in swine has been determined to be an obligate intracellular bacterium, Lawsonia intracellularis, related to the sulfatereducing bacterium Desulfovibrio desuljhricans. The intracellular agents found in the lesions of different animal species are antigenically similar. In addition, strains from the pig, ferret, and hamster have been shown to be genetically similar. In this study we performed a partial 16s ribosomal DNA sequence analysis on the intracellular agent of proliferative enteritis from a hamster, a deer, and an ostrich and compared these sequences to that of the porcine L. intracellularis isolate. Results of this study indicate that the intracellular agents from these species with proliferative enteritis have high sequence similarity, indicating that they are all in the genus Lawsonia and that they may also be the same species, L. intracellularis.proliferative enteritis (PE) is an enteric disease that occurs primarily in weanling animals but is also seen in older animals. It has been reported in a variety of species, including the pig, hamster, ferret, fox, rat, rabbit (27), horse (5, 34), deer (4), ostrich (36), and emu (17). The disease has been most thoroughly studied in the pig and the hamster (27). It may be chronic. causing weight loss and poor growth, or more acute, occasionally resulting in death. Antibiotics may be used to treat PE; however, many cases resolve spontaneously. The disease is widespread in swine. Poor weight gain and deaths during epizootics result in production loss and represent a significant cost to the swine industry (14, 27). The significance of PE in other spccies is not known. Because of the wide host range of the disease, it is likely that there are numerous reservoirs, and the disease may represent an equally serious threat to these species.PE causes hyperplasia of the intestinal mucosa, specifically the crypt epithelial cells. Large numbers of small, curved intracellular bacteria are present within the proliferating cells (27). The disease may be diagnosed by observation of these lesions at necropsy, by detecting the intracellular organism in histologic sections with silver strain (27), or by various immunologic (1.5, 20) and molecular (8,9,12, 13,19,22) methods. Serologic diagnosis of PE has not proven to be useful (10, 16). The organism has been isolated in cell cultures from hamsters and pigs with PE but has not been grown on cell-free medium (15, 3 1). Transmission studies have shown that this bacterium is the causative agent of PE in pigs (15, 23) and probably hamster5 (31). Sequence analysis of the 16s ribosomal DNA (rDNA) of this bacterium shows that it is most closely related to the sulfate-reducing bacterium Desulfovibrio desulfiricans. The organism was given the vernacular name of ileal symbiont intracellularis (7), which has recently been formalized to Lawsonia intracellularis (21). The intracellular agents of PE are morphologically identical in all species that...
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