Construction And Evaluation Of Gene Transfer Systems Derived From Equine Infectious Anemia Virus Donkey Leukocyte Attenuated Strain

Lentiviruses, like other retroviruses, with the abilities to integrate efficiently their genome into the chromosomal DNA of host cells and be stably replicated and transmitted to all of the progeny of these cells offering the potential for long-term expression, can be as gene transfer vectors to introduce the foreign target gene into host cells. In contrast to typical vectors derived from oncoretroviruses, which can only infect dividing cells, lentivirus-based vectors have received more considerable attention for gene delivery because lentiviruses are also able to infect nondividing, terminally differentiated target cells. Gene transfer systems based on human immunodeficiency virus type 1 (H1V-1) are by far the most developed lentiviral systems. Although HIV-1 vectors have been much more modified to maximize their biosafety and to minimize the potential for production of replication-competent viruses, the potential safety risks still limit HIV-1 vectors for human clinical applications. Such concerns are leading to the development of non-primate lentiviral vectors.Equine infectious anemia virus (EIAV) is a non-primate and the genetically simplest lentivirus. Chinese equine infectious anemia virus donkey leukocyte attenuated strain (E1AV-DLA) is a unique lentiviral vaccine strain used widely for prevention and control of equine infectious anemia (EIA) in China up to now. This study is to construct EIAV gene transfer vector systems based on an infectious molecular clone, pOK.8266, derived from the ElAV-DLA and to offer a new approach for gene therapy, development of vaccines, and viral pathogenic and immunological mechanisms of the EIAV and other related viruses.A series of replication-deficient EIAV gene transfer vector plasmids, envelope gene-deleted gag/pol expression plasmids and VSV-G gene expression plasmids were constructed and the gene transfer vector systems of these three plasmids were established by reverse genetic system. The results showed that the 293T cells could efficiently express the enhanced green fluorescent protein (EGFP) after transient transfection or transduction of the replication-deficient EIAV gene transfer vectors replaced EIAV-5'LTR-U3 region with human cytomegalovirus immediate-early promoter (CMV). The internal promoter and the "central polypurine tract (cPPT)/Rev responsive element (RRE) inserted into the vectors might increase expression of EGFP. The function of hepatitis B virus posttranscriptional regulatory element (HPRE) for EGFP expression is orientation-specific, HPRE inserted in reverse orientation could inhibit the EGFP expression, but this could be counteracted by cPPT/ RRE. An intron located between the CMV promoter and the VSV-G gene in VSV-G expression vector might increase the VSV-G expression, but the intron located between the CMV promoter and the EIAV gene in gag/pol expression vector was not for gag/pol expression.