Abstract
Human Immunodeficiency Virus (HIV) and the subsequent acquisition of Acquired Immunodeficiency Syndrome (AIDS) continues to affect millions of lives around the world with no effective vaccine available. Current antiretroviral treatments are effective at repressing the infection but do not cure it. The high mutation rate of HIV has made it difficult to design drugs against, however, a new gene therapy method may provide a better alternative for HIV treatment. By combining multiple highly effective anti-HIV genes into a single gene therapy vector that can be transduced into cells, a one-time treatment may provide lifelong HIV resistance. This experiment utilizes a replication-incompetent lentiviral vector encoding for three highly effective anti-HIV genes which target separate stages of the HIV lifecycle: a CCR5 shRNA (pre-entry), a chimeric TRIM5α protein (post-entry/pre-integration) and a TAR decoy (post-integration). When challenged with HIV, cultured and primary CD34+ hematopoietic progenitor cell (HPC)-derived macrophages transduced with this triple combination vector showed potent inhibition of viral pre-integration infection. The generation of mutant viral particles was blocked as well after long-term evaluation of challenged cells. When the combination anti-HIV lentiviral vector was tested in vivo in mice engineered to have elements of the human immune system, the vector proved safe and effective at inhibiting HIV infection. It was then determined that the vector must be produced in large amounts for clinical applications. During my internship at the UC Davis Institute for Regenitive Cures, I manufactured this anti-HIV lentiviral vector at a titer of 109 viral particles per mL. In addition to providing cures for infectious diseases, such as HIV, gene therapy may also provide a permanent treatment for genetic diseases such as epidermolysis bullosa (EB), through the derivation and subsequent manipulation of patient-derived induced pluripotent stem cells (iPSCs). By converting a patient’s own cells containing a genetic defect into iPSCs, the genetic defect can be corrected and then the pluripotent iPSCs can then be differentiated into any cell type in the body and grafted back into the patient. In order to derive these iPSCs, the patient’s cells must be transduced with a lentiviral vector containing genes that “reprogram” these cells back to a pluripotent state. In my project, I manufactured a single DNA vector containing the four genes: Oct4, Sox2, c-Myc and Klf4 which would, once inside a cell, cause the cell to be “reprogrammed” to a pluripotent state.