Pools of evidence have revealed a distinguishable arm of the innate immune system that reduces the efficient delivery of vector cargo into stem cells. We hypothesize that quiescent progenitor cells express innate immune factors to protect against pathogens and transduction further mediates an immune response that reduces viral pre-integration complex (PIC) formation and prevents efficient delivery and integration into host chromatin. We are currently analyzing the endogenous expression of innate immune genes in hematopoietic progenitor cultures and those transduced with VSV-G psuedotyped lentiviral vectors to correlate expression of innate genes with transduction efficiency. Innate immune gene responsiveness in CD34+ transduced cultures may confer the ability to induce and maintain a strong intrinsic antiviral response and subsequently decreasing transduction efficiency. So, by investigating which innate factors are responsive to transduction, we will target these specific factors to counteract there immune activity in order to increase transduction efficiency. The potential for improving vector delivery would contribute to more efficient clinical usage of viral vectors as tools for gene therapy. Following successful gene transfer to hematopoietic stem and progenitor cells,transgene expression may fall overtime resulting from silencing through methylation and other means. Gamma-satellite DNA has been identified in the percentromeric regions of human chromosomes. The gamma-satellite DNA is a tandem array of 220bp CG-reich repetitive units, usually forming 10-200 kb clusters. The function remains obscure. However, a recent work by NIH investigators suggests that the repetitive DNA might possess insulator activity. We hypothesize that the gamma-satellite DNA will provide stable trangene expression from SIN lentivirus vectors. We have cloned various sizes of the gamma-satellite repetitive DNA fragments and inserted these fragments into the deletion site of 3'LTR of an HIV-1 based lentiviral vector to evaluate the insulator activity with these fragments. The vector plasmids were constructed containing EGFP cDNA under the control of MSCV promoter: simple control, insertion of the control chicken HS4 insulator and insertion of several sizes of the gamma-satellite DNA. Human erythroid cell lines were transduced with these lentivirus vectors, and vector expression will be followed long term to monitor transgene expression. In order to test these lentiviral vectors in the preclinical nonhuman primate model, several modifications of HIV-1 based lentiviral vectors have been made to enable efficient transduction of rhesus macaque derived hematopoietic stem cells. These modifications include the use of the cyclophillin binding domain of the macrophage tropic strain along with portions of the simian immunodeficiency virus capsid. Testing of these constructs demonstrates improved transduction rates and these viral vectors will now be tested in the nonhuman primate model.
| Hayakawa, Jun; Ueda, Takahiro; Lisowski, Leszek et al. (2009) Transient in vivo beta-globin production after lentiviral gene transfer to hematopoietic stem cells in the nonhuman primate. Hum Gene Ther 20:563-72 |
| Uchida, Naoya; Washington, Kareem N; Hayakawa, Jun et al. (2009) Development of a human immunodeficiency virus type 1-based lentiviral vector that allows efficient transduction of both human and rhesus blood cells. J Virol 83:9854-62 |
