ALBERT

Description: AIDS remains a significant health problem worldwide despite the advent of highly active antiretroviral therapy (HAART). Although substantial efforts have been made to develop a vaccine there is still no cure and alternative strategies are needed to treat HIV infection and to control its spread. Our goal is to evaluate lentiviral vectors that inhibit HIV replication by RNA interference (RNAi) in a non-human primate SHIV model to develop a hematopoietic stem cell (HSC) gene therapy for AIDS. SHIV89.6 P is a chimeric virus comprised of an SIV genome that contains the tat, rev and env genes of HIV and infects both T lymphocytes and macrophages. Infection of non-human primates with SHIV89.6P results in significant decreases in CD4+ T cells as early as 4 weeks post infection, and is currently the best large animal model available to test gene therapy strategies for AIDS. We present here data showing efficient transduction of M. nemestrina CD34+ cells with an HIV-based lentiviral vector and RNAi-mediated inhibition of SHIV89.6 P replication in a hybrid T/B lymphocyte cell line (CEMx174). Although others reported a block to transduction of M. mulatta CD34+ cells with an HIV-based lentiviral vector, we observed efficient transduction rates (» 50%) of M. nemestrina CD34+ cells, comparable to transduction rates observed in human CD34+ cells (» 60%). To determine effectiveness of anti tat/rev shRNA to inhibit SHIV89.6P in vitro, a human T cell/B cell hybrid cell line (CEMx174) was transduced with a lentiviral vector expressing a short-hairpin RNA (shRNA) targeted to both HIV tat and rev sequences that also contained either a GFP reporter gene or a MGMT(G156A) resistance gene at MOIs of 1.3 and 3 respectively. Polyclonal populations of CEMx174 cells transduced with the GFP and MGMT(G156A) vectors were challenged with a 2.15x103 TCID50 dose of SHIV 89.6P. One week post challenge, expression of both tat and rev transcripts was reduced 88% and 97% respectively in these cultures as measured by real-time PCR. In summary, we have shown efficient HIV-based lentiviral transduction of M. nemestrina cells and efficient inhibition of SHIV infection by shRNA against HIV tat and rev thus providing a useful model to test lentiviral-mediated anti-HIV RNAi stem cell gene therapy in vivo.

Description: AIDS remains a significant health problem worldwide despite the advent of highly active antiretroviral therapy (HAART). Although substantial efforts have been made to develop a vaccine there is still no cure and alternative strategies are needed to treat HIV infection and to control its spread. Our goal is to evaluate lenti and foamy retroviral vectors that inhibit HIV replication by RNA interference (RNAi) in a non-human primate SHIV model to develop a hematopoietic stem cell (HSC) gene therapy for AIDS. SHIV is a chimeric virus comprised of an SIV genome that contains the tat, rev and env genes of HIV and infects both T lymphocytes and macrophages. Infection of non-human primates with SHIV results in significant decreases in CD4+ T cells as early as 4 weeks post infection, and is currently the best large animal model available to test gene therapy strategies for AIDS. We are developing methylguanine-DNA-methyltransferase (MGMT) selection strategies for HSCs in the primate model to allow for high level marking with vectors containing anti-SHIV/HIV transgenes. We have obtained marking levels over 90% in granulocytes and over 30% in lymphocytes. To determine the effectiveness of an anti tat/rev shRNA to inhibit SHIV in vitro, a human T cell/B cell hybrid cell line (CEMx174) was transduced with a lentiviral vector expressing a short-hairpin RNA (shRNA) targeted to both HIV tat and rev sequences that also contained either a GFP reporter gene or a MGMT(G156A) resistance gene. Polyclonal populations of CEMx174 cells transduced with the GFP and MGMT(G156A) vectors were challenged with a 2.15x103 TCID50 dose of SHIV. Expression of both tat and rev transcripts was reduced 88% and 97% respectively in these cultures as measured by real-time PCR and replication of SHIV was inhibited as evidenced by inhibition of p27 production. Although others reported a block to transduction of M. mulatta CD34+ cells with an HIV-based lentiviral vector, we observed efficient transduction rates (~45%) of M. nemestrina CD34+ cells, comparable to transduction rates observed in human CD34+ cells (~55%). Thus M. nemestrina monkeys provide a powerful model to test lenti and foamy virus mediated anti-HIV gene therapy strategies.

Description: Graft failure and graft-versus-host-disease (GvHD) remain significant complications of allogeneic stem cell transplantations. To reduce the risk of GvHD, patients often receive a graft that has been depleted of T-cells, which in turn increase the risk of graft failure. Studies have suggested that co-transplantation of ex vivo expanded marrow derived mesenchymal stem cells (MSCs) with allogeneic hematopoietic stem cells (HSC) reduces the risk of GvHD and graft rejection. Additionally, it has been shown that infusion of expanded marrow derived MSCs improves ongoing acute GvHD and facilitates engraftment across MHC-barriers. The effect of MSCs is thought to be due to their ability to suppress T-cell activation. Here we wished to examine whether inhibition of T-cells could be enhanced by MSCs from different tissues. We were particularly interested in MSCs derived from adipose tissue, because of its abundance and accessibility, which is especially important for repeat applications. We investigated and compared the immunosuppressive potential of MSCs derived from multiple mesenchymal tissues of different origins, such as adipose tissue, muscle tissue, omentum, and bone. Cells from the different tissues were enriched for MSCs and cultured and expanded for two to three weeks to deplete hematopoietic cells. MSCs from all tissues differentiated into multiple lineages and stained positive using antibodies against common MSC markers (CD29, CD44, CD105, CD106) and negative for hematopoietic markers (CD3, CD14, CD34, CD45). Mixed lymphocyte reactions (MLRs) using either a mitogen (Concanavalin A) or allogeneic T-cells as inducers of T-cell activation were performed in the presence or absence of MSCs from the different tissues. Mitogen-induced T-cell activation, as well as allogeneic T-cell responses were significantly reduced in MLRs with MSCs compared to MLRs without MSCs. The most pronounced effects were observed with MSCs from muscle tissue (90% reduction) in the mitogen-induced MLR and with MSCs from adipose tissue (85% reduction) in the allogeneic MLRs. These results suggest that MSCs from adipose tissue, which is easily optained and abundantly available, could be used to modulate GvHD and facilitate engraftment across MHC barriers. In conclusion our data demonstrate that MSCs from multiple tissues efficiently reduce T-cell activation and suggest that MSCs from adipose tissue can serve as an alternative source for MSCs to modulate GvHD or facilitate engraftment across MHC barriers. In vivo studies to test the effects on GvHD are currently under way.