ALBERT

Description: Background: Autologous stem cell transplantation (ASCT) has become an accepted treatment option for high risk or relapsed ARL. Treatment related mortality is similar to the HIV negative setting. However, ultimately further improvement in ASCT will depend on both effective anti lymphoma therapy and better control of the HIV infection. Highly active antiretroviral therapy (HAART) can lower HIV viral loads to undetectable levels in the peripheral blood, but reservoirs of HIV are still present in the tissues and acquired resistance to HAART also remains a problem. A treatment strategy that would confer intrinsic resistance to HIV could circumvent theses issues. Herein we report on one such strategy using multiplexed RNA based anti-HIV gene transfer strategies to render autologous peripheral blood progenitor cells resistant to HIV. Patients with high risk ARL deemed candidates for ASCT were eligible. Seven subjects with NHL have been enrolled. (4PR, 2 REL, 1CR2), of whom 2 failed screening phase, 1 failed product release test, 2 are pending transplant, and 2 patients have undergone successful transplantation. Median age was 43 yrs at enrollment. Four pts to date were mobilized with chemotherapy plus GCSF and cells were collected for the clinical product (Fx1) and for CD34-selection (CliniMACSª, Miltenyi) and research treatment (Fx2). (see table ) UPN # Fx1 (CD34+/kg) Fx2 (CD34+/kg) Post Selection and transductionCD34+/kg 301 2.8X106 3.5X106 .26 X106 not infused 304 3.9X106 3.6X106 1.2 X106 305 3.4X106 3.8X106 1.4X106 306 5.6X106 8.8X106 pending Three days prior to the completion of CBV (cyclophosphamide 100mg/kg, BCNU 450mg/ m2, VP16 60mg/kg) conditioning, the Fx2 cells were thawed and transduced with a lentivirus vector (LV,rHIV7-ShI-TAR-CCR5Z) encoding 3 RNA elements including short hairpin RNA (shRNA) targeted to HIV tat/rev, a nucleolar localizing TAR decoy sequence, and a ribozyme targeted to CCR5. Cell viability post transduction ranged between 52–64% in three pts. On day 0 Fx2 is given and Fx1 is given 24hrs later (day+1). UPN301 did not receive the transduced Fx2 cells due to a low cell dose. For UPN304 and 305 who received the gene modified Fx2 cells, WBC engraftment occurred at day +11, platelet engraftment at day+16, and there have been no serious adverse events. Results to date at 30 and 60 days post ASCT reveal peripheral blood marking consistent with the ratio of gene modified to unmodified cells infused. Q-PCR analysis demonstrated distribution of genetically modified cells in myeloid and lymphoid lineages, and RT-PCR evidence of shRNA in progeny cells provided further evidence of successful transduction and engraftment of progenitor cells. Follow-up data for these and subsequent patients will be presented at the meeting. Conclusion: Lentiviral vector transduction of autologous peripheral blood progenitor cells with multiplexed RNA is feasible, well tolerated, and led to successful engraftment following high dose chemotherapy for ARL.

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: Background: Optimal therapy of ARL relies on both effective anti-tumor chemotherapy and successful control of the underlying HIV infection. Management of HIV using HAART has been hampered by patient non-compliance with complex regimens, drug resistance and ongoing low level viral replication. Multiplexed RNA based anti-HIV gene transfer strategies to confer intrinsic cellular resistance may help circumvent these problems. Autologous stem cell transplantation for ARL is an ideal clinical platform for delivery of SiRNA transduced stem cells as combining gene transfer strategy with high dose antilymphoma therapy could provide control of both the HIV infection and the ARL. Methods: Gene transfer - anti-HIV RNA elements, including short hairpin RNA (shRNA) targeted to HIV tat/rev, a TAR-specific decoy sequence, and a ribozyme targeted to CCR5 were combined into a lentivirus vector (LV, rHIV7-shI-TAR-CCR5RZ). Using LV transduction methods, these anti-HIV RNAs were delivered into CD34+ hematopoietic progenitor cells (HPC). Results: Preclinical vector development - LV transduction allowed differentiation in liquid culture and in a SCID-hu model which produced macrophage and T cell progeny that were resistant to the HIV virus. Although HIV resistance can be induced in vitro with single anti-HIV shRNAs, no resistance was found in multiply passaged HIV in rHIV7-shI-TAR-CCR5RZ-transduced cells. In addition, cells were analyzed by microarray for mRNA and miRNA alterations and no significant changes were noted between CD34 transduced and untransduced cells. Copy number in transduced cells was 1–2/cell, and integration site analysis localized to transcriptionally active sites, usually away from terminal portions of gene sequences. This vector is proposed for use in ASCT for ARL. Update of ASCT in ARL: Between 1998 and 2006, 28 patients with high-risk ARL underwent ASCT at the City of Hope. Conditioning regimens included CBV (cyclophosphamide (Cy)100mg/kg, Carmustine150 mg/m2, VP16 60mg/kg) in 24 and FTBI/Cy/VP16 in 4. All patients engrafted, median time to ANC〉500 was 11 days (range, 8–19). Regimen related toxicities included grade 3–4 hepatic toxicity n=3 and interstitial pneumonitis n=2. OI’s included PCP pneumonia n=2, CMV infection (2 viremias, 1 retinitis) and 2 cases of VZV. Therapy-related MDS was seen in one patient who ultimately died of MDS while in remission from his ARL. Median HIV viral load (VL) at ASCT was 6113 gc/ml with 22 having an undetectable VL. At two years only 9 pts had an undetectable VL. Median CD4 count at ASCT was 164 (range 25–1064), this rose to 263 (95–1164) at two years post ASCT. With a median f/u of 41 months, 2yr OS is 78% (95% CI 63–87) and PFS 78% (95% CI 63–87). Conclusions: ASCT can lead to durable remission for ARL. The fluctuation in VL seen post ASCT reflects the natural history of HIV infection and limitations of current antiviral therapy. Ultimately the system of gene transfer outlined above combined with ASCT as part of our next generation clinical trial could provide the optimal chance of cure for pts with high-risk ARL by offering both effective antilymphoma and anti-HIV therapy.

Description: Increased levels of Bcr-Abl expression in chronic myelogenous leukemia (CML) cells are associated with disease progression and imatinib (IM) resistance. However, it is not clear if these associations are a direct result of elevated Bcr-Abl expression. We used a human transduction model of CML to directly investigate the role of varying Bcr-Abl expression levels in determining the phenotype and IM sensitivity of hematopoietic cells. CD34+ cells were transduced with vectors coexpressing Bcr-Abl and GFP, and cells expressing low and high levels of GFP and Bcr-Abl (BAlo and BAhi) were selected. BAhi cells demonstrated enhanced activation of downstream proliferative and antiapoptotic signaling and enhanced proliferation and survival compared to BAlo cells. Freshly isolated BAhi CD34+ cells and cell lines demonstrated increased IM-mediated growth inhibition likely reflecting Bcr-Abl dependence for growth and survival. CD34+ cells expressing BCR/ABL kinase-mutant genes demonstrated resistance to IM-mediated inhibition of proliferation and viability, which was not enhanced by increased expression of BCR/ABL kinase-mutant genes. We conclude that Bcr-Abl overexpression results in increased proliferation and antiapoptotic signaling in CD34+ cells, but may not play a direct role in IM resistance in progenitor cells expressing either wild-type or mutant BCR/ABL genes.