?-Retroviral vectors (?-RV) are powerful tools for gene therapy applications. Current clinical vectors are produced from stable producer cell lines which require minimal further downstream processing, while purification schemes for ?-RV produced by transient transfection have not been thoroughly investigated. We aimed to develop a method to purify transiently produced ?-RV for early clinical studies. Here, we report a simple one-step purification method by high-speed centrifugation for ?-RV produced by transient transfection for clinical application. High-speed centrifugation enabled the concentration of viral titers in the range of 10(7)-10(8) TU/ mL with >80% overall recovery. Analysis of research-grade concentrated vector revealed sufficient reduction in productand process-related impurities. Furthermore, product characterization of clinical-grade ?-RV by BioReliance demonstrated two-logs lower impurities per transducing unit compared with regulatory authority-approved stable producer cell line vector for clinical application. In terms of CAR T cell manufacturing, clinical-grade ?-RV produced by transient transfection and purified by high-speed centrifugation was similar to ?-RV produced from a clinical-grade stable producer cell line. This method will be of value for studies using ?-RV to bridge vector supply between early- and late-stage clinical trials.

Recent advances in the genetic modification of primary cells for ex vivo gene therapy have brought the field close to the promise envisioned almost a quarter of a century ago. The integration of vector systems that can stably transduce cells without genotoxicity, combined with superior cell processing methods, has been largely responsible for these advances. The result has been a series of ongoing clinical trials that are showing promising outcome for patients. Among the successes are adoptive T cell immunotherapy and stem cell therapies for cancer and other diseases.

Recent advances in the genetic modification of primary cells for ex vivo gene therapy have brought the field close to the promise envisioned almost a quarter of a century ago. The integration of vector systems that can stably transduce cells without genotoxicity, combined with superior cell processing methods, has been largely responsible for these advances. The result has been a series of ongoing clinical trials that are showing promising outcome for patients. Among the successes are adoptive T cell immunotherapy and stem cell therapies for cancer and other diseases.

Meeting abstracts Previous studies in adoptive T-cell transfer have suggested that persistence of the transduced T-cells is central to making this therapy a viable option. Understanding the behavior of tumor-reactive T-cells in cancer patients and measuring persistence are two objectives in a phase

Patients with Pompe disease, a rare progressive neuromuscular disorder, receive a considerable amount of informal care. In this study, we examined the impact of providing informal care to patients with Pompe disease.Caregivers were administered various instruments, which measured the (impact of) informal care in the context of Pompe disease. Patients' quality of life and use of a wheelchair and respiratory support were used to investigate the impact of disease severity on the burden and well-being of caregivers.Of all Dutch patients with Pompe disease, 88 indicated to receive informal care, of which 67 (76%; 67 caregivers) participated in this study. On average, caregivers provided 17.7 hours of informal care per week. Higher disease burden was associated with more hours of informal care. Caregivers experienced burden due to caregiving. Half of the informal caregivers reported mental health problems and problems with daily activities due to providing informal care. Physical health problems occurred in 40% of informal caregivers. Caregiver burden was higher for patients with a lower quality of life and for wheelchair dependent patients. Burden was not associated with respiratory support. Caregivers reported deriving personal fulfillment from caregiving and, on average, would become unhappier if someone else were to take over their care activities.The provision of informal care causes burden to caregivers. However, caregivers also value caring for their loved ones themselves. The study may help physicians and policy makers to design measures to support informal caregivers.

We report the safety and tolerability of 87 infusions of lentiviral vector–modified autologous CD4 T cells (VRX496-T; trade name, Lexgenleucel-T) in 17 HIV patients with well-controlled viremia. Antiviral effects were studied during analytic treatment interruption in a subset of 13 patients. VRX496-T was associated with a decrease in viral load set points in 6 of 8 subjects (P = .08). In addition, A → G transitions were enriched in HIV sequences after infusion, which is consistent with a model in which transduced CD4 T cells exert antisense-mediated genetic pressure on HIV during infection. Engraftment of vector-modified CD4 T cells was measured in gut-associated lymphoid tissue and was correlated with engraftment in blood. The engraftment half-life in the blood was approximately 5 weeks, with stable persistence in some patients for up to 5 years. Conditional replication of VRX496 was detected periodically through 1 year after infusion. No evidence of clonal selection of lentiviral vector–transduced T cells or integration enrichment near oncogenes was detected. This is the first demonstration that gene-modified cells can exert genetic pressure on HIV. We conclude that gene-modified T cells have the potential to decrease the fitness of HIV-1 and conditionally replicative lentiviral vectors have a promising safety profile in T cells.

In light of findings demonstrating that the macaque TRIM5alpha protein inhibits infection of cells by human immunodeficiency virus (HIV)-1, simian immunodeficiency virus (SIV)-based lentiviral vectors may have distinct advantages over HIV-1 vectors for the transduction of macaque hematopoietic stem cells. We evaluated the ability of an SIV vector (VRX859) encoding an antisense SIV envelope sequence and enhanced green fluorescent protein (GFP) to inhibit viral replication and to transduce rhesus CD34(+) lymphoid progenitor cells. After infection with homologous SIV strains, CD4(+) cell lines transduced with VRX859 exhibited more than 600-fold inhibition of viral replication compared with control cells. Less inhibition was observed with the divergent SIV strain SIVsmE660. Partial inhibition of a chimeric simian-human immunodeficiency virus, which contains an HIV-1 envelope in an SIV backbone, was observed, suggesting that the SIV vector also contributes to viral inhibition independent of the antisense envelope inhibitor. Transduction of rhesus CD34(+) cells with VRX859 at various multiplicities of infection resulted in transduction efficiencies comparable to those obtained with the HIV vector VRX494. However, when we evaluated transduction of rhesus T lymphocyte progenitors by examining GFP expression in CD4(+) T cells derived from transduced CD34(+) cells, we observed more efficient transduction with the SIV-based vector. GFP(+)CD4(+) T cells derived from VRX859-transduced CD34(+) cells strongly inhibited SIVmac239 replication as compared with control CD4(+) T cells. The ability of this SIV-based vector to mediate potent inhibition of SIV replication, coupled with its efficient transduction of rhesus hematopoietic progenitor cells, make it an important candidate for proof-of-principle experiments of stem cell gene therapy in the SIV-macaque model.

Gene therapy for HIV-1 infection has been proposed as an alternative to antiretroviral drug regimens due to emerging drug resistance and toxicity that raises concerns about HAART as a long term therapy. We have previously reported the successful completion of our Phase I clinical trial testing the safety and tolerability of a single dose of autologous HIV infected CD4+ T cells transduced with a lentiviral vector delivery system expressing a 937-base antisense gene against the HIV envelope (VRX496) for use in T cell therapy for HIV/AIDS. These results have lead us to initiate a Phase II clinical trial to evaluate the safety, tolerability, and biological activity of repeated infusions (4 or 8 doses) of autologous VRX496 transduced T cells. The study will enroll up to 40 male and female HIV-positive subjects in up to 8 centers. Subjects will be 18 years of age and over who have failed or are intolerant to at least one triple combination of antiretroviral drugs. Subjects will have, a viral load between 5,000 and 200,000 copies/ml and a CD4+ count of ≥150. Additionally, subjects must have a Karnofsky Performance score of 80 or higher, have no evidence of active opportunistic infection, congestive heart failure, hemodynamic instability, bleeding diathesis and no contraindications for leukapheresis. Patients will receive repeated doses by i.v. of 1*1010 genetically modified autologous T-cells, every two weeks. The primary safety objectives will include incidence of adverse events, changes in viral load, changes in CD4+ T cells, changes in TCR v beta repertoire, and tests for replication competent lentivirus (RCL). Tissue trafficking of gene modified cells will also be monitored in GALT tissue. The first part of the study is evaluating the safety and tolerability of multiple dosing using a repeat dosing design. All patients in the 4 dose cohort have received their infusions as scheduled and have reached the 3 month post-infusion visit, and one patient in the 8 dose cohort has received all of the doses as scheduled. To date, all of the doses have been well tolerated and preliminary results suggest multiple infusions are safe, with no SAE's due to the product, no changes in hematology or chemistry laboratory evaluations, and no detection of VSVG DNA or RCL. In the second part, we are determining an optimal dosing regimen for future confirmatory trials. To date 18 patients have been enrolled and 11 have started receiving infusions. The data generated from the current clinical trial demonstrates the clinical utility of lentiviral vector technology as an alternative for treatment of HIV infection. Gene therapy for HIV-1 infection has been proposed as an alternative to antiretroviral drug regimens due to emerging drug resistance and toxicity that raises concerns about HAART as a long term therapy. We have previously reported the successful completion of our Phase I clinical trial testing the safety and tolerability of a single dose of autologous HIV infected CD4+ T cells transduced with a lentiviral vector delivery system expressing a 937-base antisense gene against the HIV envelope (VRX496) for use in T cell therapy for HIV/AIDS. These results have lead us to initiate a Phase II clinical trial to evaluate the safety, tolerability, and biological activity of repeated infusions (4 or 8 doses) of autologous VRX496 transduced T cells. The study will enroll up to 40 male and female HIV-positive subjects in up to 8 centers. Subjects will be 18 years of age and over who have failed or are intolerant to at least one triple combination of antiretroviral drugs. Subjects will have, a viral load between 5,000 and 200,000 copies/ml and a CD4+ count of ≥150. Additionally, subjects must have a Karnofsky Performance score of 80 or higher, have no evidence of active opportunistic infection, congestive heart failure, hemodynamic instability, bleeding diathesis and no contraindications for leukapheresis. Patients will receive repeated doses by i.v. of 1*1010 genetically modified autologous T-cells, every two weeks. The primary safety objectives will include incidence of adverse events, changes in viral load, changes in CD4+ T cells, changes in TCR v beta repertoire, and tests for replication competent lentivirus (RCL). Tissue trafficking of gene modified cells will also be monitored in GALT tissue. The first part of the study is evaluating the safety and tolerability of multiple dosing using a repeat dosing design. All patients in the 4 dose cohort have received their infusions as scheduled and have reached the 3 month post-infusion visit, and one patient in the 8 dose cohort has received all of the doses as scheduled. To date, all of the doses have been well tolerated and preliminary results suggest multiple infusions are safe, with no SAE's due to the product, no changes in hematology or chemistry laboratory evaluations, and no detection of VSVG DNA or RCL. In the second part, we are determining an optimal dosing regimen for future confirmatory trials. To date 18 patients have been enrolled and 11 have started receiving infusions. The data generated from the current clinical trial demonstrates the clinical utility of lentiviral vector technology as an alternative for treatment of HIV infection.

We report findings from a clinical evaluation of lentiviral vectors in a phase I open-label nonrandomized clinical trial for HIV. This trial evaluated the safety of a conditionally replicating HIV-1-derived vector expressing an antisense gene against the HIV envelope. Five subjects with chronic HIV infection who had failed to respond to at least two antiviral regimens were enrolled. A single i.v. infusion of gene-modified autologous CD4 T cells was well tolerated in all patients. Viral loads were stable, and one subject exhibited a sustained decrease in viral load. CD4 counts remained steady or increased in four subjects, and sustained gene transfer was observed. Self-limiting mobilization of the vector was observed in four of five patients. There is no evidence for insertional mutagenesis after 21-36 months of observation. Immune function improved in four subjects. Lentiviral vectors appear promising for gene transfer to humans.

We have previously reported the successful completion of our Phase I clinical trial. The Phase I trial demonstrated the safety and tolerability of a single dose consisting of approximately 10 billion autologous HIV infected CD4+ T cells transduced with the lentivector VRX496 carrying a 937-base antisense targeting the HIV envelope. These encouraging results have led us to design a Phase II clinical trial to evaluate the safety, tolerability, and biological activity of four or eight repeated infusions of 5 to10 billion autologous VRX496-modified HIV+, CD4+ T cells. A major obstacle to completing this Phase II trial was manufacturing enough cells to administer multiple infusions in patients. In order to produce the required doses, major changes between the processes used for phase I, performed at the Clinical Cell and Vaccine Production Facility, University of Pennsylvania and phase II, performed at VIRxSYS were implemented. The changes focused on the methods of CD4+ cells purification (Miltenyi CliniMACS vs. BioTransplant Eligix) and expansion (Baxter Opticyte 600 bags vs. Wave 50-liter perfusion bags). Using a CD4 positive selection, we have been able to routinely recover over 1 billion CD4+ T lymphocytes from one HIV+ aphaeresis with purities over 90%. Improved CD4+ T cells recovery and purity allowed for reduced requirements of lentiviral vector while maintaining the same range of vector copy number per cell (2 copies per cell in average). Switching from multiple static 600-ml bags in a 5% CO2 incubator to a 50-liter Wave perfusion bioreactor allowed us to consistently reach the required cell numbers in 7 to 10 days post-purification. In summary, we implemented a closed, process for CD4+ cells purification, transduction and expansion, using disposable materials that proved to be consistent and robust. To date no production failures have been recorded. Furthermore, 11 patients have received their infusions as scheduled and a total of 30 doses have been infused. All of the doses have been well tolerated and preliminary results suggest multiple infusions are safe, with no changes in hematology or chemistry laboratory evaluations, and no detection of VSV-G DNA or RCL. This therapy continues to show promise for HIV patients as a long-term alternative to antiretroviral drug regimens. We have previously reported the successful completion of our Phase I clinical trial. The Phase I trial demonstrated the safety and tolerability of a single dose consisting of approximately 10 billion autologous HIV infected CD4+ T cells transduced with the lentivector VRX496 carrying a 937-base antisense targeting the HIV envelope. These encouraging results have led us to design a Phase II clinical trial to evaluate the safety, tolerability, and biological activity of four or eight repeated infusions of 5 to10 billion autologous VRX496-modified HIV+, CD4+ T cells. A major obstacle to completing this Phase II trial was manufacturing enough cells to administer multiple infusions in patients. In order to produce the required doses, major changes between the processes used for phase I, performed at the Clinical Cell and Vaccine Production Facility, University of Pennsylvania and phase II, performed at VIRxSYS were implemented. The changes focused on the methods of CD4+ cells purification (Miltenyi CliniMACS vs. BioTransplant Eligix) and expansion (Baxter Opticyte 600 bags vs. Wave 50-liter perfusion bags). Using a CD4 positive selection, we have been able to routinely recover over 1 billion CD4+ T lymphocytes from one HIV+ aphaeresis with purities over 90%. Improved CD4+ T cells recovery and purity allowed for reduced requirements of lentiviral vector while maintaining the same range of vector copy number per cell (2 copies per cell in average). Switching from multiple static 600-ml bags in a 5% CO2 incubator to a 50-liter Wave perfusion bioreactor allowed us to consistently reach the required cell numbers in 7 to 10 days post-purification. In summary, we implemented a closed, process for CD4+ cells purification, transduction and expansion, using disposable materials that proved to be consistent and robust. To date no production failures have been recorded. Furthermore, 11 patients have received their infusions as scheduled and a total of 30 doses have been infused. All of the doses have been well tolerated and preliminary results suggest multiple infusions are safe, with no changes in hematology or chemistry laboratory evaluations, and no detection of VSV-G DNA or RCL. This therapy continues to show promise for HIV patients as a long-term alternative to antiretroviral drug regimens.