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Background Lentiviral vectors are being used with increasing frequency in human clinical trials. We were the first to use lentiviral vectors in clinical trials in 2003. Our lentiviral vector encoded a long RNA antisense sequence to the HIV-1 envelope and was used in an ex vivo autologous setting to provide viral load control in HIV-1 positive subjects failing anti-HIV therapy. A total of 65 subjects have been treated in Phase 1 and Phase 2 trials in six institutions. Methods Good manufacturing practices (GMP) lots of the lentiviral vector used in our clinical trials were assayed for the presence of replication competent lentivirus (RCL). RCL assays were conducted at two stages. The first testing was performed on samples collected immediately following bulk harvest of the GMP product lot and consisted of 1 × 108 cells used in production. RCL assays were also performed on aliquots of the final fill of the vector by the inoculation of at least 5% of the GMP final fill volume into C8166 cells, passaged for at least ten passages and tested for RCL by p24 enzyme-linked immunosorbent assay and vesicular stomatitis virus-G envelope DNA. Results Following 263 infusions of autologous, transduced cells, no adverse events have been detected in these subjects, with some followed for more than 8 years following infusions. More than 4.3 × 1012 VRX496 proviral copies were administered to these 65 subjects. Conclusions Data from this small population suggest that there is no apparent risk for serious adverse events with the use of lentiviral vectors. Copyright © 2013 John Wiley & Sons, Ltd.

Lentiviral vectors are being used with increasing frequency in human clinical trials. We were the first to use lentiviral vectors in clinical trials in 2003. Our lentiviral vector encoded a long RNA antisense sequence to the HIV-1 envelope and was used in an ex vivo autologous setting to provide viral load control in HIV-1 positive subjects failing anti-HIV therapy. A total of 65 subjects have been treated in Phase 1 and Phase 2 trials in six institutions.Good manufacturing practices (GMP) lots of the lentiviral vector used in our clinical trials were assayed for the presence of replication competent lentivirus (RCL). RCL assays were conducted at two stages. The first testing was performed on samples collected immediately following bulk harvest of the GMP product lot and consisted of 1 × 10(8) cells used in production. RCL assays were also performed on aliquots of the final fill of the vector by the inoculation of at least 5% of the GMP final fill volume into C8166 cells, passaged for at least ten passages and tested for RCL by p24 enzyme-linked immunosorbent assay and vesicular stomatitis virus-G envelope DNA.Following 263 infusions of autologous, transduced cells, no adverse events have been detected in these subjects, with some followed for more than 8 years following infusions. More than 4.3 × 10(12) VRX496 proviral copies were administered to these 65 subjects.Data from this small population suggest that there is no apparent risk for serious adverse events with the use of lentiviral vectors.

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|[nbsp]|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.