A Simple and Effective Method to Generate Lentiviral Vectors forEx VivoGene Delivery to Mature Human Peripheral Blood Lymphocytes

Abstract

Human ex vivo gene therapy protocols have been used successfully to treat a variety of genetic disorders, infectious diseases, and cancer. Murine oncoretroviruses (specifically, gammaretroviruses) have served as the primary gene delivery vehicles for these trials. However, in some cases, such vectors have been associated with insertional mutagenesis. As a result, alternative vector platforms such as lentiviral vectors (LVVs) are being developed. LVVs may provide advantages compared with gammaretroviral vectors, including the ability to transduce large numbers of nondividing cells, resistance to gene silencing, and a potentially safer integration profile. The aim of this study was to develop a simplified process for the rapid production of clinical-grade LVVs. To that end, we used a self-inactivating bicistronic LVV encoding an MART (melanoma antigen recognized by T cells)-1-reactive T cell receptor containing oPRE, an optimized and truncated version of woodchuck hepatitis virus posttranslational regulatory element (wPRE). Using our simplified clinical production process, 293T cells were transiently transfected in roller bottles. The LVV supernatant was collected, treated with Benzonase, and clarified by modified step filtration. LVV produced in this manner exhibited titers and a biosafety profile similar to those of cGMP (current Good Manufacturing Practices) LVVs previously manufactured at the Indiana University Vector Production Facility in support of a phase I/II clinical trial. We describe a simple, efficient, and low-cost method for the production of clinical-grade LVV for ex vivo gene therapy protocols.

Sources of Error in Mammalian Genetic Screens

“…To minimize the effects of this contamination, we treated viral supernatants with the endonuclease Benzonase to degrade residual plasmid DNA. This method is well established in preparation of clinical-grade lentivirus for gene therapy applications ( Sastry et al 2004 ; Yang et al 2012 ; Segura et al 2013 ); however, to our knowledge, it has not been previously reported to be used in preparation of lentivirus for genetic screens or other tissue culture applications. We demonstrate that Benzonase treatment can significantly increase the fidelity of specific detection of integrated viral genomes from the genomic DNA of a population of infected cells.…”

Comparison of the efficacy of second and third generation lentiviral vector transduced CAR CD19 T cells for use in the treatment of acute lymphoblastic leukemia both in vitro and in vivo models

“…The efficacy of the gene therapy product often depends heavily on the transduction efficiency of the viral vector and the selected MOI. The lentiviral vector is the most common viral vector used to produce CAR-CD19 T cells [ 18 ]. Because of their capacity to stably integrate into the host cell genome and infect both dividing and non-dividing cells, these vectors have been extensively used for gene and cellular therapy [ 19 , 20 ].…”

“…vector used to produce CAR-CD19 T cells [18]. Because of their capacity to stably integrate into the host cell genome and infect both dividing and non-dividing cells, these vectors have been extensively used for gene and cellular therapy [19,20].…”

Potential Limitations of the NSG Humanized Mouse as a Model System to Optimize Engineered Human T cell Therapy for Cancer

“…The genetic modification of peripheral blood lymphocytes using retroviral vectors encoding T cell receptors (TCR) or chimeric antigen receptors (CAR) that effectively redirect T cells to recognize and eliminate tumor cells has been developed and can potentially overcome major technical obstacles, including the low frequency of natural tumor-specific T cells residing within the patient's T cell repertoire ( Johnson et al, 2009;Gilham et al, 2012;Uttenthal et al, 2012). Indeed, protocols to generate gene-modified T cells for clinical application have been developed (Lamers et al, 2002;Yang et al, 2008Yang et al, , 2010Yang et al, , 2012b. To date, retroviral vectors have been the primary choice for gene delivery into primary human T cells.…”