title: A simple and effective method to generate lentiviral vectors for ex vivo gene 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.
authors.affiliation: Surgery Branch, National Cancer Institute , National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Shicheng
authors.initials: S
authors.lastname: Yang
authors.forename: Neel K
authors.initials: NK
authors.lastname: Karne
authors.forename: Stephanie L
authors.initials: SL
authors.lastname: Goff
authors.forename: Mary A
authors.initials: MA
authors.lastname: Black
authors.forename: Hui
authors.initials: H
authors.lastname: Xu
authors.forename: Daniela
authors.initials: D
authors.lastname: Bischof
authors.forename: Kenneth
authors.initials: K
authors.lastname: Cornetta
authors.forename: Steven A
authors.initials: SA
authors.lastname: Rosenberg
authors.forename: Richard A
authors.initials: RA
authors.lastname: Morgan
authors.forename: Steven A
authors.initials: SA
authors.lastname: Feldman
mesh_terms: D000704:Analysis of Variance; D045744:Cell Line, Tumor; D004706:Endodeoxyribonucleases; D004722:Endoribonucleases; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000096:biosynthesis* / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008214:Lymphocytes / Q000378:metabolism; D058965:MART-1 Antigen / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D050436:Regulatory Elements, Transcriptional / Q000235:genetics
chemical_list: D058965:MART-1 Antigen; D049452:Green Fluorescent Proteins; D004706:Endodeoxyribonucleases; D004722:Endoribonucleases; C021835:Serratia marcescens nuclease