Efficient construction of producer cell lines for a SIN lentiviral vector for SCID-X1 gene therapy by concatemeric array transfection

Abstract

IntroductionHIV-based lentiviral vectors are rapidly becoming the retrovirus vector system of choice for research and clinical gene transfer applications. The enhanced ability of lentiviral vectors to transduce both quiescent stem cells 1 and nondividing terminally differentiated cells 2 has led to the development of a wide range of therapeutic gene delivery vectors, 3 as well as promising research tools such as short hairpin RNA gene knockdown libraries 4 and vectors for induction of pluripotency in terminally differentiated cells. 5 Early gamma-retroviral clinical gene therapy vectors restored immune function in patients with X-linked severe combined immunodeficiency (SCID-X1), but they were subsequently found to cause proliferative disorders via transactivation of protooncogenes. 6,7 Newer lentiviral vector designs may significantly reduce that risk, and they await clinical testing for final validation of their predicted safety. Clinical-scale production of these vectors, however, is problematic, as the generation of stable producer cell lines is made significantly more difficult by their self-inactivating (SIN) long terminal repeats (LTRs). As a result, most clinical-grade production of lentiviral vectors is currently being performed using cumbersome transient transfection processes.Insertional mutagenesis by previous gamma-retroviral gene therapy vectors occurred when strong viral enhancers within the LTR activated genes (eg, LMO2) surrounding the integrated vector. 6,7 SIN vector designs completely eliminate the viral enhancers and promoters in the LTR, and when coupled with appropriate internal promoters having less or no enhancer activity, they have been shown to significantly reduce oncogene activation. [8][9][10] Chromatin insulator sequences have also been inserted into SIN LTRs and appear to protect neighboring genes from residual transactivation from the internal promoters. 11 When inserted into the LMO2 locus in Jurkat cells, lentiviral vector genomes containing an internal EF1␣ promoter flanked by SIN LTRs and chicken HS4 chromatin insulators caused only minimal transactivation of the LMO2 promoter. 12 Clinical-scale production of such safety-modified vectors would be greatly facilitated by stable producer cell lines, which allow convenient generation of standardized, large-volume supernatants for downstream process optimization and preclinical studies. Although there have been numerous reports of lentiviral packaging cell lines, 13-22 all high-titer (Ͼ 10 7 transducing units per milliliter [TU/mL]) stable producer lines described in these publications were created by the traditional method of viral transduction of packaging cell lines using non-SIN vector supernatants, which efficiently creates populations of cells with vector genomes integrated at sites favorable for active transcription, and in multiple copies per cell. SIN vector genomes, by virtue of the inactivating deletion in the LTR, are thus incompatible with this method. "Conditional SIN" vectors, 22 which contain regulatable enhancers ...

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

“…In particular, there have been reports regarding a number of packaging cell lines for LVV production (Kafri et al, 1999;Ikeda et al, 2003;Throm et al, 2009;Stewart et al, 2011); however, use of such lines at this time remains challenging. Therefore, most current production efforts rely on large-scale transient transfection of 293T cells by a calcium phosphate (Ca-Phos)-based transfection method, primarily because of its low cost.…”

Gene Therapy for Primary Immunodeficiencies

“…This will greatly facilitate the adoption of protocol and technological refinements and perhaps eventually product registration. SIN lentiviral vectors have also been developed for SCID-X1, one of which has been successfully incorporated into a clinical grade stable producer cell line by concatemeric transfection, thereby greatly facilitating the production process (Throm et al, 2009). Similarly, lentiviral vectors using housekeeping promoter elements for ADA deficiency or natural regulatory sequences for WAS have recently entered the clinical arena in multicenter efforts (Boztug et al, 2006;Charrier et al, 2005;Montiel-Equihua et al, 2012) and are being developed for other PIDs as detailed previously.…”

Quantitative Shearing Linear Amplification Polymerase Chain Reaction: An Improved Method for Quantifying Lentiviral Vector Insertion Sites in Transplanted Hematopoietic Cell Systems

“…Transduction of CD34+ cells G-CSF mobilized human peripheral blood CD34+ cells were transduced with the CL20-4i-EF1a-hccOPT vector that was either transiently prepared in transfected 293T cells (CD34A) or produced from a lentiviral vector producer clone (CD34B) 27 at an MOI of 50 in XVivo-10 medium containing 10% fetal calf serum and 100 ng/ml each of the recombinant human cytokines stem cell factor, thrombopoietin, and ligand for FLT3. The cells were cultured for 5 days and genomic DNA was extracted for further analysis using the DNeasy blood and tissue kit (Qiagen).…”

“…Vector preparations were either generated from transient transfection of 293T cells (CD34A) or produced from our stable lentiviral vector producer clone 1A4 (CD34B). 27 After transduction, the cells were cultured for an additional 5 days and genomic DNA was extracted for qsLAM PCR. The average VCN was 2.47 and 1.92 genomes per cell for the CD34A and CD34B populations, respectively, by qPCR.…”