مقاله انگلیسی رایگان در مورد تولید وکتورهای لنتی ویروسی – الزویر 2020

Lentiviral vectors are increasingly utilized in cell and gene therapy applications because they efficiently transduce target cells such as hematopoietic stem cells and T cells. Large-scale production of current Good Manufacturing Practices-grade lentiviral vectors is limited because of the adherent, serum-dependent nature of HEK293T cells used in the manufacturing process. To optimize large-scale clinical-grade lentiviral vector production, we developed an improved production scheme by adapting HEK293T cells to grow in suspension using commercially available and chemically defined serum-free media. Lentiviral vectors with titers equivalent to those of HEK293T cells were produced from SJ293TS cells using optimized transfection conditions that reduced the required amount of plasmid DNA by 50%. Furthermore, purification of SJ293TSderived lentiviral vectors at 1 L yielded a recovery of 55% ± 14% (n = 138) of transducing units in the starting material, more than a 2-fold increase over historical yields from adherent HEK293T serum-dependent lentiviral vector preparations. SJ293TS cells were stable to produce lentiviral vectors over 4 months of continuous culture. SJ293TS-derived lentiviral vectors efficiently transduced primary hematopoietic stem cells and T cells from healthy donors. Overall, our SJ293TS cell line enables high-titer vector production in serum-free conditions while reducing the amount of input DNA required, resulting in a highly efficient manufacturing option.

Introduction

HIV-1-derived lentiviral vectors (LVs) are efficient gene transfer vehicles used in both basic and clinical research settings. The ability of LVs to efficiently shuttle DNA into mammalian cells enables researchers to explore the function of various genes of interest.1–5 Clinically, LVs are used ex vivo to deliver therapeutic genes or expression cassettes to primary target cells, such as hematopoietic stem cells (HSCs), to treat genetic disorders or infectious diseases.6–9 LVs are well suited for use in ex vivo gene therapy because they can deliver a relatively large payload (>9 kb, including intron-containing genomes), have the ability to transduce dividing and non-dividing cells, and stably integrate into the genome of a target cell to provide lifelong correction to that cell and its progeny.10–13 Further, the integration profile of LVs suggests that they are safer than gamma-retroviral vectors, and there have been no product-related malignant transformations reported to date with LVs used in clinical trials.14–16 The promise of LV cell and gene therapy has led to the initialization of nearly 200 clinical trials, with notable successes in the treatment of hemoglobinopathies, primary immunodeficiencies, and leukemias.15,17–25 In fact, LV-based cell and gene therapies have already been approved by the US Food and Drug Administration, Kymriah, and by the European Medicines Agency, Zynteglo. Furthermore, cancer immunotherapy, which currently includes the use of LV to modify T cell function, is an area of intense research. Thus, additional LV-based cellular therapies are likely to be approved in the future.