A simple and effective method to generate lentiviral vectors for ex vivo gene delivery to mature human peripheral blood lymphocytes

Shicheng Yang, Neel K. Karne, Stephanie L. Goff, Mary A. Black, Hui Xu, Daniela Bischof, Kenneth Cornetta, Steven A. Rosenberg, Richard A. Morgan, Steven A. Feldman

Research output: Contribution to journalArticle

5 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)73-83
Number of pages11
JournalHuman gene therapy methods
Volume23
Issue number2
DOIs
StatePublished - Apr 1 2012

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Serratia marcescens nuclease
Genetic Therapy
Woodchuck Hepatitis B Virus
Gammaretrovirus
MART-1 Antigen
Lymphocytes
Phase II Clinical Trials
Clinical Trials, Phase I
Inborn Genetic Diseases
Insertional Mutagenesis
HEK293 Cells
Gene Silencing
T-Cell Antigen Receptor
Genes
Communicable Diseases
Cell Count
Costs and Cost Analysis
Neoplasms

ASJC Scopus subject areas

  • Molecular Medicine
  • Applied Microbiology and Biotechnology
  • Genetics(clinical)
  • Genetics
  • Pharmacology
  • Medicine(all)

Cite this

A simple and effective method to generate lentiviral vectors for ex vivo gene delivery to mature human peripheral blood lymphocytes. / Yang, Shicheng; Karne, Neel K.; Goff, Stephanie L.; Black, Mary A.; Xu, Hui; Bischof, Daniela; Cornetta, Kenneth; Rosenberg, Steven A.; Morgan, Richard A.; Feldman, Steven A.

In: Human gene therapy methods, Vol. 23, No. 2, 01.04.2012, p. 73-83.

Research output: Contribution to journalArticle

Yang, S, Karne, NK, Goff, SL, Black, MA, Xu, H, Bischof, D, Cornetta, K, Rosenberg, SA, Morgan, RA & Feldman, SA 2012, 'A simple and effective method to generate lentiviral vectors for ex vivo gene delivery to mature human peripheral blood lymphocytes', Human gene therapy methods, vol. 23, no. 2, pp. 73-83. https://doi.org/10.1089/hgtb.2011.199
Yang, Shicheng ; Karne, Neel K. ; Goff, Stephanie L. ; Black, Mary A. ; Xu, Hui ; Bischof, Daniela ; Cornetta, Kenneth ; Rosenberg, Steven A. ; Morgan, Richard A. ; Feldman, Steven A. / A simple and effective method to generate lentiviral vectors for ex vivo gene delivery to mature human peripheral blood lymphocytes. In: Human gene therapy methods. 2012 ; Vol. 23, No. 2. pp. 73-83.
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