Integrated Strategy for the Production of Therapeutic Retroviral Vectors

Hum Gene Ther. 2011 Mar;22(3):370-9. doi: 10.1089/hum.2009.165. Epub 2011 Mar 2.

Abstract

The broad application of retroviral vectors for gene delivery is still hampered by the difficulty to reproducibly establish high vector producer cell lines generating sufficient amounts of highly concentrated virus vector preparations of high quality. To enhance the process for producing clinically relevant retroviral vector preparations for therapeutic applications, we have integrated novel and state-of-the-art technologies in a process that allows rapid access to high-efficiency vector-producing cells and consistent production, purification, and storage of retroviral vectors. The process has been designed for various types of retroviral vectors for clinical application and to support a high-throughput process. New modular helper cell lines that permit rapid insertion of DNA encoding the therapeutic vector of interest at predetermined, optimal chromosomal loci were developed to facilitate stable and high vector production levels. Packaging cell lines, cultivation methods, and improved medium composition were coupled with vector purification and storage process strategies that yield maximal vector infectivity and stability. To facilitate GMP-grade vector production, standard of operation protocols were established. These processes were validated by production of retroviral vector lots that drive the expression of type VII collagen (Col7) for the treatment of a skin genetic disease, dystrophic epidermolysis bullosa. The potential efficacy of the Col7-expressing vectors was finally proven with newly developed systems, in particular in target primary keratinocyte cultures and three-dimensional skin tissues in organ culture.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Culture Techniques
  • Cell Line
  • Collagen Type VII / genetics
  • Collagen Type VII / metabolism
  • DNA Nucleotidyltransferases / metabolism
  • Epidermolysis Bullosa Dystrophica / therapy
  • Genetic Therapy
  • Genetic Vectors / biosynthesis*
  • Genetic Vectors / genetics
  • Genetic Vectors / isolation & purification
  • Genetic Vectors / physiology*
  • HEK293 Cells
  • Humans
  • Industrial Microbiology / methods*
  • Mice
  • Mice, Inbred BALB C
  • Recombination, Genetic
  • Reproducibility of Results
  • Retroviridae / genetics
  • Retroviridae / isolation & purification
  • Retroviridae / physiology*

Substances

  • Collagen Type VII
  • DNA Nucleotidyltransferases
  • FLP recombinase