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Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells
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An experimental system for the evaluation of retroviral vector design to diminish the risk for proto-oncogene

Byoung Y Ryu1, Marguerite V Evans-Galea, John T Gray

  • 1Department of Hematology, St Jude Children's Research Hospital, Memphis, TN 38105, USA.

Blood
|November 10, 2007
PubMed

Insights

Gene therapy safety concerns, particularly LMO2 proto-oncogene activation in X-linked severe combined immunodeficiency (X-SCID) trials, were addressed. Researchers developed a system to compare vector safety, reducing proto-oncogene activation risks.

Area of Science:

  • Gene therapy
  • Oncology
  • Immunology

Background:

  • Clinical trials using oncoretroviral vectors for X-linked severe combined immunodeficiency (X-SCID) faced safety issues due to LMO2 proto-oncogene activation.
  • This activation poses a significant risk for leukemogenesis in patients.

Purpose of the Study:

  • To develop and validate a system for comparing the safety profiles of different gene therapy vector cassettes.
  • To assess the potential for proto-oncogene activation by various regulatory elements within gene therapy vectors.

Main Methods:

  • Adeno-associated virus (AAV) vector used for targeted insertion of a gamma-retroviral LTR-GFP cassette into the LMO2 gene in a human T-cell line.
  • Cre-mediated cassette exchange employed to replace the LTR-GFP cassette with insulator-flanked or globin gene regulatory cassettes.
  • Comparison of LMO2 expression levels after insertion of different vector designs, including gamma-retroviral and self-inactivating lentiviral vectors.

Main Results:

  • Insertion of the LTR-GFP cassette into the LMO2 gene's first intron strongly activated LMO2 expression.
  • Replacing the cassette with insulator-flanked elements reduced LMO2 expression significantly.
  • A self-inactivating lentiviral vector with insulator elements did not activate LMO2, unlike a gamma-retroviral vector.

Conclusions:

  • The developed system effectively compares vector cassette safety regarding proto-oncogene activation.
  • Vector design, particularly the use of insulators and self-inactivating elements, is crucial for mitigating risks like LMO2 activation in gene therapy.

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