Accession Number:

ADA279196

Title:

Correction of the Enzyme Deficiency in Hematopoietic Cells of Gaucher Patients Using a Clinically Acceptable Retroviral Supernatant Transduction Protocol

Descriptive Note:

Journal article

Corporate Author:

NAVAL MEDICAL RESEARCH INST BETHESDA MD

Report Date:

1994-01-01

Pagination or Media Count:

10.0

Abstract:

Gaucher disease is a lysosomal storage disorder caused by a deficiency of the enzyme glucocerebrosidase GC, and is an excellent candidate for gene replacement therapy. To develop a clinically acceptable protocol for this purpose, we created two amplified A high-titer retroviral vector-producer cell lines to efficiently transduce hematopoietic stem and progenitor cells. GPenvAml2A-LGSN A-LGSN, contained the GC cDNA driven by the retroviral long terminal repeat LTR and the neomycin phosphotransferase gene expressed from the simian virus 40 early promoter. GPenvAml2ALG4 A-LG4 contained only the GC gene driven by the LTR. Both A-LGSN and A-LG4 contained multiple-proviral copies and gave approximately 10-fold higher titers on 3T3 cells compared to their unamplified counterparts. These vectors were packaged in GPenvAml2 cells because vectors produced in this cell line transduced hematopoietic cells more efficiently than other packaging cells tested. Bone marrow mononuclear cells and purified CD34 cells were infected with virus supernatants four times in the presence of interleukin-3 IL-3, IL-6, and stem cell factor SCF over 96 hours in culture. Cells ,ere then plated in semisolid cultures and colony-forming unit-granulocytemacrophage CFU-GM colonies were scored for vector presence by polymerase chain reaction PCR. Transduction efficiency of CFU-GM colonies derived from CD34- cells, as improved considerably using the amplified vectors in the GPenvAm12 packaging line. For A-LGSN, A-LG4, and unamplified LGSN, transduction efficiencies were 41, 42, and 25, respectively. Gene therapy, CD34, Stem cells, CC Deficiency, Gaucher disease, Gene therapy, Retroviral supernatant, Transduction

Subject Categories:

  • Anatomy and Physiology
  • Medicine and Medical Research

Distribution Statement:

APPROVED FOR PUBLIC RELEASE