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Published on: May 27, 2011
Retroviral gene transfer into cord blood stem/progenitor cells using purified vector stocks
J Asch1, R S Weinberg, L Mueller
1Department of Pediatrics, Robert Wood Johnson Medical School, New Brunswick, New Jersey, USA.
Insights
Optimizing retroviral transduction of cord blood stem cells for gene therapy requires careful consideration of infection timing and media components. Higher efficiency was achieved with specific multiplicities of infection and autologous plasma.
Area of Science:
- Gene therapy
- Stem cell biology
- Retroviral vectors
Background:
- Cord blood (CB) progenitor/stem cells (P/SC) are crucial for treating genetic disorders via gene therapy.
- Existing retroviral transduction protocols are primarily based on adult stem cells, with limited data on CB P/SC.
Purpose of the Study:
- To investigate the impact of timing, multiplicity of infection (MOI), and polycations on retroviral transduction efficiency in CB P/SC.
- To determine optimal conditions for transducing CD34+ enriched CB cells and their derived colonies.
Main Methods:
- Utilized clinical-grade retroviral vectors with LacZ and neomycin resistance genes.
- Transduced CD34+ enriched CB cells and cells in liquid cultures (LC) at various MOIs (1-20).
- Assessed transduction efficiency using X-gal staining and PCR amplification, without drug selection.
Main Results:
- Transduction efficiency in liquid cultures ranged from 4-63%.
- Higher transduction rates were observed with MOI ≥ 10, 2 μg/ml polybrene, and 10% autologous CB plasma.
- In colonies, PCR showed 63-72% transduction, while X-gal staining indicated 32%; LTC-IC derived colonies showed 7% transduction by PCR.
Conclusions:
- Short incubations of CD34+ CB cells with retroviral stocks, polybrene, and autologous sera yield high transduction rates for committed progenitors.
- Achieved moderately low efficiencies for long-term culture-initiating cells (LTC-IC) transduction without drug selection.
Abstract:
Cord blood (CB) progenitor/stem cells (P/SC) are ideal targets for early gene therapy in individuals prenatally diagnosed with genetic disorders. Most retroviral transduction protocols were developed using adult peripheral blood stem cells (PBSC) and bone marrow (BM). Less is known about retroviral transduction of CB P/SC. We examined how timing, multiplicity of infection (MOI), and polycations in the transduction media affect transduction efficiency. Rates of transduction were determined in recently isolated CD34+ enriched CB cells and in colonies derived after various times in liquid cultures (LC). CB mononuclear cells (MNC) were separated by ficoll-hypaque centrifugation and enriched for CD34+ cells. Purity was assessed by flow cytometry. Transduction were performed with clinical-grade retroviral stocks at MOIs of 1-20. Transduction was performed with fetal bovine serum (FBS) or autologous plasma, IL-3, GM-CSF, IL-6, and SCF. The retroviral vector contained LacZ and neomycin resistance (neo) reporter genes. Transduction was determined by X-gal stain and by PCR amplification of the reporter genes. No drug selection was used. Twenty-five experiments were done. CB volumes ranged from 35-150 ml. MNC and CD34+ cell counts ranges were: 0.14-840 x 10(6) and 0.1-4.2 x 10(6), respectively. Transduction efficiency in liquid cultures ranged from 4-63%. Higher rates were seen using MOI > or = 10, 2 microg/ml polybrene, and 10% autologous CB plasma. In colonies, transduction rates were 63 to 72% by PCR and 32% by X-gal staining. In LTC-IC derived colonies, transduction was 7% by PCR. Short incubations of CD34+ CB cells with purified retroviral stocks, polybrene, and autologous sera result in high transduction rates of committed progenitors and moderately low efficiencies of transduction of LTC-IC in the absence of drug selection.
