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Enhancement of adenovirus-mediated gene transfer to human bone marrow cells
T Watanabe1, L Kelsey, A Ageitos
1Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, USA.
Abstract:
Adenovirus infection of CD34+ hematopoietic stem/progenitor cells is dependent on the multiplicity of infection (MOI), time of incubation, the volume in which the co-incubation occurs and the presence or absence of growth factors. Studies revealed that a brief co-incubation (1-8 hours), resulted in low levels of transgene expression, suggesting that adenovirus infection of CD34+ cells occurs slowly, and optimal transduction requires a 24 hour exposure to adenovirus. Infection by Ad/beta-gal or Ad/p53 at a MOI of 500:1 provided a high transduction efficiency but inhibited hematopoietic function. However, treatment at a MOI of 50-100 resulted in efficient transduction (10.7-15.7% positive) without detectable toxicity. Secondary proof of adenovirus transgene expression was demonstrated by detection of mRNA for p53 in Ad/p53 infected stem cells. We conclude that a 24 hour exposure to recombinant adenovirus encoding p53 or beta-gal, at a MOI of 50-100 is optimal for in vitro gene transfer to BM cells and has no significant effect on hematopoietic function. Adenovirus-mediated transduction of BM cells can also be modulated by growth factors (IL-3, GM-CSF and G-CSF) with improved gene delivery and maintenance of hematopoietic function. In summary, adenovirus vectors can be used to transiently transduce stem cells, and conditions have been defined to maximize expression and limit inhibitory effects on CD34+ cells. These data support continued investigation of this vector for local cytokine delivery and purging of stem cell products.
Insights
Optimal adenovirus transduction of hematopoietic stem cells (HSCs) requires 24-hour exposure at a multiplicity of infection (MOI) of 50-100. This method achieves efficient gene transfer without inhibiting HSC function, supporting further research in gene therapy applications.
Area of Science:
- Hematology
- Gene Therapy
- Molecular Biology
Background:
- Adenovirus vectors are explored for gene transfer into hematopoietic stem cells (HSCs).
- Understanding optimal infection parameters is crucial for efficient and safe gene delivery.
Purpose of the Study:
- To define optimal conditions for adenovirus-mediated gene transfer into CD34+ hematopoietic stem/progenitor cells.
- To assess the impact of infection parameters on HSC function and transgene expression.
Main Methods:
- Infection of CD34+ cells with adenovirus vectors (Ad/beta-gal, Ad/p53) at varying multiplicities of infection (MOI) and incubation times.
- Assessment of transduction efficiency via transgene expression (e.g., p53 mRNA detection).
- Evaluation of hematopoietic function post-transduction, with and without growth factors (IL-3, GM-CSF, G-CSF).
Main Results:
- Optimal transduction efficiency (10.7-15.7%) and transgene expression achieved with a 24-hour incubation at MOI 50-100.
- Higher MOI (500:1) resulted in efficient transduction but inhibited hematopoietic function.
- Growth factors improved gene delivery and maintained hematopoietic function.
- Adenovirus vectors demonstrated transient transduction without significant toxicity at optimized MOI.
Conclusions:
- A 24-hour exposure to recombinant adenovirus at MOI 50-100 is optimal for in vitro gene transfer to bone marrow (BM) cells.
- Optimized conditions maximize gene expression while minimizing inhibitory effects on CD34+ cells.
- Adenovirus vectors show potential for transient stem cell transduction, supporting applications like cytokine delivery and stem cell purging.