Retroviral Gene Transfer of Human Adenosine Deaminase into Hematopoietic Cells
We are developing a safe and efficient system of retroviral gene transfer of human ADA into hematopoietic stem cells. Inherited deficiency of ADA results in Severe Combined Immunodeficiency (SCID) and serves as a model for gene replacement therapy attempts. The vector DNN2ADA has been successful in transducing long term expression in mice via hematopoietic stem cell infection. Additional vectors with the potential to induce tissue-specific lymphoid expression have been constructed. Results obtained with one of those vectors are described.
We have developed a new system for the detection of replication competent virus which might arise from recombinational events. It involves DNA amplification of sequences located between the pol and env genes of Moloney Murine Leukemia Virus in target cells. This allows the detection of one helper-infected cell out of 104 to 105 uninfected cells.
Virus supernatant from helper free DNN2ADA producing cell lines were used to infect human bone marrow cells (BMCs). Five to 20% of unselected colony forming progenitors contained integrated copy(ies) of the vector as detectable by DNA amplification. Low copy number of the provirus were present in supernatant cells from long term bone marrow culture up to seven weeks post-infection. Transduced ADA expression was detectable following infection of BMCs derived from two ADA deficient SCID patients.
These results indicate special retroviral vectors can successfully transfer and express human ADA into mouse and human bone marrow cells.
KeywordsCodon Recombination Adenosine Sorting Thymidine
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