Sustained multilineage gene persistence and expression in dogs transplanted with CD34(+) marrow cells transduced by RD114-pseudotype oncoretrovirus vectors

Previous studies have shown that the choice of envelope protein (pseudotype ) can have a significant effect on the efficiency of retroviral gene transf er into hematopoietic stem cells. This study used a competitive repopulatio n assay in the dog model to evaluate oncoretroviral vectors carrying the en velope protein of the endogenous feline virus, RD114. CD34-enriched marrow cells were divided into equal aliquots and transduced with vectors produced by the RD114-pseudotype packaging cells FLYRD (LgGLSN and LNX) or by the g ibbon ape leukemia virus (GALV)-pseudotype packaging cells PG13 (LNY). A to tal of 5 dogs were studied. One dog died because of infection before sustai ned engraftment could be achieved, and monitoring was discontinued after 9 months In another animal that had very low overall gene-marking levels. The 3 remaining animals are alive with follow-ups at 11, 22, and 23 months. An alyses of gene marking frequencies in peripheral blood and marrow by polyme rase chain reaction revealed no significant differences between the RD114 a nd GALV-pseudotype vectors. The LgGLSN vector also contained the enhanced g reen fluorescent protein (GFP), enabling us to monitor proviral expression by flow cytometry. Up to 10% of peripheral blood cells expressed GFP shortl y after transplantation and approximately 6% after the longest follow-up of 23 months. Flow cytometric analysis of hematopoietic subpopulations showed that most of the GFP-expressing cells were granulocytes, although GFP-posi tive lymphocytes and monocytes were also detected. In summary, these result s show that RD114-pseudotype oncoretroviral vectors are able to transduce h ematopoietic longterm repopulating cells and, thus, may be useful for human stem cell gene therapy. (C) 2001 by The American Society of Hematology.