The use of granulocyte colony-stimulating factor during retroviral transduction on fibronectin fragment CH-296 enhances gene transfer into hematopoietic repopulating cells in dogs

A competitive repopulation assay in the dog was used to develop improved ge ne transfer protocols for hematopoietic stem cell gene therapy. Using this assay, we previously showed improved gene transfer into canine hematopoieti c repopulating cells when CD34-enriched marrow cells were cocultivated on g ibbon ape leukemia virus (GALV)-based retrovirus vector-producing cells. In the present study, we have investigated the use of fibronectin fragment CH -296 and 2 growth factor combinations to further improve gene transfer effi ciency. CD34-enriched marrow cells from each dog were prestimulated for 24 hours and then divided into 3 equal fractions. Two fractions were placed in to flasks coated with either CH-296 or bovine serum albumin (BSA) and virus -containing medium supplemented with growth factors, and protamine sulfate was replaced 4 times over a 48-hour period, One fraction was cocultivated o n irradiated PG13 (GALV-pseudotype) packaging cells for 48 hours. In 2 anim als, cells of the different fractions were transduced in the presence of hu man FLT-3 ligand (FLT3L), canine stem cell factor (cSCF), and human megakar yocyte growth and development factor (MGDF), and in 2 other dogs, transduct ion was performed in the presence of FLT3L, cSCF, and canine granulocyte-co lony stimulating factor (cG-CSF). The vectors used contained small sequence differences, allowing differentiation of cells genetically marked by the d ifferent vectors. After transduction, nonadherent and adherent cells from a ll 3 fractions were pooled and infused into lethally irradiated dogs. Polym erase chain reaction and Southern blot analysis were used to determine the persistence of the transferred vectors in the peripheral blood and marrow c ells after transplantation. The highest levels of gene transfer were obtain ed when cells were transduced in the presence of FLT3L, cSCF, and cG-CSF (g ene transfer levels of more than 10% for more than 8 months so far). Compar ed with the 2 animals that received cells transduced with FLT3L, cSCF, and MGDF, gene transfer levels were significantly higher when dogs received cel ls that were transduced in the presence of cG-CSF. Transduction on CH-296 r esulted in gene transfer levels that were at least as high as transduction by cocultivation. In summary, the overall levels of gene transfer obtained with these conditions should be sufficiently high to allow stem cell gene t herapy studies aimed at correcting genetic diseases in dogs as a model for human gene therapy. (C) 1999 by The American Society of Hematology.