In vivo persistence of retrovirally transduced murine long-term repopulating cells is not limited by expression of foreign gene products in the fullyor minimally myeloablated setting

Many nonmalignant hematologic disorders could potentially be treated by gen etic correction of as few as 5-10% of target lineage cells. However, immune system clearance of cells expressing gene products perceived as foreign co uld be limiting. There is evidence that tolerance to foreign proteins can r esult when myeloablative conditioning is used, but this limits the overall applicability of such techniques. Therefore, we sought to evaluate the engr aftment of hematopoietic stem cells carrying a foreign transgene after low- dose irradiation by comparing in vivo survival of murine long-term repopula ting cells (LTRC) transduced with either a retroviral vector expressing the bacterial neomycin phosphotransferase gene (neo) or a vector containing ne o gene sequences but modified to prevent protein expression (nonexpression) . First, marrow cells from congenic donors were transduced with either vect or and transplanted into recipients treated with standard dose irradiation of 800 rads. High-level engraftment and gene marking resulted, without diff erences in the marking levels or pattern of persistence of the cells betwee n cells transduced with either vector. Low-dose irradiation at 100 rads was tested using higher cell doses. Marking levels as high as 10% overall were obtained, again with no differences between mice receiving cells transduce d with the neo versus the nonexpression vectors. To investigate a potential ly more immunogenic protein, marrow cells were transduced with a vector con taining the green fluorescent protein (GFP) gene, and their persistence was studied in recipient mice receiving 100 rads. Stable GFP expression in 5-1 0% of circulating cells was observed long term. We conclude that even with very low dose conditioning, engraftment by genetically modified LTRC cells at clinically significant levels can be achieved without evidence for clear ance of cells known to be expressing immunogenic proteins.