High Efficiency Gene Transfer to Human Hematopoietic SCID-Repopulating Cells Under Serum-Free Conditions

Abstract

Stable gene transfer to human pluripotent hematopoietic stem cells (PHSCs) is an attractive strategy for the curative treatment of many genetic hematologic disorders. In clinical trials, the levels of gene transfer to this cell population have generally been low, reflecting deficiencies in both the vector systems and transduction conditions. In this study, we have used a pseudotyped murine retroviral vector to transduce human CD34+ cells purified from bone marrow (BM) and umbilical cord blood (CB) under optimized conditions. After transduction, 71% to 97% of the hematopoietic cells were found to express a low-affinity nerve growth factor receptor (LNGFR) marker gene. Six weeks after transplantation into immunodeficient NOD/LtSz-scid/scid (NOD/SCID) mice, LNGFR expression was detected in 6% to 57% of CD45+ cells in eight of nine engrafted animals. Moreover, proviral DNA was detected in 8.3% to 45% of secondary colonies derived from BM cells of engrafted NOD/SCID mice. Our data show consistent transduction of SCID-repopulating cells (SRCs) and suggest that the efficiency of gene transfer to human hematopoietic repopulating cells can be improved using existing retroviral vector systems and carefully optimized transduction conditions. © 1998 by The American Society of Hematology.

Genetic protection of repopulating hematopoietic cells with an improved MDR1‐retrovirus allows administration of intensified chemotherapy following stem cell transplantation in mice

“…32 The results shown here suggest that a limited gene transfer efficiency in the engineered graft may be sufficient to achieve protection from lethal hematotoxicity in clinical trials. Similar, if not increased, gene transfer efficiency compared to that seen in the present study has recently been reported from primate models of retroviral gene transfer, 33,34 as well as in repopulating human BM cells that have been analyzed as xenografts in immunodeficient mice, 28,35,36 and also in a clinical study. 20 The moderate transduction rate obtained in the present mouse model therefore indicates that SF1m might also be effective in human gene therapy.…”

Oncoretroviral gene transfer to NOD/SCID repopulating cells using three different viral envelopes

“…In the PG13‐MGIN SF group, all mice were engrafted with marked human cells and the mean level of marking was just below 50%, while more than 50% of human colonies derived from the bone marrow of NOD/SCID mice were GFP‐positive. This level of gene transfer to SRCs is superior to what has been reported by other investigators using GALV‐pseudotyped or amphotropic vectors5–9, 37, 38 and also slightly higher than the transduction efficiency recently reported for an oncoretroviral vector pseudotyped with the RD114 envelope39. In the latter study, Kelly et al .…”

“…Several studies have shown that serum‐free transduction of CD34 + cells with repopulating capacity using GALV‐pseudotyped vectors is feasible5, 7. However, no side‐by‐side comparison using a GALV‐pseudotyped vector with and without serum for the transduction of repopulating cells has been reported.…”

“…Recently, however, several investigators have been able to show efficient oncoretroviral gene transfer into primitive human hematopoietic cells with the capacity to repopulate NOD/SCID mice4–9. This is a significant advance in the development of gene therapy for HSCs which has been made possible through systematic improvements in the in vitro culture and transduction conditions used for the hematopoietic cells.…”

Serial Transplantations in Nonobese Diabetic/Severe Combined Immunodeficiency Mice of Transduced Human CD34+ Cord Blood Cells: Efficient Oncoretroviral Gene Transfer and Ex Vivo Expansion Under Serum-Free Conditions

“…The presence of LNGFR provirus in NOD/SCID BM was determined by polymerase chain reaction (PCR) amplifying the specific 425-bp fragment of the LNGFR gene [35].…”