Gene Transfer into Nonhuman Primate CD34⁺CD11b^–Bone Marrow Progenitor Cells Capable of Repopulating Lymphoid and Myeloid Lineages

Abstract

We investigated whether rhesus monkey CD34+CD11b- hematopoietic stem cells can be transduced with recombinant retroviruses carrying the human adenosine deaminase (hADA) gene by co-cultivation with a virus-producing cell line. Following autologous transplantation, polymerase chain reaction (PCR) analysis on peripheral blood mononuclear cells and granulocytes showed that the hADA-retrovirus was present in approximately 0.1% of the cells for at least 400 days post transplantation in 2 monkeys. Bone marrow that was harvested 16 months after transplantation carried ADA-overexpressing myeloid progenitor cells capable of in vitro colony formation. In addition, hADA activity could be demonstrated in T lymphocytes that were harvested 9 months post transplantation. Thus, in vitro transduction of CD34+CD11b- cells led to long-term repopulation of the hematopoietic system with transduced cells of lymphoid and myeloid lineages expressing the hADA gene. To investigate whether infusion of virus-producing cells into a rhesus monkey undergoing autologous bone marrow transplantation could lead to in vivo transfer of the recombinant retrovirus, 1 monkey was infused with CD34+CD11b- bone marrow cells (BMC) and a large quantity of virus-producing cells. Few provirus-carrying cells could temporarily be detected in this animal. This shows that in vivo gene transfer into a regenerating hemopoietic system can occur, albeit at very low efficiency.

Induction of cytotoxic T lymphocyte and antibody responses to enhanced green fluorescent protein following transplantation of transduced CD34+ hematopoietic cells

“…3,25 After 7 to 15 days at 37°C with 5% CO 2 , the total number of colonies was determined in duplicate using standard morphologic criteria. 26 Colonies positive for eGFP were assessed using an inverted fluorescent microscope with a mercury light source (Olympus, Mellville, NY).…”

Expression pattern of CD2 locus control region containing retroviral vectors in hemopoietic cells in vitro and in vivo

“…Furthermore, the wild-type enhancer in the MoMLV long terminal repeat (LTR) has been replaced by the enhancer of the PyF101 mutant of polyoma virus. This vector exhibited expression levels of introduced genes that equalled the levels of their endogenous counterparts in adenosine deaminase deficient T cells from a patient suffering from severe combined immunodeficiency disease (ADA − SCID) and in T cells isolated from a rhesus monkey transplanted with transduced bone marrow cells 28,29 However, when transduced bone marrow cells were transplanted into mice and 6 months later peripheral blood mononuclear cells were examined, among six mice that were positive for the provirus only two were found to express human ADA. Moreover, expression levels did not correlate with proviral copy numbers.…”

β-Galactosidase marker genes to tag and track human hematopoietic cells

“…Data obtained from different laboratories led to the hypothesis that the efficiency of transduction is not strictly related to viral titer and can differ from one producing clone to another. 71,[102][103][104][105][106][107] This hypothesis strongly argues for screening of the producing cell on the basis of its ability to infect the hematopoietic cell to be transduced rather than on the basis of titration assays performed on accessory cells such as murine fibroblasts. 107 In addition, prospects for in vivo gene therapy led to the conclusion that new packaging cell lines have to be designed that are able to produce viral particles not sensitive to the inhibitors present in human sera or able to produce targeted viral particles.…”

“…For example, the use of retroviruses to transfer the neoR gene into hematopoietic progenitors has been widely used to study the contribution of different cell populations in hematopoietic recovery in mice, 138 -143 dogs, 144 -146 or nonhuman primates. 103,147,148 Interesting data were reported by Champseix et al 149 using the expression of a cell surface marker coding sequence in a retrovirally transduced cord blood immature hematopoietic cell population reimplanted into immunoincompetent nonobese diabetic severe combined immunodeficient mice.…”