The level of mRNA encoding the amphotropic retrovirus receptor in mouse and human hematopoietic stem cells is low and correlates with the efficiency of retrovirus transduction.

Abstract

The low level of amphotropic retrovirusmediated gene transfer into human hematopoietic stem cells (HSC) has been a major impediment to gene therapy for hematopoietic diseases. In the present study, we have examined amphotropic retrovirus receptor (amphoR) and ecotropic retrovirus receptor mRNA expression in highly purified populations of mouse and human HSC. Murine HSC with low to undetectable levels of amphoR mRNA and relatively high levels of ecotropic retrovirus receptor mRNA were studied.When these HSC were analyzed simultaneously for ecotropic and amphotropic retrovirus transduction, ecotropic provirus sequences were detected in 10 of 13 long-term repopulated animals, while amphotropic proviral sequences were detected in only one recipient. A second distinct population of murine HSC were isolated that express 3-fold higher levels of amphoR mRNA. When these HSC were analyzed simultaneously for ecotropic and amphotropic retrovirus transduction, 11 of 11 repopulated mice contained ecotropic provirus and 6 of 11 contained amphotropic provirus sequences, a significant increase in the amphotropic retrovirus transduction (P = 0.018). These results indicate that, among the heterogeneous populations of HSC present in adult mouse bone marrow, the subpopulation with the highest level of amphoR mRNA is more efficiently transduced by amphotropic retrovirus. In a related study, we found low levels of human amphoR mRNA in purified populations of human HSC (CD34+ CD38-) and higher levels in committed progenitor cells (CD34+ CD38+). We conclude that the amphoR mRNA level in HSC correlates with amphotropic retrovirus transduction efficiency.

Differences among nonhuman primates in susceptibility to bone marrow progenitor transduction with retrovirus vectors

“…11,21,22 Our observed correlation between elevated progenitor transduction rates and elevated Pit2 expression in CD34-enriched marrow cells from pigtailed macaques are consistent with these studies. Whether marrow progenitors from pigtailed macaque are more susceptible to transduction with other MLV pseudotypes, such as GaLV, 11,44 is not clear.…”

“…[20][21][22] In order to determine whether the elevated progenitor transductions rates observed here for pigtailed macaques correlated with elevated Pit2 expression, CD34-enriched marrow cells were analyzed by semiquantitative RT/PCR. For this purpose we used a set of primers for a highly conserved region of Pit2, 30 and as a control, another set of primers for a highly conserved housekeeping gene, ␤-actin.…”

“…[16][17][18][19] We found that CD34-enriched marrow progenitors from pigtailed macaques were transduced at about twice the rate as progenitors from the other three species of nonhuman primates and humans using standard culture conditions. Semiquantitative RT/PCR suggests this difference may be due to differences in expression of the amphotropic receptor Pit2 (previously known as Ram1), [20][21][22] providing further evidence for the role of receptor expression in the relative susceptibility to retrovirus vector transduction. In order to optimize progenitor transduction rates, we also tested several critical parameters effecting the transduction efficiency of CD34-enriched marrow progenitors from this and other species, including the temperature and days of culture, the use of cytokines, and the use of dishes coated with the fibronectin fragment CH-296.…”

Adenovirus-mediated expresssion of the murine ecotropic receptor facilitates transduction of human hematopoietic cells with an ecotropic retroviral vector

“…Instead, amphotropic vectors used for primate stem cell gene transfer rely on a phosphate symporter that is expressed at much lower levels in primitive hematopoietic cells. 10 These differences in the level of receptor expression correlate with the efficiency of gene transfer into repopulating cells. 10 Other biological and methodological factors may also contribute to the much higher efficiency of gene transfer achieved in the murine system.…”

“…10 These differences in the level of receptor expression correlate with the efficiency of gene transfer into repopulating cells. 10 Other biological and methodological factors may also contribute to the much higher efficiency of gene transfer achieved in the murine system. Gene transfer by MuLV vectors requires cell division; mouse stem cell cycle more frequently than those of humans.…”