Gene Therapy (2000) 7, 359–367  2000 Macmillan Publishers Ltd All rights reserved 0969-7128/00 $15.00 www.nature.com/gt VIRAL TRANSFER TECHNOLOGY RESEARCH ARTICLE Differences among nonhuman primates in susceptibility to bone marrow progenitor transduction with retrovirus vectors DW Emery 1,2 RG Andrews 2,3,4 and T Papayannopoulou 1,2 University of Washington Departments of 1 Medicine and 3 Pediatrics, Seattle, WA; 4 Fred Hutchinson Cancer Research Center, Seattle, WA; and 2 University of Washington Regional Primate Research Center, Seattle, WA, USA Nonhuman primates are increasingly being used as models for pre-clinical assessment of retrovirus vector expression and function following stem and progenitor cell transduction. We compared the relative susceptibility of CD34 + marrow progenitors from four nonhuman primate species and humans to transduction with amphotropic pseudotyped retrovirus vectors containing the Neo gene. The rate of func- tional gene transfer was measured by colony formation under G418 selection. Marrow progenitors from pigtail macaques (Macaca nemestrina) were transduced at about twice the rate (19.1 ± 4.3%) as those from rhesus (11.2 ± 3.7%) and cynomolgus (7.6 ± 1.9%) macaques, baboons (7.8 ± 1.8%), and humans (9.6 ± 1.7%). Semiquan- Keywords: gene delivery; retrovirus; transduction; bone marrow; primate; culture Introduction It has long been established that retrovirus vectors based on murine leukemia virus (MLV) are able to transduce reconstituting hematopoietic stem cells (HSC) efficiently in mice, 1 with provirus routinely observed in greater then 50% of peripheral mononuclear cells. 2–4 However, initial attempts at using MLV-based vectors for transduction of HSC in large animal models such as cats, 5 dogs, 6 swine, 7 several nonhuman primates models, 8 and in human clini- cal trials, 9,10 revealed much lower rates of gene transfer, with provirus frequently present in 1–2% or less of peri- pheral mononuclear cells. Although recent studies have reported improvements in the rates of gene transfer in nonhuman primate models, 11–14 current rates still hamper the ability to assess accurately provirus levels, vector expression, or transgene function. With the goal of exploring differences among nonhu- man primate models for pre-clinical analysis of retrovirus vector expression and function, we compared the ability of CD34-enriched bone marrow progenitors from three species of macaques, baboons, and normal humans to be transduced with MLV-based reporter vectors prepared with amphotropic packaging lines. Although inde- Correspondence: DW Emery, Medicine/Medical Genetics, Box 357720, University of Washington, Seattle, WA 98195–7720, USA Received 6 May 1999; accepted 28 October 1999 titative RT/PCR analysis suggests this difference may be due to elevated expression of the amphotropic receptor Pit2 in pigtailed macaque CD34 + cells. Further, transduction rates increased an average 1.6 ± 0.4-fold when the culture temperature was lowered to 33°C, and 2.1 ± 0.3-fold when the culture dishes were coated with the fibronectin fragment CH-296. The data presented here point to important differ- ences among nonhuman primate models as well as trans- duction culture conditions, and suggest that pigtailed macaques may be particularly useful for assessing expression and function of therapeutic retrovirus vectors. Gene Therapy (2000) 7, 359–367. pendent studies have been reported for the transduction of marrow cell populations from rhesus macaques (M. mulatta), 8,13,14 baboons (P.c. anubis or P.c. cynocephalus), 11,12 and to a lesser degree cynomolgus macaques (M. fascicularis), 15 these studies used widely diverse transduction and transplantation protocols, mak- ing direct comparisons of transduction rates difficult. Further, a review of the literature revealed no previous bone marrow transduction studies with pigtailed macaques (M. nemestrina), although this species has been used extensively in other fields of biomedical research. 16–19 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 nonhu- man primates and humans using standard culture con- ditions. Semiquantitative RT/PCR suggests this differ- ence may be due to differences in expression of the amphotropic receptor Pit2 (previously known as Ram1), 20–22 providing further evidence for the role of receptor expression in the relative susceptibility to retro- virus vector transduction. In order to optimize progenitor transduction rates, we also tested several critical para- meters 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 fibronec- tin fragment CH-296. 23,24