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Transduction of Mouse Hematopoietic Stem Cells is More Efficient with 10A1 Retrovirus Vectors than with Amphotropic Vectors

(summarized by Enrico Novelli and Zhigang Gao)

The use of retroviral vectors for gene therapy of human hematopoietic diseases has been hampered by a very low frequency transduction of hematopoietic stem cells (HSC). While it is possible to retrovirally label up to 90% of the murine repopulating HSC, the efficiency of transduction in primates is low, generally <1%. Dr. Bodine noted that a clear difference between these retroviral transductions conducted in mice versus primates lies in the specific vectors employed for the different species.

For murine gene therapy, it is possible to use ecotropic retroviral vectors. The ecotropic retroviruses have a GP70 envelope protein which binds to a specific murine cell surface receptor (an amino acid transport protein). To transduce primate cells, amphotropic retroviruses are instead used. Amphotropic retroviruses have a different envelope. The amphotropic envelope protein interacts with a different transport protein receptor, named PiT-2. Dr. Bodine hypothesized that the low number of PiT-2 molecules on primate HSC, as compared to the number of ecotropic receptors on murine HSC, might result in low binding of the amphotropic virus to HSC, and thereby low transduction efficiency.

To test this hypothesis, Dr Bodine decided to evaluate mouse HSC for their level of PiT-2 expression. Mouse HSC can be subdivided by elutriation into a FR35Lin^–c-kit^high cell fraction, composed of large size cells with predominantly short-term engrafting capacity, and FR25Lin^–c-kit^high cell fraction, composed of small cells with long-term engrafting capacity. An average of 30 c-kit^high cells can repopulate 100% of recipient mice in murine transplantation studies. The level of ecotropic receptor was high on both cell types by a PCR assay devised in Dr. Bodine's lab. In contrast, the level of amphotropic receptor was lower in both cell types, and PiT-2 levels were higher in the FR35Lin^–c-kit^high fraction cells than in the FR25Lin^–c-kit^high fraction. Thus, according to Dr. Bodine's hypothesis, FR25Lin^–c-kit^high cells are likely to be transduced less efficiently by amphotropic retroviral vectors than the FR35Lin^–c-kit^high fraction cells. To test this, Dr. Bodine exposed both elutriated cell fractions to either ecotropic or amphotropic retrovirus supernatants for 96 h in culture in the presence of interleukin 3 (IL-3), IL-6, and stem cell factor (SCF). He then transplanted the transduced cells into syngeneic recipient mice. Mice were sacrificed 16 weeks post transplantation, and DNA polymerase chain reaction (PCR) analysis of blood from the animals was performed to detect the integrated retrovirus in the progeny of the repopulating HSC. All but one of the mice which received FR25Lin^–c-kit^high cells transduced with the amphotropic retrovirus did not harbor transduced repopulating cells. Conversely, high percentages of the mice that were transplanted with either (A) FR35Lin^–c-kit^high cells transduced with either retroviruses or (B) FR25Lin^–c-kit^high cells transduced with ecotropic retrovirus, contained transduced repopulating cells. This experiment confirmed the hypothesis that the low transduction efficiency observed with the amphotropic retroviruses is not due to low cycling level of the cells but to the low number of PiT-2 receptor molecules on the cell surface.

Therefore, Dr. Bodine decided to use two approaches to circumvent this problem. The first is to induce higher levels of PiT-2 expression on HSC by extending the period of in vitro culture. The second is to investigate transduction via PiT-1, the receptor used by Gibbon ape leukemia virus (GaLV). In his talk, Dr. Bodine focused on the results of his experiments aiming to induce higher levels of amphotropic retrovirus on the FR25Lin^–c-kit^high cells, which resemble human HSC. Mouse bone marrow (BM) cells were cultured in IL-3, IL-6, and SCF for up to 192 h, then FR25Lin^–c-kit^high cells were isolated and the mRNA levels of PiT-2 and PiT-1 were analyzed by reverse transcriptase-PCR. The levels of both receptors increased during the culture period. The cells were then transduced with amphotropic retrovirus and transplanted into irradiated syngeneic mice. When the cells were cultured for 0-96 h, 5/43 animals were positive for amphotropic proviral sequences, versus 7/15 animals transplanted with cells precultured for 144 h. Dr. Bodine calculated that at least six days of preculture are necessary to enhance the levels of PiT-2 to allow efficient gene transfer. Dr. Bodine drew the conclusion that the high levels of PiT-2 mRNA obtained after 144 h of preculture led to improved transduction with amphotropic retroviral vectors.

However, this study raised the concern that the extended preculture period might have partially depleted the HSC capacity of the cultured cells. A competitive repopulation assay demonstrated that mouse BM cells cultured for eight days in the same growth factors retained 60% of their initial repopulating ability. The future direction of Dr. Bodine's lab aims at developing strategies to induce the amphotropic receptor levels on murine and human HSC without depleting their repopulating capacity. Strategies may include the use of serum-free medium, different combinations of cytokines, or mobilization of HSC with a cytokine combination which leads to the induction of high levels of amphotropic and GaLV receptors on mobilized peripheral blood CD34⁺CD38^– cells. Finally, in collaboration with Donald Orlic, Dr. Bodine showed that cryopreserved human cord blood (CB) cells have much higher levels of amphotropic receptor than freshly isolated CB cells. This seems to correlate with a higher transduction efficiency; when transduced cells were tested for the ability to repopulate SCID mice, highly transduced cells of multiple lineages were recovered from the animals. Dr. Bodine will focus on elucidating the mechanisms that underlie the higher amphotropic receptor expression levels in cryopreserved HSC.

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				D. A. Williams, A. W. Nienhuis, R. G. Hawley, and F. O. Smith Gene Therapy 2000 Hematology, January 1, 2000; 2000(1): 376 - 393. [Abstract] [Full Text] [PDF]

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