Mouse Models For Retrovirusmediated Gene Transfer Into

A. Proof of Principle

The initial attempts to introduce new genetic material into mouse HSC were made using recombinant ecotropic oncore-trovirus particles. The proof-of-principle experiments targeted a primitive hematopoietic progenitor cell, the Colony Forming Unit-Spleen (CFU-S). The CFU-Ss are derived from a single cell similar to the common Myeloid progenitor (CMP) (25,35), and gives rise to macroscopic colonies in the spleen of irradiated mice that contain mature cells of all myeloid lineages (25). The vectors used for these studies contained dominant selectable marker genes, either DHFR (180) or neomycin phosphotransferase (neor) (181,182). Mouse bone marrow cells were cultured on a monolayer of virus-producing cells, often in medium conditioned with the hematopoietic growth factor Interleukin-3 (IL-3) before recovery and injection into lethally irradiated mice. Proviral DNA was identified in high-molecular-weight DNA extracted from individual spleen colonies. These studies established that primitive progenitor cells had been transduced without destroying the ability of those cells to differentiate (180,181).

Extensions of these experiments examined lethally irradiated mice repopulated with cells that had been exposed to recombinant ecotropic oncoretroviruses. Southern blot analysis of proviral insertion sites in the bone marrow spleen and thymus revealed that a single HSC could repopulate all 3 organs (Fig. 4) (181,183). Cells containing the unique proviral insertion site persisted in the peripheral blood, marrow, spleen, and thymus of these mice for periods of 12 months or more, with no changes observed after 90 days (21,184-186). These studies established that a single HSC could be transduced without destroying the ability to repopulate the lymphoid and myeloid lineages.

In these early studies, less than 10% of recipient mice had long-term persistence of retrovirus-marked cells. In the rare positive mice, 10% or less of the hematopoietic cells contained a provirus (20,185). A variety of protocols were developed which improve the transduction of mouse HSC, all of which share common elements. The best results are obtained when the donor animals are treated with 5-fluorouracil, a cell-cycle-specific drug that is toxic to cycling cells like hematopoietic progenitor cells (16,187). The depletion of progenitor cells in the host may induce cycling of their more primitive precursors, which promotes retroviral integration (183). Combining IL-3 with IL-6 and SCF in the culture medium extended the viabil

BM Ty Sp

Figure 4 Transduction of mouse HSC. Mouse peripheral blood HSC were exposed to ecotropic retrovirus particles containing a GFP gene. Sixteen weeks after transplantation DNA was extracted from the bone marrow (BM), thymus (Ty), and spleen (Sp) and digested with Eco RI for Southern Blot analysis. Eco RI cuts once in the provirus, giving a unique insertion site for each proviral integration. Most of the bands are shared in DNA from the 3 hematopoietic organs, demonstrating the transduction of HSC.

Eco Rl Digest; GFP Probe

Figure 4 Transduction of mouse HSC. Mouse peripheral blood HSC were exposed to ecotropic retrovirus particles containing a GFP gene. Sixteen weeks after transplantation DNA was extracted from the bone marrow (BM), thymus (Ty), and spleen (Sp) and digested with Eco RI for Southern Blot analysis. Eco RI cuts once in the provirus, giving a unique insertion site for each proviral integration. Most of the bands are shared in DNA from the 3 hematopoietic organs, demonstrating the transduction of HSC.

ity of PHSC in culture without significant loss of repopulating ability, and improved gene transfer (185-189). Other cyto-kines, notably FLT3 ligand in combination with SCF, IL-6, and/or IL-3 (190,191), have had similar effects.

Many protocols include 48-hour ''prestimulation'' of bone marrow cells in medium containing cytokines, prior to exposure to retrovirus particles (185,192). Recent experiments have shown that this prestimulation period appears to prevent the ''loading'' of retrovirus receptors with virus prior to the entry of HSC into cell cycle (166). Delaying the addition of virus until many HSC are competent for transduction optimizes the ability of the HSC to take up and integrate the provirus. Using the optimum combination of hematopoietic growth factors and transduction protocols, most laboratories achieve gene transfer in 100% of transplanted mice, and the proviruses are easily detected by Southern Blot analysis (Fig. 4).

B. Mouse Gene Therapy Models

Mouse models exist for many inherited diseases of the hema-topoietic system. Gene therapy using retrovirus-mediated gene transfer into HSC followed by transplantation has been evaluated in several of these models. These include severe combined immune deficiencies (193,194), inborn errors of metabolism (195), and hemoglobinopathies (21,185,196)

genes. The first successful correction of a mouse model for human disease was reported by Wolfe et al. (197), who introduced the p-glucuronidase gene into the HSC of the mouse model for Sly Syndrome (p-glucuronidase deficiency). The authors reported prolonged life spans comparable to littermate controls and a reversal of the disease phenotype seen in untreated control animals. Subsequently, oncoretrovirus-me-diated gene transfer has been used to correct both the X-linked (gp91phox deficiency) and autosomal (p47phox deficiency) forms of Chronic Granulomatous Disease (CGD) (198,199). Severe Combined Immune Deficiencies (SCID) caused by deficiencies of the Jak3 kinase and the common gamma chain (XSCID) have been cured by the transplantation of oncore-trovirus-transduced HSC (200-205). As noted above, lentivi-rus-mediated gene transfer of a human p-globin gene has significantly improved the phenotype of mouse models of p-thalassemia and Sickle Cell Disease (177,178).

The insertion of oncoretrovirus and lentivirus LTRs into the regulatory regions of specific genes is a well-described mechanism for leukemogenesis in animal models (16,17). Thousands of mice transplanted with oncoretrovirus-or lentiv-irus-transduced HSC have been evaluated in the disease models described above and related studies with other transgenes. Adverse events resulting in uncontrolled growth of hematopoietic cells have been described on two separate occasions. Bunting et al. described a myeloproliferative syndrome in mice transplanted with bone marrow cells transduced with an oncoretrovirus containing the p170 glycoprotein [or multiple drug resistance (mdr)] gene and expanded in vitro for 3 weeks. Follow-up studies demonstrated that the myelor-poliferative syndrome was the result of the prolonged in vitro culture and the overexpression of the mdr gene (206). Li et al. described the insertion of a myeloproliferative syndrome in a single mouse caused by the insertion of a recombinant provirus near the Evi-1 locus (207). In strains of mice with high levels of endogenous wild-type oncoretrovirus, the development of hematopoietic malignancies is common, and oncoretrovirus insertions near the Evi-1 locus are found in a high percentage of myeloid leukemias in the AKXD-23 strain of mice (208). It was calculated that leukemia causing insertions represented less than 1/10,000 of all of the oncoretrovirus insertions in AKXD-23 mice (208). The AKXD-23 leukemias are similar to the one described by Li et al., demonstrating that recombinant oncoretrovirus insertions can cause leukemia, albeit at a low frequency (207). These findings stress the importance of extensive follow-up of animal models and the data and need to consider every possible means to ensure the safety of any gene therapy strategy.

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