Suprasynergistic Peripheral Blood Stem Cell Mobilization in Normal and Fanconi Anemia Knockout Mice by the Combination of G-CSF Plus the CXCR4 Antagonist AMD3100 and the CXCR2 Agonist GRO β
Authors: Pelus Louis M. , Clapp D. Wade , Bridger Gary 
Published: October 2019

Abstract
In mice, the CXCR4 antagonist AMD3100 and the CXCR2 agonist GROβ rapidly mobilizes short and long term repopulating hematopoietic stem and progenitor cells (HSPC). Synergy in mobilization is observed using GRO plus G-CSF or AMD plus G-CSF, and we have recently shown synergy in rapid mobilization using AMD plus GROβ. In general, a common feature of mobilization is that only a relatively small percentage of HSPC egress from marrow. We therefore evaluated whether added benefit in HSPC mobilization could be attained by using all three mobilizers in combination. Although this alters the paradigm of rapid mobilization, it addresses shortcomings of poor mobilization response, requirements for multiple aphereses and the need for large numbers of HSPC in transplant and gene therapy applications. We mobilized BALB/c mice with AMD (5 mg/kg SC, 60 min), GROβ (2.5 mg/kg SC, 15 min), G-CSF (100 ug/kg/day, bid, SC x 4 days) or the G-CSF regimen followed by GROβ, AMD or GRO+AMD administered on day 5 and harvest of peripheral blood 15 (GRO; GRO+AMD) or 60 (AMD) min later. Significant CFU-GM/mL blood were mobilized by G-CSF (4362±996), GRO (2562±396) and AMD3100 (991±121) used alone as expected. Single administration of GRO or AMD to mice mobilized by G-CSF and harvest of blood 15 (GRO) and 60 (AMD) min later, resulted in synergistic mobilization of (12,246±2751) and (12,379±953) CFU-GM, respectively. Rapid mobilization by simultaneous injection of GRO+AMD was similar in magnitude (10,709±1041) at 15 min post administration to mobilization by GRO or AMD in combination with a multiday G-CSF regimen. Administration of the combination of GRO+AMD to mice mobilized by G-CSF resulted in suprasynergistic mobilization of 32,510±3569 CFU-GM/mL after 15 min, representing ~5% of total marrow CFU-GM, with no adverse effects. Fanconi Anemia patients mobilize poorly to G-CSF. FancC −/− mice present a phenotype similar to FancC patients and mobilize poorly to G-CSF, which can be improved by the addition of AMD. We evaluated mobilization by GRO, AMD and G-CSF alone and in combination in +/+ C57Bl and FancC −/− mice using the regimens described above. Mobilization by G-CSF was 45% lower in FancC −/− mice (858±21) compared to +/+ controls (1451±80) and AMD+G-CSF synergistically mobilized CFU-GM more effectively in FancC −/− mice (5078±597) than controls (2981±267). Similarly, CFU-GM mobilization by GRO was lower in FancC −/− mice and GRO+G-CSF synergistically mobilized CFU-GM more effectively in FancC −/− mice. The combination of GRO+AMD mobilized CFU-GM within 15 min that was similar in magnitude to mobilization by AMD+G-CSF in wild type (2077±541 vs 2511±176) as well as FancC −/− mice (4924±577 vs 5078±1597). Mobilization by addition of the rapid acting combination of GRO+AMD to mice mobilized by G-CSF was suprasynergistic reaching 44,669±2974 and 41,068±5630 CFU-GM/mL blood in wild type and −/− mice, respectively. In preliminary studies, transduction of mobilized blood cells with FancC and transplant in FancC −/−mice demonstrated durable engraftment. These studies identify highly effective, rapid GRO+AMD mobilization regimens for standalone application in normal donors and combination regimens for potential application in patients who respond poorly to G-CSF or when large quantities of HSPC are required, for example in gene therapy applications.