In Vivo Screening in SCID-X1 Mice.

Male 8- to 10-wk-old SCID-X1 mice were randomly divided into three cohorts of six: untreated, PLGA NP only, and PLGA NP plus mobilizing agents (G-CSF + Plerixafor). Untreated SCID-X1 and C57BL/6 wild-type mice were used as controls to compare baseline and expected changes in phenotypically corrected mice. Six PNA candidates were chosen for in vivo testing due to their close proximity to the target site. PLGA NPs were administered according to previously published protocols (13). Briefly, SCF (220 μg/kg per mouse, recombinant mouse SCF, carrier-free, R&D #455-mc-050/CF) was injected intraperitoneally 3 h prior to PNA treatment (2.5 mg PLGA in 150 μL PBS per dose) via retro-orbital intravenous injection every 48 h for a total of four doses. Mobilizing agent G-CSF (6.25 μg/day) was administered subcutaneously daily for 4 d and once in conjunction with plerixafor (5 mg/kg) during the last day of G-CSF administration (Fig. 6C).

Mice were anesthetized with isoflurane followed by retro-orbital bleeding (∼100 μl) and blood was analyzed at 8 and 16 wk post PNA administration by flow cytometry. BM cells were harvested at the end of study (16 wk) and likewise analyzed by flow cytometry for stem and progenitor cell correction.