Cell Engineering with Glycogen Synthase Kinase-3 Beta Inhibitor-Loaded Synthetic Nanoparticles Enhances Hematopoietic Engraftment of Bone Marrow Mononuclear Cells Following in Utero Transplantation
Authors: Loukogeorgakis Stavros P , Li Haiying , Tang Li , Kim Aimee G , Vrecenak Jesse D , Nissim Ilana , Conner Michael A , Nissim Izhtak , De Coppi Paolo , Peranteau William , Irvine Darrell J , Flake Alan W 
Published: November 2019

Abstract
Introduction
In utero hematopoietic cell transplantation (IUHCT) has tremendous potential for prenatal treatment of congenital hematological disorders, but clinical application has been limited by low engraftment levels. Host cell competition is one of the most formidable barriers to successful engraftment post IUHCT, as fetal stem cells have a proliferative advantage over their postnatal equivalents. Glycogen synthase kinase-3 beta (GSK3β) inhibition has been shown to augment expansion kinetics of bone marrow-derived hematopoietic stem cells (HSC), resulting in enhanced repopulating capacity in vivo. We have developed a strategy that allows targeted, sustained delivery of a GSK3β inhibitor to donor cells by conjugating biodegradable nanocarriers to the cell surface. The aim of the present study was to determine whether donor cell engineering with GSK3β inhibitor-loaded nanoparticles enhances hematopoietic engraftment following IUHCT.
Methods
Bone marrow mononuclear cells (MNC) were isolated from 6-week old C57BL/6TgN(act-EGFP)OsbY01 mice. GSK3β inhibitor (CHIR99021) loaded multilamelar lipid vehicles (MLV) were synthesized from simple liposomes with the addition of divalent cations to induce liposome fusion. The presence of thiol-reactive maleimide groups in MLV lipid bilayers allowed stable conjugation to the cell membrane of donor MNC. IUHCT was performed in Balb/c mice at E14 and 107 MNC were injected intravenously into each fetus. Donor cell engraftment was assessed in peripheral blood at 4 and 12 weeks of age by flow cytometry (% GFP+ cells within CD45+ population). Comparisons were made between animals that received untreated MNC (control), MNC conjugated with inhibitor-loaded MLV (CHIR99021-MLV), and MNC co-injected with a bolus dose of the inhibitor equivalent to that loaded in MLV (CHIR99021-bolus). Lineage characterization of donor cells was performed using antibodies against lymphoid (CD3, B220), and myeloid (CD11b, Gr-1) cell surface markers. Results are expressed as mean±SEM, and statistical analysis was performed using 1- or 2-way ANOVA with Bonferroni post-hoc tests. Experimental protocols were approved by the Institutional Animal Care and Use Committee at The Children’s Hospital of Philadelphia.
Results
MLV had a diameter of 489±8nm, and contained 21.7±2.1μg CHIR99021 per 1010 nanoparticles. Inhibitor release was gradual, with 92.7±0.2% of the encapsulated mass released within 7 days. 64±9 nanoparticles were conjugated on each donor cell, resulting in inhibitor dose of 1.3*10-7μg per cell (1.3μg or 4mg/kg per fetus). MLV conjugation did not affect the ability of MNC to migrate to fetal hematopoietic organs post IUHCT. Fetal survival was comparable in control (20/27; 74.1%) and CHIR99021-MLV (10/12; 83.3%) groups, but was reduced in the CHIR99021-bolus group (7/15; 46.7%; p<0.05). Sustained in vivo release of the inhibitor in CHIR99021-MLV animals resulted in increased donor cell engraftment at 4 weeks of age (52.9±2.8%) that was 3 times greater to that observed in control animals (15.4±1.4%; p<0.0001). Bolus CHIR99021 administration had no effect on engraftment (10.5±2.0%; p<0.0001 vs. CHIR99021-MLV, p=0.6 vs. control). Engraftment levels were lower at 12 weeks in control and bolus-CHIR99021 groups (control: 5.9±0.8%, bolus-CHIR99021: 4.5±1.7%; p<0.05 vs. 4 weeks), but were maintained in CHIR99021-MLV offspring (48.3±2.2%; p<0.0001 vs. control and bolus-CHIR99021). Donor cell characterization showed multi-lineage hematopoietic differentiation, with distribution similar to that of host cells and no difference between experimental groups (p=0.6). In vitro pre-treatment for 7 days with the same amount of CHIR99021 per cell resulted in enhanced HSC (lineage-/c-Kit+/Sca-1+) proliferation (p<0.01 vs. control) and augmented their ability to generate hematopoietic progenitors (p<0.05 vs. control), by modulating the Wnt, Notch and Hedgehog pathways.
Conclusion
Cell engineering with GSK3β inhibitor-loaded synthetic nanoparticles enhances hematopoietic engraftment of BM-MNC following IUHCT. Prolonged retention of the biodegradable nanocarriers on donor cell surfaces enables sustained CHIR99021 release, and allows pseudo-autocrine bioactivity. Conjugation of drug-loaded particles directly to donor cells allows targeted augmentation of HSC function, and could markedly increase the therapeutic potential of IUHCT.

Disclosures
No relevant conflicts of interest to declare.