Single Dose of Tgroβ (EN-145)/Plerixafor Safely and Effectively Mobilizes Primitive HSCs in Mouse Models for In Vivo Gene Therapy of Sickle Cell Disease

Successful granulocyte colony-stimulating factor (G-CSF)-free mobilization is crucial for HSC gene therapies for sickle cell disease (SCD). Since G-CSF is contraindicated in SCD patients, single-agent plerixafor (P) mobilization is utilized. However, P alone does not reliably yield optimal hematopoietic stem (HSC) and progenitor (HSPC) cell numbers for ex vivo gene therapies (Leonard and Weiss, Curr Opin Hematol., 2024). A single dose of CXCR2 agonist truncated GROβ (tGROβ) in combination with P has mobilized 3 to 4-fold more primitive HSCs compared to either multi-dose G-CSF or P alone in a Phase 1 healthy volunteer study (Magenta Therapeutics-Goncalves et al., TCT, 2021). This approach may enable G-CSF free mobilization for SCD, improving efficiency of ex vivo gene therapies, as well as Ensoma's in vivo HSC gene therapy, which intravenously delivers payload via a virus-like particle (VLP) derived from helper-dependent adenovirus (HDAd5/35++) to HSPC.

The goal of this work is to evaluate the safety and efficacy of Ensoma's tGROβ: EN-145, formerly MGTA-145, in combination with P in our relevant SCD mouse models.

First, we assessed mobilization in a CD46 transgenic mouse model (CD46tg), which expresses human CD46, a receptor enriched in HSCs and required for transduction of Ensoma's VLPs. Consistent with previous preclinical studies in wild-type mice and NHP (Hoggatt et al., Cell, 2018, Falahee et al., ASTCT, 2018) we demonstrate that co-administered EN-145+P improved mobilization of both mouse LSK (Lin-Sca1+cKit+ HSPCs) and long-term HSCs (LT-HSC, LSK CD48-CD150+) (n=8) compared to P only. EN-145+P mobilization improved GFP expression in LSK 3 days post-transduction with a VLP-GFP in bone marrow (BM) when compared to P only, reaching similar gene marking to that seen with G-CSF+P mobilization. This expands upon our founder Dr. Andre Lieber's data in thalassemia mouse model (Li et al., Blood Adv. 2021), reinforcing the potential of EN-145+P for in vivo HSC gene therapy.

We then characterized EN-145+P mobilization in a SCD mouse model (hCD46tgxTownes SS (CD46/SS)) that recapitulates key disease phenotypes (Li et al., Blood, 2023; internal data). Consistent with SCD patients' complications, we reported mortality with G-CSF+P mobilization in up to 15% of mice (n= 20, 3 studies). In contrast, EN-145+P was well-tolerated without observed leukocytosis and mobilized 6-fold more LT-HSCs than P only (n=4-8). Higher absolute LSK levels were observed in peripheral blood of CD46/SS compared to CD46 mice in both EN-145+P and P groups, likely due to compromised SCD BM niche.

We then evaluated the impact of EN-145+P on HSC mobilization and VLP transduction in humanized NBSGW mice (McIntosh et al., Stem cell report, 2015), a key preclinical mouse model with erythrocytic lineage response for SCD gene therapy. The ability to mobilize CD34+ cells from the humanized mouse BM microenvironment with G-CSF+P has been a contentious matter, showing either no human HSC in circulation (Choo et al, Blood Advances, 2024), or requiring an additional VLA4 inhibitor to achieve effective mobilization (Javed et al., Cell, 2022). Here we demonstrate that EN-145+P results in robust mobilization of human CD34+ cells and enriched primitive HSCs (CD34+CD90+CD45RA-) in peripheral blood within 15 minutes post simultaneous dosing (n=5). Conversely, the extended G-CSF+P dosing regimen (peak measured at 3 hours, n=5 in 2 independent studies) primarily releases lineage-committed cells and was less effective mobilizing HSC. Importantly, in vivo transduction with a VLP-GFP of EN-145+P-mobilized HSC showed significant GFP expression in both CD34+ and primitive HSC in BM at 3- and 7-days post dosing. In contrast, no GFP expression was observed in BM of G-CSF+P mobilized mice, likely due to compromised microenvironment and multiday dosing. EN-145+P mobilization did not affect the total number of human CD45+ BM cells, indicating a more favorable regimen for BM integrity and HSC homing. These findings support the use of EN-145+P mobilization in the NBSGW model for evaluating in vivo HSC gene therapy.

In conclusion, our comprehensive mouse studies further validate the in vivo safety and efficacy of single-dose EN-145+P as a robust alternative to G-CSF for primitive HSC mobilization in SCD. These data support the major therapeutic value of EN-145+P for ex vivo and Ensoma's in vivo VLP-based gene therapies in SCD.

No relevant conflicts of interest to declare.