Background.In vivo hematopoietic stem cell (HSC) gene therapy represents a simpler approach to treating hemoglobinopathies without the need for myelosuppressive conditioning and autologous HSC transplantation. We developed a helper dependent adenovirus (HDAd5/35++)-based platform that enables efficient in vivo transduction of mobilized HSCs via CD46. Transduced HSCs can be positively selected by low-dose O6BG/BCNU-treatment to achieve ~90% marking rates in peripheral blood. Initial proof-of-concept in murine models as well as in rhesus macaques demonstrates high level of g-globin expression after gene addition by a Sleeping Beauty transposase. While the current mobilization regimen-4 days of G-CSF injection followed by an injection of AMD3100/plerixafor on day 5-mobilizes HSCs from the bone marrow to the periphery, several issues exist. Despite widespread use as a mobilization agent in oncology, G-CSF is contra-indicated in patients with sickle cell disease. Additionally, G-CSF results in unselective bone marrow cell mobilization, which leads to leukocytosis and elevated numbers of cytokine-producing cells in the periphery that come into contact with HDAd particles, leading to high cytokine levels. Mobilized (committed) bone marrow cells in the periphery also sequester HDAd thus reducing the effective dose for primitive HSCs. Further, the five-day treatment regimen and high costs associated with G-CSF + plerixafor justify the development of an alternative mobilization regimen. A single-day, G-CSF-free mobilization regimen that mobilizes a high proportion of HSCs may therefore be preferred for in vivo gene therapy.

Results. Here we tested HSC mobilization by truncated MGTA-145, a CXCR2 agonist, and plerixafor in the context of in vivo HSC transduction. CD46-transgenic animals were mobilized with GCSF + plerixafor (5 days) or with MGTA-145 + plerixafor (same-day treatment) and then injected one hour later with an integrating HDAd5/35++ mgmt/GFP vector. MGTA-145 + plerixafor resulted in robust mobilization of HSCs, less leukocytosis and no significant elevation of cytokines, as observed with G-CSF + plerixafor. With both mobilization regimens, after in vivo selection with O6BG/BCNU, >90% of PBMCs expressed GFP and marking rates were stable long-term. Mice were sacrificed 12 weeks after in vivo transduction and bone marrow lineage-negative cells were harvested for transplantation into secondary recipients. Stable transgene expression (>90% at week 16 after transplantation) was observed with both mobilization regimen in secondary recipients and multilineage engraftment was observed with MGTA-145 + plerixafor in primary and secondary recipients.

Importantly, the mobilization with MGTA-145 + plerixafor worked efficiently in a mouse disease model for thalassemia (Hbbth3/CD46+/+). In this model, after in vivo transduction with an integrating HDAd5/35++mgmt/gamma-globin vector and in vivo selection, over 95% of peripheral red blood cells (RBCs) expressed human gamma-globin. The gamma-globin protein level reached 36% over mouse beta-globin. Phenotypic analyses showed a complete correction reflected by normal RBC morphology and absence of blood reticulocytosis, extramedullary hemopoiesis and hemosiderin deposition in spleen and liver sections. In secondary recipients of Lin- cells (harvested at week 14 from in vivo transduced Hbbth3/CD46+/+ mice), gamma-globin marking in RBCs was stable at 99% (currently at week 11 after transplantation). This demonstrates that MGTA145 + plerixafor mobilizes long-term repopulating HSCs.

Conclusions. These data demonstrate that the combination of MGTA-145 and plerixafor could serve as an efficient and potentially safer one-day mobilization regimen for in vivo HSC gene therapy in patients with hemoglobinopathies.


Goncalves:Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Davis:Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company. Kiem:Rocket Pharma: Membership on an entity's Board of Directors or advisory committees; Umoja: Membership on an entity's Board of Directors or advisory committees; CSL: Consultancy; Homology Medicines: Membership on an entity's Board of Directors or advisory committees; Vor Biopharma: Membership on an entity's Board of Directors or advisory committees; Enochian: Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics: Consultancy. Lieber:Ensoma, Inc: Consultancy, Research Funding.

Author notes


Asterisk with author names denotes non-ASH members.