Preclinical efficacy of a modified gamma-globin lentivirus gene therapy in Berkeley sickle cell anemia mice and human xenograft models.

We previously showed correction of sickle cell anemia (SCA) in mice utilizing a lentiviral vector (LV) expressing human γ-globin. Herein, we made a G16D mutation in the γ-globin gene to generate the G16D mutation (GbG(M)) LV to increase fetal hemoglobin formation. We also generated an insulated version of this LV, GbG(MI), inserting a 36-bp insulator from the Foamy virus in the long terminal repeats of the LV. Preclinical batches of GbG(M) and GbG(MI) LV showed both were highly efficacious in correcting SCA in mice, with sustained gene transfer in primary transplanted SCA mice and high hematopoietic stem cell (HSC) transduction in colony-forming unit-spleen in secondary transplanted mice. CRISPR-mediated targeting of the proviruses into the LMO2 proto-oncogene showed remarkably reduced LMO2 activation by both insulated and uninsulated LV, compared to the SFFV γ-RV vector targeted to the same locus. We therefore used the GbG(M) LV to perform preclinical human CD34(+) gene transfer. We assessed gene transfer and engraftment of human HSCs in two immunocompromised mouse models: persistent stable GbG(M)-transduced cell engraftment was comparable to that of untransduced cells with no detrimental effects on hematopoiesis up to 20 weeks post transplant. These robust preclinical studies in mouse and human HSCs allowed its translation into a clinical trial.