Multiple Integrated Non-clinical Studies Predict the Safety of Lentivirus-Mediated Gene Therapy for ß-Thalassemia.

Gene therapy clinical trials require rigorous non-clinical studies in the most relevant models to assess the benefit-to-risk ratio. To support the clinical development of gene therapy for ß-thalassemia, we performed in vitro and in vivo studies for prediction of safety. First we developed newly GLOBE-derived vectors that were tested for their transcriptional activity and potential interference with the expression of surrounding genes. Because these vectors did not show significant advantages, GLOBE lentiviral vector (LV) was elected for further safety characterization. To support the use of hematopoietic stem cells (HSCs) transduced by GLOBE LV for the treatment of ß-thalassemia, we conducted toxicology, tumorigenicity, and biodistribution studies in compliance with the OECD Principles of Good Laboratory Practice. We demonstrated a lack of toxicity and tumorigenic potential associated with GLOBE LV-transduced cells. Vector integration site (IS) studies demonstrated that both murine and human transduced HSCs retain self-renewal capacity and generate new blood cell progeny in the absence of clonal dominance. Moreover, IS analysis showed an absence of enrichment in cancer-related genes, and the genes targeted by GLOBE LV in human HSCs are well known sites of integration, as seen in other lentiviral gene therapy trials, and have not been associated with clonal expansion. Taken together, these integrated studies provide safety data supporting the clinical application of GLOBE-mediated gene therapy for ß-thalassemia.

Plerixafor+G-CSF-mobilized CD34+ cells represent an optimal graft source for thalassemia gene therapy.

Globin gene therapy requires abundant numbers of highly engraftable, autologous hematopoietic stem cells expressing curative levels of ß-globin on differentiation. In this study, CD34+ cells from 31 thalassemic patients mobilized with hydroxyurea+granulocyte colony-stimulating factor (G-CSF), G-CSF, Plerixafor, or Plerixafor+G-CSF were transduced with the TNS9.3.55 ß-globin lentivector and compared for transducibility and globin expression in vitro, as well as engraftment potential in a xenogeneic model after partial myeloablation. Transduction efficiency and vector copy number (VCN) averaged 48.4 ± 2.8% and 1.91 ± 0.04, respectively, whereas expression approximated the one-copy normal ß-globin output. Plerixafor+G-CSF cells produced the highest ß-globin expression/VCN. Long-term multilineage engraftment and persistent VCN and vector expression was encountered in all xenografted groups, with Plerixafor+G-CSF-mobilized cells achieving superior short-term engraftment rates, with similar numbers of CD34+ cells transplanted. Overall, Plerixafor+G-CSF not only allows high CD34+ cell yields but also provides increased ß-globin expression/VCN and enhanced early human chimerism under nonmyeloablative conditions, thus representing an optimal graft for thalassemia gene therapy.

Bone marrow as an alternative site for islet transplantation.

The liver is the current site for pancreatic islet transplantation, but has many drawbacks due to immunologic and nonimmunologic factors. We asked whether pancreatic islets could be engrafted in the bone marrow (BM), an easily accessible and widely distributed transplant site that may lack the limitations seen in the liver. Syngeneic islets engrafted efficiently in the BM of C57BL/6 mice rendered diabetic by streptozocin treatment. For more than 1 year after transplantation, these animals showed parameters of glucose metabolism that were similar to those of nondiabetic mice. Islets in BM had a higher probability to reach euglycemia than islets in liver (2.4-fold increase, P = .02), showed a compact morphology with a conserved ratio between alpha and beta cells, and affected bone structure only very marginally. Islets in BM did not compromise hematopoietic activity, even when it was strongly induced in response to a BM aplasia-inducing infection with lymphocytic choriomeningitis virus. In conclusion, BM is an attractive and safe alternative site for pancreatic islet transplantation. The results of our study open a research line with potentially significant clinical impact, not only for the treatment of diabetes, but also for other diseases amenable to treatment with cellular transplantation.