Development of Third-generation Cocal Envelope Producer Cell Lines for Robust Lentiviral Gene Transfer into Hematopoietic Stem Cells and T-cells.

Lentiviral vectors (LVs) pseudotyped with vesicular stomatitis virus envelope glycoprotein (VSV-G) have demonstrated great promise in gene therapy trials employing hematopoietic stem cell and T-cells. The VSV-G envelope confers broad tropism and stability to the vector but is toxic when constitutively expressed, which has impeded efforts to generate stable producer cell lines. We previously showed that cocal pseudotyped LVs offer an excellent alternative to VSV-G vectors because of their broad tropism and resistance to human serum inactivation. In this study, we demonstrate that cocal LVs transduce CD34(+) and CD4(+) T-cells more efficiently than VSV-G LVs and share the same receptor(s) for cell entry. 293T-cells stably expressing the cocal envelope produced significantly higher LV titers than VSV-G expressing cells. We developed cocal pseudotyped, third-generation, self-inactivating LV producer cell lines for a GFP reporter and for a WT1 tumor-specific T-cell receptor, which achieved concentrated titers above 10(8) IU/ml and were successfully adapted for growth in suspension, serum-free culture. The resulting LVs were at least as effective as standard LVs in transducing CD34(+) and CD4(+) T-cells. Our stable cocal LV producer cell lines should facilitate the production of large-scale, high titer clinical grade vectors.

No evidence of clonal dominance after transplant of HOXB4-expanded cord blood cells in a nonhuman primate model.

Umbilical cord blood transplant continues to increase in prevalence as a treatment option for various hematopoietic and immune disorders. Because of the limited number of cells available in a single cord blood unit, investigators have explored methods of increasing cell dose before transplant, including overexpression of the homeobox B4 (HOXB4) transcription factor. We have previously reported the development of leukemia in several nonhuman primate (NHP) subjects transplanted with HOXB4-expanded bone marrow cells at approximately 2 years posttransplant. Here, we provide long-term data for a NHP receiving a HOXB4-expanded cord blood graft. Longitudinal follow-up included gene marking analysis, complete blood counts, morphologic/pathologic assessment, phenotypic analysis of subsets, and retroviral integration site analysis. In each of these independent assays, we saw no indication of clonal dominance, and all signs pointed toward normal, healthy hematopoiesis. Furthermore, in-depth clonal analysis of an animal that developed leukemia after transplantation of HOXB4-modified bone marrow cells showed that dominant clones could be detected as early as 6 months posttransplant using the genomic analysis technique detailed here. Parallel analysis of the cord blood transplant macaque showed no such sites. These findings demonstrate the ability to study the use of gene-modified and expanded cord blood cells in a NHP model.