Safe and Efficient Expansion of Human HSC with Sendai Virus Vector Expressing HoxB4 in Fetal Sheep.

Homeobox B4 (HoxB4) has been shown to be a potent stem cell self-renewal gene, especially in hematopoietic stem cells (HSC). Accumulating evidence from murine studies indicates that the overexpression of HoxB4 enhances in vivo and ex vivo expansion of HSC. Although no leukemia has been observed after transplantation of HoxB4-transduced cells in murine models, the study using large animals such as dogs and non-human primates with retroviral vectors expressing HoxB4 showed the frequent development of leukemia. Regarding retroviral vectors expressing HoxB4, there is another concern, that is, insertional leukemogenesis, which has been elucidated in the hematopoietic stem cell gene therapy for X-SCID. To avoid the insertional mutagenesis, other vectors may be considered, including Epstein-Barr nuclear antigen (EBNA)-1 based episomal vectors or the transposon; however, problems are left, i.e. low transduction efficiency with EBNA vectors and unclear safety with transposon vectors. To avoid both the persistent HoxB4 expression and insertional mutagenesis leading to leukemogenesis, we have developed a new type of Sendai virus (SeV) vector; it lacks the polymerase gene, namely P-defective SeV (SeV/δP) vector. SeV is an enveloped virus with a non-segmented, negative-stranded RNA genome. SeV-based vectors are non-integrating, cytoplasmic vectors. They replicate exclusively in the cytoplasm of transduced cells, and do not go through a DNA phase; therefore, there is no concern about the unwanted integration of foreign sequences into chromosomal DNA of the host. We have previously shown that the transduction efficiency of human CD34⁺ cells with the SeV vector was very high; around 70% (100 multiplicity of infections). On the other hand, SeV/δP vectors are incapable of self-replication, thus enabling transient gene expression without spoiling their ability to efficiently transduce CD34⁺ cells. Here, using the SeV/δP vector expressing HoxB4 (SeV/δP/HoxB4 vector), we examined the effectiveness and safety of human HSC expansion after in utero transplantation to fetal sheep.

After enrichment of CD34⁺ cells from cryopreserved human umbilical cord blood, these cells were repeatedly exposed to SeV/δP/HoxB4 vector every 24 h for 4 days. The transduced cells (3.2–11.7 × 10⁵) were transplanted into the abdominal cavity of fetal sheep at 45–50 gestational days (full term, 147 days) that have premature immune system (HoxB4 group, n = 4; control group, n = 4). The engraftment of hematopoietic cells derived from human HSC in the lambs after birth was quantitatively evaluated by colony PCR of the bone marrow. The development of leukemia was assessed by regular sampling of peripheral blood and bone marrow.

The human–sheep chimeric ratio in the bone marrow of HoxB4 group was calculated 4.8-times higher than that of control group after birth, as assessed by colony PCR. The SeV genome was no longer detectable in the bone marrow and peripheral blood of lambs as assessed by RNA-PCR, confirming the SeV vectors were cleared. No leukemia developed in any of the sheep in either group at present (at 12 months after transplantation).

The SeV/δP vector would be suitable for transient expression of HoxB4 in human CD34⁺ cells, enabling 4.8-times expansion of human HSC as assessed by their repopulating ability in sheep. The expansion of human HSC with the SeV vector was comparable to that with HoxB4-retroviral vectors. In addition, the SeV/δP vector is free of concern about transgene-related and insertional leukemogenesis and should be safer than retroviral vectors.

No relevant conflicts of interest to declare.