Multi-Tissue, Long-Term Engraftment and Expansion of BCR-ABL-transduced Human Lin^-CD34⁺ Cord Blood Cells in a Large Animal Model.

Abstract 3265

Poster Board III-1

In utero transplantation of large animals (sheep and goats) with human hematopoietic cells has proven valuable in distinguishing subsets of human cells with short- and long-term repopulating activity. Transplantation of secondary and tertiary fetal sheep with cells regenerated in primary sheep has also demonstrated the ability of human hematopoietic stem cells to retain and execute their self-renewal potential in a xenogeneic setting. We now describe the novel extension of this approach to the generation of a BCR-ABL gene transfer-based in vivo model of human myeloproliferative disease. Lin^-CD34⁺ human cord blood (CB) cells were transduced with BCR-ABL retrovirus (MSCV-BCR-ABL-IRES-GFP) and the cells were then injected IP into pre-immune fetal goats at 45–55 days of gestation. This induced a high frequency of abortion among the injected fetuses: 79% (n=22) when >5×10⁴BCR-ABL- transduced CB cells were injected as compared to 64% (n=9) when control (MIG)-transduced cells were injected. Six goats transplanted with 2×10⁴ BCR-ABL-transduced cells were born alive and 3 weeks after birth, 3 of these were sacrificed so that their tissues could be analyzed. Interestingly, in the goats injected with BCR-ABL-transduced human cells, only GFP⁺(BCR-ABL⁺) human cells were detected and these were found in multiple hematopoietic and non-hematopoietic tissues, including peripheral blood, bone marrow (BM), liver, kidney, lung, heart, and skeletal and smooth muscle (1–49%) by fluorescence microscopy and confocal laser scanning microscopy, FISH and FACS. Immunohistochemical analysis allowed positive cells to also be detected in frozen sections of liver tissue. Continued follow-up of the other recipients of transduced cells showed that the 3 injected with BCR-ABL-transduced cells all developed features of early chronic phase human chronic myeloid leukemia (CML), as evidenced by a 3- to 5-fold elevation in their WBC count (up to 2.5×10¹⁰/L as compared to 5–8×10⁹/L in the recipients of MIG-transduced cells, P<0.01). Changes in the WBC count were seen as early as 3 months after birth and up to 2.5 years post-transplant and were accompanied in all 3 of these goats by the presence of GFP⁺ cells, including human CD34⁺ stem/progenitor cells, in the blood and BM. Quantitative real-time PCR analysis of genomic DNA from multiple tissues demonstrated up to 8×10⁴ copies of transgene per microgram of DNA. Microarray transcript profiling of blood and liver from BCR-ABL chimeric goats confirmed expression of many human genes, including 321 that were detected at >2.5-fold higher levels in the BCR-ABL chimeric goats as compared to both control chimeric goats and normal human CB. These over-expressed genes are from several functional categories, including tyrosine kinases and other proteins with phosphorylation activities, cell cycle control, cell adhesion, homing and differentiation, transcription, nucleotide binding and ion transport. Several were confirmed by quantitative RT-PCR. These results demonstrate long-term engraftment, but slow expansion of primitive human hematopoietic cells transduced with BCR-ABL fusion gene and transplanted in utero in a large animal model. This novel xenotransplant goat model should be useful for characterizing the early (pre-symptomatic) phase of human CML and for assessing new therapies with potential long-term benefits.