Consistent Achievement of Improved Levels of Hematopoietic Chimerism in the Canine Model Following Optimization of Haploidentical In Utero Transplantation

Abstract 2962

In utero hematopoietic cell transplantation (IUHCT) offers the potential to achieve allo-engraftment and associated donor-specific tolerance (DST) without the need for toxic conditioning regimens. In the murine model, IUHCT has been shown to uniformly achieve levels of allo-engraftment that induce DST. Levels of chimerism in these tolerant animals can subsequently be enhanced to nearly complete donor chimerism by postnatal minimal conditioning, same donor BMT. While this strategy would be clinically applicable to many congenital hematopoietic disorders, clinical attempts at IUHCT have, for the most part, failed to achieve engraftment or associated DST. Similarly, large animal models, with the exception of the sheep, have proven challenging to engraft. Over the past several years our laboratory has focused on development of the canine model as a rational preclinical, large animal model for IUHCT. Our initial efforts at canine IUHCT using intraperitoneal (IP) injection resulted in DST in approximately 33% of animals undergoing haploidentical IUHCT, as proven by the ability to enhance engraftment after birth using a minimal conditioning regimen of a single dose of busulfan for same donor BMT. However, levels of donor cell chimerism were uniformly below the limit of detection with our VNTR methodology (< 2%) and were ≤ 1.7% by FACS or PCR detection techniques when applicable. In an effort to improve upon the level of engraftment and consistency of DST in the canine model, we embarked upon a series of studies: 1) to assess the timing of critical developmental events relevant to IUHCT during hematopoietic and immune ontogeny in the dog; 2) to track the efficiency of homing and engraftment of labeled cells using different routes of IUHCT, and 3) to assess the frequency and level of engraftment in the canine model after optimization of timing and route of cell delivery.

Hematopoietic and Immune Ontogeny - The thymic expansion of double positive (CD4⁺CD8⁺) lymphocytes occurred between 39 and 42 days gestation (term=63 days). Fetal liver hematopoiesis as measured by expansion of CD45⁺ cells occurred between 37 and 46 days gestation. Population of the BM by CD45⁺ cells was first observed at around 38 days with marked expansion of BM hematopoiesis by 46 days.

Cell Tracking - For tracking studies, cells were labeled with PKH-67, allowing accurate assessment of homing and engraftment during prenatal harvests at 4, 24 and 48 hours post-injection. We performed ultrasound guided trans-uterine IP, or intracardiac (IC) injections of labeled BM cells at gestational ages (GA) between 33 and 46 days. Uptake of cells from the peritoneum proved inefficient with minimal fetal liver homing or engraftment. However, IC (intravascular) injection resulted in efficient homing and high levels of fetal liver engraftment up to the 48 hour time point.

Engraftment with optimized IUHCT - Subsequently, 60 fetuses of 14 pregnant bitches underwent ultrasound-guided IC injection of CD34 enriched or CD3 depleted maternal BM at GA 32–42 days. Post-injection viability was 85%, while survival to birth was 61%. Of 37 surviving pups, 20 demonstrated VNTR detectable chimerism after IUHCT. However, IC injection proved technically challenging prior to 40 days GA with inconsistent cell delivery. Among fetuses transplanted between 40 and 42 days GA, 14/15 (93%) were chimeric, with an average level of chimerism of 9.1% two months after birth (range 2.8%-23.6%, Figure 1). Chimerism has remained stable on long term analysis. The ability to enhance engraftment postnatally by minimal conditioning transplants (DST) in selected animals strongly correlates with VNTR measurable chimerism. No evidence of GVHD has been observed in any of the chimeric animals.

Figure 1.

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Figure 1.

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These results demonstrate that engraftment and DST can be consistently achieved with optimization of IUHCT in the canine model using a clinically applicable protocol. The critical determinant of engraftment is the intravascular delivery of a large dose of cells during a receptive stage of immune and hematopoietic ontogeny. This study supports the potential of the canine model as a pre-clinical model for IUHCT.