Robust Immune Reconstitution in Fanconi Anemia Mice Post in Utero Hematopoietic Stem Cell Transplantation

Background: Fanconi anemia (FA) is the most common inherited DNA-repair disorder and leads to bone marrow failure, immune defects and systemic malignancies in almost all patients. In utero hematopoietic stem cell transplantation (IUHSCT) presents a promising prenatal treatment for FA that avoids the toxicities associated with current postnatal therapies by leveraging fetal immune tolerance and the competitive advantage of normal HSCs over failing FA HSCs. While our prior work has demonstrated multi-lineage donor engraftment post-IUHSCT, detailed characterization of immune reconstitution has been limited. Assessing the postnatal immune repertoire (IR) as well as macrophage composition is crucial for evaluating IUHSCT safety and efficacy in ensuring a robust and functional immune recovery.

Methods: IUHSCT was performed in FA mice (Fancd2^-/- or Fanca^-/-, CD45.2) by breeding parental FA carriers and at E13.5-14.5 all fetuses received 1x10⁶ CD117⁺ enriched HSCs from bone marrow of wildtype mice (WT, CD45.1) via intrahepatic injection. At 36-weeks post-IUHSCT peripheral blood was obtained and high-throughput sequencing of the CDR3 in both the T-cell receptor (TCR) and B-cell receptor (BCR) was performed in collaboration with iRepertoire, Inc. The repertoires of FA and WT IUHSCT treated mice were compared to untreated age and sex-matched FA and WT mice (n=4 total). The sequencing and subsequent analysis utilized the iRWeb data analysis suite. To assess the diversity and clonality of T- and B-cell populations, Diversity50 (D50) scores were calculated within the TCR and BCR IR across mice providing a quantitative measure where scores closer to 50 indicate higher diversity. Donor tissue-resident macrophages were also assessed at 48-weeks post IUHSCT in skin, liver, spleen, lung, and brain by analysis of %45.1 (n=3 FA mice).

Results: IUHSCT resulted in only 1-3% donor HSC chimerism in WT and FA carrier littermates, with 18-87% donor HSC chimerism in FA mice at 48-weeks. Notable differences in IR diversity were observed between groups. Strikingly, in the non-transplanted FA setting, lower TCR diversity with D50 scores for TCRα/β/γ/δ of 20.5/36.2/41.7/13.3, respectively, and BCR heavy, kappa, and light of 2.5/1/0.9, respectively, was observed. In the FA post-IUHSCT setting, D50 scores for TCRα/β/γ/δ of 26.9/42.3/37.4/17.9, and for BCR heavy, kappa, and light chains of 2.8/1.1/0.7, was observed underscoring the selective impact on T-cell diversity enhancement. Importantly this was similar to the WT setting post IUHSCT that demonstrated D50 scores for TCRα/β/γ/δ of 26.3/42/42.9/23.8, indicating enhanced diversity compared to the non-transplanted WT setting of 24.9/40.1/40.4/12.8. For BCR heavy, kappa, and light chains, the D50 scores were 2.1/1.3/1.3 compared to 6.3/1.5/1 reflecting lower diversity in BCRs compared to WT not receiving IUHSCT. These results underscore the differential impact of IUHSCT on T-cell and B-cell repertoire diversity, with a more pronounced positive effect on T-cell diversity across both FA and WT groups. Further, donor tissue-resident macrophages were also observed in all tissues assessed with a mean (SD) 46.6% (14.1) donor chimerism (range: 15.6-84.7%) in FA mice post IUHSCT.

Conclusions: This study confirms IUHSCT as a potential transformative prenatal treatment for FA effectively addressing both hematologic and immunologic deficits. Importantly, high levels of multi-lineage donor chimerism were obtained without the need for toxic postnatal conditioning. The observed normalization of IR diversity in the FA setting post-IUHSCT indicates successful correction of hematopoietic abnormalities and robust immune reconstitution, comparable to the untreated WT control setting. The polyclonal nature of the T-cell and B-cell repertoires post-transplant and the donor macrophage engraftment illustrate the broad therapeutic impact of IUHSCT which may also aid in reducing systemic malignancies. However, the decrease in certain BCR diversities suggests that additional factors or conditions may influence the reconstitution of B-cell populations post-transplantation. Further analysis are needed to understand these dynamics fully. This research not only supports the further development of fetal therapies but also underscores their capacity to profoundly modify the progression of genetic disorders before birth.

Pan:iRepertoire Inc: Current Employment, Current equity holder in private company. Steele:iRepertoire Inc.: Current Employment, Current holder of stock options in a privately-held company. Han:iRepertoire Inc.: Current Employment, Current equity holder in private company. Czechowicz:Beam Therapeutics: Current equity holder in publicly-traded company; Global Blood Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months; Jasper Therapeutics: Patents & Royalties, Research Funding; Editas Medicine: Current equity holder in publicly-traded company, Patents & Royalties: Patent, no royalties; Prime Medicine: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Rocket Pharma: Research Funding; Spotlight Therapeutics: Consultancy, Current holder of stock options in a privately-held company; Magenta Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months, Patents & Royalties; Teiko Bio: Current holder of stock options in a privately-held company; Decibel Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months, Patents & Royalties: Patent, no royalties; Inograft Therapeutics: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; STRM.Bio: Research Funding.