Poster CC9 - In utero delivery of targeted ionizable lipid nanoparticles facilitates in vivo gene editing of hematopoietic stem cells

Introduction: Monogenic blood diseases are among the most common genetic disorders worldwide. These diseases result in significant pediatric and adult morbidity, and some can result in death prior to birth. Novel ex vivo hematopoietic stem cell (HSC) gene editing therapies hold tremendous promise to alter the therapeutic landscape but are not without potential limitations. In vivo gene editing therapies offer a safer and more accessible treatment for these diseases but are hindered by a lack of delivery vectors targeting HSCs, which reside in the difficult-to-access bone marrow niche. In this study, we propose that this biological barrier can be overcome by taking advantage of HSC residence in the easily accessible liver during fetal development.

Materials and

Methods: Our design approach leverages fetal and developmental biology to overcome conventional biological barriers in mRNA-lipid nanoparticle (LNP) delivery to HSCs. Notably, residence of fetal hematopoiesis – and consequentially fetal HSCs – within the liver during development provides a more accessible compartment for LNP-mediated gene delivery in comparison to the postnatal bone marrow niche (Figure 1A). Using a top-performing LNP from our prior study assessing the feasibility of in utero mRNA-LNP delivery to the fetal liver, we first demonstrated strong LNP-mediated transfection of the fetal liver, as expected given intrinsic LNP hepatotropism, but low-level genome modification of fetal HSCs in vivo in mice. To boost genome modification within HSCs, we conjugated CD45 antibody F(ab’)2 fragments to the surface of these LNPs using thiol-maleimide chemistry (Figure 1B, 1C). We hypothesized that targeted LNPs would engage the CD45R found expressed on the surface of hematopoietic cells residing in the fetal liver and facilitate LNP internalization into these cells. This central hypothesis was tested in vitro in liver cells and hematopoietic lineage cells and in vivo in several mouse models, including a fetal reporter mouse model, a secondary transplant mouse model, and a proof-of-concept gene editing fetal mouse model.

Results, Conclusions, and Discussions: Relative to untargeted LNPs, targeted LNPs enhanced mRNA delivery to hematopoietic lineage cells by 8-fold in vitro and genome modification in fetal HSCs by 7-fold in vivo following in utero IV administration to mid-gestation fetal mice after 60 hours (Figure 1D). Encouragingly, after 4 months, we observed that bone marrow HSCs of mice treated in utero with targeted LNPs demonstrated higher editing than mice treated in utero with untargeted LNPs (Figure 1E), and the relative difference in editing correlated with that seen at a short time point following in utero LNP delivery. To further validate that targeted LNPs durably transfect long-term HSCs, we conducted a secondary transplant study. Lethally-irradiated wild-type mice were rescued by a bone marrow transplant from donor mice treated in utero with targeted LNPs, and transfection of HSCs and hematopoietic lineage cells matched donor whole bone marrow transfection levels. Finally, we optimized our lead LNP platform for delivery of large genome editing cargos in vitro via a high-throughput design-of-experiments approach and demonstrated 3-fold greater gene editing at a proof-of-concept locus in fetal HSCs in vivo (Figure 1F). Taken together, our novel delivery strategy unlocks the possibility to treat monogenic blood diseases in utero prior to the onset of irreversible pathogenesis.

Acknowledgements (Optional):