E2 - Other Nonviral Delivery (including lipid nanoparticles and exosomes)
1753: In Utero Systemic LNP mRNA Delivery Targets the Fetal Brain and Permits Diffuse Gene Editing
Type: Poster Session
Poster Board Number: 1753
Presentation Details
Session Title: Friday Posters: Other Nonviral Delivery
No curative therapies exist for the 1% of children affected by genetic diseases of the central nervous system (CNS). Often the pathogenesis of these diseases begins before birth and is irreversible. CRISPR-Cas9 gene editing provides a unique opportunity for a single dose cure if the target cell type and organ are reached. Unfortunately, therapeutic CNS gene editing is limited by the diversity of cell types in the brain, the limited regenerative capacity of neurons and, the impenetrable blood-brain barrier (BBB) which prevents access of systemically delivered gene editing therapies. Non-viral ionizable lipid nanoparticles (LNPs) have emerged as a safe and effective means of mRNA for the correction of genetic liver diseases but they have not been shown to deliver mRNA to the brain following intravenous delivery. In addition to mitigating disease pathology before its onset, in utero gene editing via LNP delivery may take advantage of fetal developmental properties to facilitate delivery of therapeutic editing technology to the brain following systemic delivery. To address this hypothesis, we screened three LNP formulations loaded with CRE recombinase mRNA in a GFP reporter mouse, via intravenous delivery at three developmental stages: fetal, neonatal, and adult. All mice were harvested 4-6 weeks following injection and GFP expression in the brain was quantified using flow cytometry and immunohistochemistry using markers for specific cell types (neurons, endothelial cells, microglia, astrocytes, and oligodendrocytes) and other organs. We identified one LNP (LNP1) with efficient mRNA delivery to endothelial cells (~50%), macrophages (75%) and neurons (5%) in mice who were injected in utero when compared to mice injected as neonates or adults which demonstrated no editing in endothelial cells or neurons. These data suggested the fetal environment has distinct biological features that enhance mRNA delivery via LNPs to the brain. We then applied these findings and used LNP1 to assess CNS gene editing in a mouse model of Friedreich Ataxia (FRDA). FRDA is a neurodegenerative disease in which propagation of GAA repeats in the frataxin gene results in decreased frataxin protein expression. Gene editing to excise the triplet repeat is one potential therapeutic approach that has been suggested. LNP mediated systemic delivery of SpCas9 mRNA and gRNA targeting the triplet repeat to gestational day 14 FRDA fetal mice result in low level editing in the brain. These initial studies provide a foundation for an in-utero gene therapy approach for FRDA. In conclusion, we leverage our discovery related to basic fetal biology and its influences on LNP-mRNA delivery to develop clinically relevant CNS therapies.
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Plain Language Summary
Genetic disease of the central nervous system (CNS) affects nearly 100 million Americans, and no curative therapies exist. Gene editing technologies, including CRISPR-Cas9, provide the unique opportunity for a “one-and-done” cure for many of these diseases. Unfortunately, therapeutic CNS gene editing is limited by the impenetrable blood-brain barrier (BBB) which prevents access of systemically (intravascularly) delivered gene editing therapies to the target cells in the brain. The developing fetus has many properties that enhance
in vivo delivery of therapeutic gene editing tools using both viral and non-viral vehicles. In this context, mouse studies that we have performed in our lab have identified a drug delivery vehicle that targets the fetal brain, including target cell types, after systemic delivery. These studies are foundational for
our long-term goal to develop novel approaches to enhance delivery of mRNA therapeutics to both the fetal and postnatal brain.
Marco D. Carpenter, Rosa Choi
, Nicole Kus
, Andrew Mudreac
, Alanis Perez
, Hooda Said
, Mohamad Gabriel Alameh
, William Peranteau
Children's Hospital of Philadelphia, Philadelphia, PA"