Highlight: In Utero Nanoparticle Delivery Enables Brain Gene Editing

Delivering mRNA via acid-degradable nanoparticles offers a new frontier in prenatal gene editing, setting the stage for treatments targeting the root causes of neurodevelopmental disorders. A new method uses densely PEGylated lipid nanoparticles to deliver CRISPR-based mRNA editing tools into embryonic brain cells, achieving substantial genetic modifications across the brain with minimal toxicity and long-lasting results.

By: Gorm Palmgren - Oct. 28, 2024

Researchers from the University of California, Davis and the University of California, Berkeley have used a mouse model to demonstrate a high-efficiency approach to in utero gene editing. The method overcomes traditional barriers by safely transfecting neural stem cells with CRISPR-associated mRNA that can reach large swathes of the brain and introduce precise genetic edits with significant developmental benefits.

The researchers demonstrated that intracerebroventricular (ICV) injection of acid-degradable PEGylated LNPs (ADP-LNPs) leads to high transfection rates and a safety profile compatible with prenatal intervention that might be promising for tackling disorders like Angelman syndrome and Rett syndrome before they manifest (see Figure 1).

Figure 1. Graphical abstract of the new method for in utero delivery of densely PEGylated LNPs that... — Figure 1. Graphical abstract of the new method for in utero delivery of densely PEGylated LNPs that globally transfects the brain in utero with mRNA for gene editing enzymes. Reproduced under the Creative Commons license from Figure 1 in Gao et al. (2024) ACS Nano, https://doi.org/10.1021/acsnano.4c05169

While standard LNPs are effective in adult applications, they have shown limited transfection efficacy and significant toxicity in developing tissues. ADP-LNPs address these limitations with dense PEGylation, which improves cellular uptake while reducing inflammatory responses and acid degradability, which allows these particles to break down within cells after delivering their genetic payload.

In this study, ADP-LNPs containing either Cre mRNA (for the Ai9 genetic reporter system) or Cas9 mRNA paired with guide RNAs (gRNAs) targeting specific genes were injected into the brains of embryonic mice on day E15.5, a period when neural progenitor cells are actively proliferating.

The results were impressive: the ADP-LNPs achieved widespread gene editing across the brain, with approximately 30% of fetal brain cells transfected. Edited neural progenitor cells continued to proliferate throughout development, ultimately modifying over 40% of neurons in the cortex and more than 60% of neurons in the hippocampus by 10 weeks after birth (see Figure 2).

Figure 2. D) Brain section images display the distribution of td-Tomato expression, particularly... — Figure 2. D) Brain section images display the distribution of td-Tomato expression, particularly within the SVZ (b) and GE (c). (E) Detailed immunohistochemistry in the SVZ and GE regions reveals that td-Tomato expression co-localizes with the NSPC markers, suggesting ADP-LNPs were able to edit NSPCs with Cas9 mRNA and gRNA. Reproduced under the Creative Commons license from Figure 5 in Gao et al. (2024) ACS Nano, https://doi.org/10.1021/acsnano.4c05169

To potentially treat Angelman syndrome, the researchers constructed ADP-LNPs carrying Cas9 mRNA and a gRNA specific for knocking out the disease-causing Ube3a-ats locus. This locus produces an antisense transcript that silences the expression of Ube3a, a ubiquitin ligase essential for brain development (see Figure 3).

Injected into fetal mice, the ADP-LNPs were able to achieve 21% gene editing in targeted brain cells within one week postpartum and a significant reduction in Ube3a-ats expression. This is a much higher level of precision and efficiency than previous delivery systems have been able to achieve and a significant improvement in both the scope of tissue transfection and the maintenance of cell viability and function.

Figure 3. A) Schematic diagram describing the editing strategy used to upregulate the expression of... — Figure 3. A) Schematic diagram describing the editing strategy used to upregulate the expression of Ube3a by knocking out Ube3a-ats. B) The bar graph depicts the indel frequencies observed at 10 days postinjection of ADP-LNPs, demonstrating efficient editing. C) Relative expression levels of Ube3a-ats, showing a significant reduction in the treated groups. Reproduced under the Creative Commons license from Figure 6 in Gao et al. (2024) ACS Nano, https://doi.org/10.1021/acsnano.4c05169

The study’s rigorous approach included multiple assays to confirm safety and specificity. Unlike standard LNPs, which prompted high inflammation and lowered survival rates, ADP-LNPs kept cytokine levels nearly at baseline in the brain and liver. Mice treated with ADP-LNPs also showed normal growth patterns and healthy weights by 10 weeks of age.

The findings suggest that ADP-LNPs could potentially enable in utero preventive therapies that might revolutionize treatment options for many currently untreatable neurodevelopmental disorders.

Link to the original article in ACS Nano :

Gao et al. (2024) Widespread Gene Editing in the Brain via In Utero Delivery of mRNA Using Acid-Degradable Lipid Nanoparticles. ACS Nano, 24 October 2024, doi.org/10.1021/acsnano.4c05169

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ArticleCMN HighlightsNewsDeliveryDelivery - Brainin vivoLipid-based nanoparticleAdeno-associated virus (AAV)Angelman SyndromeCas9