Development of high-throughput nanoparticle screening technologies to facilitate the delivery of genetic therapies to non-liver cell types

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Sago, Cory D.
Dahlman, James E.
Santangelo, Philip J.
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Genetic drugs (such as siRNAs, mRNAs, and CRISPR/Cas9) have the potential to be curative therapies for countless diseases. However, gene therapies will only work if the genetic drug is delivered to the diseased cell type. One promising delivery method is through the use of Lipid Nanoparticles (LNPs), due to their ease of manufacture and favorable toxicity profiles. Yet, LNPs have only seen clinical success as delivery methods to liver cells. In order to treat genetic diseases outside the liver, we hypothesized that a novel methodology for LNP discovery must be applied. Utilizing DNA barcodes to allow for the testing of >100 LNPs simultaneously, we determined that the correlation between LNP biodistribution in vitro and in vivo was negligible. Motivated by these results, we developed three novel technologies to increase the efficiency of LNP development for delivery of genetic therapies to non-liver cell types. The first technology (QUANT), allows the determination of absolute biodistribution of >150 LNPs simultaneously, we applied this to reveal the genetic mechanism by which liver endothelial and Kupffer cells uptake a majority of an injected LNP dose. The second and third technologies (FIND and siDown, respectively) enable the measurement of the cytosolic delivery of a mRNA or siRNA payload by >150 LNPs simultaneously. Using FIND, we developed a LNP that specifically delivers to splenic endothelial cells, enabling CRISPR-Cas9 mediated gene editing. Using siDown, we evolved a LNP with tropism to bone marrow endothelial cells. Lastly, we developed a novel oligonucleotide-based anti-CRISPR capable of enhancing the non-liver specificity of LNP-mediated CRISPR-Cas9 editing in wildtype animals. Cumulatively, we hope that this body of work helps LNPs deliver on their promise of enabling gene therapies to extra-hepatic tissues.
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