Ates, I. | bioRxiv Science

bioRxiv2026

Exploring lipid nanoparticle design spaces using self-regulating microfluidic machines and multiplexed in vivo biodistribution

Delivering therapeutic mRNA relies on lipid nanoparticles (LNPs). Finding optimal process parameters for new lipid combinations in LNP formulations remains a challenge. In our work, we used an automated, self-regulating microfluidic platform that actively changes process parameters to tune LNP formulations for preset, desired quality standards. We tested new LNPs by swapping poly(ethylene glycol) (PEG) lipopolymers for alternatives based on poly(2-methyl-2-oxazoline) (PMeOx) and poly(2-ethyl-2-oxazoline) (PEtOx). For each lipopolymer variant, the platform independently identified optimal production conditions in four or fewer iterative cycles, yielding particles of the preset size and high mRNA encapsulation. Small-angle X-ray scattering revealed that smaller LNPs modified with PEtOx had more structural surface variety and higher mRNA loading efficiency. When multiplexing these formulations in mice, the PEtOx-containing LNPs accumulated more in bone marrow compared to those with PEG, indicating trends that the chemistry of the lipopolymer affects the biodistribution of the resulting LNPs. By combining automated formulation and in vivo multiplexed testing, our approach provides a practical way to rapidly plan, formulate, and evaluate large pharmaceutical design spaces, to select excipients and process parameters yielding optimal biological performance of LNPs. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/729611v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1a08b68org.highwire.dtl.DTLVardef@120724corg.highwire.dtl.DTLVardef@12fed98org.highwire.dtl.DTLVardef@197aa41_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

EX VIVO GENE EDITING AND CELL THERAPY FOR HEREDITARY TYROSINEMIA TYPE 1

Background & AimsWe previously demonstrated the successful use of in vivo CRISPR gene editing to delete 4-hydroxyphenylpyruvate dioxygenase (HPD) to rescue mice deficient in fumarylacetoacetate hydrolase (FAH), a disorder known as hereditary tyrosinemia type 1 (HT1). The goal of this study was to develop an ex vivo gene editing protocol and apply it as a cell therapy for HT1. MethodsWe isolated hepatocytes from wild-type (C57BL/6) and Fah-/- mice and then used an optimized electroporation protocol to deliver Hpd-targeting CRISPR-Cas9 ribonucleoproteins (RNP) into hepatocytes. Next, hepatocytes were transiently incubated in cytokine recovery media that we formulated to block apoptosis, followed by splenic injection into recipient Fah-/- mice. ResultsWe observed robust engraftment and expansion of transplanted gene-edited hepatocytes from wild-type donors in the liver of recipient mice when transient incubation with our cytokine recovery media was used after electroporation and negligible engraftment without the media (mean 46.8% and 0.83%, respectively, p = 0.0025). Thus, the cytokine recovery media was a critical component of our electroporation protocol. When hepatocytes from Fah-/- mice were used as donors for transplantation, we observed 35% and 28% engraftment for Hpd-Cas9 RNPs and Cas9 mRNA, respectively. Tyrosine, phenylalanine, and biochemical markers of liver injury normalized in both Hpd-targeting Cas9 RNP and mRNA groups independent of drug induced-inhibition of Hpd through nitisinone, indicating correction of disease indicators in Fah-/- mice. ConclusionsThe successful liver cell therapy for HT1 validates our protocol and, despite the known growth advantage of HT1, showcase ex vivo gene editing using electroporation in combination with liver cell therapy to cure a disease model. These advancements showcase the impacts of electroporation combined with transplantation as a cell therapy.

bioengineering↗