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Stokes, A. L.

Publications and source records attributed to Stokes, A. L..

2 recordsLinked to original sources
bioRxiv2025

Standardizing a Protocol for Streamlined Synthesis and Characterization of Lipid Nanoparticles to Enable Preclinical Research and Education

Lipid nanoparticles (LNPs) have revolutionized nucleic acid delivery, enabled the first FDA-approved RNAi therapy (Onpattro), and accelerated the development of mRNA vaccines during the COVID-19 pandemic. The success of LNP-based vaccines demonstrated the potential of these nanoparticles for broader therapeutic applications. As interest in LNP-based therapies expands, there is an urgent need for low-cost, reproducible synthesis protocols that can be readily implemented across a wide range of research settings. Traditional methods for LNP synthesis, such as pipette and vortex mixing, can yield inconsistent results. In contrast, microfluidic mixing offers better control over LNP properties but requires expensive equipment. This financial barrier prevents many laboratories from accessing cutting-edge LNP technology, slowing the pace of preclinical research and limiting the exploration of its full therapeutic potential. To address this, we developed a standardized protocol for microfluidic mixing using a syringe pump and a commercially available microfluidic chip. This approach offers a cost-effective and reproducible method for LNP synthesis. The protocol details the synthesis of LNPs, physical characterization via dynamic light scattering, encapsulation efficiency using the RiboGreen assay, and evaluation of in vitro transfection efficiency using the OneGlo assay, confocal microscopy, and a flow cytometer. We explored consistency through various experimental parameters, aiming to optimize the protocol and, importantly, tested user-to-user reproducibility with undergraduates without LNP experience with minimal supervision. We found consistency among assembly conditions, including mRNA/lipid concentration, flow rate, dialysis time, ionizable lipid type, and chip reusability. We tested the broader applicability of the method with four ionizable lipids (DLin-MC3-DMA, LP01, C12-200, and SM-102). High encapsulation efficiency (96-100%) was maintained across tested concentrations, with slightly larger particle sizes at lower doses. Based on the flow cytometry analysis, a 7.5 {micro}g dose was chosen for future use to conserve mRNA without sacrificing efficacy. Dialysis duration had minimal impact on encapsulation, but longer times increased particle size and reduced luminescence. The protocol consistently produced narrowly dispersed particles (PDI < 0.2), and equipment was reusable for up to six runs, potentially reducing the per-run cost. Even novice users achieved reproducible results, highlighting the protocols accessibility and potential to expand LNP research and applications.

bioengineering↗
bioRxiv2025

Beyond the Cut: Long-read sequencing reveals complex genomic and transcriptomic changes in AAV-CRISPR therapy for Duchenne Muscular Dystrophy

Adeno associated virus (AAV)-mediated delivery of CRISPR associated nucleases (AAV-CRISPR) is a promising solution to treat genetic diseases such as Duchenne Muscular Dystrophy (DMD) and is now in early clinical trials. However, genotoxicity and immunogenicity concerns have hindered clinical translation. Due to the complex etiology associated with DMD, the post-transduction consequences of double-stranded breaks induced by AAV-CRISPR in disease models are unclear. This barrier is partially conferred by conventional sequencing methods where common outcomes of AAV-CRISPR editing often escape detection. However, recent reports of novel long-read sequencing approaches permit comprehensive variant detection using a broader sequence context. Here, we comprehensively investigated genomic and transcriptomic post-AAV-CRISPR transduction consequences in myoblast cells and a DMD mouse model following intramuscular and intravenous AAV-CRISPR therapy using both long- and short-read sequencing techniques. Structural variant characterization indicates that unintended on-target large insertions and inversions are common editing outcomes. We demonstrate that combining adaptive sampling with nanopore Cas9-targeted sequencing (AS-nCATS) for long-read quantification of AAV integration is synergistic for detecting difficult-to-amplify editing events. This unbiased data suggests that full-length AAV integration is equally as probable as the on-target deletion. Further, we develop a Nanopore Rapid Amplification of cDNA Ends (nRACE-seq) pipeline for long-read detection of unknown 5 or 3 ends of edited transcripts. The nRACE-seq approach effectively detects the presence of AAV-Dmd chimeric transcripts, erroneous splicing events, and off-target AAV integration sites. In summary, our findings offer insights into the adaptation of AAV-CRISPR DSB-mediated therapeutics for monogenic diseases and promote the standardization of CRISPR evaluation. We highlight the importance of coupling polymerase-based and polymerase-free methods in long-read sequencing to assess editing outcomes as the field progresses toward clinical applications.

bioengineering↗