ARENEX aims to develop a scalable technology for producing engineered exosomes to deliver CRISPR components for personalized, therapeutic in vivo gene editing. CRISPR technology holds immense promise for addressing genetic mutations underlying various diseases. Several CRISPR-based therapies have recently gained FDA approval for treating β-hemoglobinopathies, and ongoing research explores its potential across a wide range of conditions. CRISPR's use of Cas nucleases and guide RNAs (gRNAs) allows precise genomic modifications, including gene deletion, insertion, regulation, or correction. Compared to small-molecule drugs, CRISPR offers advantages such as rational design, rapid development, and the ability to edit multiple genes simultaneously. However, in vivo therapeutic applications face critical challenges, including limited access to target cells and off-target effects that can cause tumorigenic mutations and other adverse outcomes. Consequently, clinical trials for in vivo CRISPR therapies are largely restricted to specific anatomical sites, such as the subretinal space, significantly narrowing its therapeutic scope.
Exosomes, natural nanovesicles released by cells, offer unique advantages for therapeutic delivery due to their ability to cross biological barriers, high biocompatibility, and low immunogenicity. While exosomes have shown promise for delivering drugs, siRNA, and cytokines, their use in gene editing is hindered by low production yields, inefficient cargo loading, and batch variability. This project proposes a novel manufacturing method for large-scale production of artificially engineered exosomes (AREs) that encapsulate Cas nuclease and gRNA ribonucleoprotein (RNP) complexes for in vivo gene editing. Conventional exosome production relies on labor-intensive steps like ultracentrifugation and membrane permeabilization, which are inefficient and costly for therapeutic applications, particularly for CRISPR RNP delivery. To address these limitations, the proposed approach leverages multifunctional magnetic nanoparticles (MNPs) to re-engineer the cellular exosome formation and release processes. MNPs facilitate the cellular uptake of CRISPR RNPs and drive efficient exosome production through endosome extraction mechanisms, enabling large-scale, clinical-grade ARE production. This method supports the use of patient-derived cells, reducing adverse effects associated with exogenous exosomes. Additionally, MNP-encapsulated AREs allow for image-guided delivery, enabling real-time monitoring of delivery and gene-editing efficiency. Preliminary studies demonstrate a 40-fold increase in ARE production rate and significantly enhanced cargo loading compared to existing methods. Initial therapeutic focus areas include neurological diseases and in vivo gene correction for sickle cell disease.
In vivo therapeutic gene editing holds transformative potential for numerous conditions, including substance use disorders, Alzheimer’s disease, β-hemoglobinopathies, Duchenne muscular dystrophy, and cystic fibrosis. The genome editing market, valued at $8.45 billion in 2023, is projected to grow to $40.48 billion by 2033, with the in vivo segment expected to expand at a CAGR of 19.94% from 2024 to 2030 (Grand View Research). Innovations in delivery methods, such as this ARE technology, could further accelerate market growth. This platform provides a universal delivery method for Cas nucleases and gRNAs, customizable for various in vivo gene-editing therapies. By addressing key bottlenecks in scalability and efficiency, this technology has the potential to revolutionize CRISPR-based therapeutic applications and unlock new possibilities in precision medicine.
Leader Sheng Tong: sheng.tong@uky.edu
Member Sujia Zhou: sjzhou1980@gmail.com
Project Owner
Sheng Tong