Biomaterial-Guided CRISPR Delivery for Sustained and Localized Gene Editing

Background

Genetic and epigenetic editing technologies, alongside mRNA delivery, hold transformative potential for regenerative medicine, localized gene therapy, and complex tissue engineering, such as osteochondral and cartilage repair. Steering the behavior and differentiation of therapeutic cells like human mesenchymal stem cells requires precise, localized, and sustained delivery of nucleic acid payloads within three-dimensional microenvironments. Consequently, there is a critical clinical need for advanced biomaterial platforms capable of achieving exact spatiotemporal control over genetic cargo to effectively direct cellular phenotypes and drive functional tissue regeneration.

However, current delivery approaches fail to provide safe, localized, and prolonged gene expression. Traditional viral vectors achieve high transfection efficiency but pose severe clinical safety risks, including immunogenicity and unintended genomic integration. Conversely, conventional non-viral delivery vehicles, such as standard lipid nanoparticles and unmodified cell-penetrating peptides, suffer from rapid burst clearance and non-specific biodistribution when administered in vivo. These free nanoparticles quickly diffuse away from the target site, resulting in short-lived, transient gene expression that typically peaks within three days and decays sharply inside a week. Furthermore, incorporating these vectors into standard biomaterial scaffolds without specific immobilization strategies often leads to passive protein fouling and degraded bioactivity.

Technology

Researchers at Lehigh University have developed an injectable biomaterial platform for localized, spatiotemporal genetic and epigenetic editing. The system condenses anionic nucleic acid cargo—such as mRNA, dCas9-VPR, and guide RNAs—with a blend of native RALA and engineered Snoop-RALA cell-penetrating peptides. Formulated at an optimal 20 mol% Snoop-RALA ratio, these components self-assemble into compact nanoparticles capable of efficient cellular internalization and endosomal escape. These nanoparticles are immobilized onto zwitterionic poly(sulfobetaine methacrylate) microgels co-functionalized with cell-adhesive RGD and SpyTag peptides via a bifunctional SnoopCatcher–SpyCatcher protein bridge, forming covalent isopeptide bonds for targeted delivery to encapsulated cells.

This platform is differentiated by overcoming both the safety risks of viral vectors and the rapid clearance typical of standard non-viral systems. The zwitterionic microgel prevents non-specific protein fouling, while the biorthogonal covalent tether restricts burst release, retaining nearly 60% of the nanoparticles over 14 days. Whereas conventional delivery decays within a week, this sustained bioavailability drives persistent transgene expression beyond 21 days in 3D cultures. Furthermore, when deployed for CRISPR-mediated activation of the cartilage-lubricating PRG4 gene, the tethered system achieves a 71-fold upregulation, providing prolonged, localized control for tissue regeneration.

Benefits

• Provides sustained and localized delivery of genetic cargo, maintaining gene expression for over 21 days
• Eliminates safety risks associated with viral vectors, such as immunogenicity and genomic integration
• Prevents rapid clearance and off-target biodistribution typical of conventional non-viral delivery systems
• Achieves high transfection efficiency and cellular uptake while maintaining high cell viability and low cytotoxicity
• Minimizes non-specific protein fouling and preserves nanoparticle bioactivity through a zwitterionic microgel matrix
• Enables robust and prolonged CRISPR-mediated epigenetic activation in 3D cell cultures
• Offers an injectable, shear-thinning biomaterial format well-suited for minimally invasive tissue regeneration and localized gene therapy

Applications

• Osteochondral and cartilage tissue regeneration
• Localized injectable CRISPR gene therapy
• Sustained mRNA therapeutic delivery scaffolds
• Stem cell differentiation and engineering
• Joint lubrication restoration for osteoarthritis

Additional info

Tomas Gonzalez-Fernandez’s research focuses on engineering precision cell and gene therapies for regenerative medicine. The Tomas Gonzalez-Fernandez Lab (The TGF Lab) focuses on three interconnected areas: (1) applying synthetic biology tools such as CRISPR gene editing and epigenetic editing to engineer precision stem cell products with improved therapeutic capacity, (2) developing deep learning models for rational CRISPR target discovery and predictive cell reprogramming, and (3) designing biomaterial-based delivery systems for spatiotemporally and stimuli-responsive controlled gene editing in vivo and ex vivo.