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McCarthy, H. O.

Publications and source records attributed to McCarthy, H. O..

2 recordsLinked to original sources
bioRxiv2026

Biomimetic miR-133a inhibitor activated scaffolds optimised for spinal cord repair promote neurite outgrowth and angiogenesis via neuronal cytoskeletal remodelling

Significant challenges in spinal cord injury include the loss of neural tissue, disruption of local vasculature, and intrinsic suppression of actin mobilisation in neurons, together preventing axonal regrowth. Here, we develop an implantable biomimetic microRNA (miR) inhibitor-activated scaffold that combines optimised matrix cues with transcriptomically defined RNA-based modulation of intrinsic neuronal pathways as a platform to support neuronal cell delivery and promote neurovascular repair. First, hyaluronic acid macroporous scaffolds functionalized with collagen-IV and fibronectin supported iPSC-derived neuronal spheroid formation and neurite extension. To identify a neurotrophic target, we performed analysis of public miRNA-mRNA interaction datasets, revealing that miR-133a regulates pathways involved in neuronal actin cytoskeletal organisation. MiR-133a inhibitors were complexed with the cell-penetrating peptide RALA to form nanoparticles, demonstrated >95% scaffold loading efficiency, sustained localised release over 28 days and enhanced neurite outgrowth from motor neurons and iPSC neurons. Bulk RNA-sequencing and transcriptomic analysis of iPSC neurons within the scaffolds demonstrated coordinated upregulation of actin-remodelling, cell-matrix adhesion and metabolic pathways, indicative of a cytoskeletally adaptable neuron. When employed in an ex vivo dorsal root ganglia model, scaffold-mediated miR-133a inhibition promoted neurite extension and integration of delivered iPSC neurons with injured neural tissue. Finally, miR-133a-inhibitor-activated scaffolds upregulated neurovascular genes, increased endothelial cell migration and enhanced blood vessel formation in vivo in a chick embryo assay. These findings identify miR-133a as a neurotrophic target, elucidate the underlying mechanisms of action through transcriptomic analysis and demonstrate that biomimetic scaffold-mediated inhibition of miR-133a can enhance neuronal delivery for multifaceted spinal cord repair applications. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/719922v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@13fcb26org.highwire.dtl.DTLVardef@13239c3org.highwire.dtl.DTLVardef@6e6f5eorg.highwire.dtl.DTLVardef@51ad93_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗
bioRxiv2024

Versatile Cell Penetrating Peptide for Multimodal CRISPR Gene Editing in Primary Stem Cells

CRISPR gene editing offers unprecedented genomic and transcriptomic control for precise regulation of cell function and phenotype. However, delivering the necessary CRISPR components to therapeutically relevant cell types without cytotoxicity or unexpected side effects remains challenging. Viral vectors risk genomic integration and immunogenicity while non-viral delivery systems are challenging to adapt to different CRISPR cargos, and many are highly cytotoxic. The arginine-alanine-leucine-alanine (RALA) cell penetrating peptide is an amphiphilic peptide that self-assembles into nanoparticles through electrostatic interactions with negatively charged molecules before delivering them across the cell membrane. This system has been used to deliver DNAs, RNAs, and small anionic molecules to primary cells with lower cytotoxicity compared to alternative non-viral approaches. Given the low cytotoxicity, versatility, and competitive transfection rates of RALA, we aimed to establish this peptide as a new CRISPR delivery system in a wide range of molecular formats across different editing modalities. We report that RALA was able to effectively encapsulate and deliver CRISPR in DNA, RNA, and ribonucleic protein (RNP) formats to primary mesenchymal stem cells (MSCs). Comparisons between RALA and commercially available reagents revealed superior cell viability leading to higher numbers of transfected cells and the maintenance of cell proliferative capacity. We then used the RALA peptide for the knock-in and knock-out of reporter genes into the MSC genome as well as for the transcriptional activation of therapeutically relevant genes. In summary, we establish RALA as a powerful tool for safer and effective delivery of CRISPR machinery in multiple cargo formats for a wide range of gene editing strategies.

bioengineering↗