NanoScript-Enabled Nonviral Transient Repression of Phosphatase and Tensin Homolog for Axonal Regeneration and Central Nervous System Injury Repair.

Spinal cord injury (SCI) remains a debilitating neurological disorder with limited therapeutic options, as existing treatments primarily address symptoms rather than address the complex interplay of cellular and molecular barriers to regeneration. These barriers collectively hinder functional recovery, including inhibitory glial scarring, chronic neuroinflammation, intrinsic neuronal regenerative deficits, and disruption of the blood-spinal cord barrier (BSCB). To address these limitations, we developed NanoScript-PTEN (NS-PTEN), a nonviral nanoparticle platform that delivers synthetic transcription factors to transiently suppress phosphatase and tensin homolog (PTEN) expression. PTEN negatively regulates the PI3K/AKT/mTOR signaling axis, which is a critical determinant of neuronal survival and axonal growth. By reducing PTEN levels, NS-PTEN derepresses this pro-survival pathway, promoting neuronal regeneration in the injured spinal cord. By integrating a DNA-binding domain targeting the PTEN promoter, a transcriptional repression module, and a nuclear localization signal onto a gold nanoparticle (AuNP) scaffold, NS-PTEN achieves transient control over PTEN repression, reactivating pro-regenerative signaling while minimizing the risks of tumorigenesis associated with permanent gene silencing. In a clinically relevant contusion SCI rat model, NS-PTEN induced a coordinated series of structural and microenvironmental improvements that collectively support spinal cord repair. Histologically, NS-PTEN enhanced axonal continuity and remyelination, as evidenced by denser NF-positive fibers and substantially greater MBP preservation than in both the injury and AuNP groups. Concurrently, NS-PTEN markedly attenuated astroglial and microglial reactivity, reducing GFAP(+) border formation and diminishing Iba1(+) inflammatory cell accumulation. At the vascular interface, NS-PTEN upregulated CD31 and occludin expression, indicating restored endothelial integrity and the reconstruction of tight junctions, which are critical for BSCB repair. In parallel, the inflammatory milieu shifted toward a regenerative phenotype, characterized by suppressed pro-inflammatory cytokine expression (IL-6, TNF-α, and iNOS) and elevated anti-inflammatory/neurotrophic factors (IL-10 and BDNF). These improvements are consistent with secondary, microenvironment-level benefits arising from acute neuronal PTEN repression rather than direct modification of non-neuronal cell types. Importantly, PTEN expression partially rebounded by DPI-28, aligning with the intended transient activity window of the nanoscript system and supporting its translational safety. Through this combination of precise, nonintegrative gene modulation and broad downstream remodeling, NS-PTEN addresses both intrinsic neuronal limitations and extrinsic inhibitory features of the SCI microenvironment.