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

## TL;DR

A nonviral nanoparticle system temporarily suppresses PTEN to promote spinal cord injury repair by enhancing axonal regeneration and reducing inflammation.

## Contribution

A novel nonviral nanoscript platform for transient PTEN repression that improves spinal cord injury recovery through multiple regenerative mechanisms.

## Key findings

- NS-PTEN enhanced axonal continuity and remyelination in spinal cord injury models.
- NS-PTEN reduced astroglial and microglial reactivity while promoting endothelial integrity.
- NS-PTEN shifted the inflammatory environment toward regeneration without permanent gene silencing.

## Abstract

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.