# CAG-targeted brain-permeable therapy tested in biallelic humanized polyQ mouse models

## TL;DR

This study tests a brain-targeted therapy for polyglutamine diseases using shRNAs delivered through the blood, showing reduced toxic protein with minimal side effects in mice.

## Contribution

The study demonstrates a non-invasive, brain-permeable CAG-targeted therapy with improved silencing and reduced toxicity in biallelic polyQ disease models.

## Key findings

- CAG-targeted shRNAs reduced mutant huntingtin and ataxin-3 proteins and aggregates in mouse brains.
- AAV-PHP.eB delivery achieved brain transduction with minimal toxicity compared to other shRNAs.
- The approach shows potential for targeting somatic expansion and accelerating drug development for polyQ diseases.

## Abstract

In polyglutamine (polyQ) diseases, including Huntington disease (HD) and spinocerebellar ataxia type 3 (SCA3), targeting the mutant CAG tract in mRNA could be a therapeutic strategy for lowering pathogenic protein. We explored the viability of this therapeutic strategy in vivo at the level of the reagent design, toxicity, systemic delivery, brain regions transduction, silencing efficiency, and allele preference. We designed a series of CAG-directed short hairpin RNAs (shRNAs) based on a previous A2 reagent, allele selective in vitro. Humanized HD (Hu128Q/21Q) and SCA3 (Ki150Q/21Q) mice with mutant ∼100 CAGs and normal 21 CAGs alleles were used to simulate biallelic conditions occurring in patients. We administered AAV-PHP.eB shRNAs-encoding vectors into the blood as an equivalent of non-invasive CAG-directed brain-targeted therapy crossing the blood-brain barrier. We demonstrate that optimized CAG-targeted A4(P10) and A4(P10,11) shReagents can lower mutant huntingtin and ataxin-3 protein and its aggregates by targeting brain regions selectively and with diminished toxicity compared to other tested shRNAs. The important considerations of the approach are the silencing efficiency depending on the transduction region and careful dose adjustment. Moreover, the CAG approach could be suitable to target somatic expansion. Our work paves the way toward developing the therapy for polyQ diseases, potentially shortening drug development.

Polyglutamine (polyQ) disease preclinical therapy targeted the mutant RNA with CAG tract by short hairpin RNAs (shRNAs). shRNAs were delivered to the HD and SCA3 mouse brain via the blood-brain barrier, reducing pathogenic protein and aggregates with minimal toxicity. The approach could effectively treat polyQ diseases, potentially speeding up drug development.