Finally, we evaluated Neuro-PERC in a severe murine model of Huntington’s disease. Targeting of the human mutant HTT allele produced efficient on-target editing, reduced mutant HTT levels, and extended mouse survival relative to control mice.

Neuro-PERC achieved broad putaminal coverage with clear GFP loss (indicating successful editing) within the successfully targeted area. This confirms that Neuro-PERC can be scaled to large-brained mammals using neurosurgical approaches already in clinical use.

To explore the translational potential of this approach, we performed MRI-guided CED into the putamen (a striatal region) of juvenile pigs at @mizzou. This large-animal model allowed us to evaluate delivery across a brain volume substantially larger than that of the mouse. 🐷🧠

Neuro-PERC mediated robust in vivo editing of striatal neurons, achieving ~40–60% edited volumes with high reproducibility. Independent validation at @jacksonlab recapitulated results, even after our samples were shipped coast-to-coast.

To determine whether Neuro-PERC supports editing of neurons in vivo, we performed convection-enhanced delivery (CED) into the mouse striatum across multiple reporter models (Ai9, & GFP reporters activated by Cas9 nuclease or adenine base editor).🐭🧠

We first evaluated Neuro-PERC in Ai9-derived mouse neural progenitor cells (NPCs). Peptide-enabled delivery markedly increased tdTomato reporter expression relative to Cas9 RNP alone, with peptides A5K & P55 supporting efficient editing without electroporation.

To avoid impaired protein production yields associated with N/C-terminal tags, Eric put "hairpin" pairs of NLS into the Cas9 protein backbone. If you like files *in* the computer, you'll love NLS *in* the Cas9. We call this layout hiNLS (hairpin/internal NLS).

Cas9 gets an upgrade! Check out our new paper in The CRISPR Journal: bit.ly/hiNLS-Cas9 Eric Noel (who is on the job market!) created Cas9 constructs w/ nuclear localization signals (NLS) in the backbone, boosting genome editing activity in T cells. #CRISPR

Ex vivo tx prevail because they sidestep the substantial challenges of in vivo delivery. As potent as cell therapies can be, transplant can limit access and delay treatment. Fortunately, off-the-shelf T cell therapies and in vivo delivery are poised to change this landscape.

Therapeutic genome editing of hematopoietic stem cells (HSCs) & T cells has been getting better and better over the last ~15 years, w/ progress greatly accelerated by the advent of CRISPR. Here's our timeline figure in slide-friendly "landscape" format (an online exclusive 🤗)