A self-complementary recombinant adeno-associated virus vector coding for an anchorless prion protein carrying the G127V mutation extends survival in a rodent prion disease model

ABSTRACT The replacement of a single codon in the human prion gene, causing the substitution of glycine with valine at position 127 (G127V) of the prion protein (PrP), prevents development of prion disease. We set out to explore if prion disease survival extension manifests in mice if the V127 mutant is delivered through a recombinant adeno-associated virus (rAAV) packaged as a self-complementary DNA. The notorious delivery limitations of rAAVs were overcome using a cross-correction approach that relied on the expression of the mutation in the context of glycosylphosphatidylinositoI-anchorless (ΔGPI) PrP. In this proof-of-concept study, we inoculated Rocky Mountain Laboratory (RML) prions into knock-in mice, in which the endogenous murine prion protein gene ( Prnp ) was replaced with the bank vole prion protein gene (Bv Prnp ). Prion-inoculated mice that were retro-orbitally transduced with a protective rAAV vector encoding Bv Prnp V127 ΔGPI survived ∼50 days longer than control mice that were unprotected. A deep proteomic analysis revealed that Bv Prnp V127 ΔGPI was protective by slowing perturbations to the proteome observed in late-stage RML prion disease. In addition to capturing details of synaptic decay and depletion of proteins in proximity to PrP, the proteomic dataset revealed the identity of proteins of potential diagnostic value that may be central to the brain’s attempt to fight prion disease by contributing to astrocytosis or microgliosis, by coping with calcium influx, or by enhancing the endoplasmic reticulum processing of essential proteins. Taken together, our results demonstrate that a gene therapy based on a GPI-anchorless PrP containing the G127V mutation can delay the onset of prion disease in mice, providing a framework for development of a corresponding therapy in humans. AUTHOR SUMMARY A rare change in the human prion protein, involving a single building block, has been linked to strong protection against prion diseases—fatal neurodegenerative disorders. This study tested whether that protective effect could be reproduced using gene therapy in mice. To this end, we exposed the animals to infectious prions and then delivered the protective version of the protein into mice using a viral carrier. Treated mice survived about seven weeks longer than untreated animals, showing that the approach can meaningfully slow disease progression. To understand why, we examined changes in brain proteins during disease and found that treatment helped preserve the normal protein levels of cellular proteins, particularly those involved in communication between nerve cells. The analysis also identified proteins altered in the disease that are linked to the brain’s defense responses, including inflammation, stress handling, and protein processing, some of which may serve as future disease markers. Importantly, the limited protection observed was not due to poor delivery of the therapy but likely reflects biological limits of the model used. Overall, the findings support the idea that gene therapies based on naturally protective human variants could help slow prion diseases and improve understanding of how the brain responds to them.