Engineering rAAV VP1/2 protein binding motifs to increase transduction in the CNS

Adeno-associated virus vectors have advanced within the clinical gene therapy space, where AAV 9 vectors are currently employed for spinal muscular atrophy (SMA) and giant axon neuropathy (GAN)(Mendell et al., 2018; Bailey et al., 2018), while AAV5 vectors have proven effective for hemophilia B therapy for 13 years post-treatment (Reiss et al., 2025). Given this prevalence, substantial efforts have been expended to improve various properties of the AAV vectors with a primary focus upon the external surface of the AAV capsid (Deverman et al., 2016; Hanlon et al., 2019; Büning and Srivastava, 2019)). In marked contrast, we have established that an interior portion of the AAV9 capsid interacts with promoter activity to significantly alter in vivo gene expression (Powell et al., 2020). Although previous studies established that the VP1 alternatively spliced membrane associated accessory protein (MAAP) was only associated with exosomes during virus production we discovered the absence of MAAP during virus production resulted in a five fold increase in in vivo gene expression (Powell and McCown, 2024). Now preliminary findings have emerged from this novel AAV capsid region indicating that within this AAV VP1/VP2 coding junction, a single glycine to alanine mutation at aa 141 in the AAV9 capsid or the rh10 capsid dramatically increases in vivo gene expression. Our previous findings have established that an in depth understanding of the influence of AAV capsids on in vivo gene expression can be leveraged to optimize the efficacy of AAV vectors within the CNS. Given that high vector doses remain a serious concern, the ability to significantly increase in vivo gene expression without compromising the payload limit of AAV vectors would represent a significant advance.