Intrathecal (IT) lumbar puncture delivery of recombinant adeno-associated virus serotype 9 (rAAV9) is a gene therapy approach being explored in preclinical studies and ongoing gene therapy clinical trials for neurological diseases. Few studies address IT rAAV9 vector distribution, tropism, and expression with respect to age of administration. Therefore, we IT delivered a rAAV9/GFP vector in mice at ages ranging from early postnatal development through adulthood (P10-P90). Tissues were assessed for transgene expression, cell tropism, and vector distribution. In the CNS, transduction was highest when delivered at post-natal day 10 (P10) and there was an age-dependent decline in transduction. We found higher transduction of astrocytes relative to neurons when rAAV9 was administered at younger ages and a switch to higher neuronal transduction with delivery at older timepoints. Biodistribution analysis of peripheral tissues showed that when delivered at P10, rAAV9 has the greatest distribution to the heart. Conversely, at P90 rAAV9 liver distribution was highest. As rAAV9 IT-delivered gene therapies continue to emerge for neurological diseases, careful consideration of the age of delivery should be taken in relation to the expected distribution and cell expression in animal models, and how this may translate to human studies.

The use of AAV capsid libraries coupled with various selection strategies has proven to be a remarkable approach for generating novel AAVs with enhanced and desired features. The inability to reliably sequence the complete capsid gene in a high-throughput manner has been the bottleneck of capsid engineering. As a result, many library strategies are confined to localized and modest alterations in the capsid, such as peptide insertions or single variable region (VR) alterations. The caveat of short reads by means of next-generation sequencing (NGS) hinders the diversity of capsid library construction, shifting the field away from whole-capsid modifications. We generated AAV capsid shuffled libraries of naturally occurring AAVs and applied directed evolution in both mice and non-human primates (NHPs), with the goal of yielding AAVs that are compatible across both species for translational applications. We recovered DNA from the tissues of injected animal and used single molecule real-time (SMRT) sequencing to identify variants enriched in the central nervous system (CNS). We provide insights and considerations for variant identification by comparing bulk tissue sequencing to that of isolated nuclei. Our work highlights the potential advantages of whole-capsid engineering, as well as indispensable methodological improvements for the analysis of recovered capsids, including the nuclei-enrichment step and SMRT sequencing.

SLC13A5 citrate transporter disorder is a rare epileptic encephalopathy caused by loss-of-function pathogenic variants in the SLC13A5 gene. Loss of sodium/citrate cotransporter (NaCT) function causes a severe early-life epilepsy resulting in lifelong developmental disabilities and increased extracellular citrate. Current antiseizure medications may reduce seizure frequency, yet more targeted treatments are needed to address the epileptic and neurodevelopmental SLC13A5 phenotype. We performed preclinical studies in SLC13A5-deficient (KO) mice evaluating phenotype rescue with adeno-associated virus (AAV) vector carrying a functional copy of the human SLC13A5 gene (AAV9/SLC13A5). Cerebrospinal fluid delivery of AAV9/SLC13A5 decreased extracellular citrate levels, normalized electrophysiologic and sleep architecture abnormalities, and restored resistance to chemically induced seizures and death. Treatment benefits were achieved with administration during early brain development and in young adult mice, indicating therapeutic efficacy across developmental and postdevelopmental stages. Comparison of delivery routes in young adult KO mice showed that higher brain targeting achieved with intra–cisterna magna delivery resulted in greater treatment benefit as compared with intrathecal lumbar puncture delivery. Together, these results support further development of AAV9/SLC13A5 for treating SLC13A5 citrate transporter disorder.

Tauopathies, including Alzheimer’s disease, are neurodegenerative disorders characterized by the accumulation of microtubule-associated protein tau, which is closely linked to cognitive decline. Reduction of tau is a potential and promising strategy for addressing tau-linked brain disorders. We report the development of a therapeutic approach using adeno-associated virus mediated delivery of an artificial microRNA targeting human tau. In a tauopathy mouse model, we demonstrate that a one-time intra-cisterna magna administration of vector resulted in reduced total tau, decreased pathological tau seeds, fewer tau inclusions, and amelioration of tau-related neuropathology. Notably, intervention at late disease stages, after onset of tau deposition and neurodegeneration, improved quality of life and extended survival. We further demonstrated the durability of therapeutic benefit and defined the minimally effective dose in tauopathy mice. These findings provide preclinical support for the advancement of a vectorized tau-lowering strategy as a disease-modifying approach for tauopathies and enable progression towards an investigational new drug application.

Adeno-associated virus (AAV) mediated gene therapies continue to show promise for the treatment of genetic diseases. However, delivery and safety still represent significant prohibitive barriers restricting translation of these delivery modalities to humans. We describe a novel AAV9 variant, “AAV9fyhr” or “FYHR”, identified from screening a controlled randomization point mutation AAV9 library in mice and nonhuman primates followed by directed evolution of in vivo CNS-derived libraries screened in human iPSC differentiated cortical neurons. Capsid variants of interest were identified by coupling long read sequencing and computational structure modeling. Structure-function analysis of our lead candidate reveal that two targeted amino acid changes are sufficient to result in beneficial pleiotropic effects across viral stability, cell surface attachment, and gene delivery. We developed a novel multiplexed barcoding reporter system, enabling a time and cost-effective option to gather spatially resolved single cell capsid validation data in mice and nonhuman primates. When administered intrathecally, AAV9fyhr exhibits 2-to-6-fold higher gene delivery across several tissues in the CNS while minimally altering the tropism to peripheral tissues in comparison to AAV9. These findings demonstrate the potential of whole capsid engineering for the rational development of translational next generation AAV capsids.