Andrea Boitnott

Background/Objectives: Adeno-associated virus serotype 9 (AAV9) can cross the blood–brain barrier, making it widely used as a gene delivery vector for central nervous system (CNS) applications. Despite extensive use of AAV9 in translational research, variability in study designs makes cross-comparisons difficult to interpret. We designed a study in mice to generate a resource of AAV9 biodistribution across tissues for commonly used routes of administration and treatment ages. Methods: Lumbar intrathecal, intracerebroventricular, lumbar intrathecal and intracerebroventricular combination, or intravenous injections of vehicle or AAV9/GFP were performed in C57BL/6J male and female mice on postnatal day 1, 5, 10, or 28. Organs were collected at postnatal day 56 and biodistribution of AAV9/GFP was evaluated by quantifying GFP protein expression and vector genome copy number. Results: Direct cerebrospinal fluid injections led to higher transgene expression levels in the brain and spinal cord compared to intravenous administration but did not de-target transgene expression in peripheral tissues. Lumbar intrathecal and intracerebroventricular combination injections resulted in expression throughout the CNS but did not substantially increase expression in either the spinal cord or brain beyond the levels obtained with the respective single routes. Treatment age had effects on AAV9 biodistribution regardless of the route of administration, especially in the brain, eye, and liver. Conclusions: Our results provide the necessary biodistribution data to establish a standardized benchmark for comparison of the current gold standard AAV9 to next generation viral vectors. Additionally, this body of work can provide valuable insights for the design of translational gene therapy studies.

One method to target the central nervous system for treating neurological diseases involves utilizing the lumbar intrathecal route of administration. This approach bypasses the blood-brain barrier to directly access the cerebrospinal fluid and preferentially target cells within the central nervous system. Multiple published preclinical studies employing the lumbar intrathecal injection route have contributed to the development of gene therapy clinical trials; however, the described protocols are variable and dispersed across multiple resources. Here, a comprehensive set of protocols for lumbar intrathecal injections in neonatal, juvenile, and adult mice and rats for preclinical gene therapy studies is presented. With proper training, this injection technique can be performed quickly and reliably. In addition to detailing the injection protocol at each developmental stage, associated parameters, such as injection volume, that can influence study outcomes are discussed. To demonstrate the application of lumbar intrathecal injections for targeting the central nervous system, the expression of adeno-associated virus serotype 9 in the brain, spinal cord, and peripheral tissues is presented following a successful or unsuccessful injection.

One method to target the central nervous system for treating neurological diseases involves utilizing the lumbar intrathecal route of administration. This approach bypasses the blood-brain barrier to directly access the cerebrospinal fluid and preferentially target cells within the central nervous system. Multiple published preclinical studies employing the lumbar intrathecal injection route have contributed to the development of gene therapy clinical trials; however, the described protocols are variable and dispersed across multiple resources. Here, a comprehensive set of protocols for lumbar intrathecal injections in neonatal, juvenile, and adult mice and rats for preclinical gene therapy studies is presented. With proper training, this injection technique can be performed quickly and reliably. In addition to detailing the injection protocol at each developmental stage, associated parameters, such as injection volume, that can influence study outcomes are discussed. To demonstrate the application of lumbar intrathecal injections for targeting the central nervous system, the expression of adeno-associated virus serotype 9 in the brain, spinal cord, and peripheral tissues is presented following a successful or unsuccessful injection.

Autism spectrum disorder (ASD) is a prevalent neurodevelopmental disorder characterized by impairments in social communication and social interaction, and the presence of repetitive behaviors and/or restricted interests. In the past few years, large-scale whole-exome sequencing and genome-wide association studies have made enormous progress in our understanding of the genetic risk architecture of ASD. While showing a complex and heterogeneous landscape, these studies have led to the identification of genetic loci associated with ASD risk. The intersection of genetic and transcriptomic analyses have also begun to shed light on functional convergences between risk genes, with the mid-fetal development of the cerebral cortex emerging as a critical nexus for ASD. In this review, we provide a concise summary of the latest genetic discoveries on ASD. We then discuss the studies in postmortem tissues, stem cell models, and rodent models that implicate recently identified ASD risk genes in cortical development.