Use of CRISPR/Cas9-mediated disruption of CNS cell type genes to profile transduction of AAV by neonatal intracerebroventricular delivery in mice

• Published in: Gene therapy 2021-08, Vol.28 (7-8), p.456-468
• Main Authors: Torregrosa, Tess, Lehman, Sydney, Hana, Sam, Marsh, Galina, Xu, Shanqin, Koszka, Kathryn, Mastrangelo, Nicole, McCampbell, Alexander, Henderson, Christopher E., Lo, Shih-Ching
• Format: Article
• Language: English
• Subjects: 14/32; 38/1; 38/44; 38/71; 42/41; 631/61/201; 631/61/2300/1850; Animals; Astrocytes; Biomedical and Life Sciences; Biomedicine; Blood-brain barrier; Care and treatment; Cell Biology; Cellular signal transduction; Central Nervous System; CRISPR; CRISPR-Cas Systems; Dependovirus - genetics; Dependoviruses; Expression vectors; Gene disruption; Gene Expression; Gene Therapy; Gene Transfer Techniques; Genes; Genetic aspects; Genetic vectors; Genetic Vectors - genetics; Glial fibrillary acidic protein; Human Genetics; Infants (Newborn); Methods; Mice; Nanotechnology; Neonates; Neurons; Oligodendrocyte-myelin glycoprotein; Oligodendrocytes; Reporter gene; Spinal cord; Transduction; Transduction, Genetic; Tropism
• ISSN: 0969-7128; 1476-5462
• DOI: 10.1038/s41434-021-00223-3

Adeno-associated virus (AAV) transduction efficiency and tropism are conventionally determined by high expression of a fluorescent reporter gene. Emerging data has suggested that such conventional methods may underestimate AAV transduction for cells in which reporter expression from AAV vectors is undetectable. To explore an alternative method that captures AAV transduction in cells in which low expression of a cargo is sufficient for the intended activity, we sought after CRISPR/Cas9-mediated gene disruption. In this study, we use AAV to deliver CRISPR/guide RNA designed to abolish the genes NeuN, GFAP, or MOG expressed specifically in neurons, astrocytes, or oligodendrocytes respectively in the central nervous system (CNS) of mice. Abrogated expression of these cell-type-specific genes can be measured biochemically in CNS subregions and provides quantitative assessment of AAV transduction in these CNS cell types. By using this method, we compared CNS transduction of AAV9, AAV-PHP.B, and AAV-PHP.eB delivered via intracerebroventricular injection (ICV) in neonatal mice. We found both AAV-PHP.B and AAV-PHP.eB resulted in marked disruption of the NeuN gene by CRISPR/Cas9, significantly greater than AAV9 in several brain regions and spinal cord. In contrast, only modest disruption of the GFAP gene and the MOG gene was observed by all three AAV variants. Since the procedure of ICV circumvents the blood–brain barrier, our data suggests that, independent of their ability to cross the blood–brain barrier, AAV-PHP.B variants also exhibit remarkably improved neuronal transduction in the CNS. We anticipate this approach will facilitate profiling of AAV cellular tropism in murine CNS.