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

Abstract

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.

Somatic genetics analysis of sleep in adult mice

“…In particular, Gradinaru and colleagues have used the C re- re combination-based A AV-targeted e volution (CREATE) to isolate AAV9 variants, such as AAV-PHP.B and AAV-PHP.eB, which can efficiently bypass BBB and transduce the majority of adult brain neurons and astrocytes in certain mouse strains (Chan et al, 2017; Deverman et al, 2016). Both AAV-PHP.B and AAV-PHP.eB have been successfully used to deliver systemic gene expression (Liu et al, 2021; Luoni et al, 2020; Silva-Pinheiro et al, 2020), or target gene ablation by CRISPR/Cas9 across the adult mouse brain neurons (Torregrosa et al, 2021; Xiao et al, 2021; Yamaguchi and de Lecea, 2019).…”

Dual-isoform hUBE3A gene transfer improves behavioral and seizure outcomes in Angelman syndrome model mice

“…Compared with intravascular administration, ICV injections, like other intracerebrospinal fluid routes of delivery, offer advantages in translational feasibility for CNS disorders: low doses that more readily scale from preclinical models to human, improved efficiency of CNS transduction, and the avoidance of circulating anti-AAV neutralizing antibodies (34)(35)(36). PHP.B yields robust CNS transduction via the ICV route, at least on par with the parent AAV9 capsid (37), but grants superior CNS access when delivered intravascularly in mice (30), offering greater flexibility for future preclinical studies. Following ICV delivery of 1 μL of 1.6 × 10 14 vector genomes (vg)/mL of PHP.B/hUBE3Aopt vector to neonatal AS model mice, which come to lack neuronal expression of UBE3A (38), we performed quantitative PCR (qPCR) analysis of whole-brain RNAs.…”

Efficient in vivo neuronal genome editing in the mouse brain using nanocapsules containing CRISPR-Cas9 ribonucleoproteins

“…AAV vectors have been directly injected into numerous brain regions to edit neuronal proteins [42][43][44][45][46] , including in rodent models of Huntington's disease 47,48 and Alzheimer's disease 49 . Intracerebroventricular (ICV) delivery of AAV carrying CRISPR has been explored to maximize the area of genome editing in the brain due to distribution in the cerebrospinal fluid (CSF) 16,17 . A recent study utilizing ICV delivery of AAVs showed knockdown of NeuN in multiple CNS regions 16 .…”

“…Particles with smaller sizes and neutral charges are advantageous for brain editing, as they can diffuse over longer distances in this unique extracellular matrix [11][12][13] . Viral or plasmid vectors have been successfully applied for in vivo delivery of Cas9 and single guide RNA (sgRNA) to the brain, most commonly through the use of adeno-associated virus (AAV) vectors [14][15][16][17][18] . To deliver genome editing components, AAVs depend on the host transcriptional and translational machinery of the cell to generate genome editing ribonucleoproteins (RNPs).…”