Gene Therapy. 2021 Feb 22
Tess Torregrosa, Sydney Lehman, Sam Hana, Galina Marsh, Shanqin Xu, Kathryn Koszka, Nicole Mastrangelo, Alexander McCampbell, Christopher E. Henderson, and Shih-Ching Lo
Products used in the paper Details Operation
AAV vector packaging Single-stranded viral vectors were packaged into either AAV9, AAV-PHP.B, or AAV-PHP.eB at a titer of 5E13 vg/ ml and were produced and purified (PackGene Biotech, Worcester, MA). Request Quote

Research Field: CNS

Keywords: Genetic transduction, Gene therapy

AAV Serotype: AAV9, AAV-PHP.B, or AAV-PHP.eB

Dose: On postnatal day 0 (P0), neonatal mice were cryoanesthetized and then injected with AAV-sgNeuN/ sgGFAP/sgMOG at either a low (5E10 vg) or high (20E10 vg) dose at a total volume of 4 μL. Mice injected with AAV-sgLacZ were only injected at a high dose.

Routes of Administration: intracerebroventricular (ICV) injections

Targeted organ: CNS

Animal or cell line strain: H11-Cas9 mice on C57BL/6J [B6J.129(Cg)-Igs2tm1.1 (CAG-cas9*)Mmw/J; stock #028239; laboratory of M. Winslow, Stanford University, Stanford, CA] [25] constitutively expressing Streptococcus pyogenes Cas9 (Cas9)
were purchased from Jackson Laboratory (Bar Harbor, ME).


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.

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