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.

Highlights

  • Recombinant adeno-associated viruses (AAVs) are commonly used to transfer exogenous DNA in vivo to the central nervous system (CNS) in mammals

  • Comparable degree of GFAP reduction was caused by transduction of AAV9, AAV-PHP.B and AAV-PHP.eB, indicating similar astrocyte transduction by all three AAVs. These results suggest that AAV9 and AAV-PHP.B variants are able to broadly transduce astrocytes throughout the CNS with modest efficiency, following postnatal day 0 (P0) ICV injection

  • We demonstrated the use of a CRISPR/sgRNA gene editing approach to quantitatively compare AAV9, AAV-PHP.B, and AAV-PHP.eB transduction efficiency and cellular tropism in the mouse CNS

Read more

Summary

Introduction

Recombinant adeno-associated viruses (AAVs) are commonly used to transfer exogenous DNA in vivo to the central nervous system (CNS) in mammals. The cargo DNA of AAV vectors can be designed to mediate editing in the host genome or drive transgene expression for studying the function of a gene in CNS [1, 2], modeling neurodegenerative diseases [3], or gene therapy. These authors contributed : Tess Torregrosa, Sydney Lehman

Methods
Results
Conclusion
Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call