
Use of CRISPR/Cas9-mediated disruption of CNS cell type genes to profile transduction of AAV by neonatal intracerebroventricular delivery in mice
Brief intro:
- Author: Tess Torregrosa, Sydney Lehman, Sam Hana, Galina Marsh, Shanqin Xu, Kathryn Koszka, Nicole Mastrangelo, Alexander McCampbell, Christopher E. Henderson, and Shih-Ching Lo
- Journal: Gene Therapy
- Doi: https://www.doi.org/10.1038/s41434-021-00223-3
- Publication Date: 2021 Feb 22
Products/Services used in the paper
Quotation shows PackGene: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).
Research Field:CNS
AAV Serotype:AAV9, AAV-PHP.B, or AAV-PHP.eB
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).
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.
About PackGene
PackGene Biotech is a world-leading CRO and CDMO, excelling in AAV vectors, mRNA, plasmid DNA, and lentiviral vector solutions. Our comprehensive offerings span from vector design and construction to AAV, lentivirus, and mRNA services. With a sharp focus on early-stage drug discovery, preclinical development, and cell and gene therapy trials, we deliver cost-effective, dependable, and scalable production solutions. Leveraging our groundbreaking π-alpha 293 AAV high-yield platform, we amplify AAV production by up to 10-fold, yielding up to 1e+17vg per batch to meet diverse commercial and clinical project needs. Moreover, our tailored mRNA and LNP products and services cater to every stage of drug and vaccine development, from research to GMP production, providing a seamless, end-to-end solution.
