Dataset Information

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.

SUBMITTER: Torregrosa T

PROVIDER: S-EPMC8376643 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:The non-pathogenic parvovirus, adeno-associated virus (AAV), is an efficient vector for transgene expression in vivo and shows promise for treatment of brain disorders in clinical trials. Currently, there are more than 100 AAV serotypes identified that differ in the binding capacity of capsid proteins to specific cell surface receptors that can transduce different cell types and brain regions in the CNS. In the current study, multiple AAV serotypes expressing a GFP reporter (AAV1, AAV2/1, AAVDJ, AAV8, AAVDJ8, AAV9, AAVDJ9) were screened for their infectivity in both primary murine astrocyte and neuronal cell cultures. AAV2/1, AAVDJ8 and AAV9 were selected for further investigation of their tropism throughout different brain regions and cell types. Each AAV was administered to P0-neonatal mice via intracerebroventricular injections (ICV). Brains were then systematically analyzed for GFP expression at 3 or 6 weeks post-infection in various regions, including the olfactory bulb, striatum, cortex, hippocampus, substantia nigra (SN) and cerebellum. Cell counting data revealed that AAV2/1 infections were more prevalent in the cortical layers but penetrated to the midbrain less than AAVDJ8 and AAV9. Additionally, there were differences in the persistence of viral transgene expression amongst the three serotypes examined in vivo at 3 and 6 weeks post-infection. Because AAV-mediated transgene expression is of interest in neurodegenerative diseases such as Parkinson's Disease, we examined the SN with microscopy techniques, such as CLARITY tissue transmutation, to identify AAV serotypes that resulted in optimal transgene expression in either astrocytes or dopaminergic neurons. AAVDJ8 displayed more tropism in astrocytes compared to AAV9 in the SN region. We conclude that ICV injection results in lasting expression of virally encoded transgene when using AAV vectors and that specific AAV serotypes are required to selectively deliver transgenes of interest to different brain regions in both astrocytes and neurons.

Project description:IntroductionEx vivo gene therapy for treatment of Inborn errors of Immunity (IEIs) have demonstrated significant clinical benefit in multiple Phase I/II clinical trials. Current approaches rely on engineered retroviral vectors to randomly integrate copy(s) of gene-of-interest in autologous hematopoietic stem/progenitor cells (HSPCs) genome permanently to provide gene function in transduced HSPCs and their progenies. To circumvent concerns related to potential genotoxicities due to the random vector integrations in HSPCs, targeted correction with CRISPR-Cas9-based genome editing offers improved precision for functional correction of multiple IEIs.MethodsWe compare the two approaches for integration of IL2RG transgene for functional correction of HSPCs from patients with X-linked Severe Combined Immunodeficiency (SCID-X1 or XSCID); delivery via current clinical lentivector (LV)-IL2RG versus targeted insertion (TI) of IL2RG via homology-directed repair (HDR) when using an adeno-associated virus (AAV)-IL2RG donor following double-strand DNA break at the endogenous IL2RG locus.Results and discussionIn vitro differentiation of LV- or TI-treated XSCID HSPCs similarly overcome differentiation block into Pre-T-I and Pre-T-II lymphocytes but we observed significantly superior development of NK cells when corrected by TI (40.7% versus 4.1%, p = 0.0099). Transplants into immunodeficient mice demonstrated robust engraftment (8.1% and 23.3% in bone marrow) for LV- and TI-IL2RG HSPCs with efficient T cell development following TI-IL2RG in all four patients' HSPCs. Extensive specificity analysis of CRISPR-Cas9 editing with rhAmpSeq covering 82 predicted off-target sites found no evidence of indels in edited cells before (in vitro) or following transplant, in stark contrast to LV's non-targeted vector integration sites. Together, the improved efficiency and safety of IL2RG correction via CRISPR-Cas9-based TI approach provides a strong rationale for a clinical trial for treatment of XSCID patients.

Dataset Information

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets