Madigan, V. | bioRxiv Science

bioRxiv2023

Tropism of AAV.CPP.16 in the respiratory tract and its application for a CRISPR-based gene therapy against SARS-CoV-2

Efficient gene delivery vectors are essential for developing gene therapies for respiratory diseases. Here, we report that AAV.CPP.16, a novel AAV9-derived adeno-associated virus vector, can efficiently transduce airway epithelium systems and lung parenchyma cells in both mice and non-human primates after intranasal administration. AAV.CPP.16 outperforms AAV6 and AAV9, two wild-type AAVs with demonstrated tropism to respiratory tract tissues, and can target major cell types in the respiratory tract and the lung. We also report an "all-in-one", CRISPR-Cas13d-based AAV gene therapy vector that targets the highly conserved RNA-dependent RNA polymerase (Rdrp) gene in SARS-CoV-2, and show the potential of such gene therapy against a broad range of circulating and emergent SARS-CoV-2 variants. Thus, AAV.CPP.16 could be a useful gene delivery vector for treating genetic respiratory diseases and airborne infections including for developing a potential prophilaxis to SARS-CoV-2.

bioengineering↗

bioRxiv2021

Transgenic mice for in vivo epigenome editing with CRISPR-based systems

The discovery, characterization, and adaptation of the RNA-guided clustered regularly interspersed short palindromic repeat (CRISPR)-Cas9 system has greatly increased the ease with which genome and epigenome editing can be performed. Fusion of chromatin-modifying domains to the nuclease-deactivated form of Cas9 (dCas9) has enabled targeted gene activation or repression in both cultured cells and in vivo in animal models. However, delivery of the large dCas9 fusion proteins to target cell types and tissues is an obstacle to widespread adoption of these tools for in vivo studies. Here we describe the generation and validation of two conditional transgenic mouse lines for targeted gene regulation, Rosa26:LSL-dCas9-p300 for gene activation and Rosa26:LSL-dCas9-KRAB for gene repression. Using the dCas9p300 and dCas9KRAB transgenic mice we demonstrate activation or repression of genes in both the brain and liver in vivo, and T cells and fibroblasts ex vivo. We show gene regulation and targeted epigenetic modification with gRNAs targeting either transcriptional start sites (TSS) or distal enhancer elements, as well as corresponding changes to downstream phenotypes. These mouse lines are convenient and valuable tools for facile, temporally controlled, and tissue-restricted epigenome editing and manipulation of gene expression in vivo.

genomics↗