Nat Cell Biol. 2020 Sep 3
Farzaneh Moghadam, Ryan LeGraw, Jeremy J Velazquez, Nan Cher Yeo, Chenxi Xu, Jin Park, Alejandro Chavez, Mo R Ebrahimkhani, and Samira Kiani
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AAV vector packaging AAV vectors were digested by SmaI digest to test the integrity of ITR regions before virus production. Verified AAV vectors were used to generate AAV2/1-Myd88, AAV2/1 MockgRNA and AAV2/1 GFP and AAV2/9-Pcsk9 by PackGene® Biotech, LLC. Request Quote

Research Field: an enhanced CRISPR-based transcriptional repressor to reprogram immune homeostasis in vivo

Keywords: CRISPR/Cas9, AAV, in vivo CRISPR, immunomodulation, multifunctional Cas9, endotoxemia, LPS, transcriptional repression, Myd88, AAV antibodies, Septicemia

AAV Serotype: AAV2/1 and AAV2/9

Dose: Animals were anesthetized with 3% isoflurane and virus particles were injected to the left eye with 100 microliters of AAV solution (1E+11 to 1E+12 genome copy per mouse).

Routes of Administration: AAV particles were delivered to mice through retro-orbital injection of the venous sinus.

Animal or cell line strain: Both male and female Rosa26-Cas9 knockin mice (JAX Stock number 026179) and male C57BL/6 mice (JAX Stock number: 000664) were used for AAV/CRISPR repression experiments. Mice were euthanized via CO2 inhalation. Tissue samples taken from liver, lung, bone marrow and blood were collected in RLT Plus buffer (Qiagen) and frozen or snap frozen for RNA analysis.

Abstract

Transient modulation of genes involved in immunity, without exerting a permanent change in the DNA code, can be an effective strategy to modulate the course of many inflammatory conditions. CRISPR-Cas9 technology represents a promising platform for achieving this goal. Truncation of guide RNA (gRNA) from 5’ end, enables the application of a nuclease competent Cas9 protein for transcriptional modulation of genes, allowing multi-functionality of CRISPR. Here, we introduce an enhanced CRISPR-based transcriptional repressor to reprogram immune homeostasis in vivo. In this repressor system, two transcriptional repressors heterochromatin protein 1 (HP1a) and Krüppel associated box (KRAB) are fused to MS2 coat protein and subsequently recruited by gRNA aptamer binding to a nuclease competent CRISPR complex containing truncated gRNAs. With the enhanced repressor, we demonstrate transcriptional repression of the Myeloid differentiation primary response 88 (Myd88) gene in vitro and in vivo. We demonstrate that this strategy can efficiently downregulate Myd88 expression in lung, blood and bone marrow of Cas9 transgenic mice, which receive systemic injection of adeno-associated virus- (AAV)2/1 carrying truncated gRNAs targeting Myd88 and MS2-Hp1aKRAB cassette. This downregulation is accompanied by changes in downstream signaling elements such as TNF-α and ICAM-1. Myd88 repression leads to decrease in immunoglobulin G (IgG) production against AAV2/1 and AAV2/9 and the strategy modulates IgG response against AAV cargos. It improves the efficiency of a subsequent AAV9/CRISPR treatment for repression of Proprotein convertase subtilisin/kexin type 9 (PCSK9), a gene when repressed can lower blood cholesterol levels. We also demonstrate that CRISPR-mediated Myd88 repression can act as a prophylactic measure against septicemia in both Cas9 transgenic and C57BL/6J mice. When delivered by nanoparticles, this repressor can serve as a therapeutic modality to influence the course of septicemia. Collectively, we report that CRISPR-mediated repression of endogenous Myd88 can effectively modulate host immune response against AAV-mediated gene therapy and influence the course of septicemia. The ability to control Myd88 transcript levels using a CRISPR-based synthetic repressor can be an effective strategy for AAV-based CRISPR therapies, as this pathway serves as a key node in induction of humoral immunity against AAV serotypes.

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