Synthetic immunomodulation with a CRISPR super-repressor in vivo
| Autor: | Jin Park, Samira Kiani, Ryan LeGraw, Farzaneh Moghadam, Chenxi Xu, Mo R. Ebrahimkhani, Jeremy J. Velazquez, Alejandro Chavez, Nan Cher Yeo |
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| Rok vydání: | 2019 |
| Předmět: |
Male
LPS Genetic enhancement Transgene Repressor Mice Transgenic Receptors Cell Surface Biology immunomodulation Myd88 multifunctional Cas9 Article 03 medical and health sciences Mice 0302 clinical medicine transcriptional repression CRISPR Animals Humans Clustered Regularly Interspaced Short Palindromic Repeats in vivo CRISPR Gene Psychological repression CRISPR/Cas9 030304 developmental biology Gene Editing 0303 health sciences Cas9 endotoxemia Septicemia AAV Cell Biology Proprotein convertase Cell biology Mice Inbred C57BL HEK293 Cells 030220 oncology & carcinogenesis Myeloid Differentiation Factor 88 AAV antibodies CRISPR-Cas Systems Proprotein Convertase 9 RNA Guide Kinetoplastida |
| Zdroj: | Nature cell biology |
| ISSN: | 1476-4679 |
| Popis: | Transient modulation of the 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 the 5' end enables the application of a nuclease competent Cas9 protein for transcriptional modulation of genes, allowing multifunctionality 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 Kruppel-associated box (KRAB)-are fused to the 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 that receive systemic injection of adeno-associated virus (AAV)2/1-carrying truncated gRNAs targeting Myd88 and the MS2-HP1a-KRAB cassette. This downregulation is accompanied by changes in downstream signalling elements such as TNF-α and ICAM-1. Myd88 repression leads to a decrease in immunoglobulin G (IgG) production against AAV2/1 and AAV2/9 and this strategy modulates the 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 that, when repressed, can lower blood cholesterol levels. We also demonstrate that CRISPR-mediated Myd88 repression can act as a prophylactic measure against septicaemia 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 septicaemia. Collectively, we report that CRISPR-mediated repression of endogenous Myd88 can effectively modulate the host immune response against AAV-mediated gene therapy and influence the course of septicaemia. 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 the induction of humoral immunity against AAV serotypes. |
| Databáze: | OpenAIRE |
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