Synthetic immunomodulation with a CRISPR super-repressor in vivo

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.

A Novel pH-Sensitive Multifunctional DNA Nanomedicine: An Enhanced and Harmless GD2 Aptamer-Mediated Strategy for Guiding Neuroblastoma Antitumor Therapy

“…Structured oligonucleotide sequences of RNA or DNA have strict recognition ability and high affinity for their corresponding target molecules (proteins, viruses, bacteria, cells, heavy metal ions, etc.). 46 Aptamers can fold into unique three-dimensional structures to recognize and bind to their targets. Interestingly, in addition to their well-known structure, aptamers also have more complex structures, such as the i-motif and A-motif.…”

CRISPR-based strategies for targeted transgene knock-in and gene correction

“…These include strategies to recruit endogenous catalytic domains (for example, base editor, HDR enhancer, RNA editor, or chromatin modifier) for gene knock-in and gene correction and controlled timing of CRISPR/Cas9 delivery and expression to minimize the off-target effects, toxicity, and immunogenicity associated with CRISPR components. Immunogenicity issue can also be circumvented by transient modulation of the genes involved in immunity by using a CRISPR-based synthetic repressor 191 . Other strategies to minimize undesired off-target mutagenesis are using light-activated, chemical-inducible, or multiple-input logic gate genetic circuits to spatiotemporal and conditional control of CRISPR/Cas9 activities 192 ; cell type- or tissue-specific promoters and delivery vehicles (for example, specific viral serotypes 16 , 17 , 193 or ligand-targeted cargos 192 ), engineered high-fidelity Cas9 variants 194 – 200 , and rational chemical modifications to the sgRNA 192 ; a chimeric DNA-RNA guide 201 ; and engineered truncated sgRNA 202 , 203 and sgRNA secondary structures 204 .…”

From Mesenchymal Stromal/Stem Cells to Insulin-Producing Cells: Immunological Considerations

“…Recently, gene editing has emerged as a powerful alternative approach ( 157 ). Moghadan and associates developed a CRISPR-based system that could effectively modulate the immune responses of the host ( 158 ). To this end, the authors made use of the transcriptional repressors heterochromatin protein 1 (HPIα) and Kruppel-associated box protein (KRAB).…”