Synthetic immunomodulation with a CRISPR super-repressor in vivo

Area of Science:

• Immunology
• Gene Therapy
• Molecular Biology

Background:

• Transient gene modulation offers a strategy for managing inflammatory conditions without permanent DNA alterations.
• CRISPR-Cas9 technology provides a versatile platform for gene modulation.
• Truncated guide RNA (gRNA) enables CRISPR-Cas9 for transcriptional modulation, enhancing its functionality.

Purpose of the Study:

• To introduce an enhanced CRISPR-based transcriptional repressor for reprogramming immune homeostasis in vivo.
• To demonstrate the repression of the Myeloid differentiation primary response 88 (Myd88) gene using this novel system.
• To evaluate the impact of Myd88 repression on immune responses to adeno-associated virus (AAV) vectors and its potential in treating sepsis.

Main Methods:

• Developed a CRISPR repressor system fusing heterochromatin protein 1 (HP1a) and Krüppel-associated box (KRAB) repressors to the MS2 coat protein.
• Utilized truncated gRNAs for recruitment of the repressor complex to a nuclease-competent CRISPR complex.
• Administered adeno-associated virus (AAV)2/1 carrying truncated gRNAs and the repressor cassette to Cas9 transgenic mice for in vivo gene repression.

Main Results:

• Successfully demonstrated transcriptional repression of Myd88 in vitro and in vivo, leading to downregulation of Myd88 expression in various tissues.
• Observed reduced downstream signaling elements (TNF-α, ICAM-1) and decreased immunoglobulin G (IgG) production against AAV vectors.
• Showcased improved efficiency of subsequent AAV9/CRISPR treatment for PCSK9 repression and demonstrated prophylactic effects against sepsis.

Conclusions:

• CRISPR-mediated repression of endogenous Myd88 effectively modulates host immune responses against AAV gene therapy.
• This strategy can influence the course of sepsis and serves as a therapeutic modality when delivered via nanoparticles.
• Controlling Myd88 transcript levels via a CRISPR-based synthetic repressor is a viable strategy for enhancing AAV-based CRISPR therapies.