AAV-Split CRISPR Repressors for In Vivo Gene Silencing

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Solution Overview

Problem

The challenge of delivering CRISPR/Cas9-based gene repressors in vivo is hindered by the size of the S. pyogenes dCas9 and KRAB domain fusion exceeding the packaging limits of standard AAV vectors, limiting their use in therapeutic applications.

Innovation Solution

A modified RNA-guided dCas9-based repressor is developed, which is customized to target any endogenous gene, packaged efficiently in AAV vectors, and used for in vivo gene regulation, utilizing a fusion protein with a dCas9 molecule fused to a modulator like KRAB, enabling stable gene repression.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If S. pyogenes dCas9-KRAB fusion is used for gene repression, then gene silencing potency is improved, but vector packaging capacity is exceeded

Engineering Contradiction:

Improvegene silencing potencyVSAvoidvector packaging capacity

Core Design Contradiction:

ReliabilityVSVolume of stationary object

Solution Approach 1:

The dCas9-KRAB fusion protein is divided into two separate components: dCas9 is packaged in one AAV vector while KRAB is packaged in another AAV vector. This segmentation allows each component to fit within the packaging capacity of individual vectors while maintaining the ability to function together as a repressor system when both vectors transduce the same target cells.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The gRNA molecule serves as an intermediary that bridges the two separate dCas9 and KRAB components. The gRNA guides both dCas9 and KRAB to the same target gene sequence, enabling the repressor complex to form at the target locus even though the protein components are delivered separately by different viral vectors.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Adaptability or versatility

If dCas9-KRAB fusion is delivered in vivo, then gene regulation capability is improved, but delivery efficiency is reduced

Engineering Contradiction:

Improvegene regulation capabilityVSAvoiddelivery efficiency

Core Design Contradiction:

Adaptability or versatilityVSProductivity

Solution Approach 1:

The delivery system is segmented into two separate AAV vectors, each with optimized packaging and delivery characteristics. This allows each vector to be independently optimized for efficient transduction while avoiding the packaging constraints that would limit delivery of the full-length fusion protein.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The system uses universal AAV vectors that can transduce multiple tissue types and cell types efficiently. By using well-established AAV delivery platforms for both the dCas9 and KRAB components, the system leverages the proven efficiency and versatility of AAV-mediated gene delivery while maintaining the ability to target any gene of interest through custom gRNA design.

Inventive Principle:

Principle #6Universality (Multi-functionality)

Data Source

PatentUS20260055386A1Crispr/cas9-based repressors for silencing gene targets in vivo and methods of use

Publication Date: 2026.02.26 DUKE UNIV

AI summary

The present disclosure provides Crispr/cas9-based repressors for silencing gene targets in vivo and methods of use.