Donor Template System for Large DNA Knock-in

Solution Overview

Problem

Current gene editing techniques face challenges in efficiently knocking in large DNA fragments into precise genomic loci, achieving long-term stable expression, and using Good Manufacturing Practice (GMP) compatible reagents, particularly with low efficiency and high cost for large genes.

Innovation Solution

A donor template system comprising a payload with homology arms and cleavage sites, combined with a nuclease targeting system like CRISPR-Cas, is used to facilitate efficient integration of large DNA fragments into specific genomic loci, utilizing a pseudovirus with a mutant integrase for integration-deficient lentivirus, and employing polycistronic elements for stable expression.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Length of moving object

If traditional gene editing methods are used to knock in large DNA fragments, then the size of DNA that can be inserted is limited, but the efficiency of knock-in decreases significantly for large genes

Engineering Contradiction:

Improvesize of DNA fragmentVSAvoidknock-in efficiency

Core Design Contradiction:

Length of moving objectVSProductivity

Solution Approach 1:

The donor template is designed with specific structural elements (homology arms, payload, cleavage sites) that are processed in a coordinated manner. The nuclease system processes multiple cleavage sites to generate appropriate ends for integration, segmenting the complex integration process into manageable steps that facilitate efficient knock-in of large DNA fragments

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

A donor template with homology arms serves as an intermediary structure that mediates between the nuclease-cleaved genomic DNA and the large DNA fragment to be inserted. The homology arms provide binding specificity while the payload carries the large genetic information, enabling efficient integration of large DNA fragments

Inventive Principle:

Principle #24Intermediary (Mediator)

2Reliability

If genes are knocked in using traditional methods, then integration can be achieved, but long-term stable high expression is not maintained due to cellular silencing mechanisms

Engineering Contradiction:

Improveexpression stabilityVSAvoidduration of transgene expression

Core Design Contradiction:

ReliabilityVSDuration of action of stationary object

Solution Approach 1:

The donor template is designed with specific structural elements (homology arms, payload, cleavage sites) that are processed in a coordinated manner. The nuclease system processes multiple cleavage sites to generate appropriate ends for integration, segmenting the complex integration process into manageable steps that facilitate efficient knock-in of large DNA fragments

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

A donor template with homology arms serves as an intermediary structure that mediates between the nuclease-cleaved genomic DNA and the large DNA fragment to be inserted. The homology arms provide binding specificity while the payload carries the large genetic information, enabling efficient integration of large DNA fragments

Inventive Principle:

Principle #24Intermediary (Mediator)

3Reliability

If GMP-grade materials like AAV are used for knock-in procedures, then manufacturing quality is improved, but production cost increases significantly

Engineering Contradiction:

Improvemanufacturing qualityVSAvoidproduction cost

Core Design Contradiction:

ReliabilityVSQuantity of substance

Solution Approach 1:

The invention uses a disposable donor template system that can be designed and synthesized at lower cost compared to GMP-grade AAV vectors. The template is used once for integration and then discarded, eliminating the need for expensive viral production, purification, and quality control processes while maintaining integration efficiency

Inventive Principle:

Principle #27Cheap short-living objects (Disposable)

Solution Approach 2:

The invention replaces the complex biological system of viral transduction (AAV) with a simpler biochemical system based on nuclease-mediated cleavage and homologous recombination. This substitution eliminates multiple steps in the viral production chain, reducing cost while maintaining manufacturing quality through precise control of the molecular biology reactions

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

This approach significantly enhances the efficiency of knocking in large DNA fragments, achieves stable long-term expression, and reduces costs by using GMP-compatible reagents, improving viability and expression levels in mammalian cells.

Implementation Method 1

one or more cleavage sites comprising nucleotide sequences, wherein the nucleotide sequences can be bound or cleaved by a nuclease

Methodology Applied

Scientific EffectNuclease cleavage: Enzyme

Implementation Method 2

one or more homology arms comprising nucleotide sequences, wherein the nucleotide sequences are substantially identical to at least one locus in a genome

Methodology Applied

Scientific EffectHomologous recombination: Chemical Bonding

Data Source

PatentUS20240018493A1Knock-in of large DNA for long-term high genomic expression

Publication Date: 2024.01.18 THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIV

AI summary

The present disclosure provides compositions, systems, and methods for genome editing, efficient knock-in of large DNA fragments, and long-term, stable, high expression of integrated transgenes. Also provided are modified cells, vaccines comprising modified cells, and methods of using such cells to induce an immune response.