Linear Donor Constructs for Targeted Genomic Integration

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Solution Overview

Problem

Current methods for targeted integration into genomic sequences, such as those using zinc finger nucleases, rely on circular plasmid constructs with long homology arms, which are time-consuming to construct and can stably insert into host cells, whereas there is a need for shorter, linear exogenous polynucleotides that resist exonuclease degradation and facilitate efficient targeted integration.

Innovation Solution

The development of linear exogenous nucleic acids with homology arms of 50-100 base pairs, modified with phosphorothioate phosphodiester bonds to resist degradation, which are integrated into the genome using zinc finger nucleases or meganucleases for targeted double-strand cleavage and recombination.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If circular plasmid constructs with long homology arms are used for targeted integration, then integration efficiency is improved, but construction time increases and stable insertion of donor molecules occurs

Engineering Contradiction:

Improveintegration efficiencyVSAvoidconstruction time

Core Design Contradiction:

ReliabilityVSLoss of time

Solution Approach 1:

The donor molecule is segmented into two separate oligonucleotides (first and second oligonucleotides) that each contain portions of the homology arms and the sequence of interest. These segmented oligonucleotides are then ligated together to form the complete linear donor molecule, thereby reducing construction time while maintaining integration efficiency

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

Instead of using circular plasmid constructs as the traditional donor format, the invention inverts the approach by using linear oligonucleotide-based donor molecules. This inversion eliminates the stable insertion problem associated with circular plasmids while maintaining targeted integration capability through homology-directed repair

Inventive Principle:

Principle #13The other way round (Inversion)

2Reliability

If circular plasmid constructs are used for targeted integration, then integration efficiency is improved, but donor molecule stability in host cells worsens due to stable insertion

Engineering Contradiction:

Improveintegration efficiencyVSAvoiddonor molecule stability

Core Design Contradiction:

ReliabilityVSStability of the object's composition

Solution Approach 1:

The invention inverts the traditional circular plasmid donor format to use linear oligonucleotide-based donor molecules. This inversion prevents stable insertion of the donor molecule into the host genome while maintaining targeted integration efficiency through homology-directed repair mechanisms

Inventive Principle:

Principle #13The other way round (Inversion)

Solution Approach 2:

The linear oligonucleotide donor molecules are designed to be transient and disposable, serving their purpose in facilitating targeted integration and then being naturally degraded by cellular exonucleases. This prevents long-term stable insertion of donor molecules while maintaining integration efficiency

Inventive Principle:

Principle #27Cheap short-living objects (Disposable)

3Loss of time

If linear exogenous polynucleotides are used for targeted integration, then construction time is reduced and stable insertion is minimized, but resistance to exonuclease degradation must be achieved

Engineering Contradiction:

Improveconstruction timeVSAvoidexonuclease degradation

Core Design Contradiction:

Loss of timeVSObject-affected harmful factors

Solution Approach 1:

The oligonucleotides are synthesized with composite chemical structures including phosphorothioate modifications at terminal positions. This composite material approach combines natural phosphodiester bonds in the interior with chemically modified phosphorothioate bonds at the ends, providing exonuclease resistance while maintaining ligation efficiency and integration capability

Inventive Principle:

Principle #40Composite materials

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 allows for rapid and efficient targeted integration of exogenous sequences into specific genomic locations, reducing construction time and minimizing stable insertion of donor molecules into host cells, while maintaining stability and functionality of the integrated sequences.

Implementation Method 1

the linear donor molecule is modified to resist exonucleolytic cleavage, for example by placing one or more phosphorothioate phosphodiester bonds between one or more base pairs on the ends of the donor molecule

Methodology Applied

Scientific EffectPhosphorothioate modification:

Implementation Method 2

Integration of the exogenous nucleic acid sequences into the genome is facilitated by targeted double-strand cleavage of the genome (chromosome) in the region of interest

Methodology Applied

Scientific EffectTargeted cleavage:

Implementation Method 3

Attempts have been made to alter genomic sequences in cultured cells by taking advantage of the natural phenomenon of homologous recombination

Methodology Applied

Scientific EffectHomologous recombination:

Data Source

PatentUS9376685B2Linear donor constructs for targeted integration

Publication Date: 2016.06.28 SANGAMO THERAPEUTICS INC

AI summary

Disclosed herein are linear donor molecules comprising homology arms of 50-750 base pairs (e.g., 50-100 base pairs) flanking one or more sequences of interest. The donor molecules and/or compositions comprising these molecules can be used in methods for targeted integration of an exogenous sequence into a specified region of interest in the genome of a cell.