Circular DNA Donor for Targeted Genome Editing in hPSCs

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Solution Overview

Problem

Current genome editing protocols for human pluripotent stem cells (hPSCs) are limited by low editing frequencies, necessitating the development of optimal strategies for achieving high donor concentrations in larger cell populations while maintaining safety, especially for therapeutic applications.

Innovation Solution

A complex comprising a circular DNA molecule with a backbone sequence and an insertion sequence flanked by nucleic acid sequences with homology to a genomic sequence of interest, combined with guide RNAs (gRNAs) and an effector molecule like Cas-Clover, which facilitates targeted nucleic acid insertion and increased nuclear localization of the DNA molecule.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If conventional genome editing protocols are used for hPSCs, then the basic editing function is achieved, but the editing frequency is low

Engineering Contradiction:

Improveediting frequencyVSAvoidediting efficiency

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The circular DNA molecule is divided into distinct functional segments: a backbone sequence for nuclear localization, insertion sequences for genomic integration, and flanking homology arms for targeted recombination. This segmentation allows each component to perform its specific function efficiently, thereby increasing overall editing frequency and reliability

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The circular DNA molecule serves as an intermediary carrier that delivers the insertion sequence to the genome. By using this intermediate vector with specific structural features (backbone, insertion sequences, homology arms), the editing process achieves higher frequency and reliability than direct DNA delivery methods

Inventive Principle:

Principle #24Intermediary (Mediator)

2Productivity

If higher donor concentrations are introduced into cell populations, then the editing frequency increases, but cell toxicity increases

Engineering Contradiction:

Improveediting frequencyVSAvoidcell toxicity

Core Design Contradiction:

ProductivityVSObject-affected harmful factors

Solution Approach 1:

The circular DNA molecule is designed with localized functional regions: the backbone sequence is optimized for nuclear localization, the insertion sequences are positioned for specific genomic targets, and the homology arms are configured for controlled recombination. This local optimization allows efficient editing at target sites without the toxicity associated with high concentrations of foreign DNA throughout the cell

Inventive Principle:

Principle #3Local quality

Solution Approach 2:

The patent modifies key parameters of the DNA molecule structure (circular topology, sequence composition, homology arm length) to enhance editing efficiency. By changing these structural parameters, the system achieves high editing frequency with reduced toxicity compared to conventional linear plasmid vectors

Inventive Principle:

Principle #35Parameter changes

3Adaptability or versatility

If larger gene fragments and amounts of donor DNA are introduced, then the editing capability is enhanced, but the safety and functional response are compromised

Engineering Contradiction:

Improvegene insertion capabilityVSAvoidsafety and functional response

Core Design Contradiction:

Adaptability or versatilityVSReliability

Solution Approach 1:

The circular DNA is segmented into a backbone sequence for safe delivery and multiple insertion sequences for targeted integration. This segmentation allows the system to handle larger gene fragments through multiple controlled insertion events rather than single uncontrolled events, maintaining safety and functional response reliability

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The circular DNA molecule is pre-designed with specific structural features (backbone, insertion sequences, homology arms) before introducing it into cells. This preliminary configuration ensures that when the DNA enters the cell, it automatically directs its integration to safe genomic locations, preventing off-target effects and maintaining functional safety

Inventive Principle:

Principle #10Preliminary action

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 frequency of targeted nucleic acid insertions in hPSCs, achieving higher yields of modified cells with reduced toxicity, thereby improving the efficiency and safety of genome editing for therapeutic applications.

Implementation Method 1

a DNA donor template can be provided, which the cell can use via endogenous DNA damage repair pathways to introduce modifications ranging from single-base-pair substitutions to large insertions through homology-directed repair (HDR)

Methodology Applied

Scientific EffectHomology-directed repair:

Implementation Method 2

at least one effector molecule comprising a fusion peptide, wherein the fusion peptide comprises (i) an inactivated Cas9 (dCas9) or an inactivated nuclease domain thereof, (ii) a Clo051 or a nuclease domain thereof

Methodology Applied

Scientific EffectNuclease activity: Enzyme

Data Source

PatentUS20250171809A1Genetically modified cells and methods of use thereof

Publication Date: 2025.05.29 POSEIDA THERAPEUTICS INC

AI summary

Disclosed are methods and compositions for obtaining cells (e.g. T cells, iPSCs cells, NK cells) and derivative cells with stable and functional genetic insertions at selected sites. Also provided are cell populations or clonally differentiated cell derived from modified cells, which comprise targeted integration of one or more exogenous polynucleotides, and/or indels in one or more selected gene loci.