CRISPR HDR Gene Editing in Hematopoietic Cells
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Solution Overview
Problem
Current methods for genetic modification of cells, particularly mammalian cells like human hematopoietic cells, face challenges in achieving efficient and precise correction of genomic mutations associated with genetic diseases using homology-directed repair (HDR).
Innovation Solution
The use of a CRISPR-Cas system in conjunction with HDR-promoting agents and expansion agents to facilitate targeted gene editing in hematopoietic cells. This approach involves contacting hematopoietic cells with a CRISPR-Cas system, a guide RNA, and a template polynucleotide to induce double-stranded breaks and promote the integration of corrective DNA sequences through HDR.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If CRISPR-Cas system is used for targeted gene editing in hematopoietic cells, then gene editing precision is improved, but HDR efficiency deteriorates
Solution Approach 1:
The patent employs small molecule compounds that modify cellular parameters to enhance HDR efficiency. Specifically, compounds like SCR7 (inhibitor of DNA ligase IV) and NU7441 (inhibitor of DNA-PK) alter the DNA repair pathway parameters to favor HDR over NHEJ, while compounds like SR1 and UM171 modify cell cycle parameters to prolong the S/G2 phases where HDR is active, thereby resolving the contradiction between precise CRISPR editing and low HDR efficiency in hematopoietic cells
Solution Approach 2:
The patent introduces small molecule compounds as intermediary substances that mediate between the CRISPR-Cas editing system and the cellular HDR machinery. These compounds act as chemical mediators that modulate the cellular environment to promote HDR, bridging the gap between the precise but inefficient CRISPR system and the low-efficiency HDR pathway in hematopoietic cells
2Productivity
If HDR-promoting agents are used to increase HDR efficiency, then gene editing efficiency is improved, but cell viability deteriorates
Solution Approach 1:
The patent applies partial action by using transient exposure to HDR-promoting compounds during the critical editing window, rather than continuous treatment. The compounds are added at specific time points around electroporation and removed after a defined period, providing sufficient HDR promotion while limiting cytotoxic exposure, thus achieving high editing efficiency without severe viability loss
Solution Approach 2:
The patent implements beforehand cushioning by pre-treating cells with expansion-promoting compounds like SR1 and UM171 before and during the HDR-enhancing treatment. These compounds cushion the cells against the cytotoxic effects of HDR-promoting agents by promoting cell survival and expansion, allowing high editing efficiency to be achieved without compromising overall cell viability
3Reliability
If expansion agents are used to maintain cell population, then cell viability is improved, but editing precision deteriorates
Solution Approach 1:
The patent applies preliminary action by expanding the cell population with SR1 and UM171 before CRISPR electroporation to ensure sufficient starting material and robust cell health. This preliminary expansion establishes a viable cell base that can withstand the subsequent editing process, preventing viability loss while maintaining precision through proper cell state preparation
Solution Approach 2:
The patent uses periodic action by alternating between expansion phases (with SR1/UM171) and editing phases (with HDR promoters) in a timed sequence. Cells are expanded for a defined period, then subjected to CRISPR electroporation with HDR promoters for a shorter period, then returned to expansion conditions. This periodic cycling maintains viability while concentrating editing events during the appropriate window, preserving precision
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 method achieves high efficiency in HDR-mediated gene editing and maintains high viability in edited cell populations, particularly in human hematopoietic stem cells, making it suitable for therapeutic applications in correcting genetic diseases.
Implementation Method 1
the sequence-specific nuclease activity of CRISPR/Cas systems
Implementation Method 2
homology-directed repair (HDR), enabling targeted integration of sequences from a template polynucleotide at a target sequence
Implementation Method 3
a guide RNA (gRNA) comprising a nucleotide sequence that hybridizes to a target DNA in the genome
Data Source
AI summary
Provided herein are methods and compositions for genetically engineering a cell (e.g., a hematopoietic cell) using CRISPR/Cas systems and homology-directed repair, genetically engineered cells produced by such methods, and methods involving administering such genetically engineered cells to a subject, such as a subject having a genetic disease.