Primary Cell Genome Editing With AAV Donor and Modified sgRNA
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Solution Overview
Problem
Existing genome editing methods in hematopoietic stem cells (HSCs) fail to achieve high frequencies of edited cells post-transplantation, despite efficient editing in vitro, due to challenges in achieving stable gene modifications and homologous recombination.
Innovation Solution
A method involving the use of modified single guide RNA (sgRNA), CRISPR-associated protein (Cas) polypeptide, and a homologous donor adeno-associated viral (AAV) vector to induce stable gene modification via homologous recombination in primary cells, enhancing targeted integration and gene correction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If genome editing is performed in hematopoietic stem cells using conventional methods, then editing can be achieved in vitro, but the frequency of edited cells remains low after transplantation
Solution Approach 1:
The patent modifies the sgRNA by introducing chemical modifications (2'-O-methyl and phosphorothioate) to change its physical and chemical parameters, thereby improving stability and reducing degradation. This resolves the contradiction by enhancing the durability of the editing machinery in primary cells while maintaining high editing frequency
Solution Approach 2:
The patent uses a composite delivery system combining AAV vectors with chemically modified sgRNA and Cas9 protein. This composite approach leverages the long-term expression capability of AAV while incorporating stable modified nucleic acids, achieving both high editing frequency and reliable stable gene modification post-transplantation
2Manufacturing precision
If homologous recombination is used for precise genome editing, then precise nucleotide changes can be made, but the efficiency is low in primary cells
Solution Approach 1:
The patent changes the chemical parameters of sgRNA by introducing 2'-O-methyl and phosphorothioate modifications, which enhance the stability and binding affinity of the guide RNA. This improvement in sgRNA parameters increases the efficiency of homologous recombination in primary cells while maintaining precise nucleotide editing capability
Solution Approach 2:
The patent uses AAV vectors as an intermediary delivery system to efficiently transport the editing components into primary cells. The AAV vector serves as a mediator that facilitates high-efficiency delivery of the modified sgRNA and Cas9 protein, thereby increasing homologous recombination efficiency while preserving precision
3Ease of manufacture
If conventional sgRNA is used for CRISPR/Cas9 targeting, then the system can be easily engineered, but off-target effects occur
Solution Approach 1:
The patent modifies the chemical parameters of sgRNA by introducing 2'-O-methyl and phosphorothioate modifications at specific positions. These parameter changes enhance the specificity of target recognition while maintaining the ease of system engineering, thereby reducing off-target effects without compromising the simplicity of CRISPR/Cas9 system construction
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 leads to stable and efficient gene modification in primary cells, enabling long-term multi-lineage reconstitution and disease correction in transplanted subjects, with improved editing frequencies and reduced off-target effects.
Implementation Method 1
Target identification relies on RNA-DNA Watson-Crick hybridization between a 20-nucleotide stretch of the sgRNA and the DNA target site, which then guides Cas9 to cleave both DNA strands
Implementation Method 2
CRISPR/Cas9 consists of the Cas9 endonuclease and a 100-nucleotide (nt) single guide RNA (sgRNA)... which then guides Cas9 to cleave both DNA strands
Implementation Method 3
DSB formation subsequently triggers one of two highly conserved competing repair mechanisms, canonical non-homologous end-joining (NHEJ) or homologous recombination (HR)... genome editing by HR requires the delivery of a donor molecule to serve as an undamaged DNA molecule that the HR machinery uses to repair the break by a 'copy and paste' method
Data Source
AI summary
In certain aspects, the present invention provides methods for inducing a stable gene modification of a target nucleic acid via homologous recombination in a primary cell, such as a primary blood cell and/or a primary mesenchymal cell. In certain other aspects, the present invention provides methods for enriching a population of genetically modified primary cells having targeted integration at a target nucleic acid. The methods of the present invention rely on the introduction of a DNA nuclease such as a Cas polypeptide and a homologous donor adeno-associated viral (AAV) vector into the primary cell to mediate targeted integration of the target nucleic acid. Also provided herein are methods for preventing or treating a disease in a subject in need thereof by administering to the subject any of the genetically modified primary cells or pharmaceutical compositions described herein to prevent the disease or ameliorate one or more symptoms of the disease.