Electroporation Buffer System for T Cell Polynucleotide Delivery
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Solution Overview
Problem
Non-viral genome editing for targeted insertion into T cells faces challenges due to severe cellular toxicity and low delivery efficiency caused by electroporation-mediated DNA delivery, particularly in human T cells.
Innovation Solution
A new buffer system comprising succinate, mannitol, glucose, glutamine, and an antioxidant is developed to facilitate the delivery of polynucleotides into cell nuclei with reduced toxicity, including the use of specific concentrations of sodium succinate, mannitol, glucose, L-alanyl-L-glutamine, and sodium pyruvate, and applying a pulse of electricity in an aqueous solution with controlled osmotic pressure to enhance nuclear uptake.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If electroporation-mediated DNA delivery is used to achieve non-viral genome editing, then virus-free and fast reprogramming of T cells is achieved, but severe cellular toxicity occurs
Solution Approach 1:
The patent applies parameter changes by optimizing the electroporation buffer composition with specific concentrations of succinate (5-30 mM), mannitol (1-30 mM), glucose (3-30 mM), glutamine (50-800 mg/L), and antioxidants (0.1-0.6 mM sodium pyruvate). These parameter optimizations reduce cellular toxicity while maintaining delivery effectiveness, directly resolving the contradiction between achieving virus-free reprogramming and avoiding severe cellular toxicity.
2Ease of manufacture
If electroporation-mediated DNA delivery is used to achieve non-viral reprogramming, then lentivirus and AAV preparation are circumvented, but delivery efficiency into human T cells remains low
Solution Approach 1:
The patent improves delivery efficiency through parameter changes in buffer composition, specifically optimizing osmotic pressure (using 1-30 mM mannitol and 3-30 mM glucose), pH buffering capacity (5-30 mM succinate), and cellular metabolism support (50-800 mg/L glutamine). These optimized parameters enable efficient polynucleotide delivery into human T cells without requiring lentivirus or AAV preparation, thus resolving the contradiction between manufacturing simplicity and delivery efficiency.
3Quantity of substance
If conventional electroporation buffer is used, then DNA delivery can be performed, but cell viability is significantly reduced
Solution Approach 1:
The patent introduces an intermediary buffer system comprising succinate, mannitol, glucose, glutamine, and antioxidants that mediates between the electroporation process and the cells. This intermediary buffer protects cells from electroporation-induced damage by providing metabolic support and reducing oxidative stress, thereby enabling effective polynucleotide delivery while maintaining high cell viability.
Solution Approach 2:
The patent applies beforehand cushioning by incorporating antioxidants (0.1-0.6 mM sodium pyruvate) and metabolic substrates (glutamine, glucose, succinate) into the electroporation buffer before the electroporation process. These components cushion the cells against the harmful effects of electroporation in advance, protecting cell viability while allowing effective DNA delivery.
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
The buffer system significantly reduces cellular toxicity and enhances the efficiency of polynucleotide delivery into the nuclei of T cells, leading to improved gene editing and CAR-T cell generation with increased tumor killing activity and viability, surpassing conventional methods in both in vitro and in vivo applications.
Implementation Method 1
applying a pulse of electricity to a sample comprising the polynucleotide and the cell in an aqueous solution with controlled osmotic pressure to enhance nuclear uptake
Data Source
AI summary
Provided are compositions and methods for effective delivery of polynucleotides into the nuclei of cells through electroporation. A new buffer system is provided that facilitates the delivery with greatly reduced cell toxicity. The buffer may include succinate, mannitol, a sugar, glutamine or an analog, and an antioxidant. Also provided are methods that are particularly suitable for delivering an RNA or protein into a cell by electrophoresis in a solution having an osmotic pressure greater than 310 mOsmol/kg.