Non-viral B Cell Engineering via Plasmid Delivery
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Solution Overview
Problem
Current methods for genetic modification of B cells, such as those using recombinant adeno-associated viral vectors (rAAV), are limited by small cargo capacity, high production costs, long turnaround times, and immune responses leading to rapid clearance of transduced cells.
Innovation Solution
The use of non-viral based methods, specifically introducing plasmids, nanoplasmids, or mini-circles into B cells to edit their genome using nuclease-dependent cleavage systems like CRISPR/Cas, allowing for the insertion of larger therapeutic DNA templates and overcoming the limitations of viral vectors.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If recombinant adeno-associated viral vectors (rAAV) are used for genetic modification of B cells, then delivery of therapeutic DNA template is achieved, but cargo capacity is limited to 4.7 kb
Solution Approach 1:
The patent extracts the viral vector component from the gene delivery system and replaces it with non-viral alternatives (plasmids, nanoplasmids, mini-circles). This removes the 4.7 kb cargo capacity constraint inherent to rAAV while maintaining the essential function of delivering therapeutic DNA templates to B cells for genome editing.
2Productivity
If rAAV vectors are used for B cell modification, then gene delivery is achieved, but production cost is high and turnaround time is long
Solution Approach 1:
The patent employs disposable non-viral vectors (plasmids, nanoplasmids, mini-circles) that are inexpensive to produce and do not require complex viral manufacturing processes. These vectors can be rapidly prepared and used for B cell transfection, dramatically reducing both production cost and turnaround time compared to rAAV vector production.
3Reliability
If rAAV vectors are used for B cell transduction, then gene delivery is achieved, but immune response leads to rapid clearance of transduced cells
Solution Approach 1:
The patent converts the harmful immune response to viral vectors into a benefit by using non-viral vectors that do not trigger the same immune reactions. The plasmid, nanoplasmid, and mini-circle vectors avoid viral-associated immune recognition, allowing transduced B cells to persist longer in the patient without being rapidly cleared by the immune system, thereby improving treatment reliability.
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 achieves high engineering efficiencies, with over 15% gene editing efficiency and greater than 60% B cell viability after 3 days, enabling the use of genome-edited B cells as therapeutics for various conditions without the drawbacks of viral vector methods.
Implementation Method 1
a targeting site for a nuclease dependent cleavage system targeting molecule
Implementation Method 2
The gene of interest integrates into the B cell genome via homology directed repair (HDR), homology-mediated end joining (HMEJ) or a combination of HDR/HMEJ
Data Source
AI summary
The present disclosure relates, in general, to non-viral methods for generating engineered B cell, and use of such B cells as cell-based therapeutics to treat disease.