Primary B Cell Genome Editing via Nucleoplasmin-Mediated Donor DNA Delivery

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Solution Overview

Problem

Current methods for genome editing in primary human B cells are inefficient, toxic, and lack selectivity, with low efficiency and high off-target effects due to the use of plasmids and lentiviral vectors, which are not suitable for achieving homologous recombination in these cells.

Innovation Solution

The use of RNA- and protein-based transfection with designer nucleases such as zinc finger nucleases, TALEN, homing endonucleases, and CRISPR/Cas systems, combined with recombinant adeno-associated virus (AAV) vectors for efficient delivery of donor DNA templates, to facilitate homologous recombination and prevent somatic hypermutation, enabling the selective editing and expansion of B cells into long-lived plasma cells.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Ease of manufacture

If plasmid-based methods are used for DNA delivery to primary B cells, then the process is simple to implement, but the efficiency is extremely low and toxicity is high

Engineering Contradiction:

Improveease of implementationVSAvoidtransfection efficiency

Core Design Contradiction:

Ease of manufactureVSProductivity

Solution Approach 1:

The patent uses nucleoplasmin protein as an intermediary carrier to deliver donor DNA into the nucleus of primary B cells. This mediator approach achieves high transfection efficiency (>30%) while maintaining low toxicity, resolving the contradiction between ease of implementation and productivity by providing a biocompatible delivery mechanism that is both simple to use and highly effective

Inventive Principle:

Principle #24Intermediary (Mediator)

2Reliability

If lentiviral vectors are used for gene delivery, then integration into the genome is achieved, but selectivity is poor and off-target effects are high

Engineering Contradiction:

Improvegenomic integrationVSAvoidoff-target effects

Core Design Contradiction:

ReliabilityVSObject-affected harmful factors

Solution Approach 1:

The patent employs site-specific nucleases (CRISPR-Cas9, TALENs, or homing endonucleases) to create targeted double-strand breaks at specific genomic loci, enabling precise local modification. Donor DNA is delivered to these specific sites via nucleoplasmin-mediated transport, achieving high selectivity and minimal off-target effects while maintaining reliable genomic integration through homology-directed repair at the targeted site

Inventive Principle:

Principle #3Local quality

3Productivity

If random integration methods are used, then gene delivery is efficient, but oncogenic potential increases

Engineering Contradiction:

Improvegene delivery efficiencyVSAvoidoncogenic risk

Core Design Contradiction:

ProductivityVSObject-affected harmful factors

Solution Approach 1:

The patent performs preliminary targeting by using site-specific nucleases to create double-strand breaks at predetermined safe harbor loci before delivering donor DNA. This preliminary action guides the integration process to occur only at these pre-selected, safe genomic locations, maintaining high delivery efficiency while eliminating random integration and associated oncogenic risks through controlled, site-specific homology-directed repair

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 achieves high efficiency in genome editing, with over 30% of primary human B cells undergoing homologous recombination and minimal off-target effects, allowing for the production of engineered plasma cells that can be used for therapeutic protein expression with enhanced safety and efficacy.

Implementation Method 1

Methodology Applied

Scientific EffectHomologous recombination:

Implementation Method 2

recombinant adeno-associated virus (AAV) vectors for efficient delivery of donor DNA templates

Methodology Applied

Scientific EffectViral transduction:

Implementation Method 3

RNA- and protein-based transfection to facilitate delivery of candidate designer nucleases

Methodology Applied

Scientific EffectTransfection:

Data Source

PatentUS20240287452A1Engraftable cell-based immunotherapy for long-term delivery of therapeutic proteins

Publication Date: 2024.08.29 SEATTLE CHILDRENS HOSPITAL (DBA SEATTLE CHILDRENS RES INST)

AI summary

The present application relates to plasma cells and plasma cell precursors that express a macromolecule, such as a protein, protein mimetic or a peptide and compositions comprising these plasma cells or plasma cell precursors. The application further relates to methods of using and making the plasma cells and plasma cell precursors that express the macromolecule. Methods of treatment comprising administering the plasma cells or plasma cell precursors are also contemplated.