Lipid Nanoparticle Delivery for Multiplex T Cell Genome Editing

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Solution Overview

Problem

Current methods for gene editing in cells, particularly for multiplex editing in immune cells like T cells, face challenges such as reduced cell survival, increased translocations, and decreased efficiency due to cumulative toxicity, making it difficult to achieve multiple genetic edits while maintaining cell viability and phenotype.

Innovation Solution

The use of lipid nucleic acid assembly compositions, such as lipid nanoparticles (LNPs), for safer and more efficient delivery of genome editing tools, allowing for simultaneous or sequential administration to perform multiple genome edits with reduced toxicity and improved cell survival and expansion.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If sequential electroporation editing is used to perform multiple genome edits, then gene editing capability is improved, but cell survival and viability deteriorate due to cumulative toxicity

Engineering Contradiction:

Improvegene editing capabilityVSAvoidcell survival

Core Design Contradiction:

Adaptability or versatilityVSReliability

Solution Approach 1:

Lipid nanoparticles serve as an intermediary delivery vehicle for CRISPR/Cas9 components, replacing the direct electroporation method. The LNPs protect the nucleic acid components during delivery and facilitate their entry into cells through a less toxic mechanism, thereby maintaining gene editing capability while significantly improving cell survival rates after multiple editing events

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The invention changes the delivery parameter from electroporation (electrical field application) to lipid nanoparticle-mediated delivery (chemical/biological mechanism). This parameter change fundamentally alters the toxicity profile while maintaining editing efficiency, allowing sequential edits without cumulative toxicity

Inventive Principle:

Principle #35Parameter changes

2Productivity

If multiple CRISPR/Cas9 edits are performed simultaneously, then editing efficiency is improved, but translocation frequency increases impairing product quality and safety

Engineering Contradiction:

Improveediting efficiencyVSAvoidtranslocation frequency

Core Design Contradiction:

ProductivityVSObject-generated harmful factors

Solution Approach 1:

The invention segments the delivery of multiple CRISPR/Cas9 components into separate lipid nanoparticle formulations, each carrying specific components (e.g., one LNP for Cas9 mRNA, another for sgRNA). This segmentation allows controlled, staged delivery that maintains high editing efficiency while reducing the simultaneous DNA breaks that cause translocations

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The lipid nanoparticles are pre-formulated with optimized compositions and delivery characteristics before use. This preliminary preparation ensures that the CRISPR components are delivered in a controlled manner that minimizes off-target effects and chromosomal translocations while maintaining high on-target editing efficiency

Inventive Principle:

Principle #10Preliminary action

3Adaptability or versatility

If traditional electroporation methods are used for cell engineering, then genetic modification capability is improved, but manufacturing complexity and time increase

Engineering Contradiction:

Improvegenetic modification capabilityVSAvoidmanufacturing complexity

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

The invention replaces the mechanical/electrical electroporation system with a chemical/biological lipid nanoparticle delivery system. This substitution eliminates the need for complex electroporation equipment and optimization parameters, simplifying the manufacturing process while maintaining genetic modification capability

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Solution Approach 2:

The lipid nanoparticle platform serves multiple functions: it protects nucleic acid components, facilitates cellular uptake, enables controlled release, and can be formulated for different cell types. This multi-functionality consolidates multiple process steps into a single delivery mechanism, reducing overall manufacturing complexity

Inventive Principle:

Principle #6Universality (Multi-functionality)

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 enhances the efficiency of multiplex genome editing in T cells, increasing cytokine production, maintaining a favorable memory phenotype, and promoting continued proliferation, thereby improving the quality and yield of engineered cells for therapeutic applications.

Implementation Method 1

The cell is contacted with at least a first lipid nanoparticle (LNP) composition and a second LNP composition... wherein the first LNP composition comprises a first guide RNA (gRNA) directed to a first target sequence and optionally a genome editing tool

Methodology Applied

Scientific EffectEndocytosis:

Data Source

PatentUS20230183753A1Methods of in Vitro Cell Delivery

Publication Date: 2023.06.15 INTELLIA THERAPEUTICS INC

AI summary

Compositions and methods for multiplex delivery and gene editing in vitro are provided.