Guide-Binding Expression Vector for Single-Step Gene Editing Control
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Solution Overview
Problem
Current methods for expressing an exogenous polypeptide in a cell and simultaneously disrupting endogenous gene expression are cumbersome and inefficient, particularly in applications like CAR T cell therapy, where sequential steps result in diminishing returns.
Innovation Solution
An expression vector with a guide-binding sequence complementary to a nucleic acid guide, allowing simultaneous or concurrent control of exogenous polypeptide expression and endogenous gene disruption through a single nucleic acid guide and gene editing tool, such as Cas9, enabling co-editing in a single step.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If sequential steps are used for CAR integration and endogenous gene silencing, then each step can be performed with dedicated optimization, but the overall process becomes slow and inefficient with diminishing returns
Solution Approach 1:
The patent combines CAR integration and endogenous gene silencing into a single simultaneous step by incorporating a guide-binding sequence into the expression vector. This allows the nucleic acid guide to direct gene editing at both the integration site and endogenous gene loci concurrently, eliminating sequential processing steps and overcoming the contradiction between reliability and productivity.
Solution Approach 2:
The expression vector is designed with multi-functionality, serving both as a carrier for the CAR gene and as a template for guide RNA production. The vector simultaneously enables CAR expression and guides CRISPR-Cas9-mediated silencing of endogenous genes, allowing one tool to perform multiple functions and resolve the efficiency-speed contradiction.
2Manufacturing precision
If only a small fraction of cells efficiently express CAR and show effective silencing in sequential steps, then purification can be performed, but the fraction of successfully modified cells diminishes with each step
Solution Approach 1:
By merging CAR integration and gene silencing into a single simultaneous step, the patent ensures that cells receiving the expression vector undergo both modifications together. This prevents loss of cells between sequential purification steps and maintains a higher fraction of successfully modified cells while achieving the desired purity.
Solution Approach 2:
The expression vector is designed to simultaneously provide CAR gene integration and generate guide RNA for silencing before cell purification. This preliminary combined action ensures that both modifications are established in the same cells prior to selection, preventing diminishment of the successful cell fraction.
3Quantity of substance
If constitutive expression is used for the exogenous polypeptide, then expression levels are high, but control over expression timing and conditions is lost
Solution Approach 1:
The patent implements dynamic control of polypeptide expression by placing the coding sequence under the control of an inducible promoter rather than a constitutive promoter. This allows expression levels to be adjusted and timing to be controlled based on therapeutic needs, resolving the contradiction between high expression levels and controllability.
Solution Approach 2:
The use of inducible promoters allows dynamic parameter changes in gene expression based on external signals or cellular conditions. This enables the system to maintain low baseline expression and induce high expression only when needed, achieving both quantity control and operational ease.
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
Enables precise and efficient control of exogenous polypeptide expression and endogenous gene silencing in a single step, improving the efficacy of therapies like CAR T cell therapy by enhancing the fraction of cells expressing the desired polypeptide and silencing unwanted genes.
Implementation Method 1
a guide-binding sequence located upstream of the coding sequence, wherein the guide-binding sequence comprises a sequence complementary to a nucleic acid guide; wherein binding of a nucleic acid guide to the guide-binding sequence directs a mutation
Implementation Method 2
The mutation initiated by the nucleic acid guide is a frameshift mutation which shifts the coding sequence of the polypeptide of interest into frame with a start codon, resulting in expression of the polypeptide of interest
Data Source
AI summary
Provided herein is an expression vector comprising a coding nucleic acid sequence encoding a polypeptide of interest, wherein the polypeptide of interest is not constitutively expressed from the vector; and a guide-binding sequence located upstream of the coding sequence, wherein the guide-binding sequence comprises a sequence complementary to a nucleic acid guide; wherein binding of a nucleic acid guide to the guide-binding sequence directs a mutation in a nucleic acid sequence of the vector resulting in expression of the polypeptide of interest. Also provided are a combination comprising the expression vector and a nucleic acid guide, a cell comprising the expression vector and/or nucleic acid guide and associated medical methods and uses.