CRISPR Editing of CD34+ Cells for Stable FOXP3 Expression
Solution Overview
Problem
Current methods for expressing and regulating FOXP3 in primary human lymphocytes are unstable, particularly under inflammatory conditions, which can lead to the conversion of regulatory T cells into pro-inflammatory cells, exacerbating autoimmune diseases.
Innovation Solution
The use of CRISPR/Cas systems, including specific guide RNAs and AAV donor templates, to edit the genome of CD34+ cells for constitutive or regulated FOXP3 expression, ensuring stable integration and long-term engraftment of FOXP3-expressing cells, thereby providing a therapeutic approach for autoimmune diseases like IPEX syndrome.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If peripheral regulatory T cells (pTregs) are used for therapy, then the cells can be generated in the periphery, but FOXP3 expression is unstable under inflammatory conditions leading to conversion to pro-inflammatory cells
Solution Approach 1:
The patent applies preliminary action by performing genome editing of the FOXP3 gene in T cells before the cells are deployed for therapy. Specifically, the invention uses CRISPR/Cas9 to insert a functional FOXP3 coding sequence into the T cell genome in advance, ensuring that stable FOXP3 expression is established before the cells encounter inflammatory conditions in the patient. This preemptive genetic modification prevents the instability and phenotypic conversion that would occur if using unmodified pTregs.
Solution Approach 2:
The patent applies copying by creating a functional copy of the FOXP3 gene through CRISPR/Cas9-mediated insertion of a functional FOXP3 coding sequence into the T cell genome. This copied gene serves as a stable template that continuously produces functional FOXP3 protein, replacing the unstable endogenous FOXP3 expression in pTregs and ensuring long-term regulatory function throughout the cell's lifespan.
2Reliability
If thymic regulatory T cells (tTregs) are used, then stable FOXP3 expression is achieved, but the cells require thymic development and may not be optimal for certain therapeutic applications
Solution Approach 1:
The patent applies the taking out principle by extracting the essential functional element (FOXP3 coding sequence) from thymic development and incorporating it directly into peripheral T cells through CRISPR/Cas9 editing. Instead of requiring T cells to develop in the thymus where FOXP3 expression is naturally stable, the invention extracts and inserts the functional FOXP3 gene into mature peripheral T cells, achieving stable expression without requiring thymic development. This enables therapeutic applications where thymic-derived cells are not suitable.
3Ease of manufacture
If lentiviral gene transfer of FOXP3 is used, then FOXP3 expression can be restored in T cells, but the expression is not stable under inflammatory conditions
Solution Approach 1:
The patent applies copying by using CRISPR/Cas9 to insert a functional FOXP3 coding sequence directly into the T cell genome, creating a permanent genetic copy that is replicated and expressed stably throughout the cell's life. This genomic integration approach replaces the transient expression provided by lentiviral vectors, ensuring that the copied FOXP3 gene maintains stable expression even under the stress of inflammatory conditions, thereby resolving the instability issue associated with viral vector-based therapies.
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 enables efficient editing and sustained expression of FOXP3 in human hematopoietic stem cells, offering a promising therapy for autoimmune diseases by stabilizing regulatory T cell function and preventing conversion to pro-inflammatory phenotypes.
Implementation Method 1
The use of CRISPR/Cas systems, including specific guide RNAs and AAV donor templates, to edit the genome of CD34+ cells for constitutive or regulated FOXP3 expression
Implementation Method 2
a guide RNA (gRNA) comprising a spacer sequence that is complementary to a sequence within a FOXP3 gene, AAVS1 locus, or a TRA gene in a CD34+ cell
Implementation Method 3
a DNA endonuclease or nucleic acid encoding the DNA endonuclease; In some embodiments, the DNA endonuclease is a Cas9
Implementation Method 4
a donor template comprising a nucleic acid sequence encoding a FOXP3 or a functional derivative thereof
Implementation Method 5
In some embodiments, the donor template is encoded in an adeno-associated virus (AAV) vector
Implementation Method 6
Expression of FOXP3 in edited CD34+ cells... providing constitutive or regulated FOXP3 expression in the edited CD34+ cells or cells derived therefrom, such as T cells
Implementation Method 7
to provide constitutive or regulated FOXP3 expression in the edited CD34+ cells
Data Source
AI summary
Aspects of the invention described herein concern the incorporation of a FOXP3 cDNA (e.g., full-length human codon-optimized cDNA) into a FOXP3 gene or a non-FOXP3 locus so as to provide constitutive or regulated FOXP3 expression in a primary human CD34+ cells or cells derived from edited CD34+ cells. In some embodiments, guide RNA sequences that are directed to FOXP3, AAVS1, or other candidate loci are used for CRISPR/Cas9-mediated gene regulation, and gene delivery cassettes for HDR based gene-modification are provided.