Sequential IDLV Donor Delivery for Hematopoietic Gene Editing
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Solution Overview
Problem
Current methods for gene editing in hematopoietic stem and progenitor cells face challenges such as low gene transfer efficiency, limited proficiency of homology directed DNA repair (HDR), and high activity of error-prone non-homologous end joining (NHEJ), which affect the safety and efficacy of genetic modification therapies.
Innovation Solution
The use of an Integration-Defective Lentiviral Vector (IDLV) combined with cyclosporin H (CsH) and a p53 inhibitor, along with an adenoviral protein, enhances transduction and gene editing efficiencies in hematopoietic cells by reducing DNA load and preserving clonogenic capacity, thereby improving HDR-mediated gene editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If viral vectors are used for gene transfer in hematopoietic stem cells, then gene transfer efficiency is improved, but DNA damage and genotoxicity increase
Solution Approach 1:
The patent divides the gene transfer process into two separate steps: first delivering the donor template DNA using an integration-defective lentiviral vector (IDLV), then inducing double-strand breaks with nucleases. This segmentation prevents simultaneous integration of large viral genomes that cause DNA damage, while still achieving efficient gene editing through the sequential process.
Solution Approach 2:
The patent extracts the integrase function from the lentiviral vector by using an integration-defective variant (IDLV). This removal of the integration capability eliminates the genotoxic effects associated with random genomic integration while preserving the vector's ability to efficiently deliver the donor template DNA to the target cells.
2Manufacturing precision
If homology directed repair (HDR) is enhanced for precise gene editing, then manufacturing precision is improved, but productivity decreases due to low HDR proficiency in HSCs
Solution Approach 1:
The patent performs preliminary delivery of the donor template DNA using IDLV before inducing the double-strand break with nucleases. This preliminary action ensures that the repair template is already present in the cell when the break occurs, maximizing HDR efficiency and precision without requiring multiple rounds of transduction or prolonged culture that would reduce productivity.
Solution Approach 2:
The patent uses an integration-defective lentiviral vector as an intermediary delivery system for the donor template DNA. This intermediary approach overcomes the low HDR proficiency of HSCs by efficiently delivering the template through viral transduction, which then serves as the substrate for HDR when nucleases create the break, achieving both precision and productivity.
3Object-affected harmful factors
If DNA load is reduced to improve safety, then object-affected harmful factors decrease, but gene transfer efficiency worsens
Solution Approach 1:
The patent segments the gene editing components into separate delivery steps: donor template DNA is delivered first via IDLV at low multiplicity of infection, then nucleases are introduced separately. This segmentation allows each component to be delivered at optimal concentrations without the cumulative DNA load of simultaneous delivery, reducing genotoxicity while maintaining high gene transfer efficiency for each step.
Solution Approach 2:
The patent extracts the integrase function from the viral vector system, creating an integration-defective lentiviral vector. This extraction prevents the formation of large integrated viral genomes that cause DNA damage, while the vector retains sufficient capacity to efficiently deliver the donor template DNA sequence needed for gene editing.
Data Source
AI summary
Use of a combination of (a) cyclosporin H (CsH) or a derivative thereof, and (b) a p53 inhibitor and/or an adenoviral protein, or one or more nucleotide sequences encoding therefor, for increasing the efficiency of gene editing of an isolated population of cells when transduced by a viral vector and/or increasing the efficiency of transduction of an isolated population of cells by a viral vector.