Base Editor Guide RNA for Antigen-Negative Cell Generation
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Solution Overview
Problem
Current therapies for acute myeloid leukemia (AML) targeting cell surface antigens like CD33, CD123, CD47, CD45, and CLL1 face challenges due to their expression on both cancerous and normal cells, leading to myelosuppression and adverse reactions, necessitating a method to reduce toxicity while maintaining therapeutic efficacy.
Innovation Solution
The development of a gene editing technology using specifically designed guide RNA sequences and a CRISPR-associated protein, nucleobase deaminase, and helper single-guide RNA to disrupt the expression of these antigens in cells, reducing their biological activity and minimizing off-target mutations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If therapies target cell surface antigens like CD33, CD123, CD47, CD45, and CLL1 in AML, then therapeutic efficacy is improved, but toxicity to normal myeloid cells increases
Solution Approach 1:
The patent applies local quality by creating a heterogeneous cell population where only leukemic cells retain surface antigen expression while normal myeloid cells are protected through selective pressure during therapy. This allows the therapeutic to act locally on tumor cells without affecting normal cells that have been edited to lack the target antigen.
Solution Approach 2:
The patent inverts the conventional approach by instead of trying to protect normal cells from antigen-targeted therapy, it edits normal cells to lack the antigen entirely, making them inherently resistant to therapy-induced toxicity while maintaining their normal function.
2Reliability
If Cas9 nuclease is used to disrupt CD33 gene in primary cells, then resistance to CD33-targeted therapy is achieved, but DNA double-strand breaks and cell death occur
Solution Approach 1:
The patent extracts the harmful DNA cleavage function from the CRISPR system by using base editors that lack nuclease activity. These base editors can still recognize and bind to the target DNA sequence through the guide RNA and Cas protein, but they only perform base substitution without causing double-strand breaks or triggering cell death pathways.
Solution Approach 2:
The patent substitutes the mechanical DNA cutting mechanism of Cas9 nuclease with a chemical base substitution mechanism. Instead of physically breaking the DNA backbone, the base editor uses a deaminase enzyme to chemically convert a cytosine base to uracil, which is then processed by cellular repair mechanisms to create a permanent C-to-T substitution without DNA damage responses.
3Reliability
If base editors are used to avoid DNA double-strand breaks, then cell viability is improved, but specificity is compromised due to random ssDNA formation
Solution Approach 1:
The patent introduces an intermediary mechanism where the guide RNA forms a stable RNA-DNA hybrid only at the precise complementary target sequence. This hybrid structure recruits the base editing machinery specifically to that location, preventing random off-target editing even though the deaminase can act on ssDNA. The guide RNA acts as a molecular address label that directs the editing activity to the correct genomic location.
Solution Approach 2:
The patent changes the parameter of enzyme activity regulation by designing base editors where the deaminase domain is activated only upon proper guide RNA targeting. The enzyme remains inactive or has very low activity until the guide RNA binds to the complementary DNA sequence, at which point the local structural changes activate the deaminase function specifically at the target site.
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 the generation of surface antigen-negative cells, reducing toxicity associated with AML therapies and widening the therapeutic index of treatment modalities, such as monoclonal antibodies and antibody-drug conjugates, by achieving high specificity and efficiency in base editing without inducing DNA double-strand breaks or unwanted nucleotide insertions.
Implementation Method 1
The combination of CRISPR-Cas9 and cytidine deaminases leads to cytosine base editors (CBEs) for programmable cytosine to thymine (C-T) substitution
Implementation Method 2
specifically designed and tested guide RNA sequences for improved base editors
Data Source
AI summary
Provided are gene editing technologies, including specifically designed and tested guide RNA sequences for improved base editors, useful for disrupting the expression of genes, such as CD33, CD123, CD47, CD45 and CLL1, in a cell. Such methods and edited cells are useful in reducing the toxicity associated with therapies targeting such cell surface antigens, such as those for treating acute myeloid leukemia.