Polycistronic miRNA Constructs for Checkpoint Silencing Specificity
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Solution Overview
Problem
Existing immune checkpoint inhibitor therapies, such as antibodies and gene editing, face challenges in consistency across cancer types, limited access to the tumor microenvironment, and increased complexity and cost, while polycistronic miRNA constructs risk unintended off-target gene silencing and recombination.
Innovation Solution
A polycistronic miRNA construct is designed with non-complementary pre-miRNA sequences separated by at least 10 nucleotides, targeting different genes or regions, and incorporating backbone sequences from naturally-occurring miRNAs to ensure appropriate folding and reduce RNAi toxicity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If multiple pre-miRNA structures are used in a polycistronic construct to achieve robust knockdown, then gene silencing effectiveness is improved, but the risk of alternate folding and off-target gene silencing increases
Solution Approach 1:
The patent divides the polycistronic construct into multiple independent pre-miRNA units, each targeting different genes or regions. By segmenting the construct, each pre-miRNA can be designed and optimized independently to avoid unintended interactions, reducing off-target effects while maintaining comprehensive gene silencing coverage.
Solution Approach 2:
The patent applies local quality by designing each pre-miRNA with specific structural characteristics tailored to its target. Each pre-miRNA unit has optimized stem-loop structures and guiding sequences customized for its specific target gene, ensuring precise and selective silencing without cross-reactivity with other genes.
2Reliability
If repeating pre-miRNA structures are used to achieve robust knockdown, then gene silencing effectiveness is improved, but recombination within the vector increases leading to impure vector population
Solution Approach 1:
The patent employs asymmetry by designing pre-miRNA structures with non-repeating, unique sequences and orientations. Each pre-miRNA unit has distinct structural features that prevent symmetric recombination events, thereby maintaining vector homogeneity and purity in the final product while preserving effective gene silencing.
3Adaptability or versatility
If gene editing is used to restrict checkpoint inhibitor blockade to engineered cells, then therapy specificity is improved, but manufacturing complexity and cost increase
Solution Approach 1:
The patent extracts the gene silencing function from complex gene editing systems and implements it through polycistronic miRNA constructs that can be delivered via simpler vectors. This extraction maintains therapy specificity by restricting miRNA expression to engineered cells while eliminating the need for complex CRISPR-Cas9 systems and associated manufacturing steps.
Solution Approach 2:
The patent uses transient, non-integrating miRNA constructs that provide temporary but sufficient gene silencing activity. This approach replaces permanent, complex gene editing with shorter-lived but simpler miRNA-based therapy, reducing manufacturing complexity while maintaining adequate therapeutic effect duration.
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
The construct effectively inhibits immune checkpoint proteins like CTLA4, CD70, PD-1, and TIGIT, enhancing T cell activity against cancer cells with reduced off-target effects and manufacturing complexity.
Implementation Method 1
MicroRNAs (miRNAs) are small non-coding RNA molecules that bind mRNA molecules produced from a targeted gene, affecting their translation to proteins. By such action, the miRNA silences the gene.
Data Source
AI summary
A ribonucleic acid comprising two non-natural pre-miRNA sequences, wherein each pre-miRNA sequence comprises a guide miRNA that inhibits the expression of an immune checkpoint protein. The pre-miRNA sequences may target a different gene or target a different regions of the same gene. A deoxyribonucleic acid encoding the aforementioned ribonucleic acid. The deoxyribonucleic acid may further encode a protein such as a chimeric antigen receptor, a cytokine, a cell tag, and/or an immune checkpoint inhibitor. A vector comprising the aforementioned ribonucleoic acid or the aforementioned deoxyribonucleic acid. A method for modifying the expression of a gene in a cell, wherein the method comprises introducing the aforementioned ribonucleic acid or the aforementioned deoxyribonucleic acid to the cell. A method for producing a genetically-engineered cell, wherein the method comprises introducing the aforementioned ribonucleic acid or the aforementioned deoxyribonucleic acid to the cell. A genetically-modified cell comprising the aforementioned ribonucleic acid or the aforementioned deoxyribonucleic acid. A composition comprising the aforementioned ribonucleic acid or the aforementioned deoxynbonucleic acid. A kit comprising the aforementioned ribonucleic acid or the aforementioned deoxyribonucleic acid. A method of treating a disease or disorder in a subject, comprising administering the aforementioned ribonucleic acid or the aforementioned deoxyribonucleic acid to the subject. A method of treating a disease or disorder in a subject, comprising administering the aforementioned cell to the subject. The use of the aforementioned ribonucleic acid or the aforementioned deoxyribonucleic acid in the manufacture of a medicament for modifying the expression of a gene. The use of the aforementioned ribonucleic acid or the aforementioned deoxyribonucleic acid in the manufacture of a medicament for the treatment of a disease or disorder in a subject.