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Principal Investigator: Xiaojing J Gao
Organization: STANFORD UNIVERSITY
Fiscal Year: 2024
Award: $231,600
Funding agency: National Institute of Biomedical Imaging and Bioengineering
Abstract
There is a critical need for RNA sensors in living mammalian cells. With the advent of single-cell RNA
sequencing, the transcriptome of any cell type is readily obtainable if not already available. In contrast, we are
still in urgent need for a universal method to act on such transcriptomic information. If we can genetically express
arbitrary effector proteins in specific cell types according to their transcriptional markers, we would transform
large swaths of basic research and biomedical applications, such as immunology, neuroscience, and cancer
therapy. In addition, we would like such sensors to be programmable and operate at the post-transcription level.
One promising use case that would benefit from such sensors is cancer ablation, using an approach
dubbed “circuits as medicine”, where a genetic vector encoding an entire “circuit” (metaphor for a collection of
biomolecules engineered to regulate each other and implement specific functions) is delivered intracellularly.
The circuit will sense the cellular states based on hallmarks of cancer (i.e., the overexpression of specific RNAs
or the presence of specific mutations), process the signals, and deliver specific therapeutic payloads accordingly
in cancer cells, directly killing them while educating the immune system to search and destroy other cancer cells.
Previous efforts largely relied on strand displacement, a successful strategy for nucleic acid-based signal
processing outside cells. However, their functionality has remained inadequate inside living mammalian cells,
most likely because the double-stranded RNA (dsRNA) formed during strand displacement signals viral infection
and are actively engaged by mammalian proteins in the immune pathways. We hypothesize that, because it is
impossible to evade the omnipresent dsRNA-interacting proteins, it is wiser to embrace them. In this proposal,
we will leverage endogenous human enzymes that recognize and specifically edit dsRNA, to create sensors that
can be programmed to respond to arbitrary RNA transcripts (“triggers”).
First, we will use fast design-build-test cycles in vitro to optimize sensor performance. We will focus on
increasing sensor output in response to triggers by engineering the sensor configuration and its sequence choice,
and we will characterize how the sensor affects and is affected by the cellular context. Second, to enable the
quantitative distinction of different trigger levels and the integration of multiple triggers, we will engineer
threshold-setting modifications and AND logic gates. Third, leveraging the unique post-transcriptional nature of
such sensors and gates, we will combine them with mRNA or an oncolytic RNA virus as delivery vectors, which
has traditionally been difficult to control. Last by not least, we will validate the performance and the therapeutic
potential of the sensors, gates, and the RNA vectors in cancer cell lines.
The future directions of the proposed project include continual optimization of the sensors, logic gates,
and vectors, testing them in more realistic cancer models including mouse models and patient-derived organoids,
and applying the tools to other fields.
Terms: <Ablation><Adenosine><Affect><Assay><Basic Research><Basic Science><Binding><Bioassay><Biological Assay><Biomedical Research><Cancer Detection><Cancer Model><Cancer Treatment><Cancer cell line><CancerModel><Cancers><Cell Body><Cell Communication and Signaling><Cell Signaling><Cells><Collection><DNA><Deoxyribonucleic Acid><Detection><Double-Stranded RNA><Engineering><Enzyme Gene><Enzymes><Exhibits><Future><Gene Transcription><Genes><Genetic Alteration><Genetic Change><Genetic Transcription><Genetic Vectors><Genetic defect><Goals><Guanine><Health Benefit><Human><Immune><Immune system><Immunes><Immunology><In Vitro><Inosine><Intervention><Intervention Strategies><Intracellular Communication and Signaling><Libraries><Logic><Malignant Cell><Malignant Neoplasm Therapy><Malignant Neoplasm Treatment><Malignant Neoplasms><Malignant Tumor><Mammalian Cell><Medicine><Messenger RNA><Metaphor><Methods><Mice><Mice Mammals><Modern Man><Modification><Molecular><Molecular Interaction><Murine><Mus><Mutation><Names><Nature><Neurosciences><Non-Polyadenylated RNA><Nucleic Acids><Oncolytic><Oncolytic viruses><Organism><Organoids><Outcome><Output><Pathway interactions><Patients><Performance><Printing><Process><Protein Secretion><Proteins><Public Health><RNA><RNA Expression><RNA Gene Products><RNA Viruses><RNA and protein interaction><RNA-Protein Interaction><Reporter><Reporting><Ribonucleic Acid><Signal Transduction><Signal Transduction Systems><Signaling><Specificity><Testing><Therapeutic><Transcript><Transcription><Translations><VSV><Variant><Variation><Vesicular Stomatitis Virus><Vesicular stomatitis Indiana virus><Viral><Viral Diseases><Viral Gene Products><Viral Gene Proteins><Viral Proteins><Viral Vector><Virus Diseases><Virus Replication><adenosine deaminase that acts on RNA><anti-cancer therapy><biological signal transduction><cancer biomarkers><cancer cell><cancer classification><cancer markers><cancer therapy><cancer virotherapy><cancer-directed therapy><cell type><clinical relevance><clinically relevant><conventional therapy><conventional treatment><dADAR><delivery vector><delivery vehicle><design><design,build,test><designing><differential expression><differentially expressed><double-stranded RNA-specific adenosine deaminase><dsRNA><dsRNA adenosine deaminase><dsRNA-specific adenosine deaminase><genome mutation><global gene expression><global transcription profile><improved><in vivo><interventional strategy><living system><mRNA><malignancy><mouse model><murine model><name><named><naming><neoplasm/cancer><oncolytic viral therapeutic><oncolytic viral therapy><oncolytic virotherapeutic><oncolytic virotherapy><oncolytic virus therapy><operation><operations><overexpress><overexpression><pathway><pharmacologic><plasmid vaccine><posttranscriptional><response><scRNA-seq><senescent cell><sensor><signal processing><single cell RNA-seq><single cell RNAseq><single cell expression profiling><single cell transcriptomic profiling><single-cell RNA sequencing><tool><transcriptional differences><transcriptome><transcriptomics><transfection vector><translation><vector><vector vaccine><viral infection><viral multiplication><viral replication><virus infection><virus multiplication><virus protein><virus-induced disease>