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Publication Number
US-11268089-B2
Publication Date
2022-03-08
Expiration Date
Abstract
The present application relates to a system for designing promoters for selective expression of genes. Thereby identified transcription regulatory elements are selected according to a specific methodology and used to create a library of transcription regulatory elements, which are then used to construct specific promoters, especially tissue-specific promoters.
Core Innovation
The invention relates to a promoter-construction platform that derives transcription factor regulatory elements (TFREs) from functional genomics datasets by associating each TFRE with genes identified as being differentially expressed in a particular cell type or tissue type or under a particular condition. TFRE selection is performed based on proximity to the differentially expressed genes and a SYN value larger than 0.3, where the SYN value is defined as frequency (1/length), with frequency representing the frequency of occurrence of the TFRE within 20 kilobases of any of the plurality of genes and length representing the length in nucleotides of the TFRE.
The platform builds a vector comprising a combined promoter cassette having randomly combined elements and a minimum promoter, together with a reporter gene. The randomly combined elements are selected TFREs that satisfy the stated sequence-identity, proximity, and SYN constraints, and the resulting order of the combined elements does not occur in nature.
The constructs are defined to include isolated promoter and genetic construct embodiments in which the combined promoter cassette is linked to a heterologous gene and associated with host cells, enabling measurement of promoter activity using reporter gene readouts. The documented embodiments include libraries of randomly ligated TFRE elements cloned upstream of a minimum promoter and reporter gene, with vector embodiments including plasmid and viral vectors, including self-inactivating retroviral and adenoviral libraries.
Claims Coverage
The independently stated claim defines a vector with a combined promoter cassette comprising randomly combined TFRE elements and a minimum promoter, together with a reporter gene, where each element satisfies sequence-identity, proximity, and SYN-value constraints, and where the element order does not occur in nature. Dependent claims further refine reporter gene choice, orientation of TFRE frequency calculation, exclusion of additional promoter or regulatory sequences, plurality of vectors, and a cancer condition.
Combined promoter cassette with randomly combined TFRE elements and minimum promoter
A vector comprising a combined promoter cassette comprising randomly combined elements and a minimum promoter, and a reporter gene.
TFRE elements defined by sequence identity, proximity, and SYN threshold
Each element contains more than 80% sequence identity with a TFRE that is within 20 kilobases of more than fifty percent of a plurality of genes identified as being differentially expressed in a particular cell type or tissue type or under a particular condition and has a SYN value larger than 0.3, where SYN is defined as frequency (1/length).
Naturally non-occurring order of combined elements
The order of the combined elements does not occur in nature.
Reporter gene embodiment
The reporter gene is selected from LacZ or GFP.
Sense-strand definition for TFRE frequency calculation within 20 kilobases
The TFRE frequency of occurrence within 20 kilobases is determined on the sense strand.
Exclusion of additional promoters or regulatory sequences
The vector includes only the promoter-cassette promoter/regulatory elements and does not contain any other promoter or regulatory sequences.
Vector library embodiment as a plurality of vectors
The claim covers a vector library made up of multiple vectors as defined in the claim.
Cancer condition for differential expression
The disease condition targeted is cancer.
Overall claim coverage centers on vectors having a combined promoter cassette with randomly combined TFRE elements and a minimum promoter, where TFRE elements match via more than 80% sequence identity, lie within 20 kilobases of differentially expressed genes meeting a more than 50% proximity criterion, and yield a SYN value larger than 0.3 under the frequency(1/length) metric, while the element order is constrained to not occur in nature. Dependent claims further specify reporter gene embodiments, orientation-specific frequency definition, exclusion of other promoter/regulatory sequences, plural-vector library form, and a cancer disease-condition framework.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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