Method for Engineering Synthetic Cis-Regulatory DNA
OwnerGaetano Gargiuloinventor
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Abstract
The invention relates to methods for generating cell-type specific expression cassettes and reporter vectors, as well as nucleic acid constructs that can be generated by such methods. The cell-type specific expression cassettes and reporter vectors are characterized synthetic cis-regulatory DNA, also termed synthetic locus regions (sLCRs). sLCRs allow for a cell-type specific expression of reporter or effector genes. The invention further relates to various uses of the reporter vectors, including the determination of a property of a cell, preferably a cell type, state or fate transition, in gene and viral therapy, drug discovery or validation.
Claims21
A method for generating a cell-type specific expression cassette, comprising the steps of:
- a) Providing a gene expression profile of a cell type of interest,
- b) Providing genomic sequence data of said cell type of interest,
- c) Selecting a set of signature genes from the gene expression profile, wherein said signature genes are (i) differentially regulated compared to a reference cell type or (ii) selected according to a gene expression level,
- d) Identifying genes encoding a transcription factor within the set of signature genes selected in c),
- e) Determining a set of genomic regions from the genomic sequence data, wherein each genomic region comprises a sequence encoding a signature gene identified in c) and additional genomic sequence adjacent to the sequence encoding said signature gene,
- f) Identifying multiple genomic sub-regions of comparable and limited size, preferably equal size, within the set of genomic regions determined in e), wherein said genomic sub-regions comprise one or more binding sites for one or more of the transcription factors identified in d),
- g) Selecting a minimal set of genomic sub-regions from those determined in f), wherein the set of genomic sub-regions is selected to comprise transcription factor binding sites for a predetermined percentage of all transcription factors identified in d), and
- h) Generating a cell-type specific expression cassette comprising the set of genomic sub-regions selected in step g) operably coupled with a reporter or effector gene, wherein the genomic sub-regions are configured to regulate the expression of said reporter or effector gene.
The cell-type specific reporter vector, comprising a synthetic regulatory region comprising 2 to 10 genomic sub-regions of 100 bp to 1000 bp, positioned adjacently, without a linker or with a linker sequence of less than 100 bp positioned between said sub-regions, wherein said sub-regions originate from separate and non-adjacent locations in the same genome of a cell type, wherein the sub-regions cumulatively comprise binding sites for at least 5 transcription factors, and
- a reporter or effector gene,
- wherein the genomic sub-regions are operably coupled with the reporter or effector gene to regulate the expression of said reporter or effector gene.
(canceled)
Dependencies are inferred from the claim text.