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Publication Number
US-10851388-B2
Publication Date
2020-12-01
Expiration Date
Abstract
A method for expression of transcribable unit(s) in a target cell is provided. The method comprises the steps of: a) providing a target cell expressing a site-specific recombinase, b) providing a DNA vector characterized by a 5′ to 3′ vector sequence orientation. The DNA vector comprises a plurality of recombination units, wherein a single recombination unit comprises at least one transcribable unit and a first type and a second type of target site that are recognizable by the site-specific recombinase. Recombination can only occur between two target sites of the same type and the first type of target site is located at the 5′ start of the recombination unit and the second type of target site is located at the 3′ end of the recombination unit. For all recombination units comprised within the DNA vector, the orientation of all of the first type of target sites are the same, and the orientation of all of the second type of target sites are the same. Step c) comprises introducing the DNA vector into the target cell.
Core Innovation
The invention provides an in vivo method for determining phenotype alteration by a plurality of transcribable units in a non-human tissue of interest. The method administers to the tissue of interest in vivo a DNA expression vector in 5′ to 3′ orientation that comprises a plurality of recombination units, where a single recombination unit comprises at least one transcribable unit and a first and second target site both recognizable by a site-specific recombinase.
Within the DNA vector, recombination occurs only between two target sites of the same type, with the first target site located at the 5′ start site of each recombination unit and the second target site located at the 3′ end of the recombination unit. For all recombination units comprised within the DNA vector, the orientation of all first target sites is the same, and the orientation of all second target sites is the same.
After administration, the method harvests the tissue of interest, separates it into individual cells, and sorts the individual cells according to phenotype of interest. The method identifies the transcribable unit(s) expressed in each sorted individual cell and assays for the altered phenotype that results from expression of the transcriptional unit(s) in the individual cells.
The document also describes implementation using a tissue-selective transgene expression system with target cells expressing a site-specific recombinase such as Cre or FLP and a DNA vector containing multiple recombination units with uniform orientation of target sites. An example uses AAV9 delivery into adult rodents with Cre-inducible organ specificity, and another example uses multi-shRNA constructs for fatty liver disease, where FACS-sorted lipid-containing hepatocytes enable identification of shRNAs that reduce lipid accumulation.
Claims Coverage
The independent claim is clm-00001, which covers an in vivo phenotype-determination workflow that links SSR-driven recombination of a plurality of transcribable units to single-cell phenotype sorting and identification of expressed transcribable units. The independent claim includes core inventive design constraints on recombination and uniform orientation of target-site types across all recombination units, and dependent claims add constraints on unit plurality size, specific recombinases, viral vector types, an internal transcriptional terminator placement, and phenotype characterization features for sorting.
In vivo phenotype determination using SSR-responsive plurality of transcribable units
An in vivo method for determining phenotype alteration by a plurality of transcribable units in a non-human tissue of interest, including administering a DNA expression vector in 5′ to 3′ orientation with a plurality of recombination units to a non-human tissue of interest that expresses a site-specific recombinase, harvesting the tissue, separating into individual cells, sorting cells according to phenotype of interest, identifying transcribable unit(s) expressed in each sorted individual cell, and assaying for the altered phenotype resulting from expression of the transcriptional unit(s).
Same-type-only recombination with defined 5′ and 3′ target-site placement
For each recombination unit, recombination can only occur between two target sites of the same type, with the first target site located at the 5′ start site of the recombination unit and the second target site located at the 3′ end of the recombination unit.
Uniform orientation of first-type and second-type target sites across the vector
For all recombination units comprised within the DNA vector, the orientation of all first target sites are the same, and the orientation of all second target sites are the same.
Transcribable-unit plurality size constraint
The plurality of recombination units includes between 2 and 80 recombination units.
Site-specific recombinase selected as Cre or FLP
The site-specific recombinase is selected from Cre-recombinase or FLP.
Viral vector implementation for the DNA expression vector
The DNA expression vector is implemented as a viral vector derived from a selected virus among adeno-associated virus, adenovirus, lentivirus, retrovirus, and baculovirus.
Internal transcriptional terminator placement within recombination units
Each recombination unit in the DNA vector includes a transcriptional terminator positioned between the transcribable unit nearest the 3′ end and the second type of target site.
Phenotype sorting based on cellular features
A phenotype of interest is characterized by one or more cellular features including cell size, cell morphology, cell staining, or a cell marker.
Across the independent claim, the method couples an SSR-responsive plurality of recombination units on a DNA expression vector to single-cell phenotype sorting, followed by identification of expressed transcribable unit(s) per cell and assaying for resulting altered phenotype. Dependent claims refine the system by constraining the number of recombination units, selecting recombinases (Cre or FLP), specifying viral vector types, defining internal terminator placement, and specifying phenotype-characterization features used for sorting.
Stated Advantages
Enables determination of phenotype alteration based on which transcribable unit(s) are expressed in individually sorted cells.
Supports in vivo assessment of many transcribable units for phenotype variation through tissue harvest, phenotype-based single-cell sorting, identification of expressed transcribable units, and correlation to phenotypic outcomes.
Documented Applications
Fatty liver disease application in which FACS-sorted lipid-containing hepatocytes enable identification of shRNAs that reduce lipid accumulation.
Orthotropic disease gene identification using a ROSA26-based multi-transgene mouse system.
Organ-specific targeting example using AAV9 delivery into adult rodents with Cre-inducible organ specificity.
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