Cell surface tag exchange (CSTE) system for tracing and manipulation of cells during recombinase mediated cassette exchange integration of nucleic acid sequences to engineered receiver cells

1 . A combined system comprising two separate components,

wherein a first component is a tag-exchange donor vector (TEDV) encoding a first cell surface tag (CST) exon flanked by a 3′ intron fragment and a gene in an antisense orientation, the TEDV containing no promoter sequences, and a second component is an engineered cell containing within its genome a tag-exchange receiver site (TERS) encoding a second CST exon, which is different from the first CST exon and adjoined by a full intron sequence to an exon encoding a transmembrane domain, and also encoding a reporter gene in an antisense orientation,

wherein paired recombinase mediated cassette exchange (RMCE) elements are encoded by each of the TEDV and TERS such that execution of RMCE between the TEDV and TERS results in exchange of the reporter gene for the gene encoded by the TEDV, and exchange of the first CST exon for the second CST exon, generating a derivative engineered cell, wherein the derivative engineered cell expresses the first CST exon and gene, in place of the second CST exon and the reporter gene.

2 . The combined system according to claim 1 , wherein the TEDV comprises:

a first RMCE element;

a 3′ intron fragment;

the first CST exon;

a first transcriptional terminator;

a second transcriptional terminator;

the gene;

a Kozak sequence; and

a second RMCE element,

wherein the first CST exon and first transcriptional terminator are encoded in an antisense orientation from the gene and associated transcriptional terminator and Kozak sequences.

3 . The combined system according to claim 2 , wherein the second component is a TERS comprising:

a transcriptional promoter element;

a Kozak sequence;

a transmembrane domain exon;

the full intron sequence;

a first RMCE element encoded within the intron sequence;

the second CST exon;

a first transcriptional terminator;

a second transcriptional terminator; a reporter gene;

a Kozak sequence;

a second RMCE element; and

a second transcriptional promoter element,

wherein the transmembrane domain exon and the second CST exon are encoded in an antisense orientation from the reporter gene, such that the first transcriptional promoter element drives transcription of both the transmembrane domain and the second CST exon, and the second transcriptional promoter element drives transcription of the reporter gene.

4 . The combined system according to claim 1 , wherein the TERS comprises:

a transcriptional promoter element; a Kozak sequence;

a transmembrane domain exon;

the full intron sequence;

a first RMCE element encoded within the full intron sequence;

the second CST exon;

a first transcriptional terminator;

a second transcriptional terminator a reporter gene;

a Kozak sequence;

a second RMCE element; and

a second transcriptional promoter element,

wherein the transmembrane domain exon and the second CST exon are encoded in an antisense orientation from the reporter gene, such that the first transcriptional promoter element drives transcription of both the transmembrane domain and the second CST exon, and the second transcriptional promoter element drives transcription of the reporter gene.

5 . The combined system according to claim 1 , wherein the first RMCE element of the TEDV is paired with the first RMCE element of the TERS, and the second RMCE element of the TEDV is paired with the second RMCE element of the TERS.

6 . The combined system according to claim 1 , wherein each CST exon comprises a sequence encoding one or more molecular affinity tags, wherein the CST exons encoded by the TEDV and TERS are different.

7 . The combined system according to claim 1 , wherein the engineered cell contains a single TERS in its genome.

8 . A method for generating derivative engineered cells expressing a TEDV-encoded gene from a TERS, said method comprising,

generating a TEDV encoding a first CST exon and a gene and containing no promoter sequences;

delivering said TEDV to an engineered cell line containing a paired TERS encoding a second CST exon and also encoding a reporter gene in an antisense orientation, along with a recombinase enzyme, wherein the recombinase enzyme mediates cassette exchange between the matching RMCE elements encoded in the TEDV and the TERS;

contacting at least one cell of the engineered cell line with two or more affinity reagents specific for both the TEDV-encoded CST exon and the TERS-encoded CST exon;

selecting at least one derivative engineered cell on the basis of diminished expression of the reporter gene and TERS-encoded CST exon, and increased expression of the TEDV-encoded CST exon.

9 . The method according to claim 8 , wherein the affinity reagents are fluorescently labelled to detect diminished expression of TERS-encoded CST exon and increased expression of TEDV-encoded CST exon, to enable cell partitioning and selection based on said expression by way of florescence activated cell sorting.

10 . The method according to claim 8 , wherein the affinity reagents are immobilised on a substrate such that cells expressing TERS-encoded CST exon may be depleted, or cells expressing TEDV-encoded CST exon may be enriched, in a target cell population using substrate affinity methods.

11 . A method for generating multiple derivative engineered cells expressing a range of TEDV-encoded gene, from a pool of TEDVs, said method comprising,

generating a library of two or more TEDVs, each encoding a unique gene sequence, each with a unique TEDV-encoded CST exon, and each containing no promoter sequences; delivering said library of TEDVs as a pool to an engineered cell line containing a paired TERS encoding a unique CST exon and also encoding a reporter gene in an antisense orientation, along with a recombinase enzyme, wherein the recombinase enzyme mediates cassette exchange between matching RMCE elements encoded in the TEDVs and TERS; contacting cells with three or more affinity reagents specific for both the multiple TEDV-encoded CST exons and the TERS-encoded CST exon;

selecting at least one derivative engineered cell on the basis of diminished expression of the reporter gene and TERS-encoded CST exon, and increased expression of each of the unique TEDV-encoded CST exons.

12 . A method for cell lineage tracing of derivative engineered cells expressing a range of TEDV-encoded gene, within a pool of engineered cells generated by generating a library of two or more TEDVs, each encoding a unique gene sequence, each with a unique TEDV-encoded CST exon, and each containing no promoter sequences and delivering said library of TEDVs as a pool to an engineered cell line containing a paired TERS encoding a unique CST exon and also encoding a reporter gene in antisense orientation, along with a recombinase enzyme, wherein the recombinase enzyme mediates cassette exchange between matching RMCE elements encoded in the TEDVs and the TERS, said method comprising:

contacting cells with two or more affinity reagents specific for the multiple TEDV-encoded CST exons; and

analyzing the TEDV-encoded genes of derivative engineered cells on the basis of expression of each of the unique TEDV-encoded CST exons.

13 . A tag-exchange donor vector (TEDV) encoding a cell surface tag (CST) exon flanked by a 3′ intron fragment, a gene in an antisense orientation, and containing no promoter sequences.

14 . The tag-exchange donor vector (TEDV) according to claim 13 , comprising:

a first RMCE element;

a 3′ intron fragment;

a CST exon;

a first transcriptional terminator;

a second transcriptional terminator;

a gene;

a Kozak sequence; and

a second RMCE element,

wherein the CST exon and first transcriptional terminator are encoded in an antisense orientation from the gene and associated transcriptional terminator and Kozak sequences.

15 . An engineered cell containing within its genome a tag-exchange receiver site (TERS), encoding a cell surface tag (CST) exon adjoined by a full intron sequence to an exon encoding a transmembrane domain, and also encoding a reporter gene in an antisense orientation, wherein

recombinase mediated cassette exchange (RMCE) elements are included in the TERS such that execution of RMCE between the TERS and a tag-exchange donor vector (TEDV) containing no promoter sequences results in exchange of the reporter gene for a gene encoded by the TEDV.

16 . The engineered cell according to claim 15 , wherein said TERS comprises:

a first transcriptional promoter element;

a Kozak sequence;

a transmembrane domain exon; an intron;

a first RMCE element; a CST exon;

a first transcriptional terminator;

a second transcriptional terminator; a reporter gene;

a Kozak sequence;

a second RMCE element; and

a second transcriptional promoter element,

wherein the transmembrane domain exon and CST exon are encoded in an antisense orientation from the reporter gene, such that the first transcriptional promoter element drives transcription of the combined transmembrane domain and CST exon, and the second transcriptional promoter element drives transcription of the reporter gene.