US-20260125691-A1 - Tcd34 And Other Markers For Identification And Sorting Of Cells And For Use As In Vivo Tracking Assistant

US20260125691A1US 20260125691 A1US20260125691 A1US 20260125691A1US-20260125691-A1

Abstract

Use of one or more markers to identify and sort cells that have been engineered to comprise a synthetic chromosome encoding the marker(s), with said cells expressing said marker(s) encoded on the synthetic chromosome. The marker is typically but not limited to a cluster of differentiation (CD), wherein the CD has been truncated or otherwise modified so that the biological function of the CD is lost or remarkably reduced. The modified CD is typically used to sort cells to obtain a desired population of cells containing the synthetic chromosome, or i) to ensure that a cell population administered to a subject, e.g., suffering from cancer, reaches the target tissue, ii) to ensure that a cell population administered to a subject, e.g., suffering from cancer, stays at the desired site within the body of the subject during a treatment regimen, iii) to make it possible to turn on a kill switch comprised in the synthetic chromosomes of the cells of the cell population if the cell population is present at undesired sites in the body, or iv) to use as a diagnostic tool.

Inventors

Edward L PERKINS
Julia REMNESTÅL
Amy L GREENE
Dominique Broccoli
Kara Pascarelli MANNE
Ola Winqvist
John Andersson
Marton KESZEI
Katarina LYBERG
Maria EKOFF

Assignees

CARRYGENES BIOENGINEERING, LLC

Dates

Publication Date: 20260507
Application Date: 20220826

Claims (15)

1 . A synthetic chromosome comprising a nucleic acid sequence encoding a surface marker.
2 . A synthetic chromosome according to claim 1 , wherein the surface marker is a cluster of differentiation.
3 . A synthetic chromosome according to claim 2 , wherein the cluster of differentiation is truncated.
4 . A synthetic chromosome according to any one of the preceding claims , wherein the surface marker, when expressed by a cell i) contains the antigen epitope, and ii) does not induce signaling in the cell, and/or iii) does not bind to the putative ligand.
5 . A synthetic chromosome according to any one of the preceding claims , wherein the surface marker is select from CD34, trCD34, CD20, trCD20, CD19, trCD19, CD14, and trCD14.
6 . A synthetic chromosome according to any one of the preceding claims , wherein the surface marker is trCD34.
7 . A synthetic chromosome according to any one of the preceding claims comprising two or more nucleic acid sequences encoding two or more surface markers, wherein the surface markers may be the same or different.
8 . A synthetic chromosome according to any one of the preceding claims comprising one or more inducible promotors independently controlling expression of one or more surface markers.
9 . A synthetic chromosome according to any one of the preceding claims comprising one or more insulators.
9 . A synthetic chromosome according to any one of the preceding claims for use in identifying and/or sorting cells expressing a surface marker.
10 . A synthetic chromosome according to any one of the preceding claims for use in in vivo tracking of cells expressing a surface marker.
11 . A cell comprising a synthetic chromosome as defined in any one of the preceding claims .
12 . A cell according to any of the preceding claims for medical use, veterinary use, or diagnostic use.
13 . A composition comprising a synthetic chromosome as defined in any one of claim 1-10 and an additive.
14 . A composition comprising a cell as defined in claim 11 or 12 and an additive.

Description

FIELD OF THE INVENTION The present invention relates to the use of one or more markers to identify and sort cells that have been engineered to comprise a synthetic chromosome encoding the marker(s), with said cells expressing said marker(s) encoded on the synthetic chromosome. The marker is typically but not limited to a cluster of differentiation (CD), wherein the CD has been truncated or otherwise modified so that the biological function of the CD is lost or remarkably reduced. The modified CD is typically used to sort cells to obtain a desired population of cells containing the synthetic chromosome. The present invention also relates to the use of tCD34 and other markers that are controllably expressed by cells comprising synthetic chromosomes. The markers are used i) to ensure that a cell population administered to a subject, e.g., suffering from cancer, reaches the target tissue, ii) to ensure that a cell population administered to a subject, e.g., suffering from cancer, stays at the desired site within the body of the subject during a treatment regimen, iii) to make it possible to turn on a kill switch comprised in the synthetic chromosomes of the cells of the cell population if the cell population is present at undesired sites in the body, or iv) to use as a diagnostic tool. BACKGROUND OF THE INVENTION In chromosome-based cell therapies it is vital to have the possibility to identify and enrich the cell population for chromosome carrying cells. Cell surface proteins may be used to isolate a specific cell type by using a unique epitope of said cell surface protein to label the cell and separate labelled cells from nonlabelled cells (lacking the cell surface protein epitope). SUMMARY OF THE INVENTION Specific embodiments of the invention appear from the appended claims, wherein 1. A synthetic chromosome comprising a nucleic acid sequence encoding a surface marker.2. A synthetic chromosome according to claim 1, wherein the surface marker is a cluster of differentiation.3. A synthetic chromosome according to claim 2, wherein the cluster of differentiation is truncated.4. A synthetic chromosome according to any one of the preceding claims, wherein the surface marker, when expressed by a celli) contains the antigen epitope, andii) does not induce signaling in the cell, and/oriii) does not bind to the putative ligand.5. A synthetic chromosome according to any one of the preceding claims, wherein the surface marker is select from CD34, trCD34, CD20, trCD20, CD19, trCD19, CD14, and trCD14.6. A synthetic chromosome according to any one of the preceding claims, wherein the surface marker is trCD34.7. A synthetic chromosome according to any one of the preceding claims comprising two or more nucleic acid sequences encoding two or more surface markers, wherein the surface markers may be the same or different.8. A synthetic chromosome according to any one of the preceding claims comprising one or more inducible promotors independently controlling expression of one or more surface markers.9. A synthetic chromosome according to any one of the preceding claims comprising one or more insulators.9. A synthetic chromosome according to any one of the preceding claims for use in identifying and/or sorting cells expressing a surface marker.10. A synthetic chromosome according to any one of the preceding claims for use in in vivo tracking of cells expressing a surface marker.11. A cell comprising a synthetic chromosome as defined in any one of the preceding claims.12. A cell according to any of the preceding claims for medical use, veterinary use, or diagnostic use.13. A composition comprising a synthetic chromosome as defined in any one of claim 1-10 and an additive.14. A composition comprising a cell as defined in claim 11 or 12 and an additive. DETAILED DESCRIPTION OF THE INVENTION Herein is presented a synthetic chromosome-based strategy, wherein a synthetic chromosome encodes one or more markers that can be used to sort and identify cells carrying the chromosome and/or as an in vivo tracking assistant. The methods described herein may employ, unless otherwise indicated, conventional techniques and descriptions of molecular biology (including recombinant techniques), cell biology, biochemistry, and cellular engineering technology, all of which are within the skill of those who practice in the art. Such conventional techniques include oligonucleotide synthesis, hybridization and ligation of oligonucleotides, transformation and transduction of cells, engineering of recombination systems, creation of transgenic animals and plants, and human gene therapy. Specific illustrations of suitable techniques can be had by reference to the examples herein. However, equivalent conventional procedures can, of course, also be used. Such conventional techniques and descriptions can be found in standard laboratory manuals such as Genome Analysis: A Laboratory Manual Series (Vols. I-IV) (Green, et al., eds., 1999); Genetic Variation: A Lab