5' S/MAR APPLICATIONS

Abstract

The instant invention relates to a therapeutic cell comprising an episomal polynucleotide comprising a promoter and an expressible sequence, wherein said episomal polynucleotide further comprises an S/MAR element upstream of said promoter. The present invention further relates to expression constructs, polynucleotides, animal host cells, expression constructs, vectors, and/or polynucleotides comprising a cargo sequence related thereto.

Claims

1. A therapeutic cell comprising an episomal polynucleotide comprising a promoter and an expressible sequence, wherein said episomal polynucleotide further comprises an S/MAR element upstream of said promoter.

2. The therapeutic cell of claim 1, wherein said expressible sequence encodes a therapeutic gene product, preferably a therapeutic polypeptide.

3. The therapeutic cell of claim 1, wherein said expressible sequence encodes a therapeutic polypeptide, and wherein said therapeutic polypeptide is an antibody, a T Cell Receptor (TCR), a Chimeric Antigen Receptor (CAR), a cytokine, and/or a polypeptide lacking in cells affected with a genetic disease.

4. The therapeutic cell of claim 1, wherein said therapeutic polypeptide is a TCR.

5. The therapeutic cell of claim 1, wherein said therapeutic polypeptide is a CAR.

6. The therapeutic cell of claim 1, wherein said therapeutic polypeptide is a polypeptide lacking in cells affected with a genetic disease.

7. The therapeutic cell of claim 1, wherein said episomal polynucleotide further comprises a selectable marker gene.

8. The therapeutic cell of claim 1, wherein said S/MAR element comprises a nucleic acid sequence being at least 70% identical to SEQ ID NO:1.

9. The therapeutic cell of claim 1, wherein said S/MAR element comprises a nucleic acid sequence being at least 70% identical to SEQ ID NO:2.

10. The therapeutic cell of claim 1, wherein said S/MAR element comprises a nucleic acid sequence being at least 70% identical to SEQ ID NO:3.

11. The therapeutic cell of claim 1, wherein said therapeutic cell or host cell is a vertebrate cell, preferably a mammalian cell, more preferably a human cell.

12. An expression construct comprising an expressible sequence and a promoter, wherein said expression construct further comprises an S/MAR element upstream of said promoter, and wherein said expression construct replicates episomally in a mammalian cell.

13. A polynucleotide comprising an S/MAR element, wherein said S/MAR element comprises a nucleic acid sequence being at least 93%, preferably at least 95%, identical to SEQ ID NO:3.

14. The polynucleotide of claim 10, further comprising an expressible sequence and a promoter, preferably a mammalian promoter, wherein said S/MAR sequence is located upstream of said promoter.

15. (canceled)

16. (canceled)

17. (canceled)

18. (canceled)

19. (canceled)

20. (canceled)

21. A method for treating a subject suffering from cancer, autoimmune disease, and/or genetic disease, comprising contacting said subject with a therapeutic cell according to claim 1 and, thereby, treating said cancer, autoimmune disease, and/or genetic disease.

22. The method of claim 21, wherein said treating is immunotherapy.

23. The method of claim 21, wherein said genetic disease is a monogenic recessive disease, preferably is phenylketonuria, alkaptonuria, Leber's Congenital Amaurosis, Choroideremia, or Stargardt disease.

Description

FIGURE LEGENDS

[0094] FIG. 1: Colony forming assay with S/MAR constructs; 1=GFP-β-interferon MAR, 2=Mar1 (SEQ ID NO:5)-GFP, 3=Mar2 (SEQ ID NO:3)-GFP, 4=Mar3 (SEQ ID NO:6)-GFP, 5-Mar4 (SEQ ID NO.7)-GFP; y-Axis number of resistant colonies.

[0095] FIG. 2: Number of transgene expressing (GFP+) cells and medium fluorescent intensity (MFI) of cell populations carrying various constructs, 1=unmodified cells, 2=GFP-β-interferon MAR, 3=Mar2-GFP.

[0096] FIG. 3: Detection of episomal replication by Southern blot; a=Mar2-GFP control vector, b=total DNA extracted from Hek293T established with Mar2-GFP; the GFP gene was detected.

[0097] FIG. 4: Nucleic acid sequences of nt 220 to 382 of Mar2 (1, SEQ ID NO: 1), nt 40 to 110 of Mar2 (2, SEQ ID NO:2), and Mar2 (3, SEQ ID NO:3).

[0098] The following Examples shall merely illustrate the invention. They shall not be construed, whatsoever, to limit the scope of the invention.

EXAMPLE 1

Colony formation

[0099] Efficiency of generating stably expressing cells was evaluated by a colony forming assay (FIG. 1). Following delivery a vector comprising a GFP reporter gene and a puromycin resistance gene into Hek293T, cells positive for GFP transgene expression were isolated via FACS sorting (FACS Aria II) and plated into a 6 cm cell culture dish. They were then cultured for 3 weeks in presence of 1 μg/ml Puromycin. After 3 weeks the cells were fixed with PFA, stained with Crystal Violet and counted. The number of colonies is considered as the efficiency of vector establishment. Vector number 3 containing the S/MAR sequence 2 (Mar2, SEQ ISD NO:3) 5′ of the GFP gene generated a number of resistant colonies comparable to the control vector carrying B-interferon S/MAR 3′ of the GFP gene, whereas vectors 2, 4 and 5 did not yield in any resistant cell.

EXAMPLE 2

Expression Levels

[0100] Transgene cells of Example 1 were analyzed by FACS for the relative number of cells expressing GFP (GFP+) and for the medium fluorescent intensity (MFI) of the established populations) FIG. 2). Both the control vector carrying β-interferon S/MAR 3′ of the GFP gene (2) and the vector containing the S/MAR sequence 2 5′ of the GFP gene generated cell lines in which the majority (>95%) of the cell expressed the transgene with a comparable intensity.

EXAMPLE 3

Episomal Maintenance

[0101] Total DNA was extracted from from Hek293T cells carrying the vector containing the S/MAR sequence 2 5′ of the GFP gene 35 days post DNA delivery, and subjected to digestion with the restriction enzyme BamHI for 12 h at 37°. The DNA fragments were resolved on a 0.8% agarose gel and transferred onto a nylon membrane. Simultaneously, plasmid DNA from the maxi preparation used to transfect the cells before establishment was treated with the same approach. The reporter gene GFP was used to generate the radioactive probe for testing the control (a), and the vector in the cell population (b) The plasmid has the same size when compared to the correspondent reference vector which demonstrate its episomal maintenance.

LITERATURE

[0102] Geest et al. (2004), Plant Biotech J 2:13 [0103] Haase et al. (2010), BMC Biotechnology 10:20 [0104] Haase et al (2013), PLOS One 8(11):879262 [0105] Liebeich et al. (2002), NAR 30(15): 3433 [0106] Rupprecht et al. (2010), Gene 466(1-2):36 [0107] U.S. Pat. No. 6,410,314 B1 [0108] WO 2019/057773 A1 [0109] WO 2019/057774 A1 [0110] WO 2019/060253 A1 [0111] Yusufzai & Felsenfeld (2004), PNAS 101(23), 8620