EXPRESSION CONSTRUCTS FOR THE GENETIC MODIFICATION OF CELLS

Abstract

The present invention relates to a polynucleotide comprising at least one promoter, at least one expressible construct, and an S/MAR element, wherein said polynucleotide is an integration construct or a non-integrative vector construct, wherein said S/MAR element is located downstream of said promoter and of said expressible construct, and wherein said S/MAR element is flanked by a splice donor and a splice acceptor. The present invention also relates to a composition and a host cell comprising said polynucleotide, as well as to uses and methods related thereto.

Claims

1. A polynucleotide comprising at least one promoter, at least one expressible construct, and an S/MAR element, wherein said polynucleotide is an integration construct, wherein said S/MAR element is located downstream of said promoter and of said expressible construct, and wherein said S/MAR element is flanked by a splice donor and a splice acceptor.

2. The polynucleotide of claim 1, wherein said polynucleotide is an integration construct comprising at least one integration signal.

3. The polynucleotide of claim 1, wherein said integration signal is a free terminus of a linear polynucleotide, a viral integration signal, or a transposable element.

4. The polynucleotide of claim 1, wherein said expressible construct comprises at least one coding sequence encoding a polypeptide, a sequence encoding a siRNA, a sequence encoding an miRNA, a sequence encoding an antisense RNA, and/or a sequence encoding a ribozyme.

5. The polynucleotide of claim 4, wherein said polypeptide is a therapeutic polypeptide, preferably a human T Cell Receptor (TCR), Chimeric Antigen Receptor (CAR), preferably MART1 TCR.

6. The polynucleotide of claim 1, wherein a transcript is transcribed from said promoter, from which transcript the sequence of the S/MAR element is spliced out.

7. (canceled)

8. A host cell comprising the polynucleotide according to claim 1 integrated into its genome.

9. (canceled)

10. (canceled)

11. (canceled)

12. A method for increasing expression of a eukaryotic expression construct comprising a promoter and an expressible construct, the method comprising including an S/MAR element flanked by a splice donor and a splice acceptor downstream of said expressible construct, wherein said eukaryotic expression is an integration construct.

13. (canceled)

14. (canceled)

15. (canceled)

16. A method for treating genetic disease in a subject, comprising a) contacting said subject with a polynucleotide according to claim 1, and b) thereby, treating genetic disease in said subject.

17. The method of claim 16, wherein said polynucleotide is comprised in a host cell.

18. The method of claim 16, wherein said genetic disease is causally linked to one or more epigenetic changes and/or to one or more genetic mutations.

19. The method of claim 16, wherein the genetic disease is cancer, phenylketonuria, alkaptonuria, Leber's Congenital Amaurosis, Choroideremia, Haemophilia, Ushers disease, or Stargardt disease.

Description

[0083] In view of the above, the following embodiments are preferred:

[0084] 1. A polynucleotide comprising at least one promoter, at least one expressible construct, and an S/MAR element, wherein said polynucleotide is an integration construct or a non-integrative vector construct, wherein said S/MAR element is located downstream of said promoter and of said expressible construct, and wherein said S/MAR element is flanked by a splice donor and a splice acceptor.

[0085] 2. The polynucleotide of embodiment 1, wherein said polynucleotide is an integration construct or a non-integrative viral vector construct.

[0086] 3. The polynucleotide of embodiment 1 or 2, wherein said polynucleotide is an integration construct.

[0087] 4. The polynucleotide of any one of embodiments 1 to 3, wherein said integration construct comprises at least one integration signal.

[0088] 5. The polynucleotide of embodiment 4, wherein said integration signal is a free terminus of a linear polynucleotide, a recombinase recognition sequence, a viral integration signal, or a transposable element.

[0089] 6. The polynucleotide of any one of embodiments 1 to 5, wherein said polynucleotide is devoid of a eukaryotic origin of replication, preferably is devoid of an origin of replication.

[0090] 7. The polynucleotide of any one of embodiments 1 to 6, wherein said polynucleotide does not replicate episomally in a host cell, preferably in a mammalian cell.

[0091] 8. The polynucleotide of embodiment 1 or 3, wherein said polynucleotide is a non-integrative viral vector construct.

[0092] 9. The polynucleotide of embodiment 8, wherein said non-integrative viral vector construct is a non-integrative lentivirus construct, an adeno-associated virus construct, a simian virus 40 construct, a papillomavirus construct, an adenovirus construct, a hepatitis virus construct, or a herpesvirus construct.

[0093] 10. The polynucleotide of embodiment 8 or 9, wherein said polynucleotide replicates episomally in a host cell, preferably in a mammalian cell.

[0094] 11. The polynucleotide of any one of embodiments 1 to 10, wherein said expressible construct comprises at least one coding sequence encoding a polypeptide, a sequence encoding a siRNA, a sequence encoding an miRNA, a sequence encoding an antisense RNA, and/or a sequence encoding a ribozyme.

[0095] 12. The polynucleotide of any one of embodiments 1 to 11, wherein said polypeptide is a therapeutic polypeptide, preferably a human T Cell Receptor (TCR), Chimeric Antigen Receptor (CAR), preferably MART1 TCR.

[0096] 13. The polynucleotide of any one of embodiments 1 to 12, wherein said expressible construct comprises at least one coding sequence encoding a selectable marker.

[0097] 14. The polynucleotide of any one of embodiments 1 to 13, wherein said expressible construct comprises at least two coding sequences, preferably of which one encodes a selectable marker.

[0098] 15. The polynucleotide of any one of embodiments 1 to 14, wherein said expressible construct comprises a coding sequence encoding a selectable marker (selectable marker sequence), wherein said promoter and said selectable marker sequence together constitute a selectable marker gene, and wherein said selectable marker is a selectable marker of a eukaryotic cell.

[0099] 16. The polynucleotide embodiment 15, wherein said selectable marker gene is a puromycin resistance gene, a blasticidin resistance gene, a neomycin resistance gene, or a zeocin resistance gene, preferably is a puromycin resistance gene.

[0100] 17. The polynucleotide of any one of embodiments 1 to 16, wherein a transcript is transcribed from said promoter, from which transcript the sequence of the S/MAR element is spliced out.

[0101] 18. The polynucleotide of any one of embodiments 1 to 17, wherein a poly-A signal downstream of the S/MAR element is retained in said splicing.

[0102] 19. The polynucleotide of any one of embodiments 1 to 18, wherein said host cell is a mammalian cell, preferably a human cell.

[0103] 20. The polynucleotide of any one of embodiments 1 to 19, wherein said polynucleotide is devoid of any centromere and/or telomere sequence.

[0104] 21. The polynucleotide of any one of embodiments 1 to 20, wherein said polynucleotide comprises a transcriptional insulator element upstream of said promoter.

[0105] 22. The polynucleotide of embodiment 21, wherein said insulator element is an element-40 and/or an S/MAR element.

[0106] 23. The polynucleotide of any one of embodiments 1 to 22, wherein said promoter and expressible construct are insulated from the residual sequences comprised in the polynucleotide by the presence of at least one insulation element, more preferably by being flanked by insulation elements.

[0107] 24. A composition comprising a polynucleotide according to any one of embodiments 1 to 23.

[0108] 25. A host cell comprising the polynucleotide according to any one of embodiments 1 to 23, preferably integrated into its genome.

[0109] 26. The host cell of embodiment 25, wherein said host cell is a CD34+ Progenitor Cell; a CD61+ Thrombocyte; a CD19+ B-Lymphocyte; a CD14+ Monocyte; a CD15+ Granulocyte; a CD3+ Cytotoxic T-Lymphocyte, preferably also positive for CD8 and CD45; a CD3+ Helper T-Lymphocyte, preferably also positive for CD4 and CD45; a CD3+ activated T-Lymphocyte, preferably also positive for CD25 and CD45, a Tumor infiltrating Lymphocyte, a Natural Killer (NK) cell, an embryonic stem (ES) cell, an induced pluripotent stem cell (IPS) cell, an airway epithelial cell, a fibroblast, or a retinal epithelial cell.

[0110] 27. The host cell of embodiment 25 or 26, wherein said polynucleotide is covalently bonded to a chromosome of said host cell.

[0111] 28. A polynucleotide according to any one of embodiments 1 to 23, a composition according to embodiment 24, and/or a host cell according to any one of embodiments 25 to 27 for use in medicine,

[0112] 29. A polynucleotide according to any one of embodiments 1 to 23, a composition according to embodiment 24, and/or a host cell according to any one of embodiments 25 to 27 for use in treating genetic disease.

[0113] 30. A device comprising the polynucleotide according to any one of embodiments 1 to 23, the composition according to embodiment 24, and/or the host cell according to any one of embodiments 25 to 27

[0114] 31. A method for stably expressing an expressible construct in a host cell, comprising

[0115] a) contacting said host cell with a polynucleotide according to any one of embodiments 1 to 23 and/or a composition according to embodiment 24, and,

[0116] b) thereby, stably expressing an expressible construct in a host cell.

[0117] 32. A method for treating genetic disease in a subject, comprising

[0118] a) contacting said subject with a polynucleotide according to any one of embodiments 1 to 23, a composition according to embodiment 24, and/or a host cell according to any one of embodiments 25 to 27, and,

[0119] b) thereby, treating genetic disease in said subject.

[0120] 33. Use of a polynucleotide according to any one of embodiments 1 to 23 and/or a composition according to embodiment 24, for stably genetically modifying a host cell.

[0121] 34. Use of a polynucleotide according to any one of embodiments 1 to 23, a composition according to embodiment 24, and/or a host cell according to any one of embodiments 25 to 27, for stably genetically modifying a host cell.

[0122] 35. Use of a polynucleotide according to any one of embodiments 1 to 23, a composition according to embodiment 24, and/or a host cell according to any one of embodiments 25 to 27 for the manufacture of a medicament.

[0123] 36. Use of a polynucleotide according to any one of embodiments 1 to 23, a composition according to embodiment 24, and/or a host cell according to any one of embodiments 25 to 27 for the manufacture of a medicament for treating genetic disease, preferably monogenic disease, more preferably monogenic recessive disease, most preferably phenylketonuria, alkaptonuria, Leber's Congenital Amaurosis, Choroideremia, or Stargardt disease.

[0124] 37. A method for increasing expression of a eukaryotic expression construct comprising a promoter and an expressible construct, comprising including an S/MAR element flanked by a splice donor and a splice acceptor downstream of said expressible construct.

[0125] 38. The method of embodiment 37, wherein said eukaryotic expression construct further comprises a transcriptional terminator.

[0126] 39. The method of embodiment, wherein said S/MAR element is included between said expressible construct and said transcriptional terminator.

[0127] 40. The method of any one of embodiments 37 to 39, wherein said wherein said S/MAR element is spliced out of a primary transcript initiated at said promotor.

[0128] 41. The method of any one of embodiments 37 to 40, wherein said method comprises providing a polynucleotide according to any one of embodiments 1 to 23 and/or a composition according to embodiment 24.

[0129] 42. The method of any one of embodiments 37 to 41, wherein said method further comprises contacting a host cell with a polynucleotide comprising said expression construct, preferably a polynucleotide according to any one of embodiments 1 to 23 and/or a composition according to embodiment 24.

[0130] 43. Use of an S/MAR element flanked by a splice donor and a splice acceptor for increasing expression of a eukaryotic expression construct.

[0131] 44. The use of embodiment 43, wherein said eukaryotic expression construct comprises a promoter and an expressible construct.

[0132] 45. The use of embodiment 44, wherein said use comprises including said S/MAR element downstream of said expressible construct.

[0133] 46. The use of embodiment 44 or 45, wherein said eukaryotic expression construct further comprises a transcriptional terminator.

[0134] 47. The use of embodiment 46, wherein said use comprises including said S/MAR element between said expressible construct and said transcriptional terminator.

[0135] 48. The use of any one of embodiments 43 to 47, wherein said use comprises providing a polynucleotide according to any one of embodiments 1 to 23 and/or a composition according to embodiment 24.

[0136] 49. The use of any one of embodiments 43 to 48, wherein said use further comprises contacting a host cell with a polynucleotide comprising said expression construct, preferably a polynucleotide according to any one of embodiments 1 to 23 and/or a composition according to embodiment 24.

[0137] All references cited in this specification are herewith incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned in this specification.

FIGURE LEGENDS

[0138] FIG. 1: Schematic depiction of the expression cassettes. A) GFP-S/MAR vector. The expression cassette consists of a ubiquitous promoter (Promoter) that drives the expression of the reporter gene GFP and the selectable marker Puromycin divided by the self-cleavage sequence P2A. The expression cassette is followed by an S/MAR sequence and a transcriptional terminator (PolyA). B) The expression cassette consists of the same elements, but the S/MAR sequence is flanked by a splicing donor (SD) and a splicing acceptor (SA) site.

[0139] FIG. 2: Hek293T cell populations established with the GFP-S/MAR and the GFP-S/MARsplice vector were analysed for transgene expression 35 days post DNA delivery and selection in Puromycin (0.5 ug/ml) by Flow Cytometry (A). The relative expression of the transgene GFP was evaluated and normalised to the expression of the housekeeping gene GAPDH (B). The figures show that the introduction of splicing sequences improve the transgene expression by enhancing the RNA amount in the cells.

[0140] FIG. 3: Schematic depiction of transposon mediated expression cassettes. A) GFP-S/MAR vector. The expression cassette consists of a ubiquitous promoter (Promoter) that drives the expression of the reporter gene GFP and the selectable marker Puromycin divided by the self-cleavage sequence P2A. The 5′ and 3′ ITRs are the sequences that mediated the expression cassette transposition and integration into the cell target genome. The expression cassette is followed by an S/MAR sequence and a transcriptional terminator (PolyA). B) The expression cassette consists of the same elements, but the S/MAR sequence is flanked by a splicing donor (SD) and a splicing acceptor (SA) site.

[0141] FIG. 4: Colony forming assay (A) and analysis of the transgene expression in established cells (B). The efficiency of generating stably expressing cells was evaluated by a colony forming assay. Following DNA delivery 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. The generation of stable cells lines is significantly more effective with the construct number 2 where the MAR sequences is flanked by splicing sites (p<0.00001). The expression of transgene GFP was evaluated in established Heks293T as the fluorescent intensity of the cell populations. The expression of the reporter gene GFP is significantly higher (p<0.05) in Hek293T cells modified with the construct 2; 1=GFP-Puro-S/MAR, 2=GFP-Puro-S/MAR splice.

[0142] The following Examples shall merely illustrate the invention. They shall not be construed, whatsoever, to limit the scope of the invention.

[0143] Example 1: Linear constructs as shown in FIG. 1 were purified and transfected into Hek293T cells. Cells were selected for 35 days in medium containing puromycin (0.5 μg/ml), whereafter relative GFP expression (amount of GFP RNA compared to GAPDH) and mean fluorescence intensity (MFI) in a FACS were determined. As shown in FIG. 2, transgene mRNA and protein levels are increased by the splicing sites flanking the S/MAR sequence.