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1. (WO2019028273) DNA VECTORS AND ELEMENTS FOR SUSTAINED GENE EXPRESSION IN EUKARYOTIC CELLS
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CLAIMS

What is claimed is:

A: Vector GPS

1. A method for constructing an expression vector, the method comprising: a. selecting a plurality of sequence element groups and, for each

respective sequence element group in said plurality of sequence element groups, selecting one or more element sequences; wherein the possible combinations of the plurality of sequence element groups and the one or more element sequences for each respective sequence element group in the plurality of sequence element groups collectively define an expression vector sequence-space;

b. constructing a first plurality of variants of expression vectors thereby forming a variant set, wherein said variant set comprises a subset of said polynucleotide vector sequence-space, each expression vector comprising an open reading frame or RNA-encoding sequence in operable linkage to one or more element sequences;

c. measuring expression of the open reading frame from all or a portion of the variants in the variant set;

d. modeling, using a suitably programmed computer, a sequence-activity relationship between (i) one or more element sequences in one or more element groups of the nucleic acid construct represented by the variant set and (ii) the expression of the open reading frame or RNA encoding sequence measured for all or the portion of the variants in the variant set, wherein the sequence-activity relationship has the form Y= (wixi+ W2X2,+. . . wiXi); wherein

Y is a quantitative measure of gene expression; xi is a descriptor of a sequence element, a combination of sequence elements, or a component of one or more sequence elements, in one or more element groups; and

wi is a weight of the descriptor; and wherein

the modeling comprises:

i) optimizing, using a suitably programmed computer, the sequence-activity relationship by adjusting

individual weights wi for each said descriptor xi using a refinement algorithm that minimizes the difference between the predicted values and the real values of Y from partial data, wherein the partial data is the first plurality of variants with individual sequences left out on a random basis, and

ii) repeating the optimizing i) a plurality of times thereby obtaining, for each respective element or combination of elements or component of elements xi (a) an average value for the weight wi describing a relative or absolute contribution of the respective element or combination of elements or component of elements xi to Y, and (b) a standard deviation, variance or other measure of variability of the weight wi describing the relative or absolute contribution of the respective element or combination of elements or component of elements xi to Y; and

e. constructing a new expression vector by selecting at least one element for which a descriptor xi has a weight wi whose mean value minus its standard deviation is greater than zero.

2. The method of claim 1 , wherein the first variant set of vectors comprise at least 10 vectors, which collectively show variation in at least two sequence element groups.

3. The method of claim 1 wherein each vector in the variant vector set further comprises a sequence selected from SEQ ID NOs: 1-6.

B: AAV vectors

4. A polynucleotide comprising a sequence selected from SEQ ID NO: 323-432.

5. A polynucleotide comprising a sequence selected from SEQ ID NO: 433-495.

6. A vector comprising a DNA sequence selected from SEQ ID NOs: 317-322.

7. A polynucleotide comprising a sequence selected from SEQ ID NOs: 1-6 and at least 100 contiguous bases from a sequence selected from SEQ ID Nos: 496-499.

8. A method for expressing a an open reading frame or RNA-encoding sequence comprising introducing into a mammalian cell the polynucleotide of claim 4, wherein the polynucleotide is in operable linkage with the open reading frame or RNA-encoding sequence and the open reading frame or RNA-encoding sequence is expressed.

9. A mammalian cell comprising the polynucleotide of claim 4.

C: PRE elements

10. A polynucleotide comprising a sequence selected from SEQ ID NOs 242-292.

11. A polynucleotide comprising SEQ ID NO: 316 operably linked to a

heterologous promoter.

12. A polynucleotide comprising an alpha posttranscriptional regulatory element (PRE sub-element comprising a sequence selected from SEQ ID NOs 301- 307, operably linked to a heterologous promoter.

13. A polynucleotide comprising a beta PRE sub-element comprising a sequence selected from SEQ ID NOs 308-316 operably linked to a heterologous promoter.

14. A polynucleotide comprising a gamma PRE sub-element comprising a

sequence selected from SEQ ID NOs 297-300, operably linked to a heterologous promoter.

15. A polynucleotide comprising a delta PRE sub-element comprising a sequence selected from SEQ ID NOs 293-296, operably linked to a heterologous promoter.

16. A polynucleotide comprising an alpha, beta, gamma or delta PRE sub element derived from one natural source, and an alpha, beta, gamma or delta PRE sub element derived from a different natural source.

17. A polynucleotide comprising two open reading frames or RNA-encoding sequences, wherein each open reading frame or RNA-encoding sequence is operably linked to a different PRE element.

18. The polynucleotide of claim 17, wherein one of the PRE elements comprises a sequence selected from SEQ ID NOs: 228-292.

19. The polynucleotide of claim 17, wherein one of the PRE elements comprises a sequence selected from SEQ ID NOs: 305-307 or 312-316.

20. A method for expressing an open reading frame or RNA-encoding sequence comprising introducing into a mammalian cell the polynucleotide of claim 10 operably linked to the open reading frame or RNA-encoding sequence, wherein the open reading frame or RNA-encoding sequence is expressed.

21. A mammalian cell comprising the polynucleotide of claim 10.

D: promoter / intron elements

22. A polynucleotide comprising SEQ ID NO: 25 operably linked to a

heterologous promoter.

23. The polynucleotide of claim 22 wherein the heterologous promoter comprises a sequence selected from SEQ ID NO: 29-52 or 57-78.

24. A polynucleotide comprising SEQ ID NO: 25 operably linked to a

heterologous intron, wherein the intron has at least 95% sequence identity to a sequence selected from SEQ ID Nos: 99-113 or 122-135.

25. A polynucleotide comprising SEQ ID NO: 76, operably linked to a

heterologous open reading frame or RNA encoding sequence.

26. The polynucleotide of claim 25 wherein the polynucleotide further comprises a sequence selected from SEQ ID NOs: 71-74 operably linked to the open reading frame or RNA encoding sequence.

27. The polynucleotide of claim 25, further comprising an intron having at least 95% sequence identity to a sequence selected from SEQ ID NOs: 122-135.

28. A polynucleotide comprising an intron having at least 95% sequence identity to a sequence selected from SEQ ID NO: 99-105 operably linked to a heterologous open reading frame.

29. A polynucleotide comprising an enhancer sequence with at least 95%

sequence identity to SEQ ID NO: 26 and a heterologous open reading frame or RNA-encoding sequence in operable linkage.

30. A polynucleotide comprising a promoter sequence with at least 95% sequence identity to SEQ ID NO: 58 and a heterologous open reading frame or RNA- encoding sequence in operable linkage.

31. A polynucleotide comprising a regulatory sequence with at least 95%

sequence identity to SEQ ID NO: 142-191.

32. The polynucleotide of claim 22, further comprising a sequence selected from SEQ ID NO: 581-584.

33. A method for expressing an open reading frame or RNA-encoding sequence comprising introducing into a mammalian cell the polynucleotide of claim 22 operably linked to the open reading frame, wherein the open reading frame or RNA-encoding sequence is expressed.

34. A mammalian cell comprising the polynucleotide of claim 22.

E: GS-related

35. A polynucleotide encoding a glutamine synthetase comprising an amino acid sequence selected from SEQ ID NO: 505-506 or 513-518.

36. A polynucleotide encoding a polypeptide comprising a glutamine synthetase and a peptide of SEQ ID NO: 502.

37. A polynucleotide encoding a glutamine synthetase comprising a sequence selected from SEQ ID NO: 509-512.

38. The polynucleotide of claim 35 further comprising a promoter operably linked to a heterologous open reading frame that does not encode the glutamine synthetase.

39. The polynucleotide of claim 35 further comprising a first promoter operably linked to a first heterologous open reading frame and a second promoter operably linked to a second open reading frame, wherein neither the first nor the second heterologous open reading frame encodes the glutamine synthetase.

40. The polynucleotide of claim 35 further comprising a sequence selected from SEQ ID NO: 7-10.

41. The polynucleotide of claim 35 further comprising a sequence selected from SEQ ID NO: 500-501 operably linked to the open reading frame encoding the glutamine synthetase.

42. The polynucleotide of claim 35 further comprising a promoter sequence

selected from SEQ ID NO: 79-90 in operable linkage with the open reading frame encoding the glutamine synthetase.

43. The polynucleotide of claim 35 further comprising SEQ ID NO: 141 operably linked to an open reading frame encoding the glutamine synthetase.

44. The polynucleotide of claim 35 further comprising a sequence selected from SEQ ID NO: 1 1-28 operably linked to an open reading frame encoding the glutamine synthetase.

45. The polynucleotide of claim 35 further comprising a sequence selected from SEQ ID NO: 29-78 in operable linkage with a second heterologous open reading frame.

46. The polynucleotide of claim 35 further comprising an intron sequence at least 95% identical to a sequence selected from SEQ ID NO: 91 -140.

47. A polynucleotide comprising a sequence selected from SEQ ID NO: 510-512 or 519-556.

48. A method for expressing a glutamine synthetase comprising introducing into a mammalian cell the polynucleotide of claim 35, wherein the glutamine synthetase is expressed.

49. A mammalian cell comprising the polynucleotide of claim 35.

50. The polynucleotide of claim 35, wherein the glutamine synthetase is encoded by a cDNA sequence.

51. The polynucleotide of claim 35, wherein the glutamine synthetase is encoded by a sequence including at least one intron.