ARTIFICIAL NUCLEIC ACID MOLECULES FOR IMPROVED PROTEIN OR PEPTIDE EXPRESSION
20200332293 · 2020-10-22
Assignee
Inventors
Cpc classification
A61P29/00
HUMAN NECESSITIES
C12N2320/52
CHEMISTRY; METALLURGY
A61K39/00
HUMAN NECESSITIES
C12N15/113
CHEMISTRY; METALLURGY
A61K48/00
HUMAN NECESSITIES
A61P43/00
HUMAN NECESSITIES
A61K48/0008
HUMAN NECESSITIES
C12N15/67
CHEMISTRY; METALLURGY
International classification
C12N15/113
CHEMISTRY; METALLURGY
A61K39/00
HUMAN NECESSITIES
A61K48/00
HUMAN NECESSITIES
C12N15/67
CHEMISTRY; METALLURGY
Abstract
The invention relates to an artificial nucleic acid molecule comprising at least one 5UTR element which is derived from a TOP gene, at least one open reading frame, and preferably at least one histone stem-loop. Optionally the artificial nucleic acid molecule may further comprise, e.g. a poly(A)sequence, a polyadenylation signal, and/or a 3UTR. The invention further relates to the use of such an artificial nucleic acid molecule in gene therapy and/or genetic vaccination.
Claims
1. An artificial nucleic acid molecule comprising: a. at least one 5-untranslated region element (5UTR element) which comprises or consists of a nucleic acid sequence which is derived from the 5UTR of a TOP gene or which is derived from a variant of the 5UTR of a TOP gene; and b. at least one open reading frame (ORF).
2. The artificial nucleic acid molecule according to claim 1, further comprising: c. at least one histone stem-loop.
3. The artificial nucleic acid molecule according to claim 1 or 2, wherein the 5UTR element and the open reading frame are heterologous.
4. The artificial nucleic acid molecule according to any one of claims 1-3, wherein the 5UTR element is suitable for increasing protein production from the artificial nucleic acid molecule.
5. The artificial nucleic acid molecule according to any one of claims 2-4, wherein the 5UTR element and the histone stem-loop act together, preferably at least additively, to increase protein production from the artificial nucleic acid molecule.
6. The artificial nucleic acid molecule according to any one of claims 1-5, wherein the 5UTR element does not comprise a TOP-motif, preferably wherein the nucleic acid sequence which is derived from a 5UTR of a TOP gene, preferably the 5UTR element, starts at its 5-end with a nucleotide located at position 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 downstream of the polypyrimidine tract.
7. The artificial nucleic acid molecule according to any one of claims 1-6, wherein the nucleic acid sequence which is derived from a 5UTR of a TOP gene, preferably the 5UTR element terminates at its 3-end with a nucleotide located at position 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 upstream of the start codon of the gene it is derived from.
8. The artificial nucleic acid molecule according to any one of claims 1-7, wherein the 5UTR element does not comprise a start codon or an open reading frame.
9. The artificial nucleic acid molecule according to any one of claims 1-8, wherein the nucleic acid sequence which is derived from the 5UTR of a TOP gene is derived from the 5UTR of a eukaryotic TOP gene or from a variant thereof, preferably from the 5UTR of a plant or animal TOP gene or from a variant thereof, more preferably from the 5UTR of a chordate TOP gene or from a variant thereof, even more preferably from the 5UTR of a vertebrate TOP gene or from a variant thereof, most preferably from the 5UTR of a mammalian TOP gene, such as a human TOP gene, or from a variant thereof.
10. The artificial nucleic acid molecule according to any one of claims 2-9, wherein the at least one histone stem-loop is selected from following formulae (I) or (II): ##STR00007## wherein: stem1 or stem2 bordering elements N.sub.1-6 is a consecutive sequence of 1 to 6, preferably of 2 to 6, more preferably of 2 to 5, even more preferably of 3 to 5, most preferably of 4 to 5 or 5 N, wherein each N is independently from another selected from a nucleotide selected from A, U, T, G and C, or a nucleotide analogue thereof; stem1 [N.sub.0-2GN.sub.3-5] is reverse complementary or partially reverse complementary with element stem2, and is a consecutive sequence between of 5 to 7 nucleotides; wherein N.sub.0-2 is a consecutive sequence of 0 to 2, preferably of 0 to 1, more preferably of 1 N, wherein each N is independently from another selected from a nucleotide selected from A, U, T, G and C or a nucleotide analogue thereof; wherein N.sub.3-5 is a consecutive sequence of 3 to 5, preferably of 4 to 5, more preferably of 4 N, wherein each N is independently from another selected from a nucleotide selected from A, U, T, G and C or a nucleotide analogue thereof, and wherein G is guanosine or an analogue thereof, and may be optionally replaced by a cytidine or an analogue thereof, provided that its complementary nucleotide cytidine in stem2 is replaced by guanosine; loop sequence [N.sub.0-4(U/T)N.sub.0-4] is located between elements stem1 and stem2, and is a consecutive sequence of 3 to 5 nucleotides, more preferably of 4 nucleotides; wherein each N.sub.0-4 is independent from another a consecutive sequence of 0 to 4, preferably of 1 to 3, more preferably of 1 to 2 N, wherein each N is independently from another selected from a nucleotide selected from A, U, T, G and C or a nucleotide analogue thereof; and wherein U/T represents uridine, or optionally thymidine; stem2 [N.sub.3-5CN.sub.0-2] is reverse complementary or partially reverse complementary with element stem1, and is a consecutive sequence between of 5 to 7 nucleotides; wherein N.sub.3-5 is a consecutive sequence of 3 to 5, preferably of 4 to 5, more preferably of 4 N, wherein each N is independently from another selected from a nucleotide selected from A, U, T, G and C or a nucleotide analogue thereof; wherein N.sub.0-2 is a consecutive sequence of 0 to 2, preferably of 0 to 1, more preferably of 1 N, wherein each N is independently from another selected from a nucleotide selected from A, U, T, G and C or a nucleotide analogue thereof; and wherein C is cytidine or an analogue thereof, and may be optionally replaced by a guanosine or an analogue thereof provided that its complementary nucleotide guanosine in stem1 is replaced by cytidine; wherein stem1 and stem2 are capable of base pairing with each other forming a reverse complementary sequence, wherein base pairing may occur between stem1 and stem2, or forming a partially reverse complementary sequence, wherein an incomplete base pairing may occur between stem1 and stem2.
11. The artificial nucleic acid molecule according to any one of claims 2-10, wherein the at least one histone stem-loop is selected from at least one of following formulae (Ia) or (IIa): ##STR00008##
12. The artificial nucleic acid molecule according to any one of claims 1-11, further comprising d. a poly(A) sequence and/or a polyadenylation signal.
13. The artificial nucleic acid molecule according to claim 12, wherein the poly(A) sequence comprises or consists of a sequence of about 25 to about 400 adenosine nucleotides, preferably a sequence of about 50 to about 400 adenosine nucleotides, more preferably a sequence of about 50 to about 300 adenosine nucleotides, even more preferably a sequence of about 50 to about 250 adenosine nucleotides, most preferably a sequence of about 60 to about 250 adenosine nucleotides.
14. The artificial nucleic acid molecule according to claim 12 or 13, wherein the polyadenylation signal comprises the consensus sequence NN(U/T)ANA, with N=A or U, preferably AA(U/T)AAA or A(U/T)(U/T)AAA.
15. The artificial nucleic acid molecule according to any of claims 1-14, further comprising: e. a poly(C) sequence.
16. The artificial nucleic acid molecule according to claim 15, wherein the poly(C) sequence comprises, preferably consists of, about 10 to about 200 cytidine nucleotides, more preferably about 10 to about 100 cytidine nucleotides, more preferably about 10 to about 50 cytidine nucleotides, even more preferably about 20 to about 40 cytidine nucleotides.
17. The artificial nucleic acid molecule according to any one of claims 1-16, further comprising: f. at least one 3UTR element.
18. The artificial nucleic acid molecule according to claim 17, wherein the at least one 3UTR element comprises or consists of a nucleic acid sequence which is derived from a 3UTR of a gene providing a stable mRNA or from a variant of the 3UTR of a gene providing a stable mRNA.
19. The artificial nucleic acid molecule according to claim 17 or 18, wherein the at least one 3UTR element and the at least one 5UTR element act at least additively, preferably synergistically to increase protein production from said artificial nucleic acid molecule.
20. The artificial nucleic acid molecule according to any one of claims 1-19, wherein the 5UTR element comprises or consists of a nucleic acid sequence which is derived from a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 1-1363, SEQ ID NO. 1435, SEQ ID NO. 1461 or SEQ ID NO. 1462, from the homologs of any of SEQ ID NOs. 1-1363, SEQ ID NO. 1435, SEQ ID NO. 1461 or SEQ ID NO. 1462, or from a variant thereof.
21. The artificial nucleic acid molecule according to any one of claims 1-20, wherein the 5UTR element comprises or consists of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to a nucleic acid sequence selected from SEQ ID NOs. 1-1363, SEQ ID NO. 1435, SEQ ID NO. 1461 or SEQ ID NO. 1462, or to a corresponding RNA sequence, or wherein the at least one 5UTR element comprises or consists of a fragment of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to a nucleic acid sequence selected from SEQ ID NOs. 1-1363, SEQ ID NO. 1435, SEQ ID NO. 1461 or SEQ ID NO. 1462, or to a corresponding RNA sequence, preferably lacking the 5TOP motif.
22. The artificial nucleic acid molecule according to any one of claims 1-21, wherein the 5UTR element comprises or consists of a nucleic acid sequence which is derived from a 5UTR of a TOP gene encoding a ribosomal protein or from a variant of a 5UTR of a TOP gene encoding a ribosomal protein, preferably from a 5UTR of a nucleic acid sequence according to any of SEQ ID NOs: 170, 232, 244, 259, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291, 1292, 1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300, 1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312, 1313, 1314, 1315, 1316, 1317, 1318, 1319, 1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327, 1328, 1329, 1330, 1331, 1332, 1333, 1334, 1335, 1336, 1337, 1338, 1339, 1340, 1341, 1342, 1343, 1344, 1346, 1347, 1348, 1349, 1350, 1351, 1352, 1353, 1354, 1355, 1356, 1357, 1358, 1359, or 1360; a corresponding RNA sequence, a homolog thereof, or a variant thereof, preferably lacking the 5TOP motif.
23. The artificial nucleic acid molecule according to any one of claims 1-22, wherein the 5UTR element comprises or consists of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the 5UTR of a nucleic acid sequence according to any of SEQ ID NOs: 170, 232, 244, 259, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291, 1292, 1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300, 1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312, 1313, 1314, 1315, 1316, 1317, 1318, 1319, 1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327, 1328, 1329, 1330, 1331, 1332, 1333, 1334, 1335, 1336, 1337, 1338, 1339, 1340, 1341, 1342, 1343, 1344, 1346, 1347, 1348, 1349, 1350, 1351, 1352, 1353, 1354, 1355, 1356, 1357, 1358, 1359, or 1360; or to a corresponding RNA sequence, preferably lacking the 5TOP motif, or wherein the at least one 5UTR element comprises or consists of a fragment of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the 5UTR of a nucleic acid sequence according to SEQ ID NOs: 170, 232, 244, 259, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291, 1292, 1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300, 1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312, 1313, 1314, 1315, 1316, 1317, 1318, 1319, 1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327, 1328, 1329, 1330, 1331, 1332, 1333, 1334, 1335, 1336, 1337, 1338, 1339, 1340, 1341, 1342, 1343, 1344, 1346, 1347, 1348, 1349, 1350, 1351, 1352, 1353, 1354, 1355, 1356, 1357, 1358, 1359, or 1360; or to a corresponding RNA sequence, preferably lacking the 5TOP motif.
24. The artificial nucleic acid molecule according to any one of claims 1-23, wherein the 5UTR element is derived from a 5UTR of a TOP gene encoding a ribosomal Large protein (RPL) or from a variant of a 5UTR of a TOP gene encoding a ribosomal Large protein (RPL), preferably from a 5UTR of a nucleic acid sequence according to any of SEQ ID NOs: 67, 259, 1284-1318, 1344, 1346, 1348-1354, 1357, 1461 and 1462, a corresponding RNA sequence, a homolog thereof, or a variant thereof, preferably lacking the 5TOP motif.
25. The artificial nucleic acid molecule according to any one of claims 1-24, wherein the 5UTR element comprises or consists of a nucleic acid sequence having an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the 5UTR of a nucleic acid sequence according to any of SEQ ID NOs: 67, 259, 1284-1318, 1344, 1346, 1348-1354, 1357, 1461 and 1462, or to a corresponding RNA sequence, preferably lacking the 5TOP motif, or wherein the at least one 5UTR element comprises or consists of a fragment of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the 5UTR of a nucleic acid sequence according to SEQ ID No. SEQ ID NOs: 67, 259, 1284-1318, 1344, 1346, 1348-1354, 1357, 1461 and 1462 or to a corresponding RNA sequence, preferably lacking the 5TOP motif.
26. The artificial nucleic acid molecule according to any one of claims 1-25, wherein the 5UTR element comprises or consists of a nucleic acid sequence which is derived from the 5UTR of a ribosomal protein Large 32 gene or from a variant thereof, preferably from the 5UTR of a vertebrate ribosomal protein Large 32 (L32) gene or from a variant thereof, more preferably from the 5UTR of a mammalian ribosomal protein Large 32 (L32) gene or from a variant thereof, most preferably from the 5UTR of a human ribosomal protein Large 32 (L32) gene or from a variant thereof, wherein preferably the 5UTR element, preferably the artificial nucleic acid molecule does not comprise the 5TOP of said gene.
27. The artificial nucleic acid molecule according to any one of claims 1-26, wherein the 5UTR element comprises or consists of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the nucleic acid sequence according to SEQ ID NOs. 1368 or 1452-1460, or a corresponding RNA sequence, or wherein the at least one 5UTR element comprises or consists of a fragment of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the nucleic acid sequence according to SEQ ID NOs. 1368 or 1452-1460, or to a corresponding RNA sequence.
28. The artificial nucleic acid molecule according to any one of claims 21, 23, 25 and 27, wherein the fragment consists of a continuous stretch of nucleotides corresponding to a continuous stretch of nucleotides in the full-length sequence, which represents at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, and most preferably at least 90% of the full-length sequence the fragment is derived from.
29. The artificial nucleic acid molecule according to any one of claims 1-28, wherein the at least one 5UTR element exhibits a length of at least about 20 nucleotides, preferably of at least about 30 nucleotides, more preferably of at least about 40 nucleotides.
30. The artificial nucleic acid molecule according to any one of claims 1-29, wherein the 5UTR element comprises or consists of a nucleic acid sequence which is derived from the 5UTR of a TOP gene selected from RPSA, RPS2, RPS3, RPS3A, RPS4, RPS5, RPS6, RPS7, RPS8, RPS9, RPS10, RPS11, RPS12, RPS13, RPS14, RPS15, RPS15A, RPS16, RPS17, RPS18, RPS19, RPS20, RPS21, RPS23, RPS24, RPS25, RPS26, RPS27, RPS27A, RPS28, RPS29, RPS30, RPL3, RPL4, RPL5, RPL6, RPL7, RPL7A, RPL8, RPL9, RPL10, RPL10A, RPL11, RPL12, RPL13, RPL13A, RPL14, RPL15, RPL17, RPL18, RPL18A, RPL19, RPL21, RPL22, RPL23, RPL23A, RPL24, RPL26, RPL27, RPL27A, RPL28, RPL29, RPL30, RPL31, RPL32, RPL34, RPL35, RPL35A, RPL36, RPL36A, RPL37, RPL37A, RPL38, RPL39, RPL40, RPL41, RPLP0, RPLP1, RPLP2, RPLP3, UBA52 or from a variant thereof.
31. The artificial nucleic acid molecule according to any one of claims 2-30, wherein the at least one histone stem-loop comprises or consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 1391-1433, preferably from the group consisting of SEQ ID NOs. 1403-1433.
32. The artificial nucleic acid molecule according to any one of claims 2-31, wherein the histone stem-loop comprises or consists of a nucleic acid sequence having a sequence identity of at least about 75%, preferably of at least about 80%, preferably of at least about 85%, more preferably of at least about 90%, even more preferably of at least about 95% to the sequence according to SEQ ID NO. 1433 or to the corresponding RNA sequence, wherein preferably positions 6, 13 and 20 of the sequence having a sequence identity of at least about 75%, preferably of at least about 80%, preferably at least about 85%, more preferably at least about 90%, even more preferably at least about 95% to the sequence according to SEQ ID NO. 1433 or to the corresponding RNA sequence are conserved, i.e. are identical to the nucleotides at positions 6, 13 and 20 of SEQ ID NO. 1433 or to the corresponding RNA nucleotides.
33. The artificial nucleic acid molecule according to any one of claims 17-32, wherein the 3UTR element comprises or consists of a nucleic acid sequence derived from a 3UTR of a gene selected from the group consisting of an albumin gene, an -globin gene, a -globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, and a collagen alpha gene, or from a variant of a 3UTR of a gene selected from the group consisting of an albumin gene, an -globin gene, a -globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, and a collagen alpha gene.
34. The artificial nucleic acid molecule according to any one of claims 17-33, wherein the at least one 3UTR element comprises or consists of a nucleic acid sequence which is derived from the 3UTR of a vertebrate albumin gene or from a variant thereof, preferably from the 3UTR of a mammalian albumin gene or from a variant thereof, more preferably from the 3UTR of a human albumin gene or from a variant thereof, even more preferably from the 3UTR of the human albumin gene according to GenBank Accession number NM_000477.5 or from a variant thereof.
35. The artificial nucleic acid molecule according to any one of claims 17-33, wherein the at least one 3UTR element comprises or consists of a nucleic acid sequence which is derived from the 3UTR of a vertebrate -globin gene or from a variant thereof, preferably from the 3UTR of a mammalian -globin gene or from a variant thereof, more preferably from the 3UTR of a human -globin gene or from a variant thereof.
36. The artificial nucleic acid molecule according to any one of claims 17-33, wherein the at least one 3UTR element comprises or consists of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to a nucleic acid sequence selected from SEQ ID NOs. 1369-1377 and 1434 or to a corresponding RNA sequence, or wherein the at least one 3UTR element comprises or consists of a fragment of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to a nucleic acid sequence selected from SEQ ID NOs. 1369-1377 and 1434 or to a corresponding RNA sequence.
37. The artificial nucleic acid molecule according to claim 36, wherein the fragment consists of a continuous stretch of nucleotides corresponding to a continuous stretch of nucleotides in the full-length sequence, which represents at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, and most preferably at least 90% of the full-length sequence the fragment is derived from.
38. The artificial nucleic acid molecule according to any one of claims 17-37, wherein the 3UTR element exhibits a length of at least about 40 nucleotides, preferably of at least about 50 nucleotides, preferably of at least about 75 nucleotides, more preferably of at least about 100 nucleotides, even more preferably of at least about 125 nucleotides, most preferably of at least about 150 nucleotides.
39. The artificial nucleic acid molecule according to any one of claims 1-38, wherein the artificial nucleic acid molecule, preferably the open reading frame, is at least partially G/C modified, preferably wherein the G/C content of the open reading frame is increased compared to the wild type open reading frame.
40. The artificial nucleic acid molecule according to any one of claims 1-39, wherein the open reading frame comprises a codon-optimized region, preferably, wherein the open reading frame is codon-optimized.
41. The artificial nucleic acid molecule according to any one of claims 1-40, which is an RNA, preferably an mRNA molecule.
42. A vector comprising: a. at least one 5-untranslated region element (5UTR element) which comprises or consists of a nucleic acid sequence which is derived from the 5UTR of a TOP gene or which is derived from a variant of the 5UTR of a TOP gene; and b. at least one open reading frame (ORF) and/or at least one cloning site.
43. The vector according to claim 42, further comprising: c. at least one histone-stem loop.
44. The vector according to claim 42 or 43, wherein the 5UTR element and the open reading frame are heterologous.
45. The vector according to any one of claims 42-44, wherein the 5UTR element is suitable for increasing protein production from the vector.
46. The vector to any one of claims 43-45, wherein the 5UTR element and the histone stem-loop act together, preferably at least additively, to increase protein production from the vector.
47. The vector according to any one of claims 42-46, wherein the 5UTR element does not comprise a TOP-motif, preferably wherein the nucleic acid sequence which is derived from a 5UTR of a TOP gene, preferably the 5UTR element starts at its 5-end with a nucleotide located at position 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 downstream of the polypyrimidine tract.
48. The vector according to any one of claims 42-47, wherein the nucleic acid sequence which is derived from a 5UTR of a TOP gene, preferably the 5UTR element terminates at its 3-end with a nucleotide located at position 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 upstream of the start codon of the gene it is derived from.
49. The vector according to any one of claims 42-48, wherein the 5UTR element does not comprise a start codon or an open reading frame.
50. The vector according to any one of claims 42-49, wherein the nucleic acid sequence which is derived from the 5UTR of a TOP gene is derived from the 5UTR of a eukaryotic TOP gene or from a variant thereof, preferably from the 5UTR of a plant or animal TOP gene or from a variant thereof, more preferably from the 5UTR of a chordate TOP gene or from a variant thereof, even more preferably from the 5UTR of a vertebrate TOP gene or from a variant thereof, most preferably from the 5UTR of a mammalian TOP gene, such as a human TOP gene, or from a variant thereof.
51. The vector according to any one of claims 43-50, wherein the at least one histone stem-loop is selected from following formulae (I) or (II): ##STR00009## wherein: stem1 or stem2 bordering elements N.sub.1-6 is a consecutive sequence of 1 to 6, preferably of 2 to 6, more preferably of 2 to 5, even more preferably of 3 to 5, most preferably of 4 to 5 or 5 N, wherein each N is independently from another selected from a nucleotide selected from A, U, T, G and C, or a nucleotide analogue thereof; stem1 [N.sub.0-2GN.sub.3-5] is reverse complementary or partially reverse complementary with element stem2, and is a consecutive sequence between of 5 to 7 nucleotides; wherein N.sub.0-2 is a consecutive sequence of 0 to 2, preferably of 0 to 1, more preferably of 1 N, wherein each N is independently from another selected from a nucleotide selected from A, U, T, G and C or a nucleotide analogue thereof; wherein N.sub.3-5 is a consecutive sequence of 3 to 5, preferably of 4 to 5, more preferably of 4 N, wherein each N is independently from another selected from a nucleotide selected from A, U, T, G and C or a nucleotide analogue thereof, and wherein G is guanosine or an analogue thereof, and may be optionally replaced by a cytidine or an analogue thereof, provided that its complementary nucleotide cytidine in stem2 is replaced by guanosine; loop sequence [N.sub.0-4(U/T)N.sub.0-4] is located between elements stem1 and stem2, and is a consecutive sequence of 3 to 5 nucleotides, more preferably of 4 nucleotides; wherein each N.sub.0-4 is independent from another a consecutive sequence of 0 to 4, preferably of 1 to 3, more preferably of 1 to 2 N, wherein each N is independently from another selected from a nucleotide selected from A, U, T, G and C or a nucleotide analogue thereof; and wherein U/T represents uridine, or optionally thymidine; stem2 [N.sub.3-5CN.sub.0-2] is reverse complementary or partially reverse complementary with element stem1, and is a consecutive sequence between of 5 to 7 nucleotides; wherein N.sub.3-5 is a consecutive sequence of 3 to 5, preferably of 4 to 5, more preferably of 4 N, wherein each N is independently from another selected from a nucleotide selected from A, U, T, G and C or a nucleotide analogue thereof; wherein N.sub.0-2 is a consecutive sequence of 0 to 2, preferably of 0 to 1, more preferably of 1 N, wherein each N is independently from another selected from a nucleotide selected from A, U, T, G and C or a nucleotide analogue thereof; and wherein C is cytidine or an analogue thereof, and may be optionally replaced by a guanosine or an analogue thereof provided that its complementary nucleotide guanosine in stem1 is replaced by cytidine; wherein stem1 and stem2 are capable of base pairing with each other forming a reverse complementary sequence, wherein base pairing may occur between stem1 and stem2, or forming a partially reverse complementary sequence, wherein an incomplete base pairing may occur between stem1 and stem2.
52. The vector according to any one of claims 43-51, wherein the at least one histone stem-loop is selected from at least one of following formulae (Ia) or (IIa): ##STR00010##
53. The vector according to any one of claims 42-52, further comprising d. a poly(A) sequence and/or a polyadenylation signal.
54. The vector according to claim 53, wherein the poly(A) sequence comprises or consists of a sequence of about 25 to about 400 adenosine nucleotides, preferably a sequence of about 50 to about 400 adenosine nucleotides, more preferably a sequence of about 50 to about 300 adenosine nucleotides, even more preferably a sequence of about 50 to about 250 adenosine nucleotides, most preferably a sequence of about 60 to about 250 adenosine nucleotides.
55. The vector according to claim 53 or 54, wherein the polyadenylation signal comprises the consensus sequence NN(U/T)ANA, with N=A or U, preferably AA(U/T)AAA or A(U/T)(U/T)AAA.
56. The vector according to any one of claims 42-55, further comprising: e. a poly(C) sequence.
57. The vector according to claim 56, wherein the poly(C) sequence comprises, preferably consists of, about 10 to about 200 cytidine nucleotides, more preferably about 10 to about 100 cytidine nucleotides, more preferably about 10 to about 50 cytidine nucleotides, even more preferably about 20 to about 40 cytidine nucleotides.
58. The vector according to any one of claims 42-57, further comprising: f. at least one 3UTR element.
59. The vector according to claim 58, wherein the at least one 3UTR element comprises or consists of a nucleic acid sequence which is derived from a 3UTR of a gene providing a stable mRNA or from a variant of the 3UTR of a gene providing a stable mRNA.
60. The vector according to claim 58 or 59, wherein the at least one 3UTR element and the at least one 5UTR element act at least additively, preferably synergistically to increase protein production from said vector.
61. The vector according to any one of claims 42-60, wherein the 5UTR element comprises or consists of a nucleic acid sequence which is derived from a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 1-1363, SEQ ID NO. 1435, SEQ ID NO. 1461 or SEQ ID NO. 1462, from the homologs of any of SEQ ID NOs. 1-1363, SEQ ID NO. 1435, SEQ ID NO. 1461 or SEQ ID NO. 1462, or from a variant thereof.
62. The vector according to any one of claims 42-61, wherein the 5UTR element comprises or consists of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to a nucleic acid sequence selected from SEQ ID NOs. 1-1363, SEQ ID NO. 1435, SEQ ID NO. 1461 or SEQ ID NO. 1462, or to a corresponding RNA sequence, preferably lacking the 5TOP motif, or wherein the at least one 5UTR element comprises or consists of a fragment of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to a nucleic acid sequence selected from SEQ ID NOs. 1-1363, SEQ ID NO. 1435, SEQ ID NO. 1461 or SEQ ID NO. 1462, or to a corresponding RNA sequence, preferably lacking the 5TOP motif.
63. The vector according to any one of claims 42-62, wherein the 5UTR element comprises or consists of a nucleic acid sequence which is derived from a 5UTR of a TOP gene encoding a ribosomal protein or from a variant of a 5UTR of a TOP gene encoding a ribosomal protein, preferably from a 5UTR of a nucleic acid sequence according to any of SEQ ID NOs: 170, 232, 244, 259, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291, 1292, 1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300, 1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312, 1313, 1314, 1315, 1316, 1317, 1318, 1319, 1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327, 1328, 1329, 1330, 1331, 1332, 1333, 1334, 1335, 1336, 1337, 1338, 1339, 1340, 1341, 1342, 1343, 1344, 1346, 1347, 1348, 1349, 1350, 1351, 1352, 1353, 1354, 1355, 1356, 1357, 1358, 1359, or 1360, a corresponding RNA sequence, a homolog thereof, or a variant thereof as described herein, preferably lacking the 5TOP motif.
64. The vector according to any one of claims 42-63, wherein the 5UTR element comprises or consists of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the 5UTR of a nucleic acid sequence according to any of SEQ ID NOs: 170, 232, 244, 259, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291, 1292, 1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300, 1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312, 1313, 1314, 1315, 1316, 1317, 1318, 1319, 1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327, 1328, 1329, 1330, 1331, 1332, 1333, 1334, 1335, 1336, 1337, 1338, 1339, 1340, 1341, 1342, 1343, 1344, 1346, 1347, 1348, 1349, 1350, 1351, 1352, 1353, 1354, 1355, 1356, 1357, 1358, 1359, or 1360 or to a corresponding RNA sequence, preferably lacking the 5TOP motif, or wherein the at least one 5UTR element comprises or consists of a fragment of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the 5UTR of a nucleic acid sequence according to SEQ ID NOs: 170, 232, 244, 259, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291, 1292, 1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300, 1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312, 1313, 1314, 1315, 1316, 1317, 1318, 1319, 1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327, 1328, 1329, 1330, 1331, 1332, 1333, 1334, 1335, 1336, 1337, 1338, 1339, 1340, 1341, 1342, 1343, 1344, 1346, 1347, 1348, 1349, 1350, 1351, 1352, 1353, 1354, 1355, 1356, 1357, 1358, 1359, or 1360 or to a corresponding RNA sequence, preferably lacking the 5TOP motif.
65. The vector according to any one of claims 42-64, wherein the 5UTR element is derived from a 5UTR of a TOP gene encoding a ribosomal Large protein (RPL) or from a variant of a 5UTR of a TOP gene encoding a ribosomal Large protein (RPL), preferably from a 5UTR of a nucleic acid sequence according to any of SEQ ID NOs: 67, 259, 1284-1318, 1344, 1346, 1348-1354, 1357, 1461 and 1462, a corresponding RNA sequence, a homolog thereof, or a variant thereof, preferably lacking the 5TOP motif.
66. The vector according to any one of claims 42-65, wherein the 5UTR element comprises or consists of a nucleic acid sequence having an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the 5UTR of a nucleic acid sequence according to any of SEQ ID NOs: 67, 259, 1284-1318, 1344, 1346, 1348-1354, 1357, 1461 and 1462 or to a corresponding RNA sequence, preferably lacking the 5TOP motif, or wherein the at least one 5UTR element comprises or consists of a fragment of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the 5UTR of a nucleic acid sequence according to SEQ ID No. SEQ ID NOs: 67, 259, 1284-1318, 1344, 1346, 1348-1354, 1357, 1461 and 1462 or to a corresponding RNA sequence, preferably lacking the 5TOP motif.
67. The vector according to any one of claims 42-66, wherein the 5UTR element comprises or consists of a nucleic acid sequence which is derived from the 5UTR of a ribosomal protein Large 32 gene, preferably from the 5UTR of a vertebrate ribosomal protein Large 32 (L32) gene or from a variant thereof, more preferably from the 5UTR of a mammalian ribosomal protein Large 32 (L32) gene or from a variant thereof, most preferably from the 5UTR of a human ribosomal protein Large 32 (L32) gene or from a variant thereof, wherein preferably the 5UTR element does not comprise the 5TOP of said gene.
68. The vector according to any one of claims 42-67, wherein the 5UTR element comprises or consists of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the nucleic acid sequence according to SEQ ID NOs. 1368 or 1452-1460 or to a corresponding RNA sequence, or wherein the at least one 5UTR element comprises or consists of a fragment of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the nucleic acid sequence according to SEQ ID NOs. 1368 or 1452-1460 or to a corresponding RNA sequence.
69. The vector according to any one of claims 62, 64, 66 and 68, wherein the fragment consists of a continuous stretch of nucleotides corresponding to a continuous stretch of nucleotides in the full-length sequence, which represents at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, and most preferably at least 90% of the full-length sequence the fragment is derived from.
70. The vector according to any one of claims 42-69, wherein the at least one 5UTR element exhibits a length of at least about 20 nucleotides, preferably of at least about 30 nucleotides, more preferably of at least about 40 nucleotides.
71. The vector according to any one of claims 42-70, wherein the 5UTR element comprises or consists of a nucleic acid sequence which is derived from the 5UTR of a TOP gene selected from RPSA, RPS2, RPS3, RPS3A, RPS4, RPS5, RPS6, RPS7, RPS8, RPS9, RPS10, RPS11, RPS12, RPS13, RPS14, RPS15, RPS15A, RPS16, RPS17, RPS18, RPS19, RPS20, RPS21, RPS23, RPS24, RPS25, RPS26, RPS27, RPS27A, RPS28, RPS29, RPS30, RPL3, RPL4, RPL5, RPL6, RPL7, RPL7A, RPL8, RPL9, RPL10, RPL10A, RPL11, RPL12, RPL13, RPL13A, RPL14, RPL15, RPL17, RPL18, RPL18A, RPL19, RPL21, RPL22, RPL23, RPL23A, RPL24, RPL26, RPL27, RPL27A, RPL28, RPL29, RPL30, RPL31, RPL32, RPL34, RPL35, RPL35A, RPL36, RPL36A, RPL37, RPL37A, RPL38, RPL39, RPL40, RPL41, RPLP0, RPLP1, RPLP2, RPLP3, UBA520r from a variant thereof.
72. The vector according to any one of claims 43-71, wherein the at least one histone stem-loop comprises or consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 1391-1433, preferably from the group consisting of SEQ ID NOs. 1403-1433.
73. The vector according to any one of claims 43-72, wherein the histone stem-loop comprises or consists of a nucleic acid sequence having a sequence identity of at least about 75%, preferably of at least about 80%, preferably at least about 85%, more preferably at least about 90%, even more preferably at least about 95% to the sequence according to SEQ ID NO. 1433 or to the corresponding RNA sequence, wherein preferably positions 6, 13 and 20 of the sequence having a sequence identity of at least about 75%, preferably of at least about 80%, preferably at least about 85%, more preferably at least about 90%, even more preferably at least about 95% to the sequence according to SEQ ID NO. 1433 or to the corresponding RNA sequence are conserved, i.e. are identical to the nucleotides at positions 6, 13 and 20 of SEQ ID NO. 1433 or to the corresponding RNA nucleotides.
74. The vector according to any one of claims 58-73, wherein the 3UTR element comprises or consists of a nucleic acid sequence derived from a 3UTR of a gene selected from the group consisting of an albumin gene, an -globin gene, a -globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, and a collagen alpha gene, or from a variant of a 3UTR of a gene selected from the group consisting of an albumin gene, an -globin gene, a -globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, and a collagen alpha gene.
75. The vector according to any one of claims 58-74, wherein the at least one 3UTR element comprises or consists of a nucleic acid sequence which is derived from the 3UTR of a vertebrate albumin gene or from a variant thereof, preferably from the 3UTR of a mammalian albumin gene or from a variant thereof, more preferably from the 3UTR of a human albumin gene or from a variant thereof, even more preferably from the 3UTR of the human albumin gene according to GenBank Accession number NM_000477.5 or from a variant thereof.
76. The vector according to any one of claims 58-74, wherein the at least one 3UTR element comprises or consists of a nucleic acid sequence which is derived from the 3UTR of a vertebrate -globin gene or from a variant thereof, preferably from the 3UTR of a mammalian -globin gene or from a variant thereof, more preferably from the 3UTR of a human -globin gene or from a variant thereof.
77. The vector according to any one of claims 58-74, wherein the at least one 3UTR element comprises or consists of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to a nucleic acid sequence selected from SEQ ID NOs. 1369-1377 and 1434 or to a corresponding RNA sequence, or wherein the at least one 3UTR element comprises or consists of a fragment of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to a nucleic acid sequence selected from SEQ ID NOs. 1369-1377 and 1434 or to a corresponding RNA sequence.
78. The vector according to claim 77, wherein the fragment consists of a continuous stretch of nucleotides corresponding to a continuous stretch of nucleotides in the full-length sequence, which represents at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, and most preferably at least 90% of the full-length sequence the fragment is derived from.
79. The vector according to any one of claims 58-78, wherein the 3UTR element exhibits a length of at least about 40 nucleotides, preferably of at least about 50 nucleotides, preferably of at least about 75 nucleotides, more preferably of at least about 100 nucleotides, even more preferably of at least about 125 nucleotides, most preferably of at least about 150 nucleotides.
80. The vector according to any one of claims 42-79, wherein the vector, preferably the open reading frame, is at least partially G/C modified, preferably wherein the G/C content of the open reading frame is increased compared to the wild type open reading frame.
81. The vector according to any one of claims 42-80, wherein the open reading frame comprises a codon-optimized region, preferably, wherein the open reading frame is codon-optimized.
82. The vector according to any one of claims 42-81, which is a DNA vector.
83. The vector according to any one of claims 42-82, which is a plasmid vector or a viral vector, preferably a plasmid vector.
84. The vector according to any one of claims 42-83, which comprises or codes for an artificial nucleic acid molecule according to any one of claims 1-41.
85. The vector according to any one of claims 42-84, which is a circular molecule.
86. A cell comprising the artificial nucleic acid molecule according to any one of claims 1-41 or the vector according to any one of claims 42-85.
87. The cell according to claim 86, which is a mammalian cell.
88. The cell according to claim 86 or 87, which is a cell of a mammalian subject, preferably an isolated cell of a mammalian subject, preferably of a human subject.
89. A pharmaceutical composition comprising the artificial nucleic acid molecule according to any one of claims 1-41, the vector according to any one of claims 42-85, or the cell according to any one of claims 86-88.
90. The pharmaceutical composition according to claim 89, further comprising one or more pharmaceutically acceptable diluents and/or excipients and/or one or more adjuvants.
91. The artificial nucleic acid molecule according to any one of claims 1-41, the vector according to any one of claims 42-85, the cell according to any one of claims 86-88, or the pharmaceutical composition according to claim 89 or 90 for use as a medicament.
92. The artificial nucleic acid molecule according to any one of claims 1-41, the vector according to any one of claims 42-85, the cell according to any one of claims 86-88, or the pharmaceutical composition according to claim 89 or 90 for use as a vaccine or for use in gene therapy.
93. A method for treating or preventing a disorder comprising administering the artificial nucleic acid molecule according to any one of claims 1-41, the vector according to any one of claims 42-85, the cell according to any one of claims 86-88, or the pharmaceutical composition according to claim 89 or 90 to a subject in need thereof.
94. A method of treating or preventing a disorder comprising transfection of a cell with the artificial nucleic acid molecule according to any one of claims 1-41 or the vector according to any one of claims 42-85.
95. The method according to claim 94, wherein transfection of a cell is performed in vitro/ex vivo and the transfected cell is administered to a subject in need thereof, preferably to a human patient.
96. The method according to claim 95, wherein the cell which is to be transfected in vitro is an isolated cell of the subject, preferably of the human patient.
97. The method according to any one of claims 93-96, which is a vaccination method or a gene therapy method.
98. A method for increasing protein production from an artificial nucleic acid molecule, comprising the step of providing the artificial nucleic acid molecule with i. at least one 5-untranslated region element (5UTR element) which comprises or consists of a nucleic acid sequence which is derived from the 5UTR of a TOP gene or which is derived from a variant of the 5UTR of a TOP gene; ii. preferably at least one histone stem-loop; and iii. optionally, a poly(A) sequence and/or a polyadenylation signal.
99. Use of a 5UTR element which comprises or consists of a nucleic acid sequence which is derived from the 5UTR of a TOP gene or which is derived from a variant of the 5UTR of a TOP gene and preferably at least one histone stem-loop for increasing protein production from a nucleic acid molecule.
100. A kit or kit of parts comprising an artificial nucleic acid molecule according to any one of claims 1-41, the vector according to any one of claims 42-85, the cell according to any one of claims 86-88, and/or the pharmaceutical composition according to claim 89 or 90.
101. The kit according to claim 100, further comprising instructions for use, cells for transfection, an adjuvant, a means for administration of the pharmaceutical composition, a pharmaceutically acceptable carrier and/or a pharmaceutically acceptable solution for dissolution or dilution of the artificial nucleic acid molecule, the vector or the pharmaceutical composition.
Description
[0390] The following Figures, Sequences and Examples are intended to illustrate the invention further. They are not intended to limit the subject-matter of the invention thereto.
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[0430] SEQ ID No. 1-1363. 1435, and 1461-1462 sequences comprising 5UTRs of TOP genes [0431] SEQ ID No. 1364 PpLuc(GC)-ag-A64 (
Examples
[0549] 1. Preparation of DNA-Templates
[0550] A vector for in vitro transcription was constructed containing a T7 promoter followed by a GC-enriched sequence coding for Photinus pyralis luciferase (PpLuc(GC)) and an A64 poly(A) sequence. The poly(A) sequence was followed by a restriction site used for linearization of the vector before in vitro transcription. mRNA obtained from this vector accordingly by in vitro transcription is designated as PpLuc(GC)-A64.
[0551] This vector was modified to include untranslated sequences 5 or 3 of the open reading frame. In summary, vectors comprising the following mRNA encoding sequences have been generated:
[0552] SEQ ID No. 1364 PpLuc(GC)-ag-A64 (
[0553] SEQ ID No. 1365 RPL32-PpLuc(GC)-ag-A64-C30-histoneSL (
[0554] SEQ ID NO. 1436 RPL35-PpLuc(GC)-albumin7-A64-C30-histoneSL (
[0555] SEQ ID NO. 1437 RPL21-PpLuc(GC)-albumin7-A64-C30-histoneSL (
[0556] SEQ ID NO. 1438 ATP5A1-PpLuc(GC)-albumin7-A64-C30-histoneSL (
[0557] SEQ ID NO. 1439 HSD17B4-PpLuc(GC)-albumin7-A64-C30-histoneSL (
[0558] SEQ ID NO. 1440 AIG1-PpLuc(GC)-albumin7-A64-C30-histoneSL (
[0559] SEQ ID NO. 1441 COX6C-PpLuc(GC)-albumin7-A64-C30-histoneSL (
[0560] SEQ ID NO. 1442 ASAH1-PpLuc(GC)-albumin7-A64-C30-histoneSL (
[0561] SEQ ID NO. 1443 mRPL21-PpLuc(GC)-albumin7-A64-C30-histoneSL (
[0562] SEQ ID NO. 1444 mRPL35A-PpLuc(GC)-albumin7-A64-C30-histoneSL (
[0563] SEQ ID NO. 1445 RPL35-PpLuc(GC)-A64-C30-histoneSL
[0564] SEQ ID NO. 1446 RPL21-PpLuc(GC)-A64-C30-histoneSL
[0565] SEQ ID NO. 1447 ATP5A1-PpLuc(GC)-A64-C30-histoneSL
[0566] SEQ ID NO. 1448 HSD 17B4-PpLuc(GC)-A64-C30-histoneSL
[0567] SEQ ID NO. 1449 AIG1-PpLuc(GC)-A64-C30-histoneSL
[0568] SEQ ID NO. 1450 COX6C-PpLuc(GC)-A64-C30-histoneSL
[0569] SEQ ID NO. 1451 ASAH1-PpLuc(GC)-A64-C30-histoneSL
[0570] SEQ ID NO. 1463 RPL32-PpLuc(GC)-ag-A64 (
[0571] SEQ ID NO. 1464 PpLuc(GC)-ag-A64-histoneSL (
[0572] SEQ ID NO. 1465 PpLuc(GC)-albumin7-A64-C30-histoneSL
[0573] SEQ ID NO. 1466 RPL35-PpLuc(GC)-ag-A64 (
[0574] SEQ ID NO. 1467 RPL21-PpLuc(GC)-ag-A64 (
[0575] SEQ ID NO. 1468 atp5a1-PpLuc(GC)-ag-A64 (
[0576] SEQ ID NO. 1469 HSD17B4-PpLuc(GC)-ag-A64 (
[0577] SEQ ID NO. 1470 AIG1-PpLuc(GC)-ag-A64 (
[0578] SEQ ID NO. 1471 COX6C-PpLuc(GC)-ag-A64 (
[0579] SEQ ID NO. 1472 ASAH1-PpLuc(GC)-ag-A64 (
[0580] SEQ ID NO. 1473 RPL35-PpLuc(GC)-ag-A64-histoneSL (
[0581] SEQ ID NO. 1474 RPL21-PpLuc(GC)-ag-A64-histoneSL (
[0582] SEQ ID NO. 1475 atp5a1-PpLuc(GC)-ag-A64-histoneSL (
[0583] SEQ ID NO. 1476 HSD17B4-PpLuc(GC)-ag-A64-histoneSL (
[0584] SEQ ID NO. 1477 AIG1-PpLuc(GC)-ag-A64-histoneSL (
[0585] SEQ ID NO. 1478 COX6C-PpLuc(GC)-ag-A64-histoneSL (
[0586] SEQ ID NO. 1479 ASAH1-PpLuc(GC)-ag-A64-histoneSL (
[0587] SEQ ID NO. 1480 RPL32-PpLuc(GC)-ag-A64-histoneSL (
[0588] SEQ ID NO. 1481 RPL32-PpLuc(GC)-albumin7-A64-C30-histoneSL (
[0589] 2. In Vitro Transcription
[0590] The DNA-template according to Example 1 was linearized and transcribed in vitro using T7-Polymerase. The DNA-template was then digested by DNase-treatment. mRNA transcripts contained a 5-CAP structure obtained by adding an excess of N7-Methyl-Guanosine-5-Triphosphate-5-Guanosine to the transcription reaction. mRNA thus obtained was purified and resuspended in water.
[0591] 3. Luciferase Expression by mRNA Lipofection
[0592] Human dermal fibroblasts (HDF) were seeded in 24 well plates at a density of 510.sup.4 cells per well. The following day, cells were washed in opti-MEM and then transfected with 50 ng per well of Lipofectamine2000-complexed PpLuc-encoding mRNA in opti-MEM. As a control, mRNA not coding for PpLuc was lipofected separately. mRNA coding for Renilla reniformis luciferase (RrLuc) was transfected together with PpLuc mRNA to control for transfection efficiency (20 ng of RrLuc mRNA per well). 90 minutes after start of transfection, opti-MEM was exchanged for medium. 24, 48, 72 hours after transfection, medium was aspirated and cells were lysed in 200 l of lysis buffer (25 mM Tris, pH 7.5 (HCl), 2 mM EDTA, 10% glycerol, 1% Triton X-100, 2 mM DTT, 1 mM PMSF). Lysates were stored at 20 C. until luciferase activity was measured.
[0593] Alternatively, HDF were seeded in 96 well plates one to three days before transfection at a density of 10.sup.4 cells per well. Immediately before lipofection, cells were washed in optiMEM. Cells were lipofected with 25 ng of PpLuc-encoding mRNA per well complexed with Lipofectamine2000. In some experiments, mRNA coding for Renilla reniformis luciferase (RrLuc) was transfected together with PpLuc mRNA to control for transfection efficiency (2.5 ng of RrLuc mRNA per well). 90 minutes after start of transfection, opti-MEM was exchanged for medium. At various time points post transfection, medium was aspirated and cells were lysed in 100 l of lysis buffer (Passive Lysis Buffer, Promega). Lysates were stored at 80 C. until luciferase activity was measured.
[0594] 4. Luciferase Measurement
[0595] Luciferase activity was measured as relative light units (RLU) in a BioTek SynergyHT plate reader. PpLuc activity was measured at 15 seconds measuring time using 50 l of lysate and 200 l of luciferin buffer (75 l VI luciferin, 25 mM Glycylglycin, pH 7.8 (NaOH), 15 mM MgSO4, 2 mM ATP). RrLuc activity was measured at 15 seconds measuring time using 50 l of lysate and 200 l of coelenterazin buffer (40 M coelenterazin in phosphate buffered saline adjusted to 500 mM NaCl).
[0596] Alternatively, luciferase activity was measured as relative light units (RLU) in a Hidex Chameleon plate reader. PpLuc activity was measured at 2 seconds measuring time using 20 l of lysate and 50 l of luciferin buffer (Beetle-Juice, PJK GmbH). RrLuc activity was measured at 2 seconds measuring time using 20 l of lysate and 50 l of coelenterazin buffer (Renilla-Juice, PJK GmbH).
[0597] Results
[0598] 5.1 the Combination of 5UTR Elements Derived from 5UTRs of TOP Genes and Histone Stem-Loop Increases Protein Expression Strongly.
[0599] To investigate the effect of the combination of a 5UTR element derived from a 5UTR of a TOP gene and a histone stem-loop (histoneSL) on protein expression from mRNA, mRNAs with different UTRs were synthesized: mRNAs either lacked both 5UTR element and histoneSL, or contained both 5UTR element and histoneSL. Luciferase-encoding mRNAs or control mRNA were transfected into human dermal fibroblasts (HDF). Luciferase levels were measured at 24 hours after transfection (see following Table 1 and
TABLE-US-00006 TABLE 1 mRNA RLU at 24 hours control RNA 588 PpLuc(GC) - ag - A64 12246 RPL32 - PpLuc(GC) - ag - 319840 A64 - C30 - histoneSL
[0600] Luciferase was clearly expressed from mRNA having neither 5UTR element nor histoneSL. Strikingly however, the combination of 5UTR element and histoneSL strongly increased the luciferase level. The magnitude of the rise in luciferase level due to combining 5UTR element and histoneSL in the same mRNA indicates that they are acting synergistically.
[0601] 5.2 the Combination of 5UTR Elements Derived from 5UTRs of TOP Genes and Histone Stem-Loop Increases Protein Expression from mRNA in a Synergistic Manner.
[0602] To investigate the effect of the combination of a 5UTR element derived from a 5UTR of a TOP gene and histone stem-loop on protein expression from mRNA, mRNAs with different UTRs were synthesized: mRNAs either lacked both 5UTR element and histone stem-loop, or contained either a 5UTR element or a histone stem-loop, or both 5UTR element and histone stem-loop. Luciferase-encoding mRNAs were transfected into human dermal fibroblasts (HDF). Luciferase levels were measured at 8, 24, and 48 hours after transfection (see following Table 2 and
TABLE-US-00007 TABLE 2 RLU at 8 RLU at 24 RLU at 48 mRNA hours hours hours PpLuc(GC)-ag-A64 13110 25861 14362 PpLuc(GC)-ag-A64-histoneSL 88640 97013 57026 rpl32-PpLuc(GC)-ag-A64 155654 212245 102528 rpl32-PpLuc(GC)-ag-A64-histoneSL 301384 425825 161974
[0603] Luciferase was clearly expressed from mRNA having neither 5UTR element nor histone stem-loop. Both, either the histone stem-loop or the 5UTR element increased luciferase levels compared to mRNA lacking both these elements. Strikingly however, the combination of 5UTR element and histone stem-loop further strongly increased the luciferase level, much above the level observed with either of the individual elements. The magnitude of the rise in luciferase level due to combining 5UTR element and histone stem-loop in the same mRNA demonstrates that they are acting synergistically.
[0604] The synergy between 5UTR element and histone stem-loop was quantified by dividing the signal from mRNA combining both elements by the sum of the signal from mRNA lacking both elements plus the rise in signal effected by the 5UTR element plus the rise in signal effected by the histone stem-loop. This calculation was performed for the three time points individually and for total protein expressed from 0 to 48 hours calculated from the area under the curve (AUC) (see following Table 3).
TABLE-US-00008 TABLE 3 RLU predicted rpl32 histoneSL RLU RLU (additive) synergy 8 h 13110 + 88640 75530 + 155654 142544 + + 301384 231184 1.30 24 h 25861 + 97013 71152 + 212245 186384 + + 425825 283397 1.50 48 h 14362 + 57026 42664 + 102528 88166 + + 161974 145192 1.12 AUC 0-48 hours 846881 + 3688000 2841119 + 7343000 6496119 + + 14080000 10184119 1.38
[0605] The synergy thus calculated specifies how much higher the luciferase level from mRNA combining 5UTR element and histone stem-loop is than would be expected if the effects of 5UTR element and histone stem-loop were purely additive. This result confirms that the combination of 5UTR element and histone stem-loop effects a markedly synergistic increase in protein expression.
[0606] 5.3 5UTR Elements Derived from 5UTRs of TOP Genes Increase Protein Expression from mRNA.
[0607] To investigate the effect of 5UTR elements derived from 5UTRs of TOP genes on protein expression from mRNA, mRNAs with one of different 5UTR elements were synthesized. In addition, mRNAs contained the albumin? 3UTR element. Luciferase-encoding mRNAs were transfected into human dermal fibroblasts (HDF). Luciferase levels were measured at 24, 48, and 72 hours after transfection (see following Table 4 and
TABLE-US-00009 TABLE 4 5UTR RLU at 24 hours RLU at 48 hours RLU at 72 hours none 114277 121852 68235 rpl32 332236 286792 114148 rpl35 495917 234070 96993 rpl21 563314 352241 156605 atp5a1 1000253 538287 187159 HSD17B4 1179847 636877 299337 AIG1 620315 446621 167846 COX6C 592190 806065 173743 ASAH1 820413 529901 198429
[0608] Luciferase was clearly expressed from mRNA lacking a 5UTR element. Strikingly however, all 5UTR elements strongly increased the luciferase level.
[0609] 5.4 the Combination of 5UTR Elements Derived from 5UTRs of TOP Genes and Histone Stem-Loop Increases Protein Expression from mRNA in a Synergistic Manner.
[0610] To investigate the effect of the combination of 5UTR elements derived from the 5UTRs of TOP genes and histone stem-loop on protein expression from mRNA, mRNAs with different UTRs were synthesized: mRNAs either lacked both 5UTR element and histone stem-loop, or contained a histone stem-loop, or contained one of different 5UTR elements derived from 5UTRs of TOP genes, or contained both one of different 5UTR elements and a histone stem-loop. In addition, mRNAs contained the alpha-globin 3UTR element. Luciferase-encoding mRNAs were transfected into human dermal fibroblasts (HDF). Luciferase levels were measured at 8, 24, and 48 hours after transfection (see
[0611] The synergy between 5UTR element and histone stem-loop was quantified by dividing the signal from mRNA combining both elements by the sum of the signal from mRNA lacking both elements plus the rise in signal effected by the 5UTR element plus the rise in signal effected by the histone stem-loop. This calculation was performed for total protein expressed from 0 to 48 hours calculated from the area under the curve (AUC) (see following Table 5).
TABLE-US-00010 TABLE 5 TOP 5UTR Synergy with histone stem-loop 35L 2.50 21L 3.25 atp5a1 3.00 HSD17B4 3.55 AIG1 1.52 COX6C 3.19
[0612] The synergy thus calculated specifies how much higher the luciferase level from mRNA combining 5UTR element and histone stem-loop is than would be expected if the effects of 5UTR element and histone stem-loop were purely additive. The luciferase level from mRNA combining 5UTR element and histone stem-loop was up to more than three times higher than if their effects were purely additive. This result confirms that the combination of 5UTR element and histone stem-loop effects a markedly synergistic increase in protein expression.
[0613] 5.5 5UTR Elements Derived from 5UTRs of Mouse TOP Genes Increase Protein Expression from mRNA.
[0614] To investigate the effect of TOP 5UTR elements derived from 5UTRs of mouse TOP genes on protein expression from mRNA, mRNAs with two different mouse 5UTR elements were synthesized. In addition, mRNAs contained the albumin? 3UTR element. Luciferase-encoding mRNAs were transfected into human dermal fibroblasts (HDF). For comparison, mRNA containing the human rpl32 5UTR element was transfected. Luciferase levels were measured at 24, 48, and 72 hours after transfection (see following Table 6 and
TABLE-US-00011 TABLE 6 5UTR RLU at 24 hours RLU at 48 hours RLU at 72 hours none 114277 121852 68235 rpl32 332236 286792 114148 mrpl21 798233 351894 139249 mrpl35A 838609 466236 174949
[0615] Luciferase was clearly expressed from mRNA lacking a 5UTR element. Both mouse 5UTR elements strongly increased the luciferase level, similarly as the human 5UTR element.
TABLE-US-00012 Lengthy table referenced here US20200332293A1-20201022-T00001 Please refer to the end of the specification for access instructions.
TABLE-US-LTS-00001 LENGTHY TABLES The patent application contains a lengthy table section. A copy of the table is available in electronic form from the USPTO web site (). An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3).