US2006002893A1PendingUtilityA1
Targeted adenovirus vectors for delivery of heterologous genes
Est. expiryAug 27, 2018(expired)· nominal 20-yr term from priority
A61P 31/04A61P 31/18A61P 9/10A61P 37/00A61P 35/00A61P 25/00A61P 11/06A61P 21/04C12N 2810/851C07K 14/501C12N 2710/10345C12N 2810/55C12N 2710/10322C12N 2770/32622A61K 48/00C07K 2319/00C12N 2810/855C12N 2810/80C12N 2810/6018C12N 2810/60C12N 2810/857C12N 2710/10343C12N 2810/40C07K 14/78C12N 15/86C12N 2770/32634C12N 9/6462C07K 14/005C12Y 304/21073C12N 15/861
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Claims
Abstract
Modification of internal sites of the adenovirus fiber protein and hexon protein permit effective targeting of adenovirus vectors. Accessible sites to redirect adenovirus targeting were identified. The HVR5 loop of the hexon protein and the HI loop of the fiber protein (knob) were highly permissive for the insertion of foreign protein sequences, which apparently did not impact on the viability and productivity of corresponding viruses. Accessibility and functionality of the epitope strongly depend on the size of the neighboring spacers. Other results suggest that short targeting peptides can be effectively fused to the C-terminus of the fiber protein. In a specific embodiment, a series of adenovirus vectors modified at the HVR5 site, the fiber protein HI loop, or the fiber protein C-terminus to target urokinase-type plasminogen activator receptor bearing cells were prepared. Such vectors are particularly useful for targeting the vasculature, e.g., for gene therapy of cancers or cardiovascular conditions.
Claims
exact text as granted — not AI-modified1 . An adenovirus comprising a hexon HRV5 loop from which at least a part of the hexon HRV5 loop is replaced with a binding peptide, or targeting sequence, flanked by connecting amino acid spacers so as to functionally display its binding specificity at the capsid surface.
2 . The adenovirus according to claim 1 wherein about 6 to 17 amino acids of the hexon HVR5 loop are replaced.
3 . The adenovirus according to claim 2 wherein no more than 14 amino acids of the hexon HVR5 loop are replaced.
4 . The adenovirus according to claim 3 wherein about 13 amino acids from the hexon HVR5 loop corresponding to about amino acid residue 269 to about amino acid residue 281 of adenovirus serotype 5 (Ad5) are replaced.
5 . The adenovirus according to claim 4 , wherein the spacers comprise an amino acid selected from the group consisting of glycine, serine, threonine, alanine, cysteine, aspartate, asparagine, methionine and proline.
6 . The adenovirus according to claim 5 , wherein the spacers comprise an amino acid selected from the group consisting of glycine and serine.
7 . The adenovirus according to claim 5 , wherein the first amino acid in at least one of the spacers is an amino acid selected from the group consisting of glycine, serine, threonine, alanine, cysteine, aspartate, asparagine, methionine and proline.
8 . The adenovirus according to claim 7 , wherein the first amino acid in the spacers is an amino acid selected from the group consisting of glycine, serine, threonine, alanine, cysteine, aspartate, asparagine, methionine and proline.
9 . The adenovirus according to claim 8 , wherein the first amino acid in at least one of the spacers is a glycine residue.
10 . The adenovirus according to claim 8 , wherein the first amino acid of the spacers is a glycine residue.
11 . The adenovirus according to claim 5 wherein at least one of the spacers is a dipeptide.
12 . The adenovirus according to claim 11 wherein at least one of the spacers is a Gly-Ser dipeptide.
13 . The adenovirus according to claim 4 , wherein the targeting sequence is a ligand epitope for a urokinase-type plasminogen activator receptor (UPAR).
14 . The adenovirus according to claim 13 wherein the targeting sequence is selected from the group consisting of LNGGTCVSNKYFSNIHWCN (SEQ ID NO: 1); LNGGTAVSNKYFSNIHWCN (SEQ ID NO: 2); AEPMPHSLNFSQYLWT (SEQ ID NO: 3); AEPMPHSLNFSQYLWYT (SEQ ID NO: 4); RGHSRGRNQNSR (SEQ ID NO: 5); and NQNSRRPSRA (SEQ ID NO: 6).
15 . The adenovirus according to claim 12 wherein the targeting sequence, including the spacers, is selected from the group consisting of:
(SEQ ID NO: 7);
A.
gly-ser-LNGGTCVSNKYFSNIHWCN-gly-ser;
(SEQ ID NO: 8)
B.
gly-ser-LNGGTAVSNKYFSNIHWCN-gly-ser;
(SEQ ID NO: 9)
C.
gly-ser-AEPMPHSLNFSQYLWT-gly-ser;
(SEQ ID NO: 10);
D.
gly-ser-AEPMPHSLNFSQYLWYT-gly-ser;
(SEQ ID NO: 11)
E.
gly-ser-RGHSRGRNQNSR-gly-ser;
(SEQ ID NO: 12)
F.
gly-ser-NQNSRRPSRA-gly-ser;
(SEQ ID NO: 13)
G.
gly-ser-CDCRGDCFC-gly-ser;
(SEQ ID NO: 14)
H.
gly-ser-DCRGDCF-gly-ser;
and
(SEQ ID NO: 15)
I.
gly-ser-KKKKKKK-gly-ser.
16 . The adenovirus according to claim 4 which is derived from human adenovirus serotype.
17 . The adenovirus according to claim 16 which is derived from human adenovirus subgroup C.
18 . The adenovirus according to claim 17 which is derived from human adenovirus serotype 5.
19 . The adenovirus according to claim 4 , wherein said adenovirus comprises a fiber protein comprising a fiber shaft, wherein said fiber shaft is modified to be shorter than a wild-type fiber shaft.
20 . The adenovirus according to claim 19 wherein said fiber shaft has been shortened by an in-frame deletion.
21 . The adenovirus according to claim 19 wherein said fiber shaft has been shortened by replacement with a shaft from another serotype.
22 . The adenovirus according to claim 19 wherein said fiber shaft is from human subgroup C (Ad2 or Ad5) and has been shortened by replacement with a shaft from serotype 3 (Ad3)
23 . The adenovirus according to claim 19 wherein said fiber shaft contains repeats 1 to 3 and 17 to 22 of Ad5; repeats 1 to 3 and 20 to 22 of Ad5; or an adenovirus serotype 3 (Ad3) shaft.
24 . An adenovirus comprising a hexon HI loop from which at least a part of the hexon HI loop is replaced with a targeting sequence, flanked by connecting amino acid spacers so as to functionally display the targeting sequence's binding specificity at the capsid surface.
25 . The adenovirus according to claim 24 wherein about 6 to 17 amino acids from the hexon HI loop are replaced.
26 . The adenovirus according to claim 25 , wherein no more than 11 amino acids from the hexon HI loop are replaced.
27 . The adenovirus according to claim 26 wherein about 11 amino acids from the hexon HI loop corresponding to about amino acid residue 538 to about amino acid residue 548 of adenovirus serotype 5 (Ad5) are replaced.
28 . The adenovirus according to claim 27 , wherein the spacers comprise an amino acid selected from the group consisting of glycine; serine, threonine, alanine, cysteine, aspartate, asparagine, methionine and proline.
29 . The adenovirus according to claim 28 , wherein the spacers comprise an amino acid selected from the group consisting of glycine and serine.
30 . The adenovirus according to claim 27 , wherein the first amino acid in at least one of the spacers is an amino acid selected from the group consisting of glycine, serine, threonine, alanine, cysteine, aspartate, asparagine, methionine and proline.
31 . The adenovirus according to claim 30 , wherein the first amino acid in the spacers is an amino acid selected from the group consisting of glycine, serine, threonine, alanine, cysteine, aspartate, asparagine, methionine and proline.
32 . The adenovirus according to claim 31 , wherein the first amino acid in at least one of the spacers is a glycine residue.
33 . The adenovirus according to claim 32 , wherein the first amino acid of the spacers is a glycine residue.
34 . The adenovirus according to claim 27 wherein at least one of the spacers is a tripeptide.
35 . The adenovirus according to claim 34 wherein the at least one of the spacers is a Gly-Ser-Ser tripeptide.
36 . The adenovirus according to claim 27 , wherein the targeting sequence is a ligand epitope for a urokinase-type plasminogen activator receptor (UPAR).
37 . The adenovirus according to claim 36 wherein the targeting sequence is selected from the group consisting of LNGGTCVSNKYFSNIHWCN (SEQ ID NO: 1); LNGGTAVSNKYFSNIHWCN (SEQ ID NO: 2); AEPMPHSLNFSQYLWT (SEQ ID NO: 3); AEPMPHSLNFSQYLWYT (SEQ ID NO: 4); RGHSRGRNQNSR (SEQ ID NO: 5); and NQNSRRPSRA (SEQ ID NO: 6).
38 . The adenovirus according to claim 27 wherein the targeting sequence, including the spacers, is selected from the group consisting of:
A.
gly-ser-ser-LNGGTCVSNKYFSNIHWC
(SEQ ID NO: 16)
N-gly-ser-ser;
B.
gly-ser-ser-LNGGTAVSNKYFSNIHWC
(SEQ ID NO: 17)
N-gly-ser-ser;
C.
gly-ser-ser-AEPMPHSLNFSQYLWT-
(SEQ ID NO: 18)
gly-ser-ser;
D.
gly-ser-ser-AEPMPHSLNFSQYLWYT-
(SEQ ID NO: 19)
gly-ser-ser;
E.
gly-ser-ser-RGHSRGRNQNSR-gly-
(SEQ ID NO: 20)
ser-ser;
F.
gly-ser-ser-NQNSRRPSRA-gly-
(SEQ ID NO: 21)
ser-ser;
G.
gly-ser-ser-CDCRGDCFC-gly-
(SEQ ID NO: 22)
ser-ser;
H.
gly-ser-ser-DCRGDCF-gly-
(SEQ ID NO: 23)
ser-ser;
and
I.
gly-ser-ser-KKKKKKK-gly-
(SEQ ID NO: 24)
ser-ser;
J.
ser-ser-RGHSRGRNQNSRRPSRA-
(SEQ ID NO: 143)
gly-ser;
K.
tyr-ser-glu-RGHSRGRNQNSR-
(SEQ ID NO: 144)
gly-ser;
L.
tyr-gln-glu-RGHSRGRNQNSR-
(SEQ ID NO: 145)
gly-ser;
M.
ser-ser-ser-RGHSRGRNQNSR-
(SEQ ID NO: 146)
gly-ser;
and
N.
ser-ser-RGHSRGRNQNSR-gly-gly.
(SEQ ID NO: 147)
39 . The adenovirus according to claim 27 which is derived from human adenovirus serotype.
40 . The adenovirus according to claim 39 which is derived from human adenovirus subgroup C.
41 . The adenovirus according to claim 40 which is derived from human adenovirus serotype 5.
42 . The adenovirus according to claim 27 wherein said adenovirus comprises a fiber protein comprising a fiber shaft, wherein said fiber shaft is modified to be shorter than a wild-type fiber shaft.
43 . The adenovirus according to claim 42 wherein said fiber shaft has been shortened by an in-frame deletion.
44 . The adenovirus according to claim 42 wherein said fiber shaft has been shortened by replacing it with the shaft from another serotype
45 . The adenovirus according to claim 44 wherein the fiber shaft is from human subgroup C (Ad2 or Ad5) and has been shortened by replacing it with the shaft from serotype 3 (Ad3)
46 . The adenovirus according to claim 42 wherein the fiber shaft contains repeats 1 to 3 and 17 to 22 of Ad5; repeats 1 to 3 and 20 to 22 of Ad5; or an adenovirus serotype 3 (Ad3) shaft
47 . A recombinant adenovirus comprising a fiber protein wherein a binding peptide, or targeting sequence, is connected to the C-terminus of the fiber protein by a connecting spacer, or linker, so as to functionally display its binding specificity at the capsid surface.
48 . The recombinant adenovirus according to claim 47 wherein the connecting spacer comprise an amino acid selected from the group consisting of glycine, serine, threonine, alanine, cysteine, aspartate, asparagine, methionine and proline.
49 . The recombinant adenovirus according to claim 48 wherein the first amino acid in the spacer is a proline.
50 . The recombinant adenovirus according to claim 47 which is derived from a human adenovirus serotype.
51 . The recombinant adenovirus according to claim 50 which is derived from human adenovirus subgroup C.
52 . The recombinant adenovirus according to claim 50 which is derived from human adenovirus serotype 5.
53 . The recombinant adenovirus according to claim 47 wherein the fiber protein is modified to have a fiber shaft that is shorter than a wild-type fiber shaft.
54 . The recombinant adenovirus according to claim 53 wherein said fiber shaft has been shortened by an in-frame deletion.
55 . The recombinant adenovirus according to claim 53 wherein said fiber shaft has been shortened by replacing it with the shaft from another serotype.
56 . The recombinant adenovirus according to claim 54 wherein said fiber shaft is from subgroup C and comprises an in-frame deletion encompassing repeats 4 to 16 or repeats 4 to 19.
57 . The recombinant adenovirus according to claim 53 wherein said fiber shaft is from subgroup C and has been shortened by replacing it with the shaft from serotype 3 (Ad3).
58 . The adenovirus according to claim 57 comprising a spacer or linker peptide comprising from 5 to 30 amino acids.
59 . The adenovirus according to claim 58 , wherein the spacer or linker peptide comprises the sequence PKRARPGS (SEQ ID NO:149).
60 . The adenovirus according to claim 59 , wherein the targeting sequence is a ligand epitope for a urokinase-type plasminogen activator receptor (UPAR).
61 . The adenovirus according to claim 59 , wherein the targeting sequence is a peptide fragment from FGF-1 binding to heparin, comprising between 7 and 15 amino acids.
62 . The adenovirus according to claim 59 , wherein the targeting sequence is composed of 5 to 10 lysine residues.
63 . The adenovirus according to claim 59 , wherein the targeting sequence is composed of almost 7 lysine residues.
64 . The adenovirus according to claim 59 , wherein the targeting sequence is composed of between 5 and 10 Arg-Arg and Leu-Leu motifs.
65 . The adenovirus according to claim 59 , wherein the targeting sequence is selected from the group consisting of LNGGTCVSNKYFSNIHWCN (SEQ ID NO: 1); LNGGTAVSNKYFSNIHWCN (SEQ ID NO: 2); AEPMPHSLNFSQYLWT (SEQ ID NO: 3); AEPMPHSLNFSQYLWYT (SEQ ID NO: 4); RGHSRGRNQNSR (SEQ ID NO: 5); NQNSRRPSRA (SEQ ID NO: 6); RRLLRRLLRR (SEQ ID NO: 133); and KRGPRTHYGQK (SEQ ID NO: 134);
66 . The adenovirus according to claim 59 , wherein the targeting sequence including the linker peptide comprises the sequence PKRARPGSKKKKKKK (SEQ ID NO:132).
67 . The adenovirus according to claim 59 , wherein the targeting sequence including the linker peptide comprises the sequence PKRARPGSRRLLRRLLRR (SEQ ID NO:141).
68 . The adenovirus according to claim 59 , wherein the targeting sequence including the linker peptide comprises the sequence PKRARPGSKRGPRTHYGQK (SEQ ID NO:140).
69 . A method for modifying the cellular tropism of an adenovirus vector, comprising A. deleting a native amino acid sequence from a site in a capsid protein of the adenovirus; and B. inserting a targeting peptide sequence connected by a first spacer at the N-terminus and a second spacer at the C-terminus of the targeting sequence; and wherein the targeting peptide is inserted in a deletion site selected from the group consisting of about 13 amino acids from the hexon HVR5 loop corresponding to about amino acid residue 269 to about amino acid residue 281 of adenovirus Ad5; and about 11 amino acids from the fiber protein HI loop corresponding to about amino acid residue 538 to about amino acid residue 548 of Ad5.
70 . The method according to claim 69 , wherein the first spacer comprises an amino acid selected from the group consisting of glycine and serine.
71 . The method according to claim 69 , wherein the second spacer comprises an amino acid selected from the group consisting of glycine and serine.
72 . The method according to claim 69 , wherein the targeting sequence is a ligand epitope for a urokinase-type plasminogen activator receptor (UPAR).
73 . The method according to claim 72 , wherein the targeting sequence is selected from the group consisting of LNGGTCVSNKYFSNIHWCN (SEQ ID NO: 1); LNGGTAVSNKYFSNIHWCN (SEQ ID NO: 2); AEPMPHSLNFSQYLWT (SEQ ID NO: 3); AEPMPHSLNFSQYLWYT (SEQ ID NO: 4); RGHSRGRNQNSR (SEQ ID NO: 5); and NQNSRRPSRA (SEQ ID NO: 6).
74 . The method according to claim 69 , wherein targeting sequence, including the spacers, is inserted in the HVR5 loop and is selected from the group consisting of:
(SEQ ID NO: 7)
A.
gly-ser-LNGGTCVSNKYFSNIHWCN-gly-ser;
(SEQ ID NO: 8)
B.
gly-ser-LNGGTAVSNKYFSNIHWCN-gly-ser;
(SEQ ID NO: 9)
C.
gly-ser-AEPMPHSLNFSQYLWT-gly-ser;
(SEQ ID NO: 10)
D.
gly-ser-AEPMPHSLNESQYLWYT-gly-ser;
(SEQ ID NO: 11)
E.
gly-ser-RGHSRGRNQNSR-gly-ser;
(SEQ ID NO: 12)
F.
gly-ser-NQNSRRPSRA-gly-ser;
(SEQ ID NO: 13)
G.
gly-ser-CDCRGDCFC-gly-ser;
(SEQ ID NO: 14)
H.
gly-ser-DCRGDCF-gly-ser;
and
(SEQ ID NO: 15)
I.
gly-ser-KKKKKKiK-gly-ser
75 . The method according to claim 69 , wherein the targeting sequence, including the spacers, is inserted in the fiber protein HI loop and is selected from the group consisting of:
A.
gly-ser-ser-LNGGTCVSNKYFSNIHWC
(SEQ ID NO: 16)
N-gly-ser-ser;
B.
gly-ser-ser-LNGGTAVSNKYFSNIHWC
(SEQ ID NO: 17)
N-gly-ser-ser;
C.
gly-ser-ser-AEPMPHSLNFSQYLWT-
(SEQ ID NO: 18)
gly-ser-ser;
D.
gly-ser-ser-AEPMPHSLNFSQYLWYT-
(SEQ ID NO: 19)
gly-ser-ser;
E.
gly-ser-ser-RGHSRGRNQNSR-gly-
(SEQ ID NO: 20)
ser-ser;
F.
gly-ser-ser-NQNSRRPSRA-gly-
(SEQ ID NO: 21)
ser-ser;
G.
gly-ser-ser-CDCRGDCFC-gly-
(SEQ ID NO: 22)
ser-ser;
H.
gly-ser-ser-DCRGDCF-gly-
(SEQ ID NO: 23)
ser-ser;
and
I.
gly-ser-ser-KKKKKKK-gly-
(SEQ ID NO: 24)
ser-ser;
J.
ser-ser-RGHSRGRNQNSRRPSRA-
(SEQ ID NO: 143)
gly-ser;
K.
tyr-ser-glu-RGHSRGRNQNSR-
(SEQ ID NO: 144)
gly-ser;
L.
tyr-gln-glu-RGHSRGRNQNSR-
(SEQ ID NO: 145)
gly-ser;
M.
ser-ser-ser-RGHSRGRNQNSR-
(SEQ ID NO: 146)
gly-ser;
and
N.
ser-ser-RGHSRGRNQNSR-gly-gly.
(SEQ ID NO: 147)
76 . The method according to claim 69 , further comprising shortening the fiber protein shaft.
77 . The method according to claim 69 , wherein the fiber shaft comprises repeats 1 to 3 and 17 to 22 of Ad5; repeats 1 to 3 and 20 to 22 of Ad5; or an Ad3 shaft.
78 . An adenovirus hexon comprising a deletion of about 13 amino acids from the HVR5 loop corresponding to about amino acid residue 269 to about amino acid residue 281 of adenovirus serotype 5 (Ad5) and an insertion at the site of the deletion of a targeting peptide sequence connected by a first spacer at the N-terminus and a second spacer at the C-terminus of the targeting peptide sequence.
79 . An adenovirus hexon protein comprising a deletion of about 11 amino acids from the HI loop corresponding to about amino acid residue 538 to about amino acid residue 548 of adenovirus serotype 5 (Ad5) and an insertion at the site of the deletion of a targeting peptide sequence connected by a first spacer at the N-terminus and a second spacer at the C-terminus of the targeting peptide sequence.
80 . An adenovirus fiber protein comprising a linker peptide and a targeting peptide at its C-terminus.
81 . A method for preferentially expressing a gene in a target cell comprising contacting a population of cells containing the target cell with an adenovirus of claim 1 , wherein the targeting sequence is a ligand epitope for a receptor on the target cell.
82 . A method for preferentially expressing a gene in a target cell that expresses a UPAR comprising contacting a population of cells containing the target cell with a targeted adenovirus vector of claim 13 .
83 . The method according to claim 81 , wherein the targeted adenovirus vector comprises a heterologous gene encoding a gene for treatment of a tumor.
84 . The method according to claim 81 , wherein the adenovirus comprises a gene for the treatment of restenosis.
85 . A method for the treatment of a disease by gene therapy comprising the step of administering an adenovirus of claim 1 .
86 . (canceled)
87 . (canceled)
88 . A pharmaceutical composition comprising an adenovirus of claim 1 and an efficient quantity of a pharmaceutically active excipient.Join the waitlist — get patent alerts
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