U.S. patent application number 11/038980 was filed with the patent office on 2006-01-05 for targeted adenovirus vectors for delivery of heterologous genes.
Invention is credited to Jean-Francois Dedieu, Martine Latta, Michel Perricaudet, Emmanuelle Vigne, Patrice Yeh.
Application Number | 20060002893 11/038980 |
Document ID | / |
Family ID | 22266396 |
Filed Date | 2006-01-05 |
United States Patent
Application |
20060002893 |
Kind Code |
A1 |
Vigne; Emmanuelle ; et
al. |
January 5, 2006 |
Targeted adenovirus vectors for delivery of heterologous genes
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.
Inventors: |
Vigne; Emmanuelle;
(L'Hay-Les-Roses, FR) ; Dedieu; Jean-Francois;
(Paris, FR) ; Latta; Martine; (St. Maurice,
FR) ; Yeh; Patrice; (Gif Sur Yvette, FR) ;
Perricaudet; Michel; (Ecrosnes, FR) |
Correspondence
Address: |
SYNNESTVEDT & LECHNER, LLP
2600 ARAMARK TOWER
1101 MARKET STREET
PHILADELPHIA
PA
191072950
US
|
Family ID: |
22266396 |
Appl. No.: |
11/038980 |
Filed: |
January 20, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09791524 |
Feb 22, 2001 |
6911199 |
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11038980 |
Jan 20, 2005 |
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PCT/IB99/01524 |
Aug 27, 1999 |
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09791524 |
Feb 22, 2001 |
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60098028 |
Aug 27, 1998 |
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Current U.S.
Class: |
424/93.2 ;
435/235.1; 435/456 |
Current CPC
Class: |
C12N 9/6462 20130101;
C12N 2810/855 20130101; C12Y 304/21073 20130101; C12N 2810/40
20130101; C12N 2810/55 20130101; C12N 2810/857 20130101; A61P 11/06
20180101; A61K 48/00 20130101; C07K 14/501 20130101; C12N
2770/32622 20130101; C07K 14/78 20130101; C07K 2319/00 20130101;
C12N 2710/10322 20130101; A61P 31/04 20180101; C12N 15/86 20130101;
C07K 14/005 20130101; C12N 2710/10345 20130101; A61P 37/00
20180101; C12N 2810/6018 20130101; A61P 21/04 20180101; A61P 25/00
20180101; C12N 2710/10343 20130101; A61P 31/18 20180101; C12N
2810/80 20130101; C12N 2770/32634 20130101; C12N 2810/851 20130101;
A61P 9/10 20180101; A61P 35/00 20180101; C12N 2810/60 20130101 |
Class at
Publication: |
424/093.2 ;
435/456; 435/235.1 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12N 7/00 20060101 C12N007/00; C12N 15/861 20060101
C12N015/861 |
Claims
1. 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: TABLE-US-00038 (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: TABLE-US-00039 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: TABLE-US-00040 (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:
TABLE-US-00041 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.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to effective targeting of
adenovirus vectors by modifying surface accessible sites of the
fiber or hexon proteins to include a targeting amino acid sequence.
The key to success of the present invention lies in the discovery
that additional spacer amino acid residues at the N-terminus and
C-terminus of the inserted targeting sequence are critical to
providing a recognizable binding structure of the targeting
sequence. Thus, accessibility and functionality of the targeting
sequence as well as structure of the modified protein strongly
depend on the size and nature of the neighboring spacers. Other
results suggest that short targeting peptides can be effectively
fused to the C-terminus of the fiber protein. The invention further
relates to the use of such vectors for delivering therapeutic genes
to specific target cells in vitro and in vivo.
BACKGROUND OF THE INVENTION
Adenovirus Vectors
[0002] Adenoviruses exhibit certain properties which are
particularly advantageous for use as vector for the transfer of
genes in gene therapy. In particular, they have a fairly broad host
spectrum, are capable of infecting quiescent cells, do not
integrate into the genome of the infected cell, and have not been
associated, up until now, with major pathologies in man.
Adenoviruses have thus been used to transfer genes of interest into
the muscle (Ragot et al., 1993, Nature 361:647), the liver (Jaffe
et al., 1992, Nature Genetics 1:372), the nervous system (Alki et
al., 1993, Nature Genetics 3:224), tumors (Griscelli et al., 1998,
PNAS 95:6367), intact or injured vascular endotheliums (van Belle
et al., 1998, Human Gene Therapy 9:1013), synovial tissue
(Ghivizzani et al., 1998, PNAS 95:4613), and the like. Adenovirus
vectors efficiently transfer genes to both replicating and
non-replicating cells (see, e.g., Crystal, 1995, Science
270:404-410).
Adenovirus Capsid
[0003] Characteristics of the adenovirus capsid are well known
(see, e.g., International Patent Publication WO 98/07877).
[0004] Various references describe the adenovirus hexon protein,
and permit some estimation of accessible sites. For example,
Athapilly et al. (1994, J. Mol. Biol. 242:430-455) describes its
refined crystal structure at 2.9 A resolution. Crawford-Miksza et
al. (1996, J. Virol. 70: 1836-1844) reports the location and
structure of seven hexon protein hypervariable regions containing
serotype-specific residues. Indeed, there has been a report of
expression of a foreign epitope on the surface of the adenovirus
hexon (Crompton et al., 1994, J. Gen. Virol. 75: 133-139). However,
as shown in the examples, infra, the article by Crompton et al.
does not teach a reproducible method of targeting adenovirus by
modifying the hexon protein.
[0005] Additional references provide information about the fiber
protein (Chroboczek et al., 1995, In Doerfler W. & P. Bohm
(Eds.), The molecular repertoire of adenoviruses, pp. 163-200,
Springer-Verlag; Stewart and Burnett, ibid., pp. 25-38; Xia et al.,
1995, ibid., pp. 39-46 Fender et al., 1995 Virol., 214:110-117;
Hong and Engler, 1996, J. Virol., 70: 7071-7078). Inhibition of
cell adhesion to the virus by synthetic peptides of fiber knob of
human adenovirus serotypes 2 and 3 and virus neutralization by
anti-peptide antibodies has been reported (Liebermann et al., 1996,
Virus Research, 45:111-121). Xia et al. (1994, Structure,
2:1259-1270) report the crystal structure of the receptor-binding
domain of adenovirus type 5 fiber protein at 1.7 A resolution.
[0006] Adenoviruses are nonenveloped, regular icosahedrons of about
65 to 80 nm in diameter. The adenoviral capsid comprises 252
capsomers, of which 240 are hexons and 12 are pentons. The hexons
and pentons are derived from three different viral polypeptides
(Maizel et al., 1968, Virology, 36: 115-125; Weber et al., 1997,
Virology: 76, 709-724. The Ad5 hexon comprises three identical
polypeptides of 967 amino acids each, namely polypeptide II
(Roberts et al., 1986, Science, 232: 1148-1151). The penton
comprises a penton base, which provides a point of attachment to
the capsid, and a trimeric fiber protein, which is noncovalently
bound to and projects from the penton base.
[0007] The fiber protein comprises three identical proteinaceous
subunits of polypeptide IV (582 amino acids) and comprises a tail,
a shaft and a knob (Devaux et al., 1990, J. Molec. Biol., 215:
567-588). The fiber shaft comprises pseudorepeats of 15 amino
acids, which are believed to form two alternating .beta.-strands
and .alpha.-bends (Green et al., 1983, EMBO J., 2: 1357-1365). The
overall length of the fiber shaft and the number of 15 amino acid
repeats varies between adenoviral serotypes. For example, the Ad2
fiber shaft is 37 nm long and comprises 22 repeats, whereas the Ad3
fiber is 11 nm long and comprises 6 repeats. Sequencing of over ten
fiber proteins from different adenoviral serotypes has revealed a
greater sequence diversity than that observed among other
adenoviral proteins. For example, the knob regions of the fiber
proteins from the closely related Ad2 and Ad5 serotypes are only
63% similar at the amino acid level (Chroboczek et al., 1992,
Virology, 186: 280-285), whereas their penton base sequences are
99% identical. Ad2 and Ad5 fiber proteins, however, both likely
bind to the same cellular receptor, since they cross-block each
other's binding. In contrast, Ad2 and Ad3 fibers are only 20%
identical (Signas et al., 1985, J. Virol., 53:672-678), and bind to
different receptors (Defer et al., 1990, J. Virol., 64(8),
3661-3673).
[0008] Adenovirus serotype 2 has been shown to use the fiber and
the penton base to interact with distinct cellular receptors to
attach to and efficiently infect a cell (Wickham et al., 1993,
Cell, 73: 309-319). First, the virus uses a receptor binding domain
localized in the fiber knob (Henry et al., 1994, J. Virol., 68(8):
5239-5246) to attach to one of at least two cell-surface receptors
(Hong et al., 1997, EMBO J., 16:2294-2306; Bergelson et al., 1997,
Science, 275:1320-1323; Phillipson et al., 1968, J. Virol., 2:
1064-1075; Wickham et al., 1993 supra.; Svensson et al., 1981, J.
Virol., 38: 70-81; and DiGuilmi et al., 1995, Virus Res., 38:
71-81). Then, following viral attachment, the penton base binds to
a specific member of a family of heterodimeric cell-surface
receptors called integrins. For the Ad2 and Ad5 serotypes, which
possess the long-shafted fibers, the penton base is not
significantly involved in the initial viral attachment to host
cells (Wickham et al., 1993, supra).
[0009] Most integrins recognize short linear stretches of amino
acids in a ligand, such as the tripeptide RGD, which is found in
the majority of extracellular matrix ligands. The integrin
.alpha..sub.IIb.beta..sub.3 binds fibrinogen via the amino acid
sequence KQAGD (SEQ ID NO:) (Kloczewiak et al., 1984, Biochemistry,
23, 1767-1774), and .alpha..sub.4.beta..sub.1 binds fibronectin via
the core sequence EILDV (SEQ ID NO:) (Komoriya et al., 1991, J.
Biol. Chem., 266: 15075-15079). Another structural motif, NPXY (SEQ
ID NO:), which is present in the .beta. subunits of
.alpha..sub.v-containing integrins, also has been shown to be
important for integrin-mediated internalization (Suzuki et al.,
1990, Proc. Natl. Acad. Sci. USA, 87: 5354).
[0010] Once Ad2 or Ad5 attaches to a cell via its fiber, it
undergoes receptor-mediated internalization into clathrin-coated
endocytic vesicles by penton base binding to integrins. Ultimately,
the viral particles are transported to the nuclear pore complex of
the cell, where the viral genome enters the nucleus, thereby
initiating expression from the virus chromosome.
[0011] A drawback to the use of adenovirus in gene therapy,
however, is that all cells that comprise receptors for the
adenoviral fiber and penton base will internalize the adenovirus,
and, consequently, the gene(s) being administered--not just the
cells in need of therapeutic treatment. Also, cells that lack
either the fiber receptor or the penton base receptor will be
impaired in adenoviral-mediated gene delivery. Cells that appear to
lack an adenoviral fiber receptor, are transduced by adenovirus, if
at all, with a very low efficiency (Curiel et al., 1992, supra;
Cotton et al., 1990, Proc. Natl. Acad. Sci. USA, 87: 4033-4037;
Wattel et al., 1996, Leukemia, 10: 171-174). Accordingly,
effectively directing adenoviral entry to specific cells and in
some cases expanding the repertoire of cells amenable to
adenovirus-mediated gene therapy, represents an important goal to
improve current vectors. Both approaches also could potentially
reduce the amount of adenoviral vector that is necessary to obtain
gene expression in the targeted cells and, thus, potentially reduce
side effects and complications associated with higher doses of
adenovirus.
Adenovirus Targeting Strategies
[0012] Various strategies have been employed to modify adenovirus
tropism, i.e., to target adenovirus to specific cell types not
normally infected efficiently by wild-type adenovirus vectors (see,
e.g., International Patent Publication WO 98/07877). Fiber protein,
hexon protein, and penton base protein modification strategies have
been employed.
[0013] U.S. Pat. No. 5,559,099 describes a recombinant virus
comprising a chimeric penton base protein with a nonpenton amino
acid sequence specific for a receptor, antibody, or epitope in
addition to or in place of a wild-type penton base sequence, and a
therapeutic gene.
[0014] U.S. Pat. No. 5,543,328 claims an adenovirus wherein the
adenovirus fiber includes a ligand specific for a receptor located
on a desired cell type. Such an adenovirus fiber can be prepared by
removing all or a portion of the fiber protein head portion and
replacing it with ligand, or by creating a fusion between a full
length fiber protein and a ligand.
[0015] International Patent Publication WO 95/26412 discloses
modification of adenovirus full length fiber protein to contain a
C-terminal linker for attachment of a ligand. Inclusion of a linker
is stated to avoid steric interference with formation of a fiber
protein homotrimer.
[0016] International Patent Publication WO 96/26281 discloses a
recombinant adenovirus comprising (a) a chimeric fiber protein with
a normative amino acid sequence in addition to or in place of a
native fiber sequence, (b) a therapeutic gene, and, optionally, (c)
a normative trimerization domain in place of the native fiber amino
acid trimerization domain. The normative amino acid sequence can be
a protein binding sequence, and may be located at the
C-terminus.
[0017] International Patent Publication WO 97/20051 discloses a
chimeric adenovirus coat protein with a normative amino acid
sequence that is able to direct vector entry into cells more
efficiently than a vector with wild-type coat protein. The
normative sequence can be inserted into or in place of an internal
coat protein sequence, at or near the C-terminus of the coat
protein, or in an exposed loop of the coat protein. The coat
protein can be a fiber protein, a penton base protein, or a hexon
protein. A spacer sequence can be included.
[0018] Other targeting techniques rely on post-expression
modification of the viral coat protein, e.g., by covalent or
non-covalent binding of bridging targeting groups or targeting
groups (see, e.g., International Patent Application No. WO
97/05266; International Patent Publication WO 97/23608;
International Patent Publication WO 97/32026).
[0019] Despite these efforts, there remains a need in the art to
identify suitable modes of insertion of the binding peptide so that
it is accurately displayed at the capsid surface to allow virus
growth and specific binding to its cognate receptor(s).
[0020] There is a further need to define critical parameters that
permit recognition of such sequences. Yet another need in the art
is to provide specific targeting peptide sequences suitable for
directing adenovirus vectors to target cells in vivo. These and
other needs of the art are addressed by the present invention.
[0021] The citation of any reference herein should not be construed
as an admission that such reference is available as "Prior Art" to
the instant application. Each of the references disclosed herein is
incorporated by reference in the application in its entirety.
SUMMARY OF THE INVENTION
[0022] The present invention advantageously provides an effective
targeted adenovirus vector. The targeted vector of the invention is
characterized by an appropriate deletion of amino acids from an
effective site in either the hexon protein or the fiber
protein.
[0023] Thus, in a more specific embodiment the invention relates to
an adenovirus 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 functionnaly display its
binding specificity at the capsid surface. In a specific embodiment
the adenovirus comprises a deletion of about 6 to 17 amino acids
from the hexon HVR5 loop preferably not exceeding 14 amino
acids.
[0024] In an other specific embodiment the invention relates to an
adenovirus from which at least a part of the fiber HI loop is
replaced with a binding peptide, or targeting sequence, flanked by
connecting amino acid spacers so as to functionnaly display its
binding specificity at the capsid surface. In a specific embodiment
the adenovirus comprises a deletion of about 6 to 17 amino acids
from the hexon HI loop preferably not exceeding 11 amino acids.
[0025] In a further embodiment the invention relates to a
recombinant adenovirus vector wherein a binding peptide, or
targeting sequence, is connected to the C-terminus of the fiber by
a connecting spacer, or linker, so as to functionaly display its
binding specificity at the capsid surface.
[0026] In a more specific embodiment, about 13 amino acids are
deleted from the hexon HVR5 loop corresponding to about amino acid
residue 269 to about amino acid residue 281 of adenovirus serotype
5 (Ad5). In another specific embodiment, about 11 amino acids are
deleted from the fiber protein HI loop corresponding to about amino
acid residue 538 to about amino acid residue 548 of adenovirus
serotype 5 (Ad5). A targeting peptide is inserted at the site of
the deletion. A particular advantage of the invention is the
discovery that the targeting peptide sequence should be connected
by a first spacer at the N-terminus and a second spacer at the
C-terminus of the targeting sequence, wherein the spacers comprise
a flexible amino acid residue. Preferably, the first spacer or the
second spacer, or both, comprises an amino acid selected from the
group consisting of glycine and serine.
[0027] In a specific aspect, the targeted adenovirus modified in
the hexon protein advantageously employs dipeptide spacers
consisting of flexible amino acid residues. In a specific
embodiment, the first and second spacers are a Gly-Ser dipeptide.
In a further specific embodiment of this aspect, the first spacer
is a glycine residue.
[0028] In an aspect of the invention in which the fiber protein HI
loop is modified, the first and second spacers are advantageously
tri-peptides consisting of flexible amino acid residues. In a
specific embodiment of this aspect of the invention, the first and
second spacers are a Gly-Ser-Ser tri-peptide.
[0029] Preferably, the targeting sequence is a ligand epitope for a
urokinase-type plasminogen activator receptor (UPAR). In
particular, the targeting sequence can be 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).
[0030] In an alternative embodiment in which the hexon protein is
modified, the targeting sequence including the spacers is selected
from the group consisting of: TABLE-US-00001
gly-ser-LNGGTCVSNKYFSNIHWCN-gly-ser; (SEQ ID NO:7)
gly-ser-LNGGTAVSNKYFSNIHWCN-gly-ser; (SEQ ID NO:8)
gly-ser-AEPMPHSLNFSQYLWT-gly-ser; (SEQ ID NO:9)
gly-ser-AEPMPHSLNFSQYLWYT-gly-ser; (SEQ ID NO:10)
gly-ser-RGHSRGRNQNSR-gly-ser; (SEQ ID NO:11)
gly-ser-NQNSRRPSRA-gly-ser; (SEQ ID NO:12)
gly-ser-CDCRGDCFC-gly-ser; (SEQ ID NO:13) gly-ser-DCRGDCF-gly-ser;
(SEQ ID NO:14) and gly-ser-KKKKKKK-gly-ser. (SEQ ID NO:15)
[0031] In an alternative embodiment in which the fiber protein is
modified, the targeting sequence including the spacers is selected
from the group consisting of: TABLE-US-00002 (SEQ ID NO:16 )
gly-ser-ser-LNGGTCVSNKYFSNIHWCN-gly-ser-ser; (SEQ ID NO:17 )
gly-ser-ser-LNGGTAVSNKYFSNIHWCN-gly-ser-ser; (SEQ ID NO:18 )
gly-ser-ser-AEPMPHSLNFSQYLWT-gly-ser-ser; (SEQ ID NO: 19 )
gly-ser-ser-AEPMPIISLNFSQYLWYT-gly-ser-ser; (SEQ ID NO:20 )
gly-ser-ser-RGHSRGRNQNSR-gly-ser-ser; (SEQ ID NO:21:)
gly-ser-ser-NQNSRRPSRA-gly-ser-ser; (SEQ ID NO:22 )
gly-ser-ser-CDCRGDCFC-gly-ser-ser; (SEQ ID NO:23 )
gly-ser-ser-DCRGDCF-gly-ser-ser; (SEQ ID NO:24 )
gly-ser-ser-KKKKKKK-gly-ser-ser (SEQ ID NO: 143)
ser-ser-RGHSRGRNQNSRRPSRA-gly-ser; (SEQ ID NO: 144)
tyr-ser-glu-RGFISRGRNQNSR-gly-ser; (SEQ ID NO: 145)
tyr-gln-glu-RGHSRGRNQNSR-gly-ser; (SEQ ID NO: 146)
ser-ser-ser-RGHSRGRNQNSR-gly-ser; and (SEQ ID NO: 147)
ser-ser-RGHSRGRNQNSR-gly-gly.
[0032] Preferably the connecting spacer or linker comprises an
amino acid selected from the group consisting of glycine, serine,
threonine, alanine, cysteine, aspartate, asparagine, methionine and
proline. In a refered embodiment the first amino acid in the spacer
is a proline.
[0033] The recombinant adenovirus can be derived from a human
adenovirus serotype, in particular from human adenovirus subgroup
C, such as human adenovirus serotype 5.
[0034] The fiber protein can be modified to have a fiber shaft that
is shorter than a wild-type fiber shaft, in particular by an
in-frame deletion or by replacing it with the shaft from another
serotype. The fiber shaft can be from subgroup C and comprises an
in-frame deletion encompassing repeats 4 to 16 or repeats 4 to 19
or from subgroup C and has been shortened by replacing it with the
shaft from serotype 3 (Ad3)
[0035] According to either aspect of the invention (modification of
the hexon or the fiber), the fiber protein can be modified to be
shorter than in the wild-type sequence. For example, the fiber
protein can be modified to contain only 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 in place of the endogenous Ad5 shaft.
[0036] In a specific embodiment, exemplified infra, the adenovirus
is a serotype 5 adenovirus.
[0037] In a further embodiment, the present invention provides a
specific targeted adenovirus vector comprising a linker peptide and
a targeting peptide at the C-terminus of the fiber protein.
[0038] Preferably, the targeting sequence is a ligand for a UPAR,
such as CD87, a peptide fragment from FGF-1 binding to heparin,
comprising between 7 and 15 amino acids, is composed of 5 to 10
lysine residues, preferably of almost 7 lysine residues, or is
composed of between 5 and 10 Arg-Arg and Leu-Leu motifs.
[0039] Preferably, the targeting sequence is selected from the
group consisting of TABLE-US-00003 LNGGTCVSNKYFSNIHWCN; (SEQ ID NO:
1) LNGGTAVSNKYFSNIHWCN; (SEQ ID NO: 2) AEPMPHSLNFSQYLWYT; (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)
[0040] Preferably the linker peptide comprises the sequence
PKRARPGS (SEQ ID NO.149) and the targeting sequence including the
linker peptide comprises the sequences PKRARPGSKKKKKKK (SEQ ID
NO.132), PKRARPGSRRLLRRLLRR (SEQ ID NO.141) or PKRARPGSKRGPRTHYGQK
(SEQ ID NO.140).
[0041] Naturally, given the targeted adenoviruses disclosed above
and in greater detail herein, the present invention provides a
method for modifying the cellular tropism of an adenovirus vector,
comprising deleting a native amino acid sequence from a site in a
capsid protein of the adenovirus; and 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, wherein the
spacers comprise a flexible amino acid residue. According to this
aspect of the invention, 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. In a preferred embodiment, the first spacer comprises an amino
acid selected from the group consisting of glycine and serine. In
another preferred embodiment, the second spacer comprises an amino
acid selected from the group consisting of glycine and serine.
[0042] Are also encompassed by the present invention: [0043] 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 sequence, wherein the
first and second spacers comprise a flexible amino acid residue.
[0044] an adenovirus fiber 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 sequence,
wherein the first and second spacers comprise a flexible amino acid
residue. [0045] an adenovirus fiber protein comprises a linker
peptide and a targeting peptide at its C-terminus.
[0046] The invention further specifically provides a method for
targeting cells that express a urokinase-type plasminogen activator
receptor (UPAR). In particular, this method comprises using
adenoviruses modified to expose at the capsid surface the specific
UPAR targeting peptides disclosed above. Alternatively, the
invention provides for modifying the hexon HVR5 loop or the fiber
protein HI loop by inserting the specific sequences, including
spacer groups, defined above.
[0047] The method for targeting a specific cell type in accordance
with the invention can be further enhanced by shortening the fiber
protein shaft, e.g., such that the fiber shaft only contains
repeats 1 to 3 and 17 to 22 of Ad5; repeats 1 to 3 and 20 to 22 of
Ad5; or with an Ad3 shaft. Repeats are as described by Chroboczek
et al (1995, Current Topics in Microbiology and Immunology,
Springer Verlag, 199 :163-200).
[0048] The invention further provides a method for preferentially
expressing a gene in a target cell comprising contacting a
population of cells containing the target cell with the targeted
adenovirus vector of the invention, wherein the targeting sequence
is a ligand epitope for a receptor on the target cell. In
particular, the invention provides 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
the targeted adenovirus vectors of the invention that are modified
to display a UPAR binding peptide. In this embodiment, the targeted
adenovirus vector preferably comprises a heterologous therapeutic
gene or nucleic acid for transduction of actively dividing and/or
motile cells, including tumor cells and its metastases, tumor
vasculature, activated endothelial cells, activated smooth muscle
cells . . . However, UPAR targeted vectors can also be considered
as candidate vectors for transduction of other tissues as muscle,
brain, heart, etc. More particularly, the nucleic acid encodes a
therapeutic polypeptide which acts as an angiogenesis inhibitor, an
angiogenic factor, a conditional suicide effector, a tumor
suppressor, a growth-arrest protein (GAX), or any secreted
polypeptide.
[0049] In particular, the invention provides a method for
preferentially expressing a gene in a target cell that expresses an
integrin comprising contacting a population of cells containing the
target cell with the targeted adenovirus vector of the invention
that are modified to display an integrin binding peptide. In this
embodiment, the targeted adenovirus vector preferably comprises a
therapeutic gene or nucleic acid for transduction of actively
dividing and/or motile cells, including tumor cells and its
metastases, tumor vasculature, activated endothelial cells,
activated smooth muscle cells . . . However, integrin targeted
vectors can also be considered as candidate vectors for
transduction of other tissues as skeletal muscle, brain, heart,
hematopoietic cells, ischemic tissues, etc. More particularly, the
nucleic acid encodes a therapeutic polypeptide which acts as an
angiogenesis inhibitor, an angiogenic factor, a conditional suicide
effector, a tumor suppressor, a growth-arrest protein (GAX), a cell
survival-promoting factor (in particular members of the Akt/PKB
family) or any secreted polypeptide.
[0050] Thus a further object of the present invention is a method
for the treatment of a disease by gene therapy comprising the step
of administering a targeted adenovirus vector as disclosed
hereabove, or obtained according to the method disclosed
hereabove.
[0051] Still an object of the present invention is a medecine
containing a targeted adenovirus vector as disclosed hereabove, or
obtained according to the method as disclosed hereabove and the use
of such a targeted adenovirus vector for manufacturing a medecine
for the treatment of a disease by gene therapy. A further object of
the present invention is a pharmaceutical composition containing
such targeted adenovirus vectors and a efficient quantity of a
pharmaceutically active excipient.
[0052] Thus, an object of the invention is to provide for
tropism-modified adenovirus vectors without adversely impacting
productivity of the vectors.
[0053] A further object of the invention is to identify suitable
mode of insertion so that the binding peptide is accessible and
effectively recognizes its specific receptors.
[0054] Still another object of the invention is to identify the
number of amino acid residues that can be deleted from the native
adenovirus capsid protein so that the targeting peptide sequence
can be effectively inserted.
[0055] Yet another object of the invention is to provide the
effective size and characteristics of spacer sequences to be
included at the ends of the inserted targeting peptide to permit
adoption by the targeting peptide of an accessible conformation
that permits effective binding to the target receptor.
[0056] These and other objects of the invention are further
provided by the accompanying Drawings and the following Detailed
Description of the Invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0057] FIG. 1. Neutralization assay on W162 cells with virus
modified in the hexon HVR5 region.
[0058] FIG. 2. Structure of the shortened fiber construct in which
the adenovirus serotype 3 (Ad3) shaft is inserted in place of the
Ad5 shaft.
[0059] FIG. 2A: General description of the hybrid Ad3/5 fiber;
[0060] FIG. 2B: Detailed description of the hybrid Ad3/5 fiber
[0061] FIG. 3: Knob competition in 293 cells.
[0062] FIG. 4: Infection of hSMC with various viruses in presence
of increasing doses of soluble heparin.
[0063] FIG. 5: Infection of hSMC with viruses preincubated with
increasing doses of soluble uPAR.
[0064] FIGS. 6 and 7: Gax expression in human SMC infected with
targeted adenovirus.
[0065] FIGS. 8A to 8C and 9: Infection of Hs578T with different
targeted viruses.
[0066] FIG. 10: Infection of Hs578T with virus AE43 preincubated
with increasing doses of soluble uPAR.
[0067] FIG. 11: Infection of Hs578T with virus AE43 preincubated
with increasing doses of soluble uPAR or soluble knob.
[0068] FIG. 12: Infection of Hs578T with Vn4 containing
viruses.
[0069] FIG. 13: Infection of NIH-3T3 with a large range of targeted
viruses.
[0070] FIGS. 14A and 14B: Infection of Hs578T with virus viruses
BC15X (A) and AE43 (B) preincubated with increasing doses of
soluble uPAR.
DETAILED DESCRIPTION OF THE INVENTION
[0071] As noted above, there has been keen interest in the art to
modify adenovirus tropism so as to permit targeting adenovirus
vectors to specific target cells, including those cells that are
not efficiently infected by adenoviruses. While there have been
some successes in achieving this goal, the present inventors
recognized that a practical solution to this problem, i.e., a
solution that would not adversely affect viral productivity and
that would permit a satisfactory increase in cell specificity, had
not been achieved. In particular, optimum sites for incorporation
of a targeting peptide in a viral capsid protein, the size of a
deletion from the native capsid protein (if any), the size of an
inserted targeting sequence, and the presence and nature of any
linker sequences joining the targeting sequence to the capsid
protein, are not described in the prior art. The present invention
advantageously addresses these issues, and provides highly
effective targeting by: providing optimized sites for insertion of
a targeting sequence, including the size of the native sequence to
be deleted to make room for the targeting peptide; identifying an
appropriate size for the targeting sequence; and disclosing the
size and characteristics of linkers that permit accessibility and
specific recognition of the targeting peptide; and identifying
interesting cell marker as a target receptor.
[0072] In particular, the present inventors set out to introduce an
accessible foreign peptide on the surface of the adenoviral capsid,
and thus to modify the natural tropism of the virus. With this in
mind, a series of constructs with modified hexons or fibers
incorporating a neutralizing epitope from poliovirus type 1 were
designed. The poliovirus sequence was chosen as cognate
neutralizing antibodies were readily available to document in great
details its accessibility and functionality.
[0073] Modification of the hexon and fiber to restrict infection to
specific target cells requires that the adenovirus fiber not be
able to efficiently interact with its native cellular receptors In
the case of hexon modified capsid, it also requires that the
binding peptide can interact directly with its cognate receptor on
the cell surface without steric hindrance from the fiber. For these
purposes, the possibility of shortening the shaft of the fiber was
investigated.
[0074] The invention is based, in part, on experiments that
identified sites suitable for insertion of functional targeting
sequences in the hexon protein and in the fiber protein. It was
discovered that replacement of a portion of the hexon HVR5 loop and
fiber HI loop permitted insertion of targeting sequences without
adversely affecting viral productivity. Furthermore, the data
showed that incorporation of flexible linker peptides at the ends
of the inserted sequences was critical to accessibility or
recognition of the targeting sequence. Moreover, incorporation of
ligands specific for uPAR or for some integrins was successfully
achieved in terms of accessibility of the ligand, productivity of
the modified viruses, transduction efficiency of the target cell
types as shown in in vitro and in vivo experiments. Finally, it was
also shown that shortening of the fiber efficiently reduces virus
affinity for natural host cell, which makes this strategy
attractive for ablation of natural tropism of Ad5.
[0075] In a preferred embodiment, the different approaches are
combined. Preferably, the fiber protein is shortened in a virus
having a modified hexon HVR5 loop or fiber HI loop. In a further
example, a virus with a shortened fiber could have an insertion in
HVR5 or in HI to target UPAR as the primary step of the infection,
and an insertion in HI loop or in HVR5 (respectively) to target an
integrin to favor the internalization of the virus in the endosome.
Any suitable membrane receptor can also be targeted using the same
strategy, i.e. incorporation of high affinity ligands in the hexon
and/or the fiber in combination with shortening of the fiber.
[0076] The Detailed Description of the Invention is further
elaborated in sections relating to specific definitions, adenovirus
vectors, targeting peptide sequences, and uses of the targeted
adenovirus vectors. The various headers and organization of the
sections are provided for the sake of clarity and convenience, and
are not in any way to be deemed limiting.
Definitions
[0077] Various terms are used throughout the specification and
claims. Where not otherwise defined, the following definitions
apply:
[0078] As used in the art, a <<vector>> is any means
for the transfer of a nucleic acid according to the invention into
a host cell. For purposes of the present invention, the term vector
is used to modify "adenovirus" so as to reflect that the adenovirus
has been genetically engineered to transfer a nucleic acid of
interest (a gene under control of, or operably linked to, an
expression control sequence) into the target cell. Adenovirus
vectors of the invention are described in greater detail,
infra.
[0079] A cell has been "transfected" or "infected" by an adenovirus
vector of the invention when viral DNA has been introduced inside
the cell. A cell has been "transformed" by exogenous or
heterologous DNA when the transfected DNA effects a phenotypic
change.
[0080] The term "corresponding to" is used herein to refer similar
or homologous sequences, whether the exact position is identical or
different from the molecule to which the similarity or homology is
measured. A nucleic acid or amino acid sequence alignment may
include spaces. Thus, the term "corresponding to" refers to the
sequence similarity, and not the numbering of the amino acid
residues or nucleotide bases. Examples include hexon proteins or
fiber proteins from other adenovirus serotypes (2, 3, etc.) besides
the type 5 adenovirus (Ad5) exemplified herein. Those of ordinary
skill in the art are familiar with homologous adenovirus capsid
proteins.
[0081] In other words, the present invention provides for
modification of homologous capsid proteins from other adenovirus
species using the optimized parameters defined herein for Ad5. As
used herein, the term "homologous" in all its grammatical forms and
spelling variations refers to the relationship between proteins
that possess a "common evolutionary origin," including proteins
from superfamilies (e.g., the immunoglobulin superfamily) and
homologous proteins from different species (e.g., myosin light
chain, etc.) (Reeck et al., 1987, Cell 50:667). Such proteins (and
their encoding genes) have sequence homology, as reflected by their
high degree of sequence similarity.
[0082] The term "deletion" refers to the removal of native amino
acid residues from a defined region of an adenovirus capsid
protein, i.e., a hexon or fiber protein. According to the
invention, a preferred size for such a deletion is between about 10
and about 20 amino acids. More preferably, the size of the deletion
is between about 10 and about 15 amino acids. In specific
embodiments, 11 and 13 amino acid sequences were deleted.
[0083] The term "spacer" or "spacer peptide" or
<<linker>> or <<linker peptide>> is used
herein to refer to a sequence of about one to about three amino
acids that is included to connect the binding peptide to its capsid
carrier protein. The spacer or the linker is preferably made up of
amino acid residues with high degrees of freedom of rotation, which
permits the targeting peptide to adopt a conformation that is
recognized by its binding partner (e.g., receptor). Preferably no
more than three amino acids are included in the spacer; more
preferably, the spacer consists of two amino acids. Preferred amino
acids for the spacer are glycine and serine. In specific
embodiments, the spacer is a peptide having the sequence Gly-Ser or
Gly-Ser-Ser.
[0084] As used herein, the term "about" or "approximately" means
within 20%, preferably within 10%, and more preferably within 5% of
a given value or range.
Adenovirus Vectors
[0085] Adenoviruses are eukaryotic DNA viruses that can be modified
to efficiently deliver a nucleic acid of the invention to a variety
of cell types. Various serotypes of adenovirus exist. Of these
serotypes, preference is given, within the scope of the present
invention, to using type 2 or type 5 human adenoviruses (Ad 2 or Ad
5) or adenoviruses of animal origin (see WO94/26914). Those
adenoviruses of animal origin which can be used within the scope of
the present invention include adenoviruses of canine, bovine,
murine (example: Mav1, Beard et al., Virology 75 (1990) 81), ovine,
porcine, avian, and simian (example: SAV) origin. Preferably, the
adenovirus of animal origin is a canine adenovirus, more preferably
a CAV2 adenovirus (e.g., Manhattan or A26/61 strain (ATCC
VR-800)).
[0086] Adenoviral vectors are commonly used for in vitro, in vivo
or ex vivo transfection and gene therapy procedures. Preferably,
the adenoviral vectors are replication defective, that is, they are
unable to replicate autonomously in the target cell. In general,
the genome of the replication defective viral vectors within the
scope of the present invention lack at least one region which is
necessary for the replication of the virus in the infected cell.
These regions can either be eliminated (in whole or in part), be
rendered non-functional by any technique known to a person skilled
in the art. These techniques include the total removal,
substitution (by other sequences, in particular by the inserted
nucleic acid), partial deletion or addition of one or more bases to
a region required for virus propagation. Such techniques may be
performed in vitro (on the isolated DNA) or in situ, using the
techniques of genetic manipulation or by treatment with mutagenic
agents. For purposes of the present invention, the replication
defective virus retains the sequences of its genome which are
necessary for encapsidating the viral particles. Defective viruses,
which entirely or almost entirely lack viral genes, may also be
used.
[0087] The replication defective adenoviral vectors of the
invention comprise at least the ITRs, an encapsidation sequence and
the nucleic acid of interest. Preferably, at least the E1 region of
the adenoviral vector is rendered non-functional. The deletion in
the E1 region preferably extends from nucleotides 455 to 3329 in
the sequence of the Ad5 adenovirus (PvuII-BglII fragment) or 382 to
3446 (HinfII-Sau3A fragment), or 382-3512 (HinfI-RsaI fragment).
Other regions may also be modified, in particular the E3 region
(WO95/02697), the E2 region (WO94/28938), the E4 region
(WO94/28152, WO94/12649 and WO95/02697), the IVa2 region
(WO96/10088) or in any of the late genes L1-L5.
[0088] In a preferred embodiment, the adenoviral vector has a
deletion in the E1 region (Ad 1.0). Examples of E1-deleted
adenoviruses are disclosed in EP 185,573, and in FR 97/14383, filed
11 Nov. 1997, the contents of which are incorporated herein by
reference. In another preferred embodiment, the adenoviral vector
has a deletion in the E1 and E4 regions (Ad 3.0). Examples of
E1/E4-deleted adenoviruses are disclosed in WO95/02697 and
WO96/22378, the contents of which are incorporated herein by
reference. In still another preferred embodiment, the adenoviral
vector has a deletion in the E1 region into which an E4 functional
region and the nucleic acid sequence are inserted (see FR94 13355,
the contents of which are incorporated herein by reference).
Another adenovirus vector for use in the invention is described in
WO 96/10088.
[0089] The replication defective recombinant adenoviruses according
to the invention can be prepared by any technique known to the
person skilled in the art (Levrero et al., 1991, Gene 101: 195; EP
185 573; Graham, 1984, EMBO J. 3: 2917). In particular, they can be
prepared by homologous recombination between an adenovirus and a
plasmid which carries, inter alia, the DNA sequence of interest.
The homologous recombination is effected following cotransfection
of the said adenovirus and plasmid into an appropriate cell line.
The cell line which is employed should preferably (i) be
transformable by the said elements, and (ii) contain the sequences
which are able to complement the part of the genome of the
replication defective adenovirus, preferably in integrated form in
order to avoid the risks of recombination. Examples of cell lines
which may be used are the human embryonic kidney cell line 293
(Graham et al., 1977, J. Gen. Virol. 36: 59) which contains the
left-hand portion of the genome of an Ad5 adenovirus (12%)
integrated into its genome, PER.C6 (Bout et al., 1997, Cancer Gene
Therapy 4:324; Fallaux et al, 1998, Hum Gen Ther 9: 1909-1917), and
cell lines which are able to complement the E1 and E4 functions, as
described in applications WO94/26914 and WO95/02697. In a preferred
embodiment, an E. coli vector system is used to generate the
adenovirus backbone (International Patent Publication WO 96/25506;
Crouzet et al. 1997, Proc. Natl. Acad. Sci. 94:1414-1419).
[0090] Indeed, the general techniques for preparing adenoviruses of
the invention are well known in the art. Such techniques are
explained fully in the literature. See, e.g., Sambrook, Fritsch
& Maniatis, Molecular Cloning: A Laboratory Manual, Second
Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (herein "Sambrook et al., 1989"); DNA Cloning: A
Practical Approach, Volumes I and II (D. N. Glover ed. 1985);
Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid
Hybridization [B. D. Hames & S. J. EHiggins eds. (1985)];
Transcription And Translation [B. D. Hames & S. J. Higgins,
eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)];
Immobilized Cells And Enzymes [IRL Press, (1986)]; B. EPerbal, A
Practical Guide To Molecular Cloning (1984); F. M. Ausubel et al.
(eds.), Current Protocols in Molecular Biology, John Wiley &
Sons, Inc. (1994). Incorporation of cassette insertion sites,
whether for insertion of a heterologous gene or for insertion of
the targeting sequence in the hexon or fiber protein (as
exemplified infra) facilitates such genetic manipulations. A
"cassette" refers to a segment of DNA that can be inserted into a
vector at specific restriction sites. The segment of DNA encodes a
polypeptide of interest, and the cassette and restriction sites are
designed to ensure insertion of the cassette in the proper reading
frame for transcription and translation.
[0091] Recombinant adenoviruses are recovered and purified using
standard molecular techniques, which are well known to one of
ordinary skill in the art (see, e.g., International Patent
Publication WO 98/00524, International Patent Publicaiton WO
96/27677; and International Patent Publication WO 97/08298).
[0092] Expression of heterologous genes by adenovirus vectors of
the invention. The adenovirus vectors of the invention preferably
contain a DNA coding sequence for a heterologous gene. A DNA
"coding sequence" is a double-stranded DNA sequence which is
transcribed and translated into a polypeptide in a cell in vitro or
in vivo when placed under the control of appropriate regulatory
sequences. The boundaries of the coding sequence are determined by
a start codon at the 5' (amino) terminus and a translation stop
codon at the 3' (carboxyl) terminus. A coding sequence can include,
but is not limited to, prokaryotic sequences, cDNA from eukaryotic
mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA,
and even synthetic DNA sequences. If the coding sequence is
intended for expression in a eukaryotic cell, a polyadenylation
signal and transcription termination sequence will usually be
located 3' to the coding sequence. A coding sequence is "under the
control" of transcriptional and translational control sequences in
a cell when RNA polymerase transcribes the coding sequence into
mRNA, which is then trans-RNA spliced and translated into the
protein encoded by the coding sequence. In another embodiment, the
nucleic acid of interest is not translated into a polypeptide but
acts as a specific anti-sense therapeutic molecule, or as a
therapeutic decoy.
[0093] Transcriptional and translational control sequences are DNA
regulatory sequences, such as promoters, enhancers, terminators,
and the like, that provide for the expression of a coding sequence
in a host cell. In eukaryotic cells, polyadenylation signals are
control sequences. A "promoter sequence" is a DNA regulatory region
capable of binding RNA polymerase in a cell and initiating
transcription of a downstream (3' direction) coding sequence. For
purposes of defining the present invention, the promoter sequence
is bounded at its 3' terminus by the transcription initiation site
and extends upstream (5' direction) to include the minimum number
of bases or elements necessary to initiate transcription at levels
detectable above background. Within the promoter sequence will be
found a transcription initiation site (conveniently defined for
example, by mapping with nuclease S1), as well as protein binding
domains (consensus sequences) responsible for the binding of RNA
polymerase.
[0094] Expression of a heterologous protein may be controlled by
any promoter/enhancer element known in the art, but these
regulatory elements must be functional in the target cell selected
for expression. Promoters which may be used to control gene
expression include, but are not limited to, the cytomegalovirus
immediate early (CMV-IE or CMV) promoter, the SV40 early promoter
region (Benoist and Chambon, 1981, Nature 290:304-310), the
promoter contained in the 3' long terminal repeat of Rous sarcoma
virus (Yamamoto, et al., 1980, Cell 22:787-797), the herpes
thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad.
Sci. U.S.A. 78:1441-1445), the regulatory sequences of the
metallothionein gene (Brinster et al., 1982, Nature 296:39-42); and
the animal transcriptional control regions, which exhibit tissue
specificity and have been utilized in transgenic animals: elastase
I gene control region which is active in pancreatic acinar cells
(Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold
Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987,
Hepatology 7:425-515); insulin gene control region which is active
in pancreatic beta cells (Hanahan, 1985, Nature 315:115-122),
immunoglobulin gene control region which is active in lymphoid
cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al.,
1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol.
7:1436-1444), mouse mammary tumor virus control region which is
active in testicular, breast, lymphoid and mast cells (Leder et
al., 1986, Cell 45:485-495), albumin gene control region which is
active in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276),
alpha-fetoprotein gene control region which is active in embryonic
liver and hepatomas (Krumlauf et al., 1985, Mol. Cell. Biol.
5:1639-1648; Hammer et al., 1987, Science 235:53-58), alpha
1-antitrypsin gene control region which is active in the liver
(Kelsey et al., 1987, Genes and Devel. 1:161-171), beta-globin gene
control region which is active in myeloid cells (Mogram et al.,
1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94),
myelin basic protein gene control region which is active in
oligodendrocyte cells in the brain (Readhead et al., 1987, Cell
48:703-712), myosin light chain-2 gene control region which is
active in skeletal muscle (Sani, 1985, Nature 314:283-286), and
gonadotropic releasing hormone gene control region which is active
in the hypothalamus (Mason et al., 1986, Science
234:1372-1378).
[0095] A "signal sequence" is included at the beginning of the
coding sequence of a protein to be exported at the cell surface, or
secreted. This sequence encodes a signal peptide, N-terminal to the
mature polypeptide, that directs the host cell to translocate the
polypeptide into the secretion pathway/compartment. The term
"translocation signal sequence" is used herein to refer to this
sort of signal sequence. Translocation signal sequences can be
found associated with a variety of proteins native to eukaryotes
and prokaryotes, and are often functional in both types of
organisms.
[0096] Specific heterologous genes are discussed in the section
relating to uses of the vectors of the invention, infra.
Targeting Peptide Sequences
[0097] Any known targeting sequence can be incorporated in a hexon
HVR5 loop or fiber protein in accordance with the present
invention. Examples of targeting peptides are ample in the
literature. In general, any peptide ligand can provide a targeting
sequence based on the receptor-binding sequence of the ligand. In
immunology, such a sequence is referred to as an epitope, and the
term epitope may be used herein to refer to the sequence of a
ligand recognized by a receptor. Specifically, the term "ligand
epitope of a receptor" refers to the sequence of a protein or
peptide that is recognized by a binding partner on the surface of a
target cell, which for the sake of convenience is termed a
receptor. However, it should be understood that for purposes of the
present invention, the term "receptor" encompasses
signal-transducing receptors (e.g., receptors for hormones
steroids, cytokines, insulin, and other growth factors),
recognition molecules (e.g., MHC molecules, B- or T-cell
receptors), nutrient uptake receptors (such as transferrin
receptor), lectins, ion channels, adhesion molecules, extracellular
matrix binding proteins, and the like that are located and
accessible at the surface of the target cell. Targeting peptides of
the invention can bind to polypeptide or carbohydrate moieties on
such receptors.
[0098] The size of the targeting peptide can vary within certain
parameters. As shown in the examples, inserting peptides longer
than the deleted sequence did not adversely impact viral
productivity. Thus, the invention contemplates using a targeting
sequence that is several fold longer than the deleted segment;
preferably, the inserted peptide is no more than 200% longer than
the deleted segment, and more preferably the inserted peptide is
about the same size as the deleted segment.
[0099] Due to the degeneracy of nucleotide coding sequences, other
DNA sequences which encode substantially the same amino acid
sequence as a targeting peptide sequence may be used in the
practice of the present invention. These include but are not
limited to allelic variants, homologous variants from other
species, variants in which a conservative amino acid substitution
is effected, and variants in which a highly polymorphic amino acid
residue (which is presumably not important for binding specificity)
is changed. For example, one or more amino acid residues within the
sequence can be substituted by another amino acid of a similar
polarity, which acts as a functional equivalent, resulting in a
silent alteration. Substitutes for an amino acid within the
sequence may be selected from other members of the class to which
the amino acid belongs. For example, the nonpolar (hydrophobic)
amino acids include alanine, leucine, isoleucine, valine, proline,
phenylalanine, tryptophan and methionine. Amino acids containing
aromatic ring structures are phenylalanine, tryptophan, and
tyrosine. The polar neutral amino acids include glycine, serine,
threonine, cysteine, tyrosine, asparagine, and glutamine. The
positively charged (basic) amino acids include arginine, lysine and
histidine. The negatively charged (acidic) amino acids include
aspartic acid and glutamic acid. Such alterations will not be
expected to significantly affect apparent molecular weight as
determined by polyacrylamide gel electrophoresis, or isoelectric
point.
[0100] Particularly preferred substitutions are: [0101] Lys for Arg
and vice versa such that a positive charge may be maintained;
[0102] Glu for Asp and vice versa such that a negative charge may
be maintained; [0103] Ser for Thr such that a free --OH can be
maintained; and [0104] Gln for Asn such that a free CONH.sub.2 can
be maintained.
[0105] Such variants may be encoded by highly similar nucleic
acids. Such nucleic acids will, in general, hybridize to a nucleic
acid (including an oligonucleotide probe) that encodes the native
amino acid sequence. As demonstrated in the examples, infra,
various modifications can be introduced by single base changes that
do not affect hybridization (of PCR primers), but that can create
an endonuclease cleavage site or a altered amino acid residue.
[0106] A nucleic acid molecule is "hybridizable" to another nucleic
acid molecule, such as a cDNA, genomic DNA, or RNA, when a single
stranded form of the nucleic acid molecule can anneal to the other
nucleic acid molecule under the appropriate conditions of
temperature and solution ionic strength (see Sambrook et al.,
supra). The conditions of temperature and ionic strength determine
the "stringency" of the hybridization. For preliminary screening
for homologous nucleic acids, low stringency hybridization
conditions, corresponding to a T.sub.m of 55.degree. C., can be
used, e.g., 5.times.SSC, 0.1% SDS, 0.25% milk, and no formamide; or
30% formamide, 5.times.SSC, 0.5% SDS). Moderate stringency
hybridization conditions correspond to a higher T.sub.m, e.g., 40%
formamide, with 5.times. or 6.times.SCC. High stringency
hybridization conditions correspond to the highest T.sub.m, e.g.,
50% formamide, 5.times. or 6.times.SCC. Hybridization requires that
the two nucleic acids contain complementary sequences, although
depending on the stringency of the hybridization, mismatches
between bases are possible. The appropriate stringency for
hybridizing nucleic acids depends on the length of the nucleic
acids and the degree of complementation, variables well known in
the art. The greater the degree of similarity or homology between
two nucleotide sequences, the greater the value of T.sub.m for
hybrids of nucleic acids having those sequences. The relative
stability (corresponding to higher T.sub.m) of nucleic acid
hybridizations decreases in the following order: RNA:RNA, DNA:RNA,
DNA:DNA. For hybrids of greater than 100 nucleotides in length,
equations for calculating T.sub.m have been derived (see Sambrook
et al., supra, 9.50-0.51). For hybridization with shorter nucleic
acids, i.e., oligonucleotides, the position of mismatches becomes
more important, and the length of the oligonucleotide determines
its specificity (see Sambrook et al., supra, 11.7-11.8). Preferably
a minimum length for a hybridizable nucleic acid is at least about
10 nucleotides; preferably at least about 15 nucleotides; and more
preferably the length is at least about 20 nucleotides. In a
specific embodiment, the term "standard hybridization conditions"
refers to a T.sub.m of 55.degree. C., and utilizes conditions as
set forth above. In a preferred embodiment, the T.sub.m is
60.degree. C.; in a more preferred embodiment, the T.sub.m is
65.degree. C.
[0107] Integrin-binding peptides. Targeting peptide sequences
include the well known integrin-binding peptides (see, e.g., U.S.
Pat. No. 4,517,686; U.S. Pat. No. 4,589,881; U.S. Pat. No.
4,661,111; U.S. Pat. No. 4,578,079; U.S. Pat. No. 4,614,517; U.S.
Pat. No. 5,453,489; U.S. Pat. No. 5,627,263). Other useful
targeting peptides are described in International Patent
Publication WO 98/17242 and European Patent Application EP 773
441.
[0108] Concerning the targeting of integrins, as noted above there
are many publications describing peptides that bind specific
integrins, some of which are overexpressed in different tumor or
cell types. In a specific embodiment, .alpha.v integrins (and
consequently actively dividing and/or motile cells including tumor
cells and its metastases, tumor vasculature, activated endothelial,
activated smooth muscle cells, skeletal muscle cells, etc.) are
targeted, e.g., through a peptide selected by phage display.
Naturally, any peptide specific of integrins, e.g., as described in
the literature, can be used in the invention. The targeting of
integrins could also favor the internalization of the virus, and
could be a mean to restore the infectivity of Ad with shortened
fibers.
[0109] Urokinase receptor targeted peptides. In a specific
embodiment exemplified infra, peptides that target a receptor for
urokinase-type plasminogen activator (UPAR; e.g., CD87) were
selected from various publications. Naturally, there is a much more
exhaustive list of peptides binding a UPAR. Any peptide binding
UPAR could be used.
[0110] Basic targeting peptides. Specific embodiments of the
invention, with particular targeting peptides, are described in
detail in Examples 4-6, infra. For example, a targeting peptide
comprising primarily basic amino acid residues, e.g., lysine,
arginine, and histidine, and more preferably either lysine or
arginine, or both (see, e.g., WO 97/20051) was used in the
examples, infra. In another example, ligands binding to heparan
sulfate proteoglycans such as the arginine-leucine repeated motif
RRLLRRLLRR, described in the RPR patent application WO95/21931, and
the peptide fragment KRGPRTHYGQK from the FGF-1 binding domain to
heparin (Digabriele et al, 1998, Science, 393 :812-817) were
used.
[0111] Also, sequences that bind to heparin or glycosaminoglycans
may be involved in binding to a heparin-like receptor (Sawitzky et
al., 1993, Med. Microbiol. Immunol., 182: 285-92). Similarly,
so-called <<heparin binding sequences>> may mediate the
interaction of the peptide or protein in which they are contained
with other cell surface binding sites, such as with cell surface
heparan sulfate proteoglycan (Thompson et al., 1994, J. Biol.
Chem., 269: 2541-9).
[0112] Alternatively, the targeting amino acid sequence comprises
two basic amino acids (frequently Arg) located about 20 Angstroms
apart, facing in opposite directions of an alpha helix (Margalit et
al., 1993, J. Biol. Chem., 268: 19228-31; Ma et al., 1994, J. Lipid
Res., 35: 2049-2059). Other basic amino acids can be dispersed
between these two residues, facing one side, while nonpolar
residues face the other side, forming a helical amphipathic
structure with basic residues segregated to one side.
[0113] Also, the targeting sequence can comprise common heparin
binding motifs present in fibronectin and heat shock proteins
(Hansen et al., 1995, Biochim. Biophys. Acta, 1252: 135-45);
insertions of 7 residues of either Lys or Arg, or mixtures of Lys
and Arg (Fromm et al., 1995, Arch. Biochem. Biophys., 323: 279-87);
the common basic C-terminal region of IGFBP-3 and IGFBP-5 of about
18 amino acids and which comprises a heparin binding sequence
(Booth et al., 1995, Growth Regul., 5: 1-17); either one or both of
the two hyaluronan (HA) binding motifs located within a 35 amino
acid region of the C-terminus of the HA receptor RHAMM (Yang et
al., 1994, J. Cell Biochem., 56: 455-68); a synthetic peptide
(Ala347-Arg361) modeled after the heparin-binding form of
Staphylococcus aureus vitronectin comprising heparin-binding
consensus sequences (Liang et al., 1994, J. Biochem., 116: 457-63);
any one or more of five heparin binding sites between amino acid
129 and 310 of bovine herpesvirus 1 glycoprotein gIII or any one of
four heparin binding sites between amino acids 90 and 275 of
pseudorabies virus glycoprotein gIII (Liang et al., 1993, Virol.,
194: 233-43); amino acids 134 to 141 of pseudorabies virus
glycoprotein gIII (Sawitzky et al., 1993, Med. Microbiol. Immunol.,
182: 285-92); heparin binding regions corresponding to charged
residues at positions 279-282 and 292-304 of human lipoprotein
lipase (Ma et al., supra); a synthetic 22 residue peptide, N22W,
with a sequence modeled after fibronectin and which exhibits
heparin binding properties (Ingham et al., 1994, Arch. Biochem.
Biophys., 314: 242-246); the motif present in the ectodomain zinc
binding site of the Alzheimer beta-amyloid precursor protein (APP),
as well as various other APP-like proteins, which modulates heparin
affinity (Bush et al, 1994, J. Biol. Chem., 229: 26618-21), 8 amino
acid residue peptides derived from the cross-region of the laminin
A chain (Tashiro et al., 1994, Biochem. J., 302: 73-9); peptides
based on the heparin binding regions of the serine protease
inhibitor antithrombin III including peptides F123-G148 and
K121-A134 (Tyler-Cross et al., 1994, Protein Sci., 3: 620-7); a 14
K N-terminal fragment of APP and a region close to the N-terminus
(i.e., residues 96-110) proposed as heparin binding regions (Small
et al., 1994, J. Neurosci., 14: 2117-27); a stretch of 21 amino
acids of the heparin binding epidermal growth factor-like growth
factor (HB-EGF) characterized by a high content of lysine and
arginine residues (Thompson et al., 1994, J. Biol. Chem, 269:
2541-9); a 17 amino acid region comprising the heparin binding
region of thrombospondin and corresponding to a hep 1 synthetic
peptide (Murphy-Ullrich et al., 1993, J. Biol. Chem., 268:
26784-9); a 23 amino acid sequence (Y565-A587) of human von
Willebrand factor that binds heparin (Tyler-Cross et al., 1993,
Arch. Biochem. Biophys., 306: 528-33); the fibronectin-derived
peptide PRARI, and larger peptides comprising this motif, that bind
heparin (Woods et al., 1993, Mol. Biol. Cell., 4:605-613); the
heparin binding region of platelet factor 4 (Sato et al., Jpn. J.
Cancer Res., 84: 485-8); and K18K sequence in the fibroblast growth
factor receptor tyrosine kinase transmembrane glycoprotein (Kan et
al., 1993, Science 259: 1918-21).
[0114] Identification of targeting peptide sequences. In another
embodiment, targeting peptides can be derived from various types of
combinatorial libraries, using well known strategies for
identifying ligands (see U.S. Pat. No. 5,622,699 and International
Patent Application No. PCT/US96/14600). One approach uses
recombinant bacteriophage to produce large libraries. Using the
"phage method" (Scott and Smith, 1990, Science 249:386-390; Cwirla,
et al., 1990 Proc. Natl. Acad. Sci., 87:6378-6382; Devlin et al.,
1990, Science, 249:404-406), very large libraries can be
constructed (10.sup.6-10.sup.8 chemical entities). A second
approach uses primarily chemical methods, of which the Geysen
method [Geysen et al., 1986, Molecular Immunology 23:709-715;
Geysen et al., 1987, J. Immunologic Method 102:259-274) and the
method of Fodor et al. (1991, Science 251:767-773) are examples.
Furka et al. (1988, 14th International Congress of Biochemistry,
Volume E5, Abstract FR:013; Furka, 1991, Int. J. Peptide Protein
Res. 37:487-493), Houghton (U.S. Pat. No. 4,631,211, issued
December 1986) and Rutter et al. (U.S. Pat. No. 5,010,175, issued
Apr. 23, 1991) describe methods to produce a mixture of peptides
that can be tested as targeting sequences. In another aspect,
synthetic libraries (Needels et al., 1993, Proc. Natl. Acad. Sci.
USA 90:10700-4; Ohlmeyer et al., 1993, Proc. Natl. Acad. Sci. USA
90:10922-10926; Lam et al., International Patent Publication No. WO
92/00252; Kocis et al., International Patent Publication No. WO
9428028), and the like can be used to screen for targeting
peptides.
[0115] Uses of the Vectors of the Invention
[0116] The references provided above regarding preparation of
adenovirus vectors describe various uses for such vectors.
[0117] Targeted introduction of therapeutic genes into malignant
cells in vivo can provide an effective treatment of tumors. Several
treatment modalities have been attempted. For example, one
treatment involves the delivery of normal tumor suppressor genes
(e.g., p53, retinoblastoma protein, p16, etc.) and/or inhibitors of
activated oncogenes into tumor cells. A second treatment involves
the enhancement of immunogeneity of tumor cells in vivo by the
introduction of cytokine genes. A third treatment involves the
introduction of genes that encode enzymes capable of conferring to
the tumor cells sensitivity to chemotherapeutic agents. The herpes
simplex virus-thymidine kinase (HSV-tk) gene can specifically
convert a nucleoside analog (ganciclovir) into a toxic intermediate
and cause death in dividing cells. It has recently been reported by
Culver et al. (Science, 1992, 256:1550-1552) that after delivery of
the HSV-tk gene by retroviral transduction, subsequent ganciclovir
treatment effectively caused brain tumor regression in laboratory
animals. U.S. Pat. No. 5,631,236 by Woo et al. describes gene
therapy for solid tumors with an adenovirus vector that encodes
HSV-tk or VZV-tk.
[0118] In a preferred embodiment, a vector of the invention can be
used to target a nucleic acid of interest to the tumor itself, its
metastases or the tumor vasculature, e.g., by using peptides that
bind to a UPAR. In a preferred embodiment, such vectors encode
genes for inhibitors of angiogenesis or an anti-angiogenic factor.
An "anti-angiogenic factor" is a molecule that inhibits
angiogenesis, particularly by blocking endothelial cell migration.
Such factors include fragments of angiogenic proteins that are
inhibitory (such as the amino-terminal fragment of urokinase),
angiogenesis inhibitory factors, such as angiostatin and
endostatin; soluble receptors of angiogenic factors, such as the
urokinase type receptor or FGF/VEGF receptor; molecules which block
endothelial cell growth factor receptors [O'Reilly et. al. Cell
88:277-285 (1997); O'Reilly, Nat. Med. 2:689-692 (1996)], and Tie-1
or Tie-2 inhibitors. Generally, an anti-angiogenic factor for use
in the invention is a protein or polypeptide, which may be encoded
by a gene transfected into tumors using the vectors of the
invention. For example, the vectors of the invention can be used to
deliver a gene encoding an anti-angiogenic protein into a tumor,
its metastases or the tumor vasculature in accordance with the
invention. Examples of anti-angiogenic factors include, but are not
limited to, the amino terminal fragment (ATF) of urokinase,
containing the EGF-like domain (e.g., amino acid residues about 1
to about 135 of ATF); ATF provided as a fusion protein, e.g., with
immunoglobulin or human serum albumin [WO93/15199]; angiostatin
[O'Reilly et al., Cell 79:315-328 (1994)]; tissue inhibitors of
metalloproteinases [Johnson et al., J. Cell. Physiol. 160:194-202
(1994)]; or inhibitors of FGF or VEGF such as soluble forms of
receptors for angiogenic factors, including but not limited to
soluble VGF/VEGF receptors, and soluble urokinase receptors [Wilhem
et al., FEBS Letters 337:131-134 (1994)].
[0119] In another preferred embodiment, a vector of the invention
can be used to target migrating smooth muscle cells to inhibit
post-angioplastic restenosis. An example of the use of an
adenovirus to inhibit restenosis by delivery of a suicide gene is
disclosed in WO96/05321. Use of an adenovirus encoding a GAX
protein (growth arrest protein) to inhibit vascular smooth muscle
cell proliferation and restenosis is disclosed in WO96/30385. Other
genes can be cytotoxic genes (HSV thymidine kinase),
metalloproteinases inhibitors (TIMP), endothelial NOS or
atherosclerose protecting factors (e.g. ApoE).
[0120] A vector of the invention in which a muscle or a brain
specific peptide has been included can also be used to selectively
deliver protecting or regenerating growth factors for central
nervous system (CNS) disorders. Examples of the use of adenoviruses
to deliver genes to the CNS are disclosed in WO94/08026, WO95/25804
and WO95/26408.
[0121] A vector of the invention in which a skeletal muscle- or
cardiac-specific peptide has been included can also be used to
selectively deliver angiogenic factors (e.g; members of the VEGF,
FGF, angiopoietin famillies), cell survival-promoting factors (e.g.
members of the akt/PKB familly), genes involved in the energetic
metabolism (e.g. phospholamban or adenylyl cyclase), cytokins and
their receptors (e.g. IL-6, IL 10, CXCR4, CXCR1, sdf1, MCP 1,
GM-CSF and genes protecting against apoptose (akt) useful for the
treatment of peripheral artery diseases or coronary artery
diseases.
[0122] In a more general way the vectors of the present invention
to deliver to targeting cells genes enzymes, blood derivatives,
hormones such as insulin or growth hormone, lymphokines:
interleukins, interferons, TNF, and the like (French Patent No. 92
03120), growth factors, for example angiogenic factors such as VEGF
or FGF, neurotransmitters or precursors thereof or synthesis
enzymes, trophic, in particular neurotrophic, factors for the
treatment of neurodegenerative diseases, of traumas which have
damaged the nervous system, or of retinal degeneration: BDNF, CNTF,
NGF, IGF, GMF, IFGF, NT3, NT5, HARP/pleiotrophin, or bone growth
factors, haematopoietic factors, and the like, dystrophin or a
minidystrophin (French Patent No. 91 11947), genes encoding factors
involved in coagulation: for example, factors VII, VIII and IX,
suicide genes (e.g. thymidine kinase and cytosine deaminase), genes
for haemoglobin or other protein carriers, genes corresponding to
the proteins involved in the metabolism of lipids, of the
apolipoprotein type chosen from apolipoproteins A-I, A-II, A-IV, B,
C-I, C-II, C-III, D, E, F, G, H, J and apo(a), metabolic enzymes
such as, for example, lipoprotein lipase, hepatic lipase,
lecithin-cholesterol acyltransferase, 7-alpha-cholesterol
hydroxylase, phosphatidyl acid phosphatase, or lipid transfer
proteins such as the cholesterol ester transfer protein and the
phospholipid transfer protein, an HDL-binding protein or a receptor
chosen, for example, from the LDL receptors, the remnant
chylomicron receptors and the scavenger receptors, and the like. It
is possible to add, in addition, leptin for the treatment of
obesity.
[0123] Among the other proteins or peptides which may be encoded by
the gene of the targeted vector, it is important to underline
antibodies, the variable fragments of single-chain antibody (ScFv)
or any other antibody fragment possessing recognition capacities
for its use in immunotherapy, for example for the treatment of
infectious diseases, of tumours, of autoimmune diseases such as
multiple sclerosis (which may involve the use of antiidiotype
antibodies). Other proteins of interest are, in a nonlimiting
manner, soluble receptors such as, for example, the soluble CD4
receptor or the soluble receptor for TNF which may be used for
example for anti-HIV therapy, the soluble receptor for
acetylcholine which may be used for example for the treatment of
myasthenia; substrate peptides or enzyme inhibitors, or peptides
which are agonists or antagonists of receptors or of adhesion
proteins such as, for example, for the treatment of asthma,
thrombosis and restenosis; artificial, chimeric or truncated
proteins. Among the hormones of interest, there may be mentioned
insulin in the case of diabetes, growth hormone and calcitonin.
[0124] The said gene may also be replaced by an antisense sequence
or gene whose expression in the target cell makes it possible to
control the expression of genes or the transcription of cellular
mRNAs. Such sequences may, for example, be transcribed in the
target cell into RNA complementary to cellular mRNAs and thus block
their translation into protein, according to the technique
described in European Patent No. 140 308. The therapeutic genes may
also comprise the sequences encoding ribozymes, which are capable
of selectively destroying target RNAs (European Patent No. 321
201).
EXAMPLES
[0125] The present invention may be better understood by reference
to the following non-limiting Examples, which are provided as
exemplary of the invention. All the modifications presented in the
examples can be combined when not cited.
Example 1
Manipulation of the Hexon HVR5 Loop of Ad5
[0126] The present example demonstrates that manipulation of the
HVR5 loop of Ad5 hexon from amino acids (aa) 269 to 281, keeping
intact the most conserved residues at the extremities of the loop
(a serine in position 268, and a proline in position 282),
unexpectedly provides for effective adenovirus tropism engineering.
The sequence removed from the Ad5 hexon was: TTEAAAGNGDNLT (SEQ ID
NO: 25) (i.e., our constructs naturally display a threonine to
alanine substitution at hexon residue 273 as compared to the
published refence sequence of Ad5), and the flanking sequences
conserved were FFS (upstream) and PKVV (downstream).
Materials and Methods
[0127] Construction of shuttle plasmids for the manipulation of the
HVR5 loop of the hexon. A first intermediate plasmid IE28
containing the flanking regions of the HVR5 loop and in which the
HVR5 loop was replaced with the xylE marker gene from Pseudomones
putida (Zukowski et al., 1983, PNAS 80:1101-1105) was made using
the following two primer pairs: TABLE-US-00004 hex-19243G
(5'ATGGGATGAAGCTGCTACTG- 3') (SEQ ID NO:26) and hex-19623D
(5'tcgcgaGAAAAATTGCATTTCCACTT-3'), (SEQ ID NO:27) and hex-19685G
(5'CCTAAGGTGGTATTGTACAG-3') (SEQ ID NO:28) and hex-20065D
(5'AGCAGTAATTTGGAAGTTCA-3'). (SEQ ID NO:29)
[0128] These primers were used to amplify portions of the hexon
gene corresponding respectively to nucleotides 19245 to 19639 and
19685 to 20084 of the Ad5 genome (Genbank access number M73260)
according to standard PCR techniques. Primer hex-19623D contains
the restriction site NruI and the primer hex-19685G was slightly
modified with respect to Ad5 sequence to create a Bsu36I site
without modifying the protein sequence of the hexon (nucleotide
19690: A to G). Each PCR product was cloned in the plasmid pCRII
(Invitrogen) to generate the plasmids IE21 and IE22, respectively.
Proper cloning was confirmed by DNA sequencing.
[0129] The plasmid p.alpha.xylE.OMEGA. (Frey et al., 1988, Gene
62:237-247) containing the expression cassette of the xylE gene was
restricted with EcoRI, blunt-ended with the T4 DNA polymerase, and
digested with HindIII. This DNA fragment was cloned into the
blunt-ended BamHI-HindIII IE21 plasmid, resulting in plasmid IE26.
Finally, the HindIII-XbaI fragment from IE26 and the XhoI-HindIII
fragment from IE22 were cloned into the SalI-XbaI cleaved plasmid
pXL2756 previously described (Crouzet et al., 1997, PNAS 94:
1414-1419) to generate the shuttle plasmid IE28.
[0130] All the plasmids modified in the HVR5 loop were derived from
plasmid IE28 by replacement of the xylE gene with double-stranded
oligonucleotides. Briefly, complementary single-stranded
oligonucleotides were annealed to form duplexes and cloned into the
NruI-Bsu36I digested IE28, except for the IE31 plasmid, which was
obtained by ligation of the double-stranded oligonucleotide with
the BsrGI-NruI cleaved IE28. Phenotypic screening based on the
yellow staining of bacteria expressing xylE after spraying with
0.5M catechol was used (Zukowski et al., 1983 supra.). The
following table indicates the list of the oligonucleotides used and
the names of the corresponding shuttle plasmids: TABLE-US-00005
TABLE 1 Oligonucleotides used to produce specific hexon insert
plasmid constructs. SEQ shuttle ID plasmid oligonucleotides NO:
IE30 (Ad2 HVR5) 5'- AATACTACCTCTTTGAACGACCGGCAAGGCAATGCTACTAAACC-3'
30 5'- TTAGGTTTAGTAGCATTGCCTTGCCGGTCGTTCAAAGAGGTAGTATT-3' 31 IE31
(epitope
5'-AATCTAGACTCTTTGGAACAACCTACTACTCGCGCTCAAAAACCACGTCTAGATTT-3' 32
from poliovirus
5'-GTACAAATCTAGACGTGGTTTTTGAGCGCGAGTAGTAGGTTGTTCCAAAGAGTCTAGATT-3'
33 type 3)* IE32 5'- TCAACCACTATAAACATTCC-3' 34 (Ad 30 HVRS 5'-
TTAGGAATGTTTATAGTGGTTGA-3' 35 IE33
5'-ACTCCTGGCGCAAATCCTCCAGCAGGCGGTAGTGGAAACGAAGAATACAAACC-3' 36 (Ad
19 HVR5)
5'-TTAGGTTTGTATTCTTCGTTTCCACTACCGCCTGCTAGGAGGATTTGCGCCAGGAGT-3' 37
IE35(epitope from 5'-GATAACCCAGCGTCGACCACGAATAAGGATAAGCTACC-3' 38
poliovirus type 1) 5'-TTAGGTAGCTTATCCTTATTCGTGGTCGACGCTGGGTTATC-3'
39 (5)) IE37(epitope from
5'-GGAGATAACCCAGCGTCGACCACGAATAAGGATAAGCC-3' 40 poliovirus type 1
5'-TTAGGCTTATCCTTATTCGTGGTCGACGCTGGGTTATCTCC-3' 41 IE40(epitope
from 5'-TCTGATAACCCAGCGTCGACCACGAATAAGGAAAGCC-3' 42 poliovirus type
1) 5'-TTAGGCTTATCCTTATTCGTGGTCGACGCTGGGTTATCAGA-3' 43 IE41 (epitope
from 5'- GGATCTGATAACCCAGCGTCGACCACGAATAAGGATAAGCC-3' 44 poliovirus
type 1) 5'-TTAGGCTTATCCTTATTCGTGGTCGACGCTGGGTTATCAGATCC-3' 45
IE43(epitope from
5'-GGAGATAACCCAGCGTCGACCACGAATAAGGATAAGCTAGGTGGCCC-3' 46 poliovirus
type 1) 5'-TTAGGGCCACCTAGCTTATCCTTATTCGTGGTCGACGCTGGGTTATCTCC-3' 47
IE44(epitope from
5'-GGAGATAACCCAGCGTCGACCACGAATAAGGATAAGCTAGGTTCTCC-3' 48 poliovirus
type 1) 5'-TTAGGAGAACCTAGCTTATCCTTATTCGTGGTCGACGCTGGGTTATCTCC-3' 49
IE45(epitope from
5'-GGAGATAACCCAGCGTCGACCACGAATAAGGATAAGCTATCTCC-3' 50 poliovirus
type 1) 5'-TTAGGAGATAGCTTATCCTTATTCGTGGTCGACGCTGGGTTATCTCC-3' 51
IE46(epitope from
5'-GGAGATAACCCAGCGTCGACCACGAATAAGGATAAGCTATCTCC-3' 52 poliovirus
type 1) 5'-TTAGGACCAGATAGCTTATCCTTATTCGTGGTCGACGCTGGGTTATCTCC-3' 53
IE47(epitope from
5'-GGAGATAACCCAGCGTCGACCACGAATAAGGATAAGCTATCTAGTCC-3' 52 poliovirus
type 1) 5'-TTAGGACTAGATAGCTTATCCTTATTCGTGGTCGACCGCTGGGTTATCTCC-3'
55 *Crompton, et al., 1994, J. Gen. Vir. 75:133-139.
[0131] Construction of the associated plasmid backbones and
viruses. An intermediate plasmid backbone containing the xylE gene
instead of the HVR5 loop of the hexon gene was constructed to
facilitate subsequent manipulation of the HVR5 loop. For this
purpose, the shuttle plasmid IE28 was recombined with the plasmid
backbone pXL3006 (this plasmid contains a PacI-excisable
E1E3-deleted adenoviral genome with a CMV-lacZ expression cassette
in place of the E1 region) in the G4977 bacterial strain according
to the method described by Crouzet et al. (1997 supra.) to obtain
the plasmid backbone IE28c, which differs from plasmid backbone
pXL3006 by the xylE-containing HVR5 sequence.
[0132] All the shuttle plasmids IE30 to IE47 were then recombined
with the plasmid backbone IE28c using the "xylE screening" to get
the plasmid backbones IE30c to IE47c. After cleavage with the PacI
enzyme, 2 .mu.g (or 5 .mu.g) of these digested backbones were
transfected in the 911 cells (or 293 cells) using Lipofectamine
(Gibco BRL) to generate the corresponding viruses AdIE30 to AdIE47.
These HVR5-modified E1E3-deleted adenoviruses therefore express the
same CMV-lacZ expression cassette.
[0133] All other HVR5-modified adenoviruses (e.g., displaying uPAR-
or integrin-binding peptides; see examples thereafter) were
constructed by the same strategy.
[0134] Cells and antibodies. 293 and W162 cells were maintained in
MEM (Gibco BRL) supplemented with 10% fetal calf serum. 911 cells
(Fallaux et al., 1996, Hum. Gene Ther. 7:215) were grown in DMEM
supplemented with 10% fetal calf serum. The C3 monoclonal antibody
(C3 mAb) directed against the poliovirus type 1 described in
Blondel et al, 1983, Virology 126:707 was provided by Dr. R.
Crainic (Pasteur Institute, Paris, France). L5 rabbit polyclonal
antibodies directed against the whole Ad5 capsid were produced in
RPR-Gencell's facilities (RPR SA, Vitry, France).
[0135] Viruses. All the viruses were amplified in
E1-transcomplementing cells (e.g., 293 cells) according to
classical methods. The pattern/identity of the viruses was
controlled by restriction analysis and sequencing of the inserts on
viral DNA obtained using the Hirt procedure. Viral stocks were
prepared in 293 cells, purified by CsCl gradient, desalted using
PD10 columns (Pharmacia) and stored in PBS supplemented with 10%
glycerol at -80.degree. C. Biological quantification was carried
out by numbering the plaque-forming units (PFU) on 911 cells and/or
numbering the lacZ-transducing units (TDU) two days post-infection
of W162 cells following X-Gal staining (Dedieu et al. 1997, J.
Virol. 71 :4626). Physical quantification was carried out by
anion-exchange by numbering the viral particles (VP).
[0136] Neutralization test. One-half .mu.l of anti poliovirus C3
monoclonal antibody were incubated in PBS for 1 h at 37.degree. C.
with 10.sup.5 TDU of purified virus, and the mix was then absorbed
onto W162 cells in a 6 wells-plate for a further 1 h at 37.degree.
C. Cells were then washed twice with PBS, and fresh medium was
added to the cells which were incubated at 37.degree. C. for 2
days. Cell monolayers were fixed with formaldehyde
(0.37%)-glutaraldehyde (0.2%), X-Gal stained, and blue cells were
counted.
[0137] Immunoprecipitation protocol Viral particles (10.sup.10) of
CsCl-purified virus were resuspended in 400 .mu.l of non-denaturing
incubation buffer (50 mM Tris pH7.5, 150 mM NaCl, 0.05% NP40) and
then incubated with 0.1 .mu.l of anti poliovirus C3 monoclonal
antibody for 1 h at 4.degree. C. Four hundred .mu.l of protein
A-Sepharose previously equilibrated with incubation buffer was then
added, and further incubated for 1 h at 4.degree. C. Following
incubation, the mix was spun down by brief centrifugation in a
microcentrifuge. The pellet was washed twice with 1 ml of
incubation buffer and once with 1 ml of 10 mM Tris pH7.5, 0.1% NP40
for 20 min at 4.degree. C. The pellet containing the protein
A-antibody-virus complex was resuspended in 50 .mu.l of Laemmli
buffer 1.times., boiled 2 min and the supernatant was collected
after a brief centrifugation. Ten .mu.l of supernatant was analyzed
by SDS-polyacrylamide gel electrophoresis (Novex). Western blot was
carried out using the L5 rabbit polyclonal serum directed against
the whole Ad5 capsid according to the ECL procedure (Amersham).
Results
[0138] Construction strategy for modification of the HVR5 loop. Two
series of constructions were made. The first one was designed to
assess the "capacity" of the Ad5 HVR5 loop for foreign sequences,
by replacement of this loop by the HVR5 loops from other Ad
serotypes, which greatly differ in size. TABLE-US-00006 TABLE 2
Ad5-based adenoviruses with heterospecific HVR5 loops. HVR5
sequence replacing the SEQ Virus sequence of HVR5 loop of Ad5 ID
NO: Ad IE 30 Ad2:14 aa NTTSLNDRQGNATK 56 Ad IE 32 Ad30:6 aa STTINI
57 Ad IE 33 Ad 19:17 aa TPGANPPAGGSGNEEYK 58
[0139] As a viability control, we introduced the modification
published by Crompton et al. (1994, J. Gen. Virol. 75:133-139) in
which a poliovirus type 3 epitope was introduced in place of a
larger deletion encompassing HVR5. Importantly, the crompton virus
was initially constructed by homologous recombination between a
plasmid containing the Ad2 modified hexon and an Ad5-based
adenoviral genome. This virus therefore displays a chimeric hexon
protein between Ad2 and Ad5 in which a 4-residue larger HVR5
deletion has been substituted for a foreign peptide. To reproduce
the Crompton et al. construct as closely as possible, an Ad5-based
virus (Ad IE31) was constructed by the EDRAG technology in which
the foreign peptide of Crompton et al. was introduced in place of
hexon residues 269 to 285 (TTEAAAGNGDNLTPKVV; SEQ ID NO:59) of Ad5
instead of TTEAAAGNGDNLT (SEQ ID NO:60). TABLE-US-00007 TABLE 3
Insert of control virus AdIE31. Virus sequence replacing the HVR5
loop of AdS Ad IE31 NLDSLEQPTTRAQKPRLD (SEQ ID NO:61 )
[0140] In the second series of constructs, the HVR5 loop (aa 269 to
281) was replaced by a neutralizing linear epitope of poliovirus
type 1 (DNPASTTNKDK; SEQ ID NO. 62). This model binding-peptide was
inserted in various neighboring contexts (i.e., various linkers
composed of leucine and/or glycine and/or serine residues were
used) to assess their importance for virus viability and peptide
accessibility. TABLE-US-00008 TABLE 4 Insertions of a poliovirus
type 1 epitope in the HVR5 loop. upstream downstream SEQ Virus
linker epitope used linker ID NO: Ad IE35 none DNPASTTNKDK L 63 Ad
IE37 G DNPASTTNKDK none 64 Ad IE40 S DNPASTTNKDK none 65 Ad IE41 G
S DNPASTTNKDK none 66 Ad IE43 G DNPASTTNKDK LG G 67 Ad IE44 G
DNPASTTNKDK LG S 68 Ad IE45 G DNPASTTNKDK LS 69 Ad IE46 G
DNPASTTNKDK LSG 70 Ad IE47 G DNPASTTNKDK LS S 71
[0141] Viability of the viruses. The three viruses with Ad2, Ad19,
or Ad30 HVR5 loops instead of the Ad5 HVR5 loop, and the nine
chimeric virus containing the poliovirus type 1 epitope were
viable. No loss of productivity was observed. Unexpectidely, the
control virus Ad IE31 could not be recovered despite intensive
efforts.
[0142] Assessment of poliovirus epitope accessibility by
immunoprecipitation assay. Immunoprecipitation experiments were
performed on the viruses carrying the poliovirus epitope using a
cognate antipoliovirus monoclonal antibody (C3 mAb) in
non-denaturing conditions. The following table 5 summarizes the
data obtained: TABLE-US-00009 TABLE 5 Immunoprecipitation with C3
mAb. Virus peptide inserted in the HVR5 loop Immunoprecipitation Ad
IE35 DNPASTTNKDK-L - Ad IE37 G-DNPASTTNKDK - Ad IE40 S-DNPASTTNKDK
- Ad IE41 GS-DNPASTTNKDK - Ad IE43 G-DNPASTTNKDK-LGG ++ Ad IE44
G-DNPASTTNKDK-LGS ++ Ad IE45 G-DNPASTTNKDK-LS ++ Ad IE46
G-DNPASTTNKDK-LSG ++ Ad IE47 G-DNPASTTNKDK-LSS ++
[0143] The presence of a linker downstream of the poliovirus
epitope was thus found critical for C3 mAb binding to the modified
viral capsids, most likely because it allows proper presentation
and/or accessibility of the binding peptide at the hexon surface.
When the modified viruses were denatured prior to
immunoprecipitation, all of them were efficiently
immunoprecipitated by C3 mAb (not shown).
[0144] Assessment of poliovirus epitope functionality by
neutralization assay. As the C3 mAb is neutralizing for poliovirus
infection, we anticipated that it could also neutralize the
infectivity of Ad carrying the poliovirus epitope. Incubation of C3
mAb with the whole series of Ad IE viruses was performed in PBS
(i.e., under native conditions) prior to infection of W162 monkey
cells. The data are shown in FIG. 1. These data correlate perfectly
with the immunoprecipitation results, i.e., all the viruses that
could bind C3 mAb in non-denaturating conditions were also
neutralized by this antibody.
Discussion
[0145] The data show that modification of the HVR5 loop of
adenovirus can allow functional and specific interaction of the
modified hexons with a specific binding protein. Particular modes
of insertion are however required as the data showed that
accessibility (immunoprecipitation assay) and functionality
(neutralization assay) of the binding peptide epitope were
dependent on minimal spacer/neighboring sequences.
[0146] Contrasting with the conclusion of Crompton et al., our
results also show that the deletion of aa 269 to 285 of Ad5 hexon
is deleterious for virus growth and/or viability. This can be
explained by the fact that the residues 282 to 285 are localized in
the b ta-strand located downstream of the HVR5 loop, which is
probably essential for the structure of the hexon as a monomer or a
trimer. Therefore, the present approach, which is more precise and
rigorous than that described in Crompton et al., unexpectedly
overcame the disadvantages of that reference and provided viable
viruses equipped with a modified tropism (see below).
Example 2
Manipulation of the Fiber HI Loop
[0147] Deletion of the HI loop of the Ad5 fiber was performed: the
sequence removed from the fiber knob includes residues 538 to 548
(i.e., sequence GTQETGDTTPS) (SEQ ID NO:72). Its flanking sequences
are thus TLN (upstream) and AYS (downstream).
Materials and Methods
[0148] Construction of shuttle plasmids for the manipulation of the
HI loop of the fiber. A first intermediate plasmid pJD3 containing
the flanking regions of the HI loop and in which the HI loop was
replaced with the xylE gene was made using the following two primer
pairs: TABLE-US-00010 (SEQ ID NO:73 ) HIgul
(5'-CAGCTCCATCTCCTAACTGTAGACTAAATG-3') and (SEQ ID NO:74 ) HIgul
(5'-GGTTACCGGTTTAGTTTTGTCTCCGTTTAA-3') and (SEQ ID NO:75 ) HIdul
(5'-AGCGCTTACTCTATGTCATTTTCATGGGAC-3') and (SEQ ID NO:76 ) HIddl
(5'-GAGTTTATTAATATCACTGATGAGCGTTTG-3').
[0149] These primer pairs were used to amplify portions of the
fiber and E4orf7 genes corresponding respectively to nucleotides
32255 to 32634 and 32712 to 33090 of the Ad5 genome (Genbank access
number M73260) according to standard PCR techniques. The primers
HIgd1 and HIdu1 are designed in such a way that they create the
restriction sites BstEII and Eco47III, respectively, without
modifying the fiber protein sequence at the immediate vicinity of
the HI loop. These sites were further used for direct cloning of
foreign peptides into HI (see below and table 5).
[0150] Each PCR product was cloned into the plasmid PCR2.1
(Invitrogen) to generate the plasmids PCR2.1-H4 and PCR2.1-I2,
respectively, and sequenced.
[0151] The plasmid p.alpha.xylE.OMEGA. containing the expression
cassette for the xylE gene was restricted with EcoRI, blunt-ended
with the T4 DNA polymerase, and digested with HindIII. This DNA
fragment was subcloned into HindIII-restricted PCRII (Invitrogen)
resulting in the plasmid IE23. A NsiI-XhoI fragment of IE23 was
introduced into the NsiI-XhoI digested PCR2.1-H4 plasmid, resulting
in the plasmid pJD2. Finally, the SacI-XbaI fragment from pJD2 and
the SpeI-XhoI fragment from PCR2.1-I2 were cloned into the
previously described (Crouzet et al., 1997, supra.) plasmid pXL2756
cleaved by SacI-SalI to generate the shuttle plasmid pJD3.
[0152] All the plasmids modified in the HI loop were derived from
plasmid pJD3 by replacement of the xylE gene with double-stranded
oligonucleotides. Briefly, complementary single-stranded
oligonucleotides were annealed to form duplexes and cloned into
BstEII-Eco47III digested pJD3. A phenotypic screening based on the
yellow staining of bacteria expressing xylE after spraying with
0.5M catechol was used. The following table indicates the list of
the oligonucleotides used and the name of the corresponding shuttle
plasmids: TABLE-US-00011 TABLE 5 Examples of oligonucleotides used
to replace the HI loop. SEQ Shuttle ID plasmid oligonucleotides NO:
pJD7 (HI 5'-
GTAACACTAACCATTACACTAAACGGTACCCAGGAAACAGGAGACACAACTCCAAGT-3' 77
loop from 5'-
ACTTGGAGTTGTGTCTCCTGTTTCCTGGGTACCGTTTAGTGTAATGGTTAGT-3' 78 Ad5)
pJDS(HI 5'-
GTAACACAACCATTACACTAAACGGTACCAGTGAATCCACAGAAACTAGCGAGGTAAGC-3' 79
loop from 5'-
GCTTACCTCGCTAGTTTCTGTGGATTCACTGGTACCGTTTAGTGTAATGGTTAGT-3' 80 Ad2)
pJD6(HI 5'- GTAACACTAACCATTACACTAAACCAAGAAACACAATGTGAA-3' 81 loop
from 5'- TTCACATTGTGTTTCTTGGTTTAGTGTAATGGTTAGT-3' 82 Ad9) pCF1
5'-GTAACCCTAACCATTACACTAAACGGTGATAACCCAGCGTCGACCACGAATAAGGATAAGAGC-3'
83 (epitope
5'-GCTCTTATCCTTATTCGTGGTCGACGCTGGGTTATCACCGTTTAGTCTAATGGTTAGG-3' 84
from poliovirus type 1) pCF2 5'-
GTAACCCTAACCATTACACTAAACGGTGATAACCCAGCGTCGACCACGAATAAGGATAAGGGAAG-
C-3' 85 (epitope 5'-
GCTTCCCTTATCCTTATTCGTGGTCGACGCTGGGTTATCACCGTTTAGTGTAATGGTTAGG-3'3
86 from poliovirus type I) pCF3
5'-GTAACCCTAACCATTACACTAAACGGTGATAACCCAGCGTCGACCACGAATAAGGATAAGTCAAGC-
-3' 87 (epitope
5'-GCTTGACTTATCCTTATTCGTGGTCGACGCTGGGTTATCACCGTTTAGTGTAATGGTTAGG--
3' 88 from poliovirus type 1 pCF4
5'-GTAACCCTAACCATTACACTAAACGGTGATAACCCAGCGTCGACCACGAATAAGGATAAGGGCGGA-
AGC-3' 89 (epitope
5'-GCTTCCGCCCTTATCCTTATTCGTGGTCGACGCTGGGTTATCACCGTTTAGTGTAATGGTTA-
GG-3' 90 from poliovirus type 1) pCF5
5'-GTAACCCTAACCATTACACTAAACGGTGATAACCCAGCGTCGACCACGAATAAGGATAAGTCATCT-
AGC-3' 91 (epitope
5'-GCTAGATGACTTATCCTTATTCGTGGTCGACGCTGGGTTATCACCGTTTAGTGTAATGGTTA-
GG-3' 92 from poliovirus type 1) pCF6
5'-GTAACCCTAACCATTACACTAAACGGTGATAACCCAGCGTCGACCACGAATAAGGATAAGGGATCC-
AGC-3' 93 (epitope
5'-GCTGGATCCCTTATCCTTATTCGTGGTCGACGCTGGGTTATCACCGTTTAGTGTAATGGTTA-
GG-3' 94 from poliovirus type 1) pCF7
5'-GTAACCCTAACCATTACACTAAACGGTGATAACCCAGCGTCGACCACGAATAAGGATAAGTCAGGA-
AGC-3' 95 (epitope
5'-GCTTCCTGACTTATCCTTATTCGTGGTCGACGCTGGGTTATCACCGTTTAGTGTAATGGTTA-
GG-3' 96 from poliovirus type I) pCF8
5'-GTAACCCTAACCATTACACAAACGGTGATAACCCAGCGTCGACCACCACGAATAAGGATAAG-3'
97 (epitope
5'-CTTATCCTTATTCGTGGTCGACGCTGGGTTATCACCGTTTAGTGTAATGGTTAGG-3' 98
from poliovirus type I)
[0153] Construction of the associated plasmid backbones and
viruses. An intermediate plasmid backbone containing the xylE gene
instead of the HI loop of the fiber gene was first constructed to
facilitate subsequent screening of any plasmid backbone displaying
a modified HI loop. For this purpose, the shuttle plasmid pJD3 was
recombined with the plasmid backbone pXL3006 in the G4977 bacterial
strain according to the method described by Crouzet et al., supra,
to obtain the plasmid backbone pBX, which therefore displays a
PacI-excisable E1 E3-deleted adenoviral genome containing a
CMV-lacZ expression cassette in place of E1, as well as the xylE
marker in the HI loop.
[0154] Shuttle plasmids pJD5, 7 and 6, and pCF1 to pCF8 were then
recombined with the plasmid backbone pBX using the "xylE screening"
to get the plasmid backbones pBV2, 5 and 9, and pBC1 to pBC8,
respectively. After cleavage with the PacI enzyme, 2 .mu.g (or 5
.mu.g) of DNA were transfected in the 911 cells (or 293 cells)
using Lipofectamine (Gibco BRL) to generate the corresponding
viruses vBV2, 5 and 9, and vBC1 to vBC8.
[0155] Other methods. The cells, antibodies, viruses,
immunoprecipitation assays, and neutralization assays were as
described in Example 1, supra.
Results
[0156] Construction strategy for the HI loop insertion vectors. Two
series of constructions were made. The first one was designed to
assess the "capacity" of the Ad5 HI loop for foreign sequences, by
replacement of this loop by the HI loops of other adenovirus
serotypes, with some variations in size. TABLE-US-00012 TABLE 6
Capacity insertions in the fiber protein HI loop. HI sequence
replacing SEQ Virus sequence of the HI loop of Ad5 ID NO: VBV2 Ad2:
12 aa GTSESTETSEVS 99 VBV5 Ad5: 11 aa GTQETGDTTPS 100 VBV9 Ad9: 6
aa QETQCE 101
[0157] In the second series of constructs, the HI loop was replaced
by a neutralizing epitope of poliovirus type 1 (DNPASTTNKDK) (SEQ
ID NO:62). Due to the very close 3D structures at the N-terminal
sides of the Ad5 HI loop and the poliovirus sequences in their
native environment/protein, a minimal one residue linker (glycine)
was added usptream of the epitope. This was not the case downstream
of the insertion site for which spacers of different length and
composition were included to assess their impact on virus growth
and peptide accessibility. TABLE-US-00013 TABLE 7 Insertions of a
poliovirus type 1 epitope in the fiber protein HI loop. downstream
Virus epitope used linker SEQ ID NO: vBC1 DNPASTTNKDK S 102 vBC2
DNPASTTNKDK G S 103 vBC3 DNPASTTNKDK S S 104 vBC4 DNPASTTNKDK G G S
105 vBC5 DNPASTTNKDK S S S 106 vBC6 DNPASTTNKDK G S S 107 vBC7
DNPASTTNKDK S G S 108 vBC8 DNPASTTNKDK none 109
[0158] Viability of the viruses. The three control viruses with
Ad2, Ad5 or Ad9 HI loops instead of the Ad5 HI loop and the 8
chimaeric virus containing the poliovirus epitope were viable. No
loss of productivity was observed.
[0159] Assessment of poliovirus epitope accessibility by
immunoprecipitation assay. Immunoprecipitation experiments of the
viruses carrying the poliovirus epitope were performed with the
cognate antipoliovirus C3 mAb in non-denaturing conditions. The
following table summarizes the data: TABLE-US-00014 TABLE 8
Recognition of the targeting sequence as detected by
immunoprecipitation with C3 mAb. Virus Peptide inserted in the HI
loop Immunoprecipitation VBC1 G-DNPASTTNKDK-S - VBC2
G-DNPASTTNKDK-GS +/- VBC3 G-DNPASTTNKDK-SS + VBC4 G-DNPASTTNKDK-GGS
++ VBC5 G-DNPASTTNKDK-SSS ++ VBC6 G-DNPASTTNKDK-GSS ++ VBC7
G-DNPASTTNKDK-SGS ++ VBC8 G-DNPASTTNKDK -
[0160] These data show that, as for HVR5 loop insertion, binding of
the C3 mAb antibody to the modified capsids was criticaly dependent
on the presence of a spacer sequences of minimal length. In
particular, a downstream linker of 3 residues conferred the most
efficient binding. When the viruses were denatured prior to the
immunoprecipitation assay, they were all able to bind the C3 mAb
antibody (not shown).
[0161] Assessment of poliovirus epitope functionality by
neutralisation assay. The ability of C3 mAb to neutralize
infectivity of the fiber-modified viruses was then assessed to
determine if it also correlated with the immunoprecipitation data
as exemplified in Example 1 for the hexon-modified capsids.
However, unlike the results with hexon modification, there was no
inhibition of infectivity following incubation of any of the
fiber-modified virus with C3 mAb (data not shown). Different
explanations can be suggested. In particular, there are only 12
fiber trimers on the virion surface versus 240 hexon trimers.
Therefore, the density of antibody binding to the hexon-modified
capsids may have decreased virus infectivity either directly, or
following steric hindrance of the RGD motif of the penton base so
integrin-mediated internalisation was affected. In any case, C3mAb
specific binding to the HI-modified capsids did not prevent native
interaction between the virus and its target cells.
Discussion
[0162] The data demonstrate that the HI loop of adenovirus can be
replaced by a foreign peptide and that the corresponding viruses
are: i) viable, and ii) with no drastic effect on virus
productivity. The data also demonstrate that specific recognition
of the introduced peptide requires suitable neighboring linkers
(i.e., flexible linkers of minimal size).
Example 3
Modification of Fiber Protein Length--Short Fibers
[0163] Taking into account the comparison of the cell-binding
pathway of Ad5 (a long-fiber serotype) with other serotypes such as
Ad9 (a short-fiber serotype), the length of the shaft of the fiber
was modified by designing either an intertypic fiber (substitution
of the Ad5 shaft by the Ad3, another short-fiber serotype, shaft),
or a shortened Ad5 shaft that retained only 6 or 9 repeats instead
of 22 in the native protein.
Materials and Methods
[0164] Construction of shuttle plasmids with shortened fibers.
Three shuttle plasmids containing shortened fibers were
constructed. Two of them (pSF1 and pSF2) display major deletions
within the fiber shaft, whereas the third one (pIF1) harbors the
Ad3 shaft instead of that of Ad5. All constructs therefore retain
the Ad5 tail and knob domains.
[0165] To construct pSF1, two pairs of primers were used:
TABLE-US-00015 (SEQ ID NO:110 ) 5M3g
(5'-ATTTCTGTCGACTTTATTCAGCAGCACCTC-3') and (SEQ ID NO:111 ) 5M3d
(5'-GTTTGACTTGGTTTTTTTGAGAGGTGGGCT-3'), and (SEQ ID NO:112 ) 5M17g
(5'-TTGGATATTAACTACAACAAAGGCCTTTAC-3') and (SEQ ID NO:113 ). 5M17d
(5'-GAAACTGGAGCTCGTATTTGACTGCCACAT-3').
[0166] These primers were used to amplify portions of the fiber
gene corresponding respectively to nucleotides 30885 to 31329 and
31936 to 32351 of the Ad5 genome (Genbank access number M73260)
according to standard PCR techniques. The primers 5M3g and 5M17d
slightly differ from the Ad5 sequence by respectively containing
the restriction sites SalI and SacI. After digestion by SalI or
SacI, these PCR products were ligated and cloned into the SacI-SalI
cleaved pXL2756, resulting in the pSF1 plasmid which displays an in
frame deletion encompassing shaft repeats 4 to 16.
[0167] To construct pSF2, two pairs of primers were used:
TABLE-US-00016 (SEQ ID NO: 114 ) 5M3g
(5'-ATTTCTGTCGACTTTATTCAGCAGCACCTC-3') and (SEQ ID NO: 115 ) 5M3d
(5'-GTTTGACTTGGTTTTTTTGAGAGGTGGGCT-3'), and (SEQ ID NO: 116 ) 5M20g
(5'-CTCAAAACAAAAATTGGCCATGGCCTAGAA-3') and (SEQ ID NO: 117) 5M20d
(5'-ATCCAAGAGCTCTTGTATAGGCTGTGCCTT-3').
[0168] These primers were used to amplify portions of the fiber
gene corresponding respectively to nucleotides 30885 to 31329 and
32110 to 32530 of the Ad5 genome according to standard PCR
techniques. The primers 5M3g and 5M20d slightly differ from the Ad5
sequence by respectively containing the restriction sites SalI and
SacI. After digestion by SalI or SacI, these PCR products were
ligated and cloned into the SacI-SalI cleaved pXL2756, resulting in
the pSF2 plasmid which displays an in frame deletion encompassing
shaft repeats 4 to 19.
[0169] The pIF1 plasmid was made using the gene SOEing method
described by Horton et al (4) consisting of recombining DNA
sequences by PCR without relying on restriction sites. Five
successive steps were necessary to construct the pIF1 plasmid,
which contains an intertypic fiber gene, composed of the Ad5 tail
and knob and the Ad3 shaft. A first PCR product containing the Ad5
fiber tail was amplified from the Ad5 genome using the primers:
TABLE-US-00017 (SEQ ID NO:118 ) SOE35Tg
(5'-TACAAGTCGACAACCAAGCGTCAGAAATTG-3') and (SEQ ID NO:119 ) SOE35Td
(5'-AAGACTTAAAACCCCAGGGGGACTCTCTTG-3')
[0170] The primer SOE35Tg nearly matches the Ad5 nucleotides 30660
to 30689 with a slight modification resulting in the creation of a
SalI site. The 15 underlined bases in the SOE35Td primer match with
the sequence of the first repeat of the Ad3 fiber shaft and the 15
remaining bases correspond to the sequence of the fiber tail end of
Ad5.
[0171] A second PCR product containing the Ad3 fiber shaft was
amplified from the Ad3 fiber gene using the primers: TABLE-US-00018
(SEQ ID NO: 120 ) SOE35Sg (5'-GAGAGTCCCCCTGGGGTTTTAAGTCTTAAA-3')
and (SEQ ID NO: 121 ) SOE35Sd
(5'-GGTCCACAAAGTGTTATTTTTCAGTGCAAT-3').
[0172] The underlined bases in both primers are contained in the
Ad5 fiber gene (respectively within the tail and knob subdomains),
the remaining ones are from the Ad3 shaft.
[0173] A third PCR product containing part of the Ad5 fiber knob
was amplified from the Ad5 fiber gene using the primers
TABLE-US-00019 (SEQ ID NO: 122 ) SOE35Kg
(5'-CTGAAAAATAACACTTTGTGGACCACACCA-3') and (SEQ ID NO: 123 )
SOE35Kd (5'-TCCTGAGCTCCGTTTAGTGTAATGGTTAGT-3').
[0174] The underlined bases in the SOE35Kg primer fit with the
sequence of the last repeat of the Ad3 fiber shaft, whereas the
remaining ones correspond to the first nucleotides of the Ad5 fiber
knob. The primer SOE35Kd nearly matches the Ad5 nucleotides 32634
to 32663 with a slight modification resulting in the creation of a
SacI site. The two first PCR products were mixed, denatured and
reannealed under PCR conditions, so that the top strand of the
first product and the bottom strand of the second one overlap and
act as primers on one another. The hybrid product was formed when
this overlap was extended by polymerase. Inclusion of the primers
SOE35Tg and SOE35Sd in the SOE reaction caused the recombinant
product to be PCR amplified right after it is formed (see figure of
Horton et al, previously cited). This resulted in a PCR product
containing the Ad5 tail followed by the Ad3 shaft. Finally, the
same SOE procedure was carried out with this last PCR product and
the third PCR product using the primers SOE35Tg and SOE35Kd, giving
rise to a DNA fragment containing an intertypic fiber composed of
the Ad5 tail, the Ad3 shaft and part of the Ad5 knob, and flanked
with the unique restriction sites SalI and SacI (FIG. 2). After
digestion with these enzymes, the final PCR product was cloned into
the SalI-SacI cleaved pXL2756 plasmid, resulting in the pIF1
shuttle plasmid.
[0175] Plasmids pSF1, pSF2 and pIF1 were sequenced.
[0176] Construction of the associated plasmid backbones and
viruses. The three shuttle plasmids pSF1, pSF2 and pIF1 were
recombined with the plasmid backbone pXL3006 (contains a
PacI-excisable E1 E3-deleted recombinant adenoviral genome with a
CMV-lacZ expression cassette in place of the E1 region) in the
G4977 bacterial strain according to the method described by Crouzet
et al., supra, to obtain the plasmid backbones pBS1, pBS2 and pBI1,
respectively. After cleavage with PacI, 2 .mu.g (or 5 .mu.g) of DNA
were transfected in the 911 cells (or 293 or PER.6 cells) using
Lipofectamine (Gibco BRL) to generate the corresponding viruses
vBS1, vBS2 and vBI1.
[0177] The intermediate plasmid backbones AE31, AE32 and AE33 were
also constructed by homologous recombination between shuttle
plasmid IE28 and the pBI1, pBS1 and pBS2 plasmid backbones. These
plasmid backbones which contain a xylE expression cassette instead
of the HVR5 hexon loop together with short-shafted fibers were
further used to facilitate the recovery of plasmid backbones
combining short-shafted fibers and HVR5-modified hexons (e.g., with
insertion of UPAR- or integrin-binding peptides; see example
10).
[0178] In parallel, the intermediate plasmid backbones AE34, AE35
and AE36 were constructed by recombination between shuttle plasmid
pJD3 and the pBI1, pBS1 and pBS2 plasmid backbones. These plasmid
backbones were further used to facilitate the recovery of plasmid
backbones combining short-shafted fibers and HI-modified fibers
(e.g., with insertion of UPAR- or integrin-binding peptides).
[0179] Other methods. The cells, antibodies, viruses,
immunoprecipitation assays, and neutralization assays were as
described in Example 1, supra.
Results and Discussion
[0180] Viruses with shortened fiber proteins are expected to
increase accessibility of binding peptides exposed at the hexon
surface and/or reducing wild-type (i.e., native) virus/cell
interactions. These constructs are summarized in Table 9.
TABLE-US-00020 TABLE 9 Shortened fiber protein adenoviruses. Virus
structure of the chimeric fiber vBI1 Ad5 tail - Ad3 shaft - Ad5
knob vBS1 Short-shafted fiber from Ad5 (deletion encompassing shaft
repeats 4-16) vBS2 Short-shafted fiber from Ad5 (deletion
encompassing shaft repeats 4-19)
[0181] Viral productivity was drastically reduced in all three
cases (although to different degrees), most likely because of the
inability of the modified fibers to interact efficiently with its
cellular receptor. That the defect occurred at such an early stage
is indeed supported by normal viral DNA replication parameters in
293-infected cells (i.e., Xgal-positive), together with normal
accumulation of viral late proteins. Also, Western blotting under
non-denaturing conditions demonstrated that proper trimerization of
the modified fibers occurred in all three cases.
[0182] Although vBS1 binds less efficiently to CAR (Coxsackie and
adenoviruses receptor)-positive cells, it could however be
amplified in PER.C6 up to a lab-scale stock. As shown in FIG. 3, it
was also observed that infection of 293 cells with vBS1 resulted in
a 10-fold decrease in cell transduction as compared to that of a
control virus displaying the native Ad5 fiber, which indicates a
significant loss in Ad receptor binding. In this experiment, 293
cells were incubated with PBS or purified fiber knob (100 .mu.g/ml)
for 30 min at RT before infection at an moi of 50 (VP/cell) for 30
min at RT. Cells were then washed twice with PBS and furter
incubated 24 h at 37.degree. C. in medium before preparation of
protein extracts. Specific lacZ expression was then quantified
(units per protein extract).
[0183] Based on these data, C57/B16 mice were injected iv with the
vBS1 construct or its control virus (unmodified shaft) to determine
the profile of lacZ expression in major organs. Liver, heart and
lung were analyzed for .beta.-galactosidase expression by
histochemistry. Results are presented in the following table 10 (%
of XGal-positive cells mean and range of values). TABLE-US-00021
TABLE 10 Profile of transgene expression in major organs injected
dose (VP) control virus vBS1 liver .sup. 3.10.sup.9 (n = 5) 15
(10-20) 0 (0-0) 10.sup.10 (n = 5) 50 (50-60) 5 (2-5) 3.10.sup.10 (n
= 5) 50 (20-80) 15 (10-20) heart .sup. 3.10.sup.9 (n = 5) 0 0
10.sup.10 (n = 5) 0 0 3.10.sup.10 (n = 5) 0 0 lung .sup. 3.10.sup.9
(n = 5) 0 0 10.sup.10 (n = 5) 0 0 3.10.sup.10 (n = 5) 0 0
[0184] These data indicate a dose-response effect for both
adenoviruses in the liver, whereas no XGal-positive cells could be
evidenced in the heart and lung tissues from either treated groups.
Most interestingly, shortening of the fiber shaft resulted in a
10-fold decrease in liver transduction emphasizing the usefulness
of manipulating the fiber shaft to direct infection mostly to the
desired organs/cells in vivo, provided the recombinant adenovirus
has been equipped with an additional, CAR-independent, entry
pathway.
Example 4
Specific Targeting of Cells Expressing a Urokinase-Type Plasminogen
Activator Receptor
[0185] Various high affinity uPAR-binding peptides were included
within the hexon HVR5 and the fiber HI loops, or added to the
C-terminal end of the fiber protein. These peptides originate
either from wild-type ATF (Rettenberg et al., 1995, Biol. Chem.
Hoppe-Seyler 376:587-594) and mutant ATF (Magdolen et al., 1996,
Eur. J. Biochem. 237:743-751), a phage library (Goodson et al.,
1994, Proc. Natl. Acad. Sci. 91:7129-7133), and an associated
mutant, or human vitronectin (Waltz et al., 1997, J. Clin. Invest.
100:58-67). All viruses contain a gene expression cassette (lacZ or
Gax) inserted in place of the E1 genes.
[0186] The methods for insertion of these peptides in the hexon
HVR5 loop and the fiber protein HI loop were as described for the
poliovirus epitopes in Examples 1 and 2, above. Shortening of the
fiber protein was achieved as described in Example 3. Further
Material and Methods are described hereunder.
[0187] Cell Culturing of PERC6 Cells
[0188] PER.C6 cells were grown in Dulbecco's modified medium
(DMEM), 10% FCS and 10 mM MgCl.sub.2 in a 10% CO.sub.2 atmosphere
at 37.degree. C. (Fallaux et al, 1998, Hum Gene Ther, 9
:1909-1917).
[0189] Recombinant Adenoviral Genomes
[0190] Recombinant adenoviral genomes were cloned into an
RK2-derived plasmid by homologous recombinations in E. coli
utilizing the EDRAG technology as described in the French
application FR 2 730 504. The technology was simplified by
replacing the ColE1 origin of replication by the origin of R6K in
the suicide shuttle, as described in WO 97/10343, which allows
recombination in any recA.sup.+ E. coli strain. Suicide plasmids
(also referred to as shuttle plasmids) were constructed by inserted
Ad5 sequence at appropriate restriction sites and by modifying
sequences by sequential PCR. The integrity of the EDRAG constructs
was assessed by restriction enzyme mapping and Southern analysis.
The regions involved in PCR amplification or homologous
recombination were verified by sequence analysis.
[0191] Plasmid backbone pXL3215 is a 57.8 kb long RK2 derivative
that contains a PacI-excisable E1 and E3-deleted Ad5-based genome
(French application FR 2 730 504) with an E. coli lacZ gene under
control of the Rous Sarcoma Virus promoter instead of the E1
region. Plasmid pXL3527 derives from pXL3215 by exchanging the E.
coli lacZ expression cassette by the human GAX expression cassette
utilizing the suicide shuttle pXL3521; it leads to the generation
of AV.sub.1.0CMV.Gax adenovirus. Plasmid pXL3497 derives from
pXL2689 (Crouzet et al, Proc Natl Acad Sci USA, 1997, 94
:1414-1419) and displays a (Gly-Ser).sub.5-(Lys).sub.7 peptide at
the C-terminus of the fiber protein. Following Pac1 restriction and
transfection in E1-transcomplementing cells, this backbone was used
to generate virus AV.sub.1.kCMV.lacZ which is identical to
AdZ.F(pK7)bgal described by Wickham et al (1997, J Virol, 71
:8221-8229).
[0192] Production and Quantitation of Adenoviruses
[0193] Transfections of adenoviral genomes in PER.C6 cells were
performed in the presence of lipofectAMINE in T25 cm.sup.2 flasks.
Briefly, 5 .mu.g of PacI-digested DNA plasmid diluted into H.sub.2O
were mixed with 23 .mu.l of Lipofectamine. After gentle mixing, the
suspension was incubated for 30 min at room temperature. In the
meantime, the cells at 50-60% confluence were washed twice with
phosphate buffered saline which was then replaced by the
LipofectAMINE/DNA mixture to which 3.8 ml of DMEM without serum
were added. The cells were incubated at 37.degree. C. for 5 to 8
hours, after which the medium was replaced by DMEM containing 10%
fetal calf serum and 10 mM MgCl.sub.2. Three days after
transfection the cells were split into one T75 cm2 flask. Cells and
supernatant were harvested at full CPE (day 10-14) and
freeze/thawed for three cycles followed by centrifugation and
collection of the supernatant. The EDRAG technology generates a
homogenous (i.e., clonal) population; therefore plaque purification
is not necessary.
[0194] Adenoviral particles were precisely quantified by
chromatography on a Sepharose type support.
[0195] PER.C6 cells were infected at a confluence of approximately
70% with recombinant adenovirus at an MOI between 10 and 100 viral
particles per cell in T150 cm.sup.2 flasks. When the cytopathic
effect was complete, cells and supernatant were harvested,
freeze/thawed for 3 cycles, centrifuged and the supernatant
collected. In certain cases, the recovery process had to be adapted
(see below).
[0196] Infectivity of the modified viruses was assessed in vitro in
primary cells of various origin (with a special emphasis for smooth
muscle cells and endothelial cells of human origin), and a panel of
human and non-human tumor cell lines that are refractory to
infection because they express limiting levels of adenovirus
receptor at their cellular surface (see examples 8 to 10).
Recombinant knob was used as a competitor when cells easily
infectable by Ad5 were used.
[0197] Viruses Modified in the HI Loop by Insertion of a Peptide
Targeting uPAR.
[0198] Residues 538 to 548 of the Ad5 fiber (GTQETGDTTPS) (SEQ ID
NO:72) were deleted and replaced with 6 different peptides flanked
with GSS linkers. Construction of the corresponding shuttle
plasmids, plasmid backbones and viruses were carried out as
detailed in Example 2. All viruses were viable, but some
(especially viruses AE43, AE44 and AE45) presented an altered
stability as compared to their unmodified control virus. It was
however possible to get yields comparable to a control virus (i.e.,
10000-20000 VP/cell) by adapting the procedure: PERC6-infected
cells were harvested 3 days post infection and lysed using a mild
buffer (Tris 10 mM pH7.5, MgCl2 1 mM, Tween 20 1%, NaCl 0.25M)
instead of successive freezing/thawing cycles; viruses were then
purified by ultracentrifugation on CsCl gradients. TABLE-US-00022
TABLE 11 HI loop insertion of uPAR targeting peptides. adeno- SEQ
viral Peptide sequence ID plasmid Selected peptide with linkers NO:
pAE42 ATF domain (aa 14 gly-ser-ser- 16 to 32 of mature
LNGGTCVSNKYFSNIHWCN- human urokinase) gly-ser-ser pAE45 mutated ATF
do- gly-ser-ser- 17 main (increased LNGGTAVSNKYFSNIHWCN- affinity
for uPAR) gly-ser-ser pAE48 peptide selected gly-ser-ser- 19 by
phage-display AEPMPHSLNFSQYLWYT- gly-ser-ser pAE46 a mutant of the
gly-ser-ser- above selected AEPMPHSLNFSQYLWT- 18 peptide
gly-ser-ser pAE43 uPAR-binding pep- gly-ser-ser- 20 tide (Vn4) from
RGHSRGRNQNSR-gly- human vitronectin ser-ser pAE44 uPAR-binding pep-
gly-ser-ser- 21 tide (Vn3) from NQNSRRPSRA- human vitronectin
gly-ser-ser
[0199] Viruses Modified in the HVR5 Loop by Insertion of a Peptide
Targeting uPAR.
[0200] Residues 269 to 281 of the Ad5 hexon (TTEATAGNGDNLT) (SEQ ID
NO:125) were replaced with the above 6 uPAR-binding peptides
flanked on both sides by suitable linkers (gly-ser). Construction
of the corresponding plasmid backbones and adenoviruses were
carried out as in Example 1. All viruses were viable. Some of the
constructs (e.g., AE27, AE28) displayed some unstability and were
purified accordingly (see above). TABLE-US-00023 TABLE 12 HVR5 loop
insertion of uPAR targeting peptides. adeno- SEQ viral Peptide
sequence ID plasmid Selected peptide with linkers NO: pAE26 ATF
domain gly-ser- 7 LNGGTCVSNKYFSNIHWCN- gly-ser pAE29 mutated ATF
gly-ser- 8 domain LNGGTAVSNKYFSNIHWCN- gly-ser pAE47 peptide
selected gly-ser- 10 by phage-display AEPMPHSLNFSQYLWYT- (Goodson
et al., gly-ser 1994,PNAS 91: 7129-14 7133 pAE30 a mutant of the
gly-ser- above selected AEPMPHSLNFSQYLWT- 9 peptide gly-ser pAE27
uPAR-binding pep- gly-ser- 11 tide (Vn4) from RGHSRGRNQNSR-gly-
human vitronectin ser pAE28 uPAR-binding pep- gly-ser- 12 tide
(Vn3) from NQNSRRPSRA- human vitronectin gly-ser
[0201] Viruses Modified in the HI Loop by Insertion of a Peptide
Targeting uPAR and Harboring Shortened Fibers.
[0202] Residues 538 to 548 of the Ad5 fiber (GTQETGDTTPS) (SEQ ID
NO:72) were deleted and replaced with 6 uPAR-binding peptides
flanked on both sides by suitable linkers (gly-ser-ser) as
described above. These modifications were combined with shortened
fiber shafts (see Example 3) as summarized in the following table:
TABLE-US-00024 TABLE 13 Class of short-shafted viruses modified in
the HI loop by insertion of a peptide targeting uPAR Tail Shaft
Knob modification Ad5 tail Ad3 shaft Ad5 HI loop insertions Ad5
tail repeats 1 to 3 and 17 to 22 Ad5 HI loop insertions of Ad5 Ad5
tail repeats 1 to 3 and 20 to 22 Ad5 HI loop insertions of Ad5
[0203] Viruses Modified in the HVR5 Loop by Insertion of a Peptide
Targeting uPAR and Harboring Shortened Fibers.
[0204] Residues 269 to 281 of the Ad5 hexon were replaced with 6
different peptides flanked by suitable linkers (gly-ser) as
described above. These modifications were further combined with the
shortening of the fiber shaft as described above.
[0205] For example, virus AE65 contains the NQNSRRPSRA peptide (SEQ
ID NO.6) flanked by gly-ser linkers in place of hexon HVR5 and
displays a shortened fiber (shaft deletion encompassing repeats 4
to 16). Another example is virus AE63 which is identical to AE65
except that it contains the DCRGDCF peptide instead of the Vn3
peptide (see Example 10, FIG. 13).
[0206] Viruses Modified for Targeting by a C-Terminal Addition of
an 8 Amino Acids Linker Followed by a Peptide Targeting uPAR.
[0207] The stop codon of the fiber is replaced with the proline
codon of the linker. The linker sequence used for all constructs
was PKRARPGS.
[0208] The 3 end of the fiber protein coding sequence was modified
to introduce an FspI site. PCR mutagenesis was used to generate a
single base substitution (nucleotide 32778) which creates a silent
mutation introducing a novel recognition site for FspI. The Ad5
fiber knob was amplified from the Ad5 genome using primers MOL1
(5'-ggaactttagaaatggagatcttactgaagg-3') (SEQ ID NO:126) and MOL3
(5'-cgattctttattcttgcgcaatgtatgaaaaag-3') (SEQ ID NO:127).
[0209] The primer MOL3 nearly matches the Ad5 nucleotides
32762-32794 with a slight modification resulting in the creation of
the FspI restriction site. This amplification product was
introduced in pCR2.1 (Invitrogen) to create pMA51.
[0210] The region downstream from the stop codon of the fiber
protein coding sequence was modified by PCR-mutagenesis to
introduce AatII, NruI, SpeI restriction sites upstream the polyA
region using the oligonucleotides MOL2
(5'-cttaagtgagctgcccggggag-3') (SEQ ID NO:128) and MOL4
(5'-ggatccaatgaacttcatcaagt-3') (SEQ ID NO: 129), and cloned in
pCR2.1 (Invitrogen) to create pMA52.
[0211] The sequence coding for the linker peptide was created by
annealing of two single-stranded oligonucleotides: MOL7
(5'-aattctgcgcaagaaccaaagagggccaggcccggatcctaagacgtct-3') (SEQ ID
NO:130) and MOL8
(5'-ctagagacgtcttaggatccgggcctggccctctttggttcttgcgcag-3') (SEQ ID
NO:131). This duplex was cloned between the EcoRI and XbaI sites of
pBSSK+ (Stratagene) creating pMA53.
[0212] Finally, the linker sequence was introduced at the 3'-end of
the fiber protein coding sequence by cloning the fragments
BglII-FspI from pMA51 and FspI-XbaI from pMA53 into the BamHI and
XbaI sites of pXL2756 to create the vector pMA55.
[0213] The shuttle vector pMA56 was constructed by cloning the
SmaI-AatII fragment of pMA52 into pMA55 SmaI-AatII restriction
sites. Shuttle plasmid pMA55 was recombined with plasmid backbone
pXL3006 in the G4977 bacterial strain according to the method
described by Crouzet et al., supra, to obtain the plasmid backbone
22.3 which contains a PacI-excisable E1E3-deleted CMV/lacZ
recombinant viral genome encoding fibers with C-terminal
modifications.
[0214] Further cloning were performed to add the uPAR-targeting
ligands at the C-ter of the fiber using a PKRARPGS linker. Briefly,
shuttle plasmids encoding these modifications were constructed and
recombined with adenoviral plasmid backbones pXL3091 (RSV-lacZ in
place of E1), pXL3006 (CMV-lacZ in place of E1) or pXL3527 (hGax
expression cassette in place of E1) in the G4977 bacterial strain
according to the EDRAG method to generate adenoviral backbones
displaying lacZ or Gax expression cassettes and C-terminally
modified fiber proteins.
[0215] With the exception of bC12x, all expected viruses were
recovered following transfection of PacI-restricted backbones into
911, 293 or PER.C6 cells. Their productivity (VP/cell) was
comparable with that of their unmodified control virus.
TABLE-US-00025 TABLE 14 Viruses modified by a C-terminal addition
of an 8 amino acids linker followed by a peptide targeting UPAR.
adenoviral SEQ backbones/ Sequence ID viruses Selected peptide of
the peptide NO: bc9x ATF domain (from LNGGTCVSNKYFSNIHWCN 1 (RSV
lacZ) aa 14 to 32) bc10x mutated ATF do- LNGGTAVSNKYFSNIHWCN 2 (RSV
lacZ) main (from aa 14 to 32) bc12x peptide selected
AEPMPHSLNFSQYLWYT 4 (RSV lacZ) by phage-display bc11x a mutant of
the AEPMPHSLNFSQYLWT 3 (RSV lacZ) above selected peptide bc13x
vitronectin uPAR- NQNSRRPSRA 6 (RSV lacZ) binding domain Vn3 bc14x
vitronectin uPAR- RGHSRGRNQNSR 5 (RSV lacZ), binding domain bc15x
Vn4 (CMV lacZ) and pXL3570 (Gax)
[0216] In additional experiments, these modifications were combined
with shortening the fiber shaft as examplified above.
Example 5
Specific Targeting of Cells Expressing an .alpha.v Integrin
Receptor
[0217] A high affinity .alpha.v integrin binding peptide (CDCRGDCFC
refered to as RGD-4C; see also Pasqualini et al., 1997, Nature
Biotech. 15:542) and a variant thereof (DCRGDCF refered to as
RGD-2C) have been included within the fiber HI hexon and the HVR5
loops, or added to the C-terminal end of the fiber protein. These
viruses contain a heterologous gene (lacZ) inserted in the E1
region, which has been deleted from the viruses
[0218] The methods for insertion of these peptides in the hexon
HVR5 loop and the fiber protein HI loop were as described for the
poliovirus epitopes as described in Examples 1 and 2, above.
[0219] Viruses Modified in the HI Loop by Insertion of a Peptide
Targeting .alpha.v Integrins, Associated or not with a Shortened
Fiber.
[0220] Residues 538 to 548 of the Ad5 fiber were deleted and
replaced with the peptide flanked with GSS linkers. Adenoviral
plasmid backbones containing a CMVlacZ expression cassette were
transfected in PER.C6 cells: virus AE60 was viable and could be
amplified in PER.C6 cells with a productivity comparable to a
control virus, whereas repeated transfection of PacI-digested pAE59
DNA did not generate the corresponding virus, suggesting that this
particular construct could not grow efficiently. TABLE-US-00026
TABLE 15 Viruses modified in the HI loop by insertion of a peptide
targeting .alpha..nu.integrins. adeno- SEQ viral Sequence at the ID
backbones Selected peptide peptide with linkers NO: pAE59 CDCRGDCFC
gly-ser-ser-CDCRGDCFC 22 (SEQ ID NO:124) -gly-ser-ser pAE60 DCRGDCF
gly-ser-ser-DCRGDCF- 23 (SEQ ID NO:148) gly-ser-ser
[0221] Viruses modified in the HI loop by insertion of a peptide
targeting .alpha.v integrins, associated with a shortened fiber are
also obtained.
[0222] Viruses Modified in the HVR5 Loop by Insertion of a Peptide
Targeting .alpha.v Integrins, Associated or not with a Shortened
Fiber.
[0223] The amino acids 269 to 281 of the Ad5 hexon were deleted and
replaced with the peptide (the same sequences as described in the
table above were used) flanked with GS linkers. Adenoviral plasmids
containing a LacZ or a hGax expression cassette were transfected in
PERC6 or 911 cells leading to the recovery of all 3 viruses. A loss
of productivity was observed for AE58 and AE63 viruses in 293
cells, whereas AE57 behaved normally: this is likely due to an
incorrect folding/stability of the hexon in the case of AE58, and
to the decrease in Ad receptor binding of the shortened fiber in
the case of AE63. TABLE-US-00027 TABLE 16 Viruses modified in the
HVR5 loop by insertion of a peptide targeting .alpha..nu.integrins,
associated or not with a shortened fiber Fea- adeno- tures viral
Sequence of SEQ of the back- Selected the peptide ID fiber bones
peptide with linkers NO: shaft pAE58 CDCRGDCFC gly-ser-CDCRGD 13
unmod- (SEQ ID NO:124) CFC-gly-ser ified pAE57 DCRGDCF
gly-ser-DCRGD 14 unmod- (LacZ) (SEQ ID NO:148) CF-gly-ser ified and
pXL3664 (Gax) pAE63 DCRGDCF gly-ser-DCRGD 14 short- (SEQ ID NO:148)
CF-gly-ser ened (re-peats 1-3 and 17-22 of Ad5)
[0224] The inclusion of the RGD-2C peptide in the hexon was also
combined with the addition of the linker-peptide sequence
PKRARPGS-K7 (SEQ ID NO.132) at the C-terminus of the fiber. The
corresponding virus was viable.
Example 6
Construction of Heparan Sulfate Proteoglycans Targeted Viruses
[0225] Fiber-modified adenoviruses containing a lacZ or Gax
expression cassette were constructed by genetic modification of the
adenoviral genome in E. coli using the EDRAG technology and
produced in 911 or PER.C6 cells (see Material and Methods of
Example 4).
[0226] Three ligands expected to bind to heparan sulfate
proteoglycans were identified: the heptalysine stretch (K7)
described by Wickham et al. (1997, J Virol, 71 :8221-8229), the
arginine-leucine repeated motif RRLLRRLLRR (SEQ ID NO.133),
described in patent application WO95/21931, and the peptide
fragment from FGF-1 binding to heparin KRGPRTHYGQK (SEQ ID NO.134)
described by Digabriele et al (1998, Science, 393 :812-817).
[0227] Among others, five viruses have the heptalysine K7 stretch
at the C-terminus of the fiber and differ by the transgene (lacZ or
Gax) and/or the identity of the connecting (no linker, (GS)5 or
PKRARPGS). Virus 3497 was included as a reference (Wickham et al.,
1997, J Virol, 71 :8221-8229). The importance of the linker and/or
peptide in terms of viral production and transduction efficacy in
vitro and in vivo was then assessed.
[0228] A polylysine stretch has also been included within the hexon
HVR5 or the fiber HI loops in viruses containing a heterologous
gene (lacZ) inserted in the E1 region TABLE-US-00028 TABLE 17
Heparan Sulfate Proteoglycans Targeted viruses adenoviral backbones
Modification of the capsid pAE61 Substitution of hexon aa 269-281
with GS-K5-GS (SEQ ID N.sup.o 135) pAE62 Substitution of fiber aa
538-548 with GSS-K7- GSS (SEQ ID N.sup.o 136) pXL3497 (lacZ) and
(GS)5-K7 (SEQ ID N.sup.o 137) added to the fiber pXL3528 (gax)
C-terminus pXL3496 (lacZ) and PKRARPGS-K7 (SEQ ID N.sup.o 138)
added to the pXL3569 (gax) fiber C-terminus pXL3631 (lacZ) K7 (SEQ
ID N.sup.o 139) added to the fiber C-terminus pXL3662 (lacZ) and
PKRARPGS-KRGPRTHYGQK (SEQ ID N.sup.o 140) pXL3665 (gax) added to
the fiber C-terminus pXL3663 (lacZ) and PKRARPGS-RRLLRRLLRR (SEQ ID
N.sup.o 141) pXL3666 (gax) added to the fiber C-terminus
[0229] All these viruses were viable and could be amplified in E1
transcomplementing cells.
[0230] The following table 18 summarizes the yield obtained in one
experiment performed in PER.6 cells after infection of the cells at
moi 10 to 100 VP/cell. TABLE-US-00029 TABLE 18 Amplification of
viruses in PER.C6 cells Virus Viral particles/cell 3497 1200 3528
2300 3496 2700 3569 1000 3631 6000 3662 12160
[0231] Productivity was differently affected by these C-terminal
fiber extensions. Overall, these and other data indicate that the
presence and identity of the connecting linker sequence added to
the fiber C-terminus greatly influences the adenovirus infection
cycle/behavior.
[0232] For a given linker sequence, the identity/nature of the
foreign peptide per se was also found to be an important parameter.
For example, the construct with the RRLLRRLLRR peptide
(AV.sub.1sCMV.lacZ or Ad3663) yielded very low titers whereas its
replacement by the KRGPRTHYGQK peptide (AV.sub.1.fCMV.lacZ or
Ad3662) restored productivity.
[0233] The recovery process had also to be optimized for most of
these viruses. For example, a total of 5.10.sup.12 VP of Ad3497 was
successfully purified by a two-step chromatography procedure and
finally resuspended in Tris 20 mM pH8.4-10% glycerol, with an
overall particle recovery of 68% as described hereabove.
Example 7
Construction of Viruses with a Vn4 Peptide within the HI Loop
[0234] As mentionned in example 4, the AE43 virus somehow displayed
some levels of is unstability that required an optimized recovery
process. To rescue its stability without loosing its advantageous
binding characteristics, the Vn4 peptide was introduced in the HI
loop in various neighboring contexts (see following table 19). The
corresponding viruses (which contained a lacZ or Gax expression
cassette in place of E1) were constructed by recombinational
cloning in E. coli and amplified in 911 or PERC6 cells as described
in Example 4. TABLE-US-00030 TABLE 19 viruses with a Vn4 peptide in
the HI loop SEQ ID virus Modification of the capsid N.sup.o AE43
Substitution of fiber aa 538-548 with GSS- 20 Vn4-GSS GL11
Substitution of fiber aa 538-548 with GSS- 143 Vn4+Vn3-GSS* GL12
Substitution of fiber aa 538-548 with GTSE- 144 Vn4-GSS GL13
Substitution of fiber aa 538-548 with GTQE- 145 Vn4-GSS GL14
Substitution of fiber aa 538-548 with GSSS- 146 Vn4-GSS GL16
Substitution of fiber aa 538-548 with GSS- 147 Vn4-GGS GL17
Substitution offiber aa 541-548 with SS- 142 Vn4-GSS 3630 (lacZ)
and Insertion of SS-Vn4-GS between fiber aa 150 3629 (Gax) 546
(Thr) and 547 (Pro) *Vn4+Vn3 = RGHSRGRNQNSRRPSRA is derived from
human vitronectin
[0235] Almost all constructs exhibited a productivity that was
comparable to that of their unmodified control virus (see following
table 20): TABLE-US-00031 TABLE 20 Virus Viral particles/cell
control virus 20000 (n = 2) AE43 20000 (n = 2) GL11 200 (n = 1)
GL12 20000 (n = 3) GL13 25000 (n = 2) GL14 20000 (n = 1) GL16 4000
(n = 2) GL17 15000 (n = 3) 3630 10000 (n = 3) 3629 14000 (n =
1)
[0236] Virus stability was also differently affected by these
modifications. In particular, some of them (e.g., AE43, GL11, GL14
and GL16) were sensitive to successive rounds of freezing/thawing
so the infected cells had to be lysed in mild conditions (Tris 10
mM pH 7.5, MgCl2 1 mM, Tween 20 1%, NaCl 0.25M) for recovery, again
emphasizing the influence of the linker sequences on the virus
behavior (see also Example 9, FIG. 12).
Example 8
Evaluation of Targeted Viruses in Human Primary Cells
Materials and Methods
Cell Culture
[0237] Primary cultures of rat and rabbit smooth muscle cells were
prepared from thoracic aortas of adult male Spraggue-Dawley rats or
of adult New Zealand White rabbits according to Mader et al (1992,
J Gerontol. Biol. Sci. 47: b32-b36). Cells were propagated at
37.degree. C. in Dulbecco's modified Eagle's medium (DMEM)
containing 10% fetal bovine serum (FBS) and
penicillin/streptomycin. Human aorta smooth muscle cells and HUVEC
were purchased from Clonetics and cultured following instructions
of the manufacturer.
Adenoviral Mediated In Vitro Gene Transfer and Gene Expression
Assays
[0238] Cells were seeded onto 24-well or 12-well plates one to two
days prior to experiments. Cells were infected at moi 100 or 1000
(VP/cell) by incubation of the adenoviral vector diluted in serum
free culture medium for one hour at 37.degree. C. Cells were then
washed and growth medium was added for 48 hours to allow
.beta.-galactosidase expression. The cells were then lysed and
assayed for .beta.-galactosidase activity by using Luminescent
.beta.-galactosidase genetic reporter system II (Clontech), or Xgal
stained.
Results
Construction Encoding .beta. Galactosidase Reporter Gene
[0239] Data presented in the two following tables 21 and 22 show
that most viruses are able to transduce hSMC more efficiently than
a control virus, and that viruses AE30, AE42, AE43, AE44, AE45,
AE57, AE58, AE61, AE62, BC15X and 3497 are good candidates for
further in vitro and in vivo studies. Experiments performed on
primary SMCs infected at an moi of 1000 VP/cell at different
passage demonstrated a very significant gain of transduction for
several of the modified viruses (examples are provided in tables 21
and 22). Extracts were prepared 48 hr post-infection at which time
protein and .beta.-galactosidase activity were quantified.
Transduction efficacy was then assessed by comparing the levels of
lacZ specific activity (RLU/protein extract). TABLE-US-00032 TABLE
21 Experiment #1 Transduction efficacy in Virus human SMC control 1
AE30 3.5 AE43 53 AE44 23 AE45 10 3497 115 BC15X 26
[0240] TABLE-US-00033 TABLE 22 Experiment #2 Transduction efficacy
in Virus human SMC control 1 AE27 16 AE29 6 AE30 133 AE42 95 AE43
825 AE48 31 AE57 846 AE58 264 AE60 52 AE61 781 AE62 772 BC15X
47
[0241] The following table 23 summarizes the data obtained in SMC
from different species: most viruses were able to efficiently
transduce non-human cells, indicating that there is no species
barrier in their entry pathway. TABLE-US-00034 TABLE 23 infection
of SMC from different species Transduction efficacy Transduction
efficacy Transduction efficacy Virus in rat SMC in pig SMC in
rabbit SMC control 1 1 1 BC15X -- 97 (n = 1) -- AE 43 77 (n = 2)
753 (n = 2) 131 (n = 3) AE 62 166 (n = 2) 2166 (n = 2) 320 (n = 5)
3497 50 (n = 2) 349 (n = 2) 55 (n = 2) 3496 295 (n = 2) 2318 (n =
2) 437 (n = 2) AE 57 -- 2960 (n = 1) -- AE 63 21 (n = 2) 293 (n =
2) 23 (n = 2) n = number of experiments
[0242] To demonstrate that the increase of transduction observed
following capsid modification was due at least in part to an
increase in infectivity, quantitative PCR was carried out on viral
DNA extracted from infected human SMC at moi 1000 VP/cell.
Simultaneously, RLU measurements were performed on protein
extracts.
[0243] Table 24 indicates that the gain in human smooth muscle
cells transduction efficiency that caracterized the best candidate
viruses correlated in all cases (although to different extents)
with an increase in DNA entry. The correlation was especially good
for viruses AE43 and BC15X. TABLE-US-00035 TABLE 24 genome delivery
and transgene expression RLU/.mu.g virus Genomes/cell.sup.a total
protein Ratio RLU Ratio genomes control 3.3 191641 1 1 AE30 211
677345 3.5 64 AE43 87 10207798 53 26.5 AE45 427 1851206 10 130 3497
124 22115125 115 38 BC15X 40 5004672 26 12.3 .sup.agenomes/cell is
the ratio between the amount of viral genomes as quantified by PCR
and the number of infected cells. .sup.bratio RLU: ratio between
the RLU level of the indicated virus and the RLU level of the
control virus .sup.cratio genomes: ratio between the genomes amount
of the indicated virus and the genomes amount of the control
virus.
[0244] The entry pathway in SMC was also analyzed in competition
with soluble heparin or soluble uPAR as illustrated in FIGS. 4 and
5.
[0245] In FIG. 4, hSMC were infected at moi 1000 (VP/cell) in
presence of increasing doses of soluble heparin. Cells were washed
in PBS before further incubation at 37.degree. C. Extracts were
prepared 48 hr post-infection at which time total proteins and
.beta.-galactosidase activity were quantified. The data show that
infection of hSMC by polylysine-containing viruses is specifically
inhibited by soluble heparin showing that these viruses likely bind
to cellular heparan sulfate proteoglycans at the cell surface. In
contrast, and as expected, AE43 does not use this particular
pathway for entry.
[0246] In FIG. 5, viruses were preincubated with increasing doses
of soluble uPAR (0 to 9, 4 .mu.g/ml) before incubation on hSMC at
moi 1000 (VP/cell). Cells were washed in PBS before further
incubation at 37.degree.. Extracts were prepared 48 hr
post-infection at which time total proteins and
.beta.-galactosidase activity were quantified. The data show that
infection of hSMC by some of the uPAR-targeting viruses (e.g.,
AE43) is specifically inhibited by recombinant soluble uPAR. That
hSMC express uPAR at their cellular surface (data not shown)
strongly suggests the use of this particular receptor for cellular
entry.
Gax Encoding Targeted Adenoviruses
[0247] Gax expression level in infected hSMC was analyzed by
Western blot (following figures). The highest increase in Gax
expression was obtained after infection with virus 3528 (K7 at the
C-terminus of the fiber). Gax protein was detected with all
modified viruses at moi 3000 VP/cell, whereas Gax expression in
cells infected with the control virus at the same moi was
undetectable.
[0248] FIG. 6 illustrates the Gax expression in human SMC infected
with targeted adenovirus. Protein extracts were prepared 24 hours
after infection at the indicated moi (VP/cell). (1)
AV.sub.1.0CMVrGax moi 3.10.sup.4, (2) AV.sub.1.0CMVrGax moi
10.sup.4, (3) 3528 moi 10.sup.4, (4) 3528 moi 3.10.sup.3, (5) 3528
moi 300, (6) 3569 moi 3.10.sup.3, (7) 3569 moi 300, (8) 3570 moi
10.sup.4 and (9) 3570 moi 3.10.sup.3.
[0249] FIG. 7 ilustrates the Gax expression after human SMC
infection with targeted adenovirus. Protein extracts were prepared
24 hours after infection at the indicated moi (VP/cell).
[0250] Left panel: (1) AV.sub.1.0CMVrGax moi 3.10.sup.4 (2)
AV.sub.1.0CMVhGax moi 3.10.sup.4, (3) AV.sub.1.0CMVhGax moi
3.10.sup.3, (4) 3528 moi 3000, (5) 3528 moi 300, (6) 3528 moi 30,
(7) 3569 moi 3000, (8) 3569 moi 300, (9) 3569 moi 30
[0251] Right panel: (1) AV.sub.1.0CMVrGax moi 3.10.sup.4 (2)
AV.sub.1.0CMVhGax moi 3.10.sup.4, (3) AV.sub.1.0CMVhGax moi
3.10.sup.3, (4) 3570 moi 3000, (5) 3570 moi 300, (6) 3570 moi 30,
(7) 3629 moi 3000, (8) 3629 moi 300, (9) 3629 moi 30
[0252] Gax expression was also evidenced by FACS analysis after
infection of hSMC at different moi. Even if the sensitivity of this
technique is much higher than Western blotting, the results
correlate with Western experiments and show the relative
superiority of viruses 3528 and 3569 over 3570 and 3629 in their
ability to efficiently transduce hSMC, as ilustrates on the
following table 25. TABLE-US-00036 TABLE 25 FACS analysis of Gax
expression after infection of human SMC.(% of Gax expressing cells
was determined 24 hours after infection.) Virus 10000 VP/cell 1000
VP/cell 100 VP/cell AV.sub.1.0CMVrGax 100% 52% -- AV.sub.1.0CMVhGax
86% 8% -- 3528 nd 99% 99% 3569 nd 99% 99% 3570 nd 99% 8% 3629 nd
99% 18%
Example 9
In Vitro Evaluation of Targeted Viruses in Human Tumoral Cells
[0253] Hs578T cells (human breast tumor cells) are quire refractory
to Ad5 infection most likely because they express limiting amounts
of the virus receptor at their cellular surface. In practice, an
moi as high as 10.sup.5 VP/cell is necessary to infect 50% of the
cells. They were tested for their ability to be transduced by a
panel of capsid-modified vectors.
[0254] FIGS. 8A, 8B, 8C and 9 illustrate infection of Hs578T with
different targeted viruses. Cell extracts were prepared 48 h post
infection. The data show that AE43, AE44, AE45 or 3497 are very
efficient in transducing this cell type.
[0255] The pathway of infection of AE43 was analyzed by competition
with soluble knob fiber or soluble uPAR (FIGS. 10 and 11).
[0256] In FIG. 10, AE43 was preincubated with soluble uPAR before
infection of Hs578T. Cell extracts were prepared 48 h post
infection.
[0257] In FIG. 11, AE43 was preincubated with soluble UPAR (10
.mu.g/2 10.sup.8 VP) or soluble knob (100 .mu.g/ml) before
infection of Hs578T. Cell extracts were prepared 48 h post
infection.
[0258] The results indicate that AE43 does not enter the cell via
the classical knob-CAR pathway but rather uses a uPAR-dependent
pathway for entry.
[0259] Finally, Vn4-containing viruses 3630, GL12, GL14 or GL17
were compared to AE43 in their ability to transduce Hs578T cells.
As shown in FIG. 12 at 48 h post infection, the nature of the
connecting linkers indeed can greatly influence the efficacy with
which a binding peptide inserted in the HI loop interacts with its
specific receptor at the cellular surface.
Example 10
In Vitro Evaluation of Targeted Viruses in Murine Tumoral Cells
[0260] Murine NIH-3T3 cells are very resistant to Ad5 infection as
more than 100000 VP/cell is necessary to infect 50% of the cells.
They were tested for their ability to be transduced by a panel of
capsid-modified viruses. Cell extracts were prepared 48 h post
infection. FIG. 13 include data from a representative experiment
which demonstrate that AE28, AE43, AE44, AE58, AE57 or AE62 are
particularly efficient.
[0261] Also, and importantly, AE63 (short fiber, RGD-2C in hexon)
was shown to be partially able to rescue the defect associated with
its short-shafted control virus (vBS1; no insertion in hexon).
Capsid modifications that impair the native entry pathway (e.g.,
fibers displaying short shafts) can therefore be combined with
capsid modifications that provide an additional, CAR-independent,
pathway for infection.
[0262] The pathway of infection of AE43 and BC15X was analyzed by
competition with soluble UPAR (FIGS. 14A and 14B). In FIGS. 14A and
14B, viruses BC15X (A) and AE43 (B) were preincubated with
increasing doses of soluble uPAR before incubation with cells (moi
1000 and 200 VP/cell, respectively). Cells were then washed and
further incubated at 37.degree. C. for 48 h before preparation of
cell extracts. These and other data indicate that these viruses use
uPAR for infection.
Example 11
In Vivo Evaluation of Targeted Viruses in a Restenosis Rabbit
Model
[0263] The in vivo evaluation of some of the targeted viruses was
performed in a an atheromatous double injury rabbit model, which is
a good model for restenosis: transfer takes place in atheromatous
iliac arteries; rabbits are fed 120 g daily of 1% cholesterol diet
and at 3 weeks a first injury by balloon angioplasty is performed
with a 2.5 mm diameter Nycomed balloon catheter. One week later,
adenoviral gene transfer is performed.
[0264] Microscopic quantification of SMC staining for
.beta.-galactosidase was used to define the efficacy of gene
transfer (histochemical analysis). Briefly, 32 sections/artery were
examined and XGal-positive cells were counted. The data are
presented as the highest score among the 32 sections for one
artery. Results are presented in the following table 26.
TABLE-US-00037 TABLE 26 In vivo evaluation of targeted viruses in a
restenosis rabbit model Virus injected 10.sup.11 VP/artery 5
10.sup.11 VP/artery Control virus positive arteries: 0/8 positive
arteries: 7/10 6 arteries with <30 stained cells 1 artery with
200-400 stained cells AE57 positive arteries: 0/6 positive
arteries: 2/2 RGD-2C in hexon 2 arteries with <30 stained cells
AE43 positive arteries: 5/6 VN4 in HI fiber 2 arteries with <30
stained cells 2 arteries with 30-100 stained cells 1 artery with
>400 stained cells BC15X positive arteries: 4/4 VN4 1 artery
with <30 stained cells at C-ter fiber 1 artery with 30-100
stained cells 1 artery with 100-200 stained cells 1 artery with
200-400 stained cells
[0265] These data indicate that adenoviruses AE43 and BC15X
transduce arterial wall with a dramatically increased efficacy as
compared to their unmodified control.
[0266] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and the accompanying figures. Such
modifications are intended to fall within the scope of the appended
claims.
[0267] It is further to be understood that all base sizes or amino
acid sizes, and all molecular weight or molecular mass values,
given for nucleic acids or polypeptides are approximate, and are
provided for description.
[0268] Various publications are cited herein, the disclosures of
which are incorporated by reference in their entireties.
Sequence CWU 1
1
165 1 19 PRT Adenovirus 1 Leu Asn Gly Gly Thr Cys Val Ser Asn Lys
Tyr Phe Ser Asn Ile His 1 5 10 15 Trp Cys Asn 2 19 PRT Adenovirus 2
Leu Asn Gly Gly Thr Ala Val Ser Asn Lys Tyr Phe Ser Asn Ile His 1 5
10 15 Trp Cys Asn 3 16 PRT Adenovirus 3 Ala Glu Pro Met Pro His Ser
Leu Asn Phe Ser Gln Tyr Leu Trp Thr 1 5 10 15 4 17 PRT Adenovirus 4
Ala Glu Pro Met Pro His Ser Leu Asn Phe Ser Gln Tyr Leu Trp Tyr 1 5
10 15 Thr 5 12 PRT Adenovirus 5 Arg Gly His Ser Arg Gly Arg Asn Gln
Asn Ser Arg 1 5 10 6 10 PRT Adenovirus 6 Asn Gln Asn Ser Arg Arg
Pro Ser Arg Ala 1 5 10 7 23 PRT Adenovirus 7 Gly Ser Leu Asn Gly
Gly Thr Cys Val Ser Asn Lys Tyr Phe Ser Asn 1 5 10 15 Ile His Trp
Cys Asn Gly Ser 20 8 23 PRT Adenovirus 8 Gly Ser Leu Asn Gly Gly
Thr Ala Val Ser Asn Lys Tyr Phe Ser Asn 1 5 10 15 Ile His Trp Cys
Asn Gly Ser 20 9 20 PRT Adenovirus 9 Gly Ser Ala Glu Pro Met Pro
His Ser Leu Asn Phe Ser Gln Tyr Leu 1 5 10 15 Trp Thr Gly Ser 20 10
21 PRT Adenovirus 10 Gly Ser Ala Glu Pro Met Pro His Ser Leu Asn
Phe Ser Gln Tyr Leu 1 5 10 15 Trp Tyr Thr Gly Ser 20 11 16 PRT
Adenovirus 11 Gly Ser Arg Gly His Ser Arg Gly Arg Asn Gln Asn Ser
Arg Gly Ser 1 5 10 15 12 14 PRT Adenovirus 12 Gly Ser Asn Gln Asn
Ser Arg Arg Pro Ser Arg Ala Gly Ser 1 5 10 13 13 PRT Adenovirus 13
Gly Ser Cys Asp Cys Arg Gly Asp Cys Phe Cys Gly Ser 1 5 10 14 11
PRT Adenovirus 14 Gly Ser Asp Cys Arg Gly Asp Cys Phe Gly Ser 1 5
10 15 11 PRT Adenovirus 15 Gly Ser Lys Lys Lys Lys Lys Lys Lys Gly
Ser 1 5 10 16 25 PRT Adenovirus 16 Gly Ser Ser Leu Asn Gly Gly Thr
Cys Val Ser Asn Lys Tyr Phe Ser 1 5 10 15 Asn Ile His Trp Cys Asn
Gly Ser Ser 20 25 17 25 PRT Adenovirus 17 Gly Ser Ser Leu Asn Gly
Gly Thr Ala Val Ser Asn Lys Tyr Phe Ser 1 5 10 15 Asn Ile His Trp
Cys Asn Gly Ser Ser 20 25 18 22 PRT Adenovirus 18 Gly Ser Ser Ala
Glu Pro Met Pro His Ser Leu Asn Phe Ser Gln Tyr 1 5 10 15 Leu Trp
Thr Gly Ser Ser 20 19 23 PRT Adenovirus 19 Gly Ser Ser Ala Glu Pro
Met Pro His Ser Leu Asn Phe Ser Gln Tyr 1 5 10 15 Leu Trp Tyr Thr
Gly Ser Ser 20 20 18 PRT Adenovirus 20 Gly Ser Ser Arg Gly His Ser
Arg Gly Arg Asn Gln Asn Ser Arg Gly 1 5 10 15 Ser Ser 21 16 PRT
Adenovirus 21 Gly Ser Ser Asn Gln Asn Ser Arg Arg Pro Ser Arg Ala
Gly Ser Ser 1 5 10 15 22 15 PRT Adenovirus 22 Gly Ser Ser Cys Asp
Cys Arg Gly Asp Cys Phe Cys Gly Ser Ser 1 5 10 15 23 13 PRT
Adenovirus 23 Gly Ser Ser Asp Cys Arg Gly Asp Cys Phe Gly Ser Ser 1
5 10 24 13 PRT Adenovirus 24 Gly Ser Ser Lys Lys Lys Lys Lys Lys
Lys Gly Ser Ser 1 5 10 25 13 PRT Adenovirus 25 Thr Thr Glu Ala Ala
Ala Gly Asn Gly Asp Asn Leu Thr 1 5 10 26 20 DNA Adenovirus 26
atgggatgaa gctgctactg 20 27 26 DNA Adenovirus 27 tcgcgagaaa
aattgcattt ccactt 26 28 21 DNA Adenovirus 28 cctaaaggtg gtattgtaca
g 21 29 20 DNA Adenovirus 29 agcagtaatt tggaagttca 20 30 44 DNA
Adenovirus 30 aatactacct ctttgaacga ccggcaaggc aatgctacta aacc 44
31 47 DNA Adenovirus 31 ttaggtttag tagcattgcc ttgccggtcg ttcaaagagg
tagtatt 47 32 56 DNA Adenovirus 32 aatctagact ctttggaaca acctactact
cgcgctacaa aaaccacgtc tagatt 56 33 60 DNA Adenovirus 33 gtacaaatct
agacgtggtt tttgagcgcg agtagtaggt tgttccaaag agtctagatt 60 34 20 DNA
Adenovirus 34 tcaaccacta taaacattcc 20 35 23 DNA Adenovirus 35
ttaggaatgt ttatagtggt tga 23 36 56 DNA Adenovirus 36 ttaggtttgt
attcttcgtt tccactaccg cctgctggag gatttgcgcc aggagt 56 37 56 DNA
Adenovirus 37 ttaggtttgt attcttcgtt tccactaccg cctgctggag
gatttgcgcc aggagt 56 38 38 DNA Adenovirus 38 gataacccag cgtcgaccac
gaataaggat aagctacc 38 39 41 DNA Adenovirus 39 ttaggtagct
tatccttatt cgtggtcgac gctgggttat c 41 40 38 DNA Adenovirus 40
ggagataacc cagcgtcgac cacgaataag gataagcc 38 41 41 DNA Adenovirus
41 ttaggcttat ccttattcgt ggtcgacgct gggttatctc c 41 42 38 DNA
Adenovirus 42 tctgataacc cagcgtcgac cacgaataag gataagcc 38 43 41
DNA Adenovirus 43 ttaggcttat ccttattcgt ggtcgacgct gggttatcag a 41
44 40 DNA Adenovirus 44 ggatctgata acccagcgtc gaccacgaat aaggataagc
40 45 44 DNA Adenovirus 45 ttaggcttat ccttattcgt ggtcgacgct
gggttatcag atcc 44 46 47 DNA Adenovirus 46 ggagataacc cagcgtcgac
cacgaataag gataagctag gtggccc 47 47 50 DNA Adenovirus 47 ttagggccac
ctagcttatc cttattcgtg gtcgacgctg ggttatctcc 50 48 47 DNA Adenovirus
48 ggagataacc cagcgtcgac cacgaataag gataagctag gttctcc 47 49 50 DNA
Adenovirus 49 ttaggagaac ctagcttatc cttattcgtg gtcgacgctg
ggttatctcc 50 50 44 DNA Adenovirus 50 ggagataacc cagcgtcgac
cacgaataag gataagctat ctcc 44 51 47 DNA Adenovirus 51 ttaggagata
gcttatcctt attcgtggtc gacgctgggt tatctcc 47 52 47 DNA Adenovirus 52
ggagataacc cagcgtcgac cacgaataag gataagctat ctggtcc 47 53 50 DNA
Adenovirus 53 ttaggaccag atagcttatc cttattcgtg gtcgacgctg
ggttatctcc 50 54 47 DNA Adenovirus 54 ggagataacc cagcgtcgac
cacgaataag gataagctat ctagtcc 47 55 50 DNA Adenovirus 55 ttaggactag
atagcttatc cttattcgtg gtcgacgctg ggttatctcc 50 56 14 PRT Adenovirus
56 Asn Thr Thr Ser Leu Asn Asp Arg Gln Gly Asn Ala Thr Lys 1 5 10
57 6 PRT Adenovirus 57 Ser Thr Thr Ile Asn Ile 1 5 58 16 PRT
Adenovirus 58 Thr Pro Gly Ala Asn Pro Pro Ala Gly Gly Ser Gly Asn
Glu Glu Tyr 1 5 10 15 59 17 PRT Adenovirus 59 Thr Thr Glu Ala Thr
Ala Gly Asn Gly Asp Asn Leu Thr Pro Lys Val 1 5 10 15 Val 60 13 PRT
Adenovirus 60 Thr Thr Glu Ala Thr Ala Gly Asn Gly Asp Asn Leu Tyr 1
5 10 61 18 PRT Adenovirus 61 Asn Leu Asp Ser Leu Glu Gln Pro Thr
Thr Arg Ala Gln Lys Pro Arg 1 5 10 15 Leu Asp 62 11 PRT Adenovirus
62 Asp Asn Pro Ala Ser Thr Thr Asn Lys Asp Lys 1 5 10 63 12 PRT
Adenovirus 63 Asp Asn Pro Ala Ser Thr Thr Asn Lys Asp Lys Leu 1 5
10 64 12 PRT Adenovirus 64 Gly Asp Asn Pro Ala Ser Thr Thr Asn Lys
Asp Lys 1 5 10 65 12 PRT Adenovirus 65 Ser Asp Asn Pro Ala Ser Thr
Thr Asn Lys Asp Lys 1 5 10 66 13 PRT Adenovirus 66 Gly Ser Asp Asn
Pro Ala Ser Thr Thr Asn Lys Asp Lys 1 5 10 67 15 PRT Adenovirus 67
Gly Asp Asn Pro Ala Ser Thr Thr Asn Lys Asp Lys Leu Gly Gly 1 5 10
15 68 15 PRT Adenovirus 68 Gly Asp Asn Pro Ala Ser Thr Thr Asn Lys
Asp Lys Leu Gly Ser 1 5 10 15 69 14 PRT Adenovirus 69 Gly Asp Asn
Pro Ala Ser Thr Thr Asn Lys Asp Lys Leu Ser 1 5 10 70 15 PRT
Adenovirus 70 Gly Asp Asn Pro Ala Ser Thr Thr Asn Lys Asp Lys Leu
Ser Gly 1 5 10 15 71 15 PRT Adenovirus 71 Gly Asp Asn Pro Ala Ser
Thr Thr Asn Lys Asp Lys Leu Ser Ser 1 5 10 15 72 11 PRT Adenovirus
72 Gly Thr Gln Glu Thr Gly Asp Thr Thr Pro Ser 1 5 10 73 30 DNA
Adenovirus 73 cagctccatc tcctaactgt agactaaatg 30 74 30 DNA
Adenovirus 74 ggttaccggt ttagttttgt ctccgtttaa 30 75 30 DNA
Adenovirus 75 agcgcttact ctatgtcatt ttcatgggac 30 76 30 DNA
Adenovirus 76 gagtttatta atatcactga tgagcgtttg 30 77 57 DNA
Adenovirus 77 gtaacactaa ccattacact aaacggtacc caggaaacag
gagacacaac tccaagt 57 78 52 DNA Adenovirus 78 acttggagtt gtgtctcctg
tttcctgggt accgtttagt gtaatggtta gt 52 79 60 DNA Adenovirus 79
gtaacactaa ccattacact aaacggtacc agtgaatcca cagaaactag cgaggtaagc
60 80 55 DNA Adenovirus 80 gcttaccctc gctagttttc tgtggatcac
tggtaccgtt tagtgtaatg ttagt 55 81 42 DNA Adenovirus 81 gtaacactaa
ccattacact aaaccaagaa acacaatgtg aa 42 82 37 DNA Adenovirus 82
ttcacattgt gtttcttggt ttagtgtaat ggttagt 37 83 60 DNA Adenovirus 83
gtaaccctaa ccattacact aaacggtgat aacccagcgt cgaccacgaa taaggataag
60 84 58 DNA Adenovirus 84 gctcttatcc ttattcgtgg tcgacgctgg
gttatcaccg tttagtgtaa tggttagg 58 85 66 DNA Adenovirus 85
gtaaccctaa ccattacact aaacggtgat aacccagcgt cgaccacgaa taaggataag
60 ggaagc 66 86 61 DNA Adenovirus 86 gcttccctta tccttattcg
tggtcgacgc tgggttatca ccgtttagtg taatggttag 60 g 61 87 66 DNA
Adenovirus 87 gtaaccctaa ccattacact aaacggtgat aacccagcgt
cgaccacgaa taaggataag 60 tcaagc 66 88 61 DNA Adenovirus 88
gcttgactta tccttattcg tggtcgacgc tgggttatca ccgtttagtg taatggttag
60 g 61 89 69 DNA Adenovirus 89 gtaaccctaa ccattacact aaacggtgat
aacccagcgt cgaccacgaa taaggataag 60 ggcggaagc 69 90 64 DNA
Adenovirus 90 gcttccgccc ttatccttat tcgtggtcga cgctgggtta
tcaccgttta gtgtaatggt 60 tagg 64 91 69 DNA Adenovirus 91 gtaaccctaa
ccattacact aaacggtgat aacccagcgt cgaccacgaa taaggataag 60 tcatctagc
69 92 64 DNA Adenovirus 92 gctagatgac ttatccttat tcgtggtcga
cgctgggtta tcaccgttta gtgtaatggt 60 tagg 64 93 69 DNA Adenovirus 93
gtaaccctaa ccattacact aaacggtgat aacccagcgt cgaccacgaa taaggataag
60 ggatccagc 69 94 64 DNA Adenovirus 94 gctggatccc ttatccttat
tcgtggtcga cgctgggtta tcaccgttta gtgtaatggt 60 tagg 64 95 69 DNA
Adenovirus 95 gtaaccctaa ccattacact aaacggtgat aacccagcgt
cgaccacgaa taaggataag 60 tcaggaagc 69 96 64 DNA Adenovirus 96
gcttcctgac ttatccttat tcgtggtcga cgctgggtta tcaccgttta gtgtaatggt
60 tagg 64 97 60 DNA Adenovirus 97 gtaaccctaa ccattacact aaacggtgat
aacccagcgt cgaccacgaa taaggataag 60 98 55 DNA Adenovirus 98
cttatcctta ttcgtggtcg acgctgggtt atcaccgttt agtgtaatgg ttagg 55 99
12 PRT Adenovirus 99 Gly Thr Ser Glu Ser Thr Glu Thr Ser Glu Val
Ser 1 5 10 100 11 PRT Adenovirus 100 Gly Thr Gln Glu Thr Gly Asp
Thr Thr Pro Ser 1 5 10 101 6 PRT Adenovirus 101 Gln Glu Thr Gln Cys
Glu 1 5 102 12 PRT Adenovirus 102 Asp Asn Pro Ala Ser Thr Thr Asn
Lys Asp Lys Ser 1 5 10 103 13 PRT Adenovirus 103 Asp Asn Pro Ala
Ser Thr Thr Asn Lys Asp Lys Gly Ser 1 5 10 104 13 PRT Adenovirus
104 Asp Asn Pro Ala Ser Thr Thr Asn Lys Asp Lys Ser Ser 1 5 10 105
14 PRT Adenovirus 105 Asp Asn Pro Ala Ser Thr Thr Asn Lys Asp Lys
Gly Gly Ser 1 5 10 106 14 PRT Adenovirus 106 Asp Asn Pro Ala Ser
Thr Thr Asn Lys Asp Lys Ser Ser Ser 1 5 10 107 14 PRT Adenovirus
107 Asp Asn Pro Ala Ser Thr Thr Asn Lys Asp Lys Gly Ser Ser 1 5 10
108 14 PRT Adenovirus 108 Asp Asn Pro Ala Ser Thr Thr Asn Lys Asp
Lys Ser Gly Ser 1 5 10 109 11 PRT Adenovirus 109 Asp Asn Pro Ala
Ser Thr Thr Asn Lys Asp Lys 1 5 10 110 30 DNA Adenovirus 110
atttctgtcg actttattca gcagcacctc 30 111 30 DNA Adenovirus 111
gtttgacttg gtttttttga gaggtgggct 30 112 30 DNA Adenovirus 112
ttggatatta actacaacaa aggcctttac 30 113 30 DNA Adenovirus 113
gaaactggag ctcgtatttg actgccacat 30 114 30 DNA Adenovirus 114
atttctgtcg actttattca gcagcacctc 30 115 30 DNA Adenovirus 115
gtttgacttg gtttttttga gaggtgggct 30 116 30 DNA Adenovirus 116
ctcaaaacaa aaattggcca tggcctagaa 30 117 30 DNA Adenovirus 117
atccaagagc tcttgtatag gctgtgcctt 30 118 30 DNA Adenovirus 118
tacaagtcga caaccaagcg tcagaaattg 30 119 30 DNA Adenovirus 119
aagacttaaa accccagggg gactctcttg 30 120 30 DNA Adenovirus 120
gagagtcccc ctggggtttt aagtcttaaa 30 121 30 DNA Adenovirus 121
ggtccacaaa gtgttatttt tcagtgcaat 30 122 30 DNA Adenovirus 122
ctgaaaaata acactttgtg gaccacacca 30 123 30 DNA Adenovirus 123
tcctgagctc cgtttagtgt aatggttagt 30 124 9 PRT Adenovirus 124 Cys
Asp Cys Arg Gly Asp Cys Phe Cys 1 5 125 13 PRT Adenovirus 125 Thr
Thr Glu Ala Thr Ala Gly Asn Gly Asp Asn Leu Thr 1 5 10 126 31 DNA
Adenovirus 126 ggaactttag aaatggagat cttactgaag g 31 127 33 DNA
Adenovirus 127 cgattcttta ttcttgcgca atgtatgaaa aag 33 128 22 DNA
Adenovirus 128 cttaagtgag ctgcccgggg ag 22 129 23 DNA Adenovirus
129 ggatccaatg aacttcatca agt 23 130 49 DNA Adenovirus 130
aattctgcgc aagaaccaaa gagggccagg cccggatcct aagacgtct 49 131 49 DNA
Adenovirus 131 ctagagacgt cttaggatcc gggcctggcc ctctttggtt
cttgcgcag 49 132 15 PRT Adenovirus 132 Pro Lys Arg Ala Arg Pro Gly
Ser Lys Lys Lys Lys Lys Lys Lys 1 5 10 15 133 10 PRT Adenovirus 133
Arg Arg Leu Leu Arg Arg Leu Leu Arg Arg 1 5 10 134 11 PRT
Adenovirus 134 Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys 1 5 10
135 9 PRT Adenovirus 135 Gly Ser Lys Lys Lys Lys Lys Gly Ser 1 5
136 13 PRT Adenovirus 136 Gly Ser Ser Lys Lys Lys Lys Lys Lys Lys
Gly Ser Ser 1 5 10 137 17 PRT Adenovirus 137 Gly Ser Gly Ser Gly
Ser Gly Ser Gly Ser Lys Lys Lys Lys Lys Lys 1 5 10 15 Lys 138 15
PRT Adenovirus 138 Pro Lys Arg Ala Arg Pro Gly Ser Lys Lys Lys Lys
Lys Lys Lys 1 5 10 15 139 8 PRT Adenovirus 139 Lys Lys Lys Lys Lys
Lys Lys Lys 1 5 140 19 PRT Adenovirus 140 Pro Lys Arg Ala Arg Pro
Gly Ser Lys Arg Gly Pro Arg Thr His Tyr 1 5 10 15 Gly Gln Lys 141
18 PRT Adenovirus 141 Pro Lys Arg Ala Arg Pro Gly Ser Arg Arg Leu
Leu Arg Arg Leu Leu 1 5 10 15 Arg Arg 142 17 PRT Adenovirus 142 Ser
Ser Arg Gly His Ser Arg Gly Arg Asn Gln Asn Ser Arg Gly Ser 1 5 10
15 Ser 143 23 PRT Adenovirus 143 Gly Ser Ser Arg Gly His Ser Arg
Gly Arg Asn Gln Asn Ser Arg Arg 1 5 10 15 Pro Ser Arg Ala Gly Ser
Ser 20 144 19 PRT Adenovirus 144 Gly Thr Ser Glu Arg Gly His Ser
Arg Gly Arg Asn Gln Asn Ser Arg 1 5 10 15 Gly Ser Ser 145 19 PRT
Adenovirus 145 Gly Thr Gln Glu Arg Gly His Ser Arg Gly Arg Asn Gln
Asn Ser Arg 1 5 10 15 Gly Ser Ser 146 19 PRT Adenovirus 146 Gly Ser
Ser Ser Arg Gly His Ser Arg Gly Arg Asn Gln Asn Ser Arg 1 5 10 15
Gly Ser Ser 147 18 PRT Adenovirus 147 Gly Ser Ser Arg Gly His Ser
Arg Gly Arg Asn Gln Asn Ser Arg Gly 1 5 10 15 Gly Ser 148 7 PRT
Adenovirus 148 Asp Cys Arg Gly Asp Cys Phe 1 5 149 8 PRT Adenovirus
149 Pro Lys Arg Ala Arg Pro Gly Ser 1 5 150 16
PRT Adenovirus 150 Ser Ser Arg Gly His Ser Arg Gly Arg Asn Gly Asn
Ser Arg Gly Ser 1 5 10 15 151 13 PRT Adenovirus 151 Gly Asp Asn Pro
Ala Ser Thr Thr Asn Lys Asp Lys Ser 1 5 10 152 14 PRT Adenovirus
152 Gly Asp Asn Pro Ala Ser Thr Thr Asn Lys Asp Lys Gly Ser 1 5 10
153 14 PRT Adenovirus 153 Gly Asp Asn Pro Ala Ser Thr Thr Asn Lys
Asp Lys Ser Ser 1 5 10 154 15 PRT Adenovirus 154 Gly Asp Asn Pro
Ala Ser Thr Thr Asn Lys Asp Lys Gly Gly Ser 1 5 10 15 155 15 PRT
Adenovirus 155 Gly Asp Asn Pro Ala Ser Thr Thr Asn Lys Asp Lys Ser
Ser Ser 1 5 10 15 156 15 PRT Adenovirus 156 Gly Asp Asn Pro Ala Ser
Thr Thr Asn Lys Asp Lys Gly Ser Ser 1 5 10 15 157 15 PRT Adenovirus
157 Gly Asp Asn Pro Ala Ser Thr Thr Asn Lys Asp Lys Ser Gly Ser 1 5
10 15 158 5 PRT Human 158 Lys Gln Ala Gly Asp 1 5 159 5 PRT Human
159 Glu Ile Leu Asp Val 1 5 160 4 PRT Human misc_feature (3)..(3)
Xaa can be any naturally occurring amino acid 160 Asn Pro Xaa Tyr 1
161 7 PRT Adenovirus 161 Pro Gly Val Leu Ser Leu Lys 1 5 162 7 PRT
Adenovirus 162 Asn Asn Thr Leu Trp Thr Thr 1 5 163 21 DNA
Adenovirus 163 cctggggttt taagtcttaa a 21 164 21 DNA Adenovirus 164
aataacactt tgtggaccac a 21 165 17 PRT Human 165 Arg Gly His Ser Arg
Gly Arg Asn Gln Asn Ser Arg Arg Pro Ser Arg 1 5 10 15 Ala
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