U.S. patent application number 10/670503 was filed with the patent office on 2004-02-26 for modified adenoviral fiber and uses.
This patent application is currently assigned to TRANSGENE, S.A.. Invention is credited to Cusack, Stephen, Legrand, Valerie, Leissner, Philippe, Mehtali, Majid, Van Raaij, Mark Johan.
Application Number | 20040038205 10/670503 |
Document ID | / |
Family ID | 9549387 |
Filed Date | 2004-02-26 |
United States Patent
Application |
20040038205 |
Kind Code |
A1 |
Van Raaij, Mark Johan ; et
al. |
February 26, 2004 |
Modified adenoviral fiber and uses
Abstract
The present invention relates to a modified fiber of an
adenovirus, comprising at least one mutation at one or more
residues within the region of said fiber stretching from pleated
sheet A to pleated sheet B, and including loop AB.
Inventors: |
Van Raaij, Mark Johan;
(Pays-Bas, NL) ; Cusack, Stephen; (Seyssinet,
FR) ; Legrand, Valerie; (Strasbourg, FR) ;
Leissner, Philippe; (Strasbourg, FR) ; Mehtali,
Majid; (Amsterdam, NL) |
Correspondence
Address: |
BURNS DOANE SWECKER & MATHIS L L P
POST OFFICE BOX 1404
ALEXANDRIA
VA
22313-1404
US
|
Assignee: |
TRANSGENE, S.A.
Strasbourg
FR
|
Family ID: |
9549387 |
Appl. No.: |
10/670503 |
Filed: |
September 26, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10670503 |
Sep 26, 2003 |
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09830391 |
Jul 30, 2001 |
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09830391 |
Jul 30, 2001 |
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PCT/FR00/02377 |
Aug 25, 2000 |
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Current U.S.
Class: |
435/5 ;
435/235.1; 530/350 |
Current CPC
Class: |
C12N 2810/40 20130101;
C07K 14/005 20130101; C12N 2810/851 20130101; C12N 15/86 20130101;
A61P 35/00 20180101; C12N 2710/10322 20130101; C12N 2810/859
20130101; C12N 2710/10343 20130101; C12N 2810/854 20130101; C12N
2810/10 20130101; C12N 2710/10345 20130101 |
Class at
Publication: |
435/5 ; 530/350;
435/235.1 |
International
Class: |
C07K 014/075; C12Q
001/70; C12N 007/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 27, 1999 |
FR |
99/10859 |
Claims
1. Modified fiber of an adenovirus, comprising at least one
mutation at one or more residues within the region of said fiber
stretching from pleated sheet A to pleated sheet B, and including
loop AB.
2. Fiber of an adenovirus according to claim 1, characterized in
that it comprises at least one mutation at one or more residues
within loop AB.
3. Fiber of an adenovirus according to claim 1 or 2, characterized
in that it allows, when it is included in a viral particle, the
production of a said viral particle having the following
properties: (i) said adenoviral particle does not substantially
attach to the natural cellular receptors; (ii) when said adenoviral
particle also comprises a ligand specific for an antiligand, said
modified particle has a novel tropism for one or more specific cell
types carrying, at their surface, said antiligand.
4. Fiber of an adenovirus according to one of claims 1 to 3,
characterized in that it derives from a fiber of a type 5
adenovirus (Ad5) comprising all or part of the sequence as shown in
sequence identifier No. 1 (SEQ ID NO: 1), and in that it comprises
at least one mutation at one or more residues of the region between
residues 400 and 428.
5. Fiber of a type 5 adenovirus according to claim 4, characterized
in that it comprises at least one mutation at one or more residues
of the region between residues 404 and 418 of SEQ ID NO: 1.
6. Fiber of a type 5 adenovirus according to claim 5, characterized
in that it comprises at least one mutation at one or more residues
of the region between residues 404 and 408 of SEQ ID NO: 1.
7. Fiber of a type 5 adenovirus according to claim 6, characterized
in that said residue is selected from the threonine residue at
position 404, the alanine residue at position 406 and the serine
residue at position 408.
8. Fiber of a type 5 adenovirus according to claim 7, characterized
in that it comprises substitution of the serine residue at position
408 with an amino acid residue having at least two carboxyl
groups.
9. Fiber of a type 5 adenovirus according to claim 8, characterized
in that said residue is selected from the group consisting of
aspartic acid and glutamic acid.
10. Fiber of a type 5 adenovirus according to claim 7,
characterized in that it comprises substitution of the threonine
residue at position 404 with a glycine residue and/or substitution
of the alanine residue at position 406 with a lysine residue.
11. Fiber of an adenovirus according to one of claims 1 to 10,
characterized in that one at least of the mutations is deletion of
at least 3 consecutive residues of a loop and/or of a pleated sheet
of said region.
12. Fiber of an adenovirus according to claim 11, characterized in
that said deleted residues are replaced with residues of an
equivalent loop and/or pleated sheet derived from a fiber of a
second adenovirus of heterologous type, capable of interacting with
a cellular receptor other than that recognized by said first
adenovirus.
13. Fiber of an adenovirus according to one of claims 1 to 12,
characterized in that it also comprises one or more mutations in:
(i) loops CD, DG, GH, HI and/or IJ and/or (ii) pleated sheets C, D,
G, H, I and/or J.
14. Fiber of an adenovirus according to one of claims 1 to 13,
characterized in that it also comprises a ligand capable of
recognizing a cellular antiligand other than the natural cellular
receptor of the nonmutated fiber.
15. Fiber of an adenovirus according to claim 14, characterized in
that the ligand is selected from the group consisting of an
antibody or an antibody fragment, a peptide, a lipid, a glycolipid,
a hormone, a polymer or a sugar.
16. Fiber of an adenovirus according to claim 14 or 15,
characterized in that the ligand is inserted at the C-terminal end
of the fiber.
17. Fiber of an adenovirus according to claim 14 or 15,
characterized in that the ligand is inserted as a replacement for
deleted residues.
18. Peptide fragment characterized in that it comprises the region
stretching from pleated sheet A to pleated sheet B, and including
loop AB, of a fiber according to any one of claims 1 to 17.
19. Peptide fragment according to claim 18, characterized in that
it is the sequence stretching from residue 388 to residue 592 of a
fiber according to any one of claims 4 to 17.
20. DNA fragment or expression vector encoding a fiber of an
adenovirus according to one of claims 1 to 17, or a peptide
fragment according to either of claims 18 and 19.
21. Cell line characterized in that it comprises, either in a form
integrated into the genome or in episome form, a DNA fragment
according to claim 20, placed under the control of the elements
allowing its expression in said cell line.
22. Cell line according to claim 21, characterized in that it is
also capable of complementing an adenovirus deficient for one or
more functions selected from the functions encoded by the E1, E2,
E4 and L1-L5 regions.
23. Cell line according to claim 21 or 22, characterized in that it
is produced using the 293 line.
24. Cell line according to claim 21 or 22, characterized in that it
is produced using the PERC6 line.
25. Adenoviral particle characterized in that it lacks a functional
native fiber, and in that it comprises a fiber according to one of
claims 1 to 17.
26. Adenoviral particle characterized in that it lacks a functional
native fiber, and in that it comprises a fiber according to one of
claims 1 to 17 and a ligand capable of recognizing a cellular
antiligand other than the natural cellular receptor for said
particle.
27. Adenoviral particle according to claim 26, characterized in
that said ligand is inserted into an adenoviral capsid protein
other than the fiber, in particular the hexon or the penton.
28. Adenoviral particle according to one of claims 25 to 27,
characterized in that it is empty.
29. Adenoviral particle according to one of claims 25 to 27,
characterized in that it contains an adenoviral genome.
30. Adenoviral particle according to claim 29, characterized in
that said adenoviral genome is a replication-defective recombinant
adenoviral genome.
31. Process for producing an adenoviral particle according to claim
29, characterized in that: (i) a said replication-defective
recombinant adenoviral genome is transfected into a suitable cell
line, (ii) said transfected cell line is cultured under suitable
conditions so as to allow the production of said adenoviral
particle, and (iii) said adenovirus is recovered from the culture
of said transfected cell line and, optionally, said adenoviral
particle is purified.
32. Process for producing an adenoviral particle containing an
adenoviral genome lacking all or part of the sequences encoding a
fiber, characterized in that: said genome is transfected into a
cell line according to one of claims 21 to 24, said transfected
cell line is cultured under suitable conditions so as to allow the
production of said adenoviral particle, and said adenoviral
particle is recovered from the culture of said transfected cell
line and, optionally, said adenoviral particle is purified.
33. Composition which comprises an adenoviral particle according to
one of claims 25 to 30, or which can be obtained using a process
according to claim 31 or 32, in combination with a support which is
acceptable from a pharmaceutical point of view.
34. Composition according to claim 33, characterized in that it
also comprises at least one compound selected from a naked nucleic
acid or a nucleic acid combined with at least one cationic
compound.
35. Use of an adenoviral particle according to one of claims 25 to
30, or which can be obtained using a process according to claim 31
or 32, for preparing a medicinal product intended for the treatment
of the human or animal body.
Description
[0001] The present invention relates, in particular, to an
adenoviral fiber mutated in the regions involved in recognizing and
binding to the natural cellular receptor for adenoviruses. It also
relates to the adenoviral particles bearing, at their surface, such
a fiber, optionally combined with a ligand which confers modified,
or even targeted, host specificity on said particles. The invention
is of most particular value in the context of the development of
vectors which can be used in the context of gene therapy.
[0002] Adenoviral vectors are widely used in many gene therapy
applications. They have been demonstrated in many animal species
and are relatively nonpathogenic, and nonintegrating, and replicate
both in dividing and in quiescent cells. In addition, they have a
broad host spectrum and are capable of infecting a very great
number of cell types, such as for example epithelial cells,
endothelial cells, myocytes, hepatocytes, nerve cells and
synoviocytes (Bramson et al., 1995. Curr. Op. Biotech. 6,
590-595).
[0003] The adenoviral genome consists of a double stranded, linear
DNA molecule of approximately 36 kb containing two inverted repeat
regions (referred to as ITRs for Inverted Terminal Repeat) framing
the genes encoding the viral proteins. The early genes are divided
into four regions dispersed in the adenoviral genome (E1 to E4; E
for early), including 6 transcriptional units provided with their
own promoters. The late genes (L1 to L5; L for late) cover, in
part, the early transcription units and are, mostly, transcribed
from the major late promoter (MLP).
[0004] Adenoviruses have been the subject of many studies and many
scientific teams have developed adenoviral vectors which are
replication-defective, i.e. in which the genome has been
manipulated such that these adenoviral vectors are incapable of
dividing or of proliferating in the cells which they infect.
Defective adenoviral vectors are in particular obtained by deleting
at least the El region (for examples of defective adenoviral
vectors, see, in particular, patent applications WO 94/28152 and WO
94/12649).
[0005] More recently, other uses of adenoviral particles have been
described, in particular in the context of implementing gene
therapy protocols.
[0006] Thus, patent application WO 95/21259 describes a method for
introducing a nucleic acid into a cell, which is based on combining
adenoviral particles and nucleic acid, more particularly naked
nucleic acid. This method is based mainly on the capacity of the
adenoviral particle to transport molecules to the cell nucleus'
after endocytosis. Curiel et al. (1992 Hum. Gene Ther., 3: 147-154)
and Wagner et al. (1992, Proc. Natl. Acad. Sci., 89; 6099-6103),
have, themselves also, shown that combining plasmid with
inactivated adenoviral particles allows the endosome to be lysed
before fusion with the lysosomes and, therefore, allows the plasmid
to escape degradation. This ingenious device makes it possible to
increase the efficiency of transfection of the plasmid 100- to
1000-fold in vitro. Preferably, in order for the cellular
transfection to be independent of the adenoviral process and to
indeed involve the use of a ligand chosen so as to allow targeting
of the transfection, an antibody which neutralizes the adenoviral
infection can be added to the complex (Michael et al.; 1993, J.
Biol. Chem., 268: 6866-6869). The contents of these publications
and patent applications are incorporated by reference in their
entirety, into the present application.
[0007] The infectious cycle of adenoviruses is based on two
essential steps. The early phase precedes replication initiation
and allows the production of the early proteins which regulate
replication and transcription of the viral DNA. Replication of the
genome is followed by the late phase during which the structural
proteins which constitute the basis of the viral particles are
synthesized. Assembly of the new virions takes place in the
nucleus. Initially, the viral proteins assemble so as to form empty
capsids of icosahedrai structure, in which the newly formed genome
is encapsidated. The adenoviruses released are capable of infecting
other permissive cells.
[0008] During infection, the fiber and the penton base of the
adenoviral particle, present at the surface of the capsids, play a
critical role in the cellular attachment of the virions and their
internalization (Wickham et al., 1993, Cell, 73, 309-319). Firstly,
the adenovirus binds to a cellular receptor (the CAR) present at
the surface of the permissive cells, via the fiber in its trimeric
form (Philipson et al., 1968, J. Virol. 2, 1064-1075; Defer et al.,
1990, J. Virol, 64, 3661-3673). The viral particle is then
internalized by endocytosis, due to binding of the penton base to
the .alpha..sub.v.beta..sub.3 and .alpha..sub.v.beta..sub.- 5
cellular integrins (Mathias et al., 1994, J. Virol. 68,
6811-6814).
[0009] The adenoviral fiber is composed of three distinct domains
(Chroboczek et al., 1995, Current Top. Microbiol. Immunol. 199,
165-200):
[0010] (a) at its N-terminal end, is the tail, the sequence of
which is very conserved from one adenoviral serotype to the other.
It interacts with the penton base and ensures the anchoring of the
molecule in the capsid;
[0011] (b) in the center, is the shaft. It is a rod-like structure
composed of a certain number of pleated-sheet repeats, the number
of which varies according to the serotypes under consideration;
[0012] (c) at its C-terminal end is the Knob, which has a spherical
globular structure containing the trimerization signals (Hong and
Engler, 1996, J. Virol. 70, 7071-7078; Novelli and Boulanger, 1991,
J. Biol. Chem. 266, 9299-9303; Novelli and Boulanger, 1991,
Virology 185, 365-376), and is responsible for the binding to
permissive cells (Henry et al., 1994, J. Virol 68, 5239-5246; Louis
et al., 1994, J. Virol. 68, 4104-4106).
[0013] Several teams have already described adenoviral particles
for which the native fiber has been modified so as to modify their
natural tropism and change the binding specificity of this fiber
such that it recognizes a different cellular receptor.
[0014] WO 94/10323 describes type 5 (Ad5) adenoviral particles in
which the fiber has been mutated so as to comprise the sequence of
a fragment of antibody specific for a given antigen (of scFv type),
inserted at the end of one of the 22 repetitive units of the shaft.
These mutants have a modified specificity of infection of the
adenoviral particles and are capable of attaching to cells
exhibiting the target antigen.
[0015] U.S. Pat. No. 5,543,328 describes a chimeric adenoviral
fiber in which the Knob domain is replaced with the tumor necrosis
factor (TNF) sequence, or that of the ApoE peptide, so as to
redirect the attachment of the modified adenoviral particles toward
cells expressing the cellular receptor for TNF or the LDL (low
density lipoprotein) receptor, respectively, present at the surface
of hepatic cells.
[0016] WO 95/26412 describes a fiber modified by incorporating a
ligand at the C-terminal end.
[0017] WO 96/26281 describes a chimeric fiber obtained by replacing
a portion of the native fiber, and in particular of the knob, with
the equivalent portion of an adenoviral fiber of another serotype
and, optionally, inserting a vitronectin-specific RGD peptide at
the C-terminal end.
[0018] In addition, French patent application FR 2758821 (97 01005)
has demonstrated the role of the class I major histocompatibility
complex antigens and of the III modules of fibronectin as a primary
receptor and a cofactor, respectively, for adenoviruses. In an
identical way, Tomko et al. (1997, Proc. Natl. Acad. Sci 94,
3352-3356), Bergelson et al. (1997, Science 275, 1320-1323) and
Roelvink et al. (1998, J. Virol. 72, 7909-7915) have described
another receptor for the fiber of various adenoviral serotypes. It
is a 46 kDa surface molecule, CAR (Coxsackie and Adenovirus
Receptor).
[0019] Finally, Xia et al. (1994, Structure 2, 1259-1270) have
determined the crystallographic three-dimensional structure of the
adenoviral knob. Each monomer includes 8 antiparallel
.beta.-pleated sheets, referred to as A to D and G to J, and 6
major loops of 8 to 55 residues. For example, loop CD connects
pleated sheet C to pleated sheet D. It is indicated that minor
pleated sheets E and F are considered to form part of loop DG
located between pleated sheets D and G. By way of indication, Table
1 gives the location of the structures in the amino acid sequence
of the Ad5 fiber, as shown in sequence identifier No. 0.1 (SEQ ID
NO: 1), the +1 representing the Met initiating residue. In general,
the pleated sheets form an organized and compact structure, whereas
the loops are more flexible. These terms are conventional in the
field of protein biochemistry, and are defined in fundamental works
(see, for example, Stryer, Biochemistry, 2nd Edition, Chap. 2, p.
11 to 39, Ed Freeman and Company, San Francisco).
1 TABLE 1 .beta. pleated sheet Loop nomenclature Residues
nomenclature Residues A 400 to 403 AB 404 to 418 B 419 to 428 -- --
C 431 to 440 CD 441 to 453 D 454 to 461 DC 462 to 514 G 515 to 521
GH 522 to 528 H 529 to 536 HI 537 to 549 I 550 to 557 IJ 558 to 572
J 573 to 578
[0020] The four .beta.-pleated sheets A, B, C and J constitute the
V pleated sheets directed toward the viral particle. The other four
(D, G, H and I) form the R pleated sheets, which are presumed to
face the cellular receptor. The V pleated sheets seem to play an
important role in the trimerization of the structure, while the R
pleated sheets are thought to be involved in the interaction with
the receptor.
[0021] The present invention provides novel mutants of the
adenoviral fiber which allow, in particular, the production of
viral particles which have the following properties:
[0022] (i) the adenoviral particle comprising said mutated fiber
does not substantially attach to the natural cellular receptors,
i.e. the host specificity of these adenoviral particles bearing the
mutated fiber is decreased, or even inhibited, in comparison to the
host specificity of the adenoviral particles carrying the
wild-type, i.e. nonmutated, fiber;
[0023] (ii) when the adenoviral particle comprising said mutated
fiber also comprises a ligand specific for an antiligand, it is
possible to confer on said modified particle a novel tropism for
one or more specific cell types bearing, at its (their) surface,
said antiligand, in comparison to the nonmutated adenoviral
particle.
[0024] The expression "the mutated fiber does not substantially
attach to the natural cellular receptors" is intended to indicate
that the fiber is modified so as to decrease or abolish its ability
to bind to the natural cellular receptor. Such a property can be
verified by studying the infectivity or the cellular binding of the
corresponding adenoviral particles, using the techniques of the
art, and, in particular, with infection competition experiments for
the virus bearing the modified fiber, carried out in the presence
of a competitor consisting of all or part of the wild type
adenoviral fiber (for more detail relating to this measuring
technique, see the Experimental Section of the present
application). The loss of the natural specificity can also be
evaluated with cell attachment studies carried out in the presence
of labeled viruses (for example labeled with .sup.3H thymidine,
according to the technique of Roelvink et al., 1996, J. Virol. 70,
7614-7621) or with studies of infectivity of permissive cells or of
cells expressing the surface molecule targeted by the ligand (see
the examples which follow). Advantageously, "a mutated fiber does
not substantially attach to the natural cellular receptors" when
the percentage of residual infection, measured with a competition
experiment as disclosed in the examples which follow, is between
approximately 0 and 60%, preferably between 0 and 40%, and entirely
preferably between 0 and 20%. In addition, according to an
advantageous embodiment, the properties of trimerization and of
binding to the penton-base of the mutated adenoviral fiber are not
affected. These properties are easily verified according to the
technique used in the examples which follow.
[0025] The present invention has, in particular, the advantage of
providing novel products, the properties of which make it possible
to decrease the therapeutic amounts of adenovirus to be
administered and to target the infection of the vector to the cells
to be treated. This specificity is particularly essential when an
adenoviral vector is used which is capable of expressing a
cytotoxic gene, in order to avoid the propagation of the cytotoxic
effect to the healthy and nontargeted cells. In addition, the
teachings of the present invention allow the development of other
targeting systems intended for developing methods of treatment by
administration of recombinant viral or nonviral vectors.
[0026] Firstly, the present invention relates to the modified fiber
of an adenovirus, comprising at least one mutation at one or more
residues within the region of said fiber stretching from pleated
sheet A to pleated sheet B, and including loop AB. More
particularly, the mutations are preferably produced at one or more
residues within loop AB.
[0027] For the purposes of the invention, the terms "residues" and
"amino acids" are synonyms. The terms "pleated sheets" and "loops"
are defined according to Xia et al. 1994, Structure 2,
1259-1270.
[0028] The term "nucleic acid sequence" is intended to refer to a
synthetic or isolated natural, linear or circular, double-stranded
or single-stranded fragment of DNA and/or of RNA and/or PNA which
refers to a specific series of nucleotides, which may or may not be
modified, making it possible to define a fragment or a region of a
nucleic acid without size limitation.
[0029] According to a preferred embodiment, it is a nucleic acid
chosen from the group consisting of a cDNA (complementary DNA); a
genomic DNA; a plasmid DNA; an RNA and a viral genome.
[0030] The term "portion" of an amino acid sequence is intended to
mean an amino acid sequence comprising a minimum of 6 consecutive
amino acids, preferably 10, more preferably 15, even more
preferably 20, and most preferably 30, and/or having the same
biological activity as the sequence from which said portion is
derived, in particular the ability to recognize and to bind to the
target cells of the virus.
[0031] The term "portion" of a nucleic acid sequence is intended to
mean a nucleic acid sequence comprising a minimum of 18 consecutive
nucleotides, preferably 30, more preferably 45, even more
preferably 60, and most preferably 90, and/or encoding an amino
acid sequence having the same biological activity as the amino acid
sequence encoded by the nucleic acid sequence from which said
portion is derived.
[0032] The fiber according to the present invention can derive from
an adenovirus of human, canine, avian, bovine, murine, ovine,
porcine or simien origin, or be hybrid and comprise fragments of
diverse origins, including fragments of heterologous origin, i.e.
not derived from an adenoviral fiber, or derived from nonadenoviral
fibers. With regard to human adenoviruses, those of serotype C and,
in particular, type 2 or 5 adenoviruses (Ad2 or Ad5) are preferably
used. The fiber of Ad2 includes 580 amino acids (aa), the sequence
of which is disclosed in Herisse et al. (1981, Nucleic Acid Res. 9,
4023-4042, incorporated into the present application by reference).
That of Ad5 has been determined by Chroboczek and Jacrot (1987,
Virology, 161, 549-554, incorporated by reference) and has 582
amino acids (see sequence identifier 1; SEQ ID NO: 1). In order to
simplify the presentation of the present application, only the
positions relating to Ad5 are given. However, it is within the
scope of those skilled in the art to identify the equivalent
positions of the various pleated sheets and loops on the basis of
the sequences of adenoviral fibers of other origins. When the fiber
of the present invention is of animal origin, use is preferably
made of bovine adenoviruses and, in particular, those of the BAV-3
strain. The latter have been the subject of many studies, and the
sequence of the fiber is disclosed in international application WO
95/16048, the content of which is incorporated by reference. Of
course, the fiber of the present invention can, besides the
modifications described in the present invention, have other
modifications with respect to the native sequence, as long as they
do not affect the characteristics of the fiber proposed in the
application. In addition, it is within the scope of those skilled
in the art to identify the adenoviral fiber sequences available on
databases such as, for example, GenBank, and to identify the
equivalent positions of the various pleated sheets and loops as
described later. By way of information, mention is made, for
example, of the GenBank references for the adenoviral fiber
sequences of human serotype 2 (# AAA92223), 3 (# CAA26029), 5
(#M18369), 31 (#CAA54050 or 41 (#X17016). The contents of the
publications or of the GenBank references mentioned above are
incorporated, in their entirety, into the present application by
reference. The invention also relates to a modified fiber according
to the present invention, which also contains other mutations, such
as for example those described in patent application WO 98/44121.
More particularly, such a fiber according to the invention is
characterized in that it also comprises one or more mutations
in:
[0033] a) loops CD, DG, GH, HI and/or IJ and/or
[0034] b) pleated sheets C, D, G, H, I and/or J.
[0035] For the purpose of the present invention, the term
"mutation" refers to a deletion, substitution or addition of one or
more residues, or a combination of these possibilities.
[0036] According to a first embodiment of the invention, the
adenoviral fiber according to the invention derives from a fiber-of
a type 5 adenovirus (Ad5) comprising all or part of the sequence as
shown in sequence identifier No. 1 (SEQ ID NO: 1), and is
characterized in that it is modified by mutation of one or more
residues of the region between residues 400 and 428 more
particularly between residues 404 and 418, and preferably between
residues 404 and 408, of SEQ ID NO: 1. Entirely preferably, such an
adenoviral fiber has the properties (i) and (ii) set out above.
[0037] Preferably, the invention relates to a fiber of a type 5
adenovirus, characterized in that the mutated residue is selected
from the threonine residue at position 404, the alanine residue at
position 406 and the serine residue at position 408.
[0038] Because of their spatial location in the native fiber, these
residues are capable of recognizing and/or interacting directly or
indirectly with the natural cellular receptor for the adenovirus in
question.
[0039] According to a particular case of the invention, the
mutation produced is a substitution of at least one amino acid. In
this capacity, mention may be made of the following examples of a
fiber of a type 5 adenovirus, for which:
[0040] the serine residue at position 408 is substituted with a
residue having at least two carboxyl groups, and in particular with
a residue selected from the group consisting of aspartic acid and
glutamic acid, and/or the threonine residue at position 404 is
substituted with a glycine residue and/or
[0041] the alanine residue at position 406 is substituted with a
lysine residue.
[0042] It is also possible to introduce several substitutions into
the targeted region of the fiber, in particular at the amino acids
forming a bend, preferable of .alpha..alpha. type.
[0043] In accordance with the invention, it is preferable not to
drastically modify the three-dimensional structure of the
adenoviral fiber; thus, the amino acids forming a bend will be
replaced with residues forming a similar structure, such as those
mentioned in Xia et al. (1994).
[0044] The fiber of the present invention can also be modified by
deletion. The region removed can concern all or part of the exposed
domain and, in particular, of loop AB.
[0045] According to an advantageous embodiment, when one at least
of the modifications is deletion of at least three consecutive
residues of a loop and/or of a pleated sheet, the deleted residues
can be replaced with residues of an equivalent loop and/or pleated
sheet derived from a fiber of a second adenovirus capable of
interacting with a cellular receptor other than that recognized by
the first adenovirus. This makes it possible to maintain the
structure of the fiber according to the invention, while at the
same time confering upon it a host specificity corresponding to
that of the second adenovirus. As indicated in Xia et al. (1994),
the infection of type 2 and type 5 adenoviruses is different from
that of type 3 and type 7 adenoviruses. Thus, the residues deleted
from an Ad5 or Ad2 fiber deleted of at least three consecutive
residues among those specified above can be substituted with the
residues derived from an equivalent region of the Ad3 or Ad7 fiber,
so as to decrease the ability of said fiber to bind the receptor
for Ad5 and to confer upon it a novel specificity toward the
cellular receptor for Ad3 or Ad7.
[0046] The present invention also relates to a fiber of an
adenovirus having a substantially decreased ability to bind to the
natural cellular receptor, as shown above, but nevertheless capable
of trimerizing. Such a property is, in particular, determined using
the technique described in the experimental section of the
application.
[0047] According to an equally advantageous embodiment, the fiber
according to the invention also comprises a ligand. For the purpose
of the present invention, the term "ligand" defines any entity
capable of recognizing and binding, preferably with high affinity,
a cellular antiligand other than the natural cellular receptor for
the nonmutated adenoviral fiber. This antiligand can be expressed
or exposed at the surface of the cell the targeting of which is
desired (cell surface marker, receptor, antigenic peptide presented
by histocompatibility antigens, etc.), naturally or subsequent to a
modification of said target aimed at making it express or expose
such an antiligand at its surface. In accordance with the aims
pursued by the present invention, a ligand can be an antibody or an
antibody fragment, a lipid, a glycolipid, a hormone, a polypeptide,
a polymer (PEG, polylysine, PEI, etc.) or a sugar. The term
"antibody" refers, in particular, to monoclonal antibodies,
antibody fragments (such as, for example, Fab fragments) and single
chain antibodies (scFv). These names and abbreviations are
conventional in the field of immunology.
[0048] In the context of the present invention, it may be
advantageous to target more particularly a tumor cell, an infected
cell, a specific cell type or a category of cells bearing a
specific surface marker. For example, if the host cell to be
targeted is a cell infected with the HIV virus (Human
Immunodeficiency Virus), the ligand can be a fragment of antibody
against fusin, the CD4 receptor or against an exposed viral protein
(envelope glycoprotein), or the portion of the HIV virus TAT
protein stretching from residues 37 to 72 (Fawell et al., 1994,
Proc. Natl. Acad. Sci. USA 91, 664-668). If it is a tumor cell, the
choice will relate to a ligand which recognizes a tumor-specific
antigen (for example the MUC-1 protein in the case of breast
cancer, or certain epitopes of the HPV papilloma virus E6 or E7
proteins) or which is overexpressed (IL-2 receptor overexpressed in
certain lymphoid tumors). If the intention is to target--T
lymphocytes, a T-cell receptor ligand can be used. Moreover,
transferrin is a good candidate for hepatic targeting. In general,
the ligands which can be used in the context of the invention are
widely described in the literature and can be cloned using standard
techniques. It is also possible to synthesize them chemically, and
to couple them to the fiber according to the invention. In this
respect, the coupling of galactosyl residues should confer hepatic
specificity due to the interaction with asialoglycoprotein
receptors. However, the preferred embodiment consists in inserting
the ligand at the C-terminal end of the fiber according to the
invention or as a replacement for the residues deleted when one at
least of the modifications is a deletion of at least 3 consecutive
residues.
[0049] Another subject of the invention relates to a peptide
fragment characterized in that it comprises the region stretching
from pleated sheet A to pleated sheet B, and including loop AB, of
a modified fiber as described above. Such a peptide fragment has,
in particular, the following properties:
[0050] (i) when this peptide fragment is incorporated in place of a
region stretching from pleated sheet A to pleated sheet B, and
including loop. AB, of a given heterologous adenoviral fiber, the
adenoviral particle comprising said mutated fiber does not
substantially attach to the natural cellular receptors
[0051] (ii) when the adenoviral particle comprising said mutated
fiber according to (i) also comprises a ligand specific for an
antiligand, it is possible to confer upon said modified particle a
novel tropism for one or more specific cell types bearing, at their
surface, said antiligand, in comparison with the adenoviral
particle which does not comprise such a mutated fiber.
[0052] The invention relates more specifically to such a peptide
fragment characterized in that it is the sequence stretching from
residue 388 to residue 592 of a fiber of a type 5 adenovirus (Ad5)
comprising all or part of the sequence as shown in sequence
identifier No. 1 (SEQ ID NO: 1) and comprising at least one
mutation at one or more residues of the region between residues 400
and 428.
[0053] The present invention also relates to an adenoviral particle
which comprises, at its surface, a mutated fiber according to the
invention and, optionally, a ligand as defined above. According to
a preferred case, this adenoviral particle lacks a functional
native fiber. The mutated fiber of the invention can be expressed
by the adenoviral genome itself, in particular when said adenoviral
particle contains such a genome, or provided in trans by a
complementation cell line, such as those defined hereinafter.
According to a particular embodiment, the adenoviral particle of
the invention is as shown above and is characterized in that said
ligand is inserted into an adenoviral capsid protein other than the
fiber, in particular the hexon or the penton.
[0054] According to a particular case of the invention, said
adenoviral particle of the invention is "empty", i.e. it contains
no nucleic acid. The use of such viral particles is in particular
illustrated in document WO 95/21259, mentioned later. When, on the
contrary, this adenoviral particle contains an adenoviral genome,
reference will preferably be made to an adenoviral virus (or
adenovirus) and, in the specific case in which said genome is also
modified, reference will more especially be made to a recombinant
adenoviral virus (or recombinant adenovirus).
[0055] Such cases are described in greater detail hereinafter. The
invention therefore also relates to such adenoviruses and
recombinant adenoviruses.
[0056] According to the invention, said ligand can be chemically
coupled to said adenoviral particle. However, preference is given
to the variant according to which the sequences encoding the ligand
are inserted into the adenoviral genome, and preferably into the
sequences encoding the modified fiber according to the invention,
and more specifically in frame in order to preserve the reading
frame. The insertion can take place at any site. However, the
preferred insertion site is upstream of the stop codon at the
C-terminal end, or in place of the deleted residues. It is also
possible to envisage introducing the sequences of the ligand into
other adenoviral sequences, in particular those encoding another
capsid protein, such as the hexon or the penton.
[0057] Advantageously, the invention relates to a recombinant
adenovirus which is replication-defective, i.e. incapable of
autonomous replication in a host cell. The deficiency is obtained
by a mutation or deletion of one or more essential viral genes and,
in particular, of all or part of the E1 region in the adenoviral
genome. Deletions in the E3 region can be envisaged in order to
increase cloning capacities. However, it may be advantageous to
conserve the sequences encoding the gp19k protein (Gooding and
Wood, 1990, Critical Reviews of Immunology 10, 53-71) in order to
modulate the immune responses of the host. Of course, the genome of
an adenovirus according to the invention can also comprise further
deletions or mutations affecting other regions, in particular the
E2, E4 and/or L1-L5 regions (see, for example, WO 94/28152 or WO
94/12649, or Ensinger et al., 1972, J. Virol. 10, 328-339,
describing the heat-sensitive mutation of the DBP gene of E2).
[0058] According to a preferred embodiment, a recombinant
adenovirus of the invention comprises one or more gene(s) of
interest placed under the control of the elements required for its
(their) expression in a host cell. The gene in question can be of
any origin, genomic, cDNA (complementary DNA) or hybrid (minigene
lacking one or more introns). It can be attained using conventional
techniques of molecular biology, or by chemical synthesis. It can
encode an antisense RNA, a ribozyme or an mRNA which will then be
translated into a polypeptide of interest. This polypeptide can be
cytoplasmic or membrane-bound, or can be secreted by the host cell.
Moreover, it can be all or part of a polypeptide as found
naturally, of a chimeric polypeptide originating from the fusion of
sequences of diverse origins, or of a polypeptide which is mutated
with respect to the native sequence and which has improved and/or
modified biological properties.
[0059] In the context of the present invention, it may be
advantageous to use the genes encoding the following
polypeptides:
[0060] cytokines or lymphokines (.alpha.-, .beta.- and
.gamma.-interferons, interleukins, and in particular IL-2, IL-6,
IL-10 or IL-12, tumor necrosis factors (TNFs), colony stimulating
factors (GM-CSF, C-CSF, M-CSF, etc.);
[0061] cellular or nuclear receptors, in particular those
recognized by pathogenic organisms (viruses, bacteria or
parasites), and preferably by the HIV virus, or their ligands;
[0062] proteins involved in a genetic disease (factor VII, factor
VIII, factor IX, dystrophin or minidystrophin, insulin, CFTR
(Cystic Fibrosis Transmembrane Conductance Regulator) protein,
growth hormones (hGH));
[0063] enzymes (urease, renin, thrombin, etc.);
[0064] enzyme inhibitors (.alpha.1-antitrypsin, antithrombin III,
viral protease inhibitors, etc.);
[0065] polypeptides with an antitumor effect, capable of
inhibiting, at least partially, the initiation or progression of
tumors or cancers (antibodies, inhibitors acting on cell division
or on transduction signals, tumor suppressor gene expression
products, for example p53 or Rb, proteins which stimulate the
immune system, etc.);
[0066] class I or II major histocompatibility complex proteins or
regulatory proteins acting on the expression of the corresponding
genes;
[0067] polypeptides capable of inhibiting a viral, bacterial or
parasitic infection or its development (antigenic polypeptides
having immunogenic properties, antigenic epitopes, antibodies,
transdominant variants capable of inhibiting the action of a native
protein by competition, etc.);
[0068] toxins (herpes simplex virus 1 thymidine kinase (TK-HSV-1),
ricin, cholera toxin, diptheria toxin, etc.) or immunotoxins;
and
[0069] markers (.beta.-galactosidase, luciferase, etc.).
[0070] It should be pointed out that this list is not limiting and
that other genes can also be used.
[0071] Moreover, a recombinant adenovirus according to the
invention can also comprise a selection gene allowing the selection
or identification of the infected cells. Mention may be made of the
neo gene (encoding neomycin phosphotransferase) which confers
resistance to the G418 antibiotic, the dhfr (dihydrofolate
reductase) gene, the CAT (chloramphenicol acetyltransferase) gene,
the pac (puromycinacetyltransfe- rase) gene or the gpt (xanthine
guanine phosphoribosyl transferase) gene. In general, the selection
genes are known to those skilled in the art.
[0072] The expression "elements required for the expression of a
gene of interest in a host cell" is intended to mean the set of
elements allowing its transcription into RNA and the translation of
an mRNA into protein. Among these, the promoter is of particular
importance. In the context of the present invention, it can derive
from any gene of eukaryotic, or even viral, origin and can be
constitutive or regulatable. Moreover, it can be modified so as to
improve the promoter activity, suppress a transcription-inhibiting
region, make a constitutive promoter regulatable or vice versa,
introduce a restriction site, etc. Alternatively, it can be the
natural promoter of the gene to be expressed. Mention may be made,
by way of examples, of the CMV (cytomegalovirus), RSV (Rous Sarcoma
Virus), HSV-L virus TK gene, SV40 virus (Simian Virus 40) early,
and MLP adenoviral viral promoters, or the eukaryotic promoters of
the murine or human PGK (phospho glycerate kinase),
.alpha.1-antitrypsin (liver-specific) and immunoglobulin
(lymphocyte-specific) genes.
[0073] Of course, a gene of interest used in the present invention
can also comprise additional elements required for expression
(intronic sequence, signal sequence, nuclear localization sequence,
transcription termination sequence, translation initiation site of
IRES or other type, etc.) or for its persistence in the host cell.
Such elements are known to those skilled in the art.
[0074] The present invention also relates to a DNA fragment
encoding a fiber or a peptide fragment according to the invention,
and also to a vector for expressing such a fiber or such a
fragment. Any type of vector can be used for this purpose, whether
of plasmid or viral, integrating or nonintegrating, origin. Such
vectors are commercially available or described in the literature.
Similarly, those skilled in the art are capable of adjusting the
regulatory elements required for the expression of the DNA fragment
according to the invention. According to one particular case of the
invention, a said vector will be an adenoviral vector capable of
producing, under suitable culturing conditions, adenoviral
particles according to the invention, namely adenoviruses or
recombinant adenoviruses as described above.
[0075] The invention also relates to a process for preparing
adenoviral particles according to the invention, in which:
[0076] the adenoviral genome encoding a modified fiber according to
the invention is transfected into a suitable cell line, for example
the 293 line;
[0077] said transfected cell line is cultured cultured under
suitable conditions so as to allow the production of said
adenovirus or of said recombinant adenovirus, and
[0078] the empty particles are recovered by purifying the cell
lysate on a density gradient, in particular a cesium chloride
gradient for example.
[0079] The empty particles sediment, for example, at 1.3 g/ml of
cesium chloride, while the recombinant adenoviruses (particles
containing the Ad genome), themselves, sediment at 1.34 g/ml
(D'Hallivin, 1995, Cur. Top. Microbiol. Immunol, 199, 47-66).
[0080] According to another process, it is possible to obtain empty
particles after transfecting an adenoviral genome carrying a
modified encapsidation sequence, and also containing a DNA fragment
encoding a modified fiber according to the invention, into suitable
cells. The modification of the encapsidation region makes it
possible to decrease, or even eliminate, the phenomenon of
encapsidation of the adenoviral genome in the particles (Grble and
Hearing, 1992, J. Virol. 66, 723-731). The production steps which
follow the culturing are identical to those described above.
[0081] The invention also relates to a process for preparing an
adenovirus or a recombinant adenovirus according to the invention,
according to which:
[0082] the genome of said adenovirus, which may or may not be
recombinant and which may or may not be replication-defective, is
transfected into a suitable cell line,
[0083] said transfected cell line is cultured under suitable
conditions so as to allow the production of said adenovirus or of
said recombinant adenovirus (it is also possible to refer to
adenoviral particles), and
[0084] said adenovirus or said recombinant adenovirus is recovered
from the culture of said transfected cell line and, optionally,
said adenovirus is purified.
[0085] The choice of cell line depends, where appropriate, on the
deficient functions of the adenovirus according to the invention. A
complementation line capable of providing the defective
function(s), in trans, will in particular be used. The 293 (ATCC
CRL 1573) or PERC6 (ECACC 96022940) lines are most particularly
suitable for complementing the E1 function (Graham et al., 1977, J.
Gen. Virol. 36, 59-72 or WO 97/00326, respectively). For an E1, and
E2 or E4 double deficiency, a cell line among those described in
French Patent Application FR 2737222 (96 04413) can be used. It is
also possible to use an auxiliary virus in order to complement the
defective adenovirus according to the invention in any host cell,
or a mixed system using a complementation cell and an auxiliary
virus, in which the elements are dependent upon each other. The
means for propagating a defective adenovirus are known to those
skilled in the art, who can refer, for example, to Graham and
Prevec, 1991 (Methods in Molecular Biology, vol. 7, p. 190-128; Ed.
E. J. Murey, The Human Press Inc.). The adenoviral genome is
preferably reconstituted in vitro in Escherichia coli (E. coli), by
ligation or homologous recombination (see, for example, French
Application FR 2727689 (94 14470)). The purification processes are
described in the state of the art. Mention may be made of the
density gradient centrifugation technique.
[0086] According to an alternative process, it is also possible to
construct "empty" adenoviral particles artificially by associating
carboxy- or amino-terminal ends of adenoviral capsid proteins,
peptides or glycoproteins, with lipids. Such modified lipids,
incorporating in particular the peptide fragments of the invention,
can then be incorporated into a liposome. Such a technique has been
described by Tikchonenko et al., 1988, Gene 63, 321-330 in the case
of liposomes bearing, at their surface, influenza virus
glycoproteins.
[0087] The present invention also relates to a cell line
comprising, either in a form integrated into the genome or in the
form of an episome, a DNA fragment encoding a fiber according to
the invention, placed under the control of the elements allowing
its expression. The said line can derive from a cell complementing
one or more adenoviral functions selected from those encoded by the
E1, E2, E4 and L1-L5 regions. It preferably derives from the 293
line or from the PERC6 line. Such a line can be used for preparing
an adenovirus, in particular a recombinant adenovirus, the genome
of which lacks all or part of the sequences encoding the fiber (so
as to produce a nonfunctional fiber or not to produce a fiber).
[0088] For this reason, the invention also relates to a process for
producing adenoviral particles containing an adenoviral genome
lacking all or part of the sequences encoding a fiber,
characterized in that:
[0089] said genome is transfected into a cell line given above,
[0090] said transfected cell line is cultured under suitable
conditions so as to allow the production of said adenoviral
particle, and
[0091] said adenoviral particle is recovered from the culture of
said transfected cell line and, optionally, said adenoviral
particle is purified.
[0092] The present invention also covers a host cell which can be
infected with an adenovirus according to the invention or which can
be obtained using a process according to the invention. It is
advantageously a mammalian cell and, in particular, a human cell.
It can be a primary or tumor cell and of any origin, for example
hematopoietic (totipotent stem cell, leukocyte, lymphocyte,
monocyte or macrophage, etc.), muscle, nasal; pulmonary, tracheal,
hepatic, epithelial or fibroblast origin.
[0093] A subject of the invention is also a composition which
comprises, as a therapeutic or prophylactic agent, a host cell, an
adenoviral particle or an adenovirus, in particular a recombinant
adenovirus, according to the invention, which can be obtained using
a process according to the invention, in combination with a support
which is acceptable from a pharmaceutical point of view. The
composition according to the invention is, in particular, intended
for the preventive or curative treatment of diseases such as
genetic diseases (hemophilia, cystic fibrosis, diabetes or
Duchenne, Becker, etc. myopathy), cancers, such as those induced by
oncogenes or viruses, viral diseases, such as hepatitis B or C and
AIDS (acquired immunodeficiency syndrome resulting from HIV
infection), and recurrent viral diseases, such as viral infections
caused by the herpesvirus.
[0094] A composition according to the invention can be manufactured
conventionally. In particular, a therapeutically effective amount
of the therapeutic or prophylactic agent is combined with a support
which is acceptable from a pharmaceutical point of view. Such a
support is nontoxic for the patient. It can be an injectable
solution, an isotonic solution, the pH of which is compatible with
use in vivo, a solution of dextrose, of glycerol, of mannitol, etc.
A composition according to the invention can be administered
locally, systemically or by aerosol, in particular via the
intragastric, subcutaneous, intracardiac, intra-muscular,
intravenous, intraperitoneal, intratumoral, intrapulmonary,
intranasal or intratracheal route. The administration can
take-place in a single dose or in a dose repeated one or more times
after a certain period of delay. The suitable route of
administration and dose vary depending on various parameters, for
example on the individual or on the disease to be treated, or on
the gene(s) of interest to be transferred. In particular, the viral
particles according to the invention can be formulated in the form
of doses of between 10.sup.4 and 10.sup.14 pfu (plaque forming
units), advantageously 10.sup.5 and 10.sup.13 pfu, and preferably
10.sup.6 and 10.sup.12 pfu. The formulation can also include an
adjuvant or an excipient which is acceptable from a pharmaceutical
point of view.
[0095] The composition according to the invention can also be
formulated in the form of a solid or semi-solid preparation, in
particular in the form of a gas, tablet, capsule, powder, gelatin
capsule, granule, cream, solution, suppository or aerosol,
depending on the route of administration selected.
[0096] In the pharmaceutical compositions of the present invention,
the composition can be formulated with conventional pharmaceutical
supports, known to those skilled in the art.
[0097] These supports comprise, in particular, a phrmaceutical
vehicle such as gelatin, starch, lactose, magnesium stearate, talc,
sucrose or gum arabic, or analogues.
[0098] It is also possible to obtain a preparation of gelatin
capsules by mixing the composition with a diluent and pouring the
mixture obtained into soft or hard gelatin capsules.
[0099] A preparation in the form of syrup or of elixir can contain
the composition together with a sweetener, an antiseptic, and also
a flavoring and a suitable colorant.
[0100] The water-dispersible powders or granules can contain the
composition as a mixture with dispersants or wetting agents, or
suspending agents, as well as with flavor enhancers or
sweeteners.
[0101] For rectal administration, use is made of suppositories
which are prepared with binders which melt at the rectal
temperature, for example cacao butter or polyethylene glycols.
[0102] The composition can also be formulated in the form of
microcapsules, optionally with one or more additive supports.
[0103] A subject of the present invention is also a composition
characterized in that it also comprises at least one compound
selected from a naked nucleic acid or a nucleic acid combined with
at least one cationic compound.
[0104] With a view to their use in vivo, the adenoviral particles
according to the invention can also be complexed with synthetic or
natural compounds. Such adenoviral particles, and also their use,
are, for example, described in O'Riordan et al., 1999, Human Gene
Therapy, 10, 1349-1358 or in patent application WO 98/44143. The
content of these documents is incorporated into the present
application by reference.
[0105] Finally, the present invention relates to the use of a
peptide fragment, of an adenoviral particle, of an adenovirus or of
a host cell according to the invention, or of an adenovirus which
can be obtained using a process according to the invention, for
preparing a medicinal product intended for the treatment of the
human or animal body. According to a first possibility, the
medicinal product can be administered directly in vivo (for example
by intravenous injection, into an accessible tumor, into the lungs
by aerosol, etc.). The ex vivo approach can also be adopted, which
consists in removing cells from the patient (bone marrow stem
cells, peripheral blood lymphocytes, muscle cells, etc.),
transfecting or infecting them in vitro according to the techniques
of the art, and readministering them to the patient.
[0106] The invention also extends to a treatment method according
to which a therapeutically effective amount of an adenovirus or of
a host cell according to the invention is administered to a patient
who needs such a treatment.
EXAMPLES
[0107] The aim of the examples which follow is to illustrate the
various subjects of the present invention and, consequently, they
are in no way limiting in nature.
[0108] The constructs described below are prepared according to the
general techniques of genetic engineering and of molecular cloning,
detailed in Maniatis et al., (1989, Laboratory Manual, Cold Spring
Harbor, Laboratory Press, Cold Spring Harbor, N.Y.) or according to
the manufacturer's recommendations when a commercial kit is used.
The cloning steps using bacterial plasmids are preferably carried
out in the E. coli strain 5K (Hubacek and Glover, 1970, J. Mol.
Biol., 50, 111-127) or BJ 5183 (Hanahan, 1983, J. Mol. Biol. 166,
557-580). The latter strain is preferably used for the homologous
recombination steps. The NM522 strain (Strategene) is suitable for
propagating the M13 phage vectors. The PCR amplification techniques
are known to those skilled in the art (see, for example, PCR
Protocols--A guide to methods and applications, 1990, edited by
Innis, Gelfand, Sninsky and White, Academic Press Inc.). As regards
the repair of restriction sites, the technique used consists in
filling the overhanging 5' ends using the large fragment of E. coli
DNA polymerase, I (Klenow). The Ad5 nucleotide sequences are those
used in the Genebank databank, under the reference M73260.
[0109] With regard to the cell biology, the cells are transfected
according to standard techniques known to those skilled in the art.
Mention may be made of the calcium phosphate technique (Maniatis et
al., above), but any other protocol can also be used, such as the
DEAE dextran technique, electroporation, methods based on osmotic
shocks, microinjection or methods based on the use of cationic
lipids. With regard to the culturing conditions, they are
conventional. In the examples which follow, use is made of the 293
human line (ATCC CRL 1573) and of the Swiss 3T3 (ATCC CCL92), NR6
(Wells et al., 1990, Science 247, 962-964) and NR6-hEGFR (Schneider
et al., 1986, Proc. Natl. Acad. Sci. USA 83, 333-336) murine lines.
It is understood that other cell lines can also be used.
EXAMPLE 1
Construction of an Adenovirus Having a Host Tropism for Cells
Expressing the Receptor for GRP (Gastrin Releasing Peptide)
[0110] A. Insertion of the Sequences Encoding the GRP Ligand
(Fiber-GRP)
[0111] The plasmid pTG6593 derives from p poly II (Lathe et al.,
1987, Gene 57, 193-201) by the introduction of the complete gene
encoding the Ad5 fiber, in the form of an EcoRI-SmaI fragment
(nucleotides (nt) 30049 to 33093). The HindIII-SmaI fragment (nt
31994-33093) is isolated and cloned into M13TG130. (Kieny et al.,
1983, Gene 26, 91-99), digested with these same enzymes, to give
M13TG6526. The latter is subjected to site-directed mutagenesis
using the oligonucleotide oTG7000 (SEQ ID NO: 2) (Sculptor in vitro
mutagenesis kit, Amersham) in order to introduce a linker encoding
a spacer arm of 12 amino acids of sequence PSASASASAPGS. The
mutated vector thus obtained, M13TG6527, is subjected to, a second
mutagenesis allowing the introduction of the sequence encoding the
10 residues of the GRP peptide (GNHWAVGHLM; Michael et al., 1995,
Gene Ther. 2, 660-668). The oligonucleotide oTG7001 (SEQ ID NO: 3)
is used for this purpose. The HindIII-SmaI fragment is isolated
from the mutated phage M13TG6528 and introduced, using the
homologous recombination technique (Chartier et al., 1996, J.
Virol. 70, 4805-4810), into the plasmid pTG6590 carrying the Ad5
adenoviral gene fragment stretching from nt 27081 to 35935 and
linearized with MunI (nt 32825). The SpeI-ScaI fragment (carrying
nt 27082 to 35935 of the Ad5 genome, modified by introducing the
spacer arm and the GRP peptide) is isolated from the vector above,
referred to as pTG8599, and is then exchanged against the
equivalent fragment of pTG6591, digested beforehand with these same
enzymes. By way of indication, pTG6591 comprises the wild-type
adenoviral sequences from positions 21562 to 35935, pTG4600 is
obtained, from which the BstEII fragment is isolated (nt 24843 to
35233). After homologous recombination with the plasmid pTG3602
which comprises the Ad5 genome (described in greater detail in
International Application WO 96/17070), the vector pTG4601 is
generated.
[0112] A cassette allowing the expression of the LacZ gene is
introduced in place of the E1 adenoviral region by homologous
recombination between the plasmid pTG4061 linearized with ClaI and
a BsrGI-PstI fragment comprising the LacZ gene encoding
.beta.-galactosidase under the control of the Ad2 MLP promoter and
the SV40 virus polyadenylation signal. This fragment is isolated
from the vector pTG8526 containing the 5' end of the viral genomic
DNA (nt 1 to 6241) in which the E1 region (nt 459 to 3328) is
replaced with the LacZ expression cassette. Its construction is
within the scope of those skilled in the art. The final vector is
referred to as pTG4628.
[0113] The corresponding viruses AdTG4601 and AdTG4628 are obtained
by transfecting the adenoviral fragments released from the plasmid
sequences by PacI digestion, into the 293 line. By way of
indication, AdTG4601 carries the complete Ad5 genome in which the
fiber gene comprises, in its 3' end, a spacer arm followed by the
GRP peptide. The recombinant virus AdTG4628 also carries the
cassette for expressing the LacZ reporter gene under the control of
the MLP adenoviral promoter.
[0114] B. Study of the Tropism of the Virus Carrying the
Fiber-GRP
[0115] The presence of the GRP peptide in the adenoviral fiber
makes it possible to target cells expressing, at their surface, the
receptor for GRP. The expression of the messages encoding the
latter is studied in 293 cells and in Swiss 3T3 murine cells
(Zachary et al., 1985, Proc. Natl. Acad. Sci. USA 82, 7616-7620) by
Northern blot. A mixture of 2 DNA fragments complementary to the
sequence encoding the receptor for GRP, labeled with the .sup.32P
isotope using conventional techniques, is used as a probe. By way
of indication, the fragments are produced by reverse PCR on total
cellular RNAs using the oligonucleotides oTG10776 (SEQ ID NO: 4)
and oTG10781 (SEQ ID NO: 5) (Battey et al., 1991, Proc. Natl. Acad.
Sci. USA 88, 395-399; Corjay et al., 1991, J. Biol. Chem. 266,
18771-18779). The intensity of the mRNAs detected is much greater
in the case of the Swiss-3T3 cells than in the case of the 293
cells, indicating the overexpression of the GRP receptor by the
murine line.
[0116] Competition experiments are carried out on the 2 cell types.
The competitor consists of the Ad5 fiber knob produced in E. coli,
the adenoviral cellular receptor-binding properties of which have
been shown (Henry et al., 1994, J. Virol 68, 5239-5246). The cells
in monolayer are pre-incubated for 30 min in the presence of PBS or
of increasing concentrations of recombinant Ad5 knob (0.1 to 100
.mu.g/ml), in DMEM medium (Gibco BRL) supplemented with 2% fetal
calf serum (FCS). Then, the virus AdTG4628, the fiber of which
contains the GRP peptide, is added at a multiplicity of infection
of 0.001 infectious units/cell, for 24 h at 37.degree. C. By way of
control, and according to the same experimental conditions, the
recombinant virus AdLacZ (Stratford-Perricaudet et al., 1992, J.
Clin. Invest. 90, 626-630), which carries a native fiber gene, is
used. The cells are then fixed and the expression of the LacZ gene
is evaluated (Sanes et al., 1986, EMBO J. 5, 3133-3142). The number
of blue cells is representative of the efficiency of the viral
infection. Competition inhibition causes a decrease in the number
of colored cells with respect to a noninfected control (PBS).
[0117] The addition of recombinant Ad5 knob at a concentration of
100 .mu.g/ml strongly inhibits the infection of the 293 cells with
the viruses AdLacZ and AdTG4628 (degree of inhibition of 95 and
98%). This suggests that the presence of the competitor prevents
the interaction of the adenoviral fiber with its natural cellular
receptor. On the other hand, the two viruses behave differently on
the Swiss-3T3 cells. The infection of the virus AdTG4628 in the
presence of 100 .mu.g/ml of competitor is only partially inhibited,
whereas, under the same experimental conditions, that of the virus
AdLacZ having the native fiber is totally inhibited. These results
suggest that the infection of the Swiss-3T3 cells with AdTG4628 is,
in part, mediated by an independent receptor, probably the GRP
receptor which these cells overexpress. In conclusion, the addition
of the GRP ligand to the C-terminal end of the fiber promotes the
infection of the cells expressing the GRP receptor, independently
of the fiber-natural cellular receptor interaction.
EXAMPLE 2
Construction of an Adenovirus Having a Tropism for Tumor Cells
Expressing Mucins
[0118] Construction insertion of the EPPT peptide, as described in
U.S. Pat. No. 5,591,593, into the C-term of the fiber. This
modification confers binding to mucins overexpressed on tumor
cells.
[0119] OTG11992: SEQ ID NO 12
[0120] mutagenesis with m13TG6527 to give m13TG6572. Homologous
recombination with pTG4213 to give pTG4278.
EXAMPLE 3
Construction of an adenovirus having a tropism for tumor cells
expressing .alpha..sub.4.beta..sub.1 Integrins
[0121] Construction: insertion of the LDV peptide, as described in
U.S. Pat. No. 5,628,979, in the C-term of the fiber. This
modification confers binding to .alpha..sub.4.beta..sub.1 integrins
overexpressed on tumor cells.
[0122] OTG 1191: SEQ ID NO 13
[0123] mutagenesis with m3TG6527 to give M13TG13265.
EXAMPLE 4
Construction of an Adenovirus Having a Host Tropism for Cells
Expressing the EGF (Epidermal Growth Factor) Receptor
[0124] This example describes a fiber carrying the EFG sequences at
its C-terminal end. For this, the oligonucleotides oTG11065 (SEQ ID
NO: 6), and oTG11066 (SEQ ID NO: 7) are used to amplify a
HindIII-XbaI fragment from the plasmid M13TG6527. The
oligonucleotides oTG11067 (SEQ. ID NO: 8), and oTG11068 (SEQ ID NO:
9) make it possible to generate an XhoI-SmaI fragment (ranging from
the stop codon up to nt 33093) from M13TG6527. The complementary
DNA of EGF, obtained from the ATCC (#59957), is amplified in the
form of an XhoI-XbaI fragment using the oligonucleotides oTG 11069
(SEQ ID NO: 10) and oTG11070(SEQ ID NO: 11). The 3 fragments
digested with the appropriate enzymes are then religated to give a
HindIII-SmaI fragment containing EGF fused to the C-terminal end of
the fiber. The same procedure of homologous recombination as that
described in example 1 is used to reposition this fragment in its
genomic context.
[0125] However, the cloning steps can be simplified by introducing
a unique BstBI site into the targeted region using conventional
mutagenesis techniques. pTG4609 is obtained. The homologous
recombination between pTG4609 linearized with BstBI and the
HindIII-SmaI fragment above generates the plasmid pTG4225 carrying
the wild-type El region. Its equivalent carrying the LacZ
expression cassette, pTG4226 is obtained by homologous
recombination with the pTG4213 digested with BstBI. The viruses
AdTG4225 and AdTG4226 can be produced conventionally by
transfecting a suitable cell line, for example, overexpressing the
receptor for EGF.
[0126] In order to test the specificity of infection of these
viruses, NR6 murine fibroblastic cells and Nr6-hEGFR cells
expressing the receptor for human EGF can be used. Competition with
the recombinant Ad5 knob or with EGF makes it possible to evaluate
the involvement of the EGF or natural cellular receptors in
mediating the infection of the viruses.
EXAMPLE 5
Modifications of the Fiber Knob so as to Eliminate the Binding to
the Natural Cellular Receptor
[0127] A. Modifications of the Fiber Sequences
[0128] The mutation of region AB (amino acid 404-418), of the
adenoviral fiber was undertaken in order to eliminate the ability
of the fiber to bind its natural receptor, and the addition of a
ligand will make it possible to modify the tropism of the
corresponding adenoviruses.
[0129] replacement, in loop AB, of the serine at position 408 with
the glutamic acid residue of serotype 3 using the oligo oTG12499
(SEQ ID NO: 14);
[0130] replacement, in loop AB, of the alanine at position 406 with
the lysine residue of serotype 3 using the oligo oTG12498 (SEQ ID
NO: 15);
[0131] replacement, in loop AB, of the threonine at position 404
with the glycine residue of serotype 3 using the oligo oTG12740
(SEQ ID NO: 16).
[0132] The mutageneses can be carried out on the vector M13TG6526
or M13TG6528. The first carries the wild-type HindIII-SmaI fragment
and the second carries the same fragment modified by inserting the
GRP sequences. The plasmids carrying the adenoviral genome can be
reconstituted as described previously for the plasmids pTG4225
(wild-type E1) and pTG4226 (LacZ in place of the E1 region) (by
homologous recombination with the plasmid pTG4609 or pTG4213). The
viruses are generated by transfecting 293 cells, 293 cells
expressing the wild-type fiber (Legrand et al., 1999; J. Virol.,
73, 907-919) or cells overexpressing the receptor which binds the
ligand in question. Such cells can be generated by transfection of
the corresponding complementary DNA. Cells which do not naturally
express the natural cellular receptor for adenoviruses, for example
the Daudi line (ATCC CCL213) are preferably used.
2 Oligo M13 Plasmid Mutation oTG- M13TG pTG- ABloop (404-418):
404TPAPS408 404GPAPS408 12740 14017 14283 404TPKPS408 12498 6587
4289 404TPAPE408 12499 6588 4291
[0133] B. Study of the Incorporation of the Modified Fiber into the
Viral Particle and of its Use in the Entry of the Corresponding
Adenovirus
[0134] In order to be sure that the mutated viruses indeed carry
the modified fiber proteins in their capsid, the viruses purified
after amplification in the 293 cells are loaded onto 10% acrylamide
gel under denaturing conditions (SDS-PAGE). The various proteins
are detected by silver nitrate staining. Alternatively, the fiber
is revealed specifically by carrying out a western blot using a
serum directed against the Ad5 fiber knob (Henry et al., 1994,
above). A strong signal with the expected size indicates that the
viruses incorporate stoichiometric amounts of the protein of
interest. Given that only the trimeric fiber is capable of binding
the penton base (Novelli and Boulanger, 1991, above) and of being
incorporated into the particle, the detection of the protein in the
experiment above indicates that the modified fiber is still capable
of forming trimers.
[0135] Use of the modified fiber to allow the entry of the
corresponding mutated virus can be studied by carrying out the
competition experiments using recombinant knob as described above
in Example 1B. An efficient infection in the presence of saturating
concentrations of the wild-type peptide indicates an infection
independent of the binding to the natural primary receptors. This
suggests a greatly decreased affinity of the modified fiber for its
receptors.
EXAMPLE 6
Insertion of the Ligand Into a Capsid Protein Other than the Fiber,
in Combination with One of the Abovementioned Modifications of the
Fiber
[0136] This example describes the insertion of the EGF ligand into
the hexon capsid protein. Of course, it is preferable for the
corresponding adenovirus to have lost its ability to attach to the
natural cellular receptor. Its genome can, for example, include a
modified fiber gene, or lack a portion, at least, of the fiber
sequences.
[0137] A transfer plasmid for the homologous recombination covering
the region of the Ad5 genome encoding the hexon (nt 18842-21700) is
constructed. The HindIII-XhoI fragment of Ad5 (nt 18836-24816) is
cloned into pBSK+ (Stratagene) digested with these same enzymes, to
give the plasmid pTG4224. The sequences encoding the EGF peptide
are introduced into the L1 hypervariable loop of the hexon by
creating chimeric fragments using PCR: hexon
(nt19043-19647)-XbaI-EGF-BsrGI-hexon (nt19699-20312). The nt19043
to 19647 fragment is obtained by PCR amplification using the
plasmid pTG3602 with the oligonucleotides oTG11102 (SEQ ID NO: 17)
and oTG11103 (SEQ ID NO: 18). The nt19699 to 20312 fragment is
amplified from the same DNA with the oligonucleotides oTG11104 (SEQ
ID NO: 19) and oTG11105 (SEQ ID NO: 20). The EGF is cloned using
the cDNA with the aid of the oligonucleotides oTG11106 (SEQ ID NO:
21) and oTG11107 (SEQ ID NO: 22) allowing the EGF coding sequence
to be placed in frame with the hexon. The PCR products are digested
with the appropriate enzymes and then religated. The chimeric
fragment can then be inserted by homologous recombination into the
plasmid pTG4224 linearized with NdeI (nt 19549), to give pTG4229.
The sequences encoding the modified hexon can be obtained by
HindIII-XhoI digestion and repositioned in their genomic context by
homologous recombination. Use may be made of the vector pTG3602,
pTG4607 or pTG4629 linearized with SgfI, or a vector carrying the
adenoviral genome deleted of the fiber sequences (such as pTG4607
described above) or expressing a modified fiber.
[0138] The adenoviral genome incapable of producing a functional
native fiber is obtained through a deletion which affects the
initiator codon, but which does not extend to the other adenoviral
ORFs. The following is carried out: the adenoviral fragment 5' of
the deletion (nt 30564 to 31041) is amplified by PCR using the
primers oTG7171 and oTG7275 (SEQ ID NO: 23 and 24). The
amplification of the fragment positioned 3' (nt 31129 to 33099)
uses the primers oTG7276 and oTG7049 (SEQ ID NO: 25 and 26). The
PCR fragments are digested with XhoI and ligated before being
introduced by homologous recombination into the vector pTG6591
linearized with NdeI, to give pTG4602. Then, the BstEII fragment
isolated from the latter is subjected to homologous recombination
with the vector pTG3602 digested with SpeI. pTG4607 is obtained.
The vector pTG4629 is equivalent to pTG4607, but also carries the
LacZ expression cassette in place of E1.
[0139] The corresponding viruses can be obtained after transfecting
293 cells or 293 cells expressing the wild-type fiber (Legrand et
al., 1999, above), or cells overexpressing the receptor for EGF.
The study of the specificity of infection may be carried out as
described previously, using EGF as a competitor.
Sequence CWU 1
1
28 1 581 PRT Mastadenovirus 5 Ad5 fiber Position on the map 31063
to 33120 of the Ad5 genome. 1 Met Lys Arg Ala Arg Pro Ser Glu Asp
Thr Phe Asn Pro Val Tyr Pro 1 5 10 15 Tyr Asp Thr Glu Thr Gly Pro
Pro Thr Val Pro Phe Leu Thr Pro Pro 20 25 30 Phe Val Ser Pro Asn
Gly Phe Gln Glu Ser Pro Pro Gly Val Leu Ser 35 40 45 Leu Arg Leu
Ser Glu Pro Leu Val Thr Ser Asn Gly Met Leu Ala Leu 50 55 60 Lys
Met Gly Asn Gly Leu Ser Leu Asp Glu Ala Gly Asn Leu Thr Ser 65 70
75 80 Gln Asn Val Thr Thr Val Ser Pro Pro Leu Lys Lys Thr Lys Ser
Asn 85 90 95 Ile Asn Leu Glu Ile Ser Ala Pro Leu Thr Val Thr Ser
Glu Ala Leu 100 105 110 Thr Val Ala Ala Ala Ala Pro Leu Met Val Ala
Gly Asn Thr Leu Thr 115 120 125 Met Gln Ser Gln Ala Pro Leu Thr Val
His Asp Ser Lys Leu Ser Ile 130 135 140 Ala Thr Gln Gly Pro Leu Thr
Val Ser Glu Gly Lys Leu Ala Leu Gln 145 150 155 160 Thr Ser Gly Pro
Leu Thr Thr Thr Asp Ser Ser Thr Leu Thr Ile Thr 165 170 175 Ala Ser
Pro Pro Leu Thr Thr Ala Thr Gly Ser Leu Gly Ile Asp Leu 180 185 190
Lys Glu Pro Ile Tyr Thr Gln Asn Gly Lys Leu Gly Leu Lys Tyr Gly 195
200 205 Ala Pro Leu His Val Thr Asp Asp Leu Asn Thr Leu Thr Val Ala
Thr 210 215 220 Gly Pro Gly Val Thr Ile Asn Asn Thr Ser Leu Gln Thr
Lys Val Thr 225 230 235 240 Gly Ala Leu Gly Phe Asp Ser Gln Gly Asn
Met Gln Leu Asn Val Ala 245 250 255 Gly Gly Leu Arg Ile Asp Ser Gln
Asn Arg Arg Leu Ile Leu Asp Val 260 265 270 Ser Tyr Pro Phe Asn Ala
Gln Asn Gln Leu Asn Leu Arg Leu Gly Gln 275 280 285 Gly Pro Leu Phe
Ile Asn Ser Ala His Asn Leu Asp Ile Asn Tyr Asn 290 295 300 Lys Gly
Leu Tyr Leu Phe Thr Ala Ser Asn Asn Ser Lys Lys Leu Glu 305 310 315
320 Val Asn Leu Ser Thr Ala Lys Gly Leu Met Phe Asn Ala Thr Ala Ile
325 330 335 Ala Ile Asn Ala Gly Asp Gly Leu Glu Phe Gly Ser Pro Asn
Ala Pro 340 345 350 Asn Thr Asn Pro Leu Lys Thr Lys Ile Gly His Gly
Leu Glu Phe Asp 355 360 365 Ser Asn Lys Ala Met Val Pro Lys Leu Gly
Thr Gly Leu Ser Phe Asp 370 375 380 Ser Thr Gly Ala Ile Thr Val Gly
Asn Lys Asn Asn Asp Lys Leu Thr 385 390 395 400 Leu Trp Thr Thr Pro
Ala Pro Ser Pro Asn Cys Arg Leu Asn Ala Glu 405 410 415 Lys Asp Ala
Lys Leu Thr Leu Val Leu Thr Lys Cys Gly Ser Gln Ile 420 425 430 Leu
Ala Thr Val Ser Val Leu Ala Val Lys Gly Ser Leu Ala Pro Ile 435 440
445 Ser Gly Thr Val Gln Ser Ala His Leu Ile Ile Arg Phe Asp Glu Asn
450 455 460 Gly Val Leu Leu Asn Asn Ser Phe Leu Asp Pro Glu Tyr Trp
Asn Phe 465 470 475 480 Arg Asn Gly Asp Leu Thr Glu Gly Thr Ala Tyr
Thr Asn Ala Val Gly 485 490 495 Phe Met Pro Asn Leu Ser Ala Tyr Pro
Lys Ser His Gly Lys Thr Ala 500 505 510 Lys Ser Asn Ile Val Ser Gln
Val Tyr Leu Asn Gly Asp Lys Thr Lys 515 520 525 Pro Val Thr Leu Thr
Ile Thr Leu Asn Gly Thr Gln Glu Thr Gly Asp 530 535 540 Thr Thr Pro
Ser Ala Tyr Ser Met Ser Phe Ser Trp Asp Trp Ser Gly 545 550 555 560
His Asn Tyr Ile Asn Glu Ile Phe Ala Thr Ser Ser Tyr Thr Phe Ser 565
570 575 Tyr Ile Ala Gln Glu 580 2 60 DNA Artificial Sequence
Primer. Synthetic oligonucleotide oTG7000 (codes for PSASASASAPGS)
2 aacgattctt tagctgccgg gagcagaggc ggaggcggag gcgctgggtt cttgggcaat
60 3 57 DNA Artificial Sequence Primer. Synthetic oligonucleotide
oTG7001 (codes for GRP). 3 aacgattctt tacatcaggt ggcccacagc
ccagtggttt ccgctgccgg gagcaga 57 4 20 DNA Artificial Sequence
Primer. Synthetic oligonucleotide oTG10776. 4 ccttccacgg gaagattgta
20 5 20 DNA Artificial Sequence Primer. Synthetic oligonucleotide
oTG10781. 5 ggggtgtctg tcttcacact 20 6 26 DNA Artificial Sequence
Primer. Synthetic oligonucleotide oTG11065. 6 gggaagcttg aggttaacct
aagcac 26 7 28 DNA Artificial Sequence Primer. Synthetic
oligonculeotide oTG11066. 7 gggtctagag ctgccgggag cagaggcg 28 8 29
DNA Artificial Sequence Primer. Synthetic oligonucleotide oTG11067.
8 gggctcgagt tatgtttcaa cgtgtttat 29 9 24 DNA Artificial Sequence
Primer. Synthetic oligonucleotide oTG11068. 9 gtgcccgggg agtttattaa
tatc 24 10 31 DNA Artificial Sequence Primer. Synthetic
oligonucleotide oTG11069 (EGF cloning) derived from Homo sapiens.
10 gcgtctagaa atagtgactc tgaatgtccc c 31 11 46 DNA Artificial
Sequence Primer. Synthetic oligonucleotide oTG11070 (EGF cloning)
derived from Homo sapiens. 11 gcgctcgagc acaaacgatt ctttagcgca
gttcccacca cttcag 46 12 72 DNA Artificial Sequence Primer.
Synthetic oligunucleotide oTG11992. 12 cataacacaa acgattcttt
atgttcgtgt tggtggttct cgagcgcaat agctgccggg 60 agcagaggcg ga 72 13
57 DNA Artificial Sequence Primer. Synthetic oligonucleotide
oTG11991. 13 cataacacaa acgattcttt aatatacgtc tagatagctg ccgggagcag
aggcgga 57 14 42 DNA Artificial Sequence Primer. Synthetic
oligonucleotide oTG12499 derived from Mastadenovirus. 14 gcatttagtc
tacagttagg ctctggagct ggtgtggtcc ac 42 15 39 DNA Artificial
Sequence Primer. Synthetic oligonucleotide oTG12498 derived from
Mastadenovirus. 15 gtctacagtt aggagatggc tttggtgtgg tccacaaag 39 16
47 DNA Artificial Sequence Primer. Synthetic oligonucleotide
oTG12740 derived from Mastadenovirus. 16 ctacagttag gagatggagc
gggcccggtc cacaaagtta gcttatc 47 17 23 DNA Artificial Sequence
Primer. Synthetic oligonucleotide oTG 11102 (hexon cloning) derived
from Mastadenovirus. 17 cggttcatcc ctgtggaccg tga 23 18 38 DNA
Artificial Sequence Primer. Synthetic oligonucleotide oTG11103
(hexon cloning) derived from Mastadenovirus. 18 ggcctctaga
gttgagaaaa attgcatttc cacttgac 38 19 23 DNA Artificial Sequence
Primer. Synthetic oligonucleotide oTG11104 (hexon cloning) derived
from Mastadenovirus. 19 ggtattgtac agtgaagatg tag 23 20 23 DNA
Artificial Sequence Primer. Synthetic oligonucleotide oTG11105
derived from Mastadenovirus. 20 cgttggaagg actgtacttt agc 23 21 38
DNA Artificial Sequence Primer. Synthetic oligonucleotide oTG11106
(cDNA EGF cloning) derived from Homo sapiens. 21 cgcgtctaga
ggcgaatagt gactctgaat gtcccctg 38 22 45 DNA Artificial Sequence
Primer. Synthetic oligonucleotide oTG11107 (cDNA EGF cloning)
derived from Homo sapiens. 22 ccactgtaca ataccacttt agggcgcagt
tcccaccact tcagg 45 23 21 DNA Artificial Sequence Primer. Synthetic
oligonucleotide oTG7171 (deletion of the fiber) derived from
Mastadenovirus. 23 atggttaact tgcaccagtg c 21 24 27 DNA Artificial
Sequence Primer. Synthetic oligonucleotide oTG7275 (deletion of the
fiber) derived from Mastadenovirus. 24 gggctcgagc tgcaacaaca
tgaagat 27 25 27 DNA Artificial Sequence Primer. Synthetic
oligonucleotide oTG7276 (deletion of the fiber) derived from
Mastadenovirus. 25 ccgctcgaga ctcctccctt tgtatcc 27 26 20 DNA
Artificial Sequence Primer. Synthetic oligonucleotide oTG7049
(deletion of the fiber) derived from Mastadenovirus. 26 ctgcccggga
gtttattaat 20 27 42 DNA Artificial Sequence Primer. Synthetic
oligonucleotide oTG7416 (deletion of pleated sheet H) derived from
Mastadenovirus. 27 tgtttcctgt gtaccgttgg atcctttagt tttgtctccg tt
42 28 64 DNA Artificial Sequence Primer. Synthetic oligonucleotide
oTG10352 (pleated sheet H5 to H3) derived from Mastadenovirus. 28
tgtttcctgt gtaccgttta gcatcacggt cacctcgaga ggtttagttt tgtctccgtt
60 taag 64
* * * * *