U.S. patent application number 11/018669 was filed with the patent office on 2005-08-04 for gene delivery vectors provided with a tissue tropism for smooth muscle cells, and/or endothelial cells.
Invention is credited to Bout, Abraham, Havenga, Menzo J. E., Vogels, Ronald.
Application Number | 20050169891 11/018669 |
Document ID | / |
Family ID | 34828606 |
Filed Date | 2005-08-04 |
United States Patent
Application |
20050169891 |
Kind Code |
A1 |
Vogels, Ronald ; et
al. |
August 4, 2005 |
Gene delivery vectors provided with a tissue tropism for smooth
muscle cells, and/or endothelial cells
Abstract
A gene delivery vehicle having been provided with at least a
tissue tropism for cells selected from the group of smooth muscle
cells, endothelial cells, and/or liver cells. The tissue tropism is
generally provided by a virus capsid, such as one comprising
protein fragments from at least two different viruses, such as two
different adenoviruses, including adenovirus of subgroup C or
subgroup B (for example, adenovirus 16). The protein fragments can
comprise a tissue tropism-determining fragment of a fiber protein
derived from a subgroup B adenovirus. Also, cells for producing
such gene delivery vehicles and pharmaceutical compositions
containing these gene delivery vehicles are provided. Further, a
method is disclosed for delivering nucleic acid to cells such as
smooth muscle cells and/or endothelial cells which involves
administering to the cells an adenovirus capsid having proteins
from at least two different adenoviruses and wherein at least a
tissue tropism-determining fragment of a fiber protein is derived
from a subgroup B adenovirus. Particular constructs are also
disclosed.
Inventors: |
Vogels, Ronald; (Linschoten,
NL) ; Havenga, Menzo J. E.; (Alphen aan de Rijn,
NL) ; Bout, Abraham; (Moerkapelle, NL) |
Correspondence
Address: |
TRASK BRITT
P.O. BOX 2550
SALT LAKE CITY
UT
84110
US
|
Family ID: |
34828606 |
Appl. No.: |
11/018669 |
Filed: |
December 20, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11018669 |
Dec 20, 2004 |
|
|
|
09444284 |
Nov 19, 1999 |
|
|
|
09444284 |
Nov 19, 1999 |
|
|
|
09348354 |
Jul 7, 1999 |
|
|
|
Current U.S.
Class: |
424/93.2 ;
435/456 |
Current CPC
Class: |
C07K 14/005 20130101;
C12N 15/86 20130101; C07K 2319/00 20130101; C12N 2710/10343
20130101; C12N 2810/6018 20130101; A61K 38/00 20130101; C12N
2710/10345 20130101; A61K 48/00 20130101; C12N 2710/10322
20130101 |
Class at
Publication: |
424/093.2 ;
435/456 |
International
Class: |
A61K 048/00; C12N
015/861 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 20, 1998 |
EP |
98203921.6 |
Claims
What is claimed is:
1. A viral gene delivery vehicle comprising at least a tissue
tropism for cells selected from the group consisting of smooth
muscle cells, endothelial cells, or smooth muscle cells and
epothelial cells.
2. The viral gene delivery vehicle of claim 1, wherein said gene
delivery vehicle has been deprived of at least a tissue tropism for
liver cells.
3. The viral gene delivery vehicle of claim 1, wherein said tissue
tropism is provided by a virus capsid.
4. The viral gene delivery vehicle of claim 3, wherein said virus
capsid comprises protein fragments from at least two different
viruses.
5. The viral gene delivery vehicle of claim 4, wherein at least one
of said viruses is an adenovirus.
6. The viral gene delivery vehicle of claim 4, wherein at least one
of said viruses is an adenovirus of subgroup B.
7. The viral gene delivery vehicle of claim 4, wherein at least one
of said protein fragments comprises a tissue tropism-determining
fragment of a fiber protein derived from a subgroup B
adenovirus.
8. The viral gene delivery vehicle of claim 6, wherein said
subgroup B adenovirus is adenovirus 16.
9. The viral gene delivery vehicle of claim 6, wherein protein
fragments not derived from an adenovirus of subgroup B are derived
from an adenovirus of subgroup C.
10. The viral gene delivery vehicle of claim 1, further comprising
a nucleic acid derived from an adenovirus.
11. The viral gene delivery vehicle of claim 1, further comprising
a nucleic acid derived from at least two different
adenoviruses.
12. The viral gene delivery vehicle of claim 10, wherein said
nucleic acid comprises at least one sequence encoding a fiber
protein comprising at least a tissue tropism-determining fragment
of a subgroup B adenovirus fiber protein.
13. The viral gene delivery vehicle of claim 10, wherein said
nucleic acid derived from adenovirus is modified such that the
capacity of said adenovirus nucleic acid to replicate in a target
cell has been reduced or disabled.
14. The viral gene delivery vehicle of claim 10, wherein said
nucleic acid derived from an adenovirus is modified such that a
host immune system's capacity to mount an immune response against
adenoviral proteins encoded by adenoviral nucleic acid has been
reduced or disabled.
15. The viral gene delivery vehicle of claim 1, further comprising
a minimal adenovirus vector or an Ad/AAV chimeric vector.
16. The viral gene delivery vehicle of claim 1, further comprising
at least one non-adenoviral nucleic acid.
17. The viral gene delivery vehicle of claim 16, wherein at least
one of said non-adenoviral nucleic acids is a gene selected from
the group of genes encoding a protein selected from the group
consisting of: an apolipoprotein, a nitric oxide synthase, a herpes
simplex virus thymidine kinase, an interleukin-3, an
interleukin-1.alpha., an (anti) angiogenesis protein, an
anti-proliferation protein, a smooth muscle cell anti-migration
protein, a vascular endothelial growth factor (VGEF), a basic
fibroblast growth factor, a hypoxia inducible factor 1.alpha.
(HIF-1.alpha.) and a PAI-1.
18. The viral gene delivery vehicle of claim 5, wherein at least
one of said viruses is an adenovirus of subgroup B.
19. The viral gene delivery vehicle of claim 7, wherein said
subgroup B adenovirus is adenovirus 16.
20. A recombinant adenovirus of a subgroup C origin having a
reduced tissue tropism for liver cells as compared to the
corresponding wild type adenovirus of subgroup C origin, said
recombinant adenovirus comprising: a capsid comprising a chimeric
fiber protein, wherein a knob domain of said chimeric fiber protein
is of an adenovirus origin selected from the group consisting of
adenovirus 12, adenovirus 16, adenovirus 28 and adenovirus
40-L.
21. A pharmaceutical composition comprising: the recombinant
adenovirus of claim 20; and a suitable vehicle.
22. The recombinant adenovirus of claim 20, wherein said reduced
tissue tropism is provided by a virus capsid.
23. The recombinant adenovirus of claim 22, wherein said virus
capsid comprises protein fragments from at least two different
viruses.
24. The recombinant adenovirus of claim 23, wherein at least one of
said at least two different viruses is an adenovirus.
25. The recombinant adenovirus of claim 24, wherein at least one of
said protein fragments comprises a tissue tropism-determining
fragment of a fiber protein of a subgroup B adenovirus origin.
26. A cell for producing a recombinant adenovirus having a tissue
tropism for smooth muscle cells, said cell comprising: means for
assembling said recombinant adenovirus; wherein said means includes
at least one adenoviral nucleic acid encoding an adenoviral fiber
protein having at least a tissue tropism-determining fragment of a
subgroup B adenoviral fiber protein.
27. The cell of claim 26, wherein said cell is or is derived from a
PER.C6 cell (ECACC deposit number 96022940).
28. An adenovirus capsid having a tissue tropism for smooth muscle
cells and/or endothelial cells wherein said adenovirus capsid
comprises proteins from at least two different adenoviruses and
wherein at least a tissue tropism-determining fragment of a fiber
protein is derived from a subgroup B adenovirus.
29. An adenovirus capsid having a reduced tropism for liver cells
as compared to the corresponding wild type adenovirus capsid, said
adenovirus capsid comprising: a fiber protein of an adenovirus of
subgroup C origin; and wherein at least a knob domain of the fiber
protein is of an adenovirus origin selected from the group
consisting of adenovirus 12, adenovirus 16, adenovirus 28, and
adenovirus 40-L.
30. A method of delivering nucleic acid to cells selected from the
group of cells consisting of smooth muscle cells, endothelial cells
and both smooth muscle and endothelial cells, said method
comprising: administering to said cells an adenovirus capsid
comprising proteins from at least two different adenoviruses,
wherein at least a tissue tropism-determining fragment of a fiber
protein is derived from a subgroup B adenovirus.
31. A recombinant adenovirus of a subgroup C origin having an
increased tropism for smooth muscle cells when compared to an
adenovirus of serotype 5 comprising: a recombinant adenovirus
capsid comprising a fiber protein, wherein at least a knob domain
of the fiber protein is of an adenovirus of a subgroup B origin;
wherein said adenovirus of subgroup B origin is selected from the
group consisting of adenovirus 11, adenovirus 16, adenovirus 35,
and adenovirus 51.
32. The recombinant adenovirus of claim 31, wherein at least one of
said at least two different viruses is an adenovirus.
33. The recombinant adenovirus of claim 31, wherein said subgroup B
adenovirus is adenovirus 16.
34. The recombinant adenovirus of claim 31, wherein at least one of
said peptides is of adenovirus subgroup C origin.
35. The recombinant adenovirus of claim 31, further comprising an
adenoviral nucleic acid incorporated within a genome of said
recombinant adenovirus.
36. The recombinant adenovirus of claim 35, wherein said adenoviral
nucleic acid comprises sequences from at least two different
adenoviruses.
37. The recombinant adenovirus of claim 35, wherein said adenoviral
nucleic acid comprises at least one sequence encoding a fiber
protein having a tissue tropism-determining fragment of a subgroup
B adenovirus fiber protein.
38. The recombinant adenovirus of claim 35, wherein said adenoviral
nucleic acid is modified such that the capacity of said adenoviral
nucleic acid to replicate in a target cell has been reduced or
disabled.
39. The recombinant adenovirus of claim 31, wherein said
recombinant adenovirus is a minimal adenovirus vector or an Ad/AAV
chimeric vector.
40. The recombinant adenovirus of claim 31, further comprising at
least one non-adenoviral nucleic acid incorporated within a genome
of said recombinant adenovirus.
41. The recombinant adenovirus of claim 40, wherein at least one of
said non-adenoviral nucleic acids is a gene encoding a protein
selected from the group of proteins consisting of: an
apolipoprotein, a nitric oxide synthase, a herpes simplex virus
thymidine kinase, an interleukin-3, an interleukin-1.alpha., an
angiogenesis protein, an anti-angiogenesis protein, an
anti-proliferation protein, a smooth muscle cell anti-migration
protein, a vascular endothelial growth factor, a basic fibroblast
growth factor, a hypoxia inducible factor 1.alpha. and a PAI-1.
42. A recombinant adenovirus capsid having an increased tropism for
smooth muscle cells as compared to the corresponding wild type
adenovirus, said recombinant adenovirus capsid comprising: a
chimeric fiber protein, wherein at least a knob domain of the
chimeric fiber protein is of an adenovirus of subgroup B origin;
and wherein the adenovirus of subgroup B origin is selected from
the group consisting of adenovirus 11, adenovirus 16, adenovirus
35, and adenovirus 51; wherein a remaining part of the chimeric
fiber protein is of an adenovirus of a subgroup C origin.
43. A recombinant adenovirus having a capsid with an increased
tropism for smooth muscle cells as compared to the corresponding
wild type adenovirus, said recombinant adenovirus comprising: a
chimeric fiber protein comprising at least the knob domain of a
fiber protein of an adenovirus selected from the group consisting
of adenovirus 11, adenovirus 16, adenovirus 35, and adenovirus 51;
wherein a remaining part of the chimeric fiber protein is of an
adenovirus of a subgroup C origin.
44. The recombinant adenovirus of claim 43, further comprising an
adenoviral nucleic acid incorporated within a genome of said
recombinant adenovirus.
45. The recombinant adenovirus of claim 44, wherein said adenoviral
nucleic acid comprises a sequence encoding the chimeric fiber
protein.
46. The recombinant adenovirus of claim 43, wherein said
recombinant adenovirus is a minimal adenovirus or an Ad/AAV
chimeric vector.
47. The recombinant adenovirus of claim 43, wherein said different
adenovirus serotype is an adenovirus serotype of subgroup C.
48. The recombinant adenovirus of claim 43, wherein said adenovirus
of subgroup C origin is adenovirus serotype 5.
49. A cell for producing a recombinant adenovirus having a tissue
tropism for smooth muscle cells, said cell comprising: means for
assembling the recombinant adenovirus; wherein said means comprises
at least one adenoviral nucleic acid encoding a chimeric adenoviral
fiber protein having at least a knob domain of a fiber protein of
adenovirus serotype 16; and wherein the remaining part of the fiber
protein is of a different adenovirus serotype; and wherein said
cell is of a PER.C6 cell (ECACC deposit number 96022940)
origin.
50. The cell of claim 49, wherein said different adenovirus
serotype is an adenovirus of subgoup C.
51. The cell of claim 50, wherein said adenovirus of subgroup C is
adenovirus serotype 5.
52. A recombinant adenovirus capsid having a reduced tropism for
liver cells as compared to the corresponding wild type adenovirus,
said recombinant adenovirus comprising: a chimeric fiber protein
comprising at least the knob domain of a fiber protein of
adenovirus serotype 16; wherein the remaining part of the fiber
protein is of an adenovirus of a subgroup C origin.
53. The adenovirus capsid of claim 52, wherein said different
adenovirus serotype is an adenovirus serotype of subgroup C.
54. The adenovirus capsid of claim 52, wherein said adenovirus of
subgroup C origin is adenovirus serotype 5.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of co-pending application
Ser. No. 09/444,284, filed Nov. 19, 1999, now U.S. Pat. No. ______,
which is a continuation-in-part of application Ser. No. 09/348,354,
filed Jul. 7, 1999, abandoned, the contents of both of which are
incorporated by this reference.
TECHNICAL FIELD
[0002] The invention relates generally to biotechnology and, more
particularly, to the field of molecular genetics and medicine. In
particular, the present invention relates to the field of gene
therapy and, more particularly, to gene therapy using
adenoviruses.
BACKGROUND
[0003] In gene therapy, genetic information is usually delivered to
a host cell in order to either correct (supplement) a genetic
deficiency in the host cell, or to inhibit an undesired function in
the host cell, or to eliminate the host cell. Of course, the
genetic information can also be intended to provide the host cell
with a desired function, e.g., to supply a secreted protein to
treat other cells of the host, etc.
[0004] Many different methods have been developed to introduce new
genetic information into cells. Although many different systems may
work on cell lines cultured in vitro, only the group of viral
vector mediated gene delivery methods seems to be able to meet the
required efficiency of gene transfer in vivo. Thus, for gene
therapy purposes, most of the attention is directed toward the
development of suitable viral vectors. Today, most of the attention
for the development of suitable viral vectors is directed toward
those vectors based on adenoviruses. These adenovirus vectors can
deliver foreign genetic information very efficiently to target
cells in vivo. Moreover, obtaining large amounts of adenovirus
vectors is for most types of adenovirus vectors not a problem.
Adenovirus vectors are relatively easy to concentrate and purify.
Moreover, studies in clinical trials have provided valuable
information on the use of these vectors in patients.
[0005] A lot of reasons exist for using adenovirus vectors for the
delivery of nucleic acid to target cells in gene therapy protocols.
However, some characteristics of the current vectors limit their
use in specific applications. For instance, endothelial cells and
smooth muscle cells are not easily transduced by the current
generation of adenovirus vectors. For many gene therapy
applications, such as applications in the cardiovascular area,
preferably these types of cells should be genetically modified. On
the other hand, in some applications, even the very good in vivo
delivery capacity of adenovirus vectors is not sufficient and
higher transfer efficiencies are required. This is the case, for
instance, when most cells of a target tissue need to be
transduced.
[0006] The present invention was made in the course of the
manipulation of adenovirus vectors. In the following section,
therefore, a brief introduction to adenoviruses is given.
[0007] Adenoviruses:
[0008] Adenoviruses contain a linear double-stranded DNA molecule
of approximately 36000 base pairs. The DNA molecule contains
identical Inverted Terminal Repeats (ITR) of approximately 90-140
base pairs with the exact length depending on the serotype. The
viral origins of replication are within the ITRs exactly at the
genome ends. The transcription units are divided into early and
late regions. Shortly after infection, the E1A and E1B proteins are
expressed and function in transactivation of cellular and
adenoviral genes. The early regions E2A and E2B encode proteins
(DNA binding protein, pre-terminal protein, and polymerase)
required for the replication of the adenoviral genome (reviewed in
van der Vliet, 1995). The early region E4 encodes several proteins
with pleiotropic functions, e.g., transactivation of the E2 early
promoter, facilitating transport and accumulation of viral mRNAs in
the late phase of infection and increasing nuclear stability of
major late pre-mRNAs (reviewed in Leppard, 1997). The early region
3 encodes proteins that are involved in modulation of the immune
response of the host (Wold et al., 1995). The late region is
transcribed from one single promoter (major late promoter) and is
activated at the onset of DNA replication. Complex splicing and
poly-adenylation mechanisms give rise to more than 12 RNA species
coding for core proteins, capsid proteins (Penton, hexon, fiber and
associated proteins), viral protease and proteins necessary for the
assembly of the capsid and shut-down of host protein translation
(Imperiale, M. J., Akusjnarvi, G. and Leppard, K. N. (1995).
Post-transcriptional control of adenovirus gene expression. In: The
molecular repertoire of adenoviruses I. P139-171. W. Doerfler and
P. Bohm (eds), springer-Verlag Berlin Heidelberg).
[0009] Interaction between Virus and Host Cell:
[0010] The interaction of the virus with the host cell has mainly
been investigated with the serotype C viruses Ad2 and Ad5. Binding
occurs via interaction of the knob region of the protruding fiber
with a cellular receptor. The receptor for Ad2 and Ad5 and probably
more adenoviruses is known as the "Coxsackievirus and Adenovirus
Receptor" or CAR protein (Bergelson et al., 1997). Internalization
is mediated through interaction of the RGD sequence present in the
penton base with cellular integrins (Wickham et al., 1993). This
may not be true for all serotypes, for example, serotypes 40 and 41
do not contain a RGD sequence in their penton base sequence (Kidd
et al., 1993).
[0011] The Fiber Protein:
[0012] The initial step for successful infection is binding of
adenovirus to its target cell, a process mediated through fiber
protein. The fiber protein has a trimeric structure (Stouten et
al., 1992) with different lengths depending on the virus serotype
(Signas et al., 1985; Kidd et al., 1993). Different serotypes have
polypeptides with structurally similar N and C termini, but
different middle stem regions. The first 30 amino acids at the N
terminus are involved in anchoring of the fiber to the penton base
(Chroboczek et al., 1995), especially the conserved FNPVYP (SEQ ID
NO:______) region in the tail (Arnberg et al., 1997). The
C-terminus, or knob, is responsible for initial interaction with
the cellular adenovirus receptor. After this initial binding,
secondary binding between the capsid penton base and cell-surface
integrins leads to internalization of viral particles in coated
pits and endocytosis (Morgan et al., 1969; Svensson and Persson,
1984; Varga et al., 1991; Greber et al., 1993; Wickham et al.,
1993). Integrins are .alpha..beta.-heterodimers of which at least
14 .alpha.-subunits and 8 .beta.-subunits have been identified
(Hynes, 1992). The array of integrins expressed in cells is complex
and will vary between cell types and cellular environment. Although
the knob contains some conserved regions, between serotypes, knob
proteins show a high degree of variability, indicating that
different adenovirus receptors exist.
[0013] Adenoviral Serotypes:
[0014] At present, six different subgroups of human adenoviruses
have been proposed, which in total encompass approximately 50
distinct adenovirus serotypes. Besides these human adenoviruses,
many animal adenoviruses have been identified (see, e.g., Ishibashi
and Yasue, 1984). A serotype is defined on the basis of its
immunological distinctiveness as determined by quantitative
neutralization with animal antiserum (horse, rabbit). If
neutralization shows a certain degree of cross-reaction between two
viruses, distinctiveness of serotype is assumed if A) the
hemagglutinins are unrelated, as shown by lack of cross-reaction on
hemagglutination-inhibition, or B) substantial
biophysical/biochemical differences in DNA exist (Francki et al.,
1991). The serotypes identified last (42-49) were isolated for the
first time from HIV infected patients (Hierholzer et al., 1988;
Schnurr et al., 1993). For reasons not well understood, most of
such immuno-compromised patients shed adenoviruses that were never
isolated from immuno-competent individuals (Hierholzer et al.,
1988, 1992; Khoo et al., 1995).
[0015] Besides differences towards the sensitivity against
neutralizing antibodies of different adenovirus serotypes,
adenoviruses in subgroup C such as Ad2 and Ad5 bind to different
receptors as compared to adenoviruses from subgroup B such as Ad3
and Ad7 (Defer et al., 1990; Gall et al., 1996). Likewise, it was
demonstrated that receptor specificity could be altered by
exchanging the Ad3 knob protein with the Ad 5 knob protein, and
vice versa (Krasnykh et al., 1996; Stevenson et al., 1995, 1997).
Serotypes 2, 4, 5 and 7 all have a natural affiliation towards lung
epithelia and other respiratory tissues. In contrast, serotypes 40
and 41 have a natural affiliation towards the gastrointestinal
tract. These serotypes differ in at least capsid proteins
(penton-base, hexon), proteins responsible for cell binding (fiber
protein), and proteins involved in adenovirus replication. It is
unknown to what extent the capsid proteins determine the
differences in tropism found between the serotypes. It may very
well be that post-infection mechanisms determine cell type
specificity of adenoviruses. It has been shown that adenoviruses
from serotypes A (Ad12 and Ad31), C (Ad2 and Ad5), D (Ad9 and
Ad15), E (Ad4) and F (Ad41) all are able to bind labeled, soluble
CAR (sCAR) protein when immobilized on nitrocellulose. Furthermore,
binding of adenoviruses from these serotypes to Ramos cells, that
express high levels of CAR but lack integrins (Roelvink et al.,
1996), could be efficiently blocked by addition of sCAR to viruses
prior to infection (Roelvink et al., 1998). However, the fact that
(at least some) members of these subgroups are able to bind to CAR
does not exclude that these viruses have different infection
efficiencies in various cell types. For example, subgroup D
serotypes have relatively short fiber shafts compared to subgroup A
and C viruses. It has been postulated that the tropism of subgroup
D viruses is to a large extent determined by the penton base
binding to integrins (Roelvink et al., 1996; Roelvink et al.,
1998). Another example is provided by Zabner et al., 1998 who
tested 14 different serotypes on infection of human ciliated airway
epithelia (CAE) and found that serotype 17 (subgroup D) was bound
and internalized more efficiently than all other viruses, including
other members of subgroup D. Similar experiments using serotypes
from subgroup A-F in primary fetal rat cells showed that
adenoviruses from subgroup A and B were inefficient Whereas viruses
from subgroup D were most efficient (Law et. al, 1998). Also, in
this case, viruses within one subgroup displayed different
efficiencies. The importance of fiber binding for the improved
infection of Ad17 in CAE was shown by Armentano et al. (PCT
International Patent Publication WO 98/22609) who made a
recombinant LacZ Ad2 virus with a fiber gene from Ad17 and showed
that the chimeric virus infected CAE more efficiently than LacZ Ad2
viruses with Ad2 fibers.
[0016] Thus, despite their shared ability to bind CAR, differences
in the length of the fiber, knob sequence and other capsid
proteins, e.g., penton base of the different serotypes may
determine the efficiency by which an adenovirus infects a certain
target cell. Of interest in this respect is the ability of Ad5 and
Ad2 fibers but not of Ad3 fibers to bind to fibronectin III and MHC
class 1 .alpha.2 derived peptides. This suggests that adenoviruses
are able to use cellular receptors other than CAR (Hong et al.,
1997). Serotypes 40 and 41 (subgroup F) are known to carry two
fiber proteins differing in the length of the shaft. The long
shafted 41L fiber is shown to bind to CAR whereas the short shafted
41S is not capable of binding CAR (Roelvink et al., 1998). The
receptor for the short fiber is not known.
[0017] Adenoviral Gene Delivery Vectors:
[0018] Most adenoviral gene delivery vectors currently used in gene
therapy are derived from the serotype C adenoviruses Ad2 or Ad5.
The vectors have a deletion in the E1 region, where novel genetic
information can be introduced. The E1 deletion renders the
recombinant virus replication defective. It has been demonstrated
extensively that recombinant adenovirus, in particular serotype 5,
is suitable for efficient transfer of genes in vivo to the liver,
the airway epithelium and solid tumors in animal models and human
xenografts in immuno-deficient mice (Bout 1996, 1997; Blaese et
al., 1995).
[0019] Gene transfer vectors derived from adenoviruses (adenoviral
vectors) have a number of features that make them particularly
useful for gene transfer:
[0020] 1) the biology of the adenoviruses is well
characterized;
[0021] 2) adenovirus is not generally associated with severe human
pathology;
[0022] 3) the virus is extremely efficient in introducing its DNA
into the host cell;
[0023] 4) the virus can infect a wide variety of cells and has a
broad host-range;
[0024] 5) the virus can be produced at high titers in large
quantities; and
[0025] 6) the virus can be rendered replication defective by
deletion of the early-region 1 (E1) of the viral genome (Brody and
Crystal, 1994).
[0026] However, there is still a number of drawbacks associated
with the use of adenoviral vectors:
[0027] 1) Adenoviruses, especially the well investigated serotypes
Ad2 and Ad5, usually elicit an immune response in the host into
which they are introduced;
[0028] 2) it is currently not feasible to target the virus to
certain cells and tissues;
[0029] 3) the replication and other functions of the adenovirus are
not always very well suited for the cells; which are to be provided
with the additional genetic material; and
[0030] 4) the serotypes Ad2 or Ad5 are not ideally suited for
delivering additional genetic material to organs other than the
liver. The liver can be particularly well transduced with vectors
derived from Ad2 or Ad5.
[0031] Delivery of such vectors via the bloodstream leads to a
significant delivery of the vectors to the cells of the liver. In
therapies where other cell types other than liver cells need to be
transduced some means of liver exclusion must be applied to prevent
uptake of the vector by these cells. Current methods rely on the
physical separation of the vector from the liver cells, most of
these methods rely on localizing the vector and/or the target organ
via surgery, balloon angioplasty or direct injection into an organ
via, for instance, needles. Liver exclusion is also being practiced
through delivery of the vector to compartments in the body that are
essentially isolated from the bloodstream, thereby preventing
transport of the vector to the liver. Although these methods mostly
succeed in avoiding gross delivery of the vector to the liver, most
of the methods are crude and still have considerable leakage and/or
have poor target tissue penetration characteristics. In some cases,
inadvertent delivery of the vector to liver cells can be toxic to
the patient. For instance, delivery of a herpes simplex virus (HSV)
thymidine kinase (TK) gene for the subsequent killing of dividing
cancer cells through administration of gancyclovir is quite
dangerous when also a significant amount of liver cells are
transduced by the vector. Significant delivery and subsequent
expression of the HSV-TK gene to liver cells is associated with
severe toxicity. Thus, a discrete need exists for an inherently
safe vector provided with the property of a reduced transduction
efficiency of liver cells.
BRIEF SUMMARY OF THE INVENTION
[0032] The present invention provides gene therapy methods,
compounds and medicines. The present invention is particularly
useful in gene therapy applications where endothelial cells and/or
smooth muscle cells form the target cell type. The present
invention relates to gene delivery vehicles provided with a tissue
tropism for at least endothelial cells and/or smooth muscle cells.
The present invention further relates to gene delivery vehicles
having been deprived of a tissue tropism for liver cells.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0033] Table I: Oligonucleotides and degenerate oligonucleotides
used for the amplification of DNA encoding fiber proteins derived
from alternative adenovirus serotypes. (Bold letters represent NdeI
restriction site (A-E), NsiI restriction site (1-6, 8), or PacI
restriction site (7).
[0034] Table II: Biodistribution of chimeric adenovirus upon
intravenous tail vein injection. Values represent luciferase
activity/.mu.g of total protein. All values below 200 Relative
light units/.mu.g protein are considered background. ND=not
determined.
[0035] Table III: Expression of CAR and integrins on the cell
surface of endothelial cells and smooth muscle cells. 70%: Cells
harvested for FACS analysis at a cell density of 70% confluency.
100%: Cells harvested for FACS analysis at a cell density of 100%
confluency. PER.C6 cells were taken as a control for antibody
staining. Values represent percentages of cells that express CAR or
either one of the integrins at levels above background. As
background control, HUVECs or HUVsmc were incubated only with the
secondary, rat-anti-mouse IgG1 labeled antibody.
[0036] Table IV: Determination of transgene expression (luciferase
activity) per .mu.g of total cellular protein, after infection of
A549 cells.
[0037] FIG. 1: Schematic drawing of the pBr/Ad.Bam-rITR
construct.
[0038] FIG. 2: Schematic drawing of the strategy used to delete the
fiber gene from the pBr/Ad.Bam-rITR construct.
[0039] FIG. 3: Schematic drawing of construct pBr/Ad.BamR.DELTA.fib
(ECACC deposit number 01121708).
[0040] FIG. 4: Sequences of the chimeric fibers Ad5 (SEQ ID NO:16)
Ad5/12 (SEQ ID NO:17), Ad5/16 (SEQ ID NO:18), Ad5/28 (SEQ ID
NO:19), and Ad5/40-L (SEQ ID NO:20).
[0041] FIG. 5: Schematic drawing of the construct
pClipsal.-Luc.
[0042] FIG. 6: Schematic drawing of the method to generate chimeric
adenoviruses using three overlapping fragments. Early (E) and late
regions (L) are indicated. L5 is the fiber coding sequence.
[0043] FIG. 7: A) Infection of HUVEC cells using different amounts
of virus particles per cell and different fiber chimeric
adenoviruses. Virus concentration: 10000 vp/cell (=white bar), 5000
vp/cell (=grey bar), 2500 vp/cell (=Black bar) 1000 vp/cell (light
grey bar, 250 and 50 vp/cell no detectable luciferase activity
above background. Luciferase activity is expressed in relative
light units (RLU) per microgram of cellular protein. B) Infection
of HUVEC cells using different concentrations of cells (22,500,
45,000, 90,000, or 135,000 cells seeded per well) and either
adenovirus serotype 5 (black bar) or the fiber 16 chimeric
adenovirus (white bar). Luciferase activity is expressed in RLU per
microgram cellular protein. C) Flow cytometric analysis on Human
aorta EC transduced with 500 (Black bar) or 5000 (grey bar) virus
particles per cell of Ad5 or the fiber 16 chimeric virus (Fib16).
Non-infected cells were used to set the background at 1% and a
median fluorescence of 5.4. The maximum shift in the median
fluorescence that can be observed on a flow cytometer is 9999. This
latter indicates that at 5000 Vp/cell both Ad5 and Fib 16 are
outside the sensitivity scale of the flow cytometer.
[0044] FIG. 8: A) Infection of HUVsmc cells using different amounts
of virus particles per cell and different fiber mutant Ad5 based
adenoviruses. Virus concentration: 5000 Vp/cell (=white bar), 2500
vp/cell (=grey bar), 1250 vp/cell (=dark grey bar), 250 vp/cell
(=black bar), or 50 vp/cell (light grey bar). Luciferase activity
is expressed as RLU per microgram cellular protein. B) Infection of
HUVsmc cells using different concentrations of cells (10,000,
20,000, 40,000, 60,000, or 80,000 cells per well) and either
adenovirus serotype 5 (white bars) or the fiber 16 chimeric
adenovirus (black bars). A plateau is observed after infection with
chimeric fiber 16 adenovirus due to the fact that transgene
expression is higher than the sensitivity range of the
bioluminometer used. C) Human umbilical vein SMC transduced with
500 vp/cell (black bar) or 5000 vp/cell (grey bar) using either Ad5
or the fiber 16 mutant (Fib 16). Non-transduced cells were used to
set a background median fluorescence of approximately 1. Shown is
the median fluorescence of GFP expression as measured by flow
cytometry. D) HUVsmc were infected with 312 (light grey bar), 625
(grey bar), 1250 (black bar), 2500 (dark grey bar), 5000 (light
grey bar), or 10,000 (white bar) virus particles per cell of either
the fiber 11, 16, 35, or 51 chimeric virus. Luciferase transgene
expression expressed as RLU per microgram protein was measured 48
hours after virus exposure. E) Macroscopic photographs of LacZ
staining on saphenous samples. Nuclear targeted LacZ (ntLacZ)
yields a deep blue color which appears black or dark grey in
non-color prints. F) Macroscopic photographs of LacZ staining on
pericard samples. Nuclear targeted LacZ (ntLacZ) gives a deep blue
color which appears black in non-color prints. G) Macroscopic
photographs of LacZ staining on right coronary artery samples.
Nuclear targeted LacZ (ntLacZ) gives a deep blue color which
appears black in non-color prints. H) LacZ staining on Left artery
descending (LAD) samples. Nuclear targeted LacZ (ntLacZ) gives a
deep blue color which appears black in non-color prints.
[0045] FIG. 9: Sequences including the gene encoding adenovirus 16
fiber protein as published in Genbank (SEQ ID NO:21) and sequence
including a gene encoding a fiber from an adenovirus 16 variant as
isolated in the present invention, wherein the sequences of the
fiber protein are from the NdeI-site (SEQ ID NO:22). FIG. 9A is a
nucleotide sequence comparison (SEQ ID NOS:21 (upper stand) and 22
(lower strand)). FIG. 9B is an amino-acid comparison (SEQ ID NOS:23
(upper strand) and 24 (lower strand)).
DETAILED DESCRIPTION OF THE INVENTION
[0046] The current invention provides materials and methods to
overcome the limitations of adenoviral vectors mentioned above. In
a broad sense, the invention provides new adenoviruses, derived in
whole or in part from serotypes different from Ad5. Specific genes
of serotypes with preferred characteristics may be combined in a
chimeric vector to give rise to a vector that is better suited for
specific applications. Preferred characteristics include, but are
not limited to, improved infection of a specific target cell,
reduced infection of non-target cells, improved stability of the
virus, reduced uptake in antigen presenting cells (APC), or
increased uptake in APC, reduced toxicity to target cells, reduced
neutralization in humans or animals, reduced or increased CTL
response in humans or animals, better and/or prolonged transgene
expression, increased penetration capacity in tissues, improved
yields in packaging cell lines, etc.
[0047] One aspect of the present invention facilitates the
combination of the low immunogenicity of some adenoviruses with the
characteristics of other adenoviruses that allow efficient gene
therapy. Such characteristics may be a high specificity for certain
host cells, a good replication machinery for certain cells, a high
rate of infection in certain host cells, low infection efficiency
in non-target cells, high or low efficiency of APC infection, etc.
The invention thus may provide chimeric adenoviruses having the
useful properties of at least two adenoviruses of different
serotypes.
[0048] Typically, two or more requirements from the above
non-exhaustive list are required to obtain an adenovirus capable of
efficiently transferring genetic material to a host cell.
Therefore, the present invention provides adenovirus vectors that
can be used as cassettes to insert different adenoviral genes from
different adenoviral serotypes at the required sites. This way, one
can obtain a vector capable of producing a chimeric adenovirus,
whereby of course also a gene of interest can be inserted (for
instance at the site of E1 of the original adenovirus). In this
manner, the chimeric adenovirus to be produced can be adapted to
the requirements and needs of certain hosts in need of gene therapy
for certain disorders. To enable this virus production, a packaging
cell will generally be needed in order to produce sufficient amount
of safe chimeric adenoviruses.
[0049] In one of its aspects, the present invention provides
adenoviral vectors comprising at least a fragment of a fiber
protein of an adenovirus from subgroup B. This fiber protein may be
the native fiber protein of the adenoviral vector or may be derived
from a serotype different from the serotype the adenoviral vector
is based on. In the latter case, the adenoviral vector according to
the invention is a chimeric adenovirus displaying at least a
fragment of the fiber protein derived from subgroup B adenovirus,
wherein the fragment comprises at least the receptor binding
sequence. Typically such a virus will be produced using a vector
(typically a plasmid, a cosmid or baculovirus vector). Such vectors
are also subject of the present invention. A preferred vector is a
vector that can be used to make a chimeric recombinant virus
specifically adapted to the host to be treated and the disorder to
be treated.
[0050] The present invention also provides a chimeric adenovirus
based on adenovirus type 5, but having at least a fragment of the
fiber sequence from adenovirus type 16, wherein the fragment of the
fiber of Ad16 comprises the fragment of the fiber protein that is
involved in binding a host cell. The present invention also
provides chimeric adenoviral vectors that show improved infection
as compared to adenoviruses from other subgroups in specific host
cells, for example, but not limited to, endothelial cells and
smooth muscle cells of human or animal origin. An important feature
of the present invention is the means to produce the chimeric
virus. Typically, one does not want an adenovirus batch to be
administered to the host cell, which contains replication competent
adenovirus. In general, therefore, it is desired to omit one or
more genes from the adenoviral genome on the vector encoding the
chimeric virus and to supply these genes in the genome of the cell
in which the vector is brought to produce chimeric adenovirus. Such
a cell is usually called a "packaging cell." The invention thus
also provides a packaging cell for producing a chimeric adenovirus
according to the invention, comprising, in trans, all elements
necessary for adenovirus production not present on the adenoviral
vector according to the invention. Typically, vector and packaging
cell are adapted to one another in that they have all the necessary
elements, but that they do not have overlapping elements which lead
to replication competent virus by recombination. Thus, the
invention also provides a kit of parts comprising a packaging cell
according to the invention and a recombinant vector according to
the invention wherein there is essentially no sequence overlap
leading to recombination resulting in the production of replication
competent adenovirus between the cell and the vector. For certain
applications, for example when the therapy is aimed at eradication
of tumor cells, adenoviral vector according to the invention may be
replication competent or capable of replicating under certain
conditions, for example, in specific cell types like tumor cells or
tumor endothelial cells.
[0051] It is within the scope of the invention to insert more
genes, or a functional part of these genes from the same or other
serotypes into the adenoviral vector replacing the corresponding
native sequences. Thus, for example, replacement of (or a
functional part of the) fiber sequences with corresponding
sequences of other serotypes may be combined with, for example,
replacements of (or a functional part of) other capsid genes like
penton base or hexon with corresponding sequences of the serotype
or of other distinct serotypes. Persons skilled in the art will
understand that other combinations not limited to genes are
possible and within the scope of the invention.
[0052] The chimeric adenoviral vector according to the invention
may originate from at least two different serotypes. This may
provide the vector with preferred characteristics such as improved
infection of target cells and/or less infection of non-target
cells, improved stability of the virus, reduced immunogenicity in
humans or animals (e.g., reduced uptake in APC, reduced
neutralization in the host and/or reduced cytotoxic T-lymphocyte
(CTL) response), increased penetration of tissue, better longevity
of transgene expression, etc. In this aspect, it is preferred to
use capsid genes, e.g., penton and/or hexon genes from less
immunogenic serotypes as defined by the absence or the presence of
low amounts of neutralizing antibodies in the vast majority of
hosts.
[0053] It is also preferred to use fiber and/or penton sequences
from serotypes that show improved binding and internalization in
the target cells. Furthermore, it is preferred to delete from the
viral vector those genes which lead to expression of adenoviral
genes in the target cells. In this aspect, a vector deleted of all
adenoviral genes is also preferred. Furthermore, it is preferred
that the promoter driving the gene of interest to be expressed in
the target cells is a cell type specific promoter.
[0054] In order to be able to precisely adapt the viral vector and
provide the chimeric virus with the desired properties at will, it
is preferred that a library of adenoviral genes be provided wherein
the genes to be exchanged are located on plasmid- or cosmid-based
adenoviral constructs wherein the genes or the sequences to be
exchanged are flanked by restriction sites. The preferred genes or
sequences can be selected from the library and inserted in the
adenoviral constructs that are used to generate the viruses.
Typically, such a method comprises a number of restriction and
ligation steps and transfection of a packaging cell. The adenoviral
vector can be transfected in one piece, or as two or more
overlapping fragments, wherein viruses are generated by homologous
recombination. For example, the adenoviral vector may be built up
from two or more overlapping sequences for insertion or
replacements of a gene of interest in, for example, the E1 region,
for insertion or replacements in penton and/or hexon sequences, and
for insertions or replacements into fiber sequences. Thus, the
invention provides a method for producing chimeric adenoviruses
having one or more desired properties like a desired host range and
diminished antigenicity, comprising providing one or more vectors
according to the invention having the desired insertion sites,
inserting into these vectors at least a functional part of a fiber
protein derived from an adenovirus serotype having the desired host
range and/or inserting a functional part of a capsid protein
derived from an adenovirus serotype having a relatively low
antigenicity and transfecting these vectors in a packaging cell
according to the invention and allowing for production of chimeric
viral particles. Of course, other combinations of other viral genes
originating from different serotypes can also be inserted as
disclosed herein before. Chimeric viruses having only one
non-native sequence in addition to an insertion or replacement of a
gene of interest in the E1 region, are also within the scope of the
invention.
[0055] An immunogenic response to adenovirus that typically occurs
is the production of neutralizing antibodies by the host. This
response is typically a reason for selecting a capsid protein like
penton, hexon and/or fiber of a less immunogenic serotype.
[0056] Of course, it may not be necessary to make chimeric
adenoviruses that have complete proteins from different serotypes.
It is well within the skill of the art to produce chimeric
proteins, for instance in the case of fiber proteins it is very
well possible to have the base of one serotype and the shaft and
the knob from another serotype. In this manner, it becomes possible
to have the parts of the protein responsible for assembly of viral
particles originate from one serotype, thereby enhancing the
production of intact viral particles. Thus, the invention also
provides a chimeric adenovirus wherein the hexon, penton, fiber
and/or other capsid proteins are chimeric proteins originating from
different adenovirus serotypes. Besides generating chimeric
adenoviruses by swapping entire wild type capsid (protein) genes
etc. or parts thereof, it is also within the scope of the present
invention to insert capsid (protein) genes etc. carrying
non-adenoviral sequences or mutations, such as point mutations,
deletions, insertions, etc., which can be easily screened for
preferred characteristics such as temperature stability, assembly,
anchoring, redirected infection, altered immune response etc.
Again, other chimeric combinations can also be produced and are
within the scope of the present invention.
[0057] It has been demonstrated in mice and rats that upon in vivo
systemic delivery of recombinant adenovirus of commonly used
serotypes for gene therapy purposes more than 90% of the virus is
trapped in the liver, (Herz et al., 1993; Kass-Eisler et al., 1994;
Huard et al., 1995). It is also known that human hepatocytes are
efficiently transduced by adenovirus serotype 5 vectors (Castell,
J. V., Hernandez, D. Gomez-Foix, A. M., Guillen, I, Donato, T. and
Gomez-Lechon, M. J. (1997). Adenovirus-mediated gene transfer into
human hepatocytes: analysis of the biochemical functionality of
transduced cells. Gene Ther. 4 (5), p 455-464). Thus, in vivo gene
therapy by systemic delivery of Ad2 or Ad5 based vectors is
seriously hampered by the efficient uptake of the viruses in the
liver leading to unwanted toxicity and less virus being available
for transduction of the target cells. Therefore, alteration of the
adenovirus serotype 5 host cell range to be able to target other
organs in vivo is a major interest of the invention.
[0058] To obtain redirected infection of recombinant adenovirus
serotype 5, several approaches have been or still are under
investigation. Wickham et al. have altered the RGD (Arg, Gly, Asp)
motif in the penton base which is believed to be responsible for
the .alpha..beta.3 and .alpha..beta.5 integrin binding to the
penton base. They have replaced this RGD motif by another peptide
motif which is specific for the .alpha..sub.4.beta..sub.1 receptor.
In this way, targeting the adenovirus to a specific target cell
could be accomplished (Wickham et al., 1995). Krasnykh et al.
(1998) have made use of the HI loop available in the knob. This
loop is, based on X-ray crystallography, located on the outside of
the knob trimeric structure and therefore is thought not to
contribute to the intramolecular interactions in the knob.
Insertion of a FLAG coding sequence into the HI loop resulted in
fiber proteins that were able to trimerize and it was further shown
that viruses containing the FLAG sequence in the knob region could
be made. Although interactions of the FLAG-containing knob with CAR
are not changed, insertion of ligands in the HI loop may lead to
retargeting of infection. Although successful introduction of
changes in the adenovirus serotype 5 fiber and penton-base have
been reported, the complex structure of knob and the limited
knowledge of the precise amino acids interacting with CAR render
such targeting approaches laborious and difficult. The use of
antibodies binding to CAR and to a specific cellular receptor has
also been described (Wickham et al., 1996; Rogers et al., 1997).
This approach is however limited by the availability of specific
antibody and by the complexity of the gene therapy product.
[0059] To overcome the limitations described above, we used
pre-existing adenovirus fibers, penton base proteins, hexon
proteins or other capsid proteins derived from other adenovirus
serotypes. By generating chimeric adenovirus serotype 5 libraries
containing structural proteins of alternative adenovirus serotypes,
we have developed a technology, which enables rapid screening for a
recombinant adenoviral vector with preferred characteristics.
[0060] The present invention provides methods for the generation of
chimeric capsids which can be targeted to specific cell types in
vitro as well as in vivo, and thus have an altered tropism for
certain cell types. The present invention further provides methods
and means by which an adenovirus or an adenovirus capsid can be
used as a protein or nucleic acid delivery vehicle to a specific
cell type or tissue.
[0061] The generation of chimeric adenoviruses based on adenovirus
serotype 5 with modified late genes is described. For this purpose,
three plasmids, which together contain the complete adenovirus
serotype 5 genome, were constructed. From one of these plasmids
part of the DNA encoding the adenovirus serotype 5 fiber protein
was removed and replaced by linker DNA sequences that facilitate
easy cloning. This plasmid subsequently served as template for the
insertion of DNA encoding fiber protein derived from different
adenovirus serotypes. The DNA sequences derived from the different
serotypes were obtained using the polymerase chain reaction
technique in combination with (degenerate) oligonucleotides. At the
former E1 location in the genome of adenovirus serotype 5, any gene
of interest can be cloned. A single transfection procedure of the
three plasmids together results in the formation of a recombinant
chimeric adenovirus. Alternatively, cloning of the sequences
obtained from the library of genes can be such that the chimeric
adenoviral vector is built up from one or two fragments. For
example, one construct contains at least the left ITR and sequences
necessary for packaging of the virus, an expression cassette for
the gene of interest and sequences overlapping with the second
construct comprising all sequences necessary for replication and
virus formation not present in the packaging cell as well as the
non-native sequences providing the preferred characteristics. This
new technology of libraries consisting of chimeric adenoviruses
thus allows for a rapid screening improved recombinant adenoviral
vectors for in vitro and in vivo gene therapy purposes.
[0062] The use of adenovirus type 5 for in vivo gene therapy is
limited by the apparent inability to infect certain cell types,
e.g., human endothelial cells and smooth muscle cells and the
preference of infection of certain organs, e.g., liver and spleen.
Specifically, this has consequences for treatment of cardiovascular
diseases like restenosis and pulmonary hypertension.
Adenovirus-mediated delivery of human ceNOS (constitutive
endothelial nitric oxide synthase) has been proposed as treatment
for restenosis after percutaneous transluminal coronary angioplasty
(PTCA). Restenosis is characterized by progressive arterial
remodeling, extracellular matrix formation and intimal hyperplasia
at the site of angioplasty (Schwartz et al., 1993; Carter et al.,
1994; Shi et al., 1996). NO is one of the vasoactive factors shown
to be lost after PTCA-induced injury to the endothelial barrier
(Lloyd Jones and Bloch, 1996). Thus, restoration of NO levels after
balloon-induced injury by means of adenoviral delivery of ceNOS may
prevent restenosis (Varenne et al., 1998). Other applications for
gene therapy whereby the viruses or chimeric viruses according to
the invention are superior to Ad2 or Ad5 based viruses, given as
non-limiting examples, are production of proteins by endothelial
cells that are secreted into the blood, treatment of hypertension,
preventive treatment of stenosis during vein grafting,
angiogenesis, heart failure, renal hypertension and others.
[0063] In one embodiment, this invention includes adenoviral
vectors that are, amongst others, especially suited for gene
delivery to endothelial cells and smooth muscle cells important for
treatment of cardiovascular disorders. The adenoviral vectors
preferably are derived from subgroup B adenoviruses or contain at
least a functional part of the fiber protein from an adenovirus
from subgroup B comprising at least the cell-binding moiety of the
fiber protein. In a further preferred embodiment, the adenoviral
vectors are chimeric vectors based on adenovirus type 5 and contain
at least a functional part of the fiber protein from adenovirus
type 16.
[0064] In another embodiment, this invention provides adenoviral
vectors or chimeric adenoviral vectors that escape the liver
following systemic administration. Preferably, these adenoviral
vectors are derived from subgroup A, B, D, or F in particular
serotypes 12, 16, 28 and 40 or contain at least the cell-binding
moiety of the fiber protein derived from the adenoviruses.
[0065] It is to be understood that in all embodiments, the
adenoviral vectors may be derived from the serotype having the
desired properties or that the adenoviral vector is based on an
adenovirus from one serotype and contains the sequences comprising
the desired functions of another serotype, these sequences
replacing the native sequences in the serotype.
[0066] In another aspect, this invention describes chimeric
adenoviruses and methods to generate these viruses that have an
altered tropism different from that of adenovirus serotype 5. For
example, viruses based on adenovirus serotype 5 but displaying any
adenovirus fiber existing in nature. This chimeric adenovirus
serotype 5 is able to infect certain cell types more efficiently,
or less efficiently in vitro and in vivo than the adenovirus
serotype 5. Such cells include, but are not limited to, endothelial
cells, smooth muscle cells, dendritic cells, neuronal cells, glial
cells, synovical cells, lung epithelia cells, hemopoietic stem
cells, monocytic/macrophage cells, tumor cells, skeletal muscle
cells, mesothelial cells, synoviocytes, etc.
[0067] In another aspect, the invention describes the construction
and use of libraries consisting of distinct parts of adenovirus
serotype 5 in which one or more genes or sequences have been
replaced with DNA derived from alternative human or animal
serotypes. This set of constructs, in total encompassing the
complete adenovirus genome, allows for the construction of unique
chimeric adenoviruses customized for a certain disease, group of
patients or even a single individual.
[0068] In all aspects of the invention, the chimeric adenoviruses
may, or may not, contain deletions in the E1 region and insertions
of heterologous genes linked either or not to a promoter.
Furthermore, chimeric adenoviruses may, or may not, contain
deletions in the E3 region and insertions of heterologous genes
linked to a promoter. Furthermore, chimeric adenoviruses may, or
may not, contain deletions in the E2 and/or E4 region and
insertions of heterologous genes linked to a promoter. In the
latter case, E2 and/or E4 complementing cell lines are used to
generate recombinant adenoviruses. In fact, any gene in the genome
of the viral vector can be taken out and supplied in trans. Thus,
in the extreme, chimeric viruses do not contain any adenoviral
genes in their genome and are by definition situation minimal
adenoviral vectors. In this case all adenoviral functions are
supplied in trans using stable cell lines and/or transient
expression of these genes. A method for producing minimal
adenoviral vectors is described in PCT International Publication
WO97/00326 and is incorporated by reference herein. In another case
Ad/AAV chimeric molecules are packaged into the adenovirus capsids
of the invention. A method for producing Ad/AAV chimeric vectors is
described in European Patent Office publication EP 1 042 494 and is
incorporated by reference herein. In principle, any nucleic acid
may be provided with the adenovirus capsids of the invention.
[0069] In one embodiment, the invention provides a gene delivery
vehicle having been provided with at least a tissue tropism for
smooth muscle cells and/or endothelial cells. In another
embodiment, the invention provides a gene delivery vehicle deprived
of a tissue tropism for at least liver cells. Preferably, the gene
delivery vehicle is provided with a tissue tropism for at least
smooth muscle cells and/or endothelial cells and deprived of a
tissue tropism for at least liver cells. In a preferred embodiment,
the gene delivery vehicle is provided with a tissue tropism for at
least smooth muscle cells and/or endothelial cells and/or deprived
of a tissue tropism for at least liver cells using a fiber protein
derived from a subgroup B adenovirus, preferably of adenovirus 16.
In a preferred aspect of the invention, the gene delivery vehicle
comprises a virus capsid. Preferably, this virus capsid comprises a
virus capsid derived in whole or in part from an adenovirus of
subgroup B, preferably from adenovirus 16, or it comprises
proteins, or parts thereof, from an adenovirus of subgroup B,
preferably of adenovirus 16. In a preferred embodiment, the virus
capsid comprises proteins, or fragments thereof, from at least two
different viruses, preferably adenoviruses. In a preferred
embodiment of this aspect of the invention, at least one of the
viruses is an adenovirus of subgroup B, preferably adenovirus
16.
[0070] In a preferred embodiment, the gene delivery vehicle
comprises an adenovirus fiber protein or fragments thereof. The
fiber protein is preferably derived from an adenovirus of subgroup
B, preferably adenovirus 16. The gene delivery vehicle may further
comprise other fiber proteins, or fragments thereof, from other
adenoviruses. The gene delivery vehicle may or may not comprise
other adenovirus proteins. Nucleic acid may be linked directly to
fiber proteins, or fragments thereof, but may also be linked
indirectly. Examples of indirect linkages include, but are not
limited to, packaging of nucleic acid into adenovirus capsids or
packaging of nucleic acid into liposomes, wherein a fiber protein,
or a fragment thereof, is incorporated into an adenovirus capsid or
linked to a liposome. Direct linkage of nucleic acid to a fiber
protein, or fragment thereof, may be performed when the fiber
protein, or a fragment thereof, is not part of a complex or when
the fiber protein or a fragment thereof, is part of a complex, such
as an adenovirus capsid.
[0071] In one embodiment, a gene delivery vehicle is provided
comprising an adenovirus fiber protein wherein the fiber protein
comprises a tissue-determining fragment of an adenovirus of
subgroup B adenovirus preferably of adenovirus 16. Adenovirus fiber
protein comprises three functional domains. One domain, the base,
is responsible for anchoring the fiber to a penton base of the
adenovirus capsid. Another domain, the knob, is responsible for
receptor recognition whereas the shaft domain functions as a spacer
separating the base from the knob. The different domains may also
have other functions. For instance, the shaft is presumably also
involved in target cell specificity. Each of the domains mentioned
above may be used to define a fragment of a fiber. However,
fragments may also be identified in another way. For instance, the
knob domain comprises a receptor binding fragment and a shaft
binding fragment. The base domain comprises of a penton base
binding fragment and a shaft binding fragment. Moreover, the shaft
comprises repeated stretches of amino acids. Each of these repeated
stretches may be a fragment.
[0072] Fiber proteins possess tissue tropism-determining
properties. The most well described fragment of the fiber protein
involved in tissue tropism is the knob domain. However, the shaft
domain of the fiber protein also possesses tissue
tropism-determining properties. However, not all of the tissue
tropism-determining properties of an adenovirus capsid are
incorporated into a fiber protein.
[0073] A "tissue tropism-determining fragment" of a fiber protein
may be a single fragment of a fiber protein or a combination of
fragments of at least one fiber protein, wherein the tissue
tropism-determining fragment, either alone or in combination with a
virus capsid, determines the efficiency with which a gene delivery
vehicle can transduce a given cell or cell type, preferably but not
necessarily in a positive way. With a "tissue tropism for liver
cells" is meant a tissue tropism for cells residing in the liver,
preferably liver parenchyma cells.
[0074] A tissue tropism for a certain tissue may be provided by
increasing the efficiency with which cells of the tissue are
transduced, alternatively, a tissue tropism for a certain tissue
may be provided by decreasing the efficiency with which other cells
than the cells of the tissue are transduced.
[0075] In a preferred embodiment, a fiber protein derived from a
subgroup B adenovirus, preferably adenovirus 16, is combined with
the non-fiber capsid proteins from an adenovirus of subgroup C,
preferably of adenovirus 5.
[0076] In one aspect of the invention, a gene delivery vehicle is
provided comprising a nucleic acid derived from an adenovirus. In a
preferred embodiment of the invention the adenovirus nucleic acid
comprises at least one nucleic acid sequence encoding a fiber
protein comprising at least a tissue tropism-determining fragment
of a subgroup B adenovirus fiber protein, preferably of adenovirus
16. In a preferred aspect, the adenovirus comprises nucleic acid
from at least two different adenoviruses. In a preferred aspect,
the adenovirus comprises nucleic acid from at least two different
adenoviruses wherein at least one nucleic acid sequence encoding a
fiber protein comprises at least a tissue tropism-determining
fragment of a subgroup B adenovirus fiber protein, preferably of
adenovirus 16.
[0077] In a preferred embodiment, the adenovirus nucleic acid is
modified such that the capacity of the adenovirus nucleic acid to
replicate in a target cell has been reduced or disabled. This may
be achieved through, for example, inactivating or deleting genes
encoding early region proteins.
[0078] In another preferred embodiment, the adenovirus nucleic acid
is modified such that the capacity of a host immune system to mount
an immune response against adenovirus proteins encoded by the
adenovirus nucleic acid has been reduced or disabled. This may be
achieved through deletion of genes encoding proteins of early
region 2 and/or early region 4. Alternatively, genes encoding early
region 3 proteins may be deleted, or on the contrary, considering
the anti-immune system function of some of the proteins encoded by
the genes in early region 3, the expression of early region 3
proteins may be enhanced for some purposes. Also, the adenovirus
nucleic acid may be altered by a combination of two or more of the
specific alterations of the adenovirus nucleic acid mentioned
above. It is clear that when essential genes are deleted from the
adenovirus nucleic acid, the genes must be complemented in the cell
that is going to produce the adenovirus nucleic acid, the
adenovirus vector, the vehicle or the chimeric capsid. The
adenovirus nucleic acid may also be modified such that the capacity
of a host immune system to mount an immune response against
adenovirus proteins encoded by the adenovirus nucleic acid has been
reduced or disabled, in other ways than mentioned above, for
instance through exchanging capsid proteins, or fragments thereof,
by capsid proteins, or fragments thereof, from other serotypes for
which humans do not have, or have low levels of, neutralizing
antibodies. Another example of this is the exchange of genes
encoding capsid proteins with the genes encoding for capsid
proteins from other serotypes. Also capsid proteins, or fragments
thereof, may be exchanged for other capsid proteins, or fragments
thereof, for which individuals are not capable of, or have a low
capacity of, raising an immune response against.
[0079] An adenovirus nucleic acid may be altered further or instead
of one or more of the alterations mentioned above, by inactivating
or deleting genes encoding adenovirus late proteins such as, but
not limited to, hexon, penton, fiber and/or protein IX.
[0080] In a preferred embodiment of the invention all genes
encoding adenovirus proteins are deleted from the adenovirus
nucleic acid, turning the nucleic acid into a minimal adenovirus
vector.
[0081] In another preferred embodiment of the invention, the
adenovirus nucleic acid is an Ad/AAV chimeric vector, wherein at
least the integration means of an adeno-associated virus (AAV) is
incorporated into the adenovirus nucleic acid.
[0082] In a preferred embodiment, a vector or a nucleic acid, which
may or may not be one and the same according to the invention,
further comprises at least one non-adenovirus gene. Preferably, at
least one of the non-adenovirus genes is selected from the group of
genes encoding: an apolipoprotein, a ceNOS, a herpes simplex virus
thymidine kinase, an interleukin-3, an interleukin-1.alpha., an
(anti) angiogenesis protein such as angiostatin, an
anti-proliferation protein, a vascular endothelial growth factor
(VGEF), a basic fibroblast growth factor (bFGF), a hypoxia
inducible factor 1.alpha. (HIF-1.alpha.), a PAI-1 and a smooth
muscle cell anti-migration protein.
[0083] In another aspect, the invention provides a cell for the
production of a gene delivery vehicle provided with at least a
tissue tropism for smooth muscle cells and/or endothelial cells. In
another aspect, the invention provides a cell for the production of
a gene delivery vehicle deprived of at least a tissue tropism for
liver cells. In another aspect, the invention provides a cell for
the production of a gene delivery vehicle provided with at least a
tissue tropism for smooth muscle cells and/or endothelial cells and
deprived of at least a tissue tropism for liver cells. In a
preferred embodiment, the cell is an adenovirus packaging cell,
wherein an adenovirus nucleic acid is packaged into an adenovirus
capsid. In one aspect of an adenovirus packaging cell of the
invention all proteins required for the replication and packaging
of an adenovirus nucleic acid, except for the proteins encoded by
early region 1, are provided by genes incorporated in the
adenovirus nucleic acid. The early region 1 encoded proteins in
this aspect of the invention may be encoded by genes incorporated
into the cells genomic DNA. In a preferred embodiment of the
invention said cell is PER.C6 (ECACG deposit number 96022940). In
general, when gene products required for the replication and
packaging of adenovirus nucleic acid into adenovirus capsid are not
provided by an adenovirus nucleic acid, they are provided by the
packaging cell, either by transient transfection, or through stable
transformation of said packaging cell. However, a gene product
provided by the packaging cell may also be provided by a gene
present on said adenovirus nucleic acid. For instance, fiber
protein may be provided by the packaging cell, for instance through
transient transfection, and may be encoded by the adenovirus
nucleic acid. This feature can among others be used to generate
adenovirus capsids comprising of fiber proteins from two different
viruses.
[0084] The gene delivery vehicles of the invention are useful for
the treatment of cardiovascular disease or disease treatable by
nucleic acid delivery to endothelial cells or smooth muscle cells.
A non-limiting example of the latter is for instance cancer, where
the nucleic acid transferred comprises a gene encoding an
anti-angiogenesis protein.
[0085] The gene delivery vehicles of the invention may be used as a
pharmaceutical for the treatment of diseases. Alternatively, gene
delivery vehicles of the invention may be used for the preparation
of a medicament for the treatment of diseases.
[0086] In one aspect, the invention provides an adenovirus capsid
with or provided with a tissue tropism for smooth muscle cells
and/or endothelial cells wherein the capsid preferably comprises
proteins from at least two different adenoviruses and wherein at
least a tissue tropism-determining fragment of a fiber protein is
derived from a subgroup B adenovirus, preferably of adenovirus 16.
In another aspect, the invention provides an adenovirus capsid
deprived of a tissue tropism for liver cells wherein the capsid
preferably comprises proteins from at least two different
adenoviruses and wherein at least a tissue tropism-determining
fragment of a fiber protein is derived from a subgroup B
adenovirus, preferably of adenovirus 16.
[0087] In one embodiment, the invention comprises the use of an
adenovirus capsid, for the delivery of nucleic acid to smooth
muscle cells and/or endothelial cells. In another embodiment, the
invention comprises the use of an adenovirus capsid for preventing
the delivery of nucleic acid to liver cells.
[0088] The adenovirus capsids of the invention may be used for the
treatment of cardiovascular disease or a disease treatable by
nucleic acid delivery to endothelial cells or smooth muscle cells.
An example of the latter is, for instance, cancer where the nucleic
acid transferred comprises a gene encoding an anti-angiogenesis
protein.
[0089] The adenovirus capsids of the invention may be used as a
pharmaceutical for the treatment of diseases. Alternatively,
adenovirus capsids of the invention may be used for the preparation
of a medicament for the treatment of diseases.
[0090] In another aspect of the invention, construct
pBr/Ad.BamR.DELTA.fib (ECACC deposit number 01121708, deposited on
Dec. 12, 2001 with the Centre for Applied Microbiology and Research
Authority (European Collection of Animal Cell Cultures), Porton
Down, Salisbury, Wiltshire SP4, OJG, United Kingdom, an
International Depository Authority, in accordance with the Budapest
Treaty is provided, comprising adenovirus 5 sequences 21562-31094
and 32794-35938.
[0091] In another aspect of the invention, construct
pBr/AdBamRfib16 (ECACC deposit number 01121710, deposited on Dec.
12, 2001 with the Centre for Applied Microbiology and Research
Authority (European Collection of Animal Cell Cultures), Porton
Down, Salisbury, Wiltshire SP4, OJG, United Kingdom, an
International Depository Authority, in accordance with the Budapest
Treaty is provided, comprising adenovirus 5 sequences 21562-31094
and 32794-3598, further comprising an adenovirus 16 gene encoding
fiber protein.
[0092] In yet another aspect of the invention, construct
pBr/AdBamR.pac/fib16 (ECACC deposit number 01121709, deposited on
Dec. 12, 2001 with the Centre for Applied Microbiology and Research
Authority (European Collection of Animal Cell Cultures), Porton
Down, Salisbury, Wiltshire SP4, OJG, United Kingdom, an
International Depository Authority, in accordance with the Budapest
Treaty, comprising adenovirus 5 sequences 21562-31094 and
32794-35938 is provided, comprising an adenovirus 16 gene encoding
fiber protein, and further comprising a unique PacI-site in the
proximity of the adenovirus 5 right terminal repeat, in the
non-adenovirus sequence backbone of the construct.
[0093] In another aspect of the invention, construct
pWE/Ad.AflIIrITRfib16 (ECACC deposit number 01121711, deposited on
Dec. 12, 2001 with the Centre for Applied Microbiology and Research
Authority (European Collection of Animal Cell Cultures), Porton
Down, Salisbury, Wiltshire SP4, OJG, United Kingdom, an
International Depository Authority is provided, in accordance with
the Budapest Treaty comprising Ad5 sequence 3534-31094 and
32794-35938, further comprising an adenovirus 16 gene encoding
fiber protein.
[0094] In another aspect of the invention, construct
pWE/Ad.AflIIrITRDE2Afib16 (ECACC deposit number 01121712, deposited
on Dec. 12, 2001 with the Centre for Applied Microbiology and
Research Authority (European Collection of Animal Cell Cultures),
Porton Down, Salisbury, Wiltshire SP4, OJG, United Kingdom, an
International Depository Authority, in accordance with the Budapest
Treaty, is provided comprising Ad5 sequences 3534-22443 and
24033-31094 and 32794-35938, further comprising an adenovirus 16
gene encoding fiber protein.
[0095] In the numbering of the sequences mentioned above, the
number is depicted until and not until plus.
[0096] In a preferred embodiment, the constructs are used for the
generation of a gene delivery vehicle or an adenovirus capsid with
a tissue tropism for smooth muscle cells and/or endothelial
cells.
[0097] In another aspect, the invention provides a library of
adenovirus vectors, or gene delivery vehicles which may be one and
the same or not, comprising a large selection of non-adenovirus
nucleic acids. In another aspect of the invention, adenovirus genes
encoding capsid proteins are used to generate a library of
adenovirus capsids comprising proteins derived from at least two
different adenoviruses, the adenoviruses preferably being derived
from two different serotypes, wherein preferably one serotype is an
adenovirus of subgroup B. In a particularly preferred embodiment, a
library of adenovirus capsids is generated comprising proteins from
at least two different adenoviruses and wherein at least a tissue
tropism-determining fragment of fiber protein is derived from an
adenovirus of subgroup B, preferably of adenovirus 16.
[0098] A fiber protein of adenovirus 16 preferably comprises the
sequence given in FIG. 9. However, within the scope of the present
invention analogous sequences may be obtained by using codon
degeneracy. Alternatively, amino-acid substitutions or insertions
or deletions may be performed as long as the tissue
tropism-determining property is not significantly altered. Such
amino-acid substitutions may be within the same polarity group or
without.
[0099] In the following, the invention is illustrated by a number
of non-limiting examples.
EXAMPLES
Example 1
Generation of Adenovirus Serotype 5 Based Viruses with Chimeric
Fiber Proteins
[0100] Generation of Adenovirus Template Clones Lacking DNA
Encoding for Fiber
[0101] The fiber coding sequence of adenovirus serotype 5 is
located between nucleotides 31042 and 32787. To remove the
adenovirus serotype 5 DNA encoding fiber we started with construct
pBr/Ad.Bam-rITR (FIG. 1; ECACC deposit P97082122). From this
construct a NdeI site was first removed. For this purpose, pBr322
plasmid DNA was digested with NdeI after which protruding ends were
filled using Klenow enzyme. This pBr322 plasmid was then
re-ligated, digested with NdeI and transformed into E. coli
DH5.alpha.. The obtained pBr/.DELTA.NdeI plasmid was digested with
ScaI and SalI and the resulting 3198 bp vector fragment was ligated
to the 15349 bp ScaI-SalI fragment derived from pBr/Ad.Bam-rITR,
resulting in plasmid pBr/Ad.Bam-rITR.DELTA.NdeI which hence
contained a unique NdeI site. Next a PCR was performed with
oligonucleotides "NY-up" and "NY-down" (FIG. 2). During
amplification, both a NdeI and a NsiI restriction site were
introduced to facilitate cloning of the amplified fiber DNAs.
Amplification consisted of 25 cycles of each for 45 seconds at
94.degree. C., 1 minute at 60.degree. C., and 45 seconds at
72.degree. C. The PCR reaction contained 25 pmol of
oligonucleotides NY-up or NY-down, 2 mM dNTP, PCR buffer with 1.5
mM MgCl.sub.2, and 1 unit of Elongase heat stable polymerase
(Gibco, The Netherlands). One-tenth of the PCR product was run on
an agarose gel which demonstrated that the expected DNA fragment of
.+-.2200 bp was amplified. This PCR fragment was subsequently
purified using Geneclean kit system (Bio101 Inc.). Then, both the
construct pBr/Ad.Bam-rITR.DELTA.NdeI as well as the PCR product
were digested with restriction enzymes NdeI and SbfI. The PCR
fragment was subsequently cloned using T4 ligase enzyme into the
NdeI and Sbf I sites thus generating pBr/Ad.BamR.DELTA.Fib (FIG.
3).
[0102] Amplification of Fiber Sequences from Adenovirus
Serotypes:
[0103] To enable amplification of the DNAs encoding fiber protein
derived from alternative serotypes degenerate oligonucleotides were
synthesized. For this purpose, first known DNA sequences encoding
for fiber protein of alternative serotypes were aligned to identify
conserved regions in both the tail region as well as the knob
region of the fiber protein. From the alignment, which contained
the nucleotide sequence of 19 different serotypes representing all
6 subgroups, (degenerate) oligonucleotides were synthesized (see
Table I, SEQ ID NOS:1-13). Also shown in Table 3 is the combination
of oligonucleotides used to amplify the DNA encoding fiber protein
of a specific serotype. The amplification reaction (50 .mu.l)
contained 2 mM dNTPs, 25 pmol of each oligonucleotide, standard
1.times.PCR buffer, 1.5 mM MgCl.sub.2, and 1 Unit Pwo heat stable
polymerase (Boehringer Mannheim) per reaction. The cycler program
contained 20 cycles, each consisting of 30 seconds at 94.degree.
C., 60 seconds at 60-64.degree. C., and 120 seconds at 72.degree.
C. One-tenth of the PCR product was run on an agarose gel to
demonstrate that a DNA fragment was amplified. From each different
template, two independent PCR reactions were performed.
[0104] Generation of Chimeric Adenoviral DNA Constructs:
[0105] All amplified fiber DNAs as well as the vector
(pBr/Ad.BamR.DELTA.Fib) (ECACC deposit number 01121708) were
digested with NdeI and NsiI. The digested DNAs were subsequently
run on an agarose gel after which the fragments were isolated from
the gel and purified using the Geneclean kit (Bio101 Inc). The PCR
fragments were then cloned into the NdeI and NsiI sites of
pBr/AdBamR.DELTA.Fib (ECACC deposit number 01121708), thus
generating pBr/AdBamRFibXx (where XX stands for the serotype number
of which the fiber DNA was isolated). The inserts generated by PCR
were sequenced to confirm correct amplification. The obtained
sequences of the different fiber genes are shown in FIG. 4.
[0106] Generation of Recombinant Adenovirus Chimeric for Fiber
Protein:
[0107] To enable efficient generation of chimeric viruses an AvrII
fragment from the pBr/AdBamRFib16 (ECACC deposit number 01121710),
pBr/AdBamRFib28, pBr/AdBamRFib40-L constructs was subcloned into
the vector pBr/Ad.Bam-rITR.pac#8 (ECACC deposit #P97082121)
replacing the corresponding sequences in this vector.
pBr/Ad.Bam-rITR.pac#8 has the same adenoviral insert as
pBr/Ad.Bam-rITR but has a PacI site near the rITR that enables the
ITR to be separated from the vector sequences. The constuct
pWE/Ad.AflII-Eco was generated as follows. PWE.pac was digested
with ClaI and the 5 prime protruding ends were filled in with
Klenow enzyme. The DNA was then digested with PacI and isolated
from agarose gel. PWE/AflIIrITR was digested with EcoRI and after
treatment with Klenow enzyme digested with PacI. The large 24 kb
fragment containing the adenoviral sequences was isolated from
agarose gel and ligated to the ClaI digested and blunted pWE.Pac
vector. Use was made of the ligation express kit from Clontech.
After transformation of XL10-gold cells from Stratagene, clones
were identified that contained the expected construct.
PWE/Ad.AlfII-Eco contains Ad5 sequences from basepairs 3534-27336.
Three constructs, pClipsal-Luc (FIG. 5) digested with SalI,
pWE/Ad.AflII-Eco digested with PacI and EcoRI and
pBr/AdBamR.pac/fibXX digested with BamHI and PacI were transfected
into adenovirus producer cells (PER.C6, Fallaux et al., 1998). FIG.
6 schematically depicts the method and fragments used to generate
the chimeric viruses. Only pBr/Ad.BamRfib12 was used without
subcloning in the PacI containing vector and therefor was not
liberated from vector sequences using PacI but was digested with
ClaI which leaves approximately 160 bp of vector sequences attached
to the right ITR. Furthermore, the pBr/Ad.BamRfib12 and
pBr/Ad.BamRfib28 contain an internal BamHI site in the fiber
sequences and were therefor digested with SalI which cuts in the
vector sequences flanking the BamHI site. For transfection, 2 .mu.g
of pCLIPsal-Luc, and 4 .mu.g of both pWE/Ad.AflII-Eco and
pBr/AdBamR.pac/fibXX were diluted in serum free DMEM to 100 .mu.l
total volume. To this DNA suspension, 100 .mu.l 2.5.times. diluted
lipofectamine (Gibco) in serum-free medium were added. After 30
minutes at room temperature, the DNA-lipofectamine complex solution
was added to 2.5 ml of serum-free DMEM which was subsequently added
to a T25 cm.sup.2 tissue culture flask. This flask contained PER.C6
cells that were seeded 24-hours prior to transfection at a density
of 1.times.10.sup.6 cells/flask. Two hours later, the
DNA-lipofectamine complex containing medium was diluted once by the
addition of 2.5 ml DMEM supplemented with 20% fetal calf serum.
Again 24 hours later, the medium was replaced by fresh DMEM
supplemented with 10% fetal calf serum. Cells were cultured for 6-8
days, subsequently harvested, and freeze/thawed 3 times. Cellular
debris was removed by centrifugation for 5 minutes at 3000 rpm at
room temperature. Of the supernatant (12.5 ml), 3-5 ml was used to
infect again PER.C6 cells (T80 cm.sup.2 tissue culture flasks).
This re-infection results in full cytopathogenic effect (CPE) after
5-6 days after which the adenovirus is harvested as described
above.
[0108] Production of Fiber Chimeric Adenovirus:
[0109] 10 ml of the above described crude lysate was used to
inoculate a 1 liter fermentor which contained
1.sup.7-1.5.times.10.sup.6 PER.C6 cells/ml growing in suspension.
Three days after inoculation, the cells were harvested and pelleted
by centrifuging for 10 minutes at 1750 rpm at room temperature. The
chimeric adenovirus present in the pelleted cells was subsequently
extracted and purified using the following downstream processing
protocol. The pellet was dissolved in 50 ml 10 mM NaPO.sub.4 and
frozen at -20.degree. C. After thawing at 37.degree. C., 5.6 ml
deoxycholate (5% w/v) was added after which the solution was
homogenated. The solution was subsequently incubated for 15 minutes
at 37.degree. C. to completely crack the cells. After homogenizing
the solution, 1875 .mu.l (1M) MgCl.sub.2 was added and 5 ml 100%
glycerol. After the addition of 375 .mu.l DNase (10 mg/ml) the
solution was incubated for 30 minutes at 37.degree. C. Cell debris
was removed by centrifugation at 1880.times.g for 30 minutes at
room temperature without the brake on. The supernatant was
subsequently purified from proteins by loading on 10 ml of freon.
Upon centrifugation for 15 minutes at 2000 rpm without brake at
room temperature, three bands are visible of which the upper band
represents the adenovirus. This band was isolated by pipetting
after which it was loaded on a Tris/HCl (1M) buffered cesium
chloride blockgradient (range: 1.2 to 1.4 g/ml). Upon
centrifugation at 21000 rpm for 2.5 hours at 10.degree. C., the
virus was purified from remaining protein and cell debris since the
virus in contrast to the other components, does not migrate into
the 1.4 g/ml cesium chloride solution. The virus band is isolated
after which a second purification using a Tris/HCl (1M) buffered
continues gradient of 1.33 g/ml of cesium chloride is performed.
After virus loading on top of this gradient, the virus is
centrifuged for 17 hours at 55000 rpm at 10.degree. C.
Subsequently, the virus band is isolated and after the addition of
30 .mu.l of sucrose (50 w/v) excess cesium chloride is removed by
three rounds of dialysis, each round comprising 1 hour. For
dialysis, the virus is transferred to dialysis slides
(Slide-a-lizer, cut off 10000 kDa, Pierce, USA). The buffers used
for dialysis are PBS which are supplemented with an increasing
concentration of sucrose (round 1 to 3: 30 ml, 60 ml, and 150 ml
sucrose (50% w/v)/1.5 liter PBS, all supplemented with 7.5 ml 2%
(w/v) CaMgCl.sub.2). After dialysis, the virus is removed from the
slide-a-lizer after which it is aliquoted in portions of 25 and 100
.mu.l upon which the virus is stored at -85.degree. C. To determine
the number of virus particles per ml, 50 .mu.l of the virus batch
is run on a high pressure liquid chromatograph (HPLC) as described
by Shamram et al. (1997). The virus titers were found to be in the
same range as the Ad5.Luc virus batch (Ad5.Luc: 2.2.times.10.sup.11
vp/ml; Ad5.LucFib12: 1.3.times.10.sup.11 vp/ml; Ad5.LucFib16:
3.1.times.10.sup.12 vp/ml; Ad5.LucFib28: 5.4.times.10.sup.10 vp/ml;
Ad5.LucFib40-L: 1.6.times.10.sup.12 vp/ml).
Example 2
Biodistribution of Chimeric Viruses after Intravenous Tail Vein
Injection of Rats
[0110] To investigate the biodistribution of the chimeric
adenoviruses carrying fiber 12, 16, 28, or 40-2, 1.times.10.sup.10
particles of each of the generated virus batches was diluted in 1
ml PBS after which the virus was injected in the tail vein of adult
male Wag/Rij rats (3 rats/virus). As a control, Ad5 carrying the
luciferase transgene was used. Forty-eight hours after the
administration of the virus, the rats were sacrificed after which
the liver, spleen, lung, kidney, heart, and brain were dissected.
These organs were subsequently mixed with 1 ml of lysis buffer (1%
Triton X-100/PBS) and minced for 30 seconds to obtain a protein
lysate. The protein lysate was subsequently tested for the presence
of transgene expression (luciferase activity) and the protein
concentration was determined to express the luciferase activity per
.mu.g of protein. The results, Shown in Table II, demonstrate that
in contrast to the Adenovirus serotype 5 control, none of the fiber
chimeras are targeted specifically to the liver or to the spleen.
This experiment shows that it is possible to circumvent the uptake
of adenoviruses by the liver by making use of fibers of other
serotypes. It also shows that the uptake by the liver is not
correlated with the length of the fiber shaft, or determined solely
by the ability of fiber knob to bind to CAR. The fibers used have
different shaft lengths and, except for fiber 16, are derived from
subgroups known to have a fiber that can bind CAR (Roelvink et al.
1998).
Example 3
Chimeric Viruses Display Differences in Endothelial and Smooth
Muscle Cell Transduction
[0111] A) Infection of Human Endothelial Cells
[0112] Human endothelial cells (HUVEC) were isolated, cultured and
characterized as described previously (Jaffe et al. 1973; Wijnberg
et al. 1997). Briefly, cells were cultured on gelatin-coated dishes
in M199 supplemented with 20 mM HEPES, pH 7.3 (Flow Lab., Irvine,
Scotland), 10% (v/v) human serum (local blood bank), 10% (v/v)
heat-inactivated newborn calf serum (NBCS) (GIBCO BRL,
Gaithersburg, Md.), 150 .mu.g/ml crude endothelial cell growth
factor, 5 U/ml heparin (Leo Pharmaceutics Products, Weesp, The
Netherlands), penicillin (100 IU/ml)/streptomycin (100 .mu.g/ml)
Boehringer Mannheim, Mannheim, Del.) at 37.degree. C. under 5%
(v/v) CO.sub.2/95% (v/v) air atmosphere. Cells used for experiments
were between passage 1-3. In a first set of experiments 40000 HUVEC
cells (a pool from 4 different individuals) were seeded in each
well of 24-well plates in a total volume of 200 .mu.l. Twenty-four
hours after seeding, the cells were washed with PBS after which 200
.mu.l of DMEM supplemented with 2% FGS was added to the cells. This
medium contained various amounts of virus (MOI=50, 250, 1000, 2500,
5000, and 10,000). The viruses used were besides the control Ad5,
the fiber chimeras 12, 16, 28, and 40-L (each infection in
triplicate). Two hours after addition of the virus the medium was
replaced by normal medium. Again forty-eight hours later cells were
washed and lysed by the addition of 100 .mu.l lysis buffer. In FIG.
7a, results are shown on the transgene expression per microgram
total protein after infection of HUVEC cells. These results show
that fiber chimeras 12 and 28 are unable to infect HUVEC cells,
that 40-L infects HUVECs with similar efficiency as the control Ad5
virus, and that fiber chimera 16 infects HUVECs significantly
better. In a next set of experiments (n=8), the fiber 16 chimera
was compared with the Ad5.Luc vector on HUVEC for luciferase
activity after transduction with 2500 virus particles per cell of
each virus. These experiments demonstrated that fiber 16 yields, on
average, 8.1 fold increased luciferase activity (SD.+-.4.6) as
compared with Ad5. In a next experiment, an equal number of virus
particles was added to wells of 24-well plates that contained
different HUVEC cell concentrations. This experiment was performed
since it is known that HUVECs are less efficiently infected with
adenovirus serotype 5 when these cells reach confluency. For this
purpose, HUVECs were seeded at 22,500, 45,000, 90,000, and 135,000
cells per well of 24-well-plates (in triplicate). Twenty-four hours
later, these cells were infected as described above with medium
containing 4.5.times.10.sup.9 virus particles. The viruses used
were besides the control adenovirus serotype 5, the chimera fiber
16. The result of the transgene expression (RLU) per microgram
protein determined 48 hours after infection (see FIG. 7b) shows
that the fiber 16 chimeric adenovirus is also better suited to
infect HUVEC cells even when these cells are 100% confluent which
better mimics an in vivo situation. Since the Luciferase marker
gene does not provide information concerning the number of cells
infected another experiment was performed with adenovirus serotype
5 and the fiber 16 chimera, both carrying a green fluorescent
protein (GFP) as a marker gene. This protein expression can be
detected using a flow cytometer which renders information about the
percentage of cells transduced as well as fluorescence per cell. In
this experiment, cells were seeded at a concentration of 40,000
cells per well and were exposed to virus for 2 hours. The virus
used was Ad5.GFP (8.4.times.10.sup.11 vp/ml) and Ad5.Fib16 GFP
(5.1.times.10.sup.11 vp/ml). Cells were exposed to a virus
concentration of 500 virus particles per cell. Flow cytometric
analysis, 48 hours after virus exposure demonstrated that the fiber
16 virus gives higher transgene expression levels per cell since
the median fluorescence, a parameter identifying the amount of GFP
expression per cell, is higher with fiber 16 as compared to Ad5
(FIG. 7c). These results are thus consistent and demonstrate that
the fiber 16 chimeric virus is better suited to infect human
primary endothelial cells as compared to Ad5.
[0113] B) Infection of Human Smooth Muscle Cells
[0114] Smooth muscle cells were isolated after isolation of EC
(Weinberg et al. 1997). The veins were incubated with medium (DMEM)
supplemented with penicillin/streptomycin) containing 0.075% (w/v)
collagenase (Worthington Biochemical Corp., Freehold, N.J., USA).
After 45 minutes, the incubation medium containing detached cells
was flushed from the veins. Cells were washed and cultured on
gelatin coated dishes in culture medium supplemented with 10% human
serum at 37.degree. C. under 5% (v/v) CO.sub.2/95% (v/v) air
atmosphere. Cells used for experiments were between passage 3-6. We
first tested the panel of chimeric fiber viruses versus the control
adenovirus serotype 5 for the infection of human smooth muscle
cells. For this purpose, 40000 human umbilical vein smooth muscle
cells (HUVsmc) were seeded in wells of 24-well plates in a total
volume of 200 .mu.l. Twenty-four hours after seeding, the cells
were washed with PBS after which 200 .mu.l of DMEM supplemented
with 2% FCS was added to the cells. This medium contained various
amounts of virus (MOI=50, 250, 1250, 2500, and 5000). The viruses
used were besides the control Ad5 the fiber chimeras 12, 16, 28 and
40-L (each infection in triplicate). Two hours after addition of
the virus the medium was replaced by normal medium. Again
forty-eight hours later, cells were washed and lysed by the
addition of 100 .mu.l lysis buffer. In FIG. 8a, results are shown
of the transgene expression per microgram total protein after
infection of HUVsmc cells. These results show that fiber chimeras
12 and 28 are unable to infect HUVsmc cells, that 40-L infects
HUVsmc with similar efficiency as the control Ad5 virus, and that
fiber chimera 16 infects HUVsmc significantly better. In a next set
of experiments, smooth muscle cells derived from saphenous vene,
arteria Iliaca, left interior mammory artery (LIMA) and aorta were
tested for infection with the fiber 16 chimera and Ad5 (both
carrying luciferase as a marker gene). These experiments (n=11)
demonstrated that, on average, the fiber 16 chimera yielded 61.4
fold increased levels in luciferase activity (SD.+-.54.8) as
compared to Ad5. The high standard deviation (SD) is obtained due
to the finding that the adenoviruses used vary in their efficiency
of infection of SMC derived from different human vessels. In a next
experiment, an equal number of virus particles was added to wells
of 24-well plates that contained different HUVsmc cell
concentrations confluency. For this purpose, HUVsmc were seeded at
10,000, 20,000, 40,000, 60,000, and 80,000 cells per well of
24-well plates (in triplicate). Twenty-four hours later these cells
were infected as described above with medium containing
2.times.10.sup.8 virus particles. The viruses used were, besides
the control adenovirus serotype 5, the chimera fiber 16. The result
of the transgene expression (RLU) per microgram protein determined
48 hours after infection (see FIG. 8b) shows that the fiber 16
chimeric adenovirus is better suited to infect smooth muscle cells
even when these cells are 100% confluent which better mimics an in
vivo situation.
[0115] To identify the number of SMCs transduced with the fiber 16
chimera and Ad5, we performed transduction experiments with Ad5.GFP
and Ad5Fib16.GFP (identical batches as used for EC infections).
Human umbilical vein SMC were seeded at a concentration of 60000
cells per well in 24-well plates and exposed for 2 hours to 500 or
5000 virus particles per cell of Ad5.GFP or Ad5Fib16.GFP.
Forty-eight hours after exposure, cells were harvested and analyzed
using a flow cytometer. The results obtained show that the fiber 16
mutant yields approximately 10 fold higher transduction of SMC
since the GFP expression measured after transduction with 5000
virus particles of Ad5.GFP is equal to GFP expression after
transduction with 500 virus particles per cell of the fiber 16
chimera (FIG. 8c).
[0116] C) Subgroup B Fiber Mutants Other than Fiber 16
[0117] The shaft and knob of fiber 16 are derived from adenovirus
serotype 16 which, as described earlier, belongs to subgroup B.
Based on hemagglutination assays, DNA restriction patterns, and
neutralization assays, the subgroup B viruses have been further
subdivided into subgroup B1 and B2 (Wadell et al. 1984). Subgroup
B1 members include serotypes 3, 7, 16, 21, and 51. Subgroup B2
members include 11, 14, 34, and 35. To test whether the increased
infection of smooth muscle cells is a trade of all fibers derived
from subgroup B or specific for one or more subgroup B fiber
molecules, we compared fiber 16 and fiber 51 (both subgroup B1)
with fiber 11 and fiber 35 (both subgroup B2). For this purpose,
HUVsmc were infected with increasing amounts of virus particles per
cell (156, 312, 625, 1250, 2500, 5000). The fiber mutants all carry
the Luciferase marker gene (Ad5Fib11.Luc: 1.1.times.10.sup.12
vp/ml; Ad5Fib35Luc: 1.4.times.10.sup.12 vp/ml; Ad5Fib35Luc:
1.4.times.10.sup.12 vp/ml; Ad5Fib51Luc: 1.0.times.10.sup.12 vp/ml).
Based on the Luciferase activity measured and shown in FIG. 8d,
efficient infection of SMC is not a general trade of all subgroup B
fiber molecules. Clearly, fiber 16 and fiber 11 are better suited
for infection of SMC than fiber 35 and fiber 51. Nevertheless, all
subgroup B fiber mutants tested infect SMC better than Ad5.
[0118] D) Organ Culture Experiments
[0119] We next identified whether the observed difference in
transduction of EC and SMC using the fiber 16 chimera or the Ad5
can also be demonstrated in organ culture experiments. Hereto, we
focused on the following tissues: 1) Human Saphenous vein: the vein
used in approximately 80% of all clinical vein grafting procedures.
2) Human pericard/epicard: for delivery of recombinant adenoviruses
to the pericardial fluid which after infection of the pericardial
or epicardial cells produce the protein of interest from the
transgene carried by the adenovirus. 3) Human coronary arteries:
percutaneous transluminal coronary angioplasty (PTCA) to prevent
restenosis. Of the coronary arteries we focused on the left artery
descending (LAD) and right coronary artery (RCA).
[0120] Parts of a human saphenous vein left over after vein graft
surgery were sliced into pieces of approximately 0.5 cm. These
pieces (n=3) were subsequently cultured for 2 hours in 200 ml of
5.times.10.sup.10 virus particles per ml. After two hours virus
exposure, the pieces were washed with PBS and cultured for another
48 hours at 37.degree. C. in a 10% CO.sub.2 incubator. The pieces
were then washed, fixated and stained for LacZ transgene
expression. The viruses were Ad5.LacZ (2.2.times.10.sup.12 vp/ml),
the fiber 16 chimera: Ad5Fib16.LacZ (5.2.times.10.sup.11 vp/ml),
and a fiber 51 chimera: Ad5Fib51.LacZ (2.1.times.10.sup.12 vp/ml).
The pieces of saphenous vein were macroscopically photographed
using a digital camera. Based on LacZ transgene expression obtained
after 2 hours of virus exposure on saphenous vein slices, both the
fiber 16 and the fiber 51 chimeric viruses give higher infection
since much more blue staining is observed using these viruses as
compared to Ad5.LacZ (FIG. 8e). Identical experiments as described
on saphenous vein were performed with human pericard and the human
coronary arteries: RCA and LAD. Results of these experiments (FIGS.
8f-8g-8h respectively) together with the experiments performed on
primary cells confirmed the superiority of the fiber 16 and 51
mutants as compared to Ad5 in infecting human cardiovascular
tissues.
[0121] E) CAR and Integrin Expression on Human EC and SMC
[0122] From the above described results, it is clear that the
chimeric adenovirus with the shaft and knob from fiber 16 is well
suited to infect endothelial cells and smooth muscle cells. Thus,
by changing the fiber protein on Ad5 viruses we are able to
increase infection of cells that are poorly infected by Ad5. The
difference between Ad5 and Ad5Fib16, although significant on both
cell types, is less striking on endothelial cells as compared to
smooth muscle cells. This may reflect differences in receptor
expression. For example, HUVsmc has significantly more
.alpha..sub.v.beta.5 integrins than HUVEC (see below).
Alternatively, this difference may be due to differences in
expression of the receptor of fiber 16. Ad5.LucFib16 infects
umbilical vein smooth muscle cells approximately 8 fold better than
umbilical vein endothelial cells whereas in case of Ad5.Luc viruses
endothelial cells are better infected than smooth muscle cells. To
test whether Ad5 infection correlated with receptor expression of
these cells the presence of CAR and .alpha.-integrins was assayed
on a flow cytometer. For this purpose 1.times.10.sup.5 HUVEC cells
or HUVsmc were washed once with PBS/0.5% BSA after which the cells
were pelleted by centrifugation for 5 minutes at 1750 rpm at room
temperature. Subsequently, 10 .mu.l of a 100 times diluted
.alpha..sub.v.beta.3 antibody (Mab 1961, Brunswick Chemie,
Amsterdam, The Netherlands), a 100 times diluted antibody
.alpha..sub.vB5 (antibody (Mab 1976, Brunswick Chemie, Amsterdam,
The Netherlands), or 2000 times diluted CAR antibody was a kind
gift of Dr. Bergelson, Harvard Medical School, Boston, Mass., USA
(Hsu et al.) was added to the cell pellet after which the cells
were incubated for 30 minutes at 4.degree. C. in a dark
environment. After this incubation, cells were washed twice with
PBS/0.5% BSA and again pelleted by centrifugation for 5 minutes at
1750 rpm room temperature. To label the cells, 10 ml of rat
anti-mouse IgG1 labeled with phycoerythrine (PE) was added to the
cell pellet upon which the cells were again incubated for 30
minutes at 4.degree. C. in a dark environment. Finally, the cells
were washed twice with PBS/0.5% BSA and analyzed on a flow
cytometer. The results of these experiments are shown in table III.
From the results it can be concluded that HUVsmc do not express
detectable levels of CAR confirming that these cells are difficult
to transduce with an adenovirus which enters the cells via the CAR
receptor.
[0123] F) Infection of Human A549 Cells
[0124] As a control for the experiments performed on endothelial
cells and smooth muscle cells, A549 cells were infected to
establish whether an equal amount of virus particles of the
different chimeric adenoviruses show significant differences in
transgene expression on cell lines that are easily infected by
adenovirus. This is to investigate whether the observed differences
in infection efficiency on endothelial and smooth muscle cells are
cell type specific. For this purpose, 10.sup.5 A549 cells were
seeded in 24-well plates in a volume of 200 PI. Two hours after
seeding, the medium was replaced by medium containing different
amounts of particles of either fiber chimera 5, 12, 16, or 40-L
(MOI=0, 5, 10, 25, 100, 500). Twenty-four hours after the addition
of virus, the cells were washed once with PBS after which the cells
were lysed by the addition of 100 .mu.l lysis buffer to each well
(1% Triton X-100 in PBS) after which transgene expression
(Luciferase activity) and the protein concentration was determined.
Subsequently, the luciferase activity per .mu.g protein was
calculated. The data, shown in Table IV, demonstrate that Ad5.Luc
viruses infect A549 cells most efficient while the infection
efficiency of Ad5LucFib16 or Ad5.LucFib40-L is a few times lower.
This means that the efficient infection of endothelial cells and
especially smooth muscle cells is due to differences in binding of
the virus to these cells and not to the amount of virus or the
quality of the viruses used.
1TABLE I Serotype Tail oligonucleotide Knob oligonucleotide 4 A 1 8
B 2 9 B 2 12 E 3 16 C 4 19p B 2 28 B 2 32 B 2 36 B 2 37 B 2 40-1 D
5 40-2 D 6 41-3 D 5 41-1 D 7 49 B 2 50 B 2 51 C 8 A: 5' -CCC GTG
TAT CCA TAT GAT GCA GAC AAC GAC CGA CC-3' (SEQ ID NO:1) B: 5' -CCC
GTC TAC CCA TAT GGC TAC GCG CGG-3' (SEQ ID NO:2) C: 5' -CCK GTS TAC
CCA TAT GAA GAT GAA AGC-3' (SEQ ID NO:3) D: 5' -CCC GTC TAC CCA TAT
GAC ACC TYC TCA ACT C-3' (SEQ ID NO:4) E 5' -CCC GTT TAC CCA TAT
GAC CCA TTT GAC ACA TCA GAC-3' (SEQ ID NO:5) 1: 5' -CCG ATG CAT TTA
TTG TTG GGC TAT ATA GGA-3' (SEQ ID NO:6) 2: 5' -CCG ATG CAT TYA TTC
TTG GGC RAT ATA GGA-3' (SEQ ID NO:7) 3: 5' -CCG ATG CAT TTA TTC TTG
GGR AAT GTA WGA AAA GGA-3' (SEQ ID NO:8) 4: 5' -CCG ATG CAT TCA GTC
ATC TTC TCT GAT ATA-3' (SEQ ID NO:9) 5: 5' -CCG ATG CAT TTA TTG TTC
AGT TAT GTA GCA-3' (SEQ ID NO:10) 6: 5' -GCC ATG CAT TTA TTG TTC
TGT TAC ATA AGA-3' (SEQ ID NO:11) 7: 5' -CCG TTA ATT AAG CCC TTA
TTG TTC TGT TAC ATA AGA A-3' (SEQ ID NO:12) 8: 5' -CCG ATG CAT TCA
GTC ATC YTC TWT AAT ATA-3' (SEQ ID NO:13)
[0125]
2TABLE II Organ Control Ad5 Fib 12 Fib 16 Fib 28 Fib 40-L Liver
740045 458 8844 419 2033 Spleen 105432 931 3442 592 16107 Lung 428
315 334 316 424 Kidney 254 142 190 209 224 Heart 474 473 276 304
302 Brain 291 318 294 323 257
[0126]
3 TABLE III Cell line .alpha..sub.V.beta.3 .alpha..sub.V.beta.5 CAR
HUVEC 70% 98.3% 18.9% 18.1% HUVEC 100% 97.2% 10.5% 7.2% HUVsmc 70%
95.5% 76.6% 0.3% HUVsmc 100% 92.2% 66.5% 0.3% PER.C6 7.8% 16.8%
99.6%
[0127]
4TABLE IV MOI (vp/Cell) Control Ad5 Fiber 12 Fiber 16 Fiber 40-L 0
0 0 0 0 5 1025 46 661 443 10 1982 183 1704 843 25 4840 200 3274
2614 100 21875 1216 13432 11907 500 203834 3296 93163 71433
REFERENCES
[0128] Arnberg, N., Mei Y. and Wadell G. (1997) Fiber genes of
adenoviruses with tropism for the eye and the genital tract.
Virology 227: 239-244.
[0129] Bergelson, J. M., Cunningham, J. A., Droguett, G.,
Kurt-Jones, E. A., Krithivas, A., Hong, J. S., Horwitz, M. S.,
Crowell, R. L. and Finberg, R. W. (1997) Isolation of a common
receptor for coxsackie B virus and adenoviruses 2 and 5. Science
275: 1320-1323.
[0130] Bout, A. (1997) Gene therapy, p. 167-182. In: D. J. A.
Crommelin and R. D. Sindelar (ed.), Pharmaceutical Biotechnology,
Harwood Academic Publishers.
[0131] Bout, A. (1996) Prospects for human gene therapy. Eur. J.
Drug Met. and Pharma., 2, 175-179.
[0132] Blaese, M., Blankenstein, T., Brenner, M., Cohen-Hagenauer,
O. Gansbacher, B., Russel, S., Sorrentino, B. and Velu, T. (1995)
Cancer Gene Ther. 2: 291-297
[0133] Brody, S. L. and Crystal, R. G. (1994) Adenovirus mediated
in vivo gene transfer. Ann. N.Y. Acad. Sci. 716: 90-101.
[0134] Carter, A. J., Laird, J. R., Farb, A., Kufs, W., Wortham, D.
C. and Virmani, R. (1994) Morphologic characteristics of lesion
formation and time course of smooth muscle cell proliferation in a
porcine proliferative restenosis model. J. Am. Coll. Cardiol. 24:
1398-1405.
[0135] Chroboczek J., Ruigrok R. W. H., and Cusack S. (1995)
Adenovirus fiber, p. 163-200. In: W. Doerfler and P. Bohm (ed.),
The molecular repertoire of adenoviruses, I. Springer-Verlag,
Berlin.
[0136] Defer C., Belin M., Caillet-Boudin M. and Boulanger P.
(1990) Human adenovirus-host cell interactions, comparative study
with members of subgroup B and C. Journal of Virology 64 (8):
3661-3673.
[0137] Fallaux, F. J., Bout., A, van der Velde, I et al. (1998) New
helper cells and matched E1-deleted adenovirus vectors prevent
generation of replication competent adenovirus. Human Gene Therapy,
9, p 1909-1917.
[0138] Francki, R. I. B., Fauquet, C. M., Knudson, D. L., and
Brown, F. (1991) Classification and nomenclature of viruses. Fifth
report of the international committee on taxonomy of viruses. Arch,
Virol. Suppl. 2: 140-144.
[0139] Gall J., Kass-Eisler A., Leinwand L., and Falck-Pedersen E.
(1996) Adenovirus type 5 and 7 capsid chimera: fiber replacement
alters receptor tropism without affecting primary immune
neutralization epitopes. Journal of Virology 70 (4): 2116-2123.
[0140] Greber, U. F., Willets, M., Webster, P., and Helenius, A.
(1993) Stepwise dismantling of adenovirus 2 during entry into
cells. Cell 75: 477-486.
[0141] Hynes, R. O. (1992) Integrins: versatility, modulation and
signaling in cell adhesion. Cell 69: 11-25.
[0142] Herz, J. and Gerard, R. D. (1993) Adenovirus-mediated
transfer of low density lipoprotein receptor gene acutely
accelerates cholesterol clearance in normal mice. Proc. Natl. Acad.
Sci. U.S.A. 96: 2812-2816.
[0143] Hierholzer, J. C. (1992) Adenovirus in the immunocompromised
host. Clin. Microbiol Rev. 5, 262-274.
[0144] Hierholzer, J. C., Wigand, R., Anderson, L. J., Adrian, T.,
and Gold, J. W. M. (1988) Adenoviruses from patients with AIDS: a
plethora of serotypes and a description of five new serotypes of
subgenus D (types 43-47). J. Infect. Dis. 158, 804-813.
[0145] Hong, S. S., Karayan, L., Tournier, J., Curiel, D. T. and
Boulanger, P. A. (1997) Adenovirus type 5 fiber knob binds to MHC
class I .alpha.2 domain at the surface of human epithelial and B
lymphoblastoid cells. EMBO J. 16: 2294-2306.
[0146] Hsu, K. H., Lonberg-Holm, K., Alstein, B. and Crowell, R. L.
(1988) A monoclonal antibody specific for the cellular receptor for
the group B coxsackieviruses, J. Virol 62(5): 1647-1652.
[0147] Huard, J., Lochmuller, H., Acsadi, G., Jani, A., Massie, B.
and Karpati, G. (1995) The route of administration is a major
determinant of the transduction efficiency of rat tissues by
adenoviral recombinants. Gene Ther. 2: 107-115.
[0148] Ishibashi, M. and Yasue, H. (1984) The adenoviruses, H. S.
Ginsberg, ed., Plenum Press, London, New York. Chapter 12,
497-561.
[0149] Jaffe, E. A., Nachman, R. L., Becker, C. G., Minick, C. R.
(1973) Culture of endothelial cells derived from umbilical veins.
Identification by morphologic and immunologic criteria. J. Clin.
Invest. 52, 2745-2756.
[0150] Kass-Eisler, A., Falck-Pederson, E., Elfenbein, D. H.,
Alvira, M., Buttrick, P. M., and Leinwand, L. A. (1994) The impact
of developmental stage, route of administration and the immune
system on adenovirus-mediated gene transfer. Gene Ther. 1:
395-402.
[0151] Khoo, S. H., Bailey, A. S., De Jong, J. C., and Mandal, B.
K. (1995) Adenovirus infections in human immunodeficiency
virus-positive patients: Clinical features and molecular
epidemiology. J. Infect. Dis 172, 629-637.
[0152] Kidd, A. H., Chroboczek, J., Cusack, S., and Ruigrok, R. W.
(1993) Adenovirus type 40 virions contain two distinct fibers.
Virology 192, 73-84.
[0153] Krasnykh, V. N., Mikheeva G. V., Douglas, J. T. and Curiel,
D. T. (1996) Generation of recombinant adenovirus vectors with
modified fibers for altering viral tropism. J. Virol. 70(10):
6839-6846.
[0154] Krasnykh, V. N., Dmitriev, I., Mikheeva, G., Miller, C. R.,
Belousova, N. and Curiel, D. T. (1998) Characterization of an
adenovirus vector containing a heterologous peptide epitope in the
HI loop of the fiber knob. J. Virol. 72(3): 1844-1852.
[0155] Law, L., Chillon, M., Bosch, A., Armentano, D., Welsh, M. J.
and Davidson, B. L. (1998) Infection of primary CNS cells by
different adenoviral serotypes: Searching for a more efficient
vector. Abstract 1st Annual Meeting American Society of Gene
Therapy, Seattle, Wash.
[0156] Leppard, K. N. (1997) E4 gene function in adenovirus,
adenovirus vector and adeno-associated virus infections. J. Gen.
Virol. 78: 2131-2138.
[0157] Lloyd Jones, D. M. and Bloch, K. D. (1996) The vascular
biology of nitric oxide and its role in atherogenesis. Annu. Rev.
Med. 47: 365-375.
[0158] Morgan, C., Rozenkrantz, H. S., and Mednis, B. (1969)
Structure and development of viruses as observed in the electron
microscope. Entry and uncoating of adenovirus. J. Virol 4,
777-796.
[0159] Roelvink, P. W., Kovesdi, I. and Wickham T. J. (1996)
Comparative analysis of adenovirus fiber-cell interaction:
Adenovirus type 2 (Ad2) and Ad9 utilize the same cellular fiber
receptor but use different binding strategies for attachment. J.
Virol. 70: 7614-7621.
[0160] Roelvink, P. W., Lizonova, A., Lee, J. G. N., Li, Y.,
Bergelson, J. M., Finberg, R. W., Brough, D. E., Kovesdi, I. and
Wickham, T. J. (1998) The coxsackie-adenovirus receptor protein can
function as a cellular attachment protein for adenovirus serotypes
from subgroups A, C, D, E, and F. J. Virol. 72: 7909-7915.
[0161] Rogers, B. E., Douglas, J. T., Ahlem, C., Buchsbaum, D. J.,
Frincke, J. and Curiel, D. T. (1997) Use of a novel cross-linking
method to modify adenovirus tropism. Gene Ther. 4: 1387-1392.
[0162] Schulick, A. H., Vassalli, G., Dunn, P. F., Dong, G., Rade,
J. J., Zamarron, C. and Dichek, D. A. (1997) Established immunity
precludes adenovirus-mediated gene transfer in rat carotid
arteries.
[0163] Schnurr, D. and Dondero, M. E. (1993) Two new candidate
adenovirus serotypes. Intevirol. 36, 79-83.
[0164] Schwartz, R. S., Edwards, W. D., Huber, K. C., Antoniudes,
L. C., Bailey, K. R., Camrud, A. R., Jorgenson, M. A. and Holmes,
D. R. Jr. (1993) Coronary restenosis: Prospects for solution and
new perspectives from a porcine model. Mayo Clin. Proc. 68:
54-62.
[0165] Shi, Y., Pieniek, M., Fard, A., O'Brien, J., Mannion, J. D.
and Zalewski, A. (1996) Adventitial remodeling after coronary
arterial injury. Circulation 93: 340-348.
[0166] Shabram, P. W., Giroux, D. D., Goudreau, A. M., Gregory, R.
J., Horn, M. T., Huyghe, B. G., Liu, X., Nunnally, M. H., Sugarman,
B. J. and Sutjipto, S. (1997) Analytical anion-exchange HPLC of
recombinant type-5 adenoviral particles. Hum. Gene Ther. 8(4):
453-465.
[0167] Signas, G., Akusjarvi, G., and Petterson, U. (1985)
Adenovirus 3 fiberpolypeptide gene: Complications for the structure
of the fiber protein. J. Virol. 53, 672-678.
[0168] Stevenson, S. C., Rollence, M., White B., Weaver L. and
McClelland, A. (1995) Human adenovirus serotypes 3 and 5 bind to
two different cellular receptors via the fiber head domain. J.
Virol 69(5): 2850-2857.
[0169] Stevenson, S. C., Rollence, M., Marshall-Neff, J. and
McClelland, A. (1997) Selective targeting of human cells by a
chimeric adenovirus vector containing a modified fiber protein. J.
Virology 71(6): 4782-4790.
[0170] Stouten, P. W. F., Sander, C., Ruigrok, R. W. H., and
Cusack, S. (1992) New triple helical model for the shaft of the
adenovirus fiber. J. Mol. Biol. 226, 1073-1084.
[0171] Svensson, V. and Persson, R. (1984) Entry of adenovirus 2
into Hela cells. J. Virol. 51, 687-694.
[0172] Van der Vliet, P. C. (1995) Adenovirus DNA replication In:
W. Doerfler and P. Bohm (eds.) The molecular repertoire of
adenoviruses II. Springer-Verlag, Berlin.
[0173] Varga, M. J., Weibull, C., and Everitt, E. (1991) Infectious
entry pathway of adenovirus type 2. J. Virol 65, 6061-6070.
[0174] Varenne, O., Pislaru, S., Gillijns, H., Van Pelt, N.,
Gerard, R. D., Zoldhelyi, P., Van de Werf, F., Collen, D. and
Janssens, S. P. (1998) Local adenovirus-mediated transfer of human
endothelial nitric oxide synthetase reduces luminal narrowing after
coronary angioplasty in pigs. Circulation 98: 919-926.
[0175] Wadell, G. (1984) Molecular Epidemiology of human
adenoviruses Curr. Top. Microbiol. Immunol. 110, 191-220.
[0176] Wickham, T. J, Carrion, M. E. and Kovesdi, I. (1995)
Targeting of adenovirus penton base to new receptors through
replacement of its RGD motif with other receptor-specific peptide
motifs. Gene Therapy 2: 750-756.
[0177] Wickham, T. J., Segal, D. M., Roelvink, P. W., Carrion, M.
E., Lizonova, A., Lee, G-M., and Kovesdi, I. (1996) Targeted
adenovirus gene transfer to endothelial and smooth muscle cells by
using bispecific antibodies. J. Virol. 70 (10), 6831-6838.
[0178] Wickham, T. J., Mathias, P., Cherish, D. A., and Nemerow, G.
R. (1993) Integrins avb3 and avb5 promote adenovirus
internalization but not virus attachment. Cell 73, 309-319.
[0179] Wijnberg, M. J., Quax, P. H. A., Nieuwenbroek, N. M. E.,
Verheijen, J. H. (1997) The migration of human smooth muscle cells
in vitro is mediated by plasminogen activation and can be inhibited
by alpha(2) macro globulin receptor associated protein. Thromb. and
Haemostas. 78, 880-886.
[0180] Wold, W. S., Tollefson, A. E. and Hermiston, T. W. (1995) E3
transcription unit of adenovirus. In: W. Doerfler and P. Bohm
(eds.). The molecular repertoire of adenoviruses I.
Springer-Verlag, Berlin.
[0181] Zabner, J., Armentano, D., Chillon, M., Wadsworth, S. C.,
and Welsh, M. J. (1998) Type 17 fiber enhances gene transfer
Abstract 1st Annual Meeting American Society of Gene Therapy,
Seattle, Wash.
* * * * *