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TECHNICAL FIELD OF THE INVENTION
The present invention relates to a recombinant adenovirus comprising a
chimeric adenoviral penton base protein and the use of a recombinant
adenovirus comprising a chimeric adenoviral penton base protein in gene
therapy.
BACKGROUND OF THE INVENTION
Adenoviruses belong to the family Adenoviridae, which is divided into two
genera, namely Mastadenovirus and Aviadenovirus. Adenoviruses are
nonenveloped, regular icosahedrons 65-80 nm in diameter (Horne et al., J.
Mol. Biol., 1, 84-86 (1959)). The capsid is composed of 252 capsomeres of
which 240 are hexons and 12 are pentons (Ginsberg et al., Virology, 28,
782-783 (1966)). The hexons and pentons are derived from three different
viral polypeptides (Maizel et al., Virology, 36, 115-125 (1968); Weber et
al., Virology, 76, 709-724 (1977)). The hexon comprises three identical
polypeptides of 967 amino acids each, namely polypeptide II (Roberts et
al., Science, 232, 1148-1151 (1986)). The penton contains a penton base,
which is bound to the capsid, and a fiber, which is noncovalently bound to
and projects from the penton base. The fiber protein comprises three
identical polypeptides of 582 amino acids each, namely polypeptide IV. The
Ad2 penton base protein is an 8.times.9 nm ring-shaped complex composed of
five identical protein subunits of 571 amino acids each, namely
polypeptide III (Boudin et al., Virology, 92, 125-138 (1979)). Proteins
IX, VI, and IIIa are also present in the adenovirus coat and are thought
to stabilize the viral capsid (Stewart et al., Cell, 67, 145-154 (1991);
Stewart et al., EMBO J., 12(7), 2589-2599 (1993)) .
Once an adenovirus attaches to a cell, it undergoes receptor-mediated
internalization into clathrin-coated endocytic vesicles of the cell
(Svensson et al., J. Virol., 51, 687-694 (1984); Chardonnet et al.,
Virology, 40, 462-477 (1970)). Virions entering the cell undergo a
stepwise disassembly in which many of the viral structural proteins are
shed (Greber et al., Cell, 75, 477-486 (1993)). During the uncoating
process, the viral particles cause disruption of the cell endosome by a
pH-dependent mechanism (Fitzgerald et al., Cell, 32, 607-617 (1983)),
which is still poorly understood. The viral particles are then transported
to the nuclear pore complex of the cell (Dales et al., Virology, 56,
465-483 (1973)), where the viral genome enters the nucleus, thus
initiating infection.
An adenovirus uses two separate cellular receptors, both of which must be
present, to attach to and efficiently infect a cell (Wickham et al., Cell,
73, 309-319 (1993)). First, the fiber protein attaches the virus to a cell
by binding to an, as yet, unidentified receptor. Then, the penton base
binds to .alpha..sub.v integrins. Integrins are a family of heterodimeric
cell surface receptors that mediate cellular adhesion to the extracellular
matrix molecules fibronectin, vitronectin, laminin, and collagen, as well
as other molecules (Hynes, Cell, 69, 11-25 (1992)). Integrins are known to
play important roles in cell signaling processes, including calcium
mobilization, protein phosphorylation, and cytoskeletal interactions
(Hynes, supra). The specificity with which an integrin binds to a
particular ligand, such as those associated with an adenovirus, is a
function of the paired .alpha. and .beta. subunits of the integrin. For
example, integrin .alpha..sub.6 .beta..sub.1 binds to laminin, integrin
.alpha..sub.2 .beta..sub.1 binds to collagen and laminin, and integrin
.alpha..sub.3 .beta..sub.1 binds to collagen, laminin, and fibronectin.
Furthermore, different tissue types may have different complements of
integrin subunits, thereby providing a mode of spatial control over
integrin-ligand signal transfer or over ligand internalization.
Accordingly, some integrins, such as those that include subunit
.alpha..sub.v, are broadly expressed on numerous cell types, whereas other
integrins have a much more narrow tissue distribution. For example, the
integrins that include subunit .beta..sub.2 are expressed only on
leukocytes, such as neutrophils and macrophages, integrins including
subunit .alpha..sub.4 are expressed only on lymphocytes and fibroblasts,
and the integrin defined by the subunits .alpha..sub.IIb 62 .sub.3 is
expressed only on platelets and megakaryocytes.
The specificity of integrin subunit complement also extends to the
infectability of cells by different serotypes of adenovirus, because the
particular .alpha. and .beta. subunits dictate whether a virus can enter a
cell. For example, the penton base of the adenovirus serotype Ad2 binds to
integrins .alpha..sub.v .beta..sub.3 and .alpha..sub.v .beta..sub.5
(Wickham et al. (1993), supra). Given that both receptors utilized by an
adenovirus are expressed on most human cells, nearly all cells in a human
body are susceptible to adenoviral infection.
A majority of integrins have been found to recognize short linear stretches
of amino acids in binding to a specific ligand. The tripeptide motif
arg-gly-asp (RGD) [SEQ ID NO:1], which is found in scores of matrix
ligands, including laminin, fibronectin, collagen, vitronectin, and
fibrinogen, has been implicated in the binding of .alpha..sub.3
.beta..sub.1, .alpha..sub.5 .beta..sub.1, .alpha..sub.IIb .beta..sub.3,
.alpha..sub.m .beta..sub.2, and most, if not all, of the five
.alpha..sub.v -containing integrins. The conformation of the RGD sequence
within a matrix ligand is thought to be a primary factor in integrin
specificity (Pierschbacher et al., J. Biol. Chem., 262, 17294-17298
(1987)). Sequences that directly flank the RGD sequence have been shown to
influence integrin specificity, presumably because of their effect on RGD
conformation (Smith et al., Proc. Natl. Acad. Sci. USA, 90, 10003-10007
(1993)). However, sequences distant from the RGD may also function in
integrin specificity as has been shown for the binding of .alpha..sub.5
.beta..sub.1 to fibronectin (Obara et al., Cell, 53, 649-657 (1988)).
Other integrins, which do not utilize RGD, have been found to bind similar
short linear stretches of amino acids within their specific ligands. For
example, integrin .alpha..sub.IIb .beta..sub.3 binds via the amino acid
sequence lys-gln-ala-gly-asp (KQAGD) [SEQ ID NO:2] in fibrinogen
(Kloczewiak et al., Biochemistry, 23, 1767-1774 (1984)), while
.alpha..sub.4 .beta..sub.1 binds via the core sequence glu-ile-leu-asp-val
(EILDV) [SEQ ID NO:3] in fibronectin (Komoriya et al., J. Biol. Chem.,
266, 15075-15079 (1991)). A structural motif (i.e., asn-pro-xaa-tyr (NPXY)
[SEQ ID NO:4]) present in the .beta. subunits of .alpha..sub.v -containing
integrins has been shown to be important for internalization (Suzuki et
al., Proc. Natl. Acad. Sci. USA, 87, 5354 (1990)).
The penton base sequence is highly conserved among serotypes of adenovirus
and contains five copies of the RGD tripeptide motif (Neumann et al.,
Gene, 69, 153-157 (1988)). The RGD tripeptide is believed to mediate
binding to .alpha..sub.v integrins because exogenously added RGD peptides
can block penton base binding and adenoviral infection (Wickham et al.
(1993), supra), and adenoviruses that have point mutations in the RGD
sequence of the penton base are restricted in their ability to infect
cells (Bai et al., J. Virol., 67, 5198-5205 (1993)).
The penton base genes from Ad2, Ad5, Ad12, and Ad40 serotypes of adenovirus
have been sequenced. Alignment of the sequences reveals a high degree of
conservation over the entire sequence, except for the N-terminus, and, in
Ad2, Ad5, and Ad12, a hypervariable region that includes the RGD sequence.
Only Ad40, one of two enteric adenoviral serotypes, does not have an RGD
sequence. Ad2 and Ad5 are identical in the hypervariable region and
contain a large insert of amino acids flanking either side of the RGD
sequence. Secondary structural analysis of the hypervariable regions of
the three RGD-containing penton bases predicts that, in each case, the RGD
is flanked by .alpha.-helices. Such structures are believed to form the
spikes seen in cryo-electron micrographic (cryo-EM) images of Ad2 penton
bases (Stewart et al. (1993), supra).
Recombinant adenoviral vectors have been used for the cell-targeted
transfer of one or more recombinant genes to diseased cells or tissue in
need of treatment. Such vectors are characterized by the further advantage
of not requiring host cell proliferation for expression of adenoviral
proteins (Horwitz et al., In Virology, Raven Press, New York, vol. 2, pp.
1679-1721 (1990); and Berkner, BioTechniques, 6, 616 (1988)), and, if the
targeted tissue for somatic gene therapy is the lung, these vectors have
the added advantage of being normally trophic for the respiratory
epithelium (Straus, In Adenoviruses, Plenan Press, New York, pp. 451-496
(1984)).
Other advantages of adenoviruses as potential vectors for human gene
therapy are as follows: (i) recombination is rare; (ii) there are no known
associations of human malignancies with adenoviral infections despite
common human infection with adenoviruses; (iii) the adenoviral genome
(which is a linear, double-stranded DNA) can be manipulated to accommodate
foreign genes that range in size; (iv) an adenoviral vector does not
insert its DNA into the chromosome of a cell, so its effect is impermanent
and unlikely to interfere with the cell's normal function; (v) the
adenovirus can infect non-dividing or terminally differentiated cells,
such as cells in the brain and lungs; and (vi) live adenovirus, having as
an essential characteristic the ability to replicate, has been safely used
as a human vaccine (Horwitz et al. (1990), supra; Berkner et al. (1988),
supra; Straus et al. (1984), supra; Chanock et al., JAMA, 195, 151 (1966);
Haj-Ahmad et al., J. Virol., 57, 267 (1986); and Ballay et al., EMBO, 4,
3861 (1985)).
The problem of using a recombinant adenovirus in gene therapy is that all
cells that express the aforementioned two receptors used by the adenovirus
to attach and infect a cell will internalize the gene(s) being
administered--not just the cells in need of therapeutic treatment.
Likewise, certain cells, such as lymphocytes, which lack the .alpha..sub.v
integrin adenoviral receptors, will be severely impaired in the uptake of
an adenovirus and will not be easily amenable to adenovirus-mediated gene
delivery. Accordingly, limiting adenoviral entry to specific cells and/or
expanding the repertoire of cells amenable to adenovirus-mediated gene
therapy would be a significant improvement over the current technology.
Targeted adenoviral gene delivery should expand the cells amenable to gene
therapy, reduce the amount of adenoviral vector that is necessary to
obtain gene expression in the targeted cells, as well as reduce side
effects and complications associated with increasing doses of an
adenovirus, such as inflammation and the transfection of normal, healthy
cells.
Attempts have been made to target a virus to specific cells by sterically
blocking adenoviral fiber protein with antibodies and chemically linking
tissue-specific antibodies to the viral particle (Cotten et al., Proc.
Natl. Acad. Sci. USA, 89, 6094-6098 (1992)). Although this approach has
demonstrated the potential of targeted gene delivery, the complexity and
reproducibility of this approach present major hurdles blocking its
application in clinical trials. The difficulties thus far encountered in
targeting the virus by these methods involve the method of synthesis
required, which is to make major alterations in the viral particles
following their purification. These alterations involve additional steps
that covalently link large molecules, such as polylysine, receptor ligands
and antibodies, to the virus. The targeted particle complexes are not
homogeneous in structure and their efficiency is sensitive to the relative
ratios of viral particles, linking molecules, and targeting molecules
used.
The present invention seeks to overcome the problem of lack of cell and
tissue specificity of recombinant adenoviral gene therapy. It is an object
of the present invention to provide a receptor-specific, preferably cell
receptor-specific/tissue receptor-specific, recombinant adenovirus. A
further object of the present invention is to provide means for generating
such a recombinant adenovirus at the level of gene expression, thereby
enabling purification of recombinant adenoviral particles by conventional
techniques. Another object of the present invention is to provide a method
of gene therapy involving the use of such a homogeneous adenovirus,
without the need for additional components or further modification. These
and other objects and advantages of the present invention, as well as
additional inventive features, will be apparent from the following
detailed description.
BRIEF SUMMARY OF THE INVENTION
The present invention provides a recombinant adenovirus comprising a
chimeric penton base protein that selectively binds to a given receptor,
preferably a cell-specific/tissue-specific receptor, an adenoviral
transfer vector comprising a recombinant penton base gene for the
generation of a chimeric penton base protein, and a method of using a
receptor-specific recombinant adenovirus comprising a therapeutic gene in
gene therapy.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a partial restriction map of an adenoviral transfer vector (pAT).
FIG. 2 is a partial restriction map of the vector pRcPB5 (.DELTA.RGD).
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides, among other things, a recombinant
adenovirus comprising a chimeric penton base protein. The chimeric penton
base protein comprises a nonpenton base amino acid sequence, which is
specific for binding to a receptor, a receptor-specific antibody domain or
epitope, in addition to or in place of a wild-type penton base amino acid
sequence, preferably an amino acid sequence that is specific for binding
to a receptor, most preferably an RGD amino acid sequence.
By "nonpenton base amino acid sequence" is meant any amino acid sequence
that is not found in a wild-type penton base. Preferably, the nonpenton
base sequence is less than ten amino acids, more preferably less than five
amino acids, and most preferably about three amino acids. By "RGD amino
acid sequence" is meant the RGD amino acid sequence and the RGD amino acid
sequence along with up to or including three amino acids flanking either
or both sides of the RGD amino acid sequence. The nonpenton base amino
acid sequence renders the adenovirus specific for a given receptor,
preferably a cell-specific or tissue-specific receptor, or specific for a
given receptor-specific antibody domain or epitope. The receptor is
preferably one that the wild-type adenovirus does not bind or one that the
wild-type adenovirus binds but with greater specificity upon the
introduction of the nonpenton base amino acid sequence into the chimeric
penton base protein.
Preferably, the RGD amino acid sequence of the penton base protein has been
replaced at the DNA level with an amino acid binding sequence for a given
receptor, receptor-specific antibody domain or epitope. Alternatively, the
RGD amino acid sequence has been rendered inactive at the DNA level by
mutation of the RGD amino acid sequence, such as by insertional
mutagenesis, for example, or rendered conformationally inaccessible in the
penton base protein, such as by insertion of a DNA sequence into or
adjacent to the adenoviral penton base gene sequence, wherein "gene
sequence" refers to the complete penton base gene sequence as well as any
lesser gene sequences that are capable of being expressed as functional
penton base protein. Preferably, the DNA sequence is inserted near the
gene sequence encoding the RGD amino acid sequence, so as to move the gene
sequence encoding the RGD amino acid sequence within the penton base gene
sequence such that in the chimeric penton base protein the RGD amino acid
sequence is conformationally inaccessible for binding to a receptor. In
the latter case, the inserted nonpenton base gene sequence that causes the
conformational inaccessibility of the RGD amino acid sequence in the
penton base protein is preferably one that encodes an amino acid sequence
that is specific for binding to a receptor, a receptor-specific antibody
domain or epitope. Such a recombinant adenovirus can be used, for example,
to study receptor binding, adenoviral attachment, and adenoviral infection
in vitro or in vivo.
In a preferred embodiment of the present invention, the above-described
recombinant adenovirus additionally comprises a gene capable of being
expressed in a cell to which the virus has attached or by which the virus
has been internalized and preferably is one having therapeutic utility. In
yet another preferred embodiment of the present invention, the recombinant
adenovirus is fiberless or further comprises a chimeric coat protein, such
as a fiber or hexon, that includes an amino acid sequence that is specific
for a receptor, a receptor-specific antibody domain or epitope, preferably
one that is specific for the same receptor, antibody domain or epitope as
the nonpenton base sequence. Such recombinant adenovirus can be used, for
example, to study the effects of expression of the gene in a given cell or
tissue in vitro or in vivo. Alternatively, the recombinant adenovirus can
be used for gene therapy.
The recombinant adenovirus comprising a chimeric penton base protein and
the recombinant adenovirus that additionally comprises a gene capable of
being expressed in a particular cell can be generated by use of a viral
transfer vector, preferably an adenoviral transfer (pAT) vector, in
accordance with the present invention. The viral transfer vector,
preferably the pAT vector, comprises a chimeric adenoviral penton base
gene sequence. The chimeric penton base gene sequence comprises a
nonpenton base sequence in place of the RGD amino acid sequence, which has
been deleted, or in addition to the RGD amino acid sequence, which has
been mutated or rendered conformationally inaccessible in the expressed
chimeric penton base protein as described above. The nonpenton base
sequence renders the adenovirus specific for binding to a receptor,
receptor-specific antibody domain or epitope also as described above.
Given that the penton base gene has been shown to be >90% conserved among
5 of the approximately 41 serotypes of adenovirus, it is expected that any
one of the serotypes of adenovirus may be used as the source of the penton
base gene. It is preferred, however, that one of the serotypes for which
the penton base gene has been sequenced is used.
Restriction sites that are unique with respect to the adenoviral genome are
introduced into the penton base gene sequence; preferably, such
restriction sites are introduced into or flanking the RGD region of the
penton base gene sequence by a suitable method, such as PCR mutagenesis.
These unique restriction sites may be any restriction site not already
present in the penton base gene, and are preferably Eco RI and/or Spe I.
Such sites facilitate the removal, inactivation, such as by sequence
alteration, of the DNA sequence encoding the RGD amino acid sequence in a
given adenoviral genome, such as a wild-type adenovirus, or the rendering
of the RGD amino acid sequence conformationally inaccessible, thereby
altering or eliminating the ability of the penton base molecule to bind an
.alpha..sub.v integrin receptor. A deleted RGD amino acid sequence can be
replaced with, or a mutated or conformationally inaccessible RGD sequence
can be accompanied by, a different DNA sequence, preferably a DNA sequence
encoding specificity for binding to a receptor, preferably a cell-specific
or tissue-specific cell-surface receptor, or to a receptor-specific
antibody domain or epitope, for example.
Preferably, the pAT vector is one into which any suitable receptor-specific
sequence can be rapidly inserted. For example, a unique Spe I restriction
site can be used to remove the RGD amino acid sequence. Alternatively,
sequences also can be inserted into the penton base gene sequence without
the need for unique restriction sites through PCR. Because a recombinant
adenovirus can be created via ligation of recombinant sequences with viral
DNA or via homologous recombination, the pAT vector preferably has either
(1) unique restriction sites that allow ligation of a vector fragment with
the complementing fragments of the remaining viral genomes, as described
in Example 4, or (2) adequate lengths of DNA on either side of the
receptor-specific or antibody domain- or epitope-specific sequence that
allow efficient homologous recombination with viral DNA, as described in
Example 5. A preferred pAT vector is shown in FIG. 1, which is a partial
restriction map of such a vector. The pAT of FIG. 1 was generated as
described in Example 1.
DNA encoding short peptide sequences or protein domains capable of binding
to a given receptor, preferably a specific cell or tissue receptor, and
capable of being internalized by the receptor, such as the receptor that
is resident on leukocytes, is preferred for insertion into the penton base
gene sequence in which the RGD amino acid sequence has been deleted,
mutated, or rendered conformationally inaccessible. However, other DNA
sequences, such as those that encode receptor-specific antibody domains
and sequences that encode antigenic epitopes recognized by specific
antibodies also may be used to replace the RGD amino acid sequence.
The size of the DNA used to replace the RGD amino acid sequence may be
constrained, for example, by impeded folding of the penton base and
improper assembly of the penton base/fiber complex. However, cryo-EM
analysis of the penton base indicates that, if the RGD amino acid sequence
exists at the spikes of the penton bases, there may be considerable room
to accommodate large inserts without folding or assembly being affected
adversely. Also, the large differences in size between the hypervariable
region of the Ad5 penton base and the Ad12 penton base indicates that the
hypervariable region may be able to accommodate inserts of various sizes.
Of course, assembly of the penton complex probably will not be an issue
with respect to viruses that are constructed without a fiber molecule.
The target receptor can be any receptor that is internalized by a cell, is
optimally cell-specific or tissue-specific, and desirably is expressed
only on those cells or tissue to be treated. Tissue-specific integrins are
preferred target receptors for chimeric penton base molecules for four
reasons. First, many integrins bind ligands via short linear stretches of
amino acids, and these stretches have been identified for many integrins.
Second, certain integrins are tissue-specific for cells amenable to
targeted gene therapy. For example, .alpha..sub.2 -containing integrins
are expressed only in leukocytes, thus allowing targeted delivery to
normally non-susceptible target cells while also preventing delivery to
normally susceptible non-target cells. Third, the wild-type penton base
uses .alpha..sub.v integrins to enter cells so that the use of a related
integrin molecule is more apt to be successful than the use of an
unrelated receptor. Fourth, integrins are efficiently internalized into
the cell (Bretscher, EMBO, 11, 405-410 (1992)), which is required for
viral infection of cells.
Changing the penton base protein such that it recognizes a receptor, such
as a cell-specific or tissue-specific cell-surface receptor, typically
will not alter viral particle attachment to cells because cell attachment
is mediated by the adenoviral fiber protein. Infection of the cells to
which the chimeric penton base protein has attached, however, will be
generally impeded because the virus requires the presence of an
.alpha..sub.v integrin to enter the cell. Therefore, an adenovirus with
altered penton bases will bind to most cells but will enter only those
cells expressing the receptor (e.g., the integrin) for the chimeric penton
base. Those cells unable to express the appropriate receptor nonetheless
will have adenoviral particles tethered on the cell membrane because the
viral particles will be unable to enter the cell. Consequently, tethered
adenoviral particles will be exposed to the immune system and disposed of
accordingly.
Following fiber-mediated viral attachment to cells, it is possible that
some virus may manage to non-specifically enter cells not expressing the
cell- or tissue-specific receptor recognized by the chimeric penton base.
Therefore, some leakiness in cell-specific or tissue-specific expression
may be observed. However, if this is the case, fiber-deficient viral
particles can be constructed that use the penton base as the primary cell
attachment protein. It is known that fiber protein is nonessential for
virion assembly or infectivity of cells (Falgout et al., J. Virology, 62,
622-625 (1988)). Such mutant adenoviral particles lacking fiber protein
adsorb onto cells at a ten-fold lower rate. The fact that the mutant
viruses do adsorb and at a lower rate than wild-type particles indicates
that the adsorption is mediated by the penton base/.alpha..sub.v integrin
interaction, which has a 30-fold lower affinity than the
fiber/fiber-receptor interaction (Wickham et al. (1993), supra).
Accordingly, both attachment and internalization can be restricted only to
those cells expressing the desired tissue-specific receptor.
By replacing the low affinity RGD amino acid sequence in a wild-type penton
base with an amino acid sequence with a high affinity for a given
receptor, such as a cell-specific or tissue-specific receptor, it is
possible to generate a fiberless viral particle with as high, or higher,
affinity for targeted cells than the wild-type fiber-expressing virus.
Fiberless viral particles containing a chimeric penton base, compared to
fiber-containing viral particles, can be targeted more effectively to
tissues because the altered virus is able to bind only to the cells
expressing the cell-specific or tissue-specific receptor recognized by the
chimeric penton base. The desired ligand and receptor characteristics are
as described above.
A recombinant chimeric penton base gene sequence can be moved from a pAT
vector into baculovirus or a suitable prokaryotic or eukaryotic expression
vector for expression and evaluation of receptor specificity, avidity, and
other biochemical characteristics. Accordingly, the present invention also
provides recombinant baculoviral and prokaryotic and eukaryotic expression
vectors comprising a chimeric adenoviral penton base gene sequence. The
chimeric penton base gene sequence includes a nonpenton base sequence in
addition to or in place of a wild-type penton base amino acid sequence,
such as an RGD amino acid sequence, which is specific for binding to a
receptor. The wild-type penton base amino acid sequence may be deleted,
mutated, or rendered conformationally inaccessible as described above with
respect to the recombinant adenovirus comprising a chimeric penton base
protein. By moving the chimeric gene from pAT to baculovirus or a
prokaryotic or eukaryotic expression vector, high protein expression is
achievable, resulting in approximately 50% of the total protein of the
host being the chimeric penton base. Accordingly, the present invention
also provides a recombinant baculovirus comprising a chimeric penton base
protein and a chimeric adenoviral penton base protein comprising a
nonpenton base amino acid sequence in addition to or in place of a
wild-type penton base amino acid sequence, such as an RGD amino acid
sequence, which is specific for binding to a receptor. The nonpenton base
amino acid sequence is specific for binding to a receptor or a
receptor-specific antibody domain or epitope as described above. For
protein characterization studies, the recombinant chimeric penton base
protein (rcPB protein, such as rcPB5) can be purified using any suitable
methods, such as those described by Wickham et al. (1993), supra.
Various characteristic parameters of the penton base protein of interest
can be assessed as follows: Adhesion assays are used to evaluate the
specificity of the interaction of the rcPB protein with its designated
receptor, using the method of Wickham et al. (1993), supra, for example.
Specificity and affinity of the receptor/rcPB interaction is assessed by
Scatchard analysis as shown previously by Wickham et al. (1993), supra,
for wild-type penton base protein. Receptor specificity is further
assessed by using antibodies and peptides specific for the targeted
receptor to block rcPB5 binding to cells, using conventional methods.
Internalization is assayed with rcPB protein and recombinant adenoviruses,
e.g., Ad5 rcPB, as described previously by Wickham et al. (1993), supra,
for example. rcPB binding to fiber protein is assessed by its ability to
precipitate radiolabeled fiber molecules when coupled to protein A-coated
beads via an antibody to the penton base molecule. Alternatively, fiber
binding can be assessed in an ELISA-based assay in which rcPB is coated
onto ELISA plates followed by incubation of immobilized rcPB with soluble
fiber molecules, using conventional methods. Fiber binding to the rcPB
protein is then assessed by a further incubation with antibody specific
for the fiber molecule, via conventional techniques.
Virus entry and gene expression are evaluated initially by using the pAT
vector containing the insert of interest to generate recombinant virus
expressing the chimeric penton base protein and a marker gene, such as
.beta.-glucuronidase. .beta.-glucuronidase expression in cells infected
with adenovirus containing the .beta.-glucuronidase gene (Ad-Gluc) can be
detected as early as two hours after adding Ad-Gluc to cells. This
procedure provides a quick and efficient analysis of cell entry of the
recombinant virus and gene expression, and is implemented readily by an
artisan of ordinary skill using conventional techniques.
A recombinant virus, which lacks a wild-type receptor binding sequence,
such as the RGD amino acid sequence, in the penton base protein, can be
produced in human embryonic cell line 293 (HEK 293), which has been shown
to be able to replicate recombinant adenoviral particles lacking the RGD
sequence, using previously described techniques (Bai et al. (1993),
supra). A recombinant virus, which lacks functional fiber proteins and in
which the penton base acts as both the viral attachment and
internalization protein, may be produced in cell lines which express the
receptor to which the rcPB5 protein is targeted. For example, Ad5 rcPB5
containing an insert specific for .alpha..sub.v .beta..sub.3 can be
produced in HeLa cells that express .alpha..sub.v .beta..sub.3 or 293
cells transfected with the gene for .alpha..sub.v .beta..sub.3.
The chimeric penton base protein may be present in the viral particle with
the fiber, without the fiber (in which case the rcPB protein functions as
the virus attachment and internalization protein), with a recombinant
fiber molecule that is specific for a receptor, such as a cell-specific or
tissue-specific receptor, or with a recombinant coat protein that is
specific for a receptor, such as a cell-specific or tissue-specific
receptor. See Examples 4-9 for a further description of these embodiments
of the invention.
Recombinant adenoviruses of the present invention can be used to treat any
one of a number of diseases by delivering to targeted cells corrective
DNA, i.e., DNA encoding a function that is either absent or impaired, or a
discrete killing agent, e.g., DNA encoding a cytotoxin that, for example,
is active only intracellularly, or DNA encoding ribozymes or antisense
molecules, for example. Accordingly, use of the term "therapeutic gene" is
intended to encompass these and other embodiments of that which is more
commonly referred to as gene therapy and is known to those of skill in the
art. Diseases that are candidates for such treatment include, for example,
cancer, e.g., melanoma or glioma, cystic fibrosis, genetic disorders, and
pathogenic infections, including HIV infection. For example, a recombinant
adenovirus having a penton base molecule recognized by .alpha..sub.v
.beta..sub.3 receptors can be used to treat melanoma or glioma, and a
recombinant adenovirus recognized by .alpha..sub.3 .beta..sub.1 receptors
and expressing the cystic fibrosis transmembrane regulator (CFTR) gene can
be used to treat cystic fibrosis by delivery to the epithelial cells of
the lungs. Furthermore, various blood-related diseases can be treated by
using a recombinant adenovirus recognized by .alpha..sub.m .beta..sub.2
receptors to target neutrophils and macrophages, a recombinant adenovirus
recognized by .alpha..sub.4 .beta..sub.1 receptors to target lymphocytes,
a recombinant adenovirus recognized by .alpha..sub.IIb .beta..sub.3
receptors to target platelets and megakaryocytes, and a recombinant
adenovirus recognized by .alpha..sub.v .beta..sub.3 integrins to target
endothelial cells undergoing angiogenesis.
One skilled in the art will appreciate that suitable methods of
administering a recombinant adenovirus of the present invention to an
animal for purposes of gene therapy (see, for example, Rosenfeld et al.,
Science, 252, 431-434 (1991); Jaffe et al., Clin. Res., 39 (2), 302A
(1991); Rosenfeld et al., Clin. Res., 39 (2) , 311A (1991); Berkner,
BioTechniques, 6, 616-629 (1988)) , chemotherapy, and vaccination are
available, and, although more than one route can be used to administer
such a recombinant adenovirus, a particular route can provide a more
immediate and more effective reaction than another route. Pharmaceutically
acceptable excipients are also well-known to those who are skilled in the
art, and are readily available. The choice of excipient will be determined
in part by the particular method used to administer the recombinant
adenovirus. Accordingly, there is a wide variety of suitable formulations
for use in the context of the present invention. The following methods and
excipients are merely exemplary and are in no way limiting.
Formulations suitable for oral administration can consist of (a) liquid
solutions, such as an effective amount of the compound dissolved in
diluents, such as water, saline, or orange juice;. (b) capsules, sachets
or tablets, each containing a predetermined amount of the active
ingredient, as solids or granules; (c) suspensions in an appropriate
liquid; and (d) suitable emulsions. Tablet forms can include one or more
of lactose, mannitol, corn starch, potato starch, microcrystalline
cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose
sodium, talc, magnesium stearate, stearic acid, and other excipients,
colorants, diluents, buffering agents, moistening agents, preservatives,
flavoring agents, and pharmacologically compatible excipients. Lozenge
forms can comprise the active ingredient in a flavor, usually sucrose and
acacia or tragacanth, as well as pastilles comprising the active
ingredient in an inert base, such as gelatin and glycerin, or sucrose and
acacia, emulsions, gels, and the like containing, in addition to the
active ingredient, such excipients as are known in the art.
The recombinant adenovirus of the present invention, alone or in
combination with other suitable components, can be made into aerosol
formulations to be administered via inhalation. These aerosol formulations
can be placed into pressurized acceptable propellants, such as
dichlorodifluoromethane, propane, nitrogen, and the like. They may also be
formulated as pharmaceuticals for non-pressured preparations such as in a
nebulizer or an atomizer.
Formulations suitable for parenteral administration include aqueous and
non-aqueous, isotonic sterile injection solutions, which can contain
anti-oxidants, buffers, bacteriostats, and solutes that render the
formulation isotonic with the blood of the intended recipient, and aqueous
and non-aqueous sterile suspensions that can include suspending agents,
solubilizers, thickening agents, stabilizers, and preservatives. The
formulations can be presented in unit-dose or multi-dose sealed
containers, such as ampules and vials, and can be stored in a freeze-dried
(lyophilized) condition requiring only the addition of the sterile liquid
excipient, for example, water, for injections, immediately prior to use.
Extemporaneous injection solutions and suspensions can be prepared from
sterile powders, granules, and tablets of the kind previously described.
Additionally, the recombinant adenovirus of the present invention may be
made into suppositories by mixing with a variety of bases such as
emulsifying bases or water-soluble bases.
Formulations suitable for vaginal administration may be presented as
pessaries, tampons, creams, gels, pastes, foams, or spray formulas
containing, in addition to the active ingredient, such carriers as are
known in the art to be appropriate.
The dose administered to an animal, particularly a human, in the context of
the present invention will vary with the gene of interest, the composition
employed, the method of administration, and the particular site and
organism being treated. However, the dose should be sufficient to effect a
therapeutic response.
In addition to the recombinant adenovirus of the present invention, the
adenoviral transfer vector also has utility in vitro. It can be used as a
research tool in the study of adenoviral attachment and infection of cells
and in a method of assaying receptor-ligand interaction. Similarly, the
recombinant penton base protein comprising a nonpenton base amino acid
sequence in addition to or in place of a wild-type receptor binding
sequence, preferably the RGD sequence, can be used in receptor-ligand
assays and as adhesion proteins in vitro or in vivo, for example.
The following examples further illustrate the present invention and, of
course, should not be construed as in any way limiting its scope.
EXAMPLE 1
This example describes the construction of the adenoviral transfer vector
(pAT) for making chimeric penton base molecules.
pAT, a partial restriction map of which is shown in FIG. 1, was created by
cloning the unique Bam HI/Pme I fragment (13259-21561) from the Ad5 genome
into pNEB 193 R1-, a minor derivative of pNEB 193 (New England Biolabs,
Beverly, Mass.), from which the unique Eco RI restriction site was removed
(SEQ ID NO:7). The resulting vector was called pNEB 193 R1- [Ad5 (Bam
HI/Pme I)].
Two pairs of PCR primers were synthesized and used to amplify a region
upstream (left side) and a region downstream (right side) from the RGD
sequence. A unique Spe I site was inserted into the antisense primer
(A5a(15147)S, SEQ ID NO:16) used to amplify the upstream region between
the RGD sequence and a Bst XI site (15017). The Bst XI site is 35 bp
upstream from the penton base start codon (15052). A unique Spe I site was
also inserted into the sense primer (A5a(15204)S, SEQ ID NO:15) to amplify
the right side of the gene between the RGD amino acid sequence and an As | | |
