Note: Descriptions are shown in the official language in which they were submitted.
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Novel Promoter Elements For Persistent Gene Fxn ion
The invention is directed to novel promoter elements for the persistent
expression of a transgene which is delivered to a target cell. The promoter
elements
are derived from the cytomegalovirus intermediate early promoter (CMV). The
invention is also directed to enhancer elements which, when placed upstream
(or 5') to
the novel promoter elements of the invention operably linked to a transgene,
increase
the levels of expression of the transgene. In particular embodiments of the
invention,
adenoviral vectors or plasmids comprising the CMV-derived promoter elements,
operatively linked to a transgene, are used to achieve persistexit expression
of the
transgene.
The transfer of a transgene to recipient cells such that a biologically
active and useful product (e.g. RNA, antisense RNA, protein, hormone, enzyme,
etc.)
is produced in the recipient cell is an important aspect of effective gene
transfer.
There are two important parameters for consideration - the effective transfer
of such
transgenes to recipient cells and tissues and the persistent or continued
expression of
the transgene in the target cell.
Transfer of transgenes to target cells has been the focus of much
inquiry and experimentation - leading to the development of various methods
and
modalities to accomplish such transfer. Transgene transfer has involved the
use and
development of viral vectors, such as those derived from retroviruses,
adenoviruses,
herpes viruses, vaccinia and adeno-associated virus, among others. Also,
transgene
transfer has been effectuated using plasmids, naked DNA, lipids and
combinations of
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all of these. For in vivo transgene delivery, much attention has focused on
the use of
viral vectors, particularly those derived from adenovirus.
Adenovirus is a non-enveloped, nuclear DNA virus with a genome size
of about 36 kb, which has been well-characterized through studies in classical
genetics
and molecular biology (Horwitz, M.S., "Adenoviridae and Their Replication," in
Virolo~,v, 2nd edition, Fields et al., eds., Raven Press, New York, 1990). The
viral
genes are classified into early (known as El-E4) and late (known as L1-L5)
transcriptional units, representing two temporal classes of viral proteins.
The
demarcation between early and late viral protein expression is viral DNA
replication.
The human adenoviruses are divided into numerous serotypes (approximately 47,
numbered accordingly and classified into 6 subgroups: A, B, C, D, E and F),
based
upon properties including hemaglutination of red blood cells, oncogenicity,
nucleic
acid and amino acid compositions and relatedness, and antigenic relationships.
Recombinant adenoviruses have several advantages for use as
transgene transfer vectors, including tropism for both dividing and non-
dividing cells,
minimal pathogenic potential, ability to replicate to high titer for
preparation of vector
stocks, and the potential to carry large inserts (Berkner, K.L., Curr. Top.
Micro.
Immunol. 158:39-66, 1992; Jolly, D., Cancer Gene Therapy 1:51-64, 1994).
The cloning capacity of an adenovirus vector is proportional to the size
of the adenovirus genome present in the vector. For example, a cloning
capacity of
about 8 kb can be created from the deletion of certain regions of the virus
genome
dispensable for virus growth, e.g., E3, and the deletion of a genomic region
such as E1
whose function may be restored in trans from 293 cells {Graham, F.L., J. Gen.
Virol.
36:59-72, 1977) or A549 cells (Imler et al., Gene Therapy 3:75-84, 1996). Such
E1-
deleted vectors are rendered replication-defective. The upper limit of vector
DNA
capacity for optimal carrying capacity is about 105%-108% of the length of the
wild-type genome. Further adenovirus genomic modifications are possible in
vector
design using cell lines which supply other viral gene products in trans, e.g.,
complementation of E2a (Zhou et al., J. Virol. 70:7030-7038, 1996),
complementation of E4 (Krougliak et al., Hum. Gene Ther. 6:1575-1586, 1995;
Wang
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et al., Gene Ther. 2:775-783, 1995), or complementation of protein IX (Allowed
U.S.
Patent Application Serial No. 08/895,194, incorporated herein by reference;
Caravokyri et al., J. Virol. 69:6627-6633, 1995; Krougliak et al., Hum. Gene
Ther.
6:1575-1586, 1995). Maximal carrying capacity can be achieved using adenoviral
S vectors deleted for all viral coding sequences (Kochanek et al., Proc. Natl.
Acad. Sci.
USA 93:5731-5736, 1996; Fisher et al., Virology 217:11-22, 1996).
Transgenes that have been expressed to date by adenoviral vectors
include, inter alia, p53 (Wills et al., Human Gene Therapy 5:1079-188, 1994);
dystrophin (Vincent et al., Nature Genetics 5:130-134, 1993; erythropoietin
(Descamps et al., Human Gene Therapy 5:979-985, 1994; ornithine
transcarbamylase
(Stratford-Perricaudet et al., Human Gene Therapy 1:241-256, 1990; We et al.,
J. Biol.
Chem. 271;3639-3646, 1996;); adenosine deaminase (Mitani et al., Human Gene
Therapy 5:941-948, 1994); interleukin-2 (Haddada et al., Human Gene Therapy
4:703-711, 1993); and al-antitrypsin (Jaffe et al., Nature Genetics 1:372-378,
1992);
thrombopoietin (Ohwada et al., Blood 88:778-784, 1996); and cytosine deaminase
(Ohwada et al., Hum. Gene Ther. 7:1567-1576, 1996).
The particular tropism of adenoviruses for cells of the respiratory tract
has relevance to the use of adenovirus in transgene transfer for cystic
fibrosis (CF),
which is the most common autosomal recessive disease in Caucasians. Mutations
in
the cystic fibrosis transmernbrane conductance regulator (CFTR) gene that
disturb the
cAMP-regulated Cl- channel in airway epithelia result in pulmonary dysfunction
(Zabner et al., Nature Genetics 6:75-83, 1994). Adenovirus vectors engineered
to
carry the CFTR gene have been developed (Rich et al., Human Gene Therapy
4:461-476, 1993) and studies have shown the ability of these vectors to
deliver CFTR
to nasal epithelia of CF patients (Zabner et al., Cell 75:207-216, 1993), the
airway
epithelia of cotton rats and primates (Zabner et al., Nature Genetics 6:75-83,
1994),
and the respiratory epithelium of CF patients (Crystal et al., Nature Genetics
8:42-51,
1994). Recent studies have shown that administering an adenoviral vector
containing
a DNA encoding CFTR to airway epithelial cells of CF patients can restore a
functioning chloride ion channel in the treated epithelial cells (Zabner et
al., J. Clin.
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Invest. 97:1504-1511, 1996; U.S. Patent No. 5,670,488, issued September 23,
1997).
Additionally, tissue specific expression can be obtained from adenovirus
vectors by
the use of tissue specific promoter sequences. See, for example, PCT
publication WO
97/04117 which describes a liver specific adenovirus expression vector
characterized
in that a therapeutic transgene which is coupled to a liver-specific promoter
consisting
of enhancer elements of the hepatitis B virus and an enhancer-less minimal
promoter
and is optionally surrounded by SAR elements is inserted in the adenovirus
genome.
The use of adenovirus vectors in transgene transfer studies to date
indicates that persistence of transgene expression is often transient. At
least some of
the limitation is due to the generation of a cellular immune response to the
viral
proteins which are expressed even from a replication-defective vector,
triggering a
pathological inflammatory response which may destroy or adversely affect the
adenovirus-infected cells (Yang et al., J. Virol. 69:2004-2015, 1995; Yang et
al., Proc.
Natl. Acad. Sci. USA 91:4407-4411, 1994; Zsengeller et al., Hum Gene Ther.
6:457-
467, 1995; Worgall et al., Hum. Gene Ther. 8:37-44, 1997; Kaplan et al., Hum.
Gene
Ther. 8:45-56, 1997). Because adenovirus does not integrate into the cell
genome,
host immune responses that destroy virions or infected cells have the
potential to limit
adenovirus-based transgene transfer. Long-term expression can be achieved if
the
recombinant adenoviral vector is introduced into nude mice or mice that are
given
both the adenoviral vector and immunosupressing agents. Dai, Y. et al., 1995,
Proc.
Natl. Acad Sci. USA Q~:1401-1405. These results bolster the notion that the
immune
response is behind the short-term expression that is usually obtained from
adenoviral
vectors. Verma, LN. and Somia, N., 1997, Science ,~,$Q:239-242. An adverse
immune
response can pose a serious obstacle for high dose administration of an
adenovirus
vector and for repeated administration (Crystal, R., Science 270:404-410,
1995).
In order to circumvent the host immune response which can contribute
to limiting the persistence of transgene expression, various strategies have
been
employed, generally involving either the modulation of the immune response
itself or
the engineering of a vector that decreases the immune response. The
administration of
immunosuppressive agents together with vector administration has been shown to
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prolong transgene expression (Fang et al., Hum. Gene Ther. 6:1039-1044, 1995;
Kay
et al., Nature Genetics 11:191-197, 1995; Zsellenger et al., Hum. Gene Ther.
6:457-
467, 1995) and allow for repeat administration of the vector. Engelhardt et
al. (Proc.
Natl. Acad. Sci. USA ,Q~:6196-6200 ( I 994)) have demonstrated that the use of
broad
5 innunosupressants can overcome the immunologic problems associated with
repeat
administration of Adenovirus vectors. Similarly, Dai et al. ((Proc. Natl. Acad
Sci.
USA x:1401-1405 (1995)) have shown similar results using cytoablative agents
to
overcome the immune response of the host to first generation adenovirus
vectors.
However, although immunosuprressive drugs can extend the duration of
expression
obtained from adenoviral vectors, it is more desirable to manipulate the
vector to
achieve prolonged expression rather than the patient. Verma, LN. and Somia,
N.,
1997, Science ~$Q:239-242.
Modifications to genomic adenoviral sequences contained in the
recombinant vector have been attempted in order to decrease the host immune
response (Yang et ai., Nature Genetics 7:362-369, 1994; Lieber et al., J.
Virol.
70:8944-8960, 1996; Gorziglia et al., J. Virol. 70:4173-4178; Kochanek et al.,
Proc.
Natl. Acad. Sci. USA 93:5731-5736, 1996; Fisher et al., Virology 217:11-22,
1996).
The adenovirus E3 gp 19K protein can complex with MHC Class I antigens and
retain
them in the endoplasmic reticulum, which prevents cell surface presentation
and
killing of infected cells by cytotoxic T-lymphocytes (CTLs) (Wold et al.,
Trends
Microbiol. 437-443, 1994), suggesting that its presence in a recombinant
adenoviral
vector may be beneficial.
The lack of persistence in the expression of adenovirus vector-
delivered transgenes may also be due to limitations imposed by the choice of
promoter
or transgene contained in an expression cassette or a transcription unit (Guo
et al.,
Gene Therapy 3:802-801, 1996; Tripathy et al., Nature Med. 2:545-550, 1996).
Experiments in which adenovirus vectors were introduced into nude
and immunocompetent mice demonstrated that the E4 region to the adenovirus
genome contributed to persistent transgene expression, especially when the
transgene
was operably linked to a wild-type CMV promoter which controlled the transgene
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,;
6
(Kaplan et al., Hum. Gene Ther. 8:45-56, 1997; Armentano et al., J. Virol.
71:2408-
2416, 1997; PCT publication WO 98/46781 ).
In addition to the use of adenoviral vectors, plasmids comprising a
transgene may be used to deliver the transgene to a target cell. It is
desirable that
plasmids for use as gene transfer vehicles also be able to replicate in the
recipient cells
or tissues of individuals, since continued presence of the plasmid could
provide
correction of the genetic defect (in the case of cystic fibrosis, a vector may
provide a
functioning CFTR protein in the cell membrane of lung epithelial cells or
other cells
to replace the non-functioning or missing endogenous protein) over an extended
period of time. There has been some concern that plasmids that cannot
replicate in the
targeted cells or tissues may be degraded after only a relatively short period
of
maintenance therein, thus requiring excessive repeat administrations.
Long term correction of a missing or faulty gene product in a cell could
perhaps be achieved using a vector designed to integrate into chromosomes in
the
targeted cells (for example, vectors patterned on retrovirus). Such a
strategy,
however, involves risks including ( 1 ) that the vector will integrate into an
essential
region of a chromosome, (2) that the vector will integrate adjacent to an
oncogene and
activate it.
Accordingly, one strategy has been to provide for continued
maintenance of plasmids in target cells by other means. One such strategy is
to
construct a plasmid capable of being maintained separately in the nucleus of a
target
(i.e. in an episome). C. McWhinney et al. ( Nucleic AcidaResea_rch, 18, 1233-
1242,
1990) have determined that the 2.4 kb HindIII-XhoI fragment that is present
immediately 5' to exon 1 of the human c-myc gene contains an origin of
replication
which when cloned into a plasmid and transfected into HeLa cells was shown to
allow
the plasmid to persist in the cells for more than 300 generations under drug
selection.
Replication was shown to be semiconservative (C. McWhinney et al.).
Approximately 5% of the plasmid population was lost per cell generation
without
drug selection demonstrating substantial stabilization which would be of
benefit with
respect to the design of therapeutic plasmids for gene therapy.
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One example of a replicating episomal vector, pCFI-CAT (PCT
publication WO 96118372 Figure 18A), may be constructed in which a copy of the
2.4
kb HindIII-XhoI fragment is placed just 5' to the CMV enhancer/promoter region
of
the pCF 1 backbone. Alternatively, between 2 and about 4 - in tandem - copies
of the
2.4 kb fragment may be similarly positioned. The increase in plasmid size that
results
from insertion of the 2.4 kb fragment (or multiple copies thereof j is
predicted to
provide an additional benefit, that is, to facilitate plasmid unwinding, thus
facilitating
the activity of DNA polymerase. See PCT publication WO 96/18372, incorporated
herein by reference. Use of this origin of replication, or multiple copies
thereof,
allows the resultant plasmid to replicate efficiently in human cells. Other
DNAs
comprising other origins of replication may also be used (for example, as
found in the
human p-globin gene, or the mouse DHFR gene). A plasmid containing the
cytomegalovirus promoter and enhancer, an intron, the CFTR cDNA, the bovine
growth hormone polyadenylation signal, the kanamycin resistance transposon
Tn903,
and 4 copies of the 2.4 kb 5' flanking region of the human c-myc gene is shown
in
Figure 20 of WO 96/18372.
Further optimization of adenoviral vectors and plasmids for persistent
transgene expression in target cells and tissues may also involve the design
of
expression control elements, such as promoters, which confer persistent
expression to
an operably linked transgene. Promoter elements which function independently
of
particular viral genes to confer persistent expression of a transgene may
allow the use
of vectors which contain reduced viral genomes, increasing the carrying
capacity of
the vector while decreasing the potential for host immune reaction or the
generation of
replication-competent viruses.
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The invention is directed to a novel promoter element far persistent
expression of an operably linked transgene. In one aspect, the element is
derived from
the cytomegalovirus intermediate early promoter (CMV). In an embodiment of the
invention, an adenoviral vector comprising a CMV-derived promoter element
operably linked to a transgene is administered to recipient cells. In another
embodiment of the invention, a piasmid comprising a CMV-derived promoter
element
of the invention operably linked to a transgene is administered to recipient
cells. The
plasmid may also be delivered to a cell in conjunction with a lipid, such as
those
disclosed in WO 96/18372 or U.S. Patent No. 5,650,096. Also within the scope
of the
invention are enhancer elements~derived from the human albumin gene whicl when
operably linked to the CMV-derived promoter elements of the invention increase
the
expression of a transgene operably linked to the promoter elements. The
invention is
also directed the use of such adenoviral vectors and plasmids comprising the
enhancer
and promoter elements of the invention in transgene transfer.
Figure 1. Sequence of the CMV intermediate early promoter, showing
nucleotides -523 to -14 (SEQ ID NO:1).
Figure 2. Sequence of a CMV-derived promoter element of the
invention, showing nucleotides -295 to -14 (SEQ ID N0:2).
Figure 3. Sequence of a CMV-derived promoter element of the
invention, showing nucleotides -299 to -19 (SEQ ID N0:3).
Figure 4. Sequence of a CMV-derived promoter element of the
invention, showing nucleotides -242 to -14 (SEQ ID N0:4)
Figure S. Sequence of a human albumin gene-derived enhancer
element of the invention showing a 65 nucleotide sequence found 1.7 kilobases
upstream from the transcription initiation start site of the human albumin
gene (SEQ
ID N0:4)
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Figure 6. Schematic representation of transcriptional repressor
binding sites in the CMV promoter.
Figure 7. Expression of ~3-galactosidase in rat hepatocytes using a
promoter of the invention.
Figure $. Expression of p-galactosidase in human hepatocytes using a
promoter of the invention.
Figure 9. Expression of ~i-galactosidase in Balb/c lungs using a
promoter of the invention.
Figure 10. Expression of CAT in mice using a promoter of the
invention.
Figure 11. Increased expression of a transgene operably linked to a
CMV-derived promoter element of the invention through the use of enhancer
elements
derived from the human albumin gene placed S' to the CMV-derived promoter
element in 293 cells and Hep3B cells.
Deta,'_led Description of t_he Invention
The present invention is directed to a novel promoter element for the
persistent expression of an operably linked transgene. In one aspect, the
element is
derived from the cytomegalovirus intermediate early promoter (CMV). In an
embodiment of the invention, an adenoviral vector comprising a CMV-derived
promoter element of the invention operatively linked to a transgene is used to
achieve
persistent expression of a transgene when administered to target cell. In
another
embodiment, a plasmid comprising CMV-derived promoter element operably linked
to a transgene is used to achieve persistent expression of a transgene when
administered to a target cell. Also within the scope of the invention are
enhancer
elements which effectuate increased expression of a transgene operably linked
to
CMV-derived promoter elements. In one aspect, the enhancer elements are
derived
from the human albumin gene. The invention is also directed to an expression
cassette or transcription unit comprising at least a CMV-derived promoter
element of
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IO _i
the present invention and a transgene. The expression cassette or
transcription unit
may also comprise an enhancer element.
A transgene is defined as a nucleic acid molecule coding for, inter alia,
a protein (e.g. an enzyme, a hormone, a cell-surface molecule), ribozyme, RNA,
and
antisense RNA heterologous to the carrier vector. Such a transgene may be
delivered
to a cell or tissue for example, but not by way of limitation , by a viral
vector, a
plasmid, a lipid, including a liposome, naked DNA, combinations thereof or
other
means known to those of skill in the art for delivery of transgenes.
Persistent
expression is defined as generating and maintaining a sustained level of
expression of
a transgene over time.
A CMV-derived promoter element of the invention is defined as a
promoter element which contains a nucleotide sequence derived from the wild-
type
cytomegalovirus {CMV) immediate early promoter {Boshart et al., Cell 41:521-
530,
1985, incorporated herein by reference) (Figure 1 ) (SEQ ID NO: I ), and
provides for
persistent expression of a transgene operably linked thereto.
Particular embodiments of the invention include a CMV-derived
promoter element containing nucleotides -295 to -14 (Figure 2) (SEQ ID N0:2),
a
CMV-derived promoter element containing nucleotides 299 to -19 of the CMV
promoter (Figure 3) (SEQ ID N0:3), and a CMV-derived promoter element
containing nucleotides -242 to -14 of the wild-type CMV promoter (Figure 4)
(SEQ
ID N0:4) (referred to as OCMV promoter elements).
Other promoter elements, which are within the scope of the invention,
are also derived from the nucleotide sequence of the CMV promoter and confer
persistent expression to an operably linked transgene in a target cell. Where
a wild-
type CMV promoter is dependent upon adenoviral E4 sequences to confer
persistent
expression (see, e.g. WO 98/46781 ), a promoter element of the invention may
be
identified by its ability to confer persistent expression of a transgene when
delivered
to a cell in an adenoviral vector lacking the E4 region. In another
embodiment,
promoter elements which are capable of conferring persistent expression may be
constructed, for example, by deletion of sites within the CMV promoter
sequence to
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11
which transcription repressor proteins can bind. Removal of such sites from
the wild-
type CMV promoter may lead to more sustained expression (see Figure 6).
Examples
of such repressor proteins include YY1 (Liu et al., Nucleic Acids Research
22:2453-
2459, 1994; Gualberto et al., Mol. Cell Biol. 12:4209-4214, I 992; Galvin et
al., Mol:
Cell Biol. 17:3723-3732, 1997); MDBP (Zhang et al., Nucleic Acids Res. 18:6253-
6260; Zhang et al., Virology 182: 865-869; Supekar et al., Nucleic Acids Res.
16:8029-8044, 1988); IE2 (Liu et al., J.Virol. 65:897-603, 1991), CREB/CREM
(Foulkes et al., Cell 64:739-749, 1991; Karpinski et al., Proc.Natl. Acad.
Sci. USA
89:4820-4824, 1992; Lamph et al., Proc.Natl.Acad.Sci.USA 87:4320-4324, 1990;
Lemaigre et al., Nucleic Acids Res. 21:2907-2911, 1993) and Drl (Kim et al.,
Proc.Natl.Acad.Sci.USA 94:820-825, 1997; White et al., Science 266:448-450,
1994).
Three YY1 binding sites are located in the wild-type CMV promoter between -300
and -522 relative to the transcriptional start site. Also, there are at least
five potential
binding sites for CREB and three binding sites for methylation-dependent
binding
protein. In addition, repressors such as Drl can also act on the core promoter
complex. One skilled in the art can readily remove any of these sites by
standard
techniques of recombinant DNA technology. Alternatively, other CMV-derived
promoter elements that are within the scope of the invention can retain or add
in any
nucleotides that correspond to transcriptional activator sites in order to
achieve
persistent expression. Such activators, include, for example, NFkappa~i
(Boshart et
al., Cell 41:521-530, 1985; Chang et al., J.Virol. 64:264-277, 1990; Neller et
al.,
Nucleic Acids Res. 19:3715-3721, 1991). Nucleotide sequences in the native CMV
promoter to which transcriptional repressor and activator proteins bind are
known to
those skilled in the art.
CMV-derived promoter elements of the invention can be engineered
using standard techniques of molecular biology, such as restriction enzyme
digestion,
polymerase chain reaction (PCR), and site-directed mutagenesis. A CMV-derived
promoter element can be operably linked to a particular transgene by standard
techniques known in molecular biology for ligating DNA fragments. Furthermore,
nucleotide substitutions within the CMV-derived promoter elements of the
invention
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that allow the promoter elements to retain the capability for persistent
expression of a
transgene are within the scope of the invention. Such nucleotide substitutions
can
include those that, for example, alter the binding sites for the
transcriptional repressor
proteins discussed above (e.g. YY1), such that the repressors can no longer
bind.
Preferred CMV-derived promoter elements of the invention, which
have capability to confer persistent expression of a transgene, include those
which
contain nucleotides -295 to -14, -299 to -19 and -213 to -14. Other
truncations of the
wild-type CMV promoter to create CMV-derived promoter elements which are
within
the scope of the invention include, but are not limited to, those containing
nucleotides
-406 to -19; -299 to -10; -299 to +1; and -299 to +31; -277 to -19; -277 to -
14; and -
213 to -19.
CMV-derived promoter elements of the invention can also comprise
transcription factor binding sites which can be added, for example, to the 5'
end of a
CMV-derived promoter element of the invention. Such sites are known to those
skilled in the art.
Additionally, CMV-derived promoter elements of the invention may
include cellular promoter sequences which contribute to persistent expression
of the
operably linked transgene. Such sequences can be derived from, for example but
not
by way of limitation, actin, mucin, and other constitutive cellular promoters.
Also within the scope of the invention are promoter elements derived
from wild-type promoters other than CMV which exhibit dependence on the
adenovirus E4 region for persistent transgene expression, such as the Rous
sarcoma
virus (RSV). For example, an RSV-derived promoter element can be constructed
to
delete or alter the serum response elements (SRE) to which the transcriptional
repressor protein YY1 can bind, so as to create a promoter element which can
confer
persistent expression to an operably linked transgene (Gualberto et al., Mol.
Cell Biol.
12:4209-4214, 1992).
Transgenes which can be delivered and expressed from a promoter
element of the invention include, but are not limited to, those encoding
enzymes,
blood derivatives, hormones, Iymphokines such as the interleukins and
interferons,
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coagulants, growth factors, neurotransmitters, tumor suppressors,
apolipmteins,
antigens, and antibodies, and other biologically active proteins. Specific
transgenes
which may be operably linked to the promoter elements of the invention
include, but
are not limited to, cystic fibrosis transmembrane conductance regulator
(CFTR),
dystrophin, glucocerebrosidase, tumor necrosis factor, p53, retinoblastoma
(Rb), von-
hippel lindau (VHL), pten tumor suppressor, p 16, Glut4, and adenosine
deaminase
(ADA). Transgenes encoding antisense molecules and ribozymes are also within
the
scope of the invention. Gene transfer vehicles of the invention, such as
adenoviral
vectors or plasmids, may contain one or more transgenes operably linked to a
CMV-
derived or other promoter element of the invention.
In accordance with the present invention, an adenoviral vector or
plasmid for gene transfer not only comprises the promoter element of the
invention
operably linked to a DNA encoding a transgene but may also comprise any other
expression control sequences such as another promoter or enhancer , a
polyadenylation element and any other regulatory elements that may be used to
modulate or increase expression or a transgene when operably linked thereto.
Non-
limiting examples of enhancer elements include apoE enhancer elements
(Shachter
N.S., etal., 1993, JLipid Res. 34:1699-707; Allan C.M. et al., 1995, J. Biol.
Chem.
270:26278-81 ), a 1 antitrypsin enhancer elements (Morgan K. et al., 1997,
Biochim.
Biophys. Acta. 1362:67-76. , human fibrinogen enhancer elements (Hu C.H. et
al.,
1995 J. Biol. Chem. 270:28342-9) the human cytochrome P4501A1 gene enhancer
elements (Kress, S. et al., Eur. J. Biochem. 258:803-812, 1998), the human
carboxyl
ester lipase gene enhancer elements (Lidberg, U. et al., J. Biol. Chem.
273:31417-
31426, 1998), porcine alpha-skeletal actin gene enhancer elements (Reecy, J.M.
et al.,
Anim. Biotechnol. 9:101-120, 1998) and human albumin gene enhancer elements
positioned at -1.7 and -6 kb upstream from the transcriptional start site of
the wild-
type human albumin gene (Hayashi, Y, et al., J. Biol. Chem. 267:14580-14585,
1992;
incorporated herein by reference).
In particular embodiments of the invention, an enhancer element of the
invention is derived from human albumin gene enhancer sequences and, when
placed
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14 ."
5' to the CMV-derived promoter elements of the invention operably linked to a
transgene, increases the expression levels of the transgene (Figure 11 ). The
use of any
other expression control sequences, or regulatory elements, which facilitate
persistent
expression of the transgene is also within the scope of the invention. Such
sequences
or elements may be capable of generating tissue-specific expression or be
susceptible
to induction by exogenous agents or stimuli. Polyadenylation signals which may
be
positioned at the 3' end of the transgene in a transcription unit or
expression cassette
include, but are not limited to, those derived from bovine growth hormone
(BGH)
and SV40.
A human albumin gene-derived enhancer element of the invention is
defined as an enhancer element which contains a nucleotide sequence derived
from
enhancer sequences found 1.7 kiiobases (TTGTCAATTAGTAACAA; SEQ ID NO:S)
and 6.0 kilobases (GCCAAACA; SEQ ID N0:6) upstream from the transcriptional
initiation site of the wild-type human albumin gene (Hayashi, Y. et al., J.
Biol. Chem.
267:14580-14585, 1992), and provides for increased expression of a transgene
operably linked to a CMV-derived promoter element of the invention.
Preferred human albumin gene-derived enhancer elements of the
invention which have the ability to increase the expression of a transgene
operably
linked to the CMV-derived promoter elements of the invention include a 65
nucleotide sequence located -1797 to -1737 bases upstream from the
transcriptional
initiation site of the wild-type human albumin gene comprising a 17 nucleotide
enhancer element (-1.7kb enhancer element) (Figure 5) (SEQ ID N0:7). Another
enhancer element within the scope of the invention is located -6 kilobases
from the
human albumin gene transcriptional start site (-6kb enhancer element) (SEQ ID
N0:6).
In a particular embodiment of the invention, adenoviral vectors can be
used to deliver a transgene which is operably linked to a CMV-derived promoter
element of the invention to target cells in order to achieve persistent
expression of a
desired protein. However, the promoter elements of the invention may also be
used
with other viral vectors useful for gene transfer, including, but not limited
to, those
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derived from retroviruses, herpesviruses, adeno-associated virus, and others
known to
those skilled in the art.
Specific examples of adenoviral vectors which can be used in the
invention include, for example, Ad2/CFTR-1 and Ad2/CFTR-2 and others described
5 in U. S. Patent No. 5,670,488, issued September 23, 1997, (incorporated
herein by
reference). Adenoviral vectors may include deletion of the E 1 region, partial
or
complete deletion of the E4 region, and deletions within, for example, the E2
and E3
regions. For example, the vectors can contain all, part or none of the E4
region of the
adenoviral genome because the CMV-derived promoter elements of the present
10 invention confer persistent expression in the absence of the E4 region.
Such vectors,
therefore, may include, if desired, the ORF3, ORF4 or ORF6 open reading frames
from the E4 region. The vectors are preferably replication-defective, that is,
they are
incapable of generating a productive infection in the host cell. Within the
scope of the
invention are also, for example, chimeric viral vectors which contain an Ad 2
15 backbone with one or more heterologous capsid proteins or fragments thereof
(see
PCT publication No. WO 98/22609, incorporated herein by reference, and allowed
U.S. application Serial No. 08/752,760, filed November 20, 1996, allowed
October
16, 1998 incorporated herein by reference). Other adenoviral vectors include
those
derived from U.S. Patent No. 5,707,618 and U.S. Patent No. 5,824,544 (both
incorporated herein by reference). In a particular embodiment, the CMV-derived
promoter elements of the invention can be used to confer persistent expression
of a
transgene in E4-deleted adenoviral vectors, allowing for the design of such
vectors
with increased carrying capacity, and reduced potential for the generation of
a host
immune response or replication-competent viruses, all of which are desirable
features
for a vector used for gene transfer in vivo.
In further preferred embodiments, adenoviral vectors can also be
constructed using adenovirus serotypes from the well-studied group C
adenoviruses,
especially Ad2 and AdS. Adl7 is also a preferred serotype. Moreover,
adenoviral
vectors for use in the invention derived from other group C or non-group C
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adenoviruses are also within the scope of the invention, including chimeric
adenovirai
vectors which contain nucleotide sequences from one or more serotypes.
In order to construct an adenoviral vector for use in the invention,
reference may be made to the substantial body of literature on how such
vectors may
be designed, constructed and propagated using techniques from molecular
biology and
microbiology that are well-known to the skilled artisan. For example, the
skilled
artisan can use the standard techniques of molecular biology to engineer a
transgene
operably linked to a promoter element, preferably a CMV-derived promoter
element,
of the invention into a backbone vector genome (Berkner, K.L., Curr. Top.
Micro.
Immunol. 158:39-66, 1992). For example, a plasmid containing a transgene and
any
operably linked CMV-derived promoter element of the invention inserted into an
adenoviral genomic fragment may be co-transfected with a linearized viral
genome
derived from an adenoviral vector of interest into a recipient cell under
conditions
whereby homologous recombination occurs between the genomic fragment and the
virus. In a preferred embodiment, the transgene and the operably linked CMV-
derived promoter element of the invention are inserted into the site of an E1
deletion.
As a result, the transgene is inserted into the adenoviral genome at the site
in which it
was cloned into the plasmid, creating a recombinant adenoviral vector. The
adenoviral
vectors may also be constructed using standard ligation techniques.
Construction of the adenoviral vectors may be based on adenovirus
DNA sequence information widely available in the field, e.g., nucleic acid
sequence
databases such as GenBank.
Preparation of replication-defective adenoviral vector stocks may be
accomplished using cell lines that complement viral genes deleted from the
vector,
e.g., 293 or A549 cells containing the deleted adenovirus E1 genomic
sequences.
HERS cells (human embryonic retinoblasts transformed by Ad 12) may also be
used.
After amplification of plaques in suitable complementing cell lines, the
viruses may
be recovered by freeze-thawing and may subsequently be purified using cesium
chloride centrifugation. Alternatively, virus purification may be performed
using
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chromatographic techniques, e.g., as disclosed in PCT Publication No. WO
97/08298,
incorporated herein by reference.
Titers of replication-defective adenoviral vector stocks may be
determined by plaque formation in a complementing cell line, e.g., 293 cells.
End-
s point dilution using an antibody to the adenoviral hexon protein may be used
to
quantitate virus production or infection efficiency of target cells (Anmentano
et ai.,
Hum. Gene Ther. 6:1343-1353, 1995, incorporated herein by reference).
An example of an adenoviral vector containing a CMV-derived
promoter element of the invention is ~CMV-~3ga1-1, which comprises a CMV-
derived
promoter element comprising nucleotides -295 to -14, operably linked to a (i-
galactosidase gene, and the SV40 polyadenylation signal, in an E1 deletion
that is
further deleted for the E4 region.
Plasmids which may be used to deliver a transgene operably linked to a
CMV-derived promoter element of the invention can be may be engineered using
standard recombinant DNA technology. Large scale production and purification
of
such plasmids may be performed using techniques known to those skilled in the
art
(see, gsg" Cu_r_re_n_t protocols in Molec ~lar Biology .Ausubel et al., eds.,
John Wiley &
Sons, Inc., New York, 1995). Plasmids may be delivered to target cells using
such
techniques as transfection, electroporation, microinjection, and other DNA
transfer
methods known to those skilled in the art. Plasmids may also be delivered in
conjunction with a lipid, e.g. a cationic lipid such as N4-spenmine
cholesteryl
carbamate and N4-spermidine cholesteryl carbamate as disclosed in U.S. Patent
No.
5,650,096 and PCT publication WO 96/18372, both incorporated herein by
reference.
The delivery of a transgene operably linked to a promoter element of the
invention to
a target cell in the form of naked DNA is also within the scope of the
invention.
Where the transgene is a masker or reporter gene, it may be used as to
determine the persistence of expression using a CMV-derived promoter element
of the
invention. A nonlimiting example is a plasmid such as pCF 1-CAT (PCT
publication
WO 96/18372 Figure 18A), containing the chloramphenicol acetyltransferase
(CAT)
gene operatively linked to the wild-type CMV promoter which may be truncated
to
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generate the CMV-derived promoter elements of the invention operably linked to
CAT. Other marker genes within the scope of the invention include, but are not
limited to, the genes encoding ~i-galactosidase and luciferase. Proteins
expressed
from marker genes may be readily detected by standard techniques.
In a preferred embodiment, the plasmid pCFA-299/-19 CAT (Example
4 below) is used as a plasmid backbone to construct a plasmid for transgene
transfer to
a target cell, in which the CAT marker gene is replaced by a transgene of
interest.
Infection of target cells by adenoviral vectors or plasmids comprising a
transgene operably linked to a CMV-derived promoter element of the invention
may
also be facilitated by the use of cationic molecules, such as cationic lipids
disclosed in
U.S. Patent No. 5,650,096 and PCT Publication No. WO 96/18372, published June
20, 1996, both incorporated herein by reference. Adenoviral vectors complexed
with
cationic molecules are also described in U.S. Application Serial No.
08/755,035, filed
November 22, 1996 and PCT Publication No. WO 98/22144, incorporated herein by
reference.
Cationic amphiphiles have a chemical structure which encompasses
both polar and non-polar domains so that the molecule can simultaneously
facilitate
entry across a lipid membrane with its non-polar domain and attach to a
biologically
useful molecule to be transported across the membrane with its cationic polar
domain.
Cationic amphiphiles which may be used to form complexes with the
adenoviral vectors or plasmids of the invention include, but are not limited
to, cationic
lipids such as those disclosed in U.S. Patent No. 5,650,096, PCT publication
No. WO
96/18372, and PCT publication No. WO 98/43994; DOTMA (Felgner et al., Proc.
Natl. Acad. Sci. USA 84:7413-7417, 1987) (N-[1-{2,3-dioletloxy)propy!]-N,N,N -
trimethylammonium chloride); DOGS (dioctadecyIamidoglycylspermine) (Behr et
al.,
Proc. Natl. Acad. Sci. USA 86:6982-6986, 1989); DMRIE (1,2-dimyristyloxypropyl-
3-dimethyl-hydroxyethyl ammonium bromide) (Felgner et al., J. Biol. Chem.
269:2550-2561, 1994; and DC-chol (3B [N-N', N'-dimethylaminoethane) -
carbamoyl]
cholesterol) (IJ.S. Patent No. 5, 283,185). The use of other cationic
amphiphiies
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recognized in the art or which may later be discovered is also within the
scope of the
invention.
In preferred embodiments of the invention, the cationic amphiphiles
useful to complex with and facilitate transfer of the vectors and plasmids of
the
invention are those lipids disclosed in U.S. Patent No. 5,650,096 and in PCT
Publication No. WO 96/18372, published June 20, 1996, both incorporated herein
by
reference. Preferred cationic amphiphiles described herein to be used in the
delivery
of the plasmids and/or viruses include, inter alia, GL-53, GL-67, GL-75, GL-87
and
GL-89, including protonated, partially protonated, and deprotonated forms
thereof as
set forth Figures 1, 7 and 9 of WO 9b/18372. Further embodiments include the
use of
non-T-shaped amphiphiles as disclosed in the aforementioned patent
publications,
including protonated, partially protonated and deprotonated forms thereof.
Most
preferably, the cationic amphiphile which can be used to deliver the vectors
and
plasmids of the invention is either N4-spermine cholesteryl carbamate (GL-67)
having
the following formula (I)
(I)
or N4-spermidine cholesteryl carbamate (GL-53) having the following formula
(II)
(II)
In the formulation of compositions comprising the adenoviral vectors
and plasmids used in the invention, one or more cationic amphiphiles may be
formulated with neutral co-lipids such as dileoylphosphatidylethanolamine
(DOPE) to
facilitate delivery of the vectors into a cell. Other co-lipids which may be
used in
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these complexes include, but are not limited to,
diphytanoylphosphatidylethanolamine, lyso-phosphatidylethanolamines, other
phosphatidylethanolamines, phosphatidylcholines, lyso-phosphatidylcholines and
cholesterol. A preferred molar ratio of cationic amphiphile to colipid is I
:1.
However, it is within the scope of the invention to vary this ratio, including
also over
a considerable range. In a particularly preferred embodiment of the invention,
the
cationic amphiphile N4-spermine cholesterol carbamate (GL-67) having the
formula
10 and the neutral co-lipid DOPE are combined in a 1:2 molar ratio,
respectively, before
compIexing with an adenoviral vector for delivery to a cell.
In the formulation of complexes containing a cationic amphiphile with
an adenoviral vector comprising the CMV-derived promoter element of the
invention,
a preferred range of 10' - 10'° infectious units of virus may be
combined with a range
15 of 104 - 106 cationic amphiphile molecules/viral particle. In the
formulation of
complexes containing a cationic amphiphile with a plasmid, a preferred range
of from
.4 mM - 1 mM of cationic amphiphile may be combined with a range of 3 mM - 8
mM of plasmid to form the complexes.
Infection efficiency from adenoviral vectors containing the CMV-
20 derived promoter elements of the invention may be assayed by standard
techniques.
Such methods include, but are not limited to, plaque formation, end-point
dilution
using, for example, an antibody to the adenoviral hexon protein, and cell
binding
assays using radiolabelled virus. Improved infection efficiency may be
characterized
as an increase in infection of at least one order of magnitude with reference
to a
control virus.
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Persistent expression of a transgene from adenoviral vectors
comprising the promoter elements of the invention following the infection of
target
cells or persistent expression from plasmids comprising the promoter elements
of the
invention following transfection, electroporation or other method of plasmid
transfer
to target cells may be assayed by standard techniques. Where an adenoviraI
vector or
plasmid comprising the promoter element of the invention encodes a marker or
other
transgene, relevant molecular assays to determine expression include the
measurement
of transgene mRNA, by, for example, but not by way of limitation, Northern
blot, S 1
analysis or reverse transcription-polymerase chain reaction (RT-PCR). The
presence
of a protein encoded by a transgene may be detected by Western blot,
immunoprecipitation, immunocytochemistry, or other techniques known to those
skilled in the art. Marker-specific assays can also be used, such as X-gal
staining of
cells infected with an adenoviral vector encoding (3-galactosidase.
Preferred target cells which can be used in tissue culture to determine
persistence of transgene expression from an adenoviral vector comprising a
transgene
operably linked to a promoter element of the invention include, but are not
limited to,
primary cells such as hepatocytes, airway epithelial cells, muscle cells and
endothelial
cells. Preferred target cells for determining the persistence of transgene
expression
from a plasmid containing a transgene operably linked to a promoter element of
the
invention include established cell lines, such as HeLa or COS cells, or
primary cells.
Any cells or cell lines which may be transfected with the plasmids or infected
with the
viruses comprising a transgene operably Linked to a promoter element of the
invention
are suitable for assays which measure the level and duration of expression of
such a
transgene. Demonstration of persistent expression of a transgene from
adenoviral
vector or plasmid comprising a transgene operably linked to a promoter element
of the
invention in, for example, animal and/or human hepatocytes can be predictive
of the
ability of such a plasmid or virus to achieve persistent expression of the
transgene in
the liver of an animal or human.
In order to determine the persistence of transgene expression and
infection efficiency in vivo using constructs and compositions according to
the present
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invention, animal models may be particularly relevant in order to assess
transgene
expression persistence against a background of potential host immune response.
Such a model may be chosen with reference to certain parameters such as ease
of
delivery, identity of transgene, relevant molecular assays, and assessment of
clinical
status. Where the transgene encodes a protein whose absence or mutation is
associated with a particular disease state, an animal model which is
representative of
the disease state may optimally be used in order to assess a specific
phenotypic result
and clinical improvement through the persistent expression of the transgene.
For example, knockout mice (e.g. Fabry knockout mice (Ohshima et
al., 1997, Proc. Natl. Acad. Sci. USA 94:2540-2544) and CFTR knockout mice
(Zeiher, B.G et al., 1995, J. Clin. Invest. 98:2051-2064)) may be infected or
transfected with vectors comprising the expression cassettes of the present
invention
which comprise at least a CMV-derived promoter element and a transgene. Such
knockout mice may be used to assess the biological activity and persistent
expression
1 S of a transgene of interest. Specifically, but not by way of limitation, an
expression
cassette of the present invention, comprising at least a CMV-derived promoter
element and a-galactosidase as the transgene, may be administered to Fabry
knockout
mice in order to assess persistent transgene expression of the gene,
biological activity
of the expressed transgene and clinical improvement of the knockout mice (see
U.S.
Patent Application Serial No. 09/182,245, filed October 29, 1998 and PCT
Application No. PCT/LJS98/22886, filed October 29, 1998, incorporated herein
by
reference). Similarly, an expression cassette of the present invention
comprising at
least a CMV derived promoter element and the CFTR as the transgene may be
administered to CFTR knockout mice to assess persistent transgene expression,
biological activity of the expressed transgene and clinical improvement of the
knockout mice. See Scaria, A. et al., 1998, Journal of virology 72:7302-7309,
U.S.
Patent Application Serial No. 08/839,553, filed April 14, 1997 and PCT
Publication
No. WO 98/467.80, incorporated herein by reference).
It is also possible that particular adenoviral vectors may display
enhanced infection efficiency only in human model systems, e.g., using primary
cell
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cultures, tissue explants, or permanent cell lines. In such circumstances
where there is
no animal model system available in which to model the infection efficiency of
an
adenoviral vector with respect to human cells, reference to art-recognized
human cell
culture models may be relevant and definitive.
Relevant animals in which the adenoviral vectors or plasmids may be
assayed include, but are not limited to, mice, rats, monkeys, and rabbits.
Suitable
mouse strains in which the vectors may be tested include, but are not limited
to, C3H,
C57BL/6 {wild-type and nude) and Balb/c (available from Taconic Farms,
Germantown, New York).
Where it is desirable to assess the host immune response to vector or
plasmid administration, testing in immunocompetent and immunodeficient animals
may be compared in order to define specific adverse responses generated by the
immune system. The use of immunodeficient animals, e.g., nude mice, may be
used
to characterize vector or plasmid performance and persistence of transgene
expression, independent of an acquired host response.
In a particular embodiment where the transgene is the gene encoding
cystic fibrosis transmembrane conductance regulator protein (CFTR) which is
administered to the respiratory epithelium of test animals, expression of CFTR
may be
assayed in the lungs of relevant animal models, for example, C57BL/6 or Balb/c
mice, cotton rats, or Rhesus monkeys.
Molecular markers which may used to determine expression include
the measurement of CFTR mRNA, by, for example, Northern blot, S 1 analysis or
RT-
PCR. The presence of the CFTR protein may be detected by Western blot,
immunoprecipitation, immunocytochemistry, or other techniques known to those
skilled in the art. Such assays may also be used in tissue culture where cells
deficient
in a functional CFTR protein which have been infected with the adenoviral
vectors
may be assessed to determine the presence of functional chloride ion channels -
indicative of the presence of a functional CFTR molecule. See, for example,
Zabner
et al., J. Clin. Invest. 97:1504-15I 1 (199b).
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The adenoviral vectors and plasmids comprising the promoter elements
of the invention have a number of in vivo and in vitro utilities. The vectors
and
plasmids can be used to transfer a normal copy of a transgene encoding a
biologically
active protein to target cells in order to remedy a deficient or dysfunctional
protein.
The vectors and plasmids can be used to transfer marked transgenes (e.g.,
containing
nucleotide alterations) which allow for distinguishing expression levels of a
transduced transgene from the levels of the corresponding endogenous gene. The
adenoviral vectors can also be used to def ne the mechanism of specific viral
protein-
cellular protein interactions that are mediated by specific virus surface
protein
sequences. The adenoviral vectors can also be used to optimize infection
efficiency of
specific target cells by adenoviral vectors, for example, but not by way of
limitation,
using a chimeric adenoviral vector containing Ad 17 fiber protein to infect
human
nasal polyp cells (e.g. PCT Publication No. WO 98/22609 incorporated herein by
reference). Where it is desirable to use an adenoviral vector for transgene
transfer to
cancer cells in an individual, an adenoviral vector can be chosen which
selectively
infects the specific type of target cancer cell and avoids promiscuous
infection.
Where primary cells are isolated from a tumor in an individual requiring
transgene
transfer, the cells may be tested against a panel of adenoviral vectors and
plasmids to
select a vector or plasmid with optimal infection efficiency for transgene
delivery.
The vectors can further be used to transfer transgenes encoding tumor antigens
to
dendritic cells which can then be delivered to an individual to elicit an anti-
tumor
immune response. The adenoviral vectors can also be used to evade undesirable
immune responses to particular adenovirus serotypes which compromise the gene
transfer capability of adenoviral vectors.
The present invention is further directed to compositions which
comprise the adenoviral vectors and plasmids comprising the promoter elements
of
the invention which can be administered to cells or tissues in an amount
effective to
deliver one or more desired transgenes to the cells of an individual in need
of such
molecules and cause expression of a transgene encoding a biologically active
protein
to achieve a specific phenotypic result or to produce the biologically active
protein.
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The cationic amphiphile-plasmid complexes or cationic amphiphile-virus
complexes
similarly may be formulated into compositions for administration to an
individual in
need of the delivery of the transgenes.
The compositions can include physiologically acceptable carriers,
5 including any relevant solvents. As used herein, but not by way of
limitation,
"physiologically acceptable carrier" includes any and all solvents, dispersion
media,
coatings, antibacterial and antifungal agents, isotonic and absorption
delaying agents,
and the like. Except insofar as any conventional media or agent is
incompatible with
the active ingredient, its use in the compositions is contemplated.
10 Routes of administration for the compositions comprising the
adenoviral vectors or plasmids of the invention include conventional and
physiologically acceptable routes such as, but not limited to, direct delivery
to a target
organ or tissue, intranasal, intravenous, intramuscular, subcutaneous,
intradermal, oral
and other parenteral routes of administration.
1 S The invention is further directed to methods for using the compositions
of the invention in in vivo or ex vivo applications in which it is desirable
to deliver one
or more transgenes into cells such that the transgene produces a biologically
active
protein for a normal biological or phenotypic effect. In vivo applications
involve the
direct administration of one ore more adenoviral vectors or plasmids
formulated into a
20 composition and delivered to the cells of an individual. Ex vivo
applications involve
the direct transfer of compositions comprising the vector or plasmid to
autoIogous
cells which are maintained in vitro, followed by readministration of the
transduced
cells to a recipient.
Dosage of the adenoviral vector or plasmid to be administered to an
25 individual for expression of a transgene encoding a biologically active
protein and to
achieve a specific phenotypic result is determined with reference to various
parameters, including the condition to be treated, the age, weight and
biological or
clinical status of the individual, and the particular molecular defect
requiring the
furnishing of a biologically active protein. The dosage is preferably chosen
so that
administration causes a specific phenotypic result, as measured by molecular
assays or
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clinical markers. For example, determination of the infection efi'xciency of
an
adenoviral vector or plasmid containing the CFTR transgene which is
administered to
an individual can be performed by molecular assays including the measurement
of
CFTR mRNA, by, for example, Northern blot, S 1 or RT-PCR analysis or the
measurement of the CFTR protein as detected by Western blot,
immunoprecipitation,
immunocytochemistry, or other techniques known to those skilled in the art.
Relevant
clinical studies which could be used to assess phenotypic results from
delivery of the
CFTR transgene include, but are not limited to, PFT assessment of lung
function and
radiological evaluation of the lung. Productive delivery of a transgene
encoding
CFTR may also be demonstrated by detecting the presence of a functional
chloride
channel in cells of an individual with cystic fibrosis to whom the vector
comprising
the transgene has been administered (Zabner et al., 1996, J. Cli». Invest.
97:1504-
1511 and Scaria, A. et al., 1998, Journal of Virology 72:7302-7309). Transgene
expression and phenotypic alteration associated with transgene expression can
be
assayed analogously, using the specific biological parameters most relevant to
the
condition.
Dosages of an adenoviral vector comprising the promoter elements of
the invention which are effective to provide expression of a transgene
encoding a
biologically active protein and achieve a specific phenotypic result range
from
approximately 108 infectious units (LU.) to 10" LU. for humans.
It is especially advantageous to formulate parenteral compositions in
dosage unit form for ease of administration and uniformity of dosage. Dosage
unit
form as used herein refers to physically discrete units suited as unitary
dosages for the
subjects to be treated, each unit containing a predetermined quantity of
active
ingredient calculated to produce the specific phenotypic effect in association
with the
required physiologically acceptable carrier. The specification for the novel
dosage
unit forms of the invention are dictated by and directly dependant on the
unique
characteristics of the adenoviral vector or plasmid and the limitations
inherent in the
art of compounding. The principal active ingredient (the adenoviral vector or
plasmid) is compounded for convenient and effective administration in
effective
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amounts with the physiologically acceptable carrier in dosage unit form as
discussed
above.
Maximum benefit and achievement of a specific phenotypic result from
administration of the adenoviral vectors and plasmids of the invention may
require
repeated administration. Such repeated administration may involve the use of
the
same adenoviral vector or plasmid, or, alternatively, may involve the use of
different
adenoviral vectors which are rotated in order to alter viral antigen
expression and
decrease host immune response.
The practice of the invention employs, unless otherwise indicated,
conventional techniques of protein chemistry, molecular virology,
microbiology,
recombinant DNA technology, and pharmacology, which are within the skill of
the
art. Such techniques are explained fully in the literature. See, e.g., Current
Protocols
11i~1~S~ Biol4~C, Ausubel et al., eds., John Wiley & Sons, Inc., New York,
1995,
and Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co.,
Easton,
PA, 1985.
The invention is further illustrated by the following specific examples
which are not intended in any way to limit the scope of the invention.
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28
EXAMP 1:
~CMV~igal-1 is based on Ad2/~igal-5 (complete E4 deletion,
Arnnentano et al. 1997, J. Virol. 71:2408-2416, 1997) and contains a promoter
element which is a truncated CMV promoter, containing nucleotide sequences -
295 to
-14 (see Figure 2; SEQ ID NO. 2). A pre-virus plasmid, pAdCMV(3ga1 (Armentano
et
al. 1997, J. Virol. 7I :2408-2416, 1997) was cut with restriction
endonucleases CIaI
and SnaBI which removes all sequences of the CMV promoter upstream of the
SnaBI
site (-242). The removed sequences were replaced with a CIaI - SnaBI
oligonucleotide adapter (containing CMV promoter sequences -295 to -242; see
Figure 4, SEQ ID N0:4) to extend promoter sequences to the -295 position. The
resulting plasmid, pAd~CMV(3ga1-l, was cut with BstBI and recombined with
PshAI
digested Ad2/(3ga1-S DNA in VK2-20 cells to generate ~CMV~igal.
Rat hepatocytes were isolated from Sprague-Dawley rats by perfusion
with .OS% collagenase, washed with Hepato-Stim media (Beckton-Dickinson)
several
times and plated in a hepatocyte differentiation environment (Becton-
Dickinson). The
following day hepatocytes were infected with Ad2/~3ga1-4, Ad2/~igal-2 or
OCMVpgal-
1 at an moi of 50. The media was changed every other day throughout the course
of
the experiment. At the indicated time points cultures were treated with
dispase to
remove cells from the extracellular matrix. See Figure 7. Cells were pelleted,
washed
with PBS, pelleted again and resuspended in lysis buffer. The supernatant was
analyzed for ~i-galactosidase activity by Galactolight assay and protein was
determined by BCA. As shown in Figure 7, expression from Ad2/(3gal-2 is
diminished and does not persist in comparison to Ad2/~igal-4. Expression from
OCMV(3gal-I is not diminished compared to Ad2/~igal-4 and also appears to
persist.
Because the OCMV~3gal-l vector is E4 deleted, the results indicate that ~i-gal
expression from the OCMV promoter does not require E4.
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29
Human hepatocytes were obtained from Clonetics and were maintained
in Hepatocyte Maintenance media (Clonetics). Cultures were infected at an moi
of 50
with Ad2/~igal-4, Ad2/~3ga1-2 or OCMV~igal-1 alone or were co-infected with
Ad2/CMVAAT, a vector that could supply E4 function in traps. Cells were
harvested
at the indicated time points (Figure 8) by incubation with dispase and
analyzed as in
Example 1 for (3-galactosidase activity.
The results in Figure $ indicate that expression from Ad2/(3ga1-4 could
persist to day 11 and was not further enhanced by the co-infection of a virus
that
supplies E4 functions in traps. Expression from Ad2/~igal-2 was diminished on
days 3
and l 1 in comparison to that observed from Ad2/~3ga1-4. In addition,
expression was
enhanced on days 3 and 11 when cultures were co-infected with Ad2/CMVAAT and
reached levels detected in Ad2/~igal-4 infected cultures. Expression from
OCMV~igai-1 on days 3 and 11 was in the range of levels seen with Ad2/~igal-4
and
was not further enhanced by co-infection with Ad2/CMVAAT. The results indicate
that the OCMV promoter element does not require E4 for maintained elevated
levels
of expression and is no longer influenced by supplying E4 in traps.
lrf~ect of a CMV-derived nrom~tPr P1P",a
, ~nt on tran_sgene expression in B ib/c iunQS.
Balb/c nude mice were intranasally instilled with 3 x 109 i.u. of
Ad2/~3gai-4 or OCMV~3ga1-1 or were co-infected with both vectors. Mice were
sacrif ced on days 3 and 14 post-instillation and the lungs were analyzed for
~i-
galactosidase activity by a Galactolight assay. Results of the single
infections and co-
infections represent n=2 and n=3 per time point respectively.
Expression from the OCMV promoter element persisted in the lungs of
Balb/c mice which indicates that this promoter element can give rise to long-
term
expression in vivo in the absence of E4 ORFs. Although the ~CMV promoter
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element can function independently of E4 in this experimental system,
expression was
enhanced on day 14 by approximately 4-fold by the co-infection of a vector
that could
supply E4 in trans (Figure 9).
5
in mice.
Plasmid pCF 1-299/-19-CAT was constructed by first digesting pCF 1-
SEAP (pCFI plasmid containing the gene for secreted alkaline phosphatase
{SEAP)
and an additional upstream polylinker called PCFA) {Yew et al., Hum Gene Ther.
10 8:575-584, 1997) with Pme I and Bgl I, blunting the ends with the Klenow
fragment
of DNA polymerase I, then religating. This vector was digested with Not I to
excise
SEAP and the CAT cDNA was ligated into the Not I site to form pCFI-299-CAT.
This vector was then digested with Sac I and Xba I blunted with Klenow, then
relegated. The promoter element in the plasmid comprises the sequence of
Figure 3
15 (SEQ ID NO. 3).
Cationic Iipid:DOPE:pDNA complexes were prepared as described
previously {Lee et al., Hum. Gene Ther. 7:1701-1717, 1996; U.S. Patent No.
5,650,096 and PCT Publication No. WO 96/18372, all incorporated herein by
reference ). Briefly, equal volumes of liposomes and plasmid DNA were mixed to
a
20 final concentration of 0.6 mM GL-67: 1.2 mM DOPE: 3.6 mM pCFA-299/-19-CAT
and allowed to sit 15 minutes at room temperature. Nude BALB/c mice were
instilled
intranasally with 100 pl of lipid:pDNA complex as described previously (Lee et
al.,
Hum. Gene Ther. 7:1701-1717, 1996; U.S. Patent No. 5,650,096, WO 96/18372).
Mice were instilled within 15 minutes of complex formation. At different days
post-
25 instillation, lungs were harvested and frozen at -80 ° C for later
processing. CAT
activity was assayed as described in the afore-mentioned references.
The results show that the initial level of expression from pCFl-299/-
19CAT is lower than pCFI-CAT (1 ng of CAT from pCFl-299/-19-CAT versus 26 ng
of CAT from pCF 1-CAT at day 2 post-instillation). However, expression from
pCF 1-
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31
299/-19-CAT is more persistent than pCF1-CAT, with approximately 40% of day 2
levels present at day 35 post-instillation versus less than 1% of day 2
expression from
pCFI-CAT (Figure 10).
Effect of a hu_m__a_n_ albumin gene-derived a h~ncer ~el~e n o ratio ly 1,
i~yr ,k,~~t
CMV-derived Dr01'riOtPr P~PmPntc nn arlPnnvira) ~iP~tnr-, rovi~ggene ex
Plasmids pBsl.7-2HI-AGAL, pBsl.7-3HI-AGAL, and pBsl.'7-SHI-
AGAL were constructed as follows: A double stranded 65bp fragment comprising
the
-l.7kb human albumin-derived enhancer element (SEQ ID N0:7) was generated by
annealing two complimentary oligos (sythesized by Operon, Alameda, CA) by
standard techniques. See, Wig,,, C~r_rent Protocols in h~olecu r Biolo Ausubel
et
al., eds., John Wiley & Sons, Inc., New York, 1995. The annealed double
stranded
65bp fragment comprising the -l.7kb human albumin-derived enhancer element has
5'
overhangs that can be ligated into a CIaI restriction site. Multiple copies
were ligated
into pBluescriptIIsk+ (Strategene, La Jolla, CA) which was digested with CIaI
restriction enzyme. PbluescriptIIsk+ vectors containing 2, 3 or 5 copies were
isolated
and were digested with EcoRV and XbaI. The digested vectors were ligated to a
SnaBI-XbaI digested fragment of the wild-type CMV promoter (-242 to -14) (SEQ
ID
N0:4), a hybrid intron (from pAD~3, Clonetech), wild-type a-galactosidase cDNA
and
the SV40 polyadenylation signal. Plasmids pBsl.7-2HI-AGAL, pBsl.7-3HI-AGAL,
and pBsl.7-SHI-AGAL contain 2, 3 and 5 copies of the 65bp -1.7 human albumin-
derived enhancer element respectively.
293 cells were obtained from Frank Graham and were maintained in
DMEM supplemented with 1 mM L-glutamine and 10% fetal bovine serum. Hep3B
cells (hepatocellular cell line; ATCC) were maintained in Eagle's minimum
essential
medium supplemented with 2 mM L-glutamine, Earle's BSS (balanced salt
solution)
to contain 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids, 1.0
mM
sodium pyruvate and 10% fetal bovine serum.
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32
Cell lines were transfected with the indicated plasmids (Figure 11 ) by
the CaP04 precipitation method. See Graham, F.L. and van der Eb, A.J., 1973,
Virology 52:456-467.
After 48 hours post-transfection the cells were harvested by
centrifugation and the media supernatant was collected. The media supernatant
was
assayed using the fluorescent substrate 4-methylumbelliferyl-a-D-
galactopyranoside
(4-mu-a-gal) as described (Desnick, et al., 1973, J. Lab. Clin. Med. 81:157
and
Johnson, D.L. et al., 1975, Clin. Chim. Acta. 63:81). This method was modified
as
described by Mayes et al. (CI in. Chim. Acta. 112:247-251 ( 1981 )) to include
inhibitors
to a-galactosidase B.
The results in Figure 11 indicate that in 293 cells no difference in a-
galactosidase activity is achieved with the -l.7kb enhancer element.
Expression levels
of constructs with a truncated CMV promoter linked to the -I.7kb enhancer
element
are comparable to that obtained with full length CMV promoter (Figure 11A).
However, in Hep3B cells, the constructs with the truncated CMV promoter (-242
to -
14) and 2, 3, or 5 copies of the -1.7kb enhancer element all gave
significantly higher
levels of expression than that obtained from the wild-type CMV promoter
lacking the
enhancer regions and the expression from the construct containing 5 copies of
the
enhancer yielded the greatest expression levels. (Figure 11 B).
SUBSTITUTE SHEET ( rule 26 )
CA 02317934 2000-07-OS
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SEQUENCE LISTING
< 110> Annentano, Donna
Yew, Nelson
Marshall, John
<120> Novel Promoter elements for persistent
gene expression
<130> 31365
<140> 60/071,673
<141> 1998-O1-16
< 160> 7
<170> FastSEQ for Windows Version 3.0
<210> 1
<211> 512
<212> DNA
<213> cytomegalovirus
<400> 1
ttgcgttaca taacttacgg taaatggccc gcctggctga GO
ccgcccaacg acccccgccc
attgacgtca ataatgacgt atgttcccat agtaacgcca 120
atagggactt tccattgacg
tcaatgggtg gactatttac ggtaaactgc ccacttggca 180
gtacatcaag tgtatcatat
gccaagtacg ccccctattg acgtcaatga cggtaaatgg 240
cccgcctggc attatgccca
gtacatgacc ttatgggact ttcctacttg gcagtacatc
tacgtattag tcatcgctat 300
taccatggtg atgcggtttt ggcagtacat caatgggcgt 360
ggatagcggt ttgactcacg
gggatttcca agtctccacc ccattgacgt caatgggagt 420
ttgttttggc accaaaatca
acgggacttt ccaaaatgtc gtaacaactc cgccccattg 480
acgcaaatgg gcggtaggcg
tgtacggtgg gaggtctata taagcagagc tc 512
<210> 2
<211> 282
<212> DNA
<213> cytomegalovirus
<400> 2
attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag 60
tcatcgctat taccatggtg atQcggtttt ggcagtacat caatgggcgt ggatagcggt 120
CA 02317934 2000-07-OS
WO 99/36557 PCTNS99/00915
7 .)
ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc 180
accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg 240
gcggtaggcg tgtacggtgg gaggtctata taagcagagc tc 282
<210> 3
<211> 281
<212> DNA
<213> cytomegalovirus
<400> 3
tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta 60
ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag 120
cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt 180
tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa 240
atgggcggta ggcgtgtacg gtgggaggtc tatataagca g 281
<210> 4
<211> 229
<212> DNA
<213> cytomegalovirus
<400> 4
gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga 60
tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg 120
ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg 180
caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctc 229
<210> 5
<211> 17
<212> DNA
<213> homo Sapiens
<400> 5
ttgtcaatta gtaacaa 17
<210> G
<211> 8
<212> DNA
<213> homo Sapiens
<400> 6
gccaaaca 8
CA 02317934 2000-07-OS
WO 99136557 PCT/US99/00915
<210> 7
<211> 65
<21 Z> DNA
<213> homo sapiens
<400> 7
cgatgcctga atttgtcaat tagtaacaat tgtattcaac agtaaggatt ttatgtttgg 60
gtagg
