Note: Descriptions are shown in the official language in which they were submitted.
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~
HYBRID ADENOVIRUS-AAV VIRUS AND METHODS OF USE THEREOF
This invention was supported by the National
Institute of Health Grant No. P30 DK 47757. The United
States government has rights in this invention.
Field of the Invention
The present invention relates to the field of
vectors useful in somatic gene therapy and the production
thereof.
Background of the Invention
Recombinant adenoviruses are capable of
providing extremely high levels of transgene delivery to
virtually all cell types, regardless of the mitotic
state. High titers (1013 plaque forming units/ml) of
recombinant virus can be easily generated in 293 cells
(the adenovirus equivalent to retrovirus packaging cell
lines) and cryo-stored for extended periods without
appreciable losses.
The primary limitation of this virus as a
vector resides in the complexity of the adenovirus
genome. A human adenovirus is comprised of a linear,
approximately 36 kb double-stranded DNA genome, which is
divided into 100 map units (m.u.), each of which is 360
bp in length. The DNA contains short inverted terminal
repeats (ITR) at each end of the genome that are required
for viral DNA replication. The gene products are
organized into early (El through E4) and late (L1 through
L5) regions, based on expression before or after the
initiation of viral DNA synthesis [see, e.g., Horwitz,
Viroloav, 2d edit., ed. B. N. Fields, Raven Press, Ltd.
New York (1990)].
A human adenovirus undergoes a highly regulated
program during its normal viral life cycle [Y. Yang et
al, Proc. Natl. Acad. Sci.. USA, 21:4407-4411 (1994)].
CA 02321215 2000-10-18
2
Virions are interr.alized by receptor-mediated endocytosis
and transported to the nucleus where the immediate early
genes, Ela and Elb, are expressed. Because these early
gene products regulate expression of a variety of host
genes (which prime the cell for virus production) and are
central to the cascade activation of early delayed genes
(e.g. E2, E3, and E4) followed by late genes (e.g. Li-5),
first generation recombinant adenoviruses for gene
therapy focused on the removal of the El domain. This
strategy was successful in rendering the vectors
replication defective, however, in vivo studies revealed
transgene expression was transient and invariably
associated with the development of severe inflammation at
the site of vector targeting (S. Ishibashi et al, J_.,
Clin, Invest., 22:1885-1893 (1994); J. M. Wilson et al,
Proc. Natl. Acad. Sci.. USA, BI:4421-4424 (1988); J. M.
Wilson et al, Clin. BiQs, 3:21-26 (1991); M. Grossman et
al, Som. Cell. and Mol. Gen., 12:601-607 (1991)].
Adeno-associated viruses (AAV) have also been
employed as vectors. AAV is a small, single-stranded
(ss) DNA virus with a simple genomic organization (4.7
kb) that makes it an ideal substrate for genetic
engineering. Two open reading frames encode a series of
rep and cap polypeptides. Rep polypeptides (rep78,
rep68, rep62 and rep40) are involved in replication,
rescue and integration of the AAV genome. The cap
proteins (VP1, VP2 and VP3) form the virion capsid.
Flanking the rep and cap open reading frames at the 5'
and 3' ends are 145 bp inverted terminal repeats (ITRs),
the first 125 bp of which are capable of forming Y- or T-
shaped duplex structures. Of importance for the
development of AAV vectors, the entire rep and cap
domains can be excised and replaced with a therapeutic or
reporter transgene [B. J. Carter, in "Handbook of
Parvoviruses", ed., P. Tijsser, CRC Press, pp.155-168
CA 02321215 2000-10-18
3
(1990)]. It has been shown that the ITRs represent the
minimal sequence required for replication, rescue,
packaging, and integration of the AAV genome.
The AAV life cycle is biphasic, composed of
both latent and lytic episodes. During a latent
infection, AAV virions enter a cell as an encapsidated
ssDNA, and shortly thereafter are delivered to the
nucleus where the AAV DNA stably integrates into a host
chromosome without the apparent need for host cell
division. In the absence of helper virus, the integrated
ss DNA AAV genome remains latent but capable of being
activated and rescued. The lytic phase of the life cycle
begins when a cell harboring an AAV provirus is
challenged with a secondary infection by a herpesvirus or
adenovirus which encodes helper functions that are
recruited by AAV to aid in its excision from host
chromatin [8. J. Carter, cited above]. The infecting
parental ssDNA is expanded to duplex replicating form
(RF) DNAs in a rep dependent manner. The rescued AAV
genomes are packaged into preformed protein capsids
(icosahedral symmetry approximately 20 nm in diameter)
and released as infectious virions that have packaged
either + or - ss DNA genomes following cell lysis.
Progress towards establishing AAV as a
tranaducing vector for gene therapy has been slow for a
variety of reasons. While the ability of AAV to
integrate in quiescent cells is important in terms of
long term expression of a potential transducing gene, the
tendency of the integrated provirus to preferentially
target only specific sites in chromosome 19 reduces its
usefulness. Additionally, difficulties surround large-
scale production of replication defective recombinants.
In contrast to the production of recombinant retrovirus
or adenovirus, the only widely recognized means for
manufacturing transducing AAV virions entails co-
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transfection with two different, yet complementing
plasmids. One of these contains the therapeutic or
reporter minigene sandwiched between the two cis acting
AAV ITRs. The AAV components that are needed for rescue
and subsequent packaging of progcny recombinant genomes
are provided in trans by a second plasmid encoding the
viral open reading frames for rep and cap proteins. The
cells targeted for transfection must also be infected
with adenovirus thus providing the necessary helper
functions. Because the yield of recombinant AAV is
dependent on the number of cells that are transfected
with the cis and trans-acting plasmids, it is desirable
to use a transfection protocol with high efficiency. For
large-scale production of high titer virus, however,
previously employed high efficiency calcium phosphate and
liposome systems are cumbersome and subject to
inconsistencies.
There remains a need in the art for the
development of vectors which overcome the disadvantages
of the known vector systems.
Summarv of the Invgntion
In one aspect, the present invention provides a
unique recombinant hybrid adenovirus/AAV virus, which
comprises an adenovirus capsid containing selected
portions of an adenovirus sequence, 5' and 3' AAV ITR
sequences which flank a selected transgene under the
control of a selected promoter and other conventional
vector regulatory components. This hybrid virus is
characterized by high titer transgene delivery to a host
cell and the ability to stably integrate the transgene
into the host cell chromosome in the presence of the rep
gene. In one embodiment, the transgene is a reporter
gene. Another embodiment of the hybrid virus contains a
therapeutic transgene. In a preferred embodiment, the
CA 02321215 2000-10-18
hybrid virus has associated therewith a polycation
sequence and the AAV rep gene. This construct is termed
the hybrid virus conjugate or trans-infection particle.
In another aspect, the present invention
5 provides a hybrid vector construct for use in producing
the hybrid virus or viral particle described above. This
hybrid vector comprises selected portions of an
adenovirus sequence, 5' and 3' AAV ITR sequences which
flank a selected transgene under the control of a
selected promoter and other conventional vector
regulatory components.
In another aspect, the invention provides a
composition comprising a hybrid viral particle for use in
delivering a selected gene to a host cell. Such a
composition may be employed to deliver a therapeutic gene
to a targeted host cell to treat or correct a genetically
associated disorder or disease.
In yet another aspect, the present invention
provides a method for producing the hybrid virus by
transfecting a suitable packaging cell line with the
hybrid vector construct of this invention. In another
embodiment the method involves co-transfecting a cell
line (either a packaging cell line or a non-packaging
cell line) with a hybrid vector construct and a suitable
helper virus.
In a further aspect, the present invention
provides a method for producing large quantities of
recombinant AAV particles with'high efficiency by
employing the above methods, employing the hybrid vector
construct of this invention and collecting the rAAV
particles from a packaging cell line transfected with the
vector.
Other aspects and advantages of the present
invention are described further in the following detailed
description of the preferred embodiments thereof.
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6
Brief DescriRtion of the Drawinas
Fig. 1A is a schematic diagram of a vector
construct pAd.AV.CMVLacZ [SEQ ID NO: 1], which contains
(from the top in clockwise order) adenovirus sequence map
units 0-1 (clear bar); the 5' AAJ ITR (solid bar); a CMV
immediate early enhancer/promoter (hatched arrow), an
SV40 intron (clear bar), an E. coif beta-galactosidase
cDNA (LacZ) (hatched line), an SV40 polyadenylation
signal (clear bar), a 3' AAV ITR (solid bar), adenovirus
sequence from map units 9-16 (clear bar), and a portion
of a pBR322 derivative plasmid (thin solid line).
Restriction endonuclease enzymes are identified by their
conventional designations; and the location of each
restriction enzyme is identification by the nucleotide
number in parentheses to the right of the enzyme
designation.
Fig. 1B is a schematic drawing demonstrating
linearization of pAd.AV.CMVLacZ [SEQ ID NO: 1] by
digestion with restriction enzyme NheI and a linear
arrangement of a C1aI digested adenovirus type 5 with
deletions from mu 0-1. The area where homologous
recombination will occur (between m.u. 9-16) in both the
plasmid and adenovirus sequences is indicated by crossed
lines.
Fig. 1C is a schematic drawing which
demonstrates the hybrid virus Ad.AV.CMVLacZ after co-
transfection of the linearized pAd.AV.CMVLacZ [SEQ ID NO:
1) and adenovirus into 293 cells followed by
intracellular homologous recombination.
Fig. 2A-2K report the top DNA strand of the
double-strand plasmid pAd.AV.CMVLacZ [SEQ ID NO: 1] (the
complementary strand can be readily derived by one of
skill in the art). With reference to SEQ ID NO: 1,
nucleotides 1-365 are adenovirus type 5 sequences; the 5'
AAV ITR sequence spans nucleotides 366-538; the CMV
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promoter/enhancer spans nucleotides 563-1157; the SV-40
intron spans nucleotides 1158-1179; the LacZ gene spans
nucleotides 1356-4827; the SV-40 poly A sequence spans
nucleotides 4839-5037; the 3' AAV ITR spans nucleotides
5053 to 5221; nucleotides 5221 i.o about 8100 are
adenovirus type 5 sequences. The remaining sequences are
non-specific/plasmid sequences.
Fig. 3 is a bar graph plotting u.v. absorbance
at 420 nm of the beta-galactosidase blue color for a
control and ten putative positive clones (D1A through
D1J) of 293 cells transfected with the recombinant hybrid
Ad.AV.CMVLacZ. Eight of the clones expressed high levels
of enzyme.
Fig. 4 is a schematic diaqram of pRep78/52 [SEQ
ID NO: 2]. This plasmid includes an AAV P5 promoter,
Rep78, Rep52 and a poly-A sequence in a pUC18 plasmid
background.
Fiqs. 5A - 5E report nucleotides 1-4910 of the
top DNA strand of the double-strand plasmid pRep78/52
(SEQ ID NO: 2] (the complementary strand can be readily
derived by one of skill in the art).
Fiq. 6 is a flow diagram of the construction of
a trans-infection particle formed by a hybrid virus, a
poly-L-lysine sequence and attached AAV rep-containing
plasmid.
Fig. 7 is a flow diagram of the hybrid virus'
life cycle, in which a trans-infection particle enters
the cell and is transported to'the nucleus. The virus is
uncoated and the rep mediates rescue of the inserted
gene, which is then integrated into the chromosome of the
host cell.
r~w~~.~=..~~ .=..~~~ /w. .. .. w~\
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8
Detailed DescriQt~on of the Invention
The present invention provides a unique gene
transfer vehicle which overcomes many of the limitations
of prior art viral vectors. This engineered hybrid virus
contains selected adenovirus dom.ins and selected AAV
domains as well as a selected transgene and regulatory
elements in a viral capsid. This novel hybrid virus
solves the problems observed with other, conventional
gene therapy viruses, because it is characterized by the
ability to provide extremely high levels of transgene
delivery to virtually all cell types (conferred by its
adenovirus sequence) and the ability to provide stable
long-term transgene integration into the host cell
(conferred by its AAV sequences). The adenovirus-AAV
hybrid virus of this invention has utility both as a
novel gene transfer vehicle and as a reagent in a method
for large-scale recombinant AAV production.
In a preferred embodiment, a trans-infection
particle or hybrid virus conjugate composed of the hybrid
Ad/AAV virus conjugated to a rep expression plasmid via a
poly-lysine bridge is provided. This trans-infection
particle is advantageous because the adenovirus carrier
can be grown to titers sufficient for high MOI infections
of a large number of cells, the adenoviral genome is
efficiently transported to the nucleus in nondividing
cells as a complex facilitating transduction into
mitotically quiescent cells, and incorporation of the rep
plasmid into the trans-infection particle provides high
but transient expression of rep that is necessary for
both rescue of rAAV DNA and efficient and site-specific
integration.
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I. Construction of tDe Sybria Vector and Virus
A. The Adenovirus Comoonent of the Vector and
Virus
The hybrid virus of this invention uses
adenovirus nucleic acid sequenc%.s as a shuttle to deliver
a recombinant AAV/transgene genome to a target cell. The
DNA sequences of a number of adenovirus types, including
type Ad5, are available from Genbank. The adenovirus
sequences may be obtained from any known adenovirus type,
including the presently identified 41 human types
[Horwitz et al, cited above]. Similarly adenoviruses
known to infect other animals may also be employed in the
vector constructs of this invention. The selection of
the adenovirus type is not anticipated to limit the
following invention. A variety of adenovirus strains are
available from the American Type Culture Collection,
Rockville, Maryland, or available by request from a
variety of commercial and institutional sources. In the
following exemplary embodiment an adenovirus, type 5
(Ad5) is used for convenience.
The adenovirus nucleic acid sequences
employed in the hybrid vector of this invention can range
from a minimum sequence amount, which requires the use of
a helper virus to produce the hybrid virus particle, to
only selected deletions of adenovirus genes, which
deleted gene products can be supplied in the hybrid viral
production process by a selected packaging cell.
Specifically, at a minimum, the adenovirus nucleic acid
sequences employed in the pAdA shuttle vector of this
invention are adenovirus genomic sequences from which all
viral genes are deleted and which contain only those
adenovirus sequences required for packaging adenoviral
genomic DNA into a preformed capsid head. More
specifically, the adenovirus sequences employed are the
cis-acting 5' and 3' inverted terminal repeat (ITR)
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sequences of an adenovirus (which function as origins of
replication) and the native 5' packaging/enhancer domain,
that contains sequences necessary for packaging linear Ad
genomes and enhancer elements for the El promoter.
5 According to this invention, the entire adenovirus 5'
sequence containing the 5' ITR and packaging/enhancer
region can be employed as the 5' adenovirus sequence in
the hybrid virus. This left terminal (5') sequence of
the Ad5 genome useful in this invention spans bp 1 to
10 about 360 of the conventional adenovirus genome, also
referred to as map units 0-1 of the viral genome, and
generally is from about 353 to about 360 nucleotides in
length. This sequence includes the 5' ITR (bp 1-103 of
the adenovirus genome); and the packaging/enhancer domain
(bp 194-358 of the adenovirus genome). Preferably, this
native adenovirus 5' region is employed in the hybrid
virus and vector in unmodified form. Alternatively,
corresponding sequences from other adenovirus types may
be substituted. These Ad sequences may be modified to
contain desired deletions, substitutions, or mutations,
provided that the desired function is not eliminated.
The 3' adenovirus sequences of the hybrid virus
include the right terminal (3') ITR sequence of the
adenoviral genome spanning about bp 35,353 - end of the
adenovirus genome, or map units -98.4-100. This sequence
is generally about 580 nucleotide in length. This entire
sequence is desirably employed as the 3' sequence of a
hybrid virus. Preferably, the native adenovirus 3'
region is employed in the hybrid virus in unmodified
form. However, as described above with respect to the 5'
sequences, some modifications to these sequences which do
not adversely effect their biological function may be
acceptable. As described below, when these 5' and 3'
adenovirus sequences are employed in the hybrid vector, a
helper adenovirus which supplies all other essential
CA 02321215 2000-10-18
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genes for viral formation alone or with a packaging cell
line is required in the production of the hybrid virus or
viral particle.
Alternative embodiments of the hybrid
virus employ adenovirus sequencus in addition to the
minimum sequences, but which contain deletions of all or
portions of adenovirus genes. For example, the
adenovirus immediate early gene Ela (which spans mu 1.3
to 4.5) and delayed early gene Elb (which spans mu 4.6 to
11.2) should be deleted from the adenovirus sequence
which forms a part of the hybrid vector construct and
virus. Alternatively, if these sequences are not
completely eliminated, at least a sufficient portion of
the Ela and Elb sequences must be deleted so as to render
the virus replication defective. These deletions,
whether complete or partial, which eliminate the
biological function of the gene are termed "functional
deletions" herein.
Additionally, all or a portion of the
adenovirus delayed early gene E3 (which spans mu 76.6 to
86.2) may be eliminated from the adenovirus sequence
which forms a part of the hybrid virus. The function of
E3 is irrelevant to the function and production of the
hybrid virus.
All or a portion of the adenovirus delayed
early gene E2a (which spans mu 67.9 to 61.5) may be
eliminated from the hybrid virus. It is also anticipated
that portions of the other delayed early genes E2b (which
spans mu 29 to 14.2) and E4 (which spans mu 96.8 to 91.3)
may also be eliminated from the hybrid virus and from the
vector.
Deletions may also be made in any of the
late genes L1 through L5, which span mu 16.45 to 99 of
the adenovirus genome. Similarly, deletions may be
useful in the intermediate genes IX which maps between mu
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9.8 and 11.2 and IVa2 which maps between 16.1 to 11.1.
Other deletions may occur in the other structural or non-
structural adenovirus.
The above discussed deletions may occur
individually, i.e., an adenovirub sequence for use in the
present invention may contain deletions of El only.
Alternatively, deletions of entire genes or portions
effective to destroy their biological activity may occur
in any combination. For example, in one exemplary hybrid
vector, the adenovirus sequence may contain deletions of
the El genes and the E3 gene, or of the El, E2a and E3
genes, or of the El and E4 genes, or of El, E2a and E4
genes, with or without deletion of E3, and so on.
The more deletions in the adenovirus
sequence up to the minimum sequences identified above
that characterize the hybrid virus, the larger the
sequence(s) of the other below-described components to be
inserted in the hybrid vector. As described above for
the minimum adenovirus sequences, those gene sequences
not present in the adenovirus portion of the hybrid virus
must be supplied by either a packaging cell line and/or a
helper adenovirus to generate the hybrid virus.
In an exemplary hybrid virus of this invention
which is described below and in Example 1, the adenovirus
genomic sequences present are from mu 0 to 1, mu 9 to
78.3 and mu 86 to 100 (deleted sequences eliminate the
Ela and Elb genes and a portion of the E3 gene). From
the foregoing information, it is expected that one of
skill in the art may construct hybrid vectors and viruses
containing more or less of the adenovirus gene sequence.
The portions of the adenovirus genome in
the hybrid virus permit high production titers of the
virus to be produced, often greater than 1x1013 pfu/ml.
This is in stark contrast to the low titers (1x106
pfu/ml) that have been found for recombinant AAV.
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B. The AAV CogMnents of the Vector and Virus
Also part of the hybrid vectors and
viruses of this invention are sequences of an adeno-
associated virus. The AAV sequences useful in the hybrid
vector are the viral sequences :rom which the rep and cap
polypeptide encoding sequences are deleted. More
specifically, the AAV sequences employed are the cis-
acting 5' and 3' inverted terminal repeat (ITR) sequences
[See, e.g., B. J. Carter, in "Handbook of Parvoviruses",
ed., P. Tijsser, CRC Press, pp.155-168 (1990)]. As
stated above, the ITR sequences are about 143 bp in
length. Substantially the entire sequences encoding the
ITRs are used in the vectors, although some degree of
minor modification of these sequences is expected to be
permissible for this use. See, e.g., WO 93/24641,
published December 9, 1993. The ability to modify these
ITR sequences is within the skill of the art. For
suitable techniques, see, e.g., texts such as Sambrook et
al, "Molecular Cloning. A Laboratory Manual.", 2d edit.,
Cold Spring Harbor Laboratory, New York (1989).
The AAV ITR sequences may be obtained from
any known AAV, including presently identified human AAV
types. Similarly, AAVs known to infect other animals may
also be employed in the vector constructs of this
invention. The selection of the AAV is not anticipated
to limit the following invention. A variety of AAV
strains, types 1-4, are available from the American Type
Culture Collection or available by request from a variety
of commercial and institutional sources. In the
following exemplary embodiment an AAV-2 is used for
convenience.
In the hybrid vector construct, the AAV
sequences are flanked by the selected adenovirus
sequences discussed above. The 5' and 3' AAV ITR
sequences themselves flank a selected transgene sequence
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and associated regulatory elements, described below.
Thus, the sequence formed by the transgene and flanking
5' and 3' AAV sequences may be inserted at any deletion
site in the adenovirus sequences of the vector. For
example, the AAV sequences are dtasirably inserted at the
site of the deleted Ela/Elb genes of the adenovirus,
i.e., after map unit 1. Alternatively, the AAV sequences
may be inserted at an E3 deletion, E2a deletion, and so
on. If only the adenovirus 5' ITR/packaging sequences
and 3' ITR sequences are used in the hybrid virus, the
AAV sequences are inserted between them.
C. The Transgene CoMponent of the Zjybrid
Vector and Vfrus
The transgene sequence of the vector and
recombinant virus is a nucleic acid sequence or reverse
transcript thereof, heterologous to the adenovirus
sequence, which encodes a polypeptide or protein of
interest. The transgene is operatively linked to
regulatory components in a manner which permits transgene
transcription.
The composition of the transgene sequence
will depend upon the use to which the resulting hybrid
vector will be put. For example, one type of transgene
sequence includes a reporter sequence, which upon
expression produces a detectable signal. Such reporter
sequences include without limitation an E. coli beta-
galactosidase (LacZ) cDNA, an alkaline phosphatase gene
and a green fluorescent protein'gene. These sequences,
when associated with regulatory elements which drive
their expression, provide signals detectable by
conventional means, e.g., ultraviolet wavelength
absorbance, visible color change, etc.
Another type of transgene sequence
includes a therapeutic gene which expresses a desired
gene product in a host cell. These therapeutic genes or
CA 02321215 2000-10-18
nucleic acid sequences typically encode products for
administration and expression in a patient fn vivo or ex
vivo to replace or correct on inherited or non-inherited
genetic defect or treat an epigenetic disorder or
5 disease. Such therapeutic geneb, which are desirable for
the performance of gene therapy include, without
limitation, a normal cystic fibrosis transmembrane
regulator (CFTR) gene, a low density lipoprotein (LDL)
gene, and a number of genes which may be readily selected
10 by one of skill in the art. The selection of the
transgene is not considered to be a limitation of this
invention, as such selection is within the knowledge of
those skilled in the art.
D. $eaulatorv Elements of the Hybr3d Vector
15 In addition to the major elements
identified above for the hybrid vector, i.e., the
adenovirus sequences, AAV sequences and the transgene,
the vector also includes conventional regulatory elements
necessary to drive expression of the transgene in a cell
transfected with the hybrid vector. Thus the vector
contains a selected promoter which is linked to the
transgene and located, with the transgene, between the
AAV ITR sequences of the vector.
Selection of the promoter is a routine
matter and is not a limitation of the hybrid vector
itself. Useful promoters may be constitutive promoters
or regulated (inducible) promoters, which will enable
control of the amount of the transgene to be expressed.
For example, a desirable promoter is that of the
cytomegalovirus immediate early promoter/enhancer [see,
e.g., Boshart et al, Cell, 11:521-530 (1985)]. Other
desirable promoters include, without limitation, the Rous
sarcoma virus LTR promoter/enhancer and the chicken 0-
actin promoter. Still other promoter/enhancer sequences
may be selected by one of skill in the art.
CA 02321215 2000-10-18
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The vectors will also desirably contain
nucleic acid sequences heterologous to the adenovirus
sequences including sequences providing signals required
for efficient polyadenylation of the transcript and
introns with functional splice d%,nor and acceptor sites.
A common poly-A sequence which is employed in the
exemplary vectors of this invention is that derived from
the papovavirus SV-40. The poly-A sequence generally is
inserted in the vector following the transgene sequences
and before the 3' AAV ITR sequence. A common intron
sequence is also derived from SV-40, and is referred to
as the SV-40 T intron sequence. A hybrid vector of the
present invention may also contain such an intron,
desirably located between the promoter/enhancer sequence
and the transgene. Selection of these and other common
vector elements are conventional and many such sequences
are available [see, e.g., Sambrook et al, and references
cited therein]. The DNA sequences encoding such
regulatory regions are provided in the plasmid sequence
of Fig. 2[SEQ ID NO: 1].
The combination of the transgene,
promoter/enhancer, the other regulatory vector elements
and the flanking 5' and 3' AAV ITRs are referred to as a
"minigene" for ease of reference herein. As above
stated, the minigene is located in the site of any
selected adenovirus deletion in the hybrid virus. The
size of this minigene depends upon the amount and number
of adenovirus sequence deletions referred to above. Such
a minigene may be about 8 kb in size in the exemplary
virus deleted in the El and E3 genes, as described in the
examples below. Alternatively, if only the minimum
adenovirus sequences are employed in the virus, this
minigene may be a size up to about 30 kb. Thus, this
hybrid vector and vector permit a great deal of latitude
in the selection of the various components of the
CA 02321215 2000-10-18
17
minigene, particularly the transgene, with regard to
size. Provided with the teachings of this invention, the
design of such a minigene can be made by resort to
conventional techniques.
E. Hy&ad Vector Assambly and PrQduction of
&brAd Virus
The material from which the sequences used
in the hybrid vector, helper viruses, if needed, and
recombinant hybrid virus (or viral particle) are derived
and the various vector components and sequences employed
in the construction of the hybrid vectors of this
invention are obtained from commercial or academic
sources based on previously published and described
materials. These materials may also be obtained from an
individual patient or generated and selected using
standard recombinant molecular cloning techniques known
and practiced by those skilled in the art. Any
modification of existing nucleic acid sequences forming
the vectors and viruses, including sequence deletions,
insertions, and other mutations are also generated using
standard techniques.
Assembly of the selected DNA sequences of
the adenovirus, the AAV and the reporter genes or
therapeutic genes and other vector elements into the
hybrid vector and the use of the hybrid vector to produce
a hybrid virus utilize conventional techniques, such as
described in Example 1. Such techniques include
conventional cloning techniques of cDNA such as those
described in texts [Sambrook et al, cited above], use of
overlapping oligonucleotide sequences of the adenovirus
and AAV genomes, polymerase chain reaction, and any
suitable method which provides the desired nucleotide
sequence. Standard transfection and co-transfection
techniques are employed, e.g., CaPO4 transfection
techniques using the complementation human embryonic
CA 02321215 2000-10-18
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kidney (HER) 293 cell line (a human kidney cell line
containing a functional adenovirus Ela gene which
provides a transacting Ela protein). Other conventional
methods employed in this invention include homologous
recombination of the viral genomtss, plaquing of viruses
in agar overlay, methods of measuring signal generation,
and the like.
As described in detail in Example 1 below
and with resort to Fig. 1, a unique hybrid virus of this
invention is prepared which contains an E1-deleted,
partially E3 deleted, adenovirus sequence associated with
a single copy of a recombinant AAV having deletions of
its rep and cap genes and encoding a selected reporter
transgene. Briefly, this exemplary hybrid virus was
designed such that the AV.CMVLacZ sequence [SEQ ID NO: 11
(a minigene containing a 5'AAV ITR, a CMV promoter, an
SV-40 intron, a LacZ transgene, an SV-40 poly-A sequence
and a 3' AAV ITR) was positioned in place of the
adenovirus type 5 (Ad5) Ela/Elb genes, making the
adenovirus vector replication defective.
Because of the limited amount of
adenovirus sequence present in the hybrid vectors of this
invention, including the pAV.CMVLacZ [SEQ ID NO: 1]
above, a packaging cell line or a helper adenovirus or
both may be necessary to provide sufficient adenovirus
gene sequences necessary for a productive viral infection
to generate the hybrid virus.
Helper viruses ugeful in this invention
contain selected adenovirus gene sequences not present in
the hybrid vector construct or expressed by the cell line
in which the hybrid vector is transfected. Optionally,
such a helper virus may contain a second reporter
minigene which enables separation of the resulting hybrid
virus and the helper virus upon purification. The
CA 02321215 2000-10-18
19
construction of desirable helper viruses is within the
skill of the art.
As one example, if the cell line employed
to produce the recombinant virus is not a packaging cell
line, and the hybrid vector conLains only the minimum
adenovirus sequences identified above, the helper virus
may be a wild type Ad virus. Thus, the helper virus
supplies the necessary adenovirus early genes El, E2a, E4
and all remaining late, intermediate, structural and non-
structural genes of the adenovirus genome. However, if,
in this situation, the packaging cell line is 293, which
supplies the El proteins, the helper virus need not
contain the El gene.
In another embodiment, when the hybrid
construct is rendered replication defective by a
functional deletion in El but contains no other deletions
in Ad genes necessary for production of an infective
viral particle, and the 293 cell line is employed, no
helper virus is necessary for production of the hybrid
virus. Additionally, all or a portion of the adenovirus
delayed early gene E3 (which spans mu 76.6 to 86.2) may
be eliminated from the helper virus useful in this
invention because this gene product is not necessary for
the formation of a functioning hybrid virus particle.
It should be noted that one of skill in
the art may design other helper viruses or develop other
packaging cell lines to complement the adenovirus
deletions in the vector construct and enable production
of the hybrid virus particle, given this information.
Therefore, this invention is not limited by the use or
description of any particular helper virus or packaging
cell line.
Thus, as described in Figs. 1A through 1C,
the circular plasmid pAd.AV.CMVLacZ [SEQ ID NO: 1)
(containing the minigene and only adenovirus sequences
CA 02321215 2000-10-18
from map unit 0 tc 1 and 9 to 16) was digested and co-
transfected with a selected Ad5 helper virus (containing
adenovirus sequences 9 to 78.4 and 86 to 100) into 293
cells. Thus, the packaging cell line provides the El
5 proteins and the helper virus pruvides all necessary
adenovirus gene sequences subsequent to map unit 16.
Homologous recombination occurs between the helper virus
and the hybrid vector, resulting in the hybrid viral
particle. Growth of this hybrid viral particle in 293
10 cells has been closely monitored for greater than 20
rounds of amplification with no indication of genome
instability. Rescue and integration of the transgene
from the hybrid virus into a host cell and further
modifications of the vector are described below. The
15 resulting hybrid virus Ad.AV.CMVLacZ combines the high
titer potential of adenovirus with the integrating
biology associated with AAV latency.
G. Hybrid Virus Polycation Conjuaates
Rep expression is required for rescue of
20 the rAAV genome to occur. A preferred approach is to
synthetically incorporate a plasmid permitting expression
of rep into the hybrid particle. To do so, the hybrid
viruses described above are further modified by resort to
adenovirus-polylysine conjugate technology. See, e.g.,
Wu et al, J. Biol. Chem., 2 A:16985-16987 (1989); and K.
J. Fisher and J. M. Wilson, Biochem. J., M : 49 (April
1, 1994), incorporated herein by reference. Using this
technology, a hybrid virus as described above is modified
by the addition of a poly-cation sequence distributed
around the capsid of the hybrid viral particle.
Preferably, the poly-cation is poly-lysine, which
attaches around the negatively-charged virus to form an
external positive charge. A plasmid containing the AAV
rep gene (or a functional portion thereof) under the
control of a suitable promoter is then complexed directly
CA 02321215 2000-10-18
21
to the hybrid cap3id, resulting in a single viral
particle containing the hybrid virus and an AAV rep gene.
The negatively charged plasmid DNA binds with high
affinity to the positively charged polylysine.
Essentially the techniques employed in constructing this
hybrid virus conjugate or trans-infection particle are as
described in detail in Example 3 below.
An alternative embodiment of the hybrid
vector and resulting viral particle is provided by
altering the rep containing plasmid to also contain an
AAV cap gene. This embodiment of the hybrid vector when
in a host cell is thus able to produce a recombinant AAV
particle, as discussed in more detail below.
The plasmids employed in these embodiments
contain conventional plasmid sequences, which place a
selected AAV sequence, i.e., rep and/or cap gene
sequences, under the control of a selected promoter. In
the example provided below, the exemplary plasmid is
pRep78/52 [SEQ ID NO: 2], a trans-acting plasmid
containing the AAV sequences that encode rep 78 kD and 52
kD proteins under the control of the AAV P5 promoter.
The plasmid also contains an SV40 polyadenylation signal.
The DNA sequence of this plasmid is provided in Fig. 8
[SEQ ID NO: 2].
In a similar manner and with resort to
plasmid and vector sequences known to the art, analogous
plasmids may be designed using both rep and cap genes,
and different constitutive or regulated promoters,
optional poly-A sequences and introns.
The availability of materials to make
these modified hybrid vectors and viruses and the AAV rep
and/or cap containing vectors and the techniques involved
in the assembly of the hybrid vector and rep and/or cap
containing plasmids are conventional as described above.
The assembly techniques for the trans-infection particle
CA 02321215 2000-10-18
22
employ the techniques described above for the hybrid
vector and the techniques of Wu et al and Fisher et al,
cited above. The use of this trans-infection particle
including rescue and integration of the transgene into
the host cell is described below.
II. luaotioa of the Hybrid virum
A. The Xvbrid Virus Infects a Target Cell
Once the hybrid virus or trans-infection
particle is constructed as discussed above, it is
targeted to, and taken up by, a selected target cell.
The selection of the target cell also depends upon the
use of the hybrid virus, i.e., whether or not the
transgene is to be replicated in vitro for production of
a recombinant AAV particle, or ex vivo for production
into a desired cell type for redelivery into a patient,
or in vivo for delivery to a particular cell type or
tissue. Target cells may therefor be any mammalian cell
(preferably a human cell). For example, in in vivo use,
the hybrid virus can target to any cell type normally
infected by adenovirus, depending upon the route of
administration, i.e., it can target, without limitation,
neurons, hepatocytes, epithelial cells and the like.
Uptake of the hybrid virus by the cell is caused by the
infective ability contributed to the vector by the
adenovirus and AAV sequences.
B. The Transgene is Rescued.
Once the hybrid Virus or trans-infection
particle is taken up by a cell, the AAV ITR flanked
transgene must be rescued from the parental adenovirus
backbone. Rescue of the transgene is dependent upon
supplying the infected cell with an AAV rep gene. Thus,
efficacy of the hybrid virus can be measured in terms of
rep mediated rescue of rAAV from the parental adenovirus
template.
CA 02321215 2000-10-18
23
Tha rep genes can be supplied to the
hybrid virus by several methods. One embodiment for
providing rep proteins in trans was demonstrated with the
exemplary hybrid virus Ad.AV.CMVLacZ by transfecting into
the target monolayer of cells piaviously infected with
the hybrid vector, a liposome enveloped plasmid pRep78/52
[SEQ ID NO: 2] containing the genes encoding the AAV rep
78 kDa and 52 kDa proteins under the control of the AAV
P5 promoter. Rescue and amplification of a double-
stranded AAV monomer and a double-stranded AAV dimer,
each containing the LacZ transgene described above, was
observed in 293 cells. This is described in detail in
Example 2.
The production of rep in trans can be
modulated by the choice of promoter in the rep containing
plasmid. If high levels of rep expression are important
early for rescue of the recombinant AAV domain, a
heterologous (non-adenovirus, non-AAV) promoter may be
employed to drive expression of rep and eliminate the
need for El proteins. Alternatively, the low levels of
rep expression from P5 that occur in the absence of
adenovirus El proteins may be sufficient to initiate
rescue and optimal to drive integration of the
recombinant AAV genome in a selected use.
More preferably for in vivo use, the AAV
rep gene may also be delivered as part of the hybrid
virus. One embodiment of this single particle concept is
the polycation conjugated hybrid virus (see Fig. 7).
Infection of this trans-infection particle is
accomplished in the same manner and with regard to the
same target cells as identified above. The polylysine
conjugate of the hybrid virus onto which was directly
complexed a plasmid that encoded the rep 78 and 52
proteins, combines all of the functional components into
a single particle structure. Thus, the trans-infection
CA 02321215 2000-10-18
24
particle permits delivery of a single particle to the
cell, which is considerably more desirable for
therapeutic use. Similar experiments to demonstrate
rescue of the transgene from the hybrid conjugate trans-
infection particle in 293 cells and in HeLa cells are
detailed in Example 4.
In another embodiment, the hybrid virus is
modified by cloning the rep cDNA directly into the
adenovirus genome portion of the hybrid vector. Because
it is known that even residual levels of rep expression
can interfere with replication of adenovirus DNA, such
incorporation of rep into the hybrid vector itself is
anticipated to requires possible mutation of the rep
genes to encode only selected domains, and the use of
inducible promoters to regulate rep expression, as well
as careful placement of the rep genes into the adenovirus
sequences of the hybrid vector.
C. Transgeae Integrates into Chromosomm
Once uncoupled (rescued) from the genome
of the hybrid virus, the recombinant AAV/transgene
minigene seeks an integration site in the host chromatin
and becomes integrated therein, providing stable
expression of the accompanying transgene in the host
cell. This aspect of the function of the hybrid virus is
important for its use in gene therapy. The AAV/
transgene minigene sequence rescued from the hybrid virus
achieves provirus status in the target cell, i.e., the
final event in the hybrid lifecycle (Fig. 7).
To determine whether the AAV minigene
rescued from the hybrid virus achieves provirus status in
a target cell, non-El expressing HeLa cells were infected
with the hybrid vector-poly-Lysine conjugate complexed
with pRep78/52 [SEQ ID NO: 2] and passaged until stable
colonies of LacZ expressing cells are evident. A
duplicate plate of cells was infected with the same
CA 02321215 2000-10-18
conjugate, but ir.stead of being complexed with the
pRep78/52 plasmid [SEQ ID NO: 2), carried an irrelevant
plasmid. Cells that receive the rep containing hybrid
particle produced a greater number of stable LacZ
5 positive colonies than cells iniected with the control
vector. This indicates multiple rescue and integration
events in cells that expressed rep proteins.
Confirmation of integration is revealed by characterizing
the recombinant AAV genome in the hybrid infected cells
10 and identifying flanking chromosomal sequences (see
Example 5).
ZIZ. ose of the 8ybrid viruses and viral Bartiales
in c,one Therapy
15 The novel hybrid virus and trans-infection
particles of this invention provide efficient gene
transfer vehicles for somatic gene therapy. These hybrid
viruses are prepared to contain a therapeutic gene in
place of the LacZ reporter transgene illustrated in the
20 exemplary vector. By use of the hybrid viruses and
trans-infection particles containing therapeutic
transgenes, these transgenes can be delivered to a
patient in vivo or ex vivo to provide for integration of
the desired gene into a target cell. Thus, these hybrid
25 viruses and trans-infection particles can be employed to
correct genetic deficiencies or defects. Two examples of
the generation of gene transfer vehicles for the
treatment of cystic fibrosis and familial
hypercholesterolemia are described in Examples 6 and 7
below. One of skill in the art can generate any number
of other gene transfer vehicles by including a selected
transgene for the treatment of other disorders. For
example, the trans-infection particles are anticipated to
be particularly advantageous in ex vivo gene therapy
CA 02321215 2000-10-18
26
where transductior. and proviral integration in a stem
cell is desired, such as in bone marrow directed gene
therapy.
The hybrid viruses and trans-infection
particles of the present inventiun may be administered to
a patient, preferably suspended in a biologically
compatible solution or pharmaceutically acceptable
delivery vehicle. A suitable vehicle includes sterile
saline. Other aqueous and non-aqueous isotonic sterile
injection solutions and aqueous and non-aqueous sterile
suspensions known to be pharmaceutically acceptable
carriers and well known to those of skill in the art may
be employed for this purpose.
The hybrid viruses and trans-infection
particles of this invention may be administered in
sufficient amounts to transfect the desired cells and
provide sufficient levels of integration and expression
of the selected transgene to provide a therapeutic
benefit without undue adverse or with medically
acceptable physiological effects which can be determined
by those skilled in the medical arts. Conventional and
pharmaceutically acceptable routes of administration
include direct delivery to the target organ, tissue or
site, intranasal, intravenous, intramuscular,
subcutaneous, intradermal, oral and other parental routes
of administration. Routes of administration may be
combined, if desired.
Dosages of the hybrid'virus and/or trans-
infection particle will depend primarily on factors such
as the condition being treated, the selected gene, the
age, weight and health of the patient, and may thus vary
among patients. A therapeutically effective human dose
of the hybrid viruses or trans-infection particles of the
present invention is believed to be in the range of from
about 20 to about 50 ml of saline solution containing
CA 02321215 2000-10-18
27
concentrations of from about 1 x 107 to 1 x 1010 pfu/ml
hybrid virus of the present invention. A preferred human
dose is about 20 ml saline solution at the above
concentrations. The dosage will be adjusted to balance
the therapeutic benefit against any side effects. The
levels of expression of the selected gene can be
monitored to determine the selection, adjustment or
frequency of dosage administration.
IV. 8igh Eftioionay Broauatioa of rA]-v
The hybrid viruses and trans-infection
particles of this invention have another desirable
utility in the production of large quantities of
recombinant AAV particles. Due to the complicated
current methods for generating AAV, there is only a
limited amount of AAV available for use in industrial,
medical and academic biotechnology procedures. The
vectors and viruses of the present invention provide a
convenient and efficient method for generating large
quantities of rAAV particles.
According to this aspect of the invention, a
trans-infection particle is constructed as described
above and in Example 3 and is employed to produce high
levels of rAAV as detailed in Example 8, with the
possible modifications described in Example 9 below.
Briefly, a plasmid is generated that contains both AAV
rep and cap genes under the control of a suitable plasmid
and is complexed to the poly-lysine exterior of the
hybrid virus as described above. This trans-infection
particle is then permitted to infect a selected host
cell, such as 293 cells. The presence of both rep and
cap permit the formation of AAV particles in the cells
and generate an AAV virus titer of about 109 virions. In
contrast, current methods involving the transfection of
multiple plasmids produce only about 107 virion titer.
CA 02321215 2000-10-18
28
The rAAV is isolated from the culture by selecting the
LacZ-containing blue plaques and purifying them on a
cesium chloride gradient.
The benefit of this procedure relates to the
fact that the cis AAV element is encoded by the parental
adenovirus genome. As a result, the trans plasmid is the
only DNA component that is needed for complex formation.
The cell is thereby loaded with significantly more copies
of the trans-acting rep and cap sequences, resulting in
improved efficiency of rescue and packaging.
Numerous comparative studies focusing on the
optimal ratio and copy number of the cis and trans
plasmids for AAV production indicated that there is a
positive correlation between the trans plasmid copy
number and yield of recombinant virus. As described in
detail in Example 8, the yield of recombinant AV.CMVLacZ
virus was increased by 5-10 fold by using the trans-
infection particle instead of a standard adenovirus
vector.
The primary limitation associated with the
production of recombinant AAV using a hybrid virus of
this invention relates to difficulties that arise in
distinguishing between the two viruses (i.e., adenovirus
and AAV) that are produced by the cell. Using the
exemplary vectors and vector components of this
invention, LacZ histochemical staining could not be used
to titer the yield of recombinant AV.CMVLacZ since any
contaminating Ad.AV.CMVLacZ hybrid would contribute to
the final count. Therefore, a rapid Southern blot
technique for quantitating yields of recombinant AAV was
incorporated. The assay that was developed enabled not
only quantitation and verification of AAV production, but
also demonstrated the removal of contaminating hybrid
virus from purified AAV stocks.
CA 02321215 2000-10-18
29
Another method for detecting contaminating
hybrid virions involves modifying the hybrid vector by
inserting a small second reporter minigene (i.e.,
reporter gene, promoter and other expression control
sequences, where desired) into Lhe E3 region of the
parental adenovirus backbone. Because this reporter is
not linked to the AAV domain, contaminating hybrid virus
that is present during purification can be monitored by
this hybrid-specific marker. Another possible reporter
gene is the nucleic acid sequence for green fluorescent
protein. With this hybrid vector containing two reporter
sequences, histochemical staining for alkaline
phosphatase (adenovirus reporter) or a-galactosidase (AAV
reporter) activity can be used to monitor each viral
domain.
The following examples illustrate the
construction and testing of the hybrid vectors of the
present invention and the use thereof in the productions
of recombinant AAV. These examples are illustrative
only, and do not limit the scope of the present
invention.
Example 1- Construction of a Hybrid Virus
A first hybrid adenovirus-AAV virus was
engineered by homologous recombination between DNA
extracted from an adenovirus and a complementing vector
according to protocols previously described [see, e.g.,
K. F. Kozarsky et al, J. Biol.'Chem., 269:13695-13702
(1994). The following description refers to the diagram
of Fig. 1.
Adenovirus DNA was extracted from CsCl purified
d17001 virions, an Ad5 (serotype subgroup C) variant that
carries a 3 kb deletion between mu 78.4 through 86 in the
nonessential E3 region (provided by Dr. William Wold,
Washington University, St. Louis, Missouri). Adenoviral
CA 02321215 2000-10-18
DNA was prepared for co-transfaction by digestion with
Clal (adenovirus genomic bp position 917) which removes
the left arm of the genome encompassing adenovirus map
units 0-2.5. See lower diagram of Fig. iB.
5 The complementing hybrid vector, pAd.AV.CMVLacZ
(see Fig. 1A and Fig. 2[SEQ ID NO: 1)) was constructed
as follows:
A parental cloning vector, pAd.BglII was
designed. It contains two segments of wild-type Ad5
10 genome (i.e., map units 0-1 and 9-16.1) separated by a
unique BglII cloning site for insertion of heterologous
sequences. The missing Ad5 sequences between the two
domains (adenovirus genome bp 361-3327) results in the
deletion of Ela and the majority of Elb following
15 recombination with viral DNA.
A recombinant AAV genome (AV.CMVLacZ) was
designed and inserted into the BglII site of pAd.BglII to
generate the complementing plasmid. The linear
arrangement of AV.CMVLacZ (SEQ ID NO: 1] (see top diagram
20 of Fig. 1B) includes:
(a) the 5' AAV ITR (bp 1-173) obtained by PCR
using pAV2 [C. A. Laughlin et al, Gene, 2d: 65-73 (1983)]
as template [nucleotide numbers 365-538 of Fig. 2[SEQ ID
NO: 1]);
25 (b) a CMV immediate early enhancer/promoter
[Boshart et al, Cell, 11:521-530 (1985); nucleotide
numbers 563-1157 of Fig. 2[SEQ ID NO: 1]),
(c) an SV40 splice donor-splice acceptor
(nucleotide numbers 1178-1179 of Fig. 2[SEQ ID NO: 1]),
30 (d) E. coii beta-galactosidase cDNA
(nucleotide numbers 1356 - 4827 of Fig. 2[SEQ ID NO:
1]),
(e) an SV40 polyadenylation signal (a 237 Bam
HI-BclI restriction fragment containing the
cleavage/poly-A signals from both the early and late
CA 02321215 2000-10-18
31
transcription units; nucleotide numbers 4839 - 5037 of
Fig. 2[SEQ ID NO: 1]) and
(f) 3'AAV ITR, obtained from pAV2 as a SnaBI-
BglII fragment (nucleotide numbers 5053 - 5221 of Fig. 2
[SEQ ID NO: 1]).
The resulting complementing hybrid vector,
pAd.AV.CMVLacZ (see Fig. lA and Fig. 2[SEQ ID NO: 1]),
contained a single copy of recombinant AV.CMVLacZ flanked
by adenovirus coordinates 0-1 on one side and 9-16.1 on
the other. Plasmid DNA was linearized using a unique
NheI site immediately 5' to adenovirus map unit zero (0)
(resulting in the top diagram of Fig. 1B).
Both the adenovirus substrate and the
complementing vector DNAs were transfected to 293 cells
[ATCC CRL1573] using a standard calcium phosphate
transfection procedure [see, e.g., Sambrook et al, cited
above]. The end result of homologous recombination
involving sequences that map to adenovirus map units 9-
16.1 is hybrid Ad.AV.CI+lVLacZ (see Fig. 1C) in which the
Ela and Elb coding regions from the d17001 adenovirus
substrate are replaced with the AV.CMVLacZ from the
hybrid vector.
Twenty-four hours later, the transfection
cocktail was removed and the cells overlayed with 0.8%
agarose containing lx BME and 2% fetal bovine serum
(FBS). Once viral plaques developed (typically 10-12
days post-transfection), plaques were initially screened
for E. coli 0-galactosidase (LacZ) activity by overlaying
the infected monolayer with agarose supplemented with a
histochemical stain for LacZ, according to the procedure
described in J. Price et al, Proc. Natl. Acad. Sci.. USA,
$g,:156-160 (1987). Positive clones (identified by the
deposit of insoluble blue dye) were isolated, subjected
to three rounds of freeze (dry ice/ethanol)- thaw (37 C)
and an aliquot of the suspended plaque was used to infect
CA 02321215 2000-10-18
32
a fresh monolayer of 293 cells seeded on duplicate 60mm
plates.
Twenty-four hours later the cells from one set
of plates were fixed and again stained for LacZ activity.
Cells from the duplicate plate w%.re harvested, suspended
in 0.5 ml 10 mM Tris-Cl, pH8.0, and lysed by performing a
series of three freeze (dry ice/ethanol)-thaw (37 C)
cycles. Cell debris was removed by centrifugation and an
aliquot of the supernatant used to measure LacZ enzyme
activity.
As indicated in Fig. 3, assays for
galactosidase activity which measured the absorbance at
420 nm of the beta-galactosidase blue color in successful
recombinants, revealed that eight of the ten isolated,
putative positive clones (D1A through D1J) expressed high
levels of enzyme. Histochemical staining produced
similar results.
Large-scale production and purification of
recombinant virus was performed as described in Kozarsky
et al, cited above, and references cited therein.
Example 2 - Functional Analysis of Hybrid Vector
The ability to rescue the AV.CMVLacZ sequence
[SEQ ID NO: 1] from the hybrid virus represented an
important feature of the hybrid vector and virus systems
of Example 1. To evaluate this feature, it was necessary
to provide the necessary AAV gene products in trans that
direct AAV excision and amplification (i.e. rep
proteins). Furthermore, this experiment was conducted in
293 cells to transcomplement the El deletion in the
Ad.AV.CMVLacZ clones, because the adenovirus El gene
proteins have been shown to be important for initiating
the lytic phase of the AAV lifecycle.
293 cells were seeded onto 6-well 35 mm plates
at a density of 1 x 106 cells/well. Twenty-four hours
CA 02321215 2000-10-18
33
later, seeding me3ia [DMEM/10% PBS supplemented with
antibiotics] was replaced with 1.0 ml DMEM/2% PBS and
infected with Ad.AV.CXVLacZ hybrid clones at an MOI of 1.
Two hours later, each well was transfected with 1 q
plasmid pRep78/52 [SEQ ID NO: 2j, a trans-acting plasmid
that encodes the sequence encoding the AAV rep 78 kD and
52 kD proteins. The rep sequences in this construct are
under the control of the AAV P5 promoter and utilize an
SV40 polyadenylation signal.
As a positive control for AAV rescue, 293 cells
seeded in a 6-well plate as above were co-transfected
with a cis-acting AAV plasmid pAV.C1MVLacZ and pRep78/52.
pAV.CMVLacZ contained AV.CMVZ.acZ, the identical sequence
encoded by pAd.AV.CMVLacZ [SEQ ID NO: 1] described in
Example 1 cloned into the Bg1II site of pSP72 (Promega).
To provide the necessary adenovirus helper
function for AAV rescue, cells were infected with either
wild-type Ad5 virus or a first generation El-deleted
virus Ad.CMhpAP at an MOI of 5, approximately 2 hours
prior to adding the transfection cocktail. Ad.CMhpAP is
identical to Ad.CMVLacZ (Example 1) with the modification
that the alkaline phosphatase sequence (which can be
obtained from Genbank) is inserted in place of the LacZ
gene.
Transfections were performed with Lipofectamine
(Life Technologies) according to the instructions
provided by the manufacturer. Thirty hours post-
transfection, the cells were harvested and episomal DNA
(Hirt extract) prepared as described by J. M. Wilson et
al, J. Biol. Chem., 2M:(16):11483-11489 (1992). Samples
were resolved on a 1.2% agarose gel and electroblotted
onto a nylon membrane. Blots were hybridized (Southern)
with a 32P random primer-labeled restriction fragment
isolated from the E. co1.f LacZ cDNA.
CA 02321215 2000-10-18
34
The full- spectrum of duplex molecular species
that appear during a lytic AAV infection (i.e., monomeric
forms of the double stranded intermediates, RFm and RFd,
respectively) were evident in transfected cells infected
with wild type and El deleted Ad,. No replicative
intermediates were detected when transfections were
performed in the absence of helper virus.
Hirt extracts from the 293 cells infected with
putative Ad.AV.CMVLacZ hybrid clones D1A and D1C revealed
a single band corresponding to the viral DNA, when probed
with a LacZ restriction fragment. In the presence of rep
proteins 78 and 52, however, the same clones yielded a
banding pattern that included not only the adenovirus
DNA, but an RF monomer and dimer of AV.CMVLacZ. A
single-stranded form of AV.CMVLacZ [SEQ ID NO: 1] was not
evident. Two additional clones gave similar banding
patterns, DiB and D1H. In all, each of the eight
Ad.AV.CMVLacZ hybrids that were found in Fig. 3 to
express high levels of Lac Z activity were positive for
rescue of the AAV domain.
With the exception of an extra band of
approximately 3.5 kb, the rescue of the AV.CIMVLacZ [SEQ
ID NO: 1] from the hybrid viral DNA was nearly identical
to results obtained from a standard cis and trans
plasmid-based approach. In these later samples,
adenovirus helper function was provided by pre-infecting
cells with either wild-type Ad5 or an El-deleted
recombinant virus Ad.CBhpAP (also termed H5.CBALP). The
Ad.CBhpAP virus has the same sequence as the Ad.CMhpAP
virus described above, except that the CNV promoter
sequence is replaced by the chicken cytoplasmic 8-actin
promoter (nucleotides +1 to +275 as described in T. A.
Kost et al, Nucl. Acids Res., 11(23):8287 (1983)]. The
level of rescue in cells infected with WT Ad5 appeared to
be greater relative to those infected with the
CA 02321215 2000-10-18
recombinant Ad.CEhpAP virus, likely due to the additional
El expression provided by the wild-type genome. The
relevance of including an El deleted adenovirus here is
to document that the level of adenovirus El proteins
5 expressed in 293 cells is suffi.-ient for AAV helper
function.
EXamDle 3 - Synthesis of Polylysine Coniuaates
Another version of the viral particle of this
10 invention is a polylysine conjugate with a rep plasmid
complexed directly to the hybrid virus capsid. This
conjugate permits efficient delivery of the rep
expression plasmid pRep78/52 [SEQ ID NO: 2] in tandem
with the hybrid virus, thereby removing the need for a
15 separate transfection step. See, Fig. 8 for a
diagrammatic outline of this construction.
Purified stocks of a large-scale expansion of
Ad.AV.CMVLacZ clone D1A were modified by coupling poly-L-
lysine to the virion capsid essentially as described by
20 K. J. Fisher and J. M. Wilson, Biochem. J., M :49-58
(1994), resulting in an Ad.AV.CMVLacZ-(Lys)n conjugate.
The procedure involves three steps. First, hybrid
virions are activated through primary amines on capsid
proteins with the heterobifunctional water-soluble cross-
25 linking agent, sulpho-SMCC (sulpho-(N-succinimidyl 4-(N-
maleimidomethyl)-cyclohexane-l-carboxylate] (Pierce).
The conjugation reaction, which contained 0.5 mg (375
nmol) of sulpho-SMCC and 6 x 1012 A260 hybrid vector
particles in 3.0 ml of HBS, was incubated at 30 C for 45
30 minutes with constant gentle shaking. This step involved
formation of a peptide bond between the active N-
hydroxysuccinimide (NHS) ester of sulpho-SMCC and a free
amine (e.g. lysine) contributed by an adenovirus protein
sequence (capsid protein) in the recombinant virus,
35 yielding a maleimide-activated viral particle.
CA 02321215 2002-12-11
36
Unincorparated, unreacted cross-linker was
removed by gel filtration on a 1 cm x 15 cm Bio-Gel*P-6DG
(Bio-Rad Laboratories) column equilibrated with 50 mM
Tris/HC1 buffer, pH 7.0, and 150 mM NaC1. Peak A260
.5 fractions containing maleimide-ac..ivated hybrid virus
were combined and placed on ice.
Second, poly-L-lysine having a molecular mass
of 58 kDa at 10 mg/mi in 50 mM triethanolamine buffer (pH
8.0), 150 mM NaC1 and 1 mM EDTA was thiolated with 2-
imminothiolane/HC1 (Traut's Reagent; Pierce) to a molar
ratio of 2 moles-SH/mole polylysine under N2; the cyclic
thioimidate reacts with the poly(L-lysine) primary amines
resulting in a thiolated polycation. After a 45 minute
incubation at room temperature the reaction was applied
to a 1 cm x 15 cm Bio-Gel P6DG column equilibrated with
50 mM Tris/HC1 buffer (pH 7.0), 150 mM NaCl and 2 mM EDTA
to remove unincorporated Traut's Reagent.
Quantification of free thiol groups was
accomplished with Ellman's reagent (5,5'-dithio-bis-(2-
nitrobenzoic acid)], revealing approximately 2 mol of -
SH/mol of poly(L-lysine). The coupling reaction was
initiated by adding 1 x 1012 A260 particles of maleimide-
activated hybrid virus/mg of thiolated poly(L-lysine) and
incubating the mixture on ice at 4 C for 15 hours under
argon. 2-mercaptoethylamine was added at the completion
of the reaction and incubation carried out at room
temperature for 20 minutes to block unreacted maleimide
sites.
Virus-polylysine conjugates, Ad.AV.CMVLacZ-
(Lys)n, were purified away from unconjugated poly(L-
lysine) by ultracentrifugation through a CsCl step
gradient with an initial composition of equal volumes of
1.45 g/ml (bottom step) and 1.2 g/ml (top step) CsCl in
10 mM Tris/HC1 buffer (pH 8.0). Centrifugation was at
90,000 g for 2 hours at 5 C. The final product was
* Trademark
CA 02321215 2000-10-18
37
dialyzed against 20 mM Hepes buffer (pH 7.8) containing
150 mM NaCl (HBS).
Complexes of Ad.AV.CMVLacZ-(Lys)n with
pRep78/52 plasmid DNA [SEQ ID NO: 2) were formed by
adding varying quantities of Ad.AV.CMVLacZ-(Lys)n in 50 1
HBS to 0.5 g of pRep78/52 plasaid DNA [SEQ ID NO: 2] in
50 1 HBS. After 30 minutes incubation at room
temperature, a complex was formed of the hybrid virus
Ad.AV.CMVLacZ-(Lys)n associated in a single particle with
the plasmid DNA containing the rep genes.
This complex, termed a trans-infection
particle, was evaluated for DNA binding capacity by gel
mobility shift assays performed as described in Fisher et
al, cited above. This analysis revealed that the plasmid
binding capacity of the purified conjugate (expressed as
the number of A260 particles Ad.AV.CMVLacZ-(Lys)n that
can neutralize the charge contributed by 1 g plasmid
DNA) was 1 g pRep78/52 plasmid DNA/6.0 x 1010 A260
particles Ad.AV.CMVLacZ-(Lys)n.
Examnle 4 - Trans-Infection Protocol to Demonstrate AAV
Excision and Amplification
Trans-infection complexes were prepared by
mixing Ad.AV.CMVLacZ-(Lys)n conjugate with pRep78/52
plasmid [SEQ ID NO: 2] and applied to 293 cells as
follows. Ad.AV.C1rIVLacZ-(Lys)n (6 x 1010 A260 particles) in
100 l D1MF.'M was added dropwise to a microfuge tube
containing 1 g plasmid DNA in 100 l DMEM. The mixture
was gently mixed and allowed to incubate at room
temperature for 10-15 minutes. The trans-infection
cocktail was added to 293 cells seeded in a 35 mm 6-well
as detailed above. Thirty hours later, cells were
harvested and Hirt extracts prepared.
CA 02321215 2000-10-18
38
Samples aere resolved on a 1.2% agarose gel and
electroblotted onto a nylon membrane. Blots were
hybridized (Southern) with a P-32 random primer-labeled
restriction fragment isolated from the E. co1l LacZ cDNA.
The Hirt extracts from 293 cells revealed a
banding pattern that suggested the AV.CMVLacZ minigene
sequence [SEQ ID NO: 1] was efficiently rescued from the
hybrid conjugate. Both an RF monomer and dimer of the
recombinant AV.CMVLacZ sequence were evident. As was
observed previously, the rescue event was dependent on
rep proteins since 293 cells that were trans-infected
with a hybrid conjugate complexed with an irrelevant
reporter plasaid expressing alkaline phosphatase (i.e.
pCMVhpAP) revealed only Ad.AV.CMVLacZ DNA. This negative
control for rescue was secondarily useful for
demonstrating the high efficiency of gene transfer to 293
cells that was achieved with the conjugate vehicle.
A duplicate set of 293 cells that received
hybrid conjugate which was further complexed with
alkaline phosphatase expression plasmid were fixed 24
hours after addition of the trans-infection cocktail and
histochemically stained for LacZ as described in Price et
al, cited above, or for alkaline phosphatase activity as
described in J. H. Schreiber et al, BioTechni es,
IA:818-823 (1993). Here LacZ was a marker for the
Ad.AV.CMVLacZ hybrid, while alkaline phosphatase served
as a reporter for the carrier plasmid. Greater than 90%
of the monolayer was transduced with both 0-galactosidase
and alkaline phosphatase transgenes, showing the high
efficiency of the conjugate delivery vehicle
(differential staining revealed a blue color for the
hybrids containing the LacZ marker and a purple color for
the plasmids bearing the AP marker).
Because of the important role El proteins have
for progression of the AAV lifecycle, it was critical to
CA 02321215 2000-10-18
39
test the efficienry of the hybrid delivery system in a
setting where El proteins are not expressed. A
trans-infection experiment using the hybrid conjugate
complexed with pRep78/52 [SEQ ID NO: 2] was therefore
conducted in HeLa cells [ATCC Ct,L2] to remove the
involvement of El proteins. The findings suggested
rescue of AV.CMVLecZ occurred evidenced by the
accumulation of RF monomers and dimers. Rescue from HeLa
cells (which unlike the 293 cells do not contain any
adenovirus El proteins) revealed lower levels of rescue
of the transgene. The expression of rep from the AAV P5
promoter is upregulated by adenovirus El and signals the
beginning of the AAV lytic cycle. In the absence of El,
rep expression from the P5 promoter is virtually silent
which is important for maintenance of the proviral latent
stages of the AAV lifecycle. It is anticipated that a
promoter not dependent on El expression will upon
substitution for P5, overcome this problem.
ExamDle 5 - Integration of the Transaene
A preliminary study has been performed to
determine whether the AAV sequence rescued from the
hybrid virus can achieve provirus status in a target cell
(Fig. 7). Briefly, HeLa cells [ATCC CCL 2] were infected
with the hybrid conjugate complexed with pRep78/52 [SEQ
ID NO: 2] and passaged until stable colonies of LacZ
expressing cells were evident. A duplicate plate of
cells was infected with the saaie conjugate, but instead
of being complexed with the pRep78/52 plasmid [SEQ ID NO:
2], carried an irrelevant plasmid. These findings
indicated that cells that received the Rep containing
hybrid particle produced a greater number of stable LacZ
positive colonies than cells that were infected with the
control virus. This could be interpreted as a reflection
of multiple rescue and integration events in cells that
CA 02321215 2000-10-18
expressed Rep proteins. However, it is possible that an
episomal form of AAV that can persist for extended
periods of time was present.
To establish the occurrence of integration into
5 the chromosome of the minigene Av.CNVLacZ from the hybrid
conjugate, the following experiment is performed. The
Ad.AV.CMVLacZ-(Lys)n conjugate carrying pRep78/52 plasmid
[SEQ ID NO: 2] is used to infect HeLa cells [ATCC CRL2]
(primary fibroblasts may also be used). The infected
10 cells are passaged for several generations. The cells
are grown to confluency, split and allowed to grow to
confluency again, split again and this cycle repeated as
desired. This permits sufficient time for uptake,
expression, replication and integration to occur. See
15 Fig. 7.
To verify that the recombinant AAV sequence
that was rescued from the hybrid genome (step III of Fig.
7) has integrated into a chromosome of the host cell
(step IV of Fig. 7), cells are separated by a
20 Fluorescence Activated Cell Sorter (FACS). By this
technique, those cells containing a stable integrated
copy of the recombinant AV.CMVLacZ minigene are separated
based on the presence of the 0-galactosidase reporter.
These cells are tagged with fluorescein-labeled
25 antibodies that recognize the P-Gal protein, and are then
separated from non-transduced cells (i.e. those that did
not receive a copy of the AAV minigene) by FACS.
DNA is isolated from this purified population
of cells and used to construct a genomic library which is
30 screened for individual clones and the sequence verified.
If integration occurs, it is documented directly by
sequence analysis.
CA 02321215 2000-10-18
41
Exa=le 6 - Gene Transfer Vehicle for Cystic gibrosis
An adenovirus-AAV-CFTR virus constructed by
modifying the hybrid Ad.AV.CMV1acZ virus described in
Example 1 to contain the cystic fibrosis transmembrane
regulator (CFTR) gene [J.R. Rioidan et al, Science.
W :1066-1073 (1989)] in place of the lacZ gene, using
known techniques. One suitable method involves producinq
a new vector using the techniques described in Example 1.
In this new vector the LacZ minigene is replaced with the
CFTR minigene. For performance of this method vectors
bearing the CFTR gene have been previously described and
can be readily constructed. This new or reconstructed
vector is used to generate a new virus through homologous
recombination as described above. The resulting hybrid
virus is termed hybrid Ad.AV.CIKVCFTR. It has the
sequence of Fig. 2[SEQ ID NO: 1], except that the LacZ
gene is replaced with CFTR. Alternatively, the LacZ gene
can be removed from the Ad.AV.CNVLacZ vector of Example 1
and replaced with the CFTR gene using known techniques.
This virus (or an analogous hybrid virus with a
different promoter, regulatory regions, etc.) is useful
in gene therapy alone, or preferably, in the form of a
conjugate prepared as described in Example 4.
Treatment of cystic fibrosis, utilizing the
viruses provided above, is particularly suited for in
vivo, lung-directed, gene therapy. Airway epithelial
cells are the most desirable targets for gene transfer
because the pulmonary complications of CF are usually its
most morbid and life-limiting. Thus, the hybrid vector
of the invention, containing the CFTR gene, is delivered
directly into the airway, e.g. by formulating the hybrid
virus above, into a preparation which can be inhaled.
For example, the hybrid virus or conjugate of the
invention containing the CFTR gene, is suspended in 0.25
CA 02321215 2000-10-18
42
molar sodium chloride. The virus or conjugate is taken
up by respiratory airway cells and the gene is expressed.
Alternatively, the hybrid viruses or conjugates
of the invention may be delivered by other suitable
means, including site-directed ii.jection of the virus
bearing the CFTR gene. In the case of CFTR gene
delivery, preferred solutions for bronchial instillation
are sterile saline solutions containing in the range of
from about 1 x l07 to 1 x 1010 pfu/ml, more particularly,
in the range of from about i x 108 to 1 x 109 pfu/ml of
the recombinant hybrid virus of the present invention.
Other suitable methods for the treatment of
cystic fibrosis by use of gene therapy recombinant
viruses of this invention may be obtained from the art
discussions of other types of gene therapy vehicles for
CF. See, for example, U. S. Patent No. 5,240,846.
Example 7 - Gene Transfer Vehicle for Familial
Hypercholesterolemia
Familial hypercholesterolemia (FH) is an
autosomal dominant disorder caused by abnormalities
(deficiencies) in the function or expression of LDL
receptors [M.S. Brown and J.L. Goldstein, Science,
232(4746):34-37 (1986); J.L. Goldstein and M.S. Brown,
"Familial hypercholesterolemia" in Metabolic Basis of
Inherited Disease., ed. C.R. Scriver et al, McGraw Hill,
New York, pp1215-1250 (1989).] Patients who inherit one
abnormal allele have moderate elevations in plasma LDL
and suffer premature life-threatening coronary artery
disease (CAD). Homozygous patients have severe
hypercholesterolemia and life-threatening CAD in
childhood.
CA 02321215 2000-10-18
43
A hybrid adenovirus-AAV-LDL virus of the
invention is constructed by replacing the lacZ gene in
the hybrid Ad.AV.CMVlacZ virus of Example 1 with an LDL
receptor gene [T. Yamamoto et al, Cell, 22:27-38 (1984)]
using known techniques and as dtsscribed analogously for
CF in the preceding example. Vectors bearing the LDL
receptor gene can be readily constructed according to
this invention. The resulting hybrid vector is termed
pAd.AV.CMVLDL.
This plasmid or its recombinant virus is useful
in gene therapy of FH alone, or preferably, in the form
of a viral conjugate prepared as described in Example 4
to substitute a normal LDL gene for the abnormal allele
responsible for the gene.
A. Ex Vivo Gene Ther=
Ex vivo gene therapy can be performed by
harvesting and establishing a primary culture of
hepatocytes from a patient. Known techniques may be used
to isolate and transduce the hepatocytes with the above
vector(s) bearing the LDL receptor gene(s). For example,
techniques of collagenase perfusion developed for rabbit
liver can be adapted for human tissue and used in
transduction. Following transduction, the hepatocytes
are removed from the tissue culture plates and reinfused
into the patient using known techniques, e.g. via a
catheter placed into the inferior mesenteric vein.
B. In Vivo Gene TheraRv
Desirably, the in vivo approach to gene
therapy, e.g. liver-directed, involves the use of the
hybrid viruses and viral conjugates described above. A
preferred treatment involves infusing a trans-infection
particle of the invention containing LDL into the
peripheral circulation of the patient. The patient is
then evaluated for change in serum lipids and liver
tissues.
CA 02321215 2000-10-18
44
The hybrid virus or viral conjugate can be
used to infect hepatocytes in vfvo by direct injection
into a peripheral or portal vein (107-108 pfu/kg) or
retrograde into the biliary tract (same dose). This
effects gene transfer into the m~jority of hepatocytes.
Treatments are repeated as necessary, e.g.
weekly. Administration of a dose of virus equivalent to
an MOI of approximately 20 (i.e. 20 pfu/hepatocyte) is
anticipated to lead to high level gene expression in the
majority of hepatocytes.
FxamDle 8- Efficient Production of Recombinant AAV using
A Hybrid Virus/Coniuaate
The following experiment demonstrated that the
AAV genome that was rescued from the Ad.AV.CMVLacZ hybrid
virus could be packaged into an AAV capsid, provided the
cap open reading frame was supplied in trans. Thus the
viruses of this invention are useful in a production
method for recombinant AAV which overcomes the prior art
complications that surround the high titer production of
recombinant AAV.
A. Trans-Infection Protocol for the
Production of rAAV
A trans-infection complex was formed
composed of the Ad.AV.CMVLacZ-(Lys)n conjugate described
above and a transcomplementing plasmid pAdAAV, which is
described in detail in R. J. Samulski et al, J. Virol.,
fLZ(9):3822-3828 (1989)). Briefly, plasmid pAdAAV encodes
the entire rep and cap open reading frames in the absence
of AAV ITRs, and has been shown to provide the necessary
AAV helper functions for replication and packaging of
recombinant AAV sequences.
Ad.AV.CMVLacZ-(Lys)n conjugate (4.5 x 1013
A260 particles) in 75 ml DMEM was added dropwise with
constant gentle swirling in 25 ml DMEM containing 750 g
CA 02321215 2000-10-18
pAdAAV helper plLsmid and incubated at room temperature
for 10-15 minutes. The complex was diluted with 450 ml
DMEM supplemented with 2% FBS and 20 ml aliquots were
added to monolayers of 293 cells seeded on 150 mm plates.
5 Forty hours post tran.i-infection, cells were
harvested, suspended in 12 ml 10 mM Tris-C1 (pH 8.0), and
stored at -80 C.
Because the anticipated outcome was the
production of hybrid virus Ad.AV.CMVLacZ and a
10 recombinant AAV virion (AV.CMVLacZ), both of which carry
a functional LacZ minigene, it was not possible to use
detection of LacZ activity as an indicator of AV.CMVLacZ
production. A novel molecular approach was developed
that could be performed in one day and permitted
15 identification of the packaged viral DNAs.
B. Purification of rAAV
Briefly, frozen cell suspensions were
subjected to three rounds of freeze-thaw cycles to
release recombinant AV.C!lVLacZ and hybrid Ad.AV.CMVLacZ.
20 On completion of the final thaw, bovine pancreatic DNAse
(2000 units) and ribonuclease (0.2 mq/mi final
concentration) was added and the extract incubated at
37 C for 30 minutes. Cell debris was removed by
centrifugation (5000xg for 10 minutes) and the clarified
25 supernatant (15 ml) applied to a 22.5 mi step gradient
composed of equal volumes of CsCl at 1.2 g/ml, 1.36 g/ml,
and 1.45 g/ml 10mM Tris-Cl, pH8Ø Viral particles were
banded at 25,000 rpm in a Beckman SW-28 rotor for 8 hours
at 4 C. One ml fractions were collected from the bottom
30 of the tube.
The fractions retrieved from the CsCl
gradient of partially purified virus are then digested to
release viral DNA from virion capsids as follows. A
5.O l sample of each fraction was transferred to a
35 microfuge tube containing 20 l capsid digestion buffer
CA 02321215 2000-10-18
46
(50mM Tris-Cl, pHC.0, 1.0mM EDTA, pH8,0, 0.5% SDS, and
1.0 mg/mi Proteinase R). The reaction was incubated at
50 C for 1 hour, allowed to cool to room temperature,
diluted with 10 l milli-Q water, and agarose gel loading
dye added.
These fractions are then analyzed by
Southern blotting. Samples were resolved on a 1.2%
agarose gel, electroblotted onto a nylon membrane. A 32P
labeled LacZ restriction fragment which was common to
both vectors was used as a hybridization probe to locate
the migration of viral DNA through the agarose gel.
Viral bands were quantitated on a Molecular Dynamics
Phosphoimager.
A sample of the extract before CsCl
banding was also tested and revealed both hybrid
Ad.AV.CMVLacZ DNA and double-stranded RF forms (monomers
and dimers) of the rescued AV.CMVLacZ sequence [SEQ ID
NO: 1]. A single-stranded monomer of AV.CMVLacZ appeared
to be present in the crude extract; however, it was not
until the virions were concentrated by buoyant density
ultracentrifugation that the single-stranded genome
became clearly evident. The single-stranded recombinant
genome of the virus was distributed over a range of CsCl
densities and revealed a biphasic banding pattern. The
two peaks of single-stranded rAAV genome occurred at
densities of 1.41 and 1.45 g/ml CsCl, consistent with the
reported buoyant densities of wild-type AAV in CsC1 [L.
M. de la Maza et al, J. Viro1.,,U:1129-1137 (1980)].
Analysis of the fractions corresponding to the two vector
forms revealed the rAAV-1.41 species was several orders
of magnitude more active for 1acZ transduction than the
denser rAAV-1.45 g/ml variant. To avoid confusion with
contaminating Ad.AAV, samples were heat inactivated (60 C
for 30 min) before being added to indicator HeLa cells.
CA 02321215 2000-10-18
47
The peak fractions of rAAV-1.41 were
combined and purified by equilibrium sedimentation in
CsCl to eliminate residual adenovirus particles and
concentrate rAAV virions. On the final round of
ultracentrifugation, a faint buc clearly visible
opalescent band was observed in the middle of the
gradient tube. Fractions that surrounded the band were
evaluated for density, absorbance at 260 nm, and IacZ
transducing particles. As the band eluted from the
gradient tube, a well defined peak of 260 nm absorbing
material was recorded, with a maximal absorbance
occurring at a density of 1.40 g/ml CsCl. Analysis for
lacZ transducing particles on HeLa cells revealed a peak
of activity that mirrored the absorbance profile. These
results indicate rAAV was produced from the hybrid Ad.AAV
virus. Furthermore, the titers achieved using the hybrid
virus were 5-10 fold elevated compared to more
conventional recombinant AAV production schemes (i.e.,
transfections with cis- and trans-acting plasmids). This
represents a significant improvement in rAAV production
and indicates that the hybrid is useful for large-scale
rAAV production.
All references recited above are incorporated
herein by reference. Numerous modifications and
variations of the present invention are included in the
above-identified specification and are expected to be
obvious to one of skill in the art. Such modifications
and alterations to the compositions and processes of the
present invention, such as those modifications permitting
optimal use of the hybrid viruses as gene therapy
vehicles or production vehicles for recombinant AAV
production, are believed to be encompassed in the scope
of the claims appended hereto.
CA 02321215 2000-10-18
48
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Trustees of the University of Pennsylvania
Wilson, James M.
Kelley, William M.
Fisher, Krishna J.
(ii) TITLE OF INVENTION: Hybrid Adenovirus-AAV Vector and
Methods of Use Thereof
(iii) NUMBER OF SEQUENCES: 2
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Howson and Howson
(B) STREET: Spring House Corporate Cntr, PO Box 457
(C) CITY: Spring House
(D) STATE: Pennsylvania
(E) COUNTRY: USA
(F) ZIP: 19477
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release 01.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/331,384
(B) FILING DATE: 28-OCT-1994
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Bak, Mary E.
(B) REGISTRATION NUMBER: 31,215
(C) REFERENCE/DOCKET NUMBER: GNVPN.007PCT
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 215-540-9200
(B) TELEFAX: 215-540-5818
CA 02321215 2000-10-18
49
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10398 base pairs
(B) TYPE: nuclsic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
GAATTCGCTA GCATCATCAA TAATATACCT TATTTTGGAT TGAAGCCAAT 50
ATGATAATGA GGGGGTGGAG TTTGTGACGT GGCGCGGGGC GTGGGAACGG 100
GGCGGGTGAC GTAGTAGTGT GGCGGAAGTG TGATGTTGCA AGTGTGGCGG 150
AACACATGTA AGCGACGGAT GTGGCAAAAG TGACGTTTTT GGTGTGCGCC 200
GGTGTACACA GGAAGTGACA ATTTTCGCGC GGTTTTAGGC GGATGTTGTA 250
GTAAATTTGG GCGTAACCGA GTAAGATTTG GCCATT'1'TCG CGGGAAAACT 300
GAATAAGAGG AAGTGAAATC TGAATAATTT TGTGTTACTC ATAGCGCGTA 350
ATATTTGTCT AGGGAGATCT GCTGCGCGCT CGCTCGCTCA CTGAGGCCGC 400
CCGGGCAAAG CCCGGGCGTC GGGCGACCTT TGGTCGCCCG GCCTCAGTGA 450
GCGAGCGAGC GCGCAGAGAG GGAGTGGCCA ACTCCATCAC TAGGGGTTCC 500
TTGTAGTTAA TGATTAACCC GCCATGCTAC TTATCTACAA TTCGAGCTTG 550
CATGCCTGCA GGTCGTTACA TAACTTACGG TAAATGGCCC GCCTGGCTGA 600
CCGCCCAACG ACCCCCGCCC ATTGACGTCA ATAATGACGT ATGTTCCCAT 650
AGTAACGCCA ATAGGGACTT TCCATTGACG TCAATGGGTG GAGTATTTAC 700
GGTAAACTGC CCACTTGGCA GTACATCAAG TGTATCATAT GCCAAGTACG 750
CCCCCTATTG ACGTCAATGA CGGTAAATGG CCCGCCTGGC ATTATGCCCA 800
GTACATGACC TTATGGGACT TTCCTACTTG GCAGTACATC TACGTATTAG 850
TCATCGCTAT TACCATGGTG ATGCGGTTTT GGCAGTACAT CAATGGGCGT 900
GGATAGCGGT TTGACTCACG GGGATTTCCA AGTCTCCACC CCATTGACGT 950
CAATGGGAGT TTGTTTTGGC ACCAAAATCA ACGGGACTTT CCAAAATGTC 1000
CA 02321215 2000-10-18
GTAACAACTC CGCCCCATTG ACGCAAATGG GCGGTAGGCG TGTACGGTGG 1050
GAGGTCTATA TAAGCAGAGC TCGTTTAGTG AACCGTCAGA TCGCCTGGAG 1100
ACGCCATCCA CGCTGTTTTG ACCTCCATAG AAGACACCGG GACCGATCCA 1150
GCCTCCGGAC TCTAGAGGAT CCGGTACTCG AGGAA..''TGAA AAACCAGAAA 1200
GTTAACTGGT AAGTTTAGTC TTTTTGTCTT TTATTTCAGG TCCCGGATCC 1250
GGTGGTGGTG CAAATCAAAG AACTGCTCCT CAGTGGATGT TGCCTTTACT 1300
TCTAGGCCTG TACGGAAGTG TTACTTCTGC TCTAAAAGCT GCGGAATTGT 1350
ACCCGCGGCC GCAATTCCCG GGGATCGAAA GAGCCTGCTA AAGCAAAAAA 1400
GAAGTCACCA TGTCGTTTAC TTTGACCAAC AAGAACGTGA TTTTCGTTGC 1450
CGGTCTGGGA GGCATTGGTC TGGACACCAG CAAGGAGCTG CTCAAGCGCG 1500
ATCCCGTCGT TTTACAACGT CGTGACTGGG AAAACCCTGG CGTTACCCAA 1550
CTTAATCGCC TTGCAGCACA TCCCCCT'1'TC GCCAGCTGGC GTAATAGCGA 1600
AGAGGCCCGC ACCGATCGCC CTTCCCAACA GTTGCGCAGC CTGAATGGCG 1650
AATGGCGCTT TGCCTGGTTT CCGGCACCAG AAGCGGTGCC GGAAAGCTGG 1700
CTGGAGTGCG ATCTTCCTGA GGCCGATACT GTCGTCGTCC CCTCAAACTG 1750
GCAGATGCAC GGTTACGATG CGCCCATCTA CACCAACGTA ACCTATCCCA 1800
TTACGGTCAA TCCGCCGTTT GTTCCCACGG AGAATCCGAC GGGTTGTTAC 1850
TCGCTCACAT TTAATGTTGA TGAAAGCTGG CTACAGGAAG GCCAGACGCG 1900
AATTATTTTP GATGGCGTTA ACTCGGCGTT TCATCTGTGG TGCAACGGGC 1950
GCTGGGTCGG TTACGGCCAG GACAGTCGTT TGCCGTCTGA ATTTGACCTG 2000
AGCGCATTTT TACGCGCCGG AGAAAACCGC CTCGCGGTGA TGGTGCTGCG 2050
TTGGAGTGAC GGCAGTTATC TGGAAGATCA GGATATGTGG CGGATGAGCG 2100
GCATTTTCCG TGACGTCTCG TTGCTGCATA AACCGACTAC ACAAATCAGC 2150
GATTTCCATG TTGCCACTCG CTTTAATGAT GATTTCAGCC GCGCTGTACT 2200
GGAGGCTGAA GTTCAGATGT GCGGCGAGTT GCGTGACTAC CTACGGGTAA 2250
CAGTTTCTTT ATGGCAGGGT GAAACGCAGG TCGCCAGCGG CACCGCGCCT 2300
CA 02321215 2000-10-18
51
TTCGGCGGTG AAATTATCGA TGAGCGTGGT GGTTATGCCG ATCGCGTCAC 2350
ACTACGTCTG AACGTCGAAA ACCCGAAACT GTGGAGCGCC GAAATCCCGA 2400
ATCTCTATCG TGCGGTGGTT GAACTGCACA CCGCCGACGG CACGCTGATT 2450
GAAGCAGAAG CCTGCGATGT CGGTTTCCGC GAGvTGCGGA TTGAAAATGG 2500
TCTGCTGCTG CTGAACGGCA AGCCGTTGCT GATTCGAGGC GTTAACCGTC 2550
ACGAGCATCA TCCTCTGCAT GGTCAGGTCA TGGATGAGCA GACGATGGTG 2600
CAGGATATCC TGCTGATGAA GCAGAACAAC TTTAACGCCG TGCGCTGTTC 2650
GCATTATCCG AACCATCCGC TGTGGTACAC GCTGTGCGAC CGCTACGGCC 2700
TGTATGTGGT GGATGAAGCC AATATTGAAA CCCACGGCAT GGTGCCAATG 2750
AATCGTCTGA CCGATGATCC GCGCTGGCTA CCGGCGATGA GCGAACGCGT 2800
AACGCGAATG GTGCAGCGCG ATCGTAATCA CCCGAGTGTG ATCATCTGGT 2850
CGCTGGGGAA TGAATCAGGC CACGGCGCTA ATCACGACGC GCTGTATCGC 2900
TGGATCAAAT CTGTCGATCC TTCCCGCCCG GTGCAGTATG AAGGCGGCGG 2950
AGCCGACACC ACGGCCACCG ATATTATTTG CCCGATGTAC GCGCGCGTGG 3000
ATGAAGACCA GCCCTTCCCG GCTGTGCCGA AATGGTCCAT CAAAAAATGG 3050
CTTTCGCTAC CTGGAGAGAC GCGCCCGCTG ATCCTTTGCG AATACGCCCA 3100
CGCGATGGGT AACAGTCTTG GCGGTTTCGC TAAATACTGG CAGGCGTTTC 3150
GTCAGTATCC CCGTTTACAG GGCGGCTTCG TCTGGGACTG GGTGGATCAG 3200
TCGCTGATTA AATATGATGA AAACGGCAAC CCGTGGTCGG CTTACGGCGG 3250
TGATTTTGGC GATACGCCGA ACGATCGCCA GTTCTGTATG AACGGTCTGG 3300
TCTTTGCCGA CCGCACGCCG CATCCAGCGC TGACGGAAGC AAAACACCAG 3350
CAGCAGTTTT TCCAGTTCCG TTTATCCGGG CAAACCATCG AAGTGACCAG 3400
CGAATACCTG TTCCGTCATA GCGATAACGA GCTCCTGCAC TGGATGGTGG 3450
CGCTGGATGG TAAGCCGCTG GCAAGCGGTG AAGTGCCTCT GGATGTCGCT 3500
CCACAAGGTA AACAGTTGAT TGAACTGCCT GAACTACCGC AGCCGGAGAG 3550
CGCCGGGCAA CTCTGGCTCA CAGTACGCGT AGTGCAACCG AACGCGACCG 3600
CA 02321215 2000-10-18
52
CATGGTCAGA AGCCGGGCAC ATCAGCGCCT GGCAGCAGTG GCGTCTGGCG 3650
GAAAACCTCA GTGTGACGCT CCCCGCCGCG TCCCACGCCA TCCCGCATCT 3700
GACCACCAGC GAAATGGATT TTTGCATCGA GCTGGGTAAT AAGCGTTGGC 3750
AATTTAACCG CCAGTCAGGC TTTCTTTCAC AGATt.I'GGAT TGGCGATAAA 3800
AAACAACTGC TGACGCCGCT GCGCGATCAG TTCACCCGTG CACCGCTGGA 3850
TAACGACATT GGCGTAAGTG AAGCGACCCG CATTGACCCT AACGCCTGGG 3900
TCGAACGCTG GAAGGCGGCG GGCCATTACC AGGCCGAAGC AGCGTTGTTG 3950
CAGTGCACGG CAGATACACT TGCTGATGCG GTGCTGATTA CGACCGCTCA 4000
CGCGTGGCAG CATCAGGGGA AAACCTTATT TATCAGCCGG AAAACCTACC 4050
GGATTGATGG TAGTGGTCAA ATGGCGATTA CCGTTGATGT TGAAGTGGCG 4100
AGCGATACAC CGCATCCGGC GCGGATTGGC CTGAACTGCC AGCTGGCGCA 4150
GGTAGCAGAG CGGGTAAACT GGCTCGGATT AGGGCCGCAA GAAAACTATC 4200
CCGACCGCCT TACTGCCGCC TGTTTTGACC GCTGGGATCT GCCATTGTCA 4250
GACATGTATA CCCCGTACGT CTTCCCGAGC GAAAACGGTC TGCGCTGCGG 4300
GACGCGCGAA TTGAATTATG GCCCACACCA GTGGCGCGGC GACTTCCAGT 4350
TCAACATCAG CCGCTACAGT CAACAGCAAC TGATGGAAAC CAGCCATCGC 4400
CATCTGCTGC ACGCGGAAGA AGGCACATGG CTGAATATCG ACGGTTTCCA 4450
TATGGGGATT GGTGGCGACG ACTCCTGGAG CCCGTCAGTA TCGGCGGAAT 4500
TACAGCTGAG CGCCGGTCGC TACCATTACC AGTTGGTCTG GTGTCAAAAA 4550
TAATAATAAC CGGGCAGGCC ATGTCTGCCC GTATTTCGCG TAAGGAAATC 4600
CATTATGTAC TATTTAAAAA ACACAAACTT TTGGATGTTC GGTTTATTCT 4650
TTTTCTTTTA CTTTTTTATC ATGGGAGCCT ACTTCCCGTT TTTCCCGATT 4700
TGGCTACATG ACATCAACCA TATCAGCAAA AGTGATACGG GTATTATTTT 4750
TGCCGCTATT TCTCTGTTCT CGCTATTATT CCAACCGCTG TTTGGTCTGC 4800
TTTCTGACAA ACTCGGCCTC GACTCTAGGC GGCCGCGGGG ATCCAGACAT 4850
GATAAGATAC ATTGATGAGT TTGGACAAAC CACAACTAGA ATGCAGTGAA 4900
CA 02321215 2000-10-18
53
AAAAATGCTT TATTTGTGAA ATTTGTGATG CTATTGCTTT ATTTGTAACC 4950
ATTATAAGCT GCAATAAACA AGTTAACAAC AACAATTGCA TTCATTI'TAT 5000
GTTTCAGGTT CAGGGGGAGG TGTGGGAGGT TTTTTCGGAT CCTCTAGAGT 5050
CGAGTAGATA AGTAGCATGG CGGGTTAATC AT'1=dACTACA AGGAACCCCT 5100
AGTGATGGAG TTGGCCACTC CCTCTCTGCG CGCTCGCTCG CTCACTGAGG 5150
CCGGGCGACC AAAGGTCGCC CGACGCCCGG GCTTTGCCCG GGCGGCCTCA 5200
GTGAGCGAGC GAGCGCGCAG CAGATCTGGA AGGTGCTGAG GTACGATGAG 5250
ACCCGCACCA GGTGCAGACC CTGCGAGTGT GGCGGTAAAC ATATTAGGAA 5300
CCAGCCTGTG ATGCTGGATG TGACCGAGGA GCTGAGGCCC GATCACTTGG 5350
TGCTGGCCTG CACCCGCGCT GAGTTTGGCT CTAGCGATGA AGATACAGAT 5400
TGAGGTACTG AAATGTGTGG GCGTGGCTTA AGGGTGGGAA AGAATATATA 5450
AGGTGGGGGT CTTATGTAGT TTTGTATCTG TTTTGCAGCA GCCGCCGCCG 5500
CCATGAGCAC CAACTCGTTT GATGGAAGCA TTGTGAGCTC ATATTTGACA 5550
ACGCGCATGC CCCCATGGGC CGGGGTGCGT CAGAATGTGA TGGGCTCCAG 5600
CATTGATGGT CGCCCCGTCC TGCCCGCAAA CTCTACTACC TTGACCTACG 5650
AGACCGTGTC TGGAACGCCG TTGGAGACTG CAGCCTCCGC CGCCGCTTCA 5700
GCCGCTGCAG CCACCGCCCG CGGGATTGTG ACTGACTTTG CTTTCCTGAG 5750
CCCGCTTGCA AGCAGTGCAG CTTCCCGTTC ATCCGCCCGC GATGACAAGT 5800
TGACGGCTCT TTTGGCACAA TTGGATTCTT TGACCCGGGA ACTTAATGTC 5850
GTTTCTCAGC AGCTGTTGGA TCTGCGCCAG CAGGTTTCTG CCCTGAAGGC 5900
TTCCTCCCCT CCCAATGCGG TTTAAAACAT AAATAAAAAA CCAGACTCTG 5950
TTTGGATTTG GATCAAGCAA GTGTCTTGCT GTCTTTATTT AGGGGTTTTG 6000
CGCGCGCGGT AGGCCCGGGA CCAGCGGTCT CGGTCGTTGA GGGTCCTGTG 6050
TATTTTTTCC AGGACGTGGT AAAGGTGACT CTGGATGTTC AGATACATGG 6100
GCATAAGCCC GTCTCTGGGG TGGAGGTAGC ACCACTGCAG AGCTTCATGC 6150
TGCGGGGTGG TGTTGTAGAT GATCCAGTCG TAGCAGGAGC GCTGGGCGTG 6200
CA 02321215 2000-10-18
54
GTGCCTAAAA ATGTCTTTCA CTAGCAAGCT GATTGCCAGG GGCAGGCCCT 6250
TGGTGTAAGT GTTTACAAAG CGGTTAAGCT GGGATGGGTG CATACGTGGG 6300
GATATGAGAT GCATCTTGGA CTGTATTTZ'i' AGGTTGGCTA TGTTCCCAGC 6350
CATATCCCTC CGGGGATTCA TGTTGTGCAG AACCesCCAGC ACAGTGTATC 6400
CGGTGCACTT GGGAAATTTG TCATGTAGCT TAGAAGGAAA TGCGTGGAAG 6450
AACTTGGAGA CGCCCTTGTG ACCTCCAAGA TTTTCCATGC ATTCGTCCAT 6500
AATGATGGCA ATGGGCCCAC GGGCGGCGGC CTGGGCGAAG ATATTTCTGG 6550
GATCACTAAC GTCATAGTTG TGTTCCAGGA TGAGATCGTC ATAGGCCATT 6600
TTTACAAAGC GCGGGCGGAG GGTGCCAGAC TGCGGTATAA TGGTTCCATC 6650
CGGCCCAGGG GCGTAGTTAC CCTCACAGAT TTGCATTTCC CACGCTTTGA 6700
GTTCAGATGG GGGGATCATG TCTACCTGCG GGGCGATGAA GAAAACGGTT 6750
TCCGGGGTAG GGGAGATCAG CTGGGAAGAA AGCAGGTTCC TGAGCAGCTG 6800
CGACTTACCG CAGCCGGTGG GCCCGTAAAT CACACCTATT ACCGGGTGCA 6850
ACTGGTAGTT AAGAGAGCTG CAGCTGCCGT CATCCCTGAG CAGGGGGGCC 6900
ACTTCGTTAA GCATGTCCCT GACTCGCATG TTTTCCCTGA CCAAATCCGC 6950
CAGAAGGCGC TCGCCGCCCA GCGATAGCAG TTCTTGCAAG GAAGCAAAGT 7000
TTTTCAACGG TTTGAGACCG TCCGCCGTAG GCATGCTTTT GAGCGTTTGA 7050
CCAAGCAGTT CCAGGCGGTC CCACAGCTCG GTCACCTGCT CTACGGCATC 7100
TCGATCCAGC ATATCTCCTC GTTTCGCGGG TTGGGGCGGC TTTCGCTGTA 7150
CGGCAGTAGT CGGTGCTCGT CCAGACGGGC CAGGGTCATG TCTTTCCACG 7200
GGCGCAGGGT CCTCGTCAGC GTAGTCTGGG TCACGGTGAA GGGGTGCGCT 7250
CCGGGCTGCG CGCTGGCCAG GGTGCGCTTG AGGCTGGTCC TGCTGGTGCT 7300
GAAGCGCTGC CGGTCTTCGC CCTGCGCGTC GGCCAGGTAG CATTTGACCA 7350
TGGTGTCATA GTCCAGCCCC TCCGCGGCGT GGCCCTTGGC GCGCAGCTTG 7400
CCCTTGGAGG AGGCGCCGCA CGAGGGGCAG TGCAGACTTT TGAGGGCGTA 7450
GAGCTTGGGC GCGAGAAATA CCGATTCCGG GGAGTAGGCA TCCGCGCCGC 7500
CA 02321215 2000-10-18
AGGCCCCGCA GACGGTCTCG CATTCCACGA GCCAGGTGAG CTCTGGCCGT 7550
TCGGGGTCAA AAACCAGGTT TCCCCCATGC TTTTTGATGC GTTTCTTACC 7600
TCTGGTTTCC ATGAGCCGGT GTCCACGCTC GGTGACGAAA AGGCTGTCCG 7650
TGTCCCCGTA TACAGACTTG AGAGGCCTGT CCTLGACCGA TGCCCTTGAG 7700
AGCCTTCAAC CCAGTCAGCT CCTTCCGGTG GGCGCGGGGC ATGACTATCG 7750
TCGCCGCACT TATGACTGTC TTCTTTATCA TGCAACTCGT AGGACAGGTG 7800
CCGGCAGCGC TCTGGGTCAT TTTCGGCGAG GACCGCTTTC GCTGGAGCGC 7850
GACGATGATC GGCCTGTCGC TTGCGGTATT CGGAATCTTG CACGCCCTCG 7900
CTCAAGCCTT CGTCACTGGT CCCGCCACCA AACGTTTCGG CGAGAAGCAG 7950
GCCATTATCG CCGGCATGGC GGCCGACGCG CTGGGCTACG TCTTGCTGGC 8000
GTTCGCGACG CGAGGCTGGA TGGCCTTCCC CATTATGATT CTTCTCGCTT 8050
CCGGCGGCAT CGGGATGCCC GCGTTGCAGG CCATGCTGTC CAGGCAGGTA 8100
GATGACGACC ATCAGGGACA GCTTCAAGGA TCGCTCGCGG CTCTTACCAG 8150
CCTAACTTCG ATCACTGGAC CGCTGATCGT CACGGCGATT TATGCCGCCT 8200
CGGCGAGCAC ATGGAACGGG TTGGCATGGA TTGTAGGCGC CGCCCTATAC 8250
CTTGTCTGCC TCCCCGCGTT GCGTCGCGGT GCATGGAGCC GGGCCACCTC 8300
GACCTGAATG GAAGCCGGCG GCACCTCGCT AACGGATTCA CCACTCCAAG 8350
AATTGGAGCC AATCAATTCT TGCGGAGAAC TGTGAATGCG CAAACCAACC 8400
CTTGGCAGAA CATATCCATC GCGTCCGCCA TCTCCAGCAG CCGCACGCGG 8450
CGCATCTCGG GCAGCGTTGG GTCCTGGCCA CGGGTGCGCA TGATCGTGCT 8500
CCTGTCGTTG AGGACCCGGC TAGGCTGGCG GGGTTGCCTT ACTGGTTAGC 8550
AGAATGAATC ACCGATACGC GAGCGAACGT GAAGCGACTG CTGCTGCAAA 8600
ACGTCTGCGA CCTGAGCAAC AACATGAATG GTCTTCGGTT TCCGTGTTTC 8650
GTAAAGTCTG GAAACGCGGA AGTCAGCGCC CTGCACCATT ATGTTCCGGA 8700
TCTGCATCGC AGGATGCTGC TGGCTACCCT GTGGAACACC TACATCTGTA 8750
TTAACGAAGC CTTTCTCAAT GCTCACGCTG TAGGTATCTC AGTTCGGTGT 8800
CA 02321215 2000-10-18
56
AGGTCGTTCG CTCCAAGCTG GGCTGTGTGC ACGAACCCCC CGTTCAGCCC 8850
GACCGCTGCG CCTTATCCGG TAACTATCGT CTTGAGTCCA ACCCGGTAAG 8900
ACACGACTTA TCGCCACTGG CAGCAGCCAC TGGTAACAGG ATTAGCAGAG 8950
CGAGGTATGT AGGCGGTGCT ACAGAGTTCT TGAAbTGGTG GCCTAACTAC 9000
GGCTACACTA GAAGGACAGT ATTTGGTATC TGCGCTCTGC TGAAGCCAGT 9050
TACCTTCGGA AAAAGAGTTG GTAGCTCTTG ATCCGGCAAA CAAACCACCG 9100
CTGGTAGCGG TGGTTTTTTT GTTTGCAAGC AGCAGATTAC GCGCAGAAAA 9150
AAAGGATCTC AAGAAGATCC TTTGATCTTT TCTACGGGGT CTGACGCTCA 9200
GTGGAACGAA AACTCACGTT AAGGGATTTT GGTCATGAGA TTATCAAAAA 9250
GGATCTTCAC CTAGATCCTT TTAAATTAAA AATGAAGTTT TAAATCAATC 9300
TAAAGTATAT ATGAGTAAAC TTGGTCTGAC AGTTACCAAT GCTTAATCAG 9350
TGAGGCACCT ATCTCAGCGA TCTGTCTATT TCGTTCATCC ATAGTTGCCT 9400
GACTCCCCGT CGTGTAGATA ACTACGATAC GGGAGGGCTT ACCATCTGGC 9450
CCCAGTGCTG CAATGATACC GCGAGACCCA CGCTCACCGG CTCCAGATTT 9500
ATCAGCAATA AACCAGCCAG CCGGAAGGGC CGAGCGCAGA AGTGGTCCTG 9550
CAACTTTATC CGCCTCCATC CAGTCTATTA ATTGTTGCCG GGAAGCTAGA 9600
GTAAGTAGTT CGCCAGTTAA TAGTTTGCGC AACGTTGTTG CCATTGCTGC 9650
AGGCATCGTG GTGTCACGCT CGTCGTTTGG TATGGCTTCA TTCAGCTCCG 9700
GTTCCCAACG ATCAAGGCGA GTTACATGAT CCCCCATGTT GTGCAAAAAA 9750
GCGGTTAGCT CCTTCGGTCC TCCGATCGTT GTCAGAAGTA AGTTGGCCGC 9800
AGTGTTATCA CTCATGGTTA TGGCAGCACT GCATAATTCT CTTACTGTCA 9850
TGCCATCCGT AAGATGCTTT TCTGTGACTG GTGAGTACTC AACCAAGTCA 9900
TTCTGAGAAT AGTGTATGCG GCGACCGAGT TGCTCTTGCC CGGCGTCAAC 9950
ACGGGATAAT ACCGCGCCAC ATAGCAGAAC TTTAAAAGTG CTCATCATTG 10000
GAAAACGTTC TTCGGGGCGA AAACTCTCAA GGATCTTACC GCTGTTGAGA 10050
TCCAGTTCGA TGTAACCCAC TCGTGCACCC AACTGATCTT CAGCATCTTT 10100
CA 02321215 2000-10-18
57
TACTTTCACC AGCGTTTCTG GGTGAGCAAA AACAGGAAGG CAAAATGCCG 10150
CAAAAAAGGG AATAAGGGCG ACACGGAAAT GTTGAATACT CATACTCTTC 10200
CTTT'iTCAAT ATTATTGAAG CATTTATCAG GGTTATTGTC TCATGAGCGG 10250
ATACATATTT GAATGTATTT AGAAAAATAA ACAAATAGGG GTTCCGCGCA 10300
CATTTCCCCG AAAAGTGCCA CCTGACGTCT AAGAAACCAT TATTATCATG 10350
ACATTAACCT ATAAAAATAG GCGTATCACG AGGCCCTTTC GTCTTCAA 10398
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4910 base pairs
(8) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG 50
GAGACGGTCA CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG 100
TCAGGGCGCG TCAGCGGGTG TTGGCGGGTG TCGGGGCTGG CTTAACTATG 150
CGGCATCAGA GCAGATTGTA CTGAGAGTGC ACCATATGCG GTGTGAAATA 200
CCGCACAGAT GCGTAAGGAG AAAATACCGC ATCAGGCGCC ATTCGCCATT 250
CAGGCTGCGC AACTGTTGGG AAGGGCGATC GGTGCGGGCC TCTTCGCTAT 300
TACGCCAGCT GGCGAAAGGG GGATGTGCTG CAAGGCGATT AAGTTGGGTA 350
ACGCCAGGGT TTTCCCAGTC ACGACGTTGT AAAACGACGG CCAGTGCCAA 400
GCTTGCATGC CTGCAGGTCG ACTCTAGAGG ATCCGAAAAA ACCTCCCACA 450
CCTCCCCCTG AACCTGAAAC ATAAAATGAA TGCAATTGTT GTTGTTAACT 500
TGTTTATTGC AGCTTATAAT GGTTACAAAT AAAGCAATAG CATCACAAAT 550
TTCACAAATA AAGCAT'1'TT'T TTCACTGCAT TCTAGTTGTG GTTTGTCCAA 600
ACTCATCAAT GTATCTTATC ATGTCTGGAT CCCCGCGGCC GCCAAATCAT 650
CA 02321215 2000-10-18
58
TTATTGTTCA AAGATGCAGT CATCCAAATC CACATTGACC AGATCGCAGG 700
CAGTGCAAGC GTCTGGCACC TTTCCCATGA TATGATGAAT GTAGCACAGT 750
TTCTGATACG CCTTTTTGAC GACAGAAACG GGTTGAGATT CTGACACGGG 800
AAAGCACTCT AAACAGTCTT TCTGTCCGTG AGTGhAGCAG ATATTTGAAT 850
TCTGATTCAT TCTCTCGCAT TGTCTGCAGG GAAACAGCAT CAGATTCATG 900
CCCACGTGAC GAGAACATTT GTTTTGGTAC CTGTCTGCGT AGTTGATCGA 950
AGCTTCCGCG TCTGACGTCG ATGGCTGCGC AACTGACTCG CGCACCCGTT 1000
TGGGCTCACT TATATCTGCG TCACTGGGGG CGGGTCZ"T1'T CTTGGCTCCA 1050
CCCTTTTTGA CGTAGAATTC ATGCTCCACC TCAACCACGT GATCCTTTGC 1100
CCACCGGAAA AAGTCTTTGA CTTCCTGCTT GGTGACCTTC CCAAAGTCAT 1150
GATCCAGACG GCGGGTGAGT TCAAATTTGA ACATCCGGTC TTGCAACGGC 1200
TGCTGGTGTT CGAAGGTCGT TGAGTTCCCG TCAATCACGG CGCACATGTT 1250
GGTGTTGGAG GTGACGATCA CGGGAGTCGG GTCTATCTGG GCCGAGGACT 1300
TGCATTTCTG GTCCACGCGC ACCTTGCTTC CTCCGAGAAT GGCTTTGGCC 1350
GACTCCACGA CCTTGGCGGT CATCTTCCCC TCCTCCCACC AGATCACCAT 1400
CTTGTCGACA CAGTCGTTGA AGGGAAAGTT CTCATTGGTC CAGTTTACGC 1450
ACCCGTAGAA GGGCACAGTG TGGGCTATGG CCTCCGCGAT GTTGGTCTTC 1500
CCGGTAGTTG CAGGCCCAAA CAGCCAGATG GTGTTCCTCT TGCCGAACTT 1550
TTTCGTGGCC CATCCCAGAA AGACGGAAGC CGCATATTGG GGATCGTACC 1600
CGTTTAGTTC CAAAATTTTA TAAATCCGAT TGCTGGAAAT GTCCTCCACG 1650
GGCTGCTGGC CCACCAGGTA GTCGGGGGCG GTTTTAGTCA GGCTCATAAT 1700
CTTTCCCGCA TTGTCCAAGG CAGCCTTGAT TTGGGACCGC GAGTTGGAGG 1750
CCGCATTGAA GGAGATGTAT GAGGCCTGGT CCTCCTGGAT CCACTGCTTC 1800
TCCGAGGTAA TCCCCTTGTC CACGAGCCAC CCGACCAGCT CCATGTACCT 1850
GGCTGAAGTT TTTGATCTGA TCACCGGCGC ATCAGAATTG GGATTCTGAT 1900
TCTCTTTGTT CTGCTCCTGC GTCTGCGACA CGTGCGTCAG ATGCTGCGCC 1950
CA 02321215 2000-10-18
59
ACCAACCGTT TACGCTCCGT sAGATTCAAA CAGGCGCTTA AATACTGTTC 2000
CATATTAGTC CACGCCCACT GGAGCTCAGG CTGGGTTTTG GGGAGCAAGT 2050
AATTGGGGAT GTAGCACTCA TCCACCACCT TGTTCCCGCC TCCGGCGCCA 2100
TTTCTGGTCT TTGTGACCGC GAACCAGTTT GGCAAAGTCG GCTCGATCCC 2150
GCGGTAAATT CTCTGAATCA GTTTTTCGCG AATCTGACTC AGGAAACGTC 2200
CCAAAACCAT GGATTTCACC CCGGTGGTTT CCACGAGCAC GTGCATGTGG 2250
AAGTAGCTCT CTCCCTTCTC AAATTGCACA AAGAAAAGGG CCTCCGGGGC 2300
CTTACTCACA CGGCGCCATT CCGTCAGAAA GTCGCGCTGC AGCTTCTCGG 2350
CCACGGTCAG GGGTGCCTGC TCAATCAGAT TCAGATCCAT GTCAGAATCT 2400
GGCGGCAACT CCCATTCCTT CTCGGCCACC CAGTTCACAA AGCTGTCAGA 2450
AATGCCGGGC AGATGCCCGT CAAGGTCGCT GGGGACCTTA ATCACAATCT 2500
CGTAAAACCC CGGCATGGCG GCTGCGCGTT CAAACCTCCC GCTTCAAPiAT 2550
GGAGACCCTG CGTGCTCACT CGGGCTTAAA TACCCAGCGT GACCACATGG 2600
TGTCGCAAAA TGTCGCAAAA CACTCACGTG ACCTCTAATA CAGGACTCTA 2650
GAGGATCCCC GGGTACCGAG CTCGAATTCG TAATCATGGT CATAGCTGTT 2700
TCCTGTGTGA AATTGTTATC CGCTCACAAT TCCACACAAC ATACGAGCCG 2750
GAAGCATAAA GTGTAAAGCC TGGGGTGCCT AATGAGTGAG CTAACTCACA 2800
TTAATTGCGT TGCGCTCACT GCCCGCTTTC CAGTCGGGAA ACCTGTCGTG 2850
CCAGCTGCAT TAATGAATCG GCCAACGCGC GGGGAGAGGC GGTTTGCGTA 2900
TTGGGCGCTC TTCCGCTTCC TCGCTCACTG ACTCGCTGCG CTCGGTCGTT 2950
CGGCTGCGGC GAGCGGTATC AGCTCACTCA AAGGCGGTAA TACGGTTATC 3000
CACAGAATCA GGGGATAACG CAGGAAAGAA CATGTGAGCA AAAGGCCAGC 3050
AAAAGGCCAG GAACCGTAAA AAGGCCGCGT TGCTGGCGTT TTTCCATAGG 3100
CTCCGCCCCC CTGACGAGCA TCACAAAAAT CGACGCTCAA GTCAGAGGTG 3150
GCGAAACCCG ACAGGACTAT AAAGATACCA GGCGTTTCCC CCTGGAAGCT 3200
CCCTCGTGCG CTCTCCTGTT CCGACCCTGC CGCTTACCGG ATACCTGTCC 3250
CA 02321215 2000-10-18
GCCTTTCTCC CTTCGGGAAG CGTGGCGCTT TCTCATAGCT CACGCTGTAG 3300
GTATCTCAGT TCGGTGTAGG TCGTTCGCTC CAAGCTGGGC TGTGTGCACG 3350
AACCCCCCGT TCAGCCCGAC CGCTGCGCCT TATCCGGTAA CTATCGTCTT 3400
GAGTCCAACC CGGTAAGACA CGACTTATCG CCACZjGCAG CAGCCACTGG 3450
TAACAGGATT AGCAGAGCGA GGTATGTAGG CGGTGCTACA GAGTTCTTGA 3500
AGTGGTGGCC TAACTACGGC TACACTAGAA GGACAGTATT TGGTATCTGC 3550
GCTCTGCTGA AGCCAGTTAC CTTCGGAAAA AGAGTTGGTA GCTCTTGATC 3600
CGGCAAACAA ACCACCGCTG GTAGCGGTGG TT'IwIT!"PGTT TGCAAGCAGC 3650
AGATTACGCG CAGAAAAAAA GGATCTCAAG AAGATCCTTT GATCTTTTCT 3700
ACGGGGTCTG ACGCTCAGTG GAACGAAAAC TCACGTTAAG GGATTTTGGT 3750
CATGAGATTA TCAAAAAGGA TCTTCACCTA GATCCTTTTA AATTAAAAAT 3800
GAAGTTTTAA ATCAATCTAA AGTATATATG AGTAAACTTG GTCTGACAGT 3850
TACCAATGCT TAATCAGTGA GGCACCTATC TCAGCGATCT GTCTATTTCG 3900
TTCATCCATA GTTGCCTGAC TCCCCGTCGT GTAGATAACT ACGATACGGG 3950
AGGGCTTACC ATCTGGCCCC AGTGCTGCAA TGATACCGCG AGACCCACGC 4000
TCACCGGCTC CAGATTTATC AGCAATAAAC CAGCCAGCCG GAAGGGCCGA 4050
GCGCAGAAGT GGTCCTGCAA CTTTATCCGC CTCCATCCAG TCTATTAATT 4100
GTTGCCGGGA AGCTAGAGTA AGTAGTTCGC CAGTTAATAG TTTGCGCAAC 4150
GTTGTTGCCA TTGCTACAGG CATCGTGGTG TCACGCTCGT CGTTTGGTAT 4200
GGCTTCATTC AGCTCCGGTT CCCAACGATC AAGGCGAGTT ACATGATCCC 4250
CCATGTTGTG CAAAAAAGCG GTTAGCTCCT TCGGTCCTCC GATCGTTGTC 4300
AGAAGTAAGT TGGCCGCAGT GTTATCACTC ATGGTTATGG CAGCACTGCA 4350
TAATTCTCTT ACTGTCATGC CATCCGTAAG ATGCTTTTCT GTGACTGGTG 4400
AGTACTCAAC CAAGTCATTC TGAGAATAGT GTATGCGGCG ACCGAGTTGC 4450
TCTTGCCCGG CGTCAATACG GGATAATACC GCGCCACATA GCAGAACTTT 4500
AAAAGTGCTC ATCATTGGAA AACGTTCTTC GGGGCGAAAA CTCTCAAGGA 4550
CA 02321215 2000-10-18
61
TCTTACCGCT GTTGAGATCC XGTTCGATGT AACCCACTCG TGCACCCAAC 4600
TGATCTTCAG CATCTTTTAC TTTCACCAGC GTTTCTGGGT GAGCAAAAAC 4650
AGGAAGGCAA AATGCCGCAA AAAAGGGAAT AAGGGCGACA CGGAAATGTT 4700
GAATACTCAT ACTCTTCCTT TTTCAATATT ATTGAAGCAT TTATCAGGGT 4750
TATTGTCTCA TGAGCGGATA CATATTTGAA TGTATTTAGA AAAATAAACA 4800
AATAGGGGTT CCGCGCACAT TTCCCCGAAA AGTGCCACCT GACGTCTAAG 4850
AAACCATTAT TATCATGACA TTAACCTATA AAAATAGGCG TATCACGAGG 4900
CCCTTTCGTC 4910
