Note: Descriptions are shown in the official language in which they were submitted.
21890~7
Wogs/29993 1~IJ~ 5
GE:NE DELIVERY VECTOR USING PDASMID DNA
PACRAGED INTO AN A _ ~llCU': AND A pA~'T~A~TT-- CELL LINE
This invention was made with government support
under grant no. U01 AI 33355 awarded by the National
5 Institutes of Health. The y~JV~ t _as certain rights in
the invention.
BACKGROUND OF THE INVENTION
A variety of dif~erent gene transfer approaches
are available to deliver recombinant genes into cells and
10 tissues. Among these are several non-viral vectors,
lnrl~tl;nrJ DNA/liposome complexes, DNA, and targeted viral
protein DNA complexes. Several viral vectors, including
adenoviruses, adeno-associated viruses, retroviruses, and
others have previously been well-described. Most viral
15 vectors have several limitations, including possible
h; r,h~7~rd from possible recombination with wild-type
vectors, low viral titer and low expression levels.
Adenoviral vectors, in contrast, are an efEective means f or
introducing genes into tissues in vivo because of their
20 high level of expression and eficient transformation of
cells both in vitro and in vivo, see Davidson, et al.,
Nature GeIle~:ics, 3:219-223 (1993), Quantin, et al.,
P.N.A.S., 89:2581-2584 ~1992) and Mastrangeli, et al., J.
Clin. Invest. 91(1) :225-34 (1993) . However, these viral
25 vectors are disadvantageous for ~-l ;n; r;ll use for two
reasons. Because of their ability to recombine with
endogenous viruses, adenoviral vectors have a potential for
the spread of the recombinant gene in an uncontrolled
fashion through the population. In addition, current
30 vectors express multiple viral genes which can be
cytopath1c and/or immunogenic, yet are not necessarily
rer~uired for the vector. Thus, a need exists for a vector
or gene delivery system which is safe and efEective for
clinical use. This i~vention satisfies this need and
3 5 provides related advantages as well .
Wo gs/29993 2 1 8 9 0 6 7 . ~ 4
SUMMARY OF T~R INVE~NTION
This invention provides a novel expression vector
useful for inserting and expressing foreign nucleic acid
molecules in a host cell. ~ The expression vector -of this
5 invention i8 derived from an adenoviral vector and has as
its components the adenoviral Inverted Terminal Repeat, an
adenoviral packaging sequence, and the DNA molecule to be
inserted. This invention also provides an adçnoviral
expression vector having a foreign or heterolo~ous DNA
10 molecule inserted within adenoviral capsid proteins. These
vectors are useful for gene therapy.
~RIEF DESCRIPTION OF TI~R FIt~.TT~R.C
Figure 1 graphically depicts a strategy for
introducing plasmid DNA into adenoviral particle. The
15 inverted terminal repeat ~ ITR) packaging sequence of the
virus is introduced into a plasmid in such a fashion that
the plasmid can be linearized and :co-~transfected with a
mutant full-lçngth virus. The production of viral proteins
occurs and allows the plasmid DNA l:o be packaged in the
2 0 particle . ~
Figure 2 shows a segment of adenoviral DNA
subcloned into a cosmid vector and linearized before co-
tr~3n~f~-~ti-~n into the packaging cell line.
Figure 3 shows the use of a pac~aging~ plasmid
25 with the packaging site deleted, but thç ITR sequence
r-~ nti~; n~rl viral genomic DNA.
Figure 4 schematically depicts purification and
cloning of adenoviral type 5, wild-type and sub 360 genomic
DNA .
Wo 95/29993 2 1 8 9 0 ~ t 174
3
Figure 5 is a restriction map of plasmid Psi RSV
beta- gal .
Figure 6 is a restriction map of RSV beta-gal.
Figure 7 is a restriction map of plasmid Psi RSV
5 beta-gal-2.
Figure 8 is a restriction map of plasmid Psi RSV
beta-gal after partial digestion with AatII, treated with
Xlenow fragment and created a unique Xba I site.
Figure 9 is a restriction map of the cosmid
10 vector Cos Psi RSV beta-gal.
Figure 10 is a restriction map of packaging
plasmid Psi RSV beta-gal LS.
Figures llA through llC are restriction maps of
cosmid vectors. Figure llA i8 the cosmid Psi RSV beta-gal
15 A2. Figure llB i8 the cosmid Psi RSV beta-gal S2 and
Figure llC is the cosmid Psi RSV beta-gal AS2.
Figures 12A through 12C are the maps of the
adenoviral expression vectors of this invention. Figure
12A is the map of Psi RSV beta-gal LSA2. Figure 12B is the
20 restriction map of Psi RSV beta-gal LSS2 and Figure 12C is
the re8triction map of Psi RSV beta-gal LSAS2.
DET~TT~ DESCRIPTION OF THE INVENTION
An obj ect of this invention is to provide
adenoviral vectors which can be grown to high titer and
25 infect cells e~liciently. These vectors also are useful
for~gene therapy because the probability of recombination
with wild-type virus is extremely low and they express no
adenoviral gene products. Thus, another object of this
_ _ _ _ . . ... . . . . . . _ . .. , _ _ _ _ _ _ _ _ _ ,
Wo 95/29993 2 18 ~ 0 67 r~ 5l74
invention is to provide an alternate method ~or introducing
recombinant genes into cells f or the purposes of treating
disease. This is accomplished through the development of
a uni~r,ue adenoviral vector that contains a plasmid DNA
5 rather than adenoviral DNA. This invention ofEers an
advantage over retroviral vectors and conventional prior
art adenoviral vectors because it can be grown to high
titer stocks, can infect cells e~Eiciently, and is
extremely unlikely to L~ .' ;n~ in the population.
This invention provides a pseudo-adenovirus
vector comprising, from the 5 ' end to the 3 ' end, a DNA
molecule corresponding to a first adenovirus Inverted
Terminal ~epeat, a DNA molecule ~nrnrl;n~ adenovirus
packaging sequence, a heterQlogous DNA, and a DNA molecule
15 corr~pnn~;nrj to a second adenovirus Inverted Terminal
Repeat. As used herein, the term "pseudo-adenovirus
vector" is ; ntPn~ l to include DNA molecules that can be
transferred into the host cell in adenovirus capsids to
express a recombinant gene. As used herein, the term
20 "expression vector" is ; nt~nfl~ to mean a vehicle that
promotes the expression qf a gene inserted into it;
typically, a restriction rL _ ~ that carries a regulatory
ser~uence for the particular gene and sequences that provide
for RNA polyadenylation and processing.
The term "heterologous DNA" is intended to
encompass a DNA polymer. For example, the heterologous DNA
comprises plasmid vector DNA or cosmid vector DNA. Prior
to insertion into the pseudo-adenoviral vector, the
heterologous DNA is in the form of a separate fragment, or
as a c. _ nn~nt of a larger DNA construct, which has been
derived from DNA isolated at least once in substilnt;;~lly
pure form, i.e., free of cnnt~m;n~lting endogenous materials
and in a quantity or concentration enabling ;~nt;~ir~tion/
manipulation, and recovery of the segment and its component
nucleotide se~auences by standard biorh~m; r~1 methods, for
W0 95/29993 218 3 0 6 7 r "~ 74
5
example, ~lsing a cloni~g vector. A8 used herein,
~re~ ;nAn~l~ is ;nt~nrl~ to mean that a particular DNA
sequence i8 the product of various combination of cloning,
restr;~-t;f~n, and ligation steps resulting in a construct
5 having a sequence distinguishable from homologous sequences
f ound in natural systems . Recombinant sequences can be
assembled from cloned fragments and short oligonucleotides
linkers, or from a series of oli~ m~ tides.
In one aspect of this invention, the pseudo-
lO adenovirus expression vector and the adenovirus capsids arederived from adenovirus type 5 virus. Other suitable
adenoviral subtypes are human types 1-41 or murine strains.
In yet another aspect of this invention, the
vector further C~ntA;nq a DNA molecule cf)ntA;n;n~
15 adenovirus packaging sequence which allows the genetic
material to be assembled and packaged into the adenoviral
particle. This sequence is comprised of multiple, (6-20)
oligonucleotide repeats derived from sequence 3 ' to the
left ITR (Grable et al- (1990) ia~L-) -
The heterologous DNA also can contain additional
DNA molecules which comprise a transcriptional initiation
region 80 that DNA molecules downstream from the i~itiation
region can be transcribed to a sequence of interest,
usually mRNA, who6e transcription and, as appropriate,
translation will result in the expression of a polypeptide,
a protein, a ribozyme and/or the regulation of other genes,
e.g. antisense, expression of transcriptional factors, etc.
There are technical considerationg in introducing
adenoviral DNA into adenoviral complexes. Among the cis-
3 0 acting DNA sequences required f or packaging are the
inverted terminal repeats (ITR), which are required for
replication of the DNA in cells that contain adenoviral
gene products . Second, the presence of the p~f'kA~; n~
_ _ _ _ _ _ _ _ . . .. . _ _ _ . _ ... . _ . _ _ _ _ _
Wo 9S/29993 2 1 8 9 0 6 7 ~ l4
sequence is required. These sequences have been~defined,
in part, by deletion analysis of minimal regions required
for packaging, and have been previou31y described (Grable
et al., J. Virol. 64:2047-2056 (1990) incorporated herein
5 by reference). Third, the length of the DNA to be packaged
within the adenoviral sequence needs to be considered. In
the present invention, several means to introduce the
recombinant DNA into the adenoviral particle have been set
f orth .
Conventionally, adenoviral packaging is
accomplished using a plasmid r~mti:;n;n~ the left end of the
adenoviral genome which is replication defective and co-
transfecting with wild type adenoviral DNA inactivated to
prevent its replication. In the present application, there
15 are three strategies that have been taken to introduce
plasmid DNA into the adenoviral particle. In the first
case (Figure 1), the ITR packaging sequence of the virus is
introduced into a plasmid in such a f ashion that the
plasmid can be linearized and co-transfected with the virus
2 0 DNA . Thus, the production of viral proteins occurs and
allows the plasmid DNA to be packaged in the particle. In
a variation of this approach (Figure 2), a segment of
adenoviral DNA is subcloned into a cosmid vector and
linearized before co-transfection into the pArkA~l nj cell
25 line, thus also allowing for packaging of the recombinant
DNA in the transfected cell line. The advantage of this
approach is that an artificial f orm of the truncated virus
is used, thus minimizing the possibility that uncut viral
DNA will be present in the cell culture and will allow for
30 the replication of wild-type adenovirus. Flnally, in the
preferred embodiment (Figure 3), the packaging plasmid is
used, together with an adenovirus in which the packaging
site has been deleted but the ITR se~ue~ce is maintained,
thus allowing for the replication of defective virus and
35 viral proteins at the same time that the plasmid DNA is
replicated within cells, allowing for the higher titer
Wo 95/29993 2 1 8 9 0 6 7 P~ 4
7
virus. A ~urther'' development of this technology is a
permanent packaging cell line which provides the viral
packaging proteins in trans, and thus require only the
transfection of the plasmid DhA with the packaging sec~uence
5 within. The present studies demonstrate the feasibility of
using a packaging sequence and ITR anti-plasmid to allow
incorporation of the DNA into the antiviral particle. The
addition of nonviral DNA sequences to further improve
eDiciency are within the scope of this invention. Other
aspects include to introduce adenoviral sec,uences to
further define the other cis-acting regulatory elements
rec~uired for packaging, and finally, to introduce
additional consensus packaging sequences into the
background of irrelevant DNA ~phage DNA) to further improve
the eiliciency of packaging of the plasmid vector.
MATERIA~S AND METHODS
Cell ~ultllre
The transformed human embryonic kidney cell line,
293, (ATCC) was ~;nt::l;nf~ in Dulbecco's Modified Eagle
Medium (D-MEM, Gibco) supplemented with 10~ Fetal Bovine
Serum (FBS, Gibco), 50 U/ml penicillin, 50 ~Lg/ml
streptomycin and 2 mM L-Glutamine.
DNA and pl ~prn; d
Puriilcation of Ad5 ~nd ~ub360 genomic DNA (Figure 4)
For preparation of Adenoviru8 type 5 wild type
and its derivative, sub360 ~genomic DNA, 293 cells were
infected with each virus lysate (10 plaque forming
units/cell). The adenovirus particles were purified by
CsCl density centrifugation (Graham, et al., VirolQcy
52:456-467 (1973) incorporated herein by reference), then
treated with 2 mg/ml of self-dige~ted Pronase E (Sigma) in
50 mM TrisCl pH 7.4, lmM EDTA and 0.59r SDS solution at 37 C
_ _ _ ~ _ . . , . ., .. _ _ .. . _ _ _ .. _ . _ _ _ _ _ ... ..
WOgs/29993 ` ~ 21~396't 1~, i4
for 45 min., ~xtracted with phenol-chloroform twice and
with chloroform once. Genomic DNA was recovered by ethanol
precipitation :
pWEsub3 6 0 ( Figure 4 )
The sub360 DNA was treated with T4 polynucleotide
kinase and Klenow f ragment to repair the ends of the
genomic DNA. Following the ligation of Xba I linkers
(Promega) to each end, the genomic DNA was digested with
Xba I The right hand fragment of sub360 was cloned into
the Xba I site of cosmid vector pWE15 (Strategene) which
was modiiled by creating a new Xba I site into the BamEII
site according to the manufacturer' s instructions .
y~RSV ,~lGal ~Figures 5, 6)
For cloning of the Ad5 tf~r~n;n~l sequence and
packaging signal sequence (Grable, et al . (1990) su~ra. ),
pAd-Bgl II plasmid (Davidson, et al., Nature Genetics,
3:219-223 (1993) inL_UL~JULClLed herein by reference) was
digested with Eco RI and repaired by Klenow fragment of E.
coli DNA polymerase. After ligation of BamEII linkers
(Boehringer) to the blunted Eco RI sites, the plasmid was
digested with BamlII and BgI II. A DNA fragment ~ nt;3;nin~
the terminal sequence and p~ck~; n~ signal sequence (370
bp) was introduced into the Ban~II site of RSV ,BGal
(Stewart, et al. ~uman Gene Therapy, 3:267-275 (1992)
incorporated herein by reference). This clone was
tentatively coded as Pack+RSV ,(~Gal. Another terminal
sequence was generated by Polymerase Chain Reaction (PCR)
using pAd-Bgl II as a DNA template. In this reaction, the
primers were designed as follows: sense primer cr~nti:l;n1ng
an Eco RI site (nucleotide number of pAd Bgl II 1-29),
5~-ACAGAATTCGCTAGCATCATr~T~T~T~rC-3', (Seq. I.D. No. 1)
Wo 95/29993 2 ~ 8 ~ ~ 6 7 ~ 174
9
and anti-sense primer (200-173) ~ nnti~;n~n~ a BamHI site,
5'-ACAGGATCCGGCG(~ GTCACTTTTGCC-3' (Seq. I.D. No
2). The PCR conditions were 94C 30 seconds; 65C 30
seconds; and 72C 30 secondæ for the first 5 cycles, then
5 9~C 30 seconds; and 72C 30 seconds for 30 cycles. The
amplified terminal sequence (212 bp) was digested with Eco
RI and BamHI and subcloned into pBluescript (Strategene).
Following introduction of a BamHI linker into the Xho I
site of this plasmid, the t~rm;n~l sequence fragment was
10 purified by BamHI digestion, and introduced into the BamHI
site of Pack+RSV ,BGal plasmid to generate an Inverted
Terminal Repeat ( ITR) . The l,b RSV ,~Gal plasmid was
propagated in E. coli, SURE Cells (Strategene).
pAd~6
To construct a pAdl~ plasmid that encoded the Ad5
left hand DNA sequence, deleted for the packaging signal
sequence, the t~rm;n~l sequence in the above pBluescript
plasmid was purified by digestion with Nhe I and BamHI, and
cloned into the Nhe I and BgI II sites of pAd Bgl II.
20 Tran8fection
Co-transfection was performed by the calcium
phosphate method (Sambrook, et al., Molec~ qr Clnn;n~: A
Laboratorv M~n-l~l (1989) Cold Spring EIarbor ~aboratory,
N.Y., incorporated herein by reference) in lO0 mm diameter
25 petri dishes, 293 cells were transfected with 10 ~Lg Eco RI
digested pAd~6, lO~Lg Nhe I digested ~6RSV ~Gal, and varying
amounts of Xba I and Cla I digested sub 360 genome, or Xba
I and Klenow fragment-treated pWEsub360. In control
experiments, lOIlg of Bam~II digested RSV ~IGal was used in
30 place of ~ RSV ,BGal. Eight days post-transfection, cells
were harve8ted, suspended in 1.5 ml8 of medium and freeze-
thawed 3 times in dry ice-ethanol. Supernatants were used
as viral lysates in the subsequent experiments.
, _ _ _ _ _ , . .. . . .. _ _ . . _ _ . . . , _ _ _ _ _ _ ~
W095/29993 2~89~67 ~ 4
Titration of Virus
rrmFlllpnt 293 cells in 60 mm ~ r~-t~r dishes were
infected with 0 . 5 ml of viral lysate for 1 hr_ After
infection, 4.5 mls of medium were overlaid, and cells were
5 cultured for 24 hours at 37C. The infected cells were
harvested, washed with PBS twice, and fixed with 1. 2596
glutaraldehyde-PBS solution for 5 min at room temperature.
Fixed cells were washed with PBS twice and stained with
Solution X [50 mM Tris ~Cl, pE~ 7.5, 2.5 mM
10 Ferrifer~ocyanide, 15 mM NaCl, lmM MgCl, and 0.5 mg/ml X-
gal] overnight in 6 well culture plates. The number of
blue stained cells and total cells in each well were
counted (Table 1).
TABLE 1
15 Adenovirus packaging sequence i~duces incorporation of
linearized plasmid DNA into virus particles - evidence of
~rz~nc~llrtion and expression.
Vector Conc.Sub360 # Positive
(~g) cells/plate
20 Experiment 1 RSV $Gal o .5~= ~ 0. 9
~RSV $Gal 122.1
RSV $Gal 1. 0 26 . 7
~RSV $Gal 23 0 . o
Bxperiment 2 RSV $Gal 0 . 5 2 . 3
y~RSV $Gal 9 . 6
RSV $Gal 1. 0 4 .1
~6RSV $Gal 5 6 . 6
$-galactosidase activity of RSV $Gal or ~RSV $Gal
co - transf ected with su~3 60 digested with Xba I and Cla
30 and pAd~ (Bxperiment 1); co-transfected with pWEsub360 and
pAd~ ( Experiment 2 ) .
Pl~
Psi RSV beta-gal plasmid (Figure 7) was used as
35 a parental plasmid to construct the large-size plasmids.
WO 9~129993 2 18 ~ 0 6 7 ~ C 0! ~
11
Psi RSV beta-gal plasmid was partially digested with AatII
and treated with Klenow fragment, then an XbaI linker
(Progega) wa6 introduced (nucleotide position, 5,775).
- This plasmid was tentatively named Psi RSV beta-galXba
5 (Figure 8).
Separately, a cosmid vector, SuperCosl
(Stratagene) was digested with XbaI and NheI, and blunt-
ends created by Klenow fragment incubation. Then, a NotI
linker (Promega) was introduced into this position. The
10 cos fragment was prepared by digestion with HinfI and ~coRI
and by treatment with Klenow fragment. This ~, _
(2,371 bp) was inserted into the blunt-ended SalI site of
Psi RSV beta-galXbaI, described above. Thi6 cosmid vector
was coded as Cos Psi RSV beta-gal (Figure 9). For the
15 ligation reaction with yeast or phage A genomic DNA, Cos
Psi RSV beta-gal plasmid was digested NotI, treated with
Calf intestinal ~lk~ ;n~ phosphatase, then, additionally
digested with XbaI. Yea6t genomic DNA was completely
dige3ted with NheI and treated with alkaline phosphatase.
20 The DNA fragments were separated on 0 . 5~ low melting
agarose gel, the fr~ t~ ranging 20-30 kb were purified.
These fragments were ligated to NotI, XbaI-digested Cos Psi
RSV beta-gal plasmid, described above, then, packaged into
lambda phage using the Gigapack II packaging kit
25 (Stratagene) . The clones, whose total sizes ranged between
20-40kb were selected, and designated packaging plasmids
Psi RSV beta-gal~S (Figure lO).
To enhance the adenoviral packaging efEciency of
these plasmids, another Psi RSV beta-gal ~S plasmids also
3 0 was constructed which had additional packaging signals .
The oligonucleotides which coded packaging signal element
AV and AVI (Grable and ~earing, ~. Vi~ol. 66:723-731 (1992)
incorporated herein by reference) were designed as follows.
Sense primer which had ApaI restriction site at 3 ' end;
-
W095/29993 2~ 89067 ~.,., ~4
12
5~ -GCGTAATAL ~ ~rr-GCCGCGGGGA~~ G~CC-3~, (Seq. I.D.
No. 3)
anti-sense primer which had ApaI site at 5'-end;
5'-CCAA~TCCCCGCGrrrT~r~r~T~TTACGCGGCC-3' (Seq. I.D. No.
5 4).
Sense primer which had SapI site at 5'-endi
5~-GCTCGTAATA~ lAGGGCCGCGGGGACTTTGG-3', (Seq. I.D. No.
5)
anti-sense primer which had SapI site at 3'-end;
10 5~-AGcccA~AGTccccGcGr~rrrT~r~r~T~TTAcG-3~ (Seq. I.D. No.
6) .
All 5'-ends of sense and anti-sense oligo-
nucleotides were phosphorylated by T4: polynucleotide kinase
and annealed. The oligonucleotides which had either ApaI
15 site or SapI site were introduced into ApaI or SapI site of
C05 Psi RSV beta-gal to create two (2) tandem copies and
also to show the same direction as that of wild-type
packaging signal in Cos Psl RSV beta-gal (Figure 11) . The
plasmid whlch c~n~;n~d oligonucleotides at ApaI site was
20 called Cos Psi RSV beta-galA2 and the Sap I site was termed
Cos Psi RSV beta-galS2. When a plasmid was constructed
which contained the oligonucleotides at both ApaI and SapI
site, the oligonucleotide which bore the SapI sequence at
the end was inserted into SapI site of Cos Psi RSV beta-
25 galA2. This plasmid was named Cos ~si RSV beta-galAS2
(Figure llC) . To increase the total length of Cos Psi RSV
beta-galA2, S2 and AS2, NheI digested-yeast genomic DNAs
were ligated into XbaI site of each plasmids, packaged irLto
lambda phage as previously described. The plasmids wl~ich
30 showed those size between 20-40 kb were selected. The
plasmids 3F~n~r~.ocl from Cos Psi RSV beta-galA2 were coded
W0 95/29993 2 1 ~ 9 0 6 7 r~
13
as Psi RSV beta-gal LSA2, from Cos Psi RSV beta-galS2 were
Psi RSV beta-galLSS2, and from Cos Psi RSV beta-galAS2 were
P6i RSV beta-galLSAS2, as well (Figure 12).
The expression vectors of this invention can be
- 5 in6erted into host cell8, for example, rr- 1 ;~n cells,
particularly primate, more particularly human, but can be
associated with any animal of interest, particularly
domesticated animals, such as equine, bovine, murine,
ovine, canine, feline, etc. Among these species, various
types of cells may be involved, such as h~ ~topoietic,
neural, mesenchymal, cutaneous, mucosal, stromal, muscle,
spleen, reticuloendothelial, epithelial, endothelial,
hepatic, kidney, gastrointestinal, pulmonary, etc. Of
particular interest are hematopoietic cells, which can
include any of the nucleated cells which may be involved
with the lymphoid or myelomonocytic lineages. Also of
particular interest are members of the T- and B-cell
lineages, macrophages and monocytes. Purther of interest
are stem and progenitor cells, such as hematopoietic
neural, stromal, muscle, hepatic, pulmonary,
gastrointestinal, etc.
The heterologous DNA also can code for receptors
which may include receptors for the ligands IL-2, IL-3,
IL-4, I~-7 (interacts with p59fyn); erythropoietin (E~POR),
~-CSF, leukemia inhibitory factor ~LIF), ciliary neutryphic
factor (CNTR), growth hormone (GH), herpesvirus thymidine
kinase, histocompatibility genes, and prolactin (PRL).
The heterologous DN~ also may contain DNA
6equences which provides for the n~ q~ry transcriptional
termination, and as appropriate, translational termination.
The heterologous DNA can contain a wide variety
of genes, where the gene encodes a protein of interest or
an antisense sequence of interest. The gene can be any
_ _ _ _ _ _ , . .. . . _ _ . . . . ,, _ _ _ ,
W0 95/29993 __ 218 ~ 0 6 7 r~ /4
14 ~ --
sequence of interçst which provides a desired phenotype.
The gene can express a burface membrane protein, a secreted
protein, a cytoplasmic protein, or there may be a plurality
of genes which may express difterent types of products.
5 The gene also can encode an antisenSe sequence ~hich may
modulate a particular pathway by inhibiting a
transcriptional regulation protein or turn on a particular
pathway by inhibiting an inhibitor of the pathway. The
proteins which are expressed, singly or in combination, may
10 involve homing, cytotoxicity, proliferation, immune
response, ;nfli tnry response, clotting or diissolving of
clots, hormonal regulation, or the like. The proteins
expressed could be naturally-occurring, mutants of
naturally-occurring proteins, unique sequences, or
15 combinations thereof.
The gene also can encode a product which is
secreted by a cell, so that the encoded product may be made
available at will, whenever desired or needed by the host.
Various secreted products include huLI ~-, such as
20 insulin, human growth hormone, glucagon, pituitary
releasing factor, ACTH, melanotropin, relaxin, etc.; growth
factors, such as EGF, IGF-1, TGF-o~ , PDGF, G-CSF, M-CSF,
GM-CSF, FGF, erythropoietin, megakaryocytic stimulating and
growth factors, etc.; interleukins, such as Il.-l to -11;
25 TNF-o~ and -,~, etc.; and enzymes, such as tissue plA~-;n~en
activator, members of the complement cascade, perforans,
superoxide dismutase, coagulation factors, anti-
thrombin-III, Factor VIIIc, Factor VIIIvW, ~-anti-trypsin,
protein C, protein S, etc. ~
The gene also can encode a surface membrane
protein Such proteins may include homing receptors, e.g.
3.-selectin (Mel-14), blood-related proteins, particularly
having a kringle structure, e.g., Factor VIIIc, Factor
VIIIvW, hematopoietic cell markers, e.g. CD3, CD~, CD8,
35 B cell receptor, TCR isubunits ~Y, ,B, 1', ~, CD10, CDl9, CD28,
WO 9~;/29993 2 1 8 9 ~ 6 7 ~ 74
15
CD33, CD3~, CD41, etc., receptors, such as the interleukin
receptors I~-2R, IL-4R, etc., channel proteins, for in~ux
or efflux of ions, e.g., H~, Ca+, K+, Na~, Cl-, etc., and the
like; CFTR, tyrosine activation motif, zeta activation
5 protein, etc.
Also, intracellular proteins may be of interest,
such as proteins in metabolic pathways, regulatory
proteins, steroid receptors, transcription factors, etc.,
particularly depending upon the nature of the ho8t cell.
10 Some of the proteins indicated above may also serve as
intracellular proteins.
The following are a few illustrations of
different genes. In T-cells, one may wish to introduce
genes encoding one or both chains of a T-cell receptor.
15 For B-cells, one could provide the heavy and light chain5
for an immunoglobulin for secretion. For cutaneous cells,
e . g . keratinocytes, one could provide f or inf ectious
protection, by secreting cY-, B- or y-interferon,
antichemotactic factors, proteases speci~c for bacterial
20 cell wall proteins, etc.
In addition to providing for expression of a gene
which may have therapeutic value, there will be many
situations where one may wish to direct a cell to a
particular site. The site may include anatomical sites,
25 such as lymph nodes, mucosal tissue, skin, synovium, lung
or other internal organs or functional sites, such as
clots, injured sites, sites of surgical -~-n;rul~tlon~
in~ammation, infection, etc. By providing for expression
of surface membrane proteins which will direct the host
30 cell to the particular site by providing for binding at the
host target site to a naturally-occurring epitope,
localized r~n~-Pntrations of a secreted product may be
achieved. Proteins of interest include homing receptors,
e.g. L-selectin, GMP140, LCAM-1, etc., or addressins, e.g.
Wo 95/29993 ~ - 2 1 8 9 0 6 7 ~ ~l74
16
ELAM-l, PNAd, LNAd, etc., clot binding proteins, or cell
surface proteins that respond to localized gradients of
chemotactic factors. There are numerous situations where
directing cells to a particular site, :where release of a
5 therapeutic product could be of great value. Among these
would be the delivery of a recombinant gene to malignant
cells for the purpose of causing cell death or in~ ;n~^
immune recognition of tumors.
An additional example is autoi ^ disease.
10 Cells of .^~rt~n~l.^d lifetime, e.g. endothelial cells could be
employed. The heterologous DNA would provide for a homing
receptor for homing to the site of AlltO; A injury and
for cytotoxic attack on cells causing the injury. The
therapy would then be directed against cells causing the
15 injury. Alternatively, one could provide for secretion of
soluble receptors or other peptide or protein, where the
secretion product would inhibit activation of the injury
causing cells or induce anergy. Another alternative would
be to secrete an anti - inflammatory product, which could
20 serve to ~l;m;n;Al~ the degenerative efEects.
The genes can be introduced in one or more DNA
molecules or expression vectors, where there will be at
least one marker and may be two or more markers, which will
allow for selection of ~ host cells which contain the
25 gene (s) . The heterologous DNA, genes and expression
vectors can be prepared in conventional ways, where the
genes and regulatory regions may be isolated, as
appropriate, ligated, cloned in an appropriate ~cloning
host, analyzed by restriçtion ~or seq-uencing~ or other
30 convenient means. Particularly, using PCR, individual DNA
fLa~ tA including all or portions of a functional unit
may be isolated, where one or more mutations may be
introduced using "primer repairr, ligation, etc. as
appropriate. See Sambrook et al. Molecular Cloninq: A
35 ~,Ah~^,ratory Manual (1989) Cold Spring Harbor Press, N.Y.,
WO95/29993 2 1 8 9 ~ 6 7 ~J~ /4
17
incorporated herein by reference. Host cells can be grown
and ~An~ in culture before introduction of the
vector(s) followed by the appropriate treatment for
- introduction of the vectors and integration of the
5 vector(s) . The cells will then be ~XrAn~ d and 6creened by
virtue of a marker present in the vector Various markers
which may be used successfully include hprt, neomycin
resistance, thymidine kinase, hygromycin resistance, etc.
The expression vectors can be introduced
10 simultaneously or consecutively, each with the same or
difEerent markers.
Depending upon the nature of the cells, the cells
may be administered in a wide variety of ways.
Hematopoietic cells may be administered by injection into
15 the vascular system, there being usually at least about 104
cells and generally not more than about lOl, more usually
not more than about 108 cells. The number of cells which
are employed will depend upon a number of circumstances,
the purpose for the introduction, the lifetime of the
20 cells, the protocol to be used, for example, the number of
administrations, the ability of the cells to multiply, the
stability of the therapeutic agent, the physiologic need
for the therapeutic agent, and the like. Alternatively,
with skin cells which may be used as a graft, the number of
25 cells would depend upon the size of the layer to be applied
to the burn or other lesion. Generally, for myoblasts or
fibroblasts, the number of cells will at least about 104 and
not more than about 108 and may be applied as a disper8ion,
generally being inj ected at or near the site of interest .
30 The cells will usually be in a physiologically-acceptable
medium .
The vectors of this invention can be used for the
treatment of a wide variety of conditions and indications.
For examp'~, B- and T-cells, antigen-presenting cells or
_ _ _ _ .. . _ . ... _ , _ .. _ _ . _ _
WO9SI29993 -: ~ 2~8~67 ~ 4
malignant cells themselves may be used in the treatment of
cancer, infectious diseases, metabolic deficiencies,
cardiovascular disease, hereditary coagulation
deiïciencies, al~toi ^ diseases, joint degenerative
5 diseases, e.g. arthritis, pulmonary disease, kidney
disease, nedocrine -ab~ormalities, etc. Various cells
involved with structure, such as fibroblasts and myoblasts,
may be used in the treatment of genetic ripfiripn~;esl such
as connective tissue deficiencies, arthritis, hepatic
10 disease, etc. Hepatocytes could be used in cases where
large amounts of a protein must be made to complement a
deficiency or to deliver a therapeutic product to the liver
or portal circulation.
This invention also provides a transgenic, non-
15 human animal whose germ cells and somatic cells contain aheterologous DNA molecule that has been introduced into the
animal, or an ancestor of the animal, at an embryonic
stage. ~hen the heterologous DN~ molecule encodes an
product which produces a pathological condition in the
20 animal, these animals are useful to test materials
suspected of treating the pathology. Alternatively, the
heterologous DNA can be used to encode a therapeutic or
prophylactic composition. These animals are useful to test
the particular therapy. Using the vectors of this
25 invention and methods well known to those of skill in the
art (for example, JJeder et al., U.S. Pate~t No. 4,736,866,
issued April 12, 1988, incorporated herein by reference),
the transgenic animals can be produced.
Although the invention has been described with
30 reference to the above embodiments, it should be understood
that various modifications can be made without departing
from the spirit of the invention Accordingly, the
invention is limited only by the following claims.
WO 9~129993 2 ~ g ~ O ~ 7 1 ~ .5~ _.,1 /4
19
SoQD~NCo LISTING
(1) GENERAL lNr~
(i) APPLICANT: THE UNl~or~ol'~ OF NICHIGAN
(iL) TITLE OF INVENTION: G3NE DELIVERY VoCTOR USING PLASMID DNA
PACKAGED INTO AN ADENOVIRUS AND A PACKAGING CELL LINE
(iii) NUMBER OF SEQUENCES: 6
(iv) C~JA,~or~ ADDRESS:
A, rnnT-~Cc~: ~ORR~SON & FOERSTER
B ST EET: 55 PAGE MILL ROAD
C CITY: PALO ALTO
D I STATE: C~LIFORNIA
E, CO JNTRY : ~SA
Fi ZIP: 94304--1018
(v) COM~UTER READABLE FORM:
(A MEDIUM TYPE: Floppy disk
(B COMPUTER: IBU PC compatible
(C I OPERATING SYSTEM: PC--DOS/MS--DOS
(D.l SOFTWARE: PatentIn Release ~1.0, Version 3~1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(vLi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/234,990
(B) FIL~NG DATE: 28--APR--1994,
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: I~ONSKI, r LO F.
(B) rUC~ ~L'rLL~W NUMBER: 34,202
(C) REFERENOE/DOCKET NUMBER: 20344--20910.40
(iX) TFT. ~;sTION INFORMATION:
(A) TELEPHONE: (415) 813-5600
(B) TELEFAX: (415) 494--0792
(C) TELEX: 706141
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE r~AT~r.. ,.~
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRr~ znrrlrgc: single
(D) TOPOLOGY: linear
(xi) SoQUENCE Uoo~ lr~ : SEQ ID NO:l:
ACAGAATTCG rTr'"'ATrAT rAATAr~TATA CC
32
( 2 ) INFORNATION FOR SEQ ID NO: 2:
(i) SEQUENCE r~ rTF~T~cTIcs
A) LENGTH: 37 bA8e pairs
B ) TYPE: nucleic acid
, C) STP~ ingle
D) TOPOLOGY: linear
W0 95129993 ~ ~ 8 g O ~ ~ r ~ . ' 14
(Xi) SEQI~E~ DESCRIPTION: S~Q ID NO:2:
ACAGGATCCG GrarDrDrr7~ AAAi~CGTCAC TTTTGCC 37
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQI~ENCE: rTT~ rT~oTcTIcs:
Al LENGTH: 38 base pairs
B TYPE: nucleie acid
C sT~ Kn~Ecc: ~ingle
ID TOPOLOGY: linear
(Xi) SEQIJENOE DESCRIPTION: SEQ ID NO:3:
GCGTAA~CATT Tr.TrT~r.nr.r rrrGr-aa~'rT TTGGGGCC 38
(2) INFORMATION FOR SEQ ID NO:4:
( i ) SEQUENCE rr~ l h ~
A) LENGTII: 38 base pairs
B) TYPE: nucleic acid
C) ~ r~ ~KIlN~ .c: single
D) TOPOLOGY: linear
(Xi) SEQUENOE DESCRIPTION: SEQ ID NO:4:
rr~ rTrrr 1 ~ .~ r''r~\n7~T/~TT ACGCGGCC 38
(2) INFORMATION FOR SEQ ID NO:S:
(i) SEQUENOE rTTDo~ N
A LENGTE: 36 base pairs
B TYPE: nucleic acid
C . sT~ )RnN-cc: ~ingle
l D I TOPOLOGY linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
GCTCGTAi~TA TTTGTCTAGG ~ CTTTGG . . 3 6
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQ'~ENCE rTT~RprT~oTcTIcs:
Al LENGT~: 36 ba~e pair~
B TYPB: nucleic acid
C ~ ST~7` : single
l D I TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
~r.rrr~ r~T ~ iG~ ~ rTDr.l~r~ T ATTACG 36
