CA2189067A1

CA2189067A1 - Gene delivery vector using plasmid dna packaged into an adenovirus and a packaging cell line

Info

Publication number: CA2189067A1
Application number: CA 2189067
Authority: CA
Inventors: Gary J. Nabel; Michael J. Imperiale; Takeshi Ohno
Original assignee: Individual
Current assignee: University of Michigan
Priority date: 1994-04-28
Filing date: 1995-04-25
Publication date: 1995-11-09
Also published as: EP0763103A1; JPH10507061A; EP0763103A4; WO1995029993A1

Abstract

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 invention is derived from an adenovirus 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 a pseudo-adenoviral expression vector having a foreign or heterologous DNA molecule inserted within adenoviral capsid proteins. These vectors are useful for gene therapy.

Description

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 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 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 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).

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! ~

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 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~

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 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 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 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 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

( 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

Claims

What is claimed is:

1. A pseudo-adenovirus expression vector, comprising, from the 5' end to the 3' end, a DNA molecule corresponding to a first adenovirus Inverted Terminal Repeat, a DNA molecule encoding adenovirus packaging sequence, a heterologous DNA, and a DNA molecule corresponding to a second adenovirus Inverted Terminal Repeat.

2. The pseudo-adenovirus expression vector of claim 1, wherein the adenovirus capsid is derived from adenovirus type 5 virus.

3. The pseudo-adenovirus expression vector of claim 1, further comprising a second DNA molecule containing adenovirus packaging sequences.

4. The pseudo-adenovirus expression vector of claim 1, wherein the heterologous DNA comprises plasmid vector DNA or cosmid vector DNA.

5. The pseudo-adenovirus expression vector of claim 1, wherein the heterologous DNA further comprises a promoter for transcription.

6. The pseudo-adenovirus expression vector of claim 1, wherein the heterologous DNA codes for a ribozyme, a protein, a polypeptide, or an antisense RNA molecule.

7. A gene expression system comprising the pseudo-adenovirus expression vector of claim 1 and a packaging defective adenovirus helper virus.

8. The gene expression system of claim 7, wherein the defective adenovirus is derived from adenovirus type 5 virus.

9. The gene expression system of claim 7, wherein the adenovirus expression vector further comprising a second DNA molecule encoding adenovirus packaging sequence.

10. The gene expression system of claim 7, wherein the heterologous DNA comprise plasmid vector DNA or cosmid vector DNA.

11. The gene expression system of claim 7, wherein the heterologous DNA further comprises a promoter for transcription.

12. The gene expression system of claim 7, wherein the heterologous DNA codes for a ribozyme, a protein, a polypeptide, or an antisense RNA molecule.

13. A pseudo-adenoviral expression vector comprising a heterologous DNA molecule and adenoviral capsid proteins, the DNA molecule being encapsulated within the capsid proteins.

4. The pseudo-adenovirus expression vector of claim 13, wherein the adenovirus capsid is derived from adenovirus type 5 virus.

15. The pseudo-adenovirus expression vector of claim 13, wherein the heterologous DNA comprises plasmid vector DNA or cosmid vector DNA.

16. The pseudo-adenovirus expression vector of claim 13, wherein the heterologous DNA further comprises a promoter for transcription.

17. The pseudo-adenovirus expression vector of claim 13, wherein the heterologous DNA codes for a ribozyme, a protein, a polypeptide, or an antisense RNA molecule.

18. A host cell comprising the pseudo-adenovirus expression vector of claim 1.

19. A host cell comprising the pseudo-adenovirus expression vector of claim 13.

20. The host cell of claim 18 or 19, wherein the pseudo-adenovirus is derived from adenovirus type 5 virus.

21. The host cell of claim 18 or 19, wherein the heterologous DNA comprises plasmid vector DNA or cosmid vector DNA.

22. The host cell of claim 18 or 19, wherein the heterologous DNA further comprises a promoter for transcription.

23. The host cell of claim 18 or 19, wherein the heterologous DNA codes for a ribozyme, a protein, a polypeptide, or an antisense RNA molecule.

24. A non-human transgenic animal comprising the pseudo-adenoviral expression vector of claim 13.

25. The non-human transgenic animal of claim 24, wherein the pseudo-adenovirus is derived from adenovirus type 5 virus.

26. The non-human transgenic animal of claim 24, wherein the heterologous DNA comprises plasmid vector DNA
or cosmid vector DNA.

27. The non-human transgenic animal of claim 24, wherein the heterologous DNA further comprises a promoter for transcription.

28. The non-human transgenic animal of claim 24, wherein the heterologous DNA codes for a ribozyme, a protein, a polypeptide, or an antisense RNA molecule.

29. A method of introducing a heterologous DNA
molecule into a cell which comprises inserting into the cell the pseudo-adenovirus expression vector of claim 1.
3C. A method of introducing a heterologous DNA
molecule into a cell which comprises contacting the cell with the pseudo-adenovirus expression vector of claim 13.