ADENOVIRUS-BASED METHODS, AND CELLS, USEFUL FOR THE EXPRESSION OF NUCLEIC ACID SEQUENCES

Abstract

This invention provides helper-dependent adenovirus cloning vectors and helper adenoviruses, and methods for making and using such preparations, wherein the helper adenoviruses contain recombinase target sites that are useful in reducing the level of contamination of helper virus in helper-dependent adenovirus vector preparations.

Description

FIELD OF THE INVENTION

[0002] The present invention relates to adenovirus vectors that have increased utility for gene transfer into mammalian cells. The vector systems described have increased capacity for insertion of foreign DNA and improved safety.

BACKGROUND OF THE INVENTION

[0003] Parent application Ser. No. 08/250,885, filed on May 31, 1994 and its parent application Ser. No. 08/080,727 disclose and claim a genus of adenovirus(Ad)-derived cell expression vectors having excellent potential as live recombinant vaccines and as transducing vectors for gene therapy. In the human Ad genome, early region 1 (E1), E3, and a site upstream of E4 have been utilized as sites for introducing foreign DNA sequences to generate adenovirus recombinants. In the absence of compensating deletions in E1 or E3, a maximum of about 2 kb can be inserted into the Ad genome to generate viable virus progeny. The E1 region is not required for viral replication in complementing 293 cells and up to 3.2 kb can be deleted in this region to generate conditional helper independent vectors with a capacity of 5.0-5.2 kb. In the E3 region, which is not required for viral replication in cultured cells, deletions of various sizes have been utilized to generate nonconditional helper independent vectors with a capacity of up to 4.5-4.7 kb.

[0004] The maximum capacity for inserts of foreign DNA in currently available helper independent Ad vectors such as those described in the parent applications is approximately 8 kb. This limited capacity arises from the use of Ad vectors which have deletions of E1 and E3 sequences and from the fact that most other regions of the viral genome must be retained in order that the viral vector may be propagated without the need for a helper virus. Besides this limited capacity for insert DNA, previous vectors retain most of the viral genome, making it possible for expression of viral genes in transduced cells or in inoculated animals, including humans, which can result in toxic or other untoward effects. In addition, previous viral vectors can recombine with Ad sequences present in cells used for propagation of the vectors or with Ad sequences that may be present in inoculated animals. Therefore, it is an objective of this invention to provide Ad cloning vectors from which all or most viral genes have been removed and which will have increased safety and capacity for larger insertions compared to currently available vectors.

SUMMARY OF THE INVENTION

[0005] It is a goal of this invention to provide a simple and useful system by which high capacity Ad5 cloning vectors may be developed. As demonstrated in concurrently filed application Ser. No. 08/486,549, entitled “Adenoviruses for Control of Gene Expression”, provision of Cre recombinase in Ad infected cells can catalyze excision or rearrangement of viral DNA sequences that contain the target sites (1oxP) for Cre mediated site specific recombination. In the present invention, use is made of this knowledge to construct Ad5 genomes in which the viral DNA packaging signals can be excised from the viral genome by action of Cre. Said excision of said packaging signal results in a viral DNA that is unable to package into virion particles. Such a viral DNA, though unable to package into virions, may encode viral functions that provide complementing functions for replication of a second, viral “vector”, that lacks substantial portions of the viral genome so that in coinfected cells, though both helper and vector DNAs may replicate, only the vector DNA can be packaged into virions.

[0006] One embodiment of the present invention provides a bacterial plasmid comprising a circularized modified human adenovirus type 5 (Ad5) genome that contains sequences that can be recognized and acted upon by a site specific recombinase known as Cre. Said bacterial plasmid is able to generate infectious Ad5 carrying the modified sequences including the sequences that can be recognized by Cre. The structure of the modified sequences in the bacterial plasmid and in viruses generated from said plasmid is such that recombination catalyzed by Cre results in excision of sequences, known as the packaging signal, near the left end of the Ad5 genome, that are required for packaging of Ad5 DNA into infectious virion particles. Optionally, certain regions of the plasimd and resulting viruses may be deleted, such as sequences from E1 or E3 that can be omitted from the viral genome without preventing the viral genome from replicating in such cells as may be permissive for replication of said viral genome in the form of infectious virus.

[0007] A second embodiment of the invention provides a bacterial plasmid comprising approximately 340 base pairs from the left end of the Ad5 genome, including the left end terminal repeat sequences of said genome and the packaging signal sequences thereof and the right terminal repeat sequences of the Ad5 genome. The left end of the left terminal repeat sequence is joined in “head to tail” configuration with the right end of the right terminal repeat. Between approximately nucleotide 35,800 near the right end of the genome, are substituted restriction enzyme sites suitable for insertion of foreign DNA sequences of up to about 35,000 base pairs in length.

[0008] A third embodiment of the invention provides a mammalian cell line, such as a human cell line, that provides the Cre recombinase enzyme. Alternatively, Cre may be provided by an Ad5 derived vector that expresses the Cre protein in suitable cells.

[0009] Other embodiments of the present invention include Ad genome constructs, known as “vectors”, containing substantial deletions of viral DNA sequences that are substituted with large insertions of foreign DNA 20-35 kb in length. Such genomes are unable to replicate as viruses in the absence of viral products provided by a second virus, hereafter called a “helper” virus.

[0010] One specific embodiment of the invention is a helper virus that can be designed, propagated, and used in such a way that when employed to support replication of a second virus, the vector, from which substantial portions of the viral genome have been deleted and substituted with foreign DNA, said “helper” virus DNA is unable to be packaged into infectious virions.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 is a diagrammatic representation of Cre mediated excision of DNA from a viral vector in which the packaging signal is flanked by 1ox P sites.

[0012] FIG. 2 is a diagrammatic representation of a method to generate helper dependent viral vectors using Cre mediated excision of the packaging signal to prevent packaging of the helper virus DNA.

[0013] FIG. 3 is a-diagrammatic representation of a plasmid derived from pBHG10 into which 1ox P sequences have been introduced at positions flanking the packaging signal.

[0014] FIG. 4 is a diagrammatic representation of a plasmid derived from pBHG10 from which most of the viral DNA has been deleted save for the left and right ITRs and the packaging signal.

[0015] FIG. 5 is a diagrammatic representation of a means to obtain coreplicating helper and helper dependent viruses by cotransfection of 293Cre Cells.

[0016] FIG. 6. Shows an HD leptin construct. (A) The DNA composite fragments of pΔSTK120-HCMV-mOb-BGHpΔ (≈19.6 kb total size) are from left to right: the left end terminus of Ad5, composed of the ITR sequences and the packaging signal Ψ (nucleotides 1-440, solid arrow); the 5,072-bp fragment of hypoxanthine guanine phophoribosyltransferase (HPRT) (nucleotides 12,373-17,853 in gb:humhprtb, striped area); the leptin expression cassette (1,835 bp), composed of the HCMV promoter, the murine leptin cDNA (500 bp) and the bovine growth hormone poly(A) tail (open area) (inserted in the complementary orientation): the HindIII 9063-bp fragment of C346 cosmid (nucleotides 12,421-21,484 in gb:L31948, checkered area); and the right end terminus of Ad5, composed of the ITR sequence (nucleotides 35,818-35,935). The ITRs are flanked by unique PmeI restriction sites used to liberate the vector fragment from the plasmid backbone before the initial transfection into 293-cre4 cells for viral rescue and propagation (released fragment is 16.7 kb). To the right of the vector structures is a represenative cesuim chloride banded HD-leptin vector stock (see Materials and Methods), at the final stage of band collection. The band is single, compact, and thick. (B) The structure and tail-to-tail concatamerizations (junction is at the 3′ ITR ends of ΔSTK120-HCMV-mOb-BGHpA), is verified by the restriction enzyme pattern of the three independently resucued viruses. The gel, labeled Vector DNA, shows 0.5 μg of DNA extracted from the HD-leptin viral stock (lane A), Ad-leptin stock (lane B) and the PmeI cut pΔSTK120-HCMV-mOb-BGHpA (lane C) compared on a 0.5% agarose gel for sizing. Both HD-leptin (33 kb) and Ad-leptin (34 kb) extracted DNA migrate, as expected, between 38.5-29.9 kb, and the cut ΔSTK120-HCMV-mOb-BGHpA (16.7 kb) migrates between 17.1 and 15.0 kb, the smaller band corresponds to the plasmid backbone (2.9 kb), and the faint band in lane A represents the trace amount of the propagated 16.7-kb linearized vector. Structures of ΔSTK120-HCMV-mOb-BGHpA (lane 1) (gel extracted after separation of plasmid and backbone by PmeI digestion) and the three HD-leptin vectors (lanes 24) are compared by restriction analysis, as described in Materials and Methods. The expected fragment sizes for HD-leptin are: for Asp-718: 15,391-single band(s), 6,296-double band(d), and 2,501-d; EagI:20,445-s 1,715-s and 6,270/6,266-d; FseI: 16,523/16,458-d; HindIII: 10,207/10,174-d, 5,845-d, and 454/450-d; PacI: 16,516/16,465-d; SmaI: 6,701-d, 5,163-d, 2,180-d, 1,715-s, and 1,589-d, and XhoI: 11,833-d, 2,964/2,953-d; and 1,701/1,697-d bp. The expected fragment sizes for ΔSTK120-HCMV-mOb-BGHpA are: for Asp-718: 7,837-s, 6,296-s, and 2,501-s; EagI: 10,364-s and 6,266-s; FseI: 16,458-s and 172-s; HindIII: 10,174-s, 5848-s, 450-s, and 158-s; PacI: 16,465-s and 165-s; SmaI: 6,701-s, 5,163-s, 2,180-s, 1,589-s and 997-s, and XhoI: 11833-s, 2964-s, 1697-s and 136-s bp. M1 and M2 are DNA markers (8-48 kb, Bio-Rad, and 1-kb DNA ladder, GIBCO/Life Technologies, Gaithersburg, Md., respectively).]

[0017] FIG. 7 shows mice which were treated with Ad-β-gal, Ad-leptin, and HD-leptin or dialysis buffer (controls). AST and ALT levels in the sera of lean control and treated mice are plotted at 1, 2, and 4 weeks posttreatment.

[0018] FIG. 8 shows photomicrographs illustrating liver histopathology in lean mice. (A) Un-treated control lean. (B) Positive Ad-β gal-treated control lean, 1 week posttreatment. (C) HD-leptin-treated lean, 1 week posttreatment. (D) Ad-leptin-treated lean, 1 week posttreatment. (E) HD-leptin-treated lean, 2 weeks posttreatment. (F) As-leptin-treated lean, 2 weeks posttreatment. (G) HD-leptin-treated lean, 4 weeks posttreatment. (H) Ad-leptin-treated lean, 4 weeks posttreatment. (Bar=100 μM.)

[0019] FIG. 9 shows HD-leptin and Ad-leptin effects in lean mice. Animals were injected via the tail vein with a single dose of 1-2×1011 particles of HD-leptin (n=5), Ad-leptin (n=10), Ad-β-gal (n=10), or the equivalent volume of dialysis buffer (n=10). The time course shows (A) serum leptin levels, collected 2-3 times weekly (ng/ml, mean±SEM); (B) weight (g, mean±SEM); (C) Southern blot analysis, the arrows refer to the single HD-leptin and Ad-leptin bands. Three internal genomic bands were also detected in treated and control DNA: one strong (uppermost, greater than 12 kb), and two faint (lower) bands. (D) Total RNA from livers of Ad-leptin-treated mice at 1-, 2-, 4-, and 8-week intervals, and at 1 and 8 weeks in HD-leptin treated mice. Arrows refer to the leptin message (≈500 bp) band, β-actin was used as the internal control (≈1 kb). E and F) Serum glucose (mg/dl) and insulin (ng/ml) were measured in all animal groups (mean±SEM).

[0020] FIG. 10 shows HD-leptin and Ad-leptin effects in ob/ob mice. Essentially as described in FIG. 4, animals were injected in the tail vein with a single dose of 1-2×1011 particles of HD-leptin (n=5), Ad-leptin (n=10), and Ad-β-gal (n=10), or the equivalent volume of dialysis buffer (n=10). Lean control values are plotted for comparison. The time course shows (A) serum leptin levels (ng/ml, mean±SEM), collected 2-3 times; (B) weight (g, mean±SEM). (C) Southern blot analysis, the arrows refer to the single HD-leptin and Ad-leptin bands. Three internal genomic bands were also detected in treated and control DNA: one strong (uppermost, >12 kb), and two faint (lower) bands. (D) Total RNA from livers of Ad-leptin-treated mice at 1-, 2-, 4-, and 8 week intervals, and in untreated mice. Arrows refer to the leptin message (≈500 bp) band, β-actin was used as the internal control (≈1 kb). (E and F) Serum glucose (mg/dl) and insulin (ng/ml) were measured in all animal groups (mean±SEM).

[0021] FIG. 11 shows phenotypic correction of HD-leptin-treated ob/ob mice. On the left is a representative ob/ob mouse treated with HD-leptin at day 54 posttreatment, next to a littermate treated with Ad-leptin. The Ad-leptin treated mouse initially lost weight during the first 2 weeks after the treatment, and subsequently gained weight. At 54 days post-Ad-leptin treatment, ob/ob mice are indistinguishable from untreated ob/ob control littermates, whereas HD-leptin-treated mice remained indistinguishable from untreated lean control mice. Untreated ob/ob and lean control mice are shown for comparison as labeled.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Any publications referenced herein are hereby incorporated by reference in this application in order to more fully describe the state of the art to which the present invention pertains.

[0023] It is important to an understanding of the present invention to note that all technical and scientific terms used herein, unless otherwise defined, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. The techniques employed herein are also those that are known to one of ordinary skill in the art, unless stated otherwise.

[0024] Reference to particular buffers, media, reagents, cells, culture conditions and the like, or to some subclass of same, is not intended to be limiting, but should be read to include all such related materials that one of ordinary skill in the art would recognize as being of interest or value in the particular context in which that discussion is presented. For example, it is often possible to substitute one buffer system or culture medium for another, such that a different but known way is used to achieve the same goals as those to which the use of a suggested method, material or composition is directed.

[0025] The terms used herein are not intended to be limiting of the invention. For example, the term “gene” includes cDNAs, RNA, or other polynucleotides that encode gene products. “Foreign gene” denotes a gene that has been obtained from an organism or cell type other than the organism or cell type in which it is expressed; it also refers to a gene from the same organism that has been translocated from its normal situs in the genome. In using the terms “nucleic acid”, “RNA”, “DNA”, etc., we do not mean to limit the chemical structures that can be used in particular steps. For example, it is well known to those skilled in the art that RNA can generally be substituted for DNA, and as such, the use of the term “DNA” should be read to include this substitution. In addition, it is known that a variety of nucleic acid analogues and derivatives is also within the scope of the present invention. “Expression” of a gene or nucleic acid encompasses not only cellular gene expression, but also the transcription and translation of nucleic acid(s) in cloning systems and in any other context. The term “recombinase” encompasses enzymes that induce, mediate or facilitate recombination, and other nucleic acid modifying enzymes that cause, mediate or facilitate the rearrangement of a nucleic acid sequence, or the excision or insertion of a first nucleic acid sequence from or into a second nucleic acid sequence. The “target site” of a recombinase is the nucleic acid sequence or region that is recognized (e.g., specifically binds to) and/or acted upon (excised, cut or induced to recombine) by the recombinase. The term “gene product” refers primarily to proteins and polypeptides encoded by other nucleic acids (e.g., non-coding and regulatory RNAs such as tRNA, sRNPs). The term “regulation of expression” refers to events or molecules that increase or decrease the synthesis, degradation, availability or activity of a given gene product.

[0026] The present invention is also not limited to the use of the cell types and cell lines used herein. Cells from different tissues (breast epithelium, colon, lymphocytes, etc.) or different species (human, mouse, etc.) are also useful in the present invention.

[0027] It is important in this invention to detect the generation and expression of recombinant nucleic acids and their encoded gene products. The detection methods used herein include, for example, cloning and sequencing, ligation of oligonucleotides, use of the polymerase chain reaction and variations thereof (e.g., a PCR that uses 7-deaza GTP), use of single nucleotide primer-guided extension assays, hybridization techniques using target-specific oligonucleotides that can be shown to preferentially bind to complementary sequences under given stringency conditions, and sandwich hybridization methods.

[0028] Sequencing may be carried out with commercially available automated sequencers utilizing labeled primers or terminators, or using sequencing gel-based methods. Sequence analysis is also carried out by methods based on ligation of oligonucleotide sequences which anneal immediately adjacent to each other on a target DNA or RNA molecule (Wu and Wallace, Genomics 4: 560-569 (1989); Landren et al., Proc. Natl. Acad. Sci. 87: 8923-8927 (1990); Barany, F., Proc. Natl. Acad. Sci. 88: 189-193 (1991)). Ligase-mediated covalent attachment occurs only when the oligonucleotides are correctly base-paired. The Ligase Chain Reaction (LCR), which utilizes the thermostable Taq ligase for target amplification, is particularly useful for interrogating late onset diabetes mutation loci. The elevated reaction temperatures permits the ligation reaction to be conducted with high stringency (Barany, F., PCR Methods and Applications 1: 5-16 (1991)).

[0029] The hybridization reactions may be carried out in a filter-based format, in which the target nucleic acids are immobilized on nitrocellulose or nylon membranes and probed with oligonucleotide probes. Any of the known hybridization formats may be used, including Southern blots, slot blots, “reverse” dot blots, solution hybridization, solid support based sandwich hybridization, bead-based, silicon chip-based and microtiter well-based hybridization formats.

[0030] The detection oligonucleotide probes range in size between 10-1,000 bases. In order to obtain the required target discrimination using the detection oligonucleotide probes, the hybridization reactions are generally run between 20°-60° C., and most preferably between 30°-50° C. As known to those skilled in the art, optimal discrimination between perfect and mismatched duplexes is obtained by manipulating the temperature and/or salt concentrations or inclusion of formanide in the stringency washes.

[0031] The cloning and expression vectors described herein are introduced into cells or tissues by any one of a variety of known methods within the art. Such methods are described for example in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1992), which is hereby incorporated by references, and in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), which is also hereby incorporated by reference. The methods include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors.

[0032] The protein products of recombined and unrecombined coding sequences may be analyzed using immune techniques. For example, a protein, or a fragment thereof is injected into a host animal along with an adjuvant so as to generate an immune response. Immunoglobulins which bind the recombinant fragment are harvested as an antiserum, and are optionally further purified by affinity chromatography or other means. Additionally, spleen cells may be harvested from an immunized mouse host and fused to mycloma cells to produce a bank of antibody-secreting hybridoma cells. The bank of hybridomas is screened for clones that secrete immunoglobulins which bind to the variant polypeptides but poorly or not at all to wild-type polypeptides are selected, either by pre-absorption with wild-type proteins or by screening of hybridoma cell lines for specific idiotypes that bind the variant, but not wild-type, polypeptides.

[0033] Nucleic acid sequences capable of ultimately expressing the desired variant polypeptides are formed from a variety of different polynucleotides (genomic or cDNA, RNA, synthetic olignucleotides, etc.) as well as by a variety of different techniques.

[0034] The DNA sequences are expressed in hosts after the sequences have been operably linked to (i.e., positioned to ensure the functioning of) an expression control sequence. These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors contain selection markers (e.g., markers based on tetracycline resistance or hygromycin resistance) to permit detection and/or selection of those cells transformed with the desired DNA sequences. Further details can be found in U.S. Pat. No. 4,704,362.

[0035] Polynucleotides encoding a variant polypeptide include sequences that facilitate transcription (expression sequences) and translation of the coding sequences such that the encoded polypeptide product is produced. Construction of such polynucleotides is well known in the art. For example, such polynucleotides include a promoter, a transcription termination site (polyadenylation site in eukaryotic expression hosts), a ribosome binding site, and, optionally, an enhancer for use in eukaryotic expression hosts, and optionally, sequences necessary for replication of a vector.

[0036] E. Coli is one prokaryotic host useful particularly for cloning DNA sequences of the present invention. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilus, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. Expression vectors are made in these prokaryotic hosts which will typically contain expression control sequences compatible with the host cell (e.g., an origin of replication). In addition, any number of a variety of well-known promoters are used, such as the lactose promoter system, a tryptophan (Trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda. The promoters typically control expression, optionally with an operator sequence, and have ribosome binding site sequences, for example, for initiating and completing transcription and translation.

[0037] Other microbes, such as yeast, are used for expression. Saccharomyces is a suitable host, with suitable vectors having expression control sequences, such a promoters, including 3-phosphoglycerate kinase or other glycotic enzymes, and an origin of replication, termination sequences, etc. as desired.

[0038] In addition to microorganisms, mammalian tissue cell culture is used to express and produce the polypeptides of the present invention. Eukaryotic cells are preferred, because a number of suitable host cell lines capable of secreting intact human proteins have been developed in the art, and include the CHO cell lines, various COS cell lines, HeLa cells, myeloma cell lines, Jurkat cells, and so forth. Expression vectors for these cells include expression control sequences, such as an origin of replication, a promoter, an enhancer, and necessary information processing sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Preferred expression control sequences are promoters derived from immunoglobin genes, SV40, Adenovirus, Bovine Papilloma Virus, and so forth. The vectors containing the DNA segments of interest (e.g., polypeptides encoding a variant polypeptide) are transferred into the host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation is useful for other cellular hosts.

[0039] The method lends itself readily to the formulation of test kits for use in diagnosis. Such a kit comprises a carrier compartmentalized to receive in close confinement one or more containers wherein a first container contains reagents useful in the localization of the labeled probes, such as enzyme substrates. Still other containers contain restriction enzymes, buffers etc., together with instructions for use.

[0040] The recombinant Ad vectors described herein are significantly different from previously described constructs. They combine the use of vectors having deletions of all or most of the viral genes with helper viruses that are designed so that, when used in coinfections with vector viruses, said helper viruses are able to complement the growth of the vectors but are unable to package their viral DNA into infectious virions. Thus vector viruses can be prepared substantially free of helper virus.

[0041] For viral DNA replication and packaging of viral DNA into virion particles, only three regions of the viral DNA are known to be required in cis. These are the left inverted terminal repeat, or ITR, (bp 1 to approximately 103) the packaging signals (approximately 194 to 358 bp) (Hearing and Shenk, 1983, Cell 33: 695-703; Grable and Hearing 1992, J. Virol. 64: 2047-2056) and the right ITR. All other regions of the viral genome appear to be required only to produce viral products that act in trans to allow viral replication and production of infectious viruses. Thus if all essential viral proteins and RNA could be provided by a helper virus, a vector could be designed and constructed that could have most of the viral DNA deleted save for those sequences mentioned above that are required in cis for viral DNA replication and packaging.

[0042] A problem with helper dependent vectors has been that preparations of such vectors are invariably contaminated with helper virus and it is technically very difficult to separate the helper from the vector. In the main embodiments of the present invention, the helper virus is designed to have two 1ox P sites near the left end of the genome, one inserted at approximately 189 bp from the extreme left end of the viral DNA, and the second, in parallel orientation with the first 1ox P, situated rightward of the packaging signals, ie rightward of bp 358 (diagrammed in FIG. 1). This virus will be able to replicate in cells that are normally permissive for growth of Ad5. However, in cells that express the Cre recombinase, or in the presence of a second virus that expresses Cre recombinase, excision of sequences between the 1ox P sites of the helper virus DNA will remove the packaging signal, and the resulting viral DNA will fail to package into infectious viral particles. Therefore, in cells coinfected with said helper and with a second virus, a vector from whose genome has been deleted all or most of the viral DNA sequences that are normally required for expression of viral products necessary in trans for viral replication, both vector and helper viral genomes will replicate, but only the vector DNA that retains the packaging signal will be packaged into virions (FIG. 2).

[0043] In one embodiment of the invention, the helper virus is derived from a plasmid similar to those described in the parent applications and as illustrated in FIGS. 3 and 4. In these examples, the Ad5 genome is present as a circular molecule containing a bacterial plasmid derived origin of DNA replication (designated “ori”) and a bacterial antibiotic resistance coding sequence (“Apr”) conferring to bacteria carrying said molecule resistance to ampicillin. In this example in which said circular form of the Ad5 genome is designated pBG17, viral sequences from regions E1 and E3 have been deleted from the viral genome, but this example is not meant to be limiting since other deletions or no deletions may equally be engineered in the circularized molecule by methods described in the parent application. The molecule designated pBG17 contains Ad5 sequences from bp 19 (left genomic end) to bp 341 with an artificially engineered BamH 1 restriction site inserted between approximately bp 188 and 189 in the Ad 5 sequences which is between “ITR” and the packaging signal, “Ψ”, and known not to interfere with viral replication (Bett, A. J., W. Haddara, L. Prevec, and F. L. Graham. 1994, Proc. Natl. Acad. Sci USA 91: 8802-8806). Ad5 sequences present in pBG17 then extend rightward of the packaging signal to approximately bp 341 at which position is located an Xba 1 restriction site followed by Ad 5 sequences from approximately bp 3534 to approximately bp 27864, then sequences comprising 1874 bp of DNA containing the pUC19 origin of replication and ampicillin resistance gene, and finally Ad5 bp 30996 to 35934 (right genomic end). By techniques that are readily employed by a person skilled in the art, 1ox P sites, which are well defined DNA sequences of about 34 bp, can be introduced into the Ad5 genome at the Bam HI and XbaI sites flanking “Ψ”. For example, synthetic double stranded oligodeoxnucleotides can be readily designed and synthesized such that they contain the 1ox P sequence recognized by Cre and are flanked by single stranded extensions that allow ligation into BamHI or XbaI cleaved DNA. Thus a person skilled in the art can readily obtain a plasmid such as that designated pBG17Lox1 (FIG. 4) having a 1ox P site introduced into the Bam HI site at nt 188, and subsequently pBG17Lox2 (FIG. 3) having an additional 1ox P site introduced into the XbaI site of PBG17Lox1. The plasmid pBG17 can be used to generate infectious virus by transfection of 293 cells. Equally, the plasmids pBG17Lox1 or pBG17Lox2, will generate infectious virus (eg. AdBG17Lox2 illustrated in FIG. 3) since insertions of up to 271 bp can be engineered between the ITR and the packaging signal without interfering with viral replication and packaging of viral DNA (Hearing et al., Journal of Virology Vol. 61, p2555, 1987). In the presence of Cre enzyme, the sequences containing the packaging signal will be excised as a result of intramolecular recombination between the two 1ox P sites (FIG. 3, bottom) resulting in a viral genome that retains all the sequences necessary for replication but lacks the sequences needed for packaging of DNA into virions. Said genome may serve as a complementing viral genome to support the replication of a second virus, a vector, that lacks all or most of the viral genes necessary for viral replication as diagrammed in FIG. 2. These examples are not meant to be limiting as it will be appreciated that modified viruses similar in DNA structure to that of FIG. 3 can be generated by other means. For example a person skilled in the art could introduce 1ox P sites into other sites in the plasmids illustrated in FIG. 3 such as the Bst B1 or Pac I sites or into such other plasmids containing Ad sequences, or other Ad viral genomes as might be desirable. Use of Cre recombinase in this and other examples is not meant to be limiting as a person skilled in the art will readily appreciate that other enzymes capable of catalyzing site specific recombination between DNA sequences recognized by said enzymes could equally be employed in place of the Cre recombinase. An example, not meant to be limiting, of such an enzyme that could be substituted for Cre is the “FLP” recombinase of yeast in combination with its target site (O'Gorman et al. Science 251, 1351, 1991).

[0044] Another embodiment of the invention provides human cells, such as 293 cells or other cells that may be deemed suitable in that they support the replication of the viral components of the invention, that express Cre recombinase and that can be transfected with the plasmids described in the previous examples to generate a helper virus from which the packaging signals have been removed through excision mediated by Cre. It will be appreciated by those skilled in the art that the requisite cell lines can be generated by transfecting 293 cells or other cells with a plasimd comprising the coding sequence for Cre under the control of suitable regulatory sequences including a promoter and polyadenylation signal and containing in addition a selectable gene encoding, for example, resistance to G418 or histidinol. A person skilled in the art can readily obtain drug resistant cells that will express the Cre recombinase in addition to the drug resistance gene used for selection.

[0045] In another embodiment of the invention, a plasmid consisting of sequences comprising the left ITR, the packaging signal, and the right ITR, and optionally containing additional viral sequences can be readily obtained. An example, which is not meant to be limiting, is illustrated in FIG. 4. In this example, pBG17Lox1 DNA is digested with restriction enzymes XbaI and SmaI which cleave the viral DNA in pBG17Lox1 at sites shown, as well as at other sites in viral DNA. The fragment containing the junction of viral termini (indicated by head to head arrows in FIG. 4) and the 1ox P site and packaging signal, can be purified and inserted into the polycloning site of a suitable cloning plasmid such as pUC18 or pUC19 to generate the plasmid designated as pPAD1. This example is not meant to be limiting as a person skilled in the art could equally insert said fragment into such other cloning plasmids as might be suitable or desirable. In the example illustrated, pPAD1 can serve as a vector for insertion of foreign DNA up to approximately 30 kb in size at one of the remaining restriction enzyme cloning sites present at the junctions of pUC and Ad5 DNA, to generate a plasmid such as pADHDV1, in which the open segment of pADHDV1 represents foreign DNA of arbitrary origin and sequence composition. The plasmid pADHDV1 contains all the Ad5 sequences needed in cis for viral DNA replication and packaging of viral DNA into virions. Provided that viral functions necessary in trans are supplied by a helper virus, therefore, pADHDV1 will have the potential to replicate as a helper dependent viral DNA molecule that will contain up to approximately 30-35 kb of foreign DNA flanked by viral DNA sequences from the left and right ends of the viral genome. It may be advantageous to include, as part of the foreign DNA inserted into pADHDV1, a DNA sequence capable of providing expression of a readily detectable reporter gene in addition to other sequences, the reporter gene providing a simple means of identifying cells or groups of cells that are infected with the virus ADHDV 1 derived from pADHDV1. As an example, which is not meant to be limiting, a person skilled in the art could include in pADHDV1, sequences coding for bacterial β-galactosidase, expression of which is readily detectable by exposure of cells to X-gal. Furthermore, in the example shown in FIG. 4, pPAD1 and pADHDV1 contain a single 1ox P site at Ad5 nt 189, that is at the same site as for one of the 1ox P insertions in pBG17Lox1&2. Although this example is not meant to be limiting, placement of a 1ox P site at this position in pPAD1, pADHDV1, and derivatives, may serve to reduce the efficiency of recombination between helper virus and vector during coreplication of the two viruses as illustrated in FIGS. 2 and 5.

[0046] In another embodiment of the invention, coreplication of helper virus comprising sequences derived from a plasmid such as pBG17Lox2 and a helper dependent virus comprising sequences derived from a plasmid such as pADHDV1 may be achieved by cotransfection of cells with said plasmids to generate replicating viral genomes. In the example illustrated in FIG. 5, which is not meant to be limiting, AdBG17Lox2 will, in the presence of Cre recombinase, be converted to AdBG17ψ- will, by excision of the sequences bracketed by 1ox P sites. The virus AdBG17ψ-will, by virtue of the removal of the packaging signals, be unable to package its genome into virions but will be able to replicate its DNA and provide viral functions necessary in trans for viral replication and thereby provide complementing functions for replication of the helper dependent virus, AdHDV1. Because AdHDV1 retains the packaging signals, the DNA of this helper dependent virus will be packaged into virions. The helper dependent virus AdHDV1 may be recovered, and optionally purified and concentrated by isopycnic centrifugation in CsC1 gradients to produce helper dependent virus preparations substantially free or totally free of contaminating helper virus.

[0047] In another embodiment of the invention, 293 cells or other human cells that do not express Cre may be transfected with a plasmid such as that designated as pBG17Lox2 in FIGS. 3 and 5 to produce a virus such as that designated as AdBG17Lox2. Said virus may replicate in said cells without undergoing excision of sequences bracketed by 1ox P and can therefore be readily propagated. Coinfection of 293Cre or equivalent cells with AdBG17Lox2 and AdHDV1 will lead to formation of AdBG17ψ- which will complement the growth of AdHDV1 resulting in coreplication of both viral genomes but packaging only of AdHDV1 DNA into viral particles.

[0048] Having generally described this invention, as disclosed and claimed herein, the following specific exemplary support is provided to demonstrate the functionality of the disclosed and claimed system both in vitro and in vivo. However, this invention should not be interpreted as being limited to the specifics of the following examples. Rather, the scope of this invention should be determined through consultation of the claims appended hereto and equivalents thereof.

EXAMPLE

[0049] An Adenoviral Vector Deleted for all Viral Coding Sequences Results in Enhanced Safety and Extended Expression of a Leptin Transgene

[0050] Experimental Synopsis and Conclusions Drawn:

[0051] Adenoviral (Ad) mediated in vivo gene transfer and expression are limited in part by cellular immune responses to viral-encoded proteins and/or transgene immunogenicity. In an attempt to diminish the former responses, we have previously developed and described helper-dependent (HD) Ad vectors in which the viral protein coding sequences are completely eliminated. These HD vectors have up to 37 kb insert capacity, are easily propagated in a Cre recombinase-based system, and can be produced to high concentration and purity (>99.9% helper-free vector). In this study, we compared safety and efficacy of leptin gene delivery mediated by an HD vector (HD-leptin) and a first-generation El-deleted Ad vector (Ad-leptin) in normal lean and ob/ob (leptin-deficient) mice. In contrast to evidence of liver toxicity, inflammation, and cellular infiltration observed with Ad-leptin delivery in mice, HD-leptin delivery was associated with a significant improvement in associated safety/toxicity and resulted in efficient gene delivery, prolonged elevation of serum leptin levels, and associated weight loss. The greater safety, efficient gene delivery, and increased insert capacity of HD vectors are significant improvements over current Ad vectors and represent favorable features especially for clinical gene therapy applications.

[0052] Background for this Example:

[0053] Adenoviral (Ad) vectors are currently among the most efficient gene transfer vehicles for both in vitro and in vivo delivery, but the utilization of current Ad vectors for many gene therapy applications is limited by the transient nature of transgene expression obtained by these vectors (Stratford-Perricaudet, L. D., Levrero, M., Chasse, J., Perricaudet, M. & Briand, P. (1990) Hum. Gene Ther. 1, 241-256; Kay, M. A., Li, Q., Liu, R. J. -J., Leland, F., Roman C., Finegold, M. & Woo, S. L. C. (1992) Hum. Gene Ther. 3, 641-647; Herz, J. & Gerard, R. D. (1993) Proc. Natl. Acad. Sci. USA 90,2812-2816; Englehardt, J. F., Simon, R. H., Yang, Y., Zepeda, M., Wber-Pendleton, S., Doranz, B., Grossman, M & Wilson, J. M. (1993) Hum. Gene Ther. 4, 757-769; Morsy, M. A., Alford, E. L., Bett, A., Graham, F. L. & Caskey, C. T. (1993) J. Clin. Invest. 92, 1580-1586; Morsy, M. A., Zhao, J. Z., Warman, A. W., O'Brien, W. E., Graham, F. L. & Caskey, C. T. (1996) J. Clin. Invest. 97, 826-832; Muzzin, P., Eisensmith, R. C., Copeland, K. C. & Woo, S. L. C. (1996) Proc. Natl. Acad. Sci. USA 93, 14804-14808). Several factors have been shown to contribute to and modulate the duration of Ad-mediated gene expression and the immunogenicity of these vectors, including “leaky” viral protein expression and the transgene that is delivered (Yang, Y., Nunes, F. A., Berencsi, K., Furth, E. E., Gonczol, E. & Wilson, J. M. (1994) Proc. Natl. Acad. Sci. USA 91, 4407-4411; Yang, Y., Ertl, H. D. & Wilson, J. M. (1995) J. Virol. 69, 2004-2015; Lochmuller, H., Petrof, B. J., Pari, G., Larochelle, N., Dodelet, V., Wang, Q., Allen, C., Prescott, S., Massie, B., Nalbantoglu, J., et al. (1996) Gene Ther. 3, 706-716; Gahery-Segard H., Juilliard, V., Gaston, J., Lengagne, R., Pavirani, A., Boulanger, P. & Guillet, J. G. (1997) Eur. J Immunol. 27, 653-659; Kajiwara, K., Byrnes, A. P., Charlton, H. M., Wood, M. J. & Wood, K. J., (1997) Hum. Gene Ther. 8, 45-56; Kaplan, J. M., Annentano, D., Sparer, T. E., Wynn, S. G., Peterson, P. A., Wadsworth, S. C., Couture, K. K, Pennington, S. E., St. George, J. A., Gooding, L. R. & Smith, A. E. (1997) Hum. Gene Ther. 8, 45-46; Worgall, S., Wolff, G., Falck-Pedersen, E. & Crystal, R. G. (1997) Hum. Gene Ther. 8, 37-44; Tripathy, S. K., Black, H. B., Goldwasser, E. & Lieden, J. M. (1996) Nat. Med. 2, 545-550.). The development of Ad vectors that are deleted in all viral protein-coding sequences offers the prospect of a potentially safer, less immunogenic vector with an insert capacity of up to 37 kb (Mitani, K., Graham, F. L., Caskey, C. T. & Kochanek, S., (1995) Proc. Natl. Acad. Sci. USA 92, 3854-3858; Kochanek, S., Clemens, P. R., Mitani, K., Chen, H. H., Campbell, K. P. & Caskey, C. T. (1996) Proc. Natl. Acad. Sci. USA 93, 5731-5736; Clemens, P. R., Kochaneck, S., Sunada, Y., Chan, S., Chen, H. H., Campbell, K. P. & Caskey, C. T. (1996) Gene Ther. 3, 965-72; Chen, H. H., Mack, L. M., Kelly, R., Ontell, M., Kochanek, S. & Clemens, P. R. (1997) Proc. Natl. Acad. Sci. USA 94, 965-972; Fisher, K. J., Choi, H., Burda, J., Chen, S. & Wilson, J. M. (1996) Virology 217, 1122; Kumar-Singh, R. & Chamberlain, J. S. (1996) Hum. Mol. Gent. 5, 913-921; Hardy, S., Kitamura, M., Harris-Stansil, T., Dai, Y. & Phipps, M. L. (1997) J. Virol. 71, 1842-1849; Lieber, A., He, C., Kirillova, I. & Kay, M. A. (1996) J. Virol. 70 8944-8960; Parks, R. J., Chen L., Anton, M., Sankar, U., Rudnicki, M. A. & Graham, F. L. (1996) Proc. Natl. Acad. Sci. USA 93, 13565-13570; Haecker, S. E., Stedman, H. H., Balice-Gordon, R. J., Smith, D. B., Greelish, J. P., Mitchell, M. A., Wells, A., Sweeney, H. L. & Wilson, J. M. (1996) Hum. Gen. Ther. 7, 1907-1914; Schiedner, G., Morral, N., Parks, R. J., Wu, Y., Koopmans, S. C., Langston, C., Graham, F. L., Beaudet, A. L. & Kochanek, S. (1998) Nat. Genet. 18, 180-183.). This vector is supplied in trans with the structural proteins required for packaging and rescue and is thus helper-dependent (HD) (Parks, R. J., Chen L., Anton, M., Sankar, U., Rudnicki, M. A. & Graham, F. L. (1996) Proc. Natl. Acad. Sci. USA 93, 13565-13570).

[0054] Leptin has been recently identified as a potent modulator of weight and food intake. Daily delivery of recombinant leptin protein was shown to induce weight reduction, supress appetite, and decrease blood insulin and glucose levels in ob/ob (leptin-deficient) mice (Halaas, J. L., Gajiwala, K. S., Maffei, M., Cohen, S. L., Chait, B. T., Rabinowitz, D., Lallone, R. L., Burley, S. K. & Friedman, J. M. (1995) Science 269, 543-546; Pellymounter, M. A., Cullen, M. J., Hecht, R., Winters, D., Boone, T. & Collins, F. (1995) Science 269, 546-549; Campfield, L. A., Smith, F. J., Guisez, Y., Devos, R. & Burn, P. (1995) Science 269, 546-549.). It has been shown that delivery of the leptin cDNA by first-generation Ad vectors (Ad-leptin) may substitute for daily recombinant leptin protein treatment, although the effects were transient in both lean and ob/ob treated mice (Muzzin, P., Eisensmith, R. C., Copeland, K. C. & Woo, S. L. C. (1996) Proc. Natl. Acad. Sci. USA 93, 14804-14808; Morsy, M. A., Gu, M., Zhao, J. Z., Holder, D. J., Rogers, I. T., Pouch, W., Motzel, S. L., Klein, H. J., Gupta, S. K., Liang, X., et al. (1998) Gene Ther. 5, 8-18.). In the present study, we delivered the leptin cDNA using the HD virus (HD-leptin), testing the hypothesis that elimination of the viral protein coding sequences would diminish the vector's cellular immunogenicity and toxicity, and hence support its longevity in vivo. Because both the viral proteins and the transgene were factors implicated in the cellular immunogenicity of recombinant Ad viruses, we designed experiments to compare the HD and Ad vectors in ob/ob mice that are naïve to leptin (in which the protein is potentially immunogenic), as well as in lean mice that normally express leptin.

[0055] In this study, we show that HD-leptin provided greater safety as reflected by absence of liver toxicity, cellular infiltrates, extended longevity of gene-expression, and stability of vector DNA in livers of treated mice over that observed with Ad-leptin treatment.

[0056] Materials and Methods

[0057] Construction of Vectors.

[0058] Construction of Ad-leptin and Ad-β-galalactosidase (β-gal) recombinant vectors has been described (Morsy, M. A., Gu, M., Zhao, J. Z., Holder, D. J., Rogers, I. T., Pouch, W., Motzel, S. L., Klein, H. J., Gupta, S. K., Liang, X., et al. (1998) Gene Ther. 5, 8-18.). The expression cassettes contain the human cytomegalovirus (HCMV) promoter (Invitrogen), the transgene, and the bovine growth hormone poly(A) sequence. First generation vectors were propagated and titered as described (Graham, F. L., Smiley, J., Russell, W. C. & Naim, R. (1977) J. Gen. Virol. 36, 59-72; Graham, F. L. & Prevec, L. (1991) in Gene Transfer and Expression Protocols, ed. Murray, E. J. (Humana, Clinton, N.J.), pp. 109-128.). The HD vector constructed for this study (HD-leptin) was prepared by releasing the linear backbone structure of HD-leptin from its plasmid pΔSTK120-HCMV-mOb-BGHpA (by PmeI digest) and transfecting the linear DNA into 293-cre4 cells followed by helper infection as described in HD-leptin propagation section below. Two different structures were used for rescuing HD viruses expressing leptin, HD-leptin (used in all the in vivo studies reported in this article), and HD-leptin-monomer. The structure of the HD-leptin plasmid is a pBluescript IIKS based plasmid that contains (in the following order) the Ad5 inverted terminal repeat (ITR) sequences and the packaging signal ψ, 440 bp, (nucleotides 1440): a 5,072-bp fragment of hypoxanthine guanine phosphoribosyltransferase (nucleotides 12,373-17,781 in gb:humhprtb); the leptin expression cassette, 1,835 bp; a HindIII 9,063-bp fragment of C346 cosmid (nucleotides 12,421-21,484 in gb:L31948); and the right-end terminus of Ad5, composed of the ITR sequence, 117 bp (nucleotides 35,818-35,935); with the intervening multiple cloning sites between junctions of the different fragments the total size is 19.6 kb including 2.9 kb of the pBluescript IIKS. The 2.9 kb of pBluescript IIKS is eliminated before HD vector rescue by linearizing the plasmid with two PmeI flanking sites. HD-leptinmonomer plasmid (PSTK120-HCMV-mOb-BGHpA) differs in that the hypoxanthine guanine phosphoribosyltransferase “stuffer” is a larger fragment of 16,054 bp (nucleotides 1,799-17,853 in gb:humhprtb), total size≈30 kb including 2.9 kb of the pBluescript IIKS, which as in the case of HD-leptin plasmid, is also eliminated by linearizing the plasmid with two PmeI flanking sites and releasing the HD-leptin-monomer fragment.

[0059] Propagation of the HD Viruses.

[0060] For propagation of the HD viruses, we used a helper virus system consisting of a modified first-generation E1-deleted vector with lox sites flanking the packaging signals (AdLC8cluc1), and a 293 cell line derivative expressing Cre recombinase (293-cre4)(Parks, R. J., Chen, L., Anton, M., Sankar, U., Rudnicki, M. A. & Graham, F. L. (1996) Proc. Natl. Acad. Sci. USA 93, 13565-13570; Chen, L., Anton, M. & Graham, F. L. (1996) Som. Cell Mol. Genet. 22, 477-488.). HD-leptin vector DNA was excised from the plasmid backbone (by PmeI digestion) and 4 μg were used to transfect semiconfluent 293-cre4 cells in 6-cm plates. After an overnight incubation, cells were infected at a multiplicity of infection of 1 with the helper virus AdLC8cluc1. Cells were monitored for complete cytopathic effect, at which point cells were collected and lysate was used for serial propagation and expansion of viral stock by slight modification over what was described (Parks, R. J., Chen, L., Anton, M., Sankar, U., Rudnicki, M. A. & Graham, F. L. (1996) Proc. Natl. Acad. Sci. USA 93, 13565-13570; Parks, R. J. & Graham, F. L. (1997) J. Virol. 71, 3293-3298.). Two ml of lysate collected from P1 (transfection/infection step) was used to infect 6-cm plates of semi-confluent 293-cre4 for 24 hours, supplemented with 1 ml of fresh medium. After the 24-hr incubation, the helper virus AdLC8cluc1 was added at a multiplicity of infection of 1, to the cells. P2 lysate was collected upon detection of cytopathic effect. The same procedure was repeated for another three propagation's (P3, P4, and P5) infecting 10-cm plates followed by 15-cm plates of semiconfluent 293-cre4 cells, respectively. Lysate collected from P4 was used to infect twenty 15-cm plates (1 ml of lysate added to 24 ml of fresh medium), and again upon detection of cytopathic effect the lysate was collected and cesium chloride banded as described (Graham, F. L. & Prevec, L. (1991) in Gene Transfer and Expression Protocols, ed. Murray, E. J. (Humana, Clinton, N.J.), pp. 109-128.). The banded viruses were analyzed by restriction mapping and the HD-leptin virus was sequenced for verification of structure.

[0061] The final stock of HD-leptin was harvested from ≈1.2×109 293-cre4 cells and the cesium chloride banded viral stock yield was ≈8×1012 particles (2×1012/ml). The helper virus (AdLC8cluc1) content in the HD-leptin stock was 1.5×107 plaque-forming units per ml. Fifty microliters (1-2×1011 OD particles per dose, containing ≈7.5×105 plaque-forming units helper, i.e., <0.1% contamination with helper per estimated infectious HD dose) of the stock were diluted with dialysis buffer to 100 μl for the mouse tail vein injections.

[0062] Repeat of HD-leptin Viral Rescue.

[0063] Three independent rescues of the HD-leptin recombinant virus, initiated at the first step (P1), which is the transfection of pΔSTK120-HCMV-mOb-BGHpA resulted in an identical, and stable structure of HD-leptin. Seven different enzymes were used for verifying the structures of the recombinant HD viruses; Asp-718, EagI, FseI, HindIII, PacI, SmaI, and XhoI. Digested viral DNA (50-100 ng) was analyzed by Southern blot analysis, fragments were radiolabeled using T4 DNA poymerase, DNA fragments were viewed on a 1.0 or 0.5% (for sizing purposes in case of undigested DNA extracted from HD-leptin and Ad-leptin) agarose gels in Tris/acetate/EDTA (TAE) buffer, and identified by radioautography or ethidium bromide staining.

[0064] PCR Amplification of the Junction Fragment and Sequencing.

[0065] A primer flanking the junction fragment was used for PCR, primer J4-F:5′-CTCTTCTTCTGTCACACCCCTCCCUC-3′ was used individually to amplify the junction-fragment of HD-leptin, the fragment generated was ≈300 bp, and was cloned into PCR 2.1 vector (Invitrogen) and sequenced.

[0066] Mouse Colony.

[0067] ob/ob (C57BL/J6-ob/ob) mice and homozygous normal lean (C57BL/J6) litter mates (age-matched females), were purchased from The Jackson Laboratory for use in this study. Animals were free of all common murine pathogens. Eight-to twelve-week-old mice (ob/ob≈70 g and lean≈28 g) were redistributed based on equal representation of weight and caged in groups of five on day 0, immediately preceding treatment. After a series of baseline blood samples were obtained by tail incision from conscious mice, animals were divided into four groups and received by tail vein injection a single 100-μl aliquot containing 1-2×1011 particles of HD-leptin, Ad-leptin, Ad-β-gal (control), or dialysis buffer (control). Body weight and food intake were measured daily, and blood was collected 2-3 times weekly, pre- and post-treatment.

[0068] Animals were killed by carbon dioxide inhalation and organs removed for immunohistochemistry and RNA analysis. All animals used in this study were maintained in accordance with the “Guide for the Care and Use of Laboratory Animals” (Institute for Laboratory Animal Resources, National Research Council, 1996). The protocol was approved by the Institutional Animal Care and Use Committee, Merck.

[0069] Histopathology Studies.

[0070] Mice (n=3 per treatment per time point) were humanely killed, and liver samples were collected and fixed in 10% buffered formalin. Tissues were routinely processed through paraffin, sectioned at 5 microns, and stained with hematoxylin and eosin. Replicate unstained slides also were prepared using standard procedures for immunohistochemistry and stained for the presence of CD3 (T cell) and CD45R (B cell) determinants on infiltrating or intrinsic cells (not shown).

[0071] Blood Measurements.

[0072] Blood samples were obtained by tail incision and collected into heparinized microhematocrit tubes (VWR Scientific) every 2-3 days during the course of the study. Tubes were centrifuged at 13,700×g for 2 min, and hematocrit values were monitored. Plasma was collected for measurement of aspartate, aminotransferase (AST), alanine aminotransferase (ALT), leptin, glucose, and insulin levels. ALT and AST were measured using ALT/serum glutamic oxaloacetic transaminase and AST/serum glutamic pyruvic transaminase, DT slides, respectively (Vitros Chemistry Products, Johnson & Johnson). Leptin and insulin levels were measured by radioimmunoassay performed by Linco Research Immunoassay (St. Charles, Mo.). Glucose levels were measured using Kodak Ektachem DT slides (Eastman Kodak).

[0073] Northern and Southern Blot Analysis.

[0074] For Northern blot analysis, total RNA was extracted (Trizol, GIBCO) from livers of Ad-leptin-treated and HD-leptin-treated mice at 1-, 2-, 4-, and 8-week intervals, and untreated mice. Leptin RNA message was detected by Northern blot analysis (Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press, Plainview, NY) using leptin cDNA as a probe that recognizes a single ≈500 bp band (Morsy, M. A., Gu, M., Zhao, J. Z., Holder, D. J., Rogers, I. T., Pouch, W., Motzel, S. L., Klein, H. J., Gupta, S. K., Liang, X., et al. (1998) Gene Ther. 5, 8-18.). A probe for β-actin was used as the internal control (≈1 kb) (Biochain, San Leandro, Calif.). Southern blot analysis (Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press, Plainview, N.Y.) was used to investigate the stability of vector DNA. Genomic DNA was extracted from livers (pooled DNA, n=3 per treatment per time point) of ob/ob and lean mice were treated with Ad-β-gal, Ad-leptin or HD-leptin. Control animals were injected with similar volumes of dialysis buffer. Pooled (n=3) genomic DNA was digested with HindIII restriction enzyme, and 20 μg of digested DNA were loaded on 0.8% TAE agarose gels. For copy number estimation, 20 μg control DNA were spiked with HD vector DNA equivalent to 2.0, 1.0, 0.2, and 0.1 copies per cell, and the mixture digested with HindIII restriction enzyme followed by Southern blot analysis. The filters were hybridized with a mouse leptin cDNA (≈500 bp) probe, which hybridized to a single HindIII fragment containing the leptin insert in both the HD-leptin (≈6 kb), and Ad-leptin (1.2 kb) vectors. Developed autoradiographs were scanned (Personal Densitometer SI, Molecular Dynamics) and the relative band densities quantitated (IMAGE QUANT software, Molecular Dynamics). To normalize DNA concentration and to estimate relative vector DNA stability between treatment time points, a detected internal leptin genomic DNA signal was used as control. Copy number equivalence were assigned based on comparisons to the relative density ratio between the internal genomic signal and the leptin signal of spiked vector DNA in a copy number control experiment.

[0075] Results and Discussion

[0076] The HD viruses rescued and propagated were analyzed both for structure verification and for helper-load contamination. HD-leptin (used in this study) was generated from a 16.7-kb vector fragment (FIG. 6). This fragment when transfected and propagated in the presence of a helper virus resulted in an HD-virus with a full length of ≈33 kb (FIG. 6). The full-length structure is a tail-to-tail concatamerization recombinant virus (FIG. 6A). This virus has several interesting characteristics. (i) The HD-leptin structure contains a duplicated 5′ITR and packaging signal sequence, one copy at each end of the recombinant virus (FIG. 6A). (ii) This structure is very stable throughout multiple propagations (originating from viral stock). In addition, repeated rescue (originating from the 16.7-kb DNA fragment) results in a single concatamerization species (FIG. 6B). The three HD-leptin recombinant viruses shown, are all independent results of a tail to tail (3′ ITR-3′ ITR) concatamerization of two ΔSTK120-HCMV-mOb-BGHpA molecules. The HD-leptin structure contains two copies of the 5′ ITR and packaging signals that flank the recombinant virus at both ends and the 3′ ITR (one copy only) at the junction of the two molecules. No other concatamerization species were identified in any of the three independent rescues (by restriction mapping and analysis of radiolabeled digestion fragments). (iii) The helper virus contamination load is consistently very low; <0.1%/infectious HD unit; <1 plaque-forming unit of helper virus per 100,000 OD particles per ml of HD stock (minimum estimated HD infectious unit: OD particle is 1:100). HD-leptin expressed leptin at levels comparable to its counterpart first generation Ad-leptin (data not shown).

[0077] In contrast, the HD-leptin monomer, containing a single copy of the packaging signal sequence at the left arm only, consistently results in at least 3-10 fold higher load of helper virus contamination (1 plaque-forming unit of helper virus per 103-104 OD particles per ml) in HD-leptin monomer stock. Given that all the various viral stocks were prepared following the same standard laboratory procedures, the consistently lower levels of helper contamination in the concatamerized HD-leptin viral stock may be attributed to either the duplicated copy of packaging signal sequence or the differences in backbone composition, possibilities now under investigation.

[0078] As a first measure of the difference between first generation Ad and HD vectors, safety studies were conducted in control and treated lean and ob/ob mice. Mice were treated with a single tail intravenous infusion of 1-2×1011 particles of either HD-leptin, Ad-leptin, control Ad-β-gal vector or an equal volume of dialysis buffer. FIG. 7 shows the levels of AST and ALT in the sera of lean mice at one, two and four weeks posttreatment (similar results were observed in treated ob/ob mice; data not shown). Liver toxicity, as reflected by the significant elevation in AST and ALT serum levels over basal control levels, was observed only in mice treated with Ad-β-gal and Ad-leptin, but not HD-leptin. Ad-vector-associated toxicity observed in both the lean and ob/ob treated mice was most significant at one week, was present but to a less significant extent at two weeks, and was resolved by 4 weeks posttreatment. In contrast, HD-treatment was not associated with liver toxicity as reflected by the AST and ALT serum levels that were essentially indistinguishable from controls.

[0079] Liver sections of HD-leptin-treated lean mice (FIG. 8) were histologically indistinguishable from control liver sections (FIG. 8A) at all timepoints tested posttreatment [1(FIG. 8C), 2(FIG. 8E), and 4(FIG. 8G) weeks). Occasional perivascular clusters of 50:50 T and B cells and small foci of cellular infiltrates in HD-leptin-treated as well as in untreated control mice were observed. In contrast, Ad-leptin and Ad-β-gal treated mice displayed hepatic pathology throughout the posttreatment intervals. At 1 week posttreatment, both Ad-β-gal (FIG. 8B) and Ad-leptin-treated (FIG. 8D) mice display degenerative hepatic pathology characterized by foci of round cell infiltration (solid arrows) composed almost entirely (>98%) of T-cells (data not shown), individual liver cell necrosis, increased liver cell mitotic activity, and dissociation of hepatic cords. At 2 weeks posttreatment, Ad-leptin-treated (FIG. 8F) mice display a similar, but less pronounced hepatic pathology. The cellular infiltration observed resolved by the fourth week posttreatment; there is almost an absence of lesions in the Ad-leptin treated (FIG. 8H) mice, with only a trace of individual cell death present, which is within normal ranges. Examination of liver sections obtained from ob/ob mice reflected similar Ad-vector associated histopathology. Similar to the observations in lean mice, evidence of toxicity associated with Ad vectors was not observed with HD-leptin treatment in ob/ob mice, however, a slight cellular infiltrate was detected, which may be attributed to the immunogenicity of leptin in these leptin-deficient mice. Nonetheless, the extent of inflammation and cellular infiltrates remained significantly less than that observed with Ad-leptin (data not shown).

[0080] In the lean mice, treatment with Ad-leptin resulted in a transient increase in serum leptin levels and weight loss that lasted for only 7-10 days (FIGS. 9A and B). In contrast, treatment with HD-leptin resulted in high serum leptin levels (6- to 10-fold over background) and >20% weight loss that persisted at least 2 months (FIGS. 9A and B). Weight loss in HD-leptin-treated mice was associated with satiety that persisted over a longer period (2-3 weeks, data not shown) than in those treated with Ad-leptin (5-7 days) (Morsy, M. A., Gu, M., Zhao, J. Z., Holder, D. J., Rogers, I. T., Pouch, W., Motzel, S. L., Klein, H. J., Gupta, S. K., Liang, X., et al. (1998) Gene Ther. 5, 8-18.). Vector DNA in the livers of Ad-leptin treated mice was rapidly lost and fewer than 0.2 copy per cell was detected, compared with 1 or 2 copies per cell after HD-leptin treatment at 8 weeks postinjection (FIG. 9C). These effects can be correlated with the duration of gene expression obtained with these two vector types. Gene expression mediated by Ad-leptin was transient and almost undetectable as early as 1 week posttreatment as seen by northern blot analysis of total liver RNA, whereas that mediated by HD-leptin persisted for at least eight weeks (FIG. 9D). No changes in serum glucose or insulin levels in the treated lean mice were detected throughout the study (FIGS. 9E and F). Vector DNA levels were stable at 1 to 2 copies per cell at 1, 2, 4, and 8 weeks posttreatment. The ob/ob mice are naive to leptin and thus transgene immunogenicity is not an unexpected finding. In these animals, similar to what was observed in the lean mice, HD-leptin was found to be more effective than the first-generation Ad-leptin vector. In the ob/ob treated with Ad-leptin, serum levels of leptin increased only for a short period during the first 4 days of treatment, returning to baseline levels within 10 days of postinjection (FIG. 9A). Increased leptin levels were associated with transient body weight loss of ≈25%, followed by weight gain, 2 weeks after treatment (FIGS. 10A and B). Similar to the results obtained in lean mice, the Ad-leptin vector DNA (FIG. 10C) was also rapidly lost (<0.2 copy per cell were detected by 2 weeks posttreatment, and undetectable by 8). In contrast, the ob/ob HD-leptin-treated mice had increased serum leptin levels up to 15 days posttreatment, after which the levels gradually dropped to baseline over the subsequent 25 days (FIG. 10A). The initial rise in leptin levels correlated with rapid weight reduction resulting in >60% weight loss (reaching normal lean weight) by 1 month (FIG. 10B). Weight loss was maintained for a period of 6-7 weeks posttreatment. As leptin levels dropped to baseline, a gradual increase in body weight was observed. Satiety was observed in association with increased leptin levels, and appetite suppression was sustained for a longer period (≈1 month) compared with the short transient effect induced by Ad-leptin (≈10 days) (data not shown). Leptin-specific antibodies were detected in the sera of ob/ob Ad-leptin and HD-leptin-treated mice (data not shown); therefore, it was essential to determine whether the drop observed in serum leptin levels was due to interference of the antibodies with the ELISA assay used to measure leptin or a loss of vector DNA and/or gene expression. Although by Southern blot analysis greater stability of HD-vector DNA was observed over Ad-vector DNA in livers of ob/ob treated mice compared at similar time points, the analysis revealed eventual loss of the HD-vector DNA over the 8 week time interval (FIG. 10C). Approximately 75% less vector DNA was detected in the livers of HD-leptin-treated ob/ob mice at 4 and 8 weeks posttreatment compared with the persistent levels found in the livers of HD-leptin-treated lean littermates at similar time points (FIGS. 10C and 9C repectively). Gene expression in ob/ob Ad-leptin treated mice correlated with the DNA findings, RNA levels were below the sensitivity level of detection at 1 week posttreatment, whereas in HD-leptin-treated mice, gene expression was detectedup to 4 weeks postinjection and was undetectable at 8 weeks (FIG. 10D). Serum glucose and insulin levels dropped during the first one or two weeks posttreatment to normal lean values in both HD-leptin- and Ad-leptin-treated mice, although the effects of HD-leptin treatment were sustained for longer periods, which parallels what was seen with weight loss, satiety, DNA stability and leptin gene expression (FIGS. 10E and F). The subsequent increase in glucose and insulin levels in both vector treatments correlated with the drop observed in serum leptin levels and eventual loss of vector DNA. The overall HD-leptin-mediated prolonged effect was also reflected in the accompanying phenotypic correction, which lasted longer than that seen in litter mates treated with Ad-leptin (6-7 vs. 2-3 weeks) (FIG. 11).

[0081] It has been reported that Ad vectors and/or immunogenic transgenes can be associated with cytotoxic T lymphocyte cell responses that result in elimination of vector DNA infected cells and loss of gene expression (Yang, Y., Nunes, F. A., Berencsi, K., Furth, E. E., Gonezol, E. & Wilson, J. M. (1994) Proc. Natl. Acad. Sci. USA 91, 4407-4411; Yang, Y., Ertl, H. D. & Wilson, J. M. (1995) J. Virol. 69, 2004-2015; Lochmuller, H., Petrof, B. J., Pari, G., Larochelle, N., Dodelet, V., Wang, Q., Allen, C., Prescott, S., Massie, B., Nalbantoglu, J., et al. (1996) Gene Ther. 3, 706-716; Gahery-Segard H., Juilliard, V., Gaston, J., Lengagne, R., Pavirani, A., Boulanger, P. & Guillet, J. G. (1997) Eur. J. Immunol. 27, 653-659; Kajiwara, K., Byrnes, A. P., Charlton, H. M., Wood, M. J. & Wood, K. J., (1997) Hum. Gene Ther. 8, 45-56; Kaplan, J. M., Armentano, D., Sparer, T. E., Wynn, S. G., Peterson, P. A., Wadsworth, S. C., Couture, K. K, Pennington, S. E., St. George, J. A., Gooding, L. R. & Smith, A. E. (1997) Hum. Gene Ther. 8, 45-46; Worgall, S., Wolff, G., Falck-Pedersen, E. & Crystal, R. G. (1997) Hum. Gene Ther. 8, 37-44; Tripathy, S. K., Black, H. B., Goldwasser, E. & Lieden, J. M. (1996) Nat. Med. 2, 545550.). In some cases the response is influenced by the mouse strain used (Tripathy, S. K., Black, H. B., Goldwasser, E. & Lieden, J. M. (1996) Nat. Med. 2, 545-550.). In this study we used littermates to control against strain variation in our comparisons of the Ad-vector vs. the HD-vector immunogenicity in both lean animals that normally express leptin and ob/ob mice that are leptin deficient. Our studies clearly illustrate that HD-leptin achieved a substantial improvement in the safety profile and longevity of gene expression over that achieved with the first-generation Ad-leptin vector. The differences observed in the extent of cellular infiltrate in the liver, together with the pronounced liver toxicity as measured by ≈10- and 5-fold increases in AST and ALT serum values, respectively, associated with Ad-leptin but not HD-leptin treatment in lean mice, can be directly attributed to the elimination of the Ad protein-coding DNA sequences, because the leptin expression cassette was identical in both vectors. The appearance of a leptin-specific antibody response, gradual loss of gene expression and vector DNA observed in the ob/ob (leptin-deficient) but not in the lean mice (leptin-wild type) treated with HD-leptin may suggest an independent immune response event related to leptin tolerance.

[0082] The leptin model used in these studies provided a very instructive animal model to investigate the influence of both vector design and transgene product on the duration of expression after gene transfer. The differences between the longevity of expression mediated by the HD-deleted vector in the lean mice in this study and the very short lived effects reported by others may reflect variations in the vector construction features (Lieber, A., He, C., Kirillova, I. & Kay, M. A. (1996) J. Virol. 70, 8944-8960; Sykes, R. C., Lin, D., Hwang, S. J., Framson, P. E. & Chinault, A. C. (1998) Mol. Gene. Genet.I 212, 301-309.).

[0083] The HD-vector system is a significant advance over existing Ad vectors with regards to safety and insert capacity (up to 37 kb). In addition to the gain of these two valuable properties, the HD vectors have not lost the features that contributed to the general attractiveness of Ad vectors that include: (i) efficient in vivo gene delivery, and (ii) high titer production. The concatamerization of the 16.7 vector fragment to generate a ≈33 kb recombinant virus is a phenomenon that has been previously observed by others (Fisher, K. J., Choi, H., Burda, J., Chen, S. & Wilson, J. M. (1996) Virology 217, 11-22; Parks, R. J., & Graham, F. L. (1996) J. Virol. 71, 3293-3298.). The recombinant virus preferentially propagates at higher efficiences when its genome length is at least 75% that of wild type (Parks, R. J., & Graham, F. L. (1996) J. Virol. 71, 3293-3298.). And although we detected traces of propagated 16.7 kb HD-leptin, the prevalence of this species was overwhelmingly surpassed by the 33-kb recombinant vector (FIG. 6B, Vector DNA A).

[0084] Replacements of leptin by other transgenes in the pΔSTK120 are ongoing to determine the universality of this vector backbone. The generation of other backbones with the duplicated left arm is being tested to determine the extent to which the two copies of packaging signal sequences is contributing to the efficient propagation and possible advantage of the HD-recombinant virus over the helper virus leading to the exceedingly low levels of helper contamination in the HD stocks. The unique characteristics of HD-leptin together with the utilization of the 293-cre4 cells and the lox containing helper virus provides a biological method for generation of highly purified HD vectors. These advanced vectors improve the prospect of Ad vehicles for wide application in clinical gene therapy.

Claims

1. A method for expressing nucleic acid sequences which comprises introducing into a host a helper-dependent adenovirus vector comprising: (i) a deletion of up to approximately 35,000 bp of the adenovirus genome but retaining sufficient left and right sequences including left and right ITRs and a packaging signal sufficient to support viral DNA replication and packaging; and (ii) a fragment or fragments of foreign DNA sequences of up to about 35,000 bp.

2. The method according to claim 1 wherein said helper-dependent adenovirus vector is produced by co-infecting an isolated cell with: (a) a helper adenovirus comprising a packaging signal flanked on both sides by 1ox P recombinase target sites; and (b) a helper-dependent adenovirus vector comprising: (i) a deletion of up to approximately 35,000 bp of the adenovirus genome but retaining an adenoviral packaging signal and sufficient left and right ITR sequences to support viral replication and packaging; and (ii) a fragment or fragments of foreign DNA sequences of up to about 35,000 bp; wherein said isolated cell supports replication of the helper adenovirus, and wherein said isolated cell additionally expresses Cre recombinase such that said Cre recombinase catalyzes the removal of the packaging signals from the helper adenovirus such that it replicates but does not package, and wherein the helper virus supports replication of the helper-dependent adenovirus vector such that the helper-dependent adenovirus vector is packaged into adenovirus virions which are recovered for introduction into a host.

3. A cell expressing Cre recombinase and adenovirus E1.

4. A human cell expressing Cre recombinase.