EP0954591A2 - Mini-adenoviraler vektor - Google Patents

Mini-adenoviraler vektor

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Publication number
EP0954591A2
EP0954591A2 EP97928961A EP97928961A EP0954591A2 EP 0954591 A2 EP0954591 A2 EP 0954591A2 EP 97928961 A EP97928961 A EP 97928961A EP 97928961 A EP97928961 A EP 97928961A EP 0954591 A2 EP0954591 A2 EP 0954591A2
Authority
EP
European Patent Office
Prior art keywords
dna molecule
gene
sequence
human
protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97928961A
Other languages
English (en)
French (fr)
Inventor
Wei-Wei Zhang
Ramon Alemany
Yifan Dai
Steven Josephs
Cristina Balague
David Ayares
Richard Schneiderman
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Baxter International Inc
Original Assignee
Baxter International Inc
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Filing date
Publication date
Application filed by Baxter International Inc filed Critical Baxter International Inc
Publication of EP0954591A2 publication Critical patent/EP0954591A2/de
Withdrawn legal-status Critical Current

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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
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    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N2710/10011—Adenoviridae
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    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
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    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/80—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates
    • C12N2810/85—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
    • C12N2830/003—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor tet inducible
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Definitions

  • This invention is related to adenoviral (Ad) vectors and their applications in the field of genetic medicine, including gene transfer, gene therapy, and gene vaccination. More specifically, this invention is related to the Ad vectors that carry the minimal cis- element of the Ad genome (mini-Ad vector) and are capable of delivering transgenes and/or heterologous DNA up to approximately 36 kb.
  • the generation and propagation of the mini-Ad vectors require tr ⁇ /u-complementation of a packaging-attenuated and replication-defective helper Ad (helper) in an Ad helper cell line.
  • This invention further comprises a methodology for generating a mini-adenoviral (mini-Ad) vector for use in gene therapy of hemophilia and animal test systems for in vivo evaluation of the Ad vectors. More specifically, this invention describes factor VIII (FVIII) Ad vectors that only contain minimal cis-elements of the Ad genome (so called mini-Ad) and comprise a human FVIII cDNA with other supporting DNA elements up to 36 kb. The FVIII mini-Ad can be generated and preferentially amplified through the assistance of a packaging-attenuated helper Ad and a helper cell line. This invention also reports designs and methods for producing transgenic mouse models that can be used for in vivo testing the mini- Ad.
  • mini-Ad factor VIII
  • the preferred system of gene delivery must possess several properties that are currently unavailable in a single gene therapy vector.
  • the preferred vector must retain adequate capacity to accommodate large or multiple transgenes including regulatory elements and be amenable to simple manipulation and scale-up for manufacturing.
  • Such a vector must also be safe and demonstrate low toxicity as well as demonstrate highly efficient and selective delivery of transgenes into target cells or tissues.
  • Such a vector must be capable of supporting appropriate retention, expression, and regulation of the transgenes in target cells.
  • the present invention encompasses a novel design of a high-capacity and highly-efficient Ad vector system and is focused on resolving the issues and concerns of those skilled in the art regarding an preferred gene delivery system.
  • Hemophilia A results from deficiencies in expression or function of clotting factor VIII (FVIII) in affected individuals.
  • Treatment of hemophilia currently involves infusion of normal FVffl protein obtained from plasma concentrates or as purified from cultured cells engineered to express recombinant FVIII protein (1).
  • Therapeutic benefit is achieved by restoration of plasma levels to 5-10% of normal plasma levels (200-300 ng or 1 unit per milliliter; Ref. 2).
  • Studies have shown that maintenance of greater than 30% of the normal plasma levels allows for a near normal lifestyle (3).
  • Gene therapeutic approaches towards treatment of hemophilia have exciting potential; however, several major challenges remain to be overcome for these treatment modalities to become reality (4).
  • the present invention provides several tools with which these difficulties may be resolved.
  • FVIII is normally produced in the liver and is comprised of heavy chain polypeptides with a range of apparent molecular weights of from 92 kDa to 210 kDa derived from the amino terminus of the nascent polypeptide and a C-terminal light chain of 80 kDa (53). It is protected from proteolysis by formation of a complex with von Willebrand's factor (vWF).
  • the activated form functions in the blood clotting cascade as a cofactor along with activated factor IX (FIXa), negatively charged phospholipids and calcium ions to convert factor X to its activated form, Xa.
  • the human cDNA is 9 kb in length and encodes a polypeptide of 2351 amino acids comprised of several domains in the order Al, A2, B, A3, CI and C2 (5-7).
  • the A and C domains are critical for functional activity whereas the majority of the B domain, consisting of approximately 980 amino acids, is dispensable for activity (8). Since the full-length FVIII cDNA exceeds the size limitations of retrovirus and adenovirus vectors, most gene therapy protocols utilized by those skilled in the art to date have utilized a FVIII cDNA having the B domain deleted, such that the remaining cDNA is approximately 4.5 kb.
  • Retroviral vectors were among the first to be studied for use in gene therapy (64, 65).
  • the size capacity for insertion of exogenous DNA is limited to approximately 7.5 kb.
  • it has been difficult to obtain high-level expression of FVIII from retroviral vectors due to problems of viral mRNA instability and difficulties of expression of the mRNA encoding the FVIII gene product (62, 63, 74).
  • infection of non-dividing cells, such as the majority of the liver cells is also problematic.
  • One way to overcome this limitation has been to perform a 2/3 partial hepatectomy prior to retrovirus infection to allow infection of actively regenerating liver cells (73).
  • long-term expression has been achieved using muscle specific enhancers but only low levels of gene product, FLX, were achieved (78).
  • Therapeutic levels of FVIII have been achieved in mice (77).
  • an El -substituted adenoviral vector comprising a B-domain deleted FVIII cDNA under control of the murine albumin promoter has been utilized to achieve therapeutic levels of human FVIII expression in mice and dogs (13-15).
  • gene expression in immunocompetent animals was limited in duration; a gradual decline in gene expression correlated with a loss of detection of the adenoviral vector DNA in liver tissue (13).
  • the decline of expression was partially overcome by lowering the vector inoculum resulting in therapeutic plasma levels of FVIII for 22 weeks following administration (16).
  • Adenoviral vectors may be preferred for delivery of FVIII due to the fact that: 1) intravenous (I.V.) injection of adenovirus results in targeted gene expression to the liver, in part due the accumulation of the adenoviral vector primarily in liver tissue; 2) expression of FVIII from the liver has resulted in a significant elevation of levels of FVIII in plasma; and, 3) the liver is a major site of synthesis of FVIII in normal individuals.
  • I.V. intravenous
  • Ad does not normally integrate into the host cell genome.
  • an Ad vector must include the elements required for host cell integration or other mechanisms of DNA retention.
  • the immune response mediated against the adenoviral vector makes re-administration of the vector very difficult (76).
  • the mini- Ad vector of the present invention has eliminated all adenovirus genes from the mini-Ad vector carrying the transgene. This at least partially eliminating any detrimental immune response that may be raised by Ad gene expression in the host cell, which may contribute to the decline of transgene expression.
  • adenoviral vector that allows for a large heterologous DNA insert has been described in international patent application WO 96/33280 (see ref. 132); this vector, however, does not provide elements for integration into the target cell genome or for episomal maintenance of the vector upon entry into a target cell.
  • the present invention provides elements that allow for retention of the delivered transgene in the host cell, either by integration into the target cell genome or by maintenance as an episomal nucleic acid.
  • One method with which this is accomplished by the present invention includes facilitation of integration of the transgene into the host cell genome using viral integration mechanisms.
  • the adeno-associated virus (AAV) genome has the capability of integrating into the DNA of cells which it infects and is the only example of an exogenous DNA that integrates at a specific site, AAVS1 at 19ql3.3-qter, in the human genome (35, 133).
  • the minimal elements for AAV integration are the inverted terminal repeat (ITR) sequences and a functional Rep 78/68 protein.
  • the present invention incorporates these integration elements for integration of the transgene into the host cell genome for sustained transgene expression.
  • the present invention also provides an adenoviral vector capable of homologous recombination into the genome of a target cell, another significant advantage over adenoviral vectors currently available to one skilled in the art.
  • the present invention also provides elements that allow episomal replication of the transgene. In vitro model systems have been developed to detect site-specific integration of
  • AAV or AAV-based vectors in immortalized cell lines 22-24, 134
  • episomal systems (25-27)
  • cell- free extracts 28
  • a comparison of transduction efficiencies of AAV using either primary human cells or immortalized cell lines demonstrated that the transduction efficiency was 10 - 60 times greater in immortalized human cells than in primary cells (29).
  • These results stress the importance of using primary cells, or even better, in vivo model systems, to accurately evaluate AAV vectors for gene therapy applications.
  • no in vivo animal model system has been developed to detect site-specific integration.
  • An animal model having the human AAVS1 sequence incorporated into its genome is provided in the present invention. This animal model will be useful for evaluation of vectors containing the AAV integration mechanism, not only to test site-specific integration, but also in vivo gene delivery, gene transduction efficiency, tissue distribution, and duration of gene expression.
  • the mini- Ad vector system of the present invention was developed based on two major findings: 1) the discovery of an Ad-SV40 hybrid (17) in which the majority of the viral genome was replaced by SV40 sequences but was able to be processed and packaged due to the presence of Ad ITR and packaging elements; and, 2) that Ad packaging may be attenuated by partial deletion of the packaging signal (18).
  • Ad packaging may be attenuated by partial deletion of the packaging signal (18).
  • Other adenoviral vector packaging systems based on inco ⁇ oration of minimal cis elements for packaging and genome replication are under development by others (19-21).
  • An objective of the present invention is to provide a modified adenoviral vector in order to provide: 1) a large capacity adenoviral (Ad) vector (a "mini- Ad" vector) having the capacity for insertion of approximately 36 kb heterologous DNA that may also include elements for controlling transgene expression, assisting in integration of exogenous DNA into target cell genomic DNA, and / or maintenance of the vector in an episomal form within a target cell; 2) a cognate helper Ad vector designed to support propagation of the of the mini-Ad vector and that has a manipulated packaging signal such that within a host producer cell the mini-Ad vector is packaged at a greater vector; and, 3) a helper cell line designed to support propagation of both the mini-Ad vector and the helper Ad vector that may also serve to control transgene expression during viral propagation and selectively attenuate packaging of the helper Ad genome.
  • Ad a large capacity adenoviral vector
  • a "mini- Ad" vector having the capacity for insertion of approximately 36
  • a problem encountered by those skilled in the art is that the amount of exogenous DNA that can be inserted into conventional vectors is limited, to a present maximum of approximately 8 kb. It is an objective of the present invention to provide a mini-Ad vector able to accommodate an insert capacity of up to approximately 37 kb, sufficient for delivery of a nucleic acid encoding a protein having a large coding sequence.
  • the present invention provides a mini-Ad vector as an isolated DNA molecule having the elements necessary for replication, packaging and sustained gene expression such as an inverted terminal repeat (ITR), a packaging signal, a transcriptional control region, an effector or reporter gene, and either a genomic integration sequence or an episomal maintenance sequence, all operatively associated for generating an infectious, replication-defective recombinant adenoviral vector wherein the remaining portion of said DNA molecule does not encode an adenoviral protein.
  • ITR inverted terminal repeat
  • a packaging signal a transcriptional control region
  • an effector or reporter gene an effector or reporter gene
  • a genomic integration sequence or an episomal maintenance sequence either operatively associated for generating an infectious, replication-defective recombinant adenoviral vector wherein the remaining portion of said DNA molecule does not encode an adenoviral protein.
  • a mini- Ad vector encoding human FVLTI is provided.
  • the present invention provides a vector having the elements required to extend the duration of expression of a transgene following introduction into a target cell.
  • the present invention thus provides a vector having an element that may aid in stabilization of transgene expression in the target cell as an episome or by facilitating integration of the introduced gene into the cell genome.
  • the present invention provides either a genomic integration element or an episomal maintenance element within the mini-Ad vector.
  • An example of such a system provides a mini-Ad vector including integration elements of AAV.
  • Another example of such a system provides a homologous recombination arm within the mini-Ad vector.
  • an El -deleted helper Ad genome having an altered packaging signal such that the El -deleted helper Ad genome is packaged at a lower frequency than the wild-type helper Ad genome is provided.
  • a helper or producer cell is provided as a cell stably transfected with an Ad El gene sequence that has no overlapping sequence with the genome of an El -deleted helper Ad genome is provided.
  • a method of generating a recombinant adenoviral vector by co-transfecting the helper or producer cell with a mini-Ad vector and an Ad-helper genome and/or infecting the cell with an Ad helper virus, and preparing a cell-free lysate of said producer cell is provided.
  • the cell-free lysate thus prepared contains said infective, replication-impaired recombinant adenoviral vector particles, the majority of which include the mini- Ad vector DNA.
  • the present invention provides an animal model system for assessment of targeted integration as directed by the AAV integration mechanisms inco ⁇ orated into the mini- Ad vectors of the present invention.
  • the present invention provides a methodology for the generation of transgenic mice harboring the human AAVS 1 integration sequence within their genome. Following injection of a mini- Ad vector into such an animal, targeted integration of the transgene into the AAVS1 site may be evaluated.
  • the present invention provides a methodology for the development of a transgenic mouse that contains a human FVIII gene operably linked to a developmentally-regulated promoter (i.e., the ⁇ - fetoprotein promoter), such that human FVIII is expressed in the mouse during development but not in the mature animal.
  • a developmentally-regulated promoter i.e., the ⁇ - fetoprotein promoter
  • the present invention further provides a human FVIII-tolerized, hemophiliac mouse breed as well as a human FVIII-tolerized, hemophiliac mouse having inco ⁇ orated into its genome the human AAVS1 pre-integration site.
  • the present invention therefore, provides the reagents and methodologies needed to overcome many of the difficulties associated with gene therapy vectors that have been encountered by those skilled in the art.
  • the objectives described above as well as other objectives of the present invention will be understood in light of the detailed description of the invention provided below.
  • FIG. 1 The principle of the mini-Ad vector system. Shown are three of the major components of the mini-Ad vector system: the helper Ad, the mini-Ad vector, and the Ad helper cell. El supplied by the helper cells allows the helper Ad to replicate itself and synthesize the late viral proteins that form the viral capsids.
  • the packaging of the helper Ad genome into the capsid is inefficient as the packaging signal of the helper Ad of the present invention has been attenuated.
  • the helper Ad supports replication of the mini-Ad vector genome, which is preferentially packaged because its wild-type packaging signal has high affinity for the helper virus packaging proteins (31). Further purification of the mini-Ad vector may be achieved using a biochemical or physical method, such as ultracentrifugation.
  • FIG. 1 Comparison of the current Ad vectors with the present invention. Depicted are the general structures and complementary mechanisms of the current Ad vectors compared to those of the mini- Ad vector system.
  • FIG. 3 The prototype of the helper virus and the mini-Ad vector.
  • A The placement of the packaging signal in reference to the left ITR of the adenovirus.
  • B The sequence of the packaging signal region of the wild type adenovirus 5 is shown. The brackets indicate the regions deleted to comprise the attenuated helper virus packaging signal.
  • C The repeated region of the sequence shown in B are listed along with the consensus repeat.
  • Figure 4. Construction of the shuttle vector to generate the packaging attenuated helper AdHO.
  • GT5000 the mutant packaging signal sequence, mt ⁇ , was amplified by PCR and substituted in a shuttle vector with an Ad5 sequence extended to 28.9 mu (GT4004).
  • a ⁇ -gal expression cassette from pTk- ⁇ was cloned into the El- deletion of GT5000 to give the shuttle vector GT5001.
  • FIG. 5 Generation of AdHO.
  • the shuttle vector GT5001 (see fig. 8) was cotransfected with pJM17 in 293 cells. Recombination in the homologous 7 Kb region between these plasmids (9.24 to 28.9 mu of Ad5) yields a packageable virus with the left arm derived from GT5001.
  • the numbers in the left region of AdH ⁇ shown at the bottom correspond to Ad5 nt sequence and indicate extension of the double deletion in the packaging signal as well as in El where the ⁇ -gal expression cassette is inserted.
  • the size of the amplified fragment, 310 bp for the wild type packaging signal (wt ⁇ ) and 177 bp for the mutant packaging signal (mt ⁇ ), can be distinguished in a 2% agarose gel as shown.
  • FIG. 7 Amplification and characterization of AdHD.
  • the packaging signal of AdH ⁇ was amplified after every passage due to the possibility that recombination with the endogenous left Ad5 sequences present in 293 cells could generate a replication competent adenovirus (RCA, E1+) or an El- adenovirus with wt ⁇ . Wt ⁇ was not detected in the passages previous to the CsCl purification (4 to 8).
  • the viral DNA content was analyzed separately for every of the five bands of the gradient. In a 1% agarose gel (bottom left), almost no vDNA is observed in the upper three bands, indicating that they are formed mostly by empty capsids.
  • Lower bands (4 and 5) are formed by full capsids. By PCR the expected mutant packaging signal is detected in all the bands (bottom right). 1 Kb ladder marker (as in figure 9) at the left lane of every gel. Gel with vDNAs also contains 1/Hind III marker.
  • FIG. 8 Construction of mini-viral plasmids. These plasmids are constructed to determine the effect of various deletions of the adenoviral genome on packaging when complemented with AdH ⁇ . All constructs contain the green fluorescence protein cDNA (GFP, striped box) driven by the CMV promoter with a ⁇ -actin enhancer (thick arrow). M7.9 (bottom right) also has the neomycin cDNA and an internal ribosome entry site (IRES). The top six are derived from M32, which is a pJM17 derivative with a 10 Kb deletion in the middle of the Ad5 genome.
  • GFP green fluorescence protein cDNA
  • IVS internal ribosome entry site
  • the bottom two are derived from pBluescript- KS (Stratagene, CA) with the minimal cis elements for replication and packaging of Ad5. Numbers correspond to Ad5 map units and indicate the deletion and insertion sites. 0/100 or 100/0 indicates the natural fusion of the inverted terminal repeats (ITR) of Ad5 DNA, Ad5 DNA, thick lane; plasmid backbone DNA, thin lane.
  • ITR inverted terminal repeats
  • FIG. 9 Schematic representation of the mini-adenoviral (mini-Ad) vectors constructed for packaging.
  • the Ad5 transcription map and map units (mu) with the early (E) and late (L) transcription regions.
  • MLP/TL major late promoter and tripartite leader.
  • the inverted terminal repeats (ITRs) and the packaging signal ( ⁇ ) are the unique common sequences in all the miniAd vectors.
  • the vectors are shown in a linear form as found after replication and in the capsid.
  • the circular plasmids used for vector generation contain the same sequences but fused head-to-tail by the ITRs. Every mimAd vector name refers to its size in Kb.
  • M32 to M20 derive from pJM17 by progressive deletion of the central adenovirus genome.
  • the plasmid backbone (pBRX, not drawn) is located at 3.7 mu.
  • M6.5 to vGnE5E3 are constructed in the pBluescript backbone (not drawn; located before the GFP-expression cassette) by insertion of neomycin cDNA and human genomic fragments from chr. 4ql 1-22.
  • FIG. 10 Two methods of complementation. To generate the mini-viral vectors two separate complementation protocols were used that gave similar yields. In the first method, the mini-Ad plasmid is cotransfected with viral DNA from AdH ⁇ , and the cells are cultured until a CPE is observed. In the second method, three days after an initial cotransfection of the mini- Ad plasmid with pBHGlO, AdH ⁇ is added as virus, and cells are cultured until a CPE is observed.
  • FIG. 11 Co-transfection of 293 cells with mini-viral plasmids. Nearly all cells were transfected by using a CaPO 4 modified protocol. Transfection with the M32 plasmid is shown 1 day after transfection. Right, bright field; left, fluorescence microscopy of the same field.
  • FIG. 1 Minivirus-containing plaque.
  • the presence of the minivirus (M32 here) is shown by the fluorescence of the plaque. Right, bright field; left, fluorescence microscopy of the same field.
  • FIG. 13 Packaging efficiency of mini-viral vectors of different sizes. Packaging efficiency and amplification of miniAd vectors of different sizes.
  • Thevirus produced after cotrasnfection is named as passage 0.
  • the crude lysate was used to infect 293 cells to produce passage 1.
  • 1 ml of crude lysate passage 1 was used to infect 106 293 cells and 24 h later the number of fluorescent cells were counted (transducing units/ml, dark columns). 24 h later CPE appeared and virus was extracted by freeze/thaw (crude lysate passage 2).
  • 1 ml of crude lysate passage 2 was used to infect 106 293 cells and 24 h later the number of fluorescent cells were counted (striped columns).
  • the difference between passage 1 and 2 indicates an amplification yield of 5x and the difference between different miniAd vectors indicates the effect of the size in the packaging.
  • FIG 14. Purification scheme of M32. After amplification of M32 through several passages, the crude extract was CsCl-separated. The first gradient resulted in four bands: three upper and one lower. These were collected separately, dialyzed and used to infect 293 cells as shown in Figure 12. Fractions were collected from second separated gradients of the upper and the lower bands, and used to infect 293 cells as shown in Figure 16.
  • AdH ⁇ co-purify in the band of higher density (number 4 or lower).
  • the same wells used to check for fluorescence are later used to fix and stain with X-gal.
  • FIG. 16 Purification of M32 mini-Ad viruses (2): fractionation of the lower band from the first CsCl gradient with a second CsCl gradient. An aliquot of 0.5 ⁇ l of every fraction was used to infect one well of a 96 well/plate with 293 cells at 60 % confluency. Initial fractions (1 to 6) did not contain M32 or AdH ⁇ (these fractions represent up to 3 ml of the gradient). 100 ⁇ l samples of fractions 7 to 16 reveal a large amount of M32 and AdH ⁇ (see panel B for ⁇ -gal staining of the same fractions shown under fluorescence in panel A). Subsequent fractions (17 to 29) show a level of M32 similar to the previous fractions but the level of AdH ⁇ is approximately 10 times lower. Therefore, fractions 17-29 represent a 10-fold enrichment of M32 with respect to AdH ⁇ .
  • FIG. 17 Ad5 packaging signal modification with GAL4 binding sites.
  • the nucleotide sequences between the Xho I and Xba I sites are shown ( design #1 and design #2 ).
  • design #1 there are two GAL4 binding sites before A repeat I and one GAL4 binding site between A repeat II and VI.
  • design #2 there two GAL4 binding sites before A repeat I and other two GAL4 binding sites after A repeat VII.
  • the sequences underlined are 17 mer GAL4 binding sites.
  • the sequences in italics are A repeats. The distance between center of each GAL4 binding site and A repeats is indicated.
  • FIG. 18 Ad5 packaging signal modification with tetO sequence.
  • the nucleotide sequences between the Xho I and Xba I sites are shown ( design #1 and design #2 ).
  • design #1 there are two tetO sequences before A repeat I and one tetO sequence between A repeat II and VI.
  • design #2 two tetO sequences are present before A repeat I and further tetO sequences after A repeat VII.
  • the sequences underlined are 19 mer tetO sequence.
  • the sequences in italics are A repeats. The distance between the center of each tetO binding site and the A repeats is indicated.
  • Figure 19 Position and sequences of synthetic oligos for Ad Pac " -GAL4 modification.
  • Gal#l to Gal#8 are synthetics oligos flanking the sequence between the Xho I and Xba I sites in design #1 and #2. The position and direction of each oligo is indicated by arrow bar. The sequences of Gal#l to Gal#8 are listed.
  • FIG. 20 Position and sequences of synthetic oligos for ad Pac ' -fetO modification.
  • tet#l to tet#10 are synthetics oligos to cover the sequence between the Xho I and Xba I sites in design #1 and #2. The position and direction of each oligo is indicated by arrow bar. The sequences of tet#l to tet#10 are listed.
  • FIG. 21 Construction of CMV-E1 mammalian expression vector.
  • Adenovirus 5 sequences 462-3537 (Aflffl-AfiTI Fragment) coding for El A and E1B were blunt-end cloned into the EcoRV site of pcDNA3.
  • FIG. 22 GFP expression and plaque formation in the El-complementing cell line, A549E1-68. After infection with an El-deleted adenovirus, Ad5CA-GFP. The clear area in the center of this plaque is evidence of the CPE caused by El -complemented virus amplification.
  • FIG. 23 Southern blot analysis of G418 r A549E1 clones. Genomic DNA was digested with Hind III and probed with a 750bp El probe (Pstl fragment). Lane 1 : lkb DNA ladder; Lane 2: A549; Lane 3: 293; Lane 4: A549E1-68; Lane 5: Subclone A549E1-68.3.
  • Figure 24 Morphology analysis of the new cell line. Mo ⁇ hological comparison of parental A549 cells (top panel) and the El -complementing cell line, A549E1-68 (bottom panel).
  • FIG. 25 Analysis of the El protein expression in transformed cell lines.
  • FIG 26 Schematic of pAlbl2.5CAT plasmid.
  • the plasmid comprises the pAlbl2.5CAT 12.5 kb human albumin promoter (EcoRI to Hindlll) operably linked upstream of the chloramphenicol acetyl transferase gene (CAT) in the pBRCAT plasmid vector.
  • the proximal promoter, and the enhancer regions (E, 7 and E 6 ) are shown.
  • Figure 27 Cloning of the 12.5 kb human albumin promoter into pBIueScript KS + vector.
  • the EcoRI to Aval 10.5 kb fragment and the 2.0 kb Aval to Hindlll albumin promoter fragments were separately isolated from pAlbl2.5CAT and simultaneously ligated into the EcoRI / Hindlll site of pBlueScript-KS + vector to generate GT4031.
  • FIG. 28 Cloning of the hFVIII expression cassette into GT4031.
  • the 7.5 kb human FVIII cassette was excised from plasmid GT2051 using Xhol and Sail and ligated into Sail site of GT4031 resulting in GT2053, comprising the human albumin promoter operably linked to the hFVIII cDNA.
  • FIG. 29 Construction of an albumin promoter-FVIII minivirus plasmid.
  • a fragment comprising the adenovirus 5' ITR and packaging signal from GT2033 was excised using Xhol and cloned into the Sail site of GT2053. Plasmids having either the forward or reverse orientation (GT2061 and GT2059, respectively) of the ITR were obtained.
  • the insert in GT2061 is oriented with the unique Sail site proximal to the FVTTI gene.
  • GT2059 contains the Ad ITRs in the opposite orientation of GT2061.
  • FIG 30 Restriction digest profiles of GT2053, GT2059 and GT2061. Digests show the expected banding patterns for BamHl, Xbal, Clal and Xhol in combination with Sail.
  • Figure 31 Diagram of the albumin/ ⁇ -fetoprotein gene region on chromosome 4.
  • the diagram illustrates three regions that may serve as 3' recombination arms for homologous recombination: 1.) Alb-E5, the 3' region of the albumin gene; 2.) AFP-3, a central region of the ⁇ -fetoprotein gene; and, 3.) EBB14, located further 3' in the ⁇ - fetoprotein gene.
  • FIG. 32 Cloning scheme. Restriction enzyme maps of three vectors (A, B, and C) comprising distinct 3' homologous recombination arms after cloning the arms into GT2061 (illustrated above panels A, B, and C).
  • FIG. 33 Cloning scheme. Detailed cloning scheme for GT2063 where the 3' 6.8 Kb Xhol fragment of the human albumin gene of clone pAlb-E5 was cloned into the Sail site of GT2061. A minivirus based on this vector is a potential in vivo therapeutic tool for FVTII gene therapy.
  • FIG. 34 Restriction enzyme mapping. Agarose gel demonstrating restriction enzyme digestion of the vectors utilized in the generation of the plasmid comprising the albumin promoter-driven hFVIII with the 3' albumin homologous recombination arm as shown in Fig. 9. EcoRI and Clal digests are shown for each of the indicated constructs.
  • FIG. 35 Scheme for generating the mini-AdFVTII virus. Shown are two schemes (A and B) for generating a hFVIII minivirus.
  • Scheme A helper virus genomic DNA and plasmid GT2063 were cotransfected by calcium phosphate precipitation into 293 cells (ATCC# CRL 1573) on day 1. Transfection was by calcium phosphate precipitation. At day 6, cytopathic effect (CPE) was observed and cell lysates prepared. Lysates were subsequently harvested every three days following infection until passage 4, at which time the virus preparation was amplified five-fold. Media was changed daily following infection.
  • CPE cytopathic effect
  • FIG. 36 Generation of a mini-Ad vector containing FVIII.
  • the mini-AdFVIII vector was generated by transfection of the mini-Ad plasmid GT2063 in 293 cells and infection with the helper AdH ⁇ .
  • CPE cytopathic effect
  • the cells and supernatant were collected (passage 0).
  • virus was extracted from the cells by several freeze/thaw cycles and utilized to infect fresh 293 cells. At each passage, 740 ⁇ l of supernatant was used to extract viral DNA by incubation in a solution comprising SDS, EDTA, and Proteinase K followed by ethanol precipitation.
  • supematants were subjected to DNase I digestion to avoid contamination from GT2063 plasmid DNA.
  • vDNA purified viral DNA
  • PCR polymerase chain reaction
  • the expected size for the FVIII mini-Ad and helper amplified regions were 177 and 310 bp, respectively.
  • DNA size marker 1 Kb ladder from Gibco (Gaithersburg, MD).
  • Supematants from untransfected 293 cells with or without additional GT2063 plasmid were used as negative control and as a control of DNase I treatment, respectively.
  • FIG 37 Southern blot analysis of vDNA from passages 0 to 21 of the mini- AdFVIII vector.
  • vDNA was purified as in Figure 14.
  • One half of the purified vDNA (corresponding to 370 ⁇ l of supernatant) was digested with Pst I, separated on a 1 % agarose gel, and blotted to a nylon membrane.
  • a probe corresponding to sequence adjacent to the right (3') ITR present in both the mini- Ad and the helper was utilized to detect the vDNA.
  • Four independent blots are shown (A, B, C and D). Specific hybridization to marker fragments was utilized for normalization.
  • FIG. 38 Dynamic fluctuation in mini-AdFVIII and helper over time during serial passage. The plot was obtained by densitometrical quantification of the bands shown in
  • Helper is labeled by the clear line with squares; mini-AdFVITI is labeled by the dark line with diamonds.
  • One unit is defined on the Y axis as the lowest amount detected (corresponding to the quantity of helper vDNA at passage 18). Other values are normalized to that unit.
  • FIG. 39 Separation of mini-AdFVIII and AdH ⁇ by CsCl gradient centrifugation.
  • the bottom band from the first gradient contained virions that were further separated by application to a second gradient.
  • the different sizes of the mini- Ad (31 kb) and the helper (37.1 kb) allowed separation that resulted in the generation of an upper fraction having a 10:1 mini- Ad/helper virus ratio and a lower fraction having a 1:10 mini- Ad/helper virus ratio as determined by Southern blot.
  • FIG 40 FVIII expression in cells transduced with the mini-Ad vector.
  • 293 cells were grown in chamber slides and infected with a diluted (1/100) 1 ⁇ l aliquot of the upper or lower fractions as shown in Figure 18C.
  • Twenty-four hours following infection, the cells were fixed and stained with a FVTII specific mAb (Cedar Lane Sheep anti- human FVIIIC, #CL20035A, Accurate Chemical and Scientific Co ⁇ oration, Westbury, NY) and subsequently a secondary antibody (biotinylated donkey anti-sheep IgG, Jackson Immunoresearch, #713-065-147) and DAB (resulting in a reddish-brown color; SIGMA Cat. No. D7679).
  • FVTII specific mAb Cedar Lane Sheep anti- human FVIIIC, #CL20035A, Accurate Chemical and Scientific Co ⁇ oration, Westbury, NY
  • secondary antibody biotinylated donkey anti-she
  • Figure 41 Functional FVIII expression in cells transduced with the mini-AdFVIII vector. Coatest chromogenic assay for functional FVLTI. Upper table shows the OD readings. The standard curve in triplicate (lanes 1, 2, and 3) from 4000 ng/ml to 62.5 ng/ml (rows A to G) is also plotted to obtain the equation to extrapolate the readings from the samples. Lanes 4,5 and 6 are triplicates of the samples.
  • A 10 ⁇ l aliquot of miniAdFVIII in 293 cells; B: 1 ⁇ l of mini-AdFVIII in 293 cells; C: 10 ⁇ l of mini- AdFVIII in HepG 2 cells; D: 1 ⁇ l of miniAdFVIII in HepG 2 cells.
  • E conditioned medium from untransduced 293 cells.
  • F conditioned medium from untransduced HepG 2 cells.
  • FIG 42 Map of clone GT2074.
  • the plasmid comprises an expression cassette [comprising the elongation factor- 1 (EF-1; ref. 52) promoter operably linked to the B- domain deleted human FVIII cDNA] excised from plasmid GT4020 by Sail digestion cloned into the unique Sal I site of GT2073.
  • the 3' proximal albumin promoter region downstream of the Pme I site in pALB12.5 including the TATA and CCAAT (32) were deleted.
  • the expression cassette contains the elongation factor I promoter linked to the B-domain deleted human FVIII cDNA.
  • FIG 43 Map of pCMV-hFVHI.
  • This plasmid comprises CMV promoter operably linked to the full-length hFVIII coding region as cloned into the Sal I site of GT2073.
  • the cytomegalovirus promoter was derived from pCMV ⁇ (Clontech, Palo Alto, CA).
  • FIG 44 Schematic representation of the plasmids used to test for integration frequency and specificity.
  • Plasmids GT9003 and GT9004 contain a neo expression cassette flanked on both sides by AAV ITR sequence;
  • GT9012 and GT9013 contain a GFP expression cassette flanked by AAV ITR sequence;
  • GT9003 and GT9012 also contain a Rep78 expression cassette upstream of the integration cassette.
  • Rep sequences from 193 to 2216 in the AAV genome were amplified by PCR (Pfu pol) from plasmid pSUB201, and cloned into pCRII (Invirrogen, CA).
  • the resulting plasmid (GT9000) was digested with Notl and Xhol and a fragment containing an SV40 polyA site (Not-Sal I) was cloned in those sites.
  • the resulting plasmid (GT9001) was digested with Xbal and blunt- ended with Klenow.
  • a PvuII-PvuII fragment containing the whole AAV genome was obtained from pSUB201 and subcloned in the blunted Xbal site in GT9001.
  • This plasmid (GT9002) was then cleaved with Xbal which removes the AAV coding sequences leaving the AAV ITRs.
  • a neo-expression cassette (BamHI-BamHI) was then subcloned into GT9002 using Xbal and BamHl adaptors, giving rise to plasmid GT9003.
  • Plasmid GT9004 was generated by removing the Rep coding sequences GT9003 using EcoRI.
  • Plasmid GT9012 and GT9013 were generated by replacing the neo sequences (Xbal-Xbal) in GT9003 and GT9004, respectively, with a GFP expression cassette (Spel-Nhel).
  • Figure 45 Design of the mini-adenoviral vector containing an integratable Factor VIII cassette.
  • Minimal Ad elements necessary for replication and packaging present in the construct are the Ad ITRs and packaging signal.
  • Factor VIII cassette is contained between two AAV ITRs.
  • a Rep expression cassette is positioned outside the integratable segment. Rep expression can be regulated by the Tet operator in a cell line stably expressing the repressor tet-KRAB. In the target cells, the expression of Rep should provide targeted integration of the sequences flanked by AAV ITRs in the AAVS1 site in chromosome 19.
  • FIG 46 Immunoprecipitation of Rep proteins in 293 and Chang liver cells.
  • Cells grown in 10-cm Petri dishes were transfected with 10 mg of plasmids GT9001, GT9003, and GT9004 (see Figure 31 for details on construction of plasmids). Untransfected and GT9004-transfected cells were used as negative controls.
  • Two days after transfection cells were lysed and Rep proteins were immunoprecipitated using an anti-Rep monoclonal antibody (clone 226.7; ARP, Belmont, MA 02178) coupled to protein G- agarose, run on a 10% polyacrylamide gel and immunoblotted with the same antibody used in immunoprecipitation. Proteins were visualized by chemoluminiscence (ECL kit, Amersham). The migration of Rep78 and Rep52 proteins is indicated.
  • FIG 47 Southern blot of 293 clones transfected with plasmids GT9003 or GT9004. Fifteen mg of genomic DNA from several neo-resistant clones as well as a neo- resistant population (indicated as pool) were digested with EcoRI, electrophoresed, blotted onto a nylon membrane (Hybond-N, Amersham) and hybridized to an AAVS1 probe, spanning an 8Kb EcoRI-EcoRI fragment. The normal AAVS1 locus is indicated (panel A). Some GT9003 clones show a shifted band corresponding to disruption of one of the AAVS1 loci. Panel B shows the same membrane rehybridized to neo sequences. Figure 48.
  • FIG. 49 Southern Blot Analysis of AAVS1 PI Genomic Clones. Plasmid DNA (1 ug) isolated from four PI genomic clones (termed PI clone 6576, PI clone 6577, PI clone 6578, and PI clone 6579) in which the AAVS1 sequence was detected by PCR [AAVS1 PCR(+)] digested with EcoRI (Fig. 23 A) or EcoRI in combination with EcoRV (Fig. 23B), electrophoresed on a 1% agarose gel, blotted onto a nylon membrane (Hybond N+, Amersham), and hybridized using the 253 bp AAVS1 PCR product as a probe. In both A and B, Lane 1 represents PI clone 6576, Lane 2 represents PI clone 6577, Lane 3 represents PI clone 6578, and Lane 4 represents PI clone 6579.
  • Figure 50 Construction of the pAAVSl-Neo Vector.
  • An 8.2 kb EcoRI fragment comprising an AAVS1 integration sequence was isolated from PI clone 6576 and ligated into the EcoRI site of the Neo expression vector, pGKneo, to create pAAVSl-Neo.
  • FIG. 51 Generation Of Transgenic Mice Harboring The Human AAVS1 Integration Sequence. Diagrammed are the sequences of steps involved in the generation of a transgenic mouse. ES cells were transfected with the AAVS 1 plasmid clone, microinjected into blastocysts which are then implanted into foster mothers. After approximately 17 days, chimeric mice were cross-bred to C57BL/6 mice and progeny tested for the presence of the AAVS1 transgene. Cross-breeding of the positive progeny was performed to generate a line that is homozygous for the transgene. These models are then utilized for testing in vivo delivery of the mini- Ad vectors modified to comprise the adeno-associated virus integration system to evaluate the efficiency of site-specific integration of the vector DNA.
  • FIG. 52 PCR Analysis of Neo R ES Cell Clones Following Transfection With pAAVSl-Neo. Genomic DNA independently isolated from 17 Neo R ES cell clones (3.1- P
  • PCR reaction samples were loaded on a 1.5% agarose gel as follows : Lane 1 - 1 kb DNA size markers (Gibco/BRL); Lane 2 - pAAVSl-Neo plasmid control; Lane 3 - Untransfected parental ES cell DNA; Lanes 4 through 20 - DNA from 17 individual pAAVSlNeo-transfected, Neo R ES cell clones.
  • FIG. 53 Southern Blot Analysis of AAVS1 PCR (+) ES Cell Clones. Genomic DNA from two AAVS1 PCR (+) ES cell clones (ES#4 and ES3.16) and from the parental ES cells was digested with EcoRI in combination EcoRV, electrophoresed on a 0.8% agarose gel, blotted onto Hybond N+ nylon membrane, and hybridized with an 8.2 kb AAVS1 probe. Lane 1 - AAVS1 ES#4; Lane 2 - AAVS1 ES#3.16; Lane 3 - Parental ES Cells. Expected fragments resulting from integration of the entire AAVS1 sequence into the ES cell genome are 5.2 and 3.0 kb.
  • FIG. 54 AAVS1 Chimeric Mice.
  • Two high-percentage male transgenic (chimeric) mice obtained from blastocyst microinjection experiments using AAVS1 ES#4 cells. In animals generated using ES stem cells, a higher degree of chimerism correlates with a higher probability of transmission of the transgene.
  • Genomic DNA was isolated from the tails of AAVS1 chimeras and, independently, from non-chimeric littermates, and screened by PCR using the AAVS1 -specific primers U2492 and L2722. PCR reactions were loaded onto a 1.5% agarose gel as follows : Lane 1 - 1 kb DNA size markers; Lane 2 - pAAVSl-Neo plasmid control ⁇ ; Lane 3 - untransfected parental ES cell DNA; Lane 4 - AAVS1 ES#4 ES cell DNA (4); Lane 5 - Tail DNA from a non-chimeric littermate; Lane 6 - Tail DNA from a low-percentage chimera (less than 10% agouti coat color); Lane 7 - Tail DNA from a high-percentage AAVS1 chimera (greater than 90% agouti coat color).
  • Figure 57 Southern Blot Analysis of mAFP-hFVHI-pGKNeo ES cell clones.
  • Genomic DNA from untransfected parental ES cells and from 4 Neo R ES clones was digested with Xba I (see Fig. 29), electrophoresed, blotted, and hybridized using a full- length hFVIII Not I fragment as a probe.
  • Expected fragment size indicating insertion of the mAFP-hFVIII cassette was 7.8 kb, as shown in the Figure.
  • FIG. 58 Construction of the mAFP-EGFP-1 vector. A 7.5 kb EcoRI/ Sal I fragment containing the entire AFP promoter/ enhancer region was cloned into the pEGFP-1 vector (Clontech) at EcoRI/ Sal I to create pAFP-EGFP- 1.
  • Figure 59 Scheme for the production of a transgenic mouse tolerized in utero to hFVIII for testing in vivo delivery of hFVIII without immune consequences.
  • ES cells transfected with the AFP-hFVffl-Neo plasmid containing the FVTTI cDNA operatively linked to the a-fetoprotein promoter are microinjected into blastocysts which are then implanted into foster mothers. After approximately 17 days, chimeric mice are cross-bred to C57BL/6 mice and progeny are tested for the presence of the AFP-hFVIII transgene. Cross-breeding of the positive progeny was performed to generate a line which is homozygous for the transgene.
  • These models are used for testing in vivo delivery of mini-Ad vectors modified to comprise the human FVIII expression cassettes.
  • FIG. 60 RIP-EGFP vector for use in the production of transgenic mice tolerized to Green Fluorescence Protein (GFP). Shown is a restriction enayme map of the RLP- pEGFP from the BS plasmid. This plasmid (4855 bp) comprises the green fluorescent protein (GFP) coding region operatively linked to the rat insulin promoter. The insulin promoter was utilized in this construct to drive expression of GFP in pancreatic tissue.
  • GFP Green Fluorescence Protein
  • Figure 61 Design of the episomal mini-adenoviral vector containing FVIII cassette.
  • the mini-Ad vector is designed to form a circularized plasmid structure that contains episomal maintenance mechanism and the FVIII expression cassette, after the viral vector enters the target cells.
  • the general structure of the vector has the following components: (a) Recombinase expression cassette; (b) Origin of replication; (c) Human FVTII cDNA; (d) Recombinase target sites; (e) Adenovirus ITRs; and (f) Stuffer DNA sequence.
  • Figure 62 General structure of the first version of the anticancer super-Ad vectors.
  • the viral vectors consist of the Ad-helper and the super- Ad that contains multiple genes for cancer suppression and anticancer immunomodulation.
  • the genes selected to be delivered are depicted in the diagram.
  • Figure 63 General structure of the second generation of the anticancer super-Ad vectors.
  • the viral vectors consist of the Ad-helper and the super-Ad that contains multiple genes for cancer suppression and anticancer immunomodulation.
  • the general structure is similar to the first version of the vectors.
  • Figure 64 Variations of the mini-Ad vector and the helper Ad vector. A.
  • An objective of the present invention is to provide a modified adenoviral vector in order to provide: 1) a large capacity adenoviral (Ad) vector (a "mini- Ad" vector) having the capacity for insertion of up to 37 kb heterologous DNA that may also include elements for controlling transgene expression, assisting in integration of exogenous DNA into target cell genomic DNA, and / or maintenance of the vector in an episomal form within a target cell; 2) a cognate helper Ad vector designed to support propagation of the of the mini-Ad vector and that has a manipulated packaging signal such that within a host producer cell the mini-Ad vector is packaged at a greater frequency than the helper Ad vector; and, 3) a helper cell line designed to support propagation of both the mini-Ad vector and the helper Ad vector that may also serve to control transgene expression during viral propagation and selectively attenuate packaging of the helper Ad genome.
  • Ad a large capacity adenoviral vector
  • a "mini- Ad" vector having the
  • the present invention comprises three components useful in generating a viral vector capable of delivering a therapeutic gene such as the FVIII cDNA to a target tissue in vivo.
  • the components consist of a helper virus, a miniviral genome, and a helper cell line.
  • the helper virus and helper cell line are utilized to package the miniviral genome into viral particles for gene delivery.
  • the miniviruses generated using this system have identical tropism and host range as the adenoviral strain from which the helper virus was derived.
  • the present invention further provides modifications of a mini-Ad vector to comprise elements derived from the adeno-associated virus (AAV). The elements are those having the ability to promote integration of genetic material into a host cell genome.
  • AAV adeno-associated virus
  • the elements are utilized to promote integration of a reporter or effector gene of a mini- Ad vector into the host cell genome. In this manner, expression of the gene is observed in the host cell for a longer period of time than that of a conventional adenoviral vector.
  • the present invention further provides a mini-Ad vector comprising elements for maintaining the vector as an episome in the host cell to prolong expression of the delivered gene or genes. It has been determined that limited replication of the viral genome of El -deleted viruses in the host cell allows for longer term expression of the gene of interest as compared to those genomes that are not able to replicate (88). Deletion of the E2 region of the adenoviral genome decreases the replication and duration of gene expression from the E2-deleted adenoviral vector. It is, therefore, an objective of this invention to inco ⁇ orate into the mini-Ad vector of the present invention DNA sequences derived from the normal cellular genome or equivalent sequences that will facilitate DNA replication of the mini-Ad genome in the target cell.
  • alphoid DNA One such sequence that facilitates DNA replication is alphoid DNA.
  • a 16.2 kb sequence of alphoid DNA repeats allows DNA replication but not segregation of the DNA as an artificial chromosome.
  • the present invention provides for the inco ⁇ oration of the 16.2 kb sequence (70-72) into the mini- Ad vector. Replication of the mini-Ad vector containing these sequences therefore extends the persistence of the mini-Ad vector DNA and expression of the gene of interest within the target cell.
  • Animal model test systems for evaluating the modified vectors of the present invention are also provided.
  • Animal models provided by the present invention include: 1.) a transgenic mouse comprising the AAVS1 sequence inco ⁇ orated into its genome for evaluating AAV-based integration mechanisms; and, 2.) a non-human transgenic animal comprising the human FVLTI gene operably linked to a developmentally-regulated promoter inserted into its genome.
  • transient expression of human FVIII in the transgenic non-human animal during development results in tolerization of the animal to human FVIII.
  • the human FVIII gene of the animal is not expressed once the animal matures and, therefore, evaluation of delivery of the human FVLTI gene to the animals is not complicated by hFVTTI expression from the transgene or by immune responses directed to hFVLTI. In this manner, then, the efficiency of gene delivery by a vector may be assessed without the added potential complication provided by an anti- hFVITI immune response by the animal.
  • the techniques utilized may be found in any of several well-known references including: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991.
  • a transcriptional regulatory region or transcriptional control region is defined as any nucleic acid element involved in regulating transcription of a gene, including but not limited to promoters, enhancers, silencers and repressors.
  • a DNA fragment is defined as segment of a single- or double-stranded DNA derived from any source.
  • a DNA construct is defined a plasmid, virus, autonomously replicating sequence, phage or linear segment of a single- or double-stranded DNA or RNA derived from any source.
  • a reporter construct is defined as a subchromosomal and purified DNA molecule comprising a gene encoding an assayable product.
  • An assavable product includes any product encoded by a gene that is detectable using an assay. Furthermore, the detection and quantitation of the assayable product is anticipated to be directly proportional to the level of expression of the gene.
  • a gene expressed in a tissue-specific manner is that which demonstrates a greater amount of expression in one tissue as opposed to one or more second tissues in an organism.
  • An effector gene is defined as any gene that, upon expression of the polypeptide encoded by the gene, confers an effect on an organism, tissue or cell.
  • Heterologous DNA is defined as DNA introduced into an adenoviral construct that was isolated from a source other than an adenoviral genome.
  • a transgene is defined as a gene that has been inserted into the genome of an organism other than that normally present in the genome of the organism.
  • a recombinant adenoviral vector is defined as a adenovirus having at least one segment of heterologous DNA included in its genome.
  • Adenoviral particle is defined as an infectious adenovirus, including both wild type or recombinant.
  • the adenovirus includes but is not limited to a DNA molecule encapsidated by a protein coat encoded within an adenoviral genome.
  • a recombinant adenoviral particle is defined as an infectious adenovirus having at least one portion of its genome derived from at least one other source, including both adenoviral genetic material as well as genetic material other than adenoviral genetic material.
  • Stable gene expression is defined as gene expression that may be consistently detected in a host for at least a period of time greater than seven days.
  • a treatable condition is defined as a condition of an organism that may be altered by administration of a form of treatment including but not limited to those treatments commonly defined as being of medicinal origin.
  • An antigen is defined as a molecule to which an antibody binds and may further include any molecule capable of stimulating an immune response, including both activation and repression or suppression of an immune response.
  • a tumor suppressor gene is defined as a gene that, upon expression of its protein product, serves to suppress the development of a tumor including but not limited to growth suppression or induction of cell death.
  • a growth suppressor gene is defined as a gene that, upon expression of its protein product, serves to suppress the growth of a cell.
  • An oncogene is defined as a cancer-causing gene.
  • An immunomodulatorv gene is defined as any gene that, upon expression of its nucleic acid or protein product, serves to alter an immune reaction.
  • a ribozvme is defined as an RNA molecule that has the ability to degrade other nucleic acid molecules.
  • a genetic condition is defined in this application as a condition of an organism that is a at least partially the result of expression of at least one specific gene including but not limited to the wild-type form of that gene and any mutant form of that gene.
  • An expression cassette is a DNA fragment comprising a coding sequence for a reporter or effector gene operably linked to a transcriptional regulatory region or a transcriptional control region sufficient for expression of the encoded protein in an appropriate cell type.
  • the mini-Ad vector system consists of three major parts: 1.) a packaging-attenuated helper Ad; 2.) a cognate Ad vector having a minimal amount of the viral genome; and, 3.) Ad helper cell lines that provide functions of El /r ⁇ ns-activation like 293 cells and/or regulation of packaging signal for the helper Ad.
  • the packaging-attenuated helper Ad comprises the viral genetic material required for self-replication as well as tr ⁇ /w-complementation of mini-Ad vector replication.
  • the helper Ad retains wild-type Ad genetic material except for an El deletion or substitution and a manipulated packaging signal useful in controlling or discriminating against packaging of the helper Ad in favor of packaging a mini- Ad vector of the present invention.
  • the mini-Ad vector comprises minimal Ad genetic material including only the inverted terminal repeats (ITRs) and a wild-type packaging signal as cw-elements that serve to promote replication and packaging of the mini- Ad vector.
  • the remainder of the mini-Ad vector comprises transgene or heterologous DNA.
  • the Ad helper cell lines of the present invention are similar to 293 cells (ATCC# CRL1573) in that the cell lines comprise the Ad El genes and provide Ad El gene products that support replication of the helper Ad.
  • the cell lines may further comprise a control mechanism for attenuating packaging of the helper Ad ( Figure 1).
  • the packaging protein of Ad is a trans- acting factor present in low amounts in an infected cell and serves as the rate-limiting factor in the packaging of Ad.
  • the wild-type packaging signal possessed by the mini ⁇ Ad vector of the present invention
  • packaging of the helper Ad genetic material is partially or completely suppressed in the presence of the mini-Ad vector. This results in preferential packaging of the mini-Ad vector.
  • the proteins for viral DNA replication and those for capsid assembly must be provided in adequate amounts.
  • the proteins may be provided from several different sources, including but not limited to a plasmid, a cell line, or a virus.
  • the proteins are provided by the helper Ad.
  • the present invention allows for the helper Ad to remain fully functional in replicating itself within a helper cell such that large quantities of Ad structural proteins are available to the mini- Ad vector.
  • the helper Ad is packaged, albeit slowly or ineffectively.
  • Viral DNA replication proteins are also required to amplify the mini-Ad vector DNA for generation of multiple copies of the mini-Ad vector.
  • the replication proteins may be provided from any of several different sources, including but not limited to a plasmid, a cell line, or a virus.
  • the proteins are provided by the helper Ad.
  • the mini-Ad vector comprising the wild-type packaging signal, is packaged into Ad virions as infective, replication-competent Ad particles.
  • helper Ad DNA is competed off by poor recognition or low affinity of the packaging protein for the manipulated packaging signal, and thus remains completely or partially free within the helper cells.
  • the mini-Ad vector produced using this system may be contaminated by low amounts of helper Ad, thus the mini- Ad particle preparation may not be 100% pure. If necessary, the contaminating helper Ad may be removed using biological, biochemical, or physical methods including but not limited to ultracentrifugation through a CsCl gradient.
  • mini- Ad vector system of the present invention makes it unique, sophisticated, and significantly advanced over Ad vectors that are currently available to one skilled in the art ( Figure 2). These features include but are not limited to the following: 1.) the mini- Ad vector exhibit minimal immunogenicity; 2.) the mini-Ad vector is virtually incapable of generating replication competent adenovirus (RCA); and, 3.) the mini- Ad vector may comprise much larger segments of heterologous DNA than conventional Ad vectors. Reduced immunogenecity and RCA generation (a major safety concern in the field of gene therapy) is possible because the mini- Ad vectors carry only a minimal amount of viral cw-element (ITRs and packaging signal), and as such, do not encode Ad proteins.
  • ITRs and packaging signal a minimal amount of viral cw-element
  • the mini-Ad vector of the present invention further provides increased capacity for heterologous DNA than convention Ad vectors.
  • Wild-type Ad has an average genome size of 36 kb.
  • the maximal packaging capacity of Ad is roughly 105% of the genome, i.e. approximately 38 kb.
  • the mini-Ad vector of the present invention may comprise less than 1 kb of Ad genetic material; therefore, the capacity of the mini-Ad vector for heterologous DNA may be 37 kb.
  • the heterologous DNA may include but is not limited to a transgene expression cassette, a regulatory element, or a transcriptional control region operatively linked to a reporter or effector gene.
  • the expression cassette may include but is not limited to single or multiple expression cassettes.
  • the regulatory element may include but is not limited to a DNA sequence for controlling transgene retention, integration, transcription, and / or vector targeting.
  • the Packaging- Attenuated Helper Ad a.
  • the prototype structure of the helper The helper Ad vector comprises a wild- type Ad genome having a manipulated packaging signal and an altered El gene.
  • the helper Ad must be defective in replication, such as the currently available El -deleted or substituted viral constructs.
  • the helper For the pu ⁇ ose of controlling packaging in the presence of the mini-Ad vector, the helper must be also defective in packaging (detailed below). Therefore, the general structure of the helper can be summarized as an Ad vector having a wild-type genome except that the El region and packaging signal are manipulated. However, the other essential regulatory genes of Ad such as E2 and E4 may also be manipulated.
  • the viral genome may be split into fragments in order to further disable the replication competence of the helper Ad or to reduce the genome size of the helper Ad in order to separate it from the mini-Ad vector using a biological, biochemical, or physical method including but not limited to ultracentrifugation through a CsCl gradient.
  • a biological, biochemical, or physical method including but not limited to ultracentrifugation through a CsCl gradient.
  • both a defect in viral replication and attenuation in packaging of the helper Ad may be included in the design of the helper Ad.
  • the general function of the helper Ad The primary function of the helper Ad is to supply the capsid proteins required to package the mini-Ad vector.
  • the helper Ad In order to provide the proteins, the helper Ad must be able to replicate within the host cell, although less efficiently than wild-type Ad. Preferably, DNA replication and transcription of the helper genome is not affected. If synthesis of the helper Ad genome were inhibited, the yield of the late gene products (the capsid proteins) would be altered and may adversely affect the titer of the mini-Ad vector (i.e., the titer will be reduced). For certain applications, removal of the helper Ad from the mini-Ad may not be necessary. In such situations, the stringency of packaging attenuation of the helper Ad may be greatly reduced. c.
  • the pmpose for attenuation of packaging the helper Ad is to reduce the potential for helper Ad contamination in preparations of the mini-Ad vector. This is especially important when a relatively pure batch of the mini- Ad vector is required for a particular application.
  • the packaging function of the helper Ad is designed to be defective but not completely disabled, because the helper Ad must be able to propagate, albeit slowly, in the absence of a mini-Ad vector.
  • the following genetic manipulations may be utilized to generate a packaging-attenuated helper Ad.
  • the Ad5 packaging signal is composed of a repeated element that is functionally redundant (18). Partial deletions of the packaging signal elements have been shown to reduce the yield of mutant Ad from several fold to approximately a hundred fold as compared to that of Ad having a wild-type packaging signal (18).
  • the design of the packaging signal mutation of the present invention may therefore inco ⁇ orate a partial deletion of the consensus adenosine-enriched motif (e.g. "A-repeat”: TAAATTTG; Fig. 3) from the wild-type Ad packaging signal.
  • the design of the packaging signal mutation of the present invention may therefore inco ⁇ orate a partial deletion of the consensus adenosine-enriched motif (e.g. "A-repeat": TAAATTTG; Fig. 3) from the wild-type Ad packaging signal.
  • Ad packaging signal is a specific DNA sequence that is recognized and bound by the packaging proteins. In order to interfere with the effective binding of the packaging proteins to the signal, other DNA sequences may be placed in proximity to or within the A-repeat array of the helper Ad packaging signal. The inserted DNA sequences allow binding by their cognate DNA binding proteins that may positionally compete off the binding of the Ad packaging proteins to the Ad packaging signal. 4.
  • the wild-type Ad packaging signal is positioned at the left end of the wild-type Ad genome. Investigators have found that the packaging signal may be located at the right end and retain its function (75) indicating that the packaging signal may be relocated. Positioning the manipulated packaging signal in a location other than wild-type may be useful to further attenuate the packaging efficiency of the helper Ad. In addition, relocation of the packaging signal to another region of the Ad genome may be helpful in minimizing the possibility of reversion of the helper Ad back to wild-type Ad through homologous recombination between the engineered packaging signal of the helper Ad and the wild-type packaging signal of the mini-Ad vectors (i.e., generation of RCA).
  • cw-elements and /r ⁇ ns-acting factors. Therefore, other possible designs may be oriented towards manipulation of either or both of these two factors.
  • An example of cw-elements that may be manipulated is the A-repeat motif.
  • An example of a trans ⁇ acting factor that may be mampulated is a packaging protein. Further consideration should be a controllable mechanism of packaging without sacrificing the high titer ou ⁇ ut of the mini-Ad vectors by the system.
  • the basic structure of the mini-Ad vector Ad vectors may be utilized as circularized plasmids by fusion of the Ad ITRs (54).
  • the simplest plasmid form of the mini-Ad vector of the present invention is a circular DNA molecule comprising an ITR fusion sequence (comprising an Ad ITR having a wild-type packaging signal), a plasmid DNA replication origin, and a polycloning site consisting of one or multiple restriction enzyme sites.
  • the ITR fusion sequence includes the left end of the wild-type Ad, preferably from map unit 0 to 1, and the right end, preferably from map unit 99 to 100.
  • An Ad DNA replication origin is located in each ITR and the wild-type packaging signal is located adjacent to the left ITR.
  • mini- Ad vectors Other DNA sequences and elements including but not limited to those listed below and illustrated in Figure 7B may be included in a mini- Ad vector.
  • An expression cassette is a basic transcription unit.
  • a simple expression cassette of a given gene generally comprises a transcriptional control region, a gene of interest (i.e., heterologous DNA, insert DNA), and a polyadenylation (polyA) signal.
  • polyA polyadenylation
  • two or more genes may be included as bi- or polycistronic units, as long as additional elements for translation or splicing of RNA are provided between the genes.
  • mini-Ad vectors comprise one or multiple expression cassettes.
  • AAV integration elements functional elements for vector DNA retention Elements that may assist in integration of the expression cassette into target cell genome (i.e., AAV integration elements) or maintain the mini-Ad vector as an episomal form in a host cell. Elements that have been shown to assist in integration are the inverted terminal repeats (ITRs) and the Rep78/68 proteins of the adeno-associated virus (AAV). AAV utilizes these elements to achieve specific integration of its genome in human chromosome 19 (19ql3.3-qter) at a site named AAVS 1. Although AAV has been considered as a candidate vector for gene therapy, several limitations have been identified by investigators.
  • AAV is limited by: 1.) low capacity for exogenous DNA (4.3 kb); 2.) difficulty in achieving high titers in large-scale preparations; and, 3.) loss of specific integration of the recombinant AAV.
  • the present invention combines the advantages of the mini-Ad vector with the integration capacity of AAV by inco ⁇ orating the AAV-ITR sequences and Rep 78/68 expression cassette (Rep expression cassette) into the vector.
  • extrachromosomal replication sequences Such sequences, comprised of either chromosomal or viral sequences, serve to enable the vector to efficiently replicate and be retained within a mammalian cell.
  • the sequences may include a replication component such as human genomic DNA and / or a retention component such as human centromere sequence or sequence derived from the Epstein-Barr virus (EBV) such as the oriP family of repeats and / or EBNA-1 (70).
  • the human human genomic DNA may comprise a telomere and / or alphoid DNA (70).
  • the mini-Ad genome will replicate to a higher copy number in the host cell, thus increasing the probability that the mini-Ad genome will be packaged at a greater effiency than that helper virus. Additionally, these sequences serve to lengthen the duration of expression of the effector or reporter gene within the host cell. Such functions would be useful in utilization of the mini- Ad vector for gene therapy. 3. Regulatory elements for control of DNA transcription Elements having transcriptional regulatory function including but not limited to enhancers, repressors, activator-binding sites, introns, and 5' or 3 '-untranslated regions.
  • Elements for vector and transgene targeting Targeting can be achieved by several methods including but not limited to vector surface modification and tissue-specific expression.
  • Tissue specific promoters may be utilized to drive gene expression in a specific cell type or tissue.
  • High-titer production of the mini-Ad vectors is another major aspect of this invention.
  • One advantage of Ad vectors over other viral vectors is that Ad particles are conducive to preparation of high- titer preparation stocks (67). High-titer propagation of Ad is possible due mainly to the large quantity of viral capsid proteins and viral genome copies produced wtihin a host cell such as a 293 cell during infection. Listed below are some of the factors that may be considered in designing methods for generating high-titer mini- Ad vectors.
  • E2 region proteins are the major trans-acting elements responsible for viral DNA replication.
  • the replication origins are the cw-elements located at the either or both ends of the viral genome.
  • a sufficient quantity of E2 proteins must be provided by the helper virus.
  • High-level expression of E2 proteins (encoded within the E2 region of Ad) is ensured by proper design of the helper virus genome.
  • Other such mechanisms for increase in copy numbers of the mini ⁇ Ad genome may also be considered. Such mechanisms may include but are not limited to insertion of the the SV40 origin of DNA replication (54) into the mini-Ad genome to increase the copy numbers of the mini-Ad, concomitant with SV40 T-Ag expression in the helper cell.
  • Enhanced packaein ⁇ siznal A higher number or more efficient packaging sequences may be utilized by, for example, inco ⁇ orating a greater number of tandem repeats at one or both ends of the mini-Ad genome, or by inco ⁇ oration of one or multiple synthetic packaging signals that function in a more efficient manner than the wild-type packaging signal.
  • the cell line of the present invention (that serves as the host cell) provides several important modifications that improve upon the conventionally utilized cell line, 293 (ATCC# CRL1573).
  • the host cell comprises a nucleic acid sequence encoding an Ad-El fragment for trans-activation of the transcription program of the helper Ad genome ( Figure 1).
  • a cell line of present invention may comprise nucleic acid sequence encoding the El fragment having no overlapping nucleic acid sequence with the helper Ad genome.
  • the present invention therefore, eliminates one of the current difficulties associated with Ad vectors: generation of wild-type Ad or replication- competent Ad (RCA) through homologous recombination.
  • Other elements may include but are not limited to genes involved in the support of high copy-number production of the mini- Ad vector, enhancing packaging of the mini-Ad vector, and / or attenuating the packaging of the helper Ad.
  • Assistance mechanisms for packaging attenuation of the helper Ad may include interference with the binding site for the packaging protein by placement of a binding site for a different protein nearby the packaging protein binding site within the helper Ad genome.
  • Such a system may include but is not limited to utilization of the tetracycline-repressor (Tet-R), a recombinase, and / or an altered packaging protein.
  • Tet-R tetracycline-repressor
  • the different protein is expressed within a host cell.
  • Tet-R may bind to a manipulated packaging signal of a helper virus comprising a binding site for Tet-R, the tet-operon (Tet-O), and thereby repress packaging by inhibiting binding of the packaging protein. Binding of Tet-R to Tet-O is controlled by tetracycline. Addition of tetracycline into the cell culture medium results in binding of tetracycline to the Tet-R and prevents it from binding tet-O. Removal of the tetracycline frees Tet-R for binding to the engineered packaging signal and serves to further attenuate packaging of the helper virus.
  • a recombinase such as Cre or Flp may also inhibit packaging provided the packaging signal of the helper virus is flanked by a recombination site, such as lox-p or FRP, respectively (66, 68).
  • Other genetic modifications within the helper virus genome may also be provided separately or in addition to those listed above to further attenuate helper virus replication.
  • the packaging protein may be altered by any of several methods including but not limited to utilization of a specific serotype or species difference in the packaging signal to differentiate packaging of the mini-Ad from the helper Ad provided the specific packaging protein of Ad is identified. Additionally, the packaging protein may be altered by genetic modification of the gene encoding the packaging protein. The modification may alter the packaging protein such that its binding preference for the wild-type packaging signal is increased. The modified packaging protein, then, may further provide preferred packaging of the mini- Ad genome.
  • Ad vectors demonstrate high levels of infectivity in cultured tumor cells and different types of solid tumor models in vivo. This characteristic of the Ad vector has been utilized in the treatment of cancer. The efficacy of treatment depends upon the genes that are delivered by the vectors. Multiple genes including but not limited to those having combined functions of tumor suppression and immunomodulation are utilized to optimize the anti-cancer effect.
  • the mini-Ad vector has the capacity to deliver multiple genes and is useful in constructing anti-cancer Ad vectors for intratumoral injection.
  • Modulation of host immunity bv genetic modification of the graft cells or tissues Transplantation requires transient or permanent suppression of the host immunity.
  • immune suppression genes into cells or tissues including but not limited to graft cells or graft tissues may be an alternative approach to the administration of immunosuppressive agents.
  • genes encoding immune suppression proteins to be utilized in the present invention may include but are not limited to TGFb, IL-10, viral proteins HSV-ICP47 and CMV-US11, and secretable Fas-ligand proteins that may be delivered alone or in combination by the mini -Ad vectors of the present invention.
  • Ad vectors have a distinct advantage over other viral vectors in that production of high titer stocks is possible, which is useful for in vivo gene therapy.
  • mini-Ad vectors contain only minimal amounts of cis-elements of the Ad genome, the immunogenicity of mini- Ad is minimized. Therefore, the mini- Ad vector will be useful for modifying target cell function or regulating target cell growth in vivo by genetic modification. e. Specific delivery of transgenes to target cells or tissues in vivo bv surface modification of the vectors
  • the genes encoding the adenoviral hexon and fiber proteins are engineered to fuse with certain epitopes or ligands (e.g. the protein A that binds to Fc fragment of IgG) present on the target cell surface.
  • modified genes are inco ⁇ orated into the recombinant viral genome for generation of the viruses having surface sites that interact with ligands that function as targeting agents on the target cell surface.
  • the viral particles thus produced have tissue or cell recognition capabilities.
  • the immunogenicity of the El -substituted Ad vectors may provide benefits, and has been used in development of Ad-based recombinant vaccines.
  • Mini-Ad vectors utilized in this type of application use the helper virus including but not limited to El -substituted Ad vectors as well as co-delivery of genes encoding antigens and immunogens that provide immunization.
  • helper virus including but not limited to El -substituted Ad vectors as well as co-delivery of genes encoding antigens and immunogens that provide immunization.
  • mini-Ad vector The characterization of the replication, packaging, and propagation efficiency of the mini-Ad will provide the field with important new information, which was previously unavailable.
  • Ad vectors have been used together with polylysine, liposome, and other conjugation materials as a gene delivery complex.
  • the mini-Ad vectors can also be used with these compounds as well as any other copound that comprise the ability to serve as a gene delivery complex.
  • j. To be used for other pu ⁇ oses in the field of gene transfer and therapy
  • the mini -Ad vector system has a great potential to be used for gene transfer and therapy in addition to what have been discussed above. The possibilities will come across along the further development of the field of gene transfer and therapy.
  • the present invention includes a methodology for directing gene expression to the liver using a transcriptional regulatory region, or promoter, capable of driving expression of a reporter or effector gene in liver tissue in combination with a modified and much improved adenoviral vector.
  • a transcriptional regulatory region, or promoter capable of driving expression of a reporter or effector gene in liver tissue in combination with a modified and much improved adenoviral vector.
  • One skilled in the art can envision a multitude of diseases caused by abnormal gene or gene product expression of a gene in the liver.
  • Abnormal gene or gene product expression may include a level of expression above or below that normally found in the liver and may be the result of a gene deletion, duplication, insertion, or alteration of the structure or function of the gene trasncript, or other alteration of either the gene itself or its protein product. Abnormal gene or gene product expression may also result from alteration of the transcriptional or translational machinery regulating expression of the gene and gene product, respectively.
  • a vector for delivery of a therapeutic or reporter gene to the liver comprising the significant advantages of the present invention. It will be understood by those skilled in the art that the present invention could be utilized to treat a multitude of diseases based on a defect in either gene or protein expression in the liver. Examples of diseases and genes that may be treated or utilized, respectively, using the present invention are summarized in Table 1. Additionally, the promoter of any of these genes may prove useful in driving gene expression in the liver for the pu ⁇ ose of driving expression of a gene or gene product in the liver. W
  • Apo A-l structural 1 1.000 Apo A-l In a few cases, causes decreased Dietarv treatment HMG-CoA mu ⁇ oons HDL-C levels with no increase in reducuse inhibitors coronary heart disease Two mutations lead to amvloidosis
  • Fanmhal type ill 1 1.000 - 5.000 Apolipoprotein E Accumulation in plasma of Dietary treatment.
  • HMG-CoA hypercholesterolcrraa popula ⁇ ons mediated endocytosis of LDL reductase inhibitors: nicot ⁇ uc causes LDL to accumulate in acid plus bile acid-bmdtng plasma Hypcr-cholesterolcmia and resins. For homozygous, may atherosclerosis result need probuco). portacaval anastomosis, plasma exchange.
  • Factor VIII deficiency 1 10.000 males Factor VIII Factor VIII fails to funcnonaa a Prophylaxis, plasma infusion, (hemophilia A) cofactor for activation ⁇ f factor X and Factor VIII treatment and impairs clotrmi cascade
  • Factor XI deficiency -1 1.000 m Faxt ⁇ r XI Deficiency of protein leads to Prophylaxis, plasma infusion. (hemophilia C) Ashkenazi Jews of impaired contact acnva ⁇ n and and Factor XI treatment Israel mild bleedmc tendency
  • Protein C deficiency I 10,000 protein C Impaired regulanon of blood Long-term anticoagulanon coagulation Predisposinonto therapy, plasma or protein C thrombosis infusion Table 1. (Continued)
  • C2 deficiency -1 10.000
  • Complement component 2 Markedly reduced aenvanon of the Replacement therapy classic pathway.
  • Phenylketonu ⁇ a (PKU) -1.10.000 births Phenylalanine Hepatic enzyme deficiency causes Low-phenylalan ⁇ ne-d ⁇ e ⁇ therapy due to PAH deficiency (considerable hydroxylase (PAH) hy ⁇ ei plieuy lalamnemia; plasma and enzyme (bactcrail regional variation) values persistently above I mM phcnlatanine ammonia lyase) associated with impaired cogmnve treatment- development. Risk of maternal hyperphenyl-alaninetma effect on fetus earned bv female proband
  • Hereditary fructose 1.20.000/Sw ⁇ tzer ⁇ an Fructose 1.6-b ⁇ sphospha ⁇ e Inges ⁇ on of fructose causes the Eliminanon from the diet of all intolerance d aldolase B accumulanon of fructose I- sources of sucrose and fructose, phosphate and hence multiple with supplement of vitamin C dysfuncnons tn small tntesttng, liver, and kidney
  • Glycogen storage disease -I I 00,000 Glucose 6-phosphatase Hypoglycerraa. hyperiipidema, Dietary reitncnon; nocrumal type la (von Gterke hyperuncemia. and hyperlacDc nasogastnc infusion in early disease) aciderma. Glycogen accumulanon infancy: ponacaval shunts, liver in liver and kidney transplantation
  • Glycogen storage disease -1.125,000 Amvlo- 1 , 6-glucos ⁇ dase A glycogen with shorter outer Dietary restncnon «yp « Ul (debrancher enzyme) chains (limit dcxt ⁇ n) in liver and/or muscle. Moderate hypoglycerraa and hypcrlipidemia. Muscle weakness mostly in adults
  • Presymptomatic copper-containing enzymes and treatment with copper causes arterial and brain hisndmate can modify the degeneration disease substannallv
  • tnentene or [membrane canon (Cu") copper leads to accumulanon in orraly admmtstered zinc salts lever (cirrhosis), cornea (Kayser- Liver tnutsplanianon may be Fletseher ⁇ ngs). and basal ganglia applied for irreversible hver ( movement disorder) damage
  • Carbar ⁇ yl phosphate 1 70.000 • 100.000 Carbamyl phosphate Impaired urea formation leads to Dietary rest ⁇ cnon . sodium synthetase deficiency svntheaae I ammonia intoxication phenylburyrate. and ei ⁇ ulhne
  • Argimnosuccintc acid 1.70,000 - 100,000 Arg ⁇ unosuccinic acid Impaired urea formaoon leads Dietary restncnon and argtnme synthetase deficiency synthetase tooammonia intoxication
  • Methylmalonic scidemia 1.20.000 Methyldmalonyl-CoA Accumulanon of methylmatonatc Dietary protein resmcoon and (2 allelic variants mutase (MITT) leads to metabolic ketoacidosis and oral annbionc therapy designated mur* and apoenzyme developmenul retardation mu-)
  • Table 1 is composed of the data and information from Ref. No.55.
  • compositions comprising a mini-Ad vector of the present invention.
  • the pharmaceutical compositions may be made up in a solid form (including granules, powders or suppositories) or in a liquid form (e.g., solutions, suspensions, or emulsions).
  • Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and
  • Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water. Such compositions may also comprise adjuvants, such as wetting sweetening, flavoring, and perfuming agents.
  • the compounds of the present invention can be used in the form of salts derived from inorganic or organic
  • vectors of the invention can be administered as the sole active pharmaceutical composition, they can also be used in combination with one or more vectors of the invention or other agents.
  • the therapeutic agents can be formulated as separate compositions that are given at the same time or different times, or the therapeutic agents can be given as a single composition.
  • the essential ci ' s-acting elements for Ad DNA replication and packaging are located at the ends of the viral genome (ITRs plus the packaging signal, less than 1 kb), the backbone of the mini-Ad vector has been modified to comprise only the essential cis- elements.
  • the remainder of the mini-Ad genome, up to its packaging limit of approximately 38 kb, may be comprised of heterologous DNA.
  • the heterologous DNA comprises a nucleic acid sequence encoding a protein having activity similar to that of human FVIII.
  • FVIII is normally produced in the liver and is comprised of heavy chain polypeptides with a range of apparent molecular weights of from 92 kDa to 210 kDa derived from the amino terminus of the nascent polypeptide and a C-terminal light chain of 80 kDa (53).
  • the activated form functions in the blood clotting cascade as a cofactor along with activated factor IX (FLXa), negatively charged phospholipids and calcium ions to convert factor X to its activated form, Xa.
  • the human cDNA is 9 kb in length and encodes a polypeptide of 2351 amino acids comprised of several domains in the order Al, A2, B, A3, CI and C2 (5-7).
  • the A and C domains are critical for functional activity whereas the majority of the B domain, consisting of approximately 980 amino acids, is dispensable for activity (8).
  • the present invention provides a mini-Ad vector comprising the human FVIII gene. It should be understood by those skilled in the art that a mini-Ad vector comprising genetic material that encodes a protein having FVIII-like activity (i.e., the ability to function as a cofactor in the conversion of factor X to its activated form, Xa) is encompassed by the present invention.
  • the FVIII mini- Ad of the present invention comprises the human FVIII cDNA as well as DNA elements (i.e., homologous recombination arms, AAV/ITR sequences, and a transcriptional control region) that support gene integration into a host cell genome and expression of human FVIII within a host cell.
  • the viral proteins required for the DNA replication and encapsidation of the FVIII mini- Ad vector are provided in trans from a helper Ad (trans-complementation).
  • helper Ad trans-complementation
  • packaging of the helper Ad genome is attenuated by modification of its packaging signal. This allows for preferential packaging of the FVIII mini-Ad vector genome in the helper cell line.
  • the FVIII mini-Ad comprises a site-specific integration mechanism.
  • the mechanism may comprise a homologous recombination sequence or an AAV/ITR targeted to a human integration sequence (AAVS1 site).
  • AAVS1 site In order to test the integration mechanism, the AAVS 1 site must be transferred into the mouse genome.
  • transgenic technology such as embryonic stem cell transformation or by direct DNA injection of a transgene comprising the AAVS1 site into the male pronucleus of a mouse single-cell ova (57-60).
  • a transgenic mouse developed by such methodology may be utilized to test the integration efficiency and specificity of the mini-Ad vector.
  • a further embodiment of the present invention addresses the potential anti-human FVIII immune response that may occur within a host animal. Such an immune response may interfere with utilization of the mini-Ad vector or analysis of the efficiency of the vector. Since the mini-Ad will deliver and drive expression of human FVi ⁇ within a mouse, an immune response of the treated mouse may complicate assessment of the duration and level of the FVIII expression. For these and other reasons, an FVIfl- deficient transgenic mouse model is provided by the present invention. It is to be understood that the present invention comprises a non-human transgenic animal into which has been transferred a reporter or effector gene of a mini-Ad vector such that the animal is tolerized to the gene product of the reporter or effector gene.
  • transgenic mouse models are provided by the present invention and generated through microinjection of genetically-modified embryonic stem (ES) cells.
  • One embodiment of the present invention provides a non- human transgenic animal inco ⁇ orating a double stranded human FVIII DNA sequence operably linked to developmentally regulated cw-acting control elements to allow transient gene expression.
  • the promoter may be derived from any developmentally regulated genetic unit capable of directing the expression of the human FVIII gene sequence transiently during development such that tolerization to the expressed exogenous protein occurs. Following tolerization and maturation of the animal, the FVIII transgene is no longer expressed.
  • the ⁇ -fetoprotein promoter was operably linked to the human FVIII cDNA and utilized to generate a transgenic mouse harboring the human FVIII cDNA within its genome.
  • Such mice express hFVIII in a developmentally regulated manner, and as such, are tolerized to human FVIII.
  • Another model comprises a transgenic animal containing the double stranded AAVS1 sequence which is the target for site specific integration of adeno-associated virus (AAV) gene therapy vectors.
  • AAV adeno-associated virus
  • Example 1 Construction and Characterization of The Packaging-Signal Mutated Helper Ad and Mini-Ad Vectors That Carry Green Fluorescence Protein (GFP) Reporter Gene
  • Mutant dllO/28 (also described as dl309-194/243:274/358) contains a deletion between nt 194 to 243 and between 274 to 358 of Ad5.
  • dllO/28 virus was generated by the method of Stow (89) by ligation of a plasmid containing the left end of Ad5 with this double mutation (pElA-10/28) and the rest of Ad5 genome (90).
  • dllO/28 showed a 143-fold decrease in virus yield in a single virus infection and, when co-infected with wild type virus, was not detected.
  • helper virus containing the same mutation as dl 10/28 we should be able to amplify the virus, although at low yields, and in the presence of mini-viral vector containing the wild type packaging signal the helper virus should remain unpacked.
  • the packaging signal was amplified by PCR from pElA-10/28 using the following primers:
  • R7 5'- GGAACACATGIAAGCGACGG
  • GT4004 derives from pXCX2 (91) by extending the Ad5 left region from Xhol site (nt 5792, 16 mu) until SnaBI site (nt 10307, 28 mu), therefore GT4004 contains the left end of Ad5 from 0 mu to 1.2 mu with the Afl III site at 0.38 mu, an El deletion from 1.2 mu to 9.2 mu with Cla I site in this deletion point and the rest of the left arm of Ad5 until 28 mu.
  • GT4004 with the wild type packaging signal substituted by the deleted one was named as GT5000.
  • the ⁇ -gal expression cassette from pTk ⁇ (Clontech, Ca.) was cut as a Sail fragment, blunted with Klenow enzyme and inserted into the blunted Clal site of GT5000.
  • the resulting plasmid, GT5001 contains therefore the double-deleted packaging signal and the El region of Ad5 replaced by the ⁇ -gal gene driven by the Tk promoter (Fig. 4).
  • This construct allows for detection of helper virus by X-gal staining.
  • Graham and Prevec was used (91) (Fig. 5).
  • DNase I was inactivated and viral capsids were opened by adding: 32 ml EDTA (0.25 M), EGTA (0.25 M), 10 ml SDS (20%), 5 ml Proteinase K (16 mg/ml) and incubating at 56°C for 2h. After one phenol:chlorophorm:isoamyl alcohol (1:1 :1/24) extraction, 1 ml of yeast tRNA (lOmg/ml) was added to help precipitation of viral DNA which was collected by centrifugation at 12000 ⁇ m in a microcentrifuge and resuspended in 20 ml of H 2 O. 5 ml were used for a PCR reaction with primers R7 and R8.
  • AdHelper- ⁇ gal or AdH ⁇ The virus was extracted at 48 h post-infection by centrifugation of the collected cells at 800 g for 5 min and three cycles of quick freeze and thaw of the cell pellet. This crude extract from X cells was used to infect 3X cells (amplification scale was 1 to 3 in contrast to 1 to 20 for a virus with wild type packaging signal) ( Figure 6). At every passage the deleted size of the packaging signal was verified by PCR of the supernatant (Fig.7). This deletion and the ⁇ -gal expression were stable in all the passages analyzed.
  • AdH ⁇ was purified by CsCl. Purification was done by three cycles of freeze-thawing, layering the lysate onto a step gradient of 0.5 ml CsCl 1.5 mg/ml + 2.5 ml CsCl 1.35 mg/ml + 2.5 ml CsCl 1.25 mg/ml, and centrifuging in a SW41 Beckman rotor at 10°C, 35000 ⁇ m, lh. The collected virus band was mixed with CsCl 1.35 mg/ml and centrifuged for 18 h as before. The virus band was dialyzed twice against PBS and once against PBS- 10% glycerol, and stored at -80 °C.
  • the virus- containing solution was serially diluted in D-MEM 10% FBS (1: 10 dilution until 10 "12 ) and used to infect 293 cells at 90% confluence (0.5 ml/well in 6 well-plates). After 1 h infection at 37 °C, the viral suspension was replaced by fresh medium. The next day, cells were overlaid with medium containing 0.5% agarose, 0.025% yeast extract and 5 mM Hepes pH 7.4. Plaques were counted after 6 to 10 days.
  • PFU plaque forming units
  • the titer obtained after amplification and purification of AdH ⁇ was about 10 9 PFU/ml (virus purified from 20 plates of 150 mm 2 and resuspended in a final volume of 1 ml). This titer is about lOOx lower than that obtained with a similar viral vector containing the wt packaging signal.
  • fhe plasmids for the mini-viral vectors. It has been shown that the linear adenovirus DNA, when covalently circularized head-to tail by its terminal ITRs can be grown as a plasmid in bacteria but it will replicate and produce virus when transferred into permissive human cells (92). Functional junctions have been naturally selected by transforming bacteria with circular DNA extracted from infected cells. Small deletions in the joints were observed which /US97/1 218
  • the basic minivirus structure is therefore a plasmid that contains the left end of Ad5 (including the 103 nt-ITR and the packaging signal until nt 358) fused to the right end of Ad5 (at least including the 103 nt-ITR).
  • the initial approach used to test the mini-viral vector system included the generation of progressive deletions in plasmid pJM17 that contains a functional ITR fusion.
  • pJM17 is a plasmid that contains the entire genome of Ad5 as a DNA molecule circularized at the ITR sequences and a pBR322 derivative, pBRX, inserted in El A (providing the bacterial replication origin and ampicilin and tetracycline resistant genes) (93).
  • pJM17 replicates but is not packaged because is too large (40.3 kb) to be packaged into the adenovirus capsid (maximum is 38 kb).
  • FIG. 5 Examples of various mini- viral vectors demonstrated in the current literature as well as that of the present invention are illustrated in Figure 5.
  • pJM17 was cut with Ascl and religated obtaining pBRX-AscI. This removed from mu 43.5 to 70.2 of Ad5 which completely deletes E2A (DNA binding protein) and L3 (hexon, hexon-associated proteins and 23K protease), and partially deletes L2 (penton base and core proteins) and L4 (hexon-associated protein, hexon-trimer scaffold protein, and 33K protein). This deletion abrogates replication and capsid formation from the circular viral DNA, rendering it completely dependent on a helper virus that provides in trans a sufficient quantity of the required replication proteins.
  • E2A DNA binding protein
  • L3 hexon, hexon-associated proteins and 23K protease
  • L2 penton base and core proteins
  • L4 hexon-associated protein, hexon-trimer scaffold protein, and 33K protein
  • pBRX-AscI contains a unique Spe I site at 75.2 mu (L4) into which a 2.7 kb DNA fragment comprising a green fluorescence-protein (GFP) expression cassette was inserted to give M32 (Minivirus of 32 kB).
  • This GFP-cassette is composed of a CMV enhancer/ ⁇ -actin promoter (CA promoter), the Aequorea victoria GFP cDNA, and a SV40 polyA signal.
  • CA promoter CMV enhancer/ ⁇ -actin promoter
  • the use of GFP in the mini-viral vector constructs was utilized in order to determine the presence of the vector in cells using the fluorescence microscopy.
  • Flourescent microscopy represents one of several methods including but not limited to flow cytometry that may be utilized to detect cells expressing GFP.
  • AdH ⁇ AdH ⁇ can be detected by the blue color of X-gal staining.
  • M32 was cut with Mlul and religated, this removes from 31.4 to 34.5 mu which partially deletes LI (52K, 55K and penton-associated proteins).
  • M28 M32 was cut with Mlul and Ascl and religated, this removes from 31.4 to 43.5 mu which completely deletes LI and the L2 portion that still remained in M32.
  • M28 M28 was cut with Rsr II and Spe I and religated, this removes from 30.9 to 75.2 mu extending the LI and L4 deletions.
  • M32 was digested with Nsi I and religated.
  • the Nsi I fragment from 32.2 mu to the CA promoter (with a Nsil site next to the fusion with 75.2 mu), containing the GFP cassette, was religated so the Nsi I site of the CA promoter ligated to 5.5 mu and the Nsi I site at 32.2 ligated at 75.3 mu. This abrogates expression of all proteins between 5.5 to 75.3 mu including E2b (terminal protein, DNA polymerase) and rVa2 proteins.
  • M23 was cut with Mlu I and Asc I, which removes the region from 34.5 to 43.5 mu of the Nsi I fragment of M23, and religated,.
  • mini-viral vectors were constructed by subcloning the minimal cis elements necessary for replication and packaging, including the ITR sequences and the packaging signal, into a small plasmid such as pBluescript (Stratagene) and progressively adding the transgene cassettes and other elements that could improve the therapeutic potential of the viral vector such as elements for episomal maintenance or chromosomal integration ( Figure 8, bottom).
  • the head-to-tail fused ITRs and the packaging signal next to the left ITR (ITR/ITR+ ⁇ ac)were cut from pBRX-AscI with Eco47III (98.7 mu) and PvuII (1.26 mu) blunted and subcloned into Smal - EcoR V of pBluescript, respectively.
  • the resulting plasmid, pBS/MinilTR or GT4007 is a 3.8 minivirus plasmid with no expression cassette and several unique restriction sites flanking the ITR/ITR+pac.
  • the GFP-expression cassette described above was subcloned into pBS/MinilTR to generate M6.5.
  • ERES internal ribosome entry site
  • neo neomycin
  • AdH ⁇ was utilized to support the replication and packaging of the various mini- Ad plasmids. It was important to determine whether the minivirus could be packaged. It was also important to determine whether the size of the minivirus affected the packaging efficiency.
  • adenovirus 100% of the wild type length of DNA is most efficiently packaged, and as the genomic size increases to a maximum of 105% or decreases below 100%, packaging becomes less efficient.
  • a lower limit of 69% (25 kb) has been suggested (94) when wild type adenovirus was used to complement the defective minivirus, but the use of an attenuated helper virus allowed the amplification of a shorter minivirus.
  • a CsCl- purified minivirus plasmid was cotransfected with the linear viral DNA extracted from purified AdH ⁇ . Note that the method utilized to purify the viral DNA is subjected to SDS and Proteinase K which destroys the terminal protein responsible for priming replication. This method was utilized to avoid giving the helper virus a replicative advantage over the minivirus plasmid which also lacks the terminal protein. Accordingly, complementation by direct infection with AdH ⁇ did not rescue minivirus.
  • Cotransfection was accomplished using CajPO, and 2 mg of mini-viral plasmid and 1 mg of viral DNA per well in a 6 well-plate with 293 cells at 50% confluence. After an overnight incubation in the transfection mixture, the medium was changed and the efficiency of transfection was assessed by examination of cells using fluorescence microscopy. With CsCl-purified plasmids this efficiency reached 100% irrespective of the size of the plasmids (Fig. 11). Six days post-cotransfection, CPE was observed and virus was harvested from the cells by three cycles of freeze and thaw.
  • the minivirus plasmid was cotransfected with pBHGlO, a circularized adenovirus plasmid similar to pJM17 incapable of being packaged due to a complete deletion of the packaging signal (95).
  • This plasmid produces all the early proteins necessary for replication as well as the late proteins that form the capsid.
  • the minivirus is present in the same cell as pBHGlO, it will also replicate and, as the minivirus contains the wild- type packaging signal, fhe miniviral vector will be the major nucleic acid encapsidated. However, when the minivirus is released to the neighbor cells it will not be amplified because is defective.
  • the cell monolayer was infected with AdH ⁇ at a multiplicity of infection (moi) of 10 plaque forming units (pfu)/cell.
  • moi multiplicity of infection
  • pfu plaque forming units
  • the lysate (passage 0 of the produced minivirus) was used to infect a fresh monolayer of 90% confluent 293 cells (using 1 to 3 amplification scale). The day after infection, the presence of minivirus was observed by fluorescence and the presence of helper confirmed by X-gal staining. If any helper virus was present in the lysate, further incubation of the cells would lead to the amplification of the mini-virus + helper mixture with the appearance of CPE (the new lysate of this monolayer will be considered as passage 1 of the minivirus). If no helper was present in the lysate, the minivirus alone would not be packaged and only by the addition of new helper would the CPE appear. Therefore the presence of the helper was assessed by X-gal staining and, with much higher sensitivity, by the appearance of CPE.
  • M6.5, M7.9 and M8.5 no fluorescent plaques were found, indicating very inefficient or absent packaging (Fig. 13). This could reflect a packaging lower limit somewhere between 8.5 Kb and 20 Kb. However, it seems more probable that packaging still might take place between these limits but, according to the linear decrease observed, the 11.5 Kb size difference would result in a 7.6 fold less packaging efficiency and amplification may not then be possible.
  • the titer increased until all cells became fluorescent following infection. This occurred, for example, at passage 4 of M32.
  • passage 8 was reached by continuously passing M32 at 1 to 3 amplification scale, enough virus was obtained to infect 75 plates of 150 mm 2 .
  • CPE CsCl gradient as described above. In the gradient four bands were observed, three upper (and therefore lighter) bands and one thicker band in the middle of the centrifuge tube (see scheme in Fig. 14). Every band was collected separately by aspiration from the top of the tube, and dialyzed.
  • fractionation through CsCl may be utilized to decrease the amount of helper virus present in the mini-Ad preparations.
  • the results indicate that the helper used with the partial deletion in the packaging signal taken from the dl 18/28 virus is able to complement the large deletions in the mini-viral vector system but it is still packaged in the presence of minivirus.
  • This helper can be used when a pure population of minivirus is not critical, for example in an antitumoral vector system where a minivirus containing several therapeutic genes (for example, interleukins and tumor-suppresser genes) can be combined with this helper containing another therapeutic gene. When higher mimi-Ad to helper ratio is required, this helper needs to be further attenuated in its packaging.
  • GAL4 is a sequence-specific DNA-binding protein that activates transcription in the yeast Saccharomyces cerevisiae.
  • tetO comes from the Tn70 -specified tetracycline-resistance operon of E. coli, in which transcription of resistance-mediating genes is negatively regulated by the tetracycline repressor (tet R) which binds a 19-bp inverted repeat sequence 5 '-TCCCTATCAGTGATAGAGA-3 ' in tet O (98, 99).
  • a synthetic sequence has been utilized to replace the sequence between Xho I and Xba I (nt 194, 0.5 mu to nt 452, 1.25 mu ) of GT5000.
  • Four synthetic sequences ( Figures 21 and 22 ) have been designed. All four synthetic sequence contain the Ad5 packaging element ( A repeats ) I, II, VI and VII.
  • Three or four repeats of 17-mer GAL4 binding sequences (5'-CGGAGTACTGTCCTCCG-3' ) (97) or 19-mer tetO sequences ( 5'- TCCCTATCAGTGATAGAGA-3') (100, 102) were introduced around or between these A repeats ( Figures 17 and 18).
  • adenoviral vectors used in gene therapy applications were designed to have deletions in the El region of the adenovirus 5 (Ad5) genome.
  • the El region, not including region DC, consists of 9% of the left end of Ad5 (1.2 - 9.8 map units), and encodes two early region proteins, El A and EIB.
  • El A/El B is required for virus replication and for expression of all other Ad5 proteins such as E2-E4 and late proteins (100). Deletion of El creates a replication-incompetent virus that, in theory, is silent for expression of all Ad5 proteins and expresses only the transgene of interest.
  • El A and EIB are also of interest for safety reasons, since these two proteins, in combination, have been implicated in oncogenic transformation of mammalian cells (101-103).
  • All of the Class I adenovirus vectors used to date in human clinical trials, as well as, the novel packaging-deficient helper virus described in Example 1 are deleted for El.
  • El -deficient adenoviral vectors are propagated in an Ad5 helper cell line called
  • 293 (104). 293 cells were derived by transforming human embryonic kidney cells with sheared fragments of Ad5 DNA. Genomic analysis revealed that 293 cells contain four to five copies per cell of the left 12% of the viral genome (including the entire El region) and approximately one copy per cell of 9% of the right end, the E4 region (105). While 293 cells are very efficient at producing high titers of El -deficient adenovirus, they have the disadvantage that, due to the presence of extraneous Ad5 sequences integrated into the 293 genome (other than the El region), recombination can occur with sequences in the El-deficient adenovirus vector causing the production of El -containing, replication- competent adenovirus (RCA).
  • RCA replication- competent adenovirus
  • RCA in 293 cells can present severe ramifications for the safety of human gene therapy trials (106).
  • recombination in 293 cells can also cause deletions and rearrangements that effect transgene expression, thereby decreasing the titer of functional adenovirus particles.
  • Ad5 helper cell line has been developed which harbors only the E1A/E1B sequences required for complementation, and does not contain any homologous sequences that overlap with regions in the El -deficient adenovirus.
  • a contiguous 2194 bp Xbal to Afl II (Ad5 bp 1343-3537) was cloned from pBRXad5XhoICl into the same vector.
  • the resultant 3075 bp El fragment (in pSL301) contains the TATA box and RNA cap site for El A, El A coding sequence, complete EIB promoter, and EIB coding sequence, including fhe stop codon for EIB p55 protein, but not including region LX.
  • the CMV-E1 expression vector (including the G418 resistance gene, neo) was transfected using Lipofectamine (Gibco/BRL) into A549 human lung carcinoma cells and G418 R colonies were isolated. Single-cell clones were screened for functional E1A/E1B expression; An El -deleted adenovirus containing a green florescence protein (GFP) expression cassette under CMV/ ⁇ -actin (CA) promoter, Ad5CA-GFP, was used to infect the A549-E1 clones. Three days post-infection, clones were screened for production of El -complemented Ad5CA-GFP adenovirus by visual examination for cytopathic effect (CPE).
  • CPE cytopathic effect
  • A549E1-68 displayed 100% CPE in 3 days (similar to that observed for 293 cells). This clone also showed high infectivity, in that virtually 100% of the cells fluoresced green 24 hrs. post-infection (Fig. 22).
  • A549 cells at sub-confluent density, grow as distinct single cells with an elongated, fibroblast-like mo ⁇ hology, whereas, the El cell line, A549E1-68, grows as colonies of cells with a more cuboidal mo ⁇ hology (Fig. 24).
  • A549E1-68 was compared with 293 cells for production of El -deleted adenovirus (Ad5CA-GFP) by plaque assay and found to produce an equivalent titer of complemented virus (7 x 10 9 PFU for A549E1-68 vs. 9 x 10 9 PFU for 293).
  • Immunoprecipitation and Western blot analysis using an El A specific antibody revealed two ElA-specific bands with apparent molecular weights of 46kd and 42kd, corresponding to products expected from E1A 13S and 12S mRNAs (6), and identical in size to those observed in 293 cells (Fig. 25 A).
  • A549E1-68 produced a band of approximately 55 kd using a monoclonal Ab specific for EIB p55. This 55 kd, E1B- specific band, as well as secondary background bands, were observed in 293 cells also (Fig. 25B). Extra “background” bands found in both experimental and control lanes have been observed by other authors and have been attributed to co-immunoprecipitation of a variety of proteins including, cyclins, p53, and Rb. Unlike A549E1-68 and 293 cells, the parental A549 cell line showed no expression of 46 kd, 42 kd, or 55 kd E1A/E1B proteins.
  • A549E1-68 not only expresses E1A and EIB, but that they are functional, since this cell line can complement for production of high titer, El -deleted, recombinant adenovirus.
  • this new Ad5 helper cell line can complement without production of RCA, we are serially passaging El -deleted adenovirus on A549E1- 68 cells and testing the virus amplified during passaging, on parental A549 cells for production of El -containing, replication-competent adenovirus (RCA) by CPE, as well as by using PCR primers specific for El A/El B sequences. This cell line will be used during propagation and scale-up of all El -deleted adenovirus vectors, to ensure that production lots are free of RCA.
  • Example 4 Expression cassette comprising the FVIII cDNA
  • the large capacity of the Ad-mini vector of the present invention for the gene of interest allows for insertion of large promoter and protein coding regions that far exceed the size capacity of the conventional Ad vector. It is preferred, for the pu ⁇ oses of the present invention, that the FVIII mini-Ad vector deliver the FVIII gene to the liver. It is, therefore, important to utilize a highly active promoter that functions in the liver.
  • One such promoter is the human albumin gene promoter (32). A 12.5 kb region of the human albumin promoter was obtained from the Dr. Tamaoki from the University of Calgary.
  • proximal region comprising the TATA box (550 bp); 2.) an enhancer region at -1.7 kb; and, 3.) a second enhancer region at -6.0 kb (Fig. 26). Combined, these regions approximate the strength of the entire 12.5 kb human albumin promoter.
  • the 10.5 kb EcoRI / Aval fragment of pAlbl2.5CAT (Fig. 2) was co- ligated with the Aval / Hindlll proximal human albumin promoter fragment into the EcoRI / Hindlll site of the pBluescript-KS + vector, to generate the recombinant plasmid GT4031 (Fig.
  • the FVIII cDNA may be operably linked to a promoter or transcriptional control element that may be synthetic, controllable or regulatable, or tissue / cell type specific.
  • a promoter or transcriptional control element that may be synthetic, controllable or regulatable, or tissue / cell type specific.
  • expression of the FVIII cDNA in the producer or helper cell is suppressed during viral production and activated following delivery to a target cell. In this manner, differential expression of the reporter or effector gene of the mini-Ad vector is achieved.
  • Such differentiated expression is accomplished by constructing a DNA molecule having the FVIII cDNA under the transcriptional control of a synthetic promoter such as one having a liver-specific enhancer operably linked to an ⁇ ,-antitrypsin ( ⁇ ,-AT) promoter or one in which the tetracycline operon (tetO) is operably linked to the cytomegalovirus (CMV) promoter (tetO-CMV), in which case a cell line is utilized that expresses the tet- KRAB transcriptional repressor protein.
  • a synthetic promoter such as one having a liver-specific enhancer operably linked to an ⁇ ,-antitrypsin ( ⁇ ,-AT) promoter or one in which the tetracycline operon (tetO) is operably linked to the cytomegalovirus (CMV) promoter (tetO-CMV), in which case a cell line is utilized that expresses the tet- KRAB transcriptional re
  • Homologous recombination arms of the FVIII expression cassette Homologous recombination may be employed to insert an exogenous gene into a the genome of a target cell resulting in stable gene expression.
  • the human FVIII cDNA may be targeted to the genomic DNA of a target cell.
  • Large segments of cellular DNA derived from the human albumin gene or human ⁇ -fetoprotein were utilized (32, 33).
  • the 12.5 kb albumin promoter in the FVIII mini-Ad vector functions as the upstream homologous recombination arm while a number of downstream fragments of greater than 6 kb were prepared as potential 3' recombination arms.
  • FIG 31 A figure of the albumin gene, an intergenic region and the ⁇ -fetoprotein gene regions utilized in the present invention is shown in Figure 31.
  • a construct (GT2063) comprising the 3' Xhol recombination arm derived from the human albumin gene and the pAlb-E5 segment cloned into the unique Sail site of GT2061 is shown in Figure 33.
  • Plasmid GT2063 was constructed by insertion of the Xhol albumin gene fragment of plasmid ⁇ E5 into the unique Sal I site of GT2061.
  • the 12.5 kb EcoRI / Hindlll human albumin promoter fragment was inserted into pBluescriptKS * (Stratagene, La Jolla, CA).
  • the human albumin promoter vector, GT4031 thus contains a unique Sail site into which the human FVIII cDNA (the region in GT2051 from Xhol to Sail comprising the SV40 early intron at the 5' end and the SV40 polyadenylation signal at the 3' end) was inserted.
  • the resulting plasmid, GT2053, contains unique Sal I and Xhol sites located 3' to the polyadenylation site ( Figure 29).
  • the Ad minimal ITR and wild type packaging sequence was excised from plasmid GT2033 by Xhol digestion and cloned into the Sail site of plasmid GT2053 to generate plasmid GT2061.
  • the 6.8 kb arm of the albumin gene was isolated from pAlb-E5 and cloned into the unique Sail site of GT2061 to generate plasmid GT2063.
  • GT2063 was transfected into 293 cells together with the helper virus DNA to generate the mini-Ad FVIII minivirus designated GTV2063.
  • helper- virus genome (2 ⁇ g) was purified from virus particles and co-transfected with the helper Ad genome (0.2 ⁇ g) into 293 cells by calcium phosphate-mediated transfection (81). Following the appearance of CPE, cell-free freeze thaw lysates were prepared and utilized to infect fresh 293 cells. Human FVIII, indicating the presence of the GT2063 mini-Ad, was detected in the cell supematants using the Coatest FVIII chromogenic assay (Pharmacia). The data are consistent with propagation of a helper / GT2063 mini-Ad vector mixture. In yet another approach (Fig.
  • the adenoviral helper plasmid, pBHGlO which lacks the Ad packaging signal and El region but encodes the remainder of the Ad proteins, was co-transfected with the mini-Ad clone GT2063 into 293 cells.
  • Rescue of the Ad-minivirus genome was achieved following infection of 293 cells with an El- substituted helper virus having attenuated packaging function.
  • Both the Ad-helper and mini-Ad genomes may be packaged, and adenoviral particles carrying either genome may be generated using the methodologies of the present invention, although the helper Ad / mini- Ad ratios is variable.
  • Helper plasmid pBHGlO (0 ⁇ g) and the mini-Ad vector comprising the human FVIII gene were co-transfected into 293 cells by calcium phosphate transfection (81).
  • Transfection into 293 cells may be performed using any of the well-known and widely available techniques such as lipofection (i.e., using Lipofectamine from GIBCO/BRL) or electroporation (i.e., using reagents and electroporator available from Bio-Rad).
  • lipofection i.e., using Lipofectamine from GIBCO/BRL
  • electroporation i.e., using reagents and electroporator available from Bio-Rad.
  • Infection of the transfected 293 cells with an attenuated helper virus was performed three days after transfection (Fig. 35B).
  • CPE cytopathic effect
  • hFVIII expression of hFVIII in 293 cells was expected to be minimal because fhe human albumin promoter is not very active in these cells. This has been determined using both CAT assays (69) and an FVm chromogenic assay (Helena Laboratories, Pharmacia) following transfection of 293 cells with GT2061 using the calcium phosphate precipitation transfection method.
  • PCR was performed using primers specific to human FVIII cDNA and amplifications were performed on virus subjected to DNAse treatment prior to DNA extraction to remove any residual non-viral contaminating plasmid DNA.
  • PCR was performed using isolated viral DNA as template (1/20 of the viral DNA isolated), FVIII primer #1 at a final concentration of 1 ⁇ M (listed as SEQ ID NO: l; ACCAGTCAAAGGGAGAAAGAAGA), FVIII primer #2 at a final concentration of 1 ⁇ M (listed as SEQ TD NO:2; CGATGGTTCCTCACAAGAAATGT), and the following conditions: annealing for one minute at 55°C, polymerization for one minute at 72°C, denaturation for one minute at 94°C for a total of 35 cycles.
  • Figure 36 demonstrates the results of PCR amplification of the packaging signal of the FVIII mini- Ad and, independently, the helper Ad.
  • PCR was also performed on the packaging signal region. PCR was performed using isolated viral DNA (1/20 of the total viral DNA isolated) as template, packaging signal primer #1 (SEQ ID NO:3; GGAACACATGTAAGCGACGG) at a final concentration of 1 ⁇ M, packaging signal primer #2 (SEQ ID NO:4; CCATCGATAATAATAAAACGCCAACTTTGACCCG) at a final concentration of 1 ⁇ M and the following conditions: annealing for one minute at 55°C, polymerization for one minute at 72°C, denaturation for one minute at 94°C for a total of 35 cycles.
  • packaging signal primer #1 SEQ ID NO:3; GGAACACATGTAAGCGACGG
  • packaging signal primer #2 SEQ ID NO:4; CCATCGATAATAATAAAACGCCAACTTTGACCCG
  • the packaging signal of the helper Ad is partially deleted, the PCR product from the packaging signal deleted helper is shorter (177 bp) than that of the mini ⁇ Ad having a wild-type packaging signal (approximately 310 bp).
  • the FVIII miniAd was not detected but its presence was increasingly detected in passages 3 to 6.
  • Identical results were obtained using Southern blot analysis ( Figure 37).
  • an Ad DNA fragment adjacent to the right ITR present in both the FVIII mini-Ad and the helper Ad was used.
  • the expected length of the detected fragments after Pst I digestion of the mini- Ad GT2063 and the AdH ⁇ is 3.3 and 2.2 Kb, respectively.
  • Figure 37 is a compilation of four Southern blots (A - D) of FVIII mini- Ad DNA independently isolated from passages 1 to 21.
  • the 3.3 Kb band corresponding to mini-AdFVIII was detected in DNA isolated from passage 5 -21.
  • a steady increase in FVIII mini- Ad DNA was detected until passage 10 which was followed by progressive decrease in FVIII mini- Ad until passage 12.
  • This cycle of increasing and decreasing levels of FVIII mini-Ad DNA was observed to occur approximately every four passages and was accompanied by a parallel cycle of the level of helper Ad DNA, which has a slightly earlier onset.
  • the amount of FVIII mini-Ad DNA and helper Ad DNA was quantified densitometrically from the Southern blots and plotted in Figure 38.
  • the observed cycles match with the well known dynamics of a virus population generated in association with a defective interfering virus (in the system of the present invention, the virus population comprises the FVIII mini-Ad) and a helper virus.
  • the understanding and control of these cycles is important to determine at which passage the mini-Ad vectors should be purified to obtain optimal titers.
  • Passages such as #18 (PI 8) result in a vector preparation enriched for the FVIII mini- Ad (i.e., P18 appears to contain 10 times more FVIII mini- Ad than helper Ad), albeit at a low titer.
  • Passages such as #20 (p20) comprise high levels of FVIII mini ⁇ Ad and helper Ad, although at an undesirable FVTII mini- Ad to helper Ad ratio of 1 : 1.
  • a large scale amplification was performed at p20.
  • a crude lysate was then prepared by three freeze/thaw cycles to extract the virus.
  • the crude lysate was cleared by centrifugation, loaded onto a step density gradient of CsCl (three layers of 1.5, 1.35, and 1.25 g/ml) and centrifuged at 35000 x g for 1 h.
  • the band corresponding to the mixture of mini- Ad and helper Ad was further purified using a second continuous CsCl gradient of 1.35 g/ml.
  • Example 8 Test of the mini-AdFVIII in cell lines
  • the FVIII mini- Ad (GT2063) was purified by CsCl as described above and utilized to demonstrate production of FVTII in host cells infected with the vector.
  • 293 and HepG2 cells were utilized due to their known ability to utilize the albumin promoter.
  • FVITI production in these cells was assayed by immunohistochemistry and functional assays 24 h after infection.
  • Purified FVIII mini- Ad vector was added to 0.5 ml of medium and used to infect 6xl0 5 293 and HepG2 cells in a 4cm 2 well. After a 4 h incubation to allow for adso ⁇ tion of the viral particles to the host cells, the infection medium was replaced with fresh medium.
  • the estimated titer in transducing units per milliliter was determined to be 6xl0 9 transducing units/ml. If an adso ⁇ tion time of 4h, an adso ⁇ tion volume of 0.5 ml in 4 cm 2 , and a non-rocking adso ⁇ tion are taken into account, the estimated titer may be reduced by a factor of 0.42, 0.56, and 0.53, respectively (49).
  • the actual titer of mini- AdFVIII vector would therefore be estimated to be 4.6x10 10 transducing unit/ml.
  • the titer determined by optical absorbance at 260 nm, which reflects the number of viral particles per milliliter was determined to be 3.6xl0 12 particles/ml for the lighter fraction of FVIII mini- Ad. Therefore, the bioactivity of the FVIII mini-Ad can be calculated to be one FVIII-transducing unit per every 78 viral particles, which falls within the levels of acceptability recommended by the Food and Drug Administration (49).
  • the amount of functional FVIII in the supernatant of transduced cells was determined using the chromogenic Coatest FVTII Test (Pharmacia, Piscataway, NJ).
  • Improvements in the vector system were accomplished by generation of a vector into which various expression cassettes may be cloned.
  • the vector GT2063 was modified by excising the proximal albumin promoter region and human FVITI gene localized between fhe Pme I and Sal I sites. This was accomplished by first converting the Pme I site of GT2063 to a Sal I site by ligating a Sail linker to the Pme site. The resulting clone, GT2072, was treated with Sal I and religated to remove the proximal albumin promoter/hFVTTI gene region thereby creating a mini- Ad vector having a unique Sal I cloning site for insertion of various expression cassettes. The expression from such cassettes may be affected by albumin gene enhancers located upstream. Each clone was analyzed to determine the level of expression of the transgene.
  • Expression cassettes were prepared for insertion into the improved vector, GT2072.
  • the expression cassettes of this example comprises the cytomegalovirus (CMV) immediate early promoter, the elongation factor I (EF-I) promoter (which are known to function in a wide variety of cell types) or the liver-specific promoter for the phosphoenol pyruvate carboxykinase (PEPCK) gene.
  • CMV cytomegalovirus
  • EF-I elongation factor I
  • PEPCK liver-specific promoter for the phosphoenol pyruvate carboxykinase
  • the EF-I and CMV promoters were each separately utilized to drive expression of either the full length FVITI cDNA or the B-domain deleted (BDD) factor VIII cDNA ( Figures 42 and 43, respectively).
  • Adeno-associated virus is a human non-pathogenic single-stranded linear parvovirus that replicates only in the presence of a helper virus like adenovirus or he ⁇ es virus. However, in the absence of helper, AAV can integrate specifically in the host genome and be maintained as a latent provirus (34). The particular locus where AAV integrates has been located to chromosome 19ql3.3-qter and named AAVS1 (22-25, 35).
  • AAV ITRs Inverted Terminal Repeats
  • Rep78/68 proteins are palindromic sequences present in both ends of the AAV genome, that fold into hai ⁇ in structures and function as origins of replication.
  • Rep78/68 proteins include sequence-specific DNA binding (36, 37), sequence and strand-specific endonuclease activity (38), and ATP-dependent helicase activity (38-40). These proteins can bind to a specific sequence in the ITR DNA and promote the process named terminal resolution by which the ITR hai ⁇ in is nicked and replicated.
  • a Rep- binding motif and a terminal resolution site have been identified in both the AAV ITR and AAVS1 and demonstrated to promote in vitro DNA replication in the presence of Rep (28). It has also been shown that Rep68 protein can mediate complex formation between the AAV ITR DNA and AAVS1 site in vitro (41). These findings suggest a model in which the DNA binding and endonuclease activity of Rep along with limited DNA synthesis at the ITRs and AA VS 1 sites would allow targeted integration of the sequences contained between fhe ITRs (27). AAV has been considered as a candidate vector for gene therapy.
  • the vector consists of a Rep expression cassette (containing the viral endogenous promoter), as well as a cassette for expression of a reporter gene flanked by two AAV ITRs.
  • the Rep expression cassette was obtained after PCR amplification of sequences 193 to 2216 in the AAV genome from plasmid pSUB201 (41). This fragment starts right after the ITR and extends through the p5 promoter and the Rep78 coding sequence.
  • a control plasmid was constructed by removing the Rep expression cassette, but keeping the reporter gene expression cassette placed between two AAV ITRs.
  • 293 cells were transfected with plasmids GT9003 or GT9004 and then selected for 12 days with G418 (0.5 mg/ml).
  • G418-resistant colonies were isolated, expanded, and genomic DNA was extracted from different colonies by fhe salt precipitation method (125).
  • Genomic DNA was digested with EcoRI and analyzed by Southern blot with a probe for AAVS1. EcoRI was chosen because fhe AAVS1 locus is contained within an 8Kb EcoRI-EcoRI fragment.
  • Figure 47 shows that 50% of the resistant colonies analyzed which derived from plasmid GT9003 (Rep-expressing plasmid) revealed rearrangements of at least one AAVS1 locus, as indicated by the presence of a shifted band in addition to the 8kb band corresponding to the normal sequence. Rearrangements were not observed in the colonies derived from plasmid GT9004, indicating that this phenomenon is dependent on the expression of Rep. These results suggested that Rep was able to drive specific integration of the transgene. The membrane was then rehybridized to a specific probe for neo ( Figure 48, panel B). The pattern of bands obtained indicated that some AAVS 1 rearrangements correspond to neo (ex. clone 2L2) but also suggested that random integration events occurred frequently in the clones analyzed, possibly favored by the selective pressure applied.
  • the previous invention comprises multiple plasmids comprising an expression cassette having a reporter gene [i.e., neo or the gene encoding green fluorescent protein (GFP)] flanked by AAV ITR sequence (hereafter referred to as the integration cassette), in combination with an upstream Rep expression cassette ( Figure 44, panels A - D).
  • a reporter gene i.e., neo or the gene encoding green fluorescent protein (GFP)
  • GFP green fluorescent protein
  • the present invention provides a hybrid vector that combines the advantages of the Ad vector (high titer preparation, large capacity for exogenous DNA, high level infectivity of multiple cell types) and the integration capabilities of AAV.
  • This hybrid virus of the present invention replicates as an adenovirus and comprises the AAV elements sufficient for integration.
  • the present invention comprises a mini-Ad vector having a Rep expression cassette and a FVIII expression cassette flanked by AAV ITRs ( Figure 44). Additional exogenous DNA (up to 36 kb) may be inserted into the vector. Additional exogenous DNA of this vector corresponds to human albumin genomic sequences (non-coding).
  • the Rep expression cassette encompasses bp 193 to 2216 bp of the AAV genome. This fragment originates immediately following the ITR and extends through the p5 promoter and the Rep78 coding sequence. For the reasons listed below, a fragment comprising seven tet operators was introduced upstream of the p5 promoter was included to allow for transcriptional repression of the rep gene by the tet-KRAB repressor (42).
  • the tet-KRAB repressor may be provided as a transcriptional switch in order inhibit expression of Rep during viral vector generation.
  • the present invention provides a 293 cell line stably expressing the tet-KRAB repressor protein. Upon entry of virus into the host cell that does not express the tet-KRAB repressor protein, Rep expression occurs due to the absence of the repressor in those cells, thus promoting integration of the sequences flanked by AAV ITRs into the cellular genome.
  • the viral vector thus generated may be tested in vitro and in vivo for the frequency and specificity of integration.
  • An emboidment of the present invention is a methodology for the generation of a transgenic mouse harboring the human AAVSl integration site for use as an in vivo animal model.
  • the animal model is to be used for testing site-specific integration of a viral vector containing fhe AAV integration mechanism described above.
  • the first step towards development of the animal model was cloning of the AAVSl site and insertion of the sequence into a mammalian expression vector.
  • the AAVSl human integration site was originally cloned by Kotin et.al. (50) as an 8.2 kb EcoRI fragment, of which the first 4067 bp have been sequenced.
  • This DNA sequence information was used to design two oligonucleotide primers, which were subsequently used to generate a 253 bp PCR product for use as an AAVSl -specific probe.
  • the upper primer, U2492 (SEQ ID NO:5: GCTGTCTGGTGCGTTTCACTGAT), is a 23 -mer that extends from basepairs 2492-2515 of the AAVSl sequence and the lower primer, L2722 (SEQ ID NO:6: TCACAAAGGGAGTTTTCCACACG), also a 23-mer extends from basepairs 2722-2745 of the AAVSl sequence.
  • PCR amplification was performed using 100 ng human genomic DNA as template and a 1 ⁇ M final concentration of the U2492 and L2722 primers (SEQ ID NO: 5 and SEQ ID NO;6, respectively) as follows : 95° C, 4 min - 1 cycle; 95° C, 0.5 min, 55°C, 0.5 min, 72°C, lmin - 35 cycles; 72°C, 7min - 1 cycle.
  • the 253 bp AAVSl -specific PCR product was sent to Genome Systems (St.Louis, MO) where it was used to screen a human PI genomic DNA library.
  • mice embryonic stem (ES) cell line comprising the AAVSl sequence for use in the generation of an AAVSl transgenic mouse
  • the pAAVSl-Neo plasmid was transfected into mouse ES cells (129 Sv agouti, Genome Systems) ( Figure 51).
  • 25 ug of pAAVSl-Neo plasmid DNA was linearized with Xbal and transfected by electroporation (975 uFd, 252 v.) into ES cells using a Biorad Gene Pulser.
  • Transfected cells were selected for one week in 250 ug/ml G418.
  • Neo R neo-resistant colonies were isolated, expanded, and characterized by mo ⁇ hology to obtain clones which were >95% "un-differentiated” in order to enrich for cell lines that maintained a totipotent phenotype.
  • Genomic DNA was isolated from 17 Neo R ES clones, as well as from the untransfected, parental ES cell line, and 100 ng of the DNA utilized as template using primers U2492 and L2722 (SEQ ID NO:5 and SEQ ID NO;6, respectively; final concentration of 1 ⁇ M) for AAVSl -specific PCR.
  • PCR of DNA from 17/17 Neo R ES clones generated the expected 253 bp AAVSl PCR product, while PCR analysis of DNA from the untransfected control ES cells did not generate detectable PCR product. Southern blot analysis was performed on control and AAVSl (+) ES cell lines to confirm that a functional AAVSl sequence had been preserved following transfection and genomic integration (Figure 53).
  • Genomic DNA from each of the AAVSl positive (as assessed by PCR) ES cell lines was digested with EcoRI in combination with EcoRV, electrophoresed, blotted, and hybridized with an 8.2 kb AAVSl probe.
  • Both ES#4 and ES#3.16 cell lines contained the expected 5.2 kb and 3.0 kb EcoRI/EcoRV fragments, indicating integration of the entire 8.2 kb AAVSl sequence (Fig. 53).
  • the untransfected parental ES cells showed no hybridizing bands using this human AAVSl -specific probe.
  • AAVSl -positive ES clones ES#4 and ES#3.16, were grown on 1° murine embryonic fibroblast feeder layers, in the presence of Leukemic Inhibitory Factor (LTF - an anti-differentiation factor), and maintained at very low passage (P.2 - P.7) in order to preserve an undifferentiated, totipotent phenotype.
  • LTF Leukemic Inhibitory Factor
  • Blastocyst-stage embryos were collected at Day 3.5 p.c. from superovulated, C57BL/6 mice, maintained in M16 embryo medium. 15-20 ES cells (AAVSl ES#4 or ES#3.16) and microinjected into the blastocoel cavity of the 3.5 day embryos using a Leitz DM-ILB Microinjection Workstation.
  • the embryos were returned to M16 medium and incubated in 5% CO 2 , 37°C for 2 hours to allow the blastocysts to re-cavitate. 10-15 injected blastocysts were subsequently transferred into the uterus of Day 2.5 post-coitus (p.c), pseudopregnant, CB6F, foster mothers. Following uterus transfer, the blastocysts implant into the uterine wall, the AAVSl -positive ES cells become inco ⁇ orated into the embryo's inner cell mass, and contribute their genetic information to the developing embryo, resulting in the birth of transgenic (chimeric) progeny approximately 17 days later.
  • p.c post-coitus
  • the conditions for PCR were: 95° C, 4 min - 1 cycle; 95° C, 0.5 min, 55°C, 0.5 min, 72°C, lmin - 35 cycles; 72°C, 7min - 1 cycle.
  • the correct 253 bp AAVSl PCR product was indeed detected in tail DNA from a high-percentage chimera ( Figure 57, lane 7), but was not detected in the tail DNA of a non-chimeric littermate or in a low percentage chimera with less than 10% agouti coat color chimerism ( Figure 55, lanes 5 and 6, respectively).
  • This homozygous line may then be used to test AAVSl site-specific integration of either AAV viral vectors, hybrid adenovirus/ AAV viral vectors, or any other plasmid vector comprising the AAV ITRs and Rep 78/68 genes necessary for integration at the AAVS 1 site.
  • the AAV transgene vectors may be delivered in vivo to the AAVSl transgenic mice either by viral infection (following I.V. injection) or by using ligand-mediated DNA/liposome complexes.
  • the frequency of site-specific integration, stability of the integrated transgene and the duration of stable protein expression i.e. human Factor VIII or Factor IX
  • human Factor VIII or Factor IX i.e. human Factor VIII or Factor IX
  • a viral vector of the present invention is injected into the intravenous or portal vein of the transgenic mouse.
  • the vector may be part of a pharmaceutical composition and may or may not be complexed with lipid such as Lipofectamine (GTBCO/BRL) and / or a liver-specific ligand (79, 80).
  • lipid such as Lipofectamine (GTBCO/BRL) and / or a liver-specific ligand (79, 80).
  • the level of expression of the effector or reporter gene of the viral vector is measured using a technique such as northern blot, RNase protection analysis, or PCR. In testing the FVIII mini-Ad, FVUJ is detected by ELISA assay. The level of expression of the effector or reporter gene in each blood sample is compared to one another in order to determine the duration of transgene expression. Also, in order to determine site-specific integration of the vector, genomic DNA is isolated from the liver tissue of the animal. PCR analysis of the genomic DNA using an AAVSl -specific primer and a primer containing sequence homologuos to sequence of the vector is then performed.
  • Site-specific integration of the vector at the AAVSl site of the genome of the transgenic animal produces a product containing both AAVSl and vector sequences.
  • the amplified PCR product provided the viral vector integrated into the AAVSl site of the animal, includes vector sequence.
  • A. FVIII transgenic mouse harboring the human AA VS1 integration sequence An AAV/ITR-Rep vector comprising either fhe neo or GFP reporter gene
  • the present invention comprises a transgenic mouse harboring the human AAVS 1 integration site within its genome.
  • the transgenic mouse is generated using embryonic stem cell manipulation technology (43) as illustrated in Figure 51.
  • An expression vector comprising the entire 8.2 kb human AAVSl sequence and neo (or Neo) selection marker is constructed.
  • the AAVS 1 / Neo vector is transfected into totipotent mouse embryonic stem (ES) cells to obtain Neo R , AAVST ES cell clones that are subsequently microinjected into mouse blastocyst-stage embryos and implanted into the uterus of a foster mother. Following implantation, the AAVST ES cells resume normal embryonic development and contribute their genetic information (including the human AAVSl sequence) to the developing embryo. Chimeric (transgenic) progeny are identified by the presence of ES cell-derived agouti-brown coat color. Chimeric founders are then bred with wild-type C57BL/6 mice to obtain germline transmission of the transgene.
  • ES mouse embryonic stem
  • FI heterozygotes are bred to obtain a homozygous mouse line which has stably inco ⁇ orated the human AAVSl integration sequence into its genome.
  • This mouse model is then injected, via the tail vein or portal vein, with the AAV-ITR / miniAd-FVITI vector to assess in vivo transduction efficiency, integration at the human AAVSl sequence, and duration of transgene expression.
  • the present invention is a FVffl-tolerized mouse model system.
  • FVUI tolerization has been achieved by transient injection of FVTII into neo- natal mice (44).
  • the present invention comprises a mouse having the human FVIII gene under the control of a promoter that functions in a developmental stage-specific manner.
  • a promoter may include but is not limited to that of the ⁇ -fetoprotein gene or the embryonic globin gene, epsilon.
  • the ⁇ -fetoprotein promoter is an example of an early developmental stage-specific promoter that is inactive in the mature animal (45).
  • the embryonic globin gene, epsilon is another example of a developmentally regulated gene that may be utilized in the present invention.
  • gene expression is limited to the liver and is dependent upon liver specific transcription factors for activation (46, 47).
  • a liver specific promoter element that is also developmentally regulated would be preferred.
  • the ⁇ -fetoprotein promoter (AFP) meets both of these criteria.
  • the ⁇ -fetoprotein promoter does not function in undifferentiated ES cells but is induced during differentiation (48); as such, it may be utilized to control hFVTII expression in transgenic mice.
  • An objective of the present invention is to provide a transgenic mouse that has been tolerized to the xenogenic human FVITI protein.
  • the transgenic mouse may be utilized to test delivery of human FVEQ in vivo using adenoviral or AAV vector systems, or using FVTfl-secreting cells in an immunoisolation device.
  • human FVITI is expressed embryonically in the developing transgenic mouse under control of the AFP promoter.
  • HFVIII then, is seen as "self by the mouse and tolerance occurs.
  • AFP mouse ⁇ -fetoprotein
  • the plasmid GT2057 comprises a 7.5 kb AFP promoter sequence originally characterized by Urano et.al. (33).
  • the cassette containing the AFP promoter and the hFVTII gene was subsequently cloned as an Aat II / Sal I fragment into the Neo expression vector, pGKNeo, at Aat II / Xho I.
  • the resultant 20.2 kb vector, mAFP- hFVIII-pGKNeo ( Figure 56), harbors the hFVIII gene under control of an embryonic promoter (AFP), and has a Neo expression cassette for selection in mammalian cells.
  • Example 17 Generation and characterization ofAFP-FVIII (+) ES cell clones
  • ES mouse embryonic stem
  • the mAFP-hFVITI- pGKNeo vector was stably transfected into mouse ES cells.
  • 20 ug of AFP-FVTII-Neo DNA was linearized with Aat II and electroporated into ES cells (975uFd, 252v.) using a BioRad Gene Pulser. Following electroporation, ES cells were propagated on embryonic fibroblast feeder layers in 250 ug/ml G418 to select for Neo R clones.
  • Neo R clones were picked, expanded, and analyzed for functional Factor VJTI protein using a Coatest kit. No FVIII was detected in tissue culture supematants from any of the 48 clones. Genomic DNA was isolated from 13 Neo R ES clones and from untransfected parental ES cells, digested with Xba I, and screened by Southern blot analysis using the 7.2 kb FVIII Not I fragment from GT2051 as a probe. 11/13 transfected clones contained the expected 7.8 kb Xba I fragment, confirming the presence of the entire hFVIII sequence as well as 500 bp of the 3' end of the AFP promoter.
  • the pAFP- EGFP-1 plasmid was transiently transfected into both ES cells and HepG2 cells (a liver cell line known to express high levels of ⁇ -fetoprotein), and examined by direct visualization using a Nikon Diaphot broad range microscope with a FITC filter after 24 hours for green fluorescent cells. GFP expression was detected in the HepG2 cell line but not in mouse ES cells (data not shown), confirming that the AFP promoter does not function in undifferentiated ES cells, but on in a differentiated liver cell line. These results are in agreement with those of Vogt et.al.
  • Human FVTII was detected at the level of 3.0 ng/ 10 6 cells/ 24 hrs., confirming that the AFP-FV ⁇ l construct was functional and that the tissue-specific and developmental-specific expression pattern of the 7.5 kb AFP promoter / enhancer element was preserved. Having demonstrated that the AFP-FVTII vector was functioning properly, one of the AFP-FVIII (+) ES clones, clone #22 (Fig. 57, lane 3), was chosen, based on its un-differentiated growth phenotype, to use for blastocyst microinjection experiments.
  • the present invention provides a transgenic mouse that has been tolerized in utero to the xenogeneic human Factor VUI protein.
  • This transgenic mouse is used to test delivery of human FVTII in vivo using adenoviral or AAV vector systems, or using FVIII-secreting cells in the TheraCyte immunoisolation device such as that described in U.S. Patent Nos. 5,314,417; 5,344,454; 5,421923; 5,453,278; 5,545,223; or 5,569,462.
  • hFVIII is expressed embryonically in the developing transgenic mouse under the control of the AFP promoter, hFVUI as delivered by fhe therapeutic vector (i.e.
  • the AAV- miniAd-hFVTJT is recognized by the immune system of the mouse to a "self antigen. As such, tolerance to the hFVIII protein results.
  • the transgenic mouse is tolerized to hFVIII, an immune reaction to the "xenogeneic" human FVIII protein will not occur, and an accurate assessment of antigenicity of the viral vector backbone and a realistic measurement of the duration of gene expression in vivo may be determined.
  • the amount of hFVm protein expressed by the liver as a result of transduction by the vector in mature transgenic mice may be accurately quantitated.
  • ES cells from the AFP-FVIII (+) ES clone #22 were microinjected into C57BL/6 blastocysts, and implanted into the uterus of foster mothers.
  • ES clone #22 four chimeric progeny have been produced using ES clone #22. They are mated with wild- type C57BL/6 mice to test for germline transmission, and germline founders bred to obtain a homozygous AFP-hFVIII transgenic mouse line.
  • chimeric progeny since the chimeric progeny, by definition, have mosaic expression of the AFP-hFVIII transgene in all of their tissues, they are also used directly for in vivo gene delivery experiments, without having to wait for production of a homozygous line.
  • Transgenic animals produced by this scheme are initially challenged by an injection of hFVIII protein, bled, and screened for antibodies to the human protein, to ensure tolerization to hFVTII.
  • the AFP-hFVIII-tolerized transgenic mice will also be tested for "leaky" expression of hFVIII in the adult animal.
  • hFVIII protein If a small amount of hFVIII protein is produced in adult transgenic animals, it is accurately quantitated so that it can be subtracted from the levels of hFVHl delivered by the therapeutic vector or protein delivery device.
  • the transgenic animal is tolerized to hFVIII and expresses insignificant levels of endogenous human protein, it can be used to test the efficiency of in vivo delivery of hFVIII, the duration of gene expression, tissue distribution, and immune reactions to elements of the delivery system, other than the transgene (i.e., vector backbone, viral coat proteins) may be analyzed. Other parameters may also be tested using the transgenic animal.
  • Example 15 Second Generation Transgenic Animal Models a. Breeding of AFP-hFVm tolerized mouse with A mouse FVIII knockout
  • a transgenic mouse strain with a targeted disruption (gene knockout) of the mouse Factor VTTI gene has been obtained through a non-exclusive, restricted-use license agreement with John Hopkins University and The University of Pennsylvania.
  • This mouse line has severe mFVUI deficiency and thus is a useful model for hemophilia A (51).
  • an accurate quantitation of hFVIII In addition, this doubly-transgenic mouse provides a useful model for the phenotypic correction of hemophilia A using gene therapy.
  • a further embodiment of this invention involves the crossing of all three above- mentioned transgenic animals to produce a "triple-transgenic" mouse model.
  • the mouse described in the previous section which is tolerized to human FVTII and deficient in mouse FVIII, is cross bred with the AAVSl transgenic mouse line.
  • This triple transgenic mouse model is preferredly suited for testing all aspects of our AAV-miniAd-hFVIII vector system including: site-specific integration at AAVSl via the AAV ITR/ Rep integration system; delivery and long-term expression of the human FVTII transgene without immune reaction to the tolerized transgene; accurate quantitation of delivered hFVIII due to a lack of adult expression of human FVITI as well as a lack of cross- reacting mouse FVTII protein; and, finally, genetic and phenotypic correction of severe FVIII deficiency (hemophilia A).
  • a transgenic mouse tolerized to green fluorescence protein It is convenient to inco ⁇ orate the GFP expression cassette into the various virus vectors as a reporter gene in new mini-Ad vectors, AAV vectors, and novel versions of helper virus are developed. Viral infection, expansion, helper complementation and in vivo delivery to target cells is easily followed by visual detection of green fluorescence. It has been shown that immune responses to transgene-encoded proteins can negatively impact the stability of gene expression following injection of adenovirus vectors (30). In order to eliminate immune responses to the GFP transgene inco ⁇ orated into the vector, which could shorten the duration of GFP expression after injection into mice, a GFP- tolerized transgenic mouse is developed.
  • the AFP-EGFP-1 vector described in Figure 58, or a similar vector comprising the Rat Insulin Promoter (RIP) for pancreas-specific expression of GFP could be used for this purpose.
  • a RTP-EGFP-1 vector (Fig. 60) was used to transfect mouse ES cells in order to develop a stable, Neo R PJP-GFP ES cell line [RIP-GFP(+) ES].
  • RJP-GFP(+)ES cells are utilized to generate a GFP-tolerized transgenic mouse, in a manner identical to that described for the generation of an AFP-hFVIII tolerized mouse model ( Figure 59), substituting the RTP-EGFP-1 vector for the AFP-hFVITI vector.
  • the RTP-GFP tolerized mouse thus produced provides a useful research tool for the development of novel adenovirus vectors or other delivery systems that utilize the GFP transgene as a reporter.
  • transgenic animal models including the AAVSl transgenic mouse provided in Example 12, the hFVIII-tolerized transgenic mouse of Example 15, and the GFP-tolerized transgenic mouse of Example 19C, may be alternatively generated by direct DNA injection of the transgene (pAAVSl-Neo, mAFP- hFVITI-pGKNeo and RTP-EGFP-1, respectively). This is accomplished by injection of the transgene into the male pronucleus of mouse single cell ova to produce transgenic mice, as an alternative to using the ES cell technology descirbed above. To one skilled in the art, this is an obvious alternative method for producing a transgenic mouse. The present inventions, therefore, may be produced by either of the methods discussed in this application (57-61).
  • the present invention provides designs for a site-specific recombinase-based system that permits excision of an auto-replicative episome from the mini-viral sequences upon infection of target cells.
  • Site-specific recombinases have been extensively used to manipulate DNA. Site- specific recombinases catalyze precise recombination between two appropriate target sequences, cleaving DNA at a specific site and ligating it to the cleaved DNA of a second site (for a review, see Ref. 111).
  • Several systems have been identified and characterized such as the cre/loxP system from bacteriophage PI (111) or FLP/FRT from yeast (112).
  • the recognition sites (loxP and FRT) for both recombinases (ere and FLP) share a common structure: they have two inverted repeat elements (recombinase binding site) flanking a central core region (site of crossing-over).
  • the orientation of the target sites (as defined by the core region) is responsible for the final outcome: recombination between two parallel sites on the same molecule results in excision of intervening sequences generating two molecules, each one with a target site. Recombination between two antiparallel sites results in inversion of the intervening sequence. Recombination between two parallel sites in diferent molecules results in the integration of sequences flanked by target sites. Since excision is an intramolecular event, it is favored over integration.
  • recombinases will be used to excise sequences having an eukaryotic origin of replication (ori).
  • Mammalian ori sequences and binding factors have not been characterized to date.
  • some viral ori sequences and viral proteins required for initiation of replication have been characterized and inco ⁇ orated in plasmid vectors, some examples of which including but not limited to SV40 ori/T-Ag from simian virus 40 (113) and oriP/EBNA-1 from Epstein-Barr virus (114). These elements have allowed the generation of plasmids that replicate autonomously in eukaryotic cells and are stably maintained upon selective pressure.
  • Plasmids containing oriP and expressing EBNA-1 protein replicate once per cell cycle (115, 116) and are lost when selective pressure is removed from cells in culture.
  • nondividing cells such as hepatocytes.
  • an episome could remain stable for a long period of time. It is believed by the inventors of the present invention that the inco ⁇ oration of ori sequences in the mini-viral DNA will permit a extended expression of the transgene in nondividing cells.
  • the episomal minivirus elements include but are not limited to (Fig.
  • Recombinase expression cassette recombinase must be expressed only in target cells, because inappropriate expression in the cells used to generate the virus will promote the excision of the sequences contained between two recombination sites. For this reason, expression is tightly controlled by either adding binding sequences for transcriptional repressors upstream of the promoter (for instance, tetO ) or through the use of tissue- specific promoters (ex: albumin promoter, factor VTTI promoter).
  • Origin of replication ori: must include the sequence to initiate or begin replication of DNA and any other element required for replication (ex: DNA binding protein recognizing origin sequences).
  • Transgene may be any therapeutic or reporter gene flanked by a recombination site (5') and a polyA signal sequence (3'). It will be expressed only in target cells upon circularization of the DNA.
  • Recombinase target sites two sites are necessary in parallel orientation, one being placed between the promoter and the recombinase cDNA and the other upstream of the therapeutic gene cDNA.
  • Adenovirus ITRs necessary for replication and packaging of the minivirus.
  • Sniffer DNA sequence if necessary to increase the size of the minivirus up to a packageable length.
  • the sniffer DNA sequence may be any DNA fragment of any length.
  • the recombinase is not expressed while amplifying the minivirus.
  • the promoter is functional, recombinase is expressed and the sequences contained between two recombinase target sites are excised and circularized.
  • the recombinase promoter turns into the transgene promoter and the presence of the origin of replication allows stable maintenance of the plasmid, therefore assuring stable expression of the transgene.
  • TGF ⁇ secretion by tumor cells may be a significant approach to cancer gene therapy (120, 121).
  • Fakhrai et al. used antisense to TGF ⁇ to inhibit fhe expression of that cytokine in a rat gliosarcoma cell line. Immunization of tumor-bearing rats with the antisense modified tumor cells resulted in significant survival of animals compared to animal's immunization with tumor cells modified with control vectors.
  • Isaka et al. was able reduce the amount fibrotic disease in rats, by transfecting skeletal muscle with a cDNA encoding decorin.
  • Decorin is a small proteoglycan that inhibits the expression of TGF ⁇ .
  • CTLA-4 a molecule also expressed on T cells, binds B7-1 and B7-2 with much higher affinities than CD28.
  • CTLA-4 acts as a negative regulator of T cell responsiveness, and raises the possibility that blocking the inhibition delivered by the CTLA-4-B7 interaction might augment the T cell response to tumor cells and enhance antitumor activity.
  • Leach et al. demonstrated that injecting antibody to CTLA-4 resulted in the rejection of tumors including pre-established tumors in a mouse model (124). This demonstrates the care must be used in designing gene transfer experiments such that the desired effects are not masked by other potential deleterious effects.
  • the genetic basis of cancer includes abnormalities in oncogenes and/or tumor suppressor genes. Both types have been the targets of cancer gene therapy. Because the cancer-related defects of tumor suppressor genes are usually mutations or deletions, the strategy in tumor suppressor gene therapy thus far developed has been gene replacement therapy, in which a wild-type tumor suppressor gene is transferred into cancer cells to restore the normal function of the defective gene or induce tumoricidal effect (124).
  • the human tumor suppressor genes that have been cloned and characterized include Rb, Wilms tumor (WT1), and neurofibromatosis (NF1), which are involved in pediatric cancers; adenomatosis polyposis coli (APQ and deleted in colon cancer (DCC), which contribute to colorectal cancer; which is found in mutated forms in a wide range of human cancers (for a review, see Ref. 125).
  • WT1 Wilms tumor
  • NF1 neurofibromatosis
  • APQ and deleted in colon cancer DCC
  • i 6 major tumor suppressor 1
  • MTSI major tumor suppressor 1
  • MTS2 major tumor suppressor 2
  • MTS2 major tumor suppressor 2
  • MTS2 major tumor suppressor 2
  • MTS2 major tumor suppressor 2
  • MTS2 major tumor suppressor 2
  • MTS2 major tumor suppressor 2
  • MTS2 major tumor suppressor 2
  • MTS2 major tumor suppressor 2
  • MTS2 major tumor suppressor 2
  • MTS2 major tumor suppressor 1
  • MTSI major tumor suppressor 1
  • MTSI major tumor suppressor 2
  • the mini-Ad vectors may carry of the multiple genes that function to suppress tumor growth or induce host anticancer immune responses. This type of vectors is called anticancer super-Ad vectors.
  • the first version of the super- Ad vector will carry four double expression cassettes for human p53 cDNA, GFP marker gene, human IL2 cDNA, human GM-CSF cDNA, human B7-1 cDNA, human IL7 cDNA and human IL12 p35 and p40 cDNA.
  • Cassette 1 includes a CMV promoter, a Human p53 cDNA, an EMC-IRES, a GFP gene and a SV40 pA.
  • Cassette 2 includes an EF promoter, a human GM-CSF cDNA, an EMC- IRES, a human TL 12 cDNA and a bovine growth hormone pA.
  • Cassette 3 includes an SV40 promoter, human B7-1 cDNA, an EMC-TRES, human TL7 cDNA and SV40 pA.
  • Cassette 4 includes a tk promoter, a Human IL12 p35 cDNA, an EMC-TRES, a human IL12 p40 cDNA and a bovine growth hormone pA (Fig. 64).
  • a second version of the anti-cancer super Ad vectors has a similar structure to that of the first version, including adenovirus inverted terminal repeats at both the 5' and 3' ends and four discrete expression cassettes.
  • Several combinations of regulatory molecules and genes may be utilized in the constrcution of anti-cancer super Ad vectors. The examples described below are not in any way limiting to the types of mini- Ad vectors that may be constructed to regulate the growth of a tumor cell.
  • Each expression cassette is flanked at the 5' end by a unique promoter.
  • each expression cassette inco ⁇ orates two genes linked by the encephalomyocarditis virus internal ribosome entry site sequence for cap independent translation of the "distal" gene.
  • the genes shown for this vector include cytokine genes as represented by IL-2, IL-7, and GM-CSF; a tumor suppresser gene as represented by p53; immune cell co-stimulatory molecules as represented by B7-1 and ICAM-1; and molecules that can reverse the immune suppression often associated with cancers, anti-TGF ⁇ and SCA to CTLA-4.
  • cytokine genes as represented by IL-2, IL-7, and GM-CSF
  • a tumor suppresser gene as represented by p53
  • immune cell co-stimulatory molecules as represented by B7-1 and ICAM-1
  • molecules that can reverse the immune suppression often associated with cancers, anti-TGF ⁇ and SCA to CTLA-4 to increase the size of the vector so that the vector will be efficiently packaged into progeny virus.
  • the sniffer DNA may include any DNA fragment of any length.
  • the general structure of the second version of the anticancer super-Ad vectors are shown in Figure 63.
  • Mini-Ad vectors having targeting capability Multiple mechanisms may be utilized to target gene expression to a specific cell type or tissue.
  • One such mechanism involves transcriptional targeting of a cell type, cell type subset or a specific tissue.
  • Transcriptional targeting includes the use of a transcriptional regulatory unit that drives gene expression in only a certain type of cell or tissue. Such a transcriptional regulatory unit is referred to as being tissue-specific.
  • a mini-ad vector is designed to inco ⁇ orate a tissue-specific transcriptional regulatory unit driving expression of a reporter or effector gene. In this manner, expression of the reporter or effector gene under control of the tissue-specific transcriptional regulatory unit will be detected at a higher level in those specific tissues in which the transcriptional regulatory unit is active.
  • tissue-specific gene expression may be utilized.
  • a helper virus that encodes a cell surface protein reactive to a ligand on a cell type of interest.
  • a helper virus may be engineered to express a ligand for a cell surface receptor.
  • a further example would include a recombinant adenovirus that expresses an antibody or a fragment of an antibody on the surface of its viral coat.
  • a recombinant virus may be produced by engineering a packaging- deficient helper virus to express an antibody or antibody fragment as a fusion or a separate protein on its viral coat.
  • a DNA molecule encoding an at least an adenoviral packaging sequence and at least one reporter or effector gene
  • recombinant adenoviral particles having an antibody or antibody fragment reactive to a cell surface molecule on a target cell are produced. In this manner, recombinant adenoviral particles will specifically bind to those cells in the host that express cell surface molecules reactive to said antibodies or antibody fragments.
  • the mini-Ad vectors that have local immune suppression function. Certain autoimmune disorders result from the inappropriate immune reactions.
  • One method that may be utilized to prevent, halt or slow the autoimmune reaction is to direct expression of immunomodulatory proteins at the site of such reactions. This may be accomplished by application of adenoviral particles constructed from a mini-Ad genome as demonstrated within this application. Genes encoding certain cytokines or chemokines may be expressed and such expression may result in an attenuation of the immune reponse. This attenuation in the immune response would then lead to an alleviation of the symptoms of the autoimmune reaction.
  • a further example may include the attenuation of an allergic reaction. An antigen known to cause an allergic reaction may be encoded by a mini-Ad vector.
  • Mini-Ad vectors that hybridize with other elements. It will also be possible to utilize the mini-Ad vectors disclosed in this application to prevent or eliminate viral infection and replication within a host. Mini-Ad vectors can be designed such that viral certain genetic processes may be interfered with or eliminated.
  • the mini-Ad vectors may be designed to express antisense nucleic acids that interfere with viral replication at the transcriptional or translational stage of infection. Interference may be promoted by the expression of antisense RNA or DNA including that which binds to messenger RNA or binds to DNA after integration of a viral genome to prevent transcription. Also, ribozymes may be designed that target certain viral transcripts for destruction. "Decoy" molecules may also be encoded by a mini-Ad vector.
  • Such decoys may function by binding to transcription factors required for viral trasncription such that the trasncription factors are no longer available for binding to and driving trasncription of genes required for viral gene expression and replication.
  • Mini- Ad vectors may be engineered to drive expression of certain antigens or immunogens that will serve to generate immunity in the organism in which expression takes place.
  • Mini-Ad vectors may be designed that drive expression of bacterial or viral genes that induce an immune reaction resulting in immunity.
  • a coat protein from a retrovirus such as HTV may be encoded by a mini-Ad vector.
  • mini-Ad vectors may also be designed to drive expression of cancer- specific antigens.
  • a recombinant adenoviral particle comprising a mini-Ad vector directing expression of a cancer antigen Upon infection of cells in a host with a recombinant adenoviral particle comprising a mini-Ad vector directing expression of a cancer antigen, immunity to that type of cancer will follow. Optimally, such immunity will result in widespread eradication of the primary tumor as well as other metastases and micrometastases that exist throughout the treated organism. Additionally, mini-Ad vectors may be designed that encode antigenic molecules derived from a parasite.
  • GENERAL INFORMATION (i) APPLICANT: BAXTER HEALTHCARE CORP.
  • a new adenoviral vector Replacement of all viral coding sequences with 28 kb of DNA independently expressing both full-length dystrophin and ⁇ - galactosidase. Proc. Natl. Acad. Sci. USA 93: 5731-5736
  • the AAV origin binding protein Rep68 is an ATP-dependent site-specific endonuclease with DNA helicase activity. Cell 61 : 447-457.
  • a maltose-binding protein/adeno-associated virus rep68 fusion protein has DNA-RNA helicase and
  • Adenovirus type 5 packaging domain is composed of a repeated element that is functionally redundant.
  • J. Virol. 64: 2047-2056. 98. Webster, N., J.R. Jin, S. Green, M. Hollis, and P. Chambon 1988.
  • the yeast UAS G is a transcriptional enhancer in human Hela cells in the presence of the Gal4 trans- activator. Cell 52: 169-178, 99. Gatz, C. and P.H. Ouau 1988. Tn/0-encodes tet repressor can regulate an operator- containing plant promoter. Proc. Natl. Acad. Sci. USA 85: 1394-1397. 100. Gossen, M. and H.
  • Kidney Cells (HEK-Ad-5 or 293). Virology 94: 460-469.
  • pl5INK4B is a potential effector of TGF- ⁇ - induced cell cycle arrest. Nature 371 : 257-261.

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