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WO2002088319A2 - Vecteur mini-adenoviral et ses methodes d'utilisation - Google Patents

Vecteur mini-adenoviral et ses methodes d'utilisation Download PDF

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Publication number
WO2002088319A2
WO2002088319A2 PCT/US2002/013661 US0213661W WO02088319A2 WO 2002088319 A2 WO2002088319 A2 WO 2002088319A2 US 0213661 W US0213661 W US 0213661W WO 02088319 A2 WO02088319 A2 WO 02088319A2
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vector
mini
dna
cells
gene
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PCT/US2002/013661
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WO2002088319A3 (fr
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Xiangming Fang
Mangala J. Hariharan
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Genstar Therapeutics Corp.
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Priority to AU2002308540A priority Critical patent/AU2002308540A1/en
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Publication of WO2002088319A3 publication Critical patent/WO2002088319A3/fr

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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39541—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against normal tissues, cells
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
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    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
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    • C12N2710/10351—Methods of production or purification of viral material
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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
    • C12N2830/005—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination repressible enhancer/promoter combination, e.g. KRAB
    • C12N2830/006—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination repressible enhancer/promoter combination, e.g. KRAB tet repressible
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    • C12N2830/00—Vector systems having a special element relevant for transcription
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    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

  • the present invention relates to adenoviral vectors for delivery of nucleic acids for expression of proteins, peptides and the like in cells.
  • the adenoviral vectors are largely or completely devoid of adenoviral protein coding sequences.
  • 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 which results from deficiencies in expression or function of clotting factor VIII (FVIII).
  • Treatment of hemophilia currently involves infusion of normal FVIII 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, Cl 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, FIX, 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 (IN.) 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.
  • 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 D ⁇ A 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.
  • This vector 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 infected cells 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.
  • ITR inverted terminal repeat
  • 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.
  • 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 incorporation of minimal cis elements for packaging and genome replication are under development by others (19-21).
  • the present invention provides a method for treating a disorder such as hemophilia.
  • a method of treating hemophilia in a mammal by administering recombinant virus virions comprising a nucleotide sequence having an adenoviral inverted terminal repeat fusion sequence, a packaging signal, a transcriptional control region, and a nucleic acid encoding a therapeutic protein such as FVIII.
  • the DNA molecule does not encode an adenoviral protein. It is preferred that the virions be administered to the mammal under conditions that result in the expression of the therapeutic protein at a level that provides a therapeutic effect in said mammal.
  • the adenoviral vector may be administered in conjunction with an immunosuppressive agent, such as an antibody.
  • FIGURES Figure 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.
  • D Additional embodiments of Mini- Ad vectors.
  • FIG. 4 Construction of the shuttle vector to generate the packaging attenuated helper AdH ⁇ .
  • 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 AdH ⁇ .
  • 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. Two weeks after cotransfection, several blue plaques were isolated and the mutation in the packaging signal was analyzed by PCR with oligos 7 an 8.
  • 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. 6 Amplification and characterization of AdH ⁇ .
  • 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. 7 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. 8 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 mini Ad 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 miniAd 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.
  • Figure 9 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. 10 Packaging efficiency of mini- viral vectors of different sizes. Packaging efficiency and amplification of miniAd vectors of different sizes.
  • the virus produced after co-transfection is indicated 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 5 and the difference between the miniAd vectors indicates the effect of the size in the packaging.
  • FIG. 11 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. M32 and AdH ⁇ co-purify in the band of higher density (number 4 or 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.
  • FIG. 12 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 the two GAL4 binding sites before A repeat I and 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. 13 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.
  • FIG. 14 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. 15 Position and sequences of synthetic oligos for ad Pac ' -tetO 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. 16 Construction of CMV-E1 mammalian expression vector.
  • Adenovirus 5 sequences 462-3537 (Afllll-Aflll Fragment) coding for EIA and EIB were blunt-end cloned into the EcoRV site of pcDNA3.
  • FIG. 1 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.
  • FIG. 1 A.
  • FIG. 19 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 1- and E 6 ) are shown.
  • Figure 20 Cloning of the 12.5 kb human albumin promoter into pBlueScript 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. 21 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 22 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 FVIII gene.
  • GT2059 contains the Ad ITRs in the opposite orientation of GT2061.
  • Figure 23 Restriction digest profiles of GT2053, GT2059 and GT2061. Digests show the expected banding patterns for BamHI, Xbal, Clal and Xhol in combination with Sail.
  • FIG. 24 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.) EBB 14, located further 3' in the ⁇ - fetoprotein gene.
  • FIG. 25 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. 26 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 FVIII gene therapy.
  • FIG. 27 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. 28 Scheme for generating the mini-AdFVHI 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. 29 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.
  • FIG. 30 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 31 Dynamic fluctuation in mini-AdFVIII and helper over time during serial passage.
  • the plot was obtained by densitometrical quantification of the bands shown in Figure 15.
  • Helper is labeled by the clear line with squares; mini-AdFVIII 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. 32 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.
  • Figure 33 MiniAd FVIII-mediated expression of human Factor VIII in vivo.
  • FIG. 34 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 35 Map of pCMV-hFVIII.
  • 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 36 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 (Invitrogen, 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 BamHI 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).
  • FIG. 37 Design of the mini-adenoviral vector containing an integratable GFP cassette.
  • Minimal Ad elements necessary for replication and packaging present in the construct are the Ad ITRs and packaging signal.
  • the GFP 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 38 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 immunob lotted with the same antibody used in immunoprecipitation.
  • an anti-Rep monoclonal antibody clone 226.7; ARP, Belmont, MA 02178
  • Figure 40 Southern blot of 293 clones transfected with plasmids GT9012 or GT9013. The conditions are as described in Figure 33.
  • FIG. 41 Southern Blot Analysis of AAVSl 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 AAVSl sequence was detected by PCR (AAVSl PCR(+)) digested with EcoRI (Fig. 23 A) or EcoRI in combination with EcoRV electrophoresed on a 1% agarose gel, blotted onto a nylon membrane (Hybond N+, Amersham), and hybridized using the 253 bp AAVSl 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 42 Construction of the pAAVSl-Neo Vector.
  • An 8.2 kb EcoRI fragment comprising an AAVSl 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 43 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 FVIII cDNA; (d) Recombinase target sites; (e) Adenovirus ITRs; and (f) Stuffer DNA sequence.
  • Figure 44 Inhibition of Anti-vector IgG Response by Pretreatment with Anti-CD4 Antibody.
  • Figure 45 Human FVIII Levels in C57BL/6 Mice After Repeated Treatment with FVIII Mini-Ad and Anti-CD4 Antibody.
  • 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.
  • 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.
  • a reporter construct is defined as a subchromosomal and purified DNA molecule comprising a gene encoding an assayable product.
  • An assayable 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • Heterologous DNA is defined as DNA introduced into an adenoviral construct that was isolated from a source other than an adenoviral genome.
  • 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.
  • 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 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 immunomodulatory gene is defined as any gene that, upon expression of its nucleic acid or protein product, serves to alter an immune reaction.
  • a ribozyme is defined as an RNA molecule that has the ability to degrade other nucleic acid molecules.
  • immunosuppressive agent refers to a compound that suppresses the function of the immune system.
  • an antibody directed against CD4 may serve to suppress the function of CD4+ T cells.
  • antibodies against other immune molecules may serve to suppress the immune system.
  • immunosuppressive agents comprise peptides, polypeptides, and small molecules, among others including but not limited to anti-CD40L (CD 154); anti-B7 (CD80/CD86); anti-CTLA4 (CD152); anti-CD3; anti-CD19; anti-CD20; anti-CD22; cyclosporin A; cyclosporin G; FK506; rapamycin; leflunomide; mizoribine (Bredinin); berquinar sodium (BQR); deoxyspergualin; and, cyclophospamide.
  • anti-CD40L CD 154
  • anti-B7 CD80/CD86
  • anti-CTLA4 CD152
  • anti-CD3 anti-CD19
  • anti-CD20 anti-CD22
  • cyclosporin A cyclosporin G
  • FK506 rapamycin
  • leflunomide mizoribine
  • Bredinin berquinar sodium
  • deoxyspergualin and, cyclopho
  • an adenoviral vector in conjunction with an immunosuppressive agent, the vector may be administered following administration of the immunosuppressive agent.
  • an adenoviral vector may administered in conjunction with an immunosuppressive agent where the vector and the agent are administered concurrently.
  • an adenoviral vector and an immunosuppressive agent may be administered in conjunction with one another where the vector is administered prior to administration of the immunosuppressive agent.
  • the present invention provides a modified adenoviral
  • Ad vector in order to provide: 1) a large capacity 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.
  • a large capacity Ad vector a "mini- 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
  • 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 in certain embodiments 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 incorporate 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 incorporation 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.
  • the mini-Ad vector system consists of three major parts: 1) a helper Ad that may be packaging-deficient; 2) a mini- Ad vector having a minimal amount of the viral genome; and, 3) an Ad helper cell line that provide functions of El tra s-activation like 293 cells and/or regulation of packaging signal for the helper Ad.
  • the helper Ad generally comprises the viral genetic material required for self-replication as well as tr ⁇ «s-complementation of mini-Ad vector replication.
  • the helper Ad retains wild-type Ad genetic material except for an El deletion or substitution and, in certain embodiments, a manipulated packaging signal for 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 typically comprises minimal Ad genetic material including only the inverted terminal repeats (ITRs) and a wild-type packaging signal as cis- 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.
  • Exemplary 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 transacting 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 miniAd vector of the present invention, is recognized by the packaging protein with higher affinity than the manipulated packaging signal of the helper Ad, 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. In the absence of the mini-Ad vector and without attenuation of the packaging signal, the helper Ad is typically 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. In a preferred embodiment of the present invention, 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.
  • a packaging-attenuated 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.
  • this system of the present invention results in preferential propagation of the mini-Ad vector.
  • 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.
  • the contaminating helper Ad may be removed using biological, biochemical, or physical methods including but not limited to ultracentrifugation through a CsCl gradient. 3.
  • mini-Ad vector system of the present invention provides significant advantages 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 exhibits minimal immunogenicity to the vector itself; 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 adenoviral 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 c/s-element (ITRs and packaging signal), and in preferred embodiments, do not encode Ad proteins.
  • ITRs and packaging signal a minimal amount of viral c/s-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 preferably comprises 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 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 purpose 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, other essential regulatory genes such as E2 and E4, for example, 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 purpose 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 incorporate a partial deletion of the consensus adenosine-enriched motif (e.g. "A-repeat”: TAAATTTG; Fig. 3) from the wild-type Ad packaging signal.
  • Ad5 packaging signal has a consensus A (adenosine) enriched motif (e.g. A-repeat: TAAATTTG), incorporation of an array of tandem repeats including but not limited to a selected A-repeat or any synthetic DNA motifs that may alter the affinity of the packaging protein for the artificial packaging signal.
  • A-repeat TAAATTTG
  • the 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. Packaging signal relocation 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.
  • 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).
  • cts-elements and trans-acting factors. Therefore, other possible designs may be oriented towards manipulation of either or both of these two factors.
  • An example of czs-elements that may be manipulated is the A-repeat motif.
  • An example of a transacting factor that may be manipulated is a packaging protein. Further consideration should be a controllable mechanism of packaging without sacrificing the high titer output 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 may be included in a mini-Ad vector.
  • 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.
  • a gene of interest i.e., heterologous DNA, insert DNA
  • 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 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 (Fig. 3B).
  • 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).
  • ITRs inverted terminal repeats
  • AAV adeno-associated virus
  • AAV utilizes these elements to achieve specific integration of its genome in human chromosome 19 (19ql3.3-qter) at a site named AAVSl.
  • 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. Each of these have proven to be difficult challenges to those skilled in the art.
  • the present invention combines the advantages of the mini-Ad vector with the integration capacity of AAV by incorporating 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).
  • 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.
  • 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. Various combinations of such elements may be incorporated into the mini-Ad to enhance or control expression of a gene of interest.
  • Tissue specific promoters may be utilized to drive gene expression in a specific cell type or tissue. Many such promoters are available to one of skill in the art.
  • Further supporting elements may include but are not limited to DNA replication origins for prokaryotic or eukaryotic cells, plasmid or vector selection markers, and vectors backbones. The skilled artisan would understand the need to incorporate one or more of such supporting elements into the mini-Ad vector as necessary.
  • 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.
  • Enhanced DNA replication Ad has its own enzymatic system for DNA replication.
  • the E2 region proteins are the major trans-acting elements responsible for viral DNA replication.
  • the replication origins are the cts-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 packaging signal A higher number or more efficient packaging sequences may be utilized by, for example, incorporating a greater number of tandem repeats at one or both ends of the mini-Ad genome, or by incorporation 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 (Fig. 3B), respectively (66, 68).
  • a recombination site such as lox-p or FRP (Fig. 3B), 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.
  • Modifications of the mini-Ad vector designed to increase the copy number of the mini-Ad genome within a host cell are useful in the development of a high-titer mini-Ad vectors.
  • Expression of the SV40 T-Ag (mutated T-Ag with no transforming activity) by the host cell may increase the copy number of the mini-Ad genome, provided a SV40 DNA replication origin is incorporated into the mini-Ad plasmid vector.
  • 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. Single or multiple genes including but not limited to those having functions of tumor suppression are utilized to optimize the anti-cancer effect.
  • the miniAd vector has the capacity to deliver multiple genes and is useful in constructing anti- cancer Ad vectors for administration.
  • Modulation of host immunity by genetic modification of the graft cells or tissues Transplantation requires transient or permanent suppression of the host immunity.
  • To deliver 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 TGF- ⁇ , 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. Because the 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 by 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 mini-Ad vector system itself has a great value for basic adenovirology studies.
  • the construction and demonstration of the feasibility and operation are already a breakthrough in the field.
  • the helper Ad and the mini-Ad provide convenient tools for study of the Ad and its potential applications. This is particularly true for the 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. h.
  • Ad vectors To be used in combination with other methodology in the field of gene transfer and therapy
  • 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 compound that comprise the ability to serve as a gene delivery complex. i. To be used for other purposes 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, j. Pharmaceutical Compositions
  • the vectors of the present invention may be administered by any suitable route including but not limited to oral, parenteral, inhalation, rectal, topical, intravenous (i.e., the portal vein), intrarterial (i.e., hepatic artery), intrapleural, nasal, intrathecaly, or direct intaorgan injection in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles.
  • parenteral as used herein includes but is not limited to subcutaneous, intravenous, intradermal, intramuscular, intrasternal, infusion techniques or intraperitoneally, among others.
  • Suppositories for rectal administration of the drug can be prepared by mixing the drug with a suitable non- irritating excipient such as cocoa butter and polyethylene glycols that are solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum and release the drug.
  • a suitable non- irritating excipient such as cocoa butter and polyethylene glycols that are solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum and release the drug.
  • Other suitable routes would be understood by one of skill in the art.
  • the dosage regimen for treating a disorder or a disease with the vectors of this invention and/or compositions of this invention is based on a variety of factors, including the type of disease, the age, weight, sex, medical condition of the patient, the severity of the condition, the route of administration, and the particular compound employed. Thus, the dosage regimen may vary widely, but can be determined routinely using standard methods.
  • the pharmaceutically active compounds (i.e., vectors) of this invention can be processed in accordance with conventional methods of pharmacy to produce medicinal agents for administration to patients, including humans and other mammals.
  • the pharmaceutical composition may be in the form of, for example, a capsule, a tablet, a suspension, or liquid.
  • the pharmaceutical composition is preferably made in the form of a dosage unit containing a given amount of DNA or viral vector particles (collectively referred to as "vector").
  • vector may contain an amount of vector from about 10 3 -10 15 viral particles, preferably from about 10 6 -10 12 viral particles.
  • a suitable daily dose for a human or other mammal may vary widely depending on the condition of the patient and other factors, but, once again, can be determined using routine methods.
  • the vector may also be administered by injection as a composition with suitable carriers including saline, dextrose, or water.
  • nucleic acids and /or vectors of the invention can be administered as the sole active pharmaceutical agent, 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 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 purpose of driving expression of a gene or gene product in the liver.
  • Apo A-I structural 1 1 ,000 Apo A-I In a few cases, causes Dietary treatment, HMG- mutations decreased HDL-C levels with CoA reductase inhibitors no increase in coronary heart disease Two mutations lead to amyloidosis
  • HMG- hypercholesterolemia populations mediated endocytosis of LDL CoA reductase inhibitors causes LDL to accumulate in nicotimc acid plus bile acid- plasma Hyper-cholesterolemia binding resins
  • atherosclerosis result homozygous may need probucol, portacaval anastomosis, plasma exchange, and liver transplantation von Willebrand disease 8,000 von Willebrand factor Abnormal platelet adhesionand Vasopressin analogue mildly to moderately reduced DDAVP for mild deficiency factor VIII levels cause Plasma infusion and vWF bleeding treatment for severe patients
  • Factor IX deficiency 1 70,000 Faxtor IX Impaired blood coagulation Prophylaxis, plasma infusion, (hemophilia B) and Factor IX treatment
  • Protein C deficiency 1 10,000 protein C Impaired regulation of blood Long-term anticoagulation coagulation Predispositionto therapy, plasma or protein C thrombosis infusion Table 1. (Continued)
  • Phenylketonu ⁇ a (P U) -1 10,000 Phenylalamne Hepatic enzyme deficiency Low-phenylalantne-diet due to PAH deficiency births hydroxylase (PAH) causes hyperphenylalamnemia, therapy and enzyme (bacterail
  • Hereditary fructose 1 20,000/Sw ⁇ tz Fructose 1 ,6- Ingestion of fructose causes the Elimination from the diet of intolerance erland bisphosphate aldolase accumulation of fructose 1 - all sources of sucrose and B phosphate and hence multiple fructose, with supplement of dysfunctions in small intesting, vitamin C liver, and kidney
  • Glycogen storage disease -1 100,000 Glucose 6-phosphatase Hypoglycemia, Dietary rest ⁇ ction, nocturnal type la (von Gierke hyper pidemia, hyperu ⁇ cemia, nasogast ⁇ c infusion in early disease) and hyperlactic acidemia infancy, portacaval shunts, Glycogen accumulation in liver liver transplantation and kidney
  • Omithine transcarbamylase 1 70,000 - Omithine Impaired urea formationleads Dietary rest ⁇ ction, sodium deficiency 100,000 transcarbamylase to ammonia intoxication phenylbutyrate, and argi ne Table 1. (Continued)
  • Methylmalonic acidemia 1 20,000 Methyldmalonyl-CoA Accumulation of Dietary protein rest ⁇ ction and
  • allehc va ⁇ ants mutase (MUT) methylmalonate leads to oral antibiotic therapy designated muC and mut-) apoenzyme metabolic ketoacidosis and developmental retardation
  • 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 granules.
  • 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 acids.
  • 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 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 (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, Cl 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 as an example of a mini-Ad vector having FVIII-like activity, provides a mini-Ad vector comprising the human FVIII gene.
  • Various naturally occurring and recombinant forms of Factor VIII have been described in patent and scientific literature such as U.S. Pat. No. 5,563,045, U.S. Pat. No. 5,451,521, U.S. Pat. No. 5,422,260, U.S. Pat. No. 5,004,803, U.S. Pat. No. 4,757,006, U.S. Pat. No. 5,661,008, U.S. Pat. No.
  • Nucleic acid sequences encoding Factor VIII may be prepared by recombinant methods, such as by screening cDNA and / or genomic DNA libraries from cells expressing Factor VIII or by deriving the sequence from a vector known to include the same.
  • the desired sequence may also be isolated directly from cells and tissues expressing FVIII mRNA, using standard techniques, such as PCR of cDNA and / or genomic DNA (See e.g., Sambrook et al., supra, for a description of techniques used to obtain and isolate DNA). Such nucleotide sequences may also be produced synthetically.
  • the sequence may be assembled from overlapping oligonucleotides and assembly of the oligos into a complete coding sequence (i.e., Edge, Nature 292:756 (1981); Nambair et al., Science 223:1299 (1984); and Jay et al., J. Biol. Chem., 259:6311 (1984)).
  • 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 (AAVSl site).
  • AAVSl site a human integration sequence
  • 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 AAVSl 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.
  • Immunosuppressive agents useful for thse applications include ab to a variety of immune system components such as anti-CD40L (CD 154); anti- B7 (CD80/CD86); anti-CTLA4 (CD152); anti-CD3; anti-CD19; anti-CD20; anti-CD22; as well as other immunosuppressive agents including but not limited to cyclosporin A; cyclosporin G; FK506; rapamycin; leflunomide; mizoribine (Bredinin); berquinar sodium (BQR); deoxyspergualin; and, cyclophospamide as well as others immunosuppressive agents known in the art.
  • 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.
  • dl 10/28 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).
  • dl 10/28 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 dllO/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'- GGAACACATGTAAGCGACGG (SEQ ID NO: 3) (nt 137 to 163 of Ad5 with Afllll site underlined) and R8: 5'- CCATCGATAATAATAAAACGCCAACTTTGACCCG (SEQ ID NO: 4) (nt 449 to 421 with Cla I site attached).
  • the amplified 133 bp fragment was cut with Afllll and Clal and used to substitute the corresponding sequence of the shuttle vector GT4004 (see Fig. 4 for this construction scheme).
  • 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. This extended left arm increases the frequency of homologous recombination used to generate recombinant virus.
  • 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.
  • Fig. 5 To generate the helper virus the method described by Graham and Prevec was used (91) (Fig. 5). An early passage of 293 cells obtained from ATCC, were grown in MEM- 10%) Horse Serum and seeded in 60 mm plates. At 30%> confluence cells were cotransfected by CaPO 4 using 2 mg of GT5001 and 4 mg of pJM17 (91) per plate. Three days after cotransfection cells were overlaid with medium containing 0.5 %> agarose and thereafter the medium above the overlay was changed every-other day. When plaques became visible, X-gal (40 mg/ml in DMSO) was directly added to the medium to 100 mg/ml an let incubating overnight. Plaques producing the desired helper virus were identified by the blue color (Fig. 6).
  • 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 rpm 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 This helper Ad is hereafter termed "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) and plaques identified by staining with X-gal.
  • the deleted size of the packaging signal was verified by PCR of the supernatant. 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 rpm, 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.
  • 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 EIA (providing the bacterial replication origin and ampicilin and tetracycline resistant genes) (93).
  • EIA providing the bacterial replication origin and ampicilin and tetracycline resistant genes
  • FIG. 1 Examples of various mini-viral vectors demonstrated in the current literature as well as that of the present invention are illustrated in Figure 1.
  • 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
  • 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 TVa2 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 ( Figures 3B and 7, bottom).
  • the head-to-tail fused ITRs and the packaging signal next to the left ITR 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.
  • An internal ribosome entry site (IRES) and a neomycin (neo) cDNA were then subcloned between the CA promoter and the GFP gene to produce M7.9.
  • a similar minivirus was generated comprising neo and GFP in two separate cassettes, M8.5: the Nru I-BstE II fragment from pREP9 (Invitrogen) containing the Tk promoter, neo cDNA and Tk pA, was blunted and subcloned into Stu I- EcoR I of M6.5.
  • M8.5 was used to construct a larger miniAd plasmid in order to test the packaging of miniAd vector with a complete substitution of the adenoviral genome by exogenous DNA.
  • 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 Ca 2 PO 4 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. 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 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.
  • the 11.5 Kb size difference would result in a 7.6 fold less packaging efficiency and amplification may not then be possible.
  • Complete substitution of the viral genome by exogenous DNA was possible and whether this would affect the packaging efficiency was tested.
  • 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 .
  • 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. 11). Every band was collected separately by aspiration from the top of the tube, and dialyzed.
  • the results indicate that the helper used with the partial deletion in the packaging signal taken from the dll8/28 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.
  • Example 2
  • GAL4 is a sequence-specific DNA-binding protein that activates transcription in the yeast Saccharomyces cerevisiae.
  • tetO comes from the Tn/0 -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 13 and 14).
  • 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 IX, consists of 9% of the left end of Ad5 (1.2 - 9.8 map units), and encodes two early region proteins, EIA and EIB.
  • EIA and EIB encodes two early region proteins
  • 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.
  • EIA 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).
  • recombination in 293 cells can also cause deletions and rearrangements that effect transgene expression, thereby decreasing the titer of functional adenovirus particles.
  • cell lines have been developed using defined Ad5 DNA fragments, including the El region, however these cell lines retain significant sequence overlap with homologous sequences in the El- deleted adenovirus vectors, which allows for undesirable homologous recombination events and the possibility for generation of RCA (107, 108).
  • Ad5 helper cell line 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 EIA, El A coding sequence, complete EIB promoter, and EIB coding sequence, including the stop codon for EIB p55 protein, but not including region IX.
  • Ad5 bp 462- 3537 The 3075 bp Afl III - Afl II E1A/E1B fragment (Ad5 bp 462- 3537) was isolated, blunted with Klenow enzyme, and blunt-end ligated into the EcoRV site of the mammalian expression vector, pCDNA3 (Invitrogen), under control of the CMV promoter/enhancer. This process generated an Ad5El expression vector, CMV-E1 (Fig. 16).
  • 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, as determined microscopically, 24 hrs. post-infection (Fig. 22). Infection with the El -deleted adenovirus, Ad5CA-GFP generated a clear area in the center of its plaque, which is evidence of the CPE caused by El -complemented virus amplification. The high infection rate as well as rapid generation of CPE induced in this cell line is strong evidence that functional E1A/E1B proteins are being produced which are capable of promoting the replication and amplification of the El -deleted Ad5CA-GFP virus.
  • 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 EIA 13S and 12S niRNAs (6), and identical in size to those observed in 293 cells (Fig. 18).
  • A549E1-68 produced a band of approximately 55 kd using a monoclonal Ab specific for EIB p55.
  • A549E1-68 not only expresses EIA 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/E1B 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.
  • the cells were further optimized and developed by a process of subcloning. Two subclones, A549E1-8 and A549E1-14, were selected. The doubling time of the subclones in serum-free culture is about 48 hr.
  • the clone A549E1-8 was adapted to serum-free culture. Burst sizes of the A549E1-8 cells (in terms of vp/cell) in the culture conditions with or without serum were found to be comparable to that of 293 cells in conventional culture condition.
  • the A549E1-8 cells were also successfully adapted into anchorage- independent culture for a production process using a suspension stirred-tank bioreactor.
  • 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 delivers the FVIII gene to the liver. It is, therefore, preferred 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.
  • Three regions within the 12.5 kb promoter segment have been determined to significantly influence promoter activity (32): 1.) the 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. 19. 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 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. 20).
  • the 7.2 kb full-length human FVIII cDNA with a 5' flanking SV40 immediate early intron and a 3' flanking SV-40 poly-adenylation signal was excised from plasmid GT2051 by Xhol / Sail digestion and was cloned into the Sail site of GT4031 to generate plasmid GT2053 (Fig. 21).
  • the Xhol fragment derived from plasmid GT2033 containing the minimal ITR region and Ad packaging signal was then cloned into the Sail site of GT2053 in either the forward or reverse orientation to generate the albumin/hFVIII minivirus plasmids GT2059 and GT2061, respectively (Fig. 22).
  • the restriction enzyme digest patterns of the GT2059 and GT2061 minivirus plasmids are shown in Figure 23.
  • 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 ⁇ i-antitrypsin (cti-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 ⁇ i-antitrypsin (cti-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
  • 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.
  • a figure of the albumin gene, an intergenic region and the ⁇ -fetoprotein gene regions utilized in the present invention is shown in Figure 24.
  • FIG. 25 The structure of the expression cassette in plasmid GT2061 comprising the 12.5 kb albumin promoter at the 5' end and several regions serving as 3' homologous recombination arms is presented in Figure 25. These vectors serve as homologous recombination replacement vectors since the orientation of the arms are in identical orientation as the sequences in the normal human genome.
  • 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 25A.
  • Plasmid GT2063 was constructed by insertion of the Xhol albumin gene fragment of plasmid pE5 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 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
  • the resulting plasmid, GT2053, contains unique Sal I and Xhol sites located 3' to the polyadenylation site ( Figure 21).
  • 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 supernatants 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-mini virus 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. 28B).
  • CPE cytopathic effect
  • hFVIII in 293 cells was expected to be minimal because the human albumin promoter is not very active in these cells. This has been determined using both CAT assays (69) and an FVIII chromogenic assay (Helena Laboratories, Pharmacia) following transfection of 293 cells with GT2061 using the calcium phosphate precipitation transfection method.
  • the viral DNA was used for PCR and Southern blot to detect mini-Ad and, independently, helper Ad virus DNA.
  • 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 (SEQ ED NO:l; ACCAGTCAAAGGGAGAAAGAAGA), FVIII primer #2 at a final concentration of 1 ⁇ M (SEQ ID 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. The results indicated that the FVIII minivirus was present in early passages (passage 3; data not shown).
  • Figure 29 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 miniAd 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 30).
  • 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 30 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 31.
  • the intensity of the bands from equal amounts of marker (1 Kb ladder marker from Gibco, Gaithersburg, MD) were used to normalize the results of the different blots.
  • 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 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 comprise high levels of FVIII mini-Ad and helper Ad, although at an undesirable FVIII mini-Ad to helper Ad ratio of 1 : 1.
  • a large scale amplification was performed at p20.
  • One hundred 15-cm dishes, each comprising approximately approximate 10 9 infected 293 cells (ATCC# CRL1573) were harvested upon completion of the CPE.
  • 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.
  • the FVIII mini-Ad (GT2063) was purified by CsCl as described above and utilized to demonstrate production of FVIII in host cells infected with the vector. To this end, 293 and HepG2 cells were utilized due to their known ability to utilize the albumin promoter. FVIII 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.
  • 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 FVIII 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).
  • FVIII specific mAb Cedar Lane Sheep anti-human FVIIIC, #CL20035A, Accurate Chemical and Scientific Co ⁇ oration, Westbury, NY
  • secondary antibody biotinylated donkey anti-sheep IgG, Jackson Immunoresearch, #713-065-14
  • DAB resulting in
  • 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 4.6xl0 10 transducing unit/ml.
  • the titer determined by optical absorbance at 260 nm, which reflects the number of viral particles
  • 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 FVIII Test (Pharmacia, Piscataway, NJ). A Coatest chromogenic assay for functional FVIII was performed.
  • a standard curve in triplicate from 4000 ng/ml to 62.5 ng/ml was plotted to obtain the equation to extrapolate the readings from the samples. Experiments were performed in triplicates. 10 ⁇ l aliquots of miniAdFVIII in 293 cells; 1 ⁇ l aliquots of mini-AdFVIII in 293 cells; 10 ⁇ l aliquots of mini-AdFVIII in HepG 2 cells; 1 ⁇ l aliquots of miniAdFVIII in HepG 2 cells; conditioned medium from untransduced 293 cells; and conditioned medium from untransduced HepG 2 cells were independently tested.
  • hFVIII protein produced by MiniAdFVIII HepG2 cells were infected with the vector and the conditioned medium was used for immunoblot analysis. Detection with a specific antibody to the heavy chain revealed several protein species ranging in size from 100 kDa to 200 kDa. A unique band of 80 kDa was detected with specific antibodies to the light chain. The pattern obtained from infected cells was comparable to that of recombinant FVIII (Hyland-Immuno, Baxter Healthcare Co ⁇ .). An observed slight difference in the intensity of the bands of the heavy chain may reflect a difference in post-translational processing in different cell lines, since the recombinant FVIII was produced from CHO cells. Taken together, the results demonstrate that hFVIII produced in vitro by the MiniAdFVIII-infected cells is biologically active and has the expected protein structure.
  • MiniAdFVIII activity studies Six to eight week old C57BL/6 and Balb/c mice were purchased from Harlan. Hemophilic mice (exon 16-disrupted FVIII knock-out mice) were obtained from the University of Pennsylvania ⁇ and bred in house. For dose- response studies, groups of three mice were injected via tail vein with different doses of MiniAdFVIII. At the designated timepoints, blood was collected by retro-orbital puncture in 0.1 volume of 0.1 M sodium citrate. Cells were removed by centrifugation and the plasma was tested for hFVIII by ELISA or functional assay.
  • mice Phenotypic correction studies. Groups of 12 mice were injected on day 1 with 2.5xl ⁇ " vp of MiniAdFVIII or vehicle (PBS), respectively. On day 3, blood was collected by retro-orbital puncture with a glass capillary tube. Tubes were broken at one minute intervals to check for clot formation and the clotting time was recorded. On day 6, a 2-cm section of the tail was clipped from each mouse to measure the bleeding time (time until bleeding stopped) and the volume of blood shed.
  • PBS vehicle
  • mice Toxicity studies. Groups of six C57BL/6 mice were injected via tail vein with the selected doses of MiniAdFVIII or with vehicle. Three and 14 days after injection, 3 mice from each group were sacrificed. Blood was collected by cardiac puncture and serum samples were tested for ALT levels (Boehringer Manheim). Tissues (liver, spleen, kidney, lung and heart) were fixed in formalin for 24 hr, paraffin-embedded and processed for histopathology. Analysis of Vector DNA in vivo. PCR assays with hFVIII specific primers
  • FIG. 6A were used for detecting DNA of the MiniAdFVIII and ancillary vectors.
  • Liver tissue (0.25g per sample) from MiniAdFVIII-treated mice was ground to a powder in a liquid nitrogen-cooled mortar and DNA was extracted using a Qiagen Tissue Kit (Qiagen Inc., Valencia, CA).
  • Each PCR reaction contained IX reaction buffer, 200 ⁇ M each deoxynucleotide triphosphate, 2.25-4.0 Mg ++ (the concentration varied with different primers), 0.4 ⁇ M of each primer and 2.5 units of Qiagen HotStar Taq polymerase in a total volume of 50 ⁇ l.
  • Amplification was carried out for one cycle of 15 min at 95°C followed by 35 cycles of 30 sec at 94°C, 30 sec at 52°C and 45 sec at 72°C with a final extension of 7 min at 72°C.
  • Aliquots of the PCR reactions (15 ⁇ l) were analyzed on 1.5% agarose gels. Amplified DNA fragments were semi-quantitated by comparing Gelstar- stained band intensities from tissue samples to those for the control plasmid (Gelstar, FMC BioProducts, Rockland, ME). Division of the copies by pg of input DNA yielded copies/pg input DNA. These values were normalized to Copies/Cell Equivalent by assuming 6 pg/cell nucleus then adjusting values accordingly.
  • ELISA assays were used to determine the antibodies against the hFVIII protein and the MiniAdFVIII vector.
  • the assay plates either coated with purified hFVIII protein or the ancillary vector in carbonate buffer, were blocked with PBS containing 4%> FBS for 1 hour at room temperature, washed once, and incubated overnight with serially diluted test plasma samples (lOO ⁇ l/well). After 5 washes, each well was incubated with lOO ⁇ l of 1 :2000 diluted goat anti-mouse IgG (H+L) conjugated to HRP (Southern Biotechnology Associates) at 37°C for 1 h.
  • MiniAdFVIII A mild-grade increased extramedullary hematopoeisis was found in all the injected mice irrespective of the virus dose. A characteristic feature was the presence of clusters of proliferating cells in the spleen. There was no dose-dependent ALT increase at any timepoint, indicating that injection of the highest dose did not cause significant liver injury. Other markers of liver injury were studied, such as hepatocyte apoptosis and expression of proliferating cell nuclear antigen (PCNA), which reflects the proliferating status of the liver. In both cases, liver sections for the highest dose group revealed similar numbers of apoptotic and proliferating cells as the control group for both timepoints in accordance with the ALT and histopathology data.
  • PCNA proliferating cell nuclear antigen
  • MiniAdFVIII preparation a toxicity study with the ancillary Ad alone was carried out in Balb/c mice.
  • Five dose groups with 5 mice per group were given tail vein injections of the ancillary Ad at 5xl0 7 , 5xl0 8 , 5xl0 9 , 5xl0 10 , and 4xl0 n vp/mouse respectively.
  • Vehicle (PBS) was used as a negative control. From days 1-14 postinjection, no differences in general condition and body weight were observed between the treated mice and the PBS control. On day 14 postinjection, the mice were sacrificed. Tissue (liver, spleen, heart, lung, and kidney) samples were collected and analyzed for histopathology changes.
  • mice were injected with a single dose of the vector at 3xl0 u vp, lxlO 11 vp, or 2.5 xlO 10 vp per mouse. Tissue samples of heart, lung, liver, spleen, and kidney were collected on days 3 and 14 p.i. for histopathology analysis. A summary of the analysis is shown in Table 3.
  • ALT serum alanine aminotransferase
  • PCNA proliferating cell nuclear antigen
  • a dose-response study of the vector was performed in C57BL/6 mice (Fig. 33B). Groups of three mice were injected via tail vein with different doses of MiniAdFVIII. At various timepoints, plasma was collected and assayed for hFVIII expression by ELISA. Doses of 4xlO ⁇ and 2xlO n vp MiniAdFVIII mediated hFVIII expression in mice at levels above the human physiological levels (>200 ng/ml).
  • hFVIII 6xl0 10 vp produced relatively lower levels of hFVIII (-100 ng/ml) and at 2xl0 10 vp hFVIII levels were below the assay detection limit.
  • the hFVIII produced from the vector was compared to the recombinant hFVIII protein in plasma samples of the treated hemophilic mice.
  • the plasma samples were collected either on days 3 and 6 p.i. of MiniAdFVIII (2.5 x 10 n vp/mouse) or at 1.5 hr post-injection of the recombinant protein (3.5 or 14 units/mouse).
  • Specific activities of the proteins derived from the two sources were calculated by dividing the values derived from the chromogenic assay for FVIII function by those derived by the ELISA assay for FVIII protein.
  • mice The results indicate that the specific activities of the transgene product and the recombinant hFVIII in mouse models are comparable.
  • the specific activities of the hFVIII protein generated from the MiniAdFVIII- infected cells in vitro were within the same range as that of recombinant hFVIII at above 4 to 8 Units/ ⁇ g protein.
  • PCR analysis was used to detect biodistribution of the vector following the systemic administration of MiniAdFVIII.
  • the vector DNA was detectable in the tissue samples from liver, spleen, lungs, kidneys, and heart of the treated mice (data not shown), with the majority detected in the liver, as previously reported (Vrancken Peeters et al. Method for multiple portal vein infusions in mice: quantitation of adenovirus-mediated hepatic gene transfer. BioTechniques 20:278-285, 1996; Huard, et al. The route of administration is a major determinant of the transduction efficiency of rat tissues by adenoviral recombinants. Gene Ther. 2:107-115, 1995; Li, et al. Assessment of recombinant adenoviral vectors for hepatic gene therapy. Hum. Gene Ther. 4:403-409, 1993).
  • the sustained expression of hFVIII indicates the presence and activity of the vector DNA in treated animals.
  • a set of five pairs of PCR primers were designed to detect different segments along the MiniAdFVIII genome, as shown in Table 3.
  • mice with long-term transgene expression (HM48, HM28, and HM46) were sacrificed and liver DNA was extracted for the PCR assays. Specific PCR products of the predicted sizes were obtained and the copy numbers of the representative fragments were quantified on a cell equivalent basis. Although the copy numbers varied between individual mice, an approximate equal number of vector copies/cell were detected within each mouse, regardless of the fragment tested. Thus, the complete vector genome persisted in the transduced cells without apparent loss or amplification of any specific portion. In contrast, no specific products were detected for either of the two control mice (data not shown). These results show that the MiniAdFVIII vector was mainly distributed in liver via i.v. administration and the vector DNA stayed likely in an episomal form in the mouse liver.
  • 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 FVIII gene localized between the 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/hFVIII 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 FVIII cDNA or the B-domain deleted (BDD) factor VIII cDNA ( Figures 34 and 35, respectively).
  • Example 11 Construction of an integratable AAV-ITR/Rep system-based vector
  • 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.
  • helper virus like adenovirus or he ⁇ es virus.
  • 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 AAVSl (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 AAVS 1 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 AAVSl site in vitro (41).
  • AAV has been considered as a candidate vector for gene therapy.
  • the limited size of exogenous DNA that it can accept (4.2 Kb)
  • the difficulty in getting high titers in large-scale preparations and the loss of specific integration of the recombinant AAV have posed problems for the use of this virus as a gene therapy vector.
  • 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 the salt precipitation method (125).
  • Genomic DNA was digested with EcoRI and analyzed by Southern blot with a probe for AAVSl. EcoRI was chosen because the AAVSl locus is contained within an 8Kb EcoRI-EcoRI fragment.
  • Figure 39 shows that 50%> of the resistant colonies analyzed which derived from plasmid GT9003 (Rep-expressing plasmid) revealed rearrangements of at least one AAVSl 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 40, panel B). The pattern of bands obtained indicated that some AAVSl 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 reporter gene is GFP (Aequorea victoria green fluorescent protein).
  • GFP Aequorea victoria green fluorescent protein
  • This reporter makes cells suitable for isolation using methods including but not limited to sorting and single-cell cloning by flow cytometry, thereby eliminating effects of selective pressure imparted by the neo expression cassette. 293 cells were transfected with either plasmid. One day after transfection, cells falling into a given range of fluorescence (thus eliminating variability due to differences in transfectability) were sorted by flow cytometry and single-cell cloned in 96-well plates. Two to three weeks after sorting, colonies were scored for fluorescence.
  • 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 42, 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. 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.
  • 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).
  • recombinases will be used to excise sequences having a 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 extended expression of the transgene in nondividing cells.
  • the episomal minivirus elements include but are not limited to (Fig. 43): a) 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 VIII promoter). b) 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.
  • Stuffer DNA sequence if necessary to increase the size of the minivirus up to a packageable length.
  • the stuffer 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.
  • Transcriptional targeting includes the use of a transcriptional regulatory unit that drives gene expression in only a certain type of cell or tissue.
  • 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. It may be preferable to restrict gene expression to a certain cell type or tissue.
  • Therapeutic genes are often toxic if expressed in high amounts. Regulation of gene expression to specific tissues, then, may serve to protect the host from the adverse effects of high level gene expression of certain therapeutic genes.
  • a further method to direct tissue-specific gene expression would be to utilize a helper virus encoding 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.
  • an recombinant adenoviral particle that binds to a receptor on the surface of a cell is produced.
  • 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.
  • Such 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 packaging-deficient helper virus to express an antibody or antibody fragment as a fusion or a separate protein on its viral coat.
  • recombinant adenoviral particles having an antibody or antibody fragment reactive to a cell surface molecule on a target cell are produced.
  • recombinant adenoviral particles will specifically bind to those cells in the host that express cell surface molecules reactive to said antibodies or antibody fragments.
  • 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 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.
  • 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.
  • the vectors of the present invention may also be utilized to treat cancer as described below.
  • 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 (APC) and deleted in colon cancer (DCC), which contribute to colorectal cancer; and p53, which is found in mutated forms in a wide range of human cancers (for a review, see Ref. 125). Recently, two major events occurred in the area of identification of new tumor suppressor genes or cancer susceptibility genes.
  • pi 6 major tumor suppressor 1, MTS1
  • MTS2 cyclin-dependent kinase
  • MTS1 major tumor suppressor 1
  • MTS2 pi 5
  • p53 cyclin-dependent kinase
  • the FVIII Mini-Ad vector is a helper-dependent adenoviral vector devoid of all viral genes and carries the full-length human FVIII (hFVIII) cDNA under the control of the human 12.5-kb albumin promoter.
  • the FVIII vector produced sustained expression of hFVIII at therapeutic levels up to one year after a single intravenous injection (Balague et al., Blood, 95:820-828, 2000).
  • cynomolgus primates were treated with a single intravenous injection of the FVIII Mini-Ad.
  • hFVIII Three vector doses were administered (4.3 x 10 11 , 1.4 x 10 12 , and 4.3 x 10 12 vp/kg). In all three groups, hFVIII was detected and the highest values ranged from 28 to 88 mU/mL. No significant adverse effects were detected in the two lower dosage groups that resulted in the expression of therapeutic hFVIII levels. In the highest dose group, thrombocytopenia and minimal elevation in liver transaminase levels were transient and similar to those previously observed in mice.
  • FVIII Mini-Ad was injected into mice with or without anti-CD4 antibodies.
  • a first-generation adenoviral vector (AdH ⁇ ) was tested in parallel.
  • the mice were all pretreated with anti-CD4 or an isotype control antibody intiaperitoneally followed by treatment with either FVIII Mini- Ad or AdH ⁇ vectors (5 x 10 12 vp/kg) ( Figure 44).
  • Two weeks following re- administration on Day 112 the mice treated with FVIII Mini-Ad plus anti-CD4 had a low total anti-vector IgG response (titer ⁇ 1:1200).
  • mice treated with FVIII MiniAd plus the isotype control antibody had a high total anti-vector IgG response (titer 1 :160,000).
  • the AdH ⁇ vector produced a substantial anti -vector response upon re-administration despite anti-CD4 antibody treatment (titer 1 :50,000) indicating superior immune response suppression with the Mini-Ad compared to earlier generation Ad vectors.
  • mice treated with the FVIIII Mini-Ad vector in conjunction with an anti-CD4 antibody were treated with either the FVIII mini-Ad vector and a control antibody or an anti-CD4 antibody, or with a control 1 st generation Ad vector (AdH ⁇ ) and anti-CD4.
  • AdH ⁇ Ad vector
  • Rep administration was also performed in certain animals.
  • mice treated twice with anti-CD4 and the FVIII vector exhibited much higher levels of human FVIII after an extended period of time (at least 60 weeks).
  • FVIII levels were increased following combined anti-CD4 antibody and FVIII Mini-Ad treatment even for the initial administration of the FVIII Mini-Ad before the generation of memory immune responses.
  • a maltose-binding protein/adeno-associated virus rep68 fusion protein has DNA-RNA helicase and ATPase activities. J. Virol. 69: 3542-3548.
  • helper-dependent adenovirus helper system removal of helper virus by cre-mediated excision of the viral packaging signal. Proc. Natl. Acad. Sci. USA 93: 13565-13570.
  • Adenovirus type 5 packaging domain is composed of a repeated element that is functionally redundant.
  • the yeast UAS G is a transcriptional enhancer in human Hela cells in the presence of the Gal4 trans- activator. Cell 52: 169-178,
  • pl5INK4B is a potential effector of TGF- ⁇ -induced cell cycle arrest. Nature 371 : 257-261.

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Abstract

La présente invention concerne une méthode de traitement d'un trouble, tel que l'hémophilie. Selon l'invention, une méthode de traitement de l'hémophilie chez un mammifère consiste à administrer des virions de virus recombinant contenant une séquence de nucléotides dotée d'une séquence hybride à répétition terminale inverse adénovirale, un signal d'encapsidation, une région de régulation transcriptionnelle, et un acide nucléique codant une protéine thérapeutique, telle que FVIII. En outre, la molécule d'ADN ne code pas une protéine adénovirale. On administre, de préférence, les virions au mammifère dans des conditions qui se soldent par l'expression de la protéine thérapeutique à un niveau qui engendre un effet thérapeutique chez ce mammifère. Par ailleurs, on administre les virions avec des agents immunodépresseurs.
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