US20220364115A1 - Enhanced promoter - Google Patents

Enhanced promoter Download PDF

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US20220364115A1
US20220364115A1 US17/731,027 US202217731027A US2022364115A1 US 20220364115 A1 US20220364115 A1 US 20220364115A1 US 202217731027 A US202217731027 A US 202217731027A US 2022364115 A1 US2022364115 A1 US 2022364115A1
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promoter
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Stefano Colloca
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GlaxoSmithKline Biologicals SA
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • A—HUMAN NECESSITIES
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    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
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    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
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    • C12N2830/00—Vector systems having a special element relevant for transcription
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    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/44—Vectors comprising a special translation-regulating system being a specific part of the splice mechanism, e.g. donor, acceptor

Definitions

  • This invention is in the field of promoters for use in vectors such as plasmids or viruses, particularly viral vectors such as adenoviral vectors.
  • the present invention is directed towards an enhanced human CMV promoter.
  • vector refers to an agent (such as a plasmid or virus) that contains or carries genetic material and can be used to introduce exogenous genes into an organism.
  • agent such as a plasmid or virus
  • An adenoviral vector is one example of a type of vector.
  • RNA can be transcribed from the delivered DNA using an RNA polymerase.
  • An RNA polymerase can recognize specific promoter elements, enabling the transcription of the DNA sequence linked to that promoter element.
  • a promoter is a nucleotide sequence that permits the binding of RNA polymerase and directs the transcription of the DNA.
  • a promoter is located in a non-coding region of the DNA, proximal to the transcriptional start site. Sequence elements within promoters that function in the initiation of transcription are often characterized by consensus nucleotide sequences.
  • Vectors are often said to comprise an “expression cassette”.
  • the expression cassette comprises the genetic material of interest operatively linked to regulatory components in a manner which permits transgene transcription, translation, and/or expression of the DNA of interest in the host cell.
  • the promoter is one of these regulatory components. If the DNA sequence of interest (e.g. a gene) is heterologous to the vector sequences flanking the gene, it can be referred to as a “transgene”.
  • promoters include, but are not limited to, promoters from bacteria, yeast, plants, viruses, and mammals, including simians and humans.
  • a great number of expression control sequences, including promoters which are internal, native, constitutive, inducible and/or tissue-specific, are known in the art.
  • promoters examples include, without limitation, the TBG promoter, the retroviral Rous sarcoma virus LTR promoter (optionally with the enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer, see, e.g., Boshart et al, Cell, 41:521-530 (1985)), the CASI promoter, the SV40 promoter, the dihydrofolate reductase promoter, the ⁇ -actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1a promoter (Invitrogen).
  • TBG promoter the retroviral Rous sarcoma virus LTR promoter (optionally with the enhancer)
  • CMV cytomegalovirus
  • CASI promoter the SV40 promoter
  • the dihydrofolate reductase promoter the ⁇ -actin promoter
  • PGK phosphoglycerol kinase
  • the CMV promoter is strong and ubiquitously active. It has the ability to drive high levels of transgene expression in many tissue types and is well known in the art.
  • the CASI promoter is a synthetic promoter described as a combination of the CMV enhancer, the chicken beta-actin promoter, and a splice donor and splice acceptor flanking the ubiquitin (UBC) enhancer (US 8865881).
  • SEQ ID NO: 2 is a polynucleotide sequence encoding the CASI promoter
  • the invention relates to a new promoter. More particularly, the invention relates to a new human CMV promoter.
  • the present invention provides a promoter comprising:
  • cell-derived means that the promoter is obtained from a eukaryotic (for example, human) cell.
  • the cell-derived enhancer sequence is an ubitquitin (UBC) enhancer sequence.
  • UBC ubitquitin
  • the components (i) to (v) of the promoter are provided in the order listed above, i.e. component (i) is first, (ii) is second, (iii) is third, (iv) is forth and (v) is fifth.
  • the order of the two enhancers i.e. components (i) and (iv)) could be swapped.
  • the promoter comprises one or more of the following sequences:
  • the promoter comprises at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more, sequence identity to SEQ ID NO: 8, SEQ ID NO:10, SEQ ID NO:11 and/or SEQ ID NO:12.
  • the parts (i) to (v) of the promoter consist of the relevant sequence.
  • the promoter comprises:
  • the promoter further comprises:
  • the fragment of the chicken beta-actin sequence preferably comprises a 5′ untranslated region of the chicken beta actin sequence and does not contain the promoter sequence.
  • the chicken beta actin sequence may have at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more, sequence identity to SEQ ID NO: 9.
  • this fragment is preferably found between the hCMV promoter region (ii) and the splice donor region (iii).
  • the present invention relates to a new promoter having at least about 84.1%, or more, identity to SEQ ID NO: 3.
  • the promoter can include a nucleic acid sequence having at least about 84.5%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, or more, sequence identity to SEQ ID NO: 3
  • the promoter can include a nucleic acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more, sequence identity to SEQ ID NO: 3. In some embodiments, the promoter comprises or consists of a nucleic acid sequence of SEQ ID NO: 3.
  • the invention in another aspect, relates to a vector, such as an adenoviral vector or a plasmid, containing the new promoter described above. All of the features described above in relation to the promoter may be incorporated into the vector.
  • the invention provides an adenoviral vector of the invention, the adenoviral vector comprises an expression cassette, wherein the expression cassette comprises a transgene and a promoter, wherein the promoter comprises:
  • a vector of the invention is an adenoviral vector comprising an expression cassette, wherein the expression cassette comprises a transgene and a promoter, wherein the promoter comprises a nucleic acid sequence having at least 84.1% identity to SEQ ID NO: 3.
  • a vector e.g. an adenoviral vector
  • each expression cassette comprises a transgene and a promoter, wherein the promoter of the first expression cassette and/or the second expression cassette is the new promoter described above.
  • the first expression cassette comprises the promoter.
  • the second expression cassette comprises the promoter.
  • an adenoviral vector of the invention comprises a first and a second expression cassette, wherein each expression cassette comprises a transgene and a promoter, wherein the promoter of the first expression cassette and/or the second expression cassette is a promoter comprising:
  • an adenoviral vector comprises a first and a second expression cassette, wherein each expression cassette comprises a transgene and a promoter, wherein the promoter of the first expression cassette and/or the second expression cassette is a promoter having at least 84.1% identity to SEQ ID NO: 3.
  • the vectors (e.g. adenoviral vectors) of the invention are useful as components of immunogenic compositions for the induction of an immune response in a subject, methods for their use in treatment and processes for manufacture.
  • the adenoviral vector of the present invention is preferably derived from a non-human simian adenovirus, also referred to as a “simian adenovirus”.
  • the simian adenoviral vector of the present invention is a chimp adenovirus (for example ChAd155 or ChAd83).
  • the present invention also provides a composition comprising the above-mentioned adenoviral vector and a pharmaceutically acceptable excipient.
  • the present invention provides the above-mentioned adenoviral vector or composition comprising such an adenoviral vector for use as a medicament, a vaccine, and/or for the therapy or prophylaxis of a disease.
  • the invention also provides a method of inducing an immune response in a subject comprising administering the the above-mentioned adenoviral vector or composition described above to the subject.
  • a vector or composition of the invention can be used in the manufacture of a medicament for the prevention or treatment of a disease
  • FIG. 1 A simian adenoviral construct according to the invention with a dual expression cassette.
  • Inverted terminal repeats flank the 3′ and 5′ ends;
  • human CMV hCMV
  • Enchanced hCMV is the enhanced cytomegalovirus promoter;
  • N-M2-1 and F ⁇ TM are the RSV antigens;
  • WPRE is the Woodchuck Hepatitis Postranscriptional Regulatory Element;
  • ⁇ E3 denotes that the early gene 3 is deleted; fiber denotes the adenoviral gene encoding the fiber protein; and Ad5E4orf6 in a substitute in the early gene 4 (E4) region.
  • the vector of FIG. 1 was constructed by inserting a first transgene expression cassette in place of the E1 region of the adenoviral genome, and a second transgene expression cassette in the HE2 region, i.e., downstream of the right ITR.
  • FIG. 2 Comparison of the expression levels of vectors expressing F ⁇ TM transgene in a MRC5 cell line, demonstrated by western blot at 48 hours and 96 hours post-infection under non-reducing conditions. Cells were infected at multiplicities of infection of 500 and 1250.
  • FIG. 3 Comparison of the expression levels of vectors expressing NM2-1 transgene in a MRC5 cell line, demonstrated by western blot at 48 hours post-infection under reducing conditions. Cells were infected at multiplicities of infection of 250 and 1250.
  • FIG. 4 Comparison of the immunogencity from ChAd155 vectors expressing the RSV antigen F ⁇ Tm. The data was collected at 4 weeks and 8 weeks after vaccination with a dose of 5 ⁇ 10 8 virus particles.
  • FIG. 5 Comparison of the immunogencity from ChAd155 vectors expressing the M2 RSV antigen. The data was collected at 3 weeks after vaccination with a dose of either 10 7 or 10 6 virus particles.
  • FIG. 6 SeAP expression in MRC5 cells by ChAd155 with different promoters.
  • FIG. 7 SeAP expression in HeLa cells by ChAd155 with different promoters.
  • SEQ ID NO: 1 Polynucleotide sequence encoding wild type ChAd155
  • SEQ ID NO: 2 Polynucleotide sequence encoding the CASI promoter
  • SEQ ID NO: 3 Polynucleotide sequence encoding the enhanced hCMV promoter
  • SEQ ID NO: 4 Polynucleotide sequence encoding the hCMV NM2 bghpolyA cassette
  • SEQ ID NO: 6 Polynucleotide sequence encoding the hCMV F0 WPRE bghpolyA cassette
  • SEQ ID NO: 8 Polynucleotide sequence encoding the hCMV promoter and enhancer sequence (nucleotides 1-650 of SEQ ID NO: 3).
  • SEQ ID NO: 9 Polynucleotide sequence encoding a Chicken Beta-Actin Fragment (nucleotides 651-809 of SEQ ID NO: 3).
  • SEQ ID NO: 10 Polynucleotide sequence encoding the Splice Donor Region (nucleotides 810-824 of SEQ ID NO: 3).
  • SEQ ID NO: 11 Polynucleotide sequence encoding the ubiquitin (UBC) enhancer (nucleotides 825-1127 of SEQ ID NO: 3).
  • SEQ ID NO: 12 Polynucleotide sequence encoding the Splice Acceptor Region (nucleotides 1128-1187 of SEQ ID NO: 3).
  • Adenoviruses are nonenveloped icosahedral viruses with a linear double stranded DNA genome of approximately 36 kb. Adenoviruses can transduce numerous cell types of several mammalian species, including both dividing and nondividing cells, without integrating into the genome of the host cell. They have been widely used for gene transfer applications due to their proven safety, ability to achieve highly efficient gene transfer in a variety of target tissues, and large transgene capacity. Human adenoviral vectors are currently used in gene therapy and vaccines but have the drawback of a high worldwide prevalence of pre-existing immunity, following previous exposure to common human adenoviruses.
  • Adenoviruses have a characteristic morphology with an icosahedral capsid comprising three major proteins, hexon (II), penton base (III) and a knobbed fiber (IV), along with a number of other minor proteins, VI, VIII, IX, IIIa and IVa2.
  • the hexon accounts for the majority of the structural components of the capsid, which consists of 240 trimeric hexon capsomeres and 12 penton bases.
  • the hexon has three conserved double barrels and the top has three towers, each tower containing a loop from each subunit that forms most of the capsid.
  • the base of the hexon is highly conserved between adenoviral serotypes, while the surface loops are variable.
  • the penton is another adenoviral capsid protein; it forms a pentameric base to which the fiber attaches.
  • the trimeric fiber protein protrudes from the penton base at each of the 12 vertices of the capsid and is a knobbed rod-like structure.
  • the primary role of the fiber protein is to tether the viral capsid to the cell surface via the interaction of the knob region with a cellular receptor. Variations in the flexible shaft, as well as knob regions of fiber, are characteristic of the different adenovral serotypes.
  • the adenoviral genome has been well characterized.
  • the linear, double-stranded DNA is associated with the highly basic protein VII and a small peptide pX (also termed mu).
  • Another protein, V is packaged with this DNA-protein complex and provides a structural link to the capsid via protein VI.
  • Each extremity of the adenoviral genome comprises a sequence known as an inverted terminal repeat (ITR), which is necessary for viral replication.
  • ITR inverted terminal repeat
  • the 5′ end of the adenoviral genome contains the 5′ cis-elements necessary for packaging and replication; i.e., the 5′ ITR sequences (which can function as origins of replication) and the native 5′ packaging enhancer domains, which contain sequences necessary for packaging linear adenoviral genomes and enhancer elements for the E1 promoter.
  • the 3′ end of the adenoviral genome includes 3′ cis-elements, including the ITRs, necessary for packaging and encapsidation.
  • the virus also comprises a virus-encoded protease, which is necessary for processing some of the structural proteins required to produce infectious virions.
  • the structure of the adenoviral genome is described on the basis of the order in which the viral genes are expressed following host cell transduction. More specifically, the viral genes are referred to as early (E) or late (L) genes according to whether transcription occurs prior to or after onset of DNA replication.
  • the E1A, E1B, E2A, E2B, E3 and E4 genes of adenovirus are expressed to prepare the host cell for viral replication.
  • the E1 gene is considered a master switch, it acts as a transcription activator and is involved in both early and late gene transcription.
  • E2 is involved in DNA replication;
  • E3 is involved in immune modulation and E4 regulates viral mRNA metabolism.
  • L1-L5 which encode the structural components of the viral particles, is activated. Late genes are transcribed from the Major Late Promoter (MLP) with alternative splicing.
  • MLP Major Late Promoter
  • HE1 and HE2 sites were identified as potential insertion sites for a transgene since the insertion in these specific points does not interrupt the coding sequences or important regulatory sequences of a chimp adenovirus, such as a Type C or E chimp adenovirus, for example, ChAd155 and ChAd83.
  • the HE1 and HE2 sites can be identified by sequence alignment in any chimp adenovirus. Therefore, cloning of expression cassettes in the HE1 and HE2 sites of the ChAd genomes doesn't impact the virus replication cycle.
  • adenovirus vaccine development has focused on defective, non-replicating vectors. They are rendered replication defective by deletion of the E1 region genes, which are essential for replication. Typically, non-essential E3 region genes are also deleted to make room for exogenous transgenes. An expression cassette comprising the transgene under the control of an exogenous promoter is then inserted. These replication-defective viruses are then produced in E1-complementing cells.
  • replication-defective or “replication-incompetent” adenovirus refers to an adenovirus that is incapable of replication because it has been engineered to comprise at least a functional deletion (or “loss-of-function” mutation), i.e. a deletion or mutation which impairs the function of a gene without removing it entirely, e.g.
  • E1A, E1B, E2A, E2B, E3 and E4 such as E3 ORF1, E3 ORF2, E3 ORF3, E3 ORF4, E3 ORF5, E3 ORF6, E3 ORF7, E3 ORF8, E3 ORF9, E4 ORF7, E4 ORF6, E4 ORF4, E4 ORF3, E4 ORF2 and/or E4 ORF1).
  • E1 and optionally E3 and/or E4 are deleted. If deleted, the aforementioned deleted gene region will suitably not be considered in the alignment when determining percent identity with respect to another sequence.
  • the adenoviral vector is a replication defective adenovirus.
  • the first expression cassette is inserted in the deleted E1 region and so these adenoviruses will be replication defective.
  • the adenoviral vector is a replication competent adenovirus.
  • replication-competent adenovirus refers to an adenovirus which can replicate in a host cell in the absence of any recombinant helper proteins comprised in the cell.
  • a “replication-competent” adenovirus comprises intact structural genes and the following intact or functionally essential early genes: E1A, E1B, E2A, E2B and E4. Wild type adenoviruses isolated from a particular animal will be replication competent in that animal.
  • Viral vectors based on non-human simian adenovirus represent an alternative to the use of human derived vectors for gene therapy and genetic vaccines.
  • Certain adenoviruses isolated from non-human simians are closely related to adenoviruses isolated from humans, as demonstrated by their efficient propagation in cells of human origin. As humans typically do not develop immunity to simian adenoviruses, they promise to provide an improved alternative to human adenoviral uses.
  • Low seroprevalence may mean having a reduced pre-existing neutralizing antibody level as compared to human adenovirus 5 (Ad5). Similarly or alternatively, “low seroprevalence” may mean less than about 40% seroprevalence, less than about 30% seroprevalence, less than about 20% seroprevalence, less than about 15% seroprevalence, less than about 10% seroprevalence, less than about 5% seroprevalence, less than about 4% seroprevalence, less than about 3% seroprevalence, less than about 2% seroprevalence, less than about 1% seroprevalence or no detectable seroprevalence. Seroprevalence can be measured as the percentage of individuals having a clinically relevant neutralizing titer (defined as a 50% neutralisation titer >200) using methods as described in Hum. Gene Ther. (2004) 15:293.
  • the adenoviral vector of the present invention is derived from a nonhuman simian adenovirus, also referred to as a “simian adenovirus.”
  • a nonhuman simian adenovirus also referred to as a “simian adenovirus.”
  • Numerous adenoviruses have been isolated from nonhuman simians such as chimpanzees, bonobos, rhesus macaques, orangutans and gorillas. Vectors derived from these adenoviruses can induce strong immune responses to transgenes encoded by these vectors.
  • vectors based on nonhuman simian adenoviruses include a relative lack of cross-neutralizing antibodies to these adenoviruses in the human target population, thus their use overcomes the pre-existing immunity to human adenoviruses.
  • some simian adenoviruses have no cross reactivity with preexisting human neutralizing antibodies and cross-reaction of certain chimpanzee adenoviruses with pre-existing human neutralizing antibodies is only present in 2% of the target population, compared with 35% in the case of certain candidate human adenovirus vectors (Sci. Transl. Med. (2012) 4:1).
  • Adenoviral vectors of the invention may be derived from a non-human adenovirus, such as a simian adenovirus, e.g., from chimpanzees ( Pan troglodytes ), bonobos ( Pan paniscus ), gorillas ( Gorilla gorilla ) and orangutans ( Pongo abelii and Pongo pygnaeus ). They include adenoviruses from Group B, Group C, Group D, Group E and Group G.
  • a non-human adenovirus such as a simian adenovirus, e.g., from chimpanzees ( Pan troglodytes ), bonobos ( Pan paniscus ), gorillas ( Gorilla gorilla ) and orangutans ( Pongo abelii and Pongo pygnaeus ). They include adenoviruses from Group B, Group C, Group D, Group E and Group G.
  • Chimpanzee adenoviruses include, but are not limited to ChAd3, ChAd19, ChAd25.2, ChAd26, ChAd27, ChAd29, ChAd30, ChAd31, ChAd32, ChAd33, ChAd34, ChAd35, ChAd37, ChAd38, ChAd39, ChAd40, ChAd63, ChAd83, ChAd155, ChAd15, SadV41 and ChAd157.
  • adenoviral vectors may be derived from nonhuman simian adenoviruses isolated from bonobos, such as PanAd1, PanAd2, PanAd3, Pan 5, Pan 6, Pan 7 (also referred to as C7) and Pan 9. Vectors may include, in whole or in part, a nucleotide encoding the fiber, penton or hexon of a non-human adenovirus.
  • the adenoviral vector has a seroprevalence of less than 40%, less than 30%, less than 20%, less than 10% or less than 5% in human subjects, preferably no seroprevalence in human subjects and more preferably no seroprevalence in human subjects that have not previously been in contact with a chimpanzee adenovirus.
  • the adenoviral DNA is capable of entering a mammalian target cell, i.e. it is infectious.
  • An infectious recombinant adenoviral vector of the invention can be used as a prophylactic or therapeutic vaccine and for gene therapy.
  • the recombinant adenoviral vector comprises an endogenous molecule for delivery into a target cell.
  • the target cell is a mammalian cell, e.g.
  • bovine cell a bovine cell, a canine cell, a caprine cell, a cervine cell, a chimpanzee cell, a chiroptera cell, an equine cell, a feline cell, a human cell, a lupine cell, an ovine cell, a porcine cell, a rodent cell, an ursine cell or a vulpine cell.
  • Theendogenous molecule for delivery into a target cell is an expression cassette.
  • the vector comprises a left ITR region, a deleted E1 region, then a deleted E3 region, and, optionally, additional enhancer elements; these are followed by a fiber region, an E4 region and a right ITR. Translation occurs in the rightward and leftward directions.
  • the first expression cassette is inserted in the deleted E1 region, and the second expression cassette is insertion in the deleted E3 region.
  • the promoters of the two expression cassettes are CMV promoters.
  • the enhancer element is the Hepatitis B Postranslational Regulatory Element (HPRE) or the Woodchuck Hepatitis Postranslational Element (WPRE).
  • the vector comprises left and right ITR regions; a deleted E1 region; at least a partially deleted E3 region; a fiber region; an E4 region; two expression cassettes, each comprising: a promoter and at least one an antigen of interest and, optionally, one or more enhancer elements.
  • the first expression cassette is inserted in the deleted E1 region, and the second expression cassette is inserted at the HE1 site, i.e., between the stop codons of the fiber gene and an E4 region (“the HE1 site”).
  • the ChAd155 HE1 insertion site is between bp 34611 and 34612 of the wild type ChAd155 sequence.
  • the ChAd83 HE1 insertion site is between bp 33535 and 33536 of the wild type ChAd83 sequence. Translation occurs in the rightward and leftward directions.
  • the promoters are CMV promoters.
  • one promoter is a CMV promoter and the other is a eCMV promoter.
  • the enhancer element is HPRE or WPRE.
  • the vector comprises left and right ITR regions; a deleted E1 region; at least a partially deleted E3 region; a fiber region; an E4 region; two expression cassettes, each comprising: a promoter, at least one antigen of interest and, optionally, one or more enhancer elements.
  • the first expression cassette is inserted in the deleted E1 region, and the second expression cassette is inserted at the HE2 site, i.e., between the end of the left ITR and the cap site of the E4 mRNA (“the HE2 site”).
  • the ChAd155 HE2 insertion site is between bp 37662 and 37663 of the wild type ChAd155 sequence.
  • the ChAd83 HE2 insertion site is between bp 36387 and 36388 of the wild type ChAd83 sequence. Translation occurs in the rightward and leftward directions.
  • the promoters are CMV promoters.
  • one promoter is a CMV promoter and the other is a eCMV promoter.
  • the enhancer element is HPRE or WPRE (the enhancer element increases expression of the transgene).
  • the HE1 and HE2 sites were identified as insertion sites for a transgene, as the insertion in these specific points does not interrupt the coding sequences or regulatory sequences of ChAd155 and ChAd83. Therefore, inserting expression cassettes in the HE1 or HE2 sites of the ChAd genome does not affect the viral replication cycle.
  • the vector is a functional or an immunogenic derivative of an adenoviral vector.
  • derivative of an adenoviral vector is meant a modified version of the vector, e.g., one or more nucleotides of the vector are deleted, inserted, modified or substituted.
  • Regulatory elements i.e., expression control sequences, in addition to promoter sequences, include appropriate transcription initiation, termination and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (poly A) signals including rabbit beta-globin polyA; tetracycline regulatable systems, microRNAs, posttranscriptional regulatory elements e.g., WPRE, posttranscriptional regulatory element of woodchuck hepatitis virus); sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of an encoded product.
  • poly A polyadenylation
  • WPRE posttranscriptional regulatory element of woodchuck hepatitis virus
  • vectors carrying transgenes encoding therapeutically useful or immunogenic products may also include selectable markers or reporter genes.
  • the reporter gene may be chosen from those known in the art. Suitable reporter genes include, but are not limited to enhanced green fluorescent protein, red fluorescent protein, luciferase and secreted embryonic alkaline phosphatase (seAP), which may include sequences encoding geneticin, hygromicin or purimycin resistance, among others.
  • selectable reporters or marker genes (which may or may not be located outside the viral genome to be packaged into a viral particle) can be used to signal the presence of the plasmids in bacterial cells, such as ampicillin resistance.
  • Other components of the vector may include an origin of replication.
  • a “posttranscriptional regulatory element,” as used herein, is a DNA sequence that, when transcribed, enhances the expression of the transgene(s) or fragments thereof that are delivered by viral vectors of the invention.
  • Postranscriptional regulatory elements include, but are not limited to the Hepatitis B Virus Postranscriptional Regulatory Element (HPRE) and the Woodchuck Hepatitis Postranscriptional Regulatory Element (WPRE).
  • HPRE Hepatitis B Virus Postranscriptional Regulatory Element
  • WPRE Woodchuck Hepatitis Postranscriptional Regulatory Element
  • a ChAd155 vector may comprise one or more of a promoter, an enhancer, and a reporter gene.
  • vectors of the invention may comprise ChAd155-enhanced hCMV-SeAP ChAd155-CASI-seAP and ChAd155-hCMV-seAP, optionally with a tetracycline on/off transcriptional control and ChAd155 —CMV-hFerL-chEF1-seAP with a tetracycline on/off transcriptional control.
  • a ChAd83 vector may comprise one or more of a promoter, an enhancer, and a reporter gene.
  • vectors of the invention may comprise ChAd155 enhanced hCMV SeAP, ChAd83 enhanced hCMV SeAP, ChAd155-CASI-seAP and ChAd83-hCMV-seAP, optionally with a tetracycline on/off transcriptional control and ChAd83-CMV-hFerL-chEF1-seAP with a tetracycline on/off transcriptional control.
  • Vectors of the invention are generated using techniques provided herein, in conjunction with techniques known to those of skill in the art. Such techniques include conventional cloning techniques of cDNA such as those described in texts, use of overlapping oligonucleotide sequences of the adenovirus genomes, polymerase chain reaction, and any suitable method which provides the desired nucleotide sequence.
  • a “transgene” is a nucleic acid sequence, heterologous to the vector sequences flanking the transgene, which encodes a polypeptide of interest.
  • the nucleic acid coding sequence is operatively linked to regulatory components in a manner which permits transgene transcription, translation, and/or expression in a host cell.
  • the vectors express transgenes at a therapeutic or a prophylactic level.
  • a “functional derivative” of a transgenic polypeptide is a modified version of a polypeptide, e.g., wherein one or more amino acids are deleted, inserted, modified or substituted.
  • the transgene may be used for prophylaxis or treatment, e.g., as a vaccine for inducing an immune response, to correct genetic deficiencies by correcting or replacing a defective or missing gene, or as a cancer therapeutic.
  • induction of an immune response refers to the ability of a protein to induce a T cell and/or a humoral antibody immune response to the protein.
  • the immune response elicited by the transgene may be an antigen specific B cell response, which produces neutralizing antibodies.
  • the elicited immune response may be an antigen specific T cell response, which may be a systemic and/or a local response.
  • the antigen specific T cell response may comprise a CD4+ T cell response, such as a response involving CD4+ T cells expressing cytokines, e.g. interferon gamma (IFN gamma), tumor necrosis factor alpha (TNF alpha) and/or interleukin 2 (IL2).
  • IFN gamma interferon gamma
  • TNF alpha tumor necrosis factor alpha
  • IL2 interleukin 2
  • the antigen specific T cell response comprises a CD8+ T cell response, such as a response involving CD8+ T cells expressing cytokines, e.g., IFN gamma, TNF alpha and/or IL2.
  • cytokines e.g., IFN gamma, TNF alpha and/or IL2.
  • the composition of the transgene sequence will depend upon the use to which the resulting vector will be put.
  • the transgene is a sequence encoding a product which is useful in biology and medicine, such as a prophylactic transgene, a therapeutic transgene or an immunogenic transgene, e.g., protein or RNA.
  • Protein transgenes include antigens.
  • Antigenic transgenes of the invention induce an immunogenic response to a disease causing organism.
  • Transgenes such rabies virus antigens, e.g., rabies glycoprotein (RG), respiratory syncytial virus (RSV) antigens, human immunodeficiency virus (HIV) antigens, or fragments thereof would be suitable for use with promoters of the invention.
  • rabies virus antigens e.g., rabies glycoprotein (RG), respiratory syncytial virus (RSV) antigens, human immunodeficiency virus (HIV) antigens, or fragments thereof
  • rabies virus antigens e.g., rabies glycoprotein (RG), respiratory syncytial virus (RSV) antigens, human immunodeficiency virus (HIV) antigens, or fragments thereof
  • rabies virus antigens e.g., rabies glycoprotein (RG), respiratory syncytial virus (RSV) antigens, human immunodeficiency virus (HIV) antigens, or fragments thereof
  • rabies virus antigens
  • a polypeptide can be encoded by a variety of different nucleic acid sequences. Coding is biased to use some synonymous codons, i.e., codons that encode the same amino acid, more than others.
  • codon optimized it is meant that modifications in the codon composition of a recombinant nucleic acid are made without altering the amino acid sequence. Codon optimization has been used to improve mRNA expression in different organisms by using organism-specific codon-usage frequencies.
  • codon pair deoptimization has been used to reduce viral virulence. For example, it has been reported that polioviruses modified to contain underrepresented codon pairs demonstrated decreased translation efficiency and were attenuated compared to wild type poliovirus (Science (2008) 320:1784). Engineering a synthetic attenuated virus by codon pair deoptimization can produce viruses that encode the same amino acid sequences as wild type but use different pairwise arrangements of synonymous codons. Viruses attenuated by codon pair deoptimization generated up to 1000-fold fewer plaques compared to wild type, produced fewer viral particles and required about 100 times as many viral particles to form a plaque.
  • polioviruses modified to contain codon pairs that are overrepresented in the human genome acted in a manner similar to wild type RNA and generated plaques identical in size to wild type RNA (Coleman et al. (2008) Science 320:1784). This occurred despite the fact that the virus with overrepresented codon pairs contained a similar number of mutations as the virus with underrepresented codon pairs and demonstrated enhanced translation compared to wild type. This observation suggests that codon pair optimized constructs would be expected to act in a manner similar to their non-codon pair optimized counterparts and would not be expected to provide a functional advantage. Without wishing to be constrained by theory, this may be because natural evolution has optimized codon pairing.
  • a construct of the invention may comprise a codon optimized nucleic acid sequence.
  • a vector of the invention comprises a codon optimized sequence of a transgene or an immunogenic derivative or fragment thereof.
  • a construct of the invention may comprise a codon pair optimized nucleic acid sequence.
  • a vector of the invention comprises or consists of a codon pair optimized sequence of a transgene or an immunogenic derivative or fragment thereof.
  • the humoral immune response is capable of neutralizing the virus and inhibiting viral replication, thereby playing a major role in protection against lower respiratory RSV infection and severe disease [Piedra, 2003].
  • Passive immunization in the form of Immunoglobulin G (IgG) RSV-neutralizing monoclonal antibodies (Synagis) given prophylactically, has been shown to prevent RSV disease to some extent in premature infants and newborns with bronchopulmonary dysplasia or underlying cardiopulmonary disease [Cardenas, 2005].
  • T cells are also involved in the control of RSV disease.
  • Lethal RSV infections have been described in patients with low CD8 T cells counts, as in the case of severe combined immunodeficiency, bone marrow and lung transplant recipients [Hertz, 1989].
  • the histopathology of fatal cases of RSV infection of newborns shows that there is a relative paucity of CD8 T cells in the lung infiltrate [Welliver, 2007].
  • CD8 T cells producing Interferon-gamma (IFN- ⁇ ) has been associated with diminished Th2 responses and reduced eosinophilia in animal models of RSV [Castilow, 2008; Stevens, 2009].
  • Suitable antigens of RSV which are useful as immunogens to immunize a human or non-human animal can be selected from: the fusion protein (F), the attachment protein (G), the matrix protein (M2) and the nucleoprotein (N).
  • F protein or “fusion protein” or “F protein polypeptide” or “fusion protein polypeptide” refers to a polypeptide or protein having all or part of an amino acid sequence of an RSV Fusion protein polypeptide.
  • G protein or “G protein polypeptide” refers to a polypeptide or protein having all or part of an amino acid sequence of an RSV Attachment protein polypeptide.
  • M protein or “matrix protein” or “M protein polypeptide” refers to a polypeptide or protein having all or part of an amino acid sequence of an RSV Matrix protein and may include either or both of the M2-1 (which may be written herein as M2.1) and M2-2 gene products.
  • N protein or “Nucleocapsid protein” or “N protein polypeptide” refers to a polypeptide or protein having all or part of an amino acid sequence of an RSV Nucleoprotein.
  • RSV F protein can be an ectodomain of an RSV F Protein (F ⁇ TM).
  • Exemplary M and N protein nucleic acids and protein sequences can be found, e.g., in US published application number 2014/0141042 (WO2012/089833), which are incorporated herein for purpose of disclosing the nucleic acid and polypeptide sequences of RSV M and N proteins suitable for use in present invention.
  • Transgene nucleic acids may encode an RSV F antigen and RSV, M and N antigens. More specifically, the nucleic acids may encode an RSV F ⁇ TM antigen (fusion (F) protein deleted of the transmembrane and cytoplasmic regions), and RSV M2-1 (transcription anti-termination) and N (nucleocapsid) antigens.
  • RSV F ⁇ TM antigen fusion (F) protein deleted of the transmembrane and cytoplasmic regions
  • RSV M2-1 transcription anti-termination
  • N nucleocapsid
  • the RSV F protein is a major surface antigen and mediates viral fusion to target cells.
  • the F protein is an antigen which is highly conserved among RSV subgroups and strains.
  • the F protein is a target for neutralizing antibodies, including the prophylactic RSV-neutralizing monoclonal antibody Synagis. Deletion of the transmembrane region and cytoplasmic tail permits secretion of the F ⁇ TM protein. Neutralizing antibodies including Synagis, that recognize this soluble form of the F protein, inhibit RSV infectivity in vitro [Magro, 2010].
  • the N protein is an internal (non-exposed) antigen, highly conserved between RSV strains and known to be a source of many T cell epitopes [Townsend, 1984].
  • the N protein is essential for the replication and transcription of the RSV genome.
  • the primary function of the N protein is to encapsulate the virus genome for the purposes of RNA transcription, replication and packaging and protects it from ribonucleases.
  • the M2-1 protein is a transcription anti-termination factor that is important for the efficient synthesis of full-length messenger RNAs (mRNAs) as well as for the synthesis of polycistronic readthrough mRNAs, which are characteristic of non-segmented negative-strand RNA viruses.
  • M2-1 is an internal (non-exposed) antigen, which is highly conserved between RSV strains and known to be a source of many T cell epitopes [Townsend, 1984].
  • a polynucleotide encoding a linker is positioned between the polynucleotide encoding an RSV N antigen, or fragment thereof, and the polynucleotide encoding an RSV M2.1 antigen, or fragment thereof.
  • an expression cassette contains a transgene which encodes a fused RSV viral protein N-linker-M2.1
  • the linker is a flexible linker, preferably a flexible linker comprising an amino acid sequence according to SEQ ID NO: 13 (Gly-Gly-Gly-Ser-Gly-Gly-Gly) or SEQ ID NO: 14 (Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Gly).
  • the recombinant adenoviral vector of the invention is administered to a subject by epicutaneous administration, intradermal administration, intramuscular injection, intraperitoneal injection, intravenous injection, nasal administration, oral administration, rectal administration, subcutaneous injection, transdermal administration or intravaginal administration.
  • the vectors can be administered intramuscularly (IM), i.e., injection directly into muscle. Muscles are well vascularized and the uptake is typically rapid.
  • IM intramuscularly
  • adjuvants are meant an agent that augments, stimulates, activates, potentiates or modulates the immune response to an active ingredient of the composition.
  • the adjuvant effect may occur at the cellular or humoral level, or both.
  • Adjuvants stimulate the response of the immune system to the actual antigen but have no immunological effect themselves.
  • adjuvented compositions of the invention may comprise one or more immunostimulants.
  • immunostimulant it is meant an agent that induces a general, temporary increase in a subject's immune response, whether administered with the antigen or separately.
  • composition of the invention may be administered with or without an adjuvant.
  • the composition may comprise, or be administered in conjunction with, one or more adjuvants (e.g. vaccine adjuvants), in particular the composition comprises an immunologically effective amount of a vector of the invention encoding a transgene.
  • adjuvants e.g. vaccine adjuvants
  • Methods are provided for inducing an immune response against a disease caused by a pathogen in a subject in need thereof comprising a step of administering an immunologically effective amount of a construct or composition as disclosed herein.
  • a construct or composition as disclosed herein.
  • Vectors of the invention may be applied for the prophylaxis, treatment or amelioration of diseases due to infection.
  • Methods of the invention include the use of a vector of the invention in medicine. They include the use of a vector of the invention for the treatment of a disease caused by a pathogen.
  • a vector of the invention can be used in the manufacture of a medicament for treating a disease caused by a pathogen.
  • Effective immunization with adenoviral vectors depends on the intrinsic immnomodulatory capability of the adenoviral vector backbone. Immunologically less potent adenoviruses induce less antigen expression. Effective immunization also depends on the ability of the promoter to drive strong and sustained transgene expression. For example, adenoviral vectors driven by the cytomegalovirus immediate-early (CMV-IE) promoter do not sustain long-term transgene expression because they induce cytokines that dampen expression.
  • CMV-IE cytomegalovirus immediate-early
  • subject is intended a vertebrate, such as a mammal e.g. a human or a veterinary mammal. In some embodiments the subject is human.
  • Vectors of the invention are generated using techniques and sequences provided herein, in conjunction with techniques known to those of skill in the art. Such techniques include conventional cloning techniques of cDNA such as those described in texts, use of overlapping oligonucleotide sequences of the adenovirus genomes, polymerase chain reaction, and any suitable method which provides the desired nucleotide sequence.
  • Example 1 Construction of a Chimpanzee Adenovirus
  • Wild type chimpanzee adenoviruses type 155 (ChAd155) (WO 2016/198621) isolated from healthy chimpanzees using standard procedures and were constructed as replication defective viruses as described in Sci Transl Med (2012) 4:1 and WO 2010/086189.
  • the ChAd155 is constructed by inserting two transgene expression cassettes into two different locations in the adeno:
  • This vector comprising a dual expression cassette is shown in FIG. 1 .
  • Ad5E4orf6 has been substituted into the early gene 4 (E4) region.
  • substitution is necessary to increase the productivity in HEK 293 cells.
  • Example 2 Transgene Expression from the Chimpanzee Adenovirus of Example 1
  • FIG. 2 The western blot analysis is shown in FIG. 2 and FIG. 3 .
  • the cells were infected with ChAd155-F ⁇ TM, ChAd155-F ⁇ TM.NM2 (“RSV”) or the ChAd155 dual cassette at a multiplicity of infection of 500 viral particles per cell.
  • cells were infected with ChAd155-F ⁇ TM.NM2 (“RSV”) at a multiplicity of infection of 1250 viral particles per cell.
  • the cells were harvested at 48 hours and 96 hours post infection, extracts prepared using standard methods and an equivalent amount of total cell extract loaded onto SDS-PAGE gels.
  • FIG. 2 shows that the ChAd155 dual cassette provides an expression level of the F antigen which is comparable to ChAd155F ⁇ TM and higher than ChAd155-F ⁇ TM.NM2 in MRC5 cells.
  • the cells were infected with ChAd155-NM2, ChAd155-F ⁇ TM.NM2 (“RSV”) or the ChAd155 dual cassette of Example 6 at a multiplicity of infection of 250 and 1250 viral particles per cell.
  • the cells were harvested at 48 hours post infection, extracts prepared using standard methods and an equivalent amount of total cell extract loaded onto SDS-PAGE gels.
  • the ChAd155 dual cassette provides NM2-1 expression level comparable to the ChAd155-NM2 single vector and higher than ChAd155-F ⁇ TM.NM2 (“RSV”) in MRC5 cells.
  • RSV ChAd155-F ⁇ TM.NM2
  • Example 3 Immunogencity of the Chimpanzee Adenovirus of Example 1
  • the immunogenicity of the dual expression cassette of Example 6 was evaluated in CD1 outbred mice (10 per group). The experiment was performed by injecting 5 ⁇ 10 8 viral particles intramuscularly into the mice. The B-cell response was measured at 4 and 8 weeks after the immunization by measuring the RSV neutralising titres. Each dot represents the response in a single mouse, and the line corresponds to the mean for each dose group. The results of this analysis are shown in FIG. 4 .
  • FIG. 4 shows that the ChAd155 dual cassette provides a B-cell response comparable to ChAd155F ⁇ TM and higher than that produced by ChAd155-F ⁇ TM.NM2 (“RSV”).
  • the immunogenicity of the dual expression cassette of Example 6 was also evaluated in BALB/c inbred mice (48, 11 or 8 per group). The experiment was performed by injecting 10 7 or 10 8 viral particles intramuscularly. The T-cell response was measured 3 weeks after the immunization by ex vivo IFN-gamma enzyme-linked immunospot (ELISpot) using a M2 peptide T cell epitope mapped in BALB/c mice. The results are shown in FIG. 11 , expressed as IFN-gamma Spot Forming Cells (SFC) per million of splenocytes. Each dot represents the response in a single mouse, and the line corresponds to the mean for each dose group. Injected dose in number of virus particles are shown on the x axis. The results are shown in FIG. 5 .
  • SFC IFN-gamma Spot Forming Cells
  • FIG. 5 shows that the ChAd155 dual cassette provides a T-cell response higher than that produced by ChAd155-F ⁇ TM.NM2 (“RSV”, the results for which are obtained from historical data). This difference in response is greater for the 10 6 dose.
  • RSV ChAd155-F ⁇ TM.NM2
  • FIG. 5 refers to “# positive mice”, i.e. the number of mice which responded to the vaccine.
  • Example 3 SeAP Expression in MRC5 Cells by ChAd155 with Different Promoters
  • the secreted embryonic alkaline phosphatase (SeAP) system is widely used to study promoter activity.
  • the SeAP reporter gene encodes a truncated for of the human placental alkaline phosphatase gene that lacks the membrane anchoring domain. Therefore, the SeAP protein is secreted into the cell supernatant and allows promoter activity to be determined without disturbing the cells.
  • FIG. 6 shows the SeAP expression in MRC5 cells from ChAd155 vectors constructed with different promoters.
  • the three different ChAd155 vectors used in this example are as follows:
  • the vectors constructed with the new enhanced hCMV promoter showed higher SeAP expression than the other two vectors at every time point measured.
  • FIG. 7 shows the SeAP expression in HeLa cells from ChAd155 vectors constructed with different promoters.
  • the three different ChAd155 vectors used in this experiment were as follows:
  • the vectors constructed with the new enhanced hCMV promoter showed higher SeAP expression than the other two vectors at every time point measured.

Abstract

A new promoter comprising: (i) an hCMV enhancer sequence; (ii) an hCMV promoter sequence; (iii) a splice donor region; (iv) a cell-derived enhancer sequence; and (v) a splice acceptor region.

Description

    CROSS-REFERENCE TO RELATED APPLICATIONS
  • This application is a Continuation of copending application Ser. No. 16/756,373, filed on Apr. 15, 2020, which is the National Phase under 35 U.S.C. § 371 of International Application No. PCT/EP2018/078242, filed on Oct. 16, 2018, which claims the benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62/572,944, filed on Oct. 16, 2017, U.S. Provisional Application No. 62/572,951, filed on Oct. 16, 2017, and U.S. Provisional Application No. 62/572,927, filed on Oct. 16, 2017, all of which are hereby expressly incorporated by reference into the present application.
    • FIELD OF THE INVENTION
  • This invention is in the field of promoters for use in vectors such as plasmids or viruses, particularly viral vectors such as adenoviral vectors. In particular, the present invention is directed towards an enhanced human CMV promoter.
    • BACKGROUND OF THE INVENTION
  • The term “vector” refers to an agent (such as a plasmid or virus) that contains or carries genetic material and can be used to introduce exogenous genes into an organism. An adenoviral vector is one example of a type of vector.
  • When a vector has delivered the genetic material to the cells of an organism, RNA can be transcribed from the delivered DNA using an RNA polymerase. An RNA polymerase can recognize specific promoter elements, enabling the transcription of the DNA sequence linked to that promoter element.
  • A promoter is a nucleotide sequence that permits the binding of RNA polymerase and directs the transcription of the DNA. Typically, a promoter is located in a non-coding region of the DNA, proximal to the transcriptional start site. Sequence elements within promoters that function in the initiation of transcription are often characterized by consensus nucleotide sequences.
  • Vectors are often said to comprise an “expression cassette”. The expression cassette comprises the genetic material of interest operatively linked to regulatory components in a manner which permits transgene transcription, translation, and/or expression of the DNA of interest in the host cell. The promoter is one of these regulatory components. If the DNA sequence of interest (e.g. a gene) is heterologous to the vector sequences flanking the gene, it can be referred to as a “transgene”.
  • Examples of promoters include, but are not limited to, promoters from bacteria, yeast, plants, viruses, and mammals, including simians and humans. A great number of expression control sequences, including promoters which are internal, native, constitutive, inducible and/or tissue-specific, are known in the art.
  • Examples of available promoters include, without limitation, the TBG promoter, the retroviral Rous sarcoma virus LTR promoter (optionally with the enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer, see, e.g., Boshart et al, Cell, 41:521-530 (1985)), the CASI promoter, the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1a promoter (Invitrogen).
  • The CMV promoter is strong and ubiquitously active. It has the ability to drive high levels of transgene expression in many tissue types and is well known in the art.
  • The CASI promoter is a synthetic promoter described as a combination of the CMV enhancer, the chicken beta-actin promoter, and a splice donor and splice acceptor flanking the ubiquitin (UBC) enhancer (US 8865881). SEQ ID NO: 2 is a polynucleotide sequence encoding the CASI promoter
  • There is a need in the art for new promoters.
    • SUMMARY OF THE INVENTION
  • The invention relates to a new promoter. More particularly, the invention relates to a new human CMV promoter.
  • The present invention provides a promoter comprising:
      • (i) an hCMV enhancer sequence;
      • (ii) an hCMV promoter sequence;
      • (ii) a splice donor region;
      • (iv) a cell-derived enhancer sequence; and
      • (v) a splice acceptor region
  • The term “cell-derived” means that the promoter is obtained from a eukaryotic (for example, human) cell.
  • In a preferred embodiment, the cell-derived enhancer sequence is an ubitquitin (UBC) enhancer sequence.
  • In another preferred embodiment, the components (i) to (v) of the promoter are provided in the order listed above, i.e. component (i) is first, (ii) is second, (iii) is third, (iv) is forth and (v) is fifth. In another embodiment, the order of the two enhancers (i.e. components (i) and (iv)) could be swapped.
  • In one embodiment, the promoter comprises one or more of the following sequences:
      • (i) the hCMV enhancer; and
      • (ii) the hCMV promoter sequences; of SEQ ID NO: 8; and/or
      • (iii) the splice donor region of SEQ ID NO:10; and/or
      • (iv) the UBC enhancer sequence of SEQ ID NO:11; and/or
      • (v) the splice acceptor region of SEQ ID NO: 12
  • In some embodiments, the promoter comprises at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more, sequence identity to SEQ ID NO: 8, SEQ ID NO:10, SEQ ID NO:11 and/or SEQ ID NO:12. In some embodiments, the parts (i) to (v) of the promoter consist of the relevant sequence.
  • In an embodiment, the promoter comprises:
      • (i) the hCMV enhancer; and
      • (ii) the hCMV promoter sequences; of SEQ ID NO: 8; and
      • (iii) the splice donor region of SEQ ID NO:10;
      • (iv) the UBC enhancer sequence of SEQ ID NO:11; and
      • (v) the splice acceptor region of SEQ ID NO: 12
  • In one embodiment, the promoter further comprises:
      • (vi) a fragment of the beta-actin sequence
  • In this embodiment comprising a fragment of the beta-actin sequence, the fragment of the chicken beta-actin sequence preferably comprises a 5′ untranslated region of the chicken beta actin sequence and does not contain the promoter sequence. In one embodiment, the chicken beta actin sequence may have at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more, sequence identity to SEQ ID NO: 9. In an embodiment comprising (vi) a fragment of the beta-actin sequence, this fragment is preferably found between the hCMV promoter region (ii) and the splice donor region (iii).
  • In another aspect, the present invention relates to a new promoter having at least about 84.1%, or more, identity to SEQ ID NO: 3. In some embodiments, the promoter can include a nucleic acid sequence having at least about 84.5%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, or more, sequence identity to SEQ ID NO: 3
  • In some embodiments, the promoter can include a nucleic acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more, sequence identity to SEQ ID NO: 3. In some embodiments, the promoter comprises or consists of a nucleic acid sequence of SEQ ID NO: 3.
  • In another aspect, the invention relates to a vector, such as an adenoviral vector or a plasmid, containing the new promoter described above. All of the features described above in relation to the promoter may be incorporated into the vector. For example, in one embodiment, the invention provides an adenoviral vector of the invention, the adenoviral vector comprises an expression cassette, wherein the expression cassette comprises a transgene and a promoter, wherein the promoter comprises:
      • (i) an hCMV enhancer sequence;
      • (ii) an hCMV promoter sequence;
      • (iii) a splice donor region;
      • (iv) a cell-derived enhancer sequence; and
      • (v) a splice acceptor region.
  • Another example of a vector of the invention is an adenoviral vector comprising an expression cassette, wherein the expression cassette comprises a transgene and a promoter, wherein the promoter comprises a nucleic acid sequence having at least 84.1% identity to SEQ ID NO: 3.
  • In a further example, a vector (e.g. an adenoviral vector) comprises a first and a second expression cassette, wherein each expression cassette comprises a transgene and a promoter, wherein the promoter of the first expression cassette and/or the second expression cassette is the new promoter described above. In one embodiment, the first expression cassette comprises the promoter. In another embodiment, the second expression cassette comprises the promoter.
  • For example, in one embodiment, an adenoviral vector of the invention comprises a first and a second expression cassette, wherein each expression cassette comprises a transgene and a promoter, wherein the promoter of the first expression cassette and/or the second expression cassette is a promoter comprising:
      • (i) an hCMV enhancer sequence;
      • (ii) an hCMV promoter sequence;
      • (iii) a splice donor region;
      • (iv) a cell-derived sequence; and
      • (v) a splice acceptor region.
  • In an additional example, an adenoviral vector comprises a first and a second expression cassette, wherein each expression cassette comprises a transgene and a promoter, wherein the promoter of the first expression cassette and/or the second expression cassette is a promoter having at least 84.1% identity to SEQ ID NO: 3.
  • The vectors (e.g. adenoviral vectors) of the invention are useful as components of immunogenic compositions for the induction of an immune response in a subject, methods for their use in treatment and processes for manufacture. The adenoviral vector of the present invention is preferably derived from a non-human simian adenovirus, also referred to as a “simian adenovirus”. Preferably, the simian adenoviral vector of the present invention is a chimp adenovirus (for example ChAd155 or ChAd83).
  • The present invention also provides a composition comprising the above-mentioned adenoviral vector and a pharmaceutically acceptable excipient. In addition, the present invention provides the above-mentioned adenoviral vector or composition comprising such an adenoviral vector for use as a medicament, a vaccine, and/or for the therapy or prophylaxis of a disease.
  • The invention also provides a method of inducing an immune response in a subject comprising administering the the above-mentioned adenoviral vector or composition described above to the subject. A vector or composition of the invention can be used in the manufacture of a medicament for the prevention or treatment of a disease
    • DESCRIPTION OF THE FIGURES
  • FIG. 1: A simian adenoviral construct according to the invention with a dual expression cassette. Inverted terminal repeats (ITR) flank the 3′ and 5′ ends; human CMV (hCMV) is the cytomegalovirus promoter; Enchanced hCMV is the enhanced cytomegalovirus promoter; N-M2-1 and FΔTM are the RSV antigens; WPRE is the Woodchuck Hepatitis Postranscriptional Regulatory Element; ΔE3 denotes that the early gene 3 is deleted; fiber denotes the adenoviral gene encoding the fiber protein; and Ad5E4orf6 in a substitute in the early gene 4 (E4) region.
  • The vector of FIG. 1 was constructed by inserting a first transgene expression cassette in place of the E1 region of the adenoviral genome, and a second transgene expression cassette in the HE2 region, i.e., downstream of the right ITR.
  • FIG. 2: Comparison of the expression levels of vectors expressing FΔTM transgene in a MRC5 cell line, demonstrated by western blot at 48 hours and 96 hours post-infection under non-reducing conditions. Cells were infected at multiplicities of infection of 500 and 1250.
  • FIG. 3: Comparison of the expression levels of vectors expressing NM2-1 transgene in a MRC5 cell line, demonstrated by western blot at 48 hours post-infection under reducing conditions. Cells were infected at multiplicities of infection of 250 and 1250.
  • FIG. 4: Comparison of the immunogencity from ChAd155 vectors expressing the RSV antigen FΔTm. The data was collected at 4 weeks and 8 weeks after vaccination with a dose of 5×108 virus particles.
  • FIG. 5: Comparison of the immunogencity from ChAd155 vectors expressing the M2 RSV antigen. The data was collected at 3 weeks after vaccination with a dose of either 107 or 106 virus particles.
  • FIG. 6: SeAP expression in MRC5 cells by ChAd155 with different promoters.
  • FIG. 7: SeAP expression in HeLa cells by ChAd155 with different promoters.
    •  ANNOTATION OF THE SEQUENCES
  • SEQ ID NO: 1—Polynucleotide sequence encoding wild type ChAd155
  • SEQ ID NO: 2—Polynucleotide sequence encoding the CASI promoter
  • SEQ ID NO: 3—Polynucleotide sequence encoding the enhanced hCMV promoter
  • SEQ ID NO: 4—Polynucleotide sequence encoding the hCMV NM2 bghpolyA cassette
  • SEQ ID NO: 5—NM2 protein sequence
  • SEQ ID NO: 6—Polynucleotide sequence encoding the hCMV F0 WPRE bghpolyA cassette
  • SEQ ID NO: 7—F0 protein sequence
  • SEQ ID NO: 8—Polynucleotide sequence encoding the hCMV promoter and enhancer sequence (nucleotides 1-650 of SEQ ID NO: 3).
  • SEQ ID NO: 9—Polynucleotide sequence encoding a Chicken Beta-Actin Fragment (nucleotides 651-809 of SEQ ID NO: 3).
  • SEQ ID NO: 10—Polynucleotide sequence encoding the Splice Donor Region (nucleotides 810-824 of SEQ ID NO: 3).
  • SEQ ID NO: 11—Polynucleotide sequence encoding the ubiquitin (UBC) enhancer (nucleotides 825-1127 of SEQ ID NO: 3).
  • SEQ ID NO: 12—Polynucleotide sequence encoding the Splice Acceptor Region (nucleotides 1128-1187 of SEQ ID NO: 3).
    • DETAILED DESCRIPTION OF THE INVENTION Adenoviruses
  • Adenoviruses are nonenveloped icosahedral viruses with a linear double stranded DNA genome of approximately 36 kb. Adenoviruses can transduce numerous cell types of several mammalian species, including both dividing and nondividing cells, without integrating into the genome of the host cell. They have been widely used for gene transfer applications due to their proven safety, ability to achieve highly efficient gene transfer in a variety of target tissues, and large transgene capacity. Human adenoviral vectors are currently used in gene therapy and vaccines but have the drawback of a high worldwide prevalence of pre-existing immunity, following previous exposure to common human adenoviruses.
  • Adenoviruses have a characteristic morphology with an icosahedral capsid comprising three major proteins, hexon (II), penton base (III) and a knobbed fiber (IV), along with a number of other minor proteins, VI, VIII, IX, IIIa and IVa2. The hexon accounts for the majority of the structural components of the capsid, which consists of 240 trimeric hexon capsomeres and 12 penton bases. The hexon has three conserved double barrels and the top has three towers, each tower containing a loop from each subunit that forms most of the capsid. The base of the hexon is highly conserved between adenoviral serotypes, while the surface loops are variable. The penton is another adenoviral capsid protein; it forms a pentameric base to which the fiber attaches. The trimeric fiber protein protrudes from the penton base at each of the 12 vertices of the capsid and is a knobbed rod-like structure. The primary role of the fiber protein is to tether the viral capsid to the cell surface via the interaction of the knob region with a cellular receptor. Variations in the flexible shaft, as well as knob regions of fiber, are characteristic of the different adenovral serotypes.
  • The adenoviral genome has been well characterized. The linear, double-stranded DNA is associated with the highly basic protein VII and a small peptide pX (also termed mu). Another protein, V, is packaged with this DNA-protein complex and provides a structural link to the capsid via protein VI. There is general conservation in the overall organization of the adenoviral genome with respect to specific open reading frames being similarly positioned, e.g. the location of the E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4 and L5 genes of each virus. Each extremity of the adenoviral genome comprises a sequence known as an inverted terminal repeat (ITR), which is necessary for viral replication. The 5′ end of the adenoviral genome contains the 5′ cis-elements necessary for packaging and replication; i.e., the 5′ ITR sequences (which can function as origins of replication) and the native 5′ packaging enhancer domains, which contain sequences necessary for packaging linear adenoviral genomes and enhancer elements for the E1 promoter. The 3′ end of the adenoviral genome includes 3′ cis-elements, including the ITRs, necessary for packaging and encapsidation. The virus also comprises a virus-encoded protease, which is necessary for processing some of the structural proteins required to produce infectious virions.
  • The structure of the adenoviral genome is described on the basis of the order in which the viral genes are expressed following host cell transduction. More specifically, the viral genes are referred to as early (E) or late (L) genes according to whether transcription occurs prior to or after onset of DNA replication. In the early phase of transduction, the E1A, E1B, E2A, E2B, E3 and E4 genes of adenovirus are expressed to prepare the host cell for viral replication. The E1 gene is considered a master switch, it acts as a transcription activator and is involved in both early and late gene transcription. E2 is involved in DNA replication; E3 is involved in immune modulation and E4 regulates viral mRNA metabolism. During the late phase of infection, expression of the late genes L1-L5, which encode the structural components of the viral particles, is activated. Late genes are transcribed from the Major Late Promoter (MLP) with alternative splicing.
  • HE1 and HE2 sites were identified as potential insertion sites for a transgene since the insertion in these specific points does not interrupt the coding sequences or important regulatory sequences of a chimp adenovirus, such as a Type C or E chimp adenovirus, for example, ChAd155 and ChAd83. The HE1 and HE2 sites can be identified by sequence alignment in any chimp adenovirus. Therefore, cloning of expression cassettes in the HE1 and HE2 sites of the ChAd genomes doesn't impact the virus replication cycle.
    • Adenoviral Replication
  • Historically, adenovirus vaccine development has focused on defective, non-replicating vectors. They are rendered replication defective by deletion of the E1 region genes, which are essential for replication. Typically, non-essential E3 region genes are also deleted to make room for exogenous transgenes. An expression cassette comprising the transgene under the control of an exogenous promoter is then inserted. These replication-defective viruses are then produced in E1-complementing cells.
  • The term “replication-defective ” or “replication-incompetent” adenovirus refers to an adenovirus that is incapable of replication because it has been engineered to comprise at least a functional deletion (or “loss-of-function” mutation), i.e. a deletion or mutation which impairs the function of a gene without removing it entirely, e.g. introduction of artificial stop codons, deletion or mutation of active sites or interaction domains, mutation or deletion of a regulatory sequence of a gene etc, or a complete removal of a gene encoding a gene product that is essential for viral replication, such as one or more of the adenoviral genes selected from E1A, E1B, E2A, E2B, E3 and E4 (such as E3 ORF1, E3 ORF2, E3 ORF3, E3 ORF4, E3 ORF5, E3 ORF6, E3 ORF7, E3 ORF8, E3 ORF9, E4 ORF7, E4 ORF6, E4 ORF4, E4 ORF3, E4 ORF2 and/or E4 ORF1). Suitably, E1 and optionally E3 and/or E4 are deleted. If deleted, the aforementioned deleted gene region will suitably not be considered in the alignment when determining percent identity with respect to another sequence.
  • In some embodiments of the invention, the adenoviral vector is a replication defective adenovirus. For example, in the embodiments of an adenoviral vector with two expression cassettes, the first expression cassette is inserted in the deleted E1 region and so these adenoviruses will be replication defective.
  • In other embodiments, the adenoviral vector is a replication competent adenovirus. The term “replication-competent” adenovirus refers to an adenovirus which can replicate in a host cell in the absence of any recombinant helper proteins comprised in the cell. Suitably, a “replication-competent” adenovirus comprises intact structural genes and the following intact or functionally essential early genes: E1A, E1B, E2A, E2B and E4. Wild type adenoviruses isolated from a particular animal will be replication competent in that animal.
    • Vectors of the Invention
  • Viral vectors based on non-human simian adenovirus represent an alternative to the use of human derived vectors for gene therapy and genetic vaccines. Certain adenoviruses isolated from non-human simians are closely related to adenoviruses isolated from humans, as demonstrated by their efficient propagation in cells of human origin. As humans typically do not develop immunity to simian adenoviruses, they promise to provide an improved alternative to human adenoviral uses.
  • “Low seroprevalence” may mean having a reduced pre-existing neutralizing antibody level as compared to human adenovirus 5 (Ad5). Similarly or alternatively, “low seroprevalence” may mean less than about 40% seroprevalence, less than about 30% seroprevalence, less than about 20% seroprevalence, less than about 15% seroprevalence, less than about 10% seroprevalence, less than about 5% seroprevalence, less than about 4% seroprevalence, less than about 3% seroprevalence, less than about 2% seroprevalence, less than about 1% seroprevalence or no detectable seroprevalence. Seroprevalence can be measured as the percentage of individuals having a clinically relevant neutralizing titer (defined as a 50% neutralisation titer >200) using methods as described in Hum. Gene Ther. (2004) 15:293.
  • In one embodiment, the adenoviral vector of the present invention is derived from a nonhuman simian adenovirus, also referred to as a “simian adenovirus.” Numerous adenoviruses have been isolated from nonhuman simians such as chimpanzees, bonobos, rhesus macaques, orangutans and gorillas. Vectors derived from these adenoviruses can induce strong immune responses to transgenes encoded by these vectors. Certain advantages of vectors based on nonhuman simian adenoviruses include a relative lack of cross-neutralizing antibodies to these adenoviruses in the human target population, thus their use overcomes the pre-existing immunity to human adenoviruses. For example, some simian adenoviruses have no cross reactivity with preexisting human neutralizing antibodies and cross-reaction of certain chimpanzee adenoviruses with pre-existing human neutralizing antibodies is only present in 2% of the target population, compared with 35% in the case of certain candidate human adenovirus vectors (Sci. Transl. Med. (2012) 4:1).
  • Adenoviral vectors of the invention may be derived from a non-human adenovirus, such as a simian adenovirus, e.g., from chimpanzees (Pan troglodytes), bonobos (Pan paniscus), gorillas (Gorilla gorilla) and orangutans (Pongo abelii and Pongo pygnaeus). They include adenoviruses from Group B, Group C, Group D, Group E and Group G. Chimpanzee adenoviruses include, but are not limited to ChAd3, ChAd19, ChAd25.2, ChAd26, ChAd27, ChAd29, ChAd30, ChAd31, ChAd32, ChAd33, ChAd34, ChAd35, ChAd37, ChAd38, ChAd39, ChAd40, ChAd63, ChAd83, ChAd155, ChAd15, SadV41 and ChAd157. Alternatively, adenoviral vectors may be derived from nonhuman simian adenoviruses isolated from bonobos, such as PanAd1, PanAd2, PanAd3, Pan 5, Pan 6, Pan 7 (also referred to as C7) and Pan 9. Vectors may include, in whole or in part, a nucleotide encoding the fiber, penton or hexon of a non-human adenovirus.
  • In an embodiment of the adenoviral vectors of the invention, the adenoviral vector has a seroprevalence of less than 40%, less than 30%, less than 20%, less than 10% or less than 5% in human subjects, preferably no seroprevalence in human subjects and more preferably no seroprevalence in human subjects that have not previously been in contact with a chimpanzee adenovirus.
  • In embodiments of the adenoviral vectors of the invention, the adenoviral DNA is capable of entering a mammalian target cell, i.e. it is infectious. An infectious recombinant adenoviral vector of the invention can be used as a prophylactic or therapeutic vaccine and for gene therapy. Thus, in an embodiment, the recombinant adenoviral vector comprises an endogenous molecule for delivery into a target cell. The target cell is a mammalian cell, e.g. a bovine cell, a canine cell, a caprine cell, a cervine cell, a chimpanzee cell, a chiroptera cell, an equine cell, a feline cell, a human cell, a lupine cell, an ovine cell, a porcine cell, a rodent cell, an ursine cell or a vulpine cell. Theendogenous molecule for delivery into a target cell is an expression cassette.
  • In an embodiment of the invention, the vector comprises a left ITR region, a deleted E1 region, then a deleted E3 region, and, optionally, additional enhancer elements; these are followed by a fiber region, an E4 region and a right ITR. Translation occurs in the rightward and leftward directions. In this embodiment, the first expression cassette is inserted in the deleted E1 region, and the second expression cassette is insertion in the deleted E3 region. In a further embodiment, the promoters of the two expression cassettes are CMV promoters. In a yet further embodiment, the enhancer element is the Hepatitis B Postranslational Regulatory Element (HPRE) or the Woodchuck Hepatitis Postranslational Element (WPRE).
  • In one embodiment of the invention, the vector comprises left and right ITR regions; a deleted E1 region; at least a partially deleted E3 region; a fiber region; an E4 region; two expression cassettes, each comprising: a promoter and at least one an antigen of interest and, optionally, one or more enhancer elements. The first expression cassette is inserted in the deleted E1 region, and the second expression cassette is inserted at the HE1 site, i.e., between the stop codons of the fiber gene and an E4 region (“the HE1 site”). The ChAd155 HE1 insertion site is between bp 34611 and 34612 of the wild type ChAd155 sequence. The ChAd83 HE1 insertion site is between bp 33535 and 33536 of the wild type ChAd83 sequence. Translation occurs in the rightward and leftward directions. In a further embodiment, the promoters are CMV promoters. In a preferred embodiment, one promoter is a CMV promoter and the other is a eCMV promoter. In a yet further embodiment, the enhancer element is HPRE or WPRE.
  • In a further embodiment, the vector comprises left and right ITR regions; a deleted E1 region; at least a partially deleted E3 region; a fiber region; an E4 region; two expression cassettes, each comprising: a promoter, at least one antigen of interest and, optionally, one or more enhancer elements. The first expression cassette is inserted in the deleted E1 region, and the second expression cassette is inserted at the HE2 site, i.e., between the end of the left ITR and the cap site of the E4 mRNA (“the HE2 site”). The ChAd155 HE2 insertion site is between bp 37662 and 37663 of the wild type ChAd155 sequence. The ChAd83 HE2 insertion site is between bp 36387 and 36388 of the wild type ChAd83 sequence. Translation occurs in the rightward and leftward directions. In a further embodiment, the promoters are CMV promoters. In a preferred embodiment, one promoter is a CMV promoter and the other is a eCMV promoter. In a yet further embodiment, the enhancer element is HPRE or WPRE (the enhancer element increases expression of the transgene).
  • The HE1 and HE2 sites were identified as insertion sites for a transgene, as the insertion in these specific points does not interrupt the coding sequences or regulatory sequences of ChAd155 and ChAd83. Therefore, inserting expression cassettes in the HE1 or HE2 sites of the ChAd genome does not affect the viral replication cycle.
  • In an embodiment of the invention, the vector is a functional or an immunogenic derivative of an adenoviral vector. By “derivative of an adenoviral vector” is meant a modified version of the vector, e.g., one or more nucleotides of the vector are deleted, inserted, modified or substituted.
    • Further Regulatory Elements
  • Regulatory elements, i.e., expression control sequences, in addition to promoter sequences, include appropriate transcription initiation, termination and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (poly A) signals including rabbit beta-globin polyA; tetracycline regulatable systems, microRNAs, posttranscriptional regulatory elements e.g., WPRE, posttranscriptional regulatory element of woodchuck hepatitis virus); sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of an encoded product.
  • Optionally, vectors carrying transgenes encoding therapeutically useful or immunogenic products may also include selectable markers or reporter genes. The reporter gene may be chosen from those known in the art. Suitable reporter genes include, but are not limited to enhanced green fluorescent protein, red fluorescent protein, luciferase and secreted embryonic alkaline phosphatase (seAP), which may include sequences encoding geneticin, hygromicin or purimycin resistance, among others. Such selectable reporters or marker genes (which may or may not be located outside the viral genome to be packaged into a viral particle) can be used to signal the presence of the plasmids in bacterial cells, such as ampicillin resistance. Other components of the vector may include an origin of replication.
  • A “posttranscriptional regulatory element,” as used herein, is a DNA sequence that, when transcribed, enhances the expression of the transgene(s) or fragments thereof that are delivered by viral vectors of the invention. Postranscriptional regulatory elements include, but are not limited to the Hepatitis B Virus Postranscriptional Regulatory Element (HPRE) and the Woodchuck Hepatitis Postranscriptional Regulatory Element (WPRE). The WPRE is a tripartite cis-acting element that has been demonstrated to enhance transgene expression driven by certain, but not all promoters
  • In embodiments of the invention, a ChAd155 vector may comprise one or more of a promoter, an enhancer, and a reporter gene. For example, vectors of the invention may comprise ChAd155-enhanced hCMV-SeAP ChAd155-CASI-seAP and ChAd155-hCMV-seAP, optionally with a tetracycline on/off transcriptional control and ChAd155 —CMV-hFerL-chEF1-seAP with a tetracycline on/off transcriptional control.
  • In embodiments of the invention, a ChAd83 vector may comprise one or more of a promoter, an enhancer, and a reporter gene. For example, vectors of the invention may comprise ChAd155 enhanced hCMV SeAP, ChAd83 enhanced hCMV SeAP, ChAd155-CASI-seAP and ChAd83-hCMV-seAP, optionally with a tetracycline on/off transcriptional control and ChAd83-CMV-hFerL-chEF1-seAP with a tetracycline on/off transcriptional control.
  • Vectors of the invention are generated using techniques provided herein, in conjunction with techniques known to those of skill in the art. Such techniques include conventional cloning techniques of cDNA such as those described in texts, use of overlapping oligonucleotide sequences of the adenovirus genomes, polymerase chain reaction, and any suitable method which provides the desired nucleotide sequence.
    • Transgenes
  • A “transgene” is a nucleic acid sequence, heterologous to the vector sequences flanking the transgene, which encodes a polypeptide of interest. The nucleic acid coding sequence is operatively linked to regulatory components in a manner which permits transgene transcription, translation, and/or expression in a host cell. In embodiments of the invention, the vectors express transgenes at a therapeutic or a prophylactic level. A “functional derivative” of a transgenic polypeptide is a modified version of a polypeptide, e.g., wherein one or more amino acids are deleted, inserted, modified or substituted.
  • The transgene may be used for prophylaxis or treatment, e.g., as a vaccine for inducing an immune response, to correct genetic deficiencies by correcting or replacing a defective or missing gene, or as a cancer therapeutic. As used herein, induction of an immune response refers to the ability of a protein to induce a T cell and/or a humoral antibody immune response to the protein.
  • The immune response elicited by the transgene may be an antigen specific B cell response, which produces neutralizing antibodies. The elicited immune response may be an antigen specific T cell response, which may be a systemic and/or a local response. The antigen specific T cell response may comprise a CD4+ T cell response, such as a response involving CD4+ T cells expressing cytokines, e.g. interferon gamma (IFN gamma), tumor necrosis factor alpha (TNF alpha) and/or interleukin 2 (IL2). Alternatively, or additionally, the antigen specific T cell response comprises a CD8+ T cell response, such as a response involving CD8+ T cells expressing cytokines, e.g., IFN gamma, TNF alpha and/or IL2.
  • The composition of the transgene sequence will depend upon the use to which the resulting vector will be put. In an embodiment, the transgene is a sequence encoding a product which is useful in biology and medicine, such as a prophylactic transgene, a therapeutic transgene or an immunogenic transgene, e.g., protein or RNA. Protein transgenes include antigens. Antigenic transgenes of the invention induce an immunogenic response to a disease causing organism.
  • Transgenes such rabies virus antigens, e.g., rabies glycoprotein (RG), respiratory syncytial virus (RSV) antigens, human immunodeficiency virus (HIV) antigens, or fragments thereof would be suitable for use with promoters of the invention. However, the invention is not limited to use with such transgenes.
  • As a result of the redundancy in the genetic code, a polypeptide can be encoded by a variety of different nucleic acid sequences. Coding is biased to use some synonymous codons, i.e., codons that encode the same amino acid, more than others. By “codon optimized,” it is meant that modifications in the codon composition of a recombinant nucleic acid are made without altering the amino acid sequence. Codon optimization has been used to improve mRNA expression in different organisms by using organism-specific codon-usage frequencies.
  • In addition to, and independently from, codon bias, some synonymous codon pairs are used more frequently than others. This codon pair bias means that some codon pairs are overrepresented and others are underrepresented. Codon pair deoptimization has been used to reduce viral virulence. For example, it has been reported that polioviruses modified to contain underrepresented codon pairs demonstrated decreased translation efficiency and were attenuated compared to wild type poliovirus (Science (2008) 320:1784). Engineering a synthetic attenuated virus by codon pair deoptimization can produce viruses that encode the same amino acid sequences as wild type but use different pairwise arrangements of synonymous codons. Viruses attenuated by codon pair deoptimization generated up to 1000-fold fewer plaques compared to wild type, produced fewer viral particles and required about 100 times as many viral particles to form a plaque.
  • In contrast, polioviruses modified to contain codon pairs that are overrepresented in the human genome acted in a manner similar to wild type RNA and generated plaques identical in size to wild type RNA (Coleman et al. (2008) Science 320:1784). This occurred despite the fact that the virus with overrepresented codon pairs contained a similar number of mutations as the virus with underrepresented codon pairs and demonstrated enhanced translation compared to wild type. This observation suggests that codon pair optimized constructs would be expected to act in a manner similar to their non-codon pair optimized counterparts and would not be expected to provide a functional advantage. Without wishing to be constrained by theory, this may be because natural evolution has optimized codon pairing.
  • A construct of the invention may comprise a codon optimized nucleic acid sequence. Alternatively or additionally, a vector of the invention comprises a codon optimized sequence of a transgene or an immunogenic derivative or fragment thereof. A construct of the invention may comprise a codon pair optimized nucleic acid sequence. Alternatively or additionally, a vector of the invention comprises or consists of a codon pair optimized sequence of a transgene or an immunogenic derivative or fragment thereof.
    • Respiratory Syncytial Virus (RSV) Transgenes
  • Infection with RSV does not confer full protective immunity. Infection in infancy is followed by symptomatic RSV re-infections which continue throughout adulthood. These re-infections generally go undiagnosed because they usually present as common acute upper respiratory tract infections. In more vulnerable persons (e.g., immunocompromised adults or elderly), re infections can however also lead to severe disease. Both arms of the immune system (humoral and cellular immunity) are involved in protection from severe disease [Guvenel, 2014].
  • The humoral immune response is capable of neutralizing the virus and inhibiting viral replication, thereby playing a major role in protection against lower respiratory RSV infection and severe disease [Piedra, 2003]. Passive immunization, in the form of Immunoglobulin G (IgG) RSV-neutralizing monoclonal antibodies (Synagis) given prophylactically, has been shown to prevent RSV disease to some extent in premature infants and newborns with bronchopulmonary dysplasia or underlying cardiopulmonary disease [Cardenas, 2005].
  • T cells are also involved in the control of RSV disease. Lethal RSV infections have been described in patients with low CD8 T cells counts, as in the case of severe combined immunodeficiency, bone marrow and lung transplant recipients [Hertz, 1989]. The histopathology of fatal cases of RSV infection of newborns shows that there is a relative paucity of CD8 T cells in the lung infiltrate [Welliver, 2007]. Moreover, the presence of CD8 T cells producing Interferon-gamma (IFN-γ) has been associated with diminished Th2 responses and reduced eosinophilia in animal models of RSV [Castilow, 2008; Stevens, 2009].
  • Suitable antigens of RSV which are useful as immunogens to immunize a human or non-human animal can be selected from: the fusion protein (F), the attachment protein (G), the matrix protein (M2) and the nucleoprotein (N). The term “F protein” or “fusion protein” or “F protein polypeptide” or “fusion protein polypeptide” refers to a polypeptide or protein having all or part of an amino acid sequence of an RSV Fusion protein polypeptide. Similarly, the term “G protein” or “G protein polypeptide” refers to a polypeptide or protein having all or part of an amino acid sequence of an RSV Attachment protein polypeptide. The term “M protein” or “matrix protein” or “M protein polypeptide” refers to a polypeptide or protein having all or part of an amino acid sequence of an RSV Matrix protein and may include either or both of the M2-1 (which may be written herein as M2.1) and M2-2 gene products. Likewise, the term “N protein” or “Nucleocapsid protein” or “N protein polypeptide” refers to a polypeptide or protein having all or part of an amino acid sequence of an RSV Nucleoprotein.
  • Two groups of human RSV strains have been described, the A and B groups, based mainly on differences in the antigenicity of the G glycoprotein. Numerous strains of RSV have been isolated to date, any of which are suitable in the context of the antigens of the immunogenic combinations disclosed herein. Exemplary strains indicated by GenBank and/or EMBL Accession number can be found in US published application number 2010/0203071 (WO2008114149), which is incorporated herein by reference for the purpose of disclosing the nucleic acid and polypeptide sequences of RSV F and G proteins suitable for use in present invention. In an embodiment, the RSV F protein can be an ectodomain of an RSV F Protein (FΔTM).
  • Exemplary M and N protein nucleic acids and protein sequences can be found, e.g., in US published application number 2014/0141042 (WO2012/089833), which are incorporated herein for purpose of disclosing the nucleic acid and polypeptide sequences of RSV M and N proteins suitable for use in present invention.
  • Transgene nucleic acids may encode an RSV F antigen and RSV, M and N antigens. More specifically, the nucleic acids may encode an RSV FΔTM antigen (fusion (F) protein deleted of the transmembrane and cytoplasmic regions), and RSV M2-1 (transcription anti-termination) and N (nucleocapsid) antigens.
    • Fusion (F) Protein Deleted of the Transmembrane and Cytoplasmic Regions (FΔTM)
  • The RSV F protein is a major surface antigen and mediates viral fusion to target cells. The F protein is an antigen which is highly conserved among RSV subgroups and strains. The F protein is a target for neutralizing antibodies, including the prophylactic RSV-neutralizing monoclonal antibody Synagis. Deletion of the transmembrane region and cytoplasmic tail permits secretion of the FΔTM protein. Neutralizing antibodies including Synagis, that recognize this soluble form of the F protein, inhibit RSV infectivity in vitro [Magro, 2010].
    • Nucleocapsid (N) Protein
  • The N protein is an internal (non-exposed) antigen, highly conserved between RSV strains and known to be a source of many T cell epitopes [Townsend, 1984]. The N protein is essential for the replication and transcription of the RSV genome. The primary function of the N protein is to encapsulate the virus genome for the purposes of RNA transcription, replication and packaging and protects it from ribonucleases.
    • Transcription Anti-Termination (M2-1) Protein
  • The M2-1 protein is a transcription anti-termination factor that is important for the efficient synthesis of full-length messenger RNAs (mRNAs) as well as for the synthesis of polycistronic readthrough mRNAs, which are characteristic of non-segmented negative-strand RNA viruses. M2-1 is an internal (non-exposed) antigen, which is highly conserved between RSV strains and known to be a source of many T cell epitopes [Townsend, 1984].
    • N-M2-1 Fusion Protein
  • A polynucleotide encoding a linker is positioned between the polynucleotide encoding an RSV N antigen, or fragment thereof, and the polynucleotide encoding an RSV M2.1 antigen, or fragment thereof. Thus, in certain preferred examples, an expression cassette contains a transgene which encodes a fused RSV viral protein N-linker-M2.1 It is preferred that the linker is a flexible linker, preferably a flexible linker comprising an amino acid sequence according to SEQ ID NO: 13 (Gly-Gly-Gly-Ser-Gly-Gly-Gly) or SEQ ID NO: 14 (Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly).
    • Delivery of Adenoviral Vectors
  • In some embodiments, the recombinant adenoviral vector of the invention is administered to a subject by epicutaneous administration, intradermal administration, intramuscular injection, intraperitoneal injection, intravenous injection, nasal administration, oral administration, rectal administration, subcutaneous injection, transdermal administration or intravaginal administration.
  • In an embodiment of the invention, the vectors can be administered intramuscularly (IM), i.e., injection directly into muscle. Muscles are well vascularized and the uptake is typically rapid.
    • Adjuvants
  • Approaches to establishing strong and lasting immunity to specific pathogens include addition of adjuvants to vaccines. By “adjuvant” is meant an agent that augments, stimulates, activates, potentiates or modulates the immune response to an active ingredient of the composition. The adjuvant effect may occur at the cellular or humoral level, or both. Adjuvants stimulate the response of the immune system to the actual antigen but have no immunological effect themselves. Alternatively or additionally, adjuvented compositions of the invention may comprise one or more immunostimulants. By “immunostimulant” it is meant an agent that induces a general, temporary increase in a subject's immune response, whether administered with the antigen or separately.
  • A composition of the invention may be administered with or without an adjuvant. Alternatively, or additionally, the composition may comprise, or be administered in conjunction with, one or more adjuvants (e.g. vaccine adjuvants), in particular the composition comprises an immunologically effective amount of a vector of the invention encoding a transgene.
    • Methods of Use/Uses
  • Methods are provided for inducing an immune response against a disease caused by a pathogen in a subject in need thereof comprising a step of administering an immunologically effective amount of a construct or composition as disclosed herein. In some embodiments are provided the use of the constructs or compositions disclosed herein for inducing an immune response to a transgenic antigen in a subject in need thereof. Vectors of the invention may be applied for the prophylaxis, treatment or amelioration of diseases due to infection.
  • Methods of the invention include the use of a vector of the invention in medicine. They include the use of a vector of the invention for the treatment of a disease caused by a pathogen. A vector of the invention can be used in the manufacture of a medicament for treating a disease caused by a pathogen.
  • Effective immunization with adenoviral vectors depends on the intrinsic immnomodulatory capability of the adenoviral vector backbone. Immunologically less potent adenoviruses induce less antigen expression. Effective immunization also depends on the ability of the promoter to drive strong and sustained transgene expression. For example, adenoviral vectors driven by the cytomegalovirus immediate-early (CMV-IE) promoter do not sustain long-term transgene expression because they induce cytokines that dampen expression.
  • By “subject” is intended a vertebrate, such as a mammal e.g. a human or a veterinary mammal. In some embodiments the subject is human.
    • General
  • Vectors of the invention are generated using techniques and sequences provided herein, in conjunction with techniques known to those of skill in the art. Such techniques include conventional cloning techniques of cDNA such as those described in texts, use of overlapping oligonucleotide sequences of the adenovirus genomes, polymerase chain reaction, and any suitable method which provides the desired nucleotide sequence.
  • Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. The term “plurality” refers to two or more. Additionally, numerical limitations given with respect to concentrations or levels of a substance, such as solution component concentrations or ratios thereof, and reaction conditions such as temperatures, pressures and cycle times are intended to be approximate. The term “about” used herein is intended to mean the amount ±10%.
  • The present invention will now be further described by means of the following non-limiting examples.
    • EXAMPLES Example 1: Construction of a Chimpanzee Adenovirus
  • Wild type chimpanzee adenoviruses type 155 (ChAd155) (WO 2016/198621) isolated from healthy chimpanzees using standard procedures and were constructed as replication defective viruses as described in Sci Transl Med (2012) 4:1 and WO 2010/086189.
  • The ChAd155 is constructed by inserting two transgene expression cassettes into two different locations in the adeno:
      • (1) The first expression cassette components comprise the classical human CMV (hCMV) promoter and N.M2-1 RSV antigens. This first expression cassette is inserted into the E1 region of the adeno (after the E1 region has been deleted).
      • (2) The second expression cassette comprises an enhanced classical human CMV (enhanced hCMV) promoter, the FΔTM RSV antigen and a WPRE enhancer. This first expression cassette is inserted into the HE2 region of the adeno (after the HE2 region has been deleted).
  • This vector comprising a dual expression cassette is shown in FIG. 1.
  • In the construct of FIG. 1, Ad5E4orf6 has been substituted into the early gene 4 (E4) region.
  • The substitution is necessary to increase the productivity in HEK 293 cells.
    • Example 2: Transgene Expression from the Chimpanzee Adenovirus of Example 1
  • Western blot analysis was performed to compare the level of transgene expression in the ChAd155 vector of Example 6 (labelled “Dual” or “Dual cassette” in the figures) in MRC5 cells with:
      • (i) a vector comprising a single F expression cassette (ChAd155-FΔTM, labelled “F0ΔTm”),
      • (ii) a vector comprising a single NM2 expression cassette (ChAd155-NM2, labelled “NM2-1”), and
      • (iii) the vector of Example 5 comprising a single expression cassette containing the F and N-M2 RSV antigens (ChAd155-FΔTM.NM2, also labelled “RSV”)
  • The western blot analysis is shown in FIG. 2 and FIG. 3.
  • As shown in FIG. 2, the cells were infected with ChAd155-FΔTM, ChAd155-FΔTM.NM2 (“RSV”) or the ChAd155 dual cassette at a multiplicity of infection of 500 viral particles per cell. In addition, cells were infected with ChAd155-FΔTM.NM2 (“RSV”) at a multiplicity of infection of 1250 viral particles per cell. The cells were harvested at 48 hours and 96 hours post infection, extracts prepared using standard methods and an equivalent amount of total cell extract loaded onto SDS-PAGE gels.
  • FIG. 2 shows that the ChAd155 dual cassette provides an expression level of the F antigen which is comparable to ChAd155FΔTM and higher than ChAd155-FΔTM.NM2 in MRC5 cells.
  • As shown in FIG. 3, the cells were infected with ChAd155-NM2, ChAd155-FΔTM.NM2 (“RSV”) or the ChAd155 dual cassette of Example 6 at a multiplicity of infection of 250 and 1250 viral particles per cell. The cells were harvested at 48 hours post infection, extracts prepared using standard methods and an equivalent amount of total cell extract loaded onto SDS-PAGE gels.
  • In FIG. 3, the ChAd155 dual cassette provides NM2-1 expression level comparable to the ChAd155-NM2 single vector and higher than ChAd155-FΔTM.NM2 (“RSV”) in MRC5 cells.
    • Example 3: Immunogencity of the Chimpanzee Adenovirus of Example 1
  • The immunogenicity of the dual expression cassette of Example 6 was evaluated in CD1 outbred mice (10 per group). The experiment was performed by injecting 5×108 viral particles intramuscularly into the mice. The B-cell response was measured at 4 and 8 weeks after the immunization by measuring the RSV neutralising titres. Each dot represents the response in a single mouse, and the line corresponds to the mean for each dose group. The results of this analysis are shown in FIG. 4.
  • FIG. 4 shows that the ChAd155 dual cassette provides a B-cell response comparable to ChAd155FΔTM and higher than that produced by ChAd155-FΔTM.NM2 (“RSV”).
  • The immunogenicity of the dual expression cassette of Example 6 was also evaluated in BALB/c inbred mice (48, 11 or 8 per group). The experiment was performed by injecting 107 or 108 viral particles intramuscularly. The T-cell response was measured 3 weeks after the immunization by ex vivo IFN-gamma enzyme-linked immunospot (ELISpot) using a M2 peptide T cell epitope mapped in BALB/c mice. The results are shown in FIG. 11, expressed as IFN-gamma Spot Forming Cells (SFC) per million of splenocytes. Each dot represents the response in a single mouse, and the line corresponds to the mean for each dose group. Injected dose in number of virus particles are shown on the x axis. The results are shown in FIG. 5.
  • FIG. 5 shows that the ChAd155 dual cassette provides a T-cell response higher than that produced by ChAd155-FΔTM.NM2 (“RSV”, the results for which are obtained from historical data). This difference in response is greater for the 106 dose.
  • FIG. 5 refers to “# positive mice”, i.e. the number of mice which responded to the vaccine.
    • Example 3: SeAP Expression in MRC5 Cells by ChAd155 with Different Promoters
  • The secreted embryonic alkaline phosphatase (SeAP) system is widely used to study promoter activity. The SeAP reporter gene encodes a truncated for of the human placental alkaline phosphatase gene that lacks the membrane anchoring domain. Therefore, the SeAP protein is secreted into the cell supernatant and allows promoter activity to be determined without disturbing the cells.
  • FIG. 6 shows the SeAP expression in MRC5 cells from ChAd155 vectors constructed with different promoters. The three different ChAd155 vectors used in this example are as follows:
      • A ChAd155 with the known human CMV (hCMV) promoter;
      • A ChAd155 with the known CASI promoter; and
      • A ChAd155 with the new enhanced hCMV promoter
  • In this experiment, the MRC5 were infected with moi=250 vp/cell, and measurement of the SeAP took place at 2 days (48 hours), 4 days (96 hours) and 7 days (1 week) post-infection with the ChAd155 viruses.
  • As can be seen from FIG. 6, the vectors constructed with the new enhanced hCMV promoter showed higher SeAP expression than the other two vectors at every time point measured.
    • Example 4: SeAP Expression in HeLa Cells by ChAd155 with Different Promoters
  • FIG. 7 shows the SeAP expression in HeLa cells from ChAd155 vectors constructed with different promoters. As with Example 3, the three different ChAd155 vectors used in this experiment were as follows:
      • A ChAd155 (d) with the known human CMV (hCMV) promoter;
      • A ChAd155 (d) with the known CASI promoter; and
      • A ChAd155 (d) with the new enhanced hCMV promoter
  • In this experiment, the HeLa were infected with moi=50 vp/cell, and measurement of the SeAP took place at 2 days (48 hours), 4 days (96 hours) and 7 days (1 week) post-infection with the ChAd155 viruses.
  • As can be seen from FIG. 7, the vectors constructed with the new enhanced hCMV promoter showed higher SeAP expression than the other two vectors at every time point measured.
  • DESCRIPTION OF THE SEQUENCES
    Polynucleotide sequence encodinq wild type ChAd155
    SEQ ID NO: 1
          CATCATCAATAATATACCTTATTTTGGATTGAAGCCAATATGATAATGAGATGGGCGGCGCGGGGCGGGAG
    GCGGGTCCGGGGGCGGGCCGGCGGGCGGGGCGGTGTGGCGGAAGTGGACTTTGTAAGTGTGGCGGATGTGACTTGCT
    AGTGCCGGGCGCGGTAAAAGTGACGTTTTCCGTGCGCGACAACGCCCACGGGAAGTGACATTTTTCCCGCGGTTTTT
    ACCGGATGTTGTAGTGAATTTGGGCGTAACCAAGTAAGATTTGGCCATTTTCGCGGGAAAACTGAAACGGGGAAGTG
    AAATCTGATTAATTTCGCGTTAGTCATACCGCGTAATATTTGTCGAGGGCCGAGGGACTTTGGCCGATTACGTGGAG
    GACTCGCCCAGGTGTTTTTTGAGGTGAATTTCCGCGTTCCGGGTCAAAGTCTCCGTTTTATTATTATAGTCAGCTGA
    CGCGGAGTGTATTTATACCCTCTGATCTCGTCAAGTGGCCACTCTTGAGTGCCAGCGAGTAGAGTTTTCTCCTCTGC
    CGCTCTCCGCTCCGCTCCGCTCGGCTCTGACACCGGGGAAAAAATGAGACATTTCACCTACGATGGCGGTGTGCTCA
    CCGGCCAGCTGGCTGCTGAAGTCCTGGACACCCTGATCGAGGAGGTATTGGCCGATAATTATCCTCCCTCGACTCCT
    TTTGAGCCACCTACACTTCACGAACTCTACGATCTGGATGTGGTGGGGCCCAGCGATCCGAACGAGCAGGCGGTTTC
    CAGTTTTTTTCCAGAGTCCATGTTGTTGGCCAGCCAGGAGGGGGTCGAACTTGAGACCCCTCCTCCGATCGTGGATT
    CCCCCGATCCGCCGCAGCTGACTAGGCAGCCCGAGCGCTGTGCGGGACCTGAGACTATGCCCCAGCTGCTACCTGAG
    GTGATCGATCTCACCTGTAATGAGTCTGGTTTTCCACCCAGCGAGGATGAGGACGAAGAGGGTGAGCAGTTTGTGTT
    AGATTCTGTGGAACAACCCGGGCGAGGATGCAGGTCTTGTCAATATCACCGGAAAAACACAGGAGACTCCCAGATTA
    TGTGTTCTCTGTGTTATATGAAGATGACCTGTATGTTTATTTACAGTAAGTTTATCATCTGTGGGCAGGTGGGCTAT
    AGTGTGGGTGGTGGTCTTTGGGGGGTTTTTTAATATATGTCAGGGGTTATGCTGAAGACTTTTTTATTGTGATTTTT
    AAAGGTCCAGTGTCTGAGCCCGAGCAAGAACCTGAACCGGAGCCTGAGCCTTCTCGCCCCAGGAGAAAGCCTGTAAT
    CTTAACTAGACCCAGCGCACCGGTAGCGAGAGGCCTCAGCAGCGCGGAGACCACCGACTCCGGTGCTTCCTCATCAC
    CCCCGGAGATTCACCCCCTGGTGCCCCTGTGTCCCGTTAAGCCCGTTGCCGTGAGAGTCAGTGGGCGGCGGTCTGCT
    GTGGAGTGCATTGAGGACTTGCTTTTTGATTCACAGGAACCTTTGGACTTGAGCTTGAAACGCCCCAGGCATTAAAC
    CTGGTCACCTGGACTGAATGAGTTGACGCCTATGTTTGCTTTTGAATGACTTAATGTGTATAGATAATAAAGAGTGA
    GATAATGTTTTAATTGCATGGTGTGTTTAACTTGGGCGGAGTCTGCTGGGTATATAAGCTTCCCTGGGCTAAACTTG
    GTTACACTTGACCTCATGGAGGCCTGGGAGTGTTTGGAGAACTTTGCCGGAGTTCGTGCCTTGCTGGACGAGAGCTC
    TAACAATACCTCTTGGTGGTGGAGGTATTTGTGGGGCTCTCCCCAGGGCAAGTTAGTTTGTAGAATCAAGGAGGATT
    ACAAGTGGGAATTTGAAGAGCTTTTGAAATCCTGTGGTGAGCTATTGGATTCTTTGAATCTAGGCCACCAGGCTCTC
    TTCCAGGAGAAGGTCATCAGGACTTTGGATTTTTCCACACCGGGGCGCATTGCAGCCGCGGTTGCTTTTCTAGCTTT
    TTTGAAGGATAGATGGAGCGAAGAGACCCACTTGAGTTCGGGCTACGTCCTGGATTTTCTGGCCATGCAACTGTGGA
    GAGCATGGATCAGACACAAGAACAGGCTGCAACTGTTGTCTTCCGTCCGCCCGTTGCTGATTCCGGCGGAGGAGCAA
    CAGGCCGGGTCAGAGGACCGGGCCCGTCGGGATCCGGAGGAGAGGGCACCGAGGCCGGGCGAGAGGAGCGCGCTGAA
    CCTGGGAACCGGGCTGAGCGGCCATCCACATCGGGAGTGAATGTCGGGCAGGTGGTGGATCTTTTTCCAGAACTGCG
    GCGGATTTTGACTATTAGGGAGGATGGGCAATTTGTTAAGGGTCTTAAGAGGGAGAGGGGGGCTTCTGAGCATAACG
    AGGAGGCCAGTAATTTAGCTTTTAGCTTGATGACCAGACACCGTCCAGAGTGCATCACTTTTCAGCAGATTAAGGAC
    AATTGTGCCAATGAGTTGGATCTGTTGGGTCAGAAGTATAGCATAGAGCAGCTGACCACTTACTGGCTGCAGCCGGG
    TGATGATCTGGAGGAAGCTATTAGGGTGTATGCTAAGGTGGCCCTGCGGCCCGATTGCAAGTACAAGCTCAAGGGGC
    TGGTGAATATCAGGAATTGTTGCTACATTTCTGGCAACGGGGCGGAGGTGGAGATAGAGACCGAAGACAGGGTGGCT
    GGGGCCCAACTTTAACGGCACGGTGTTTTTGGGGAACACCAACCTGGTCCTGCACGGGGTGAGCTTCTATGGGTTTA
    ACAACACCTGTGTGGAGGCCTGGACCGATGTGAAGGTCCGCGGTTGCGCCTTTTATGGATGTTGGAAGGCCATAGTG
    AGCCGCCCTAAGAGCAGGAGTTCCATTAAGAAATGCTTGTTTGAGAGGTGCACCTTGGGGATCCTGGCCGAGGGCAA
    CTGCAGGGTGCGCCACAATGTGGCCTCCGAGTGCGGTTGCTTCATGCTAGTCAAGAGCGTGGCGGTAATCAAGCATA
    ATATGGTGTGCGGCAACAGCGAGGACAAGGCCTCACAGATGCTGACCTGCACGGATGGCAACTGCCACTTGCTGAAG
    ACCATCCATGTAACCAGCCACAGCCGGAAGGCCTGGCCCGTGTTCGAGCACAACTTGCTGACCCGCTGCTCCTTGCA
    TCTGGGCAACAGGCGGGGGGTGTTCCTGCCCTATCAATGCAACTTTAGTCACACCAAGATCTTGCTAGAGCCCGAGA
    GCATGTCCAAGGTGAACTTGAACGGGGTGTTTGACATGACCATGAAGATCTGGAAGGTGCTGAGGTACGACGAGACC
    AGGTCCCGGTGCAGACCCTGCGAGTGCGGGGGCAAGCATATGAGGAACCAGCCCGTGATGCTGGATGTGACCGAGGA
    GCTGAGGACAGACCACTTGGTTCTGGCCTGCACCAGGGCCGAGTTTGGTTCTAGCGATGAAGACACAGATTGAGGTG
    GGTGAGTGGGCGTGGCCTGGGGTGGTCATGAAAATATATAAGTTGGGGGTCTTAGGGTCTCTTTATTTGTGTTGCAG
    AGACCGCCGGAGCCATGAGCGGGAGCAGCAGCAGCAGCAGTAGCAGCAGCGCCTTGGATGGCAGCATCGTGAGCCCT
    TATTTGACGACGCGGATGCCCCACTGGGCCGGGGTGCGTCAGAATGTGATGGGCTCCAGCATCGACGGCCGACCCGT
    CCTGCCCGCAAATTCCGCCACGCTGACCTATGCGACCGTCGCGGGGACGCCGTTGGACGCCACCGCCGCCGCCGCCG
    CCACCGCAGCCGCCTCGGCCGTGCGCAGCCTGGCCACGGACTTTGCATTCCTGGGACCACTGGCGACAGGGGCTACT
    TCTCGGGCCGCTGCTGCCGCCGTTCGCGATGACAAGCTGACCGCCCTGCTGGCGCAGTTGGATGCGCTTACTCGGGA
    ACTGGGTGACCTTTCTCAGCAGGTCATGGCCCTGCGCCAGCAGGTCTCCTCCCTGCAAGCTGGCGGGAATGCTTCTC
    CCACAAATGCCGTTTAAGATAAATAAAACCAGACTCTGTTTGGATTAAAGAAAAGTAGCAAGTGCATTGCTCTCTTT
    ATTTCATAATTTTCCGCGCGCGATAGGCCCTAGACCAGCGTTCTCGGTCGTTGAGGGTGCGGTGTATCTTCTCCAGG
    ACGTGGTAGAGGTGGCTCTGGACGTTGAGATACATGGGCATGAGCCCGTCCCGGGGGTGGAGGTAGCACCACTGCAG
    AGCTTCATGCTCCGGGGTGGTGTTGTAGATGATCCAGTCGTAGCAGGAGCGCTGGGCATGGTGCCTAAAAATGTCCT
    TCAGCAGCAGGCCGATGGCCAGGGGGAGGCCCTTGGTGTAAGTGTTTACAAAACGGTTAAGTTGGGAAGGGTGCATT
    CGGGGAGAGATGATGTGCATCTTGGACTGTATTTTTAGATTGGCGATGTTTCCGCCCAGATCCCTTCTGGGATTCAT
    GTTGTGCAGGACCACCAGTACAGTGTATCCGGTGCACTTGGGGAATTTGTCATGCAGCTTAGAGGGAAAAGCGTGGA
    AGAACTTGGAGACGCCTTTGTGGCCTCCCAGATTTTCCATGCATTCGTCCATGATGATGGCAATGGGCCCGCGGGAG
    GCAGCTTGGGCAAAGATATTTCTGGGGTCGCTGACGTCGTAGTTGTGTTCCAGGGTGAGGTCGTCATAGGCCATTTT
    TACAAAGCGCGGGCGGAGGGTGCCCGACTGGGGGATGATGGTCCCCTCTGGCCCTGGGGCGTAGTTGCCCTCGCAGA
    TCTGCATTTCCCAGGCCTTAATCTCGGAGGGGGGAATCATATCCACCTGCGGGGCGATGAAGAAAACGGTTTCCGGA
    GCCGGGGAGATTAACTGGGATGAGAGCAGGTTTCTAAGCAGCTGTGATTTTCCACAACCGGTGGGCCCATAAATAAC
    ACCTATAACCGGTTGCAGCTGGTAGTTTAGAGAGCTGCAGCTGCCGTCGTCCCGGAGGAGGGGGGCCACCTCGTTGA
    GCATGTCCCTGACGCGCATGTTCTCCCCGACCAGATCCGCCAGAAGGCGCTCGCCGCCCAGGGACAGCAGCTCTTGC
    AAGGAAGCAAAGTTTTTCAGCGGCTTGAGGCCGTCCGCCGTGGGCATGTTTTTCAGGGTCTGGCTCAGCAGCTCCAG
    GCGGTCCCAGAGCTCGGTGACGTGCTCTACGGCATCTCTATCCAGCATATCTCCTCGTTTCGCGGGTTGGGGCGACT
    TTCGCTGTAGGGCACCAAGCGGTGGTCGTCCAGCGGGGCCAGAGTCATGTCCTTCCATGGGCGCAGGGTCCTCGTCA
    GGGTGGTCTGGGTCACGGTGAAGGGGTGCGCTCCGGGCTGAGCGCTTGCCAAGGTGCGCTTGAGGCTGGTTCTGCTG
    GTGCTGAAGCGCTGCCGGTCTTCGCCCTGCGCGTCGGCCAGGTAGCATTTGACCATGGTGTCATAGTCCAGCCCCTC
    CGCGGCGTGTCCCTTGGCGCGCAGCTTGCCCTTGGAGGTGGCGCCGCACGAGGGGCAGAGCAGGCTCTTGAGCGCGT
    AGAGCTTGGGGGCGAGGAAGACCGATTCGGGGGAGTAGGCGTCCGCGCCGCAGACCCCGCACACGGTCTCGCACTCC
    ACCAGCCAGGTGAGCTCGGGGCGCGCCGGGTCAAAAACCAGGTTTCCCCCATGCTTTTTGATGCGTTTCTTACCTCG
    GGTCTCCATGAGGTGGTGTCCCCGCTCGGTGACGAAGAGGCTGTCCGTGTCTCCGTAGACCGACTTGAGGGGTCTTT
    TCTCCAGGGGGGTCCCTCGGTCTTCCTCGTAGAGGAACTCGGACCACTCTGAGACGAAGGCCCGCGTCCAGGCCAGG
    ACGAAGGAGGCTATGTGGGAGGGGTAGCGGTCGTTGTCCACTAGGGGGTCCACCTTCTCCAAGGTGTGAAGACACAT
    GTCGCCTTCCTCGGCGTCCAGGAAGGTGATTGGCTTGTAGGTGTAGGCCACGTGACCGGGGGTTCCTGACGGGGGGG
    TATAAAAGGGGGTGGGGGCGCGCTCGTCGTCACTCTCTTCCGCATCGCTGTCTGCGAGGGCCAGCTGCTGGGGTGAG
    TATTCCCTCTCGAAGGCGGGCATGACCTCCGCGCTGAGGTTGTCAGTTTCCAAAAACGAGGAGGATTTGATGTTCAC
    CTGTCCCGAGGTGATACCTTTGAGGGTACCCGCGTCCATCTGGTCAGAAAACACGATCTTTTTATTGTCCAGCTTGG
    TGGCGAACGACCCGTAGAGGGCGTTGGAGAGCAGCTTGGCGATGGAGCGCAGGGTCTGGTTCTTGTCCCTGTCGGCG
    CGCTCCTTGGCCGCGATGTTGAGCTGCACGTACTCGCGCGCGACGCAGCGCCACTCGGGGAAGACGGTGGTGCGCTC
    GTCGGGCACCAGGCGCACGCGCCAGCCGCGGTTGTGCAGGGTGACCAGGTCCACGCTGGTGGCGACCTCGCCGCGCA
    GGCGCTCGTTGGTCCAGCAGAGACGGCCGCCCTTGCGCGAGCAGAAGGGGGGCAGGGGGTCGAGCTGGGTCTCGTCC
    GGGGGGTCCGCGTCCACGGTGAAAACCCCGGGGCGCAGGCGCGCGTCGAAGTAGTCTATCTTGCAACCTTGCATGTC
    CAGCGCCTGCTGCCAGTCGCGGGCGGCGAGCGCGCGCTCGTAGGGGTTGAGCGGCGGGCCCCAGGGCATGGGGTGGG
    TGAGTGCGGAGGCGTACATGCCGCAGATGTCATAGACGTAGAGGGGCTCCCGCAGGACCCCGATGTAGGTGGGGTAG
    CAGCGGCCGCCGCGGATGCTGGCGCGCACGTAGTCATACAGCTCGTGCGAGGGGGCGAGGAGGTCGGGGCCCAGGTT
    GGTGCGGGCGGGGCGCTCCGCGCGGAAGACGATCTGCCTGAAGATGGCATGCGAGTTGGAAGAGATGGTGGGGCGCT
    GGAAGACGTTGAAGCTGGCGTCCTGCAGGCCGACGGCGTCGCGCACGAAGGAGGCGTAGGAGTCGCGCAGCTTGTGT
    ACCAGCTCGGCGGTGACCTGCACGTCGAGCGCGCAGTAGTCGAGGGTCTCGCGGATGATGTCATATTTAGCCTGCCC
    CTTCTTTTTCCACAGCTCGCGGTTGAGGACAAACTCTTCGCGGTCTTTCCAGTACTCTTGGATCGGGAAACCGTCCG
    GTTCCGAACGGTAAGAGCCTAGCATGTAGAACTGGTTGACGGCCTGGTAGGCGCAGCAGCCCTTCTCCACGGGGAGG
    GCGTAGGCCTGCGCGGCCTTGCGGAGCGAGGTGTGGGTCAGGGCGAAGGTGTCCCTGACCATGACTTTGAGGTACTG
    GTGCTTGAAGTCGGAGTCGTCGCAGCCGCCCCGCTCCCAGAGCGAGAAGTCGGTGCGCTTCTTGGAGCGGGGGTTGG
    GCAGAGCGAAGGTGACATCGTTGAAGAGGATTTTGCCCGCGCGGGGCATGAAGTTGCGGGTGATGCGGAAGGGCCCC
    GGCACTTCAGAGCGGTTGTTGATGACCTGGGCGGCGAGCACGATCTCGTCGAAGCCGTTGATGTTGTGGCCCACGAT
    GTAGAGTTCCAGGAAGCGGGGCCGGCCCTTTACGGTGGGCAGCTTCTTTAGCTCTTCGTAGGTGAGCTCCTCGGGCG
    AGGCGAGGCCGTGCTCGGCCAGGGCCCAGTCCGCGAGGTGCGGGTTGTCTCTGAGGAAGGACTTCCAGAGGTCGCGG
    GCCAGGAGGGTCTGCAGGCGGTCTCTGAAGGTCCTGAACTGGCGGCCCACGGCCATTTTTTCGGGGGTGATGCAGTA
    GAAGGTGAGGGGGTCTTGCTGCCAGCGGTCCCAGTCGAGCTGCAGGGCGAGGTCGCGCGCGGCGGTGACCAGGCGCT
    CGTCGCCCCCGAATTTCATGACCAGCATGAAGGGCACGAGCTGCTTTCCGAAGGCCCCCATCCAAGTGTAGGTCTCT
    ACATCGTAGGTGACAAAGAGGCGCTCCGTGCGAGGATGCGAGCCGATCGGGAAGAACTGGATCTCCCGCCACCAGTT
    GGAGGAGTGGCTGTTGATGTGGTGGAAGTAGAAGTCCCGTCGCCGGGCCGAACACTCGTGCTGGCTTTTGTAAAAGC
    GAGCGCAGTACTGGCAGCGCTGCACGGGCTGTACCTCATGCACGAGATGCACCTTTCGCCCGCGCACGAGGAAGCCG
    AGGGGAAATCTGAGCCCCCCGCCTGGCTCGCGGCATGGCTGGTTCTCTTCTACTTTGGATGCGTGTCCGTCTCCGTC
    TGGCTCCTCGAGGGGTGTTACGGTGGAGCGGACCACCACGCCGCGCGAGCCGCAGGTCCAGATATCGGCGCGCGGCG
    GTCGGAGTTTGATGACGACATCGCGCAGCTGGGAGCTGTCCATGGTCTGGAGCTCCCGCGGCGGCGGCAGGTCAGCC
    GGGAGTTCTTGCAGGTTCACCTCGCAGAGTCGGGCCAGGGCGCGGGGCAGGTCTAGGTGGTACCTGATCTCTAGGGG
    CGTGTTGGTGGCGGCGTCGATGGCTTGCAGGAGCCCGCAGCCCCGGGGGGCGACGACGGTGCCCCGCGGGGTGGTGG
    TGGTGGTGGCGGTGCAGCTCAGAAGCGGTGCCGCGGGCGGGCCCCCGGAGGTAGGGGGGGCTCCGGTCCCGCGGGCA
    GGGGCGGCAGCGGCACGTCGGCGTGGAGCGCGGGCAGGAGTTGGTGCTGTGCCCGGAGGTTGCTGGCGAAGGCGACG
    ACGCGGCGGTTGATCTCCTGGATCTGGCGCCTCTGCGTGAAGACGACGGGCCCGGTGAGCTTGAACCTGAAAGAGAG
    TTCGACAGAATCAATCTCGGTGTCATTGACCGCGGCCTGGCGCAGGATCTCCTGCACGTCTCCCGAGTTGTCTTGGT
    AGGCGATCTCGGCCATGAACTGCTCGATCTCTTCCTCCTGGAGGTCTCCGCGTCCGGCGCGTTCCACGGTGGCCGCC
    AGGTCGTTGGAGATGCGCCCCATGAGCTGCGAGAAGGCGTTGAGTCCGCCCTCGTTCCAGACTCGGCTGTAGACCAC
    GCCCCCCTGGTCATCGCGGGCGCGCATGACCACCTGCGCGAGGTTGAGCTCCACGTGCCGCGCGAAGACGGCGTAGT
    TGCGCAGACGCTGGAAGAGGTAGTTGAGGGTGGTGGCGGTGTGCTCGGCCACGAAGAAGTTCATGACCCAGCGGCGC
    AACGTGGATTCGTTGATGTCCCCCAAGGCCTCCAGCCGTTCCATGGCCTCGTAGAAGTCCACGGCGAAGTTGAAAAA
    CTGGGAGTTGCGCGCCGACACGGTCAACTCCTCCTCCAGAAGACGGATGAGCTCGGCGACGGTGTCGCGCACCTCGC
    GCTCGAAGGCTATGGGGATCTCTTCCTCCGCTAGCATCACCACCTCCTCCTCTTCCTCCTCTTCTGGCACTTCCATG
    ATGGCTTCCTCCTCTTCGGGGGGTGGCGGCGGCGGCGGTGGGGGAGGGGGCGCTCTGCGCCGGCGGCGGCGCACCGG
    GAGGCGGTCCACGAAGCGCGCGATCATCTCCCCGCGGCGGCGGCGCATGGTCTCGGTGACGGCGCGGCCGTTCTCCC
    GGGGGCGCAGTTGGAAGACGCCGCCGGACATCTGGTGCTGGGGCGGGTGGCCGTGAGGCAGCGAGACGGCGCTGACG
    ATGCATCTCAACAATTGCTGCGTAGGTACGCCGCCGAGGGACCTGAGGGAGTCCATATCCACCGGATCCGAAAACCT
    TTCGAGGAAGGCGTCTAACCAGTCGCAGTCGCAAGGTAGGCTGAGCACCGTGGCGGGCGGCGGGGGGTGGGGGGAGT
    GTCTGGCGGAGGTGCTGCTGATGATGTAATTGAAGTAGGCGGACTTGACACGGCGGATGGTCGACAGGAGCACCATG
    TCCTTGGGTCCGGCCTGCTGGATGCGGAGGCGGTCGGCTATGCCCCAGGCTTCGTTCTGGCATCGGCGCAGGTCCTT
    GTAGTAGTCTTGCATGAGCCTTTCCACCGGCACCTCTTCTCCTTCCTCTTCTGCTTCTTCCATGTCTGCTTCGGCCC
    TGGGGCGGCGCCGCGCCCCCCTGCCCCCCATGCGCGTGACCCCGAACCCCCTGAGCGGTTGGAGCAGGGCCAGGTCG
    GCGACGACGCGCTCGGCCAGGATGGCCTGCTGCACCTGCGTGAGGGTGGTTTGGAAGTCATCCAAGTCCACGAAGCG
    GTGGTAGGCGCCCGTGTTGATGGTGTAGGTGCAGTTGGCCATGACGGACCAGTTGACGGTCTGGTGGCCCGGTTGCG
    ACATCTCGGTGTACCTGAGTCGCGAGTAGGCGCGGGAGTCGAAGACGTAGTCGTTGCAAGTCCGCACCAGGTACTGG
    TAGCCCACCAGGAAGTGCGGCGGCGGCTGGCGGTAGAGGGGCCAGCGCAGGGTGGCGGGGGCTCCGGGGGCCAGGTC
    TTCCAGCATGAGGCGGTGGTAGGCGTAGATGTACCTGGACATCCAGGTGATACCCGCGGCGGTGGTGGAGGCGCGCG
    GGAAGTCGCGCACCCGGTTCCAGATGTTGCGCAGGGGCAGAAAGTGCTCCATGGTAGGCGTGCTCTGTCCAGTCAGA
    CGCGCGCAGTCGTTGATACTCTAGACCAGGGAAAACGAAAGCCGGTCAGCGGGCACTCTTCCGTGGTCTGGTGAATA
    GATCGCAAGGGTATCATGGCGGAGGGCCTCGGTTCGAGCCCCGGGTCCGGGCCGGACGGTCCGCCATGATCCACGCG
    GTTACCGCCCGCGTGTCGAACCCAGGTGTGCGACGTCAGACAACGGTGGAGTGTTCCTTTTGGCGTTTTTCTGGCCG
    GGCGCCGGCGCCGCGTAAGAGACTAAGCCGCGAAAGCGAAAGCAGTAAGTGGCTCGCTCCCCGTAGCCGGAGGGATC
    CTTGCTAAGGGTTGCGTTGCGGCGAACCCCGGTTCGAATCCCGTACTCGGGCCGGCCGGACCCGCGGCTAAGGTGTT
    GGATTGGCCTCCCCCTCGTATAAAGACCCCGCTTGCGGATTGACTCCGGACACGGGGACGAGCCCCTTTTATTTTTG
    CTTTCCCCAGATGCATCCGGTGCTGCGGCAGATGCGCCCCCCGCCCCAGCAGCAGCAACAACACCAGCAAGAGCGGC
    AGCAACAGCAGCGGGAGTCATGCAGGGCCCCCTCACCCACCCTCGGCGGGCCGGCCACCTCGGCGTCCGCGGCCGTG
    TCTGGCGCCTGCGGCGGCGGCGGGGGGCCGGCTGACGACCCCGAGGAGCCCCCGCGGCGCAGGGCCAGACACTACCT
    GGACCTGGAGGAGGGCGAGGGCCTGGCGCGGCTGGGGGCGCCGTCTCCCGAGCGCCACCCGCGGGTGCAGCTGAAGC
    GCGACTCGCGCGAGGCGTACGTGCCTCGGCAGAACCTGTTCAGGGACCGCGCGGGCGAGGAGCCCGAGGAGATGCGG
    GACAGGAGGTTCAGCGCAGGGCGGGAGCTGCGGCAGGGGCTGAACCGCGAGCGGCTGCTGCGCGAGGAGGACTTTGA
    GCCCGACGCGCGGACGGGGATCAGCCCCGCGCGCGCGCACGTGGCGGCCGCCGACCTGGTGACGGCGTACGAGCAGA
    CGGTGAACCAGGAGATCAACTTCCAAAAGAGTTTCAACAACCACGTGCGCACGCTGGTGGCGCGCGAGGAGGTGACC
    ATCGGGCTGATGCACCTGTGGGACTTTGTAAGCGCGCTGGTGCAGAACCCCAACAGCAAGCCTCTGACGGCGCAGCT
    GTTCCTGATAGTGCAGCACAGCAGGGACAACGAGGCGTTTAGGGACGCGCTGCTGAACATCACCGAGCCCGAGGGTC
    GGTGGCTGCTGGACCTGATTAACATCCTGCAGAGCATAGTGGTGCAGGAGCGCAGCCTGAGCCTGGCCGACAAGGTG
    GCGGCCATCAACTACTCGATGCTGAGCCTGGGCAAGTTTTACGCGCGCAAGATCTACCAGACGCCGTACGTGCCCAT
    AGACAAGGAGGTGAAGATCGACGGTTTTTACATGCGCATGGCGCTGAAGGTGCTCACCCTGAGCGACGACCTGGGCG
    TGTACCGCAACGAGCGCATCCACAAGGCCGTGAGCGTGAGCCGGCGGCGCGAGCTGAGCGACCGCGAGCTGATGCAC
    AGCCTGCAGCGGGCGCTGGCGGGCGCCGGCAGCGGCGACAGGGAGGCGGAGTCCTACTTCGATGCGGGGGCGGACCT
    GCGCTGGGCGCCCAGCCGGCGGGCCCTGGAGGCCGCGGGGGTCCGCGAGGACTATGACGAGGACGGCGAGGAGGATG
    AGGAGTACGAGCTAGAGGAGGGCGAGTACCTGGACTAAACCGCGGGTGGTGTTTCCGGTAGATGCAAGACCCGAACG
    TGGTGGACCCGGCGCTGCGGGCGGCTCTGCAGAGCCAGCCGTCCGGCCTTAACTCCTCAGACGACTGGCGACAGGTC
    ATGGACCGCATCATGTCGCTGACGGCGCGTAACCCGGACGCGTTCCGGCAGCAGCCGCAGGCCAACAGGCTCTCCGC
    CATCCTGGAGGCGGTGGTGCCTGCGCGCTCGAACCCCACGCACGAGAAGGTGCTGGCCATAGTGAACGCGCTGGCCG
    AGAACAGGGCCATCCGCCCGGACGAGGCCGGGCTGGTGTACGACGCGCTGCTGCAGCGCGTGGCCCGCTACAACAGC
    GGCAACGTGCAGACCAACCTGGACCGGCTGGTGGGGGACGTGCGCGAGGCGGTGGCGCAGCGCGAGCGCGCGGATCG
    GCAGGGCAACCTGGGCTCCATGGTGGCGCTGAATGCCTTCCTGAGCACGCAGCCGGCCAACGTGCCGCGGGGGCAGG
    AAGACTACACCAACTTTGTGAGCGCGCTGCGGCTGATGGTGACCGAGACCCCCCAGAGCGAGGTGTACCAGTCGGGC
    CCGGACTACTTCTTCCAGACCAGCAGACAGGGCCTGCAGACGGTGAACCTGAGCCAGGCTTTCAAGAACCTGCGGGG
    GCTGTGGGGCGTGAAGGCGCCCACCGGCGACCGGGCGACGGTGTCCAGCCTGCTGACGCCCAACTCGCGCCTGCTGC
    TGCTGCTGATCGCGCCGTTCACGGACAGCGGCAGCGTGTCCCGGGACACCTACCTGGGGCACCTGCTGACCCTGTAC
    CGCGAGGCCATCGGGCAGGCGCAGGTGGACGAGCACACCTTCCAGGAGATCACCAGCGTGAGCCGCGCGCTGGGGCA
    GGAGGACACGAGCAGCCTGGAGGCGACTCTGAACTACCTGCTGACCAACCGGCGGCAGAAGATTCCCTCGCTGCACA
    GCCTGACCTCCGAGGAGGAGCGCATCTTGCGCTACGTGCAGCAGAGCGTGAGCCTGAACCTGATGCGCGACGGGGTG
    ACGCCCAGCGTGGCGCTGGACATGACCGCGCGCAACATGGAACCGGGCATGTACGCCGCGCACCGGCCTTACATCAA
    CCGCCTGATGGACTACCTGCATCGCGCGGCGGCCGTGAACCCCGAGTACTTTACCAACGCCATCCTGAACCCGCACT
    GGCTCCCGCCGCCCGGGTTCTACAGCGGGGGCTTCGAGGTCCCGGAGACCAACGATGGCTTCCTGTGGGACGACATG
    GACGACAGCGTGTTCTCCCCGCGGCCGCAGGCGCTGGCGGAAGCGTCCCTGCTGCGTCCCAAGAAGGAGGAGGAGGA
    GGAGGCGAGTCGCCGCCGCGGCAGCAGCGGCGTGGCTTCTCTGTCCGAGCTGGGGGCGGCAGCCGCCGCGCGCCCCG
    GGTCCCTGGGCGGCAGCCCCTTTCCGAGCCTGGTGGGGTCTCTGCACAGCGAGCGCACCACCCGCCCTCGGCTGCTG
    GGCGAGGACGAGTACCTGAATAACTCCCTGCTGCAGCCGGTGCGGGAGAAAAACCTGCCTCCCGCCTTCCCCAACAA
    CGGGATAGAGAGCCTGGTGGACAAGATGAGCAGATGGAAGACCTATGCGCAGGAGCACAGGGACGCGCCTGCGCTCC
    GGCCGCCCACGCGGCGCCAGCGCCACGACCGGCAGCGGGGGCTGGTGTGGGATGACGAGGACTCCGCGGACGATAGC
    AGCGTGCTGGACCTGGGAGGGAGCGGCAACCCGTTCGCGCACCTGCGCCCCCGCCTGGGGAGGATGTTTTAAAAAAA
    AAAAAAAAAAGCAAGAAGCATGATGCAAAAATTAAATAAAACTCACCAAGGCCATGGCGACCGAGCGTTGGTTTCTT
    GTGTTCCCTTCAGTATGCGGCGCGCGGCGATGTACCAGGAGGGACCTCCTCCCTCTTACGAGAGCGTGGTGGGCGCG
    GCGGCGGCGGCGCCCTCTTCTCCCTTTGCGTCGCAGCTGCTGGAGCCGCCGTACGTGCCTCCGCGCTACCTGCGGCC
    TACGGGGGGGAGAAACAGCATCCGTTACTCGGAGCTGGCGCCCCTGTTCGACACCACCCGGGTGTACCTGGTGGACA
    ACAAGTCGGCGGACGTGGCCTCCCTGAACTACCAGAACGACCACAGCAATTTTTTGACCACGGTCATCCAGAACAAT
    GACTACAGCCCGAGCGAGGCCAGCACCCAGACCATCAATCTGGATGACCGGTCGCACTGGGGCGGCGACCTGAAAAC
    CATCCTGCACACCAACATGCCCAACGTGAACGAGTTCATGTTCACCAATAAGTTCAAGGCGCGGGTGATGGTGTCGC
    GCTCGCACACCAAGGAAGACCGGGTGGAGCTGAAGTACGAGTGGGTGGAGTTCGAGCTGCCAGAGGGCAACTACTCC
    GAGACCATGACCATTGACCTGATGAACAACGCGATCGTGGAGCACTATCTGAAAGTGGGCAGGCAGAACGGGGTCCT
    GGAGAGCGACATCGGGGTCAAGTTCGACACCAGGAACTTCCGCCTGGGGCTGGACCCCGTGACCGGGCTGGTTATGC
    CCGGGGTGTACACCAACGAGGCCTTCCATCCCGACATCATCCTGCTGCCCGGCTGCGGGGTGGACTTCACTTACAGC
    CGCCTGAGCAACCTCCTGGGCATCCGCAAGCGGCAGCCCTTCCAGGAGGGCTTCAGGATCACCTACGAGGACCTGGA
    GGGGGGCAACATCCCCGCGCTCCTCGATGTGGAGGCCTACCAGGATAGCTTGAAGGAAAATGAGGCGGGACAGGAGG
    ATACCGCCCCCGCCGCCTCCGCCGCCGCCGAGCAGGGCGAGGATGCTGCTGACACCGCGGCCGCGGACGGGGCAGAG
    GCCGACCCCGCTATGGTGGTGGAGGCTCCCGAGCAGGAGGAGGACATGAATGACAGTGCGGTGCGCGGAGACACCTT
    CGTCACCCGGGGGGAGGAAAAGCAAGCGGAGGCCGAGGCCGCGGCCGAGGAAAAGCAACTGGCGGCAGCAGCGGCGG
    CGGCGGCGTTGGCCGCGGCGGAGGCTGAGTCTGAGGGGACCAAGCCCGCCAAGGAGCCCGTGATTAAGCCCCTGACC
    GAAGATAGCAAGAAGCGCAGTTACAACCTGCTCAAGGACAGCACCAACACCGCGTACCGCAGCTGGTACCTGGCCTA
    CAACTACGGCGACCCGTCGACGGGGGTGCGCTCCTGGACCCTGCTGTGCACGCCGGACGTGACCTGCGGCTCGGAGC
    AGGTGTACTGGTCGCTGCCCGACATGATGCAAGACCCCGTGACCTTCCGCTCCACGCGGCAGGTCAGCAACTTCCCG
    GTGGTGGGCGCCGAGCTGCTGCCCGTGCACTCCAAGAGCTTCTACAACGACCAGGCCGTCTACTCCCAGCTCATCCG
    CCAGTTCACCTCTCTGACCCACGTGTTCAATCGCTTTCCTGAGAACCAGATTCTGGCGCGCCCGCCCGCCCCCACCA
    TCACCACCGTCAGTGAAAACGTTCCTGCTCTCACAGATCACGGGACGCTACCGCTGCGCAACAGCATCGGAGGAGTC
    CAGCGAGTGACCGTTACTGACGCCAGACGCCGCACCTGCCCCTACGTTTACAAGGCCTTGGGCATAGTCTCGCCGCG
    CGTCCTTTCCAGCCGCACTTTTTGAGCAACACCACCATCATGTCCATCCTGATCTCACCCAGCAATAACTCCGGCTG
    GGGACTGCTGCGCGCGCCCAGCAAGATGTTCGGAGGGGCGAGGAAGCGTTCCGAGCAGCACCCCGTGCGCGTGCGCG
    GGCACTTCCGCGCCCCCTGGGGAGCGCACAAACGCGGCCGCGCGGGGCGCACCACCGTGGACGACGCCATCGACTCG
    GTGGTGGAGCAGGCGCGCAACTACAGGCCCGCGGTCTCTACCGTGGACGCGGCCATCCAGACCGTGGTGCGGGGCGC
    GCGGCGGTACGCCAAGCTGAAGAGCCGCCGGAAGCGCGTGGCCCGCCGCCACCGCCGCCGACCCGGGGCCGCCGCCA
    AACGCGCCGCCGCGGCCCTGCTTCGCCGGGCCAAGCGCACGGGCCGCCGCGCCGCCATGAGGGCCGCGCGCCGCTTG
    GCCGCCGGCATCACCGCCGCCACCATGGCCCCCCGTACCCGAAGACGCGCGGCCGCCGCCGCCGCCGCCGCCATCAG
    TGACATGGCCAGCAGGCGCCGGGGCAACGTGTACTGGGTGCGCGACTCGGTGACCGGCACGCGCGTGCCCGTGCGCT
    TCCGCCCCCCGCGGACTTGAGATGATGTGAAAAAACAACACTGAGTCTCCTGCTGTTGTGTGTATCCCAGCGGCGGC
    GGCGCGCGCAGCGTCATGTCCAAGCGCAAAATCAAAGAAGAGATGCTCCAGGTCGTCGCGCCGGAGATCTATGGGCC
    CCCGAAGAAGGAAGAGCAGGATTCGAAGCCCCGCAAGATAAAGCGGGTCAAAAAGAAAAAGAAAGATGATGACGATG
    CCGATGGGGAGGTGGAGTTCCTGCGCGCCACGGCGCCCAGGCGCCCGGTGCAGTGGAAGGGCCGGCGCGTAAAGCGC
    GTCCTGCGCCCCGGCACCGCGGTGGTCTTCACGCCCGGCGAGCGCTCCACCCGGACTTTCAAGCGCGTCTATGACGA
    GGTGTACGGCGACGAAGACCTGCTGGAGCAGGCCAACGAGCGCTTCGGAGAGTTTGCTTACGGGAAGCGTCAGCGGG
    CGCTGGGGAAGGAGGACCTGCTGGCGCTGCCGCTGGACCAGGGCAACCCCACCCCCAGTCTGAAGCCCGTGACCCTG
    CAGCAGGTGCTGCCGAGCAGCGCACCCTCCGAGGCGAAGCGGGGTCTGAAGCGCGAGGGCGGCGACCTGGCGCCCAC
    CGTGCAGCTCATGGTGCCCAAGCGGCAGAGGCTGGAGGATGTGCTGGAGAAAATGAAAGTAGACCCCGGTCTGCAGC
    CGGACATCAGGGTCCGCCCCATCAAGCAGGTGGCGCCGGGCCTCGGCGTGCAGACCGTGGACGTGGTCATCCCCACC
    GGCAACTCCCCCGCCGCCGCCACCACTACCGCTGCCTCCACGGACATGGAGACACAGACCGATCCCGCCGCAGCCGC
    AGCCGCAGCCGCCGCCGCGACCTCCTCGGCGGAGGTGCAGACGGACCCCTGGCTGCCGCCGGCGATGTCAGCTCCCC
    GCGCGCGTCGCGGGCGCAGGAAGTACGGCGCCGCCAACGCGCTCCTGCCCGAGTACGCCTTGCATCCTTCCATCGCG
    CCCACCCCCGGCTACCGAGGCTATACCTACCGCCCGCGAAGAGCCAAGGGTTCCACCCGCCGTCCCCGCCGACGCGC
    CGCCGCCACCACCCGCCGCCGCCGCCGCAGACGCCAGCCCGCACTGGCTCCAGTCTCCGTGAGGAAAGTGGCGCGCG
    ACGGACACACCCTGGTGCTGCCCAGGGCGCGCTACCACCCCAGCATCGTTTAAAAGCCTGTTGTGGTTCTTGCAGAT
    ATGGCCCTCACTTGCCGCCTCCGTTTCCCGGTGCCGGGATACCGAGGAGGAAGATCGCGCCGCAGGAGGGGTCTGGC
    CGGCCGCGGCCTGAGCGGAGGCAGCCGCCGCGCGCACCGGCGGCGACGCGCCACCAGCCGACGCATGCGCGGCGGGG
    TGCTGCCCCTGTTAATCCCCCTGATCGCCGCGGCGATCGGCGCCGTGCCCGGGATCGCCTCCGTGGCCTTGCAAGCG
    TCCCAGAGGCATTGACAGACTTGCAAACTTGCAAATATGGAAAAAAAAACCCCAATAAAAAAGTCTAGACTCTCACG
    CTCGCTTGGTCCTGTGACTATTTTGTAGAATGGAAGACATCAACTTTGCGTCGCTGGCCCCGCGTCACGGCTCGCGC
    CCGTTCCTGGGACACTGGAACGATATCGGCACCAGCAACATGAGCGGTGGCGCCTTCAGTTGGGGCTCTCTGTGGAG
    CGGCATTAAAAGTATCGGGTCTGCCGTTAAAAATTACGGCTCCCGGGCCTGGAACAGCAGCACGGGCCAGATGTTGA
    GAGACAAGTTGAAAGAGCAGAACTTCCAGCAGAAGGTGGTGGAGGGCCTGGCCTCCGGCATCAACGGGGTGGTGGAC
    CTGGCCAACCAGGCCGTGCAGAATAAGATCAACAGCAGACTGGACCCCCGGCCGCCGGTGGAGGAGGTGCCGCCGGC
    GCTGGAGACGGTGTCCCCCGATGGGCGTGGCGAGAAGCGCCCGCGGCCCGATAGGGAAGAGACCACTCTGGTCACGC
    AGACCGATGAGCCGCCCCCGTATGAGGAGGCCCTGAAGCAAGGTCTGCCCACCACGCGGCCCATCGCGCCCATGGCC
    ACCGGGGTGGTGGGCCGCCACACCCCCGCCACGCTGGACTTGCCTCCGCCCGCCGATGTGCCGCAGCAGCAGAAGGC
    GGCACAGCCGGGCCCGCCCGCGACCGCCTCCCGTTCCTCCGCCGGTCCTCTGCGCCGCGCGGCCAGCGGCCCCCGCG
    GGGGGGTCGCGAGGCACGGCAACTGGCAGAGCACGCTGAACAGCATCGTGGGTCTGGGGGTGCGGTCCGTGAAGCGC
    CGCCGATGCTACTGAATAGCTTAGCTAACGTGTTGTATGTGTGTATGCGCCCTATGTCGCCGCCAGAGGAGCTGCTG
    AGTCGCCGCCGTTCGCGCGCCCACCACCACCGCCACTCCGCCCCTCAAGATGGCGACCCCATCGATGATGCCGCAGT
    GGTCGTACATGCACATCTCGGGCCAGGACGCCTCGGAGTACCTGAGCCCCGGGCTGGTGCAGTTCGCCCGCGCCACC
    GAGAGCTACTTCAGCCTGAGTAACAAGTTTAGGAACCCCACGGTGGCGCCCACGCACGATGTGACCACCGACCGGTC
    TCAGCGCCTGACGCTGCGGTTCATTCCCGTGGACCGCGAGGACACCGCGTACTCGTACAAGGCGCGGTTCACCCTGG
    CCGTGGGCGACAACCGCGTGCTGGACATGGCCTCCACCTACTTTGACATCCGCGGGGTGCTGGACCGGGGTCCCACT
    TTCAAGCCCTACTCTGGCACCGCCTACAACTCCCTGGCCCCCAAGGGCGCTCCCAACTCCTGCGAGTGGGAGCAAGA
    GGAAACTCAGGCAGTTGAAGAAGCAGCAGAAGAGGAAGAAGAAGATGCTGACGGTCAAGCTGAGGAAGAGCAAGCAG
    CTACCAAAAAGACTCATGTATATGCTCAGGCTCCCCTTTCTGGCGAAAAAATTAGTAAAGATGGTCTGCAAATAGGA
    ACGGACGCTACAGCTACAGAACAAAAACCTATTTATGCAGACCCTACATTCCAGCCCGAACCCCAAATCGGGGAGTC
    CCAGTGGAATGAGGCAGATGCTACAGTCGCCGGCGGTAGAGTGCTAAAGAAATCTACTCCCATGAAACCATGCTATG
    GTTCCTATGCAAGACCCACAAATGCTAATGGAGGTCAGGGTGTACTAACGGCAAATGCCCAGGGACAGCTAGAATCT
    CAGGTTGAAATGCAATTCTTTTCAACTTCTGAAAACGCCCGTAACGAGGCTAACAACATTCAGCCCAAATTGGTGCT
    GTATAGTGAGGATGTGCACATGGAGACCCCGGATACGCACCTTTCTTACAAGCCCGCAAAAAGCGATGACAATTCAA
    AAATCATGCTGGGTCAGCAGTCCATGCCCAACAGACCTAATTACATCGGCTTCAGAGACAACTTTATCGGCCTCATG
    TATTACAATAGCACTGGCAACATGGGAGTGCTTGCAGGTCAGGCCTCTCAGTTGAATGCAGTGGTGGACTTGCAAGA
    CAGAAACACAGAACTGTCCTACCAGCTCTTGCTTGATTCCATGGGTGACAGAACCAGATACTTTTCCATGTGGAATC
    AGGCAGTGGACAGTTATGACCCAGATGTTAGAATTATTGAAAATCATGGAACTGAAGACGAGCTCCCCAACTATTGT
    TTCCCTCTGGGTGGCATAGGGGTAACTGACACTTACCAGGCTGTTAAAACCAACAATGGCAATAACGGGGGCCAGGT
    GACTTGGACAAAAGATGAAACTTTTGCAGATCGCAATGAAATAGGGGTGGGAAACAATTTCGCTATGGAGATCAACC
    TCAGTGCCAACCTGTGGAGAAACTTCCTGTACTCCAACGTGGCGCTGTACCTACCAGACAAGCTTAAGTACAACCCC
    TCCAATGTGGACATCTCTGACAACCCCAACACCTACGATTACATGAACAAGCGAGTGGTGGCCCCGGGGCTGGTGGA
    CTGCTACATCAACCTGGGCGCGCGCTGGTCGCTGGACTACATGGACAACGTCAACCCCTTCAACCACCACCGCAATG
    CGGGCCTGCGCTACCGCTCCATGCTCCTGGGCAACGGGCGCTACGTGCCCTTCCACATCCAGGTGCCCCAGAAGTTC
    TTTGCCATCAAGAACCTCCTCCTCCTGCCGGGCTCCTACACCTACGAGTGGAACTTCAGGAAGGATGTCAACATGGT
    CCTCCAGAGCTCTCTGGGTAACGATCTCAGGGTGGACGGGGCCAGCATCAAGTTCGAGAGCATCTGCCTCTACGCCA
    CCTTCTTCCCCATGGCCCACAACACGGCCTCCACGCTCGAGGCCATGCTCAGGAACGACACCAACGACCAGTCCTTC
    AATGACTACCTCTCCGCCGCCAACATGCTCTACCCCATACCCGCCAACGCCACCAACGTCCCCATCTCCATCCCCTC
    GCGCAACTGGGCGGCCTTCCGCGGCTGGGCCTTCACCCGCCTCAAGACCAAGGAGACCCCCTCCCTGGGCTCGGGAT
    TCGACCCCTACTACACCTACTCGGGCTCCATTCCCTACCTGGACGGCACCTTCTACCTCAACCACACTTTCAAGAAG
    GTCTCGGTCACCTTCGACTCCTCGGTCAGCTGGCCGGGCAACGACCGTCTGCTCACCCCCAACGAGTTCGAGATCAA
    GCGCTCGGTCGACGGGGAGGGCTACAACGTGGCCCAGTGCAACATGACCAAGGACTGGTTCCTGGTCCAGATGCTGG
    CCAACTACAACATCGGCTACCAGGGCTTCTACATCCCAGAGAGCTACAAGGACAGGATGTACTCCTTCTTCAGGAAC
    TTCCAGCCCATGAGCCGGCAGGTGGTGGACCAGACCAAGTACAAGGACTACCAGGAGGTGGGCATCATCCACCAGCA
    CAACAACTCGGGCTTCGTGGGCTACCTCGCCCCCACCATGCGCGAGGGACAGGCCTACCCCGCCAACTTCCCCTATC
    CGCTCATAGGCAAGACCGCGGTCGACAGCATCACCCAGAAAAAGTTCCTCTGCGACCGCACCCTCTGGCGCATCCCC
    TTCTCCAGCAACTTCATGTCCATGGGTGCGCTCTCGGACCTGGGCCAGAACTTGCTCTACGCCAACTCCGCCCACGC
    CCTCGACATGACCTTCGAGGTCGACCCCATGGACGAGCCCACCCTTCTCTATGTTCTGTTCGAAGTCTTTGACGTGG
    TCCGGGTCCACCAGCCGCACCGCGGCGTCATCGAGACCGTGTACCTGCGTACGCCCTTCTCGGCCGGCAACGCCACC
    ACCTAAAGAAGCAAGCCGCAGTCATCGCCGCCTGCATGCCGTCGGGTTCCACCGAGCAAGAGCTCAGGGCCATCGTC
    AGAGACCTGGGATGCGGGCCCTATTTTTTGGGCACCTTCGACAAGCGCTTCCCTGGCTTTGTCTCCCCACACAAGCT
    GGCCTGCGCCATCGTCAACACGGCCGGCCGCGAGACCGGGGGCGTGCACTGGCTGGCCTTCGCCTGGAACCCGCGCT
    CCAAAACATGCTTCCTCTTTGACCCCTTCGGCTTTTCGGACCAGCGGCTCAAGCAAATCTACGAGTTCGAGTACGAG
    GGCTTGCTGCGTCGCAGCGCCATCGCCTCCTCGCCCGACCGCTGCGTCACCCTCGAAAAGTCCACCCAGACCGTGCA
    GGGGCCCGACTCGGCCGCCTGCGGTCTCTTCTGCTGCATGTTTCTGCACGCCTTTGTGCACTGGCCTCAGAGTCCCA
    TGGACCGCAACCCCACCATGAACTTGCTGACGGGGGTGCCCAACTCCATGCTCCAGAGCCCCCAGGTCGAGCCCACC
    CTGCGCCGCAACCAGGAGCAGCTCTACAGCTTCCTGGAGCGCCACTCGCCTTACTTCCGCCGCCACAGCGCACAGAT
    CAGGAGGGCCACCTCCTTCTGCCACTTGCAAGAGATGCAAGAAGGGTAATAACGATGTACACACTTTTTTTCTCAAT
    AAATGGCATCTTTTTATTTATACAAGCTCTCTGGGGTATTCATTTCCCACCACCACCCGCCGTTGTCGCCATCTGGC
    TCTATTTAGAAATCGAAAGGGTTCTGCCGGGAGTCGCCGTGCGCCACGGGCAGGGACACGTTGCGATACTGGTAGCG
    GGTGCCCCACTTGAACTCGGGCACCACCAGGCGAGGCAGCTCGGGGAAGTTTTCGCTCCACAGGCTGCGGGTCAGCA
    CCAGCGCGTTCATCAGGTCGGGCGCCGAGATCTTGAAGTCGCAGTTGGGGCCGCCGCCCTGCGCGCGCGAGTTGCGG
    TACACCGGGTTGCAGCACTGGAACACCAACAGCGCCGGGTGCTTCACGCTGGCCAGCACGCTGCGGTCGGAGATCAG
    CTCGGCGTCCAGGTCCTCCGCGTTGCTCAGCGCGAACGGGGTCATCTTGGGCACTTGCCGCCCCAGGAAGGGCGCGT
    GCCCCGGTTTCGAGTTGCAGTCGCAGCGCAGCGGGATCAGCAGGTGCCCGTGCCCGGACTCGGCGTTGGGGTACAGC
    GCGCGCATGAAGGCCTGCATCTGGCGGAAGGCCATCTGGGCCTTGGCGCCCTCCGAGAAGAACATGCCGCAGGACTT
    GCCCGAGAACTGGTTTGCGGGGCAGCTGGCGTCGTGCAGGCAGCAGCGCGCGTCGGTGTTGGCGATCTGCACCACGT
    TGCGCCCCCACCGGTTCTTCACGATCTTGGCCTTGGACGATTGCTCCTTCAGCGCGCGCTGCCCGTTCTCGCTGGTC
    ACATCCATCTCGATCACATGTTCCTTGTTCACCATGCTGCTGCCGTGCAGACACTTCAGCTCGCCCTCCGTCTCGGT
    GCAGCGGTGCTGCCACAGCGCGCAGCCCGTGGGCTCGAAAGACTTGTAGGTCACCTCCGCGAAGGACTGCAGGTACC
    CCTGCAAAAAGCGGCCCATCATGGTCACGAAGGTCTTGTTGCTGCTGAAGGTCAGCTGCAGCCCGCGGTGCTCCTCG
    TTCAGCCAGGTCTTGCACACGGCCGCCAGCGCCTCCACCTGGTCGGGCAGCATCTTGAAGTTCACCTTCAGCTCATT
    CTCCACGTGGTACTTGTCCATCAGCGTGCGCGCCGCCTCCATGCCCTTCTCCCAGGCCGACACCAGCGGCAGGCTCA
    CGGGGTTCTTCACCATCACCGTGGCCGCCGCCTCCGCCGCGCTTTCGCTTTCCGCCCCGCTGTTCTCTTCCTCTTCC
    TCCTCTTCCTCGCCGCCGCCCACTCGCAGCCCCCGCACCACGGGGTCGTCTTCCTGCAGGCGCTGCACCTTGCGCTT
    GCCGTTGCGCCCCTGCTTGATGCGCACGGGCGGGTTGCTGAAGCCCACCATCACCAGCGCGGCCTCTTCTTGCTCGT
    CCTCGCTGTCCAGAATGACCTCCGGGGAGGGGGGGTTGGTCATCCTCAGTACCGAGGCACGCTTCTTTTTCTTCCTG
    GGGGCGTTCGCCAGCTCCGCGGCTGCGGCCGCTGCCGAGGTCGAAGGCCGAGGGCTGGGCGTGCGCGGCACCAGCGC
    GTCCTGCGAGCCGTCCTCGTCCTCCTCGGACTCGAGACGGAGGCGGGCCCGCTTCTTCGGGGGCGCGCGGGGCGGCG
    GAGGCGGCGGCGGCGACGGAGACGGGGACGAGACATCGTCCAGGGTGGGTGGACGGCGGGCCGCGCCGCGTCCGCGC
    TCGGGGGTGGTCTCGCGCTGGTCCTCTTCCCGACTGGCCATCTCCCACTGCTCCTTCTCCTATAGGCAGAAAGAGAT
    CATGGAGTCTCTCATGCGAGTCGAGAAGGAGGAGGACAGCCTAACCGCCCCCTCTGAGCCCTCCACCACCGCCGCCA
    CCACCGCCAATGCCGCCGCGGACGACGCGCCCACCGAGACCACCGCCAGTACCACCCTCCCCAGCGACGCACCCCCG
    CTCGAGAATGAAGTGCTGATCGAGCAGGACCCGGGTTTTGTGAGCGGAGAGGAGGATGAGGTGGATGAGAAGGAGAA
    GGAGGAGGTCGCCGCCTCAGTGCCAAAAGAGGATAAAAAGCAAGACCAGGACGACGCAGATAAGGATGAGACAGCAG
    TCGGGCGGGGGAACGGAAGCCATGATGCTGATGACGGCTACCTAGACGTGGGAGACGACGTGCTGCTTAAGCACCTG
    CACCGCCAGTGCGTCATCGTCTGCGACGCGCTGCAGGAGCGCTGCGAAGTGCCCCTGGACGTGGCGGAGGTCAGCCG
    CGCCTACGAGCGGCACCTCTTCGCGCCGCACGTGCCCCCCAAGCGCCGGGAGAACGGCACCTGCGAGCCCAACCCGC
    GTCTCAACTTCTACCCGGTCTTCGCGGTACCCGAGGTGCTGGCCACCTACCACATCTTTTTCCAAAACTGCAAGATC
    CCCCTCTCCTGCCGCGCCAACCGCACCCGCGCCGACAAAACCCTGACCCTGCGGCAGGGCGCCCACATACCTGATAT
    CGCCTCTCTGGAGGAAGTGCCCAAGATCTTCGAGGGTCTCGGTCGCGACGAGAAACGGGCGGCGAACGCTCTGCACG
    GAGACAGCGAAAACGAGAGTCACTCGGGGGTGCTGGTGGAGCTCGAGGGCGACAACGCGCGCCTGGCCGTACTCAAG
    CGCAGCATAGAGGTCACCCACTTTGCCTACCCGGCGCTCAACCTGCCCCCCAAGGTCATGAGTGTGGTCATGGGCGA
    GCTCATCATGCGCCGCGCCCAGCCCCTGGCCGCGGATGCAAACTTGCAAGAGTCCTCCGAGGAAGGCCTGCCCGCGG
    TCAGCGACGAGCAGCTGGCGCGCTGGCTGGAGACCCGCGACCCCGCGCAGCTGGAGGAGCGGCGCAAGCTCATGATG
    GCCGCGGTGCTGGTCACCGTGGAGCTCGAGTGTCTGCAGCGCTTCTTCGCGGACCCCGAGATGCAGCGCAAGCTCGA
    GGAGACCCTGCACTACACCTTCCGCCAGGGCTACGTGCGCCAGGCCTGCAAGATCTCCAACGTGGAGCTCTGCAACC
    TGGTCTCCTACCTGGGCATCCTGCACGAGAACCGCCTCGGGCAGAACGTCCTGCACTCCACCCTCAAAGGGGAGGCG
    CGCCGCGACTACATCCGCGACTGCGCCTACCTCTTCCTCTGCTACACCTGGCAGACGGCCATGGGGGTCTGGCAGCA
    GTGCCTGGAGGAGCGCAACCTCAAGGAGCTGGAAAAGCTCCTCAAGCGCACCCTCAGGGACCTCTGGACGGGCTTCA
    ACGAGCGCTCGGTGGCCGCCGCGCTGGCGGACATCATCTTTCCCGAGCGCCTGCTCAAGACCCTGCAGCAGGGCCTG
    CCCGACTTCACCAGCCAGAGCATGCTGCAGAACTTCAGGACTTTCATCCTGGAGCGCTCGGGCATCCTGCCGGCCAC
    TTGCTGCGCGCTGCCCAGCGACTTCGTGCCCATCAAGTACAGGGAGTGCCCGCCGCCGCTCTGGGGCCACTGCTACC
    TCTTCCAGCTGGCCAACTACCTCGCCTACCACTCGGACCTCATGGAAGACGTGAGCGGCGAGGGCCTGCTCGAGTGC
    CACTGCCGCTGCAACCTCTGCACGCCCCACCGCTCTCTAGTCTGCAACCCGCAGCTGCTCAGCGAGAGTCAGATTAT
    CGGTACCTTCGAGCTGCAGGGTCCCTCGCCTGACGAGAAGTCCGCGGCTCCAGGGCTGAAACTCACTCCGGGGCTGT
    GGACTTCCGCCTACCTACGCAAATTTGTACCTGAGGACTACCACGCCCACGAGATCAGGTTCTACGAAGACCAATCC
    CGCCCGCCCAAGGCGGAGCTCACCGCCTGCGTCATCACCCAGGGGCACATCCTGGGCCAATTGCAAGCCATCAACAA
    AGCCCGCCGAGAGTTCTTGCTGAAAAAGGGTCGGGGGGTGTACCTGGACCCCCAGTCCGGCGAGGAGCTAAACCCGC
    TACCCCCGCCGCCGCCCCAGCAGCGGGACCTTGCTTCCCAGGATGGCACCCAGAAAGAAGCAGCAGCCGCCGCCGCC
    GCCGCAGCCATACATGCTTCTGGAGGAAGAGGAGGAGGACTGGGACAGTCAGGCAGAGGAGGTTTCGGACGAGGAGC
    AGGAGGAGATGATGGAAGACTGGGAGGAGGACAGCAGCCTAGACGAGGAAGCTTCAGAGGCCGAAGAGGTGGCAGAC
    GCAACACCATCGCCCTCGGTCGCAGCCCCCTCGCCGGGGCCCCTGAAATCCTCCGAACCCAGCACCAGCGCTATAAC
    CTCCGCTCCTCCGGCGCCGGCGCCACCCGCCCGCAGACCCAACCGTAGATGGGACACCACAGGAACCGGGGTCGGTA
    AGTCCAAGTGCCCGCCGCCGCCACCGCAGCAGCAGCAGCAGCAGCGCCAGGGCTACCGCTCGTGGCGCGGGCACAAG
    AACGCCATAGTCGCCTGCTTGCAAGACTGCGGGGGCAACATCTCTTTCGCCCGCCGCTTCCTGCTATTCCACCACGG
    GGTCGCCTTTCCCCGCAATGTCCTGCATTACTACCGTCATCTCTACAGCCCCTACTGCAGCGGCGACCCAGAGGCGG
    CAGCGGCAGCCACAGCGGCGACCACCACCTAGGAAGATATCCTCCGCGGGCAAGACAGCGGCAGCAGCGGCCAGGAG
    ACCCGCGGCAGCAGCGGCGGGAGCGGTGGGCGCACTGCGCCTCTCGCCCAACGAACCCCTCTCGACCCGGGAGCTCA
    GACACAGGATCTTCCCCACTTTGTATGCCATCTTCCAACAGAGCAGAGGCCAGGAGCAGGAGCTGAAAATAAAAAAC
    AGATCTCTGCGCTCCCTCACCCGCAGCTGTCTGTATCACAAAAGCGAAGATCAGCTTCGGCGCACGCTGGAGGACGC
    GGAGGCACTCTTCAGCAAATACTGCGCGCTCACTCTTAAAGACTAGCTCCGCGCCCTTCTCGAATTTAGGCGGGAGA
    AAACTACGTCATCGCCGGCCGCCGCCCAGCCCGCCCAGCCGAGATGAGCAAAGAGATTCCCACGCCATACATGTGGA
    GCTACCAGCCGCAGATGGGACTCGCGGCGGGAGCGGCCCAGGACTACTCCACCCGCATGAACTACATGAGCGCGGGA
    CCCCACATGATCTCACAGGTCAACGGGATCCGCGCCCAGCGAAACCAAATACTGCTGGAACAGGCGGCCATCACCGC
    CACGCCCCGCCATAATCTCAACCCCCGAAATTGGCCCGCCGCCCTCGTGTACCAGGAAACCCCCTCCGCCACCACCG
    TACTACTTCCGCGTGACGCCCAGGCCGAAGTCCAGATGACTAACTCAGGGGCGCAGCTCGCGGGCGGCTTTCGTCAC
    GGGGCGCGGCCGCTCCGACCAGGTATAAGACACCTGATGATCAGAGGCCGAGGTATCCAGCTCAACGACGAGTCGGT
    GAGCTCTTCGCTCGGTCTCCGTCCGGACGGAACTTTCCAGCTCGCCGGATCCGGCCGCTCTTCGTTCACGCCCCGCC
    AGGCGTACCTGACTCTGCAGACCTCGTCCTCGGAGCCCCGCTCCGGCGGCATCGGAACCCTCCAGTTCGTGGAGGAG
    TTCGTGCCCTCGGTCTACTTCAACCCCTTCTCGGGACCTCCCGGACGCTACCCCGACCAGTTCATTCCGAACTTTGA
    CGCGGTGAAGGACTCGGCGGACGGCTACGACTGAATGTCAGGTGTCGAGGCAGAGCAGCTTCGCCTGAGACACCTCG
    AGCACTGCCGCCGCCACAAGTGCTTCGCCCGCGGTTCTGGTGAGTTCTGCTACTTTCAGCTACCCGAGGAGCATACC
    GAGGGGCCGGCGCACGGCGTCCGCCTGACCACCCAGGGCGAGGTTACCTGTTCCCTCATCCGGGAGTTTACCCTCCG
    TCCCCTGCTAGTGGAGCGGGAGCGGGGTCCCTGTGTCCTAACTATCGCCTGCAACTGCCCTAACCCTGGATTACATC
    AAGATCTTTGCTGTCATCTCTGTGCTGAGTTTAATAAACGCTGAGATCAGAATCTACTGGGGCTCCTGTCGCCATCC
    TGTGAACGCCACCGTCTTCACCCACCCCGACCAGGCCCAGGCGAACCTCACCTGCGGTCTGCATCGGAGGGCCAAGA
    AGTACCTCACCTGGTACTTCAACGGCACCCCCTTTGTGGTTTACAACAGCTTCGACGGGGACGGAGTCTCCCTGAAA
    GACCAGCTCTCCGGTCTCAGCTACTCCATCCACAAGAACACCACCCTCCAACTCTTCCCTCCCTACCTGCCGGGAAC
    CTACGAGTGCGTCACCGGCCGCTGCACCCACCTCACCCGCCTGATCGTAAACCAGAGCTTTCCGGGAACAGATAACT
    CCCTCTTCCCCAGAACAGGAGGTGAGCTCAGGAAACTCCCCGGGGACCAGGGCGGAGACGTACCTTCGACCCTTGTG
    GGGTTAGGATTTTTTATTACCGGGTTGCTGGCTCTTTTAATCAAAGTTTCCTTGAGATTTGTTCTTTCCTTCTACGT
    GTATGAACACCTCAACCTCCAATAACTCTACCCTTTCTTCGGAATCAGGTGACTTCTCTGAAATCGGGCTTGGTGTG
    CTGCTTACTCTGTTGATTTTTTTCCTTATCATACTCAGCCTTCTGTGCCTCAGGCTCGCCGCCTGCTGCGCACACAT
    CTATATCTACTGCTGGTTGCTCAAGTGCAGGGGTCGCCACCCAAGATGAACAGGTACATGGTCCTATCGATCCTAGG
    CCTGCTGGCCCTGGCGGCCTGCAGCGCCGCCAAAAAAGAGATTACCTTTGAGGAGCCCGCTTGCAATGTAACTTTCA
    AGCCCGAGGGTGACCAATGCACCACCCTCGTCAAATGCGTTACCAATCATGAGAGGCTGCGCATCGACTACAAAAAC
    AAAACTGGCCAGTTTGCGGTCTATAGTGTGTTTACGCCCGGAGACCCCTCTAACTACTCTGTCACCGTCTTCCAGGG
    CGGACAGTCTAAGATATTCAATTACACTTTCCCTTTTTATGAGTTATGCGATGCGGTCATGTACATGTCAAAACAGT
    ACAACCTGTGGCCTCCCTCTCCCCAGGCGTGTGTGGAAAATACTGGGTCTTACTGCTGTATGGCTTTCGCAATCACT
    ACGCTCGCTCTAATCTGCACGGTGCTATACATAAAATTCAGGCAGAGGCGAATCTTTATCGATGAAAAGAAAATGCC
    TTGATCGCTAACACCGGCTTTCTATCTGCAGAATGAATGCAATCACCTCCCTACTAATCACCACCACCCTCCTTGCG
    ATTGCCCATGGGTTGACACGAATCGAAGTGCCAGTGGGGTCCAATGTCACCATGGTGGGCCCCGCCGGCAATTCCAC
    CCTCATGTGGGAAAAATTTGTCCGCAATCAATGGGTTCATTTCTGCTCTAACCGAATCAGTATCAAGCCCAGAGCCA
    TCTGCGATGGGCAAAATCTAACTCTGATCAATGTGCAAATGATGGATGCTGGGTACTATTACGGGCAGCGGGGAGAA
    ATCATTAATTACTGGCGACCCCACAAGGACTACATGCTGCATGTAGTCGAGGCACTTCCCACTACCACCCCCACTAC
    CACCTCTCCCACCACCACCACCACTACTACTACTACTACTACTACTACTACTACTACCACTACCGCTGCCCGCCATA
    CCCGCAAAAGCACCATGATTAGCACAAAGCCCCCTCGTGCTCACTCCCACGCCGGCGGGCCCATCGGTGCGACCTCA
    GAAACCACCGAGCTTTGCTTCTGCCAATGCACTAACGCCAGCGCTCATGAACTGTTCGACCTGGAGAATGAGGATGT
    CCAGCAGAGCTCCGCTTGCCTGACCCAGGAGGCTGTGGAGCCCGTTGCCCTGAAGCAGATCGGTGATTCAATAATTG
    ACTCTTCTTCTTTTGCCACTCCCGAATACCCTCCCGATTCTACTTTCCACATCACGGGTACCAAAGACCCTAACCTC
    TCTTTCTACCTGATGCTGCTGCTCTGTATCTCTGTGGTCTCTTCCGCGCTGATGTTACTGGGGATGTTCTGCTGCCT
    GATCTGCCGCAGAAAGAGAAAAGCTCGCTCTCAGGGCCAACCACTGATGCCCTTCCCCTACCCCCCGGATTTTGCAG
    ATAACAAGATATGAGCTCGCTGCTGACACTAACCGCTTTACTAGCCTGCGCTCTAACCCTTGTCGCTTGCGACTCGA
    GATTCCACAATGTCACAGCTGTGGCAGGAGAAAATGTTACTTTCAACTCCACGGCCGATACCCAGTGGTCGTGGAGT
    GGCTCAGGTAGCTACTTAACTATCTGCAATAGCTCCACTTCCCCCGGCATATCCCCAACCAAGTACCAATGCAATGC
    CAGCCTGTTCACCCTCATCAACGCTTCCACCCTGGACAATGGACTCTATGTAGGCTATGTACCCTTTGGTGGGCAAG
    GAAAGACCCACGCTTACAACCTGGAAGTTCGCCAGCCCAGAACCACTACCCAAGCTTCTCCCACCACCACCACCACC
    ACCACCATCACCAGCAGCAGCAGCAGCAGCAGCCACAGCAGCAGCAGCAGATTATTGACTTTGGTTTTGGCCAGCTC
    ATCTGCCGCTACCCAGGCCATCTACAGCTCTGTGCCCGAAACCACTCAGATCCACCGCCCAGAAACGACCACCGCCA
    CCACCCTACACACCTCCAGCGATCAGATGCCGACCAACATCACCCCCTTGGCTCTTCAAATGGGACTTACAAGCCCC
    ACTCCAAAACCAGTGGATGCGGCCGAGGTCTCCGCCCTCGTCAATGACTGGGCGGGGCTGGGAATGTGGTGGTTCGC
    CATAGGCATGATGGCGCTCTGCCTGCTTCTGCTCTGGCTCATCTGCTGCCTCCACCGCAGGCGAGCCAGACCCCCCA
    TCTATAGACCCATCATTGTCCTGAACCCCGATAATGATGGGATCCATAGATTGGATGGCCTGAAAAACCTACTTTTT
    TCTTTTACAGTATGATAAATTGAGACATGCCTCGCATTTTCTTGTACATGTTCCTTCTCCCACCTTTTCTGGGGTGT
    TCTACGCTGGCCGCTGTGTCTCACCTGGAGGTAGACTGCCTCTCACCCTTCACTGTCTACCTGCTTTACGGATTGGT
    CACCCTCACTCTCATCTGCAGCCTAATCACAGTAATCATCGCCTTCATCCAGTGCATTGATTACATCTGTGTGCGCC
    TCGCATACTTCAGACACCACCCGCAGTACCGAGACAGGAACATTGCCCAACTTCTAAGACTGCTCTAATCATGCATA
    AGACTGTGATCTGCCTTCTGATCCTCTGCATCCTGCCCACCCTCACCTCCTGCCAGTACACCACAAAATCTCCGCGC
    AAAAGACATGCCTCCTGCCGCTTCACCCAACTGTGGAATATACCCAAATGCTACAACGAAAAGAGCGAGCTCTCCGA
    AGCTTGGCTGTATGGGGTCATCTGTGTCTTAGTTTTCTGCAGCACTGTCTTTGCCCTCATAATCTACCCCTACTTTG
    ATTTGGGATGGAACGCGATCGATGCCATGAATTACCCCACCTTTCCCGCACCCGAGATAATTCCACTGCGACAAGTT
    GTACCCGTTGTCGTTAATCAACGCCCCCCATCCCCTACGCCCACTGAAATCAGCTACTTTAACCTAACAGGCGGAGA
    TGACTGACGCCCTAGATCTAGAAATGGACGGCATCAGTACCGAGCAGCGTCTCCTAGAGAGGCGCAGGCAGGCGGCT
    GAGCAAGAGCGCCTCAATCAGGAGCTCCGAGATCTCGTTAACCTGCACCAGTGCAAAAGAGGCATCTTTTGTCTGGT
    AAAGCAGGCCAAAGTCACCTACGAGAAGACCGGCAACAGCCACCGCCTCAGTTACAAATTGCCCACCCAGCGCCAGA
    AGCTGGTGCTCATGGTGGGTGAGAATCCCATCACCGTCACCCAGCACTCGGTAGAGACCGAGGGGTGTCTGCACTCC
    CCCTGTCGGGGTCCAGAAGACCTCTGCACCCTGGTAAAGACCCTGTGCGGTCTCAGAGATTTAGTCCCCTTTAACTA
    ATCAAACACTGGAATCAATAAAAAGAATCACTTACTTAAAATCAGACAGCAGGTCTCTGTCCAGTTTATTCAGCAGC
    ACCTCCTTCCCCTCCTCCCAACTCTGGTACTCCAAACGCCTTCTGGCGGCAAACTTCCTCCACACCCTGAAGGGAAT
    GTCAGATTCTTGCTCCTGTCCCTCCGCACCCACTATCTTCATGTTGTTGCAGATGAAGCGCACCAAAACGTCTGACG
    AGAGCTTCAACCCCGTGTACCCCTATGACACGGAAAGCGGCCCTCCCTCCGTCCCTTTCCTCACCCCTCCCTTCGTG
    TCTCCCGATGGATTCCAAGAAAGTCCCCCCGGGGTCCTGTCTCTGAACCTGGCCGAGCCCCTGGTCACTTCCCACGG
    CATGCTCGCCCTGAAAATGGGAAGTGGCCTCTCCCTGGACGACGCTGGCAACCTCACCTCTCAAGATATCACCACCG
    CTAGCCCTCCCCTCAAAAAAACCAAGACCAACCTCAGCCTAGAAACCTCATCCCCCCTAACTGTGAGCACCTCAGGC
    GCCCTCACCGTAGCAGCCGCCGCTCCCCTGGCGGTGGCCGGCACCTCCCTCACCATGCAATCAGAGGCCCCCCTGAC
    AGTACAGGATGCAAAACTCACCCTGGCCACCAAAGGCCCCCTGACCGTGTCTGAAGGCAAACTGGCCTTGCAAACAT
    CGGCCCCGCTGACGGCCGCTGACAGCAGCACCCTCACAGTCAGTGCCACACCACCCCTTAGCACAAGCAATGGCAGC
    TTGGGTATTGACATGCAAGCCCCCATTTACACCACCAATGGAAAACTAGGACTTAACTTTGGCGCTCCCCTGCATGT
    GGTAGACAGCCTAAATGCACTGACTGTAGTTACTGGCCAAGGTCTTACGATAAACGGAACAGCCCTACAAACTAGAG
    TCTCAGGTGCCCTCAACTATGACACATCAGGAAACCTAGAATTGAGAGCTGCAGGGGGTATGCGAGTTGATGCAAAT
    GGTCAACTTATCCTTGATGTAGCTTACCCATTTGATGCACAAAACAATCTCAGCCTTAGGCTTGGACAGGGACCCCT
    GTTTGTTAACTCTGCCCACAACTTGGATGTTAACTACAACAGAGGCCTCTACCTGTTCACATCTGGAAATACCAAAA
    AGCTAGAAGTTAATATCAAAACAGCCAAGGGTCTCATTTATGATGACACTGCTATAGCAATCAATGCGGGTGATGGG
    CTACAGTTTGACTCAGGCTCAGATACAAATCCATTAAAAACTAAACTTGGATTAGGACTGGATTATGACTCCAGCAG
    AGCCATAATTGCTAAACTGGGAACTGGCCTAAGCTTTGACAACACAGGTGCCATCACAGTAGGCAACAAAAATGATG
    ACAAGCTTACCTTGTGGACCACACCAGACCCATCCCCTAACTGTAGAATCTATTCAGAGAAAGATGCTAAATTCACA
    CTTGTTTTGACTAAATGCGGCAGTCAGGTGTTGGCCAGCGTTTCTGTTTTATCTGTAAAAGGTAGCCTTGCGCCCAT
    CAGTGGCACAGTAACTAGTGCTCAGATTGTCCTCAGATTTGATGAAAATGGAGTTCTACTAAGCAATTCTTCCCTTG
    ACCCTCAATACTGGAACTACAGAAAAGGTGACCTTACAGAGGGCACTGCATATACCAACGCAGTGGGATTTATGCCC
    AACCTCACAGCATACCCAAAAACACAGAGCCAAACTGCTAAAAGCAACATTGTAAGTCAGGTTTACTTGAATGGGGA
    CAAATCCAAACCCATGACCCTCACCATTACCCTCAATGGAACTAATGAAACAGGAGATGCCACAGTAAGCACTTACT
    CCATGTCATTCTCATGGAACTGGAATGGAAGTAATTACATTAATGAAACGTTCCAAACCAACTCCTTCACCTTCTCC
    TACATCGCCCAAGAATAAAAAGCATGACGCTGTTGATTTGATTCAATGTGTTTCTGTTTTATTTTCAAGCACAACAA
    AATCATTCAAGTCATTCTTCCATCTTAGCTTAATAGACACAGTAGCTTAATAGACCCAGTAGTGCAAAGCCCCATTC
    TAGCTTATAGATCAGACAGTGATAATTAACCACCACCACCACCATACCTTTTGATTCAGGAAATCATGATCATCACA
    GGATCCTAGTCTTCAGGCCGCCCCCTCCCTCCCAAGACACAGAATACACAGTCCTCTCCCCCCGACTGGCTTTAAAT
    AACACCATCTGGTTGGTCACAGACATGTTCTTAGGGGTTATATTCCACACGGTCTCCTGCCGCGCCAGGCGCTCGTC
    GGTGATGTTGATAAACTCTCCCGGCAGCTCGCTCAAGTTCACGTCGCTGTCCAGCGGCTGAACCTCCGGCTGACGCG
    ATAACTGTGCGACCGGCTGCTGGACGAACGGAGGCCGCGCCTACAAGGGGGTAGAGTCATAATCCTCGGTCAGGATA
    GGGCGGTGATGCAGCAGCAGCGAGCGAAACATCTGCTGCCGCCGCCGCTCCGTCCGGCAGGAAAACAACACGCCGGT
    GGTCTCCTCCGCGATAATCCGCACCGCCCGCAGCATCAGCTTCCTCGTTCTCCGCGCGCAGCACCTCACCCTTATCT
    CGCTCAAATCGGCGCAGTAGGTACAGCACAGCACCACGATGTTATTCATGATCCCACAGTGCAGGGCGCTGTATCCA
    AAGCTCATGCCGGGAACCACCGCCCCCACGTGGCCATCGTACCACAAGCGCACGTAAATCAAGTGTCGACCCCTCAT
    GAACGCGCTGGACACAAACATTACTTCCTTGGGCATGTTGTAATTCACCACCTCCCGGTACCAGATAAACCTCTGGT
    TGAACAGGGCACCTTCCACCACCATCCTGAACCAAGAGGCCAGAACCTGCCCACCGGCTATGCACTGCAGGGAACCC
    GGGTTGGAACAATGACAATGCAGACTCCAAGGCTCGTAACCGTGGATCATCCGGCTGCTGAAGGCATCGATGTTGGC
    ACAACACAGACACACGTGCATGCACTTTCTCATGATTAGCAGCTCTTCCCTCGTCAGGATCATATCCCAAGGAATAA
    CCCATTCTTGAATCAACGTAAAACCCACACAGCAGGGAAGGCCTCGCACATAACTCACGTTGTGCATGGTCAGCGTG
    TTGCATTCCGGAAACAGCGGATGATCCTCCAGTATCGAGGCGCGGGTCTCCTTCTCACAGGGAGGTAAAGGGTCCCT
    GCTGTACGGACTGCGCCGGGACGACCGAGATCGTGTTGAGCGTAGTGTCATGGAAAAGGGAACGCCGGACGTGGTCA
    TACTTCTTGAAGCAGAACCAGGTTCGCGCGTGGCAGGCCTCCTTGCGTCTGCGGTCTCGCCGTCTAGCTCGCTCCGT
    GTGATAGTTGTAGTACAGCCACTCCCGCAGAGCGTCGAGGCGCACCCTGGCTTCCGGATCTATGTAGACTCCGTCTT
    GCACCGCGGCCCTGATAATATCCACCACCGTAGAATAAGCAACACCCAGCCAAGCAATACACTCGCTCTGCGAGCGG
    CAGACAGGAGGAGCGGGCAGAGATGGGAGAACCATGATAAAAAACTTTTTTTAAAGAATATTTTCCAATTCTTCGAA
    AGTAAGATCTATCAAGTGGCAGCGCTCCCCTCCACTGGCGCGGTCAAACTCTACGGCCAAAGCACAGACAACGGCAT
    TTCTAAGATGTTCCTTAATGGCGTCCAAAAGACACACCGCTCTCAAGTTGCAGTAAACTATGAATGAAAACCCATCC
    GGCTGATTTTCCAATATAGACGCGCCGGCAGCGTCCACCAAACCCAGATAATTTTCTTCTCTCCAGCGGTTTACGAT
    CTGTCTAAGCAAATCCCTTATATCAAGTCCGACCATGCCAAAAATCTGCTCAAGAGCGCCCTCCACCTTCATGTACA
    AGCAGCGCATCATGATTGCAAAAATTCAGGTTCTTCAGAGACCTGTATAAGATTCAAAATGGGAACATTAACAAAAA
    TTCCTCTGTCGCGCAGATCCCTTCGCAGGGCAAGCTGAACATAATCAGACAGGTCCGAACGGACCAGTGAGGCCAAA
    TCCCCACCAGGAACCAGATCCAGAGACCCTATACTGATTATGACGCGCATACTCGGGGCTATGCTGACCAGCGTAGC
    GCCGATGTAGGCGTGCTGCATGGGCGGCGAGATAAAATGCAAAGTGCTGGTTAAAAAATCAGGCAAAGCCTCGCGCA
    AAAAAGCTAACACATCATAATCATGCTCATGCAGGTAGTTGCAGGTAAGCTCAGGAACCAAAACGGAATAACACACG
    ATTTTCCTCTCAAACATGACTTCGCGGATACTGCGTAAAACAAAAAATTATAAATAAAAAATTAATTAAATAACTTA
    AACATTGGAAGCCTGTCTCACAACAGGAAAAACCACTTTAATCAACATAAGACGGGCCACGGGCATGCCGGCATAGC
    CGTAAAAAAATTGGTCCCCGTGATTAACAAGTACCACAGACAGCTCCCCGGTCATGTCGGGGGTCATCATGTGAGAC
    TCTGTATACACGTCTGGATTGTGAACATCAGACAAACAAAGAAATCGAGCCACGTAGCCCGGAGGTATAATCACCCG
    CAGGCGGAGGTACAGCAAAACGACCCCCATAGGAGGAATCACAAAATTAGTAGGAGAAAAAAATACATAAACACCAG
    AAAAACCCTGTTGCTGAGGCAAAATAGCGCCCTCCCGATCCAAAACAACATAAAGCGCTTCCACAGGAGCAGCCATA
    ACAAAGACCCGAGTCTTACCAGTAAAAGAAAAAAGATCTCTCAACGCAGCACCAGCACCAACACTTCGCAGTGTAAA
    AGGCCAAGTGCCGAGAGAGTATATATAGGAATAAAAAGTGACGTAAACGGGCAAAGTCCAAAAAACGCCCAGAAAAA
    CCGCACGCGAACCTACGCCCCGAAACGAAAGCCAAAAAACACTAGACACTCCCTTCCGGCGTCAACTTCCGCTTTCC
    CACGCTACGTCACTTCCCCCGGTCAAACAAACTACATATCCCGAACTTCCAAGTCGCCACGCCCAAAACACCGCCTA
    CACCTCCCCGCCCGCCGGCCCGCCCCCGGACCCGCCTCCCGCCCCGCGCCGCCCATCTCATTATCATATTGGCTTCA
    ATCCAAAATAAGGTATATTATTGATGATG
    Polynucleotide sequence encodinq the CASI promoter
    SEQ ID NO: 2
    GGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAA
    TAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACT
    GCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGC
    CTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTA
    CCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTA
    TTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGC
    GAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTAT
    GGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCTCCCTATCAGTGATAGAGATCTCCCTATCAGTGAT
    AGAGATCGTCGACGAGCTCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCC
    TCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTAAAACAGGTAAGTCCGGCCTCCGCGCCGGGTTTTGGCGC
    CTCCCGCGGGCGCCCCCCTCCTCACGGCGAGCGCTGCCACGTCAGACGAAGGGCGCAGCGAGCGTCCTGATCCTTCC
    GCCCGGACGCTCAGGACAGCGGCCCGCTGCTCATAAGACTCGGCCTTAGAACCCCAGTATCAGCAGAAGGACATTTT
    AGGACGGGACTTGGGTGACTCTAGGGCACTGGTTTTCTTTCCAGAGAGCGGAACAGGCGAGGAAAAGTAGTCCCTTC
    TCGGCGATTCTGCGGAGGGATCTCCGTGGGGCGGTGAACGCCGATGATGCCTCTACTAACCATGTTCATGTTTTCTT
    TTTTTTTCTACAGGTCCTGGGTGACGAACAG
    Polynucleotide sequence encodinq the enhanced hCMV promoter
    SEQ ID NO: 3
    CCATTGCATACGTTGTATCCATATCATAATATGTACATTTATATTGGCTCATGTCCAACATTACCGCCATGTTGACA
    TTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTA
    CATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTT
    CCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGT
    ACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCC
    AGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGG
    TTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAA
    TGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGG
    GCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGGCGAAGCGCTCCCTATCAGTGATAGAGATCTCCCTATCAGTGA
    TAGAGATCGTCGACGAGCTCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGC
    CTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTAAAACAGGTAAGTCCGGCCTCCGCGCCGGGTTTTGGCG
    CCTCCCGCGGGCGCCCCCCTCCTCACGGCGAGCGCTGCCACGTCAGACGAAGGGCGCAGCGAGCGTCCTGATCCTTC
    CGCCCGGACGCTCAGGACAGCGGCCCGCTGCTCATAAGACTCGGCCTTAGAACCCCAGTATCAGCAGAAGGACATTT
    TAGGACGGGACTTGGGTGACTCTAGGGCACTGGTTTTCTTTCCAGAGAGCGGAACAGGCGAGGAAAAGTAGTCCCTT
    CTCGGCGATTCTGCGGAGGGATCTCCGTGGGGCGGTGAACGCCGATGATGCCTCTACTAACCATGTTCATGTTTTCT
    TTTTTTTTCTACAGGTCCTGGGTGACGAAGAG
    Polynucleotide sequence encoding the hCMV NM2 bghpolyA cassette
    SEQ ID NO: 4
    CCATTGCATACGTTGTATCCATATCATAATATGTACATTTATATTGGCTCATGTCCAACATTACCGCCATGTTGACA
    TTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTA
    CATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTT
    CCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGT
    ACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCC
    AGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGG
    TTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAA
    TGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGG
    GCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCCCTATCAGTGATAGAGATCTCCCTATCAGTGAT
    AGAGATCGTCGACGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAG
    AAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGAGA
    TCTTCCGTTTATCTAGGTACCAGATATCGCCACCATGGCCCTGAGCAAAGTGAAACTGAACGATACACTGAACAAGG
    ACCAGCTGCTGTCCAGCAGCAAGTACACCATCCAGCGGAGCACCGGCGACAGCATCGATACCCCCAACTACGACGTG
    CAGAAGCACATCAACAAGCTGTGCGGCATGCTGCTGATCACAGAGGACGCCAACCACAAGTTCACCGGCCTGATCGG
    CATGCTGTACGCCATGAGCCGGCTGGGCCGGGAGGACACCATCAAGATCCTGCGGGACGCCGGCTACCACGTGAAGG
    CCAATGGCGTGGACGTGACCACACACCGGCAGGACATCAACGGCAAAGAAATGAAGTTCGAGGTGCTGACCCTGGCC
    AGCCTGACCACCGAGATCCAGATCAATATCGAGATCGAGAGCCGGAAGTCCTACAAGAAAATGCTGAAAGAAATGGG
    CGAGGTGGCCCCCGAGTACAGACACGACAGCCCCGACTGCGGCATGATCATCCTGTGTATCGCCGCCCTGGTGATCA
    CAAAGCTGGCCGCTGGCGACAGATCTGGCCTGACAGCCGTGATCAGACGGGCCAACAATGTGCTGAAGAACGAGATG
    AAGCGGTACAAGGGCCTGCTGCCCAAGGACATTGCCAACAGCTTCTACGAGGTGTTCGAGAAGTACCCCCACTTCAT
    CGACGTGTTCGTGCACTTCGGCATTGCCCAGAGCAGCACCAGAGGCGGCTCCAGAGTGGAGGGCATCTTCGCCGGCC
    TGTTCATGAACGCCTACGGCGCTGGCCAGGTGATGCTGAGATGGGGCGTGCTGGCCAAGAGCGTGAAGAACATCATG
    CTGGGCCACGCCAGCGTGCAGGCCGAGATGGAACAGGTGGTGGAGGTGTACGAGTACGCCCAGAAGCTGGGCGGAGA
    GGCCGGCTTCTACCACATCCTGAACAACCCTAAGGCCTCCCTGCTGTCCCTGACCCAGTTCCCCCACTTCTCCAGCG
    TGGTGCTGGGAAATGCCGCCGGACTGGGCATCATGGGCGAGTACCGGGGCACCCCCAGAAACCAGGACCTGTACGAC
    GCCGCCAAGGCCTACGCCGAGCAGCTGAAAGAAAACGGCGTGATCAACTACAGCGTGCTGGACCTGACCGCTGAGGA
    ACTGGAAGCCATCAAGCACCAGCTGAACCCCAAGGACAACGACGTGGAGCTGGGAGGCGGAGGATCTGGCGGCGGAG
    GCATGAGCAGACGGAACCCCTGCAAGTTCGAGATCCGGGGCCACTGCCTGAACGGCAAGCGGTGCCACTTCAGCCAC
    AACTACTTCGAGTGGCCCCCTCATGCTCTGCTGGTGCGGCAGAACTTCATGCTGAACCGGATCCTGAAGTCCATGGA
    CAAGAGCATCGACACCCTGAGCGAGATCAGCGGAGCCGCCGAGCTGGACAGAACCGAGGAATATGCCCTGGGCGTGG
    TGGGAGTGCTGGAAAGCTACATCGGCTCCATCAACAACATCACAAAGCAGAGCGCCTGCGTGGCCATGAGCAAGCTG
    CTGACAGAGCTGAACAGCGACGACATCAAGAAGCTGAGGGACAACGAGGAACTGAACAGCCCCAAGATCCGGGTGTA
    CAACACCGTGATCAGCTACATTGAGAGCAACCGCAAGAACAACAAGCAGACCATCCATCTGCTGAAGCGGCTGCCCG
    CCGACGTGCTGAAAAAGACCATCAAGAACACCCTGGACATCCACAAGTCCATCACCATCAACAATCCCAAAGAAAGC
    ACCGTGTCTGACACCAACGATCACGCCAAGAACAACGACACCACCTGATGAGCGGCCGCGATCTGCTGTGCCTTCTA
    GTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCC
    TAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAG
    CAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG
    CMV Promoter sequence: bold
    Transgene sequence NM2: Italic
    bahpo/yA Poly A signal: italic + underline
    NM2 protein sequence
    SEQ ID NO: 5
    MALSKVKLNDTLNKDQLLSSSKYTIQRSTGDSIDTPNYDVQKHINKLCGMLLITEDANHKFTGLIGMLYAMSRLGRE
    DTIKILRDAGYHVKANGVDVTTHRQDINGKEMKFEVLTLASLTTEIQINIEIESRKSYKKMLKEMGEVAPEYRHDSP
    DCGMIILCIAALVITKLAAGDRSGLTAVIRRANNVLKNEMKRYKGLLPKDIANSFYEVFEKYPHFIDVFVHFGIAQS
    STRGGSRVEGIFAGLFMNAYGAGQVMLRWGVLAKSVKNIMLGHASVQAEMEQVVEVYEYAQKLGGEAGFYHILNNPK
    ASLLSLTQFPHFSSVVLGNAAGLGIMGEYRGTPRNQDLYDAAKAYAEQLKENGVINYSVLDLTAEELEAIKHQLNPK
    DNDVELGGGGSGGGGMSRRNPCKFEIRGHCLNGKRCHFSHNYFEWPPHALLVRQNFMLNRILKSMDKSIDTLSEISG
    AAELDRTEEYALGVVGVLESYIGSINNITKQSACVAMSKLLTELNSDDIKKLRDNEELNSPKIRVYNTVISYIESNR
    KNNKQTIHLLKRLPADVLKKTIKNTLDIHKSITINNPKESTVSDTNDHAKNNDTT
    Polynucleotide sequence encodinq the hCMV F0 WPRE bghpolyA cassette
    SEQ ID NO: 6
    CCATTGCATACGTTGTATCCATATCATAATATGTACATTTATATTGGCTCATGTCCAACATTACCGCCATGTTGACA
    TTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTA
    CATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTT
    CCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGT
    ACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCC
    AGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGG
    TTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAA
    TGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGG
    GCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGGCGAAGCGCTCCCTATCAGTGATAGAGATCTCCCTATCAGTGA
    TAGAGATCGTCGACGAGCTCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGC
    CTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTAAAACAGGTAAGTCCGGCCTCCGCGCCGGGTTTTGGCG
    CCTCCCGCGGGCGCCCCCCTCCTCACGGCGAGCGCTGCCACGTCAGACGAAGGGCGCAGCGAGCGTCCTGATCCTTC
    CGCCCGGACGCTCAGGACAGCGGCCCGCTGCTCATAAGACTCGGCCTTAGAACCCCAGTATCAGCAGAAGGACATTT
    TAGGACGGGACTTGGGTGACTCTAGGGCACTGGTTTTCTTTCCAGAGAGCGGAACAGGCGAGGAAAAGTAGTCCCTT
    CTCGGCGATTCTGCGGAGGGATCTCCGTGGGGCGGTGAACGCCGATGATGCCTCTACTAACCATGTTCATGTTTTCT
    TTTTTTTTCTACAGGTCCTGGGTGACGAACAGGATATCGCCACCATGGAACTGCTGATCCTGAAGGCCAACGCCATC
    ACCACCATCCTGACCGCCGTGACCTTCTGCTTCGCCAGCGGCCAGAACATCACCGAGGAATTCTACCAGAGCACCTG
    TAGCGCCGTGAGCAAGGGCTACCTGAGCGCCCTGAGAACCGGCTGGTACACCAGCGTGATCACCATCGAGCTGAGCA
    ACATCAAAGAAAACAAGTGCAACGGCACCGACGCCAAAGTGAAGCTGATCAAGCAGGAACTGGACAAGTACAAGAAC
    GCCGTGACCGAGCTGCAGCTGCTGATGCAGAGCACCCCCGCCACCAACAACCGGGCCAGACGGGAGCTGCCCCGGTT
    CATGAACTACACCCTGAACAACGCCAAAAAGACCAACGTGACCCTGAGCAAGAAGCGGAAGCGGCGGTTCCTGGGCT
    TTCTGCTGGGCGTGGGCAGCGCCATTGCCAGCGGCGTGGCCGTGTCTAAGGTGCTGCACCTGGAAGGCGAAGTGAAC
    AAGATCAAGAGCGCCCTGCTGAGCACCAACAAGGCCGTGGTGTCCCTGAGCAACGGCGTGAGCGTGCTGACCAGCAA
    GGTGCTGGATCTGAAGAACTACATCGACAAGCAGCTGCTGCCCATCGTGAACAAGCAGAGCTGCAGCATCAGCAACA
    TCGAGACAGTGATCGAGTTCCAGCAGAAGAACAACCGGCTGCTGGAAATCACCCGGGAGTTCAGCGTGAACGCCGGC
    GTGACCACCCCTGTGTCCACCTACATGCTGACCAACAGCGAGCTGCTGAGCCTGATCAACGACATGCCCATCACCAA
    CGACCAGAAAAAGCTGATGAGCAACAACGTGCAGATCGTGCGGCAGCAGAGCTACTCCATCATGTCCATCATCAAAG
    AAGAGGTGCTGGCCTACGTGGTGCAGCTGCCCCTGTACGGCGTGATCGACACCCCCTGCTGGAAGCTGCACACCAGC
    CCCCTGTGCACCACCAACACCAAAGAGGGCAGCAACATCTGCCTGACCCGGACCGACAGAGGCTGGTACTGCGACAA
    CGCCGGCAGCGTGTCATTCTTTCCACAGGCCGAGACATGCAAGGTGCAGAGCAACCGGGTGTTCTGCGACACCATGA
    ACAGCCTGACCCTGCCCTCCGAAGTGAACCTGTGCAACGTGGACATCTTCAACCCCAAGTACGACTGCAAGATCATG
    ACCTCCAAGACCGACGTGTCCAGCTCCGTGATCACCTCCCTGGGCGCCATCGTGTCCTGCTACGGCAAGACCAAGTG
    CACCGCCAGCAACAAGAACCGGGGCATCATCAAGACCTTCAGCAACGGCTGCGACTACGTGTCCAACAAGGGGGTGG
    ACACCGTGTCCGTGGGCAACACCCTGTACTACGTGAACAAACAGGAAGGCAAGAGCCTGTACGTGAAGGGCGAGCCC
    ATCATCAACTTCTACGACCCCCTGGTGTTCCCCAGCGACGAGTTCGACGCCAGCATCAGCCAGGTGAACGAGAAGAT
    CAACCAGAGCCTGGCCTTCATCCGGAAGTCCGACGAGCTGCTGCACAATGTGAATGCCGGCAAGTCCACCACCAACT
    GATGAGCGGCCATCTAA TCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCC
    TTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCT
    CCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACT
    GTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCC
    CCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTG
    ACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGC
    GGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCG
    GCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCT GCGGCCGCGA
    TCTG CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCAC
    TCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTG
    GGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG
    Enhanced CMV Promoter sequence: bold
    Transgene sequence FO: Italic
    WPRE sequence: underlined bold
    bahpo/yA Poly A signal: italic + underline
    F0 protein sequence
    SEQ ID NO: 7
    MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVK
    LIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAV
    SKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLL
    EITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGV
    IDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVD
    IFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQ
    EGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTN
    Polynucleotide sequence of the hCMV promoter and enhancer sequence
    SEQ ID NO: 8
    CCATTGCATACGTTGTATCCATATCATAATATGTACATTTATATTGGCTCATGTCCAACATTACCGCCATGTTGACA
    TTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTA
    CATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTT
    CCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGT
    ACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCC
    AGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGG
    TTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAA
    TGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGG
    GCGGTAGGCGTGTACGGTGGGAGGTCTATATAAG
    Polynucleotide sequence of the Chicken Beta-Actin Fraqment
    SEQ ID NO: 9
    GCGAAGCGCTCCCTATCAGTGATAGAGATCTCCCTATCAGTGATAGAGATCGTCGACGAGCTCGCGGCGGGCGGGAG
    TCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCG
    TTACT
    Polynucleotide sequence of the the Splice Donor Reqion
    SEQ ID NO: 10
    AAAACAGGTAAGTCC
    Polynucleotide sequence of the the ubiquitin (UBC) enhancer
    SEQ ID NO: 11
    GGCCTCCGCGCCGGGTTTTGGCGCCTCCCGCGGGCGCCCCCCTCCTCACGGCGAGCGCTGCCACGTCAGACGAAGGG
    CGCAGCGAGCGTCCTGATCCTTCCGCCCGGACGCTCAGGACAGCGGCCCGCTGCTCATAAGACTCGGCCTTAGAACC
    CCAGTATCAGCAGAAGGACATTTTAGGACGGGACTTGGGTGACTCTAGGGCACTGGTTTTCTTCCAGAGAGCGGAAC
    AGGCGAGGAAAAGTAGTCCCTTCTCGGCGATTCTGCGGAGGGATCTCCGTGGGGCGGTGAACGCCGATGAT
    Polynucleotide sequence of the Splice Acceptor Reqion
    SEQ ID NO: 12
    GCCTCTACTAACCATGTTCATGTTTTCTTTTTTTTTCTACAGGTCCTGGGTGACGAACAG

Claims (16)

1-18. (canceled)
19. An adenoviral vector comprising an expression cassette, wherein the expression cassette comprises a transgene and a promoter, wherein the promoter comprises:
(i) an hCMV enhancer sequence;
(ii) an hCMV promoter sequence;
(iii) a splice donor region;
(iv) a cell-derived enhancer sequence; and
(v) a splice acceptor region,
wherein the promotor comprises a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 3.
20. The adenoviral vector of claim 19, wherein the adenoviral vector comprises a first expression cassette and a second expression cassette, wherein both the first expression cassette and the second expression cassette comprise a transgene and a promoter, wherein the promoter comprises:
(i) an hCMV enhancer sequence;
(ii) an hCMV promoter sequence;
(iii) a splice donor region;
(iv) a cell-derived enhancer sequence; and
(v) a splice acceptor region,
wherein the promotor comprises a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 3.
21. The adenoviral vector of claim 19, wherein the components (i) to (v) of the promoter are provided in the order listed.
22. The adenoviral vector of claim 19, wherein the cell-derived enhancer sequence is an ubitquitin (UBC) enhancer sequence, and wherein the UBC enhancer sequence comprises the sequence of SEQ ID NO: 11.
23. The adenoviral vector of claim 19, wherein the promotor comprises a sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to one or more of SEQ ID NO: 8, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12.
24. The adenoviral vector of claim 19, wherein the promotor comprises:
(i) the hCMV enhancer sequence;
(ii) the hCMV promoter sequence of SEQ ID NO: 8;
(iii) the splice donor region of SEQ ID NO:10;
(iv) the UBC enhancer sequence of SEQ ID NO:11; and
(v) the splice acceptor region of SEQ ID NO: 12.
25. The adenoviral vector of claim 19, wherein the promotor further comprises a fragment of the beta-actin sequence, wherein the fragment of the beta-actin sequence comprises a 5′ untranslated region of the beta actin sequence and does not contain the promoter sequence.
26. The adenoviral vector of claim 25, wherein the beta-actin sequence comprises a sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 9.
27. The adenoviral vector of claim 25, wherein the fragment of the beta-actin sequence is located between the hCMV promoter region and the splice donor region.
28. The adenoviral vector of claim 19, wherein the adenoviral vector is derived from a simian adenovirus.
29. A composition comprising the adenoviral vector of claim 19 and a pharmaceutically acceptable excipient.
30. A method of inducing an immune response against a disease caused by a pathogen in a subject in need thereof comprising administering an immunologically effective amount of the composition of claim 29 to the subject.
31. A method of inducing an immune response against a disease caused by a pathogen in a subject in need thereof comprising administering an immunologically effective amount of the adenoviral vector of claim 19 to the subject.
32. A method of treating a subject infected with a disease caused by a pathogen comprising administering an effective amount of the adenoviral vector of claim 19 to the subject.
33. A method of treating a subject infected with a disease caused by a pathogen comprising administering an effective amount of the composition of claim 29 to the subject.
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