Abstract:
This invention relates to compositions and methods of delivering therapeutic agents to bone. More specifically, the invention relates to endowing a large molecule vectors i.e., adeno virus, retrovirus, liposomes, micelles, natural and synthetic polymers, or combinations thereof, with the ability to target bone tissue in vivo and with improved stability in the blood, by attaching multiple copies of acid amino acid peptides. One preferred embodiment of the invention relates to endowing an adeno-associated virus (AAV) vector with the ability to target bone-tissue in vivo and improve its stability, by the addition of multiple acidic amino acid peptides attached to the capsid of the viral vector.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims benefit of priority to U.S. Provisional Patent Application No. 61/081,711, filed Jul. 17, 2008, and is also a continuation in part of U.S. patent application Ser. No., 11/614,970, filed on Dec. 21, 2006, which claims benefit of priority to U.S. patent application Ser. No. 11/245,424, filed Oct. 7, 2005, now abandoned. This application is also a continuation in part of U.S. patent application Ser. No., 11/484,870, filed Jul. 11, 2006, which claims benefit of priority to U.S. Provisional Patent Application No. 60/725,563, filed Oct. 11, 2005. This application is also a continuation in part of U.S. patent application Ser. No., 10/864,758 filed Jun. 10, 2004. All documents above are incorporated herein in their entirety by reference. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The invention relates to compositions and methods for targeting vectors to bone tissue for the delivery of therapeutic agents, including but not limited to viral vectors, liposomes, and large synthetic and natural polymers, for the delivery of polypeptides, polynucleic acids, and other therapeutic agents. 
       BACKGROUND OF THE INVENTION 
       [0003]    Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disorder caused by deficiency of N-acetylgalatosamine-6-sulfate-sulfatase (GALNS, EC 3.1.6.4), leading to accumulation of glycosaminoglycans (GAGs), keratan sulfate (KS) and chondroitin-6-sulfate (C6S) (For review see; Neufeld et al. (2001) McGraw-Hill: New York. vol III, pp 3421-3452). Clinical manifestations vary from severe to an attenuated form characterized by systemic skeletal dysplasia, laxity of joints, hearing loss, corneal clouding, and heart valvular disease, with normal intelligence. Generally MPS IVA patients do not survive beyond second or third decade of life, although patients with an attenuated form can survive into the seventh decade of life (Montaño et al. (2007)  J Inherit Metab Dis.,  30: 165-174). Currently, no effective therapies exist for MPS IVA. Surgical interventions are used to treat some manifestations of the disease Id. Although other tissues are affected in MPS IVA patients, an ideal therapeutic agent would be efficiently distributed to bone and bone marrow. Other diseases also exist for which delivery of therapeutic agents to bone would be beneficial. One example is hypophosphatasia, for which the targeted delivery of tissue non-specific alkaline phosphatase (TNSALP) would be highly beneficial. Another example is type VII mucopolysaccharidosis, which would benefit greatly from the targeted delivery of β-glucuronidase (GUS). Gene and enzyme replacement therapy are promising treatments for bone related diseases. However, there exists a need to facilitate the delivery of therapeutic agents including polynucleotides and polypeptides to bone. The inventors provide compositions and methods to promote effective delivery of therapeutic agents to bone using large molecule vectors. 
       SUMMARY 
       [0004]    The present invention relates to methods and compositions for delivering therapeutic agents to bone. More specifically the present invention is directed to endowing large molecule vectors with capable of targeting bone by attaching acid amino acid peptides to these vectors externally. 
         [0005]    In the one embodiment, the vector is a viral vector, a liposome, a large synthetic polymer, a large natural polymer, or a polymer comprised of natural and synthetic components, with acid amino acid peptides attached externally. The vector incorporates a therapeutic agent. The therapeutic agent is a pharmaceutical, a nucleotide, or a polypeptide therapeutic agent. 
         [0006]    In a preferred embodiment, the vector is adeno-associated virus, with acid amino acid peptides attached externally, and the therapeutic polypeptide is either N-acetylgalatosamine-6-sulfate-sulfatase, tissue non-specific alkaline phosphatase, or β-glucuronidase. 
         [0007]    In a most preferred embodiment, the vector is adeno-associated virus, with acid amino acid peptides attached externally, and the therapeutic polypeptide is N-acetylgalatosamine-6-sulfate-sulfatase. 
         [0008]    In yet another embodiment, is a method of making an adeno-associated viral vector, targeted to bone, with acid amino acid peptides attached externally, and incorporating a polypeptide therapeutic agent. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0009]      FIG. 1 . Map of plasmid pAAV-CBA-GALNS. ITR: inverted terminal repeat, CBA promoter: cytomegalovirus enhancer and β-actin promoter, b-globin: rabbit β-globin polyA, polyA: Fragment containing the bovine growth hormone poly-A signal, Amp: β-lactamase gene. 
           [0010]      FIG. 2 . Map of plasmid pAAV-CMV-GALNS. ITR: inverted terminal repeat, CMV/IE: cytomegalovirus immediate early enhancer/promoter, IVS: Synthetic intron, IRES: Attenuated internal ribosome entry site (IRES) from encephalomyocarditis virus, Neo: Neomycin phosphotransferase coding sequence, polyA: fragment containing the bovine growth hormone poly-A signal, Amp: β-lactamase gene. 
           [0011]      FIG. 3 . Map of plasmid pCXN. CMV-IE: cytomegalovirus immediate early enhancer, Amp: β-lactamase, Neo: Neomycin phosphotransferase coding sequence. 
           [0012]      FIG. 4 . Scheme of the construction of the plasmid pAAV-CBA-GALNS. 
           [0013]      FIG. 5 . Insertion of sequence encoding the octapeptide of aspartic amino acids in the pXX2 plasmid. Arrows show the site for the initial codon of VP1, VP2 and VP3. 
           [0014]      FIG. 6 . Construction of pXX2-ND8 plasmid. (a) Positive clone after site-directed mutagenesis was screened by PCR using primers flanking the insertion site. The 715-bp fragment was produced for the targeted clone compared to the 691-bp fragment from pXX2 plasmid. Marker: 100 bp ladder. (b) Alignment of sequencing result of pXX2 and clone 19. A box (single strand) designates the insertion site and the nucleotide sequence encoding eight aspartic amino acids in clone 19. 
           [0015]      FIG. 7 . Transfection of HEK293 cells. HEK 193 cells were transfected with 1×10 11  vg of the unmodified native AAV capsid or the modified AAV-AAA-capsid. GALNS activity in the cell lysate was assayed after 4 days of post-transfection. 
           [0016]      FIG. 8 . Hydroxyapatite-binding assay. Hydroxyapatite beads were incubated with 5×10 11  vg (blue, n=3) or 1×10 12  vg (red, n=3) of each virus for 1 h at 37° C. After centrifugation virus titers were quantified in the supernatant by spectrophotometric method, and compared with the initial amount of virus mixed. 
           [0017]      FIG. 9 . Biodistribution experiment. Mice were sacrificed 48 h after a vein tail infusion of 1,5×10 11  vg. 1 μg of DNA samples from bone (1), liver (2), brain (3) and bone marrow (4) were subjected to PCR using specific primers for GALNS cDNA. Primers for mouse β-glucuronidase (GUS) were used as internal control to check DNA quality and absence of PCR-inhibitors 
       
    
    
     DETAILED DESCRIPTION 
       [0018]    The inventors have made the surprising discovered that 4-15 acidic amino acid polypeptides, inserted into a large molecule or vector such as adeno-associated virus (AAV)(approximately 5000 KDa), by incorporating the acidic amino acid polypeptides into the AAV capsid, will increase the affinity of this viral vector for bone. Most therapeutic agents intended for bone diseases including AAV, do not have a particular affinity to Bone (Gittensa et al. (2005)  Adv Drug Deliv Rev.  57: 1011-1036). Bone is distinguished from other tissues by the presence of hydroxyapatite (HA), which is positively charged. The inventors have utilized a peptides of 4-15 acidic amino acid residues (AAA), inserted into a virus capsid to increase the affinity for HA and enhance delivery of the vector nucleotides to bone. As disclosed below, AAA tagged AAV (AAA-AAV), showed 100% binding to HA while the untagged vector showed no binding with HA. In addition, the level of viral gene production after transduction of virus into the cells was not affected by the addition of the AAA peptide. Experiments in mice showed that 48 hours after intravenous infusion of the AAA tagged vector, the virus genome was increased between 16 and 291 fold in bone compared to mice infused with untagged vector. 
       Adeno-Associated Virus (AAV). 
       [0019]    Adeno-associated virus (AAV) are non-enveloped virus with a linear single-stranded DNA of 4.7 kb genome. AAV typically require a helper virus, usually adenovirus or herpesvirus, for replication (Flotte (2004)  Gene Ther  11: 805-810). The viral capsid protein is the first element that a cellular receptor encounters during a viral infection. Capsid structure for the serotypes AAV2, AAV4, AAV5, and AAV8 has been determined and the regions involved in host receptor interactions have been identified (see Xie et al. (2002)  Proc Natl Acad Sci USA  99: 10405-10410; Nam et al. (2007) J  Virol  81: 12260-12271; Choi et al. (2005)  Curr Gen Ther  5: 299-310). The AAV capsid is formed by 60 proteins consisting of VP1, VP2 and VP3 in a 1:1:20 ratio, respectively, which differ in their N-terminus (Flotte (2004)  Gene Ther  11: 805-810). Mutagenesis analysis has identified capsid positions which allow the insertion of peptide sequences with little effect on the DNA packaging and virus trafficking. These positions are exposed on the capsid surface (Büning, et al. (2003)  Gene Ther  10: 1142-1151). For example, in AAV2, the most studied serotype, peptides inserted after amino acid positions 138, 161, 459, 584, 587 and 588, relative to VP1 sequence, are exposed on the viral vector surface. It was seen that modified AAV2 produced viral titers similar to wild-type AAV2 (Büning, et al. (2003)  Gene Ther  10: 1142-1151)-12; Wu et al. (2000) J  Virol  74: 8635-8647; Shi et al. (2001)  Hum Gene Ther  12: 1697-1711). It was reasoned that the attachment of ligands with an affinity for a component of bone such as hydroxyapatite may endow AAV with the ability to target bone and, if attached externally, would not affect the functionality of the virus. 
       Method of Making Acid Amino Acid-Adeno-Associated Virus (AAA-AAV) 
       [0020]    Producing AAA-AAV, involves methodology that is generally known by the skilled artisan and described in detail in numerous laboratory protocols, one of which is Molecular Cloning 3rd edition, (2001) J. F. Sambrook and D. W. Russell, ed., Cold Spring Harbor University Press, incorporated by reference herein in it entirety. Many modifications and variations of the present illustrative DNA sequences and nucleotide vectors are possible. For example, the degeneracy of the genetic code allows for the substitution of nucleotides throughout polypeptide coding regions, as well as in the translational stop signal, without alteration of the encoded polypeptide coding sequence. Such substitutable sequences can be deduced from the known amino acid or DNA sequence. AAA-AAV can be constructed by following conventional synthetic or site-directed mutagenesis procedures. Synthetic methods can be carried out in substantial accordance with the procedures of Itakura et. al., (1977)  Science  198:1056; and Crea et. al. (1978)  Proc. Nati. Acad. Sci, USA  75:5765, incorporated by reference herein in their entirety. The present invention is in no way limited to the DNA sequences and plasmids specifically exemplified. 
       Plasmid Construction. 
       [0021]    The pAAV-CBA-GALNS plasmid, as illustrated in FIG.  1 ., incorporates the cytomegalovirus enhancer and β-actin promoter (CBA) to drive expression of the human N-acetylgalactosamine-6-sulphate sulphatase (GALNS). It is flanked by AAV2 ITRs. The plasmid was constructed by replacing the cytomegalovirus immediate early enhancer/promoter (CMV) in pAAV-CMV-GALNS ( FIG. 2 ) as previously constructed with a 1.8-kb fragment from pCXN ( FIG. 3 ) containing the CBA promoter. The CMV immediate early enhancer/promoter in pAAV-CMV-GALNS has been previously described (Niwa et al. (1991) Dec 15; 108(2):193-9) and is herein incorporated by reference in its entirety. The 1.8-kb fragment was ligated into the plasmid and the correct orientation of the insert was confirmed by restriction enzyme analysis ( FIG. 4 ). 
         [0022]    To produce the AAA-AAV vector which incorporates the octapeptide of aspartic acid in to the capsid protein, the pXX2 plasmid (SEQ ID NO:1) which encodes for the Rep and Cap AAV2 proteins (Xiao et al. (1998) J Virol 72: 2224-2232), was modified to produce (pXX2-ND8) (SEQ ID NO:2). This was done by inserting a sequence encoding eight aspartic amino acids (ND8) (5′-GATGATGATGATGATGATGACGAC-3′) (SEQ ID NO:3), immediately after the initial codon of the VP2 protein in the packing plasmid pXX ( FIG. 5 ). Insertion was carried out using a commercial site-directed mutagenesis kit (QuikChangee® Site-Directed Mutagenesis Kit, Stratagene, La Jolla, Calif.) according to manufacturer&#39;s instructions, by using the primers: 5-gaggaacctgttaagacgGATGATGATGATGATGATGACGACgctccgggaaaaaagagg-3 (SEQ ID NO:4) (XX2-ND8 sense) and its complement (XX2-ND8 antisense). Insertion of the sequence encoding the octapeptide sequence was first confirmed by PCR with primers XX2-ND8-4F 5′-ATCTCAACCCGTTTCTGTCG-3′ (SEQ ID NO:5) and XX2-ND8-4R 5′-GCGTCTCCAGTCTGACCAA-3′(SEQ ID NO:6), flanking the insertion site, which produced a PCR product of 691 bp with the original pXX2 plasmid and 715 bp after the insertion of sequence. The resulting plasmid (pXX2-ND8) (SEQ ID NO:2) was sequenced to ensure the presence of the eight aspartic amino acids without introduction of fortuitous mutations. 
       Production of Recombinant AAV-AAV Vectors 
       [0023]    CBA-GALNS (native capsid) or ND8/CBA-GALNS (AAA tagged capsid) were produced by calcium phosphate-mediated co-transfection of pAAV-CBA-GALNS, pXX6-80 helper plasmid (Xiao et al. (1998) J Virol 72: 2224-2232), and pXX2 or pXX2-ND8 plasmids (Zolotukhin et al. (1999).  Gene  Ther 6: 973-985). HEK 293 cells were seeded to 80-90% confluence on 15-cm culture plates and media was removed immediately before starting the transfection. The three plasmids were mixed in 18:18:54 μg ratio (1:1:1 molar ratio) with 1.25 mL of 0.25 M CaCl 2 . Then, 1.25 mL of 2×HeBS buffer (280 mM NaCl, 1.5 mM Na 2 HPO 4 , 50 mM HEPES, pH 7.1) was added and the mixture was incubated for 1 minute at room temperature. The mixture was added to 20 mL of culture media (DMEM with FBS and antibiotics) and immediately dispensed into the culture plate. Forty-eight hours after transfection, the cells were harvested, resuspended in 15 mL of AAV lysis buffer (0.15 M NaCl, 50 mM Tris-HCl pH 8.5), and lysated by three freeze/thaw cycles. The solution was clarified by centrifugation at 3,700 g at 40 for 20 minutes. The supernatant was designated the primary viral solution and stored at −80° C. for further analysis. 
         [0024]    AAV vectors were purified by iodixanol gradient (Zolotukhin et al. (1999)  Gene  Ther 6: 973-985). The gradient was prepared by combining 9 mL of 15% iodixanol (Optiprep®, Sigma-Aldrich, Saint Louis, Mo.), 1 M NaCl in PBS-MK buffer (1×PBS, 1 mM MgCl 2  and 2.5 mM KCl), 6 mL of 25% iodixanol in PBS-MK buffer with Phenol red (2.5 μL of stock solution per mL of iodixanol solution), 5 mL of 40% iodixanol in PBS-MK buffer, and 5 mL of 60% iodixanol in PBS-MK. Primary viral solution (aprox. 15 mL) was added and gradient was centrifuged at 25,000 RPM for 3 h at 18° C. Using a syringe with a 18-gauge needle, 2.5 mL were aspirated of each of the 60% and 40% phases. The virus solution was concentrated with Centricon 100 K (Millipore), desalted with 2 mL of 0.9% NaCl, and stored to −80° C. Quantification was be carried out by a spectrophotometric method, based on the extinction coefficient of the AAV2 capsid proteins and genome (Sommer et al. (2003).  Mol Ther  7:122-128). For quantification 100 μL of viral solution was incubated with 0.5 μL of 20% SDS at 75° C. for 10 minutes, and absorbance was measured at 260 and 280 nm. A solution of 0.9% NaCl with 0.5 μL of 20% SDS was used as blank. Virus genomes per mL (vg/mL) were calculated according to the equation: 
         [0000]    
       
         
           
             
               
                 
                   
                     vg 
                      
                     
                       / 
                     
                      
                     mL 
                   
                   = 
                   
                     
                       4 
                       , 
                       47 
                       × 
                       
                         10 
                         19 
                       
                        
                       
                         ( 
                         
                           
                             A 
                             260 
                           
                           - 
                           
                             0 
                             , 
                             59 
                              
                             
                                 
                             
                              
                             
                               A 
                               280 
                             
                           
                         
                         ) 
                       
                     
                     
                       MW 
                       DNA 
                     
                   
                 
               
               
                 
                   ( 
                   1 
                   ) 
                 
               
             
           
         
       
     
         [0000]    where MWDNA is the molecular weight of each viral genome based on its sequence and using the molecular weight of each nucleotide (A=312.2 Da, C=288.2 Da, G=328.2 Da y T=303.2 Da) (see Sommer et al. (2003).  Mol Ther  7: 122-128). 
       In Vitro Transfection. 
       [0025]    HEK293 cells, 1×10 5  (ATCC CRL-1573) were seeded in 12-well plates and transfected with 1×10 10  vg (1×10 5  vg/cell) of each viral genome. Cells were harvested postransfection, and resuspended in 100 μL of 1% sodium deoxycholate (Sigma-Aldrich, Saint Louis, Mo.). GALNS activity in cell lysate was assayed using the substrate 4-methylumbeliferyl-β-D-galactopyranoside-6-sulphate (Toronto Chemicals Research, North York, On, Canada), as described (van Diggelen et al. (1993)  Clin Chem Acta  187:131-140). One unit is defined as the enzyme catalyzing 1 nmol of substrate per hour. Total protein in cell lysate will be determined by micro-Lowry protein assay. 
       Hydroxyapatite-Binding Assay. 
       [0026]    Assays were carried out essentially as described (Nishioka et al. (2006)  Mol Genet Metab  88: 244-255). Hydroxyapatite beads (Sigma-Aldrich, Saint Louis, Mo.) were suspended in 25 mM Tris-HCl buffered saline, pH 7.4, at a concentration of 100 μg/μL. AAV2 (wild-type virus), CBA-GALNS and ND8/CBA-GALNS plasmids were mixed at a final concentration 5×10 11  and 1×10 12  vg. The mixture was incubated at 37° C. for 1 h, and centrifuged at 14,000 rpm for 10 minutes. The AAV titers were measured in the supernatant, and the bound AAV fraction was determined from the amount of the total and unbound AAV. Quantification of AAV vectors in the supernatant was carried out by the spectrophotometric method described above. Hydroxyapatite-binding assays for each AAV vector was carried out by triplicate. 
       Biodistribution Experiment. 
       [0027]    1.5×10 11  vg of CBA-GALNS or ND8/CBA-GALNS were injected intravenously into 7-8-weeks-old MPS IVA knock-out mice (n=3 for each group) according to Tomatsu et al. (2003)  Hum Mol Genet  12: 3349-3358, incorporated by reference herein. Control animals were injected with PBS. Mice were sacrificed 48 hours after the injection, and liver, brain, and bone (leg) were dissected and immediately frozen in dry-ice. Bone marrow was obtained by flushing the femurs with PBS. Genomic DNA was extracted by tissue homogenization in 1 ml of DNAzol (GIBCO, Grand Island, N.Y.) according to manufacturer&#39;s instructions. DNA samples from liver, brain, bone and bone marrow were analyzed for the presence of viral DNA by PCR using the primers TOMF23 5′-ACAGGGCCATTGATGGCCTCAACCTCCT-3′ (SEQ ID NO:7) and TOMF34R 5′-GCTTCGTGTGGTCTTCCAGATT GTGAGTTG-3′(SEQ ID NO:8), which were specific for human GALNS cDNA, and produced a 235 bp PCR-fragment. This pair-primers specific for human GALNS cDNA, did not amplify the genomic GALNS sequence under these conditions, because the primers annealed in exons 10 and 12, producing a 4.1 kb PCR product. Primers of mouse β-glucuronidase gene were used as an internal control to check DNA quality and absence of PCR-inhibitors. Quantification of the viral genome in bone samples was done by real-time PCR (Tomatsu, et al. (2003)  Hum Mol Genet  12: 3349-3358), with a commercial kit, the Fast SYBR® Green Master Mix (Applied Biosystems, Foster City, Calif.), according to manufacturer&#39;s instructions, using 1 μg of total DNA and the primers TOMF23 and TOMF34R. The pAAV-CBA-GALNS plasmid was used as standard. 
       DEFINITIONS 
       [0028]    The term “vector” as used herein, refers to vectors for the delivery of therapeutic agents. Examples include, but are not limited to, viral vectors, liposomes, large natural polymers, large synthetic polymers, and polymers comprised of both natural and synthetic components. 
         [0029]    The term “therapeutic agent” is intended in its broadest meaning to include not only the polypeptides and polynucleotides of the instance invention but also any agent which conveys an effect beneficial to health including but not limited to any pharmaceutical agent, including cytokines, small molecule drugs, cell-permeable small molecule drugs, hormones, chemotherapy, combinations of interleukins, lectins and other stimulating agents. 
         [0030]    The term “polypeptide therapeutic agent” as used herein, refers to any peptide, polypeptide, or protein, with out limitation with therapeutic benefits. By way of example and not of limitation are enzymes which may be useful in enzyme replacement therapy. Non-limiting examples include N-acetylgalatosamine-6-sulfate-sulfatase (GALNS), also described in U.S. patent application Ser. No. 10/864,758, and tissue non-specific alkaline phosphatase (TNSALP) also described in U.S. patent application Ser. No. 11/484,870, and β-glucuronidase (GUS), also described in 11/614,970. Polypeptide therapeutic agents may include enzymes in their native form, or functional fragments thereof. Polypeptide therapeutic agents may be used alone, or in combination or incorporated into fusion proteins. 
         [0031]    The term “acidic amino acid” or “AAA” as used herein, refers to any repeating amino acid sequence of glutamic acid or aspartic acid. As used herein AAA may comprise multiple copies of acidic amino acid peptides, in any arbitrary combination including repeating glutamic acid or aspartic acid sequences or a combination thereof. The number of acid amino acids in each AAA peptide may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. Preferably 4-15, more preferably 4-8, and most preferably 8 acid amino acids. Multiple copies of a peptide consisting of AAA may be directly attached to a vector (viral and non-viral) via a peptide bond or the like. In the present invention, though there is no specific limitation as to the method for attaching multiple copies of a AAA peptide to a vector, it is advantageous, e.g., to produce and use fusion proteins of comprising the vector and the AAA peptide. 
         [0032]    The term “large polymer” as used herein, refers to any polymer which may be used to deliver a therapeutic agent. Non-limiting examples of polymers and methods of modification may be found in International Patent Applications Nos. WO/2007/012013 and WO/2004/022099 incorporated by reference herein. 
         [0033]    In addition to HEK 293 cells described herein, any number of cell lines are know in the art are capable of expressing the various polynucleotides and phasmids in the invention. To this end, any eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript may be used. Cell culture techniques are also well known in the art. 
       Other Large Molecule Vectors. 
       [0034]    The instant invention is not limited to AAV. The surprising discovery that AAA peptides may endow large molecules with an affinity for hydroxyapatite (HA) may be applied to other virus or large molecule vectors including any virus vector, by way of example but not of limitation, adenoviruses, retro viruses, HCV, HIV, herpesvirus, papovavirus, poxvirus hepadnavirus, adeno-associated virus, parvovirus, vaccinia virus, etc. or related or derived viruses thereof. Mutant herpesviruses can for example be based on HSV1, HSV2, VZV, CMV, EBV, HHV6, HHV7, or on non-human animal herpesviruses such as PRY, IBRV/BHV, MDV, EHV, and others. Vectors may also include Lentiviruses which have been used for delivery of small interfering RNA as described (Li and Rossi (2005)  Methods Enzymol  392, 226), hereby incorporated by reference in its entirety. AAA peptides may be inserted into, capsid or coat proteins of any of the aforementioned viral vectors, as described herein for AAV, whereby the virus vector is endowed with an increased affinity for HA. 
         [0035]    Also included are any and all vectors derived from liposomes, micelles, or large natural or synthetic polymers. Methods of attaching polypeptides to liposomes are know in the art and may be adapted to the AAA peptides of the instant invention. By way of example but not of limitation, AAA peptides may be fused with transmembrane proteins using methods described in U.S. Pat. No. 5,374,548, incorporated herein by reference in its entirety. Other methods include chemical linking AAA to liposomes, using methods described in U.S. Pat. No. 5,401,511, incorporated herein by reference in its entirety. Other gene delivery vectors include liposome-derived systems, artificial viral envelopes, and other systems known in the art (See, e.g., Rossi, J. J. (1995) Br. Med. Bull. 51(1):217-225; Boado, R. J. et al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Morris, M. C. et al. (1997) Nucleic Acids Res. 25(14):2730-2736; El-Aneed, (2004) J Control Release 94, 1-14), all, herein incorporated by reference in its entirety. 
         [0036]    These same chemical linking methods may be applied to large natural and synthetic polymers. By way of example, but not of limitation, natural polymers include polymers derived proteins including collagen and fibrin, or, carbohydrates including hyaluronic acid and sulfated glycosaminoglycans, as well as polymers derived from lipids including liposome or micelles, or polymers derived from polyamino acids including poly-L-arginine, poly-L-lysine and poly-L-ornithine. By way of example but not of limitation, synthetic polymers may include poly(methyl methacrylate) (PMMA), and poly(hydroxyethyl methacrylic) poly(HEMA), or derivatives thereof. By way of example but not of limitation, polymers which are combinations of synthetic and natural polymers include HEMA-PC and pMPC as described in International Patent Application publication WO 2007/100902, and hereby incorporated by reference in its entirety. 
         [0037]    The skilled artisan will recognize that amino acid coupling to proteins or synthetic polymers differ, and conditions will be varied as necessary to promote the formation of the conjugates. Additional guidance maybe obtained from texts such as Wong, 8.S., “Chemistry of Protein Conjugation and Cross-Linking,” (CRC Press 1991), or standard texts in organic chemistry. 
         [0038]    In one embodiment is a vector with 4-15 acid amino acids attached externally, incorporating a therapeutic agent. 
         [0039]    In another embodiment is a viral vector with 4-15 acid amino acids attached externally, incorporating a nucleic acid encoding a polypeptide therapeutic agent. Examples of polypeptide therapeutic agent include, N-acetylgalatosamine-6-sulfate-sulfatase (GALNS), tissue non-specific alkaline phosphatase (TNSALP), and β-glucuronidase (GUS) alone or in combination. 
         [0040]    In one preferred embodiment is an adeno-associated virus with 4-15 acid amino acids attached externally, incorporating a nucleic acid encoding N-acetylgalatosamine-6-sulfate-sulfatase (GALNS). 
         [0041]    In another embodiment is an adeno-associated virus with 4-15 acid amino acids attached externally, incorporating a nucleic acid encoding tissue non-specific alkaline phosphatase (TNSALP). 
         [0042]    In another embodiment is an adeno-associated virus with 4-15 acid amino acids attached externally, incorporating a nucleic acid encoding p-glucuronidase (GUS). 
         [0043]    In one embodiment is a method of making a viral vector which targets bone by incorporating 4-15 acid amino acids into the viral caspid. 
         [0044]    In another embodiment is a method of treating a subject in need by administering a viral vector with 4-15 acid amino acids attached externally and incorporating a therapeutic agent. 
         [0045]    In another embodiment is a liposome with 4-15 acid amino acids attached externally, incorporating a therapeutic agent. 
         [0046]    In another embodiment is a synthetic polymer with 4-15 acid amino acids attached externally, incorporating a therapeutic agent. 
         [0047]    In another embodiment is a natural polymer with 4-15 acid amino acids attached externally, incorporating a therapeutic agent. 
         [0048]    In another embodiment is a polymer with both natural and synthetic components with 4-15 acid amino acids attached externally, incorporating a therapeutic agent. 
       Methods of Practicing the Invention 
     Administration 
       [0049]    An AAA-AAV vector of the present invention may be prepared in the form of a pharmaceutical composition containing the fusion protein dissolved or dispersed in a pharmaceutically acceptable carrier well known to those who are skilled in the art, for parenteral administration by e.g., intravenous, subcutaneous, or intramuscular injection or by intravenous drip infusion. For the pharmaceutical composition for parenteral administration, any conventional additives may be used such as excipients, binders, disintegrates, dispersing agents, lubricants, diluents, absorption enhancers, buffering agents, surfactants, solubilizing agents, preservatives, emulsifiers, isotonizers, stabilizers, solubilizers for injection, pH adjusting agents, etc. An AAA viral, liposomal, or polymer vector of the present invention, in particular a AAA-AAV viral vector and a AAA peptide attached to a viral capsid, may be used advantageously in place of the conventional untagged (native) viral vector in a substitution therapy for the treatment of bone diseases. In the treatment, the vector carrying the fusion protein may be administered intravenously, subcutaneously, or intramuscularly. Doses and frequencies of administration are to be determined by the physician in charge in accordance with the condition of his or her patient. 
         [0050]    The various embodiment described herein are water-soluble and maybe administered, by way of example, in a sterile aqueous solution, preferably a physiological solution. A pharmaceutically acceptable formulation of the present invention may be any injectable or topically applied physiological solution. A physiological solution may be comprised of isotonic balanced salts with a pH of about 7.0 to about 7.5. A preferred physiological solution may comprise isotonic saline and a pH of 7.5. For topical administration or for certain targeted applications it may be desirable to increase the viscosity of the formulation. Various carriers known to increase viscosity include but are not limited to such high molecular weight polymers such as, hyaluronic acid, hydroxypropyl methyl cellulose, as well as other carbohydrates or sugars. These are typical included in the formulation at 0.01 to 0.1 percent, 0.1 to 1.0 percent, 1 to 2 percent, 2 to 3 percent, 3 to 4 percent, 4 to 5 percent 5 to 10 percent, or 10 to 20 percent by weight. By way of example and not of limitation, recombinant viruses may be administered at a dose of 10 7 -10 12  pfu for a non-intravenous administration. 
         [0051]    Preferred embodiments of the invention are described in the following examples. Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herein. It is intended that the specification, together with the examples, be considered exemplary only, with the scope and spirit of the invention being indicated by the claims, which follow the examples. 
       EXAMPLES 
     Example 1 
     Construction of pXX2-ND8 Plasmid 
       [0052]    After site-directed mutagenesis was performed, 20 clones were obtained. Five clones out of 20 clones had an expected size of 8.3 kb. PCR with XX2-ND8-4F and XX2-ND8-4R primers showed that in three clones a PCR product of 715 bp was obtained ( FIG. 6   a ). Sequencing of those plasmids showed the presence of the precise insertional sequence in one clone ( FIG. 6   b ) without introduction of fortuitous mutations. 
       Example 2 
     In Vitro Transfection 
       [0053]    GALNS activity from transfected cells with either untagged or tagged plasmid increased to 12.24+/−3.25 U/mg or 12.53+/−2,33 U/mg respectively, compared to 0.63+/−0.55 U/mg in untransfected cells ( FIG. 7 ). These results show that the presence of the AAA in the capsid does not alter the transfection efficacy of the plasmid and expression level of the gene product. 
       Example 3 
     Hydroxyapatite-Binding Assay 
       [0054]    AAV2 wild-type and CBA-GALNS (native capsid) virus vectors were found in all 100% in the supernatant after the hydroxyapatite-binding assay indicating no binding with hydroxyapatite, while no ND8/CBA-GALNS virus vectors were was found in the supernatant, indicating 100% affinity with hydroxyapatite ( FIG. 8 ). 
       Example 4 
     Biodistribution Experiment 
       [0055]    DNA samples from bone, liver, brain and bone marrow were tested by PCR for presence of vector DNA after 48 h. After 48 h post injection, virus DNA was detected in liver, brain, and bone marrow with both CBA-GALNS and ND8/CBA-GALNS vectors. However, in bone with ND8/CBA-GALNS, the virus genome was detected while CBA-GALNS, was not detected ( FIG. 9 ). Although mouse-by-mouse variation was observed, virus genome quantification by real-time PCR in DNA samples from bone showed an increment between 16- and 291-folds in the amount of virus genome in mice infused with ND8/CBA-GALNS compared to mice infused with CBA-GALNS. No virus DNA was detected in any tissue sample from control mice with PBS. 
         [0056]    All publications and patents cited in this specification are hereby incorporated by reference in their entirety. The discussion of the references herein is intended merely to summarize the assertions made by the authors and no admission is made that any reference constitutes prior art. Applicants reserve the right to challenge the accuracy and pertinence of the cited references. 
       Sequences 
       [0057]    SEQ ID NO: 1. Complete sequence of packing plasmid pXX2 (8,3 kb). Initial codon for capsid proteins VP1, VP2 and VP3 are shown in bold. 
         [0000]    
       
         
               
             
           
               
                 CGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGAATTCCCATCATC 
               
               
                   
               
               
                 AATAATATACCTTATTTTGGATTGAAGCCAATATGATAATGAGGGGGTGG 
               
               
                   
               
               
                 AGTTTGTGACGTGGCGCGGGGCGTGGGAACGGGGCGGGTGACGTAGTAGC 
               
               
                   
               
               
                 TCTAGAGGTCCTGTATTAGAGGTCACGTGAGTGTTTTGCGACATTTTGCG 
               
               
                   
               
               
                 ACACCATGTGGTCACGCTGGGTATTTAAGCCCGAGTGAGCACGCAGGGTC 
               
               
                   
               
               
                 TCCATTTTGAAGCGGGAGGTTTGAACGCGCAGCCACCACGGCGGGGTTTT 
               
               
                   
               
               
                 ACGAGATTGTGATTAAGGTCCCCAGCGACCTTGACGAGCATCTGCCCGGC 
               
               
                   
               
               
                 ATTTCTGACAGCTTTGTGAACTGGGTGGCCGAGAAGGAATGGGAGTTGCC 
               
               
                   
               
               
                 GCCAGATTCTGACATGGATCTGAATCTGATTGAGCAGGCACCCCTGACCG 
               
               
                   
               
               
                 TGGCCGAGAAGCTGCAGCGCGACTTTCTGACGGAATGGCGCCGTGTGAGT 
               
               
                   
               
               
                 AAGGCCCCGGAGGCCCTTTTCTTTGTGCAATTTGAGAAGGGAGAGAGCTA 
               
               
                   
               
               
                 CTTCCACATGCACGTGCTCGTGGAAACCACCGGGGTGAAATCCATGGTTT 
               
               
                   
               
               
                 TGGGACGTTTCCTGAGTCAGATTCGCGAAAAACTGATTCAGAGAATTTAC 
               
               
                   
               
               
                 CGCGGGATCGAGCCGACTTTGCCAAACTGGTTCGCGGTCACAAAGACCAG 
               
               
                   
               
               
                 AAATGGCGCCGGAGGCGGGAACAAGGTGGTGGATGAGTGCTACATCCCCA 
               
               
                   
               
               
                 ATTACTTGCTCCCCAAAACCCAGCCTGAGCTCCAGTGGGCGTGGACTAAT 
               
               
                   
               
               
                 ATGGAACAGTATTTAAGCGCCTGTTTGAATCTCACGGAGCGTAAACGGTT 
               
               
                   
               
               
                 GGTGGCGCAGCATCTGACGCACGTGTCGCAGACGCAGGAGCAGAACAAAG 
               
               
                   
               
               
                 AGAATCAGAATCCCAATTCTGATGCGCCGGTGATCAGATCAAAAACTTCA 
               
               
                   
               
               
                 GCCAGGTACATGGAGCTGGTCGGGTGGCTCGTGGACAAGGGGATTACCTC 
               
               
                   
               
               
                 GGAGAAGCAGTGGATCCAGGAGGACCAGGCCTCATACATCTCCTTCAATG 
               
               
                   
               
               
                 CGGCCTCCAACTCGCGGTCCCAAATCAAGGCTGCCTTGGACAATGGGGGA 
               
               
                   
               
               
                 AAGATTATGAGCCTGACTAAAACCGCCCCCGACTACCTGGTGGGCCAGCA 
               
               
                   
               
               
                 GCCCGTGGAGGACATTTCCAGCAATCGGATTTATAAAATTTTGGAACTAA 
               
               
                   
               
               
                 ACGGGTACGATCCCCAATATGCGGCTTCCGTCTTTCTGGGATGGGCCACG 
               
               
                   
               
               
                 AAAAAGTTCGGCAAGAGGAACACCATCTGGCTGTTTGGGCCTGCAACTAC 
               
               
                   
               
               
                 CGGGAAGACCAACATCGCGGAGGCCATAGCCCACACTGTGCCCTTCTACG 
               
               
                   
               
               
                 GGTGCGTAAACTGGACCAATGAGAACTTTCCCTTCAACGACTGTGTCGAC 
               
               
                   
               
               
                 AAGATGGTGATCTGGTGGGAGGAGGGGAAGATGACCGCCAAGGTCGTGGA 
               
               
                   
               
               
                 GTCGGCCAAAGCCATTCTCGGAGGAAGCAAGGTGCGCGTGGACCAGAAAT 
               
               
                   
               
               
                 GCAAGTCCTCGGCCCAGATAGACCCGACTCCCGTGATCGTCACCTCCAAC 
               
               
                   
               
               
                 ACCAACATGTGCGCCGTGATTGACGGGAACTCAACGACCTTCGAACACCA 
               
               
                   
               
               
                 GCAGCCGTTGCAAGACCGGATGTTCAAATTTGAACTCACCCGCCGTCTGG 
               
               
                   
               
               
                 ATCATGACTTTGGGAAGGTCACCAAGCAGGAAGTCAAAGACTTTTTCCGG 
               
               
                   
               
               
                 TGGGCAAAGGATCACGTGGTTGAGGTGGAGCATGAATTCTACGTCAAAAA 
               
               
                   
               
               
                 GGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGAGC 
               
               
                   
               
               
                 CCAAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCGGAA 
               
               
                   
               
               
                 GCTTCGATCAACTACGCAGACAGGTACCAAAACAAATGTTCTCGTCACGT 
               
               
                   
               
               
                 GGGCATGAATCTGATGCTGTTTCCCTGCAGACAATGCGAGAGAATGAATC 
               
               
                   
               
               
                 AGAATTCAAATATCTGCTTCACTCACGGACAGAAAGACTGTTTAGAGTGC 
               
               
                   
               
               
                 TTTCCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCA 
               
               
                   
               
               
                 GAAACTGTGCTACATTCATCATATCATGGGAAAGGTGCCAGACGCTTGCA 
               
               
                   
               
               
                 CTGCCTGCGATCTGGTCAATGTGGATTTGGATGACTGCATCTTTGAACAA 
               
               
                   
               
               
                 TAAATGATTTAAATCAGGT ATG GCTGCCGATGGTTATCTTCCAGATTGGC 
               
               
                   
               
               
                 TCGAGGACACTCTCTCTGAAGGAATAAGACAGTGGTGGAAGCTCAAACCT 
               
               
                   
               
               
                 GGCCCACCACCACCAAAGCCCGCAGAGCGGCATAAGGACGACAGCAGGGG 
               
               
                   
               
               
                 TCTTGTGCTTCCTGGGTACAAGTACCTCGGACCCTTCAACGGACTCGACA 
               
               
                   
               
               
                 AGGGAGAGCCGGTCAACGAGGCAGACGCCGCGGCCCTCGAGCACGACAAA 
               
               
                   
               
               
                 GCCTACGACCGGCAGCTCGACAGCGGAGACAACCCGTACCTCAAGTACAA 
               
               
                   
               
               
                 CCACGCCGACGCGGAGTTTCAGGAGCGCCTTAAAGAAGATACGTCTTTTG 
               
               
                   
               
               
                 GGGGCAACCTCGGACGAGCAGTCTTCCAGGCGAAAAAGAGGGTTCTTGAA 
               
               
                   
               
               
                 CCTCTGGGCCTGGTTGAGGAACCTGTTAAG ACG GCTCCGGGAAAAAAGAG 
               
               
                   
               
               
                 GCCGGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGAA 
               
               
                   
               
               
                 AGGCGGGCCAGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAGACTGGA 
               
               
                   
               
               
                 GACGCAGACTCAGTACCTGACCCCCAGCCTCTCGGACAGCCACCAGCAGC 
               
               
                   
               
               
                 CCCCTCTGGTCTGGGAACTAATACGATGGCTACAGGCAGTGGCGCACCA A   
               
               
                   
               
               
                   TG GCAGACAATAACGAGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAAT 
               
               
                   
               
               
                 TGGCATTGCGATTCCACATGGATGGGCGACAGAGTCATCACCACCAGCAC 
               
               
                   
               
               
                 CCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAACAAATTT 
               
               
                   
               
               
                 CCAGCCAATCAGGAGCCTCGAACGACAATCACTACTTTGGCTACAGCACC 
               
               
                   
               
               
                 CCTTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACCACG 
               
               
                   
               
               
                 TGACTGGCAAAGACTCATCAACAACAACTGGGGATTCCGACCCAAGAGAC 
               
               
                   
               
               
                 TCAACTTCAAGCTCTTTAACATTCAAGTCAAAGAGGTCACGCAGAATGAC 
               
               
                   
               
               
                 GGTACGACGACGATTGCCAATAACCTTACCAGCACGGTTCAGGTGTTTAC 
               
               
                   
               
               
                 TGACTCGGAGTACCAGCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGAT 
               
               
                   
               
               
                 GCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATGGATAC 
               
               
                   
               
               
                 CTCACCCTGAACAACGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACTG 
               
               
                   
               
               
                 CCTGGAGTACTTTCCTTCTCAGATGCTGCGTACCGGAAACAACTTTACCT 
               
               
                   
               
               
                 TCAGCTACACTTTTGAGGACGTTCCTTTCCACAGCAGCTACGCTCACAGC 
               
               
                   
               
               
                 CAGAGTCTGGACCGTCTCATGAATCCTCTCATCGACCAGTACCTGTATTA 
               
               
                   
               
               
                 CTTGAGCAGAACAAACACTCCAAGTGGAACCACCACGCAGTCAAGGCTTC 
               
               
                   
               
               
                 AGTTTTCTCAGGCCGGAGCGAGTGACATTCGGGACCAGTCTAGGAACTGG 
               
               
                   
               
               
                 CTTCCTGGACCCTGTTACCGCCAGCAGCGAGTATCAAAGACATCTGCGGA 
               
               
                   
               
               
                 TAACAACAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCACCTCA 
               
               
                   
               
               
                 ATGGCAGAGACTCTCTGGTGAATCCGGGCGCGGCCATGGCAAGCCACAAG 
               
               
                   
               
               
                 GACGATGAAGAAAAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAA 
               
               
                   
               
               
                 GCAAGGCTCAGAGAAAACAAATGTGGACATTGAAAAGGTCATGATTACAG 
               
               
                   
               
               
                 ACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAGTATGGT 
               
               
                   
               
               
                 TCTGTATCTACCAACCTCCAGAGAGGCAACAGACAAGCAGCTACCGCAGA 
               
               
                   
               
               
                 TGTCAACACACAAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAGATG 
               
               
                   
               
               
                 TGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCACACACGGACGGACAT 
               
               
                   
               
               
                 TTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAACACCCTCCTCC 
               
               
                   
               
               
                 ACAGATTCTCATCAAGAACACCCCGGTACCTGCGAATCCTTCGACCACCT 
               
               
                   
               
               
                 TCAGTGCGGCAAAGTTTGCTTCCTTCATCACACAGTACTCCACGGGACAG 
               
               
                   
               
               
                 GTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAACGCTG 
               
               
                   
               
               
                 GAATCCCGAAATTCAGTACACTTCCAACTACAACAAGTCTGTTAATGTGG 
               
               
                   
               
               
                 ACTTTACTGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCCATTGGC 
               
               
                   
               
               
                 ACCAGATACCTGACTCGTAATCTGTAATTGCTTGTTAATCAATAAACCGT 
               
               
                   
               
               
                 TTAATTCGTTTCAGTTGAACTTTGGTCTCTGCGTATTTCTTTCTTATCTA 
               
               
                   
               
               
                 GTTTCCATGCTCTAGAGGTCCTGTATTAGAGGTCACGTGAGTGTTTTGCG 
               
               
                   
               
               
                 ACATTTTGCGACACCATGTGGTCACGCTGGGTATTTAAGCCCGAGTGAGC 
               
               
                   
               
               
                 ACGCAGGGTCTCCATTTTGAAGCGGGAGGTTTGAACGCGCAGCCACCACG 
               
               
                   
               
               
                 GCGGGGTTTTACGAGATTGTGATTAAGGTCCCCAGCGACCTTGACGAGCA 
               
               
                   
               
               
                 TCTGCCCGGCATTTCTGACAGCTTTGTGAACTGGGTGGCCGAGAAGGAAT 
               
               
                   
               
               
                 GGGAGTTGCCGCCAGATTCTGACATGGATCTGAATCTGATTGAGCAGGCA 
               
               
                   
               
               
                 CCCCTGACCGTGGCCGAGAAGCTGCATCGCTGGCGTAATAGCGAAGAGGC 
               
               
                   
               
               
                 CCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGC 
               
               
                   
               
               
                 GATTCCGTTGCAATGGCTGGCGGTAATATTGTTCTGGATATTACCAGCAA 
               
               
                   
               
               
                 GGCCGATAGTTTGAGTTCTTCTACTCAGGCAAGTGATGTTATTACTAATC 
               
               
                   
               
               
                 AAAGAAGTATTGCGACAACGGTTAATTTGCGTGATGGACAGACTCTTTTA 
               
               
                   
               
               
                 CTCGGTGGCCTCACTGATTATAAAAACACTTCTCAGGATTCTGGCGTACC 
               
               
                   
               
               
                 GTTCCTGTCTAAAATCCCTTTAATCGGCCTCCTGTTTAGCTCCCGCTCTG 
               
               
                   
               
               
                 ATTCTAACGAGGAAAGCACGTTATACGTGCTCGTCAAAGCAACCATAGTA 
               
               
                   
               
               
                 CGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCA 
               
               
                   
               
               
                 GCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTC 
               
               
                   
               
               
                 TTCCCTTCCTTTCTCGCGACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAA 
               
               
                   
               
               
                 TCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACC 
               
               
                   
               
               
                 CCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGA 
               
               
                   
               
               
                 TAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGG 
               
               
                   
               
               
                 ACTCTTGTTCCAAACTGGAACAACACTCAAGCCTATCTCGGTCTATTCTT 
               
               
                   
               
               
                 TTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAG 
               
               
                   
               
               
                 CTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTAC 
               
               
                   
               
               
                 AATTTAAATATTTGCTTATACAATCTTCCTGTTTTTGGGGCTTTTCTGAT 
               
               
                   
               
               
                 TATCAACCGGGGTACATATGATTGACATGCTAGTTTTACGATTACCGTTC 
               
               
                   
               
               
                 ATCGATTCTCTTGTTTGCTCCAGACTCTCAGGCAATGACCTGATAGCCTT 
               
               
                   
               
               
                 TGTAGAGACCTCTCAAAAATAGCTACCCTCTCCGGCATGAATTTATCAGC 
               
               
                   
               
               
                 TAGAACGGTTGAATATCATATTGATGGTGATTTGACTGTCTCCGGCCTTT 
               
               
                   
               
               
                 CTCACCCGTTTGAATCTTTACCTACACATTACTCAGGCATTGCATTTAAA 
               
               
                   
               
               
                 ATATATGAGGGTTCTAAAAATTTTTATCCTTGCGTTGAAATAAAGGCTTC 
               
               
                   
               
               
                 TCCCGCAAAAGTATTACAGGGTCATAATGTTTTTGGTACAACCGATTTAG 
               
               
                   
               
               
                 CTTTATGCTCTGAGGCTTTATTGCTTAATTTTGCTAATTCTTTGCCTTGC 
               
               
                   
               
               
                 CTGTATGATTTATTGGATGTTGCAATTCCTGATGCGGTATTTTCTCCTTA 
               
               
                   
               
               
                 CGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATC 
               
               
                   
               
               
                 TGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGC 
               
               
                   
               
               
                 TGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAG 
               
               
                   
               
               
                 CTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCA 
               
               
                   
               
               
                 CCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGT 
               
               
                   
               
               
                 TAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGG 
               
               
                   
               
               
                 GAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAAT 
               
               
                   
               
               
                 ATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTG 
               
               
                   
               
               
                 AAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCT 
               
               
                   
               
               
                 TTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTG 
               
               
                   
               
               
                 AAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGA 
               
               
                   
               
               
                 ACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAAC 
               
               
                   
               
               
                 GTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTA 
               
               
                   
               
               
                 TCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTC 
               
               
                   
               
               
                 TCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGG 
               
               
                   
               
               
                 ATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGAT 
               
               
                   
               
               
                 AACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCT 
               
               
                   
               
               
                 AACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTT 
               
               
                   
               
               
                 GGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACG 
               
               
                   
               
               
                 ATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACT 
               
               
                   
               
               
                 ACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATA 
               
               
                   
               
               
                 AAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATT 
               
               
                   
               
               
                 GCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGC 
               
               
                   
               
               
                 ACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGG 
               
               
                   
               
               
                 GGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGT 
               
               
                   
               
               
                 GCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATAT 
               
               
                   
               
               
                 ACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGA 
               
               
                   
               
               
                 AGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCG 
               
               
                   
               
               
                 TTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGA 
               
               
                   
               
               
                 TCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGC 
               
               
                   
               
               
                 TACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCG 
               
               
                   
               
               
                 AAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGT 
               
               
                   
               
               
                 GTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACAT 
               
               
                   
               
               
                 ACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAG 
               
               
                   
               
               
                 TCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCA 
               
               
                   
               
               
                 GCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAA 
               
               
                   
               
               
                 CGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCC 
               
               
                   
               
               
                 ACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGT 
               
               
                   
               
               
                 CGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAAcGCCTGGTATC 
               
               
                   
               
               
                 TTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTG 
               
               
                   
               
               
                 TGATGCTCGTCAGGGGGGCGGAGCGTATGGAAAAACGCCAGCAACGCGGG 
               
               
                   
               
               
                 CTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTC 
               
               
                   
               
               
                 CTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGA 
               
               
                   
               
               
                 GCTGATACCGCTCGCCGGAGCCGAACGACCGAGCGCAGCGAGTCAGTGAG 
               
               
                   
               
               
                 CGAGGAAGCGGAAGAGCGCCCAATACGCAAAC 
               
             
          
         
       
     
         [0058]    SEQ ID NO: 2. Complete sequence of packing plasmid pXX2 with the bone-tag sequence (pXX2-ND8-8,4 kb). Initial codon for capsid proteins VP1, VP2 and VP3 are shown in bold. Sequence encoding for the amino acidic octapeptide is underlined. 
         [0000]    
       
         
               
             
           
               
                 CGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGAATTCCCATCATC 
               
               
                   
               
               
                 AATAATATACCTTATTTTGGATTGAAGCCAATATGATAATGAGGGGGTGG 
               
               
                   
               
               
                 AGTTTGTGACGTGGCGCGGGGCGTGGGAACGGGGCGGGTGACGTAGTAGC 
               
               
                   
               
               
                 TCTAGAGGTCCTGTATTAGAGGTCACGTGAGTGTTTTGCGACATTTTGCG 
               
               
                   
               
               
                 ACACCATGTGGTCACGCTGGGTATTTAAGCCCGAGTGAGCACGCAGGGTC 
               
               
                   
               
               
                 TCCATTTTGAAGCGGGAGGTTTGAACGCGCAGCCACCACGGCGGGGTTTT 
               
               
                   
               
               
                 ACGAGATTGTGATTAAGGTCCCCAGCGACCTTGACGAGCATCTGCCCGGC 
               
               
                   
               
               
                 ATTTCTGACAGCTTTGTGAACTGGGTGGCCGAGAAGGAATGGGAGTTGCC 
               
               
                   
               
               
                 GCCAGATTCTGACATGGATCTGAATCTGATTGAGCAGGCACCCCTGACCG 
               
               
                   
               
               
                 TGGCCGAGAAGCTGCAGCGCGACTTTCTGACGGAATGGCGCCGTGTGAGT 
               
               
                   
               
               
                 AAGGCCCCGGAGGCCCTTTTCTTTGTGCAATTTGAGAAGGGAGAGAGCTA 
               
               
                   
               
               
                 CTTCCACATGCACGTGCTCGTGGAAACCACCGGGGTGAAATCCATGGTTT 
               
               
                   
               
               
                 TGGGACGTTTCCTGAGTCAGATTCGCGAAAAACTGATTCAGAGAATTTAC 
               
               
                   
               
               
                 CGCGGGATCGAGCCGACTTTGCCAAACTGGTTCGCGGTCACAAAGACCAG 
               
               
                   
               
               
                 AAATGGCGCCGGAGGCGGGAACAAGGTGGTGGATGAGTGCTACATCCCCA 
               
               
                   
               
               
                 ATTACTTGCTCCCCAAAACCCAGCCTGAGCTCCAGTGGGCGTGGACTAAT 
               
               
                   
               
               
                 ATGGAACAGTATTTAAGCGCCTGTTTGAATCTCACGGAGCGTAAACGGTT 
               
               
                   
               
               
                 GGTGGCGCAGCATCTGACGCACGTGTCGCAGACGCAGGAGCAGAACAAAG 
               
               
                   
               
               
                 AGAATCAGAATCCCAATTCTGATGCGCCGGTGATCAGATCAAAAACTTCA 
               
               
                   
               
               
                 GCCAGGTACATGGAGCTGGTCGGGTGGCTCGTGGACAAGGGGATTACCTC 
               
               
                   
               
               
                 GGAGAAGCAGTGGATCCAGGAGGACCAGGCCTCATACATCTCCTTCAATG 
               
               
                   
               
               
                 CGGCCTCCAACTCGCGGTCCCAAATCAAGGCTGCCTTGGACAATGCGGGA 
               
               
                   
               
               
                 AAGATTATGAGCCTGACTAAAACCGCCCCCGACTACCTGGTGGGCCAGCA 
               
               
                   
               
               
                 GCCCGTGGAGGACATTTCCAGCAATCGGATTTATAAAATTTTGGAACTAA 
               
               
                   
               
               
                 ACGGGTACGATCCCCAATATGCGGCTTCCGTCTTTCTGGGATGGGCCACG 
               
               
                   
               
               
                 AAAAAGTTCGGCAAGAGGAACACCATCTGGCTGTTTGGGCCTGCAACTAC 
               
               
                   
               
               
                 CGGGAAGACCAACATCGCGGAGGCCATAGCCCACACTGTGCCCTTCTACG 
               
               
                   
               
               
                 GGTGCGTAAACTGGACCAATGAGAACTTTCCCTTCAACGACTGTGTCGAC 
               
               
                   
               
               
                 AAGATGGTGATCTGGTGGGAGGAGGGGAAGATGACCGCCAAGGTCGTGGA 
               
               
                   
               
               
                 GTCGGCCAAAGCCATTCTCGGAGGAAGCAAGGTGCGCGTGGACCAGAAAT 
               
               
                   
               
               
                 GCAAGTCCTCGGCCCAGATAGACCCGACTCCCGTGATCGTCACCTCCAAC 
               
               
                   
               
               
                 ACCAACATGTGCGCCGTGATTGACGGGAACTCAACGACCTTCGAACACCA 
               
               
                   
               
               
                 GCAGCCGTTGCAAGACCGGATGTTCAAATTTGAACTCACCCGCCGTCTGG 
               
               
                   
               
               
                 ATCATGACTTTGGGAAGGTCACCAAGCAGGAAGTCAAAGACTTTTTCCGG 
               
               
                   
               
               
                 TGGGCAAAGGATCACGTGGTTGAGGTGGAGCATGAATTCTACGTCAAAAA 
               
               
                   
               
               
                 GGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGAGC 
               
               
                   
               
               
                 CCAAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCGGAA 
               
               
                   
               
               
                 GCTTCGATCAACTACGCAGACAGGTACCAAAACAAATGTTCTCGTCACGT 
               
               
                   
               
               
                 GGGCATGAATCTGATGCTGTTTCCCTGCAGACAATGCGAGAGAATGAATC 
               
               
                   
               
               
                 AGAATTCAAATATCTGCTTCACTCACGGACAGAAAGACTGTTTAGAGTGC 
               
               
                   
               
               
                 TTTCCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCA 
               
               
                   
               
               
                 GAAACTGTGCTACATTCATCATATCATGGGAAAGGTGCCAGACGCTTGCA 
               
               
                   
               
               
                 CTGCCTGCGATCTGGTCAATGTGGATTTGGATGACTGCATCTTTGAACAA 
               
               
                   
               
               
                 TAAATGATTTAAATCAGGTATGGCTGCCGATGGTTATCTTCCAGATTGGC 
               
               
                   
               
               
                 TCGAGGACAGTCTCTCTGAAGGAATAAGACAGTGGTGGAAGCTCAAACCT 
               
               
                   
               
               
                 GGCCCACCACCACCAAAGCCCGCAGAGCGGCATAAGGACGACAGCAGGGG 
               
               
                   
               
               
                 TCTTGTGCTTCCTGGGTACAAGTACCTCGGACCCTTCAACGGACTCGACA 
               
               
                   
               
               
                 AGGGAGAGCCGGTCAACGAGGCAGACGCCGCGGCCCTCGAGCACGACAAA 
               
               
                   
               
               
                 GCCTACGACCGGCAGCTCGACAGCGGAGACAACCCGTACCTCAAGTACAA 
               
               
                   
               
               
                 CCACGCCGACGCGGAGTTTCAGGAGCGCCTTAAAGAAGATACGTCTTTTG 
               
               
                   
               
               
                 GGGGCAACCTCGGACGAGCAGTCTTCCAGGCGAAAAAGAGGGTTCTTGAA 
               
               
                   
               
               
                 CCTCTGGGCCTGGTTGAGGAACCTGTTAAG ACG   GATGATGATGATGATGA   
               
               
                   
               
               
                   TGACGAC GCTCCGGGAAAAAAGAGGCCGGTAGAGCACTCTCCTGTGGAGC 
               
               
                   
               
               
                 CAGACTCCTCCTCGGGAACCGGAAAGGCGGGCCAGCAGCCTGCAAGAAAA 
               
               
                   
               
               
                 AGATTGAATTTTGGTCAGACTGGAGACGCAGACTCAGTACCTGACCCCCA 
               
               
                   
               
               
                 GCCTCTCGGACAGCCACCAGCAGCCCCCTCTGGTCTGGGAACTAATACGA 
               
               
                   
               
               
                 TGGCTACAGGCAGTGGCGCACCA ATG GCAGACAATAACGAGGGCGCCGAC 
               
               
                   
               
               
                 GGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGATGGG 
               
               
                   
               
               
                 CGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACA 
               
               
                   
               
               
                 ACAACCACCTCTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGAC 
               
               
                   
               
               
                 AATCACTACTTTGGCTACAGCACCCCTTGGGGGTATTTTGACTTCAACAG 
               
               
                   
               
               
                 ATTCCACTGCCACTTTTCACCACGTGACTGGCAAAGACTCATCAACAACA 
               
               
                   
               
               
                 ACTGGGGATTCCGACCCAAGAGACTCAACTTCAAGCTCTTTAACATTCAA 
               
               
                   
               
               
                 GTCAAAGAGGTCACGCAGAATGACGGTACGACGACGATTGCCAATAACCT 
               
               
                   
               
               
                 TACCAGCACGGTTCAGGTGTTTACTGACTCGGAGTACCAGCTCCCGTACG 
               
               
                   
               
               
                 TCCTCGGCTCGGCGCATCAAGGATGCCTCCCGCCGTTCCCAGCAGACGTC 
               
               
                   
               
               
                 TTCATGGTGCCACAGTATGGATACCTCACCCTGAACAACGGGAGTCAGGC 
               
               
                   
               
               
                 AGTAGGACGCTCTTCATTTTACTGCCTGGAGTACTTTCCTTCTCAGATGC 
               
               
                   
               
               
                 TGCGTACCGGAAACAACTTTACCTTCAGCTACACTTTTGAGGACGTTCCT 
               
               
                   
               
               
                 TTCCACAGCAGCTACGCTCACAGCCAGAGTCTGGACCGTCTCATGAATCC 
               
               
                   
               
               
                 TCTCATCGACCAGTACCTGTATTACTTGAGCAGAACAAACACTCCAAGTG 
               
               
                   
               
               
                 GAACCACCACGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGTGAC 
               
               
                   
               
               
                 ATTCGGGACCAGTCTAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGCA 
               
               
                   
               
               
                 GCGAGTATCAAAGACATCTGCGGATAACAACAACAGTGAATACTCGTGGA 
               
               
                   
               
               
                 CTGGAGCTACCAAGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCGG 
               
               
                   
               
               
                 GGCCCGGCCATGGCAAGCCACAAGGACGATGAAGAAAAGTTTTTTCCTCA 
               
               
                   
               
               
                 GAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAACAAATGTGG 
               
               
                   
               
               
                 ACATTGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGACAACCAAT 
               
               
                   
               
               
                 CCCGTGGCTACGGAGCAGTATGGTTCTGTATCTACCAACCTCCAGAGAGG 
               
               
                   
               
               
                 CAACAGACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCCAG 
               
               
                   
               
               
                 GCATGGTCTGGCAGGAGAGAGATGTGTACCTTCAGGGGCCCATCTGGGCA 
               
               
                   
               
               
                 AAGATTGCACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGG 
               
               
                   
               
               
                 ATTCGGACTTAAACACCCTCCTCCACAGATTCTCATCAAGAACACCCCGG 
               
               
                   
               
               
                 TACCTGCGAATCCTTCGACCACCTTCAGTGCGGCAAAGTTTGCTTCCTTC 
               
               
                   
               
               
                 ATCACACAGTACTCCACGGGACAGGTCAGCGTGGAGATCGAGTGGGAGCT 
               
               
                   
               
               
                 GCAGAAGGAAAACAGCAAACGCTGGAATCCCGAAATTCAGTACACTTCCA 
               
               
                   
               
               
                 ACTACAACAAGTCTGTTAATGTGGACTTTACTGTGGACACTAATGGCGTG 
               
               
                   
               
               
                 TATTCAGAGCCTCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTA 
               
               
                   
               
               
                 ATTGCTTGTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACTTTGGT 
               
               
                   
               
               
                 CTCTGCGTATTTCTTTCTTATCTAGTTTCCATGCTCTAGAGGTCCTGTAT 
               
               
                   
               
               
                 TAGAGGTCACGTGAGTGTTTTGCGACATTTTGCGACACCATGTGGTCACG 
               
               
                   
               
               
                 CTGGGTATTTAAGCCCGAGTGAGCACGCAGGGTCTCCATTTTGAAGCGGG 
               
               
                   
               
               
                 AGGTTTGAACGCGCAGCCACCACGGCGGGGTTTTACGAGATTGTGATTAA 
               
               
                   
               
               
                 GGTCCCCAGCGACCTTGACGAGCATCTGCCCGGCATTTCTGACAGCTTTG 
               
               
                   
               
               
                 TGAACTGGGTGGCCGAGAAGGAATGGGAGTTGCCGCCAGATTCTGACATG 
               
               
                   
               
               
                 GATCTGAATCTGATTGAGCAGGCACCCCTGACCGTGGCCGAGAAGCTGCA 
               
               
                   
               
               
                 TCGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGT 
               
               
                   
               
               
                 TGCGCAGCCTGAATGGCGAATGGCGATTCCGTTGCAATGGCTGGCGGTAA 
               
               
                   
               
               
                 TATTGTTCTGGATATTACCAGCAAGGCCGATAGTTTGAGTTCTTCTACTC 
               
               
                   
               
               
                 AGGCAAGTGATGTTATTACTAATCAAAGAAGTATTGCGACAACGGTTAAT 
               
               
                   
               
               
                 TTGCGTGATGGACAGACTCTTTTACTCGGTGGCCTCACTGATTATAAAAA 
               
               
                   
               
               
                 CACTTCTCAGGATTCTGGCGTACCGTTCCTGTCTAAAATCCCTTTAATCG 
               
               
                   
               
               
                 GCCTCCTGTTTAGCTCCCGCTCTGATTCTAACGAGGAAAGCACGTTATAC 
               
               
                   
               
               
                 GTGCTCGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCG 
               
               
                   
               
               
                 CGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCC 
               
               
                   
               
               
                 CTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGC 
               
               
                   
               
               
                 CGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGAT 
               
               
                   
               
               
                 TTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGT 
               
               
                   
               
               
                 TCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTT 
               
               
                   
               
               
                 GGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACAC 
               
               
                   
               
               
                 TCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATT 
               
               
                   
               
               
                 TCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAA 
               
               
                   
               
               
                 TTTTAACAAAATATTAACGTTTACAATTTAAATATTTGCTTATACAATCT 
               
               
                   
               
               
                 TCCTGTTTTTGGGGCTTTTCTGATTATCAACCGGGGTACATATGATTGAC 
               
               
                   
               
               
                 ATGCTAGTTTTACGATTACCGTTCATCGATTCTCTTGTTTGCTCCAGACT 
               
               
                   
               
               
                 CTCAGGCAATGACCTGATAGCCTTTGTAGAGACCTCTCAAAAATAGCTAC 
               
               
                   
               
               
                 CCTCTCCGGCATGAATTTATCAGCTAGAACGGTTGAATATCATATTGATG 
               
               
                   
               
               
                 GTGATTTGACTGTCTCCGGCCTTTCTCACCCGTTTGAATCTTTACCTACA 
               
               
                   
               
               
                 CATTACTCAGGCATTGCATTTAAAATATATGAGGGTTCTAAAAATTTTTA 
               
               
                   
               
               
                 TCCTTGCGTTGAAATAAAGGCTTCTCCCGCAAAAGTATTACAGGGTCATA 
               
               
                   
               
               
                 ATGTTTTTGGTACAACCGATTTAGCTTTATGCTCTGAGGCTTTATTGCTT 
               
               
                   
               
               
                 AATTTTGCTAATTCTTTGCCTTGCCTGTATGATTTATTGGATGTTGCAAT 
               
               
                   
               
               
                 TCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGC 
               
               
                   
               
               
                 ATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCA 
               
               
                   
               
               
                 GCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGC 
               
               
                   
               
               
                 TCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATG 
               
               
                   
               
               
                 TGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCT 
               
               
                   
               
               
                 CGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTT 
               
               
                   
               
               
                 AGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGT 
               
               
                   
               
               
                 TTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACC 
               
               
                   
               
               
                 CTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAAC 
               
               
                   
               
               
                 ATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTT 
               
               
                   
               
               
                 TTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTT 
               
               
                   
               
               
                 GGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCC 
               
               
                   
               
               
                 TTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAA 
               
               
                   
               
               
                 GTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCA 
               
               
                   
               
               
                 ACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCAC 
               
               
                   
               
               
                 CAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGC 
               
               
                   
               
               
                 AGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGAC 
               
               
                   
               
               
                 AACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGG 
               
               
                   
               
               
                 ATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATA 
               
               
                   
               
               
                 CCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTT 
               
               
                   
               
               
                 GCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAAT 
               
               
                   
               
               
                 TAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCG 
               
               
                   
               
               
                 GCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCG 
               
               
                   
               
               
                 TGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCC 
               
               
                   
               
               
                 GTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGA 
               
               
                   
               
               
                 AATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACT 
               
               
                   
               
               
                 GTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATT 
               
               
                   
               
               
                 TTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACC 
               
               
                   
               
               
                 AAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGA 
               
               
                   
               
               
                 AAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCT 
               
               
                   
               
               
                 GCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGAT 
               
               
                   
               
               
                 CAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCA 
               
               
                   
               
               
                 GATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCA 
               
               
                   
               
               
                 AGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCA 
               
               
                   
               
               
                 GTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAG 
               
               
                   
               
               
                 ACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGT 
               
               
                   
               
               
                 GCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTA 
               
               
                   
               
               
                 CAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGA 
               
               
                   
               
               
                 CAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGC 
               
               
                   
               
               
                 TTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCAC 
               
               
                   
               
               
                 CTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCT 
               
               
                   
               
               
                 ATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCT 
               
               
                   
               
               
                 GGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGAT 
               
               
                   
               
               
                 AACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAAC 
               
               
                   
               
               
                 GACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATAC 
               
               
                   
               
               
                 GCAAAC