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 is a continuation of U.S. patent application Ser. No. 12/497,612, filed Jul. 3, 2009, which claims priority to U.S. Provisional Patent Application No. 61/081,711, filed Jul. 17, 2008. 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-acetylgalactosamine-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-acetylgalactosamine-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-acetylgalactosamine-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 done after site-directed mutagenesis was screened by PCR using primers flanking the insertion site. The 715-bp fragment was produced for the targeted done 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. HEK293 cells were transfected with 1×10 10  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 Nati Aced 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  Viral  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. Natl. 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) December 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 sire-directed mutagenesis kit (QuikChange® 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 4° 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-acetylgalactosamine-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 P-glucuronidase (GUS), also described in Ser. No. 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 4-0.8, 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-acetylgalactosamine-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-acetylgalactosamine-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 β-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, including U.S. patent application Ser. Nos., 12/497,612, 61/081,711, 11/614,970, 11/245,424, 11/484,870, 60/725,563, and 10/864,758, 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]    
       
         
               
               
             
           
               
                 CGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGAATTCCCATCATCAATAATA 
                   
               
               
                   
               
               
                 TACCTTATTTTGGATTGAAGCCAATATGATAATGAGGGGGTGGAGTTTGTGACGTGG 
               
               
                   
               
               
                 CGCGGGGCGTGGGAACGGGGCGGGTGACGTAGTAGCTCTAGAGGTCCTGTATTAGA 
               
               
                   
               
               
                 GGTCACGTGAGTGTTTTGCGACATTTTGCGACACCATGTGGTCACGCTGGGTATTTA 
               
               
                   
               
               
                 AGCCCGAGTGAGCACGCAGGGTCTCCATTTTGAAGCGGGAGGTTTGAACGCGCAGC 
               
               
                   
               
               
                 CACCACGGCGGGGTTTTACGAGATTGTGATTAAGGTCCCCAGCGACCTTGACGAGCA 
               
               
                   
               
               
                 TCTGCCCGGCATTTCTGACAGCTTTGTGAACTGGGTGGCCGAGAAGGAATGGGAGTT 
               
               
                   
               
               
                 GCCGCCAGATTCTGACATGGATCTGAATCTGATTGAGCAGGCACCCCTGACCGTGGC 
               
               
                   
               
               
                 CGAGAAGCTGCAGCGCGACTTTCTGACGGAATGGCGCCGTGTGAGTAAGGCCCCGG 
               
               
                   
               
               
                 AGGCCCTTTTCTTTGTGCAATTTGAGAAGGGAGAGAGCTACTTCCACATGCACGTGC 
               
               
                   
               
               
                 TCGTGGAAACCACCGGGGTGAAATCCATGGTTTTGGGACGTTTCCTGAGTCAGATTC 
               
               
                   
               
               
                 GCGAAAAACTGATTCAGAGAATTTACCGCGGGATCGAGCCGACTTTGCCAAACTGG 
               
               
                   
               
               
                 TTCGCGGTCACAAAGACCAGAAATGGCGCCGGAGGCGGGAACAAGGTGGTGGATG 
               
               
                   
               
               
                 AGTGCTACATCCCCAATTACTTGCTCCCCAAAACCCAGCCTGAGCTCCAGTGGGCGT 
               
               
                   
               
               
                 GGACTAATATGGAACAGTATTTAAGCGCCTGTTTGAATCTCACGGAGCGTAAACGGT 
               
               
                   
               
               
                 TGGTGGCGCAGCATCTGACGCACGTGTCGCAGACGCAGGAGCAGAACAAAGAGAAT 
               
               
                   
               
               
                 CAGAATCCCAATTCTGATGCGCCGGTGATCAGATCAAAAACTTCAGCCAGGTACATG 
               
               
                   
               
               
                 GAGCTGGTCGGGTGGCTCGTGGACAAGGGGATTACCTCGGAGAAGCAGTGGATCCA 
               
               
                   
               
               
                 GGAGGACCAGGCCTCATACATCTCCTTCAATGCGGCCTCCAACTCGCGGTCCCAAAT 
               
               
                   
               
               
                 CAAGGCTGCCTTGGACAATGCGGGAAAGATTATGAGCCTGACTAAAACCGCCCCCG 
               
               
                   
               
               
                 ACTACCTGGTGGGCCAGCAGCCCGTGGAGGACATTTCCAGCAATCGGATTTATAAA 
               
               
                   
               
               
                 ATTTTGGAACTAAACGGGTACGATCCCCAATATGCGGCTTCCGTCTTTCTGGGATGG 
               
               
                   
               
               
                 GCCACGAAAAAGTTCGGCAAGAGGAACACCATCTGGCTGTTTGGGCCTGCAACTAC 
               
               
                   
               
               
                 CGGGAAGACCAACATCGCGGAGGCCATAGCCCACACTGTGCCCTTCTACGGGTGCG 
               
               
                   
               
               
                 TAAACTGGACCAATGAGAACTTTCCCTTCAACGACTGTGTCGACAAGATGGTGATCT 
               
               
                   
               
               
                 GGTGGGAGGAGGGGAAGATGACCGCCAAGGTCGTGGAGTCGGCCAAAGCCATTCTC 
               
               
                   
               
               
                 GGAGGAAGCAAGGTGCGCGTGGACCAGAAATGCAAGTCCTCGGCCCAGATAGACCC 
               
               
                   
               
               
                 GACTCCCGTGATCGTCACCTCCAACACCAACATGTGCGCCGTGATTGACGGGAACTC 
               
               
                   
               
               
                 AACGACCTTCGAACACCAGCAGCCGTTGCAAGACCGGATGTTCAAATTTGAACTCA 
               
               
                   
               
               
                 CCCGCCGTCTGGATCATGACTTTGGGAAGGTCACCAAGCAGGAAGTCAAAGACTTTT 
               
               
                   
               
               
                 TCCGGTGGGCAAAGGATCACGTGGTTGAGGTGGAGCATGAATTCTACGTCAAAAAG 
               
               
                   
               
               
                 GGTGGAGCCAAGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGAGCCCAAAC 
               
               
                   
               
               
                 GGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATCAAC 
               
               
                   
               
               
                 TACGCAGACAGGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTG 
               
               
                   
               
               
                 TTTCCCTGCAGACAATGCGAGAGAATGAATCAGAATTCAAATATCTGCTTCACTCAC 
               
               
                   
               
               
                 GGACAGAAAGACTGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTC 
               
               
                   
               
               
                 GTCAAAAAGGCGTATCAGAAACTGTGCTACATTCATCATATCATGGGAAAGGTGCC 
               
               
                   
               
               
                 AGACGCTTGCACTGCCTGCGATCTGGTCAATGTGGATTTGGATGACTGCATCTTTGA 
               
               
                   
               
               
                 ACAATAAATGATTTAAATCAGGT ATG GCTGCCGATGGTTATCTTCCAGATTGGCTCG 
               
               
                   
               
               
                 AGGACACTCTCTCTGAAGGAATAAGACAGTGGTGGAAGCTCAAACCTGGCCCACCA 
               
               
                   
               
               
                 CCACCAAAGCCCGCAGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTTCCTGG 
               
               
                   
               
               
                 GTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGG 
               
               
                   
               
               
                 CAGACGCCGCGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTCGACAGCGGA 
               
               
                   
               
               
                 GACAACCCGTACCTCAAGTACAACCACGCCGACGCGGAGTTTCAGGAGCGCCTTAA 
               
               
                   
               
               
                 AGAAGATACGTCTTTTGGGGGCAACCTCGGACGAGCAGTCTTCCAGGCGAAAAAGA 
               
               
                   
               
               
                 GGGTTCTTGAACCTCTGGGCCTGGTTGAGGAACCTGTTAAG ACG GCTCCGGGAAAA 
               
               
                   
               
               
                 AAGAGGCCGGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGAAA 
               
               
                   
               
               
                 GGCGGGCCAGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAGACTGGAGACGCAG 
               
               
                   
               
               
                 ACTCAGTACCTGACCCCCAGCCTCTCGGACAGCCACCAGCAGCCCCCTCTGGTCTGG 
               
               
                   
               
               
                 GAACTAATACGATGGCTACAGGCAGTGGCGCACCA ATG GCAGACAATAACGAGGG 
               
               
                   
               
               
                 CGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGATGG 
               
               
                   
               
               
                 GCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAAC 
               
               
                   
               
               
                 CACCTCTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGACAATCACTACTTT 
               
               
                   
               
               
                 GGCTACAGCACCCCTTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCAC 
               
               
                   
               
               
                 CACGTGACTGGCAAAGACTCATCAACAACAACTGGGGATTCCGACCCAAGAGACTC 
               
               
                   
               
               
                 AACTTCAAGCTCTTTAACATTCAAGTCAAAGAGGTCACGCAGAATGACGGTACGAC 
               
               
                   
               
               
                 GACGATTGCCAATAACCTTACCAGCACGGTTCAGGTGTTTACTGACTCGGAGTACCA 
               
               
                   
               
               
                 GCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGATGCCTCCCGCCGTTCCCAGCAGA 
               
               
                   
               
               
                 CGTCTTCATGGTGCCACAGTATGGATACCTCACCCTGAACAACGGGAGTCAGGCAGT 
               
               
                   
               
               
                 AGGACGCTCTTCATTTTACTGCCTGGAGTACTTTCCTTCTCAGATGCTGCGTACCGGA 
               
               
                   
               
               
                 AACAACTTTACCTTCAGCTACACTTTTGAGGACGTTCCTTTCCACAGCAGCTACGCTC 
               
               
                   
               
               
                 ACAGCCAGAGTCTGGACCGTCTCATGAATCCTCTCATCGACCAGTACCTGTATTACT 
               
               
                   
               
               
                 TGAGCAGAACAAACACTCCAAGTGGAACCACCACGCAGTCAAGGCTTCAGTTTTCT 
               
               
                   
               
               
                 CAGGCCGGAGCGAGTGACATTCGGGACCAGTCTAGGAACTGGCTTCCTGGACCCTG 
               
               
                   
               
               
                 TTACCGCCAGCAGCGAGTATCAAAGACATCTGCGGATAACAACAACAGTGAATACT 
               
               
                   
               
               
                 CGTGGACTGGAGCTACCAAGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCCG 
               
               
                   
               
               
                 GGCCCGGCCATGGCAAGCCACAAGGACGATGAAGAAAAGTTTTTTCCTCAGAGCGG 
               
               
                   
               
               
                 GGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAACAAATGTGGACATTGAAAAGG 
               
               
                   
               
               
                 TCATGATTACAGACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAG 
               
               
                   
               
               
                 TATGGTTCTGTATCTACCAACCTCCAGAGAGGCAACAGACAAGCAGCTACCGCAGA 
               
               
                   
               
               
                 TGTCAACACACAAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCT 
               
               
                   
               
               
                 TCAGGGGCCCATCTGGGCAAAGATTCCACACACGGACGGACATTTTCACCCCTCTCC 
               
               
                   
               
               
                 CCTCATGGGTGGATTCGGACTTAAACACCCTCCTCCACAGATTCTCATCAAGAACAC 
               
               
                   
               
               
                 CCCGGTACCTGCGAATCCTTCGACCACCTTCAGTGCGGCAAAGTTTGCTTCCTTCATC 
               
               
                   
               
               
                 ACACAGTACTCCACGGGACAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGA 
               
               
                   
               
               
                 AAACAGCAAACGCTGGAATCCCGAAATTCAGTACACTTCCAACTACAACAAGTCTG 
               
               
                   
               
               
                 TTAATGTGGACTTTACTGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCCATTG 
               
               
                   
               
               
                 GCACCAGATACCTGACTCGTAATCTGTAATTGCTTGTTAATCAATAAACCGTTTAATT 
               
               
                   
               
               
                 CGTTTCAGTTGAACTTTGGTCTCTGCGTATTTCTTTCTTATCTAGTTTCCATGCTCTAG 
               
               
                   
               
               
                 AGGTCCTGTATTAGAGGTCACGTGAGTGTTTTGCGACATTTTGCGACACCATGTGGT 
               
               
                   
               
               
                 CACGCTGGGTATTTAAGCCCGAGTGAGCACGCAGGGTCTCCATTTTGAAGCGGGAG 
               
               
                   
               
               
                 GTTTGAACGCGCAGCCACCACGGCGGGGTTTTACGAGATTGTGATTAAGGTCCCCAG 
               
               
                   
               
               
                 CGACCTTGACGAGCATCTGCCCGGCATTTCTGACAGCTTTGTGAACTGGGTGGCCGA 
               
               
                   
               
               
                 GAAGGAATGGGAGTTGCCGCCAGATTCTGACATGGATCTGAATCTGATTGAGCAGG 
               
               
                   
               
               
                 CACCCCTGACCGTGGCCGAGAAGCTGCATCGCTGGCGTAATAGCGAAGAGGCCCGC 
               
               
                   
               
               
                 ACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGATTCCGTTGC 
               
               
                   
               
               
                 AATGGCTGGCGGTAATATTGTTCTGGATATTACCAGCAAGGCCGATAGTTTGAGTTC 
               
               
                   
               
               
                 TTCTACTCAGGCAAGTGATGTTATTACTAATCAAAGAAGTATTGCGACAACGGTTAA 
               
               
                   
               
               
                 TTTGCGTGATGGACAGACTCTTTTACTCGGTGGCCTCACTGATTATAAAAACACTTCT 
               
               
                   
               
               
                 CAGGATTCTGGCGTACCGTTCCTGTCTAAAATCCCTTTAATCGGCCTCCTGTTTAGCT 
               
               
                   
               
               
                 CCCGCTCTGATTCTAACGAGGAAAGCACGTTATACGTGCTCGTCAAAGCAACCATAG 
               
               
                   
               
               
                 TACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGT 
               
               
                   
               
               
                 GACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTT 
               
               
                   
               
               
                 CTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGG 
               
               
                   
               
               
                 TTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGT 
               
               
                   
               
               
                 TCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCC 
               
               
                   
               
               
                 ACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCG 
               
               
                   
               
               
                 GTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATG 
               
               
                   
               
               
                 AGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTT 
               
               
                   
               
               
                 AAATATTTGCTTATACAATCTTCCTGTTTTTGGGGCTTTTCTGATTATCAACCGGGGT 
               
               
                   
               
               
                 ACATATGATTGACATGCTAGTTTTACGATTACCGTTCATCGATTCTCTTGTTTGCTCC 
               
               
                   
               
               
                 AGACTCTCAGGCAATGACCTGATAGCCTTTGTAGAGACCTCTCAAAAATAGCTACCC 
               
               
                   
               
               
                 TCTCCGGCATGAATTTATCAGCTAGAACGGTTGAATATCATATTGATGGTGATTTGA 
               
               
                   
               
               
                 CTGTCTCCGGCCTTTCTCACCCGTTTGAATCTTTACCTACACATTACTCAGGCATTGC 
               
               
                   
               
               
                 ATTTAAAATATATGAGGGTTCTAAAAATTTTTATCCTTGCGTTGAAATAAAGGCTTCT 
               
               
                   
               
               
                 CCCGCAAAAGTATTACAGGGTCATAATGTTTTTGGTACAACCGATTTAGCTTTATGC 
               
               
                   
               
               
                 TCTGAGGCTTTATTGCTTAATTTTGCTAATTCTTTGCCTTGCCTGTATGATTTATTGGA 
               
               
                   
               
               
                 TGTTGCAATTCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCG 
               
               
                   
               
               
                 CATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCG 
               
               
                   
               
               
                 ACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGC 
               
               
                   
               
               
                 TTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTC 
               
               
                   
               
               
                 ATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAA 
               
               
                   
               
               
                 TGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCG 
               
               
                   
               
               
                 CGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGA 
               
               
                   
               
               
                 CAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCA 
               
               
                   
               
               
                 ACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTC 
               
               
                   
               
               
                 ACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTG 
               
               
                   
               
               
                 GGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAA 
               
               
                   
               
               
                 GAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCC 
               
               
                   
               
               
                 GTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACT 
               
               
                   
               
               
                 TGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGA 
               
               
                   
               
               
                 GAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTG 
               
               
                   
               
               
                 ACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTGCACAACATGGGGGATCA 
               
               
                   
               
               
                 TGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACG 
               
               
                   
               
               
                 AGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACT 
               
               
                   
               
               
                 GGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGAT 
               
               
                   
               
               
                 AAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGAT 
               
               
                   
               
               
                 AAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGA 
               
               
                   
               
               
                 TGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGA 
               
               
                   
               
               
                 TGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAAC 
               
               
                   
               
               
                 TGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATT 
               
               
                   
               
               
                 TAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACG 
               
               
                   
               
               
                 TGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTG 
               
               
                   
               
               
                 AGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACC 
               
               
                   
               
               
                 AGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGG 
               
               
                   
               
               
                 CTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCA 
               
               
                   
               
               
                 CCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACC 
               
               
                   
               
               
                 AGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATA 
               
               
                   
               
               
                 GTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCA 
               
               
                   
               
               
                 GCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAA 
               
               
                   
               
               
                 AGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGG 
               
               
                   
               
               
                 TCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTAT 
               
               
                   
               
               
                 AGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAG 
               
               
                   
               
               
                 GGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCC 
               
               
                   
               
               
                 TTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAA 
               
               
                   
               
               
                 CCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCG 
               
               
                   
               
               
                 CAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAC 
               
             
          
         
       
     
         [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]    
       
         
               
               
             
           
               
                 CGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGAATTCCCATCATCAATAATA 
                   
               
               
                   
               
               
                 TACCTTATTTTGGATTGAAGCCAATATGATAATGAGGGGGTGGAGTTTGTGACGTGG 
               
               
                   
               
               
                 CGCGGGGCGTGGGAACGGGGCGGGTGACGTAGTAGCTCTAGAGGTCCTGTATTAGA 
               
               
                   
               
               
                 GGTCACGTGAGTGTTTTGCGACATTTTGCGACACCATGTGGTCACGCTGGGTATTTA 
               
               
                   
               
               
                 AGCCCGAGTGAGCACGCAGGGTCTCCATTTTGAAGCGGGAGGTTTGAACGCGCAGC 
               
               
                   
               
               
                 CACCACGGCGGGGTTTTACGAGATTGTGATTAAGGTCCCCAGCGACCTTGACGAGCA 
               
               
                   
               
               
                 TCTGCCCGGCATTTCTGACAGCTTTGTGAACTGGGTGGCCGAGAAGGAATGGGAGTT 
               
               
                   
               
               
                 GCCGCCAGATTCTGACATGGATCTGAATCTGATTGAGCAGGCACCCCTGACCGTGGC 
               
               
                   
               
               
                 CGAGAAGCTGCAGCGCGACTTTCTGACGGAATGGCGCCGTGTGAGTAAGGCCCCGG 
               
               
                   
               
               
                 AGGCCCTTTTCTTTGTGCAATTTGAGAAGGGAGAGAGCTACTTCCACATGCACGTGC 
               
               
                   
               
               
                 TCGTGGAAACCACCGGGGTGAAATCCATGGTTTTGGGACGTTTCCTGAGTCAGATTC 
               
               
                   
               
               
                 GCGAAAAACTGATTCAGAGAATTTACCGCGGGATCGAGCCGACTTTGCCAAACTGG 
               
               
                   
               
               
                 TTCGCGGTCACAAAGACCAGAAATGGCGCCGGAGGCGGGAACAAGGTGGTGGATG 
               
               
                   
               
               
                 AGTGCTACATCCCCAATTACTTGCTCCCCAAAACCCAGCCTGAGCTCCAGTGGGCGT 
               
               
                   
               
               
                 GGACTAATATGGAACAGTATTTAAGCGCCTGTTTGAATCTCACGGAGCGTAAACGGT 
               
               
                   
               
               
                 TGGTGGCGCAGCATCTGACGCACGTGTCGCAGACGCAGGAGCAGAACAAAGAGAAT 
               
               
                   
               
               
                 CAGAATCCCAATTCTGATGCGCCGGTGATCAGATCAAAAACTTCAGCCAGGTACATG 
               
               
                   
               
               
                 GAGCTGGTCGGGTGGCTCGTGGACAAGGGGATTACCTCGGAGAAGCAGTGGATCCA 
               
               
                   
               
               
                 GGAGGACCAGGCCTCATACATCTCCTTCAATGCGGCCTCCAACTCGCGGTCCCAAAT 
               
               
                   
               
               
                 CAAGGCTGCCTTGGACAATGCGGGAAAGATTATGAGCCTGACTAAAACCGCCCCCG 
               
               
                   
               
               
                 ACTACCTGGTGGGCCAGCAGCCCGTGGAGGACATTTCCAGCAATCGGATTTATAAA 
               
               
                   
               
               
                 ATTTTGGAACTAAACGGGTACGATCCCCAATATGCGGCTTCCGTCTTTCTGGGATGG 
               
               
                   
               
               
                 GCCACGAAAAAGTTCGGCAAGAGGAACACCATCTGGCTGTTTGGGCCTGCAACTAC 
               
               
                   
               
               
                 CGGGAAGACCAACATCGCGGAGGCCATAGCCCACACTGTGCCCTTCTACGGGTGCG 
               
               
                   
               
               
                 TAAACTGGACCAATGAGAACTTTCCCTTCAACGACTGTGTCGACAAGATGGTGATCT 
               
               
                   
               
               
                 GGTGGGAGGAGGGGAAGATGACCGCCAAGGTCGTGGAGTCGGCCAAAGCCATTCTC 
               
               
                   
               
               
                 GGAGGAAGCAAGGTGCGCGTGGACCAGAAATGCAAGTCCTCGGCCCAGATAGACCC 
               
               
                   
               
               
                 GACTCCCGTGATCGTCACCTCCAACACCAACATGTGCGCCGTGATTGACGGGAACTC 
               
               
                   
               
               
                 AACGACCTTCGAACACCAGCAGCCGTTGCAAGACCGGATGTTCAAATTTGAACTCA 
               
               
                   
               
               
                 CCCGCCGTCTGGATCATGACTTTGGGAAGGTCACCAAGCAGGAAGTCAAAGACTTTT 
               
               
                   
               
               
                 TCCGGTGGGCAAAGGATCACGTGGTTGAGGTGGAGCATGAATTCTACGTCAAAAAG 
               
               
                   
               
               
                 GGTGGAGCCAAGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGAGCCCAAAC 
               
               
                   
               
               
                 GGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATCAAC 
               
               
                   
               
               
                 TACGCAGACAGGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTG 
               
               
                   
               
               
                 TTTCCCTGCAGACAATGCGAGAGAATGAATCAGAATTCAAATATCTGCTTCACTCAC 
               
               
                   
               
               
                 GGACAGAAAGACTGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTC 
               
               
                   
               
               
                 GTCAAAAAGGCGTATCAGAAACTGTGCTACATTCATCATATCATGGGAAAGGTGCC 
               
               
                   
               
               
                 AGACGCTTGCACTGCCTGCGATCTGGTCAATGTGGATTTGGATGACTGCATCTTTGA 
               
               
                   
               
               
                 ACAATAAATGATTTAAATCAGGT ATG GCTGCCGATGGTTATCTTCCAGATTGGCTCG 
               
               
                   
               
               
                 AGGACACTCTCTCTGAAGGAATAAGACAGTGGTGGAAGCTCAAACCTGGCCCACCA 
               
               
                   
               
               
                 CCACCAAAGCCCGCAGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTTCCTGG 
               
               
                   
               
               
                 GTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGG 
               
               
                   
               
               
                 CAGACGCCGCGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTCGACAGCGGA 
               
               
                   
               
               
                 GACAACCCGTACCTCAAGTACAACCACGCCGACGCGGAGTTTCAGGAGCGCCTTAA 
               
               
                   
               
               
                 AGAAGATACGTCTTTTGGGGGCAACCTCGGACGAGCAGTCTTCCAGGCGAAAAAGA 
               
               
                   
               
               
                 GGGTTCTTGAACCTCTGGGCCTGGTTGAGGAACCTGTTAAG ACG   GATGATGATGATG   
               
               
                   
               
               
                   ATGATGACGAC GCTCCGGGAAAAAAGAGGCCGGTAGAGCACTCTCCTGTGGAGCCA 
               
               
                   
               
               
                 GACTCCTCCTCGGGAACCGGAAAGGCGGGCCAGCAGCCTGCAAGAAAAAGATTGAA 
               
               
                   
               
               
                 TTTTGGTCAGACTGGAGACGCAGACTCAGTACCTGACCCCCAGCCTCTCGGACAGCC 
               
               
                   
               
               
                 ACCAGCAGCCCCCTCTGGTCTGGGAACTAATACGATGGCTACAGGCAGTGGCGCAC 
               
               
                   
               
               
                 CA ATG GCAGACAATAACGAGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGG 
               
               
                   
               
               
                 CATTGCGATTCCACATGGATGGGCGACAGAGTCATCACCACCAGCACCCGAACCTG 
               
               
                   
               
               
                 GGCCCTGCCCACCTACAACAACCACCTCTACAAACAAATTTCCAGCCAATCAGGAG 
               
               
                   
               
               
                 CCTCGAACGACAATCACTACTTTGGCTACAGCACCCCTTGGGGGTATTTTGACTTCA 
               
               
                   
               
               
                 ACAGATTCCACTGCCACTTTTCACCACGTGACTGGCAAAGACTCATCAACAACAACT 
               
               
                   
               
               
                 GGGGATTCCGACCCAAGAGACTCAACTTCAAGCTCTTTAACATTCAAGTCAAAGAG 
               
               
                   
               
               
                 GTCACGCAGAATGACGGTACGACGACGATTGCCAATAACCTTACCAGCACGGTTCA 
               
               
                   
               
               
                 GGTGTTTACTGACTCGGAGTACCAGCTCCCGTACGTCCTCGGCTCGGCGCATCAAGG 
               
               
                   
               
               
                 ATGCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATGGATACCTCAC 
               
               
                   
               
               
                 CCTGAACAACGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACTGCCTGGAGTACTT 
               
               
                   
               
               
                 TCCTTCTCAGATGCTGCGTACCGGAAACAACTTTACCTTCAGCTACACTTTTGAGGA 
               
               
                   
               
               
                 CGTTCCTTTCCACAGCAGCTACGCTCACAGCCAGAGTCTGGACCGTCTCATGAATCC 
               
               
                   
               
               
                 TCTCATCGACCAGTACCTGTATTACTTGAGCAGAACAAACACTCCAAGTGGAACCAC 
               
               
                   
               
               
                 CACGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACATTCGGGACCAGTC 
               
               
                   
               
               
                 TAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTATCAAAGACATCTGC 
               
               
                   
               
               
                 GGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCACCTCAATG 
               
               
                   
               
               
                 GCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGCCACAAGGACGATGAA 
               
               
                   
               
               
                 GAAAAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAA 
               
               
                   
               
               
                 ACAAATGTGGACATTGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGACAAC 
               
               
                   
               
               
                 CAATCCCGTGGCTACGGAGCAGTATGGTTCTGTATCTACCAACCTCCAGAGAGGCAA 
               
               
                   
               
               
                 CAGACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCATGGTCT 
               
               
                   
               
               
                 GGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCACACACG 
               
               
                   
               
               
                 GACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAACACCCTCCT 
               
               
                   
               
               
                 CCACAGATTCTCATCAAGAACACCCCGGTACCTGCGAATCCTTCGACCACCTTCAGT 
               
               
                   
               
               
                 GCGGCAAAGTTTGCTTCCTTCATCACACAGTACTCCACGGGACAGGTCAGCGTGGAG 
               
               
                   
               
               
                 ATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAACGCTGGAATCCCGAAATTCAGTA 
               
               
                   
               
               
                 CACTTCCAACTACAACAAGTCTGTTAATGTGGACTTTACTGTGGACACTAATGGCGT 
               
               
                   
               
               
                 GTATTCAGAGCCTCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAATTGCT 
               
               
                   
               
               
                 TGTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACTTTGGTCTCTGCGTATTTCT 
               
               
                   
               
               
                 TTCTTATCTAGTTTCCATGCTCTAGAGGTCCTGTATTAGAGGTCACGTGAGTGTTTTG 
               
               
                   
               
               
                 CGACATTTTGCGACACCATGTGGTCACGCTGGGTATTTAAGCCCGAGTGAGCACGCA 
               
               
                   
               
               
                 GGGTCTCCATTTTGAAGCGGGAGGTTTGAACGCGCAGCCACCACGGCGGGGTTTTAC 
               
               
                   
               
               
                 GAGATTGTGATTAAGGTCCCCAGCGACCTTGACGAGCATCTGCCCGGCATTTCTGAC 
               
               
                   
               
               
                 AGCTTTGTGAACTGGGTGGCCGAGAAGGAATGGGAGTTGCCGCCAGATTCTGACAT 
               
               
                   
               
               
                 GGATCTGAATCTGATTGAGCAGGCACCCCTGACCGTGGCCGAGAAGCTGCATCGCT 
               
               
                   
               
               
                 GGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTG 
               
               
                   
               
               
                 AATGGCGAATGGCGATTCCGTTGCAATGGCTGGCGGTAATATTGTTCTGGATATTAC 
               
               
                   
               
               
                 CAGCAAGGCCGATAGTTTGAGTTCTTCTACTCAGGCAAGTGATGTTATTACTAATCA 
               
               
                   
               
               
                 AAGAAGTATTGCGACAACGGTTAATTTGCGTGATGGACAGACTCTTTTACTCGGTGG 
               
               
                   
               
               
                 CCTCACTGATTATAAAAACACTTCTCAGGATTCTGGCGTACCGTTCCTGTCTAAAATC 
               
               
                   
               
               
                 CCTTTAATCGGCCTCCTGTTTAGCTCCCGCTCTGATTCTAACGAGGAAAGCACGTTAT 
               
               
                   
               
               
                 ACGTGCTCGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGC 
               
               
                   
               
               
                 GGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGC 
               
               
                   
               
               
                 TCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTC 
               
               
                   
               
               
                 TAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCA 
               
               
                   
               
               
                 AAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTT 
               
               
                   
               
               
                 TTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGG 
               
               
                   
               
               
                 AACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATT 
               
               
                   
               
               
                 TCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAAC 
               
               
                   
               
               
                 AAAATATTAACGTTTACAATTTAAATATTTGCTTATACAATCTTCCTGTTTTTGGGGC 
               
               
                   
               
               
                 TTTTCTGATTATCAACCGGGGTACATATGATTGACATGCTAGTTTTACGATTACCGTT 
               
               
                   
               
               
                 CATCGATTCTCTTGTTTGCTCCAGACTCTCAGGCAATGACCTGATAGCCTTTGTAGAG 
               
               
                   
               
               
                 ACCTCTCAAAAATAGCTACCCTCTCCGGCATGAATTTATCAGCTAGAACGGTTGAAT 
               
               
                   
               
               
                 ATCATATTGATGGTGATTTGACTGTCTCCGGCCTTTCTCACCCGTTTGAATCTTTACC 
               
               
                   
               
               
                 TACACATTACTCAGGCATTGCATTTAAAATATATGAGGGTTCTAAAAATTTTTATCCT 
               
               
                   
               
               
                 TGCGTTGAAATAAAGGCTTCTCCCGCAAAAGTATTACAGGGTCATAATGTTTTTGGT 
               
               
                   
               
               
                 ACAACCGATTTAGCTTTATGCTCTGAGGCTTTATTGCTTAATTTTGCTAATTCTTTGC 
               
               
                   
               
               
                 CTTGCCTGTATGATTTATTGGATGTTGCAATTCCTGATGCGGTATTTTCTCCTTACGC 
               
               
                   
               
               
                 ATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGC 
               
               
                   
               
               
                 CGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGG 
               
               
                   
               
               
                 CTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCA 
               
               
                   
               
               
                 TGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTG 
               
               
                   
               
               
                 ATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTG 
               
               
                   
               
               
                 GCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTC 
               
               
                   
               
               
                 AAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAA 
               
               
                   
               
               
                 AAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCA 
               
               
                   
               
               
                 TTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAA 
               
               
                   
               
               
                 GATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGAT 
               
               
                   
               
               
                 CCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCT 
               
               
                   
               
               
                 GCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCG 
               
               
                   
               
               
                 CATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCT 
               
               
                   
               
               
                 TACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATA 
               
               
                   
               
               
                 ACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCT 
               
               
                   
               
               
                 TTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTG 
               
               
                   
               
               
                 AATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAAC 
               
               
                   
               
               
                 AACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATT 
               
               
                   
               
               
                 AATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCC 
               
               
                   
               
               
                 GGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTAT 
               
               
                   
               
               
                 CATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGAC 
               
               
                   
               
               
                 GGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCT 
               
               
                   
               
               
                 CACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTG 
               
               
                   
               
               
                 ATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCT 
               
               
                   
               
               
                 CATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGA 
               
               
                   
               
               
                 AAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAA 
               
               
                   
               
               
                 ACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAAC 
               
               
                   
               
               
                 TCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCT 
               
               
                   
               
               
                 AGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCT 
               
               
                   
               
               
                 CGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTAC 
               
               
                   
               
               
                 CGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGG 
               
               
                   
               
               
                 GGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATAC 
               
               
                   
               
               
                 CTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACA 
               
               
                   
               
               
                 GGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGG 
               
               
                   
               
               
                 GGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCG 
               
               
                   
               
               
                 TCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACG 
               
               
                   
               
               
                 CGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCG 
               
               
                   
               
               
                 TTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTC 
               
               
                   
               
               
                 GCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCG 
               
               
                   
               
               
                 CCCAATACGCAAAC