Patent Publication Number: US-2022233719-A1

Title: Recombinant adeno-associated virus vectors to target medullary thyroid carcinoma

Description:
RELATED APPLICATIONS 
     This application is a continuation application of U.S. application Ser. No. 15/741,253, filed Dec. 30, 2017, entitled “RECOMBINANT ADENO-ASSOCIATED VIRUS VECTORS TO TARGET MEDULLARY THYROID CARCINOMA”, which is a National Stage Application of PCT/US2016/040687, filed Jul. 1, 2016, which claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application Ser. No. 62/188,191, filed Jul. 2, 2015, entitled “RECOMBINANT ADENO-ASSOCIATED VIRUS VECTORS TO TARGET MEDULLARY THYROID CARCINOMA”, the entire contents of each of which is incorporated by reference herein. 
    
    
     BACKGROUND OF THE INVENTION 
     Medullary thyroid carcinomas (MTC) originate in parafollicular, or C cells, of the thyroid. MTC often presents with metastatic disease and once metastasized, are incurable. 
     SUMMARY OF THE INVENTION 
     Aspects of the disclosure relate to nucleic acids, recombinant adeno-associated virus (rAAV) particles, compositions, and methods related to gene therapy for medullary thyroid carcinoma (MTC). 
     As described herein, it was found that a truncated calcitonin promoter was capable of driving higher expression levels in MTC cells than the full wild-type calcitonin promoter and was specific for MTC cells. Further it was determined that capsid-modified rAAV2 viral particles containing one or more mutations in surface-exposed tyrosine, serine, or threonine residues transduced MTC cells with higher efficiency than wild-type rAAV2 viral particles. Lastly, it was shown that capsid-modified rAAV2 viral particles encapsidating a rAAV nucleic acid vector containing the truncated calcitonin promoter were capable of transducing MTC cells in vivo. These results demonstrate that such rAAV particles and rAAV nucleic acid vectors are useful, e.g., to target MTC cells both in vitro and in vivo. 
     In some embodiments, aspects of the description relate to a nucleic acid comprising an expression construct containing a truncated calcitonin promoter operably linked to a coding sequence of a gene of interest. In some embodiments, the expression construct is flanked on each side by an inverted terminal repeat sequence. In some embodiments, the truncated calcitonin promoter comprises a proximal promoter region of a calcitonin gene having the coordinates −185 to +125, relative to the transcription start site of a calcitonin coding sequence of the calcitonin gene, and a tissue-specific enhancer region of the calcitonin gene having the coordinates −1080 to −860, relative to the transcription start site of the calcitonin coding sequence. In some embodiments, the truncated calcitonin promoter comprises the sequence of SEQ ID NO: 1. In some embodiments, the gene of interest is a selected gene that is therapeutically useful for treating cancer or other conditions (e.g., medullary thyroid cancer or other medullary thyroid condition). In some embodiments, the nucleic acid is a recombinant adeno-associated virus (rAAV) vector. In some embodiments, the nucleic acid is a single-stranded or self-complementary rAAV nucleic acid vector. In some embodiments, the gene of interest is CYP2B6. 
     In some embodiments, aspects of the description relate to a recombinant adeno-associated virus (rAAV) particle comprising a nucleic acid described herein. In some embodiments, the rAAV particle is an rAAV2 particle. In some embodiments, the rAAV2 particle comprises a modified capsid protein comprising a non-native amino acid substitution at a position that corresponds to a surface-exposed amino acid in a wild-type AAV2 capsid protein. 
     In some embodiments of an rAAV particle, the non-native amino acid substitution is selected from: 
     (a) a non-tyrosine amino acid at Y730, 
     (b) a non-serine amino acid at S662, 
     (c) a non-threonine amino acid at T491, 
     (d) a non-serine amino acid at S662 and a non-threonine amino acid at T491, 
     (e) a non-tyrosine amino acid at Y444, a non-tyrosine amino acid at Y500, and a non-tyrosine amino acid at Y730, or 
     (f) a non-tyrosine amino acid at Y444, a non-tyrosine amino acid at Y500, a non-tyrosine amino acid at Y730 and a non-threonine amino acid at T491. 
     In some embodiments of an rAAV particle, the non-native amino acid substitution is selected from: 
     (a) Y730F, 
     (b) S662V, 
     (c) T491V, 
     (d) S662V and T491V, 
     (e) Y444F, Y500F, and Y730F, or 
     (f) Y444F, Y500F, Y730F and T491V. 
     In some embodiments of an rAAV particle, the non-native amino acid substitution is selected from (a) Y730F and (e) Y444F, Y500F, and Y730F. 
     In some embodiments, aspects of the description provide a composition that includes a plurality of rAAV particles described herein. In some embodiments, a composition includes a pharmaceutically acceptable carrier. 
     In some embodiments, aspects of the description provide a method of delivering a nucleic acid to a medullary thyroid carcinoma cell by administering an rAAV particle or composition described herein to a medullary thyroid carcinoma cell. In some embodiments, the cell is a cell in a subject. In some embodiments, a method of treating or assisting in the treatment of medullary thyroid carcinoma includes administering an rAAV particle or composition described herein to a subject having medullary thyroid carcinoma. 
     In some embodiments, a method of delivering a nucleic acid to a medullary thyroid carcinoma cell includes administering to a medullary thyroid carcinoma cell a rAAV2 particle comprising:
         (a) a modified capsid protein comprising a non-native amino acid substitution at a position that corresponds to a surface-exposed amino acid in a wild-type AAV2 capsid protein; and   (b) a nucleic acid comprising an expression construct containing a promoter operably linked to a coding sequence of a gene of interest.       

     In some embodiments, the non-native amino acid substitution is selected from: 
     (a) a non-tyrosine amino acid at Y730, 
     (b) a non-serine amino acid at S662, 
     (c) a non-threonine amino acid at T491, 
     (d) a non-serine amino acid at S662 and a non-threonine amino acid at T491, 
     (e) a non-tyrosine amino acid at Y444, a non-tyrosine amino acid at Y500, and a non-tyrosine amino acid at Y730, or 
     (f) a non-tyrosine amino acid at Y444, a non-tyrosine amino acid at Y500, a non-tyrosine amino acid at Y730 and a non-threonine amino acid at T491. 
     In some embodiments, the non-native amino acid substitution is selected from: 
     (a) Y730F, 
     (b) S662V, 
     (c) T491V, 
     (d) S662V and T491V, 
     (e) Y444F, Y500F, and Y730F, or 
     (f) Y444F, Y500F, Y730F and T491V. 
     In some embodiments, the non-native amino acid substitution is selected from (a) Y730F and (e) Y444F, Y500F, and Y730F. 
     These and other aspects are described in more detail herein. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. 
         FIGS. 1A and 1B  are graphs showing evaluation of wild-type (WT) and capsid mutant AAV2 expression in the MTC cell line TT. ( FIG. 1A ) Quantification of transduction efficiency of wild-type AAV2 in HeLa vs. TT cells as measured by eGFP expression. ( FIG. 1B ) Quantification of transduction efficiency of capsid mutants compared to wild-type AAV2 in the TT cell line. All cells were analyzed at 48 h post-transduction at MOI of 2×10 3  vector genomes per cell. **P&lt;0.01 and ***P&lt;0.001 compared to wild-type AAV2 expression. 
         FIGS. 2A, 2B, and 2C  are a diagram and two graphs that show characterization of the calcitonin promoter and enhancer regions in MTC and non-MTC cell lines. ( FIG. 2A ) Schematic representation of the calcitonin promoter with enhancer and proximal promoter regions highlighted. The shaded area downstream represents the location of the transgene expressed. “E”, “P”, and “PS” denote sequence areas used in subsequent experiments. ( FIG. 2B ) Relative luciferase expression levels of the full-length calcitonin promoter from −1738 to +125 in MTC cell line TT, and 7 non-MTC cell lines. The data are shown as percentage of Firefly luciferase relative to co-transfected SV40- renilla  expression. ( FIG. 2C ) Evaluation of expression from truncated promoter and enhancer regions of the calcitonin promoter compared to the full length promoter. Proximal promoter regions P encompass −185 to +125, PS −140 to +125, and enhancer region −860 to −1080, with numbers representing single or repeated promoter or enhancer regions. n=3 for all experiments. ***P&lt;0.001 TT vs. other cell lines, **P&lt;0.01 compared to full-length calcitonin promoter. 
         FIGS. 3A, 3B, 3C and 3D  are a series of photographs and graphs showing flow cytometry analysis of dsAAV2-Y730E-CBA or -Calcitonin P1.E2 transduced cells. ( FIG. 3A ) Representative eGFP expression images of cell lines. ( FIG. 3B ) Quantification of eGFP-positive cells (n=4). ( FIGS. 3C-3D ) Flow cytometry images of infected cells—black represents untreated, grey mock infected, and green eGFP infected cells. All analyses were done at 48 hours post-transduction at an MOI of 2×10 3  vector genomes per cell. *P&lt;0.05, **P&lt;0.01, ***P&lt;0.001. 
         FIGS. 4A, 4B, 4C, and 4D  are a series of photographs that show histological analysis of rAAV-injected MTC xenograft tumors. ( FIG. 4A ) H&amp;E staining of tumor tissue demonstrated MTC morphology and vascularization of tumors. ( FIG. 4B ) Strong calcitonin expression was seen within the C cells of the MTC xenografts. ( FIG. 4C ) Positive immunohistochemical staining was observed in dsAAV2-Y730E-CP1.E2 injected tumors. ( FIG. 4D ) Expression of eGFP was absent in IgG controls. Scale bars represent 50 μm. 
         FIG. 5  is a graph showing relative change in GFP expression in cells infected with wild-type rAAV2, rAAV2(Y730F), rAAV2 (S662V), rAAV2(T491V), rAAV2(S662V+T491V), rAAV2(Y444F+Y500F+Y730F) (M3), and rAAV2(M3+T491V). 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Provided herein are nucleic acids, recombinant adeno-associated virus (rAAV) particles, compositions, and methods related in part to gene therapy for medullary thyroid carcinoma. 
     Medullary thyroid carcinomas (MTC) originate in parafollicular, or C cells, of the thyroid. MTC often presents with metastatic disease and once metastasized, are incurable. There remains a need for therapeutics for MTC. 
     The study described herein was used to determine if recombinant adeno-associated viral vectors (rAAV) could specifically target MTC. It was postulated that gene therapeutic approaches could target MTC cells. First, rAAV viral particles capable of targeting MTC cells were identified. In the MTC cell line TT, rAAV2 with mutated surface amino acids within the capsid proteins showed improved transduction compared to wild-type virus. Next, a truncated calcitonin promoter was used to achieve tissue-specific expression. Transgene expression from the truncated calcitonin promoter was specific to MTC cells compared to other non-thyroid cell lines in vitro. Finally, expression of a capsid-modified rAAV viral particle containing a nucleic acid vector comprising a truncated calcitonin promoter was evaluated in vivo in a mouse xenograft model of MTC. By assessing transduction in vivo, eGFP expression was observed following intratumor rAAV injections in a human MTC xenograft model. The results of the study described herein demonstrate that modified rAAV particles (e.g., rAAV2 containing one or more mutations in a surface-exposed residue in the capsid protein), optionally combined with a truncated calcitonin promoter, can be used to selectively target MTC. 
     Recombinant Adeno-Associated Virus (rAAV) Particles and Nucleic Acids 
     Aspects of the disclosure relate to recombinant AAV (rAAV) particles and nucleic acids. 
     In some embodiments, a nucleic acid is provided, the nucleic acid comprising an expression construct containing a truncated calcitonin promoter operably linked to a coding sequence of a gene of interest. As used herein, a truncated calcitonin promoter is a promoter that comprises enhancer and/or basal promoter elements from a calcitonin promoter but is not a full-length calcitonin promoter. In some embodiments, the truncated calcitonin promoter is less than 1.6 kb in length, less than 1.5 kb in length, less than 1.4 kb in length, less than 1.3 kb in length, less than 1.2 kb in length, less than 1.1 kb in length, less than 1 kb in length, or less than 900 bp in length. In some embodiments, the expression construct is flanked on each side by an inverted terminal repeat sequence. In some embodiments, the truncated calcitonin promoter is a human truncated calcitonin promoter. 
     In some embodiments, the truncated calcitonin promoter comprises a proximal promoter region of a calcitonin gene having the coordinates −185 to +125, relative to the transcription start site of a calcitonin coding sequence of the calcitonin gene, and a tissue-specific enhancer region of the calcitonin gene having the coordinates −1080 to −860, relative to the transcription start site of the calcitonin coding sequence. 
     Full Calcitonin Upstream Region 
       
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 3) 
               
               
                   
                 gatccggggctcattgtgcccaagatccgccatcca 
               
               
                   
               
               
                   
                 agccctgctctgcgcgcagcttgcctgtttcacgc 
               
               
                   
               
               
                   
                 tctgcgcctgacacgcgccggtgtcctcccgggcc 
               
               
                   
               
               
                   
                 agttccagtcccgggtcctgtggccgccctgccgg 
               
               
                   
               
               
                   
                 cggaccctgcggagagcgagtcttagatacccagt 
               
               
                   
               
               
                   
                 ccccagccccgagttgttattccctcgctgtagtt 
               
               
                   
               
               
                   
                 aagaaggaggagatcaattaagggcatcttagaag 
               
               
                   
               
               
                   
                 ttaggcgttcccgctgcctcctttgagcacggagg 
               
               
                   
               
               
                   
                 ccaccaaccccctagggggaagagatgtagcgcga 
               
               
                   
               
               
                   
                 ggcaggggtgtcgtgctaagaaatttcgacgcttc 
               
               
                   
               
               
                   
                 tggggactgaggacaaaggtgcggacacgaccccg 
               
               
                   
               
               
                   
                 gggtacctggagttccgtgactcgcgccacggacg 
               
               
                   
               
               
                   
                 gcacacctaggggctaatttctgctctgcctcaaa 
               
               
                   
               
               
                   
                 gaacctcaagctagagtccttgcctccgcccacag 
               
               
                   
               
               
                   
                 ccccgggatgccgctgctgcgctcaccgcacaggc 
               
               
                   
               
               
                   
                 agcgcccggaccggctgcagcagatcgcgcgctgc 
               
               
                   
               
               
                   
                 gcgttccaccgggagatggtggagacgctgaaaag 
               
               
                   
               
               
                   
                 cttctttcttgccactctggacgctgtgggcggca 
               
               
                   
               
               
                   
                 agcgccttagtccctacctctgctgagctgaacgc 
               
               
                   
               
               
                   
                 tcaggcacagtggaactgaaacccggttctgcggg 
               
               
                   
               
               
                   
                 atgtgagagctgttgaggtcacgcgtaattgggtg 
               
               
                   
               
               
                   
                 tgatggagggcgcctgttcgtgatgtgtgcaggtt 
               
               
                   
               
               
                   
                 tgatgcaagcaggtcatcgtcgtgcgagtgtgtgg 
               
               
                   
               
               
                   
                 atgcgaccgcccgagagactcggaggcaggcttgg 
               
               
                   
               
               
                   
                 gacacgtttgagtgaacacctcaggatactcttct 
               
               
                   
               
               
                   
                 ggccagtatctgttttttagtgtctgtgattcaga 
               
               
                   
               
               
                   
                 gtgggcacatgttgggagacagtaatgggtttggg 
               
               
                   
               
               
                   
                 tgtgtgtaaatgagtgtgaccggaagc gagtgtga   
               
               
                   
               
               
                   
                 
                   gcttgatctaggcagggaccacacagcactgtcac 
                 
               
               
                   
               
               
                   
                 
                   acctgcctgctctttagtagaggactgaagtgcgg 
                 
               
               
                   
               
               
                   
                 
                   gggtgggggtacggggccggaatagaatgtctctg 
                 
               
               
                   
               
               
                   
                 
                   ggacatcttggcaaacagcagccggaagcaaaggg 
                 
               
               
                   
               
               
                   
                 
                   gcagctgtgcaaacggctcaggcaggtgatggatg 
                 
               
               
                   
               
               
                   
                 
                   gcagggtaggaagggggaggtccagaggtctggat 
                 
               
               
                   
               
               
                   
                   ggaggcttc cgcatctgtaccttgcaactcacccc 
               
               
                   
               
               
                   
                 tcaggcccagcaggtcatcggccccctcctcacac 
               
               
                   
               
               
                   
                 atgtaatggatctgaagagtaccccgggacagtcc 
               
               
                   
               
               
                   
                 ggggagatggagattcggaaagtatccatggagat 
               
               
                   
               
               
                   
                 cttacagaatcccctgtgcggaccaggaaactctt 
               
               
                   
               
               
                   
                 gtagatccctgcctatctgaggcccaggcgctggg 
               
               
                   
               
               
                   
                 ctgtttctcacaatattccttcaagatgagattgt 
               
               
                   
               
               
                   
                 ggtccccatttcaaagatgagtacactgagcctct 
               
               
                   
               
               
                   
                 gtgaagttacttgcccatgatcacacaaccaggaa 
               
               
                   
               
               
                   
                 ttgggccaactgtaattgaactcctgtctaacaaa 
               
               
                   
               
               
                   
                 gttcttgctcccagctccgtctcttgtttcccacg 
               
               
                   
               
               
                   
                 agccctggccctctgtgggtaataccagctactgg 
               
               
                   
               
               
                   
                 agtcagatttcttgggcccagaacccacccttagg 
               
               
                   
               
               
                   
                 ggcattaacctttaaaatctcacttgggcaggggt 
               
               
                   
               
               
                   
                 ctgggatcagagttggaagagtccctacaatcctg 
               
               
                   
               
               
                   
                 gaccctttccgccaaatcgtgaaaccaggggtgga 
               
               
                   
               
               
                   
                 gtggggcgagggttcaaaaccaggccggactgaga 
               
               
                   
               
               
                   
                 ggtgaaattcaccatgacgtcaaactgccctcaaa 
               
               
                   
               
               
                   
                 ttcccgctcactttaagggcgttacttgttggtgc 
               
               
                   
               
               
                   
                 ccccaccatcccccaccat ttccatcaatgacctc   
               
               
                   
               
               
                   
                 
                   aatgcaaatacaagtgggacggtcctgctggatcc 
                 
               
               
                   
               
               
                   
                 
                   tccaggttctggaagcatgagggtgacgcaaccca 
                 
               
               
                   
               
               
                   
                 
                   ggggcaaaggacccctccgcccattggttgctgtg 
                 
               
               
                   
               
               
                   
                 
                   cactggcggaactttcccgacccacagcggcggga 
                 
               
               
                   
               
               
                   
                 
                   ataagagcagtcgctggcgctgggaggc 
                   
                   gag 
                 
               
               
                   
               
               
                   
                 
                   acactgcccagcccaagtgtcgccgccgcttccac 
                 
               
               
                   
               
               
                   
                 
                   agggctctggctggacgccgccgccgccgctgcca 
                 
               
               
                   
               
               
                   
                 
                   ccgcctctgatccaagccacctcccgccaggtgag 
                 
               
               
                   
               
               
                   
                 
                   ccccgagatcctggctcaggtatatgtctctccct 
                 
               
               
                   
               
               
                   
                 
                   cc 
                 
               
               
                   
                     = transcription start site 
               
               
                   
                 italicized text = P1 
               
               
                   
                 Underlined text = E 
               
            
           
         
       
     
     In some embodiments, the truncated calcitonin promoter comprises or consists of the sequence of SEQ ID NO: 1. In some embodiments, the truncated calcitonin promoter comprises the sequence of SEQ ID NO: 1 and is no more than 1000, 900, or 800 nucleotides in length. In some embodiments, the truncated calcitonin promoter comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to the sequence of SEQ ID NO: 1 and optionally has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, or more of the activity (e.g., promotion of transcription of a gene, such as in a medullary thyroid carcinoma cell) compared to a truncated calcitonin promoter having the sequence of SEQ ID NO: 1: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 1) 
               
               
                   
                 TTCCATCAATGACCTCAATGCAAATACAAGTGGGAC 
               
               
                   
               
               
                   
                 GGTCCTGCTGGATCCTCCAGGTTCTGGAAGCATGA 
               
               
                   
               
               
                   
                 GGGTGACGCAACCCAGGGGCAAAGGACCCCTCCGC 
               
               
                   
               
               
                   
                 CCATTGGTTGCTGTGCACTGGCGGAACTTTCCCGA 
               
               
                   
               
               
                   
                 CCCACAGCGGCGGGAATAAGAGCAGTCGCTGGCGC 
               
               
                   
               
               
                   
                 TGGGAGGCATCAGAGACACTGCCCAGCCCAAGTGT 
               
               
                   
               
               
                   
                 CGCCGCCGCTTCCACAGGGCTCTGGCTGGACGCCG 
               
               
                   
               
               
                   
                 CCGCCGCCGCTGCCACCGCCTCTGATCCAAGCCAC 
               
               
                   
               
               
                   
                 CTCCCGCCA 
               
            
           
         
       
     
     The coding sequence of a gene of interest may be any coding sequence of any gene that is appropriate for use in gene therapy. In some embodiments, the gene of interest is a gene that encodes a cytochrome protein. In some embodiments, the gene of interest is a CYP2B6 gene (e.g., a human CYP2B6 gene), which encodes the protein Cytochrome P450 2B6 (e.g., human Cytochrome P450 2B6). 
     
       
         
           
               
               
            
               
                   
                 An example of a CYP2B6 cDNA 
               
               
                   
                 sequence is as follows: 
               
               
                   
                 (SEQ ID NO: 4) 
               
               
                   
                 caggaccatggaactcagcgtcctcctcttccttg 
               
               
                   
               
               
                   
                 cactcctcacaggactcttgctactcctggttcag 
               
               
                   
               
               
                   
                 cgccaccctaacacccatgaccgcctcccaccagg 
               
               
                   
               
               
                   
                 gccccgccctctgccccttttgggaaaccttctgc 
               
               
                   
               
               
                   
                 agatggatagaagaggcctactcaaatcctttctg 
               
               
                   
               
               
                   
                 aggttccgagagaaatatggggacgtcttcacggt 
               
               
                   
               
               
                   
                 acacctgggaccgaggcccgtggtcatgctgtgtg 
               
               
                   
               
               
                   
                 gagtagaggccatacgggaggcccttgtggacaag 
               
               
                   
               
               
                   
                 gctgaggccttctctggccggggaaaaatcgccat 
               
               
                   
               
               
                   
                 ggtcgacccattcttccggggatatggtgtgatct 
               
               
                   
               
               
                   
                 ttgccaatggaaaccgctggaaggtgcttcggcga 
               
               
                   
               
               
                   
                 ttctctgtgaccactatgagggacttcgggatggg 
               
               
                   
               
               
                   
                 aaagcggagtgtggaggagcggattcaggaggagg 
               
               
                   
               
               
                   
                 ctcagtgtctgatagaggagcttcggaaatccaag 
               
               
                   
               
               
                   
                 ggggccctcatggaccccaccttcctcttccagtc 
               
               
                   
               
               
                   
                 cattaccgccaacatcatctgctccatcgtctttg 
               
               
                   
               
               
                   
                 gaaaacgattccactaccaagatcaagagttcctg 
               
               
                   
               
               
                   
                 aagatgctgaacttgttctaccagactttttcact 
               
               
                   
               
               
                   
                 catcagctctgtattcggccagctgtttgagctct 
               
               
                   
               
               
                   
                 tctctggcttcttgaaatactttcctggggcacac 
               
               
                   
               
               
                   
                 aggcaagtttacaaaaacctgcaggaaatcaatgc 
               
               
                   
               
               
                   
                 ttacattggccacagtgtggagaagcaccgtgaaa 
               
               
                   
               
               
                   
                 ccctggaccccagcgcccccaaggacctcatcgac 
               
               
                   
               
               
                   
                 acctacctgctccacatggaaaaagagaaatccaa 
               
               
                   
               
               
                   
                 cgcacacagtgaattcagccaccagaacctcaacc 
               
               
                   
               
               
                   
                 tcaacacgctctcgctcttctttgctggcactgag 
               
               
                   
               
               
                   
                 accaccagcaccactctccgctacggcttcctgct 
               
               
                   
               
               
                   
                 catgctcaaataccctcatgttgcagagagagtct 
               
               
                   
               
               
                   
                 acagggagattgaacaggtgattggcccacatcgc 
               
               
                   
               
               
                   
                 cctccagagcttcatgaccgagccaaaatgccata 
               
               
                   
               
               
                   
                 cacagaggcagtcatctatgagattcagagatttt 
               
               
                   
               
               
                   
                 ccgaccttctccccatgggtgtgccccacattgtc 
               
               
                   
               
               
                   
                 acccaacacaccagcttccgagggtacatcatccc 
               
               
                   
               
               
                   
                 caaggacacagaagtatttctcatcctgagcactg 
               
               
                   
               
               
                   
                 ctctccatgacccacactactttgaaaaaccagac 
               
               
                   
               
               
                   
                 gccttcaatcctgaccactttctggatgccaatgg 
               
               
                   
               
               
                   
                 ggcactgaaaaagactgaagcttttatccccttct 
               
               
                   
               
               
                   
                 ccttagggaagcggatttgtcttggtgaaggcatc 
               
               
                   
               
               
                   
                 gcccgtgcggaattgttcctcttcttcaccaccat 
               
               
                   
               
               
                   
                 cctccagaacttctccatggccagccccgtggccc 
               
               
                   
               
               
                   
                 cagaagacatcgatctgacaccccaggagtgtggt 
               
               
                   
               
               
                   
                 gtgggcaaaatacccccaacataccagatccgctt 
               
               
                   
               
               
                   
                 cctgccccgctgaaggggctgagggaagggggtca 
               
               
                   
               
               
                   
                 aaggattccagggtcatttcagtgttccccgcctc 
               
               
                   
               
               
                   
                 tgttagacaatggctctgactcccccgcaacttcc 
               
               
                   
               
               
                   
                 ttgcctctgagagaccttgctacaagccagcttcc 
               
               
                   
               
               
                   
                 ttcccctccatggcaccagttgtctgaggtcacat 
               
               
                   
               
               
                   
                 tgcaagtgagtgcaggagtgagattatcgaaaatt 
               
               
                   
               
               
                   
                 ataatatacaaaatcatatatatatatatgttctt 
               
               
                   
               
               
                   
                 gttttttgagacagagtctcacactgttgcccagg 
               
               
                   
               
               
                   
                 ctggagtgcagtggcgtgatctcggctcactgcaa 
               
               
                   
               
               
                   
                 cctccacccccggggatcaagcaactctcctgcct 
               
               
                   
               
               
                   
                 cagcctccctagtagctgggattacaggcatgcac 
               
               
                   
               
               
                   
                 taccacgcttggctaatttttgttatttttagtag 
               
               
                   
               
               
                   
                 agatggggtttcactgtgtaggccaggctggtctc 
               
               
                   
               
               
                   
                 gaactcctgaactcaagtgattcacccaccttagc 
               
               
                   
               
               
                   
                 ctcccaaagtgctgggattacaggcgtgagtcacc 
               
               
                   
               
               
                   
                 gtgcccagccatgtatatatataattttaaaaatt 
               
               
                   
               
               
                   
                 aagctgaaattcacataacataaaattagctgttt 
               
               
                   
               
               
                   
                 taaagttgtaaaatttagtggcgtgtggttcattc 
               
               
                   
               
               
                   
                 acaaagctgtacaaccaccaccatctagttccaaa 
               
               
                   
               
               
                   
                 cattttctttttttctgagatggagtctcactctg 
               
               
                   
               
               
                   
                 tcacccaggttcgagttcagtggtgccatctcttg 
               
               
                   
               
               
                   
                 tccactgcaacctccacatcctgggttcaagtgat 
               
               
                   
               
               
                   
                 tctcctgcctcagcctctggaggagctggtatcac 
               
               
                   
               
               
                   
                 aggcgtcccccaccacgcctggctaaattttgtat 
               
               
                   
               
               
                   
                 ttttaggtggtcttgaactcctgatgtcaggtgat 
               
               
                   
               
               
                   
                 tctcctagctccaaatgttttcattatctctcccc 
               
               
                   
               
               
                   
                 caacaaaacccatacctatcaagctgtcactcccc 
               
               
                   
               
               
                   
                 ataccccattctctttttcatctcggcccctgtca 
               
               
                   
               
               
                   
                 atctggtttttgtcactatggacttaccaattctg 
               
               
                   
               
               
                   
                 aatatttcccataaacagaatcatacaatatttga 
               
               
                   
               
               
                   
                 ttttttttttttttttttgaaactaagccttgctc 
               
               
                   
               
               
                   
                 tgtctcccaggctggagtgctatggtgcaattttt 
               
               
                   
               
               
                   
                 gttcactgcaacctctgccttccaagatcaagaga 
               
               
                   
               
               
                   
                 ttctccagtctcagctcccaagtagctgggattac 
               
               
                   
               
               
                   
                 aggcatgtactaccatgcctggctaattttcttgt 
               
               
                   
               
               
                   
                 agttttagtagggacatgttggccaggctggtggt 
               
               
                   
               
               
                   
                 gagctcctggcctcaggtgatccacccacctcagt 
               
               
                   
               
               
                   
                 gttccaaagtgctgatattacaggcataatatgtg 
               
               
                   
               
               
                   
                 atcttttgtgtctggttgctttcatgttgaattgc 
               
               
                   
               
               
                   
                 tatttttgaggttcatgcctgttgtagaccacagt 
               
               
                   
               
               
                   
                 cacacactgctgtagtcttccccagtcctcattcc 
               
               
                   
               
               
                   
                 cagctgcctcttcctactgcttccgtctatcaaaa 
               
               
                   
               
               
                   
                 agcccccttggcccaggttccctgagctgtgggat 
               
               
                   
               
               
                   
                 tctgcactggtgctttggattccctgatatgttcc 
               
               
                   
               
               
                   
                 ttcaaatctgctgagaattaaattaaacatcttct 
               
               
                   
               
               
                   
                 aaagcctgacctccccacgtc 
               
            
           
         
       
     
     In some embodiments, the gene of interest is a detectable reporter gene. Exemplary detectable reporter genes include genes encoding a fluorescent protein (e.g., GFP, YFP, RFP, CFP, BFP and variants thereof), luciferase, β-galactosidase, chloramphenicol acetyltransferase and/or alkaline phosphatase. 
     In some embodiments, the expression construct comprises one or more regions comprising a sequence that facilitates expression of the coding sequence of the gene of interest, e.g., expression control sequences operably linked to the coding sequence. Non-limiting examples of expression control sequences include promoters, insulators, silencers, response elements, introns, enhancers, initiation sites, termination signals, and poly(A) tails. Any combination of such control sequences is contemplated herein (e.g., a promoter and an enhancer). In some embodiments, the promoter is a truncated calcitonin promoter as described herein. 
     In some embodiments, the nucleic acid is a plasmid (e.g., a circular nucleic acid comprising one or more of an origin of replication, a selectable marker, and a reporter gene). In some embodiments, a nucleic acid described herein, such as a plasmid, may also contain marker or reporter genes, e.g., LacZ or a fluorescent protein, and an origin of replication. In some embodiments, the plasmid is transfected into a producer cell that produces AAV particles containing the expression construct. 
     In some embodiments, the nucleic acid is a nucleic acid vector such as a recombinant adeno-associated virus (rAAV) vector. Exemplary rAAV nucleic acid vectors useful according to the disclosure include single-stranded (ss) or self-complementary (sc) AAV nucleic acid vectors. 
     In some embodiments, a recombinant rAAV particle comprises a nucleic acid vector, such as a single-stranded (ss) or self-complementary (sc) AAV nucleic acid vector. In some embodiments, the nucleic acid vector contains an expression construct as described herein and one or more regions comprising inverted terminal repeat (ITR) sequences (e.g., wild-type ITR sequences or engineered ITR sequences) flanking the expression construct. In some embodiments, the nucleic acid is encapsidated by a viral capsid. 
     Accordingly, in some embodiments, a rAAV particle comprises a viral capsid and a nucleic acid vector as described herein, which is encapsidated by the viral capsid. In some embodiments, the viral capsid comprises 60 capsid protein subunits comprising VP1, VP2 and VP3. In some embodiments, the VP1, VP2, and VP3 subunits are present in the capsid at a ratio of approximately 1:1:10, respectively. 
     The ITR sequences of a nucleic acid or nucleic acid vector described herein can be derived from any AAV serotype (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) or can be derived from more than one serotype. In some embodiments of the nucleic acid or nucleic acid vector provided herein, the ITR sequences are derived from AAV2. ITR sequences and plasmids containing ITR sequences are known in the art and commercially available (see, e.g., products and services available from Vector Biolabs, Philadelphia, Pa.; Cellbiolabs, San Diego, Calif.; Agilent Technologies, Santa Clara, Clif.; and Addgene, Cambridge, Mass.; and Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein. Kessler P D, Podsakoff G M, Chen X, McQuiston S A, Colosi P C, Matelis L A, Kurtzman G J, Byrne B J. Proc Natl Acad Sci USA. 1996 November 26; 93(24):14082-7; and Curtis A. Machida. Methods in Molecular Medicine™. Viral Vectors for Gene Therapy Methods and Protocols. 10.1385/1-59259-304-6:201 © Humana Press Inc. 2003. Chapter 10. Targeted Integration by Adeno-Associated Virus. Matthew D. Weitzman, Samuel M. Young Jr., Toni Cathomen and Richard Jude Samulski; U.S. Pat. Nos. 5,139,941 and 5,962,313, all of which are incorporated herein by reference). An exemplary AAV2 ITR sequence is shown below. 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 5) 
               
               
                   
                 TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCAC 
               
               
                   
               
               
                   
                 TGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGG 
               
               
                   
               
               
                   
                 GCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCG 
               
               
                   
               
               
                   
                 CGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGG 
               
               
                   
               
               
                   
                 TTCCT 
               
            
           
         
       
     
     In some embodiments, the expression construct is no more than 7 kilobases, no more than 6 kilobases, no more than 5 kilobases, no more than 4 kilobases, or no more than 3 kilobases in size. In some embodiments, the expression construct is between 4 and 7 kilobases in size. 
     The rAAV particle may be of any AAV serotype (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), including any derivative (including non-naturally occurring variants of a serotype) or pseudotype. In some embodiments, the rAAV particle is an rAAV2 particle. Non-limiting examples of derivatives and pseudotypes include AAV2-AAV3 hybrid, AAVrh.10, AAVhu.14, AAV3a/3b, AAVrh32.33, AAV-HSC15, AAV-HSC17, AAVhu.37, AAVrh.8, CHt-P6, AAV2.5, AAV6.2, AAV2i8, AAV-HSC15/17, AAVM41, AAV9.45, AAV6(Y445F/Y731F), AAV2.5T, AAV-HAE1/2, AAV clone 32/83, AAVShH10, AAV2 (Y-&gt;F), AAV8 (Y733F), AAV2.15, AAV2.4, AAVM41, and AAVr3.45. Such AAV serotypes and derivatives/pseudotypes, and methods of producing such derivatives/pseudotypes are known in the art (see, e.g., Mol Ther. 2012 April; 20(4):699-708. doi: 10.1038/mt.2011.287. Epub 2012 Jan. 24. The AAV vector toolkit: poised at the clinical crossroads. Asokan Al, Schaffer D V, Samulski R J.). 
     In some embodiments, the rAAV particle comprises a capsid that includes modified capsid proteins (e.g., capsid proteins comprising a modified VP3 region). Methods of producing modified capsid proteins are known in the art (see, e.g., U.S Patent Publication Number US20130310443, which is incorporated herein by reference in its entirety). In some embodiments, the rAAV particle comprises a modified capsid protein comprising a non-native amino acid substitution at a position that corresponds to a surface-exposed amino acid in a wild-type capsid protein (e.g., wild-type AAV2 capsid protein, such as SEQ ID NO: 2). In some embodiments, the rAAV particle comprises a modified capsid protein comprising a non-tyrosine amino acid (e.g., a phenylalanine) at a position that corresponds to a surface-exposed tyrosine amino acid in a wild-type capsid protein, a non-threonine amino acid (e.g., a valine) at a position that corresponds to a surface-exposed threonine amino acid in the wild-type capsid protein, a non-lysine amino acid (e.g., a glutamic acid) at a position that corresponds to a surface-exposed lysine amino acid in the wild-type capsid protein, a non-serine amino acid (e.g., a valine) at a position that corresponds to a surface-exposed serine amino acid in the wild-type capsid protein, or a combination thereof. Exemplary surface-exposed tyrosine amino acids include positions that correspond to Y252, Y272, Y444, Y500, Y700, Y704, or Y730 of the wild-type AAV2 capsid protein. Exemplary surface-exposed serine amino acids include positions that correspond to S261, S264, S267, S276, S384, S458, S468, S492, S498, S578, S658, S662, S668, S707, or S721 of the wild-type AAV2 capsid protein. Exemplary surface-exposed threonine amino acids include positions that correspond to T251, T329, T330, T454, T455, T503, T550, T592, T581, T597, T491, T671, T659, T660, T701, T713, or T716 of the wild-type AAV2 capsid protein. Exemplary surface-exposed lysine amino acids include positions that correspond to K258, K321, K459, K490, K507, K527, K572, K532, K544, K549, K556, K649, K655, K665, or K706 of the wild-type AAV2 capsid protein. 
     An exemplary, non-limiting wild-type AAV2 capsid protein sequence is provided below. 
     Exemplary Wild-Type AAV2 Capsid Protein 
       
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 2) 
               
               
                   
                   1 MAADGYLPDW LEDTLSEGIR QWWKLKPGPP 
               
               
                   
               
               
                   
                     PPKPAERHKD DSRGLVLPGY 
               
               
                   
               
               
                   
                  51 KYLGPFNGLD KGEPVNEADA AALEHDKAYD 
               
               
                   
               
               
                   
                     RQLDSGDNPY LKYNHADAEF 
               
               
                   
               
               
                   
                 101 QERLKEDTSF GGNLGRAVFQ AKKRVLEPLG 
               
               
                   
               
               
                   
                     LVEEPVKTAP GKKRPVEHSP 
               
               
                   
               
               
                   
                 151 VEPDSSSGTG KAGQQPARKR LNFGQTGDAD 
               
               
                   
               
               
                   
                     SVPDPQPLGQ PPAAPSGLGT 
               
               
                   
               
               
                   
                 201 NTMATGSGAP MADNNEGADG VGNSSGNWHC 
               
               
                   
               
               
                   
                     DSTWMGDRVI TTSTRTWALP 
               
               
                   
               
               
                   
                 251 TYNNHLYKQI SSQSGASNDN HYFGYSTPWG 
               
               
                   
               
               
                   
                     YFDFNRFHCH FSPRDWQRLI 
               
               
                   
               
               
                   
                 301 NNNWGFRPKR LNFKLFNIQV KEVTQNDGTT 
               
               
                   
               
               
                   
                     TIANNLTSTV QVFTDSEYQL 
               
               
                   
               
               
                   
                 351 PYVLGSAHQG CLPPFPADVF MVPQYGYLTL 
               
               
                   
               
               
                   
                     NNGSQAVGRS SFYCLEYFPS 
               
               
                   
               
               
                   
                 401 QMLRTGNNFT FSYTFEDVPF HSSYAHSQSL 
               
               
                   
               
               
                   
                     DRLMNPLIDQ YLYYLSRTNT 
               
               
                   
               
               
                   
                 451 PSGTTTQSRL QFSQAGASDI RDQSRNWLPG 
               
               
                   
               
               
                   
                     PCYRQQRVSK TSADNNNSEY 
               
               
                   
               
               
                   
                 501 SWTGATKYHL NGRDSLVNPG PAMASHKDDE 
               
               
                   
               
               
                   
                     EKFFPQSGVL IFGKQGSEKT 
               
               
                   
               
               
                   
                 551 NVDIEKVMIT DEEEIRTTNP VATEQYGSVS 
               
               
                   
               
               
                   
                     TNLQRGNRQA ATADVNTQGV 
               
               
                   
               
               
                   
                 601 LPGMVWQDRD VYLQGPIWAK IPHTDGHFHP 
               
               
                   
               
               
                   
                     SPLMGGFGLK HPPPQILIKN 
               
               
                   
               
               
                   
                 651 TPVPANPSTT FSAAKFASFI TQYSTGQVSV 
               
               
                   
               
               
                   
                     EIEWELQKEN SKRWNPEIQY 
               
               
                   
               
               
                   
                 701 TSNYNKSVNV DFTVDTNGVY SEPRPIGTRY 
               
               
                   
               
               
                   
                     LTRNL* 
               
            
           
         
       
     
     In some embodiments, the non-native amino acid substitution in the capsid protein is a non-native amino acid substitution in a AAV2 capsid protein selected from: 
     (a) a non-tyrosine amino acid at Y730, 
     (b) a non-serine amino acid at S662, 
     (c) a non-threonine amino acid at T491, 
     (d) a non-serine amino acid at S662 and a non-threonine amino acid at T491, 
     (e) a non-tyrosine amino acid at Y444, a non-tyrosine amino acid at Y500, and a non-tyrosine amino acid at Y730, or 
     (f) a non-tyrosine amino acid at Y444, a non-tyrosine amino acid at Y500, a non-tyrosine amino acid at Y730 and a non-threonine amino acid at T491. 
     In some embodiments, the non-native amino acid substitution in the capsid protein is a non-native amino acid substitution in a AAV2 capsid protein selected from: 
     (a) Y730F, 
     (b) S662V, 
     (c) T491V, 
     (d) S662V and T491V, 
     (e) Y444F, Y500F, and Y730F, or 
     (f) Y444F, Y500F, Y730F and T491V. In some embodiments, the non-native amino acid substitution is Y730F in an AAV2 capsid protein. In some embodiments, the non-native amino acid substitution is S662V in an AAV2 capsid protein. In some embodiments, the non-native amino acid substitution is T491V in an AAV2 capsid protein. In some embodiments, the non-native amino acid substitution is S662V and T491V in an AAV2 capsid protein. In some embodiments, the non-native amino acid substitution is Y444F, Y500F, and Y730F in an AAV2 capsid protein. In some embodiments, the non-native amino acid substitution is Y444F, Y500F, Y730F and T491V in an AAV2 capsid protein. 
     Methods of producing rAAV particles and nucleic acid vectors are also known in the art and commercially available (see, e.g., Zolotukhin et al. Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors. Methods 28 (2002) 158-167; and U.S. Patent Publication Numbers US20070015238 and US20120322861, which are incorporated herein by reference; and plasmids and kits available from ATCC and Cell Biolabs, Inc.). For example, the nucleic acid vector (e.g., as a plasmid) may be combined with one or more helper plasmids, e.g., that contain a rep gene (e.g., encoding Rep78, Rep68, Rep52 and Rep40) and a cap gene (encoding VP1, VP2, and VP3), and transfected into a producer cell line such that the rAAV particle can be packaged and subsequently purified. 
     In some embodiments, the one or more helper plasmids includes a first helper plasmid comprising a rep gene and a cap gene and a second helper plasmid comprising other genes that assist in AAV production, such as a Ela gene, a E1b gene, a E4 gene, a E2a gene, and a VA gene. In some embodiments, the rep gene is a rep gene derived from AAV2 and the cap gene is derived from AAVS. Helper plasmids, and methods of making such plasmids, are known in the art and commercially available (see, e.g., pDM, pDG, pDP1rs, pDP2rs, pDP3rs, pDP4rs, pDP5rs, pDP6rs, pDG(R484E/R585E), and pDP8.ape plasmids from PlasmidFactory, Bielefeld, Germany; other products and services available from Vector Biolabs, Philadelphia, Pa.; Cellbiolabs, San Diego, Calif.; Agilent Technologies, Santa Clara, Ca; and Addgene, Cambridge, Mass.; pxx6; Grimm et al. (1998), Novel Tools for Production and Purification of Recombinant Adenoassociated Virus Vectors, Human Gene Therapy, Vol. 9, 2745-2760; Kern, A. et al. (2003), Identification of a Heparin-Binding Motif on Adeno-Associated Virus Type 2 Capsids, Journal of Virology, Vol. 77, 11072-11081.; Grimm et al. (2003), Helper Virus-Free, Optically Controllable, and Two-Plasmid-Based Production of Adeno-associated Virus Vectors of Serotypes 1 to 6, Molecular Therapy, Vol. 7, 839-850; Kronenberg et al. (2005), A Conformational Change in the Adeno-Associated Virus Type 2 Capsid Leads to the Exposure of Hidden VP1 N Termini, Journal of Virology, Vol. 79, 5296-5303; and Moullier, P. and Snyder, R. O. (2008), International efforts for recombinant adenoassociated viral vector reference standards, Molecular Therapy, Vol. 16, 1185-1188). 
     An exemplary, non-limiting, rAAV particle production method is described next. One or more helper plasmids are produced or obtained, which comprise rep and cap ORFs for the desired AAV serotype and the adenoviral VA, E2A (DBP), and E4 genes under the transcriptional control of their native promoters. HEK293 cells (available from ATCC®) are transfected via CaPO4-mediated transfection, lipids or polymeric molecules such as Polyethylenimine (PEI) with the helper plasmid(s) and a plasmid containing a nucleic acid vector described herein. Alternatively, in another example, Sf9-based producer stable cell lines are infected with a single recombinant baculovirus containing the nucleic acid vector. As a further alternative, in another example HEK293 or BHK cell lines are infected with a HSV containing the nucleic acid vector and optionally one or more helper HSVs containing rep and cap ORFs as described herein and the adenoviral VA, E2A (DBP), and E4 genes under the transcriptional control of their native promoters. The HEK293, BHK, or Sf9 cells are then incubated for at least 60 hours to allow for rAAV particle production. The rAAV particles can then be purified using any method known in the art or described herein, e.g., by iodixanol step gradient, CsCl gradient, chromatography, or polyethylene glycol (PEG) precipitation. 
     The disclosure also contemplates host cells that comprise at least one of the disclosed rAAV particles, expression constructs, or nucleic acid vectors. Such host cells include mammalian host cells, with human host cells being preferred, and may be either isolated, in cell or tissue culture. In the case of genetically modified animal models (e.g., a mouse), the transformed host cells may be comprised within the body of a non-human animal itself. 
     Compositions 
     Aspects of the disclosure relate to compositions comprising rAAV particles or nucleic acids described herein. In some embodiments, rAAV particles described herein are added to a composition, e.g., a pharmaceutical composition. 
     In some embodiments, the composition comprises a pharmaceutically acceptable carrier. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the rAAV particle is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum oil such as mineral oil, vegetable oil such as peanut oil, soybean oil, and sesame oil, animal oil, or oil of synthetic origin. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers. Non-limiting examples of pharmaceutically acceptable carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline, syrup, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, polyacrylic acids, lubricating agents (such as talc, magnesium stearate, and mineral oil), wetting agents, emulsifying agents, suspending agents, preserving agents (such as methyl-, ethyl-, and propyl-hydroxy-benzoates), and pH adjusting agents (such as inorganic and organic acids and bases). Other examples of carriers include phosphate buffered saline, HEPES-buffered saline, and water for injection, any of which may be optionally combined with one or more of calcium chloride dihydrate, disodium phosphate anhydrous, magnesium chloride hexahydrate, potassium chloride, potassium dihydrogen phosphate, sodium chloride, or sucrose. Other examples of carriers that might be used include saline (e.g., sterilized, pyrogen-free saline), saline buffers (e.g., citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins (for example, serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, and glycerol. USP grade carriers and excipients are particularly useful for delivery of rAAV particles to human subjects. Such compositions may further optionally comprise a liposome, a lipid, a lipid complex, a microsphere, a microparticle, a nanosphere, or a nanoparticle, or may be otherwise formulated for administration to the cells, tissues, organs, or body of a subject in need thereof. Methods for making such compositions are well known and can be found in, for example, Remington: The Science and Practice of Pharmacy, 22 nd  edition, Pharmaceutical Press, 2012. 
     Typically, such compositions may contain at least about 0.1% of the therapeutic agent (e.g., rAAV particle) or more, although the percentage of the active ingredient(s) may, of course, be varied and may conveniently be between about 1 or 2% and about 70% or 80% or more of the weight or volume of the total formulation. Naturally, the amount of therapeutic agent(s) (e.g., rAAV particle) in each therapeutically-useful composition may be prepared in such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable. 
     In some embodiments, a composition described herein may be administered to a subject in need thereof, such as a subject having medullary thyroid carcinoma. In some embodiments, a method described herein may comprise administering a composition comprising rAAV particles as described herein to a subject in need thereof. In some embodiments, the subject is a human subject. In some embodiments, the subject has or is suspected of having a disease that may be treated with gene therapy, such as medullary thyroid carcinoma. In some embodiments, the subject has been diagnosed with medullary thyroid carcinoma. 
     Methods 
     Aspects of the disclosure relate to methods of delivering a nucleic acid to a medullary thyroid carcinoma cell. In some embodiments, the method comprises administering a rAAV particle as described herein or a composition as described herein. The method may be performed in vitro or in vivo. In some embodiments, the cell is a human medullary thyroid carcinoma cell. In some embodiments, the cell is in a subject (e.g., a human subject). 
     Other aspects of the disclosure relate to treatment of treating medullary thyroid carcinoma. In some embodiments, the method comprises administering a therapeutically effective amount of an rAAV particle or a composition as described herein to a subject having medullary thyroid carcinoma. 
     To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject. The compositions described above or elsewhere herein are typically administered to a subject in an effective amount, that is, an amount capable of producing a desirable result. The desirable result will depend upon the active agent being administered. For example, an effective amount of rAAV particles may be an amount of the particles that are capable of transferring an expression construct to a host organ, tissue, or cell. A therapeutically acceptable amount may be an amount that is capable of treating a disease, e.g., medullary thyroid carcinoma. As is well known in the medical and veterinary arts, dosage for any one subject depends on many factors, including the subject&#39;s size, body surface area, age, the particular composition to be administered, the active ingredient(s) in the composition, time and route of administration, general health, and other drugs being administered concurrently. 
     The rAAV particle or nucleic acid vector may be delivered in the form of a composition, such as a composition comprising the active ingredient, such as a rAAV particle described herein, and a pharmaceutically acceptable carrier as described herein. The rAAV particles or nucleic acid vectors may be prepared in a variety of compositions, and may also be formulated in appropriate pharmaceutical vehicles for administration to human or animal subjects. 
     In some embodiments, the rAAV particles administered to a subject may be provided in a composition having a concentration on the order ranging from 10 6  to 10 14  particles/m1 or 10 3  to 10 15  particles/ml, or any values therebetween for either range, such as for example, about 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , or 10 14  particles/ml. In one embodiment, rAAV particles of higher than 10 13  particles/ml are to be administered. In some embodiments, the number of rAAV particles administered to a subject may be on the order ranging from 10 6  to 10 14  vector genomes (vgs)/m1 or 10 3  to 10 15  vgs/ml, or any values therebetween for either range, such as for example, about 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , or 10 14  vgs/ml. In one embodiment, rAAV particles of higher than 10 13  vgs/ml are to be administered. The rAAV particles can be administered as a single dose, or divided into two or more administrations as may be required to achieve therapy of the particular disease or disorder being treated. In some embodiments, 0.0001 ml to 10 mls are delivered to a subject. In some embodiments, the number of rAAV particles administered to a subject may be on the order ranging from 10 6 -10 14  vg/kg, or any values therebetween, such as for example, about 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , or 10 14  vgs/kg. In some embodiments, the number of rAAV particles administered to a subject may be on the order ranging from 10 12 -10 14  vgs/kg. 
     If desired, rAAV particles may be administered in combination with other agents or therapies as well, such as, e.g., proteins or polypeptides or various pharmaceutically-active agents, including one or more systemic or topical administrations of therapeutic polypeptides, biologically active fragments, or variants thereof. In fact, there is virtually no limit to other components that may also be included, given that the additional agents do not cause a significant adverse effect upon contact with the target cells or host tissues. The rAAV particles may thus be delivered along with various other agents as required in the particular instance. In some embodiments, the other agent is cyclophosphamide. In some embodiments, rAAV particles comprising a vector encoding CYP2B6 are delivered along with (either together or separately) cyclophosphamide. Such compositions may be purified from host cells or other biological sources, or alternatively may be chemically synthesized. In some embodiments, one or more other genes that can be used to treat cancer or other conditions (e.g., medullary thyroid cancer or other medullary thyroid conditions) can be delivered (e.g., alone or along with an additional agent). In some embodiments, rAAV particles as described herein (e.g., comprising a vector encoding CYP2B6) are administered in combination with one or more of thyroidectomy, radiation therapy (e.g., with radiolabeled iodine), or thyroid hormone treatment (e.g., with a synthetic thyroid hormone such as levothyroxine). 
     In certain circumstances it will be desirable to deliver the rAAV particles in suitably formulated pharmaceutical compositions disclosed herein either subcutaneously, intraocularly, intravitreally, subretinally, parenterally, intravenously, intracerebro-ventricularly, intramuscularly, intrathecally, orally, intraperitoneally, by oral or nasal inhalation, or by direct injection to one or more cells, tissues, or organs. In some embodiments, the direct injection is performed ex vivo. In some embodiments, the administration is a route suitable for systemic delivery, such as by intravenous injection or infusion. The pharmaceutical forms of the rAAV particle compositions suitable for injectable use include sterile aqueous solutions or dispersions. In some embodiments, the form is sterile and fluid to the extent that easy syringability exists. In some embodiments, the form is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, saline, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In some embodiments, rAAV particles are delivered IV, e.g., for the treatment of metastatic disease. 
     For administration of an injectable aqueous solution, for example, the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, intravitreal, subretinal, subcutaneous and intraperitoneal administration. In this connection, a sterile aqueous medium that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, “Remington&#39;s Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, and the general safety and purity standards as required by, e.g., FDA Office of Biologics standards. 
     Sterile injectable solutions are prepared by incorporating the rAAV particles in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization or another sterilization technique. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. 
     The amount of rAAV particle or nucleic acid vector compositions and time of administration of such compositions will be within the purview of the skilled artisan having benefit of the present teachings. It is likely, however, that the administration of therapeutically-effective amounts of the disclosed compositions may be achieved by a single administration, such as for example, a single injection of sufficient numbers of infectious particles to provide therapeutic benefit to the patient undergoing such treatment. Alternatively, in some circumstances, it may be desirable to provide multiple, or successive administrations of the rAAV particle compositions, either over a relatively short, or a relatively prolonged period of time, as may be determined by the medical practitioner overseeing the administration of such compositions. 
     The composition may include rAAV particles, either alone, or in combination with one or more additional active ingredients, which may be obtained from natural or recombinant sources or chemically synthesized. 
     Toxicity and efficacy of the compositions utilized in methods of the disclosure can be determined by standard pharmaceutical procedures, using either cells in culture or experimental animals to determine the LD50 (the dose lethal to 50% of the population). The dose ratio between toxicity and efficacy is the therapeutic index and it can be expressed as the ratio LD50/ED50. Those compositions that exhibit large therapeutic indices are preferred. While those that exhibit toxic side effects may be used, care should be taken to design a delivery system that minimizes the potential damage of such side effects. The dosage of compositions as described herein lies generally within a range that includes an ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. 
     Subjects 
     Aspects of the disclosure relate to methods for use with a subject, such as human or non-human primate subjects. Non-limiting examples of non-human primate subjects include macaques (e.g., cynomolgus or rhesus macaques), marmosets, tamarins, spider monkeys, owl monkeys, vervet monkeys, squirrel monkeys, baboons, gorillas, chimpanzees, and orangutans. In some embodiments, the subject is a human subject. Other exemplary subjects include domesticated animals such as dogs and cats; livestock such as horses, cattle, pigs, sheep, goats, and chickens; and other animals such as mice, rats, guinea pigs, and hamsters. 
     In some embodiments, the subject has or is suspected of having a disease that may be treated with gene therapy. In some embodiments, the subject has or is suspected of having medullary thyroid carcinoma. Medullary thyroid carcinoma (MTC) is a form of thyroid cancer which originates from C cells (parafollicular cells). MTC makes up approximately 3-4% of all thyroid cancer cases. If the MTC metastasizes, the 5-year survival rate drops to about 28%. In some embodiments, the subject has been diagnosed as having MTC. MTC can be identified by a skilled medical practitioner using methods known in the art, e.g., by measuring serum concentration of calcitonin, serum concentration of carcinoembryonic antigen (CEA), biopsy (e.g., fine needle aspiration), ultrasound, CT scan, or MRI, or any combination thereof. 
     Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present disclosure to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein. 
     EXAMPLES 
     Example 1. Recombinant AAV Vector Targeting of Medullary Thyroid Carcinoma 
     Thyroid cancers principally develop from two types of cells within the thyroid: thyroid epithelial cells and parafollicular, or C cells (1). Epithelial cell-derived cancers, which include papillary, follicular, and anaplastic carcinomas, are the most common type of thyroid cancers and account for approximately 90% of all thyroid cancer. Differentiated thyroid cancers generally have a good prognosis (2). In contrast, medullary thyroid carcinomas (MTC), which arise from C cells, account for less than 12% of thyroid cancers (3). If confined within the thyroid, MTC can be treated surgically (4). Yet most patients with MTC present with or develop metastatic disease, which is generally not curable (5-6). Tyrosine kinase inhibitors can stop MTC progression, but are not curative (7). Thus, new approaches for the treatment of MTC are needed. 
     One potential novel approach for cancer treatment is through the use of gene therapy to target malignant cells using viral vectors (8). Of potential viral vectors, recombinant adeno-associated viruses (rAAVs) have recently shown to hold potential for such therapeutic approaches (9-11). There are a large number of rAAV serotypes, and different serotypes can selectively transduce different cell types (12-13). By utilizing tissue-specific promoters in these viral vectors, it is possible to achieve tissue-specific expression (14). The use of a calcitonin promoter can confer MTC specificity due to its strong and specific expression in the C cells of the thyroid (15). Viral vectors can also be utilized to incorporate genes that encode for cytotoxic factors or enzymes that convert selective agents to cytotoxic chemicals to eliminate tumor tissue (16). For example, it is possible to insert the CYP2B6 gene, which converts cyclophosphamide to acrolein and phosphamide mustard, into such vectors resulting in cell death. 
     It is postulated that it will be possible to develop novel gene therapy-based approaches for MTC. It is postulated that it would be possible to utilize the calcitonin promoter to achieve specific expression in MTC tissues. The study herein describes the development of rAAV vectors that transduce MTC cells in vitro and in vivo. 
     Materials and Methods 
     Cell Culture 
     Human MTC cell line TT, lung carcinoma cell line A549, cervical adenocarcinoma cell line HeLa, medulloblastoma cell line Daoy, breast adenocarinoma cell line MCF7, and human papillomavirus transformed kidney cell line HK-2 were obtained from American Type Culture Collection (ATCC; Manassas, Va., USA). The human epidermal carcinoma cell line A431 was obtained from Sigma-Aldrich. Normal thyroid epithelial cell line Nthy-ori 3-1 (SV40-immortalized) was obtained from European Collection of Cell Cultures (ECACC; Salisbury, UK). Cells were grown at 37° C. and 5% CO 2 . All cell lines except HK-2 were grown in their recommended media supplemented with 10% fetal bovine serum (Gemini Bio-Products, West Sacramento, Calif., USA). HK-2 was grown in keratinocyte serum-free media supplemented with bovine pituitary extract and human recombinant epidermal growth factor (Life Technologies). 
     AAV Vector Production 
     Recombinant AAV vectors were generated as described (17) and contained eGFP driven by the CBA promoter or calcitonin modified P1.E2 promoter. Briefly, HEK293 cells were triple transfected with polyethylenimine (PEI, linear; Polysciences, Inc., Warrington, Pa., USA). Vectors were purified 72 hours post transfection by iodixanol gradient centrifugation (Sigma-Aldrich, St. Louis, Mo., USA) and ion exchange column chromatography using HiTrap SP HP (GE Healthcare, Piscataway, N.J., USA). Titers were determined using a quantitative PCR assay amplifying the transgene region (18). 
     Construction of Plasmids 
     The calcitonin promoter was amplified by polymerase chain reaction (PCR) from the MTC cell line TT using the forward 5′-CAGGGGTGTCGTGCTAAGAA-3′ (SEQ ID NO: 6) and reverse 5′-CCAGAATCTCGGGGCTCACCT-3′(SEQ ID NO: 7) primers corresponding to −1738 to +125 bases relative to the transcriptional start site (19). The PCR product was cloned into the pGEM vector (Promega, Madison, Wis., USA) and sequenced by Sanger Sequencing at the University of Florida Interdisciplinary Center for Biotechnology Research (ICBR) DNA Sequencing Core facility. The promoter was cloned into the pGL3 luciferase reporter vector (Promega). Synthesis of the modified calcitonin promoter sequence was performed by Life Technologies (Grand Island, N.Y., USA) 
     The chicken beta actin (CBA) promoter was removed and the P1.E2 promoter containing the proximal calcitonin promoter and duplicate enhancer sequences were cloned into the double-stranded AAV backbone expressing enhanced GFP (eGFP) using Kpnl and Ncol sites. 
     Luciferase Assays 
     Transfections were performed in 96-well plates. Cells were transfected at 70% density that was equivalent to approximately 5×10 3 -10 4  cells per well. Each well was transfected with 50 of serum-free media containing 0.1 μg of luciferase reporter plasmid, 0.1 μg pRL-SV40- Renilla  control, and 0.3 μl of FuGene HD transfection Reagent (Promega). 48 hours post-transfection, media was removed, and cells were briefly rinsed in PBS and lysed. All experiments were analyzed using the Dual-Luciferase Reporter Assay System (Promega). Luciferase activity in each well was measured twice following addition of 100 μl of firefly luciferase substrate then 100 μl Stop &amp; Glo  Renilla  substrate in a Biotek Synergy HT plate reader. Results are expressed as a percentage of the promoter activity compared to the SV40- Renilla  co-transfected control. 
     Recombinant AAV Vector In Vitro Transduction Assays 
     Cells were transduced with 2×10 3  vector genomes (vgs) per cell of wild-type (WT) or capsid mutant double stranded (ds) AAV2 vectors expressing eGFP. 48 hours post-transduction, transgene expression was assessed by total area of green fluorescence pixels per visual field or by flow cytometry. For pixel analysis, 4 wells in a 96-well plate were transduced, and fluorescent images taken at 5× magnification using a Leica DM IRB fluorescence microscope. Transgene expression was assessed as the total area of green fluorescence per visual field examined for each well (pixe12). Image analysis was performed using Image J (NIH, USA). For flow cytometry, cells were plated in a 6-well plate, detached from the plates with trypsin, and suspended in PBS containing 5% fetal calf serum. Cell sorting for eGFP expression was performed on a FACS Calibur (BD Biosciences, San Jose, Calif., USA) and analyzed using Cellquest Version 3.3. Each experiment was run with 10,000 cells, and untreated, mock, and AAV-transduced cells were compared. Pixel count analysis was performed in triplicate and flow cytometry in quadruplicate. 
     Mouse Xenograft Model of MTC and Administration of Recombinant AAV2 Mutant Vector 
     Subcutaneous injections of 1×10 7  TT cells into the lower dorsal region were performed in 6-week-old NSG mice (Jackson Laboratories, Bar Harbor, Me., USA). Cells were suspended in PBS and mixed at a 1:1 ratio with matrigel prior to injection (BD Biosciences, San Jose, Calif., USA). When tumors reached 9-10 mm in diameter, 2×10 10  vgs of ds AAV2 with capsid mutated Y730F containing the CBA (dsAAV2-Y730-CBA-eGFP; n=3) or the modified calcitonin CP1.E2 promoter (dsAAV2-Y730E-CP1.E2-eGFP; n=3) expressing eGFP were injected into the tumor. Mock control animals were injected with an equal volume of phosphate buffered saline (n=2). Two days after vector injection, tumors were resected. 
     Immunohistochemical Staining of TT MTC Xenograft Tumors for eGFP and Calcitonin 
     Immunohistochemical (IHC) staining was performed to evaluate eGFP expression following vector injection. All tumors were sectioned in half at the site of vector injection, fixed in 4% paraformaldehyde overnight, dehydrated, and embedded in paraffin. 5 μm thick tissue sections were prepared for staining. Hematoxylin and eosin (H&amp;E) and IHC staining was performed using a Rabbi anti-eGFP (1:100; Origene, Rockville, Md., USA) or Rabbit anti-Calcitonin (1:250; AbCam, Cambridge, Mass., USA) antibody as described (19-20). All images were taken on a Zeiss Axio Vert.A1 inverted light microscope (Zeiss, Thornwood, N.Y., USA). 
     Statistical Analysis 
     All bar graph results are presented as mean±standard deviation. Differences between WT- and mutant virus-transduced samples and promoter activity were assessed using analysis of variance (ANOVA). Flow cytometry analysis comparing cells transduced with rAAV2-Y730F with a CBA or CP1.E2 promoter were analyzed using the t-test. P-values &lt;0.05 were considered statistically significant. 
     Results 
     Transduction of MTC Cells by rAAV2 Capsids 
     Delivery of transgenes to MTC cells by rAAV2 vectors has been demonstrated in only one previous publication (11). To determine if other AAV serotypes could more efficiently transduce MTC cells, the TT cell line was transduced with rAAV1-6 in vitro. Wild-type (WT) rAAV2 capsid vectors showed significantly increased transduction compared to other serotypes as measured by eGFP expression (data not shown). When transduced with WT dsAAV2-eGFP, TT cells showed low eGFP expression that was 4-fold lower than those observed in HeLa cells ( FIG. 1A ) 
     Previous work in HEK293 cells has shown that mutation of amino acids on the surface of AAV capsids increases transduction efficiency (21). To determine if capsid mutations could increase transduction by rAAV in MTC cells, WT and mutant dsAAV-eGFP-expressing vectors were transduced into TT cells, and fluorescent gene expression was compared. Variations in eGFP expression by mutant rAAV2 vectors was also compared by transducing TT cells with WT or 6 other rAAV2 capsid mutant vectors. These capsids had different mutated surface-exposed tyrosines (Y), serines (S), or threonines (T) (21). These included single amino acid substitutions: Y730F, S662V, T491V; double substitutions at S662V+T491V; triple substitutions at Y444F+Y500F+Y730F (M3); and quadruple substitutions: M3+T491V. As shown in  FIG. 1B , single mutation Y730F and triple mutant M3 showed significantly increased transduction compared to WT virus in the TT cell line in vitro as measured by differences in eGFP expression. The level of transduction rivaled that seen in HeLa. These data support the hypothesis that capsid mutated dsAAV2 can improve transduction efficiency in MTC-derived cells. 
     A Truncated Calcitonin Promoter Region Confers C Cell Specificity In Vitro 
     To improve MTC-specific expression, different variants of the calcitonin promoter were examined in TT cells compared to other non-C cell-derived cells. Modified enhancer (E; −1080 to −860) and proximal promoter (P; −185 to +125) regions were synthesized with MluI, Nhel, and BglII sites incorporated upstream, Xbal between the promoter and enhancer, and EcoRV and HindIII sites incorporated downstream. The proximal promoter region −185 to +125, labeled as P1, was digested with Xbal and HindIII and cloned into the pGL3 vector using the Nhel and HindIII sites. A shortened version of the proximal promoter region −140 to +125, labeled PS1, was digested with BamHI and HindIII and cloned into the pGL3 vector using BglII and HindIII. The tissue-specific enhancer region −1080 to −860, labeled E1 (single) and E2 (double), were cloned upstream of the proximal promoters using the MluI and Nhel sites, respectively, upstream and the Xbal downstream site. Non-limiting examples of promoter sequences include: 
     
       
         
           
               
               
            
               
                   
                 P1.E1 
               
               
                   
                 (SEQ ID NO: 8) 
               
               
                   
                 GATCTGAGCTTGATCTAGGCAGGGACCACACAGCA 
               
               
                   
               
               
                   
                 CTGTCACACCTGCCTGCTCTTTAGTAGAGGACTGA 
               
               
                   
               
               
                   
                 AGTGCGGGGGTGGGGGTACGGGGCCGGAATAGAAT 
               
               
                   
               
               
                   
                 GTCTCTGGGACATCTTGGCAAACAGCAGCCGGAAG 
               
               
                   
               
               
                   
                 CAAAGGGGCAGCTGTGCAAACGGCTCAGGCAGGTG 
               
               
                   
               
               
                   
                 ATGGATGGCAGGGTAGGAAGGGGGAGCCAGAGGTC 
               
               
                   
               
               
                   
                 TGGATGGAGGTTCTAGATTTTCCATCAATGACCTC 
               
               
                   
               
               
                   
                 AATGCAAATACAAGTGGGACGGTCCTGCTGGATCC 
               
               
                   
               
               
                   
                 TCCAGGTTCTGGAAGCATGAGGGTGACGCAACCCA 
               
               
                   
               
               
                   
                 GGGGCAAAGGACCCCTCCGCCCATTGGTTGCTGTG 
               
               
                   
               
               
                   
                 CACTGGCGGAACTTTCCCGACCCACAGCGGCGGGA 
               
               
                   
               
               
                   
                 ATAAGAGCAGTCGCTGGCGCTGGGAGGCATCAGAG 
               
               
                   
               
               
                   
                 ACACTGCCCAGCCCAAGTGTCGCCGCCGCTTCCAC 
               
               
                   
               
               
                   
                 AGGGCTCTGGCTGGACGCCGCCGCCGCCGCTGCCA 
               
               
                   
               
               
                   
                 CCGCCTCTGATCCAAGCCACCTCCCGCCACTAGTG 
               
               
                   
               
               
                   
                 ATATCAP1.E2 
               
               
                   
                 (SEQ ID NO: 9) 
               
               
                   
                 GATCTGAGCTTGATCTAGGCAGGGACCACACAGCA 
               
               
                   
               
               
                   
                 CTGTCACACCTGCCTGCTCTTTAGTAGAGGACTGA 
               
               
                   
               
               
                   
                 AGTGCGGGGGTGGGGGTACGGGGCCGGAATAGAAT 
               
               
                   
               
               
                   
                 GTCTCTGGGACATCTTGGCAAACAGCAGCCGGAAG 
               
               
                   
               
               
                   
                 CAAAGGGGCAGCTGTGCAAACGGCTCAGGCAGGTG 
               
               
                   
               
               
                   
                 ATGGATGGCAGGGTAGGAAGGGGGAGCCAGAGGTC 
               
               
                   
               
               
                   
                 TGGATGGAGGTTCTAGCAGATCTGAGCTTGATCTA 
               
               
                   
               
               
                   
                 GGCAGGGACCACACAGCACTGTCACACCTGCCTGC 
               
               
                   
               
               
                   
                 TCTTTAGTAGAGGACTGAAGTGCGGGGGTGGGGGT 
               
               
                   
               
               
                   
                 ACGGGGCCGGAATAGAATGTCTCTGGGACATCTTG 
               
               
                   
               
               
                   
                 GCAAACAGCAGCCGGAAGCAAAGGGGCAGCTGTGC 
               
               
                   
               
               
                   
                 AAACGGCTCAGGCAGGTGATGGATGGCAGGGTAGG 
               
               
                   
               
               
                   
                 AAGGGGGAGCCAGAGGTCTGGATGGAGGTTCTAGA 
               
               
                   
               
               
                   
                 TTTTCCATCAATGACCTCAATGCAAATACAAGTGG 
               
               
                   
               
               
                   
                 GACGGTCCTGCTGGATCCTCCAGGTTCTGGAAGCA 
               
               
                   
               
               
                   
                 TGAGGGTGACGCAACCCAGGGGCAAAGGACCCCTC 
               
               
                   
               
               
                   
                 CGCCCATTGGTTGCTGTGCACTGGCGGAACTTTCC 
               
               
                   
               
               
                   
                 CGACCCACAGCGGCGGGAATAAGAGCAGTCGCTGG 
               
               
                   
               
               
                   
                 CGCTGGGAGGCATCAGAGACACTGCCCAGCCCAAG 
               
               
                   
               
               
                   
                 TGTCGCCGCCGCTTCCACAGGGCTCTGGCTGGACG 
               
               
                   
               
               
                   
                 CCGCCGCCGCCGCTGCCACCGCCTCTGATCCAAGC 
               
               
                   
               
               
                   
                 CACCTCCCGCCACTAGTGATATCA 
               
               
                   
               
               
                   
                 PS1.E1 
               
               
                   
                 (SEQ ID NO: 10) 
               
               
                   
                 GATCTGAGCTTGATCTAGGCAGGGACCACACAGCA 
               
               
                   
               
               
                   
                 CTGTCACACCTGCCTGCTCTTTAGTAGAGGACTGA 
               
               
                   
               
               
                   
                 AGTGCGGGGGTGGGGGTACGGGGCCGGAATAGAAT 
               
               
                   
               
               
                   
                 GTCTCTGGGACATCTTGGCAAACAGCAGCCGGAAG 
               
               
                   
               
               
                   
                 CAAAGGGGCAGCTGTGCAAACGGCTCAGGCAGGTG 
               
               
                   
               
               
                   
                 ATGGATGGCAGGGTAGGAAGGGGGAGCCAGAGGTC 
               
               
                   
               
               
                   
                 TGGATGGAGGTTTGGATCCTCCAGGTTCTGGAAGC 
               
               
                   
               
               
                   
                 ATGAGGGTGACGCAACCCAGGGGCAAAGGACCCCT 
               
               
                   
               
               
                   
                 CCGCCCATTGGTTGCTGTGCACTGGCGGAACTTTC 
               
               
                   
               
               
                   
                 CCGACCCACAGCGGCGGGAATAAGAGCAGTCGCTG 
               
               
                   
               
               
                   
                 GCGCTGGGAGGCATCAGAGACACTGCCCAGCCCAA 
               
               
                   
               
               
                   
                 GTGTCGCCGCCGCTTCCACAGGGCTCTGGCTGGAC 
               
               
                   
               
               
                   
                 GCCGCCGCCGCCGCTGCCACCGCCTCTGATCCAAG 
               
               
                   
               
               
                   
                 CCACCTCCCGCCACTAGTGATATCA 
               
               
                   
               
               
                   
                 PS1.E2 
               
               
                   
                 (SEQ ID NO: 11) 
               
               
                   
                 GATCTGAGCTTGATCTAGGCAGGGACCACACAGCA 
               
               
                   
               
               
                   
                 CTGTCACACCTGCCTGCTCTTTAGTAGAGGACTGA 
               
               
                   
               
               
                   
                 AGTGCGGGGGTGGGGGTACGGGGCCGGAATAGAAT 
               
               
                   
               
               
                   
                 GTCTCTGGGACATCTTGGCAAACAGCAGCCGGAAG 
               
               
                   
               
               
                   
                 CAAAGGGGCAGCTGTGCAAACGGCTCAGGCAGGTG 
               
               
                   
               
               
                   
                 ATGGATGGCAGGGTAGGAAGGGGGAGCCAGAGGTC 
               
               
                   
               
               
                   
                 TGGATGGAGGTTCTAGCAGATCTGAGCTTGATCTA 
               
               
                   
               
               
                   
                 GGCAGGGACCACACAGCACTGTCACACCTGCCTGC 
               
               
                   
               
               
                   
                 TCTTTAGTAGAGGACTGAAGTGCGGGGGTGGGGGT 
               
               
                   
               
               
                   
                 ACGGGGCCGGAATAGAATGTCTCTGGGACATCTTG 
               
               
                   
               
               
                   
                 GCAAACAGCAGCCGGAAGCAAAGGGGCAGCTGTGC 
               
               
                   
               
               
                   
                 AAACGGCTCAGGCAGGTGATGGATGGCAGGGTAGG 
               
               
                   
               
               
                   
                 AAGGGGGAGCCAGAGGTCTGGATGGAGGTTTGGAT 
               
               
                   
               
               
                   
                 CCTCCAGGTTCTGGAAGCATGAGGGTGACGCAACC 
               
               
                   
               
               
                   
                 CAGGGGCAAAGGACCCCTCCGCCCATTGGTTGCTG 
               
               
                   
               
               
                   
                 TGCACTGGCGGAACTTTCCCGACCCACAGCGGCGG 
               
               
                   
               
               
                   
                 GAATAAGAGCAGTCGCTGGCGCTGGGAGGCATCAG 
               
               
                   
               
               
                   
                 AGACACTGCCCAGCCCAAGTGTCGCCGCCGCTTCC 
               
               
                   
               
               
                   
                 ACAGGGCTCTGGCTGGACGCCGCCGCCGCCGCTGC 
               
               
                   
               
               
                   
                 CACCGCCTCTGATCCAAGCCACCTCCCGCCACTAG 
               
               
                   
               
               
                   
                 TGATATCA 
               
               
                   
               
               
                   
                 P1 
               
               
                   
                 (SEQ ID NO: 12) 
               
               
                   
                 TTCCATCAATGACCTCAATGCAAATACAAGTGGGA 
               
               
                   
               
               
                   
                 CGGTCCTGCTGGATCCTCCAGGTTCTGGAAGCATG 
               
               
                   
               
               
                   
                 AGGGTGACGCAACCCAGGGGCAAAGGACCCCTCCG 
               
               
                   
               
               
                   
                 CCCATTGGTTGCTGTGCACTGGCGGAACTTTCCCG 
               
               
                   
               
               
                   
                 ACCCACAGCGGCGGGAATAAGAGCAGTCGCTGGCG 
               
               
                   
               
               
                   
                 CTGGGAGGCATCAGAGACACTGCCCAGCCCAAGTG 
               
               
                   
               
               
                   
                 TCGCCGCCGCTTCCACAGGGCTCTGGCTGGACGCC 
               
               
                   
               
               
                   
                 GCCGCCGCCGCTGCCACCGCCTCTGATCCAAGCCA 
               
               
                   
               
               
                   
                 CCTCCCGCCAGGTGAGCCCCGAGATCCT 
               
               
                   
               
               
                   
                 PS1 
               
               
                   
                 (SEQ ID NO: 13) 
               
               
                   
                 TGGATCCTCCAGGTTCTGGAAGCATGAGGGTGACG 
               
               
                   
               
               
                   
                 CAACCCAGGGGCAAAGGACCCCTCCGCCCATTGGT 
               
               
                   
               
               
                   
                 TGCTGTGCACTGGCGGAACTTTCCCGACCCACAGC 
               
               
                   
               
               
                   
                 GGCGGGAATAAGAGCAGTCGCTGGCGCTGGGAGGC 
               
               
                   
               
               
                   
                 ATCAGAGACACTGCCCAGCCCAAGTGTCGCCGCCG 
               
               
                   
               
               
                   
                 CTTCCACAGGGCTCTGGCTGGACGCCGCCGCCGCC 
               
               
                   
               
               
                   
                 GCTGCCACCGCCTCTGATCCAAGCCACCTCCCGCC 
               
               
                   
               
               
                   
                 AGGTGAGCCCCGAGATCCT 
               
               
                   
               
               
                   
                 E 
               
               
                   
                 (SEQ ID NO: 14) 
               
               
                   
                 GAGCTTGATCTAGGCAGGGACCACACAGCACTGTC 
               
               
                   
               
               
                   
                 ACACCTGCCTGCTCTTTAGTAGAGGACTGAAGTGC 
               
               
                   
               
               
                   
                 GGGGGTGGGGGTACGGGGCCGGAATAGAATGTCTC 
               
               
                   
               
               
                   
                 TGGGACATCTTGGCAAACAGCAGCCGGAAGCAAAG 
               
               
                   
               
               
                   
                 GGGCAGCTGTGCAAACGGCTCAGGCAGGTGATGGA 
               
               
                   
               
               
                   
                 TGGCAGGGTAGGAAGGGGGAGGTCCAGAGGTCTGG 
               
               
                   
               
               
                   
                 ATGGAGGCTTC 
               
               
                   
               
               
                   
                 Full Calcitonin Promoter Tested 
               
               
                   
                 (SEQ ID NO: 15) 
               
               
                   
                 GGGTGTCGTGCTAAGAAATTTCGACGCTTCTGGGG 
               
               
                   
               
               
                   
                 ACTGAGGACAAAGGTGCGGACACGACCCCGGGGTA 
               
               
                   
               
               
                   
                 CCTGGAGTTCCGTGACTCGCGCCACGGACGGCACA 
               
               
                   
               
               
                   
                 CCTAGGGGCTAATTTCTGCTCTGCCTCAAAGAACC 
               
               
                   
               
               
                   
                 TCAAGCTAGAGTCCTTGCCTCCGCCCACAGCCCCG 
               
               
                   
               
               
                   
                 GGATGCCGCTGCTGCGCTCACCGCACAGGCAGCGC 
               
               
                   
               
               
                   
                 CCGGACCGGCTGCAGCAGATCGCGCGCTGCGCGTT 
               
               
                   
               
               
                   
                 CCACCGGGAGATGGTGGAGACGCTGAAAAGCTTCT 
               
               
                   
               
               
                   
                 TTCTTGCCACTCTGGACGCTGTGGGCGGCAAGCGC 
               
               
                   
               
               
                   
                 CTTAGTCCCTACCTCTGCTGAGCTGAACGCTCAGG 
               
               
                   
               
               
                   
                 CACAGTGGAACTGAAACCCGGTTCTGCGGGATGTG 
               
               
                   
               
               
                   
                 AGAGCTGTTGAGGTCACGCGTAATTGGGTGTGATG 
               
               
                   
               
               
                   
                 GAGGGCGCCTGTTCGTGATGTGTGCAGGTTTGATG 
               
               
                   
               
               
                   
                 CAAGCAGGTCATCGTCGTGCGAGTGTGTGGATGCG 
               
               
                   
               
               
                   
                 ACCGCCCGAGAGACTCGGAGGCAGGCTTGGGACAC 
               
               
                   
               
               
                   
                 GTTTGAGTGAACACCTCAGGATACTCTTCTGGCCA 
               
               
                   
               
               
                   
                 GTATCTGTTTTTTAGTGTCTGTGATTCAGAGTGGG 
               
               
                   
               
               
                   
                 CACATGTTGGGAGACAGTAATGGGTTTGGGTGTGT 
               
               
                   
               
               
                   
                 GTAAATGAGTGTGACCGGAAGCGAGTGTGAGCTTG 
               
               
                   
               
               
                   
                 ATCTAGGCAGGGACCACACAGCACTGTCACACCTG 
               
               
                   
               
               
                   
                 CCTGCTCTTTAGTAGAGGACTGAAGTGCGGGGGTG 
               
               
                   
               
               
                   
                 GGGGTACGGGGCCGGAATAGAATGTCTCTGGGACA 
               
               
                   
               
               
                   
                 TCTTGGCAAACAGCAGCCGGAAGCAAAGGGGCAGC 
               
               
                   
               
               
                   
                 TGTGCAAACGGCTCAGGCAGGTGATGGATGGCAGG 
               
               
                   
               
               
                   
                 GTAGGAAGGGGGAGGTCCAGAGGTCTGGATGGAGG 
               
               
                   
               
               
                   
                 CTTCCGCATCTGTACCTTGCAACTCACCCCTCAGG 
               
               
                   
               
               
                   
                 CCCAGCAGGTCATCGGCCCCCTCCTCACACATGTA 
               
               
                   
               
               
                   
                 ATGGATCTGAAGAGTACCCCGGGACAGTCCGGGGA 
               
               
                   
               
               
                   
                 GATGGAGATTCGGAAAGTATCCATGGAGATCTTAC 
               
               
                   
               
               
                   
                 AGAATCCCCTGTGCGGACCAGGAAACTCTTGTAGA 
               
               
                   
               
               
                   
                 TCCCTGCCTATCTGAGGCCCAGGCGCTGGGCTGTT 
               
               
                   
               
               
                   
                 TCTCACAATATTCCTTCAAGATGAGATTGTGGTCC 
               
               
                   
               
               
                   
                 CCATTTCAAAGATGAGTACACTGAGCCTCTGTGAA 
               
               
                   
               
               
                   
                 GTTACTTGCCCATGATCACACAACCAGGAATTGGG 
               
               
                   
               
               
                   
                 CCAACTGTAATTGAACTCCTGTCTAACAAAGTTCT 
               
               
                   
               
               
                   
                 TGCTCCCAGCTCCGTCTCTTGTTTCCCACGAGCCC 
               
               
                   
               
               
                   
                 TGGCCCTCTGTGGGTAATACCAGCTACTGGAGTCA 
               
               
                   
               
               
                   
                 GATTTCTTGGGCCCAGAACCCACCCTTAGGGGCAT 
               
               
                   
               
               
                   
                 TAACCTTTAAAATCTCACTTGGGCAGGGGTCTGGG 
               
               
                   
               
               
                   
                 ATCAGAGTTGGAAGAGTCCCTACAATCCTGGACCC 
               
               
                   
               
               
                   
                 TTTCCGCCAAATCGTGAAACCAGGGGTGGAGTGGG 
               
               
                   
               
               
                   
                 GCGAGGGTTCAAAACCAGGCCGGACTGAGAGGTGA 
               
               
                   
               
               
                   
                 AATTCACCATGACGTCAAACTGCCCTCAAATTCCC 
               
               
                   
               
               
                   
                 GCTCACTTTAAGGGCGTTACTTGTTGGTGCCCCCA 
               
               
                   
               
               
                   
                 CCATCCCCCACCATTTCCATCAATGACCTCAATGC 
               
               
                   
               
               
                   
                 AAATACAAGTGGGACGGTCCTGCTGGATCCTCCAG 
               
               
                   
               
               
                   
                 GTTCTGGAAGCATGAGGGTGACGCAACCCAGGGGC 
               
               
                   
               
               
                   
                 AAAGGACCCCTCCGCCCATTGGTTGCTGTGCACTG 
               
               
                   
               
               
                   
                 GCGGAACTTTCCCGACCCACAGCGGCGGGAATAAG 
               
               
                   
               
               
                   
                 AGCAGTCGCTGGCGCTGGGAGGCATCAGAGACACT 
               
               
                   
               
               
                   
                 GCCCAGCCCAAGTGTCGCCGCCGCTTCCACAGGGC 
               
               
                   
               
               
                   
                 TCTGGCTGGACGCCGCCGCCGCCGCTGCCACCGCC 
               
               
                   
               
               
                   
                 TCTGATCCAAGCCACCTCCCGCCAGGTGAGCCCCG 
               
               
                   
               
               
                   
                 AGATCCTG 
               
            
           
         
       
     
       FIG. 2A  depicts the calcitonin promoter regions based on previously published sequence analysis, with relevant transcription factor binding sites and regions highlighted (22). A previous report demonstrated that truncated versions of the calcitonin core promoter combined with tissue-specific enhancer (TSE) regions could increase expression in C cells (23). To confirm specificity of the calcitonin promoter for C cells, the promoter region 1.7 kb upstream of the transcriptional start site was cloned into a luciferase expression vector and tested in TT cells compared to non-C cell-derived cells. As shown in  FIG. 2B , luciferase expression from the full calcitonin promoter was significantly higher in TT cells compared to cells derived from the lung, thyroid epithelial, cervix, brain, breast, skin, and kidney. Values are shown as a percentage of the calcitonin promoter divided by co-transfected SV40- Renilla . Changes in expression levels were evaluated in TT cells transfected with a luciferase expression vector containing either the full length calcitonin promoter, the proximal promoter with a single or duplicate enhancer region (P1.E1 and P1.E2), or a truncated proximal promoter region lacking the CRE/Octl binding sites and single or duplicate enhancer regions (PS1.E1 or PS1.E2). The proximal promoter region (P) containing duplicate enhancer sequences (P1.E2) increased luciferase expression approximately 5-fold in TT cells compared to the full calcitonin promoter region ( FIG. 2C ). This promoter construct with duplicate enhancer regions provides significantly improved expression in MTC-derived cells. The P1.E2 calcitonin promoter (CP1.E2) was then packaged into a dsAAV2-Y730F mutant vector expressing eGFP. TT cells, as well as A549, Nthy-ori 3-1, and HeLa cells, were evaluated by flow cytometry for specific transduction from the dsAAV2-Y730E-CP1.E2-eGFP vector. Visualization of eGFP expression from this vector is shown compared to a dsAAV2-Y730E-CBA-eGFP positive control ( FIG. 3A ). Quantification of flow cytometry results are shown in  FIG. 3B . 
     Approximately 10% of TT cells were eGFP positive when transduced with either vector, with no significant difference seen between promoters. While the number of TT cells transduced is relatively low, the expression level of eGFP in positive cells was similar when expressed from either the CBA or CP1.E2 promoters and was of a similar intensity to eGFP expression from the CBA promoter visualized in other cell lines ( FIG. 3A ). 
     In the three other non-C cell lines examined, there was a significant decrease in eGFP positive cells when transduced with the CP1.E2 vector compared to the CBA control ( FIG. 3B ). Whereas flow cytometry detected eGFP positive cells in the A549 and HeLa cell lines, expression levels were low and hardly visible by fluorescence microscopy. Representative histograms for each cell line and vector are shown in  FIGS. 3C-3D .  FIG. 5  provides further data showing infection efficiencies (as a relative change in GFP expression compared to wild-type rAAV2) of rAAV2(Y730F), rAAV2 (S662V), rAAV2(T491V), rAAV2(S662V+T491V), rAAV2(Y444F+Y500F+Y730F) (M3), and rAAV2(M3+T491V). 
     These data demonstrate that rAAV2 mutant capsids combined with a modified calcitonin promoter can achieve expression levels similar to the control CBA promoter but also with specificity for MTC-derived cells. 
     Expression and Localization of Mutant rAAV2 Expressing eGFP In Vivo 
     To determine whether the capsid-mutated rAAV2 vector containing a modified calcitonin promoter was expressed in vivo, TT cells were used to generate mice containing human MTC tumor xenografts. Tumors of 9-10 mm in diameter (approximately 6-8 weeks post-TT cell inoculation) were injected with 2×10 10  vgs of dsAAV2-Y730E-CBA-eGFP or dsAAV2-Y730E-CP1.E2 or an equal volume of PBS. Two days following vector injection, tumors were resected and evaluated. 
     Staining of the tumor tissue by H&amp;E demonstrated analogous histological features to MTC with notable vascularization and strong staining for calcitonin ( FIGS. 4A and 4B ). When evaluated for eGFP expression, positive staining was observed in both dsAAV2-Y730E-CBA and -CP1.E2 injected tumors. Positively stained cells with the strongest expression were observed near the injection site, with sporadic staining of positive cells throughout the tumor section equivalent to approximately 10-15% of MTC cells.  FIG. 4C  shows a representative image of positive eGFP IHC staining in a dsAAV-Y730E-CP1.E2-eGFP injected tumor, with lack of staining observed in the IgG control ( FIG. 4D ). All mock-injected tumors were negative for eGFP staining. This observation demonstrates that capsid-mutated rAAV containing a modified calcitonin promoter was capable of expressing in TT cells both in vitro and in vivo. 
     In some aspects, the results reported here reveal a novel rAAV vector that transduces MTC both in vitro and in vivo. The use of capsid mutant rAAV2 vectors increased transduction in the MTC cell line TT. Development of a shortened, modified calcitonin promoter further increased transgene expression and conferred specificity for MTC cells. These vectors were then shown to transduce MTC cells in a tumor xenograft model. This is believed to be the first demonstration of a rAAV vector transduction of MTC in vivo. Expression of the rAAV in vivo demonstrates this vector could be further optimized for the delivery of therapeutic genes to tumor tissues. 
     Approximately 10-15% of TT cells were transduced in vitro and in vivo by the vectors tested here. A previous report examining rAAV transduction in TT cells in vitro reported 40% efficiency, but this was performed with an adenovirus co-infection (11). Although the transduction efficiency is lower for the rAAV vectors reported here, transgene expression with rAAV alone is more pertinent and safer for clinical application. Furthermore, previous work has demonstrated that the parent cell line TT contains multiple cell types and expresses proteins at varied levels, including differences in calcitonin expression (24). Additional studies examining the single cell types within the cell line TT may improve the targeting and transduction, and ultimately treatment, of MTC with rAAV. 
     The use of the truncated calcitonin promoter containing repeated enhancer elements gives improved, specific expression in MTC cells in vitro. The small size of approximately 800 base pairs of this modified calcitonin promoter compared to the 1.7 kb full calcitonin upstream region allows for the capability of packaging larger therapeutic genes in a double-stranded vector. Furthermore, the use of repeated enhancer elements increased expression from transgenes packaged with this promoter as shown in  FIG. 1C . This modified promoter could be utilized to strongly express a wide range of transgenes specifically in MTC cells. Multiple methods have been proposed for gene therapy approaches for treatment of thyroid carcinomas including targeting of specific pathways, reintroduction of the sodium iodine symporter, immune modulation, and gene-directed enzyme/prodrug therapy (GDEPT; 8). As the RET gene is commonly mutated in both hereditary and sporadic MTC, drug treatment inhibiting the RET pathway has been used (25-27). Improved, specific methods of inhibiting RET could be delivered using rAAV vectors but would require a high percentages of cells being transduced. Many GDEPT methods not only induce cell death in targeted cells, but result in bystander killing of surrounding cells due to the release of toxic metabolites (28). While the rAAV vector reported here specifically targets MTC cells, only approximately 10% of cells showed transgene expression. A GDEPT method may be beneficial in targeting tumors using rAAV without 100% transduction efficiency being necessary for tumor cytotoxicity. 
     In some aspects, this study reports use of a rAAV vector for in vivo expression in MTC. Accordingly, in some embodiments, rAAV vectors described herein allow for improved targeting and expression in primary and metastatic MTC tumors, the latter particularly important for developing novel cures for MTC (29). Reliable and specific expression can be achieved in MTC cells due to the use of the modified calcitonin reporter employed here. 
     REFERENCES 
     
         
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         7. Massicotte M H, Brassard M, Claude-Desroches M, Borget I, Bonichon F, Giraudet A L, Do Cao C, Chougnet C N, Leboulleux S, Baudin E, Schlumberger M, de la Fouchardière C 2014 Tyrosine kinase inhibitor treatments in patients with metastatic thyroid carcinomas: a retrospective study of the TUTHYREF network. Eur J Endocrinol 170(4): 575-82. 
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         13. Rabinowitz J E, Rolling F, Li C, Conrath H, Xiao W, Xiao X, Samulski R J 2002 Cross-packaging of a single adeno-associated virus (AAV) type 2 vector genome into multiple AAV serotypes enables transduction with broad specificity. J Virol 76(2): 791-801. 
         14. Gray S J, Foti S B, Schwartz J W, Bachaboina L, Taylor-Blake B, Coleman J, Ehlers M D, Zylka M J, McCown T J, Samulski R J 2011 Optimizing promoters for recombinant adeno-associated virus-mediated gene expression in the peripheral and central nervous system using self-complementary vectors. Hum Gene Ther 22(9): 1143-53. 
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         23. Messina M, Yu D M, Learoyd D L, Both G W, Molloy P L, Robinson B G 2000 High level, tissue-specific expression of a modified calcitonin/calcitonin gene-related peptide promoter in a human medullary thyroid carcinoma cell line. Mol Cell Endocrinol 164(1-2): 219-24. 
         24. Berger C L, de Bustros A, Roos B A, Leong S S, Mendelsohn G, Gesell M S, Baylin S B 1984 Human medullary thyroid carcinoma in culture provides a model relating growth dynamics, endocrine cell differentiation, and tumor progression. J Clin Endocrinol Metab 59(2): 338-43. 
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         26. Elisei R, Cosci B, Romei C, Bottici V, Renzini G, Molinaro E, Agate L, Vivaldi A, Faviana P, Basolo F, Miccoli P, Berti P, Pacini F, Pinchera A 2008 Prognostic significance of somatic RET oncogene mutations in sporadic medullary thyroid cancer: a 10-year follow-up study. J Clin Endocrinol Metab 93(3): 682-7. 
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         30. Frendo J L, Delage-Mourroux R, Cohen R, Pichaud F, Pidoux E, Guliana J M, Jullienne A 1998 Calcitonin receptor mRNA is expressed in human medullary thyroid carcinoma. Thyroid 8(2): 141-7. 
       
    
     Other Embodiments 
     All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features. 
     From the above description, one skilled in the art can easily ascertain the essential characteristics of the present disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the disclosure to adapt it to various usages and conditions. Thus, other embodiments are also within the claims. 
     EQUIVALENTS 
     While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure. 
     All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms. 
     All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document. 
     The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” 
     The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc. 
     As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law. 
     As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc. 
     It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited. 
     In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g., “comprising”) are also contemplated, in alternative embodiments, as “consisting of” and “consisting essentially of” the feature described by the open-ended transitional phrase. For example, if the disclosure describes “a composition comprising A and B”, the disclosure also contemplates the alternative embodiments “a composition consisting of A and B” and “a composition consisting essentially of A and B”.