Patent Publication Number: US-2015079051-A1

Title: Tfeb gene therapy of alpha-1-antitrypsin deficiency

Description:
TECHNICAL FIELD 
     The present invention relates to a vector comprising a TFEB coding sequence under the control of a promoter able to efficiently express said TFEB coding sequence for use in the gene therapy of a pathological condition characterized by a deficiency of alpha-1-antitrypsin (AAT), to a host cell comprising said vector, pharmaceutical compositions and uses thereof. 
     BACKGROUND ART 
     Alpha-1-antitrypsin (AAT) is a member of the serine protease inhibitor (SERPIN) superfamily of structurally conserved proteins that inhibit serine proteases. AAT is synthesized in the liver and released into the plasma where it is the most abundant circulating protease inhibitor. AAT deficiency caused by missense mutation (lysine for glutamate at amino acid position 342) that alters protein folding is the most common genetic cause of liver disease in children′. It is also responsible for chronic liver disease and hepatocellular carcinoma in adults 2-3 . This mutated form of AAT is also known as ATZ. ATZ is prone to aggregate in the endoplasmic reticulum of hepatocytes causing liver injury. The only curative treatment available is liver transplantation. Therefore there is the need for alternative less invasive therapeutic strategies. Intracellular liver inclusions have been identified with other mutated form of AAT characterized by polymer formation, including S iiyama  (phenylalanine for serine at amino acid position 53) and M malton  (deletion of phenylalanine at amino acid position 52) 34 . 
     Recent findings have shown that stimulation of autophagy may reduce accumulation of hepatotoxic ATZ 4-5 . Transcription factor EB (TFEB, NCBI GeneID=7942; Accession no.: nt=NM — 007162.2, protein=NP — 009093.1 and variants thereof) is a master gene that regulates the number and function of lysosomes and autophagy by direct binding to a palindromic 10-base pair regulatory motif (CLEAR element) that is highly enriched in autophagy and lysosomal genes promoters 6-8 . 
     The document WO 2010/092112 relates to TFEB protein, synthetic or biotechnological functional derivative thereof, peptide fragments thereof, chimeric molecules comprising the TFEB protein, synthetic or biotechnological functional derivative thereof acting either direcly or indirectly on a CLEAR element. However the document is silent concerning pathological condition characterized by a deficiency of alpha-1-antitrypsin. 
     SUMMARY OF THE INVENTION 
     In the present study, the authors investigated the therapeutic potential of liver-directed gene transfer of transcription factor EB (TFEB), a master gene that regulates lysosomal function and autophagy, in the PiZ transgenic mice, recapitulating the human hepatic disease. The authors have investigated efficiency of hepatic gene transfer of TFEB at increasing clearance of mutant hepatotoxic alpha-1-antitrypsin (ATZ). The authors injected a helper-dependent adenoviral (HDAd) vector expressing the TFEB under the control of a liver-specific promoter (HDAd-TFEB) in the PiZ mouse model, a transgenic mouse expressing the human ATZ gene and recapitulating the features of liver disease observed in humans. Three-month old PiZ mice were injected intravenously with HDAd-TFEB at the dose of 1×10 13  vp/kg or, as controls, with the same dose of a HDAd vector expressing the unrelated alpha-fetoprotein (AFP) gene under the control of the same expression cassette (HDAd-AFP) or saline. Compared to saline or HDAd-AFP, mice injected with HDAd-TFEB showed a dramatic reduction in hepatic ATZ accumulation, as demonstrated by marked reduction of periodic acid-Schiff (PAS) staining and ATZ-containing globules. As expected, TFEB gene transfer resulted in an increase in hepatic LC3-II, a marker of autophagic activity. Taken together, these results demonstrate that hepatic gene transfer of TFEB reduces accumulation of ATZ by enhancement of autophagy in the liver. ATZ serum levels were reduced in mice injected with HDAd-TFEB vector as compared to baseline levels of the same mice. Moreover, a marked, statistically significant decrease in both ATZ monomer and polymer was observed in HDAd-TFEB injected mouse livers as compared to either saline or HDAd-AFP injected control mice, thus indicating that TFEB hepatic expression enhances disposal of both insoluble and soluble hepatic ATZ. In addition, the authors showed that HDAd-TFEB injected mice showed a reduction in hepatocyte apoptosis and hepatic fibrosis, which are key features of the hepatic disease of AAT deficiency. In summary, the authors showed that TFEB-mediated hepatocyte expression results in clearance of ATZ, improvement of the liver phenotype and therefore, is an attractive gene-based strategy for the treatment of alpha-1-antitrypsin deficiency hepatic disease. 
     Therefore, hepatocyte TFEB gene transfer resulted in a dramatic reduction of hepatic ATZ and reduced liver apoptosis and fibrosis, which are key features of AAT deficiency. Moreover, TFEB enhanced hepatic LC3, a marker of autophagy, and increased ATZ degradation by autophagolysosomes. For in vivo hepatocyte gene transfer, the authors used helper-dependent adenoviral (HDAd) vectors which are the most efficient vectors for liver-directed gene therapy 9 . 
     TFEB gene transfer is a novel strategy for treatment of liver disease of AAT deficiency. The present invention confirms the application of TFEB gene transfer for treatment of a wide spectrum of human disorders due to accumulation of toxic proteins. 
     In the present invention TFEB coding sequence means a sequence coding for the entire TFEB protein or for TFEB functional fragment(s), said fragment(s) preferably being able to act on a CLEAR element and having a transcription activation domain, and/or maintaining the clearance activity. 
     Object of the present invention is a vector comprising a TFEB coding sequence under the control of a promoter able to efficiently express said TFEB coding sequence for use in the gene therapy of a pathological condition characterized by a deficiency of alpha-1-antitrypsin (AAT). 
     Preferably the TFEB coding sequence is hTFEB consisting essentially of the sequence of SEQ ID No. 3. 
     In a preferred embodiment the vector is a viral vector. Preferably the viral vector belongs to the group of: adenoviral vectors, lentiviral vectors, retroviral vectors, Adeno associated vectors (AAV) or naked plasmid DNA vectors. 
     Still preferably the vector belongs to the group of helper-dependent adenoviral vectors. 
     In a preferred embodiment the deficiency of AAT is due to a mutation of the AAT gene. 
     Preferably the mutation of the AAT gene causes a substitution of glutamate into lysine at amino acid position 342 and/or a substitution of serine into phenylalanine at amino acid position 53 (S iiyama ) and/or a deletion of phenylalanine at amino acid position 52 (M malton ) of the AAT protein (SEQ ID No. 9). 
     Still preferably the AAT deficiency is characterized by an accumulation of a wild type and/or mutated AAT protein in a tissue. 
     Yet preferably the accumulation of the wild type and/or mutated AAT protein further comprises the formation of wild type and/or mutated AAT aggregates in the tissue. 
     Preferably the tissue is liver. 
     Still preferably, the vector as described above comprises a liver specific promoter and, optionally, regulatory sequences. 
     Preferably the liver specific promoter is phosphoenolpyruvate carboxykinase (PEPCK) promoter consisting essentially of the sequence of SEQ ID No. 1. 
     Still preferably the liver regulatory sequence is the liver specific enhancer Locus Control Region (LCR) from the apoE locus consisting essentially of the sequence of SEQ ID No. 6. 
     In a preferred embodiment the vector of the invention comprises essentially the nucleotide sequence of SEQ ID No. 8. 
     It is a further object of the invention a host cell transformed by the vector as defined above. 
     It is a further object of the invention a viral particle containing the vector as defined above. 
     It is a further object of the invention a pharmaceutical composition comprising the vector of the invention or the host cell of the invention or the viral particle of the invention for use in the gene therapy of a pathological condition characterized by a deficiency of alpha-1-antitrypsin (AAT). 
     It is a further object of the invention a method for gene therapy of a pathological condition characterized by a deficiency of alpha-1-antitrypsin (AAT) of a subject in need thereof, said method comprising administering a suitable amount of the pharmaceutical composition as defined above. 
     It is a further object of the invention the TFEB protein, synthetic or biotechnological functional derivative thereof, peptide fragments thereof, chimeric molecules comprising the TFEB protein, synthetic or biotechnological functional derivative thereof for use in the treatment of a pathological condition characterized by a deficiency of alpha-1-antitrypsin (AAT). 
     Preferably, the TFEB protein consists essentially of the amino acid sequence of SEQ ID No. 4. 
     In the present invention a synthetic or biotechnological functional derivative of a protein, peptide fragments of a protein, chimeric molecules comprising the TFEB protein, synthetic or biotechnological functional derivative thereof are defined as molecules able to maintain the therapeutic effect of TFEB, i.e. the treatment of a pathological condition characterized by a deficiency of alpha-1-antitrypsin (AAT). 
     In the present invention, the viral vector may be selected from the group of: adenoviral vectors, adeno-associated viral (AAV) vectors, pseudotyped AAV vectors, herpes viral vectors, retroviral vectors, lentiviral vectors, baculoviral vectors. Pseudotyped AAV vectors are those which contain the genome of one AAV serotype in the capsid of a second AAV serotype; for example an AAV2/8 vector contains the AAV8 capsid and the AAV 2 genome 35 . Such vectors are also known as chimeric vectors. Naked plasmid DNA vectors and other vectors known in the art may be used to deliver a TFEB gene according to the present invention 36 . Other examples of delivery systems include ex vivo delivery systems, which include but are not limited to DNA transfection methods such as electroporation, DNA biolistics, lipid-mediated transfection, compacted DNA-mediated transfection. Typically, a viral vector can accommodate a transgene (i.e., a TFEB gene described herein) and regulatory elements. 
     Various methods may be used to deliver viral vectors encoding a TFEB gene described herein into a subject in need of treatment. For example, a viral vector may be delivered through intravenous or intravascular injection. Other routes of systemic administration include, but are not limited to, intra-arterial, intra-cardiac, intraperitoneal and subcutaneous or via local administration such as muscle injection or intramuscular administration. 
     The vector of the invention, in particular the HDAd vector may be injected at a dose range between 1×10e10 viral particles (vp)/kg and 1×10e13 vp/kg. 
     A dose range between 1×10e11 and 1×10e12 vp/kg is more likely to be effective in humans because these doses are expected to result in large transduction efficiency of the liver. 
     The vector of the invention, in particular the AAV vector may be injected at doses between 1×10e11 vector genomes (vg)/kg and 1×10e13 vg/kg are expected to provide high liver transduction 26 . 
     Adenoviral vector genomes do not integrate into the genome of the transduced cells and therefore vector genomes are lost in actively dividing cells 37 . Should TFEB expression fade over time, to maintain phenotypic correction it would be possible to re-administer a vector with a different serotype to overcome the neutralizing anti-Ad antibody elicited with the first administration 38,39 . 
     The present invention provides pharmaceutical compositions comprising: a) an effective amount of a vector as described herein or an effective amount of a transformed host cell as described herein, and b) a pharmaceutically acceptable carrier, which may be inert or physiologically active. 
     As used herein, “pharmaceutically-acceptable carriers” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, and the like that are physiologically compatible. Examples of suitable carriers, diluents and/or excipients include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combination thereof. In many cases, it will be preferable to include isotonic agents, such as sugars, polyalcohols, or sodium chloride in the composition. In particular, relevant examples of suitable carrier include: (1) Dulbecco&#39;s phosphate buffered saline, pH˜7.4, containing or not containing about 1 mg/ml to 25 mg/ml human serum albumin, (2) 0.9% saline (0.9% w/v sodium chloride (NaCl)), and (3) 5% (w/v) dextrose; and may also contain an antioxidant such as tryptamine and a stabilizing agent such as Tween 20. 
     The pharmaceutical compositions encompassed by the present invention may also contain a further therapeutic agent for the treatment of a pathological condition characterized by a deficiency of alpha-1-antitrypsin (AAT). 
     The compositions of the invention may be in a variety of forms. These include for example liquid, semi-solid, but the preferred form depends on the intended mode of administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions. The preferred mode of administration is parenteral (e.g. intravenous, intramuscular, intraperinoneal, subcutaneous). In a preferred embodiment, the compositions of the invention are administered intravenously as a bolus or by continuous infusion over a period of time. In another preferred embodiment, they are injected by intramuscular, subcutaneous, intraarticular, intrasynovial, intratumoral, peritumoral, intralesional, or perilesional routes, to exert local as well as systemic therapeutic effects. 
     Sterile compositions for parenteral administration can be prepared by incorporating the vector or host cell as described in the present invention in the required amount in the appropriate solvent, followed by sterilization by micro filtration. As solvent or vehicle, there may be used water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combination thereof. In many cases, it will be preferable to include isotonic agents, such as sugars, polyalcohols, or sodium chloride in the composition. These compositions may also contain adjuvants, in particular wetting, isotonizing, emulsifying, dispersing and stabilizing agents. Sterile compositions for parenteral administration may also be prepared in the form of sterile solid compositions which may be dissolved at the time of use in sterile water or any other injectable sterile medium. 
     There may be used pharmaceutically acceptable solutions, suspensions, emulsions, syrups and elixirs containing inert diluents such as water, ethanol, glycerol, vegetable oils or paraffin oil. These compositions may comprise substances other than diluents, for example wetting, sweetening, thickening, flavoring or stabilizing products. 
     The doses depend on the desired effect, the duration of the treatment and the route of administration used and may be determined easiy by the skilled person in the art using known methods. 
     As well known in the medical arts, dosages for any one patient depends upon many factors, including the patient&#39;s size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. 
    
    
     
       The present invention will be illustrated by means of non-limiting examples referring to the following figures. 
         FIG. 1 . (A) ATZ stably transfected mouse Hepa1,6 cell lysates were immunoprecipitated with anti-AAT antibody (which recognize also ATZ) following pulse labeling. The intracellular, newly synthesized ATZ decreased more rapidly in TFEB transfected cells as compared to control untreated cells. Kinetics of disappearance of intracellular ATZ determined by densitometric analysis from 2 independent experiments showed statistical significant reduction at 120 and 300 minutes of chase (*p&lt;0.05). HeLa cells stably over-expressing TFEB (HeLa-CF7) secrete reduced amount of ATZ in the media (B) and accumulate less ATZ intracellularly (C) 24 hours after transfection with a plasmid expressing ATZ. Two independent samples from HeLa and HeLa-CF7 cells are shown. Media ATZ and ELISA and band quantifications were performed on n=3 per condition (*p&lt;0.05). 
         FIG. 2 . (A) PAS staining was markedly reduced in livers from mice injected with HDAd-TFEB as compared to saline or HDAd-AFP injected mice (magnification 20×). (B) Immunofluorescence of livers from HDAd-TFEB injected mice showed a marked reduction of ATZ globules as compared to controls (magnification 20×). (C) Co-staining of liver specimens for LAMP-1 (red) and ATZ (green) shows an increase in LAMP-1 staining in HDAd-TFEB injected mice. For HDAd-TFEB injected livers the selected field presented shows a region of increased LAMP-1 signal and absent ATZ surrounding an area of increased ATZ staining (magnification 63×). (D) Western blot analysis show a significant reduction in ATZ band intensities in livers of HDAd-TFEB injected PiZ mice as compared to HDAd-AFP and saline injected controls. Three of five representative mice are shown in the western blot analysis. Quantification of band intensities was performed on n=5 mice per group (*p&lt;0.05). (E) ELISA for ATZ on hepatic extracts show a statistically significant (*p&lt;0.05) reduction in the amount of ATZ in HDAd-TFEB injected mice (n=5 per group). (F) Western blot for LC-3 showed an increase in LC3-I in mice injected with HDAd-TFEB as compared to controls. Quantification of band intensities is shown in  FIG. 8 . 
         FIG. 3 . (A) Effect of hepatocyte TFEB expression on serum levels of human ATZ in PiZ mice. Serum levels were determined by ELISA specific for human AAT (n=5 per group; *p&lt;0.05). (B) Monomer-polymer analysis of mouse livers. Representative bands from 3 mice for each treatment group are shown. The graph shows densitometric quantification of n=5 mice from each treatment group. A marked, statistically significant decrease in both ATZ monomer and polymer was observed in HDAd-TFEB injected mouse livers as compared to either saline or HDAd-AFP injected control mice (*p&lt;0.05). 
         FIG. 4 . Livers from either saline injected (A-D) or HDAd-TFEB injected (E-H) mice were prepared for EM as described in online Methods. (A-C) Arrows show membrane bound inclusions in the hepatocyte cytoplasm, while asterisks indicate nuclei. (D) This panel shows the area corresponding to the region outlined by dash box in C. Arrow indicates the place where inclusion (asterisk) is connected with RER membranes. (E, F) Hepatocytes in HDAd-TFEB injected animals do not exhibit inclusions in cytoplasm. Asterisks indicate nuclei. (G) Example of hepatocyte that still contains inclusion (asterisk). (H) The image shows the area corresponding to the region outlined by dash box in G. Arrows indicate double membrane around inclusion (asterisk). Scale bar: 1.5 μm (A-C), 340 nm (D), 1.3 μm (E, F), 950 nm (G), 380 nm (H)  FIG. 5 . Livers from either saline-injected (A, B, F, G) or HDAd-TFEB injected (C-E) mice were prepared for immuno-EM of ATZ as described in online Methods. (A) Region of the hepatocyte cytoplasm exhibits large membrane bound inclusion (asterisk) similar to that shown in  FIG. 4B . (B) Higher magnification from the region outlined by dash box in A reveals dense gold ATZ labeling over inclusion body (asterisk). (C) Arrows indicate diffuse ATZ signal along the RER membranes of hepatocyte in HDAd-TFEB-treated mice. (D, G) Liver cells from HDAd-TFEB-injected mice exhibit ATZ-corresponding gold particles within the lysosome-like structures (arrows) where ATZ signal is frequently associated with intraluminal vesicles (arrowheads). (E, F) Lysosomes (asterisks) and their intraluminal vesicles (arrowheads) show little or no ATZ in control animals. (H) Immuno-gold ATZ labeling densities (n=20 cells) in lysosome-like structures were quantified as described in online Methods and expressed in arbitrary units (AU). Scale bar: 1.8 μm (A), 520 nm (B), 320 nm (C), 250 nm (D), 220 nm (E-G). 
         FIG. 6 . Sirius red staining (A) (magnification 20×) and hepatic hydroxyproline content (B) showed a reduction of fibrosis in PiZ mice injected with HDAd-TFEB compared to saline ore HDAd-AFP injected mice (n=5 per group; *p&lt;0.05). (C) Western blot analysis showed that PiZ mice injected with HDAd-TFEB have a significant reduction of cleaved caspase-12 compared to saline or HDAd-AFP injected mice (representative bands from 3 independent animals are shown). Band densities of cleaved 42 KDa caspase-12 band normalized for β-actin levels showed a statistical significant (*p&lt;0.05) reduction in HDAd-TFEB injected mice (n=5 per group). (D) Western blot analysis showed that PiZ mice injected with HDAd-TFEB have a significant reduction of cleaved PARP-1 compared to saline or HDAd-AFP injected mice (representative bands from 3 independent animals are shown). Densities of cleaved 89 and 24 KDa PARP-1 bands normalized for β-actin levels showed a statistical significant reduction in HDAd-TFEB injected mice (n=5 per group; *p&lt;0.05, **p&lt;0.01). 
         FIG. 7 . HDAd-TFEB vector. ITR means sequences necessary for virus replication. ψ means the packaging sequence that ensures packaging of the vector DNA into virions. “stuffer” means human genomic DNA sequences or other non-transcribed DNA sequences used to increase the vector insert size. “WPRE” means the mRNA-stabilizing post-transcriptional regulatory element from the woodchuck hepatitis virus. “BGHpA” means the bovine growth hormone polyadenylation signal. This sequence represents a specialized termination sequence for protein expression in eukaryotic cells. HDAd-TFEB contains the human TFEB transgene under the control of a liver-specific PEPCK promoter. The expression cassette contains the ApoAI intron, the woodchuck hepatitis post-transcriptional regulatory element (WPRE), the Locus Control Region (LCR) from the apoE locus and the human growth hormone poly A (GHpA). Adenoviral inverted terminal repeats (ITR) and packaging signal (Ψ) are shown. Not drawn to scale. 
         FIG. 8 . Quantification of LC3 in HDAd-TFEB injected mice. Densitometric analysis of LC3 bands on Western blot performed on n=5 mice per group. No change in LC3-II was detected while LC3-I was increased in HDAd-TFEB injected mouse livers. **p&lt;0.01; NS=not statistically significant. 
     
    
    
       
     
       
         
           
               
               
            
               
                 SEQUENCES: 
                   
               
               
                 PEPCK PROMOTER 
               
               
                 (SEQ ID No. 1) 
                   
               
               
                 ctttggggagtcctaagagggcagctggcaatggacacctagcagtccctttgagacttatttcagatggagctgtagaaagatgccatggc 
                   
               
               
                 tcacagtgcctccctgggaagggggcagagggctgcccagtgaggcctcttgcgagcaggaaatcaccagagacaaggaaagaccag 
               
               
                 accccaggatgacctcagttaggccttgcccgactgtcctcagagtcccattctctgtgtcctggttcttttagaagatcatggacctccaggtc 
               
               
                 atttcgtaaccggaatctgcctgcggggggttttgacaagctatggtatagtgtatgtgggggtactgacgaattggaagatcatggagaccc 
               
               
                 cttctcctcctccatcattggtctgccacatccctcccaggcgactcacagcagagagaccttggatgtatgtagggtgctttaaaactccagc 
               
               
                 tgagttacagtctctcctttctgttttcaccttaaccttccagggatgcaaacccacgacaggtttagcagcagagtggaggctggccatgaat 
               
               
                 ctcagagaaagtgctcactggaaaggctggtttagcccaggcctgatgtggaggcactgagctggacgttctageggggttgacacccaa 
               
               
                 cagtttacatagggggaggccacccctcctgagcagtctcggtgacttgaagaggaagccgcttcttctgtaccaacacagaagctccagc 
               
               
                 gaacccccagaatgctggcagtgtgggtgctatgtaaaagtatttacatagctttgtagagtgagccaagcccagtctgtttgggatgactctt 
               
               
                 cacagtgcctcgaatctgtcacacgtcttagtaagcagagtcacagagtttctgtcacatcatcctcctgcctacagggaagtaggccatgtc 
               
               
                 cctgccccctactctgagcccagctgtgggagccagccctgcccaatgggctctctctgattggcttctcactcacttctaaactccagtgag 
               
               
                 caacttctctcggctcgttcaattggcgtgaaggtctgtgtcttgcagagaaggttcttcacaactgggataaaggtctcgctgctcaagtgta 
               
               
                 gcccagtagaactgccaagccccttcccctcctctccctagactcttggatgcaagaagaatccaggcagctccaagggtgattgtgtccaa 
               
               
                 cctagaatgtcttgaaaaagacattaaggggactagagaagacaggggatccaacggttctctgcagcccagcctgactgacatgtaactct 
               
               
                 tctggttctcaccagccagctggacctgettagtattctttctgcctcagtttcccagcctgtacccagggctgtcatagttccatttcaggcagt 
               
               
                 agtaatgaatgagctgacataaaacatttagagcaggggtcagtatgtatatagagtgattattctatatcaggcattgcctcctcggaatgaag 
               
               
                 cttacaatcacccctccctctgcagttcatcttggggtggccagaggatccagcagacacctagtggggtaacacaccccagccaactcgg 
               
               
                 ctgttgcagactttgtctagaagtttcacgtctcagagctgaattcccttctcatgacctttggccgtgggagtgacacctcacagctgtggtgtt 
               
               
                 ttgacaaccagcagccactggcacacaaaatgtgcagccagcagcatatgaagtccaagaggcgtcccggccagccctgtccttgacccc 
               
               
                 cacctgacaattaaggcaagagcctatagtttgcatcagcaacagtcacggtcaaagtttagtcaatcaaacgttgtgtaaggactcaactatg 
               
               
                 gctgacacgggggcctgaggcctcccaacattcattaacaacagcaagttcaatcattatctccccaaagtttattgtgttaggtcagttccaa 
               
               
                 accgtgctgaccatggctatgatccaaaggccggccccttacgtcagaggcgagcctccaggtccagctgaggggcagggctgtcctcc 
               
               
                 cttctgtatactatttaaagcgaggagggctagctaccaagcacggttggccttccctctgggaacacacccttggccaacaggggaaatcc 
               
               
                 ggcgagacgctctgag 
               
               
                   
               
               
                 APOA1 INTRON 
               
               
                 (SEQ ID No. 2) 
                   
               
               
                 atcctgcgagaaggaggtgcgtcctgctgcctgccccggcactctggctccccagctcaaggttcaggccttgccccaggccgggcctct 
                   
               
               
                 gggtacctgaggtcttctcccgctctgtgcccttctc 
               
               
                   
               
               
                 HUMAN TFEB (NCBI GeneID = 7942; Accession no.: nt = NM_007162.2, protein = NP_009093.1) 
               
               
                 (SEQ ID No. 3) 
                   
               
               
                 atggcgtcacgcatagggttgcgcatgcagctcatgcgggagcaggcgcagcaggaggagcagcgggagcgcatgcagcaacaggct 
                   
               
               
                 gtcatgcattacatgcagcagcagcagcagcagcaacagcagcagctcggagggccgcccaccccggccatcaatacccccgtccactt 
               
               
                 ccagtcgccaccacctgtgcctggggaggtgttgaaggtgcagtcctacctggagaatcccacatcctaccatctgcagcagtcgcagcat 
               
               
                 cagaaggtgcgggagtacctgtccgagacctatgggaacaagtttgctgcccacatcagcccagcccagggctctccgaaacccccacc 
               
               
                 agccgcctccccaggggtgcgagctggacacgtgctgtcctcctccgctggcaacagtgctcccaatagccccatggccatgctgcacatt 
               
               
                 ggctccaaccctgagagggagttggatgatgtcattgacaacattatgcgtctggacgatgtccttggctacatcaatcctgaaatgcagatg 
               
               
                 cccaacacgctacccctgtccagcagccacctgaatgtgtacagcagcgacccccaggtcacagcctccctggtgggcgtcaccagcag 
               
               
                 ctcctgccctgcggacctgacccagaagcgagagctcacagatgctgagagcagggccctggccaaggagcggcagaagaaagacaa 
               
               
                 tcacaacttaattgaaaggagacgaaggttcaacatcaatgaccgcatcaaggagttgggaatgctgatccccaaggccaatgacctggac 
               
               
                 gtgcgctggaacaagggcaccatcctcaaggcctctgtggattacatccggaggatgcagaaggacctgcaaaagtccagggagctgga 
               
               
                 gaaccactctcgccgcctggagatgaccaacaagcagctctggctccgtatccaggagctggagatgcaggctcgagtgcacggcctcc 
               
               
                 ctaccacctccccgtccggcatgaacatggctgagctggcccagcaggtggtgaagcaggagctgcctagcgaagagggcccagggga 
               
               
                 ggccctgatgctgggggctgaggtccctgaccctgagccactgccagctctgcccccgcaagccccgctgcccctgcccacccagccac 
               
               
                 catccccattccatcacctggacttcagccacagcctgagctttgggggcagggaggacgagggtcccccgggctaccccgaacccctg 
               
               
                 gcgccggggcatggctccccattccccagcctgtccaagaaggatctggacctcatgctcctggacgactcactgctaccgctggcctctg 
               
               
                 atccacttctgtccaccatgtcccccgaggcctccaaggccagcagccgccggagcagcttcagcatggaggagggcgatgtgctgtga 
               
               
                   
               
               
                 HUMAN TFEB protein 
               
               
                 (SEQ ID No. 4) 
                   
               
               
                 MASRIGLRMQLMREQAQQEEQRERMQQQAVMHYMQQQQQQQQQQLGGPPTPAINTP 
                   
               
               
                 VHFQSPPPVPGEVLKVQSYLENPTSYHLQQSQHQKVREYLSETYGNKFAAHISPAQGSP 
               
               
                 KPPPAASPGVRAGHVLSSSAGNSAPNSPMAMLHIGSNPERELDDVIDNIMRLDDVLGYI 
               
               
                 NPEMQMPNTLPLSSSHLNVYSSDPQVTASLVGVTSSSCPADLTQKRELTDAESRALAKE 
               
               
                 RQKKDNHNLIERRRRFNINDRIKELGMLIPKANDLDVRWNKGTILKASVDYIRRMQKDL 
               
               
                 QKSRELENHSRRLEMTNKQLWLRIQELEMQARVHGLPTTSPSGMNMAELAQQVVKQE 
               
               
                 LPSEEGPGEALMLGAEVPDPEPLPALPPQAPLPLPTQPPSPFHHLDFSHSLSFGGREDEGP 
               
               
                 PGYPEPLAPGHGSPFPSLSKKDLDLMLLDDSLLPLASDPLLSTMSPEASKASSRRSSFSME 
               
               
                 EGDVL 
               
               
                   
               
               
                 WPRE 
               
               
                 (SEQ ID No. 5) 
                   
               
               
                 cgcgaatcaacctctggattacaaaatttctgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaat 
                   
               
               
                 gcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctatttatgaggagttgtggcccgt 
               
               
                 tgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcaactcctttccgggactta 
               
               
                 cgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgt 
               
               
                 ggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcg 
               
               
                 gccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgactcggat 
               
               
                 ctccctttgggccgcctccccgc 
               
               
                   
               
               
                 LCR 
               
               
                 (SEQ ID No. 6) 
                   
               
               
                 ggcgcgccgacgcgcatgctcctctagactcgaggaattcggtaccccgggttcgaaatcgataagcttgatatcgaattcctgcaggctca 
                   
               
               
                 gaggcacacaggagtttctgggctcaccctgcccccttccaacccctcagttcccatcctccagcagctgtttgtgtgctgcctctgaagtcc 
               
               
                 acactgaacaaacttcagcctactcatgtccctaaaatgggcaaacattgcaagcagcaaacagcaaacacacagccctccctgcctgctg 
               
               
                 accttggagctggggcagaggtcagagacctctctgggcccatgccacctccaacatccactcgaccccttggaatttcggtggagagga 
               
               
                 gcagaggttgtcctggcgtggtttaggtagtgtgagagggtccgggttcaaaaccacttgctgggtggggagtcgtcagtaagtggctatgc 
               
               
                 cccgaccccgaagcctgtttccccatctgtacaatggaaatgataaagacgcccatctgatagggtttttgtggcaaataaacatttggttttttt 
               
               
                 gttttgttttgttttgttttttgagatggaggtttgctctgtcgcccaggctggagtgcagtgacacaatctcatctcaccacaaccttcccctgc 
               
               
                 ctcagcctcccaagtagctgggattacaagcatgtgccaccacacctggctaattttctatttttagtagagacgggtttctccatgttggtcagc 
               
               
                 ctcagcctcccaagtaactgggattacaggcctgtgccaccacacccggctaattttttctatttttgacagggacggggtttcaccatgttggtc 
               
               
                 aggctggtctagaactcctgacctcaaatgatccacccacctaggcctcccaaagtgcacagattacaggcgtgggccaccgcacctggcc 
               
               
                 aaatttttaatttttttctagagatagggtcttactgtgttgcccaggctggtgtcaaactcctgggctcaagcagatcctcctgcctcagcttcc 
               
               
                 caaagtggtgggattataggtgtgagccactgcgcccagtcagtagccccctctttgcccctcactgagccctactggatgttcttggttgtgtg 
               
               
                 acagtttccccatctattaaacagaaacccctatagcagaggggaggatgaggttggaaaatcaggagcattgttattctattcttgtgggatc 
               
               
                 ggggaagcagacatctgggtggatgtttggggaatgctgggctcagttgaggaagtaggggggcccctggggcttacagggactggaa 
               
               
                 gctctgagctggccagagggatgttgcaatcctgccagggtcttgtctatgctgtccctttcacaaccatccccctaccgccaggctgacacg 
               
               
                 tggttgtgggggcacaaggccagccgaactagagtctgaggctgggctgaggacaccctccccatcagctgccagggtcactggcggtc 
               
               
                 aaaggcagctggtggggaaggaattggactccagccctgggggacggatgtggtgatggtgggaagcaggcttggtgccaggagggg 
               
               
                 catcagagggtgaataagagcagatagagtgtttgggggaggtagccagccaaagggggtgaggcccggtggaagggaagaagggg 
               
               
                 catacactcagagctttgcagctgaaggttttaattttttgagatggggtctcactctgtctcaccaggctggagtgcagtggcgcaatcacag 
               
               
                 ctcactgcagcccgggggatccggagagctcgtcgacggcgcgcc 
               
               
                   
               
               
                 GHpA 
               
               
                 (SEQ ID No. 7) 
                   
               
               
                 Aattcctgcagcccgggtggcatccctgtgacccctccccagtgcctctcctggccctggaagttgccactccagtgcccaccagccttgtc 
                   
               
               
                 ctaataaaattaagttgcatcattttgtctgactaggtgtccttctataatattatggggtggaggggggtggtatggagcaaggggcaagttgg 
               
               
                 gaagacaacctgtagggcctgcggggtctattgggaaccaagctggagtgcagtggcacaatcttggctcactgcaatctccgcctcctgg 
               
               
                 gttcaagcgattctcctgcctcagcctcccgagttgttgggattccaggcatgcatgaccaggctcagctaatttttgtttttttggtagagacgg 
               
               
                 ggtttcaccatattggccaggctggtctccaactcctaatctcaggtgatctacccaccttggcctcccaaattgctgggattacaggcgtgaa 
               
               
                 ccactgctcccttccctgtccttctgattttaaaataactataccagcaggaggacgtccagacacagcataggctacctggccatgcccaac 
               
               
                 cggtgggacatttgagttgcttgcttggcactgtcctctcatgcgttgggtccactcagtagatgcct 
               
               
                   
               
               
                 Construct 
               
               
                 (SEQ ID No. 8) 
                   
               
               
                 AAACATCATCAATAATATACCTTATTTTGGATTGAAGCCAATATGATAATGAGGGGGTGGAGTTTGTG 
                   
               
               
                 ACGTGGCGCGGGGCGTGGGAACGGGGCGGGTGACGTAGTAGTGTGGCGGAAGTGTGATGTTGCAAGT 
               
               
                 GTGGCGGAACACATGTAAGCGACGGATGTGGCAAAAGTGACGTTTTTGGTGTGCGCCGGTGTACACA 
               
               
                 GGAAGTGACAATTTTCGCGCGGTTTTAGGCGGATGTTGTAGTAAATTTGGGCGTAACCGAGTAAGATT 
               
               
                 TGGCCATTTTCGCGGGAAAACTGAATAAGAGGAAGTGAAATCTGAATAATTTTGTGTTACTCATAGCG 
               
               
                 CGTAATATTTGTCTAGGGCCGCGGGGACTTTGACCGTTTACGTGGAGACTCGCCCAGGTGTTTTTCTCA 
               
               
                 GGTGTTTTCCGCGTTCCGGGTCAAAGTTGGCGTTTTGATATCAAGCTTATCGATACCGTAAACAAGTCT 
               
               
                 TTAATTCAAGCAAGACTTTAACAAGTTAAAAGGAGCTTATGGGTAGGAAGTAGTGTTATGATGTATGG 
               
               
                 GCATAAAGGGTTTTAATGGGATAGTGAAAATGTCTATAATAATACTTAAATGGCTGCCCAATCACCTA 
               
               
                 CAGGATTGATGTAAACATGGAAAAGGTCAAAAACTTGGGTCACTAAAATAGATGATTAATGGAGAGG 
               
               
                 ATGAGGTTGATAGTTAAATGTAGATAAGTGGTCTTATTCTCAATAAAAATGTGAACATAAGGCGAGTT 
               
               
                 TCTACAAAGATGGACAGGACTCATTCATGAAACAGCAAAAACTGGACATTTGTTCTAATCTTTGAAGA 
               
               
                 GTATGAAAAATTCCTATTTTAAAGGTAAAACAGTAACTCACAGGAAATACCAACCCAACATAAAATCA 
               
               
                 GAAACAATAGTCTAAAGTAATAAAAATCAAACGTTTGCACGATCAAATTATGAATGAAATTCACTACT 
               
               
                 AAAATTCACACTGATTTTGTTTCATCCACAGTGTCAATGTTGTGATGCATTTCAATTGTGTGACACAGG 
               
               
                 CAGACTGTGGATCAAAAGTGGTTTCTGGTGCGACTTACTCTCTTGAGTATACCTGCAGTCCCCTTTCTT 
               
               
                 AAGTGTGTTAAAAAAAAAGGGGGATTTCTTCAATTCGCCAATACTCTAGCTCTCCATGTGCTTTCTAGG 
               
               
                 AAACAAGTGTTAACCCACCTTATTTGTCAAACCTAGCTCCAAAGGACTTTTGACTCCCCACAAACCGA 
               
               
                 TGTAGCTCAAGAGAGGGTATCTGTCACCAGTATGTATAGTGAAAAAAGTATCCCAAGTCCCAACAGCA 
               
               
                 ATTCCTAAAAGGAGTTTATTTAAAAAACCACACACACCTGTAAAATAAGTATATATCCTCCAAGGTGA 
               
               
                 CTAGTTTTAAAAAAACAGTATTGGCTTTGATGTAAAGTACTAGTGAATATGTTAGAAAAATCTCACTG 
               
               
                 TAACCAAGTGAAATGAAAGCAAGTATGGTTTGCAGAGATTCAAAGAAAATATAAGAAAACCTACTGT 
               
               
                 TGCCACTAAAAAGAATCATATATTAAATATACTCACACAATAGCTCTTCAGTCTGATAAAATCTACAG 
               
               
                 TCATAGGAATGGATCTATCACTATTTCTATTCAGTGCTTTGATGTAATCCAGCAGGTCAGCAAAGAATT 
               
               
                 TATAGCCCCCCTTGAGCACACAGAGGGCTACAATGTGATGGCCTCCCATCTCCTTCATCACATCTCGAG 
               
               
                 CAAGACGTTCAGTCCTACAGAAATAAAATCAGGAATTTAATAGAAAGTTTCATACATTAAACTTTATA 
               
               
                 ACAAACACCTCTTAGTCATTAAACTTCCACACCAACCTGGGCAATATAGTGAGACCCCATGCCTGCAA 
               
               
                 AAAAAAAAAAATTAGCCAGGCATGGTAGCATGTACCTGTAGTCCCAGCTACTTGAGAGGTGAGGTGG 
               
               
                 GAAAATCACTTTAGTGCAGGATGTTGAGGCTGGAGTGAACTGTGATTGTGCCACTGCACTCCAGCCTG 
               
               
                 GACAATAGAGCAAGACCTTGTCTCAAAAAAATGCATTAAAAATTTTTTTTAAATCTTCCACGTATCAC 
               
               
                 ATCCTTTGCCCTCATGTTTCATAAGGTAAAAAATTTGATACCTTCAAAAAAACCAAGCATACCACTATC 
               
               
                 ATAATTTTTTTTAAATGCAAATAAAAACAAGATACCATTTTCACCTATCAGACTGGCAGGTTCTGATTA 
               
               
                 AATGAAATTTTCTGGATAATATACAATATTAAGAGAGACTGTAGAAACTGGGCCAGTGGCTCATGCCT 
               
               
                 GTAATCCCAGCACTTTGGGAGGCTGGGTAACATGGCGAACCCTGTTTCTACAAAATAAAAATATTAGC 
               
               
                 TGGGAGTGGTGGCGCACACCTATAGTCCCAGCTACTCAGGAGGCTGAGGTGGAAGGATCGCTTGAAC 
               
               
                 CCAGGAGGTTGAGACTGCAGTGAACTGTGATCATTCTGCTGCACTGCACCCCAGCCTGGGCAACAGAG 
               
               
                 ACCTTGTCTCAAAAAAAAAAAAAAAAGAGACAAATTGTGAAGAGAAAGGTACTCTCATATAACATCA 
               
               
                 GGAGTATAAAATGATTCAACTTCTTAGAGGAAAATTTGGCAATACCAAAATATTCAATAAACTCTTTC 
               
               
                 CCCTTGACCCAGAAATTCCACTTGAATAAAGCTGAACAAGTACCAAACATGTAAAAGAATGTTTCTTC 
               
               
                 TAGTACAGTCGGTAAGAACAAAATAGTGTCTATCAATAGTGGACTGGTTAAATCAGTTATGGTATCTC 
               
               
                 CATAAGACAGAATGCTATGCAACCTTTAAAATATATTAGATAGCTCTAGACACACTAATATTAAAAGT 
               
               
                 GTCCAATAACATTTAAAACTATACTCATACGTTAAAATATAAATGTATATATGTACTTTTGCATATAGT 
               
               
                 ATACATGCATAGGCCAGTGCTTGAGAAGAAATGTGTACAGAAGGCTGAAAGGAGAGAACTTTAGTCT 
               
               
                 TCTTGTTTATGGCCTCCATAGTTAGAATATTTTATAACACAAATATTTTGATATTATAATTTTAAAATAA 
               
               
                 AAACACAGAATAGCCAGACATACAATGCAAGCATTCAATACCAGGTAAGGTTTTTCACTGTAATTGAC 
               
               
                 TTAACAGAAAATTTTCAAGCTAGATGTGCATAATAATAAAAATCTGACCTTGCCTTCATGTGATTCAGC 
               
               
                 CCCAGTCCATTACCCTGTTTAGGACTGAGAAATGCAAGACTCTGGCTAGAGTTCCTTCTTCCATCTCCC 
               
               
                 TTCAATGTTTACTTTGTTCTGGTCCCTACAGAGTCCCACTATACCACAACTGATACTAAGTAATTAGTA 
               
               
                 AGGCCCTCCTCTTTTATTTTTAATAAAGAAGATTTTAGAAAGCATCAGTTATTTAATAAGTTGGCCTAG 
               
               
                 TTTATGTTCAAATAGCAAGTACTCAGAACAGCTGCTGATGTTTGAAATTAACACAAGAAAAAGTAAAA 
               
               
                 AACCTCATTTTAAGATCTTACTTACCTGTCCATAATTAGTCCATGAGGAATAAACACCCTTTCCAAATC 
               
               
                 CTCAGCATAATGATTAGGTATGCAAAATAAATCAAGGTCATAACCTGGTTCATCATCACTAATCTGAA 
               
               
                 AAAGAAATATAGCTGTTTCAATGAGAGCATTACAGGATACAAACATTTGATTGGATTAAGATGTTAAA 
               
               
                 AAATAACCTTAGTCTATCAGAGAAATTTAGGTGTAAGATGATATTAGTAACTGTTAACTTTGTAGGTAT 
               
               
                 GATAATGAATTATGTAAGAAAACAACAGGCCGGGCGGGTTGGTTCACACGTGTAATCCCAGCACTTTG 
               
               
                 GGAGGCTGAGGCAGGCAGACTGCCTGAGCTCAGGAGTTCGAGACCAGCCTGGGCAACACGGTGAAAT 
               
               
                 CCCGTCTCTACTAAAAATACAAAAAAATTAGCCGGGTGTGGTGACACATGCCTGTAGTCCCAGCTACT 
               
               
                 TGGGAGGCTGAGGCAGGAGAATCACTTGAACCTGGGAGGTGAAGGTTGCAGTGAGCCAAGATGGCAC 
               
               
                 CACTTCACTCCAGCCTGGGAAACAGAGCAAGACTCTGTCTCTGAGCTGAGATGGCACCACTTCACTCC 
               
               
                 AGCCTGGGAAACAGAGCAAGACTCTGTCTCAAAAAAAACAAAACACACAAACAAAAAAACAGGCTG 
               
               
                 GGCGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGTGGATCACCTGAGGTCA 
               
               
                 GGAGTTCCAGACCAGCCTTGTCAACATGGTGAAACCTCCCCCCGCCGTCTCTACTAAAAATACAAAAA 
               
               
                 TTAGCCAGGCGTGGTGGCAGGAGCCTGTAATCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATCGCTT 
               
               
                 GTACCCAGAAGGCAGAGGTTGCACTGAGCTGAGATGGCACCATTGCACTCCAGCCTGGGGGACAAGA 
               
               
                 GCGAGATTTCGTCTTTAAAAAACAAAAACAAAACAAAAAACCATGTAACTATATGTCTTAGTCATCTT 
               
               
                 AGTCAAGAATGTAGAAGTAAAGTGATAAGATATGGAATTTCCTTTAGGTCACAAAGAGAAAAAGAAA 
               
               
                 AATTTTAAAGAGCTAAGACAAACGCAGCAAAATCTTTATATTTAATAATATTCTAAACATGGGTGATG 
               
               
                 AACATACGGGTATTCATTATACTATTCTCTCCACTTTTGAGTATGTTTGAAAATTTAGTAAAACAAGTT 
               
               
                 TTAACACACTGTAGTCTAACAAGATAAAATATCACACTGAACAGGAAAAACTGGCATGGTGTGGTGG 
               
               
                 CTCACACTTGTAATCCCAGTGCTTTGGGAGGCTGAGACAGGAGAGTTGCTTGAGGCCAGGAGTTCAAG 
               
               
                 ACCGACATGGGGAATGTAGCAAGACCCCGTCCCTACAAAAAACTTTGTAAAAATTTGCCAGGTATGGT 
               
               
                 GGTGCATACCTGTAGTCCCAGCTACTCGGGAGGCGGAGGCAGAAGGAATCACTTGAGCCCAGGAGTT 
               
               
                 TGAGGCTGCAGTGAGCTACGATCATACCACAGCACTCCAGCGTGGACAACAGAGTAAGACCCTATCTC 
               
               
                 AAAAACAAAACAAAACAAAACAAACAAAAAAAACCACAAGAAAAACTGCTGGCTGATGCAGCGGCT 
               
               
                 CATGCCTGTAATCCCAGTATTTTGGGAGGCCCAGGTGGGCGTATCACCTGAGGTCAGGAGTTAGAGAC 
               
               
                 CAGCCTGGCCAACATGGTGAAACCCCATCTCTACTAAAAATACAAAATTAGCCAGGCATGTGGCACGC 
               
               
                 GCCTGTAGTCCCAGTTACTGGGAGGCTGAAGCAGGAGGATCACCTGAGCCCGGGAGGTGGAGGTTGC 
               
               
                 AGTGAGCCGAGATCACACCACTGCACTCCAGCCTGGGTGACACAGCAATACCCTACCTCAAAATAAA 
               
               
                 AAAGAAAAAGAAAAGAAAAGTTGCTGTCCCCGCTACCCCAATCCCAAATCCAAACAGCCTCTCTCATC 
               
               
                 TCACAGTAAGGGGGAAAAATCACCCAAAAAAGCTAAGTGATCTTTTGAAAACCCAAACTCTTAGAAG 
               
               
                 TCTAAGATTATTATAGTCAACTCATGAAGTGTCATCATAAAAGATACTCTAATATTATTTAAGTAGAAC 
               
               
                 CACATATTGGTTGTCTTGGTATGTCTAGCCCCTGGCATACAAAATATTTAATAACACTGATATGGTACC 
               
               
                 TGTGATGTGAAAATGTACTATGAGTACAGCTTTATAAATACTATATATGTACCTATATACAGAAAAAA 
               
               
                 ATACAACAAAATCATAAAAGCACTTATCTTTGAAAGAGGAGTTACAGCAATTTTATTTAGTTCTTTATT 
               
               
                 GCTTTGCTATATATTCTAAATTTTTTTCAATGAATATATATCACTTTTAAAAAAATTCAATGGTCTTTCT 
               
               
                 TATAAATTATCTTTGGCAGCATGCGTTTTTATATATACATATAAAATGTATGGGAAATTTTTAAAGGAT 
               
               
                 ACATTAAATTAAAGCAAAATATACAAACAAAAAATCAGAATACAAAAAGATAAAAAGATTGGGAAG 
               
               
                 GGAGGGAGGGAGTAAGGAGGAAGGGTGGGTGGGTATAGAGAAATATACCAAATAATGGTAAGAAGT 
               
               
                 GGGGTCTTGACACTTTCTACACTTTTTTTAAATAAAAAAAATTTTTTTCTCTCTCTTTTTTTTTTTTAGAG 
               
               
                 ACGAAGTCTCGCTATGTTGCCCAGGCTGGTCTTGAACTCCTGGGATCAAGAGATCCTCCTGCCTCAGCC 
               
               
                 TCCCAAGGTGCTTGGATTACAGGTGTGAGCCACCACGCCTGGTCACTTTCTACACTTTAATATATATAT 
               
               
                 TTTTTCATTTTCAATGTCATTTTTATTAGTTAATTTATAATACCCATTCACCATTATATTCAAAGTCTATT 
               
               
                 TGAAGAAATAAACCAGAAAGAATGAAATACTCTAGCTCACATGCTATTCAATACTAAATTACCTTTCA 
               
               
                 AATCACATTCAAGAAGCTGATGATTTAAGCTTTGGCGGTTTCCAATAAATATTGGTCAAACCATAATT 
               
               
                 AAATCTCAATATATCAGTTAGTACCTATTGAGCATCTCCTTTTACAACCTAAGCATTGTATTAGGTGCT 
               
               
                 TAAATACAAGCAGCTTGACTTTTAATACATTTAAAAATACATATTTAAGACTTAAAATCTTATTTATGG 
               
               
                 AATTCAGTTATATTTTGAGGTTTCCAGTGCTGAGAAATTTGAGGTTTGTGCTGTCTTTCAGTCCCCAAA 
               
               
                 GCTCAGTTCTGAGTTCTCAGACTTTGGTGGAACTTCATGTATTGTCAGGTTGGCCCGTAATACCTGTGG 
               
               
                 GACAACTTCAGCCCCTGTGCACATGGCCAGGAGGCTGGTTGCAAACATTTTCAGGTAGGTGGACCAGG 
               
               
                 ACATGCCCCTGGTCATGGCCAGGTGGAGGCATAGTGCTATACAGCAGGCAGAAGTCAATATTGATTTG 
               
               
                 TTTTTAAAGAAACATGTACTACTTTCATAAGCAGAAAAAATTTCTATTCTTGGGGGAAAAGATTATGC 
               
               
                 CAGATCCTCTAGGATTAAATGCTGATGCATCTGCTAAACCTTCACATATCAGAACATATTTACTATAGA 
               
               
                 AAGAATGAAAATGGGACATTTGTGTGTCACCTATGTGAACATTCCAAAAATATTTTACAACAACTAAG 
               
               
                 TATTTTATAAATTTTATGAACTGAAATTTAGTTCAAGTTCTAGGAAAATACAAACCTTGCTAGATATTA 
               
               
                 TAAAAATGATACAATATATATTCATTTCAGGCTCATCAGAATATATCTGTTATCACTTGACAAGAATGA 
               
               
                 AAATGCACCATTTTGTAGTGCTTTAAAATCAGGAAGATCCAGAGTACTAAAAATGACTTCTTCCTTGA 
               
               
                 AGCTTACTCACCAACTTCCTCCCAGTTACTCACTGCTTCTGCCACAAGCATAAACTAGGACCCAGCCAG 
               
               
                 AACTCCCTTGAAATATACACTTGCAACGATTACTGCATCTATCAAAATGGTTCAGTGCCTGGCTACAG 
               
               
                 GTTCTGCAGATCGACTAAGAATTTGAAAAGTCTTGTTTATTTCAAAGGAAGCCCATGTGAATTCTGCCC 
               
               
                 AGAGTTCATCCCAGATATGCAGTCTAAGAATACAGACAGATCAGCAGAGATGTATTCTAAAACAGGA 
               
               
                 ATTCTGGCAATATAACAAATTGATTTCCAATCAAAACAGATTTACATACCATACTTATGTCAAGAAGTT 
               
               
                 GTTTTGTTTTATTGCATCCTAGATTTTATTTTTTTGATTTATGGTTTACTTTAAGCATAAAAAATTTGTCA 
               
               
                 ATACAACTCTTCCCAAAAGGCATAAACAAAAATTCATAAAACTTGCATCACTTGAGATACTTCAGGTA 
               
               
                 TGAATTCACAACTTTGTTACAACTTACTATATATATGCACACATATATATATATTTGGGTATATTGGGG 
               
               
                 GGGTTCTAATTTAAGAAATGCATAATTGGCTATAGACAGACAGTTGTCTGGAATGAAAATCAATACTT 
               
               
                 TTGCTATAATCGATTACTGAAATAATTTTACTTTCCAGTAAAACTGGCATTATAATTTTTTTTAATTTTT 
               
               
                 AAAACTTCATAATTTTTTGCCAGACTGACCCATGTAAACATACAAATTACTAATAATTATGCACGTCAC 
               
               
                 ATCTGTAATAATGGCCTTCATGTAAACATTTTTGTGGTTTACACATAAAATCTCTAATTACAAAGCTAT 
               
               
                 ATTATCTAAAATTACAGTAAGCAAGAAAATTAATCCAAGCTAAGACAATACTTGCAACATCAATTCAT 
               
               
                 CATCTGTGACAAGGACTGCTTAAGTCTCTTTGTGGTTAAAAAGGAAAAAAAAAAAAAAGACATGTTG 
               
               
                 GCCAGATGCGGTGGCTCACACCTGTAATCCCAGCACTTTGGGAGGCTGAGGTGGGCGGATCACCCCTG 
               
               
                 GCCTGCCCAACATGGTGAAACCCCGTCTCTACTAAAAACACAAAAATTAGCTGGGCGTGGTGGCGGGC 
               
               
                 GCCTGTAATTCCAGCTACTCGGGAGGCTGAGGCAGGAGAATTGCTAGAACCCAGGAGGCAGAGATTG 
               
               
                 CAGTGAGCTGAGATTGCACCATTGCACTACAGTCTGGGCAACAAAAGTGAAACTCCATCTTAAAAAAA 
               
               
                 AAAAGACAATGTTCGTGGGTCCAAACAAGACTTAATGGAAGTGAGTCTAAAAATGAGCTATGTGGGC 
               
               
                 CAGGCGTAGTGGCTCCCACCTGTAATCCCAGCACTTTGGGAGGCCGAAGCAGGCAGATCATGAGGTCA 
               
               
                 GGAGATGGAGACCATCCTGGCCAACACGGTGAAATCCTGTCTCTACAAAAATTAGCTGGGCGTGGTGG 
               
               
                 TGCCTGCCTGTAATCCCAGCTACTCAGAAGGCTCAGGCAGGAGAATCGCTTGAACCAGGGAGTCGGTG 
               
               
                 GCTAGAGTGAGCCGAGATTTGCATCACTGCACTCCTGCCTGGTGACAGAGCAAGACTCCATCTCAAAA 
               
               
                 AAAACAAACAAAAATAAAAGATAAAAATGAGCTATGTGAATTAAAAGAGGTATAACAATAGATAAA 
               
               
                 CCATATTTTATTTAATTCCTAGTAATGAGTAATATTTCCAAACTTCTGGAATGGGCAGAAATTGCTAGT 
               
               
                 TGGCATATTTTTACCTTTTATATTCAGATACATTAAAATTCTCAAAAAAAAACACCTCAAAGCAGATGA 
               
               
                 TCCGCCATCTCCTTGGATAATTTGTGTTAACTCAGGATAACAGAAAACCAAAATTATGAGTTACTGAT 
               
               
                 GCAATATTCCTAAATGTAAAAATAATTAAAGCTAATAGTAGATTCATCTTCCAATTTCATATCAGTCTT 
               
               
                 ACAAATAAACTACATATATAACTTGCTTGCCTTCCCTTCTGAGGGATAAAGCTGTTAGAAGAATTAAA 
               
               
                 ATCAGCATTCTTGACTATTCAACCAAGGGAGGGATAAATTATTACTCATTCTAGGGACATGGGCTCAT 
               
               
                 AACTACTACATGTGTAAGGACATGAATTTACCCAATATTACAATTTTTCCTTTTATTAGTGTGTACAGT 
               
               
                 GGAAGAATAGACATGTTCACTCTGGACAAAAAAAAAATTATACTTATCAGTTATCAGAAGCACAATGC 
               
               
                 TGAAGACAGTAGTTCCATAACAATTTGAAGTATGTGATCGAACTAGTAGATTATCTTAGTAGTAGTGA 
               
               
                 ATTATTGTAAATGTTAGTAATTTGGCAGCCACTGGGCAGAAAAATAAGAATTGAGGCTCAATATTGAT 
               
               
                 ATTAATGGTGGTGATTGACACATAAATTTTATCAAGTCTACACAATATAAAATTACAGAAAGGTAGAA 
               
               
                 GAGTATACCAGTACAACTTCAACATATCTTCACTACAAGGGAGTAAAATGACATGGCCTAGTTACTAT 
               
               
                 CTAATGAACTGCAGAAAACTAAAAGAAAACTCCAAGGCAACTCTTCTCTGCTGATCTGGTTGGTCCTT 
               
               
                 TTCCTACCTTTTGCAATACCCAGATACAAACAATGGATAGAAAACAAAGTAGACTTGTAGTATGCAGG 
               
               
                 TCACAGTGCTAAATTCACAGAAAGAAACCCCTGAACTGAACTGCTCTATTTCCTGGTGGTCACAAAGA 
               
               
                 GTAATTCTGGTTTACACCTACAGATTGATGTCAATCTACACCCTGTTGATAACAGTGTGGCCAAGGAC 
               
               
                 AAAAAAAAGGTGCTCCGTTTTACCAATTCTGTAAAAAATTATTGGCAGGGTAAGCTCGGCTAGGGCAG 
               
               
                 GATTACATTTCTAGGACTACCATCCCCGAAATTTAGAAGATATTATATCCACATAAAGCATATCTTTCA 
               
               
                 CATTAATTTGCAAAAATCTAAAAGCTTTTTCTTAGCTCAAGTGTGTCCAAGTTTACCCTGGCAGTTTAA 
               
               
                 AACGATAGTTACAAGCAGCATGGGTTGTATCAGACACATTTGAGGGCCAATTTCATGTAAGTGATATT 
               
               
                 GGGCAAGTTACTTCAACTATCTGTGCCTCCAAGGTCATACTAGTGTTTATTTACCTAAAGGGTACCTGT 
               
               
                 TATGTAACTTTAGGGTGTTTACATTAGATAATGCCTGCAAAATATTTACTTCAACGCCTAAAACATAGT 
               
               
                 TAAGTATTCAATAAATACCTACTATTGTCACTACTAACTTAAAAGTTTAGAGATTAAGAGCAGAATCT 
               
               
                 GGGGTGAGACAAACTTAGGTTCAAATCCTAGTATTGTTGGGTAATCTTGGGCAAGTTACTTAACCTCTC 
               
               
                 TGATTTGTGTAATTTAAAAAATTAGTTAATATACATAACAGGGCTTAGAAGAGTATCTAGCACATAGC 
               
               
                 ACCATTTAAGCATTTGTTATTGCTAACATGCAAACAATTTAAGGGAAAGAAATTTTTTAAAAAGGAAG 
               
               
                 AGGGATTTGCAAACTAAAAACAATGAGTATCTTATGTTCAAAGAAAACTAACAAACAGCCAGCTCTA 
               
               
                 GCAATAATTAAATTCACTATATACTGGGGCAGGCATCACACCCCAAAGCTAAAAGCGTCTACCTAGGC 
               
               
                 CAGGCACGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAAGCAGAGGCGGGCAGATCGCTTGAGCT 
               
               
                 CAGGAGTTCAAGACCAGCCTGGACAACATGGCAAAACACCATCTCTACAAAAAATACAAATATTAGG 
               
               
                 CCGGGCGCAGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCAAGGCGGGTGGATCACCTGAG 
               
               
                 ATCAGGAGTTCGAGAGTAGCCTGGCCAACATGGTGAAACCTCGTCTCTATTAAAAATACAAAAAATTA 
               
               
                 GCCAGGCATGGTGGCAGGCGCCTGTAATCCCAGCTACTCAGGGGGATGAGGTAGGAGAATCGCTTGA 
               
               
                 ACCCGGGAGGCAGAGGTTGCACTGAGCCGAGATCATGCCACTGTACTCCAGCCCGGGCAACAAGAGC 
               
               
                 GAAACTCCATCTCAAAAAATAAATAAATAAATAAATAAAATAAAGTACAAATATTAGCCAGGGATGG 
               
               
                 TGGTGCGCACCTGTAGTCCCAGCTACTTGGGAGGCTGAAGTGGGAGAATCCCCTGAGCCTGGGGAGA 
               
               
                 ATCACCCGAGCCCGGGAAGTCGAGGCTGCAGTGAGCAGTGATTGTGCCACTGCACTCCATCCTAGGTG 
               
               
                 ACAGAGTGAGACCCTGTCTCAAAAAAAAGAAATTGGCAGAATTAAGTAAGTTGATGTTTAGAGATGA 
               
               
                 AAAATCAACATTTTTTCCTCAGCAACTGAATAAAAACAACAGCCACTACCATTTTTTTGAGTACCTATT 
               
               
                 TGTAGCCTATTTTTTAACTGGTATTACTCGAGAGAGAGAGAGCTAGGTTCGAGACAGAGCTCCTTCTCT 
               
               
                 TAATAACTGTATGACCTAGGGTATGTCTGTTAGCCTCTCTGAGGCTTCAAAGGTTCCTCATCTGTAAAA 
               
               
                 TGGTAATAATCATACCATTGCTACAGGGCTGTTTTGAAGACTAATTAGGACTATGTAAGTAAACATGA 
               
               
                 TGATGGCTATTATTACTGTTCCCCGCCAGGGGCCATGCAAGGGTTGCTGATTCACATAGACTGTCTTAT 
               
               
                 AATCCTCTCAATAACTCCAAGAGGTAGCCAGCACCTCAGATATACATAAAATGACTTAAGCCCAGAGA 
               
               
                 GGTGAAGTAAGTTGCCCACAGCCACACAACTAGTAAATAGCCCAAACAAGCTGGATTCCCAGTTAGA 
               
               
                 CTCCGTTAATAGCACTGCTCTTTACCTTAAGTCATTACAATGCCTAATATGAAATAGAATCGCTTCTTT 
               
               
                 CTTAGGGTTCAAGTGGTTAATTATTTAATGTATTCATTCAACAAACCATCATCGAGGACCTCTTACAAG 
               
               
                 CCAAGTACTGTGCTAAGTGCTAGAGTTACGGCGGTGATTCCTGCCCTTAAAAAGTTTTAGTGGGAGAA 
               
               
                 ACAACAGGTAACCAGGTCATTGCCAAAACAACAAAAATAATCATAATAAAGCAGGCTAAAGCATATT 
               
               
                 TAACTGGCCGGGGTTTTGACTATTTTAGCAAGCATGATCAGAACGGTTGAGGAGGGAGGCCAGCAGCT 
               
               
                 TGGCCGGTTCAACAAACAAGAAAAAACCAGTGAGGGTGGAGCTAAGATACCAGAGGCTGATTACGGT 
               
               
                 TAAGAATGTTCTTGAAGGTAAGGACCAGATTCTCATTTTCTATATCCTGGGGCATCGGTCAGCATGGA 
               
               
                 ATCTGGATTCTAGCACATGTGAATTTCGGCTTGAAATGACCTAATGCCTTTTCCCTAGTTCCTTCGTGT 
               
               
                 GTCAAATACGCATGGTTACCGCTACCAGAGCTGTAGTGGGGCTTCAATGAGGCCATGAGCATCTCCAT 
               
               
                 AAAGATGAACTACAGTGTGTGCAAAACTAAAGGCAAAACCTGGTCCCCACACGCCCTCCCAGGTGGT 
               
               
                 CGCTTTCCGTGCCGAGGCCCCTCCAGAGGTGCCCCGAGAACCTCACCATCGCACCCCAAACTTCCAGG 
               
               
                 GAAGGGCCTCTCCCGAGAAAGCCCCCACGCCCCCACCCCGCGCCATCATTCCCGAATCTGCCCTCGGC 
               
               
                 CCCTCCCCGCAGCACGCTCGCAGGCGGCACATGTCAACCAAAACGCCATTTCCACCTTCTCTTCCCACA 
               
               
                 CGCAGTCCTCTTTTCCCAGGGCTCCCCCGAGGAGGGACCCACCCCAAACCCCGCCATTCCGTCCTCCCT 
               
               
                 GCCGCCCTCGCGTGACGTAAAGCCGAACCCGGGAAACTGGCCGCCCCCGCCTGCGGGGTTCCCTGGGC 
               
               
                 CCGGCCGCTCTAGAACTAGTGGATCCCAATTGAAGGCCTGGTCTAAATGACTCCAAAATCACCACTTA 
               
               
                 ATTCAAGAGACTGATTTCCCTGAGTCAGGCCCCTTAAAGCAGCTATTTCAATGGGACAGGGAAACAAC 
               
               
                 CCTAGGATCTGGATTAGAATCACTTGGGGGCTGCCACACCCCCAGGGCTCTGATCCTGCCCTTCTCCCA 
               
               
                 CACGCACATTCACATACTGCTGCAGTGACCTTCCATTTCTAATGGGTTCCTGGGCCATCTGTCAGGTAT 
               
               
                 AGGGAATGGAAAAGGGGTTGGGGAGGCTCTGCTTCAGAAAGTTTGTGTCAGGGGCTCCCAGAGCCTC 
               
               
                 CACAGATAGATAGCAGGGGTCCCCACCCTACCATGGCAGCTATAAATGTGATCAACATTTATTGGCCT 
               
               
                 AGGATACAGCAGTTAGCAAAATGCCTGATGTAGTTCCCACTCCGTGGAGGTTGCAGGCTAGCTCTTTC 
               
               
                 CTAATGAGCTTTACAGCAGAAGCTGTTTTATCGTTAAGTGCCCCACAGAGACACTTTACCAGGAGGCT 
               
               
                 GGGAGAGTTCTCCAGATTTGGGAGAGGCGCAGAGACAGTGTGTGAGCCGAGCCCTGTCTCAGCAATC 
               
               
                 CACCTGGAGGAGCTAGAGTATCCTCCTCCCTTTACCATTCAGACCGAGAGAAAAAGCCCAGCTTGTGT 
               
               
                 GCACCCTCGTGGGGTTAAGGCGAGCTGTTCCTGGTTTAAAGCCTTTCAGTATTTGTTTTGATGTAAGGC 
               
               
                 TCTGTGGTTTGGGGGGGAACATCTGTAAACATTATTAGTTGATTTGGGGTTTGTCTTTGATGGTTTCTAT 
               
               
                 CTGCAATTATCGTCATGTATATTTAAGTGTCTGTTATAGAAAACCCACACCCACTGTCCTGTAAACTTT 
               
               
                 TCTCAGTGTCCAGACTTTCTGTAATCACATTTTAATTGCCACCTCGTATTTCACCTCTACATTTGAAATC 
               
               
                 TGGCGTCTGTTTCAAGCCAGTGTGTTTTTTCTTCGTTCTGTAATAAACAGCCAGGAGAAAAGTGCCTCT 
               
               
                 ATGTTTTTATTTTTCAAGGGAGTATTCAGTACCTACAAACCCAAGTCAGGAAGCCTGCTAGTGGCTTTG 
               
               
                 GTTCTTTCAGAGGCTGCTCGATGCCTTGTGTGTCAGAAAGAAAGATTCAGCAGTTTTGCATCATGGCA 
               
               
                 AAGAAGCCTGTTATTTTGGGGCTCAGCCCCTCATTTTATAGAGGATGAAACAGAGGGGGATGGGAGGT 
               
               
                 CACAAAGACAACTGCCCCGGGAGCAGGTGTGGGGGAGACTTGCCCTGAGGGTCTAGACGCTCTGCAC 
               
               
                 CACCGTCCTGTCTCCCTTGCTGAAGACCACACATGCCCTTCTTTGACCAGACCCTGCCACCTGATAGGC 
               
               
                 CAGGACCTGGTAGGCGGGTACCCAGGTTTCATGGATGGAACCACATCTCCCCAAAAGTGGGGAGGTA 
               
               
                 GCTACTGGGATGCACGCCTCCCGCCATGTGCTATAGGAGAGCAGCTGAAGCAACAGTTGGGATCAGAT 
               
               
                 GTAGTCACAATTGAATGCATCATCACATTTATCCCTCTAAGTGGCTGGGAGAGTTGATATCCTCATCCC 
               
               
                 TAAGGTACAAAATGTTCCAATTTGATCAGTGGCTTTCAGGAGCTGAGAAAGGCATGTGCTCTGAGGCA 
               
               
                 GAGCTGTTATGTCCCGCAGAGCCTAAAAATGCTCTAAGAACATGCTCCCTGCCAAAATTCTCAATGGC 
               
               
                 TGTGACAAGGGACAACGATCGACCAATGGGGGTGGAAGCAGACCTCCGCAGTCCAGGGGCCAGAGCT 
               
               
                 AGGACAGAGGGGTCGGAGAAAGAGTCATTTTCCCAACACTCCAGCTCTTGGCCAGTCCTCACACAGTC 
               
               
                 CCCTCCTGCTTCCTGCTGAGAGAGATATCCTCATAGGTCTGGGTAAAGTCCTTCAGTCAGCTTTCATTC 
               
               
                 CCTGTCACCAACTTTGTCTCTGTTCTCCCTGCCCGTCTCAGGCAGCACTCCTCAGGAAACCTCTCCAAG 
               
               
                 AGCCAGCCTCACTGCAGCGCCCACTATTGTCCCTCTGCCTCAAGTGTCCCATCCATGCCAGGCCCCAGG 
               
               
                 CAGGCTGCAGCTTTCCCTCAGGGCCACACCAAAGCACTTGGGCTCAGCTGTGCTGTCCCCCTCCATCAC 
               
               
                 TGAGCTCAGGGGCAGCAGGGGTGGGGTGCCAGGAGGCCCATTCACCCTTCTCTGGCTCTGTGTTGGAC 
               
               
                 CCACCTGCCCAGCCACTGCTGCTTAGAACCTACCCGCTGGGAAAATGAAGCCCTCCCGGAGGGGCCAC 
               
               
                 CTCAACCTGAGAGCCTCACGGATCACAGTTGTCCCCACTCAGCTCTGCCAGCCCTCAGAGACCCATAG 
               
               
                 ATAAAAGCTGAGCTTGGCTCGCAGAGCTGGTTCCATCTTCCATTCCCAGAGGGTTCAACTTCCTACCCC 
               
               
                 AACCACACAGGGAACCTCAAGGCTGAGCCAGTGTGGGCTGCAGTGCAGACCAGCTTCCTGGACACGT 
               
               
                 CCTGCCACCTGACCCCAGGCTGGCCTCACTGCCCCTGGCACTCCTGACCCTATCCTCATTCCTCCTGGC 
               
               
                 AGTGCGTGTTCTGCCATTCCGCTTTCCCTTAGCTGTCCTCTCACTGTACTGTCAGCTTCTCCTTTTCCAG 
               
               
                 GTGCCCCCCAGGGGCTTTCCACATGACCCTGTCACCCCACAGCCCATCCAGCACCAATTCCAGCTCTCT 
               
               
                 GCCACCCTTCAAAGGAGTGACAGTGCCCTGCTTCACCTCCCACTCACCCCTCAACCCAGAGCAATCTG 
               
               
                 GCTCCAGTCTTGCCTCCTTCCCCCTAAGTACTCTAGTCACAGTTCCAAATTCCTCCTGGTCATAAAGCC 
               
               
                 AAATGAAGCTTCCTGGTCCTCAGCGGACTTGCCACTTCAGCAGTACTGGACTCTCTCCTCCCAGAAACC 
               
               
                 TGTTTCCCCTTGGCTCCTGGAGCCCACACTCTGCTGGAATCCTTCTGCCTCTCTGGCCTGTAGCCTGGCC 
               
               
                 CTCTCTCCCAACCTGAGGTCCATTCTCTCCTGCTCCTCCACAAGATGTTGCTCCTTCCATTACTTCCTCC 
               
               
                 CTCTCAACCAAAGCTCCTTCATTAGCTCTTTATCTTCTGGTTTCTTCCCCTGGGCAGACGAATGGATTCA 
               
               
                 AGAGCCTGTGGCCCAGCAGCCCAGCACTCCAGGATCTCAGCACTTCAGCATCCCAGTACCCTAGCATC 
               
               
                 TCAATACCCCAGCACCCCAGCACCATAGTATTCCAGCACCCCATTGTCCAAGCATCTCAGCACTCCAG 
               
               
                 CATCCCAGCACCCCAACACTCCAGCAGCCCAGAATCTCAGCACCCTAGCACTGCAGCATCTCAGGACC 
               
               
                 CCAGCACTTCAGCATCCCAGCACACTAGTACTCCAGCATCTCGGCACCCCAGCACCTAGGCATCCCAA 
               
               
                 CACCCAGCACCCCAGCACTTAAGCATCCCACCACTACAGTATCTCAACACTCCAGCACCCCAGCACCA 
               
               
                 TAGTGTTCCAGCACCCCAGCATCCCAACACCCCAGCACTTAAGCATCCCAACACCTCGGCATCCCAAC 
               
               
                 ACCCCAGCACTGCAGCATCTCAGCACCTTAGCATCCCAGTGCCCTAGCATCTCAATGCTCCAGCACAC 
               
               
                 CAGTACTACAGTATTCCAGCACCCCAGCACTCCAGCATCTCAGCACTGCAGCACTGCAGCACTCCAGC 
               
               
                 ATCCCAAAATCCCAGCATCCCAACACCCCAGCAGACCAGCAGACCAGCATCTCAGCACCGCAGCATCC 
               
               
                 AAGGACTATCCCAGCATCCCAGCAACCCAGCACCTCAGCATCCCAACACCCCAGCATTTCAGCATGGC 
               
               
                 AACACCCCAGTACCCCAGCACTTCAGCACCCCAGTATCCCAGCATCTCAGCGACCCAGTATCACAAAA 
               
               
                 CCTCAGCATCCTAGCACCCCAGCACCCCAGCACCTTAGCACCTTAGCATCCCAGCATCTCAGCGCCTC 
               
               
                 AGCATCTTGATATTCTGGCTGAGGTCAGCGTGGTGTATCTAGTCAGGGTCCTAACTTTCACTTCGCAGG 
               
               
                 GAAATGCTGCTGGACTGGGTCTCATGTTGGGCTGAAGCTCTCTAGACCCCTTGAAGACAGCATAAAAG 
               
               
                 AGCTTGGAGACGCTGGGTGTCCCCCATGGAAGAGTTCACTCTCATCCTGCTTTGACAACAGCCTTCTCT 
               
               
                 GGGGTCCCTCACGGGCCCCTCTTTCTTACTGCAAGTTTGTCTCTGAGAAGACTGTGATGCAGAAGTCAC 
               
               
                 TCAGCTGCCTGTGGCTCCTGAAGAGCTGAAGGTGGAGGCCTGTAGGCCTCCCTATGAGAGGCGCAGAA 
               
               
                 AAAACCATGATTGCTAGTGGGGAGGTGCTCCCTCTACAACCCACTCCATAATCTGCCCCCGCCCAGCT 
               
               
                 CTGAGGCCAGCCCCAGGGGAAAATGCCAGATCCCCAGGGAGGTGTGTGAGACCTCAGGGGCTCCCTC 
               
               
                 CTCCCTTACAGCAGGCTCAGGCCCCTGGGGGCCTCAGGGCCAAGGTCTGTGGGTAAGCTACTATCTCT 
               
               
                 CACTTGTCCTCTAGCCACAAAAGCCAGGGAGATCTGGCAATGGACATGAGGTTCTGAAGAAGCACAT 
               
               
                 ATGACTGGCTTCCTAATGCGTGGTTGTTCAGTGATTCAATAAACACGCATGGGCCAGGCATGGGGAAA 
               
               
                 TAGACAAACATGATCCCCAACCTCTCCCAGAGTGAACTGGGAGGGAGGAGTGTTCATCCCTCAGGATT 
               
               
                 ACACCAGAGAAACAAACCAGCAGGAGATATATATGGTTTTGGGGGGTCAAGAAAGAGGAAAAACCTG 
               
               
                 GCAAGGCAAGTCCAAAATCATAGGACAGGCTGTCAGGAAGGGCAGCCTGGAACCTCTCAAGCAGGAG 
               
               
                 CTGATGCTGCAGTCCACAGGCAGAATTTCTTCTTCCTCGGGGAAATCTCAGCTTTGTTCTTAAGGCCTT 
               
               
                 TCAACTGATTGGCTGAGGTCTGCCCCTTCCCCCACATTCTCCAGGATAATCTTCCTTACTTAAAGTCAA 
               
               
                 CTATTAATCACAGCTACAAAATCCCTTCACAGCTACACATAGATCAGTGTTTGATTGACGAACAGCCC 
               
               
                 CTACAGCCTAGCCAAGTTGACACATAAAACTAACCATCACAGGGGGACAAATGATGTAAACACATCA 
               
               
                 ACAAATAAAACAGTAACAAGTTAAGGTCTATGGAAAAAACACAGAAGGGGCAGAGAGAAAGAAAGC 
               
               
                 AAGAAGGAGAGTCCCAGTTTGCTAGGGCTTGTGGGAAGTGGGGAGCAGTTCTCTTTAGCTAGGATATT 
               
               
                 TGGGAAAGGCATATCTGAAGGAGTGATATTTGAGCTTAGATTAAAAGATGGGAAGGAGCAAGCCATG 
               
               
                 CAAAGAGCTAGGATGTTCCAAGCAGAGACGGAACAGCAAGTGCAAATGTCAGGAGGAATAGAAGGA 
               
               
                 GGCTGGTGGGTGGGGTCCAGTGAGCAAGAGGAGGGCAGGCAGGAGAGGGGATGGGGAGGTGGGCAG 
               
               
                 GCCCAGACCACCCAGGGCCCTGGAGACTATCCTGATCCAACAAGGGAAGCCTTGAGTCACTTCAGTGT 
               
               
                 CCATGTGGAGAATGGACCTCAGACTGAATGAGGGAGGCAGTAAGGAGGGCCTCTACCTCCAGGGCTT 
               
               
                 CGCCCTGTGGACTGCGCATAGACATCTCCAACTCAGAAAGTCTGAACCAAACTTTCCATAGTTCCCCC 
               
               
                 AAGTCTGGGCATCCTCCTACTCAGTGAAAGGCAGCCATCACACCTCCCTGCCCTGCTCCCGGATGCCC 
               
               
                 CAAATCCTCTTGGTCTCCAAGTCCAGAACCTGAGACTTGTCCTTGATGTTTGTCTTTCCCTCACCCTTTC 
               
               
                 TGTATTCTGGGAAGATGGGTTTTTTTCCCCCAGATGAATCTGTAAAACTTCTGTGATCACAATAAAAAT 
               
               
                 TCTGGCAGTATTATTTTCTGGAACATGACAAAGTGATTCAAAATTATTTATCTGGAAGACTACAAAAC 
               
               
                 AAGAATAGCCAGGAAATTTCTAAAAAGAAAGAAGAAGGAGGAGGAGAAAGAAGGAGGAGGAAAAG 
               
               
                 GAGGAGAAGAAGAAAAGAAAAAGAACCAAGAAAGGGTTCTAGCTCTACCAAATATTAAAACATATC 
               
               
                 ATGAAGCTATTTAAAACAATATGGTTGTGGATACTGAAAAAGATGTGAATAAAGTGGAAGGAAAATA 
               
               
                 AATAGAAATGCACATGGGGATTGAGACTGTGAAAAAGGCAGCATCTCACATCAGTGAGGGATGTTCA 
               
               
                 ACACCTGGTGTTGGGAAAACTGGCTAGTCATTTAAACCAAACAACTGGGTCCTCTACCTCACTCCTGA 
               
               
                 CATTAAGATACATTTAGATGATTCAAAGAGTAAGACAGAAAAAATAACACGTGAAAACACTATCAGA 
               
               
                 AAACAACGTGGGCCAGGTGTGGTGGGTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCAGAC 
               
               
                 AGATCACCTGAGGTGGGGAGTTCAAGACCAGCCTGACCAACATGGTGAAATCCTGTCTCTACTAAAAA 
               
               
                 TACAAAATTAGCTGAGCGTGGTGGCGCATGCCTGTAATCCCAGCTACTCAGGAGGCCGAGGCAGGAG 
               
               
                 AATCACTTGAACCTGGGAGGCAGAGGTTGTGGTGAGCCGAGATCACGCCATTGCACTCCAGCCTGGGC 
               
               
                 AACAAGAGTGAAAATCCATCTAAAAAAAAAAAAAAAAGCCAAGGTGGATATTTTTATAGTATCAGGG 
               
               
                 TAGATCAAGCTTCTCCAATCATGACATGAAACCCAGAAACCATAAAAGAAAAGAATGATAAAATTGC 
               
               
                 CCACGTAAAGTAAAAAGCTTGCACACAGAAAAACACCATACAGGTTACAAGATGAGCAGCAAAATCA 
               
               
                 GAGAAAAAACATTGCAATTCAGGACACACAGAGGCTATTGTTCCTAATATTTAAAAATAAAAGTAGTG 
               
               
                 GATTGTCTACAAAAAGATGAAGACAAGAATTTCAGAAAACCAAATACTGCATGTTTTCACTTACAAGT 
               
               
                 GGAAGCTAAACACTGAGTACACGTGTACACAAAGAATGGAACCATAGGCCAGGCACCGTGGCTCACG 
               
               
                 CCTGTAATCCCAGTACTTTGCGAGGCCGAAGCGGGCGGATCACCTGAGGTGAGGAGTTCGAGACCATC 
               
               
                 CTGGCCAACATGGTGAAACCCAGTCTCTACTAAAAATACAAAAATTAGCCGGGCGTGGTGGTGGGTGC 
               
               
                 CTGTAATCCCAGCTACTCGGGAGGCTGCGGCAGTAGAATCGCTTGAACCCTGGAGGTGGACCTTGCAG 
               
               
                 TGAGCCGAGATCGCACCACTGCACTCCAGCCTGGGCAACAGAGTGAGACTCCATCTCAAAAAAAAAA 
               
               
                 AAAAGGAATAGAACAATAGACACTGGGGCCTACTTGAGGGAGGAGGGTGAGGATCAAAAACCTGCCT 
               
               
                 ATCAGGTACTATGCTTATTACCTGGGTGGTGAAATAATCTGTACACCAAACCCCAGTGACATGCAATT 
               
               
                 TACCGATGTAACAAACCTGCCCATGTACCCGCTGAACCTAAAATAAAAGTTGGAAAAAAATATAGAA 
               
               
                 ATTTTCTTTGTAATAGCCAAAAACTGCAAACAGCCCAGGTGTCTATTAGTAGAATGCATAAACAAACT 
               
               
                 CGGGCATGTTCATACAATGTAAAACTACTCATCAATAAAAAGTGATACTTCTCAGCAATGAAAAGAAA 
               
               
                 CTAGCTACTGATACCAGCTACAACATGGATGGATTTCAAGTGCTTTATGATGAGAGCAAGAAGCCAGA 
               
               
                 CACAAAAGTGTCTATATATATATACAGTATATATACGTATATATACACATATATACAGTATATATATAC 
               
               
                 ATATACATGTATATATATACTGTATATATACTGTATATATATACACAGTATATATATACATATATACAG 
               
               
                 TGTATATATACTGTGTATATATACATGTATATATACTGTGTATATATACATGTATATATACTGTGTATAT 
               
               
                 ATACATGTATATATACTGTGTATATATACATGTATATATATGTATACTGTATATATACTGTATATATAT 
               
               
                 ATACACATATATACAGTATATATATACAGTATATACTGTATATATACAGTATATACGTGTATATATACA 
               
               
                 TATATACAGTATATATGTAAATATACATATATACAGTATATATGTAAATATACATATATACATGTATAT 
               
               
                 ATATACACTATATATATACATATATAGTGTATATATACATATATACATGTATATATTTACTATATGATT 
               
               
                 CCATTTATATAAAGTGCCAAAACAGTCAAAAATAATCTATGTGGAAAAAATCAACAAAGGGATCCCC 
               
               
                 CGGGCTGCAGGAATTCGAT GGCGCGCC CTTTGGGGAGTCCTAAGAGGGCAGCTGGCAATGGACACCT 
               
               
                 AGCAGTCCCTTTGAGACTTATTTCAGATGGAGCTGTAGAAAGATGCCATGGCTCACAGTGCCTCCCTG 
               
               
                 GGAAGGGGGCAGAGGGCTGCCCAGTGAGGCCTCTTGCGAGCAGGAAATCACCAGAGACAAGGAAAG 
               
               
                 ACCAGACCCCAGGATGACCTCAGTTAGGCCTTGCCCGACTGTCCTCAGAGTCCCATTCTCTGTGTCCTG 
               
               
                 GTTCTTTTAGAAGATCATGGACCTCCAGGTCATTTCGTAACCGGAATCTGCCTGCGGGGGGTTTTGACA 
               
               
                 AGCTATGGTATAGTGTATGTGGGGGTACTGACGAATTGGAAGATCATGGAGACCCCTTCTCCTCCTCC 
               
               
                 ATCATTGGTCTGCCACATCCCTCCCAGGCGACTCACAGCAGAGAGACCTTGGATGTATGTAGGGTGCT 
               
               
                 TTAAAACTCCAGCTGAGTTACAGTCTCTCCTTTCTGTTTTCACCTTAACCTTCCAGGGATGCAAACCCA 
               
               
                 CGACAGGTTTAGCAGCAGAGTGGAGGCTGGCCATGAATCTCAGAGAAAGTGCTCACTGGAAAGGCTG 
               
               
                 GTTTAGCCCAGGCCTGATGTGGAGGCACTGAGCTGGACGTTCTAGCGGGGTTGACACCCAACAGTTTA 
               
               
                 CATAGGGGGAGGCCACCCCTCCTGAGCAGTCTCGGTGACTTGAAGAGGAAGCCGCTTCTTCTGTACCA 
               
               
                 ACACAGAAGCTCCAGCGAACCCCCAGAATGCTGGCAGTGTGGGTGCTATGTAAAAGTATTTACATAGC 
               
               
                 TTTGTAGAGTGAGCCAAGCCCAGTCTGTTTGGGATGACTCTTCACAGTGCCTCGAATCTGTCACACGTC 
               
               
                 TTAGTAAGCAGAGTCACAGAGTTTCTGTCACATCATCCTCCTGCCTACAGGGAAGTAGGCCATGTCCC 
               
               
                 TGCCCCCTACTCTGAGCCCAGCTGTGGGAGCCAGCCCTGCCCAATGGGCTCTCTCTGATTGGCTTCTCA 
               
               
                 CTCACTTCTAAACTCCAGTGAGCAACTTCTCTCGGCTCGTTCAATTGGCGTGAAGGTCTGTGTCTTGCA 
               
               
                 GAGAAGGTTCTTCACAACTGGGATAAAGGTCTCGCTGCTCAAGTGTAGCCCAGTAGAACTGCCAAGCC 
               
               
                 CCTTCCCCTCCTCTCCCTAGACTCTTGGATGCAAGAAGAATCCAGGCAGCTCCAAGGGTGATTGTGTCC 
               
               
                 AACCTAGAATGTCTTGAAAAAGACATTAAGGGGACTAGAGAAGACAGGGGATCCAACGGTTCTCTGC 
               
               
                 AGCCCAGCCTGACTGACATGTAACTCTTCTGGTTCTCACCAGCCAGCTGGACCTGCTTAGTATTCTTTC 
               
               
                 TGCCTCAGTTTCCCAGCCTGTACCCAGGGCTGTCATAGTTCCATTTCAGGCAGTAGTAATGAATGAGCT 
               
               
                 GACATAAAACATTTAGAGCAGGGGTCAGTATGTATATAGAGTGATTATTCTATATCAGGCATTGCCTC 
               
               
                 CTCGGAATGAAGCTTACAATCACCCCTCCCTCTGCAGTTCATCTTGGGGTGGCCAGAGGATCCAGCAG 
               
               
                 ACACCTAGTGGGGTAACACACCCCAGCCAACTCGGCTGTTGCAGACTTTGTCTAGAAGTTTCACGTCT 
               
               
                 CAGAGCTGAATTCCCTTCTCATGACCTTTGGCCGTGGGAGTGACACCTCACAGCTGTGGTGTTTTGACA 
               
               
                 ACCAGCAGCCACTGGCACACAAAATGTGCAGCCAGCAGCATATGAAGTCCAAGAGGCGTCCCGGCCA 
               
               
                 GCCCTGTCCTTGACCCCCACCTGACAATTAAGGCAAGAGCCTATAGTTTGCATCAGCAACAGTCACGG 
               
               
                 TCAAAGTTTAGTCAATCAAACGTTGTGTAAGGACTCAACTATGGCTGACACGGGGGCCTGAGGCCTCC 
               
               
                 CAACATTCATTAACAACAGCAAGTTCAATCATTATCTCCCCAAAGTTTATTGTGTTAGGTCAGTTCCAA 
               
               
                 ACCGTGCTGACCATGGCTATGATCCAAAGGCCGGCCCCTTACGTCAGAGGCGAGCCTCCAGGTCCAGC 
               
               
                 TGAGGGGCAGGGCTGTCCTCCCTTCTGTATACTATTTAAAGCGAGGAGGGCTAGCTACCAAGCACGGT 
               
               
                 TGGCCTTCCCTCTGGGAACACACCCTTGGCCAACAGGGGAAATCCGGCGAGACGCTCTGAGATCCTGC 
               
               
                 GAGAAGGAGGTGCGTCCTGCTGCCTGCCCCGGCACTCTGGCTCCCCAGCTCAAGGTTCAGGCCTTGCC 
               
               
                 CCAGGCCGGGCCTCTGGGTACCTGAGGTCTTCTCCCGCTCTGTGCCCTTCTCATGGCGTCACGCATAGG 
               
               
                 GTTGCGCATGCAGCTCATGCGGGAGCAGGCGCAGCAGGAGGAGCAGCGGGAGCGCATGCAGCAACA 
               
               
                 GGCTGTCATGCATTACATGCAGCAGCAGCAGCAGCAGCAACAGCAGCAGCTCGGAGGGCCGCCCACC 
               
               
                 CCGGCCATCAATACCCCCGTCCACTTCCAGTCGCCACCACCTGTGCCTGGGGAGGTGTTGAAGGTGCA 
               
               
                 GTCCTACCTGGAGAATCCCACATCCTACCATCTGCAGCAGTCGCAGCATCAGAAGGTGCGGGAGTACC 
               
               
                 TGTCCGAGACCTATGGGAACAAGTTTGCTGCCCACATCAGCCCAGCCCAGGGCTCTCCGAAACCCCCA 
               
               
                 CCAGCCGCCTCCCCAGGGGTGCGAGCTGGACACGTGCTGTCCTCCTCCGCTGGCAACAGTGCTCCCAA 
               
               
                 TAGCCCCATGGCCATGCTGCACATTGGCTCCAACCCTGAGAGGGAGTTGGATGATGTCATTGACAACA 
               
               
                 TTATGCGTCTGGACGATGTCCTTGGCTACATCAATCCTGAAATGCAGATGCCCAACACGCTACCCCTGT 
               
               
                 CCAGCAGCCACCTGAATGTGTACAGCAGCGACCCCCAGGTCACAGCCTCCCTGGTGGGCGTCACCAGC 
               
               
                 AGCTCCTGCCCTGCGGACCTGACCCAGAAGCGAGAGCTCACAGATGCTGAGAGCAGGGCCCTGGCCA 
               
               
                 AGGAGCGGCAGAAGAAAGACAATCACAACTTAATTGAAAGGAGACGAAGGTTCAACATCAATGACCG 
               
               
                 CATCAAGGAGTTGGGAATGCTGATCCCCAAGGCCAATGACCTGGACGTGCGCTGGAACAAGGGCACC 
               
               
                 ATCCTCAAGGCCTCTGTGGATTACATCCGGAGGATGCAGAAGGACCTGCAAAAGTCCAGGGAGCTGG 
               
               
                 AGAACCACTCTCGCCGCCTGGAGATGACCAACAAGCAGCTCTGGCTCCGTATCCAGGAGCTGGAGATG 
               
               
                 CAGGCTCGAGTGCACGGCCTCCCTACCACCTCCCCGTCCGGCATGAACATGGCTGAGCTGGCCCAGCA 
               
               
                 GGTGGTGAAGCAGGAGCTGCCTAGCGAAGAGGGCCCAGGGGAGGCCCTGATGCTGGGGGCTGAGGTC 
               
               
                 CCTGACCCTGAGCCACTGCCAGCTCTGCCCCCGCAAGCCCCGCTGCCCCTGCCCACCCAGCCACCATC 
               
               
                 CCCATTCCATCACCTGGACTTCAGCCACAGCCTGAGCTTTGGGGGCAGGGAGGACGAGGGTCCCCCGG 
               
               
                 GCTACCCCGAACCCCTGGCGCCGGGGCATGGCTCCCCATTCCCCAGCCTGTCCAAGAAGGATCTGGAC 
               
               
                 CTCATGCTCCTGGACGACTCACTGCTACCGCTGGCCTCTGATCCACTTCTGTCCACCATGTCCCCCGAG 
               
               
                 GCCTCCAAGGCCAGCAGCCGCCGGAGCAGCTTCAGCATGGAGGAGGGCGATGTGCTGTGAGAATTCC 
               
               
                 GCGAATCAACCTCTGGATTACAAAATTTCTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTT 
               
               
                 ACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCT 
               
               
                 CCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCG 
               
               
                 TGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAACTCCTTT 
               
               
                 CCGGGACTTACGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCT 
               
               
                 GGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCA 
               
               
                 TGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTC 
               
               
                 AATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGC 
               
               
                 CCTCAGACGACTCGGATCTCCCTTTGGGCCGCCTCCCCGCGGCGCGCCGACGCGCATGCTCCTCTAGA 
               
               
                 CTCGAGGAATTCGGTACCCCGGGTTCGAAATCGATAAGCTTGATATCGAATTCCTGCAGGCTCAGAGG 
               
               
                 CACACAGGAGTTTCTGGGCTCACCCTGCCCCCTTCCAACCCCTCAGTTCCCATCCTCCAGCAGCTGTTT 
               
               
                 GTGTGCTGCCTCTGAAGTCCACACTGAACAAACTTCAGCCTACTCATGTCCCTAAAATGGGCAAACAT 
               
               
                 TGCAAGCAGCAAACAGCAAACACACAGCCCTCCCTGCCTGCTGACCTTGGAGCTGGGGCAGAGGTCA 
               
               
                 GAGACCTCTCTGGGCCCATGCCACCTCCAACATCCACTCGACCCCTTGGAATTTCGGTGGAGAGGAGC 
               
               
                 AGAGGTTGTCCTGGCGTGGTTTAGGTAGTGTGAGAGGGTCCGGGTTCAAAACCACTTGCTGGGTGGGG 
               
               
                 AGTCGTCAGTAAGTGGCTATGCCCCGACCCCGAAGCCTGTTTCCCCATCTGTACAATGGAAATGATAA 
               
               
                 AGACGCCCATCTGATAGGGTTTTTGTGGCAAATAAACATTTGGTTTTTTTGTTTTGTTTTGTTTTGTTTTT 
               
               
                 TGAGATGGAGGTTTGCTCTGTCGCCCAGGCTGGAGTGCAGTGACACAATCTCATCTCACCACAACCTT 
               
               
                 CCCCTGCCTCAGCCTCCCAAGTAGCTGGGATTACAAGCATGTGCCACCACACCTGGCTAATTTTCTATT 
               
               
                 TTTAGTAGAGACGGGTTTCTCCATGTTGGTCAGCCTCAGCCTCCCAAGTAACTGGGATTACAGGCCTGT 
               
               
                 GCCACCACACCCGGCTAATTTTTTCTATTTTTGACAGGGACGGGGTTTCACCATGTTGGTCAGGCTGGT 
               
               
                 CTAGAACTCCTGACCTCAAATGATCCACCCACCTAGGCCTCCCAAAGTGCACAGATTACAGGCGTGGG 
               
               
                 CCACCGCACCTGGCCAAATTTTTAATTTTTTTCTAGAGATAGGGTCTTACTGTGTTGCCCAGGCTGGTG 
               
               
                 TCAAACTCCTGGGCTCAAGCAGATCCTCCTGCCTCAGCTTCCCAAAGTGGTGGGATTATAGGTGTGAG 
               
               
                 CCACTGCGCCCAGTCAGTAGCCCCCTCTTTGCCCCTCACTGAGCCCTACTGGATGTTCTTGGTTGTGTG 
               
               
                 ACAGTTTCCCCATCTATTAAACAGAAACCCCTATAGCAGAGGGGAGGATGAGGTTGGAAAATCAGGA 
               
               
                 GCATTGTTATTCTATTCTTGTGGGATCGGGGAAGCAGACATCTGGGTGGATGTTTGGGGAATGCTGGG 
               
               
                 CTCAGTTGAGGAAGTAGGGGGGCCCCTGGGGCTTACAGGGACTGGAAGCTCTGAGCTGGCCAGAGGG 
               
               
                 ATGTTGCAATCCTGCCAGGGTCTTGTCTATGCTGTCCCTTTCACAACCATCCCCCTACCGCCAGGCTGA 
               
               
                 CACGTGGTTGTGGGGGCACAAGGCCAGCCGAACTAGAGTCTGAGGCTGGGCTGAGGACACCCTCCCC 
               
               
                 ATCAGCTGCCAGGGTCACTGGCGGTCAAAGGCAGCTGGTGGGGAAGGAATTGGACTCCAGCCCTGGG 
               
               
                 GGACGGATGTGGTGATGGTGGGAAGCAGGCTTGGTGCCAGGAGGGGCATCAGAGGGTGAATAAGAGC 
               
               
                 AGATAGAGTGTTTGGGGGAGGTAGCCAGCCAAAGGGGGTGAGGCCCGGTGGAAGGGAAGAAGGGGC 
               
               
                 ATACACTCAGAGCTTTGCAGCTGAAGGTTTTAATTTTTTGAGATGGGGTCTCACTCTGTCTCACCAGGC 
               
               
                 TGGAGTGCAGTGGCGCAATCACAGCTCACTGCAGCCCGGGGGATCCGGAGAGCTCGTCGACGGCGCG 
               
               
                 CCAATTCCTGCAGCCCGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGC 
               
               
                 CACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTT 
               
               
                 CTATAATATTATGGGGTGGAGGGGGGTGGTATGGAGCAAGGGGCAAGTTGGGAAGACAACCTGTAGG 
               
               
                 GCCTGCGGGGTCTATTGGGAACCAAGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGC 
               
               
                 CTCCTGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATGACCAG 
               
               
                 GCTCAGCTAATTTTTGTTTTTTTGGTAGAGACGGGGTTTCACCATATTGGCCAGGCTGGTCTCCAACTC 
               
               
                 CTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGATTACAGGCGTGAACCACTGCTCC 
               
               
                 CTTCCCTGTCCTTCTGATTTTAAAATAACTATACCAGCAGGAGGACGTCCAGACACAGCATAGGCTAC 
               
               
                 CTGGCCATGCCCAACCGGTGGGACATTTGAGTTGCTTGCTTGGCACTGTCCTCTCATGCGTTGGGTCCA 
               
               
                 CTCAGTAGATGCCTGGCGCGCCTACCAGTAAAAAAGAAAACCTATTAAAAAAACACCACTCGACACG 
               
               
                 GCACCAGCTCAATCAGTCACAGTGTAAAAAAGGGCCAAGTGCAGAGCGAGTATATATAGGACTAAAA 
               
               
                 AATGACGTAACGGTTAAAGTCCACAAAAAACACCCAGAAAACCGCACGCGAACCTACGCCCAGAAAC 
               
               
                 GAAAGCCAAAAAACCCACAACTTCCTCAAATCGTCACTTCCGTTTTCCCACGTTACGTCACTTCCCATT 
               
               
                 TTAAGAAAACTACAATTCCCAACACATACAAGTTACTCCGCCCTAAAACCTACGTCACCCGCCCCGTT 
               
               
                 CCCACGCCCCGCGCCACGTCACAAACTCCACCCCCTCATTATCATATTGGCTTCAATCCAAAATAAGGT 
               
               
                 ATATTATTGATGATGTTT. 
               
               
                   
               
               
                 Mature ATT protein 
               
               
                 (SEQ ID No. 9) 
                   
               
               
                 EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIF FS PVSIATAFAML 
                   
               
               
                 SLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVD 
               
               
                 KFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKG 
               
               
                 KWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPD 
               
               
                 EGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSG 
               
               
                 VTEEAPLKLSKAVHKAVLTID E KGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLF 
               
               
                 MGKVVNPTQK 
               
            
           
         
       
     
     The highlighted amino acids are the positions mutated/deleted in the mutated protein ATZ. 
     Materials and Methods 
     Cell Culture Studies. 
     The cDNA for human AAT was inserted in the pcDNA3.1 plasmid (Invitrogen). The pcDNA3.1-ATZ was generated by site-directed mutagenesis of the pcDNA3.1-AAT. Mouse hepatoma Hepa 1-6 cells (ATCC Catalog No. CRL-1830™) stably transfected with human ATZ (clone ATZ13) were cultured in DMEM with 10% fetal bovine serum (FBS), 5% penicillin/streptomycin, and 1 mg/ml of G418. ATZ13 cells were incubated in Met/Cys-free medium for 1 h at 37° C. followed by pulse labeling with 150 μCi/ml of Easy Tag Express Protein Labeling Mix (Perkin Elmer) in pulse medium for 30 min at 37° C. Cells were then rinsed with DMEM 5% FBS with Met and Cys (chase medium) and chased for different time points. Cells were lysed in Lysis Buffer (50 mM Tris-HCl pH7.4, 200 mM NaCl, 1% Triton X-100, 1 mM EDTA, 50 mM Hepes, 1× protease inhibitor) for 1 h at 4° C. Cell lysates were rotated overnight at 4° C. with the polyclonal rabbit anti-human AAT (Dako, cod. A0012). After addition of protein A sepharose conjugated (Sigma), the samples were incubated for 2 h at 4° C. After four washes with lysis buffer, samples were resuspended in Laemmli Buffer 2× (24 mM Tris-HCl pH 6.8, 0.8% SDS, 4% glycerol, 2.5% β-mercaptoethanol, 0.004% bromophenol blue) and boiled for 5 min. Cell extracts were loaded onto a 10% SDS-PAGE gel. 
     TFEB-3xFLAG HeLa stable cell lines (HeLa-CF7) 6  and HeLa untransfected cells were cultured in DMEM with 10% fetal bovine serum (FBS) and 5% penicillin/streptomycin and transiently transfected with pcDNA3.1-ATZ using Lipofectamine 2000 (Invitrogen). 24 hours after transfection, media were harvested for ELISA and cells were washed once with cold phosphate-buffered saline (PBS) and scraped with Ripa buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA pH 8.0, 0.1% SDS) containing complete protease inhibitor cocktail (Sigma). Samples were incubated for 20 min at 4° C. and centrifuged at 13,200 rpm for 10 min. The pellet was discarded and cell lysates were used for Western blot analysis. After transfer to PVDF membrane, blots were blocked with TBS-Tween 20 containing 5% non-fat milk for 1 hr at room temperature followed by incubation with rabbit-anti human AAT (Dako) overnight at 4° C. Donkey anti-rabbit IgG-HRP (GE Healthcare, cod. NA934) and ECL (Pierce) were used for detection of ATZ. Equal gel loading was confirmed with immunoblot for actin (Novus Biological, cod. NB600-501). 
     To detect human ATZ, ELISA was performed on five independent liver specimens from each mouse. Nunc Maxisorp plates were coated with Cappel Goat anti-human AAT (MP Biomedicals, cod. 55111), and then blocked in PBS-0.1% Tween20 containing 5% nonfat milk. 0.1-1 μg of total protein from liver samples were loaded into the wells. Serial dilutions of purified human AAT were loaded to build a standard curve. Rabbit-anti human AAT (Dako) was used as capturing antibody and goat anti-rabbit IgG-HRP (Dako, cod. PO448) as secondary antibody. 
     HDAd Vectors. 
     HDAd-TFEB and HDAd-AFP both bear a PEPCK-WL expression cassette 11,30  driving the expression of baboon alpha-fetoprotein (AFP) or human TFEB, respectively. HDAd was produced in 116 cells with the helper virus AdNG163 as described elsewhere 30 . Helper virus contamination levels were determined as described elsewhere and were found to be &lt;0.05% 30 . DNA analyses of HDAd genomic structure was confirmed as described elsewhere 30 . 
     Mice and Injections. 
     The PiZ transgenic mice 31  were maintained on a C57/BL6 background. Injections of HDAd-TFEB, HDAd-AFP, or saline were performed in the retrorbital plexus of 3-month-old PiZ mice. Blood samples were collected at baseline, 1 and 4 weeks post-injection by retrorbital bleeding. Mice were sacrificed at 4 weeks post-injection for harvesting of liver samples. 
     Analyses of Serum and Liver Samples. 
     Serum samples were analyzed by ELISA for ATZ detection. For ATZ immunoblot, ATZ ELISA and western blots, liver samples were snap frozen in liquid nitrogen and stored at −80° C. Liver specimens were fixed in 4% PFA for 12 h and stored in 70% EtOH until process and embedded into paraffin blocks and cut into 10 μm sections. The sections were rehydrated and treated with amylase solution 0.5% (a-amylase type VI-B, Sigma) for 20 minutes and then stained with PAS reagent according to manufacturer&#39;s instructions (Bio-Optica). For ATZ immunofluorescence, 6-um sections were rehydrated, blocked, incubated overnight at 4° C. with polyclonal rabbit anti-human AAT (Dako) and then with donkey anti-rabbit 488 (AlexaFluor, cod. A-21206) for one hour at room temperature. 
     For co-staining LAMP-1 and ATZ, 6-μm thick paraffin sections of livers were de-waxed by standard techniques, hydrated in PBS pH7.4 and permeabilized with PBS, 0.2% Triton. Heat Induced Epitope Retrieval (HIER) using citrate buffer method (pH 6.0) for LAMP-1 and Proteolytic Induced Epitope Retrieval (PIER) by Proteinase K for ATZ, were performed to retrieve the antigen sites. The sections were then covered for 30 min with 75 mM NH 4 Cl/PBS to reduce quenching and incubated for 1 h at room temperature with blocking solution (3% BSA, 5% donkey serum, 20 mM MgCl 2 , 0.3% Tween 20 in PBS pH 7.4). The primary antibodies used were: rat monoclonal LAMP-1 (1D4B) (Santa Cruz Biotechnology, cod. sc-19992) and polyclonal rabbit anti-human AAT (Dako). The incubation for LAMP-1 was carried out overnight at 4° C. whereas that for AAT was done for 1 h at room temperature. The secondary antibodies made in donkey, were: AlexaFluor-594 anti-rat (Invitrogen, cod. A-21209) for LAMP-1 and AlexaFluor-488 (Invitrogen, cod. A-21206) for AAT. Nuclei were counterstained with dapi (Invitrogen). Finally, the stained liver sections were mounted in mowiol, cover-slipped and examined under a Zeiss LSM 710 confocal laser-scanning microscope. At least 3 animals of each type were used for the experiments in triplicate and representative images are shown. 
     Liver specimens were homogenized in Ripa buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA pH 8.0, 0.1% SDS) and complete protease inhibitor cocktail (Sigma). Western blot and ELISA on hepatic extracts and serum were performed following the protocol used for detecting AAT in cell media reported above. For LC-3 western blotting, 20 μg of total protein were loaded on 12% SDS-PAGE. After transfer to PVDF membrane, the blots were blocked in TBS-Tween20 containing 1% BSA for 1 hr at RT, then rabbit-anti LC-3 (Novus Biological, cod NB100-2220) was applied overnight at 4° C. Donkey anti-rabbit IgG-HRP (GE Healthcare, cod. NA934) and ECL (Pierce) was used for detection of AT. Equal gel loading was confirmed with immunoblot for actin (Novus Biological, cod NB600-501). 
     Momomer-polymer analysis was performed according to previous method 14 . Sirius red staining was performed on 10 μm liver sections which were rehydrated and stained for one hour in picro-sirius red solution (0.1% Sirius red in saturated aqueous solution of picric acid). After two changes of acidified water (5 ml acetic acid glacial in 1 liter of water), the sections were dehydrate in three changes of 100% ethanol, cleared in xylene and mounted in a resinous medium. Hydroxyproline content was measured by a spectrophotometric assay as an assessment of liver collagen content as previously described 32  and expressed as micrograms of hydroxyproline per microgram of liver. 
     For caspase-12 and PARP western blotting, 20 μg of total protein were loaded on 10% SDS-PAGE and after transfer to PVDF membrane, the blots were blocked in TBS-Tween20 containing 5% non-fat milk or 1% BSA for 1 hr at room temperature followed by overnight 4° C. incubation with rat-anti caspase-12 (Sigma, cod. C7611) or rabbit-anti PARP-1 (Alexis Biochemical, ALX-210-219-R100). Goat anti-rat IgG-HRP (GE Healthcare, cod. NA935) or donkey anti-rabbit IgG-HRP (GE Healthcare, cod. NA934) were used for detection. 
     Electron Microscopy (EM) Studies. 
     For routine EM analysis the small pieces of liver were excised from PiZ mice injected with either saline or control HDAd-AFP vector or HDAd-TFEB and fixed in 1% glutaraldehyde in 0.2 M HEPES buffer. Then small blocks of the liver tissue were post-fixed in uranyl acetate and in OsO 4 . After dehydration through a graded series of ethanol, the tissue samples were cleared in propylene oxide, embedded in the Epoxy resin (Epon 812) and polymerized at 60° C. for 72 h. From each sample, thin sections were cut with a Leica EM UC6 ultramicrotome. For immuno-EM analysis of ATZ distribution in hepatocytes, small pieces of liver tissue were fixed in a mixture of 4% paraformaldehyde and 0.4% glutaraldehyde in 0.2 M PHEM buffer, infused with 2.3 M sucrose, frozen in liquid nitrogen and sectioned in Leica EM FC7 cryoultratome. Cryosections were incubated with antibodies against ATT and then with protein A conjugated with 10 nm gold particles. Both cryo and Epon-812 plastic sections were further investigated using a FEI Tecnai-12 (FEI, Einhoven, The Netherlands) electron microscope equipped with an Veletta CCD camera for digital image acquisition. Quantification of ATT gold labeling densities over the lysosome-like organelle in hepatocytes was performed using iTEM software (Olympus SYS, Germany) according the previously describe method 33 . Briefly, morphometric grid with 50 nm mesh was placed over profiles of lysosome-like structures. “Touch count” module of the iTEM software was used to quantify (i) number of gold particles and (ii) number of grid nodes inside the lysosome profile. Gold density was expressed in arbitrary units (gold particles per node). The organelle was defined as “lysosome” on the basis of the round/oval shape and presence of intraluminal vesicles as well as disorganized electron-dense and membrane material in the lumen 15 . 
     Statistical Analyses. 
     Data are expressed as mean values±standard deviation. Statistical significance was computed using the Student&#39;s 2 tail t-test. A p&lt;0.05 was considered statistically significant. 
     Results 
     To investigate whether TFEB-mediated enhancement of lysosomal degradation pathways and autophagy ameliorates the liver phenotype of AAT deficiency, the authors transfected a mouse hepa-1,6 cell line stably expressing the human ATZ protein (ATZ13 cell line) with a plasmid that expresses TFEB under the control of the CMV promoter. The ATZ13 cells were subjected to a pulse-chase radiolabeling with  35 S-labeled Cys and Met and the resulting cell lysates were analyzed by immunoprecipitation followed by SDS-PAGE analysis. This experiment showed that newly synthesized intracellular ATZ decreased more rapidly in TFEB-transfected cells as compared to control untreated cells ( FIG. 1A ). HeLa cells stably overexpressing TFEB (HeLa-CF7 cell line) 6  and control HeLa cells were transfected with the plasmid expressing ATZ and after 24 hours media and cells were harvested for detection by ELISA and western blot, respectively, of the ATZ protein, which was reduced in HeLa-CF7 cells compared to control cells ( FIGS. 1B and 1C ). 
     The authors next generated an HDAd vector that expresses the human TFEB cDNA under the control of a liver-specific promoter (phosphoenolpyruvate carboxykinase (PEPCK)-promoter) derived from rat and a liver-specific enhancer (Locus Control Region (LCR) from the apoE locus) 10  derived from human (HDAd-TFEB;  FIG. 7 ) to investigate the therapeutic potential of HDAd-TFEB vector in the PiZ mouse, a transgenic mouse that expresses the human ATZ gene under control of its endogenous regulatory regions and recapitulates the features of liver disease observed in humans, i.e. intrahepatocytic ATZ-containing globules, inflammation/regenerative activity, and fibrosis 4 . 
     Human liver-specific promoter, as human phosphoenolpyruvate carboxykinase (PEPCK)-promoter can be similarly used. 
     The authors injected 3-month old PiZ mice (at least n=5 for each group) intravenously with the HDAd-TFEB vector at the dose of 1×10 13  vp/kg. Control mice were injected with either saline or with the same dose of 1×10 13  vp/kg of HDAd vector that expresses the unrelated, non-immunogenic, non-toxic alpha-fetoprotein (AFP) reporter gene under the control of the same expression cassette and within the same vector backbone 11  as the HDAd-TFEB vector (HDAd-AFP). No changes in appearance, behavior, and body weight were noted in mice injected with HDAd-TFEB compared to the two groups of control mice up to the time of sacrifice at 4 weeks post-injection. The livers of animals injected with HDAd-TFEB showed a dramatic reduction of both ATZ accumulation and ATZ-containing globules by periodic acid-Schiff (PAS) staining and by immunofluorescence, respectively, compared to saline or HDAd-AFP injected mice (FIGS.  2 A and B). Consistent with TFEB-mediated activation of lysosome biogenesis 6,8 , high levels of LAMP-1 were observed in livers of HDAd-TFEB injected animals ( FIG. 2C ). Interestingly, a negative correlation was noted between ATZ and LAMP-1 immunostaining signals and the few areas positive for ATZ signals did not show an increase in LAMP-1 expression ( FIG. 2C ). The reduction of ATZ protein levels was confirmed by both western blot ( FIG. 2D ) and ELISA on hepatic protein extracts ( FIG. 2E ). The effect of HDAd-TFEB was specific, and HDAd-AFP had no effect on hepatic ATZ levels ( FIGS. 2A , B, and D). Besides the increase in LAMP-1 ( FIG. 2C ), an increase in LC3 levels was observed in the livers of mice injected with HDAd-TFEB, compared to saline or HDAd-AFP injected mice ( FIG. 2F  and  FIG. 8 ). Taken together, these results demonstrated that hepatic gene transfer of TFEB enhances autophagy and lysosome biogenesis in the liver and reduces accumulation of ATZ in the liver of PiZ mice. 
     ATZ serum levels were reduced in mice injected with HDAd-TFEB vector at 4 weeks post-injection compared to baseline levels of the same mice. Animals injected with either saline or HDAd-AFP vector showed serum ATZ levels, which were not statistically different from baseline levels ( FIG. 3 ). 
     Monomeric ATZ molecules bind together forming long, polymeric chains that reside in the endoplasmic reticulum (ER) of the cells in a conformation with a very long half-life. In the present study, PiZ mice were injected at the age of 3 months, when a significant hepatic accumulation of ATZ is already established, as shown by PAS staining and ATZ immunostaining 12-13 . To determine the effect of HDAd-mediated gene transfer of TFEB on monomer and polymer ATZ pools, the authors analyzed liver samples from HDAd-TFEB injected mice and the corresponding controls using a previously published assay 14 . First, ATZ polymers were isolated from the monomers in liver lysates under non-denaturing conditions and then the separated polymer and monomer fractions were denatured and compared by quantitative immunoblot. The denaturation step reduces the polymers to monomers and the resulting bands can be compared at the same molecular weight 14 . A statistically significant decrease in both ATZ monomer and polymer was observed in HDAd-TFEB injected mouse livers compared to either saline or HDAd-AFP injected control mice ( FIG. 3 ). 
     Electron microscopy (EM) analysis of thin sections from livers of animals injected with saline or control HDAd-AFP vector revealed numerous membrane bound inclusions in hepatocyte cytoplasm ( FIG. 4A-D ). These inclusions ranged from smaller (0.3-1 μm; see  FIG. 4A ) to larger sizes which were comparable to nuclei (up to 10 μm; see  FIG. 4B ), and exhibited membrane continuity with cisternae of rough endoplasmic reticulum (RER) decorated by ribosomes ( FIG. 4C , D). These features suggested that such structures are the sites of newly-synthesized ATZ which accumulates and aggregates in the RER. Indeed, immunolabelling of ATZ in thin cryosections indicated a strong concentration of ATZ in the inclusions ( FIG. 5A , B). In contrast, most of the hepatocytes in HDAd-TFEB injected animals lack the inclusions observed in control PiZ mice ( FIG. 4E , F), with exception of few cells that still contained large ATZ aggregates ( FIG. 4G ). Notably, these remaining aggregates were frequently surrounded by double membrane ( FIG. 4G , H), that indicates their transformation into an autophagic vacuole. Immuno-EM revealed ATZ to be diffusely distributed along the RER profiles in the hepatocytes of HDAd-TFEB injected mice ( FIG. 5C ). In addition, significant amounts of the protein were detected within several multi-vesicular body (MVB)-like structures ( FIG. 5D , E), which correspond to “lysosomes” or “autolysosomes” based on their ultrastructural features (see online Methods). The elevated ATZ signal in MVB-like structures indicates the activation of ATZ degradation by the lysosomal pathway upon TFEB gene transfer. Indeed, gold particles in lysosome-like organelles were frequently associated with intraluminal vesicles that are actively involved in lysosome degradation 15 . Notably, similar MVB-like structures in control saline-treated animals exhibited little or no ATZ compared to HDAd-TFEB injected mice ( FIG. 5F , G), as shown by morphometric quantitative analysis ( FIG. 5H ). Taken together these data, showed that TFEB hepatic expression enhances degradation of insoluble hepatic ATZ in autolysosomes. 
     Hepatic fibrosis is a key feature of the hepatic disease that characterizes AAT deficiency and is secondary to hepatocyte apoptosis. Therefore, the authors next investigated whether TFEB gene transfer reduced ATZ-induced liver fibrosis. Collagen deposition was determined by Sirius red staining and by measurement of hepatic hydroxyproline content. HDAd-TFEB injection resulted in a reduction of Sirius red staining ( FIG. 6A ) and of hydroxyproline content of approximately 44% in the livers of HD-TFEB injected mice compared to saline injected animals ( FIG. 6B ). 
     Caspase-12 is related to ER stress-induced apoptosis in ATZ expressing cells and livers 16 . Therefore, the authors next investigated whether the reduction in the ATZ load of HDAd-TFEB injected mice resulted in reduced activation of caspase-12. Western blot analysis showed that the ˜42 KDa cleavage product that corresponds to activated caspase-12 is significantly reduced in HDAd-TFEB injected mouse livers compared to control livers ( FIG. 6C ). The authors also observed a reduction in caspase-cleaved 89 KDa and 24 KDa fragments of poly(ADP-ribose) polymerase-1 (PARP-1), which are generated during the execution of apoptotic program ( FIG. 6D ). 
     In summary, TFEB hepatic gene transfer reduced detrimental activation of liver apoptosis and fibrosis which underlines the pathogenesis of neonatal hepatitis, cirrhosis and hepatocellular carcinoma in AAT deficiency 17 . 
     Discussion 
     Gene therapy strategies for liver disease of AAT deficiency have been investigated so far to avoid liver transplantation and have been designed with the goal of downregulating endogenous hepatocyte ATZ levels 18 . Previous attempts to correct the liver phenotype of AAT deficiency have been focused on the use of short hairpin RNA (shRNA) to silence the mutant ATZ 19-20 . However, the use of shRNA delivered by gene therapy vectors has raised concerns because severe toxicity and lethality have been reported in mice 21 . shRNA-mediated saturation of the exportin-5 pathway, which shuttles cellular micro-RNA (miRNA) from the nucleus to the cytoplasm, has been proposed as the mechanism responsible for this severe toxic response 21 . To overcome this problem, strategies based on viral vector-mediated transfer of miRNA sequences targeting the AAT gene have been developed 22 . 
     In the present study, the authors have investigated a novel strategy to correct the hepatic disease of AAT deficiency, based on clearance of ATZ accumulation mediated by TFEB gene transfer. The current view is that ATZ is degraded by both proteasomal and autophagic pathway. The proteasome is responsible for degrading the soluble forms of ATZ by means of ER-associated degradation while autophagy is involved in the disposal of the insoluble ATZ polymers and aggregates 23 . Autophagy has been previously shown to be involved in ATZ degradation 4,13,24-25 . In the present study, the authors showed that TFEB gene transfer in liver resulted in the reduction of polymeric ATZ accumulation upon increased autophagy in the liver, as indicated by increased LAMP-1 staining ( FIG. 2C ) and by immunogold EM that showed increased ATZ signals within autolysosomes ( FIG. 5 ). 
     The results of the present study clearly indicate that HDAd vector-mediated TFEB hepatic expression resulted in reduction of ATZ aggregates ( FIG. 2 ) and liver injury, as shown by decrease in hepatic apoptosis and fibrosis ( FIG. 6 ). Liver injury in patients with AAT deficiency is a direct consequence of hepatic accumulation of polymerized ATZ and therefore, TFEB gene transfer has tremendous potential for the treatment of liver disease in patients with AAT deficiency. 
     Previous work demonstrated that overexpression of TFEB promotes reduction of glycosaminoglycans by lysosomal exocytosis in a mouse model of lysosomal storage disorder due to deficiency of lysosomal enzymes 8 . In the present study, the authors show that TFEB gene transfer results in clearance of mutant, toxic protein which accumulates in a cell compartment that is not the lysosome. Therefore, the authors&#39; data indicate that TFEB gene transfer may be effective for treatment of a wide spectrum of human disorders due to accumulation of toxic proteins, including neurodegenerative disorders. 
     Although the results of the present study were obtained with the HDAd vector, similar outcomes are expected with TFEB gene transfer achieved by other gene therapy vectors, such as AAV that has recently generated encouraging results in humans 26 . Ultimately, the choice of the vector for clinical liver gene therapy will be dictated by a careful evaluation of efficacy and safety profile between the available vectors. 
     Up to one month after the injection, the HDAd-TFEB mice appeared in general good health and were undistinguishable from the control groups. Further studies are needed to investigate the safety of hepatic long-term TFEB expression. Nevertheless, the results of the present study illustrate the great potential of TFEB gene transfer, or possibly of pharmacological approaches resulting in TFEB activation, for therapy of liver disease caused by hepatotoxic ATZ. TFEB colocalizes with master growth regulator mTOR complex 1 (mTORC1) on the lysosomal membrane and in the presence of nutrients TFEB phosphorylation by mTORC1 inhibits TFEB activity 27 . Conversely, pharmacological inhibition of mTORC1, as well as starvation and lysosomal disruption, activates TFEB by promoting its nuclear translocation. Small molecules inducing TFEB nuclear translocation and activity have been identified and could be used for therapeutic applications 27 . Therefore, besides gene transfer, pharmacological induction of TFEB or TFEB target gene activation can be exploited to promote clearance of ATZ. 
     Genetic or environmental modifiers predispose a subgroup of homozygotes for the classical form of AAT deficiency to develop liver disease and/or protect the remainder population from hepatic disease 3 . Moreover, ATZ protein appears to also act as a modifier gene that exacerbates other forms of liver disease 28-29 . The results of the authors&#39; study suggest that genetically determined differences in the level of activity of the TFEB-autophagy-lysosome axis may play a role in favoring or protecting from the development of liver disease. 
     In conclusion, the results of this study show the efficacy of TFEB gene transfer for therapy of AAT deficiency liver disease by ATZ disposal through the autophagolysosome system. TFEB gene transfer might provide an innovative therapeutic strategy for treatment of hepatic damage caused by AAT deficiency, which is a common cause of liver injury. 
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