Patent Publication Number: US-2023136699-A1

Title: Gene therapy for maple syrup urine disease

Description:
FIELD OF THE INVENTION 
     The present invention is in the field of medicine, in particular in rare diseases. 
     BACKGROUND OF THE INVENTION 
     Maple syrup urine disease (MSUD, MIM: 248600) is a rare autosomal recessive disease with an incidence of one in 185,000 live births. This disorder is caused by a defective activity of the branched-chain 2-keto acid dehydrogenase (BCKD) leading to accumulation of branched-chain amino acids (BCAA) leucine, isoleucine, valine and their corresponding alpha-ketoacids (BCKA) in tissues and body fluids (Strauss et al., 2013). The BCKD enzyme is a multi-enzyme complex with four components, branched-chain keto acid decarboxylase alpha and beta subunits (E1 α  and E1 β ), dihydrolipoyl transacylase (E2) subunit and dihydrolipoamide dehydrogenase (E3) subunit. MSUD is due to mutations in BCKDHA, BCKDHB and DBT genes respectively coding for E1 α , E1 β  and E2 subunits and accounting for 45%, 35% and 20% of MSUD patients respectively (Strauss et al., 2013). Neurotoxicity in MSUD was shown to be related to accumulation of leucine and α-ketoisocaproic acid (αKIC, the ketoacid derived from leucine) (Muelly et al., 2013). In the classical severe form of MSUD, with less than 3% residual enzyme activity, this accumulation causes coma and cerebral edema shortly after birth with early death in the absence of appropriate management. 
     MSUD represents an unmet clinical need. Current MSUD treatment is limited to very severe and life-long BCAA dietary restriction associated with an oral BCAA-free amino acids mixture. Such treatment is difficult to maintain on the long-term, largely incompatible with a normal professional life. Further, it does not prevent long-term neurocognitive (Bouchereau et al., 2017) and psychiatric issues (Abi-Wardé et al., 2017). Orthotopic liver transplantation (OLT), was shown to be an effective therapy for MSUD allowing removal of dietary restrictions, complete protection from acute decompensations during illness (Bodner-Leidecker et al., 2000; Wendel et al., 1999), arrest (although not reversion) of neurocognitive impairment progression (Mazariegos et al., 2012; Muelly et al., 2013), prevention of life-threatening cerebral edema (Muelly et al., 2013), metabolic and clinical stability (Mazariegos et al., 2012). However OLT is a therapeutic option available only for a few patients and is associated with the potential (though low) risk of death and graft failures (Mazariegos et al., 2012). As a monogenic disease MSUD represents an ideal target for liver-directed gene therapy since clinical OLT data suggests that the restoration of liver BCKD enzyme activity (only contributing to 9-13% of whole-body BCKD activity (Suryawan et al., 1998)) is fully therapeutic. This was an incentive to test liver gene transfer in an MSUD mouse model. 
     Among the available gene-delivery vehicles, adeno-associated virus (AAV) vectors are the most suitable for liver gene transfer. AAV liver gene therapy achieved a major milestone with the proof of safety and long-term efficacy in a clinical trial for haemophilia B (Nathwani et al., 2014). Inborn errors of metabolism are good candidates for AAV gene therapy (Ginocchio et al., 2019). Proofs of concept of efficacy were obtained in mice for urea cycle disorders (Baruteau et al., 2018; Chandler et al., 2013; Cunningham et al., 2009; Lee et al., 2012), organic acidemias (Chandler and Venditti, 2019) or phenylketonuria (Grisch-Chan et al., 2019) and human clinical trials are currently being conducted for ornithine transcarbamylase deficiency (OTC) (NCT02991144), glycogen storage disease type 1a (NCT03517085), mucopolysaccharidosis type VI (MPSVI) (NCT03173521) and Pompe disease (NCT03533673). 
     Mouse models of MSUD with mutations in the Dbt gene have been developed and characterized (Homanics et al., 2006; S Sonnet et al., 2016; Zinnanti et al., 2009). While the majority of patients harbour mutations in BCKDHA and BCKDHB genes, there is, to our knowledge, no characterised mouse model of MSUD involving the Bckdha or Bckdhb genes. Recently, a mouse model with tissue-specific Bckdha knockout in brown adipose tissue was described and showed a reduced tolerance of BCAA loading but no other phenotypic features of MSUD (Yoneshiro et al., 2019). 
     SUMMARY OF THE INVENTION 
     As defined by the claims, the present invention relates to a method of treating Maple syrup urine disease (MSUD) by gene therapy. 
     DETAILED DESCRIPTION OF THE INVENTION 
     The inventors herein characterized the Bckdha−/− mouse, recapitulating the classical form of MSUD. As a proof of concept, they developed a (liver-directed) AAV gene therapy based on the transfer of human BCKDHA (hBCKDHA) mediated by AAV8 during immediate neonatal period in Bckdha−/− mice. The inventors demonstrated that hBCKDHA gene transfer completely rescued the lethal early-onset phenotype of Bckdha−/− mice allowing long-term survival to 12 months without overt phenotypic abnormalities. Mice were systematically sacrificed at the age of 12 months. 
     The first object of the present invention relates to a recombinant nucleic acid molecule comprising a transgene encoding for the branched-chain keto acid decarboxylase alpha or beta subunit wherein the transgene is operatively linked to a promoter. 
     As used herein, the term “nucleic acid molecule” has its general meaning in the art and refers to a DNA molecule. 
     As used herein, the term “transgene” refers to any nucleic acid that shall be expressed in a mammal cell. 
     In some embodiments, the transgene comprises a nucleic acid sequence having at least 80% of identity with SEQ ID NO:1 or SEQ ID NO:2. 
     
       
         
           
               
               
            
               
                   
                 &gt; Coding sequence of human branched 
               
               
                   
                 chain keto acid dehydrogenase El, 
               
               
                   
                 alpha polypeptide (BCKDHA), 
               
               
                   
                 denominated BCKDHA CDS WT. 
               
               
                   
                 SEQ ID NO: 1 
               
               
                   
                 
                   ggccgccatggcggtagcgatcgctgcagcgagggtctgg 
                 
               
               
                   
                   
               
               
                   
                 
                   cggctaaaccgtggtttgagccaggctgccctcctgctgc 
                 
               
               
                   
                   
               
               
                   
                 
                   tgcggcagcctggggctcggggactggctagatctcaccc 
                 
               
               
                   
                   
               
               
                   
                 
                   ccccaggcagcagcagcagttttcatctctggatgacaag 
                 
               
               
                   
                   
               
               
                   
                 
                   ccccagttcccaggggcctcggcggagtttatagataagt 
                 
               
               
                   
                   
               
               
                   
                 
                   tggaattcatccagcccaacgtcatctctggaatccccat 
                 
               
               
                   
                   
               
               
                   
                 
                   ctaccgcgtcatggaccggcaaggccagatcatcaacccc 
                 
               
               
                   
                   
               
               
                   
                 
                   agcgaggacccccacctgccgaaggagaaggtgctgaagc 
                 
               
               
                   
                   
               
               
                   
                 
                   tctacaagagcatgacactgcttaacaccatggaccgcat 
                 
               
               
                   
                   
               
               
                   
                 
                   cctctatgagtctcagcggcagggccggatctccttctac 
                 
               
               
                   
                   
               
               
                   
                 
                   atgaccaactatggtgaggagggcacgcacgtggggagtg 
                 
               
               
                   
                   
               
               
                   
                 
                   ccgccgccctggacaacacggacctggtgtttggccagta 
                 
               
               
                   
                   
               
               
                   
                 
                   ccgggaggcaggtgtgctgatgtatcgggactaccccctg 
                 
               
               
                   
                   
               
               
                   
                 
                   gaactattcatggcccagtgctatggcaacatcagtgact 
                 
               
               
                   
                   
               
               
                   
                 
                   tgggcaaggggcgccagatgcctgtccactacggctgcaa 
                 
               
               
                   
                   
               
               
                   
                 
                   ggaacgccacttcgtcactatctcctctccactggccacg 
                 
               
               
                   
                   
               
               
                   
                 
                   cagatccctcaggcggtgggggcggcgtacgcagccaagc 
                 
               
               
                   
                   
               
               
                   
                 
                   gggccaatgccaacagggtcgtcatctgttacttcggcga 
                 
               
               
                   
                   
               
               
                   
                 
                   gggggcagccagtgagggggacgcccatgccggcttcaac 
                 
               
               
                   
                   
               
               
                   
                 
                   ttcgctgccacacttgagtgccccatcatcttcttctgcc 
                 
               
               
                   
                   
               
               
                   
                 
                   ggaacaatggctacgccatctccacgcccacctctgagca 
                 
               
               
                   
                   
               
               
                   
                 
                   gtatcgcggcgatggcattgcagcacgaggccccgggtat 
                 
               
               
                   
                   
               
               
                   
                 
                   ggcatcatgtcaatccgcgtggatggtaatgatgtgtttg 
                 
               
               
                   
                   
               
               
                   
                 
                   ccgtatacaacgccacaaaggaggcccgacggcgggctgt 
                 
               
               
                   
                   
               
               
                   
                 
                   ggcagagaaccagcccttcctcatcgaggccatgacctac 
                 
               
               
                   
                   
               
               
                   
                 
                   aggatcgggcaccacagcaccagtgacgacagttcagcgt 
                 
               
               
                   
                   
               
               
                   
                 
                   accgctcggtggatgaggtcaattactgggataaacagga 
                 
               
               
                   
                   
               
               
                   
                 
                   ccaccccatctcccggctgcggcactatctgctgagccaa 
                 
               
               
                   
                   
               
               
                   
                 
                   ggctggtgggatgaggagcaggagaaggcctggaggaagc 
                 
               
               
                   
                   
               
               
                   
                 
                   agtcccgcaggaaggtgatggaggcctttgagcaggccga 
                 
               
               
                   
                   
               
               
                   
                 
                   gcggaagcccaaacccaaccccaacctactcttctcagac 
                 
               
               
                   
                   
               
               
                   
                 
                   gtgtatcaggagatgcccgcccagctccgcaagcagcagg 
                 
               
               
                   
                   
               
               
                   
                 
                   agtctctggcccgccacctgcagacctacggggagcacta 
                 
               
               
                   
                   
               
               
                   
                 
                   cccactggatcacttcgataagtgaa 
                 
               
               
                   
                   
               
               
                   
                 &gt; Coding sequence of human branched 
               
               
                   
                 chain alpha-keto acid dehydrogenase 
               
               
                   
                 El-beta subunit (BCKDHB), denominated 
               
               
                   
                 BCKDHB CDS WT. 
               
               
                   
                 SEQ ID NO: 2 
               
               
                   
                 
                   ggccgccatggcggttgtagcggcggctgccggctggcta 
                 
               
               
                   
                   
               
               
                   
                 
                   ctcaggctcagggcggcaggggctgaggggcactggcgtc 
                 
               
               
                   
                   
               
               
                   
                 
                   ggcttcctggcgcggggctggcgcggggctttttgcaccc 
                 
               
               
                   
                   
               
               
                   
                 
                   cgccgcgactgtcgaggatgcggcccagaggcggcaggtg 
                 
               
               
                   
                   
               
               
                   
                 
                   gctcattttactttccagccagatccggagccccgggagt 
                 
               
               
                   
                   
               
               
                   
                 
                   acgggcaaactcagaaaatgaatcttttccagtctgtaac 
                 
               
               
                   
                   
               
               
                   
                 
                   aagtgccttggataactcattggccaaagatcctactgca 
                 
               
               
                   
                   
               
               
                   
                 
                   gtaatatttggtgaagatgttgcctttggtggagtcttta 
                 
               
               
                   
                   
               
               
                   
                 
                   gatgcactgttggcttgcgagacaaatatggaaaagatag 
                 
               
               
                   
                   
               
               
                   
                 
                   agtttttaataccccattgtgtgaacaaggaattgttgga 
                 
               
               
                   
                   
               
               
                   
                 
                   tttggaatcggaattgcggtcactggagctactgccattg 
                 
               
               
                   
                   
               
               
                   
                 
                   cggaaattcagtttgcagattatattttccctgcatttga 
                 
               
               
                   
                   
               
               
                   
                 
                   tcagattgttaatgaagctgccaagtatcgctatcgctct 
                 
               
               
                   
                   
               
               
                   
                 
                   ggggatctttttaactgtggaagcctcactatccggtccc 
                 
               
               
                   
                   
               
               
                   
                 
                   cttggggctgtgttggtcatggggctctctatcattctca 
                 
               
               
                   
                   
               
               
                   
                 
                   gagtcctgaagcattttttgcccattgcccaggaatcaag 
                 
               
               
                   
                   
               
               
                   
                 
                   gtggttatacccagaagccctttccaggccaaaggacttc 
                 
               
               
                   
                   
               
               
                   
                 
                   ttttgtcatgcatagaggataaaaatccttgtatattttt 
                 
               
               
                   
                   
               
               
                   
                 
                   tgaacctaaaatactttacagggcagcagcggaagaagtc 
                 
               
               
                   
                   
               
               
                   
                 
                   cctatagaaccatacaacatcccactgtcccaggccgaag 
                 
               
               
                   
                   
               
               
                   
                 
                   tcatacaggaagggagtgatgttactctagttgcctgggg 
                 
               
               
                   
                   
               
               
                   
                 
                   cactcaggttcatgtgatccgagaggtagcttccatggca 
                 
               
               
                   
                   
               
               
                   
                 
                   aaagaaaagcttggagtgtcttgtgaagtcattgatctga 
                 
               
               
                   
                   
               
               
                   
                 
                   ggactataataccttgggatgtggacacaatttgtaagtc 
                 
               
               
                   
                   
               
               
                   
                 
                   tgtgatcaaaacagggcgactgctaatcagtcacgaggct 
                 
               
               
                   
                   
               
               
                   
                 cccttgacaggcggctttgcatcggaaatcagctctacag 
               
               
                   
                   
               
               
                   
                 
                   ttcaggaggaatgtttcttgaacctagaggctcctatatc 
                 
               
               
                   
                   
               
               
                   
                 
                   aagagtatgtggttatgacacaccatttcctcacattttt 
                 
               
               
                   
                   
               
               
                   
                 
                   gaaccattctacatcccagacaaatggaagtgttatgatg 
                 
               
               
                   
                   
               
               
                   
                 
                   cccttcgaaaaatgatcaactattgag 
                 
               
            
           
         
       
     
     According to the invention a first nucleic acid sequence having at least 80% of identity with a second nucleic acid sequence means that the first sequence has 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity with the second nucleic acid sequence. 
     As used herein, the term “sequence identity,” as used herein, has the standard meaning in the art. As is known in the art, a number of different programs can be used to identify whether a nucleic acid sequence has sequence identity or similarity to another nucleic acid sequence. Sequence identity or similarity may be determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith &amp; Waterman, Adv. Appl. Math. 2:482 (1981), by the sequence identity alignment algorithm of Needleman &amp; Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson &amp; Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.), the Best Fit sequence program described by Devereux et al., Nucl. Acid Res. 12:387 (1984), preferably using the default settings, or by inspection. An example of a useful algorithm is the BLAST algorithm, described in Altschul et al., J. Mol. Biol. 215:403 (1990) and Karlin et al., Proc. Natl. Acad. Sci. USA 90:5873 (1993). A particularly useful BLAST program is the WU-BLAST-2 program which was obtained from Altschul et al., Meth. Enzymol., 266:460 (1996); blast.wustl/edu/blast/README.html. WU-BLAST-2 uses several search parameters, which are preferably set to the default values. The parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity. 
     In some embodiments, the sequence of the transgene is codon-optimized. As used herein, the term “codon-optimized” refers to nucleic sequence that has been optimized to increase expression by substituting one or more codons normally present in a coding sequence with a codon for the same (synonymous) amino acid. In this manner, the protein encoded by the gene is identical, but the underlying nucleobase sequence of the gene or corresponding mRNA is different. In some embodiments, the optimization substitutes one or more rare codons (that is, codons for tRNA that occur relatively infrequently in cells from a particular species) with synonymous codons that occur more frequently to improve the efficiency of translation. For example, in human codon-optimization one or more codons in a coding sequence are replaced by codons that occur more frequently in human cells for the same amino acid. Codon optimization can also increase gene expression through other mechanisms that can improve efficiency of transcription and/or translation. Strategies include, without limitation, increasing total GC content (that is, the percent of guanines and cytosines in the entire coding sequence), decreasing CpG content (that is, the number of CG or GC dinucleotides in the coding sequence), removing cryptic splice donor or acceptor sites, and/or adding or removing ribosomal entry sites, such as Kozak sequences. Desirably, a codon-optimized gene exhibits improved protein expression, for example, the protein encoded thereby is expressed at a detectably greater level in a cell compared with the level of expression of the protein provided by the wildtype gene in an otherwise similar cell. 
     In some embodiments, the transgene comprises the nucleic acid sequence of SEQ ID NO:3 or SEQ ID NO:4. 
     
       
         
           
               
               
            
               
                   
                 &gt; Codon optimised coding sequence of 
               
               
                   
                 human branched chain keto acid 
               
               
                   
                 dehydrogenase El, alpha polypeptide 
               
               
                   
                 (BCKDHA), denominated BCKDHAco1. 
               
               
                   
                 SEQ ID NO: 3 
               
               
                   
                 
                   ggccgccatggccgttgctatcgctgccgcgagagtatgg 
                 
               
               
                   
                   
               
               
                   
                 
                   cgacttaacaggggtctttcacaagctgctcttcttcttt 
                 
               
               
                   
                   
               
               
                   
                 
                   tgcgacagccaggcgcgcgggggcttgcccggagccatcc 
                 
               
               
                   
                   
               
               
                   
                 
                   cccccggcagcaacaacagttcagtagccttgacgataaa 
                 
               
               
                   
                   
               
               
                   
                 
                   ccgcaattcccaggcgcttcagcagagttcattgataagc 
                 
               
               
                   
                   
               
               
                   
                 
                   tggaattcattcaacccaacgtaatttccggcattcctat 
                 
               
               
                   
                   
               
               
                   
                 
                   ttatcgcgtaatggatagacaggggcaaataattaacccg 
                 
               
               
                   
                   
               
               
                   
                 
                   agcgaggatccacatcttcccaaggaaaaagttcttaaat 
                 
               
               
                   
                   
               
               
                   
                 
                   tgtataagtctatgaccttgcttaacacgatggaccgaat 
                 
               
               
                   
                   
               
               
                   
                 
                   actctatgaatctcagcggcagggcaggattagtttctat 
                 
               
               
                   
                   
               
               
                   
                 
                   atgacaaattatggcgaagaaggaacccacgtcgggtccg 
                 
               
               
                   
                   
               
               
                   
                 
                   cagcggccttggataacaccgacttggtctttggacagta 
                 
               
               
                   
                   
               
               
                   
                 
                   ccgggaggcaggtgttcttatgtaccgggactatcccctt 
                 
               
               
                   
                   
               
               
                   
                 
                   gagctgttcatggctcaatgttatgggaacattagtgatc 
                 
               
               
                   
                   
               
               
                   
                 
                   tggggaaaggccgacaaatgcccgtgcattacggatgtaa 
                 
               
               
                   
                   
               
               
                   
                 
                   agaaaggcattttgtaactatctcaagtcctcttgctact 
                 
               
               
                   
                   
               
               
                   
                 
                   caaataccgcaggccgtaggtgcggcgtatgctgctaaga 
                 
               
               
                   
                   
               
               
                   
                 
                   gggcaaacgccaatagagttgtgatatgctacttcggtga 
                 
               
               
                   
                   
               
               
                   
                 
                   gggggctgcaagcgagggagatgcccacgccgggttcaac 
                 
               
               
                   
                   
               
               
                   
                 
                   tttgcagcgacactggagtgtcccattatatttttttgtc 
                 
               
               
                   
                   
               
               
                   
                 
                   gaaacaatggctatgcgatctctaccccaacatcagagca 
                 
               
               
                   
                   
               
               
                   
                 
                   gtacagaggagatgggattgcagcacggggccccggttat 
                 
               
               
                   
                   
               
               
                   
                 
                   ggaatcatgtctatacgcgtggatgggaacgacgtctttg 
                 
               
               
                   
                   
               
               
                   
                 
                   ccgtatataacgctactaaagaggccagaagacgagccgt 
                 
               
               
                   
                   
               
               
                   
                 
                   ggccgagaatcaacccttccttatagaggccatgacttac 
                 
               
               
                   
                   
               
               
                   
                 
                   agaattggtcatcactctacgtccgatgattcttcagctt 
                 
               
               
                   
                   
               
               
                   
                 
                   accgcagcgtggacgaggtaaattactgggataaacagga 
                 
               
               
                   
                   
               
               
                   
                 
                   ccatcctatttcacgacttcggcattatctcctcagccag 
                 
               
               
                   
                   
               
               
                   
                 
                   ggctggtgggacgaagaacaagaaaaggcatggagaaaac 
                 
               
               
                   
                   
               
               
                   
                 
                   aatctagaagaaaggttatggaggcctttgagcaggcaga 
                 
               
               
                   
                   
               
               
                   
                 
                   acgcaaaccaaaaccaaatcccaatcttcttttcagcgac 
                 
               
               
                   
                   
               
               
                   
                 
                   gtgtaccaggaaatgccagcccagctgcggaaacagcaag 
                 
               
               
                   
                   
               
               
                   
                 
                   aaagcctggcgagacatcttcagacctacggggaacatta 
                 
               
               
                   
                   
               
               
                   
                 
                   cccactggatcactttgacaaatgaa 
                 
               
               
                   
                   
               
               
                   
                 &gt; Codon optimised coding sequence of human 
               
               
                   
                 branched chain keto acid dehydrogenase El, 
               
               
                   
                 alpha polypeptide (BCKDHA), denominated 
               
               
                   
                 BCKDHAco2. 
               
               
                   
                 SEQ ID NO: 4 
               
               
                   
                 
                   ggccgccatggctgttgctattgctgctgcgagagtatgg 
                 
               
               
                   
                   
               
               
                   
                 
                   cgacttaacaggggtctttcacaagctgctcttcttcttt 
                 
               
               
                   
                   
               
               
                   
                 
                   tgaggcagccaggagccagagggcttgccagaagccatcc 
                 
               
               
                   
                   
               
               
                   
                 
                   ccccagacagcaacaacagttcagtagccttgatgataaa 
                 
               
               
                   
                   
               
               
                   
                 
                   ccccaattcccaggagcttcagcagagttcattgataagc 
                 
               
               
                   
                   
               
               
                   
                 
                   tggaattcattcaacccaatgtaatttctggcattcctat 
                 
               
               
                   
                   
               
               
                   
                 
                   ttatagagtaatggatagacaggggcaaataattaacccc 
                 
               
               
                   
                   
               
               
                   
                 
                   tccgaggatccacatcttcccaaggaaaaagttcttaaat 
                 
               
               
                   
                   
               
               
                   
                 
                   tgtataagtctatgaccttgcttaacaccatggacaggat 
                 
               
               
                   
                   
               
               
                   
                 
                   actctatgaatctcagagacagggcaggattagtttctat 
                 
               
               
                   
                   
               
               
                   
                 
                   atgacaaattatggagaagaaggaacccacgtggggagcg 
                 
               
               
                   
                   
               
               
                   
                 
                   cagccgccttggataacaccgacttggtctttggacagta 
                 
               
               
                   
                   
               
               
                   
                 
                   cagggaggcaggtgttcttatgtacagggactatcccctt 
                 
               
               
                   
                   
               
               
                   
                 
                   gagctgttcatggctcaatgttatgggaacattagtgatc 
                 
               
               
                   
                   
               
               
                   
                 
                   tggggaaaggccgacaaatgcccgtgcattacggatgtaa 
                 
               
               
                   
                   
               
               
                   
                 
                   agaaaggcattttgtaactatctcaagtcctcttgctact 
                 
               
               
                   
                   
               
               
                   
                 
                   caaataccccaggctgtaggtgccgcctatgctgctaaga 
                 
               
               
                   
                   
               
               
                   
                 
                   gggcaaacgccaatagagttgtgatatgctacttcggtga 
                 
               
               
                   
                   
               
               
                   
                 
                   gggggctgcaagcgagggagatgcccacgctgggttcaac 
                 
               
               
                   
                   
               
               
                   
                 
                   tttgcagccacactggagtgtcccattatatttttttgta 
                 
               
               
                   
                   
               
               
                   
                 ggaacaatggctatgccatctctaccccaacatcagagca 
               
               
                   
                   
               
               
                   
                 
                   gtacagaggagatgggattgcagcaagaggccccggttat 
                 
               
               
                   
                   
               
               
                   
                 
                   ggaatcatgtctataagggtggatgggaacgacgtctttg 
                 
               
               
                   
                   
               
               
                   
                 
                   ccgtgtataacgctactaaagaggccagaagaagggctgt 
                 
               
               
                   
                   
               
               
                   
                 
                   ggctgagaatcaacccttccttatagaggccatgacttac 
                 
               
               
                   
                   
               
               
                   
                 
                   agaattggtcatcactctacctccgatgattcttcagctt 
                 
               
               
                   
                   
               
               
                   
                 
                   acagatccgtggatgaggtaaattactgggataaacagga 
                 
               
               
                   
                   
               
               
                   
                 
                   ccatcctatttcaagacttaggcattatctcctcagccag 
                 
               
               
                   
                   
               
               
                   
                 
                   ggctggtgggacgaagaacaagaaaaggcatggagaaaac 
                 
               
               
                   
                   
               
               
                   
                 
                   aatctagaagaaaggttatggaggcctttgagcaggcaga 
                 
               
               
                   
                   
               
               
                   
                 
                   aaggaaaccaaaaccaaatcccaatcttcttttctccgac 
                 
               
               
                   
                   
               
               
                   
                 
                   gtgtaccaggaaatgccagcccagctgaggaaacagcaag 
                 
               
               
                   
                   
               
               
                   
                 
                   aaagcctggccagacatcttcagacctacggggaacatta 
                 
               
               
                   
                   
               
               
                   
                 
                   cccactggatcactttgacaaatgaa 
                 
               
            
           
         
       
     
     In some embodiments, the transgene comprises the nucleic acid sequence of SEQ ID NO:5 or SEQ ID NO:6. 
     
       
         
           
               
            
               
                 SEQ ID NO: 5 
               
               
                 &gt; Codon optimised coding sequence of the human 
               
               
                 branched chain alpha-ketoacid dehydrogenase E1- 
               
               
                 beta subunit(BCKDHB), denominated BCKDHBco1. 
               
               
                 
                   ggccgccatggcagttgtggcagccgcagcgggctggttgttgcgactca 
                 
               
               
                   
               
               
                 
                   gagcagccggtgcagaaggccattggagacggttgccgggtgcgggactg 
                 
               
               
                   
               
               
                 
                   gcgcgcggctttctccaccccgcagcgactgtagaagacgcagcccaaag 
                 
               
               
                   
               
               
                 
                   acgacaggtcgctcacttcacattccagcctgatcccgagccacgagaat 
                 
               
               
                   
               
               
                 
                   acgggcaaacgcaaaaaatgaatctctttcagtccgtaacatctgctttg 
                 
               
               
                   
               
               
                 
                   gataatagtcttgcaaaagatccaacagctgtaattttcggggaagatgt 
                 
               
               
                   
               
               
                 
                   agcgtttggcggtgtcttccgatgtaccgtcgggctgagggataagtacg 
                 
               
               
                   
               
               
                 
                   ggaaagatagagtatttaatacccccctgtgcgagcagggtatagtcgga 
                 
               
               
                   
               
               
                 
                   tttgggattggaatagccgtaacgggagcaacagcgattgccgaaataca 
                 
               
               
                   
               
               
                 
                   atttgccgactatatcttcccggcgtttgaccaaattgttaacgaggctg 
                 
               
               
                   
               
               
                 
                   cgaaatatcggtatcgctccggcgacttgtttaattgcggtagcctcaca 
                 
               
               
                   
               
               
                 
                   attagaagtccttgggggtgcgttggacacggtgcgctctatcacagtca 
                 
               
               
                   
               
               
                 
                   atctccagaagcttttttcgcacattgtccaggcatcaaagtagtgattc 
                 
               
               
                   
               
               
                 
                   cccgaagcccatttcaggcgaaaggtctcttgctctcctgtatagaagat 
                 
               
               
                   
               
               
                 
                   aaaaacccatgtatcttttttgagcctaaaatcctgtaccgcgccgcagc 
                 
               
               
                   
               
               
                 
                   tgaggaagtccctatagagccatacaacatcccactctcacaggcagaag 
                 
               
               
                   
               
               
                 
                   ttatacaagaagggagtgacgtgacactcgtagcatgggggacgcaggtt 
                 
               
               
                   
               
               
                 
                   catgtgatcagagaggtagccagtatggcaaaagagaaattgggagtttc 
                 
               
               
                   
               
               
                 
                   ttgtgaagttatcgatctccgaacaataatcccttgggatgtagatacca 
                 
               
               
                   
               
               
                 
                   tttgtaagtctgttatcaaaactggtaggctcctcatatctcatgaggcc 
                 
               
               
                   
               
               
                 
                   ccgttgacgggtgggttcgcgtccgaaatttcatcaactgttcaagagga 
                 
               
               
                   
               
               
                 
                   gtgctttctcaacctggaagcgccgatctctagagtctgcggatatgata 
                 
               
               
                   
               
               
                 
                   cccccttcccacacatatttgagcctttttatatcccggacaaatggaag 
                 
               
               
                   
               
               
                 
                   tgttacgacgcccttcgaaaaatgataaattattgag 
                 
               
               
                   
               
               
                 SEQ ID NO: 6 
               
               
                 &gt; Codon optimised coding sequence of the human 
               
               
                 branched chain alpha-ketoacid dehydrogenase E1- 
               
               
                 beta subunit(BCKDHB), denominated BCKDHBco2. 
               
               
                 
                   ggccgccatggcagttgtggcagctgcagcaggctggttgttgcgcctca 
                 
               
               
                   
               
               
                 
                   gagcagctggtgcagaaggccattggagaaggttgcctggtgccggactg 
                 
               
               
                   
               
               
                 
                   gcccgcggctttctccaccccgcagccactgtagaagatgcagcccaaag 
                 
               
               
                   
               
               
                 
                   aagacaggtcgctcacttcacattccagcctgatcccgagccaagagaat 
                 
               
               
                   
               
               
                 
                   atgggcaaacccaaaaaatgaatctctttcagtccgtaacatctgctttg 
                 
               
               
                   
               
               
                 
                   gataatagtcttgcaaaagatccaacagctgtaattttcggggaagatgt 
                 
               
               
                   
               
               
                 
                   agcatttggaggtgtcttcaggtgtacagtcgggctgagggataagtacg 
                 
               
               
                   
               
               
                 
                   ggaaagatagagtatttaatacccccctgtgtgagcagggtatagtggga 
                 
               
               
                   
               
               
                 
                   tttgggattggaatagctgtaacgggagcaacagcaattgctgaaataca 
                 
               
               
                   
               
               
                 
                   atttgctgactatatcttcccggcatttgaccaaattgttaacgaggctg 
                 
               
               
                   
               
               
                 
                   caaaatataggtataggtccggagacttgtttaattgtggtagcctcaca 
                 
               
               
                   
               
               
                 
                   attagaagtccttgggggtgtgttggacatggtgcactctatcacagtca 
                 
               
               
                   
               
               
                 
                   atctccagaagcttttttcgcacattgtccaggcatcaaagtagtgattc 
                 
               
               
                   
               
               
                 
                   ccaggagcccatttcaggcaaaaggtctcttgctctcctgtatagaagat 
                 
               
               
                   
               
               
                 
                   aaaaacccatgtatcttttttgagcctaaaatcctgtacagagctgcagc 
                 
               
               
                   
               
               
                 
                   tgaggaagtccctatagagccatacaacatcccactctcacaggcagaag 
                 
               
               
                   
               
               
                 
                   ttatacaagaagggagtgatgtgacactggtagcatgggggacccaggtt 
                 
               
               
                   
               
               
                 
                   catgtgatcagagaggtagccagtatggcaaaagagaaattgggagtttc 
                 
               
               
                   
               
               
                 
                   ttgtgaagttatcgatctccgaacaataatcccttgggatgtagatacca 
                 
               
               
                   
               
               
                 
                   tttgtaagtctgttatcaaaactggtaggctcctcatatctcatgaggcc 
                 
               
               
                   
               
               
                 
                   ccgttgaccggtgggttcgcatccgaaatttcatcaactgttcaagagga 
                 
               
               
                   
               
               
                 
                   gtgctttctcaacctggaagcaccaatctctagagtctgtggatatgata 
                 
               
               
                   
               
               
                 
                   cccccttcccacacatatttgagcctttttatatcccagacaaatggaag 
                 
               
               
                   
               
               
                 
                   tgttacgatgcccttagaaaaatgataaattattgag 
                 
               
            
           
         
       
     
     As used herein, the terms “promoter” has its general meaning in the art and refers to a segment of a nucleic acid sequence, typically but not limited to DNA that controls the transcription of the nucleic acid sequence to which it is operatively linked. The promoter region includes specific sequences that are sufficient for RNA polymerase recognition, binding and transcription initiation. In addition, the promoter region can optionally include sequences which modulate this recognition, binding and transcription initiation activity of RNA polymerase. The skilled person will be aware that promoters are built from stretches of nucleic acid sequences and often comprise elements or functional units in those stretches of nucleic acid sequences, such as a transcription start site, a binding site for RNA polymerase, general transcription factor binding sites, such as a TATA box, specific transcription factor binding sites, and the like. Further regulatory sequences may be present as well, such as enhancers, and sometimes introns at the end of a promoter sequence. 
     Typically, the promoter may be an ubiquitous or tissue-specific promoter, in particular a promoter able to promote expression in cells or tissues in which expression of the transgene is desirable such as in cells or tissues in which the transgene expression is desirable. 
     In some embodiments, the promoter is a liver-specific promoter such as the alpha-1 antitrypsin promoter (hAAT), the transthyretin promoter, the albumin promoter, the thyroxine-binding globulin (TBG) promoter, the LSP promoter (comprising a thyroid hormone-binding globulin promoter sequence, two copies of an alphal-microglobulin/bikunin enhancer sequence, and a leader sequence—34.111, C. R., et al. (1997).  Optimization of the human factor VIII complementary DNA expression plasmid for gene therapy of hemophilia A. Blood Coag. Fibrinol.  8: S23-S30.), etc. Other useful liver-specific promoters are known in the art, for example those listed in the Liver Specific Gene Promoter Database compiled the Cold Spring Harbor Laboratory (http://rulai.cshl.edu/LSPD/). 
     In some embodiments, the promoter is the hAAT promoter. As used herein, the term “hAAT” as its general meaning in the art and refers to the promoter of the gene encoding for the human alpha 1-antitrypsin. 
     In some embodiments, the hAAT promoter comprises the nucleic acid sequence of SEQ ID NO:7. 
     
       
         
           
               
            
               
                 SEQ ID NO: 7 
               
               
                 &gt; promoter of the gene encoding for human alpha 1- 
               
               
                 antitrypsin 
               
               
                 gatcttgctaccagtggaacagccactaaggattctgcagtgagagcaga 
               
               
                   
               
               
                 gggccagctaagtggtactctcccagagactgtctgactcacgccacccc 
               
               
                   
               
               
                 ctccaccttggacacaggacgctgtggtttctgagccaggtacaatgact 
               
               
                   
               
               
                 cctttcggtaagtgcagtggaagctgtacactgcccaggcaaagcgtccg 
               
               
                   
               
               
                 ggcagcgtaggcgggcgactcagatcccagccagtggacttagcccctgt 
               
               
                   
               
               
                 ttgctcctccgataactggggtgaccttggttaatattcaccagcagcct 
               
               
                   
               
               
                 cccccgttgcccctctggatccactgcttaaatacggacgaggacagggc 
               
               
                   
               
               
                 cctgtctcctcagcttcaggcaccaccactgacctgggacagt 
               
            
           
         
       
     
     Other tissue-specific or non-tissue-specific promoters may be useful in the practice of the invention. 
     In some embodiments, the promoter is a ubiquitous promoter. Representative ubiquitous promoters include the cytomegalovirus enhancer/chicken beta actin (CAG) promoter, the cytomegalovirus enhancer/promoter (CMV), the PGK promoter, the SV40 early promoter, etc. 
     In some embodiments, the promoter is the EF1a promoter. As used herein, the term “EF1a promoter” has its general meaning in the art and refers to the promoter of the gene encoding for elongation factor-1 alpha. 
     In some embodiments, the EF1a promoter comprises the nucleic acid sequence of SEQ ID NO:8. 
     
       
         
           
               
            
               
                 SEQ ID NO: 8 
               
               
                 &gt; promoter of the gene encoding for elongation 
               
               
                 factor-1 alpha 
               
               
                 
                   ctagcgctccggtgcccgtcagtgggcagagcgcacatcgcccacagtcc 
                 
               
               
                   
               
               
                 
                   ccgagaagttggggggaggggtcggcaattgaaccggtgcctagagaagg 
                 
               
               
                   
               
               
                 
                   tggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttt 
                 
               
               
                   
               
               
                 
                   tcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacg 
                 
               
               
                   
               
               
                 
                   ttctttttcgcaacgggtttgccgccagaacacag 
                 
               
            
           
         
       
     
     In some embodiments, the EF1a promoter of the present invention further comprises an extra intronic sequence that will increase the expression of the transgene by the promoter. Typically, said extra intronic sequence consists of the nucleic acid sequence of SEQ ID NO:9. 
     
       
         
           
               
            
               
                 SEQ ID NO: 9 
               
               
                 &gt; This extra intronic sequence gives a higher 
               
               
                 level of expression than the core EF1a promoter 
               
               
                 alone. 
               
               
                 
                   gtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatgg 
                 
               
               
                   
               
               
                 
                   cccttgcgtgccttgaattacttccacgcccctggctgcagtacgtgatt 
                 
               
               
                   
               
               
                 
                   cttgatcccgagcttcgggttggaagtgggtgggagagttcgaggccttg 
                 
               
               
                   
               
               
                 
                   cgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcctgggc 
                 
               
               
                   
               
               
                 
                   gctggggccgccgcgtgcgaatctggtggcaccttcgcgcctgtctcgct 
                 
               
               
                   
               
               
                 
                   gctttcgataagtctctagccatttaaaatttttgatgacctgctgcgac 
                 
               
               
                   
               
               
                 
                   gctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcaca 
                 
               
               
                   
               
               
                 
                   ctggtatttcggtttttggggccgcgggcggcgacggggcccgtgcgtcc 
                 
               
               
                   
               
               
                 
                   cagcgcacatgttcggcgaggcggggcctgcgagcgcggccaccgagaat 
                 
               
               
                   
               
               
                 
                   cggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcg 
                 
               
               
                   
               
               
                 
                   cgccgccgtgtatcgccccgccctgggcggcaaggctggcccggtcggca 
                 
               
               
                   
               
               
                 
                   ccagttgcgtgagcggaaagatggccgcttcccggccctgctgcagggag 
                 
               
               
                   
               
               
                 
                   ctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcaccca 
                 
               
               
                   
               
               
                 
                   cacaaaggaaaagggcctttccgtcctcagccgtcgcttcatgtgactcc 
                 
               
               
                   
               
               
                 
                   acggagtaccgggcgccgtccaggcacctcgattagttctcgagcttttg 
                 
               
               
                   
               
               
                 
                   gagtacgtcgtctttaggttggggggaggggttttatgcgatggagtttc 
                 
               
               
                   
               
               
                 
                   cccacactgagtgggtggagactgaagttaggccagcttggcacttgatg 
                 
               
               
                   
               
               
                 
                   taattctccttggaatttgccctttttgagtttggatcttggttcattct 
                 
               
               
                   
               
               
                 
                   caagcctcagacagtggttcaaagtttttttcttccatttcag 
                 
               
            
           
         
       
     
     As used herein, the terms “operably linked”, or “operatively linked” are used interchangeably herein, and refer to the functional relationship of the nucleic acid sequences with regulatory sequences of nucleotides, such as promoters, enhancers, transcriptional and translational stop sites, and other signal sequences and indicates that two or more DNA segments are joined together such that they function in concert for their intended purposes. For example, operative linkage of nucleic acid sequences, typically DNA, to a regulatory sequence or promoter region refers to the physical and functional relationship between the DNA and the regulatory sequence or promoter such that the transcription of such DNA is initiated from the regulatory sequence or promoter, by an RNA polymerase that specifically recognizes, binds and transcribes the DNA. In order to optimize expression and/or in vitro transcription, it may be necessary to modify the regulatory sequence for the expression of the nucleic acid or DNA in the cell type for which it is expressed. The desirability of, or need of, such modification may be empirically determined. 
     In some embodiments, further regulatory sequences may also be added to the recombinant nucleic acid molecule of the present invention. 
     As used herein, the term “regulatory sequence” is used interchangeably with “regulatory element” herein and refers to a segment of nucleic acid, typically but not limited to DNA, that modulate the transcription of the nucleic acid sequence to which it is operatively linked, and thus acts as a transcriptional modulator. A regulatory sequence often comprises nucleic acid sequences that are transcription binding domains that are recognized by the nucleic acid-binding domains of transcriptional proteins and/or transcription factors, enhancers or repressors etc. 
     In some embodiments, the nucleic acid molecule of the present invention comprises a Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE) sequence that is a DNA sequence that, when transcribed creates a tertiary structure enhancing expression, by stabilization of the messenger RNA. Typically, the WPRE sequence is inserted downstream to the transgene. In some embodiments, the recombinant acid molecule of the present invention comprises a WPRE sequence devoid of X protein open reading frames (ORFs), that allows to remove oncogenic side effect without significant loss of RNA enhancement activity (Schambach, A. et al.  Woodchuck hepatitis virus post - transcriptional regulatory element deleted from X protein and promoter sequences enhances retroviral vector titer and expression. Gene Ther.  13, 641-645 (2006)). 
     In some embodiments, the WPRE sequence comprises the nucleic acid sequence of SEQ ID NO: 10. 
     
       
         
           
               
            
               
                 SEQ ID NO: 10 
               
               
                 &gt; Woodchuck hepatitis virus post-transcriptional 
               
               
                 regulatory element (WPRE) is a sequence that 
               
               
                 stimulates the expression of transgenes via 
               
               
                 increased nuclear export. 
               
               
                 
                   aatcaacctctggattacaaaatttgtgaaagattgactggtattcttaa 
                 
               
               
                   
               
               
                 
                   ctatgttgctccttttacgctatgtggatacgctgctttaatgcctttgt 
                 
               
               
                   
               
               
                 
                   atcatgctattgcttcccgtatggctttcattttctcctccttgtataaa 
                 
               
               
                   
               
               
                 
                   tcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacg 
                 
               
               
                   
               
               
                 
                   tggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggca 
                 
               
               
                   
               
               
                 
                   ttgccaccacctgtcagctcctttccgggactttcgctttccccctccct 
                 
               
               
                   
               
               
                 
                   attgccacggcggaactcatcgccgcctgccttgcccgctgctggacagg 
                 
               
               
                   
               
               
                 
                   ggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcat 
                 
               
               
                   
               
               
                 
                   cgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcggg 
                 
               
               
                   
               
               
                 
                   acgtccttctgctacgtcccttcggccctcaatccagcggaccttccttc 
                 
               
               
                   
               
               
                 
                   ccgcggcctgctgccggctctgcggcctcttccgcgtcttcg 
                 
               
            
           
         
       
     
     In some embodiments, the recombinant nucleic acid molecule of the present invention comprises a polyadenylation signal sequence inserted downstream to the transgene. 
     As used herein, the term “polyadenylation signal sequence” has its general meaning in the art and refers to a nucleic acid sequence that mediates the attachment of a polyadenine stretch to the 3′ terminus of the mRNA. Suitable polyadenylation signals include the SV40 early polyadenylation signal, the SV40 late polyadenylation signal, the HSV thymidine kinase polyadenylation signal, the protamine gene polyadenylation signal, the adenovirus 5 EIb polyadenylation signal, the bovine growth hormone polyadenylation signal, the human variant growth hormone polyadenylation signal and the like. 
     In some embodiments, the polyadenylation sequence comprises the nucleic acid sequence of SEQ ID NO: 11. 
     
       
         
           
               
            
               
                 SEQ ID NO: 11 
               
               
                 &gt; Bovine growth hormone polyA signal is a termi- 
               
               
                 nator it&#39;s role is to define the end of a 
               
               
                 transcriptional unit (such as a gene) and 
               
               
                 initiate the process of releasing the newly 
               
               
                 synthesized RNA from the transcription machinery. 
               
               
                 
                   ctgtgccttctagttgccagccatctgttgtttgcccctcccccgtgcct 
                 
               
               
                   
               
               
                 
                   tccttgaccctggaaggtgccactcccactgtcctttcctaataaaatga 
                 
               
               
                   
               
               
                 
                   ggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtg  
                 
               
               
                   
               
               
                 
                   gggtggggcaggacagcaagggggaggattgggaagacaatagcaggcat 
                 
               
               
                   
               
               
                 
                   gctgggga 
                 
               
            
           
         
       
     
     In some embodiments, the recombinant nucleic acid molecule of the present invention comprises inverted terminal repeats (ITRs) sequences that are required for genome replication and packaging. In some embodiments, the recombinant nucleic acid molecule of the present invention comprises the AAV2 inverted terminal repeat sequences of SEQ ID NO:12. 
     
       
         
           
               
            
               
                 SEQ ID NO: 12 
               
               
                 &gt; Inverted terminal repeats (ITRs) that are 
               
               
                 required for genome replication and packaging. 
               
               
                 
                   ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcg 
                 
               
               
                   
               
               
                 
                   ggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagg 
                 
               
               
                   
               
               
                 
                   gagtggccaactccatcactaggggttcct 
                 
               
            
           
         
       
     
     In some embodiments, the recombinant nucleic acid molecule of the present invention comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:13 to SEQ ID NO:24. 
     
       
         
           
               
               
            
               
                 SEQ ID NO: 13 
                   
               
               
                 &gt; ITR-EF1a-BCKDHA-WT-ITR 
                   
               
               
                 ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccg 
               
               
                   
               
               
                 gcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttccttgtagtta 
               
               
                   
               
               
                 atgattaacccgccatgctacttatctacgtagccatgctctaggaagatcggaattcgcccttaagct 
               
               
                   
               
               
                 agcgctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggagggg 
               
               
                   
               
               
                 tcggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggc 
               
               
                   
               
               
                 tccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttc 
               
               
                   
               
               
                 gcaacgggtttgccgccagaacacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgg 
               
               
                   
               
               
                 gttatggcccttgcgtgccttgaattacttccacgcccctggctgcagtacgtgattcttgatcccgag 
               
               
                   
               
               
                 cttcgggttggaagtgggtgggagagttcgaggccttgcgcttaaggagccccttcgcctcgtgcttga 
               
               
                   
               
               
                 gttgaggcctggcctgggcgctggggccgccgcgtgcgaatctggtggcaccttcgcgcctgtctcgct 
               
               
                   
               
               
                 gctttcgataagtctctagccatttaaaatttttgatgacctgctgcgacgctttttttctggcaagat 
               
               
                   
               
               
                 agtcttgtaaatgcgggccaagatctgcacactggtatttcggtttttggggccgcgggcggcgacggg 
               
               
                   
               
               
                 gcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggccaccgagaatcggacgg 
               
               
                   
               
               
                 gggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcgccccgccctgg 
               
               
                   
               
               
                 gcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagatggccgcttcccggccctgctgca 
               
               
                   
               
               
                 gggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaaggaaaagg 
               
               
                   
               
               
                 gcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctcgat 
               
               
                   
               
               
                 tagttctcgagcttttggagtacgtcgtctttaggttggggggaggggttttatgcgatggagtttccc 
               
               
                   
               
               
                 cacactgagtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgcc 
               
               
                   
               
               
                 ctttttgagtttggatcttggttcattctcaagcctcagacagtggttcaaagtttttttcttccattt 
               
               
                   
               
               
                 caggtgtcgtgaagcaattgcgcGGCCGCCATGGCGGTAGCGATCGCTGCAGCGAGGGTCTGGCGGCTA 
               
               
                   
               
               
                 AACCGTGGTTTGAGCCAGGCTGCCCTCCTGCTGCTGCGGCAGCCTGGGGCTCGGGGACTGGCTAGATCT 
               
               
                   
               
               
                 CACCCCCCCAGGCAGCAGCAGCAGTTTTCATCTCTGGATGACAAGCCCCAGTTCCCAGGGGCCTCGGCG 
               
               
                   
               
               
                 GAGTTTATAGATAAGTTGGAATTCATCCAGCCCAACGTCATCTCTGGAATCCCCATCTACCGCGTCATG 
               
               
                   
               
               
                 GACCGGCAAGGCCAGATCATCAACCCCAGCGAGGACCCCCACCTGCCGAAGGAGAAGGTGCTGAAGCTC 
               
               
                   
               
               
                 TACAAGAGCATGACACTGCTTAACACCATGGACCGCATCCTCTATGAGTCTCAGCGGCAGGGCCGGATC 
               
               
                   
               
               
                 TCCTTCTACATGACCAACTATGGTGAGGAGGGCACGCACGTGGGGAGTGCCGCCGCCCTGGACAACACG 
               
               
                   
               
               
                 GACCTGGTGTTTGGCCAGTACCGGGAGGCAGGTGTGCTGATGTATCGGGACTACCCCCTGGAACTATTC 
               
               
                   
               
               
                 ATGGCCCAGTGCTATGGCAACATCAGTGACTTGGGCAAGGGGCGCCAGATGCCTGTCCACTACGGCTGC 
               
               
                   
               
               
                 AAGGAACGCCACTTCGTCACTATCTCCTCTCCACTGGCCACGCAGATCCCTCAGGCGGTGGGGGCGGCG 
               
               
                   
               
               
                 TACGCAGCCAAGCGGGCCAATGCCAACAGGGTCGTCATCTGTTACTTCGGCGAGGGGGCAGCCAGTGAG 
               
               
                   
               
               
                 GGGGACGCCCATGCCGGCTTCAACTTCGCTGCCACACTTGAGTGCCCCATCATCTTCTTCTGCCGGAAC 
               
               
                   
               
               
                 AATGGCTACGCCATCTCCACGCCCACCTCTGAGCAGTATCGCGGCGATGGCATTGCAGCACGAGGCCCC 
               
               
                   
               
               
                 GGGTATGGCATCATGTCAATCCGCGTGGATGGTAATGATGTGTTTGCCGTATACAACGCCACAAAGGAG 
               
               
                   
               
               
                 GCCCGACGGCGGGCTGTGGCAGAGAACCAGCCCTTCCTCATCGAGGCCATGACCTACAGGATCGGGCAC 
               
               
                   
               
               
                 CACAGCACCAGTGACGACAGTTCAGCGTACCGCTCGGTGGATGAGGTCAATTACTGGGATAAACAGGAC 
               
               
                   
               
               
                 CACCCCATCTCCCGGCTGCGGCACTATCTGCTGAGCCAAGGCTGGTGGGATGAGGAGCAGGAGAAGGCC 
               
               
                   
               
               
                 TGGAGGAAGCAGTCCCGCAGGAAGGTGATGGAGGCCTTTGAGCAGGCCGAGCGGAAGCCCAAACCCAAC 
               
               
                   
               
               
                 CCCAACCTACTCTTCTCAGACGTGTATCAGGAGATGCCCGCCCAGCTCCGCAAGCAGCAGGAGTCTCTG 
               
               
                   
               
               
                 GCCCGCCACCTGCAGACCTACGGGGAGCACTACCCACTGGATCACTTCGATAAGTGAAagcttggatcc 
               
               
                   
               
               
                 aatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacg 
               
               
                   
               
               
                 ctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcc 
               
               
                   
               
               
                 tccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtg 
               
               
                   
               
               
                 gtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttcc 
               
               
                   
               
               
                 gggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctgg 
               
               
                   
               
               
                 acaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttgg 
               
               
                   
               
               
                 ctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaat 
               
               
                   
               
               
                 ccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgagatctgcct 
               
               
                   
               
               
                 cgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaag 
               
               
                   
               
               
                 gtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcatt 
               
               
                   
               
               
                 ctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctg 
               
               
                   
               
               
                 gggactcgagttaagggcgaattcccgataaggatcttcctagagcatggctacgtagataagtagcat 
               
               
                   
               
               
                 ggcgggttaatcattaactacaaggaacccctagtgatggagttggccactccctctctgcgcgctcgc 
               
               
                   
               
               
                 tcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagc 
               
               
                   
               
               
                 gagcgagcgcgcag 
               
               
                   
               
               
                 SEQ ID NO: 14 
                   
               
               
                 &gt; ITR-EF1a-BOKDHA-co1-ITR 
                   
               
               
                 ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccg 
               
               
                   
               
               
                 gcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttccttgtagtta 
               
               
                   
               
               
                 atgattaacccgccatgctacttatctacgtagccatgctctaggaagatcggaattcgcccttaagct 
               
               
                   
               
               
                 agcgctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggagggg 
               
               
                   
               
               
                 tcggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggc 
               
               
                   
               
               
                 tccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttc 
               
               
                   
               
               
                 gcaacgggtttgccgccagaacacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgg 
               
               
                   
               
               
                 gttatggcccttgcgtgccttgaattacttccacgcccctggctgcagtacgtgattcttgatcccgag 
               
               
                   
               
               
                 cttcgggttggaagtgggtgggagagttcgaggccttgcgcttaaggagccccttcgcctcgtgcttga 
               
               
                   
               
               
                 gttgaggcctggcctgggcgctggggccgccgcgtgcgaatctggtggcaccttcgcgcctgtctcgct 
               
               
                   
               
               
                 gctttcgataagtctctagccatttaaaatttttgatgacctgctgcgacgctttttttctggcaagat 
               
               
                   
               
               
                 agtcttgtaaatgcgggccaagatctgcacactggtatttcggtttttggggccgcgggcggcgacggg 
               
               
                   
               
               
                 gcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggccaccgagaatcggacgg 
               
               
                   
               
               
                 gggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcgccccgccctgg 
               
               
                   
               
               
                 gcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagatggccgcttcccggccctgctgca 
               
               
                   
               
               
                 gggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaaggaaaagg 
               
               
                   
               
               
                 gcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctcgat 
               
               
                   
               
               
                 tagttctcgagcttttggagtacgtcgtctttaggttggggggaggggttttatgcgatggagtttccc 
               
               
                   
               
               
                 cacactgagtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgcc 
               
               
                   
               
               
                 ctttttgagtttggatcttggttcattctcaagcctcagacagtggttcaaagtttttttcttccattt 
               
               
                   
               
               
                 caggtgtcgtgaagcaattgcgcGGCCGCCATGGCCGTTGCTATCGCTGCCGCGAGAGTATGGCGACTT 
               
               
                   
               
               
                 AACAGGGGTCTTTCACAAGCTGCTCTTCTTCTTTTGCGACAGCCAGGCGCGCGGGGGCTTGCCCGGAGC 
               
               
                   
               
               
                 CATCCCCCCCGGCAGCAACAACAGTTCAGTAGCCTTGACGATAAACCGCAATTCCCAGGCGCTTCAGCA 
               
               
                   
               
               
                 GAGTTCATTGATAAGCTGGAATTCATTCAACCCAACGTAATTTCCGGCATTCCTATTTATCGCGTAATG 
               
               
                   
               
               
                 GATAGACAGGGGCAAATAATTAACCCGAGCGAGGATCCACATCTTCCCAAGGAAAAAGTTCTTAAATTG 
               
               
                   
               
               
                 TATAAGTCTATGACCTTGCTTAACACGATGGACCGAATACTCTATGAATCTCAGCGGCAGGGCAGGATT 
               
               
                   
               
               
                 AGTTTCTATATGACAAATTATGGCGAAGAAGGAACCCACGTCGGGTCCGCAGCGGCCTTGGATAACACC 
               
               
                   
               
               
                 GACTTGGTCTTTGGACAGTACCGGGAGGCAGGTGTTCTTATGTACCGGGACTATCCCCTTGAGCTGTTC 
               
               
                   
               
               
                 ATGGCTCAATGTTATGGGAACATTAGTGATCTGGGGAAAGGCCGACAAATGCCCGTGCATTACGGATGT 
               
               
                   
               
               
                 AAAGAAAGGCATTTTGTAACTATCTCAAGTCCTCTTGCTACTCAAATACCGCAGGCCGTAGGTGCGGCG 
               
               
                   
               
               
                 TATGCTGCTAAGAGGGCAAACGCCAATAGAGTTGTGATATGCTACTTCGGTGAGGGGGCTGCAAGCGAG 
               
               
                   
               
               
                 GGAGATGCCCACGCCGGGTTCAACTTTGCAGCGACACTGGAGTGTCCCATTATATTTTTTTGTCGAAAC 
               
               
                   
               
               
                 AATGGCTATGCGATCTCTACCCCAACATCAGAGCAGTACAGAGGAGATGGGATTGCAGCACGGGGCCCC 
               
               
                   
               
               
                 GGTTATGGAATCATGTCTATACGCGTGGATGGGAACGACGTCTTTGCCGTATATAACGCTACTAAAGAG 
               
               
                   
               
               
                 GCCAGAAGACGAGCCGTGGCCGAGAATCAACCCTTCCTTATAGAGGCCATGACTTACAGAATTGGTCAT 
               
               
                   
               
               
                 CACTCTACGTCCGATGATTCTTCAGCTTACCGCAGCGTGGACGAGGTAAATTACTGGGATAAACAGGAC 
               
               
                   
               
               
                 CATCCTATTTCACGACTTCGGCATTATCTCCTCAGCCAGGGCTGGTGGGACGAAGAACAAGAAAAGGCA 
               
               
                   
               
               
                 TGGAGAAAACAATCTAGAAGAAAGGTTATGGAGGCCTTTGAGCAGGCAGAACGCAAACCAAAACCAAAT 
               
               
                   
               
               
                 CCCAATCTTCTTTTCAGCGACGTGTACCAGGAAATGCCAGCCCAGCTGCGGAAACAGCAAGAAAGCCTG 
               
               
                   
               
               
                 GCGAGACATCTTCAGACCTACGGGGAACATTACCCACTGGATCACTTTGACAAATGAAagcttggatcc 
               
               
                   
               
               
                 aatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacg 
               
               
                   
               
               
                 ctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcc 
               
               
                   
               
               
                 tccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtg 
               
               
                   
               
               
                 gtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttcc 
               
               
                   
               
               
                 gggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctgg 
               
               
                   
               
               
                 acaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttgg 
               
               
                   
               
               
                 ctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaat 
               
               
                   
               
               
                 ccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgagatctgcct 
               
               
                   
               
               
                 cgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaag 
               
               
                   
               
               
                 gtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcatt 
               
               
                   
               
               
                 ctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctg 
               
               
                   
               
               
                 gggactcgagttaagggcgaattcccgataaggatcttcctagagcatggctacgtagataagtagcat 
               
               
                   
               
               
                 ggcgggttaatcattaactacaaggaacccctagtgatggagttggccactccctctctgcgcgctcgc 
               
               
                   
               
               
                 tcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagc 
               
               
                   
               
               
                 gagcgagcgcgcagc 
               
               
                   
               
               
                 SEQ ID NO: 15 
                   
               
               
                 &gt; ITR-EF1a-BCKDHA-co2-ITR 
                   
               
               
                 ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccg 
               
               
                   
               
               
                 gcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttccttgtagtta 
               
               
                   
               
               
                 atgattaacccgccatgctacttatctacgtagccatgctctaggaagatcggaattcgcccttaagct 
               
               
                   
               
               
                 agcgctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggagggg 
               
               
                   
               
               
                 tcggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggc 
               
               
                   
               
               
                 tccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttc 
               
               
                   
               
               
                 gcaacgggtttgccgccagaacacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgg 
               
               
                   
               
               
                 gttatggcccttgcgtgccttgaattacttccacgcccctggctgcagtacgtgattcttgatcccgag 
               
               
                   
               
               
                 cttcgggttggaagtgggtgggagagttcgaggccttgcgcttaaggagccccttcgcctcgtgcttga 
               
               
                   
               
               
                 gttgaggcctggcctgggcgctggggccgccgcgtgcgaatctggtggcaccttcgcgcctgtctcgct 
               
               
                   
               
               
                 gctttcgataagtctctagccatttaaaatttttgatgacctgctgcgacgctttttttctggcaagat 
               
               
                   
               
               
                 agtcttgtaaatgcgggccaagatctgcacactggtatttcggtttttggggccgcgggcggcgacggg 
               
               
                   
               
               
                 gcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggccaccgagaatcggacgg 
               
               
                   
               
               
                 gggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcgccccgccctgg 
               
               
                   
               
               
                 gcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagatggccgcttcccggccctgctgca 
               
               
                   
               
               
                 gggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaaggaaaagg 
               
               
                   
               
               
                 gcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctcgat 
               
               
                   
               
               
                 tagttctcgagcttttggagtacgtcgtctttaggttggggggaggggttttatgcgatggagtttccc 
               
               
                   
               
               
                 cacactgagtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgcc 
               
               
                   
               
               
                 ctttttgagtttggatcttggttcattctcaagcctcagacagtggttcaaagtttttttcttccattt 
               
               
                   
               
               
                 caggtgtcgtgaagcaattgcgcGGCCGCCATGGCTGTTGCTATTGCTGCTGCGAGAGTATGGCGACTT 
               
               
                   
               
               
                 AACAGGGGTCTTTCACAAGCTGCTCTTCTTCTTTTGAGGCAGCCAGGAGCCAGAGGGCTTGCCAGAAGC 
               
               
                   
               
               
                 CATCCCCCCAGACAGCAACAACAGTTCAGTAGCCTTGATGATAAACCCCAATTCCCAGGAGCTTCAGCA 
               
               
                   
               
               
                 GAGTTCATTGATAAGCTGGAATTCATTCAACCCAATGTAATTTCTGGCATTCCTATTTATAGAGTAATG 
               
               
                   
               
               
                 GATAGACAGGGGCAAATAATTAACCCCTCCGAGGATCCACATCTTCCCAAGGAAAAAGTTCTTAAATTG 
               
               
                   
               
               
                 TATAAGTCTATGACCTTGCTTAACACCATGGACAGGATACTCTATGAATCTCAGAGACAGGGCAGGATT 
               
               
                   
               
               
                 AGTTTCTATATGACAAATTATGGAGAAGAAGGAACCCACGTGGGGAGCGCAGCCGCCTTGGATAACACC 
               
               
                   
               
               
                 GACTTGGTCTTTGGACAGTACAGGGAGGCAGGTGTTCTTATGTACAGGGACTATCCCCTTGAGCTGTTC 
               
               
                   
               
               
                 ATGGCTCAATGTTATGGGAACATTAGTGATCTGGGGAAAGGCCGACAAATGCCCGTGCATTACGGATGT 
               
               
                   
               
               
                 AAAGAAAGGCATTTTGTAACTATCTCAAGTCCTCTTGCTACTCAAATACCCCAGGCTGTAGGTGCCGCC 
               
               
                   
               
               
                 TATGCTGCTAAGAGGGCAAACGCCAATAGAGTTGTGATATGCTACTTCGGTGAGGGGGCTGCAAGCGAG 
               
               
                   
               
               
                 GGAGATGCCCACGCTGGGTTCAACTTTGCAGCCACACTGGAGTGTCCCATTATATTTTTTTGTAGGAAC 
               
               
                   
               
               
                 AATGGCTATGCCATCTCTACCCCAACATCAGAGCAGTACAGAGGAGATGGGATTGCAGCAAGAGGCCCC 
               
               
                   
               
               
                 GGTTATGGAATCATGTCTATAAGGGTGGATGGGAACGACGTCTTTGCCGTGTATAACGCTACTAAAGAG 
               
               
                   
               
               
                 GCCAGAAGAAGGGCTGTGGCTGAGAATCAACCCTTCCTTATAGAGGCCATGACTTACAGAATTGGTCAT 
               
               
                   
               
               
                 CACTCTACCTCCGATGATTCTTCAGCTTACAGATCCGTGGATGAGGTAAATTACTGGGATAAACAGGAC 
               
               
                   
               
               
                 CATCCTATTTCAAGACTTAGGCATTATCTCCTCAGCCAGGGCTGGTGGGACGAAGAACAAGAAAAGGCA 
               
               
                   
               
               
                 TGGAGAAAACAATCTAGAAGAAAGGTTATGGAGGCCTTTGAGCAGGCAGAAAGGAAACCAAAACCAAAT 
               
               
                   
               
               
                 CCCAATCTTCTTTTCTCCGACGTGTACCAGGAAATGCCAGCCCAGCTGAGGAAACAGCAAGAAAGCCTG 
               
               
                   
               
               
                 GCCAGACATCTTCAGACCTACGGGGAACATTACCCACTGGATCACTTTGACAAATGAAagcttggatcc 
               
               
                   
               
               
                 aatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacg 
               
               
                   
               
               
                 ctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcc 
               
               
                   
               
               
                 tccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtg 
               
               
                   
               
               
                 gtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttcc 
               
               
                   
               
               
                 gggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctgg 
               
               
                   
               
               
                 acaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttgg 
               
               
                   
               
               
                 ctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaat 
               
               
                   
               
               
                 ccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgagatctgcct 
               
               
                   
               
               
                 cgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaag 
               
               
                   
               
               
                 gtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcatt 
               
               
                   
               
               
                 ctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctg 
               
               
                   
               
               
                 gggactcgagttaagggcgaattcccgataaggatcttcctagagcatggctacgtagataagtagcat 
               
               
                   
               
               
                 ggcgggttaatcattaactacaaggaacccctagtgatggagttggccactccctctctgcgcgctcgc 
               
               
                   
               
               
                 tcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagc 
               
               
                   
               
               
                 gagcgagcgcgcag 
               
               
                   
               
               
                 SEQ ID NO: 16 
                   
               
               
                 &gt; ITR-EF1a-BCKDHB-WT-ITR 
                   
               
               
                 ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccg 
               
               
                   
               
               
                 gcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttccttgtagtta 
               
               
                   
               
               
                 atgattaacccgccatgctacttatctacgtagccatgctctaggaagatcggaattcgcccttaagct 
               
               
                   
               
               
                 agcgctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggagggg 
               
               
                   
               
               
                 tcggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggc 
               
               
                   
               
               
                 tccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttc 
               
               
                   
               
               
                 gcaacgggtttgccgccagaacacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgg 
               
               
                   
               
               
                 gttatggcccttgcgtgccttgaattacttccacgcccctggctgcagtacgtgattcttgatcccgag 
               
               
                   
               
               
                 cttcgggttggaagtgggtgggagagttcgaggccttgcgcttaaggagccccttcgcctcgtgcttga 
               
               
                   
               
               
                 gttgaggcctggcctgggcgctggggccgccgcgtgcgaatctggtggcaccttcgcgcctgtctcgct 
               
               
                   
               
               
                 gctttcgataagtctctagccatttaaaatttttgatgacctgctgcgacgctttttttctggcaagat 
               
               
                   
               
               
                 agtcttgtaaatgcgggccaagatctgcacactggtatttcggtttttggggccgcgggcggcgacggg 
               
               
                   
               
               
                 gcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggccaccgagaatcggacgg 
               
               
                   
               
               
                 gggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcgccccgccctgg 
               
               
                   
               
               
                 gcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagatggccgcttcccggccctgctgca 
               
               
                   
               
               
                 gggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaaggaaaagg 
               
               
                   
               
               
                 gcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctcgat 
               
               
                   
               
               
                 tagttctcgagcttttggagtacgtcgtctttaggttggggggaggggttttatgcgatggagtttccc 
               
               
                   
               
               
                 cacactgagtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgcc 
               
               
                   
               
               
                 ctttttgagtttggatcttggttcattctcaagcctcagacagtggttcaaagtttttttcttccattt 
               
               
                   
               
               
                 caggtgtcgtgaagcaattgcgcGGCCGCCATGGCGGTTGTAGCGGCGGCTGCCGGCTGGCTACTCAGG 
               
               
                   
               
               
                 CTCAGGGCGGCAGGGGCTGAGGGGCACTGGCGTCGGCTTCCTGGCGCGGGGCTGGCGCGGGGCTTTTTG 
               
               
                   
               
               
                 CACCCCGCCGCGACTGTCGAGGATGCGGCCCAGAGGCGGCAGGTGGCTCATTTTACTTTCCAGCCAGAT 
               
               
                   
               
               
                 CCGGAGCCCCGGGAGTACGGGCAAACTCAGAAAATGAATCTTTTCCAGTCTGTAACAAGTGCCTTGGAT 
               
               
                   
               
               
                 AACTCATTGGCCAAAGATCCTACTGCAGTAATATTTGGTGAAGATGTTGCCTTTGGTGGAGTCTTTAGA 
               
               
                   
               
               
                 TGCACTGTTGGCTTGCGAGACAAATATGGAAAAGATAGAGTTTTTAATACCCCATTGTGTGAACAAGGA 
               
               
                   
               
               
                 ATTGTTGGATTTGGAATCGGAATTGCGGTCACTGGAGCTACTGCCATTGCGGAAATTCAGTTTGCAGAT 
               
               
                   
               
               
                 TATATTTTCCCTGCATTTGATCAGATTGTTAATGAAGCTGCCAAGTATCGCTATCGCTCTGGGGATCTT 
               
               
                   
               
               
                 TTTAACTGTGGAAGCCTCACTATCCGGTCCCCTTGGGGCTGTGTTGGTCATGGGGCTCTCTATCATTCT 
               
               
                   
               
               
                 CAGAGTCCTGAAGCATTTTTTGCCCATTGCCCAGGAATCAAGGTGGTTATACCCAGAAGCCCTTTCCAG 
               
               
                   
               
               
                 GCCAAAGGACTTCTTTTGTCATGCATAGAGGATAAAAATCCTTGTATATTTTTTGAACCTAAAATACTT 
               
               
                   
               
               
                 TACAGGGCAGCAGCGGAAGAAGTCCCTATAGAACCATACAACATCCCACTGTCCCAGGCCGAAGTCATA 
               
               
                   
               
               
                 CAGGAAGGGAGTGATGTTACTCTAGTTGCCTGGGGCACTCAGGTTCATGTGATCCGAGAGGTAGCTTCC 
               
               
                   
               
               
                 ATGGCAAAAGAAAAGCTTGGAGTGTCTTGTGAAGTCATTGATCTGAGGACTATAATACCTTGGGATGTG 
               
               
                   
               
               
                 GACACAATTTGTAAGTCTGTGATCAAAACAGGGCGACTGCTAATCAGTCACGAGGCTCCCTTGACAGGC 
               
               
                   
               
               
                 GGCTTTGCATCGGAAATCAGCTCTACAGTTCAGGAGGAATGTTTCTTGAACCTAGAGGCTCCTATATCA 
               
               
                   
               
               
                 AGAGTATGTGGTTATGACACACCATTTCCTCACATTTTTGAACCATTCTACATCCCAGACAAATGGAAG 
               
               
                   
               
               
                 TGTTATGATGCCCTTCGAAAAATGATCAACTATTGAGgatccaatcaacctctggattacaaaatttgt 
               
               
                   
               
               
                 gaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcct 
               
               
                   
               
               
                 ttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtct 
               
               
                   
               
               
                 ctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacc 
               
               
                   
               
               
                 cccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctatt 
               
               
                   
               
               
                 gccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgac 
               
               
                   
               
               
                 aattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggatt 
               
               
                   
               
               
                 ctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctg 
               
               
                   
               
               
                 ctgccggctctgcggcctcttccgcgtcttcgagatctgcctcgactgtgccttctagttgccagccat 
               
               
                   
               
               
                 ctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaat 
               
               
                   
               
               
                 aaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcagg 
               
               
                   
               
               
                 acagcaagggggaggattgggaagacaatagcaggcatgctggggactcgagttaagggcgaattcccg 
               
               
                   
               
               
                 ataaggatcttcctagagcatggctacgtagataagtagcatggcgggttaatcattaactacaaggaa 
               
               
                   
               
               
                 cccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaag 
               
               
                   
               
               
                 gtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcag 
               
               
                   
               
               
                 SEQ ID NO: 17 
                   
               
               
                 &gt; ITR-EF1a-BCKDHB-co1-ITR 
                   
               
               
                 ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccg 
               
               
                   
               
               
                 gcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttccttgtagtta 
               
               
                   
               
               
                 atgattaacccgccatgctacttatctacgtagccatgctctaggaagatcggaattcgcccttaagct 
               
               
                   
               
               
                 agcgctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggagggg 
               
               
                   
               
               
                 tcggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggc 
               
               
                   
               
               
                 tccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttc 
               
               
                   
               
               
                 gcaacgggtttgccgccagaacacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgg 
               
               
                   
               
               
                 gttatggcccttgcgtgccttgaattacttccacgcccctggctgcagtacgtgattcttgatcccgag 
               
               
                   
               
               
                 cttcgggttggaagtgggtgggagagttcgaggccttgcgcttaaggagccccttcgcctcgtgcttga 
               
               
                   
               
               
                 gttgaggcctggcctgggcgctggggccgccgcgtgcgaatctggtggcaccttcgcgcctgtctcgct 
               
               
                   
               
               
                 gctttcgataagtctctagccatttaaaatttttgatgacctgctgcgacgctttttttctggcaagat 
               
               
                   
               
               
                 agtcttgtaaatgcgggccaagatctgcacactggtatttcggtttttggggccgcgggcggcgacggg 
               
               
                   
               
               
                 gcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggccaccgagaatcggacgg 
               
               
                   
               
               
                 gggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcgccccgccctgg 
               
               
                   
               
               
                 gcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagatggccgcttcccggccctgctgca 
               
               
                   
               
               
                 gggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaaggaaaagg 
               
               
                   
               
               
                 gcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctcgat 
               
               
                   
               
               
                 tagttctcgagcttttggagtacgtcgtctttaggttggggggaggggttttatgcgatggagtttccc 
               
               
                   
               
               
                 cacactgagtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgcc 
               
               
                   
               
               
                 ctttttgagtttggatcttggttcattctcaagcctcagacagtggttcaaagtttttttcttccattt 
               
               
                   
               
               
                 caggtgtcgtgaagcaattgcgcGGCCGCCATGGCAGTTGTGGCAGCCGCAGCGGGCTGGTTGTTGCGA 
               
               
                   
               
               
                 CTCAGAGCAGCCGGTGCAGAAGGCCATTGGAGACGGTTGCCGGGTGCGGGACTGGCGCGCGGCTTTCTC 
               
               
                   
               
               
                 CACCCCGCAGCGACTGTAGAAGACGCAGCCCAAAGACGACAGGTCGCTCACTTCACATTCCAGCCTGAT 
               
               
                   
               
               
                 CCCGAGCCACGAGAATACGGGCAAACGCAAAAAATGAATCTCTTTCAGTCCGTAACATCTGCTTTGGAT 
               
               
                   
               
               
                 AATAGTCTTGCAAAAGATCCAACAGCTGTAATTTTCGGGGAAGATGTAGCGTTTGGCGGTGTCTTCCGA 
               
               
                   
               
               
                 TGTACCGTCGGGCTGAGGGATAAGTACGGGAAAGATAGAGTATTTAATACCCCCCTGTGCGAGCAGGGT 
               
               
                   
               
               
                 ATAGTCGGATTTGGGATTGGAATAGCCGTAACGGGAGCAACAGCGATTGCCGAAATACAATTTGCCGAC 
               
               
                   
               
               
                 TATATCTTCCCGGCGTTTGACCAAATTGTTAACGAGGCTGCGAAATATCGGTATCGCTCCGGCGACTTG 
               
               
                   
               
               
                 TTTAATTGCGGTAGCCTCACAATTAGAAGTCCTTGGGGGTGCGTTGGACACGGTGCGCTCTATCACAGT 
               
               
                   
               
               
                 CAATCTCCAGAAGCTTTTTTCGCACATTGTCCAGGCATCAAAGTAGTGATTCCCCGAAGCCCATTTCAG 
               
               
                   
               
               
                 GCGAAAGGTCTCTTGCTCTCCTGTATAGAAGATAAAAACCCATGTATCTTTTTTGAGCCTAAAATCCTG 
               
               
                   
               
               
                 TACCGCGCCGCAGCTGAGGAAGTCCCTATAGAGCCATACAACATCCCACTCTCACAGGCAGAAGTTATA 
               
               
                   
               
               
                 CAAGAAGGGAGTGACGTGACACTCGTAGCATGGGGGACGCAGGTTCATGTGATCAGAGAGGTAGCCAGT 
               
               
                   
               
               
                 ATGGCAAAAGAGAAATTGGGAGTTTCTTGTGAAGTTATCGATCTCCGAACAATAATCCCTTGGGATGTA 
               
               
                   
               
               
                 GATACCATTTGTAAGTCTGTTATCAAAACTGGTAGGCTCCTCATATCTCATGAGGCCCCGTTGACGGGT 
               
               
                   
               
               
                 GGGTTCGCGTCCGAAATTTCATCAACTGTTCAAGAGGAGTGCTTTCTCAACCTGGAAGCGCCGATCTCT 
               
               
                   
               
               
                 AGAGTCTGCGGATATGATACCCCCTTCCCACACATATTTGAGCCTTTTTATATCCCGGACAAATGGAAG 
               
               
                   
               
               
                 TGTTACGACGCCCTTCGAAAAATGATAAATTATTGAGgatccaatcaacctctggattacaaaatttgt 
               
               
                   
               
               
                 gaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcct 
               
               
                   
               
               
                 ttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtct 
               
               
                   
               
               
                 ctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacc 
               
               
                   
               
               
                 cccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctatt 
               
               
                   
               
               
                 gccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgac 
               
               
                   
               
               
                 aattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggatt 
               
               
                   
               
               
                 ctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctg 
               
               
                   
               
               
                 ctgccggctctgcggcctcttccgcgtcttcgagatctgcctcgactgtgccttctagttgccagccat 
               
               
                   
               
               
                 ctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaat 
               
               
                   
               
               
                 aaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcagg 
               
               
                   
               
               
                 acagcaagggggaggattgggaagacaatagcaggcatgctggggactcgagttaagggcgaattcccg 
               
               
                   
               
               
                 ataaggatcttcctagagcatggctacgtagataagtagcatggcgggttaatcattaactacaaggaa 
               
               
                   
               
               
                 cccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaag 
               
               
                   
               
               
                 gtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcag 
               
               
                   
               
               
                 SEQ ID NO: 18 
                   
               
               
                 &gt; ITR-EF1a-BCKDHB-co2-ITR 
                   
               
               
                 ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccg 
               
               
                   
               
               
                 gcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttccttgtagtta 
               
               
                   
               
               
                 atgattaacccgccatgctacttatctacgtagccatgctctaggaagatcggaattcgcccttaagct 
               
               
                   
               
               
                 agcgctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggagggg 
               
               
                   
               
               
                 tcggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggc 
               
               
                   
               
               
                 tccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttc 
               
               
                   
               
               
                 gcaacgggtttgccgccagaacacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgg 
               
               
                   
               
               
                 gttatggcccttgcgtgccttgaattacttccacgcccctggctgcagtacgtgattcttgatcccgag 
               
               
                   
               
               
                 cttcgggttggaagtgggtgggagagttcgaggccttgcgcttaaggagccccttcgcctcgtgcttga 
               
               
                   
               
               
                 gttgaggcctggcctgggcgctggggccgccgcgtgcgaatctggtggcaccttcgcgcctgtctcgct 
               
               
                   
               
               
                 gctttcgataagtctctagccatttaaaatttttgatgacctgctgcgacgctttttttctggcaagat 
               
               
                   
               
               
                 agtcttgtaaatgcgggccaagatctgcacactggtatttcggtttttggggccgcgggcggcgacggg 
               
               
                   
               
               
                 gcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggccaccgagaatcggacgg 
               
               
                   
               
               
                 gggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcgccccgccctgg 
               
               
                   
               
               
                 gcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagatggccgcttcccggccctgctgca 
               
               
                   
               
               
                 gggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaaggaaaagg 
               
               
                   
               
               
                 gcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctcgat 
               
               
                   
               
               
                 tagttctcgagcttttggagtacgtcgtctttaggttggggggaggggttttatgcgatggagtttccc 
               
               
                   
               
               
                 cacactgagtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgcc 
               
               
                   
               
               
                 ctttttgagtttggatcttggttcattctcaagcctcagacagtggttcaaagtttttttcttccattt 
               
               
                   
               
               
                 caggtgtcgtgaagcaattgcgcGGCCGCCATGGCAGTTGTGGCAGCTGCAGCAGGCTGGTTGTTGCGC 
               
               
                   
               
               
                 CTCAGAGCAGCTGGTGCAGAAGGCCATTGGAGAAGGTTGCCTGGTGCCGGACTGGCCCGCGGCTTTCTC 
               
               
                   
               
               
                 CACCCCGCAGCCACTGTAGAAGATGCAGCCCAAAGAAGACAGGTCGCTCACTTCACATTCCAGCCTGAT 
               
               
                   
               
               
                 CCCGAGCCAAGAGAATATGGGCAAACCCAAAAAATGAATCTCTTTCAGTCCGTAACATCTGCTTTGGAT 
               
               
                   
               
               
                 AATAGTCTTGCAAAAGATCCAACAGCTGTAATTTTCGGGGAAGATGTAGCATTTGGAGGTGTCTTCAGG 
               
               
                   
               
               
                 TGTACAGTCGGGCTGAGGGATAAGTACGGGAAAGATAGAGTATTTAATACCCCCCTGTGTGAGCAGGGT 
               
               
                   
               
               
                 ATAGTGGGATTTGGGATTGGAATAGCTGTAACGGGAGCAACAGCAATTGCTGAAATACAATTTGCTGAC 
               
               
                   
               
               
                 TATATCTTCCCGGCATTTGACCAAATTGTTAACGAGGCTGCAAAATATAGGTATAGGTCCGGAGACTTG 
               
               
                   
               
               
                 TTTAATTGTGGTAGCCTCACAATTAGAAGTCCTTGGGGGTGTGTTGGACATGGTGCACTCTATCACAGT 
               
               
                   
               
               
                 CAATCTCCAGAAGCTTTTTTCGCACATTGTCCAGGCATCAAAGTAGTGATTCCCAGGAGCCCATTTCAG 
               
               
                   
               
               
                 GCAAAAGGTCTCTTGCTCTCCTGTATAGAAGATAAAAACCCATGTATCTTTTTTGAGCCTAAAATCCTG 
               
               
                   
               
               
                 TACAGAGCTGCAGCTGAGGAAGTCCCTATAGAGCCATACAACATCCCACTCTCACAGGCAGAAGTTATA 
               
               
                   
               
               
                 CAAGAAGGGAGTGATGTGACACTGGTAGCATGGGGGACCCAGGTTCATGTGATCAGAGAGGTAGCCAGT 
               
               
                   
               
               
                 ATGGCAAAAGAGAAATTGGGAGTTTCTTGTGAAGTTATCGATCTCCGAACAATAATCCCTTGGGATGTA 
               
               
                   
               
               
                 GATACCATTTGTAAGTCTGTTATCAAAACTGGTAGGCTCCTCATATCTCATGAGGCCCCGTTGACCGGT 
               
               
                   
               
               
                 GGGTTCGCATCCGAAATTTCATCAACTGTTCAAGAGGAGTGCTTTCTCAACCTGGAAGCACCAATCTCT 
               
               
                   
               
               
                 AGAGTCTGTGGATATGATACCCCCTTCCCACACATATTTGAGCCTTTTTATATCCCAGACAAATGGAAG 
               
               
                   
               
               
                 TGTTACGATGCCCTTAGAAAAATGATAAATTATTGAGgatccaatcaacctctggattacaaaatttgt 
               
               
                   
               
               
                 gaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcct 
               
               
                   
               
               
                 ttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtct 
               
               
                   
               
               
                 ctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacc 
               
               
                   
               
               
                 cccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctatt 
               
               
                   
               
               
                 gccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgac 
               
               
                   
               
               
                 aattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggatt 
               
               
                   
               
               
                 ctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctg 
               
               
                   
               
               
                 ctgccggctctgcggcctcttccgcgtcttcgagatctgcctcgactgtgccttctagttgccagccat 
               
               
                   
               
               
                 ctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaat 
               
               
                   
               
               
                 aaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcagg 
               
               
                   
               
               
                 acagcaagggggaggattgggaagacaatagcaggcatgctggggactcgagttaagggcgaattcccg 
               
               
                   
               
               
                 ataaggatcttcctagagcatggctacgtagataagtagcatggcgggttaatcattaactacaaggaa 
               
               
                   
               
               
                 cccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaag 
               
               
                   
               
               
                 gtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcag 
               
               
                   
               
               
                 SEQ ID NO: 19 
                   
               
               
                 &gt; ITR-hAAT-BCKDHA-WT-ITR 
                   
               
               
                 ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccg 
               
               
                   
               
               
                 gcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttccttgtagtta 
               
               
                   
               
               
                 atgattaacccgccatgctacttatctacgtagccatgctctaggaagatcggaattcgcccttaagCT 
               
               
                   
               
               
                 AGCAGGCTCAGAGGCACACAGGAGTTTCTGGGCTCACCCTGCCCCCTTCCAACCCCTCAGTTCCCATCC 
               
               
                   
               
               
                 TCCAGCAGCTGTTTGTGTGCTGCCTCTGAAGTCCACACTGAACAAACTTCAGCCTACTCATGTCCCTAA 
               
               
                   
               
               
                 AATGGGCAAACATTGCAAGCAGCAAACAGCAAACACACAGCCCTCCCTGCCTGCTGACCTTGGAGCTGG 
               
               
                   
               
               
                 GGCAGAGGTCAGAGACCTCTCTGGGCCCATGCCACCTCCAACATCCACTCGACCCCTTGGAATTTCGGT 
               
               
                   
               
               
                 GGAGAGGAGCAGAGGTTGTCCTGGCGTGGTTTAGGTAGTGTGAGAGGGGATCTTGCTACCAGTGGAACA 
               
               
                   
               
               
                 GCCACTAAGGATTCTGCAGTGAGAGCAGAGGGCCAGCTAAGTGGTACTCTCCCAGAGACTGTCTGACTC 
               
               
                   
               
               
                 ACGCCACCCCCTCCACCTTGGACACAGGACGCTGTGGTTTCTGAGCCAGGTACAATGACTCCTTTCGGT 
               
               
                   
               
               
                 AAGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGCGTCCGGGCAGCGTAGGCGGGCGACTCAGATCCC 
               
               
                   
               
               
                 AGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCTTGGTTAATATTCACCAGCAG 
               
               
                   
               
               
                 CCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGACAGGGCCCTGTCTCCTCAGCTT 
               
               
                   
               
               
                 CAGGCACCACCACTGACCTGGGACAGTGAATGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCA 
               
               
                   
               
               
                 ATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGA 
               
               
                   
               
               
                 CATCCACTTTGCCTTTCTCTCCACAGcaattgcGCGGCCGCCATGGCGGTAGCGATCGCTGCAGCGAGG 
               
               
                   
               
               
                 GTCTGGCGGCTAAACCGTGGTTTGAGCCAGGCTGCCCTCCTGCTGCTGCGGCAGCCTGGGGCTCGGGGA 
               
               
                   
               
               
                 CTGGCTAGATCTCACCCCCCCAGGCAGCAGCAGCAGTTTTCATCTCTGGATGACAAGCCCCAGTTCCCA 
               
               
                   
               
               
                 GGGGCCTCGGCGGAGTTTATAGATAAGTTGGAATTCATCCAGCCCAACGTCATCTCTGGAATCCCCATC 
               
               
                   
               
               
                 TACCGCGTCATGGACCGGCAAGGCCAGATCATCAACCCCAGCGAGGACCCCCACCTGCCGAAGGAGAAG 
               
               
                   
               
               
                 GTGCTGAAGCTCTACAAGAGCATGACACTGCTTAACACCATGGACCGCATCCTCTATGAGTCTCAGCGG 
               
               
                   
               
               
                 CAGGGCCGGATCTCCTTCTACATGACCAACTATGGTGAGGAGGGCACGCACGTGGGGAGTGCCGCCGCC 
               
               
                   
               
               
                 CTGGACAACACGGACCTGGTGTTTGGCCAGTACCGGGAGGCAGGTGTGCTGATGTATCGGGACTACCCC 
               
               
                   
               
               
                 CTGGAACTATTCATGGCCCAGTGCTATGGCAACATCAGTGACTTGGGCAAGGGGCGCCAGATGCCTGTC 
               
               
                   
               
               
                 CACTACGGCTGCAAGGAACGCCACTTCGTCACTATCTCCTCTCCACTGGCCACGCAGATCCCTCAGGCG 
               
               
                   
               
               
                 GTGGGGGCGGCGTACGCAGCCAAGCGGGCCAATGCCAACAGGGTCGTCATCTGTTACTTCGGCGAGGGG 
               
               
                   
               
               
                 GCAGCCAGTGAGGGGGACGCCCATGCCGGCTTCAACTTCGCTGCCACACTTGAGTGCCCCATCATCTTC 
               
               
                   
               
               
                 TTCTGCCGGAACAATGGCTACGCCATCTCCACGCCCACCTCTGAGCAGTATCGCGGCGATGGCATTGCA 
               
               
                   
               
               
                 GCACGAGGCCCCGGGTATGGCATCATGTCAATCCGCGTGGATGGTAATGATGTGTTTGCCGTATACAAC 
               
               
                   
               
               
                 GCCACAAAGGAGGCCCGACGGCGGGCTGTGGCAGAGAACCAGCCCTTCCTCATCGAGGCCATGACCTAC 
               
               
                   
               
               
                 AGGATCGGGCACCACAGCACCAGTGACGACAGTTCAGCGTACCGCTCGGTGGATGAGGTCAATTACTGG 
               
               
                   
               
               
                 GATAAACAGGACCACCCCATCTCCCGGCTGCGGCACTATCTGCTGAGCCAAGGCTGGTGGGATGAGGAG 
               
               
                   
               
               
                 CAGGAGAAGGCCTGGAGGAAGCAGTCCCGCAGGAAGGTGATGGAGGCCTTTGAGCAGGCCGAGCGGAAG 
               
               
                   
               
               
                 CCCAAACCCAACCCCAACCTACTCTTCTCAGACGTGTATCAGGAGATGCCCGCCCAGCTCCGCAAGCAG 
               
               
                   
               
               
                 CAGGAGTCTCTGGCCCGCCACCTGCAGACCTACGGGGAGCACTACCCACTGGATCACTTCGATAAGTGA 
               
               
                   
               
               
                 Aagcttggatccaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgtt 
               
               
                   
               
               
                 gctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggct 
               
               
                   
               
               
                 ttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcagg 
               
               
                   
               
               
                 caacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgt 
               
               
                   
               
               
                 cagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgcctt 
               
               
                   
               
               
                 gcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcg 
               
               
                   
               
               
                 tcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtccct 
               
               
                   
               
               
                 tcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtctt 
               
               
                   
               
               
                 cgagatctgcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttcct 
               
               
                   
               
               
                 tgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctga 
               
               
                   
               
               
                 gtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaata 
               
               
                   
               
               
                 gcaggcatgctggggactcgagttaagggcgaattcccgataaggatcttcctagagcatggctacgta 
               
               
                   
               
               
                 gataagtagcatggcgggttaatcattaactacaaggaacccctagtgatggagttggccactccctct 
               
               
                   
               
               
                 ctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcg 
               
               
                   
               
               
                 gcctcagtgagcgagcgagcgcgcag 
               
               
                   
               
               
                 SEQ ID NO: 20 
                   
               
               
                 &gt; ITR-hAAT-BCKDHA-co1-ITR 
                   
               
               
                 ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccg 
               
               
                   
               
               
                 gcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttccttgtagtta 
               
               
                   
               
               
                 atgattaacccgccatgctacttatctacgtagccatgctctaggaagatcggaattcgcccttaagCT 
               
               
                   
               
               
                 AGCAGGCTCAGAGGCACACAGGAGTTTCTGGGCTCACCCTGCCCCCTTCCAACCCCTCAGTTCCCATCC 
               
               
                   
               
               
                 TCCAGCAGCTGTTTGTGTGCTGCCTCTGAAGTCCACACTGAACAAACTTCAGCCTACTCATGTCCCTAA 
               
               
                   
               
               
                 AATGGGCAAACATTGCAAGCAGCAAACAGCAAACACACAGCCCTCCCTGCCTGCTGACCTTGGAGCTGG 
               
               
                   
               
               
                 GGCAGAGGTCAGAGACCTCTCTGGGCCCATGCCACCTCCAACATCCACTCGACCCCTTGGAATTTCGGT 
               
               
                   
               
               
                 GGAGAGGAGCAGAGGTTGTCCTGGCGTGGTTTAGGTAGTGTGAGAGGGGATCTTGCTACCAGTGGAACA 
               
               
                   
               
               
                 GCCACTAAGGATTCTGCAGTGAGAGCAGAGGGCCAGCTAAGTGGTACTCTCCCAGAGACTGTCTGACTC 
               
               
                   
               
               
                 ACGCCACCCCCTCCACCTTGGACACAGGACGCTGTGGTTTCTGAGCCAGGTACAATGACTCCTTTCGGT 
               
               
                   
               
               
                 AAGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGCGTCCGGGCAGCGTAGGCGGGCGACTCAGATCCC 
               
               
                   
               
               
                 AGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCTTGGTTAATATTCACCAGCAG 
               
               
                   
               
               
                 CCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGACAGGGCCCTGTCTCCTCAGCTT 
               
               
                   
               
               
                 CAGGCACCACCACTGACCTGGGACAGTGAATGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCA 
               
               
                   
               
               
                 ATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGA 
               
               
                   
               
               
                 CATCCACTTTGCCTTTCTCTCCACAGcaattgcGCGGCCGCCATGGCCGTTGCTATCGCTGCCGCGAGA 
               
               
                   
               
               
                 GTATGGCGACTTAACAGGGGTCTTTCACAAGCTGCTCTTCTTCTTTTGCGACAGCCAGGCGCGCGGGGG 
               
               
                   
               
               
                 CTTGCCCGGAGCCATCCCCCCCGGCAGCAACAACAGTTCAGTAGCCTTGACGATAAACCGCAATTCCCA 
               
               
                   
               
               
                 GGCGCTTCAGCAGAGTTCATTGATAAGCTGGAATTCATTCAACCCAACGTAATTTCCGGCATTCCTATT 
               
               
                   
               
               
                 TATCGCGTAATGGATAGACAGGGGCAAATAATTAACCCGAGCGAGGATCCACATCTTCCCAAGGAAAAA 
               
               
                   
               
               
                 GTTCTTAAATTGTATAAGTCTATGACCTTGCTTAACACGATGGACCGAATACTCTATGAATCTCAGCGG 
               
               
                   
               
               
                 CAGGGCAGGATTAGTTTCTATATGACAAATTATGGCGAAGAAGGAACCCACGTCGGGTCCGCAGCGGCC 
               
               
                   
               
               
                 TTGGATAACACCGACTTGGTCTTTGGACAGTACCGGGAGGCAGGTGTTCTTATGTACCGGGACTATCCC 
               
               
                   
               
               
                 CTTGAGCTGTTCATGGCTCAATGTTATGGGAACATTAGTGATCTGGGGAAAGGCCGACAAATGCCCGTG 
               
               
                   
               
               
                 CATTACGGATGTAAAGAAAGGCATTTTGTAACTATCTCAAGTCCTCTTGCTACTCAAATACCGCAGGCC 
               
               
                   
               
               
                 GTAGGTGCGGCGTATGCTGCTAAGAGGGCAAACGCCAATAGAGTTGTGATATGCTACTTCGGTGAGGGG 
               
               
                   
               
               
                 GCTGCAAGCGAGGGAGATGCCCACGCCGGGTTCAACTTTGCAGCGACACTGGAGTGTCCCATTATATTT 
               
               
                   
               
               
                 TTTTGTCGAAACAATGGCTATGCGATCTCTACCCCAACATCAGAGCAGTACAGAGGAGATGGGATTGCA 
               
               
                   
               
               
                 GCACGGGGCCCCGGTTATGGAATCATGTCTATACGCGTGGATGGGAACGACGTCTTTGCCGTATATAAC 
               
               
                   
               
               
                 GCTACTAAAGAGGCCAGAAGACGAGCCGTGGCCGAGAATCAACCCTTCCTTATAGAGGCCATGACTTAC 
               
               
                   
               
               
                 AGAATTGGTCATCACTCTACGTCCGATGATTCTTCAGCTTACCGCAGCGTGGACGAGGTAAATTACTGG 
               
               
                   
               
               
                 GATAAACAGGACCATCCTATTTCACGACTTCGGCATTATCTCCTCAGCCAGGGCTGGTGGGACGAAGAA 
               
               
                   
               
               
                 CAAGAAAAGGCATGGAGAAAACAATCTAGAAGAAAGGTTATGGAGGCCTTTGAGCAGGCAGAACGCAAA 
               
               
                   
               
               
                 CCAAAACCAAATCCCAATCTTCTTTTCAGCGACGTGTACCAGGAAATGCCAGCCCAGCTGCGGAAACAG 
               
               
                   
               
               
                 CAAGAAAGCCTGGCGAGACATCTTCAGACCTACGGGGAACATTACCCACTGGATCACTTTGACAAATGA 
               
               
                   
               
               
                 Aagcttggatccaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgtt 
               
               
                   
               
               
                 gctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggct 
               
               
                   
               
               
                 ttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcagg 
               
               
                   
               
               
                 caacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgt 
               
               
                   
               
               
                 cagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgcctt 
               
               
                   
               
               
                 gcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcg 
               
               
                   
               
               
                 tcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtccct 
               
               
                   
               
               
                 tcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtctt 
               
               
                   
               
               
                 cgagatctgcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttcct 
               
               
                   
               
               
                 tgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctga 
               
               
                   
               
               
                 gtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaata 
               
               
                   
               
               
                 gcaggcatgctggggactcgagttaagggcgaattcccgataaggatcttcctagagcatggctacgta 
               
               
                   
               
               
                 gataagtagcatggcgggttaatcattaactacaaggaacccctagtgatggagttggccactccctct 
               
               
                   
               
               
                 ctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcg 
               
               
                   
               
               
                 gcctcagtgagcgagcgagcgcgcag 
               
               
                   
               
               
                 SEQ ID NO: 21 
                   
               
               
                 &gt; ITR-hAAT-BCKDHA-co2-ITR 
                   
               
               
                 ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccg 
               
               
                   
               
               
                 gcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttccttgtagtta 
               
               
                   
               
               
                 atgattaacccgccatgctacttatctacgtagccatgctctaggaagatcggaattcgcccttaagCT 
               
               
                   
               
               
                 AGCAGGCTCAGAGGCACACAGGAGTTTCTGGGCTCACCCTGCCCCCTTCCAACCCCTCAGTTCCCATCC 
               
               
                   
               
               
                 TCCAGCAGCTGTTTGTGTGCTGCCTCTGAAGTCCACACTGAACAAACTTCAGCCTACTCATGTCCCTAA 
               
               
                   
               
               
                 AATGGGCAAACATTGCAAGCAGCAAACAGCAAACACACAGCCCTCCCTGCCTGCTGACCTTGGAGCTGG 
               
               
                   
               
               
                 GGCAGAGGTCAGAGACCTCTCTGGGCCCATGCCACCTCCAACATCCACTCGACCCCTTGGAATTTCGGT 
               
               
                   
               
               
                 GGAGAGGAGCAGAGGTTGTCCTGGCGTGGTTTAGGTAGTGTGAGAGGGGATCTTGCTACCAGTGGAACA 
               
               
                   
               
               
                 GCCACTAAGGATTCTGCAGTGAGAGCAGAGGGCCAGCTAAGTGGTACTCTCCCAGAGACTGTCTGACTC 
               
               
                   
               
               
                 ACGCCACCCCCTCCACCTTGGACACAGGACGCTGTGGTTTCTGAGCCAGGTACAATGACTCCTTTCGGT 
               
               
                   
               
               
                 AAGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGCGTCCGGGCAGCGTAGGCGGGCGACTCAGATCCC 
               
               
                   
               
               
                 AGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCTTGGTTAATATTCACCAGCAG 
               
               
                   
               
               
                 CCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGACAGGGCCCTGTCTCCTCAGCTT 
               
               
                   
               
               
                 CAGGCACCACCACTGACCTGGGACAGTGAATGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCA 
               
               
                   
               
               
                 ATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGA 
               
               
                   
               
               
                 CATCCACTTTGCCTTTCTCTCCACAGcaattgcGCGGCCGCCATGGCTGTTGCTATTGCTGCTGCGAGA 
               
               
                   
               
               
                 GTATGGCGACTTAACAGGGGTCTTTCACAAGCTGCTCTTCTTCTTTTGAGGCAGCCAGGAGCCAGAGGG 
               
               
                   
               
               
                 CTTGCCAGAAGCCATCCCCCCAGACAGCAACAACAGTTCAGTAGCCTTGATGATAAACCCCAATTCCCA 
               
               
                   
               
               
                 GGAGCTTCAGCAGAGTTCATTGATAAGCTGGAATTCATTCAACCCAATGTAATTTCTGGCATTCCTATT 
               
               
                   
               
               
                 TATAGAGTAATGGATAGACAGGGGCAAATAATTAACCCCTCCGAGGATCCACATCTTCCCAAGGAAAAA 
               
               
                   
               
               
                 GTTCTTAAATTGTATAAGTCTATGACCTTGCTTAACACCATGGACAGGATACTCTATGAATCTCAGAGA 
               
               
                   
               
               
                 CAGGGCAGGATTAGTTTCTATATGACAAATTATGGAGAAGAAGGAACCCACGTGGGGAGCGCAGCCGCC 
               
               
                   
               
               
                 TTGGATAACACCGACTTGGTCTTTGGACAGTACAGGGAGGCAGGTGTTCTTATGTACAGGGACTATCCC 
               
               
                   
               
               
                 CTTGAGCTGTTCATGGCTCAATGTTATGGGAACATTAGTGATCTGGGGAAAGGCCGACAAATGCCCGTG 
               
               
                   
               
               
                 CATTACGGATGTAAAGAAAGGCATTTTGTAACTATCTCAAGTCCTCTTGCTACTCAAATACCCCAGGCT 
               
               
                   
               
               
                 GTAGGTGCCGCCTATGCTGCTAAGAGGGCAAACGCCAATAGAGTTGTGATATGCTACTTCGGTGAGGGG 
               
               
                   
               
               
                 GCTGCAAGCGAGGGAGATGCCCACGCTGGGTTCAACTTTGCAGCCACACTGGAGTGTCCCATTATATTT 
               
               
                   
               
               
                 TTTTGTAGGAACAATGGCTATGCCATCTCTACCCCAACATCAGAGCAGTACAGAGGAGATGGGATTGCA 
               
               
                   
               
               
                 GCAAGAGGCCCCGGTTATGGAATCATGTCTATAAGGGTGGATGGGAACGACGTCTTTGCCGTGTATAAC 
               
               
                   
               
               
                 GCTACTAAAGAGGCCAGAAGAAGGGCTGTGGCTGAGAATCAACCCTTCCTTATAGAGGCCATGACTTAC 
               
               
                   
               
               
                 AGAATTGGTCATCACTCTACCTCCGATGATTCTTCAGCTTACAGATCCGTGGATGAGGTAAATTACTGG 
               
               
                   
               
               
                 GATAAACAGGACCATCCTATTTCAAGACTTAGGCATTATCTCCTCAGCCAGGGCTGGTGGGACGAAGAA 
               
               
                   
               
               
                 CAAGAAAAGGCATGGAGAAAACAATCTAGAAGAAAGGTTATGGAGGCCTTTGAGCAGGCAGAAAGGAAA 
               
               
                   
               
               
                 CCAAAACCAAATCCCAATCTTCTTTTCTCCGACGTGTACCAGGAAATGCCAGCCCAGCTGAGGAAACAG 
               
               
                   
               
               
                 CAAGAAAGCCTGGCCAGACATCTTCAGACCTACGGGGAACATTACCCACTGGATCACTTTGACAAATGA 
               
               
                   
               
               
                 Aagcttggatccaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgtt 
               
               
                   
               
               
                 gctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggct 
               
               
                   
               
               
                 ttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcagg 
               
               
                   
               
               
                 caacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgt 
               
               
                   
               
               
                 cagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgcctt 
               
               
                   
               
               
                 gcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcg 
               
               
                   
               
               
                 tcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtccct 
               
               
                   
               
               
                 tcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtctt 
               
               
                   
               
               
                 cgagatctgcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttcct 
               
               
                   
               
               
                 tgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctga 
               
               
                   
               
               
                 gtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaata 
               
               
                   
               
               
                 gcaggcatgctggggactcgagttaagggcgaattcccgataaggatcttcctagagcatggctacgta 
               
               
                   
               
               
                 gataagtagcatggcgggttaatcattaactacaaggaacccctagtgatggagttggccactccctct 
               
               
                   
               
               
                 ctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcg 
               
               
                   
               
               
                 gcctcagtgagcgagcgagcgcgcag 
               
               
                   
               
               
                 SEQ ID NO: 22 
                   
               
               
                 &gt; ITR-hAAT-BCKDHB-WT-ITR 
                   
               
               
                 ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccg 
               
               
                   
               
               
                 gcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttccttgtagtta 
               
               
                   
               
               
                 atgattaacccgccatgctacttatctacgtagccatgctctaggaagatcggaattcgcccttaagCT 
               
               
                   
               
               
                 AGCAGGCTCAGAGGCACACAGGAGTTTCTGGGCTCACCCTGCCCCCTTCCAACCCCTCAGTTCCCATCC 
               
               
                   
               
               
                 TCCAGCAGCTGTTTGTGTGCTGCCTCTGAAGTCCACACTGAACAAACTTCAGCCTACTCATGTCCCTAA 
               
               
                   
               
               
                 AATGGGCAAACATTGCAAGCAGCAAACAGCAAACACACAGCCCTCCCTGCCTGCTGACCTTGGAGCTGG 
               
               
                   
               
               
                 GGCAGAGGTCAGAGACCTCTCTGGGCCCATGCCACCTCCAACATCCACTCGACCCCTTGGAATTTCGGT 
               
               
                   
               
               
                 GGAGAGGAGCAGAGGTTGTCCTGGCGTGGTTTAGGTAGTGTGAGAGGGGATCTTGCTACCAGTGGAACA 
               
               
                   
               
               
                 GCCACTAAGGATTCTGCAGTGAGAGCAGAGGGCCAGCTAAGTGGTACTCTCCCAGAGACTGTCTGACTC 
               
               
                   
               
               
                 ACGCCACCCCCTCCACCTTGGACACAGGACGCTGTGGTTTCTGAGCCAGGTACAATGACTCCTTTCGGT 
               
               
                   
               
               
                 AAGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGCGTCCGGGCAGCGTAGGCGGGCGACTCAGATCCC 
               
               
                   
               
               
                 AGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCTTGGTTAATATTCACCAGCAG 
               
               
                   
               
               
                 CCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGACAGGGCCCTGTCTCCTCAGCTT 
               
               
                   
               
               
                 CAGGCACCACCACTGACCTGGGACAGTGAATGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCA 
               
               
                   
               
               
                 ATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGA 
               
               
                   
               
               
                 CATCCACTTTGCCTTTCTCTCCACAGcaattgcGCGGCCGCCATGGCGGTTGTAGCGGCGGCTGCCGGC 
               
               
                   
               
               
                 TGGCTACTCAGGCTCAGGGCGGCAGGGGCTGAGGGGCACTGGCGTCGGCTTCCTGGCGCGGGGCTGGCG 
               
               
                   
               
               
                 CGGGGCTTTTTGCACCCCGCCGCGACTGTCGAGGATGCGGCCCAGAGGCGGCAGGTGGCTCATTTTACT 
               
               
                   
               
               
                 TTCCAGCCAGATCCGGAGCCCCGGGAGTACGGGCAAACTCAGAAAATGAATCTTTTCCAGTCTGTAACA 
               
               
                   
               
               
                 AGTGCCTTGGATAACTCATTGGCCAAAGATCCTACTGCAGTAATATTTGGTGAAGATGTTGCCTTTGGT 
               
               
                   
               
               
                 GGAGTCTTTAGATGCACTGTTGGCTTGCGAGACAAATATGGAAAAGATAGAGTTTTTAATACCCCATTG 
               
               
                   
               
               
                 TGTGAACAAGGAATTGTTGGATTTGGAATCGGAATTGCGGTCACTGGAGCTACTGCCATTGCGGAAATT 
               
               
                   
               
               
                 CAGTTTGCAGATTATATTTTCCCTGCATTTGATCAGATTGTTAATGAAGCTGCCAAGTATCGCTATCGC 
               
               
                   
               
               
                 TCTGGGGATCTTTTTAACTGTGGAAGCCTCACTATCCGGTCCCCTTGGGGCTGTGTTGGTCATGGGGCT 
               
               
                   
               
               
                 CTCTATCATTCTCAGAGTCCTGAAGCATTTTTTGCCCATTGCCCAGGAATCAAGGTGGTTATACCCAGA 
               
               
                   
               
               
                 AGCCCTTTCCAGGCCAAAGGACTTCTTTTGTCATGCATAGAGGATAAAAATCCTTGTATATTTTTTGAA 
               
               
                   
               
               
                 CCTAAAATACTTTACAGGGCAGCAGCGGAAGAAGTCCCTATAGAACCATACAACATCCCACTGTCCCAG 
               
               
                   
               
               
                 GCCGAAGTCATACAGGAAGGGAGTGATGTTACTCTAGTTGCCTGGGGCACTCAGGTTCATGTGATCCGA 
               
               
                   
               
               
                 GAGGTAGCTTCCATGGCAAAAGAAAAGCTTGGAGTGTCTTGTGAAGTCATTGATCTGAGGACTATAATA 
               
               
                   
               
               
                 CCTTGGGATGTGGACACAATTTGTAAGTCTGTGATCAAAACAGGGCGACTGCTAATCAGTCACGAGGCT 
               
               
                   
               
               
                 CCCTTGACAGGCGGCTTTGCATCGGAAATCAGCTCTACAGTTCAGGAGGAATGTTTCTTGAACCTAGAG 
               
               
                   
               
               
                 GCTCCTATATCAAGAGTATGTGGTTATGACACACCATTTCCTCACATTTTTGAACCATTCTACATCCCA 
               
               
                   
               
               
                 GACAAATGGAAGTGTTATGATGCCCTTCGAAAAATGATCAACTATTGAGgatccaatcaacctctggat 
               
               
                   
               
               
                 tacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgct 
               
               
                   
               
               
                 gctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcc 
               
               
                   
               
               
                 tggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgttt 
               
               
                   
               
               
                 gctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttc 
               
               
                   
               
               
                 cccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctg 
               
               
                   
               
               
                 ttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgtt 
               
               
                   
               
               
                 gccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttcct 
               
               
                   
               
               
                 tcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgagatctgcctcgactgtgccttcta 
               
               
                   
               
               
                 gttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactg 
               
               
                   
               
               
                 tcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtg 
               
               
                   
               
               
                 gggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggactcgagttaag 
               
               
                   
               
               
                 ggcgaattcccgataaggatcttcctagagcatggctacgtagataagtagcatggcgggttaatcatt 
               
               
                   
               
               
                 aactacaaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggcc 
               
               
                   
               
               
                 gggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcag 
               
               
                   
               
               
                 SEQ ID NO: 23 
                   
               
               
                 &gt; ITR-hAAT-BCKDHB-co1-ITR 
                   
               
               
                 ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccg 
               
               
                   
               
               
                 gcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttccttgtagtta 
               
               
                   
               
               
                 atgattaacccgccatgctacttatctacgtagccatgctctaggaagatcggaattcgcccttaagCT 
               
               
                   
               
               
                 AGCAGGCTCAGAGGCACACAGGAGTTTCTGGGCTCACCCTGCCCCCTTCCAACCCCTCAGTTCCCATCC 
               
               
                   
               
               
                 TCCAGCAGCTGTTTGTGTGCTGCCTCTGAAGTCCACACTGAACAAACTTCAGCCTACTCATGTCCCTAA 
               
               
                   
               
               
                 AATGGGCAAACATTGCAAGCAGCAAACAGCAAACACACAGCCCTCCCTGCCTGCTGACCTTGGAGCTGG 
               
               
                   
               
               
                 GGCAGAGGTCAGAGACCTCTCTGGGCCCATGCCACCTCCAACATCCACTCGACCCCTTGGAATTTCGGT 
               
               
                   
               
               
                 GGAGAGGAGCAGAGGTTGTCCTGGCGTGGTTTAGGTAGTGTGAGAGGGGATCTTGCTACCAGTGGAACA 
               
               
                   
               
               
                 GCCACTAAGGATTCTGCAGTGAGAGCAGAGGGCCAGCTAAGTGGTACTCTCCCAGAGACTGTCTGACTC 
               
               
                   
               
               
                 ACGCCACCCCCTCCACCTTGGACACAGGACGCTGTGGTTTCTGAGCCAGGTACAATGACTCCTTTCGGT 
               
               
                   
               
               
                 AAGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGCGTCCGGGCAGCGTAGGCGGGCGACTCAGATCCC 
               
               
                   
               
               
                 AGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCTTGGTTAATATTCACCAGCAG 
               
               
                   
               
               
                 CCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGACAGGGCCCTGTCTCCTCAGCTT 
               
               
                   
               
               
                 CAGGCACCACCACTGACCTGGGACAGTGAATGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCA 
               
               
                   
               
               
                 ATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGA 
               
               
                   
               
               
                 CATCCACTTTGCCTTTCTCTCCACAGcaattgcGCGGCCGCCATGGCAGTTGTGGCAGCCGCAGCGGGC 
               
               
                   
               
               
                 TGGTTGTTGCGACTCAGAGCAGCCGGTGCAGAAGGCCATTGGAGACGGTTGCCGGGTGCGGGACTGGCG 
               
               
                   
               
               
                 CGCGGCTTTCTCCACCCCGCAGCGACTGTAGAAGACGCAGCCCAAAGACGACAGGTCGCTCACTTCACA 
               
               
                   
               
               
                 TTCCAGCCTGATCCCGAGCCACGAGAATACGGGCAAACGCAAAAAATGAATCTCTTTCAGTCCGTAACA 
               
               
                   
               
               
                 TCTGCTTTGGATAATAGTCTTGCAAAAGATCCAACAGCTGTAATTTTCGGGGAAGATGTAGCGTTTGGC 
               
               
                   
               
               
                 GGTGTCTTCCGATGTACCGTCGGGCTGAGGGATAAGTACGGGAAAGATAGAGTATTTAATACCCCCCTG 
               
               
                   
               
               
                 TGCGAGCAGGGTATAGTCGGATTTGGGATTGGAATAGCCGTAACGGGAGCAACAGCGATTGCCGAAATA 
               
               
                   
               
               
                 CAATTTGCCGACTATATCTTCCCGGCGTTTGACCAAATTGTTAACGAGGCTGCGAAATATCGGTATCGC 
               
               
                   
               
               
                 TCCGGCGACTTGTTTAATTGCGGTAGCCTCACAATTAGAAGTCCTTGGGGGTGCGTTGGACACGGTGCG 
               
               
                   
               
               
                 CTCTATCACAGTCAATCTCCAGAAGCTTTTTTCGCACATTGTCCAGGCATCAAAGTAGTGATTCCCCGA 
               
               
                   
               
               
                 AGCCCATTTCAGGCGAAAGGTCTCTTGCTCTCCTGTATAGAAGATAAAAACCCATGTATCTTTTTTGAG 
               
               
                   
               
               
                 CCTAAAATCCTGTACCGCGCCGCAGCTGAGGAAGTCCCTATAGAGCCATACAACATCCCACTCTCACAG 
               
               
                   
               
               
                 GCAGAAGTTATACAAGAAGGGAGTGACGTGACACTCGTAGCATGGGGGACGCAGGTTCATGTGATCAGA 
               
               
                   
               
               
                 GAGGTAGCCAGTATGGCAAAAGAGAAATTGGGAGTTTCTTGTGAAGTTATCGATCTCCGAACAATAATC 
               
               
                   
               
               
                 CCTTGGGATGTAGATACCATTTGTAAGTCTGTTATCAAAACTGGTAGGCTCCTCATATCTCATGAGGCC 
               
               
                   
               
               
                 CCGTTGACGGGTGGGTTCGCGTCCGAAATTTCATCAACTGTTCAAGAGGAGTGCTTTCTCAACCTGGAA 
               
               
                   
               
               
                 GCGCCGATCTCTAGAGTCTGCGGATATGATACCCCCTTCCCACACATATTTGAGCCTTTTTATATCCCG 
               
               
                   
               
               
                 GACAAATGGAAGTGTTACGACGCCCTTCGAAAAATGATAAATTATTGAGgatccaatcaacctctggat 
               
               
                   
               
               
                 tacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgct 
               
               
                   
               
               
                 gctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcc 
               
               
                   
               
               
                 tggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgttt 
               
               
                   
               
               
                 gctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttc 
               
               
                   
               
               
                 cccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctg 
               
               
                   
               
               
                 ttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgtt 
               
               
                   
               
               
                 gccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttcct 
               
               
                   
               
               
                 tcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgagatctgcctcgactgtgccttcta 
               
               
                   
               
               
                 gttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactg 
               
               
                   
               
               
                 tcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtg 
               
               
                   
               
               
                 gggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggactcgagttaag 
               
               
                   
               
               
                 ggcgaattcccgataaggatcttcctagagcatggctacgtagataagtagcatggcgggttaatcatt 
               
               
                   
               
               
                 aactacaaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggcc 
               
               
                   
               
               
                 gggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcag 
               
               
                   
               
               
                 SEQ ID NO: 24 
                   
               
               
                 &gt; ITR-hAAT-BCKDHB-co2-ITR 
                   
               
               
                 ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccg 
               
               
                   
               
               
                 gcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttccttgtagtta 
               
               
                   
               
               
                 atgattaacccgccatgctacttatctacgtagccatgctctaggaagatcggaattcgcccttaagcT 
               
               
                   
               
               
                 AGCAGGCTCAGAGGCACACAGGAGTTTCTGGGCTCACCCTGCCCCCTTCCAACCCCTCAGTTCCCATCC 
               
               
                   
               
               
                 TCCAGCAGCTGTTTGTGTGCTGCCTCTGAAGTCCACACTGAACAAACTTCAGCCTACTCATGTCCCTAA 
               
               
                   
               
               
                 AATGGGCAAACATTGCAAGCAGCAAACAGCAAACACACAGCCCTCCCTGCCTGCTGACCTTGGAGCTGG 
               
               
                   
               
               
                 GGCAGAGGTCAGAGACCTCTCTGGGCCCATGCCACCTCCAACATCCACTCGACCCCTTGGAATTTCGGT 
               
               
                   
               
               
                 GGAGAGGAGCAGAGGTTGTCCTGGCGTGGTTTAGGTAGTGTGAGAGGGGATCTTGCTACCAGTGGAACA 
               
               
                   
               
               
                 GCCACTAAGGATTCTGCAGTGAGAGCAGAGGGCCAGCTAAGTGGTACTCTCCCAGAGACTGTCTGACTC 
               
               
                   
               
               
                 ACGCCACCCCCTCCACCTTGGACACAGGACGCTGTGGTTTCTGAGCCAGGTACAATGACTCCTTTCGGT 
               
               
                   
               
               
                 AAGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGCGTCCGGGCAGCGTAGGCGGGCGACTCAGATCCC 
               
               
                   
               
               
                 AGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCTTGGTTAATATTCACCAGCAG 
               
               
                   
               
               
                 CCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGACAGGGCCCTGTCTCCTCAGCTT 
               
               
                   
               
               
                 CAGGCACCACCACTGACCTGGGACAGTGAATGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCA 
               
               
                   
               
               
                 ATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGA 
               
               
                   
               
               
                 CATCCACTTTGCCTTTCTCTCCACAGcaattgcGCGGCCGCCATGGCAGTTGTGGCAGCTGCAGCAGGC 
               
               
                   
               
               
                 TGGTTGTTGCGCCTCAGAGCAGCTGGTGCAGAAGGCCATTGGAGAAGGTTGCCTGGTGCCGGACTGGCC 
               
               
                   
               
               
                 CGCGGCTTTCTCCACCCCGCAGCCACTGTAGAAGATGCAGCCCAAAGAAGACAGGTCGCTCACTTCACA 
               
               
                   
               
               
                 TTCCAGCCTGATCCCGAGCCAAGAGAATATGGGCAAACCCAAAAAATGAATCTCTTTCAGTCCGTAACA 
               
               
                   
               
               
                 TCTGCTTTGGATAATAGTCTTGCAAAAGATCCAACAGCTGTAATTTTCGGGGAAGATGTAGCATTTGGA 
               
               
                   
               
               
                 GGTGTCTTCAGGTGTACAGTCGGGCTGAGGGATAAGTACGGGAAAGATAGAGTATTTAATACCCCCCTG 
               
               
                   
               
               
                 TGTGAGCAGGGTATAGTGGGATTTGGGATTGGAATAGCTGTAACGGGAGCAACAGCAATTGCTGAAATA 
               
               
                   
               
               
                 CAATTTGCTGACTATATCTTCCCGGCATTTGACCAAATTGTTAACGAGGCTGCAAAATATAGGTATAGG 
               
               
                   
               
               
                 TCCGGAGACTTGTTTAATTGTGGTAGCCTCACAATTAGAAGTCCTTGGGGGTGTGTTGGACATGGTGCA 
               
               
                   
               
               
                 CTCTATCACAGTCAATCTCCAGAAGCTTTTTTCGCACATTGTCCAGGCATCAAAGTAGTGATTCCCAGG 
               
               
                   
               
               
                 AGCCCATTTCAGGCAAAAGGTCTCTTGCTCTCCTGTATAGAAGATAAAAACCCATGTATCTTTTTTGAG 
               
               
                   
               
               
                 CCTAAAATCCTGTACAGAGCTGCAGCTGAGGAAGTCCCTATAGAGCCATACAACATCCCACTCTCACAG 
               
               
                   
               
               
                 GCAGAAGTTATACAAGAAGGGAGTGATGTGACACTGGTAGCATGGGGGACCCAGGTTCATGTGATCAGA 
               
               
                   
               
               
                 GAGGTAGCCAGTATGGCAAAAGAGAAATTGGGAGTTTCTTGTGAAGTTATCGATCTCCGAACAATAATC 
               
               
                   
               
               
                 CCTTGGGATGTAGATACCATTTGTAAGTCTGTTATCAAAACTGGTAGGCTCCTCATATCTCATGAGGCC 
               
               
                   
               
               
                 CCGTTGACCGGTGGGTTCGCATCCGAAATTTCATCAACTGTTCAAGAGGAGTGCTTTCTCAACCTGGAA 
               
               
                   
               
               
                 GCACCAATCTCTAGAGTCTGTGGATATGATACCCCCTTCCCACACATATTTGAGCCTTTTTATATCCCA 
               
               
                   
               
               
                 GACAAATGGAAGTGTTACGATGCCCTTAGAAAAATGATAAATTATTGAGgatccaatcaacctctggat 
               
               
                   
               
               
                 tacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgct 
               
               
                   
               
               
                 gctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcc 
               
               
                   
               
               
                 tggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgttt 
               
               
                   
               
               
                 gctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttc 
               
               
                   
               
               
                 cccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctg 
               
               
                   
               
               
                 ttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgtt 
               
               
                   
               
               
                 gccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttcct 
               
               
                   
               
               
                 tcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgagatctgcctcgactgtgccttcta 
               
               
                   
               
               
                 gttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactg 
               
               
                   
               
               
                 tcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtg 
               
               
                   
               
               
                 gggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggactcgagttaag 
               
               
                   
               
               
                 ggcgaattcccgataaggatcttcctagagcatggctacgtagataagtagcatggcgggttaatcatt 
               
               
                   
               
               
                 aactacaaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggcc 
               
               
                   
               
               
                 gggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcag 
               
            
           
         
       
     
     In some embodiments, the recombinant nucleic acid molecule of the present invention is inserted in a viral vector, more particularly in an AAV vector. 
     As used herein the term “AAV” refers to the more than 30 naturally occurring and available adeno-associated viruses, as well as artificial AAVs. Typically the AAV capsid, ITRs, and other selected AAV components described herein, may be readily selected from among any AAV, including, without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, rh10, AAVrh64R1, AAVrh64R2, rh8, variants of any of the known or mentioned AAVs or AAVs yet to be discovered or variants or mixtures thereof. See, e.g., WO 2005/033321. The genomic and protein sequences of various serotypes of AAV, as well as the sequences of the native terminal repeats (TRs), Rep proteins, and capsid subunits including VP1 protein are known in the art. Such sequences may be found in the literature or in public databases such as GenBank or Protein Data Bank (PDB). See, e.g., GenBank and PDB Accession Numbers NC_002077 and 3NG9 (AAV-1), AF043303 and 1LP3 (AAV-2), NC_001729 (AAV-3), U89790 and 2G8G (AAV-4), NC_006152 and 3NTT (AAV-5), 3OAH (AAV6), AF513851 (AAV-7), NC_006261 and 2QA0 (AAV-8), AY530579 and 3UX1 (AAV-9 (isolate hu.14)); the disclosures of which are incorporated by reference herein for teaching AAV nucleic acid and amino acid sequences. See also, e.g., Srivistava et al. (1983) J. Virology 45:555; Chiorini et al. (1998) J. Virology 71:6823; Chiorini et al. (1999) J. Virology 73: 1309; Bantel-Schaal et al. (1999) J. Virology 73:939; Xiao et al. (1999) J. Virology 73:3994; Muramatsu et al. (1996) Virology 221:208; Shade et al., (1986) J. Virol. 58:921; Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99: 11854; Moris et al. (2004) Virology 33:375-383; international patent publications WO 00/28061, WO 99/61601, WO 98/11244; and U.S. Pat. Nos. 6,156,303 and 7,906,111. 
     In some embodiments, the AAV vector is used in association with exosomes (exo-AAV) as described in WO2017136764 and in Hudry, Eloise, et al. “Exosome-associated AAV vector as a robust and convenient neuroscience tool.” Gene therapy 23.4 (2016): 380. 
     In some embodiments, the recombinant nucleic acid molecule of the present invention is inserted in a recombinant AAV8 viral particle. 
     As used herein, the term “recombinant AAV8 viral particle” refers to a viral particle that has an AAV8 capsid, the capsid having packaged therein the expression cassette comprising the recombinant nucleic molecule of the present invention. 
     As used herein, “AAV8 capsid” refers to the AAV8 capsid having the encoded amino acid sequence of GenBank accession:YP_077180, which is incorporated by reference herein and reproduced in SEQ ID NO:25. 
     
       
         
           
               
               
            
               
                   
                 SEQ ID NO: 25 
               
               
                   
                 &gt; capsid protein [Adeno-associated virus - 8] 
               
               
                   
                 MAADGYLPDW LEDNLSEGIR EWWALKPGAP KPKANQQKQD 
               
               
                   
                   
               
               
                   
                 DGRGLVLPGY KYLGPFNGLD KGEPVNAADA AALEHDKAYD 
               
               
                   
                   
               
               
                   
                 QQLQAGDNPY LRYNHADAEF QERLQEDTSF GGNLGRAVFQ 
               
               
                   
                   
               
               
                   
                 AKKRVLEPLG LVEEGAKTAP GKKRPVEPSP QRSPDSSTGI 
               
               
                   
                   
               
               
                   
                 GKKGQQPARK RLNFGQTGDS ESVPDPQPLG EPPAAPSGVG 
               
               
                   
                   
               
               
                   
                 PNTMAAGGGA PMADNNEGAD GVGSSSGNWH CDSTWLGDRV 
               
               
                   
                   
               
               
                   
                 ITTSTRTWAL PTYNNHLYKQ ISNGTSGGAT NDNTYFGYST 
               
               
                   
                   
               
               
                   
                 PWGYFDFNRF HCHFSPRDWQ RLINNNWGFR PKRLSFKLFN 
               
               
                   
                   
               
               
                   
                 IQVKEVTQNE GTKTIANNLT STIQVFTDSE YQLPYVLGSA 
               
               
                   
                   
               
               
                   
                 HQGCLPPFPA DVFMIPQYGY LTLNNGSQAV GRSSFYCLEY 
               
               
                   
                   
               
               
                   
                 FPSQMLRTGN NFQFTYTFED VPFHSSYAHS QSLDRLMNPL 
               
               
                   
                   
               
               
                   
                 IDQYLYYLSR TQTTGGTANT QTLGFSQGGP NTMANQAKNW 
               
               
                   
                   
               
               
                   
                 LPGPCYRQQR VSTTTGQNNN SNFAWTAGTK YHLNGRNSLA 
               
               
                   
                   
               
               
                   
                 NPGIAMATHK DDEERFFPSN GILIFGKQNA ARDNADYSDV 
               
               
                   
                   
               
               
                   
                 MLTSEEEIKT TNPVATEEYG IVADNLQQQN TAPQIGTVNS 
               
               
                   
                   
               
               
                   
                 QGALPGMVWQ NRDVYLQGPI WAKIPHTDGN FHPSPLMGGF 
               
               
                   
                   
               
               
                   
                 GLKHPPPQIL IKNTPVPADP PTTFNQSKLN SFITQYSTGQ 
               
               
                   
                   
               
               
                   
                 VSVEIEWELQ KENSKRWNPE IQYTSNYYKS TSVDFAVNTE 
               
               
                   
                   
               
               
                   
                 GVYSEPRPIG TRYLTRNL 
               
            
           
         
       
     
     The expression cassette of the recombinant AAV8 viral particle typically contains an AAV2 inverted terminal repeat sequence flanking the recombinant nucleic acid molecule of the present invention, in which the transgene sequence is operably linked to expression control sequences. Such a rAAV viral particle is termed “pharmacologically active” when it delivers the transgene to a host cell which is capable of expressing the desired gene product carried by the expression cassette. 
     Numerous methods are known in the art for production of rAAV vectors, including transfection, stable cell line production, and infectious hybrid virus production systems which include Adenovirus-AAV hybrids, herpesvirus-AAV hybrids and baculovirus-AAV hybrids. See, e.g., G Ye, et al, Hu Gene Ther Clin Dev, 25: 212-217 (December 2014); R M Kotin, Hu Mol Genet, 2011, Vol. 20, Rev Issue 1, R2-R6; M. Mietzsch, et al, Hum Gene Therapy, 25: 212-222 (March 2014); T Virag et al, Hu Gene Therapy, 20: 807-817 (August 2009); N. Clement et al, Hum Gene Therapy, 20: 796-806 (August 2009); DL Thomas et al, Hum Gene Ther, 20: 861-870 (August 2009). rAAV production cultures for the production of rAAV virus particles may require; 1) suitable host cells, including, for example, human-derived cell lines such as HeLa, A549, or 293 cells, or insect-derived cell lines such as SF-9, in the case of baculovirus production systems; 2) suitable helper virus function, provided by wild type or mutant adenovirus (such as temperature sensitive adenovirus), herpes virus, baculovirus, or a nucleic acid construct providing helper functions in trans or in cis; 3) functional AAV rep genes, functional cap genes and gene products; 4) a transgene (such as a therapeutic transgene) flanked by AAV ITR sequences; and 5) suitable media and media components to support rAAV production. A variety of suitable cells and cell lines have been described for use in production of AAV. The cell itself may be selected from any biological organism, including prokaryotic (e.g., bacterial) cells, and eukaryotic cells, including, insect cells, yeast cells and mammalian cells. Particularly desirable host cells are selected from among any mammalian species, including, without limitation, cells such as A549, WEHI, 3T3, 10T1/2, BHK, MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, VERO, WI38, HeLa, a HEK 293 cell (which express functional adenoviral E1), Saos, C2C12, L cells, HT1080, HepG2 and primary fibroblast, hepatocyte and myoblast cells derived from mammals including human, monkey, mouse, rat, rabbit, and hamster. rAAV vector particles may be harvested from rAAV production cultures by lysis of the host cells of the production culture or by harvest of the spent media from the production culture, provided the cells are cultured under conditions known in the art to cause release of rAAV particles into the media from intact cells, as described more fully in U.S. Pat. No. 6,566,118). Suitable methods of lysing cells are also known in the art and include for example multiple freeze/thaw cycles, sonication, microfluidization, and treatment with chemicals, such as detergents and/or proteases. In some embodiments, the rAAV production culture harvest is clarified to remove host cell debris. Suitably, the rAAV production culture harvest is treated with a nuclease, or a combination of nucleases, to digest any contaminating high molecular weight nucleic acid present in the production culture. The mixture containing full rAAV particles may be isolated or purified using one or more of the following purification steps: tangential flow filtration (TFF) for concentrating the rAAV particles, heat inactivation of helper virus, rAAV capture by hydrophobic interaction chromatography, buffer exchange by size exclusion chromatography (SEC), and/or nanofiltration. These steps may be used alone, in various combinations, or in different orders. 
     The recombinant AAV8 viral particle of the present invention is particularly suitable for the treatment of maple syrup urine disease (MSUD). 
     Therefore, a further object of the present invention relates to a method of treating MSUD in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the recombinant AAV8 viral particle of the present invention. 
     As used herein, the term “maple syrup urine disease” or “MSUD” has its general meaning in the art and refers to an inherited disorder in which the body is unable to process certain protein building blocks (amino acids) properly. The condition gets its name from the distinctive sweet odor of affected infants&#39; urine. It is also characterized by poor feeding, vomiting, lack of energy (lethargy), abnormal movements, and delayed development. If untreated, maple syrup urine disease can lead to seizures, coma, and death. Maple syrup urine disease is often classified by its pattern of signs and symptoms. The most common and severe form of the disease is the classic type, which becomes apparent soon after birth. Variant forms of the disorder become apparent later in infancy or childhood and are typically milder, but they still lead to delayed development and other health problems if not treated. 
     As used herein, the term “treatment” or “treat” refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By a “therapeutically effective amount” is meant a sufficient amount of cells generated with the present invention for the treatment of the disease at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total usage of these cells will be decided by the attending physicians within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and survival rate of the cells employed; the duration of the treatment; drugs used in combination or coincidental with the administered cells; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of cells at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. 
     By a “therapeutically effective amount” is meant a sufficient amount of the vector to treat the maple syrup urine disease at a reasonable benefit/risk ratio. It will be understood that the total daily usage of the vector will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. Thus, the doses of vectors may be adapted depending on the disease condition, the subject (for example, according to his weight, metabolism, etc.), the treatment schedule, etc. Typically, the doses of AAV vectors to be administered in humans may range from 5.10 11  to 5.10 14  vg/kg. 
     In some embodiments, the recombinant AAV8 viral particle of the present invention is administered to the subject intravenously. 
     The recombinant AAV8 viral particle of the present invention is thus formulated into pharmaceutical compositions. These compositions may comprise, in addition to the vector, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient (i.e. the vector of the invention). The precise nature of the carrier or other material may be determined by the skilled person according to the route of administration, i.e. here intravitreal injection. The pharmaceutical composition is typically in liquid form. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, magnesium chloride, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. For injection, the active ingredient will be in the form of an aqueous solution, which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer&#39;s Injection, Lactated Ringer&#39;s Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required. For delayed release, the vector may be included in a pharmaceutical composition, which is formulated for slow release, such as in microcapsules formed from biocompatible polymers or in liposomal carrier systems according to methods known in the art. Typically, the pharmaceutical composition of the present invention is supplied in a prefilled syringe. A “ready-to-use syringe” or “prefilled syringe” is a syringe which is supplied in a filled state, i.e. the pharmaceutical composition to be administered is already present in the syringe and ready for administration. Prefilled syringes have many benefits compared to separately provided syringe and vial, such as improved convenience, affordability, accuracy, sterility, and safety. The use of prefilled syringes results in greater dose precision, in a reduction of the potential for needle sticks injuries that can occur while drawing medication from vials, in pre-measured dosage reducing dosing errors due to the need to reconstituting and/or drawing medication into a syringe, and in less overfilling of the syringe helping to reduce costs by minimising drug waste. In some embodiments the pH of the liquid pharmaceutical composition of the present invention is in the range of 5.0 to 7.0, 5.1 to 6.9, 5.2 to 6.8, 5.3 to 6.7 or 5.4 to 6.6. 
     The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. 
    
    
     
       FIGURES 
         FIG.  1   . Bckdha −/−  mouse model recapitulates the severe human MSUD phenotype. a) Kaplan-Meier curves showing the survival probability during the first 30 days of life (Bckdha −/−  N=15, Bckdha +/−  N=44, Bckdha +/+  N=26); **** P&lt;0.0001 for the comparison Bckdha −/−  vs Bckdha +/+  and Bckdha −/−  vs Bckdha +/− , log-rank Mantel-Cox test. b) Weight curves showing major growth delay for Bckdha −/−  (Bckdha −/−  N=1, Bckdha +/−  N=2, Bckdha +/+  N=3); data are means±SD. c) Weights at 1 week; data are means±SD; **** P&lt;0.0001 for the comparison Bckdha −/−  vs Bckdha +/+  and Bckdha −/−  vs Bckdha +/− , one-way analysis of variance (ANOVA) with Tukey&#39;s post hoc. d) Leucine concentrations in plasma at 3 days; data are means±SD; **** P&lt;0.0001 for the comparison Bckdha −/−  vs Bckdha +/+  and Bckdha +/−  vs Bckdha +/− , one-way analysis of variance (ANOVA) with Tukey&#39;s post hoc. e) Alloisoleucine concentrations in plasma at 3 days; data are means±SD. 
         FIG.  2   . Scheme of the optimised AAV expression cassettes coding for either human BCKDHA or BCKDHB with (a) the Human elongation factor-1 alpha promoter (EF-1 alpha) and (b) the Human alpha anti-trypsin promoter (hAAT). 
         FIG.  3   . High dose gene therapy allows long-term rescue of severe MSUD phenotype of Bckdha−/− mice with EF1α hBCKDHA transgene. a) Weight curves for males (Bckdha −/−  N=6, Bckdha−/− N=3, Bckdha +/+  N=3); data are means±SD. b) Weight curves for females (Bckdha −/−  N=3, Bckdha +/−  N=8, Bckdha +/+  N=6); data are means±SD. c) Leucine concentrations in plasma ((Bckdha −/−  non injected N=3, Bckdha −/−  injected N=5, Bckdha +/−  injected N=5, Bckdha +/+  injected N=5); data are means±SD; Western blot analyses of Bckdha −/−  mice sacrificed at 6 months (N=5) and 1 non injected Bckdha −/−  mice died at 1 week and 1 non injected Bckdha +/+ . Histograms represent 3 technical replicates per individual. Data are means and SEM. d) Liver. e) Heart. f) Brain. g) Muscle. 
         FIG.  4   . Reducing EF1a hBCKDHA dosage allows partial albeit transient rescue of the MSUD phenotype in Bckdha−/− mice. a) Weight curves (Bckdha −/−  injected with 10 13  vg/kg and sacrificed &lt;4 weeks N=2, Bckdha −/−  injected with 10 13  vg/kg and sacrificed at 4 weeks N=5, Bckdha −/−  injected with 10 14  vg/kg and sacrificed at 4 weeks N=2, Bckdha +/−  N=18, Bckdha +/+  N=9); data are means±SD. b) Leucine concentrations in plasma at sacrifice (Bckdha −/−  injected with 10 13  vg/kg and sacrificed 4 weeks N=2, Bckdha −/−  injected with 10 13  vg/kg and sacrificed at 4 weeks N=5, Bckdha −/−  injected with 10 14  vg/kg and sacrificed at 4 weeks N=2, Bckdha +/−  N=18, Bckdha +/+  N=9) or at 4 weeks (Bckdha −/−  injected with 10 13  vg/kg from the previous experiment N=3); data are means±SD. Western blot analyses of Bckdha −/−  mice injected with 10 13  vg/kg and sacrificed at 4 weeks (N=7), Bckdha −/−  mice injected with 10 14  vg/kg and sacrificed at 4 weeks (N=2) and 1 non injected Bckdha −/−  mice died at 1 week and 1 non injected Bckdha +/+ . Histograms represent 3 technical replicates per individual. Data are means and SEM. c) Liver. d) Heart. e) Brain. f) Muscle. 
         FIG.  5   . Gene therapy with hAAT hBCKDHA transgene allows transient rescue of the MSUD phenotype in Bckdha −/−  mice. Weight curves showing a growth arrest and weight loss after day 14 leading to sacrifice at day 19 or 21 (Bckdha −/−  with initial normal growth N=3, Bckdha −/−  with growth on a lower curve N=2, Bckdha +/−  N=10, Bckdha +/+  N=6); data are means±SD. 
         FIG.  6   . Bckdhb −/−  mouse model recapitulates the severe human MSUD phenotype. a) Kaplan-Meier curves showing the survival probability during the first 16 days of life (Bckdhb −/−  N=7, Bckdhb +/+  N=8, Bckdhb +/+  N=4). b) Leucine and c) alloisoleucine concentrations in plasma at 1 days (Bckdhb −/−  N=7, Bckdhb +/−  N=8, Bckdhb +/+  N=4). Data are means±SD. 
         FIG.  7   . High dose gene therapy allows rescue of severe MSUD phenotype of Bckdhb −/−  mice with EF1α hBCKDHB transgene. Weight curves for a) males (Bckdhb −/−  N=5, Bckdhb +/−  N=4) and b) females (Bckdhb −/−  N=2, Bckdhb +/−  N=4, Bckdhb +/+  N=1). c) Leucine concentrations in plasma at 28 and 56 days post injection (Bckdha −/−  N=7, Bckdha +/−  N=8, Bckdha +/+  N=1). Data are means±SD. 
     
    
    
     EXAMPLE 
     Example 1 
     Bckdha−/− Mice Recapitulate the Severe Human MSUD Phenotype 
     We generated Bckdha−/− mice by crossing commercial heterozygous Bckdha+/− males and females, which did not display any particular phenotype. Bckdha−/− mice showed a lethal early-onset phenotype. Fifty percent of mice died before P3, 50% around P7 with a maximum life expectancy of 12 days ( FIG.  1   a   ). Bckdha−/− mice had a major growth delay ( FIG.  1   b, c   ). Mice that survived for more than a week showed reduced activity and abnormal response in the hindlimb test. At the biochemical level, Bckdha−/− mice displayed a major increase of branched-chain amino acids ( FIG.  1   d   ) and accumulation of alloisoleucine, a pathognomonic marker of MSUD in humans ( FIG.  1   e   ) in blood. In wild-type (WT) individuals, western blot showed that Bckdha protein was not expressed in skeletal muscle (quadriceps) ( FIG.  3   g   ) and showed mild expression in brain ( FIG.  3   f   ). In Bckdha−/− mice, Bckdha protein was absent in liver and heart, confirming the null nature of the model ( FIG.  3   d, e   ). 
     Design and In Vitro Validation of a Viral Vector for the Treatment of MSUD 
     To maximize transgene expression in liver in vivo we developed optimised AAV expression cassettes coding for either human BCKDHA or BCKDHB. We generated 3 variants of each gene coding sequence (CDS): the wild-type version (WT) and 2 different codon-optimised versions, the first one denominated co1 is a classic optimisation to increase protein expression and the second one, denominated co2 has a reduced CpG content ( FIG.  2   a, b   ). This is due to the fact that the reduction or elimination of immunostimulatory CpG sequences in plasmid expression vectors prevents the stimulation of transgene product-specific immune responses without necessarily reducing transgene expression. 
     The capsid serotype of choice was AAV8 due to its tropism to the liver; two different promoters, one ubiquitous, the Human elongation factor-1 alpha promoter (EF-1 alpha) ( FIG.  2   a   ) and one liver specific, the Human alpha anti-trypsin promoter (hAAT) were chosen to compare the protein expression ( FIG.  2   b   ), vector genome copy number (VGCN) in different tissues along with mRNA expression. 
     Intravenous EF1a hBCKDHA Allows Long-Term and Sustainable Rescue of Severe MSUD phenotype of Bckdha−/− Mice 
     In order to establish a proof of concept of treatment efficacy, we performed systemic intra-temporal injection of hBCKDHA transgene under the control of the ubiquitous promoter EF1α encapsulated in AAV8 at 10 14  vg/kg (further referred to as high dose) at P0, immediately after birth in mice pups. All the pups of the litters were injected, prior to get the genotype results. One pup died at P2 without corpse for genotyping and was not further included in the study. Genotypes were performed at P10. Nine Bckdha−/− pups from 3 litters were injected. Compared to their wild-type and heterozygous littermates they exhibited similar survival and a normal growth ( FIG.  3   a, b   ). At age 6 months these 9 pups were still alive without overt phenotypic abnormalities ( FIG.  3   a, b   ). The biochemical phenotype was dramatically improved ( FIG.  3   c   ). We sacrificed 5 Bckdha−/− mice at 6 months of age. At that age, hBCKDHA protein was detectable mainly in the liver and the heart and present albeit at lower levels in brain and skeletal muscle ( FIG.  3     d, e, f, g ). The remaining 4 Bckdha−/− mice were still alive without overt phenotypic abnormalities at age 12 months. 
     Reducing EF1a hBCKDHA Dosage Allows Partial Though Transient Rescue of the MSUD Phenotype in Bckdha−/− Mice 
     We performed the same experiment reducing EF1α hBCKDHA in mice to 10 13  vg/kg. Three litters were injected at P0 with EF1α hBCKDHA at 10 13  vg/kg and two as control with EF1α hBCKDHA at 10 14  vg/kg. In the litters injected at 10 13  vg/kg, one pup died at P3 without corpse, one Bckdha −/−  died at P1 probably due to injection failure and one Bckdha −/−  died at P7 of traumatic urine sampling, leaving for the analysis 7 Bckdha −/− , 9 Bckdha +/−  and 5 Bckdha +/+  mice. In the litters injected at 10 14  vg/kg, one Bckdha −/−  died at P2 probably due to injection failure and one Bckdha +/−  died at P7 in a context of major growth retardation, leaving for the analysis 2 Bckdha −/− , 9 Bckdha +/−  and 4 Bckdha +/+  mice. With the injections at 10 13  vg/kg, we observed a partial and transient recue of the MSUD phenotype in Bckdha−/− mice (N=7) with important inter-individual variability. Five out of seven Bckdha−/− mice showed a normal growth without obvious neurological signs during the first 3 weeks but then stopped growing and developed neurological signs (chiefly ataxia with frequent falls), urging us to sacrifice them at age 4 weeks ( FIG.  4   a   ). Two out of seven Bckdha−/− mice showed a more severe evolution, with a growth arrest during the third week followed by weight loss and the development of neurological signs evolving towards a moribund state requiring an anticipated sacrifice before age 4 weeks ( FIG.  4   a   ). As expected, the Bckdha−/− mice injected with EF1α hBCKDHA at 10 14  vg/kg displayed a normal growth without any neurological signs ( FIG.  4   a    N=2 Bckdha−/− injected at 10 14  vg/kg and sacrificed at 4 weeks). We observed a similar dose effect at the biochemical level: while the Bckdha−/− mice injected at 10 14  vg/kg displayed normal-high plasma leucine concentrations, the Bckdha−/− mice injected at 10 13  vg/kg and sacrificed at 4 weeks displayed a marked increase in leucine concentrations ( FIG.  4   b   ). The two Bckdha−/− mice injected at 10 13  vg/kg and sacrificed before 4 weeks showed a massive increase in leucine concentrations consistent with their worse clinical state, suggesting a loss of treatment efficacy and a relapse of the disease. Western blot analysis showed an important increase of hBCKDHA expression in liver for the two dosages, an increase of hBCKDHA expression in heart with a dose effect and a detectable hBCKDHA expression only at the 10 14  vg/kg dosage in the brain ( FIG.  4     c, d, e, f ). 
     Intravenous hAAT hBCKDHA Allows Transient Rescue of the MSUD Phenotype in Bckdha −/−  Mice 
     To evaluate the contribution of liver and extra-hepatic tissues to the whole-body BCKDHA enzyme activity responsible for the phenotypic rescue of mice treated with the EF1α hBCKDHA transgene at 10 14  vg/kg, we tested a non-ubiquitous liver-specific promotor (hAAT) with a dosage of 10 13  vg/kg that would be equivalent to 10 14  vg/kg with EF1α in terms of “liver” targeting. We performed systemic intra-temporal injections at P0, immediately after birth in three litters. One Bckdha −/−  died at P1, probably due to injection failure and one Bckdha +/−  died at P12 with a major growth retardation and was not included in the analysis, leaving 5 Bckdha −/− , 10 Bckdha +/−  and 6 Bckdha +/+  mice. This treatment allowed a transient rescue of the MSUD phenotype as 5/5 Bckdha −/−  mice survived more than 14 days without overt clinical symptoms. 3/5 Bckdha −/−  mice exhibited a strictly normal growth until P14, followed by a rapid weight loss, appearance of clinical signs (ataxia, frequent falls) evolving towards a moribund state requiring sacrifice at P19 or P21 ( FIG.  5   ). The 2 last Bckdha −/−  mice displayed growth on a lower curve during the 2 first weeks followed by a growth arrest during the third week with neurological deterioration and weight loss at P20 requiring sacrifice at P21. These results suggest that of non-liver tissues effectively contribute to the whole-body BCKDHA activity responsible for the phenotypic rescue of mice treated with the EF1α hBCKDHA transgene at 10 14  vg/kg. 
     Example 2 
     Bckdhb −/−  Mouse Model Recapitulates the Severe Human MSUD Phenotype 
     Bckdhb −/−  mouse model recapitulates the severe human MSUD phenotype, displaying a lethal early phenotype ( FIG.  6   a   ) with major accumulation of MSUD markers, leucine ( FIG.  6   b   ) and alloisoleucine, in plasma ( FIG.  6   c   ). 
     High dose gene therapy allows rescue of severe MSUD phenotype of Bckdhb −/−  mice with EF1α hBCKDHB transgene. In order to establish a proof of concept of treatment efficacy, we performed systemic intra-temporal injection of hBCKDHB transgene under the control of the ubiquitous promoter EF1a encapsulated in AAV8 at 10 14  vg/kg at P0, immediately after birth in mice pups. Compared to their wild-type and heterozygous littermates Bckdhb −/−  mice exhibited similar survival and a normal growth ( FIG.  7   a, b   ) without overt phenotypic abnormalities at age 3 months, with a dramatic improvement of the biochemical phenotype ( FIG.  7   c   ). 
     REFERENCES 
     Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.
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