COMBINED THERAPY FOR MUCOPOLYSACCHARIDOSIS TYPE VI (MAROTEAUX-LAMY-SYNDROME)
20190343929 ยท 2019-11-14
Assignee
Inventors
Cpc classification
H01F2027/2814
ELECTRICITY
A61K9/0019
HUMAN NECESSITIES
A61K48/0058
HUMAN NECESSITIES
H01F2027/2819
ELECTRICITY
A61K38/465
HUMAN NECESSITIES
C12Y301/06012
CHEMISTRY; METALLURGY
International classification
A61K9/00
HUMAN NECESSITIES
Abstract
The present invention relates to a method for the treatment of MPS VI comprising administering an arylsulfatase B by gene therapy to a subject in need thereof, wherein said subject is administered with an arylsulfatase B enzyme replacement therapy (ERT) less frequently than once a week.
Claims
1. (canceled)
2. A method for the treatment of MPS VI comprising: a) administering to a subject in need thereof a vector comprising a nucleic acid encoding an arylsulfatase B and b) administering to said subject an arylsulfatase B enzyme replacement therapy (ERT), wherein the ERT is administered less frequently than once a week.
3. The method of claim 2 wherein the nucleic acid encodes a wild-type arylsulfatase B.
4. The method of claim 3 wherein the wild-type arylsulfatase B comprises SEQ ID No. 2 or SEQ ID No. 4.
5. The method of claim 2 wherein the nucleic acid comprises SEQ ID No. 1.
6. The method of claim 2 wherein the nucleic acid is operably linked to a liver-specific promoter.
7. The method of claim 6 wherein the liver-specific promoter is selected from the group consisting of: thyroxine-binding globulin (TBG) promoter, alfa-1-antitripsin promoter, and albumin promoter.
8. The method of claim 7 wherein the thyroxine-binding globulin (TBG) promoter comprises SEQ ID No. 11, the alfa-1-antitripsin promoter comprises SEQ ID No. 12 and the albumin promoter comprises SEQ ID No. 13.
9. The method of claim 2 wherein the vector comprises SEQ ID No. 3.
10. The method of claim 2 wherein the vector is selected from the group consisting of: an adenoviral vector, lentiviral vector, retroviral vector, adeno associated vector (AAV) and a naked plasmid DNA vector.
11. The method of claim 2 wherein the vector is an adeno-associated viral (AAV) vector.
12. The method of claim 11 wherein the AAV vector is of serotype 8.
13. The method of claim 2 wherein the vector comprises SEQ ID No. 8.
14. The method of claim 2 wherein the dosage of the vector is of from 110.sup.9 to 210.sup.16 gc/kg.
15. The method of claim 2 wherein the vector is administered intravenously.
16. The method of claim 2 wherein the arylsulfatase B in the ERT comprises SEQ ID No. 2 or SEQ ID No. 4.
17. The method of claim 2 wherein the arylsulfatase B in the ERT is a recombinant arylsulfatase B.
18. The method of claim 2 wherein the arylsulfatase B in the ERT is administered at a dose range of 0.001 mg/kg to 5 mg/kg.
19. The method of claim 2 wherein the arylsulfatase B in the ERT is administered intravenously.
20. The method of claim 2, wherein the arylsulfatase B enzyme replacement therapy is administered less frequently than once every 2 weeks.
21. The method of claim 2, wherein the vector is administered at a dose ranging from 210.sup.11 gc/kg to 210.sup.12 gc/kg and the arylsulfatase B enzyme replacement therapy (ERT) is administered at a dose of 1 mg/kg and less frequently than once a week, preferably once a month.
22. The method of claim 2 wherein the vector and the arylsulfatase B enzyme replacement therapy are administered at different times.
23. The method of claim 2 wherein the vector is administered prior to the initiation of the arylsulfatase B enzyme replacement therapy.
24. The method of claim 2 wherein the vector is administered simultaneously with initiation of the arylsulfatase B enzyme replacement therapy.
25. The method of claim 2 wherein the vector is administered only once.
26. The method of claim 2 wherein the vector is administered after the initiation on the arylsulfatase B enzyme replacement therapy.
Description
DETAILED DESCRIPTION OF THE INVENTION
Figures
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[0110] Beta-glucuronidase (GUSB) activity was measured in liver (a) and kidney (b) of treated MPS VI mice (gray bars), and of normal (NR, white bars) and affected (AF, black bars) controls. GUSB activity was averaged for all animals within the same group of treatment and the resulting value is reported as meanSE. The number of animals is 5 per each group. Statistical comparisons were made using the one-way ANOVA and the Tukey post hoc test. The p value vs AF is: *<0.05 and **<<0.01. The exact p-values obtained are indicated in the Material and Methods section. Abbreviations: AAV, AAV2/8.TBG.hARSB; ERT, monthly ERT.
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TABLE-US-00002 SEQUENCES SEQIDNo.1hARSBpolynucleotidecodingsequence ATGGGTCCGCGCGGCGCGGCGAGCTTGCCCCGAGGCCCCGGACCTCGGCGGCTGCTCCTCCCCGTCGTCCTCCCGC TGCTGCTGCTGCTGTTGTTGGCGCCGCCGGGCTCGGGCGCCGGGGCCAGCCGGCCGCCCCACCTGGTCTTCTTGCT GGCAGACGACCTAGGCTGGAACGACGTCGGCTTCCACGGCTCCCGCATCCGCACGCCGCACCTGGACGCGCTGGCG GCCGGCGGGGTGCTCCTGGACAACTACTACACGCAGCCGCTGTGCACGCCGTCGCGGAGCCAGCTGCTCACTGGCC GCTACCAGATCCGTACAGGTTTACAGCACCAAATAATCTGGCCCTGTCAGCCCAGCTGTGTTCCTCTGGATGAAAA ACTCCTGCCCCAGCTCCTAAAAGAAGCAGGTTATACTACCCATATGGTCGGAAAATGGCACCTGGGAATGTACCGG AAAGAATGCCTTCCAACCCGCCGAGGATTTGATACCTACTTTGGATATCTCCTGGGTAGTGAAGATTATTATTCCC ATGAACGCTGTACATTAATTGACGCTCTGAATGTCACACGATGTGCTCTTGATTTTCGAGATGGCGAAGAAGTTGC AACAGGATATAAAAATATGTATTCAACAAACATATTCACCAAAAGGGCTATAGCCCTCATAACTAACCATCCACCA GAGAAGCCTCTGTTTCTCTACCTTGCTCTCCAGTCTGTGCATGAGCCCCTTCAGGTCCCTGAGGAATACTTGAAGC CATATGACTTTATCCAAGACAAGAACAGGCATCACTATGCAGGAATGGTGTCCCTTATGGATGAAGCAGTAGGAAA TGTCACTGCAGCTTTAAAAAGCAGTGGGCTCTGGAACAACACGGTGTTCATCTTTTCTACAGATAACGGAGGGCAG ACTTTGGCAGGGGGTAATAACTGGCCCCTTCGAGGAAGAAAATGGAGCCTGTGGGAAGGAGGCGTCCGAGGGGTGG GCTTTGTGGCAAGCCCCTTGCTGAAGCAGAAGGGCGTGAAGAACCGGGAGCTCATCCACATCTCTGACTGGCTGCC AACACTCGTGAAGCTGGCCAGGGGACACACCAATGGCACAAAGCCTCTGGATGGCTTCGACGTGTGGAAAACCATC AGTGAAGGAAGCCCATCCCCCAGAATTGAGCTGCTGCATAATATTGACCCGAACTTCGTGGACTCTTCACCGTGTC CCAGGAACAGCATGGCTCCAGCAAAGGATGACTCTTCTCTTCCAGAATATTCAGCCTTTAACACATCTGTCCATGC TGCAATTAGACATGGAAATTGGAAACTCCTCACGGGCTACCCAGGCTGTGGTTACTGGTTCCCTCCACCGTCTCAA TACAATGTTTCTGAGATACCCTCATCAGACCCACCAACCAAGACCCTCTGGCTCTTTGATATTGATCGGGACCCTG AAGAAAGACATGACCTGTCCAGAGAATATCCTCACATCGTCACAAAGCTCCTGTCCCGCCTACAGTTCTACCATAA ACACTCAGTCCCCGTGTACTTCCCTGCACAGGACCCCCGCTGTGATCCCAAGGCCACTGGGGTGTGGGGCCCTTGG ATGTAG SEQIDNo.2humanARSBpolypeptidesequence MGPRGAASLPRGPGPRRLLLPVVLPLLLLLLLAPPGSGAGASRPPHLVFLLADDLGWNDVGFHGSRIRTPHLDALA AGGVLLDNYYTQPLCTPSRSQLLTGRYQIRTGLQHQIIWPCQPSCVPLDEKLLPQLLKEAGYTTHMVGKWHLGMYR KECLPTRRGFDTYFGYLLGSEDYYSHERCTLIDALNVTRCALDFRDGEEVATGYKNMYSTNIFTKRAIALITNHPP EKPLFLYLALQSVHEPLQVPEEYLKPYDFIQDKNRHHYAGMVSLMDEAVGNVTAALKSSGLWNNTVFIFSTDNGGQ TLAGGNNWPLRGRKWSLWEGGVRGVGFVASPLLKQKGVKNRELIHISDWLPTLVKLARGHTNGTKPLDGFDVWKTI SEGSPSPRIELLHNIDPNFVDSSPCPRNSMAPAKDDSSLPEYSAFNTSVHAAIRHGNWKLLTGYPGCGYWFPPPSQ YNVSEIPSSDPPTKTLWLFDIDRDPEERHDLSREYPHIVTKLLSRLQFYHKHSVPVYFPAQDPRCDPKATGVWGPW M SEQIDNo.3hARSBexpressioncassette CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAG TGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCC ATGCTACTTATCTACCAGGGTAATGGGGATCCTCTAGAACTATAGCTAGAATTCGCCCTTAAGCTAGCAGGTTAAT TTTTAAAAAGCAGTCAAAAGTCCAAGTGGCCCTTGGCAGCATTTACTCTCTCTGTTTGCTCTGGTTAATAATCTCA GGAGCACAAACATTCCAGATCCAGGTTAATTTTTAAAAAGCAGTCAAAAGTCCAAGTGGCCCTTGGCAGCATTTAC TCTCTCTGTTTGCTCTGGTTAATAATCTCAGGAGCACAAACATTCCAGATCCGGCGCGCCAGGGCTGGAAGCTACC TTTGACATCATTTCCTCTGCGAATGCATGTATAATTTCTACAGAACCTATTAGAAAGGATCACCCAGCCTCTGCTT TTGTACAACTTTCCCTTAAAAAACTGCCAATTCCACTGCTGTTTGGCCCAATAGTGAGAACTTTTTCCTGCTGCCT CTTGGTGCTTTTGCCTATGGCCCCTATTCTGCCTGCTGAAGACACTCTTGCCAGCATGGACTTAAACCCCTCCAGC TCTGACAATCCTCTTTCTCTTTTGTTTTACATGAAGGGTCTGGCAGCCAAAGCAATCACTCAAAGTTCAAACCTTA TCATTTTTTGCTTTGTTCCTCTTGGCCTTGGTTTTGTACATCAGCTTTGAAAATACCATCCCAGGGTTAATGCTGG GGTTAATTTATAACTAAGAGTGCTCTAGTTTTGCAATACAGGACATGCTATAAAAATGGAAAGATGTTGCTTTCTG AGAGACTGCAGAAGTTGGTCGTGAGGCACTGGGCAGGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAAT AGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGACATCCACTT TGCCTTTCTCTCCACAGGTGTCCAGGCCCGGAGCCGCCATGGGTCCGCGCGGCGCGGCGAGCTTGCCCCGAGGCCC CGGACCTCGGCGGCTGCTCCTCCCCGTCGTCCTCCCGCTGCTGCTGCTGCTGTTGTTGGCGCCGCCGGGCTCGGGC GCCGGGGCCAGCCGGCCGCCCCACCTGGTCTTCTTGCTGGCAGACGACCTAGGCTGGAACGACGTCGGCTTCCACG GCTCCCGCATCCGCACGCCGCACCTGGACGCGCTGGCGGCCGGCGGGGTGCTCCTGGACAACTACTACACGCAGCC GCTGTGCACGCCGTCGCGGAGCCAGCTGCTCACTGGCCGCTACCAGATCCGTACAGGTTTACAGCACCAAATAATC TGGCCCTGTCAGCCCAGCTGTGTTCCTCTGGATGAAAAACTCCTGCCCCAGCTCCTAAAAGAAGCAGGTTATACTA CCCATATGGTCGGAAAATGGCACCTGGGAATGTACCGGAAAGAATGCCTTCCAACCCGCCGAGGATTTGATACCTA CTTTGGATATCTCCTGGGTAGTGAAGATTATTATTCCCATGAACGCTGTACATTAATTGACGCTCTGAATGTCACA CGATGTGCTCTTGATTTTCGAGATGGCGAAGAAGTTGCAACAGGATATAAAAATATGTATTCAACAAACATATTCA CCAAAAGGGCTATAGCCCTCATAACTAACCATCCACCAGAGAAGCCTCTGTTTCTCTACCTTGCTCTCCAGTCTGT GCATGAGCCCCTTCAGGTCCCTGAGGAATACTTGAAGCCATATGACTTTATCCAAGACAAGAACAGGCATCACTAT GCAGGAATGGTGTCCCTTATGGATGAAGCAGTAGGAAATGTCACTGCAGCTTTAAAAAGCAGTGGGCTCTGGAACA ACACGGTGTTCATCTTTTCTACAGATAACGGAGGGCAGACTTTGGCAGGGGGTAATAACTGGCCCCTTCGAGGAAG AAAATGGAGCCTGTGGGAAGGAGGCGTCCGAGGGGTGGGCTTTGTGGCAAGCCCCTTGCTGAAGCAGAAGGGCGTG AAGAACCGGGAGCTCATCCACATCTCTGACTGGCTGCCAACACTCGTGAAGCTGGCCAGGGGACACACCAATGGCA CAAAGCCTCTGGATGGCTTCGACGTGTGGAAAACCATCAGTGAAGGAAGCCCATCCCCCAGAATTGAGCTGCTGCA TAATATTGACCCGAACTTCGTGGACTCTTCACCGTGTCCCAGGAACAGCATGGCTCCAGCAAAGGATGACTCTTCT CTTCCAGAATATTCAGCCTTTAACACATCTGTCCATGCTGCAATTAGACATGGAAATTGGAAACTCCTCACGGGCT ACCCAGGCTGTGGTTACTGGTTCCCTCCACCGTCTCAATACAATGTTTCTGAGATACCCTCATCAGACCCACCAAC CAAGACCCTCTGGCTCTTTGATATTGATCGGGACCCTGAAGAAAGACATGACCTGTCCAGAGAATATCCTCACATC GTCACAAAGCTCCTGTCCCGCCTACAGTTCTACCATAAACACTCAGTCCCCGTGTACTTCCCTGCACAGGACCCCC GCTGTGATCCCAAGGCCACTGGGGTGTGGGGCCCTTGGATGTAGCTCGAATCAAGCTTATCGATTCTAGTAGATCT GCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTG CCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGG GGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGACTCGAGTTAAGGG CGAATTCCCGATAAGGATCTTCCTAGAGCATGGCTACAATTCGTTGATCTGAATTTCGACCACCCATAATACCCAT TACCCTGGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCC TCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCT CAGTGAGCGAGCGAGCGCGCAG Legend 5 ITR:nt1-130 3 ITR:nt3085-3214 Humanthyroxine-bindingglobulin(TBG)promoter:nt220-917(of whichAMBPenhancer(alpha-1-microglobulin/bikuninprecursor) genetranscriptionregulatoryregion,:nt220-426) Intron:nt950-1081 Kozaksequence:nt1089-1102 hARSBCodingsequence:nt1103-2704 BovineGrowthHormone(BGH)polyA:nt2744-2951 SEQIDNo.4galsulfase SGAGASRPPHLVFLLADDLGWNDVGFHGSRIRTPHLDALAAGGVLLDNYYTQPLCTPSRS QLLTGRYQIRTGLQHQIIWPCQPSCVPLDEKLLPQLLKEAGYTTHMVGKWHLGMYRKECL PTRRGFDTYFGYLLGSEDYYSHERCTLIDALNVTRCALDFRDGEEVATGYKNMYSTNIFT KRAIALITNHPPEKPLFLYLALQSVHEPLQVPEEYLKPYDFIQDKNRHHYAGMVSLMDEA VGNVTAALKSSGLWNNTVFIFSTDNGGQTLAGGNNWPLRGRKWSLWEGGVRGVGFVASPL LKQKGVKNRELIHISDWLPTLVKLARGHTNGTKPLDGFDVWKTISEGSPSPRIELLHNID PNFVDSSPCPRNSMAPAKDDSSLPEYSAFNTSVHAAIRHGNWKLLTGYPGCGYWFPPPSQ YNVSEIPSSDPPTKTLWLFDIDRDPEERHDLSREYPHIVTKLLSRLQFYHKHSVPVYFPA QDPRCDPKATGVWGPWM SEQIDNo.55 ITR CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAG TGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT SEQIDNo.63 ITR AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGT CGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAG SEQIDNo.7BGHpolyAsequence CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCC CACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGG GTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGA SEQIDNo.8pAAV2.1.TBG-hARSB CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAG TGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCC ATGCTACTTATCTACCAGGGTAATGGGGATCCTCTAGAACTATAGCTAGAATTCGCCCTTAAGCTAGCAGGTTAAT TTTTAAAAAGCAGTCAAAAGTCCAAGTGGCCCTTGGCAGCATTTACTCTCTCTGTTTGCTCTGGTTAATAATCTCA GGAGCACAAACATTCCAGATCCAGGTTAATTTTTAAAAAGCAGTCAAAAGTCCAAGTGGCCCTTGGCAGCATTTAC TCTCTCTGTTTGCTCTGGTTAATAATCTCAGGAGCACAAACATTCCAGATCCGGCGCGCCAGGGCTGGAAGCTACC TTTGACATCATTTCCTCTGCGAATGCATGTATAATTTCTACAGAACCTATTAGAAAGGATCACCCAGCCTCTGCTT TTGTACAACTTTCCCTTAAAAAACTGCCAATTCCACTGCTGTTTGGCCCAATAGTGAGAACTTTTTCCTGCTGCCT CTTGGTGCTTTTGCCTATGGCCCCTATTCTGCCTGCTGAAGACACTCTTGCCAGCATGGACTTAAACCCCTCCAGC TCTGACAATCCTCTTTCTCTTTTGTTTTACATGAAGGGTCTGGCAGCCAAAGCAATCACTCAAAGTTCAAACCTTA TCATTTTTTGCTTTGTTCCTCTTGGCCTTGGTTTTGTACATCAGCTTTGAAAATACCATCCCAGGGTTAATGCTGG GGTTAATTTATAACTAAGAGTGCTCTAGTTTTGCAATACAGGACATGCTATAAAAATGGAAAGATGTTGCTTTCTG AGAGACTGCAGAAGTTGGTCGTGAGGCACTGGGCAGGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAAT AGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGACATCCACTT TGCCTTTCTCTCCACAGGTGTCCAGGCCCGGAGCCGCCATGGGTCCGCGCGGCGCGGCGAGCTTGCCCCGAGGCCC CGGACCTCGGCGGCTGCTCCTCCCCGTCGTCCTCCCGCTGCTGCTGCTGCTGTTGTTGGCGCCGCCGGGCTCGGGC GCCGGGGCCAGCCGGCCGCCCCACCTGGTCTTCTTGCTGGCAGACGACCTAGGCTGGAACGACGTCGGCTTCCACG GCTCCCGCATCCGCACGCCGCACCTGGACGCGCTGGCGGCCGGCGGGGTGCTCCTGGACAACTACTACACGCAGCC GCTGTGCACGCCGTCGCGGAGCCAGCTGCTCACTGGCCGCTACCAGATCCGTACAGGTTTACAGCACCAAATAATC TGGCCCTGTCAGCCCAGCTGTGTTCCTCTGGATGAAAAACTCCTGCCCCAGCTCCTAAAAGAAGCAGGTTATACTA CCCATATGGTCGGAAAATGGCACCTGGGAATGTACCGGAAAGAATGCCTTCCAACCCGCCGAGGATTTGATACCTA CTTTGGATATCTCCTGGGTAGTGAAGATTATTATTCCCATGAACGCTGTACATTAATTGACGCTCTGAATGTCACA CGATGTGCTCTTGATTTTCGAGATGGCGAAGAAGTTGCAACAGGATATAAAAATATGTATTCAACAAACATATTCA CCAAAAGGGCTATAGCCCTCATAACTAACCATCCACCAGAGAAGCCTCTGTTTCTCTACCTTGCTCTCCAGTCTGT GCATGAGCCCCTTCAGGTCCCTGAGGAATACTTGAAGCCATATGACTTTATCCAAGACAAGAACAGGCATCACTAT GCAGGAATGGTGTCCCTTATGGATGAAGCAGTAGGAAATGTCACTGCAGCTTTAAAAAGCAGTGGGCTCTGGAACA ACACGGTGTTCATCTTTTCTACAGATAACGGAGGGCAGACTTTGGCAGGGGGTAATAACTGGCCCCTTCGAGGAAG AAAATGGAGCCTGTGGGAAGGAGGCGTCCGAGGGGTGGGCTTTGTGGCAAGCCCCTTGCTGAAGCAGAAGGGCGTG AAGAACCGGGAGCTCATCCACATCTCTGACTGGCTGCCAACACTCGTGAAGCTGGCCAGGGGACACACCAATGGCA CAAAGCCTCTGGATGGCTTCGACGTGTGGAAAACCATCAGTGAAGGAAGCCCATCCCCCAGAATTGAGCTGCTGCA TAATATTGACCCGAACTTCGTGGACTCTTCACCGTGTCCCAGGAACAGCATGGCTCCAGCAAAGGATGACTCTTCT CTTCCAGAATATTCAGCCTTTAACACATCTGTCCATGCTGCAATTAGACATGGAAATTGGAAACTCCTCACGGGCT ACCCAGGCTGTGGTTACTGGTTCCCTCCACCGTCTCAATACAATGTTTCTGAGATACCCTCATCAGACCCACCAAC CAAGACCCTCTGGCTCTTTGATATTGATCGGGACCCTGAAGAAAGACATGACCTGTCCAGAGAATATCCTCACATC GTCACAAAGCTCCTGTCCCGCCTACAGTTCTACCATAAACACTCAGTCCCCGTGTACTTCCCTGCACAGGACCCCC GCTGTGATCCCAAGGCCACTGGGGTGTGGGGCCCTTGGATGTAGCTCGAATCAAGCTTATCGATTCTAGTAGATCT GCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTG CCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGG GGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGACTCGAGTTAAGGG CGAATTCCCGATAAGGATCTTCCTAGAGCATGGCTACAATTCGTTGATCTGAATTTCGACCACCCATAATACCCAT TACCCTGGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCC TCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCT CAGTGAGCGAGCGAGCGCGCAGCCTTAATTAACCTAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAA CCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGC ACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGG CGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCC TTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGT GCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGG TTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCC TATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAA CAAAAATTTAACGCGAATTTTAACAAAATCATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCG CGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCG AAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTG CCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATC TCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTT ATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGG ATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAA CAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACA AACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGAT CCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTAT CAAAAAGGATCTTCACCTAGATCCTTTTGATCCTCCGGCGTTCAGCCTGTGCCACAGCCGACAGGATGGTGACCAC CATTTGCCCCATATCACCGTCGGTACTGATCCCGTCGTCAATAAACCGAACCGCTACACCCTGAGCATCAAACTCT TTTATCAGTTGGATCATGTCGGCGGTGTCGCGGCCAAGACGGTCGAGCTTCTTCACCAGAATGACATCACCTTCCT CCACCTTCATCCTCAGCAAATCCAGCCCTTCCCGATCTGTTGAACTGCCGGATGCCTTGTCGGTAAAGATGCGGTT AGCTTTTACCCCTGCATCTTTGAGCGCTGAGGTCTGCCTCGTGAAGAAGGTGTTGCTGACTCATACCAGGCCTGAA TCGCCCCATCATCCAGCCAGAAAGTGAGGGAGCCACGGTTGATGAGAGCTTTGTTGTAGGTGGACCAGTTGGTGAT TTTGAACTTTTGCTTTGCCACGGAACGGTCTGCGTTGTCGGGAAGATGCGTGATCTGATCCTTCAACTCAGCAAAA GTTCGATTTATTCAACAAAGCCGCCGTCCCGTCAAGTCAGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAA TTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTT TGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGT CTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAA ATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGC CAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGAC GAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGC ATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTG AGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTA GTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGG CTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCA GCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTAT TACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATT TTGAGACACCATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGAT ACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAAC CGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGA GCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATG TTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAGATTTAATTAAG GCCTTAATTAGG Legend 5 ITR:nt1-130 3 ITR:nt3085-3214 Humanthyroxine-bindingglobulin(TBG)promoter:nt220-917(of whichAMBPenhancer(alpha-1-microglobulin/bikuninprecursor) genetranscriptionregulatoryregion,:nt220-426) Intron:nt950-1081 Kozaksequence:nt1089-1102 hARSBCodingsequence:nt1103-2704 BovineGrowthHormone(BGH)polyA:nt2744-2951 Vectorbackbonent3215-6472 SEQIDNo.9pAAV2.1.TBG-eGFP AGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGA CTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTT ATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATT ACGCCAGATTTAATTAAGGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACC TTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGT AGTTAATGATTAACCCGCCATGCTACTTATCTACGTAGCCATGCTCTAGGAAGATCGGAATTCGCCCTTAAGCTAG CAGGTTAATTTTTAAAAAGCAGTCAAAAGTCCAAGTGGCCCTTGGCAGCATTTACTCTCTCTGTTTGCTCTGGTTA ATAATCTCAGGAGCACAAACATTCCAGATCCAGGTTAATTTTTAAAAAGCAGTCAAAAGTCCAAGTGGCCCTTGGC AGCATTTACTCTCTCTGTTTGCTCTGGTTAATAATCTCAGGAGCACAAACATTCCAGATCCGGCGCGCCAGGGCTG GAAGCTACCTTTGACATCATTTCCTCTGCGAATGCATGTATAATTTCTACAGAACCTATTAGAAAGGATCACCCAG CCTCTGCTTTTGTACAACTTTCCCTTAAAAAACTGCCAATTCCACTGCTGTTTGGCCCAATAGTGAGAACTTTTTC CTGCTGCCTCTTGGTGCTTTTGCCTATGGCCCCTATTCTGCCTGCTGAAGACACTCTTGCCAGCATGGACTTAAAC CCCTCCAGCTCTGACAATCCTCTTTCTCTTTTGTTTTACATGAAGGGTCTGGCAGCCAAAGCAATCACTCAAAGTT CAAACCTTATCATTTTTTGCTTTGTTCCTCTTGGCCTTGGTTTTGTACATCAGCTTTGAAAATACCATCCCAGGGT TAATGCTGGGGTTAATTTATAACTAAGAGTGCTCTAGTTTTGCAATACAGGACATGCTATAAAAATGGAAAGATGT TGCTTTCTGAGAGACTGCAGAAGTTGGTCGTGAGGCACTGGGCAGGTAAGTATCAAGGTTACAAGACAGGTTTAAG GAGACCAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGA CATCCACTTTGCCTTTCTCTCCACAGGTGTCCAGGCGGCCGCCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGG GTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCG ATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGT GACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCC GCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGG TGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCT GGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAG GTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCA TCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGA GAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAG TAATAAGCTTGGATCCAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCT CCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCT CCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTG CACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCT TTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGG GCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGAT TCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCG GCTCTGCGGCCTCTTCCGCGTCTTCGAGATCTGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCC CTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCG CATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACA ATAGCAGGCATGCTGGGGACTCGAGTTAAGGGCGAATTCCCGATTAGGATCTTCCTAGAGCATGGCTACGTAGATA AGTAGCATGGCGGGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCG CTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGA GCGCGCAGCCTTAATTAACCTAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCC AACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTC CCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTT ACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCA CGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCT CGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCCGATAGACGGTTTTTCGCCCTTTG ACGCTGGAGTTCACGTTCCTCAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATT CTTTTGATTTATAAGGGATTTTTCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGC GAATTTTAACAAAATATTAACGTTTATAATTTCAGGTGGCATCTTTCGGGGAAATGTGCGCGGAACCCCTATTTGT TTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAA AAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTT TGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTG GATCTCAATAGTGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTC TGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAA TGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCT GCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTT TTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGA CGAGCGTGACACCACGATGCCTGTAGTAATGGTAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTA GCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGG CTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGA TGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATC GCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATT TAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACG TGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGC GTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTC TTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCA CCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGC GATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGG GTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAG CGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGG GAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTT TGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTG CTGCGGTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGA GCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAG Legend 5 ITR:nt248-377 3 UTR:nt2919-3048 CodingsequenceeGFP:nt1336-2052 TBGpromoter:nt678-1154 Vecorbackbone:nt3052-5612 PROMOTERS SEQIDNo.10TBGpromoter AGGGCTGGAAGCTACCTTTGACATCATTTCCTCTGCGAATGCATGTATAATTTCTACAGAACCTATTAGAAAGGAT CACCCAGCCTCTGCTTTTGTACAACTTTCCCTTAAAAAACTGCCAATTCCACTGCTGTTTGGCCCAATAGTGAGAA CTTTTTCCTGCTGCCTCTTGGTGCTTTTGCCTATGGCCCCTATTCTGCCTGCTGAAGACACTCTTGCCAGCATGGA CTTAAACCCCTCCAGCTCTGACAATCCTCTTTCTCTTTTGTTTTACATGAAGGGTCTGGCAGCCAAAGCAATCACT CAAAGTTCAAACCTTATCATTTTTTGCTTTGTTCCTCTTGGCCTTGGTTTTGTACATCAGCTTTGAAAATACCATC CCAGGGTTAATGCTGGGGTTAATTTATAACTAAGAGTGCTCTAGTTTTGCAATACAGGACATGCTATAAAAATGGA AAGATGTTGCTTTCTGAGAGA SEQIDNo.11alfa-1-antitripsinpromoter GATCTTGCTACCAGTGGAACAGCCACTAAGGATTCTGCAGTGAGAGCAGAGGGCCAGCTAAGTGGTACTCTCCCAG AGACTGTCTGACTCACGCCACCCCCTCCACCTTGGACACAGGACGCTGTGGTTTCTGAGCCAGGTACAATGACTCC TTTCGGTAAGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGCGTCCGGGCAGCGTAGGCGGGCGACTCAGATCCC AGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCTTGGTTAATATTCACCAGCAGCCTCCCC CGTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGACAGGGCCCTGTCTCCTCAGCTTCAGGCACCACCACT GACCTGGGACAGT SEQIDNo.12albumin GCATGCTTCCATGCCAAGGCCCACACTGAAATGCTCAAATGGGAGACAAAGAGATTAAGCTCTTATGTAAAATTTG CTGTTTTACATAACTTTAATGAATGGACAAAGTCTTGTGCATGGGGGTGGGGGTGGGGTTAGAGGGGAACAGCTCC AGATGGCAAACATACGCAAGGGATTTAGTCAAACAACT TTTTGGCAAAGATGGTATGATTTTGTAATGGGGTAGGAACCAATGAAATGCGAGGTAAGTATGGTTAATGATCTAC AGTTATTGGTTAAAGAAGTATATTAGAGCGAGTCTTTCTGCACACAGATCACCTTTCCTATCAACCCC SEQIDNo.13phosphoglyceratekinasepromoter CACGGGGTTGGGGTTGCGCCTTTTCCAAGGCAGCCCTGGGTTTGCGCAGGGACGCGGCTGCTCTGGGCGTGGTTCC GGGAAACGCAGCGGCGCCGACCCTGGGTCTCGCACATTCTTCACGTCCGTTCGCAGCGTCACCCGGATCTTCGCCG CTACCCTTGTGGGCCCCCCGGCGACGCTTCCTGCTCCGCCCCTAAGTCGGGAAGGTTCCTTGCGGTTCGCGGCGTG CCGGACGTGACAAACGGAAGCCGCACGTCTCACTAGTACCCTCGCAGACGGACAGCGCCAGGGAGCAATGGCAGCG CGCCGACCGCGATGGGCTGTGGCCAATAGCGGCTGCTCAGCGGGGCGCGCCGAGAGCAGCGGCCGGGAAGGGGCGG TGCGGGAGGCGGGGTGTGGGGCGGTAGTGTGGGCCCTGTTCCTGCCCGCGCGGTGTTCCGCATTCTGCAAGCCTCC GGAGCGCACGTCGGCAGTCGGCTCCCTCGTTGACCGAATCACCGACCTCTCTCCCCAGGG SEQIDNo.14CMVpromoter TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACG GTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAA CGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGT GTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATG ACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGC AGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAG TTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGT AGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGT
Materials and Methods
Animal Colony
[0114] MPS VI mice were maintained at the Cardarelli Hospital Animal House (Naples, Italy). Animals were raised in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines for the care and use of animals in research. This mouse model carries a targeted disruption of the ARSB locus.sup.64 and is made immune-tolerant to human ARSB by transgenic insertion of the C91S hARSB mutant, resulting in the production of inactive hARSB.sup.65. Six out of 38 MPS VI mice from the same genetic background had the C91S hARSB transgene inserted into the ROSA26 locus.sup.66, while the remaining presented random integrations of this transgene.sup.15. Genotype analysis was performed by polymerase chain reaction (PCR) on genomic DNA obtained from the tail, as previously described 15.
Plasmid and Vector Production
[0115] The plasmid pAAV2.1.TBG-hARSB (see
Treatment Administration
[0116] MPS VI mice were treated with gene therapy and/or ERT through intravenous retro-orbital injections, starting from p30 to avoid vector dilution due to hepatocyte proliferation.sup.13, 56, 57 and were followed up to 6-7 months (180-210 post-natal days) of age. MPS VI mice were treated with a single injection of the AAV2/8.TBG.hARSB vector at either 210.sup.11 or 610.sup.11 gc/kg and/or monthly injections of 1 mg/kg rhARSB protein (Naglazyme, BioMarin Europe, London, UK), appropriately diluted in phosphate buffered saline (PBS). As control, MPS VI mice either received monthly administrations of PBS (n=1) or a single injection of the control AAV2/8.TBG.eGFP vector (n=1) or were left untreated (n=8). Both male and female mice are included in each group of treatment.
Blood, Urine, and Tissue Collection
[0117] Blood was collected each month from treated and control mice and before ERT administration in mice receiving ERT with or without gene therapy. Serum samples were collected via eye bleeding and centrifuged at 10,000g in a microcentrifuge (Z 216 MK, HERMLE) for 10 min at 4 C. to obtain the serum.
[0118] Urine was also collected monthly before each ERT administration in mice receiving ERT with or without gene therapy. Specifically, mice were put in metabolic cages for 24 hours. The urine samples were briefly centrifuged to remove debris and stored at 20 C.
[0119] Mice were sacrificed 5 or 6 months following the start of treatment and 1 month after the last ERT administration. A cardiac perfusion with PBS was performed, and the liver, kidney, spleen and heart were collected. Tissue samples were fixed in a methacarn solution (30% chloroform, 60% methanol, 10% acetic acid) for 24 h or frozen in dry ice (for ARSB activity and GAG quantitative assays).
Immune Capture Assay for Determination of Serum ARSB Levels?
[0120] Serum ARSB levels were measured by an immune capture assay based on the use of a specific anti-hARSB polyclonal antibody (Covalab, Villeurbanne, France). Ninety-six-well plates (Nunclon, Roskilde, Denmark) were coated with 5 g/ml in 0.1 M NaHCO.sub.3(100 l/well) and incubated overnight (0/N) at 4 C. The following day, plates were washed twice with 0.25 M NaCl/0.02 M Tris, pH 7, and then blocked with 1% milk in 0.25 M NaCl/0.02 M Tris, pH 7 (blocking solution), for 2 h at room temperature. Plates were washed again, as described above, and then 50 l of standard and unknown samples (diluted 1:10) were added to each well. Plates were shaken for 1 h at room temperature and then incubated at 4 C. O/N. The following day, plates were shaken for 1 h at room temperature and then washed 2 with 0.25 M NaCl/0.02 M Tris, pH 7. In total, 100 l of 5 mM 4-methylumbelliferylsulfate potassium salt (4-MUS, Sigma-Aldrich, Milan, Italy) substrate was added to each well and then incubated at 37 C. for 4 h. The reaction was stopped by the addition of 100 l/well of stop solution (glycine 0.2 M). Plates were shaken for 10 min at room temperature and the fluorescence was read (excitation 365 nm/emission 460 nm) on a multiplate fluorimeter (TECAN Infinite F200, Mnnedorf, Switzerland). Serum ARSB was determined based on a rhARSB (Naglazyme, BioMarin Europe, London, UK) standard curve and expressed as pg/mL.
AAV Vector Genome Distribution
[0121] Genomic DNA was extracted from the livers using the DNeasy Blood and Tissue Extraction kit (Qiagen). Real-time PCR analysis was performed on 100 ng of genomic DNA using a set of primers/probe (Fw 5-TCTAGTTGCCAGCCATCTGTTGT-3 (SEQ ID NO. 15), Rev 5-TGGGAGTGGCACCTTCCA-3 (SEQ ID NO. 16), Probe 5-TCCCCCGTGCCTTCCTTGACC-3 (SEQ ID NO. 17)) specific for the viral genome and Taq-Man universal PCR master mix (Applied Biosystems, Foster City, Calif., USA). Amplification was run on a 7300 Real-Time PCR system (Applied Biosystems) with standard cycles. All the reactions were performed in triplicate.
Assay for ARSB and GASB Enzymatic Activity Evaluation in Tissues
[0122] Tissues, i.e liver, kidney and spleen, were homogenized in water and protein concentrations were determined with the bicinchoninic acid (BCA) protein assay kit (Pierce Protein Research Products, Thermo Fisher Scientific, Rockford, Ill., USA). The ARSB assay was performed, as previously described.sup.67. Briefly, 30 g of protein was incubated with 40 l of the fluorogenic4-methylumbelliferyl sulfate substrate (12.5 mM; Sigma-Aldrich, Saint Louis, Mo., USA) for 3 h at 37 C. in the presence of 40 l silver nitrate (0.75 mM; Carlo Erba, Milan, Italy), which is known to inhibit the activity of other sulfatases. The reaction was stopped by adding 200 l of carbonate glycine stop buffer and the fluorescence of the 4-methylumbelliferone liberated was measured on a multiplate reader (TECAN Infinite F200) at 365 nm (excitation) and 460 nm (emission).
[0123] -glucuronidase (GUSB) assay was performed, as previously described.sup.68. Briefly, 200 g of protein was incubated with 400 l of GUS assay buffer (50 mM NaPO.sub.4 pH 7, 5 mM DTT, 1 mM EDTA, 0.1% Triton X-100) and 100 l of the fluorogenic 4-methylumbelliferyl--d-glucuronide (MUG) substrate (5 mM; Sigma-Aldrich, Saint Louis, Mo., USA) for 30 min at 37 C. The reaction was stopped by adding 900 l of stop buffer (0.2 mM Na.sub.2CO.sub.3, pH 9.5) to 50 l of sample. The fluorescence was measured on a multiplate reader (GloMax-Multi detection system Promega) at 388 nm (excitation) and 480 nm (emission).
[0124] Enzyme activities were calculated with a standard curve of the fluorogenic4-methylumbelliferone product (12.5 mM; Sigma-Aldrich, Saint Louis, Mo., USA). For tissue lysates the activity was expressed as nanomoles per milligram of protein per hour (nmol/mg/h).
Quantitative Analyses of GAG Accumulation in Tissues and Urine
[0125] Urine samples were diluted 1:50 in water to measure GAG content. One hundred microliters of diluted urine or 250 g of protein extract from the liver, spleen, and kidney was used for the GAG assay, as previously described.sup.69. GAG concentrations were determined on the basis of a dermatan sulfate standard curve (Sigma-Aldrich, Saint Louis, Mo., USA). Tissue GAGs were expressed as micrograms of GAG per milligram of protein (g GAG/mg protein). Urinary GAGs were normalized to creatinine content which was measured with a creatinine assay kit (Quidel, San Diego, Calif., USA). Thus, the units of urinary GAGs are micrograms per micromole of creatinine (g GAG/mol creatinine). Urinary GAGs were reported as percentage of AF control mice. The urinary GAG levels measured at each time point were averaged for each group.
Alcian Blue Staining
[0126] After methacarn fixation, all the tissues (liver, spleen, kidney, heart) were dehydrated through immersion in alcohols at increasing concentration (70%, 80%, 90%, 100%) and then in xylene. Tissues were embedded in paraffin and sectioned into 7-m-thick serial sections on a microtome. Tissue sections were de-paraffinized, rehydrated through immersion in alcohols at decreasing concentration (100%, 95%, 80% and 70%) and then water and stained with 1% Alcian blue (Sigma-Aldrich, Saint Louis, Mo., USA) in hydrochloric acid for 10 seconds. Counterstaining was performed for 1 min with nuclear-fast red (Sigma-Aldrich, Saint Louis, Mo., USA). Alcian blue staining in heart valves and myocardium was quantified to provide a measure of GAG storage. Specifically, Alcian blue staining was quantified by measuring RGB intensity on histological section using the Image J software. RGB may assume integer values from 0 to 255. The more intense is the Alcian Blue staining, the lower is the RBG value. Four different areas were randomly selected in each valve. As far as myocardium, five areas corresponding to Alcian blue spots were randomly selected per each section. Where Alcian blue spots were not present, as in NR and some treated mice, five equivalent areas were randomly selected. RGB was measured per each area and then averaged for each animal and each group of animals, as reported in
GAG Levels
Sample Preparations
Urine
[0127] Keep urine frozen (20/80). Before use, spin urines at 200 g for 2 minutes to remove debris and collect supernatant. Urine samples should be diluted between 1:50 in water. Depending on the assay used, use 50 ul of sample (microplate assay) or 100 ul sample (cuvette assay).
Tissue Lysates
[0128] Homogenize tissue in the tissue lyser (QIAGEN) in water with LAP (protease inhibitor cocktail tablets) (Roche), Pellet cell debris (14000 rpr, 15-20 min, 4 C.), Collect supernatant, Measure protein concentration, Dilute tissue at 5 ug/ul.
[0129] 250 ug is loaded in a final volume of 100 ul (cuvette assay); 125 ug is loaded in a final volume of 50 ul (multiplate). (Please note that the amount of protein to be tested depends on the concentration of the sample and the expected GAG concentration).
Materials
[0130] Dermatan Sulfate stock (2 mg/ml) also known as chondroitin sulphate B (Sigma C3788); Dimethylmethylene Blue (DMB) Reagent (10.7 mg DMB in 55 mM formate buffer, pH 3.3);
[0131] 2M Tris (base);
[0132] F96 Maxisorp Nunc-immuno plate (Nalge Nunc part n. 442404) for multiplate protocol.
[0133] For cuvette assay:
[0134] Plastic cuvettes;
[0135] 90% formic acid;
[0136] 1,9-Dimethyl-methylene blue (DMB; Sigma-Aldrich 341088);
[0137] Tris base (MW 121,14);
[0138] Tissue culture filter 0.22 um PES
Preparation of Solutions
Dmb Formate:
[0139] 1. mix 897.2 ml H.sub.2O with 2.8 ml of 90% formic acid in a 1 L cylinder
2. measure pH of the solution while stirring. Add NaOH to bring the solution to a pH of 3.29
3. Bring the solution to 1 L with H2O. This will increase pH to 3.33
4. Wrap a 1 L sterilized glass bottle with aluminium to protect from light.
5. Add about 500 ml of Formate buffer in the bottle
6. Slowly add 10.7 mg (0.0107 g) of DMB to the Formate buffer during 2 minutes while stirring
7. Continue to stir for about 1 hour and then add the other 500 ml of formate buffer
8. Stir the solution for about 8 hours to overnight
2M Tris
[0140] Weight 60.57 g of tris and add about 200 ml water
Dissolve Tris by stirring (if needed can heat the solution to allow dissolution)
Bring to 250 ml with water
Filter the solution with 0.2 um filters
Measure pH (11.34)
Procedure
[0141] Prepare the DS standard from the 2 mg/ml stock. The DS should be diluted with water to obtain concentrations of 40, 20, 10, 5, 2.5, and 1.25 ug/ml (standard can be stored at 20 C.)
TABLE-US-00003 STANDARD DS Water A) 40 ug/ml 10 ul stock 490 B) 20 ug/ml 150 ul standard A 150 C) 10 ug/ml 150 ul standard B 150 D) 5 ug/ml 150 ul standard C 150 E) 2.5 ug/ml 150 ul standard D 150 F) 1.25 ug/ml 150 ul standard E 150
Prepare Dilutions of the Samples (in Water)
[0142] If done in multiplate [0143] load in each plate well a blank (water), the standard and the diluted samples in duplicate (50 ul/well) [0144] Calculate the amount of DMB-Tris reagent required for all samples (each sample require 275 ul of reagent). Prepare the DMB-Tris reagent by combining ten parts of the DMB reagent with one part of 2M Tris (Es. 500 ul DMB+50 ul Tris). The resultant solution is only stable for about 15 minutes. [0145] Add 275 ul of DMB-Tris to each well (use multichannel pipette to be fast). Read the plate at 520 nm one minute after adding the DMB-Tris reagent to the last column.
[0146] If done in cuvette [0147] prepare as much plastic cuvettes as the number of samples-standard you have (in duplicate) [0148] load in the cuvettes a blank (water), the standard and the diluted samples in duplicate (100 ul/each). [0149] Calculate the amount of DMB-Tris reagent required for all samples (each sample require 550 ul of reagent). Prepare the DMB-Tris reagent by combining ten parts of the DMB reagent with one part of 2M Tris (Es. 500 ul DMB+50 ul Tris). The resultant solution is only stable for about 15 minutes. [0150] Add 550 ul of DMB-Tris to each cuvette (since the reaction is stable for few minutes, we usually add the buffer to 3-5 cuvettes and read absorbance; then we add buffer to additional 3-5 cuvette and so on). Mix cuvette and read absorbance at 520 nm (spectrophotometer).
Results Analysis
Tissue Lysates
[0151] Calculate GAG concentration of the diluted sample form the standard curve (GAG concentration in ug/ml) [0152] Divide this concentration by the protein concentration of the diluted sample (You will obtain ug GAG/mg protein)
Urine Samples
[0153] Normalize on creatinine concentration: on the same urine dilution used for GAG determination, measure creatinine concentration with the QUIDEL Microvue Creatinine Assay Kit. Divide GAG concentration (ug/ml) for Creatinine concentration (umol/ml) to obtain ug GAG/umol of creatinine.
Creatinine Assay
[0154] Follow Creatinine Assay Kit (Quidel San Diego, Calif.; catalog number 8009) protocol. Dilute standards and controls (from the kit) 1:40.
[0155] For samples the same dilution used for GAG measurement should be used. If samples are diluted differently, the different dilution should be kept in mind before normalyzing. Calculate creatinine concentration from standard curve. Add lower point of the creatinine curve if necessary, i.e 2.5 and 1.25 mmol/L.
Statistical Analyses
[0156] All results are expressed as meanstandard error (SE). Statistical comparisons were made using either t-test or one-way analysis of variance (ANOVA); the Tukey post hoc test was used. Statistical significance was considered if p<0.05. The exact p value for each comparison follows:
[0157] Table 1. Serum ARSB: The ANOVA p value is 2.00 e.sup.16; the p value of NR vs AF is: 4.88e.sup.13; the p value of ERT vs AF is: 1.00; the p value of AAV 210.sup.11 vs AF is: 0.99; the p value of AAV 210.sup.11+ERT vs AF is: 0.83; the p value of AAV 610.sup.11 vs AF is: 0.02; the p value of AAV 610.sup.11+ERT vs AF is: 6.90e.sup.4;
[0158] Liver genome copies: The ANOVA p value is 4.85e.sup.8; the p value of AAV 210.sup.11 vs AAV 610.sup.11 is: 2.20e.sup.6; the p value of AAV 210.sup.11+ERT vs AAV 610.sup.11+ERT is: 3.70e.sup.6; the p value of AAV 210.sup.11 vs AAV 210.sup.11+ERT is: 0.98; the p value of AAV 610.sup.11 vs AAV 610.sup.11+ERT is: 0.99;
[0159] Liver ARSB activity: The ANOVA p value is 3.75e.sup.9; the p value of ERT vs AF is: 0.58; the p value of AAV 210.sup.11 vs AF is: 0.03; the p value of AAV 210.sup.11+ERT vs AF is: 1.00e.sup.3; the p value of AAV 610.sup.11 vs AF is: 1.00e.sup.7; the p value of AAV 610.sup.11+ERT vs AF is: <3.75e.sup.9; the p value of AAV 210.sup.11 vs AAV 610.sup.11 is: 1.00e.sup.3; the p value of AAV 210.sup.11+ERT vs AAV 610.sup.11+ERT is: 6.00e.sup.3; the p value of NR vs AF is 2.17e.sup.8.
[0160] Liver GAGs: The ANOVA p value is 7.93e.sup.21; the p value of NR vs AF is <7.93e.sup.21; the p value of ERT vs AF is: <7.93e.sup.21; the p value of AAV 210.sup.11 vs AF is: <7.93e.sup.21; the p value of AAV 210.sup.11+ERT vs AF is: <7.93e.sup.21; the p value of AAV 610.sup.11 vs AF is: <7.93e.sup.21; the p value of AAV 610.sup.11+ERT vs AF is: <7.93e.sup.21; the p value of ERT vs NR is: 0.99; the p value of AAV 210.sup.11 vs NR is: 0.99; the p value of AAV 210.sup.11+ERT vs NR is: 0.99; the p value of AAV 610.sup.11 vs NR is: 0.99; the p value of AAV 610.sup.11+ERT vs NR is: 0.99;
[0161] Kidney ARSB activity: The ANOVA p value is 1.28e.sup.5; the p value of ERT vs AF is: 0.22; the p value of AAV 210.sup.11 vs AF is: 2.00e.sup.3; the p value of AAV 210.sup.11+ERT vs AF is: 8.20e.sup.5; the p value of AAV 610.sup.11 vs AF is: 8.30e.sup.5; the p value of AAV 610.sup.11+ERT vs AF is: 1.00e.sup.3; the p value of NR vs AF is 1.80e.sup.8.
[0162] Kidney GAGs: The ANOVA p value is 1.16e.sup.15; the p value of NR vs AF is <1.16e.sup.15; the p value of ERT vs AF is: 1.00e.sup.7; the p value of AAV 210.sup.11 vs AF is: 4.00e.sup.7; the p value of AAV 210.sup.11+ERT vs AF is: <1.16e.sup.15; the p value of AAV 610.sup.11 vs AF is: <1.16e.sup.15; the p value of AAV 610.sup.11+ERT vs AF is: <1.16e.sup.15; the p value of ERT vs NR is: 0.42; the p value of AAV 210.sup.11 vs NR is: 0.03; the p value of AAV 210.sup.11+ERT vs NR is: 0.99; the p value of AAV 610.sup.11 vs NR is: 0.99; the p value of AAV 610.sup.11+ERT vs NR is: 0.99;
[0163] Spleen ARSB activity: The ANOVA p value is 5.73e.sup.6; the p value of ERT vs AF is: 0.20; the p value of AAV 210.sup.11 vs AF is: 7.00e.sup.3; the p value of AAV 210.sup.11+ERT vs AF is: 1.12e.sup.4; the p value of AAV 610.sup.11 vs AF is: 2.46e.sup.4; the p value of AAV 610.sup.11+ERT vs AF is: 2.46e.sup.5; the p value of NR vs AF is 4.28e.sup.9.
Spleen GAGs: The ANOVA p value is 1.49e.sup.18; the p value of NR vs AF is <1.49e.sup.18; the p value of ERT vs AF is: 1.08e.sup.11; the p value of AAV 210.sup.11 vs AF is: 1.67e.sup.10; the p value of AAV 210.sup.11+ERT vs AF is: <1.49e.sup.18; the p value of AAV 610.sup.11 vs AF is: <1.49e.sup.8; the p value of AAV 610.sup.11+ERT vs AF is: <1.49e.sup.18; the p value of ERT vs NR is: 0.66; the p value of AAV 210.sup.11 vs NR is: 0.04; the p value of AAV 210.sup.11+ERT vs NR is: 0.98; the p value of AAV 610.sup.11 vs NR is: 0.99; the p value of AAV 610.sup.11+ERT vs NR is: 0.99.
[0164]
[0165]
[0166]
[0167]
[0168]
[0169]
[0170] Kidney -glucuronidase activity (b): The ANOVA p value is 7.16e.sup.7; the p value of NR vs AF is 1.00e.sup.6; the p value of ERT vs AF is 2.50e.sup.4; the p value of AAV 610.sup.11 vs AF is 8.99e.sup.5; the p value of AAV 610.sup.11+ERT vs AF is 2.30e.sup.6; the p value of ERT vs NR is 0.09; the p value of AAV 610.sup.11 vs NR is 0.21; the p value of AAV 610.sup.11+ERT vs NR is 0.99.
EXAMPLES
Example 1: Increased Serum ARSB Levels in MPS VI Transgenic Mice Treated with Combined Monthly ERT and Gene Therapy
[0171] MPS VI mice received at postnatal day 30 (p30) a single intravenous (i.v) administration of either 210.sup.11 or 610.sup.11 gc/kg of AAV2/8.TBG.hARSB, which encodes human ARSB (hARSB) under the control of the liver-specific thyroxine-binding globulin (TBG) promoter, and/or monthly i.v injections of 1 mg/kg rhARSB (Naglazyme, BioMarin Europe, London, UK), which is the dose currently used in MPS VI patients management (canonical ERT schedule).sup.6, 7, 49-52. As control, MPS VI mice were either left untreated or received a combination of monthly administrations of ERT and a single injection of the control AAV2/8.TBG.eGFP vector, which encodes the enhanced green fluorescence protein (eGFP) under the control of the TBG promoter. Serum ARSB was undetectable in affected control (AF) mice (Table 1).
TABLE-US-00004 TABLE 1 Liver vector genome copies, serum and peripheral tissue ARSB and GAGs in MPS VI mice receiving low doses of gene therapy and/or monthly ERT. Serum Liver Kidney Spleen ARSB ARSB ARSB ARSB Groups levels gc activity GAGs activity GAGs activity GAGs NR 11825 334** 96.7 7.9** 4.5 0.4** 148.0 12.0** 7.4 0.3** 61.2 4.6** 3.0 0.3** AF 0 0 57.0 6.2 0 46.9 7.8 0 49.5 5.4 ERT 0 2.4 0.5 7.3 2.3** 0.6 0.3 15.1 3.5** 2.7 1.2 9.2 2.5** AAV 2 376 67 0.015 0.003 4.5 1.2* 8.6 3.3** 1.0 0.3** 18.9 4.6** 4.0 1.0** 14.2 4.9** 10.sup.11 AAV 2 726 82 0.034 0.009 6.0 1.6** 5.1 0.5** 1.3 0.2** 7.3 0.4** 5.4 0.5** 6.0 0.9** 10.sup.11 + ERT AAV 6 2035 98* 0.131 0.038 11.1 1.5** 3.4 0.5** 1.4 0.2** 7.3 1.0** 5.4 0.5** 5.4 0.7** 10.sup.11 AAV 6 2500 84** 0.105 0.019 11.8 0.6** 3.3 0.4** 1.1 0.1** 6.0 0.6** 6.3 1.1** 5.1 0.2** 10.sup.11 + ERT
[0172] Abbreviations: AAV, adeno-associated viral vector; AF, affected MPS VI untreated mice; ARSB, arylsulfatase B; GAGs, glycosaminoglycans; ERT, enzyme replacement therapy; gc, genome copies; NR, normal untreated mice; n.a, not applicable. Dashed lines refer to values below the detection limit of the assay. Each serum ARSB value is the mean of all the time points measured in that group over time and is expressed as pg/ml. Measurements in tissues were done at the time of sacrifice (180 or 210 days of age). ARSB activity in tissues is expressed as nmol/mg protein/hour; GAG levels in tissues are expressed as g GAG/mg protein; genome copies in livers are expressed as genome copies/molecule of diploid genome. The AAV vector dose used (gc/kg) is reported per each group. The number of animals within each treated group is: ERT, n=5 except for serum ARSB (n=4); AAV 210.sup.11, n=6 except for serum ARSB (n=5); AAV 210.sup.11+ERT, n=6 except for serum ARSB (n=7); AAV 610.sup.11, n=5 except for serum ARSB n=4; AAV 610.sup.11+ERT, n=5. The number of animals in the NR group is: 16-24 for serum ARSB [23 at post-natal day 30 (p30) and p150, 24 at p60, p90 and p120, 22 at p180 and 16 at p210];-23 for ARSB activity and GAGs levels in tissues. The number of animals in the AF group is: 9, except for serum ARSB (3-6). Genome copies in liver were analyzed in 3 un-injected (2NR and 1 ERT) mice as control. Values are represented as meanSE. Statistical comparisons were made using the one-way ANOVA and the Tukey post hoc test. The p value vs. AF is: * <0.05 and ** <0.01. The exact p values obtained are indicated in the Material and Methods section.
[0173] MPS VI mice receiving either 210.sup.11 or 610.sup.11 gc/kg of AAV2/8.TBG.hARSB vector, with or without monthly ERT, showed a dose-dependent increase of serum ARSB levels, with a tendency to decrease at the end of the study (Table 1 and
[0174] To confirm long-term transgene expression the inventors measured ARSB enzyme activity and AAV vector genome copies (gc) in the livers from treated and control mice at the end of the study, i.e 180 or 210 days of age (Table 1). Persistence of liver transduction was confirmed by the presence of detectable AAV vector gc in mice receiving gene therapy.
Example 2: Increased Urinary GAG Reduction in Mice Receiving Gene Therapy in Combination with ERT
[0175] Reduction of urinary GAGs is a sensitive and reliable biomarker of lysosomal storage clearance and therapeutic efficacy in LSDs.sup.7, 49-52.
[0176] Urinary GAGs were measured monthly in MPS VI-treated mice as well as in age-matched NR and AF controls, from p60, i.e. one month after the start of treatment. Urinary GAG levels measured at each time point were averaged for each group and the resulting value was reported as a percentage (%) of age-matched AF controls (
[0177] Overall, urinary GAGs significantly decreased compared to AF controls in all groups of treatment (
[0178] More importantly, urinary GAGs decreased more in mice receiving the combined therapy than in those receiving the corresponding single treatments. Indeed, urinary GAGs were significantly lower (61% of AF) in mice treated with both 210.sup.11 gc/kg of AAV and ERT than in mice treated with either monthly ERT (82% of AF, p value: <<0.01) or 210.sup.11 gc/kg of AAV (73% of AF, p value: <<0.01). Likewise, a greater reduction of urinary GAGs was observed in mice receiving both 610.sup.11 gc/kg of AAV and ERT (41% of AF) compared to mice treated with either ERT (p value: <<0.01) or gene therapy at the same dose (53% of AF, p value: <<0.01) (
Example 3: Amelioration of Biochemical, Visceral and Cardiac Abnormalities in MPS VI Transgenic Mice Treated with Combined Monthly ERT and Gene Therapy
[0179] ARSB activity and GAG levels were measured in the liver, kidney, and spleen of MPS VI-treated and control mice (Table 1). ARSB activity was undetectable in tissues of AF controls. MPS VI mice receiving ERT (with or without gene therapy) were sacrificed one month after the last injection of rhARSB to measure the residual tissue enzymatic activity. Although ARSB activity was almost undetectable in the serum of mice treated with ERT alone, the inventors found ARSB activity in tissues up to 1 month after injection (Table 1), although at levels lower than those previously measured in mice receiving weekly ERT.sup.15.
[0180] Increased ARSB activity was observed in the liver of all treated mice; detectable activity was variably observed in the spleen and kidney of treated mice, although at levels lower than those measured in the liver (Table 1). Specifically, a statistically significant increase in ARSB activity compared to AF was observed in all treated groups but the one that received monthly ERT.
[0181] Beta-glucuronidase (GUSB) activity has been found to be secondarily increased in tissues from MPS VI cats as result of ARSB deficiency.sup.53. As further therapeutic endpoint, GUSB activity was thus evaluated in tissues of MPS VI mice treated with either the combination of 610.sup.11 gc/kg of AAV and ERT or single therapies, and in NR and AF controls. GUSB activity was significantly increased in liver and kidney but not in spleen of MPS VI mice compared to NR (
[0182] More importantly, a statistically significant reduction of GAGs storage was observed in these organs, regardless of treatment and ARSB levels (Table 1), similarly to what was observed in mice administered with weekly ERT.sup.15. This supports previous data indicating that low enzymatic levels are sufficient to improve the MPS VI visceral phenotype.sup.13-15. Specifically, tissue GAGs in all groups were not statistically different than in normal controls, except for the spleen and kidney of mice receiving 210.sup.11 gc/kg (p value<0.05). In particular, GAGs were completely normalized in the visceral organs of all mice receiving 210.sup.11 gc/kg and ERT or gene therapy at the dose of 610.sup.11 gc/kg with ERT. Alcian blue staining of tissues confirmed the reduction of lysosomal GAG storage in the liver, kidney and spleen of MPS VI-treated mice (
[0183] Cardiomyopathy and heart valve involvement are serious clinical complications of MPS VI that often negatively affect its prognosis.sup.54. The MPS VI mouse mimics the human MPS VI cardiac phenotype.sup.15, 55.
[0184] The inventors performed Alcian blue staining on heart histological sections from treated and control mice (
[0185] In the present invention, the inventors show that therapeutic efficacy can be obtained by combining gene therapy with rarified ERT. In particular, this was demonstrated by a great reduction of both urinary GAGs and storage in myocardium and heart valves observed in mice receiving combined monthly ERT and gene therapy. These levels of correction were similar to normal controls.
[0186] The inventors demonstrate that gene therapy may be regarded as a means to decrease the frequency of ERT infusions. While gene therapy could provide baseline enzyme levels to taper GAG levels, the high intracellular levels of therapeutic enzyme achieved with ERT can be used only occasionally to help clear tissues from any GAGs storage in excess. The use of a rarified ERT schedule should lead to several important advantages, including reduction of both allergic reaction associated with the frequent infusion of recombinant enzyme and the costs of ERT, that range between euro 150,000 and euro 450,000 per patient/year.sup.8 (depending on the patient weight) in the case of MPS VI. The high costs may limit the access to the therapy to patients living in less developed countries and where therapies are not supported by the public health system.sup.4, 8. This scenario may change if a single administration of low dose gene therapy allows rarifying the ERT schedule.
[0187] An additional potential advantage of combining gene and protein delivery is that liver-directed gene therapy has been demonstrated to either prevent the generation of humoral immunity to the transgene product in several models of LSDs.sup.59 or to eradicate it, if already present.sup.69-62. Notably, immune-modulatory gene therapy with a sub-therapeutic dose of vector was shown to enhance the efficacy of ERT in murine Pompe disease by preventing the generation of humoral immunity to recombinant alfa-glucosidase.sup.20, 63. Therefore, gene therapy may also positively impact on ERT therapeutic efficacy and safety by avoiding the generation of inhibitors to therapeutic proteins, which is a limit to the successful treatment of several inherited diseases. Last but not least, this study helps managing patients with LSDs for which ERT is available and who are enrolled in gene therapy clinical trials. The inventors are indeed developing a phase I/II study to test the efficacy of gene therapy for MPS VI (http://meusix.tigem.it). If efficacy is observed that is inferior to that observed during ERT, these patients who have received gene therapy could be put on a rarified rather than on the canonical highly frequent ERT schedule. In this study the inventors show in a mouse model the therapeutic efficacy of a novel combinatorial gene therapy/ERT approach for MPS VI, and potentially other LSDs. By taking advantage of the different pharmacokinetics and dynamics of either approach, this combination has the potential to reduce the risks and costs associated with gene therapy and ERT, respectively.
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