                         SEQUENCE LISTING

<110>  ESTEVE PHARMACEUTICALS, S.A.
       UNIVERSITAT AUTNOMA DE BARCELONA
 
<120>  ANIMAL MODEL OF MUCOPOLYSACCHARIDOSES TYPE IV A

<130>  ESTA-1810-WO

<160>  11    

<170>  PatentIn version 3.5

<210>  1
<211>  1569
<212>  DNA
<213>  Homo sapiens


<220>
<221>  mutation
<222>  (1156)..(1156)
<223>  cytosine at position 1156 is substituted by thymine (1156C>T)

<400>  1
atggcggcgg ttgtcgcggc gacgaggtgg tggcagctgt tgctggtgct cagcgccgcg       60

gggatggggg cctcgggcgc cccgcagccc cccaacatcc tgctcctgct catggacgac      120

atgggatggg gtgacctcgg ggtgtatgga gagccctcca gagagacccc gaatttggac      180

cggatggctg cagaagggct gcttttccca aacttctatt ctgccaaccc tctgtgctcg      240

ccatcgaggg cggcactgct cacaggacgg ctacccatcc gcaatggctt ctacaccacc      300

aacgcccatg ccagaaacgc ctacacaccg caggagattg tgggcggcat cccagactcg      360

gagcagctcc tgccggagct tctgaagaag gccggctacg tcagcaagat tgtcggcaag      420

tggcatctgg gtcacaggcc ccagttccac cccctgaagc acggatttga tgagtggttt      480

ggatccccca actgccactt tggaccttat gacaacaagg ccaggcccaa catccctgtg      540

tacagggact gggagatggt tggcagatat tatgaagaat ttcctattaa tctgaagacg      600

ggggaagcca acctcaccca gatctacctg caggaagccc tggacttcat taagagacag      660

gcacggcacc accccttttt cctctactgg gctgtcgacg ccacgcacgc acccgtctat      720

gcctccaaac ccttcttggg caccagtcag cgagggcggt atggagacgc cgtccgggag      780

attgatgaca gcattgggaa gatactggag ctcctccaag acctgcacgt cgcggacaac      840

accttcgtct tcttcacgtc ggacaacggc gctgccctca tttccgcccc cgaacaaggt      900

ggcagcaacg gcccctttct gtgtgggaag cagaccacgt ttgaaggagg gatgagggag      960

cctgccctcg catggtggcc agggcacgtc actgcaggcc aggtgagcca ccagctgggc     1020

agcatcatgg acctcttcac caccagcctg gcccttgcgg gcctgacgcc gcccagcgac     1080

agggccattg atggcctcaa cctcctcccc accctcctgc agggccggct gatggacagg     1140

cctatcttct attaccgtgg cgacacgctg atggcggcca ccctcgggca gcacaaggct     1200

cacttctgga cctggaccaa ctcctgggag aacttcagac agggcattga tttctgccct     1260

gggcagaacg tttcaggggt cacaactcac aatctggaag accacacgaa gctgcccctg     1320

atcttccacc tgggacggga cccaggggag aggttccccc tcagctttgc cagcgccgag     1380

taccaggagg ccctcagcag gatcacctcg gtcgtccagc agcaccagga ggccttggtc     1440

cccgcgcagc cccagctcaa cgtgtgcaac tgggcggtca tgaactgggc acctccgggc     1500

tgtgaaaagt tagggaagtg tctgacacct ccagaatcca ttcccaagaa gtgcctctgg     1560

tcccactag                                                             1569


<210>  2
<211>  522
<212>  PRT
<213>  Homo sapiens


<220>
<221>  MUTAGEN
<222>  (386)..(386)
<223>  Arginine at position 386 is substituted by Cysteine (R386C)

<400>  2

Met Ala Ala Val Val Ala Ala Thr Arg Trp Trp Gln Leu Leu Leu Val 
1               5                   10                  15      


Leu Ser Ala Ala Gly Met Gly Ala Ser Gly Ala Pro Gln Pro Pro Asn 
            20                  25                  30          


Ile Leu Leu Leu Leu Met Asp Asp Met Gly Trp Gly Asp Leu Gly Val 
        35                  40                  45              


Tyr Gly Glu Pro Ser Arg Glu Thr Pro Asn Leu Asp Arg Met Ala Ala 
    50                  55                  60                  


Glu Gly Leu Leu Phe Pro Asn Phe Tyr Ser Ala Asn Pro Leu Cys Ser 
65                  70                  75                  80  


Pro Ser Arg Ala Ala Leu Leu Thr Gly Arg Leu Pro Ile Arg Asn Gly 
                85                  90                  95      


Phe Tyr Thr Thr Asn Ala His Ala Arg Asn Ala Tyr Thr Pro Gln Glu 
            100                 105                 110         


Ile Val Gly Gly Ile Pro Asp Ser Glu Gln Leu Leu Pro Glu Leu Leu 
        115                 120                 125             


Lys Lys Ala Gly Tyr Val Ser Lys Ile Val Gly Lys Trp His Leu Gly 
    130                 135                 140                 


His Arg Pro Gln Phe His Pro Leu Lys His Gly Phe Asp Glu Trp Phe 
145                 150                 155                 160 


Gly Ser Pro Asn Cys His Phe Gly Pro Tyr Asp Asn Lys Ala Arg Pro 
                165                 170                 175     


Asn Ile Pro Val Tyr Arg Asp Trp Glu Met Val Gly Arg Tyr Tyr Glu 
            180                 185                 190         


Glu Phe Pro Ile Asn Leu Lys Thr Gly Glu Ala Asn Leu Thr Gln Ile 
        195                 200                 205             


Tyr Leu Gln Glu Ala Leu Asp Phe Ile Lys Arg Gln Ala Arg His His 
    210                 215                 220                 


Pro Phe Phe Leu Tyr Trp Ala Val Asp Ala Thr His Ala Pro Val Tyr 
225                 230                 235                 240 


Ala Ser Lys Pro Phe Leu Gly Thr Ser Gln Arg Gly Arg Tyr Gly Asp 
                245                 250                 255     


Ala Val Arg Glu Ile Asp Asp Ser Ile Gly Lys Ile Leu Glu Leu Leu 
            260                 265                 270         


Gln Asp Leu His Val Ala Asp Asn Thr Phe Val Phe Phe Thr Ser Asp 
        275                 280                 285             


Asn Gly Ala Ala Leu Ile Ser Ala Pro Glu Gln Gly Gly Ser Asn Gly 
    290                 295                 300                 


Pro Phe Leu Cys Gly Lys Gln Thr Thr Phe Glu Gly Gly Met Arg Glu 
305                 310                 315                 320 


Pro Ala Leu Ala Trp Trp Pro Gly His Val Thr Ala Gly Gln Val Ser 
                325                 330                 335     


His Gln Leu Gly Ser Ile Met Asp Leu Phe Thr Thr Ser Leu Ala Leu 
            340                 345                 350         


Ala Gly Leu Thr Pro Pro Ser Asp Arg Ala Ile Asp Gly Leu Asn Leu 
        355                 360                 365             


Leu Pro Thr Leu Leu Gln Gly Arg Leu Met Asp Arg Pro Ile Phe Tyr 
    370                 375                 380                 


Tyr Arg Gly Asp Thr Leu Met Ala Ala Thr Leu Gly Gln His Lys Ala 
385                 390                 395                 400 


His Phe Trp Thr Trp Thr Asn Ser Trp Glu Asn Phe Arg Gln Gly Ile 
                405                 410                 415     


Asp Phe Cys Pro Gly Gln Asn Val Ser Gly Val Thr Thr His Asn Leu 
            420                 425                 430         


Glu Asp His Thr Lys Leu Pro Leu Ile Phe His Leu Gly Arg Asp Pro 
        435                 440                 445             


Gly Glu Arg Phe Pro Leu Ser Phe Ala Ser Ala Glu Tyr Gln Glu Ala 
    450                 455                 460                 


Leu Ser Arg Ile Thr Ser Val Val Gln Gln His Gln Glu Ala Leu Val 
465                 470                 475                 480 


Pro Ala Gln Pro Gln Leu Asn Val Cys Asn Trp Ala Val Met Asn Trp 
                485                 490                 495     


Ala Pro Pro Gly Cys Glu Lys Leu Gly Lys Cys Leu Thr Pro Pro Glu 
            500                 505                 510         


Ser Ile Pro Lys Lys Cys Leu Trp Ser His 
        515                 520         


<210>  3
<211>  1575
<212>  DNA
<213>  Rattus norvegicus


<220>
<221>  mutation
<222>  (1162)..(1162)
<223>  cytosine at position 1162 is substituted by thymine (1162C>T)

<400>  3
atgacggcct gttccacagc gatcagggcc cagcagctac tgctgccggt actgagtgct       60

ctaggactct tggctgcagg tgctccgcag ccccccaaca ttgtgctact gctcatggac      120

gatatggggt ggggtgacct gggtgtgtat ggagaacctt ccagagagac cccaaattta      180

gaccggatgg ctgcagaagg gatgcttttc ccaagcttct attctgccaa ccctttgtgc      240

tcaccatcta gggcagccct gctcacagga cggttgccca tccgcaatgg cttctacaca      300

accaacgcac atgcaaggaa tgcttacaca cctcaggaga tcatgggcgg gatccctaac      360

tcagaacacc tcctgcctga gctcctgaag aaagcaggct ataccaacaa gatcgtgggc      420

aaatggcatc tgggtcatag accccagttc caccccctga agcatggatt tgatgagtgg      480

tttggatccc ccaattgtca ttttggacca tacgacaaca aggtcaagcc caacatccct      540

gtgtacaggg actgggaaat ggttggcaga ttttatgaag agttcccaat caacctaaag      600

accggggaag ctaacctcac ccaactctac ttacaagaag ctctggactt catccggaca      660

caacatgcaa ggcagagccc cttcttcctc tactgggcca tcgacgccac acacgcacca      720

gtgtatgcct caaaacagtt cctgggtacc agccttcgag ggcgttatgg cgatgctgtc      780

cgggaaatag atgacagtgt tggaaagatc ctgagccttc tgcagaactt gggcatcagt      840

aagaacacat ttgtcttctt tacgtctgac aatggtgcag cgctcatctc tgctcccaaa      900

gaaggtggca gcaatggtcc cttcctgtgt gggaagcaga ctacgtttga aggcgggatg      960

agggagcctg caatcgcttg gtggccaggg cacattgccg caggccaggt cagccaccag     1020

ctgggaagca tcatggacct cttcaccacc agcctgtccc ttgcaggcct gaagcccccc     1080

agtgacaggg taattgatgg ccttgacctc ctccccacca tgctccaggg ccacatcata     1140

gacaggccca tattttatta ccgtggcaac acactgatgg cagtcactct tggccagtac     1200

aaagcacacc tctggacttg gaccaactcc tgggaggagt tcagacaggg cattgacttc     1260

tgccccgggc agaatgtttc aggagtcaca acccacaccc aggaagagca cacggagcta     1320

cccctgatct tccacctggg acgtgaccca ggggagagat tcccactcag gttcaccagc     1380

aatgagtacc aggatgccct cagcaggacc acccaggtca tccagcaaca ccaaaagtct     1440

ttggtccctg gacagcccca gctcaatgtg tgtaaccagg ctgtcatgaa ttgggcgcct     1500

ccaggctgtg aaaaactagg gaagtgtctg acacctcctg agtctgtccc cgagaagtgt     1560

ttctgggccc attag                                                      1575


<210>  4
<211>  524
<212>  PRT
<213>  Rattus norvegicus


<220>
<221>  MUTAGEN
<222>  (388)..(388)
<223>  Arginine at position 388 is substituted by Cysteine (R388C)

<400>  4

Met Thr Ala Cys Ser Thr Ala Ile Arg Ala Gln Gln Leu Leu Leu Pro 
1               5                   10                  15      


Val Leu Ser Ala Leu Gly Leu Leu Ala Ala Gly Ala Pro Gln Pro Pro 
            20                  25                  30          


Asn Ile Val Leu Leu Leu Met Asp Asp Met Gly Trp Gly Asp Leu Gly 
        35                  40                  45              


Val Tyr Gly Glu Pro Ser Arg Glu Thr Pro Asn Leu Asp Arg Met Ala 
    50                  55                  60                  


Ala Glu Gly Met Leu Phe Pro Ser Phe Tyr Ser Ala Asn Pro Leu Cys 
65                  70                  75                  80  


Ser Pro Ser Arg Ala Ala Leu Leu Thr Gly Arg Leu Pro Ile Arg Asn 
                85                  90                  95      


Gly Phe Tyr Thr Thr Asn Ala His Ala Arg Asn Ala Tyr Thr Pro Gln 
            100                 105                 110         


Glu Ile Met Gly Gly Ile Pro Asn Ser Glu His Leu Leu Pro Glu Leu 
        115                 120                 125             


Leu Lys Lys Ala Gly Tyr Thr Asn Lys Ile Val Gly Lys Trp His Leu 
    130                 135                 140                 


Gly His Arg Pro Gln Phe His Pro Leu Lys His Gly Phe Asp Glu Trp 
145                 150                 155                 160 


Phe Gly Ser Pro Asn Cys His Phe Gly Pro Tyr Asp Asn Lys Val Lys 
                165                 170                 175     


Pro Asn Ile Pro Val Tyr Arg Asp Trp Glu Met Val Gly Arg Phe Tyr 
            180                 185                 190         


Glu Glu Phe Pro Ile Asn Leu Lys Thr Gly Glu Ala Asn Leu Thr Gln 
        195                 200                 205             


Leu Tyr Leu Gln Glu Ala Leu Asp Phe Ile Arg Thr Gln His Ala Arg 
    210                 215                 220                 


Gln Ser Pro Phe Phe Leu Tyr Trp Ala Ile Asp Ala Thr His Ala Pro 
225                 230                 235                 240 


Val Tyr Ala Ser Lys Gln Phe Leu Gly Thr Ser Leu Arg Gly Arg Tyr 
                245                 250                 255     


Gly Asp Ala Val Arg Glu Ile Asp Asp Ser Val Gly Lys Ile Leu Ser 
            260                 265                 270         


Leu Leu Gln Asn Leu Gly Ile Ser Lys Asn Thr Phe Val Phe Phe Thr 
        275                 280                 285             


Ser Asp Asn Gly Ala Ala Leu Ile Ser Ala Pro Lys Glu Gly Gly Ser 
    290                 295                 300                 


Asn Gly Pro Phe Leu Cys Gly Lys Gln Thr Thr Phe Glu Gly Gly Met 
305                 310                 315                 320 


Arg Glu Pro Ala Ile Ala Trp Trp Pro Gly His Ile Ala Ala Gly Gln 
                325                 330                 335     


Val Ser His Gln Leu Gly Ser Ile Met Asp Leu Phe Thr Thr Ser Leu 
            340                 345                 350         


Ser Leu Ala Gly Leu Lys Pro Pro Ser Asp Arg Val Ile Asp Gly Leu 
        355                 360                 365             


Asp Leu Leu Pro Thr Met Leu Gln Gly His Ile Ile Asp Arg Pro Ile 
    370                 375                 380                 


Phe Tyr Tyr Arg Gly Asn Thr Leu Met Ala Val Thr Leu Gly Gln Tyr 
385                 390                 395                 400 


Lys Ala His Leu Trp Thr Trp Thr Asn Ser Trp Glu Glu Phe Arg Gln 
                405                 410                 415     


Gly Ile Asp Phe Cys Pro Gly Gln Asn Val Ser Gly Val Thr Thr His 
            420                 425                 430         


Thr Gln Glu Glu His Thr Glu Leu Pro Leu Ile Phe His Leu Gly Arg 
        435                 440                 445             


Asp Pro Gly Glu Arg Phe Pro Leu Arg Phe Thr Ser Asn Glu Tyr Gln 
    450                 455                 460                 


Asp Ala Leu Ser Arg Thr Thr Gln Val Ile Gln Gln His Gln Lys Ser 
465                 470                 475                 480 


Leu Val Pro Gly Gln Pro Gln Leu Asn Val Cys Asn Gln Ala Val Met 
                485                 490                 495     


Asn Trp Ala Pro Pro Gly Cys Glu Lys Leu Gly Lys Cys Leu Thr Pro 
            500                 505                 510         


Pro Glu Ser Val Pro Glu Lys Cys Phe Trp Ala His 
        515                 520                 


<210>  5
<211>  22
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  gRNA1 guide

<400>  5
cccatatttt attaccgtgg ca                                                22


<210>  6
<211>  22
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  gRNA2 guide

<400>  6
taccgtggca acacactgat gg                                                22


<210>  7
<211>  120
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Donor DNA


<220>
<221>  misc_feature
<222>  (1)..(50)
<223>  Left homology arm

<220>
<221>  misc_feature
<222>  (51)..(84)
<223>  gRNAs target sequence

<220>
<221>  mutation
<222>  (59)..(59)
<223>  Single silent mutations generating a new MboII restriction site

<220>
<221>  mutation
<222>  (66)..(66)
<223>  Missense mutation: cytosine (C) at position 66 is substituted by 
       thymine (T) (66C>T)

<220>
<221>  misc_feature
<222>  (85)..(120)
<223>  Right homology arm

<400>  7
tgtaaacaca cagacttgat ctctactctt tctctcctca acacaacagg cccatcttct       60

attactgtgg caacacactg atggcagtca ctcttggcca gtacaaagca cacctctgga      120


<210>  8
<211>  20
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Common forward primer

<400>  8
tttgtcagcc ccatttccta                                                   20


<210>  9
<211>  20
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  Common reverse primer

<400>  9
tgtggtgtga ccattcacct                                                   20


<210>  10
<211>  915
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  PCR amplicon WT sequence (Galns+/+)

<400>  10
tttgtcagcc ccatttccta gggcctggct ttgcctccac agggatgagg gcctgccagg       60

ggatgactca ggtcagttcc tgtagccttg ttgtcacaca taccaggcca gtaggaccca      120

ggacctcatg cgtgctggga aatcaccctc ctgagctgtg caccccaatt cccccctgcc      180

ttttgttctc tgtggcctta gcacatcttt aaagtatcgg gtgatacttt aaagtgggca      240

acttgccact cccagccagg agctccacat gtaaacacac agacttgatc tctactcttt      300

ctctcctcaa cacaacaggc ccatatttta ttaccgtggc aacacactga tggcagtcac      360

tcttggccag tacaaagcac acctctggac ttggaccaac tcctgggagg agttcagaca      420

ggtacaggcc tctgaataca gcttctttgg gagggggcac cactgggagg ccgctggaga      480

actgggttga atctctcagc taccaacaaa ggaggcacca ttttcctcac agcctccctc      540

ccttgtgtgt ccctatgggt cctgcccagt cttagttttc acgtccagat gtctagatgg      600

agatggagat tgggcacatg gctctggaaa attctagggg tgtttgagtg gtaccctgct      660

aggggcaaat attctctcct aggatgcact gccctcatag gtggtcttca gagagcgcaa      720

ggtccaggtt caggcagagg gcctctctac ccaccatacc cctaaggctc cacttagcac      780

tcttctcttg ctccctccag gctggcagca tcaggggtgg cctgcccagg tccagatgct      840

gccggccaca actgagacat cagtgtctac atcctggtgc ctgctcaagg ccagaaggtg      900

aatggtcaca ccaca                                                       915


<210>  11
<211>  915
<212>  DNA
<213>  Artificial Sequence

<220>
<223>  PCR amplicon Galns-/-

<400>  11
tttgtcagcc ccatttccta gggcctggct ttgcctccac agggatgagg gcctgccagg       60

ggatgactca ggtcagttcc tgtagccttg ttgtcacaca taccaggcca gtaggaccca      120

ggacctcatg cgtgctggga aatcaccctc ctgagctgtg caccccaatt cccccctgcc      180

ttttgttctc tgtggcctta gcacatcttt aaagtatcgg gtgatacttt aaagtgggca      240

acttgccact cccagccagg agctccacat gtaaacacac agacttgatc tctactcttt      300

ctctcctcaa cacaacaggc ccatcttcta ttactgtggc aacacactga tggcagtcac      360

tcttggccag tacaaagcac acctctggac ttggaccaac tcctgggagg agttcagaca      420

ggtacaggcc tctgaataca gcttctttgg gagggggcac cactgggagg ccgctggaga      480

actgggttga atctctcagc taccaacaaa ggaggcacca ttttcctcac agcctccctc      540

ccttgtgtgt ccctatgggt cctgcccagt cttagttttc acgtccagat gtctagatgg      600

agatggagat tgggcacatg gctctggaaa attctagggg tgtttgagtg gtaccctgct      660

aggggcaaat attctctcct aggatgcact gccctcatag gtggtcttca gagagcgcaa      720

ggtccaggtt caggcagagg gcctctctac ccaccatacc cctaaggctc cacttagcac      780

tcttctcttg ctccctccag gctggcagca tcaggggtgg cctgcccagg tccagatgct      840

gccggccaca actgagacat cagtgtctac atcctggtgc ctgctcaagg ccagaaggtg      900

aatggtcaca ccaca                                                       915


