IT202300004443A1 - HUMAN ALPHA GALACTOSIDASE CODING SEQUENCE FOR THE TREATMENT OF FABRY DISEASE - Google Patents

HUMAN ALPHA GALACTOSIDASE CODING SEQUENCE FOR THE TREATMENT OF FABRY DISEASE Download PDF

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IT202300004443A1
IT202300004443A1 IT102023000004443A IT202300004443A IT202300004443A1 IT 202300004443 A1 IT202300004443 A1 IT 202300004443A1 IT 102023000004443 A IT102023000004443 A IT 102023000004443A IT 202300004443 A IT202300004443 A IT 202300004443A IT 202300004443 A1 IT202300004443 A1 IT 202300004443A1
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vector
aav
nucleic acid
gla
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Himanshi Saxena
Andrés F Muro
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Int Centre For Genetic Engineering And Biotechnology Icgeb
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Description

SEQUENZA?CODIFICANTE?PER?LA?ALFA?GALATTOSIDASI?A?UMANA?PER? IL?TRATTAMENTO?DELLA? MALATTIA?DI?FABRY? HUMAN ALPHA GALACTOSIDASE CODING SEQUENCE FOR THE TREATMENT OF FABRY DISEASE

SETTORE?DELL'INVENZIONE? SECTOR?OF THE INVENTION?

La?presente?invenzione?riguarda?una?sequenza?codificante?per?l'alfa?galattosidasi?A?(GLA)?umana?che? pu??essere?utile?per? il?trattamento?della?malattia?di?Fabry.?Anche?vettori,?plasmidi,?cellule?ospiti?e? composizioni?farmaceutiche?comprendenti?tale?sequenza?sono?oggetti?dell'invenzione.? The present invention relates to a sequence encoding human alpha-galactosidase A (GLA) which may be useful for the treatment of Fabry disease. Vectors, plasmids, host cells and pharmaceutical compositions comprising such a sequence are also objects of the invention.

SFONDO?DELL'INVENZIONE? BACKGROUND?OF THE INVENTION?

Malattia?di?Fabry? Fabry's disease?

La?malattia?di?Fabry?(OMIM?301500)?(FD)???una?malattia?rara? lisosomiale?spesso?conosciuta?come? malattia?di?Anderson?Fabry?dai?ricercatori?che?l'hanno?portata?alla?luce?(Anderson,?1898).?Pu??anche? essere?chiamata?deficit?di?alfa?galattosidasi?A?o?deficienza?di?GLA,?angiocheratoma?corporis?diffusum,? angiocheratoma?diffuso,?ceramide?triesossidosi?e?lipidosi?distopica?ereditaria.?Si?tratta?di?un?errore? congenito? del?metabolismo? legato? al? cromosoma? X? che? si? verifica? a? causa? della? carenza? di?Alfa? galattosidasi? destinata? a? catabolizzare? la? Globotrioasilceramide? o? Gb3,? invece? la? accumula? progressivamente?nei? lisosomi? .? I?disturbi?da?accumulo? lisosomiale? (LSD)? sono? un? gruppo? di? oltre? 70? disturbi?metabolici? ereditari? con? una? frequenza? collettiva?di?1?su?5000?pazienti,?anche?se?individualmente?sono?rari?nella?popolazione.?La?malattia?di? Fabry???la?seconda?LSD?pi??comune?e?in?cima?alla?lista?c'??la?malattia?di?Gaucher.?Le?altre?includono? la?malattia?di?Niemann?Pick,? la?sindrome?di?Hunter,? la?malattia?di?Pompe?e? la?malattia?di?Tay?Sach? Fabry disease (OMIM 301500) (FD) is a rare lysosomal disease often known as Anderson Fabry disease by the researchers who first brought it to light (Anderson, 1898). It may also be called alpha-galactosidase A deficiency or GLA deficiency, angiokeratoma corporis diffuse, diffuse angiokeratoma, ceramide trioxide oxidosis, and hereditary dystopic lipidosis. It is an inborn error of X-linked metabolism that occurs because of a deficiency of alpha-galactosidase, which is designed to catabolize the Globotrioasylceramide? or? Gb3,? instead? progressively? accumulates? in? lysosomes? .? Lysosomal? storage? disorders? (LSD)? are? a? group? of? over? 70? inherited? metabolic? disorders? with? a? collective? frequency? of? 1? in? 5000? patients,? although? individually? they? are? rare? in? the? population.? Fabry? disease? is? the? second? most? common? LSD? and? at? the? top? of? the? list? is? Gaucher? disease.? Others? include? Niemann? Pick? disease,? Hunter? syndrome,? Pompe? disease? and? Tay? Sach? disease?

La?FD???causata?da?mutazioni?nel?gene?GLA.?Secondo?fabry?database.org,?fino?al?2019,?nel?gene?sono? state?riportate?1993?mutazioni,?tra?cui?1518?missenso?e?297?nonsense?che?rappresentano?una?grande? percentuale?delle?mutazioni? ( Tuttavia,???fondamentale?notare?che?non?tutte?portano?a?una?proteina?GLA?difettosa,?ma?pi??di?800? mutazioni?sono?gi??state? ritenute? responsabili?della?malattia? .?Fisiologicamente,? l'enzima?galattosidasi?alfa???attivo?nel?lisosoma?dove?funziona?per?degradare?le?molecole?lipidiche? globotriaosilceramide? o?Gb3? in? particelle? pi?? piccole? come? parte? del? processo? di? riciclaggio? del? lisosoma.?Nella?condizione?di?malattia,?il?gene?GLA?mutato?si?traduce?in?una?proteina?disfunzionale,? portando?alla?deficienza?dell'enzima?alfa?galattosidasi?che?limita?la?scissione?delle?molecole?Gb3?e? quindi?provoca?l'accumulo?di?queste?frazioni?lipidiche?nelle?cellule?di?vari?organi? FD is caused by mutations in the GLA gene. According to Fabry database.org, as of 2019, 1,993 mutations have been reported in the gene, including 1,518 missense and 297 nonsense mutations, representing a large percentage of all GLA mutations. (However, it is important to note that not all of them lead to a defective GLA protein, but more than 800 mutations have already been implicated in the disease. Physiologically, the enzyme galactosidase alpha is active in the lysosome, where it functions to break down the lipid molecules globotriaosylceramide or Gb3 into smaller particles as part of the lysosome recycling process. In the disease state, the mutated GLA gene results in a dysfunctional protein, leading to a deficiency of the enzyme alpha-galactosidase, which limits the breakdown of Gb3 molecules and thus causes the accumulation of these lipid fractions in the cells of various organs.

? ?

La?malattia?di?Fabry?(FD)?colpisce? la?maggior?parte?degli?organi? in?modo?non?specifico,?alcuni?con? sintomi? comuni? per? rendere? la?malattia? non? rintracciabile? e? quindi? in? gran? parte? inosservata.? I? pazienti?sofferenti?avvertono?sensazioni?di?bruciore?ed?episodi?di?dolore?agli?arti,?macchie?rosso?scuro? sulla? pelle,? compromissione? degli? occhi,? perdita? dell'udito? e? della? capacit?? di? sudare.? La? FD? si? accompagna?a?varie?complessit??gastrointestinali?e?cardiache.?Esistono?due?varianti?di?FD:?la?variante? classica?o?FD?classica?e?la?FD?atipica?o?ad?esordio?tardivo.?La?forma?classica?di?FD?comprende?i?pazienti? con?mutazioni?che?risultano?in?un'attivit??enzimatica?residua?minima?o?per?lo?pi??nulla?che?influisce? sulla?qualit??della?vita?fin?dalla?tenera?et??e?riduce?l'aspettativa?di?vita.?I?bambini?con?la?forma?classica? di?FD?soffrono?di?intolleranza?al?calore,?anomalie?della?pelle,?problemi?gastrointestinali?e?dolore?agli? arti?noti?come?crisi?di?Fabry?che?spesso?si?estendono?a?sintomi?renali?e?cardiaci?verso?l'et??adulta? Fabry's disease (FD) affects most organs in a nonspecific manner, some with common symptoms, making the disease undetectable and therefore largely undetected. Sufferers experience burning sensations and episodes of pain in the limbs, dark red spots on the skin, impaired vision, hearing loss and the ability to sweat. FD is accompanied by various gastrointestinal and cardiac complications. There are two variants of FD: the classic?or?classic?FD?and?atypical?or?late-onset?FD.?The?classic?form?of?FD?includes?patients?with?mutations?that?result?in?little?or?no?residual?enzyme?activity?that?affects?the?quality?of?life?from?an?early?age?and?reduces?life?expectancy.?Children?with?the?classic?form?of?FD?suffer?from?heat?intolerance,?skin?abnormalities,?gastrointestinal?problems?and?pain?in?the?extremities?known?as?Fabry?crises?that?often?extend?to?kidney?and?cardiac?symptoms?in?adulthood?

La?variante?ad?esordio?tardivo?della?malattia?di?Fabry?si?riferisce?a?pazienti?con?attivit????Gal?residua? (3?30%).?Questa?variante???generalmente?etichettata?con?manifestazioni?organo?specifiche? in?et?? adulta?come? il?cuore.?Le?varianti? renali?e?cardiache?costituiscono?una?percentuale? importante?di? pazienti?con?FD?a?esordio?tardivo? ? The late-onset variant of Fabry disease refers to patients with residual Gal activity (3-30%). This variant is generally labeled with organ-specific manifestations in adulthood, such as the heart. Renal and cardiac variants constitute a significant percentage of patients with late-onset FD.

Epidemiologia? Epidemiology?

La?malattia?di?Fabry???una?malattia? rara?da?accumulo? lisosomiale? con?un'incidenza?da?1:8.454?a? 1:117.000?nei?maschi,?che???tuttavia?altamente?sottostimata.?Poich??la?malattia?coinvolge?pi??organi? e?varie?manifestazioni?cliniche?con?variabili?come?l'et??e?il?sesso,?spesso?rimane?sottodiagnosticata?in? una? popolazione? comune.? La? reale? prevalenza? della?malattia? dovrebbe? essere? studiata? a? livello? regionale?a?causa?della?sua?natura?pan?etnica,?che?crea?allarmanti?differenze?nel?numero?di?casi?a? livello?globale.?A?Taiwan,?uno?screening?neonatale?prevede?un?inaspettato?1?su?1.500?maschi?affetti? da?FD,?mentre?in?Italia?i?numeri?raggiungono?fino?a?1?su?3.100?maschi? ?Un?altro? studio?nei?Paesi?Bassi?che?coinvolge? la?diagnosi?prenatale?e?postnatale?rappresenta?1?su?500.000? pazienti?con?FD.?Numeri?simili?sono?stati?diagnosticati?nel?Regno?Unito;?anche?1?su?300.000.?Questa? discrepanza?ha?portato?a?una?stima?errata?dei?dati?sulla?prevalenza?nella?comunit?? Fabry?disease?is?a?rare?lysosomal?storage?disease?with?an?incidence?of?1:8,454?to?1:117,000?in?males,?which?is?highly?underestimated.?Because?the?disease?involves?multiple?organs?and?various?clinical?manifestations?with?variables?such?as?age?and?gender,?it?often?remains?underdiagnosed?in?a?common?population.?The?real?prevalence?of?the?disease?should?be?studied?at?the?regional?level?due?to?its?pan?ethnic?nature,?which?creates?alarming?differences?in?the?number?of?cases?at? global?level.?In?Taiwan,?a?newborn?screening?predicts?an?unexpected?1?in?1,500?males?with?FD,?while?in?Italy?the?numbers?reach?up?to?1?in?3,100?males? ?Another? study?in?the?Netherlands?involving?prenatal?and?postnatal?diagnosis?represents?1?in?500,000?patients?with?FD.?Similar?numbers?have?been?diagnosed?in?the?UK;?even?1?in?300,000.?This?discrepancy?has?led?to?an?incorrect?estimate?of?the?prevalence?data?in?the?European?community?

Diagnosi? Diagnosis?

A? causa?dei? complessi? sintomi?della?malattia? che?variano?da? individuo?a? individuo,? la?diagnosi??? spesso?ritardata?e?per?lo?pi??trascurata.???cruciale?e?forse?raro?per?un?medico?generico?identificare?i? sintomi?di?un?paziente?come?quelli?della?malattia?di?Fabry.?Nel?caso?in?cui?si?sospetti?che?il?paziente? sia?affetto?dalla?malattia?di?Fabry,?vengono?effettuate?valutazioni?genetiche?ed?enzimatiche.? I?test? Because of the complex symptoms of the disease, which vary from individual to individual, diagnosis is often delayed and mostly overlooked. It is crucial and perhaps rare for a general practitioner to identify a patient's symptoms as those of Fabry disease. If a patient is suspected of having Fabry disease, genetic and enzymatic evaluations are performed. Tests

? ?

clinici?per?l'attivit??dell'enzima???galattosidasi?nel?plasma?vengono?eseguiti?sia?per?la?FD?classica?che? per? quella? non? classica.? I?maschi? affetti? dalla? forma? classica? di? FD? indicano? l'1?3%? dell'attivit?? enzimatica?mentre?nei?pazienti?con?attivit??FD?ad?insorgenza?tardiva?i?valori?vanno?dal?3?al?30%?nel? qual?caso?la?diagnosi?confermata?richiede?una?valutazione?genetica.?Il?test?genetico?conferma?il?tipo? di?mutazione?nel?locus?enzimatico?e?le?sue?ripercussioni.?Questo?tipo?di?test?genetico???raccomandato? per?le?femmine?poich????molto?probabile?che?il?test?enzimatico?indichi?un'attivit??residua.?Inoltre,?la? consulenza?genetica?o?gli?studi? familiari?hanno?aiutato?a?diagnosticare? la?malattia?di?Fabry.?Nelle? famiglie?con?questo?disturbo?legato?all'X,?la?valutazione?prenatale?viene?effettuata?mediante?l'analisi? dei? liquidi?amniotici?e? il? sequenziamento?genico?per? sospette?mutazioni? Clinical tests for plasma galactosidase enzyme activity are performed for both classical and non-classical FD. Males with classical FD show 1.3% enzyme activity, whereas patients with late-onset FD activity show values ranging from 3 to 30%, in which case a confirmed diagnosis requires genetic evaluation. Genetic testing confirms the type of mutation in the enzyme locus and its repercussions. This type of genetic testing is recommended for?females?because?the?enzyme?test?is?very?likely?to?indicate?residual?activity.?In?addition,?genetic?counseling?or?family?studies?have?helped?diagnose?Fabry?disease.?In?families?with?this?X-linked?disorder,?prenatal?evaluation?is?performed?by?analysis?of?amniotic?fluids?and?gene?sequencing?for?suspected?mutations?

Trattamenti?disponibili? Treatments?Available?

Attualmente,? la?terapia?enzimatica?sostitutiva?(ERT)? ?e,?pi??recentemente,? prodotti?di?terapia?chaperonica?hanno?riscontrato?un?certo?successo?nel?trattamento?della?FD.?Le? terapie?ERT? ?Replagal?di?Shire?(Agalsidasi?alpha) ?e?Fabrazyme? di? Sanofi? (Agalsidasi? beta)? ? costano,? approssimativamente,?$?300.000?315.000/anno.? Il?costo?annuale?di?Galafold,? la?prima?capsula?di? terapia?chaperonica?orale?di?Amicus?Therapeutics,???simile?all?ERT?(Markham,?2016;?Moran,?2018),? ma?pu??essere?indirizzato?solo?a?un?sottogruppo?di?pazienti.?Il?costo?di?questi?trattamenti???un?fattore? preoccupante?che?incoraggia?la?necessit??di?trattamenti?nuovi?e?pi??economici?che?possano?essere? accessibili?alla?gente?comune?(Motabar?et?al.,?2010).? Currently, enzyme replacement therapy (ERT) and, more recently, chaperone therapy products have seen some success in treating FD. The ERT therapies Shire’s Replagal (Agalsidase alpha) and Sanofi’s Fabrazyme (Agalsidase beta) cost approximately $300,000-$315,000/year. The annual cost of Galafold, Amicus Therapeutics’ first oral chaperone therapy capsule, similar to ERT (Markham, 2016; Moran, 2018), is approximately $100,000-$150,000. but?it?can?only?be?addressed?to?a?subgroup?of?patients.?The?cost?of?these?treatments?is?a?worrying?factor?that?encourages?the?need?for?new?and?cheaper?treatments?that?can?be?accessible?to?common?people?(Motabar?et?al.,?2010).?

Pertanto,?permane? la?necessit??di?un?trattamento?per? la?malattia?di?Fabry?che?sia?efficiente?e?pi?? conveniente?economicamente?rispetto?alle?attuali?terapie.? Therefore, there remains a need for a treatment for Fabry disease that is both effective and more cost-effective than current therapies.

RIASSUNTO?DELL'INVENZIONE? SUMMARY?OF THE INVENTION?

??stata?ora?trovata?una?sequenza?di?cDNA?con?codoni?ottimizzati?che?esprime?GLA?umano?in?grado?di? produrre? efficacemente? l'enzima? in? vivo.? In? particolare,? l'uso? di? questa? sequenza? migliora? vantaggiosamente?la?traduzione?dell'enzima?GLA?di?circa?4?6?volte?rispetto?alla?sequenza?wild?type? che?codifica?per?GLA.? ??a?codon-optimized?cDNA?sequence?expressing?human?GLA?has?now?been?found?that?is?capable?of?effectively?producing?the?enzyme?in?vivo.? In?particular,?the?use?of?this?sequence?beneficially?enhances?the?translation?of?the?GLA?enzyme?by?approximately?4?6?fold?compared?to?the?wild?type?sequence?coding?for?GLA.?

Costituisce? oggetto? dell'invenzione? un? acido? nucleico? comprendente? o? costituito? dalla? seguente? sequenza?di?SEQ?ID?N.1:? The subject of the invention is a nucleic acid comprising or consisting of the following sequence of SEQ ID No. 1:

ATGCAGCTGCGCAACCCCGAGCTGCACCTGGGCTGCGCCCTGGCCCTGCGCTTCCTGGCCCTGGTCAGCTGG ATGCAGCTGCGCAACCCCGAGCTGCACCTGGGCTGCGCCCTGGCCCTGCGCTTCCTGGCCCTGGTCAGCTGG

? ?

GACATCCCCGGCGCCCGCGCCCTGGACAACGGCCTGGCCCGCACCCCCACCATGGGCTGGCTGCACTGGGA GCGCTTCATGTGCAACCTGGACTGCCAGGAGGAGCCCGACAGCTGCATCAGCGAGAAGCTGTTTATGGAGAT GGCCGAGCTGATGGTCAGCGAGGGCTGGAAGGACGCCGGCTACGAGTACCTGTGCATCGACGACTGCTGGA TGGCCCCCCAGCGCGACAGCGAGGGCCGCCTGCAGGCCGACCCCCAGCGCTTCCCCCACGGAATCCGCCAG CTGGCCAACTACGTGCACAGCAAGGGCCTGAAGCTGGGCATCTACGCCGACGTGGGCAACAAGACCTGCGC CGGCTTCCCCGGCAGCTTCGGCTACTACGACATCGACGCCCAGACCTTCGCCGACTGGGGCGTGGACCTGCT GAAGTTCGACGGCTGCTACTGCGACAGCCTGGAGAACCTGGCCGACGGCTACAAGCACATGAGCCTGGCCC TGAACCGCACCGGCCGCAGCATCGTGTACAGCTGCGAGTGGCCCCTGTATATGTGGCCCTTCCAGAAGCCCAA CTACACCGAGATCCGCCAGTACTGCAACCACTGGCGCAACTTCGCCGACATCGACGACAGCTGGAAGAGCAT CAAGAGCATCCTGGACTGGACCAGCTTCAACCAGGAGCGCATCGTGGACGTGGCCGGCCCCGGCGGCTGGA ACGACCCCGACATGCTGGTGATCGGCAACTTCGGCCTGAGCTGGAACCAGCAGGTGACCCAGATGGCCCTGT GGGCCATTATGGCCGCCCCCCTGTTTATGAGCAACGACCTGCGCCACATCAGCCCCCAGGCCAAGGCCCTGCT GCAGGACAAGGACGTGATCGCTATCAACCAGGACCCCCTGGGCAAGCAGGGCTACCAGCTGCGCCAGGGCG ACAACTTCGAGGTCTGGGAGCGCCCCCTGAGCGGCCTGGCCTGGGCCGTGGCTATGATCAACCGCCAGGAG ATCGGCGGCCCCCGCAGCTACACCATCGCCGTGGCCAGCCTGGGCAAGGGCGTGGCCTGCAACCCCGCCTG CTTCATCACCCAGCTGCTGCCCGTGAAGCGCAAGCTGGGCTTCTACGAGTGGACCAGCCGCCTGCGCAGCCA CATCAACCCCACCGGCACCGTGCTGCTGCAGCTGGAGAACACAATGCAGATGAGCCTGAAGGACCTGCTGTA GACATCCCCGGCGCCCGCGCCCTGGACAACGGCCTGGCCCGCACCCCCACCATGGGCTGGCTGCACTGGGA GCGCTTCATGTGCAACCTGGACTGCCAGGAGGAGCCCGACAGCTGCATCAGCGAGAAGCTGTTTATGGAGAT GGCCGAGCTGATGGTCAGCGAGGGCTGGAAGGACGCCGGCTACGAGTACCTGTGCATCGACGACTGCTGGA TGGCCCCCCAGCGCGACAGCGAGGCCGCCTGCAGGCCGACCCCCAGCGCTTCCCCCACGGAATCCGCCAG CTGGCCAACTACGTGCACAGCAAGGGCCTGAAGCTGGGCATCTACGCCGACGTGGGCAACAAGACCTGCGC CGGCTTCCCCGGCAGCTTCGGCTACTACGACATCGACGCCCAGACCTTCGCCGACTGGGGCGTGGACCTGCT GAAGTTCGACGGCTGCTACTGCGACAGCCTGGAGAACCTGGCCGACGGCTACAAGCACATGAGCCTGGCCC TGAACCGCACCGGCCGCAGCATCGTGTACAGCTGCGAGTGGCCCCTGTATATGTGGCCCTTCCAGAAGCCCAA CTACACCGAGATCCGCCAGTACTGCAACCACTGGCGCAACTTCGCCGACATCGACGACAGCTGGAAGAGCAT CAAGAGCATCCTGGACTGGACCAGCTTCAACCAGGAGCGCATCGTGGACGTGGCCGGCCCCGGCGGCTGGA ACGACCCCGACATGCTGGTGATCGGCAACTTCGGCCTGAGCTGGAACCAGCAGGTGACCCAGATGGCCCTGT GGGCCATTATGGCCGCCCCCCTGTTTATGAGCAACGACCTGCGCCACATCAGCCCCCAGGCCAAGGCCCTGCT GCAGGACAAGGACGTGATCGCTATCAACCAGGACCCCCTGGGCAAGCAGGGCTACCAGCTGCGCCAGGGCG ACAACTTCGAGGTCTGGGAGCGCCCCCTGAGCGGCCTGGCCTGGGCCGTGGCTATGATCAACCGCCAGGAG ATCGGCGGCCCCCGCAGCTACACCATCGCCGTGGCCAGCTTGGGCAAGGGCGTGGCCTGCAACCCCGCCTG CTTCATCACCCAGCTGCTGCCCGTGAAGCGCAAGCTGGGCTTCTACGAGTGGACCAGCCGCCTGCGCAGCCA CATCAACCCCACCGGCACCGTGCTGCTGCAGCTGGAGAACACAATGCAGATGAGCCTGAAGGACCTGCTGTA

A?[SEQ?ID?N.1],? A?[SEQ?ID?N.1],?

oppure?comprendente?o?costituito?da?una?sequenza?avente?almeno?il?79%?di?identit??con?la?sequenza? di?SEQ?ID?N.1.? or?comprising?or?consisting?of?a?sequence?having?at?least?79%?identity?with?the?sequence?of?SEQ?ID?N.1.?

Preferibilmente,?detto?acido?nucleico?comprende?o???costituito?da?una?sequenza?avente?80,?85,?90,? 91,?92,?93,?94,?95,?96,?97,?98,?99?o?100%?di?identit??con?la?sequenza?di?SEQ?ID?N.1.? Preferably, said nucleic acid comprises or consists of a sequence having 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with the sequence of SEQ ID No. 1.

Preferibilmente,?detto?acido?nucleico?comprende?o???costituito?dalla?seguente?sequenza?con?SEQ?ID? N.2:? Preferably, said nucleic acid comprises or consists of the following sequence with SEQ ID No. 2:

ATGCAGTTGAGAAACCCAGAGCTCCACCTGGGCTGTGCCCTGGCACTGAGGTTCCTGGCCCTTGTGAGCTGG GATATCCCTGGGGCCAGGGCCTTGGACAACGGCTTGGCCCGCACCCCCACAATGGGCTGGCTGCACTGGGA ACGCTTTATGTGCAATCTGGACTGCCAGGAGGAGCCTGACAGCTGTATCAGCGAGAAGCTCTTTATGGAGATG GCAGAGCTGATGGTGTCTGAGGGATGGAAGGACGCCGGCTACGAATACCTGTGCATTGACGATTGCTGGATG GCTCCACAGAGGGACTCAGAAGGACGCCTGCAGGCTGATCCCCAGAGATTCCCCCATGGAATCCGCCAGCTG GCCAACTATGTGCACAGCAAAGGCCTGAAGCTGGGCATCTACGCCGACGTGGGCAACAAGACCTGTGCTGG CTTCCCTGGCTCCTTTGGATATTACGATATCGACGCTCAGACCTTTGCTGACTGGGGAGTGGATCTCCTCAAGT ATGCAGTTGAGAAACCCAGAGCTCCACCTGGGCTGTGCCCTGGCACTGAGGTTCCTGGCCCTTGTGAGCTGG GATATCCCTGGGGCCAGGGCCTTGGACAACGGCTTGGCCCGCACCCCCACAATGGGCTGGCTGCACTGGGA ACGCTTTATGTGCAAATCTGGACTGCCAGGAGGAGCCTGACAGCTGTATCAGCGAGAAGCTCTTTATGGAGATG GCAGAGCTGATGGTGTCTGAGGGATGGAAGGACGCCGGCTACGAATACCTGTGCATTGACGATTGCTGGATG GCTCCACAGAGGGACTCAGAAGGACGCCTGCAGGCTGATCCCCAGAGATTCCCCCATGGAATCCGCCAGCTG GCCAACTATGTGCACAGCAAAGGCCTGAAGCTGGGCATCTACGCCGACGTGGGCAACAAGACCTGTGCTGG CTTCCCTGGCTCCTTTGGATATTACGATATCGACGCTCAGACCTTTGCTGACTGGGGAGTGGATCTCCTCAAGT

? ?

TTGACGGCTGCTACTGTGACTCTCTGGAAAACCTGGCAGATGGCTACAAGCACATGTCCCTGGCTCTGAACAG AACAGGCCGCAGCATTGTGTACAGCTGCGAGTGGCCCCTGTATATGTGGCCCTTCCAGAAGCCCAACTACACA GAGATCAGGCAGTACTGCAACCACTGGAGGAACTTTGCCGACATTGACGACTCCTGGAAATCTATCAAGTCTA TCCTGGATTGGACATCCTTCAACCAAGAGCGGATCGTGGACGTGGCTGGACCTGGAGGCTGGAATGATCCAG ATATGCTGGTGATTGGAAACTTCGGGCTGTCTTGGAACCAGCAGGTCACTCAGATGGCGCTGTGGGCCATCAT GGCCGCCCCCCTCTTTATGAGCAACGACCTGCGCCACATTTCTCCTCAAGCCAAGGCCCTGCTCCAGGACAAG GACGTCATCGCCATTAATCAGGATCCTCTGGGGAAGCAGGGCTACCAGCTTAGACAGGGAGACAATTTTGAG GTGTGGGAGAGGCCTCTCTCTGGACTTGCCTGGGCTGTGGCTATGATCAACCGGCAGGAAATTGGTGGCCCC CGCTCCTACACCATTGCTGTTGCCTCCTTGGGCAAGGGCGTGGCCTGTAACCCTGCCTGCTTCATCACCCAGCT CCTGCCTGTGAAGAGAAAACTGGGATTCTACGAGTGGACCAGCCGGCTGCGGAGCCACATCAATCCCACCGG CACCGTGCTGCTTCAGCTGGAGAACACCATGCAGATGTCACTGAAAGATCTGCTGTGA?[SEQ?ID?N.2]? TTGACGGCTGCTACTGTGACTCTCTGGAAAACCTGGCAGATGGCTACAAGCACATGTCCCTGGCTCTGAACAG AACAGGCCGCAGCATTGTGTACAGCTGCGAGTGGCCCCTGTATATGTGGCCCTTCCAGAAGCCCAACTACACA GAGATCAGGCAGTACTGCAACCACTGGAGGAACTTTGCCGACATTGACGACTCCTGGAAATCTATCAAGTCTA TCCTGGATTGGACATCCTTCAACCAAGAGCGGATCGTGGACGTGGCTGGACCTGGAGGCTGGAATGATCCAG ATATGCTGGTGATTGGAAACTTCGGGCTGTCTTGGAACCAGCAGGTCACTCAGATGGCGCTGTGGGCCATCAT GGCCGCCCCCCTCTTTATGAGCAACGACCTGCGCCACATTTCTCCTCAAGCCAAGGCCCTGCTCCAGGACAAG GACGTCATCGCCATTAATCAGGATCCTCTGGGGAAGCAGGGCTACCAGCTTAGACAGGGAGACAATTTTGAG GTGTGGGAGAGGCCTCTCTCTGGACTTGCCTGGGCTGTGGCTATGATCAACCGGCAGGAAATTGGTGGCCCC CGCTCCTACACCATTGCTGTTGCCTCCTTGGGCAAGGGCGTGGCCTGTAACCCTGCCTGCTTCATCACCCAGCT CCTGCCTGTGAAGAGAAAACTGGGATTCTACGAGTGGACCAGCCGGCTGCGGAGCCACATCAATCCCACCGG CACCGTGCTGCTTCAGCTGGAGAACACCATGCAGATGTCACTGAAAGATCTGCTGTGA?[SEQ?ID?N.2]?

o?dalla?seguente?sequenza?con?SEQ?ID?N.3:? or?from?the?following?sequence?with?SEQ?ID?N.3:?

ATGCAGCTCCGCAACCCAGAGCTCCATCTTGGGTGTGCTCTCGCTCTTCGATTCCTTGCACTGGTCAGTTGGG ATATCCCGGGAGCTAGAGCTTTGGATAACGGTCTCGCACGCACTCCCACAATGGGATGGCTTCACTGGGAGC GATTTATGTGCAACCTGGACTGCCAGGAAGAGCCGGATAGCTGTATATCTGAGAAGCTTTTTATGGAGATGGC GGAATTGATGGTCAGTGAAGGCTGGAAAGACGCGGGCTACGAATATCTCTGTATCGACGATTGTTGGATGGC ACCACAACGCGATAGCGAAGGCAGGCTCCAGGCTGATCCACAGAGGTTTCCCCACGGAATACGACAGCTGG CTAACTATGTGCACAGCAAGGGCCTCAAACTGGGAATCTACGCTGACGTGGGCAATAAGACGTGCGCCGGTT TCCCGGGGTCTTTCGGTTACTACGACATTGACGCCCAAACTTTTGCTGACTGGGGTGTGGATCTTCTCAAGTT TGACGGCTGTTACTGCGACTCCCTCGAAAATTTGGCTGATGGTTACAAGCACATGTCTCTTGCCTTGAATCGCA CCGGCCGCTCCATCGTGTACTCTTGCGAGTGGCCGTTGTATATGTGGCCCTTTCAAAAACCGAACTACACAGA AATAAGACAGTATTGCAACCACTGGAGAAACTTCGCTGATATCGACGATAGCTGGAAATCTATTAAATCTATTCT TGATTGGACGAGTTTTAATCAAGAGCGAATTGTGGACGTTGCGGGGCCGGGAGGGTGGAACGACCCCGATA TGCTGGTTATCGGAAATTTTGGCCTTTCCTGGAATCAGCAGGTTACCCAGATGGCCCTGTGGGCTATTATGGCC GCTCCACTCTTCATGAGCAATGATTTGCGCCACATCAGTCCACAAGCGAAGGCTCTCTTGCAGGATAAGGATG TGATTGCTATCAACCAAGATCCGCTGGGCAAGCAGGGGTATCAGTTGAGACAAGGAGATAACTTCGAAGTTT GGGAGCGGCCCCTGAGTGGTTTGGCCTGGGCAGTGGCGATGATAAATCGACAAGAAATAGGAGGACCCAG GAGTTATACTATTGCTGTAGCATCCCTTGGGAAAGGTGTCGCGTGTAACCCCGCTTGTTTTATTACACAACTGCT GCCTGTTAAGAGAAAACTGGGCTTTTACGAGTGGACCTCTCGGCTCAGATCCCACATCAACCCGACAGGCAC CGTTCTTCTGCAACTGGAGAATACGATGCAGATGAGCCTCAAGGACTTGTTGTAA?[SEQ?ID?N.3]? ATGCAGCTCCGCAACCCAGAGCTCCATCTTGGGTGTGCTCTCGCTCTTCGATTCCTTGCACTGGTCAGTTGGG ATATCCCGGGAGCTAGAGCTTTGGATAACGGTCTCGCACGCACTCCCACAATGGGATGGCTTCACTGGGAGC GATTTATGTGCAACCTGGACTGCCAGGAAGAGCCGGATAGCTTGTATATCTGAGAAGCTTTTTATGGAGATGGC GGAATTGATGGTCAGTGAAGGCTGGAAAGACGCGGGCTACGAATATCTCTGTATCGACGATTGTTGGATGGC ACCACAACGCGATAGCGAAGGCAGGCTCCAGGCTGATCCACAGAGGTTTCCCCACGGAATACGACAGCTGG CTAACTATGTGCACAGCAAGGGCCTCAAACTGGGAATCTACGCTGACGTGGGCAATAAGACGTGCGCCGGTT TCCCGGGGTCTTTCGGTTACTACGACATTGACGCCCAAACTTTTGCTGACTGGGGTGTGGATCTTCTCAAGTT TGACGGCTGTTACTGCGACTCCCTCGAAAATTTGGCTGATGGTTACAAGCACATGTCTCTTGCCTTGAATCGCA CCGGCCGCTCCATCGTGTACTCTTGCGAGTGGCCGTTGTATATGTGGCCCTTTCAAAAACCGAACTACACAGA AATAAGACAGTATTGCAACCACTGGAGAAACTTCGCTGATATCGACGATAGCTGGAAATCTATTAAATCTATTCT TGATTGGACGAGTTTTAATCAAGAGCGAATTGTGGACGTGCGGGGCCGGGAGGGTGGAACGACCCCGATA TGCTGGTTATCGGAAATTTTGGCCTTTCCTGGAATCAGCAGGTTACCCAGATGGCCCTGTGGGCTATTATGGCC GCTCCACTCTTCATGAGCAATGATTTGCGCCACATCAGTCCACAAGCGAAGGCTCTCTTGCAGGATAAGGATG TGATTGCTATCAACCAAGATCCGCTGGGCAAGCAGGGGTATCAGTTGAGACAAGGAGATAACTTCGAAGTTT GGGAGCGGCCCCTGAGTGGTTTGGCCTGGGCAGTGGCGATGATAAATCGACAAGAAATAGGAGGACCCAG GAGTTATACTATTGCTGTAGCATCCCTTGGGAAAGGTGTCGCGTGTAACCCCGCTTGTTTTATTACACAACTGCT GCCTGTTAAGAGAAAACTGGGCTTTTACGAGTGGACCTCTCGGCTCAGATCCCACATCAACCCGACAGGCAC CGTTCTTCTGCAACTGGAGAATACGATGCAGATGAGCCTCAAGGACTTGTTGTAA?[SEQ?ID?N.3]?

In?una?forma?di?realizzazione,?detto?acido?nucleico?comprende?o???costituito?da?una?sequenza?avente? In one embodiment, said nucleic acid comprises or consists of a sequence having

? ?

80,?85,?90,?91,?92,?93,?94,?95,?96,?97,?98,?99?o?100%?di?identit??con?la?sequenza?di?SEQ?ID?N.2.? 80,?85,?90,?91,?92,?93,?94,?95,?96,?97,?98,?99?or?100%?identity?with?the?SEQ?ID?sequence?N.2.?

In?una?forma?di?realizzazione,?detto?acido?nucleico?comprende?o???costituito?da?una?sequenza?avente? 80,?85,?90,?91,?92,?93,?94,?95,?96,?97,?98,?99?o?100%?di?identit??con?la?sequenza?di?SEQ?ID?N.3.? In one embodiment, said nucleic acid comprises or consists of a sequence having 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identity with the sequence of SEQ ID No. 3.

Costituisce? anche?oggetto?dell'invenzione?detto? acido?nucleico?per? l'espressione? in? vivo?dell'alfa? galattosidasi? A? (GLA)? umana,? preferibilmente? in? una? cellula,? pi?? preferibilmente? in? una? cellula? umana.? In? questa? forma? di? realizzazione,? detto? acido? nucleico? preferibilmente? comprende? o? ?? costituito?da?una?sequenza?con?SEQ?ID?N.?1?o?2.? The invention also provides said nucleic acid for the in vivo expression of human alpha-galactosidase A (GLA), preferably in a cell, more preferably in a human cell. In this embodiment, said nucleic acid preferably comprises or consists of a sequence with SEQ ID No. 1 or 2.

?? anche? oggetto? dell'invenzione? l'uso? di? detto? acido? nucleico? per? l'espressione? in? vitro? dell'alfa? galattosidasi? A? (GLA)? umana,? preferibilmente? in? una? cellula,? pi?? preferibilmente? in? una? cellula? umana.? In? questa? forma? di? realizzazione,? detto? acido? nucleico? preferibilmente? comprende? o? ?? costituito?da?una?sequenza?con?SEQ?ID?N.3.? ?? also? the? object? of? the? invention? is? the? use? of? said? nucleic? acid? for? the? in? vitro? expression? of? human? alpha? galactosidase? A? (GLA),? preferably? in? a? cell,? more? preferably? in? a? human? cell.? In? this? embodiment,? said? nucleic? acid? preferably? comprises? or? consists? of? a? sequence? with? SEQ? ID? No. 3.?

Detto?acido?nucleico?pu??essere?utilizzato?per?esprimere?l'enzima?GLA?in?vitro?o?in?vivo,?in?particolare? utilizzando?opportuni?vettori?per?la?trasfezione?cellulare.? This?nucleic?acid?can?be?used?to?express?the?GLA?enzyme?in?vitro?or?in?vivo,?in?particular?using?appropriate?vectors?for?cellular?transfection.?

Un?ulteriore?oggetto?dell'invenzione???un?costrutto?di?acido?nucleico?comprendente:? A further object of the invention is a nucleic acid construct comprising:

??una?sequenza?promotore?e? ??a?promoter?sequence?and?

?? una? sequenza? codificante? del? gene? dell'alfa?galattosidasi? A? (GLA)? sotto? il? controllo? di? detto? promotore,? in? cui? detta? sequenza? codificante? ?? l'acido? nucleico? sopra? descritto,? preferibilmente? comprendente?o?costituito?da?una?sequenza?con?SEQ?ID?N.1,?SEQ?ID?N.2?o?SEQ?ID?N.3.? ?? a? coding? sequence? of? the? alpha? galactosidase? A? (GLA)? gene? under? the? control? of? said? promoter,? in? which? said? coding? sequence? is? the? nucleic? acid? described? above,? preferably? comprising? or? consisting? of? a? sequence? with? SEQ? ID? No.1,? SEQ? ID? No.2? or? SEQ? ID? No.3.?

Preferibilmente,?detta?sequenza?promotore???operativamente? legata?alla?porzione?terminale?5'?di? detta?sequenza?codificante.? Preferably, said promoter sequence is operationally linked to the 5'-terminal portion of said coding sequence.

Il?promotore?pu??essere,?ad?esempio,?un?promotore?ubiquitario?o?un?promotore?tessuto?specifico.? The promoter may be, for example, a ubiquitous promoter or a tissue-specific promoter.

In?una?forma?di?realizzazione,?detto?promotore???un?promotore?specifico?per?l'espressione?epatica,? preferibilmente?il?promotore?dell'alfa?1?antitripsina?umana?(hAAT).? In one embodiment, called the promoter is a promoter specific for hepatic expression, preferably the human alpha-1 antitrypsin (hAAT) promoter.

In?una?forma?di?realizzazione?esemplificativa,?detto?promotore???il?promotore?dell'alfa?1?antitripsina? umana?(hAAT)?e?una?molecola?di?acido?nucleico?che?comprende?il?promotore?hAAT,?un?sito?di?inizio? della?trascrizione?e?una?porzione?del?primo?esone?comprende?o???costituita?da?una?sequenza?con? SEQ?ID?N.?4.? In?an?exemplary?embodiment,?called?the?promoter?the?human?alpha?1?antitrypsin?(hAAT)?promoter?is?a?nucleic?acid?molecule?that?comprises?the?hAAT?promoter,?a?transcription?start?site?and?a?portion?of?the?first?exon?comprises?or?consists?of?a?sequence?with?SEQ?ID?NO.?4.?

In?una?particolare? forma?di? realizzazione,? il?promotore???associato?ad?una? sequenza??enhancer?,? preferibilmente?un??enhancer??derivato?dal?gene?dell'apolipoproteina?E,?preferibilmente?la?regione? In?a?particular? embodiment,? the?promoter?is?associated?with?an?enhancer?sequence,?preferably?an?enhancer?derived?from?the?apolipoprotein?gene?E,?preferably?the?region?

? ?

di?controllo?dell'ApoE?umana.? of?human?ApoE?control.?

Detto? costrutto? di? acido? nucleico? pu?? essere? una? cassetta? di? espressione? comprendente? l'acido? nucleico?dell'invenzione?legato?operativamente?a?una?o?pi??sequenze?di?controllo?dell'espressione?o? altre?sequenze?che?migliorano?l'espressione?di?un?transgene,?come?noto?nell'arte.? Said nucleic acid construct may be an expression cassette comprising the nucleic acid of the invention operably linked to one or more expression control sequences or other sequences that enhance the expression of a transgene, as known in the art.

Preferibilmente,?detto?costrutto?di?acido?nucleico???inserito?in?un?vettore.? Preferably,?this?nucleic?acid?construct?inserted?in?a?vector.?

??un?oggetto?dell'invenzione?un?vettore?comprendente?l'acido?nucleico?o?il?costrutto?di?acido?nucleico? sopra?definito,?detto?vettore?che?comprende?preferibilmente?un?acido?nucleico?comprendente?o? costituito?da?una?sequenza?con?SEQ?ID?N.1,?SEQ?ID?N.2?o?SEQ?ID?N?.3.? ??an?object?of?the?invention?is?a?vector?comprising?the?nucleic?acid?or?nucleic?acid?construct?defined?above,?said?vector?preferably?comprising?a?nucleic?acid?comprising?or?consisting?of?a?sequence?with?SEQ?ID?No.1,?SEQ?ID?No.2?or?SEQ?ID?No.3.?

Preferibilmente?detto?vettore???un?vettore?virale.?Esempi?di?vettori?virali?sono?vettori?adenovirali,? vettori?virali?adeno?associati?(AAV),?vettori?virali?dell'herpes,?vettori?retrovirali,?vettori? lentivirali?e? vettori? baculovirali? virus? vaccinici,? virus? schiumosi,? citomegalovirus,? virus? della? foresta? Semliki,? poxvirus,?vettore?di?virus?a?RNA?e?vettore?di?virus?a?DNA.?Preferibilmente?il?vettore?virale?deriva?da? parvovirus? non? patogeno? come? virus? adeno?associato? (AAV),? retrovirus? come? gammaretrovirus,? spumavirus?e?lentivirus,?adenovirus,?poxvirus?e?un?herpes?virus.?Pi??preferibilmente,?il?vettore?virale? ??scelto?nel?gruppo?costituito?da:?vettori?adenovirali,?vettori? lentivirali,?vettori?retrovirali?e?vettori? virali?adeno?associati?(AAV).? Preferably?called?a?viral?vector.?Examples?of?viral?vectors?are?adenoviral?vectors,?adeno?associated?viral?vectors?(AAV),?herpes?viral?vectors,?retroviral?vectors,?lentiviral?vectors?and?baculoviral?vectors?vaccinal?viruses,?foamy?viruses,?cytomegaloviruses,?Semliki?forest?viruses,?poxviruses,?RNA?virus?vectors?and?DNA?virus?vectors.?Preferably?the?viral?vector?is?derived?from?non-pathogenic?parvoviruses?such?as?adeno?associated?viruses?(AAV),?retroviruses?such?as?gammaretroviruses,? spumavirus?and?lentivirus,?adenovirus,?poxvirus?and?a?herpes?virus.?Most preferably,?the?viral?vector?is?chosen?from?the?group?consisting?of?adenoviral?vectors,?lentiviral?vectors,?retroviral?vectors?and?adeno?associated?viral?vectors?(AAV).?

In?un?altra?forma?di?realizzazione,?il?vettore???un?vettore?non?virale?come?veicoli?di?rilascio?a?base?di? polimeri,?particelle,? lipidi,?peptidi?o? loro?combinazioni,?come?polimeri?cationici,?micelle,? liposomi,? esosomi,?microparticelle?e?nanoparticelle?incluse?nanoparticelle?lipidiche?(LNP).? In?another?embodiment,?the?vector?is?a?non-viral?vector?as?delivery?vehicles?based?on?polymers,?particles,?lipids,?peptides?or?combinations?thereof,?such?as?cationic?polymers,?micelles,?liposomes,?exosomes,?microparticles?and?nanoparticles?including?lipid?nanoparticles?(LNP).?

In?un?altra?forma?di?realizzazione,?detto?vettore?virale?comprende?inoltre?uno?o?pi??di:? In another embodiment, said viral vector further comprises one or more of:

- una?sequenza?5'??inverted?terminal?repeat??(ITR)?di?AAV,?preferibilmente?localizzata? all'estremit??5'?del?promotore;? - a 5'??inverted?terminal?repeat??(ITR)?sequence of?AAV,?preferably?localized?at?the?5'?end?of?the?promoter;?

- un?elemento?enhancer,?preferibilmente?localizzato?all'estremit??5'?del?promotore;? - an enhancer element, preferably located at the 5' end of the promoter;

- una?sequenza?promotore;? - a?promoter?sequence;?

- una?sequenza?di?Kozak,?preferibilmente?localizzata?all'estremit??5'?della?sequenza? codificante?per?GLA?e?legata?operativamente?a?detta?sequenza;? - a Kozak sequence, preferably located at the 5' end of the GLA-coding sequence and operationally linked to said sequence;

- una?sequenza?di?terminazione?della?trascrizione?preferibilmente?localizzata?all'estremit??3'? della?sequenza?codificante?per?GLA;? - a transcription termination sequence preferably located at the 3' end of the GLA coding sequence;

- una?sequenza?di?3???inverted?terminal?repeat??(ITR)?di?AAV,?preferibilmente?localizzata? all'estremit??3'?della?sequenza?di?terminazione?della?trascrizione.? - a 3-inverted terminal repeat (ITR) sequence of AAV, preferably located at the 3' end of the transcription termination sequence.

? ?

Preferibilmente?il?vettore?comprende?in?una?direzione?5'?3':? Preferably?the?vector?includes?in?a?direction?5'?3':?

??una?sequenza?5???inverted?terminal?repeat??di?AAV?(5'?ITR);? ??a?5???inverted?terminal?repeat?sequence?of?AAV?(5'?ITR);?

??una?sequenza?enhancer;? ??an?enhancer?sequence;?

??una?sequenza?promotore;? ??a?promoter?sequence;?

??una?sequenza?di?Kozak;? ??a?sequence?of?Kozak;?

??la?sequenza?codificante?per?GLA?sopra?descritta?sotto?il?controllo?di?detto?promotore;? ??the?GLA?coding?sequence?described?above?under?the?control?of?the?said?promoter;?

??una?sequenza?di?terminazione?della?trascrizione;?e? ??a?transcription?termination?sequence;?and?

??una?sequenza?3???inverted?terminal?repeat??di?AAV?(3'?ITR).? ??a?3???inverted?terminal?repeat?sequence?of?AAV?(3'?ITR).?

Preferibilmente?detto?elemento?enhancer???un?enhancer?derivato?dal?gene?dell'apolipoproteina?E.? Preferably?called?enhancer?element?an?enhancer?derived?from?the?apolipoprotein?E?gene.?

Preferibilmente? detta? sequenza? di? terminazione? della? trascrizione? ?? una? sequenza? segnale? di? poliadenilazione,?preferibilmente?il?segnale?di?poliadenilazione?dell'emoglobina?beta?umana?a?(HHB? poliA).? Preferably? this? transcription? termination? sequence? is? a? polyadenylation? signal? sequence,? preferably? the? human? hemoglobin? beta? a? (HHB? polyA)? polyadenylation? signal.?

Preferibilmente? gli? ITR? derivano? dallo? stesso? sierotipo? virale? o? da? diversi? sierotipi? virali,? preferibilmente?il?virus???un?AAV,?preferibilmente?del?sierotipo?2.? Preferably? ITRs? are derived? from? the? same? viral? serotype? or? from? different? viral? serotypes,? preferably? the? virus? is? an? AAV,? preferably? of? serotype? 2.?

Facoltativamente,? il? vettore? comprende? inoltre? un? introne,? preferibilmente? l'introne? sintetico? derivato? dall'emoglobina? beta? umana? (HBB2).? Detto? introne? ?? preferibilmente? operativamente? legato?al?3'?della?sequenza?promotore.? Optionally,? the? vector? also? comprises? an? intron,? preferably? the? synthetic? intron? derived? from? human? beta? hemoglobin? (HBB2).? Said? intron? is? preferably? operably? linked? to? the? 3'? of? the? promoter? sequence.?

Tutti?questi?elementi?sono?noti?nel?settore?e?le?loro?sequenze?possono?essere?ottenute?dall'esperto? secondo?le?conoscenze?generali?nel?settore.? All?these?elements?are?known?in?the?field?and?their?sequences?can?be?obtained?by?the?expert?according?to?the?general?knowledge?in?the?field.?

In?una?forma?di?realizzazione?esemplificativa,?detto?elemento?enhancer???un?enhancer?della?regione? di?controllo?di?ApoE?e?comprende?o???costituito?da?una?sequenza?con?SEQ?ID?N.?5.? In?an?exemplary?embodiment,?said?enhancer?element?is?an?ApoE?control?region?enhancer?and?comprises?or?consists?of?a?sequence?with?SEQ?ID?No.?5.?

In?una?forma?di?realizzazione?esemplificativa,?detta?sequenza?di?terminazione?della?trascrizione???il? segnale?di?poliadenilazione?dell'emoglobina?beta?umana?a?(HHB?poliA)?e?comprende?o???costituita? da?una?sequenza?con?SEQ?ID?N.?6.? In?an?exemplary?embodiment,?this?transcription?termination?sequence?is?the?human?hemoglobin?beta?a?polyadenylation?signal?and?comprises?or?consists?of?a?sequence?with?SEQ?ID?NO.?6.?

In?una?forma?di?realizzazione?esemplificativa,?detta?sequenza?5'ITR?deriva?da?AAV2?e?comprende?o??? costituita?da?una?sequenza?con?SEQ?ID?N.?7.? In?an?exemplary?embodiment,?said?5'ITR?sequence?is?derived?from?AAV2?and?comprises?or?consists?of?a?sequence?with?SEQ?ID?NO.?7.?

In?una?forma?di?realizzazione?esemplificativa,?detta?sequenza?3'ITR?deriva?da?AAV2?e?comprende?o? consiste?in?una?sequenza?con?SEQ?ID?N.?8.? In?an?exemplary?embodiment,?said?3'ITR?sequence?is?derived?from?AAV2?and?comprises?or?consists?of?a?sequence?with?SEQ?ID?NO.?8.?

? ?

In? una? forma? di? realizzazione? esemplificativa,? detto? introne? ?? l'introne? sintetico? derivato? dalla? emoglobina?beta?umana?(HBB2)?e?comprende?o???costituito?da?una?sequenza?con?SEQ?ID?N.?9.? Preferibilmente,?il?vettore?pu??essere?ottenuto?come?descritto?in?Ronzitti?et?al.?2016,?sostituendo?il? cDNA?codificante?per?UGT1A1?con?la?sequenza?nucleotidica?codificante?per?GLA?qui?descritta.? Un?ulteriore?oggetto?dell'invenzione???un?plasmide?comprendente:? ??una?sequenza?promotore,? In? an? exemplary? embodiment,? said? intron? is? the? synthetic? intron? derived? from? human? beta? hemoglobin? (HBB2)? and? comprises? or? consists? of? a? sequence? with? SEQ? ID? No.? 9.? Preferably,? the? vector? may? be? obtained? as? described? in? Ronzitti? et? al.? 2016,? replacing? the? cDNA? encoding? UGT1A1? with? the? nucleotide? sequence? encoding? GLA? described? herein.? A? further? object? of? the? invention? is? a? plasmid? comprising:? a? promoter? sequence,?

?? una? sequenza? codificante? del? gene? dell'alfa?galattosidasi? A? (GLA)? sotto? il? controllo? di? detto? promotore,? in? cui? detta? sequenza? codificante? ?? l'acido? nucleico? sopra? descritto,? preferibilmente? comprendente?o?costituito?da?una?sequenza?con?SEQ?ID?N.1,?SEQ?ID?N.2?o?SEQ?ID?N.3.? Detto?plasmide?pu??inoltre?comprendere?uno?o?pi??di:? ??una?sequenza?5'??inverted?terminal?repeat??(ITR)?di?AAV,?preferibilmente?localizzata?all'estremit??5'? del?promotore;? ??un?elemento??enhancer?,?preferibilmente?localizzato?all'estremit??5'?del?promotore;? ??una?sequenza?promotore;? ??una?sequenza?di?Kozak,?preferibilmente?localizzata?all'estremit??5'?della?sequenza?codificante?GLA? e?legata?operativamente?a?detta?sequenza;? ??una?sequenza?di?terminazione?della?trascrizione?preferibilmente? localizzata?all'estremit??3'?della? sequenza?codificante?GLA;? ??una?sequenza?3???inverted?terminal?repeat??(ITR)?di?AAV,?preferibilmente?localizzata?all'estremit??3'? della?sequenza?di?terminazione?della?trascrizione.? Preferibilmente?il?plasmide?comprende?in?una?direzione?5'?3':? ??una?sequenza?5???inverted?terminal?repeat??di?AAV?(5'?ITR);? ?? a? coding? sequence? of? the? alpha? galactosidase? A? (GLA)? gene? under? the? control? of? said? promoter,? in? which? said? coding? sequence? the? nucleic? acid? described above,? preferably? comprising? or? consisting? of? a? sequence? with? SEQ? ID? No. 1,? SEQ? ID? No. 2? or? SEQ? ID? No. 3.? Said? plasmid? may? further? comprise? one? or? more? of:? an? AAV? 5'? inverted? terminal? repeat? (ITR)? sequence,? preferably? located? at? the? 5'? end? of? the? promoter;? ??an?enhancer?element?,?preferably?located?at?the?5'?end?of?the?promoter?;? ??a?promoter?sequence;? ??a?Kozak?sequence,?preferably?located?at?the?5'?end?of?the?GLA?coding?sequence?and?operationally?linked?to?said?sequence;? ??a?transcription?termination?sequence?preferably?located?at?the?3'?end?of?the?GLA?coding?sequence;? ??an?AAV?3?inverted?terminal?repeat?(ITR)?sequence,?preferably?localized?at?the?3'?end?of?the?transcription?termination?sequence.? Preferably, the plasmid comprises in a 5'-3' direction: a 5-inverted terminal repeat sequence of AAV (5'-ITR);

??una?sequenza??enhancer?;? ??an?enhancer?sequence?;?

??una?sequenza?promotore;? ??a?promoter?sequence;?

??una?sequenza?di?Kozak;? ??la?sequenza?codificante?GLA?sopra?descritta?sotto?il?controllo?di?detto?promotore;? ??una?sequenza?nucleotidica?di?una?sequenza?di?terminazione?della?trascrizione;?e? ??a?Kozak?sequence;? ??the?GLA?coding?sequence?described?above?under?the?control?of?the?said?promoter;? ??a?nucleotide?sequence?of?a?transcription?termination?sequence;?and?

? ?

??una?sequenza?3???inverted?terminal?repeat??di?AAV?(3'?ITR).? ??a?3???inverted?terminal?repeat?sequence?of?AAV?(3'?ITR).?

Facoltativamente,? il? plasmide? comprende? inoltre? un? introne,? preferibilmente? l'introne? sintetico? derivato? dall'emoglobina? beta? umana? (HBB2).? Detto? introne? ?? preferibilmente? operativamente? legato?al?3'?della?sequenza?promotore.? Optionally,? the? plasmid? also? comprises? an? intron,? preferably? the? synthetic? intron? derived? from? human? beta? hemoglobin? (HBB2).? Said? intron? is? preferably? operably? linked? to? the? 3'? of? the? promoter? sequence.?

Il?plasmide?generalmente?comprende?inoltre?elementi?strutturali?che?sono?tipicamente?richiesti?per? la?produzione?di?plasmidi?su?larga?scala?nei?batteri,?come?origine?batterica?di?replicazione,?promotore? batterico,?gene?di?resistenza?agli?antibiotici.? The?plasmid?generally?also?includes?structural?elements?that?are?typically?required?for?large-scale?plasmid?production?in?bacteria,?such?as?bacterial?origin?of?replication,?bacterial?promoter,?antibiotic?resistance?gene.?

In?una?forma?di?realizzazione?preferita,?il?plasmide?comprende?o???costituito?da?una?sequenza?con? SEQ?ID?N.10.? In a preferred embodiment, the plasmid comprises or consists of a sequence with SEQ ID No. 10.

Un? ulteriore? oggetto? dell'invenzione? ?? l'uso? di? detto? plasmide? per? la? generazione? di? un? vettore? secondo? l'invenzione.?Preferibilmente?detto? vettore???un? vettore?AAV?o?un? vettore?di? lentivirus.? Preferibilmente,???un?vettore?come?descritto?sopra.?Preferibilmente? il?virus?adeno?associato???del? sierotipo?8.? A further object of the invention is the use of said plasmid for the generation of a vector according to the invention. Preferably said vector is an AAV vector or a lentivirus vector. Preferably a vector as described above. Preferably adeno-associated virus serotype 8.

In?una?forma?di?realizzazione,?l'acido?nucleico?o?l'invenzione???incluso?in?un?vettore?integrativo,?cio?? un? vettore? per? l'integrazione?mirata? di? un? cDNA? terapeutico? nel? locus? dell'albumina.? I? vettori? integrativi?sono?noti?nel?settore,?si?veda?ad?esempio?Barzel?et?al.,?2015;?De?Caneva?et?al.,?2019;?Porro? et?al.,?2017.? In one embodiment, the nucleic acid or the invention is included in an integrative vector, i.e., a vector for the targeted integration of a therapeutic cDNA into the albumin locus. Integrative vectors are known in the art, see, for example, Barzel et al., 2015; De Caneva et al., 2019; Porro et al., 2017.

In?questa?forma?di?realizzazione,?il?vettore?comprende?almeno:? In this embodiment, the carrier comprises at least:

??sequenza?codificante?del?gene?dell'alfa?galattosidasi?A?(GLA),? in?cui?detta?sequenza?codificante??? l'acido?nucleico?sopra?descritto,?preferibilmente?comprendente?o?costituita?da?una?sequenza?con? SEQ?ID?N.1,?SEQ?ID?N.2?o?SEQ?ID?N.3?,?e? ??sequence?coding?for?the?alpha?galactosidase?A?(GLA)?gene,?wherein?said?sequence?coding?the?above?described?nucleic?acid,?preferably?comprising?or?consisting?of?a?sequence?with?SEQ?ID?No.1,?SEQ?ID?No.2?or?SEQ?ID?No.3?,?and?

??una?o?pi??sequenze?dell?albumina.? ??one?or?more?albumin?sequences.?

Dette? sequenze? dell?albumina? possono? provenire? da? geni? dell'albumina? di? qualsiasi? origine,? preferibilmente?provengono?da?geni?dell'albumina?di?topo?o?umani.?Preferibilmente,?sono?sequenze? genomiche?di?albumina,?preferibilmente?comprendono?l'esone?13,?14?e/o?15?del?gene?dell'albumina? umana.? These albumin sequences may originate from albumin genes of any origin, preferably from mouse or human albumin genes. Preferably, they are albumin genomic sequences, preferably comprising exon 13, 14 and/or 15 of the human albumin gene.

Preferibilmente,?il?vettore?comprende?la?sequenza?codificante?GLA?all'interno?di?una?prima?e?di?una? seconda?sequenza?genomica?di?albumina.? Preferably, the vector comprises the GLA coding sequence within a first and second albumin genomic sequence.

In?una?forma?di?realizzazione,?detto?vettore?comprende?inoltre?uno?o?pi??di:? In one embodiment, said carrier further comprises one or more of:

? ?

??una?sequenza?5'??inverted?terminal?repeat??(ITR)?di?AAV,?preferibilmente?localizzata?all'estremit??5'? della?prima?sequenza?di?albumina;? ??a?5'?inverted?terminal?repeat?(ITR)?sequence?of?AAV,?preferably?localized?at?the?5'?end?of?the?first?albumin?sequence;?

??una?sequenza?di?skipping?ribosomiale,?preferibilmente?localizzata?tra?la?prima?sequenza?di?albumina? e?la?sequenza?codificante?GLA,?preferibilmente???in?frame?con?la?sequenza?di?albumina;? ??a?ribosomal?skipping?sequence,?preferably?located?between?the?first?albumin?sequence?and?the?GLA?coding?sequence,?preferably?in?frame?with?the?albumin?sequence;?

??una?sequenza?3'??inverted?terminal?repeat??(ITR)?di?AAV,?preferibilmente?localizzata?all'estremit??3'? della?seconda?sequenza?di?albumina.? ??a?3'?inverted?terminal?repeat?(ITR)?sequence?of?AAV,?preferably?localized?at?the?3'?end?of?the?second?albumin?sequence.?

Preferibilmente,? la? sequenza? di? skipping? ribosomiale? ?? una? sequenza? T2A,? P2A,? E2A,? F2A,? preferibilmente? P2A.?Questa? sequenza? quando? espressa? in? una? cellula? permette? di? ottenere? la? proteina?di?interesse?separata?dall'albumina.? Preferably,? the? ribosomal? skipping? sequence? is? a? sequence? T2A,? P2A,? E2A,? F2A,? preferably? P2A.? This? sequence? when? expressed? in? a? cell? allows? to? obtain? the? protein? of? interest? separated? from? albumin.?

Preferibilmente?detto? vettore? comprende? anche?una? sequenza?motivo? adiacente? al?protospacer? (protospacer?adjacent?motif)? (PAM),?preferibilmente?una?sequenza?PAM8,?preferibilmente?situata? all'interno?di?una?sequenza?di?albumina.? Preferably, said vector also comprises a protospacer-adjacent motif (PAM) sequence, preferably a PAM8 sequence, preferably located within an albumin sequence.

In?una?forma?di?realizzazione,?detto?vettore?virale?comprende:? In one embodiment, said viral vector comprises:

??una?sequenza?5???inverted?terminal?repeat??di?AAV?(5'?ITR);? ??a?5???inverted?terminal?repeat?sequence?of?AAV?(5'?ITR);?

??una?prima?sequenza?genomica?di?albumina;? ??a?first?genomic?sequence?of?albumin;?

??una?sequenza?di??skipping??ribosomiale,?preferibilmente?P2A,? ??a?ribosomal?skipping?sequence,?preferably?P2A,?

??una?sequenza?codificante?GLA?comprendente?o?costituita?da?una?sequenza?con?SEQ?ID?N.1,?SEQ?ID? N.2?o?SEQ?ID?N.3;? ??a?GLA?coding?sequence?comprising?or?consisting?of?a?sequence?with?SEQ?ID?N.1,?SEQ?ID?N.2?or?SEQ?ID?N.3;?

??una?seconda?sequenza?genomica?di?albumina?comprendente?una?sequenza?PAM;? ??a?second?albumin?genomic?sequence?including?a?PAM?sequence;?

??una?sequenza?3???inverted?terminal?repeat??di?AAV?(3'?ITR).? ??a?3???inverted?terminal?repeat?sequence?of?AAV?(3'?ITR).?

Preferibilmente,?la?sequenza?codificante?GLA???priva?del?primo?codone,?cio??ha?la?sequenza?di?SEQ? ID?N.1,?2?o?3?senza?l'ATG?iniziale,?al?fine?di?ridurre?il?rischio?di?espressione?fuori?bersaglio.? Preferably, the GLA coding sequence should be missing the first codon, i.e. it should have the SEQ ID sequence No. 1, 2 or 3 without the initial ATG, in order to reduce the risk of off-target expression.

Preferibilmente,?detti?elementi?sono?nell'ordine?5'?3'?come?elencato?ma?altri?ordini?possono?essere? ugualmente?adatti.? Preferably,?these?elements?are?in?the?order?5'?3'?as?listed?but?other?orders?may?be?equally?suitable.?

Il?vettore?pu??inoltre?comprendere?sequenze?virali?aggiuntive,?come?sequenze?AAV?aggiuntive.? The vector may also include additional viral sequences, such as additional AAV sequences.

Tutti?questi?elementi?sono?noti?nel?settore?e?le?loro?sequenze?possono?essere?ottenute?dall'esperto? secondo?le?conoscenze?generali?nel?settore.? All?these?elements?are?known?in?the?field?and?their?sequences?can?be?obtained?by?the?expert?according?to?the?general?knowledge?in?the?field.?

In?una?forma?di?realizzazione?esemplificativa,?detta?sequenza?5'ITR?deriva?da?AAV2?e?comprende?o? In an exemplary embodiment, said 5'ITR sequence is derived from AAV2 and comprises either

? ?

consiste?in?una?sequenza?con?SEQ?ID?N.?11.? consists of a sequence with SEQ ID No. 11.

In?una?forma?di?realizzazione?esemplificativa,?detta?prima?sequenza?genomica?di?albumina?deriva? dall'albumina?di?topo?e?comprende?o???costituita?da?una?sequenza?con?SEQ?ID?N.?12.? In?an?exemplary?embodiment,?said?first?albumin?genomic?sequence?is?derived?from?mouse?albumin?and?comprises?or?consists?of?a?sequence?with?SEQ?ID?NO.?12.?

In? una? forma? di? realizzazione? esemplificativa,? detta? sequenza? di? skipping? ribosomiale? ?? P2A? e? comprende?o???costituita?da?una?sequenza?con?SEQ?ID?N.?13.? In? an? exemplary? embodiment,? said? P2A? ribosomal? skipping? sequence? comprises? or? consists? of? a? sequence? with? SEQ? ID? No.? 13.?

In?una?forma?di?realizzazione?esemplificativa,?detta?seconda?sequenza?genomica?di?albumina?deriva? dall'albumina?di?topo?e?comprende?o???costituita?da?una?sequenza?con?SEQ?ID?N.?14.?? In?an?exemplary?embodiment,?said?second?albumin?genomic?sequence?is?derived?from?mouse?albumin?and?comprises?or?consists?of?a?sequence?with?SEQ?ID?NO.?14.??

In?una?forma?di?realizzazione?esemplificativa,?detta?sequenza?3'ITR?deriva?da?AAV2?e?comprende?o??? costituita?da?una?sequenza?con?SEQ?ID?N.?19.? In?an?exemplary?embodiment,?said?3'ITR?sequence?is?derived?from?AAV2?and?comprises?or?consists?of?a?sequence?with?SEQ?ID?NO.?19.?

Preferibilmente,?il?vettore?pu??essere?ottenuto?come?descritto?in?De?Caneva?et?al.,?2019,?sostituendo? il?cDNA?codificante?per?UGT1A1?con?la?sequenza?nucleotidica?codificante?per?GLA?qui?descritta.? Preferably, the vector can be obtained as described in De Caneva et al., 2019, replacing the cDNA encoding UGT1A1 with the nucleotide sequence encoding GLA described here.

In?una?forma?di?realizzazione,?detto?vettore???somministrato?insieme?ad?un?vettore?che?esprime?una? nucleasi.?Detto?vettore?esprimente?una?nucleasi?comprende?un?acido?nucleico?codificante?per?una? nucleasi.? In one embodiment, said vector is administered together with a vector expressing a nuclease. Said nuclease-expressing vector comprises a nucleic acid encoding a nuclease.

Pertanto,?rientra?nell'ambito?dell'invenzione?anche?una?composizione?farmaceutica?comprendente? un?vettore?secondo?l'invenzione?e?un?vettore?che?esprime?una?nucleasi.? Therefore,?the?scope?of?the?invention?also?includes?a?pharmaceutical?composition?comprising?a?vector?according?to?the?invention?and?a?vector?expressing?a?nuclease.?

Preferibilmente,? detto? acido? nucleico? codificante? per? una? nucleasi? ?? un? costrutto? di? DNA? comprendente?un?acido?nucleico?codificante?per?Cas9?o?spCas9?o?SaCas9?preferibilmente?sotto? il? controllo?di?un?promotore? tessuto? specifico,? ad? es.?un?promotore? specifico?del? fegato? come?un? promotore? epatico? ibrido? del? fegato? (HLP).? Detto? costrutto? pu?? inoltre? comprendere? un? poliA,? opportunamente? un? breve? poliA? sintetico? (sh? polyA).? Tutti? questi? elementi? sono? ben? noti? nella? tecnica?e?possono?avere?sequenze?nucleotidiche?convenzionali.? Preferably,? said? nucleic? acid? encoding? a? nuclease? ?? a? DNA? construct? comprising? a? nucleic? acid? encoding? Cas9? or? spCas9? or? SaCas9? preferably? under? the? control? of? a? tissue? specific? promoter,? e.g.? a? liver? specific? promoter? such? as? a? hybrid? liver? promoter? (HLP).? Said? construct? may? further? comprise? a? polyA,? conveniently? a? synthetic? short? polyA? (sh? polyA).? All? of? these? elements? are? well? known? in? the? art? and? may? have? conventional? nucleotide? sequences.?

Detta?nucleasi???preferibilmente? scelta? tra:?una?nucleasi?CRISPR,?una?TALEN,?una?nucleasi?DNA-guidata,?una?meganucleasi?e?una?nucleasi?Zinc?Finger,?preferibilmente?detta?nucleasi???una?nucleasi? CRISPR?scelta?dal?gruppo?costituito?da:?Cas9,?Cpfl,?Casl2b?(C2cl?),?Casl3a?(C2c2),?Cas3,?Csf1,?Casl3b? (C2c6)?e?C2c3?o?loro?varianti?come?SaCas9,?VQR?Cas9?HF1?o?dcas9.? Said nuclease?preferably? chosen? from:?a?CRISPR?nuclease,?a?TALEN,?a?DNA-guided?nuclease,?a?meganuclease?and?a?Zinc?Finger?nuclease,?preferably?said?nuclease?a?CRISPR?nuclease?chosen?from?the?group?consisting?of:?Cas9,?Cpfl,?Casl2b?(C2cl?),?Casl3a?(C2c2),?Cas3,?Csf1,?Casl3b? (C2c6)?and?C2c3?or?their?variants?such as?SaCas9,?VQR?Cas9?HF1?or?dcas9.?

In?una?forma?di?realizzazione,?detto?vettore?che?esprime?una?nucleasi?comprende? inoltre?un?RNA? guida.?Un?RNA?guida???una?sequenza?di?RNA?che?ibrida?con?una?sequenza?mirata?e?guida?la?nucleasi? al?sito?di?taglio.?L'RNA?guida?pu??comprendere?un?RNA?CRISPR?trans?attivante?(tracrRNA)?che?fornisce? In one embodiment, said vector expressing a nuclease further comprises a guide RNA. A guide RNA is an RNA sequence that hybridizes with a targeted sequence and guides the nuclease to the cleavage site. The guide RNA may comprise a trans-activating CRISPR RNA (tracrRNA) that provides

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la?struttura??stem?loop??e?un?RNA?CRISPR?specifico?per?il?bersaglio?(crRNA)?progettato?per?scindere? il?sito?di? interesse?del?gene?bersaglio.? Il?tracrRNA?e? il?crRNA?possono?essere?appaiati,?ad?esempio? riscaldandoli?a?95?C?per?5?minuti?e?lasciandoli?raffreddare?lentamente?a?temperatura?ambiente?per? 10?minuti.?Preferibilmente,?l'RNA?guida???un?singolo?RNA?guida?(sgRNA)?che?consiste?sia?del?crRNA? che?del?tracrRNA?come?un?singolo?costrutto.? Un?esempio?di?vettore?che?codifica?per?una?nucleasi?e?un?RNA?guida???descritto?in?De?Caneva?et?al.,? 2019.? In?un?altra?forma?di?realizzazione,?detto?vettore?viene?somministrato?insieme?a?uno?o?pi??farmaci?che? aumentano?la?frequenza?di?targeting?genico.? Pertanto,?rientra?nell'ambito?dell'invenzione?anche?una?composizione?farmaceutica?comprendente? un?vettore?secondo?l'invenzione?e?uno?o?pi??farmaci?che?potenziano?la?frequenza?di?targeting?genico.? Un?ulteriore?oggetto?dell'invenzione???un?plasmide?comprendente:? ??sequenza?codificante?del?gene?dell'alfa?galattosidasi?A?(GLA)? in?cui?detta?sequenza?codificante??? l'acido?nucleico?sopra?descritto,?preferibilmente?comprendente?o?costituito?da?una?sequenza?con? SEQ?ID?N.1,?SEQ?ID?N.2?o?SEQ?ID?N.3,?e? ??una?o?pi??sequenze?dell?albumina,?come?sopra?descritto.? In?una?forma?di?realizzazione,?detto?plasmide?comprende:? ??una?sequenza?5???inverted?terminal?repeat??di?AAV?(5'?ITR);? ??una?prima?sequenza?genomica?di?albumina;? ??una?sequenza?di?skipping?ribosomiale,?preferibilmente?P2A,? ??una?sequenza?codificante?GLA?comprendente?o?costituita?da?una?sequenza?con?SEQ?ID?N.1,?2?o?3;? ??una?seconda?sequenza?genomica?di?albumina?comprendente?una?sequenza?PAM;? ??una?sequenza?3???inverted?terminal?repeat??di?AAV?(3'?ITR).? Preferibilmente,?la?sequenza?codificante?GLA???priva?del?primo?codone,?cio??ha?la?sequenza?di?SEQ? ID?N.1,?2?o?3?senza?l'ATG?iniziale,?al?fine?di?ridurre?il?rischio?di?espressione?fuori?bersaglio.? Preferibilmente,?detti?elementi?sono?nell'ordine?5'?3'?come?elencato?ma?altri?ordini?possono?essere? ugualmente?adatti.? the?stem?loop?structure?and?a?target-specific?CRISPR?RNA?(crRNA)?designed?to?cleave?the?site?of?interest?in?the?target?gene.? The?tracrRNA?and?the?crRNA?can?be?paired,?for?example?by?heating?them?at?95?C?for?5?minutes?and?allowing?them?to?cool?slowly?to?room?temperature?for?10?minutes.?Preferably,?the?guide?RNA?is?a?single?guide?RNA?(sgRNA)?that?consists?of?both?the?crRNA?and?the?tracrRNA?as?a?single?construct.? An example of a vector encoding a nuclease and a guide RNA is described in De Caneva et al., 2019. In another embodiment, said vector is administered together with one or more drugs that increase the frequency of gene targeting. Therefore, the scope of the invention also includes a pharmaceutical composition comprising a vector according to the invention and one or more drugs that enhance the frequency of gene targeting. A further object of the invention is a plasmid comprising: a sequence encoding the alpha-galactosidase A (GLA) gene. wherein said sequence encoding the above-described nucleic acid, preferably comprising or consisting of a sequence with SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3, and one or more albumin sequences as described above. In one embodiment, said plasmid comprises: an AAV 5-inverted terminal repeat sequence (5' ITR); a first albumin genomic sequence; a ribosomal skipping sequence, preferably P2A, ??a?GLA?coding?sequence?comprising?or?consisting?of?a?sequence?with?SEQ?ID?No.1,?2?or?3;??a?second?albumin?genomic?sequence?comprising?a?PAM?sequence;??an?AAV?3?inverted?terminal?repeat?sequence?(3'?ITR).? Preferably,?the?GLA?coding?sequence?lacks?the?first?codon,?i.e.?has?the?sequence?with?SEQ?ID?No.1,?2?or?3?without?the?initial?ATG,?in?order?to?reduce?the?risk?of?off?target?expression.? Preferably,?these?elements?are?in?the?order?5'?3'?as?listed?but?other?orders?may?be?equally?suitable.?

Il?plasmide?di? solito?comprende? inoltre?elementi? strutturali?che? sono? tipicamente? richiesti?per? la? The?plasmid?usually?also?includes?structural?elements?that?are?typically?required?for?the?

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produzione?di?plasmidi?su?larga?scala?nei?batteri,?come?origine?batterica?di?replicazione,?promotore? batterico,?gene?di?resistenza?agli?antibiotici.? large-scale production of plasmids in bacteria, such as bacterial origin of replication, bacterial promoter, antibiotic resistance gene.

In?una?forma?di?realizzazione?preferita,?il?plasmide?comprende?o???costituito?da?una?sequenza?con? SEQ?ID?N.15.? In a preferred embodiment, the plasmid comprises or consists of a sequence with SEQ ID No. 15.

Un? ulteriore? oggetto? dell'invenzione? ?? l'uso? di? detto? plasmide? per? la? generazione? di? un? vettore?? secondo? l'invenzione.? Preferibilmente? detto? vettore? ?? un? vettore? AAV? o? un? vettore? lentivirus.? Preferibilmente,???un?vettore?come?descritto?sopra.? A further object of the invention is the use of said plasmid for the generation of a vector according to the invention. Preferably said vector is an AAV vector or a lentivirus vector. Preferably a vector as described above.

Un?ulteriore?oggetto?dell'invenzione???una?particella?virale?contenente?il?vettore?virale?come?sopra? definito.? A further object of the invention is a viral particle containing the viral vector as defined above.

Costituisce? anche? oggetto? dell'invenzione? una? cellula? ospite? trasformata? con? il? vettore? secondo? l'invenzione.? The invention also includes a host cell transformed with the vector according to the invention.

In?una?forma?di?realizzazione,?detta?cellula?ospite?comprende?il?vettore?dell'invenzione?come?vettore? episomiale,?cio??l'acido?nucleico?dell'invenzione?non???integrato?nel?genoma?della?cellula?ospite.? In one embodiment, said host cell comprises the vector of the invention as an episomal vector, i.e., the nucleic acid of the invention not integrated into the genome of the host cell.

In?un?altra? forma?di? realizzazione,?detta?cellula?ospite?comprende? l'acido?nucleico?dell'invenzione? all'interno?del?suo?genoma?ed?esprime?detto?acido?nucleico.? In another embodiment, said host cell comprises the inventive nucleic acid within its genome and expresses said nucleic acid.

??anche?oggetto?dell'invenzione?una?composizione?farmaceutica?comprendente?l'acido?nucleico,?il? costrutto?di?acido?nucleico,?il?vettore?o?la?cellula?ospite?secondo?l'invenzione?e?almeno?un?veicolo?e/o? eccipiente?farmaceuticamente?accettabile.? ??also?the?invention?is?a?pharmaceutical?composition?comprising?the?nucleic?acid,?the?nucleic?acid?construct,?the?vector?or?the?host?cell?according?to?the?invention?and?at?least?one?pharmaceutically?acceptable?vehicle?and/or?excipient.?

??un?oggetto?dell'invenzione?l'acido?nucleico,?il?costrutto?di?acido?nucleico,?il?vettore?o?la?cellula?ospite? o?la?composizione?farmaceutica?dell'invenzione?per?l'uso?come?medicamento.? ??an?object?of?the?invention?is?the?nucleic?acid,?the?nucleic?acid?construct,?the?carrier?or?host?cell?or?the?pharmaceutical?composition?of?the?invention?for?use?as?a?medicinal?agent.?

??un?oggetto?dell'invenzione?l'acido?nucleico,?il?costrutto?di?acido?nucleico,?il?vettore?o?la?cellula?ospite? o?la?composizione?farmaceutica?dell'invenzione?per?l'uso?nella?terapia?genica.? ??an?object?of?the?invention?is?the?nucleic?acid,?the?nucleic?acid?construct,?the?vector?or?the?host?cell?or?the?pharmaceutical?composition?of?the?invention?for?use?in?gene?therapy.?

Preferibilmente,? detto? acido? nucleico,? costrutto? di? acido? nucleico,? vettore? o? cellula? ospite? o? composizione?farmaceutica???per?l'uso?nel?trattamento?della?malattia?di?Fabry.? Preferably,? so-called? nucleic? acid,? nucleic? acid? construct,? vector? or? host? cell? or? pharmaceutical? composition? for? use? in? the? treatment? of? Fabry? disease.?

Preferibilmente,?il?vettore?viene?veicolato?per?via?sistemica.? Preferably, the vector is delivered systemically.

Preferibilmente,? il? vettore? viene? somministrato? mediante? iniezione? endovenosa? o? iniezione? retroorbitale.? Preferably,? the? vector? is? administered? by? intravenous? injection? or? retro-orbital? injection.?

In?una? forma?di?realizzazione,? il?vettore?comprende?un?promotore?specifico?per? l'espressione?del? fegato.?In?questa?forma?di?realizzazione,?il?transgene???specificamente?espresso?nel?fegato?trattando? In one embodiment, the vector comprises a liver-specific promoter. In this embodiment, the transgene is specifically expressed in the liver by treating

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cos?? la?malattia?mediante? la? produzione? epatica? dell'enzima?GLA.? Vantaggiosamente,? l'aumento? dell'enzima?GLA?nel?fegato?porta?ad?un?abbassamento?dei?livelli?circolanti?di?liso?Gb3?nel?plasma?e? ad?una?clearance?completa?o?quasi?completa?di?liso?Gb3?nei?tessuti.? what? the? disease? is? caused? by? the? hepatic? production? of? the? enzyme? GLA.? Advantageously,? the? increase? of? the? enzyme? GLA? in? the? liver? leads? to? a? decrease? in? the? circulating? levels? of? lyso? Gb3? in? the? plasma? and? to? a? complete? or? almost? complete? clearance? of? lyso? Gb3? in? the? tissues.?

??anche?oggetto?dell'invenzione?un?metodo?per?il?trattamento?della?malattia?di?Fabry?comprendente? la?somministrazione?a?un?soggetto?che?ne?ha?bisogno?di?una?quantit??efficace?dell'acido?nucleico?o? del?costrutto?di?acido?nucleico?o?del?vettore?o?della?cellula?ospite?o?della?composizione?farmaceutica? secondo?l'invenzione.? ??also?the?invention?is?a?method?for?the?treatment?of?Fabry?disease?comprising?the?administration?to?a?subject?in?need?of?an?effective?amount?of?the?nucleic?acid?or?nucleic?acid?construct?or?vector?or?host?cell?or?pharmaceutical?composition?according?to?the?invention.?

In?una?forma?di?realizzazione,?il?vettore?integrativo?come?sopra?descritto?viene?somministrato?ad?un? soggetto?neonato?affetto?dalla?malattia?di?Fabry?per?trattare?la?malattia?nelle?sue?fasi?iniziali.? In one embodiment, the integrative vector as described above is administered to a neonatal subject with Fabry disease to treat the disease in its early stages.

L'invenzione?fornisce?anche?un?metodo?per?aumentare?l'espressione?dell'enzima?alfa?galattosidasi?A? comprendente? la? somministrazione? a? un? soggetto? che? ne? ha? bisogno? dell'acido? nucleico? o? del? costrutto?di?acido?nucleico?secondo? l'invenzione,? il?vettore?secondo? l'invenzione,? la?cellula?ospite? secondo?l'invenzione?o?la?composizione?farmaceutica?secondo?l'invenzione.? The invention also provides a method for increasing the expression of the enzyme alpha-galactosidase A comprising administering to a subject in need of the nucleic acid or the nucleic acid construct of the invention, the vector of the invention, the host cell of the invention or the pharmaceutical composition of the invention.

L'invenzione?fornisce?anche?un?metodo?per?abbassare?il?livello?circolante?di?liso?Gb3?comprendente? la?somministrazione?a?un?soggetto?che?ne?ha?bisogno?dell'acido?nucleico?o?del?costrutto?di?acido? nucleico,?del?vettore,?della?particella?virale,?della?cellula?ospite?o?della?composizione?farmaceutica? secondo?l'invenzione.? The invention also provides a method for lowering the circulating level of lyso-Gb3 comprising administering to a subject in need the nucleic acid or nucleic acid construct, vector, viral particle, host cell or pharmaceutical composition according to the invention.

DESCRIZIONE?DETTAGLIATA?DELL'INVENZIONE? DETAILED DESCRIPTION OF THE INVENTION?

Definizioni? Definitions?

I?termini?"comprendente",?"comprende"?e?"composto?da"?utilizzati?nel?presente?documento?sono? sinonimi?di?"che?include"?o?"include";?o?"contenenti"?o?"contiene",?e?sono?inclusivi?o?aperti?e?non? escludono? membri,? elementi? o? passaggi? aggiuntivi? non? recitati.? I? termini? "comprendente",? "comprende"?e?"composto?da"?includono?anche?il?termine?"costituito?da".? The terms "including", "comprises" and "consisting of" used in this document are synonymous with "including" or "includes" or "containing" or "contains", and are inclusive or open-ended and do not exclude additional members, elements or passages that are not recited. The terms "including", "comprises" and "consisting of" also include the term "consisting of".

Nella?presente?invenzione?"almeno?l'80%?di?identit?"?significa?che?l'identit??pu??essere?almeno?l'80%,? o?l'85%?o?il?90%?o?il?95%?o?il?100%?di?identit??di?sequenza?rispetto?alle?sequenze?indicate.?Questo? vale?per?tutte?le?%?di?identit??citate.?Nella?presente?invenzione?"almeno?il?95%?di?identit?"?significa? che? l'identit??pu??essere?almeno?del?95%,?96%,?97%,?98%,?99%?o?100%?di? identit??di? sequenza? rispetto? alle? sequenze? indicate.? Questo? vale? per? tutte? le? %? di? identit?? citate.? Nella? presente? invenzione?"almeno? il?98%?di? identit?"?significa?che? l'identit??pu??essere?almeno?del?98%,?99%?o? 100%?di?identit??di?sequenza?rispetto?alle?sequenze?indicate.?Questo?vale?per?tutte?le?%?di?identit?? In the present invention, "at least 80% identity" means that the identity can be at least 80%, or 85%, or 90%, or 95%, or 100% sequence identity with respect to the designated sequences. This applies to all % of the identities mentioned. In the present invention, "at least 95% identity" means that the identity can be at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the designated sequences. This applies to all % of the identities? cited.? In? the? present? invention? "at? least? 98%? identity?"? means? that? the? identity? can? be? at? least? 98%,? 99%? or? 100%? sequence? identity? to? the? sequences? indicated.? This? applies? to? all? percent? of? identities?

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citate.?Preferibilmente,?la?%?di?identit??si?riferisce?all'intera?lunghezza?della?sequenza?di?riferimento.? quote.?Preferably,?the?%?of?identity?refers?to?the?entire?length?of?the?reference?sequence.?

Come? qui? utilizzati,? i? termini? "acido? nucleico"? e? "sequenza? polinucleotidica"? e? "costrutto"? si? riferiscono?a?un?polimero?deossiribonucleotidico?o? ribonucleotidico? in? forma?a? singolo?o?doppio? filamento?e,?se?non?diversamente? limitato,?comprenderebbero?analoghi?noti?di?nucleotidi?naturali? che? possono? funzionare? in? modo? simile? ai? nucleotidi? presenti? in? natura.? Le? sequenze? polinucleotidiche?includono?sia?sequenze?a?lunghezza?intera?che?sequenze?pi??brevi?derivate?dalle? sequenze?a? lunghezza? intera.?Resta? inteso? che?una?particolare? sequenza?polinucleotidica? include? codoni?degenerati?della?sequenza?o?delle?sequenze?native?che?possono?essere?introdotti?per?fornire? preferenza?di?codone? in?una?specifica?cellula?ospite.?Le?sequenze?polinucleotidiche?che?rientrano? nell'ambito?dell'invenzione?in?oggetto?includono?inoltre?sequenze?che?si?ibridano?in?modo?specifico? con?le?sequenze?che?codificano?per?un?peptide?dell'invenzione.?Il?polinucleotide?include?sia?i?filamenti? senso?che?antisenso?come?singoli?filamenti?o?nel?duplex.? As? used? here,? the? terms? "nucleic? acid"? and? "polynucleotide? sequence"? and? "construct"? refer? to? a? deoxyribonucleotide? or? ribonucleotide? polymer? in? single? or? double? stranded? form? and,? unless? otherwise? limited,? would? include? known? analogues? of? naturally occurring? nucleotides? that? may? function? similarly? to? naturally occurring? nucleotides.? Polynucleotide? sequences? include? both? full-length? sequences? and? shorter? sequences? derived? from? full-length? sequences.? It? is? understood? that? a? particular? polynucleotide? sequence? includes? degenerate codons of the native sequence or sequences that can be introduced to provide codon preference in a specific host cell. Polynucleotide sequences within the scope of the subject invention further include sequences that specifically hybridize with sequences encoding a peptide of the invention. The polynucleotide includes both sense and antisense strands as single strands or in duplex.

Sono? incluse?nella?presente? invenzione?anche?sequenze?di?acido?nucleico?derivate?dalle?sequenze? nucleotidiche?qui?menzionate,?per?es.?frammenti?funzionali,?mutanti,?varianti,?derivati,?analoghi?e? sequenze?aventi?una?%?di?identit??di?almeno?l'80%?con?le?sequenze?qui?menzionate,?nella?misura?in? cui?tali?frammenti,?mutanti,?varianti,?derivati?e?analoghi?mantengono?la?funzione?della?sequenza?da? cui?derivano.? Also included in the present invention are nucleic acid sequences derived from the nucleotide sequences mentioned herein, e.g., functional fragments, mutants, variants, derivatives, analogues, and sequences having an identity of at least 80% with the sequences mentioned herein, to the extent that such fragments, mutants, variants, derivatives, and analogues retain the function of the sequence from which they are derived.

Il?termine?"inverted?terminal?repeat"?indica?sequenze?che?si?ripetono?ad?entrambe?le?estremit??di? una?sequenza?nucleotidica?nell'orientamento?opposto?(complementare?inverso).? The term "inverted terminal repeat" refers to sequences that are repeated at both ends of a nucleotide sequence in the opposite orientation (reverse complementary).

Con? il? termine? ?malattia? di? Fabry?? (FD)? si? intende? una? rara?malattia? lisosomiale? identificata? nel? database?Online?Mendelian?Inheritance?in?Man?(OMIM)?con?l'ID?OMIM?301500?e?caratterizzata?da? una?o?pi??mutazioni?nel?gene?dell'alfa?galattosidasi?A?che?determinano?una?carenza?dell'enzima?alfa? galattosidasi?A.?Deficienza? di? alfa?galattosidasi?A,? deficienza?di?GLA,?malattia? di?Anderson?Fabry,? angiocheratoma? corporis? diffusum,? angiocheratoma? diffuso,? ceramide? triesossidosi? e? lipidosi? distopica?ereditaria?sono?sinonimi.? The term Fabry disease (FD) refers to a rare lysosomal disease identified in the Online Mendelian Inheritance in Man (OMIM) database with the OMIM ID 301500 and characterized by one or more mutations in the alpha-galactosidase A gene that result in a deficiency of the alpha-galactosidase A enzyme. Alpha-galactosidase A deficiency, GLA deficiency, Anderson Fabry disease, angiokeratoma corporis diffusum, diffuse angiokeratoma, ceramide trihexyoxidosis, and hereditary dystopic lipidosis are synonymous.

Il?termine?"alfa?galattosidasi?A"?o?"GLA"?si?riferisce?qui?all'enzima?umano?alfa?galattosidasi?A,?per? esempio?codificato?da?cDNA?wild?type?avente?la?sequenza?di?SEQ?ID?N.16.? The term "alpha-galactosidase A" or "GLA" refers here to the human enzyme alpha-galactosidase A, for example encoded by wild-type cDNA having the sequence SEQ ID No. 16.

Il?termine?"codone?ottimizzato"?significa?qui?che?un?codone?che?esprime?un??bias??per?gli?umani? viene?modificato?in?un?codone?sinonimo?che?non?esprime?un??bias??per?gli?umani.?Il?cambiamento? nel?codone?non?si?traduce?in?un?cambiamento?dell'amminoacido?nella?proteina?codificata.? The term "optimized codon" here means that a codon that expresses a bias for humans is changed to a synonymous codon that does not express a bias for humans. The change in the codon does not result in a change in the amino acid in the encoded protein.

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Figure? Figures?

Figura? 1.? Valutazione? comparativa? delle? sequenze? con? codoni? ottimizzati? in? vitro.? A?? Disegno? sperimentale?per?la?valutazione?delle?sequenze?con?codoni?ottimizzati.?Queste?sequenze?sono?state? clonate?in?un?vettore?episomiale?che???stato?trasfettato?in?cellule?HuH7?tramite?lipofectamina?2000.? 48?ore?dopo?la?trasfezione?l'estratto?cellulare?e?il?surnatante?sono?stati?raccolti?per?l'analisi.?B????stata? eseguita?una?reazione?enzimatica?per?valutare?l'attivit??enzimatica?relativa?delle?versioni?con?codoni? ottimizzati?e?della?sequenza?hGLA?wild?type.?Questi?dati?rappresentano?l'attivit??relativa?dell'enzima? hGLA? con? tre? saggi? replicati? individuali? (n=2)? in? cui? i? cerchi? neri? e? i? quadrati? grigi? indicano? rispettivamente?l'estratto?cellulare?e?il?surnatante?cellulare?in?ciascun?saggio.? Figure? 1.? Comparative? evaluation? of? codon-optimized? sequences? in? vitro.? A? Experimental? design? for? the? evaluation? of? codon-optimized? sequences.? These? sequences? were? cloned? into? an? episomal? vector? which? was? transfected? into? HuH7? cells? using? lipofectamine? 2000.? 48? hours? after? transfection? the? cell? extract? and? supernatant? were? collected? for? analysis.? B? An? enzymatic? reaction? was? performed? to? evaluate? the? relative? enzymatic? activity? of? the? codon-optimized? versions. optimized?and?the?wild?type?hGLA?sequence.?These?data?represent?the?relative?activity?of?the?hGLA?enzyme?with?three?individual?replicate?assays?(n=2)?where?the?black?circles?and?gray?squares?indicate?the?cell?extract?and?cell?supernatant?in?each?assay,?respectively.?

Figura?2.?Valutazione?comparativa?in?vitro?delle?sequenze?con?codoni?ottimizzati.?A?Il?saggio?western? blot???stato?eseguito?per? l'estratto?cellulare?e? il?surnatante?utilizzando?un?anticorpo?specifico?per? hGLA.?B??Il?grafico?a?barre?mostra?una?rappresentazione?quantitativa?di?tre?repliche?tecniche?(n=2).? Media???SEM?(*?p?<0,?05).?L'estratto?cellulare?e?il?surnatante?sono?indicati?rispettivamente?da?barre? nere?e?grigie.? Figure?2.?In?vitro?comparative?evaluation?of?sequences?with?optimized?codons.?A?Western?blot?assay?was?performed?for?the?cell?extract?and?supernatant?using?an?antibody?specific?to?hGLA.?B?The?bar?graph?shows?a?quantitative?representation?of?three?technical?replicates?(n=2).?Mean?SEM?(*?p?<0.?05).?The?cell?extract?and?supernatant?are?indicated?by?black?and?gray?bars,?respectively.?

Figura?3.?Studi? in?vivo? in?topi?WT?C57Bl/6.?A??Schema?del?vettore?AAV?episomiale?specifico?per? il? fegato.?Le?varianti?WT?e?CO02?hGLA?cDNA?sono?trascritte?sotto?il?controllo?di?un?promotore?specifico? per?il?fegato.?B?Topi?C57BL6/WT?(P30)?sono?stati?trattati?con?vettori?episomiali?AAV8_hGLA?(pSMD2? hGLA_WT,?AAV8_pSMD2_CO02?e?AAV8_pSMD2_CO03)?tramite?iniezioni?retroorbitali?con?una?dose? di?3E+12vg/kg.?Il?fegato???stato?prelevato?e?il?sangue???stato?raccolto?3?settimane?dopo?l'iniezione? (P51).?I?topi?WT?non?trattati?sono?stati?considerati?come?controllo.?Il?sangue???stato?processato?per? ottenere?il?plasma?mentre?le?proteine?sono?state?estratte?dai?tessuti?del?fegato.?C????stato?eseguito? un?test?enzimatico?per?determinare?l'attivit??di?GLA?avviando?una?reazione?tra?i?campioni?contenenti? l'enzima;?plasma? (1:10.000)? (barre? rosse)?e?proteine?epatiche? (3ng)? (barre?blu)?e? il? substrato? (4-metilumbelliferil???D?galattopiranoside??4MUG)?per?1?ora?a?37?C.?Il?prodotto?fluorescente?rilasciato;? il?4?metilumbelliferone?(4MU)???stato?misurato?con?eccitazione?a?365?nm?ed?emissione?a?450?nm.? Diverse?concentrazioni?di?4MU?(da?2?pmoli?a?100?pmoli)?sono?state?utilizzate?per?ottenere?una?curva? standard?per?determinare? l'attivit??enzimatica?(EA).?Un'unit??di?attivit??enzimatica???stata?definita? come? la?quantit??di?enzima?che?rilascia?1?nmol?di?4?MU?per?milligrammo/millilitro?di?enzima?e? in? un'ora.?Fegato?e?plasma?sono?indicati?rispettivamente?da?barre?nere?e?grigie.? Figure?3.?In?vivo?studies?in?WT?C57Bl/6?mice.?A?Schematic?of?the?liver-specific?episomal?AAV?vector.?The?WT?and?CO02?hGLA?cDNA?variants?are?transcribed?under?the?control?of?a?liver-specific?promoter.?B?C57BL6/WT?(P30)?mice?were?treated?with?AAV8_hGLA?episomal?vectors?(pSMD2? hGLA_WT,?AAV8_pSMD2_CO02?and?AAV8_pSMD2_CO03)?via?retroorbital?injections?with?a?dose? of?3E+12vg/kg.?Liver?was?removed?and?blood?was?collected?3?weeks?after?injection? (P51).?Untreated?WT?mice?were?considered?as?controls.?Blood?was?processed?to?obtain?plasma?while?proteins?were?extracted?from?liver?tissues.?An?enzyme?assay?was?performed?to?determine?GLA?activity?by?initiating?a?reaction?between?samples?containing?the?enzyme;?plasma?(1:10,000)? (red?bars)?and?liver?proteins?(3ng)? (blue?bars)?and?the?substrate? (4-methylumbelliferyl???D?galactopyranoside??4MUG)?for?1?hour?at?37?C.?The?released?fluorescent?product;?4?methylumbelliferone?(4MU)?was?measured?with?excitation?at?365?nm?and?emission?at?450?nm.? Different?concentrations?of?4MU?(from?2?pmoles?to?100?pmoles)?were?used?to?obtain?a?standard?curve?to?determine?the?enzyme?activity?(EA).?A?unit?of?enzyme?activity?was?defined?as?the?amount?of?enzyme?that?releases?1?nmol?of?4?MU?per?milligram/milliliter?of?enzyme?and?in? one hour.?Liver?and?plasma?are?indicated?by?black?and?gray?bars,?respectively.?

Figura?4.?Studi?in?vivo?in?topi?WT?C57Bl/6.?A?e?B??L'analisi?Western?blot???stata?eseguita?per?tutti?gli? animali?trattati?e?non?trattati?utilizzando?proteine?epatiche?(barre?blu)?e?plasma?(barre?rosse).?HSP70? Figure?4.?In?vivo?studies?in?WT?C57Bl/6?mice.?A?and?B?Western?blot?analysis?was?performed?for?all?treated?and?untreated?animals?using?liver?proteins?(blue?bars)?and?plasma?(red?bars).?HSP70?

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(per?fegato)?e?mIgG?(plasma)?sono?stati?usati?come?geni?di?riferimento.?La?quantificazione?ha?indicato? livelli? di? espressione? proteica? pi?? elevati? nel? caso? pSMD2_hGLA_CO02? e? pSMD2_hGLA_CO03? rispetto?a?pSMD2_hGLA_WT?nel?fegato?e?nel?plasma,?il?che?suggerisce?la?migliore?efficienza?in?vivo? di?queste?due?sequenze?con?codoni?ottimizzati.?Fegato?e?plasma?sono? indicati?rispettivamente?da? barre?nere?e?grigie.? (liver) and mIgG (plasma) were used as reference genes. Quantification indicated higher protein expression levels for pSMD2_hGLA_CO02 and pSMD2_hGLA_CO03 than for pSMD2_hGLA_WT in liver and plasma, suggesting the improved in vivo efficiency of these two codon-optimized sequences. Liver and plasma are indicated by black and gray bars, respectively.

Figura?5.?Trattamento?di?topi?Fabry?con?vettori?AAV?episomiali?specifici?per?il?fegato.?A??I?topi?sono? stati?trattati?a?P30?con?AAV8_pSMD2_WT?o?AAV8_pSMD2_CO02?a?diverse?dosi?di?AAV,?che?vanno?da? 3.0E11?vg/kg?a?3.0E13?vg/kg,?e?sacrificati?a?P150?(M5).?B?e?C??Determinazione?dell'attivit??del?GLA? (B)?e?del?liso?Bb3?(C)?plasmatico.?Topi?WT?non?trattati?e?topi?MUT?Fabry?KO?non?trattati?sono?stati? usati?come?controlli.?D??Determinazione?delle?copie?del?genoma?virale?nel?fegato.? Figure?5.?Treatment?of?Fabry?mice?with?liver-specific?episomal?AAV?vectors.?A?Mice?were?treated?at?P30?with?AAV8_pSMD2_WT?or?AAV8_pSMD2_CO02?at?different?AAV?doses,?ranging?from?3.0E11?vg/kg?to?3.0E13?vg/kg,?and?sacrificed?at?P150?(M5).?B?and?C?Determination?of?plasma?GLA?(B)?and?lyso?Bb3?(C)?activity.?Untreated?WT?mice?and?untreated?MUT?Fabry?KO?mice?were? used?as?controls.?D?Determination?of?copies?of?the?viral?genome?in?the?liver.?

Figura? 6.? Trattamento? di? topi? Fabry? con? vettori? AAV? episomiali? specifici? per? il? fegato.? A?? Determinazione? dell'attivit?? di?GLA?nei? tessuti.?B??Determinazione? dell'accumulo? di? liso?Gb3? nei? tessuti.? Figure? 6.? Treatment? of? Fabry? mice? with? liver-specific? episomal? AAV? vectors.? A? Determination? of? GLA? activity? in? tissues.? B? Determination? of? lyso? Gb3? accumulation? in? tissues.?

Figura?7.?Trattamento?di?topi?Fabry?con?l?approccio?di?targeting?genico?nel?fegato.?A??I?topi?sono?stati? trattati?a?P5?con?AAV8?Alb?hGLA?WT?o?AAV8?Alb?hGLA?CO02,?pi??il?vettore?AA8?pX602,?che?esprime? SaCas9? e? sgRNA,? a? due? diverse? dosi? di? AAV,? e? sacrificati? a? P150? (M5).? B? e? C?? Determinazione? dell'attivit??del?GLA?(B)?e?del?liso?Bb3?(C)?plasmatico.?Topi?WT?non?trattati?e?topi?MUT?Fabry?KO?non? trattati?sono?stati?usati?come?controlli.?D??Determinazione?della?frequenza?di?integrazione?mediante? ddPCR?nel?fegato.? Figure?7.?Treatment?of?Fabry?mice?with?the?liver?gene?targeting?approach.?A?Mice?were?treated?at?P5?with?AAV8?Alb?hGLA?WT?or?AAV8?Alb?hGLA?CO02,?plus?the?AA8?pX602?vector,?which?expresses?SaCas9?and?sgRNA,?at?two?different?doses?of?AAV,?and?sacrificed?at?P150?(M5).?B?and?C?Determination?of?plasma?GLA?(B)?and?lyso?Bb3?(C)?activity.?Untreated?WT?mice?and?MUT?Fabry?KO?mice? treated?were?used?as?controls.?D??Determination?of?the?frequency?of?integration?by?ddPCR?in?the?liver.?

Figura?8.?Trattamento?di?topi?Fabry?con?approccio?di?targeting?genico?del?fegato.?A??Determinazione? dell'attivit??del?GLA?nei?tessuti.?B??Determinazione?dell'accumulo?di?liso?Gb3?nei?tessuti.? Figure?8.?Treatment?of?Fabry?mice?with?liver?gene?targeting?approach.?A??Determination?of?GLA?activity?in?tissues.?B??Determination?of?lyso?Gb3?accumulation?in?tissues.?

TERAPIA?GENICA? GENE THERAPY?

Negli?ultimi?dieci?anni,?la?terapia?genica???stata?applicata?al?trattamento?della?malattia?in?centinaia?di? studi?clinici.?Vari?strumenti?sono?stati?sviluppati?per?fornire?geni?nelle?cellule?umane.?Nella?presente? invenzione?i?vettori?possono?essere?somministrati?ad?un?paziente.?Un?lavoratore?qualificato?sarebbe? in?grado?di?determinare?l'intervallo?di?dosaggio?appropriato.? Over the past decade, gene therapy has been applied to the treatment of disease in hundreds of clinical studies. Various tools have been developed to deliver genes into human cells. In the present invention, vectors can be administered to a patient. A trained worker would be able to determine the appropriate dosage range.

La?terapia?genica?pu??essere?diretta?a?un?tessuto?specifico?per?correggere?un?difetto?genetico.? Gene therapy can be directed at a specific tissue to correct a genetic defect.

Nella?presente?invenzione,?un?deficit?di?galattosidasi?A?viene?corretto?mediante?la?riduzione?dei?livelli? di?liso?Gb3?nel?plasma?e?nel?tessuto?ottenuta?attraverso?l'espressione?dell'enzima?GLA.? In the present invention, a deficiency of galactosidase A is corrected by reducing the levels of lyso-Gb3 in plasma and tissue by expression of the GLA enzyme.

ELEMENTI?REGOLATORI? ELEMENTS?REGULATORS?

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L'invenzione? in? oggetto? riguarda? anche? un? vettore? che? pu?? includere? elementi? regolatori? che? sono? funzionali?nella?cellula?ospite?in?cui?il?vettore?deve?essere?espresso.?Una?persona?esperta?nella?tecnica?pu?? selezionare? elementi? regolatori? per? l'uso? in? cellule? ospiti? appropriate,? per? esempio? cellule? ospiti? di? mammifero? o? umane.? Gli? elementi? regolatori? includono,? per? esempio,? promotori,? sequenze? di? terminazione?della?trascrizione,?sequenze?di?terminazione?della?traduzione,?enhancer,?peptidi?segnale,? segnali?di?degradazione?ed?elementi?di?poliadenilazione.? The subject? invention? also? relates? to? a? vector? that? may? include? regulatory? elements? that? are? functional? in? the? host? cell? in? which? the? vector? is? to? be? expressed.? A? person? skilled? in? the? art? may? select? regulatory? elements? for? use? in? appropriate? host? cells,? for? example? mammalian? or? human? host? cells.? Regulatory? elements? include,? for? example,? promoters,? transcription? termination? sequences,? translation? termination? sequences,? enhancers,? signal? peptides,? degradation? signals? and? polyadenylation? elements.?

Un? vettore? dell'invenzione? pu?? facoltativamente? contenere? una? sequenza? di? terminazione? della? trascrizione,?una?sequenza?di?terminazione?della?traduzione,?una?sequenza?del?peptide?segnale,?siti?di?inizio? interni?per?i?ribosomi?(IRES),?elementi?enhancer?e/o?elementi?regolatori?post?trascrizione?come?l?elemento? regolatorio?post?trascrizionale?del?virus?dell'epatite?Woodchuck?(WHV)?(WPRE).?Le?regioni?di?terminazione? della? trascrizione?possono? tipicamente?essere?ottenute?dalla?regione?3'?non? tradotta?di?una?sequenza? genica?eucariotica?o?virale.?Le?sequenze?di?terminazione?della?trascrizione?possono?essere?posizionate?a? valle?di?una?sequenza?codificante?per?fornire?una?terminazione?efficiente.?Nel?sistema?dell'invenzione??? tipicamente?incluso?un?sito?di?terminazione?della?trascrizione.? A vector of the invention may optionally contain a transcription termination sequence, a translation termination sequence, a signal peptide sequence, internal ribosome initiation sites (IRES), enhancer elements, and/or post-transcriptional regulatory elements such as the Woodchuck Hepatitis Virus (WHV) post-transcriptional regulatory element (WPRE). Transcription termination regions may typically be obtained from the 3' untranslated region of a eukaryotic or viral gene sequence. Transcription termination sequences may be positioned at downstream?of?a?coding?sequence?to?provide?efficient?termination.?In?the?inventive?system?typically?a?transcription?termination?site?is?included.?

PROMOTORI? PROMOTERS?

Il? costrutto?di?acido?nucleico?o? il? vettore?dell'invenzione?pu?? comprendere?una? sequenza?promotore? operativamente? legata? alla? sequenza? nucleotidica? che? codifica? il? polipeptide? desiderato.? Il? termine? "operativamente?legata"?significa?che?le?parti?(ad?es.?transgene?e?promotore)?sono?collegate?insieme?in?un? modo?che?consente?a?entrambe?di?svolgere?la?loro?funzione?sostanzialmente?senza?ostacoli.? The? nucleic? acid? construct? or? vector? of? the? invention? may? comprise? a? promoter? sequence? operably? linked? to? the? nucleotide? sequence? encoding? the? desired? polypeptide.? The? term? "operably? linked"? means? that? the? parts? (e.g.,? transgene? and? promoter)? are? linked? together? in? a? manner? that? allows? both? to? perform? their? function? substantially? without? hindrance.?

Un?promotore?nel?significato?della?presente?invenzione?pu??essere?un?promotore?ubiquitario,?nel?senso? che?guida?l'espressione?del?gene?in?un'ampia?gamma?di?cellule?e?tessuti.?Un?ulteriore?promotore?all'interno? della?presente?invenzione???un?promotore?tessuto?specifico?che?mostra?attivit??selettiva?in?uno?o?in?un? gruppo?di?tessuti?ma???meno?attivo?o?non?attivo?in?altri?tessuti.?Il?promotore?pu??mostrare?un'espressione? inducibile?in?risposta?alla?presenza?di?un?altro?fattore,?per?esempio?un?fattore?presente?in?una?cellula?ospite.? A promoter within the meaning of the present invention may be a ubiquitous promoter, meaning that it drives expression of the gene in a broad range of cells and tissues. A further promoter within the present invention is a tissue-specific promoter that exhibits selective activity in one or a group of tissues but is less active or inactive in other tissues. The promoter may exhibit inducible expression in response to the presence of another factor, for example a factor present in a host cell.

Laddove? il? vettore? comprendente? il? costrutto? viene? somministrato?per? la? terapia,???preferibile? che? il? promotore?sia?funzionale?nella?cellula?bersaglio?(per?esempio?cellula?retinica?o?cellula?epatica).? Where? the? vector? comprising? the? construct? is? administered? for? therapy,? it? is? preferable? that? the? promoter? is? functional? in? the? target? cell? (for? example? retinal? cell? or? liver? cell).?

I?promotori?contemplati?per?l'uso?nella?presente?invenzione?includono,?ma?non?sono?limitati?a,?promotori? di?geni?nativi?o?loro?frammenti?come?il?promotore?del?citomegalovirus?(CMV)?(KF853603.1,?bp?149?735),?il? promotore?della?globulina?legante?la?tiroxina?(TBG),?il?promotore?chimerico?dell'OAT?CMV/beta?actina?di? pollo?(CBA)?e? la?forma?troncata?del?promotore?CBA?(smCBA)?(US8298818?e?Light?Driven?Cone?Arrestin? Translocation?in?Cones?of?Postnatal?Guanylate?Cyclase?1?Knockout?Mouse?Retina?Treated?with?AAVGC1),? Promoters contemplated for use in the present invention include, but are not limited to, native gene promoters or fragments thereof such as the cytomegalovirus (CMV) promoter (KF853603.1, bp 149,735), the thyroxine-binding globulin (TBG) promoter, the chimeric OAT CMV/chicken beta-actin (CBA) promoter, and the?truncated?form?of?the?CBA?promoter?(smCBA)?(US8298818?and?Light?Driven?Cone?Arrestin? Translocation?in?Cones?of?Postnatal?Guanylate?Cyclase?1?Knockout?Mouse?Retina?Treated?with?AAVGC1),?

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promotore? della? Rhodopsina? (NG_009115,? bp? 4205?5010),? promotore? della? proteina? legante? interfotorecettori?retinoidi?(NG_029718.1,?bp?4777?5011),?promotore?della?distrofia?maculare?vitelliforme? 2?(NG_009033.1,?bp?4870?5470),?recettore?chinasi?1?accoppiato?a?proteine?G?umane?specifico?per?PR?(? hGRK1;?AY327580.1?bp1793?2087?o?bp?1793?1991)? ( ?brevetto?U.S.?n.?8,298,818).? Tuttavia,?pu??essere?utilizzato?qualsiasi?promotore?adatto?noto?nell'arte.? Rhodopsin promoter (NG_009115, bp 4205-5010), retinoid interphotoreceptor binding protein promoter (NG_029718.1, bp 4777-5011), vitelliform macular dystrophy 2 promoter (NG_009033.1, bp 4870-5470), PR-specific human G protein-coupled receptor kinase 1 (hGRK1; AY327580.1 bp 1793-2087 or bp 1793-1991) (U.S. Patent Nos. 8,298,818). However, any suitable promoter known in the art may be used.

In?una?forma?di?realizzazione?preferita,?il?promotore???un?promotore?specifico?per?l'espressione?epatica,? preferibilmente?il?promotore?dell'alfa?1?antitripsina?umana?(hAAT).? In a preferred embodiment, the promoter is a promoter specific for hepatic expression, preferably the human alpha-1 antitrypsin (hAAT) promoter.

I?promotori?possono?essere?incorporati?in?un?costrutto?o?in?un?vettore?utilizzando?tecniche?standard?note? nell'arte.?Copie?multiple?di?promotori?o?promotori?multipli?possono?essere?usate?in?un?costrutto?o?in?un? vettore?dell'invenzione.?In?una?forma?di?realizzazione,?il?promotore?pu??essere?posizionato?all'incirca?alla? stessa?distanza?dal?sito?di? inizio?della?trascrizione?come? lo???dal?sito?di? inizio?della?trascrizione?nel?suo? ambiente?genetico?naturale.???consentita?una?certa?variazione?di?questa?distanza?senza?una?sostanziale? diminuzione?dell'attivit??del?promotore.? Promoters may be incorporated into a construct or vector using standard techniques known in the art. Multiple copies of promoters or multiple promoters may be used in a construct or vector of the invention. In one embodiment, the promoter may be positioned approximately the same distance from the transcription start site as it would be from the transcription start site in its natural genetic environment. Some variation in this distance is permitted without substantially decreasing the activity of the promoter.

INTRONE? INTRO?

Gli?introni?possono?essere?inclusi?in?un?costrutto?di?acido?nucleico?o?in?un?vettore.?In?particolare,?un? introne? posto? tra? il? promotore? e? la? sequenza? codificante? aumenta? la? stabilit?? dell'mRNA? e? la? produzione?di?proteina,?aumentando?cos??l'espressione?del?transgene.? Introns can be included in a nucleic acid construct or a vector. In particular, an intron placed between the promoter and the coding sequence increases mRNA stability and protein production, thereby increasing transgene expression.

Pu?? essere? utilizzato? qualsiasi? introne? adatto,? la? cui? selezione? pu?? essere? facilmente? effettuata? dall'esperto.? Any suitable intron can be used, the selection of which can be easily performed by the expert.

Un?introne?preferito???l'introne?sintetico?derivato?da?emoglobina?beta?(HBB2).? A preferred intron is the synthetic intron derived from hemoglobin beta (HBB2).

SEQUENZA?DI?POLIADENILAZIONE? POLYADENYLATION SEQUENCE

Il? vettore? della? presente? invenzione? pu?? comprendere? una? sequenza? di? poliadenilazione.? Opportunamente,? il? transgene? ?? operativamente? legato? ad? una? sequenza? di? poliadenilazione.? Una? sequenza?di?poliadenilazione?pu??essere? inserita?a?valle?del?transgene?per?migliorare? l'espressione?del? transgene.? The vector of the present invention may comprise a polyadenylation sequence. Suitably, the transgene is operably linked to a polyadenylation sequence. A polyadenylation sequence may be inserted downstream of the transgene to enhance expression of the transgene.

Una? sequenza? di? poliadenilazione? comprende? tipicamente? un? segnale? di? poliadenilazione,? un? sito? di? poliadenilazione?e?un?elemento?a?valle:?il?segnale?di?poliadenilazione?comprende?il?motivo?di?sequenza? riconosciuto?dal?complesso?di?scissione?dell'RNA;?il?sito?di?poliadenilazione???il?sito?di?scissione?in?cui?una? coda?di?poli?A?viene?aggiunta?all'mRNA;?l'elemento?a?valle???una?regione?ricca?di?GT?che?di?solito?si?trova? appena?a?valle?del?sito?di?poliadenilazione,?che???importante?per?un?processamento?efficiente.? A polyadenylation sequence typically comprises a polyadenylation signal, a polyadenylation site, and a downstream element: the polyadenylation signal comprises the sequence motif recognized by the RNA cleavage complex; the polyadenylation site is the cleavage site where a polyA tail is added to the mRNA; the downstream element is a GT-rich region that is usually located just downstream of the polyadenylation site, which is important for efficient processing.

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In? forme? di? realizzazione? preferite,? la? sequenza? di? poliadenilazione? ?? il? segnale? di? poliadenilazione? dell'emoglobina?umana?beta?(HHB?poliA)?o?un?suo?frammento?che?conserva? la?funzione?naturale?della? sequenza?di?poliadenilazione.? In? preferred? embodiments,? the? polyadenylation? sequence? is? the? human? hemoglobin? beta? (HHB? polyA)? polyadenylation? signal? or? a? fragment? thereof? that? retains? the? natural? function? of? the? polyadenylation? sequence.?

ELEMENTI?REGOLATORI?POST?TRASCRIZIONALI? REGULATORY?POST?TRANSCRIPTIONAL?ELEMENTS?

Il? vettore? della? presente? invenzione? pu?? comprendere? elementi? regolatori? post?trascrizionali.? Opportunamente,? la?sequenza?codificante? la?proteina???operativamente?collegata?a?uno?o?pi??ulteriori? elementi?regolatori?post?trascrizionali?che?possono?migliorare?l'espressione?genica.? The vector of the present invention may comprise post-transcriptional regulatory elements. Suitably, the protein-coding sequence is operatively linked to one or more additional post-transcriptional regulatory elements that may enhance gene expression.

SEQUENZA?DI?KOZAK? SEQUENCE?OF?KOZAK?

Il? vettore? della? presente? invenzione? pu?? comprendere? una? sequenza? di? Kozak.?Opportunamente,? la? sequenza?codificante?GLA???collegata?operativamente?a?una?sequenza?di?Kozak.?Una?sequenza?di?Kozak? pu??essere?inserita?prima?del?codone?di?inizio?per?migliorare?l'inizio?della?traduzione.? The? vector? of? the? present? invention? may? comprise? a? Kozak? sequence.? Suitably,? the? GLA? coding? sequence? is? operatively? linked? to? a? Kozak? sequence.? A? Kozak? sequence? may? be? inserted? prior? to? the? start? codon? to? enhance? the? initiation? of? translation.?

Sequenze?di?Kozak?adatte?saranno?ben?note?all'esperto?(si?veda,?ad?esempio,?Kozak25).? Suitable Kozak sequences will be well known to the expert (see, for example, Kozak25).

SEQUENZE?DI?SKIPPING?RIBOSOMIALE:?PEPTIDI?AUTO?CLIVANTI?2A? Sequenza?di?skipping?ribosomiale???qui?usata?come?sinonimo?di?peptide?auto?clivante?2A?o?peptide? 2A.? RIBOSOMAL SKIPPING SEQUENCES: 2A SELF-CLIVATING PEPTIDES Ribosomal skipping sequence is used here as a synonym for 2A self-cleaving peptide or 2A peptide.

Questi?sono?peptidi? lunghi?18?22?aa?che?possono? indurre? la?scissione?delle?proteine?ricombinanti? nella?cellula.?I?peptidi?2A?derivano?dalla?regione?2A?nel?genoma?del?virus.? Quattro?membri?della?famiglia?dei?peptidi?2A?sono?spesso?utilizzati?nella?ricerca?sulle?scienze?della? vita.?Sono?P2A,?E2A,?F2A?e?T2A.?F2A?deriva?dal?virus?18?dell'afta?epizootica;?E2A???derivato?dal?virus? A?della?rinite?equina;?P2A?deriva?dal?teschovirus?suino?1?2A;?T2A???derivato?dal?virus?thosea?asigna? 2.? These are 18-22 aa long peptides that can induce the cleavage of recombinant proteins in the cell. 2A peptides are derived from the 2A region in the virus genome. Four members of the 2A peptide family are often used in life sciences research. They are P2A, E2A, F2A and T2A. F2A is derived from foot-and-mouth disease virus 18; E2A is derived from equine rhinitis virus A; P2A is derived from porcine testes virus 1 2A; T2A is derived from thosea asign 2 virus.

Qualsiasi? sequenza? di? skipping? ribosomiale? pu?? essere? utilizzata? nell'ambito? della? presente? invenzione.?Uno?preferito???P2A.?I?peptidi?dello?skipping?ribosomiale,?ad?esempio?i?peptidi?2A,?sono? preferibilmente?localizzati?tra?la?sequenza?dell'albumina?e?la?sequenza?del?DNA?esogeno.? SEQUENZE?ACCETTATRICI?DI?SPLICING? Lo?splicing?dell'RNA???una?forma?di?processamento?dell'RNA?in?cui?un?trascritto?di?RNA?messaggero? precursore?(pre?mRNA)?appena?prodotto?viene?trasformato?in?un?RNA?messaggero?maturo?(mRNA).? Durante? lo? splicing,? gli? introni? (regioni? non? codificanti)? vengono? rimossi? e? gli? esoni? (regioni? codificanti)?vengono?uniti?insieme.? All'interno?degli?introni,?per?lo?splicing?sono?necessari?un?sito?donatore?(estremit??5'?dell'introne),? un? sito? di? diramazione? (vicino? all'estremit?? 3'? dell'introne)? e? un? sito? accettore? (estremit?? 3'? Any? ribosomal?skipping? sequence? may? be? used? within? the? present? invention.? A? preferred? one? is? P2A.? Ribosomal?skipping? peptides,? for? example? 2A? peptides,? are? preferably? located? between? the? albumin? sequence? and? the? exogenous? DNA? sequence.? SPLICING? ACCEPTANT? SEQUENCES? RNA? splicing? is? a? form? of? RNA? processing? in? which? a? newly? produced? precursor? messenger? RNA? transcript? (pre? mRNA)? is? transformed? into? a? mature? messenger? RNA? (mRNA).? During? splicing,? introns? (non? coding? regions)? are? removed? and? the? exons? (coding? regions)? are? joined? together.? Within? introns,? splicing? requires? a? donor? site? (5' end of the intron),? a? branch? site? (near the 3' end of the intron)? and? an? acceptor? site? (3' end?

? ?

dell'introne).?Il?sito?donatore?di?splicing? include?una?sequenza?quasi? invariante?GU?all'estremit??5'? dell'introne,?all'interno?di?una?regione?pi??ampia?e?meno?altamente?conservata.?Il?sito?accettore?di? splicing?all'estremit??3'?dell'introne?termina?l'introne?con?una?sequenza?AG?quasi?invariante.?A?monte? (direzione?5')?dall'AG?c'??una?regione?ad?alto?contenuto?di?pirimidine?(C?e?U),?o?tratto?polipirimidinico.? Pi??a?monte?del?tratto?polipirimidinico?si?trova?il?punto?di?diramazione.? Una?"sequenza?accettore?di?splicing"???una?sequenza?nucleotidica?che?pu?? funzionare?come?sito? accettore?all'estremit??3'?dell'introne.?Le?sequenze?di?consenso?e?le?frequenze?delle?regioni?del?sito? di?splicing?umano?sono?descritte?in ?PLoS?One,?10(6),?p.e0130729.? Opportunamente,? la?sequenza?accettore?di?giunzione?pu??comprendere? la?sequenza?nucleotidica? (Y)nNYAG,?dove?n???10?20,?o?una?variante?con?almeno?il?90%?o?almeno?il?95%?di?identit??di?sequenza.? Opportunamente,?la?sequenza?accettore?di?giunzione?pu??comprendere?la?sequenza?(Y)nNCAG,?dove? n???10?20,?o?una?variante?con?almeno?il?90%?o?almeno?il?95%?di?identit??di?sequenza.? SEQUENZE?DI?ALBUMINA? In?alcune?forme?di?realizzazione,?il?vettore?dell'invenzione?comprende?anche?sequenze?di?albumina.? Le?sequenze?di?albumina?sono?sequenze?genomiche?che?codificano?per?l'albumina.?Preferibilmente? provengono?da?topo?o?genoma?umano.? of the intron).?The?donor?splice?site?includes?a?nearly?invariant?GU?sequence?at?the?5'?end?of?the?intron,?within?a?larger?and?less?highly?conserved?region.?The?acceptor?splice?site?at?the?3'?end?of?the?intron?terminates?the?intron?with?a?nearly?invariant?AG?sequence.?Upstream?(5'?direction)?from?the AG?is?a?region?of?high?pyrimidine?content?(C?and?U),?or?polypyrimidine?tract.?Further?upstream?of?the?polypyrimidine?tract?is?the?branch?point.? A "splice acceptor sequence" is a nucleotide sequence that can function as an acceptor site at the 3' end of an intron. The consensus sequences and frequencies of human splice site regions are described in PLoS One, 10(6), p.e0130729. Appropriately, the splice acceptor sequence may include the nucleotide sequence (Y)nNYAG, where n is 10 20, or a variant with at least 90% or at least 95% sequence identity. Suitably, the splice acceptor sequence may comprise the (Y)nNCAG sequence, where n???10?20, or a variant with at least 90% or at least 95% sequence identity. ALBUMIN SEQUENCES In some embodiments, the vector of the invention also comprises albumin sequences. Albumin sequences are genomic sequences encoding albumin. Preferably, they originate from mouse or human genomes.

Una? sequenza? genomica? di? albumina? comprende? almeno? un? introne? e? almeno? un? esone.? Preferibilmente?comprende?esoni?13,?14?e/o?15?e?introni?adiacenti.? In?una?forma?di?realizzazione,?il?vettore?dell'invenzione?comprende?una?prima?sequenza?di?albumina? che???a?monte?della?sequenza?codificante?GLA?e?comprende?l'esone?13?e?una?porzione?dell'esone?14? e?introni?adiacenti,?e?una?seconda?sequenza?di?albumina?che???a?valle?della?sequenza?codificante?GLA? e?comprende?una?porzione?dell'esone?14,?l'esone?15?e?introni?adiacenti.?Ci??consente?di?introdurre? la?sequenza?codificante?GLA?nel?gene?dell'albumina?del?genoma?della?cellula?trasfettata.? Preferibilmente,?il?vettore?comprende?il?peptide?P2A?in?frame?con?la?prima?sequenza?di?albumina.? SITI?DI?INSERIMENTO?NEL?GENOMA? Nella?forma?di?realizzazione?di?un?vettore?integrativo,?il?sito?della?rottura?del?doppio?filamento?(DSB)? pu??essere?introdotto?specificamente?mediante?qualsiasi?tecnica?adatta,?ad?esempio?utilizzando?un? sistema?CRISPR/Cas9.? In?una?forma?di?realizzazione,?la?nucleasi???diretta?ai?siti?di?inserimento?preferibilmente?da?un?RNA? guida.? An albumin genomic sequence comprises at least one intron and at least one exon. Preferably, it comprises exons 13, 14, and/or 15 and adjacent introns. In one embodiment, the vector of the invention comprises a first albumin sequence that is upstream of the GLA encoding sequence and comprises exon 13 and a portion of exon 14 and adjacent introns, and a second albumin sequence that is downstream of the GLA encoding sequence. and?comprises?a?portion?of?exon?14,?exon?15?and?adjacent?introns.?This?allows?the?GLA?encoding?sequence?to?be?introduced?into?the?albumin?gene?of?the?transfected?cell?genome.? Preferably,?the?vector?comprises?the?P2A?peptide?in?frame?with?the?first?albumin?sequence.? SITES?FOR?INSERTION?IN?THE?GENOME? In?an?integrative?vector?embodiment,?the?double-stranded?break?(DSB)?site?may?be?specifically?introduced?by?any?suitable?technique,?for?example?using?a?CRISPR/Cas9?system.? In one embodiment, the nuclease is directed to the insertion sites, preferably by a guide RNA.

CRISPR? CRISPR?

Il?sistema?di?nucleasi?CRISPR?(Clustered?Regularly?Interspaced?Short?Palindromic?Repeats)/Cas?(proteina? The?CRISPR?(Clustered?Regularly?Interspaced?Short?Palindromic?Repeats)/Cas?(protein?) nuclease system

? ?

associata?a?CRISPR)???un?sistema?di?nucleasi?ingegnerizzato?basato?su?un?sistema?batterico?utilizzato?per? l'ingegneria?del?genoma.?Si?basa?in?parte?sulla?risposta?immunitaria?adattativa?di?molti?batteri?e?archaea.? Quando?un?virus?o?un?plasmide?invade?un?batterio,?i?segmenti?del?DNA?dell'invasore?sono?convertiti?in? CRISPR?RNA?(crRNA)?dalla?risposta?"immune".?Il?crRNA?si?associa?quindi,?attraverso?una?regione?di?parziale? complementarit?,?con?un?altro?tipo?di?RNA?chiamato?tracrRNA?per?guidare?la?nucleasi?Cas?(ad?esempio,? Cas9)?verso?una?regione?omologa?al?crRNA?nel?DNA?bersaglio?chiamata?"protospacer".?La?nucleasi?Cas?(ad? es.?Cas9)? scinde? il?DNA?per? generare?estremit?? smussate? in? corrispondenza?della? rottura?del?doppio? filamento?nei? siti? specificati?da?una? sequenza?di? filamenti? complementari?di? 20?nucleotidi? contenuta? all'interno?del?trascritto?del?crRNA.?La?nucleasi?Cas?(ad?esempio,?Cas9)?in?alcune?forme?di?realizzazione,? richiede?sia? il?crRNA?che? il?tracrRNA?per? il?riconoscimento?e? la?scissione?del?DNA?sito?specifici.?Questo? sistema???stato?ora?ingegnerizzato?in?modo?tale?che,?in?alcune?forme?di?realizzazione,?il?crRNA?e?il?tracrRNA? sono?combinati?in?una?molecola?(il?"singolo?RNA?guida"?o?"sgRNA"),?e?la?porzione?crRNA?equivalente?del? singolo?RNA?guida???ingegnerizzata?per?guidare?la?nucleasi?Cas?(ad?esempio,?Cas9)?per?mirare?a?qualsiasi? sequenza?desiderata?(vedi,?ad?esempio,? (2012)?Science?337:816?821;? ?(2013)?eLife? 2:e00471; ?(2013)?eLife?2:?e00563).? (CRISPR-associated) is an engineered nuclease system based on a bacterial system used for genome engineering. It is based in part on the adaptive immune response of many bacteria and archaea. When a virus or plasmid invades a bacterium, segments of the invader's DNA are converted into CRISPR RNA (crRNA) by the immune response. The crRNA then associates with a region of partial complementarity, with another type of RNA called tracrRNA to guide the Cas nuclease (e.g., Cas9) to a region homologous to the crRNA in the target DNA called a protospacer. The Cas nuclease (e.g., Cas9) cleaves DNA to generate blunt ends at double-strand breaks at sites specified by a complementary 20-nucleotide strand sequence contained within the crRNA transcript. The Cas nuclease (e.g., Cas9) in some embodiments requires both crRNA and tracrRNA to site-specific DNA recognition and cleavage. This system has now been engineered so that, in some embodiments, the crRNA and tracrRNA are combined into one molecule (the "single guide RNA" or sgRNA), and the equivalent crRNA portion of the single guide RNA is engineered to guide the Cas nuclease (e.g., Cas9) to target any target molecule. desired sequence (see, for example, (2012) Science 337:816-821; (2013) eLife 2:e00471; (2013) eLife 2:e00563).

Come?qui?utilizzato,?il?tracRNA???anche?definito?come?scaffold?gRNA.?Pertanto,?il?sistema?CRISPR/Cas?pu?? essere? ingegnerizzato? per? creare? una? rottura? a? doppio? filamento? in? corrispondenza? di? un? bersaglio? desiderato? in?un?genoma?di?una?cellula?e?sfruttare? i?meccanismi?endogeni?della?cellula?per?riparare? la? rottura? indotta?mediante?riparazione?diretta?dall'omologia?(HDR)?o?unione?di?estremit??non?omologhe? (NHEJ).?In?alcune?forme?di?realizzazione,?la?nucleasi?Cas?ha?attivit??di?scissione?del?DNA.?La?nucleasi?Cas,?in? alcune? forme?di?realizzazione,?dirige? la?scissione?di?uno?o?entrambi? i? filamenti? in?una?posizione? in?una? sequenza?di?DNA?bersaglio.?Ad?esempio,? in?alcune?forme?di?realizzazione,? la?nucleasi?Cas???una?nickasi? avente?uno?o?pi??domini?catalitici? inattivati?che? scinde?un? singolo? filamento?di?una? sequenza?di?DNA? bersaglio.?Esempi?non?limitativi?di?nucleasi?Cas?includono?Casl,?CaslB,?Cas2,?Cas3,?Cas4,?Cas5,?Cas6,?Cas7,? Cas8,?Cas9?(noto?anche?come?Csnl?e?Csxl2),?CaslO,?,?Cpfl,?C2c3,?C2c2?e?C2clCsyl,?Csy2,?Csy3?,?Csel,?Cse2,?Cscl,? Csc2,?Csa5,?Csn2,?Csm2,?Csm3,?Csm4,?Csm5,?Csm6,?Cmrl,?Cmr3,?Cmr4,?Cmr5,?Cmr6,?Cpfl,?Csbl,?Csb2,?Csb3,? Csxl7,?Csxl4,?CsxlO,?Csxl6,?CsaX?,?Csx3,?Csxl,?Csxl5,?Csfl,?Csf2,?Csf3,?Csf4,?loro?omologhi,?loro?varianti,?loro? mutanti?e?loro?derivati.?Esistono?tre?tipi?principali?di?nucleasi?Cas?(tipo?I,?tipo?II?e?tipo?III)?e?10?sottotipi?che? includono?5?proteine?di?tipo?I,?3?di?tipo?II?e?2?di?tipo?III?(vedere,?ad?esempio,?Hochstrasser?e?Doudna,?Trends? Biochem?Sci,?2015:40(l):58?66).?Le?nucleasi?Cas?di?tipo?II?includono,?ma?non?sono?limitate?a,?Casl,?Cas2,?Csn2? e?Cas9.?Queste?nucleasi?Cas?sono?note?agli?esperti?del?ramo.?Per?esempio,?la?sequenza?amminoacidica?del? polipeptide?Cas9?wild?type?di?Streptococcus?pyogenes??? riportata,?per?esempio,? in?NBCI?Ref.?Seg.?NP? As?used?here,?tracRNA?is?also?referred?to?as?a?gRNA?scaffold.?Therefore,?the?CRISPR/Cas?system?can?be?engineered?to?create?a?double-stranded?break?at?a?desired?target?in?a?cell?genome?and?exploit?the?cell’s?endogenous?mechanisms?to?repair?the?induced?break?by?homology-directed?repair?(HDR)?or?non-homologous?end?joining?(NHEJ).?In?some?forms,?the?Cas?nuclease?has?DNA?cleavage?activity.?The?Cas?nuclease,?in?some?forms,?directs? the?cleavage?of?one?or?both?strands?at?one?position?in?a?target?DNA?sequence.?For?example,?in?some?embodiments,?Cas?nuclease?is?a?nickase?having?one?or?more?inactivated?catalytic?domains?that?cleaves?a?single?strand?of?a?target?DNA?sequence.?Non-limiting?examples?of?Cas?nucleases?include?Casl,?CaslB,?Cas2,?Cas3,?Cas4,?Cas5,?Cas6,?Cas7,? Cas8,?Cas9?(also?known?as?Csnl?and?Csxl2),?CaslO,?,?Cpfl,?C2c3,?C2c2?and?C2clCsyl,?Csy2,?Csy3?,?Csel,?Cse2,?Cscl,? Csc2,?Csa5,?Csn2,?Csm2,?Csm3,?Csm4,?Csm5,?Csm6,?Cmrl,?Cmr3,?Cmr4,?Cmr5,?Cmr6,?Cpfl,?Csbl,?Csb2,?Csb3,? Csxl7,?Csxl4,?CsxlO,?Csxl6,?CsaX?,?Csx3,?Csxl,?Csxl5,?Csfl,?Csf2,?Csf3,?Csf4,?their?homologs,?their?variants,?their?mutants?and?their?derivatives.?There?are?three?main?types?of?Cas?nucleases?(type?I,?type?II?and?type?III)?and?10?subtypes?that? include?5?type?I,?3?type?II?and?2?type?III?proteins?(see,?for?example,?Hochstrasser?and?Doudna,?Trends? Biochem?Sci,?2015:40(l):58?66).?Type?II?Cas?nucleases?include,?but?are?not?limited?to,?Casl,?Cas2,?Csn2?,?and?Cas9.?These?Cas?nucleases?are?known?to?those?of?skill?in?the?art.?For?example,?the?amino?acid?sequence?of?the?wild?type?Cas9?polypeptide?from?Streptococcus?pyogenes???is?reported,?for?example,?in?NBCI?Ref.?Seg.?NP?

? ?

269215,?e? la?sequenza?amminoacidica?del?polipeptide?Cas9?wild?type?di?Streptococcus?thermophilus??? riportata,?ad?esempio,?in?NBCI?Ref.?Seg.?N.?WP_011681470.?Le?nucleasi?Cas,?ad?esempio?i?polipeptidi?Cas9,? in?alcune?forme?di?realizzazione,?derivano?da?una?variet??di?specie?batteriche.?"Cas9"?si?riferisce?a?una? proteina?nucleasi?legante?il?DNA?a?doppio?filamento?guidata?da?RNA?o?una?proteina?nickasi.?La?nucleasi? Cas9?wild?type?ha?due?domini?funzionali,?ad?esempio?RuvC?e?HNH,?che?tagliano?diversi?filamenti?di?DNA.? Cas9?pu??indurre?rotture?a?doppio?filamento?nel?DNA?genomico?(DNA?bersaglio)?quando?entrambi?i?domini? funzionali? sono? attivi.? L'enzima?Cas9,? in? alcune? forme?di? realizzazione,? comprende?uno?o?pi??domini? catalitici?di?una?proteina?Cas9?derivata?da?batteri?appartenenti?al?gruppo? costituito?da?Corynebacter,? Sutterella,?Legionella,?Treponema,?Filif?attore,?Eubacterium,?Streptococcus,?Lactobacillus,?Mycoplasma,? Bacteroides,? Flaviivola,? Flavobacterium,? Sphaerochaeta,? Azospirillum,? Gluconacetobacter,? Neisseria,? Roseburia,?Parvibaculum,?Staphylococcus,?Nitratifractor?e?Campylobacter.?In?alcune?forme?di?realizzazione,? Cas9???una?proteina?di?fusione,?ad?es.?i?due?domini?catalitici?derivano?da?diverse?specie?batteriche.?Varianti? utili?della?nucleasi?Cas9?includono?un?singolo?dominio?catalitico?inattivo,?come?un?enzima?RuvC?o?HNH?o? una?nickasi.?Una?nickasi?Cas9?ha?solo?un?dominio?funzionale?attivo?e,?in?alcune?forme?di?realizzazione,?taglia? solo?un?filamento?del?DNA?bersaglio,?creando?cos??una?rottura?o?intaccatura?del?filamento?singolo.?In?alcune? forme?di?realizzazione,?la?nucleasi?Cas9?mutante?avente?almeno?una?mutazione?D10A???una?nickasi?Cas9.? In?altre?forme?di?realizzazione,?la?nucleasi?Cas9?mutante?avente?almeno?una?mutazione?H840A???una?nickasi? Cas9.?Altri?esempi?di?mutazioni?presenti?in?una?nickasi?Cas9?includono,?senza?limitazioni,?N854A?e?N863?A.? Viene?introdotta?una?rottura?a?doppio?filamento?utilizzando?una?nickasi?Cas9?se?vengono?utilizzati?almeno? due?RNA?mirati?al?DNA?che?mirano?a?filamenti?di?DNA?opposti.?Una?rottura?a?doppio?filamento?indotta?da? doppio? taglio? viene? riparata? da?NHEJ? o?HDR.?Questa? strategia? di? editing? genetico? favorisce? l'HDR? e? diminuisce?la?frequenza?delle?mutazioni?indel?nei?siti?del?DNA?fuori?bersaglio.?La?nucleasi?o?nickasi?Cas9,?in? alcune?forme?di?realizzazione,???ottimizzata?per?i?codoni?per?la?cellula?bersaglio?o?l'organismo?bersaglio.?In? alcune? forme? di? realizzazione,? la? nucleasi? Cas? ?? un? polipeptide? Cas9? che? contiene? due?mutazioni? di? silenziamento?dei?domini?nucleasici?RuvCl?e?HNH?(D10A?e?H840A),?a?cui?si?fa?riferimento?come?dCas9.?In? una? forma?di? realizzazione,? il?polipeptide?dCas9?da? Streptococcus?pyogenes? comprende? almeno?una? mutazione? in?posizione?D10,?G12,?G17,? E762,?H840,?N854,?N863,?H982,?H983,?A984,?D986,?A987,?o? qualsiasi?loro?combinazione.?Le?descrizioni?di?tali?polipeptidi?dCas9?e?loro?varianti?sono?fornite,?ad?esempio,? nella?pubblicazione?di?brevetto? internazionale?n.?WO?2013/176772.?L'enzima?dCas9? in?alcune?forme?di? realizzazione?contiene?una?mutazione?in?D10,?E762,?H983?o?D986,?nonch??una?mutazione?in?H840?o?N863.? In? alcuni? casi,? l'enzima?dCas9? contiene?una?mutazione?D10A?o?DION.? Inoltre,? l'enzima?dCas9? include? alternativamente?una?mutazione?H840A,?H840Y?o?H840N.?In?alcune?forme?di?realizzazione,?l'enzima?dCas9? 269215,?and?the?amino acid?sequence?of?the?wild?type?Cas9?polypeptide?from?Streptococcus?thermophilus??? reported,?for?example,?in?NBCI?Ref.?Seg.?N.?WP_011681470.?Cas?nucleases,?for?example?Cas9?polypeptides,?in?some?forms?of?embodiment,?are?derived?from?a?variety?of?bacterial?species.?"Cas9"?refers?to?an?RNA-guided?double-stranded?DNA?binding?protein?nuclease?or?protein?nickase.?The?nuclease? Wild-type Cas9 has two functional domains, i.e., RuvC and HNH, that cut different DNA strands. Cas9 can induce double-strand breaks in genomic DNA (target DNA) when both functional domains are active. The Cas9 enzyme, in some embodiments, comprises one or more catalytic domains of a Cas9 protein derived from bacteria belonging to the group consisting of Corynebacter, Sutterella, Legionella, Treponema, Filif?actor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta,? Azospirillum,? Gluconacetobacter,? Neisseria,? Roseburia,? Parvibaculum,? Staphylococcus,? Nitratifractor? and? Campylobacter.? In? some? embodiments,? Cas9? is? a? fusion? protein,? i.e.,? the? two? catalytic? domains? are derived? from? different? bacterial? species.? Useful? variants? of? the? Cas9? nuclease? include? a? single? inactive? catalytic? domain,? such? as? a? RuvC? or? HNH? enzyme,? or? a? nickase.? A? Cas9? nickase? has? only? one? functional? active? domain? and,? in? some? embodiments,? cuts? only?one?strand?of?the?target?DNA,?thereby?creating?a?single-strand?break?or?nick.?In?some?embodiments,?the?mutant?Cas9?nuclease?having?at?least?one?D10A?mutation?is?a?Cas9?nickase.? In?other?embodiments,?the?mutant?Cas9?nuclease?having?at?least?one?H840A?mutation?is?a?Cas9?nickase.?Other?examples?of?mutations?present?in?a?Cas9?nickase?include,?but?not?limited?to,?N854A?and?N863?A.?A?double-strand?break?is?introduced?using?a?Cas9?nickase?if?at?least?one?D10A?mutation?is?used. two DNA-targeting RNAs that target opposite DNA strands. A double-strand break induced by a double cut is repaired by NHEJ or HDR. This gene editing strategy promotes HDR and decreases the frequency of indel mutations at off-target DNA sites. The Cas9 nuclease or nickase, in some embodiments, is optimized for the codons of the target cell or organism. In some embodiments, the Cas9 nuclease is a Cas9 polypeptide that contains two indel mutations. silencing of the RuvCl and HNH nuclease domains (D10A and H840A), which are referred to as dCas9. In one embodiment, the dCas9 polypeptide from Streptococcus pyogenes comprises at least one mutation at positions D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, A987, or any combination thereof. Descriptions of such dCas9 polypeptides and variants thereof are provided, for example, in U.S. Pat. International?No.?WO?2013/176772.?The?dCas9? enzyme?in?some?embodiments?contains?a?mutation?in?D10,?E762,?H983?or?D986,?as?well?as?a?mutation?in?H840?or?N863.? In?some?instances,?the?dCas9? enzyme?contains?a?mutation?D10A?or?DION.? Additionally,?the?dCas9? enzyme?alternatively?includes?a?mutation?H840A,?H840Y?or?H840N.?In?some?embodiments,?the?dCas9? enzyme?

? ?

della?presente?invenzione?comprende?D10A?e?H840A;?D10A?e?H840Y;?D10A?e?H840N;?DION?e?H840A;?DION? e?H840Y;?o?sostituzioni?DION?e?H840N.?Le?sostituzioni?sono?in?alternativa?sostituzioni?conservative?o?non? conservative?per?rendere?il?polipeptide?Cas9?cataliticamente?inattivo?e?in?grado?di?legarsi?al?DNA?bersaglio.? Per? i?metodi?di?modifica?del?genoma,? la?nucleasi?Cas? in?alcune? forme?di?realizzazione?comprende?una? proteina?di?fusione?Cas9?come?un?polipeptide?comprendente?il?dominio?catalitico?dell'enzima?di?restrizione? di?tipo?IIS,?Fokl,?legato?a?dCas9.?La?proteina?di?fusione?FokI?dCas9?(fCas9)?pu??utilizzare?due?RNA?guida?per? legarsi?a?un?singolo?filamento?di?DNA?bersaglio?per?generare?una?rottura?a?doppio?filamento.? of the present invention comprises D10A and H840A; D10A and H840Y; D10A and H840N; DION and H840A; DION and H840Y; or DION and H840N substitutions. The substitutions are either conservative or non-conservative substitutions to render the Cas9 polypeptide catalytically inactive and capable of binding to the target DNA. For genome editing methods, the Cas nuclease in some embodiments comprises a Cas9?fusion?protein?as?a?polypeptide?comprising?the?catalytic?domain?of?the?type?IIS?restriction?enzyme,?FokI,?linked?to?dCas9.?The?FokI?dCas9?(fCas9)?fusion?protein?can?use?two?guide?RNAs?to?bind?to?a?target?single?strand?of?DNA?to?generate?a?double-strand?break.?

Sequenze?bersaglio? Sequences? target?

Le?sequenze?bersaglio?nel?presente?documento?sono?sequenze?di?acido?nucleico?riconosciute?e?tagliate?da? una?nucleasi.?In?alcune?forme?di?realizzazione,?la?sequenza?bersaglio???lunga?da?circa?9?a?circa?12?nucleotidi,? da?circa?12?a?circa?18?nucleotidi?di?lunghezza,?da?circa?18?a?circa?21?nucleotidi?di?lunghezza,?da?circa?21?a? circa?40?nucleotidi?di?lunghezza,?da?circa?40?a?circa?80?nucleotidi?di?lunghezza,?o?qualsiasi?combinazione?di? sottointervalli? (ad? esempio,? 9?18,? 9?21,? 9?40? e? 9?80? nucleotidi).? In? alcune? forme? di? realizzazione,? la? sequenza? bersaglio? comprende? un? sito? di? legame? della? nucleasi.? In? alcune? forme? di? realizzazione,? la? sequenza? bersaglio? comprende? un? sito? di? nick/taglio.? In? alcune? forme? di? realizzazione,? la? sequenza? bersaglio? comprende? una? sequenza? di?motivo? adiacente? al? protospacer? (PAM).? In? alcune? forme? di? realizzazione,?la?sequenza?di?acido?nucleico?bersaglio?(ad?esempio?protospacer)???di?20?nucleotidi.?In?alcune? forme? di? realizzazione,? l'acido? nucleico? bersaglio? ?? inferiore? a? 20? nucleotidi.? In? alcune? forme? di? realizzazione,?l'acido?nucleico?bersaglio???di?almeno?5,?10,?15,?16,?17,?18,?19,?20,?21,?22,?23,?24,?25,?30?o?pi?? nucleotidi.?L'acido?nucleico?bersaglio,?in?alcune?forme?di?realizzazione,???al?massimo?5,?10,?15,?16,?17,?18,? 19,?20,?21,?22,?23,?24,?25,?30?o?pi??nucleotidi.?In?alcune?forme?di?realizzazione,?la?sequenza?di?acido?nucleico? bersaglio???16,?17,?18,?19,?20,?21,?22?o?23?basi?immediatamente?5'?del?primo?nucleotide?del?PAM.?In?alcune? forme?di? realizzazione,? la? sequenza?di?acido?nucleico?bersaglio???16,?17,?18,?19,?20,?21,?22?o?23?basi? immediatamente?3'?dell'ultimo?nucleotide?del?PAM.?In?alcune?forme?di?realizzazione,?la?sequenza?di?acido? nucleico?bersaglio???di?20?basi? immediatamente?5'?del?primo?nucleotide?del?PAM.? In?alcune? forme?di? realizzazione,?la?sequenza?di?acido?nucleico?bersaglio???di?20?basi?immediatamente?3'?dell'ultimo?nucleotide? del?PAM.?In?alcune?forme?di?realizzazione,?la?sequenza?di?acido?nucleico?bersaglio???5'?o?3'?del?PAM.?Una? sequenza?bersaglio,?in?alcune?forme?di?realizzazione?include?sequenze?di?acido?nucleico?presenti?in?un?acido? nucleico? bersaglio? a? cui? si? lega? un? segmento? che? bersaglia? un? acido? nucleico? di? un? filamento? complementare?di?un?acido?nucleico..?Ad?esempio,?le?sequenze?bersaglio,?in?alcune?forme?di?realizzazione,? includono?sequenze?per? le?quali?un?acido?nucleico?a?filamento?complementare???progettato?per?avere? appaiamento?di?basi.?Le?sequenze?bersaglio?includono?i?siti?di?taglio?per?le?nucleasi.?Una?sequenza?bersaglio,? The target sequences herein are recognized nucleic acid sequences cleaved by a nuclease. In some embodiments, the target sequence is about 9 to about 12 nucleotides in length, about 12 to about 18 nucleotides in length, about 18 to about 21 nucleotides in length, about 21 to about 40 nucleotides in length, about 40 to about 80 nucleotides in length, or any combination of subranges. (e.g., 9-18, 9-21, 9-40, and 9-80 nucleotides). In some embodiments, the target sequence includes a nuclease binding site. In some embodiments, the target sequence includes a nick/cleavage site. In some embodiments, the target sequence includes a protospacer motif (PAM) sequence. In some embodiments, the target nucleic acid sequence (e.g., protospacer) is 20 nucleotides. In some embodiments, the target nucleic acid is less than 20 nucleotides. In some embodiments, the embodiment,?the target?nucleic?acid?of?at?least?5,?10,?15,?16,?17,?18,?19,?20,?21,?22,?23,?24,?25,?30?or?more?nucleotides.?The?target?nucleic?acid,?in?some?embodiments,?at?most?5,?10,?15,?16,?17,?18,? 19,?20,?21,?22,?23,?24,?25,?30?or?more?nucleotides.?In?some?embodiments,?the?sequence?of?the?nucleic?acid? target???16,?17,?18,?19,?20,?21,?22?or?23?bases?immediately?5'?of?the?first?nucleotide?of?the?PAM.?In?some?embodiments,?the?target?nucleic?acid?sequence???16,?17,?18,?19,?20,?21,?22?or?23?bases?immediately?3'?of?the?last?nucleotide?of?the?PAM.?In?some?embodiments,?the?target?nucleic?acid?sequence???20?bases?immediately?5'?of?the?first?nucleotide?of?the?PAM.? In?some?embodiments,?the?target?nucleic?acid?sequence???20?bases?immediately?5'?of?the?first?nucleotide?of?the?PAM.? embodiment,?the?target?nucleic?acid?sequence?of?20?bases?immediately?3'?of?the?last?nucleotide?of?the?PAM.?In?some?embodiments,?the?target?nucleic?acid?sequence?is?5'?or?3'?of?the?PAM.?A?target?sequence,?in?some?embodiments,?includes?nucleic?acid?sequences?present?in?a?target?nucleic?acid?to?which?a?segment?that?targets?a?nucleic?acid?of?a?complementary?strand?of?a?nucleic?acid.?For?example,?target?sequences,?in?some?embodiments,?include?sequences?for? which?is?a?complementary?stranded?nucleic?acid?designed?to?have?base?pairing.?Target?sequences?include?the?cleavage?sites?for?nucleases.?A?target?sequence,?

? ?

in?alcune?forme?di?realizzazione,???adiacente?ai?siti?di?taglio?per?le?nucleasi.?La?nucleasi?taglia?l'acido?nucleico,? in?alcune?forme?di?realizzazione,?in?un?sito?all'interno?o?all'esterno?della?sequenza?di?acido?nucleico?presente? nell'acido? nucleico? bersaglio? a? cui? si? lega? la? sequenza? bersaglio? di? acido? nucleico? del? filamento? complementare.?Il?sito?di?taglio,?in?alcune?forme?di?realizzazione,?include?la?posizione?di?un?acido?nucleico? in?corrispondenza?del?quale?una?nucleasi?produce?una?rottura?a?singolo?filamento?o?una?rottura?a?doppio? filamento.?Ad?esempio,? la? formazione?di?un?complesso?nucleasico?comprendente?un?acido?nucleico?a? filamento?complementare?ibridato?con?una?sequenza?di?riconoscimento?di?proteasi?e?complessato?con?una? proteasi?determina?il?taglio?di?uno?o?entrambi?i?filamenti?all'interno?o?in?prossimit??(ad?esempio,?entro?1,? 2,?3,?4,?5,?6,?7,?8,?9,?10,?19,?20,?23,?50?o?pi??paia?di?basi?dalla)?della?sequenza?di?acido?nucleico?presente?in? un? acido? nucleico? bersaglio? a? cui? si? lega? una? regione? spaziatrice? di? un? acido? nucleico? a? filamento? complementare.?Il?sito?di?scissione,? in?alcune?forme?di?realizzazione,?si?trova?solo?su?un?filamento?o?su? entrambi?i?filamenti?di?un?acido?nucleico.?In?alcune?forme?di?realizzazione,?i?siti?di?taglio?si?trovano?nella? stessa?posizione?su?entrambi?i?filamenti?dell'acido?nucleico?(producendo?estremit??smussate)?o?si?trovano? in? siti?diversi? su? ciascun? filamento? (producendo?estremit?? sfalsate).? Il? taglio? sito?specifico?di?un?acido? nucleico? bersaglio? da? parte? di? una? nucleasi,? in? alcune? forme? di? realizzazione,? si? verifica? in? posizioni? determinate? dalla? complementarit?? di? appaiamento? di? basi? tra? l'acido? nucleico? del? filamento? complementare?e?l'acido?nucleico?bersaglio.?Il?taglio?sito?specifico?di?un?acido?nucleico?bersaglio?da?parte? di?una?proteina?nucleasi,?in?alcune?forme?di?realizzazione,?si?verifica?in?posizioni?determinate?da?un?breve? motivo,?chiamato?motivo?adiacente?al?protospacer?(PAM),?nell'acido?nucleico?bersaglio.?Ad?esempio,? il? PAM? fiancheggia? la? sequenza? di? riconoscimento? della? nucleasi? all'estremit?? 3'? della? sequenza? di? riconoscimento.? In? alcuni? casi,? il? taglio? produce? estremit?? smussate.? In? alcuni? casi,? il? taglio? produce? estremit??sfalsate?o?appiccicose?(sticky)?con?sporgenze?nell?estremit??5'.?In?alcuni?casi,?il?taglio?produce? estremit??sfalsate?o?appiccicose?(sticky)?con?sporgenze?nell?estremit???3'.?Gli?ortologhi?di?varie?proteine? nucleasi? utilizzano? diverse? sequenze? PAM.? Ad? esempio? diverse? proteine? Cas,? in? alcune? forme? di? realizzazione,?riconoscono?diverse?sequenze?PAM.?Ad?esempio,?in?S.?pyogenes,?il?PAM???una?sequenza? nell'acido?nucleico?bersaglio?che?comprende? la?sequenza?5'?XRR?3',?dove?R???A?o?G,?dove?X???qualsiasi? nucleotide?e?X???immediatamente?3'?della?sequenza?bersaglio?dell'acido?nucleico?bersaglio?della?sequenza? spaziatrice.?La?sequenza?PAM?di?S.?pyogenes?Cas9?(SpyCas9)???5'?XGG?3',?dove?X???qualsiasi?nucleotide?di? DNA? ed? ?? immediatamente? 3'? della? sequenza? di? riconoscimento? della? nucleasi? del? filamento? non? complementare?del?DNA?bersaglio.?Il?PAM?di?Cpfl???5'?TTX?3',?dove?X???qualsiasi?nucleotide?di?DNA?ed??? immediatamente? 5'? della? sequenza? di? riconoscimento? della? nucleasi.? Preferibilmente,? il? complesso? Cas9/sgRNA?introduce?DSB?3?paia?di?basi?a?monte?della?sequenza?PAM?nella?sequenza?bersaglio?genomica,? in?some?embodiments,?adjacent?to?the?nuclease?cutting?sites.?The?nuclease?cuts?the?nucleic?acid,?in?some?embodiments,?at?a?site?within?or?outside?the?nucleic?acid?sequence?in?the?target?nucleic?acid?sequence?of?the?complementary?strand?binds.?The?cutting?site,?in?some?embodiments,?includes?the?location?on?a?nucleic?acid?at?which?a?nuclease?produces?a?single-strand?break?or?a?double-strand?break.?For?example,?the? formation?of?a?nuclease?complex?comprising?a?complementary?stranded?nucleic?acid?hybridized?within?a?protease?recognition?sequence?and?complexed?within?a?protease?results?in?cleavage?of?one?or?both?strands?within?or?close?to?(e.g.,?within?1,?2,?3,?4,?5,?6,?7,?8,?9,?10,?19,?20,?23,?50?or?more?base?pairs?of)?the?nucleic?acid?sequence?present?in?a?target?nucleic?acid?to?which?a?spacer?region?of?a?stranded?nucleic?acid?binds complementary.?The?cleavage?site,?in?some?embodiments,?is?found?on?only?one?strand?or?both?strands?of?a?nucleic?acid.?In?some?embodiments,?the?cleavage?sites?are?found?at?the?same?location?on?both?strands?of?the?nucleic?acid?(producing?blunt?ends)?or?are?found?at?different?sites?on?each?strand?(producing?staggered?ends).?Site-specific?cleavage?of?a?target?nucleic?acid?by?a?nuclease,?in?some?embodiments,?occurs?at?positions?determined?by?complementary?base?pairing?between?the?acid?and? nucleic acid of the complementary strand and the target nucleic acid. Site-specific cleavage of a target nucleic acid by a protein nuclease, in some embodiments, occurs at positions determined by a short motif, called a protospacer adjacent motif (PAM), in the target nucleic acid. For example, the PAM flanks the nuclease recognition sequence at the 3' end of the recognition sequence. In some cases, the cleavage produces blunt ends. In some cases, the cleavage produces staggered? or?sticky? ends? with? protrusions? at? the? 5' end.? In? some? cases,? the? cleavage? produces? staggered? or? sticky? ends? with? protrusions? at? the? 3' end.? Orthologs? of? various? nuclease? proteins? utilize? different? PAM? sequences.? For? example? different? Cas? proteins,? in? some? embodiments,? recognize? different? PAM? sequences.? For? example,? in? S.? pyogenes,? the? PAM? is? a? sequence? in? the? target? nucleic acid? that? includes? the? 5'? sequence? XRR? 3',? where? R??? A? or? G,? where? X??? any? nucleotide?and?X???immediately?3'?of?the?target?sequence?of?the?nucleic?acid?target?spacer?sequence.?The?PAM?sequence?of?S.?pyogenes?Cas9?(SpyCas9)?5'?XGG?3',?where?X???is?any?DNA?nucleotide?and?is?immediately?3'?of?the?nuclease?recognition?sequence?of?the?non-complementary?strand?of?the?target?DNA?target.?The?PAM?of?Cpfl?5'?TTX?3',?where?X???is?any?DNA?nucleotide?and?is?immediately?5'?of?the?nuclease?recognition?sequence.? Preferably,?the?complex? Cas9/sgRNA?introduces?3?base?pair?DSB?upstream?of?the?PAM?sequence?in?the?genomic?target?sequence,?

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risultando?in?due?estremit??smussate.?La?stessa?identica?sequenza?target?Cas9/sgRNA?viene?caricata?sul? DNA?del?donatore?nella?direzione?opposta.? I? loci?genomici?bersagliati,?cos??come? il?DNA?del?donatore,? vengono?tagliati?da?Cas9/gRNA?e?i?DNA?del?donatore?linearizzati?vengono?integrati?nei?siti?bersaglio?tramite? la?via?di?riparazione?DSB?NHEJ.?Se?il?DNA?del?donatore???integrato?nell'orientamento?corretto,?le?sequenze? di?giunzione?sono?protette?da?un'ulteriore?clivaggio?da?parte?da?Cas9/gRNA.?Se? il?DNA?del?donatore?si? integra?nell'orientamento?inverso,?Cas9/gRNA?elimina?il?DNA?del?donatore?integrato?a?causa?della?presenza? di?siti?bersaglio?Cas9/gRNA?intatti.? resulting in two blunt ends. The exact same Cas9/sgRNA target sequence is loaded onto the donor DNA in the opposite direction. The targeted genomic loci, as well as the donor DNA, are cut by Cas9/gRNA and the linearized donor DNA is integrated into the target sites via the NHEJ DSB repair pathway. If the donor DNA is integrated in the correct orientation, the junction sequences are protected from further cleavage by Cas9/gRNA. If the donor DNA is integrates?in?the?reverse?orientation,?Cas9/gRNA?eliminates?the?integrated?donor?DNA?due?to?the?presence?of?intact?Cas9/gRNA?target?sites.?

VETTORI? VECTORS?

La?presente?invenzione?riguarda?anche?un?vettore?che?comprende?l'acido?nucleico?o?il?costrutto?di?acido? nucleico?qui?descritto.?? The present invention also relates to a vector comprising the nucleic acid or nucleic acid construct described herein.

Tale? vettore? pu?? quindi? contenere? uno? qualsiasi? degli? elementi? sopra? descritti.? In? particolare,? pu?? contenere,?oltre?all'acido?nucleico?che?codifica?per?GLA,?uno?o?pi??elementi?regolatori,?tra?cui,?ad?esempio,? promotori,? sequenze? di? terminazione? della? trascrizione,? sequenze? di? terminazione? della? traduzione,? enhancer,?peptidi?di?segnale,?segnali?di?degradazione?ed?elementi?di?poliadenilazione,?in?particolare?come? sopra?definiti.?Vettori?adatti?per?la?somministrazione?e?l'espressione?di?acidi?nucleici?in?cellule?per?la?terapia? genica?sono?compresi?nella?presente?invenzione.? Such? vector? may? therefore? contain? any? of? the? elements? described? above.? In? particular,? it? may? contain,? in? addition? to? the? nucleic? acid? encoding? GLA,? one? or? more? regulatory? elements,? including,? for? example,? promoters,? transcription? termination? sequences,? translation? termination? sequences,? enhancers,? signal? peptides,? degradation? signals? and? polyadenylation? elements,? in? particular? as? defined? above.? Suitable? vectors? for? the? delivery? and? expression? of? nucleic? acids? in? cells? for? gene? therapy? are? included? in? the? present? invention.?

I?vettori?dell'invenzione?includono?vettori?virali?e?non?virali.? The vectors of the invention include viral and non-viral vectors.

I?vettori?non?virali? includono?agenti?non?virali?comunemente?usati?per? introdurre?o?mantenere? l'acido? nucleico?nelle?cellule.?Detti?agenti?includono?in?particolare?veicoli?di?rilascio?a?base?di?polimeri,?particelle,? lipidi,?peptidi?o?loro?combinazioni,?come?polimeri?cationici,?micelle,?liposomi,?esosomi,?microparticelle?e? nanoparticelle?comprese?le?nanoparticelle?lipidiche?(LNP).? Non-viral carriers include non-viral agents commonly used to deliver or maintain nucleic acid in cells. Such agents include in particular delivery vehicles based on polymers, particles, lipids, peptides or combinations thereof, such as cationic polymers, micelles, liposomes, exosomes, microparticles and nanoparticles, including lipid nanoparticles (LNPs).

Tra?i?vettori?virali,?i?virus?geneticamente?modificati,?inclusi?i?virus?adeno?associati,?sono?attualmente?tra?gli? strumenti?pi??popolari?per? la?veicolazione?dei?geni.? Il? concetto?di?veicolazione?genica?basata? su?virus? consiste?nell'ingegnerizzare?il?virus?in?modo?che?possa?esprimere?il?gene?o?i?geni?di?interesse?o?le?sequenze? regolatrici?come?promotori?e?introni.?A?seconda?dell'applicazione?specifica?e?del?tipo?di?virus,?la?maggior? parte?dei?vettori?virali?contiene?mutazioni?che?ostacolano?la?loro?capacit??di?replicarsi?liberamente?come? virus?wild?type?nell'ospite.?I?virus?di?diverse?famiglie?sono?stati?modificati?per?generare?vettori?virali?per?la? somministrazione?di?geni.?Questi?virus?includono?retrovirus,?lentivirus,?adenovirus,?virus?adeno?associati,? herpes?virus,?baculovirus,?picornavirus?e?alfavirus.? Among viral vectors, genetically engineered viruses, including adeno-associated viruses, are currently among the most popular tools for gene delivery. The concept of virus-based gene delivery involves engineering the virus to express the gene or genes of interest or regulatory sequences such as promoters and introns. Depending on the specific application and the type of virus, most viral vectors contain mutations that hinder their ability to replicate freely as normal. wild-type?viruses?in?the?host.?Viruses?from?several?families?have?been?modified?to?generate?viral?vectors?for?gene?delivery.?These?viruses?include?retroviruses,?lentiviruses,?adenoviruses,?adeno?associated?viruses,?herpes?viruses,?baculoviruses,?picornaviruses?and?alphaviruses.?

I?vettori?virali?dell'invenzione?possono?essere?derivati?da?parvovirus?non?patogeni? come?virus?adeno? The viral vectors of the invention may be derived from non-pathogenic parvoviruses such as adenoviruses.

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associato? (AAV),?retrovirus?come?gammaretrovirus,?spumavirus?e? lentivirus,?adenovirus,?poxvirus?e?un? virus?dell'herpes.? associated? (AAV),?retroviruses?such as?gammaretrovirus,?spumavirus?and? lentivirus,?adenovirus,?poxvirus?and?a?herpes?virus.?

Virus?particolarmente?preferiti?secondo?la?presente?invenzione?sono?virus?adeno?associati.? Particularly preferred viruses according to the present invention are adeno-associated viruses.

I?vettori?virali?sono?per?loro?natura?in?grado?di?penetrare?all'interno?delle?cellule?e?di?veicolare?acidi?nucleici? di?interesse?nelle?cellule,?secondo?un?processo?noto?come?trasduzione?virale.? Viral vectors are by their very nature capable of penetrating cells and delivering nucleic acids of interest into cells, through a process known as viral transduction.

Come? usato? qui,? il? termine? "vettore? virale"? si? riferisce? a? un? virus? non? replicante? e? non? patogeno? ingegnerizzato? per? la? veicolazione? di?materiale? genetico? nelle? cellule.? I? geni? virali? essenziali? per? la? replicazione?e? la?virulenza?sono?sostituiti?con?una?cassetta?di?espressione?per? il?transgene?d?interesse.? Pertanto,?il?genoma?del?vettore?virale?comprende?la?cassetta?di?espressione?del?transgene?affiancata?dalle? sequenze?virali?necessarie?per?la?produzione?del?vettore?virale.? As? used? here,? the? term? "viral? vector"? refers? to? a? non-replicating? and? non-pathogenic? virus? engineered? for? the? delivery? of? genetic? material? into? cells.? Viral? genes? essential? for? replication? and? virulence? are? replaced? with? an? expression? cassette? for? the? transgene? of? interest.? Thus,? the? genome? of? the? viral? vector? comprises? the? transgene? expression? cassette? flanked? by? the? viral? sequences? required? for? production? of? the? viral? vector.?

Con?il?termine?"particella?di?virus"?o?"particella?virale"?si?intende?la?forma?extracellulare?di?un?virus?non? patogeno,? in?particolare?un?vettore?virale,?composto?da?materiale?genetico? costituito?da?DNA?o?RNA? circondato?da?un?mantello?proteico,?chiamato?capside,?e?in?alcuni?casi?da?un?involucro?derivato?da?porzioni? di?membrane?di?cellule?ospiti?e?comprendente?glicoproteine?virali.?? The term "virus particle" or "viral particle" refers to the extracellular form of a non-pathogenic virus, in particular a viral vector, composed of genetic material consisting of DNA or RNA, surrounded by a protein coat called the capsid and, in some cases, an envelope derived from portions of host cell membranes and comprising viral glycoproteins.

Come?qui?utilizzato,?un?vettore?virale?si?riferisce?anche?a?una?particella?di?vettore?virale.?? As used here, a viral vector also refers to a viral vector particle.

I?vettori?virali?compresi?nella?presente?invenzione?sono?adatti?alla?terapia?genica.?? The viral vectors included in the present invention are suitable for gene therapy.

Le?particelle?virali?possono?essere?ottenute,?ad?esempio,?utilizzando?vettori?in?grado?di?ospitare?i?geni?di? interesse?e?cellule?helper? in?grado?di? fornire? le?proteine?strutturali?virali?e?gli?enzimi?per?consentire? la? generazione?di?particelle?virali?infettive?contenenti?il?vettore.? Viral particles can be obtained, for example, by using vectors that can host the genes of interest and helper cells that can provide the viral structural proteins and enzymes to enable the generation of infectious viral particles containing the vector.

Virus?adeno?associati?(AAV)?? Adeno?associated?viruses?(AAV)??

Il?virus?adeno?associato???una?famiglia?di?virus?che?differisce?per?sequenza?nucleotidica?e?amminoacidica,? struttura?del?genoma,?patogenicit??e?gamma?di?ospiti.?Questa?diversit??offre?l'opportunit??di?utilizzare?virus? con?caratteristiche?biologiche?diverse?per?sviluppare?diverse?applicazioni?terapeutiche.?? Adeno-associated virus is a family of viruses that differ in nucleotide and amino acid sequence, genome structure, pathogenicity, and host range. This diversity provides the opportunity to use viruses with different biological characteristics to develop different therapeutic applications.

Un? vettore? ideale? basato? su? virus? adeno?associati? per? la? veicolazione? di? geni? deve? essere? efficiente,? specifico?per?le?cellule,?regolato?e?sicuro.?L'efficienza?della?veicolazione?pu??determinare?l'efficacia?della? terapia.?Gli?sforzi?attuali?mirano?a?ottenere?un'infezione?e?un'espressione?genica?specifiche?per?ogni?tipo?di? cellula?con?vettori?virali?adeno?associati.?Inoltre,?si?stanno?sviluppando?vettori?virali?adeno?associati?per? regolare? l'espressione?del?gene?di? interesse,?poich?? la? terapia?pu?? richiedere?un'espressione?di? lunga? durata?o?regolata.?? An ideal adeno-associated virus (AAV)-based vector for gene delivery must be efficient, cell-specific, regulated, and safe. Delivery efficiency can determine the efficacy of therapy. Current efforts are aimed at achieving cell-type-specific infection and gene expression with AAV vectors. Additionally, AAV vectors are being developed to regulate gene expression, since therapy may require long-lasting or regulated expression.

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Il?virus?adeno?associato?(AAV)???un?piccolo?virus?che?infetta?l'uomo?e?alcune?specie?di?primati.?Attualmente? non???noto?che?l'AAV?causi?malattie?e?di?conseguenza?il?virus?provoca?una?risposta?immunitaria?molto?lieve.? I?vettori?per?la?terapia?genica?che?utilizzano?l'AAV?possono?infettare?sia?le?cellule?in?divisione?che?quelle? quiescenti?e?persistere?in?uno?stato?extracromosomico?senza?integrarsi?nel?genoma?della?cellula?ospite.? Queste?caratteristiche?rendono?l'AAV?un?candidato?molto?attraente?per?la?creazione?di?vettori?virali?per?la? terapia?genica?e?per?la?creazione?di?modelli?isogenici?di?malattie?umane.?? Adeno-associated virus (AAV) is a small virus that infects humans and some primate species. Currently, AAV is not known to cause disease, and as a result, the virus elicits a very mild immune response. Gene therapy vectors that use AAV can infect both dividing and quiescent cells and persist in an extrachromosomal state without integrating into the host cell's genome. These characteristics make AAV a very attractive candidate for the creation of viral vectors for gene therapy and for the creation of isogenic models of human diseases.

L'AAV?wild?type?ha?attirato?un?notevole?interesse?da?parte?dei?ricercatori?di?terapia?genica?grazie?a?una? serie?di?caratteristiche.?La?principale???l'apparente?mancanza?di?patogenicit??del?virus.?Inoltre,?pu??infettare? cellule?non?in?divisione?e?ha?la?capacit??di?integrarsi?stabilmente?nel?genoma?della?cellula?ospite?in?un?sito? specifico?(designato?AAVS1)?nel?cromosoma?umano?19.?Lo?sviluppo?degli?AAV?come?vettori?per?la?terapia? genica,?tuttavia,?ha?eliminato?questa?capacit??integrativa?rimuovendo?i?geni?rep?e?cap?dal?DNA?del?vettore.? Il?gene?desiderato,? insieme?a?un?promotore?che?ne?guida? la? trascrizione,?viene? inserito? tra?gli? ITR?che? favoriscono?la?formazione?di?concatameri?nel?nucleo?dopo?che?il?DNA?del?vettore?a?singolo?filamento?viene? convertito?dai?complessi?di?DNA?polimerasi?della?cellula?ospite?in?DNA?a?doppio?filamento.?I?vettori?per?la? terapia?genica?basati?su?AAV?formano?concatameri?episomali?nel?nucleo?della?cellula?ospite.?Nelle?cellule? non?in?divisione,?questi?concatenamenti?rimangono?intatti?per?tutta?la?vita?della?cellula?ospite.?Nelle?cellule? in?divisione,?il?DNA?dell?AAV?viene?perso?durante?la?divisione?cellulare,?poich??il?DNA?episomale?non?viene? replicato? insieme? al? DNA? della? cellula? ospite.? L'integrazione? casuale? del? DNA? dell?AAV? nel? genoma? dell'ospite? ?? rilevabile,? ma? avviene? con? una? frequenza? molto? bassa.? Gli? AAV? presentano? anche? un'immunogenicit??molto?bassa,?apparentemente? limitata?alla?generazione?di?anticorpi?neutralizzanti,? mentre?non? inducono?una?risposta?citotossica?chiaramente?definita.?Questa?caratteristica,? insieme?alla? capacit??di?infettare?cellule?quiescenti,?rende?gli?AAV?particolarmente?adatti?alla?terapia?genica?umana.? Wild-type AAV has attracted considerable interest from gene therapy researchers because of a number of features. Chief among them is the virus's apparent lack of pathogenicity. Additionally, it can infect non-dividing cells and has the ability to stably integrate into the host cell's genome at a specific site (designated AAVS1) on the human chromosome. 19 However, the development of AAVs as gene therapy vectors has eliminated this integrative ability by removing the rep and cap genes from the vector's DNA. The desired gene, along with a promoter that drives its transcription, is inserted between ITRs that promote the formation of concatamers in the nucleus after the single-stranded vector DNA is converted into double-stranded DNA by host cell DNA polymerase complexes. AAV-based gene therapy vectors form episomal concatamers in the host cell nucleus. In non-dividing cells, these concatamers remain intact for the life of the host cell. In non-dividing cells, these concatamers are maintained throughout the life of the host cell. in?division,?AAV?DNA?is?lost?during?cell?division,?because?episomal?DNA?is?not?replicated?with?host?cell?DNA.? Random?integration?of?AAV?DNA?into?the?host?genome?is?detectable,?but?occurs?at?a?very?low?frequency.?AAVs?also?display?a?very?low?immunogenicity,?apparently?limited?to?the?generation?of?neutralizing?antibodies,?while?they?do?not?induce?a?clearly?defined?cytotoxic?response.?This?feature,?together?with?the?ability?to?infect?quiescent?cells,?makes?AAVs?particularly?suitable?for?human?gene?therapy.?

Il?genoma?dell'AAV???costituito?da?acido?desossiribonucleico?a?singolo?filamento?(ssDNA),?a?orientamento? positivo?o?negativo,? lungo?circa?4,7?kilobasi.?Il?genoma?comprende?ripetizioni?terminali? invertite?(ITR)?a? entrambe? le?estremit??del? filamento?di?DNA?e?due?open? reading? frames? (ORF):? rep?e?cap.? Il?primo??? composto?da?quattro?geni?sovrapposti?che?codificano?le?proteine?Rep?necessarie?per?il?ciclo?vitale?dell'AAV,? mentre?il?secondo?contiene?sequenze?nucleotidiche?sovrapposte?di?proteine?del?capside:?VP1,?VP2?e?VP3,? che?interagiscono?tra?loro?per?formare?un?capside?a?simmetria?icosaedrica.?? The AAV genome is composed of single-stranded deoxyribonucleic acid (ssDNA), either in positive or negative orientation, approximately 4.7 kilobases long. The genome includes inverted terminal repeats (ITRs) at both ends of the DNA strand and two open reading frames (ORFs): rep and cap. The former is composed of four overlapping genes encoding the Rep proteins required for the AAV life cycle, while the latter contains overlapping nucleotide sequences of capsid proteins: VP1, VP2, and VP3, which interact with each other to form a capsid with icosahedral symmetry.

Le?sequenze?Inverted?Terminal?Repeat?(ITR)?hanno?ricevuto?il?loro?nome?a?causa?della?loro?simmetria,?che? ??stata?dimostrata?essere?necessaria?per?un'efficiente?moltiplicazione?del?genoma?AAV.?Un'altra?propriet?? di?queste?sequenze???la?loro?capacit??di?formare?una?forcina,?che?contribuisce?al?cosiddetto?self?priming? Inverted Terminal Repeat (ITR) sequences received their name because of their symmetry, which has been shown to be necessary for efficient multiplication of the AAV genome. Another property of these sequences is their ability to form a hairpin, which contributes to so-called self-priming.

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che?consente?la?sintesi?indipendente?dalla?primasi?del?secondo?filamento?di?DNA.???stato?inoltre?dimostrato? che? le? ITR? sono?necessarie?per?un'efficiente? incapsidazione?del?DNA?dell?AAV?e?per? la?generazione?di? particelle?AAV?completamente?assemblate?e?resistenti?alla?desossiribonucleasi.?? which?allows?primase-independent?synthesis?of?the?second?strand?DNA.?It?has?also?been?demonstrated?that?ITRs?are?required?for?efficient?encapsidation?of?AAV?DNA?and?for?the?generation?of?fully?assembled?and?deoxyribonuclease-resistant?AAV?particles.??

Per?quanto?riguarda?la?terapia?genica,?le?ITR?sembrano?essere?le?uniche?sequenze?richieste?in?cis?accanto? al?gene?terapeutico:?i?geni?strutturali?(cap)?e?di?packaging?(rep)?possono?essere?veicolati?in?trans.?Partendo? da?questo?presupposto,?sono?stati?messi?a?punto?molti?metodi?per?la?produzione?efficiente?di?vettori?AAV? ricombinanti?(rAAV)?contenenti?un?gene?reporter?o?terapeutico.?? With regard to gene therapy, ITRs appear to be the only sequences required in cis next to the therapeutic gene: structural (cap) and packaging (rep) genes can be delivered in trans. Based on this assumption, several methods have been developed for the efficient production of recombinant AAV vectors (rAAV) containing a therapeutic reporter gene.

Il?vettore?AAV?comprende?un?capside?AAV?in?grado?di?trasdurre?le?cellule?bersaglio?d?interesse.?Il?capside? AAV?pu??appartenere?a?uno?o?pi??sierotipi?AAV?naturali?o?artificiali.?? The AAV vector comprises an AAV capsid capable of transducing the target cells of interest. The AAV capsid may belong to one or more natural or artificial AAV serotypes.

Gli?AAV?possono?essere?indicati?in?termini?di?sierotipo.?Un?sierotipo?corrisponde?a?una?sottospecie?variante? di?AAV?che,?a?causa?del?suo?profilo?di?espressione?degli?antigeni?di?superficie?del?capside,?ha?una?reattivit?? distintiva?che?pu??essere?utilizzata?per?distinguerla?da?altre?sottospecie?varianti.?In?genere,?una?particella? di?vettore?AAV?con?un?particolare?sierotipo?AAV?non?reagisce?efficacemente?con?anticorpi?neutralizzanti? specifici?per?qualsiasi?altro?sierotipo?AAV.?? AAVs can be designated in terms of a serotype. A serotype corresponds to a variant AAV subtype that, due to its capsid surface antigen expression profile, has a distinctive reactivity that can be used to distinguish it from other variant AAV subtypes. Typically, an AAV vector particle with a particular AAV serotype will not react effectively with neutralizing antibodies specific for any other AAV serotype.

Tutti? i? sierotipi? conosciuti?possono? infettare? cellule?di?diversi? tipi?di? tessuto.? La? specificit?? tissutale? ?? determinata?dal?sierotipo?del?capside?e? la?pseudotipizzazione?dei?vettori?AAV?per?alterarne? il?tropismo? influisce?sul?loro?uso?in?terapia.?? All? known? serotypes? can? infect? cells? of? different? tissue? types.? Tissue? specificity? is? determined? by? the? capsid? serotype? and? pseudotyping? of? AAV? vectors? to? alter? their? tropism? influences? their? use? in? therapy.?

Le?sequenze?di?ripetizione?terminale?invertite?(ITR)?utilizzate?in?un?sistema?di?vettori?AAV?della?presente? invenzione?possono?essere?ITR?di?qualsiasi?AAV.?Gli?ITR?utilizzati?in?un?vettore?AAV?possono?essere?uguali?o? diversi.?Ad?esempio,?un?vettore?pu??comprendere?un?ITR?del?sierotipo?2?di?AAV?e?un?ITR?del?sierotipo?5?di? AAV.?In?una?forma?di?realizzazione?di?un?vettore?dell'invenzione,?un?ITR?appartiene?al?sierotipo?2,?4,?5?o?8? di?AAV.?Nella?presente?invenzione?sono?preferiti?gli?ITR?del?sierotipo?2?di?AAV.?Le?sequenze?ITR?di?AAV?sono? ben?note?nell'arte? (ad?esempio,?per? ITR2,? si? vedano? i?n.?di?acc.?GenBank?AF043303.1;?NC_001401.2;? J01901.1;?JN898962.1;?per?ITR5,?si?veda?il?n.?di?acc.?GenBank?NC_006152.1).? The inverted terminal repeat (ITR) sequences used in an AAV vector system of the present invention may be ITRs of any AAV. The ITRs used in an AAV vector may be the same or different. For example, a vector may comprise an ITR of AAV serotype 2 and an ITR of AAV serotype 5. In one embodiment of a vector of the invention, an ITR belongs to AAV serotype 2, 4, 5, or 8. of?AAV.?In?the?present?invention?the?ITRs?of?AAV?serotype?2?are?preferred.?The?ITR?sequences?of?AAV?are?well?known?in?the?art?(e.g.,?for?ITR2,?see?GenBank?Access?Nos.?AF043303.1;?NC_001401.2;?J01901.1;?JN898962.1;?for?ITR5,?see?GenBank?Access?No.?NC_006152.1).?

Il?sierotipo?2?(AAV2)???stato?finora? il?pi??ampiamente?esaminato.?AAV2?presenta?un?tropismo?naturale? verso?muscoli?scheletrici,?neuroni,?cellule?muscolari?lisce?vascolari?ed?epatociti.?? Serotype 2 (AAV2) has been the most extensively studied to date. AAV2 has a natural tropism for skeletal muscle, neurons, vascular smooth muscle cells, and hepatocytes.

Sono?stati?descritti?tre?recettori?cellulari?per?AAV2:?il?proteoglicano?eparan?solfato?(HSPG),?l'integrina?aV?5? e?il?recettore?del?fattore?di?crescita?dei?fibroblasti?1?(FGFR?1).?Il?primo?funziona?come?recettore?primario,? mentre?gli?ultimi?due?hanno?un'attivit??di?co?recettore?e?consentono?all'AAV?di?entrare?nella?cellula?tramite? endocitosi?mediata?dal?recettore.?L'HSPG?funziona?come?recettore?primario,?anche?se?la?sua?abbondanza? Three cellular receptors for AAV2 have been described: heparan sulfate proteoglycan (HSPG), integrin aV5, and fibroblast growth factor receptor 1 (FGFR1). The former functions as a primary receptor, while the latter two have co-receptor activity and allow AAV to enter the cell via receptor-mediated endocytosis. HSPG functions as a primary receptor, although its abundance is low.

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nella?matrice?extracellulare?pu??eliminare?le?particelle?di?AAV?e?compromettere?l'efficacia?dell'infezione.?? in the extracellular matrix can eliminate AAV particles and compromise the effectiveness of the infection.

Sebbene?l'AAV2?sia?il?sierotipo?pi??popolare?in?varie?ricerche?basate?sull'AAV,???stato?dimostrato?che?altri? sierotipi?possono?essere?efficaci?come?vettori?per?la?somministrazione?di?geni.?Ad?esempio,?l'AAV6?sembra? essere?molto?pi??efficace?nell'infettare? le?cellule?epiteliali?delle?vie?aeree,? l'AAV7?presenta?un? tasso?di? trasduzione?molto?elevato?delle?cellule?muscolari?scheletriche?murine?(analogamente?all'AAV1?e?all'AAV5),? l'AAV8???eccellente?nella?trasduzione?degli?epatociti?e?dei?fotorecettori,?mentre?l'AAV1?e?l'AAV?5?si?sono? dimostrati?molto?efficienti?nella?somministrazione?di?geni?alle?cellule?endoteliali?vascolari.?Nel?cervello,?la? maggior?parte?dei?sierotipi?AAV?mostra?un?tropismo?neuronale,?mentre?AAV5?trasduce?anche?gli?astrociti.? L'AAV6,?un?ibrido?di?AAV1?e?AAV2,?mostra?anche?una?minore?immunogenicit??rispetto?all'AAV2.?? Although AAV2 is the most popular serotype in various AAV-based studies, other serotypes have been shown to be effective as gene delivery vectors. For example, AAV6 appears to be much more effective in infecting airway epithelial cells, AAV7 has a very high transduction rate of mouse skeletal muscle cells (similar to AAV1 and AAV5), AAV8 is excellent at transducing hepatocytes and photoreceptors, while AAV1 and AAV5 have been shown to be have been shown to be highly efficient in gene delivery to vascular endothelial cells. In the brain, most AAV serotypes exhibit neuronal tropism, while AAV5 also transduces astrocytes. AAV6, a hybrid of AAV1 and AAV2, also shows lower immunogenicity than AAV2.

I?sierotipi?possono?differire?per?quanto?riguarda?i?recettori?a?cui?sono?legati.?Ad?esempio,?la?trasduzione?di? AAV4?e?AAV5?pu??essere?inibita?da?acidi?sialici?solubili?(di?forma?diversa?per?ciascuno?di?questi?sierotipi)?e? AAV5?ha?dimostrato?di?entrare?nelle?cellule?attraverso? il?recettore?del?fattore?di?crescita?derivato?dalle? piastrine.?? Serotypes may differ in the receptors to which they bind. For example, AAV4 and AAV5 transduction can be inhibited by soluble sialic acids (different in shape for each of these serotypes), and AAV5 has been shown to enter cells via the platelet-derived growth factor receptor.

Metodi?per?preparare?virus?e?virioni?contenenti?un?polinucleotide?o?un?costrutto?eterologo?sono?noti? nell'arte.?Nel?caso?dell'AAV,?le?cellule?possono?essere?coinfettate?o?trasfettate?con?adenovirus?o?costrutti? polinucleotidici?comprendenti?geni?di?adenovirus?adatti?alla?funzione?di?AAV?helper.?Esempi?di?materiali?e? metodi?sono?descritti,?ad?esempio,?nei?brevetti?USA?n.?8.137.962?e?6.967.018.?Un?virus?AAV?o?un?vettore? AAV?dell'invenzione?pu??appartenere?a?qualsiasi?sierotipo?AAV,?compresi,?ma?non? limitati?a,? i?sierotipi? AAV1,?AAV2,?AAV3,?AAV4,?AAV5,?AAV6,?AAV7,?AAV8,?AAV9,?AAV10?e?AAV11,?AAV?PhP.B?e?AAV?PhP.eB.?? Methods?for?preparing?viruses?and?virions?containing?a?polynucleotide?or?a?heterologous?construct?are?known?in?the?art.?In?the?case?of?AAV,?the?cells?may?be?co-infected?or?transfected?with?adenoviruses?or?polynucleotide?constructs?comprising?adenovirus?genes?adaptable?to?function?as?an?AAV?helper.?Examples?of?materials?and?methods?are?described?for?example?in?U.S.?Patents?Nos.?8,137,962?and?6,967,018.?An?AAV?virus?or?AAV?vector?of?the?invention?may?be?any?AAV?serotype,?including,?but?not?limited?to,? the?serotypes? AAV1,?AAV2,?AAV3,?AAV4,?AAV5,?AAV6,?AAV7,?AAV8,?AAV9,?AAV10?and?AAV11,?AAV?PhP.B?and?AAV?PhP.eB.??

In?una?specifica?forma?di?realizzazione,?viene?utilizzato?un?sierotipo?AAV2?o?AAV5?o?AAV7?o?AAV8?o?AAV9.? Di?preferenza,?si?utilizza?l'AAV8.? In?a?specific?embodiment,?an?AAV2?or?AAV5?or?AAV7?or?AAV8?or?AAV9?serotype is?used.? Preferably,?AAV8?is?used.?

In?maniera?opportuna,?il?genoma?AAV?viene?derivatizzato?allo?scopo?di?somministrarlo?ai?pazienti.?Tale? derivatizzazione? ?? standard? nell'arte? e? l'invenzione? comprende? l'uso? di? qualsiasi? derivato? noto? di? un? genoma?AAV?e?di?derivati?che?potrebbero?essere?generati?applicando?tecniche?note?nell'arte.?Il?genoma? AAV?pu??essere?un?derivato?di?qualsiasi?AAV?presente? in?natura.?Preferibilmente,? il?genoma?AAV???un? derivato?di?AAV1,?AAV2,?AAV3,?AAV4,?AAV5,?AAV6,?AAV7,?AAV8,?AAV9,?AAV10?o?AAV11.?? In?a?suitable?manner,?the?AAV?genome?is?derivatized?for?the?purpose?of?administration?to?patients.?Such? derivatization?is?standard?in?the?art?and?the?invention?comprises?the?use?of?any?known?derivative?of?an?AAV?genome?and?derivatives?that?could?be?generated?by?application?of?techniques?known?in?the?art.?The?AAV?genome?may?be?a?derivative?of?any?naturally?occurring?AAV.?Preferably,?the?AAV?genome?is?a?derivative?of?AAV1,?AAV2,?AAV3,?AAV4,?AAV5,?AAV6,?AAV7,?AAV8,?AAV9,?AAV10?or?AAV11.??

I?derivati?di?un?genoma?AAV?comprendono?forme?tronche?o?modificate?di?un?genoma?AAV?che?consentono? l'espressione?in?vivo?di?un?transgene?da?un?vettore?AAV?dell'invenzione.?In?una?forma?di?realizzazione,?il? sierotipo?AAV?prevede?una?o?pi??mutazioni?da?tirosina?a?fenilalanina?(Y?F)?sulla?superficie?del?capside.?? Derivatives of an AAV genome include truncated or modified forms of an AAV genome that allow for the in vivo expression of a transgene from an AAV vector of the invention. In one embodiment, the AAV serotype includes one or more tyrosine-to-phenylalanine (Y-F) mutations on the capsid surface.

Il?plasmide?sopra?descritto?pu??essere?utilizzato?per?generare?il?vettore?AAV?dell'invenzione.?Il?vettore?AAV? The above-described plasmid can be used to generate the AAV vector of the invention. The AAV vector

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pu??essere?prodotto,?ad?esempio,?mediante?tripla?trasfezione?di?cellule?produttrici,?come?le?cellule?HEK293,? un?metodo?noto?nel?settore?in?cui?il?plasmide?che?comprende?il?gene?d?interesse,?l'acido?nucleico?con?ID? SEQ?N.1?nel?caso?in?questione,?viene?trasfettato?insieme?a?due?plasmidi?aggiuntivi?in?una?cellula?produttrice? in?cui?verranno?poi?prodotte?le?particelle?virali.? can be produced, for example, by triple transfection of producer cells, such as HEK293 cells, a well-known method in the field whereby the plasmid carrying the gene of interest, the nucleic acid with SEQ ID No. 1 in this case, is transfected together with two additional plasmids into a producer cell in which the viral particles will then be produced.

PLASMIDE? PLASMID?

l'invenzione?prevede?anche?un?plasmide?per?la?generazione?di?un?vettore?virale?come?qui?definito.?? the invention also provides a plasmid for the generation of a viral vector as defined herein.

Il?plasmide?pu??comprendere?costrutti?di?DNA?come?sopra?descritti.?Il?plasmide?di?solito?comprende? anche?elementi?strutturali?che?sono? tipicamente?necessari?per? la?produzione?di?plasmidi?su? larga? scala?nei?batteri,?come?l'origine?di?replicazione?batterica,?il?promotore?batterico,?il?gene?di?resistenza? agli?antibiotici.?? The?plasmid?may?include?DNA?constructs?as?described?above.?The?plasmid?usually?also?includes?structural?elements?that?are?typically?required?for?large-scale?plasmid?production?in?bacteria,?such?as?the?bacterial?origin?of?replication,?the?bacterial?promoter,?the?antibiotic?resistance?gene.??

Rientra?nell'invenzione?l'uso?di?detto?plasmide?per?la?generazione?di?un?vettore?secondo?l'invenzione.? The invention includes the use of said plasmid for the generation of a vector according to the invention.

Il?vettore,?ad?esempio?un?vettore?AAV,?pu??essere?prodotto?ad?esempio?tramite?tripla?trasfezione?di? cellule? produttrici,? come? le? cellule?HEK293,? un?metodo? noto? nel? settore? in? cui? il? plasmide? che? comprende?i?costrutti?di?DNA?d?interesse?viene?trasfettato?insieme?a?due?plasmidi?aggiuntivi?in?una? cellula?produttrice?in?cui?verranno?poi?prodotte?le?particelle?virali.?Altri?metodi?noti?nell'arte?per?la? generazione?di?vettori?sono?ugualmente?adatti.? The vector, for example an AAV vector, may be produced, for example, by triple transfection of producer cells, such as HEK293 cells, a method known in the art wherein the plasmid comprising the DNA constructs of interest is transfected along with two additional plasmids into a producer cell in which the viral particles will then be produced. Other methods known in the art for generating vectors are equally suitable.

CELLULA?OSPITE? GUEST CELL?

L'invenzione?riguarda?anche?una?cellula?ospite?che?contiene? il?vettore?virale?dell'invenzione.?La?cellula? ospite?pu??essere?una?cellula?coltivata?o?una?cellula?primaria,?cio??isolata?direttamente?da?un?organismo,? ad?esempio?umano.?La?cellula?ospite?pu??essere?una?cellula?aderente?o?una?cellula?sospesa,?cio??una?cellula? che?cresce?in?sospensione.?Le?cellule?ospiti?adatte?sono?note?nell'arte?e?comprendono,?ad?esempio,?cellule? DH5?,?E.?coli,?cellule?ovariche?di?criceto?cinese,?cellule?VERO?di?scimmia,?cellule?COS,?cellule?HEK293?e?simili.? La?cellula?pu??essere?umana?o?di?un?altro?animale.?La?cellula?pu??essere?ad?esempio?una?cellula?epatica,?in? particolare?un?epatocita.?La?selezione?di?un?ospite?appropriato???ritenuta?di?competenza?di?coloro?che?sono? esperti?dell'arte?in?base?agli?insegnamenti?del?presente?documento.?Preferibilmente,?tale?cellula?ospite??? una?cellula?animale,?e?pi??preferibilmente?una?cellula?umana.?La?cellula?pu??esprimere?una?sequenza? nucleotidica?fornita?nel?vettore?virale?dell?invenzione.? The invention also relates to a host cell that contains the viral vector of the invention. The host cell may be a cultured cell or a primary cell, i.e., isolated directly from an organism, e.g., a human. The host cell may be an adherent cell or a suspended cell, i.e., a cell that grows in suspension. Suitable host cells are known in the art and include, for example, DH5 cells, E. coli, Chinese hamster ovary cells, VERO monkey cells, COS cells, HEK293 cells, and the like. The?cell?may?be?human?or?of?another?animal.?The?cell?may?be?for?example?a?liver?cell,?in?particular?a?hepatocyte.?The?selection?of?an?appropriate?host?is?deemed?to?be?within?the?competence?of?those?skilled?in?the?art?based?on?the?teachings?herein.?Preferably,?such?host?cell?is?an?animal?cell,?and?more?preferably?a?human?cell.?The?cell?may?express?a?nucleotide?sequence?provided?in?the?viral?vector?of?the?invention.?

L?esperto?nell'arte?conosce?bene?i?metodi?standard?per?incorporare?un?polinucleotide?o?un?vettore?in?una? cellula?ospite,?ad?esempio?la?trasfezione,?la?lipofezione,?l'elettroporazione,?la?microiniezione,?l'infezione? virale,?lo?shock?termico,?la?trasformazione?dopo?permeabilizzazione?chimica?della?membrana?o?la?fusione? The person skilled in the art is familiar with standard methods for incorporating a polynucleotide or vector into a host cell, such as transfection, lipofection, electroporation, microinjection, viral infection, heat shock, transformation after chemical membrane permeabilization, or fusion.

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cellulare.?? mobile phone.??

Come?usato?nel?presente?documento,?il?termine?"cellula?ospite?o?cellula?ospite?geneticamente?modificata"? si? riferisce? a? cellule? ospiti? che? sono? state? trasdotte,? trasformate? o? trasfettate? con? il? vettore? virale? dell'invenzione.? As used herein, the term "host cell or genetically modified host cell" refers to host cells that have been transduced, transformed, or transfected with the viral vector of the invention.

COMPOSIZIONI? COMPOSITIONS?

La?presente?invenzione?fornisce?anche?una?composizione?farmaceutica?per?il?trattamento?di?un?individuo? mediante?terapia?genica,?in?cui?la?composizione?comprende?una?quantit??terapeuticamente?efficace?del? vettore?della?presente?invenzione?comprendente?la?sequenza?codificante?GLA?terapeutica?qui?descritta?o? una?particella?virale?prodotta?o?ottenuta?dalla?stessa.? The present invention also provides a pharmaceutical composition for the treatment of an individual by gene therapy, wherein the composition comprises a therapeutically effective amount of the present invention vector comprising the therapeutic GLA encoding sequence described herein or a viral particle produced or obtained therefrom.

Composizioni?farmaceutiche?ai?sensi?della?presente?invenzione?comprendono?il?vettore?o?la?cellula?ospite? dell'invenzione,?facoltativamente?in?combinazione?con?almeno?un?vettore?farmaceuticamente?accettabile,? un?diluente,?un?eccipiente?o?un?adiuvante.?La?scelta?del?vettore?farmaceutico,?dell'eccipiente?o?del?diluente? pu??essere?effettuata?in?base?alla?via?di?somministrazione?prevista?e?alla?pratica?farmaceutica?standard.?Le? composizioni? farmaceutiche?possono?contenere,?come?o? in?aggiunta?al?vettore,?eccipiente?o?diluente,? qualsiasi? legante,? lubrificante,?agente? sospensivo,?agente?di? rivestimento,?agente? solubilizzante?e?altri? agenti?di? trasporto?che?possono? favorire?o?aumentare? l'ingresso?del?virus?nel?sito?bersaglio? (come?ad? esempio?un?sistema?di?rilascio?lipidico).?Il?vettore?pu??essere?somministrato?in?vivo?o?ex?vivo.?? Pharmaceutical compositions according to the present invention comprise the carrier or host cell of the invention, optionally in combination with at least one pharmaceutically acceptable carrier, a diluent, an excipient or an adjuvant. The choice of the pharmaceutical carrier, excipient or diluent may be made based on the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may contain, as or in addition to the carrier, excipient or diluent, any binder, lubricant, suspending agent, coating agent, solubilizing agent and other transport agents that can promote or enhance the entry of the virus into the target site (such as a lipid delivery system). The vector may be administered in vivo or ex vivo.

Per?la?somministrazione?parenterale,?le?composizioni?possono?essere?utilizzate?al?meglio?sotto?forma?di? soluzione?acquosa?sterile?che?pu??contenere?altre?sostanze,?ad?esempio?sali?o?monosaccaridi?sufficienti?a? rendere? la? soluzione? isotonica? con? il? sangue.? In? una? forma? di? realizzazione? preferita,? il? vettore? o? la? composizione? farmaceutica? vengono? somministrati? per? via? sistemica,? ad? esempio? tramite? iniezione? endovenosa.?? For?parenteral?administration,?the?compositions?are?best?used?in?the?form?of?a?sterile?aqueous?solution?which?may?contain?other?substances,?for?example?salts?or?monosaccharides?sufficient?to?make?the?solution?isotonic?with?the?blood.? In?a?preferred?embodiment?the?carrier?or?the?pharmaceutical?composition?is?administered?systemically,?for?example?by?intravenous?injection.?

I?metodi?della?presente? invenzione?possono?essere?utilizzati? con? l'uomo?e? con?altri?animali.? I? termini? "paziente"?e?"soggetto"?sono?qui?usati? in?modo? intercambiabile?e?si?riferiscono?a?specie?umane?e?non? umane.?Allo?stesso?modo,?i?metodi?in?vitro?della?presente?invenzione?possono?essere?eseguiti?su?cellule?di? tali?specie?umane?e?non?umane.? The?methods?of?the?present?invention?may?be?used?with?humans?and?other?animals.? The?terms?"patient"?and?"subject"?are?used?herein?interchangeably?and?refer?to?human?and?non-human?species.?Likewise,?the?in?vitro?methods?of?the?present?invention?may?be?performed?on?cells?of?such?human?and?non-human?species.?

KIT? KIT?

L'invenzione?riguarda?anche?i?kit?comprendenti?il?vettore?virale?o?le?cellule?ospiti?dell'invenzione?in?uno?o? pi?? contenitori.? I? kit? dell'invenzione? possono? facoltativamente? includere? vettori? e/o? diluenti? farmaceuticamente?accettabili.?In?una?forma?di?realizzazione,?un?kit?dell'invenzione?include?uno?o?pi??altri? The invention also relates to kits comprising the viral vector or host cells of the invention in one or more containers. The kits of the invention may optionally include pharmaceutically acceptable vectors and/or diluents. In one embodiment, a kit of the invention includes one or more other

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componenti,?coadiuvanti?o?additivi?come?qui?descritti.?In?una?forma?di?realizzazione,?un?kit?dell'invenzione? include? istruzioni? o? materiali? di? confezionamento? che? descrivono? come? somministrare? un? sistema? vettoriale?del?kit.? I?contenitori?del?kit?possono?essere?di?qualsiasi?materiale?adatto,?ad?esempio?vetro,? plastica,? metallo,? ecc.? e? di? qualsiasi? dimensione,? forma? o? configurazione? adatta.? In? una? forma? di? realizzazione,?il?vettore?virale?o?la?cellula?ospite?dell'invenzione?sono?forniti?nel?kit?sotto?forma?di?solido?In? un?altra?forma?di?realizzazione,?il?vettore?virale?o?la?cellula?ospite?dell'invenzione?sono?forniti?nel?kit?come? liquido?o?soluzione.?In?una?forma?di?realizzazione,?il?kit?comprende?una?fiala?o?una?siringa?contenente?il? vettore?virale?o?la?cellula?ospite?dell'invenzione?in?forma?liquida?o?di?soluzione.? components, adjuvants, or additives as described herein. In one embodiment, a kit of the invention includes instructions or packaging materials describing how to administer a vector system of the kit. Containers of the kit may be made of any suitable material, e.g., glass, plastic, metal, etc., and of any suitable size, shape, or configuration. In one embodiment, the viral vector or host cell of the invention is provided in the kit as a solid. In another embodiment, the viral vector or host cell of the invention is provided in the kit as a liquid?or?solution.?In?one?embodiment,?the?kit?comprises?a?vial?or?syringe?containing?the?viral?vector?or?host?cell?of?the?invention?in?liquid?or?solution?form.?

USI?MEDICI?E?METODI?DI?TRATTAMENTO? MEDICAL?USES?AND?TREATMENT?METHODS?

In? un? aspetto? l'invenzione? fornisce? l'acido? nucleico,? il? vettore,? la? cellula,? il? kit? o? la? composizione? dell'invenzione?per?l'uso?come?medicinale.?? In? one? aspect? the? invention? provides? the? nucleic? acid,? the? vector,? the? cell,? the? kit? or? the? composition? of the invention? for? use? as? a? medicinal product.??

In?un?altro?aspetto? l'invenzione? fornisce? l'acido?nucleico,? il?vettore,? la? cellula,? il?kit?o? la?composizione? dell'invenzione?per?l'uso?nel?trattamento?della?malattia?di?Fabry.?? In?another?aspect?the?invention?provides?the?nucleic?acid,?the?vector,?the?cell,?the?kit?or?the?composition?of?the?invention?for?use?in?the?treatment?of?Fabry?disease.??

Tipicamente,?un?medico?di?ordinaria?esperienza?determiner??il?dosaggio?effettivo?che?sar??pi??adatto?a?un? singolo? soggetto,? che?varier?? in?base?all'et?,?al?peso?e?alla? risposta?del? singolo? individuo?e?alla?via?di? somministrazione.?Un? intervallo?di?dosi? compreso? tra?1x10e9?e?1x10e15? copie?di?genoma?di? ciascun? vettore/kg,?preferibilmente? tra?1x10e11?e?1x10e13? copie?di?genoma?di? ciascun?vettore/kg,?dovrebbe? essere?efficace?nell'uomo.?? Typically, an experienced physician will determine the actual dosage that will be most appropriate for an individual subject, which will vary with the age, weight, and response of the individual and the route of administration. A dose range of between 1x10e9 and 1x10e15 genome copies of each vector/kg, preferably between 1x10e11 and 1x10e13 genome copies of each vector/kg, should be effective in humans.

I? regimi? di? dosaggio? e? le? quantit?? efficaci? da? somministrare? possono? essere? determinati? da?medici? normalmente?esperti.?La?somministrazione?pu??avvenire?sotto?forma?di?dose?singola?o?di?dosi?multiple,? preferibilmente? come? dose? singola.? I? metodi? generali? per? eseguire? la? terapia? genica? utilizzando? polinucleotidi,?costrutti?di?espressione?e?vettori?sono?noti?nell'arte?(si?veda,?ad?esempio,?Gene?Therapy:? Principles?and?Applications,?Springer?Verlag?199926;?e?i?brevetti?statunitensi?n.?6,461?,606;?6,204,251?e? 6,106,826).?? Dosage regimens and effective amounts to be administered may be determined by ordinarily skilled practitioners. Administration may be as a single dose or as multiple doses, preferably as a single dose. General methods for performing gene therapy using polynucleotides, expression constructs and vectors are known in the art (see, for example, Gene Therapy: Principles and Applications, Springer Verlag, 1999, 26; and U.S. Patent Nos. 6,461, 606; 6,204,251 and 6,106,826).

Il?vettore?per?l'uso?secondo?la?presente?invenzione?pu??essere?utilizzato?da?solo?o?in?combinazione?con?altri? trattamenti?o?componenti?del?trattamento.?Ad?esempio,?pu??essere?utilizzato?insieme?ad?altri?trattamenti? utili?per?la?malattia?di?Fabry,?come?terapie?enzimatiche?sostitutive?o?terapie?con?chaperoni?orali.?? The carrier for use according to the present invention may be used alone or in combination with other treatments or treatment components. For example, it may be used in conjunction with other treatments useful for Fabry disease, such as enzyme replacement therapies or oral chaperone therapies.

In?una? forma? di? realizzazione,? il?vettore?per? l'uso?dell'invenzione?viene? somministrato?al? fegato,?per? esempio?mediante?iniezione?intravenosa.?? In one embodiment, the vector for use in the invention is administered to the liver, for example by intravenous injection.

In? una? forma? di? realizzazione,? il? vettore? ?? un? vettore? integrativo? come? sopra? descritto? e? viene? In? one? embodiment,? the? vector? is? an? integrative? vector? as? described? above? and? is?

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somministrato?a?un?soggetto?neonatale?o?giovane?insieme?a?un?vettore?che?esprime?una?nucleasi,?come? Cas9,?o? insieme? a?uno?o?pi?? farmaci? che? aumentano? la? frequenza?di? targeting? genico.?Ci?? consente? vantaggiosamente?di?trattare?la?malattia?di?Fabry?nelle?sue?fasi?iniziali,?integrando?la?sequenza?codificante? GLA?funzionante?nel?genoma?del?soggetto.?? administered?to?a?neonate?or?young?subject?together?with?a?vector?that?expresses?a?nuclease,?such?as?Cas9,?or?together?with?one?or?more?drugs?that?increase?the?frequency?of?gene?targeting.?This?advantageously?allows?to?treat?Fabry?disease?in?its?early?stages,?integrating?the?functional?GLA?coding?sequence?into?the?subject's?genome.?

In?un?altra? forma?di? realizzazione,? il?vettore???un?vettore?non?integrativo?e?viene?somministrato?a?un? soggetto?di?qualsiasi?et?.? In another embodiment, the vector is a non-supplemental vector and is administered to a subject of any age.

??contemplato?l'uso?di?qualsiasi?metodo?di?veicolazione?adatto?per?la?somministrazione?delle?composizioni? della?descrizione.?? ??the use?of?any?method?of?delivery?suitable?for?the?administration?of?the?compositions?of?the?description?is?envisaged.??

Quando???utilizzato,?un?vettore? integrativo,? i?singoli?componenti?del?sistema?di?editing?del?genoma?(ad? esempio,? gRNA,? nucleasi? e/o? sequenza? di?DNA? esogeno),? in? alcune? forme? di? realizzazione,? vengono? somministrati? simultaneamente? o? separati? temporalmente.? La? scelta? del?metodo? di? editing? genetico? dipende?dal? tipo?di? cellula?da? trasformare?e/o?dalle? circostanze? in? cui? avviene? la? trasformazione? (ad? esempio,?in?vitro,?ex?vivo?o?in?vivo).?Una?discussione?generale?di?questi?metodi?si?trova?in? When?an?integrative?vector?is?used,?the?individual?components?of?the?genome?editing?system?(e.g.,?gRNA,?nuclease,?and/or?exogenous?DNA?sequence),?in?some?embodiments,?are?administered?simultaneously?or?temporally?separated.?The?choice?of?gene?editing?method?depends?on?the?type?of?cell?to?be?transformed?and/or?the?circumstances?under?which?transformation?occurs?(e.g.,?in?vitro,?ex?vivo,?or?in?vivo).?A?general?discussion?of?these?methods?can?be?found?in?

Short?Protocols?in?Molecular?Biology,?3rd?ed.,?Wiley?&?Sons,?1995.? Short?Protocols?in?Molecular?Biology,?3rd?ed.,?Wiley?&?Sons,?1995.?

Come?qui?utilizzato,?il?termine?"somministrare"?include?la?somministrazione?orale,?il?contatto?topico,?la? somministrazione? come? supposta,? la? somministrazione?endovenosa,? intraperitoneale,? intramuscolare,? intralesionale,? intratecale,? intranasale?o?sottocutanea?a?un?soggetto.?La?somministrazione?avviene?per? qualsiasi?via,?compresa?quella?parenterale?e? transmucosa? (ad?esempio,?buccale,? sublinguale,?palatale,? gengivale,?nasale,? vaginale,? rettale?o? transdermica).? La? somministrazione?parenterale? comprende,? ad? esempio,? quella? endovenosa,? intramuscolare,? intra?arteriolare,? intradermica,? sottocutanea,? intraperitoneale,?intraventricolare?e?intracranica.?Altre?modalit??di?somministrazione?includono,?ma?non? solo,?l'uso?di?formulazioni?liposomiali,?l'infusione?endovenosa,?i?cerotti?transdermici,?ecc.? As used herein, the term "administer" includes oral, topical, suppository, intravenous, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal, or subcutaneous administration to a subject. Administration occurs by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarteriolar, intradermal, subcutaneous, intraperitoneal,?intraventricular?and?intracranial.?Other?routes?of?administration?include,?but?not?limited?to,?the?use?of?liposomal?formulations,?intravenous?infusion,?transdermal?patches,?etc.?

Il?termine?"trattamento"?si?riferisce?a?un?approccio?per?ottenere?risultati?benefici?o?desiderati,?compresi,? ma? non? limitati? a,? un? beneficio? terapeutico? e/o? un? beneficio? profilattico.? Per? beneficio? terapeutico? s?intende?qualsiasi?miglioramento?o?effetto?terapeuticamente?rilevante?su?una?o?pi??malattie,?condizioni?o? sintomi? in? trattamento.? Il? rallentamento? della? progressione? di? una? malattia? ?? considerato? un? miglioramento? terapeutico? ai? sensi? della? presente? invenzione.? A? scopo? profilattico,? le? composizioni? possono?essere?somministrate?a?un?soggetto?a?rischio?di?sviluppare?una?particolare?malattia,?condizione?o? sintomo,?o?a?un?soggetto?che?presenta?uno?o?pi??sintomi?fisiologici?di?una?malattia,?anche?se?la?malattia,?la? condizione? o? il? sintomo? non? si? sono? ancora?manifestati.? Il? termine? "quantit?? efficace"? o? "quantit?? sufficiente"? si? riferisce?alla?quantit??di?un?agente? (ad?esempio,?nucleasi?del?DNA,?ecc.)?sufficiente?per? The term "treatment" refers to an approach to achieving beneficial or desired results, including, but not limited to, a therapeutic benefit and/or a prophylactic benefit. A therapeutic benefit means any therapeutically relevant improvement or effect on one or more diseases, conditions, or symptoms being treated. The slowing of the progression of a disease is considered a therapeutic improvement within the meaning of this invention. For prophylactic purposes, the compositions may be administered to a subject at risk of developing a particular disease, condition, or symptom. symptom,?or?to?a?subject?who?presents?one?or?more?physiological?symptoms?of?a?disease,?even?if?the?disease,?condition?or?symptom?has?not?yet?manifested?itself.?The?term?"effective?amount"?or?"sufficient?amount"?refers?to?the?amount?of?an?agent?(e.g.,?DNA?nuclease,?etc.)?that?is?sufficient?to?

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ottenere?risultati?benefici?o?desiderati.?La?quantit??terapeuticamente?efficace?pu??variare?a?seconda?di?uno? o?pi??fattori:?il?soggetto?e?la?condizione?patologica?da?trattare,?il?peso?e?l'et??del?soggetto,?la?gravit??della? condizione?patologica,?la?modalit??di?somministrazione?e?simili,?che?possono?essere?facilmente?determinati? da?una?persona?di?comune?competenza?nel?settore.?La?quantit??specifica?pu??variare?in?base?a?uno?o?pi?? dei?seguenti?fattori:? l?agente?particolare?scelto,? il?tipo?di?cellula?bersaglio,? la? localizzazione?della?cellula? bersaglio?nel?soggetto,?il?regime?di?dosaggio?da?seguire,?l'eventuale?somministrazione?in?combinazione?con? altri?composti,?i?tempi?di?somministrazione?e?il?sistema?fisico?di?somministrazione?in?cui?viene?eseguito.?? to obtain beneficial or desired results. The therapeutically effective amount may vary depending on one or more factors: the subject and the medical condition being treated, the weight and age of the subject, the severity of the medical condition, the mode of administration, and the like, which may be readily determined by a person of ordinary skill in the field. The specific amount may vary depending on one or more of the following factors: the particular agent chosen, the type of target cell, the location of the target cell, target?in?the?subject,?the?dosage?regime?to?be?followed,?the?possible?administration?in?combination?with?other?compounds,?the?timing?of?administration?and?the?physical?system?of?administration?in?which?it?is?performed.??

Il? termine? "vettore? farmaceuticamente? accettabile"? si? riferisce? a? una? sostanza? che? favorisce? la? somministrazione?di?un?agente?(ad?esempio,?nucleasi?del?DNA,?ecc.)?a?una?cellula,?a?un?organismo?o?a?un? soggetto.?Per?"vettore? farmaceuticamente?accettabile"?si? intende?un?vettore?o?un?eccipiente?che?pu?? essere?incluso?in?una?composizione?o?formulazione?e?che?non?causa?effetti?tossicologici?avversi?significativi? sul? paziente.? Esempi? non? limitativi? di? vettore? farmaceuticamente? accettabile? includono? acqua,?NaCl,? soluzioni? saline? normali,? Ringer? lattato,? saccarosio? normale,? glucosio? normale,? leganti,? riempitivi,? disintegranti,?lubrificanti,?rivestimenti,?edulcoranti,?aromi?e?colori?e?simili.?Un?esperto?dell'arte?riconoscer?? che?altri?veicoli?farmaceutici?sono?utili?per?la?presente?invenzione.?? The? term? "pharmaceutically? acceptable? carrier"? refers? to? a? substance? that? promotes? the? delivery? of? an? agent? (e.g.,? DNA? nuclease,? etc.)? to? a? cell,? organism? or? subject.? A? "pharmaceutically? acceptable? carrier"? means? a? carrier? or? excipient? that? may? be? included? in? a? composition? or? formulation? and? that? does? not? cause? significant? adverse? toxicological? effects? on? the? patient.? Non-limiting? examples? of? a? pharmaceutically? acceptable? carrier? include? water,? NaCl,? normal? saline,? Ringer's? lactate,? normal? sucrose,? normal? glucose,? binders,? fillers,? disintegrants,?lubricants,?coatings,?sweeteners,?flavors?and?colors?and?the?like.?A?skilled?art?will?recognize?that?other?pharmaceutical?vehicles?are?useful?for?the?present?invention.??

L'invenzione?sar??ora?descritta?dai?seguenti?esempi.? The invention will now be described by the following examples.

ESEMPI? EXAMPLES?

Per?valutare?la?maggiore?traducibilit??del?cDNA?ottimizzato,?sono?stati?eseguiti?test?in?vitro?e?in?vivo,? nell'ordine?seguente:?? To?evaluate?the?higher?translatability?of?the?optimized?cDNA,?in?vitro?and?in?vivo?tests?were?performed,?in?the?following?order:??

1???In?silico:?ottimizzazione?dei?codoni?del?cDNA?dell'a?galattosidasi?A?(GLA)?umana? 1???In?silico:?Optimization?of?human?galactosidase?A?(GLA)?cDNA?codons?

2???Clonaggio?delle?varianti?ottimizzate?in?un?vettore?di?espressione?specifico?per?il?fegato? 2???Cloning?of?optimized?variants?into?a?liver-specific?expression?vector?

3???Test?in?vitro?in?una?linea?cellulare?epatica?umana?HUH7? 3???In vitro tests in a human liver cell line HUH7?

4???Produzione?di?stock?AAV?dei?costrutti?pi??attivi? 4???Production?of?AAV?stock?of?the?most?active?constructs?

5???Test?in?vivo?in?topi?WT?C57?Bl/6?maschi?giovani?(P30)? 5???In vivo?test?in?young?male?WT?C57?Bl/6?mice?(P30)?

6???Test?in?vivo?in?topi?Fabry?maschi?giovani?(P30)? 6???In vivo?test?in?young?male?Fabry?mice?(P30)?

I?test? in?vivo?sono?stati?effettuati? in?topi?WT?e? in?topi?Fabry?(modello?murino?B6;?129?Glatm1kul/J? Fabry?KO,?numero?di?stock?del?Jackson?lab?003535).?I?topi?Fabry?provengono?dal?Jackson?Laboratory,? Maine?USA;?i?topi?wild?type?provengono?da? .?Sono?state?testate?due?strategie? terapeutiche,?entrambe?basate?sulla?somministrazione?del?DNA?mediata?da?virus?adeno?associati? In vivo tests were performed in WT and Fabry mice (B6; 129 Glatm1kul/J Fabry KO mouse model, Jackson Lab Stock Number 003535). Fabry mice were from Jackson Laboratory, Maine, USA; wild type mice were from . Two therapeutic strategies were tested, both based on adeno-associated virus-mediated DNA delivery.

? ?

(AAV):?approcci?non? integrativi?e? integrativi.? I?principali?marcatori?di?malattia? sono? stati? l'attivit?? enzimatica?e?la?diminuzione?dell'accumulo?di?liso?Gb3,?il?metabolita?tossico,?nel?plasma?e?nei?tessuti? bersaglio.? (AAV):?non?integrative?and?integrative?approaches.?The?main?disease?markers?were?enzyme?activity?and?decreased?accumulation?of?lyso?Gb3,?the?toxic?metabolite,?in?plasma?and?target?tissues.?

Esempio?1? Example?1?

Studio?comparativo?per?plasmidi?hGLA?con?codoni?ottimizzati?in?vitro? Comparative study for hGLA plasmids with optimized codons in vitro

Costruzione?di?sequenze?hGLA?con?codoni?ottimizzati? Construction of hGLA sequences with optimized codons

Per?aumentare?i?livelli?di?espressione?del?cDNA?di?hGLA,?la?sequenza?wild?type???stata?ottimizzata?per? il?codone.? To?increase?the?expression?levels?of?hGLA?cDNA,?the?wild?type?sequence?was?optimized?for?the?codon.?

Il?trascritto?201?della?sequenza?hGLA?wild?type???stato?preso?come?sequenza?madre?e?diverse?varianti? sono? state? generate? utilizzando? strumenti? di? ottimizzazione? dei? codoni? online:? IDT? Codon? Optimization? tool? (https://eu.idtdna.com/CodonOpt),? JCAT? o? Java? Codon? Adaptation? Tool? (http://www.jcat.de/)?e?COOL?o?Codon?Optimization?On?Line?della?National?University?of?Singapore.?? The?transcript?201?of?the?wild?type?hGLA?sequence?was?taken?as?the?parent?sequence?and?several?variants?were?generated?using?online?codon?optimization?tools:?IDT?Codon?Optimization?tool?(https://eu.idtdna.com/CodonOpt),?JCAT?or?Java?Codon?Adaptation?Tool?(http://www.jcat.de/)?and?COOL?or?Codon?Optimization?On?Line?from?the?National?University?of?Singapore.??

Le?sequenze?ottimizzate?per?i?codoni?generate?online?sono?state?poi?valutate?per?parametri?quali?il? contenuto?di?GC,?le?isole?CpG,?i?siti?di?splicing?criptici?e?la?presenza?di?OFR?alternativi.?? The codon-optimized sequences generated online were then evaluated for parameters such as GC content, CpG islands, cryptic splice sites, and the presence of alternative OFRs.

Queste?sequenze?sono?state?modificate?manualmente?per?ottenere?valori?migliori?per? i?parametri? considerati? e? alla? fine? sono? state? selezionate? quattro? sequenze,? denominate? hGLA_CO01,? hGLA_CO02,?hGLA_CO03,?hGLA_CO04.?Per?migliorare?ulteriormente?l'efficacia?della?traduzione,?la? regione?del?codone?d?inizio???stata?modificata?seguendo? il?consenso?della?sequenza?di?Kozak.? ?Per? facilitare?il?clonaggio?nel?vettore?AAV,?sono?stati?introdotti?i?siti?di?clonaggio?NheI?e?SalI.?I?cDNA?sono? stati?sintetizzati?da?un'azienda.?Nella?prima? fase,?queste?sequenze?hGLA?sono?state?clonate? in?un? plasmide?episomale?non?integrativo?pSMD2?ApoE?hAAT?(ampicillina),?specifico?per?il?fegato,?per?la? valutazione?in?vitro?e?in?vivo,?con?la?seguente?struttura:?? These?sequences?were?manually?edited?to?obtain?better?values?for?the?considered?parameters?and?finally?four?sequences?were?selected,?named?hGLA_CO01,?hGLA_CO02,?hGLA_CO03,?hGLA_CO04.?To?further?improve?the?translation?efficiency,?the?start?codon?region?was?edited?following?the?Kozak?sequence?consensus.?To?facilitate?cloning?into?the?AAV?vector,?the?NheI?and?SalI?cloning?sites?were?introduced.?The?cDNAs?were?synthesized?by?a?company.?In?the?first? phase,?these?hGLA?sequences?were?cloned?into?a?non-integrative?episomal?plasmid?pSMD2?ApoE?hAAT?(ampicillin),?specific?for?in?vitro?and?in?vivo?evaluation,?with?the?following?structure:??

5'?SalI??Kozak?seq?ATG?hGLA??STOP??NheI?EcoR1?3'?? 5'?SalI??Kozak?seq?ATG?hGLA??STOP??NheI?EcoR1?3'??

Saggio?enzimatico?GLA? GLA enzyme assay?

Il? saggio? enzimatico? ?? stato? ottimizzato? sfruttando? il? substrato? 4?metilumbelliferil? ??D? galattopiranoside?(4?MUG)?che,?quando?reagisce?con?l'enzima?Alfa?galattosidasi?A?(GLA),?d??luogo?a? un?prodotto?fluorescente;?4?Metilumbelliferone?(4MU)?che?pu??essere?analizzato?a?una?lunghezza? d'onda?di?365?nm?(eccitazione)?e?450?nm?(emissione).?Pertanto,?2,46?mM?di?substrato?in?un?tampone? citrato?fosfato?(pH?4,5)?sono?stati?incubati?con?25??l?di?campioni?contenenti?l'enzima?GLA?per?1h?a? The? enzyme? assay? was? optimized? by? exploiting? the? substrate? 4?methylumbelliferyl? D? galactopyranoside? (4?MUG)? which,? when? reacting? with? the? enzyme? Alpha? galactosidase? A? (GLA),? gives? a? fluorescent? product;? 4? Methylumbelliferone? (4MU)? which? can? be? analyzed? at? a? wavelength? of? 365? nm? (excitation)? and? 450? nm? (emission).? Therefore,? 2.46? mM? substrate? in? a? citrate? phosphate? buffer? (pH? 4.5)? were? incubated? with? 25? l? of? samples? containing? the? GLA? enzyme? for? 1? h? at?

? ?

37?C?su?un?agitatore,?e?successivamente?la?reazione???stata?fermata?utilizzando?200?mM?di?NaOH-Glicina?(pH?10,4).?Sono?stati?realizzati?standard?di?4MU?da?2?pmoli?a?100?pmoli?per?ottenere?una? curva?standard.? I?campioni?sono?stati?misurati?a?365?450?nm?e?analizzati?per? l'attivit??enzimatica? (nmoli/mg/h).? 37?C?on?a?stirrer,?and?the?reaction?was?stopped?using?200?mM?NaOH-Glycine?(pH?10.4).?4MU?standards?from?2?pmoles?to?100?pmoles?were?made?to?obtain?a?standard?curve.?Samples?were?measured?at?365?450?nm?and?analyzed?for?enzyme?activity? (nmoles/mg/h).?

Per?determinare?se? le?sequenze?di?cDNA?hGLA?ottimizzate?per? il?codone? fossero? funzionali?e?pi?? efficienti? rispetto? alla? controparte? hGLA? wild?type,? i? plasmidi? pSMD2_hGLA,? sia? la? versione? ottimizzata? per? il? codone? che? quella?wild?type,? sono? stati? trasfettati? in? cellule?HuH7? utilizzando? Lipofectamina? (per?normalizzare? l'efficienza?di?trasfezione,???stato?co?trasfettato?un?plasmide?che? esprimeva?EGFP).?Le?cellule?non?trattate?sono?state?considerate?un?controllo?Mock?per?il?test.?Dopo? un?periodo?d?incubazione?di?48?ore,???stato?raccolto?il?surnatante?delle?cellule?e?l'estratto?cellulare?in? PBS.?Le?proteine?sono?state?estratte?dalle?cellule?e?dal?surnatante.?5??g?di?proteine?cellulari?e?5??l?di? surnatante?cellulare?sono?stati?utilizzati?per?reagire?con?4MUG?per?1h;?successivamente?la?reazione? ??stata?interrotta?e?la?fluorescenza???stata?misurata?(Figura?1A).?? To determine whether the codon-optimized hGLA cDNA sequences were functional and more efficient than their wild-type hGLA counterparts, pSMD2_hGLA plasmids, both the codon-optimized and wild-type versions, were transfected into HuH7 cells using Lipofectamine (an EGFP-expressing plasmid was co-transfected to normalize transfection efficiency). The untreated cells were considered as mock controls for the assay. After an incubation period of 48 hours, the cell supernatant and the cell extract were collected in PBS. Proteins were extracted from the cells and supernatant. 5 g of cell proteins and 5 l of cell supernatant were used to react with 4MUG for 1 h; the reaction was then stopped and the fluorescence was measured (Figure 1A).

?? stato?evidente,?con? tre? repliche? tecniche?del? saggio?e?due? repliche?biologiche?ciascuna,?che? le? sequenze?hGLA?ottimizzate?per?il?codone?erano?pi??efficienti?della?sequenza?wild?type,?mostrando? livelli?pi??elevati?di?attivit??enzimatica?GLA?(escluso?CO04;?Figura?1B).?Inoltre,???stato? interessante? osservare?che? la?GLA?presente?nel?surnatante?(4?volte?pi??alta)?era?superiore? in?termini?di?attivit?? rispetto?all'estratto?cellulare?(2?volte?pi??alta),?dimostrando?che?una?percentuale?maggiore?di?GLA?si? trova?nella?forma?secreta.?? ?? it was evident,? with? three? technical? replicates? of? the? assay? and? two? biological? replicates? each,? that? the? codon-optimized? hGLA? sequences? were? more efficient? than? the? wild-type? sequence,? showing? higher? levels? of? GLA? enzymatic? activity? (except? CO04;? Figure? 1B).? Furthermore,? it was? interesting? to? observe? that? the? GLA? present? in? the? supernatant? (4? fold? higher)? was? superior? in? activity? compared? to? the? cell? extract? (2? fold? higher),? demonstrating? that? a? higher? percentage? of? GLA? is? in? the? secreted? form.??

Per? confermare? la?presenza?della?proteina?hGLA?nei? campioni,??? stata?necessario?un'analisi?dell?? espressione.? Pertanto,? sono? stati? eseguiti? gel? SDS?PAGE? per? l'estratto? cellulare? e? il? surnatante? separatamente.?L'analisi?Western?blot???stata?eseguita?per?confrontare?i?livelli?proteici?delle?sequenze? di?hGLA?ottimizzata?per? il?codone?e?wild?type? in?entrambi? i?casi,?utilizzando?eGFP?come?controllo? dell'efficienza?di?trasfezione?(Figura?2A).?L'anticorpo?anti?GLA?(1:3000;?49?KDa;?Sino?Biological?Cat#? 12078?R001)???stato?utilizzato?per?ottenere?bande?specifiche?di?hGLA.?? To? confirm? the? presence? of? hGLA? protein? in? the? samples,? expression? analysis? was? necessary.? Therefore,? SDS? PAGE? gels? were? performed? for? the? cell? extract? and? the? supernatant? separately.? Western? blot? analysis? was? performed? to? compare? the? protein? levels? of? the? codon-optimized? and? wild? type? hGLA? sequences? in? both? cases,? using? eGFP? as? a? control? of transfection efficiency (Figure 2A). The anti-GLA antibody (1:3000; 49 KDa; Sino Biological Cat# 12078 R001) was used to obtain specific hGLA bands.

La?Figura?2A? rappresenta? l'analisi?dei?western?blot?dove???evidente? che? le? sequenze? con? codoni? ottimizzati?esprimono?una?proteina?5?6?volte?pi??alta?rispetto?a?quelle?wild?type?nel?surnatante?e?~2? volte?pi??alta?nell'estratto?cellulare?(n=2).?Questi?dati?coincidono?con? le? informazioni?ottenute?dal? saggio? enzimatico? e? quindi? si? pu?? affermare? con? sicurezza? che? le? sequenze? hGLA? con? codoni? ottimizzati?erano?pi??efficienti?della?sequenza?hGLA?wild?type?(escluso?CO04)?(Figura?2).? Figure 2A represents the Western blot analysis where it is evident that the sequences with optimized codons expressed a protein 5-6 times higher than the wild type sequences in the supernatant and ~2 times higher in the cell extract (n=2). These data coincide with the information obtained from the enzymatic assay and therefore it can be stated with certainty that the hGLA sequences with optimized codons were more efficient than the wild type hGLA sequence (except CO04) (Figure 2).

Esempio?2? Example?2?

? ?

Studio?comparativo?dei?vettori?hGLA?con?codoni?ottimizzati?in?vivo? Comparative study of hGLA vectors with optimized codons in vivo

Con? la? fiducia? nei? risultati? dei? saggi? in? vitro,? in? cui? le? sequenze? con? codoni? ottimizzati? hanno? dimostrato?una?migliore?efficienza?rispetto?al?wild?type?sia?in?termini?di?espressione?proteica?che?di? attivit??enzimatica,???stato?necessario?convalidare?lo?stesso?in?vivo.?? With? confidence? in? the? results? of? in? vitro? assays,? in? which? sequences? with? optimized? codons? have? demonstrated? better? efficiency? than? the? wild? type? both? in? terms? of? protein? expression? and? enzymatic? activity,? it? was? necessary? to? validate? the? same? in? vivo.??

A? tale? scopo,? sono? stati?preparati? stock?di?virus?AAV8?pSMD2?hGLA?per?hGLA?CO02?e?CO03? con? codoni?ottimizzati?e?per?hGLA?wild?type?(Figura?3A).?Il?vettore?contiene?il?promotore?specifico?per?il? fegato?del?gene?alfa1?AAT.?? For? this? purpose,? stocks? of? AAV8? pSMD2? hGLA? viruses? were? prepared? for? hGLA? CO02? and? CO03? with? optimized? codons? and? for? wild? type? hGLA? (Figure? 3A).? The? vector? contains? the? liver? specific? promoter? of? the? alpha1? AAT? gene.?

Il?cDNA?di?GLA?umano?wild?type?ha? la?sequenza?con?SEQ? ID?N.?16.?La?sequenza?ottimizzata?per? il? codone?CO2?ha?SEQ?ID?N.1;?la?sequenza?ottimizzata?per?il?codone?CO3?ha?SEQ?ID?N.2.?? The wild-type human GLA cDNA has the sequence with SEQ ID No. 16. The optimized sequence for the CO2 codon has SEQ ID No. 1; the optimized sequence for the CO3 codon has SEQ ID No. 2.

Topi?C57BL6/WT?di?un?mese? (giorno?post?natale?30,?P30)? (n=5)? sono? stati? trattati? con?una?dose? elevata? di? vettori? episomali? (3,0E+12? vg/kg)? tramite? iniezioni? retro?orbitali,? con? l?attesa? di? un'espressione?di?hGLA?molto?pi??elevata?rispetto?alla?mGLA?endogena.?Gli?animali?non?trattati?sono? stati? considerati? come? controllo.?Questi? topi? sono? stati? sacrificati? tre? settimane?dopo? l'iniezione? (P51).?Il?fegato???stato?prelevato?e?trattato?per?l'estrazione?delle?proteine?e?la?loro?quantificazione? mediante?il?saggio?di?Bradford,?mentre?il?sangue???stato?raccolto?per?l'analisi?del?plasma?(Figura?3B).?? One-month-old (postnatal day 30, P30) C57BL6/WT mice (n=5) were treated with a high dose of episomal vectors (3.0E+12 vg/kg) via retro-orbital injections, with the expectation of significantly higher hGLA expression than endogenous mGLA. Untreated animals were considered as controls. These mice were sacrificed three weeks after injection (P51). Livers were harvested and processed for protein extraction and quantification. using the Bradford assay, while blood was collected for plasma analysis (Figure 3B).

Per?determinare?l'attivit??della?GLA,?il?saggio?enzimatico?fluorescente???stato?eseguito?con?3?ng?di? proteine?epatiche?e?plasma?diluito?1:10000?in?PBS.?? To determine the activity of GLA, the fluorescent enzyme assay was performed with 3 ng of liver protein and plasma diluted 1:10000 in PBS.

?? stato? osservato? un? notevole? aumento? dei? livelli? di? attivit?? (nmole/mg/ora? per? il? fegato? e? nmoli/ml/ora?per? il?plasma)?dell'enzima?hGLA?nel? caso?di?CO02?e?CO03?nel? fegato?e?nel?plasma? rispetto?ai?campioni?trattati?con?hGLA?wild?type? (Figura?3C).? I? livelli?di?CO02?e?CO03?raggiungono? 6000?8000?nmoli/ml/ora?nel?plasma,?mentre?i?valori?del?wild?type?si?attestano?a?~2000?nmoli/ml/ora.? Nel? caso? del? tessuto? epatico,? l'attivit?? delle? sequenze? con? codoni? ottimizzati? (CO02? e? CO03)? ?? aumentata? fino?a?2000?3000?nmoli/mg/ora?da?quella?di?1000?nmoli/mg/ora?della?sequenza?wildtype?(Figura?3C).??? ?? a? significant? increase? in? hGLA? enzyme? activity? levels? (nmole/mg/h? for? liver? and? nmol/ml/h? for? plasma)? was? observed? in? the? case? of? CO02? and? CO03? in? liver? and? plasma? compared? to? samples? treated? with? wild? type? hGLA? (Figure? 3C).? CO02? and? CO03? levels? reach? 6000? 8000? nmol/ml/h? in? plasma,? while? the? values? of? the? wild? type? are? at? ~2000? nmol/ml/h.? In? the? case? of? liver? tissue,? the? activity? of? sequences? with? optimized? codons? (CO02? and? CO03)? ?? increased? up? to? 2000? 3000? nmol/mg/h? from? that? of? 1000? nmol/mg/h? of? the? wildtype? sequence? (Figure? 3C).???

Per? un? risultato? pi?? rassicurante,? l'espressione? della? proteina? hGLA? ?? stata? analizzata?mediante? western?blot? (Figura?4).?Le?proteine?del?plasma?e?del? fegato?degli?animali? trattati?e?dei?gruppi?di? controllo?non? trattati? sono? state?analizzate? su?un?gel?SDS?PAGE?e? trasferite? su?una?membrana?di? nitrocellulosa?(Figura?4A).?L'anticorpo?specifico?per?GLA???stato?utilizzato?insieme?a?HSP70?(fegato)?e? mIgG?(plasma)?per?la?normalizzazione.?Secondo?la?quantificazione?effettuata?dalle?bande?ottenute,? hGLA?CO02?e?hGLA?CO03? indicano?un?aumento?dell'espressione? relativa?di?~1?volta? rispetto?alla? For? a? more? reassuring? result,? hGLA? protein? expression? was? analyzed? by? western? blot? (Figure? 4).? Proteins? from? plasma? and? liver? of? treated? and? untreated? control? groups? were? analyzed? on? an? SDS? PAGE? gel? and? transferred? onto? a? nitrocellulose? membrane? (Figure? 4A).? The? GLA? specific? antibody? was? used? together? with? HSP70? (liver)? and? mIgG? (plasma)? for? normalization.? According? to? the? quantitation? of? the? obtained? bands,? hGLA? CO02? and? hGLA? CO03? indicate? an? increase? in? the? expression? relative?of?~1?time?with respect?to?the?

? ?

sequenza?wild?type?sia?nel?tessuto?epatico?che?nel?plasma,?che?ha?seguito?lo?stesso?andamento?della? loro?attivit??enzimatica?(Figura?4B).?? wild-type sequence in both liver tissue and plasma, which followed the same pattern of their enzymatic activity (Figure 4B).

Considerando?i?dati?ottenuti?dal?saggio?enzimatico?e?dal?western?blot,?si???concluso?che?CO02?e?CO03? avevano?un?potenziale?migliore?rispetto?al?wild?type?nei?topi?C57BL/6.?E??stata?data?la?preferenza?per? gli?esperimenti?successivi?a?CO02?in?base?al?suo?notevole?aumento?dei?livelli?di?attivit??enzimatica.? Considering?the?data?obtained?from?the?enzyme?assay?and?the?western?blot,?it?was?concluded?that?CO02?and?CO03?had?better?potential?than?the?wild?type?in?C57BL/6?mice.?CO02?was?given?preference?for?subsequent?experiments?based?on?its?significantly?increased?levels?of?enzyme?activity.?

Esempio?3? Example?3?

Terapia?genica?episomale?epato?specifica?mediata?da?AAV?in?topi?Fabry?KO?? AAV-mediated liver-specific episomal gene therapy in Fabry KO mice

Modello?di?topo?Fabry?[B6;129?Glatm1kul/J]? Fabry mouse model [B6;129?Glatm1kul/J]?

Il?modello?di?topo?Fabry?KO?B6;129?Glatm1kul/J???stato?acquistato?dal?The?Jackson?laboratory,?Maine,? USA,?ed???stato?stabilito?e?alloggiato?secondo?le?linee?guida?internazionali?approvate?dalla?struttura? per? animali? dell'ICGEB,? Trieste,? ricevendo? dieta? standard? e? acqua? ad? libitum.? La? colonia? ?? stata? propagata?per?ottenere?maschi?emizigoti?per?la?sperimentazione?e?maschi?wild?type?come?controlli.? The Fabry KO B6;129 Glatm1kul/J mouse model was purchased from The Jackson Laboratory, Maine, USA, and was established and housed according to international guidelines approved by the ICGEB Animal Facility, Trieste, Italy, and received a standard diet and water ad libitum. The colony was propagated to obtain hemizygous males for the experiments and wild-type males as controls.

Risultati? Results?

In?questo?studio?i?cDNA?di?hGLA?WT?e?CO2?sono?stati?somministrati?a?topi?Fabry?di?un?mese?di?et?? (P30)?mediante? iniezione?retro?orbitale.? Il?vettore?AAV???quello?descritto?nella?Figura?3A,? in?cui? il? cDNA?hGLA?viene?trascritto?sotto?il?controllo?di?un?promotore?specifico?per?il?fegato?(Ronzitti?et?al.,? 2016).?Il?vettore?pSMD2?wild?type?contenente?il?cDNA?di?hGLA?wild?type?ha?la?sequenza?SEQ?ID?N.18? e?il?vettore?pSMD2?CO2?hGLA?contenente?il?cDNA?di?hGLA?CO02?ha?sequenza?con?SEQ?ID?N.10.?? In this study, hGLA WT and CO2 cDNAs were administered to one-month-old (P30) Fabry mice by retro-orbital injection. The AAV vector is the one described in Figure 3A, in which hGLA cDNA is transcribed under the control of a liver-specific promoter (Ronzitti et al., 2016). The wild-type pSMD2 vector containing wild-type hGLA cDNA has the SEQ ID No. 18. and?the?pSMD2?CO2?hGLA?vector?containing?the?cDNA?of?hGLA?CO02?has?sequence?with?SEQ?ID?N.10.??

I?due?vettori?di?terapia?genica?non? integrativa?AAV8?pSMD2?hGLA?WT?o?AAV8?pSMD2?hGLA?CO02? sono?stati?iniettati?a?dosi?comprese?tra?3,0E11?e?3,0E13vg/kg.?I?mutanti?non?trattati?e?gli?animali?wildtype?non?trattati?sono?stati?utilizzati?come?gruppi?di?controllo.?Tutti?gli?animali?sono?stati?pesati?e?fatti? sanguinare?mediante?puntura?della?vena?sottomandibolare?ogni?30?giorni?per?estrarre?il?plasma.?A?5? mesi?di?et??(P150)?tutti?gli?animali?sono?stati?sacrificati?e?sono?stati?prelevati?fegato,?reni?e?cuore;? anche?il?sangue???stato?raccolto?per?il?plasma?(Figura?5B).?? The two non-supplemental gene therapy vectors AAV8 pSMD2 hGLA WT or AAV8 pSMD2 hGLA CO02 were injected at doses ranging from 3.0E11 to 3.0E13 vg/kg. Untreated mutants and untreated wildtype animals served as controls. All animals were weighed and bled by puncture of the submandibular vein every 30 days to extract plasma. At 5? months of age (P150)?All animals were sacrificed and liver, kidneys, and heart were removed; blood was also collected for plasma (Figure 5B).

Utilizzando? il? plasma? raccolto? in? diversi?momenti? del? trattamento,? ?? stato? eseguito? un? saggio? enzimatico?per?determinare?l'efficacia?dei?vettori.?Una?diluizione?1:10.000?di?plasma?in?PBS???stata? utilizzata?per?impostare?il?saggio?per?1?ora?(nmoli/ml/ora).?Gli?animali?trattati?con?i?vettori?episomali? AAV8?pSMD2?hGLA?WT?e?AAV8?pSMD2?hGLA?CO02?hanno?mostrato?livelli?sovrafisiologici?di?attivit?? enzimatica,?raggiungendo?valori?~10000x?superiori?rispetto?agli?animali?wild?type?nei?gruppi?trattati? con?le?dosi?AAV?pi??elevate?(Figura?5A).?? Using plasma collected at different time points during treatment, an enzyme assay was performed to determine the efficacy of the vectors. A 1:10,000 dilution of plasma in PBS was used to set up the assay for 1 hour (nmol/ml/hour). Animals treated with the AAV8pSMD2hGLAWT and AAV8pSMD2hGLACO02 episomal vectors showed supraphysiological levels of enzyme activity, reaching values of ~10,000x higher than wild-type animals in the AAV8pSMD2hGLACO02-treated groups. with the highest AAV doses (Figure 5A).

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L'attivit??GLA???persistita?per?4?mesi?dopo?il?trattamento,?con?una?diminuzione?nel?primo?mese?nel? caso?di?pSMD2?hGLA?CO02,?a?causa?dell'eccessiva?produzione?di?GLA?e?dello?stress?ER?combinato?con? un?aumento?dei?marcatori?di?infiammazione.?? GLA activity persisted for 4 months after treatment, with a decrease in the first month in the case of pSMD2 hGLA CO02, due to excessive GLA production and ER stress combined with increased inflammation markers.

Abbiamo? osservato? la? completa? eliminazione? di? liso?Gb3? nel? plasma? negli? animali? trattati? con? entrambi? i? vettori? alla?dose?pi?? alta?di?AAV? (Figura? 5C).?Abbiamo?osservato?un? chiaro? aumento? dell'attivit?? GLA? nei? gruppi? trattati? con? il? vettore? AAV8? pSMD2? hGLA? CO02? (66x,? 74x? e? 93x,? rispettivamente?nei?gruppi?1,0E13?vg/kg,?3,0E12?vg/kg?e?3,0E11?vg/kg;?Figura?5A).?Anche?l'analisi?con? spettrometria? di? massa? dell'accumulo? di? liso?Gb3? nel? plasma? ha? mostrato? un'impressionante? differenza?tra?i?gruppi?trattati?con?WT?e?CO2.?Mentre?il?trattamento?con?la?dose?di?3,0E11?vg/kg?ha? comportato?una?riduzione?di?circa?il?50%?dei?livelli?di?liso?Gb3?nel?gruppo?trattato?con?il?cDNA?hGLA? WT,?la?riduzione???stata?di?circa?il?90%?negli?animali?trattati?con?hGLA?CO2?(Figura?5C).???importante? notare?che? il?numero?di?copie?del?vettore?nel? fegato?era?simile?nei?gruppi?WT?e? trattati?con?CO2? (Figura?5D).? We observed complete elimination of lyso-Gb3 in plasma in animals treated with both vectors at the highest dose of AAV (Figure 5C). We observed a clear increase in GLA activity in groups treated with the AAV8 vector pSMD2 hGLA CO02 (66x, 74x and 93x in the 1.0E13 vg/kg, 3.0E12 vg/kg and 3.0E11 vg/kg groups, respectively; Figure 5A). Mass spectrometry analysis of lyso-Gb3 accumulation in plasma also showed an impressive difference?between?the?WT?and?CO2?treated?groups.?While?treatment?with?the?dose?of?3.0E11?vg/kg?resulted?in?an?approximately?50%?reduction?in?lyso?Gb3?levels?in?the?WT?hGLA?cDNA?treated?group,?the?reduction?was?approximately?90%?in?the?hGLA?CO2?treated?animals?(Figure?5C).?It?is?important?to?note?that?the?number?of?vector?copies?in?the?liver?was?similar?in?the?WT?and?CO2?treated?groups?(Figure?5D).?

La?distribuzione?e?l?assorbimento?di?GLA?da?parte?dei?tessuti?sono?stati?considerati?anche?misurando? l'attivit??di?GLA?e?l'analisi?di?Lyso?Gb3?nel?fegato,?nel?rene?e?nel?cuore.?Come?nel?plasma,?a?tutte?le? dosi? testate? e? con? entrambi? i? vettori?WT? e? CO2,? tutti? i? tessuti? hanno?mostrato? un'attivit??GLA? sovrafisiologica?rispetto?agli?animali?WT?non?trattati?(Figura?6A).?L'attivit??nei?tessuti?degli?animali? trattati?con?il?cDNA?di?hGLA?CO2?ha?presentato?un'attivit??enzimatica?pi??elevata,?pi??evidente?nei? gruppi? trattati? con? le? dosi? di? AAV? pi?? basse,? a? causa? probabilmente? della? sovrasaturazione? del? meccanismo?di?cattura?dell'enzima?hGLA?presente?nei?tessuti,?nei?gruppi?di?animali?trattati?con? le? dosi?di?AAV?pi??elevate.?? Tissue distribution and uptake of GLA were also considered by measuring GLA activity and Lyso Gb3 assay in liver, kidney and heart. As in plasma, at all tested doses and with both WT and CO2 vectors, all tissues showed supraphysiological GLA activity compared to untreated WT animals (Figure 6A). Tissue activity from animals treated with hGLA CO2 cDNA showed higher enzymatic activity, which was more evident in the hGLA CO2-treated groups. the lower? doses? of? AAV,? probably? due? to? the? supersaturation? of? the? hGLA? enzyme? capture? mechanism? present? in? the? tissues,? in? the? groups? of? animals? treated? with? the? higher? doses? of? AAV.??

La?liso?Gb3???stata?completamente?eliminata?nei?gruppi?trattati?con?la?dose?pi??alta,?sia?per?gli?animali? WT?che?per?quelli?trattati?con?CO2?(Figura?6B).?Nei?gruppi?trattati?con?la?dose?di?AAV?pi??bassa?(3.? 0E11?vg/kg)?abbiamo?osservato?una?clearance?completa?o?quasi?completa?della?liso?Gb3?nei?tessuti? dei?topi?trattati?con? il?vettore?AAV8?pSMD2?hGLA?CO02?(98%,?90%?e?85%? in?fegato,?rene?e?cuore,? rispettivamente,?Figura?6B),?mentre?nei?topi?trattati?con?il?vettore?AAV8?pSMD2?hGLA?WT?abbiamo? osservato?una?diminuzione?dell'accumulo?di?liso?Gb3?del?65%,?40%?e?40%?in?fegato,?rene?e?cuore,? rispettivamente?(Figura?6B).? Lyso?Gb3?was?completely?eliminated?in?the?highest?dose?treated?groups,?both?in?WT?and?CO2?treated?animals?(Figure?6B).?In?the?lowest?AAV?dose?(3.? 0E11?vg/kg)?we?observed?a?complete?or?almost?complete?clearance?of?lyso?Gb3?in?tissues?of?mice?treated?with? the?AAV8?pSMD2?hGLA?CO02?vector?(98%,?90%?and?85%?in?liver,?kidney?and?heart,? respectively,?Figure?6B),?while?in?mice?treated?with?the?AAV8?pSMD2?hGLA?WT?vector?we?observed?a?decrease?in?lyso?Gb3?accumulation?of?65%,?40%?and?40%?in?liver,?kidney?and?heart,? respectively?(Figure?6B).?

Esempio?4? Example?4?

Clonaggio?di?hGLA? in?un?vettore? integrativo?e?trattamento?della?malattia?di?Fabry?a? insorgenza? precoce?mediante?targeting?di?animali?Fabry?KO?neonatali? Cloning of hGLA into a Complementary Vector and Treatment of Early-Onset Fabry Disease by Targeting Neonatal Fabry KO Animals

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Considerando?i?risultati?ottenuti?dai?dati?in?vitro?e?in?vivo?in?cui?abbiamo?confrontato?i?costrutti?con? codoni?ottimizzati,???risultato?evidente?che?hGLA_CO02?ha?il?potenziale?per?funzionare?meglio?del? costrutto?wild?type.?? Considering the results obtained from the in vitro and in vivo data in which we compared the codon-optimized constructs, it is clear that hGLA_CO02 has the potential to perform better than the wild-type construct.

Pertanto,?abbiamo?testato?un?altro?approccio?terapeutico?basato?sull'integrazione?mirata?del?cDNA? terapeutico?nel?locus?dell'albumina?(Barzel?et?al.,?2015;?De?Caneva?et?al.,?2019;?Porro?et?al.,?2017).? Pertanto,?il?vettore?wild?type?e?la?versione?CO02?del?cDNA?di?hGLA?sono?stati?clonati?in?un?vettore? integrativo;? struttura?del?pAB288?affiancato?da?bracci?di?omologia?dell'albumina? su?entrambe? le? estremit?,?una?sequenza?peptidica?P2A?e?una?sequenza?PAM8?modificata.?Il?vettore?ha?regioni?ITR? per?essere?impacchettato?in?vettori?AAV.?Il?DNA?plasmidico???stato?preparato?dai?vettori?clonati?e?i? vettori?virali?AAV8?sono?stati?preparati?dal?servizio?AAV?dell'ICGEB?di?Trieste.?Il?vettore?pAB288?wild? type?contenente?il?cDNA?di?hGLA?wild?type?ha?la?sequenza?con?SEQ?ID?N.17?e?il?vettore?pAB288?CO2? hGLA?contenente?il?cDNA?di?hGLA?CO02?ha?la?sequenza?con?SEQ?ID?N.15.? Therefore, we tested another therapeutic approach based on targeted integration of the therapeutic cDNA into the albumin locus (Barzel et al., 2015; De Caneva et al., 2019; Porro et al., 2017). Therefore, the wild type vector and the CO02 version of the hGLA cDNA were cloned into an integrative vector; pAB288 structure flanked by albumin homology arms on both ends, a P2A peptide sequence and a modified PAM8 sequence. The vector has ITR regions to be packaged into AAV vectors. The plasmid DNA was prepared from the cloned vectors and the AAV8 viral vectors were prepared by the AAV service of ICGEB in Trieste. The wild type pAB288 vector containing the wild type hGLA cDNA has the sequence with SEQ ID No. 17 and the hGLA pAB288 CO2 vector containing the hGLA CO02 cDNA has the sequence with SEQ ID No. 15.

Per? trattare? una? malattia? nelle? sue? fasi? iniziali? o? in? fase? neonatale,? l'approccio? integrativo? ?? fondamentale?e?pu??rivelarsi?efficace.?Pertanto,???stato?progettato?un?esperimento?in?cui?topi?maschi? Fabry?KO?neonati?a?P5?sono?stati?trattati?con?il?vettore?donatore?WT?e?con?CO02?accoppiato?a?Cas9? (5:1)?a?due?dosi?diverse,?ossia?3E+13vg/kg?e?1E+14vg/kg.?Gli?animali?sono?stati?sacrificati?a?5?mesi?di? et?.?Fegato,?reni?e?cuore?sono?stati?prelevati?al?momento?del?sacrificio,?insieme?al?sangue?e?al?plasma? raccolti?a?intervalli?di?tempo?intermedi?per?l'analisi?MS?dell'attivit??enzimatica?della?GLA?e?della?liso-Gb3?(Figura?7A).?? To treat a disease in its early or neonatal stages, an integrative approach is essential and can prove effective. Therefore, an experiment was designed in which neonatal male Fabry KO mice at P5 were treated with the donor WT vector and with CO02 coupled to Cas9 (5:1) at two different doses, namely 3E+13vg/kg and 1E+14vg/kg. The animals were sacrificed at 5 months of age. Liver, kidneys and heart were collected at the time of sacrifice, along with blood and plasma. collected at intermediate time intervals for MS analysis of GLA and lyso-Gb3 enzymatic activity (Figure 7A).

Il? livello?di?attivit??plasmatica?di?GLA?era? superiore?a?quello?dei? topi?WT? in? tutti? i? topi? trattati.??? importante?notare?che? i?topi?trattati?con? il?vettore?donatore?CO02?hanno?presentato?un?aumento? dell'attivit??della?GLA,?pi??evidente?nei?gruppi?trattati?con? la?dose?pi??bassa?(aumento?di?circa?10? volte;?Figura?7B).?La?determinazione?con?spettrometro?di?massa?dell'accumulo?di?liso?Gb3?nel?plasma? ha?mostrato? la?completa?correzione?alla?dose?pi??alta?di?AAV?per?entrambi? i?cDNA.?Negli?animali? trattati?con?la?dose?inferiore,?la?riduzione???stata?completa?per?il?vettore?CO02,?mentre?nel?gruppo? trattato?con?il?cDNA?WT?i?livelli?di?liso?Gb3?hanno?presentato?una?riduzione?del?75%,?rispetto?ai?topi? mutanti?non? trattati? (Figura?7C).?La?determinazione?del? tasso?d?integrazione?mediante?ddPCR?ha? indicato?valori?simili?per?entrambi?i?costrutti?donatori?(Figura?7D).? The? level? of? plasma? GLA? activity? was? higher? than? that? of? WT? mice? in? all? treated? mice.? Importantly? it? is? noted? that? mice? treated? with? the? donor? vector? CO02? showed? an? increase? in? GLA? activity,? more? evident? in? the? groups? treated? with? the? lower? dose? (approximately? 10-fold? increase;? Figure? 7B).? Mass? spectrometric? determination? of? accumulation? of? lyso? Gb3? in? plasma? showed? complete? correction? at? the? higher? dose? of? AAV? for? both? cDNAs.? In? the? animals? treated with the lower dose, the reduction was complete for the CO02 vector, while in the group treated with the WT cDNA, Gb3 lysosome levels showed a 75% reduction, compared to the untreated mutant mice (Figure 7C). Determination of the integration rate by ddPCR indicated similar values for both donor constructs (Figure 7D).

L'attivit??GLA?e?l'accumulo?di?liso?Gb3?sono?stati?determinati?nei?tessuti?dei?topi?trattati?(Figura?8).? L'attivit??nei?tessuti???risultata?in?tutti?i?casi?pi??elevata?nei?topi?trattati?con?il?cDNA?hGLA?CO02?(Figura? 8A).?In?linea?con?questi?risultati,?abbiamo?osservato?una?completa?eliminazione?dell'accumulo?di?liso? GLA activity and Gb3 lysate accumulation were determined in tissues of treated mice (Figure 8). Tissue activity was in all cases higher in mice treated with hGLA CO02 cDNA (Figure 8A). Consistent with these results, we observed a complete elimination of Gb3 lysate accumulation.

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Gb3? nei? topi? trattati? con? entrambe? le? dosi? del? vettore? donatore? cDNA? di? hGLA? CO02,?mentre? Gb3? in? mice? treated? with? both? doses? of? the? donor? cDNA? vector? of? hGLA? CO02,? while?

l'eliminazione?non???stata?completa?negli?animali?trattati?con?il?vettore?donatore?di?hGLA?WT?(Figura? elimination was not complete in animals treated with the hGLA WT donor vector (Figure

8B).? 8B).?

SEQUENZE? SEQUENCES?

cDNA?di?GLA?umano?wild?type? cDNA?of?human?GLA?wild?type?

ATGCAGCTGAGGAACCCAGAACTACATCTGGGCTGCGCGCTTGCGCTTCGCTTCCTGGCCCTCGTTTCCTGGG ACATCCCTGGGGCTAGAGCACTGGACAATGGATTGGCAAGGACGCCTACCATGGGCTGGCTGCACTGGGAG CGCTTCATGTGCAACCTTGACTGCCAGGAAGAGCCAGATTCCTGCATCAGTGAGAAGCTCTTCATGGAGATG GCAGAGCTCATGGTCTCAGAAGGCTGGAAGGATGCAGGTTATGAGTACCTCTGCATTGATGACTGTTGGATG GCTCCCCAAAGAGATTCAGAAGGCAGACTTCAGGCAGACCCTCAGCGCTTTCCTCATGGGATTCGCCAGCTG GCTAATTATGTTCACAGCAAAGGACTGAAGCTAGGGATTTATGCAGATGTTGGAAATAAAACCTGCGCAGGCT TCCCTGGGAGTTTTGGATACTACGACATTGATGCCCAGACCTTTGCTGACTGGGGAGTAGATCTGCTAAAATTT GATGGTTGTTACTGTGACAGTTTGGAAAATTTGGCAGATGGTTATAAGCACATGTCCTTGGCCCTGAATAGGA CTGGCAGAAGCATTGTGTACTCCTGTGAGTGGCCTCTTTATATGTGGCCCTTTCAAAAGCCCAATTATACAGAA ATCCGACAGTACTGCAATCACTGGCGAAATTTTGCTGACATTGATGATTCCTGGAAAAGTATAAAGAGTATCTT GGACTGGACATCTTTTAACCAGGAGAGAATTGTTGATGTTGCTGGACCAGGGGGTTGGAATGACCCAGATAT GTTAGTGATTGGCAACTTTGGCCTCAGCTGGAATCAGCAAGTAACTCAGATGGCCCTCTGGGCTATCATGGCT GCTCCTTTATTCATGTCTAATGACCTCCGACACATCAGCCCTCAAGCCAAAGCTCTCCTTCAGGATAAGGACGT AATTGCCATCAATCAGGACCCCTTGGGCAAGCAAGGGTACCAGCTTAGACAGGGAGACAACTTTGAAGTGTG GGAACGACCTCTCTCAGGCTTAGCCTGGGCTGTAGCTATGATAAACCGGCAGGAGATTGGTGGACCTCGCTCT TATACCATCGCAGTTGCTTCCCTGGGTAAAGGAGTGGCCTGTAATCCTGCCTGCTTCATCACACAGCTCCTCCC TGTGAAAAGGAAGCTAGGGTTCTATGAATGGACTTCAAGGTTAAGAAGTCACATAAATCCCACAGGCACTGTT TTGCTTCAGCTAGAAAATACAATGCAGATGTCATTAAAAGACTTACTTTAA?[SEQ?ID?N.16]? ATGCAGCTGAGGAACCCAGAACTACATCTGGGCTGCGCGCTTGCGCTTCGCTTCCTGGCCCTCGTTTCCTGGG ACATCCCTGGGGCTAGAGCACTGGACAATGGATTGGCAAGGACGCCTACCATGGGCTGGCTGCACTGGGAG CGCTTCATGTGCAACCTTGACTGCCAGGAAGAGCCAGATTCCTGCATCAGTGAGAAGCTCTTCATGGAGATG GCAGAGCTCATGGTCTCAGAAGGCTGGAAGGATGCAGGTTATGAGTACCTCTGCATTGATGACTGTTGGATG GCTCCCCAAAGAGATTCAGAAGGCAGACTTCAGGCAGACCCTCAGCGCTTTCCTCATGGGATTCGCCAGCTG GCTAATTATGTTCACAGCAAAGGACTGAAGCTAGGGATTTATGCAGATGTTGGAAATAAAACCTGCGCAGGCT TCCCTGGGAGTTTTGGATACTACGACATTGATGCCCAGACCTTTGCTGACTGGGGAGTAGATCTGCTAAAATTT GATGGTTGTTACTGTGACAGTTTGGAAAATTTGGCAGATGGTTATAAGCACATGTCCTTGGCCCTGAATAGGA CTGGCAGAAGCATTGTGTACTCCTGTGAGTGGCCTCTTTATATGTGGCCCTTTCAAAAGCCCAATTATACAGAA ATCCGACAGTACTGCAATCACTGGCGAAATTTTGCTGACATTGATGATTCCTGGAAAAGTATAAAGAGTATCTT GGACTGGACATCTTTTAACCAGGAGAGAATTGTTGATGTTGCTGGACCAGGGGGTTGGAATGACCCAGATAT GTTAGTGATTGGCAACTTTGGCCTCAGCTGGAATCAGCAAGTAACTCAGATGGCCCTCTGGGCTATCATGGCT GCTCCTTTATTCATGTCTAATGACCTCCGACACATCAGCCCTCAAGCCAAAGCTCTCCTTCAGGATAAGGACGT AATTGCCATCAATCAGGACCCCTTGGGCAAGCAAGGGTACCAGCTTAGACAGGGAGACAACTTTGAAGTGTG GGAACGACCTCTCTCAGGCTTAGCCTGGGCTGTAGCTATGATAAACCGGCAGGAGATTGGTGGACCTCGCTCT TATACCATCGCAGTTGCTTCCCTGGGTAAAGGAGTGGCCTGTAATCCTGCCTGCTTCATCACACAGCTCCTCCC TGTGAAAAGGAAGCTAGGGTTCTATGAATGGACTTCAAGGTTAAGAAGTCACATAAATCCCCAGGCACTGTT TTGCTTCAGCTAGAAAATACAATGCAGATGTCATTAAAAGACTTACTTTAA?[SEQ?ID?N.16]?

cDNA?di?GLA?umano?con?codoni?ottimizzati?02?(CO02)? Human GLA cDNA with optimized codons 02 (CO02)

ATGCAGCTGCGCAACCCCGAGCTGCACCTGGGCTGCGCCCTGGCCCTGCGCTTCCTGGCCCTGGTCAGCTGG GACATCCCCGGCGCCCGCGCCCTGGACAACGGCCTGGCCCGCACCCCCACCATGGGCTGGCTGCACTGGGA GCGCTTCATGTGCAACCTGGACTGCCAGGAGGAGCCCGACAGCTGCATCAGCGAGAAGCTGTTTATGGAGAT GGCCGAGCTGATGGTCAGCGAGGGCTGGAAGGACGCCGGCTACGAGTACCTGTGCATCGACGACTGCTGGA TGGCCCCCCAGCGCGACAGCGAGGGCCGCCTGCAGGCCGACCCCCAGCGCTTCCCCCACGGAATCCGCCAG CTGGCCAACTACGTGCACAGCAAGGGCCTGAAGCTGGGCATCTACGCCGACGTGGGCAACAAGACCTGCGC CGGCTTCCCCGGCAGCTTCGGCTACTACGACATCGACGCCCAGACCTTCGCCGACTGGGGCGTGGACCTGCT GAAGTTCGACGGCTGCTACTGCGACAGCCTGGAGAACCTGGCCGACGGCTACAAGCACATGAGCCTGGCCC TGAACCGCACCGGCCGCAGCATCGTGTACAGCTGCGAGTGGCCCCTGTATATGTGGCCCTTCCAGAAGCCCAA CTACACCGAGATCCGCCAGTACTGCAACCACTGGCGCAACTTCGCCGACATCGACGACAGCTGGAAGAGCAT CAAGAGCATCCTGGACTGGACCAGCTTCAACCAGGAGCGCATCGTGGACGTGGCCGGCCCCGGCGGCTGGA ACGACCCCGACATGCTGGTGATCGGCAACTTCGGCCTGAGCTGGAACCAGCAGGTGACCCAGATGGCCCTGT GGGCCATTATGGCCGCCCCCCTGTTTATGAGCAACGACCTGCGCCACATCAGCCCCCAGGCCAAGGCCCTGCT ATGCAGCTGCGCAACCCCGAGCTGCACCTGGGCTGCGCCCTGGCCCTGCGCTTCCTGGCCCTGGTCAGCTGG GACATCCCCGGCGCCCGCGCCCTGGACAACGGCCTGGCCCGCACCCCCACCATGGGCTGGCTGCACTGGGA GCGCTTCATGTGCAACCTGGACTGCCAGGAGGAGCCCGACAGCTGCATCAGCGAGAAGCTGTTTATGGAGAT GGCCGAGCTGATGGTCAGCGAGGGCTGGAAGGACGCCGGCTACGAGTACCTGTGCATCGACGACTGCTGGA TGGCCCCCCAGCGCGACAGCGAGGGCCGCCTGCAGGCCGACCCCCAGCGCTTCCCCCACGGAATCCGCCAG CTGGCCAACTACGTGCACAGCAAGGGCCTGAAGCTGGGCATCTACGCCGACGTGGGCAACAAGACCTGCGC CGGCTTCCCCGGCAGCTTCGGCTACTACGACATCGACGCCCAGACCTTCGCCGACTGGGGCGTGGACCTGCT GAAGTTCGACGGCTGCTACTGCGACAGCCTGGAGAACCTGGCCGACGGCTACAAGCACATGAGCCTGGCCC TGAACCGCACCGGCCGCAGCATCGTGTACAGCTGCGAGTGGCCCCTGTATATGTGGCCCTTCCAGAAGCCCAA CTACACCGAGATCCGCCAGTACTGCAACCACTGGCGCAACTTCGCCGACATCGACGACAGCTGGAAGAGCAT CAAGAGCATCCTGGACTGGACCAGCTTCAACCAGGAGCGCATCGTGGACGTGGCCGGCCCCGGCGGCTGGA ACGACCCCGACATGCTGGTGATCGGCAACTTCGGCCTGAGCTGGAACCAGCAGGTGACCCAGATGGCCCTGT GGGCCATTATGGCCGCCCCCCTGTTTATGAGCAACGACCTGCGCCACATCAGCCCCCAGGCCAAGGCCCTGCT

? ?

GCAGGACAAGGACGTGATCGCTATCAACCAGGACCCCCTGGGCAAGCAGGGCTACCAGCTGCGCCAGGGCG ACAACTTCGAGGTCTGGGAGCGCCCCCTGAGCGGCCTGGCCTGGGCCGTGGCTATGATCAACCGCCAGGAG ATCGGCGGCCCCCGCAGCTACACCATCGCCGTGGCCAGCCTGGGCAAGGGCGTGGCCTGCAACCCCGCCTG CTTCATCACCCAGCTGCTGCCCGTGAAGCGCAAGCTGGGCTTCTACGAGTGGACCAGCCGCCTGCGCAGCCA CATCAACCCCACCGGCACCGTGCTGCTGCAGCTGGAGAACACAATGCAGATGAGCCTGAAGGACCTGCTGTA GCAGGACAAGGACGTGATCGCTATCAACCAGGACCCCCTGGGCAAGCAGGGCTACCAGCTGCGCCAGGGCG ACAACTTCGAGGTCTGGGAGCGCCCCCTGAGCGGCCTGGCCTGGGCCGTGGCTATGATCAACCGCCAGGAG ATCGGCGGCCCCCGCAGCTACACCATCGCCGTGGCCAGCTTGGGCAAGGGCGTGGCCTGCAACCCCGCCTG CTTCATCACCCAGCTGCTGCCCGTGAAGCGCAAGCTGGGCTTCTACGAGTGGACCAGCCGCCTGCGCAGCCA CATCAACCCCACCGGCACCGTGCTGCTGCAGCTGGAGAACACAATGCAGATGAGCCTGAAGGACCTGCTGTA

A?[SEQ?ID?N.?1]? A?[SEQ?ID?N.?1]?

cDNA?di?GLA?umano?con?codoni?ottimizzati?03?(CO03)? Human GLA cDNA with optimized codons 03 (CO03)

ATGCAGTTGAGAAACCCAGAGCTCCACCTGGGCTGTGCCCTGGCACTGAGGTTCCTGGCCCTTGTGAGCTGG GATATCCCTGGGGCCAGGGCCTTGGACAACGGCTTGGCCCGCACCCCCACAATGGGCTGGCTGCACTGGGA ACGCTTTATGTGCAATCTGGACTGCCAGGAGGAGCCTGACAGCTGTATCAGCGAGAAGCTCTTTATGGAGATG GCAGAGCTGATGGTGTCTGAGGGATGGAAGGACGCCGGCTACGAATACCTGTGCATTGACGATTGCTGGATG GCTCCACAGAGGGACTCAGAAGGACGCCTGCAGGCTGATCCCCAGAGATTCCCCCATGGAATCCGCCAGCTG GCCAACTATGTGCACAGCAAAGGCCTGAAGCTGGGCATCTACGCCGACGTGGGCAACAAGACCTGTGCTGG CTTCCCTGGCTCCTTTGGATATTACGATATCGACGCTCAGACCTTTGCTGACTGGGGAGTGGATCTCCTCAAGT TTGACGGCTGCTACTGTGACTCTCTGGAAAACCTGGCAGATGGCTACAAGCACATGTCCCTGGCTCTGAACAG AACAGGCCGCAGCATTGTGTACAGCTGCGAGTGGCCCCTGTATATGTGGCCCTTCCAGAAGCCCAACTACACA GAGATCAGGCAGTACTGCAACCACTGGAGGAACTTTGCCGACATTGACGACTCCTGGAAATCTATCAAGTCTA TCCTGGATTGGACATCCTTCAACCAAGAGCGGATCGTGGACGTGGCTGGACCTGGAGGCTGGAATGATCCAG ATATGCTGGTGATTGGAAACTTCGGGCTGTCTTGGAACCAGCAGGTCACTCAGATGGCGCTGTGGGCCATCAT GGCCGCCCCCCTCTTTATGAGCAACGACCTGCGCCACATTTCTCCTCAAGCCAAGGCCCTGCTCCAGGACAAG GACGTCATCGCCATTAATCAGGATCCTCTGGGGAAGCAGGGCTACCAGCTTAGACAGGGAGACAATTTTGAG GTGTGGGAGAGGCCTCTCTCTGGACTTGCCTGGGCTGTGGCTATGATCAACCGGCAGGAAATTGGTGGCCCC CGCTCCTACACCATTGCTGTTGCCTCCTTGGGCAAGGGCGTGGCCTGTAACCCTGCCTGCTTCATCACCCAGCT CCTGCCTGTGAAGAGAAAACTGGGATTCTACGAGTGGACCAGCCGGCTGCGGAGCCACATCAATCCCACCGG CACCGTGCTGCTTCAGCTGGAGAACACCATGCAGATGTCACTGAAAGATCTGCTGTGA?[SEQ?ID?N.2]? ATGCAGTTGAGAAACCCAGAGCTCCACCTGGGCTGTGCCCTGGCACTGAGGTTCCTGGCCCTTGTGAGCTGG GATATCCCTGGGGCCAGGGCCTTGGACAACGGCTTGGCCCGCACCCCCACAATGGGCTGGCTGCACTGGGA ACGCTTTATGTGCAAATCTGGACTGCCAGGAGGAGCCTGACAGCTGTATCAGCGAGAAGCTCTTTATGGAGATG GCAGAGCTGATGGTGTCTGAGGGATGGAAGGACGCCGGCTACGAATACCTGTGCATTGACGATTGCTGGATG GCTCCACAGAGGGACTCAGAAGGACGCCTGCAGGCTGATCCCCAGAGATTCCCCCATGGAATCCGCCAGCTG GCCAACTATGTGCACAGCAAAGGCCTGAAGCTGGGCATCTACGCCGACGTGGGCAACAAGACCTGTGCTGG CTTCCCTGGCTCCTTTGGATATTACGATATCGACGCTCAGACCTTTGCTGACTGGGGAGTGGATCTCCTCAAGT TTGACGGCTGCTACTGTGACTCTCTGGAAAACCTGGCAGATGGCTACAAGCACATGTCCCTGGCTCTGAACAG AACAGGCCGCAGCATTGTGTACAGCTGCGAGTGGCCCCTGTATATGTGGCCCTTCCAGAAGCCCAACTACACA GAGATCAGGCAGTACTGCAACCACTGGAGGAACTTTGCCGACATTGACGACTCCTGGAAATCTATCAAGTCTA TCCTGGATTGGACATCCTTCAACCAAGAGCGGATCGTGGACGTGGCTGGACCTGGAGGCTGGAATGATCCAG ATATGCTGGTGATTGGAAACTTCGGGCTGTCTTGGAACCAGCAGGTCACTCAGATGGCGCTGTGGGCCATCAT GGCCGCCCCCCTCTTTATGAGCAACGACCTGCGCCACATTTCTCCTCAAGCCAAGGCCCTGCTCCAGGACAAG GACGTCATCGCCATTAATCAGGATCCTCTGGGGAAGCAGGGCTACCAGCTTAGACAGGGAGACAATTTTGAG GTGTGGGAGAGGCCTCTCTCTGGACTTGCCTGGGCTGTGGCTATGATCAACCGGCAGGAAATTGGTGGCCCC CGCTCCTACACCATTGCTGTTGCCTCCTTGGGCAAGGGCGTGGCCTGTAACCCTGCCTGCTTCATCACCCAGCT CCTGCCTGTGAAGAAAACTGGGATTCTACGAGTGGACCAGCCGGCTGCGGAGCCACATCAATCCCACCGG CACCGTGCTGCTTCAGCTGGAGAACACCATGCAGATGTCACTGAAAGATCTGCTGTGA?[SEQ?ID?N.2]?

cDNA?di?GLA?umano?con?codoni?ottimizzati?01?(CO01)? Human GLA cDNA with optimized codons 01 (CO01)

ATGCAGCTCCGCAACCCAGAGCTCCATCTTGGGTGTGCTCTCGCTCTTCGATTCCTTGCACTGGTCAGTTGGG ATATCCCGGGAGCTAGAGCTTTGGATAACGGTCTCGCACGCACTCCCACAATGGGATGGCTTCACTGGGAGC GATTTATGTGCAACCTGGACTGCCAGGAAGAGCCGGATAGCTGTATATCTGAGAAGCTTTTTATGGAGATGGC GGAATTGATGGTCAGTGAAGGCTGGAAAGACGCGGGCTACGAATATCTCTGTATCGACGATTGTTGGATGGC ACCACAACGCGATAGCGAAGGCAGGCTCCAGGCTGATCCACAGAGGTTTCCCCACGGAATACGACAGCTGG ATGCAGCTCCGCAACCCAGAGCTCCATCTTGGGTGTGCTCTCGCTCTTCGATTCCTTGCACTGGTCAGTTGGG ATATCCCGGGAGCTAGAGCTTTGGATAACGGTCTCGCACGCACTCCCACAATGGGATGGCTTCACTGGGAGC GATTTATGTGCAACCTGGACTGCCAGGAAGAGCCGGATAGCTTGTATATCTGAGAAGCTTTTTATGGAGATGGC GGAATTGATGGTCAGTGAAGGCTGGAAAGACGCGGGCTACGAATATCTCTGTATCGACGATTGTTGGATGGC ACCACAACGCGATAGCGAAGGCAGGCTCCAGGCTGATCCACAGAGGTTTCCCCACGGAATACGACAGCTGG

? ?

CTAACTATGTGCACAGCAAGGGCCTCAAACTGGGAATCTACGCTGACGTGGGCAATAAGACGTGCGCCGGTT CTAACTATGTGCACAGCAAGGGCCTCAAACTGGGAATCTACGCTGACGTGGGCAATAAGACGTGCGCCGGTT

TCCCGGGGTCTTTCGGTTACTACGACATTGACGCCCAAACTTTTGCTGACTGGGGTGTGGATCTTCTCAAGTT TCCCGGGGTCTTTCGGTTACTACGACATTGACGCCCAAACTTTTGCTGACTGGGGTGTGGATCTTCTCAAGTT

TGACGGCTGTTACTGCGACTCCCTCGAAAATTTGGCTGATGGTTACAAGCACATGTCTCTTGCCTTGAATCGCA TGACGGCTGTTACTGCGACTCCCTCGAAAATTTGGCTGATGGTTACAAGCACATGTCTCTTGCCTTGAATCGCA

CCGGCCGCTCCATCGTGTACTCTTGCGAGTGGCCGTTGTATATGTGGCCCTTTCAAAAACCGAACTACACAGA CCGGCCGCTCCATCGTGTACTCTTGCGAGTGGCCGTTGTATATGTGGCCCTTTCAAAAACCGAACTACACAGA

AATAAGACAGTATTGCAACCACTGGAGAAACTTCGCTGATATCGACGATAGCTGGAAATCTATTAAATCTATTCT AATAAGACAGTATTGCAACCACTGGAGAAACTTCGCTGATATCGACGATAGCTGGAAATCTATTAAATCTATTCT

TGATTGGACGAGTTTTAATCAAGAGCGAATTGTGGACGTTGCGGGGCCGGGAGGGTGGAACGACCCCGATA TGATTGGACGAGTTTTAATCAAGAGCGAATTGTGGACGTGCGGGGCCGGGAGGGTGGAACGACCCCGATA

TGCTGGTTATCGGAAATTTTGGCCTTTCCTGGAATCAGCAGGTTACCCAGATGGCCCTGTGGGCTATTATGGCC TGCTGGTTATCGGAAATTTTGGCCTTTCCTGGAATCAGCAGGTTACCCAGATGGCCCTGTGGGCTATTATGGCC

GCTCCACTCTTCATGAGCAATGATTTGCGCCACATCAGTCCACAAGCGAAGGCTCTCTTGCAGGATAAGGATG GCTCCACTCTTCATGAGCAATGATTTGCGCCACATCAGTCCACAAGCGAAGGCTCTCTTGCAGGATAAGGATG

TGATTGCTATCAACCAAGATCCGCTGGGCAAGCAGGGGTATCAGTTGAGACAAGGAGATAACTTCGAAGTTT TGATTGCTATCAACCAAGATCCGCTGGGCAAGCAGGGGTATCAGTTGAGACAAGGAGATAACTTCGAAGTTT

GGGAGCGGCCCCTGAGTGGTTTGGCCTGGGCAGTGGCGATGATAAATCGACAAGAAATAGGAGGACCCAG GGGAGCGGCCCCTGAGTGGTTTGGCCTGGGCAGTGGCGATGATAAATCGACAAGAAATAGGAGGACCCAG

GAGTTATACTATTGCTGTAGCATCCCTTGGGAAAGGTGTCGCGTGTAACCCCGCTTGTTTTATTACACAACTGCT GAGTTATACTATTGCTGTAGCATCCCTTGGGAAAGGTGTCGCGTGTAACCCCGCTTGTTTTATTACACAACTGCT

GCCTGTTAAGAGAAAACTGGGCTTTTACGAGTGGACCTCTCGGCTCAGATCCCACATCAACCCGACAGGCAC GCCTGTTAAGAGAAAACTGGGCTTTTACGAGTGGACCTCTCGGCTCAGATCCCACATCAACCCGACAGGCAC

CGTTCTTCTGCAACTGGAGAATACGATGCAGATGAGCCTCAAGGACTTGTTGTAA?[SEQ?ID?N.3]? CGTTCTTCTGCAACTGGAGAATACGATGCAGATGAGCCTCAAGGACTTGTTGTAA?[SEQ?ID?N.3]?

Elementi?del?vettore?pSMD2:? Elements of the pSMD2 vector:

? ?

5??AAV2?ITR? Ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcg cagagagggagtggccaactccatcactaggggttcct?[SEQ?ID?N.7]? 5??AAV2?ITR? Ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcg cagagagggagtggccaactccatcactaggggttcct?[SEQ?ID?N.7]?

? ?

Enhancer???regione?di?controllo?di?ApoE?? Enhancer???ApoE?control?region?

Aaggctcagaggcacacaggagtttctgggctcaccctgcccccttccaacccctcagttcccatcctccagcagctgtttgtgtgctgcctctga agtccacactgaacaaacttcagcctactcatgtccctaaaatgggcaaacattgcaagcagcaaacagcaaacacacagccctccctgcctg ctgaccttggagctggggcagaggtcagagacctctctgggcccatgccacctccaacatccactcgaccccttggaatttcggtggagagga gcagaggttgtcctggcgtggtttaggtagtgtgagaggg?[SEQ?ID?N.5]? Aaggctcagaggcacacaggagtttctgggctcaccctgcccccttccaacccctcagttcccatcctccagcagctgtttgtgtgctgcctctga agtccacactgaacaaacttcagcctactcatgtccctaaaatgggcaaacattgcaagcagcaaacagcaaacacacagccctccctgcctg ctgaccttggagctggggcagaggtcagagacctctctgggcccatgccacctccaacatccactcgaccccttggaatttcggtggagagga gcagaggttgtcctggcgtggtttaggtagtgtgagaggg?[SEQ?ID?N.5]?

? ?

Promotore?hAAT?e?primo?esone? hAAT promoter and first exon

GATCTTGCTACCAGTGGAACAGCCACTAAGGATTCTGCAGTGAGAGCAGAGGGCCAGCTAAGTGGTACTCTC CCAGAGACTGTCTGACTCACGCCACCCCCTCCACCTTGGACACAGGACGCTGTGGTTTCTGAGCCAGGTACA ATGACTCCTTTCGGTAAGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGCGTCCGGGCAGCGTAGGCGGG CGACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCTTGGTTAATATTC ACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGACAGGGccctgtctcctcagctt caggcaccaccactgacctgggacagtgaat?[SEQ?ID?N.4]? GATCTTGCTACCAGTGGAACAGCCACTAAGGATTCTGCAGTGAGAGCAGAGGGCCAGCTAAGTGGTACTCTC CCAGAGACTGTCTGACTCACGCCACCCCCTCCACCTTGGACACAGGACGCTGTGGTTTCTGAGCCAGGTACA ATGACTCCTTTCGGTAAGTGCAGTGGGAAGCTGTACACTGCCCAGGCAAAGCGTCCGGGCAGCGTAGGCGGG CGACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTGCTCCTCCGATAACTGGGGTGACCTTGGTTAATATTC ACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGACAGGGccctgtctcctcagctt caggcaccaccactgacctgggacagtgaat?[SEQ?ID?N.4]?

? ?

Introne?dell'emoglobina?beta?umana?(HBB2)?modificato? Modified human hemoglobin beta (HBB2) intron

? ?

? ?

Gtacacatattgaccaaatcagggtaattttgcatttgtaattttaaaaaatgctttcttcttttaatatacttttttgtttatcttatttctaatactttc cctaatctctttctttcagggcaatattgatacaatgtatcttgcctctttgcaccattctaaagaataacagtgataatttctgggttaaggcaata gcaatatttctgcatataaatatttctgcatataaattgtaactgatgtaagaggtttcatattgctaatagcagctacaatccagctaccattctg cttttattttctggttgggataaggctggattattctgagtccaagctaggcccttttgctaatcttgttcatacctcttatcttcctcccacagctcct gggcaacctgctggtctctctgctggcccatcactttggcaaag?[SEQ?ID?N.9]? Gtacacatattgaccaaatcagggtaattttgcatttgtaattttaaaaaatgctttcttcttttaatatacttttttgtttatcttatttctaatactttc cctaatctctttctttcagggcaatattgatacaatgtatcttgcctctttgcaccattctaaagaataacagtgataatttctgggttaaggcaata gcaatatttctgcatataaatatttctgcatataaattgtaactgatgtaagaggtttcatattgctaatagcagctacaatccagctaccattctg cttttattttctggttgggataaggctggattattctgagtccaagctaggcccttttgctaatcttgttcatacctcttatcttcctcccacagctcct gggcaacctgctggtctctctgctggcccatcactttggcaaag?[SEQ?ID?N.9]?

? ?

HBB2?poli?A? HBB2?poli?A?

Gaattcaccccaccagtgcaggctgcctatcagaaagtggtggctggtgtggctaatgccctggcccacaagtatcactaagctcgctttcttg ctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaat aaaaaacatttattttcattgcaatgatgtatttaaattatttctgaatattttactaaaaagggaatgtgggaggtcagtgcatttaaaacataa agaaatgaagagctagttcaaaccttgggaaaatacactatatcttaaactccatgaaagaaggtgaggctgcaaacagctaatgcacattg gcaacagcccctgatgcctatgccttattcatccctcagaaaaggattcaagtagaggcttgatttggaggttaaagtttggctatgctgtatttt acattacttattgttttagctgtcctcatgaatgtcttttcactacccatttgcttatcctgcatctctcagccttgactccactcagttctcttgcttag agataccacctttcccctgaagtgttccttccatgttttacggcgagatggtttctcctcgcctggccactcagccttagttgtctctgttgtcttata gaggtctacttgaagaaggaaaaacagggggcatggtttgactgtcctgtgagcccttcttccctgcctcccccactcacagtgacccggaatc? [SEQ?ID?N.6]? Gaattcaccccaccagtgcaggctgcctatcagaaagtggtggctggtgtggctaatgccctggcccacaagtatcactaagctcgctttcttg ctgtccaatttctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaat aaaaaacatttattttcattgcaatgatgtatttaaattatttctgaatattttactaaaaagggaatgtgggaggtcagtgcatttaaaacataa agaaatgaagagctagttcaaaccttgggaaaatacactatatcttaaactccatgaaagaaggtgaggctgcaaacagctaatgcacattg gcaacagcccctgatgcctatgccttattcatccctcagaaaaggattcaagtagaggcttgatttggaggttaaagtttggctatgctgtatttt acattacttattgttttagctgtcctcatgaatgtcttttcactacccatttgcttatcctgcatctctcagccttgactccactcagttctcttgcttag agataccacctttcccctgaagtgttccttccatgttttacggcgagatggtttctcctcgcctggccactcagccttagttgtctctgttgtcttata gaggtctacttgaagaaggaaaaacagggggcatggtttgactgtcctgtgagcccttcttccctgcctccccccactcacagtgacccggaatc? [SEQ?ID?N.6]?

? ?

3??AAV2?ITR? 3??AAV2?ITR?

Aggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggct ttgcccgggcggcctcagtgagcgagcgagcgcgc?[SEQ?ID?N.8]? Aggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggct ttgcccgggcggcctcagtgagcgagcgagcgcgc?[SEQ?ID?N.8]?

? ?

Elementi?del?vettore?pAB288?modificato:? Elements of the modified pAB288 vector:

? ?

5??AAV2?ITR? 5??AAV2?ITR?

Ttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtg agcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcct?[SEQ?ID?N.11]? Ttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtg agcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcct?[SEQ?ID?N.11]?

? ?

albumina?di?topo???braccio?di?omologia?sinistro? mouse?albumin?left?homology?arm?

? ?

tgtacataggaggttcgaaccctgctgaagggagaggttccaatactacaaaatgtagcgggatattgtcatcacctttggggacatgtcatca tggtccccagacagagttacaaaactcatcccctacacagcactatgtctctggtactgtttgttctacagatgtcaacaacagaggcccagcca tctcctattgcttggcttgtcagtctttctagcctccccattattaatttcaaatggggcaggtgttaggagggcaaaaatccacatattaagtgca aagcctttcaggagatttcctgaaactagacaaaacccgtgtgactggcatcgattattctatttgatctagctagtcctagcaaagtgacaact gctactcccctcctacacagccaagattcctaagttggcagtggcatgcttaatcctcaaagccaaagttacttggctccaagatttatagcctta aactgtggcctcacattccttcctatcttactttcctgcactggggtaaatgtctccttgctcttcttgctttctgtcctactgcagGGCTCTTGCT GAGCTGGTGAAGCACAAGCCCAAGGCTACAGCGGAGCAACTGAAGACTGTCATGGATGACTTTGCACAGTT CCTGGATACATGTTGCAAGGCTGCTGACAAGGACACCTGCTTCTCGACTGAGgtcagaaacgtttttgcattttgacgat gttcagtttccattttctgtgcacgtggtcaggtgtagctctctggaactcacacactgaataactccaccaatctagatgttgttctctacgtaact gtaatagaaactgacttacgtagcttttaatttttattttctgccacactgctgcctattaaatacctattatcactatttggtttcaaatttgtgacac agaagagcatagttagaaatacttgcaaagcctagaatcatgaactcatttaaaccttgccctgaaatgtttctttttgaattgagttattttacac atgaatggacagttaccattatatatctgaatcatttcacattccctcccatggcctaacaacagtttatcttcttattttgggcacaacagatgtca gagagcctgctttaggaattctaagtagaactgtaattaagcaatgcaaggcacgtacgtttactatgtcattgcctatggctatgaagtgcaaa tgtacataggaggttcgaaccctgctgaagggagaggttccaatactacaaaatgtagcgggatattgtcatcacctttggggacatgtcatca tggtccccagacagagttacaaaactcatcccctacacagcactatgtctctggtactgtttgttctacagatgtcaacaacagaggcccagcca tctcctattgcttggcttgtcagtctttctagcctccccattattaatttcaaatggggcaggtgttaggagggcaaaaatccacatattaagtgca aagcctttcaggagatttcctgaaactagacaaaacccgtgtgactggcatcgattattctatttgatctagctagtcctagcaaagtgacaact gctactcccctcctacacagccaagattcctaagttggcagtggcatgcttaatcctcaaagccaaagttacttggctccaagatttatagcctta aactgtggcctcacattccttcctatcttactttcctgcactggggtaaatgtctccttgctcttcttgctttctgtcctactgcagGGCTCTTGCT GAGCTGGTGAAGCACAAGCCCAAGGCTACAGCGGAGCAACTGAAGACTGTCATGGATGACTTTGCACAGTT CCTGGATACATGTTGCAAGGCTGCTGACAAGGACACCTGCTTCTCGACTGAGgtcagaaacgtttttgcattttgacgat gttcagtttccattttctgtgcacgtggtcaggtgtagctctctggaactcacacactgaataactccaccaatctagatgttgttctctacgtaact gtaatagaaactgacttacgtagcttttaatttttattttctgccacactgctgcctattaaatacctattatcactatttggtttcaaatttgtgacac agaagagcatagttagaaatacttgcaaagcctagaatcatgaactcatttaaaccttgccctgaaatgtttctttttgaattgagttattttacac atgaatggacagttaccattatatatctgaatcatttcacattccctcccatggcctaacaacagtttatcttcttattttgggcacaacagatgtca gagagcctgctttaggaattctaagtagaactgtaattaagcaatgcaaggcacgtacgtttactatgtcattgcctatggctatgaagtgcaaa

? ?

tcctaACAGTCCTGCTAATACTTTTCtaacatccatcatttctttgttttcagGGTCCAAACCTTGTCACTAGATGCAAAGAC GCCTTAGCC?[SEQ?ID?N.12]? tcctaACAGTCCTGCTAATACTTTTCtaacatccatcatttctttgttttcagGGTCCAAACCTTGTCACTAGATGCAAAGAC GCCTTAGCC?[SEQ?ID?N.12]?

? ?

P2A? P2A?

Ggaagcggcgccaccaatttcagcctgctgaaacaggccggcgacgtggaagagaaccctggccct?[SEQ?ID?N.13]? Ggaagcggcgccaccaatttcagcctgctgaaacaggccggcgacgtggaagagaaccctggccct?[SEQ?ID?N.13]?

? ?

albumina?di?topo???braccio?di?omologia?destro?? mouse?albumin?right?homology?arm?

ttagccTAAacacatcacaaccacaaccttctcaggtaactatacttgggacttaaaaaacataatcataatcatttttcctaaaacgatcaag actgataaccatttgacaagagccatacagacaagcaccagctggcTCTCGAGCGTCTTCACGTATGGTCATCagtttgggttccat ttgtagataagaaactgaacatataaaggtctaggttaatgcaatttacacaaaaggagaccaaaccagggagagaaggaaccaaaattaa aaattcaaaccagagcaaaggagttagccctggttttgctctgacttacatgaaccactatgtggagtcctccatgttagcctagtcaagcttatc ctctggatgaagttgaaaccatatgaaggaatatttggggggtgggtcaaaacagttgtgtatcaatgattccatgtggtttgacccaatcattct gtgaatccatttcaacagaagatacaacgggttctgtttcataataagtgatccacttccaaatttctgatgtgccccatgctaagctttaacaga atttatcttcttatgacaaagcagcctcctttgaaaatatagccaactgcacacagctatgttgatcaattttgtttataatcttgcagaagagaat tttttaaaatagggcaataatggaaggctttggcaaaaaaattgtttctccatatgaaaacaaaaaacttatttttttattcaagcaaagaacct atagacataaggctatttcaaaattatttcagttttagaaagaattgaaagttttgtagcattctgagaagacagctttcatttgtaatcataggta atatgtaggtcctcagaaatggtgagacccctgactttgacacttggggactctgagggaccagtgatgaagagggcacaacttatatcacac atgcacgagttggggtgagagggtgtcacaacatctatcagtgtgtcatctgcccaccaagtaacagatgtcagctaagactaggtcatgtgta ggctgtctacaccagtgaaaatcgcaaaaagaatctaagaaattccacatttctagaaaataggtttggaaaccgtattccattttacaaagga cacttacatttctctttttgttttccagGCTACCCTGAGAAAAAAAGACATGAAGACTCAGGACTCATCTTTTCTGTTGGT GTAAAATCAACACCCTAAGGAACACAAATTTCTTTAAACATTTGACTTCTTGTCTCTGTGCTGCAATTAATAAAA AATGGAAAGAATCTACtctgtggttcagaactctatcttccaaaggcgcgcttcaccctagcagcctctttggctcagaggaatccctgcc tttcctcccttcatctcagcagagaatgtagttccacatggg?[SEQ?ID?N.14]? ? ttagccTAAacacatcacaaccacaaccttctcaggtaactatacttgggacttaaaaaacataatcataatcatttttcctaaaacgatcaag actgataaccatttgacaagagccatacagacaagcaccagctggcTCTCGAGCGTCTTCACGTATGGTCATCagtttgggttccat ttgtagataagaaactgaacatataaaggtctaggttaatgcaatttacacaaaaggagaccaaaccagggagagaaggaaccaaaattaa aaattcaaaccagagcaaaggagttagccctggttttgctctgacttacatgaaccactatgtggagtcctccatgttagcctagtcaagcttatc ctctggatgaagttgaaaccatatgaaggaatatttggggggtgggtcaaaacagttgtgtatcaatgattccatgtggtttgacccaatcattct gtgaatccatttcaacagaagatacaacgggttctgtttcataataagtgatccacttccaaatttctgatgtgccccatgctaagctttaacaga atttatcttcttatgacaaagcagcctcctttgaaaatatagccaactgcacacagctatgttgatcaattttgtttataatcttgcagaagagaat tttttaaaatagggcaataatggaaggctttggcaaaaaaattgtttctccatatgaaaacaaaaaacttatttttttattcaagcaaagaacct atagacataaggctatttcaaaattatttcagttttagaaagaattgaaagttttgtagcattctgagaagacagctttcatttgtaatcataggta atatgtaggtcctcagaaatggtgagacccctgactttgacacttggggactctgagggaccagtgatgaagagggcacaacttatatcacac atgcacgagttggggtgagagggtgtcacaacatctatcagtgtgtcatctgcccaccaagtaacagatgtcagctaagactaggtcatgtgta ggctgtctacaccagtgaaaatcgcaaaaagaatctaagaaattccacatttctagaaaataggtttggaaaccgtattccattttacaaagga cacttacatttctctttttgttttccagGCTACCCTGAGAAAAAAAGACATGAAGACTCAGGACTCATCTTTTCTGTTGGT GTAAAATCAACACCCTAAGGAACACAAATTTCTTTAAACATTTGACTTCTTGTCTCTGTGCTGCAATTAATAAAA AATGGAAAGAATCTACtctgtggttcagaactctatcttccaaaggcgcgcttcaccctagcagcctctttggctcagaggaatccctgcc tttcctcccttcatctcagcagagaatgtagttccacatggg?[SEQ?ID?N.14]? ?

? ?

3??AAV2?ITR?? 3??AAV2?ITR??

Aggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacct ttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaa?[SEQ?ID?N.19]? ? Aggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacct ttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaa?[SEQ?ID?N.19]? ?

? ?

plasmide?pAB?hGLA?WILD?TYPE? plasmid?pAB?hGLA?WILD?TYPE?

ttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtga gcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctggaggggtggagtcgtgacgtaaagatctgatatcatcgat cgcgatgcattaattaagcggccgctgtacataggaggttcgaaccctgctgaagggagaggttccaatactacaaaatgtagcgggatattgt catcacctttggggacatgtcatcatggtccccagacagagttacaaaactcatcccctacacagcactatgtctctggtactgtttgttctacag atgtcaacaacagaggcccagccatctcctattgcttggcttgtcagtctttctagcctccccattattaatttcaaatggggcaggtgttaggag ggcaaaaatccacatattaagtgcaaagcctttcaggagatttcctgaaactagacaaaacccgtgtgactggcatcgattattctatttgatct agctagtcctagcaaagtgacaactgctactcccctcctacacagccaagattcctaagttggcagtggcatgcttaatcctcaaagccaaagt tacttggctccaagatttatagccttaaactgtggcctcacattccttcctatcttactttcctgcactggggtaaatgtctccttgctcttcttgcttt ctgtcctactgcagGGCTCTTGCTGAGCTGGTGAAGCACAAGCCCAAGGCTACAGCGGAGCAACTGAAGACTGTC ATGGATGACTTTGCACAGTTCCTGGATACATGTTGCAAGGCTGCTGACAAGGACACCTGCTTCTCGACTGAGg ttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtga gcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctggaggggtggagtcgtgacgtaaagatctgatatcatcgat cgcgatgcattaattaagcggccgctgtacataggaggttcgaaccctgctgaagggagaggttccaatactacaaaatgtagcgggatattgt catcacctttggggacatgtcatcatggtccccagacagagttacaaaactcatcccctacacagcactatgtctctggtactgtttgttctacag atgtcaacaacagaggcccagccatctcctattgcttggcttgtcagtctttctagcctccccattattaatttcaaatggggcaggtgttaggag ggcaaaaatccacatattaagtgcaaagcctttcaggagatttcctgaaactagacaaaacccgtgtgactggcatcgattattctatttgatct agctagtcctagcaaagtgacaactgctactcccctcctacacagccaagattcctaagttggcagtggcatgcttaatcctcaaagccaaagt tacttggctccaagatttatagccttaaactgtggcctcacattccttcctatcttactttcctgcactggggtaaatgtctccttgctcttcttgcttt ctgtcctactgcagGGCTCTTGCTGAGCTGGTGAAGCACAAGCCCAAGGCTACAGCGGAGCAACTGAAGACTGTC ATGGATGACTTTGCACAGTTCCTGGATACATGTTGCAAGGCTGCTGACAAGGACACCTGCTTCTCGACTGAGg

? ?

tcagaaacgtttttgcattttgacgatgttcagtttccattttctgtgcacgtggtcaggtgtagctctctggaactcacacactgaataactccacc aatctagatgttgttctctacgtaactgtaatagaaactgacttacgtagcttttaatttttattttctgccacactgctgcctattaaatacctattat cactatttggtttcaaatttgtgacacagaagagcatagttagaaatacttgcaaagcctagaatcatgaactcatttaaaccttgccctgaaat gtttctttttgaattgagttattttacacatgaatggacagttaccattatatatctgaatcatttcacattccctcccatggcctaacaacagtttat cttcttattttgggcacaacagatgtcagagagcctgctttaggaattctaagtagaactgtaattaagcaatgcaaggcacgtacgtttactatg tcattgcctatggctatgaagtgcaaatcctaACAGTCCTGCTAATACTTTTCtaacatccatcatttctttgttttcagGGTCCAAAC CTTGTCACTAGATGCAAAGACGCCTTAGCCggaagcggcgccaccaatttcagcctgctgaaacaggccggcgacgtggaagag aaccctggccctGCTAGCCAGCTGAGGAACCCAGAACTACATCTGGGCTGCGCGCTTGCGCTTCGCTTCCTGGCC CTCGTTTCCTGGGACATCCCTGGGGCTAGAGCACTGGACAATGGATTGGCAAGGACGCCTACCATGGGCTGG CTGCACTGGGAGCGCTTCATGTGCAACCTTGACTGCCAGGAAGAGCCAGATTCCTGCATCAGTGAGAAGCTC TTCATGGAGATGGCAGAGCTCATGGTCTCAGAAGGCTGGAAGGATGCAGGTTATGAGTACCTCTGCATTGATG ACTGTTGGATGGCTCCCCAAAGAGATTCAGAAGGCAGACTTCAGGCAGACCCTCAGCGCTTTCCTCATGGGA TTCGCCAGCTGGCTAATTATGTTCACAGCAAAGGACTGAAGCTAGGGATTTATGCAGATGTTGGAAATAAAAC CTGCGCAGGCTTCCCTGGGAGTTTTGGATACTACGACATTGATGCCCAGACCTTTGCTGACTGGGGAGTAGAT CTGCTAAAATTTGATGGTTGTTACTGTGACAGTTTGGAAAATTTGGCAGATGGTTATAAGCACATGTCCTTGGC CCTGAATAGGACTGGCAGAAGCATTGTGTACTCCTGTGAGTGGCCTCTTTATATGTGGCCCTTTCAAAAGCCCA ATTATACAGAAATCCGACAGTACTGCAATCACTGGCGAAATTTTGCTGACATTGATGATTCCTGGAAAAGTATA AAGAGTATCTTGGACTGGACATCTTTTAACCAGGAGAGAATTGTTGATGTTGCTGGACCAGGGGGTTGGAAT GACCCAGATATGTTAGTGATTGGCAACTTTGGCCTCAGCTGGAATCAGCAAGTAACTCAGATGGCCCTCTGGG CTATCATGGCTGCTCCTTTATTCATGTCTAATGACCTCCGACACATCAGCCCTCAAGCCAAAGCTCTCCTTCAGG ATAAGGACGTAATTGCCATCAATCAGGACCCCTTGGGCAAGCAAGGGTACCAGCTTAGACAGGGAGACAACT TTGAAGTGTGGGAACGACCTCTCTCAGGCTTAGCCTGGGCTGTAGCTATGATAAACCGGCAGGAGATTGGTG GACCTCGCTCTTATACCATCGCAGTTGCTTCCCTGGGTAAAGGAGTGGCCTGTAATCCTGCCTGCTTCATCACA CAGCTCCTCCCTGTGAAAAGGAAGCTAGGGTTCTATGAATGGACTTCAAGGTTAAGAAGTCACATAAATCCCA CAGGCACTGTTTTGCTTCAGCTAGAAAATACAATGCAGATGTCATTAAAAGACTTACTTTAAtgagctAGCttagcc TAAacacatcacaaccacaaccttctcaggtaactatacttgggacttaaaaaacataatcataatcatttttcctaaaacgatcaagactgat aaccatttgacaagagccatacagacaagcaccagctggcTCTCGAGCGTCTTCACGTATGGTCATCagtttgggttccatttgtag ataagaaactgaacatataaaggtctaggttaatgcaatttacacaaaaggagaccaaaccagggagagaaggaaccaaaattaaaaattc aaaccagagcaaaggagttagccctggttttgctctgacttacatgaaccactatgtggagtcctccatgttagcctagtcaagcttatcctctgg atgaagttgaaaccatatgaaggaatatttggggggtgggtcaaaacagttgtgtatcaatgattccatgtggtttgacccaatcattctgtgaa tccatttcaacagaagatacaacgggttctgtttcataataagtgatccacttccaaatttctgatgtgccccatgctaagctttaacagaatttat cttcttatgacaaagcagcctcctttgaaaatatagccaactgcacacagctatgttgatcaattttgtttataatcttgcagaagagaatttttta aaatagggcaataatggaaggctttggcaaaaaaattgtttctccatatgaaaacaaaaaacttatttttttattcaagcaaagaacctataga cataaggctatttcaaaattatttcagttttagaaagaattgaaagttttgtagcattctgagaagacagctttcatttgtaatcataggtaatatgt aggtcctcagaaatggtgagacccctgactttgacacttggggactctgagggaccagtgatgaagagggcacaacttatatcacacatgcac gagttggggtgagagggtgtcacaacatctatcagtgtgtcatctgcccaccaagtaacagatgtcagctaagactaggtcatgtgtaggctgt ctacaccagtgaaaatcgcaaaaagaatctaagaaattccacatttctagaaaataggtttggaaaccgtattccattttacaaaggacactta catttctctttttgttttccagGCTACCCTGAGAAAAAAAGACATGAAGACTCAGGACTCATCTTTTCTGTTGGTGTAAA ATCAACACCCTAAGGAACACAAATTTCTTTAAACATTTGACTTCTTGTCTCTGTGCTGCAATTAATAAAAAATGG AAAGAATCTACtctgtggttcagaactctatcttccaaaggcgcgcttcaccctagcagcctctttggctcagaggaatccctgcctttcctcc cttcatctcagcagagaatgtagttccacatgggactagtgtacacgcgtgatatcagatctgttacgtagataagtagcatggcgggttaatca ttaactacaaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcg tcagaaacgtttttgcattttgacgatgttcagtttccattttctgtgcacgtggtcaggtgtagctctctggaactcacacactgaataactccacc aatctagatgttgttctctacgtaactgtaatagaaactgacttacgtagcttttaatttttattttctgccacactgctgcctattaaatacctattat cactatttggtttcaaatttgtgacacagaagagcatagttagaaatacttgcaaagcctagaatcatgaactcatttaaaccttgccctgaaat gtttctttttgaattgagttattttacacatgaatggacagttaccattatatatctgaatcatttcacattccctcccatggcctaacaacagtttat cttcttattttgggcacaacagatgtcagagagcctgctttaggaattctaagtagaactgtaattaagcaatgcaaggcacgtacgtttactatg tcattgcctatggctatgaagtgcaaatcctaACAGTCCTGCTAATACTTTTCtaacatccatcatttctttgttttcagGGTCCAAAC CTTGTCACTAGATGCAAAGACGCCTTAGCCggaagcggcgccaccaatttcagcctgctgaaacaggccggcgacgtggaagag aaccctggccctGCTAGCCAGCTGAGGAACCCAGAACTACATCTGGGCTGCGCGCTTGCGCTTCGCTTCCTGGCC CTCGTTTCCTGGGACATCCCTGGGGCTAGAGCACTGGACAATGGATTGGCAAGGACGCCTACCATGGGCTGG CTGCACTGGGAGCGCTTCATGTGCAACCTTGACTGCCAGGAAGAGCCAGATTCCTGCATCAGTGAGAAGCTC TTCATGGAGATGGCAGAGCTCATGGTCTCAGAAGGCTGGAAGGATGCAGGTTATGAGTACCTCTGCATTGATG ACTGTTGGATGGCTCCCCAAAGAGATTCAGAAGGCAGACTTCAGGCAGACCCTCAGCGCTTTCCTCATGGGA TTCGCCAGCTGGCTAATTATGTTCACAGCAAAGGACTGAAGCTAGGGATTTATGCAGATGTTGGAAATAAAAC CTGCGCAGGCTTCCCTGGGAGTTTTGGATACTACGACATTGATGCCCAGACCTTTGCTGACTGGGGAGTAGAT CTGCTAAAATTTGATGGTTGTTACTGTGACAGTTTGGAAAATTTGGCAGATGGTTATAAGCACATGTCTTGGC CCTGAATAGGACTGGCAGAAGCATTGTGTACTCCTGTGAGTGGCCTCTTTATATGTGGCCCTTTCAAAAGCCCA ATTATACAGAAATCCGACAGTACTGCAATCACTGGCGAAATTTTGCTGACATTGATGATTCCTGGAAAAGTATA AAGAGTATCTTGGACTGGACATCTTTTAACCAGGAGAGAATTGTTGATGTTGCTGGACCAGGGGGTTGGAAT GACCCAGATATGTTAGTGATTGGCAACTTTGGCCTCAGCTGGAATCAGCAAGTAACTCAGATGGCCCTCTGGG CTATCATGGCTGCTCCTTTATTCATGTCTAATGACCTCCGACACATCAGCCCTCAAGCCAAAGCTCTCCTTCAGG ATAAGGACGTAATTGCCATCAATCAGGACCCCTTGGGCAAGCAAGGGTACCAGCTTAGACAGGGAGACAACT TTGAAGTGTGGGAACGACCTCTCTCAGGCTTAGCCTGGGCTGTAGCTATGATAAACCGGCAGGAGATTGGTG GACCTCGCTCTTATACCATCGCAGTTGCTTCCCTGGGTAAAGGAGTGGCCTGTAATCCTGCCTGCTTCATCACA CAGCTCCTCCCTGTGAAAAGGAAGCTAGGGTTCTATGAATGGACTTCAAGGTTAAGAAGTCACATAAATCCCA CAGGCACTGTTTTGCTTCAGCTAGAAAATACAATGCAGATGTCATTAAAAGACTTACTTTAAtgagctAGCttagcc TAAacacatcacaaccacaaccttctcaggtaactatacttgggacttaaaaaacataatcataatcatttttcctaaaacgatcaagactgat aaccatttgacaagagccatacagacaagcaccagctggcTCTCGAGCGTCTTCACGTATGGTCATCagtttgggttccatttgtag ataagaaactgaacatataaaggtctaggttaatgcaatttacacaaaaggagaccaaaccagggagagaaggaaccaaaattaaaaattc aaaccagagcaaaggagttagccctggttttgctctgacttacatgaaccactatgtggagtcctccatgttagcctagtcaagcttatcctctgg atgaagttgaaaccatatgaaggaatatttggggggtgggtcaaaacagttgtgtatcaatgattccatgtggtttgacccaatcattctgtgaa tccatttcaacagaagatacaacgggttctgtttcataataagtgatccacttccaaatttctgatgtgccccatgctaagctttaacagaatttat cttcttatgacaaagcagcctcctttgaaaatatagccaactgcacacagctatgttgatcaattttgtttataatcttgcagaagagaatttttta aaatagggcaataatggaaggctttggcaaaaaaattgtttctccatatgaaaacaaaaaacttatttttttattcaagcaaagaacctataga cataaggctatttcaaaattatttcagttttagaaagaattgaaagttttgtagcattctgagaagacagctttcatttgtaatcataggtaatatgt aggtcctcagaaatggtgagacccctgactttgacacttggggactctgagggaccagtgatgaagagggcacaacttatatcacacatgcac gagttggggtgagagggtgtcacaacatctatcagtgtgtcatctgcccaccaagtaacagatgtcagctaagactaggtcatgtgtaggctgt ctacaccagtgaaaatcgcaaaaagaatctaagaaattccacatttctagaaaataggtttggaaaccgtattccattttacaaaggacaactta catttctctttttgttttccagGCTACCCTGAGAAAAAAAGACATGAAGACTCAGGACTCATCTTTTCTGTTGGTGTAAA ATCAACACCCTAAGGAACACAAATTTCTTTAAACATTTGACTTCTTGTCTCTGTGCTGCAATTAATAAAAAATGG AAAGAATCTACtctgtggttcagaactctatcttccaaaggcgcgcttcaccctagcagcctctttggctcagaggaatccctgcctttcctcc cttcatctcagcagagaatgtagttccacatgggactagtgtacacgcgtgatatcagatctgttacgtagataagtagcatggcgggttaatca ttaactacaaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcg

? ?

ggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaaagcgcgcagctgcctgcaggtcgactctagag gatccccgggtaccgagctcgaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagc acatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgcct gatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatacgtcaaagcaaccatagtacgcgccctgtagcggcgcattaagcgc ggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgcttagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcg ccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgatttgggtg atggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactgg aacaacactcaactctatctcgggctattcttttgatttataagggattttgccgatttcggtctattggttaaaaaatgagctgatttaacaaaaa tttaacgcgaattttaacaaaatattaacgtttacaattttatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgac acccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtg tcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttc ttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaa taaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgcct tcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaac agcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattg acgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatg gcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaagg agctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagc gtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaataga ctggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgt gggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatg aacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaa aacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtca gaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggt ggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagcc gtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagt cgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttgg agcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatcc ggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacct ctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttg ctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagcc gaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcatta atgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggc tttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagct tgcatgcctgcaggcagctgcgcgctcgaacttcatgcctgccgaccttccccaggtcacgatccggacggcgggtgagttcacattttarcagc cggacgtgcaractccgctggtggtctaacgtcggttaggtcccttgaatcacgggacatatgttggtgttggaggt?[SEQ?ID?N.17]? ggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaaagcgcgcagctgcctgcaggtcgactctagag gatccccgggtaccgagctcgaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagc acatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgcct gatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatacgtcaaagcaaccatagtacgcgccctgtagcggcgcattaagcgc ggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgcttagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcg ccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgatttgggtg atggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactgg aacaacactcaactctatctcgggctattcttttgatttataagggattttgccgatttcggtctattggttaaaaaatgagctgatttaacaaaaa tttaacgcgaattttaacaaaatattaacgtttacaattttatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgac acccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtg tcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttc ttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaa taaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgcct tcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaac agcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattg acgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatg gcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaagg agctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagc gtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaataga ctggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgt gggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatg aacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaa aacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtca gaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggt ggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagcc gtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagt cgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttgg agcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatcc ggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacct ctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttg ctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagcc gaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcatta atgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggc tttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagct tgcatgcctgcaggcagctgcgcgctcgaacttcatgcctgccgaccttccccaggtcacgatccggacggcgggtgagttcacattttarcagc cggacgtgcaractccgctggtggtctaacgtcggttaggtcccttgaatcacgggacatatgttggtgttggaggt?[SEQ?ID?N.17]?

plasmide?pAB?hGLA?CO02? plasmid?pAB?hGLA?CO02?

ttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtga gcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctggaggggtggagtcgtgacgtaaagatctgatatcatcgat cgcgatgcattaattaagcggccgctgtacataggaggttcgaaccctgctgaagggagaggttccaatactacaaaatgtagcgggatattgt ttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtga gcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctggaggggtggagtcgtgacgtaaagatctgatatcatcgat cgcgatgcattaattaagcggccgctgtacataggaggttcgaaccctgctgaagggagaggttccaatactacaaaatgtagcgggatattgt

? ?

catcacctttggggacatgtcatcatggtccccagacagagttacaaaactcatcccctacacagcactatgtctctggtactgtttgttctacag atgtcaacaacagaggcccagccatctcctattgcttggcttgtcagtctttctagcctccccattattaatttcaaatggggcaggtgttaggag ggcaaaaatccacatattaagtgcaaagcctttcaggagatttcctgaaactagacaaaacccgtgtgactggcatcgattattctatttgatct agctagtcctagcaaagtgacaactgctactcccctcctacacagccaagattcctaagttggcagtggcatgcttaatcctcaaagccaaagt tacttggctccaagatttatagccttaaactgtggcctcacattccttcctatcttactttcctgcactggggtaaatgtctccttgctcttcttgcttt ctgtcctactgcagGGCTCTTGCTGAGCTGGTGAAGCACAAGCCCAAGGCTACAGCGGAGCAACTGAAGACTGTC ATGGATGACTTTGCACAGTTCCTGGATACATGTTGCAAGGCTGCTGACAAGGACACCTGCTTCTCGACTGAGg tcagaaacgtttttgcattttgacgatgttcagtttccattttctgtgcacgtggtcaggtgtagctctctggaactcacacactgaataactccacc aatctagatgttgttctctacgtaactgtaatagaaactgacttacgtagcttttaatttttattttctgccacactgctgcctattaaatacctattat cactatttggtttcaaatttgtgacacagaagagcatagttagaaatacttgcaaagcctagaatcatgaactcatttaaaccttgccctgaaat gtttctttttgaattgagttattttacacatgaatggacagttaccattatatatctgaatcatttcacattccctcccatggcctaacaacagtttat cttcttattttgggcacaacagatgtcagagagcctgctttaggaattctaagtagaactgtaattaagcaatgcaaggcacgtacgtttactatg tcattgcctatggctatgaagtgcaaatcctaACAGTCCTGCTAATACTTTTCtaacatccatcatttctttgttttcagGGTCCAAAC CTTGTCACTAGATGCAAAGACGCCTTAGCCggaagcggcgccaccaatttcagcctgctgaaacaggccggcgacgtggaagag aaccctggccctGCTAGCcagctgcgcaaccccgagctgcacctgggctgcgccctggccctgcgcttcctggccctggtcagctgggacat ccccggcgcccgcgccctggacaacggcctggcccgcacccccaccatgggctggctgcactgggagcgcttcatgtgcaacctggactgcc aggaggagcccgacagctgcatcagcgagaagctgtttatggagatggccgagctgatggtcagcgagggctggaaggacgccggctacga gtacctgtgcatcgacgactgctggatggccccccagcgcgacagcgagggccgcctgcaggccgacccccagcgcttcccccacggaatcc gccagctggccaactacgtgcacagcaagggcctgaagctgggcatctacgccgacgtgggcaacaagacctgcgccggcttccccggcagc ttcggctactacgacatcgacgcccagaccttcgccgactggggcgtggacctgctgaagttcgacggctgctactgcgacagcctggagaac ctggccgacggctacaagcacatgagcctggccctgaaccgcaccggccgcagcatcgtgtacagctgcgagtggcccctgtatatgtggccc ttccagaagcccaactacaccgagatccgccagtactgcaaccactggcgcaacttcgccgacatcgacgacagctggaagagcatcaagag catcctggactggaccagcttcaaccaggagcgcatcgtggacgtggccggccccggcggctggaacgaccccgacatgctggtgatcggca acttcggcctgagctggaaccagcaggtgacccagatggccctgtgggccattatggccgcccccctgtttatgagcaacgacctgcgccacat cagcccccaggccaaggccctgctgcaggacaaggacgtgatcgctatcaaccaggaccccctgggcaagcagggctaccagctgcgccag ggcgacaacttcgaggtctgggagcgccccctgagcggcctggcctgggccgtggctatgatcaaccgccaggagatcggcggcccccgcag ctacaccatcgccgtggccagcctgggcaagggcgtggcctgcaaccccgcctgcttcatcacccagctgctgcccgtgaagcgcaagctggg cttctacgagtggaccagccgcctgcgcagccacatcaaccccaccggcaccgtgctgctgcagctggagaacacaatgcagatgagcctga aggacctgctgtaatgagctAGCttagccTAAacacatcacaaccacaaccttctcaggtaactatacttgggacttaaaaaacataatcat aatcatttttcctaaaacgatcaagactgataaccatttgacaagagccatacagacaagcaccagctggcTCTCGAGCGTCTTCACGT ATGGTCATCagtttgggttccatttgtagataagaaactgaacatataaaggtctaggttaatgcaatttacacaaaaggagaccaaacca gggagagaaggaaccaaaattaaaaattcaaaccagagcaaaggagttagccctggttttgctctgacttacatgaaccactatgtggagtcc tccatgttagcctagtcaagcttatcctctggatgaagttgaaaccatatgaaggaatatttggggggtgggtcaaaacagttgtgtatcaatga ttccatgtggtttgacccaatcattctgtgaatccatttcaacagaagatacaacgggttctgtttcataataagtgatccacttccaaatttctgat gtgccccatgctaagctttaacagaatttatcttcttatgacaaagcagcctcctttgaaaatatagccaactgcacacagctatgttgatcaattt tgtttataatcttgcagaagagaattttttaaaatagggcaataatggaaggctttggcaaaaaaattgtttctccatatgaaaacaaaaaactt atttttttattcaagcaaagaacctatagacataaggctatttcaaaattatttcagttttagaaagaattgaaagttttgtagcattctgagaaga cagctttcatttgtaatcataggtaatatgtaggtcctcagaaatggtgagacccctgactttgacacttggggactctgagggaccagtgatga agagggcacaacttatatcacacatgcacgagttggggtgagagggtgtcacaacatctatcagtgtgtcatctgcccaccaagtaacagatgt cagctaagactaggtcatgtgtaggctgtctacaccagtgaaaatcgcaaaaagaatctaagaaattccacatttctagaaaataggtttggaa accgtattccattttacaaaggacacttacatttctctttttgttttccagGCTACCCTGAGAAAAAAAGACATGAAGACTCAGGA CTCATCTTTTCTGTTGGTGTAAAATCAACACCCTAAGGAACACAAATTTCTTTAAACATTTGACTTCTTGTCTCT catcacctttggggacatgtcatcatggtccccagacagagttacaaaactcatcccctacacagcactatgtctctggtactgtttgttctacag atgtcaacaacagaggcccagccatctcctattgcttggcttgtcagtctttctagcctccccattattaatttcaaatggggcaggtgttaggag ggcaaaaatccacatattaagtgcaaagcctttcaggagatttcctgaaactagacaaaacccgtgtgactggcatcgattattctatttgatct agctagtcctagcaaagtgacaactgctactcccctcctacacagccaagattcctaagttggcagtggcatgcttaatcctcaaagccaaagt tacttggctccaagatttatagccttaaactgtggcctcacattccttcctatcttactttcctgcactggggtaaatgtctccttgctcttcttgcttt ctgtcctactgcagGGCTCTTGCTGAGCTGGTGAAGCACAAGCCCAAGGCTACAGCGGAGCAACTGAAGACTGTC ATGGATGACTTTGCACAGTTCCTGGATACATGTTGCAAGGCTGCTGACAAGGACACCTGCTTCTCGACTGAGg tcagaaacgtttttgcattttgacgatgttcagtttccattttctgtgcacgtggtcaggtgtagctctctggaactcacacactgaataactccacc aatctagatgttgttctctacgtaactgtaatagaaactgacttacgtagcttttaatttttattttctgccacactgctgcctattaaatacctattat cactatttggtttcaaatttgtgacacagaagagcatagttagaaatacttgcaaagcctagaatcatgaactcatttaaaccttgccctgaaat gtttctttttgaattgagttattttacacatgaatggacagttaccattatatatctgaatcatttcacattccctcccatggcctaacaacagtttat cttcttattttgggcacaacagatgtcagagagcctgctttaggaattctaagtagaactgtaattaagcaatgcaaggcacgtacgtttactatg tcattgcctatggctatgaagtgcaaatcctaACAGTCCTGCTAATACTTTTCtaacatccatcatttctttgttttcagGGTCCAAAC CTTGTCACTAGATGCAAAGACGCCTTAGCCggaagcggcgccaccaatttcagcctgctgaaacaggccggcgacgtggaagag aaccctggccctGCTAGCcagctgcgcaaccccgagctgcacctgggctgcgccctggccctgcgcttcctggccctggtcagctgggacat ccccggcgcccgcgccctggacaacggcctggcccgcacccccaccatgggctggctgcactgggagcgcttcatgtgcaacctggactgcc aggaggagcccgacagctgcatcagcgagaagctgtttatggagatggccgagctgatggtcagcgagggctggaaggacgccggctacga gtacctgtgcatcgacgactgctggatggccccccagcgcgacagcgagggccgcctgcaggccgacccccagcgcttcccccacggaatcc gccagctggccaactacgtgcacagcaagggcctgaagctgggcatctacgccgacgtgggcaacaagacctgcgccggcttccccggcagc ttcggctactacgacatcgacgcccagaccttcgccgactggggcgtggacctgctgaagttcgacggctgctactgcgacagcctggagaac ctggccgacggctacaagcacatgagcctggccctgaaccgcaccggccgcagcatcgtgtacagctgcgagtggcccctgtatatgtggccttccagaagcccaactacaccgagatccgccagtactgcaaccactggcgcaacttcgccgacatcgacgacagctggaagagcatcaagag catcctggactggaccagcttcaaccaggagcgcatcgtggacgtggccggccccggcggctggaacgaccccgacatgctggtgatcggca acttcggcctgagctggaaccagcaggtgacccagatggccctgtgggccattatggccgcccccctgtttatgagcaacgacctgcgccacat cagcccccaggccaaggccctgctgcaggacaaggacgtgatcgctatcaaccaggaccccctgggcaagcagggctaccagctgcgccag ggcgacaacttcgaggtctgggagcgccccctgagcggcctggcctgggccgtggctatgatcaaccgccaggagatcggcggcccccgcag ctacaccatcgccgtggccagcctgggcaagggcgtggcctgcaaccccgcctgcttcatcacccagctgctgcccgtgaagcgcaagctggg cttctacgagtggaccagccgcctgcgcagccacatcaaccccaccggcaccgtgctgctgcagctggagaacacaatgcagatgagcctga aggacctgctgtaatgagctAGCttagccTAAacacatcacaaccacaaccttctcaggtaactatacttgggacttaaaaaacataatcat aatcatttttcctaaaacgatcaagactgataaccatttgacaagagccatacagacaagcaccagctggcTCTCGAGCGTCTTCACGT ATGGTCATCagtttgggttccatttgtagataagaaactgaacatataaaggtctaggttaatgcaatttacacaaaaaggagaccaaacca gggagagaaggaaccaaaattaaaaattcaaaccagagcaaaggagttagccctggttttgctctgacttacatgaaccactatgtggagtcc tccatgttagcctagtcaagcttatcctctggatgaagttgaaaccatatgaaggaatatttggggggtgggtcaaaacagttgtgtatcaatga ttccatgtggtttgacccaatcattctgtgaatccatttcaacagaagatacaacgggttctgtttcataataagtgatccacttccaaatttctgat gtgccccatgctaagctttaacagaatttatcttcttatgacaaagcagcctcctttgaaaatatagccaactgcacacagctatgttgatcaattt tgtttataatcttgcagaagagaattttttaaaatagggcaataatggaaggctttggcaaaaaaattgtttctccatatgaaaacaaaaaactt atttttttattcaagcaaagaacctatagacataaggctatttcaaaattattcagttttagaaagaattgaaagttttgtagcattctgagaaga cagctttcatttgtaatcataggtaatatgtaggtcctcagaaatggtgagacccctgactttgacacttggggactctgagggaccagtgatga agagggcacaacttatatcacacatgcacgagttggggtgagagggtgtcacaacatctatcagtgtgtcatctgcccaccaagtaacagatgt cagctaagactaggtcatgtgtaggctgtctacaccagtgaaaatcgcaaaaagaatctaagaaattccacatttctagaaaataggtttggaa accgtattccatttacaaaggacacttacatttctctttttgttttccagGCTACCCTGAGAAAAAAAGACATGAAGACTCAGGA CTCATCTTTTCTGTTGGTGTAAAATCAACACCCTAAGGAACACAAATTTCTTTAAACATTTGACTTCTTGTCTCT

? ?

GTGCTGCAATTAATAAAAAATGGAAAGAATCTACtctgtggttcagaactctatcttccaaaggcgcgcttcaccctagcagcctc tttggctcagaggaatccctgcctttcctcccttcatctcagcagagaatgtagttccacatgggactagtgtacacgcgtgatatcagatctgtta cgtagataagtagcatggcgggttaatcattaactacaaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactg aggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaaag cgcgcagctgcctgcaggtcgactctagaggatccccgggtaccgagctcgaattcactggccgtcgttttacaacgtcgtgactgggaaaacc ctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaaca gttgcgcagcctgaatggcgaatggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatacgtcaaagcaaccata gtacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgcttagcgcccgctcctt tcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggc acctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgt tctttaatagtggactcttgttccaaactggaacaacactcaactctatctcgggctattcttttgatttataagggattttgccgatttcggtctatt ggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaattttatggtgcactctcagtacaatctgct ctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagaca agctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttt tataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaata cattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgt cgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgca cgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagtt ctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactc accagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaactt acttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggag ctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactactt actctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttat tgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctaca cgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaa gtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatccct taacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgc aaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgca gataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgtta ccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacg gggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcc cgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggt atctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagca acgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctt tgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaacc gcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtg agttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacagga aacagctatgaccatgattacgccaagcttgcatgcctgcaggcagctgcgcgctcgaacttcatgcctgccgaccttccccaggtcacgatcc ggacggcgggtgagttcacattttarcagccggacgtgcaractccgctggtggtctaacgtcggttaggtcccttgaatcacgggacatatgtt ggtgttggaggt?[SEQ?ID?N.15]? GTGCTGCAATTAATAAAAAATGGAAAGAATCTACtctgtggttcagaactctatcttccaaaggcgcgcttcaccctagcagcctc tttggctcagaggaatccctgcctttcctcccttcatctcagcagagaatgtagttccacatgggactagtgtacacgcgtgatatcagatctgtta cgtagataagtagcatggcgggttaatcattaactacaaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactg aggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaaag cgcgcagctgcctgcaggtcgactctagaggatccccgggtaccgagctcgaattcactggccgtcgttttacaacgtcgtgactgggaaaacc ctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaaca gttgcgcagcctgaatggcgaatggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatacgtcaaagcaaccata gtacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgcttagcgcccgctcctt tcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggc acctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgt tctttaatagtggactcttgttccaaactggaacaacactcaactctatctcgggctattcttttgatttataagggattttgccgatttcggtctatt ggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaattttatggtgcactctcagtacaatctgct ctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagaca agctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttt tataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaata cattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgt cgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgca cgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagtt ctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactc accagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaactt acttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggag ctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactactt actctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttat tgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctaca cgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaa gtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatccct taacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgc aaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctctttttccgaaggtaactggcttcagcagagcgca gataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgtta ccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacg gggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcc cgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggt atctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagca acgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctt tgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaacc gcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtg agttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacagga aacagctatgaccatgattacgccaagcttgcatgcctgcaggcagctgcgcgctcgaacttcatgcctgccgaccttccccaggtcacgatcc ggacggcgggtgagttcacattttarcagccggacgtgcaractccgctggtggtctaacgtcggttaggtcccttgaatcacgggacatatgtt ggtgttggaggt?[SEQ?ID?N.15]?

? ?

pSMD2?hGLA?WT? pSMD2?hGLA?WT?

? ?

AGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGC CCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTT AATGATTAACCCGCCATGCTACTTATCTACGTAGCCATGCTCTAGAGGATCAAGGCTCAGAGGCACACAGGAG TTTCTGGGCTCACCCTGCCCCCTTCCAACCCCTCAGTTCCCATCCTCCAGCAGCTGTTTGTGTGCTGCCTCTGA AGTCCACACTGAACAAACTTCAGCCTACTCATGTCCCTAAAATGGGCAAACATTGCAAGCAGCAAACAGCAAA CACACAGCCCTCCCTGCCTGCTGACCTTGGAGCTGGGGCAGAGGTCAGAGACCTCTCTGGGCCCATGCCACC TCCAACATCCACTCGACCCCTTGGAATTTCGGTGGAGAGGAGCAGAGGTTGTCCTGGCGTGGTTTAGGTAGT GTGAGAGGGGTACCCGGGGATCTTGCTACCAGTGGAACAGCCACTAAGGATTCTGCAGTGAGAGCAGAGGG CCAGCTAAGTGGTACTCTCCCAGAGACTGTCTGACTCACGCCACCCCCTCCACCTTGGACACAGGACGCTGTG GTTTCTGAGCCAGGTACAATGACTCCTTTCGGTAAGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGCGTC CGGGCAGCGTAGGCGGGCGACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGG GTGACCTTGGTTAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGA CAGGGCCCTGTCTCCTCAGCTTCAGGCACCACCACTGACCTGGGACAGTGAATAGATCCTGAGAACTTCAGG GTGAGTCTATGGGACCCTTGATGTTTTCTTTCCCCTTCTTTTCTATGGTTAAGTTCATGTCATAGGAAGGGGAG AAGTAACAGGGTACACATATTGACCAAATCAGGGTAATTTTGCATTTGTAATTTTAAAAAATGCTTTCTTCTTTT AATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATATTGATACAATGTATC TTGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATA AATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATT CTGCTTTTATTTTCTGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCTTGTTCA TACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACCTGCTGGTCTCTCTGCTGGCCCATCACTTTGGCAAAGCA CGCGTCgacgccgccaccATGCAGCTGAGGAACCCAGAACTACATCTGGGCTGCGCGCTTGCGCTTCGCTTCCTG GCCCTCGTTTCCTGGGACATCCCTGGGGCTAGAGCACTGGACAATGGATTGGCAAGGACGCCTACCATGGGC TGGCTGCACTGGGAGCGCTTCATGTGCAACCTTGACTGCCAGGAAGAGCCAGATTCCTGCATCAGTGAGAAG CTCTTCATGGAGATGGCAGAGCTCATGGTCTCAGAAGGCTGGAAGGATGCAGGTTATGAGTACCTCTGCATTG ATGACTGTTGGATGGCTCCCCAAAGAGATTCAGAAGGCAGACTTCAGGCAGACCCTCAGCGCTTTCCTCATG GGATTCGCCAGCTGGCTAATTATGTTCACAGCAAAGGACTGAAGCTAGGGATTTATGCAGATGTTGGAAATAA AACCTGCGCAGGCTTCCCTGGGAGTTTTGGATACTACGACATTGATGCCCAGACCTTTGCTGACTGGGGAGTA GATCTGCTAAAATTTGATGGTTGTTACTGTGACAGTTTGGAAAATTTGGCAGATGGTTATAAGCACATGTCCTT GGCCCTGAATAGGACTGGCAGAAGCATTGTGTACTCCTGTGAGTGGCCTCTTTATATGTGGCCCTTTCAAAAG CCCAATTATACAGAAATCCGACAGTACTGCAATCACTGGCGAAATTTTGCTGACATTGATGATTCCTGGAAAAG TATAAAGAGTATCTTGGACTGGACATCTTTTAACCAGGAGAGAATTGTTGATGTTGCTGGACCAGGGGGTTGG AATGACCCAGATATGTTAGTGATTGGCAACTTTGGCCTCAGCTGGAATCAGCAAGTAACTCAGATGGCCCTCT GGGCTATCATGGCTGCTCCTTTATTCATGTCTAATGACCTCCGACACATCAGCCCTCAAGCCAAAGCTCTCCTTC AGGATAAGGACGTAATTGCCATCAATCAGGACCCCTTGGGCAAGCAAGGGTACCAGCTTAGACAGGGAGAC AACTTTGAAGTGTGGGAACGACCTCTCTCAGGCTTAGCCTGGGCTGTAGCTATGATAAACCGGCAGGAGATT GGTGGACCTCGCTCTTATACCATCGCAGTTGCTTCCCTGGGTAAAGGAGTGGCCTGTAATCCTGCCTGCTTCAT CACACAGCTCCTCCCTGTGAAAAGGAAGCTAGGGTTCTATGAATGGACTTCAAGGTTAAGAAGTCACATAAAT CCCACAGGCACTGTTTTGCTTCAGCTAGAAAATACAATGCAGATGTCATTAAAAGACTTACTTTAAtgagctAGC TCGAGAGATCTGAATTCACCCCACCAGTGCAGGCTGCCTATCAGAAAGTGGTGGCTGGTGTGGCTAATGCCCT GGCCCACAAGTATCACTAAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAA CTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTG AGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGC CCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTT AATGATTAACCCGCCATGCTACTTATCTACGTAGCCATGCTCTAGAGGATCAAGGCTCAGAGGCACACAGGAG TTTCTGGGCTCACCCTGCCCCCTTCCAACCCCTCAGTTCCCATCCTCCAGCAGCTGTTTGTGTGCTGCCTCTGA AGTCCACACTGAACAAACTTCAGCCTACTCATGTCCCTAAAAATGGCAAACATTGCAAGCAGCAAACAGCAAA CACACAGCCCTCCCTGCCTGCTGACCTTGGAGCTGGGGCAGAGGTCAGAGACCTCTCTGGGCCCATGCCACC TCCAACATCCACTCGACCCCTTGGAATTTCGGTGGAGAGGAGCAGAGGTTGTCCTGGCGTGGTTTAGGTAGT GTGAGAGGGGTACCCGGGGATCTTGCTACCAGTGGAACAGCCACTAAGGATTCTGCAGTGAGAGCAGAGGG CCAGCTAAGTGGTACTCTCCCAGAGACTGTCTGACTCACGCCACCCCCTCCACCTTGGACACAGGACGCTGTG GTTTCTGAGCCAGGTACAATGACTCCTTTCGGTAAGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGCGTC CGGGCAGCGTAGGCGGGCGACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTGCTCCTCCGATAACTGGG GTGACCTTGGTTAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGA CAGGGCCCTGTCTCCTCAGCTTCAGGCACCACCACTGACCTGGGACAGTGAATAGATCCTGAGAACTTCAGG GTGAGTCTATGGGACCCTTGATGTTTTCTTTCCCCTTCTTTTCTATGGTTAAGTTCATGTCATAGGAAGGGGAG AAGTAACAGGGTACACATATTGACCAAATCAGGGTAATTTTGCATTTGTAATTTTAAAAAATGCTTTCTTCTTTT AATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATATTGATACAATGTATC TTGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATA AATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATT CTGCTTTTATTTTCTGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCTTGTTCA TACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACCTGCTGGTCTCTCTGCTGGCCCATCACTTTGGCAAAGCA CGCGTCgacgccgccaccATGCAGCTGAGGAACCCAGAACTACATCTGGGCTGCGCGCTTGCGCTTCGCTTCCTG GCCCTCGTTTCCTGGGACATCCCTGGGGCTAGAGCACTGGACAATGGATTGGCAAGGACGCCTACCATGGGC TGGCTGCACTGGGAGCGCTTCATGTGCAACCTTGACTGCCAGGAAGAGCCAGATTCCTGCATCAGTGAGAAG CTCTTCATGGAGATGGCAGAGCTCATGGTCTCAGAAGGCTGGAAGGATGCAGGTTATGAGTACCTCTGCATTG ATGACTGTTGGATGGCTCCCCAAAGAGATTCAGAAGGCAGACTTCAGGCAGACCCTCAGCGCTTTCCTCATG GGATTCGCCAGCTGGCTAATTATGTTCACAGCAAAGGACTGAAGCTAGGGATTTATGCAGATGTTGGAAATAA AACCTGCGCAGGCTTCCCTGGGAGTTTTGGATACTACGACATTGATGCCCAGACCTTTGCTGACTGGGGAGTA GATCTGCTAAAATTTGATGGTTGTTACTGTGACAGTTTTGGAAAATTTGGCAGATGGTTATAAGCACATGTCCTT GGCCCTGAATAGGACTGGCAGAAGCATTGTGTACTCCTGTGAGTGGCCTCTTTATATGTGGCCCTTTCAAAAG CCCAATTATACAGAAATCCGACAGTACTGCAATCACTGGCGAAATTTTGCTGACATTGATGATTCCTGGAAAAG TATAAAGAGTATCTTGGACTGGACATCTTTTAACCAGGAGAGAATTGTTGATGTTGCTGGACCAGGGGGTTGG AATGACCCAGATATGTTAGTGATTGGCAACTTTGGCCTCAGCTGGAATCAGCAAGTAACTCAGATGGCCCTCT GGGCTATCATGGCTGCTCCTTTATTCATGTCTAATGACCTCCGACACATCAGCCCTCAAGCCAAAGCTCTCCTTC AGGATAAGGACGTAATTGCCATCAATCAGGACCCCTTGGGCAAGCAAGGGTACCAGCTTAGACAGGGAGAC AACTTTGAAGTGTGGGAACGACCTCTCTCAGGCTTAGCCTGGGCTGTAGCTATGATAAACCGGCAGGAGATT GGTGGACCTCGCTCTTATACCATCGCAGTTGCTTCCCTGGGTAAAGGAGTGGCCTGTAATCCTGCCTGCTTCAT CACACAGCTCCTCCCTGTGAAAAGGAAGCTAGGGTTCTATGAATGGACTTCAAGGTTAAGAAGTCACATAAAT CCCACAGGCACTGTTTTGCTTCAGCTAGAAAATACAATGCAGATGTCATTAAAAGACTTACTTTAAtgagctAGC TCGAGAGATCTGAATTCACCCCACCAGTGCAGGCTGCCTATCAGAAAGTGGTGGCTGGTGTGGCTAATGCCCT GGCCCACAAGTATCACTAAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAA CTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTG

? ?

CAATGATGTATTTAAATTATTTCTGAATATTTTACTAAAAAGGGAATGTGGGAGGTCAGTGCATTTAAAACATAA AGAAATGAAGAGCTAGTTCAAACCTTGGGAAAATACACTATATCTTAAACTCCATGAAAGAAGGTGAGGCTGC AAACAGCTAATGCACATTGGCAACAGCCCCTGATGCCTATGCCTTATTCATCCCTCAGAAAAGGATTCAAGTAG AGGCTTGATTTGGAGGTTAAAGTTTGGCTATGCTGTATTTTACATTACTTATTGTTTTAGCTGTCCTCATGAATGT CTTTTCACTACCCATTTGCTTATCCTGCATCTCTCAGCCTTGACTCCACTCAGTTCTCTTGCTTAGAGATACCACC TTTCCCCTGAAGTGTTCCTTCCATGTTTTACGGCGAGATGGTTTCTCCTCGCCTGGCCACTCAGCCTTAGTTGTC TCTGTTGTCTTATAGAGGTCTACTTGAAGAAGGAAAAACAGGGGGCATGGTTTGACTGTCCTGTGAGCCCTTC TTCCCTGCCTCCCCCACTCACAGTGACCCGGAATCCCTCGACATGGCAGTCTAGAGCATGGCTACGTAGATAA GTAGCATGGCGGGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCT CGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAG CGAGCGAGCGCGCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCC TGAATGGCGAATGGCGATTCCGTTGCAATGGCTGGCGGTAATATTGTTCTGGATATTACCAGCAAGGCCGATA GTTTGAGTTCTTCTACTCAGGCAAGTGATGTTATTACTAATCAAAGAAGTATTGCGACAACGGTTAATTTGCGT GATGGACAGACTCTTTTACTCGGTGGCCTCACTGATTATAAAAACACTTCTCAGGATTCTGGCGTACCGTTCCT GTCTAAAATCCCTTTAATCGGCCTCCTGTTTAGCTCCCGCTCTGATTCTAACGAGGAAAGCACGTTATACGTGCT CGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGC GTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGC CGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACC CCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGAC GTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTC TTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGC GAATTTTAACAAAATATTAACGCTTACAATTTAAATATTTGCTTATACAATCTTCCTGTTTTTGGGGCTTTTCTGAT TATCAACCGGGGTACATATGATTGACATGCTAGTTTTACGATTACCGTTCATCGATTCTCTTGTTTGCTCCAGACT CTCAGGCAATGACCTGATAGCCTTTGTAGAGACCTCTCAAAAATAGCTACCCTCTCCGGCATGAATTTATCAGC TAGAACGGTTGAATATCATATTGATGGTGATTTGACTGTCTCCGGCCTTTCTCACCCGTTTGAATCTTTACCTAC ACATTACTCAGGCATTGCATTTAAAATATATGAGGGTTCTAAAAATTTTTATCCTTGCGTTGAAATAAAGGCTTC TCCCGCAAAAGTATTACAGGGTCATAATGTTTTTGGTACAACCGATTTAGCTTTATGCTCTGAGGCTTTATTGCT TAATTTTGCTAATTCTTTGCCTTGCCTGTATGATTTATTGGATGTTGGAATCGTCCTGATGCGGTATTTTCTCCTTA CGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCC AGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGAC AAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAA AGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTT TTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGAC AATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTA TTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAA GATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGC CCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGC CGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAA AAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGG CCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATG TAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGC CTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTA CAATGATGTATTTAAATTATTTCTGAATATTTTACTAAAAAGGGAATGTGGGAGGTCAGTGCATTTAAAACATAA AGAAATGAAGAGCTAGTTCAAACCTTGGGAAAATACACTATATCTTAAACTCCATGAAAGAAGGTGAGGCTGC AAACAGCTAATGCACATTGGCAACAGCCCCTGATGCCTATGCCTTATTCATCCCTCAGAAAAGGATTCAAGTAG AGGCTTGATTTGGAGGTTAAAGTTTGGCTATGCTGTATTTTACATTACTTATTGTTTTAGCTGTCCTCATGAATGT CTTTTCACTACCCATTTGCTTATCCTGCATCTCTCAGCCTTGACTCCACTCAGTTCTCTTGCTTAGAGATACCACC TTTCCCCTGAAGTGTTCCTTCCATGTTTTACGGCGAGATGGTTTCTCCTCGCCTGGCCACTCAGCCTTAGTTGTC TCTGTTGTCTTATAGAGGTCTACTTGAAGAAGGAAAAACAGGGGGCATGGTTTGACTGTCCTGTGAGCCCTTC TTCCCTGCCTCCCCCACTCACAGTGACCCGGAATCCCTCGACATGGCAGTCTAGAGCATGGCTACGTAGATAA GTAGCATGGCGGGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCT CGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAG CGAGCGAGCGCGCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCCTTCCCAACAGTTGCGCAGCC TGAATGGCGAATGGCGATTCCGTTGCAATGGCTGGCGGTAATATTGTTCTGGATATTACCAGCAAGGCCGATA GTTTGAGTTCTTCTACTCAGGCAAGTGATGTTATTACTAATCAAAGAAGTATTGCGACAACGGTTAATTTGCGT GATGGACAGACTCTTTTACTCGGTGGCCTCACTGATTATAAAAACACTTCTCAGGATTCTGGCGTACCGTTCCT GTCTAAAATCCCTTTAATCGGCCTCCTGTTTAGCTCCCGCTCTGATTCTAACGAGGAAAGCACGTTATACGTGCT CGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGC GTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGC CGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACC CCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGAC GTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTC TTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGC GAATTTTAACAAAATATTAACGCTTACAATTTAAATATTTGCTTATACAATCTTCCTGTTTTTGGGGCTTTTCTGAT TATCAACCGGGGTACATATGATTGACATGCTAGTTTTACGATTACCGTTCATCGATTCTCTTGTTTGCTCCAGACT CTCAGGCAATGACCTGATAGCCTTTGTAGAGACCTCTCAAAAATAGCTACCCTCTCCGGCATGAATTTATCAGC TAGAACGGTTGAATATCATATTGATGGTGATTTGACTGTCTCCGGCCTTTCTCACCCGTTTGAATCTTTACCTAC ACATTACTCAGGCATTGCATTTAAAATATATGAGGGTTCTAAAAATTTTTATCCTTGCGTTGAAATAAAGGCTTC TCCCGCAAAAGTATTACAGGGTCATAATGTTTTTGGTACAACCGATTTAGCTTTATGCTCTGAGGCTTTATTGCT TAATTTTGCTAATTCTTTGCCTTGCCTGTATGATTTATTGGATGTTGGAATCGTCCTGATGCGGTATTTTCTCCTTA CGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCC AGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGAC AAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAA AGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTT TTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGAC AATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTA TTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAA GATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGC CCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGC CGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGTTGAGTACTCACCAGTCACAGAA AAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGG CCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATG TAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGC CTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAAACAATTA

? ?

ATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATT GCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCC TCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGA TAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACT TCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTT TTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAA TCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCT TTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGC CACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAG TGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTG AACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTG AGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGG AACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCC ACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACG CGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTG TGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGT CAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAAT ATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATT GCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCC TCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGA TAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACT TCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTT TTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAA TCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCT TTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGC CACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAG TGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTG AACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTG AGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGG AACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCC ACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACG CGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTG TGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGT CAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGTTGGCCGATTCATTAAT

G?[SEQ?ID?N.18]? G?[SEQ?ID?N.18]?

? ?

pSMD2?hGLA?CO02? pSMD2?hGLA?CO02?

AGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGC CCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTT AATGATTAACCCGCCATGCTACTTATCTACGTAGCCATGCTCTAGAGGATCAAGGCTCAGAGGCACACAGGAG TTTCTGGGCTCACCCTGCCCCCTTCCAACCCCTCAGTTCCCATCCTCCAGCAGCTGTTTGTGTGCTGCCTCTGA AGTCCACACTGAACAAACTTCAGCCTACTCATGTCCCTAAAATGGGCAAACATTGCAAGCAGCAAACAGCAAA CACACAGCCCTCCCTGCCTGCTGACCTTGGAGCTGGGGCAGAGGTCAGAGACCTCTCTGGGCCCATGCCACC TCCAACATCCACTCGACCCCTTGGAATTTCGGTGGAGAGGAGCAGAGGTTGTCCTGGCGTGGTTTAGGTAGT GTGAGAGGGGTACCCGGGGATCTTGCTACCAGTGGAACAGCCACTAAGGATTCTGCAGTGAGAGCAGAGGG CCAGCTAAGTGGTACTCTCCCAGAGACTGTCTGACTCACGCCACCCCCTCCACCTTGGACACAGGACGCTGTG GTTTCTGAGCCAGGTACAATGACTCCTTTCGGTAAGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGCGTC CGGGCAGCGTAGGCGGGCGACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGG GTGACCTTGGTTAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGA CAGGGCCCTGTCTCCTCAGCTTCAGGCACCACCACTGACCTGGGACAGTGAATAGATCCTGAGAACTTCAGG GTGAGTCTATGGGACCCTTGATGTTTTCTTTCCCCTTCTTTTCTATGGTTAAGTTCATGTCATAGGAAGGGGAG AAGTAACAGGGTACACATATTGACCAAATCAGGGTAATTTTGCATTTGTAATTTTAAAAAATGCTTTCTTCTTTT AATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATATTGATACAATGTATC TTGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATA AATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATT CTGCTTTTATTTTCTGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCTTGTTCA TACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACCTGCTGGTCTCTCTGCTGGCCCATCACTTTGGCAAAGCA CGCgtcgacgccgccaccatgcagctgcgcaaccccgagctgcacctgggctgcgccctggccctgcgcttcctggccctggtcagctggga AGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGC CCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTT AATGATTAACCCGCCATGCTACTTATCTACGTAGCCATGCTCTAGAGGATCAAGGCTCAGAGGCACACAGGAG TTTCTGGGCTCACCCTGCCCCCTTCCAACCCCTCAGTTCCCATCCTCCAGCAGCTGTTTGTGTGCTGCCTCTGA AGTCCACACTGAACAAACTTCAGCCTACTCATGTCCCTAAAAATGGCAAACATTGCAAGCAGCAAACAGCAAA CACACAGCCCTCCCTGCCTGCTGACCTTGGAGCTGGGGCAGAGGTCAGAGACCTCTCTGGGCCCATGCCACC TCCAACATCCACTCGACCCCTTGGAATTTCGGTGGAGAGGAGCAGAGGTTGTCCTGGCGTGGTTTAGGTAGT GTGAGAGGGGTACCCGGGGATCTTGCTACCAGTGGAACAGCCACTAAGGATTCTGCAGTGAGAGCAGAGGG CCAGCTAAGTGGTACTCTCCCAGAGACTGTCTGACTCACGCCACCCCCTCCACCTTGGACACAGGACGCTGTG GTTTCTGAGCCAGGTACAATGACTCCTTTCGGTAAGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGCGTC CGGGCAGCGTAGGCGGGCGACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTGCTCCTCCGATAACTGGG GTGACCTTGGTTAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGA CAGGGCCCTGTCTCCTCAGCTTCAGGCACCACCACTGACCTGGGACAGTGAATAGATCCTGAGAACTTCAGG GTGAGTCTATGGGACCCTTGATGTTTTCTTTCCCCTTCTTTTCTATGGTTAAGTTCATGTCATAGGAAGGGGAG AAGTAACAGGGTACACATATTGACCAAATCAGGGTAATTTTGCATTTGTAATTTTAAAAAATGCTTTCTTCTTTT AATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAATCTCTTTCTTTCAGGGCAATATTGATACAATGTATC TTGCCTCTTTGCACCATTCTAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATA AATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATT CTGCTTTTATTTTCTGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCTTGTTCA TACCTCTTATCTTTCCTCCCACAGCTCCTGGGCAACCTGCTGGTCTCTCTGCTGGCCCATCACTTTGGCAAAGCA CGCgtcgacgccgccaccatgcagctgcgcaaccccgagctgcacctgggctgcgccctggccctgcgcttcctggccctggtcagctggga

? ?

catccccggcgcccgcgccctggacaacggcctggcccgcacccccaccatgggctggctgcactgggagcgcttcatgtgcaacctggactg ccaggaggagcccgacagctgcatcagcgagaagctgtttatggagatggccgagctgatggtcagcgagggctggaaggacgccggctac gagtacctgtgcatcgacgactgctggatggccccccagcgcgacagcgagggccgcctgcaggccgacccccagcgcttcccccacggaat ccgccagctggccaactacgtgcacagcaagggcctgaagctgggcatctacgccgacgtgggcaacaagacctgcgccggcttccccggca gcttcggctactacgacatcgacgcccagaccttcgccgactggggcgtggacctgctgaagttcgacggctgctactgcgacagcctggaga acctggccgacggctacaagcacatgagcctggccctgaaccgcaccggccgcagcatcgtgtacagctgcgagtggcccctgtatatgtggc ccttccagaagcccaactacaccgagatccgccagtactgcaaccactggcgcaacttcgccgacatcgacgacagctggaagagcatcaag agcatcctggactggaccagcttcaaccaggagcgcatcgtggacgtggccggccccggcggctggaacgaccccgacatgctggtgatcgg caacttcggcctgagctggaaccagcaggtgacccagatggccctgtgggccattatggccgcccccctgtttatgagcaacgacctgcgcca catcagcccccaggccaaggccctgctgcaggacaaggacgtgatcgctatcaaccaggaccccctgggcaagcagggctaccagctgcgc cagggcgacaacttcgaggtctgggagcgccccctgagcggcctggcctgggccgtggctatgatcaaccgccaggagatcggcggcccccg cagctacaccatcgccgtggccagcctgggcaagggcgtggcctgcaaccccgcctgcttcatcacccagctgctgcccgtgaagcgcaagct gggcttctacgagtggaccagccgcctgcgcagccacatcaaccccaccggcaccgtgctgctgcagctggagaacacaatgcagatgagcc tgaaggacctgctgtaatgagctagcTCGAGAGATCTGAATTCACCCCACCAGTGCAGGCTGCCTATCAGAAAGTGGT GGCTGGTGTGGCTAATGCCCTGGCCCACAAGTATCACTAAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGG TTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTA ATAAAAAACATTTATTTTCATTGCAATGATGTATTTAAATTATTTCTGAATATTTTACTAAAAAGGGAATGTGGGA GGTCAGTGCATTTAAAACATAAAGAAATGAAGAGCTAGTTCAAACCTTGGGAAAATACACTATATCTTAAACTC CATGAAAGAAGGTGAGGCTGCAAACAGCTAATGCACATTGGCAACAGCCCCTGATGCCTATGCCTTATTCATC CCTCAGAAAAGGATTCAAGTAGAGGCTTGATTTGGAGGTTAAAGTTTGGCTATGCTGTATTTTACATTACTTAT TGTTTTAGCTGTCCTCATGAATGTCTTTTCACTACCCATTTGCTTATCCTGCATCTCTCAGCCTTGACTCCACTCA GTTCTCTTGCTTAGAGATACCACCTTTCCCCTGAAGTGTTCCTTCCATGTTTTACGGCGAGATGGTTTCTCCTCG CCTGGCCACTCAGCCTTAGTTGTCTCTGTTGTCTTATAGAGGTCTACTTGAAGAAGGAAAAACAGGGGGCATG GTTTGACTGTCCTGTGAGCCCTTCTTCCCTGCCTCCCCCACTCACAGTGACCCGGAATCCCTCGACATGGCAGT CTAGAGCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGT TGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCT TTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGC CCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGATTCCGTTGCAATGGCTGGCGGTAATATTGTTCTG GATATTACCAGCAAGGCCGATAGTTTGAGTTCTTCTACTCAGGCAAGTGATGTTATTACTAATCAAAGAAGTATT GCGACAACGGTTAATTTGCGTGATGGACAGACTCTTTTACTCGGTGGCCTCACTGATTATAAAAACACTTCTCA GGATTCTGGCGTACCGTTCCTGTCTAAAATCCCTTTAATCGGCCTCCTGTTTAGCTCCCGCTCTGATTCTAACGA GGAAAGCACGTTATACGTGCTCGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGG GTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCC TTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTA GTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATA GACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACA CTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAG CTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAAATATTTGCTTATACAATCT TCCTGTTTTTGGGGCTTTTCTGATTATCAACCGGGGTACATATGATTGACATGCTAGTTTTACGATTACCGTTCAT CGATTCTCTTGTTTGCTCCAGACTCTCAGGCAATGACCTGATAGCCTTTGTAGAGACCTCTCAAAAATAGCTAC CCTCTCCGGCATGAATTTATCAGCTAGAACGGTTGAATATCATATTGATGGTGATTTGACTGTCTCCGGCCTTTC TCACCCGTTTGAATCTTTACCTACACATTACTCAGGCATTGCATTTAAAATATATGAGGGTTCTAAAAATTTTTAT catccccggcgcccgcgccctggacaacggcctggcccgcacccccaccatgggctggctgcactgggagcgcttcatgtgcaacctggactg ccaggaggagcccgacagctgcatcagcgagaagctgtttatggagatggccgagctgatggtcagcgagggctggaaggacgccggctac gagtacctgtgcatcgacgactgctggatggccccccagcgcgacagcgagggccgcctgcaggccgacccccagcgcttcccccacggaat ccgccagctggccaactacgtgcacagcaagggcctgaagctgggcatctacgccgacgtgggcaacaagacctgcgccggcttccccggca gcttcggctactacgacatcgacgcccagaccttcgccgactggggcgtggacctgctgaagttcgacggctgctactgcgacagcctggaga acctggccgacggctacaagcacatgagcctggccctgaaccgcaccggccgcagcatcgtgtacagctgcgagtggcccctgtatatgtggc ccttccagaagcccaactacaccgagatccgccagtactgcaaccactggcgcaacttcgccgacatcgacgacagctggaagagcatcaag agcatcctggactggaccagcttcaaccaggagcgcatcgtggacgtggccggccccggcggctggaacgaccccgacatgctggtgatcgg caacttcggcctgagctggaaccagcaggtgacccagatggccctgtgggccattatggccgcccccctgtttatgagcaacgacctgcgcca catcagcccccaggccaaggccctgctgcaggacaaggacgtgatcgctatcaaccaggaccccctgggcaagcagggctaccagctgcgc cagggcgacaacttcgaggtctgggagcgccccctgagcggcctggcctgggccgtggctatgatcaaccgccaggagatcggcggcccccg cagctacaccatcgccgtggccagcctgggcaagggcgtggcctgcaaccccgcctgcttcatcacccagctgctgcccgtgaagcgcaagct gggcttctacgagtggaccagccgcctgcgcagccacatcaaccccaccggcaccgtgctgctgcagctggagaacacaatgcagatgagcc tgaaggacctgctgtaatgagctagcTCGAGAGATCTGAATTCACCCCACCAGTGCAGGCTGCCTATCAGAAAGTGGT GGCTGGTGTGGCTAATGCCCTGGCCCACAAGTATCACTAAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGG TTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTA ATAAAAAACATTTATTTTCATTGCAATGATGTATTTAAATTATTTCTGAATATTTTACTAAAAAGGGAATGTGGGA GGTCAGTGCATTTAAAACATAAAGAAATGAAGAGCTAGTTCAAACCTTGGGAAAATACACTATATCTTAAACTC CATGAAAGAAGGTGAGGCTGCAAACAGCTAATGCACATTGGCAACAGCCCCTGATGCCTATGCCTTATTCATC CCTCAGAAAAGGATTCAAGTAGAGGCTTGATTTGGAGGTTAAAGTTTGGCTATGCTGTATTTTACATTACTTAT TGTTTTAGCTGTCCTCATGAATGTCTTTTCACTACCCATTTGCTTATCCTGCATCTCTCAGCCTTGACTCCACTCA GTTCTCTTGCTTAGAGATACCACCTTTCCCCTGAAGTGTTCCTTCCATGTTTTACGGCGAGATGGTTTCTCCTCG CCTGGCCACTCAGCCTTAGTTGTCTCTGTTGTCTTATAGAGGTCTACTTGAAGAAGGAAAAACAGGGGGCATG GTTTGACTGTCCTGTGAGCCCTTCTTCCCTGCCTCCCCCACTCACAGTGACCCGGAATCCCTCGACATGGCAGT CTAGAGCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGAACCCCTAGTGATGGAGT TGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCT TTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGC CCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGATTCCGTTGCAATGGCTGGCGGTAATATTGTTCTG GATATTACCAGCAAGGCCGATAGTTTGAGTTCTTCTACTCAGGCAAGTGATGTTATTACTAATCAAAGAAGTATT GCGACAACGGTTAATTTGCGTGATGGACAGACTCTTTTACTCGGTGGCCTCACTGATTATAAAAACACTTCTCA GGATTCTGGCGTACCGTTCCTGTCTAAAATCCCTTTAATCGGCCTCCTGTTTAGCTCCCGCTCTGATTCTAACGA GGAAAGCACGTTATACGTGCTCGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGG GTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCC TTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTA GTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATA GACGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACA CTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAAATGAG CTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAAATATTTGCTTATACAATCT TCCTGTTTTTGGGGCTTTTCTGATTATCAACCGGGGTACATATGATTGACATGCTAGTTTTACGATTACCGTTCAT CGATTCTCTCTTGTTTGCTCCAGACTCTCAGGCAATGACCTGATAGCCTTTGTAGAGACCTCTCAAAAATAGCTAC CCTCTCCGGCATGAATTTATCAGCTAGAACGGTTGAATATCATATTGATGGTGATTTGACTGTCTCCGGCCTTTC TCACCCGTTTGAATCTTTACCTACACATTACTCAGGCATTGCATTTAAAATATATGAGGGTTCTAAAAATTTTTAT

? ?

CCTTGCGTTGAAATAAAGGCTTCTCCCGCAAAAGTATTACAGGGTCATAATGTTTTTGGTACAACCGATTTAGC TTTATGCTCTGAGGCTTTATTGCTTAATTTTGCTAATTCTTTGCCTTGCCTGTATGATTTATTGGATGTTGGAATC GTCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATC TGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTC TGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTC ATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGG TTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATT CAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTA TTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGC TGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCG GTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGC GCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGT TGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATA ACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTT TGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACG ACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTAC TCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCC CTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCAC TGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACG AAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATA TACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGAC CAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGA GATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCC GGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTT CTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCT GTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGAT AAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCG AACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTAT CCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTT ATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCT ATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTC CTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGA ACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCG CGCGTTGGCCGATTCATTAATG?[SEQ?ID?N.10]? CCTTGCGTTGAAATAAAGGCTTCTCCCGCAAAAGTATTACAGGGTCATAATGTTTTTGGTACAACCGATTTAGC TTTATGCTCTGAGGCTTTATTGCTTAATTTTGCTAATTCTTTGCCTTGCCTGTATGATTTATTGGATGTTGGAATC GTCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATC TGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTC TGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTC ATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGG TTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCCGGAACCCCTATTTGTTTATTTTTCTAAATACATT CAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTA TTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGC TGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCG GTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGC GCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGT TGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATA ACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTT TGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACG ACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTAC TCTAGCTTCCCGGCAAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCC CTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCAC TGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACG AAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATA TACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGAC CAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGA GATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCC GGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTT CTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCT GTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGAT AAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCG AACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTAT CCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTT ATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCT ATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTC CTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGA ACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCG CGCGTTGGCCGATTCATTAATG?[SEQ?ID?N.10]?

Claims (15)

RIVENDICAZIONI?CLAIMS? 1. Un?acido?nucleico?che?comprende?o?consiste?di?una? sequenza?con?SEQ? ID?N.1,?o?che? comprende?o?consiste?di?una?sequenza?avente?almeno?il?79%?di?identit??con?la?sequenza? con?SEQ?ID?N.1.?1. A nucleic acid that comprises or consists of a sequence with SEQ ID No. 1, or that comprises or consists of a sequence having at least 79% identity with the sequence with SEQ ID No. 1. 2. L'acido?nucleico?secondo?la?rivendicazione?1?che?comprende?o?consiste?di?una?sequenza? con?SEQ?ID?N.2?o?che?comprende?o?consiste?di?una?sequenza?con?SEQ?ID?N.3.?2. The nucleic acid according to claim 1 which comprises or consists of a sequence with SEQ ID No. 2 or which comprises or consists of a sequence with SEQ ID No. 3. 3. L'acido? nucleico? secondo? una? qualsiasi? delle? rivendicazioni? 1? o? 2? per? l?uso? per? l'espressione?in?vivo?dell'alfa?galattosidasi?A?(GLA)?umana,?preferibilmente?in?una?cellula? umana.?3. The nucleic acid according to any one of claims 1 or 2 for use in the in vivo expression of human alpha-galactosidase A (GLA), preferably in a human cell. 4. Un?costrutto?di?acido?nucleico?comprendente:?4. A nucleic acid construct comprising: - una?sequenza?promotore?e??- a?promoter?sequence?and?? - una?sequenza?codificante?del?gene?dell'alfa?galattosidasi?A?(GLA)?sotto?il?controllo?di? detto?promotore,?? - a?sequence?coding?for?the?alpha?galactosidase?A?(GLA)?gene?under?the?control?of?the?said?promoter,?? in?cui?detta?sequenza?codificante???l'acido?nucleico?della?rivendicazione?1?o?2.?wherein said sequence encoding the nucleic acid of claim 1 or 2. 5. Un?vettore?comprendente?l'acido?nucleico?della?rivendicazione?1?o?2?o?il?costrutto?di?acido? nucleico?della?rivendicazione?4,?preferibilmente?detto?vettore???un?vettore?virale?scelto? nel?gruppo?costituito?da:?vettori?adenovirali,?vettori?lentivirali,?vettori?retrovirali?e?vettori? virali?adenoassociati?(AAV).??5. A vector comprising the nucleic acid of claim 1 or 2 or the nucleic acid construct of claim 4, preferably said vector being a viral vector selected from the group consisting of: adenoviral vectors, lentiviral vectors, retroviral vectors and adeno-associated viral (AAV) vectors. 6. Il?vettore?secondo?la?rivendicazione?5?che?comprende?ulteriormente?uno?o?pi??di:?? 6. The carrier according to claim 5 which further comprises one or more of:?? - una? sequenza? 5'? ?inverted? terminal? repeat?? (ITR)? di? un? AAV,? preferibilmente? localizzata?all'estremit??5'?del?promotore;?- a? 5'? inverted? terminal? repeat? (ITR)? sequence? of? an? AAV,? preferably? located? at? the? 5' end? of? the? promoter;? - un?elemento?enhancer,?preferibilmente?localizzato?all'estremit??5'?del?promotore;? - an enhancer element, preferably located at the 5' end of the promoter; - una?sequenza?promotore;?- a?promoter?sequence;? - una? sequenza?di?Kozak,?preferibilmente? localizzata?all'estremit??5'?della? sequenza? codificante? per? GLA? della? rivendicazione? 1? o? 2? e? operativamente? legata? a? detta? sequenza;??- a Kozak sequence, preferably located at the 5' end of the GLA coding sequence of claim 1 or 2, and operatively linked to said sequence; - una? sequenza? di? terminazione? della? trascrizione,? preferibilmente? localizzata? all'estremit??3'?della?sequenza?codificante?per?GLA?della?rivendicazione?1?o?2;?? - una? sequenza? 3'? ?inverted? terminal? repeat?? (ITR)? di? un? AAV,? preferibilmente? localizzata?all'estremit??3'?della?sequenza?di?terminazione?della?trascrizione,?? detto?vettore?comprendente?preferibilmente?in?una?direzione?5'?3':?? ??una?sequenza?5'??inverted?terminal?repeat??(5'?ITR)?di?AAV;? ??una?sequenza?enhancer;?- a transcription termination sequence, preferably located at the 3' end of the GLA coding sequence of claim 1 or 2; - a 3' inverted terminal repeat (ITR) sequence of an AAV, preferably located at the 3' end of the transcription termination sequence, said vector preferably comprising in a 5' 3' direction: a 5' inverted terminal repeat (5' ITR) sequence of an AAV; an enhancer sequence; ??una?sequenza?promotore;???a?promoter?sequence;? ??una?sequenza?di?Kozak;? ??l'acido?nucleico?della?rivendicazione?1?o?2?come?sequenza?codificante?per?GLA;? ??una?sequenza?di?terminazione?della?trascrizione;?e? ??una?sequenza?3'??inverted?terminal?repeat??(3'?ITR)?di?AAV.???a?Kozak?sequence;? ??the?nucleic?acid?of?claim?1?or?2?as?the?GLA?coding?sequence;? ??a?transcription?termination?sequence;?and? ??a?3'?inverted?terminal?repeat?(3'?ITR)?sequence?of?AAV.? 7. Il?vettore?secondo? la? rivendicazione?6? in?cui?detto?elemento?enhancer???un?enhancer? derivato? dal? gene? dell'apolipoproteina? E,? e/o? detta? sequenza? di? terminazione? della? trascrizione???una?sequenza?di?segnale?di?poli?adenilazione,?preferibilmente?il?segnale?di? poli?adenilazione?dell'emoglobina?beta?umana? (HHB?poliA),?e/o?gli? ITR?derivano?dallo? stesso? sierotipo? virale? o? da? sierotipi? virali? diversi,? preferibilmente? il? virus? ?? un? AAV,? preferibilmente? del? sierotipo? 2,? e/o? il? vettore? comprende? ulteriormente? un? introne,? preferibilmente? l'introne? sintetico? derivato? dall'emoglobina? beta? umana? (HBB2),? preferibilmente?legato?operativamente?al?3'?della?sequenza?promotore.?7. The vector according to claim 6 wherein said enhancer element is an enhancer derived from the apolipoprotein gene; and, and/or said transcription termination sequence, a polyadenylation signal sequence, preferably the human hemoglobin beta (HHB polyA) polyadenylation signal, and/or the ITRs are derived from the same viral serotype or different viral serotypes, preferably the virus is an AAV, preferably serotype 2, and/or the vector further comprises an intron, preferably the synthetic intron derived from human hemoglobin beta (HBB2), preferably?operationally linked?to?the?3'?of?the?promoter?sequence.? 8. Un?vettore?integrativo?comprendente??almeno:? ??una?sequenza?codificante?del?gene?dell'alfa?galattosidasi?A?(GLA),?in?cui?detta?sequenza? codificante???l'acido?nucleico?della?rivendicazione?1?o?2,?e? ??una?o?pi??sequenze?di?albumina,??8. An integrative vector comprising at least: a sequence encoding the alpha-galactosidase A (GLA) gene, wherein said sequence encoding the nucleic acid of claim 1 or 2, and one or more albumin sequences, preferibilmente?detto?vettore?comprendente:?? ??una?sequenza?5'??inverted?terminal?repeat??(5'?ITR)?di?AAV;? ??una?prima?sequenza?di?albumina?genomica;? ??una?sequenza?di?skipping?ribosomiale,?preferibilmente?P2A;? ??l'acido?nucleico?della?rivendicazione?1?o?2?come?sequenza?codificante?per?GLA;?preferably?said?vector?comprising:?? a?5'?inverted?terminal?repeat?(5'?ITR)?sequence?of?AAV;? a?first?genomic?albumin?sequence;? a?ribosomal?skipping?sequence,?preferably?P2A;? the?nucleic?acid?of?claim?1?or?2?as?GLA?encoding?sequence;? ? ? ??una?seconda?sequenza?di?albumina?genomica?comprendente?un?motivo?adiacente?al? protospacer?(PAM);???a?second?albumin?genomic?sequence?comprising?a?protospacer?adjacent?motif?(PAM);? ??una?sequenza?3'???inverted?terminal?repeat??(3'?ITR)?di?AAV.???a?3'???inverted?terminal?repeat??(3'?ITR)?sequence?of?AAV.? 9. Un?plasmide?comprendente:?9. A plasmid comprising: ??una?sequenza?promotore,?e? ??a?promoter?sequence,?and? ??una?sequenza?codificante?del?gene?dell'alfa?galattosidasi?A? (GLA)?sotto? il?controllo?di? detto?promotore,?in?cui?detta?sequenza?codificante???l'acido?nucleico?della?rivendicazione? 1?o?2,?preferibilmente?detto?plasmide?che?comprende?o?che?consiste?di?una?sequenza?con? SEQ?ID?N.10.???a?sequence?coding?for?the?alpha?galactosidase?A? (GLA)?gene?under?the?control?of?said?promoter,?wherein?said?sequence?coding?the?nucleic?acid?of?claim?1?or?2,?preferably?said?plasmid?comprising?or?consisting?of?a?sequence?with?SEQ?ID?No.10.? 10. Un?plasmide?comprendente:?10. A plasmid comprising: ?? la?sequenza?codificante?del?gene?dell'alfa?galattosidasi?A?(GLA),? in?cui?detta?sequenza? codificante???l'acido?nucleico?della?rivendicazione?1?o?2,?e? ?? the?coding?sequence?of?the?alpha?galactosidase?A?(GLA)?gene,?wherein?said?sequence?coding?the?nucleic?acid?of?claim?1?or?2,?is? ??una?o?pi??sequenze?di?albumina,?? ??one?or?more?albumin?sequences,?? preferibilmente?detto?plasmide?che?comprende??o?che?consiste?di?una?sequenza?con?SEQ? ID?N.15.?preferably?called?plasmid?which?comprises?or?consists?of?a?sequence?with?SEQ? ID?N.15.? 11. Una?cellula?ospite?trasformata?con?il?vettore?secondo?una?qualsiasi?delle?rivendicazioni?5-8?o?con?il?plasmide?secondo?una?qualsiasi?delle?rivendicazioni?9?10.?11. A host cell transformed with the vector according to any of claims 5-8 or with the plasmid according to any of claims 9 or 10. 12. Una?composizione?farmaceutica?che?comprende?l'acido?nucleico?della?rivendicazione?1?o? 2?o?il?costrutto?di?acido?nucleico?della?rivendicazione?4?o?il?vettore?secondo?una?qualsiasi? delle?rivendicazioni?5?8?o?il?plasmide?secondo?una?qualsiasi?delle?rivendicazioni?9?10?o?la? cellula? ospite? secondo? la? rivendicazione? 11? e? almeno? un? veicolo? e/o? eccipiente? farmaceuticamente?accettabile.?12. A pharmaceutical composition comprising the nucleic acid of claim 1 or 2, or the nucleic acid construct of claim 4, or the vector of any of claims 5 or 8, or the plasmid of any of claims 9 or 10, or the host cell of claim 11, and at least one pharmaceutically acceptable carrier and/or excipient. 13. L'acido? nucleico? della? rivendicazione? 1? o? 2? o? il? costrutto? di? acido? nucleico? della? rivendicazione?4?o?il?vettore?secondo?una?qualsiasi?delle?rivendicazioni?5?8?o?il?plasmide? secondo? una? qualsiasi? delle? rivendicazioni? 9?10? o? la? cellula? ospite? secondo? la? rivendicazione?11?o?la?composizione?farmaceutica?della?rivendicazione?12?per?l'uso?come? medicamento.?13. The nucleic acid of claim 1 or 2, or the nucleic acid construct of claim 4, or the vector of any one of claims 5 or 8, or the plasmid of any one of claims 9 or 10, or the host cell of claim 11, or the pharmaceutical composition of claim 12 for use as a medicament. 14. L'acido? nucleico? della? rivendicazione? 1? o? 2? o? il? costrutto? di? acido? nucleico? della? rivendicazione?4?o?il?vettore?secondo?una?qualsiasi?delle?rivendicazioni?5?8?o?il?plasmide? 14. The nucleic acid of claim 1 or 2 or the nucleic acid construct of claim 4 or the vector of any of claims 5 or 8 or the plasmid ? ? secondo? una? qualsiasi? delle? rivendicazioni? 9?10? o? la? cellula? ospite? secondo? la? rivendicazione?11?o?la?composizione?farmaceutica?della?rivendicazione?12?per?uso?nella? terapia?genica.?according to? any? of? claims? 9?10? or? the? host? cell? according to? claim? 11? or? the? pharmaceutical? composition? of? claim? 12? for? use? in? gene? therapy.? 15. L'acido? nucleico? della? rivendicazione? 1? o? 2? o? il? costrutto? di? acido? nucleico? della? rivendicazione?4?o?il?vettore?secondo?una?qualsiasi?delle?rivendicazioni?5?8?o?il?plasmide? secondo? una? qualsiasi? delle? rivendicazioni? 9?10? o? la? cellula? ospite? secondo? la? rivendicazione?11?o? la?composizione? farmaceutica?della? rivendicazione?12?per?uso?nel? trattamento?della?malattia?di?Fabry.?15. The nucleic acid of claim 1 or 2, or the nucleic acid construct of claim 4, or the vector of any one of claims 5 or 8, or the plasmid of any one of claims 9 or 10, or the host cell of claim 11, or the pharmaceutical composition of claim 12, for use in the treatment of Fabry disease. ? ?
IT102023000004443A 2023-03-09 2023-03-09 HUMAN ALPHA GALACTOSIDASE CODING SEQUENCE FOR THE TREATMENT OF FABRY DISEASE IT202300004443A1 (en)

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