Basi molecolari delle malattieBasi molecolari delle malattie

Nuove strategie terapeutiche nella cura dell’emofil ia A:Nuove strategie terapeutiche nella cura dell’emofil ia A:Spliceosome Mediated RNA transSpliceosome Mediated RNA trans --splicing (SMaRT)splicing (SMaRT)Spliceosome Mediated RNA transSpliceosome Mediated RNA trans --splicing (SMaRT)splicing (SMaRT)

HEMOPHILIAHEMOPHILIA

•• An XAn X--linked bleeding disorder caused by mutations in the linked bleeding disorder caused by mutations in the gene for coagulation Factor VIII (hemophilia A).gene for coagulation Factor VIII (hemophilia A).

•• HA affects 1:5000 males worldwide.HA affects 1:5000 males worldwide.

•• Severe cases are characterized by frequent spontaneous Severe cases are characterized by frequent spontaneous •• Severe cases are characterized by frequent spontaneous Severe cases are characterized by frequent spontaneous bleeding episodes (joints). bleeding episodes (joints).

•• Hemorrhagic events in untreated severe patients can be Hemorrhagic events in untreated severe patients can be fatal.fatal.

The coagulation cascadeThe coagulation cascade

The coagulation cascadeThe coagulation cascade

La diagnosi di HALa diagnosi di HA

LaLa diagnosidiagnosi dell’emofiliadell’emofilia AA èè ottenuta,ottenuta, inin primaprima fase,fase, attraversoattraverso unun semplicesemplice testtestdelladella coagulazione,coagulazione, denominatodenominato aPTTaPTT (tempo(tempo didi tromboplastinatromboplastina parziale),parziale), checherisultarisulta allungatoallungato;; inin secondaseconda fase,fase, ilil dosaggiodosaggio direttodiretto deldel FVIIIFVIII (test(test cromogenico)cromogenico)permettepermette didi quantificarnequantificarne ilil deficitdeficit

Clinical classification:Clinical classification:66--30% Mild30% Mild66--30% Mild30% Mild11--5% Moderate5% Moderate< 1% Severe< 1% Severe

NelleNelle formeforme lievilievi ee moderatemoderate gligli episodiepisodi didi sanguinamentosanguinamento sisi verificanoverificano dopodopotraumitraumi importantiimportanti oo interventiinterventi chirurgicichirurgici;; nellenelle formeforme piùpiù severe,severe, episodiepisodi emorragiciemorragiciaa livellolivello muscolaremuscolare oo intramuscolareintramuscolare possonopossono insorgereinsorgere spontaneamentespontaneamente oo aaseguitoseguito didi traumitraumi lievilievi..

LaLa mutazionemutazione piùpiù frequente,frequente, presentepresentenelnel 4040%% deidei pazientipazienti affettiaffetti dada emofiliaemofiliaA,A, èè l’l’inversioneinversione dell’intronedell’introne 2222 (gene(geneA)A) deldel genegene perper ilil FVIIIFVIII perperricombinazioniricombinazioni concon sequenzesequenze omologheomologheripetuteripetute situatesituate aa 500500 KbKb didi distanzadistanza..

II casicasi cheche nonnon presentanopresentano questaquestamutazionemutazione sono,sono, invece,invece, associatiassociati adad ununmutazionemutazione sono,sono, invece,invece, associatiassociati adad ununinsiemeinsieme assaiassai eterogeneoeterogeneo didi alterazionialterazionigenetichegenetiche

Intrinsic & Extrinsic Xase ComplexIntrinsic & Extrinsic Xase Complex

10%10% 90%90%

Current Treatment for HemophiliaCurrent Treatment for Hemophilia•• Infusion of either recombinant or plasmaInfusion of either recombinant or plasma--derived derived

clotting factor concentrates in response to bleeds clotting factor concentrates in response to bleeds

((Kogenate®, Helixate®, Ricombinase®Kogenate®, Helixate®, Ricombinase®, , Refacto®, Refacto®,

NovoSeven®)NovoSeven®)

•• DisadvantagesDisadvantages•• DisadvantagesDisadvantages–– ProteinProtein hashas aa shortshort halfhalf--lifelife–– RisksRisks ofof plasmaplasma--derivedderived productsproducts–– OngoingOngoing tissuetissue damagedamage becausebecause bleedsbleeds areare treatedtreated

ratherrather thanthan preventedprevented–– ExpenseExpense (>(> USUS 100100K/year)K/year)

Antibody formation to FVIII (Inhibitors, IgG antiAntibody formation to FVIII (Inhibitors, IgG anti-- FVIII)FVIII)

•• MinimalMinimal elevationelevation inin thethe factorfactor levellevel (>(>11%%)) isisclinicallyclinically beneficialbeneficial

•• SuccessfulSuccessful genegene transfertransfer wouldwould resultresult inincontinuouscontinuous maintenancemaintenance ofof clottingclotting factorfactor levelslevels

Advantages in gene therapyAdvantages in gene therapy

continuouscontinuous maintenancemaintenance ofof clottingclotting factorfactor levelslevelsadequateadequate toto preventprevent ratherrather thanthan treattreat mostmost bleedsbleedsafterafter theythey havehave occurredoccurred

•• More convenient (no intravenous injection)More convenient (no intravenous injection)

•• Risks of blood products avoidedRisks of blood products avoided

Animal model for HAAnimal model for HA

DogsDogs

-- ExpensiveExpensive-- Long generation timeLong generation time

MICE (E16 MICE (E16 –– E17)E17)

-- Ablation of ex.16 and ex.17Ablation of ex.16 and ex.17-- No light chain No light chain -- No functional FVIIINo functional FVIII

MoAbsMoAbs to to hFVIIIhFVIII

-- Ablation of ex.16 and ex.17Ablation of ex.16 and ex.17-- No light chain No light chain -- No functional FVIIINo functional FVIII

EE--1616 andand EE--1717 micemicehavehave aa mildmild phenotypephenotypeMoAbs to hFVIIIMoAbs to hFVIII

-- Ablation of ex.16 and ex.17Ablation of ex.16 and ex.17-- No light chain No light chain -- No functional FVIIINo functional FVIII

SplicingSplicing

5’ splice5’ splice--sitesiteAG/AG/GUGURAGURAGU

BranchpointBranchpointCURCURAACYNYCYNY

PolypyrimidinePolypyrimidinetracttract

3’ splice site3’ splice siteYYAGAG lGlG

SplicingSplicing

TransTrans--SplicingSplicing

ciscis transtrans

Esempi in naturaEsempi in natura

TRANSTRANS--SPLICINGSPLICING--Hybrid coding/nonHybrid coding/non--coding RNAscoding RNAs

-- RibozymesRibozymesselfself--catalytic RNA moleculescatalytic RNA molecules

-- tRNAtRNA endonucleaseendonuclease ((archaebacteriumarchaebacterium))

-- SMaRTSMaRTSpliceosome Mediated RNA TransSpliceosome Mediated RNA Trans--splicingsplicing

SMaRT: spliceosome mediated RNA SMaRT: spliceosome mediated RNA transtrans--splicingsplicing

SMaRT: spliceosome mediated RNA SMaRT: spliceosome mediated RNA transtrans--splicingsplicing

PMT: prePMT: pre--transtrans--splicing moleculesplicing molecule

PTMs structurePTMs structure

Prototype PTMs comprise three domains:

PTMs structurePTMs structure

Prototype PTMs comprise three domains:

Binding domaincomplementary to the target intron

PTMs structurePTMs structure

Prototype PTMs comprise three domains:

Binding domaincomplementary to the target intron

Splicing domaincontaining necessary splicing elements Donor; Branch point; Polypirimidin tract; Acceptor

TSDTSDTransTrans--splicing domainsplicing domain

PTMs structurePTMs structure

Prototype PTMs comprise three domains:

Binding domaincomplementary to the target intron

Splicing domaincontaining necessary splicing elements Donor; Branch point; Polypirimidin tract; Acceptor

Coding domainto be trans-spliced to the target

TSDTSDTransTrans--splicing domainsplicing domain

PTMs structurePTMs structure

TheThe lengthlength andand compositioncomposition ofof thethe bindingbinding domainsdomains cancan bebemodifiedmodified toto alteralter efficiency,efficiency, specificity,specificity, andand targetingtargeting locationlocation withinwithinaa prepre--mRNAmRNA..

TheThe splicingsplicing domaindomain isis designeddesigned toto maximizemaximize transtrans--splicingsplicingactivityactivity and,and, therefore,therefore, typicallytypically containscontains aa potentpotent branchpointbranchpointsequencesequence (UACUAAC)(UACUAAC) andand aa longlong pyrimidinepyrimidine--richrich tracttract..

PositionSplice site type

MotifConsensus value (0-100)

+3 Acceptor CTTCAAGgtgagcg 69.56

+7 Donor AAGgtgagc 95.71

+31 Acceptor atctgggtcgaggg 76.4

SPLICING ELEMENT DEFINITIONSPLICING ELEMENT DEFINITION

CTTCAAG gtgagcg ccctc Ac Py cacagCTATC7 150

+31 Acceptor atctgggtcgaggg 76.4

+34 Donor tgggtcgag 88.7

+56 Acceptor ccttcctcgcaggg 94.13

+61 Acceptor ctcgcagggcagag 80.9

+92 Donor gaggtgtag 74.18

+94 Acceptor ggtgtagcgcaggc 81.05

+147 Acceptor ttctctgcacagCT 89.78

DependingDepending onon thethe TransTrans--splicingsplicing moleculemolecule ((55’,’,33’’ oror 55’’--33’)’) splicingsplicing elementelement positionpositionhadhad toto bebe defineddefined alsoalso forfor thethe presencepresence ofof putativeputative cripticcriptic sitesite nearbynearby

Branch Point position Branch Point motif Consensus va lue (0-100)

61 ccctgAc 95.07

75 tcctcAg 95.75

89 gcctcAa 91.75

SPLICING ELEMENT DEFINITIONSPLICING ELEMENT DEFINITION

CTTCAAG gtgagcg ccctc Ac Py cacagCTATC7 150

89 gcctcAa 91.75

94 ctctcAg 93.91

107 ccctcAc 100

120 ttctgAc 90.17

Branch points are normally located within 50bp upstream of the Branch points are normally located within 50bp upstream of the acceptor site of introns acceptor site of introns

PTMs VALIDATIONPTMs VALIDATIONRNA secondary structure predictionRNA secondary structure prediction

AA BB

PrePre--TransTrans--splicing Moleculessplicing Molecules

Correction of HA in ECorrection of HA in E--16 mice (Naked DNA injection)16 mice (Naked DNA injection)

mFVIIImFVIII mRNAmRNA

The coagulation cascadeThe coagulation cascade

mFVIII activitymFVIII activity

Correction of HA in ECorrection of HA in E--16 mice (Naked DNA injection)16 mice (Naked DNA injection)

mFVIII mRNAmFVIII mRNA

Correction of HA in ECorrection of HA in E--16 mice (Naked DNA injection)16 mice (Naked DNA injection)

mFVIII activitymFVIII activity

mFVIII mRNAmFVIII mRNA

mFVIII protein (light chain)mFVIII protein (light chain)

Correction of HA in ECorrection of HA in E--16 mice (Adenovirus vector)16 mice (Adenovirus vector)

FVIII protein (light chain)FVIII protein (light chain)

TransTrans--splicing advantages..........splicing advantages..........

-- ReductionReduction ofof transgenetransgene sizesizereplacementreplacement onlyonly ofof mutatedmutated portionportion

-- GeneGene expressionexpression underunder thethe controlcontrol ofof endougenousendougenousregulatoryregulatory elementselements

-- TissueTissue specificspecific expressionexpression-- TissueTissue specificspecific expressionexpressionRepairedRepaired productsproducts areare limitedlimited byby thethe presencepresence ofofthethe endogenousendogenous targettarget

TransTrans--splicing advantages..........splicing advantages..........

-- ReductionReduction ofof transgenetransgene sizesizereplacementreplacement onlyonly ofof mutatedmutated portionportion

-- GeneGene expressionexpression underunder thethe controlcontrol ofof endougenousendougenousregulatoryregulatory elementselements

-- TissueTissue specificspecific expressionexpression-- TissueTissue specificspecific expressionexpressionRepairedRepaired productsproducts areare limitedlimited byby thethe presencepresence ofofthethe endogenousendogenous targettarget

-- LowLow efficiencyefficiency

...........and disadvantages...........and disadvantages

Examples of successful Trans-splicing

- Cystic Fibrosis (CFTR) in vivo

-Spinal muscular atrophy (SMA) in vitro

- Epidermolysis bullosa simplex with muscular dystrophy - Epidermolysis bullosa simplex with muscular dystrophy (EBS-MD) in vitro

ReferencesReferences

-- Fang, H., L. Wang, et al. (2007). "The protein structure and effect of factor Fang, H., L. Wang, et al. (2007). "The protein structure and effect of factor VIII." VIII." Thromb ResThromb Res 119(1): 1119(1): 1--13.13.-- Chao, H. and C. E. Walsh (2006). "RNA repair for haemophilia A." Chao, H. and C. E. Walsh (2006). "RNA repair for haemophilia A." Expert Rev Expert Rev Mol MedMol Med 88(1): 1(1): 1--8.8.-- Fay, P. J. and P. V. Jenkins (2005). "Mutating factor VIII: lessons from structure Fay, P. J. and P. V. Jenkins (2005). "Mutating factor VIII: lessons from structure to function." to function." Blood RevBlood Rev 1919(1): 15(1): 15--27.27.-- Fay, P. J. (2004). "Activation of factor VIII and mechanisms of cofactor action." Fay, P. J. (2004). "Activation of factor VIII and mechanisms of cofactor action." Blood RevBlood Rev 1818(1): 1(1): 1--15.15.-- Mansfield, S. G., H. Chao, et al. (2004). "RNA repair using spliceosomeMansfield, S. G., H. Chao, et al. (2004). "RNA repair using spliceosome--mediated RNA transmediated RNA trans--splicing." splicing." Trends Mol MedTrends Mol Med 1010(6): 263(6): 263--8.8.mediated RNA transmediated RNA trans--splicing." splicing." Trends Mol MedTrends Mol Med 1010(6): 263(6): 263--8.8.-- Chao, H., S. G. Mansfield, et al. (2003). "Phenotype correction of hemophilia A Chao, H., S. G. Mansfield, et al. (2003). "Phenotype correction of hemophilia A mice by spliceosomemice by spliceosome--mediated RNA transmediated RNA trans--splicing." splicing." Nat MedNat Med 99(8): 1015(8): 1015--9.9.-- GarciaGarcia--Blanco, M. A. (2003). "Messenger RNA reprogramming by Blanco, M. A. (2003). "Messenger RNA reprogramming by spliceosomespliceosome--mediated RNA transmediated RNA trans--splicing." splicing." J Clin InvestJ Clin Invest 112112(4): 474(4): 474--80.80.-- Puttaraju, M., J. DiPasquale, et al. (2001). "Messenger RNA repair and Puttaraju, M., J. DiPasquale, et al. (2001). "Messenger RNA repair and restoration of protein function by spliceosomerestoration of protein function by spliceosome--mediated RNA transmediated RNA trans--splicing." splicing." Mol TherMol Ther 44(2): 105(2): 105--14.14.-- Bi, L., R. Sarkar, et al. (1996). "Further characterization of factor VIIIBi, L., R. Sarkar, et al. (1996). "Further characterization of factor VIII--deficient deficient mice created by gene targeting: RNA and protein studies." mice created by gene targeting: RNA and protein studies." BloodBlood 8888(9): 3446(9): 3446--50.50.