Dario Balestra University of Ferrara
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1 ABERRANT mrna SPLICING IN COAGULATION FACTOR DEFICIENCIES: FROM MOLECULAR MECHANISMS TO RNA-BASED THERAPEUTIC APPROACHES Dario Balestra University of Ferrara
2 mrna SPLICING
3 Several sequence elements, both conserved and exon-specific, are required for proper definition of the exons.
4 Sequence elements define the proper assembly of the complex Spliceosome machinery
5 Key role of the U1snRNP in the earliest splicing step, and thus in exon definition
6 This complexity makes the spliceosome susceptible to derangements Protein isoforms or unproductive splicing 80% of human genes undergo alternative splicing
7 Mutations affecting mrna splicing are frequent cause of severe human genetic disease forms (>20-30%) Gene Mutation impairing splicing mrna Correct processing Exon skipping intron inclusion Functional protein Altered protein, if any Physiological Function Genetic Disease
8 Molecular mechanisms of genetic disorders Modulation of pre-mrna splicing Gene Mutation impairing splicing mrna Correct processing RNA-based Therapeutic Approaches Functional protein Rescue of physiological function
9 Coagulation factor disorders as models splicing mutations are relatively frequent in severe coagulation factor deficiencies even tiny increase in activity of plasma proteins could significantly increase the coagulation efficiency and thus ameliorate the bleeding phenotype in patients protein and activity levels of clotting factors can be evaluated by enzymatic assays (hardly feasible for most of the other human diseases).
10 IVS7 +5GA (FVII Lazio) A IVS7 5 ss is in the 1st of highly homologous 37bp repeats containing identical cryptic 5'ss (*) TGG / GTGGGTACC (Pinotti et al, Blood, 1998) * * * * * Exon 6 Exon 7 EXON Exon 8 8
11 IVS7 +5GA (FVII Lazio) A TGG / GTGGGTACC * * * * * Exon 6 Exon 7 EXON Exon VNTR-modulating splicing efficiency (Pinotti et al, Blood, 2000)
12 What s the molecular mechanism of the IVS7+5G/A mutation?
13 Expression studies in eukaryotic cells: Minigene approach 1a 1b IVS7 Minigene (ex.6-8) Ex.6 Ex7 Ex8 Gene region of interest Expression in mammalian cells and studies at the mrna level Pinotti et al, Blood 2008
14 The mutation induces exon skipping or 1 repeat inclusion 6F IVS7+5GA 8R pcdna3 Ex.6 Ex7 * Ex8 Wt IVS7+5A HD 1 2 Ex.6 Ex.6 Ex7 Ex7 Ex8 Ex8 637 bp 600 bp 3 Ex.6 Ex8 480 bp RT-PCR 6F-8R Causing frameshift and premature termination of translation Pinotti et al, Blood 2008
15 Fluorescent labeling of RT-PCR products Luxex7 7 8 R8 Wt (237bp) 7 8 1st repeat inclusion (274 bp) 7 8 Wt IVS7+5G IVS7+5G/A (5x) The mutation impairs but not abrogates splicing (0.3±0.1%), thus accounting for residual FVII levels in patients Pinotti et al, Blood 2008
16 IVS7+5GA U1snRNA Ex.6 Ex7 Ex8? Restore exon definition by compensatory U1 snrna changes Could this rescue FVII splicing impaired by the IVS7+5G/A?
17 Ex7 Ex8 We constructed expression vectors for U1snRNAs, designed to bind the mutated 5'ss or different sequences in repeats
18 Modified U1snRNAs reduced exon 7 skipping M Wt IVS7+5A + U1+5A U1Mut1 U1Mut2 Wt-U1 RT-PCR 6F-8R Ex.6 Ex.6 Ex7 HD Ex.6 Ex7 Ex8 Ex8 Ex8 Only U1+5A, binding the mutated 5'ss, partially rescued splicing M Wt IVS7+5A + U1+5A U1Mut1 U1Mut2 Wt-U1 Ex7 Ex7 Ex8 Ex8 RT-PCR 7F-8R Pinotti et al, Blood 2008
19 The snu1+5a was able to partially rescue correct splicing.and in a dose-dependent manner Wt +U1-A5 (1x) IVS7+5A +U1-A5 (1.5x) The correctly spliced form corresponded to 15% of the aberrant forms Pinotti et al, Blood 2008
20 Does the rescue of correct mrna produce an increase in FVII protein expression? a XbaI TGG/gtgggtac * XbaI ex1 ex2 ex3 ex4 ex5 ex6 ex7 ex8 HYBRID cdna- GENE CONSTRUCT Expression in Cos-7 cells and studies at the mrna and protein levels
21 Fluorogenic Assays Tissue Factor (TF), phospholipids and Ca 2+) Conditioned medium containing rfvii TF-FVIIa FX FXa FXa specific fluorogenic substrate Fluorescence
22 Coagulation Assays Prothrombin time (PT) Human FVII deficient plasma supplemented with rfvii Tissue factor Phospholipid, Calcium Coagulation pathway Fibrin clot T I M E
23 Pinotti et al, Blood 2009
24 . to a level that would be, in vivo, well beyond the therapeutic threshold
25 Modified U1snRNA are able to re-direct the spliceosome assembly and restore exon definition in cellular models IVS7+5G/A Ex.6 Ex7 Ex8 U1snRNA Enginnered U1+5a What about correction efficacy in vivo?
26 CREATION OF THE MOUSE MODEL OF HUMAN FVII DEFICIENCY CAUSED BY SPLICING MUTATION Liver-restricted expressionof the human FVII cassette in wtmice Evaluation of hfviiin hepatocytes and plasma by speciesspecific assays paav-fvii FVII+5G>A ClaI ClaI XhoI ITR phaat SV40pA ITR
27 ASSESSMENT OF THE U1+5A-MEDIATED RESCUE Liver-restricted expressionof the human FVII cassette in wtmice Co-expression of the U1+5a EVALUATION OF RESCUE paav-fvii FVII+5G>A ClaI ClaI XhoI ITR phaat SV40pA ITR paav-u1+5a ITR BamHI pu1 U1+5a BamHI ITR
28 HYDRODYNAMIC INJECTION STUDIES paav2-haat-fvii+5a paav8-u1+5a
29 The expression of the FVII+5A variant did not resulted in appreciable plasma hfvii levels hfvii antigen (µg/ml) X 2X paav-fviiwt paav-fvii+5a paav-u1+5a n= 4 mice/group Molar ratio U1+5a/FVII+5A=1,5
30 U1+5a rescue of circulating hfvii protein levels hfvii antigen (µg/ml) X 2X paav-fviiwt paav-fvii+5a paav-u1+5a n= 4 mice/group Molar ratio U1+5a/FVII+5A=1,5 Compared to levels in mice injected with FVII wt plasmid, the correction obtained after injection of U1+5a was ~8,5 %
31 U1+5a-mediated rescue of hfvii expression in mouse liver hfvii protein (HI) paav-u1+5a paav-fvii+5a paav-fviiwt +
32 U1+5a-mediated rescue of hfvii expression in mouse liver hfvii protein (HI) hfvii mrna (RT-PCR) paav-fviiwt bp M paav-fviiwt paav-fvii+5a + paav-u1+5a 2 h7f h8r a n paav-u1+5a paav-fvii+5a + % total hfvii n= 4 mice/group Molar ratio U1+5a/FVII+5A=1,5 a1 n 2
33 PROLONGED RESCUE BY ADENOASSOCIATED VIRAL VECTORS AAV2-hAAT-FVII+5A AAV8-U1+5a
34 STUDIES WITH ADENOASSOCIATED VIRAL VECTORS AAV2-hAAT-FVII+5A AAV8-U1+5a AAV2-FVII+5A (vg/mouse) AAV8-U1+5a (vg/mouse) 1.2* *10 11 U1+5a-mediated rescue of circulating hfvii levels was appreciable and prolonged
35 STUDIES WITH ADENOASSOCIATED VIRAL VECTORS AAV2-hAAT-FVII+5A AAV8-U1+5a and the correction extent was dose-dependent
36 U1+5a-MEDIATED RESCUE IN MOUSE LIVER + AAV8-U1+5a AAV2-FVII+5A 1.2*10 12 vg/mouse + AAV8-U1+5a hfvii mrna (RT-PCR) bp M 6.0*10 11 vg/mouse 1.2*10 11 vg/mouse n a % total hfvii % a n AAV2-FVII+5A (1.2*10 12 vg/mouse) AAV2-FVII+5A (1.2*10 12 vg/mouse) + AAV8-U1+5a (6.0*10 11 vg/mouse) hfvii protein (HI)
37 LIMITATION OF THE MOUSE MODEL AAV2-FVII+5a AAV8-U1+5a No rescue No rescue Rescue
38 LIMITATION OF THE MOUSE MODEL AAV2-FVII+5a AAV8-U1+5a No rescue No rescue Rescue IF TRUE, INCREASING THE SUBSTRATE WOULD RESULT IN INCREASED RESCUE
39 INCREASING THE DOSE OF THE hfvii SUBSTRATE RESULTED IN INCREASED RESCUE BY THE U1+5a hfvii antigen (ng/ml) AAV2-FVII+5A (vg/mouse) AAV8-U1+5a (vg/mouse) 1.2* * * *10 11 therefore, rescue efficiency in patients, expressing the FVII mrna in all hepatocytes, should be much more pronounced
40 CONCLUSION I Engineered U1snRNA are capable to re-direct the spliceosome assembly to the mutated exon-intron juntion and rescue mrna processing and secretion of functional FVII; For the first time, we provide the «proof-of-principle» for the U1-mediated correction in vivo
41 HOWEVER the approach implies one modified U1snRNA for each splicing mutation, thus limiting the potential applicability In vivo, a liver-toxicity has been observed with the highest doses. This could be due to Off-target effect Oversaturation of UsnRNA pathway (Grimm, Streetz et al. 2006) (Vickers, Sabripour et al. 2011) Can we be more sequence specific? Reduction of amount of modified U1snRNA
42 TF FVIIa FIX FX FIXa FVIIIa FXa FVa Hemophilia B model PT TH Alanis et al, HMG 2012
43 TF FVIIa FIX FX FIXa FVIIIa FXa FVa F9 Exon 5 is poorly defined 3 ss 5 ss ,94 0,21 0,99 0,79 PT TH Alanis et al, HMG 2012
44 Expression studies with hybrid F9 minigenes ptb NdeI FIX ex5 NdeI NdeI FIX IVS4 FIX IVS5 Glob ex1 Glob ex2 Glob ex3a FIX ex5 Glob ex3b Fibr ex1 Fibr ex2 ptb NdeI FIX ex5 in vitro exon 5 skipping 482 bp 611 bp messaggero wt Exon 5 is poorly defined Alanis et al, HMG 2012
45 Is this due to the hybrid minigene features?
46 Exon 5 is poorly defined in vivo ptb NdeI FIX ex5 in vitro exon 5 skipping IN VIVO Human Liver FIX mrna 482 bp 611 bp 299 bp 418 bp messaggero wt Exon 5 is poorly defined Alanis et al, HMG 2012
47 U2AFs U1wt The mutations, reducing exon 5 definition, are candidate to induce exon 5 skipping
48 All mutations induced exon 5 skipping
49 U1FIXwt GTCCAGTAT Rescue by U1snRNA targeting the FIX donor splice site Alanis et al, HMG 2012
50 U1FIXwt GTCCAGTAT A single U1snRNA rescued mutations at either donor or acceptor sites Alanis et al, HMG 2012
51 However, U1snRNA targeting 5 ss might not ensure enough sequence specificity
52 Exon specific U1snNAs (ExSpeU1) targeting intronic sequences Design of ExSpeU1 F. Pagani ICGEB, Trieste Alanis et al, HMG 2012
53 Rescue by Exon specific U1snNAs Design of ExSpeU1 FIX-IVS5-2C variant Exon-specific U1 rescued splicing to appreciable levels Alanis et al, HMG 2012
54 Rescue by Exon specific U1snNAs Design of ExSpeU1 FIX-IVS5-2C variant ExSpeU1 FIX9 and FIX10 Alanis et al, HMG 2012
55 Rescue of FIX biosynthesis and function: Alanis et al, HMG 2012
56 Differential impact on Splicing (exon skipping) RNA Protein profile (deleted FIX) Protein Isoforms Secretion (impaired) Secreted levels Activity (abolished) Coagulant Act. The deleted variant, lacking EGF2, is secreted but inactive
57 RNA The deleted variant, lacking EGF2, is secreted Protein Isoforms Secreted levels but inactive Coagulant Act. The deleted variant, lacking EGF2, is secreted but inactive
58 U1snRNA mediated rescue of: Splicing RNA protein pattern Protein Isoforms Secretion Secreted levels activity Coagulant Act. The deleted variant, lacking EGF2, is secreted but inactive
59 RNA Protein Isoforms Secreted levels Coagulant Act. A unique ExSpeU1 completely rescued FIX function in the presence of different mutations at either the donor or acceptor splice sites
60 REPLACEMENT GENE THERAPY ADVANTAGES DNA encoding disease protein Mutation Viral/non viral delivery of therapeutic gene INTERVENTION AT pre-mrna LEVEL Therapy Maintainance of the gene regulation Correction in physiological tissues only Small size of the therapeutic expression cassette
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