Ana Terron-Kwiatkowski, MRCPath part I course, London 2010

1. Explain with examples how our current understanding of gene structure and function has led to the development of treatment strategies for genetic diseases Ana Terron-Kwiatkowski, MRCPath part I course, London 2010

2. Gene structure, function and therapeutic strategies 1- CF (AR) - CFTR functions - Type of mutations - Therapies: * Gene replacement * Aminoglycosides read-through stops * Correctors * Potentiators * Stimulation of alternative Cl- channnels 2- DMD (X-linked) - Severe DMD - PTC: lack of dystrophin (NMD) - Milder BMD - In-frame deletions: residual function - Therapies: * Antisense oligonucleotides: exon-skipping * Regenerative therapy: myostatin inhibition 3- HD (AD) - HTT - CAG triplet expansion - transcriptional dysregulation (hypoacetylation of histones) - polyQ aggregates in brain - Therapies: * siRNAs  HTT expression * HDAC inhibitors * Tricyclic pyrone compounds

3. Gene structure, function and therapeutic strategies 4- Marfan syndrome (AD) - FBN1 point mutations: dominant-negative effect - Alter TGFß signal pathway - Therapies: * Ribozymes to specifically target mutant alleles * TGFß antagonists: losartan 5- CML: t(9;22)- chromosome Philadelphia • BCR-ABL fusion gene- potent TK activity • Therapy: ABL specific TK inhibitor (imatinib, Gleevec) 6- Melanoma: B-RAF mutations - protein kinase: RAF/MEK/ERK pathway - Therapy: inhibitor of oncogenic B-RAF (PLX4032)

6. Cystic fibrosis: gene defects, altered functions and therapeutic strategies • CFTR replacement therapy: - successful gene transfer into airway epithelial cells - short term expression * viral vectors: more efficacious more likely adverse effects transient effect  repeated dosing  immune response * adeno-associated-virus based gene therapy: successful transfer and improved FEV * cationic-lipid based vector trial underway in UK (2009)

7. Therapeutic strategies for CF • Aminoglycosides (Gentamicin) - Target class I mutations - PTC • Induce read-through premature stop codons • Reduce translation fidelity by inhibiting ribosomal ‘proofreading’ • Susceptibility to supression depends on stop-codon and surrounding sequences • Result in full-length functional CFTR • Topical application to nasal epithelium • Concentrations required are toxic (ototoxicity) • PTC 124- derivative that lacks antibiotic function and toxicity

8. Therapeutic strategies for CF • Class II mutations (Phe508del) - CFTR trafficking impaired due to proteosomic degradation of misfolded CFTR ‘Correctors’ - small molecules (eg 4-phenylbutirate) that improve CFTR processing - facilitating exit from RE - may have adverse effects (degradation of misfolded proteins important intracellular control mechanism) • Class III mutations - reduce opening probability of CFTR channel (G551D) ‘ Potentiators’ - compounds that activate CFTR channel * VX770 - good efficacy and safety profile, oral admin - clinical trials: improved CFTR function

9. DMD gene defects, altered functions and therapeutic strategies Dystrophin • 20-30% expression of dystrophin gene constructs shown in mice challenge efficient delivery and long-term expression in skeletal muscle • Dystrophin function: essential component of dystrophin-glycoprotein complex (DGC) maintaining the membrane integrity of muscle fibers • DMD - PTC: absence of dystrophin- continuous loss of muscle fibers- severe phenotype (premature death) • BMD - In-frame deletions: residual function – milder phenotype

10. Therapeutic strategies for DMD • Aim to restore reading frame: severe DMD → milder BMD * Using antisense oligo(ribo)nucleotides (AONs) - modulate pre-mRNA splicing - inducing skipping of specific exons (with fs mutations) - restore protein synthesis

11. Therapeutic strategies for DMD • AONs in DMD gene therapy (GT): • - dystrophin expression in 75% transfected myotubes • - specific and highly efficient (targeting ESEs/ERS) • - simultaneous correction of most affected isoforms • - applicable to a variety of DMD mutations: • 10 exons > 65% DMD deletions • relatively safe in comparison with GT based on viral vectors

12. Antisense-based manipulation of pre-mRNA splicing

13. Therapeutic strategies for DMD • Pharmacological strategies that enhance muscle regeneration myostatin (TGF-β family) - potent, negative regulator of muscle growth - limiting excessive growth of skeletal muscle - spontaneous mutations → absence of myostatin → hypermuscular phenotype * Blockade of myostatin – ↑ muscle growth and strength - beneficial effect on disease progression (mdx mice) - monoclonal antibodies - inactivation with propeptide - inhibition by transgenic expression of follistatin

14. Huntington disease: gene defects, altered functions and therapeutic strategies HD pathogenesis • AD dominant disorder • Caused by CAG expansion in HTT gene ( 36 repeats)  polyQ repeats huntingtin  toxic gain of function • HTT ubiquitous expression: Striatal and cortical neurons primarily affected Polyglutamine aggregates in brain • Transcriptional dysregulation - interaction with specific transcription factors (Sp1)  alter expression of genes (repression) - hypoacetylation of histones  condensed chromatin structure

15. Therapeutic strategies for HD • siRNAs  HTT expression • 2 types of artificial inhibitory RNAs: • - Synthesized ds siRNA - transient effect, repeated administration for long term target supression • - Promoter expressed shRNAs - more enduring effect • Preclinical studies: • - AAV1-shRNA mediated HD silencing improved motor and neuropathology in rodents • - Lipid encapsulated siRNAs  transient supression  long lasting behavioural improvements • - Chemically modified and cholesterol conjugated siRNAs - reduced aggregation

16. Therapeutic strategies for HD • siRNAs  HTT expression • siRNAs power relies on its sequence specificity • Non-specific off-target effects, sources: - Immunoestimulation - Unintended sequence-based silencing of non-targets - Inhibitory RNA over-expression that disrupts natural miRNA biogenesis • Is long-term expression required? - Viral vector delivered shRNA or miRNAs - Major drawback is control: cannot be turned off. Long term effects unknown. - Conditional shRNA expression using doxycycline-inducible vectors - Non-viral systems that allow dosage control- require repeated administration • Decades long mutant HTT accumulation required to produce HD symptoms - Transient silencing  cellular protein turnover mechanisms to normalize toxic HTT levels  delay manifestation of symptoms • Possible to preferentially suppress mutant HTT allele by targeting disease linked SNPs

17. Therapeutic strategies for HD • Histone deacetylase (HDAC) inhibitors • Previous - SAHA, phenylbutyrate - limited by toxicity • Benzamide-type HDAC inhibitors (HDAci 4b) - low toxicity - prevent motor deficits and neurodegeneration (mouse models) - ameliorate gene expression abnormalities (dopamine D2 receptor and enkephalin) detected by microarrays

18. Therapeutic strategies for HD • Long-polyQ tracts prone to aggregate - affect vital cellular functions  cell death • Tricyclic pyrone (TP) compounds efficiently inhibited formation of -amyloid aggregates in cell and mouse models of Alzheimer’s disease - Small molecules with anti-aggregate properties - Could ameliorate cellular defects that preclude aggregate formation • TP4- ability to penetrate blood-brain barrier

19. Marfan syndrome: gene defects, altered functions and therapeutic strategies Marfan syndrome (MFS) • Common AD disorder of connective tissue • Caused by mutations in fibrillin 1 gene (FBN1): many dominant-negative effect • Fibrillin 1- 347KDa glycoprotein - elastin-associated microfibrils component of ECM • Additional role as regulator of cytokine TGF • Reducing amount of mutant FBN1 might be therapeutic for MFS

20. Therapeutic approaches to Marfan syndrome Ribozymes Catalytic RNA molecules that can be designed to cleave a specific mutant mRNA target High specificity: discriminate single bp Delivery of FBN1-Rz into dermal fibroblasts:  FBN1- mRNA and deposition of fibrillin in ECM (in vitro) Kilpatrick et al. 1996; Hum Mol Genet 5:1939-44.

21. Therapeutic approaches to Marfan syndrome • Manifestations of MFS - aortic aneurysm- - associated with increased TGF signalling (Figure?) - Other aneurysm syndromes - Loeys-Dietz syndrome -caused by mutations in TGFBR1 or TGFBR2 • Can be prevented with TGF antagonists: - TGF- neutralising antibody - Angiotensin II type 1 receptor (AT1) blocker: losartan - clinical use for hypertension - AT1 blockade with losartan appeared to achieve full correction of phenotypic abnormalities in the aortic wall of Fbn1C1039G/+ mice - exceptional tolerance in adults and children  clinical trials

22. Chronic myeloid leukemia: genetic defects, altered functions and therapeutic strategies CML - due to reciprocal translocation t(9;22)(q34;q11) - fusion gene BCR-ABL:  tyrosine kinase activity ability to transform cells  cell proliferation or  apoptosis: immature myeloid cells in blood genetic instability resulting  disease progression via activation of RAS

23. CML: Therapeutic strategies Specific kinase inhibitors: Imatinib blocks binding of ATP to BCR-ABL - Overall survival with imatinib at 5 years is 89% - 93% imatinib-treated patients remain free from disease progression - Side effects mild-moderate. Well tolerated, oral therapy - Achieved complete cytogenetic response but majority have levels of BCR-ABL detectable by RT-PCR  CML as long term controllable illness Imatinib also inhibits PDGFR and KIT tyrosine kinases (GIST) can be used in BCR-ABL positive ALL and in other translocations Mechanisms of relapse: Mutations in ABL kinase domain prevent blocking by imatinib Second generation of BCR-ABL inhibitors

24. Melanoma: gene defects, altered functions and therapeutic strategies B-RAF is most frequently mutated protein kinase in human cancers BRAF oncogenic mutations common in melanoma PLX4032 (RG7204) potent inhibitor of oncogenic BRAF activity - Selective block RAF/MEK/ERK pathway (inhibition of P-ERK) in BRAF mutant cells + inhibition of tumour growth - Modest preference V660E , V600K best responders - Effect dose-dependent - Well tolerated, no adverse effects - Durability of response under evaluation - Median progression-free survival in Phase I estimated 7 months

25. BRAF inhibitors can induce tumour growth A percentage of patients treated with RAF inhibitors developed skin lesions: SCC, keratoacanthoma type - Lesions appear within months of treatment in sun-exposed skin - Suggest pre-existing oncogenic mutations that may potentiate the RAF inhibitor effects - Paradoxical activation of RAF/MEK/ERK pathway by RAF kinase inhibitors Heidorn et al. Cell 2010; 209-21

26. References • Ratjen. Cystic Fibrosis: Pathogenesis and Future Treatment Strategies. Respiratory Care 2009; 54: 595-605 • Dietz. New therapeutic approaches to Mendelian disorders. New Eng J Medicine 2010; 363:852-63. • Wilschanski et al. Gentamicin-induced correction of CFTR function in patients with CF and CFTR stop codon mutations. New Eng J Medicine 2003; 349:1433. • Van Deutekom et al. Antisense-induced exon-skipping restores dystrophin expression in DMD patient derived muscle cells. Hum Mol Genetics 2001; 10:1547-54. • Mozzetta et al. Regenerative pharmacology in the treatment of genetic diseases: the paradigm of muscular dystrophy. Int J Biochem Cell Biol 2009; 41:701-10. • Wood et al. Modulating the expression of disease genes with RNA-based therapy. PLoS Genetics 2007; 3. • Harper et al. Progress and Challenges in RNA interference for HD. Arch Neurol 66:933. • Thomas et al. The HDAC inhibitor 4b ameliorates the disease phenotype and transcriptional abnormalities in HD disease mice. PNAS 2008; 105. • Habashi et al. Losartan, an AT1 antagonist, prevents aortic aneurism in a mouse model of Marfan syndrome. Science 2006; 7:117-21. • Druker et al. Translation of Philadelphia chromosome into therapy for CML. Blood 2008; 112:480815. • Bollag et al. Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma. Nature 2010; 596-599.