Dr Alessandro Fatica (on behalf of Prof. Irene Bozzoni)

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1 Dr Alessandro Fatica (on behalf of Prof. Irene Bozzoni) non coding RNAs in human diseases ROUND TABLE ITALY RUSSIA Dubna, December 2010

2 Where is the information that programs our complexity? The biggest surprise of the genome sequencing projects was the discovery that the number of protein-coding genes does not scale strongly or consistently with complexity. Humans (and other vertebrates) have approximately the same number of protein-coding genes (~20,000) as C. elegans, and less than those of plants (Arabidopsis ~28,000, rice ~40,000) and protozoa (~30,000). Most of the proteins are orthologous and have similar functions from nematodes to humans, and many are common with yeast.

3 The proportion of non-coding DNA broadly increases with developmental complexity >90% 40%-70% <1% Vertebrates Ciona (urochordate) Invertebrates Plants Complex fungi (Neurospora) Simple eukaryotes (yeasts, plasmodium, Dictyostelium) Prokaryotes Vertebrates Urochordate Invertebrates Plants Complex fungi Simple eukaryotes Prokaryotes Mattick et al. Trends Genetics (2010).

4 Non coding RNAs (ncrnas) in human Genome >90% RNA 2% 98% mrnas non coding RNAs 5000 nt length Proteins rrnas large long ncrnas with mostly unkonwn function ncrnas ~100 nt trnas snrnas snornas rrnas small nt micrornas piwirnas endo-sirna. tiny

5 Functions of regulatory ncrnas Chromatin modification and epigenetic memory Transcriptional regulation Splicing RNA modification and editing Control of mrna turnover Control of mrna translation Amaral et al., Science

6 Our interests 1. ncrnas as therapeutic tools 2. ncrnas in gene regulatory networks

7 1.ncRNAs as therapeutic tools ncrna can be engineered in order to produce molecules that can interfere with gene expression in a sequencespecific way Therapeutic ncrnas: Antisense Aptamers Ribozymes Modifying RNAs RNA interference micrornas

8 1.ncRNAs as therapeutic tools ncrna can be engineered in order to produce molecules that can interfere with gene expression in a sequencespecific way Therapeutic ncrnas: Antisense Aptamers Ribozymes Modifying RNAs RNA interference micrornas

9 Antisense RNAs as therapeutic molecules in Duchenne Muscular Dystrophy

10 Duchenne Muscular Dystrophy (DMD) is a severe disorder characterized by rapid progression of muscle degeneration, leading to loss of ambulation and death. X-linked recessive disorder that affects 1 in 3500 live males Histopathology of a Duchenne muscle

11 Dystrophin-The LONGEST GENE of our genome Patients with DMD are deficient in dystrophin, a protein that connects the cytoskeleton of a muscle fiber to the surrounding extraxellular matrix this deficiency causes sarcolemmal instability and muscle degeneration 2,5 Mb genomic locus 14 kb mature mrna (79 exons) 427 kd protein

12 Duchenne Muscular Dystrophy - the deletion - DMD is caused by mutations in the Dystrophin gene that alter the pre-mrna splicing and disrupt the open reading frame of the proteins, producing premature stop codons and mrna degradation CUA AG CUG AAU G CAA Pre-mRNA Splicing out of frame fusion STOP CUA AGCUGAAU G CAA mrna DMD Translation Protein

13 The Exon skypping By skypping the out of frame exon, we can produce a shorter dystrophin protein, converting the DMD phenotype to a Becker phenotype (BMD), a milder form of muscular dystrophy with near-normal life expectancy CUA AG CUG AAU G CAA STOP Splicing Pre-mRNA in frame fusion CUA AGG CAA mrna BMD Translation Protein

14 U1 snrna U2 snrna U4 snrna U5 snrna U6 snrna Pre-mRNA Splicing

15 RNA technology applied to the correction of DMD mutations Antisense RNA can be used to mask motifs involved in normal splicing to induce removal of specific exons snrnp antisense RNA U 1 Ex Exon 1 Exon 2 Exon 3 Pre-mRNA 5 3 Exon 1 Exon 3 mrna w/o exon 2 75% of all known dystrophin mutations can be cured by exon skipping

16 Chimeric U1-exon skippingconstructs STOP - Nuclear localization - Localizes at splice sites - Short expression cassette - Stable expression - No immunogen

17 Exon skyppping in mdx mouse U1#23 Exon 22 Exon 23 Exon 24 Exon 22 Exon 24

18 Dystrophin rescue upon AAV-U1#23 delivery AAV-U1#23 WT mdx Dystrophin Specific force of single muscle fibers Denti et al., Hum Gene Therapy; Denti et al., 2006 PNAS; Denti et al., Hum Gene Therapy

19 Towards clinical trilas on human U1-antisense patent licensed to AMT (Amsterdam Molecular Therapeutics) September 2009 Orphan drug designation by EMEA October Orphan drug designation by FDA

20 Work in progress Improving AAV serotypes and production (AMT) Toxicity and delivery studies in pigs (AMT) Testing new constructs for different human mutations (Sapienza Univ.) Testing exon skipping in human primary cells from DMD patients (Sapienza Univ.)

21 micrornas

22 Human micrornas A novel class of ~20-22 nt long ncrnas. Unknown before 2001 in human distinct micrornas have been identified in human since 2001 (data from MiRBase Release 16.0) Negatively regulate the translation/stability of mrnas By pairing with mrnas they can target almost 50% of all human coding genes mirnas participate in the regulation of almost every cellular process investigated so far Changes in their expression are associated with many human pathologies

23 mirna biogenesis mismatched interaction perfect complementarity

24 mirnas and their targets form complex regulatory networks One microrna can control hundreds different mrnas etc. A single mrna can be controlled by more than one microrna AAAAA

25 mirnas and their targets form complex regulatory networks Different combinations of micrornas can regulate different sets of mrnas etc. mrna-1 mrna-1 mrna-1 mrna-2 mrna-2 mrna-2 mrna-3 mrna-3 mrna-3 mrna-4 mrna-4 mrna-4 Major advantages of microrna regulation Networking and fine-tuning of gene expression Rapid repression

26 1. microrna expression profiling under physiological and pathological conditions: - microarray - qrt-pcr cards - deep sequencing 1. microrna expression regulation (e.g. promoter identification,trans acting factor involvement): - Chromatin immuprecipitation - Luc Reporters Lab interests 2. identification of microrna targets - Stable isotope labeling with amino acids in cell culture (SILAC) - Luc Reporters

27 Model systems for microrna function A. MicroRNA profiling 1.Muscle cells: Disease: normal vs pathological Duchenne muscular dystrophy 2. Hematopoietic cells : normal vs pathological Acute Myeloid Leukemia 3. Neuronal cells: normal vs pathological Neuroblastoma tumors Amyotrophic lateral sclerosis

28 micrornas as biomarkers E.g. profiling of muscle specific microrna in the serum of DMD patients as diagnostic and prognostic tools correlation with clinical assessments monitoring gene therapy fold with respect to WT

29 Model systems for microrna function B. Gene regulatory network controlled by micrornas 1.Myoblast differentiation: Myoblasts Myotubes 2. Myeloid differentiation: Leukemia cell lines Granulocytes or Moncytes 3. Neuronal differentiation: Neuroblastoma cell lines Neurons

30 Identifying networks 1) Forward Differentiation mirna profiling Relevant mirna(s) Computational prediction Gain of function Loss of funtion Target Gene(s) ID 2) Reverse Phenotype Gain of function Loss of funtion Relevant mirna(s) Computational prediction Gene function

31 Identification of mirna regulatory networks in muscle cell differentiation mir-206 mir- 31 Pax7 Dys mir-206: promote muscle stem cell differentiation mir-31: avoid the expression of late differentiation markers Cacchiarelli et al., Cell Metabolism.

32 Identification of mirna regulatory networks in myeloid differentiation AML CD34+ Granulocyte Monocyte 1. Transcription factors microrna gene Differentiation 1. C/EBPα PU.1 c-myc 2. micrornas 3. relevant target mrnas 3. NFIA 2. mir-223 mir-424 mir-342 mir-26 De Marchis et al., Leukemia; Rosa et al., PNAS; Fazi et al., Cell

33 Identification of mirna regulatory networks in neuronal differentiation neuroblastoma cells 1. Transcription factors microrna gene Differentiation neurons 1. REST CREB SP1 2. micrornas 3. relevant mrna targets 2. mir-9 mir-125a mir-125b mir-103 mir-100 mir-324 mir trkc ID2 Smo Laneve et al., NAR; Ferretti et al., Int J Cancer; Ferretti et al., EMBO J; Laneve et al J Biol Chem; Laneve et al., PNAS.

34 Prof. Irene Bozzoni group Duchenne Muscular Dystrophy Acute Myeloid Leukemia Amyotrophic lateral sclerosis Neuroblastoma Davide Cacchiarelli Julie Martone Marcella Cesana Valentina Cazzella Chiara Pinnarò Ivano legnini Olga Sthandier Beatrice Salvatori Arianna Mangiavacchi Marcella Marchioni Alessandro Fatica Stefano Dini Modigliani Giulia Torrelli Alessandro Rosa Mariangela Morlando Ubaldo Gioia Valerio Di Carlo Valeria Bevilacqua Antonella Cinquino Elisa Caffarelli