Protusmisleni oligonukleotidi u lijeenju spinalne miine atrofije Prof

Protusmisleni oligonukleotidi u liječenju spinalne mišićne atrofije Prof. dr. sci. Goran Šimić, dr. med. Laboratorij za razvojnu neuropatologiju Zavod za neuroznanost Hrvatski institut za istraživanje mozga Medicinski fakultet Sveučilišta u Zagrebu Republika Hrvatska IP-2014 -09 -9730

Spinalna mišićna atrofija (SMA) • Vodeći genetski uzrok smrti djece karakteriziran progresivnim propadanjem -motoneurona leđne moždine • Druga najčešća AR bolest • Druga najčešća NM bolest • Jedna od češćih rijetkih bolesti (rare diseases), ali nije „orphan disease” • >95% svih SMA bolest nastaje zbog homozigotne mutacije ili delecije eksona 7 SMN 1 gena na 5. kromosomu (Lefebvre et al. , Cell, 1995) Lefebvre S et al. (1995) Cell 80: 155 -165. (Judith Melki, Institut Necker, Paris)

Klinička slika SMA neonatalna hipotonija („floppy baby”) noge u abdukciji i vanjskoj rotaciji (frog-leg posture), a ruke u fleksiji, smanjeni spontani pokreti 4 y hipotonija u suspenziji („ragdoll sign”) 6 y značajno zaostajanje glave pri trakciji progresivni gubitak mišićne mase SMA simetrično zahvaća proksimalne skupine mišića ruku i nogu, interkostalni mišići su uvijek zahvaćeni, a dijafragma obično nije, što omogućuje dovoljnu spontanu respiraciju sve dok ne nastupi respiratorna infekcija ili aspiracija Haliloglu G (2002) Neuropediatrics 33: 314 -9. Iannaconne ST et al. (2004) Spinal muscular atrophy. Curr Neurol Neurosci Rep 4: 74 -80.

Neuropatološka slika SMA: dominira gubitak -motoneurona normalna kontrola SMA-1 Nissl bojanje Šimić G, Šešo-Šimić Đ, et al. (2000) Ultrastructural analysis and TUNEL demonstrate motor neuron apoptosis in Werdnig–Hoffmann disease. J Neuropathol Exp Neurol 59: 398– 407.

Neuropatološka slika SMA: neurogena atrofija mišićnih vlakana Neurogena atrofija: - skupine atrofiranih mišićnih vlakana - mala angulirana vlakna - oba tipa vlakana su zahvaćena (tip 1 i tip 2) - reinervirana mišićna vlakna SMA-1 punch biopsija m. biceps brachii (H-E bojanje)

Klinička dijagnostika SMA Elektromiografska (EMG) analiza akcijskih potencijala motoričkih jedinica pomoću mišićne elektrode: motoričke jedinice normalan nalaz SMA SMN 1 SMN 2 SMA miopatija (DMD, BMD) Ilustracija preuzeta s http: //emedicine. medscape. com/ con Analiza SMN 1 gena pomoću RLFP metode - znakovi neurogenog oštećenja: fibrilacija i fascikulacije mišića brzina provođenja normalna, u teškim oblicima može biti usporena (zbog retrogradne Waalerove degeneracije uslijed propadanja DMN i sekundarne demijelinizacije) normalan nalaz ne isključuje mogućnost SMA (treba ponoviti pretragu) potencijali imaju velike amplitude, ali nisku frekvenciju Markowitz JA, Tinkle MB, Fischbeck KH (2004) Spinal muscular atrophy in the neonate. JOGNN 33: 12 -20 Sertić J, Barišić N, Šoštarko M, Bošnjak N, Ćulić V, Cvitanović Lj, Ferenčak G, Brzović Z, Stavljenić-Rukavina A (1997) Deletions in the SMN and NAIP genes in patients with spinal muscular atrophy in Croatia. Coll Antropol 21: 487 -492

Tipovi SMA modificirano prema Monani UR (2005) Spinal muscular atrophy. A deficiency in a ubiqutous protein: a motor neuron specific disease. Neuron 48: 885– 896 0 – kongenitalni oblik Werdnig-Hoffmann Intermedijarni (kronični infantilni) oblik Wohlfart-Kugelberg-Welander 4 – adultni oblik Wohlfart Gunnar (1942) Zwei Fälle von Dystrophia musculorum progressiva mit fibrillären Zuckungen und atypischem Muskelbefund. Deutsche Z Nervenheilk 153: 189 -204 Kugelberg Erik, Welander Lisa (1956) Heredofamilial juvenile muscular atrophy simulating muscular dystrophy. Arch Neurol 75: 500. Dubowitz V (1999) Very severe spinal muscular atrophy (SMA type 0): an expanding clinical phenotype. Eur J Paediatr Neurol 3: 65 -72.

SMN 2 se razlikuje od SMN 1 gena u samo jednoj, translacijski tihoj mutaciji nukleotida eksona 7: umjesto citozina u kodonu 280 je timin, koji „smeta“ normalnom izrezivanju i uključivanju eksona 7 u SMN 2 m. RNA. Stoga u 80 -90% SMN 2 glasničke RNA nedostaje ekson 7 zbog čega je ona nestabilna, a samo 1020% njezinog proteina funkcionalno (full length, FL) 100% FL SMN 10 -20% FL SMN Burghes AHM, Beattie CE (2009) Nat Rev Neurosci 10: 597 -609 Monani UR et al. (1999) Hum Mol Genet 8: 1177 -1183 Lorson CL, Androphy EJ (2000) Hum Mol Genet 9: 259 -265

Težina bolesti ovisi o broju kopija SMN 2 gena odnosno količini SMN proteina % of the fl-SMN protein razina SMN proteina potrebna za normalnu f-ju -motoneurona (oko 23%) Schmalbruch H, Haase G. Brain Pathol 2001 10 -20% SMN + 100% SMN 10 -20% SMN + 0% SMN 30% SMN 40% SMN Incidencija na 100. 000 živorođene djece Učestalost nositelja 4 1/90 Italija (svi tipovi) 7, 8 1/57 Italija (SMA-1) 4, 1 Njemačka 10 1/50 S. A. D. 8. 3 1/54 Zemlja Engleska SMN 1+/+ SMN 1+/- SMN 1 -/4 -8 SMN 2 SMN 1 -/3 -4 SMN 2 SMN 1 -/2 -3 SMN 2 Misli se kako različit broj kopija SMN 2 gena (1 -4) nastaje slučajnom duplikacijom, a u manjeg broja SMA bolesnika SMN 1 gen se zamjenom eksona 7 i 8 konvertira u SMN 2. U SMA-2 i SMA-3 su točkaste mutacije s pogrešnim značenjem puno češće nego u SMA tipu 1. Feldkötter M, Schwarzer V, Wirth R, Wienker TF, Wirth B (2002) Quantitative analyses of SMN 1 and SMN 2 based on realtime lightcycler PCR: fast and highly reliable carrier testing and prediction of severity of spinal muscular atrophy. Am J Hum Genet 70: 358– 368.

Funkcija SMN proteina (1 -11)

(1) SMN je ubikvitaran, visoko-konzerviran i multifunkcionalan protein Nucleic acid binding Self-oligomerization SMN protein nalazi se i u jezgri i u citoplazmi, a najveći mu je izražaj u -motoneuronima. Selenko P, Sprangers R, Stier G, Bühler D, Fischer U, Sattler M (2001) SMN Tudor domain structure and its interaction with Sm proteins. Nature Struct Biol 8: 27– 31 Wan L, Ottinger E, Sungchan S, Dreyfuss G (2008) Inactivation of the SMN complex by oxidative stress. Mol Cell 31: 244 -254

SMN protein functions (2) • Within the nucleus, the SMN protein forms heteromeric complexes For simplicity the SMN complex is illustrated in dimeric form although, from its size (3060 S), it most likely is larger oligomeric structure. Fischer U, Liu Q, Dreyfuss G (1997) The SMN-SIP 1 complex has an essential role in spliceosome biogenesis. Cell 90: 1023– 1029

SMN protein functions (3) • These SMN complexes play an important role in biogenesis of small nuclear ribonucleoproteins (sn. RNPs), which are important for prem. RNA processing (splicing) (small nuclear Ribo. Nucleic Acids) spliceosome Kolb SJ, Battle DJ, Dreyfuss G (2007) Molecular functions of the SMN complex. J Child Neurol 22: 990 -994. Zhang Z, Lotti F, Dittmar K, Younis I, Wan L, Kasim M, Dreyfuss G (2008) SMN deficiency causes tissue-specific perturbations in the repertoire of sn. RNAs and widespread deficits in splicing. Cell 133: 585– 600

SMN protein functions (4) • • SMN protein is also a component of the nuclear domains called Cajal bodies (CBs) that contain coiled threads of the marker protein (coilin) SMN protein seems to be essential for both the assembly and delivery of sn. RNPs to the CBs are involved in the biogenesis and recycling of splicing sn. RNPs, sno. RNPs (nucleolar) and 3 eukaryotic. RNA polymerases However, since CBs are deficient in DNA, nascent prem. RNA, and non-sn. RNP essential splicing factors, they are probably not active sites for transcription or splicing 1 2 CBs 3 4 U sn. RNP receptor ICG – interchromatine granule clusters (“speckles”) Eggert C, Chari A, Laggerbauer B, Fischer U (2006) Spinal muscular atrophy: the RNP connection. Trends Mol Med 12: 113– 121 Morris GE (2008) The Cajal body. Biochim Biophys Acta (in press)

SMN protein functions (5) • • In SMA, low SMN protein levels result in altered CBs composition and a notable separation of SMN protein into distinct nuclear bodies called Gems (“gemini of Cajal bodies”); in severe cases of SMA, a dramatic reduction in nuclear gems can be observed Otherwise, Gems and CBs mostly colocalize in some cell lines and adult tissues but are separated in fetal tissues - this indicates that Gems and Cajal bodies (as well as ICGs) are distinct nuclear structures that have a dynamic functional relationship CBs Dundr M, Hebert MD, Karpova TS, Stanek D, Xu H, Shpargel KB, Meier UT, Neugebauer KM, Matera AG, Misteli T (2004) In vivo kinetics of Cajal body components. J Cell Biol 164: 831– 42 Gubitz AK, Feng W, Dreyfuss G (2004) The SMN complex. Exp Cell Res 296: 51 -56 Morris GE (2008) The Cajal body. Biochim Biophys Acta

Widespread splicing deficits can not explain the selectivity of motoneuron death in SMA (6) • With a false discovery rate (FDR) set at less than 0. 1, 259 genes from spinal cord, 73 from brain, and 633 from kidney were identified as having splicing pattern changes • Since only a large degree of SMN decrease (>80%) is required to cause a significant change in the levels of sn. RNAs or cause cell death in cultured cells, this suggests that cells normally contain a large excess capacity of SMN complex to maintain their normal inventory of sn. RNAs • Therefore, most experts in the field think that splicing deficit hypothesis can not adequately answer the question of selective motoneuron cell death in SMA Zhang Z, Lotti F, Dittmar K, Younis I, Wan L, Kasim M, Dreyfuss G (2008) SMN Deficiency Causes Tissue-Specific Perturbations in the Repertoire of sn. RNAs and Widespread Defects in Splicing. Cell 133: 585 -600

SMN protein interactions (7) SMN protein has an enormous number of interactions with other proteins, but in the last several years the role of SMN protein in the control of actin dynamics has been documented as probably the most important for motoneuron survival Wirth B, Brichta L, Hahnen E (2006) Spinal muscular atrophy: from gene to therapy. Sem Pediat Neurol 13: 121– 131 Fuller H, Morris GE (2010) SMN complexes of nucleus and cytoplasm: a proteomic study for SMA therapy. Transl Neurosci 2010

SMN protein functions (8) The first suggestion that SMN protein might have other important functions than sn. RNP assembly came from EM analysis of mouse spinal cord that revealed SMN protein present in dendrites and axons (Pagliardini et al. 2000) Actually, further studies confirmed that SMN accumulates in growth cone and filopodia in both neuronal- and glial-like cells; since SMN was present at the leading edge of neurite outgrowths, this suggests its specific role in axono- and dendrogenesis (Fan and Simard, 2002) Moreover, the axonal-SMN isoform which is found to be selectively expressed in developing spinal cord motoneurons has recently been identified (Setola et al. , 2007) Pagliardini S, Giavazzi A, Setola V, Lizier C, Di Luca M, De. Biasi S, Battaglia G (2000) Subcellular localization and axonal transport of the survival motor neuron (SMN) in the developing rat spinal cord. Hum Mol Genet 9: 47– 56 Fan L, Simard LR (2002) Survival motor neuron (SMN) protein: role in neurite outgrowth and neuromuscular maturation during neuronal differentiation and development. Hum Mol Genet 11: 1605– 1614 Setola V, Terao M, Locatelli D, Bassanini S, Garattini E, Battaglia G (2007) Axonal-SMN (a-SMN), a protein isoform of the survival motor neuron gene, is specifically involved in axonogenesis. Proc Natl Acad Sci USA 104: 1959– 1964

SMN protein functions (9) • in-situ hybridization analysis using anti-sense probe against actin m. RNA in PC 12 cells and motoneurons confirmed that SMN protein (Smn in mouse) colocalizes with hn. RNP-R (protein that transports RNA along axons) in cell bodies and neurite-like proceses Rossoll W, Kroning AK, Ohndorf UM, Steegborn C, Jablonka S, Sendtner M (2002) Specific interaction of Smn, the spinal muscular atrophy determining gene product, with hn. RNP-R and gry-rbp/hn. RNPQ: a role for Smn in RNA processing in motor axons? Hum Mol Genet 11: 93– 105

SMN protein functions (10) wt morpholino-injected embryos 50 hours post-fertilization • Injecting synthetic anti-sense RNA oligonucleotydes (morpholinos) knock-outs the expression of zebrafish smn causing severe truncation of motoneurons’ axons Beattie CE, Carrel TE, Mc. Whorter ML (2007) Fishing for a mechanism: using zebrafish to understand spinal muscular atrophy. J Child Neurol 22: 995– 1003

SMN protein f-on: accumulated evidence (11) Rat spinal cord motoneurons cultured for 3 days (SMN – red, tau – green) EGFP-SMN in motoneuron processes, both dendrites and axons EM-immunogold detectioni of SMN in growth cones Chick motor neurons in culture (SMN – red) Rat motoneuron: colocalization of SMN (green) with ribosomal RNA (red: biotin-labeled oligonucleotide probe) Zhang HL, Pan F, Hong D, Shenoy SM, Singer RH, Bassell GJ (2003) Active Transport of the Survival Motor Neuron Protein and the Role of Exon-7 in Cytoplasmic Localization. J Neurosci 23: 6627 -6637

: Genotype-phenotype relationship in SMA PLASTIN 3 (PLS 3) CORO 1 C I jedan i drugi prenose -aktin do čunjića rasta umjesto SMN!? !? Oprea GE, Kröber S, Mc. Whorter ML, Rossoll W, Müller S, Krawczak M, Bassell GJ, Beattie CE, Wirth B (2008) Plastin 3 Is a Protective Modifier of Autosomal Recessive Spinal Muscular Atrophy. Science 320: 524 -527 Hosseinibarkooie S et al. The power of human protective modifiers: PLS 3 and CORO 1 C unravel impaired endocytosis in spinal muscular atrophy and rescue SMA phenotype. Am J Hum Gen 2016; 99: 647 -665.

PLS 3 as a SMA-modifing gene • • • In rare instances, some children are asymptomatic despite carrying the same SMN 1 mutations as their affected siblings! Earlier this year it has been discovered in 6 disconcordant SMA families that 8 unaffected SMN 1 -deleted children were exclusively females: GWAS search revealed that these girls are having a significantly higher expression of the PLS 3 gene (the nature of this gender-specific protective effect is not known) Overexpression of PLS 3 rescues axonogenesis through the increase of the F-actin levels in both mouse and zebrafish models: Oprea GE, Kröber S, Mc. Whorter ML, Rossoll W, Müller S, Krawczak M, Bassell GJ, Beattie CE, Wirth B (2008) Plastin 3 Is a Protective Modifier of Autosomal Recessive Spinal Muscular Atrophy. Science 320: 524 -527

Kratka „povijest” nusinersena u intronu 7 SMN 2 gena kombinatornom selekcijom in vivo 2004. godine otkriven inhibitorni slijed CCAGCAUUAUGAAAG (intronic splicing silencer N 1, ISS -N 1) koji sprječava uključivanje eksona 7 u SMN 2 m. RNA. Protusmisleni oligonukleotid koji se veže na navedeni slijed ispravlja deficit izrezivanja tj. uključivanja eksona 7 u SMN 2 m. RNA, što je višestruko potvrđeno te kulminiralo odobravanjem nusinersena (Spinraza TM, Ionis Pharmaceuticals / Biogen Inc. ) za liječenje svih tipova SMA (US FDA 23. 12. 2016). Lorson et al. , PNAS, 1999 u tijeku je klin. studija (NCT 02386553) u kojoj se oboljeloj djeci nusinersen ubrizgava lumbalnom punkcijom u likvor već u prodromalnom stadiju bolesti Ottesen EW. ISS-N 1 makes the first FDA-approved drug for spinal muscular atrophy. Transl Neurosci 2017; 8: 1 -6

Kratka „povijest” nusinersena Sličnosti s C 9 ORF 72 (G 4 C 2 hexanucleotide expansion) i protein loss of fon in FTLD/ALS Lorson et al. , PNAS, 1999 Ottesen EW. ISS-N 1 makes the first FDA-approved drug for spinal muscular atrophy. Transl Neurosci 2017; 8: 1 -6

Ograničenja th nusinersenom (Spinrazom) • Uzak terapijski prozor (th nakon nastanka izraženih simptoma nije učinkovita, a neonatalni genetički screening se obično provodi tek nakon uočavanja simptoma) • SMA je sistemska bolest, pa se pokušava razviti sistemska th s Adeno-Associated Viral vectors (AAV gene th) Druga najčešća NM bolest • Th samo smanjuje simptome, ali ne liječi bolest u potpunosti (mišićna aktivnost i tjelesna težina nikad ne dostižu normalne vrijednosti) • Skupoća lijeka!

Jutarnji list, 29. srpnja 2017.

Očekivanja (ciljevi) u bližoj budućnosti: • Razviti takve načine davanja nusinersena koji će djelovati ranije • Omogućiti svima neonatalni probir na SMA analizom SMN 1 gena • Shvatiti koji su glavni nizvodni signalni putovi u motoneuronima i drugim živčanim stanicama zahvaćeni nedostatkom SMN proteina (PTEN, RSK 2, Rho-kinazni put, ERK/AKT, mi. R-189/m. TOR) • Razumjeti zaštitno djelovanje modificirajućih gena PLS 3 i CORO 1 C u asimptomatske djece s homozigotnom delecijom eksona 7 SMN 1 gena

Suradnici Patrick R. Hof New York Božo Krušlin Zagreb Nina Barišić, Zagreb Paul J. Lucassen Amsterdam Jadranka Sertić Zagreb Đurđica ŠešoŠimić, Zagreb Glenn E. Morris and Carolyne Sewry, Oswestry Atiqul Islam, Stockholm

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Sažetak (Pediatria Croatica 2017; 61 (Suppl. 2): 78 -79. Spinalna mišićna atrofija (SMA) je vodeći genetski uzrok smrti djece karakteriziran progresivnom degeneracijom -motoneurona leđne moždine. Uzrokovana je niskom razinom SMN (survival motor neuron) proteina uslijed homozigotne delecije ili mutacije eksona 7 SMN 1 gena na kromosomu 5, što je otkriveno 1995. godine. Zbog obrnute duplikacije na kromosomu 5 u ljudi postoje još i 1 -4 kopije gotovo identičnog SMN 2 gena. SMN 2 se razlikuje od SMN 1 gena u samo jednoj, translacijski tihoj mutaciji nukleotida eksona 7: umjesto citozina u kodonu 280 je timin, koji „smeta“ normalnom izrezivanju i uključivanju eksona 7 u SMN 2 m. RNA. Stoga u 80 -90% SMN 2 glasničke RNA nedostaje ekson 7 zbog čega je ona nestabilna, a samo 10 -20% njezinog proteina funkcionalno. Ipak, zbog činjenice da djeca s homozigotnom mutacijom eksona 7 SMN 1 gena koja imaju veći broj kopija SMN 2 gena proizvode i sveukupno veću količinu SMN proteina pune dužine, njihova je klinička slika bolesti blaža, a izuzetno rijetko uopće nemaju simptoma ako razina proizvodnje putem SMN 2 gena dostiže 23% koncentracije normalne vrijednosti SMN proteina. Na tragu te činjenice je u intronu 7 SMN 2 gena kombinatornom selekcijom in vivo 2004. godine otkriven inhibitorni slijed CCAGCAUUAUGAAAG (intronic splicing silencer N 1, ISS-N 1) koji sprječava uključivanje eksona 7 u SMN 2 m. RNA. Protusmisleni oligonukleotid koji se veže na navedeni slijed ispravlja deficit izrezivanja tj. uključivanja eksona 7 u SMN 2 m. RNA, što je dokazano u brojnim prekliničkim ispitivanjima od 2011. godine naovamo, te kulminiralo odobravanjem nusinersena (Spinraza. TM, Ionis Pharmaceuticals/Biogen Inc. ) za liječenje svih tipova SMA (US Food and Drug Administration, 23. prosinca 2016. godine). U tijeku je registracija toga lijeka u EU te kliničko ispitivanje (NCT 02386553) u kojem se oboljeloj djeci nusinersen ubrizgava lumbalnom punkcijom u cerebrospinalnu tekućinu već u prodromalnom stadiju bolesti. Zaključno, nusinersen je prvi protusmisleni oligonukleotid uopće kojim se uspješno liječi deficit izrezivanja, odnosno uključivanja eksona nekoga gena u njegovu m. RNA, a ujedno ukazuje na golemi potencijal intronskih dijelova gena kao terapijskih ciljeva za liječenje genetski uvjetovanih bolesti. Rad autora podupire Hrvatska zaklada za znanost (IP-2014 -09 -9730).