US 20160244766A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0244766A1 Bettencourt et al. (43) Pub. Date: Aug. 25, 2016 (54) COMPOSITIONS AND METHODS FOR Related U.S. Application Data INHIBITING EXPRESSION OF THE ALAS1 (60) Provisional application No. 61/983,720, filed on Apr. GENE 24, 2014, provisional application No. 61/887,288, (71) Applicants: ALNYLAM PHARMACEUTICALS, filed on Oct. 4, 2013. INC., Cambridge, MA (US); ICAHN SCHOOL OF MEDCINEAT MOUNTSINAI, New York, NY (US) Publication Classification (72) Inventors: Brian Bettencourt, Groton, MA (US); (51) Int. Cl. Kevin Fitzgerald, Brookline, MA (US); CI2N IS/II3 (2006.01) William Querbes, Cambridge, MA CI2O I/68 (2006.01) (US); Robert J. Desnick, New York, NY A619/00 (2006.01) (US); Makiko Yasuda, New York, NY (52) U.S. Cl. (US) CPC .......... CI2N 15/I 137 (2013.01); A61 K9/0019 (2013.01); C12O 1/6876 (2013.01); C12N (73) Assignees: ALNYLAM PHARMACEUTICALS, 23 10/14 (2013.01); C12N 23.10/321 (2013.01); INC., Cambridge, MA (US); ICAHN CI2N 23.10/315 (2013.01); C12N 23.10/351 SCHOOL OF MEDCINEAT (2013.01) MOUNTSINAI, New York, NY (US) (21) Appl. No.: 15/027,176 (57) ABSTRACT (22) PCT Filed: Oct. 3, 2014 The invention relates to double-stranded ribonucleic acid (86). PCT No.: PCT/US2O14/05916O (dsRNA) compositions targeting the ALAS1 gene, and meth S371 (c)(1), ods of using Such dsRNA compositions to alter (e.g., inhibit) (2) Date: Apr. 4, 2016 expression of ALAS1. Patent Application Publication Aug. 25, 2016 Sheet 1 of 61 US 2016/024476.6 A1 Mitochondria Cytoplasm GOO CH goo SUCCINYLCOA C(H2 COO ALA COO CH COAS YS O ALA-SYNTHASE CH DEHYDRATASE CH S.CH 2 H B CoASH do, H2 HOy NH-CH-NH H-C-NH C=O H COO - H-(-NH PORPHOBILINOGEN GLYCINE SYNTHASEHMB p-4 NH Wi (PBG Deaminase) i CH AMINOLEVULINIC Pr CH Vi ACID AC-( N N-4 Pr FEEDBACK REPRESSION HO CH CH Pr Pr Pr HEME HYDROXYMETHYBILANE 2H URO- HO Fe FERROCHELATASE | SYNTHASE 2 Vi CH Pr AC CH Vi AC Pr CH CH AC AC Pr Pr Pr Pr PROTOPORPHYRINIX UROPORPHYRINOGEN III URO 4 H 6H - PROTO-OXIDASE DECARBOXYLASE 4CO, Vi CH Pr (H, CH Vi CH Pr COPRO-OXIDASE -e y y CH CH 2CO, 2H CH CH Pr Pr Pr Pr PROTOPORPHYRINOGENIX COPROPORPHYRINOGEN III Fig. 1 Patent Application Publication Aug. 25, 2016 Sheet 2 of 61 US 2016/024476.6 A1 FIG. 2A Enzyme, Reaction Catalyzed Associated Type of Typical Typical Chromosomal Porphyria Porphyria Inheritanc Symptoms location e Pattern 6- Glycine + SuccinylCoA aminolevulinate (ALA) synthase 1 s 6-aminolevulinic acid 3p21 (ALA) 6- Glycine + SuccinylCoA X-linked Erythropoietic X-linked aminolevulinate sideroblastic (ALA) synthase 2 x- anemia (XLSA), (ALAS2) 6-aminolevulinic acid X-linked (ALA) protoporphyria (erythroid (XLP) specific) Xp11.21 6- 6-aminolevulinic acid ALA dehydratase Hepatic Autosomal Abdominal aminolevulinate (ALA) deficiency recessive pain, dehydratase porphyria (ADP or neuropathy (ALAD) x- DOSS porphyria) Porphobilinogen (PBG) 9q34 PBG deaminase Porphobilinogen (PBG) || Acute intermittent Hepatic Autosomal Periodic (PBGD) porphyria (AIP) dominant abdominal O x- pain, Hydroxymethylbi Hydroxymethylbilane peripheral lane Synthase (HMB) neuropathy, (HMBS) psychiatric disorders, tachycardia 11d23 Patent Application Publication Aug. 25, 2016 Sheet 3 of 61 US 2016/024476.6 A1 FIG.2B Uroporphyrino | Hydroxymethylbilane Congenital Erythropoietic AutoSomal Severe gen III Synthase erythropoietic recessive photosensit ( ) N- porphyria (CEP) ivity with UROS Uroporphyrinogen III erythema, Swelling 10q26 (URO) and blistering. Hemolytic anemia, Splenomega ly Uroporphyrino Uroporphyrinogen III Porphyria cutanea Hepatic Autosomal Photosensit gen (URO) tarda (PCT) dominant ivity with decarboxylase or sporadic vesicles and (UROD) N- bullae Coprophyrinogen III 1q34 Coproporphyrin Coprophyrinogen II Hereditary Hepatic Autosomal Photosensit ogen III oxidase (COPRO) Coproporphyria dominant ivity, (CPOX)3q12 (HCP) neurologic N- Symptoms, Protoporphyrinogen IX Colic Protoporphyrin Protoporphyrinogen IX Variegate Mixed Autosomal Photosensit ogen oxidase (PROTO) prophyria (VP) dominant ivity, (PPOX) neurologic N- Symptoms, 1d.14 Protoporphyrin IX developme ntal delay Ferrochelatase Protoporphyrin IX Erythropoietic Erythropoietic Autosomal Photosensit protoporphyria recessive ivity with 18q21.3 N- (EPP) skin lesions. Henne Gallstones, mild liver dysfunction Patent Application Publication Aug. 25, 2016 Sheet 5 of 61 US 2016/024476.6 A1 FIG. 3B 2281 Ctta acccca ggcc attatc at atccagat ggit Cttcaga gttgtctitta tatgttgaatt 2341 aagttatatt aaattittaat Citat agtaala aa Catagt CC toggaaataaa tt CttgCtta 24 Ol 3a tCC, tC (S EO ID NO : 1) Patent Application Publication Aug. 25, 2016 Sheet 7 of 61 US 2016/024476.6 A1 FIG. 4B 2281 Caagttggta t. Ctgct Cagg CCtgag catg acct Caatta titt Cacttaa CCCC aggc.ca 2341 titat catatc. Cagatggtct tcagagttgt Ctttatatgt gaattaagtt at attaaatt 2401 ttaatctata gtaaaaacat agt cct ggaa at aaattctt gcttaaatgg to aaaaaa (SEO ID NO : 382) Patent Application Publication Aug. 25, 2016 Sheet 8 of 61 US 2016/024476.6 A1 FIG. 5 Patent Application Publication Aug. 25, 2016 Sheet 9 of 61 US 2016/024476.6 A1 *; *--*-- *-- *-- *-- *-- *-- ||2 Patent Application Publication Aug. 25, 2016 Sheet 10 of 61 US 2016/024476.6 A1 FIG 7 a PBS R w:on ALASl CA Y St. e se ^emow e s E Patent Application Publication Aug. 25, 2016 Sheet 11 of 61 US 2016/024476.6 A1 O i- - - - - - - - - - - - - - -N.N. Y c : is Seine At ASE siRNA FCS RNA P&n of 3:3 treat &d Patent Application Publication Aug. 25, 2016 Sheet 12 of 61 US 2016/024476.6 A1 FIG. 9 & 2 E - 8. 6 4. O ||;;}£ z-?zºwi?? ?%8-9 -|-+-- ~~~~?;;--~~~~~~~~~~~~~ -??+-- ?!!!-08-?zºw??? ************************************************************************************************************************************ ?OTÁG-G?-I?-º?º?-£ 4GT-gº-?zº??? Patent Application Publication Aug. 25, 2016 Sheet 13 of 61 US 2016/024476.6 A1 ****************************************************************************************************************** FIG. 10 Patent Application Publication Aug. 25, 2016 Sheet 14 of 61 US 2016/024476.6 A1 FIG. 11 ||;··································---- Phenobati) retail Patent Application Publication Aug. 25, 2016 Sheet 15 of 61 US 2016/024476.6 A1 e e S r s: 3s e s o .N.N. ...N.N. Baseline Baseline PBS ALAS1 siRNA ALAS1 siRNA Patent Application Publication US 2016/024476.6 A1 |—^)#|----- ·|-?=??;-š;{|—————- ?|----- « ||—~~~~~|| Patent Application Publication Aug. 25, 2016 Sheet 17 of 61 US 2016/024476.6 A1 s & e - E. & : * . J. &xw wŠw AAS l 4 ... S. xSas R & At AS - - - - - - - - - - - - - - - - - - - - - - - - - - - -RSSS i- Sax Patent Application Publication Aug. 25, 2016 Sheet 18 of 61 US 2016/024476.6 A1 FG, 15 AA 28 BG Baseline Saline ALAS-GaNac : 20mpk w/ Phenobarbitol Patent Application Publication Aug. 25, 2016 Sheet 19 of 61 US 2016/024476.6 A1 FG 16 ... 3 s E. S. R s 6 s ba . See Patent Application Publication Aug. 25, 2016 Sheet 20 of 61 US 2016/024476.6 A1 FIG. 17 ALAS siRNA PB Plasa-ALAPEG PE Plasma-ALAPBG PE Plasma-ALAPEG " " | " "I I I I I I I I I I I I I 1 - 3 4 5 1516 17 18 9 3CD31 D32 WWeek Week 2 Week 4 2 e s ... O a. k s S. 0 Easeline "c Wee Week 2 Patent Application Publication Aug. 25, 2016 Sheet 21 of 61 US 2016/024476.6 A1 FIG. 18 PB+ DDC P x 3 Heme or siRNA D1 D2 D3 t t Fiasrna-AA/FB3, D4 C-SS: was wlan ALA Lic & marl ALA S1 , siRNA or hemin &mdan ALA ham 50.00 X a. O - C s 30.00 R x8. t A 20.00 1000 Š O.C. --~~~~~~~~ Gr 1r 13 ar Hrs after last PB/DDC dose O. - ww.capiti I.I. siRNA or hemin s\Sa mgar pig alas1 S *x innear pbg home 50.00 Š 540.00 s ? s s & &^ 30.00 s^ys. & ^\S sa yŠas \a *.--- ---,T s & YS ^ &* .x* 20.00 - N ' s N N Ša 10.00 Ya,YS 0.00 i-s-s-s- r 1 hr 13 24r Hrs after last PB/DDC dose Patent Application Publication Aug. 25, 2016 Sheet 22 of 61 US 2016/024476.6 A1 FIG. 19 30 10 AD-53632 Patent Application Publication Aug. 25, 2016 Sheet 23 of 61 US 2016/024476.6 A1 FIG. 20 ALAS1-GalNAc sERPI.V s 4 Daily IP PB s injections X->Measure. SŠ 1. Urinary ALA/PBG 2. ALAS-1 and PBGD - H mRNA D1 D4 D5 D6 D7 D8 24h Urine Patent Application Publication Aug. 25, 2016 Sheet 24 of 61 US 2016/024476.6 A1 FG. 21 1.2 1.0 O.8 O.6 0.4 0.2 O.O PBGD SiRNA - -- -- + ALAS-1-GalNAC - 30mg/kg 10mg/kg PhenObarb + -- -- + : O.O Š PBGD SiRNA - -- -- -- ALAS-1-GalNAC 30mg/kg 10mg/kg PhenObarb -- -- -- -- Patent Application Publication Aug. 25, 2016 Sheet 25 of 61 US 2016/024476.6 A1 FG. 22 300 15 . CDS an as ?al SD 200 DOg S ES S 9R S 5. 1 OO O PBGD SiRNA - -- -- -- ALAS1-GalNAc - 30mg/kg 10mg/kg Phenobarb + -- -- -- i PBGD SiRNA - -- -- -- 30mg/kg 10mg/kg PhenObarb + -- -- -- Patent Application Publication Aug. 25, 2016 Sheet 26 of 61 US 2016/024476.6 A1 FIG. 23 |—) |--){|—~~'; |—~~'; Patent Application Publication Aug. 25, 2016 Sheet 29 of 61 US 2016/024476.6 A1 FIG. 26 1. ae1. } A.11s AO-53) D-63091 AC-802 A-512S E-63(893 i).309 $895 19/21 19/21 N. f 31 23 NE | 19.19 1 2 3 in?t hf.cymir. Orhang. hydrid, Sye haig hymir. heydmit ve rhing y Patent Application Publication Aug. 25, 2016 Sheet 30 of 61 US 2016/024476.6 A1 WNUU LSW eNhee Patent Application Publication Aug. 25, 2016 Sheet 32 of 61 US 2016/024476.6 A1 FIG. 29 Study Day: 1 2 3 4 5 7 9 11 13 15, 17 19 21 23 25 27 29 S.