IL240982A

Cftr mrna compositions and related methods and uses

Abstract

This record has no abstract on file.

IL240982A, drawing sheet 1 — Sheet 1 of 26

Term

No projected expiry on record.

20 claims: 9 independent, 11 dependent

1
CLAIMS 1. An in vitro transcribed mRNA comprising a coding sequence at least 80% identical to SEQ ID NO:3 for use in treating cystic fibrosis in a mammal.
4
The mRNA for use according to any one of the preceding claims, wherein the mRNA comprises a 5’ untranslated region (UTR), a 3’ UTR, a signal peptide coding sequence, a cap structure and/or a tail structure.
7
The mRNA for use according to any one of claims 4-6, wherein the cap structure is a capl structure.
8
The mRNA for use according to any one of the preceding claims, wherein the mRNA comprises one or more nonstandard nucleotide(s), optionally wherein the one or more nonstandard nucleotide(s) is/are chosen from 5-methyl-cytidine, pseudouridine, and 2-thiouridine. 112
9
The mRNA for use according to any one of the preceding claims, wherein the composition is administered to the lung of the mammal by inhalation, intranasal administration, aerosolization, or nebulization.
10
The mRNA for use according to any one of the preceding claims, wherein the mRNA comprises a coding sequence which encodes a human cystic fibrosis transmembrane conductance regulator (CFTR) protein of SEQ ID NO:1, optionally wherein the human CFTR protein is expressed in the epithelial cells of the lung.
11
The mRNA for use according to any one of the preceding claims, wherein the mRNA is administered with a carrier.
15
The mRNA for use according to any one of claims 11-14, wherein the carrier is a liposome.
16
A composition comprising an mRNA and a carrier, wherein the mRNA is an in vitro transcribed mRNA and has a coding sequence at least 80% identical to SEQ ID NO:3.

Independent claims9

2,436 paragraphs in 219 sections, as filed

RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Application Serial No.

61/783,663, filed March 14, 2013, the disclosure of which is hereby incorporated by reference.

BACKGROUND [0002] The present invention relates to cystic fibrosis transmembrane regulator (CFTR) mRNA compositions, uses of same, and methods of making and using same.

[0003] Cystic fibrosis is an autosomal inherited disorder resulting from mutation of the

CFTR gene, which encodes a chloride ion channel believed to be involved in regulation of multiple other ion channels and transport systems in epithelial cells. Loss of function of CFTR results in chronic lung disease, aberrant mucus production, and dramatically reduced life expectancy. See generally Rowe et al., New Engl. J. Med. 352, 1992-2001 (2005).

[0004] Despite cloning of the CFTR gene in 1989, effective therapy for replacing CFTR for the treatment of cystic fibrosis has yet to be developed. The literature has documented numerous difficulties encountered in attempting to induce expression of CFTR in the lung. For example, viral vectors comprising CFTR DNA triggered immune responses and CF symptoms persisted after administration. Conese et al., J. Cyst. Fibros. 10 Suppl 2, SI 14-28 (2011); Rosenecker et al., Curr. Opin. Mol. Ther. 8,439-45 (2006). Non-viral delivery of DNA, including CFTR DNA, has also been reported to trigger immune responses. Alton et al., Lancet 353, 947-54 (1999); Rosenecker et al., J Gene Med. 5, 49-60 (2003). Furthermore, non-viral DNA vectors encounter the additional problem that the machinery of the nuclear pore complex does not ordinarily import DNA into the nucleus, where transcription would occur. Pearson, Nature 460,164-69 (2009).

[0005] Another source of difficulties in inducing CFTR expression in the lung is the lung environment itself. Pulmonary surfactant has been reported to reduce transfection efficiency for cationic lipid transfer vehicles such as Lipofectamine (DOSPA:DOPE).

Page 1 of 121

WO 2014/153052

PCT/US2014/028849 [0006] Ernst et al., J. Gene Med. 1, 331-40 (1999). Also, Rosenecker et al., 2003, supra, identified multiple inhibitory components present in the airway surface liquid which can interfere with either polymer-mediated or lipid-mediated transfection. Messenger RNA therapy has been proposed as a general approach for inducing expression of a therapeutic or replacement protein. The concept of introduction of messenger RNA (mRNA) as a means of protein production within a host has been reported previously (Yamamoto, A. et al. Eur. J. Pharm. 2009, 71, 484-489; Debus, H. et al. J. Control Rel. 2010, 148, 334-343). However, apparent lung-specific difficulties have been reported for mRNA delivery using certain lipoplexes formulations. For example, a comparison of in vitro and in vivo performance of lipoplexes carrying mRNA or DNA revealed that even though the mRNA composition gave higher expression in cultured cells, measureable expression was detected only with the DNA composition when administered intranasally to mouse lung. Andries et al., Mol. Pharmaceut. 9, 2136-45 (2012).

[0007] It should also be noted that CFTR is a relatively large gene relative to model or reporter genes such as firefly luciferase (FFL), Compare the lengths of the wild-type CFTR coding sequence (SEQ ID NO: 2) and the FFL coding sequence (SEQ ID NO: 7). The difference in length can impact stability under some circumstances, and therefore whether and how much protein expression any given dose of mRNA will produce. Furthermore, although in vitro synthesis of mRNA is generally preferable to synthesis by cells due to the absence of normal cellular mRNA and other cellular components which constitute undesirable contaminants, in vitro synthesis of mRNA with a long coding sequence, such as CFTR mRNA, is substantially more difficult to achieve than in vitro synthesis of mRNA with a relatively short coding sequence such as FFL.

[0008] PCT patent publication W02007/024708 and US patent publications

US2010/0203627 andUS2011/0035819 discuss the therapeutic administration of CFTR mRNA but provide neither a demonstrated reduction to practice of production of functional CFTR in the lung following administration of CFTR mRNA or sufficient guidance for overcoming the difficulties associated with inducing CFTR expression in the lung using in vz7ro~transcribed CFTR mRNA. These include difficulties with achieving in vitro synthesis of the mRNA and difficulties specific to the interactions of mRNA compositions with lung-specific substances that investigators such as Andries ct al., supra, have found to render mRNA compositions ineffective

Page 2 of 121 i

WO 2014/153052

PCT/US2014/028849 for induction, of expression even while corresponding DNA-based compositions did provide some level of expression.

[0009] Thus, there is a need for improved materials, formulations, production methods, and methods for delivery of CFTR mRNA for induction of CFTR expression, including in the mammalian lung, for the treatment of cystic fibrosis.

SUMMARY OF THE INVENTION [0010] The present invention is based, in part, on the development of formulations of

CFTR mRNA and non-naturally occurring CFTR mRNA and methods of administration thereof that can induce functional CFTR expression in vivo. The compositions, methods, and uses according to the invention can provide CFTR expression in the lung of a large mammal with a favorable safety profile suitable for effective treatment of cystic fibrosis.

[0011] Thus, in one aspect, the present invention provides a method of in vivo production of CFTR, in particular, in the lung of a subject (e.g,, a mammal) in need of delivery by delivering an mRNA encoding a CFTR protein. In some embodiments, the mRNA encoding a CFTR protein is delivered directly to the lung of the subject. As used herein, a “CFTR protein” encompasses any full length, fragment or portion of a CFTR protein which can be used to substitute for naturally-occurring CFTR protein activity and/or reduce the intensity, severity, and/or frequency of one or more symptoms associated with Cystic fibrosis. For example, a suitable CFTR protein according to the present invention may have an amino acid sequence identical to the wild-type human CFTR protein (SEQ ID NO: 1). In some embodiments, a suitable CFTR protein according to the present invention may have an amino acid sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the wild-type human CFTR protein (SEQ ID NO: 1).

[0012] In one embodiment, the invention provides a method of inducing CFTR expression in epithelial cells in a lung of a mammal, the method comprising contacting the epithelial cells in the lung of the mammal with a composition, wherein: the composition is a pharmaceutical composition comprising an in vitro transcribed mRNA;the in vitro transcribed mRNA comprises a coding sequence which encodes SEQ ID NO: 1. In another embodiment, the

Page 3 of 121

WO 2014/153052

PCT/US2014/028849 in vitro transcribed mRNA comprises a coding sequence which encodes an amino acid sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1.

[0013] In one embodiment, the invention provides a method of inducing CFTR expression in a mammalian target cell, the method comprising contacting the mammalian target cell with a composition, the composition comprising an in vitro transcribed mRNA encoding the amino acid sequence of SEQ ID NO: 1. In another embodiment, the in vitro transcribed mRNA comprises a coding sequence which encodes an amino acid sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1.

[0014] In another embodiment, the invention provides a non-naturally occurring mRNA molecule comprising a coding sequence, a 5’-UTR, and a 3’-UTR, wherein the coding sequence encodes the amino acid sequence of SEQ ID NO: 1 and the coding sequence is at least 80% identical to SEQ ID NO: 3. In another embodiment, the coding sequence encodes the amino acid sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 and/or the coding sequence is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3.

[0015] In another embodiment, the invention provides a non-naturally occurring mRNA molecule comprising a coding sequence, a 5’-UTR, and a 3’-UTR, wherein the coding sequence encodes the amino acid sequence of SEQ ID NO: 1 and the coding sequence comprises at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the non-wild-type bases listed in Table 1 at the positions of the coding sequence listed in Table 1 relative to the wild-type coding sequence of SEQ ID NO:

[0016] In another embodiment, the invention provides a non-naturally occurring mRNA molecule comprising a coding sequence, a 5!-UTR, and a 3’-UTR, wherein the coding sequence encodes the amino acid sequence of SEQ ID NO: 1 and the coding sequence comprises at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the non-wild-type bases listed in Table 2 at the corresponding positions of the coding sequence listed in Table 2 relative to the wild-type coding sequence of SEQ ID NO: 2.

Page 4 of 121

WO 2014/153052

PCT/US2014/028849 [0017] In some embodiments, the invention provides a non-naturally occurring mRNA molecule comprising a coding sequence for a signal peptide. In a particular embodiment, the invention provides a non-naturally occurring mRNA comprising a coding sequence for a growth hormone leader sequence. In certain embodiments, the invention provides a non-naturally occurring mRNA comprising a coding sequence of SEQ ID NO: 18 or SEQ ID NO:19. In some embodiments, the invention provides a non-naturally occurring mRNA comprising a coding sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% to SEQ ID NO: 18 or SEQ ID NO:19.

[0018] In some embodiments, the invention provides a non-naturally occurring mRNA molecule comprising a sequence of SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ UD NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17. In some embodiments, the invention provides a non-naturally occurring mRNA molecule comprising a sequence at least about 50%, 55%, 60%o, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% to any of SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO; 11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO:14, SEQ UD NO:15, SEQ ID NO:16, or SEQ ID NO:17.

[0019] In another embodiment, the invention provides a polynucleotide comprising a sequence complementary to the sequence of an mRNA according to the invention.

[0020] In another embodiment, the invention provides a composition comprising the polynucleotide according to the invention, an RNA polymerase, and nucleoside triphosphates.

[0021] In another embodiment, the invention provides a pharmaceutical composition comprising an mRNA according to the invention.

[0022] In another embodiment, the invention provides a nebulization or aerosolization apparatus loaded with a pharmaceutical composition according to the invention.

[0023] In another embodiment, the invention provides a cultured cell comprising an mRNA according to the invention and functional CFTR expressed from the mRNA.

[0024] In another embodiment, the invention provides a use of a pharmaceutical composition according to the invention for the induction of expression of functional CFTR.

[0025] In another embodiment, the invention provides a method of inducing CFTR expression in epithelial cells in a lung of a mammal, the method comprising contacting the

Page5 of 121

WO 2014/153052

PCT/US2014/028849 epithelial cells with a composition, wherein the composition is a pharmaceutical composition comprising an mRNA according to the invention.

[0026] In another embodiment, the invention provides a method of inducing CFTR expression in a mammalian target cell, the method comprising contacting the mammalian target cell with a composition, the composition comprising an mRNA according to the invention.

[0027] In another embodiment, the present invention provides a method of treating cystic fibrosis by administering to a subject in need of treatment an mRNA encoding a CFTR protein as described herein. In one embodiment, the mRNA is administered to the lung of the subject. In one embodiment, the mRNA is administered by inhalation, nebulization, intranasal administration or aerosolization. In various embodiments, administration of the mRNA results in expression of CFTR in the lung of the subject.

[0028] In a particular embodiment, the present invention provides a method of treating cystic fibrosis by administering to the lung of a subject in need of treatment an. mRNA comprising a coding sequence which encodes SEQ ID NO: 1. In some embodiments, the present invention provides a method of treating cystic fibrosis by administering to the lung of a subject in need of treatment an mRNA comprising a coding sequence which encodes an amino acid sequence at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the wild-type human CFTR protein (SEQ ID NO:1). In another particular embodiment, the present invention provides a method of treating cystic fibrosis by administering to the lung of a subject in need of treatment an mRNA comprising a coding sequence of SEQ ID NO:3. In some embodiments, the present invention provides a method of treating cystic fibrosis by administering to the lung of a subject in need of treatment an mRNA comprising a coding sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3.

[0029] In yet another aspect, the present invention provides methods for making an mRNA encoding a CFTR protein as described herein. In one embodiment, the invention provides a method of making CFTR mRNA in vitro, comprising contacting an isolated polynucleotide with an RNA polymerase in the presence of nucleoside triphosphates, wherein: the isolated polynucleotide and RNA polymerase are not contained within a cell; the isolated polynucleotide is a template for the RNA polymerase; the isolated polynucleotide comprises a Page 6 of 121

WO 2014/153052

CHO and COS-7 (A) and BHK and PKC (B) cells transfected with hCFTR coding constructs. Protein lysates were prepared 24 hrs post transfection and screened using MAI-935 as primary antibody. Arrow indicates putative CFTR. See the discussion of MAI-935 specificity in Example 6.

[0049] Figure 13. Cross reactivity of different anti-human CFTR antibodies. (A) Mouse anti-human CFTR MAI-935 (Chemicon): (B) - Mouse anti-human CFTR AB57O (Cystic Fibrosis Foundation): (C) - Mouse anti-human CFTR AB596 (Cystic Fibrosis Foundation): (D) Rabbit anti-human CFTR G449 (Rockefeller University). Arrow indicates CFTR.

[0050] Figure 14. Immunoprecipitation of human CFTR using three different antibodies (R29, R66/17 and R66/16) followed by immunodetection using AB596. Lane 1: T84 cells (positive control), Lane 2: untreated pig lung tissue (300 mg), Lane 3: treated pig lung tissue (697 mg), Lane 4: treated pig lung tissue (163 mg).

[0051] Figure 15. Immunoprecipitation and Western blotting of a mouse at 24 hrs post

IT spray application of 20 pg hCFTR SNIM RNA/10 pg FFL SNIM RNA each in the HGT5001 Formulation of Example 6. T84 cells served a positive control showing the mature glycosylated C-band and the mannosylated B -band of hCFTR. “supernatant” remaining cellular extract fraction without immunoprecipitated fraction. “IP” immunoprecipitated fraction.

[0052] Figure 16. Immunoprecipitation of hCFTR from T84 cells using MAB25031 followed by immunodetection using AB570 (A) and ΜΑΒΙ660 (B).

[0053] Figure 17. Immunoprecipitation of CFTR from NIH3T3 cells at 72 hrs post transfection with different constructs.

[0054] Figure 18. Immunoprecipitation of CFTR from NIH3T3 cells at 72 hrs post transfection with different constructs using 500 ug protein and ΜΑΒΙ 660 (left and center panels) and increased amount of total protein (8 mg) using MAB25031 (right panel).

[0055] Figure 19. Immunoprecipitation of hCFTR using MAB25031 and subsequent immunodetection using AB570 from pig lung samples post hCFTR SNIM RNA delivery in the

Page 10 of 121

WO 2014/153052

PCT/US2014/028849

PEI Formulation of Example 6. Lane 1: sample from luciferase-negative left caudal lobe of pig #2, Lane 2: sample from luciferase-positive lung regions of pig #1.

[0056] Figure 20. Nebulisation was performed on anesthetized and ventilated pigs (left).

The nebulizer was connected in-line to the ventilation system (right, see white arrow).

[0057] Figure 21. Luciferase expression measured in homogenates of pig tissue specimens from different lung regions after aerosol administration of 1 mg FFL SNIM RNA in the PEI Formulation of Example 6 with the EFlow mesh nebulizer. Lung specimens were ex vivo cultured overnight before luciferase measurements (pg luciferase/mg lung tissue).

[0058] Figure 22. BLI of luciferase expression in representative pig tissue specimens from different lung regions after aerosol administration of 1 mg FFL SNIM RNA in the PEI Formulation of Example 6. Lung specimens were ex vivo cultured overnight before measurements.

[0059] Figure 23. BLI imaging of luciferase expression in representative pig tissue specimens from different lung regions after aerosol administration of 1 mg FFL SNIM RNA in the PEI Formulation of Example 6 using a PARI BOY jet nebulizer. Lung specimens were ex vivo cultured overnight before measurements.

[0060] Figure 24. BLI of luciferase expression in representative pig tissue specimens from different lung regions after aerosol administration of each 1 mg FFL SNIM RNA and hCFTR mRNA in the PEI Formulation of Example 6 using an Acroneb mesh nebulizer. Lung specimens were ex vivo cultured overnight before measurements.

[0061] Figure 25. BLI of luciferase expression in representative pig tissue specimens from different lung regions after aerosol administration of 1 mg FFL SNIM RNA in “SHIRE Formulation #3” (HGT5001 :DOPE:Chol:DMGPEG2K (50:25:20:5) (mol ratio) using an Aeroneb mesh nebulizer. Lung specimens were ex vivo cultured overnight before measurements.

[0062] Figure 26. BLI of luciferase expression in pig tissue specimens from different lung regions from one untreated control pig. The other untreated control pig showed the same result (data not shown).

Page 11 of 121

WO 2014/153052

PCT/US2014/028849 [0063] Figure 27. BLI of luciferase expression in lung specimens of once-treated pigs #3 and #6. Aerosol administration of each 1 mg FFL SNIM RNA and hCFTR SNIM RNA in the PEI Formulation of Example 6 was performed using an Aeroneb mesh nebulizer. Slices of the entire pig lung are shown. Upper three rows: pig #3, lower three rows: pig #6.

[0064] Figure 28. BLI of luciferase expression in lung specimens of twice-treated pigs #4 and #8. Aerosol administration of each 1 mg FFL SNIM RNA and hCFTR SNIM RNA in the PEI Formulation of Example 6 was performed using an Aeroneb mesh nebulizer. Slices of the entire pig lung are shown. Upper three rows: pig #4, lower three rows: pig #8.

[0065] Figure 29. BLI of luciferase expression in lung specimens of three times-treated pigs #1 and #2. Aerosol administration of each 1 mg FFL SNIMRNA and hCFTR-mRNA SNIM RNA in the PEI Formulation of Example 6 were performed using an Aeroneb mesh nebulizer. Slices of the entire pig lung are shown. Upper three rows: pig #1, lower three rows: pig #2.

[0066] Figure 30. Luciferase 1HC on lung tissue of three times treated pig #1. Aerosol administration of each 1 mg FFL SNIM RNA and hCFTR SNIM RNA in the PEI Formulation of Example 6 was performed using an Aeroneb mesh nebulizer. Luciferase expression appeared in reddish-pink colour (Anti-Luciferase pAb 1:300, G7451, Promega, Refine AP-Kit, chromogen: New fuchsine).

[0067] Figure 31. Highly BLI-positive lung tissue of threefold treated pig #1 was subjected to hCFTR IP/WB. Lane 1 :T84 cells (positive control), Lane 2: untreated pig lung tissue (300 mg), Lane 3: treated pig lung tissue (697 mg), Lane 4: treated pig lung tissue (163 mg). Mature complex-glycosylated hCFTR appeared as the disperse so-called C-band. Mannoserich hCFTR appeared as the more dense so-called B-band. hCFTR expression was observed in T84 cells and pig lung tissue of hCFTR SNIM RNA treated pig #1, whereas no hCFTR expression was observed in untreated pigs.

[0068] Figure 32. Immunoprecipitation of hCFTR using MAB25031 and subsequent immunodetection using AB570 from pig lung samples post hCFTR SNIM RNA delivery in the PEI Formulation of Example 6. Lane 1: sample from luciferase-negative left caudal lobe of pig #2, Lane 2: sample from luciferase-positive lung regions of pig #1.

Page 12 of 121

WO 2014/153052

PCT/US2014/028849 [0069] Figure 33 A&B. In vitro transfection of HEK 293T cells with C-terminal Hisw tagged (CO-CFTR-C-Hisio) and non-tagged (CO-CFTR) codon optimized human CFTR SNIM RNA. Following transfection, whole cell lysate was collected and analyzed for human CFTR expression by Western blot using (A) anti-CFTR antibody #217 and (B) anti-His antibody 1187. Transfected samples were compared to non-transfection HEK 293T control lysate (Lane 3).

[0070] Figure 33 C. In vitro transfection of HEK 293T cells with SNIM RNA encoding codon optimized human CFTR with a growth hormone leader sequence and a (GH-CO-CFTR ) or SNIM RNA encoding a C-tcrminal Hisjo tagged codon optimized human CFTR (CO-CFTRC-Hisio). Following transfection, whole cell lysate was collected and analyzed for human CFTR expression by Western blot using anti-CFTR antibody #217. Transfected samples were compared to non-transfection HEK293T control lysate (Lane 3).

[0071] Figure 34. In vivo transfection of CFTR knockout mice with C-terminal Hisio tagged codon optimized human CFTR SNIM RNA encapsulated within either a lipid (cKK-E12) or polymeric (PEI) nanoparticle formulation . Following nebulized delivery of each respective mRNA formulation, Right and Left lung tissue lysate was collected and analyzed for CFTR expression by Western blot using anti-His antibody 1187. Control CFTR knockout lung tissue and CFTR-Hisio HEK293 lysate was used as a negative and positive controls respectively.

[0072] Figure 35. Bioluminescent detection of FFL expression in porcine lung samples collected following nebulization with water for injection.

[0073] Figure 36. Bioluminescent detection of FFL expression in porcine lung samples collected following nebulization with 1 mg FFL SNIM RNA + 1 mg CO-CFTR SNIM RNA in a branched 25 kDa PEI formulation.

[0074] Figure 37. Bioluminescent detection of FFL expression in porcine lung samples collected following nebulization with 1 mg FFL SNIM RNA + 5 mg CO-CFTR SNIM RNA in a branched 25 kDa PEI formulation.

[0075] Figure 38. Bioluminescent detection of FFL expression in porcine lung samples collected following nebulization with 1 mg FFL SNIM RNA + 10 mg CO-CFTR SNIM RNA in a branched 25 kDa PEI formulation.

Page 13 of 121

WO 2014/153052

PCT/US2014/028849 [0076] Figure 39. Relative quantification of CFTR expression in different chorots.

Band intensities were normalized to 150kDa band in the protein ladder.

[0077] Figure 40. Representative example of a “CFTR-positive” bronchi with at least one epithelial cell detected within the epithelial cell layer and displaying a clear membrane localized CFTR signal via CFTR immunohistochemical staining using an anti-CFTR antibody.

[0078] Figure 41. Immunohistochemical staining of CFTR in porcine lung following aerosol delivery of control (WFI) or 5 mg CO-CFTR SNIM RNA.

[0079] Figure 42. Represents a “low” CFTR expression level, assayed in porcine lung by immunohistochemical staining with anti-CFTR following aerosol delivery of 5 mg CO-CFTR SNIM RNA.

[0080] Figure 43. Represents a “medium” CFTR expression level, assayed in porcine lung by immunohistochemical staining with anti-CFTR following aerosol delivery of 5 mg COCFTR SNIM RNA.

[0081] Figure 44. Represents a “high” CFTR expression level, assayed in porcine lung by immunohistochemical staining with anti-CFTR following aerosol delivery of 5 mg CO-CFTR SNIM RNA.

[0082] Figure 45. Immunohistochemical staining of CFTR in porcine lung following aerosol delivery of control (WFI) or 10 mg CO-CFTR SNIM RNA.

[0083] Figure 46. Quantification of relative numbers of CFTR-positive bronchi / bronchioles per animal. Analysis of each cohort (WFI; and lmg, 5mg, lOmg human CFTR SNIM RN A) 24 hours post aerosol administration. CFTR expression normalized to signal intensity for 150 kDa protein standard. (WFI=9.4±5.6%, 1MG=15.2±6.6%, 5MG=25.4±14.1%, 10MG=20.9±3.7%; WFI vs 5MG p=0.0281, WFI vs 10MG p—0.0174) [0084] Figure 47. Illustrates multiplex nucleic acid in situ detection of (A) ubiquitin C and (B) dap B in porcine lung, post aerosol delivery of water for injection by nebulizer.

[0085] Figure 48. Illustrates multiplex nucleic acid in situ detection of (A) ubiquitin C and (B) dap B in porcine lung, post aerosol delivery of 1 mg FFL SNIM RNA + 10 mg COCFTR SNIM RNA in a branched 25 kDa PEI formulation.

Page 14 of 121

WO 2014/153052

PCT/US2014/028849 [0086] Figure 49. Illustrates multiplex nucleic acid in situ detection of (A) right cranialis and (B) left cranialis in porcine, post aerosol delivery of water for injection by nebulizer.

[0087] Figure 50. Illustrates multiplex nucleic acid in situ detection of (A) right cranialis and (B) left cranialis in. porcine, post aerosol delivery of 1 mg FF.L SNIM RNA + 1 mg CO-CFTR SNIM RNA in a branched 25 kDa PEI formulation.

[0088] Figure 51. Illustrates multiplex nucleic acid in situ detection of (A) right cranialis and (B) left cranialis in porcine, post aerosol delivery of 1 mg FFL SNIM RNA + 5 mg CO-CFTR SNIM RNA in a branched 25 kDa PEI formulation.

[0089] Figure 52. Illustrates multiplex nucleic acid in situ detection of (A) right cranialis and (B) left cranialis in porcine, post aerosol delivery of 1 mg FFL SNIM RNA + 10 mg CO-CFTR SNIM RNA in a branched 25 kDa PEI formulation.

[0090] Figure 53. Illustrates positive detection of active firefly luciferase (FFL) protein in a treated pig lung via luminescence upon exposure to FFL/CO-CFTR-C-HislO mRNA encapsulated cKK-E12 lipid nanoparticles. Pigs were treated with 1 mg FFL + 9 mg CO-CFTRC-HislO mRNA encapsulated lipid nanoparticles via nebulization using a Pari jet nebulizer and sacrificed 24 hours post-treatment. FFL luminescence was visualized using an JVIS bioluminometer.

[0091] Figure 54. Illustrates exemplary results of hCFTR expression in HER cells transfected using neubilized complexes given to pigs 10, 11 and 12 (1 mg dose).

[0092] Figure 55. Illustrates exemplary results of hCFTR expression in HER cells transfected using neubilized complexes given to pigs 13, 14 and 15 (5 mg dose) and in HEK cells transfected using neubilized complexes given to pigs 19, 20 and 21 (10 mg dose).

[0093] Figure 56. Illustrates exemplary results of hCFTR expression in HEK cells transfected using neubilized complexes given to pig 16 (5 mg dose), 22 (10 mg dose) and 67 (1 mg dose).

[0094] Figure 57. Illustrates exemplary results of hCFTR expression in HEK cells transfected using neubilized complexes given to pigs 17, 18 (5 mg dose), 23, 24 (10 mg dose) and 68, 69 (1 mg dose).

Page 15 of 121

WO 2014/153052

PCT/US2014/028849

DETAILED DESCRIPTION OF THE INVENTION

Definitions [0095] As used herein, the term “polynucleotide” is generally used to refer to a nucleic acid (e.g., DNA or RNA). The terms polynucleotide, nucleic acid, DNA, RNA, and mRNA include such molecules that are comprised of: standard or unmodified residues; nonstandard or modified residues; and mixtures of standard and nonstandard residues.

[0096] As used herein, the term “mRNA” is used to refer to modified and unmodified

RNA including both a coding region and a noncoding region.

[0097] As used herein, the phrase “coding region” of an mRNA generally refers to that portion that when translated results in the production of an expression product, such as a polypeptide, protein, or enzyme.

[0098] A “nonstandard nucleobase” is a base moiety other than the natural bases adenine (A), cytosine (C), guanine (G), thymine (T), or uracil (U). The nonstandard nucleobase is an analog of a specific nucleobase (A, C, G, T, or U) when its base pairing properties in a nucleic acid double helix and locus of incorporation by DNA or RNA polymerases in a nucleic acid double helix (including a local RNA-DNA helix such as that formed during transcription of a DNA template by RNA polymerase) are most similar to one of the five previously listed nucleobases, with the exception that analogs of T will generally also be analogs of U and vice versa. For purposes of determining percentage identity of a first sequence relative to a second sequence, an analog of a base is not a mismatch to the natural base; for example, pseudouridinc matches uridine, 5-methylcytidine matches cytidine, etc.

[0099] The term “nonstandard” used in conjunction with terms including but not limited to “nucleoside”, “base”, “nucleotide”, or “residue” is to be interpreted in the same manner as if it were used in conjunction with “nucleobase.” [0100] GC content” is the fraction or percentage of total nucleobase residues in a nucleic acid sequence that are guanine residues, cytosine residues, or analogs thereof. For

Page 16 of 121

WO 2014/153052

PCT/US2014/028849 example, a 100 nt sequence that contains exactly 30 cytosines, exactly 30 guanines, exactly one cytosine analog, and exactly one guanine analog has a GC richness of 62%.

[0101] As used herein, a “disfavored codon” refers to a codon which is translated less efficiently or rapidly by mammalian cells than another codon for the same amino acid residue. Disfavored codons generally include codons with an A or U in the 3rd or “wobble” position of the codon. For a discussion of disfavored codons, see, e.g., U.S. Patent Publication 2009/0069256 Al.

[0102] A “non-naturally occurring mRNA molecule” is an mRNA that is not produced through normal transcription and splicing processes of wild-type cells. An mRNA may qualify as non-naturally occurring by virtue of its sequence (e.g., a series of codons and/or one or more UTRs that do not present in any naturally-occurring CFTR mRNA) and/or because it includes nonstandard nucleotide residues. A non-naturally occurring mRNA molecule may be in vitro synthesized.

[0103] In each of Tables 1 and 2 below, the NWT column indicates the non-wild-type base at the position (Pos.) in the CFTR coding sequence (see, e.g,, SEQ ID NO: 3), and the WT column indicates the wild-type base at the same position (see, e.g., SEQ ID NO: 2 or the RefSeq entry for human CFTR (accession no. NM_000492.3, Feb. 10, 2013, version, available from GenBank; note that the sequence of NM_000492.3 contains noncoding sequence such that the coding sequence occurs at position 133 to 4575, such that, for example, position 7 in the tables below corresponds to position 139 of the NM 000492.3 sequence).

Non-naturally occurring CFTR mRNA [0104] In addition to providing methods of producing functional CFTR in vivo using naturally occurring or wild-type CFTR mRNA (and compositions comprising that mRNA), the invention also provides non-naturally occurring CFTR mRNA that encodes CFTR protein (e.g., SEQ ID NO :1). In some embodiments, the non-naturally occurring CFTR mRNA is purified or isolated.

[0105] In other embodiments, the non-naturally occurring CFTR mRNA is present in a cell. In some embodiments, the cell comprising the non-naturally occurring CFTR mRNA did not synthesize the non-naturally occurring CFTR mRNA and/or does not comprise DNA

Page 17 of 121

WO 2014/153052

PCT/US2014/028849 complementary to the non-naturally occurring CFTR mRNA and/or a functional CFTR gene; the cell may optionally comprise an inactive CFTR gene, such as a CFTR gene with a nonsense, missense, frameshift, insertion, or deletion mutation that renders the expression product of the gene nonfunctional. In some embodiments, the cell comprising the non-naturally occurring CFTR mRNA further comprises functional CFTR protein translated from the non-naturally occurring CFTR mRNA. The cell may be, e.g., a lung epithelial cell, a liver cell, or a kidney cell. In some embodiments, the cell is in a cell culture.

CFTR Coding Sequence

10106] In some embodiments, CFTR mRNA according to the invention comprises a coding sequence with fewer complements of cryptic promoters than SEQ ID NO: 2 (i.e., the coding sequence of wild-type human CFTR), fewer direct and/or inverted repeats than SEQ ID NO: 2, fewer disfavored codons than SEQ ID NO: 2, and/or the GC content of the coding sequence is lower than the GC content of SEQ ID NO: 2.

[0107] Cryptic promoters, direct and/or inverted repeats and/or disfavored codons of a sequence may be reegnized by one skilled in the art using routine methods. For example, the direct and/or inverted repeat content of a sequence can be determined by sequence analysis (Liu et al., Journal of Theoretical Biology (2014) 344: 19-30). The cryptic promoter content of a sequence can also be determined by sequence analysis, e.g., presence of Shine-Dalgamo sequences within construct or the like.

[0108] In some embodiments, CFTR mRNA according to the invention is in vitrotranscribed, i.e., the mRNA was synthesized in an artificial setting not within a biological cell (e.g., a cell free in vitro transcription system). Generally, in vitro transcription involves providing a DNA template comprising a promoter and a sequence complementary to the desired mRNA (which may be circular or linear), an RNA polymerase, and nucleoside triphosphates in suitable reaction conditions (salts, buffers, and temperature). RNase inhibitors, reducing agents, and/or pyrophosphatase may be present in the reaction mixture. In some embodiments, the RNA polymerase is T7 RNA polymerase.

[0109] In some embodiments, the CFTR mRNA according to the invention comprises a coding sequence comprising at least 50%, at least 55%, at least 60%, at least 65%, at least 70%,

Page 18 of 121

WO 2014/153052

PCT/US2014/028849 at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the non-wild-type bases listed in Table 1 at the positions of the coding sequence listed in Table 1 relative to the wildtype coding sequence of SEQ ID NO: 2.

Table 1. Non-wild-type bases that can be used in the coding sequence of mRNA encoding

CFTR.

WO 2014/153052

PCT/US2014/028849 [0111] In some embodiments, the present invention comprises a non-naturally occurring

CFTR mRNA comprising a coding sequence of SEQ ID NO: 3. Additional exemplary nonnaturally occurring CFTR mRNA coding sequences arc described in the Brief Description of Sequences section, such as, for example, SEQ ID NOs:9, 10, 11, 12, 13, 14, 15, 16, or 17. In some embodiments, the present invention provides a CFTR mRNA comprising a coding sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NO: 3, 9, 10, 11, 12, 13, 14, 15, 16, or 17. In some embodiments, a non-naturally occurring CFTR mRNA comprises a 5’UTR, 3’UTR, a signal peptide coding sequence or a cap or tail structure as described below.

[0112] The above-described CFTR mRNAs comprising coding sequence which differs from wild-type CFTR coding sequence can provide advantages with respect to efficacy and ease of preparation. For example, in vitro transcription reactions using a polynucleotide comprising template sequence complementary to the CFTR coding sequence can give greater RNA yield; a polynucleotide comprising said template sequence can be more stable (i.e., less prone to mutation) during growth in a host cell, reducing the amount of purification needed to generate template usable in a reaction; and the in vivo translation of an mRNA comprising the coding sequence can be higher.

Signal Peptide Sequence [0113] In some embodiments, an mRNA encoding a CFTR protein incorporates a nucleotide sequence encoding a signal peptide. As used herein, the term “signal peptide” refers to a peptide present at a newly synthesized protein that can target the protein towards the secretory pathway. In some embodiments, the signal peptide is cleaved after translocation into the endoplasmic reticulum following translation of the mRNA. Signal peptide is also referred to as signal sequence, leader sequence or leader peptide. Typically, a signal peptide is a short (e.g., 5-30, 5-25, 5-20, 5-15, or 5-10 amino acids long) peptide. A signal peptide may be present at the N-terminus of a newly synthesized protein. Without wishing to be bound by any particular

Page 35 of 121

WO 2014/153052

PCT/US2014/028849 theory, the incorporation of a signal peptide encoding sequence on a CFTR encoding mRNA may facilitate the secretion and/or production of the CFTR protein in vivo.

[0114] A suitable signal peptide for the present invention can be a heterogeneous sequence derived from various eukaryotic and prokaryotic proteins, in particular secreted proteins. In some embodiments, a suitable signal peptide is a leucine-rich sequence. See Yamamoto Y et al. (1989), Biochemistry, 28:2728-2732, which is incorporated herein by reference. A suitable signal peptide may be derived from a human growth hormone (hGH), serum albumin preproprotein, Ig kappa light chain precursor, Azurocidin preproprotein, cystatinS precursor, trypsinogen 2 precursor, potassium channel blocker, alpha conotoxin lpl.3, alpha conotoxin, alfa-galactosidase, cellulose, aspartic proteinase nepenthesin-1, acid chitinase, K28 prepro-toxin, killer toxin zygocin precursor, and Cholera toxin. Exemplary signal peptide sequences are described in Kober, et al., Biotechnol. Bioeng., 110: 1164-73, 2012, which is incorporated herein by reference.

[0115] In some embodiments, a CFTR encoding mRNA may incorporate a sequence encoding a signal peptide derived from human growth hormone (hGH), or a fragment thereof, A non-limiting nucleotide sequence encoding a hGH signal peptide is show below.

5’ human growth hormone (hGH) sequence (SEQ ID NO :18):

AUGGCCACUGGAUCAAGAACCUCACUGCUGCUCGCUUUUGGACUGCUUUGCCUGC CCUGGUUGCAAGAAGGAUCGGCUUUCCCGACCAUCCCACUCUCC

PCT/US2014/028849 nanoparticulates, silicon dioxide nanoparticulates, nano crystalline particulates, semiconductor nanoparticulates, dry powders, poly(D-arginine), nanodendrimers, starch-based delivery systems, micelles, emulsions, sol-gels, niosomes, plasmids, viruses, calcium phosphate nucleotides, aptamers, peptides, peptide conjugates, small-molecule targeted conjugates, and other vectorial tags. Also contemplated is the use of bionanocapsules and other viral capsid proteins assemblies as a suitable carrier. (Hum. Gene Ther. 2008 Sep; 19(9):887-95).

[0125] In some embodiments, the carrier comprises an organic cation, such as a cationic lipid or a cationic organic polymer. If present, the cationic lipid may be a component of liposomal vesicles encapsulating the mRNA.

Biophys. Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997); U.S. Pat. No. 5,744,335), or ICE.

PEGylated Lipids [0133] In some embodiments, a suitable lipid nanoparticle comprises one or more

PEGylated lipids. For example, the use of polyethylene glycol (PEG)-modified phospholipids and derivatized lipids such as dcrivatized ceramides (PEG-CER), including N-OctanoylSphingosine-l-[Succinyl(Methoxy Polyethylene Glycol)-2000] (C8 PEG-2000 ceramide) is contemplated by the present invention in combination with one or more of the cationic and, in some embodiments, other lipids. In some embodiments, suitable PEGylated lipids comprise PEG-ceramides having shorter acyl chains (e.g., C]4 or Cis). In some embodiments, the PEGylated lipid DSPE-PEG-Maleimide-Lectin may be used. Other contemplated PEG-modified lipids include, but arc not limited to, a polyethylene glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of Ct.-Cho length. Without wishing to be bound by a particular theory, it is contemplated that the addition of PEGylated lipids may prevent complex aggregation and increase circulation lifetime to facilitate the delivery of the lipsome encapsulated mRNA to the target cell.

[0134] In certain embodiments, the composition comprises one of the following combinations of lipids:

[0143] The in vitro transcribed mRNA for uses and methods according to the invention can comprise a 5’-UTR, 3’UTR, poly-A, poly-U and/or poly-C tail, cap, and/or nonstandard nucleotide residues, as discussed in the section above concerning such features.

Pharmaceutical Compositions for Uses and Methods [0144] Pharmaceutical compositions for use according to the invention may comprise mRNA for uses and methods according to the invention as discussed in the preceding section and additional ingredients as discussed in the section above regarding compositions comprising CFTR mRNA. Thus, use and/or administration of pharmaceutical compositions comprising any of the carriers discussed above is contemplated.

[0145] In some preferred embodiments, pharmaceutical compositions comprise PEI, such as branched PEI having a molecular weight ranging from 10-40 kDa, for example, 25 kDa.

[0146] In other preferred embodiments, pharmaceutical compositions comprise a cationic lipid, a pegylated lipid, and an additional lipid (such as a neutral lipid). The cationic lipid, pegylated lipid, and/or additional lipid may be chosen from those listed in the section above regarding compositions comprising CFTR mRNA.

Page 47 of 121

WO 2014/153052

PCT/US2014/028849

Routes of administration for induction of expression in lung [0147] In some embodiments of methods and uses for induction of CFTR expression in a lung of a mammal, a pharmaceutical composition as described above is administered by a route chosen from intratracheal instillation, nebulization, and aerosolization. The apparatus for administering the composition can be chosen from the apparatuses listed, in the section above regarding apparatuses loaded with a pharmaceutical composition.

[0148] In preferred embodiments, the composition is administered via nebulization or aerosolization. Some lipid formulations may have a tendency to aggregate when nebulization is attempted but it is generally possible to solve aggregation issues by adjusting the formulation, e.g., by substituting the cationic lipid.

Treatment of cystic fibrosis [0149] Among other things, the present invention can be used for treating cystic fibrosis.

In some embodiments, the present invention provides a method of treating cystic fibrosis by administering to a subject in need of treatment an mRNA encoding a CFTR protein as described herein or a pharmaceutical composition containing the mRNA. The mRNA or a pharmaceutical composition containing the mRNA may be administered directly to the lung of the subject. Various administration routes for pulmonary delivery may be used. In some embodiments, an mRNA or a composition containing an mRNA described herein is administered by inhalation, nebulization or aerosolization. In various embodiments, administration of the mRNA results in expression of CFTR in the lung of the subject (e.g., epithelial cells of the lung).

[0150] In a parti cular embodiment, the present invention provides a method of treating cystic fibrosis by administering to the lung of a subject in need of treatment an mRNA comprising a coding sequence which encodes SEQ ID NO:1, In certain embodiments, the present invention provides a method of treating cystic fibrosis by administering to the lung of a subject in need of treatment an mRNA comprising a coding sequence which encodes an amino

Page 48 of 121

WO 2014/153052

Page 57 of 121

WO 2014/153052

PCT/US2014/028849 [0187] Upon further magnification (40x), positive detection of human. CFTR mRNA was observed within the apical cytoplasm of bronchial epithelial cells and well as intracellular terminal alveolar regions (Figure 11).

[0188] In total, successful delivery of synthetic human CFTR messenger RNA can be achieved utilizing both polymeric (PEI) and lipid nanoparticle-based (C12-200) delivery systems. These systems afforded intracellular accumulation of the drug substance within the target cells of the mice. Further, substantial amounts of hCFTR mRNA were present in these target cells 24 hours post-administration.

Example 6: Validation of Human CFTR Expression Using Specific Antibody [0189] Antibody validation for human CFTR protein detection in mouse, pig, and cultured cells. Experiments were performed to identify an antibody which is specific for the hCFTR protein, which does not cross-react with the mouse and swine analogue and which is available in sufficient supply for future experiments. Briefly, testing of various anti-hCFTR antibodies from academic and commercial sources led to identification of a combination of antihCFTR antibodies which were capable of detecting human CFTR protein after immunoprecipitation and Western blotting (IP/WB) without cross-reactivity for either murine or porcine CFTR. Thus, suitable anti-hCFTR antibodies for detection of hCFTR protein without cross-reactivity for either murine or porcine CFTR were identified based on IP/WB results.

[0190] Cells were transfected with hCFTR mRNA and protein lysates were prepared using ProteoExtract Transmembrane Kit (Merck) at 24 hrs post transfection and transmembrane fraction was screened by Western blotting for hCFTR using mouse anti-human CFTR antibody (MAI-935). Lysates from 16HBE cells were used as positive control. Figure 12A presents the data from CHO and COS-7 cells.

[0191] Baby Hamster Kidney cells (BHK), described as CFTR-negative in the literature, were transfected similar to CHO and COS-7 cells and protein lysates screened by Western blot.

Page 58 of 121

WO 2014/153052

PCT/US2014/028849

Tn contrast to the previously published reports, a clear positive signal for CFTR could be observed using the mouse monoclonal anti-CFTR antibody (Figure 12B). To test the specificity of antibody used in Western blot analysis, Pig Kidney Cells from CFTR-knockout pig (PKC), kindly provided by Prof. Eckhardt Wolf (Ludwig Maximilians University, Munich), were used in transfection experiments and protein lysates screened for CFTR expression. As was evident in Figure 12B, no signal for CFTR could be detected in PKC cells. However, transfection did not result in any detectable hCFTR expression either. Using luciferase as a control for transfection, PKC cells were found to express luciferase several fold less efficient when compared to CHO or COS-7 cells. As no significant difference in the intensity of hCFTR band could be detected in any of the screened cell lines post transfection, extensive screening for other hCFTR antibodies with higher sensitivities and specificity towards hCFTR was performed.

[0192] Antibody Screening via Western Blots. Protein lysates were prepared from human bronchial epithelial cell line (BEAS-2B), human embryonic kidney cell line (HEK), mouse lungs and pig lungs using ProteoExtract Transmembrane Kit (Merck) and transmembrane fraction used for immunoblotting using different primary antibodies (MAI-935 from Thermo Scientific Pierce Antibodies, Rockford, IL, USA, AB596 from the Cystic Fibrosis Consortium, University of Pennsylvania, PA, USA, and AB570 from the Cystic Fibrosis Consortium, University of Pennsylvania, PA, USA). The data are summarized as Figure 13.

[0193] Whereas MAI-935 detected CFTR in all the three species, AB596 detects human and murine CFTR but not porcine and antibody G449 detects only human CFTR specifically. With AB570, it was not clear if the slightly low molecular weight bands observed with murine and porcine samples are indeed CFTR or non-specific products. In subsequent experiments (data not shown), it was found that MAI-935 recognizes a band which is not CFTR. Therefore, in general, MAI-935 results were considered as confirming results generated using other antibodies, but experiments in which the only anti-CFTR antibody used was MA 1-935 were not considered conclusive.

Page 59 of 121

WO 2014/153052

PCT/US2014/028849 [0194] Immunoprecipitation ofhCFTR (IP-hCFTR) from Tissue samples. Given that all the screened antibodies produced several non-specific bands and none of them produced the characteristic banding pattern ofhCFTR (C-band representing the fully glycosylated protein and B-band representing the core mannosylatcd form), immunoprecipitation (IP) ofhCFTR and subsequent detection by Western blot was established to increase the sensitivity and specificity of detection thereby increasing the signal to noise ratio.

[0195] Initial IP experiments were performed in collaboration with Prof. Burkhard

Tummler (Medizinsche Hochschule Hannover) using protocols and antibodies published by van Barneveld et al. 2012, Immunochemical analysis of Mutant CFTR in Lung explants, Cell Physiol. Biochem. 30, 587-595 (2012)). Human colon carcinoma cells (T84) which overexpress hCFTR were used as positive controls for IP experiments.

[0196] Immunoprecipitation ofhCFTR using three different antibodies (R29, R66/17 and

R66/16) followed by immunodetection with AB596 resulted in specific detection ofhCFTR in protein lysates from lungs of pigs treated with an aerosol ofhCFTR SNIM RNA as described in Example 8 below (Figure 14).

[0197] HGT5001 Formulation. Aerosol experiments using hCFTR SNIM RNA in a formulation of HGT5001:DOPE;Chol;PEGDMG2K (relative amounts 50:25:20:5 (mg:mg:mg:mg)) (“HGT5001 Formulation”) were performed in mice and protein lysates from the isolated lungs at 24 hrs post mRNA delivery were also analysed by IP using the same antibodies and conditions as for the pig lysates. However, no characteristic mature CFTR banding pattern could be detected for mouse samples (Figure 15).

[0198] Immunoprecipitation ofhCFTR (IP-hCFTR) from in vitro-transfected cells. Initial

IP results using tissue material from pigs provided the evidence for the technical feasibility of hCFTR detection post transcript delivery in vivo. However, as none of the antibodies used in immunoprecipitating CFTR (R29, R66/17 and R66/16) arc commercially available, other commercially available antibodies were screened for their efficacy in IP reactions. Two antibodies from R&D systems (MAB25031 and MAB1660) were tested.

Page 60 of 121

WO 2014/153052

PCT/US2014/028849 [0199] Protein lysates were prepared from T84 cells and 500 pg of total protein was used in the IP reaction using different concentrations of MAB25031 antibody. The amount of hCFTR protein immunoprecipitated was then detected by immunobloting using AB5 70 (Cystic Fibrosis Foundation). AB596 under these conditions resulted in much higher background and so was not tested further. As revealed in Figure 16A, there was no further increase in the amount of CFTR protein precipitated when the concentration of IP antibody was increased from 2 pg/ml to 4 pg/ml. Both the fully glycosylated and only core glycosylated forms (C- and B-band, respectively) were detected. The same immunoprccipitatcs were also screened using MAB1660 as primary antibody in western blot. With this antibody however, only band-C was visible (Figure 16B).

[0200] After the successful detection of endogenous hCFTR from T84 immunoprecipitates using MAB25031 antibody, experiments were performed in NIH3T3 cells with the aim to detect hCFTR protein post transfection. NIH3T3 cells were transfected with hCFTR SNIM RNA. Protein lysates were prepared at 72 hrs post transfection and protein amounts quantified using BCA method. Human CFTR protein was immunoprecipitated from 500 pg of total protein lysate using MAB25031 antibody at 2 pg/ml followed by immunoblottmg using AB570 (Figure 17). However, no CFTR could be detected. Cells transfected with LacZ encoding mRNA were analysed as control samples for the effect of transfection per se on amount of CFTR protein.

[0201] Increasing the amount of total protein used in immunoprecipitation from 500 pg to 8 mg did not result in any detectable hCFTR protein post immunodetection with AB 570. Another hCFTR specific antibody, MAB1660 (R&D Systems), was also screened for immunoprecipitation (Figure 18). However, this antibody does not precipitate CFTR as effectively as MAB25031. Therefore all future immunoprecipitations were performed with MAB25031.

[0202] Lack of hCFTR detection in mRNA transfected samples may not necessarily mean lack of functionality of the tested mRNAs as kinetic experiments using luciferase as

Page 61 of 121

WO 2014/153052

PCT/US2014/028849 marker gene have shown that maximum expression with mRNA is observed at 24 hrs post transfection. Lack of hCFTR detection is rather due to insufficient hCFTR concentration in the tested samples or lack of specificity of the applied antibodies.

[0203] PEI Formulation. The established conditions were tested for their feasibility to detect hCFTR after hCFTR SNIM RNA in delivery to pigs (see Example 7) of a nanoparticle formulation with 25 kDa branched PEI (“PEI Formulation”) prepared as follows.. The required amount of SNIM RNA was diluted just before application in water for injection (Braun, Melsungen) to a total volume of 4 ml and added quickly to 4 ml of an aqueous solution of branched PEI 25 kDa using a pipette at an N/P ratio of 10. The solution was mixed by pipetting up and down ten times and nebulized as two separate 4.0 ml fractions one after another to the pig lungs using the indicated nebulizer. One sample from the luciferase expressing lung areas from pig #1 and another from the caudal lobe of pig #2, where no luciferase activity could be detected, thus indicating lack of mRNA delivery and/or expression, were selected as positive and negative controls. Protein lysates prepared from these samples were immunoprecipitated using MAB25031 (R&D Systems) and hCFTR protein detected using AB570. As shown in Figure 19, luciferase expression correlated with the expression of hCFTR mRNA. Sample from the left caudal lobe from pig #2 where no luciferase activity was detectable, was also negative for hCFTR (lane 1) whereas hCFTR could be detected in samples from pig #1 which were positive for luciferase (lane 2).

Example 7: Aerosol Delivery of mRNA [0204] Establishment of encapsulated mRNA aerosol delivery to the lungs ofpigs.

Aerosol administration of firefly luciferase (FFL) SNIM RNA to the pig lungs was established by a stepwise experimental procedure. In a first step FFL SNIM RNA formulations were nebulized to anaesthetized pigs during controlled ventilation. In a second step lungs were excised immediately after aerosol administration was completed and lung specimens were

Page 62 of 121

WO 2014/153052

PCT/US2014/028849 incubated in cell culture medium overnight before ex vivo luciferase measurement was performed on lung specimens by BL1.

[0205] Pigs of the German Landrace were obtained from Technical University Munich,

Weihenstephan, Germany. The pigs had a body weight ranging from 35-90 kg. Each treatment was performed on one pig. In total five pigs were heated. The first pig (90 kg weight) was heated with FFL SNIM RNA in the PEI Formulation of Example 6 using an EFlow mesh nebulizer and measurement of luciferase activity in lung homogenates. The second pig (60 kg weight) was heated with FFL SNIM RNA in the PEI Formulation of Example 6 using an EFlow mesh nebulizer and measurement of luciferase activity in lung specimens by BLI. The third pig (80 kg weight) was treated with FFL SNIM RNA in the PEI Formulation of Example 6 using a PARI BOY jet nebulizer and measurement of luciferase activity in lung specimens by BLI. The fourth pig (60 kg weight) was heated with FFL SNIM RNA/hCFTR mRNA in the PEI Formulation of Example 6 using an Aeroneb mesh nebulizer and measurement of luciferase activity in lung specimens by BLI. The fifth pig (35 kg weight) was heated with FFL SNIM RNA in the HGT5001 Formulation of Example 6 using an Aeroneb mesh nebulizer and measurement of luciferase activity in lung specimens by BLI.

[0206] Sedation in pigs was initiated by premedication with azaperone 2 mg/kg body weight, ketamine 15 mg/kg body weight, atropine 0.1 mg/kg body weight and followed by insertion of an intravenous line to the lateral auricular vein. Pigs were anesthetized by inhavenous injection of propofol 3-5 mg/kg body weight as required. Anesthesia was maintained by isoflurane (2-3%) with 1% propofol bolus injection at 4 to 8 mg/kg body weight to enhance anesthesia as required. Duration of the anesthesia was approximately 1-3 hrs. Pigs were killed with bolus injection of pentobarbital (100 mg/kg body weight) and potassium chloride via the lateral ear vein. Lungs were excised and tissue specimens were collected from various lung regions followed by incubation in cell culture medium overnight. For measurement of luciferase activity tissue specimens were either homogenized and analyzed in a tube

Page 63 of 121

WO 2014/153052

PCT/US2014/028849 luminometer or incubated in a medium bath comprising D-Luciferin. substrate and subjected to ex vivo luciferase BLI.

[0207] Details and Results for Pig #1. The experimental set up is illustrated in Figure 20.

For aerosol administration an EFlow mesh nebulizer was connected in-line to the ventilation tubing of the respirator. Aerosol administration took approximately 60 min and was longer than expected from control experiments with an open system. This was apparently caused by increased back pressure during nebulisation as evidenced by aerosol outflow at the reservoir of the mesh nebulizer. Eight milliliters of the PEI Formulation of Example 6 comprising 1 mg FFL SNIM RNA in water for injection were prepared as described in WP5 and were nebulized in two separate 4 ml portions one after another. Luciferase measurement was performed in tissue homogenates of excised lung specimens of various lung regions after overnight incubation in cell culture medium. Expression values were mapped according to the origin of the lung specimens (Figure 21).

[0208] The results showed successful luciferase expression in pig lung tissue. Luciferase expression was highest in central parts of the lung and declined towards more distal regions of the lung. The expression pattern correlated with the expected deposition pattern of the inhaled FFL SNIM RNA-PEI nanoparticles according to the chosen ventilation parameters. Levels of luciferase expression were in the same range as observed in mouse experiments in WP5 using the same the PET Formulation of Example 6.

PCT/US2014/028849 [0217] Repeated aerosol administration of SNIM RNA in the PEI Formulation to the lungs of pigs was established. Groups of two pigs were treated one, two, or three times at weakly intervals with FFL SNIM RNA/hCFTR SNIM RNA in the PEI Formulation of Example

6. Two untreated pigs served as controls. Lungs were excised 24 hrs after treatment and ex vivo luciferase activity was measured in isolated lung specimens by BLI. Expression ofhCFTR protein was analysed using IP/WB. Immunohistochemistry (1HC) was performed for detection of luciferase expression on the cellular level. Toxicology was investigated by measurement of inflammatory cytokines in serum and blood chemistry. Histopathology was performed on lung samples. The study protocol “Pilot project: Repeated application of modified mRNA to establish an animal model for aerosol therapy of cystic fibrosis in pigs” was approved by the local authorities before the start of the experiments (Animal experiments license Nr.: 0-045-12).

[0218] Experimental Design. Pigs, German Landrace, female approximately 6 weeks old (—25 kg body mass in average) at nebulisation, were purchased from Technical University Munich, Weihenstephan, Germany. Pigs were randomized and treated according to the scheme below (Table 3). Treatment groups of each two pigs were as follows:

Group 0 - Control group without treatment

Group I - Aerosol administration of 1 mg FFL SNIM RNA and 1 mg hCFTR SNIM RNA in the PEI Formulation of Example 6 on day 1.

Group II - Aerosol administration of 2 mg hCFTR SNIM RNA in the PEI Formulation of Example 6 on day 1 and 1 mg FFL SNIM RNA and 1 mg hCFTR SNIM RNA in the PEI Formulation of Example 6 on day 8.

Group III - Aerosol administration of 2 mg hCFTR SNIM RNA (6379-186) in the PEI Formulation of Example 6 on day 1 and day 8, aerosol administration of 1 mg FFL SNIM RNA and 1 mg hCFTR SNIM RNA in the PEI Formulation of Example 6 on day 15.

Page 67 of 121

WO 2014/153052

PCT/US2014/028849 [0219| The scheme for treatment and evaluation of each group is shown in Table 3. In addition to the illustrated interventions, physical examination of the pigs was done on a daily basis.

[0220] Table 3. Time-line diagram of different treatment groups.

[0221] Experimental procedure. Sedation in pigs was initiated by premedication with azaperone 2 mg/kg body weight, ketamine 15 mg/kg body weight, atropine 0.1 mg/kg body

Page 68 of 121

WO 2014/153052

PCT/US2014/0 28849 weight and followed by insertion of an intravenous line to the lateral auricular vein. Pigs were anesthetized by intravenous injection of propofol 3-5 mg/kg body weight as required. Anesthesia was maintained with continuous intravenous infusion of 1% propofol as required. Ventilation parameters were matched with endexpiratory carbon dioxide and adjusted if necessary. Anesthesia, respiratory and cardiovascular parameters were monitored continuously using pulse oximetry, capnography, rectal temperature probe and reflex status. Animals received infusion of balanced electrolyte solution at 10 ml/kg/h. Duration of the anesthesia was approximately 80-120 min. Pigs were extubated after onset of sufficient spontaneous breathing. Pigs were killed with bolus injection of pentobarbital 100 mg/kg of body weight via the lateral ear vein after sedation. Lungs were excised and sliced approximately 1 cm thick tissue specimens were collected from various lung regions followed by incubation in cell culture. For measurement of luciferase activity tissue specimens were incubated in a medium bath comprising D-Luciferin substrate and subjected to ex vivo luciferase BLI.

Luciferase expression in treatment groups by BLI.

[0222] For Group 0 (Control group without treatment), no luciferase activity was observed in lung slices (Figure 26).

[0223] For Group 1 (Aerosol administration of 1 mg FFL SNIM RNA and 1 mg hCFTR

SNIM RNA in the PEI Formulation of Example 6), Luciferase activity was clearly detected in lung specimens of one time treated pigs #3 and #6 (Figure 27). Luciferase expression was highest in central parts of the lungs.

[0224] For Group II (Aerosol administration of 2 mg hCFTR SNIM RNA in the PEI

Formulation of Example 6 on day 1 and 1 mg FFL SNIM RNA and 1 mg hCFTR SNIM RNA in the PEI Formulation of Example 6 on day 8), Luciferase activity was clearly detected in lung specimens of twice-treated pigs #4 and #8 (Figure 28). Luciferase expression was highest in central parts of the lungs. It has to be considered that samples were stored for additional 10 hours in cell culture medium before measurement because of a power blackout on the day of the measurements and resulting technical problems with the BLI system.

Page 69 of 121

WO 2014/153052

PCT/US2014/028849 [0225] For Group III (Aerosol administration of 2 mg hCFTR SNIM RNA in the PEI

Formulation of Example 6 on day 1 and day 8, aerosol administration of 1 mg FFL SNIM RNA and 1 mg hCFTR SNIM RNA in the PEI Formulation, of Example 6 on day 15), Luciferase activity was clearly detected in lung specimens of thrice-treated pigs #1 and #2 (Figure 29). Luciferase expression was highest in central parts of the lungs.

[0226] Properties of SNIMRNA-PEI nanoparticles. Particle size and zeta potential was measured for SNIM RNA-PEI formulations before nebulisation (Table XI). The SNIM RNAPEI nanoparticles could be reproducibly formed with a size ranging from 25-37 nm and zeta potentials ranging from 30-49 mV.

Table XI. Particle size and Zeta Potential measurements

<td> Pig#</td><td> T reatment #</td><td> Radius ± S.D.(nm)</td><td> Zeta potential ± S.D.(mV)</td>

[0227] Luciferase expression in treatment groups by IHC. 1HC for FFL was performed on tissue specimens of lung slices (Sophistolab AG, Eglisau, Switzerland) which were positive by BLI and compared with lung tissue of an untreated pig and luciferase-positive mouse tumor tissue as positive control. As expected a strong signal was seen in the luciferase-positive mouse tumor tissue, whereas lung tissue of the untreated pig did not show specific staining. A clearly detectable staining pattern could be observed in the lung tissue of pig #1 which received three

PCT/US2014/028849 of pigs without loss of activity after each treatment cycle and without adverse events. Luciferase expression was found in central parts of the lung tissue but hardly detected in distal lung areas. The regional pattern of luciferase expression correlated with the expected deposition pattern of the the PEI Formulation of Example 6 according to settings used for controlled ventilation. Immunohistochemistry on selected lung samples form treated pigs showed luciferase expression predominantly in the bronchial epithelium of large and small airways. IP/WB clearly demonstrated expression of complex-glycosylated C-band of mature human CFTR in treated pig lung which was absent in untreated pig lung and luciferase-negative lung specimens. Expression of hCFTR in pig lung tissue after hCFTR SNIM RNA aerosol treatment was comparable to the hCFTR expression in healthy human lung when compared to published reports using the identical protocol for hCFTR protein detection. Adverse events grade 1 or 2 were very rare and showed no correlation to the aerosol application of SNIM RNA in the PEI Formulation. Thus, expression of hCFTR protein was successfully demonstrated in lungs of pigs treated with SNIM hCFTR mRNA.

Example 9: CFTR Encoding mRNA Containing Signal Peptide [0239] This example demonstrates that a CFTR protein may be cffectivey expressed from a CFTR encoding mRNA with a signal peptide encoding sequence.

[0240] Messenger RNA Synthesis. For the experiment, C-terminal Hisjo tagged codon optimized human cystic fibrosis transmembrane conductance regulator (CO-CFTR-CHisio)(SEQ ID NO: 15), a codon optimized human CFTR with a growth hormone signal sequence leader (GH-CO-CFTR)(SEQ ID NO: 16) and codon optimized human CFTR (CO-CFTR)(SEQ ID NO: 17) SNIM RNA were synthesized by in vitro transcription from a plasmid DNA template using standard methods. Cells and CFTR transfection. Human embryonic kidney HEK293T cells were grown in DMEM (Invitrogen Cat # 11965-092) supplemented with 10% fetal bovine serum, 2 mM L-Glutaminc, 100 U/ml penicillin and 100 pg/ml streptomycin. The day before

Page 74 of 121

WO 2014/153052

PCT/US2014/028849 transfection, cells were plated on 6-wcll plates at 50-60% confluence and incubated under normal tissue culture conditions (36°C in a humidified atmosphere of 5% CO2, 95% air). In preparation for transfection, 60 μ.1 Lipofectamine 2000 (Invitrogen Cat # 11668019) was diluted in OptiMem reduced scrum media (Invitrogen Cat # 31985-062) and gently vortexed. For the experiment 4 pg of either CO-CFTR, GH-CO-CFTR or CO-CFTR-C-Hisio SNIM RNA was diluted in 900 pl OptiMem media. The mRNA was immediately added to the diluted Lipofectamine® and incubated at room temperature for 30 minutes. The plating media was gently aspirated and replaced with 1 ml OptiMem Reduced Serum Medium and 300 pl of each respective mRNA/Lipofectamine® complex. Cells were incubated under standard tissue culture conditions.

[0241] Western Analysis. Approximately 48 post transfection, cells were removed from their respective plates and lysed. Whole cell lysate was subjected to separation by SDS-PAGE and probed by Western blot. As shown in Figure 33, robust expression of human CFTR protein was detected following CO-CFTR, GH-CO-CFTR and human CO-CFTR-C-Hisio mRNA transfection, by anti-CFTR (A&B) or anti-His (C) antibodies (Figure 33).

Example 10: In Vivo CO-CFTR-C-HislO mRNA Delivery to CFTR Knockout Mice [0242] Analysis of human CFTR protein produced via intratracheal administered niRNAloaded nanoparticles. All studies were performed using CFTR KO mice. CFTR mRNA formulation or vehicle control was introduced using a PARI Boy jet nebulizer. Mice were sacrificed and perfused with saline, after a predetermined period of time, to allow for protein expression from the mRNA.

[0243] Messenger RNA Synthesis. In the example, C-terminal Hisio tagged codon optimized human cystic fibrosis transmembrane conductance regulator (CO-CFTR-C-Hisjo) SNIM RNA and codon-optimized FFL SNIM RNA were synthesized by in vitro transcription from plasmid DNA templates.

Page 75 of 121

WO 2014/153052

PCT/US2014/028849 [0244J PEI Formulation. For the approach, delivery and expression of CO-CFTR-CHisio mRNA in the lungs of CFTR knockout mice was evaluated using both polymeric and lipidbased nanoparticle forluations. Polymeric nanoparticle formulations with 25 kDa branched PEI prepared as follows. The required amount of SNIM RNA was diluted just before application in water for injection (Braun, Melsungen) to a total volume of 4 ml and added quickly to 4 ml of an aqueous solution of branched PEI 25 kDa using a pipette at an N/P ratio of 10. The solution was mixed by pipetting up and down ten times and nebulized as two separate 4.0 ml fractions one after another to the mouse lungs using the indicated nebulizer.

[0245] cKK-E12 Formulation. For the lipid-based nanoparticle experiment, a lipid formulation was created using CO-CFTR-C-HislO SNIM RNA in a formulation of cKKE12:DOPE;Chol:PEGDMG2K (relative amounts 50:25:20:5 (mg:mg:mg:mg). The solution was nebulized to the mouse lungs using the indicated nebulizer.

[0246] Nebulization (Aerosol) Administration of Human CO-CFTR-C-HislO mRNA.

CFTR test materials were administered by a single aerosol inhalation via PARI Boy jet nebulizer (nomin al dose volume of up to 8 mL/group). The test material was delivered to a box containing the whole group of animals (n=4) and connected to oxygen flow and scavenger system.

[0247] Administration of Human CO-CFTR-C-His jo mRNA. CFTR mRNA was prepared in the manner described above. Four CFTR knockout mice were placed in an aerosol chamber box and exposed to 2 mg total codon optimized unmodified human CFTR mRNA (comprising the coding sequence of SEQ ID NO: 3) via nebulization (Pari Boy jet nebulizer) over the course of approximately one hour. Mice were sacrificed 24 hours post-exposure.

[0248] Euthanasia. Animals were euthanized by CO2 asphyxiation at representative times post-dose administration (± 5%) followed by thoracotomy and exsanguinations. Whole blood (maximal obtainable volume) was collected via cardiac puncture and discarded.

[0249] Perfusion. Following exsanguination, all animals underwent cardiac perfusion with saline. In brief, whole body intracardiac perfusion was performed by inserting 23/21 gauge

Page 76 of 121

WO 2014/153052

PCT/US2014/028849 needle attached to 10 mL syringe containing saline set into the lumen of the left ventricle for perfusion. The right atrium was incised to provide a drainage outlet for perfusate. Gentle and steady pressure was applied to the plunger to perfuse the animal after the needle had been positioned in the heart. Adequate flow of the flushing solution was ensured when the exiting perfusate flows clear (free of visible blood) indicating that the flushing solution has saturated the body and the procedure was complete.

[0250] Tissue Collection. Following perfusion, all animals had their lungs (right and left) harvested. Both (right and left) lungs were snap frozen in liquid nitrogen and stored separately at nominally -70°C.

[0251] Expression of human CFTR from CO-CFTR-C-His w mRNA in CFTR knockout mice. CFTR expression was detected by Western blot analysis of tissue lysate collected from CFTR mRNA-treated mouse lungs. Mature “C” band was detected in left and right lungs of all treated mice, for both the lipid-based and polymeric-based formulations (Figure 34). Expression of the mature “C” band was verified by comparison with lysate collected from HEK 293T human CO-CFTR-C-Hisio positive cells as described in Example 9. In contrast, no detectable signal was observed in lysate collected from wild type untreated control mice (Figure 34). Taken together, these data suggest that both polymeric and lipid based formulations (such as the cKKE12 formulation listed above) are effective for lung delivery of CFTR mRNA, e.g., via inhalation, and that once delivered, the codon optimized CFTR mRNA can effectively express human CFTR protein.

Example 11: In Vivo Dose Escalation Study [0252] Dose Escalation of PEI encapsulated mRNA aerosol delivery to the lungs ofpigs.

Aerosol administration of a combination of firefly luciferase (FFL) SNIM RNA and codon optimized human CFTR (CO-CFTR) SNIM RNA at varying concentrations to pig lungs was established by a stepwise experimental procedure. In a first step the FFL/CO-CFTR SNIM RNA

Page 77 of 121

WO 2014/153052

PCT/US2014/028849 formulation was nebulized to anaesthetized pigs during controlled ventilation. In a second step, the animals were sacrificed by bolus injection of pentobarbital (100 mg/kg of body weight) and potassium chloride via the lateral ear vein after sedation 24 hours after aerosol administration was completed. Lungs were excised and sliced to approximately 1 cm thick tissue specimens. For measurement of luciferase activity, tissue specimens were incubated in a medium bath comprising D-Luciferin substrate and subjected to ex vivo luciferase BLI. After BLI, samples from luciferase-positive and luciferasc-ncgativc regions were taken for histopathology, immunohistochemistry and in situ hybridization. The residual specimens were shock-frozen in liquid nitrogen and subsequently stored at -80°C until analysis by IP/WB and Elisa.

[0253] Messenger RNA Synthesis. In the example, codon optimized human cystic fibrosis transmembrane conductance regulator (CO-CFTR) SNIM RNA, codon-optimized FFL mRNA SNIM RNA were synthesized by in vitro transcription from plasmid DNA templates using standard methods.

[0254] Experimental Design. Pigs of the German Landrace were obtained from Technical

University Munich, Weihenstephan, Germany. The pigs had a body weight ranging from 35-90 kg. The study was designed using both age and weight-matched pigs to control for variability. A single cohort of 6 pigs (3 male and 3 female) was established for each experimental group of the 4-arm study. The first cohort was treated with water for injection (WFI) alone, which was administered using a Aeroneb mesh nebulizer. The second cohort was treated with a solution of 1 mg FFL SNIM RNA and 1 mg of codon optimized human CFTR (CO-CFTR) SNIM RNA in the PEI Formulation described below, using an Aeroneb mesh nebulizer. The third cohort received 1 mg of FFL SNIM RNA and 5 mg of codon optimized human CFTR (CO-CFTR) SNIM RNA in the PEI Formulation described below. The fourth cohort was treated with 1 mg of FFL SNIM RNA and 10 mg of codon optimized human CFTR (CO-CFTR) SNIM RNA in the PEI Formulation described below. The scheme for treatment and evaluation of each group is shown in Table 4 below.

Page 78 of 121

WO 2014/153052

PCT/US2014/028849

Table 4. Experimental Design for Dose Escalation Study

<td> Cohort</td><td> Pigs (No. and Sex)</td><td> Treatment</td><td> Formulation</td>

<td> 1</td><td> 6 (3 male + 3 female)</td><td> N/A</td><td> WFI</td>

<td> 2</td><td> 6 (3 male + 3 female)</td><td> 1 mg FFL + 1 mg CO-CFTR</td><td> Branched 25 kDa PEI + WFI</td>

<td> 3</td><td> 6 (3 male +3 female)</td><td> 1 mg FFL + 5 mg CO-CFTR</td><td> Branched 25 kDa PEI + WFI</td>

<td> 4</td><td> 6 (3 male + 3 female)</td><td> 1 mg FFL + 10 mg CO-CFTR</td><td> Branched 25 kDa PEI + WFI</td>

[0255] mRNA - PEI formulation. An exemplary standardized formulation procedure described below was performed just before treatment of the animals.

Materials:

Syringe pump (Mixing device):

Manufacturer: KD Scientific

Type: KDS-210-CE

Syringe:

Manufacturer: B.Braun

Type: Omni fix, 20mL or 30 mL / Luer Lock Solo

Ref.: 4617207V

Tubing:

Manufacturer: B.Braun

Type: Safeflow Extension Set

Ref.: 4097154

Needle:

Manufacturer: B.Braun

Type: Sterican, 20G x 1 Vi ”

Ref.: . 4657519

Mixing valve:

Manufacturer: B.Braun

Type: Discofix C 3SC

Ref.: 16494C

Water for injection:

Manufacturer: B.Braun

Page 79 of 121

WO 2014/153052

PCT/US2014/028849

Type: Aqua

Ref.: 82423E [0256] Examplary method for the preparation of polyplexes containing lmg hCFTR

SNIM RNA and 1 mg FFL SNIM RNA N/P 10 in a volume of 8mL: 3mL water for injection and 3mL RNA stock solution (c: lmg/mL in water; 1,5mL FFL mRNA + 1,5mL CFTR mRNA) were filled into a 15mL falcon tube. In a second falcon tube 5.61mL water for injection were mixed with 0.39mL brPEI stock solution (c: 1 Omg/mL in water). Two 20mL syringes were fixed in the mixing device. Each of them was connected to a needle via a tubing. One syringe was filled with the RNA- and the other with the PEl-solution using the withdrawal function of the syringe pump. (Settings: Diameter: 20.1mm, Flow: 5mL/min, Volume: 5.9mL). The needles were removed and the tubes connected to the mixing valve. It was important to connect the syringe containing the RNA-solution to the angled position of the valve. To control the outlet diameter, a needle was connected. The mixing was performed using the infusion function of the syringe pump (Settings: Diameter: 20.1mm, Flow: 40mL/min, Volume: 5.8mL). To achieve a reproducible polydispersity index, the samples were fractionated manually during mixing. The first few μΤ until the flow was stable (100-200pL) and the last few μΕ sometimes containing air bubbles were collected, in a separate tube. The mixture was incubated for 30min at room temperature for polyplex formation and afterwards stored on ice. For different doses, the parameters were modified and adapted as shown in Table 5.

Page 80 of 121

WO 2014/153052

PCT/US2014/028849

Table 5: Exemplary volumes and settings for different mixing volumes

<td></td><td> mR</td><td colspan="2"> NA component</td><td></td><td colspan="2"> ’El component</td>

<td> Cohort</td><td> V (FFL SNIM RNA 1 mg/mL) (mL)</td><td> V (hCFTR SNIM RNA lmg/mL) (mL)</td><td> Water (mL)</td><td> V (brPEI stock; 10 mg/mL) (mL)</td><td> Water (mL)</td><td> Aerosolized volume (ml)</td>

<td> Cohort</td><td> V(withdrawal) (ml)</td><td> V (infusion) (ml)</td>

V(withdrawal) and V(infusion) designate the setting on the syringe pump for aspiration and dispension, respectively, of the mRNA and PEI components.

[0262] CFTR Expression Analysis by In Situ Hybridization (ISH). FFL positive tissues samples were excised (minimum of 10 samples for each pig within a cohort) and subjected to manual in situ hybridization analysis using the RNAscope® (Advanced Cell Diagnostic) “ZZ” probe technology. Probes were generated based on the codon-optimized sequence of codon optimized human CFTR SNIM RNA (SEQ ID NO: 17). Briefly, the RNAscope® assay is an in situ hybridication assay designed to visualize single RNA molecules per cell in formalin-fixed, paraffin-embedded (FFPE) tissue mounted on slides. Each embedded tissue sample was pretreated according to the manufacturers protocol and incubated with a target specific human CFTR specific RNA probe. The hCFTR probe was shown bind CFTR, with cross reactivity to human, mouse, rat, pig and monkey. Once bound, the probe is hybridized to a cascade of signal amplification molecules, through a series of 6 consecutive rounds of amplification. The sample was then treated with an HRP-labeled probe specific to the signal amplification cassette and Page 84 of 121

WO 2014/153052

PCT/US2014/028849 assayed by chromatic visualization using 3,3’-diaminobenzidine (DAB). A probe specific for Ubiquitin C was used as the positive control (Figures 47A and 48A), while dapD was used as the negative control (Figures 47B and 4SB). Positive CFTR signal was compared to that of untreated and vehicle control treated porcine lung tissue (Figure 49 A and B). Stained samples were visualized under a standard bright field microscope. The data demonstrates that treatment with 1 mg of codon optimized human CFTR SNIM RNA resulted in a dramatic increase in CFTR expression in both the right (A) and left (B) lung tissue of corhort 2, when compared to vehicle control (Figure 49 and 50 A &B) Furthermore, a further increase in CFTR expression was observed for the 5 mg and 10 mg treatment groups, as demonstrated by a dramatic increase staining observed within the right (A) and left (B) lung samples analyzed for cohorts 3 and 4 (Figures 51 and 52 A&B). Taken together, these data strongly supports the effective delivery of mRNA via inhalation and expression of human CFTR within both lobes of the lung and their various tissues.

[0263] Conclusion. The results demonstrated that both luciferase and CFTR mRNA can be effectively delivered in vivo to lung tissues. Luciferase expression was observed throughout various tissue samples collected from different regions within both the right and left lobs of the lungs. Thus suggestions, that nebulization is an effective approach for administering mRNA and results in fairly uniform distribution. Furthermore, in addition to luciferase, CFTR mRNA was also efficiently delivered to the lungs, resulting in enhanced protein expression. Expression and protein activity was verified by IP-WB, immunohistochemistry and in situ hybridization. Each approach clearly demonstrated a dose dependent increase in mRNA delivery and CFTR expression and/or activity, within the tissues of the lung. Taken together, the experiments highlight the overall practicality and feasibility for delivering CFTR mRNA to the lung of a human subject and demonstrate the effectiveness of in vivo CFTR protein production for therapeutic use.

Example 12: in Vivo Expression in the Lung

Page 85 of 121

WO 2014/153052

PCT/US2014/0 28849 [0264] This example desmonstrates successful in vivo expression, in the lung following aerosol delivery of mRNA-loaded nanoparticles. All studies were performed using pigs of the German Landrace, obtained from Technical University Munich, Weihenstephan, Germany. The pigs had a body weight ranging from 35-90 kg. FFL/CO-CFTR-C-HislO mRNA formulation or vehicle control was introduced using a Pari jet nebulizer. Pigs were sacrificed and perfused with saline, after a predetermined period of time, to allow for protein expression from the mRNA.

[0265] Messenger RNA Synthesis. In the example, codon optimized fire fly luciferase (CO-FFL) mRNA was synthesized by in vitro transcription from plasmid DNA templates.

[0266] cKK-E12 Formulation. For the lipid-based nanoparticle experiment, a lipid formulation was created using 1 mg FFL + 9 mg of CO-CFTR-C-HislO mRNA encapsulated in a formulation of cKK-E12:DOPE:Chol:PEGDMG2K (relative amounts 40:30:25:5 (mol ratio). The solution was nebulized to the Pig lungs using the indicated nebulizer.

[0267] Aerosol Application. The aerosol (Saline or CO-FFL cKK-E12 formulation) was nebulized and inhaled into the anaesthetized pig. Sedation in pigs was initiated by premedication with azaperone 2 mg/kg body weight, ketamine 15 mg/kg body weight, atropine 0.1 mg/kg body weight and followed by insertion of an intravenous line to the lateral auricular vein. Pigs were anesthetized by intravenous injection of propofol 3-5 mg/kg body weight as required. Anesthesia was maintained by isoflurane (2-3%) with 1% propofol bolus injection at 4 to 8 mg/kg body weight to enhance anesthesia as required. Duration of the anesthesia was approximately 1-3 hrs. Pigs were killed with bolus injection of pentobarbital (100 mg/kg body weight) and potassium chloride via the lateral ear vein. Lungs were excised and tissue specimens were collected from various lung regions followed by incubation in cell culture medium overnight. The stored samples were subjected to bioluminescence detection.

[0268] Bioluminescence Analysis. For measurement of luciferase activity tissue specimens were either homogenized and analyzed in a tube luminometer or incubated in a medium bath comprising D-Luciferin substrate and subjected to ex vivo luciferase BLI. A strong bioluminescence signal was observed for each of the (A) FFL/CO-CFTR-C-HislO mRNA

Page 86 of 121

WO 2014/153052

PCI7US2014/028849 treated pigs, when compared to (B) control lung tissue samples from control pigs (Saline vehicle control) (Figure 53 A&B).

[0269] These data illustrate that FFL/CFTR mRNA were successfully delivered to and expressed in the lung by aerosol administration.

BRIEF DESCRIPTION OF SEQUENCES

SEQ ID NO 1. Wild-type CFTR amino acid sequence.

SEQ ID NO 2. Wild-type CFTR mRNA coding sequence.

SEQ ID NO 3. Non-naturally occurring CFTR mRNA coding sequence #1.

SEQ ID NO 4. CFTR mRNA 5’-UTR.

SEQ ID NO 5. CFTR mRNA 3’-UTR #1.

SEQ ID NO 6. FFL 5’ UTR.

SEQ ID NO 7. FFL coding sequence.

SEQ ID NO 8. FFL 3’UTR.

SEQ ID NO 9. Non-naturally occurring CFTR mRNA coding sequence 42.

SEQ ID NO 10. Non-naturally occurring CFTR mRNA coding sequence #3.

SEQ ID NO 11. Non-naturally occurring CFTR mRNA coding sequence #4.

SEQ ID NO 12. Non-naturally occurring CFTR mRNA coding sequence #5.

SEQ ID NO 13. Non-naturally occurring CFTR mRNA coding sequence #6.

SEQ ID NO 14. Non-naturally occurring CFTR mRNA coding sequence #7.

SEQ ID NO 15. Codon Optimized Human CFTR C-terminal Hisio fusion mRNA coding sequence.

Page 87 of 121

RECTIFIED SHEET (RULE 91) ISA/EP

WO 2014/153052

PCT/US2014/028849

SEQ ID NO 16, Codon Optimized Human CFTR mRNA coding sequence with a Growth Hormone Leader Sequence.

SEQ ID NO 17. Codon Optimized Human CFTR mRNA

SEQ ID NO 18. mRNA Leader Sequence #1

SEQ ID NO 19. mRNA Leader Sequence #2

SEQ ID NO 20. CFTR mRNA 3’-UTR #2.

SEQ ID NO: 1 mqrsplekasvvsklffswtrpilrkgyrqrlelsdiyqipsvd sadnlseklerewdrelaskknpklinalrrcffwrfmfygiflylgevtkavqplll griiasydpdnkeersiaiylgiglcllfivrtlllhpaifglhhigmqmrtamfsli ykktlklssrvldkisigqlvsllsnnlnkfdeglalahfvwiaplqvallmgliwel LQASAFCGLGFLrVLALFQAGLGRMMMKYRDQRAGKISERLVITSEMIENIQSVKAYC WEEAMEKMIENLRQTELKLTRKAAYVRYFNSSAFFFSGFFVVFLSVLPYALIKGIILR KTFTTISFCIVLRMAVTRQFPWAVQTWYDSLGAINKIQDFLQKQEYKTLEYNLTTTEV VMENVTAFWEEGFGELFEKAKQNNNNRKTSNGDDSLFFSNFSLLGTPVLKDINFKIER GQLLAVAGSTGAGKTSLLMVIMGELEPSEGKIKHSGRISFCSQFSWIMPGTIKENnF GVSYDEYRYRSVIKACQLEED1SKFAEKDNIVLGEGGITLSGGQRARISLARAVYKJDA DLYLLDSPFGYLDVLTEKEIFESCVCKLMANKTRILVTSKMEHLKKADKILILHEGSS YFYGTFSELQNLQPDFSSKLMGCDSFDQFSAERRNS1LTETLHRESLEGDAPVSWTET KKQSFKQTGEFGEKRKNSILNPINSIRKFSIVQKTPLQMNGIEEDSDEPLERRLSLVP DSEQGEAILPRISVISTGPTLQARRRQSVLNLMTHSVNQGQNIHRKTTASTRKVSLAP QANLTELDIYSRRLSQETGLE1SEEINEEDLKECFFDDMESIPA VTTWNTYLRYIT VH KSLIFVL1WCLVIFLAEVAASLVVLWLLGNTPLQDKGNSTHSRNNSYAVHTSTSSYY VFYIYVGVADTLLAMGFFRGLPLVHTLnVSKILHHKMLHSVLQAPMSTLNTLKAGGI LNRFSKDIAILDDLLPLTIFDF1QLLL1VIGAIAVVAVLQPYIFVATVPVIVAFIMLR AYFLQTSQQLKQLESEGRSPrFTHLVTSLKGLWTLRAFGRQPYFETLFtLKALNLHTAN WFLYLSTLRWFQMRIEMIFVIFFlAVTFISILTTG'EGEGRVGIlLTLAMNIMSTLQWA . VNSSIDVDSLMRSVSRVFKFIDMPTEGKPTKSTKPYKNGQLSKVMIIENSHVKKDDIW PSGGQMTVKDLTAKYTEGGNAILENISFSISPGQRVGLLGRTGSG.KSTLLSAFLRLLN TEGEIQIDGVSWDSITLQQW.RKAFGVIPQKVFIFSGTFRKNLDPYEQWSDQEIWK.VAD EVGLRSVIEQFPGKLDFVLVDGGCVLSHGHKQLMCLARSVLSKAKILLLDEPSAHLDP VTYQHRRTLKQAFADCTV1LCEHRIEAMLECQQFLVIEENKVRQYDSIQKLLNERSL FRQAISPSDRVKLFPHRNSSKCKSKPQ1AALKEETEEEVQDTRL (SEQ IDNO:1)

SEQ ID NO: 2

Page 88 of 121

RECTIFIED SHEET (RULE 91) ISA/EP

WO 2014/153052

PCT/US2014/028849

AUGCAGAGGUCGCCUCUGGAAAAGGCCAGCGUUGUCUCCAAACUUUUUUUCAGCUGGACC AGACCAAUUUUGAGGAAAGGAUACAGACAGCGCCUGGAAUUGUCAGACAUAUACCAAAU CCCUUCUGUUGAUUCUGCUGACAAUCUAUCUGAAAAAUUGGAAAGAGAAUGGGAUAGAG AGCUGGCUUCAAAGAAAAAUCCUAAACUCAUUAAUGCCCUUCGGCGAUGUUUUULICUGG AGAUUUAUGUUCUAUGGAAUCLUUUUAUAUUUAGGGGAAGUCACCAAAGCAGUACAGCC UCUCUUACUGGGAAGAAUCAUAGCUUCCUAUGACCCGGAUAACAAGGAGGAACGCUCUA UCGCGAUUUAUCUAGGCAUAGGCUUAUGCCUUCUCUUUAUUGUGAGGACACUGCUCCUAC ACCCAGCCAULTUTJUGGCCUUCAUCACAUUGGAAUGCAGAUGAGAAUAGCUAUGUUUAGU UUGAUUUAUAAGAAGACUUUAAAGCUGUCAAGCCGUGUUCUAGAUAAAAUAAGUAUUGG ACAACUUGUUAGUCUCCUUUCCAACAACCUGAACAAAUUUGAUGAAGGACUUGCAUUGG CACAUUUCGUGUGGAUCGCUCCUUUGCAAGUGGCACUCCUCAUGGGGCUAAUCUGGGAGU UGUUACAGGCGUCUGCCUUCUGUGGACUUGGUUUCCUGAUAGUCCUUGCCCUUUULICAGG CUGGGCUAGGGAGAAUGAUGAUGAAGUACAGAGAUCAGAGAGCUGGGAAGAUCAGUGAA AGACUUGUGAUUACCUCAGAAAUGAUUGAAAAUAUCCAAUCUGUUAAGGCAUACUGCUG GGAAGAAGCAAUGGAAAAAAUGAUUGAAAACUUAAGACAAACAGAACUGAAACUGACUC GGAAGGCAGCCUAUGUGAGAUACUUCAAUAGCUCAGCCUUCUUCUUCUCAGGGUUCUUU GUGGGGUUUUUAUCUGUGCUUCCCUAUGCACUAAUCAAAGGAAUCAUCCUCCGGAAAAU AULTCACCACCAUCUCAUUCUGCAUUGUUCUGCGCAUGGCGGUCACUCGGCAAUUUCCCUG GGCUGUACAAACAUGGUAUGACUCUCUUGGAGCAAUAAACAAAAUACAGGAUUUCUUAC AAAAGCAAGAAUAUAAGACAUUGGAAUAUAACUUAACGACUACAGAAGUAGUGAUGGAG AAUGUAACAGCCUUCUGGGAGGAGGGAUUUGGGGAAUUAUUUGAGAAAGCAAAACAAAA CAAUAACAAUAGAAAAACUUCUAAUGGUGAUGACAGCCUCLUCUUCAGUAAUUUCUCAC uucuugguacuccuguccugaaagauauuaauuucaagauagaaagaggacaguuguug GCGGUUGCUGGAUCCACUGGAGCAGGCAAGACUUCACUUCUAAUGAUGAUUAUGGGAGA acuggagccuucagaggguaaaauuaagcacaguggaagaauuucauucuguucucagu UUUCCUGGAUUAUGCCUGGCACCAUUAAAGAAAAUAUCAUCUUUGGUGLfUUCCUAUGAU GAAUAUAGAUACAGAAGCGUCAUCAAAGCAUGCCAACUAGAAGAGGACAUCUCCAAGUU UGCAGAGAAAGACAAUAUAGLJUCUUGGAGAAGGUGGAAUCACACUGAGUGGAGGUCAAC GAGCAAGAAUUUCUUUAGCAAGAGCAGUAUACAAAGAUGCUGAUUUGUAUUUAUUAGAC UCUCCUUUUGGAUACCUAGAUGUUUUAACAGAAAAAGAAAUAUUUGAAAGCUGUGUCUG U AAACUGAUGGCUAAC A A AACUAG GAUUUUGG UCACUUCUAAAAUGGAACAUUUA AAGA AAGCUGACAAAAUAUUAAUUUUGAAUGAAGGUAGCAGCUAUUUUUAUGGGACAUUUUCA GAACUCCAAAAUCUACAGCCAGACUUUAGCUCAAAACUCAUGGGAUGUGAIJUCUUUCGAC CAAUUUAGUGCAGAAAGAAGAAAUUCAAUCCUAACUGAGACCUUACACCGUUUCUCAUD AGAAGGAGAUGCUCCUGUCUCCUGGACAGAAACAAAAAAACAAUCUUUUAAACAGACUG gagaguuuggggaaaaaaggaagaauucuauucucaauccaaucaacucuauacgaaaa UUUUCCAUUGUGCAAAAGACUCCCUUACAAAUGAAUGGCAUCGAAGAGGAUUCUGAUGA GCCUUUAGAGAGAAGGCUGUCCUUAGUACCAGAUUCUGAGCAGGGAGAGGCGAUACUGC CUCGCAGCAGCGUGAUCAGCACUGGCCCCACGCUUCAGGCACGAAGGAGGCAGUCUGUCC UGAACCUGAUGACACACUCAGUUAACCAAGGUCAGAACAUUCACCGAAAGACAACAGCAU CCACACGAAAAGUGUCACUGGCCCCUCAGGCAAACUUGACUGAACUGGAUAUAGAUUCAA GAAGGUUAUCUCAAGAAACUGGCUUGGAAAUAAGUGAAGAAAUUAACGAAGAAGACUUA AAGGAGUGCCUUDUUGAUGAUAUGGAGAGCAUACCAGCAGUGACUACAUGGAACACAUA CCUUCGAUAUAUUACUGUCCACAAGAGCUUAAUUGUUGUGCUAAUUUGGUGCUUAGUAA UUUUUCUGGCAGAGGUGGCUGCUUCUUUGGUUGUGCUGUGGCUCCUUGGAAACACUCCU

Page 89 of 121

RECTIFIED SHEET (RULE 91) ISA/EP

WO 2014/153052

PCT/US2014/028849

CUUCAAGACAAAGGGAAUAGUACUCAUAGUAGAAAUAACAGCUAUGCAGUGAUUAUCAC CAGCACCAGUUCGUAUUAUGUGUUUUACAUUUACGUGGGAGUAGCCGACACUUUGCUUG CUAUGGGAUUCUUCAGAGGUCUACCACUGGUGCAUACUCUAAUCACAGUGUCGAAAAUU UUACACCACAAAAUGUUACAUUCUGUGCUUCAAGCACCUAUGUCAACCCUCAACACGUUG AAAGCAGGUGGGAUUCUUAAUAGAUUCUCCAAAGAUAUAGCAAUUUIJGGAUGACCUUCU GCCUCUUACCAUAUUUGACUUCAUCCAGUUGUUAUUAAUUGUGAUGGGAGCUAUAGCAG UUGUCGCAGUUUUACAACCCUACAUCUUUGUUGCAACAGUGCCAGUGAUAGUGGCUUUU AHUAUGUUGAGAGCAUAUUUCCUCCAAACCUCACAGCAACUCAAACAACUGGAAUCUGAA GGCAGGAGUCCAAUUUUCACUCAUCUUGUUACAAGCUUAAAAGGACUAUGGACACUUCG UGCCUUCGGACGGCAGCCUUACUUUGAAACUCUGUUCCACAAAGCGCUGAAUUUACAUAC UGCCAACUGGUUCUUGUACCUGUCAACACUGCGCUGGUUCCAAAUGAGAAUAGAAAUGA UUUUUGUCAUCUUCUUCAUUGCUGUUACCUUCAUUUCCAUUUUAACAACAGGAGAAGGA GAAGGAAGAGUUGGUAUUAUCCUGACUUUAGCCAUGAAUAUCAUGAGUACAUUGCAGUG GGCUGUAAACUCCAGCAUAGAUGUGGAUAGCUUGAUGCGAUCUGUGAGCCGAGUCUUUA AGUUCAUUGACAUGCCAACAGAAGGUAAACCUACCAAGUCAACCAAACCAUACAAGAAUG GCCAACUCUCGAAAGUUAUGAUUAUUGAGAAUUCACACGUGAAGAAAGAUGACAUCUGG CCCUCAGGGGGCCAAAUGACUGUCAAAGAUCUCACAGCAAAAUACACAGAAGGUGGAAA UGCCAUAUUAGAGAACAUUUCCUUCUCAAUAAGUCCUGGCCAGAGGGUGGGCCUCUUGG GAAGAACUGGAUCAGGGAAGAGUACUUUGUUAUCAGCUUUUUUGAGACUACUGAACACU GAAGGAGAAAUCCAGAUCGAUGGUGUGUCUUGGGAUUCAAUAACUUUGCAACAGUGGAG GAAAGCCUUUGGAGUGAUACCACAGAAAGUAUUUAUUUUUUCUGGAACAUUUAGAAAAA ACUUGGAUCCCUAGGAACAGGGGAGUGAUCAAGAAAUAUGGAAAGUUGCAGAUGAGGUU gggcucagaucugugauagaacaguuuccugggaagcuugacuuuguccuuguggaugg GGGCUGUGGCCUAAGCCAUGGCCACAAGCAGUUGAUGUGCUUGGCUAGAUCUGUUCUCA GUAAGGCGAAGAUCUUGCUGCUUGAUGAACCCAGUGCUCAUUUGGAUCCAGUAACAUAC CAAAUAAUUAGAAGAACUCUAAAACAAGCAUUUGCUGAUUGCACAGUAAUUCUCUGUGA ACACAGGAUAGAAGCAAUGCUGGAAUGCCAACAAUUUUUGGUCAUAGAAGAGAACAAAG UGCGGCAGUACGAUUCCAUCCAGAAACUGCUGAACGAGAGGAGCCUCUUCCGGCAAGCCA UCAGCCCCUCCGACAGGGUGAAGCUCUUUCCCCACCGGAACUCAAGCAAGUGCAAGUCUA AGCCCCAGAUUGCUGCUCUGAAAGAGGAGACAGAAGAAGAGGUGCAAGAUACAAGGCUU UAG (SEQ ID NO:2)

SEQ ID NO:3

AUGCAGCGGUCCCCGCUCGAAAAGGCCAGUGUCGUGUCCAAACUCUUCUUCUCAUGGACU CGGCCUAUCCUUAGAAAGGGGUAUCGGCAGAGGCUUGAGUUGUCGGACALrCUACCAGAU CCCCUCGGUAGAUUCGGCGGAUAACCUCUCGGAGAAGCUCGAACGGGAAUGGGACCGCGA ACUCGCGUCUAAGAAAAACCCGAAGCUCAUCAACGCACUGAGAAGGUGCULICUUCUGGCG GUUCAUGUUCUACGGUAUCUUCUUGUAUCUCGGGGAGGUCACAAAAGCAGUCCAACCCCU GUUGUUGGGUCGCAUUAUCGCCUCGUACGACCCCGAUAACAAAGAAGAACGGAGCAUCGC GAUCUACCUCGGGAUCGGACLFGUGUUUGCUUOUCAUCGUCAGAACACUUUUGUUGCAUCC AGCAAUCUUCGGCCUCCAUCACAUCGGUAUGCAGAUGCGAAUCGCUAUGUUUAGCUUGAU CUACAAAAAGACACUGAAACUCUCGUCGCGGGUGUUGGAUAAGAUUUCCAUCGGUCAGU UGGUGUCCCUGCUUAGUAAUAACCUCAACAAAUUCGAUGAGGGACUGGCGCUGGCACAU UUCGUGUGGAUUGCCCCGUUGCAAGUCGCCCUUUUGAUGGGCCUUAGGUGGGAGCUGUU

Page 90 of 121

RECTIFIED SHEET (RULE 91) ISA/EP

WO 2014/153052

PCT/US2014/028849

GCAGGCAUCUGCCUUUUGUGGCCUGGGAUUUCUGAUUGUGUUGGCAUUGUUUCAGGCUG GGCUUGGGCGGAUGAUGAUGAAGUAUCGCGACCAGAGAGCGGGUAAAAUCUCGGAAAGA CUCGUCAUCACUUCGGAAAUGAUCGAAAACAUCCAGUCGGUCAAAGCCUAUUGCUGGGAA GAAGCUAUGGAGAAGAUGAUUGAAAACCUCCGCCAAACUGAGCUGAAACUGACCCGCAA GGCGGCGUAUGUCCGGUAUUUCAAUUCGUCAGCGUUCUUCUUUUCCGGGUUCUUCGUUG UCUUUCUCUCGGUUUUGCCUUAUGCCUUGAUUAAGGGGAUUAUCCUCCGCAAGAUUUUC ACCACGAU U UCGUUCUGCAUUGUAUUGCGCAUGGCAGUGACACGGCAAUUUCCGUGGGCC GUGCAGACAU GGUAUGACUCGCUUGGAGCGAUC AACAAA AUCC A AGACUUCUUGCAAAA GCAAGAGUACAAGACCCUGGAGUACAAUCUUACUACUACGGAGGUAGUAAUGGAGAAUG UGACGGCUUUUUGGGAAGAGGGUUUUGGAGAACUGUUUGAGAAAGCAAAGCAGAAUAAC AACAACCGCAAGACCUCAAAUGGGGACGAUUCCCUGUUUUUCUCGAACUUCUCCCUGCUC GGAACACCCGUGUUGAAGGACAUCAAUUUCAAGAUUGAGAGGGGACAGCUUCUCGCGGU AGCGGGAAGCACUGGUGCGGGAAAAACUAGCCUCUUGAUGGUGAUUAUGGGGGAGCIJUG AGCCCAGCGAGGGGAAGAUUAAACACUCCGGGCGUAUCUCAUUCUGUAGCCAGUUUUCAU GGAUCAUGCCCGGAACCAUUAAAGAGAACAUCAUUUUCGGAGUAUCCUAUGAUGAGUAC CGAUACAGAUCGGUCAUUAAGGCGUGCCAGUUGGAAGAGGACAULTUCUAAGUUCGCCGA GAAGGAUAACAUCGUCUUGGGAGAAGGGGGUAUUACAUUGUCGGGAGGGCAGCGAGCGC GGAUCAGCCUCGCGAGAGCGGUAUACAAAGAUGCAGAUUUGUAUCUGCUUGAUUCACCG UUUGGAUACCUCGACGUAUUGACAGAAAAAGAAAUCUUCGAGUCGUGCGUGUGUAAACU UAUGGCCJAAUAAGACGAGAAUCCUGGUGACAUCAAAAAUGGAACACCUUAAGAAGGCGG ACAAGAUCCUGAUCCUCCACGAAGGAUCGUCCUACUUUUACGGCACUUUCUCAGAGUUGC AAAACUUGCAGCCGGACUUCUCAAGCAAACUCAUGGGGUGUGACUCAUUCGACCAGUUCA GCGCGGAACGGCGGAACUCGAUCUUGACGGAAACGCUGCACCGAUUCUCGCUUGAGGGUG AUGCCCCGGUAUCGUGGACCGAGACAAAGAAGCAGGCGUUUAAGCAGACAGGAGAAUUU GGUGAGAAAAGAAAGAACAGUAGCUUGAAUCCUAUUAACUCAAUUCGCAAGUUCUCAAU CGUCCAGAAAACUCCACUGCAGAUGAAUGGAAUUGAAGAGGAUUCGGACGAACCCCUGG AGCGCAGGCUUAGCCUCGUGCCGGAUUCAGAGCAAGGGGAGGCCAUUCUUCCCCGGAUUU CGGUGAUUUCAACCGGACCUACACUUCAGGCGAGGCGAAGGCAAUCCGUGCUCAACCUGA UGACGCAUUCGGUAAACCAGGGGCAAAACAUUCACCGCAAAACGACGGCCUCAACGAGAA AAGUGUCACUUGCACCCCAGGCGAAUUUGACUGAACUCGACAUCUACAGCCGUAGGCUUU CGCAAGAAACCGGACUUGAGAUCAGCGAAGAAAUCAAUGAAGAAGAUUUGAAAGAGUGU WCUUUGAUGACAUGGAAUCAAUCCCAGCGGUGACAACGUGGAACACAUACUUGCGUUA CAUCACGGGGCACAAGUCCUUGAUUUDCGUCCUCAUCUGGUGUCUCGUGAUCUUDCUCGC UGAGGUCGCAGCGUCACUUGUGGUCCUCUGGCUGCUUGGUAAUACGCCCUUGCAAGACAA AGGCAAUUCUACACACUCAAGAAACAAUUCCUAUGCCGUGAUUAUCACUUCUACAAGCUC GUAUUACGUGUUUUACAUCUACGUAGGAGUGGCCGACACUCUGCUCGCGAUGGGUUUCU UCCGAGGACUCCCACUCGUUCACACGCUUAUCACUGUCUCCAAGAUUCUCCACCAUAAGA UGCUUCAUAGCGUACUGCAGGCUCCCAUGUCCACCUUGAAUACGCUCAAGGCGGGAGGUA UUUUGAAUCGCUUCUCAAAAGAUAUUGCAAUUUUGGAUGACCUUCUGCCCCUGACGAUC UUCGACUUCAUCCAGUUGUUGCUGAUCGUGAUUGGGGCUAGUGCAGUAGUCGCUGUCCU CCAGCCGUACAUUUiJUGUCGCGACCGUUCCGGUGAUCGUGGCGUUUAUCAUGCUGCGGGC CUAUUUCUUGCAGACGUCACAGCAGCUUAAGCAACUGGAGUCUGAAGGGAGGUCGCCUA UCUUUACGCAUCUUGUGACCAGUUUGAAGGGAUUGUGGACGUUGCGCGCCUUUGGCAGG CAGCCCUACUUUGAAACACUGUUCCACAAAGCGCUGAAUCUCCAUACGGCAAAUUGGUUU (JUGUAUUGGAGUACCCUCCGAUGGUUUCAGAUGCGCAUUGAGAUGAUUUUUGUGAUCUU

Page 91 of 121

RECTIFIED SHEET (RULE 91) ISA/EP

WO 2014/153052

PCT/ES2014/028849

CUUUAUCGCGGUGACUUUUAUCUCCAUCUUGACCACGGGAGAGGGCGAGGGACGGGUCG G U AUU AUCCUG AC ACU CGCC AUG A ACAUUAUG AGC ACUUUGC AGUGGGC AGUG A A C AG C UCGAUUGAUGUGGAUAGCCUGAUGAGGUCCGUUUCGAGGGUCUUUAAGUUCAUCGACAU GCCGACGGAGGGAAAGCCCACAAAAAGUACGAAACCCUAUAAGAAUGGGCAAUUGAGUA AGGCJAAUGAUCAUCGAGAACAGUCACGUGAAGAAGGAUGACAUCUGGCCUAGCGGGGGU CAGAUGACCGUGAAGGACCUGACGGCAAAAUACACCGAGGGAGGGAACGCAAUCCUUGA AAACAUCUCGUUCAGCAUUAGCCCCGGUCAGCGUGUGGGGUUGCUCGGGAGGACCGGGUC AGGAAAAUCGACGUUGCUGUCGGCCUUCUUGAGACUUCUGAAUACAGAGGGUGAGAUCC AGAUCGACGGCGUUUCGUGGGAUAGCAUCACCUUGCAGCAGUGGCGGAAAGCGUUUGGA GUAAUCCCCCAAAAGGUCUUUAUCUUUAGCGGAACCUUCCGAAAGAAUCUCGAUCCUGAU GAACAGUGGUCAGAUCAAGAGAUUUGGAAAGUCGCGGACGAGGUUGGCCUUCGGAGUGU AAUCGAGCAGUUUCCGGGAAAACUCGACUUUGUCCUUGUAGAUGGGGGAUGCGUCCUGU CGCAUGGGCACAAGCAGCUCAUGUGCCUGGCGCGAUCCGUCCUCUCUAAAGCGAAAAUUC UGCUCUUGGACJGAACCUUCGGCCCAUCUGGACCCGGUAACGUAUCAGAUCAUCAGAAGGA CACUUAAGCAGGCGUUUGCCGACUGCACGGUGAUUCUCUGUGAGCAUCGUAUCGAGGCCA UGCGCGAAUGCCAGCAAUUUCUUGUCAUCGAAGAGAAUAAGGUCCGCCAGUACGACUCCA UCCAGAAGCUGCUUAAUGAGAGAUCAUUGUUCCGGCAGGCGAUUUCACCAUCCGAUAGG GUGAAACUUUUUCCACACAGAAAUUCGUCGAAGUGCAAGUCCAAACCGCAGAUCGCGGCC UUGAAAGAAGAGACUGAAGAAGAAGUUCAAGACACGCGUCUUUAA (SEQ ID NOG)

SEQ ED NO: 4

GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGAC CGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGU GACUCACCGUCCUUGACACG (SEQ ID NO:4)

SEQ ID NO: 5

CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCDCCUGGCCCUGGAAGUUGCCACUCC

AGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC (SEQ ID NOG)

SEQ ID NO: 6

GGGAUCCUACC (SEQ ID NO:6)

SEQ ID NO: 7

AUGGAAGAUGCCAAAAACAUUAAGAAGGGCCCAGCGCCAUUCUACCCACUCGAAGACGGG ACCGCCGGCGAGCAGCUGCACAAAGCCAUGAAGCGCUACGCCCUGGUGCCCGGCACCAUC

GCCUUUACCGACGCACAUAUCGAGGUGGACAUUACCUACGCCGAGUACUUCGAGAUGAGC GUUCGGCUGGCAGAAGCUAUGAAGCGCUAUGGGCUGAAUACAAACCAUCGGAUCGUGGU GUGCAGCGAGAAUAGCUUGCAGUUCUUCAUGCCCGUGUUGGGUGCCCUGUUCAUCGGUG

UGGCUGUGGCCCCAGCUAACGACAUCUACAACGAGCGCGAGCUGCUGAACAGCAUGGGCA

UCAGCCAGCCCACCGUCGUAUUCGUGAGCAAGAAAGGGCUGCAAAAGAUCCUCAACGUGC

AAAAGAAGCUACCGAUCAUACAAAAGAUCAUCAUCAUGGAUAGCAAGACCGACUACCAG

Page 92 of 121

RECTIFIED SHEET (RULE 91) ISA/EP

WO 2014/153052

PCT/US2014/028849

GGCUUCCAAAGCAUGUACACCUUCGUGACUUCCCAUUUGCCACCCGGCUUCAACGAGUAC GACUUCGUGCCCGAGAGCUUCGACCGGGACAAAACCAUCGCCCUGAUCAUGAACAGUAGU GGCAGUACCGGAUUGCCCAAGGGCGUAGCCCUACCGCACCGCACCGCUUGUGUCCGAUUC AGUCAUGCCCGCGACCCCAUCUUCGGCAACCAGAUCAUCCCCGACACCGCUAUCCUCAGC GUGGUGCCAtrUUCACCACGGCUUCGGCAUGUUCACCACGCUGGGCUACUUGAUCUGCGGC UUUCGGGUCGUGCUCAUGUACCGCUUCGAGGAGGAGCUAUUCUUGCGCAGCUUGCAAGAC UAUAAGAUGCAAUCUGCCCUGCUGGUGCCCACACUAUUUAGCUUCUUCGCUAAGAGCACU CUCAUCGACAAGUACGACCUAAGCAACUUGCACGAGAUCGCCAGCGGCGGGGCGCCGCUC AGCAAGGAGGUAGGUGAGGCCGUGGCCAAACGCUUCCACCUACCAGGCAUCCGCCAGGGC UACGGCCUGACAGAAACAACCAGCGCCAUUCUGAUCACCCCCGAAGGOGACGACAAGCCU GGCGCAGDAGGCAAGGUGGDGCCCUUCUUCGAGGCUAAGGUGGUGGACUUGGACACCGG UAAGACACUGGGUGUGAACCAGCGCGGCGAGCUGUGCGUCCGUGGCCCCAUGAUCAUGAG CGGCUACGUUAACAACCCCGAGGCUACAAACGCUCUCAUCGACAAGGACGGCUGGCUGCA CAGCGGCGACAUCGCCUACUGGGACGAGGACGAGCACLiUCUUCAUCGUGGACCGGCUGAA GAGCCUGAUCAAAUACAAGGGCUACCAGGUAGCCCCAGCCGAACUGGAGAGCAUCCUGCU GCAACACCCCAACAUCUUCGACGCCGGGGUCGCCGGCCUGCCCGACGACGAUGCCGGCGA GCUGCCCGCCGCAGUCGUCGUGCUGGAACACGGUAAAACCAUGACCGAGAAGGAGAUCGU GGACUAUGUGGCCAGCCAGGUUACAACCGCCAAGAAGCUGCGCGGUGGUGUUGUGUUCG UGGACGAGGUGCCUAAAGGACUGACCGGCAAGUUGGACGCCCGCAAGAUCCGCGAGAUUC UCAUUAAGGCCAAGAAGGGCGGCAAGAUCGCCGUGUA (SEQ ID NO:7)

SEQ ID NO: 8

UUUGAAUU (SEQ ID NO:8)

SEQ ID NO: 9

AUGCAGAGAAGCCCCCUGGAAAAGGCCAGCGUGGUGUCCAAGCUGUUCUUCAGCUGGACC

AGACCCAUCCUGAGAAAGGGCUACAGACAGAGACUGGAACUGAGCGACAUCUACCAGAUC CCCAGCGUGGACAGCGCCGACAACCUGAGCGAGAAGCUGGAAAGAGAGUGGGACAGAGA

GCUGGCUAGCAAGAAGAACCCCAAGCUGAUCAACGCCCUGAGGCGGUGCUUCUUCUGGCG GDUUAUGUUCUACGGCAUCUUCCUGUACCUGGGCGAAGUGACAAAGGCCGUGCAGCCCCU

GCUCCUGGGCAGAAUCAUUGCCAGCUACGACCCCGACAACAAAGAGGAAAGAUCUAUCGC

CAUCUACCUGGGCAUCGGCCUGUGCCUGCUGUUCAUCGUGCGGACACUGCUGCUGCACCC CGCCAUCUUCGGCCUGCACCACAUCGGCAUGCAGAUGAGAAUCGCCAUGUDCAGCCUGAU CUACAAGAAAACCCUGAAGCUGAGCAGCAGGGUGCUGGACAAGAUCAGCAUCGGACAGCU GGUGQCCCUGCUGAGCAACAACCUGAACAAGUUCGACGAGGGACUGGCCCUGGCUCACUU CGUGUGGAUCGCUCCACUGCAGGUCGCCCUGCUGAUGGGCCUGAUCUGGGAGCUGCUGCA GGCCAGCGCUUUCUGCGGCCUGGGCUUUCUGAUUGUGCUGGCCCUGUUUCAGGCUGGCCU GGGCAGGAUGAUGAUGAAGUACAGGGACCAGAGAGCCGGCAAGAUCAGCGAGAGACUGG UCAUCACCAGCGAGAUGAUCGAGAACAUCCAGAGCGUGAAGGCCUACUGCUGGGAAGAG GCCAUGGAAAAGAUGAUCGAAAACCUGAGACAGACCGAGCUGAAGCUGACCAGAAAGGC CGCCUACGUGCGGUACUUCAACAGCAGCGCCUUCUUCUUCUCCGGCUUCUUCGUGGUGUU CCUGUCCGUGCUGCCCUACGCCCUGAUCAAGGGCAUCAUCCUGAGGAAGAUCUUCACCAC

Page 93 of 121

RECTIFIED SHEET (RULE 91) ISA/EP

WO 2014/153052

PCT/US2014/028849

CAUUUCUUUCUGCAUCGUGCUGAGAAUGGCCGUGACCAGACAGUUCCCCUGGGCCGUGCA GACUUGGUACGACAGCCUGGGCGCCAUCAACAAGAUCCAGGACUUCCUGCAGAAGCAGGA GUACAAGACCCUCGAGUACAACCUGACCACCACCGAGGUGGUCAUGGAAAACGUGACCGC CUUCUGGGAGGAAGGCUUCGGCGAGCUGUUCGAGAAGGCCAAGCAGAACAACAACAACA GAAAGACCAGCAACGGCGACGACUCCCUGUUCUUCUCCAACUUCUCCCUGCUGGGCACCC CCGUGCUGAAGGACAUCAACUUCAAGAUCGAGAGAGGCCAGCUGCUCGCCGUGGCCGGCU CUACAGGCGCUGGCAAGACCUCUCUGCGGAUGGUCAUCAUGGGCGAGCUGGAACCCAGCG AGGGCAAGAUCAAGCACAGCGGCAGAAUCAGCUUCUGCAGCCAGUUCAGCUGGAUCAUGC CCGGCACCAUCAAAGAGAACAUCAUCUUCGGCGUGUCCUACGACGAGUACAGAUACAGAA GCGUGAUCAAGGCCUGCCAGCUGGAAGAGGACAUCAGCAAGUUCGCCGAGAAGGACAACA UCGUGCUGGGCGAGGGCGGCAUCACCCUGUCUGGCGGCCAGAGAGCCAGAAUCAGCCGGG CCAGAGCCGUGUACAAGGACGCCGACCUGUACCUGCUGGACAGCCCCUUCGGCUACCUGG ACGUGCUGACCGAGAAAGAGAUCUUCGAGAGCUGCGUGUGCAAGCUGAUGGCCAACAAG ACCAGAAUCCUGGUCACCAGCAAGAUGGAACACCUGAAGAAGGCCGACAAGAUCCUGAUC CUGCACGAGGGCAGCAGCUACUUCUACGGCACAUUCAGCGAGCUGCAGAACCUGCAGCCC GACUUCAGCAGCAAACUGAUGGGCUGCGACAGCUUCGACCAGUUCAGCGCCGAGAGAAGA AACAGCAUCCUGACCGAGACACUGCACAGAUUCAGCCUGGAAGGCGACGCCCCCGUGUCU UGGACCGAGACAAAGAAGCAGAGCUUCAAGCAGACCGGCGAGUUCGGCGAGAAGAGAAA GAACUCCAUCCUGAACCCCAUCAACAGCAUCCGGAAGUUCAGCAUCGUGCAGAAAACCCC CCUGCAGAUGAACGGCAUCGAAGAGGACAGCGACGAGCCCCUGGAAAGACGGCUGAGCCU GGUGCCUGACAGCGAGCAGGGCGAGGCCAUCCUGCCUAGAAUCAGCGUGAUCAGCACCGG CCCCACCCUGCAGGCUAGAAGGCGGCAGAGCGUGCUGAACCUGAUGACCCACAGCGUGAA CCAGGGCCAGAACAUCCACCGCAAGACCACCGCCAGCACCAGAAAGGUGUCCCUGGCUCC UCAGGCCAACCUGACCGAGCUGGACAUCUACAGCAGAAGGCUGAGCCAGGAAACCGGCCU GGAAAUCAGCGAGGAAAUCAACGAAGAGGACCUGAAAGAGUGCUUCGUCGACGACAUGG AAUCCAUCCCCGCCGUGACCACCUGGAACACCUACCUGCGGUACAUCACCGUGCACAAGA GCCUGAUCUUCGUGCUGAUCUGGUGCCUGGUCAUCUUCCUGGCCGAGGUGGCCGCCAGCC UGGUGGUGCUGUGGCUCCUGGGAAACACCCCUCUGCAGGACAAGGGCAACAGCACCCACA GCAGAAACAACAGCUACGCCGUGAUCAUCACCUCCACCAGCUCCUACLTACGUGUUCUACA GCUACGUGGGCGUGGCCGACACCCUGCUGGCUAUGGGCUUCUUCAGAGGCCUGCCCCUGG ugcacacccugaucaccguguccaagauccugcaccauaagaugcugcacagcgugcugc AGGCUCCCAUGAGCACCCUGAACACACUGAAGGCUGGCGGCAUCCUGAACAGGUGCAGCA AGGAUAUCGCCAUCCUGGACGACCUGCUGCCUCUGACCAUCUUCGACUUCAUCCAGCUGC UGCUGAUCGUGAUCGGCGCUAUCGCCGUGGUGGCCGUGCUGCAGCCCUACAUCUUCGUGG CCACCGUGCCCGUGAUCGUGGCCUUCAGUAUGCUGAGAGCCUACUCUCUGCAGACCAGCC AGCAGCUGAAGCAGCUGGAAAGCGAGGGCAGAAGCCCCAUCUUCACCCACCUCGUGACCA GCCUGAAGGGCCUGUGGACCCUGAGAGCCUUCGGCAGACAGCCCUACUUCGAGACACUGU UCCACAAGGCCCUGAACCUGCACACCGCCAACUGGUUUCUGUACCUGUCCACCCUGAGAU GGUUCCAGAUGAGGAUCGAGAUGAUCUUCGUCAUCUUCUUUAUCGCCGCGACCUUCAUCLI CUAUCCUGACCACCGGCGAGGGCGAGGGAAGAGUGGGAAUCAGCCUGACCCUGGCCAUGA ACAUCAUGAGCACACUGCAGUGGGCCGUGAACAGCAGCAUCGACGUGGACAGCCUGAUGA GAAGCGUGUCCAGAGUGLTUCAAGUUCAUCGACAUGCCUACCGAGGGCAAGCCCACCAAGA GCACCAAGCCCUACAAGAACGGCCAGCGGAGCAAAGUGAUGAUCAUCGAGAACAGCCACG UCAAGAAGGACGACAUCUGGCCCAGCGGCGGACAGAUGACCGUGAAGGACCUGACCGCCA AGUACACAGAGGGCGGCAACGCUAUCCUGGAAAACAUCAGCUUCAGCAUCAGCCCAGGCC

Page 94 of 121

RECTIFIED SHEET (RULE 91) ISA/EP

WO 2014/153052

PCT/US2014/028849

AGAGAGUGGGCCUGCUGGGGAGAACAGGCAGCGGCAAGUCUACCCUGCUGUCCGCCUUCC UGAGACUGCUGAACACCGAGGGCGAGAUCCAGAUCGAUGGCGUGUCCUGGGACLICCAUCA CCCUGCAGCAGUGGCGCAAGGCCUUCGGCGUGAUCCCCCAGAAGGUGUUCAUCUUCAGCG GCACCUUCAGAAAGAACCUGGACCCCUACGAGCAGUGGUCCGACCAGGAAAUCUGGAAGG UCGCCGAUGAAGUGGGCCUGAGAUCCGUGAUCGAGCAGUUCCCCGGCAAGCUGGACUUCG UGCUGGUGGACGGCGGCUGCGUGGUGAGCCACGGCCACAAGCAGCUGAUGUGUCUGGCCC GCUCCGUGCUGAGCAAGGCUAAGAUUCUGCUGCUGGACGAGCCUAGCGCCCACCUGGACC CUGUGACCUACCAGAUCAUCAGAAGGACCCUGAAGCAGGCCUUCGCCGACUGCACCGUGA UCCTJGUGCGAGCACAGAAUCGAGGCCAUGCUGGAAUGCCAGCAGUUCCUGGUCAUCGAAG AGAACAAAGUGCGGCAGUACGACAGCAUCCAGAAGCUGCUGAACGAGAGAAGCCUGUUC AGACAGGCCAUCAGCCCCAGCGACAGAGUGAAGCUGLTUCCCCCACCGCAACAGCAGCAAG UGCAAGAGCAAGCCCCAGAUCGCCGCCCUGAAAGAAGAGACUGAGGAAGAGGUGCAGGAC ACCAGACUGUGA (SEQ ID NO:9)

SEQIDNOilO

AUGCAGCGGUCCCCGCUCGAAAAGGCCAGUGUCGUGUCCAAACUCUUCUUCUCAUGGACU CGGCCUAUCCUUAGAAAGGGGUAUCGGCAGAGGCUUGAGUUGUCUGACAUCUACCAGAU CCCCUCGGUAGAUUCGGCGGAUAACCUCUCGGAGAAGCUCGAACGGGAAUGGGACCGCGA

ACUCGCGUCUAAGAAAAACCCGAAGCUCAUCAACGCACUGAGAAGGUGCUUCUUCUGGCG GUUCAUGUUCUACGGUAUCUUCUUGUAUCUCGGGGAGGUCACAAAAGCAGUCCAACCCCU GUUGUUGGGUCGCAUUAUCGCCUCGUACGACCCCGAUAACAAAGAAGAACGGAGCAUCGC GAUCUACCUCGGGAUCGGACUGGGUUUGCUGGUCAUCGUCAGAACACUUUUGUUGCAUCC AGCAAUCUUCGGCCUCCAUCACAUCGGUAUGCAGAUGCGAAUCGCUAUGIJUUAGCUUGAU CUACAAAAAGACACUGAAACUCUCGUCGCGGGUGUUGGAUAAGAUUUCCAUCGGUCAGU UGGUGUCCCUGCUUAGUAAUAACCUCAACAAAUUCGAUGAGGGACUGGCGCUGGCACALT UUCGUGUGGAUUGCCCCGCUGCAAGUCGCACUGCUUAGGGGACUGAUUUGGGAACUGGU

GCAGGCCAGCGCCUUUUGCGGCCUGGGAUUGCUCAUUGUGCUUGCACUUUUCCAAGCAGG GCUCGGCAGAAUGAUGAUGAAGUACAGGGACCAGAGAGCCGGAAAGAUCUCAGAACGGC UCGUGAUUACUUCAGAAAUGAUCGAGAACAUUCAAUCGGUGAAAGCGUACUGCUGGGAA GAGGCGAUGGAAAAGAUGAUCGAAAACCLTCAGACAGACCGAGUUGAAGCUGACCCGGAA GGCCGCGUACGUCAGAUACUUCAACAGCAGCGCUUUCUUCUUCUCGGGCUUCUUCGUCGU GUUCCUGUCGGUGCUGCCGUAUGCCCUCAUUAAGGGAAUUAUCUUGCGGAAGAUCUUUA CUACUAUCUCAUUUUGCAUCGUCCUUCGGAUGGCGGUCACUCGGCAGUUCCCGUGGGCCG UGCAGACCUGGUACGACAGCCUCGGGGCCAUCAACAAGAUCCAAGACUUUCUCCAAAAGC AAGAGUACAAAACCCUCGAAUACAACCUCACCACUACUGAAGUGGUCAUGGAAAACGUGA CCGCCUUUUGGGAAGAAGGCUUCGGAGAACUGUUCGAGAAGGCGAAGCAAAACAACAAU

CACUUGCACCGCAGGCAAACCUGACCGAACUCGACAUCUACAGCAGACGGCUCUCACAAG AAACUGGAUUGGAGAUCAGCGAAGAGAUCAACGAAGAAGAUCUCAAAGAAUGCUUCUUC GACGAUAUGGAGUCCAUCCCAGCAGUCACUACGUGGAAUACCUACCUCCGCUACAUCACU GUGCACAAGAGCCUGAUUUUCGUGUGGAUCUGGUGCCUGGUCAUCUUCUUGGCCGAGGtr GGCCGCGAGCCUCGUGGUCCUCUGGCUGCUCGGCAAUACGCCGCUGCAAGAUAAGGGAAA UUCCACGCAUAGCAGAAACAACUCAUACGCAGUGAUCAUCACUAGCACUUCAUCGUACUA CGUGUUCUACAUCUACGUGGGGGUGGCCGAUACUCUGUDGGCAAUGGGAUUCUUDAGAG GGCUGCCUCUGGUGCAUACGCUGAUCACUGUGUCCAAGAUCCUCCACCACAAGAUGCUCC ACUCCGUGCUUCAGGCCCCUAUGUCAACUCUCAACACCCUCAAGGCCGGAGGUAUUCUUA AUCGCUUDQCCAAGGACAUCGCCAUUCUCGAUGACUUGCUUCCCCUGACUAUCUUCGACU UUAUCCAGUUGCGGCUGAUUGUGAUCGGCGCUAUUGCCGUCGUCGCAGUGCUGCAACCGU ACAUCUUUGUGGCUACCGUCCCAGUCAUUGUGGCCUUCAUCAUGCUCAGGGCAUACUUUC UCCAGACCAGCCAGCAGCUCAAGCAGCUCGAAUCCGAAGGCAGAUCGCCGAUCUUCACCC ACCUCGUCACUUCGCUCAAGGGCCUCUGGACCCUGCGCGCCUUCGGUCGCCAGCCGUAUU OCGAAACCCUGUUCCAUAAAGCACUGAACCUCCAUACUGCGAACUGGUUUCUCUACCUUU CAACCCUGAGGUGGUUCCAGAUGAGAAUCGAGAUGAUCUUUGUGAUCUUCUUUAUCGCLr GUGACGUUCAUCUCCAUUCUCACUACCGGCGAGGGAGAGGGCAGAGUGGGGAUUAUCCUC ACGCUGGCCAUGAAUAUCAUGAGCACGCUGCAGUGGGCCGUCAAUAGCAGCAUCGACGUG GACUCCCUGAUGCGGUCCGUGUCGAGAGUGUUUAAGUUCAUCGAUAUGCCUACUGAAGG GAAACCGACCAAGUCGA.CCAAGCCGUACAAGAAUGGGCAGCUGAGCAAGGUGAUGAUUA UUGAGAACUCCCAUGUGAAGAAGGACGACAUCUGGCCCAGCGGAGGCCAGAUGACCGUGA AGGACUUGACCGCUAAGUACACUGAGGGUGGAAAUGCCAUUCUUGAGAAUAUCAGCUUC UCGAUCUCGCCGGGACAACGCGUGGGAUUGCUCGGGCGCACUGGCAGCGGCAAAUCCACC CUGCUUAGCGCUUUUCUGAGGCUGCUGAACACUGAAGGUGAAAUUCAAAUCGAUGGAGU GUCGUGGGAUAGCAUCACCCUUCAACAGUGGCGCAAGGCCUUCGGCGUGAUCCCUCAAAA GGUCUUUAUCUUCUCGGGGACGUUCCGGAAAAAUCUCGACCCCUACGAACAGUGGUCAGA CCAAGAGAUUUGGAAAGUCGCAGAUGAGGUCGGACDGCGCUCAGUGAUCGAACAGUUUC CGGGUAAACUUGACUUCGUGCUCGUCGAUGGAGGUUGCGUCCUGUCCCACGGACAUAAGC AGCUGAUGUGUCUGGCGCGCUCGGUCCUCUCCAAAGCGAAGAUCCUGCUGCUCGAUGAAC CGUCCGCCCACCUUGAUCCAGUGACCUAUCAGAUCAUUCGGAGAACUUUGAAGCAAGOCU UCGCUGACUGCACCGUCAUCCUCUGCGAACACCGGAUCGAGGCAAUGCUGGAGUGCCAAC AGUUUCUGGUCAUCGAAGAAAACAAAGUGCGCCAGUAUGACUCGAUCCAAAAACUUCUG AACGAGCGCUCCCUCUUCCGGCAGGCAAUCAGCCCAUCCGACCGCGUGAAGUUGUUCCCU CAUCGGAAUAGCUCCAAAUGCAAAUCGAAGCCGCAGAUCGCUGCCUUGAAAGAAGAAACC GAAGAAGAAGUCCAAGACACUAGGUUGUAG (SEQ ID NO: 11)

SEQIDNO:12

AUGCAGCGGDCCCCUCUGGAGAAGGCUUCCGUGGUCAGCAAGCUGUUCUUCUCGUGGACC

AGACCUAUCCUCCGCAAGGGAUACCGCCAGCGCCUGGAGCUGUCAGAQAUCUACCAGAUC

CCAAGCGUGGACUCAGCCGACAALrcUGAGCGAAAAGCUGGAACGGGAGUGGGACCGGGA GCUCGCCUCCAAGAAGAAUCCGAAGUUGAUCAAUGCGCUGCGCAGAUGCUUCUUCUGGCG

GUUUAUG UU U UACGGCAUCUUUCUGUAUCUCGGAGAAGUGACCAAAGCCGUGCAGCCGC UGCUCUUGGGUAGGAUCAUUGCUUCGUACGACCCGGACAACAAAGAAGAACGCUCCAUCG

CCAUCUACCUCGGAAUCGGUCUGUGCCDGCUCUUUAUCGUGCGCACUCUCCUGCUGCAUC

Page 98 of 121

RECTIFIED SHEET (RULE 91) ISA/EP

WO 2014/153052

PCT/US2014/028849

CGGCGAUCUUCGGACUGCACCACAUCGGCAUGCAAAUGCGGAUCGCAAUGUUCUCACUGA UCUACAAAAAGACUCUGAAGCUCAGCUCCAGAGUGCUGGAUAAGAUCUCGAUCGGGCAAC UCGUCAGCCUGCUGUCGAACAAUCUGAAGAAGUUCGACGAAGGGUUGGCCCUCGCACAUU UCGUGUGGAUCGCACCGCUGCAAGUGGCGCUCCUGAUGGGACUCAUUUGGGAACUGCUCC AAGCCAGCGCGUUUUGCGGACUCGGAUUCCUGAUCGUGCUCGCCCUGUUCCAAGCCGGAC UGGGGCGCAUGAGGAUGAAGUACCGCGAUCAGCGGGCAGGAAAGAUCUCCGAGCGGUUG GUGAUCACUUCCGAAAUGAUCGAGAAUAUUCAGUCCGUGAAGGCCUACUGCUGGGAAGA AGCUAUGGAAAAGAUGAUUGAAAACUUGCGGCAAACUGAGCUGAAAUUGACUCGCAAAG CGGCAUACGUCCGCUACUUCAAUAGCAGCGCCUUCUUCUUUUCGGGCUUUUUCGUGGUGU UUCUGAGCGUGCUGCCCUACGCUCUGAUCAAGGGAAUCAUCCUCCGGAAAAUCUUCACCA CCAUUUCGUUCUGUAUCGUGUUGCGCAUGGCCGUGACUCGCCAGUUCCCCUGGGCGGUGC AGACCGGGUACGACAGCUUGGGGGCAAUCAAUAAGAUUCAAGACUUCUUGCAAAAGCAG GAGUACAAGACUCUGGAGUACAACCUGACCACCACUGAAGUCGUGAUGGAGAACGUGACC GCCUUUUGGGAAGAGGGUUUUGGAGAACUGUUUGAGAAAGCAAAGCAGAAUAACAACAA CCGCAAGACCUCAAAUGGGGACGAUUCCCUGUUUUUCUCGAACUUCUCCCUGCUCGGAAC ACCCGUGUUGAAGGACAUCAAUUUCAAGAUUGAGAGGGGACAGCUUCUCGCGGUAGCGG GAAGCACUGGUGCGGGAAAAACUAGCCUCGUGAUGGUGAUUAUGGGGGAGCUUGAGCCC AGCGAGGGGAAGAUUAAACACUCCGGGCGUAUCUCAUUCUGUAGCCAGUUUUCAUGGAU CAUGCCCGGAACCAUUAAAGAGAACAUCAUUUUCGGAGUAUCCUAUGAUGAGUACCGAU ACAGAUCGGUCAUUAAGGCGUGCCAGUUGGAAGAGGACAUUUCUAAGUUCGCCGAGAAG GAUAACAUCGUCUUGGGAGAAGGGGGUAUUACAUUGUCGGGAGGGCAGCGAGCGCGGAU CAGCCUCGCGAGAGCGGUAUACAAAGAUGCAGAUUUGUAUCUGCUUGAUUCACCGUUUG GAUACCUCGACGUAUUGACAGAAAAAGAAAUCUUCGAGUCGLJGCGUGUGUAAACGUAUG GCUAAUAAGACGAGAAUCCUGGUGACUUCCAAAAUGGAGCAUCUCAAGAAGGCGGACAA GAUCCUGAUUCUGCAUGAGGGAUCAAGCUAUUUCUACGGAACUUUUUCCGAGCUGCAGA ACCUCCAGCCGGAUUUUAGCUCCAAGCUGAUGGGUtJGCGACUCAUUCGACCAAUUCUCGG CUGAGCGGCGGAACUCAAUCCUGACCGAAACCCUGCAUCGCUUCUCCCUUGAGGGAGAUG CCCCGGUGUCGUGGACUGAGACUAAAAAGCAGUCGUUUAAGCAAACUGGCGAAUUCGGC GAAAAGCGGAAGAAUAGCAUCCUCAACCCAAUCAACAGCAUUCGGAAGUUCAGCAUCGUC CAAAAGACCCCGCUCCAGAUGAACGGCAUUGAAGAGGACUCAGACGAGCCAUUGGAAAGA CGCCUGUCACUGGUCCCAGAUUCGGAGCAGGGUGAAGCAAUUCUGCCUCGGAUCUCGGUC AUCUCGACUGGGCCCACUCUCCAAGCUCGGCGGAGACAGAGCGUGCUUAACUUGAUGACC CACUCCGUGAACCAGGGUCAGAACAUCCACCGCAAAACCACCGCCUCCACCAGGAAGGUG UCACUGGCCCCUCAAGCCAAUCUGACUGAGUUGGAUAUCUACUCCAGAAGGCUCAGCCAG GAAACCGGACUGGAAAUCUCGGAAGAGAUCAACGAAGAGGAUCUCAAAGAGUGCJUUCUU CGACGACAUGGAAUCAAUCCCUGCUGUCACUACUUGGAACACCUAUCUCCGCUACAUUAC CGUGCACAAGUCACUCAUCGUCGUCCUGAUCUGGUGCCUCGGGAUCUUCCUGGCCGAGGU CGCAGCAUCGCUGGUCGUGCUGUGGCUGCUCGGCAACACCCCACUCCAAGACAAAGGCAA CAGCACCCAUUCCCGCAACAACUCCUACGCGGUGAUCAUCACUUCAACUUCGUCCUACUA CGUCUUUUACAUCUACGUGGGCGUGGCGGACACGCUCCUGGCUAUGGGGUUCUUUCGCGG GCUGCCUCUGGUCCACACGCUCAUCACUGUGUCAAAGAUUCUCCACCACAAAAUGCUGCA CUCCGUGCUCCAGGCCCCUAUGUCGACUUGGAACACGCUUAAGGCCGGAGGCAUCCUUAA CAGAUUCUCGAAAGAUAUCGCGAUCDGGGACGAUCUUCUGCCGCUGACUAUCUUUGACUG CAUCCAACUCCUGCUGAUCGUCAUCGGUGCCAUCGCAGUGGUCGCGGUGCUCCAACCGUA CAUUUUCGUGGCGACUGUGCCGGUGAUCGUGGCGUUCAUCAUGCUGCGGGCUUACUUUCU

Page 99 of 121

RECTIFIED SHEET (RULE 91) ISA/EP

WO 2014/153052

SEQ ID NO:16

AUGGCCACUGGAUCAAGAACCUCACUGCUGCUCGCUUUUGGACUGCUUUGCCUGCCCUGGU UGCAAGAAGGAUCGGCUUUCCCGACCAUCCCACUCUCCAGGCAGCGGUCCCCGCUCGAAAA GGCCAGUGUCGUGUCCAAACUCUUCUUCUCAUGGACDCGGCCIJAUCCUUAGAAAGGGGUA UCGGCAGAGGCUUGAGUUGUCUGACAUCUACCAGAUCCCCUCGGUAGAUUCGGCGGAUAA CCUCUCGGAGAAGCUCGAACGGGAAUGGGACCGCGAACUCGCGUCUAAGAAAAACCCGAA GCUCAUCAACGCACUGAGAAGGUGCUUCLTUCUGGCGGUUCAUGUUCUACGGUAUCUUCUU GUAUCUCGGGGAGGUCACAAAAGCAGUCCAACCCCUGUUGUUGGGUCGCAUUAUCGCCUC GUACGACCCCGAUAACAAAGAAGAACGGAGCAUCGCGAUCUACCUCGGGAUCGGACUGUG UUUGCUUUUCAUCGUCAGAACACUUUUGUUGCAUCCAGCAAUCUUCGGCCUCCAUCACAU CGGUAUGCAGAUGCGAAUCGCUAUGUUUAGCUUGAUCUACAAAAAGACACUGAAACUCU

Page 105 of 121

RECTIFIED SHEET (RULE 91) ISA/EP

WO 2014/153052

PCT/US2014/028849

CGUCGCGGGOGUUGGAUAAGAUUUCCAUCGGUCAGUUGGUGUCCCUGCUUAGUAAUAAC CUCAACAAAUUCGAUGAGGGACUGGCGCUGGCACAUUUCGUGUGGAUUGCCCCGUUGCAA GUCGCCCUUUUGAUGGGCCUUAUUUGGGAGCUGGUGCAGGCAUCUGCCUUUUGUGGCCU GGGAUUUCUGAUUGUGUUGGCAUUGUUUCAGGCUGGGCUUGGGCGGAUGAUGAUGAAGU AUCGCGACCAGAGAGCGGGUAAAAUCUCGGAAAGACUCGUCAUCACUUCGGAAAUGAUC GAAAACAUCCAGUCGGUCAAAGCCUAUUGCUGGGAAGAAGCUAUGGAGAAGAUGAUUGA AAACCUCCGCCAAACUGAGCUGAAACUGACCCGCAAGGCGGCGUAUGUCCGGUAUUUCAA UUCGUCAGCGUUCUUCUUUUCCGGGUUCUUCGUUGUCUUUCUCUCGGGUUUGCCUUAUGC CUUGAUUAAGGGGAUUAUCCUCCGCAAGAUUUUCACCACGALUUCGUUCUGCAUUGUAU UGCGCAUGGCAGUGACACGGCAAUUUCCGUGGGCCGUGCAGACAUGGUAUGACUCGCUUG GAGCGAUCAACAAAAUCCAAGACUUCUUGCAAAAGCAAGAGUACAAGACCCUGGAGUAC AAUCUUACUACUACGGAGGUAGUAAUGGAGAAUGUGACGGCUUUUUGGGAAGAGGGUUU UGGAGAACUGUUUGAGAAAGCAAAGCAGAAUAACAACAACCGCAAGACCUCAAAUGGGG ACGALUCCCUGUUUUUCUCGAACUUCUCCCUGCUCGGAACACCCGUGUUGAAGGACAUCA AUOUCAAGAUUGAGAGGGGACAGCUUCUCGCGGUAGCGGGAAGCACUGGUGCGGGAAAA ACUAGCCUCUUGAUGGUGAUUAUGGGGGAGCUUGAGCCCAGCGAGGGGAAGAUUAAACA CUCCGGGCGUAUCUCAUUCUGUAGCCAGUUUUCAUGGAUCALTGCCCGGAACCAUUAAAGA GAACAUCAUUGUCGGAGUAUCCUAUGAUGAGUACCGAUACAGAUCGGUCAUUAAGGCGU GCCAGUUGGAAGAGGACAUUUCUAAGUUCGCCGAGAAGGAUAACAUCGUCUUGGGAGAA GGGGGUAUUACAUUGUCGGGAGGGCAGCGAGCGCGGAUCAGCCUCGCGAGAGCGGUAUA CAAAGAUGCAGAUUUGUAUCUGCUUGAUUCACCGUUUGGAUACCUCGACGUAUCJGACAG AAAAAGAAAUCUUCGAGUCGUGCGUGUGUAAACUUAUGGCUAAUAAGACGAGAAUCCUG GUGACAUCAAAAAUGGAACACCUUAAGAAGGCGGACAAGAUCCUGAUCCUCCACGAAGG AUCGUCCUACUUUUACGGCACUUUCUCAGAGUUGCAAAACUUGCAGCCGGACUUCUCAAG CAAACUCAUGGGGUGUGACUCAUUCGACCAGUUCAGCGCGGAACGGCGGAACUCGAUCUU GACGGAAACGCUGCACCGAUUCUCGCUUGAGGGUGAUGCCCCGGVAUCGUGGACCGAGAC aaagaagcagucguuuaagcagacaggagaauuuggugagaaaagaaagaacaguaucu UGAAUCCUAUUAACUCAAUUCGCAAGUUCUCAAUCGUCCAGAAAACUCCACUGCAGAUGA AUGGAAUUGAAGAGGAUUCGGACGAACCCCUGGAGCGCAGGCUUAGCCUCGUGCCGGAU UCAGAGCAAGGGGAGGCCAUUCUUCCCCGGAUUUCGGUGAUUUCAACCGGACCGACACUU CAGGCGAGGCGAAGGCAAUCCGUGCUCAACCUCAUGACGCAUUCGGUAAACCAGGGGCAA AACAUUCACCGCAAAACGACGGCCUCAACGAGAAAAGUGUCACUUGCACCCCAGGCGAAU UUGACUGAACUCGACAUCUACAGCCGUAGGCUUtJCGCAAGAAACCGGACUUGAGAUCAGC GAAGAAAUCAAUGAAGAAGAUUUGAAAGAGUGUUUCUUUGAUGACAUGGAALTCAAUCCC AGCGGUGACAACGUGGAACACAUACUUGCGUUACAUCACGGUGCACAAGUCCUUGAUUU UCGUCCUCAUCUGGUGUCUCGUGAUCUUVCUCGCUGAGGUCGCAGCGUCACUUGUGGUCC UCUGGCUGCUUGGUAAUACGCCCUUGCAAGACAAAGGCAAUUCUACACACUCAAGAAACA AUUCCUAUGCCGUGAUUAUCACUUCUACAAGCUCGUAUUACGUGUUUUACAUCUACGUA GGAGUGGCCGACACUCUGCUCGCGAUGGGUUUCUUCCGAGGACUCCCACUCGUUCACACG CUUAUCACUGUCUCCAAGAUUCUCCACCAUAAGAUGCUUCALTAGCGUACUGCAGGCUCCC AUGUCCACCUUGAAUACGCUCAAGGCGGGAGGUAUUUUGAAUCGCUUCUCAAAAGAUAU UGCAAUUUUGGAUGACCUUCUGCCCCUGACGAUCUUCGACUUCAUCCAGUUGUUGCUGAU CGUGAUUGGGGCUAUGGCAGUAGUCGCUGUCCUCCAGCCUUACAUUUUUGUCGCGACCGU UCCGGUGAUCGUGGCGUUUAUCAUGCUGCGGGCCUAUUUCUUGCAGACGUCACAGCAGCU UAAGCAACUGGAGUCUGAAGGGAGGUCGCCUAUCUUUACGCAUCUUGUGACCAGUUGGA

Page 106 of 121

RECTIFIED SHEET (RULE 91) ISA/EP

WO 2014/153052

PCT/US2014/028849

AGGGAUUGIJGGACGUUGCGCGCCUUUGGCAGGCAGCCCUACUUUGAAACACUGUUCCACA AAGCGCUGAAUCUCCAUACGGCAAAOUGGUUUUXJGUAUUUGAGUACCCUCCGAUGGUUU CAGAUGCGCAUUGAGAUGAUUUUUGUGAUCUUCUUUAUCGCGGUGACUUUUAUCUCCAU CIJUGACCACGGGAGAGGGCGAGGGACGGGUCGGUAUUAUCCUGACACUCGCCAUGAACAU UAUGAGCACUUUGCAGUGGGCAGUGAACAGCUCGAUUGAUGUGGAUAGCCUGAUGAGGU CCGUUUCGAGGGUCUUUAAGUUCAUCGACAUGCCGACGGAGGGAAAGCCCACAAAAAGU ACGAAACCCUAUAAGAAUGGGCAAUUGAGUAAGGUAAUGAUCAUCGAGAACAGUCACGU GAAGAAGGAUGACAUCUGGCCUAGCGGGGGUCAGAUGACCGUGAAGGACCUGACGGCAA AAUACACCGAGGGAGGGAACGCAAUCCUUGAAAACAUCUCGUUCAGCAUUAGCCCCGGUC AGCGUGUGGGGUUGCUCGGGAGGACCGGGUCAGGAAAAUCGACGUUGCUGUCGGCCUUC UUGAGACUUCUGAAUACAGAGGGUGAGAUCCAGAUCGACGGCGUUUCGUGGGAUAGCAU CACCUUGCAGCAGUGGCGGAAAGCGUUUGGAGUAAUCCCCCAAAAGGUCUtlUAUCUUUA GCGGAACCUUCCGAAAGAAUCUCGAUCCUUAUGAACAGUGGUCAGAUCAAGAGAUUUGG AAAGUCGCGGACGAGGUUGGCCUUCGGAGUGUAAUCGAGCAGUUUCCGGGAAAACUCGA CUUUGUCCUUGUAGAUGGGGGAUGCGUCCUGUCGCAUGGGCACAAGCAGCUCAUGUGCCU GGCGCGAUCCGUCCUCUCUAAAGCGAAAAUUCCJUCUCUUGGALiGAACCUUCGGCCCAUCU GGACCCGGUAACGUAUCAGAUCAUCAGAAGGACACUUAAGCAGGCGUUUGCCGACUGCAC GGUGAUUCUCUGUGAGCAUCGUAUCGAGGCCAUGCUCGAAUGCCAGCAAUUUCUUGUCA UCGAAGAGAAUAAGGUCCGCCAGUACGACUCCAUCCAGAAGCUGCUUAAUGAGAGAUCA UUGUUCCGGCAGGCGAUUUCACCAUCCGAUAGGGUGAAACUUUUUCCACACAGAAAUUCG UCGAAGUGCAAGUCCAAACCGCAGAUCGCGGCCUUGAAAGAAGAGACUGAAGAAGAAGU UCAAGACACGCGUCUUUAA (SEQ ID NO: 16)

SEQ ID NO: 17

AUGCAGCGGUCCCCGCUCGAAAAGGCCAGUGUCGUGUCCAAACUCUUCUUCUCAUGGACU CGGCCUAUCCUUAGAAAGGGGUAUCGGCAGAGGCUUGAGUUGUCUGACAUCUACCAGAU CCCCUCGGUAGAUUCGGCGGAUAACCUCUCGGAGAAGCUCGAACGGGAAUGGGACCGCGA ACUCGCGUCUAAGAAAAACCCGAAGCUCAUCAACGCACUGAGAAGGUGCUUCUUCUGGCG GUUCAUGQUCUACGGUAUCUUCUUGUAUCUCGGGGAGGUCACAAAAGCAGUCCAACCCCU GUUGUUGGGUCGCAUUAUCGCCUCGUACGACCCCGAUAACAAAGAAGAACGGAGCAUCGC GAUCUACCUCGGGAUCGGACUGUGUUUGCUUUUCAUCGUCAGAACACUUUUGUUGCAUCC AGCAAUCUUCGGCCUCCAUCACAUCGGUAUGCAGAUGCGAAUCGCUAUGUUUAGCUUGAU CUACAAAAAGACACUGAAACUCUCGUCGCGGGUGUUGGAUAAGAUUUCCAUCGGUCAGU UGGUGUCCCUGCUUAGUAAUAACCUCAACAAAUUCGAUGAGGGACQGGCGCUGGCACAU UUCGUGUGGAUUGCCCCGUQGCAAGUCGCCCUUUUGAUGGGCCIJUAUUUGGGAGCUGDU GCAGGCAUCUGCCLTUUUGUGGCCUGGGAUUUCUGAUUGUGUUGGCAUUGUUUCAGGCUG GGCUUGGGCGGAUGAUGAUGAAGUAUCGCGACCAGAGAGCGGGUAAAAUCUCGGAAAGA CUCGUCAUCACUUCGGAAAUGAUCGAAAACAUCCAGUCGGUCAAAGCCUAUUGCUGGGAA GAAGCUAUGGAGAAGAUGAUUGAAAACCUCCGCCAAACUGAGCUGAAACUGACCCGCAA GGCGGCGUAUOLrCCGGUAUUUCAAUUCGUCAGCGUUCLrUCQUUQCCGGGUUCUUCGUtrG UCUUUCUCUCGGUUUUGCCUUAUGCCUUGAUUAAGGGGAUUAUCCUCCGCAAGAUUUUC accacgauuucguucugcauuguauugcgcauggcagugacacggcaauuuccgugggcc GUGCAGACAUGGUAUGACUCGCDUGGAGCGAUCAACAAAAUCCAAGACUUCUUGCAAAA GCAAGAGUACAAGACCCUGGAGUACAAUCUUACUACUACGGAGGUAGUAAUGGAGAAUG

Page 107 of 121

RECTIFIED SHEET (RULE 91) ISA/EP

WO 2014/153052

PCT/US2014/028849

UGACGGCUULTUUGGGAAGAGGGUUUUGGAGAACUGUUUGAGAAAGCAAAGCAGAAUAAC AACAACCGCAAGACCUCAAAUGGGGACGAUUCCCUGUUUUUCUCGAACUUCUCCCUGCUC GGAACACCCGUGUUGAAGGACAUCAAUUUCAAGAUUGAGAGGGGACAGCUUCUCGCGGU AGCGGGAAGCACUGGUGCGGGAAAAACUAGCCUCUUGAUGGUGAUUAUGGGGGAGCUUG AGCCCAGCGAGGGGAAGAUUAAACACUCCGGGCGUAUCUCAUUCUGUAGCCAGUUUUCAU GGAUCAUGCCCGGAACCAUUAAAGAGAACAUCAUUUUCGGAGUAUCCUAUGAUGAGUAC CGAUACAGAUCGGUCAUUAAGGCGUGCCAGUUGGAAGAGGACAUUUCUAAGUUCGCCGA GAAGGAUAACAUCGUCUUGGGAGAAGGGGGUAUUACAUUGUCGGGAGGGCAGCGAGCGC GGAUCAGCCUCGCGAGAGCGGUAUACAAAGAUGCAGAUUUGUAUCUGCUUGAUUCACCG UUUGGAUACCUCGACGUAUUGACAGAAAAAGAAAUCUUCGAGUCGUGCGUGUGUAAACU UAUGGCUAAUAAGACGAGAAUCCUGGUGACAUCAAAAAUGGAACACCUUAAGAAGGCGG ACAAGAUCCUGAUCCUCCACGAAGGAUCGUCCUACUUUUACGGCACUUUCUCAGAGUUGC AAAACUUGCAGCCGGACUUCUCAAGCAAACUCAUGGGGUGUGACUCAGUCGACCAGUUCA GCGCGGAACGGCGGAACUCGAUCUUGACGGAAACGCUGCACCGAUUCUCGCUUGAGGGUG AUGCCCCGGUAUCGUGGACCGAGACAAAGAAGCAGUCGULlUAAGCAGACAGGAGAAUGU GGUGAGAAAAGAAAGAACAGUAUCUUGAAUCCUAUGAACUCAAGUCGCAAGUUCUCAAU CGUCCAGAAAACUCCACUGCAGAUGAAUGGAAUUGAAGAGGAUUCGGACGAACCCCUGG AGCGCAGGCUUAGCCUCGUGCCGGAUUCAGAGCAAGGGGAGGCCAUUCUUCCCCGGAUUU CGGGGAUUUCAACCGGACCUACACUUCAGGCGAGGCGAAGGCAAUCCGLJGCUCAACCUCA UGACGCAUUCGGUAAACCAGGGGCAAAACAUUCACCGCAAAACGACGGCCUCAACGAGAA AAGUGUCACUUGCACCCCAGGCGAAUUUGACUGAACUCGACAUCUACAGCCGUAGGCUUU CGCAAGAAACCGGACUUGAGAUCAGCGAAGAAAUCAAUGAAGAAGAUUUGAAAGAGUGG UUCUUUGAUGACAUGGAAUCAAUCCCAGCGGUGACAACGUGGAACACAUACUUGCGUUA CAUCACGGUGCACAAGUCCUUGAUUUGCGUCCUCAUCUGGUGUCUCGUGAUCUUUCUCGC UGAGGUCGCAGCGUCACUUGUGGUCCUCUGGCUGCUUGGUAAUACGCCCUUGCAAGACAA AGGCAAUUCUACACACUCAAGAAACAAUUCCUAUGCCGUGAUUAUCACUUCUACAAGCUC GUAUUACGUGUUUUACAGCUACGUAGGAGUGGCCGACACUCUGCUCGCGAUGGGUUUCU UCCGAGGACUCCCACUCGUUCACACGCUUA.UCACUGUCUCCAAGAUUCUCCACCAUAAGA UGCUUCAUAGCGUACUGCAGGCUCCCAUGUCCACCUUGAAUACGCUCAAGGCGGGAGGUA UUUUGAAUCGCUUCUCAAAAGAUAUUGCAAGUUUGGAUGACCUUCUGCCCCUGACGAGC UUCGACUUCAUCCAGUUGUUGCUGAUCGUGAUUGGGGCUAUUGCAGUAGUCGCUGUCCU CCAGCCUUACAUUUUUGUCGCGACCGUUCCGGUGAUCGUGGCGUUUAUCAUGCUGCGGGC CUAUUUCUUGCAGACGUCACAGCAGCUUAAGCAACUGGAGGCUGAAGGGAGGUCGCCUA UC'UUUACGCAUCUUGUGACCAGUUUGAAGGGAUUGUGGACGUUGCGCGCCUUUGGCAGG CAGCCCUACUUUGAAACACUGUUCCACAAAGCGCUGAAUCUCCAUACGGCAAAUUGGUUU UUGUAUUUGAGUACCCUCCGAUGGLTLTUCAGAUGCGCAUUGAGAUGAUUUUUGUGAUCUU CULTUAUCGCGGUGACUUUUAUCUCCAUCUUGACCACGGGAGAGGGCGAGGGACGGGUCG GLiAUUAUCCUGACACUCGCCAUGAACAUUAUGAGCACUUUGCAGUGGGCAGUGAACAGC UCGAUUGAUGUGGAUAGCCUGAUGAGGUCCGUUUCGAGGGUCUUUAAGUUCAUCGACAU GCCGACGGAGGGAAAGCCCACAAAAAGUACGAAACCCUAUAAGAAUGGGCAAUUGAGUA AGGUAAUGAUCAUCGAGAACAGUCACGUGAAGAAGGAUGACAUCUGGCCUAGCGGGGGU CAGAUGACCGUGAAGGACCUGACGGCAAAAUACACCGAGGGAGGGAACGCAAUCCUUGA AAACAUCUCGUUCAGCAUUAGCCCCGGUCAGCGUGUGGGGUUGCUCGGGAGGACCGGGUC AGGAAAAUCGACGUUGCUGUCGGCCOUCUUGAGACUUCUGAAUACAGAGGGUGAGAUCC AGAUCGACGGCGUUUCGUGGGAUAGCAUCACCUUGCAGCAGGGGCGGAAAGCGUUGGGA

Page 108 of 121

RECTIFIED SHEET (RULE 91) ISA/EP

WO 2014/153052

PCT/US2014/028849

GUAAUCCCCCAAAAGGUCUUUAUCUUUAGCGGAACCUUCCGAAAGAAUCUCGAUCCUUAU GAACAGUGGUCAGAUCAAGAGAUUUGGAAAGUCGCGGACGAGGUUGGCCUUCGGAGUGU AAUCGAGCAGUUUCCGGGAAAACUCGACUUUGUCCUUGUAGAUGGGGGAUGCGUCCUGU CGCAUGGGCACAAGCAGCUCAUGUGCCUGGCGCGAUCCGUCCUCUCUAAAGCGAAAAUUC UUCUCUUGGAUGAACCUUCGGCCCAUCUGGACCCGGUAACGUAUCAGAUCAUCAGAAGGA CACUUAAGCAGGCGUUUGCCGACUGCACGGUGAUUCUCUGUGAGCAUCGUAUCGAGGCCA UGCUCGAAUGCCAGCAAUUUCUUGUCAUCGAAGAGAAUAAGGUCCGCCAGUACGACUCCA UCCAGAAGCUGCUUAAUGAGAGAUCAUUGUUCCGGCAGGCGAUUUCACCAUCCGAUAGG GUGAAACU U U UUCCACACAGAAAUUCGUCGAAGUGCAAGUCCAAACCGCAGAUCGCGGCC UUGAAAGAAGAGACUGAAGAAGAAGUUCAAGACACGCGUCLfUUAA (SEQ ED NO: 17)

SEQ ID NO: 18

AUGGCCACUGGAUCAAGAACCUCACUGCUGCUCGCULJUUGGACUGCUUUGCCUGC CCUGGUUGCAAGAAGGAUCGGCUUUCCCGACCAUCCCACUCUCC (SEQ ID NO: 18)

SEQ ID NO: 19 AUGGCAACUGGAUCAAGAACCUCCCUCCUGCUCGCAUUCGGCCUGCUCUGUCUCC CAUGGCUCCAAGAAGGAAGCGCGUUCCCCACUAUCCCCCUCUCG (SEQ ID NO: 19)

SEQ ID NO: 20

CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCC ACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUCAAGCU

Page 109 of 121

RECTIFIED SHEET (RULE 91) ISA/EP

WO 2014/153052

PCT/US2014/028849

Equivalents [0270] The specification is most thoroughly understood in light of the teachings of the references cited within the specification. The embodiments within the specification provide an illustration of embodiments of the invention and should not be construed to limit the scope of the invention. The skilled artisan readily recognizes that many other embodiments arc encompassed by the invention. All publications and patents cited in this disclosure are incorporated by reference in their entirety. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material. The citation of any references herein is not an admission that such references are prior art to the present invention.

[0271] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification, including claims, are to be understood as approximations and may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches. The recitation of series of numbers with differing amounts of significant digits in the specification is not to be construed as implying that numbers with fewer significant digits given have the same precision as numbers with more significant digits given.

[0272] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” [0273] Unless otherwise indicated, the term “at Least” preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or

Page 110 of 121

WO 2014/153052

PCT/US2014/028849 be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[0274] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials arc now described.

[0275] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0276] Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein, ft is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Contents219

83 members in 28 offices

This recordMember, by publication datePriority claim18 more offices are in the list below

Priority claims6

Priority claims
Document	Office	Kind	Date
201361783663	United States of America	P	2013-03-14
2014028849	United States of America	W	2014-03-14
61783663	–	–	–
PCTUS2014028849	–	–	–
US201361783663P	–	–	–
WO2014US28849	–	–	–

Members83

Family members, oldest first
Document	Office	Kind
CA2904151A1	Canada	A1	2014-09-25
WO2014153052A2	World Intellectual Property Organization (WIPO)	A2	2014-09-25
US2015038556A1	United States of America	A1	2015-02-05
WO2014153052A9	World Intellectual Property Organization (WIPO)	A9	2015-08-06
AU2014236305A1	Australia	A1	2015-09-24
WO2014153052A3	World Intellectual Property Organization (WIPO)	A3	2015-09-24
SG11201507391VA	Singapore	A	2015-10-29
US9181321B2	United States of America	B2	2015-11-10
IL240982A0	Israel	A0	2015-11-30
IL240982D0	Israel	D0	2015-11-30
CN105142676A	China	A	2015-12-09
PE20151773A1	Peru	A1	2015-12-20
EP2968586A2	European Patent Office (EPO)	A2	2016-01-20
KR20160010398A	Republic of Korea	A	2016-01-27
EA201591477A1	Eurasian Patent Organization (EAPO)	A1	2016-03-31
US2016106772A1	United States of America	A1	2016-04-21
MX2015012333A	Mexico	A	2016-05-31
CL2015002675A1	Chile	A1	2016-06-10
JP2016517437A	Japan	A	2016-06-16
HK1218068A	Hong Kong, China	A	2017-02-03
HK1218068A1	Hong Kong, China	A1	2017-02-03
HK1220137A	Hong Kong, China	A	2017-04-28
HK1220137A1	Hong Kong, China	A1	2017-04-28
US9713626B2	United States of America	B2	2017-07-25
BR112015022868A2	Brazil	A2	2017-11-07
US2018015116A1	United States of America	A1	2018-01-18
SG10201710253QA	Singapore	A	2018-01-30
JP6316930B2	Japan	B2	2018-04-25
UA117008C2	Ukraine	C2	2018-06-11
JP2018100307A	Japan	A	2018-06-28
EP2968586B1	European Patent Office (EPO)	B1	2018-07-25
DK2968586T3	Denmark	T3	2018-10-08
PT2968586T	Portugal	T	2018-11-13
ES2689523T3	Spain	T3	2018-11-14
LT2968586T	Lithuania	T	2018-11-26
IL240982AThis record	Israel	A	2018-11-29
IL240982B	Israel	B	2018-11-29
IL262984D0	Israel	D0	2018-12-31
RS57739B1	Serbia	B1	2018-12-31
SI2968586T1	Slovenia	T1	2018-12-31
AU2014236305B2	Australia	B2	2019-01-17
PL2968586T3	Poland	T3	2019-01-31
HRP20181580T1	Croatia	T1	2019-02-22
EP3446712A1	European Patent Office (EPO)	A1	2019-02-27
EP3446712A8	European Patent Office (EPO)	A8	2019-04-10
MX365409B	Mexico	B	2019-05-31
AU2019202582A1	Australia	A1	2019-07-25
HUE042640T2	Hungary	T2	2019-07-29
US10420791B2	United States of America	B2	2019-09-24
CY1120790T1	Cyprus	T1	2019-12-11
US2020046752A1	United States of America	A1	2020-02-13
JP6738366B2	Japan	B2	2020-08-12
JP2020169218A	Japan	A	2020-10-15
NZ711657A	New Zealand	A	2021-01-29
KR102248744B1	Republic of Korea	B1	2021-05-06
KR20210049986A	Republic of Korea	A	2021-05-06
AU2019202582B2	Australia	B2	2021-05-27
IL262984A	Israel	A	2021-05-31
IL262984B	Israel	B	2021-05-31
EA037922B1	Eurasian Patent Organization (EAPO)	B1	2021-06-07
IL282453D0	Israel	D0	2021-06-30
NZ751462A	New Zealand	A	2021-07-30
AU2021215095A1	Australia	A1	2021-08-26
KR102311614B1	Republic of Korea	B1	2021-10-08
KR20210122917A	Republic of Korea	A	2021-10-12
JP2022022419A	Japan	A	2022-02-03
IL282453A	Israel	A	2022-03-01
IL282453B	Israel	B	2022-03-01
EA202190410A1	Eurasian Patent Organization (EAPO)	A1	2022-03-31
IL290953A	Israel	A	2022-04-01
IL290953D0	Israel	D0	2022-04-01
CN105142676B	China	B	2022-06-28
CN115154620A	China	A	2022-10-11
US11510937B2	United States of America	B2	2022-11-29
JP7236525B2	Japan	B2	2023-03-09
JP2023054353A	Japan	A	2023-04-13
BR112015022868B1	Brazil	B1	2023-05-16
IL290953B1	Israel	B1	2023-09-01
CA2904151C	Canada	C	2023-09-12
US2023302039A1	United States of America	A1	2023-09-28
IL305374A	Israel	A	2023-10-01
IL290953B2	Israel	B2	2024-01-01
AU2021215095B2	Australia	B2	2024-09-19

3 legal events, as the office reported them to INPADOC

Over the term

Point at a mark for the event

Events

Legal events, newest first
	Event	Code
2020-03-31	Patent renewedKB	KB
2019-05-30	Patent renewedKB	KB
2019-03-31	Patent grantedGrantedFF	FF

Abstract

Term