RS57739B1

Cftr mrna compositions and related methods and uses

Abstract

This record has no abstract on file.

RS57739B1, drawing sheet 1 — Sheet 1 of 58

Term

7.5 yearsto projected expiry

Projected expiry 14 March 2034, counted from filing; an application has no term until it is granted.

15 claims: 9 independent, 6 dependent

1
Patentni zahtevi 1. In vitro prepisana iRNK obuhvata kodirajuću sekvencu koja je najmanje 80% identična SEQ ID N0:3 za upotrebu u lečenju cistične fibroze kod sisara.
2
iRNK za upotrebu na osnovu patentnog zahteva 1, što je kodirajuća sekvenca najmanje 90% ili 100% identična sa SEQ ID NO:
3
iRNK za upotrebu na osnovu patentnih zahteva 1 ili 2, što iRNK obuhvata 5’ netranslatirani region (UTR), 3’ UTR, kodirajuću skevencu za signalni peptid, strukturu kape i/ili strukturu repa.
4
iRNK za upotrebu na osnovu patentnog zahteva 3,što 5’-UTR sadrži SEQ ID N0:4 i/ili 3’UTR sadrži SEQ ID N0:5.
5
iRNK za upotrebu prema patentnim zahtevima 3 ili 4, što je struktura repa poli-A rep dužine od najmanje 70, 100, 120, 150, 200, ili 250 ostataka.
6
iRNK za upotrebu na osnovu bilo kojeg od patentnih zahteva 3-5, što je struktura kape cap1 struktura.
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iRNK za upotrebu na osnovu bilo kojeg od prethodnih patentnih zahteva, što iRNK obuhvata jedan ili više nestandardnih nukleotida, opcionalno gdejedan ili više nestandardnih nukleotida je/su odabrani od 5- metil-citidina, pseudouridina, i 2-tio-uridina.
8
iRNK za upotrebu prema bilo kojem od prethodnih patentnih zahteva, što se kompozicija daje ргеко pluća sisara putem inhalacije, intranazalne davanja, aerosolizacije ili raspršivanja.
9
iRNK za upotrebu na osnovu bilo kojeg od prethodnih patentnih zahteva, što iRNK sadrži kodirajuću sekvencu koja kodira humani protein regulator transmembranske provodljivosti cistične fibroze (CFTR) koji ima sekvencu SEQ ID N0:1, opcionalno kada se humani CFTR eksprimira u epitelijalnim ćelijama pluća.
10
iRNK za upotrebu na osnovu bilo kojeg od prethodnih patentnih zahteva, što se iRNK daje sa nosačem.
11
Kompozicija koja sadrži iRNK i nosač, što je iRNK prepisana iRNK i ima kodirajuću sekvencu koja je najmanje 80% identična sa SEQ ID NO:3.
12
iRNKza upotrebu prema patentnom zahtevu 10 ili kompozicija iz patentnog zahteva 11, što nosač sadrži organski katjon kao katjonski lipid ili katjonski organski polimer.
13
iRNKza upotrebu prema patentnom zahtevu 12 ili kompozicija iz patentnog zahteva 12, što je katjonski organski polimer odabran iz grupe koja se sastoji od polietilenimina (PEI), protamina, PEGilovanog protamina, poli-L-lizina (PLL), i PEGilovanog PLL. 57739 Β1
14
iRNKza upotrebu prema patentnom zahtevu 13 ili kompozicija iz patentnog zahteva 13, što je PEI razgranat PEI sa molekulskom težinom u opsegu od 10 kDa do 40 kDa.
15
iRNKza upotrebu prema bilo kojem od patentnih zahteva 10 i 12-14 ili kompozicija iz bilo kog od patentnih zahteva11-14, što je nosač lipozom.

Independent claims15

2,310 paragraphs in 10 sections, as filed

Description

BACKGROUND OF THE INVENTION The present invention relates to a cystic fibrosis transmembrane regulator (CFTR), mRNA compositions, uses thereof, and methods for their production and use.

Cystic fibrosis is an autosomal inherited disorder caused by a mutation within the CFTR gene that encodes a chlorine ion channel that is believed to participate in the regulation of many other ion channels and transport systems in epithelial cells. Loss of CFTR function results in chronic lung disease, impaired mucus production, and a dramatic reduction in life expectancy. See generally Rowe et al., New Engl. J. Med. 352, 1992-2001 (2005).

Despite the cloning of the CFTR gene back in 1989, no effective therapies have yet been developed to replace CFTR and treat cystic physbrosis. Literature data have documented numerous problems encountered during attempts to induce CFTR expression in the lungs. For example, viral vectors containing CFTR DNA elicited immune responses and CF symptoms continued to exist after administration of the vector. Conese et al., J. Cyst. Fibros. 10 Suppl 2, S114-28 (2011); Rosenecker et al., Curr. Opin. Mol. Ther. 8, 439-45 (2006). Non-viral DNA administration, including CFTR DNA, has also been described to induce immune responses. Alton et al., Lancet 353, 947-54 (1999); Rosenecker et al., J Gene Med. 5, 49-60 (2003). Furthermore, non-viral vectors face an additional problem, which is that the machinery of the nucleus horn complex usually does not introduce DNA into the nucleus, where transcription (transcription) should take place. Pearson, Nature460, 164-69 (2009).

Another source of problems for inducing CFTR expression in the lungs is the lung environment alone. Pulmonary surfactant has been reported to reduce the efficacy of cation transfer agents for lipids such as lipoectamine (DOSPAOOPE).

Emst et al., J. Gene Med. 1, 331-40 (1999). Also, Rosenecker et al., 2003, supra, identified several inhibitory components present in the airway surface fluid that may interfere with either polymer-mediated or lipid-mediated transfection. Information RNA therapy has been proposed as a general approach to induce the expression of a therapeutic or replacement protein. The concept of introducing messenger RNA (mRNA) as a mode of protein production in a host has been previously reported (Yamamoto, A. et al., Eur. J. Pharm. 2009, 71, 484-489;

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Debus, Η. et al. J. Control Rel. 2010, 148, 334-343). However, obvious lung-specific difficulties have been reported for mRNA delivery using certain lipocomplex formulations. For example, a comparison of in vitro and in vivo performance of lipocomplexes carrying mRNA or DNA revealed that, although the mRNA composition gives greater expression in cultured cells, the expression measurable was detected only with the DNA composition when administered intranasally to mouse lungs. Andries et al., Mol. Pharmaceut. 9, 2136-45 (2012).

It should also be noted that CFTR is a large gene relative to a model or reporter gene such as luciferase switch (FFL). Compare the lengths of the wild-type (WT) CFTR coding sequences (SEQ ID NO: 2) and the FFL coding sequences (SEQ ID NO: 7). The difference in length can affect stability in certain circumstances, and thus whether it will be expressed and how much is expressed by any dose of mRNA and protein production. Furthermore, although in vitro synthesis of mRNA by cells is preferred due to the lack of normal cellular mRNA and other cellular components that represent unwanted contaminants, in vitro synthesis of mRNA with a long coding sequence such as CFTR mRNA is significantly more difficult than in vitro synthesis of short mRNA. by a coding sequence such as FFL.

PCT Patent Publication VV02007 / 024708 and US (US) Patent Publications US2010 / 0203627 and US2011 / 0035819 discuss the therapeutic administration of CFTR mRNA, but do not provide a demonstrated reduction to the practice of producing functional CFTR in the lung after administration of CFTR mRNA or does not provide sufficient guidance. how to solve problems related to induction of CFTR expression in the lungs using in vitro transcribed CFTR mRNA. These include difficulties in achieving in vitro mRNA synthesis and difficulties specific to the interactions of mRNA compositions with lung-specific substances that researchers such as Andries et al., Supra, have found to make mRNA composition ineffective in inducing expression even when appropriate compositions are based. on DNA provide some level of expression. WO 2008/045548 describes codoptimized depletion for CpG-CFTR RNA.

Therefore, there is a need for improved materials, formulations, manufacturing methods and methods for delivering CFTR mRNA to induce CFTR expression, including mammalian lungs for the treatment of cystic fibrosis.

SUMMARY OF THE INVENTION The present invention is based, in part, on the development of non-naturally occurring formulations of CFTR mRNA and CFTR mRNA and a method for their administration that can induce the expression of functional CFTR in vivo. The compositions, methods, and uses described herein may provide CFTR expression in the lungs of large mammals with a favorable safety profile for the effective treatment of cystic fibrosis.

Specifically, the present invention encompasses an in vitro transcribed mRNA comprising a coding sequence that is at least 80% identical to SEQ. The invention further provides a composition comprising an mRNA and a carrier, wherein the mRNA is an in vitro transcribed mRNA and has a coding sequence that is at least 80% identical to SEQ ID NO: 3.

57739 Opis1 This disclosure also provides a method for the in vivo production of CFTR, in particular, in the lungs of a subject (e.g., a mammal) to whom delivery is required by administration of an mRNA encoding a CFTR protein. The mRNA encoding the CFTR protein can be deposited directly into the subject's lungs. As used herein, a CFTR protein encompasses any whole, fragment, or portion of a CFTR protein that can be used to replace the activity of a native CFTR protein and / or reduce the intensity, severity, and / or frequency of one or more symptoms associated with cystic fibrosis. For example, a suitable CFTR protein may have an amino acid sequence identical to the natural human CFTR protein (SEQ ID NO: 1).

The disclosure also provides a method of inducing CFTR expression in mammalian lung epithelial cells, a method comprising contacting mammalian lung epithelial cells 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 encoding SEQ ID NO: 1.

The disclosure also provides a method for inducing CFTR expression in mammalian target cells, a method comprising contacting mammalian lung epithelial cells with a composition, the composition being a pharmaceutical composition comprising an in vitro transcribed mRNA encoding SEQ ID NO: 1.

The disclosure provides a non-naturally occurring mRNA molecule comprising a coding sequence, a 5'-UTR, and a 3'-UTR, wherein the coding sequence is at least 80% identical to SEQ ID NO: 3. In one example, the coding sequence is about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3.

In another example, the disclosure provides a composition comprising a polynucleotide according to the invention, an RNA polymerase, and a nucleoside triphosphate.

In another example, the disclosure provides a pharmaceutical composition comprising mRNA based on a description.

The disclosure provides an apparatus for nebulization or aerosolization filled with a pharmaceutical composition described herein.

The disclosure provides a culture cell comprising mRNA as described herein and a functional CFTR expressed by mRNA.

The disclosure provides the use of a pharmaceutical composition as described herein for inducing the expression of functional CFTR.

In another example, the disclosure provides a method for inducing CFTR expression in epithelial cells in a mammalian lung, a method comprising contacting the epithelial cells with a composition, wherein the composition is a pharmaceutical composition comprising mRNA as described herein.

In another example, the disclosure provides a method of inducing CFTR expression in a target mammalian cell, a method comprising contacting the target mammalian cells with a composition, the composition comprising the mRNA described herein.

57739 Drugomί In another aspect, the present invention provides a composition for use in 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 aspect, the mRNA is delivered to the lungs of the subject. In one aspect, the mRNA is delivered by inhalation, nebulization, intranasal delivery, or aerosolization. In various aspects of administration, the mRNA results in the expression of CFTR in the lungs of the subject.

The present invention provides a composition for use in a method of treating cystic fibrosis by administering a lung to a subject in need of treatment, an mRNA comprising a coding sequence encoding SEQ ID NO: 1. In another particular aspect, the present invention provides a composition for use in a method of treating cystic fibrosis by administering to a subject in need of treatment an mRNA comprising a coding sequence encoding SEQ ID NO: 3. In another aspect, the present invention provides a composition for use in a method of treating cystic fibrosis by administering via the lung to a subject in need of treatment, an mRNA comprising a coding sequence that is at least 80%, 85%, 90%, 95%, 96 %, 97%, 98%, or 99% identical to SEQ ID NO: 3.

In another embodiment, the disclosure provides a method of making an CFTR of mRNA in vitro, comprising an isolated polynucleotide as described herein with RNA polymerase in the presence of a nucleoside triphosphate, wherein: the isolating polynucleotide and RNA polymerase are not contained in the cell; the isolated polynucleotide is a template for RNA polymerase; the isolated polynucleotide comprises a promoter operably linked to a template sequence, and RNA polymerase synthesizes mRNA comprising the coding sequence encoding SEQ ID NO: 1.

Additional objects and advantages of the invention will be presented in the descriptive part which follows, and will be in part apparent from the description, or may be learned and learned by application of the invention. The objects and advantages of the invention may be realized and achieved in terms of the elements and combinations particularly emphasized in the claims.

It is to be understood that both the foregoing description and the following details are exemplary and illustrative only and are not restrictive of the invention as claimed in the claims.

The drawings accompanying the text, which are incorporated into the text and form an integral part of the specification, illustrate several aspects of the invention and together with the description serve to explain the principles of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS The above-mentioned aspects and advantages of the present invention may become apparent from the detailed description which follows together with the accompanying drawings.

SlikalA. Detection of mature C band for human CFTR protein 24 hours after transfection with human CFTR mRNA. Successful protein production was observed for both unmodified and modified (SNIM) mRNA (containing 5% 2-thiouridine and 5-methylcytidine). Immuoprecipitation was performed with R&D Systems MAB25031 antibody and detection was done with Ab570.

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Figure 1Β. Western Blot analysis of CFTR KO of mouse lungs 24 hours after exposure to PEI / unmodified human CFTR mRNA nanoparticles. Mice were treated with a nebulizer (Pari Vou jet nebulizer) for a period of approximately one hour. Immunoprecipitation of human CFTR protein obtained on the basis of the provided procedure was performed. Mature C band was detected in all treated mice but was not detected in control mice.

Figure 2. Current-voltage diagram of 8-Br-cAMP awakened currents of treated (4 ug hCFTR mRNA) and untreated HEK293T cells. A large current was induced within the hCFTR mRNAs that were transfected cells compared to untreated cells. Treated cells that were exposed to a specific CFTR protein inhibitor, CFTRinh-172, showed a marked reduction (~ 89%) in the Cl-ion current.

Figure 3. Histogram diagrams of 8-Vg-sAMR excited currents of treated (4 ug hCFTR mRNA) and untreated HEK293T cells after application of + 80mV membrane potential. A large current was induced within the hCFTR mRNAs that were transfected cells compared to untreated cells. Treated cells that were exposed to a specific CFTR protein inhibitor, CFTRinh-172, showed a marked reduction (~ 89%) in the Cl-ion current.

Figure 4. Diagram of comparative current-voltage profiles HEK 293 native cells, those exposed to forxoline and GlyH-101. No significant changes in electricity were recorded in any of the scenarios.

Figure 5. Current-voltage diagram of currents awakened by forxoline-treated (4 ug hCFTR mRNA) and untreated HEK293T cells. High current was induced within hCFTR mRNA of transfected cells compared to untreated cells. Treated cells that were exposed to a specific CFTR protein inhibitor, CFTRinh-172, showed a marked reduction (~ 95%) in the Cl-ion current as shown in the diagram step (+100 mV) on the right side of the graph.

Figure 6. In situ hybridization of human CFTR mRNA in untreated (PBS) (left) and treated (right) CFTR KO mouse lungs. Mice were exposed to 30 ug encapsulated unmodified CFTR mRNA in PEI nanoparticles via intratracheal delivery. Significant positive staining was observed through both lungs 24 hours after delivery.

Figure 7. In situ hybridization of human CFTR mRNA-treated CFTR KO mouse lungs under different magnifications (up to 20x magnification). Mice were exposed to 30 μg of encapsulated unmodified CFTR mRNA in PEI nanoparticles via intratracheal administration.

Figure 8. High magnification (40x) of representative lung cross sections show in situ hybridization of human CFTR mRNA treated (right) CFTR KO mouse lungs. Human CFTR mRNA was detected in the pical cytoplasm of target bronchial epithelial cells 24 hours after administration. Mice were exposed to 30 encapsulated unmodified CFTR mRNA and PEI nanoparticles via intratracheal administration.

Figure 9. Comparison of in situ hybridization staining with human CFTR mRNA-treated CFTR KO mouse lungs at 6 hours (left) and 24 hours (right) after administration. They are mice

57739 Β1 exposed to 30 ug encapsulated unmodified CFTR mRNA in PEI nanoparticles via intratracheal delivery. Intense positive staining was observed within six hours through the complete lungs within the bronchial and alveolar regions while significant positive staining was still visible 24 hours after delivery.

Figure 10. In situ hybridization of human CFTR mRNA in untreated (PBS) and treated (bottom) CFTR KO mouse lungs. Mice were exposed to up to 15 μg of encapsulated unmodified hCFTR mRNA in C12-200 lipid nanoparticles via intratracheal delivery. Significant positive staining was seen through both lungs 6 hours after administration.

Figure 11. High magnification (40x) of representative lung cross-sections show in situ hybridization of human CFTR mRNA-treated CFTR KO mouse lungs. Human CFTR mRNA was detected in the apical cytoplasm of the target bronchial epithelium (left) as well as in the intracellular alveolar regions (right) six hours after administration. Mice were exposed to 15 μg of encapsulated unmodified hCFTR mRNA in S12-200 lipid nanoparticles via intratracheal delivery.

Figure 12. Search of different cell lines for hCFTR expression. Immunoblot analysis of CHO and COS-7 (A) iBHK and PKC (B) cells transfected with hCFTR coding constructs. Protein lysate was prepared 24 hours after transfection and examined with MA1-935 as the primary antibody. The arrow indicates the default CFTR. See in the discussion MA1-935 specificity in example 6

Figure 13. Cross-reactivity of different anti-human CFTR antibodies. (A) - mouse anti-human CFTR MA1-935 (Chemicon): (B) - mouse anti-human CFTR AB570 (Cystic Fibrosis Foundation): (C) - mouse anti-human CFTR AB596 (Cystic Fibrosis Foundation): (D ) rabbit anti-human CFTR G449 (Rockefeller University). The arrow shows the CFTR.

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

Figure 15. Immunoprecipitation and Western blot analysis of mice 24 hours after IT spray given 20 cd hCFTR SNIM RNA / 10 cd FFL SNIM RNA each in the HGT5001 formulation of Example 6. T84 cells served as a positive control showing mature glycosylated C band and manosylated B band of hCFTR. supernatant residue of cell extract fraction without immunoprecipitated fraction. IP immunoprecipitated fraction.

Figure 16. Immunoprecipitation of hCFTR from T84 cells by MAB25031 followed by immunodetection by AB570 (A) and MAB1660 (B).

Figure 17. Immunoprecipitation of CFTR from NIH3T3 cells 72 hours after transfection with different constructs.

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Figure 18. Immunoprecipitation of CFTR from NIH3T3 cells 72 hours after transfection with different constructs using 500 μg protein and MAB1660 (left and center panels) and increased total protein (8 mg) using MAB25031 (right panel).

Figure 19. Immunoprecipitation of CFTR using MAB25031 and further immunodetection with AB570 from pig lung samples after delivery of hCFTR SNIMRNA in PEI formulation from Example 6. Column 1: samples from left caudal lobe of pig luciferase negative # 2, Column 2: samples from pig region positive for luciferase # 1.

Figure 20. Spraying was performed on pigs that were under anesthesia and ventilation (left). The nebulizer was connected in line with the ventilation system (right, see white arrow).

Figure 21. Luciferase expression measured in homogenates of porcine tissue samples from different lung areas after aerosol administration of 1 mg FFL SNIM RNA in PEI Formulation of Example 6 with EFIow air spray. Lung samples were grown ex vivo overnight for luciferase measurements (pg luciferase / mg lung tissue).

Figure 22. BLI expression of luciferase in representative samples of porcine tissue from different regions of the lung after aerosol administration of 1 mg FFL SNIM RNA in PEI Formulation Example 6. Lung samples were exh vivo grown overnight after the measurement.

Figure 23. BLI representation of luciferase expression in representative samples of porcine tissue from different regions of the lung after aerosol administration of 1 mg FFL SNIM RNA in PEI Formulation of Example 6 using PARI BOI nebulizers. Examples of lungs were ex vivo grown overnight after measurements.

Figure 24. BLI representation of luciferase expression in representative samples of porcine tissue from different regions of the lung after aerosol administration of each of 1 mg of FFL SNIM RNA and hCFTR mRNA in the PEI Formulation of Example 6 using an Aeroneb air spray. Lung samples were grown ex vivo overnight before measurement.

Figure 25. BLI expression of luciferase in representative porcine tissue samples 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 Aeroneb air nebulizer (nebulizer) Lung samples were grown ex vivo overnight before measurement.

Figure 26. BLI expression of luciferase in pig tissue samples from different regions of the lung from one untreated control pig. Another untreated control pig showed the same result (data not shown).

Figure 27. BLI expression of luciferase in lung samples of single-treated pigs # 3 and # 6. Aerosol administration for each 1 mg of FFL SNIM RNA and hCFTR SNIM RNA in the PEI Formulation of Example 6 was performed using an Aeroneb air nebulizer (nebulizer). Sections of whole pig lungs are shown. Top three rows: pig # 3, bottom three rows: pig # 6.

Figure 28. BLI expression of luciferase in lung samples of double-treated pigs # 4 and # 8. Aerosol application for each 1 mg FFL SNIM RNA and hCFTR SNIM RNA in PEI

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The formulation of Example 6 was performed using an Aeroneb air spray. Sections of whole pig lungs are shown. Top three rows: pig # 4, bottom three rows: pig # 8.

Figure 29. BLI expression of luciferase in lung samples of triple-treated pigs # 1 and # 2. The administration of the aerosol for each 1 mg of FFL SNIM RNA and hCFTR-mRNA SNIM RNA in the PEI Formulation of Example 6 was performed using an Aeroneb air nebulizer (nebulizer). Sections of whole pig lungs are shown. Upper three rows: pig # 1, lower three rows: pig # 2.

Figure 30. Luciferase IHC on lung tissue of triple-treated pig # 1. Aerosol administration of each 1 mg of FFLSNIM RNA and hCFTR SNIM RNA in the PEI Formulation of Example 6 was performed using an Aeroneb air nebulizer (nebulizer). Luciferase expression is detected as a red-pink color (Anti-Luciferase pAb 1: 300, G7451, Promega, Refine AP-Kit, chromogen: New fuchsin).

Figure 31. High BLI-positive lung tissue of triple-treated pig # 1 was subjected to hCFTR IP / VB. Line 1: T84 cells (positive control), Line 2: untreated pig lung tissue (300 mg), Line 3: treated pig lung tissue (697 mg), Line 4: treated pig lung tissue (163 mg). Mature complex-glycosylated hCFTR appeared as a dispersed so-called. C-strip. Mannose-rich HCFTR, appeared as a much denser so-called. B-bar. Expression of hCFTR was observed in T84 cells and lung tissue hCFTR SNIM RNA of treated pig # 1, while in untreated pigs hCFTR expression was not observed.

Figure 32. Immunoprecipitation of hCFTR by MAB25031 and subsequent immunodetection by AB570 from pig lung samples after hCFTR RECORD RNA delivery to PEI Formulation Example 6. Line 1: sample from luciferase-negative left caudal lobe of pig # 2, Line 2: sample from positive pig lung region # 1.

Figure 33 A & B. In vitro transfection of HEK 293T cells with C-terminally labeled His-ιο (CO-CFTR-C-His10) and unlabeled (CO-CFTR) codon-optimized human CFTR SNIM RNA. After transfection, whole cell lysate was collected and analyzed by Western blot for human CFTR expression using (A) anti-CFTR antibody # 217 and (B) anti-His antibody 1187. Transfected samples were compared with untransfected HEK293T control lysate. 3).

Figure 33 C. In vitro transfection of HEK 293T cells with SNIM RNA encoding codonoptimized human CFTR with growth hormone leader sequence and (GH-CO-CFTR) or SNIM RNA encoding C-terminally labeled Hisio codon optimized human CFTR (CO -CFTR- C-His10). After transfection, whole cell lysate was collected and analyzed for human CFTR expression using Western blot using antiCFTR antibody # 217. Transfected samples were compared with non-transfected HEK 293T control lysate (Line 3).

Figure 34. In vivo transfection of mouse CFTR nails with a C10 terminal His10 tagged codon optimized with human CFTR SNIM RNA encapsulated within a lipid (cKK-E12) or polymeric (PEI) nanoparticle formulation. After dispersal of each appropriate mRNA formulation, left and right wing lysates were collected and analyzed for CFTR expression by Western blot using anti

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His antibody 1187. Control CFTR “knockout” of lung tissue and CFTR-Hisio HEK293 lysate were used as negative and positive controls.

Figure 35. Detection of FFL expression by bioluminescence in pig lung samples collected after spraying with water for injection.

Figure 36. Bioluminescent detection of FFL expression in pig lung samples collected after spraying with 1 mg FFL SNIM RNA + 1 mg CO-CFTR SNIM RNA in a branched 25 kDa PEI formulation.

Figure 37. Bioluminescent detection of FFL expression in pig lung samples collected after spraying with 1 mg FFL SNIM RNA + 5 mg CO-CFTR SNIM RNA in a branched 25 kDa PEI formulation.

Figure 38. Bioluminescent detection of FFL expression in pig lung samples collected after spraying with 1 mg FFL SNIM RNA + 10 mg CO-CFTR SNIM RNA in a branched 25 kDa PEI formulation.

Figure 39. Relative quantification of CFTR expression in different groups. The intensity of the bands was normalized to 150 kDa bands in the protein scale.

Figure 40. Representative example of CFTR-positive bronchi with at least one epithelial cell detected within the epithelial cell layer and showing a pure, membrane-localized CFTR signal via CFTR immunohistochemical staining, obtained using anti-CFTR antibodies.

Figure 41. Immunohistochemical staining of CFTR in pig lungs after aerosol delivery control (WFI) or 5 mg CO-CFTR SNIM RNA.

Figure 42. Represents a low level of CFTR expression, analyzed in pig lungs by immunohistochemical staining with anti-CFTR after aerosol delivery of 5 mg CO-CFTR SNIM RNA.

Figure 43. Represents the mean level of CFTR expression, analyzed in pig lungs by immunohistochemical staining with anti-CFTR after aerosol delivery of 5 mg CO-CFTR SNIM RNA.

Figure 44. Represents a high level of CFTR expression, analyzed in pig lungs using immunohistochemical staining with anti-CFTR after aerosol delivery of 5 mg CO-CFTR SNIM RNA.

Figure 45. Immunohistochemical staining of CFTR in pig lungs after aerosol delivery control (WFI) or 10 mg CO-CFTR SNIM RNA.

Figure 46. Quantification of relative numbers of CFTR-positive bronchi / bronchioles per animal. Analysis of each group (WFI; and 1mg, 5mg, 10mg human CFTR SNIM RNA) 24 hours after aerosol administration. CFTR expression normalized to signal intensity for 150 kDa protein standard. (WFI = 9.465.6%, 1MG = 15.266.6%, 5MG = 25.4614.1%, 10MG = 20.963.7%, VFI vs 5MG p = 0.0281, VFI vs 10MG p = 0.0174).

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Figure 47. Shows multiple in situ detection of nucleic acid (A) ubiquitin C and (B) dap B in pig lungs, after aerosol delivery of water for injection using a nebulizer.

Figure 48. Shows multiple in situ detection of nucleic acid (A) ubiquitin C and (B) dap B in pig lungs, after aerosol delivery of 1 mg FFL SNIM RNA +10 mg CO-CFTR SNIM RNA in a branched 25 kDa PEI formulation.

Figure 49. Shows multiple in situ detection of nucleic acid (A) of the right cranial and (B) left cranial incision of the pig, after aerosol delivery of water for injection using a nebulizer.

Figure 50. Shows multiple in situ detection of nucleic acid (A) of the right cranial and (B) left cranial incision of a pig, after aerosol delivery of 1 mg FFL SNIM RNA + 1 mg CO-CFTR SNIM RNA in a branched 25 kDa PEI formulation.

Figure 51. Shows multiple in situ detection of nucleic acid (A) of the right cranial and (B) left cranial incision of a pig, after aerosol delivery of 1 mg FFL SNIM RNA + 5 mg CO-CFTR SNIM RNA in a branched 25 kDa PEI formulation.

Figure 52. Shows multiple in situ detection of nucleic acid (A) of the right cranial and (B) left cranial incision of a pig, after aerosol delivery of 1 mg FFL SNIM RNA + 10 mg CO-CFTR SNIM RNA in a branched 25 kDa PEI formulation.

Figure 53. Shows the positive detection of active luciferase (FFL) protein in treated pig lungs of rheumatoid luminescence and after exposure to lipid nanoparticles CKK-E12 with encapsulated FFL / CO-CFTR-C-His10 mRNA. Pigs were treated with 1 mg FFL + 9 mg CO-CFTR-C-His10 mRNA encapsulated lipid nanoparticles by nebulization using a Pari jet nebulizer and sacrificed 24 hours after treatment.

Figure 54. Illustrates the results as an example of hCFTR expression in HEK cells transfected with nebulozovanin complexes given to pigs 10, 11, and 12 (dose 1 mg).

Figure 55. Illustrates the results as an example of hCFTR expression in HEK cells transfected with nebulozovanin complex given to pigs 13, 14, and 15 (5 mg dose) and in HEK cells transfected with nebulozovanin complex given to pigs 19, 20, and 21 (dose 10 mg).

Figure 56. Illustrates the results as an example of hCFTR expression in HEK cells transfected with nebulozovanin complex given to pigs 16 (5 mg doses), 22 (10 mg doses) and 67 (1 mg doses).

Figure 57. Illustrates the results as an example of hCFTR expression in HEK cells transfected using nebulozovanin complexes given to pigs 17, 18 (5 mg doses), 23, 24 (10 mg doses) and 68, 69 (1 mg dose).

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DETAILED DESCRIPTION OF THE INVENTION

Definitions As used herein, the term polynucleotide is generally used to refer to a nucleic acid (on either DNA or RNA). The terms polynucleotide, nucleic acid, DNA, RNA and mRNA include such molecules consisting of: standard or unmodified residues; non-standard or modified residues; and mixtures of standard and non-standard residues.

As used herein, the term mRNA is used herein to refer to modified and unmodified RNA including both coding and non-coding regions.

As used herein, the term encoding an mRNA region generally refers to a moiety that, when translated, results in the production of an expression product, such as a polypeptide, protein, or enzyme.

A non-standard nucleobase is a base that is second to the natural bases of adenine (A), cytosine (C), guanine (G), thymine (T), or uracil (U). A non-standard nucleobase is an analog of a specific nucleobase (A, C, G, T, or U) when its pairing properties are within the double helix of nucleic acids and the site of incorporation of DNA or RNA polymerases within the double helix of nucleic acids (including local DNA-RNA helix as the one formed during the transcription of the DNA template by RNA polymerase) most similar to one of the five nucleobases listed above, with the exception that T analogs will also be U and vice versa analogs. For the purpose of determining the percentage of identity of the first sequence relative to the second sequence, the base analog is not a mismatch with the natural base; for example, pseudouridine corresponds to uridine, 5-methylcytidine corresponds to cytidine, and so on.

The term non-standard when used in conjunction with terms including but not limited to a nucleoside, nucleotide, or residue should be interpreted in the same manner as used in conjunction with a nucleobase.

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 example, a sequence of 100 nucleotides containing exactly 30 cytosines, exactly 30 guanines, exactly one cytosine analog, and exactly one guanine analog has a GC content of 62%.

As used herein, a less favored codon that is translated less efficiently or less rapidly by a mammalian cell than another codon of the same amino acid residue. Less favored codons generally include codons with A or U at the third or wobble position of the codon. For a discussion of less favored codons, see for example U.S. patent publication 2009/0069256 A1.

A non-naturally occurring RNA molecule is an mRNA that is not produced during normal transcription (transcription) and processing (splicing) in the original (wild type) cells. An mRNA can be qualified as one that cannot be found in nature in terms of its sequence (for example a series of codons and / or one or more UTRs that are not in the natural CFTR of the mRNA and / or include non-standard nucleotide residues. RNA molecules which is not found in nature can be synthesized in vitro.

57739 Β1 In each of Tables 1 and 2 below, the NWT column indicates a non-wild base at position (Pos) in the CFTR coding seven (see rg: SEQ ID NO: 3), And the WT column points to a wild-type base at the same position (see rg. SEQ ID NO: 2 or RefSeq entry for human CFTR (access number NM_000492.3, Feb. 10, 2013, version, available at GenBank; note that the sequence NM_000492.3 contains a non-coding sequence so that the coding sequence appears at positions 133 to 4575, so that for example, position 7 in the table below corresponds to position 139 NM_000492.3).

Non-naturally occurring CFTR mRNA molecules In addition to providing methods for producing CFTR in vivo using natural or wild (wild type) CFTR mRNA (and compositions containing that mRNA), the disclosure also provides non-naturally occurring CFTR mRNA. in nature encoding a CFTR protein (e.g. SEQ ID NO: 1). In some embodiments, the non-naturally occurring CFTR mRNA molecule is purified or isolated.

In other embodiments, a non-naturally occurring CFTR mRNA molecule is present in the cell. In some embodiments, a cell comprising a non-naturally occurring CFTR mRNA molecule has not synthesized a non-naturally occurring CFTR mRNA molecule and / or does not contain DNA complementary to a naturally occurring CFTR mRNA molecule and / or a functional CFTR gene; the cell may optionally contain an inactive CFTR gene such as a CFTR gene with a nonsense, missense, frameshift, insertion or deletion mutation leading to a non-functional gene product. In some embodiments, a cell comprising a non-naturally occurring CFTR mRNA further comprises a functional CFTR protein that is translated from a naturally occurring CFTR mRNA molecule. The cell may be, for example, an epithelial cell, a liver cell, or a kidney cell. In some embodiments, the cell is a cell culture.

CFTR coding sequence The CFTR mRNA described herein contains a coding sequence with fewer crypt promoter complements than SEQ ID NO: 2 (i.e., a wild-type human CFTR coding sequence), fewer direct and / or inverted repeats than SEQ ID NO: 2, fewer non-favored codons than SEQ ID NO: 2 and / or a GC content of the coding sequence that is lower than the GC content of SEQ ID NO: 2.

Crypto promoters, direct and / or inverted repeats and / or unfavored codon sequences can be recognized by those skilled in the art using routine procedures. For example, direct and / or inverted repeats found in a sequence can be determined by sequence analysis (Liu et al., JouRNKI of Theoretical Biology (2014) 344: 19-30). The content of the cryptic promoter in the sequence can also be determined by sequence analysis, for example the presence of a Shine-Dalgarno sequence in the construct or the like.

The CFTR mRNA according to the invention is transcribed (transcribed) in vitro, i.e. the mRNA is synthesized in an artificial setting and not in a living cell (e.g. an in vitro cell-free transcription system). In general, in vitro transcription involves providing a DNA template containing a promoter and sequence to complement the desired mRNA (which may be circular or linear), RNA polymerase, nucleoside

57739 Β1 triphosphate in a suitable reaction environment (salts, buffers, and temperature). RNA inhibitors, reducing agents, and / or polyphosphates may also be present in the reaction mixture. In some embodiments, the RNA polymerase is a T7 RNA polymerase.

Table 1. Non-wild-type databases that can be used to encode the CFR mRNA sequence. Pos - position; WT- wild strain; NWT- non-wild strain

57739 Yes

57739 Β1

57739 Β1

57739 Β1

57739 Β1

57739 Yes

57739 Β1

57739 Β1

57739 Β1

57739 Yes

Table 2. Subset of non-wild-type databases that can be used to encode the CFTR mRNA sequence. Pos - position; WT- wild strain; NWT- non-wild strain

57739 Yes

57739 Β1

57739 Yes

57739 Β1

57739 Β1

The present invention encompasses a non-naturally occurring CFTR mRNA comprising the coding sequence of SEQ ID NO: 3 for use in the treatment of cystic fibrosis in a mammal. Additional examples of non-naturally occurring coding sequences of CFTR mRNA are described in the Summary of the Sequences, 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 that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NO: 3. In in some embodiments, the non-naturally occurring CFTR mRNA comprises a 5'UTR, 3'UTR, coding sequence for a signal peptide or cap or tail structure as described below.

The CFTR mRNA described above comprising a coding sequence that differs from the wild-type CFTR coding sequence may provide advantages in terms of efficiency and ease of preparation. For example, in vitro transcription reactions using a polynucleotide comprising a sequence template that complements the CFTR coding sequence can yield higher RNA yields; a polynucleotide comprising said sequence of the template being more stable (i.e., less prone to mutations) during growth in the host cell, reducing the amount of purification necessary to generate the template used in the reaction; and in vivo translation of the mRNA comprising the coding sequence may be greater.

Signal Peptide Sequence In some embodiments, the CFR protein mRNA incorporates a nucleotide sequence.

57739 Β1 encoding a signal peptide. As used herein, the term signal peptide refers to a preptide present in a newly synthesized protein that can direct the protein toward the secretory pathway. In some embodiments, the signal peptide is excised after translocation into the endoplasmic reticulum after mRNA translation. The signal peptide is also referred to as a signal sequence, sequence leader or leader peptide. Typically, the signal peptide is a short (e.g., 5-30, 5-25, 5-20, 5-15, or 5-10 amino acid long) peptide. The signal peptide may be present at the N terminus (end) of the newly synthesized protein. Without wishing to be bound by any particular theory, the incorporation of a coding sequence for a signal peptide into a mRNA encoding a CFTR may facilitate the secretion and / or production of CFTR protein in vivo.

A suitable signal peptide for use in the present invention may be a heterogeneous sequence derived from different 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: 27282732. A suitable signal peptide can be derived from human growth hormone (hGH), serum albumin preprotein, Ig kappa light chain precursor, azuroricidin proprotein, cystatin-S precursor, trypsinogen precursor 2, potassium channel blocker, alpha conotoxin 1p1.3, alpha galactosidase, cellilase, aspartic proteinase nepentensin-1, acid chitinase, K28 prepro toxin, precursor killer toxin zygocin, and cholera toxin. Examples of the peptide sequence are described in Kober, et al., Biotechnol. Bioeng., 110: 1164-73, 2012.

In some embodiments, the mRNA encoding the CFTR may comprise a sequence encoding a signal peptide derived from human growth hormone (hGH), or a portion thereof. A non-limiting coding nucleotide sequence encoding the hGH signal peptide is shown below.

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

AUGGCCACUGGAUCAAGAACCUCACUGCUGCUCGCUUUUGGACUGCUUUGCCUGC CCUGGUUGCAAGAAGGAUCGGCUUUCCCGACCAUCCCACUCUCC

Alternative sequence 5 'of human growth hormone (hGH) (SEQ ID NO: 19):

AUGGCAACUGGAUCAAGAACCUCCCUCCUGCUCGCAUUCGGCCUGCUCUGUCUCC CAUGGCUCCAAGAAGGAAGCGCGUUCCCCACUAUCCCCCUCUCGG In some embodiments, the mRNA of the present invention may, at least,%,%,%, 75% of the sequence, encoding a sequence peptide encoding a sequence of , 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 18orSEQ ID NO: 19.

5'-UTR, 3'-UTR, Poly-A tail, kappa and non-standard nucleotide residues In some embodiments, the mRNA comprises a sequence at its 5'-UTR end that is identical to SEQ ID NO: 4 or 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%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 4.

57739 In some embodiments, the mRNA comprises a sequence at its 3'-UTR end that is identical to SEQ ID NO: 5 or 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%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 5.

In some embodiments, the mRNA comprises a poly-A rep. In some embodiments, the polyA ger has a length of at least 70, 100, 120, 150, 200, 250, 300, 400, or 500 residues. In some embodiments, the poly-A ger has a length in the range of 70 to 100, 100 to 120, 120 to 150, 150 to 200, or 200 to 300, 300 to 400, or 400 to 500 residues. Poly A tails can be added using various techniques known in the art art. For example, long poly A tails can be added to synthetic or in vitro transcribed RNA using poly A polymerase Uokoe, et al. Nature Biotechnology. 1996; 14: 1252-1256). The transcription vector can also encode long poly A tails. Additionally, poly A tails can be added by transcription directly from the PCR product (polymerase chain reaction). Poly A can be ligated for 3 'kgaj sense (read) RNA with RNA ligase (see for example, Molecular Cloning A Laboratory Manual, 2nd Ed., Ed. By Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory) Press: 1991 edition)). In some embodiments, the pol-U or poly-C ger may be used in place of or in addition to the poly-A beet. For example, CFTR-encoding RNA may include a 3 'poly (C) gera structure. A suitable poly-C ger at the 3 'terminus of the mRNA typically comprises about 10 to 200 cytosine nucleotides (for example about 10 to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides). The poly-C ger can be added to the poly-A tail or the poly-A ger can be ground.

In some embodiments, the mRNA comprises a 5'-cap, for example, a cap1 structure. For enzymes that add a cap to mRNA and appropriate procedures see for example, Fechter, P .; Brownlee, GG Recognition of mRNA cap structures by viral and cellular proteins J. Gen. Virology 2005, 86, 1239-1249; European patent publication 2 010 659 A2; U.S. Pat. 6,312,926. The 5 'kappa is typically added as follows: first the RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates: guanosine triphosphate (GTP) is then added to the terminal phosphates of rgeco guanylyl transferase, producing 5'5 ' 5 triphosphate bonds; And 7-nitrogen guanine is then methylated by methyl transferase. Examples of cap structures include, but are not limited to, m7G (5 ') ppp (5' (A, G (5 ') ppp (5') A and G (5 ') ppp (5') G.

In some embodiments, the mRNA comprises one or more non-standard nucleotide residues. Non-standard nucleotide residues may include, for example, 5-methyl-cytidine (5 mC), pseudouridine (JU), and / or 2-thio-uridine (2 cm 3). See, e.g., U.S. Pat. 8,278,036 or VV02011012316 for a discussion of such residues and their incorporation into mRNA. In some embodiments, the mRNA may be SNIM RNA. As used herein, SNIM RNA is an acronym for stabilized nonimmunogenic information RNA, certain information RNA produced by in vitro transcription (IVT) including certain percentages of modified nucleotides in the ICT reaction as described in PC/012 Publication WO . The SNIM RNA used in the examples disclosed herein produced IVT in which 25% of the U residues were 2-thio.uridine and 25% of the C residues were 5-methylcytidine. Absence

57739 Β1 non-standard nucleotide residues can make mRNA more stable and / or less immunogenic than control mRNA with the same sequence but in content only with standard residues. In further embodiments, the mRNA may comprise one or more non-standard nucleotide residues selected from isocytosine, pseudo isocytosine, 5-bromouracial, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, and 2-chloro-c-6-aminopurine. of these modifications and other nucleobase modifications. Certain embodiments may further include additional modifications to the furanose ring or nucleobase. Additional modifications may include, for example, sugar modifications or substitutions (e.g., one or more 2'-Oalkyl modifications, locked nucleic acid (LNA)). In some embodiments, the RNA may be complexed or hybridized with added polynucleotides and / or peptide polynucleotides (PNAs). In embodiments where the sugar modification is a 2'-O-alkyl modification, such modification may include, but is not limited to, 2'-deoxy-2'-fluoro modification, and 2'-O-methyl modification, 2O-methoxyethyl modification, and 2'-deoxy modification. In certain embodiments of this modification, 0-100% of the nucleotides may be present, for example more than 0%, 1%, 10%, 25%, 50%, 75%, 85%, 90%, 95%, or 100% of the individual constituent nucleotides or in combination.

Compositions Containing CFTR MRNA In certain embodiments, the mRNA molecules may be administered as naked or unpackaged mRNA. In some embodiments, the administration of mRNA in the compositions of the invention may be facilitated by the inclusion of a suitable carrier. In certain embodiments, the carrier is selected based on its ability to facilitate transfection of target cells with one or more mRNAs.

As used herein, the term carrier includes any standard pharmaceutical carriers, vehicles, diluents, excipients and the like generally used in connection with the administration of biologically active agents, including mRNA.

In certain embodiments, carriers used in the compositions of the invention may comprise a liposomal carrier or other means that facilitate the transfer of mRNA to target cells and / or tissues. Suitable carriers include, but are not limited to, polymer-based carriers such as polyethyleneimine (PEI), and polymers with multiple domain blocks, lipid nanoparticles and liposomes, nanolipoomes, ceramide-containing nanoliposomes, proteoliposomes, including natural and synthetically derived exosomes, natural, synthetic and semi-synthetic lamellar bodies, nanoparticles, calcium phosphorsilicate nanoparticles, calcium phosphate nanoparticles, silicon dioxide nanoparticles, nanocrystalline particles, semiconductor nanoparticles, dry powders, poly (Darginin), nanodendrimers, starch-based delivery systems, micelles, emulsions, salt gels, nisomes, plasmids, viruses, calcium phosphate nucleotides, aptamers, peptimides, peptides molecules, and other vector additions. The use of bionanocapsules and other formations of viral capsid proteins as a suitable carrier has also been discussed in detail. (Hum. Gene Ther. 2008 Sep; 19 (9): 887-95).

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 that encapsulate mRNA.

57739 In certain embodiments of the invention, the carrier is formulated with the polymer as carrier, alone or in combination with other carriers. Suitable polymers may include, for example, polyacrylates, polyalkyanoacrylate, polylactide, polylactide-polyglycolide copolymers, polycaprolactone, dextran, albumin, gelatin, alginate, collagen, chitosan, cyclodextrins, protamine, PEGylated protamine, PLL and PL, PL, PL, PI. When PEI is present, a branched PEI molecular weight can be in the range of 10 to 40 kDa, for example, a 25 kDa branched PEI (Sigma # 408727). Additional examples of polymers suitable for the present invention include those described in PCT Publication WO2013182683.

The use of liposomal carriers to facilitate delivery of polynucleotides to target cells is contemplated in the present invention. Liposomes (e.g., lipzoomal lipid nanoparticles) are generally useful in a variety of applications in research, industry, and medicine, especially for their use as carriers of diagnostic or therapeutic components in vivo (Lasic, Trends Biotechnol., 16: 307-321, 1998; Drummond et al., Pharmacol. Rev., 51: 691-743, 1999) and are most often characterized by microscopic vesicles that have an inner water space that is separated from the outer medium by a membrane of one or two layers. The bilayer membranes of liposomes are typically formed by amphiphilic molecules, such as lipids of either natural or synthetic origin that contain spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16: 307-321, 1998). Two-layer membranes of liposomes can also be formed by amphiphilic polymers and surfactants (for example polymerosomes, niosomes, etc.).

In certain embodiments, the mRNA is complexed with nanoparticles to facilitate delivery to the target cell. In certain embodiments, the compositions of the invention may be combined with a multi-component lipid mixture using one or more cationic lipids, additional lipids such as non-cationic lipids (also referred to as auxiliary lipids), cholesterol-based lipids, and / or PEGylated lipids. for mRNA encapsulation.

Cationic Lipids In some embodiments, a suitable nanoparticle comprises a cationic lipid. As used herein, the phrase cationic lipid refers to any of a number of types of lipids that have a positive total charge at a selected pH, such as physiological pH. Some cationic lipids, in particular those known as titratable and pH-titratable cationic lipids, are particularly effective in mRNA delivery. Several cationic (e.g. titratable) lipids have been described in the literature and many are commercially available. Particularly suitable cationic lipids for use in the compositions of the invention include those described in International Patent Publication WO 2010/053572 (and in particular, C12200 described in paragraph [00225]) and WO 2012/170930. In some embodiments, the cationic lipid CKK-E12 is used (disclosed in WO 2013/063468). In some embodiments, a cationic lipid, N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride, or DOTMA is used. (Feigner et al. (Rgos. Nat'l Acad. Sci. 84, 7413 (1987); U.S. Pat. No. 4,897,355). DOTMA may be formulated alone or in combination with a neutral lipid, Dioleoylphosphatidyl-ethanolamine or DOPE or other cationic or non-cationic lipids in a liposomal transfer medium or lipid nanoparticle, and such liposomes may be used to enhance the delivery of nucleic acids to the target cell. Other suitable cationic lipids include, for example, 533

57739 Karί carboxypermylglycindioctad-cilam id or DOGS, 2,3-dioleyloxy-N- [2 (spermincarboxamido) ethyl] -N, N-dimethyl-1-propanaminium or DOSPA [Behr et al. Proc. Nat.'l Acad. Sci. 86, 6982 (1989); U.S. Pat. No. 5,171,678; U.S. Pat. No. 5,334,761), 1,2-Dioleoyl-3-Dimethylammonium Propane or DODAP, 1,2-Dioleoyl-3-Trimethylammonium Propane or DOTAP. Contemplated cationic lipids also include 1,2-distearyloxyN, N-dimethyl-3-aminopropane or DSDMA, 1,2-dioleyloxy-N, N-dimethyl-3-aminopropane or DODMA, 1,2-dilinoleyloxy-N, N-dimethyl -3-aminopropane or DLinDMA, 1,2-dilinolenyloxy-N, N-dimethyl-3-aminopropane or DLenDMA, N-dioleyl-N, N-dimethylammonium chloride or DODAC, N, N-distearyl-N, N-dimethylaminonium bromide or DDAB, N - (1,2-dimyristyloxyprop-3-yl) -N, N-dimethyl-N-hydroxyethylammonium bromide or DMRIE, 3-dimethylamino-2- (cholest-5-ene-3-beta-oxybutane-4-oxy) -1- (cis, cis-9,12-octadecadienoxy) propane or CLinDMA, 2- [5 '- (cholest-5- en-3-beta-oxy) -3'-oxapentoxy) -3-dimethyl-1- (cis, cis-9 ', 1-2'-octadecadienoxy) propane or Cp-pyridine, N, N-dimethyl-3,4- dioleyloxybenzylamine or DMOBA, 1,2-N, N'-dioleylcarbamyl-3dimethylaminopropane or DOcarbDAP, 2,3-Dilinoleoyloxy-N, N-dimethylpropylamine or DLinDAP, 1,2-N, N-Dilinoleylcarbamyl 3-dimethylaminopropane or 1,2-Dilinoleylcarbamyl-3-dimethylaminopropane or DLinCDAP, 2,2-dilinoleyl-4-dimethylaminomethyl- [1,3] -dioxolane or DLin- -DMA, 2,2-dilinoleyl-4-dimethylaminoethyl [1,3] -dioxolane or DLin- K-XTC2-DMA, and 2- (2,2-di (9Z, 12Z) -octadeca-9,12-dien-1-yl) -1,3-dioxolan-4-yl) -N, N-dimethylethanamine DLin-KC2-DMA)) (See, WO 2010/042877; Semple et al., Nature Biotech. 28: 172-176 (2010)), or mixtures thereof. (Heyes, J., et al., J Controlled Release 107: 276-287 (2005); Morrissey, DV., Et al., Nat. Biotechnol. 23 (8): 1003-1007 (2005); PCT Publication

VVO2005 / 121348A1).

In certain embodiments, the compositions of the invention use lipid nanoparticles comprising the ionizing cationic lipids described in U.S. Provisional Patent Application 61 / 617,468, filed 29. March 2013, such as (15Z, 18Z) -N, N-dimethyl-6- (9Z, 12Z) -octa-9,12-dien-1-yl) tetracose-15,18-dien-1-amine HGT5000), (15Z, 18Z) -N, N-dimethyl-6 - ((9Z, 12Z) -octadeca-9, 12-dien-1-yl) tetracose4,15,18-trien-1-amine (HGT5001) , and (15Z, 18Z) -N, N-dimethyl-6 - ((9Z, 12Z) -octadeca-9, 12-dien-1-yl) tetracose-5,15,18-trien-1-amine (HGT5002 ).

In some embodiments, one or more cationic lipids present in such a composition comprise at least one of the imidazole, dialkylamino, or guanidinium moieties. In a preferred embodiment, the one or more cationic lipids do not contain a cauter amine.

Non-cationic / auxiliary lipids In some embodiments, a suitable lipid nanoparticle comprises one or more non-cationic (auxiliary) lipids. As used herein, a non-cationic lipid refers to any neutral,<zwitteri ionic or anionic lipid. As used herein, the term anionic lipid refers to any of a number of lipid species that carry a negative charge (batch) at a selected pH, such as physiological pH. In some embodiments, the non-cationic lipid is a neutral lipid that is a lipid that does not carry a total batch (loading) under the conditions in which the composition is formulated and / or administered. Nekatjonski lipids include but are not limited, distearoilfosfatidilcholin (DSPC) dioleoilfosfatidilcholin dioleoylphosphatidylglycerol dioleoyl-phosphatidylethanolamine (DOPC), (DOPG), (DOPE), DiPalma itoilfosfatidilholin dipalmitoilfofatidilglicerol palmitoiloleoilfosfatidilholin (DPPC), (DPPG) (POPC)

57739 Palί palmitoiloleoylphosphatidylethanolamine (ΡΟΡΕ), dioleoyl-phosphatidylethanolamine 4- (Νmaleimidomethyl) -cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), DM-dilaminoyl 16-O-dimethyl PE, 181-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), or a mixture thereof.

Cholesterol-Based Lipids In some embodiments, suitable pipeid nanoparticles comprise one or more cholesterol-based lipids. For example, suitable cholesterol-based cationic lipids include, for example, cholesterol, PEGylated cholesterol, DC-Choi (N, N-dimethyl-Netylcarboxamidocholesterol), 1,4-bis (3-N-oleylamino-propyl) piperazine (Gao, et al. Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997); U.S. Pat. No. 5,744,335), or ICE.

PEGylated Lipids In some embodiments, suitable lipid nanoparticles comprise one or more PEGylated lipids. For example, the use of polyethylene glycol (PEG) modified phospholipids and derivatized lipids such as derivatized ceramides (PEG-CER) including N-Octanoyl-Sphingosine-1- [Succinyl (Methoxy Polyethylene Glycol) -2000] (C8 PEG-2000 ceramide) is contemplated in the present invention in combination with one or more cationic and in some embodiments other lipids. In some embodiments, suitable PEGylated lipids include PEG-ceramide having shorter acyl chains (e.g., S-jd or C-1g). In some embodiments, the PEGylated lipid DSPEPEG-Maleimide-Lectin may be used. Other contemplated PEG-modified lipids include, but are not limited to, polyethylene glycol chains up to 5 kDa in length that are conventionally linked to the lipid via an alkyl chain (s) of C6-C20 length. Without wishing to be bound by any theory, it has been contemplated that the addition of PEGylated lipid may prevent complex aggregation and increase circulation life time to facilitate delivery of mRNA encapsulated in the liposome to the target cell.

In certain embodiments, the composition comprises one of the following lipid combinations:

S12-200, DOPE, cholesterol, DMG-PEG2K; DODAP, DOPE, cholesterol, DMG-PEG2K; HGT5000, DOPE, cholesterol, DMG-PEG2K; HGT5001, DOPE, cholesterol, DMG-PEG2K; HTS, DSPC, cholesterol, PEG-DMG; MSZ, DSPC, cholesterol, PEG-DMG;

ALNY-100, DSPC, cholesterol, PEG-DSG; CKK-E12, DOPE, Chol, PEGDMG2K.

In some embodiments, the lipid: mRNA ratios can be 5: 1 (mg: mg), 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, and more up to 30: 1 (mg: mg). mg) or more. The N / P ratio can be in the range of 1.1: 1 to 10: 1 or more. Examples of lipid ratios are 40: 30: 20: 10, 55: 20: 20: 5, 50: 25: 20: 5 (cationic lipid: auxiliary lipid: chol: PEG lipid).

57739 In some embodiments, the pharmaceutical compositions of the invention do not comprise mucolytic agents (e.g., N-acetylcysteine, erdostein, bromhexine, carbocysteine, guiaphenesin, or iodinated glycerol).

Apparatus Filled with Pharmaceutical Composition In some embodiments, the pharmaceutical composition of the invention, such as a cationic lipid or PEI-based composition, comprises a naturally occurring CFTR mRNA provided in an apparatus for administration to a subject's respiratory system. The apparatus may be, for example, an instillation, aerosolization or nebulizer. Suitable devices include, for example, a PARI Vou jet nebulizer, an Aeroneb® Lab nebulizer, a MicroSprayer®, or an EFIow mesh nebulizer. Alternatively, dry powder inhalers or aerosolization devices such as portable inhalers may be used.

Uses and Methods of MRNA for Applications and Methods of the Invention Among other things, the present invention provides methods for the in vivo production of CFTR proteins, particularly in mammalian lungs. In some embodiments, the invention provides methods for inducing CFTR expression in epithelial cells in mammalian lungs, comprising contacting epithelial cells with a pharmaceutical composition comprising in vitro transcribed mRNA, wherein the in vitro transcribed mRNA comprises a coding sequence encoding SEQ ID NO: 1. amino acid sequence of human CFTR-wild type). The disclosure also allows the use of pharmaceutical compositions comprising in vitro transcribed mRNA, wherein the in vitro transcribed iRNA comprises a coding sequence encoding SEQ ID NO: 1., for the induction of CFTR expression in epithelial cells in mammalian lungs.

The description further provides methods of inducing CFTR expression in a mammalian target cell, a method comprising contacting a mammalian target cell with a composition, a composition comprising an in vitro transcribed mRNA encoding the amino acid sequence of SEQ ID NO: 1. The description further provides the use of a composition contains in vitro transcribed mRNA encoding the amino acid sequence of SEQ ID NO: 1., for the induction of CFTR expression in mammalian target cells.

It has been observed that vectors containing SEQ ID NO: 2 are often subject to insertion / deletion / displacement type mutations in host cells under typical growth conditions resulting in a heterogeneous population of vectors that cannot be used directly for in vitro transcription. Host cells in the growth phase under conditions such as lower temperature, attenuated light and / or a small number of cells such as CopiCutter® have been found to reduce but not eliminate the occurrence of the mutation. Accordingly, it may be recommended for in vitro transcription reactions with mRNA containing a coding sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to SEQ ID N0: 2 to use a template obtained by dissolving the vector as described above, collecting and linearizing the vector and purifying the desired species for use in transcription. Kogak purification can be, in rg., Size exclusion chromatography or weak anion exchange.

57739 Inί The in vitro transcribed mRNA described herein for use and methods may comprise 5'-UTR, 3'-UTR, poly-A, poly-U and / or poly-C ger, cap, and / or non-standard nucleotide residues, such as which is stated in the above-mentioned part related to these characteristics.

Pharmaceutical Preparations for Use and Methods The pharmaceutical preparations for use according to the invention may contain mRNA for use and methods as discussed in the previous section and additional ingredients as set forth in the previous section in connection with compositions containing CFTR mRNA. Thus, the use and / or administration of pharmaceutical compositions containing any of the carriers discussed above is contemplated.

In some preferred embodiments, the pharmaceutical compositions comprise PEI, such as branched PEI having a molecular weight in the range of 10-40 kDa, for example, 25 kDa.

[0078]. In other preferred embodiments, the 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 selected from those listed in the previous section in connection with preparations containing CFTR mRNA.

Routes of Administration for Induction of Pulmonary Expression In some embodiments, methods and uses for inducing CFTR expression in mammalian lungs, the pharmaceutical composition, as previously described, is administered by intratracheal instillation (dropwise instillation), nebulization, and aerosolization. The preparation device can be selected from the devices listed in the previous section in connection with the devices filled with the pharmaceutical preparation.

In preferred embodiments, the composition is administered by spraying or aerosolization. Some lipid formulations may tend to aggregate when attempting to disperse, but it is generally possible to solve aggregation problems by adjusting the formulation, e.g. by replacing the cationic lipid.

Treatment of Cystic Fibrosis Among other things, the present invention can be used to treat cystic fibrosis. In some embodiments, the present invention provides a composition for use in a method of treating cystic fibrosis by administering to a subject in need of treatment with an mRNA encoding a CFTR protein as described herein or a pharmaceutical composition comprising an mRNA. The mRNA or pharmaceutical composition containing the mRNA can be administered directly to the lungs of the subject. Different pathways can be used for transmission to the lungs. In some embodiments, the mRNA or mRNA-containing composition described herein is administered by inhalation, nebulization, or aerosolization. In various embodiments, administration of the mRNA results in the expression of CFTR in the subject's lungs (e.g., lung epithelial cells).

57739 In a specific embodiment, the present invention provides a composition for use in a method of treating cystic fibrosis by administering to a subject in need of treatment with an mRNA comprising the coding sequence of SEQ ID NO: 1. In particular, the present invention provides a composition for use in a method of treatment cystic fibrosis by administration to the lungs of a subject in need of mRNA containing the coding sequence of SEQ ID NO: 3 for treatment. In other embodiments, the present invention provides a composition for use in a method of treating cystic fibrosis by administering to a lung to a subject requiring treatment with an mRNA comprising a coding sequence that is at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO. : 3. Additional examples of non-natural CFTR mRNAs (not found in nature) that can be used to treat cystic fibrosis are described in the Brief Description of the Sequence section, such as, for example, SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, 16 or 17.

EXAMPLES The following specific examples serve solely by way of illustration, and are not limiting to the remainder of the invention in any way. Without further elaboration, it is believed that a person skilled in the art can use the present invention to its fullest extent based on this description herein.

Unless otherwise indicated, CFTR mRNA AND RNA RNA coated in the examples disclosed herein included 5 'UTR with SEQ ID NO: 4, coding sequence (CDS) with SEQ ID NO: 3, and 3' UTR with the sequence SEQ ID NO: 5.mFFL mRNA and SNIM RNA used in the examples disclosed herein included 5 'UTR, CDS, and 3' UTR with the sequences SEQ ID NOS: 6, 7, and 8.

Example 1: In Vitro synthesized mRNA encoding CFTR Synthesis of information RNA. Human transmembrane cystic fibrosis regulator and mRNA switch luciferase (FFL) mRNA were synthesized in vitro by transcription from a plasmid DNA template encoding the gene, followed by the addition of a 5 'cap structure (Cap 1) (Fechter, P .; Brownlee, GG Recognition of mRNA cap structures by viral and cellular proteins J. Gen. Virology 2005, 86, 1239-1249) and 3 'poly (A) gera of about 200 nucleotides in length as determined by gel electrophoresis. 5 'and 3' non-translational regions were present in each mRNA product.

Examples of non-naturally occurring CFTR mRNA include 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 ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17 described in the Brief Description and Sequences section.

Example 2: CFTR expression and activity in HEK cells This example shows that a fully functional CFTR protein is expressed from synthetic CFTR mRNA delivered to the cells.

Cells and CFTR transfection. Human embryonic renal HEK293T cells were grown in DMEM (Invitrogen Cat # 11965-092) supplemented with 10% fetal bovine serum, 2 mM L-Glutamine, 100 U / ml penicillin, and 100 pg / ml streptomycin. On the day of transfection, the cells were seeded on plates with 6 wells at a density of 50-60%

57739 Β1 confluences and incubated under normal conditions for tissue cultures (36 ° C in a humid atmosphere 5% CO2, 95% air). 60 μΙ Lipofectamine 2000 (Invitrogen Cat # 11668019) was diluted in 900 μΙ OptiMem reduced serum medium (Invitrogen Cat # 31985-062) and gently vortexed. 24 μg CFTR iRNA (4 mg per plate) was diluted with 900 μΙ OptiMem medium. The mRNA was immediately added to the diluted Lipofectamine and incubated at room temperature for 30 minutes. Plate medium was lightly aspirated with HEK293T cells and replaced with 1 ml of OptiMem Reduced Serum Medium. 300 μΙ iRNA / Lipofectamine complex was added to each well and the cells were allowed to rest under normal tissue culture conditions for 24 hours after which they were sieved onto plates by mechanical separation on poly-L-lysine coated glass cover plates (BD Biosciences , BD Biocoat) so that the cells could be easily transferred to the recording chamber and electrophoretic recording. The cells were incubated under normal tissue culture conditions for a minimum of 24 hours and used within 48 hours of final plating.

Electrophysiological imaging. Whole cell imaging and patch-clamp imaging were performed at room temperature using an Axopatch 200B amplifier with 5-8 ΜΩ electrodes. The data were digitized (50 kHz) and filtered (5 kHz) accordingly. The series resistance is compensated (70-80%) to minimize voltage errors. 'Voltage-clamp' recording was performed with a pipette and a solution of the following composition: 140 mM NMDG-CI; 5 mM EGTA; 1 mM MgCl 2; 10 mM HEPES; pH 7.2; 310 mOsm / l. The bath solution contained: 140 mM NaCl, 3 mM KCl, 2 mM MgCl 2, 2 mM CaCl 2, and 10 mM HEPES; pH 7.3, adjusted to 315 mOsm / l with D-glucose. 'Voltage clamp' recordings started 3-5 minutes after the whole cell configuration was established.

Cells were 'voltage-clamped' with a holding potential of either -60 mV or 0 mV and a series of positive and negative voltage cogs (either -80 mV to +80 mV or -100 to +100 mV at 20 mV increments) injected into recorded HEK293T cells to induce CFTR-induced whole-cell (CI-) chloride currents. A membrane permeable analog of cAMPu, 8-Vg-cAMP (500 μΜ, Sigma Aldrich) was applied for 4 minutes to the recorded cells to facilitate CFTR current. The gold standard CFTR blocker, CFTRinh-172 (10 μΜ, Sigma) was applied at the end of each recording to block the CFTR-induced Cl-current. Control recordings were performed in untransfected HEK293T cells.

Compound test. Test compounds were used using a DAD-16VC rapid perfusion system (ALA Scientific Instruments, USA) with an ejection pipette placed approximately 200 μπη from the imaging cell. 8-Vg-sAMR was made as a 500 mM stock solution in DMSO. All compounds were stored at -20 ° C and rapidly thawed and dissolved to the final desired concentration immediately before use.

Analysis. All analyzes were performed using Clampfit (MDS Analytical Technologies) and Excel (Microsoft) software. All values were the maximum evoked-peak current amplitude. Statistical differences in the data were checked by Student's t test, paired or unpaired according to needs and were considered significant at P <0.05.

57739 Β1 [0093] Ιη Vitro production of human CFTR Protein. Production of human CFTR protein via hCFTR iRNA was achieved by re-transfection of human HEK293T cells with CFTR mRNA as described herein. Treated and untreated cells were harvested and subjected to immunoprecipitation procedures 24 hours after transfection. Detection of human CFTR protein by Western Western blot analysis demonstrates that the complete complex of glycosylated CFTR protein (labeled as a C band) is produced from synthetic information RNA (Figure 1).

In Vitro activity of human CFTR protein. To determine the activity of CFTR protein obtained from synthetic CFTR mRNA after transfection, complete cell patch assays were performed in both HEK 293 and HEK 293T cells. Treated cells as well as control cells (untreated and falsely transfected) were subjected to activator (8-Br-cAMP, forskolin) and inhibitory (CFTRinh-172, GlyH-101) substrates to facilitate the determination of changes in current flow (chloride ion transport) HEK293Ts were transfected with 4 μg hCFTR mRNA and analyzed 24 hours after transfection. Whole-cell ‘Clamp’ assays were performed to measure the current flow as shown rgeko the transport of chloride ions after application of a set voltage. The current vs voltage diagram as a result of ramp voltages from -80 mV to +80 mV (shown in Figure 2) demonstrates significant differences in current when comparing untreated versus cells treated with hCFTR mRNA. This increase in current after exposure to 8-Br-cAMP, a known activator of CFTR protein, indicates that human CFTR protein is present in cells. After treatment of these pre-transfected cells with a known specific CFTR inhibitor, CFTRinh-172, the corresponding current drops back almost to control levels (<89% reduction). This reduction after exposure to the inhibitor strongly supports the presence of human CFTR protein. These results demonstrate that synthetic hCFTR mRNA can produce active human CFTR protein.

Separately, the complete cell activity assay was performed using an automated system (lonVWorks) in HEK293 cells. As described above, treated cells as well as control cells (untreated and falsely treated) were subjected to activator and inhibitory substrates to determine changes in current flow (chloride ion transport). In these studies, forskolin was used as a CFTR protein activator and part of the hCFTR mRNA of transfected cells was further exposed to a specific CFTR inhibitor GlyH-101. GlyH-101 is believed to act as a horn CFTR blocker that acts after the extracellular membrane side of the protein. This action and mechanism is different from that of CFTRinh-172, which has been reported to function on the intracellular side of CFTR protein.

Figure 4 is a current and voltage diagram of parental (source) HEK293 cell lines treated with forskolin as well as GlyH-101. No significant change in current was observed, suggesting that these specific CFTR activators and inhibitors have no effect on endogenous proteins present in cell lines.

Current diagram vs. voltage as a result of a voltage ramp of -100 mV to +100 mV (shown in Figure 5) demonstrates significant current differences when comparing untreated HEK293 cells with cells transfected with hCFTR mRNA. This increase in current after exposure to forskolin, a known activator of CFTR protein, indicates this

57739 Β1 that human CFTR protein is present in these cells. After treatment of these pre-transfected cells with another known specific CFTR inhibitor, GlyH-101, the corresponding current drops back almost to control levels (<95% reduction). This reduction after exposure to the inhibitor strongly supports the presence of human CFTR protein.

In total, these inhibition data, which are the result of two different separate mechanisms, strongly support the identity of the fully functional CFTR protein derived from the synthetic human CFTR messenger RNA.

Example 3: In Vivo Expression of CFTR This example demonstrates that the CFTR protein is efficiently expressed in vivo from CFTR encoding mRNA delivered via pulmonary administration.

Formulation Protocol 1. Aliquots of 50 mg / mL ethenolic solutions of C12-200, DOPE, Chol and DMG-PEG2K were mixed and dissolved and diluted with ethanol to a final volume of 3 mL. Separate aqueous buffer solution (10 mM citrate / 150 mM NaCl, pH 4.5) CFTR mRNA was prepared from stock at 1 mg / mL. The lipid solution was injected rapidly into the aqueous mRNA solution and stirred until the final suspension in 20% ethanol. The resulting nanoparticle suspension was filtered, diafiltered with 1x PBS (pH 7.4), then water, concentrated and stored at 2-8 ° C. Final concentration = 1.09 mg / mL CFTR mRNA (encapsulated). Zave = 80.2 nm (Dv (5Q) = 55.5 nm; Dv (gQ) = 99.6 nm).

Formulation Protocol 2. Aliquots of 2.0 mg / mL aqueous PEI (branched, 25 kDa) were mixed with aqueous CFTR mRNA solution (1.0 mg / mL). The resulting complex mixture was pipetted up and down a couple of purts and removed from the side 20 minutes before injection. Final concentration = 0.60 mg / mL CFTR mRNA (encapsulated Zave = 75.9 nm (Dv (50) = 57.3 nm; Dv (go) = 92.1 nm).

Analysis of FFL and CFTR proteins produced via intratracheally administered iRNA-filled nanoparticles. All studies were performed using either female BALB / C mice or CFTR KO mice. FFL samples were introduced via either direct instillation (MicroSprayer®) or nebulization (PARI Vou or Aeroneb) of the appropriate dose of encapsulated FFL mRNA. CFTR IRNK was inserted using a PARI Vou jet nebulizer. Mice were sacrificed and perfusion was done with saline after the time allowed for expression.

Intratracheal administration of FFL mRNA. FFL-tested materials were administered by a single intratracheal aerosol administration via MicrosprayerTM (50 pL / animal) while the animals were anesthetized with an intraperiotone injection of a mixture of ketamine 50-100 mg / kg and xylazine 5-15 mg / kg.

Spray (aerosol) administration of FFL iRNA. FFL test materials were administered by single intratracheal aerosol administration via an Aeroneb® Lab nebulizer (nominal dose volume up to 8 mL / group). The test material was delivered to a box containing the whole group of animals (n = 4) and was connected to the oxygen flow and scavenger system.

57739 Daί [0106] Giving CFTR iRNA. CFTR mRNA was prepared as described in Figure 6 below. Four CFTR 'knockout' mice were placed in an aerosol chamber and exposed to 2 mg of total codon-optimized unmodified human CFTR mRNA (comprising the coding sequence of SEQ ID NO: 3) for nebulization (Pari Vou jet nebulizer) for a period of approximately one hour. Mice were sacrificed 24 hours after exposure.

Euthanasia. Animals were euthanized with CO2 asphyxia at representative time points after dosing (± 5%) after thoracotomy and bloodletting. Complete blood (maximum volume) was collected by cardiac puncture and discarded.

Perfusion. After blood release, all animals were subjected to cardiac prefusion with saline. Briefly, whole-body intracardiac perfusion was performed by inserting a 23/21 gavage needle attached to a 10 ml syringe containing saline adjusted to the volume of the left ventricle for perfusion. The right atrium was incised to provide a drainage site for the perfusate. Lightly and gently and calmly, pressure was applied to the plunger to perfuse the animal after the needle was placed in the heart. Adequate flow of the rinsing solution was provided when the existing perfusate became clear (without visible blood) indicating that the rinsing solution saturated the body and the procedure was completed.

Tissue collection. After perfusion, the animals had their livers and lungs (left and right wings) collected. Selected groups were subjected to rapid freezing of approximately half of the liver and both left and right lungs in liquid nitrogen and stored separately nominally at -70 ° C. Selected groups underwent histological cassette preparation approximately half of the lungs were placed on one histological cassette per animal. Additionally, the lungs were inflated with 10% NBF through a cannula inserted into the trachea. The trachea is ligature-bound and the trachea and lungs (left and right) are placed intact on a single animal hostage cassette. All histological cassettes were stored at ambient temperature in 10% NBF for 24 hours and then transferred to 70% ethanol.

FFL expression in FFL-treated mice. After analysis of tissue samples, FFL expression was detected in FFL-treated mice (data not shown).

CFTR expression in CFTR 'knockout' mice. CFTR expression was detected by immunoprecipitation and Western blot analysis of CFTR IRNK of treated mouse lungs. Mature C band was detected in the left and right lungs in all treated mice until it was observed in untreated mice (Figure 1B). The antibodies used were MAB25031 (R&D Systems) for immunoprecipitation and SAB4501942 (Sigma) for detection via Western blot analysis. The results shown herein indicate that the CFTR protein can be successfully expressed in vivo based on lung mRNA delivery. Furthermore, the fact that CFTR mRNA has been successfully delivered to the lungs of CFTR ‘knockout’ mice and has resulted in effective protein production in the lungs suggests that mRNA-based CFTR protein production can be used to treat CFTR protein deficiency.

57739 Yes

Example 4: Administration of CFTR mRNA via the lungs using polymer nanoparticles Delivery of human CFTR information RNA to the lungs of mice can be achieved either by direct inhalation as well as by spraying. Using in situ hybridization procedures, human CFTR mRNA can be successfully detected after intratracheal administration of nanoparticles loaded with CFTR mRNA mice. Administration can be achieved using lipid-based nanoparticles (e.g., S12-200) as well as polymeric nanoparticles (e.g., polyethyleneimine, PEI).

Administration of CFTR mRNA using polymer nanoparticles. CFTR KO mice were treated with polyethyleneimine (PEI) treated CFTR mRNAs that filled the nanoparticles via inatratracheal administration (30 μg encapsulated mRNA). Treated mice were sacrificed six hours and 24 hours after administration and the lungs were harvested and fixed in 10% neutral buffered formalin (NFB). In situ hybridization was used to detect exogenous human CFTR mRNA (Figure 6). Significant staining was observed 24 hours after administration with wide distribution in mouse lungs of CFTR KO-treated mice until staining was observed in control mice treated with PBS.

Analysis of treated lungs at higher magnification (up to 20H magnification) revealed strongly positive intracellular staining through the alveolar and bronchial regions of both lungs (Fig. 7). After further magnification (40x), positive staining was observed in the cytoplasm of the target apical bronchial epithelial cells (Fig. 8). Therefore, it can be concluded that the RNA API information has been successfully delivered to the apical bronchial epithelial cells. Further while significant staining was observed 6 hours after administration. Significant positive staining was observed 24 hours after administration (Figure 9).

Significant positive intracellular staining was observed in both lungs in the bronchial and alveolar regions 24 hours after administration.

Example 5: Administration of CFTR mRNA via the lungs using lipid-based nanoparticles (nanoparticles) Administration of CFTR mRNA using lipid-based nanocarriers. As already mentioned, successful delivery of human CFTR mRNA to the lungs can be achieved by delivery using lipid nanoparticles. Examples of cationic lipid nanoparticles filled with hCFTR mRNA using C12-200 as a cationic lipid component are disclosed herein.

Successful detection of human CFTR mRNA in the lungs of CFTR KO mice was achieved by in situ hybridization. Knockout mice were treated with 15 [mu] g hCFTR mRNA, encapsulated in S12-200-based lipid nanoparticles and sacrificed 6 hours after administration. Positive detection of hCFTR mRNA was observed in the entire bronchial and alveolar region of both lungs compared with control mice treated with PBS (Figure 10).

Under further magnification (40k), positive detection of human CFTR mRNA was observed in the apical cytoplasm of bronchial epithelial cells as well as in the intracellular terminal alveolar regions (Figure 11).

57739 Β1 CFTR of information RNA can be achieved using both polymer-based (PEI) and lipid-based nanoparticles (S12-200). These systems allow the intracellular accumulation of the drug substance within the target cells of mice. Further, significant amounts of hCFTR mRNA were present in these target cells 24 hours after administration.

Example 6: Validation of human CFTR expression using a specific antibody Validation of antibodies for the detection of human CFTR protein in mice, pigs and cultured cells. The experiments were performed to identify an antibody that is specific for the hCFTR protein, which does not cross-react with the analogue in mice and pigs, and which is available in sufficient quantity for future experiments. Briefly, testing of various anti-hCFTR antibodies from academic and commercial sources led to the identification of a combination of anti-hCFTR antibodies that were capable of detecting human CFTR protein after immunoprecipitation and Western blotting (IP / WB) without cross-reactivity to either mice or porcine CFTR. Therefore, based on the IP / WB results, suitable anti-hCFTR antibodies were identified for the detection of hCFTR protein without cross-reactivity to mouse or porcine CFTR.

Cells were transfected with hCFTR mRNA and protein lysates were prepared using a ProteoExtract transmembrane kit (Megsk) at 24 h after transfection, and the transmembrane fraction was screened by Western blotting for hCFTR using murine anti-human CFTR antibodies (MA1-). Lysates from 16HBE cells were used as a positive control. Figure 12A shows data from CHO and COS-7 cells.

Hamster kidney (BHK) kidney cells, described as CFTR-negative in the literature, were transfected similarly to CHO and COS-7 cells and Western blot-tested protein lysates. In contrast to previously published reports, a clear positive signal for CFTR can be seen with the help of a monoclonal anti-CFTR antibody (Figure 12B). To test the specificity of the antibodies used in the Western blot analysis, Pig Kidney (porcine kidney) cells from CFTR- "knockout" pig (PKC), kindly provided by Prof. Eckhardt Wolf (Ludwig Makimilians University, Munich), were used in transfection experiments and to search for protein lysates for CFTR expression. As seen in Figure 12B, no CFTR signal can be detected in PKC cells. However, transfection did not result in any hCFTR expression. Using luciferase as a transfection control, PKC cells were found to express luciferase several times less efficiently compared to CHO or COS-7 cells. As no significant difference in hCFTR band intensity could be detected in any of the examined cell lines after transfection, a large screening was performed for other hCFTR antibodies with higher sensitivity and specificity to hCFTR.

Search for antibodies by Western Blot. Protein lysates were prepared from human bronchial epithelial cell line (BEAS-2B), human embryonic kidney cell line (HEK), mouse lung and porcine lung using ProteoExtract transmembrane kit (Merck), and the transmembrane fraction used for MA1 immunolabeling by various primers. - 935 from Thermo Scientific Pierce Antibodies, Rockford, IL, USA, AB596 from the Cystic Fibrosis Consortium, University of Pennsylvania, PA, USA and AB570 from the Cistic Consortium

57739 Β1

Fibrosis, University of Pennsylvania, PA, USA). Data are summarized as Figure 13.

While MA1-935 detects CFTR in all three species, AB596 detects human and mouse but not porcine CFTR, and antibody G449 specifically detects only human CFTR. With AB570, it was not clear whether the low molecular weight weak bands obtained from rodent and pig samples were really CFTR or non-specific products. In subsequent experiments (data not shown), it was found that MA1-935 recognizes a band other than CFTR. Therefore, in general, the results for MA1-935 were considered as confirmed results generated using other antibodies, but experiments in which only the anti-CFTR antibody used was MA1-935 were not considered convincing.

Immunoprecipitation of hCFTR (IP-hCFTR) from tissue samples. Since all antibodies screened produced several nonspecific bands, none of which gave the characteristic band position for hCFTR (C-band representing fully glycosylated protein and B-band representing nucleus of mannosylated form), immunoprecipitation ( IP) hCFTR and subsequent Western blot detection were established to increase the sensitivity and specificity of the detection, amplifying the signal above the noise level.

[0127] Initial IP experiments were performed in collaboration with Prof. Burkhard Tummler (Medizinsche Hochschule Hanover) 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 cancer cells (T84) that overexpressed hCFTR were used as positive controls for IP experiments.

Immunoprecipitation of hCFTR by three different antibodies (R29, R66 / 17 and R66 / 16) followed by immunodextication with AB596 led to specific detection of hCFTR in protein lysates from pig lungs treated with hCFTR SNIM RNA aerosol as described Example 8 below (Figure 14).

Formulation HGT5001. Aerosol experiments using hCFTR SNIM RNA in the formulation HGT5001: DOPE; Chol; PEGDMG2K (relative amounts 50: 25: 20: 5 (mg: mg: mg: mg)) (HGT5001 formulation) were performed in mice and protein lysates from isolated lungs in 24 h post-mRNA delivery were also analyzed by IP using the same antibodies and conditions as in porcine lysates. However, a characteristic mature CFTR band arrangement could not be detected in mouse samples (Fig. 15).

Immunoprecipitation of hCFTR (IP-hCFTR) from in vitro transfected cells. Initial IP results, using pig tissue material, provided evidence of technical feasibility for the hCFTR post-transcript to be delivered in vivo. However, since none of the antibodies used in immunoprecipitation of CFTR (R29, R66 / 17 and R66 / 16) are commercially available, other commercially available antibodies have been shown for their efficacy in IP reactions. Two antibodies from the R&D system (MAB25031 and MAB1660) were tested.

Protein lysates were prepared from T84 cells and 500 mg of total protein was used in the IP reaction using different concentrations of MAB25031 antibody. The amount of immunoprecipitated hCFTR protein was then detected

57739 Β1 by immunolabeling with AB570 (Cystic Fibrosis Foundation). AB596 under these conditions resulted in a much higher background and was not further tested. As shown in Figure 16A, there was no further increase in the amount of CFTR protein precipitated when the IP antibody concentration was increased from 2 mg / ml to 4 mg / ml. Forms with glycosylated nuclei (C- and B-bands, respectively) were also detected and fully glycosylated. The same immunoprecipitates were also screened with MAB1660 as the primary antibody by Western blotting. With this antibody, however, only the C-band is visible (Figure 16B).

After successful detection of endogenous hCFTR from T84 immunoprecipitates using MAB25031 antibodies, experiments were performed in NIH3T3 cells to detect hCFTR protein after transfection. NIH3T3 cells were transfected with hCFTR SNIM RNA. Protein lysates were prepared 72 hours after transfection and the amount of protein quantified by the BCA method. Human CFTR protein was immunoprecipitated from 500 cd of total protein lysate using antibody MAB25031 at 2 pg / ml, followed by immunolabeling using AB570 (Figure 17). However, CFTR could not be detected. Cells transfected with LacZ encoded by mRNA were analyzed as controls for transfection on the amount of CFTR protein.

An increase in the amount of total proteins used in immunoprecipitation, from 500 cd to 8 mg, did not result in any detectable hCFTR protein after immunodetection with AB570. Another hCFTR-specific antibody, MAB1660 (R&D system), was also used for immunoprecipitation (Figure 18). However, this antibody does not precipitate (precipitate) CFTR successfully as MAB25031. Therefore, all future immunoprecipitation was performed with MAB25031.

The lack of hCFTR detection in mRNA transfected samples does not necessarily mean the lack of functionality of the tested mRNAs because kinetic experiments using luciferase as a marker gene showed that the maximum expression with mRNA was observed in transfection after 24 hours. The lack of hCFTR detection is quite due to the insufficient concentration of hCFTR in the tested samples or the lack of specificity of the antibodies used.

PEI Formulation. The established conditions were tested for their ability (feasibility) to detect hCFTR after hCFTR SNIM RNA delivery in pigs (see Example 7) nanoparticle formulation with 25 kDa branched PEI (PEI Formulation) prepared as follows. The required amount of SNIM RNA was diluted immediately after use in water for injection (Braun, Melsungen) to a total volume of 4 ml and quickly added to 4 ml of aqueous solution of branched PEI 25 kDa using a pipette in the ratio N / P of 10. The solution was mixed by pipetting the dog down ten times and dispersing as two separate fractions of 4.0 ml one after the other into the pig lungs using the indicated nebulizer. One sample from the lung region where luciferase from pig # 1 is expressed, and the other from caudal lobe of pig # 2, where luciferase activity could not be detected, indicating a 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 hCFTR mRNA expression. The sample from the left caudal lobe of pig # 2, where there was no detectable luciferase activity, was also negative for hCFTR (lane 1) while hCFTR could be detected in samples from

57739 Svί pigs # 1, which were positive for luciferase (lane 2).

Example 7: Aerosol mRNA delivery Establishing aerosol delivery of encapsulated mRNA to the lungs of pigs. Aerosol administration of luminous luciferase (FFL) to SNIM RNA to porcine lungs was established by a gradual experimental procedure. In the first step FFL SNIM RNA formulations were dispersed into anesthetized pigs during controlled ventilation. In the second step, the lungs were removed by incision immediately upon completion of aerosol administration, and the lung samples were incubated in cell culture medium rgeko night rge ex vivo luciferase measurement on lung samples using BLI.

German Landrace pigs were obtained from the Technical University of Munich, Weihenstephan, Germany. The pigs weighed 35-90 kg. Each treatment was performed on one pig. A total of 5 pigs were treated. The first pig (weight 90 kg) was treated with FFL SNIM RNA in the PEI formulation of Example 6 using an EFIow aqueous nebulizer and by measuring luciferase activity in lung homogenates. The second pig (weight 60 kg) was treated with FFL SNIM RNA in the PEI formulation of Example 6 using an EFIow air nebulizer and measurement of luciferase activity in lung samples by BLI. A third pig (weight 80 kg) was treated with FFL SNIM RNA in PEI Formulation of Example 6 using a PARI m jet nebulizer and measurement of luciferase activity in lung samples by BLI. A fourth pig (60 kg weight) was treated with mRNA SNIM RNA / hCFTR mFNA in PEI Formulation Example 6 using an Aeroneb network nebulizer and by measuring luciferase activity in lung samples by BLI. A fifth pig (weight 35 kg) was treated with FFL SNIM RNA in the HGT5001 formulation of Example 6 using an Aeroneb network nebulizer and measuring luciferase activity in lung samples by BLI.

Slaughter of 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 followed by insertion of an intravenous catheter into the lateral auricular vein. Pigs were anesthetized by intravenous injection of propofol 3-5 mg / kg body weight, as needed. Anesthesia was maintained with isoflurane (2-3%) with a 1% propofol “bolus” injection of 4 to 8 mg / kg body weight to improve anesthesia, as needed. The duration of anesthesia was approximately 1-3 hours. The pigs were killed by a "bolus" injection of pentobarbital (100 mg / kg body weight) and potassium chloride in the lateral vein of the ear. Lungs were removed and tissue samples were collected from different regions of the lung, followed by incubation in cell culture medium overnight. To measure luciferase activity, tissue samples were either homogenized and analyzed in a tubular luminometer or incubated in medium containing D-luciferin substrate and subjected to ex vivo BLI luciferase.

Details and results for pig # 1. The experimental setup is illustrated in Figure 20. For aerosol delivery, an EFIow air nebulizer is connected in line to the respirator vent tube. The aerosol administration lasted about 60 minutes and was longer than expected in open system control experiments. This is apparently caused by the increased back pressure during spraying, which proves the aerosol outflow in the air nebulizer tank. Eight milliliters of the PEI formulation of Example 6 containing 1 mg of FFL SNIM RNA in water for injection was prepared as described in WP5 and distributed in two separate portions of 4

57739 Β1 ml one after the other. Measurement of luciferase was performed in tissue homogenates from removed lung samples from different regions of the lung after incubation overnight in cell culture medium. Expression values were mapped according to the origin of lung samples (Figure 21).

The results showed successful expression of luciferase in pig lung tissue. Luciferase expression was highest in the central parts of the lungs and decreased towards the more distal regions of the lungs. The expression schedule was correlated with the expected distribution of inhaled FFL SNIM RNA-PEI nanoparticles according to the selected ventilation parameters. Luciferase expression levels were in the same range as observed in experiments with mice in WP5 using the same PEI Formulation of Example 6.

Details and results for pig # 2. Aerosol administration of FFL SNIM RNA in the PEI formulation of Example 6 in pig # 2 was performed as in pig # 1, but luciferase activity was measured in lung samples using a bioluminescent imaging (BLI). This experiment was performed to establish an ex vivo measurement of luciferase of cultured organ lung samples using BLI. Megepje luciferase was clearly observed in individual tissue samples from different regions of the lung of the treated pig (Figure 22). The experiment confirmed the results obtained in pig # 1.

Details and results for pig # 3. Administration of aerosol in pigs # 1 and # 2 using an EFIow air nebulizer revealed some technical difficulties and inadequate nebulization times. Therefore, pig # 3 was treated using a PARI ne jet nebulizer connected to the ventilation tube of the rgeko T-connector. Aerosol administration lasted longer (approximately 80 minutes) than with the EFIow air nebulizer and aerosol administration was unsatisfactory. Very low luciferase activity was detected in excised lung samples from different lung regions of the treated pig (Figure 23).

Details and Results for Pig # 4. The results of previous experiments have shown that an air nebulizer is more suitable for administering an aerosol to the lungs of pigs in a selected setting compared to a jet nebulizer. For this reason, another air nebulizer was tested for this purpose, which satisfactorily dispersed the PEI formulation of Example 6 when tested in an open system. Pig # 4 was treated using an Aeroneb air nebulizer connected in-line to the respirator tube. In this experiment, 1 mg of hCFTR mRNA was co-delivered with 1 mg of FFL SNIM RNA in the PEI formulation of Example 6. This was done to test the stability of the formulation and the nebulability of the co-formulated FFL SNIM RNA / hCFTR iRNAPEI nanoparticle versus repeated dosing. should be performed in Example 8. The formulation is stable and did not reveal incompatibility with spraying. Luciferase activity was clearly observed in individual tissue samples from different regions of the lung of the treated pig (Figure 24).

The experiment confirmed the results obtained for pig # 1 and pig # 2, although higher levels of expression were obtained. The experiment showed that the Aeroneb air nebulizer was the most suitable for delivering PEI Formulation Example 6 to the lungs of pigs. Moreover, the experiment showed that FFL SNIM RNA was still active when delivered together with hCFTR mRNA.

57739 Β1 [0145] Details and results for pig # 5. Pig # 5 was treated with 1 mg of FFL SNIM RNA in the formulation HGT5001 from Example 6 aerosolized with an Aeroneb air nebulizer. The formulation can be aerosolized without technical difficulties. Luciferase activity was apparently observed in individual tissue samples from different regions of the lung of the treated pig (Figure 25).

The experiment showed that the aerosolized FFL SNIM RNA in the formulation of HGT5001 in Example 6 was active in pig lung tissue, although expression levels were approximately 15-20 times lower than in pigs treated with the PEI Formulation of Example 6.

Conclusion. Successful results were obtained using an Aeroneb air nebulizer with PEI Formulation Example 6. Four pigs were treated with PEI Formulation Example 6 to identify the optimal experimental setting for aerosol delivery. The results showed that luciferase expression can be detected in pig lung homogenates and with BLI. Luciferase expression was highest in the central parts of the lungs and was barely seen in the distal parts of the lungs. Aeroneb air nebulizer was found to give the best results along with the shortest delivery time. Based on these experiments, the second pig was treated with FFL SNIM RNA encapsulated in the HGT5001 formulation in Example 6. Although luciferase expression was clearly observed in some parts of the pig lungs, expression levels were lower than in FFL SNIM RNA in the PEI Formulation of Example 6. The results of this work package clearly showed that the delivery of SNIM RNA to the lungs of pigs as a large preclinical animal model is feasible using different formulations such as a polymer-based formulation (e.g., PEI) and a lipid-based formulation (e.g., HGT5001). The results of this example prove the concept for the successful delivery of SNIM RNA to the lungs of a large animal that closely mimics the situation in human patients using a nebulizer used in clinical practice.

Example 8: In vivo mRNA delivery (weekly dose) A study was performed to evaluate the practicality of aerosol administration once a week in pigs. Convenience is defined as performing three aerosol applications of modified mRNA at one-week intervals without induction of lung disease (absence of adverse events higher than grade 2). Additional objectives were to assess i) the degree of stress of the animals, ii) adverse events that occurred during laboratory or clinical evaluation of pigs, and iii) measurement of induced proteins (luciferase and hCFTR).

Repeated aerosol administration of SNIM RNA in PEI Formulation to pig lungs was established. Groups of two pigs were treated one, two or three times at low intervals with FIM SNIM RNA / hCFTR SNIM RNA in PEI Formulation of Example 6. Two untreated pigs served as controls. Lungs were removed 24 hours after treatment, and ex vivo luciferase activity was measured in isolated lung samples using BLI. HCFTR protein expression was analyzed using ΙΡΛ / Β. Immunohistochemistry (IHC) was used to detect luciferase expression at the cellular level. Toxicology was examined by measuring inflammatory cytokines in the serum and performing chemical parameters in the blood. Histopathology was performed on lung samples. Research protocol Pilot project: Repeated use of modified mRNA to establish an animal model for aerosol therapy of cystic fibrosis in pigs was approved by the local authorities at the beginning of the experiments (Animal Experiment License No .: 0-045-12).

57739 Β1 [0150] Draft experiment. Pigs, German Landrace, females about 6 weeks old (~ 25 kg body weight on average) when sprayed, were procured from the Technical University of Munich, Weihenstephan, Germany. The pigs were randomly distributed and treated according to the scheme below (Table 3). The treatment groups, two pigs each, 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 PEI formulation of Example 6 on day 1 and 1 mg FFL SNIM RNA and 1 mg hCFTR SNIM RNA in PEI formulation of Example 6 on day 8.

Group III - Aerosol administration of 2 mg hCFTR SNIM RNA (6379-186) in 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 PEI Formulation of Example 6 on day 15.

A treatment and assessment scheme for each group is shown in Table 3. In addition to the illustrated interventions, a medical examination of the pigs was performed daily.

Table 3. Time diagram of groups on different treatments.

57739 Β1

ΑΑ Euth dE d2, d3, d4, d5, d6, d7, d8, d9

Group 3 (3rd aerosol application; survival 15 d):

st Jst ^ nđ ^ rci

Bw Bw Bw Bw Bw Bw

ΑΑ ΑΑ AA Euth dl ~, dIdVd4, d5, d6, d7, d8, d9, dlO, dll, d! 2, d! 3, d! 4, d! 5, d! 6 [0152] Experimental procedure. Calming 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 followed by intravenous intravenous catheter into the lateral auricular vein. Pigs were anesthetized by intravenous injection of propofol 3-5 mg / kg body weight, as needed. Anesthesia was maintained by continuous intravenous infusion with 1% propofol, as needed. Ventilation parameters are adjusted with endepiratory carbon dioxide and adjusted, if necessary. Anesthesia, respiratory and cardiovascular parameters were continuously monitored by pulse oximetry, capnography, measurement of rectal temperature and reflex status. Animals received an infusion of a balanced electrolyte solution of 10 ml / kg / h. The duration of anesthesia was about 80-120 min. Pigs were extubated after the onset of satisfactory spontaneous respiration. Pigs were killed by bolus injection of pentobarbital 100 mg / kg body weight in the lateral vein of the ear, after sedation. Lungs were removed and cut to approximately 1 cm thick tissue samples collected from different regions of the lung and later incubated in cell culture. To measure luciferase activity, tissue samples were incubated in medium containing D-luciferin substrate and subjected to ex vivo BLI luciferase.

Luciferase expression in treated groups using BLI.

For Group 0 (untreated control group), no luciferase activity was observed in lung sections (Figure 26).

For Group I (aerosol administration of 1 SNF RNL SNIM RNA and 1 mg hCFTR SNIM RNA in the PEI Formulation of Example 6) Luciferase activity was clearly detected in lung samples of one-year-old treated pigs 3 and 6 (Figure 27). Luciferase expression was highest in the central parts of the lungs.

For Group II (aerosol administration of 2 mg hCFTR SNIM RNA in PEI Formulation of Example 6 on day 1 and 1 mg FFL SNIM RNA and 1 mg hCFTR SNIM RNA in PEI Formulation of Example 6 on day 8) Luciferase activity detected in lung samples doubled treated pigs 4 and 8 (Fig. 28). Luciferase expression was highest in the central parts of the lungs. It should be noted that the samples were stored for another 10 hours in

57739 Muί medium of cell culture rge measurement due to darkening of electricity on the day of measurement and resulting technical problems with BLI sis [0156]. For Group III (Aerosol administration of 2 mg hCFTR SNIM RNA in PEI Formulation of Example 6 on days 1 and 8, aerosol administration of 1 mg FFL SNIM RNA and 1 mg hCFTR SNIM RNA in PEI Formulation of Example 6 on day 15), luciferase activity is clear detected in lung samples of three-treated pigs # 1 and # 2 (Fig. 29). Luciferase expression was highest in the central parts of the lungs.

Properties of SNIM RNA-PEI nanoparticles. Particle size and zeta potential were measured for SNIM RNA-PEI spraying formulations (Table X1). SNIM RNA-PEI nanoparticles can be reproducibly formed with a size in the range of 25-37 nm and zeta potentials in the range of 30-49 mV.

Table X1. Particle size and zeta potential measurements.

<td>Pig #</td><td>Treatment #</td><td>Radius ± SD (nm)</td><td>Zeta potential 6 SD (mV)</td>

Luciferase expression in IHC-treated groups. IHC for FFL was performed on tissue samples of lung sections (Sophistolab AG, Eglisau, Switzerland), which were positive for BLI and compared with the lung tissue of untreated pigs and luciferase-positive mouse tumor tissue as positive controls. As expected, a strong signal was seen in the luciferase-positive tumor tissue of the mouse, while the lung tissue of the untreated pig showed no specific staining. Clearly colored detectable signals can be seen in the lung tissue of pig # 1, which received three treatments. FFL expression was most prominent in the bronchial epithelium of the large and small airways (Fig. 30).

Detection of hCFTR protein in the lung tissue of a treated pig by IP / VB. High BLI-positive lung tissue of triple-treated pig # 1 was subjected to hCFTR ΙΡΛ / Β according to the protocol described by van Barneveld A et al., Cell Physiol Biochem. 30, 587-95 (2012) (Fig. 31). Mature complex-glycosylated hCFTR appears as dispersed so-called. C-strip. Mannose-rich hCFTR appears as a more compact so-called. B-bar. Clear hCFTR expression was observed in T84 positive control cells and lung tissue of pigs # 1 treated with hCFTR SNIM RNA in the PEI formulation of Example 6. hCFTR protein expression was not observed in untreated pigs. Comparison of hCFTR protein expression in human lung tissue from published

57739 Β1 studies, using an identical protocol (van Barneveld A et al., Supra) suggest that hCFTR expression in porcine lung tissue, after hCFTR SNIM aerosol treatment, is similar to hCFTR expression in healthy human lungs.

This finding was further confirmed using a different set of antibodies for the detection of hCFTR protein by IP / VB in the treated pig lungs (see Example 6). One sample from the region where luciferase is expressed from pig # 1 and the other from the caudal lobe of pig # 2, where luciferase activity cannot be detected, indicates the inability of mRNA delivery and / or expression, chosen 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 32, luciferase expression correlated with hCFTR mRNA expression. The sample from the left caudal lobe of pig # 2, where there was no detectable luciferase activity, was also negative for hCFTR (lane 1), while hCFTR could be detected in samples from pig # 1, which were positive for luciferase (lane 2). ).

Toxicology: Preliminary histological evaluation of lung samples. Histological evaluation of lung samples taken after euthanasia of three animals was performed. After insertion into paraffin molds, lung samples were stained with hematoxylin-eosin for morphological evaluation. The findings were consistent on samples from three pigs, of which two (pig # 1 and pig # 2) received three aerosol applications, and the third (pig # 7) was an untreated control without aerosolization.

Toxicology: Stress. Only pig # 2 and pig # 1 showed mild signs of stress 2-4 days after the first treatment. Therefore, three three-week aerosol applications caused only mild stress.

Toxicology: Adverse Events. The type and frequency of adverse events (AE) were analyzed by laboratory parameters (blood, MBS and BAL) and medical examination of pigs (defined as a secondary target in this study).

Serum and whole blood samples were taken at time points defined by the study protocol. Twelve representative parameters (hemoglobin, hematocrit, AP, ALT, AST, CK, bilirubin, creatinine, glucose, potassium, platelets, and white blood cells) that are indicative to indicate organ-specific pathology (blood, bone marrow, and kidney) were selected and test results obtained from VetMedl_ab, Ludwigsburg, Germany classified according to VCOG, 2011 version.

The results show that no serious adverse events (AE) were observed in pigs (grade 3, 4, and 5 AEs would qualify as serious). There was no deterioration in laboratory parameters after aerosol administration of SNIM RNA in the PEI formulation of Example 6. For small changes in some parameters (e.g., CK or liver enzymes), changes are more likely to be caused by an experimental procedure per se (e.g., injections and anesthesia). Also, no adverse effect of repeated administration was detected - during the third administration, pigs of group 3 did not show AE more than AE grade 2. Even grades AE 1 or 2 are rare and did not show any correlation with aerosol application of SNIM RNA in the PEI formulation of Primer 6.

In addition to repeated blood samples, two other parameters were evaluated to assess pathological processes in the lungs: i) Brocho-Alveolar-Lavage fluid (BALF)

57739 Uzetiί taken after euthanasia, ii) microbiological samples (MBS) (tracheal swab - taken during anesthesia). BALF was taken from each pig during the autopsy and stored at -80 ° C for further examination. Tracheal swabs were taken from each aerosol application and microbiologically examined. These examinations revealed a wide range of pathogens including Bordetella bronchiospectica (a common pathogen of the porcine respiratory tract) and Escherichia coli. Pigs were treated once with tulatromycin injection into the gluteus muscle (1 ml Drakkin® 10%).

Medical examination. In addition to laboratory parameters, medical examinations of pigs were performed in the observation periods between aerosol applications (for more details see 1.1.2 of Annex 1 and Annex 4 of the study protocol). As there is no system for documenting, assessing and attributing AE, either for intervention or something else defined for pigs, the Common Toxicology Criterion (CTC) system established for dogs and cats was used (published by VOCG in 2011). Specific types of ULN (upper normal limits) and LLN (lower normal limits) were used to evaluate laboratory parameters. Clinical evaluations were performed in the following six AE categories: (1) allergic / immunological events; (2) pulmonary / respiratory; (3) structural (essential) clinical signs; (4) dermatological / dermal; (5) gastrointestinal; and (6) pulmonary / respiratory.

The results show that no serious AE (no grade 3, 4 or 5) was observed in pigs. There was no deterioration of the parameters assessed by medical examination after aerosol application of SNIM RNA in PEI formulation. Two pigs from group 3 showed AE levels 1 and 2 in the case of three respiratory parameters (bronchospasm / wheezing, laryngeal edema and dyspnea), but these mild or moderate findings were limited to one or two days. Since these observations only occurred after the first anesthesia / intubation / aerosol application in these two pigs, but not after the second or third aerosol application in the same or any other pig, it is unlikely that these findings were caused by the test substance.

Conclusion. The results of this example showed that a PEI formulation encoding FFL and hCFTR SNIM RNA can be successfully aerosolized multiple times in the lungs of pigs without loss of activity after each treatment cycle and without adverse events. Luciferase expression is found in the central parts of the lung tissue, but is difficult to find in the distal areas of the lungs. The regional occurrence of luciferase expression correlated with the expected distribution of the PEI formulation of Example 6 based on the settings used for controlled ventilation. Immunohistochemistry on selected lung samples from treated powders showed luciferase expression predominantly in the bronchial epithelium of the large and small airways. IP / VB clearly showed the expression of complex-glycosylated C-tract of mature CFTR in treated pig lungs that was absent in untreated pig lungs and luciferase-negative lung samples. Expression of hCFTR in pig lung tissue after hCFTR SNIM RNA aerosol treatment was comparable to hCFTR expression in healthy human lungs compared to published reports using an identical protocol for hCFTR protein detection. Adverse events rated 1 or 2 were very rare and showed no correlation with aerosol application of SNIM RNA in the PEI formulation. Therefore, hCFTR protein expression has been successfully demonstrated in pig lungs treated with SNIM hCFTR mRNA.

Example 9: CFTR encoding an mRNA containing a signal peptide

57739 Β1 This example demonstrates that a CFTR protein can be effectively expressed from a CFTR encoded by an mRNA with a signal peptide coding sequence.

Synthesis of RNA information. For the experiment, a C-terminal Hisio-labeled codon optimized human cystic fibrous transmembrane conductivity regulator (CO-CFTR-C-His10), a codon optimized human CFTR with a signal leader sequence for growth hormone (GH-CO -CFTR) (SEQ ID NO: 16) and codon optimized human CFTR (CO-CFTR) (SEQ ID NO: 17) SNIM RNA was synthesized by in vitro transcription from plasmid DNA samples using standard procedures.

Cells and CFTR transfection. Human embryonic renal HEK293T cells were grown in DMEM (Invitrogen Cat No. # 11965-092) with the addition of 10% fetal bovine serum, 2 mM L-glutamine, 100 U / ml penicillin and 100 mg / ml streptomycin. On the day of transfection, cells were cultured in plates with 6 wells at 50-60% density and incubated under normal tissue culture conditions (36 ° C in 5% CO2, 95% air humidity). In preparation for transfection, 60 ml lipofectamine 2000 (Invitrogen Cat no. # 11668019) was diluted in OptiMem Reduced Serum medium (Invitrogen Cat No. # 31985-062) and slightly vortexed. For the experiment 4 mg of either COCFTR, GH-CO-CFTR or CO-CFTR-C-Hisi0 SNIM RNA was diluted in 900 plOptiMem medium. The mRNA was immediately added to diluted Lipofectamine® and incubated at room temperature for 30 minutes. The culture medium was lightly collected and replaced with 1 ml of OptiMem Reduced Serum medium and 300 μΙ of each of the corresponding mRNA / Lipofectamine® complexes. The cells were incubated under standard tissue culture conditions.

Western analysis. Approximately 48 post-transfections, cells were removed from appropriate plates and lysed. Whole cell lysate was separated on SDSPAGE and examined by Western blot. As shown in Figure 33, robust expression of human CFTR protein was detected after transfection with CO-CFTR, GH-CO-CFTR and human CO-CFTR-C-Hisio mRNA, anti-CFTR (A and B) or anti-His (C) antibodies (Figure 33).

Example 10: In vivo delivery of CO-CFTR-C-His10 mRNA to CFTR “Knockout” (targeted inactivated) mouse Analysis of human CFTR protein produced by nanoparticles containing intratracheally delivered packaged mRNA. All studies were performed using CFTR KO mice. The CFTR mRNA formulation or control vehicle was introduced using a PARI Vou inhaler. Mice were sacrificed and perfused with saline, after a predetermined period of time, to allow expression of the protein by mRNA.

Synthesis of RNA information. In this example, C-terminal His10 labeled codon optimized human cystic fibrous transmembrane conductance regulator (CO-CFTR-C-Hisio) SNIM RNA and optimized FFL SNIM RNA were synthesized by in vitro transcription of isa plasmid DNA patterns.

PEI Formulation. For this approach, delivery and expression of CO-CFTR-C-His10 mRNA into the lungs of CFTR “knockout” mice was evaluated using both polymeric and lipid-based nanoparticle formulations. Polymer nanoparticle formulations with 25 kDa branched PEI were prepared as follows. Required amount of SNIM

57739 Β1

RNA was diluted directly after use in water for injection (Braun, Melsungen) to a total volume of 4 ml and added rapidly to 4 ml of aqueous 25 kDa branched PEI solution using a pipette in the N / P ratio of 10. The solution was mixed by pipetting up and down ten times and inhaled (dispersed) as two separate 4.0 ml fractions one after the other into the mouse lung using a designated inhaler (nebulizer).

CKK-E12 formulation. For the experiment with lipid-based nanoparticles, a lipid formulation was made using CO-CFTR-C-His10 SNIM RNA in the formulation CKK-E12: DOPE: Chol: PEGDMG2K (relative amounts 50: 25: 20: 5 (mg: mg: mg: mg)) The solution was dispersed into mouse lungs using a designated nebulizer.

Administration of dispersed (aerosol) human CO-CFTR-C-His10 mRNA. CFTR-tested materials were administered by a single inhalation of a rgeko PARI Vou jet nebulizer aerosol (nominal dose volume up to 8 mL / group). The test material was delivered to a box containing the whole group of animals (n = 4) and which was connected to the oxygen flow system and the cleaning system.

Administration of human CO-CFTR-C-His10 mRNA. CFTR mRNA was prepared as described above. Four CFTR “knockout” mice were placed in an aerosol chamber and exposed to a total of 2 mg codon optimized unmodified human HRTR mRNA (containing the coding sequence SEQ ID NO: 3) by spraying (Pari Vou jet nebulizer) for a period of approximately one hour. Mice were sacrificed 24 hours after exposure.

Euthanasia. Animals were euthanized by suffocation with CO2 in a representative period after dose administration (6 5%), followed by thoracotomy and exanguination (bleeding). Whole blood (the maximum amount that can be obtained) was collected by cardiac puncture and discarded.

Perfusion (injection). After exanguination, all animals were subjected to cardiac perfusion with saline. Briefly, whole tissue perfusion was performed by inserting a 23/21 diameter needle attached to a 10 ml syringe containing saline into the lumen of the left perfusion chamber. The right atrium is incised to provide an outlet for perfusion drainage. Gentle and constant pressure was applied to the plunger to ensure injection into the animal after the needle was placed in the heart. Adequate flow of the rinsing solution was provided when the outlet perfusate was clean (no visible blood) indicating that the rinsing solution had saturated the body and the procedure was complete.

Tissue collection. After perfusion, the lungs (right and left) were collected from all animals. Both (right and left) lungs were frozen in liquid nitrogen and stored separately marked at -70 ° C.

Expression of human CFTR from CO-CFTR-C-His10 mRNA in CFTR knockout mice. CFTR expression was detected by Wester blot analysis of tissue lysates collected from CFTR mRNA-treated mouse lungs. Mature C band was detected in the left and right lungs of all treated mice, both in the case of the lipid-based formulation and in the case of the polymer-based formulation (Figure 34). Mature C band expression was verified by comparison with lysate collected from HEK

57739 Β1

293Τ human CO-CFTR-C-HiS10 positive cells as described in Example 9. In contrast, there was no detectable signal in the lysate collected from untreated wild-type control mice (Figure 34). Overall, these data suggest that both polymer and lipid-based formulations (such as the CKK-E12 formulation listed above) are also effective for delivering CFTR mRNA to the lungs, e.g. by inhalation, and once delivered, codon-optimized CFTR mRNA can efficiently express the CFTR protein.

Example 11: In vivo dose increase study Dose increase in PEI encapsulated mRNA and aerosol deliveries to pig lungs. Aerosol delivery of the combination of luminous luciferase (FFL) SNIM RNA and codon optimized CFTR (CO-CFTR) SNIM RNA in different concentrations to the lungs of pigs was determined by a gradual experimental procedure. In the first step, the FFL / CO-CFTR 35 SNIM RNA formulation was dispersed into anesthetized pigs during controlled ventilation. In the second step, the animals were sacrificed by "bolus" by injecting pentobarbital (100 mg / kg body weight) and potassium chloride into the lateral vein of the ear after sedation, and 24 hours after aerosol delivery. The lungs were removed and cut into approximately 1 cm wide tissue samples. To measure luciferase activity, tissue samples were incubated in medium containing D-luciferin substrate and offered ex vivo BLI luciferase. Following BLI, samples from luciferase-positive and luciferase-negative regions were taken for histopathology, immunohistochemistry, and in situ hybridization. The remaining samples were frozen in liquid nitrogen and later stored at -80 ° C until analysis by ΙΡΛ / Β and “Elisa”.

Synthesis of RNA information. In the example, codon optimized human cystic fibrous transmembrane conductivity regulator (CO-CFTR) SNIM RNA, codon optimized FFL mRNA SNIM RNA was synthesized by in vitro transcription from plasmid DNA patterns using standard procedures.

Experimental design. German Landrace pigs were obtained from the Technical University of Munich, Weihen Stephan, Germany. The pigs weighed 35-90 kg. The study was designed to match the age and weight of the pigs to control variability. Individual groups of 6 pigs (3 males and 3 females) were selected for each experimental group of this quadruple study. The first group was treated only with water for injection (WFI), which was given using a network inhaler Aeroneb. The second group was treated with a solution containing 1 mg of FFL SNIM RNA and 1 mg codon of optimized human CFTR (CO-CFTR) SNIM RNA in the PEI formulation described below, using an Aeroneb network inhaler. The third group received 1 mg of SNF RNA FFL and 5 mg of optimized HRTR CFR (CO-CFTR) SNIM RNA in the PEI formulation described below. The fourth group was treated with 1 mg FFL SNIM RNA and 10 mg optimized coded CFIM (CO-CFTR) SNIM RNA in the PEI formulation described below. The treatment and assessment scheme of each group is shown in Table 4, below

57739 Yes

Table 4. Experimental design for dose increase testing.

<td>Group</td><td>Pigs (Number 1 pole)</td><td>Treatman</td><td>Formulation</td>

<td> 1</td><td>6 (3 males + 3 females)</td><td>N / A</td><td>WFI</td>

<td> 2</td><td>6 (3 males + 3 females)</td><td>1 mg FFL + 1 mg CO-CFTR</td><td>Forked 25 kDa PEI + WFI</td>

<td> 3</td><td>6 (3 males + 3 females)</td><td>1 mg FFL + 5 mg CO-CFTR</td><td>Forked 25 kDa PEI + WFI</td>

<td> 4</td><td>6 (3 males + 3 females)</td><td>1 mg FFL + 10 mg CO-CFTR</td><td>Forked 25 kDa PEI + WFI</td>

MRNA - PEI formulation. The examples of standardized formulation procedures described below have been prepared directly for the treatment of animals.

Material [0187]

Injection pump (mixing device):

Manufacturer: KD Scientific

Type: KDS-210-CE

Syringe [0188]

Manufacturer: B. Brown

Type: Omnifix, 20ml_ or 30 mL / Luer Lock Solo

Ref: 4617207V

Hose [0189]

Manufacturer: B. Brown

Type: “Safeflow” Extension Set

Ref: 4097154

Needle [0190]

Manufacturer: B. Brown

Type: Sterican, 20G x 1 1/2

Ref: 4657519

Mixing valve [0191]

Manufacturer: B. Brown

Type: Discofix C 3SC

Ref: 16494C

Water for injections [0192]

Manufacturer: B. Brown

Type: Water

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Ref: 82423Ε Example of a process for the preparation of a polypex (polymer complex and RNA) comprising 1 mg of hCFTR SNIM RNA and 1 mg of FFL SNIM RNA N / P 10 in a volume of 8 ml: 3 mL of water for injection and 3 mL of RNA stock solution (c: 1 mg / mL in water; 1.5 mL FFL mRNA + 1.5 mL CFTR mRNA) were poured into 15 mL “falcon” tubes. In another falcon tube, 5.61 mL of water for injection was mixed with 0.39 mL of brPEI stock solution (c: 10 mg / mL in water). Two 20 ml syringes were fixed in the mixing device. Each of them was connected with a needle of a small intestine. One syringe was filled with RNA and the other with PEI solution, by pulling the pump on the syringes. (Settings: Diameter: 20.1 mm, Flow: 5mL / min, Volume: 5.9mL). The needles have been removed and the tubes connected to the mixing valve. It is important to connect the syringe containing RNA solution in the appropriate position of the valve angle. A needle is attached to control the outlet diameter. Mixing was performed using the infusion function of a syringe pump (Settings: Diameter: 20.1mm, Flow: 40mL / min, Volume: 5.8mL). To achieve a reproductive polydispersion index, the samples were fractionated manually during mixing. The first few mL while the flow was stable (100-200ml_) and the last few mL that sometimes contained air bubbles were collected in a separate hose. The mixture was incubated for 30 minutes at room temperature to form a polypex and then stored on ice. For different doses, the parameters were modified and adjusted as shown in Table 5.

Table 5. Examples of volumes and settings for different mixtures of volumes.

<td></td><td colspan="3">mRNA component</td><td colspan="3">PEI component</td>

<td>Group a</td><td>V (FFL SNIM RNA 1mg / mL) (mL)</td><td>V (hCFTR RECORD RNA 1mg / 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>Group</td><td>V (withdrawal) (ml)</td><td>V (infusion) (ml)</td>

V (withdrawal) and V (infusion) denote the setting on the pump of aspiration and dispersion syringes, respectively, of the mRNA and PEI components.

Transfection of HEK cells to check the functionality of the spray complex. After spraying, an aliquot of the complex (80ml) was used to transfect HEK cells. One day of rge transfection, 1x106 the cell is arranged in 6 plates. On the day of transfection, the medium was removed from the cells, the cells were washed with one PBS after which 80 ml of the complex was added to the well together with 920 μΙ of serum-free MEM medium. Triplicate wells were prepared for each complex. The cells were incubated with the complexes for 4 hours under standard cell culture conditions. At the end of the incubation, the medium-containing complex was removed and serum containing MEM medium (1 ml) per well was added. The plates were incubated under standard cell culture conditions. Within 24 hours after transfection, protein lysates were prepared using the same protocol and buffers used for animal tissues, excluding the degree of homogenization. Cells from the three wells were pooled for analysis. Human CFTR expression was detected using immunoprecipitation with antibodies R24.1 (R&D Systems) and Western Blot combination 217, 432 and 596.

57739 Antί antibodies (all from the Cystic Fibrosis Consortium, University of Pennsylvania, PA, USA). hCFTR can be detected for all pig-dispersed complexes (see Figures 54-57).

Aerosol application. The aerosol (WFI 44 ml alone, modified mRNA PEI formulation in WFI: 8, 24 and 44 ml) was sprayed and inhaled into the anesthetized pig using an Aeroneb® network inhaler. Sedation (calming) in pigs began with pretreatment with azaperone 2 mg / kg body weight, ketamine 15 mg / kg body weight, atropine 0.1 mg / kg body weight followed by insertion of an intravenous catheter into the lateral auricular vein. Pigs were anesthetized by intravenous injection of propofol 3-5 mg / kg body weight, as needed. Anesthesia was maintained by isoflurane (23%) with 1% propofol injection of a "bolus" of 4 to 8 mg / kg body weight to improve anesthesia as needed. The duration of anesthesia is about 1-3 hours. Pigs were sacrificed by "bolus" injection of pentobarbital (100 mg / kg body weight) and potassium chloride through the lateral vein of the ear 24 hours after completion of aerosolization. Lungs were removed and tissue samples were collected from different lung regions. Stored samples were subjected to various evaluation procedures such as bioluminescence, histopathology, IP / Western Blot and Elisa.

Bioluminescence analysis. To measure luciferase activity, tissues were either homogenized and analyzed in a tube luminometer or incubated in medium containing D-luciferin substrate and subjected to ex vivo BLI luciferase. The data show that a strong bioluminescent signal was observed in the 2-4 group (1 mg, 5, mg, and 10 mg, respectively), compared to control lung tissue samples from group 1 (WFI vehicle control) (Figures 35-38).

CFTR expression analysis using Western Blot and immunohistochemistry. FFL-positive tissue samples were cut (minimum 10 samples for each pig within the group) and analyzed by immunoprecipitation / Western blotting (IP-WB) and immunohistochemistry for human CFTR. Briefly, protein lysates were prepared from pig lungs as follows: Between 300-400 mg of lung tissue was used for analysis. Tissue was homogenized in basic buffer (20mM Tris, 150mM NaCl, pH 8.0) containing protease inhibitors using LisingMatrikA (MPBiomedicals, Ref: 6910-500) and Homogenizer FastPrep24 (MP Biomedicals). The entire tissue mix was transferred to a new tube with a 2 ml safety cap and then 25 ml of iodoacetamide (Sigma: 16125) and 1 μl of Omni cleave (enzyme) (1: 5 diluted in Omni Cleave buffer) (Epicenter: OC7810K) were added. The samples were then incubated for 5 minutes on ice, and then 26ml of 10% SDS solution was added. The samples were further incubated with stirring at 4 ° C for 60 min. Subsequent incubation, 260ml of lysis buffer (basic buffer 850ml + 10% TritonX-100 + 5% sodium deoxycholate) was added to the samples and they were incubated with stirring at 4 ° C for 90 minutes. Finally, the protein lysates were centrifuged at 13,000 rpm at 4 ° C for 10-20 min, and the supernatant was transferred to a new Eppendorf tube. Protein concentration was quantified using BCA Protein Assay. Samples were divided to contain 10 mg of total protein, and the final volume was adjusted with basic buffer up to 1 ml per sample. Based on the data shown in Example 6, CFTR immunoprecipitation was performed using R24.1 antibody, followed by Western Blot immunodetection of CFTR using a triple combination of three different antibodies obtained from the Cystic Fibrosis Consortium, University of Pennsylvania, PA, USA (antibody 217 , 432, 596). To control intra-group variability among different animals and variability in CFTR expression, a 150 kDa protein standard was set

57739 Β1 is as a reference, and the instance bars of different groups are normalized to this value. As shown in Figure 39, only 16% of the tissue samples analyzed in control pigs from group 1 showed a higher level of CFTR expression than the baseline. In contrast, the results for groups 3 and 4, which represent the therapeutic groups of 5 mg and 10 mg, each, show that in more than 30% of the samples, positive for the CFTR expression level was higher than the baseline (Figure 39). Moreover, the increase in CFTR expression observed within groups 3 and 4 was almost twice as large as the control.

CFTR immunohistochemistry analysis was performed by quantification of CFTR-positive bronchi and bronchioles. Bronchi / bronchioles are considered positive if at least one epithelial cell is detected within the epithelial cell layer showing a clear CFTR signal located on the membrane. A representative representation of the positive sample is shown in Figure 40. Codings for CFTR immunohistochemistry were optimized by assessing the specificity of available antibodies to CFTR using one antibody or a combination of up to three antibodies, respectively. Clear CFTR-specific signals were observed after incubation of antibody 596. Data show that CFTR-positive epithelial cells were detected in parts of lung tissue of all four groups, indicating that the immunohistochemical procedure demonstrates the detection of human and porcine CFTR (Figure 41 and 45). While low (Fig. 42), medium (Fig. 43) and high (Fig. 44) CFTR expression levels were observed for group 3, the overall results show that treatment with 5 mg codon-optimized CFTR SNIM RNA resulted in a higher number of CFTR positive cells and the overall intensity of the CFTR signal relative to the control carrier. The data also illustrate another increase in CFTR expression after treatment with 10 mg, showing a clear effect of the dose response (Figure 45). Quantification of absolute and relative numbers of CFTR-positive bronchi / bronchioles further supports these findings, revealing a significantly higher number in animals treated with 5 or 10 mg of human CFTR SNIM RNA compared to vehicle control (Figure 46). Demonstrating total elevation of CFTR expression levels after treatment with human CFTR SNIM RNA.

CFTR expression analysis by in situ hybridization (ISH). FFL positive tissue samples were cut (minimum 10 samples for each pig within the group) and subjected to manual in situ hybridization analysis using RNAscope® (Advanced Cell Diagnostic) ZZ technology. Probes were made based on the codon optimized sequence of the codon optimized human CFTR SNIM RNA (SEK ID NO: 17). Briefly, the RNAscope® test is an in situ hybridization test designed to visualize individual RNA molecules per cell in formalin-fixed, paraffin-imprinted (FFPE) tissue mounted on slides. Each sample of imprinted tissue was pretreated according to the manufacturer's instructions and incubated with a target-specific human CFTR-specific RNA probe. The hCFTR assay binds to CFTR and also shows cross-reactivity to human, mouse, rat, swine, and monkey. Once bound, the probe was hybridized to a cascade of signal amplified molecules, through a series of 6 consecutive amplification cycles. The sample was then treated with an HRP-labeled probe specific for the signal amplified cassette and analyzed by chromatic visualization using 3,3'-diaminobenzidine (DAB). The ubiquitin C specific assay was used as a positive control (Figures 47A and 48A), while dapB was used as a negative control (Figures 47B and 48B). The positive CFTR signal was compared to the signal of lung tissue of pigs both untreated and treated with the control vehicle (Fig. 49A and B). Stained samples were visualized under a standard light microscope. The data show that treatment with 1 mg codon-optimized human CFTR SNIM RNA resulted

57739 Βί a dramatic increase in CFTR expression in the right (A) and left (B) lung tissue of group 2, compared to the control (Figures 49 and 50 A&B) Moreover, an additional increase in CFTR expression was observed for the groups treated with 5 mg and 10 mg , which was observed by a dramatic increase in color within the right (A) and left (B) lung samples analyzed in dgiraZ and 4 (Figures 51 and 52 A & B). Taken together, these data strongly support efficient mRNA delivery through inhalation and expression of human CFTR in both lobes of the lung and their various tissues.

[0200] Conclusion. The results showed that both luciferase and CFTR mRNA could be efficiently delivered in vivo to lung tissue. Expression Luciferase expression has been observed in different tissue samples collected from different regions within the right and left lobes of the lung. Thus, it suggests that scattering is an efficient way to deliver mRNA and results in a fairly even distribution. Furthermore, in addition to luciferase, CFTR mRNA is also efficiently delivered to the lungs, leading to increased protein expression. Protein expression and activity were confirmed by IP-WB, immunohistochemistry, and in situ hybridization. Each approach clearly demonstrated a dose-dependent increase in mRNA delivery and CFTR expression and / or dose-dependent activity, within lung tissue. Taken together, the experiments emphasize the practicality and feasibility of delivering CFTR mRNA to the lungs of a human subject and demonstrate the efficiency of in vivo production of CFTR protein for therapeutic use.

Example 12: In vivo expression in the lungs This example demonstrates successful in vivo expression in the lungs after aerosol delivery of nanoparticles filled with mRNA. All studies were performed using German Landrace pigs obtained from the Technical University of Munich, Weihenstephan, Germany. The pigs weighed 35-90 kg. The FFL / CO-CFTR-C-His10 mRNA formulation or control vehicle was introduced using a Pari jet nebulizer. Pigs were sacrificed and perfused with saline, after a predetermined period of time, to allow protein expression with mRNA.

[0202] Synthesis of RNA information. In the example, codon-optimized mRNA luminous luciferase (CO-FFL) was synthesized by in vitro transcription from plasmid DNA samples.

CKK-E12 formulation. For the lipid-based nanoparticle experiment, a lipid formulation was created using 1 mg FFL + 9 mg CO-CFTR-C-His10 mRNA encapsulated in CKK-E12: DOPE: Chol: PEGDMG2K formulation (relative ratios 40: 30: 25: 5 (molar The solution was sprayed to the lungs of pigs using a designated inhaler.

Aerosol application. The aerosol (saline or CO-FFL CKK-E12 formulation) was sprayed and inhaled into the anesthetized pig. Sedation (calming) in pigs began with pretreatment using azaperone 2 mg / kg body weight, ketamine 15 mg / kg body weight, atropine 0.1 mg / kg body weight followed by insertion of an intravenous catheter into the lateral auricular vein. Pigs were anesthetized by intravenous injection of propofol 3-5 mg / kg body weight, as needed. Anesthesia was maintained by isoflurane (2-3%) with a 1% propofol bolus injection of 4 to 8 mg / kg body weight to improve anesthesia as needed. Duration of anesthesia

57739 Β1 was approximately 1-3 hours. Pigs were killed by a "bolus" injection of pentobarbital (100 mg / kg body weight) and potassium chloride through the lateral vein of the ear. Lung excision was performed and tissue samples were collected from different regions of the lung, followed by incubation in cell culture medium overnight. The preserved samples were subjected to bioluminescence detection.

Bioluminescence analysis. To measure luciferase activity, tissues were either homogenized and analyzed in a tube luminometer or incubated in medium containing D-luciferin substrate and subjected to ex vivo BLI luciferase. A strong bioluminescent signal was observed in all (A) pigs treated with FFL / CO-CFTR-C-His10 mRNA, compared to (B) control samples of lung tissue from control pigs (saline delivered as a control) (Figure 53 A&B).

These data illustrate that FFL / CFTR mRNA has been successfully delivered and expressed in the lungs using aerosols.

57739 Β1

SHORT DESCRIPTION SEQUENCES [0207]

SEQ ID NO: 1. CFTR wild strain - amino acid sequence.

SEQ ID NO: 2. CFTR wild-type - 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.

SEQID N0 8. FFL3 'UTR.

SEQ ID NO 9. Non-naturally occurring CFTR mRNA coding sequence # 2.

SEQ ID NO 10. Non-naturally occurring CFTR mRNA coding sequence # 3. SEQ ID NO 11. 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 His10 fused mRNA coding sequence.

SEQ ID NO: 16. Codon optimized human CFTR mRNA coding sequence with growth hormone leader sequence.

SEQ ID N0 18. mRNA leader # 1.

SEOID N0 19. mRNA leader # 2.

SEQ ID NO: 20. CFTR mRNA 3'-UTR # 2.

[0208]

MQRSPLEKASWSKLFFSWTRPILRKGYRQRLELSDIYQIPSVD SADNLSEKLEREWDRELASKKNPKLINALRRCFFWRFMFYGIFLYLGEVTKAVQPLLL GRIIASYDPDNKEERSIAIYLGIGLCLLFIVRTLLLHPAIFGLHHIGMQMRIAMFSLI YKKTLKLSSRVLDKISIGQLVSLLSNNLNKFDEGLALAHFVWIAPLQVALLMGLIWEL LQASAFCGLGFLIVLALFQAGLGRMMMKYRDQRAGKISERLVITSEMIENIQSVKAYC WEEAMEKMFENLRQTELKLTRKAAYVRYFNSSAFFFSGFFWFLSVLPYALIKGIILR KIFTTISFCIVLRMAVTRQFPWAVQTWYDSLGAINKIQDFLQKQEYKTLEYNLTTTEV VMENVTAFWEEGFGELFEKAKQNNNNRKTSNGDDSLFFSNFSLLGTPVLKDINFKIER GQLLAVAGSTGAGKTSLLMVIMGELEPSEGKIKHSGRISFCSQFSWIMPGTIKENIIF GVSYDEYRYRSVIKACQLEEDISKFAEKDNIVLGEGGITLSGGQRARISLARAVYKDA DLYLLDSPFGYLDVLTEKEIFESCVCKLMANKTRILVTSKMEHLKKADKILILHEGSS YFYGTFSELQNLQPDFSSKLMGCDSFDQFSAERRNSILTETLHRFSLEGDAPVSWTET KKQSFKQTGEFGEKRKNSILNPINSIRKFSIVQKTPLQMNGIEEDSDEPLERRLSLVP DSEQGEAILPRISVISTGPTLQARRRQSVLNLMTHSVNQGQNIHRKTTASTRKVSLAP QANLTELDIYSRRLSQETGLEISEEINEEDLKECFFDDMESIPAVTTWNTYLRYITVH KSLIFVLIWCLVIFLAEVAASLVVLWLLGNTPLQDKGNSTHSRNNSYAVIITSTSSYY VFYIYVGVADTLLAMGFFRGLPLVHTLITVSKILHHKMLHSVLQAPMSTLNTLKAGGI LNRFSKDIAILDDLLPLTIFDFIQLLLIVIGAIAWAVLQPYIFVATVPVIVAFIMLR AYFLQTSQQLKQLESEGRSPIFTHLVTSLKGLWTLRAFGRQPYFETLFHKALNLHTAN WFLYLSTLRWFQMRIEMIFVIFFIAVTFISILTTGEGEGRVGIILTLAMNIMSTLQWA VNSSIDVDSLMRSVSRVFKFIDMPTEGKPTKSTKPYKNGQLSKVMIIENSHVKKDDIW PSGGQMTVKDLTAKYTEGGNAILENISFSISPGQRVGLLGRTGSGKSTLLSAFLRLLN TEGEIQIDGVSWDSITLQQWRKAEGVIPQKVFIFSGTFRKNLDPYEQWSDQEIWKVAD EVGLRSVIEQFPGKLDFVLVDGGCVLSHGHKQLMCLARSVLSKAKILLLDEPSAHLDP VTYQIIRRTLKQAFADCTVILCEHRIEAMLECQQFLVIEENKVRQYDSIQKLLNERSL FRQAISPSDRVKLFPHRNSSKCKSKPQIAALKEETEEEVQDTRL (SEQ ID N0: 1)

57739 Yes

SEQ ID NO: 2 [0209]

AUGCAGAGGUCGCCUCUGGAAAAGGCCAGCGUUGUCUCCAAACUUUUUUUCAGCUGGACC AGACCAAUUUUGAGGAAAGGAUACAGACAGCGCCUGGAAUUGUCAGACAUAUACCAAAU CCCUUCUGUUGAUUCUGCUGACAAUCUAUCUGAAAAAUUGGAAAGAGAAUGGGAUAGAG AGCUGGCUUCAAAGAAAAAUCCUAAACUCAUUAAUGCCCUUCGGCGAUGUUUUUUCUGG AGAUUUAUGUUCUAUGGAAUCUUUUUAUAUUUAGGGGAAGUCACCAAAGCAGUACAGCC UCUCUUACUGGGAAGAAUCAUAGCUUCCUAUGACCCGGAUAACAAGGAGGAACGCUCUA UCGCGAUUUAUCUAGGCAUAGGCUUAUGCCUUCUCUUUAUUGUGAGGACACUGCUCCUAC ACCCAGCCAUUUUUGGCCUUCAUCACAUUGGAAUGCAGAUGAGAAUAGCUAUGUUUAGU UUGAUUUAUAAGAAGACUUUAAAGCUGUCAAGCCGUGUUCUAGAUAAAAUAAGUAUUGG ACAACUUGUUAGUCUCCUUUCCAACAACCUGAACAAAUUUGAUGAAGGACUUGCAUUGG CACAUUUCGUGUGGAUCGCUCCUUUGCAAGUGGCACUCCUCAUGGGGCUAAUCUGGGAGU UGUUACAGGCGUCUGCCUUCUGUGGACUUGGUUUCCUGAUAGUCCUUGCCCUUUUUCAGG CUGGGCUAGGGAGAAUGAUGAUGAAGUACAGAGAUCAGAGAGCUGGGAAGAUCAGUGAA AGACUUGUGAUUACCUCAGAAAUGAUUGAAAAUAUCCAAUCUGUUAAGGCAUACUGCUG GGAAGAAGCAAUGGAAAAAAUGAUUGAAAACUUAAGACAAACAGAACUGAAACUGACUC GGAAGGCAGCCUAUGUGAGAUACUUCAAUAGCUCAGCCUUCUUCUUCUCAGGGUUCUUU GUGGUGUUUUUAUCUGUGCUUCCCUAUGCACUAAUCAAAGGAAUCAUCCUCCGGAAAAU AUUCACCACCAUCUCAUUCUGCAUUGUUCUGCGCAUGGCGGUCACUCGGCAAUUUCCCUG GGCUGUACAAACAUGGUAUGACUCUCUUGGAGCAAUAAACAAAAUACAGGAUUUCUUAC AAAAGCAAGAAUAUAAGACAUUGGAAUAUAACUUAACGACUACAGAAGUAGUGAUGGAG AAUGUAACAGCCUUCUGGGAGGAGGGAUUUGGGGAAUUAUUUGAGAAAGCAAAACAAAA CAAUAACAAUAGAAAAACUUCUAAUGGUGAUGACAGCCUCUUCUUCAGUAAUUUCUCAC UUCUUGGUACUCCUGUCCUGAAAGAUAUUAAUUUCAAGAUAGAAAGAGGACAGUUGUUG GCGGUUGCUGGAUCCACUGGAGCAGGCAAGACUUCACUUCUAAUGAUGAUUAUGGGAGA ACUGGAGCCUUCAGAGGGUAAAAUUAAGCACAGUGGAAGAAUUUCAUUCUGUUCUCAGU UUUCCUGGAUUAUGCCUGGCACCAUUAAAGAAAAUAUCAUCUUUGGUGUUUCCUAUGAU GAAUAUAGAUACAGAAGCGUCAUCAAAGCAUGCCAACUAGAAGAGGACAUCUCCAAGUU UGCAGAGAAAGACAAUAUAGUUCUUGGAGAAGGUGGAAUCACACUGAGUGGAGGUCAAC GAGCAAGAAUUUCUUUAGCAAGAGCAGUAUACAAAGAUGCUGAUUUGUAUUUAUUAGAC UCUCCUUUUGGAUACCUAGAUGUUUUAACAGAAAAAGAAAUAUUUGAAAGCUGUGUCUG UAAACUGAUGGCUAACAAAACUAGGAUUUUGGUCACUUCUAAAAUGGAACAUUUAAAGA AAGCUGACAAAAUAUUAAUUUUGAAUGAAGGUAGCAGCUAUUUUUAUGGGACAUUUUCA GAACUCCAAAAUCUACAGCCAGACUUUAGCUCAAAACUCAUGGGAUGUGAUUCUUUCGAC CAAUUUAGUGCAGAAAGAAGAAAUUCAAUCCUAACUGAGACCUUACACCGUUUCUCAUU AGAAGGAGAUGCUCCUGUCUCCUGGACAGAAACAAAAAAACAAUCUUUUAAACAGACUG GAGAGUUUGGGGAAAAAAGGAAGAAUUCUAUUCUCAAUCCAAUCAACUCUAUACGAAAA UUUUCCAUUGUGCAAAAGACUCCCUUACAAAUGAAUGGCAUCGAAGAGGAUUCUGAUGA GCCUUUAGAGAGAAGGCUGUCCUUAGUACCAGAUUCUGAGCAGGGAGAGGCGAUACUGC CUCGCAUCAGCGUGAUCAGCACUGGCCCCACGCUUCAGGCACGAAGGAGGCAGUCUGUCC UGAACCUGAUGACACACUCAGUUAACCAAGGUCAGAACAUUCACCGAAAGACAACAGCAU CCACACGAAAAGUGUCACUGGCCCCUCAGGCAAACUUGACUGAACUGGAUAUAUAUUCAA GAAGGUUAUCUCAAGAAACUGGCUUGGAAAUAAGUGAAGAAAUUAACGAAGAAGACUUA AAGGAGUGCCUUUUUGAUGAUAUGGAGAGCAUACCAGCAGUGACUACAUGGAACACAUA CCUUCGAUAUAUUACUGUCCACAAGAGCUUAAUUUUUGUGCUAAUUUGGUGCUUAGUAA UUUUUCUGGCAGAGGUGGCUGCUUCUUUGGUUGUGCUGUGGCUCCUUGGAAACACUCCU

57739 Β1

CUUCAAGACAAAGGGAAUAGUACUCAUAGUAGAAAUAACAGCUAUGCAGUGAUUAUCAC CAGCACCAGUUCGUAUUAUGUGUUUUACAUUUACGUGGGAGUAGCCGACACUUUGCUUG CUAUGGGAUUCUUCAGAGGUCUACCACUGGUGCAUACUCUAAUCACAGUGUCGAAAAUU UUACACCACAAAAUGUUACAUUCUGUUCUUCAAGCACCUAUGUCAACCCUCAACACGUUG AAAGCAGGUGGGAUUCUUAAUAGAUUCUCCAAAGAUAUAGCAAUUUUGGAUGACCUUCU GCCUCUUACCAUAUUUGACUUCAUCCAGUUGUUAUUAAUUGUGAUUGGAGCUAUAGCAG UUGUCGCAGUUUUACAACCCUACAUCUUUGUUGCAACAGUGCCAGUGAUAGUGGCUUUU AUUAUGUUGAGAGCAUAUUUCCUCCAAACCUCACAGCAACUCAAACAACUGGAAUCUGAA GGCAGGAGUCCAAUUUUCACUCAUCUUGUUACAAGCUUAAAAGGACUAUGGACACUUCG UGCCUUCGGACGGCAGCCUUACUUUGAAACUCUGUUCCACAAAGCUCUGAAUUUACAUAC UGCCAACUGGUUCUUGUACCUGUCAACACUGCGCUGGUUCCAAAUGAGAAUAGAAAUGA UUUUUGUCAUCUUCUUCAUUGCUGUUACCUUCAUUUCCAUUUUAACAACAGGAGAAGGA GAAGGAAGAGUUGGUAUUAUCCUGACUUUAGCCAUGAAUAUCAUGAGUACAUUGCAGUG GGCUGUAAACUCCAGCAUAGAUGUGGAUAGCUUGAUGCGAUCUGUGAGCCGAGUCUUUA AGUUCAUUGACAUGCCAACAGAAGGUAAACCUACCAAGUCAACCAAACCAUACAAGAAUG GCCAACUCUCGAAAGUUAUGAUUAUUGAGAAUUCACACGUGAAGAAAGAUGACAUCUGG CCCUCAGGGGGCCAAAUGACUGUCAAAGAUCUCACAGCAAAAUACACAGAAGGUGGAAA UGCCAUAUUAGAGAACAUUUCCUUCUCAAUAAGUCCUGGCCAGAGGGUGGGCCUCUUGG GAAGAACUGGAUCAGGGAAGAGUACUUUGUUAUCAGCUUUUUUGAGACUACUGAACACU GAAGGAGAAAUCCAGAUCGAUGGUGUGUCUUGGGAUUCAAUAACUUUGCAACAGUGGAG GAAAGCCUUUGGAGUGAUACCACAGAAAGUAUUUAUUUUUUCUGGAACAUUUAGAAAAA ACUUGGAUCCCUAUGAACAGUGGAGUGAUCAAGAAAUAUGGAAAGUUGCAGAUGAGGUU GGGCUCAGAUCUGUGAUAGAACAGUUUCCUGGGAAGCUUGACUUUGUCCUUGUGGAUGG GGGCUGUGUCCUAAGCCAUGGCCACAAGCAGUUGAUGUGCUUGGCUAGAUCUGUUCUCA GUAAGGCGAAGAUCUUGCUGCUUGAUGAACCCAGUGCUCAUUUGGAUCCAGUAACAUAC CAAAUAAUUAGAAGAACUCUAAAACAAGCAUUUGCUGAUUGCACAGUAAUUCUCUGUGA ACACAGGAUAGAAGCAAUGCUGGAAUGCCAACAAUUUUUGGUCAUAGAAGAGAACAAAG UGCGGCAGUACGAUUCCAUCCAGAAACUGCUGAACGAGAGGAGCCUCUUCCGGCAAGCCA UCAGCCCCUCCGACAGGGUGAAGCUCUUUCCCCACCGGAACUCAAGCAAGUGCAAGUCUA AGCCCCAGAUUGCUGCUCUGAAAGAGGAGACAGAAGAAGAGGUGCAAGAUACAAGGCUU UAG (SEQ ID N0: 2)

SEQ ID NO: 3

57739 Yes

GCAGGCAUCUGCCUUUUGUGGCCUGGGAUUUCUGAUUGUGUUGGCAUUGUUUCAGGCUG GGCUUGGGCGGAUGAUGAUGAAGUAUCGCGACCAGAGAGCGGGUAAAAUCUCGGAAAGA CUCGUCAUCACUUCGGAAAUGAUCGAAAACAUCCAGUCGGUCAAAGCCUAUUGCUGGGAA GAAGCUAUGGAGAAGAUGAUUGAAAACCUCCGCCAAACUGAGCUGAAACUGACCCGCAA GGCGGCGUAUGUCCGGUAUUUCAAUUCGUCAGCGUUCUUCUUUUCCGGGUUCUUCGUUG UCUUUCUCUCGGUUUUGCCUUAUGCCUUGAUUAAGGGGAUUAUCCUCCGCAAGAUUUUC ACCACGAUUUCGUUCUGCAUUGUAUUGCGCAUGGCAGUGACACGGCAAUUUCCGUGGGCC GUGCAGACAUGGUAUGACUCGCUUGGAGCGAUCAACAAAAUCCAAGACUUCUUGCAAAA GCAAGAGUACAAGACCCUGGAGUACAAUCUUACUACUACGGAGGUAGUAAUGGAGAAUG UGACGGCUUUUUGGGAAGAGGGUUUUGGAGAACUGUUUGAGAAAGCAAAGCAGAAUAAC AACAACCGCAAGACCUCAAAUGGGGACGAUUCCCUGUUUUUCUCGAACUUCUCCCUGCUC GGAACACCCGUGUUGAAGGACAUCAAUUUCAAGAUUGAGAGGGGACAGCUUCUCGCGGU AGCGGGAAGCACUGGUGCGGGAAAAACUAGCCUCUUGAUGGUGAUUAUGGGGGAGCUUG AGCCCAGCGAGGGGAAGAUUAAACACUCCGGGCGUAUCUCAUUCUGUAGCCAGUUUUCAU GGAUCAUGCCCGGAACCAUUAAAGAGAACAUCAUUUUCGGAGUAUCCUAUGAUGAGUAC CGAUACAGAUCGGUCAUUAAGGCGUGCCAGUUGGAAGAGGACAUUUCUAAGUUCGCCGA GAAGGAUAACAUCGUCUUGGGAGAAGGGGGUAUUACAUUGUCGGGAGGGCAGCGAGCGC GGAUCAGCCUCGCGAGAGCGGUAUACAAAGAUGCAGAUUUGUAUCUGCUUGAUUCACCG UUUGGAUACCUCGACGUAUUGACAGAAAAAGAAAUCUUCGAGUCGUGCGUGUGUAAACU UAUGGCUAAUAAGACGAGAAUCCUGGUGACAUCAAAAAUGGAACACCUUAAGAAGGCGG ACAAGAUCCUGAUCCUCCACGAAGGAUCGUCCUACUUUUACGGCACUUUCUCAGAGUUGC AAAACUUGCAGCCGGACUUCUCAAGCAAACUCAUGGGGUGUGACUCAUUCGACCAGUUCA GCGCGGAACGGCGGAACUCGAUCUUGACGGAAACGCUGCACCGAUUCUCGCUUGAGGGUG AUGCCCCGGUAUCGUGGACCGAGACAAAGAAGCAGUCGUUUAAGCAGACAGGAGAAUUU GGUGAGAAAAGAAAGAACAGUAUCUUGAAUCCUAUUAACUCAAUUCGCAAGUUCUCAAU CGUCCAGAAAACUCCACUGCAGAUGAAUGGAAUUGAAGAGGAUUCGGACGAACCCCUGG AGCGCAGGCUUAGCCUCGUGCCGGAUUCAGAGCAAGGGGAGGCCAUUCUUCCCCGGAUUU CGGUGAUUUCAACCGGACCUACACUUCAGGCGAGGCGAAGGCAAUCCGUGCUCAACCUCA UGACGCAUUCGGUAAACCAGGGGCAAAACAUUCACCGCAAAACGACGGCCUCAACGAGAA AAGUGUCACUUGCACCCCAGGCGAAUUUGACUGAACUCGACAUCUACAGCCGUAGGCUUU CGCAAGAAACCGGACUUGAGAUCAGCGAAGAAAUCAAUGAAGAAGAUUUGAAAGAGUGU UUCUUUGAUGACAUGGAAUCAAUCCCAGCGGUGACAACGUGGAACACAUACUUGCGUUA CAUCACGGUGCACAAGUCCUUGAUUUUCGUCCUCAUCUGGUGUCUCGUGAUCUUUCUCGC UGAGGUCGCAGCGUCACUUGUGGUCCUCUGGCUGCUUGGUAAUACGCCCUUGCAAGACAA AGGCAAUUCUACACACUCAAGAAACAAUUCCUAUGCCGUGAUUAUCACUUCUACAAGCUC GUAUUACGUGUUUUACAUCUACGUAGGAGUGGCCGACACUCUGCUCGCGAUGGGUUUCU UCCGAGGACUCCCACUCGUUCACACGCUUAUCACUGUCUCCAAGAUUCUCCACCAUAAGA UGCUUCAUAGCGUACUGCAGGCUCCCAUGUCCACCUUGAAUACGCUCAAGGCGGGAGGUA UUUUGAAUCGCUUCUCAAAAGAUAUUGCAAUUUUGGAUGACCUUCUGCCCCUGACGAUC UUCGACUUCAUCCAGUUGUUGCUGAUCGUGAUUGGGGCUAUUGCAGUAGUCGCUGUCCU CCAGCCUUACAUUUUUGUCGCGACCGUUCCGGUGAUCGUGGCGUUUAUCAUGCUGCGGGC CUAUUUCUUGCAGACGUCACAGCAGCUUAAGCAACUGGAGUCUGAAGGGAGGUCGCCUA UCUUUACGCAUCUUGUGACCAGUUUGAAGGGAUUGUGGACGUUGCGCGCCUUUGGCAGG CAGCCCUACUUUGAAACACUGUUCCACAAAGCGCUGAAUCUCCAUACGGCAAAUUGGUUU UUGUAUUUGAGUACCCUCCGAUGGUUUCAGAUGCGCAUUGAGAUGAUUUUUGUGAUCUU

57739 Β1

CUUUAUCGCGGUGACUUUUAUCUCCAUCUUGACCACGGGAGAGGGCGAGGGACGGGUCG GUAUUAUCCUGACACUCGCCAUGAACAUUAUGAGCACUUUGCAGUGGGCAGUGAACAGC UCGAUUGAUGUGGAUAGCCUGAUGAGGUCCGUUUCGAGGGUCUUUAAGUUCAUCGACAU GCCGACGGAGGGAAAGCCCACAAAAAGUACGAAACCCUAUAAGAAUGGGCAAUUGAGUA AGGUAAUGAUCAUCGAGAACAGUCACGUGAAGAAGGAUGACAUCUGGCCUAGCGGGGGU CAGAUGACCGUGAAGGACCUGACGGCAAAAUACACCGAGGGAGGGAACGCAAUCCUUGA AAACAUCUCGUUCAGCAUUAGCCCCGGUCAGCGUGUGGGGUUGCUCGGGAGGACCGGGUC AGGAAAAUCGACGUUGCUGUCGGCCUUCUUGAGACUUCUGAAUACAGAGGGUGAGAUCC AGAUCGACGGCGUUUCGUGGGAUAGCAUCACCUUGCAGCAGUGGCGGAAAGCGUUUGGA GUAAUCCCCCAAAAGGUCUUUAUCUUUAGCGGAACCUUCCGAAAGAAUCUCGAUCCUUAU GAACAGUGGUCAGAUCAAGAGAUUUGGAAAGUCGCGGACGAGGUUGGCCUUCGGAGUGU AAUCGAGCAGUUUCCGGGAAAACUCGACUUUGUCCUUGUAGAUGGGGGAUGCGUCCUGU CGCAUGGGCACAAGCAGCUCAUGUGCCUGGCGCGAUCCGUCCUCUCUAAAGCGAAAAUUC UUCUCUUGGAUGAACCUUCGGCCCAUCUGGACCCGGUAACGUAUCAGAUCAUCAGAAGGA CACUUAAGCAGGCGUUUGCCGACUGCACGGUGAUUCUCUGUGAGCAUCGUAUCGAGGCCA UGCUCGAAUGCCAGCAAUUUCUUGUCAUCGAAGAGAAUAAGGUCCGCCAGUACGACUCCA UCCAGAAGCUGCUUAAUGAGAGAUCAUUGUUCCGGCAGGCGAUUUCACCAUCCGAUAGG GUGAAACUUUUUCCACACAGAAAUCGUCGAAGUGCAAGUCCAAACCGCAGAUCGCGGCC

SEQ ID NO: 4 [0211]

GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGAC CGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGU GACUCACCGUCCUUGACACG: 4 SEQ ID

SEQ ID NO: 5 [0212]

CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCC

AGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC (SEQID N0: 5)

SEQ ID NO: 6 [0213] GGGAUCCUACC (SEQ ID NO: 6)

SEQ ID NO: 7 [0214]

AUGGAAGAUGCCAAAAACAUUAAGAAGGGCCCAGCGCCAUUCUACCCACUCGAAGACGGG ACCGCCGGCGAGCAGCUGCACAAAGCCAUGAAGCGCUACGCCCUGGUGCCCGGCACCAUC GCCUUUACCGACGCACAUAUCGAGGUGGACAUUACCUACGCCGAGUACUUCGAGAUGAGC GUUCGGCUGGCAGAAGCUAUGAAGCGCUAUGGGCUGAAUACAAACCAUCGGAUCGUGGU GUGCAGCGAGAAUAGCUUGCAGUUCUUCAUGCCCGUGUUGGGUGCCCUGUUCAUCGGUG UGGCUGUGGCCCCAGCUAACGACAUCUACAACGAGCGCGAGCUGCUGAACAGCAUGGGCA UCAGCCAGCCCACCGUCGUAUUCGUGAGCAAGAAAGGGCUGCAAAAGAUCCUCAACGUGC AAAAGAAGCUACCGAUCAUACAAAAGAUCAUCAUCAUGGAUAGCAAGACCGACUACCAG

57739 Β1

GGCUUCCAAAGCAUGUACACCUUCGUGACUUCCCAUUUGCCACCCGGCUUCAACGAGUAC GACUUCGUGCCCGAGAGCUUCGACCGGGACAAAACCAUCGCCCUGAUCAUGAACAGUAGU GGCAGUACCGGAUUGCCCAAGGGCGUAGCCCUACCGCACCGCACCGCUUGUGUCCGAUUC AGUCAUGCCCGCGACCCCAUCUUCGGCAACCAGAUCAUCCCCGACACCGCUAUCCUCAGC GUGGUGCCAUUUCACCACGGCUUCGGCAUGUUCACCACGCUGGGCUACUUGAUCUGCGGC UUUCGGGUCGUGCUCAUGUACCGCUUCGAGGAGGAGCUAUUCUUGCGCAGCUUGCAAGAC UAUAAGAUUCAAUCUGCCCUGCUGGUGCCCACACUAUUUAGCUUCUUCGCUAAGAGCACU CUCAUCGACAAGUACGACCUAAGCAACUUGCACGAGAUCGCCAGCGGCGGGGCGCCGCUC AGCAAGGAGGUAGGUGAGGCCGUGGCCAAACGCUUCCACCUACCAGGCAUCCGCCAGGGC UACGGCCUGACAGAAACAACCAGCGCCAUUCUGAUCACCCCCGAAGGGGACGACAAGCCU GGCGCAGUAGGCAAGGUGGUGCCCUUCUUCGAGGCUAAGGUGGUGGACUUGGACACCGG UAAGACACUGGGUGUGAACCAGCGCGGCGAGCUGUGCGUCCGUGGCCCCAUGAUCAUGAG CGGCUACGUUAACAACCCCGAGGCUACAAACGCUCUCAUCGACAAGGACGGCUGGCUGCA CAGCGGCGACAUCGCCUACUGGGACGAGGACGAGCACUUCUUCAUCGUGGACCGGCUGAA GAGCCUGAUCAAAUACAAGGGCUACCAGGUAGCCCCAGCCGAACUGGAGAGCAUCCUGCU GCAACACCCCAACAUCUUCGACGCCGGGGUCGCCGGCCUGCCCGACGACGAUGCCGGCGA GCUGCCCGCCGCAGUCGUCGUGCUGGAACACGGUAAAACCAUGACCGAGAAGGAGAUCGU GGACUAUGUGGCCAGCCAGGUUACAACCGCCAAGAAGCUGCGCGGUGGUGUUGUGUUCG UGGACGAGGUGCCUAAAGGACUGACCGGCAAGUUGGACGCCCGCAAGAUCCGCGAGAUUC UCAUUAAGGCCAAGAAGGGCGGCAAGAUCGCCGUGUA (SEQ ID N0: 7)

SEQ ID NO: 8 UUUGAAUU (SEQ ID NO: 8)

SEOIDNO: 9 [216]

AUGCAGAGAAGCCCCCUGGAAAAGGCCAGCGUGGUGUCCAAGCUGUUCUUCAGCUGGACC AGACCCAUCCUGAGAAAGGGCUACAGACAGAGACUGGAACUGAGCGACAUCUACCAGAUC CCCAGCGUGGACAGCGCCGACAACCUGAGCGAGAAGCUGGAAAGAGAGUGGGACAGAGA GCUGGCUAGCAAGAAGAACCCCAAGCUGAUCAACGCCCUGAGGCGGUGCUUCUUCUGGCG GUUUAUGUUCUACGGCAUCUUCCUGUACCUGGGCGAAGUGACAAAGGCCGUGCAGCCCCU GCUCCUGGGCAGAAUCAUUGCCAGCUACGACCCCGACAACAAAGAGGAAAGAUCUAUCGC CAUCUACCUGGGCAUCGGCCUGUGCCUGCUGUUCAUCGUGCGGACACUGCUGCUGCACCC CGCCAUCUUCGGCCUGCACCACAUCGGCAUGCAGAUGAGAAUCGCCAUGUUCAGCCUGAU CUACAAGAAAACCCUGAAGCUGAGCAGCAGGGUGCUGGACAAGAUCAGCAUCGGACAGCU GGUGUCCCUGCUGAGCAACAACCUGAACAAGUUCGACGAGGGACUGGCCCUGGCUCACUU CGUGUGGAUCGCUCCACUGCAGGUCGCCCUGCUGAUGGGCCUGAUCUGGGAGCUGCUGCA GGCCAGCGCUUUCUGCGGCCUGGGCUUUCUGAUUGUGCUGGCCCUGUUUCAGGCUGGCCU GGGCAGGAUGAUGAUGAAGUACAGGGACCAGAGAGCCGGCAAGAUCAGCGAGAGACUGG UCAUCACCAGCGAGAUGAUCGAGAACAUCCAGAGCGUGAAGGCCUACUGCUGGGAAGAG GCCAUGGAAAAGAUGAUCGAAAACCUGAGACAGACCGAGCUGAAGCUGACCAGAAAGGC CGCCUACGUGCGGUACUUCAACAGCAGCGCCUUCUUCUUCUCCGGCUUCUUCGUGGUGUU CCUGUCCGUGCUGCCCUACGCCCUGAUCAAGGGCAUCAUCCUGAGGAAGAUCUUCACCAC

57739 Β1

CAUUUCUUUCUGCAUCGUGCUGAGAAUGGCCGUGACCAGACAGUUCCCCUGGGCCGUGCA GACUUGGUACGACAGCCUGGGCGCCAUCAACAAGAUCCAGGACUUCCUGCAGAAGCAGGA GUACAAGACCCUCGAGUACAACCUGACCACCACCGAGGUGGUCAUGGAAAACGUGACCGC CUUCUGGGAGGAAGGCUUCGGCGAGCUGUUCGAGAAGGCCAAGCAGAACAACAACAACA GAAAGACCAGCAACGGCGACGACUCCCUGUUCUUCUCCAACUUCUCCCUGCUGGGCACCC CCGUGCUGAAGGACAUCAACUUCAAGAUCGAGAGAGGCCAGCUGCUCGCCGUGGCCGGCU CUACAGGCGCUGGCAAGACCUCUCUGCUGAUGGUCAUCAUGGGCGAGCUGGAACCCAGCG AGGGCAAGAUCAAGCACAGCGGCAGAAUCAGCUUCUGCAGCCAGUUCAGCUGGAUCAUGC CCGGCACCAUCAAAGAGAACAUCAUCUUCGGCGUGUCCUACGACGAGUACAGAUACAGAA GCGUGAUCAAGGCCUGCCAGCUGGAAGAGGACAUCAGCAAGUUCGCCGAGAAGGACAACA UCGUGCUGGGCGAGGGCGGCAUCACCCUGUCUGGCGGCCAGAGAGCCAGAAUCAGCCUGG CCAGAGCCGUGUACAAGGACGCCGACCUGUACCUGCUGGACAGCCCCUUCGGCUACCUGG ACGUGCUGACCGAGAAAGAGAUCUUCGAGAGCUGCGUGUGCAAGCUGAUGGCCAACAAG ACCAGAAUCCUGGUCACCAGCAAGAUGGAACACCUGAAGAAGGCCGACAAGAUCCUGAUC CUGCACGAGGGCAGCAGCUACUUCUACGGCACAUUCAGCGAGCUGCAGAACCUGCAGCCC GACUUCAGCAGCAAACUGAUGGGCUGCGACAGCUUCGACCAGUUCAGCGCCGAGAGAAGA AACAGCAUCCUGACCGAGACACUGCACAGAUUCAGCCUGGAAGGCGACGCCCCCGUGUCU UGGACCGAGACAAAGAAGCAGAGCUUCAAGCAGACCGGCGAGUUCGGCGAGAAGAGAAA GAACUCCAUCCUGAACCCCAUCAACAGCAUCCGGAAGUUCAGCAUCGUGCAGAAAACCCC CCUGCAGAUGAACGGCAUCGAAGAGGACAGCGACGAGCCCCUGGAAAGACGGCUGAGCCU GGUGCCUGACAGCGAGCAGGGCGAGGCCAUCCUGCCUAGAAUCAGCGUGAUCAGCACCGG CCCCACCCUGCAGGCUAGAAGGCGGCAGAGCGUGCUGAACCUGAUGACCCACAGCGUGAA CCAGGGCCAGAACAUCCACCGCAAGACCACCGCCAGCACCAGAAAGGUGUCCCUGGCUCC UCAGGCCAACCUGACCGAGCUGGACAUCUACAGCAGAAGGCUGAGCCAGGAAACCGGCCU GGAAAUCAGCGAGGAAAUCAACGAAGAGGACCUGAAAGAGUGCUUCUUCGACGACAUGG AAUCCAUCCCCGCCGUGACCACCUGGAACACCUACCUGCGGUACAUCACCGUGCACAAGA GCCUGAUCUUCGUGCUGAUCUGGUGCCUGGUCAUCUUCCUGGCCGAGGUGGCCGCCAGCC UGGUGGUGCUGUGGCUCCUGGGAAACACCCCUCUGCAGGACAAGGGCAACAGCACCCACA GCAGAAACAACAGCUACGCCGUGAUCAUCACCUCCACCAGCUCCUACUACGUGUUCUACA UCUACGUGGGCGUGGCCGACACCCUGCUGGCUAUGGGCUUCUUCAGAGGCCUGCCCCUGG UGCACACCCUGAUCACCGUGUCCAAGAUCCUGCACCAUAAGAUGCUGCACAGCGUGCUGC AGGCUCCCAUGAGCACCCUGAACACACUGAAGGCUGGCGGCAUCCUGAACAGGUUCAGCA AGGAUAUCGCCAUCCUGGACGACCUGCUGCCUCUGACCAUCUUCGACUUCAUCCAGCUGC UGCUGAUCGUGAUCGGCGCUAUCGCCGUGGUGGCCGUGCUGCAGCCCUACAUCUUCGUGG CCACCGUGCCCGUGAUCGUGGCCUUCAUUAUGCUGAGAGCCUACUUUCUGCAGACCAGCC AGCAGCUGAAGCAGCUGGAAAGCGAGGGCAGAAGCCCCAUCUUCACCCACCUCGUGACCA GCCUGAAGGGCCUGUGGACCCUGAGAGCCUUCGGCAGACAGCCCUACUUCGAGACACUGU UCCACAAGGCCCUGAACCUGCACACCGCCAACUGGUUUCUGUACCUGUCCACCCUGAGAU GGUUCCAGAUGAGGAUCGAGAUGAUCUUCGUCAUCUUCUUUAUCGCCGUGACCUUCAUCU CUAUCCUGACCACCGGCGAGGGCGAGGGAAGAGUGGGAAUCAUCCUGACCCUGGCCAUGA ACAUCAUGAGCACACUGCAGUGGGCCGUGAACAGCAGCAUCGACGUGGACAGCCUGAUGA GAAGCGUGUCCAGAGUGUUCAAGUUCAUCGACAUGCCUACCGAGGGCAAGCCCACCAAGA GCACCAAGCCCUACAAGAACGGCCAGCUGAGCAAAGUGAUGAUCAUCGAGAACAGCCACG UCAAGAAGGACGACAUCUGGCCCAGCGGCGGACAGAUGACCGUGAAGGACCUGACCGCCA AGUACACAGAGGGCGGCAACGCUAUCCUGGAAAACAUCAGCUUCAGCAUCAGCCCAGGCC

57739 Β1

AGAGAGUGGGCCUGCUGGGGAGAACAGGCAGCGGCAAGUCUACCCUGCUGUCCGCCUUCC UGAGACUGCUGAACACCGAGGGCGAGAUCCAGAUCGAUGGCGUGUCCUGGGACUCCAUCA CCCUGCAGCAGUGGCGCAAGGCCUUCGGCGUGAUCCCCCAGAAGGUGUUCAUCUUCAGCG GCACCUUCAGAAAGAACCUGGACCCCUACGAGCAGUGGUCCGACCAGGAAAUCUGGAAGG UCGCCGAUGAAGUGGGCCUGAGAUCCGUGAUCGAGCAGUUCCCCGGCAAGCUGGACUUCG UGCUGGUGGACGGCGGCUGCGUGCUGAGCCACGGCCACAAGCAGCUGAUGUGUCUGGCCC GCUCCGUGCUGAGCAAGGCUAAGAUUCUGCUGCUGGACGAGCCUAGCGCCCACCUGGACC CUGUGACCUACCAGAUCAUCAGAAGGACCCUGAAGCAGGCCUUCGCCGACUGCACCGUGA UCCUGUGCGAGCACAGAAUCGAGGCCAUGCUGGAAUGCCAGCAGUUCCUGGUCAUCGAAG AGAACAAAGUGCGGCAGUACGACAGCAUCCAGAAGCUGCUGAACGAGAGAAGCCUGUUC AGACAGGCCAUCAGCCCCAGCGACAGAGUGAAGCUGUUCCCCCACCGCAACAGCAGCAAG UGCAAGAGCAAGCCCCAGAUCGCCGCCCUGAAAGAAGAGACUGAGGAAGAGGUGCAGGAC ACCAGACUGUGA (SEQ ID N0: 9)

SEQ ID NO: 10 [0217]

57739 Yes

UACCUCGACGUGCUGACCGAAAAAGAAAUCUUCGAGAGCUGCGUGUGUAAGCUGAUGGC UAAUAAGACUAGAAUCCUCGUGACGUCCAAAAUGGAACAUCUUAAGAAGGCGGAUAAGA UUCUCAUUCUUCACGAGGGGUCGAGCUACUUCUACGGGACUUUUAGCGAGCUGCAGAAU UUGCAGCCGGACUUCAGCUCAAAGCUCAUGGGCUGCGACUCGUUCGAUCAGUUCAGCGCC GAACGGCGCAAUUCGAUCUUGACGGAAACCCUGCACAGAUUCUCGCUGGAGGGAGAUGCA CCUGUCUCGUGGACCGAAACCAAGAAGCAGUCCUUCAAGCAGACGGGAGAGUUCGGAGAA AAGCGGAAGAACUCAAUCCUCAACCCAAUCAACUCCAUUCGCAAAUUCUCAAUCGUGCAG AAAACUCCACUGCAGAUGAACGGUAUCGAAGAGGAUUCGGACGAGCCACUUGAGCGGAG ACUGUCGCUGGUGCCAGAUUCAGAACAGGGGGAGGCAAUCCUGCCGCGCAUUUCCGUGAU CAGCACUGGGCCGACCCUCCAAGCUAGACGCAGGCAAUCAGUGCUGAAUCUCAUGACCCA CUCCGUCAACCAGGGACAGAAUAUCCACCGCAAGACCACCGCGUCGACUAGAAAGGUGUC AUUGGCACCGCAAGCAAAUUUGACUGAACUUGACAUCUACUCACGGCGCCUCUCCCAAGA AACCGGAUUGGAAAUCUCCGAAGAGAUUAACGAAGAAGAUUUGAAAGAGUGUUUCUUCG ACGAUAUGGAGUCGAUCCCCGCAGUGACCACUUGGAAUACGUAUCUUCGGUACAUCACCG UGCACAAGAGCCUGAUCUUCGUCCUCAUCUGGUGCCUGGUGAUCUUUCUGGCCGAAGUCG CCGCUUCGCUGGUCGUGCUGUGGCUGCUCGGUAAUACCCCGCUCCAAGACAAAGGCAAUU CCACUCACUCGCGCAACAACAGCUACGCUGUGAUUAUCACGUCAACCUCGUCGUACUAUG UGUUCUACAUCUACGUGGGAGUCGCGGACACUCUGCUCGCUAUGGGCUUCUUUCGCGGAC UGCCCCUGGUCCACACUCUCAUCACGGUGAGCAAGAUCCUCCAUCAUAAGAUGCUCCAUU CCGUGCUGCAGGCCCCGAUGAGCACUCUCAACACUCUGAAGGCGGGUGGAAUCUUGAACA GAUUUUCCAAAGACAUCGCGAUUCUGGACGAUCUGCUCCCACUCACUAUCUUCGACUUCA UCCAACUGCUGCUGAUCGUCAUCGGAGCUAUCGCCGUGGUGGCUGUCCUCCAGCCGUAUA UCUUCGUGGCCACUGUGCCGGUGAUUGUCGCUUUCAUCAUGUUGCGCGCGUACUUCUUGC AAACCUCGCACtCAACUCAAGCAACUGGAGUCCGACtGCiCCCtGAGCCCAAUCUUUACCCAUC UGGUGACUUCACUGAAAGGUCUGUGGACCCUCCGCGCCUUUGGUCGCCAGCCUUACUUCG AAACUCUCUUUCACAAAGCACUGAAUCUCCACACUGCAAACUGGUUCUUGUACCUGUCCA cccugcggugguuccaaaugcggaucgagaugaucuuugucaucuucuucaucgccguga CUUUUAUCUCCAUCCUCACCACCGGCGAGGGAGAGGGGAGAGUGGGAAUCAUCCUGACGC UGGCGAUGAAUAUCAUGUCCACUUUGCAGUGGGCCGUCAAUUCGAGCAUCGACGUGGAU ucgcugaugcgcagcgugucgcgcguguucaaguucaucgauaugcccaccgaagguaaa CCCACCAAGAGCACGAAGCCUUACAAGAACGGGCAGCUCUCAAAGGUGAUGAUUAUCGAG AACUCCCAUGUGAAGAAGGACGACAUCUGGCCAUCCGGAGGACAGAUGACCGUGAAGGAC CUGACCGCCAAAUACACGGAGGGCGGAAAUGCAAUCCUCGAAAACAUCUCGUUCUCCAUC UCGCCUGGCCAAAGGGUGGGACUUUUGGGACGCACUGGAUCCGGAAAGAGCACCCUGCUU AGCGCCUUCUUGAGGCUCUUGAACACCGAGGGCGAAAUCCAGAUCGAUGGCGUGUCGUGG GAUUCGAUCACCCUGCAGCAGUGGAGAAAGGCCUUCGGGGUGAUCCCGCAAAAAGUGUUC AUCUUCUCCGGAACGUUUCGGAAAAACCUUGACCCAUACGAACAAUGGUCGGAUCAAGAG AUUUGGAAGGUCGCCGACGAAGUGGGGCUGCGCUCCGUGAUCGAGCAGUUUCCGGGAAA ACUGGACUUCGUCUUGGUCGACGGCGGAUGCGUCCUGUCCCACGGACAUAAGCAGCUGAU GUGCCUGGCCCGCAGCGUCCUUUCAAAAGCUAAGAUCCUGCUGCUGGAUGAACCUUCAGC ACACCUCGACCCGGUCACCUACCAGAUCAUCAGACGGACCCUGAAACAGGCCUUUGCGGA UUGUACUGUGAUCUUGUGUGAACACCGCAUUGAAGCCAUGCUGGAGUGCCAGCAGUUCC UGGUCAUCGAAGAGAACAAAGUGCGGCAGUACGAUUCCAUCCAAAAACUGCUCAAUGAG CGGUCCCUGUUCAGACAGGCAAUUAGCCCGAGCGACAGGGUCAAAUUGUUCCCCCAUAGA

AAUUCGUCGAAAUGUAAGUCAAAGCCUCAGAUCGCGGCACUGAAAGAAGAAACUGAAGA AGAGGUGCAAGACACCAGACUGUGA (SEQ ID N0: 10)

SEQ ID NO: 11 [0218]

57739 Yes

AUGCAGAGAAGCCCACUGGAAAAGGCGUCGGUGGUGUCAAAGCUGUUCUUUAGCUGGAC CAGACCUAUCUUGCGGAAGGGAUACCGCCAACGCCUGGAGCUGUCGGACAUCUACCAGAU UCCGUCAGUGGAUUCAGCAGACAAUCUCUCCGAAAAGCUGGAACGCGAAUGGGACAGAG AGUUGGCGUCAAAGAAGAACCCAAAGUUGAUCAAUGCCCUGCGCCGCUGCUUCUUCUGGC GGUUCAUGUUCUACGGAAUCUUUCUGUACCUCGGCGAAGUCACCAAGGCUGUGCAACCGC UUCUGCUGGGACGCAUCAUCGCCUCAUACGACCCGGACAACAAGGAAGAACGCUCCAUCG CAAUCUACCUCGGGAUCGGCCUCUGCCUGCUGUUUAUCGUGCGGACGCUGCUGCUCCAUC CAGCCAUUUUCGGACUGCACCACAUUGGCAUGCAAAUGCGGAUCGCCAUGUUCAGCCUGA UCUACAAAAAGACCCUGAAGUUGAGCUCACGGGUGUUGGAUAAGAUUUCGAUCGGACAG CUGGUGUCGCUGCUCUCCAACAACCUCAACAAGUUUGACGAAGGCCUGGCACUGGCCCAC UUCGUGUGGAUUGCCCCGUUGCAAGUCGCCCUUUUGAUGGGCCUUAUUUGGGAGCUGUU GCAGGCAUCUGCCUUUUGUGGCCUGGGAUUUCUGAUUGUGUUGGCAUUGUUUCAGGCUG GGCUUGGGCGGAUGAUGAUGAAGUAUCGCGACCAGAGAGCGGGUAAAAUCUCGGAAAGA CUCGUCAUCACUUCGGAAAUGAUCGAAAACAUCCAGUCGGUCAAAGCCUAUUGCUGGGAA GAAGCUAUGGAGAAGAUGAUUGAAAACCUCCGCCAAACUGAGCUGAAACUGACCCGCAA GGCGGCGUAUGUCCGGUAUUUCAAUUCGUCAGCGUUCUUCUUUUCCGGGUUCUUCGUUG UCUUUCUCUCGGUUUUGCCUUAUGCCUUGAUUAAGGGGAUUAUCCUCCGCAAGAUUUUC ACCACGAUUUCGUUCUGCAUUGUAUUGCGCAUGGCAGUGACACGGCAAUUUCCGUGGGCC GUGCAGACAUGGUAUGACUCGCUUGGAGCGAUCAACAAAAUCCAAGACUUCUUGCAAAA GCAAGAGUACAAGACCCUGGAGUACAAUCUUACUACUACGGAGGUAGUAAUGGAGAAUG UGACGGCUUUUUGGGAGGAAGGAUUCGGCGAAUUGUUCGAAAAGGCUAAGCAGAACAAC AACAAUCGGAAAACCUCCAAUGGGGACGAUUCGCUGUUCUUCUCGAAUUUCUCCCUGCUG GGAACGCCCGUGCUUAAAGACAUCAACUUCAAGAUCGAACGGGGCCAGCUGCUCGCGGUC GCGGGCAGCACUGGAGCGGGAAAGACUUCCCUGCUCAUGGUCAUCAUGGGAGAGCUGGA GCCCUCGGAGGGCAAAAUCAAGCACUCGGGGAGGAUCUCAUUUUGCAGCCAGUUCUCGUG GAUCAUGCCCGGUACUAUCAAAGAAAACAUCAUCUUUGGAGUCAGCUAUGACGAGUACC GCUACCGGUCGGUGAUCAAGGCCUGCCAGCUGGAAGAAGAUAUCUCCAAGUUCGCCGAAA AGGACAACAUUGUGCUGGGAGAAGGUGGAAUCACUCUCUCGGGAGGCCAGCGCGCACGG AUCUCACUCGCAAGGGCCGUGUACAAGGAUGCCGAUUUGUACCUGUUGGAUUCGCCGUUC GGUUAUCUUGAUGUCCUCACUGAGAAAGAGAUUUUUGAGUCGUGCGUCUGUAAGCUGAU GGCCAACAAAACCCGCAUCCUGGUGACCUCGAAGAUGGAGCACUUGAAGAAGGCCGACAA AAUCCUUAUCCUCCAUGAGGGUAGCUCAUACUUCUACGGCACCUUUUCGGAACUGCAGAA UCUGCAGCCCGACUUCUCAUCAAAACUGAUGGGAUGUGACUCGUUCGAUCAGUUCUCGGC GGAGCGGCGGAACUCGAUCCUCACCGAAACUCUCCACCGGUUCAGCCUCGAGGGAGAUGC CCCAGUCAGCUGGACCGAAACUAAGAAGCAGUCCUUCAAACAGACCGGAGAGUUCGGAGA AAAACGCAAGAACUCCAUCCUCAAUCCAAUCAACAGCAUCCGCAAGUUCAGCAUCGUGCA GAAAACUCCACUUCAGAUGAACGGAAUCGAAGAGGAUAGCGACGAGCCGCUUGAGCGGA GAUUGUCACUGGUGCCGGACAGCGAGCAAGGGGAAGCGAUUCUGCCGCGGAUCUCCGUGA UCUCGACUGGCCCUACCCUCCAAGCUCGCAGACGCCAGAGCGUGCUGAAUCUCAUGACCC ACUCAGUCAACCAGGGACAAAACAUCCAUAGAAAGACCACCGCUUCAACCCGGAAAGUGU

57739 Β1

CACUUGCACCGCAGGCAAACCUGACCGAACUCGACAUCUACAGCAGACGGCUCUCACAAG AAACUGGAUUGGAGAUCAGCGAAGAGAUCAACGAAGAAGAUCUCAAAGAAUGCUUCUUC GACGAUAUGGAGUCCAUCCCAGCAGUCACUACGUGGAAUACCUACCUCCGCUACAUCACU GUGCACAAGAGCCUGAUUUUCGUGUUGAUCUGGUGCCUGGUCAUCUUCUUGGCCGAGGU GGCCGCGAGCCUCGUGGUCCUCUGGCUGCUCGGCAAUA CGCCGCUGCAAGAUAAGGGAAA UUCCACGCAUAGCAGAAACAACUCAUACGCAGUGAUCAUCACUAGCACUUCAUCGUACUA CGUGUUCUACAUCUACGUGGGGGUGGCCGAUACUCUGUUGGCAAUGGGAUUCUUUAGAG GGCUGCCUCUGGUGCAUACUCUGAUCACUGUGUCCAAGAUCCUCCACCACAAGAUGCUCC ACUCCGUGCUUCAGGCCCCUAUGUCAACUCUCAACACCCUCAAGGCCGGAGGUAUUCUUA AUCGCUUUUCCAAGGACAUCGCCAUUCUCGAUGACUUGCUUCCCCUGACUAUCUUCGACU UUAUCCAGUUGCUGCUGAUUGUGAUCGGCGCUAUUGCCGUCGUCGCAGUGCUGCAACCGU ACAUCUUUGUGGCUACCGUCCCAGUCAUUGUGGCCUUCAUCAUGCUCAGGGCAUACUUUC UCCAGACCAGCCAGCAGCUCAAGCAGCUCGAAUCCGAAGGCAGAUCGCCGAUCUUCACCC ACCUCGUCACUUCGCUCAAGGGCCUCUGGACCCUGCGCGCCUUCGGUCGCCAGCCGUAUU UCGAAACCCUGUUCCAUAAAGCACUGAACCUCCAUACUGCGAACUGGUUUCUCUACCUUU CAACCCUGAGGUGGUUCCAGAUGAGAAUCGAGAUGAUCUUUGUGAUCUUCUUUAUCGCU GUGACGUUCAUCUCCAUUCUCACUACCGGCGAGGGAGAGGGCAGAGUGGGGAUUAUCCUC ACGCUGGCCAUGAAUAUCAUGAGCACGCUGCAGUGGGCCGUCAAUAGCAGCAUCGACGUG GACUCCCUGAUGCGGUCCGUGUCGAGAGUGUUUAAGUUCAUCGAUAUGCCUACUGAAGG GAAACCGACCAAGUCGACCAAGCCGUACAAGAAUGGGCAGCUGAGCAAGGUGAUGAUUA UUGAGAACUCCCAUGUGAAGAAGGACGACAUCUGGCCCAGCGGAGGCCAGAUGACCGUGA AGGACUUGACCGCUAAGUACACUGAGGGUGGAAAUGCCAUUCUUGAGAAUAUCAGCUUC UCGAUCUCGCCGGGACAACGCGUGGGAUUGCUCGGGCGCACUGGCAGCGGCAAAUCCACC CUGCUUAGCGCUUUUCUGAGGCUGCUGAACACUGAAGGUGAAAUUCAAAUCGAUGGAGU GUCGUGGGAUAGCAUCACCCUUCAACAGUGGCGCAAGGCCUUCGGCGUGAUCCCUCAAAA GGUCUUUAUCUUCUCGGGGACGUUCCGGAAAAAUCUCGACCCCUACGAACAGUGGUCAGA CCAAGAGAUUUGGAAAGUCGCAGAUGAGGUCGGACUGCGCUCAGUGAUCGAACAGUUUC CGGGUAAACUUGACUUCGUGCUCGUCGAUGGAGGUUGCGUCCUGUCCCACGGACAUAAGC AGCUGAUGUGUCUGGCGCGCUCGGUCCUCUCCAAAGCGAAGAUCCUGCUGCUCGAUGAAC CGUCCGCCCACCUUGAUCCAGUGACCUAUCAGAUCAUUCGGAGAACUUUGAAGCAAGCCU UCGCUGACUGCACCGUCAUCCUCUGCGAACACCGGAUCGAGGCAAUGCUGGAGUGCCAAC AGUUUCUGGUCAUCGAAGAAAACAAAGUGCGCCAGUAUGACUCGAUCCAAAAACUUCUG AACGAGCGCUCCCUCUUCCGGCAGGCAAUCAGCCCAUCCGACCGCGUGAAGUUGUUCCCU CAUCGGAAUAGCUCCAAAUGCAAAUCGAAGCCGCAGAUCGCUGCCUUGAAAGAAGAAACC GAAGAAGAAGUCCAAGACACUAGGU

SEQ ID NO: 12 [0219]

AUGCAGCGGUCCCCUCUGGAGAAGGCUUCCGUGGUCAGCAAGCUGUUCUUCUCGUGGACC AGACCUAUCCUCCGCAAGGGAUACCGCCAGCGCCUGGAGCUGUCAGAUAUCUACCAGAUC CCAAGCGUGGACUCAGCCGACAAUCUGAGCGAAAAGCUGGAACGGGAGUGGGACCGGGA GCUCGCCUCCAAGAAGAAUCCGAAGUUGAUCAAUGCGCUGCGCAGAUGCUUCUUCUGGCG GUUUAUGUUUUACGGCAUCUUUCUGUAUCUCGGAGAAGUGACCAAAGCCGUGCAGCCGC UGCUCUUGGGUAGGAUCAUUGCUUCGUACGACCCGGACAACAAAGAAGAACGCUCCAUCG CCAUCUACCUCGGAAUCGGUCUGUGCCUGCUCUUUAUCGUGCGCACUCUCCUGCUGCAUC

57739 Β1

CGGCGAUCUUCGGACUGCACCACAUCGGCAUGCAAAUGCGGAUCGCAAUGUUCUCACUGA UCUACAAAAAGACUCUGAAGCUCAGCUCCAGAGUGCUGGAUAAGAUCUCGAUCGGGCAAC UCGUCAGCCUGCUGUCGAACAAUCUGAAUAAGUUCGACGAAGGGUUGGCCCUCGCACAUU UCGUGUGGAUCGCACCGCUGCAAGUGGCGCUCCUGAUGGGACUCAUUUGGGAACUGCUCC AAGCCAGCGCGUUUUGCGGACUCGGAUUCCUGAUCGUGCUCGCCCUGUUCCAAGCCGGAC UGGGGCGCAUGAUGAUGAAGUACCGCGAUCAGCGGGCAGGAAAGAUCUCCGAGCGGUUG GUGAUCACUUCCGAAAUGAUCGAGAAUAUUCAGUCCGUGAAGGCCUACUGCUGGGAAGA AGCUAUGGAAAAGAUGAUUGAAAACUUGCGGCAAACUGAGCUGAAAUUGACUCGCAAAG CGGCAUACGUCCGCUACUUCAAUAGCAGCGCCUUCUUCUUUUCGGGCUUUUUCGUGGUGU UUCUGAGCGUGCUGCCCUACGCUCUGAUCAAGGGAAUCAUCCUCCGGAAAAUCUUCACCA CCAUUUCGUUCUGUAUCGUGUUGCGCAUGGCCGUGACUCGCCAGUUCCCCUGGGCGGUGC AGACCUGGUACGACAGCUUGGGGGCAAUCAAUAAGAUUCAAGACUUCUUGCAAAAGCAG GAGUACAAGACUCUGGAGUACAACCUGACCACCACUGAAGUCGUGAUGGAGAACGUGACC GCCUUUUGGGAAGAGGGUUUUGGAGAACUGUUUGAGAAAGCAAAGCAGAAUAACAACAA CCGCAAGACCUCAAAUGGGGACGAUUCCCUGUUUUUCUCGAACUUCUCCCUGCUCGGAAC ACCCGUGUUGAAGGACAUCAAUUUCAAGAUUGAGAGGGGACAGCUUCUCGCGGUAGCGG GAAGCACUGGUGCGGGAAAAACUAGCCUCUUGAUGGUGAUUAUGGGGGAGCUUGAGCCC AGCGAGGGGAAGAUUAAACACUCCGGGCGUAUCUCAUUCUGUAGCCAGUUUUCAUGGAU CAUGCCCGGAACCAUUAAAGAGAACAUCAUUUUCGGAGUAUCCUAUGAUGAGUACCGAU ACAGAUCGGUCAUUAAGGCGUGCCAGUUGGAAGAGGACAUUUCUAAGUUCGCCGAGAAG GAUAACAUCGUCUUGGGAGAAGGGGGUAUUACAUUGUCGGGAGGGCAGCGAGCGCGGAU CAGCCUCGCGAGAGCGGUAUACAAAGAUGCAGAUUUGUAUCUGCUUGAUUCACCGUUUG GAUACCUCGACGUAUUGACAGAAAAAGAAAUCUUCGAGUCGUGCGUGUGUAAACUUAUG GCUAAUAAGACGAGAAUCCUGGUGACUUCCAAAAUGGAGCAUCUCAAGAAGGCGGACAA GAUCCUGAUUCUGCAUGAGGGAUCAAGCUAUUUCUACGGAACUUUUUCCGAGCUGCAGA ACCUCCAGCCGGAUUUUAGCUCCAAGCUGAUGGGUUGCGACUCAUUCGACCAAUUCUCGG CUGAGCGGCGGAACUCAAUCCUGACCGAAACCCUGCAUCGCUUCUCCCUUGAGGGAGAUG CCCCGGUGUCGUGGACUGAGACUAAAAAGCAGUCGUUUAAGCAAACUGGCGAAUUCGGC GAAAAGCGGAAGAAUAGCAUCCUCAACCCAAUCAACAGCAUUCGGAAGUUCAGCAUCGUC CAAAAGACCCCGCUCCAGAUGAACGGCAUUGAAGAGGACUCAGACGAGCCAUUGGAAAGA CGCCUGUCACUGGUCCCAGAUUCGGAGCAGGGUGAAGCAAUUCUGCCUCGGAUCUCGGUC AUCUCGACUGGCCCCACUCUCCAAGCUCGGCGGAGACAGAGCGUGCUUAACUUGAUGACC CACUCCGUGAACCAGGGUCAGAACAUCCACCGCAAAACCACCGCCUCCACCAGGAAGGUG UCACUGGCCCCUCAAGCCAAUCUGACUGAGUUGGAUAUCUACUCCAGAAGGCUCAGCCAG GAAACCGGACUGGAAAUCUCGGAAGAGAUCAACGAAGAGGAUCUCAAAGAGUGUUUCUU CGACGACAUGGAAUCAAUCCCUGCUGUCACUACUUGGAACACCUAUCUCCGCUACAUUAC CGUGCACAAGUCACUCAUCUUCGUCCUGAUCUGGUGCCUCGUGAUCUUCCUGGCCGAGGU CGCAGCAUCGCUGGUCGUGCUGUGGCUGCUCGGCAACACCCCACUCCAAGACAAAGGCAA CAGCACCCAUUCCCGCAACAACUCCUACGCGGUGAUCAUCACUUCAACUUCGUCCUACUA CGUCUUUUACAUCUACGUGGGCGUGGCGGACACGCUCCUGGCUAUGGGGUUCUUUCGCGG GCUGCCUCUUGUCCACACGCUCAUCACUGUGUCAAAGAUUCUCCACCACAAAAUGCUGCA CUCCGUGCUCCAGGCCCCUAUGUCGACUUUGAACACGCUUAAGGCCGGAGGCAUCCUUAA CAGAUUCUCGAAAGAUAUCGCGAUCUUGGACGAUCUUCUGCCGCUGACUAUCUUUGACUU CAUCCAACUCCUGCUGAUCGUCAUCGGUGCCAUCGCAGUGGUCGCGGUGCUCCAACCGUA CAUUUUCGUGGCGACUGUGCCGGUGAUCGUGGCGUUCAUCAUGCUGCGGGCUUACUUUCU

57739 Β1

UCAGACCUCACAGCAGCUGAAGCAACUCGAAUCGGAGGGUAGAUCACCAAUCUUUACCCA CCUCGUCACCUCGCUGAAGGGACUCUGGACCCUGCGCGCAUUUGGACGGCAACCGUACUU CGAGACUCUCUUCCAUAAGGCCCUGAAUCUGCAUACGGCGAAUUGGUUUCUUUACCUCUC GACGCUCCGCUGGUUCCAGAUGCGCAUUGAGAUGAUUUUCGUCAUCUUUUUCAUCGCGGU GACCUUCAUCUCCAUCCUCACCACGGGUGAGGGAGAGGGCAGAGUCGGAAUUAUCCUCAC UCUGGCCAUGAACAUCAUGUCCACUCUGCAGUGGGCCGUCAACUCAUCCAUUGACGUGGA CUCGCUGAUGCGCUCCGUGUCGAGAGUGUUCAAGUUCAUCGAUAUGCCGACCGAGGGAAA GCCAACUAAGUCGACCAAGCCGUACAAAAACGGACAGCUGAGCAAGGUCAUGAUCAUCGA AAACUCCCACGUGAAAAAGGAUGACAUCUGGCCGUCCGGUGGACAGAUGACGGUGAAGG AUCUGACUGCGAAGUACACUGAGGGAGGGAAUGCCAUCCUCGAAAACAUCUCAUUCUCAA UCUCCCCUGGACAGAGGGUCGGGCUGCUGGGCCGCACUGGCUCGGGGAAGUCGACUCUUC UUUCGGCAUUUCUGCGCUUGCUCAAUACCGAGGGAGAAAUCCAGAUCGAUGGAGUGUCA UGGGACUCGAUCACCCUGCAGCAGUGGCGCAAGGCUUUUGGCGUCAUCCCGCAAAAGGUG UUCAUCUUCUCGGGCACUUUUAGAAAGAAUCUGGAUCCCUACGAACAGUGGUCAGAUCA AGAGAUUUGGAAAGUCGCAGACGAAGUGGGCCUCCGGUCCGUGAUUGAACAGUUUCCGG GAAAGCUCGACUUCGUGCUUGUGGACGGAGGAUGUGUGCUGAGCCACGGCCACAAACAGC UCAUGUGCCUGGCUCGGUCGGUCCUGUCGAAAGCAAAGAUCCUGCUGCUGGACGAACCGU CGGCACACCUCGAUCCAGUGACGUACCAGAUCAUCCGGCGGACCCUGAAGCAGGCCUUCG CAGACUGCACUGUCAUUUUGUGUGAACACAGAAUCGAAGCUAUGUUGGAGUGCCAGCAG UUCCUGGUCAUCGAAGAAAACAAAGUCCGCCAGUACGAUUCGAUUCAGAAGCUGCUGAAC GAACGGAGCCUCUUCAGACAGGCGAUCAGCCCCAGCGAUCGGGUCAAGUUGUUCCCGCAU CGGAACAGCAGCAAGUGUAAGUCAAAGCCUCAGAUCGCUGCACUCAAAGAAGAGACUGA AGAAGAAGUGCAAGACACCAGACUCUGA (SEQ ID N0: 12)

SEQ ID NO: 13 [0220]

AUGCAGCGCUCGCCUCUGGAGAAAGCCUCAGUCGUGUCAAAACUGUUCUUUAGCUGGACU CGCCCGAUUCUCCGGAAGGGUUAUAGACAGCGCUUGGAGCUCUCCGACAUCUACCAAAUC CCUUCCGUGGACUCCGCCGACAACCUGUCGGAGAAGCUCGAACGCGAGUGGGACCGGGAA CUCGCGUCCAAAAAGAAUCCAAAACUCAUUAAUGCACUGCGCCGCUGCUUCUUCUGGCGC UUUAUGUUUUACGGUAUCUUUCUCUACCUGGGCGAGGUGACGAAAGCAGUGCAGCCGCU CCUGCUUGGCAGAAUUAUCGCCUCGUACGAUCCGGAUAACAAAGAAGAACGCUCAAUCGC UAUCUACCUCGGUAUCGGAUUGUGCCUGCUUUUCAUCGUGCGCACCCUGUUGCUGCACCC GGCGAUUUUCGGACUCCACCACAUCGGAAUGCAAAUGAGAAUUGCAAUGUUCUCAUUGA UCUACAAAAAGACCCUUAAACUGUCGUCCCGCGUCCUCGACAAGAUUUCAAUCGGCCAGC UGGUGUCGCUUCUUUCGAAUAAUCUUAACAAGUUCGAUGAAGGACUCGCGCUCGCCCAUU UCGUGUGGAUCGCACCACUUCAAGUCGCACUGCUCAUGGGACUGAUUUGGGAGUUGCUGC AGGCUUCCGCCUUUUGCGGCCUGGGAUUCCUGAUCGUCCUGGCUUUGUUCCAGGCUGGAC UGGGCAGAAUGAUGAUGAAGUACCGGGACCAGCGGGCAGGAAAGAUCAGCGAAAGGCUC GUGAUCACUAGCGAAAUGAUCGAGAACAUCCAAUCCGUCAAGGCGUACUGCUGGGAAGA AGCGAUGGAGAAGAUGAUCGAAAAUCUUCGCCAGACCGAACUCAAACUCACUAGAAAGG CUGCCUACGUGCGCUACUUUAACAGCUCAGCAUUUUUCUUCUCCGGAUUUUUCGUGGUGU UCCUGUCGGUGCUGCCAUACGCCCUGAUCAAGGGGAUCAUUCUUCGCAAAAUCUUCACCA CGAUCUCAUUCUGCAUUGUCCUCCGGAUGGCCGUGACGCGGCAGUUCCCUUGGGCAGUGC AAACUUGGUACGAUUCGACUGUAGUG

57739 Β1

GAGUACAAAACCCUGGAGUACAAUCUGACCACUACGGAAGUCGUGAUGGAAAACGUGAC UGCUUUUUGGGAGGAAGGCUUCGGCGAACUUUUUGAAAAGGCAAAGCAAAACAAUAACA ACAGAAAGACGUCAAACGGCGAUGACUCGCUGUUCUUCUCCAAUUUCUCCCUGCUCGGCA CCCCUGUGCUGAAGGACAUCAACUUCAAAAUUGAACGCGGACAGCUGCUGGCCGUGGCGG GAUCGACCGGGGCUGGGAAAACCUCGUUGUUGAUGGUGAUCAUGGGAGAACUCGAACCC UCGGAGGGAAAGAUUAAGCAUAGCGGACGGAUCAGCUUCUGUUCCCAGUUCUCGUGGAU CAUGCCGGGAACCAUUAAGGAAAACAUCAUCUUCGGCGUGUCCUACGACGAGUACCGGUA UAGGUCGGUGAUCAAGGCCUGCCAGUUGGAAGAGGACAUCUCCAAGUUCGCUGAGAAGG ACAACAUCGUGCUCGGUGAAGGGGGCAUUACUCUGUCCGGUGGCCAGCGCGCGAGAAUUU CGCUGGCUCGCGCGGUGUACAAAGAUGCGGAUCUCUAUCUGCUGGAUUCGCCCUUCGGAU ACCUCGAUGUCCUCACGGAGAAGGAGAUCUUCGAAUCGUGCGUGUGCAAGUUGAUGGCG AACAAGACUAGGAUCCUGGUCACUUCCAAGAUGGAGCACUUGAAGAAGGCCGAUAAGAU CUUGAUCCUCCAUGAAGGAUCGAGCUACUUUUACGGAACUUUCUCAGAGCUGCAGAACUU GCAGCCGGACUUCUCAAGCAAACUGAUGGGUUGCGACUCGUUCGACCAGUUUUCGGCAGA ACGGCGGAACUCGAUCCUGACUGAGACUCUGCAUCGCUUUUCGCUGGAAGGCGAUGCCCC UGUGUCCUGGACUGAAACCAAGAAGCAAUCCUUCAAACAAACUGGAGAAUUCGGAGAAA AGCGGAAGAACUCCAUCCUUAACCCCAUCAAUAGCAUCCGGAAGUUCUCAAUCGUCCAAA AGACCCCGCUGCAGAUGAAUGGCAUCGAAGAAGAUAGCGACGAACCUCUUGAAAGACGGC UGUCCUUGGUGCCAGACUCAGAACAGGGAGAAGCUAUCCUGCCGCGGAUCUCCGUGAUCA GCACCGGACCGACUCUGCAGGCUCGCAGACGCCAGAGCGUGCUCAACCUGAUGACCCACU CCGUGAACCAGGGACAAAACAUCCAUAGAAAGACCACGGCCUCCACCAGAAAAGUCUCCC UGGCACCGCAAGCCAACCUGACUGAACUGGACAUCUACAGCAGAAGGCUCAGCCAAGAAA CCGGACUGGAGAUUUCAGAAGAAAUCAACGAGGAAGAUCUUAAAGAGUGCUUCUUCGAC GACAUGGAAUCGAUCCCAGCCGUGACCACUUGGAAUACCUAUCUGAGAUACAUCACCGUG CACAAAUCCCUGAUCUUCGUGCUGAUCUGGUGCCUGGUGAUCUUCCUGGCUGAGGUGGCC GCCUCACUGGUGGUGCUUUGGUUGCUGGGGAAUACGCCGCUCCAAGACAAGGGAAACUCC ACGCACUCCAGAAACAACUCGUACGCCGUGAUCAUCACGUCGACUUCGUCGUACUACGUG UUCUACAUCUACGUCGGUGUGGCAGACACUCUCUUGGCGAUGGGCUUUUUCCGGGGACUG CCACUGGUCCACACCCUGAUCACCGUGUCCAAAAUCUUGCACCACAAGAUGCUCCACAGC GUGCUGCAAGCCCCGAUGAGCACCCUGAAUACCCUCAAAGCGGGAGGCAUCCUCAACAGA UUCAGCAAGGACAUCGCCAUCCUCGACGACCUGUUGCCCCUGACCAUCUUCGAUUUCAUC CAGCUUCUUCUCAUCGUGAUCGGGGCAAUCGCUGUCGUGGCGGUGCUGCAGCCGUACAUC UUCGUGGCGACUGUGCCAGUGAUCGUCGCCUUUAUCAUGCUGCGGGCCUACUUUCUCCAA ACUUCCCAACAGCUGAAACAACUGGAGUCGGAGGGCCGCAGCCCUAUCUUCACCCAUCUG GUGACCAGCCUCAAAGGACUGUGGACUCUGAGGGCUUUCGGGAGGCAGCCAUACUUCGAG ACUCUCUUUCACAAGGCCCUGAAUCUCCAUACGGCAAAUUGGUUUUUGUAUUUGAGUACC CUCCGAUGGUUUCAGAUGCGCAUUGAGAUGAUUUUUGUGAUCUUCUUUAUCGCGGUGAC UUUUAUCUCCAUCUUGACCACGGGAGAGGGCGAGGGACGGGUCGGUAUUAUCCUGACAC UCGCCAUGAACAUUAUGAGCACUUUGCAGUGGGCAGUGAACAGCUCGAUUGAUGUGGAU AGCCUGAUGAGGUCCGUUUCGAGGGUCUUUAAGUUCAUCGACAUGCCGACGGAGGGAAA GCCCACAAAAAGUACGAAACCCUAUAAGAAUGGGCAAUUGAGUAAGGUAAUGAUCAUCG AGAACAGUCACGUGAAGAAGGAUGACAUCUGGCCUAGCGGGGGUCAGAUGACCGUGAAG GACCUGACGGCAAAAUACACCGAGGGAGGGAACGCAAUCCUUGAAAACAUCUCGUUCAGC AUUAGCCCCGGUCAGCGUGUGGGGUUGCUCGGGAGGACCGGGUCAGGAAAAUCGACGUU GCUGUCGGCCUUCUUGAGACUUCUGAAUACAGAGGGUGAGAUCCAGAUCGACGGCGUUU

57739 Β1

CGUGGGAUAGCAUCACCUUGCAGCAGUGGCGCAAGGCGUUCGGAGUCAUUCCCCAAAAGG UGUUCAUCUUUUCGGGAACCUUCCGCAAGAAUCUGGAUCCGUACGAACAGUGGAGCGACC AAGAGAUUUGGAAAGUGGCAGAUGAAGUGGGAUUGCGGAGCGUCAUCGAACAGUUUCCG GGAAAGCUCGAUUUCGUCCUUGUGGACGGUGGAUGUGUGCUGUCGCACGGCCAUAAGCA GCUGAUGUGUCUCGCCCGCUCGGUGCUGUCAAAGGCGAAGAUCCUCUUGCUGGAUGAGCC AUCAGCCCAUCUGGACCCGGUGACGUACCAGAUCAUUAGACGGACGCUGAAACAGGCAUU CGCGGACUGCACUGUGAUCCUCUGUGAACAUCGGAUCGAGGCCAUGCUGGAGUGUCAACA AUUCUUGGUCAUCGAAGAGAACAAAGUGCGGCAGUACGACAGCAUCCAAAAGCUGCUGA ACGAGAGGUCCCUCUUCCGCCAGGCCAUCUCCCCAUCCGACCGGGUCAAGCUGUUCCCUC ACCGCAACAGCUCAAAGUGCAAAUCCAAACCCCAGAUCGCAGCGCUGAAAGAAGAAACUG AAGAAGAAGUGCAAGACACUAGACUGUGA: 13

SEQ ID NO: 14 [0221]

AUGCAAAGGUCCCCAUUGGAGAAGGCCUCAGUGGUGUCGAAGCUGUUCUUCUCGUGGACC AGGCCUAUCCUCCGGAAGGGAUACAGACAGCGGCUGGAACUGUCCGAUAUCUACCAGAUC CCCAGCGUGGACAGCGCCGAUAAUCUCAGCGAAAAGCUGGAACGGGAAUGGGACCGCGAA CUCGCUUCGAAGAAGAACCCGAAGCUGAUUAAUGCUCUGCGGAGAUGUUUCUUUUGGCG GUUCAUGUUUUACGGAAUCUUUCUGUACUUGGGAGAGGUCACGAAGGCUGUGCAGCCUC UGCUGCUGGGACGGAUUAUCGCGUCGUAUGACCCCGACAAUAAGGAAGAACGCAGCAUCG CAAUCUACCUGGGCAUCGGAUUGUGCCUGCUGUUCAUCGUGAGAACUCUCCUGCUGCAUC CAGCCAUCUUCGGACUCCACCACAUUGGAAUGCAGAUGAGAAUCGCAAUGUUCUCCCUGA UCUACAAGAAAACGCUCAAGCUCAGCAGCCGCGUGCUCGAUAAGAUCAGCAUCGGUCAAU UGGUGUCCCUGCUGUCGAAUAACCUCAACAAGUUCGACGAAGGGUUGGCCCUCGCUCACU UCGUGUGGAUCGCACCUCUGCAAGUGGCCCUGCUGAUGGGACUGAUUUGGGAGCUGCUGC AGGCUUCCGCUUUCUGCGGCCUGGGAUUUCUUAUCGUGCUUGCUCUGUUCCAGGCGGGAC UGGGACGCAUGAUGAUGAAGUACCGGGACCAACGGGCUGGAAAGAUCAGCGAACGGCUG GUGAUCACUUCCGAAAUGAUUGAGAAUAUCCAGUCAGUCAAGGCGUACUGCUGGGAAGA GGCUAUGGAAAAGAUGAUUGAAAAUCUGAGACAAACCGAGCUGAAGCUGACUCGGAAAG CGGCCUACGUCAGAUACUUCAAUAGCUCAGCUUUCUUUUUCUCGGGGUUUUUCGUCGUGU UCCUGUCGGUGCUUCCCUAUGCCCUGAUUAAGGGCAUCAUUCUGCGCAAGAUCUUCACUA CGAUCUCAUUCUGCAUCGUGCUGCGCAUGGCUGUGACCAGACAAUUCCCGUGGGCCGUGC AAACCUGGUACGAUUCACUGGGAGCCAUCAACAAGAUCCAAGACUUUCUCCAAAAACAGG AGUAUAAGACCCUGGAGUACAACCUGACUACUACCGAGGUGGUGAUGGAGAACGUGACU GCGUUUUGGGAAGAAGGGUUCGGCGAACUGUUUGAAAAGGCCAAGCAGAACAAUAACAA CAGAAAGACUUCAAACGGAGAUGACUCGCUGUUCUUUUCGAACUUCAGCCUGCUGGGUAC CCCAGUGUUGAAAGAUAUCAACUUCAAGAUUGAGAGAGGACAGCUGCUGGCUGUGGCGG GAUCCACCGGAGCAGGAAAAACUUCACUCCUGAUGGUGAUCAUGGGAGAACUCGAACCGU CAGAGGGGAAGAUUAAACACUCGGGAAGAAUCUCAUUUUGCUCCCAAUUUUCAUGGAUU AUGCCGGGAACCAUUAAAGAAAACAUUAUCUUCGGCGUGUCCUACGACGAGUACCGCUAC AGAUCGGUGAUCAAAGCAUGCCAGCUGGAAGAGGACAUCUCGAAAUUCGCUGAAAAAGA CAAUAUCGUGCUCGGGGAAGGCGGCAUCACCCUCAGCGGAGGACAACGGGCACGGAUUUC GCUCGCACGCGCAGUCUACAAAGACGCCGAUCUCUACCUCUUGGACAGCCCAUUCGGGUA UCUGGACGUGCUCACCGAGAAAGAGAUCUUCGAAAGCUGCGUCUGCAAGCUCAUGGCCAA CAAGACCCGCAUCCUCGUGACGUCGAAGAUGGAACAUCUUAAGAAGGCUGACAAGAUUCU

57739 Yes

CAUUCUCCAUGAAGGGAGCUCAUACUUCUACGGCACCUUUUCCGAGCUCCAGAAUCUGCA ACCGGACUUCUCGUCCAAGCUGAUGGGCUGCGAUUCGUUUGAUCAGUUCUCCGCCGAGCG GAGAAACAGCAUUCUGACGGAAACCCUGCACCGGUUCUCGCUGGAAGGCGAUGCACCGGU GUCGUGGACCGAAACUAAGAAGCAAUCGUUCAAGCAGACGGGAGAGUUUGGAGAGAAGC GGAAAAACUCCAUCCUCAACCCGAUCAACAGCAUCCGGAAGUUCAGCAUCGUGCAAAAGA CCCCGCUCCAGAUGAAUGGCAUUGAAGAGGACUCCGACGAACCUUUGGAACGCAGACUGA GCCUCGUGCCGGAUUCAGAACAGGGAGAAGCCAUUCUGCCACGGAUCUCCGUGAUCAGCA CUGGGCCAACUCUCCAAGCACGGCGGAGGCAGUCCGUGCUGAAUCUUAUGACGCACAGCG UGAACCAAGGGCAGAACAUCCAUAGAAAAACGACCGCUUCGACCAGGAAAGUCUCCCUCG CCCCACAAGCUAACCUCACGGAACUGGAUAUCUACUCCCGCAGACUGUCGCAAGAGACUG GCCUUGAGAUCUCCGAAGAGAUUAACGAAGAAGAUCUCAAAGAAUGUUUCUUCGAUGAU AUGGAAUCAAUCCCGGCAGUGACCACUUGGAACACCUACUUGCGCUAUAUCACUGUGCAC AAAAGCCUUAUCUUCGUCCUCAUCUGGUGCCUCGUCAUCUUCCUGGCUGAGGUCGCAGCC UCGCUGGUCGUGCUCUGGUUGCUCGGAAACACUCCGCUGCAGGAUAAGGGGAAUUCGACU CACUCGCGGAACAAUUCGUACGCUGUCAUUAUCACCUCGACGUCGUCAUACUACGUGUUU UACAUCUACGUGGGAGUGGCUGACACUCUGUUGGCUAUGGGGUUCUUUCGCGGCCUGCCA CUGGUCCAUACUCUCAUUACUGUGUCCAAAAUCCUUCAUCACAAGAUGUUGCAUUCAGUG CUGCAAGCACCGAUGUCCACCCUCAAUACCCUUAAGGCUGGCGGGAUUCUCAACCGCUUC UCGAAAGACAUCGCCAUCCUCGAUGAUCUUCUGCCUCUCACCAUCUUUGAUUUCAUCCAG CUGCUCCUGAUCGUGAUCGGAGCGAUUGCCGUGGUGGCAGUGUUGCAGCCGUACAUCUUU GUCGCAACUGUGCCGGUCAUCGUCGCCUUCAUCAUGCUGCGCGCCUACUUCUUGCAAACG UCACAGCAACUGAAGCAGCUUGAAUCCGAGGGAAGAUCACCUAUCUUCACCCACCUCGUG ACUUCGCUGAAGGGGCUGUGGACGCUGCGCGCAUUUGGAAGGCAACCGUACUUCGAGACU UUGUUCCACAAGGCGCUCAAUCUUCACACUGCCAAUUGGUUCUUGUACCUGUCAACGCUG AGAUGGUUUCAGAUGCGGAUCGAAAUGAUCUUCGUGAUCUUCUUUAUCGCGGUGACUUU CAUCUCGAUCCUGACUACCGGAGAGGGAGAAGGACGGGUGGGUAUUAUCCUCACUCUGGC GAUGAACAUCAUGUCGACGCUUCAGUGGGCGGUGAAUAGCUCAAUCGAUGUCGACUCGC UGAUGCGCUCCGUGAGCCGGGUGUUUAAGUUCAUCGACAUGCCAACUGAAGGGAAGCCG ACCAAGUCGACCAAACCGUACAAAAACGGACAGCUCUCCAAGGUGAUGAUUAUCGAGAAU ucccacgugaaaaaggacgacaucuggccauccgguggacagaugaccgugaaggaccug ACCGCGAAGUACACUGAGGGAGGCAACGCAAUCCUUGAGAACAUCAGCUUCUCCAUCUCG CCCGGUCAGAGGGUGGGCCUUCUUGGCCGGACCGGAUCGGGAAAGUCCACUCUUCUGUCG gccuuucuucgccucuugaauacugaaggggaaauccagaucgacggagugucguggga UAGCAUCACUCUGCAGCAGUGGCGGAAAGCGUUUGGAGUAAUCCCCCAAAAGGUCUUUA UCUUUAGCGGAACCUUCCGAAAGAAUCUCGAUCCUUAUGAACAGUGGUCAGAUCAAGAG AUUUGGAAAGUCGCGGACGAGGUUGGCCUUCGGAGUGUAAUCGAGCAGUUUCCGGGAAA ACUCGACUUUGUCCUUGUAGAUGGGGGAUGCGUCCUGUCGCAUGGGCACAAGCAGCUCAU GUGCCUGGCGCGAUCCGUCCUCUCUAAAGCGAAAAUUCUUCUCUUGGAUGAACCUUCGGC CCAUCUGGACCCGGUAACGUAUCAGAUCAUCAGAAGGACACUUAAGCAGGCGUUUGCCGA CUGCACGGUGAUUCUCUGUGAGCAUCGUAUCGAGGCCAUGCUCGAAUGCCAGCAAUUUCU UGUCAUCGAAGAGAAUAAGGUCCGCCAGUACGACUCCAUCCAGAAGCUGCUUAAUGAGA GAUCAUUGUUCCGGCAGGCGAUUUCACCAUCCGAUAGGGUGAAACUUUUUCCACACAGAA AUUCGUCGAAGUGCAAGUCCAAACCGCAGAUCGCGGCCUUGAAAGAAGAGACUGAAGAA GAAGUUCAAGACACGCGUCUUUAA (SEQ IDN0: 14)

SEQ ID NO: 15 [0222]

57739 Β1

AUGCAGCGGUCCCCGCUCGAAAAGGCCAGUGUCGUGUCCAAACUCUUCUUCUCAUGGACU CGGCCUAUCCUUAGAAAGGGGUAUCGGCAGAGGCUUGAGUUGUCUGACAUCUACCAGAU CCCCUCGGUAGAUUCGGCGGAUAACCUCUCGGAGAAGCUCGAACGGGAAUGGGACCGCGA ACUCGCGUCUAAGAAAAACCCGAAGCUCAUCAACGCACUGAGAAGGUGCUUCUUCUGGCG GUUCAUGUUCUACGGUAUCUUCUUGUAUCUCGGGGAGGUCACAAAAGCAGUCCAACCCCU GUUGUUGGGUCGCAUUAUCGCCUCGUACGACCCCGAUAACAAAGAAGAACGGAGCAUCGC GAUCUACCUCGGGAUCGGACUGUGUUUGCUUUUCAUCGUCAGAACACUUUUGUUGCAUCC AGCAAUCUUCGGCCUCCAUCACAUCGGUAUGCAGAUGCGAAUCGCUAUGUUUAGCUUGAU CUACAAAAAGACACUGAAACUCUCGUCGCGGGUGUUGGAUAAGAUUUCCAUCGGUCAGU UGGUGUCCCUGCUUAGUAAUAACCUCAACAAAUUCGAUGAGGGACUGGCGCUGGCACAU UUCGUGUGGAUUGCCCCGUUGCAAGUCGCCCUUUUGAUGGGCCUUAUUUGGGAGCUGUU GCAGGCAUCUGCCUUUUGUGGCCUGGGAUUUCUGAUUGUGUUGGCAUUGUUUCAGGCUG GGCUUGGGCGGAUGAUGAUGAAGUAUCGCGACCAGAGAGCGGGUAAAAUCUCGGAAAGA CUCGUCAUCACUUCGGAAAUGAUCGAAAACAUCCAGUCGGUCAAAGCCUAUUGCUGGGAA GAAGCUAUGGAGAAGAUGAUUGAAAACCUCCGCCAAACUGAGCUGAAACUGACCCGCAA GGCGGCGUAUGUCCGGUAUUUCAAUUCGUCAGCGUUCUUCUUUUCCGGGUUCUUCGUUG UCUUUCUCUCGGUUUUGCCUUAUGCCUUGAUUAAGGGGAUUAUCCUCCGCAAGAUUUUC ACCACGAUUUCGUUCUGCAUUGUAUUGCGCAUGGCAGUGACACGGCAAUUUCCGUGGGCC GUGCAGACAUGGUAUGACUCGCUUGGAGCGAUCAACAAAAUCCAAGACUUCUUGCAAAA GCAAGAGUACAAGACCCUGGAGUACAAUCUUACUACUACGGAGGUAGUAAUGGAGAAUG UGACGGCUUUUUGGGAAGAGGGUUUUGGAGAACUGUUUGAGAAAGCAAAGCAGAAUAAC AACAACCGCAAGACCUCAAAUGGGGACGAUUCCCUGUUUUUCUCGAACUUCUCCCUGCUC GGAACACCCGUGUUGAAGGACAUCAAUUUCAAGAUUGAGAGGGGACAGCUUCUCGCGGU AGCGGGAAGCACUGGUGCGGGAAAAACUAGCCUCUUGAUGGUGAUUAUGGGGGAGCUUG AGCCCAGCGAGGGGAAGAUUAAACACUCCGGGCGUAUCUCAUUCUGUAGCCAGUUUUCAU GGAUCAUGCCCGGAACCAUUAAAGAGAACAUCAUUUUCGGAGUAUCCUAUGAUGAGUAC CGAUACAGAUCGGUCAUUAAGGCGUGCCAGUUGGAAGAGGACAUUUCUAAGUUCGCCGA GAAGGAUAACAUCGUCUUGGGAGAAGGGGGUAUUACAUUGUCGGGAGGGCAGCGAGCGC GGAUCAGCCUCGCGAGAGCGGUAUACAAAGAUGCAGAUUUGUAUCUGCUUGAUUCACCG UUUGGAUACCUCGACGUAUUGACAGAAAAAGAAAUCUUCGAGUCGUGCGUGUGUAAACU UAUGGCUAAUAAGACGAGAAUCCUGGUGACAUCAAAAAUGGAACACCUUAAGAAGGCGG ACAAGAUCCUGAUCCUCCACGAAGGAUCGUCCUACUUUUACGGCACUUUCUCAGAGUUGC AAAACUUGCAGCCGGACUUCUCAAGCAAACUCAUGGGGUGUGACUCAUUCGACCAGUUCA GCGCGGAACGGCGGAACUCGAUCUUGACGGAAACGCUGCACCGAUUCUCGCUUGAGGGUG AUGCCCCGGUAUCGUGGACCGAGACAAAGAAGCAGUCGUUUAAGCAGACAGGAGAAUUU GGUGAGAAAAGAAAGAACAGUAUCUUGAAUCCUAUUAACUCAAUUCGCAAGUUCUCAAU CGUCCAGAAAACUCCACUGCAGAUGAAUGGAAUUGAAGAGGAUUCGGACGAACCCCUGG AGCGCAGGCUUAGCCUCGUGCCGGAUUCAGAGCAAGGGGAGGCCAUUCUUCCCCGGAUUU CGGUGAUUUCAACCGGACCUACACUUCAGGCGAGGCGAAGGCAAUCCGUGCUCAACCUCA UGACGCAUUCGGUAAACCAGGGGCAAAACAUUCACCGCAAAACGACGGCCUCAACGAGAA AAGUGUCACUUGCACCCCAGGCGAAUUUGACUGAACUCGACAUCUACAGCCGUAGGCUUU CGCAAGAAACCGGACUUGAGAUCAGCGAAGAAAUCAAUGAAGAAGAUUUGAAAGAGUGU UUCUUUGAUGACAUGGAAUCAAUCCCAGCGGUGACAACGUGGAACACAUACUUGCGUUA CAUCACGGUGCACAAGUCCUUGAUUUUCGUCCUCAUCUGGUGUCUCGUGAUCUUUCUCGC

57739 Yes

UGAGGUCGCAGCGUCACUUGUGGUCCUCUGGCUGCUUGGUAAUACGCCCUUGCAAGACAA AGGCAAUUCUACACACUCAAGAAACAAUUCCUAUGCCGUGAUUAUCACUUCUACAAGCUC GUAUUACGUGUUUUACAUCUACGUAGGAGUGGCCGACACUCUGCUCGCGAUGGGUUUCU UCCGAGGACUCCCACUCGUUCACACGCUUAUCACUGUCUCCAAGAUUCUCCACCAUAAGA UGCUUCAUAGCGUACUGCAGGCUCCCAUGUCCACCUUGAAUACGCUCAAGGCGGGAGGUA UUUUGAAUCGCUUCUCAAAAGAUAUUGCAAUUUUGGAUGACCUUCUGCCCCUGACGAUC UUCGACUUCAUCCAGUUGUUGCUGAUCGUGAUUGGGGCUAUUGCAGUAGUCGCUGUCCU CCAGCCUUACAUUUUUGUCGCGACCGUUCCGGUGAUCGUGGCGUUUAUCAUGCUGCGGGC CUAUUUCUUGCAGACGUCACAGCAGCUUAAGCAACUGGAGUCUGAAGGGAGGUCGCCUA UCUUUACGCAUCUUGUGACCAGUUUGAAGGGAUUGUGGACGUUGCGCGCCUUUGGCAGG CAGCCCUACUUUGAAACACUGUUCCACAAAGCGCUGAAUCUCCAUACGGCAAAUUGGUUU UUGUAUUUGAGUACCCUCCGAUGGUUUCAGAUGCGCAUUGAGAUGAUUUUUGUGAUCUU CUUUAUCGCGGUGACUUUUAUCUCCAUCUUGACCACGGGAGAGGGCGAGGGACGGGUCG GUAUUAUCCUGACACUCGCCAUGAACAUUAUGAGCACUUUGCAGUGGGCAGUGAACAGC UCGAUUGAUGUGGAUAGCCUGAUGAGGUCCGUUUCGAGGGUCUUUAAGUUCAUCGACAU GCCGACGGAGGGAAAGCCCACAAAAAGUACGAAACCCUAUAAGAAUGGGCAAUUGAGUA AGGUAAUGAUCAUCGAGAACAGUCACGUGAAGAAGGAUGACAUCUGGCCUAGCGGGGGU CAGAUGACCGUGAAGGACCUGACGGCAAAAUACACCGAGGGAGGGAACGCAAUCCUUGA AAACAUCUCGUUCAGCAUUAGCCCCGGUCAGCGUGUGGGGUUGCUCGGGAGGACCGGGUC AGGAAAAUCGACGUUGCUGUCGGCCUUCUUGAGACUUCUGAAUACAGAGGGUGAGAUCC AGAUCGACGGCGUUUCGUGGGAUAGCAUCACCUUGCAGCAGUGGCGGAAAGCGUUUGGA GUAAUCCCCCAAAAGGUCUUUAUCUUUAGCGGAACCUUCCGAAAGAAUCUCGAUCCUUAU GAACAGUGGUCAGAUCAAGAGAUUUGGAAAGUCGCGGACGAGGUUGGCCUUCGGAGUGU AAUCGAGCAGUUUCCGGGAAAACUCGACUUUGUCCUUGUAGAUGGGGGAUGCGUCCUGU CGCAUGGGCACAAGCAGCUCAUGUGCCUGGCGCGAUCCGUCCUCUCUAAAGCGAAAAUUC UUCUCUUGGAUGAACCUUCGGCCCAUCUGGACCCGGUAACGUAUCAGAUCAUCAGAAGGA CACUUAAGCAGGCGUUUGCCGACUGCACGGUGAUUCUCUGUGAGCAUCGUAUCGAGGCCA UGCUCGAAUGCCAGCAAUUUCUUGUCAUCGAAGAGAAUAAGGUCCGCCAGUACGACUCCA UCCAGAAGCUGCUUAAUGAGAGAUCAUUGUUCCGGCAGGCGAUUUCACCAUCCGAUAGG GUGAAACUUUUUCCACACAGAAAUUCGUCGAAGUGCAAGUCCAAACCGCAGAUCGCGGCC UUGAAAGAAGAGACUGAAGAAGAAGUUCAAGACACGCGUCUUCACCAUCACCAUCACCAU CACCAUCACCAUUAA (SEQ ID N0: 15)

SEQ ID NO: 16 [0223]

A UGGCCA CUGGA UCAA GAA CCUCACUGCUGCUCGCUUUUGGA CUGCUUUGCCUGCCCUGGU UGCA GAAGGA UCGGCUUUCCCGA CCA17CCC4 C17C17CCAUGCAGCGGUCCCCGCUCGAAAA GGCCAGUGUCGUGUCCAAACUCUUCUUCUCAUGGACUCGGCCUAUCCUUAGAAAGGGGUA UCGGCAGAGGCUUGAGUUGUCUGACAUCUACCAGAUCCCCUCGGUAGAUUCGGCGGAUAA CCUCUCGGAGAAGCUCGAACGGGAAUGGGACCGCGAACUCGCGUCUAAGAAAAACCCGAA GCUCAUCAACGCACUGAGAAGGUGCUUCUUCUGGCGGUUCAUGUUCUACGGUAUCUUCUU GUAUCUCGGGGAGGUCACAAAAGCAGUCCAACCCCUGUUGUUGGGUCGCAUUAUCGCCUC GUACGACCCCGAUAACAAAGAAGAACGGAGCAUCGCGAUCUACCUCGGGAUCGGACUGUG UUUGCUUUUCAUCGUCAGAACACUUUUGUUGCAUCCAGCAAUCUUCGGCCUCCAUCACAU CGGUAUGCAGAUGCGAAUUCAGACUA

57739 Β1

CGUCGCGGGUGUUGGAUAAGAUUUCCAUCGGUCAGUUGGUGUCCCUGCUUAGUAAUAAC CUCAACAAAUUCGAUGAGGGACUGGCGCUGGCACAUUUCGUGUGGAUUGCCCCGUUGCAA GUCGCCCUUUUGAUGGGCCUUAUUUGGGAGCUGUUGCAGGCAUCUGCCUUUUGUGGCCU GGGAUUUCUGAUUGUGUUGGCAUUGUUUCAGGCUGGGCUUGGGCGGAUGAUGAUGAAGU AUCGCGACCAGAGAGCGGGUAAAAUCUCGGAAAGACUCGUCAUCACUUCGGAAAUGAUC GAAAACAUCCAGUCGGUCAAAGCCUAUUGCUGGGAAGAAGCUAUGGAGAAGAUGAUUGA AAACCUCCGCCAAACUGAGCUGAAACUGACCCGCAAGGCGGCGUAUGUCCGGUAUUUCAA UUCGUCAGCGUUCUUCUUUUCCGGGUUCUUCGUUGUCUUUCUCUCGGUUUUGCCUUAUGC CUUGAUUAAGGGGAUUAUCCUCCGCAAGAUUUUCACCACGAUUUCGUUCUGCAUUGUAU UGCGCAUGGCAGUGACACGGCAAUUUCCGUGGGCCGUGCAGACAUGGUAUGACUCGCUUG GAGCGAUCAACAAAAUCCAAGACUUCUUGCAAAAGCAAGAGUACAAGACCCUGGAGUAC AAUCUUACUACUACGGAGGUAGUAAUGGAGAAUGUGACGGCUUUUUGGGAAGAGGGUUU UGGAGAACUGUUUGAGAAAGCAAAGCAGAAUAACAACAACCGCAAGACCUCAAAUGGGG ACGAUUCCCUGUUUUUCUCGAACUUCUCCCUGCUCGGAACACCCGUGUUGAAGGACAUCA AUUUCAAGAUUGAGAGGGGACAGCUUCUCGCGGUAGCGGGAAGCACUGGUGCGGGAAAA ACUAGCCUCUUGAUGGUGAUUAUGGGGGAGCUUGAGCCCAGCGAGGGGAAGAUUAAACA CUCCGGGCGUAUCUCAUUCUGUAGCCAGUUUUCAUGGAUCAUGCCCGGAACCAUUAAAGA GAACAUCAUUUUCGGAGUAUCCUAUGAUGAGUACCGAUACAGAUCGGUCAUUAAGGCGU GCCAGUUGGAAGAGGACAUUUCUAAGUUCGCCGAGAAGGAUAACAUCGUCUUGGGAGAA GGGGGUAUUACAUUGUCGGGAGGGCAGCGAGCGCGGAUCAGCCUCGCGAGAGCGGUAUA CAAAGAUGCAGAUUUGUAUCUGCUUGAUUCACCGUUUGGAUACCUCGACGUAUUGACAG AAAAAGAAAUCUUCGAGUCGUGCGUGUGUAAACUUAUGGCUAAUAAGACGAGAAUCCUG GUGACAUCAAAAAUGGAACACCUUAAGAAGGCGGACAAGAUCCUGAUCCUCCACGAAGG AUCGUCCUACUUUUACGGCACUUUCUCAGAGUUGCAAAACUUGCAGCCGGACUUCUCAAG CAAACUCAUGGGGUGUGACUCAUUCGACCAGUUCAGCGCGGAACGGCGGAACUCGAUCUU GACGGAAACGCUGCACCGAUUCUCGCUUGAGGGUGAUGCCCCGGUAUCGUGGACCGAGAC AAAGAAGCAGUCGUUUAAGCAGACAGGAGAAUUUGGUGAGAAAAGAAAGAACAGUAUCU UGAAUCCUAUUAACUCAAUUCGCAAGUUCUCAAUCGUCCAGAAAACUCCACUGCAGAUGA AUGGAAUUGAAGAGGAUUCGGACGAACCCCUGGAGCGCAGGCUUAGCCUCGUGCCGGAU UCAGAGCAAGGGGAGGCCAUUCUUCCCCGGAUUUCGGUGAUUUCAACCGGACCUACACUU CAGGCGAGGCGAAGGCAAUCCGUGCUCAACCUCAUGACGCAUUCGGUAAACCAGGGGCAA AACAUUCACCGCAAAACGACGGCCUCAACGAGAAAAGUGUCACUUGCACCCCAGGCGAAU UUGACUGAACUCGACAUCUACAGCCGUAGGCUUUCGCAAGAAACCGGACUUGAGAUCAGC GAAGAAAUCAAUGAAGAAGAUUUGAAAGAGUGUUUCUUUGAUGACAUGGAAUCAAUCCC AGCGGUGACAACGUGGAACACAUACUUGCGUUACAUCACGGUGCACAAGUCCUUGAUUU UCGUCCUCAUCUGGUGUCUCGUGAUCUUUCUCGCUGAGGUCGCAGCGUCACUUGUGGUCC UCUGGCUGCUUGGUAAUACGCCCUUGCAAGACAAAGGCAAUUCUACACACUCAAGAAACA AUUCCUAUGCCGUGAUUAUCACUUCUACAAGCUCGUAUUACGUGUUUUACAUCUACGUA GGAGUGGCCGACACUCUGCUCGCGAUGGGUUUCUUCCGAGGACUCCCACUCGUUCACACG CUUAUCACUGUCUCCAAGAUUCUCCACCAUAAGAUGCUUCAUAGCGUACUGCAGGCUCCC AUGUCCACCUUGAAUACGCUCAAGGCGGGAGGUAUUUUGAAUCGCUUCUCAAAAGAUAU UGCAAUUUUGGAUGACCUUCUGCCCCUGACGAUCUUCGACUUCAUCCAGUUGUUGCUGAU CGUGAUUGGGGCUAUUGCAGUAGUCGCUGUCCUCCAGCCUUACAUUUUUGUCGCGACCGU UCCGGUGAUCGUGGCGUUUAUCAUGCUGCGGGCCUAUUUCUUGCAGACGUCACAGCAGCU UAAGCAACUGGAGUCUGAAGGGAGGUCGCCUAUCUUUACGCAUCUUGUGACCAGUUUGA

57739 Yes

AGGGAUUGUGGACGUUGCGCGCCUUUGGCAGGCAGCCCUACUUUGAAACACUGUUCCACA AAGCGCUGAAUCUCCAUACGGCAAAUUGGUUUUUGUAUUUGAGUACCCUCCGAUGGUUU CAGAUGCGCAUUGAGAUGAUUUUUGUGAUCUUCUUUAUCGCGGUGACUUUUAUCUCCAU CUUGACCACGGGAGAGGGCGAGGGACGGGUCGGUAUUAUCCUGACACUCGCCAUGAACAU UAUGAGCACUUUGCAGUGGGCAGUGAACAGCUCGAUUGAUGUGGAUAGCCUGAUGAGGU CCGUUUCGAGGGUCUUUAAGUUCAUCGACAUGCCGACGGAGGGAAAGCCCACAAAAAGU ACGAAACCCUAUAAGAAUGGGCAAUUGAGUAAGGUAAUGAUCAUCGAGAACAGUCACGU GAAGAAGGAUGACAUCUGGCCUAGCGGGGGUCAGAUGACCGUGAAGGACCUGACGGCAA AAUACACCGAGGGAGGGAACGCAAUCCUUGAAAACAUCUCGUUCAGCAUUAGCCCCGGUC AGCGUGUGGGGUUGCUCGGGAGGACCGGGUCAGGAAAAUCGACGUUGCUGUCGGCCUUC UUGAGACUUCUGAAUACAGAGGGUGAGAUCCAGAUCGACGGCGUUUCGUGGGAUAGCAU CACCUUGCAGCAGUGGCGGAAAGCGUUUGGAGUAAUCCCCCAAAAGGUCUUUAUCUUUA GCGGAACCUUCCGAAAGAAUCUCGAUCCUUAUGAACAGUGGUCAGAUCAAGAGAUUUGG AAAGUCGCGGACGAGGUUGGCCUUCGGAGUGUAAUCGAGCAGUUUCCGGGAAAACUCGA CUUUGUCCUUGUAGAUGGGGGAUGCGUCCUGUCGCAUGGGCACAAGCAGCUCAUGUGCCU GGCGCGAUCCGUCCUCUCUAAAGCGAAAAUUCUUCUCUUGGAUGAACCUUCGGCCCAUCU GGACCCGGUAACGUAUCAGAUCAUCAGAAGGACACUUAAGCAGGCGUUUGCCGACUGCAC GGUGAUUCUCUGUGAGCAUCGUAUCGAGGCCAUGCUCGAAUGCCAGCAAUUUCUUGUCA UCGAAGAGAAUAAGGUCCGCCAGUACGACUCCAUCCAGAAGCUGCUUAAUGAGAGAUCA UUGUUCCGGCAGGCGAUUUCACCAUCCGAUAGGGUGAAACUUUUUCCACACAGAAAUUCG UCGAAGUGCAAGUCCAAACCGCAGAUCGCGGCCUUGAAAGAAGAGACUGAAGAAGAAGU UCAAGACACGCGUCUUUAA (SEQ IDN0: 16)

SEOIDNO: 17 [0224]

57739 Yes

UGACGGCUUUUUGGGAAGAGGGUUUUGGAGAACUGUUUGAGAAAGCAAAGCAGAAUAAC AACAACCGCAAGACCUCAAAUGGGGACGAUUCCCUGUUUUUCUCGAACUUCUCCCUGCUC GGAACACCCGUGUUGAAGGACAUCAAUUUCAAGAUUGAGAGGGGACAGCUUCUCGCGGU AGCGGGAAGCACUGGUGCGGGAAAAACUAGCCUCUUGAUGGUGAUUAUGGGGGAGCUUG AGCCCAGCGAGGGGAAGAUUAAACACUCCGGGCGUAUCUCAUUCUGUAGCCAGUUUUCAU GGAUCAUGCCCGGAACCAUUAAAGAGAACAUCAUUUUCGGAGUAUCCUAUGAUGAGUAC CGAUACAGAUCGGUCAUUAAGGCGUGCCAGUUGGAAGAGGACAUUUCUAAGUUCGCCGA GAAGGAUAACAUCGUCUUGGGAGAAGGGGGUAUUACAUUGUCGGGAGGGCAGCGAGCGC GGAUCAGCCUCGCGAGAGCGGUAUACAAAGAUGCAGAUUUGUAUCUGCUUGAUUCACCG UUUGGAUACCUCGACGUAUUGACAGAAAAAGAAAUCUUCGAGUCGUGCGUGUGUAAACU UAUGGCUAAUAAGACGAGAAUCCUGGUGACAUCAAAAAUGGAACACCUUAAGAAGGCGG ACAAGAUCCUGAUCCUCCACGAAGGAUCGUCCUACUUUUACGGCACUUUCUCAGAGUUGC AAAACUUGCAGCCGGACUUCUCAAGCAAACUCAUGGGGUGUGACUCAUUCGACCAGUUCA GCGCGGAACGGCGGAACUCGAUCUUGACGGAAACGCUGCACCGAUUCUCGCUUGAGGGUG AUGCCCCGGUAUCGUGGACCGAGACAAAGAAGCAGUCGUUUAAGCAGACAGGAGAAUUU GGUGAGAAAAGAAAGAACAGUAUCUUGAAUCCUAUUAACUCAAUUCGCAAGUUCUCAAU CGUCCAGAAAACUCCACUGCAGAUGAAUGGAAUUGAAGAGGAUUCGGACGAACCCCUGG AGCGCAGGCUUAGCCUCGUGCCGGAUUCAGAGCAAGGGGAGGCCAUUCUUCCCCGGAUUU CGGUGAUUUCAACCGGACCUACACUUCAGGCGAGGCGAAGGCAAUCCGUGCUCAACCUCA UGACGCAUUCGGUAAACCAGGGGCAAAACAUUCACCGCAAAACGACGGCCUCAACGAGAA AAGUGUCACUUGCACCCCAGGCGAAUUUGACUGAACUCGACAUCUACAGCCGUAGGCUUU CGCAAGAAACCGGACUUGAGAUCAGCGAAGAAAUCAAUGAAGAAGAUUUGAAAGAGUGU UUCUUUGAUGACAUGGAAUCAAUCCCAGCGGUGACAACGUGGAACACAUACUUGCGUUA CAUCACGGUGCACAAGUCCUUGAUUUUCGUCCUCAUCUGGUGUCUCGUGAUCUUUCUCGC UGAGGUCGCAGCGUCACUUGUGGUCCUCUGGCUGCUUGGUAAUACGCCCUUGCAAGACAA AGGCAAUUCUACACACUCAAGAAACAAUUCCUAUGCCGUGAUUAUCACUUCUACAAGCUC GUAUUACGUGUUUUACAUCUACGUAGGAGUGGCCGACACUCUGCUCGCGAUGGGUUUCU UCCGAGGACUCCCACUCGUUCACACGCUUAUCACUGUCUCCAAGAUUCUCCACCAUAAGA UGCUUCAUAGCGUACUGCAGGCUCCCAUGUCCACCUUGAAUACGCUCAAGGCGGGAGGUA UUUUGAAUCGCUUCUCAAAAGAUAUUGCAAUUUUGGAUGACCUUCUGCCCCUGACGAUC UUCGACUUCAUCCAGUUGUUGCUGAUCGUGAUUGGGGCUAUUGCAGUAGUCGCUGUCCU CCAGCCUUACAUUUUUGUCGCGACCGUUCCGGUGAUCGUGGCGUUUAUCAUGCUGCGGGC CUAUUUCUUGCAGACGUCACAGCAGCUUAAGCAACUGGAGUCUGAAGGGAGGUCGCCUA UCUUUACGCAUCUUGUGACCAGUUUGAAGGGAUUGUGGACGUUGCGCGCCUUUGGCAGG CAGCCCUACUUUGAAACACUGUUCCACAAAGCGCUGAAUCUCCAUACGGCAAAUUGGUUU UUGUAUUUGAGUACCCUCCGAUGGUUUCAGAUGCGCAUUGAGAUGAUUUUUGUGAUCUU CUUUAUCGCGGUGACUUUUAUCUCCAUCUUGACCACGGGAGAGGGCGAGGGACGGGUCG GUAUUAUCCUGACACUCGCCAUGAACAUUAUGAGCACUUUGCAGUGGGCAGUGAACAGC UCGAUUGAUGUGGAUAGCCUGAUGAGGUCCGUUUCGAGGGUCUUUAAGUUCAUCGACAU GCCGACGGAGGGAAAGCCCACAAAAAGUACGAAACCCUAUAAGAAUGGGCAAUUGAGUA AGGUAAUGAUCAUCGAGAACAGUCACGUGAAGAAGGAUGACAUCUGGCCUAGCGGGGGU CAGAUGACCGUGAAGGACCUGACGGCAAAAUACACCGAGGGAGGGAACGCAAUCCUUGA AAACAUCUCGUUCAGCAUUAGCCCCGGUCAGCGUGUGGGGUUGCUCGGGAGGACCGGGUC AGGAAAAUCGACGUUGCUGUCGGCCUUCUUGAGACUUCUGAAUACAGAGGGUGAGAUCC AGAUCGACGGCGUUUCGUGGGAUAGCAUCACCUUGCAGCAGUGGCGGAAAGCGUUUGGA

GUAAUCCCCCAAAAGGUCUUUAUCUUUAGCGGAACCUUCCGAAAGAAUCUCGAUCCUUAU GAACAGUGGUCAGAUCAAGAGAUUUGGAAAGUCGCGGACGAGGUUGGCCUUCGGAGUGU AAUCGAGCAGUUUCCGGGAAAACUCGACUUUGUCCUUGUAGAUGGGGGAUGCGUCCUGU CGCAUGGGCACAAGCAGCUCAUGUGCCUGGCGCGAUCCGUCCUCUCUAAAGCGAAAAUUC UUCUCUUGGAUGAACCUUCGGCCCAUCUGGACCCGGUAACGUAUCAGAUCAUCAGAAGGA CACUUAAGCAGGCGUUUGCCGACUGCACGGUGAUUCUCUGUGAGCAUCGUAUCGAGGCCA UGCUCGAAUGCCAGCAAUUUCUUGUCAUCGAAGAGAAUAAGGUCCGCCAGUACGACUCCA UCCAGAAGCUGCUUAAUGAGAGAUCAUUGUUCCGGCAGGCGAUUUCACCAUCCGAUAGG GUGAAACUUUUUCCACACAGAAAUUCGUCGAAGUGCAAGUCCAAACCGCAGAUCGCGGCC UUGAAAGAAGAGACUGAAGAAGAAGUUCAAGACACGCGUCUUUAA (SEQ IDN0: 17)

57739 Β1

SEQ ID NO: 18 [0225]

AUGGCCACUGGAUCAAGAACCUCACUGCUGCUCGCUUUUGGACUGCUUUGCCUGC

CCUGGUUGCAAGAAGGAUCGGCUUUCCCGACCAUCCCACUCUCC (SEQ IDNO: 18)

SEQ ID NO: 19 [0226]

AUGGCAACUGGAUCAAGAACCUCCCUCCUGCUCGCAUUCGGCCUGCUCUGUCUCC

CAUGGCUCCAAGAAGGAAGCGCGUUCCCCACUAUCCCCCUCUCG (SEQ ID NO: 19)

SEQ ID NO: 20 [0227]

CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCC

ACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUCAAGCU The use of the singular when used in conjunction with the term encompasses 'contains' in the claims and / or specification may mean one but may also be consistent with the meaning of one or more, at least one, and one or more. The use of the terms either in the claims means and / or unless it is explicitly stated that it refers to alternatives only or mutually exclusive alternatives, although the disclosure supports the definition that it refers to only alternatives ii / or.

Unless otherwise stated, the expression at least preceding a series of elements should be understood to refer to each individual element in the series.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those used in the art by ordinary scientists in the field to which the invention relates.

Contents10

83 members in 28 offices

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

Priority claims12

Priority claims
Document	Office	Kind	Date
201361783663	United States of America	P	2013-03-14
201361783663	United States of America	P	2013-03-14
14718284	European Patent Office (EPO)	A	2014-03-14
14718284	European Patent Office (EPO)	A	2014-03-14
2014028849	United States of America	W	2014-03-14
2014028849	United States of America	W	2014-03-14
147182844	–	–	–
201361783663P	–	–	–
EP20140718284	–	–	–
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
IL240982A	Israel	A	2018-11-29
IL240982B	Israel	B	2018-11-29
IL262984D0	Israel	D0	2018-12-31
RS57739B1This record	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
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Cftr mrna compositions and related methods and uses

Abstract

Term

DETAILED DESCRIPTION OF THE INVENTION

SEQ ID NO: 3

CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCC

AGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC (SEQID N0: 5)

SEQ ID NO: 8 UUUGAAUU (SEQ ID NO: 8)

AUGGCCACUGGAUCAAGAACCUCACUGCUGCUCGCUUUUGGACUGCUUUGCCUGC

CCUGGUUGCAAGAAGGAUCGGCUUUCCCGACCAUCCCACUCUCC (SEQ IDNO: 18)

AUGGCAACUGGAUCAAGAACCUCCCUCCUGCUCGCAUUCGGCCUGCUCUGUCUCC

CAUGGCUCCAAGAAGGAAGCGCGUUCCCCACUAUCCCCCUCUCG (SEQ ID NO: 19)

CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCC

Priority claims12

Members83

Numbers

Titles2

Classification