AU2015221515B2

AU2015221515B2 - Organic compositions to treat Beta-ENaC-related diseases

Info

Publication number: AU2015221515B2
Application number: AU2015221515A
Authority: AU
Inventors: Antonin De Fougerolles; John L. Diener; Emma Hickman; Gregory Hinkle; Stuart Milstein; Anne-Marie Pulichino; Andrew Sprague
Original assignee: Arrowhead Pharmaceuticals Inc
Current assignee: Arrowhead Pharmaceuticals Inc
Priority date: 2010-04-23
Filing date: 2015-09-03
Publication date: 2016-12-22
Anticipated expiration: 2031-04-20
Also published as: AU2017201838A1; AU2021240326A1; AU2019203596A1; AU2015221515A1

Abstract

Abstract The present disclosure relates to RNAi agents useful in methods of treating Beta ENaC-related diseases such as cystic fibrosis, pseudohypoaldosteronism type I (PHA1), Liddle's syndrome, hypertension, alkalosis, hypokalemia, and obesity-associated hypertension, using a therapeutically effective amount of a RNAi agent to Beta-ENaC.

Description

1 2015221515 03 Sep 2015

ORGANIC COMPOSITIONS TO TREAT BETA-ENaC-RELATED DISEASES

[001] This application claims benefit under 35 U.S.C. § 119(a)-(d) of U.S. Provisional 5 Application 61/327,379, filed April 23, 2010, and U.S. Provisional Application 61/333,398, filed May 11, 2010, the contents of which are incorporated herein by reference in their entirety.

[001 A] This is a divisional of Australian patent application No, 2011244335, the entire content of which is incorporated herein by reference. 10

BACKGROUND OF THE INVENTION

[002] The mucosa! surface between the environment and the body has many protective mechanisms. One form of defense is cleansing the surface with liquid. The quantity of liquid 15 reflects the balance between epithelial liquid secretion (which often reflects anion secretion coupled with water and a cation counter-ion) and liquid absorption (which often reflects Na+ absorption, coupled with water and counter anion). Many diseases of mucosal surfaces are caused by too little liquid, as caused by an imbalance between secretion (too little) and absorption (too much). One method to balance the liquid layer is to decrease Na+ channel-20 mediated liquid absorption.

[003] Nonvoltage-gated, amiloride-sensitive sodium channels control fluid and electrolyte transport across epithelia in many organs. The apical membranes of many tight epithelia contain sodium channels that are primarily characterized by their high affinity to the diuretic blocker amiloride. These channels mediate the first step of active sodium 25 reabsorption essential for the maintenance of body salt and water homeostasis. In vertebrates, the channels control reabsorption of sodium in the kidney, colon, lung and sweat glands; they also play a role in taste perception.

[004] The rate-limiting step of Na+ and liquid absorption is mediated by the epithelial sodium (Na+) channel (ENaC). These sodium channels are heteromeric complexes 30 consisting of 3 subunits: Alpha-ENaC, Beta-ENaG, and Gamma-ENaC.

[005] Beta-ENaC (also known as SCNN1B) encodes the beta subunit of this sodium channel, and mutations in and/or altered expression of this gene have been associated with several diseases (and/or associated with treatments of diseases), including cystic fibrosis, pseudohypoaldosteronism type 1 (PHA1), Liddle’s syndrome, hypertension, alkalosis, 35 hypokalemia, and obesity-associated hypertension.

[006] There exists the need for treatments related to Beta-ENaC-related diseases. 2 2015221515 03 Sep 2015

BRIEF SUMMARY OF THE INVENTION

[007] The present disclosure encompasses RNAi agents to Beta-ENaC, which are useful in the treatment of Beta-ENaC-reiated diseases, such as cystic fibrosis, 5 pseudohypoaldosteronism type 1 (PHA1), Liddle’s syndrome, hypertension, alkalosis, hypokalemia, and obesity-associated hypertension. The present disclosure also encompasses a method of treating a human subject having a pathological state mediated at least in part by aipha-ENaC expression, the method comprising the step of administering to the subject a therapeutically effective amount of a RNAi agent Beta-ENaC. 10 [008] The present disclosure provides specific RNAi agents and methods that are useful in reducing Beta-ENaC levels in a subject, e.g., a mammal, such as a human. The present disclosure specifically provides double-stranded RNAi agents comprising at least 15 or more contiguous nucleotides of Beta-ENaC. in particular, the present disclosure provides agents comprising sequences of 15 or more contiguous nucleotides differing by 0, 1, 2 or 3 15 from those of the RNAi agents provided, e.g., in Table 1. The RNAi agents particularly can in one embodiment comprise less than 30 nucleotides per strand, e.g., such as 18-23 nucleotides, and/or 19-21 nucleotides, and/or such as those provided, e.g., in Table 1.

[009] The double-stranded RNAi agents can have blunt ends or overhangs of 1, 2, 3 or 4 nucleotides (i.e., 1-4 nt) from one or both 3' and/or 5’ ends. The double-stranded RNAi 20 agents can also optionally comprise one or two 3’ caps and/or one or more modified nucleotides. Modified variants of sequences as provided herein include those that are otherwise identical but contain substitutions of a naturally occurring nucleotide for a corresponding modified nucleotide.

[0010] Further, the RNAi agent can either contain only naturally-occurring ribonucleotide 25 subunits, or one or more modifications to the sugar, phosphate or base of one or more of the replacement nucleotide subunits, whether they comprise ribonucleotide subunits or deoxyribonucleotide subunits. In one embodiment, modified variants of the disclosed RNAi agents include RNAi agents with the same sequence, but with one or more modifications to one or more of the sugar, phosphate or base of one or more of the nucleotide subunits. In 30 one embodiment, the modifications improve efficacy, stability and/or reduce immunogenicity of the RNAi agent. One aspect of the present disclosure relates to a double-stranded oligonucleotide comprising at least one non-natural nucleobase. In certain embodiments, the non-natural nucleobase is difluorotoiyi, nitroindolyl, nitropyrrolyl, or nitroimidazolyl. In a particular embodiment, the non-natural nucleobase is difluorotoiyi. In certain embodiments, 35 only one of the two oligonucleotide strands contains a non-natural nucleobase. In certain embodiments, both of the oligonucleotide strands contain a non-natural nucleobase.

[0011] The RNAi agent(s) can optionally be attached to a ligand selected to improve one or more characteristic, such as, e.g., stability, distribution and/or cellular uptake of the agent, 3 2015221515 03 Sep 2015 e.g., choiestero! or a derivative thereof. The RNAi agent(s) can be isolated or be part of a pharmaceutical composition used for the methods described herein. Particularly, the pharmaceutical composition can be formulated for delivery to the lungs or nasal passage or formulated for parental administration. The pharmaceutical compositions can optionally 5 comprise two or more RNAi agents, each one directed to the same or a different segment of the Beta-ENaC mRNA. Optionally, the pharmaceutical compositions can further comprise or be used in conjunction with any known treatment for any Beta-ENaC-related disease.

[0012] The present disclosure further provides methods for reducing the level of Beta-ENaC mRNA in a cell, particularly in the case of a disease characterized by over-expression 10 or hyper-activity of ENaC. The present disclosure also encompasses a method of treating a human subject having a pathological state mediated at least in part by Beta-ENaC expression, the method comprising the step of administering to the subject a therapeutically effective amount of a RNAi agent Beta-ENaC. Such methods comprise the step of administering one of the RNAi agents of the present disclosure to a subject, as further 15 described below. The present methods utilize the cellular mechanisms involved in RNA interference to selectively degrade the target RNA in a cell and are comprised of the step of contacting a cell with one of the RNAi agents of the present disclosure. Such methods can be performed directly on a cell or can be performed on a mammalian subject by administering to a subject one of the RNAi agents/pharmaceuticai compositions of the 20 present disclosure. Reduction of target Beta-ENaC RNA in a cell results in a reduction in the amount of encoded Beta-ENaC protein produced. In an organism, this can result in reduction of epithelial potential difference, balanced fluid absorption and increased mucociliary clearance.

[0013] The methods and compositions of the present disclosure, e.g., the methods and 25 Beta-ENaC RNAi agent compositions, can be used with any dosage and/or formulation described herein, as well as with any route of administration described herein.

[0014] The details of one or more embodiments of the present disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the present disclosure will be apparent from this description, the drawings, 30 and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

[0015] Figures 1A-1B depict the ability of RNAi agents AD20807, AD20826, AD20832, AS20834, AD20848, and AD20861 to knock-down Beta-ENaC activity in vivo, 35 [0016] Figures 2A -- 2C depict the in vitro effect of Beta-ENaC RNAi Agent AD20832 on ENaC channel functional activity in human bronchial epithelial cells.

DETAILED DESCRIPTION OF THE INVENTION 4 2015221515 03 Sep 2015 [0017] The present disclosure encompasses RNAi agents to Beta-ENaC, which are useful in treatment of Beta-ENaC-related diseases (e.g., diseases associated with mutations in and/or altered expression, level and/or activity of Beta-ENaC, and/or diseases treatable by modulating the expression, level and/or activity of Beta-ENaC), such as cystic fibrosis, 5 pseudohypoaldosteronism type 1 (PHA1), Liddle’s syndrome, hypertension, alkalosis, hypokalemia, and obesity-associated hypertension. The present disclosure also provides methods of treating a human subject having a pathological state mediated at least in part by Beta-ENaC expression, the method comprising the step of administering to the subject a therapeutically effective amount of a RNAi agent Beta-ENaC. 10 [0018] Various embodiments of the present disclosure include: [0019] A RNAi agent comprising an antisense strand described herein, [0020] In one embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense 15 strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of a RNAi agent specific to Beta-ENaC (or any set of overlapping RNAi agents specific to Beta-ENaC) provided, e.g., in Table 1. in another embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense and an anti-sense strand, wherein the anti-sense strand comprises at least 15 contiguous nucleotides 20 differing by 0, 1, 2, or 3 nucleotides from the anti-sense strand of a RNAi agent from any sequence provided herein. In another embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a first strand and a second strand, wherein the first strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the sequence of the first strand, and the second strand comprises at least 25 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the sequence of the second strand of any RNAi agent provided herein.

[0021] Particular duplexes include the following, wherein each duplex comprises a set of SEQ ID NOs, wherein the first SEQ ID NO corresponds to a first strand (e.g., a sense strand), and the second SEQ ID NO corresponds to a second strand (e.g., an anti-sense 30 strand): AD-20805 (SEQ ID NOs. 111 and 112); AD-20806 (SEQ ID NOs. 113 and 114); AD-20807 (SEQ ID NOs. 115 and 116); AD-20808 (SEQ ID NOs. 117 and 118); AD-20809 (SEQ ID NOs. 119 and 120); AD-20810 (SEQ ID NOs. 121 and 122); AD-20811 (SEQ ID NOs. 123 and 124); AD-20812 (SEQ ID NOs. 125 and 126); AD-20813 (SEQ ID NOs. 127 and 128); AD-20814 (SEQ ID NOs. 129 and 130); AD-20815 (SEQ ID NOs. 131 and 132); AD-20816 35 (SEQ ID NOs. 133 and 134); AD-20817 (SEQ ID NOs. 135 and 136); AD-20818 (SEQ ID NOs. 137 and 138); AD-20819 (SEQ ID NOs. 139 and 140); AD-20820 (SEQ ID NOs. 141 and 142); AD-20821 (SEQ ID NOs. 143 and 144); AD-20822 (SEQ ID NOs. 145 and 146); AD-20823 (SEQ ID NOs. 147 and 148); AD-20824 (SEQ ID NOs. 149 and 150); AD-20825 5 2015221515 03 Sep 2015 (SEQ ID NOs. 151 and 152); AD-20826 (SEQ iD NOs. 153 and 154); AD-20827 (SEQ ID NOs. 155 and 156); AD-20828 (SEQ ID NOs. 157 and 158); AD-20829 (SEQ ID NOs. 159 and 160); AD-20830 (SEQ ID NOs. 161 and 162); AD-20831 (SEQ ID NOs. 163 and 164); AD-20832 (SEQ ID NOs. 165 and 166); AD-20833 (SEQ ID NOs. 167 and 168); AD-20834 5 (SEQ ID NOs. 169 and 170); AD-20835 (SEQ ID NOs. 171 and 172); AD-20836 (SEQ ID NOs. 173 and 174); AD-20837 (SEQ ID NOs. 175 and 176); AD-20838 (SEQ ID NOs. 177 and 178); AD-20839 (SEQ ID NOs. 179 and 180); AD-20840 (SEQ ID NOs. 181 and 182); AD-20841 (SEQ ID NOs. 183 and 184); AD-20842 (SEQ ID NOs. 185 and 186); AD-20843 (SEQ ID NOs. 187 and 188); AD-20844 (SEQ ID NOs. 189 and 190); AD-20845 (SEQ ID 10 NOs. 191 and 192); AD-20846 (SEQ ID NOs. 193 and 194); AD-20847 (SEQ ID NOs. 195 and 196); AD-20848 (SEQ ID NOs. 197 and 198); AD-20849 (SEQ ID NOs. 199 and 200); AD-20850 (SEQ ID NOs. 201 and 202); AD-20851 (SEQ ID NOs. 203 and 204); AD-20852 (SEQ ID NOs. 205 and 206); AD-20861 (SEQ ID NOs. 207 and 208); AD-20862 (SEQ ID NOs. 209 and 210); AD-20863 (SEQ ID NOs. 211 and 212); AD-20864 (SEQ ID NOs. 213 15 and 214); AD-20865 (SEQ ID NOs. 215 and 216); AD-20866 (SEQ ID NOs. 217 and 218); and AD-20867 (SEQ ID NOs. 219 and 220), and modified variants thereof.

[0022] One embodiment provides modified variants of particular duplexes, wherein each duplex comprises a set of SEQ ID NOs, wherein the first SEQ ID NO corresponds to a first strand (e.g., a sense strand), and the second SEQ ID NO corresponds to a second strand 20 (e.g., an anti-sense strand) that are selected from the group consisting of: AD-20805 (SEQ ID NOs. 1 and 2); AD-20806 (SEQ ID NOs. 3 and 4); AD-20807 (SEQ ID NOs. 5 and 6); AD-20808 (SEQ ID NOs. 7 and 8); AD-20809 (SEQ ID NOs. 9 and 10); AD-20810 (SEQ ID NOs. 11 and 12); AD-20811 (SEQ ID NQs, 13 and 14); AD-20812 (SEQ ID NOs. 15 and 16); AD-20813 (SEQ ID NOs. 17 and 18); AD-20814 (SEQ ID NOs. 19 and 20); AD-20815 (SEQ ID 25 NOs. 21 and 22); AD-20816 (SEQ ID NOs. 23 and 24); AD-20817 (SEQ ID NOs. 25 and 26); AD-20818 (SEQ ID NOs. 27 and 28); AD-20819 (SEQ ID NOs. 29 and 30); AD-20820 (SEQ ID NOs. 31 and 32); AD-20821 (SEQ ID NOs. 33 and 34); AD-20822 (SEQ ID NOs. 35 and 36); AD-20823 (SEQ ID NOs. 37 and 38); AD-20824 (SEQ ID NOs. 39 and 40); AD-20825 (SEQ ID NOs. 41 and 42); AD-20826 (SEQ ID NOs. 43 and 44); AD-20827 (SEQ ID NOs. 45 30 and 46); AD-20828 (SEQ ID NOs. 47 and 48); AD-20829 (SEQ ID NOs. 49 and 50); AD-20830 (SEQ ID NOs. 51 and 52); AD-20831 (SEQ ID NOs. 53 and 54); AD-20832 (SEQ ID NOs. 55 and 56); AD-20833 (SEQ ID NOs. 57 and 58); AD-20834 (SEQ ID NQs. 59 and 60); AD-20835 (SEQ ID NOs. 61 and 62); AD-20836 (SEQ ID NOs. 63 and 64); AD-20837 (SEQ ID NOs. 65 and 66); AD-20838 (SEQ ID NOs. 67 and 68); AD-20839 (SEQ ID NOs. 69 and 35 70); AD-20840 (SEQ ID NOs. 71 and 72); AD-20841 (SEQ ID NOs. 73 and 74); AD-20842 (SEQ ID NOs. 75 and 76); AD-20843 (SEQ ID NOs. 77 and 78); AD-20844 (SEQ ID NOs. 79 and 80); AD-20845 (SEQ ID NOs. 81 and 82); AD-20846 (SEQ ID NOs. 83 and 84); AD- 6 2015221515 03 Sep 2015 20847 (SEQ ID NOs. 85 and 86); AD-20848 (SEQ ID NOs. 87 and 88); AD-20849 (SEQ ID NOs. 89 and 90); AD-20850 (SEQ ID NOs. 91 and 92); AD-20851 (SEQ ID NOs. 93 and 94); AD-20852 (SEQ ID NOs, 95 and 96); AD-20861 (SEQ ID NOs. 97 and 98); AD-20862 (SEQ ID NOs. 99 and 100); AD-20863 (SEQ ID NOs. 101 and 102); AD-20864 (SEQ ID NOs. 103 5 and 104); AD-20865 (SEQ ID NOs. 105 and 106); AD-20866 (SEQ ID NOs. 107 and 108); and AD-20867 (SEQ ID NOs. 109 and 110).

[0023] Particular compositions [0024] In one embodiment, the present disclosure relates to a composition comprising a 10 RNAi agent comprising an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of a RNAi agent to Beta-ENaC selected from any sequence (or overlapping set of sequences) provided in a table here (e.g., Table 1). In one embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense strand and an antisense strand, 15 wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of a RNAi agent to Beta-ENaC selected from any sequence (or overlapping set of sequences) provided in a table here (e.g., Table 1). In another embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense and an anti-sense strand, wherein the anti-sense strand comprises 20 at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the anti-sense strand of a RNAi agent from any sequence provided herein. In another embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a first strand and a second strand, wherein the first strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the sequence of the first strand, and the second 25 strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the sequence of the second strand of any RNAi agent provided herein. Particular duplexes include those specific duplexes provided above and as listed in any one or more of Table 1. Additional modified sequences (e.g., sequences comprising one or more modified base) of each of the compositions above are also contemplated as part of the present disclosure. 30 [0025] Table A1, below, provides the SEQ ID NOs for the unmodified and an example modified sequence of the sense and an anti-sense strands of various RNAi agents to Beta-ENaC. The base composition of each is specific sequence represented by the SEQ ID NOs is provided in more detail in Table 1, and portions thereof are provided in Table 2.

35 Table A1. SEQ ID NOs for a first and a second strand (e.g., sense (“SS”) and anti-sense (“AS”) strand) for RNAi agents to Beta-ENaC

RNAi agent --duplex name Modified sequence SEQ ID NO Unmodified sequence SEQ ID NO 7 2015221515 03 Sep 2015 AD-20805 Sense 1 111 Anti-Sense 2 112 AD-20808 Sense 3 113 Anti-Sense 4 114 AD-20807 Sense 5 115 Anti-Sense 8 116 AD-20808 Sense i 117 Anti-Sense 8 118 AD-20809 Sense 9 119 Anti-Sense 10 120 AD-20810 Sense 11 121 Anti-Sense 12 122 AD-20811 Sense 13 123 Anti-Sense 14 124 AD-20812 Sense 15 125 Anti-Sense 18 126 AD-20813 Sense 17 127 Anti-Sense 18 128 AD-20814 Sense 19 129 Anti-Sense 20 130 AD-20815 Sense 21 131 Anti-Sense 22 132 AD-20816 Sense 23 133 Anti-Sense 24 134 AD-20817 Sense 25 135 Anti-Sense 26 136 AD-20818 Sense 27 137 Anti-Sense 28 138 AD-20819 Sense 29 139 Anti-Sense 30 140 AD-20820 Sense 31 141 Anti-Sense 32 142 AD-20821 Sense 33 143 Anti-Sense 34 144 AD-20822 Sense 35 145 Anti-Sense 36 146 AD-20823 Sense 37 147 Anti-Sense 38 148 AD-20824 Sense 39 149 Anti-Sense 40 150 AD-20825 Sense 41 151 Anti-Sense 42 152 AD-20826 Sense 43 153 Anti-Sense 44 154 AD-20827 Sense 45 155 Anti-Sense 46 156 2015221515 03 Sep 2015 AD-20828 Sense 47 157 Anti-Sense 48 158 AD-20829 Sense 49 159 Anti-Sense 50 160 AD-20830 Sense 51 161 Anti-Sense 52 162 AD-20831 Sense 53 163 Anti-Sense 54 164 AD-20832 Sense 55 165 Anti-Sense 56 166 AD-20833 Sense 57 167 Anti-Sense 58 168 AD-20834 Sense 59 169 Anti-Sense 60 170 AD-20835 Sense 61 171 Anti-Sense 62 172 AD-20838 Sense 63 173 Anti-Sense 84 174 AD-20837 Sense 85 175 Anti-Sense 88 176 AD-20838 Sense 87 177 Anti-Sense 68 178 AD-20839 Sense 69 179 Anti-Sense 70 180 AD-20840 Sense 71 181 Anti-Sense 72 182 AD-20841 Sense 73 183 Anti-Sense 74 184 AD-20842 Sense 75 185 Anti-Sense 76 186 AD-20843 Sense 77 187 Anti-Sense 78 188 AD-20844 Sense 79 189 Anti-Sense 80 190 AD-20845 Sense 81 191 Anti-Sense 82 192 AD-20846 Sense 83 193 Anti-Sense 84 194 AD-20847 Sense 85 195 Anti-Sense 86 196 AD-20848 Sense 87 197 Anti-Sense 88 198 AD-20849 Sense 89 199 Anti-Sense 90 200 AD-20850 Sense 91 201 Anti-Sense 92 202 2015221515 03 Sep 2015 9 AD-20851 Sense 93 203 Anti-Sense 94 204 AD-20852 Sense 95 205 Anti-Sense 96 206 AD-20861 Sense 97 207 Anti-Sense 98 208 AD-20862 Sense 99 209 Anti-Sense 100 210 AD-20863 Sense 101 211 Anti-Sense 102 212 AD-20864 Sense 103 213 Anti-Sense 104 214 AD-20865 Sense 105 215 Anti-Sense 106 216 AD-20866 Sense 107 217 Anti-Sense 108 218 AD-20867 Sense 109 219 Anti-Sense 110 220 [0026] For example, in Table A1, an exemplary modified sequence of RNAi agent AD-20805 is represented by SEQ ID NO: 1 (the sense strand) and SEQ ID NO: 2 (the anti-sense strand). The unmodified sequence of AD-20805 is represented by SEQ ID NO: 111 (the 5 sense strand) and SEQ ID NO: 112 (the anti-sense strand). Thus, Table A1 presents the SEQ ID NO identifiers of a first and second strand of the unmodified sequence and at least one exemplary modified sequence for each of the various RNAi agents to Beta-ENaC.

[0027] An RNAi agent comprising an anti-sense strand described herein 10 [0028] In one particular specific embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising an anti-sense strand, wherein the anti-sense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the anti-sense strand of a RNAi agent to Beta-ENaC selected from those anti-sense strands in the specific duplexes provided herein and as listed, e.g., in Table 1. 15 [0029] Various specific embodiments of this embodiment are described below.

[0030] In one embodiment, the composition further comprises a second RNAi agent to Beta-ENaC. in various embodiments, the second RNAi agent is physically separate from the first, or the two are physically connected (e.g., covalently linked or otherwise conjugated).

[0031] In one embodiment, the antisense strand is about 30 or fewer nt in length. 20 [0032] In one embodiment, the sense strand and the antisense strand form a duplex region of about 15 to about 30 nucleotide pairs in length.

[0033] In one embodiment, the antisense strand is about 15 to about 36 nt in length, including about 18 to about 30 nt in length, and further including about 19 to about 23 nt in 10 2015221515 03 Sep 2015 length. In one embodiment, the antisense strand has at least the length selected from about 15 nt, about 16 nt, about 17 nt, about 18 nt, about 19 nt, about 20 nt, about 21 nt, about 22 nt, about 23 nt, about 24 nt, about 25 nt, about 26 nt, about 27 nt, about 28 nt, about 29 nt and about 30 nt. 5 [0034] In one embodiment, the RNAi agent comprises a modification that causes the RNAi agent to have increased stability in a biological sample or environment, e.g., blood serum or intestinal lavage fluid.

[0035] In one embodiment, the RNAi agent comprises at least one sugar backbone modification (e.g., phosphorothioate linkage) and/or at least one 2’-modified nucleotide, in 10 one embodiment, all the pyrimidines are 2’ O-methyl-modified nucleotides.

[0036] In one embodiment, the RNAi agent comprises: at least one 5’-uridine-adenine-3’ (5’-ua-3’) dinucleotide, wherein the uridine is a 2’-modified nucleotide; and/or at least one 5’-uridine-guanine-3’ (5’-ug-3’) dinucleotide, wherein the 5’-uridine is a 2’-modified nucleotide; and/or at least one 5’-cytidine-adenine-3’ (5’-ca~3’) dinucleotide, wherein the 5’-cytidine is a 15 2’-modified nucleotide; and/or at least one 5’~uridine-uridine-3! (5!-uu~3’) dinucieotide, wherein the 5’-uridine is a 2’-modified nucleotide.

[0037] In one embodiment, the RNAi agent comprises a 2'-modification selected from the group consisting of: 2'-deoxy, 2'~deoxy-2’-fiuoro, 2’-0-methyl, 2'-Omethoxyethy! (2'-0-MOE), 2'-0-aminopropyl (2'~0-AP), 2'~0-dimethyiaminoetbyi (2'~G-DMAOE), 2!-0- 20 dimethylaminopropyl (2'-0-DMAP), 2'-0-dimethylaminoethyloxyethyl (2'-0-DMAE0E), and 2'-O-N-methylacetamido (2-O-NMA). In one embodiment all the pyrimidines are 2’ O-methyl-modified nucleotides.

[0038] In one embodiment, the RNAi agent comprises a blunt end, [0039] In one embodiment, the RNAi agent comprises an overhang having 1 to 4 25 unpaired nucleotides.

[0040] In one embodiment, the RNAi agent comprises an overhang at the 3’-end of the antisense strand of the RNAi agent, [0041] In one embodiment, the RNAi agent is ligated to one or more diagnostic compound, reporter group, cross-linking agent, nuclease-resistance conferring moiety, 30 natural or unusual nucleobase, lipophilic molecule, cholesterol, lipid, lectin, steroid, uvaol, hecigenin, diosgenin, terpene, triterpene, sarsasapogenin, Friedelin, epifriedeianol-derivatized iithocholic acid, vitamin, carbohydrate, dextran, pullulan, chitin, chitosan, synthetic carbohydrate, oligo lactate 15-mer, natural polymer, low- or medium-molecular weight polymer, inulin, cyclodextrin, hyaluronic acid, protein, protein-binding agent, integrin-35 targeting molecule, polycationic, peptide, polyamine, peptide mimic, and/or transferrin.

[0042] In one embodiment, the RNAi agent is capable of inhibiting expression of the Beta-ENaC gene by at least about 60% at a concentration of 10 nM in H441 cells in vitro. 11 2015221515 03 Sep 2015 [0043] in one embodiment, the RNAi agent is capable of inhibiting expression of the Beta-ENaC gene by at least about 70% at a concentration of 10 nM in H441 cells in vitro.

[0044] in one embodiment, the RNAi agent is capable of inhibiting expression of the Beta-ENaC gene by at least about 80% at a concentration of 10 nM in H441 cells in vitro. 5 [0045] In one embodiment, the RNAi agent is capable of inhibiting expression of the Beta-ENaC gene by at least about 90% at a concentration of 10 nM in H441 cells in vitro.

[0046] In one embodiment, the RNAi has an EC50 of no more than about 0.1 nM.

[0047] In one embodiment, the RNAi has an EC50 of no more than about 0.01 nM.

[0048] In one embodiment, the RNAi has an EC50 of no more than about 0.001 nM. 10 [0049] A RNAi agent comprising a first and a second strand described herein [0050] In one particular specific embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a first strand and a second strand, wherein the first strand and second strand comprise at least 15 contiguous nucleotides, each differing 15 by 0, 1, 2, or 3 nucleotides from the first and second strand, respectively, of a RNAi agent to Beta-ENaC selected from the specific duplexes provided herein and listed, e.g., in Table 1.

[0051] Various specific embodiments of this embodiment are described below.

[0052] In one embodiment, the composition further comprises a second RNAi agent to Beta-ENaC. In various embodiments, the second RNAi agent is physically separate from the 20 first, or the two are physically connected (e.g., covalently linked or otherwise conjugated).

[0053] In one embodiment, the antisense strand is about 30 or fewer nt in length.

[0054] In one embodiment, the sense strand and the antisense strand form a duplex region of about 15 to about 30 nt pairs in length.

[0055] In one embodiment, the antisense strand is about 15 to about 36 nt in length, 25 including about 18 to about 23 nt in length, and including about 19 to about 23 nt in length.

In one embodiment, the antisense strand has at least the length selected from about 15 nt, about 16 nt, about 17 nt, about 18 nt, about 19 nt, about 20 nt, about 21 nt, about 22 nt, about 23 nt, about 24 nt, about 25 nt, about 26 nt, about 27 nt, about 28 nt, about 29 nt and about 30 nt. 30 [0056] In one embodiment, the RNAi agent comprises a modification that causes the RNAi agent to have increased stability in a biological sample or environment, e.g., blood serum or intestinal lavage fluid.

[0057] In one embodiment, the RNAi agent comprises at least one sugar backbone modification (e.g., phosphorothioate linkage) and/or at least one 2’-modified nucleotide. In 35 one embodiment, all the pyrimidines are 2’ O-methyi-modified nucleotides.

[0058] In one embodiment, the RNAi agent comprises: at least one 5’-uridine-adenine-3’ (5’-ua-3’) dinucleotide, wherein the uridine is a 2’~modified nucleotide; and/or at least one 5’-uridine-guanine-3’ (5’-ug-3’) dinucleotide, wherein the 5’~uridine is a 2’-modified nucleotide; 12 2015221515 03 Sep 2015 and/or at least one 5’-cytidine-adenine-3’ (5’-ca-3’) dinucleotide, wherein the 5’~cytidine is a 2’-modified nucleotide; and/or at least one 5,-uridine-uridine-3, (5’-uu-3’) dinucleotide, wherein the 5’-uridine is a 2’-modified nucleotide.

[0059] In one embodiment, the RNAi agent comprises a 2'-modification selected from 5 the group consisting of: 2'-deoxy, 2’-deoxy-2’-f!uoro, 2’-0-methyl, 2'-0-methoxyethyl (2'-0- MOE), 2'-0-aminopropy! (2’-0-AP), 2‘-0-dimethyiaminoethyl (2'-0-DMAOE), 2'-0-dimethylaminopropyl (2'~0-DMAP), 2'-0-dimethylaminoethyloxyethyl (2'-0-DMAE0E), and 2'~ O-N-methylacetamido (2-O-NMA).

[0060] In one embodiment, the RNAi agent comprises a blunt end. 10 [0061] In one embodiment, the RNAi agent comprises an overhang having 1 to 4 unpaired nucleotides.

[0062] In one embodiment, the RNAi agent comprises an overhang at the 3’-end of the antisense strand of the RNAi agent.

[0063] In one embodiment, the RNAi agent is ligated to one or more diagnostic 15 compound, reporter group, cross-linking agent, nuclease-resistance conferring moiety, natural or unusual nucieobase, lipophilic molecule, cholesterol, lipid, lectin, steroid, uvaol, hecigenin, diosgenin, terpene, triterpene, sarsasapogenin, Friedelin, epifriedelanol-derivatized lithocholic acid, vitamin, carbohydrate, dextran, pullulan, chitin, chitosan, synthetic carbohydrate, oligo lactate 15-mer, natural polymer, low- or medium-molecular 20 weight polymer, inulin, cyclodextrin, hyaluronic acid, protein, protein-binding agent, integrin-targeting molecule, polycationic, peptide, polyamine, peptide mimic, and/or transferrin.

[0064] In one embodiment, the RNAi agent is capable of inhibiting expression of the Beta-ENaC gene by at least about 60% at a concentration of 10 nM in H441 cells in vitro.

[0065] In one embodiment, the RNAi agent is capable of inhibiting expression of the 25 Beta-ENaC gene by at least about 70% at a concentration of 10 nM in H441 cells in vitro.

[0066] In one embodiment, the RNAi agent is capable of inhibiting expression of the Beta-ENaC gene by at least about 80% at a concentration of 10 nM in H441 cells in vitro.

[0067] In one embodiment, the RNAi agent is capable of inhibiting expression of the

Beta-ENaC gene by at least about 90% at a concentration of 10 nM in H441 cells in vitro. 30 [0068] In one embodiment, the RNAi has an EC50 of no more than about 0.1 nM.

[0069] In one embodiment, the RNAi has an EC50 of no more than about 0.01 nM.

[0070] In one embodiment, the RNAi has an EC50 of no more than about 0.001 nM.

[0071] A method of treatment using a RNAi agent described herein 35 [0072] In one particular specific embodiment, the present disclosure relates to a method of treating a Beta-ENaC-related disease in an individual, comprising the step of administering to the individual a therapeutically effective amount of a composition comprising a RNAi agent comprising at least an antisense strand, wherein the antisense strand comprises at least 15 13 2015221515 03 Sep 2015 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of a RNAi agent to Beta-ENaC selected from the specific duplexes provided herein and as listed, e.g., in Table 1. In another embodiment, the present disclosure relates to such method, wherein the composition comprising a RNAi agent further comprises a sense strand, wherein 5 the sense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the sense strand of a RNAi agent to Beta-ENaC selected from the specific duplexes provided herein and as listed, e.g., in Table 1.

[0073] Various particular specific embodiments of this embodiment are described below. 10 [0074] in one embodiment, the Beta-ENaC-related disease is cystic fibrosis, pseudohypoaldosteronism type 1 (PHA1), Liddle’s syndrome, hypertension, aikaiosis, hypokalemia, and/or obesity-associated hypertension.

[0075] in one embodiment, the Beta-ENaC-related disease is cystic fibrosis.

[0076] in one embodiment, the method further comprises the administration of an 15 additional treatment. In one embodiment, the additional treatment is a therapeutically effective amount of a composition.

[0077] In one embodiment, the additional treatment is a method (or procedure), [0078] In one embodiment, the additional treatment and the RNAi agent can be administered in any order, or can be administered simultaneously. 20 [0079] In one embodiment, the method further comprises the step of administering an additional treatment for cystic fibrosis, pseudohypoaldosteronism type 1 (PHA1), Liddle’s syndrome, hypertension, alkalosis, hypokalemia, and/or obesity-associated hypertension.

[0080] In one embodiment, the method further comprises the step of administering an additional treatment or therapy selected from the list of an additional antagonist to ENaC, a 25 potassium-sparing diuretic, amiloride, triamterene, regulation of dietary salt intake, antibiotics, DNase therapy, albutrol, N-acetylcysteine, breathing therapy, percussive therapy, and aerobic exercise.

[0081] In one embodiment, the composition comprises a second RNAi agent to Beta-ENaC. In various embodiments, the second RNAi agent is physically separate from the first, 30 or the two are physically connected (e.g., covalently linked or otherwise conjugated).

[0082] In one embodiment, the method further comprises the step of administering an additional RNAi agent which comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of a RNAi agent to Beta-ENaC selected from the specific duplexes provided herein and as listed, e.g., in Table 1. 35 14 2015221515 03 Sep 2015 [0083] A method of inhibiting the expression of Beta-ENaC, using a RNAi agent described herein [0084] in one particular specific embodiment, the present disclosure relates to a method of inhibiting the expression of the Beta-ENaC gene in an individual, comprising the step of 5 administering to the individual a therapeutically effective amount of a composition comprising a RNAi agent of the present disclosure. In one embodiment, the RNAi agent comprises at least an anti-sense strand, and/or comprises a sense and an anti-sense strand, wherein the anti-sense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the anti-sense strand of a RNAi agent to Beta-ENaC selected from those 10 specific duplex provided herein and as listed, e.g., in Table 1.

[0085] Various embodiments of this aspect of the invention are described below.

[0086] In one embodiment, the individual is afflicted with or susceptible to a Beta-ENaC-related disease.

[0087] In one embodiment, the Beta-ENaC-related disease is cystic fibrosis, 15 pseudohypoaldosteronism type 1 (PHA1), Liddle’s syndrome, hypertension, alkalosis, hypokalemia, and/or obesity-associated hypertension.

[0088] In one embodiment, the Beta-ENaC-related disease is cystic fibrosis.

[0089] In one embodiment, the method further comprises the administration of an additional treatment. In one embodiment, the additional treatment is a therapeutically 20 effective amount of a composition.

[0090] In one embodiment, the additional treatment is a method (or procedure).

[0091] In one embodiment, the additional treatment and the RNAi agent can be administered in any order or can be administered simultaneously.

[0092] In one embodiment, the method further comprises the step of administering an 25 additional treatment for cystic fibrosis, pseudohypoaldosteronism type 1 (PHA1), Liddle’s syndrome, hypertension, alkalosis, hypokalemia, and/or obesity-associated hypertension.

[0093] In one embodiment, the method further comprises the step of administering an additional treatment or therapy selected from the list of an additional antagonist to ENaC, a potassium-sparing diuretic, amiioride, triamterene, regulation of dietary salt intake, 30 antibiotics, DNase therapy, albutrol, N-acetylcysteine, breathing therapy, percussive therapy, and aerobic exercise.

[0094] In one embodiment, the composition comprises a second RNAi agent to Beta-ENaC. In various embodiments, the second RNAi agent is physically separate from the first, or the two are physically connected (e.g., covalently linked or otherwise conjugated). 35 [0095] In one embodiment, the method further comprises the step of administering an additional RNAi agent which comprises at least 15 contiguous nucleotides differing by 0, 1,2, 15 2015221515 03 Sep 2015 or 3 nucleotides from the antisense strand of a RNAi agent to Beta-ENaC selected from the specific duplexes provided herein and as listed, e.g., in Table 1.

[0096] Pharmaceutical formulations of a RNAi agent to Beta-ENaC 5 [0097] In one particular specific embodiment, the present disclosure relates to a composition comprising a RNAi agent of the present disclosure. In one embodiment, the RNAi agent comprises at least an anti-sense strand, and/or comprises a sense and an anti-sense strand, wherein the anti-sense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the anti-sense strand of a RNAi agent to Beta-10 ENaC selected from those specific duplex provided herein and as listed, e.g., in Table 1, wherein the composition is in a pharmaceutically effective formulation.

[0098] In one embodiment, the present disclosure pertains to the use of a RNAi agent in the manufacture of a medicament for treatment of a Beta-ENaC-related disease, wherein the RNAi agent comprises a sense strand and an antisense strand, wherein the antisense strand 15 comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of a RNAi agent to Beta-ENaC selected from those specific duplex provided herein and as listed, e.g., in Table 1.

[0099] ENaC 20 [00100] By “ENaC” is meant the epithelial sodium channel, a membrane protein made of three different but homologous subunits (Alpha, Beta and Gamma).

[00101] ENaC is present in the apical membrane of epithelial cells of the distal nephron (cortical and medullary collecting tubule) and distal colon and in the airways and in the excretory ducts of several glands. ENaC is also expressed in placenta, brain and urinary 25 bladder. It provides a controlled entry pathway for Na+ from the lumen of these organs into the epithelial cells, and, together with the Na+/K+-ATPase located in the basolateral membrane of the same cells, and is responsible for the active, vectorial transport of Na+ from the external medium through the epithelial cells into the extracellular fluid and toward the blood. ENaC is located on the apical membrane facing the lumen, and allows movement of 30 sodium from the lumen into the epithelial cell. The sodium reabsorbed via ENaC is then extruded from the epithelial cell back into the bloodstream by the NaVlC-ATPase. The reabsorption of sodium by the ENaC is accompanied by an osmotic uptake of water to maintain a constant extracellular Na+ concentration. This changes blood volume and consequently affects blood pressure. Thus, ENaC plays an important role in electrolyte 35 homeostasis and the control of blood volume and blood pressure. See, e.g., Saxena et al. 1998 Biochem. Biophys. Res. Comm. 252: 208-213.

[00102] ENaC has different functional roles in various organs in which it is expressed. In the kidney (collecting tubule), the modulated reabsorption of Na+ through ENaC provides the 16 2015221515 03 Sep 2015 primary mechanism of the regulation of urinary Na+ excretion and thus allows the fine control of the whole organism Na+ balance under the hormonal control of aldosterone. By its depolarizing effect on the apical membrane potential, the Na+ channel also provides the driving force for tubular K+ secretion. 5 [00103] Specific inhibitors of ENaC promote urinary Na* excretion and inhibit K* secretion; these drugs (including amiioride and triamterene), are therefore used as K+-sparing diuretics. ENaC has a similar functional role in the distal colon, preventing excessive Na+ loss in the stools, in airways, an important role is the reabsorption of the fluid that fills the airways at birth, promoting the shift from fluid secretion (before birth) to fluid reabsorption 10 (postnatal).

[00104] With the cystic fibrosis transmembrane conductance regulator, it also participates in the delicate regulation of the fluid balance in the airways that maintains a thin mucosal fluid film necessary for mucus clearance, in the excretory ducts of the salivary and sweat glands, the activity of ENaC tends to decrease the luminal Na+ concentration, allowing the 15 excretion of a less salty saliva and preventing major loss of Na+ in the sweat fluid. See, for example, Hummier et al. 1999 Am. J. Physiol. Gastrointest. Liver Physiol. 276: 567-571 and references cited therein.

[00105] Alterations and mutations in the sequence and/or expression of ENaC can lead to over-expression or hyper-activity of ENaC. Providing RNAi agents of this disclosure restores 20 balance to the modulated reabsorption of Na+ by reducing the level of the Beta-ENaC.

[00106] Beta-ENaC

[00107] By “Beta-ENaC” is meant the gene or protein amiloride-sensitive sodium channel subunit beta (or any nucleic acid encoding this protein), also variously designated: sodium 25 channel, nonvoltage-gated 1, beta; SCNN1B; bENaC; ENaCb; ENaC-beta; SCNEB, or β-ENaC. Additional identifiers include: OMIM: 600760; MGI: 104696; HomoloGene: 284; and GeneCards: SCNN1B. Additional information can be found: Human: Entrez 6338; Ensembl ENSG00000168447; UniProt P51168; RefSeq (mRNA) NM_000336; RefSeq (protein) NP_000327; Location (UCSC) Chr 16: 23.22 - 23.3 Mb. Mouse: Entrez 20277; Ensembl 30 ENSMUSG00000030873; UniProt Q3TP51; RefSeq (mRNA) NM_011325 RefSeq (protein) NPJ335455; Location (UCSC) Chr 7: 121.66-121.71 Mb.

[00108] The amino acid sequence of human Beta-ENaC is provided in Saxena et al. 1998 Biochem. Biophys. Res. Comm. 252: 208-213.

[00109] The functional domains of Beta-ENaC have been delineated. The protein has an 35 intracellular N-termina! domain [amino acids (“aa”) 1 to 50], a first transmembrane domain (aa 51 to 71), an extracellular loop (aa 72 to 533), a second transmembrane domain (aa 534 to 553), and a C-terminal intracellular domain (aa 554 to 640). 17 2015221515 03 Sep 2015 [00110] The Otermina! intracellular domain contains two regions wherein mutations relate to Liddle’s syndrome and other diseases: in the region from amino acid 564 to 595 and the “PY” motif [with the amino acid consensus sequence PPXY at aa (amino acids) 615 to 618], See, e.g., Saxena et al. 1998. 5 [00111] The Beta-ENaC RNAi agent of the present disclosure can interact with a portion of the mRNA corresponding to a specific functional domain or domains of Beta-ENaC. In various embodiments, the RNAi agents herein specifically bind to Beta-ENaC mRNA, in a sequence corresponding to a functional domain, e.g., in the N-terminal intracellular domain, in the first transmembrane domain, in the extracellular loop, in the second transmembrane 10 domain, or in the C-terminal intracellular domain, or, more specifically, in the region from amino acid 564 to 595, or in the PY motif (amino acids 615 to 618).

[00112] In various embodiments, the RNAi agents of the present disclosure bind to the 5’ or 3’ UTR (i.e., untranslated region(s)).

[00113] In various embodiments, the RNAi agents of the present disclosure bind to Beta-15 ENaC mRNA, but not in a sequence corresponding to a particular functional domain, e.g., not in the N-terminal intracellular domain, not in the first transmembrane domain, not in the extracellular loop, not in the second transmembrane domain, or not in the C-terminal intracellular domain, or, more specifically, not in the region from amino acid 564 to 595, or not in the PY motif (amino acids 615 to 618). 20 [00114] In embodiments herein, binding of a RNAi agent to a particular region of the Beta-ENaC mRNA leads to reduced expression, level and/or activity of Beta-ENaC.

[00115] The efficacy of a RNAi agent in reducing the level of Beta-ENaC can be measured directly, e.g., by measuring the levels of Beta-ENaC mRNA abundance or levels of the protein itself. Alternatively, the efficacy of the RNAi can be measured indirectly by 25 measuring the level of any one or more of the known activities of Beta-ENaC or by measuring changes in the activities of pathway components downstream of Beta-ENaC.

[00116] The protein’s chief activity is to form, along with Alpha-ENaC and Gamma-ENaC, and, possibly at times, Delta-ENaC, the sodium channel ENaC. Beta-ENaC, Gamma-ENaC and Delta-ENaC may also form a particular type of channel found in the pancreas, testes and 30 ovaries. Beta-ENaC has also been shown to interact with WWP2 and NEDD4. See., e.g., McDonald et al. (2002). Am. J. Physiol. Renal Physiol. 283 (3): F431-6; Harvey et al. 2001. J. Biol. Chem. 276 (11): 8597-601; Farr et al, (2000). Biochem. J. 345 Ft 3: 503-9. The activity of Beta-ENaC can be measured, for example, by its ability to bind and form functional units with these other biological components. The efficacy of a RNAi agent can also be measured 35 indirectly by measuring the amount of surface liquid on mucus membranes, and via histological studies of tissues expressing Beta-ENaC.

50 Cyno Beta-ENaC Human Beta-ENaC 18 2015221515 03 Sep 2015 [00117] Beta-ENaC sequences in various species [00118] A RNAi agent specific to Beta-ENaC can be designed such that the sequence thereof completely matches that of the mRNA corresponding to the human Beta-ENaC gene and the homologous gene from a test animal. Thus, the exact same RNAi agent can be 5 used in both test animals (e.g., rat, mouse, cynomolgus monkey, etc.) and humans. The sequences for the various ENaC genes have been determined in many species, including humans, mouse, rat, bovine and chicken, as described in, inter alia, Garty et ai. 1997 Physiol. Rev. 77: 359-396; and Ahn et al. 1999 Am. J. Physiol. 277:F121-F129. [00119] The Beta-ENaC sequence in cynomolgus monkey (Macaca fascicularis, or 10 “cyno”) has been determined. [00120] The alignment of the cyno Beta-ENaC mRNA (SEQ ID NO: 221) and human Beta-ENaC mRNA (SEQ ID NO: 222) sequences is shown below.

Cyno Beta-ENaC -------------------------------------------------- 15 Human Beta-ENaC GTGCTTCCCCGCCCCTGAACCTGCTCCCTCCCAGTCGGTCTCGCCGCGCT 50 20

Cyno Beta-ENaC -----------------------------------GGTACCCAGCTTGCT 15 Human Beta-ENaC CGCCGGGTGTCCCAGTGTCACCAACACTCGGCCGCCGCCGCCAGCTTGGC 10C * ********

Cyno Beta-ENaC TGTTCTTTTTGCAGAAGCTCAGAATAAACGCTCAACTTTGGCAGATCAAT 65 Human Beta-ENaC GCGCACCGCCGCCTCCGCCACCGCCGACAGCGCGCATCCTCCGTGTCCCC 150 * * ** * ** * * * * * 25

Cyno Beta -ENaC Human Beta-ENaC TCCCCGGGGATCCGA-ATTCGCCACCATGCACGTGAAGAAGTACCTGCTG GCTCCGCCGCCCGAGCAGGTGCCACTATGCACGTGAAGAAGTACCTGCTG * *** * * * ***** ************************ 114 ο η n

30 Cyno Beta-ENaC Human Beta-ENaC AAGTGCCTGCACCGGCTGCAGAAGGGCCCCGGCTACACGTACAAGGAGCT 16 4 AAGGGCCTGCATCGGCTGCAGAAGGGCCCCGGCTACACGTACAAGGAGCT 2 5 0 * * * ******* **************************************

Cyno Beta-ENaC 35 Human Beta-ENaC GCTGGTGTGGTACTGCGATAACACCAACACCCACGGCCCCAAGCGTATCA 214 GCTGGTGTGGTACTGCGACAACACCAACACCCACGGCCCCAAGCGCATCA 3 0 0 ****************** ************************** * * * *

Cyno Beta-ENaC Human Beta-ENaC 40 TCTGCGAGGGGCCCAAGAAGAAAGCCGTGTGGTTCCTGCTCACCCTGCTC 264 TCTGTGAGGGGCCCAAGAAGAAAGCCATGTGGTTCCTGCTCACCCTGCTC 3 5 0 * * * * ********************* ***********************

Cyno Beta-ENaC Human Beta-ENaC 45

Cyno Beta-ENaC Human Beta-ENaC TTCACTGCTCTCGTCTGCTGGCAGTGGGGCATCTTCATCAGGACCTACTT 314 TTCGCCGCCCTCGTCTGCTGGCAGTGGGGCATCTTCATCAGGACCTACTT 400 *** * ** ***************************************** GAGCTGGGAGGTCAGCGTCTCCCTCTCCGTAGGCTTCAAGACCATGGACT 364 GAGCTGGGAGGTCAGCGTCTCCCTCTCCGTAGGCTTCAAGACCATGGACT 450 ************************************************** TCCCCGCCGTCACCATCTGCAATGCTAGCCCCTTCAAGTATTCCAAAGTC 414 TCCCTGCCGTCACCATCTGCAATGCTAGCCCCTTCAAGTATTCCAAAATC 5 0 0 * * * * ************************************* ***·* * * *

Cyno Beta-ENaC AAGCATTTGCTGAAGGACCTGGATGAGCTGATGGAAGCTGTCCTGGAGAG 464 19 2015221515 03 Sep 2015

Human Beta-ENaC AAGCATTTGCTGAAGGACCTGGATGAGCTGATGGAAGCTGTCCTGGAGAG 550 **************************************************

Cyno Beta-ENaC 5 Human Beta-ENaC 10

Cyno Beta-ENaC Human Beta-ENaC

20 Cyno Beta-ENaC Human Beta-ENaC

Cyno Beta-ENaC 25 Human Beta-ENaC 35 AATCCTGGCTCCTGAGCTAAGCCATGCCAATGCCACCAGGACCCTGAACT 514 AATCCTGGCTCCTGAGCTAAGCCATGCCAATGCCACCAGGAACCTGAACT 600 •k k k k k k k * * k k k k k k k * k k k k k k k * k * Ά· *· * * * * * * Ά· k * * * * **· ·Α· Ά· * * k k CTTCCATCTGGAACCACACACCACTGGTCCTTATTGATGAACGGAACCCC 564 TCTCCATCTGGAACCACACACCCCTGGTCCTTATTGATGAACGGAACCCC 6 5 0 kkkkkkkkkkkkkkkkkkkk k k k k k k k k k k k k k k k k k k k k k k k k k k k CACCACCCCATGGTCCTCGATCTCTTTGGAGATAACCACAATGGCTTAAC 614 CACCACCCCATGGTCCTTGATCTCTTTGGAGACAACCACAATGGCTTAAC 700 kk kkkk kkkkk****** ******** ** ** * * * ******* * ******** AAACAGCTCAGCATCAGAAAAGATCTGTAATGCCCATGGGTGCAAAATGG 664 AAGCAGCTCAGCATCAGAAAAGATCTGTAATGCCCACGGGTGCAAAATGG 750 ** ********************·^************ **·^*******·^** CCATGAGACTATGTAGCCTCAACGGGACCCAGTGCACCTTCCGGAACTTC 714 CCATGAGACTATGTAGCCTCAACAGGACCCAGTGTACCTTCCGGAACTTC 800 *********************** ********** *************** ACCAGCGCTACCCAGGCAGTGACAGAGTGGTACAGCCTGCAGGCCACCAA 764 ACCAGTGCTACCCAGGCATTGACAGAGTGGTACATCCTGCAGGCCACCAA 850 kkkkk kkkkkkkkkkkk kkkkkkkkkkkkkkk kkkkkkkkkkkkkkk

Cyno Beta-ENaC CATCTTTGCGCAGGTGCCGCAGCAGGAGCTGGTGGAGATGAGCTACCCCG 814 Human Beta-ENaC CATCTTTGCACAGGTGCCACAGCAGGAGCTAGTAGAGATGAGCTACCCCG 900 ****** * * * ******** *********** ** ****************

Cyno Beta-ENaC Human Beta-ENaC

Cyno Beta-ENaC Human Beta-ENaC GCGAGCAGATGATCCTGGCCTGCCTGTTTGGAGCTGAGCCCTGCAACTAC 864 GCGAGCAGATGATCCTGGCCTGCCTATTCGGAGCTGAGCCCTGCAACTAC 950 ****************** * * ***** * * ***** * * ****** * * ****** CGGAACTTCACGTCCATCTTCTACCCTCACTATGGCAACTGTTACATCTT 914 CGGAACTTCACGTCCATCTTCTACCCTCACTATGGCAACTGTTACATCTT 1000 **************************************************

40 Cyno Beta-ENaC Human Beta-ENaC CAACTGGGGCATGACAGAGAAGGCACTTCCTTCGGCCAACCCTGGACCTG 964 CAACTGGGGCATGJSlCAGAGAAGGCACTTCCTTCGGCCAACCCTGGJSlACTG 1050

Cyno Beta-ENaC 45 Human Beta-ENaC AATTTGGCCTGAAGTTGATCCTGGACATAGGCCAGGAAGACTACGTCCCC 1014 AATTCGGCCTGAAGTTGATCCTGGACATAGGCCAGGAAGACTACGTCCCC .1.10 0 **** *********************************************

Cyno Beta-ENaC TTCCTCGCGTCCACGGCTGGGGTCAGGCTGATGCTTCACGAGCAGAGGTC 1064 Human Beta-ENaC TTCCTTGCGTCCACGGCCGGGGTCAGGCTGATGCTTCACGAGCAGAGGTC 115 0 ***** *********** ********************************

Cyno Beta-ENaC Human Beta-ENaC 55

Cyno Beta-ENaC Human Beta-ENaC ATACCCCTTCATCAGAGACGAGGGCATCTATGCCATGTCGGGGACAGAGA 1114 ATACCCCTTCATCAGAGATGAGGGCATCTACGCCATGTCGGGGACAGAGA 1200 ****************** *********** ******************* CGTCCATCGGGGTACTCGTGGACAAGCTTCAGCGCATGGGGGAGCCCTAC 1164 CGTCCATCGGGGTACTCGTGGACAAGCTTCAGCGCATGGGGGAGCCCTAC 1250 kkkkkkkkkkkkkkkkkkkkkkkkkkkkkkk-*kkkkkkkkkkkkkkkkkk

60 Cyno Beta-ENaC Human Beta-ENaC AGCCCGTGCACCGTGAATGGCTCCGAGGTCCCCGTCCAAAACTTCTACAG 1214 AGCCCGTGCACCGTGAATGGTTCTGAGGTCCCCGTCCAAAACTTCTACAG 1300 ******************** ** ************************** 20 2015221515 03 Sep 2015 5 10 15 20 25 30 35 40 45 50 55 80

Cyno Beta-ENaC Human Beta-ENaC TGACTACAACACGACCTACTCCATCCAGGCCTGTCTTCGCTCCTGCTTCC TGACTACAACACGACCTACTCCATCCAGGCCTGTCTTCGCTCCTGCTTCC * * * * ********* ******* ************************ ****** 1264 1350 Cvno Beta-ENaC Human Beta-ENaC AAGACCACATGATCCGTAGCTGCAAGTGTGGGCACTACCTCTACCCACTG AAGACCACATGATCCGTAACTGCAACTGTGGCCACTACCTGTACCCACTG ****************+* ****** ***** ******** ********* 1314 14 0 0 Cyno Beta-SNaC Human Beta-ENaC CCCCGTGGGGAGAAATACTGCAACAACCGGGACTTCCCAGACTGGGCCCA CCCCGTGGGGAGAAATACTGCAACAACCGGGACTTCCCAGACTGGGCCCA ************************************************** 13 64 1450 Cyno Beta-ENaC Human Beta-ENaC TTGCTACTCAGATCTGCAGATGAGCGTGGCGCAGAGAGAGACCTGCATTG TTGCTACTCAGATCTACAGATGAGCGTGGCGCAGAGAGAGACCTGCATTG *************** ********************************** .1414 15 0 0 Cyno Beta-ENaC Human Beta-ENaC GCATGTGCAAGGAATCCTGCAATGACACCCAGTACAAGATGACTATCTCC G CATGTGCAAGGAGTCCTGCAATGACACCCAGTACAAGATGACCATCTCC ************* ***************************** ****** 1464 155 0 Cyno Beta-ENaC Human Beta-ENaC ATGGCTGACTGGCCTTCTGAGGCCTCTGAGGACTGGATTTTCCACGTCTT ATGGCTGACTGGCCTTCTGAGGCCTCCGAGGACTGGATTTTCCACGTCTT ************ ************** ******* * * * * * * * * * ****** * 1514 1600 Cyno Beta-ENaC Human Beta-ENaC GTCTCAGGAGCGGGACCAAAGCACCAATATCACCCTGAGCAGGAAGGGAA GTCTCAGGAGCGGGACCAAAGCACCAATATCACCCTGAGCAGGAAGGGAA ************************************************** 1564 1650 Cyno Beta-ENaC Human Beta-ENaC TTGTCAAGCTCAACATCTACTTCCAAGAATTTAACTATCGCACCATTGAA TTGTCAAGCTCAACATCTACTTCCAAGAATTTAACTATCGCACCATTGAA ************************************************** 1614 1700 Cyno Beta-ENaC Human Beta-ENaC GAATCAGCAGCCAATAACCTCGTCTGGCTGCTCTCAAATCTGGGTGGCCA GAATCAGCAGCCAATAACATCGTCTGGCTGCTCTCGAATCTGGGTGGCCA ****************** **************** ************** 1664 1750 Cyno Beta-ENaC Human Beta-ENaC GTTTGGCTTCTGGATGGGGGGCTCTGTGCTGTGCCTCATCGAGTTTGGGG GTTTGGCTTCTGGATGGGGGGCTCTGTGCTGTGCCTCATCGAGTTTGGGG ************************************************** 1714 18 00 Cyno Beta-ENaC Wnman Beta-ENaC AGATCATCATCGACTTTGTGTGGATCACCATCATCAAGCTGGTGGCCTTG AGATCATCATCGACTTTGTGTGGATCACCATCATCAAGCTGGTGGCCTTG ************************************************** 1764 1850 Cyno Beta-ENaC Human Beta-ENaC GCCAAGAGCCTCCGGCAGCGGCGAGCCCAAGCCAGCTACTCCGGCCCACC GCCAAGAGCCTACGGCAGCGGCGA.GCCCAAGCCAGCTACGCTGGCCCA.CC *********** *************************** * ******** 1814 1900 Cyno Beta-SNa.C Huma.n Beta-ENaC GCCCACGGTGGCTGAGCTGGTGGAGGCCCACACCAACTTCGGCTACCAGC GCCCACCGTGGCCGAGCTGGTGGAGGCCCACACCAACTTTGGCTTCCAGC ****** ***** ************************** **** ***** 1864 1950 Cyno Beta-ENaC Human Beta-ENaC CTGACACGGCCCCCCGCAGCCCCAACACCGGGCCCTACCCCAGTGAGCAG CTGACACGGCCCCCCGCAGCCCCAACACTGGGCCCTACCCCAGTGAGCAG **************************** ********************* 1914 2000 Cyno Beta-ENaC Human Beta-ENaC GCCCTGCCCATCCCGGGCACCCCGCCCCCCAACTATGACTCCCTGCGTCT GCCCTGCCCATCCCAGGCACCCCGCCCCCCAACTATGACTCCCTGCGTCT ************** *********************************** 1964 2 050 Cyno Beta-ENaC GCAGCCACTGGACGTCATCGAGTCTGACAGTGAGGGTGATGCCATCTAA- 2 013 21 2015221515 03 Sep 2015

Human Beta-ENaC GCAGCCGCTGGACGTCATCGAGTCTGACAGTGAGGGTGATGCCATCTAAC 2100 ****** ******************************************

Cyno Beta-ENaC 5 Human Beta-ENaC

Cyno Beta-ENaC Human Beta-ENaC 10 15

Cyno Beta-ENaC Human Beta-ENaC

20 Cyno Beta-ENaC Human Beta-ENaC

Cyno Beta-ENaC 25 Human Beta-ENaC -----GCGGCCGCCTAG AAATAGCTTGATCTGGTTA- -CCACTAAACCA 2055 CCTGCCCCTGCCCACCCCGGGCGGCTGAAACTCACTGAGCAGCCAAGACT 215 0 * * ***** * * * * * * * * * * * * GC--CTCAAGAACAC-CCGAATGGAGTCTCT------AAGCTACATAATACC 2 098 GTTGCCCGAGGCCTCACTGTATGGTGCCCTCTCCAAAGGGTCGGGAGGGT 2200 * * * * * ********** *** * * AACTTACACTTTAjCAJLAATGTTGTCCCCCAA-AATGTAGCCATTCGTATC 2147 AGCTCTCCAGGCCAGAGCTTGTGTCCTTCAACAGAGAGGCCAGCGGCAAC 2250 * * * * * * ***** **** * **** * * * TGCTCCTAATAAAAAGAAAGTTTCTTCACATTCTAAAAAAAAAAAAAAAA 2197 TGGTCCGTTACTGGCCAAGGGCTCTGTAGAATCACGGTGCTGGTACAGGA 2300 ** *** ** * *** * * ** * * * AAAAAAAAAAAAAAAAAACCCCCCCC--CCCCCCCCCCTGCAGAGATCTG 2245 TGCAGGAATAAATTGTATCTTCACCTGGTTCCTACCCTCGTCCCTACCTG 2350 * ** *** * * * ** ** *** * * *** CTAGCTTGAGTATTCTATAGAGTCACCTAAATACT---------------------- 228 0 TCCTGATCCTGGTCCTGAAGACCCCTCGGAACACCCTCTCCTGGTGGCAG 2400 * *** *** * * * * * *

Cyno Beta-ENaC Human Beta-ENaC 30 Cyno Beta-ENaC Human Beta-ENaC Cyno Beta-ENaC 35 Human Beta.-ENaC Cyno Beta -ENaC Human Beta--ENaC 40 Cyno Beta-ENaC Human Beta-ENaC [00121] The start (ATG) and stop (TAA) codons of the cyno and human sequences are underlined. Nucleotides matching between the human and cyno sequences are marked 45 with an asterick (*). [00122] In one embodiment, the Beta-ENaC RNAi agent of the present disclosure comprises a sequence which is identical in the human, rat and cyno Beta-ENaC mRNAs. This sequence identity facilitates animal testing prior to human testing. In another embodiment, the Beta-ENaC RNAi agent comprises a sequence which is identical in the 50 human, mouse and cyno Beta-ENaC mRNAs. GCCACTTCCCTCCCAGTGCCAGTCTCCATCCACCCCAGAGAGGAACAGGC 2450 GGGTGGGCCATGTGGTTTTCTCCTTCCTGGCCTTGGCTGGCCTCTGGGGC 2500 AGGGGTGGTGGAGAGATGGAAGGGCATCAGGTGTAGGGACCCTGCCAAGT 2550 GGCACCTGATTTACTCTAGAAAATAAAAGTAGAAAATACTGAGTCCA 259" (SEQ ID NO: 221) (SEQ ID NO: 222) [00123] Additional embodiments of a RNAi agent to Beta-ENaC [00124] In one embodiment, the Beta-ENaC RNAi agent comprises a sequence which does not match that of any other mRNA or gene. In one embodiment, the Beta-ENaC RNAi 22 2015221515 03 Sep 2015 agent comprises a sequence which differs from all other known non-Beta-ENaC mRNAs or genes by at least 0, 1, 2 or 3 nucleotides.

[00125] In one embodiment, the Beta-ENaC RNAi agent of the present disclosure is administered to a patient in need thereof (e.g., a patient suffering from cystic fibrosis, 5 pseudohypoaidosteronism type 1 (PHA1), Liddle’s syndrome, hypertension, alkalosis, hypokalemia, and obesity-associated hypertension).

[00126] The patient can also be administered more than one RNAi agent specific to Beta-ENaC. In one embodiment, the Beta-ENaC RNAi agent(s) of the present disclosure can optionally be administered along with one or more additional pharmaceutical agent 10 appropriate for that disease. In one embodiment, the Beta-ENaC RNAi agent(s) of the present disclosure can be optionally administered along with any other appropriate additional treatment, wherein the additional treatment can be a composition or a method.

[00127] In the case of cystic fibrosis, pseudohypoaidosteronism type 1 (PHA1), Liddie’s syndrome, hypertension, alkalosis, hypokalemia, and/or obesity-associated hypertension, the 15 RNAi agent(s) and additional disease treatment(s) can be administered in any order, simultaneously or sequentially, or in one or multipie doses over time.

[00128] Definitions [00129] For convenience, the meaning of certain terms and phrases used in the 20 specification, examples, and appended claims, are provided below. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definition in this section shall prevail.

[00130] RNAi Agent 25 [00131] In one embodiment, the present disclosure pertains to a Beta-ENaC RNAi agent or other composition comprising at least an antisense nucleic acid sequence complementary to a Beta-ENaC nucleic acid (or portion thereof), or pertains to a recombinant expression vector encoding the siRNA or composition comprising the antisense nucleic acid that can function as an RNAi as defined below. As used herein, an "antisense" nucleic acid 30 comprises a nucleotide sequence complementary to a "sense" nucleic acid encoding the Beta-ENaC protein (e.g., complementary to the coding strand of a double-stranded DNA, complementary to an mRNA or complementary to the coding strand of a Beta-ENaC gene or nucleic acid).

[00132] As used herein, the term “RNAi agent to Beta-ENaC,” “RNAi agent specific to 35 Beta-ENaC,” “iRNA agent to Beta-ENaC,” “siRNA to Beta-ENaC”, “Beta-ENaC siRNA” and the like refer to a siRNA (short inhibitory RNA), shRNA (short or small hairpin RNA), iRNA (interference RNA) agent, RNAi (RNA interference) agent, dsRNA (double-stranded RNA), microRNA, and the like, and refer to a composition which specifically targets, is specific to, 23 2015221515 03 Sep 2015 and/or binds to a Beta-ENaC mRNA. As used herein, the term “antisense nucleic acid” or “composition comprising an anti-sense nucleic acid” and the like is broadly meant to encompass any composition comprising at least one nucleic acid strand which is anti-sense to its target; this includes, but is not limited to, any siRNA, shRNA, iRHA, dsRNA, microRNA, 5 antisense oligonucleotide, and any other composition comprising an anti-sense nucleic acid. As used herein, the terms “iRNA” and “RNAi” refers to an agent that contains RNA (or a derivative thereof), and which mediates the targeted cleavage of another RNA transcript via an RNA-induced silencing complex (RISC) pathway. In one embodiment, the RNAi agent is an oligonucleotide composition that activates the RISC complex/pathway. In another 10 embodiment, the RNAi agent comprises an antisense strand sequence (antisense oligonucleotide).

[00133] The RNAi agent(s) of the present disclosure target (e.g., bind to, anneal to, etc.) the Beta-ENaC mRNA. The use of the RNAi agent specific to Beta-ENaC results in a decrease of Beta-ENaC activity, level and/or expression, e.g., a “knock-down” or “knock-out” 15 of the target gene or target sequence. Particularly in one embodiment, in the case of a disease state characterized by over-expression or hyper-activity of Beta-ENaC, administration of a RNAi agent to Beta-ENaC knocks down the Beta-ENaC target enough to restore a normal level of Beta-ENaC activity and/or a norma! level of Na+ reabsorption.

[00134] In one embodiment, the RNAi comprises a single strand (such as an shRNA, as 20 described herein).

[00135] In various embodiments, one or both strands are nicked.

[00136] In one embodiment, a single-stranded RNAi agent oligonucleotide or polynucleotide can comprise the sense and/or antisense strand. See, e.g., Sioud 2005 J.

Mol. Biol. 348:1079-1090, and references cited therein. Thus the present disclosure 25 encompasses RNAi agents with a single strand comprising either the sense or antisense strand of a RNAi agent described herein.

[00137] siRNAs that are particularly useful for this disclosure include those which can bind specifically to a region of the Beta-ENaC mRNA, and have one or more of the following qualities: binding in the coding segment of Beta-ENaC; binding at or near the junction of the 30 5' untranslated region and the start of the coding segment; binding at or near the translational start site of the mRNA; binding at, across or near junctions of exons and introns; little or no binding to the mRNAs or transcripts of other genes (little or no “off-target effects”); binding to the Beta-ENaC mRNA in or near a region or regions that is not double-stranded or a stem region, e.g., in a loop or single-stranded portion; eliciting little or no immunogenicity; 35 binding in a segment of the Beta-ENaC mRNA sequence which is conserved among various animal species (including human, mouse, rat, cyno, etc.), as the presence of a conserved sequence facilitates testing using various laboratory animals; binding to double-stranded 24 2015221515 03 Sep 2015 region(s) of the mRNA; binding to an AT-rich region (e.g., at least about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60% AT-rich); and/or lacking particular sequences known or suspected to decrease siRNA activity, e.g,, the presence of a GG sequence at the 5’ end, which may decrease separation of the double-stranded portion of the siRNA. in one embodiment, the 5 RNAi agent specific to Beta-ENaC can be a double-stranded RNA having any one or more of these qualities.

[00138] The term “double-stranded RNA” or “dsRNA,” as used herein, refers to a RNAi agent comprising a first and a second strand; e.g., a composition that includes an RNA molecule or complex of molecules having a hybridized duplex region (i.e., a region where the 10 nucleotide bases from the first strand and the second strand are paired) that comprises two anti-parallel and substantially complementary nucleic acid strands, which will be referred to as having “sense” and “antisense” orientations with respect to a target RNA. The antisense strand, with respect to the mRNA target, is also called the “guide” strand, and the sense strand is also called the “passenger” strand. The passenger strand can include at least one 15 or more of the following: one or more extra nucleotides (e.g., a bulge or 1 nt loop) compared to the other strand, a nick, a gap, etc., compared to the other strand. In various embodiments, the RNAi agent comprises a first strand and a second strand. In various embodiments, the first strand is the sense strand and the second strand is the anti-sense strand, in other embodiments, the first strand is the anti-sense strand, and the second 20 strand is the sense strand.

[00139] The duplex region can be of any length that permits specific degradation of a desired target RNA through a RISC pathway, but will typically range from 9 to 36 base pairs (“bp”) in length, e.g., 15-30 bp in length. Considering a duplex between 9 and 36 bp, the duplex can be any length in this range, for example, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 25 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 bp and any sub-range therebetween, including, but not limited to 15-30 bp, 15-26 bp, 15-23 bp, 15-22 bp, 15-21 bp, 15-20 bp, 15-19 bp, 15-18 bp, 15-17 bp, 18-30 bp, 18-26 bp, 18-23 bp, 18-22 bp, 18-21 bp, 18-20 bp, 19-30 bp, 19-26 bp, 19-23 bp, 19-22 bp, 19-21 bp, 19-20 bp, 19 bp, 20-30 bp, 20- 26 bp, 20-25 bp, 20-24 bp, 20-23 bp, 20-22 bp, 20-21 bp, 20 basepairs, 21-30 bp, 21-26 bp, 30 21-25 bp, 21-24 bp, 21-23 bp, 21-22 bp, 21 bp, 22 bp, or 23 bp. The dsRNAs generated in the cell by processing with Dicer and similar enzymes are generally in the range of about 19 to about 22 bp in length. One strand of the duplex region of a dsRNA comprises a sequence that is substantially complementary to a region of a target RNA. The two strands forming the duplex structure can be from a single RNA molecule having at least one self-complementary 35 duplex region, or can be formed from two or more separate RNA molecules that hybridize to form the duplex. Where the duplex region is formed from two self-complementary regions of a single molecule, the molecule can have a duplex region separated by a single-stranded 25 2015221515 03 Sep 2015 chain of nucleotides (herein referred to as a "hairpin loop", e.g., such as found in an shRNA construct) between the 3’-end of one strand and the 5’-end of the respective other strand forming the duplex structure. The hairpin loop can comprise at least one unpaired nucleotide; in some embodiments the hairpin loop can comprise at least 3, at ieast 4, at least 5, at least 5 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides. Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not, but can be covalently connected. Where the two strands are connected covalently by a hairpin loop, the construct is generally referred to herein and in the art as a “shRNA”. Where the two strands are connected 10 covalently by means other than a hairpin loop, the connecting structure is referred to as a “linker.” [00140] RNA Interference [00141] RNA interference (RNAi) is a post-transcriptional, targeted gene-silencing 15 technique that uses double-stranded RNA (dsRNA) to degrade messenger RNA (mRNA) containing the same sequence as the dsRNA. The process of RNAi occurs when ribonudease III (Dicer) cleaves the longer dsRNA into shorter fragments called siRNAs. siRNAs (small interfering RNAs) are typically about 21 to 23 nucleotides long and comprise about 19 base pair duplexes. The smaller RNA segments then mediate the degradation of 20 the target mRNA. Dicer has also been implicated in the excision of 21- and 22-nucleotide small temporal RNAs (stRNAs) from precursor RNA of conserved structure that are implicated in translational control. Hutvagner et al. 2001, Science, 293, 834. The RNAi response also features an endonuclease complex, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single-stranded mRNA 25 complementary to the antisense strand of the siRNA. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex.

[00142] In one aspect, an RNA interference agent includes a single-stranded RNA that interacts with a target RNA sequence to direct the cleavage of the target RNA. Without wishing to be bound by theory, long double-stranded RNA introduced into plants and 30 invertebrate cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al., Genes Dev. 2001, 15:485). Dicer, a ribonuclease-lll-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3’ overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA 35 duplex, enabling one of the now unpaired siRNA strands to act as a “guide” strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding of the antisense guide strand to the appropriate target mRNA, one or more endonucleases within the RISC cleaves the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one 26 2015221515 03 Sep 2015 aspect the present disclosure relates to a single-stranded RNA that promotes the formation of a RISC complex to effect silencing of the target gene.

[00143] RNA interference has also been studied in a variety of systems. Work in Drosophila embryonic lysates (Elbashir et a!. 2001 EMBO J. 20: 6877 and Tuschl et ai. 5 International PCT Publication No. WO 01/75164) has revealed certain requirements for siRNA length, structure, chemical composition, and sequence that are essentia! to mediate efficient RNAi activity in a variety of systems, including especially mammals. These studies have shown that 21-nucleotide siRNA duplexes are most active when containing 3'-terminal dinucleotide overhangs. Substitution of the S'-termina! siRNA overhang nucleotides with 2'-10 deoxy nucleotides (2'-H) was tolerated. In addition, a 5'-phosphate on the targetcomplementary strand of a siRNA duplex is usually required for siRNA activity. Most importantly for therapeutic use, siRNA duplexes shorter than 50 bp or so do not activate the interferon response in mammalian cells. See, e.g., Tuschl et a!., WO 01/752164.

[00144] The dsRNA molecules (RNAi agents) described herein are thus useful in RNA 15 interference of Beta-ENaC.

[00145] Features of a RNAi Agent: Sense strand, Antisense Strand and (Optional)

Overhangs [00146] In various embodiments, the RNAi agents comprise a first strand and a second 20 strand, e.g., a sense strand and an antisense strand and, optionally, one or both ends of the duplex containing unpaired nucleotides referred to herein as overhangs.

[00147] The term “antisense strand” refers to the strand of a RNAi agent which includes a region that is substantially complementary to a target sequence. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially 25 complementary to a sequence, for example a target sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches may be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5’ and/or 3’ terminus. 30 [00148] The term “sense strand,” as used herein, refers to the strand of a RNAi agent that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein.

[00149] The sequence of a gene may vary from individual to individual, especially at wobble positions within the coding segment, or in the untranslated region; individuals may 35 also differ from each other in coding sequence, resulting in additional differences in mRNA. The sequence of the sense and antisense strands of the RNAi agent can thus be designed to correspond to that of an individual patient, if and where needed. RNAi agents can also be modified in sequence to reduce immunogenicity, binding to undesired mRNAs (e.g., “off- 27 2015221515 03 Sep 2015 target effects”) or to increase stability in the blood. These sequence variants are independent of chemical modification of the bases or 5’ or 3’ or other end-caps of the RNAi agents.

[00150] The RNAi agents can also have overhangs of 0, 1, or 2 overhangs; in the case of 5 a 0 nt overhang, they are blunt-ended. A RNAi agent can have 0, 1 or 2 blunt ends. In a “blunt-ended RNAi agent" both strands terminate in a base-pair; thus a blunt-ended molecule lacks either 3’ or 5’ single-stranded nucleotide overhangs.

[00151] As used herein, the term “overhang” or “nucleotide overhang” refer to at least one unpaired nucleotide that protrudes from the end of at least one of the two strands of the 10 duplex structure of a RNAi agent. For example, when a 3’-end of one strand of a dsRNA extends beyond the 5'-end of the other strand, or vice versa, the unpaired nucleotide(s) form the overhang. A dsRNA can comprise an overhang of at least one nucleotide; alternatively the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. An overhang can comprise or consist of a 15 nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) may be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucieotide(s) of an overhang can be present on the 5' end, 3' end or both ends of either an antisense or sense strand of a dsRNA.

[00152] The RNAi agent can also optionally comprise a cap. The term "cap” and the like 20 include a chemical moiety attached to the end of a double-stranded nucleotide duplex, but is used herein to exclude a chemical moiety that is a nucleotide or nucleoside. A “3’ Cap” is attached at the 3’ end of a nucleotide or oligonucleotide. A “5! Cap” is attached at the 5’ end of a nucleotide or oligonucleotide. In one embodiment, 3’ end caps are as disclosed in, for example, WO 2005/021749 and WO 2007/128477. 25 [00153] The present disclosure thus contemplates a RNAi agent specific to Beta-ENaC comprising an antisense strand (which may be contiguous or connected via a linker or loop) in a RNAi agent. In a more specific embodiment, an RNAi agent comprises an antisense strand and a sense strand which together comprise a double-stranded or complementary region, in one embodiment, it can also optionally comprise one or two overhangs and/or one 30 or two caps. The RNAi agent is used to induce RNA interference of Beta-ENaC.

[00154] Target and complementary sequences [00155] The RNAi agents of the present disclosure target (e.g., specifically bind to, anneal to, etc.) the mRNA encoding the gene Beta-ENaC. The use of the RNAi agent 35 specific to Beta-ENaC results in a decrease of Beta-ENaC activity, level and/or expression, e.g., a “knock-down” or “knock-out” of the target gene or target sequence. Particularly in one embodiment, in the case of a disease state characterized by over-expression or hyperactivity of Beta-ENaC, administration of a RNAi agent to Beta-ENaC knocks down the Beta- 28 2015221515 03 Sep 2015 ENaC gene enough to restore a normal level of Beta-ENaC activity and/or a normal level of Na+ reabsorption, [00156] As used herein, “target sequence” or “target gene” refer to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a gene, 5 e.g., a Beta-ENaC gene, including mRNA that is a product of RNA processing of a primary transcription product. The target portion of the sequence will be at least Song enough to serve as a substrate for iRNA-directed cleavage at or near that portion. For example, the target sequence will generally be from 9-36 nucleotides (“nt”) in length, e.g., 15-30 nt in length, including all sub-ranges therebetween. As non-limiting examples, the target sequence can be 10 from 15-30 nt, 15-26 nt, 15-23 nt, 15-22 nt, 15-21 nt, 15-20 nt, 15-19 nt, 15-18 nt, 15-17 nt, 18-30 nt, 18-26 nt, 18-23 nt, 18-22 nt, 18-21 nt, 18-20 nt, 19-30 nt, 19-26 nt, 19-23 nt, 19-22 nt, 19-21 nt, 19-20 nt, 19 nt, 20-30 nt, 20-26 nt, 20-25 nt, 20-24 nt, 20-23 nt, 20-22 nt, 20-21 nt, 20 nt, 21-30 nt, 21-26 nt, 21-25 nt, 21-24 nt, 21-23 nt, or 21-22 nt, 21 nt, 22 nt, or 23 nt. The sense and antisense strands of the RNAi comprise a sequence complementary to that of 15 the target nucleic acid, Beta-ENaC.

[00157] As used herein, and unless otherwise indicated, the term “complementary” refers to the ability of an oligonucleotide or polynucleotide comprising a first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising a second nucleotide sequence. Such conditions can, for example, 20 be stringent, e.g., 400 mM NaCI, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides. 25 [00158] “Complementary” sequences, as used herein, may also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs includes, but are not limited to, G:U Wobble or Hoogstein base pairing. 30 [00159] The terms “complementary,” “fully complementary” and “substantially complementary” herein may furthermore be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of a RNAi agent and a target sequence, as will be understood from the context of their use.

[00160] As used herein, a polynucleotide that is “substantially complementary to at least 35 part of a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding Beta-ENaC). For example, a polynucleotide is complementary to at least a part of a Beta- 29 2015221515 03 Sep 2015 ENaC mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding Beta-ENaC.

[00161] Complementary sequences within a RNAi agent, e.g., within a dsRNA as described herein, include base-paired oligonucleotides or polynucleotides comprising a first 5 nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they may form one or 10 more, but generally not more than 5, 4, 3 or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression via a RISC pathway. However, where two oligonucleotides are designed to form, upon hybridization, one or more single-stranded overhangs, such overhangs shall not be regarded as mismatches with 15 regard to the determination of complementarity. For example, a duplex comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as “fully complementary” for the purposes described herein. The term overhang describes an 20 unpaired nucleotide at the 3' or 5' end of a double-stranded nucleotide duplex, as described above. In one embodiment, the overhang is 0 to 4 nt long and is on the 3’ end.

[00162] Thus, the RNAi agent of the present disclosure is complimentary or substantially complimentary to a target sequence in the target Beta-ENaC and is double-stranded, comprising a sense and an antisense strand (which can be contiguous, linked via a loop, or 25 otherwise joined), where the double-stranded region an be 9 to 36 bp long (particularly for example, 19-22 bp or 19-23 bp long), and can furthermore optionally comprise a 3’ or 5’ overhang, and the RNAi agent can furthermore comprise a 3’ cap. The RNAi agent mediates RNA interference, down-regulating or inhibiting the level, expression and/or activity of Beta-ENaC, and/or establishing or re-establishing an approximately normal level of ENaC and/or 30 Beta-ENaC activity, or other biological function related to ENaC.

[00163] RNAi Agents Lowering Beta-ENaC Level, Expression And/Or Activity [00164] RNAi agents for targeting Beta-ENaC include those which bind to a Beta-ENaC sequence provided herein and which work to reduce Beta-ENaC through a RNAi 35 mechanism. Exemplary siRNAs to Beta-ENaC are provided, e.g., in Table 1.

[00165] The RNAi agents of the present disclosure silence, inhibit the expression of, down-regulate the expression of, and/or suppress the expression of the Beta-ENaC gene, 30 2015221515 03 Sep 2015 such that an approximately normal level of Beta-ENaC activity, expression and/or level and/or Na+ reabsorption is achieved.

[00166] In addition, in various embodiments, depending on the disease condition and biological context, it is acceptable to use the RNAi agents of the present disclosure to 5 establish a level of Beta-ENaC expression, activity and/or level which is below the normal level, or above the normal level.

[00167] Any method known in the art can be use to measure changes in Beta-ENaC activity, level and/or expression induced by a Beta-ENaC siRNA. Measurements can be performed at multiple timepoints, prior to, during and after administration of the siRNA, to 10 determine the effect of the siRNA.

[00168] The terms “silence,” “inhibit the expression of,” “down-regulate the expression of,” "suppress the expression of,” and the like, in so far as they refer to a Beta-ENaC gene, herein refer to the at least partial suppression of the expression of a Beta-ENaC gene, as manifested by a reduction of the amount of Beta-ENaC mRNA which may be isolated from or 15 detected in a first cell or group of cells in which a Beta-ENaC gene is transcribed and which has or have been treated such that the expression of a Beta-ENaC gene is inhibited, as compared to a second ceil or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells). The degree of inhibition is usually expressed in terms of (mRNA in control cells) - (mRNA in treated cells) --------------------------:— ..................................................— ® 100% 20 (mRNAin control cells) Equation 1 [00169] Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to Beta-ENaC gene expression, e.g., the amount of protein encoded by a Beta-ENaC gene, alteration in lung fluid levels or mucus levels, etc. In 25 principle, Beta-ENaC gene silencing may be determined in any cell expressing Beta-ENaC, either constitutiveiy or by genomic engineering, and by any appropriate assay. However, when a reference or control is needed in order to determine whether a given RNAi agent inhibits the expression of the Beta-ENaC gene by a certain degree and therefore is encompassed by the instant disclosure, the assays provided in the Examples below shall 30 serve as such reference.

[00170] For example, in certain instances, expression of a Beta-ENaC gene is suppressed by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of a RNAi agent featured in the present disclosure, in some embodiments, a Beta-ENaC gene is suppressed by at least about 60%, 70%, or 80% by administration of a 35 RNAi agent featured in the present disclosure. In some embodiments, a Beta-ENaC gene is suppressed by at least about 85%, 90%, or 95% or more by administration of a RNAi agent, as described herein. 31 2015221515 03 Sep 2015 [00171] The ability of a RNAi agent to suppress Beta-ENaC can be first tested in vitro (e.g,, using test cells such as H441).

[00172] RNAi agents which can suppress Beta-ENaC in vitro can then be tested for immunostimulation using, for example, a PBMC (peripheral blood mononuclear cell) assay. 5 RNAi agents can also be tested in animal tests. Test and control animals include those which over-express or under-express Beta-ENaC, as described in, for example, Hummer et al. 2005 J. Am. Soc. Nephrol. 16: 3160-3166; Randrianarison et ai. 2007 Am. J. Physiol,

Lung Cell. Mol. Physiol. 294: 409-416; Cao et ai. 2006 Am. J. Physiol. Renal Physiol., and references cited therein. RNAi agents which suppress or alter the level, activity and/or 10 expression of Beta-ENaC can be used in medicaments to treat various Beta-ENaC-reSated diseases.

[00173] By “lower” in the context of Beta-ENaC or a symptom of a Beta-ENaC-reiated disease is meant a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more. If, for a particular 15 disease, or for an individual suffering from a particular disease, the levels or expression of Beta-ENaC are elevated, treatment with a Beta-ENaC RNAi agent of the present disclosure can particularly reduce the level or expression of Beta-ENaC to a level considered in the literature as within the range of normal for an individual without such disorder. The level or expression of Beta-ENaC can be measured by evaluation of mRNA (e.g., via Northern blots

20 or PCR), or protein (e.g., Western blots). The effect of a RNAi agent on Beta-ENaC expression can be determined by measuring Beta-ENaC gene transcription rates (e.g., via Northern blots; or reverse transcriptase polymerase chain reaction or real-time polymerase chain reaction). RT-PCR has been used to show that mRNA levels of Beta-ENaC are high in kidney, pancreas and prostate, and medium in liver and spleen. Brauner-Osbome et al. 25 2001. Biochim. Biophys. Acta 1518: 237-248. Direct measurements can be made of levels of Beta-ENaC (which is expressed by the ceil surface), e.g. by Western blots of tissues in which Beta-ENaC is expressed.

[00174] As used herein, “down-regulates” refers to any statistically significant decrease in a biological activity and/or expression of Beta-ENaC, including full blocking of the activity 30 (i.e., complete inhibition) and/or expression. For example, “down-regulation” can refer to a decrease of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 % in Beta-ENaC level, activity and/or expression.

[00175] As used herein, the term "inhibit” or "inhibiting” Beta-ENaC refers to any statistically significant decrease in biological level, activity and/or expression of Beta-ENaC, 35 including full blocking of the activity and/or expression. For example, “inhibition” can refer to a decrease of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 % in Beta-ENaC level, activity and/or expression. As used herein, the term “inhibit” similarly refers to a significant 32 2015221515 03 Sep 2015 decrease in level, activity and/or expression, while referring to any other biological agent or composition.

[00176] By “level”, it is meant that the Beta-ENaC RNAi agent can alter the level of Beta-ENaC, e.g., the level of Beta-ENaC mRNA or the level of Beta-ENaC protein, or the level of 5 activity of Beta-ENaC.

[00177] Some diseases, such as cystic fibrosis, are characterized by excessive ENaC-mediated Na+ absorption. Particularly in one embodiment, in the case of a disease characterized by over-expression and/or hyper-activity of Beta-ENaC, administration of a RNAi agent to Beta-ENaC reduces the level, expression and/or activity of Beta-ENaC. 10 However, excessively low levels of Beta-ENaC can also lead to impairment of lung fluid clearance and renal dysfunction. Randrianarison et a!. 2007 Am. J. Physiol. Lung Cell. Mol. Physiol. 294: 409-416. Thus, in various embodiments, administration of a RNAi agent to Beta-ENaC particularly establishes or re-establishes a normal or approximately normal level of Beta-ENaC activity, expression and/or level. 15 [00178] By “normal” or “approximately normal” in terms of level, expression and/or activity, is meant at least: about 50%, about 60%, about 70%, about 80%, about 90%, and/or about 100%; and/or no more than: about 100%, about 120%, about 130%, about 140%, or about 150% of the level, expression or activity of Beta-ENaC in a healthy cell, tissue, or organ. This can be measured using, for example, lung or kidney homogenates, as described 20 in Gambling et al. 2004 Kidney Inti. 65: 1774-1781. Particularly in one embodiment, administration of the appropriate amount of the appropriate Beta-ENaC RNAi agent restores Beta-ENaC level, activity and/or expression and/or Na+ reabsorption levels to about 50% to about 150%, more particularly about 60% to about 140%, more particularly to about 70% to about 130%, more particularly to about 80% to about 120%, more particularly to about 90% 25 to about 110%, and most particularly to about 100% of that of a healthy cell, tissue or organ. The level of Beta-ENaC activity can also be indirectly measured by lung fluid balance. Lung fluid balance can be estimated by calculating bloodless, wet-to-dry lung weight ratios, which reflect the amount of extra-vascular lung water. Randrianarison et al. 2007 Am. J. Physiol. Lung Celi. Mol. Physioi. 294: 409-416. The level of Beta-ENaC activity can aiso be indirectly 30 measured by histological studies of the lung, particularly the bronchioles, alveolar ducts, alveolar epithelium, and blood vessels. Randrianarison et al. 2007; and Zhou et al. 2008 Am. J. Resp. Crit. Care Med. 178: 1245-1256. Administration of a Beta-ENaC RNAi to a patient with a Beta-ENaC-reiated disease thus particularly restores the ievel, activity, and/or expression of Beta-ENaC and the level of Na+ reabsorption to an approximately normal level, 35 as determined by direct measurements of Beta-ENaC mRNA or protein levels, or indirect determinations, such as analyses of histological samples or levels of lung fluid. 33 2015221515 03 Sep 2015 [00179] !n addition, in various embodiments, depending on the disease condition and biological context, it is acceptable to use the RNAi agents of the present disclosure to establish a level of Beta-ENaC expression, activity and/or level which is below the normal level, or above the normal level. 5 [00180] Various factors are known to alter the level of ENaC or, specifically, Beta-ENaC. Hormones that increase the physiological activity of ENaC include aldosterone, vasopressin and insulin. Beta-ENaC is specifically up-regulated by vasopressin and water restriction, as well as during sodium-bicarbonate loading in rats. These various factors can be used as controls in determining the effect of a RNAi agent on Beta-ENaC level. 10 [00181] Types of RNAi Agents and Modification Thereof [00182] The use of RNAi agents or compositions comprising an antisense nucleic acid to down-modulate the expression of a particular protein in a cell is well known in the art. A RNAi agent comprises a sequence complementary to, and is capable of hydrogen binding to, the 15 coding strand of another nucleic acid (e.g., an mRNA). Thus, in various embodiments, the RNAi agents of the present disclosure encompass any RNAi agents which target (e.g., are complementary, capable of hydrogen binding to, etc.) any sequence presented, e.g., in Table 1.

[00183] Antisense sequences complementary to an mRNA can be complementary to the 20 coding region, the 5' or 3' untranslated region of the mRNA, and/or a region bridging the coding and untranslated regions, and/or portions thereof. Furthermore, a RNAi agent or a portion thereof can be complementary to a regulatory region of the gene encoding the mRNA, for instance a transcription or translation initiation sequence or regulatory element. Particularly, a RNAi agent or a portion thereof can be complementary to a region preceding 25 or spanning the initiation codon on the coding strand or in the 3' untranslated region of an mRNA.

[00184] RNAi agent molecules can be designed according to the rules of Watson and Crick base pairing. The RNAi agent can be complementary to the entire coding region of Beta-ENaC mRNA, but more particularly is an oligonucleotide which is antisense to only a 30 portion of the coding or non-coding region of Beta-ENaC mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of Beta-ENaC mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45 or 50 nucleotides in length.

[00185] The RNAi agent may have modifications internally, or at one or both ends. The 35 modifications at the ends can help stabilize the RNAi agent, protecting it from degradation by nucleases in the blood. The RNAi agents may optionally be directed to regions of the Beta-ENaC mRNA known or predicted to be near or at splice sites of the gene; e.g., exon-intron junctions (as described in, for example, Saxena et ai. 1998). 34 2015221515 03 Sep 2015 [00186] The RNAi agents can also optionally be designed to annea! to known or predicted exposed and/or single-stranded regions of the mRNA (e.g., loops), [00187] A RNAi agent can be constructed using chemicai synthesis and enzymatic ligation reactions using procedures known in the art. For example, RNAi agent can be 5 chemically synthesized using naturally-occurring nucleotides or variously modified nucleotides designed to decrease off-target effects, and/or increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g,, phosphorothioate derivatives and acridine substituted nucleotides can be used. 10 [00188] “G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as a base, respectively. However, the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide or a surrogate replacement moiety. The skilled person is weli aware that guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing 15 properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of dsRNA featured in the present disclosure by a nucleotide containing, for example, inosine. in another example, 20 adenine and cytosine anywhere in the oligonucieotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the present disclosure.

[00189] The skilled artisan will recognize that the term “RNA molecule” or “ribonucleic 25 acid molecule” encompasses not oniy RNA molecules as expressed or found in nature (i.e., are naturally occurring), but also non-naturaliy occurring analogs and derivatives of RNA comprising one or more ribonucleotide/ribonucleoside analogs or derivatives as described herein or as known in the art. Strictly speaking, a “ribonucleoside” includes a nucleoside base and a ribose sugar, and a “ribonucleotide” is a ribonucieoside with one, two or three 30 phosphate moieties. However, the terms “ribonucleoside” and “ribonucleotide” can be considered to be equivalent as used herein. The RNA can be modified in the nucieobase structure or in the ribose-phosphate backbone structure, e.g., as described herein below. However, the molecules comprising ribonucleoside analogs or derivatives must retain the ability to form a duplex. As non-limiting examples, an RNA molecule can also include at least 35 one modified ribonucleoside, including but not limited to a 2'-0-methyl modified nucleotide, a nucleoside comprising a 5' phosphorothioate group, a terminal nucleoside linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, a locked nucleoside, an 35 2015221515 03 Sep 2015 abasic nucleoside, a 2'-deoxy-2’-fiuoro modified nucleoside, a 2'-amino-modified nucleoside, 2’-alkyl-modified nucleoside, morpholine nucleoside, an unlocked ribonucleotide (e.g., an acyclic nucleotide monomer, as described in WO 2008/147824), a phosphoramidate or a non-natural base comprising nucleoside, or any combination thereof. Alternatively, an RNA 5 molecule can comprise at least two modified ribonucleosides, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or more, up to the entire length of the dsRNA molecule. The modifications need not be the same for each of such a plurality of modified ribonucleosides in an RNA molecule. In one embodiment, modified RNAs contemplated for use in methods and compositions described herein are 10 peptide nucleic acids (RNAs) that have the ability to form the required duplex structure and that permit or mediate the specific degradation of a target RNA via a RISC pathway.

[00190] Examples of modified nucleotides which can be used to generate the RNAi agent include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetyicytosine, 5~(carboxyhydroxylmethy!) uracil, 5-carboxymethylaminomethyi-2-thiouridine, 15 5-carboxymethyiaminomethyluraciI, dihydrouracii, beta-D-gaiactosylqueosine, inosine, N6-isopentenyiadenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methyiguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracii, 5-methoxyuracil, 2-methylthio-N6-20 isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- oxyacetic acid methylester, uracii-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.

[00191] In one embodiment, the present disclosure encompasses modified any modified 25 variant of any RNAi agent disclosed herein. The modified variant contains the same sequence, but can be modified to contain modifications in the phosphate, sugar, base, nucleotide, etc. For example, the modified variant can contain one or more of the modified nucleotides listed herein, for example a C replaced by a 2’-modified C.

[00192] In one aspect, a modified ribonucleoside includes a deoxyribonucleoside. In such 30 an instance, a RNAi agent can comprise one or more deoxynucleosides, including, for example, a deoxynucleoside overhang(s), or one or more deoxynucleosides within the double-stranded portion of a dsRNA. However, it is self-evident that under no circumstances is a double-stranded DNA molecule encompassed by the term “RNAi agent.” [00193] Replacing the 3'-terminal nucleotide overhanging segments of a 21-mer siRNA 35 duplex having two-nucleotide 3'-overhangs with deoxyribonucleotides does not have an adverse effect on RNAi activity. Replacing up to four nucleotides on each end of the siRNA with deoxyribonucleotides has been well tolerated, whereas complete substitution with 36 2015221515 03 Sep 2015 deoxyribonucleotides results in no RNAi activity. International PCI Publication No. WO 00/44914, and Beach et al. International PCT Publication No. WO 01/68836 preliminarily suggest that siRNA may include modifications to either the phosphate-sugar backbone or the nucleoside to include at least one of a nitrogen or sulfur heteroatom. Kreutzer et al. Canadian 5 Patent Application No. 2,359,180, also describe certain chemical modifications for use in dsRNA constructs in order to counteract activation of double-stranded RNA-dependent protein kinase PKR, specifically 2'-amino or 2'-0-methyl nucleotides, and nucleotides containing a 2'-0 or 4'-C methylene bridge. Additional 3’-terminai nucleotide overhangs include dT (deoxythimidine), 2’-0,4’-C-ethylene thymidine (eT), and 2-hydroxyethyl 10 phosphate (hp).

[00194] Parrish et al. 2000 Molecular Cell 6: 1077-1087 tested certain chemical modifications targeting the unc-22 gene in C. elegans using long (>25 nt) siRNA transcripts. The authors describe the introduction of thiophosphate residues into these siRNA transcripts by incorporating thiophosphate nucleotide analogs with T7 and T3 RNA polymerase and 15 observed that RNAs with two phosphorothioate modified bases also had substantial decreases in effectiveness as RNAi. Further, Parrish etai. reported that phosphorothioate modification of more than two residues greatly destabilized the RNAs in vitro such that interference activities could not be assayed. Id. at 1081. The authors also tested certain modifications at the 2'-position of the nucleotide sugar in the long siRNA transcripts and 20 found that substituting deoxynucleotides for ribonucieotides produced a substantial decrease in interference activity, especially in the case of Uridine to Thymidine and/or Cytidine to deoxy-Cytidine substitutions. Id. In addition, the authors tested certain base modifications, including substituting, in sense and antisense strands of the siRNA, 4-thiouracil, 5-bromouracil, 5-iodouracii, and 3-(aminoa!lyl) uracil for uracii, and inosine for guanosine. 25 Whereas 4-thiouracil and 5-bromouracil substitution appeared to be tolerated, Parrish reported that inosine produced a substantial decrease in interference activity when incorporated in either strand. Parrish also reported that incorporation of 5-iodouracil and 3-(aminoaiiyl)uracil in the antisense strand resulted in a substantial decrease in RNAi activity as well. 30 [00195] Those skilled in the art will appreciate that it is possible to synthesize and modify the siRNA as desired, using any conventional method known in the art (see Henschel et al. 2004 DEQOR: a web-based tool for the design and quality control of siRNAs. Nucleic Acids Research 32 (Web Server Issue): W113-W12G). Further, it will be apparent to those skilled in the art that there are a variety of regulatory sequences (for example, constitutive or inducible 35 promoters, tissue- specific promoters or functional fragments thereof, etc.) which are useful for the antisense oligonucleotide, siRNA, or shRNA expression construct/vector. 37 2015221515 03 Sep 2015 [00196] There are several examples in the art describing sugar, base, phosphate and backbone modifications that can be introduced into nucleic acid molecules with significant enhancement in their nuclease stability and efficacy. For example, oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease 5 resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-0-methyi, 2-O-allyl, 2'-H, nucleotide base modifications (for a review see Usman and Cedergren 1992 TIBS. 17: 34; Usman etal. 1994 Nucleic Acids Symp. Ser. 31: 163; Burgin etal. 1996 Biochemistry 35: 14090). Sugar modification of nucleic acid molecules are extensively described in the art.

[00197] Additional modifications and conjugations of RNAi agents have been described. 10 Soutschek et al. 2004 Nature 432: 173-178 presented conjugation of cholesterol to the 3’- end of the sense strand of a siRNA molecule by means of a pyrrolidine linker, thereby generating a covalent and irreversible conjugate. Chemical modifications (including conjugation with other molecules) of RNAi agents may also be made to improve the in vivo pharmacokinetic retention time and efficiency. 15 [00198] In various embodiments, the RNAi agent to Beta-ENaC comprises at least one 5’-uridine-adenine-3’ (5’-ua-3’) dinucleotide, wherein the uridine is a 2’-modified nucleotide; at least one 5’-uridine-guanine-3! (5’-ug-35) dinucleotide, wherein the S’-uridine is a 2’-modified nucleotide; at least one 5’-cytidine-adenine-3’ (5’-ca-3’) dinucleotide, wherein the 5’-cytidine is a 2’-modified nucleotide; and/or at least one 5’-uridine-uridine-3’ (5’-uu-3!) dinucleotide, 20 wherein the 5’-uridine is a 2’-modified nucleotide.

[00199] In various embodiments, the RNAi agent comprises a 2'-modification selected from the group consisting of: 2'-deoxy, 2’-deoxy-2’-fluoro, 2’-0-methyl, 2'-0-methoxyethyl (2’-O-MOE), 2'-0-aminopropyl (2'-0-AP), 2'-0-dimethyiaminoethyl (2'-0-DMAOE), 2'-0-dimethylaminopropyi (2'-0-DMAP), 2'-0-dimethylaminoethyloxyethyl (2'-0-DMAEOE), and 2'- 25 O-N-methylacetamido (2-O-NMA).

[00200] In another embodiment, the RNAi comprises a gap or missing base. For example, the phosphate-sugar backbone may be present, but the base missing.

[00201] In another embodiment, the RNAi agent has a single-stranded nick (e.g., a break or missing bond in the backbone). In various embodiments, a single-stranded nick can be in 30 either the sense or anti-sense strand, or both.

[00202] This nick can be, for example, in the sense strand, producing a small internally segmented interfering RNA, or sisiRNA, which may have less off-target effects than the corresponding RNAi agent without a nick.

[00203] The antisense nucleic acid or RNAi agent can also have an alternative backbone 35 such as locked nucleic acids (LNA), Morpholinos, peptidic nucleic acids (PNA), threose nucleic acid (TNA), or glycol nucleic acid (GNA), and/or it can be labeled (e.g., radiolabeled or otherwise tagged). 38 2015221515 03 Sep 2015 [00204] One or both strands can comprise an alternative backbone [00205] In yet another embodiment, the RNAi agent employed by the methods of the present disclosure can include an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, 5 contrary to the usual β-units, the strands run parallel to each other. Gaultier et al. 1987 Nucleic Acids. Res. 15: 6625-8641.

[00206] The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucieotide (Inoue etal. 1987 Nucleic Acids Res. 15: 6131-6148) ora chimeric RNA-DNA analogue (inoue et al. 1987 FEBS Lett. 215: 327-330). 10 [00207] In still another embodiment, a RNAi agent is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes [e.g., hammerhead ribozymes (described in Haselhoff et al. 1988, Nature 334: 585-591)] can be used to catalyticaliy cleave Beta-ENaC mRNA transcripts to thereby inhibit 15 translation of Beta-ENaC mRNA.

[00208] Alternatively, gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of Beta-ENaC (e.g., the promoter and/or enhancers) to form triple helical structures that prevent transcription of the Beta-ENaC gene. See generally, Helene 1991 Anticancer Drug Des. 8(8): 569-84; Helene et ai. 1992 Ann. N.Y. 20 Acad. Sci. 660: 27-36; and Maher 1992, Bioassays 14(12): 807-15.

[00209] Production of RNAi Agents [00210] The RNAi agent can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed 25 from the inserted nucleic acid will be in an antisense orientation to a target nucleic acid of interest). The RNAi agent can also be produced biologically using an expression vector into which a nucleic acid has been subcloned as an shRNA construct (i.e., RNA transcribed from the inserted nucleic acid will have a first region in an antisense orientation to a target nucleic acid of interest, a second region that comprises a loop or hinge, and a third region in a sense 30 orientation to the target nucleic acid of interest, wherein the first and third regions of the transcript preferably hybridizes with itself, thereby forming a stem-and-loop structure).

[00211] Methods of producing RNAi agents are well-known in the art and available to persons of ordinary skill in the art.

[00212] Kits for synthesis of RNAi are commercially available from, e.g., New England 35 Biolabs and Ambion. 39 2015221515 03 Sep 2015 [00213] Delivery of RNAi Agents [00214] RNAi agents of the present disclosure can be delivered or introduced (e.g., to a cell in vitro, to a test animal, or to a human) by any means known in the art.

[00215] The RNAi agents of the present disclosure are typically administered to a subject 5 or generated in situ such that they hybridize with cellular mRNA and/or genomic DNA encoding Beta-ENaC, and inhibit expression by inhibiting transcription and/or translation. An example of a route of administration of the RNAi agent includes direct injection at a tissue site. Alternatively, RNAi agents can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules 10 can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using vectors well known in the art and described in, for example, US20070111230, the entire contents of which are incorporated herein. To 15 achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.

[00216] “Introducing into a cell,” when referring to a RNAi agent, means facilitating or effecting uptake or absorption into the cell, as is understood by those skilled in the art. 20 Absorption or uptake of a RNAi agent can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; a RNAi agent may also be "introduced into a cell,” wherein the cell is part of a living organism, in such an instance, introduction into the cell will include the delivery to the organism. For example, for in vivo delivery, a RNAi agent can be injected into a tissue site or 25 administered systemically. In vivo delivery can also be by a beta-giucan delivery system, such as those described in U.S. Patent Nos. 5,032,401 and 5,607,677, and U.S. Publication No. 2005/0281781. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein or known in the art.

[00217] Delivery of RNAi agent to tissue is a problem both because the material must 30 reach the target organ and must also enter the cytoplasm of target cells. RNA cannot

penetrate cellular membranes, so systemic delivery of naked RNAi agent is unlikely to be successful. RNA is quickly degraded by RNAse activity in serum. For these reasons, other mechanisms to deliver RNAi agent to target cells has been devised. Methods known in the art include but are not limited to: viral delivery (retrovirus, adenovirus, lentivirus, bacuiovirus, 35 AAV); liposomes (Lipofectamine, cationic DOTAP, neutral DOPC) or nanoparticles (cationic polymer, PEI), bacterial delivery (tkRNAi), and also chemical modification (LNA) of siRNA to improve stability. Xia et al. 2002 Nat. Biotechnol. 20 and Devroe et ai. 2002. BMC 40 2015221515 03 Sep 2015

Biotechnol. 2 1:15, disclose incorporation of siRNA into a viral vector. Other systems for delivery of RNAi agents are contemplated and the RNAi agents of the present disclosure can be delivered by various methods yet to be found and/or approved by the FDA or other regulatory authorities. RNAi agents of the present disclosure can delivered in a suitable 5 pharmaceutical composition.

[00218] Pharmaceutical Compositions of RNAi Agents [00219] As used here, a “pharmaceutical composition” comprises a pharmaceutically effective amount of one or more Beta-ENaC RNAi agent, a pharmaceutically acceptable 10 carrier, and, optionally, an additional disease treatment which works synergistically with the RNAi agent. As used herein, “pharmacologically effective amount,” “therapeutically effective amount” or simply “effective amount” refers to that amount of a RNAi agent effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective where there is at least a 10% reduction in a 15 measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 10% reduction in that parameter, in this embodiment, a therapeutically effective amount of a RNAi agent targeting Beta-ENaC can reduce Beta-ENaC protein ievels by at least 10%. in additional embodiments, a given clinical treatment is considered effective 20 where there is at least a 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% reduction in a measurable parameter associated with a disease or disorder, and the therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% reduction, respectively, in that parameter. 25 [00220] The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients 30 such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, 35 stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract. Agents included in drug formulations are described further herein. 41 2015221515 03 Sep 2015 [00221] The pharmaceutical compositions comprising a Beta-ENaC RNAi agent can be in solid form, for example, powders, granules, tablets, pills, gelcaps, gelatin capsules, liposomes, suppositories, chewabie forms, or patches. The pharmaceutical compositions comprising a Beta-ENaC RNAi agent can also be presented in liquid form, for example, 5 solutions, emulsions, suspensions, elixirs, or syrups. Appropriate liquid supports can be, for example, water, organic solvents such as polyol, such as glycerol or glycols, including propylene glycol and polyethylene glycol, or ethanol, Cremophor EL, or mixtures thereof, in varying proportions, in water. The compositions can comprise nano-sized amorphous or crystalline granules coated with albumin or a surfactant. 10 [00222] Appropriate supports can include, for example, antibacterial and antifungal agents, buffering agents, calcium phosphate, cellulose, methyl cellulose, chlorobutanol, cocoa butter, colorings, dextrin, emulsifiers, enteric coatings, flavorings, gelatin, isotonic agents, lecithin, magnesium stearate, perfuming agents, polyalcohols such as mannitol, injectable organic esters such as ethyl oleate, paraben, phenol sorbic acid, polyethylene 15 glycol, poiyvinylpyrrolidine, phosphate buffered saline (PBS), preserving agents, propylene glycol, sodium carboxymethylcellulose, sodium chloride, sorbitol, various sugars (including, but not limited to, sucrose, fructose, galactose, lactose and trehalose), starch, suppository wax, talc, vegetable oils, such as olive oil and corn oil, vitamins, wax, and/or wetting agents. For Beta-ENaC RNAi agents, a preferred support comprises dextran and water, e.g. 5% 20 dextrose in water (D5W).

[00223] The biologically inert portion of the pharmaceutical composition can optionally be erodible, allowing timed release of the RNAi agent.

[00224] The pharmaceutical composition can comprise additional components which aid in delivery, stability, efficacy, or reduction of immunogenicity. 25

[00225] Pharmaceutical Composition Comprising a RNAi Agent to Beta-ENaC

[00226] Additional components of a pharmaceutical composition comprising a RNAi Agent to Beta-ENaC can be added to aid in delivery, stability, efficacy, or reduction of immunogenicity. 30 [00227] Liposomes have been used previously for drug delivery (e.g., delivery of a chemotherapeutic). Liposomes (e.g., cationic liposomes) are described in PCT publications WO02/100435A1, W003/015757A1, and WO04029213A2; U.S. Pat. Nos. 5,962,016; 5,030,453; and 6,680,068; and U.S. Patent Application 2004/0208921. A process of making liposomes is also described in W004/002453A1. Furthermore, neutral lipids have been 35 incorporated into cationic liposomes (e.g., Farhood et ai. 1995).

[00228] Cationic liposomes have been used to deliver RNAi agent to various cell types (Sioud and Sorensen 2003; U.S. Patent Application 2004/0204377; Duxbury et ai., 2004; Donze and Picard, 2002), 42 2015221515 03 Sep 2015 [00229] Use of neutral liposomes disclosed in Miller et a!. 1998, and U.S. Patent Application 2003/0012812.

[00230] As used herein, the term "SNALP" refers to a stable nucleic acid-lipid particle. A SNALP represents a vesicle of lipids coating a reduced aqueous interior comprising a nucleic 5 acid such as an iRNA or a plasmid from which an IRMA is transcribed. SNALPs are described, e.g., in U.S, Patent Application Publication Nos. 20080240093, 20070135372, and in International Application No. WO 2009082817.

[00231] Chemical transfection using lipid-based, amine-based and polymer-based techniques, is disclosed in products from Ambion Inc., Austin, Tex.; and Novagen, EMD 10 Biosciences, Inc, an Affiliate of Merck KGaA, Darmstadt, Germany); Ovcharenko D (2003) “Efficient delivery of siRNAs to human primary cells,” Ambion Tech Notes 10 (5): 15-16). Additionally, Song et ai. (Nat Med. published online (Fete I 0, 2003) doi: 10.1038/nm828) and others [Caplen et al. 2001 Proc. Natl. Acad. Sci. (USA), 98: 9742-9747; and McCaffrey et al. Nature 414: 34-39] disclose that liver cells can be efficiently transfected by injection of the 15 siRNA into a mammal's circulatory system.

[00232] A variety of molecules have been used for cell-specific RNAi agent delivery. For example, the nucleic acid-condensing property of protamine has been combined with specific antibodies to deliver siRNAs. Song et al. 2005 Nat Biotech. 23: 709-717. The self-assembly PEGylated polycation polyethylenimine (PEI) has also been used to condense and protect 20 siRNAs. Schiffelers et al. 2004 Nucl. Acids Res. 32: el49, 141-1 10.

[00233] The siRNA-containing nanoparticles were then successfully delivered to integrin-overexpressing tumor neovasculature. Hu-Lieskovan et al. 2005 Cancer Res. 65: 8984-8992.

[00234] The RNAi agents of the present disclosure can be delivered via, for example,

Lipid nanoparticles (LNP); neutral liposomes (NL); polymer nanoparticles; double-stranded 25 RNA binding motifs (dsRBMs); or via modification of the RNAi agent (e.g., covalent attachment to the dsRNA).

[00235] Lipid nanoparticles (LNP) are self-assembling cationic lipid based systems.

These can comprise, for example, a neutral lipid (the liposome base); a cationic lipid (for siRNA loading); cholesterol (for stabilizing the liposomes); and PEG-lipid (for stabilizing the 30 formulation, charge shielding and extended circulation in the bloodstream).

[00236] The cationic lipid can comprise, for example, a headgroup, a linker, a tail and a cholesterol tail. The LNP can have, for example, good tumor delivery, extended circulation in the blood, small particles (e.g., less than 100 nm), and stability in the tumor microenvironment (which has low pH and is hypoxic). 35 [00237] Neutral liposomes (NL) are non-cationic lipid based particles.

[00238] Polymer nanoparticles are self-assembling polymer-based particles. 43 2015221515 03 Sep 2015 [00239] Double-stranded RNA binding motifs (dsRBMs) are self-assembling RNA binding proteins, which will need modifications.

[00240] In various embodiments, the RNAi agent to Beta-ENaC is ligated to one or more diagnostic compound, reporter group, cross-linking agent, nuclease-resistance conferring 5 moiety, natural or unusual nucleobase, lipophilic molecule, cholesterol, lipid, lectin, steroid, uvaol, hecigenin, diosgenin, terpene, triterpene, sarsasapogenin, Friedelin, epifriedelanol-derivatized lithocholic acid, vitamin, carbohydrate, dextran, puilulan, chitin, chitosan, synthetic carbohydrate, oligo lactate 15-mer, natural polymer, low- or medium-molecular weight polymer, inulin, cyciodextrin, hyaluronic acid, protein, protein-binding agent, integrin-10 targeting molecule, polycationic, peptide, polyamine, peptide mimic, and/or transferrin.

[00241] The RNAi agents of the present disclosure can be prepared in a pharmaceutical composition comprising various components appropriate for the particular method of administration of the RNAi agent. 15 [00242] Administration of a RNAi agent [00243] The pharmaceutical composition comprising a Beta-ENaC can be administered by buccal, inhalation (including insufflation and deep inhalation), nasal, oral, parenteral, implant, injection or infusion via epidural, intra-arterial, intra-articular, intra-capsular, intracardiac, intra-cerebroventricular, intracranial, intradermal, intramuscular, intra-orbital, 20 intraperitoneal, intra-spinal, intrasternai, intrathecal, intravenous, subarachnoid, sub- capsular, subcutaneous, sub-cuticular, transendothelial, transtracheal, transvascular, rectal, sublingual, topical, and/or vaginai routes. This may be by injection, infusion, dermal patch, or any other method known in the art. The formulation can be powdered, nebulized, aerosolized, granulized or otherwise appropriately prepared for delivery. The administration, 25 if liquid, may be slow or via bolus, though, under some circumstances known in the art, bolus injections may lead to loss of material through the kidneys.

[00244] The pharmaceutical compositions comprising a Beta-ENaC RNAi agent can be administered with medical devices known in the art. For example, in a particular embodiment, a RNAi agent can be administered with a needle-less hypodermic injection 30 device, such as the devices disclosed in U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556. Examples of well-known implants and modules useful in the present disclosure include: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4.,486,194, which discloses a therapeutic device for administering 35 medications through the skin; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system having multi- 44 2015221515 03 Sep 2015 chamber compartments; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. Many other such implants, delivery systems, and modules are known to those skilled in the art.

[00245] In certain embodiments, the pharmaceutical compositions comprising a RNAi 5 agent can be formulated to ensure proper distribution in vivo. Administration of a RNAi agent to Beta-ENaC can be systemic (whole-body) or, particularly, targeted to tissues or organs that express (or over-express or demonstrate a hyper-activity of) Beta-ENaC, such as lung, kidney, colon, and glands. Methods for targeting these particular tissues or organs are described herein, and/or are known in the art. For example, they can be formulated in 10 liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Patents 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., V.V. Ranade (1989) J. Clin. Pharmacol. 29: 685).

[00246] Example targeting moieties include folate or biotin (see, e.g., U.S. Patent 15 5,416,016 to Low et al.); mannosides (Umezawa et a!,, (1988) Biochem. Biophys. Res.

Commun. 153: 1038); antibodies (P.G. Bloeman etai. (1995) FEBS Lett. 357: 140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39: 180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233: 134), different species of which may comprise the formulations of the present disclosures, as well as components of the invented molecules; 20 p120 (Schreier et al. (1994) J. Biol. Chem. 269: 9090); see also K. Keinanen; M.L.

Laukkanen (1994) FEBS Lett. 346: 123; J.J. Killion; I.J. Fidler (1994) Immunomethods 4: 273.

[00247] The present disclosure thus encompasses pharmaceutical compositions comprising one or more RNAi agents to Beta-ENaC, which can optionally comprise various 25 modifications and/or additional components, for use in treatment of Beta-ENaC-related diseases.

[00248] Beta-ENaC-Related Diseases [00249] The present disclosure encompasses RNAi agents to Beta-ENaC and 30 administration of the RNAi agents to humans and non-human animals to treat various Beta-ENaC-related diseases.

[00250] By “Beta-ENaC-related disease” is meant any disease related to a dysfunction in the level, expression and/or activity of Beta-ENaC, and/or any disease which can be treated and/or ameliorated by modulating the level, expression and/or activity of Beta-ENaC. In 35 particular, it includes cystic fibrosis, pseudohypoaldosteronism type 1 (PHA1), Liddie’s syndrome, hypertension, alkalosis, hypokalemia, and obesity-associated hypertension.

[00251] By “cystic fibrosis” or “CF” is meant the common hereditary disease associated with mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. 45 2015221515 03 Sep 2015 CFTR encodes a cAMP-dependent Cl- channel and regulates the ENaC. In CF airway epithelia, CFTR-mediated Cl- secretion is defective and ENaC-mediated Na+ absorption is increased. These ion transport defects in CF airways cause airway surface liquid (ASL) volume depletion, defective mucus clearance, and mucus adhesion, suggesting that ASL 5 volume depletion is a key mechanism in the pathogenesis of CF lung disease. In experimental mice, airway-specific over-expression of Beta-ENaC demonstrates that accelerated Na+ transport alone is sufficient to produce ASL volume depletion and CF-like lung disease, including airway mucus obstruction, goblet cell metaplasia, chronic neutrophilic airway inflammation, impaired clearance of bacterial pathogens, and ultimately mortality. 10 See Zhou et al. 2008, and references cited therein.

[00252] By “Liddle’s syndrome” is meant an autosomal dominant hereditary form of hypertension, characterized by an early and severe hypertension, often accompanied by metabolic alkalosis and hypokalemia, al! signs that are characteristic of an excess of aldosterone (Conn’s syndrome). 15 [00253] The plasma levels of aldosterone are low, however. Thus, Liddle’s syndrome is also called pseudoaldosteronism. This severe form of hypertension is responsive to treatment with a low-salt diet and Na+ channel inhibitors (KC-sparing diuretics), suggesting a primary defective regulation of the ENaC. The disease is related to mutations in Gamma- ENaC, and also several mutations in Beta-ENaC (P615S, P616L, and Y618H in the “PY” 20 motif which has a consensus sequence of PPXY; and also R564st, W574st, 579del32, G589st, T592fr, A593fr, and R595fr, where “fr” is a frameshift, “del” is a deletion, and “st” is a premature stop codon).

[00254] These mutations cause an overexpression of the Na+ channels that are hyperactive compared to the wild-type ENaC. The mutations also prevent the 25 downregulation of the channel that normally occurs with a rise in intracellular Na+; ENaC channels with the Liddle’s mutation remain in a highly active state despite a high intracellular Na+ concentration. Thus, the level and/or activity of a mutated ENaC with Liddie’s Syndrome can be modulated by a siRNA to Beta-ENaC, or such a siRNA in combination with known treatments for Liddle’s syndrome, such as a low-salt diet, and Na+ channel inhibitors (K+- 30 sparing diuretics).

[00255] For additional information on Beta-ENaC-reiated diseases, see, for example, Hummler et al. 1999. Am. J. Physiol. Gastrointest. Liver Physiol. 276: 567-571.

[00256] By “obesity-associated hypertension” is meant hypertension related or associated with obesity, and the like. Obesity is associated with hypertension. Multiple mechanisms 35 have been proposed to explain this correlation, including (in the obese) increased sympathetic activity; increased activity of the renin-angiotensin-aldosterone system; increased cardiac output; and increased mechanical pressure from interstitial fat around 46 2015221515 03 Sep 2015 organs, hyperinsulinemia, and/or insulin resistance. Sodium retention by the kidney could result from any of these mechanisms. In the connecting tubule and the collecting duct, sodium reabsorption occurs through the ENaC. Levels of Beta-ENaC were increased in the kidney in Zucker rats (a model animal for obesity). 5 [00257] Bickel et al. 2001 Am. J. Physiol. Renal Physiol. 281: 639-648. The relative increases in abundance of this and other sodium transporters, without decreases in the other sodium transporters, likely results in enhanced tubular sodium reabsorption. As a result, these alterations in renal sodium transporter abundance might play a role in the development and/or maintenance of elevated blood pressures in obese mammals, including humans. 10 [00258] By “pseudohypoaldosteronism type Τ’, “PHA1”, “PHA-1 ” and the like is meant a heterologous clinical syndrome characterized by mineralocorticoid end organ resistance, i.e., urinary loss of Na+ and reduced K+ excretion despite an elevated levei of aldosterone. A severe form of this syndrome is inherited as an autosomal recessive trait, resulting in sometimes lethal episodes of hyponatremia, hypotension, and hyperkalemia, and shows 15 alteration of Na+ transport in several organs, kidney, salivary glands, sweat glands, and colon. In several families showing this form of PHA-1, links to mutations in any one of the three ENaC subunits are found (including G37S in Beta-ENaC).

[00259] A less severe form of PHA-1 with an autosomal dominant mode of inheritance is symptomatic mostly during infancy and improves with age. See Hummier et al. 1999. Am, J, 20 Physiol. Gastrointest. Liver Physiol. 276: 567-571.

[00260] RNAi agents to Beta-ENaC can be used to treat Beta-ENaC-related diseases, particularly those diseases associated with altered expression, activity and/or levels of Beta-ENaC. 25 [00261] Use of RNAi Agents for Treatment of Beta-ENaC-Related Diseases [00262] The RNAi agents to Beta-ENaC described herein can be formulated into pharmaceutical compositions which can be administered to humans or non-human animals. These compositions can comprise one or more RNAi agents, and, optionally, additional treatments useful for treating Beta-ENaC-related diseases. They can be administered as part 30 of an early/preventative treatment, and can be administered in a therapeutically-effective dosage. The pharmaceutical composition can comprise a pharmaceutical carrier and can be administered by any method known in the art. These various aspects of the present disclosure are described in additional detail below.

[00263] RNAi agents to Beta-ENaC can be administered to humans and non-human 35 animals for treatment of Beta-ENaC-related diseases.

[00264] In one embodiment of the present disclosure, the compositions comprising a Beta-ENaC RNAi agent can be administered to non-human animals. For example, the compositions can be given to chickens, turkeys, livestock animals (such as sheep, pigs, 47 2015221515 03 Sep 2015 horses, cattie, etc.), companion animals (e.g., cats and dogs) and can have efficacy in treatment of cystic fibrosis, pseudohypoaldosteronism type 1 (PHA1), Liddle’s syndrome, hypertension, alkalosis, hypokalemia, and obesity-associated hypertension and similar diseases, in each case, the RNAi agent to Beta-ENaC wouid be selected to match the 5 sequence of the Beta-ENaC of the genome of the animal, and to, particularly, contain at least 1 nt mismatch from all other genes in that animal’s genome. The RNAi agents of the present disclosure can thus be used in treatment of Beta-ENaC-related diseases in humans and nonhuman animals.

[00265] As used herein in the context of Beta-ENaC expression, the terms “treat,” 10 “treatment,” and the like, refer to relief from or alleviation of pathological processes mediated by Beta-ENaC expression. In the context of the present disclosure insofar as it relates to any of the other conditions recited herein beiow (other than pathological processes mediated by Beta-ENaC expression), the terms “treat,” “treatment,” and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the 15 progression or anticipated progression of such condition, such as slowing the progression of a lipid disorder, such as atherosclerosis.

[00266] By “treatment” is also meant prophylaxis, therapy, cure, or any other change in a patient’s condition indicating improvement or absence of degradation of physical condition.

By “treatment” is meant treatment of Beta-ENaC-related disease (e.g., cystic fibrosis, 20 pseudohypoaldosteronism type 1 (PHA1), Liddle’s syndrome, hypertension, alkalosis, hypokalemia, and obesity-associated hypertension), or any appropriate treatment of any other ailment the patient has. As used herein, the terms “treatment” and “treat” refer to both prophylactic and preventative treatment and curative or disease-modifying treatment, including treatment of patients at risk of contracting a disease or suspected of having a 25 disease, as well as patients already ill or diagnosed as suffering from a condition. The terms “treatment” and “treat” also refer to the maintenance and/or promotion of health in an individual not suffering from a disease but who may be susceptible to developing an unhealthy condition, such as nitrogen imbalance or muscle loss, in one embodiment, “treatment” does not encompass prevention of a disease state. Thus, the present disclosure 30 is useful for suppressing expression of the Beta-ENaC gene and/or treating a Beta-ENaC-related disease in an individual afflicted by a Beta-ENaC-related disease, or an individual susceptible to a Beta-ENaC-related disease. An individual “afflicted” by a Beta-ENaC-related disease has demonstrated detectable symptoms characteristics of the disease, or had otherwise been shown clinically to have been exposed to or to carry Beta-ENaC-related 35 disease pathogens or markers. As non-limiting examples, an individual afflicted by a Beta-ENaC-related disease can show outward symptoms; or can show no outward symptoms but 48 2015221515 03 Sep 2015 can be shown with a clinical test to carry protein markers associated with a Beta-ENaC-reiated disease, or proteins or genetic materia! associated with a pathogen in the blood.

[00267] Early treatment of some Beta-ENaC-related diseases may be more efficacious if administered early rather than later. Preventative early administration of amiloride (an ENaC 5 inhibitor) was useful in treating CF model mice, while later administration was not. Similarly, early intervention with antimicrobial agents in CF was more effective than treatment after infection was established. Zhou et al. 2008. Thus, in one particular embodiment, the RNAi agent to Beta-ENaC is administered early, prior to disease manifestation, and/or as a preventative agent, rather than administered after disease establishment. 10 [00268] Treatments of Beta-ENaC-related diseases can comprise various treatments, comprising a Beta ENaC RNAi agent, and optionally further comprising an additional treatment, which can be a method (or procedure), or an additional composition (e.g., an agent or additional RNAi agent). 15 [00269] Dosages and Effective Amounts of RNAi Agents [00270] The RNAi agents of the present disclosure are administered in a dosage of a therapeutically effective amount to a patient in need thereof.

[00271] An "effective amount” or a "therapeutically effective amount” is an amount that treats a disease or medical condition of an individual, or, more generally, provides a 20 nutritional, physiological or medical benefit to an individual. As used herein, the phrases “therapeutically effective amount” and “prophylactically effective amount” refer to an amount that provides a therapeutic benefit in the treatment, prevention, or management of pathological processes mediated by Beta-ENaC expression or an overt symptom of pathological processes mediated by Beta-ENaC expression. The specific amount that is

25 therapeutically effective can be readily determined by an ordinary medical practitioner, and may vary depending on factors known in the art, such as, for example, the type of pathological processes mediated by Beta-ENaC expression, the patient’s history and age, the stage of pathological processes mediated by Beta-ENaC expression, and the administration of other agents that inhibit pathological processes mediated by Beta-ENaC 30 expression.

[00272] In various embodiments of the present disclosure, the patient is at least about 1, 3, 6, or 9 months, or 1, 5, 10, 20, 30, 40, 50, 55, 60, 65, 70, or 75 years of age. In various embodiments, the patient is no more than about 1, 3, 6, or 9 months, or 1, 5, 10, 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 90, or 100 years of age. In various embodiments the patient has a 35 body weight of at least about 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380 or 400 lbs. In various embodiments, the patient has a body weight of no more than about 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380 or 400 lbs. 49 2015221515 03 Sep 2015 [00273] !n various embodiments of the present disclosure, the dosage [measuring only the active ingredient(s)] can be at least about 1, 5, 10, 25, 50, 100, 200, 250, 300, 250, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 ng, 1, 5, 10, 25, 50, 100, 200, 250, 300, 250, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 5 micrograms, 1, 5, 10, 25, 50, 100, 200, 250, 300, 250, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 mg. In various embodiments, the dosage can be no more than about 10, 25, 50, 100, 200, 250, 300, 250, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 mg. in various embodiments, the dosage can be administered at least more than once a day, daily, more than once a weekly, weekly, bi-weekly, monthly, and/or 10 every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or a combination thereof.

[00274] in various embodiments, the dosage is correlated to the body weight or body surface area of the individual· The actual dosage level can be varied to obtain an amount of active agent which is effective for a particular patient, composition and mode of administration, without being toxic to the patient. The selected dose will depend on a variety 15 of pharmacokinetic factors, including the activity of the particular RNAi agent employed, the route of administration, the rate of excretion of the RNAi agent, the duration of the treatment, other drugs, compounds and/or materials used in combination with the RNAi agent, the age, sex, weight, condition, general health and prior medical history of the patient, and like factors well known in the medical arts. A physician or veterinarian having ordinary skill in the art can 20 readily determine the effective amount of the RNAi agent required. A suitable dose will be that amount which is the lowest dose effective to produce a therapeutic effect, or a dose low enough to produce a therapeutic effect without causing side effects.

[00275] In addition to a therapeuticaliy-effective dosage of one or more RNAi agents to Beta-ENaC, the pharmaceutical compositions of the present disclosure can comprise or be 25 used in conjunction with an additional disease treatment which works synergistically with the RNAi agent. For example, the pharmaceutical composition can comprise an additional antagonist to ENaC, such as potassium-sparing diuretics, amiloride and triamterene. Additional treatments can be administered along with the pharmaceutical composition, including, as a non-limiting example, regulation of dietary salt intake. When used to treat 30 cystic fibrosis, the pharmaceutical composition can be used in conjunction with various medicaments and therapies known in the art, including, but not limited to, antibiotics, DNase therapy, albutrol, N-acetylcysteine, breathing therapy, percussive therapy, aerobic exercise, and various medicaments and therapies to treat ailments associated with cystic fibrosis (e.g., diarrhea, osteoporosis, diabetes, bleeding, etc.). 35

[00278] Additional Embodiments of RNAi Agents to Beta-ENaC

[00277] In a particular embodiment, the present disclosure encompasses a composition comprising one or more Beta-ENaC RNAi agents. In one embodiment, the present disclosure 50 2015221515 03 Sep 2015 comprises a RNAi agent comprising a sense strand and an antisense strand, in one embodiment, the antisense strand consists of, consists essentialiy of, or comprises the sequence of the antisense strand of a RNAi agent listed, e g., in Table 1. In one embodiment, the antisense strand consists of, consists essentially of, or comprises a 5 sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of any RNAi agent listed, e.g., in Table 1. in one embodiment the antisense strand consists of the sequence of the antisense strand of a RNAi agent listed, e.g., in Table 1, and further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, in one embodiment, the antisense strand consists of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of a RNAi agent 10 listed, e.g., in Table 1, and further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[00278] In another embodiment, the composition of the claimed disclosure does not comprise any particular individual RNAi agent listed, e.g., in Table 1. in another embodiment of the present disclosure, the RNAi agent to Beta-ENaC does not comprise a sequence of any Beta-ENaC RNAi agent disclosed in the patent or scientific literature, e.g., U.S. Patent 15 App. No. 60/346,069 (PCT/US02/41850), and Hyde et a!. 2009, The 23rd North American Cystic Fibrosis Conference, Minneapolis, October 14-17, 2009; or that available as sc-42418 (and related products) from Santa Cruz Biotechnology, Santa Cruz, CA.

[00279] Specific Embodiments of RNAi Agents to Beta-ENaC 20 [00280] Various specific embodiments of a RNAi agent to Beta-ENaC are disclosed herein. Example duplex sequences are provided herein and, e.g., in Table 1. Specific embodiments of the present disclosure include RNAi agents which comprise sequences differing by 0, 1, 2, or 3 nt or bp (e.g., with 0, 1, 2 or 3 mismatches) from those of the RNAi agents listed, e.g., in Table 1. 25 [00281] A mismatch is defined herein as a difference between the base sequence or length when two sequences are maximally aligned and compared. A mismatch is defined as a position wherein the base of one sequence does not match the base of the other sequence. Thus, a mismatch is counted, for example, if a position in one sequence has a particular base (e.g., A), and the corresponding position on the other sequence has a 30 different base (e.g., G).

[00282] A mismatch is also counted, e.g., if a position in one sequence has a base (e.g., A), and the corresponding position on the other sequence has no base (e.g., that position is an abasic nucleotide which comprises a phosphate-sugar backbone but no base). A single-stranded nick in either sequence (or in the sense or antisense strand) is not counted as 35 mismatch. Thus, as a non-limiting example, no mismatch would be counted if one sequence comprises the sequence AG, but the other sequence comprises the sequence AG with a single-stranded nick between the A and the G. A base modification is also not considered a 51 2015221515 03 Sep 2015 mismatch. Thus, if one sequence comprises a C, and the other sequence comprises a modified C (e.g., 2’-modification) at the same position, no mismatch would be counted.

[00283] in one particular embodiment, the present disclosure comprises a RNAi agent comprising a antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 5 2, or 3 nt from the antisense strand of: AD-20807 (SEQ ID NOs: 5 and 6, or SEQ ID NOs:115 and 116).

[00284] in another particular embodiment, the siRNA also further comprises a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand of AD-20807. 10 [00285] In another particular embodiment, the siRNA comprises AD-20807.

[00286] In another particular embodiment, the siRNA has a sequence consisting of that of AD-20807.

[00287] In one particular embodiment, the present disclosure comprises a RNAi agent comprising a antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 15 2, or 3 nt from the antisense strand of: AD-20826 (SEQ ID NOs: 43 and 44, or SEQ ID NOs: 153 and 154).

[00288] in another particular embodiment, the siRNA also further comprises a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand of AD-20826. 20 [00289] In another particular embodiment, the siRNA comprises AD-20826.

[00290] In another particular embodiment, the siRNA has a sequence consisting of that of AD-20626.

[00291] In one particular embodiment, the present disclosure comprises a RNAi agent comprising a antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 25 2, or 3 nt from the antisense strand of: AD-20832, which comprises SEQ ID NOs: 55 and 56, or SEQ ID NOs:165 and 166.

[00292] In another particular embodiment, the siRNA also further comprises a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the sense strand of AD-20832. 30 [00293] In another particular embodiment, the siRNA comprises AD-20832.

[00294] In another particular embodiment, the siRNA has a sequence consisting of that of AD-20832.

[00295] In one particular embodiment, the present disclosure comprises a RNAi agent comprising a antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 35 2, or 3 nt from the antisense strand of: AD-20834, which comprises SEQ ID NOs: 59 and 60, or SEQ ID NOs:169 and 170. 52 2015221515 03 Sep 2015 [00296] In another particular embodiment, the siRNA also further comprises a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the sense strand of AD-20834.

[00297] In another particular embodiment, the siRNA comprises AD-20834. 5 [00298] In another particular embodiment, the siRNA has a sequence consisting of that of AD-20834.

[00299] In one particular embodiment, the present disclosure comprises a RNAi agent comprising a antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of: AD-20848, which comprises SEQ ID NOs: 87 and 88, 10 or SEQ ID NOs: 197 and 198.

[00300] In another particular embodiment, the siRNA also further comprises a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand of AD-20848.

[00301] In another particular embodiment, the siRNA comprises AD-20848. 15 [00302] In another particular embodiment, the siRNA has a sequence consisting of that of AD-20848.

[00303] In one particular embodiment the present disclosure comprises a RNAi agent comprising a antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of: AD-20861, which comprises SEQ ID NQs: 97 and 98, 20 or SEQ ID NOs:2Q7 and 208.

[00304] In another particular embodiment, the siRNA also further comprises a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 ntfrom the sense strand of AD-20861.

[00305] In another particular embodiment, the siRNA comprises AD-20861. 25 [00306] In another particular embodiment, the siRNA has a sequence consisting of that of AD-20861.

[00307] In one embodiment, the present disclosure comprises a RNAi agent demonstrating at least about 80% knockdown (no more than about 20% residual gene activity) of the Beta-ENaC gene at an in vitro concentration of 10 nM in H441 ceils. 30 [00308] Thus, in one particular embodiment, the present disclosure comprises a RNAi agent comprising a antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of: AD-20832; AD-20848; AD-20807; AD-20826; AD-20837; AD-20861; or AD-20834.

[00309] In another particular embodiment, the siRNA also further comprises a sense 35 strand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the sense strand of AD-20832; AD-20848; AD-20807; AD-20826; AD-20837; AD-20861; or AD-20834. 53 2015221515 03 Sep 2015 [00310] In another particular embodiment, the siRNA comprises AD-20832; AD-20848; AD-20807; AD-20826; AD-20837; AD-20861; or AD-20834.

[00311] In another particular embodiment, the siRNA has a sequence consisting of that of AD-20832; AD-20848; AD-20807; AD-20826; AD-20837; AD-20861; or AD-20834. 5 [00312] In one embodiment, the present disclosure comprises a RNAi agent demonstrating at least about 70% knockdown (no more than about 30% residual gene activity) of the Beta-ENaC gene at an in vitro concentration of 10 nM in H441 cells.

[00313] Thus, in one particular embodiment, the present disclosure comprises a RNAi agent comprising a antisense strand comprising at least 15 contiguous nucleotides differing 10 by 0, 1, 2, or 3 nt from the antisense strand of: AD-20832; AD-20848; AD-20807; AD-20826; AD-20837; AD-20861; AD-20834; AD-20806; AD-20851; AD-20865; AD-20811; AD-20819; AD-20839; AD-20835; AD-20825;or AD-20867.

[00314] In another particular embodiment, the siRNA also further comprises a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 ntfrom the 15 sense strand of AD-20832; AD-20848; AD-20807; AD-20826; AD-20837; AD-20861; AD-20834; AD-20806; AD-20851; AD-20865; AD-20811; AD-20819; AD-20839; AD-20835; AD-20825;or AD-20867.

[00315] In another particular embodiment, the siRNA comprises AD-20832; AD-20848; AD-20807; AD-20826; AD-20837; AD-20861; AD-20834; AD-20806; AD-20851; AD-20865; 20 AD-20811; AD-20819; AD-20839; AD-20835; AD-20825; or AD-20867.

[00316] In another particular embodiment, the siRNA has a sequence consisting of that of AD-20832; AD-20848; AD-20807; AD-20826; AD-20837; AD-20861; AD-20834; AD-20806; AD-20851; AD-20865; AD-20811; AD-20819; AD-20839; AD-20835; AD-20825; or AD-20867. 25 [00317] In one embodiment, the present disclosure comprises a RNAi agent demonstrating at least about 60% knockdown (no more than about 40% residual gene activity) of the Beta-ENaC gene at an in vitro concentration of 10 nM in H441 cells.

[00318] Thus, in one particular embodiment, the present disclosure comprises a RNAi agent comprising a antisense strand comprising at least 15 contiguous nucleotides differing 30 by 0, 1, 2, or 3 nt from the antisense strand of: AD-20832; AD-20848; AD-20807; AD-20826: AD-20837; AD-20861; AD-20834; AD-20806; AD-20851; AD-20865; AD-20811; AD-20819; AD-20839; AD-20835; AD-20825; AD-20867; AD-20813; AD-20823; AD-20805; AD-20831; AD-20862; AD-20808; or AD-20827.

[00319] In another particular embodiment, the siRNA also further comprises a sense 35 strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand of AD-20832; AD-20848; AD-20807; AD-20826; AD-20837; AD-20861; AD-20834; AD-20806; AD-20851; AD-20865; AD-20811; AD-20819; AD-20839: AD-20835: AD- 54 2015221515 03 Sep 2015 20825; AD-20867; AD-20813; AD-20823; AD-20805; AD-20831; AD-20862; AD-20808; or AD-20827.

[00320] In another particular embodiment, the siRNA comprises: AD-20832; AD-20848; AD-20807; AD-20826; AD-20837; AD-20861; AD-20834; AD-20806; AD-20851; AD-20865; 5 AD-20811; AD-20819; AD-20839; AD-20835; AD-20825; AD-20867; AD-20813; AD-20823; AD-20805; AD-20831; AD-20862; AD-20808; or AD-20827.

[00321] In another particular embodiment, the siRNA has a sequence consisting of that of AD-20832; AD-20848; AD-20807; AD-20826; AD-20837; AD-20861; AD-20834; AD-20806; AD-20851; AD-20865; AD-20811; AD-20819; AD-20839; AD-20835; AD-20825; AD-20867; 10 AD-20813; AD-20823; AD-20805; AD-20831; AD-20862; AD-20808; or AD-20827.

[00322] In one embodiment, the present disclosure comprises a RNAi agent demonstrating at least about 50% knockdown (no more than about 50% residual gene activity) of the Beta-ENaC gene at an in vitro concentration of 10 nM in H441 cells.

[00323] Thus, in one particular embodiment, the present disclosure comprises a RNAi 15 agent comprising a antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of: AD-20832: AD-20848; AD-20807; AD-20826; AD-20837; AD-20861; AD-20834; AD-20806; AD-20851; AD-20865; AD-20811; AD-20819; AD-20839; AD-20835; AD-20825; AD-20867; AD-20813; AD-20823; AD-20805; AD-20831; AD-20862; AD-20808; AD-20827; AD-20828; AD-20812; AD-20836; or AD-20822. 20 [00324] In another particular embodiment, the siRNA also further comprises a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand of AD-20832; AD-20848; AD-20807; AD-20826; AD-20837; AD-20861; AD-20834; AD-20806; AD-20851; AD-20865; AD-20811; AD-20819; AD-20839; AD-20835; AD-20825; AD-20867; AD-20813; AD-20823; AD-20805; AD-20831; AD-20862; AD-20808; AD-25 20827; AD-20828; AD-20812; AD-20836; or AD-20822.

[00325] In another particular embodiment the siRNA comprises AD-20832; AD-20848; AD-20807; AD-20826; AD-20837; AD-20861; AD-20834; AD-20806; AD-20851; AD-20865; AD-20811; AD-20819; AD-20839; AD-20835; AD-20825; AD-20867; AD-20813; AD-20823; AD-20805; AD-20831; AD-20862; AD-20808; AD-20827; AD-20828; AD-20812; AD-20836: 30 or AD-20822.

[00326] In another particular embodiment, the siRNA has a sequence consisting of that of AD-20832; AD-20848; AD-20807; AD-20826; AD-20837; AD-20861; AD-20834; AD-20806; AD-20851; AD-20865; AD-20811; AD-20819; AD-20839; AD-20835; AD-20825; AD-20867; AD-20813; AD-20823; AD-20805; AD-20831; AD-20862; AD-20808; AD-20827; AD-20828; 35 AD-20812; AD-20836; or AD-20822. 55 2015221515 03 Sep 2015

[00327] Various Embodiments of a RNAi Agent to Beta-ENaC

[00328] !n one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides 5 differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20805.

[00329] in one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20806. 10 [00330] In one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20807.

[00331] In one embodiment, the present disclosure comprises a RNAi agent comprising 15 a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20808.

[00332] In one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from 20 the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20809.

[00333] In one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 ntfrom the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides 25 differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20810.

[00334] In one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20811. 30 [00335] In one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20812.

[00336] In one embodiment, the present disclosure comprises a RNAi agent comprising 35 a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20813. 56 2015221515 03 Sep 2015 [00337] In one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20814. 5 [00338] In one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 ntfrorn the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20815.

[00339] In one embodiment, the present disclosure comprises a RNAi agent comprising 10 a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20816.

[00340] In one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from 15 the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20817.

[00341] In one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides 20 differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20818.

[00342] In one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20819. 25 [00343] in one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20820.

[00344] In one embodiment, the present disclosure comprises a RNAi agent comprising 30 a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20821.

[00345] In one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from 35 the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20822. 57 2015221515 03 Sep 2015 [00346] In one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20823. 5 [00347] In one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 ntfrom the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20824.

[00348] In one embodiment, the present disclosure comprises a RNAi agent comprising 10 a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20825.

[00349] In one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from 15 the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20826.

[00350] In one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides 20 differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20827.

[00351] In one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20828. 25 [00352] In one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20829.

[00353] in one embodiment, the present disclosure comprises a RNAi agent comprising 30 a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20830.

[00354] In one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from 35 the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20831. 58 2015221515 03 Sep 2015 [0035S] In one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20832. 5 [00358] In one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 ntfrom the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20833.

[00357] In one embodiment, the present disclosure comprises a RNAi agent comprising 10 a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20834.

[00358] In one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from 15 the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20835.

[00359] In one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides 20 differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20836.

[00360] In one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20837. 25 [00381] In one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20838.

[00362] In one embodiment, the present disclosure comprises a RNAi agent comprising 30 a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20839.

[00383] In one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from 35 the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20840. 59 2015221515 03 Sep 2015 [00384] In one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20841. 5 [00365] In one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 ntfrom the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20842.

[00386] In one embodiment, the present disclosure comprises a RNAi agent comprising 10 a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20843.

[00367] In one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from 15 the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20844.

[00368] In one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides 20 differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20845.

[00369] In one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20846. 25 [00370] In one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20847.

[00371] In one embodiment, the present disclosure comprises a RNAi agent comprising 30 a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20848.

[00372] In one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from 35 the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20849. 60 2015221515 03 Sep 2015 [00373] In one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20850. 5 [00374] In one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 ntfrom the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20851.

[00375] In one embodiment, the present disclosure comprises a RNAi agent comprising 10 a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20852.

[00378] In one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from 15 the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20861.

[00377] In one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides 20 differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20862.

[00378] In one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20863. 25 [00379] In one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20864.

[00380] In one embodiment, the present disclosure comprises a RNAi agent comprising 30 a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20865.

[00381] In one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from 35 the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20866. 61 2015221515 03 Sep 2015 [00382] !n one embodiment, the present disclosure comprises a RNAi agent comprising a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 ni from the sense strand and/or an antisense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of AD-20867. 5

[00383] Various Embodiments of a RNAi Agent to Beta-ENaC

[00384] In one embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from 10 the antisense strand of: AD-20805, which comprises SEQ ID NOs. 111-112, and modified variants thereof.

[00385] in one embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from 15 the antisense strand of: AD-20806, which comprises SEQ ID NOs. 113-114, and modified variants thereof.

[00386] In one embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from 20 the antisense strand of: AD-20807, which comprises SEQ ID NOs. 115 -116, and modified variants thereof.

[00387] In one embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from 25 the antisense strand of: AD-20808, which comprises SEQ ID NOs. 117-118, and modified variants thereof.

[00388] In one embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from 30 the antisense strand of: AD-20809, which comprises SEQ ID NOs. 119-120, and modified variants thereof.

[00389] In one embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from 35 the antisense strand of: AD-20810, which comprises SEQ ID NOs. 121 -122, and modified variants thereof.

[00390] in one embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense 62 2015221515 03 Sep 2015 strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20811, which comprises SEQ ID NOs. 123 - 124, and modified variants thereof.

[00391] In one embodiment, the present disclosure relates to a composition comprising a 5 RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20812, which comprises SEQ ID NOs. 125 - 126, and modified variants thereof.

[00392] In one embodiment, the present disclosure relates to a composition comprising a 10 RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20813, which comprises SEQ ID NOs. 127 - 128, and modified variants thereof.

[00393] In one embodiment, the present disclosure relates to a composition comprising a 15 RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20814, which comprises SEQ ID NOs. 129 -130, and modified variants thereof.

[00394] In one embodiment, the present disclosure relates to a composition comprising a 20 RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20815, which comprises SEQ ID NOs. 131 -132, and modified variants thereof.

[00395] In one embodiment, the present disclosure relates to a composition comprising a 25 RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20816, which comprises SEQ ID NOs. 133 -134, and modified variants thereof.

[00396] In one embodiment, the present disclosure relates to a composition comprising a 30 RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20817, which comprises SEQ ID NOs. 135 -136, and modified variants thereof.

[00397] In one embodiment, the present disclosure relates to a composition comprising a 35 RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from 63 2015221515 03 Sep 2015 the antisense strand of: AD-20818, which comprises SEQ ID NOs. 137 - 138, and modified variants thereof.

[00398] In one embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense 5 strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20819, which comprises SEQ ID NOs. 139 - 140, and modified variants thereof.

[00399] In one embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense 10 strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20820, which comprises SEQ ID NOs. 141 -142, and modified variants thereof.

[00400] In one embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense 15 strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20821, which comprises SEQ ID NOs. 143 - 144, and modified variants thereof.

[00401] In one embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense 20 strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20822, which comprises SEQ ID NOs. 145 -146, and modified variants thereof.

[00402] In one embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense 25 strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20823, which comprises SEQ ID NOs. 147 - 148, and modified variants thereof.

[00403] in one embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense 30 strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20824, which comprises SEQ ID NOs. 149 -150, and modified variants thereof.

[00404] In one embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense 35 strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20825, which comprises SEQ ID NOs. 151 -152, and modified variants thereof. 64 2015221515 03 Sep 2015 [00405] In one embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20826, which comprises SEQ ID NOs. 153 - 154, and modified 5 variants thereof.

[00406] In one embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20827, which comprises SEQ ID NQs. 155 - 156, and modified 10 variants thereof.

[00407] In one embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20828, which comprises SEQ ID NOs. 157 -158, and modified 15 variants thereof.

[00408] In one embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20829, which comprises SEQ ID NOs. 159 -160, and modified 20 variants thereof.

[00409] In one embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at ieast 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucieotides from the antisense strand of: AD-20830, which comprises SEQ ID NOs. 161 -162, and modified 25 variants thereof.

[00410] In one embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20831, which comprises SEQ ID NOs. 163 -164, and modified 30 variants thereof.

[00411] In one embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20832, which comprises SEQ ID NOs. 165 -166, and modified 35 variants thereof.

[00412] In one embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense 65 2015221515 03 Sep 2015 strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20833, which comprises SEQ ID NOs. 167 - 168, and modified variants thereof.

[00413] In one embodiment, the present disclosure relates to a composition comprising a 5 RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20834, which comprises SEQ ID NOs. 169 -170, and modified variants thereof.

[00414] In one embodiment, the present disclosure relates to a composition comprising a 10 RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20835, which comprises SEQ ID NOs. 171-172, and modified variants thereof.

[00415] In one embodiment, the present disclosure relates to a composition comprising a 15 RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20836, which comprises SEQ ID NOs. 173 -174, and modified variants thereof.

[00416] In one embodiment, the present disclosure relates to a composition comprising a 20 RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20837, which comprises SEQ ID NOs. 175 -176, and modified variants thereof.

[00417] In one embodiment, the present disclosure relates to a composition comprising a 25 RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20838, which comprises SEQ ID NOs. 177 - 178, and modified variants thereof.

[00418] In one embodiment, the present disclosure relates to a composition comprising a 30 RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20839, which comprises SEQ ID NOs. 179 -180, and modified variants thereof.

[00419] In one embodiment, the present disclosure relates to a composition comprising a 35 RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from 66 2015221515 03 Sep 2015 the antisense strand of: AD-20840, which comprises SEQ ID NOs. 181 -182, and modified variants thereof.

[00420] In one embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense 5 strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20841, which comprises SEQ ID NOs. 183 - 184, and modified variants thereof.

[00421] In one embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense 10 strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20842, which comprises SEQ ID NOs. 185 - 186, and modified variants thereof.

[00422] In one embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense 15 strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20843, which comprises SEQ ID NOs. 187 - 188, and modified variants thereof.

[00423] In one embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense 20 strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20844, which comprises SEQ ID NOs. 189 -190, and modified variants thereof.

[00424] In one embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense 25 strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20845, which comprises SEQ ID NOs. 191 -192, and modified variants thereof.

[00425] In one embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense 30 strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20848, which comprises SEQ ID NOs. 193 -194, and modified variants thereof.

[00428] In one embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense 35 strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20847, which comprises SEQ ID NOs. 195 -196, and modified variants thereof. 67 2015221515 03 Sep 2015 [00427] In one embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20848, which comprises SEQ ID NOs. 197 - 198, and modified 5 variants thereof.

[00428] In one embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20849, which comprises SEQ ID NQs. 199 - 200, and modified 10 variants thereof.

[00429] In one embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20850, which comprises SEQ ID NOs. 201 - 202, and modified 15 variants thereof.

[00430] In one embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20851, which comprises SEQ ID NOs. 203 - 204, and modified 20 variants thereof.

[00431] In one embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20852, which comprises SEQ ID NOs. 205 - 206, and modified 25 variants thereof.

[00432] In one embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20861, which comprises SEQ ID NOs. 207 - 208, and modified 30 variants thereof.

[00433] In one embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20862, which comprises SEQ ID NOs. 209 - 210, and modified 35 variants thereof.

[00434] In one embodiment, the present disclosure relates to a composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense 68 2015221515 03 Sep 2015 strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20863, which comprises SEQ ID NOs. 211 - 212, and modified variants thereof.

[00435] In one embodiment, the present disclosure relates to a composition comprising a 5 RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20864, which comprises SEQ ID NOs. 213 - 214, and modified variants thereof.

[00436] In one embodiment, the present disclosure relates to a composition comprising a 10 RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20865, which comprises SEQ ID NOs. 215 - 216, and modified variants thereof.

[00437] in one embodiment, the present disclosure relates to a composition comprising a 15 RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20866, which comprises SEQ ID NOs. 217 - 218, and modified variants thereof.

[00438] In one embodiment, the present disclosure relates to a composition comprising a 20 RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of: AD-20867, which comprises SEQ ID NOs. 219 - 220, and modified variants thereof.

25 [00439] Various Embodiments of a RNAi Agent to Beta-ENaC

[00440] In one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20805, which comprises SEQ ID NOs. 111 and 112. 30 [00441] in one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20806, which comprises SEQ ID NOs. 113 and 114 [00442] In one embodiment, the composition comprises a RNAi agent comprising a sense 35 strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20807, which comprises SEQ ID NOs. 115 and 116. 69 2015221515 03 Sep 2015 [00443] !n one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20808, which comprises SEQ i D NOs. 117 and 118. 5 [00444] In one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20809, which comprises SEQ ID NOs. 119 and 120.

[00445] In one embodiment, the composition comprises a RNAi agent comprising a 10 sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20810, which comprises SEQ ID NOs. 121 and 122.

[00446] In one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20811, which comprises SEQ ID NQs. 123 and 124.

[00447] In one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20812, 20 which comprises SEQ ID NOs. 125 and 126.

[00448] In one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20813, which comprises SEQ ID NOs. 127 and 128. 25 [00449] In one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20814, which comprises SEQ ID NOs. 129 and 130.

[00450] in one embodiment, the composition comprises a RNAi agent comprising a 30 sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20815, which comprises SEQ ID NOs. 131 and 132.

[00451] In one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 35 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20816, which comprises SEQ ID NOs. 133 and 134. 70 2015221515 03 Sep 2015 [00452] In one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at feast 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20817, which comprises SEQ ID NOs. 135 and 136. 5 [00453] In one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20818, which comprises SEQ ID NOs. 137 and 138.

[00454] In one embodiment, the composition comprises a RNAi agent comprising a 10 sense strand and an antisense strand, wherein the antisense strand comprises at feast 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20819, which comprises SEQ ID NOs. 139 and 140.

[00455] In one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20820, which comprises SEQ ID NQs. 141 and 142.

[00456] In one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20821, 20 which comprises SEQ ID NOs. 143 and 144.

[00457] In one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20822, which comprises SEQ ID NOs. 145 and 146. 25 [00458] In one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20823, which comprises SEQ ID NOs. 147 and 148.

[00459] In one embodiment, the composition comprises a RNAi agent comprising a 30 sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20824, which comprises SEQ ID NOs. 149 and 150.

[00460] in one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 35 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20825, which comprises SEQ ID NOs. 151 and 152. 71 2015221515 03 Sep 2015 [00461] In one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20828, which comprises SEQ ID NOs. 153 and 154. 5 [00462] In one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20827, which comprises SEQ ID NOs. 155 and 156.

[00463] In one embodiment, the composition comprises a RNAi agent comprising a 10 sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20828, which comprises SEQ ID NOs. 157 and 158.

[00464] In one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20829, which comprises SEQ ID NQs. 159 and 160.

[00465] In one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20830, 20 which comprises SEQ ID NOs. 161 and 162.

[00466] In one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20831, which comprises SEQ ID NOs. 163 and 164. 25 [00467] In one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20832, which comprises SEQ ID NOs. 165 and 166.

[00468] In one embodiment, the composition comprises a RNAi agent comprising a 30 sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20833, which comprises SEQ ID NOs. 167 and 188.

[00469] in one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 35 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20834, which comprises SEQ ID NOs. 169 and 170. 72 2015221515 03 Sep 2015 [00470] In one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20835, which comprises SEQ ID NOs. 171 and 172. 5 [00471] In one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20836, which comprises SEQ ID NOs. 173 and 174.

[00472] In one embodiment, the composition comprises a RNAi agent comprising a 10 sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20837, which comprises SEQ ID NOs. 175 and 176.

[00473] In one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20838, which comprises SEQ ID NOs. 177 and 178.

[00474] In one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20839, 20 which comprises SEQ ID NOs. 179 and 180.

[00475] In one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20840, which comprises SEQ ID NOs. 181 and 182. 25 [00476] In one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20841, which comprises SEQ ID NOs. 183 and 184.

[00477] In one embodiment, the composition comprises a RNAi agent comprising a 30 sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20842, which comprises SEQ ID NOs. 185 and 186.

[00478] In one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 35 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20843, which comprises SEQ ID NOs. 187 and 188. 73 2015221515 03 Sep 2015 [00479] In one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20844, which comprises SEQ ID NOs. 189 and 190. 5 [00480] In one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20845, which comprises SEQ ID NOs. 191 and 192.

[00481] In one embodiment, the composition comprises a RNAi agent comprising a 10 sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20848, which comprises SEQ ID NOs. 193 and 194.

[00482] In one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20847, which comprises SEQ ID NOs. 195 and 196.

[00483] In one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20848, 20 which comprises SEQ ID NOs. 197 and 198.

[00484] in one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20849, which comprises SEQ ID NOs. 199 and 200. 25 [00485] In one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20850, which comprises SEQ ID NOs. 201 and 202.

[00486] In one embodiment, the composition comprises a RNAi agent comprising a 30 sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20851, which comprises SEQ ID NOs. 203 and 204.

[00487] In one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 35 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20852, which comprises SEQ ID NOs. 205 and 206. 74 2015221515 03 Sep 2015 [00488] !n one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20861, which comprises SEQ ID NOs. 207 and 208. 5 [00489] in one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20862, which comprises SEQ ID NOs. 209 and 210.

[00490] In one embodiment, the composition comprises a RNAi agent comprising a 10 sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20863, which comprises SEQ ID NOs. 211 and 212.

[00491] in one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20864, which comprises SEQ ID NOs. 213 and 214.

[00492] In one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20865, 20 which comprises SEQ ID NOs. 215 and 216.

[00493] In one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20866, which comprises SEQ ID NOs. 217 and 218. 25 [00494] and in one embodiment, the composition comprises a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0 nucleotides from the antisense strand of: AD-20867, which comprises SEQ ID NOs. 219 and 220.

30 [00495] Various Embodiments of a RNAi Agent to Beta-ENaC

[00496] In one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nucleotides from the antisense sequence of: AD-20805, which comprises SEQ ID NOs. 1 35 and 2.

[00497] In one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 75 2015221515 03 Sep 2015 nucleotides from the antisense sequence of: AD-20806, which comprises SEQ ID NOs. 3 and 4.

[00498] In one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the 5 antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20807, which comprises SEQ ID NOs. 5 and 6, [00499] In one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the 10 antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20808, which comprises SEQ ID NOs. 7 and 8.

[00500] In one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the 15 antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20809, which comprises SEQ ID NOs. 9 and 10.

[00501] In one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the 20 antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20810, which comprises SEQ ID NOs. 11 and 12.

[00502] in one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the 25 antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20811, which comprises SEQ ID NOs. 13 and 14.

[00503] In one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the 30 antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20812, which comprises SEQ ID NOs. 15 and 16.

[00504] in one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the 35 antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20813, which comprises SEQ ID NOs. 17 and 18. 76 2015221515 03 Sep 2015 [00505] In one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20814, which comprises SEQ ID NOs. 19 5 and 20.

[00506] In one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20815, which comprises SEQ ID NOs. 21 10 and 22.

[00507] In one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20816, which comprises SEQ SD NOs. 23 15 and 24.

[00508] In one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20817, which comprises SEQ ID NOs. 25 20 and 26.

[00509] In one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20818, which comprises SEQ ID NOs. 27 25 and 28.

[00510] In one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20819, which comprises SEQ ID NOs. 29 30 and 30.

[00511] In one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20820, which comprises SEQ ID NOs. 31 35 and 32.

[00512] In one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the 77 2015221515 03 Sep 2015 antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20821, which comprises SEQ ID NOs. 33 and 34.

[00513] in one embodiment, the composition comprises a modified variant of a RNAi 5 agent, wherein the variant comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20822, which comprises SEQ ID NOs. 35 and 36.

[00514] In one embodiment, the composition comprises a modified variant of a RNAi 10 agent, wherein the variant comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20823, which comprises SEQ ID NOs. 37 and 38.

[00515] In one embodiment, the composition comprises a modified variant of a RNAi 15 agent, wherein the variant comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20824, which comprises SEQ ID NOs. 39 and 40.

[00516] In one embodiment, the composition comprises a modified variant of a RNAi 20 agent, wherein the variant comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20825, which comprises SEQ ID NOs. 41 and 42.

[00517] In one embodiment, the composition comprises a modified variant of a RNAi 25 agent, wherein the variant comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20826, which comprises SEQ ID NOs. 43 and 44.

[00518] In one embodiment, the composition comprises a modified variant of a RNAi 30 agent, wherein the variant comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20827, which comprises SEQ ID NOs. 45 and 46.

[00519] In one embodiment, the composition comprises a modified variant of a RNAi 35 agent, wherein the variant comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 78 2015221515 03 Sep 2015 nucleotides from the antisense sequence of: AD-20828, which comprises SEQ ID NOs. 47 and 48.

[00520] In one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the 5 antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20829, which comprises SEQ ID NOs. 49 and 50.

[00521] In one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the 10 antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20830, which comprises SEQ ID NOs. 51 and 52.

[00522] In one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the 15 antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20831, which comprises SEQ ID NOs. 53 and 54.

[00523] In one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the 20 antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20832, which comprises SEQ ID NOs. 55 and 56.

[00524] in one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the 25 antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20833, which comprises SEQ ID NOs. 57 and 58.

[00525] In one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the 30 antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20834, which comprises SEQ ID NOs. 59 and 60.

[00526] in one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the 35 antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20835, which comprises SEQ ID NOs. 61 and 62. 79 2015221515 03 Sep 2015 [00527] In one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20836, which comprises SEQ ID NOs. 63 5 and 64.

[00528] In one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20837, which comprises SEQ ID NOs. 65 10 and 66.

[00529] In one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20838, which comprises SEQ ID NOs. 67 15 and 68.

[00530] In one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20839, which comprises SEQ ID NOs. 69 20 and 70.

[00531] In one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20840, which comprises SEQ ID NOs. 71 25 and 72.

[00532] In one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20841, which comprises SEQ ID NOs. 73 30 and 74.

[00533] In one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20842, which comprises SEQ ID NOs. 75 35 and 76.

[00534] In one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the 80 2015221515 03 Sep 2015 antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20843, which comprises SEQ ID NOs. 77 and 78.

[00535] In one embodiment, the composition comprises a modified variant of a RNAi 5 agent, wherein the variant comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20844, which comprises SEQ ID NOs. 79 and 80.

[00536] In one embodiment, the composition comprises a modified variant of a RNAi 10 agent, wherein the variant comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20845, which comprises SEQ ID NOs. 81 and 82.

[00537] In one embodiment, the composition comprises a modified variant of a RNAi 15 agent, wherein the variant comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20846, which comprises SEQ ID NOs. 83 and 84.

[00538] In one embodiment, the composition comprises a modified variant of a RNAi 20 agent, wherein the variant comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20847, which comprises SEQ ID NOs. 85 and 86.

[00539] In one embodiment, the composition comprises a modified variant of a RNAi 25 agent, wherein the variant comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20848, which comprises SEQ ID NOs. 87 and 88.

[00540] In one embodiment, the composition comprises a modified variant of a RNAi 30 agent, wherein the variant comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20849, which comprises SEQ ID NOs. 89 and 90.

[00541] In one embodiment, the composition comprises a modified variant of a RNAi 35 agent, wherein the variant comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 81 2015221515 03 Sep 2015 nucleotides from the antisense sequence of: AD-20850, which comprises SEQ ID NOs. 91 and 92.

[00542] In one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the 5 antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20851, which comprises SEQ ID NOs. 93 and 94.

[00543] In one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the 10 antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20852, which comprises SEQ ID NOs. 95 and 96.

[00544] In one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the 15 antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20861, which comprises SEQ ID NOs. 97 and 98.

[00545] In one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the 20 antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20862, which comprises SEQ ID NOs. 99 and 100.

[00546] in one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the 25 antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20863, which comprises SEQ ID NOs. 101 and 102.

[00547] In one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the 30 antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20864, which comprises SEQ ID NOs. 103 and 104.

[00548] in one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the 35 antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20865, which comprises SEQ ID NOs. 105 and 106. 82 2015221515 03 Sep 2015 [00549] !n one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucieotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20866, which comprises SEQ ID NOs. 107 5 and 108.

[00550] and In one embodiment, the composition comprises a modified variant of a RNAi agent, wherein the variant comprises a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense sequence of: AD-20867, which comprises SEQ ID NOs. 109 10 and 110.

[00551] Various Embodiments of a RNAi Agent to Beta-ENaC

[00552] In one embodiment, the present disclosure comprises AD-20805 (SEQ ID NOs: 1 and 2, or SEQ ID NOs: 111 and 112). 15 [00553] In one embodiment, the present disclosure comprises AD-20806 (SEQ ID NOs: 3 and 4, or SEQ ID NOs: 113 and 114).

[00554] In one embodiment, the present disclosure comprises AD-20807 (SEQ ID NOs: 5 and 6, or SEQ ID NOs: 115 and 116).

[00555] In one embodiment, the present disclosure comprises AD-20808 (SEQ ID NOs: 7 20 and 8, or SEQ ID NOs: 117 and 118).

[00556] In one embodiment, the present disclosure comprises AD-20809 (SEQ ID NOs: 9 and 10, or SEQ ID NOs: 119 and 120).

[00557] In one embodiment, the present disclosure comprises AD-20810 (SEQ ID NOs: 11 and 12, or SEQ ID NOs: 121 and 122). 25 [00558] In one embodiment, the present disclosure comprises AD-20811 (SEQ ID NOs: 13 and 14, or SEQ ID NOs: 123 and 124).

[00559] In one embodiment, the present disclosure comprises AD-20812 (SEQ ID NOs: 15 and 16, or SEQ ID NOs: 125 and 126).

[00560] In one embodiment, the present disclosure comprises AD-20813 (SEQ ID NOs: 30 17 and 18, or SEQ ID NOs: 127 and 128).

[00561] In one embodiment, the present disclosure comprises AD-20814 (SEQ ID NOs: 19 and 20, or SEQ ID NOs: 129 and 130).

[00562] In one embodiment, the present disclosure comprises AD-20815 (SEQ ID NOs: 21 and 22, or SEQ ID NOs: 131 and 132). 35 [00563] In one embodiment, the present disclosure comprises AD-20816 (SEQ ID NOs: 23 and 24, or SEQ !D NOs: 133 and 134).

[00564] in one embodiment, the present disclosure comprises AD-20817 (SEQ iD NOs: 25 and 26, or SEQ iD NOs: 135 and 136). 83 2015221515 03 Sep 2015 [00565] !n one embodiment, the present 27 and 28, or SEQ !D NOs: 137 and 138), [00566] In one embodiment, the present 29 and 30, or SEQ ID NOs: 139 and 140). 5 [00567] In one embodiment, the present 31 and 32, or SEQ ID NQs: 141 and 142).

[00568] In one embodiment, the present 33 and 34, or SEQ ID NOs: 143 and 144).

[00569] In one embodiment, the present 10 35 and 36, or SEQ ID NQs: 145 and 146).

[00570] In one embodiment, the present 37 and 38, or SEQ ID NOs: 147 and 148).

[00571] In one embodiment, the present 39 and 40, or SEQ ID NQs: 149 and 150). 15 [00572] In one embodiment, the present 41 and 42, or SEQ ID NQs: 151 and 152).

[00573] In one embodiment, the present 43 and 44, or SEQ ID NOs: 153 and 154).

[00574] In one embodiment, the present 20 45 and 46, or SEQ ID NQs: 155 and 156).

[00575] In one embodiment, the present 47 and 48, or SEQ ID NOs: 157 and 158).

[00576] In one embodiment, the present 49 and 50, or SEQ ID NOs: 159 and 160). 25 [00577] In one embodiment, the present 51 and 52, or SEQ ID NOs: 161 and 162).

[00578] In one embodiment, the present 53 and 54, or SEQ ID NOs: 163 and 164).

[00579] In one embodiment, the present 30 55 and 56, or SEQ ID NOs: 165 and 166).

[00580] In one embodiment, the present 57 and 58, or SEQ ID NOs: 167 and 168).

[00581] in one embodiment, the present 59 and 60, or SEQ ID NOs: 169 and 170). 35 [00582] In one embodiment, the present 61 and 62, or SEQ ID NOs: 171 and 172), disclosure comprises AD-20818 (SEQ ID NOs disclosure comprises AD-20819 (SEQ ID NOs disclosure comprises AD-20820 (SEQ ID NOs disclosure comprises AD-20821 (SEQ ID NOs disclosure comprises AD-20822 (SEQ ID NOs disclosure comprises AD-20823 (SEQ ID NOs disclosure comprises AD-20824 (SEQ ID NOs disclosure comprises AD-20825 (SEQ ID NOs disclosure comprises AD-20826 (SEQ ID NOs disclosure comprises AD-20827 (SEQ ID NOs disclosure comprises AD-20828 (SEQ ID NOs disclosure comprises AD-20829 (SEQ ID NOs disclosure comprises AD-20830 (SEQ ID NOs disclosure comprises AD-20831 (SEQ ID NOs disclosure comprises AD-20832 (SEQ ID NOs disclosure comprises AD-20833 (SEQ ID NOs disclosure comprises AD-20834 (SEQ ID NOs disclosure comprises AD-20835 (SEQ ID NOs 2015221515 03 Sep 2015 84 [00583] In one embodiment, the present 63 and 64, or SEQ ID NOs: 173 and 174).

[00584] In one embodiment, the present 65 and 66, or SEQ ID NOs: 175 and 176). 5 [00585] In one embodiment, the present 67 and 68, or SEQ ID NOs: 177 and 178).

[00586] In one embodiment, the present 69 and 70, or SEQ ID NOs: 179 and 180).

[00587] In one embodiment, the present 10 71 and 72, or SEQ ID NOs: 181 and 182).

[00588] In one embodiment, the present 73 and 74, or SEQ ID NOs: 183 and 184).

[00589] In one embodiment, the present 75 and 76, or SEQ ID NOs: 185 and 186). 15 [00590] in one embodiment, the present 77 and 78, or SEQ ID NOs: 187 and 188).

[00591] in one embodiment, the present 79 and 80, or SEQ ID NOs: 189 and 190).

[00592] In one embodiment, the present 20 81 and 82, or SEQ ID NQs: 191 and 192).

[00593] In one embodiment, the present 83 and 84, or SEQ ID NOs: 193 and 194).

[00594] In one embodiment, the present 85 and 86, or SEQ ID NOs: 195 and 196). 25 [00595] In one embodiment, the present 87 and 88, or SEQ ID NOs: 197 and 198).

[00596] In one embodiment, the present 89 and 90, or SEQ ID NOs: 199 and 200), [00597] In one embodiment, the present 30 91 and 92, or SEQ ID NOs: 201 and 202).

[00598] In one embodiment, the present 93 and 94, or SEQ ID NOs: 203 and 204).

[00599] In one embodiment, the present 95 and 96, or SEQ ID NOs: 205 and 206). 35 [00600] In one embodiment, the present 97 and 98, or SEQ ID NOs: 207 and 208), disclosure comprises AD-20836 (SEQ ID NOs disclosure comprises AD-20837 (SEQ ID NOs disclosure comprises AD-20838 (SEQ ID NOs disclosure comprises AD-20839 (SEQ ID NOs disclosure comprises AD-20840 (SEQ ID NOs disclosure comprises AD-20841 (SEQ ID NOs disclosure comprises AD-20842 (SEQ ID NOs disclosure comprises AD-20843 (SEQ ID NOs disclosure comprises AD-20844 (SEQ ID NOs disclosure comprises AD-20845 (SEQ ID NOs disclosure comprises AD-20846 (SEQ ID NOs disclosure comprises AD-20847 (SEQ ID NOs: disclosure comprises AD-20848 (SEQ ID NOs: disclosure comprises AD-20849 (SEQ ID NQs disclosure comprises AD-20850 (SEQ ID NOs: disclosure comprises AD-20851 (SEQ ID NOs: disclosure comprises AD-20852 (SEQ ID NOs: disclosure comprises AD-20861 (SEQ ID NQs 85 2015221515 03 Sep 2015 [00601] In one embodiment, the present disclosure comprises AD-20862 (SEQ ID NOs: 99 and 100, or SEQ ID NOs: 209 and 210).

[00602] In one embodiment, the present disclosure comprises AD-20863 (SEQ ID NOs: 101 and 102, or SEQ ID NOs: 211 and 212). 5 [00603] In one embodiment, the present disclosure comprises AD-20864 (SEQ ID NOs: 103 and 104, or SEQ ID NOs: 213 and 214).

[00604] In one embodiment, the present disclosure comprises AD-20865 (SEQ ID NOs: 105 and 106, or SEQ ID NOs: 215 and 216).

[00605] In one embodiment, the present disclosure comprises AD-20866 (SEQ ID NOs: 10 107 and 108, or SEQ ID NOs: 217 and 218).

[00606] In one embodiment, the present disclosure comprises AD-20867 (SEQ ID NOs: 109 and 110, or SEQ ID NOs: 219 and 220).

[00607] In one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20805. 15 [00608] In one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20806.

[00609] In one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20807.

[00610] In one embodiment, the RNAi agent comprises an antisense strand that is the 20 exact sequence and length of the antisense strand of AD-20808.

[00611] In one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20809.

[00612] in one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20810. 25 [00613] in one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20811.

[00614] In one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20812, [00615] In one embodiment, the RNAi agent comprises an antisense strand that is the 30 exact sequence and length of the antisense strand of AD-20813.

[00616] In one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20814.

[00617] In one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20815. 35 [00618] In one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20816, 86 2015221515 03 Sep 2015 [00619] in one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20817, [00620] in one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20818, 5 [00621] In one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20819.

[00622] In one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20820.

[00623] In one embodiment, the RNAi agent comprises an antisense strand that is the 10 exact sequence and length of the antisense strand of AD-20821.

[00624] In one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20822.

[00625] In one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20823. 15 [00626] In one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20824.

[00627] In one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20825.

[00628] In one embodiment, the RNAi agent comprises an antisense strand that is the 20 exact sequence and length of the antisense strand of AD-20826.

[00629] In one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20827.

[00630] In one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20828. 25 [00631] In one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20829.

[00632] In one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20830, [00633] In one embodiment, the RNAi agent comprises an antisense strand that is the 30 exact sequence and length of the antisense strand of AD-20831.

[00634] In one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20832.

[00635] In one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20833. 35 [00636] In one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20834. 87 2015221515 03 Sep 2015 [00637] In one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20835, [00638] In one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20836. 5 [00639] in one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20837.

[00640] in one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20838.

[00641] in one embodiment, the RNAi agent comprises an antisense strand that is the 10 exact sequence and length of the antisense strand of AD-20839.

[00642] In one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20840.

[00643] In one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20841. 15 [00644] in one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20842.

[00645] In one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20843.

[00646] In one embodiment, the RNAi agent comprises an antisense strand that is the 20 exact sequence and length of the antisense strand of AD-20844.

[00647] In one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20845.

[00648] In one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20846. 25 [00649] In one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20847.

[00650] In one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20848.

[00651] In one embodiment, the RNAi agent comprises an antisense strand that is the 30 exact sequence and length of the antisense strand of AD-20849.

[00652] In one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20850.

[00653] In one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20851. 35 [00654] In one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20852, 88 2015221515 03 Sep 2015 [00655] !n one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20861.

[00656] In one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20862. 5 [00657] In one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20863.

[00658] In one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20864.

[00659] in one embodiment, the RNAi agent comprises an antisense strand that is the 10 exact sequence and length of the antisense strand of AD-20865, [00660] In one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20866.

[00661] In one embodiment, the RNAi agent comprises an antisense strand that is the exact sequence and length of the antisense strand of AD-20867, in these various 15 embodiments, a RNAi agent comprising an antisense strand that is the exact sequence and length of a recited antisense strand of a recited RNAi agent can comprise modified nucleotides, 3-end caps, and/or other modifications which do not alter the sequence or length of the RNAi agent.

20 [00662] Various Embodiments of a RNAi Agent to Beta-ENaC

[00663] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20805.

[00664] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20806. 25 [00665] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20807.

[00666] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20808.

[00667] In one embodiment, the RNAi agent comprises an antisense strand consisting 30 of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20809.

[00668] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20810.

[00669] in one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20811. 35 [00670] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20812.

[00671] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20813. 89 2015221515 03 Sep 2015 [00672] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20814.

[00673] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20815. 5 [00674] in one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20818.

[00675] in one embodiment the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20817.

[00676] in one embodiment, the RNAi agent comprises an antisense strand consisting 10 of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20818.

[00677] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20819.

[00678] in one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1,2, or 3 mismatches from that of the antisense strand of AD-20820. 15 [00679] in one embodiment the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20821.

[00680] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20822.

[00681] in one embodiment, the RNAi agent comprises an antisense strand consisting 20 of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20823.

[00682] in one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20824.

[00683] in one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20825. 25 [00684] in one embodiment the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20826.

[00685] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20827.

[00686] in one embodiment, the RNAi agent comprises an antisense strand consisting 30 of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20828.

[00687] in one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20829.

[00688] In one embodiment the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20830. 35 [00689] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20831. 90 2015221515 03 Sep 2015 [00690] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20832.

[00691] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20833. 5 [00692] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20834.

[00693] in one embodiment the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20835.

[00694] in one embodiment, the RNAi agent comprises an antisense strand consisting 10 of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20836.

[00695] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20837.

[00696] in one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1,2, or 3 mismatches from that of the antisense strand of AD-20838. 15 [00697] in one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20839.

[00698] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20840.

[00699] In one embodiment, the RNAi agent comprises an antisense strand consisting 20 of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20841.

[00700] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20842.

[00701] in one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20843. 25 [00702] in one embodiment the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20844.

[00703] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20845.

[00704] In one embodiment, the RNAi agent comprises an antisense strand consisting 30 of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20846.

[00705] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20847.

[00706] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20848. 35 [00707] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20849. 91 2015221515 03 Sep 2015 [00708] in one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20850.

[00709] in one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20851. 5 [00710] in one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1,2, or 3 mismatches from that of the antisense strand of AD-20852.

[00711] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20861.

[00712] in one embodiment, the RNAi agent comprises an antisense strand consisting 10 of a sequence with 0, 1,2, or 3 mismatches from that of the antisense strand of AD-20862.

[00713] in one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20863.

[00714] in one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1,2, or 3 mismatches from that of the antisense strand of AD-20864. 15 [00715] in one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20865.

[00716] in one embodiment the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20866.

[00717] in one embodiment, the RNAi agent comprises an antisense strand consisting 20 of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20867.

[00718] Various Embodiments of a RNAi Agent to Beta-ENaC

[00719] in one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20805, 25 wherein the antisense strand optionaily further comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any range thereof, e.g., 0-1, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, or 2-5 nt, etc.).

[00720] in one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20806, 30 wherein the antisense strand optionaily further comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any range thereof).

[00721] in one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20807, wherein the antisense strand optionaily further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt 35 (or any range thereof].

[00722] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20808, 92 2015221515 03 Sep 2015 wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any range thereof).

[00723] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20809, 5 wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 8, 7, 8, 9, or 10 nt (or any range thereof).

[00724] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20810, wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt 10 (or any range thereof).

[00725] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20811, wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any range thereof). 15 [00726] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20812, wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any range thereof).

[00727] In one embodiment, the RNAi agent comprises an antisense strand consisting of 20 a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20813, wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any range thereof).

[00728] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20814, 25 wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any range thereof).

[00729] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20815, wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt 30 (or any range thereof).

[00730] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20816, wherein the antisense strand optionally further comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any range thereof). 35 [00731] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20817, 93 2015221515 03 Sep 2015 wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any range thereof).

[00732] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20818, 5 wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 8, 7, 8, 9, or 10 nt (or any range thereof).

[00733] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20819, wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt 10 (or any range thereof).

[00734] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20820, wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any range thereof). 15 [00735] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20821, wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any range thereof).

[00736] In one embodiment, the RNAi agent comprises an antisense strand consisting of 20 a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20822, wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any range thereof).

[00737] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20823, 25 wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any range thereof).

[00738] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20824, wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt 30 (or any range thereof).

[00739] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20825, wherein the antisense strand optionally further comprises 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any range thereof). 35 [00740] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20826, 94 2015221515 03 Sep 2015 wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any range thereof).

[00741] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20827, 5 wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 8, 7, 8, 9, or 10 nt (or any range thereof).

[00742] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20828, wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt 10 (or any range thereof).

[00743] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20829, wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any range thereof). 15 [00744] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20830, wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any range thereof).

[00745] In one embodiment, the RNAi agent comprises an antisense strand consisting of 20 a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20831, wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any range thereof).

[00746] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20832, 25 wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any range thereof).

[00747] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20833, wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt 30 (or any range thereof).

[00748] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20834, wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any range thereof). 35 [00749] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20835, 95 2015221515 03 Sep 2015 wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any range thereof).

[00750] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20836, 5 wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any range thereof).

[00751] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20837, wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt 10 (or any range thereof).

[00752] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20838, wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any range thereof). 15 [00753] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20839, wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any range thereof).

[00754] In one embodiment, the RNAi agent comprises an antisense strand consisting of 20 a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20840, wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any range thereof).

[00755] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20841, 25 wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any range thereof).

[00756] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20842, wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt 30 (or any range thereof).

[00757] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20843, wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any range thereof). 35 [00758] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20844, 96 2015221515 03 Sep 2015 wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any range thereof).

[00759] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20845, 5 wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any range thereof).

[00760] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20846, wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt 10 (or any range thereof).

[00761] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20847, wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any range thereof). 15 [00762] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20848, wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any range thereof).

[00763] In one embodiment, the RNAi agent comprises an antisense strand consisting of 20 a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20849, wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any range thereof).

[00764] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20850, 25 wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any range thereof).

[00765] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20851, wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt 30 (or any range thereof).

[00766] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20852, wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any range thereof). 35 [00767] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20861, 97 2015221515 03 Sep 2015 wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any range thereof).

[00768] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20862, 5 wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any range thereof).

[00769] in one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20863, wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt 10 (or any range thereof).

[00770] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20864, wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any range thereof). 15 [00771] in one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20865, wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any range thereof).

[00772] In one embodiment, the RNAi agent comprises an antisense strand consisting of 20 a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20866, wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any range thereof).

[00773] In one embodiment, the RNAi agent comprises an antisense strand consisting of a sequence with 0, 1, 2, or 3 mismatches from that of the antisense strand of AD-20867, 25 wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nt (or any range thereof).

[00774] Additional particular specific embodiments [00775] in various embodiments, the disclosure comprises a RNAi agent comprising a 30 sense and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of any RNAi agent disclosed herein.

[00776] Thus, in various embodiments: [00777] The disclosure comprises a RNAi agent comprising a sense and an antisense 35 strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of any one or more of the following duplexes, or modified or unmodified variants thereof: AD-20832; AD-20848; AD-20807; AD-20826; AD-20837; AD-20861; AD-20834, AD-20805, AD-20806, AD-20808, AD-20809, AD-20810, AD- 98 2015221515 03 Sep 2015 20811, AD-20812, AD-20813, AD-20814, AD-20815, AD-20816, AD-20817, AD-20818, AD-20819, AD-20820, AD-20821, AD-20822, AD-20823, AD-20824, AD-20825, AD-20827, AD-20828, AD-20829, AD-20830, AD-20831, AD-20833, AD-20835, AD-20836, AD-20838, AD-20839, AD-20840, AD-20841, AD-20842, AD-20843, AD-20844, AD-20845, AD-20846, AD-5 20847, AD-20849, AD-20850, AD-20851, AD-20852, AD-20862, AD-20863, AD-20864, AD-20865, AD-20866, AD-20867, or modified or unmodified variants thereof.

[00778] Additional particular specific embodiments [00779] In various embodiments, the disclosure comprises a RNAi agent comprising a 10 first and a second strand, wherein the sequence of the first strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sequence of the first strand of, and the sequence of the second strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the sequence of the second strand, of any RNAi agent, disclosed herein. 15 [00780] Thus, in various embodiments: [00781] The disclosure comprises a RNAi agent comprising a sense and an antisense strand, wherein the sequence of the first strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sequence of the first strand of, and the sequence of the second strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 20 nucleotides from the sequence of the second strand, of any one or more of the following duplexes, or modified or unmodified variants thereof: AD-20832; AD-20848; AD-20807; AD-20826; AD-20837; AD-20861; AD-20834, AD-20805, AD-20806, AD-20808, AD-20809, AD-20810, AD-20811, AD-20812, AD-20813, AD-20814, AD-20815, AD-20816, AD-20817, AD-20818, AD-20819, AD-20820, AD-20821, AD-20822, AD-20823, AD-20824, AD-20825, AD-25 20827, AD-20828, AD-20829, AD-20830, AD-20831, AD-20833, AD-20835, AD-20836, AD-20838, AD-20839, AD-20840, AD-20841, AD-20842, AD-20843, AD-20844, AD-20845, AD-20846, AD-20847, AD-20849, AD-20850, AD-20851, AD-20852, AD-20862, AD-20863, AD-20864, AD-20865, AD-20866, AD-20867, or modified or unmodified variants thereof. 30 [00782] Additional particular embodiments [00783] In various embodiments, the disclosure comprises a RNAi agent comprising a sense and an antisense strand, wherein the antisense strand comprises or consists of the antisense strand of any RNAi agent disclosed herein.

[00784] Thus, the following are provided as examples of the various embodiments. 35 [00785] The disclosure comprises a RNAi agent comprising a sense and an antisense strand, wherein the antisense strand comprises or consists of the antisense strand of: AD-20832; AD-20848; AD-20807; AD-20826; AD-20837; AD-20861; AD-20834, AD-20805, AD-20806, AD-20808, AD-20809, AD-20810, AD-20811, AD-20812, AD-20813, AD-20814, AD- 99 2015221515 03 Sep 2015 20815, AD-20816, AD-20817, AD-20818, AD-20819, AD-20820, AD-20821, AD-20822, AD-20823, AD-20824, AD-20825, AD-20827, AD-20828, AD-20829, AD-20830, AD-20831, AD-20833, AD-20835, AD-20836, AD-20838, AD-20839, AD-20840, AD-20841, AD-20842, AD-20843, AD-20844, AD-20845, AD-20846, AD-20847, AD-20849, AD-20850, AD-20851, AD-5 20852, AD-20862, AD-20863, AD-20864, AD-20865, AD-20866, AD-20867, or modified or unmodified variants thereof.

[00786] In various embodiments, the disclosure comprises a RNAi agent comprising a sense and an antisense strand, wherein the antisense strand comprises at least 15 10 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of any RNAi agent disclosed herein, or modified or unmodified variants thereof, wherein the antisense strand optionally further comprises 0, 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nt (or any range thereof, e.g., 0-1, 1-2, 1-3, 1-4 nt, etc.).

[00787] Thus, in various embodiments, the disclosure comprises a RNAi agent 15 comprising a sense and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the antisense strand of: AD-20832; AD-20848; AD-20807; AD-20826; AD-20837; AD-20861; AD-20834, AD-20805, AD-20806, AD-20808, AD-20809, AD-20810, AD-20811, AD-20812, AD-20813, AD-20814, AD-20815, AD-20816, AD-20817, AD-20818, AD-20819, AD-20820, AD-20821, AD-20822, AD-20 20823, AD-20824, AD-20825, AD-20827, AD-20828, AD-20829, AD-20830, AD-20831, AD-20833, AD-20835, AD-20836, AD-20838, AD-20839, AD-20840, AD-20841, AD-20842, AD-20843, AD-20844, AD-20845, AD-20846, AD-20847, AD-20849, AD-20850, AD-20851, AD-20852, AD-20862, AD-20863, AD-20864, AD-20865, AD-20866, AD-20867, or modified or unmodified variants thereof, wherein the antisense strand optionally further comprises 0, 1, 25 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nt (or any range thereof, e.g., 0-1, 1-2, 1-3, 1-4 nt, etc.).

[00788] In various embodiments, the disclosure comprises a RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises or consists of the sequence of the first strand of, and the sequence of the second strand comprises or 30 consists of the sequence of the second strand of any RNAi agent disclosed herein, or modified or unmodified variants thereof.

[00789] Thus, in various embodiments, the disclosure comprises a RNAi agent comprising a first and a second strand, wherein the sequence of the first strand comprises or consists of the sequence of the first strand of, and the sequence of the second strand 35 comprises or consists of the sequence of the second strand of: AD-20832; AD-20848; AD-20807; AD-20826; AD-20837; AD-20861; AD-20834, AD-20805, AD-20806, AD-20808, AD-20809, AD-20810, AD-20811, AD-20812, AD-20813, AD-20814, AD-20815, AD-20816, AD-20817, AD-20818, AD-20819, AD-20820, AD-20821, AD-20822, AD-20823, AD-20824, AD- 100 2015221515 03 Sep 2015 20825, AD-20827, AD-20828, AD-20829, AD-20830, AD-20831, AD-20833, AD-20835, AD-20836, AD-20838, AD-20839, AD-20840, AD-20841, AD-20842, AD-20843, AD-20844, AD-20845, AD-20846, AD-20847, AD-20849, AD-20850, AD-20851, AD-20852, AD-20862, AD-20863, AD-20864, AD-20865, AD-20866, AD-20867. 5 [00790] In one embodiment, the disclosure comprises any one or more RNAi agent listed herein.

[00791] Overlapping sets of RNAi agents to Beta-ENaC

[00792] In various embodiments, the present disclosure relates to groups of RNAi agents 10 to Beta-ENaC with overlapping sequences. Thus, the present disclosure encompasses groups of RNAi agents wherein each RNAi agent in the group overlaps with each other RNAi agent in the same group by at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or more nucleotides. Particularly, in one embodiment, the overlap is at least 12 nt.

[00793] Some of the RNAi agents listed herein overlap each other in sequence. Table 2 15 presents a compilation of some of these groups of overlapping RNAi agents, wherein each member of a group overlaps with each other member of the same group by at least 12 nt. A 12-nt portion of the overlap of the sense and anti-sense strand are presented.

[00794] Thus, for example, as shown in Table 2, the sequences of RNAi agents AD-20807 and AD-20832 overlap, wherein the overlap in the sense strand comprises the 20 sequence UGAAGAAGUACC (SEQ ID NO: 223); these RNAi agents also overlap in the anti-sense strand sequence, wherein the overlap comprises the sequence GGUACUUCUUCA (SEQ ID NO: 224). The RNAi agents AD-20807, AD-20862 and AD-20832 all overlap in the sense strand, wherein the overlap comprises the sequence GAAGAAGUACCU (SEQ ID NO: 225); these RNAi agents also overlap in the anti-sense strand, wherein the overlap 25 comprises the sequence AGGUACUUCUUC (SEQ ID NO: 226). Thus, these and other various sets of overlapping RNAi agents presented in Table 2 share common technical features, for example, the overlap in the sense and anti-sense strand.

[00795] Particular sets of overlapping RNAi agents to Beta-ENaC are provided below in Table 2. 30 [00796] The present disclosure thus encompasses any group or subgroup of RNAi agents comprising a common technical feature, wherein the common technical feature is an overlap (e.g., of at least 12 nt) of a sequence in the sense or anti-sense strand.

[00797] Thus: [00798] The present disclosure encompasses a RNAi agent comprising: an antisense 35 strand comprising at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nt from the antisense strand, and/or a sense strand comprising at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the sense strand, of any of the group of: AD-20807 and AD-20832 (or any other group presented in Table 2). 101 2015221515 03 Sep 2015 [00799] The present disclosure encompasses a RNAi agent comprising a first and a second strand, wherein the first strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the first strand of, and/or the second strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nt from the second strand, of any of the group 5 of: AD-20807 and AD-20832 (or any other group presented in Table 2).

[00800] The present disclosure encompasses a RNAi agent comprising a first and a second strand, wherein the first strand comprises or consists of the sequence of a first strand of, and/or the second strand comprises or consists of the sequence of, any of the group of: AD-20807 and AD-20832 (or any other group presented in Table 2). 10 [00801] The disclose encompasses a RNAi agent comprising a first and a second strand (wherein the first and second strand may optionally be covalently linked, linked via a loop or linker, or contiguous), and wherein the first and/or second strand comprise, consist essentially of, or consist of sequences with 0, 1, 2, or 3 nt or bp mismatches of any of the group of: AD-20807 and AD-20832 (or any other group presented in Table 2), optionally 15 further comprising 0-10 nt or bp.

[00802] The present disclosure similarly encompasses various embodiments encompassing groups of overlapping RNAi agents presented in Table 2.

[00803] Additional Definitions 20 [00804] The articles “a” and “an” as used herein and in the claims refer to one or more than one (at least one) of the grammatical object of the article.

[00805] The terms “RNAi agent,” “RNAi agents”, “RNAi agent(s)” and the like all refer without limitation to one or more RNAi agents of the present disclosure.

[00806] The designations of particular example duplexes of RNAi agents to Beta-ENaC 25 disclosed herein on occasion have the suffix “b” followed by a number. This indicates a batch number. Thus, the suffix “b1” indicates “batch 1.” Thus, a RNAi duplex designated, for example, “AD-20807-b1” is specifically from batch 1 and has the same sequence as any RNAi agent designated “AD-20807”. 30 [00807] Unless defined otherwise, the technical and scientific terms used herein have the same meaning as that usually understood by a specialist familiar with the field to which the present disclosure belongs.

[00808] Unless indicated otherwise, all methods, steps, techniques and manipulations that are not specifically described in detail can be performed and have been performed in a 35 manner known per se, as will be clear to the skilled person. Reference is for example again made to the standard handbooks and the general background art mentioned herein and to the further references cited therein.

[00809] Claims to the present disclosure are non-limiting and are provided below. 102 2015221515 03 Sep 2015 [00810] Although particular embodiments and claims have been disclosed herein in detail, this has been done by way of example for purposes of illustration only, and is not intended to be limiting with respect to the scope of the appended claims, or the scope of subject matter of claims of any corresponding future application, in particular, it is 5 contemplated by the inventors that various substitutions, alterations, and modifications may be made to the present disclosure without departing from the spirit and scope of the present disclosure as defined by the claims. The choice of nucleic acid starting material, clone of interest, or library type is believed to be a matter of routine for a person of ordinary skill in the art with knowledge of the embodiments described herein. Other aspects, advantages, and 10 modifications considered to be within the scope of the following claims. Redrafting of claim scope in later-filed corresponding applications may be due to limitations by the patent laws of various countries and should not be interpreted as giving up subject matter of the claims.

[00811] Various additional formulations and obvious variants of the described RNAi agents to Beta-ENaC can be devised by those of ordinary skill in the art. Non-limiting 15 example RNAi agents to Beta-ENaC are described in the Examples below, which do not limit the scope of the present disclosure as described in the claims.

EXAMPLES 20 EXAMPLE 1

Bioinformatics and Beta-ENaC RNAi Agent (siRNA) sequences [00812] Beta-ENaC oligonucleotide design is carried out to identify siRNAs targeting mRNAs encoding the Beta-ENaC gene [“sodium channel, nonvoltage-gated 1 beta” from human (NCBI human symbol SCNN1B) and the orthologous sequences from cynomolgus 25 monkey (Macaca fascicularis) and rat (Rattus norvegicus)]. The design process uses the SCNNB1 transcripts NM_000336.2 from human (NCBI Geneld 6338), NM_012648.1 from rat (NCBI Geneld 24767), and a full length cynomolgus monkey sequence (described herein).

[00813] All siRNA duplexes are designed to have 100% identity to ali three SCNNB1 transcripts. All sequences are from Transcript NM_00Q336. 30 [00814] Unmodified and modified sequences are listed in Table 1. Unmodified sequences include both the sense and antisense sequences which are listed as SEQ ID NO: 111 to 220. The relative positions of the first residue as compared to the human Beta-ENaC transcript in SEQ ID NO: 222 are also provided.

[00815] As described below, Table 1 also provides example modified variants of these 35 sequences (SEQ ID NO: 1 to 110). For the Table 1 columns, “S” represents the sense strand, “AS” represents the antisense strand, and “Pos’n” represents the position of the first nucleotide. Modified nucleotides, as indicated by lower case letters (e.g., “c” and “u”) are as described in Table 1A, below. 103 2015221515 03 Sep 2015 [00816] In the sequences in Table 1, the modified and unmodified sequences can optionally comprise the sequence “dTsdT” at the 3’ end. Thus, for example, AD-20805 can optionally have the modified sequence cAGuGAcuAcAAcAcGAccdTsdT (SEG ID NO: 429) in the sense strand and GGUCGUGUUGuAGUcACUGdTsdT (SEQ ID NO: 430) in the anti-5 sense strand. As noted in Table 1A, below, dT is 2'-deoxy-thymidine-5’-phosphate and sdT is 2'-deoxy Thymidine S'-phosphorothioate.

Table 1. Beta-ENaC sequences

Duplex ID SEQ ID Modified sequence qi?n ID Unmodified sequence Pos' n AD- 20805 S 1 cAGuGAcuAcAAcAcGAcC 111 CAGUGACUACAACACGACC 1298 AS 2 GGUCGUGUUGuAGUcACUG 112 GGU CGUGUUGUAGU CACUG 1298 AD- 20806 S 3 AuGAcAGAGAAGGcAcuuc 113 AUGACAGAGAAGGCACUUC 1011 AS 4 GAAGUGCCOUCUCUGUcAU 114 vjAAGTJGCuuUCUCUG 1.JCAU 1011 AD- 20807 s 5 GuGAAGAAGuAc cuGCuGA 115 GUGAAGAAGUACCUGCUGA 183 AS 6 UcAGcAGGuACUUCUUcAC 116 ϋ CAGCAGGUACUU CUU CAC 183 AD- 20808 S 7 GuGAcuAcAAcAcGAcCuA 117 GUGACUACAACACGAC CUA 1300 AS 8 uAGGUCGUGUUGuAGUcAC 118 UAGGUCGUGUUGUAGUCAC 1300 AD- 20809 S 9 GGuGGAGGcccAcAc cAAc 119 GGUGGAGGCCCACACCAAC 1919 AS 10 GUUGGUGUGGGC CUCcACC 120 GUUGGUGUGGGC CUC CAC C 1919 AD- 2 0810 S 11 uGGuGGAGGc ccAcAccAA 121 UGGUGGAGGC CCACACCAA 1918 AS 12 UUGGUGUGGGCCUCcACcA 122 UUGGUGUGGGCCUCCACCA 1918 AD- 2 0811 S 13 uuccAAGAccAcAuGAucC 123 UUC CAAGAC CACAUGAUC C .1347 AS 14 GGAUcAUGUGGUCUUGGAA 124 GGAU CAUGUGGU CUUGGAA 1347 AD- 20812 S 15 AGcuGGGAGGucAGcGucu 125 AG CJUGGG AGGU C AGCGU CU 4 02 AS 16 AGA CGCUGACCUCCcAGCU 12 6 AGACGCUGAC CUC C C AGCU 402 AD- 20813 S 17 GGGAGAAAuAc uGcAAcAA 12 7 GGGAGAAAUACUGCAACAA 14 08 AS 18 UUGUUGcAGuAUUUCUC C C 12 8 UUGUUGCAGUAUUUCUCCC 14 08 AD- 20814 S 19 c cAGuuuGGcuucuGGAuG 12 9 CCAGUUUGGCUUCUGGAUG 1748 AS 20 cAUCcAGAAGCcAAACUGG 130 CAUCCAGAAGCCAAACUGG 1748 AD- 20815 S 21 AGuGAcuAcAAcAcGAc cu 131 AGUGACUACAACACGACCU 1299 AS 22 AGGU CGUGUUGuAGU cACU 132 AGGU CGUGUUGUAGUCACU 1299 AD- 20816 S 23 AAuAucAcccuGAGcAGGA 133 AAUAUCAC C CUGAGCAGGA 1626 AS 24 UC CUGCU cAGGGUGAuAUU 134 DC CUGCU CAGGGUGAUAUU 1626 104 2015221515 03 Sep 2015 AD-2 0817 s 25 c cuGcAGGc cAccAAcAuc 135 C CUGCAGGCCACCAACAUC 836 AS 2 6 GAUGUUGGUGGCCUGcAGG 136 GAUGUUGGUGGCCUGCAGG 836 AD -20818 s 27 An c Ac c c uGAGcAGGAAGG 13 7 AUCAC C CUGAGCAGGAAGG 1629 AS 2 8 C CUUC CUGCucAGGGUGAU 138 C CUU C CUGCUCAGGGUGAU 1629 AD- 20819 S 2 9 G c uGGGAGGucAGcGucuc 139 GCUGgGAGGUCAgCGJCUC 4 03 AS 3 0 GAGACGCUGACCUCCcAGO 14 0 GAGACGCUGAC CUC C CAGC 4 03 AD- 20820 S 31 GAG c uGGGAGGucAG cGuc 141 GAGCUGGGAGGUCAGCGUC 4 01 AS 32 GACGCUGACCUCCcAGCUC 142 GACGCUGACCUCCCAGCUC 4 01 AD- 20821 S 33 GuGG c cAGuuuGGcuucuG 143 GUGGCCAGUUUGGCUUCUG 1744 AS 34 cAGAAGCcAAACUGGCcAC 144 CAGAAGC C AAACUGGC CA CJ 1744 AD- 20822 S 35 c AGu uuGG c uucuGGAuGG 145 CAGUUUGGCUUCUGGAUGG 174 9 AS 3 6 C cAUCcAGAAGC cAAACUG 14 6 C CAUC CAGAAG C CAAACUG 174 9 AD- 20823 S 3 7 GGc cAGuuuGGcuucuGGA 14 7 GGCCAGUUUGGCUUCUGGA 174 6 AS 3 8 TJC c AGAAGC c AAACUGGC C 14 8 UCCAGAAGCCAAACUGGCC 174 6 AD- 20824 S 3 9 cuGGGuGGc cAGuuuGGcu 14 9 CUGGGUGGCCAGUUUGGCU 174 0 AS 4 0 AGG cAAACUGGC cAC C cAG 150 AGC CAAACUGGC CACCCAG 1740 AD- 20825 S 41 ucuAcAGuGAcuAcAAcAc 151 UCUACAGUGACUACAACAC 1294 AS 42 GUGUUGuAGUcACUGuAGA 152 GUGUUGUAGUCACUGUAGA 1294 AD- 20826 s 43 GcAuGAcAGAGAAGGcAcu 153 GCAUGACAGAGAAGGCACU 10 0 9 AS 44 AGUGC CUUCuCUGUcAUGC 154 AGUGC CUUCUCUGUCAUGC 10 0 9 AD- 20827 s 45 AuAucAcc cuGAGcAGGAA 155 AUAUC A CJ C CUGAGCAGGAA 1627 AS 4 6 UU C CUGCU cAGGGUGAuAU 156 UU C CUGCU CAGGGUGAUAU 1627 AD -20828 s 47 cuAcAGuGAcuAcAAcAcG 157 CUACAGUGACUACAACACG 12 95 AS 4 8 CGUGUUGuAGUc ACUGuAG 158 CGUGUUGUAGUCACUGUAG 12 95 AD- 20829 S 4 9 uAucAcc cuGAGcAGGAAG 159 UAUCAC C CUGAGCAGGAAG 1628 AS 5 0 CUU C CUGCU cAGGGUGAuA 16 0 CUU C CUGCU CAGGGUGAUA 1628 AD- 20830 S 51 uGcAGGc cAccAAcAucuu 161 UG CAGGC CAC CAACAUCUU 838 AS 52 AAGAUGUUGGUGGCCUGcA 162 AAGAUGUUGGUGGCCUGCA 838 AD- 20831 S 53 cAuGAcAGAGAAGGcAcuu 163 CAUGACAGAGAAGGCACUU 1010 AS 54 AAGUG CCUUCUC UGUcAUG 164 AAGUGC CUUCUCUGUCAUG 1010 AD- 20832 S 55 uGAAGAAGuAc ciuGc uGAA 165 UGAAGAAGUA C CUGCUGAA 184 AS 56 UUcAGcAGGuACUUCUUcA 16 6 UU CAGCAGGUACUUCUUCA 184 AD- 20833 S 57 GcuGGuGGAGGc c cAcAcc 16 7 G CUGGUGGAGG C C CACACC 1916 105 2015221515 03 Sep 2015 AS 58 GGUGUGGGCCUCcACcAGC 168 GGUGUGGGCCUCCACCAGC 1916 AD- 20834 s 59 UAcAGuGAcuAcAAcAcGA 169 UACAGUGACUACAACACGA 1296 AS 6 0 UCGUGUUGuAGUcACUGuA 17 0 UCGUGUUGUAGUCACUGUA 1296 AD-2 083 5 s 61 AcAGAGAAGGcAcuuccuu 171 ACAGAGAAGGCACUUCCUU 1014 AS 62 AAGGAAGUGC CUUCUCUGU 172 AAGGAAGUGCCUUCUCUGU 1014 AD -20836 s 63 AcAGuGA CllAcAAcA c: GAc 173 ACAGUGACUACAACACGAC 1297 AS 64 GUCGUGUUGuAGUcACUGU 174 GU CGUGUUGUAGUCACUGU 1297 AD-2 0837 S 6 5 uG AG c uGGG AGGu c AG cGu 175 UGAGCUGGGAGGUCAGCGU 4 0 0 AS 6 6 ACGCUGACCUCCcAGCUcA 176 ACGCUGAC CUC C CAGCUCA 4 0 0 AD- 20838 S 6 7 uGGecAGuuuGGcuucuGG 177 UGGCCAGUUUGGCUUCUGG 174 5 AS 68 C cAGAAGC cAAACUGGCcA 178 C CAGAAGC CAAACUGGCCA 174 5 AD- 20839 S 69 uGucucAGGAGcGGGAccA 17 9 UG UCUCAGGAGCGGGACCA 1600 AS 70 T.TGGUC C CGCTJC CUGAGAcA 18 0 UGGU C C CGCU CJ CUGAGACA 16 0 0 AD- 20840 S 71 GuGGAGGec cAcAccAAcu 181 GUGGAGGCCCACACCAACU 192 0 AS 72 AGUUGGUGUGGG C CUC cAC 182 AGUUGGUGUGGGC CUC CAC 192 0 AD- 20841 S 73 GGGu GG c c AGuu uGG G uu c 183 GGGUGGCCAGUUUGGCUUC 1742 AS 74 GAAGC GAAACUGGCGACCC 184 GAAGCCAAACUGGCCACCC 1742 AD- 20842 S 75 GGuGGe cAGuuuGGcuucu 18 5 GGUGGCCAGUUUGGCUUCU 1743 AS 76 AGAAGC cAAACUGGC cACC 18 6 AGAAGC CAAACUGG C CACC 1743 AD- 20843 S 77 ucAcc cuGAGeAGGAAGGG 187 UCAC C C UGAGCAGGAAGGG 1630 AS 78 CCCUUCCUGCUCAGGGuga 188 C C CUU C CUGCUCAGGGUGA 1630 AD-2 0844 s 7 9 Gc cAGuuuGGc uucuGGAu 189 GCCAGUUUGGCUUCUGGAU 174 7 AS 8 0 AUCcAGAAGCcAAACUGGC 19 0 AUCCAGAAGCCAAACUGGC 174 7 AD -2 084 5 s 81 AGguGGuGGAGGcc cAcAc 191 AGCUGGUGGAGGCCCACAC 1915 AS 82 G u GUGGG CCwC c AC cAGCIJ 192 GUGUGGGC CU C CAC CAGCU 1915 AD -20846 s 83 Au c a c c Au GG c aG A c u GG c 193 AUCUC CAUGGCUGACUGGC 154 5 AS 84 GCgAGUcAGCgAUGGAGAU 194 GCCAGUCAGCCAUGGAGAU 1545 AD- 20847 S 8 5 GGgAuGAcAGAGAAGGcAg 195 GGCAUGACAGAGAAGGCAC 10 08 AS 8 6 GUGCCUUCUCUGUcAUGCC 196 GUGCCUUCUCUGUCAUGCC 10 08 AD- 20848 S 8 7 GGAGAAAuAcuGcAAcAAc 197 GGAGAAAUACUGCAACAAC 14 0 9 AS 88 GUUG UUGcAGuAUUUCUCC 198 GUUGUUGCAGUAUUUCUCC 1409 AD- 20849 S 89 uGGGuGGc c AGuuuGGc uu 199 UGGGUGGCCAGUUUGGCUU 1741 AS 90 AAGCcAAACUGGCcACCcA 2 00 AAG C CAAACUGGCCACC C A 1741 2015221515 03 Sep 2015 106 AD- 20850 s 91 GAGcuGGuGGAGGcccAcA 2 01 GAGCUGGUGGAGGCCCACA 1914 AS 92 UGUGGGCCUCcACcAGCUC 2 02 UGUGGG CJ CUC C AC CAGCUC 1914 AD- 20851 s 93 GAcAGAGAAGGcAcuuc cu 2 03 GACAGAGAAGGCACUUCCU 1013 AS 94 AGGAAGUGC CUUCUCUGUC 204 AGGAAGUGC CUU CU CUGU C 1013 AD- 20852 S 95 AGuuuGG cuucuGGAuGGG 2 05 AGUUUGGCUUCUGGAUGGG 1750 AS 96 C C GAUC CAGAAGCcAAACU 2 06 C C CAUC CAGAAGC CAAACU 1750 AD- 20861 S 97 uGAcAGAGAAGGcAcuuc c 2 07 UGACAGAGAAGGCACUU C C 1012 AS 98 GGAAGUGC CUU CU CUGU cA 2 08 GGAAGUGC CUU CU CUGU CA 1012 AD- 20862 S 99 GAAGAAGuAc cuGcuGAAG 2 09 GAAGAAGUACCUGCUGAAG 185 AS 100 CUU cAGcAGGuACUUCUU C 210 CUU CAGCAGGUACUU CUU CJ 185 AD- 20863 S 101 ucuc CAuGGcuGAcuGGc c 211 U CU C CAUGGCUGACUGGCC 1546 AS 102 GGCcAGUcAGCcAUGGAGA 212 GGCCAGUCAGCCAUGGAGA 1546 AD- 20864 S 103 cuGGuGGAGGc c: c Ac Ac cA 213 CUGGUGG AGG C C C AC AC C A 1917 AS 104 UGGUGUGGGC CUC cACcAG 214 UGGUGUGGGC CUC CAC CAG 1917 AD- 20865 S 105 cAGAGAAGGcAcuuccuuc 215 CAGAGAAGGCACUU C CUU C 1015 AS 106 GAAGGAAGUGCCUUCUCUG 216 GAAGGAAGUGCCUUCUCUG 1015 AD- 20866 S 107 CuGcAGGc CAc cAAcAucu 217 CUGCAGGCCACCAACAUCU 837 AS 108 AGAUGUUGGUGGCCUGcAG 218 AGAUGUUGGUGGCCUGCAG 837 AD- 20867 s 109 GGGcAuGAcAGAGAAGGcA 219 GGGCAUGACAGAGAAGGCA 1007 AS 110 UGC CUU CU CUGU cAUG CCC 2 2 0 UGC CUUCUCUGUCAUGC C C 1007 [00817] Modifications of the sequences of RNAi agents of SEQ ID NO: 111 to 220 are easily conceived by one of skill in the art. Examples and non-limiting modifications of these sequences are conceived and are also listed in Table 1, e.g., the sense and antisense (AS) 5 sequences in SEQ ID NO: 1 to 110.

[00818] Some modifications are placed at sites predicted to be sensitive to endonucleases. Some modifications are designed to eliminate an immune response to the siRNA while preserving activity. In general, the sense strand is heavily modified, and the antisense strand lightly modified. Some modifications serve more than one purpose. 10 [00819] The sequences in Table 1 and other tables are represented by these abbreviations:

TABLE 1 A. ABBREVIATIONS

Abbreviation Nucleotide(s) A adenosine-5’-phosphate C cytidine-5’-phosphate 107

Abbreviation Nucleotide(s) G guanosine-5’-phosphate dT 2'-deoxy-thymidine-5’-phosphate U uridine-5’-phosphate c 2'-0-methy!cytidine-5’-phosphate u 2‘-0-methy!uridine-5’-phosphate sdT 2'-deoxy Thymidine S'-phosphorothioate 2015221515 03 Sep 2015 [00820] siRNA sequence selection [00821] A total of 55 sense and 55 antisense human SCNNBI-derived siRNA oligos (RNAi agents to Beta-ENaC) are synthesized, as described in Example 2. The sense and 5 their respective antisense oligos are annealed into duplexes.

EXAMPLE1A

Overlapping sets of Beta-ENaC RNAi Agents 10 [00822] The present disclosure also relates to groups of RNAi agents to Beta-ENaC with overlapping sequences. Thus, the present disclosure encompasses groups of RNAi agents wherein each RNAi agent in the group overlaps with each other RNAi agent in the same group by at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or more nucleotides.

Particularly, in one embodiment, the overlap is at least 12 nt. 15 [00823] Some of the RNAi agents listed herein overlap each other in sequence. Table 2 presents a compilation of some of these groups of overlapping RNAi agents, wherein each member of a group overlaps with each other member of the same group by at least 12 nt. A 12-nt portion of the overlap of the sense and anti-sense strand are presented.

[00824] Thus, for example, as shown in Table 2, the sequences of RNAi agents AD-20 20807 and AD-20832 overlap, wherein the overlap in the sense strand comprises the sequence UGAAGAAGUACC (SEQ ID NO: 223); these RNAi agents also overlap in the anti-sense strand sequence, wherein the overlap comprises the sequence GGUACUUCUUCA (SEQ ID NO: 224). The RNAi agents AD-20807, AD-20862 and AD-20832 all overlap in the sense strand, wherein the overlap comprises the sequence GAAGAAGUACCU (SEQ ID NO: 25 225); these RNAi agents also overlap in the anti-sense strand, wherein the overlap comprises the sequence AGGUACUUCUUC (SEQ ID NO: 226). Thus, these and other various sets of overlapping RNAi agents presented in Table 2 share common technical features, for example, the overlap in the sense and anti-sense strand.

[00825] Particular sets of overlapping RNAi agents to Beta-ENaC are provided below in 30 Table 2. 108 2015221515 03 Sep 2015 [00826] The present disclosure thus encompasses any group or subgroup of RNAi agents comprising a common technical feature, wherein the common technical feature is an overlap (e.g,, of at least 12 nt) of a sequence in the sense or anti-sense strand. 5 Table 2.

Pos Sense overlap SEQ ID Anti-sense overlap SEQ ID Overlapping RNAi agents to Beta-ENaC 183 UGAAGAAGUACC 223 GGLiACUUC UTICA 224 AD-20807, AD-20832 184 GAAGAAGUACCU 225 AGGUACUUCUUC 226 AD-20807, AD-20862, AD- 20832 185 AAGAAGUACCUG 227 C AGGUACUUCUU 228 AD-20807, AD-20862, AD-20832 186 AGAAGUACCUGC 229 GCAGGUACUUCU 230 AD-20807, AD-20862, AD-20832 187 GAAGUACCUGCU 231 AGCAGGUACUuC 232 AD-20807, AD-20862, AD-20832 188 AAGUACCUGCUG 233 CAGCAGGUACUU 234 AD-20807, AD-20862, AD-20832 189 AGUACCUGCUGA 235 U CAGCAGGUACU 236 AD-20807, AD-20862, AD-20832 190 GUACCUGCUGAA 237 UUCAGCAGGUAC 238 AD-20862, AD-20832 400 GAGCUGGGAGGU 239 ACCUCCCAGCUC 240 AD-20820, AD-20837 401 AGCUGGGAGGUC 241 GACCUCCCAGCU 242 AD-20820, AD-20812, AD-20837 402 GCUGGGAGGUCA 243 UGACCUCCCAGC 244 AD-20820, AD-20819, AD-20812, AD-20837 403 CUGGGAGGUCAG 245 CUGACCUCCCAG 246 AD-20819, AD-20812, AD-20837 404 U GGGAGGU GAG C 247 GCUGACCUCCCA 248 AD-20820, AD-20819, AD-20837 405 GGGAGGUCAGC G 249 CGCUGACCUCCC 250 AD-20820, AD-20819, AD-20812. AD-20837 406 GGAGGUCAGCGU 251 ACGCUGACCUCC 252 AD-20819. AD-20837 407 rGAGGUCAGCGUC 253 GACGCUGACCUC 254 AD-20820, AD-20819, AD-20812 408 AGGUCAGCGUCU 255 AGACGCUGACCU 256 AD-20819, AD-20812 836 CUGCAGGCCACC 257 GGUGGCCTJGCAG 258 AD-20866. AD-20817 837 UGCAGGCCACCA 259 UGGUGGCCUGCA 260 AD-20866, AD-20830, AD-20817 838 GCAGGCCACCAA 261 UUGGUGGCCUGC 262 AD-20866, AD-20830, AD-20817 839 CAGGCCACCAAC 263 GUUGGUGGCCUG 264 AD-20866, AD-20830, AD-20817 840 AGGCCACCAACA 265 UGU’JGGUGGCCU 266 AD-20866, AD-20830, AD-20817 841 GGCCACCAACAU 267 AUGUIJ GGUGGC C 268 AD-20866, AD-20830, AD-20817 2015221515 03 Sep 2015 109 842 GCCACCAACAUC 269 GAU GUUGGUGGC 270 AD-20866, AD-20830, AD-20817 843 CCACCAACAUCU 271 AGAUGUUGGUGG 272 AD-20866, AD-20830 1007 GGCAUGACAGAG 273 CUCTJGUCAUGCC 274 AD-20847, AD-20867 1008 GCAUGACAGAGA 275 UCUCUGUCAUGC 276 AD-20826, AD-20867 1009 CAUGACAGAGAA 277 UUCUCUGUCAUG 278 AD-20826, AD-20831, AD-20867 1010 AUGACAGAGAAG 279 C UU CUCUGUCAU 280 AD-20826, AD-20831, AD-20867, AD-20806 1011 UGACAGAGAAGG 281 C CUUCUCUG UC A 282 AD-20826, AD-20831, AD-20867, AD-20806, AD-20861 1012 GACAGAGAAGGC 283 GCCUUCUCUGUC 284 AD-20851, AD-20847, AD-20826, AD-20831, AD-20867, AD-20806, AD-20861 1013 ACAGAGAAGG CA 285 UGCCUUCUCUGU 286 AD-20851, AD-20835, AD-20847, AD-20826, AD-20831, AD-20867, AD-20806, AD-20861 1014 CAGAGAAGGCAC 287 GUGCCUUCUCUG 288 AD-20851, AD-20835, AD-20865, AD-20826, AD-20831, AD-20806, AD-20861 1015 AGAGAAGGCACU 289 AGUGCCUUCUCU 290 AD-20851, AD-20835, AD-20865, AD-20826, AD-20831, AD-20806, AD-20861 1016 GAGAAGGCACUU 291 AAGUGCCUUCUC 292 AD-20851, AD-20835, AD-20865, AD-20831, AD-20806, AD-20861 1017 AGAAGGCACUUC 293 GAAGUGCCUUCU 294 AD-20851, AD-20835, AD-20865, AD-20806, AD-20861 1018 GAAGGCACUUCC 295 GGAAGUGCCUUC 296 AD-20851, AD-20835, AD-20865, AD-20861 1019 AAGGCACUUCCU 297 AGGAAGUGC C UU 298 AD-20851, AD-20835, AD-20865 1020 AGGCACUUCCUU 299 AAGGAAGUGCCU 300 AD-20835, AD-20865 1294 CUACAGUGACUA 301 UAGUCACUGUAG 302 AD-20828, AD-20825 1295 UACAGUGACUAC 303 GUAGUCACUGUA 304 AD-20834, AD-20825 1296 ACAGUGACUACA 305 UGUAGUCACUGU 306 AD-20828, AD-20834, AD-20825, AD-20836 1297 CAGUGAC UACAA 307 UUGUAGUCACUG 308 AD-20834, AD-20805, AD-20825 1298 AGUGACUACAAC 309 GUUGUAGUCACU 310 AD-20828, AD-20834, AD-20805, AD-20825, AD-20836 1299 GUGACUACAACA 311 UGUuGUAGUCAC 312 AD-20834, AD-20805, AD-20808, AD-20825 1300 UGACUACAACAC 313 GUGUUGUAGUCA 314 AD-20828, AD-20834, AD-20805, AD-20808, AD-20825, AD-20815, AD-20836 1301 HgACUAC A.A.C A C G 315 CGUGUUGUAGUC 316 AD-20828, AD-20834, AD- 2015221515 03 Sep 2015 110 20805, AD-20808, AD-20836 1302 ACUACAACACGA 317 UCGUGUUGUAGU 318 AD-20834, AD-20805, AD-20808 1303 CUACAACACGAC 319 GUCGUGUUGUAG 320 AD-20805, AD-20808, AD-20815, AD-20836 1304 UACAACACGAC C 321 GGUCGUGUUGUA 322 AD-20805, AD-20808 1305 ACAACAC GA C CU 323 AGGUCGUGUUGU 324 AD-20808, AD-20815 1408 GGAGAAAUACUG 325 CAGUAUUUCUCC 326 AD-20813, AD-20848 1409 GAGAAAUACUGC 327 GCAGUAUUUCUC 328 AD-20813, AD-20848 1410 AGAAAUACUGCA 329 UGCAGUAUUUCU 330 AD-20813, AD-20848 1411 GAAAUACUGCAA 331 UUGCAGUAUUUC 332 AD-20813, AD-20848 1412 AAAUACUGCAAC 333 GUUGCAGUAUUU 334 AD-20813, AD-20848 1413 AAUACUGCAACA 335 UGUUGCAGUAUU 336 AD-20813, AD-20848 1414 AUACUGCAACAA 337 IJUGUUGCAGUAIJ 338 AD-20813, AD-20848 1545 UCUCCAUGGCUG 339 CAGCCAUGGAGA 340 AD-20846, AD-20863 1546 CUC CAUGGCUGA 341 UCAGCCAUGGAG 342 AD-20846, AD-20863 1547 iJCCAUGGCUGAC 343 GUCAGCCAUGGA 344 AD-20846, AD-20863 1548 CCAUGGCUGACU 345 AGUCAGCCAUGG 346 AD-20846, AD-20863 1549 CAUGGCUGACUG 347 CAGUCAGCCAUG 348 AD-20846, AD-20863 1550 AUGGCUGACUGG 349 CCAGUCAGCCAU 350 AD-20846, AD-20863 1551 UGGCUGACUGGC 351 GCCAGUCAGCCA 352 AD-20846, AD-20863 1626 AUAUCACCCUGA 353 UCAGGGUGAUAU 354 AD-20816, AD-20827 1627 UAUCACCCUGAG 355 CUCAGGGUGAUA 356 AD-20816, AD-20827, AD-20829 1628 AUCACCCUGAGC 357 GCUCAGGGUGAU 358 AD-20816, AD-20827, AD-20829, AD-20818 1629 UCACCCUGAGCA 359 UGCUCAGGGUGA 360 AD-20816, AD-20827, AD-20829, AD-20843, AD-20818 1630 CACCCTJGAGCAG 361 CUGCUCAGGGUG 362 AD-20816, AD-20827, AD-20829, AD-20843, AD-20818 1631 ACCCUGAGCAGG 363 CCUGCUCAGGGU 364 AD-20816, AD-20827, AD-20829, AD-20843, AD-20818 1632 CCCUGAGCAGGA 365 UCCUGCUCAGGG 366 AD-20816, AD-20827, AD-20829, AD-20843, AD-20818 1633 C CUGAGCAGGAA 367 UUCCUGCUCAGG 368 AD-20827, AD-20829, AD-20843, AD-20818 1634 CUGAGCAGGAAG 369 CUUCCUGCUCAG 370 AD-20829, AD-20843, AD-20818 1635 UGAGCAGGAAGG 371 CCUUCCUGCUCA 372 AD-20843, AD-20818 1740 1741 UGGGUGGCCAGIJ 373 ACUGGCCACCCA 374 AD-20824, AD-20849 GGGUGGCCAGUIJ 375 AACUGGC CACC C 376 AD-20824, AD-20841, AD-20849 1742 GGUGGCCAGUUU 377 AAACUGGCCACC 378 AD-20824, AD-20842, AD-20841, AD-20849 1743 GUGGC CAGUUUG 379 CAAACUGGCCAC 380 AD-20824, AD-20842, AD-20821, AD-20841, AD-20849 1744 UGGCCAGUUUGG 381 CCAAACUGGCCA 382 AD-20824, AD-20842, AD- 2015221515 03 Sep 2015 111 20821, AD-20838, AD-20841, AD-20849 1745 GGC CAGUTJLT GGC 383 GCCAAACUGGCC 384 AD-20824, AD-20842, AD-20821, AD-20838, AD-20841, AD-20823, AD-20849 1746 GCCAGUUUGGCU 385 AGCCAAACUGGC 386 AD-20844, AD-20824, AD-20842, AD-20821, AD-20838, AD-20841, AD-20823, AD-20849 1747 CCAlG(JUTJGGCUU 387 AAGCCAAACUGG 388 AD-20814, AD-20844, AD-20842, AD-20821, AD-20838, AD-20841, AD-20823, AD-20849 1748 CAGUTJUGGCULTC 389 GAAGCCAAACUG 390 AD-20814, AD-20844, AD-20842, AD-20821, AD-20838, AD-20841, AD-20822, AD-20823 1749 AGLJUTJ GG CUU C U 391 AGAAGCCAAACU 392 AD-20814, AD-20844, AD-20842, AD-20821, AD-20852, AD-20838, AD-20822, AD-20823 1750 GUUUGGCUUCUG 393 CAGAAGCCAAAC 394 AD-20814, AD-20844, AD-20821, AD-20852, AD-20838, AD-20822, AD-20823 1751 UUUGGCUUCUGG 395 CCAGAAGCCAAA 396 AD-20814, AD-20844, AD-20852, AD-20838, AD-20822, AD-20823 1752 UUGGCUUCUGGA 397 UCCAGAAGCCAA 398 AD-20814, AD-20844, AD-20852. AD-20822. AD-20823 1753 UGGCUUCUGGAU 399 AUCCAGAAGCCA 400 AD-20814, AD-20844, AD-20852, AD-20822 1754 GGCUUCUGGAUG 401 CAUCCAGAAGCC 402 AD-20814, AD-20852, AD- 20822 1755 GCUUCUGGAUGG 403 CCAUCCAGAAGC 404 AD-20852, AD-20822 1914 AGCUGGUGGAGG 405 CCUCCACCAGCU 406 AD-20850, AD-20845 1915 GCUGGUGGAGGC 407 GCCUCCACCAGC 408 AD-20850, AD-20845, AD-20833 1916 CUGGUGGAGGCC 409 4ΪΪ GGCCUCCACCAG 410 AD-20850, AD-20845, AD-20833, AD-20864 1917 UGGUGGAGGCCC GGGCCUCCACCA 412 AD-20810, AD-20850, AD-20845, AD-20833, AD-20864 1918 GGUGGAGGCCCA 413 UGGGCCUCCACC 414 AD-20809, AD-20810, AD-20850, AD-20845, AD-20833, AD-20864 1919 GUGGAGGCCCAC 415 GUGGGCCUCCAC 416 AD-20809, AD-20810, AD-20850, AD-20845, AD-20833, AD-20864, AD-20840 1920 UGGAGGCCCACA 417 UGUGGGCCUCCA 418 AD-20809, AD-20810, AD- 112 20850, AD-20845, AD-20833, AD-20864, AD-20840 1921 GGAGGCCCACAC 419 GUGUGGGCCUCC 420 AD-20809, AD-20810, AD-20845, AD-20833, AD-20864, AD-20840 1922 GAGGCCCACACC 421 GGUGUGGGC C U C 422 AD-20809, AD-20810, AD-20833, AD-20864, AD-20840 1923 AGGCCCACACCA 423 UGGUGUGGGCCU 424 AD-20809, AD-20810, AD-20864, AD-20840 1924 GGCCCACACCAA 425 UUGGUGUGGGCC 426 AD-20809, AD-20810, AD-20840 1925 GCCCACACCAAC 427 GITJGGUGUGGGC 428 AD-20809, AD-20840 [00827] The position (“Pos”) in NM_000336,2 is indicated. 12 exemplary nt of the overlap in the sense and anti-sense strand are presented; in many cases, the overlap is actually longer. 2015221515 03 Sep 2015 5 EXAMPLE 2

Synthesis of Beta-ENaC RNAi Agent Sequences [00828] The modified Beta-ENaC RNAi agent sequences listed as SEQ ID NO: 1 to 110 10 in Table 1 are synthesized on MerMade 192 synthesizer at 1 .iimol scale.

[00829] For ail the sequences in the list, 'endolighf chemistry is applied as detailed below.

[00830] All pyrimidines (cytosine and uridine) in the sense strand contain 2’-0-Methyi bases (2’ Ο-Methyl C and 2’-0-Methyi U). 15 [00831] In the antisense strand, pyrimidines adjacent to (i.e., towards the 5’ position) ribo A nucleoside are replaced with their corresponding 2-O-Methyl nucleosides.

[00832] A two-base dTsdT extension at 3’ end of both sense and anti sense sequences is introduced.

[00833] The sequence file is converted to a text file to make it compatible for loading in 20 the MerMade 192 synthesis software.

[00834] Synthesis, Cleavage and deprotection: [00835] The synthesis of Beta-ENaC sequences uses solid supported oligonucleotide synthesis using phosphoramidite chemistry.

[00836] The synthesis of the above sequences is performed at 1 urn scale in 96 well 25 plates. The amidite solutions are prepared at 0.1M concentration and ethyl thio tetrazole (0.6M in Acetonitrile) is used as activator.

[00837] The synthesized sequences are cleaved and de-protected in 96 well plates, using methylamine in the first step and fluoride reagent in the second step. The crude sequences 113 2015221515 03 Sep 2015 are precipitated using acetone: ethanol (80:20) mix and the pellets are re-suspended in 0.2M sodium acetate buffer. Samples from each sequence are analyzed by LC-MS to confirm the identity, UV for quantification and a selected set of samples by I EX chromatography to determine purity. 5 [00838] Purification and desalting: [00839] Beta-ENaC sequences are purified on AKTA explorer purification system using Source 15Q column. A column temperature of 65 C is maintained during purification.

Sample injection and collection is performed in 96 well (1.8mL -deep well) plates. A single peak corresponding to the full length sequence is collected in the eluent. The purified 10 sequences are desalted on a Sephadex G25 column using AKTA purifier. The desalted Beta-ENaC sequences are analyzed for concentration (by UV measurement at A260) and purity (by ion exchange HPLC).

[00840] The single strands are then submitted for annealing.

[00841] A detailed list of Beta-ENaC single strands and duplexes are shown in Table 1, 15 above. The duplexes are used in in vitro screening to test their ability to knock down Beta- ENaC gene level. EXAMPLE 3.

In vitro screening of Beta-ENaC RNAi Agents 20 [00842] The Beta-ENaC RNAi agents are screened in vitro to determine their ability to knock down Beta-ENaC gene level· [00843] Cell culture and transfections: [00844] H441 (ATCC, Manassas, VA) cells are grown to near confluence at 37°C in an atmosphere of 5% C02 in RPMI 1640 (ATCC) supplemented with 10% FBS, streptomycin, 25 and glutamine (ATCC) before being released from the plate by trypsinization. Reverse transfection is carried out by adding 5 μΙ of Opti-MEM to 5 μ! of siRNA duplexes per weli into a 96-well plate along with 10 μΙ of Opti-MEM plus 0.2 μ! of Lipofectamine RNAIMax per well (Invitrogen, Carlsbad CA. cat # 13778-150) and incubated at room temperature for 15 minutes. 80 μ! of complete growth media without antibiotic containing 2.0 x104 H441 cells are 30 then added. Ceils are incubated for 24 hours prior to RNA purification. Experiments are performed at 0.1 or 10 nM final duplex concentration for single dose screens with each of the 55 Beta-ENaC duplexes. Each siRNA is transfected 3 times at each of the doses tested.

The results are shown in Table 3.

[00845] A subset of duplexes that shows robust silencing in the 10 nM and 0.1 nM 35 screens is assayed over a range of concentrations from 10 nM to 10 fM using serial dilutions to determine their IC50. The results are shown in Table 4. 114 2015221515 03 Sep 2015 [00846] Total RNA isolation: [00847] Cells are harvested and lysed in 140 μ! of Lysis/Binding Solution then mixed for 1 minute at 850 rpm using an Eppendorf Thermomixer (the mixing speed was the same throughout the process). 5 [00848] A MagMAX-96 Total RNA Isolation Kit (Applied Biosystem, Foster City CA, part #: AM 1830) is used to isolate total RNA. Twenty micro liters of magnetic beads and Lysis/Binding Enhancer mixture are added into cell-lysate and mixed for 5 minutes.

Magnetic beads are captured using magnetic stand and the supernatant is removed without disturbing the beads. After removing supernatant, magnetic beads are washed with Wash 10 Solution 1 (isopropanol added) and mixed for 1 minute. Beads are capture again and supernatant removed. Beads are then washed with 150 μΙ Wash Solution 2 (Ethanol added), captured and supernatant is removed. 50 ul of DNase mixture (MagMax turbo DNase Buffer and Turbo DNase) is then added to the beads and they are mixed for 10 to 15 minutes. After mixing, 100 μΙ of RNA Rebinding Solution is added and mixed for 3 minutes. Supernatant is 15 removed and magnetic beads are washed again with 150 μ! Wash Solution 2 and mixed for 1 minute and supernatant is removed completely. The magnetic beads are mixed for 2 minutes to dry before RNA is eluted with 50 μ! of water.

[00849] cDNA synthesis: [00850] ABI High capacity cDNA reverse transcription kit (Applied Biosystems, Foster 20 City, CA, Cat #4368813) is used for cDNA synthesis. A master mix of 2 ui 10X Buffer, 0.8 ui 25X dNTPs, 2 μΙ Random primers, 1 μ! Reverse Transcriptase, 1 ui RNase inhibitor and 3.2 μί of H20 per reaction are added into 10 μ! total RNA. cDNA is generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, CA) through the following steps: 25°C 10 min, 37°C 120 min, 85°C 5 sec, 4°C hold, 25 [00851] Real time PCR: [00852] 2 μΙ of cDNA are added to a master mix containing 0.5 u! GAPDH TaqMan Probe (Applied Biosystems. Cat # 4326317E), 0.5 μ! Beta-ENaC TaqMan probe (Applied Biosysfems cat # Hs00165722_m1) and 5 μί Roche Probes Master Mix (Roche Cat # 04887301001) in a total of 10 μ! perweil in a LightCycler480 384 well plate (Roche cat# 30 0472974001). Real time PCR is done in a LightCycler 480 Real Time PCR machine (Roche). Each duplex is tested in two independent transfections and each transfections is assayed in duplicate.

[00853] Real time data are analyzed using the AACt method. Each sample is normalized to GAPDH expression and knockdown is assessed relative to cells transfected with the non- 35 targeting duplex AD-1955. IC50s are defined using a 4 parameter fit model in XLfit, [00854] The results are shown below. Table 3 shows the results of experiments performed at 0.1 nM or 10 nM final duplex concentrations for single dose screens with each 115 2015221515 03 Sep 2015 of the 55 Beta-ENaC duplexes. The “Fraction message remaining” indicates the residual gene level, at 10 nm or 0.1 nM. Thus “0.17” in the second column for AD-20832-b1 indicates that, at a concentration of 10 nM, there was 17% residual gene level, or 83% knockdown of expression. Note also that the suffix “b1” indicates “batch 1.” Thus, for example, a RNAi 5 agent with the designation “AD-20832-b1” has the same sequence as a RNAi agent designated “AD-20832”.

Table 3. 10 nM and 0.1 nM knockdown of Beta-ENaC

Fraction message remaining At 10 nM Fraction message remaining At 0.1 nM Standard deviation At 10 nM Standard deviation At 0.1 nM AD-20832-b1 0.17 0.33 0.04 0.03 AD-20848-b1 0.17 0.49 0.01 0.04 AD-20807-b1 0,18 0.26 0.02 0.05 AD-20826-b1 0.19 0.49 0.02 0.22 AD-20837-b1 0,19 0.51 0.04 0.04 AD-20861-b1 0.19 0.71 0.02 0.29 AD-20834-b1 0.20 0.34 0.06 0.05 AD-20806-b1 0.22 0.60 0.02 0.15 AD-20851-b1 0.23 0.55 0.04 0.07 AD-20865-b1 0.24 0.64 0.02 0.05 AD-20811-b1 0.25 0.52 0.17 0.23 AD-20819-b1 0.27 0.60 0.01 0.07 AD-20839~b1 0.27 0.55 0.06 0.05 AD-20835-b1 0.28 0.63 0.07 0.21 AD-20825-b1 0.30 0.72 0.11 0.15 AD-20867-b1 0.30 0.68 0.00 0.20 AD~2Q813~b1 0.34 0.56 0.17 0.36 AD-20823-b1 0.34 0.75 0.05 0.05 AD-20805-b1 0.36 0.86 0.02 0.09 AD-20831-b1 0.36 0.60 0.01 0.21 AD-20862-b1 0.38 0.93 0.02 0.29 AD-20808-b1 0.40 0.81 0.13 0.16 AD-20827-b1 0.40 2.55 0.07 1.44 AD-20828-b1 0.42 0.89 0.11 0.25 AD-20812-b1 0.47 0.74 0.32 0.36 AD-20836~b1 0.48 1.07 0.11 0.27 AD-2QS22-b1 0.49 0.94 0.11 0.09 AD-20810-b1 0.53 0.87 0.25 0.20 AD-20824-b1 0.54 1.12 0.08 0.33 AD-20844-b1 0.55 0.98 0.07 0.28 AD-20814-b1 0.60 1.30 0.09 0.12 2015221515 03 Sep 2015 116 AD-2QS38-b1 0.65 1.18 0.07 0.18 AD~2Q816~b1 0.66 1.38 0,05 0.17 AD-2G845-b1 0,72 1.18 0.01 0.27 AD~2Q82G~b1 0.75 0.89 0,06 0.14 AD-2G83Q-b1 0,75 0,94 0.04 0.24 AD-2Q866-b1 0.77 1.24 0.03 0.57 AD-2Q8Q9-b1 0.78 1.05 0.05 0.03 AD-20833-b1 0.79 0.99 0.01 0.35 AD-20821-b1 0.80 0.99 0.07 0.14 AD-20846-b1 0.83 1.13 0.10 0.15 AD-20818-b1 0.88 1.36 0.04 0.62 AD-20817-b1 0.89 1.11 0.11 0.19 AD-20843-b1 0.92 1.64 0.11 0.16 AD-2Q840-b1 0.93 1.13 0.15 0.30 AD-20847-b1 0.94 0.99 0.64 0.12 AD-20815-b1 0,96 2.06 0.23 0.99 AD-20842-b1 0.96 1.37 0.16 0,28 AD-20852-b1 0,96 1.30 0.17 0.17 AD-20863-b1 0.99 0.84 0.24 0,11 AD-20864-b1 0.99 1.36 0.05 0.74 AD-20850-b1 1.00 1.22 0.14 0.14 AD-20829-b1 1.08 1.39 0.26 0.70 AD-20849-b1 1.11 1.31 0.27 0.17 AD-20841-b1 1.12 1.37 0.10 0.48 [00855] Ail the RNAi agents to Beta-ENaC used in these experiments were the modified sequences (SEQ ID NO: 1 to 110) listed in Table 1.

[00858] Table 4 shows the results of experiments wherein a subset of duplexes that show 5 robust silencing in the 10 nM and 0.1 nM screens is assayed over a range of concentrations from 10 nM to 10 fM using serial dilutions to determine their iCSO.

Table 4. Beta-ENaC dose response screen H441 New (Average of 4 replicates) H441 Old (Average of 8 replicates) DuplexJD ICSOnM ICSO Standard deviation ICSOnM ICSO Standard deviation AD-20807 0.05 0.03 0.04 0.06 AD-20826 0.14 0.05 0.05 0.07 AD-20832 0.05 0.02 0.04 0.05 AD-20834 0.06 0.03 0.03 0.06 AD-20848 0.25 0.14 0,13 0.17

Table 5.

Group Rat Numbers Formulation Concentration Rats per group 1 1-5 D5W NA 5 2 6-10 AD1955 1Qmg/kg 5 3 11-15 AD20191 10mg/kg 5 4 16-20 AD20807 10mg/kg 5 5 12-25 AD20807 3mg/kg 5 6 26-30 AD20807 1 mg/kg 5 7 31-35 AD20832 1Qmg/kg 4* 8 36-40 AD20832 3mg/kg 5 9 41-45 AD20832 1 mg/kg 5 *ln the group of 4, 5 rats are initially dosed, but 1 in each group does not survive the 15 experiment and is not included in the final data. 117 2015221515 03 Sep 2015 AD-20861 0.13 0.08 0.09 0.06 EXAMPLE 4 in vivo anaiysis of Beta-ENaC RNAi Agents AD-20807 and AD-20832 5 [00857] In in vivo experiments, two Beta-ENaC RNAi agents, AD-20807 and AD-20832, are tested for the ability to knock down Beta-ENaC gene level in whole lungs in rats. The purpose is to determine the dose responses, immunostimuiation is also measured. [00858] The Rat strain used is Sprague-Dawiey; individuals have an approximate weight of 280-300 grams. Rats are dosed once a day for two days. They are then sacrificed about 10 24 hrs after the second dose. The left lung is taken and ground for qPCR determination of Beta-ENaC levels; the right lung frozen and stored.

[00859] Both RNAi agents to Beta-ENaC, AD20807 and AD20832, show reductions in Beta-ENaC levels in a dose-dependent manner. For AD20807, the level of Beta-ENaC is reduced by approximately 30%, 40%, and 50% at dosages of 1, 3 and 10 mg/kg, 20 respectively.

[00860] In contrast, the Beta-ENaC (bENaC) RNAi agents do not decrease the level of Alpha-ENaC (aENaC). There is, however, an increase in Alpha-ENaC with administration of AD20832.

[00861] Negative controls include: 25 [00862] D5W: a solution of 5% dextrose in water; it is the vehicle used to dilute the siRNA when dosing; AD1955: a siRNA which does not specifically target either Alpha- or Beta-ENaC, but targets firefly luciferase; and AD20191: a siRNA which does not bind to Beta-ENaC, but targets rat Alpha-ENaC; and AD-9201, which targets Alpha-ENaC (not used in this particular example). 118 2015221515 03 Sep 2015 [00863] Thus, specific knock-down of Beta-ENaC is seen with RNAi agent AD20807 and AD20832 in this experiment. 5 EXAMPLE 5

In vivo analysis of Beta-ENaC AD-20834 [00864] in in vivo experiments, Beta-ENaC RNAi agent AD20834 is tested for its ability to knock down Beta-ENaC gene level in whole lungs in rats. The purpose is to determine the dosage responses. Immunostimulation is also measured. 10 [00865] The rat strain is Sprague-Dawley; individuals have an approximate weight of 280-300 grams. Rats are dosed once a day for two days. They are then sacrificed about 24 hours after the second dose. The left lung is taken and ground for qPCR determination of Beta-ENaC levels; the right lung is frozen and stored. 15

Table 6.

Group rat #s Formulation concentration rats per group 1 1-5 D5W NA 5 2 6-10 AD1955 10mg/kg 4* 3 11-15 AD20191 10mg/kg 5 4 16-20 AD20834 1Gmg/kg 5 5 21-25 AD20834 3mg/kg 5 6 26-30 AD20834 1 mg/kg 4* *in the groups of 4, 5 rats are initially dosed, but 1 in each group does not survive the experiment and is not included in the final data.

[00866] Assuming a weight of SOOgrams (0.3kg) the following dilutions are made:

10mg/kg = 3mg of siRNA in a 200uL volume = 15mg/mL 3mg/kg = 1 mg of siRNA in a 2Q0uL volume = 5mg/mL 1 mg/kg = G,3mg of siRNA in a 200uL volume = 1.5mg/mL

[00867] The data are normalized to PPIB [Peptidyl-prolyl cis-trans isomerase B, used as 20 25 a housekeeping (normalization) gene], [00868] The experiments show that Beta-ENaC RNAi agent AD20834 demonstrates an approximately 40% reduction in Beta-ENaC level in Sprague-Dawley rats. This effect is specific to Beta-ENaC.

[00869] The controls are as follows: D5W (5% dextrose in water) is a negative control, not showing an effect on Alpha-ENaC or Beta-ENaC levels. AD1955, a control siRNA which does not bind to Alpha- or Beta-ENaC, also showing little effect on Alpha- or Beta-ENaC 119 2015221515 03 Sep 2015 level. The positive control siRNA AD20191, which targets Alpha- but not Beta-ENaC, demonstrates an approximately 50% reduction in Alpha-ENaC level, but not Beta-ENaC.

[00870] Thus, a dosage of 10 mg/kg of Beta-ENaC RNAi agent AD20834 demonstrates at least about 40% inhibition of Beta-ENaC gene expression in Sprague-Dawley rats. 5 EXAMPLE 6

Analysis of Beta-ENaC RNAi Agents [00871] Additional experimentation is done with Beta-ENaC RNAi agents AD20807, AD20832, AD20834, AD20848, and AD20861 in vivo in Sprague-Dawiey rats. Rats are 10 dosed at 10 mg/kg in D5W on day 1 and day 2, and are sacrificed on day 3, and the lungs are collected.

[00872] The results are shown in Figure 1. The results show qPCR data from the left lung, normalized to the control gene PPIB.

[00873] The controls in Figure 1 are as follows: D5W (5% dextrose in water) is a negative 15 control, not showing an effect on Alpha-ENaC or Beta-ENaC levels. The positive control is AD-9201, which targets Alpha-ENaC (aENaC).

[00874] The results, shown in Figure 1, show a statistically significant and specific knockdown of Beta-ENaC (bENaC) by AD20807, AD20832, AD20834, AD20848, and AD20861. The expression of the Beta-ENaC gene is inhibited by at least about 40% at a concentration 20 of 10 mg/kg of these RNAi agents in Sprague-Dawiey rats. EXAMPLE 7

In vitro effect of Beta-ENaC RNAi Agent AD20832 on ENaC Channel Functional Activity in Human Bronchia! Epithelial Cells 25 [00875] Human Bronchial Epithelial Cells (HBEC) are transfected with the indicated siRNA, including Beta-ENaC RNAi agent AD20832. Transfected cells are seeded onto Snapwell inserts and cultured for 24 hours. Subsequently, the apical culture medium is removed from each insert and the ceils cultured at Air Liquid interface (ALI). Cells are assayed for ENaC and CFTR activity at Day 8 post-ALI as described. To control for cell 30 viability, ENaC function is normalized to CFTR activity and the data presented as a percentage relative to the untransfected control (Figure 2A). As an additional viability control, trans-membrane resistance is also measured (Figure 2B). Expression analysis of alpha and beta ENaC subunit mRNA is performed for each insert and normalized to GAPDH expression. (Figure 2C). The data demonstrate that a 70% inhibition of mRNA expression is 35 sufficient to generate a 50% functional inhibition of ENaC channel. This is true for knockdown of either alpha or beta subunits, where each is compared to untransfected (neg) and nonspecific (ns) siRNA controls. The data also show that beta ENaC siRNA does not inhibit alpha ENaC mRNA expression and vice versa. 120 2015221515 03 Sep 2015 [00876] Methods: ENaC functional! activity in Human Bronchial Epithelial Cells [00877] Human Bronchial Epithelial Cells (HBEC) are purchased from Lonza and passaged once before freezing in growth media (BEGM plus singlequots - Lonza). Subsequently, cells are thawed, expanded to confluence and split 1:10 for transfection. Once 5 at 80% confluence, each flask of cells is transfected with the indicated siRNA at 30nM, using 2pL/mL Lipofectamine 2000 in a total volume of 3GmL transfection media (1:1 mix of BEGM (Lonza) and DMEM high glucose (Gibco) with no additives). At 24 hours post-transfection, cells are seeded onto 6 well Snapweil inserts (Costar) at 2.5x10“ celis/insert in differentiation medium (50:50 mix of BEBM and DMEM/high glucose with singlequots (minus the tri-10 iodoibreonine and retinoic acid supplements, with all-trans retinoic acid added separately at 50nM). Cells are supplemented with 0.5mL differentiation media apicaliy and 2.5mL differentiation media basolateraliy. Following a further 24 hours of culture on the inserts the basolateral media is replaced and the apical media is removed, thus taking the cells to Air Liquid Interface (ALI) culture. Cells are assayed for ENaC and CFTR activity at Day 8 (D8) 15 Post-ALI.

[00878] To assess the ion transport phenotype of the transfected cells the Snapweil inserts are mounted in Vertical Diffusion Chambers (Costar) and are bathed continuously with gassed Ringer solution (5% C02 in 02; pH 7.4) maintained at 37°C containing (in mM): 120 NaCI, 25 NaHC03, 3.3 KH2P04, 0.8 K2HP04> 1.2 CaCI2, 1.2 MgCI2, and 10 glucose 20 (Osmoiarity maintained between 280 and 300 mosmol/l). Cells are voltage clamped to 0 mV (model EVC4000; WPI). Trans-membrane resistance (TM res) is measured by applying a 2-mV pulse at 30-s intervals and calculating TM res using Ohm's law. Short circuit current data are recorded using a PowerLab workstation (ADI Instruments). Activity of the ENaC channel in each group is assessed by the change in short-circuit current following the apical addition 25 of 10μΜ of the ENaC blocker Amiloride (Amiloride-sensitive current). Chloride secretion via CFTR is assessed by the change in short circuit current following apical and basolateral addition of 0.6μΜ Forskolin which is known to activate CFTR (Forskoiin response). For each insert the Amiloride-sensitive current Is normalized to the Forskolin response and the data presented as a percentage relative to the untransfected control. At the end of the study each 30 insert is lysed for RNA analysis (300 pL RLT Buffer -- Qiagen) and samples retained for subsequent analysis of mRNA knockdown by rtPCR as described.

Abbreviations: ALI 35 BEGM D6, D8 DMEM HBEC TM res

Air-Liquid Interface

Bronchial Epithelial Growth Medium

Day 8, Day 8

Duibecco’s Modified Eagle Medium Human Bronchial Epithelial Cells Trans-membrane resistance 121 2015221515 03 Sep 2015

EQUIVALENTS

[00879] A composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides 5 differing by 0, 1, 2, or 3 nucleotides from the antisense strand of a RNAi agent specific to Beta-ENaC provided in Table 1.

[00880] The composition of paragraph [00879], wherein the composition further comprises a second RNAi agent to Beta-ENaC, [00881] The composition of paragraph [00879], wherein the antisense strand is 30 or 10 fewer nucleotides in length.

[00882] The composition of paragraph [00879], wherein the sense strand and the antisense strand form a duplex region 15 to 30 nucleotide pairs in length.

[00883] The composition of paragraph [00879], wherein the antisense strand and the sense strand are independently 19 to 23 nucleotides in length. 15 [00884] The composition of paragraph [00879], wherein the RNAi agent comprises a modification that causes the RNAi agent to have increased stability in a biological sample or environment.

[00885] The composition of paragraph [00879], wherein the RNAi agent comprises at least one modified backbone and/or at least one 2’-modified nucleotide. 20 [00886] The composition of paragraph [00879], wherein the RNAi agent comprises: a) at least one 5’-uridine-adenine-3’ (5’-ua-3’) dinucleotide, wherein the uridine is a 2’-modified nucleotide; and/or b) at least one 5’-uridine-guanine-3’ (5’-ug-3’) dinucleotide, wherein the S’-uridine is a 2’-modified nucleotide; and/or 25 c) at least one 5’-cytidine-adenine-3’ (5’-ca-3’) dinucleotide, wherein the 5’- cytidine is a 2’-modified nucleotide; and/or d) at least one 5’-uridine-uridine-3’ (5’-uu-3’) dinucleotide, wherein the 5’-uridine is a 2’-modified nucleotide.

[00887] The composition of paragraph [00879], wherein the RNAi agent comprises a 2'-30 modification selected from the group consisting of: 2'-deoxy, 2'-deoxy-2’-fluoro, 2’-0-methyi, 2'-0-methoxyethy! (2'-0-MOE), 2'-0-aminopropyl (2'-0-AP), 2'-0-dimethylaminoethyl (2'-0-DMAOE), 2'-0-dimethylaminopropyl (2-O-DMAP), 2'-0-dimethylaminoethyloxyethyl (2'-0-DMAEOE), and 2'-0-N-methylacetamido (2'~0-NMA).

[00888] The composition of cla paragraph [00879] 1, wherein the RNAi agent comprises a 35 blunt end.

[00889] The composition of paragraph [00879], wherein the RNAi agent comprises an overhang having 1 to 4 unpaired nucleotides. 122 2015221515 03 Sep 2015 [00890] The composition of paragraph [00879], wherein the RNAi agent comprises an overhang at the 3’~end of the antisense strand of the RNAi agent.

[00891] The composition of paragraph [00879], wherein the RNAi agent is ligated to one or more agent selected from: diagnostic compound, reporter group, cross-linking agent, 5 nuclease-resistance conferring moiety, natural or unusual nucieobase, lipophilic molecule, cholesterol, lipid, lectin, steroid, uvaol, hecigenin, diosgenin, terpene, triterpene, sarsasapogenin, Friedelin, epifriedelanol-derivatized lithocholic acid, vitamin, carbohydrate, dextran, pullulan, chitin, chitosan, synthetic carbohydrate, oligo lactate 15-mer, natural polymer, low- or medium-molecular weight polymer, inulin, cyciodextrin, hyaluronic acid, 10 protein, protein-binding agent, integrin-targeting molecule, poiycationic, peptide, polyamine, peptide mimic, and/or transferrin.

[00892] The composition of paragraph [00879], wherein the RNAi agent is capable of inhibiting expression of the Beta-ENaC gene by at least about 80% at a concentration of 10 nM in H441 cells in vitro. 15 [00893] The composition of paragraph [00879], wherein the RNAi agent is capable of inhibiting expression of the Beta-ENaC gene by at least about 70% at a concentration of 10 nM in H441 cells in vitro.

[00894] The composition of paragraph [00879], wherein the RNAi agent is capable of inhibiting expression of the Beta-ENaC gene by at least about 80% at a concentration of 10 20 nM in H441 cells in vitro.

[00895] The composition of paragraph [00879], wherein the RNAi agent is capable of inhibiting expression of the Beta-ENaC gene by at least about 90% at a concentration of 10 nM in H441 cells in vitro.

[00896] The composition of paragraph [00879], wherein the RNAi has an EC50 of no 25 more than about 0.1 nM.

[00897] The composition of paragraph [00879], wherein the RNAi has an EC50 of no more than about 0.01 nM.

[00898] The composition of paragraph [00879], wherein the RNAi has an EC50 of no more than about 0.001 nM. 30 [00899] A composition comprising a RNAi agent comprising a first strand and a second strand, wherein the first strand and second strand comprise at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the first and second strand, respectively, of a RNAi agent specific to Beta-ENaC provided in Table 1.

[00900] The composition of paragraph [00899], wherein the composition comprises a 35 second RNAi agent to Beta-ENaC.

[00901] The composition of paragraph [00899], wherein the second strand is 30 or fewer nucleotides in length. 123 2015221515 03 Sep 2015 [00902] The composition of paragraph [00899], wherein the first strand and the second strand form a duplex region 15 to 30 nucleotide pairs in length.

[00903] The composition of paragraph [00899], wherein the first strand and the second strand are independently 19 to 23 nucleotides in length. 5 [00904] The composition of paragraph [00899], wherein the RNAi agent comprises a modification that causes the RNAi agent to have increased stability in a biological sample or environment.

[00905] The composition of paragraph [00899], wherein the RNAi agent comprises a phosphorothioate and/or a 2'-modified nucleotide. 10 [00906] The composition of paragraph [00899], wherein the RNAi agent comprises: at least one 5’-uridine-adenine-3’ (5’-ua-3’) dinucleotide, wherein the uridine is a 2’-modified nucleotide; and/or at least one 5’-uridine-guanine-3’ (5’-ug-3’) dinucieotide, wherein the 5’-uridine is a 2'-modified nucleotide; and/or at least one 5s-cytidine-adenine-3’ (5’-ca-3’) dinucieotide, wherein the 5’-15 cytidine is a 2’-modified nucleotide; and/or at least one 5’-uridine-uridine-3’ (5’-uu-3’) dinucieotide, wherein the 5’-uridine is a 2’-modified nucleotide.

[00907] The composition of paragraph [00899], wherein the RNAi agent comprises one or more 2'-modifications selected from the group consisting of: 20 2'-deoxy, 2'-deoxy-2’-fluoro, 2’-0-methyi, 2-O-methoxyethyl (2'-0-MOE), 2-0- aminopropyl (2-O-AP), 2'-0-dimethylaminoethyl (2'-0-DMAOE), 2'-0-dimethyiaminopropyl (2'-0-DMAP), 2'-0-dimethylaminoethyioxyethyi (2-O-DMAEOE), and 2'-0-N-methylacetamido (2'-0-NMA).

[00908] The composition of paragraph [00899], wherein the RNAi agent comprises a 25 blunt end.

[00909] The composition of paragraph [00899], wherein the RNAi agent comprises an overhang having 1 to 4 unpaired nucleotides.

[00910] The composition of paragraph [00899], wherein the RNAi agent comprises an overhang at the 3’-end of the antisense strand. 30 [00911] The composition of paragraph [00899], wherein the RNAi agent is ligated to one or more agents, the agent selected from a: diagnostic compound, reporter group, cross-linking agent, nuclease-resistance conferring moiety, natural or unusual nucleobase, lipophilic molecule, cholesterol, lipid, lectin, steroid, uvaoi, hecigenin, diosgenin, terpene, triterpene, sarsasapogenin, Friedelin, epifriedelanol-derivatized iithocholic acid, vitamin, 35 carbohydrate, dextran, pulluian, chitin, chitosan, synthetic carbohydrate, oiigo lactate 15-mer, natural polymer, low- or medium-molecular weight polymer, inulin, cyclodextrin, hyaluronic 124 2015221515 03 Sep 2015 acid, protein, protein-binding agent, integrin-targeting molecule, polycationic, peptide, polyamine, peptide mimic, and/or transferrin.

[00912] The composition of paragraph [00899], wherein the RNAi agent is capable of inhibiting expression of the Beta-ENaC gene by at least about 60% at a concentration of 10 5 nM in H441 cells in vitro.

[00913] The composition of paragraph [00899], wherein the RNAi agent is capable of inhibiting expression of the Beta-ENaC gene by at least about 70% at a concentration of 10 nM in H441 cells in vitro.

[00914] The composition of paragraph [00899], wherein the RNAi agent is capable of 10 inhibiting expression of the Beta-ENaC gene by at least about 80% at a concentration of 10 nM in H441 cells in vitro.

[00915] The composition of paragraph [00899], wherein the RNAi agent is capable of inhibiting expression of the Beta-ENaC gene by at least about 90% at a concentration of 10 nM in H441 cells in vitro. 15 [00918] The composition of paragraph [00899], wherein the RNAi has an EC50 of no more than about 0.1 nM.

[00917] The composition of paragraph [00899], wherein the RNAi has an EC50 of no more than about 0.01 nM.

[00918] The composition of paragraph [00899], wherein the RNAi has an EC50 of no 20 more than about 0.001 nM.

[00919] A method of treating a Beta-ENaC-related disease in an individual, comprising the step of administering to the individual a therapeutically effective amount of a composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 25 nucleotides from the antisense strand of a RNAi agent specific to Beta-ENaC provided in Table 1.

[00920] The method of paragraph [00919], wherein the Beta-ENaC-related disease is cystic fibrosis, pseudohypoaldosteronism type 1 (PHA1), Liddle’s syndrome, hypertension, alkalosis, hypokalemia, and/or obesity-associated hypertension. 30 [00921] The method of paragraph [00919], wherein the Beta-ENaC-related disease is cystic fibrosis.

[00922] The method of paragraph [00919], wherein the method further comprises the step of administering an additional treatment for cystic fibrosis, pseudohypoaldosteronism type 1 (PHA1), Liddle’s syndrome, hypertension, alkalosis, hypokalemia, and/or obesity-associated 35 hypertension.

[00923] The method of paragraph [00919], wherein the composition comprises a second RNAi agent to Beta-ENaC. 125 2015221515 03 Sep 2015 [00924] The method of paragraph [00919], wherein the method further comprises the step of administering an additionai RNAi agent to Beta-ENaC.

[00925] The method of paragraph [00919], further comprising the administration of an additionai treatment. 5 [00926] The method of paragraph [00919], wherein the additionai treatment is a composition.

[00927] The method of paragraph [00919], wherein the additionai treatment is a method.

[00928] The method of paragraph [00919], wherein the additionai treatment and the RNAi agent can be administered in any order. 10 [00929] A method of inhibiting the expression of the Beta-ENaC gene in an individual, comprising the step of administering to the individuai a therapeuticaily effective amount of a composition comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of a RNAi agent specific to Beta-ENaC 15 provided in Table 1.

[00930] The method of paragraph [00929], wherein the individual is afflicted with or susceptible to a Beta-ENaC-related disease.

[00931] The method of paragraph [00929], wherein the Beta-ENaC-related disease is cystic fibrosis, pseudohypoaldosteronism type 1 (PHA1), Liddle’s syndrome, hypertension, 20 alkalosis, hypokalemia, and/or obesity-associated hypertension.

[00932] The method of paragraph [00929], wherein the Beta-ENaC-related disease is cystic fibrosis.

[00933] The method of paragraph [00932], further comprising the administration of an additional treatment. 25 [00934] The method of paragraph [00933], wherein the additional treatment is a composition.

[00935] The method of paragraph [00933], wherein the additional treatment is a method.

[00936] The method of paragraph [00933], wherein the additional treatment and the RNAi agent can be administered in any order. 30 [00937] A medicament for use in an RNAi formulation comprising a RNAi agent comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the antisense strand of a RNAi agent specific to Beta-ENaC provided in Table 1.

[00938] Any composition above in a pharmaceutically effective formulation. 35 [00939] The composition according to paragraph [00879], for use in a method of treating a Beta-ENaC-related disease in an individual, the method comprising the step of 2015221515 03 Sep 2015 126 administering to the individual a therapeuticaliy effective amount of a composition according to paragraph [00879].

[00940] The use of a composition according to paragraph [00879], in the manufacture of a medicament for the treatment of a Beta-ENaC-reiated disease. 5 [00941] The use of paragraph [00940], wherein the Beta-ENaC-reiated disease is cystic fibrosis, pseudohypoaidosteronism type 1 (PHA1), Liddle’s syndrome, hypertension, alkalosis, hypokalemia, and/or obesity-associated hypertension.

[00942] The composition of paragraph [00879], wherein ali the pyrimidines are 2’ O-methyi-modified nucleotides. 10 [00943] The composition of paragraph [00899], wherein ali the pyrimidines are 2’ O-methyi-modified nucleotides.

[00944] Throughout this specification and the claims which foilow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", wili be understood to imply the inclusion of a stated integer or step or group of 15 integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[00945] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or 20 information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A composition comprising a RNAi agent comprising a first strand and a second strand, wherein: the sequence of the first strand comprises at least 15 contiguous nucleotides differing by 0, 1, 2, or 3 nucleotides from the sequence of SEQ ID NO: 116 or SEQ ID NO: 166, wherein the length of the first and second strand are each at least about 15 nucleotides and no more than about 30 nucleotides; and wherein the first and/or second strand is modified or unmodified.
2. The composition of claim 1, wherein the composition further comprises a second RNAi agent to Beta-ENaC.
3. The composition of claim 1, wherein the RNAi agent comprises at least one modified backbone and/or at least one 2'-modified nucleotide.
4. The composition of claim 1, wherein the RNAi agent is ligated to one or more agents, wherein the one or more agents are selected from: diagnostic compound, reporter group, cross-linking agent, nuclease-resistance conferring moiety, natural or unusual nucleobase, lipophilic molecule, cholesterol, lipid, lectin, steroid, uvaol, hecigenin, diosgenin, terpene, triterpene, sarsasapogenin, Friedelin, epifriedelanol-derivatized lithocholic acid, vitamin, carbohydrate, dextran, pullulan, chitin, chitosan, synthetic carbohydrate, oligo lactate 15-mer, natural polymer, low- or medium-molecular weight polymer, inulin, cyclodextrin, hyaluronic acid, protein, protein-binding agent, integrin-targeting molecule, polycationic, peptide, polyamine, peptide mimic, and transferrin.
5. A composition comprising a RNAi agent comprising a first strand and a second strand, wherein: the sequence of the first strand comprises the sequence of SEQ ID NO: 116 or SEQ ID NO: 166; wherein the length of the first and second strand are each no more than about 30 nucleotides; and wherein the first and/or second strand is modified or unmodified.
6. The composition of claim 5, wherein the composition further comprises a second RNAi agent to Beta-ENaC.
7. The composition of claim 5, wherein the RNAi agent comprises a phosphorothioate and/or a 2'-modified nucleotide.
8. The composition of claim 5, wherein the RNAi agent is ligated to one or more agents, wherein the one or more agents are selected from: diagnostic compound, reporter group, cross-linking agent, nuclease-resistance conferring moiety, natural or unusual nucleobase, lipophilic molecule, cholesterol, lipid, lectin, steroid, uvaol, hecigenin, diosgenin, terpene, triterpene, sarsasapogenin, Friedelin, epifriedelanol-derivatized lithocholic acid, vitamin, carbohydrate, dextran, pullulan, chitin, chitosan, synthetic carbohydrate, oligo lactate 15-mer, natural polymer, low- or medium-molecular weight polymer, inulin, cyclodextrin, hyaluronic acid, protein, protein-binding agent, integrin-targeting molecule, polycationic, peptide, polyamine, peptide mimic, and transferrin.
9. A method of reducing the level and/or expression of Beta-ENaC in an individual, the method comprising the step of administering to the individual a composition comprising a pharmaceutically acceptable carrier and a RNAi agent comprising a first strand and a second strand, wherein: the sequence of the first strand comprises at least 15 contiguous nucleotides differing by 0, 1,2, or 3 nucleotides from the sequence of SEQ ID NO: 116 or SEQ ID NO: 166; wherein the length of the first and second strand are each at least about 15 nucleotides and no more than about 30 nucleotides; and wherein the first and/or the second strand are modified or unmodified.
10. The method of claim 9, wherein the method further comprises the step of administering an additional RNAi agent to Beta-ENaC.
11. The method of claim 9, wherein the RNAi agent comprises one or more 2'-modifications selected from the group consisting of: 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-0-methoxyethyl (2'-0-M0E), 2'-0-aminopropyl (2'-0-AP), 2'-0-dimethylaminoethyl (2'-0-DMA0E), 2'-0-dimethylaminopropyl (2'-0-DMAP), 2'-0-dimethylaminoethyloxyethyl (2'-0-DMAE0E), and 2'-0-N-methylacetamido (2'-0-NMA).
12. The method of claim 9, wherein the RNAi agent is ligated to one or more agents, wherein the one or more agents are selected from: diagnostic compound, reporter group, cross-linking agent, nuclease-resistance conferring moiety, natural or unusual nucleobase, lipophilic molecule, cholesterol, lipid, lectin, steroid, uvaol, hecigenin, diosgenin, terpene, triterpene, sarsasapogenin, Friedelin, epifriedelanol-derivatized lithocholic acid, vitamin, carbohydrate, dextran, pullulan, chitin, chitosan, synthetic carbohydrate, oligo lactate 15-mer, natural polymer, low- or medium-molecular weight polymer, inulin, cyclodextrin, hyaluronic acid, protein, protein-binding agent, integrin-targeting molecule, polycationic, peptide, polyamine, peptide mimic, and transferrin.
13. The method of claim 9, wherein the RNAi agent comprises a phosphorothioate and/or a 2'-modified nucleotide.
14. A composition comprising a therapeutically effective amount of a composition of any one of claims 1 to 4 and a pharmaceutically acceptable carrier.
15. A composition comprising a therapeutically effective amount of a composition of any one of claims 5 to 8 and a pharmaceutically acceptable carrier.
16. A composition comprising a RNAi agent comprising a first strand and a second strand, wherein: the sequence of the first strand is the sequence of SEQ ID NO: 116 or SEQ ID NO: 166; and wherein the first and/or second strand are modified or unmodified.
17. A composition comprising a RNAi agent comprising a first strand and a second strand, wherein: the sequence of the first strand comprises at least 15 contiguous nucleotides from the sequence of SEQ ID NO: 116 or SEQ ID NO: 166, wherein the length of the first and second strand are each at least about 19 nucleotides and no more than about 30 nucleotides; and wherein the first and/or second strand is modified or unmodified.