WO2025075868A1

WO2025075868A1 - Ppp1r15b rna interference agents

Info

Publication number: WO2025075868A1
Application number: PCT/US2024/048763
Authority: WO
Inventors: Sarah Ashley BRISKEY; Selene Hernandez BUQUER; Rebecca R. Miles; Douglas Raymond Perkins; Ricardo J. SAMMS
Original assignee: Eli Lilly and Co
Current assignee: Eli Lilly and Co
Priority date: 2023-10-02
Filing date: 2024-09-27
Publication date: 2025-04-10
Anticipated expiration: 2026-04-02
Also published as: CN121941769A; TW202532087A; AR133998A1; CO2026003999A2; AU2024354329A1; DOP2026000082A; IL327234A

Abstract

Provided herein are PPP1R15B RNAi agents and compositions comprising a PPP1R15B RNAi agent. Also provided herein are methods of using the PPP1R15B RNAi agents or compositions comprising a PPP1R15B RNAi agent for reducing PPP1R15B expression, and/or treating PPP1R15B associated metabolic disorder in a subject.

Description

PPP1R15B RNA INTERFERENCE AGENTS

SEQUENCE LISTING

[0001] The present application is being filed along with a Sequence Listing in ST.26 XML format. The Sequence Listing is provided as a file titled 30554_WO.xnd created September 10.

2024, and is 2.26 megabytes in size. The Sequence Listing information in the ST.26 XML format is incorporated herein by reference in its entirety.

BACKGROUND

[0002] PPP1R15B gene encodes the protein phosphatase 1 regulatory subunit 15B (also known as constitutive repressor of eIF2a phosphorylation or CReP), which is a critical subunit of the phosphatase complex that promotes the dephosphorylation of eukaryotic translation initiation factor 2A (eIF2a). eIF2a is a central component of the integrated stress response (ISR), an evolutionarily conserved intracellular signaling system that helps the cell, tissue, organism to adapt to different environmental and pathological conditions and restore balance (Mauro Costa- Mattioli and Peter Walter, Science. 2020;368(6489): eaat5314). PPP1R15B /CReP ablation causes constitutive eIF2a phosphorylation in the liver, which leads to activation of the activation transcription factor 4 (ATF4) transcriptional program (Xu, et al., Hepatology. 2018; 68(6): 2167- 2181).

[0003] ISR activation has been observed in metabolic diseases including diabetes, hepatic steatosis, steatohepatitis and obesity. It has been reported that PPP1R15B /CReP ablation causes constitutive eIF2a phosphorylation in the liver, which leads to activation of the ATF4 transcriptional program (Xu, et al., Hepatology. 2018; 68(6): 2167-2181). Liver-specific PPP1 R15B /CReP knockout mice exhibited marked browning of white adipose tissue, increased energy expenditure and insulin sensitivity (Xu, et al., Hepatology. 2018; 68(6): 2167-2181). [0004] RNA interference (RNAi) is a highly conserved regulatory mechanism in which RNA molecules are involved in sequence-specific suppression of gene expression by double-stranded RNA molecules (dsRNA) (Fire et al., Nature 391 :806-811, 1998)

[0005] There remains a need for therapeutic agents that can inhibit or adjust the expression of PPPlR15B/CReP for treating PPP1R15B associated metabolic disorders, e.g., by utilizing RNAi. SUMMARY OF INVENTION

[0006] Provided herein are PPP1R15B RNAi agents and compositions comprising a PPP1R15B RNAi agent. Also provided herein are methods of using the PPP1R15B RNAi agents or compositions comprising a PPP1R15B RNAi agent for treating PPP1R15B associated metabolic disorder, such as obesity, Type 2 diabetes (T2D), or nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), dyslipidemia, or metabolic syndrome in a subject.

[0007] In one aspect, provided herein are PPP1R15B RNAi agent comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a duplex, and wherein the antisense strand is complementary to a region of PPP1R15B mRNA. In some embodiments, provided herein are PPP1R15B RNAi agent comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a duplex, wherein the sense strand comprises a nucleic acid sequence selected from any one of SEQ ID NOs: 81-159, and the antisense strand comprises a nucleic acid sequence selected from any one of SEQ ID NOs: 160-238, wherein optionally one or more nucleotides of the sense strand and the antisense strand are independently modified nucleotides, and wherein optionally one or more internucleotide linkages of the sense strand and the antisense strand are modified internucleotide linkages.

[0008] In some embodiments, the sense strand is linked to a delivery moiety, e.g., a lipid or a carbohydrate. In some embodiments, the delivery moiety is linked to the 3’ end of the sense strand, optionally via a linker (L). In some embodiments, the delivery moiety comprises a GalNAc (N-acetyl galactosamine) moiety. In some embodiments, the delivery moiety comprises Formula (II) (Ac means acetyl):

[0009] In another aspect, provided herein are PPP1 R l 5B RNAi agent comprising Formula (I): R-L-D, wherein R is a double stranded RNA (dsRNA) comprising a sense stand and an antisense strand, wherein the antisense strand is complementary to PPP1R15B mRNA, wherein L is a linker, or optionally absent, and wherein D is a delivery moiety comprising Formula (II):

[00010] Exemplary sense strand and antisense strand sequences of the dsRNA of PPP 1 Rl 5B RNAi agent are provided in Tables 1 and 2. In some embodiments, the sense strand comprises a nucleic acid sequence selected from any one of SEQ ID NOs: 2-80, wherein optionally one or more nucleotides of the sense strand are independently modified nucleotides, and wherein optionally one or more internucleotide linkages of the sense strand are modified internucleotide linkages. In some embodiments, the sense strand comprises a nucleic acid sequence selected from any one of SEQ ID NOs: 81-159, and the antisense strand comprises a nucleic acid sequence selected from any one of SEQ ID NOs: 160-238, wherein optionally one or more nucleotides of the sense strand and the antisense strand are independently modified nucleotides, and wherein optionally one or more internucleotide linkages of the sense strand and the antisense strand are modified internucleotide linkages.

[00011] In some embodiments, the sense strand and the antisense strand comprise a pair of nucleic acid sequences of the sense strand and the antisense strand of any one of dsRNA No. 1 - 79, wherein optionally one or more nucleotides of the sense strand and the antisense strand are independently modified nucleotides, and wherein optionally one or more internucleotide linkages of the sense strand and the antisense strand are modified intemucleotide linkages.

[00012] In some embodiments, the sense strand and the antisense strand comprise a pair of nucleic acid sequences selected from the group consisting of:

(a) the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 93, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 172, (b) the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 123, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 202;

(c) the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 125, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 204;

(d) the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 129, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 208;

(e) the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 132, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 211,

(f) the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 136, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 215, and

(g) the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 157, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 236, wherein optionally one or more nucleotides of the sense strand and the antisense strand are independently modified nucleotides, and wherein optionally one or more internucleotide linkages of the sense strand and the antisense strand are modified intemucleotide linkages.

[00013] The PPP1R15B RNAi agents described herein may include modifications. The modifications can be made to one or more nucleotides of the sense strand and/or antisense strand or to the internucleotide linkages. In some embodiments, one or more nucleotides of the sense strand are modified nucleotides. In some embodiments, each nucleotide of the sense strand is a modified nucleotide. In some embodiments, one or more nucleotides of the antisense strand are modified nucleotides. In some embodiments, each nucleotide of the antisense strand is a modified nucleotide. In some embodiments, the modified nucleotide is a 2'-fluoro modified nucleotide, 2 -O-methyl modified nucleotide, 2’ deoxy nucleotide (D\ A), or 2 -O-alkyl modified nucleotide.

[00014] In some embodiments, the sense strand has four 2’-fluoro modified nucleotides, e.g., at positions 7, 9, 10, I l from the 5’ end of the sense strand In some embodiments, the other nucleotides of the sense strand are 2'-O-methyl modified nucleotides. In some embodiments, the antisense strand has four 2’-fluoro modified nucleotides, e.g., at positions 2, 6, 14, 16 from the 5’ end of the antisense strand. In some embodiments, the other nucleotides of the antisense strand are 2'-O-methyl modified nucleotides. [00015] In some embodiments, the sense strand has three 2'-fluoro modified nucleotides, e.g., at positions 9, 10, 11 from the 5’ end of the sense strand. In some embodiments, the other nucleotides of the sense strand are 2'-O-methyl modified nucleotides. In some embodiments, the antisense strand has five 2'-fluoro modified nucleotides, e.g., at positions 2, 5, 7, 14, 16 from the 5’ end of the antisense strand. In some embodiments, the antisense strand has five 2'-fluoro modified nucleotides, e.g., at positions 2, 5, 8, 14, 16 from the 5¹ end of the antisense strand. In some embodiments, the antisense strand has five 2'-fluoro modified nucleotides, e.g , at positions 2, 3, 7, 14, 16 from the 5’ end of the antisense strand. In some embodiments, the other nucleotides of the antisense strand are 2'-O-methyl modified nucleotides.

[00016] In some embodiments, the 5’ end of the antisense strand has 5’ phosphate or 5’ vinylphosphonate (5’-VP).

[00017] In some embodiments, the sense strand or the antisense strand comprises an abasic moiety or inverted abasic moiety.

[00018] In some embodiments, the sense strand and the antisense strand have one or more modified internucleotide linkages. In some embodiments, the modified internucleotide linkage is phosphorothioate linkage. In some embodiments, the sense strand has four or five phosphorothioate linkages. In some embodiments, the antisense strand has four or five phosphorothioate linkages. In some embodiments, the sense strand and the antisense strand each has four or five phosphorothioate linkages. In some embodiments, the sense strand has four phosphorothioate linkages and the antisense strand has five phosphorothioate linkages.

[00019] Exemplary modified sense strand and antisense strand nucleic acid sequences of PPP1R15B RNAi agent are provided in Table 3. In some embodiments, the sense strand and the antisense strand comprise a pair of nucleic acid sequences of the sense strand and the antisense strand of any one of dsRNA No. 80-186.

[00020] In some embodiments, the sense strand and the antisense strand comprise a pair of nucleic acid sequences selected from the group consisting of:

(a) the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 251, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 340,

(b) the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 281, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 370; (c) the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 283, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 372:

(d) the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 287, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 376;

(e) the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 290, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 379;

(f) the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 294, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 383, and

(g) the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 315, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 404.

[00021] In some embodiments, L (linker) is present and comprises Formula (III) or Formula (IV):

[00022] In another aspect, provided herein are pharmaceutical compositions comprising an PPP1R15B RNAi agent described herein and a pharmaceutically acceptable carrier. Also provided herein are pharmaceutical compositions comprising a means for reducing PPP1R15B expression in a cell and a pharmaceutically acceptable carrier.

[00023] In another aspect, provided herein are methods of treating a PPP1R.15B associated metabolic disorder, such as obesity, Type 2 diabetes, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), dyslipidemia, or metabolic syndrome in a patient in need thereof, and such method comprises administering to the patient an effective amount of a PPP1R15B RNAi agent or a pharmaceutical composition described herein. In some embodiments, the PPP1 R15B RN Ai agent or the pharmaceutical composition is administered to the patient intravenously or subcutaneously. In some embodiments, such methods further comprise administering a second therapeutic, agent (e.g., an incretin) to the patient.

[00024] Also provided herein are methods of reducing PPP1R15B expression in a cell, such methods comprising: introducing a PPP1R15B RNAi agent described herein into the cell; and incubating the cell for a time sufficient for degradation of PPP1R15B mRNA, thereby reducing PPP1R15B expression in the cell.

[00025] In another aspect, provided herein are PPP1R15B RNAi agents or the pharmaceutical composition comprising an PPP1R15B RNAi agent for use in a therapy. Also provided are PPP1R15B RNAi agents or pharmaceutical compositions comprising a PPP!R15B RNAi agent for use in the treatment of a PPP1R15B associated metabolic disorder, such as obesity, Type 2 diabetes, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), dyslipidemia, or metabolic syndrome. Also provided herein are uses of PPP1R15B RNAi agents in the manufacture of a medicament for the treatment of a PPP1R15B associated metabolic disorder, such as obesity, Type 2 diabetes, nonalcoholic steatohepatitis (NASH), nonalcoholic faty liver disease (NAFLD), dyslipidemia, or metabolic syndrome.

BRIEF DESCRIPTION OF THE DRAWINGS

[00026] Figure 1 is a bar graph showing the percent body weight change in diet-induced obese (DIO) mice up to day 1 1 after a single dose of a PPP1R15B RNAi agent. (dsRNA No. 122), a daily GLP-1R agonist, or a combination of the PPP1R15B RNAi agent (dsRNA No 12.2) and daily GLP-1R agonist.

[00027] Figure 2 is a line graph illustrating energy expenditure in DIO mice up to day 10 after a single dose of a PPP1RI5B RNAi agent (dsRNA No. 122) as compared with vehicle.

DETAILED DESCRIPTION

[00028] Provided herein are PPP1R15B RNAi agents and compositions comprising a PPP1R15B RNAi agent. Also provided herein are methods of using the PPP1R15B RNAi agents or compositions comprising a PPP1R15B RNAi agent for treating PPP1R15B associated metabolic disorder, such as obesity, Type 2 diabetes, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), dyslipidemia, or metabolic syndrome, in a subject. [00029] In one aspect, provided herein are PPP1R15B RNAi agent comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a duplex, and wherein the antisense strand is complementary to a region of PPP1R15B mRNA. In some embodiments, the sense strand and the antisense strand are each 15-30 nucleotides in length, e.g,, 20-25 nucleotides in length. In some embodiments, provided herein are PPP1R15B RNAi agents comprising a sense strand of 21 nucleotides and an antisense strand of 23 nucleotides. In some embodiments, the sense strand and antisense strand of the PPP1R15B RNAi agent may have overhangs at either the 5’ end or the 3’ end (i.e., 5’ overhang or 3’ overhang). For example, the sense strand and the antisense strand may have 5’ or 3’ overhangs of 1 to 5 nucleotides.

[00030] Exemplary sense strand and antisense strand sequences of PPP1R15B RNAi agent are provided in Tables 1 and 2.

[00031] In some embodiments, provided herein are PPP1 R15B RNAi agent comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a duplex, wherein the sense strand comprises a nucleic acid sequence selected from any one of SEQ ID NOs: 81-159, and the antisense strand comprises a nucleic acid sequence selected from any one of SEQ ID NOs: 160-238, wherein optionally one or more nucleotides of the sense strand and the antisense strand are independently modified nucleotides, and wherein optionally one or more intemucleotide linkages of the sense strand and the antisense strand are modified internucleotide linkages

[00032] In some embodiments, the sense strand is linked to a delivery moiety, e g., a lipid or a carbohydrate. The delivery moiety can facilitate the entry of RNAi agent into the cells. In some embodiments, the delivery moiety is linked to the 3’ end of the sense strand, optionally via a linker. In some embodiments, the delivery moiety comprises a GalNAc (N-acetylgalactosamine) moiety. In some embodiments, the delivers' moiety comprises Formula (II) (Ac means acetyl):

[00033] In another aspect, provided herein are PPP1R15B RNAi agent comprising Formula (I): R-L-D, wherein R is a double stranded RNA (dsRNA) comprising a sense stand and an antisense strand, wherein the antisense strand is complementary to PPP1R15B mRNA, wherein L is a linker, or optionally absent, and wherein D is a delivery moiety comprising Formula (II):

[00034] Exemplary sense strand and antisense strand sequences of the dsRNA of PPP1R15B RNAi agent are provided in Tables 1 and 2. In some embodiments, the sense strand comprises a nucleic acid sequence selected from any one of SEQ ID NOs: 2-80, wherein optionally one or more nucleotides of the sense strand are independently modified nucleotides, and wherein optionally one or more internucleotide linkages of the sense strand are modified internucleotide linkages. In some embodiments, the sense strand comprises a nucleic acid sequence selected from any one of SEQ ID NOs: 81-159, and the antisense strand comprises a nucleic acid sequence selected from any one of SEQ ID NOs: 160-238, wherein optionally one or more nucleotides of the sense strand and the antisense strand are independently modified nucleotides, and wherein optionally one or more internucleotide linkages of the sense strand and the antisense strand are modified internucleotide linkages.

[00035] In some embodiments, the sense strand and the antisense strand comprise a pair of nucleic acid sequences of the sense strand and the antisense strand of any one of dsRNA No. 1 - 79, wherein optionally one or more nucleotides of the sense strand and the antisense strand are independently modified nucleotides, and wherein optionally one or more internucleotide linkages of the sense strand and the antisense strand are modified intemucleotide linkages.

[00036] In some embodiments, the sense strand and the antisense strand comprise a pair of nucleic acid sequences selected from the group consisting of: (a) the sense strand comprises a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 93, and the antisense strand comprises a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 172;

(b) the sense strand comprises a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 123, and the antisense strand comprises a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 202;

(c) the sense strand comprises a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 125, and the antisense strand comprises a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 204;

(d) the sense strand comprises a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 129, and the antisense strand comprises a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 208;

(e) the sense strand comprises a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 132, and the antisense strand comprises a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 211;

( f) the sense strand comprises a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 136, and the antisense strand comprises a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 215; and

(g) the sense strand comprises a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 157, and the antisense strand comprises a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 236, wherein optionally one or more nucleotides of the sense strand and the antisense strand are independently modified nucleotides, and wherein optionally one or more internucleotide linkages of the sense strand and the antisense strand are modified intemucleotide linkages.

[00037] In some embodiments, the sense strand and the antisense strand comprise a pair of nucleic acid sequences selected from the group consisting of:

(a) the sense strand comprises a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 93, and the antisense strand comprises a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 172; (b) the sense strand comprises a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 123, and the antisense strand comprises a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 202;

(c) the sense strand comprises a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 125, and the antisense strand comprises a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 204;

(d) the sense strand comprises a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 129, and the antisense strand comprises a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 208;

(e) the sense strand comprises a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 132, and the antisense strand comprises a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 211 ;

(f) the sense strand comprises a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 136, and the antisense strand comprises a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 215; and

(g) the sense strand comprises a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 157, and the antisense strand comprises a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 236, wherein optionally one or more nucleotides of the sense strand and the antisense strand are independently modified nucleotides, and wherein optionally one or more internucleotide linkages of the sense strand and the anti sense strand are modified internucleotide linkages.

[00038] In some embodiments, the sense strand and the antisense strand comprise a pair of nucleic acid sequences selected from the¹ group consisting of:

(a) the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 93, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 172;

(b) the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 123, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 202,

(d) the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 129, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 208; (e) the sense strand comprises a first nucleic acid sequence of SEQ !D NO: 132, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 211;

(fl the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 136, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 215; and

Table 1: Exemplary sense strand sequences of PPP1R15B RNAi agents

1

Table 2: Unmodified sense strand and antisense strand sequences of PPP1R15B RNAi agents

[00039] The PPP1R15B RNAi agents described herein may include modifications. The modifications can be made to one or more nucleotides of the sense strand and/or antisense strand or to the internucleotide linkages. In some embodiments, one or more nucleotides of the sense strand are modified nucleotides. In some embodiments, each nucleotide of the sense strand is a modified nucleotide. In some embodiments, one or more nucleotides of the antisense strand are modified nucleotides. In some embodiments, each nucleotide of the antisense strand is a modified nucleotide. In some embodiments, the modified nucleotide is a 2'-fluoro modified nucleotide, 2'-O-methyl modified nucleotide, 2’ deoxy nucleotide (DNA), or 2'-O-alkyl modified nucleotide.

[00040] In some embodiments, the sense strand has four 2'-fluoro modified nucleotides, e.g., at positions 7, 9, 10, 11 from the 5’ end of the sense strand. In some embodiments, the other nucleotides of the sense strand are 2'-O-methyl modified nucleotides. Ln some embodiments, the antisense strand has four 2'-fluoro modified nucleotides, e.g., at positions 2, 6, 14, 16 from the 5’ end of the antisense strand. In some embodiments, the other nucleotides of the antisense strand are 2’-O-methyl modified nucleotides.

[00041] In some embodiments, the sense strand has three 2'-fluoro modified nucleotides, e.g., at positions 9, 10, 11 from the 5^; end of the sense strand. In some embodiments, the other nucleotides of the sense strand are 2'-O-methyl modified nucleotides. In some embodiments, the antisense strand has five 2'-fluoro modified nucleotides, e.g., at positions 2, 5, 7, 14, 16 from the 5’ end of the antisense strand. In some embodiments, the antisense strand has five 2’-fluoro modified nucleotides, e.g., at positions 2, 5, 8, 14, 16 from the 5’ end of the antisense strand. In some embodiments, the antisense strand has five 2'-fluoro modified nucleotides, e.g., at positions 2, 3, 7, 14, 16 from the 5’ end of the antisense strand. In some embodiments, the other nucleotides of the antisense strand are 2'-O-methyl modified nucleotides.

[00042] In some embodiments, the 5’ end of the antisense strand has 5’ phosphate or 5’ vi ny I phosphonate (5 ’ - VP) .

[00043] In some embodiments, the sense strand or the antisense strand comprises an abasic moiety or inverted abasic moiety, e.g., a moiety in Table 4

[00044] In some embodiments, the sense strand and the antisense strand have one or more modified internucleotide linkages. In some embodiments, the modified internucleotide linkage is phosphorothioate linkage. In some embodiments, the sense strand has four or five phosphorothioate linkages. In some embodiments, the antisense strand has four or five phosphorothioate linkages. In some embodiments, the sense strand and the antisense strand each has four or five phosphorothioate linkages. In some embodiments, the sense strand has four phosphorothioate linkages and the antisense strand has five phosphorothioate linkages.

[00045] Exemplary modified sense strand and antisense strand nucleic acid sequences of ppp I Kj 5B RNAi agent are provided in Table 3. In some embodiments, the sense strand and the antisense strand comprise a pair of nucleic acid sequences of the sense strand and the antisense strand of any one of dsRNA No. 80-186.

[00046] In some embodiments, the sense strand and the antisense strand comprise a pair of nucleic acid sequences selected from the group consisting of:

(a) the sense strand comprises a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 251 , and the antisense strand comprises a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 340;

(b) the sense strand comprises a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 281, and the antisense strand comprises a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 370,

(c) the sense strand comprises a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 283, and the antisense strand comprises a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 372;

(d) the sense strand comprises a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 287, and the antisense strand comprises a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 376;

(e) the sense strand comprises a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 290, and the antisense strand comprises a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 379;

(f) the sense strand comprises a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 294, and the antisense strand comprises a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 383; and

(g) the sense strand comprises a first nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 315, and the antisense strand comprises a second nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 404. [00047] In some embodiments, the sense strand and the antisense strand comprise a pair of nucleic acid sequences selected from the group consisting of:

(a) the sense strand comprises a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 251. and the antisense strand comprises a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 340;

(b) the sense strand comprises a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 281 , and the antisense strand comprises a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 370;

(c) the sense strand comprises a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 283, and the antisense strand comprises a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 372,

(d) the sense strand comprises a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 287, and the antisense strand comprises a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 376;

(e) the sense strand comprises a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 290, and the antisense strand comprises a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 379;

(f) the sense strand comprises a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 294, and the antisense strand comprises a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 383; and

(g) the sense strand comprises a first nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 315, and the antisense strand comprises a second nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 404

[00048] In some embodiments, the sense strand and the antisense strand comprise a pair of nucleic acid sequences selected from the group consisting of:

(b) the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 281, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 370;

(c) the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 283, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 372; (d) the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 287, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 376:

(f) the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 294, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 383; and

Table 3a: Modified sense strand and antisense strand sequences of PPP1R15B RNAi agents

Abbreviations - “m” indicates 2’-0Me; "‘f ’ indicates 2’-fluoro; indicates phosphorothioate linkage; “P” indicates 5 ’-phosphate; “S” means the sense strand; “AS” means the antisense strand.

Table 3b: Modified sense strand and antisense strand sequences of PPP1R15B RNAi agents

Abbreviations - “m” indicates 2’-OMe; “f” indicates 2’-fluoro; indicates phosphorothioate linkage; “P” indicates S’-phosphate, “S” means the sense strand, “AS” means the antisense strand.

Table 4. Abasic and inverted abasic (iAb) moieties

“5”’ and “3^?” indicate the 5’ to 3’ direction of the sequences.

[00049] In some embodiments, the delivery moiety (D) is conjugated to the 3’ end of the sense stand via a linker (L). Suitable linkers are known in the art.. In some embodiments, linker (L) comprises Formula (III) or Formula (IV):

[00050] The sense strand and antisense strand of PPP1R15B RNAi agent can be synthesized using any nucleic acid polymerization methods known in the art, for example, solid-phase synthesis by employing phosphoramidite chemistry' methodology (e.g., Current Protocols in Nucleic Acid Chemistry, Beaucage, S.L. et al, (Edrs ), John Wiley & Sons, Inc., New York, NY, USA), H-phosphonate, phosphortriester chemistry, or enzymatic synthesis. Automated commercial synthesizers can be used, for example, MerMade™ 12 from LGC Biosearch Technologies, or other synthesizers from BioAutomation or Applied Biosystems.

Phosphorothioate linkages can be introduced using a sulfurizing reagent such as phenylacetyl disulfide or DDTT (((dimethylaminomethylidene) amino)-3H~l,2,4-dithiazaoline-3-thione). It is well known to use similar techniques and commercially available modified amidites and controlled -pore glass (CPG) products to synthesize modified oligonucleotides or conjugated oligonucleotides.

[00051] Purification methods can be used to exclude the unwanted impurities from the final oligonucleotide product. Commonly used purification techniques for single stranded oligonucleotides include reverse-phase ion pair high performance liquid chromatography (RP- IP-HPLC), capillary gel electrophoresis (CGE), anion exchange HPLC (AX-HPLC), and size exclusion chromatography (SEC). After purification, oligonucleotides can be analyzed by mass spectrometry and quantified by spectrophotometry at a wavelength of 260 nra. The sense strand and antisense strand can then be annealed to form a duplex.

[00052] In another aspect, provided herein are pharmaceutical compositions comprising an PPP1R15B RNAi agent described herein and a pharmaceutically acceptable carrier. Also provided herein are pharmaceutical compositions comprising a means for reducing PPP1R15B expression in a cell and a pharmaceutically acceptable carrier. Such pharmaceutical compositions can also comprise one or more pharmaceutically acceptable excipient, diluent, or carrier Pharmaceutical compositions can be prepared by methods well known in the art (e.g , Remington: The Science and Practice of Pharmacy, 23rd edition (2020), A. Loyd et al., Academic Press).

[00053] In a further aspect, provided herein are methods of reducing PPP1R15B expression in a cell, such methods can include introducing a PPP1R15B RNAi agent described herein into the cell; and incubating the cell for a time sufficient for degradation of PPP1RI5B mRNA, thereby reducing PPP1R15B expression in the cell. The PPP1R15B RNAi agent can be introduced into the cell using a method known in the art, e.g., transfection, electroporation, microinjection, or uptake by the cell via natural transport mechanisms.

[00054] In another aspect, provided herein are methods of treating a PPP I R15B associated metabolic disorder, such as obesity, Type 2 diabetes, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), dyslipidemia, or metabolic syndrome in a patient in need thereof, and such method comprises administering to the patient an effective amount of a PPP1R15B RNAi agent or a pharmaceutical composition described herein. In some embodiments, the PPP1R15B RNAi agent or the pharmaceutical composition is administered to the patient intravenously or subcutaneously.

[00055] In some embodiments, such methods further comprise administering a second therapeutic agent to the patient. The PPP1R15B RNAi agent described herein may be used in simultaneous, separate, and sequential combinations with a second therapeutic agent selected from incretin, metformin, a thiazolidinedione, a sulfonylurea, a dipeptidyl peptidase 4 inhibitor, a sodium glucose co-transporter, a SGLT-2 inhibitor, a growth differentiation factor 15 modulator (“GDF15”), a peptide tyrosine tyrosine modulator (“PYY”), a modified insulin, amylin, a dual amylin calcitonin receptor agonist, or oxyntomodulin agonist (“OXM”) for the treatment of a condition selected from the group consisting of T2D, obesity, NASH, NAFLD, dyslipidemia, and metabolic syndrome.

[00056] In some embodiments, such methods comprise administering a PPP1R15B RNAi agent and an incretin to the patient in simultaneous, separate, and sequential combinations. Examples of incretin include glucagon like peptide- 1 (GLP-1) or GLP-1 analogs, glucosedependent insulinotropic polypeptide (GIP) or GIP analogs, oxyntomodulin or oxyntomodulin analogs; dual GIP and GLP-1 receptor agonists; GCG, and GIP receptor agonist and GLP-1 receptor tri-agonists. In certain embodiments, the incretin may be selected from semaglutide, dulaglutide, tirzepatide, exenatide, liraglutide, albiglutide, lixisenatide, or retatrutide.

[00057] In some embodiments, a PPP1R15B RNAi agent described here is for use in simultaneous, separate and sequential combinations with a second therapeutic agent selected from incretin, metformin, a thiazolidinedione, a sulfonylurea, a dipeptidyl peptidase 4 inhibitor, a sodium glucose co-transporter, a SGLT-2 inhibitor, GDFI5, PYY, a modified insulin, amylin, a dual amylin calcitonin receptor agonist, OXM, a glucagon like peptide-1 (GLP-1) or GLP-1 analog, a glucose-dependent insulinotropic polypeptide (GIP) or GIP analog, oxyntomodulin or an oxyntomodulin analog, a dual GIP and GLP-1 receptor agonist; a GCG, and GIP receptor agonist and GLP-1 receptor tri-agonists; semaglutide, dulaglutide, tirzepatide, exenatide, liraglutide, albiglutide, lixisenatide, and retatrutide in the treatment of a condition selected from the group consisting of T2D, NASH, obesity, NAFLD, dyslipidemia, and metabolic syndrome [00058] RNAi dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.

[00059] Dosage values may vary with the type and severity of the condition to be alleviated. It is further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.

[00060] In another aspect, provided herein are PPP1R15B RNAi agents or the pharmaceutical composition comprising an PPP1R15B RNAi agent for use in a therapy. Also provided are PPP1R15B RNAi agents or pharmaceutical compositions comprising a PPP1R15B RNAi agent for use in the treatment of a PPPIR15B associated metabolic disorder, such as obesity, Type 2 diabetes, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), dyslipidemia, or metabolic syndrome. Also provided herein are uses of PPP1R15B RNAi agents in the manufacture of a medicament for the treatment of a PPP1R15B associated metabolic disorder, such as obesity. Type 2 diabetes, or nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), dyslipidemia, or metabolic syndrome.

[00061] As used herein, the terms “a,” “an,” “the,” and similar terms used in the context of the present disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context

[00062] As used herein, the term “alkyl” means saturated linear or branched-chain monovalent hydrocarbon radical, containing the indicated number of carbon atoms. For example, “C1-C20 alkyl” means a radical having 1-20 carbon atoms in a linear or branched arrangement.

[00063] As used herein, “antisense strand” means a single-stranded oligonucleotide that is complementary to a region of a target sequence. Likewise, and as used herein, “sense strand” means a single-stranded oligonucleotide that is complementary to a region of an antisense strand.

[00064] As used herein, “complementary'” means a structural relationship between two nucleotides (e.g, on two opposing nucleic acids or on opposing regions of a single nucleic acid strand, e.g., a hairpin) that permits the two nucleotides to form base pairs with one another For example, a purine nucleotide of one nucleic acid that is complementary to a pyrimidine nucleotide of an opposing nucleic acid may base pair together by forming hydrogen bonds with one another Complementary nucleotides can base pair in the Watson-Crick manner or in any other manner that allows for the formation of stable duplexes Likewise, two nucleic acids may have regions of multiple nucleotides that are complementary with each other to form regions of complementarity, as described herein.

[00065] As used herein, a “delivery moiety” refers to a chemical moiety that facilitates the entry of an oligonucleotide or RNAi agent into a cell. The delivery moiety can be lipid, cholesterol, vitamin E, carbohydrate (e.g., GalNAc), amino sugar, or polypeptide.

[00066] As used herein, “duplex,” in reference to nucleic acids or oligonucleotides, means a structure formed through complementary' base pairing of two antiparallel sequences of nucleotides (i.e , in opposite directions), whether formed by two separate nucleic acid strands or by a single, folded strand (e.g., via a hairpin).

[00067] An “effective amount” refers to an amount necessary (for periods of time and for the means of administration) to achieve the desired therapeutic result. An effective amount of a RNAi agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the RNAi agent to elicit a desired response in the individual An effective amount is also one in which any toxic or detrimental effects of the RNAi agent are outweighed by the therapeutically beneficial effects.

[00068] The term “knockdown” or “expression knockdown” refers to reduced niRNA or protein expression of a gene after treatment of a reagent, e.g,, a RNAi agent

[00069] As used herein, “modified internucleotide linkage” means an internucleotide linkage having one or more chemical modifications when compared with a reference internucleotide linkage having a phosphodi ester bond. A modified internucleotide linkage can be a non- naturally occurring linkage. In some embodiments, the modified internucleotide linkage is ph osph oroth i oate li nkage .

[00070] As used herein, “modified nucleotide” refers to a nucleotide having one or more chemical modifications when compared with a corresponding reference nucleotide selected from: adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide, and thymidine deoxyribonucleotide. A modified nucleotide can have, for example, one or more chemical modification in its sugar, nucleobase, and/or phosphate group. Additionally, or alternatively, a modified nucleotide can have one or more chemical moieties conjugated to a corresponding reference nucleotide.

[00071] As used herein, ‘"nucleotide” means an organic compound having a nucleoside (a nucleobase, e.g., adenine, cytosine, guanine, thymine, or uracil, and a pentose sugar, e.g,, ribose or 2^!-deoxyribose) linked to a phosphate group. A “nucleotide” can serve as a monomeric unit of nucleic acid polymers such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[00072] As used herein, “oligonucleotide” means a polymer of linked nucleotides, each of which can be modified or unmodified. An oligonucleotide is typically less than about 100 nucleotides in length.

[00073] As used herein, “overhang” means the unpaired nucleotide or nucleotides that protrude from the duplex structure of a double stranded oligonucleotide. An overhang may include one or more unpaired nucleotides extending from a duplex region at the 5’ terminus or 3’ terminus of a double stranded oligonucleotide. The overhang can be a 3’ or 5’ overhang on the antisense strand or sense strand of a double stranded oligonucleotide.

[00074] The term “patient”, as used herein, refers to a human patient,

[00075] As used herein, “phosphate analog” means a chemical moiety that mimics the electrostatic and/or steric properties of a phosphate group. In some embodiments, a phosphate analog is positioned at the 5’ terminal nucleotide of an oligonucleotide in place of a 5’- phosphate, which is often susceptible to enzymatic removal. A 5’ phosphate analog can include a phosphatase-resistant linkage. Examples of phosphate analogs include 5’ methylene phosphonate (5’-MP) and 5’ -(E)-vinylphosphonate (5’-VP). In some embodiments, the phosphate analog is 5’-VP.

[00076] The term “% sequence identity'” or “percentage sequence identity'” with respect to a reference nucleic acid sequence is defined as the percentage of nucleotides, nucleosides, or nucleobases in a candidate sequence that are identical with the nucleotides, nucleosides, or nucleobases in the reference nucleic acid sequence, after optimally aligning the sequences and introducing gaps or overhangs, if necessary', to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software programs, for example, those described in Current Protocols in Molecular Biology (Ausubel et al., eds., 1987, Supp. 30, section 7.7.18, Table 7.7.] ), and including BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), Clustal W2.0 or Clustal X2.0 software. Those skill ed in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Percentage of “sequence identity” can be determined by comparing two optimally aligned sequences over a comparison window, where the fragment of the nucleic acid sequence in the comparison window may comprise additions or deletions (e.g., gaps or overhangs) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions at which the identical nucleotide, nucleoside, or nucleobase occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. The output is the percent identity of the subject sequence with respect to the query sequence.

[00077] As used herein, “RNAi,” “RNAi agent,” “iRNA,” “iRNA agent,” and “RNA interference agent” means an agent that mediates sequence-specific degradation of a target mRNA by RNA interference, e.g., via RNA-induced silencing complex (RISC) pathway. In some embodiments, the RNAi agent has a sense strand and an antisense strand, and the sense strand and the antisense strand form a duplex (e.g., a double stranded RNA). In some embodiments, the sense strand has a delivery moiety conjugated to the 3’ end of the sense strand or a nucl eotide of the sense strand.

[00078] As used herein, “strand” refers to a single, contiguous sequence of nucleotides linked together through internucleotide linkages (e.g., phosphodiester linkages or phosphorothioate linkages). A strand can have tw⁷o free ends (e.g., a 5’ end and a 3’ end).

[00079] As used herein, PPP1R15B refers to a PPP1R15B mRNA transcript. The nucleic acid sequence of human PPP1R15B mRNA can be found at NM_032833.5 (SEQ II) NO: 1). The amino acid sequence of human PPP1R15B protein can be found at NP 116222.4. The nucleic acid sequence of mouse PPP1R15B mRNA can be found at NM_133819.3 (SEQ ID NO: 425). The amino acid sequence of mouse PPP1R15B protein can be found at NP_598580.1.

[00080] As used herein, “PPP1R15B associated metabolic disorder” means a metabolic disorder associated with abnormal PPP1R15B expression, activity, or function [00081 ] As used herein, “subject” means a mammal, including cat, dog, mouse, rat, chimpanzee, ape, monkey, and human. Preferably the subject is human.

[00082] As used herein, “'treatment” or “treating” refers to all processes wherein there may be a slowing, controlling, delaying, or stopping of the progression of the disorders or disease disclosed herein, or ameliorating disorder or disease symptoms, but does not necessarily indicate a total elimination of all disorder or disease symptoms. Treatment includes administration of a protein or nucleic acid or vector or composition for treatment of a disease or condition in a patient, particularly in a human.

EXAMPLES

Example 1. Synthesis of PPP1R15B RNAi Agents

[00083] Single strands (sense and antisense) of the dsRNA were typically synthesized on solid support via a MerMade™ 12 (LGC Biosearch Technologies) or a similar automated oligonucleotide synthesizer The sequences of the sense and antisense strands were shown in Tables 2 and 3. The sense strands were synthesized using an appropriate CPG such as 3'- Cholesterol-TEG CNA CPG 500 (LGC Biosearch Technologies) or phthalamido amino C6 Icaa CPG 500 A (Chemgenes) whereas the antisense strands used standard support (LGC Biosearch Technologies). The oligonucleotides were synthesized via phosphoramidite chemistry at an appropriate scale for in-vitro or in-vivo experimentation.

[00084] Standard reagents were used in the oligo synthesis (Table 5), where 0.1M xanthane hydride in pyridine was used as the sulfurization reagent and 20% DEA in ACN was used as an auxiliary wash post synthesis. All monomers (Table 6) were made at 0. IM in ACN and contained a molecular sieves trap bag. The structure of linked cholesterol is shown in Table 7. [00085] The antisense strands were typically cleaved and deprotected (C/D) at 45 °C for 16- 24 hours. The sense strands were ty pically cleaved and deprotected from the CPG using cold 50% (methylamine/ammonia hydroxide 28-30%) at RT for 2-3 hrs, whereas 3% DEA in ammonia hydroxide (28-30%, cold) was typically used for the antisense strands. C/D was determined complete by IP-RP LCMS when the resulting mass data confirmed the identity of sequence. RNA hydroxy desilylation may be carried out using triethylamine tri hydrofluoride in DM SO. Dependent on scale, the CPG was filtered via 0.45 um PVDF syringeless filter, 0.22 pm PVDF Steriflip® vacuum filtration or 0.22 gm PVDF Stericup® Quick release. The CPG was typically back washed/rinsed with either 30% EtOH/RNAse free water then filtered through the same filtering device and combined with the first filtrate. This was repeated twice. The material was then divided evenly into conical centrifuge tubes to remove organics via Genevac™. After concentration, the crude oligonucleotides were diluted back to synthesized scale with RNAse free water and filtered either by 0.45 pm PVDF syringeless filter, 0,22 pm PVDF Sterillip® vacuum filtration or 0.22 gm PVDF Stericup® Quick release.

[00086] The crude oligonucleotides were purified via AKTA™ Pure purification system using anion-exchange (AEX) or reverse-phase (RP) chromatography. For AEX, an ES Industry Source™ 15Q column with MPA: 20mM NaftPCfi, 15% ACN, pH 7.4 and MPB: 20 mM NaH₂PO₄, IM NaBr, 15% ACN, pH 7.4. For RP, an ES Industry Source™ 15RPC with MPA: 50mM sodium acetate, 10% ACN and MPB: 80% ACN. Fractions which contained a mass purity greater than 85% without impurities >5% where combined.

[00087] The purified oligonucleotides were desalted using 15 mL 3K MWCO centrifugal spin tubes at 3500xg for ~30 min. The oligonucleotides were rinsed with RNAse free water until the eluent conductivity reached < 100 usemi/cm. After desalting was complete, 2-3 mL of RNAse free water was added then aspirated lOx, the retainment was transferred to a 50 mL falcon tube, this was repeated until complete transfer of oligo by measuring concentration of compound on filter via nanodrop. The final oligonucleotide was then nano filtered 2x via 15 mL 100K MWCO centrifugal spin tubes at 3500xgfor 2 min. Cholesterol-linked oligonucleotides were annealed at this stage to give cholesterol conjugated dsRNA by mixing equimolar aliquots of sense and antisense strands at room temperature for 30 minutes The final desalted oligonucleotides were analyzed for concentration (nano drop at A260), characterized by IP-RP LC/MS for mass purity and LJPLC for UV-purity

[00088] RNAi agents comprising a GalNAc moiety, e.g., Formula (II), can be synthesized according to the methods described in WO2022/271806.

Table 5 ~ Oligonucleotide Synthesis Reagents

Cap B (1 -.Methylimidazole in THF, 16:84)

Oxidation Solution (0.02M Iodine in THF/Pyridine/Water, 70:20:10)

Deblock Solution, 3% TCA in DCM (w/v)

Acetonitrile (Anhydrosolv, Water max. 10 ppm)

Xanthane Hydride (O IM in Pyridine)

Di ethylamine (20% in Acetonitrile)

Table 6 - Phosphoramidites

Phosphoramidite Abbreviation Supplier Catalog # CAS

DMT-2'-F-A(Bz)-CE fA Hongene PD 1-001 136834-22-5

Phosphoamidite

DMT-2'-F-C(Ac)-CE fC Hongene PD3-001 159414.99-0

Phosphoamidite

DMT-2'-F-G(iBu)-CE iG Hongene PD2-002 144089-97-4

Phosphoamidite

DMT-2'-F-U-CE fXj Hongene PD5-001 146954-75-8

Phosphoamidite

DMT-2'-O-Me-A(Bz)~ mA Hongene PR 1-001 110782-31-5

CE Phosphoamidite

DMT-2'-O-Me-C(Ac)~ mC Hongene PR3-001 199593-09-4

CE Phosphoamidite

DMT-2'-O-Me-G(iBu)- mG Hongene PR2-002 150780-67-9

CE Phosphoamidite

Table 7. Linked Cholesterol Structures

Example 2. In vitro Characterization of PPP1R15B RNAi Agents

[00089] Selected PPP1R15B RNAi agents were tested in vitro for PPP1R15B knock down in cultured cells. Knockdown of PPP1R15B expression by these RNAi agents is assayed using the following procedure: human Hep3b cells were plated and each RNAi agent was added directly to an individual well The RNAi agents were added at. concentrations of 500 nM and 50 nM, in duplicate. Treated cells were lysed and an analysis of changes in gene expression in RNAi agent treated cells was measured using Cells-to-CT Kits following the manufacturer’s protocol (ThermoFisher A35377). Predesigned gene expression assays (supplied as 20X mixtures) were selected from Applied Bio-systems (Foster City, CA, USA) The efficiencies of these assays (ThermoFisher Hs03044848_m 1 PPP1R15B and ThermoFisher Hs99999905_ml GAPDH) were characterized with a dilution series of cDNA. RT-QPCR was performed in MicroAmp Optical 384-well reaction plates using QuantStudio 7 Flex system. The delta-delta CT method of normalizing to the housekeeping gene GAPDH was used to determine relative amounts of gene expression. GraphPad Prism was used to determine ICso with a three-parameter logistic fit. Select siRNzA agents were further tested at multiple concentrations: 1000, 200, 40, 8, 1.6, 0.32, 0.064, and 0.0128 nM of cholesterol conjugated siRNA was used. Results are shown as percent knockdown (%KD) of the PPP1R15B transcript

[00090] Knockdown of PPPlR15B expression by top 7 RNAi agents at 1000, 200, 40, 8, 1 6, 0.32, 0.064, and 0.0128 nM was assayed in HepalClC7 (mouse) cells using the same procedure described in Hep3b cells Predesigned gene expression assays (supplied as 20X mixtures) were selected from .Applied Bio-systems (Foster City, CA, USA). The efficiencies of these assays (ThermoFisher Mm00551747 ml PPP1R15B and ThermoFisher 435234 IE beta-actin) were characterized with a dilution series of cDNA. RT-QPCR was performed in MicroAmp Optical 384-well reaction plates using QuantStudio 7 Flex system. The delta-delta CT method of normalizing to the housekeeping gene beta-actin was used to determine relative amounts of gene expression. GraphPad Prism was used to determine IC50 with a three-parameter logistic fit. Results are shown as percent knockdown (%KD) of the PPP1R15B transcript.

Results

[00091 ] Table 8 shows the percentage knockdown of PPP1 R15B mRNA in human Hep3b cells by the tested PPP1R15B RNAi agents. Table 9 shows the percentage knockdown of PPP1R.15B in human Hep3b cells by selected PPP1 R15B RNAi agents at different concentrations. Table 10 shows the percentage knockdown of PPPIR15B in mouse HepalCIC7 cells by selected PPP1R15B RNAi agents.

Table 8: In vitro knock down of PPP1R15B mRNA in human Hep3b cells by PPP1R15B

RNAi agents.

Table 9: In vitro knock down of PPP1R15B in human Hep3b cells by PPP1R15B RNAi agents

Table 10: In vitro knock down of PPP1R15B in mouse HepalClC7 ceils by PPP1R15B RNAi agents.

Example 3, In vivo Characterization of Selected PPP1R15B RNAi Agents in Mice

[00092] The efficacy of selected PPP1R15B RNAi agents was studied in mice.

PPP1R15B RNAi potency study

[00093] Animals were individually housed in a temperature-controlled facility with 12h/12h light/dark cycle and fed a standard chow diet (TD2014; Teklad Diets). Animal protocols were approved by the Eli Lilly and Co., Animal Use and Care Committee (Protocol No. 20-025). C57BL/6 male mice were dosed subcutaneously with vehicle control (PBS) or an RNAi agent (5 mg/kg), n=6 per group. Animals were sacrificed 14 days post dose and livers were harvested. RNA was isolated from liver tissue using TRIzol reagent (Invitrogen) and the PureLink Pro 96 Total RNA purification kit (Invitrogen) RN A was transcribed into cDNA using a High- Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Taqman assays for RPLP0 (Mm01974474_gH, Applied Biosystems), PPP1R15B (Mm00551747_m l, Applied Biosystems) were used to measure gene expression using QuanStudio 7 Flex and normalized to RPLP0 rnRNA.

[00094] The results from this study are shown in Table 11 as the percent knockdown (%KD) of PPP1R15B relative to vehicle group.

Table 11: In vivo knockdown of PPP1RI5B in monse liver by GalNAc conjugated PPP1R15B RNAi agents

Efficacy of PPP1 R15B RNAi agent in diet induced obese (DIO) mice

[00095] Animals were individually housed at thermoneutrality with 12h/12h light/dark cycle and fed 60% high-fat diet (D12492, Research Diets). C57B1/6J male mice (lax 380050, The Jackson Laboratory) were treated with RNAi agent or vehicle (PBS, Invitrogen) control subcutaneously at 10 tng/kg (n=4 for vehicle, n~6 for siRNA group). Animals were euthanized 14 or 28 days post dose and livers were harvested for confirmation of gene knockdown. RNA isolation and gene expression were tested as described above.

[00096] The results from this study are shown in Table 12 as the percent knockdown (%KD) of PPP1R15B relative to vehicle group.

Table 12: Knockdown of PPP1R15B in liver samples collected from DIO mice after PPP1R15B RNAi agent treatment.

Example 4. PPP1R15B RNAi agent enhances GLP-1R agonist induced body weight loss in DIO mice

[00097] Animals were individually housed at thermoneutrality with 12h/12h light/dark cycle and fed 60% high-fat diet (DI 2492; Research Diets), C57B1/6J male mice (Jax 380050, The Jackson Laboratory) were dosed subcutaneously with a non-targeting control (NIC, 10 mg/kg), PPP1R15B RNAi agent (10 mg/kg), GLP-1R agonist (3 nmol/kg/day), or a combination of PPP1R15B RNAi agent and GLP-1R agonist (n=6 for each agent). Animals were monitored daily for body weight, food intake and were euthanized 18 days post dose when livers were harvested for confirmation of gene knockdown. RNA was isolated from liver tissue using TRIzol reagent (Invitrogen) and the RNeasy Plus mini kit (Qiagen). RNA was transcribed into cDNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Taqman assays for RPLPO (MmO 1974474 gH, Applied Biosystems), PPP1R15B (Mm00551747 ml, Applied Biosystems) were used to measure gene expression using QuanStudio 7 Flex and normalized to RPLPO mRNA.

[00098] Results from this study showed that a single dose of RNAi agent or daily dosing with GLP-1R agonist reduced body weight by 13% and 8% respectively after 11 days, while the combination treatment led to -32% body weight loss (Figure 1).

Example 5. PPP1R15B RNAi agent enhances energy expenditure in DIO mice

[00099] To investigate the effect of the PPP1R15B RNAi agent on whole-body metabolic rate, animals were individually housed in metabolic cages (Sable Systems) at thermoneutrality with 12h/12h light/dark cycle and fed 60% high-fat diet (HFD, D12492; Research Diets).

Animals had ad libitum access to water and HFD when in metabolic cages. Energy expenditure was determined in obese mice treated with vehicle (n = 6) or the PPP1R15B RNAi agent (dsRNA No. 122; 10 mg/kg, n 6) using an open respirometer system (Sable Systems). Energy expenditure was calculated by standard methods using oxygen consumption (VO?, mL/kg/h) and carbon dioxide production (VCO?, mL/kg/h) to arrive at the energy expenditure in kilocalories (kcal) per day (1.440 (3.9 VO₂ + 1.1 VCO₂, Weir. 1949, J. Physiol. 109: 1 -9).

[000100] As shown in Figure 2, mice given PPP1R15B RNAi agent showed increased daily energy expenditure when compared to the vehicle control group in statistically significant fashion starting on day 2 and continuing throughout the 10-day treatment period.

SEQUENCE LISTING

Claims

1 . A PPP1R15B RNAi agent comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a duplex, wherein the sense strand comprises a nucleic acid sequence selected from any one of SEQ ID NOs: 81-159, and the antisense strand comprises a nucleic acid sequence selected from any one of SEQ ID NOs: 160-238, wherein optionally one or more nucleotides of the sense strand and the antisense strand are independently modified nucleotides, and wherein optionally one or more internucleotide linkages of the sense strand and the antisense strand are modified internucl eotide linkages.

2. The PPP1R15B RNAi agent of claim 1, wherein the sense strand is linked to a delivery' moiety.

3 The PPP1R15B RNAi agent of claim 2, wherein the delivery moiety is linked to the 3’ end of the sense strand, optionally via a linker (L).

4. The PPP1R15B RNAi agent of any one of claims 2-3, wherein the delivery moiety comprises aN-acetylgalactosamine (GalNAc) moiety.

5. The PPP1R15B RNAi agent of any one of claims 2-4, wherein the delivery moiety comprises the Formula of:

6. A PPP1 R15B RNAi agent comprising the Formula of: R-L-D, wherein R is a double stranded RNA (dsRNA) comprising a sense stand and an antisense strand, wherein the antisense strand is complementary to PPP1R15B mRNA, wherein L is a linker, or optionally absent, and wherein D is a delivery moiety comprising the Formula of

7. The PPP1R15B RNAi agent of claim 6, wherein the sense strand comprises a nucleic acid sequence selected from any one of SEQ ID NOs: 2-80, wherein optionally one or more nucleotides of the sense strand are independently modified nucleotides, and wherein optionally one or more internucleotide linkages of the sense strand are modified internucleotide linkages

8 The PPP1R15B RNAi agent of claim 6, wherein the sense strand comprises a nucleic acid sequence selected from any one of SEQ ID NOs: 81 -159, and the antisense strand comprises a nucleic acid sequence selected from any one of SEQ ID NOs: 160-238, wherein optionally one or more nucleotides of the sense strand and the antisense strand are independently modified nucleotides, and wherein optionally one or more internucleotide linkages of the sense strand and the antisense strand are modified internucleotide linkages.

9. The PPP1R15B RNAi agent of any one of claims 1-8, wherein the sense strand and the antisense strand comprise a pair of nucleic acid sequences of the sense strand and the antisense strand of any one of dsRNA No. 1 -79, wherein optionally one or more nucleotides of the sense strand and the antisense strand are independently modified nucleotides, and wherein optionally one or more intemucleotide linkages of the sense strand and the antisense strand are modified intemucleotide linkages.

10. The PPP1R15B RNAi agent of any one of claims 1-9, wherein the sense strand and the antisense strand comprise a pair of nucleic acid sequences selected from the group consisting of:

(a) the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 93, and the antisense strand comprises a second nucleic acid sequence of SEQ ED NO: 172;

(c) the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 12.5, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 204;

(d) the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 129, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 208,

(e) the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 132, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 21 1;

(g) the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 157, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 236, wherein optionally one or more nucleotides of the sense strand and the antisense strand are independently modified nucleotides, and wherein optionally one or more intemucleotide linkages of the sense strand and the antisense strand are modified i nternucl eoti de 1 i n kages .

11. The PPP1R15B RNAi agent of any one of claims 1-10, wherein one or more nucleotides of the sense strand are modified nucleotides.

12. The PPP1R15B RNAi agent of claim 11, wherein each nucleotide of the sense strand is a modified nucleotide.

13. The PPP1 R15B RNAi agent of any one of claims 1 -12, wherein one or more nucleotides of the antisense strand are modified nucleotides.

14. The PPP1R15B RNAi agent of claim 13, wherein each nucleotide of the antisense strand is a modified nucleotide.

15 The PPP1R15B RNAi agent of any one of claims 11-14, wherein the modified nucleotide is a 2'-fluoro modified nucleotide, 2'-O-methyl modified nucleotide, 2’ deoxy nucleotide (DNA), or 2'-O-alkyl modified nucleotide.

16. The PPP1R15B RNAi agent of any one of claims 11-15, wherein the sense strand has four 2'-fluoro modified nucleotides at positions 7, 9, 10, and 11 from the 5’ end of the sense strand.

17. The PPPlR15B RNAi agent of claim 16, wherein nucleotides at positions other than positions 7, 9, 10, and 11 of the sense strand are 2'-O-methyl modified nucleotides

18. The PPP1R15B RNAi agent of any one of claims 1 1 -17, wherein the antisense strand has four 2'-fluoro modified nucleotides at positions 2, 6, 14, and 16 from the 5’ end of the antisense strand.

19. The PPP1R15B RNAi agent of claim 18, wherein nucleotides at positions other than positions 2, 6, 14 and 16 of the antisense strand are 2'-O-methyl modified nucleotides.

20 The PPP1R15B RNAi agent of any one of claims 11-15, wherein the sense strand has three 2'-fluoro modified nucleotides at positions 9, 10, and 11 from the 5’ end of the sense strand

21 The PPP] R15B RNAi agent of claim 20, wherein nucleotides at positions other than positions 9, 10, and 1 1 of the sense strand are 2'-O-methyl modified nucleotides.

22. The PPPlR15B RNAi agent of any one of claims 11-17 and 20-21, wherein the antisense strand has five 2'-fluoro modified nucleotides at positions 2, 5, 7, 14, and 16 from the 5’ end of the antisense strand.

23. The PPP1R15B RNAi agent of claim 22, wherein nucleotides at positions other than positions 2, 5, 1, 14, and 16 of the antisense strand are 2'-O-methyl modified nucleotides.

24. The PPP1R15B RNAi agent of any one of claims 1 1-17 and 20-21, wherein the antisense strand has five 2'-fluoro modified nucleotides at positions 2, 5, 8, 14, and 16 from the 5’ end of the antisense strand.

25. The PPP1R15B RNAi agent of claim 24, wherein nucleotides at positions other than positions 2, 5, 8, 14, and 16 of the antisense strand are 2'-O-methyl modified nucleotides.

26. The PPP1R15B RNAi agent of any one of claims 11-17 and 20-21 , wherein the antisense strand has five 2'-fluoro modified nucleotides at positions 2, 3, 7, 14, and 16 from the 5’ end of the antisense strand.

27. The PPP1R15B RNAi agent, of claim 26, wherein nucleotides at positions other than positions 2, 3, 7, 14, and 16 of the antisense strand are 2'-O-methyl modified nucleotides.

28. The PPP1R15B RNAi agent of any one of claims 1-27, wherein the sense strand and the antisense strand have one or more modified internucleotide linkages.

29. The PPP1R15B RNAi agent of claim 28, wherein the modified internucleotide linkage is phosphorothioate linkage.

30. The PPP1R15B RNAi agent of claim 28 or 29, wherein the sense strand has four or five phosphorothioate linkages.

31 The PPP1 R15B RNAi agent of any one of claims 28-30, wherein the antisense strand has four or five phosphorothioate linkages

32. The PPPlR15B RNAi agent of any one of claims 1-31, wherein the anti sense strand has

5" phosphate or 5’ vinylphosphonate.

33. The PPP1R15B RN Ai agent of any one of claims 1 -32, wherein the sense strand comprises an abasic moiety or inverted abasic moiety.

34. The PPP1R15B RNAi agent of any one of claims 1-15 or 28-33, wherein the sense strand and the antisense strand comprise a pair of nucleic acid sequences of the sense strand and the antisense strand of any one of dsRNA No. 80-186.

35. The PPP1R15B RNAi agent of any one of claims 1-15 or 28-34, wherein the sense strand and the antisense strand comprise a pair of nucleic acid sequences selected from the group consisting of:

(a) the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 251, and the antisense strand comprises a second nucleic acid sequence of SEQ ED NO: 340;

(b) the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 281, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 370,

(c) the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 283, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 372;

(d) the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 287, and the antisense strand comprises a second nucleic acid sequence of SEQ ID NO: 376,

(f) the sense strand comprises a first nucleic acid sequence of SEQ ID NO: 294, and the antisense strand comprises a second nucleic acid sequence of SEQ ED NO: 383; and

36. The PPP1R15B RNAi agent of any one of claims 3-35, wherein L (linker) is present and comprises any one of the following Formulas:

37 A pharmaceutical composition comprising the PPP1R15B RNAi agent of any one of claims 1-36 and a pharmaceutically acceptable carrier.

38. A method of treating a PPP1R15B associated metabolic disorder in a patient in need thereof, the method comprising administering to the patient an effective amount of the PPP1R15B RNAi agent of any one of claims 1-36, or the pharmaceutical composition of claim 37.

39. The method of claim 38, wherein the PPP1R15B associated metabolic disorder is obesity, Type 2 diabetes, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), dyslipidemia, or metabolic syndrome.

40. The method of claim 38 or 39, wherein the PPP1R15B RNAi agent or the pharmaceutical composition is administered to the patient intravenously or subcutaneously.

41. The method of any one of claims 38-40 further comprising administering a second therapeutic agent to the patient.

42. The method of claim 41, wherein the second therapeutic agent is an incretin.

43. A method of reducing PPP1R15B expression in a cell, the method comprising: introducing the PPP1R15B RNAi agent of any one of claims 1-36 into the cell; and incubating the cell for a time sufficient for degradation of PPP1R15B mRNA, thereby reducing PPP1R15B expression in the cell.

44. The PPP1R15B RNAi agent of any one of claims 1 -36, or the pharmaceutical composition of claim 37, for use in a therapy.

45. The PPP1R15B RNAi agent of any one of claims 1-36, or the pharmaceutical composition of claim 37, for use in the treatment of a PPP1R15B associated metabolic disorder.

46. The PPP1R15B RNAi agent or pharmaceutical composition for use of claim 45, wherein the PPP1R15B associated metabolic disorder is obesity. Type 2 diabetes, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), dyslipidemia, or metabolic syndrome.

47. Use of the PPP1R15B RNAi agent of any one of claims 1-36 in the manufacture of a medicament for treating a PPP1R15B associated metabolic disorder.

48 The use of claim 47, wherein the PPP1R15B associated metabolic disorder is obesity, Type 2 diabetes, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), dyslipidemia, or metabolic syndrome.

49. The PPP1R15B RNAi agent for use of any one of claim 45-46 or the use of any one of claims 47-48 further comprising administering a second therapeutic agent to the patient.

50. The PPP1R15B RNAi agent for use or the use of 49, wherein the second therapeutic agent is an incretin.