Compositions and methods for treating mash

Abstract

The invention is directed to nucleic acid compositions and methods for targeting ODC1.

Description

Docket No: 2932719-000287-W01Date of Filing: December 11, 2025 COMPOSITIONS AND METHODS FOR TREATING MASH

[0001] This application is an International Application, which claims the benefit of priority from U.S. provisional patent application no. 63 / 730,830, filed on December 11, 2024, the entire contents of each which are incorporated herein by reference in their entireties.

[0002] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.

[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U. S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.FIELD OF THE INVENTION

[0004] The invention is directed to nucleic acid compositions and methods for treating metabolic dysfunction-associated steatohepatitis (MASH).BACKGROUND OF THE INVENTION

[0005] Affecting over one-third of the global population, metabolic dysfunction-associated steatotic liver disease (MASLD) has become the most common cause of chronic liver disease worldwide. Approximately 20-30% of individuals with MASLD develop metabolic dysfunction-associated steatohepatitis (MASH), characterized by lobular inflammation and hepatocellular ballooning. Unmitigated progression of MASH leads to hepatic fibrosis, which is the main contributor to death in for individuals with this disease. Although the recent approval of resmetirom represents a significant advancement for the treatment of MASH, its effectiveness is limited - benefiting only 10-15% of patients after placebo-adjustment. Moreover, the interaction of resmetirom with statins, fibrates, and anticoagulants, along with its uncertain long-term safety profile, complicates its use in clinical settings where patients often require multiple therapies over extended periods.DocketNo: 2932719-000287-W01Date of Filing: December 11, 2025 SUMMARY OF THE INVENTION

[0006] Aspects of the invention are drawn towards a nucleic acid molecule comprising a sense oligonucleotide and an antisense oligonucleotide. In embodiments, the sense oligonucleotide or the antisense oligonucleotide is complementary to a target nucleic acid sequence of a gene or gene product encoding ornithine decarboxylase 1 (ODC1).

[0007] In embodiments, the sense oligonucleotide is complimentary to the antisense oligonucleotide.

[0008] In embodiments, the nucleic acid molecule can comprise one or more N-acetylgalactosamine (GalNAc) derivatives.

[0009] In embodiments, the nucleic acid molecule can comprise one or more nucleotides with a modification chosen from a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, or both.

[0010] In embodiments, the nucleic acid molecule can comprise one or more nucleotides connected to one another by way of phosphodiester or phosphorothioate linkages.

[0011] In embodiments, the nucleic acid molecule can comprise a small interfering RNA (siRNA) molecule.

[0012] In embodiments, the antisense oligonucleotide can comprise the sequence of:a. mUfUmAmUmAfCmGmCmCmGmAmGmCfAmCfGmUmCmGmUmCmAmU; b. AACACAGCGGGCAUCAGAG;c. AAUGAUGGCGUCUAUGGAUCAUU;d. AACAUUAUCCUCUUGUGCUAAUU;e. AAUACAAUGUGAUAAGUUGACUU;f. AAGUUGACUUGAAGAUUACAGUU;g. AAAGCUUAGUGUUGUGACCUGUU;h. AACUGUAAUACUCAGUAGCCGUU; oran antisense oligonucleotide having 70%, 75%, 80%, 85%, 90% or 95% nucleic acid sequence identity to any of the same.

[0013] In embodiments, the sense oligonucleotide can comprise the sequence of:a. mG*mA*mCmGmAmCfGmUfGfCfUmCmGmGmCmGmUmAmUmAmA;b. CUCUGAUGCCCGCUGUGUU;c. UGAUGGCGUCUAUGGAUCA;Docket No: 2932719-000287-W01Date of Filing: December 11, 2025 d. CAUUAUCCUCUUGUGCUAA;e. UACAAUGUGAUAAGUUGAC;f. GUUGACUUGAAGAUUACAG;g. AGCUUAGUGUUGUGACCUG;h. CUGUAAUACUCAGUAGCCG; ora sense oligonucleotide having 70%, 75%, 80%, 85%, 90% or 95% nucleic acid sequence identity to any of the same.

[0014] In embodiments, the nucleic acid molecule can inhibit expression of ODC1. For example, the nucleic acid molecule can inhibit the expression of ODC1 mRNA by at least 50%.

[0015] In embodiments, the nucleic acid molecule can comprise at least five 2'-O-methyl modified nucleotides and at least five 2'-fluoro modified nucleotides.

[0016] In embodiments, the one or more GalNAc derivatives is attached to the 3' end of the sense strand.

[0017] In embodiments, the one or more GalNAc derivatives can comprise a biantennaiy or a triantennary GalNAc ligand.

[0018] In embodiments, the GalNAc ligand is attached to the 3' end of the sense strand.

[0019] In embodiments, the GalNAc ligand is attached to the 3' end of the sense strand via linker.

[0020] Aspects of the invention are further drawn to a nucleic acid according to the sequence of:a. mUfUmAmUmAfCmGmCmCmGmAmGmCfAmCfGmUmCmGmUmCmAmU; b. AACACAGCGGGCAUCAGAG;c. AAUGAUGGCGUCUAUGGAUCAUU;d. AACAUUAUCCUCUUGUGCUAAUU;e. AAUACAAUGUGAUAAGUUGACUU;f. AAGUUGACUUGAAGAUUACAGUU;g. AAAGCUUAGUGUUGUGACCUGUU;h. AACUGUAAUACUCAGUAGCCGUU; ora nucleic acid having 70%, 75%, 80%, 85%, 90% or 95% nucleic acid sequence identity to any of the same.

[0021] Aspects of the invention are further drawn to a nucleic acid according to the sequence of:a. mG*mA*mCmGmAmCfGmUfGfCfUmCmGmGmCmGmUmAmUmAmA;Docket No: 2932719-000287-W01Date of Filing: December 11, 2025 b. CUCUGAUGCCCGCUGUGUU;c. UGAUGGCGUCUAUGGAUCA;d. CAUUAUCCUCUUGUGCUAA;e. UACAAUGUGAUAAGUUGAC;f. GUUGACUUGAAGAUUACAG;g. AGCUUAGUGUUGUGACCUG;h. CUGUAAUACUCAGUAGCCG; ora nucleic acid having 70%, 75%, 80%, 85%, 90% or 95% nucleic acid sequence identity to any of the same.

[0022] Aspects of the invention are further drawn to a pharmaceutical composition comprising the nucleic acid molecule described herein.

[0023] In embodiments, the pharmaceutical composition can be formulated for intravenous or subcutaneous administration.

[0024] Still further, aspects of the invention are drawn to a method of treating chronic liver disease. In embodiments, the method can comprise administering to a subject the nucleic acid molecule described herein.

[0025] In embodiments, the subject is at risk for developing, or is diagnosed with, chronic liver disease. For example, chronic liver disease can comprise metabolic dysfunction-associated steatotic liver disease (MASLD), metabolic dysfunction-associated steatohepatitis (MASH), hepatic fibrosis alcoholic liver disease, chronic hepatitis B, chronic hepatitis C, autoimmune hepatitis, primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), hemochromatosis, Wilson's disease, alpha- 1 antitrypsin deficiency, hepatocellular carcinoma, or a combination thereof.

[0026] In embodiments, the method can decrease a level of hepatic putrescine in the subject.

[0027] Aspects of the invention are further drawn to a method of reducing hepatic putrescine levels in a subject. In embodiments, the method can comprise administering to the subject the nucleic acid molecule described herein.BRIEF DESCRIPTION OF THE FIGURES

[0028] FIG. 1 provides the structure of polyamines putrescine, spermidine, and spermine.DocketNo: 2932719-000287-W01Date of Filing: December 11, 2025

[0029] FIG. 2 provides a schematic showing the molecular pathway of polyamine biosynthesis. Ornithine is de-carboxylated by Odel in putrescine. Spermidine is synthesized from putrescine using an aminopropyl group donated from decarboxylated S-adenosyl-L-methionine (S-adeno met-DC) catalyzed by spermidine synthase. Spermine is synthesized from spermidine with S-adeno met-DC catalyzed by spermine synthase.

[0030] FIG.3 provides data showing circulating and hepatic putrescine in humans and mice. (A) Liver sections from mice fed a control or MASH diet for 24 weeks were stained with H& E and picrosirius red. Samples from these mice (n=8-10 / group) were analyzed for (B) hepatic putrescine.(C) Liver sections from individuals with or without MASH were stained with H& E and picrosirius red. Samples from these individuals (n=10-44 / group) were analyzed for (D) hepatic putrescine. Data are mean ± SEM, P values are indicated.

[0031] FIG.4 provides data showing putrescine drives fibrosis in MASH. (A) Hepatic putrescine levels from mice fed a MASH-inducing diet and putrescine-enriched water (3 mM) or normal water for 20 weeks. (B) Sirius red staining of livers from mice described in Panel A. Student’s t-test (AB); n=7 / group. Graphs are mean ± SEM with P values indicated.

[0032] FIG. 5 provides data showing feasibility of siOdcl-GalNAcs and potential to reduce liver injury. (A) Livers from ScrRNA-GalNAc and siOdcl-GalNAc treated mice were immunoblotted for ODC1 and GAPDH (6 representative samples from each group are shown). (B) Hepatic putrescine levels from mice in Panel A. (C-D) Plasma from mice in Panel A were measured for ALT and AST (n=7-8 / group). Data are mean ± SEM with P values indicated.

[0033] FIG.6 provides data showing siOdcl-GalNAcs reduce hepatic fibrosis and the expression of collagen genes. (A) Liver crosssections from ScrRNA-GalNAc and siOdcl-GalNAc treated mice were stained for picrosirius red and quantified as fold change relative to ScrRNA-GalNAc controls (representative images shown; n=7-8 / group). (B-E) Quantitative PCR of RNA isolated from livers were performed for Collal, Colla2, Col4al, and Col4a2 (n=7-8 / group). Data are mean ± SEM with P values indicated.

[0034] FIG. 7 provides a schematic showing the progression of MASLD to hepatocellular carcinoma.

[0035] FIG. 8 provides data showing AST and ALT levels in healthy and MASH individuals.

[0036] FIG. 9 provides a schematic showing the polyamine metabolic pathway.

[0037] FIG. 10 provides data showing dysregulation in polyamines during MASH.DocketNo: 2932719-000287-W01Date of Filing: December 11, 2025

[0038] FIG. 11 provides data showing putrescine positively correlates with MASH indices in humans and mice.

[0039] FIG. 12 provides data showing AMD1 is decreased in hepatocytes in MASH.

[0040] FIG. 13 provides data showing AMD1 silencing leads to putrescine accumulation.

[0041] FIG. 14 provides data showing AMD1 downregulation worsens fibrosis in MASH.

[0042] FIG. 15 provides data showing mitochondrial respiration is lower in AMD 1 -deficient HepG2 cells.

[0043] FIG. 16 data showing genes associated with FAO are decreased in livers from shAMDl mice.

[0044] FIG. 17 provides data showing genes associated with FAO are decreased in AMD1-deficient HepG2 cells.

[0045] FIG. 18 provides data showing FAO oxidation is unaffected in AMD 1 -deficient cells treated with a CPT1 inhibitor.

[0046] FIG. 19 provides data showing hepatic AMD1 silencing drives hepatic fibrosis in MASH.

[0047] FIG. 20 provides data showing validation of AMD1 deletion and hepatocyte specificity is provided.

[0048] FIG. 21 provides data showing putrescine drives hepatic fibrosis in MASH.

[0049] FIG. 22 provides data showing the effect overexpressing AMD1 has on fibrosis during MASH.

[0050] FIG. 23 provides data showing the effects of IHH upregulation by putrescine.

[0051] FIG. 24 provides data showing putrescine promotes Mcf2 mRNA stability.

[0052] FIG. 25 provides data showing levels of fibrosis in mice.

[0053] FIG. 26 provides data showing targeting putrescine using a N-acetyl-galactosamines (GalNAc)-based therapeutic approach.

[0054] FIG. 27 provides data showing putrescine metabolism is dysregulated in human and murine MASH. (A) Representative images of H& E and picrosirius red stained liver sections from mice fed a standard diet or with a MASH-inducing diet for 12 or 24 weeks. (B-D) Hepatic polyamines (putrescine, spermidine, and spermine) were quantified by UPLC-MS / MS (n=6 mice / group). (E) Correlation of hepatic putrescine levels with fibrosis score (n=18 mice). (F) Representative images of H& E and picrosirius red stained liver sections of liver specimens from healthy individuals or from patients with MASH. (G-I) Hepatic polyamines from humans in FDocket No: 2932719-000287-W01Date of Filing: December 11, 2025 were quantified as in B-D (n=10 healthy donors and n=15 patients form MASH). (J) Correlation between hepatic putrescine and fibrosis score in human MASH patients (n=25 individuals). (K-M) HepG2 cells were treated with palmitate for 24 hours (200 pM) followed by polyamine analysis (n=4 / group). (N) Representative image of a primary hepatocyte immunostained for ASGPR1. (O-Q) Primary mouse hepatocytes were treated with palmitate and quantified for polyamines as in K-M (n=4 / group). Data are mean + SEM, P values are shown.

[0055] FIG. 28 provides data showing putrescine is sufficient to drive hepatic fibrosis in MASH.(A) Schematic of the experimental design showing mice fed a MASH-inducing diet for 20 weeks and provided with normal drinking water or water supplemented with 3 mM putrescine (n=7 mice / group). (B) Hepatic putrescine levels were quantified from mice in A. (C-G) Representative H& E staining and quantitative assessment of steatosis, lobular inflammation, hepatocellular ballooning, and NAS scores (n=7 mice / group). (H) Liver section from mice in A were stained for picrosirius red and quantified for picrosirius red+ areas of total area (n=7 mice / group). (I) Hepatic hydroxyproline from mice in A was quantified using a quantitative colorimetric detection assay (n=7 mice / group). (J) Total RNA was isolated from the livers of mice in A followed by cDNA synthesis and qPCR analysis for Collal, Colla2, Col4al, Col4a5, and Col4a6 (n=7 mice / group). Data are mean ± SEM, P values are shown.

[0056] FIG. 29 provides data showing AMD1 is downregulated in hepatocytes during MASH in both humans and mice. (A-C) HepG2 cells were treated with palmitate (200 pM) for 24 hours and analyzed for AMD1, ODC1, and SRM by qPCR (n=6 / group). (D-F) Primary mouse hepatocytes were treated with palmitate and analyzed as in A-C (n=4 / group). (G-I) RNA from human liver specimens was analyzed for AMD1, ODC1, and SRM by qPCR (n=10-15 individuals / group).(J) Tissue sections from human livers were immunostained for AMD 1 and HepParl and quantified for AMD1 MFI within HepParl+ regions (n=9-17 individuals / group). (K-M) RNA isolated from mouse livers was analyzed for Amdl, Odel, and Srm by qPCR (n=6 mice / group). (N) Liver sections from mice fed a standard diet or with a MASH-inducing diet for 12 or 24 weeks were immunostained for AMD1 and ARG1 and quantified for AMD1 MFI within ARG1+ regions (n=5 / group). (O-P) HepG2 cells were silenced for AMD1 followed by qPCR analysis o AMD 1 and quantification for putrescine (n=4 / group). (Q-R) Primary mouse hepatocytes were transfected with siAmdl and quantified for Amdl and putrescine as in O-P (n=4 / group). Data are mean ± SEM, P values are shown.Docket No: 2932719-000287-W01Date of Filing: December 11, 2025

[0057] FIG. 30 provides data showing silencing liver AMD1 accelerates fibrosis in MASH.(A) Schematic of the liver-targeted AAV8-U6 shRNA approach used to silence AMD1 in mice maintained on a MASH-inducing diet for 12 weeks. (B) Liver sections from mice in A were immunostained and quantified as in FIG. 29N (n=7-8 / group). (C) Liver specimens were measured for putrescine as in FIG. 27 (n=7-8 / group). (D) Circulating ALT was measured from plasma in mice from A (n=7-8 / group). (E) RNA from livers from mice in A were analyzed by RNAseq. Pathway analysis and associated genes with overrepresented genes are shown (n=4 / group, Fisher’s exact test and Benjamini -Hochberg multiple testing adjustment). (F) Total RNA was isolated from the livers of mice in A followed by qPCR analysis for Collal, Colla2, Col4al, Col4a5, and Col4a6 (n=7-8 mice / group). (G) Liver fibrosis was quantified as in FIG. 28H (n=7-8 mice / group).(H) Hydroxyproline content was quantified as in FIG. 28H (n=7-8 mice / group). (I) Liver sections from mice in A were immunostained for aSMA and quantified for aSMA+ area of total area relative to shScr (n=7-8 mice / group). Data are mean ± SEM, P values are shown.

[0058] FIG. 31 provides data showing hepatocyte-specific AMD1 deletion promotes fibrosis in MASH. (A) Amdl^ mice were injected retro-orbitally with 2xlOnviral genomes of AAV8-TBG-Cre or AAV8-TBG-GFP and maintained on chow for four weeks. Livers were then isolated and immunostained for AMD1 and ARG1. Representative images of liver sections are shown, with non-parenchymal cells from AAV8-TBG-Cre injected mice retaining AMD1 signal shown in yellow boxes and expanded to the right. (B) Primary hepatocytes were isolated from livers described in A and quantified for Amdl by qPCR (n=7 mice / group). (C) Schematic of the experimental design for control and hepatocyte-specific AMD1 knockout mice maintained on a MASH-inducing diet for 12 weeks. (D) Liver specimens were measured for putrescine as in FIG.27 (n=10-l 1 mice / group). (E) Circulating ALT was measured from plasma in mice from C (n=10-11 mice / group). (F) Liver fibrosis stained for picrosirius red and quantified as in FIG. 28H (n=10-11 mice / group). (G) Hydroxyproline content was quantified as in FIG. 28H (n=10- l 1 mice / group). Total RNA was isolated from the livers of mice in C followed by qPCR analysis for Collal, Colla2, Col4al, Col4a5, Col4a6, and Col6a5 (n=10-ll mice / group). Data are mean ± SEM, P values are shown.

[0059] FIG.32 provides data showing hepatocyte-specific AMD1 overexpression reduces fibrosis in MASH. (A) Schematic of mice being fed a MASH-inducing diet for 12 weeks followed by hepatocytetargeted AMD1 overexpression while being maintained on diet for another 12 weeks.Docket No: 2932719-000287-W01Date of Filing: December 11, 2025 (B) Liver sections from mice in A were immunostained and quantified as in FIG. 29N (n=7 mice / group). (C) Liver specimens were measured for putrescine as in FIG. 27 (n=7 mice / group).(D) Circulating ALT was measured from plasma in mice (n=7 mice / group). (E) Liver fibrosis stained for picrosirius red and quantified as in FIG. 28H (n=7 mice / group). (F) Hydroxyproline content was quantified as in FIG. 28H (n=7 mice / group). (G) Total RNA was isolated from the livers of mice in followed by qPCR analysis for Collal, Colla2, Col4al, Col4a5, Col4a6, and Col6a5 (n=7 mice / group). (H) Liver sections from mice in A were immunostained for aSMA and quantified for aSMA+ area of total area relative to mice injected with AAV8-TBG-AMD1 (n=7 mice / group). Data are mean ± SEM, P values are shown.

[0060] FIG. 33 provides data showing putrescine-induced IHH by hepatocytes activates stellate cells to drive fibrosis. (A) RNAseq data from FIG. 30C was analyzed for Tgfb, Pdgfb, Shh, Tnf, and Ihh (n=4 mice / group). (B-D) ScrRNA or si AMD 1 -transfected (B) HepG2 cells and (C) primary mouse hepatocytes along with (D) Putr-treated HepG2 cells were quantified for IHH mRNA by qPCR (n=4-6 / group). (E-G) Cells treated as in A-C were quantified for IHH secretion in conditioned media by ELISA (n=4-6 / group). (H-K) HepG2 and primary mouse hepatocytes were treated with putrescine or transfected with si AMD 1 and analyzed for Ihh by qPCR (n=4-5 / group). RNA isolated from (H) mice treated with putrescine-containing water, (I) silenced for Amdl using AAV8-U6-shAmdl, (J) treated with AAV8-TBG-Cre to delete AMD1, or (K) AAV8-TBG-AMD1 to overexpress AMD1 were analyzed by qPCR for GH2, Gli3, Ccndl, Ccnl (n=7-l 1 mice / group). (L-O) Conditioned media (CM) was harvested from ScrRNA- or siAMDl -transfected HepG2 cells followed by immunodepletion in siAMDl -transfected CM using a bead-immobilized anti-IHH antibody. LX-2 cells were cultured for 24 hours with the indicated groups of CM (50% native media / 50% CM) and analyzed for (L) COL1A1, (M) COL1A2, (N) COL4A1, and (O) COL4A5 by qPCR (n=4 / group). (P-S) LX-2 cells were transfected with siSMO and treated with siAMDl- conditioned media as in L-O. LX-2 cells were then lysed and analyzed for (P) COL1A1 (Q) COL1A2 (R) COL4A1 and (S) COL4A5 by qPCR (n=3 / group). Data are mean ± SEM, P values are shown.

[0061] FIG. 34 provides data showing therapeutic lowering of hepatocyte putrescine reduces hepatocyte / stellate cell crosstalk and mitigates fibrosis in MASH. (A) Mice were fed a MASH-inducing diet for 12 weeks then received weekly intraperitoneal injections of GalNAc-conjugated siRNA against ODC1 (siODCl-GalNAc, 1 mg / kg) or control ScrRNA. (B) RepresentativeDocket No: 2932719-000287-W01Date of Filing: December 11, 2025 immunoblots for ODC1, IHH, and GAPDH. (C) Livers from mice in A were quantified for liver putrescine (n=7 mice / group). (D) Circulating ALT was measured from plasma in mice (n=7 mice / group). (E) Total RNA was isolated from the livers of mice in followed by qPCR analysis for Col1a1, Colla2, Col4al, Col4a5, and Col4a6 (n=7 mice / group). (F) RNA from livers were analyzed by qPCR for Gli2, Gli3, Ccndl, Ccnl (n=7 mice / group). (G) Liver fibrosis stained for picrosirius red and quantified as in FIG. 28H (n=7 mice / group). (H) Hydroxyproline content was quantified as in FIG. 28H (n=7 mice / group). (I) Liver sections from mice in A were immunostained for aSMA and quantified for aSMA+ area of total area relative to mice injected with AAV8-TBG-AMD1 (n=7 mice / group). Data are mean ± SEM, P values are shown.DETAILED DESCRIPTION OF THE INVENTION

[0062] Detailed descriptions of one or more embodiments are provided herein. It is to be understood, however, that the invention can be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the invention in any appropriate manner.

[0063] The singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification can mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0064] Wherever any of the phrases “for example,” “such as,” “including” and the like are used herein, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise. Similarly, “an example,” “exemplary” and the like are understood to be non-limiting.

[0065] The term “substantially” allows for deviations from the descriptor that do not negatively impact the intended purpose. Descriptive terms are understood to be modified by the term “substantially” even if the word “substantially” is not explicitly recited.

[0066] The terms “comprising” and “including” and “having” and “involving” (and similarly “comprises”, “includes,” “has,” and “involves”) and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of “comprising” and is therefore interpreted to be an open termDocket No: 2932719-000287-W01Date of Filing: December 11, 2025 meaning “at least the following,” and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, “a process involving steps a, b, and c” means that the process includes at least steps a, b and c. Wherever the terms “a” or “an” are used, “one or more” is understood, unless such interpretation is nonsensical in context.

[0067] As used herein, the term “about” can refer to approximately, roughly, around, or in the region of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower).

[0068] Aspects of the invention are directed towards a nucleic acid molecule(s) for targeting ornithine decarboxylase 1 (ODC1). For example, the nucleic acid molecule(s) comprises a sense oligonucleotide and an antisense oligonucleotide, wherein the sense oligonucleotide or the antisense oligonucleotide is complementary to a target nucleic acid sequence of a gene or gene product encoding ODC1.

[0069] In embodiments, the nucleic acid molecule(s) comprises a sense oligonucleotide and an antisense oligonucleotide, wherein the sense oligonucleotide or the antisense oligonucleotide is complementary to a target nucleic acid sequence of a gene or gene product encoding ODC1. The nucleic acid sequence for ODC1 can comprise the sequence of or a fragment thereof according to:GTCAGTCCCTCCTGTAGCCGCCGCCGCCGCCGCCCGCCGCCCCTCTGCCAGCAG CTCCGGCGCCACCTCGGGCCGGCGTCTCCGGCGGGCGGGAGCCAGGCGCTGACG GGCGCGGCGGGGGCGGCCGAGCGCTCCTGCGGCTGCGACTCAGGCTCCGGCGTC TGCGCTTCCCCATGGGGCTGGCCTGCGGCGCCTGGGCGCTCTGAGGTGAGGGAC TCCCCGGCCGCGGAGGAAGGGAGGGAGCGAGGGCGGGAGCCGGGGCGGGCTGCG GGCCCCGGGCCCCGGGCACGTGTGCGGCGCGCCTCGCCGGCCTGCGGAGACACG TGGTCGCCGAGCGGGCCACGACCTTGAGGCGCCGCTTCCTCCCGGCCCGGGGTT CTCCCGCGGCTGGATAAGGGTGATCCGGGCGCCTCGTTCTGCCCCCGTCTTCAC AGCTCGGGGCTGGAGGGGCCTAGGGGAGACCCACCCGGAGACCCTGCGGCCCCG CGCCGGCCTCTTTCCCAACCCTTCGGCGGCCGCGCGCTGGCCGGGGAGCCGTTG GGGAGGCCCTGGCGGCCGCGCAGCAGGTGCAGGGGCGCAGAGCCCGGGCTCGCC TTGGTACAGACGAGCGGGCCCCGGCCTTGGCGCCTTCAGTTTCCTTCCAGTTTT TATTTTCGCTGTGTC T AC AGAGC AGAT GAG AC C AAT T T GGAAAC C C G C GAGAG T GGGTAGAGCTAAGATAGTCTTGCTGTAGTAGCTGTGATATTAGATGCTCGGCCA TGACTTAGAGGTGTTTATTTAAGGACTGTGAATGACTCGGTGATTTCGGAAAAG CTTGGCTTAGATGAACGGACATACACAGGGGAGACAGCCCTAAGGTTTGCAGAA AAGGCTGATTGTGCTGTTTGCGAAGTCGAAATAATTGGTGAAAGTGTAGAAGGC AGAACCTCTCAGGAATGTCTGGGGAGGACAAAGAATGTGTTGGCTGACTTTGTT TAAAC AT AAAAT T G G GC AGAC T T T AAT T GAT T T G T GAAAT T T T T T T C AAAG T T T GTTTGAATTAGCCCCTATCTCTTCTAACATTATCCTCTTGTGCTAATTGATTGADocket No: 2932719-000287-W01Date of Filing: December 11, 2025 C C AT T T T AAT AC T T AG C T G T T AC AGAAAGAC C GAAAGG T G T T C T T GAG T AAA AT AT AT T C AAG T AAG T T AC T T AAG T AAC G C C T T AAAAGAT AC AGAAAAG C AAAA AAGTAT T GGCGTAT TAAAAAGAAAT CAAAAC T T T CCAAGT T TAGGCC T GAACAT T G C C T T AAAAAT AT T T AAT AAGG C C T CAAAT GAC C C AG T C C GAGAC T G C AT GAG CCTATTTAT T T T AAAT T G T AAAT AT T C T T C AT AT AAAC AAAAT AT AT AC C A TGTCTGTAACAAAAATGGTTTTGCTAGCGTTGTTACTCTCTTCCCTTCTCCGAG GGGTGATTTAGGCAACTTCGGAGGTTGACAATGCCAAGCAGTCACAATAGATAG AGCTTTAAAGCAAATTCTATGCATGGGTTTGGATTTATGACAGGCCCGTCACCC TGGGCCTGTCATAGTACCCCATGCCAGAGCAAACTGTGTCCCCGAACCATTGCC TGGCCTCTGTGCCCGTAGGCTGCTGGCACTGAAGTGGGTTGCACAGTGGAAAAG AAGAAAGC T CTACC T GGCAGAAAT T T T TAAAGGT TAAAATAAATAAT T T TAAGA AAGC TGGT T CACAAGGT GCCACAT T T GAT GAAAGCAAAATACAGT GGC T T T TAT TGTTACTAGAGTGATGTTCTTGCTTGTTTTTCTTTTTTGGTGAAGTTAGCCCCA AT T T T C T CAT AGC T AAGCAAAT AC GAGAG T GAC T G T AAGGACAG T T GGC T T CCCGGAATTGCTAAACTTGGTAGGCAACGCTGGTTTAAGAATACTGAGTTCTAG CCGGGCGTGGTGGCTCACGCCTGTAATCCCAACACTTTGGGAGGCTGAGGCAGG CGGATCACCTGAGGTCGGGAGTTGGAGACCAGCCTGACTAACATGGAGAAACGC CATCTCCACTAAAAATATAAAATTAGCCAGGCCCCGGGTGTGGTGGCACATGCC GGTAATCCCAGCTACTCGGGAGACTGAGGCAGGAGAATCGCTTGAACCCAGGAG GCGGAGGTTGAGGTGAGCCGAGATCATGCCATTGCACTCCAGCCTGGGCAACAA GAG T AAAAC T C T G T C T C AAAAAAAAAAAAAAAAAAT AC T GAAT T C T GAT C AG G T AAC AGC AAC T G T AAT AC AAT G T GAT AAG T T GAC T T GAAGAT T ACAG T T T T T AAG AAG T T AT AC C C AG C T AT AC T GAAAAT T AAC T C G T AAAAT C T C AAT GC T C C AGAC AT T T C CAT GATGCCTGTTGGT C AG T AAAAAT C AT T C T AAGAC T T AG T G GA AG T AGG AAT GTTTGTATGGCTGTG T T AAG GC TAT AT G T AAT C C C AGC C T TTGGAAGACCGAGGCGGGTGGATCACCTGGGGTCAGGAGTTTGAGACCCACCTG GAC AAC G T G G T GAAAT C C T G T C T C T AC T AAAAC C AAAAAT T AG C C G G GC AT G GTGGCAGGCGCCTGTAATCCCAGCTGCTGGGGAGGCTGAGGCAGGAGAATCGCT TGAACCCGGGAGGCAGAGGTTGCAGTGAGCCAAGATTGCACCGCTGCACTCCAG C C T G GG T GACAG C G T GAGAC T C T G T C T CAAAAAAAAT AAAAAAG T C TATAAT G C T AT T T T AAG T T T C T AAG GAAC T GAAAC T G C T C T GAAAT AAAT C AGAC CAT TAT A AGACTTTTTTCCATATCAGTGAGCTAAGTGCAGATAAGCTTCTGAAACTTGCAT GCTAGATTTTTTTGGTACAAATATTTGAAATGCTTAGTGTGCTGCCTTGGAAAA ACCTGGTATTTTTTGTTGTGTCCTTATACTGCCAAGGTTTATGGAATCATGTAC CTTATGCCTAGTAATAATTAGGATGACCAGGCCAGTGAGTGGTTCATATCCGGG GCATGATTAGCTCTGCGTGTGCTCAGCCAGTGCCCCATCTTCAACTCGATGTGT TCCTAAGGTAGACAGCAAATTCCCTATTTTATTTCTCAGATTGTCACTGCTGTT CCAAGGGCACACGCAGAGGGATTTGGAATTCCTGGAGAGTTGCCTTTGTGAGAA GCTGGAAATATTTCTTTCAATTCCATCTCTTAGTTTTCCATGTAAGTATTCAGT TTACATTTATGTTGCAGGTTAATCTTAAGAATTGTATTGCTAAGGCTTCTAAGT GAAT T T C T CCAC T C TAT T T GCAT T T T GT T GCAT T T CAGAGGAACAT CAAGAAAT CAT GAACAAC T T T GG T AAT GAAGAG T T T GAC T GC CAC T T C C T C GAT GAAGG T T T T AC T GC CAAG GAC AT T C T G GAC C GAAAAT T AAT GAAG T T T C T T C T T C T G T AG TATATGAGGCCCATGCTGGCAGTGCAGCTGAGAGTGCCAGGCAAGTGGAAAACT TTGGCAAGGTCTAAGGAAGAGCAATGAGGCTTACATGTCTTGTTATGGAATGTA GAAAT TAAT TCAC T GGT GGTAAAT TAATAGT GAT AAT GGT GAT ACT CATAT CAG TGGCTAGACTCAAAAGAGCAGGATTCATTGTGACTGATGGGAATGAAGGTCGCTDocket No: 2932719-000287-W01Date of Filing: December 11, 2025 GGCTATTGGTGTGGTGTGTGGTGAGGCTGCTAGTGAGTCACCTGTGACCACTCT TGTTTCAGGATGATAAGGATGCCTTCTATGTGGCAGACCTGGGAGACATTCTAA AGAAACATCTGAGGTGGTTAAAAGCTCTCCCTCGTGTCACCCCCTTTTATGCAG TCAAATGTAATGATAGCAAAGCCATCGTGAAGACCCTTGCTGCTACCGGGACAG GATTTGACTGTGCTAGCAAGGTAAGCGATAGCAGCAGGCCTCAAAAGCGTTGTA TAAAATGGGCCTGGTATTCCCCACGAGGCAGATACAAGTTGTGTTTTTTGGGCA ATAAATGCTCACTAAAGGCAAATGGGGCGGGGGGGTACATGACAACTTCCCATG CTTTTCTGTTTATTC GAG G T G T T AAG C GAG AT AT G GAT AG CAT GAG AC GAG T C T TCTTTTTCAGACTGAAATACAGTTGGTGCAGAGTCTGGGGGTGCCTCCAGAGAG GATTATCTATG C AAAT C C T T G T AAAC AAG T AT C T C AAAT T AAG TAT G C T GC T AA T AAT GGAG T C C AGAT GAT GAG T T T T GAT AG T GAAG T T GAG T T GAT GAAAG T T G C CAGAGCACATCCCAAAGCAAAGTGAGTTATTCCCCCATCTGAGGGCAAGATCGG GAG C AT AAGAT AT GTGGATTCTTAT C AAAC AAAC T T AAAT TTCTGATTATTATA TTTCTATACTTTAG T AGAAAG T AG T T GAAAG C C C C AT T GAG T C AT GAAG C C T G G GAG T GAAAG T AC AGAAT AT AT GAG C GAG AG T AT T T AGAAC AG GATTGTTTTTAT TTTAATTGTGGCTATAAGTGAACATCTATCATGAGACATTTGCTGCACTTTCCT TGCTTGTAGGTTGGTTTTGCGGATTGCCACTGATGATTCCAAAGCAGTCTGTCG TCTCAGTGTGAAATTCGGTGCCACGCTCAGAACCAGCAGGCTCCTTTTGGAACG GGCGAAAGAGCTAAATATCGATGTTGTTGGTGTCAGGTGAGATTTTGGTGGGAT AGCTAGAGGTCAAGACATTGAACAGTTTGAGTTTTACAGGCTTTCTCCTAGTGT T T G C T AT T AT T T T AAGAAAT AC TAAGAC AC AG T G T C T C G T C T C T T T AT T T T AC C CCAGCTTCCATGTAGGAAGCGGCTGTACCGATCCTGAGACCTTCGTGCAGGCAA TCTCTGATGCCCGCTGTGTTTTTGACATGGGGGTGAGTATACGTGACCCTGTTA GGGAAGGGCGGGACACAAC T GACAATAAC TAGTC T TAAT T C TAGAGT TAAC T T T TTATGGCAGTTGGTTCTGTATTACATGGGTTTCAGCCTATCTGCTGCATACATT TTTGTTATTAGCTGTGGATCTGGCTGACTTATTTTCTTGATTCTAGGCTGAGGT TGGTTTCAGCATGTATCTGCTTGATATTGGCGGTGGCTTTCCTGGATCTGAGGA T G T GAAAG T TAAAT T T GAAGAGG T AAT T T AGAACAAAAC T G TAAT AC T GAG TAG CCGTTCTAATAAATTCCTTTTTGGAATATTTCAAAATTTAAGTGTCTTAACTAA TACCACAATGGGCTGAAGTGTCTTGGTGTGATATTTTGAGTGATTTCTTTGTGC TGTCTGACATTACACTTGATACCATTTGGTTTTCTAAAGTGTGAATCAGCTTTC C C AGAAG T C T T G GATAAT T G G T T AC AT T G GAAAT C AT G GC T C ACAC C T G T AAT C CAGCACTTGGGGAGGCCAAGGTGGTAGGATCACTTGAGCCCAGGAGTTTGAGAC C AG C C T G G G GAAG C AG T GAGAC C C C AT C T C T AC AAAAAAAAT T T T AAAAT TAG CCTGGTGTGGTGGCGGGCACCTGTAATCCCAGCTACTTGGAAGGCTGAGGTGGG AGGATCACTTGAGCCCAGGAGGTTGAGGCTGCAGTGAGCCATGATCATGCCACT GCACTCAGCCTGGGCTACAGAGTGAGACCCTGTCTCAAAAAAAAAAAAGAAAAA GCATGTTGCTGTGGGCTTCCTAGAGAATATGCTGACTGTAGCACATCATCACCC CAAATGTGCTTTGCTAGACCTATGCTTCCTCTCCTTAAAATACTTGAAATGTTT AG T GAG T T AG GAAG T T AAG C CAT TATATTGGTGCTT GAAT T T AT AAAAT AT AT C CACATGGTTTGTTAAAATCATGACGTAGGCAGAATAGGATTTTTATCCTGTTGG CATGTATTTGTTAAAATGTTTTGACATCTTGATGCCTTCCTAGGTAGTAGTTAG TTGCGTACTGTTCTTT GAT AAAAAT CAT AC C C AT AAG AT C C T AAAG GAGATAG G GTGCCTGGAGGGGAATGAAAACGAGCCACCTGGGATATGTAGCCTGGTTTTCAG GGAGATGTTGATGTTTTTTTGCTTTTGTTACTTTAATGATAAACCTGTCTGTTG ATGCCTGGTCTCATGATGTCATGTCACAAGGCCCTGTGATGTTACTCCCCCATG TGAATTTCCCACAATGAAGGCTGCTCTTTCTTTTCTGTTTCACTCTCTTAGATCDocketNo: 2932719-000287-W01Date of Filing: December 11, 2025 ACCGGCGTAATCAACCCAGCGTTGGACAAATACTTTCCGTCAGACTCTGGAGTG AGAATCATAGCTGAGCCCGGCAGATACTATGTTGCATCAGCTTTCACGCTTGCA GTTAATATCATTGCCAAGAAAATTGTATTAAAGGAACAGACGGGCTCTGATGGT AT G T AT AAAG GAG GAAT C AC T T C AT G T AT AAC T GAAAG C T GAT G C AAAAAG T C A T T AGAT TGTTGATCTGCCTTTC T AGAC GAAGAT GAG T C GAG T GAG C GAC C T T TAT GTAT TATGT GAATGAT GGCGT C TAT GGAT CAT T TAAT T GCATAC T C TAT GA CCACGCACATGTAAAGCCCCTTCTGCAAAAGGTAATTTCTGAGCATACTGTATA AAAC AAT T AAGAG GAC T G G T C AC AAC AC G T G T AAT T AAG TAGTACTTCCTCTCT CCGTCTCTT T AT AT GAGAC C T AAAC CAGAT GAGAAG TATTATTCATC C AG CAT ATGGGGACCAACATGTGATGGCCTCGATCGGATTGTTGAGCGCTGTGACCTGCC TGAAATGCATGTGGGTGATTGGATGCTCTTTGAAAACATGGGCGCTTACACTGT TGCTGCTGCCTCTACGTTCAATGGCTTCCAGAGGCCGACGATCTACTATGTGAT GTCAGGGCCTGCGTGGTAAGTAAGCCATGCATGTTGATGGTGCTGCCAAGAATA GGCACCTTCTTGGATGTGTGCTTCTTGTCTAGACGAATAAGAAATTGTCTTGCC T AAGAT T AAAT AT AT AT GGATATTTTTCC T AAGAAAAG T T T T AGAAAAGAC T GA TGAGTGTATTTCTATGTAATTGGAATATATTTAAGTTCATGCCATGTGTCTTGT GGTTTCCTTATTACCAAAACGGTGACTGAAGAAACGCTTGCTTTAGAAATACAT TGAATTGGCCAGGTGTGCTGGCTCACACCTGAAATCACAACACATTGGGAGGCC AAGGCAGAAGGATCACTTGAGCCCAGGAGTTCGAGCCTGGGCAACATAGTGAGA CCCTGTCTCTACAAAAAATTAAAAAATTAGTTGGCCATGGTAGTGGGCGCCTGT AGTCCCAGCTGCTTGGCTAAGGTGAGAGGTTTGCTTGAGCCTGGGAGGTTGAGG CTGCGGTGAGCTATGATAGCACCATTGTATTCCAGCCTGAGTAACAGAGAAAGA CCC T GT C T CAGAAAAAAAAAAAATACAT T GAAT T GT T T CC T GATGGGAAGTAAA TACTCTCATGCCCAGTTAGGAGTGAGTCAGGGTTTTTAATATGCCACTTTTTCT TTCTCAGGCAACTCATGCAGCAATTCCAGAACCCCGACTTCCCACCCGAAGTAG AGGAACAGGATGCCAGCACCCTGCCTGTGTCTTGTGCCTGGGAGAGTGGGATGA AACGCCACAGAGCAGCCTGTGCTTCGGCTAGTATTAATGTGTAGATAGCACTCT GGTAGCTGTTAACTGCAAGTTTAGCTTGAATTAAGGGATTTGGGGGGACCATGT AACTTAATTACTGCTAGTTTTGAAATGTCTTTGTAAGAGTAGGGTCGCCATGAT GCAGCCATATGGAAGACTAGGATATGGGTCACACTTATCTGTGTTCCTATGGAA AC TAT T T GAAT AT T T GT T T TAT AT GGAT T T T TAT T CAC T C T T CAGACAC GC T AC TCAAGAGTGCCCCTCAGCTGCTGAACAAGCATTTGTAGCTTGTACAATGGCAGA AT G G GC C AAAAG C T T AG T G T T G T GAC C T G T T T T TAAAAT AAAG T AT C T T GAAAT AATTAGGCATTGGGACGTTTTTATGGTGTGTTCATTCCAGACAGTTCACGAATC CCGTATAGCTCGCTCTGATTCT C AGAGAAC AAT GAG T G GG T C CAC C CAC AC AC A GGTAGGAGGACAGGTGAGACGGAAGCCCCATCCTCCCATGTGGACGGTGCACAT CTGCTCAGCCCACCCCACATGTCCAGAGTTGGCTGCAAACTCCTTGTCCAGAGC CTCTGGTGGTGGGACCTACTTAAGTCTGACGGACCTGTCCTGTCCAGGCCAGTG CCCAGGGAAGGTGTGGGAGGCCCTTTGAGCCTGGCCTGCAGAGACCATCCGTGT CCCCTCCCACCTTCATGCCTGTGAGAAGTTAGGAATGTATACGGTACCACATTT GGCAGTCAGCTTATTTTAATAAATTCAGCAACAGCAAGTCCCTA

[0070] The nucleic acid sequence for the nucleic acid molecules described herein are provided in Table 1.Table 1: Nucleic Acid Sequences for ODC1 siRNAs.Docket No: 2932719-000287-W01Date of Filing: December 11, 2025 ODC1 siRNA Sense Strand Sequence Antisense Strand SequenceMouse GalNAC mG * mA* mCmGmAmC f GmU f G f mU f UmAmUmAf CmGmCmCmG Sequence C f UmCmGmGmCmGmUmAmUm mAmGmC f AmC f GmUmCmGmU AmA (GalNAc) mCmAmUValidated Human CUCUGAUGCCCGCUGUGUU AACACAGCGGGCAUCAGAG siRNA SequenceHuman siRNA UGAUGGCGUCUAUGGAUCA AAUGAUGGCGUCUAUGGAUCAUU Sequence 1Human siRNA CAUUAUCCUCUUGUGCUAA AACAUUAUCCUCUUGUGCUAAUU Sequence 2Human siRNA UACAAUGUGAUAAGUUGAC AAUACAAUGUGAUAAGUUGACUU Sequence 3Human siRNA GUUGACUUGAAGAUUACAG AAGUUGACUUGAAGAUUACAGUU Sequence 4Human siRNA AGCUUAGUGUUGUGACCUG AAAGCUUAGUGUUGUGACCUGUU Sequence 5Human siRNA CUGUAAUACUCAGUAGC C G AACUGUAAUACUCAGUAGC C GUU Sequence 62'-O-methvl modifications are indicated bv “m”. 2'-fluoro moc ifications are indicated bv “f”.

[0071] In embodiments, the nucleic acid molecule described herein can comprise a small interfering RNA (siRNA). The term “small interfering RNA” or “siRNA” can refer to a synthetic, double-stranded RNAs of approximately 20-25 nucleotides in length, which silence translation of a target mRNA through binding to the mRNA and promoting its degradation. siRNAs can be introduced into cells and tissues of animals through a variety of transfection techniques and can efficiently produce a specific knockdown of targeted genes. Therefore, siRNA is an important tool for the study of gene function and drug targets.

[0072] The term "double-stranded RNA" or "dsRNA," as used herein, can refer to an RNA molecule or complex of molecules having a hybridized duplex region that comprises two antiparallel and substantially complementary nucleic acid strands, which will be referred to as havingDocketNo: 2932719-000287-W01Date of Filing: December 11, 2025 "sense" and "antisense" orientations with respect to a target RNA. The duplex region can be of any length that permits specific degradation of a target RNA.

[0073] The term " RNA molecule" or "ribonucleic acid molecule" encompasses not only RNA molecules as expressed or found in nature, but also analogs and derivatives of RNA comprising one or more ribonucleotide / ribonucleoside analogs or derivatives as described herein or as known in the art. A "ribonucleoside" includes a nucleoside base and a ribose sugar, and a "ribonucleotide" is a ribonucleoside with one, two or three phosphate moieties. However, the terms "ribonucleoside" and "ribonucleotide" can be considered to be equivalent as used herein. The RNA can be modified in the nucleobase structure or in the ribose-phosphate backbone structure, e.g., as described herein. 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 one modified ribonucleotide including but not limited to a 2'-O-methyl modified nucleotide, 2'-fluoro modified nucleotide, a nucleotide comprising a 5' phosphorothioate group, a terminal nucleoside linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, a locked nucleoside, an abasic nucleoside, a 2'-deoxy-2'-fluoro modified nucleoside, a 2'-amino-modified nucleoside, 2'-alkylmodified nucleoside, morpholino nucleoside, a phosphoramidate or a non-natural base comprising nucleoside, or any combination thereof. Alternatively, an RNA molecule can comprise at least two modified ribonucleotides, 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 ribonucleotides in an RNA molecule.

[0074] As described herein, embodiments can comprise nucleic acid molecule(s) targeting a mRNA encoding ODC1 or a fragment thereof, and uses of the same. A “nucleic acid” or “polynucleotide” is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term "polynucleotide sequence" or “nucleic acid sequence” is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.

[0075] The terms "polynucleotide" and "oligonucleotide" can be used interchangeably and can refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides orDocket No: 2932719-000287-W01Date of Filing: December 11, 2025 ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non- limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term can also refer to both double- and singlestranded molecules. Unless otherwise specified or required, any embodiment of this disclosure that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double- stranded form.

[0076] " G," " C," " A," " T" and " U" each stand for a nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as a base, respectively. However, it will be understood that the term "ribonucleotide" or "nucleotide" can also refer to a modified nucleotide, as further detailed herein, or a surrogate replacement moiety. The skilled person is well aware that guanine, cytosine, adenine, and uracil can be replaced by other moi eties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base can base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine can be replaced in the nucleotide sequences of dsRNA featured in the invention by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide 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 invention.

[0077] The term "encode" as it is applied to polynucleotides can refer to a polynucleotide which is said to "encode" a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and / or translated to produce the mRNA for theDocket No: 2932719-000287-W01Date of Filing: December 11, 2025 polypeptide and / or a fragment thereof. The antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.

[0078] The terms “functional gene”, and “gene” can refer to a polynucleotide that contains a RNA coding sequence for one or more proteins that is operably linked to a promoter sequence and other transcriptional regulatory sequences to direct the correct transcription of the coding sequence into RNA and which also contains any of a number of translational regulatory sequences that can be necessary we are or are desirable to direct the correct translation of RNA into the protein in a given host cell. A translational start codon (e.g., ATG) and a ribosome binding site are required in RNA for translation occurring in prokaryotic and eukaryotic cells. Translational regulatory sequences can also be used, depending on the host cell, including, but not limited to, an RNA splicing site and a polyadenylation site.

[0079] The term "recombinant" is used in this application to describe altered nucleic acids or nucleic acids that have been manipulated, nucleic acids isolated from the environment in which they are found in nature, host cells transfected or otherwise manipulated to contain exogenous nucleic acids, or proteins expressed synthetically by manipulating isolated DNA or transforming host cells. “Recombinant” is a term that especially encompasses DNA molecules that have been constructed in vitro using genetic engineering methods, and the use of the term “recombinant” as an adjective to describe a molecule, construct, vector, cell, protein, polypeptide or polynucleotide specifically excludes such natural molecules, constructs, vectors, cells, proteins, polypeptides or polynucleotides.

[0080] In embodiments, the nucleic acid molecule(s) can comprise an antisense oligonucleotide complementary to a target mRNA molecule. For example, an antisense strand can be complementary to a target nucleic acid sequence of a gene or gene product encoding ODC1. The term “antisense oligonucleotide,” "antisense strand," or "guide strand" can refer to the strand of a nucleic acid molecule described herein, e.g., a siRNA, which includes a region that is substantially complementary to a target sequence. As used herein, the term "region of complementarity" can refer to the region on the antisense strand that is substantially 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 can be in the internal or terminal regions of the molecule. The most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, orDocketNo: 2932719-000287-W01Date of Filing: December 11, 2025 2 nucleotides of the 5' and / or 3' terminus. In embodiments, the antisense oligonucleotide can comprise the sequence of:a. mU f UmAmUmAf CmGmCmCmGmAmGmC f AmC f GmUmCmGmUmCmAmU; b. AACACAGCGGGCAUCAGAG;c. AAUGAUGGCGUCUAUGGAUCAUU;d. AACAUUAUCCUCUUGUGCUAAUU;e. AAUACAAUGUGAUAAGUUGACUU;f. AAGUUGACUUGAAGAUUACAGUU;g. AAAGCUUAGUGUUGUGACCUGUU;h. AACUGUAAUACUCAGUAGCCGUU; oran antisense oligonucleotide having 70%, 75%, 80%, 85%, 90% or 95% nucleic acid sequence identity to any of the same.

[0081] In embodiments, the nucleic acid molecule can comprise a sense oligonucleotide complimentary to the antisense strand. The term “sense oligonucleotide,” "sense strand," or "passenger strand" as used herein, can refer to the strand of a nucleic acid molecule described herein, e.g., a siRNA, that includes a region that is substantially complementary to a region of the antisense oligonucleotide as that term is defined herein. In embodiments, the sense oligonucleotide can comprise the sequence of:a. mG * mA* mCmGmAmC f GmU f G f C f UmCmGmGmCmGmUmAmUmAmA; b. CUCUGAUGCCCGCUGUGUU;c. UGAUGGCGUCUAUGGAUCA;d. CAUUAUCCUCUUGUGCUAA;e. UACAAUGUGAUAAGUUGAC;f. GUUGACUUGAAGAUUACAG;g. AGCUUAGUGUUGUGACCUG;h. CUGUAAUACUCAGUAGCCG; ora sense oligonucleotide having 70%, 75%, 80%, 85%, 90% or 95% nucleic acid sequence identity to any of the same.

[0082] As used herein, and unless otherwise indicated, the term "complementary," when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, can refer to theDocketNo: 2932719-000287-W01Date of Filing: December 11, 2025 ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person.

[0083] The terms "complementary," "fully complementary" and "substantially complementary" herein can 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 an siRNA agent and a target sequence, as will be understood from the context of their use.

[0084] As used herein, a polynucleotide that is "substantially complementary to at least part of a messenger RNA (mRNA) can refer to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding an ODC1 protein). For example, a polynucleotide is complementary to at least a part of an ODC1 mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding ODC1. As another example, a polynucleotide is complementary to at least a part of an ODC1 mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding ODC1.

[0085] The nucleic acid molecule(s) described herein target ODC1. For example, the nucleic acid molecule(s) comprises a sense oligonucleotide and an antisense oligonucleotide, wherein the sense oligonucleotide or the antisense oligonucleotide is complementary to a target nucleic acid sequence of a gene or gene product encoding ODC1. Some embodiments feature siRNA molecules that have a specified percentage identity or similarity to the amino acid or nucleotide sequences of the ODC1 siRNA molecules described herein.

[0086] The terms “homology” or “identity” or “similarity” can refer to sequence similarity between two peptides or between two nucleic acid molecules. To determine the percent homology of two amino acid sequences or two nucleic acids, the sequences are aligned for optimal comparison purposes (for example, gaps can be introduced into the sequence of the first amino acid sequence or nucleic acid for optimal alignment with the second compared amino acid sequence or nucleic acid sequence). Then compare amino acid residues or nucleotides at the corresponding positions of amino acids or nucleotides. In those cases where the position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous in this position (for example,Docket No: 2932719-000287-W01Date of Filing: December 11, 2025 as used in the description, the "homology" of amino acids or nucleic acids is equivalent to "identity " Amino acids or nucleic acid). Homology of nucleic acid sequences can be defined as the degree of identity between two sequences. Homology can be determined using computer programs known in the art, such as the GAP software provided in the GCG software package. See Needleman and Wunsch, J. Mol. Biol, 48: 443-453 (1970). For example, the mouse ODC1 sequence or fragment thereof can be homologous to the human ODC1 sequence or fragment thereof.

[0087] Homology can be determined by comparing a position in each sequence, which can be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. For example, the nucleic acid molecules can have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity when compared to a specified region or the full length of any one of the nucleic acid molecules described herein. Sequence identity or similarity to the nucleic acids and proteins of the invention can be determined by sequence comparison and / or alignment by methods using software programs known in the art, such as those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. For example, sequence comparison algorithms (i.e. BLAST or BLAST 2.0), manual alignment or visual inspection can be utilized to determine percent sequence identity or similarity for the nucleic acids and proteins of the invention.

[0088] In embodiments, the nucleic acid molecule(s) can inhibit expression of ornithine decarboxylase 1 (ODC1). ODC1 catalyzes the first and rate-limiting step of polyamine biosynthesis that converts ornithine into putrescine, which is the precursor for the polyamines, spermidine and spermine. As used herein, the term “polyamine” can refer to an organic compound comprising carbon, nitrogen, and hydrogen, and contains two or more amino groups. For example, polyamines of the disclosure can comprise putrescine and metabolites thereof. Putrescine is a biogenic polyamine generated through the decarboxylation of ornithine or related metabolic pathways. Polyamines are essential for cell proliferation and are implicated in cellular processes, ranging from DNA replication to apoptosis.

[0089] In embodiments, putrescine can comprise hepatic putrescine. The term “hepatic putrescine” can refer to putrescine present in, produced by, or accumulated within liver tissue. Hepatic putrescine can encompass endogenous putrescine synthesized by hepatocytes, putrescineDocketNo: 2932719-000287-W01Date of Filing: December 11, 2025 derived from systemic circulation and taken up by the liver, and putrescine formed through polyamine interconversion or catabolic processes within hepatic cells.

[0090] In embodiments, putrescine can comprise circulating putrescine. The term “circulating putrescine” can refer to putrescine present within the systemic circulation of a subject, including but not limited to whole blood, serum, plasma, or other blood-derived fractions

[0091] Metabolism of the amino acids arginine, ornithine, and methionine generates byproducts that feed into the polyamine biosynthetic pathway. Polyamines are small, linear polycations comprised of putrescine, spermidine, and spermine (FIG.2). The poly amine pathway is initiated by the conversion of arginine into ornithine via arginase 1 (ARG1), which then becomes decarboxylated by ornithine decarboxylase (ODC1) to give rise to putrescine. The conversion of putrescine into spermidine and spermine follows linear reactions carried out by the enzymes spermidine synthase (SRM) and spermine synthase (SMS). Essential to SRM and SMS activity is decarboxylated S-adenosyl-L -methionine (S-adeno met-DC), synthesized by adenosylmethionine decarboxylase (AMD1), which donates an n-propylamine residue for spermidine and spermine. Dysregulation in the expression of polyamine biosynthetic genes leads to imbalances in polyamine homeostasis, causing overexuberant inflammatory responses in macrophages, an inability to resolve inflammation, and unchecked proliferation and migration of tumor cells.

[0092] In one embodiment, the nucleic acid molecule(s) can reduce or inhibit expression of an ODC1 gene, or the level of one or more polyamines (e g., putrescine), by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more compared to a reference, (e.g., an untreated cell or a cell treated with a non-targeting control nucleic acid molecule). Without being bound by theory, ODC1 catalyzes the first and rate-limiting step of polyamine biosynthesis that converts ornithine into putrescine. Thus, reducing expression of the ODC1 gene can reduce the level of one or more polyamines.

[0093] Putrescine is a polyamine compound ((CH₂)₄(NH₂)₂) that, together with spermine and spermidine, play a role in cell growth.

[0094] Spermidine is a polyamine compound (C₇H₁₉N₃) found in ribosomes and living tissues and having various metabolic functions within organisms. Spermidine is a precursor to other polyamines, such as spermine.

[0095] Spermine is a polyamine compound (NH₂(CH₂)₃NH(CH₂)₄NH(CH₂)₃NH₂) that is involved in cellular metabolism.Docket No: 2932719-000287-W01Date of Filing: December 11, 2025

[0096] As used herein, the term "modulate the expression of," can refer to an at least partial "inhibition" or partial "activation" of an ODC1 gene expression in a cell treated with a composition as described herein compared to the expression of ODC1 in a control cell. A control cell includes an untreated cell, or a cell treated with a non-targeting control nucleic acid molecule.

[0097] The terms "silence," "inhibit expression of," "down-regulate expression of," or "suppress expression of can refer to the at least partial suppression of the expression of an ODC1 gene, as assessed, e.g., based on ODC1 mRNA expression, OCD1 protein expression, or another parameter functionally linked to ODC1 gene expression. For example, inhibition of ODC1 expression can be manifested by a reduction of the amount of ODC1 mRNA which can be isolated from or detected in a first cell or group of cells in which an ODC1 gene is transcribed and which has or have been treated such that the expression of an ODC1 gene is inhibited, as compared to a control. The control can be a second cell or group of cells substantially identical to the first cell or group of cells, except that the second cell or group of cells have not been so treated (control cells).

[0098] In embodiments, the nucleic acid molecule(s) can comprise one or more N-acetylgalactosamine (GalNAc) derivatives. The term “N-acetylgalactosamine derivative” or “GalNAc derivative” can refer to a chemically modified version of N-acetylgalactosamine (GalNAc), a sugar molecule that acts as a targeting ligand for liver cells due to its high affinity for the asialoglycoprotein receptor (ASGPR) on hepatocytes. Essentially, a GalNAc derivative can refer to a modified form of GalNAc used to specifically deliver drugs or therapeutic molecules to the liver by attaching to them and facilitating uptake through the ASGPR. In embodiments, the GalNAc derivative described herein can comprise a biantennary or a triantennary GalNAc ligand. In embodiments, the GalNAc ligand is attached to the 3' end of the sense strand. In embodiments, the GalNAc ligand is attached to the 3' end of the sense strand via linker.

[0099] In some embodiments, the nucleic acid molecule(s) can comprise at least one modified nucleotide.

[0100] In some embodiments, at least one of the modified nucleotides is chosen from the group comprising: a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a nucleotide comprising a 5'-phosphorothioate group, a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, or a combination thereof. The nucleic acid molecule can comprise at least two modified ribonucleotides, 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 entireDocketNo: 2932719-000287-W01Date of Filing: December 11, 2025 length of the nucleic acid molecule. The modifications need not be the same for each of such a plurality of modified ribonucleotides in an RNA molecule.

[0101] In some embodiments, the nucleic acid molecule(s) can comprise one or more nucleotides connected to one another by way of phosphodiester or phosphorothioate linkages. A “phosphodiester linkage” can refer to a covalent linkage between the phosphate of one nucleotide and the hydroxyl (OH) group attached to the 3' carbon of the deoxyribose sugar in an adjacent nucleotide, forming what is known as the “sugar-phosphate backbone” of DNA. A “phosphorothioate linkage” can refer to a chemical modification in a nucleic acid backbone where a non-bridging oxygen atom in a phosphodiester bond is replaced by a sulfur atom, essentially making the molecule more resistant to degradation by nucleases while maintaining its ability to bind to complementary strands.

[0102] In one aspect, the invention provides an isolated cell containing the nucleic acid molecule(s) described herein. The cell is a mammalian cell, such as a human cell. In some embodiments, the cell is an erythroid cell. In other embodiments, the cell is a liver cell (e.g., a hepatocyte).

[0103] The term "isolated" as used herein with respect to cells, proteins, and nucleic acids (e.g., DNA or RNA) can refer to molecules separated from other cells, proteins, or nucleic acids, respectively, that are present in the natural source of the macromolecule. The term "isolated" as used herein also can refer to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Moreover, an "isolated nucleic acid" can refer to a nucleic acid fragments which are not naturally occurring as fragments and cannot be found in the natural state. The term "isolated" can also be used herein to refer to cells or polypeptides which are isolated from other cellular proteins or tissues. Isolated polypeptide can refer to both purified and recombinant polypeptides.

[0104] In an aspect provided herein is a pharmaceutical composition comprising a nucleic acid molecule described herein. The phrase "pharmaceutical composition" or a “pharmaceutical formulation” can refer to a composition or pharmaceutical composition suitable for administration to a subject, such as a mammal, especially a human and that can refer to the combination of an active agent(s), or ingredient with a pharmaceutically acceptable carrier or excipient, making the composition suitable for diagnostic, therapeutic, or preventive use in vitro, in vivo, or ex vivo. ADocket No: 2932719-000287-W01Date of Filing: December 11, 2025 “pharmaceutical composition” can be sterile and can be free of contaminants that can elicit an undesirable response within the subject (e.g., the compound(s) in the pharmaceutical composition is pharmaceutical grade). Pharmaceutical compositions can be designed for administration to subjects or patients in need thereof via a number of different routes of administration including oral, intranasal, topical, intravenous, buccal, rectal, parenteral, intraperitoneal, intradermal, intratracheal, intramuscular, subcutaneous, by stent-eluting devices, catheters-eluting devices, intravascular balloons, inhalational and the like.

[0105] Aspects of the invention are further drawn to methods for treating, preventing or managing chronic liver disease (e.g., metabolic dysfunction-associated steatohepatitis (MASH)). In one embodiment, the method includes administering to a subject, e.g., a patient in need of such treatment, prevention or management, an effective (e.g., a therapeutically or prophylactically effective) amount of one or more of the nucleic acid molecules described herein.

[0106] As used herein, the term “liver disease” and “hepatic disease” can be used interchangeably and can refer to damage to, a disorder involving, or a disease of the liver. Nonlimiting examples of liver disease include intrahepatic cholestasis (e.g., Alagille syndrome, biliary liver cirrhosis), fatty liver (e.g., alcoholic fatty liver, Reye's syndrome), hepatic vein thrombosis, hepatolenticular degeneration (i.e., Wilson's disease), hepatomegaly, liver abscess (e.g., amebic liver abscess), liver cirrhosis (e.g., alcoholic, biliary, and experimental liver cirrhosis), alcoholic liver diseases (e.g., fatty liver, hepatitis, cirrhosis), parasitic liver disease (e.g., hepatic echinococcosis, fascioliasis, amebic liver abscess), jaundice (e.g., hemolytic, hepatocellular, cholestatic jaundice), cholestasis, portal hypertension, liver enlargement, ascites, hepatitis (e.g., alcoholic hepatitis, animal hepatitis, chronic hepatitis (e.g., autoimmune, hepatitis B, hepatitis C, hepatitis D, drug induced chronic hepatitis), toxic hepatitis, viral human hepatitis (e.g., hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E), granulomatous hepatitis, secondary biliary cirrhosis, hepatic encephalopathy, varices, primary biliary cirrhosis, primary sclerosing cholangitis, hepatic steatosis or steatohepatitis, hepatocellular adenoma, hemangiomas, bile stones, liver failure (e.g., hepatic encephalopathy, acute liver failure), angiomyolipoma, calcified liver metastases, cystic liver metastases, fibrolamellar hepatocarcinoma, hepatic adenoma, hepatoma, hepatic cysts (e.g., Simple cysts, Polycystic liver disease, hepatobiliary cystadenoma, choledochal cyst), mesenchymal tumors (mesenchymal hamartoma, infantile hemangioendothelioma, hemangioma, peliosis hepatis, lipomas, inflammatory pseudotumor), epithelial tumors (e.g., bileDocketNo: 2932719-000287-W01Date of Filing: December 11, 2025 duct hamartoma, bile duct adenoma), focal nodular hyperplasia, nodular regenerative hyperplasia, hepatoblastoma, hepatocellular carcinoma, cholangiocarcinoma, cystadenocarcinoma, tumors of blood vessels, angiosarcoma, Karposi's sarcoma, hemangioendothelioma, embryonal sarcoma, fibrosarcoma, leiomyosarcoma, rhabdomyosarcoma, carcinosarcoma, teratoma, carcinoid, squamous carcinoma, primary lymphoma, peliosis hepatis, erythrohepatic porphyria, hepatic porphyria (e.g., acute intermittent porphyria, porphyria cutanea tarda), and Zellweger syndrome. In embodiments, the liver disease can comprise fibrosis, a liver disease characterized in that the liver has excess cholesterol, triglycerides or other lipids that is illustrative of liver diseases such as MASLD, MASH or alcoholic related steatosis of the liver, liver cirrhosis or liver inflammation or hepatocellular carcinoma.

[0107] The terms "treat”, or "treatment" can refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progression of a metabolic disease. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.

[0108] Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

[0109] By "subject" or "individual" or "animal" or "patient" or "mammal," is meant any subject, such as a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sport, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on.

[0110] Phrases such as "to a patient in need of treatment" or "a subject in need of treatment" includes subjects, such as mammalian subjects, that can benefit from administration of a composition as described herein.

[0111] Treatment can be used to treat subjects diagnosed with chronic liver disease. Nonlimiting examples of chronic liver disease can comprise metabolic dysfunction-associated steatotic liver disease (MASLD), metabolic dysfunction-associated steatohepatitis (MASH), hepaticDocketNo: 2932719-000287-W01Date of Filing: December 11, 2025 fibrosis alcoholic liver disease, chronic hepatitis B, chronic hepatitis C, autoimmune hepatitis, primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), hemochromatosis, Wilson's disease, alpha-1 antitrypsin deficiency, hepatocellular carcinoma, or a combination thereof. For example, embodiments comprise administering to a subject an effective amount of a pharmaceutical formulation or composition as described herein for the treatment of the condition.

[0112] Embodiments as described herein can further comprise administering to the subject the nucleic acid molecule(s) described herein.

[0113] The term "administering" can refer to introducing a substance into a subject. Any route of administration can be utilized including, for example, intranasal, topical, oral, parenteral, intravitreal, intraocular, ocular, subretinal, intrathecal, intravenous, subcutaneous, transcutaneous, intracutaneous, intracranial and the like administration. For example, “parenteral administration” can refer to administration via injection or infusion. Parenteral administration includes, but is not limited to, subcutaneous administration, intravenous administration, and intramuscular administration. For example, the nucleic acid molecule can be administered by injection (e.g., intravenous or subcutaneous).

[0114] In embodiments, "administering" can also refer to providing a therapeutically effective amount of a formulation or pharmaceutical composition to a subject. The formulation or pharmaceutical composition can be administered alone, but can be administered with other compounds, excipients, fillers, binders, carriers or other vehicles selected based upon the chosen route of administration and standard pharmaceutical practice.

[0115] In embodiments, the nucleic acid can be administered alone, or can be administered as a pharmaceutical composition together with other compounds, excipients, carriers, diluents, fillers, binders, or other vehicles selected based upon the chosen route of administration and standard pharmaceutical practice. Administration can be by way of carriers or vehicles, such as injectable solutions, including sterile aqueous or non-aqueous solutions, or saline solutions; creams; lotions; capsules; tablets; granules; pellets; powders; suspensions, emulsions, or microemulsions; patches; micelles; liposomes; vesicles; implants, including microimplants; eye drops; other proteins and peptides; synthetic polymers; microspheres; nanoparticles; and the like.

[0116] Embodiments can be administered to a subject in one or more doses. The dose level can vary as a function of the specific composition or pharmaceutical composition administered, the severity of the symptoms and the susceptibility of the subject to side effects. Dosages for aDocket No: 2932719-000287-W01Date of Filing: December 11, 2025 given compound are readily determinable by a variety of means. For example, dosages can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can be employed to help identify optimal dosage ranges. The precise dose to be employed can also depend on the route of administration and can be decided according to the judgment of the practitioner and each patient's circumstances.

[0117] In an embodiment, multiple doses of the pharmaceutical composition can be administered. The frequency of administration and the duration of administration of the pharmaceutical composition can vary depending on any of a variety of factors, e.g., patient response, severity of the symptoms, and the like. For example, in an embodiment, the pharmaceutical composition can be administered once per month, twice per month, three times per month, every other week (qow), once per week (qw), twice per week (biw), three times per week (tiw), four times per week, five times per week, six times per week, every other day (qod), daily (ad), twice a day (qid), three times a day (tid), or four times a day. In an embodiment, the pharmaceutical composition can be administered 1 to 4 times a day over a period of time, such as 1 to 10-day time period, or longer than a 10-day period of time.

[0118] In embodiments, the pharmaceutical composition can be administered in combination with one or more additional active agents. For example, a first agent (e.g., a prophylactic or therapeutic agent) can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second agent (e.g., a prophylactic or therapeutic agent) to a subject with a disease or disorder or a symptom thereof.

[0119] In an embodiment, the pharmaceutical composition is formulated for administration according to a dosage regimen described herein, e.g., not more than once every four weeks, not more than once every three weeks, not more than once every two weeks, or not more than once every week. In another embodiment, the administration of the pharmaceutical composition can be maintained for a month or longer, e.g., one, two, three, or six months, or one year or longer.DocketNo: 2932719-000287-W01Date of Filing: December 11, 2025

[0120] Aspects of the invention are directed to a method of reducing hepatic putrescine levels in a subject. In embodiments, the method comprises administering to the subject the nucleic acid molecule described herein.

[0121] In embodiments, the concentration of putrescine can be reduced to a level such that homeostasis is restored. For example, the concentration of putrescine in circulating serum or blood can be decreased to below about 150 pM, below about 140 pM, below about 130 pM, below about 120 pM, below about 110 pM, below about 100 pM, below about 90 pM, below about 95 pM, below about 90 pM, below about 80 pM, below about 85 pM, below about 80 pM, below about 75 pM, below about 70 pM, below about 65 pM, below about 60 pM, below about 55 pM, below about 50 pM, below about 45 pM, below about 40 pM, below about 35 pM, below about 30 pM, below about 25 pM, below about 20 pM, below about 15 pM, below about 10 pM, below about 5 pM, and below about 1 pM in the subject.

[0122] In embodiments, the nucleic acid molecule(s) can be administered to a subject in an amount sufficient to effect beneficial or desired clinical results. For example, the “effective amount”, “sufficient amount” or “therapeutically effective amount” can be sufficient to reduce the frequency of symptoms associated with chronic liver disease, such as but not limited to, reduction of liver function, abdominal pain, weakness, fatigue, nausea, vomiting, loss of appetite, unexplained weight loss,jaundice, dark-colored urine, pale or clay-colored stools, muscle wasting, fever, chills, night sweats, bruising, bleeding, ascites, edema, enlarged liver, enlarged spleen, spider veins, hypotension, hepatic encephalopathy, cirrhosis, portal hypertension, pruritus, gynecomastia, testicular atrophy, menstrual irregularities, palmar erythema, cognitive impairment, irritability, personality changes, confusion, drowsiness, slurred speech, or a combination thereof.

[0123] In embodiments of the invention, a biological sample, or “sample”, can be isolated from a subject. A “biological sample” can refer to any sample that is obtained or otherwise derived from a biological subject, including sample of biological tissue or fluid origin obtained in vivo or in vitro. Non-limiting examples of biological samples that can be isolated from a subject, and thus used in methods of the invention, include tissue biopsy, stool, blood, plasma, serum, cord blood, neonatal blood, cerebral spinal fluid (CSF), tears, vomit, saliva, urine, feces, and meconium.

[0124] Embodiments described herein can involve “obtaining” or “isolating” a biological sample from the subject, such as a subject afflicted with a metabolic disease. As used herein, the phrase “obtaining a biological sample” or “isolating a biological sample” can refer to any processDocketNo: 2932719-000287-W01Date of Filing: December 11, 2025 for directly or indirectly acquiring a biological sample from a subject. For example, a biological sample can be obtained (e.g., at a point-of-care facility, e.g., a physician's office, a hospital, laboratory facility) by procuring a tissue or fluid sample (e.g., blood draw, marrow sample, spinal tap) from a subject. Alternatively, a biological sample can be obtained by receiving the biological sample (e.g., at a laboratory facility) from one or more persons who procured the sample directly from the subject. The biological sample can be, for example, a tissue (e.g., blood), cell (e.g., hematopoietic cell such as hematopoietic stem cell, leukocyte, or reticulocyte, stem cell, or plasma cell), vesicle, biomolecular aggregate or platelet from the subject.

[0125] Any of the therapeutic applications described herein can be applied to any subject or patient in need of such therapy, including, for example, a mammal such as a mouse, a rat, a dog, a cat, a cow, a horse, a rabbit, a monkey, a pig, a sheep, a goat, or a human. In some embodiments, the subject is a mouse, rat, pig, or human.

[0126] Aspects of the invention are further directed towards a medical kit suitable for the treatment of chronic liver disease. In embodiments, the kit can comprise printed instructions for administering a formulation or composition as described herein to a subject in need thereof; a pharmaceutical composition as described herein, and / or a pharmaceutically acceptable carrier. A "kit" or "medical kit" of the disclosure comprises a dosage form of the nucleic acid molecule(s) or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, prodrug, or clathrate thereof. A kit can also include two or more compositions as described herein, either in combination, such as in a single tablet, or provided separately, such as in two or more tablets.

[0127] Kits can further comprise additional active agents, examples of which are described herein. Kits of the disclosure can further comprise devices that are used to administer the active ingredients. Examples of such devices include, but are not limited to, syringes, drip bags, patches, and inhalers. Kits can also comprise printed instructions for administering the formulation to a subject.

[0128] Kits of the invention can further comprise pharmaceutically acceptable vehicles that can be used to administer one or more active ingredients. For example, if an active ingredient is provided in a solid form that must be reconstituted for parenteral administration, the kit can comprise a sealed container of a suitable vehicle in which the active ingredient can be dissolved to form a particulate-free sterile solution that is suitable for parenteral administration. Examples of pharmaceutically acceptable vehicles include, but are not limited to: Water for Injection USP;Docket No: 2932719-000287-W01Date of Filing: December 11, 2025 aqueous vehicles such as, but not limited to, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.EXAMPLES

[0129] The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the invention in any fashion. One skilled in the art will appreciate readily that the invention is well adapted to carry out various embodiments of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.

[0130] Examples are provided herein to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.EXAMPLE 1

[0131] This invention pertains to the use of an N-Acetylgalactosamine (GalNAc)-based platform designed to specifically target ornithine decarboxylase 1 (ODC1), the rate-limiting enzyme responsible for the biosynthesis of putrescine, as a new treatment for metabolic dysfunction-associated steatohepatitis (MASH). GalNAcs are an established platform in therapeutic development, such as in the targeted delivery of oligonucleotides to hepatocytes via the asialoglycoprotein receptor (ASGPR). The GalNAc platform is currently FDA-approved for several diseases, including Givosiran for the treatment of Acute Hepatic Porphyria (AHP), Lumasiran for the treatment of Primary Hyperoxaluria Type 1 (PHI), Inclisiran for the treatment of hypercholesterolemia, and Fitusiran for the treatment of Hemophilia A and B. These drugs reveal the clinical applicability of GalNAc-based therapies for metabolic diseases, such as in liver-related diseases. The new element of this invention lies in the strategic application of the GalNAc platform to target ODC1. This protein is the rate-limiting enzyme for putrescine synthesis,DocketNo: 2932719-000287-W01Date of Filing: December 11, 2025 catalyzing the decarboxylation of ornithine to produce putrescine. By utilizing the GalNAc platform to specifically target ODC1, this invention offers a targeted therapeutic approach that effectively lowers putrescine levels in the liver. The platform’s specificity for liver cells ensures that ODC1 silencing by GalNAcs minimizes off-target effects and enhances therapeutic efficacy.

[0132] Background and Significance

[0133] Metabolic Dysfunction- Associated Steatotic Liver Disease (MASLD) has Reached Epidemic Proportions with Limited Pharmacological Therapies.

[0134] Affecting over one-third of the global population, MASLD has become the most common cause of chronic liver disease worldwide1,2. Approximately 20-30% of individuals with MASLD develop metabolic dysfunction-associated steatohepatitis (MASH), characterized by lobular inflammation and hepatocellular ballooning3. Unmitigated progression of MASH leads to hepatic fibrosis, which is the main contributor to death in for individuals with this disease4’13. Although the recent approval of resmetirom represents a significant advancement for the treatment of MASH14, its effectiveness is limited - benefiting only 10-15% of patients after placeboadjustment. Moreover, the interaction of resmetirom with statins, fibrates, and anticoagulants, along with its uncertain long-term safety profile, complicates its use in clinical settings where patients often require multiple therapies over extended periods15. Here, we discovered a new metabolic pathway involving putrescine, revealing its potential as a therapeutic target, setting the stage for transformative advancements in the management of MASH.

[0135] Metabolic Regulation of Polyamines.

[0136] Considerable advancements, such as in metabolomics, have expanded our understanding of MASH pathogenesis and progression. Whereas impaired lipid and carbohydrate metabolism are well-established features of MASH16, recent discoveries from our group and others highlighted the importance of dysregulated amino acid metabolism in MASH and related comorbidities17’24. Metabolism of the amino acids arginine, ornithine, and methionine generate byproducts that feed into the polyamine biosynthetic pathway. Polyamines are a family of small, linear polycations comprised of putrescine, spermidine, and spermine (FIG. 1) that direct multiple molecular and cellular functions, such as proliferation, gene transcription, mRNA stability, and protein translation25,26. The polyamine pathway is initiated by the decarboxylation of ornithine into putrescine by ornithine decarboxylase 1 (ODC1). The conversion of putrescine into spermidine and spermine follows linear reactions carried out by spermidine synthase (SRM) and spermineDocket No: 2932719-000287-W01Date of Filing: December 11, 2025 synthase (SMS), respectively. Decarboxylated S-adenosyl-L-methionine (dcSAM), which is synthesized by adenosylmethionine decarboxylase 1 (AMD1), donates an n-propylamine residue to putrescine and spermidine to form spermidine and spermine, respectively (FIG. 2). While polyamine metabolism and the therapeutic potential of targeting this pathway have been studied in cancer, aging, and cardiovascular diseases25,27'29, their role in MASLD / MASH and hepatic fibrosis has yet to be systematically and rigorously explored.

[0137] Supporting Data

[0138] Hepatic Putrescine Increases in MASH in Humans and Mice.

[0139] As the first step to identify a role for polyamines in MASH, we established an LC- MS / MS-based platform (verified with standards) at LSUHS to determine the levels of polyamines in mice and humans. We fed C57BL / 6J mice a standard laboratory diet or our established MASH-inducing diet for 24 weeks, as we reported19,30,31. Important features of MASH, such as steatohepatitis and fibrosis, were confirmed by H& E and picrosirius red staining (FIG. 3, panel A). We discovered that hepatic putrescine levels were significantly increased in MASH (FIG. 3, panel B), whereas spermidine, spermine, and total polyamines were unchanged. To determine the clinical relevance of these findings, we measured polyamines in the liver of patients with biopsy-proven MASH versus disease-free, age, sex, and race-matched healthy individuals (FIG.3, panel C). Similar to our findings in mice, hepatic putrescine showed a significant increase in patients with MASH (FIG. 3, panel D) whereas spermidine, spermine, and total polyamines were unchanged.

[0140] Putrescine Drives Hepatic Fibrosis in MASH,

[0141] To test if increasing hepatic putrescine exogenously exacerbates hepatic fibrosis, C57BL / 6J mice were fed the MASH-inducing diet while concurrently receiving putrescine-enriched (3 mM, a dose we and others have used to increase putrescine levels in mice22,27,32) or normal drinking water for 20 weeks. We confirmed elevations in hepatic putrescine (FIG.4, panel A), with no significant changes in other polyamines. Importantly, hepatic fibrosis was significantly enhanced in putrescine-treated mice compared to controls (FIG. 4, panel B), indicating that hepatic putrescine accumulation is a key driver of MASH-associated fibrosis.

[0142] Therapeutic Strategies to Lower Putrescine Mitigate Hepatic Fibrosis in MASH,

[0143] To test the translational value of lowering putrescine, we used Odel -targeting siRNA conjugated to GalNAcs. This delivery system, which targets asialoglycoprotein receptorsDocket No: 2932719-000287-W01Date of Filing: December 11, 2025 highly expressed on hepatocytes, ensures hepatocyte-specific siRNA delivery and is FDA-approved for multiple diseases33'36. Here, C57BL / 6J mice were fed a MASH-inducing diet for 12 weeks followed by weekly injections of ScrRNA-GalNAcs or siOdcl-GalNAcs for 8 weeks (S. Q. injection, 5 mg / kg37-39). Livers were collected, immunoblotted for 0DC1 and GAPDH, and measured for putrescine content (FIG. 5, panels A-B). Suppressing putrescine synthesis using siOdcl-GalNAcs in mice with established MASH (i.e., intervention) lowered ALT and AST levels (FIG. 5, panels C-D), surrogates for liver injury. Most importantly, picrosirius red staining of liver cross-sections from these mice revealed a significant decrease in hepatic fibrosis (FIG. 6, panel A), which was associated with a suppression in the collagen genes Collal, Colla2, Col4al, and Col4a2 (FIG. 6, panels B-E). Together, these data represent a significant advancement by leveraging the FDA-approved GalNAc platform to target a critical enzyme in putrescine biosynthesis specifically in hepatocytes, offering a new avenue for the treatment of MASH. In this Disclosure, we submit that strategies for lowering hepatic putrescine, such as those aimed at reducing its rate-limiting enzyme 0DC1, blunt fibrosis and can be a suitable treatment to curb the progression of MASH.

[0144] References Cited in this Example:1. Riazi K, Azhari H, Charette JH, Underwood FE, King JA, Afshar EE, Swain MG, Congly SE, Kaplan GG, Shaheen AA. The prevalence and incidence of NAFLD worldwide: a systematic review and metaanalysis. Lancet Gastroenterol Hepatol. 2022;7:851-861. doi: 10.1016 / S2468-1253(22)00165-02. Cheemerla S, Balakrishnan M. Global Epidemiology of Chronic Liver Disease. Clin Liver Dis (Hoboken). 2021;17:365-370. doi: 10.1002 / cld.10613. Chalasani N, Younossi Z, Lavine JE, Charlton M, Cusi K, Rinella M, Harrison SA, Brunt EM, Sanyal AJ. The diagnosis and management of nonalcoholic fatty liver disease: Practice guidance from the American Association for the Study of Liver Diseases. Hepatology. 2018;67:328-357. doi: 10.1002 / hep.293674. Puche JE, Saiman Y, Friedman SL. Hepatic stellate cells and liver fibrosis. Compr Physiol.2013;3:1473-1492. doi: 10.1002 / cphy.cl200355. Angulo P, Kleiner DE, Dam -Larsen S, Adams LA, Bjornsson ES, Charatcharoenwitthaya P, Mills PR, Keach JC, Lafferty HD, Stabler A, et al. Liver Fibrosis, but No Other Histologic Features, Is Associated With Long-term Outcomes of Patients With Nonalcoholic Fatty LiverDocket No: 2932719-000287-W01Date of Filing: December 11, 2025 Disease. Gastroenterology. 2015;149:389-397 e310. doi: 10.1053 / j.gastro.2015.04.0436. Singh S, Allen AM, Wang Z, Prokop LJ, Murad MH, Loomba R. Fibrosis progression in nonalcoholic fatty liver vs nonalcoholic steatohepatitis: a systematic review and meta-analysis of paired-biopsy studies. Clin Gastroenterol Hepatol. 2015;13:643-654 e641-649; quiz e639-640. doi: 10.1016 / j.cgh.2014.04.0147. Vilar-Gomez E, Calzadilla-Bertot L, Wai-Sun Wong V, Castellanos M, Aller-de la Fuente R, Metwally M, Eslam M, Gonzalez-Fabian L, Alvarez-Quinones Sanz M, Conde-Martin AF, et al. Fibrosis Severity as a Determinant of Cause-Specific Mortality in Patients With Advanced Nonalcoholic Fatty Liver Disease: A Multi-National Cohort Study. Gastroenterology.2018;155:443-457 e417. doi: 10.1053 / j.gastro.2018.04.0348. Neuschwander-Tetri BA. Hepatic lipotoxicity and the pathogenesis of nonalcoholic steatohepatitis: the central role of nontriglyceride fatty acid metabolites. Hepatology. 2010;52:774-788. doi: 10.1002 / hep.237199. Cusi K. Role of obesity and lipotoxicity in the development of nonalcoholic steatohepatitis: pathophysiology and clinical implications. Gastroenterology. 2012;142:711-725 e716. doi: 10.1053 / j.gastro.2012.02.00310. Chaurasia B, Summers SA. Ceramides - Lipotoxic Inducers of Metabolic Disorders: (Trends in Endocrinology and Metabolism 26, 538-550; 2015). Trends Endocrinol Metab. 2018;29:66-67. doi: 10.1016 / j.tem.2017.09.00511. Begriche K, Igoudjil A, Pessayre D, Fromenty B. Mitochondrial dysfunction in NASH: causes, consequences and possible means to prevent it. Mitochondrion. 2006;6:1-28. doi: 10.1016 / j.mito.2005.10.00412. Koliaki C, Szendroedi J, Kaul K, Jelenik T, Nowotny P, Jankowiak F, Herder C, Carstensen M, Krausch M, Knoefel WT, et al. Adaptation of hepatic mitochondrial function in humans with non-alcoholic fatty liver is lost in steatohepatitis. Cell Metab. 2015;21:739-746. doi: 10.1016 / j.cmet.2015.04.00413. Masarone M, Rosato V, Dallio M, Gravina AG, Aglitti A, Loguercio C, Federico A, Persico M. Role of Oxidative Stress in Pathophysiology of Nonalcoholic Fatty Liver Disease. Oxid Med Cell Longev. 2018;2018:9547613. doi: 10.1155 / 2018 / 954761314. Harrison SA, Bedossa P, Guy CD, Schattenberg JM, Loomba R, Taub R, Labriola D, Moussa SE, Neff GW, Rinella ME, et al. A Phase 3, Randomized, Controlled Trial of Resmetirom inDocket No: 2932719-000287-W01Date of Filing: December 11, 2025 NASH with Liver Fibrosis. N Engl J Med. 2024;390:497-509. doi: 10.1056 / NEJMoa2309000 15. Petta S, Targher G, Romeo S, Pajvani UB, Zheng MH, Aghemo A, Valenti LVC. The first MASH drug therapy on the horizon: Current perspectives of resmetirom. Liver Int. 2024. doi: 10.1111 / liv.1593016. Parekh S, Anania FA. Abnormal lipid and glucose metabolism in obesity: implications for nonalcoholic fatty liver disease. Gastroenterology. 2007;132:2191-2207. doi: 10.1053 / j.gastro.2007.03.05517. Gaggini M, Carli F, Rosso C, Buzzigoli E, Marietti M, Della Latta V, Ciociaro D, Abate ML, Gambino R, Cassader M, et al. Altered amino acid concentrations in NAFLD: Impact of obesity and insulin resistance. Hepatology. 2018;67:145-158. doi: 10.1002 / hep.2946518. Yamakado M, Tanaka T, Nagao K, Imaizumi A, Komatsu M, Daimon T, Miyano H, Tani M, Toda A, Yamamoto H, et al. Plasma amino acid profile associated with fatty liver disease and cooccurrence of metabolic risk factors. Sci Rep. 2017;7: 14485. doi: 10.1038 / s41598-017-14974-w 19. Rom O, Liu Y, Liu Z, Zhao Y, Wu J, Ghrayeb A, Villacorta L, Fan Y, Chang L, Wang L, et al. Glycinebased treatment ameliorates NAFLD by modulating fatty acid oxidation, glutathione synthesis, and the gut microbiome. Sci Transl Med. 2020;12. doi: 10.1126 / scitranslmed.aaz2841 20. Mardinoglu A, Bjornson E, Zhang C, Klevstig M, Soderlund S, Stahlman M, Adiels M, Hakkarainen A, Lundbom N, Kilicarslan M, et al. Personal model-assisted identification of NAD(+) and glutathione metabolism as intervention target in NAFLD. Mol Syst Biol.2017;13:916. doi: 10.15252 / msb.2016742221. Simon J, Nunez-Garcia M, Fernandez-Tussy P, Barbier-Torres L, Fernandez-Ramos D, Gomez-Santos B, Buque X, Lopitz-Otsoa F, Goikoetxea-Usandizaga N, Serrano-Macia M, et al. Targeting Hepatic Glutaminase 1 Ameliorates Non-alcoholic Steatohepatitis by Restoring Very-Low-Density Lipoprotein Triglyceride Assembly. Cell Metab. 2020;31:605-622 e610. doi: 10.1016 / j.cmet.2020.01.01322. Yurdagul A, Jr., Subramanian M, Wang X, Crown SB, Ilkayeva OR, Darville L, Kolluru GK, Rymond CC, Gerlach BD, Zheng Z, et al. Macrophage Metabolism of Apoptotic Cell-Derived Arginine Promotes Continual Efferocytosis and Resolution of Injury. Cell Metab. 2020;31:518-533 e510. doi: 10.1016 / j.cmet.2020.01.00123. Liu Y, Zhao Y, Shukha Y, Lu H, Wang L, Liu Z, Liu C, Zhao Y, Wang H, Zhao G, et al. Dysregulated oxalate metabolism is a driver and therapeutic target in atherosclerosis. Cell Rep.DocketNo: 2932719-000287-W01Date of Filing: December 11, 2025 2021;36: 109420. doi: 10.1016 / j.celrep.2021.10942024. Rom O, Liu Y, Finney AC, Ghrayeb A, Zhao Y, Shukha Y, Wang L, Rajanayake KK, Das S, Rashdan NA, et al. Induction of glutathione biosynthesis by glycine-based treatment mitigates atherosclerosis. Redox Biol. 2022;52:102313. doi: 10.1016 / j.redox.2022.10231325. Casero RA, Jr., Murray Stewart T, Pegg AE. Polyamine metabolism and cancer: treatments, challenges and opportunities. Nat Rev Cancer. 2018;18:681-695. doi: 10.1038 / s41568-018-0050-326. Wallace HM, Fraser AV, Hughes A. A perspective of polyamine metabolism. Biochem J.2003;376:1-14. doi: 10.1042 / BJ2003132727. Eisenberg T, AbdellatifM, Schroeder S, PrimessnigU, Stekovic S, Pendl T, Harger A, Schipke J, Zimmermann A, Schmidt A, et al. Cardioprotection and lifespan extension by the natural polyamine spermidine. Nat Med. 2016;22:1428-1438. doi: 10.1038 / nm.422228. Eisenberg T, Knauer H, Schauer A, Buttner S, Ruckenstuhl C, Carmona-Gutierrez D, Ring J, Schroeder S, Magnes C, Antonacci L, et al. Induction of autophagy by spermidine promotes longevity. Nat Cell Biol. 2009;11:1305-1314. doi: 10.1038 / ncbl97529. Morselli E, Marino G, Bennetzen MV, Eisenberg T, Megalou E, Schroeder S, Cabrera S, Benit P, Rustin P, Criollo A, et al. Spermidine and resveratrol induce autophagy by distinct pathways converging on the acetylproteome. J Cell Biol. 2011;192:615-629. doi: 10.1083 / jcb.201008167 30. Rom O, Xu G, Guo Y, Zhu Y, Wang H, Zhang J, Fan Y, Liang W, Lu H, Liu Y, et al. Nitrofatty acids protect against steatosis and fibrosis during development of nonalcoholic fatty liver disease in mice. EBioMedicine. 2019;41:62-72. doi: 10.1016 / j.ebiom.2019.02.01931. Qu P, Rom O, Li K, Jia L, Gao X, Liu Z, Ding S, Zhao M, Wang H, Chen S, et al. DT-109 ameliorates nonalcoholic steatohepatitis in nonhuman primates. Cell Metab. 2023;35:742-757 e710. doi: 10.1016 / j.cmet.2023.03.01332. Yurdagul A, Jr., Kong N, Gerlach BD, Wang X, Ampomah P, Kuriakose G, Tao W, Shi J, Tabas I. ODC (Ornithine Decarboxylase)-Dependent Putrescine Synthesis Maintains MerTK (MER Tyrosine-Protein Kinase) Expression to Drive Resolution. Arterioscler Thromb Vase Biol.2021;41:el44-el59. doi: 10.1161 / ATVBAHA.120.31562233. Balwani M, Sardh E, Ventura P, Peiro PA, Rees DC, Stolzel U, Bissell DM, Bonkovsky HL, Windyga J, Anderson KE, et al. Phase 3 Trial of RNAi Therapeutic Givosiran for Acute Intermittent Porphyria. N Engl J Med. 2020;382:2289-2301. doi: 10.1056 / NEJMoal913147Docket No: 2932719-000287-W01Date of Filing: December 11, 2025 34. Garrelfs SF, Frishberg Y, Hulton SA, Koren MJ, O'Riordan WD, Cochat P, Deschenes G, Shasha-Lavsky H, Saland JM, Van't Hoff WG, et al. Lumasiran, an RNAi Therapeutic for Primary Hyperoxaluria Type i. N Engl J Med. 2021;384:1216-1226. doi: 10.1056 / NEJMoa2021712 35. Adams D, Tournev IL, Taylor MS, Coelho T, Plante-Bordeneuve V, Berk JL, Gonzalez-Duarte A, Gillmore JD, Low SC, Sekijima Y, et al. Efficacy and safety of vutrisiran for patients with hereditary transthyretinmediated amyloidosis with polyneuropathy: a randomized clinical trial. Amyloid. 2023;30:1-9. doi: 10.1080 / 13506129.2022.209198536. Ray KK, Wright RS, Kailend D, Koenig W, Leiter LA, Raal FJ, Bisch JA, Richardson T, Jaros M, Wijngaard PLJ, et al. Two Phase 3 Trials of Inclisiran in Patients with Elevated LDL Cholesterol. N Engl J Med. 2020;382:1507-1519. doi: 10.1056 / NEJMoal91238737. Wang X, Sommerfeld MR, Jahn-Hofmann K, Cai B, Filliol A, Remotti HE, Schwabe RF, Kannt A, Tabas I. A Therapeutic Silencing RNA Targeting Hepatocyte TAZ Prevents and Reverses Fibrosis in Nonalcoholic Steatohepatitis in Mice. Hepatol Commun. 2019;3:1221-1234. doi: 10.1002 / hep4.140538. Godinho B, Knox EG, Hildebrand S, Gilbert JW, Echeverria D, Kennedy Z, Haraszti RA, Ferguson CM, Coles AH, Biscans A, et al. PK-modifying anchors significantly alter clearance kinetics, tissue distribution, and efficacy of therapeutics siRNAs. Mol Ther Nucleic Acids.2022;29:116-132. doi: 10.1016 / j.omtn.2022.06.00539. Kim Y, Jo M, Schmidt J, Luo X, Prakash TP, Zhou T, Klein S, Xiao X, Post N, Yin Z, et al. Enhanced Potency of GalNAc-Conjugated Antisense Oligonucleotides in Hepatocellular Cancer Models. Mol Ther. 2019;27:1547-1557. doi: 10.1016 / j.ymthe.2019.06.009EXAMPLE 2

[0145] Dysregulation in Putrescine Metabolism Drives Hepatic Fibrosis in MASH

[0146] Metabolic dysfunction-associated steatotic liver disease (MASLD) is driven by excessive caloric intake and dyslipidemia, accelerated by genetic predispositions (i.e., PNPLA3), and can progress to MASH, which is characterized by steatosis, inflammation, ballooning, and fibrosis.

[0147] Referring to FIG. 7, a schematic showing the progression of MASLD to hepatocellular carcinoma is provided.DocketNo: 2932719-000287-W01Date of Filing: December 11, 2025

[0148] The prevalence of MASLD is 30-35% and is now the leading cause of chronic liver disease worldwide. Prevalence of MASLD has increased by 50% within the past 3 decades.

[0149] There are limited therapeutic options available to treat MASH:• Low efficacy - 10-15% of patients show a response after placebo adjustment• Drug interactions with statins, fibrates, and anticoagulants• Long-term safety profiles are unknown

[0150] Accordingly, diagnosing individuals with this disease remains challenging.

[0151] Biomarkers to diagnose MASH are lacking. Transaminases (e.g., ALT and AST) -markers of hepatocyte death and liver injury - increase in patients with MASH, and up to 50% of MASLD patients can have normal ALT and AST levels. However, no single markers or multimarker scores met the acceptable AUC to replace biopsy for detecting MASH with clinically significant fibrosis.

[0152] Referring to FIG. 8, data showing AST and ALT levels in healthy and MASH individuals is provided.

[0153] Polyamines are polycations and inherently interact with negatively charged molecules, such as DNA, RNA or proteins.• Gene transcription and translation• DNA stabilization• Signal transduction• Cell growth and proliferation

[0154] We were provided with >150 plasma and liver specimens from individuals with ALD, AiD, MASLD / MASH, HCC, HCV.

[0155] Referring to FIG. 9, a schematic showing the polyamine metabolic pathway is provided.

[0156] Referring to FIG. 10, data showing dysregulation in polyamines during MASH is provided.

[0157] Referring to FIG. 11, data showing putrescine positively correlates with MASH indices in humans and mice is provided.

[0158] Referring to FIG. 12, data showing AMD1 is decreased in hepatocytes in MASH is provided.DocketNo: 2932719-000287-W01Date of Filing: December 11, 2025

[0159] Referring to FIG. 13, data showing AMD1 silencing leads to putrescine accumulation is provided.

[0160] Referring to FIG. 14, data showing AMD1 downregulation worsens fibrosis in MASH is provided.

[0161] Referring to FIG. 15, data showing mitochondrial respiration is lower in AMD1-deficient HepG2 cells is provided.

[0162] Referring to FIG. 16, data showing genes associated with FAO (fatty acid oxidation) are decreased in livers from shAMDl mice is provided.

[0163] Referring to FIG. 17, data showing genes associated with FAO are decreased in AMD 1 -deficient HepG2 cells.

[0164] Referring to FIG. 18, data showing FAO is unaffected in AMD 1 -deficient cells treated with a CPT1 inhibitor is provided.

[0165] Referring to FIG. 19, data showing hepatic AMD1 silencing drives hepatic fibrosis in MASH is provided.

[0166] Referring to FIG. 20, data showing validation of AMD1 deletion and hepatocyte specificity is provided.

[0167] Referring to FIG. 21, data showing putrescine drives hepatic fibrosis in MASH is provided.

[0168] Referring to FIG.22, data showing the effect overexpressing AMD1 has on fibrosis during MASH is provided.

[0169] Referring to FIG. 23, data showing the effects of IHH upregulation by putrescine is provided.

[0170] Referring to FIG. 24, data showing putrescine promotes Mcf2 mRNA stability is provided.

[0171] Referring to FIG. 25, data showing levels of fibrosis in mice is provided.

[0172] Referring to FIG. 26, data showing targeting putrescine using a N-acetyl-galactosamines (GalNAc)-based therapeutic approach is provided. GalNAcs bind to the asialoglycoprotein receptors (ASGPRs), which are abundantly expressed by hepatocytes.

[0173] In conclusion, putrescine is elevated in human and mouse MASH and is associated with hepatic fibrosis. AMD1 suppression accounts for increased putrescine during this disease. Reducing or overexpressing AMD1 promotes and mitigates MASH, respectively, through IHHDocket No: 2932719-000287-W01Date of Filing: December 11, 2025 mRNA stability. Therapeutic strategies using GalNAcs targeting 0DC1 suppresses putrescine biosynthesis and blunts hepatic fibrosis.EXAMPLE 3

[0174] Dysregulated Putrescine Metabolism Drives Hepatic Fibrosis in Metabolic Dysfunction-Associated Steatohepatitis

[0175] Summary

[0176] Metabolic dysfunction-associated steatohepatitis (MASH) has emerged as a leading cause of chronic liver disease worldwide. Although our understanding of MASH pathogenesis has advanced, available therapies remain limited and focus on steatosis, leaving fibrosis, the main contributor to death in this disease, untreated. Here, we identify dysregulated polyamine metabolism as a previously unrecognized driver of hepatic fibrosis in MASH independent of steatosis. Metabolomics of human and murine MASH livers revealed selective accumulation of putrescine, caused by hepatocyte downregulation of adenosylmethionine decarboxylase 1 (AMD1). Hepatocyte-specific AMD1 deletion exacerbated fibrosis, whereas restoring AMD1 was protective. Mechanistically, excess putrescine in hepatocytes increased IHH secretion, thereby amplifying fibrogenic Hedgehog signaling in stellate cells. Furthermore, silencing the rate-limiting enzyme for putrescine biosynthesis in hepatocytes using an FDA-approved platform attenuated fibrosis in MASH. These findings define dysregulated putrescine metabolism as a key driver of hepatic fibrosis and highlight putrescine lowering in hepatocytes as a promising therapeutic strategy.

[0177] Introduction

[0178] Metabolic dysfunction-associated steatotic liver disease (MASLD) has emerged as the most common chronic liver disease worldwide, affecting nearly one-third of the global population1,2, and approximately 20-30% of these individuals will develop metabolic dysfunction-associated steatohepatitis (MASH). MASH, characterized by lobular inflammation and hepatocellular ballooning, frequently progresses to hepatic fibrosis, the major driver of cirrhosis, hepatocellular carcinoma, and liver-related mortality3'7. Although the recent approval of pharmacologic agents highlights therapeutic progress8,9, treatment options for MASH remain limited. Despite these therapeutic advances, prevalence of this disease is projected to continueDocketNo: 2932719-000287-W01Date of Filing: December 11, 2025 rising through 205010. Thus, there is an urgent need to uncover new drivers of MASH progression that can be therapeutically targeted.

[0179] Considerable advances in metabolomics have expanded our understanding of MASH pathogenesis and progression. Whereas impaired lipid and carbohydrate metabolism are well-established features of MASH11, recent discoveries from our group and others highlighted the importance of dysregulated amino acid metabolism in MASH and related comorbidities12’22. Metabolism of the amino acids ornithine and methionine generate intermediates that feed into the polyamine biosynthetic pathway. Polyamines are a family of small linear polycations comprised of putrescine, spermidine, and spermine that direct multiple molecular and cellular functions, such as proliferation, gene transcription, mRNA stability, and protein translation23,24. The polyamine pathway is initiated by the decarboxylation of ornithine into putrescine by ornithine decarboxylase 1 (ODC1). The conversion of putrescine into spermidine and spermine follows linear reactions carried out by spermidine synthase (SRM) and spermine synthase (SMS), respectively. Decarboxylated S-adenosyl-L-methionine (dcSAM), which is synthesized by adenosylmethionine decarboxylase 1 (AMD1), donates an n-propylamine residue to putrescine and spermidine to form spermidine and spermine, respectively. While polyamine metabolism and the therapeutic potential of targeting this pathway have been studied in cancer, aging, and cardiovascular diseases23,25’27, their role in MASH and hepatic fibrosis has yet to be systematically and rigorously explored, representing a critical mechanistic gap and therapeutic opportunity.

[0180] Here, we identified that among the polyamines, only putrescine correlated with fibrosis severity, which can be explained by hepatic suppression of AMD1 in humans and mice. Genetic and therapeutic modulation of this pathway revealed that hepatocyte-specific AMD1 loss exacerbates, whereas AMD1 restoration attenuates fibrosis by regulating putrescine-dependent IHH secretion and Hedgehog signaling in stellate cells. Finally, silencing the rate-limiting enzyme for putrescine biosynthesis in hepatocytes using an FDA-approved platform reduced fibrosis in MASH, establishing hepatocyte putrescine lowering as a promising antifibrotic strategy.

[0181] Results

[0182] Polyamine Metabolism Is Dysregulated in Mice and Humans with MASH

[0183] Dysregulated amino acid metabolism contributes to many chronic diseases, including MASH. Because polyamines are understudied amino acid-derived metabolites and are perturbed in non-resolving diseases, we sought to determine whether altered polyamineDocket No: 2932719-000287-W01Date of Filing: December 11, 2025 metabolism also occurs in MASH. We fed C57BL / 6J mice a standard laboratory diet (10% fat, LabDiet) or our established MASH-inducing diet (Research Diets #D17010103, 40% fat and 22% fructose) for 12 and 24 weeks, as we previously reported14’20'22’28,29, and performed targeted metabolomics (Agilent XDB Cl 8 columns, Orbitrap Exploris 480) to quantify hepatic polyamines. Steatohepatitis and fibrosis were confirmed by H& E and picrosirius red staining (FIG. 27, panel A). Hepatic putrescine levels increased significantly with MASH progression (FIG.27, panel B), whereas spermidine and spermine remained unchanged (FIG. 27, panels C and D). Putrescine also showed a strong positive correlation with fibrosis severity (FIG. 27, panel E). To assess clinical relevance, we quantified hepatic polyamines in individuals with biopsy-proven MASH versus age- and sex-matched healthy individuals (FIG. 27, panel F). As in mice, hepatic putrescine was significantly elevated in MASH (FIG. 27, panel G), while spermidine and spermine were unchanged (FIG. 27, panels H and I), and putrescine again positively correlated with fibrosis (FIG. 27, panel J). We next treated HepG2 cells with the MASH-relevant fatty acid palmitate and observed a marked increase in putrescine (FIG. 27, panel K) without changes in spermidine or spermine (FIG.27, panels L and M). We then isolated primary mouse hepatocytes, confirmed by ASGR1 immunostaining (FIG. 27, panel N), and similarly found that palmitate increased putrescine (FIG.27, panel O) while leaving spermidine and spermine unchanged (FIG.27, panels P and Q).

[0184] To directly evaluate the contribution of putrescine to MASH pathogenesis, we fed mice a MASH-inducing diet for 20 weeks, with one group receiving 3 mM putrescine supplementation in drinking water and the control group receiving water alone (FIG. 28, panel A). Quantitative analysis confirmed a marked accumulation of hepatic putrescine in supplemented mice compared with controls (FIG. 28, panel B), indicating efficient uptake of exogenous putrescine. Despite comparable degrees of steatosis, lobular inflammation, hepatocyte ballooning, NAS score, body weight, and fasting blood glucose (FIG. 28, panels C-G), histological examination revealed a striking increase in hepatic fibrosis severity in mice supplemented with exogenous putrescine (FIG. 28, panel H). Consistently, hepatic hydroxyproline levels, a biochemical measure of collagen content, was significantly elevated in putrescine-supplemented mice (FIG. 28, panel I). Quantitative PCR further confirmed upregulation of key profibrotic genes, including Collal, Colla2 Col4al, Col4a5, and Col4a6 (FIG.28, panel J). Together, theseDocket No: 2932719-000287-W01Date of Filing: December 11, 2025 data demonstrate that sustained elevation of putrescine drives extracellular matrix deposition during MASH, thereby exacerbating fibrosis independently of steatosis or inflammation.

[0185] To begin exploring the mechanisms underlying enhanced hepatic putrescine in MASH, we next examined whether the levels of the polyamine biosynthetic enzymes ODC1 (ornithine decarboxylase 1), AMD1 (adenosylmethionine decarboxylase 1), or SRM (spermidine synthase) were altered by lipid loading. These enzymes were selected because ODC1 decarboxylates ornithine into putrescine, AMD1 generates the amine donor for putrescine to be converted into spermidine, and SRM utilizes the amine donor from AMD1 to synthesize spermidine from putrescine23. Thus, altered expression in any of these enzymes can augment putrescine levels. We treated human HepG2 cells, a hepatoma cell line used for MASH-related studies14,30,31, with 200 µM palmitate-BSA complexes or BSA and assessed ODC1, AMD1, and SRM expression by qPCR. Notably, only AMD1 was decreased by lipid loading (FIG. 29, panel A) whereas ODC1 and SRM were unaffected (FIG. 29, panels B and C). Primary mouse hepatocytes isolated from 10-week-old C57BL / 6J mice also showed downregulation of Amdl upon lipid loading with 200 µM palmitate-BSA (FIG. 29, panel D), which was not observed for Odc1 or Srm (FIG. 29, panels E and F). Importantly, these findings were corroborated in liver samples from patients with MASH compared to age-and sex-matched healthy specimens collected from livers donated for transplantation (FIG. 29, panels G-I). To address AMD1 protein expression in hepatocytes in vivo, we immunostained liver sections from humans and mice using a validated anti-AMDl antibody and antibodies targeting the hepatocyte markers ARG1 and HepParl32’33, and we found AMD1 was significantly downregulated (FIG. 29, panel J).Consistently, analysis of mRNA expression in the livers of mice fed a standard diet or a MASH-inducing diet for 12 and 24 weeks revealed that Amdl was significantly downregulated in MASH (FIG. 29, panel K), whereas Odel and Srm were unaffected (FIG.29, panels L and M), and was confirmed at the protein level as measured by immunostaining (FIG. 29, panel N). To determine whether the loss of AMD1 leads to an accumulation of putrescine, we transfected HepG2 cells and primary mouse hepatocytes with siRNA targeting AMD 1 (siAMDl / siAmdl) or control scrambled siRNA (ScrRNA). These data indicate that putrescine levels increase in MASH via AMD1 downregulation. Indeed, AMD1 silencing in HepG2 cells and primary mouse hepatocytes (-75% silencing efficiency, FIG.29, panels O and Q) significantly elevated putrescine levels (FIG. 29, panels P and R).Docket No: 2932719-000287-W01Date of Filing: December 11, 2025

[0186] L iver Deficiency in AMD1 Drives Hepatic Fibrosis in MASH

[0187] Based on our in vitro analyses of human and murine liver tissue, which identified AMD1 deficiency as a critical determinant of aberrant putrescine accumulation during MASH, we next sought to define the functional consequences of AMD1 loss in vivo. To this end, we employed a liver-targeted shRNA strategy using AAV8-U6 strategies (FIG. 30, panel A). Livers from these mice showed significant downregulation of AMD1 in hepatocytes (FIG. 30, panel B) and a marked increase in hepatic putrescine levels (FIG. 30, panel C). Serum ALT levels were also significantly elevated in liver AMD 1 -deficient mice (FIG.30, panel D). To investigate the broader consequences of AMD1 loss during MASH progression, we performed unbiased RNA-sequencing of liver tissue. AMD1 silencing resulted in strong induction of fibrosis-associated pathways (FIG.30, panel E), including significant upregulation of Col1a1, Col1a2, Col4a1, Col4a5, Col4a6, and Col6a5 (FIG. 30, panel F). These transcriptional alterations were accompanied by pronounced histological evidence of fibrotic remodeling, as reflected by increased collagen deposition on picrosirius red staining (FIG. 30, panel G) and hydroxyproline content (FIG. 30, panel H).Furthermore, there were more aSMA positive cells in the livers of these mice, indicating expansion of the fibrogenic myofibroblast population (FIG.30, panel I).

[0188] To achieve hepatocyte-specific AMD1 deletion, we generated Amdl fl ox mice and deleted AMD1 selectively in hepatocytes using AAV8-TBG-Cre viruses. Four weeks after AAV8-TBG-Cre injections, hepatocytes showed near complete absence of immunofluorescence signal in ARG1+ cells (FIG. 31, panel A). Furthermore, isolated primary hepatocytes showed over 95% reductions in Amdl expression (FIG. 31, panel B). Mice were then injected with control AAV8-TBG-GFP viruses or AAV8-TBG-Cre viruses followed by MASH diet feeding for 12 weeks (FIG.31, panel C). Livers from hepatocyte-specific AMD1 knockout mice showed significant elevations in putrescine content (FIG. 31, panel D). These mice also exhibited marked increases in ALT levels (FIG.31, panel E). Furthermore, picrosirius red staining demonstrated significantly increased collagen deposition after hepatocyte-specific AMD1 deletion (FIG.31, panel F), which was further confirmed by hydroxyproline measurements (FIG. 31, panel G) and elevated expression of collagen genes (FIG.31, panel H).

[0189] AMD1 Restoration in Hepatocytes Attenuates Fibrosis in MASH

[0190] Our findings thus far led us to determine whether restoring hepatocyte AMD1 can mitigate hepatic fibrosis in MASH. To assess the impact of AMD1 overexpression on MASHDocketNo: 2932719-000287-W01Date of Filing: December 11, 2025 progression, mice were fed a MASH-inducing diet for 12 weeks, followed by retro-orbital injection of AAV8-TBG-GFP (control) or AAV8-TBG-AMD1 (1 x 1011viral genomes per mouse, (FIG.32, panel A). Immunostaining confirmed robust AMD1 expression in ARG1+hepatocytes (FIG.32, panel B). Consistent with its role in polyamine metabolism, hepatic putrescine levels were significantly reduced following AMD1 overexpression (FIG. 32, panel C). Serum ALT levels were also significantly decreased (FIG. 32, panel D). Picrosirius red staining revealed substantial reductions in liver collagen content (FIG. 32, panel E), which was confirmed by hydroxyproline assays (FIG.32, panel F). qPCR further demonstrated significant downregulation of the collagen genes Collal, Colla2, Col4al, Col4a5, Col4a6, and Col6a5 (FIG.32, panel G). aSMA staining also revealed a significant reduction in stellate cells in the liver of mice with AMD1 overexpression (FIG. 32, panel H)

[0191] Putrescine Triggers Hepatocyte IHH Signaling to Activate Stellate Cells and Drive Liver Fibrosis

[0192] TGFβ1, PDGF, SHH, and TNFα are well-known profibrogenic mediators that drive fibrosis through stellate cell activation and collagen deposition34'41. However, we did not observe any changes in TGFβ, PDGF, SHH, or TNFα signaling in our shAMDl group. In contrast, Indian Hedgehog (IHH), a hepatocyte-derived morphogen linked to the TAZ pathway that directly activates HSCs, was significantly upregulated42,43(FIG. 33, panel A). Additionally, silencing AMD1 in HepG2 cells and primary mouse hepatocytes or treating HepG2 cells with putrescine increased IHH expression (FIG. 33, panels B-D) and its subsequent secretion into cell culture media, as measured by ELISA (FIG.33, panels E-G).

[0193] Because HSCs are the most dominant ECM-producing cell type in the liver and are responsive to IHH, we next sought to determine the impact of IHH secreted from AMD 1 -silenced hepatocytes on collagen gene expression in HSCs. To test this, we collected conditioned media from ScrRNA- or siAMDl -transfected HepG2 cells and further performed IHH immunodepletion on the latter using a bead-immobilized anti-IHH antibody (advanced verification from Invitrogen). When exposed to this conditioned media, LX-2 cells, a human HSC line that retains key features of HSC signaling and fibrogenesis, showed an increase in COL1A1 and COL1A2, which was abolished when IHH was immunodepleted. We next evaluated IHH gene expression in livers from putrescine-treated mice, shAMDl group mice, and in vitro hepatocytes with siAMDl silencing. Notably, IHH expression was significantly elevated in the groups (FIG. 33, panels B-D). SinceDocketNo: 2932719-000287-W01Date of Filing: December 11, 2025 IHH acts through secreted hepatocyte-derived signals, we next assessed secreted IHH levels. HepG2 cells were treated with putrescine or subjected to siAMDl silencing, and mouse primary hepatocytes were similarly analyzed. IHH levels were measured by ELISA as previously described42. Interestingly, we observed a significant increase in secreted IHH across the groups (FIG. 33, panels E-G). We then evaluated the expression of IHH-dependent genes (Gli2, Gli3, Ccnd1, and Ccn1) in livers and found upregulation in these genes in models where putrescine was experimentally enhanced (FIG. 33, panels H-J), and downregulation in mice with AMD1 overexpression (FIG. 33, panel K).

[0194] To determine the role of IHH in stellate cell activation, we depleted IHH from the conditioned media of AMD 1 -silenced hepatocytes. The LX2 stellate cell line was then treated with these IHH-depleted or control media for 24 hours, after which collagen gene expression was assessed by qPCR. Depletion of IHH from hepatocyte-conditioned media abolished the induction of COL1A1, COL1A2, COL4A1, and COL4A5 (FIG. 33, panels L-O). To determine whether SMO, the obligatory intermediate present on stellate cells, mediates IHH-dependent fibrogenic signaling in hepatic stellate cells, we silenced SMO in LX2 cells and treated them with conditioned media from siAMDl- or ScrRNA-transfected HepG2 cells. Conditioned media from AMD1-silenced HepG2 cells induced robust expression of fibrotic genes in control LX2 cells, whereas media from si AMD 1 -transfected HepG2 cells elicited minimal fibrogenic responses when SMO was silenced in LX2 cells (FIG. 33, panels P-S).

[0195] Therapeutic Lowering of Hepatocyte Putrescine using GalN Ac-Conjugated ODC1 siRNA Mitigates Liver Fibrosis in MASH

[0196] We designed a GalN Ac-conjugated siRNA against ODC1 (siODCl-GalNAc) and administered weekly intraperitoneal injections (15 mg / kg per mouse) in mice with established MASH (12 weeks of diet feeding prior to GalNAc initiation) for 8 weeks (FIG. 34, panel A).Immunoblotting confirmed efficient ODC1 silencing and reduced IHH expression (FIG.34, panel B). Liver putrescine levels were significantly reduced in the siODCl -treated cohort (FIG. 34, panel C). Furthermore, si ODC 1 -GalNAc treatments significantly lowered ALT levels compared to controls (FIG. 34, panel D), and collagen gene expression by qPCR was similarly reduced (FIG. 34E). Notably, IHH-dependent genes were also significantly downregulated (FIG. 34, panel F). Picrosirius red staining demonstrated reduced collagen deposition (FIG. 34, panel G), confirmed by hydroxyproline quantification (FIG.34, panel H). aSMA immunostaining revealedDocket No: 2932719-000287-W01Date of Filing: December 11, 2025 diminished activation of hepatic stellate cells, consistent with reduced fibrogenesis following 0DC1 -targeted therapy (FIG. 34, panel I).

[0197] Discussion

[0198] Metabolic dysfunction-associated steatotic liver disease (MASLD) and its progressive inflammatory form, MASH, have rapidly become leading causes of cirrhosis, hepatocellular carcinoma, and liver-related mortality worldwide1,2. While recently approved therapies primarily target steatosis, fibrosis remains the strongest predictor of adverse outcomes and a major unmet clinical need. In this context, our study identifies dysregulated hepatocyte polyamine metabolism, specifically, selective accumulation of putrescine driven by AMD1 suppression as a previously unrecognized, steatosis-independent driver of hepatic fibrosis in MASH. Across human biopsy specimens and multiple mouse models, we found that putrescine, but not spermidine or spermine, is consistently elevated and tightly correlated with fibrosis severity. Genetic loss- and gain-of-function approaches in hepatocytes, coupled with exogenous putrescine supplementation and hepatocyte-targeted ODC1 silencing, converge on a model in which excess putrescine amplifies hepatocyte IHH production, thereby activating Hedgehog (Hh) signaling in hepatic stellate cells (HSCs) and promoting matrix remodeling.

[0199] Fibrosis in MASH arises from complex cellular crosstalk involving hepatocytes, stellate cells, and immune cells3'7,44'46. Among the profibrotic pathways engaged in this milieu, the Hh pathway has emerged as a central regulator of HSC activation and survival47'51. Prior work has demonstrated that Hedgehog ligands produced by injured hepatocytes and cholangiocytes bind Patched receptors on HSCs, relieve repression of Smoothened (SMO), and enable GLI transcription factors to drive fibrogenic gene programs47'51. Our data extend this paradigm by positioning hepatocyte polyamine metabolism upstream of the IHH-SMO signaling axis. We show that hepatocyte-specific AMD1 deficiency, whether induced by liver-targeted shRNA or conditional deletion, leads to robust putrescine accumulation, increased IHH expression and secretion, upregulation of canonical Hh target genes, and exacerbated fibrosis. Conversely, restoring AMD1 in hepatocytes normalizes putrescine levels, dampens IHH-Hh signaling, and attenuates collagen deposition and αSMA+myofibroblast expansion.

[0200] Our putrescine supplementation experiments support a direct, profibrotic role for this polyamine species. Chronic administration of putrescine to mice with diet-induced MASH increased hepatic putrescine content and markedly worsened fibrosis, as evidenced by picrosiriusDocketNo: 2932719-000287-W01Date of Filing: December 11, 2025 red staining, hydroxyproline levels, and induction of multiple collagen genes. Notably, these changes occurred without significant alterations in steatosis, lobular inflammation, or hepatocellular ballooning, underscoring that putrescine primarily acts as a fibrogenic signal rather than a broad driver of liver injury or metabolic derangement. This selective fibrotic phenotype parallels our human and murine metabolomics data and aligns with the concept that distinct polyamine species exert nonredundant and context-dependent biological effects.

[0201] These findings can be interpreted within the broader reports on polyamines in liver disease. Several reports have indicated that spermidine supplementation ameliorates metabolic syndrome features, attenuates steatosis, and improves systemic metabolic indices in high-fat diet models31,52'54However, more recent data have challenged uniformly beneficial roles of spermidine, including evidence that prolonged supplementation can promote hepatocarcinogenesis in certain settings55. In striking contrast to these spermidine-focused studies, the role of putrescine in MASH has been largely overlooked, with only limited in vitro data indicating modest effects on lipotoxic cell death and no mechanistic or in vivo work addressing fibrosis36. By systematically quantifying hepatic polyamines in well-characterized human and murine MASH cohorts and combining this with hepatocyte-specific genetic manipulation and therapeutic targeting, our study fills this critical gap and establishes putrescine, rather than spermidine or spermine, as a key polyamine mediator of MASH-associated fibrosis.

[0202] Our work also has important translational implications by demonstrating that hepatocyte-directed lowering of putrescine can mitigate fibrosis in established MASH. Using a GalN Ac-conjugated siRNA targeting ODC 1-the rate-limiting enzyme for putrescine biosynthesis, we achieved efficient, hepatocyte-selective ODC1 silencing, reduced hepatic putrescine levels, and attenuated MASH-associated fibrosis. This intervention decreased serum ALT, collagen gene expression, picrosirius red staining, hydroxyproline content, HSC activation, and expression of IHH and its downstream Hh target genes. These results are compelling given that GalNAc-siRNA platforms have already been validated and approved for multiple liver-directed therapies in humans57'60, providing a clinically relevant framework for targeting hepatocyte polyamine metabolism. Compared with viral strategies, GalNAc-siRNA offers transient, titratable, and highly hepatocyte-specific gene silencing, potentially reducing off-target effects and long-term safety concerns.DocketNo: 2932719-000287-W01Date of Filing: December 11, 2025

[0203] At the same time, several unanswered questions warrant consideration. Although our data strongly support a hepatocyte-centric mechanism in which AMD1 downregulation and putrescine accumulation drive Hh-dependent fibrogenesis, other liver-resident cell types can also contribute to altered polyamine metabolism and Hh signaling in MASH. Kupffer cells, infiltrating monocyte-derived macrophages, cholangiocytes, and liver sinusoidal endothelial cells each express components of the polyamine machinery and Hh pathway to varying degrees. Whether putrescine directly modulates their function or indirectly shapes the inflammatory and fibrotic microenvironment remains to be defined.

[0204] In summary, our study defines dysregulated hepatocyte putrescine metabolism as a key driver of hepatic fibrosis in MASH and uncovers a putrescine / IHH / SMO / stellate cell activation axis that mechanistically links metabolic stress in hepatocytes to HSC activation and matrix remodeling. By integrating human metabolomics, mechanistic hepatocyte-stellate cell crosstalk studies, hepatocyte-specific genetic perturbations, and an FDA-aligned GalNAc-siRNA therapeutic strategy, we provide both conceptual and translational advances. These findings position hepatocyte putrescine lowering as a promising antifibrotic approach that is mechanistically distinct from current steatosis-focused therapies and support further development of polyamine-targeted interventions for patients with MASH.

[0205] References Cited in this Example

[0206] 1. Riazi, K., Azhari, H., Charette, J. H., Underwood, F. E., King, J. A., Afshar, E. E., Swain, M. G., Congly, S. E., Kaplan, G. G., and Shaheen, A. A. (2022). The prevalence and incidence of NAFLD worldwide: a systematic review and meta-analysis. Lancet Gastroenterol Hepatol 7, 851-861. 10.1016 / S2468-1253(22)00165-0.

[0207] 2. Cheemerla, S., and Balakrishnan, M. (2021). Global Epidemiology of Chronic Liver Disease. Clin Liver Dis (Hoboken) 17, 365-370. 10.1002 / cld.1061.

[0208] 3. Sanyal, A. J., Van Natta, M. L., Clark, J., Neuschwander-Tetri, B. A., Diehl, A., Dasarathy, S., Loomba, R., Chalasani, N., Kowdley, K., Hameed, B., et al. (2021). Prospective Study of Outcomes in Adults with Nonalcoholic Fatty Liver Disease. N Engl J Med 385, 1559-1569. 10.1056 / NEJMoa2029349.

[0209] 4. Younossi, Z. M., Mangla, K. K., Berentzen, T. L., Grau, K., Kjaer, M. S., Ladelund, S., Nitze, L. M., Coolbaugh, C., Hsu, C. Y., and Hagstrom, H. (2024). Liver histology isDocket No: 2932719-000287-W01Date of Filing: December 11, 2025 associated with long-term clinical outcomes in patients with metabolic dysfunctionassociated steatohepatitis. Hepatol Commun 8. 10.1097 / HC9.0000000000000423.[002101 5. Li, Y. Y., Zheng, T. L., Xiao, S. Y., Wang, P., Yang, W. J., Jiang, L. L., Chen, L. L., Sha, J. C., Jin, Y., Chen, S. D., et al. (2023). Hepatocytic ballooning in non-alcoholic steatohepatitis: Dilemmas and future directions. Liver Int 43, 1170-1182. 10.1111 / liv.15571.

[0211] 6. Dulai, P. S., Singh, S., Patel, J., Soni, M., Prokop, L. J., Younossi, Z., Sebastiani, G., Ekstedt, M., Hagstrom, H., Nasr, P., et al. (2017). Increased risk of mortality by fibrosis stage in nonalcoholic fatty liver disease: Systematic review and meta-analysis. Hepatology 65, 1557-1565. 10.1002 / hep.29085.

[0212] 7. Angulo, P., Kleiner, D. E., Dam-Larsen, S., Adams, L. A., Bjornsson, E. S., Charatcharoenwitthaya, P., Mills, P. R., Keach, J. C., Lafferty, H. D., Stabler, A., et al. (2015). Liver Fibrosis, but No Other Histologic Features, Is Associated With Long-term Outcomes of Patients With Nonalcoholic Fatty Liver Disease. Gastroenterology 149, 389-397 e310.10.1053 / j.gastro.2015.04.043.

[0213] 8. Harrison, S. A., Bedossa, P., Guy, C. D., Schattenberg, J. M., Loomba, R., Taub, R., Labriola, D., Moussa, S. E., Neff, G. W., Rinella, M. E., et al. (2024). A Phase 3, Randomized, Controlled Trial of Resmetirom in NASH with Liver Fibrosis. N Engl J Med 390, 497-509.10.1056 / NEJMoa2309000.

[0214] 9. Sanyal, A. J., Newsome, P. N., Kliers, I., Ostergaard, L. H., Long, M. T., Kjaer, M. S., Cali, A. M. G., Bugianesi, E., Rinella, ME., Roden, M., et al. (2025). Phase 3 Trial of Semaglutide in Metabolic Dysfunction- Associated Steatohepatitis. N Engl J Med 392, 2089-2099.10.1056 / NEJMoa2413258.

[0215] 10. Le, P., Tatar, M., Dasarathy, S., Alkhouri, N., Herman, W. H., Taksler, G. B., Deshpande, A., Ye, W., Adekunle, O. A., McCullough, A., and Rothberg, M. B. (2025). Estimated Burden of Metabolic Dysfunction-Associated Steatotic Liver Disease in US Adults, 2020 to 2050. JAMA Netw Open 8, e2454707. 10.1001 / jamanetworkopen.2024.54707.

[0216] 11. Parekh, S., and Anania, F. A. (2007). Abnormal lipid and glucose metabolism in obesity: implications for nonalcoholic fatty liver disease. Gastroenterology 132, 2191-2207.10.1053 / j.gastro.2007.03.055.

[0217] 12. Gaggini, M., Carli, F., Rosso, C., Buzzigoli, E., Marietti, M., Della Latta, V., Ciociaro, D., Abate, M. L., Gambino, R., Cassader, M., et al. (2018). Altered amino acidDocketNo: 2932719-000287-W01Date of Filing: December 11, 2025 concentrations in NAFLD: Impact of obesity and insulin resistance. Hepatology 67, 145- 158.10.1002 / hep.29465.

[0218] 13. Yamakado, M., Tanaka, T., Nagao, K., Imaizumi, A., Komatsu, M., Daimon, T., Miyano, H., Tani, M., Toda, A., Yamamoto, H., et al. (2017). Plasma amino acid profile associated with fatty liver disease and co-occurrence of metabolic risk factors. Sci Rep 7, 14485.10.1038 / s41598-017-14974-w.

[0219] 14. Rom, O., Liu, Y., Liu, Z., Zhao, Y., Wu, J., Ghrayeb, A., Villacorta, L., Fan, Y., Chang, L., Wang, L., et al. (2020). Glycine-based treatment ameliorates NAFLD by modulating fatty acid oxidation, glutathione synthesis, and the gut microbiome. Sci Transl Med 12. 10.1126 / scitranslmed.aaz2841.

[0220] 15. Mardinoglu, A., Bjomson, E., Zhang, C., Klevstig, M., Soderlund, S., Stahlman, M., Adiels, M., Hakkarainen, A., Lundbom, N., Kilicarslan, M., et al. (2017). Personal modelassisted identification of NAD(+) and glutathione metabolism as intervention target in NAFLD. Mol SystBiol 13, 916. 10.15252 / msb.20167422.

[0221] 16. Simon, J., Nunez-Garcia, M., Fernandez-Tussy, P., Barbier-Torres, L., Fernandez-Ramos, D., Gomez-Santos, B., Buque, X., Lopitz-Otsoa, F., Goikoetxea-Usandizaga, N., Serrano-Macia, M., et al. (2020). Targeting Hepatic Glutaminase 1 Ameliorates Nonalcoholic Steatohepatitis by Restoring Very -Low-Density Lipoprotein Triglyceride Assembly. Cell Metab 31, 605-622 e610. 10.1016 / j.cmet.2020.01.013.

[0222] 17. Yurdagul, A., Jr., Subramanian, M., Wang, X., Crown, S B., Ilkayeva, O R., Darville, L., Kolluru, G. K., Rymond, C. C., Gerlach, B. D., Zheng, Z., et al. (2020). Macrophage Metabolism of Apoptotic Cell-Derived Arginine Promotes Continual Efferocytosis and Resolution of Injury. Cell Metab 31, 518-533 e510. 10.1016 / j.cmet.2020.01.001.

[0223] 18. Liu, Y, Zhao, Y., Shukha, Y., Lu, H., Wang, L., Liu, Z., Liu, C., Zhao, Y., Wang, H., Zhao, G., et al. (2021). Dysregulated oxalate metabolism is a driver and therapeutic target in atherosclerosis. Cell Rep 36, 109420. 10.1016 / j.celrep.2021.109420.

[0224] 19. Rom, O., Liu, Y., Finney, A C, Ghrayeb, A., Zhao, Y, Shukha, Y., Wang, L., Rajanayake, K. K., Das, S., Rashdan, N. A., et al. (2022). Induction of glutathione biosynthesis by glycine-based treatment mitigates atherosclerosis. Redox Biol 52, 102313.10.1016 / j.redox.2022.102313.Docket No: 2932719-000287-W01Date of Filing: December 11, 2025

[0225] 20. Das, S., Finney, A C., Anand, S. K., Rohilla, S., Liu, Y, Pandey, N., Ghrayeb, A., Kumar, D., Nunez, K., Liu, Z., et al. (2024). Inhibition of hepatic oxalate overproduction ameliorates metabolic dysfunction-associated steatohepatitis. Nat Metab 6, 1939-1962.10.1038 / s42255-024-01134-4.

[0226] 21. Ghrayeb, A., Finney, A. C., Agranovich, B., Peled, D., Anand, S. K., McKinney, M. P., Sarji, M., Yang, D., Weissman, N., Drucker, S., et al. (2024). Serine synthesis via reversed SHMT2 activity drives glycine depletion and acetaminophen hepatotoxicity in MASLD. Cell Metab 36, 116-129 ell7. 10.1016 / j.cmet.2023.12.013.

[0227] 22. Qu, P., Rom, O, Li, K., Jia, L., Gao, X, Liu, Z., Ding, S., Zhao, M„ Wang, H., Chen, S., et al. (2023). DT-109 ameliorates nonalcoholic steatohepatitis in nonhuman primates. Cell Metab 35, 742-757 e710. 10.1016 / j.cmet.2023.03.013.

[0228] 23. Casero, R. A., Jr., Murray Stewart, T., and Pegg, A. E. (2018). Polyamine metabolism and cancer: treatments, challenges and opportunities. Nat Rev Cancer 18, 681-695.10.1038 / s41568-018-0050-3.

[0229] 24. Wallace, H. M., Fraser, A. V., and Hughes, A. (2003). A perspective of polyamine metabolism. Biochem J 376, 1-14. 10.1042 / BJ20031327.

[0230] 25. Eisenberg, T., Abdellatif, M., Schroeder, S., Primessnig, U., Stekovic, S., Pendl, T., Harger, A., Schipke, J., Zimmermann, A., Schmidt, A., et al. (2016). Cardioprotection and lifespan extension by the natural poly amine spermidine. Nat Med 22, 1428-1438.10.1038 / nm.4222.

[0231] 26. Eisenberg, T., Knauer, H., Schauer, A., Buttner, S., Ruckenstuhl, C., Carmona- Gutierrez, D., Ring, J., Schroeder, S., Magnes, C., Antonacci, L., et al. (2009). Induction of autophagy by spermidine promotes longevity. Nat Cell Biol 11, 1305-1314. 10.1038 / ncb1975.

[0232] 27. Morselli, E., Marino, G., Bennetzen, M. V., Eisenberg, T., Megalou, E., Schroeder, S., Cabrera, S., Benit, P., Rustin, P., Criollo, A., et al. (2011). Spermidine and resveratrol induce autophagy by distinct pathways converging on the acetylproteome. J Cell Biol 192, 615-629. 10.1083 / jcb.201008167.

[0233] 28. Rom, O, Xu, G., Guo, Y., Zhu, Y., Wang, H., Zhang, J., Fan, Y., Liang, W., Lu, H., Liu, Y., et al. (2019). Nitro-fatty acids protect against steatosis and fibrosis during development of nonalcoholic fatty liver disease in mice. EBioMedicine 41, 62-72.10.1016 / j.ebiom.2019.02.019.DocketNo: 2932719-000287-W01Date of Filing: December 11, 2025

[0234] 29. Ma, F., Longo, M., Meroni, M., Bhattacharya, D., Paolini, E., Mughal, S., Hussain, S., Anand, S. K., Gupta, N., Zhu, Y., et al. (2025). EHBP1 suppresses liver fibrosis in metabolic dysfunction-associated steatohepatitis. Cell Metab 37, 1152-1170 e1157.10.1016 / j.cmet.2025.01.020.

[0235] 30. Jain, M. R., Giri, S. R., Bhoi, B., Trivedi, C., Rath, A., Rathod, R., Ranvir, R., Kadam, S., Patel, H., Swain, P., et al. (2018). Dual PPARalpha / gamma agonist saroglitazar improves liver histopathology and biochemistry in experimental NASH models. Liver Int 38, 1084-1094. 10.1111 / liv.13634.

[0236] 31. Ma, L., Ni, Y., Hu, L., Zhao, Y., Zheng, L., Yang, S., Ni, L., and Fu, Z. (2021). Spermidine ameliorates high-fat diet-induced hepatic steatosis and adipose tissue inflammation in preexisting obese mice. Life Sci 265, 118739. 10.1016 / j.lfs.2020.118739.

[0237] 32. Yan, B. C., Gong, C., Song, J., Krausz, T., Tretiakova, M., Hyjek, E., AL Ahmadie, H., Alves, V., Xiao, S. Y., Anders, R. A., and Hart, J. A. (2010). Arginase-1: a new immunohistochemical marker of hepatocytes and hepatocellular neoplasms. Am J Surg Pathol 34, 1147-1154. 10.1097 / PAS.0b013e3181e5dffa. 33. Haruna, Y., Saito, K., Spaulding, S., Nalesnik, M. A., and Gerber, M. A. (1996). Identification of bipotential progenitor cells in human liver development. Hepatology 23, 476-481. 10.1002 / hep.510230312.

[0238] 34. Wang S, L. F., Han M, Chaudhary R, Asimakopoulos A, Liebe R, Yao Y, Hammad S, Dropmann A, Krizanac M, Rubie C, Feiner LK, Glanemann M, Ebert MPA, Weiskirchen R, Henis YI, Ehrlich M, Dooley S. The Interplay of TGF-pi and Cholesterol Orchestrating Hepatocyte Cell Fate, EMT, and Signals for HSC Activation. Cell Mol Gastroenterol Hepatol. 2024;17(4):567-587. doi: 10.1016 / j.jcmgh.2023.12.012. Epub 2023 Dec 27. PMID: 38154598; PMCID: PMC10883985.

[0239] 35. Ghafoory S, V. R., Robison T, Kouzbari K, Woolington S, Murphy B, Xia L, Ahamed J. Platelet TGF-pi deficiency decreases liver fibrosis in a mouse model of liver injury. Blood Adv. 2018 Mar 13;2(5):470-480. doi: 10.1182 / bloodadvances.2017010868. PMID: 29490978; PMCID: PMC5851416.

[0240] 36. Sasaki R, K. T., Nakamura M, Nakamoto S, Haga Y, Wu S, Shirasawa H, Yokosuka O. Possible Involvement of Hepatitis B Virus Infection of Hepatocytes in the Attenuation of Apoptosis in Hepatic Stellate Cells. PLoS One. 2016 Jan 5; 11(1):e0146314. doi: 10.1371 / journal. pone.0146314. PMID: 26731332; PMCID: PMC4701422.Docket No: 2932719-000287-W01Date of Filing: December 11, 2025

[0241] 37. Yoshida S, IN., Liu SB, Peng ZW, Chung J, Sverdlov DY, Miyamoto M, Kim YO, Ogawa S, Arch RH, Schuppan D, Popov Y. Extrahepatic platelet-derived growth factor-p, delivered by platelets, promotes activation of hepatic stellate cells and biliary fibrosis in mice. Gastroenterology. 2014 Dec; 147(6): 1378-92. doi: 10.1053 / j.gastro.2014.08.038. Epub 2014 Aug 28. PMID: 25173753.

[0242] 38. Fickert P, T. A., Moustafa T, Silbert D, Gumhold J, Tsybrovskyy O, Lebofsky M, Jaeschke H, Denk H, Trauner M. The role of osteopontin and tumor necrosis factor alpha receptor- 1 in xenobiotic-induced cholangitis and biliary fibrosis in mice. Lab Invest. 2010 Jun;90(6): 844-52. doi: 10.1038 / labinvest.2010.61. Epub 2010 Apr 5. PMID: 20368698; PMCID: PMC4285781.

[0243] 39. Verdelho Machado M, D. A. Role of Hedgehog Signaling Pathway in NASH. Int I Mol Sci. 2016 Jun 1;17(6):857. doi: 10.3390 / ijmsl7060857. PMID: 27258259; PMCID: PMC4926391.

[0244] 40. Tarrats N, M. A., Morales A, Garcia-Ruiz C, Fernandez-Checa JC, Mari M. Critical role of tumor necrosis factor receptor 1, but not 2, in hepatic stellate cell proliferation, extracellular matrix remodeling, and liver fibrogenesis. Hepatology. 2011 Jul;54(l):319-27. doi: 10.1002 / hep.24388. PMID: 21523796; PMCID: PMC3125435.

[0245] 41. Yang YM, S. E. T.i.l.f. Curr Pathobiol Rep. 2015 Dec;3(4):253-261. doi: 10.1007 / s40139-015-0093-z. Epub 2015 Sep 30. PMID: 26726307; PMCID: PMC4693602.

[0246] 42. Moore MP, W. X., Shi H, Meroni M, Cherubini A, Ronzoni L, Parks EJ, Ibdah J A, Rector RS, Valenti L, Dongiovanni P, Tabas I. Circulating indian hedgehog is a marker of the hepatocyte-TAZ pathway in experimental NASH and is elevated in humans with NASH. JHEP Rep. 2023 Feb 26;5(5): 100716. doi: 10.1016 / j.jhepr.2023.100716. PMID: 37035456; PMCID: PMC 10074197.

[0247] 43. Wang X, Z. Z., Caviglia JM, Corey KE, Herfel TM, Cai B, Masia R, Chung RT, Lefkowitch JH, Schwabe RF, Tabas I. Hepatocyte TAZ / WWTR1 Promotes Inflammation and Fibrosis in Nonalcoholic Steatohepatitis. Cell Metab. 2016 Dec 13;24(6):848-862. doi: 10.1016 / j.cmet.2016.09.016. Epub 2016 Oct 27. PMID: 28068223; PMCID: PMC5226184.

[0248] 44. Cai, X., Wang, J., Wang, J., Zhou, Q., Yang, B., He, Q., and Weng, Q. (2020). Intercellular crosstalk of hepatic stellate cells in liver fibrosis: New insights into therapy. Pharmacol Res 155, 104720. 10.1016 / j.phrs.2020.104720.DocketNo: 2932719-000287-W01Date of Filing: December 11, 2025

[0249] 45. Xiong, X, Kuang, H., Ansari, S., Liu, T„ Gong, J., Wang, S., Zhao, X. Y., Ji, Y., Li, C., Guo, L., et al. (2019). Landscape of Intercellular Crosstalk in Healthy and NASH Liver Revealed by Single-Cell Secretome Gene Analysis. Mol Cell 75, 644-660 e645.10.1016 / j.molcel.2019.07.028.

[0250] 46. Subramanian, P., Hampe, J., Tacke, F., and Chavakis, T. (2022). Fibrogenic Pathways in Metabolic Dysfunction Associated Fatty Liver Disease (MAFLD). Int J Mol Sci 23.10.3390 / ijms23136996.

[0251] 47. Khajehahmadi, Z., Mohagheghi, S., Nikeghbalian, S., Geramizadeh, B., Khodadadi, I., Karimi, J., Ghaffari, M. E., and Tavilani, H. (2019). Downregulation of hedgehog ligands in human simple steatosis may protect against nonalcoholic steatohepatitis: Is TAZ a crucial regulator? IUBMB Life 71, 1382-1390. 10.1002 / iub.2068.

[0252] 48. Moore, M. P., Wang, X., Shi, H, Meroni, M., Cherubini, A., Ronzoni, L., Parks, E. J., Ibdah, J. A., Rector, R. S., Valenti, L., et al. (2023). Circulating indian hedgehog is a marker of the hepatocyte-TAZ pathway in experimental NASH and is elevated in humans with NASH. JHEPRep 5, 100716. 10.1016 / j.jhepr.2023.100716.

[0253] 49. Wang, X., Cai, B., Yang, X., Sonubi, O. O., Zheng, Z., Ramakrishnan, R., Shi, H., Valenti, L., Pajvani, U. B., Sandhu, J., et al. (2020). Cholesterol Stabilizes TAZ in Hepatocytes to Promote Experimental Non-alcoholic Steatohepatitis. Cell Metab 31, 969-986 e967.10.1016 / j.cmet.2020.03.010.

[0254] 50. Wang, X., Sommerfeld, M. R., Jahn-Hofmann, K., Cai, B., Filliol, A., Remotti, H. E., Schwabe, R. F., Kannt, A., and Tabas, I. (2019). A Therapeutic Silencing RNA Targeting Hepatocyte TAZ Prevents and Reverses Fibrosis in Nonalcoholic Steatohepatitis in Mice. Hepatol Commun 3, 1221-1234. 10.1002 / hep4.1405.

[0255] 51. Wang, X., Zheng, Z, Caviglia, J. M., Corey, K. E., Herfel, T. M., Cai, B., Masia, R., Chung, R. T., Lefkowitch, J. H., Schwabe, R. F., and Tabas, I. (2016). Hepatocyte TAZ / WWTR1 Promotes Inflammation and Fibrosis in Nonalcoholic Steatohepatitis. Cell Metab 24, 848-862.10.1016 / j.cmet.2016.09.016.

[0256] 52. Gao, M., Zhao, W., Li, C., Xie, X., Li, M., Bi, Y., Fang, F., Du, Y., and Liu, X. (2018). Spermidine ameliorates non-alcoholic fatty liver disease through regulating lipid metabolism via AMPK. Biochem Biophys Res Commun 505, 93-98. 10.1016 / j.bbrc.2018.09.078.DocketNo: 2932719-000287-W01Date of Filing: December 11, 2025

[0257] 53 Wang, D., Yin, J., Zhou, Z, Tao, Y, Jia, Y., Jie, H, Zhao, J, Li, R., Li, Y., Guo, C., et al. (2021). Oral Spermidine Targets Brown Fat and Skeletal Muscle to Mitigate Diet-Induced Obesity and Metabolic Disorders. Mol Nutr Food Res 65, e2100315.10.1002 / mnfr.202100315.

[0258] 54. Zhou, J., Pang, J., Tripathi, M., Ho, J. P., Widjaja, A. A., Shekeran, S. G., Cook, S. A., Suzuki, A., Diehl, A. M., Petretto, E., et al. (2022). Spermidine-mediated hypusination of translation factor EIF5A improves mitochondrial fatty acid oxidation and prevents nonalcoholic steatohepatitis progression. Nat Commun 13, 5202. 10.1038 / s41467-022-32788-x.

[0259] 55. Szydlowska, M., Lasky, G., Oldham, S., Rivera, C., Ford, M., Sellman, B. R., Rhodes, C. J., and Cohen, T. S. (2023). Restoring polyamine levels by supplementation of spermidine modulates hepatic immune landscape in murine model of NASH. Biochim Biophys Acta Mol Basis Dis 1869, 166697. 10.1016 / j.bbadis.2023.166697.

[0260] 56. Nunez-Sanchez, M. A., Martinez-Sanchez, M. A., Sierra-Cruz, M., Lambertos, A., Rico-Chazarra, S., Oliva-Bolarin, A., Balaguer-Roman, A., Yuste, J. E., Martinez, C. M., Mika, A., et al. (2024). Increased hepatic putrescine levels as a new potential factor related to the progression of metabolic dysfunction-associated steatotic liver disease. J Pathol 264, 101-111.10.1002 / path.6330.

[0261] 57. Balwani, M., Sardh, E., Ventura, P., Peiro, P. A., Rees, D. C., Stolzel, U., Bissell, D. M., Bonkovsky, H. L., Windyga, J., Anderson, K. E., et al. (2020). Phase 3 Trial of RNAi Therapeutic Givosiran for Acute Intermittent Porphyria. N Engl J Med 382, 2289-2301.10.1056 / NEJMoal913147.

[0262] 58. Garrelfs, S. F., Frishberg, Y., Hulton, S. A., Koren, M. J., O'Riordan, W. D., Cochat, P., Deschenes, G., Shasha-Lavsky, H., Saland, J. M., Van't Hoff, W. G., et al. (2021). Lumasiran, an RNAi Therapeutic for Primary Hyperoxaluria Type 1. N Engl J Med 384, 1216-1226. 10.1056 / NEJMoa2021712.

[0263] 59. Adams, D., Tournev, I. L., Taylor, M. S., Coelho, T., Plante-Bordeneuve, V., Berk, J. L., Gonzalez-Duarte, A., Gillmore, J. D., Low, S. C., Sekijima, Y., et al. (2023). Efficacy and safety of vutrisiran for patients with hereditary transthyretin-mediated amyloidosis with polyneuropathy: a randomized clinical trial. Amyloid 30, 1-9. 10.1080 / 13506129.2022.2091985.

[0264] 60. Ray, K. K., Wright, R. S., Kaliend, D., Koenig, W., Leiter, L. A., Raal, F. J., Bisch, J. A., Richardson, T., Jaros, M., Wijngaard, P L. J., et al. (2020). Two Phase 3 Trials ofDocket No: 2932719-000287-W01Date of Filing: December 11, 2025 Inclisiran in Patients with Elevated LDL Cholesterol. N Engl J Med 382, 1507-1519.10.1056 / NEJMoal912387.EQUIVALENTS

[0265] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention, and are covered by the following claims.

Claims

1. DocketNo: 2932719-000287-W012.Date of Filing: December 11, 2025 CLAIMS3.What is claimed is:

1. A nucleic acid molecule comprising a sense oligonucleotide and an antisense oligonucleotide, wherein the sense oligonucleotide or the antisense oligonucleotide is complementary to a target nucleic acid sequence of a gene or gene product encoding ODC1.

2. The nucleic acid molecule of claim 1, wherein the sense oligonucleotide is complimentary to the antisense oligonucleotide.

3. The nucleic acid molecule of claim 1, wherein the molecule comprises one or more N- acetylgalactosamine (GalNAc) derivatives.

4. The nucleic acid molecule of claim 1, wherein the molecule comprises one or more nucleotides with a modification chosen from a 2'-O-methyl modified nucleotide, a 2'- fluoro modified nucleotide, or both.

5. The nucleic acid molecule of claim 1, wherein the molecule comprises one or more nucleotides connected to one another by way of phosphodiester or phosphorothioate linkages.

6. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule comprises a small interfering RNA (siRNA) molecule.

7. The nucleic acid molecule of claim 1, wherein the antisense oligonucleotide comprises the sequence of11.a. mUfUmAmUmAfCmGmCmCmGmAmGmCfAmCfGmUmCmGmUmCmAmU; b. AACACAGCGGGCAUCAGAG;12.c. AAUGAUGGCGUCUAUGGAUCAUU;13.d. AACAUUAUCCUCUUGUGCUAAUU;14.e. AAUACAAUGUGAUAAGUUGACUU;15.f AAGUUGACUUGAAGAUUACAGUU;16.g. AAAGCUUAGUGUUGUGACCUGUU;17.h. AACUGUAAUACUCAGUAGCCGUU; or DocketNo: 2932719-000287-W0118.Date of Filing: December 11, 2025 an antisense oligonucleotide having 70%, 75%, 80%, 85%, 90% or 95% nucleic acid sequence identity to any of the same.

8. The nucleic acid molecule of claim 1, wherein the sense oligonucleotide comprises the sequence of:20.a. mG*mA*mCmGmAmCfGmUfGfCfUmCmGmGmCmGmUmAmUmAmA; b. CUCUGAUGCCCGCUGUGUU;21.c. UGAUGGCGUCUAUGGAUCA;22.d. CAUUAUCCUCUUGUGCUAA;23.e. UACAAUGUGAUAAGUUGAC;24.f. GUUGACUUGAAGAUUACAG;25.g. AGCUUAGUGUUGUGACCUG;26.h. CUGUAAUACUCAGUAGCCG; or27.a sense oligonucleotide having 70%, 75%, 80%, 85%, 90% or 95% nucleic acid sequence identity to any of the same.

9. The nucleic acid molecule of claim 1, wherein the molecule inhibits the expression of ODC1.

10. The nucleic acid molecule of claim 9, wherein the molecule inhibits the expression of ODC1 mRNA by at least 50%.

11. The nucleic acid molecule of claim 4, wherein the molecule comprises at least five 2'-O- methyl modified nucleotides and at least five 2'-fluoro modified nucleotides.

12. The nucleic acid molecule of claim 3, wherein the one or more GalNAc derivatives is attached to the 3' end of the sense oligonucleotide.

13. The nucleic acid molecule of claim 3, wherein the one or more GalNAc derivatives is a biantennaiy or a triantennary GalNAc ligand.

14. The nucleic acid molecule of 13, wherein the GalNAc ligand is attached to the 3' end of the sense oligonucleotide.Docket No: 2932719-000287-W0134.Date of Filing: December 11, 2025 15. The nucleic acid molecule of 13, wherein the GalNAc ligand is attached to the 3' end of the sense oligonucleotide via linker.

16. A nucleic acid according to the sequence of:36.a. mUfUmAmUmAfCmGmCmCmGmAmGmCfAmCfGmUmCmGmUmCmAmU; b. AACACAGCGGGCAUCAGAG;37.c. AAUGAUGGCGUCUAUGGAUCAUU;38.d. AACAUUAUCCUCUUGUGCUAAUU;39.e. AAUACAAUGUGAUAAGUUGACUU;40.f. AAGUUGACUUGAAGAUUACAGUU;41.g. AAAGCUUAGUGUUGUGACCUGUU;42.h. AACUGUAAUACUCAGUAGCCGUU; or43.a nucleic acid having 70%, 75%, 80%, 85%, 90% or 95% nucleic acid sequence identity to any of the same.

17. A nucleic acid according to the sequence of45.a. mG*mA*mCmGmAmCfGmUfGfCfUmCmGmGmCmGmUmAmUmAmA; b. CUCUGAUGCCCGCUGUGUU;46.c. UGAUGGCGUCUAUGGAUCA;47.d. CAUUAUCCUCUUGUGCUAA;48.e. UACAAUGUGAUAAGUUGAC;49.f. GUUGACUUGAAGAUUACAG;50.g. AGCUUAGUGUUGUGACCUG;51.h. CUGUAAUACUCAGUAGCCG; or52.a nucleic acid having 70%, 75%, 80%, 85%, 90% or 95% nucleic acid sequence identity to any of the same.

18. A pharmaceutical composition comprising the nucleic acid molecule of claim 1.

19. A method of treating chronic liver disease comprising administering to a subject the nucleic acid molecule of claim 1.Docket No: 2932719-000287-W0155.Date of Filing: December 11, 2025 20. The method of claim 19, wherein the subject is at risk for developing, or is diagnosed with, chronic liver disease.

21. The method of claim 19, wherein chronic liver disease comprises metabolic dysfunction- associated steatotic liver disease (MASLD), metabolic dysfunction-associated steatohepatitis (MASH), hepatic fibrosis alcoholic liver disease, chronic hepatitis B, chronic hepatitis C, autoimmune hepatitis, primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), hemochromatosis, Wilson's disease, alpha- 1 antitrypsin deficiency, hepatocellular carcinoma, or a combination thereof.

22. The method of claim 19, wherein the method decreases a level of hepatic putrescine in the subject.

23. A method of reducing hepatic putrescine levels in a subject, the method comprising administering to the subject the nucleic acid molecule of claim 1.

Images