Modified short interfering nucleic acid (siNA) molecules and uses thereof
Inventors
Beigelman, Leonid • Rajwanshi, Vivek Kumar • Hossbach, Markus • Pandey, Rajendra K. • Hong, Jin • Eltepu, Laxman • MONTERO, Saul MARTINEZ • De Costa, N. Tilani S.
Assignees
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Publication Number
US-12129469-B2
Publication Date
2024-10-29
Expiration Date
Abstract
Disclosed herein are short interfering nucleic acid (siNA) molecules comprising modified nucleotides and uses thereof. The siNA molecules may be double stranded and comprise modified nucleotides selected from 2′-O-methyl nucleotides and 2′-fluoro nucleotides. Further disclosed herein are siNA molecules comprising (a) a phosphorylation blocker, conjugated moiety, or 5′-stabilized end cap; and (b) a short interfering nucleic acid (siNA).
Core Innovation
The invention relates to treating hepatitis B virus (HBV) by administering double stranded short interfering nucleic acid (siNA) or siNA molecules to a subject with HBV. The siNA includes sense and antisense strands with nucleic acid sequences defined relative to SEQ ID NO: 40 or consisting of defined SEQ ID numbers, and the strands have specified lengths in different claim sets. The disclosure further includes modified nucleotides selected from 2′-O-methyl nucleotides and 2′-fluoro nucleotides, with specified counts and positional patterns on both strands.
The siNA embodiments further include structural features such as 5′-stabilized end caps, phosphorothioate internucleoside linkages, phosphodiester, phosphorodithioate, and PS-mimic linkages, optional overhangs made of 1 or 2 nucleotides, blunt ends, and end-cap formulas. Additional disclosed features include thermally destabilizing nucleotides, d2vd3 nucleotides, and linker types including HEG and TEG.
In particular embodiments, the siNA includes N-acetylgalactosamine (GalNAc) or galactosamine conjugation, including attachment to the 3′ end of the sense strand, with GalNAc described by Formula (VII) with n=1 and R=OH in at least one embodiment. The content also describes 5′-vinyl phosphonate variants, including deuterated vinyl phosphonate variants, and target selection criteria for viral hepadnavirus regions including HBV, S gene, and X gene.
Claims Coverage
The partial content identifies multiple independent claims directed to treating HBV by administering double stranded siNA or siNA molecules. Across the independent claims, the main inventive features cluster around strand length and sequence identity/complementarity requirements, specific 2′-O-methyl and 2′-fluoro nucleotide modification distributions, defined sense/antisense sequence pairings, and optional GalNAc conjugation and additional structural features.
HBV treatment by double stranded siNA with SEQ ID NO: 40 similarity and balanced 2′-O-methyl/2′-fluoro modifications
A method of treating HBV comprising administering a double stranded siNA with a sense strand 19–21 nucleotides and at least 80% identical to SEQ ID NO: 40, and an antisense strand 19–21 nucleotides and at least 80% complementary to SEQ ID NO: 40, where each strand has 15 or more modified nucleotides independently selected from 2′-O-methyl and 2′-fluoro nucleotides, with at least 11 2′-O-methyl nucleotides and at least 4 2′-fluoro nucleotides on the sense strand and at least 11 2′-O-methyl nucleotides and 4 to 6 2′-fluoro nucleotides on the antisense strand.
HBV treatment by defined 2′-fluoro and 2′-O-methyl positional patterning on sense and antisense strands
A method of treating HBV comprising administering a double stranded siNA including a sense strand comprising 19 nucleotides with 2′-fluoro nucleotides at positions 5 and 7–9 from the 5′ end and 2′-O-methyl nucleotides at positions 1–4, 6, and 10–19 from the 5′ end, and an antisense strand comprising 21 nucleotides with 2′-fluoro nucleotides at positions 2, 6, 14, and 16 from the 5′ end and 2′-O-methyl nucleotides at positions 1, 3–5, 7–13, 15, and 17–21 from the 5′ end.
HBV treatment by siNA with sense and antisense strand sequences consisting of selected SEQ ID numbers
A method of treating HBV comprising administering an siNA molecule including a sense strand comprising a nucleic acid sequence consisting of SEQ ID NO: 438 or SEQ ID NO: 435, and an antisense strand comprising a nucleic acid sequence consisting of any one of SEQ ID NO: 501, SEQ ID NO: 505, SEQ ID NO: 506, SEQ ID NO: 537, SEQ ID NO: 538, and SEQ ID NO: 539.
GalNAc-conjugated siNA with defined modified sequence pairings
A method of treating HBV comprising administering a double stranded siNA comprising a sense strand of 5′-mGpsmUpsmGmGfUmGfGfAfCmUmUmCmUmCmUmCmAmAmU-p-ps2-GalNAc-3′ (SEQ ID NO: 438) and an antisense strand selected among specified modified antisense sequences, wherein GalNAc comprises a structure of Formula (VII) with n=1 and R=OH.
Across the independent claims, HBV treatment is performed by administering siNA where the sense and antisense strands have defined lengths and sequence identity/complementarity or consist of specific SEQ ID numbers, and where the strands include modified nucleotides selected from 2′-O-methyl and 2′-fluoro nucleotides with defined minimum counts or defined positional distributions. The claim set also includes GalNAc-conjugated sequence pairings and additional structural features such as phosphorothioate internucleoside linkages, blunt ends, overhangs, and 5′-stabilized end cap structures.
Stated Advantages
Sustained reduction of serum HBsAg after single dosing in an AAV-HBV mouse model.
Improved potency/durability for ds-siNAs incorporating a deuterated vinyl phosphonate.
HBsAg serum reduction.
HBsAg reduction improved with a 5′-stabilized end cap on the antisense compared to constructs without the cap.
Enhanced HBsAg reduction in combination therapy, including synergy with ds-siNA-0160 plus ASO 1.
Documented Applications
Treating hepatitis B virus (HBV) by administering a double stranded short interfering nucleic acid (siNA) or siNA molecule to a subject with HBV.
In vivo evaluation in an AAV-HBV mouse model, including measurement of serum HBsAg and comparison of ds-siNA formulations after dosing.
In vitro and in vivo evaluation using EC50/CC50 and ELISA of HBsAg, including HepG2.2.15 cells in vitro evaluation context as described in the partial content.
In vivo HBV efficacy evaluation in an AAV-HBV mouse model, including HBsAg serum reduction measured for multiple ds-siNA constructs with defined 2′-fluoro/2′-O-methyl nucleotide patterns and GalNAc conjugation at the 3′ end of the sense strand.
Use of ds-siNA constructs in combination therapy with an ASO (ASO 1), described with synergy analysis and enhanced HBsAg reduction.
Treating hepatitis B virus (HBV) using siNA compositions comprising chemically modified siNA as described, optionally combined with additional HBV agents.
Treating liver diseases including NAFLD, NASH, and hepatocellular carcinoma (HCC) using the described siNA compositions.
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