WO2022189861A1

WO2022189861A1 - Carbohydrate conjugates of tlr3 ligands and uses thereof

Info

Publication number: WO2022189861A1
Application number: PCT/IB2022/000123
Authority: WO
Inventors: Marc Bonnin; Philippe GUILLOT-CHENE; Dominique Rigal
Original assignee: Tollys
Priority date: 2021-03-08
Filing date: 2022-03-08
Publication date: 2022-09-15

Abstract

The present technology relates to conjugates, or pharmaceutically acceptable salts thereof, comprising a carbohydrate covalently linked to a double-stranded RNA. The double-stranded RNA comprised in the conjugate has a sense strand and an antisense strand, wherein the sense strand comprises at least one block of poly A comprising at least 15 A, and the antisense strand comprises at least one complementary block poly U comprising at least 15 U, and each of the sense and antisense strands have a length of between 50 and 200 nucleotides. The present technology further relates to pharmaceutical compositions comprising the conjugates of the present technology for use, and methods of use thereof, for the treatment of subjects having an HBV infection, HBV-associated disorder and/or hepatocellular carcinoma.

Description

CARBOHYDRATE CONJUGATES OF TLR3 LIGANDS AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of and priority to U.S. provisional patent application No. 62/158,065, filed on March 8, 2021; the content of all of which is herein incorporated in entirety by reference.

FIELD OF TECHNOLOGY

[0002] The present technology generally relates to conjugates comprising carbohydrates covalently linked to double-stranded RNAs (dsRNA) and uses thereof.

BACKGROUND INFORMATION

[0003] Hepatitis B virus (HBV) infection is a major global health problem with 2 billion people infected worldwide and 350 million suffering from chronic HBV infection. Among the longer-term complications of HBV infection cirrhosis and hepatocellular carcinoma cause a large disease burden. Particularly, hepatocellular carcinoma (HCC) progresses rapidly, and since treatment options are limited, the outcome is generally poor. Indeed, worldwide, the incidence of HCC has increased and presently constitutes the fifth most common cancer. [0004] Currently treatments of HBV generally aim at permanently suppressing HBV replication and prevent the development of cirrhosis and hepatocellular carcinoma in patients to prolong survival. However, HBV infection cannot be completely eradicated due to persistence of a particular form of viral covalently closed circular DNA (cccDNA) in the nuclei of infected hepatocytes. Furthermore, persistent production of the 3 primary viral proteins HBsAg, HBeAg and HBcAg, which all process immunoinhibitory properties, further prevents eradication of the disease by allowing infectious viral particles to escape immune detection. [0005] Therefore, there is a needed for effective treatment against HBV infection which can inhibit viral replication and restore immunological control in patients.

[0006] TLR3 agonists may be promising candidates for the treatment of HBV. TLR3 is a pattern recognition receptor expressed mostly in endo-lysosomes that appears to be dedicated to the detection of viral infection through the binding of dsRNA. DsRNAs produced by virus either as their genomic material (dsRNA viruses) or as intermediates of their life cycle (ssRNA viruses, DNA viruses) have been shown to activate TLR3.

[0007] Previously, the inventors of the present technology demonstrated that TLR3 agonists such as Poly (I:C)-HMW dsRNAs comprising long strands of inosine poly(I) homopolymer annealed to strands of cytidine poly(C) homopolymer, and having an average size of between about 1.5 kb and about 8 kb, or Riboxxol^®, a perfectly annealed RNA duplex of 50bp, were able to reduce several HBV parameters, including % cccDNA, total intracellular HBV DNA, and HBeAg secretion in HBV infected hepatocytes in vitro. Furthermore, little or no rebound effect was observed after treatment arrest, implying a long-lasting effect. However, these TLR3 agonists, are not suitable for systemic delivery and require the assistance of additional modifications or delivery systems such as nanoparticle technology to protect them from degradation and to specifically deliver them to the liver, and thereby prevent systemic exposure and potential off target and/or adverse effects.

[0008] Therefore, there is a need for TLR3 agonists which can overcome at least some of the above-described drawbacks.

SUMMARY

[0009] From one aspect, the present technology relates to conjugates, or pharmaceutically acceptable salts thereof, comprising a carbohydrate covalently linked to a double-stranded RNA (dsRNA), the double-stranded RNA having a sense strand and an antisense strand. In some instances, the sense strand comprises at least one block of poly A. In some instances, the block of poly A comprises at least 15 A. In some instances, the antisense strand comprises at least one complementary block of poly U. In some instances, the complementary block of poly U comprises at least 15 U. In some instances, each of the sense and antisense strands have a length of between 50 and 200 nucleotides.

[0010] From one aspect, the present technology relates to conjugates, or pharmaceutically acceptable salts thereof, comprising a carbohydrate covalently linked to a double-stranded RNA (dsRNA), the double-stranded RNA having a sense strand and an antisense strand, the sense strand comprises at least one block of poly A comprising at least 15 A. The antisense strand comprises at least one complementary block of poly U comprising at least 15 U. Each of the sense and antisense strands have a length of between 50 and 200 nucleotides.

[0011] In certain embodiments, the sense strand comprises a sequence of formula (I): wherein:

Q is a A or U; b is an integer selected from 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, and 90, and more; a and c may be independently 0 or any integer such that a + b + c = 50 to 200;

P is at least one nucleotide selected from A, U, G, I and C, and combinations thereof; and R is at least one nucleotide selected from A, U, G, I and C; and combinations thereof.

[0012] In other embodiments, the dsRNA comprises a sequence of formula (II):

wherein Y and Z comprise the complementary nucleotides of P and R and are selected from A, U, G, I and C and combinations thereof.

[0013] In certain implementations of any one of the aforementioned embodiments, P and R are independently selected from combinations of at least two different nucleotides, a block a single nucleotide, at least two blocks of different nucleotides, or a mixture of at least one block of a nucleotide and at least one other nucleotide. In other implementations, P and R are a block of a single nucleotide. In further implementations, P and R are identical. In other embodiments, P is I.

[0014] In some embodiments, a and c are equivalent. In other embodiments, a+b+c is equivalent to 70. In yet other embodiments, a=10, b=50 and c=10. In further embodiments, Q is A, P is I, R is I, a is 10, b is 50, c is 10. In yet further embodiments, Y and Z are both C.

[0015] In other embodiments, the dsRNA comprises a sequence of formula (III):

wherein Z comprises the complementary nucleotides of R selected from A, U, G, I and C and combinations thereof.

[0016] In certain embodiments of the above formula, R is selected from random combinations of at least two different nucleotides, a block a single nucleotide, at least two blocks of different nucleotides, or a mixture of at least one block of a nucleotides and at least one other nucleotide. In certain implementations of these embodiments, R is a block of a single nucleotide. In other implementations, R is I.

[0017] In some embodiments, b+c is equivalent to 70. In other embodiments, b is 35 and c is 35. In further embodiments, R is I, b is 35 and c is 35 and Z is C.

[0018] In certain embodiments, the conjugates of the present technology comprise double- stranded RNAs which are TLR3 agonists.

[0019] In some embodiments, the carbohydrate in the conjugate of the present technology is attached to one end of the double-stranded RNA. In certain implementations of these embodiments, the carbohydrate is attached to the 3’ or the 5’ end of the anti-sense strand. In yet other implementations, the carbohydrate is attached to the 5’ end of the anti-sense strand. [0020] In some embodiments, the carbohydrate is a N-acetyl-galactosamine derivative and comprises monovalent N-acetyl-galactosamine units sequentially conjugated to the dsRNA by non-nucleosidic linkers. In other embodiments, the carbohydrate comprises at least three monovalent N-acetylgalactosamine units sequentially conjugated to the dsRNA by non- nucleosidic linkers. In certain implementations of the latter embodiment, the carbohydrate structure is as represented in Formula A, Formula B or Formula C:

Formula C.

[0021] In yet further embodiments, the carbohydrate is an ASPG-R ligand. [0022] Another aspect of the present technology relates to pharmaceutical compositions comprising the conjugates of the present technology, and one or more pharmaceutically acceptable vehicles, carriers or excipients.

[0023] In certain embodiments, the pharmaceutical compositions of the present technology are for use in the treatment of an HBV-associated disease which include hepatitis D virus infection, delta hepatitis, acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; liver fibrosis; end-stage liver disease; and hepatocellular carcinoma.

[0024] In other embodiment, the pharmaceutical compositions of the present technology are for use in the treatment of hepatocellular carcinoma.

[0025] In yet other aspects, the present technology relates to a treat treating a subject having an HBV infection, comprising administering to the subject a therapeutically effective amount of the conjugates or the pharmaceutical compositions of the present technology.

[0026] In yet further aspects, the present technology relates to a method of treating a subj ect having hepatocellular carcinoma, comprising administering to the subject a therapeutically effective amount of the conjugates or the pharmaceutical compositions of present technology. [0027] Other aspects and features of the present disclosure will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0028] All features of embodiments which are described in this disclosure are not mutually exclusive and can be combined with one another. For example, elements of one embodiment can be utilized in the other embodiments without further mention. A detailed description of specific embodiments is provided herein below with reference to the accompanying drawings in which:

[0029] FIG.1A & IB illustrate conjugate profiles on native acrylamide gels in which 200 ng of conjugated and unconjugated dsRNAs according to certain embodiments of the present technology were loaded on 6% PAGE (TBE IX) and stained with ethidium bromide.

[0030] FIG. 2 illustrates antiviral activity of conjugated and unconjugated dsRNAs according to certain embodiments of the present technology as measured by HBeAg ELISA in an in vitro model of HBV-infected primary human hepatocytes.

[0031] FIG. 3 illustrates antiviral activity of the conjugated and unconjugated dsRNAs of FIG. 2 as measured by HBsAg ELISA.

[0032] FIG. 4 illustrates antiviral activity of the conjugated and unconjugated dsRNAs of FIG. 2 as measured by Q-PCR of secreted HBV virions. [0033] FIG. 5 illustrates cell viability following treatment with the conjugated and unconjugated dsRNAs of FIG. 2 as assessed by Red Neutral staining.

[0034] FIG. 6 illustrates cell viability following treatment with the conjugated and unconjugated dsRNAs of FIG. 2 as assessed by Sulforhodamine staining.

[0035] FIG. 7 illustrates secretion of human Interferon gamma-induced protein 10 (hIPIO) following treatment with the conjugated and unconjugated dsRNAs of FIG. 2 as assessed by hIP 10 ELISA.

[0036] FIG. 8 illustrates secretion of human Interleukin 6 (hIL-6) following treatment with the conjugated and unconjugated dsRNAs of FIG. 2 as assessed by hIL-6 ELISA.

[0037] FIG. 9A illustrates antiviral activity of conjugated and unconjugated dsRNAs according to certain embodiments of the present technology as measured by HBeAg and HBsAg ELISA, as well as Q-PCR of secreted HBV virions in an in vitro model of HBV- infected primary human hepatocytes. Cytotoxicity of the dsRNAs in these experiments was assessed by Red Neutral and Sulforhodamine staining. FIG. 9B illustrates secretion of hIP10 and hIL-6 as assessed by hIP10 and IL-6 ELISA following treatment with the conjugated and unconjugated dsRNAs of FIG. 9 A.

[0038] FIG. 10 illustrates Flow Cytometry graphs showing expression of the ASGP- receptor (ASPGR) in HepG2 wt hepatoma cells compared to the NCI-H292 wt lung cancer cells.

[0039] FIG. 11 illustrates comparisons of the apoptotic activity of conjugated and unconjugated dsRNAs of FIG. 2 in NCI-H292 wt lung cancer cells, which is a non-hepatocyte in vitro cell model.

[0040] FIG. 12 illustrates comparisons of the cell viability reduction effects of the conjugated and unconjugated dsRNAs of FIG. 11.

DETAILED DESCRIPTION

[0041] The present technology is explained in greater detail below. This description is not intended to be a detailed catalog of all the different ways in which the technology may be implemented, or all the features that may be added to the instant technology. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure which variations and additions do not depart from the present technology. Hence, the following description is intended to illustrate some particular embodiments of the technology, and not to exhaustively specify all permutations, combinations and variations thereof.

[0042] The use of “including”, “comprising”, or “having”, “containing”, “involving” and variations thereof herein, is meant to encompass the items listed thereafter as well as, optionally, additional items. In the following description, the same numerical references refer to similar elements.

[0043] It must be noted that, as used in this specification and the appended claims, the singular form “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0044] As used herein, the term “about” in the context of a given value or range refers to a value or range that is within 20%, preferably within 10%, and more preferably within 5% of the given value or range.

[0045] As used herein, the term “and/or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

[0046] The recitation herein of numerical ranges by endpoints is intended to include all numbers subsumed within that range (e.g., a recitation of 1 to 5 includes 1, 1.25, 1.33, 1.5, 2, 2.75, 3, 3.80, 4, 4.32, and 5).

[0047] As used herein, the expression "double-stranded" means a portion where ribonucleotides are hydrogen bonded (base-paired) to complementary ribonucleotides to form a double-stranded structure by canonical Watson-Crick or non-canonical Hoogsteen base pairs. One may speak of overlap where both strands are paired. In certain embodiments, the entire strands are paired (100% of the complementary strands are paired). However, the invention encompasses dsRNA having at least about 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or 99.5% of strand length paired (double-stranded conformation). In a same composition it is possible to have dsRNA of varying % of pairing. The determination of what percentage of the dsRNA is in a double-stranded conformation is achieved by dividing the number of nucleotides that are base-paired by the total number of nucleotides in a molecule. Thus, a 21 base-paired molecule containing 2 nucleotide overhangs at both the 3' and 5' end would have 42 nucleotides that are base-paired and 4 nucleotides that are not base-paired, making it 42/46 or 91 .3% double-stranded or paired.

[0048] "TLR3", "TLR3 protein" and "TLR3 receptor", used interchangeably, are used herein to refer to Toll Like Receptor 3, a member of the Toll-like receptor (TLRs) family. Its amino acid sequence of is shown in NCBI gene ID 7098. Toll Like Receptor 3 is a member of the Toll-like receptor (TLR) family which plays a fundamental role in pathogen recognition and activation of innate immunity. This receptor is expressed by epithelial and endothelial cells, and is restricted to some immune cells population such as dendritic cells and macrophages. It recognizes dsRNA associated with viral infection and induces the activation of NF-kB and IRF3 leading to the production of type I interferons and other inflammatory cytokines.

[0049] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.

[0050] "G", "C", "A", "T", "U" and “I” each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine, uracil and inosine as a base, respectively. However, it will be understood that the term "ribonucleotide" or "nucleotide" can also refer to a modified nucleotide, or a surrogate replacement moiety known in the arts.

[0051] As used herein, "carbohydrate" refers to a compound which is either a carbohydrate per se made up of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which can be linear, branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. [0052] As used herein, a "pharmaceutically acceptable excipient" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type and are further described below. [0053] As used herein, "Hepatitis B virus," used interchangeably with the term "HBV" refers to the well-known noncytopathic, liver-tropic DNA virus belonging to the Hepadnaviridae family.

[0054] As used herein, the term "subject" or “patient” refers to a warm-blooded animal such as a mammal, animal or human, in particular a human, who is afflicted with, or has the potential to be afflicted with one or more diseases and conditions described herein. The identification of subjects who are in need of treatment for the herein-described diseases and conditions is well within the ability and knowledge of one skilled in the art. A clinician skilled in the art can readily identify, by the use of clinical tests, physical examination and medical/family history, those subjects who are in need of such treatment. [0055] The terms "treat", “treating”, “treated” or "treatment", as used herein, refer to therapeutic treatment wherein the object is to eliminate or lessen symptoms. Beneficial or desired clinical results include, but are not limited to, elimination of symptoms, alleviation of symptoms, diminishment of extent of condition, stabilized (i.e., not worsening) state of condition, delay or slowing of progression of the condition, to the prevention of the onset, recurrence or spread of a disease or disorder, or of one or more symptoms thereof. In certain embodiments, the terms refer to the treatment with administration of a compound provided herein prior to the onset of symptoms. The terms encompass the inhibition or reduction of a symptom of the particular disease. Subjects with familial history of a disease in particular are candidates for treatment regimens in certain embodiments. Also, subjects in whom a genetic disposition for the particular disease has been shown are candidates for treatment regimens in certain embodiments. In addition, subjects who have a history of recurring symptoms are also potential candidates for the treatment. The term “treatment” also includes “prophylactic treatment”.

[0056] The present disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The disclosure is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

[0057] The present technology stems from the elucidation by the present inventors of conjugates comprising carbohydrates covalently linked to double-stranded RNAs (dsRNA) which activate the TLR3 pathway in the liver and have anti-HBV and anti-cancer effects. The present inventors had previously screened a series of synthetic dsRNAs with unique base compositions and lengths for their capacity to activate TLR3 signaling which they demonstrated induced inflammatory responses in RAW mouse macrophage cells lines and cancer cells, and further resulted in cancer cell death.

The present inventors have now developed conjugates of these dsRNAs (Table 1) covalently linked to a carbohydrate in order to target the dsRNAs to the liver. More specifically, the inventors tested the sequential conjugation of three monovalent N-acetylgalactosamine units (also known as (1+1+1) GalNac) to the dsRNAs of the present technology by non-nucleosidic linkers.

[0058] To date, the only nucleic acids coupled to GalNac are antisense oligonucleotides (ASOs). DsRNA, including the TLR3 agonists described herein, and more particularly those having blunt ends at both ends, have never been modified with GalNac, and have never been tested for their effect on primary hepatocytes and cancer cells.

[0059] The present inventors have demonstrated the ability of conjugates of the present technology to result in anti-HBV and anti-cancer effects.

[0060] In some embodiments, the present technology thus relates to conjugates comprising a carbohydrate covalently linked to a double-stranded RNA having a sense strand and an antisense strand, wherein the sense strand comprises at least one block of poly A comprising at least 15 A, and the antisense strand comprises at least one complementary block of poly U comprising at least 15 U, and wherein each of the sense and antisense strands have a length of between 50 and 200 nucleotides. In some implementations of these embodiments the carbohydrate is a GalNac covalently linked to a double-stranded RNA. In some implementations the double-stranded RNA is about 70bp in length and having the A:U-rich chimeric sequences.

[0061] Other examples of the conjugates of the present technology include conjugates comprising carbohydrates covalently linked to double-stranded RNAs (dsRNA) disclosed in WO2019/211492, incorporated herein by reference.

Conjugates of the present technology

[0062] In one embodiment, the present technology relates to a conjugate, or pharmaceutically acceptable salt thereof, comprising a carbohydrate covalently linked to a double-stranded RNA. i) Sequence of the dsRNAs

[0063] The double-stranded RNA has a sense strand and an antisense strand, wherein the sense strand comprises at least one block of poly A comprising at least 15 A, and the antisense strand comprises at least one complementary block of poly U comprising at least 15 U. In some implementations, each block of poly A or poly U may comprise at least 20, 25 or 30 A, or U. In some embodiments, each of the sense and antisense strands have a length of between about 50 and about 200 nucleotides. In other embodiments, each of the sense and antisense strands have a length of between about 55 and about 100, about 55 and about 150, or about 55 and about 200 nucleotides. In certain implementations, each of the sense and antisense strands have a length of about 60, about 70, about 80, about 90 or about 100 nucleotides. Advantageously, all the strands or substantially all the strands in the conjugate have the same length.

[0064] In some embodiments, the strands of the dsRNAs are synthetic. Synthesis is the privileged mode of production of the dsRNA strands of short and determined length. Standard synthesis methods, such as chemical synthesis on a solid phase, in vitro enzymatic synthesis and the like may be used to produce the dsRNAs comprised in the conjugates of the present technology. In some embodiments, the dsRNAs suitable for the conjugates of the present technology are synthesized using solid phase RNA synthesis methods which include, but are not limited to, methods using 2'-0-tert-butyldimethylsilyl, 2-O-triisopropylsilyloxymethyl, 2'- bis(2-Acetoxyethoxy)methyl, and 2'-Thiomorpholine-4-carbothioate protecting groups. In certain implementations of these embodiments, the dsRNAs suitable for the conjugates of the present technology are synthesized by solid phase synthesis using the 2'-0-tert- butyldimethylsilyl method. Methods such as electrophoresis on gel, such as the acrylamide gel used in Example 1 below, may be used to check purity, and the length of the strands. [0065] Methods of synthesis may advantageously allow to produce the short and defined dsRNA strands in the conjugate of the present technology. Advantageously, these methods allow one to produce dsRNA strands having the exact length wished and result in conjugates wherein all the strands are of the same length and/or wherein all the dsRNA are made of strands having the same length. However, a certain and limited variation of the length of some strands may be acceptable. Such variations are acceptable and encompassed in the present technology so long as they do not substantially change the function or the efficacy of the conjugate. As such, in certain embodiments, the conjugate comprises variant dsRNAs which still activate the TLR3 pathway, have anti-HVB effects and induce cell death at substantially the same level than the conjugates wherein all the strands are of the same length and/or wherein all the dsRNA are made of strands having the same length. In certain embodiments, the dsRNAs comprised in the conjugates of the present technology comprise a significant and efficient proportion, equal to or above 95, 96, 97, 98, 99, or 99.5, or of about 100%, of strands having the determined length.

[0066] In certain embodiments, the poly A and the poly U blocks may be combined with one or more bases among A, U, G, I, C, poly A, poly U, poly G, poly I or poly C, and the complementary nucleotides or blocks. Exemplary structures for the sense strand include: poly A - poly I, poly A - poly C, poly I - poly A, poly C - Poly A, poly I - poly A - poly I, poly C- poly A- poly C, poly A - poly I - poly A, poly A - poly C - Poly A.

[0067] Poly A/I or A/C and a complementary strand made of blocks of Poly U/C or U/I are thus encompassed. They may also be designated as poly A/I: poly U/C or Poly A/C: Poly U/I. In some embodiments, a poly A/I or poly U/C strand may have a predetermined length of between about 50 to about 200 nucleotides. In other embodiments, the length of said strand is between about 55 and about 200 nucleotides. In yet other embodiments, the length of said strand is between about 60 and about 100 bases. [0068] In certain embodiments, the block of Poly A may comprise a homopolymer of polyA. By homopolymer it is meant a sequence of at least two identical and contiguous bases. In other embodiments, the sense or antisense strands may comprise one or more blocks of A or U comprising at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or 90 A, or U; such block may optionally contain less than 20, 15, 14, 13, 12, 1 1 , 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 % of another base among A, U, G, I and C. In yet other embodiments, the sense or antisense strands may comprise one block of A or U comprising at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or 90 A, or U. In further embodiments, the sense or antisense strands may comprise two blocks of A or U comprising at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or 90 A, or U.

[0069] In certain embodiment, the conjugates of the present technology comprise dsRNAs of a length of between 50 and about 200 nucleotides. In other embodiments, the dsRNA may be of a length of between about 55 to about 100, or about 150 to about 200 nucleotides. In other embodiments, the dsRNA is between about 60 and about 120 nucleotides. In yet other embodiments, the dsRNA is of a length of between about 70 and about 100 nucleotides. In certain implementations of this embodiment, the dsRNA may be of a length of about 60, about 70, about 80, or about 90 nucleotides.

[0070] In certain embodiments, the conjugate of the present technology comprises dsRNAs having a sense strand which comprise a sequence of formula (I)

(I) [P]a [Q]b [R]c wherein Q is a A or U; b is an integer selected from 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, and 90, and more; a and c may be independently 0 or any integer such that a + b + c = 50 to 200;

P is at least one nucleotide selected from A, U, G, I and C, and combinations thereof; and

R is at least one nucleotide selected from A, U, G, I and C; and combinations thereof a, b and c represent a number of nucleotides, so a+b+c represents the length of the strand or the dsRNA.

[0071] In certain embodiments, a +b+c may be between about 60 and 120 or between about 70 and about 100. In other embodiments, a+b+c is 70.

[0072] In certain embodiments, a, b, and c, may be equal. In other embodiments, a, b, and c, may be different. [0073] In certain embodiments, a and c are independently selected from an integer between 5 and 50, between 5 and 45, between5 and 40, between 5 and 35, between 5 and 30, between 5 and 25, between 5 and 20, between 5 and 15, between 5 and 10, between 10 and 50, between 10 and 45, between 10 and 40, between 10 and 35, between 10 and 30, between 10 and 25, between 10 and 20, between 10 and 15, between 15 and 50, between 15 and 45, between 15 and 40, between 15 and 35, between 15 and 30, between 15 and 25, between 15 and 20, between 20 and 50, between 20 and 45, between 20 and 40, between 20 and 35, between 20 and 30, between 20 and 25, between 25 and 50, between 52 and 45, between 25 and 40, between 25 and 35, between 25 and 30, between 30 and 50, between 30 and 45, between 30 and 40, between 30 and 35, between 35 and 50, between 35 and 45, between 35 and 40, between 40 and 50, between 40 and 45, or between 45 and 50. In certain embodiments, a and c may selected to be different from one another. In other embodiments, a and c may be equal. In one embodiment a and c are both 10.

[0074] In certain embodiments, wherein a = 1, and Q = A, P may be made of one base among U, G, I and C; as a result of complementarity, in case Q = U, P is made of one base among A, G, I and C. In other embodiments, wherein a > 1 , P is made of at least one or two bases among A, U, G, I and C, under one of these configurations: random combination of at least two of these bases, one block of a base among A, U, G, I and C, at least two blocks of different bases among A, U, G, I and C, or a mixture of at least one block of base among A, U, G, I and C and at least one other base among A, U, G, I and C.

[0075] In certain embodiments, wherein c = 1, and Q = A, R is made of one base among U, G, I and C; as a result of complementarity, in case Q = U, R is made of one base among A, G, I and C. In other embodiments, wherein if c > 1 , R is made of at least one or two bases among A, U, G, I and C, under one of these configurations: random combination of at least two of these bases, one block of a base among A, U, G, I and C, at least two blocks of different bases among A, U, G, I and C, or a mixture of at least one block of base among A, U, G, I and C and at least one other base among A, U, G, I and C.

[0076] In certain implementations of these embodiments, P and R may be selected to be identical. In other implementations, P and R may be selected to be different. In certain embodiments, a and c may be equal, meaning that P and R may be of the same length. In other embodiments, a and c may be different; meaning that P and R may have different lengths. In one embodiment, P and R are both a block of single nucleotides comprised of I and are 10 nucleotides in length (i.e., a=c=10). [0077] In certain embodiments, b is an integer of at least 10. In other embodiments, b may be between about 10 and about 100, between about 10 and about 90, between about 10 and about 80, between about 10 and about 70, between about 10 and about 60, between about 10 and about 50, between about 10 and about 40, between about 10 and about 30, between about 10 and about 20, between about 20 and about 100, between about 20 and about 90, between about 20 and about 80, between about 20 and about 70, between about 20 and about 60, between about 20 and about 50, between about 20 and about 40, between about 20 and about 30, between about 30 and about 100, between about 30 and about 90, between about 30 and about 80, between about 30 and about 70, between about 30 and about 60, between 30 about and about 50, between about 30 and about 40, between about 40 and about 100, between about 40 and about 90, between about 40 and about 80, between about 40 and about 70, between about 40 and about 60, between about 40 and about 50, between about 50 and about 100, between about 50 and about 90, between about 50 and about 80, between about 50 and about 70, between about 50 and about 60, between about 60 and about 100, between about 60 and about 90, between about 60 and about 80, between about 60 and about 70, between about 70 and about 100, between about 70 and about 90, between about 70 and about 80, between about 80 and about 100, between about 80 and about 90, or between about 90 and about 100 nucleotides. In one embodiment b is 50 nucleotides. In another embodiment, b is 35 nucleotides, or is 40 nucleotides, or is 50 nucleotides, or is 60 nucleotides, or is 70 nucleotides, or is 75 nucleotides, or is 80 nucleotides, or is 90 nucleotides, or is 100 nucleotides, or is 110 nucleotides, or is 120 nucleotides, or is 125 nucleotides, or is 130 nucleotides, or is 140 nucleotides or is 150 nucleotides.

[0078] In certain embodiments, a + b + c is at least 50, or at least 55, or at least 60, or at least 65, or at least 70, or at least 75, or at least 80, or at least 85, or at least 90, or at least 95, or at least 100. In some embodiments, a+ b + c is between 70 and 150. In some embodiments, a+ b + c is 75, or 80, or 90, or 100, or 110, or 120, or 125, or 130, or 135, or 140, or 144, or 150

[0079] In other embodiments, any one of Q, P and R may be selected from modified nucleotide of A, U, I, G, C, such as the O-methylated nucleotides, the phosphorothioate nucleotides, and may include others modifications including 2’ribose substitutions, ribose modifications, bridge nucleic acids modifications, and the like.

[0080] In certain embodiments, the conjugate of the present technology comprises dsRNAs having a sense strand which comprise a sequence of formula (I(a))

(I(a)) [P]a [Q]b [R]c wherein Q is a A; b is an integer selected from 30 to 60, particularly 35 to 55, more particularly 50;

P and R are I and a and c are equal and are an integer selected from 1 to 20, particularly 5 to 15, more particularly 10.

[0081] In a certain embodiment, the conjugates of the present technology comprise dsRNAs comprising a sequence of Formula (II):

(II) [P]a [A]b [R]c [Y]a [U]b [Z]c wherein a, b, c, P and R have the same meaning as in formula (I), described above, and Y and Z comprise the complementary nucleotides of P and R and are selected from A, U, G, I and C and combinations thereof. In other embodiments, the conjugates of the present technology comprise dsRNAs comprising the sequence of Formula (II), wherein P and R are independently chosen among I and C, Y and Z are the complementary bases, and b is an integer selected from 20, 25, 30, 50 and 100. In further embodiments, one of a or c may be 0. In yet further embodiments, where a and c are not equal to 0 at the same time, a and c may be between about 10 and about 50.

[0082] In certain embodiments, the conjugates of the present technology comprise a double-stranded RNA (dsRNA) having two complementary strands, wherein the dsRNA comprises at least one block or homopolymer of poly A and the complementary block or homopolymer of poly U, each block comprising at least 15 A, or U, and each strand having a determined length of between 50 and 200 bases, the dsRNA being of formula (III):

[P]a [A]b [R]c [Y]a [U]b [Z]c, wherein P and R are independently chosen among I and C, and Y and Z are the complementary bases, b is an integer between 20 and 100, and a and c independently are between 5 and 50.

[0083] In a certain embodiment, the conjugates of the present technology comprise dsRNAs comprising a sequence of Formula (II) wherein:

P and R are I and Y and Z are C b is an integer selected from 30 to 60, particularly 35 to 55, more particularly 50; and a and c are equal and are an integer selected from 1 to 20, particularly 5 to 15, more particularly 10.

[0084] Other dsRNAs suitable for the conjugates of the present technology include, dsRNAs comprising the sequences of Formula (IV)

(IV) [A]b

[U]b wherein b has the same meaning as in formula (I), described above; or Formula (V)

(V) [P]a [A]b [Y]a [U]b wherein a and b have the same meaning as in formula (I) and P is chosen among U, G, I and/or C, and Y comprises the complementary nucleotides of P.

[0085] Therefore, by way of example, in embodiments wherein P and R are selected as a block of a single nucleotide, Y and Z would also be blocks of a single nucleotide comprising the complementary nucleotides selected for P and R.

[0086] As such, in certain embodiments where P and R are blocks of I, for example, Y and Z would be blocks of C.

[0087] In other embodiments, P and R are independently selected from G, I and C and combinations thereof. In one embodiment, P is I, R is I, a is 10, b is 50, c is 10 and Y and Z are both C.

[0088] In other embodiments, a may be 0 and thus the conjugates of the present technology may comprise dsRNAs comprising a sequence of Formula (III):

(III) [A]b [R]c

[U]b [Z]c wherein b, c, and R have the same meaning as in formula (I), described above, and Z comprises the complementary nucleotides of R selected from A, U, G, I and C and combinations thereof.

[0089] In certain embodiments, in the sequence of Formula (III) R is I and Z is C.

[0090] In other particular embodiments, the conjugates of the present technology may comprise dsRNAs comprising a sequence of Formula (III) disclosed above wherein b and c are integers independently selected from 20 to 60, particularly 30 to 50, more particularly 25 to 40. [0091] In yet other embodiments, R is selected from G, I and C and combinations thereof. In another embodiment, R is I, b is 35, c is 35 and Z is C.

[0092] In some embodiments of formulae (II), (III), (IV), (V), (VI), the block or homopolymer of poly A or the blocks or homopolymers of poly A may contain less than 20, 15, 14, 13, 12, 1 1 , 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 % of another base among U, G, I and C and the complementary block(s) of U comprise the complementary bases. [0093] In one embodiment, the conjugate comprises dsRNA having a poly A/I sense strand and a poly antisense U/C strand wherein both strands have a length of more than 50 to about 200 nucleotides or more than about 55 and about 200 nucleotides. In other embodiments, the sense and antisense strands have a length of between about 60 and about 100 nucleotides. For example, each strand has a length of about 60, about 70, about 80, about 90 or about 100 nucleotides.

[0094] In certain embodiments, the conjugate of the present technology comprises a dsRNA having more than 95, 96, 97, 98, 99 or 99.5% pairing or fully (about 100%) paired sense and antisense strands. For example, the dsRNA comprises a Poly A/I strand and a Poly U/C strand, wherein both strands have the same length and the sense and antisense strands are fully paired (ie there are no overhangs at the 3’ nor the 5’ end of the dsRNA). ii) TLR3 agonization

[0095] In some embodiments, the conjugate of the present technology comprises dsRNAs disclosed above which serve as agonists of the TLR3 receptor. The dsRNAs suitable for the conjugate of the present technology, serving as agonists of the TLR3 receptor, have been previously described in WO2019211492, US2020/0080084, EP2235177B1,

W02009150156A1, EP3083962A1, US10023871, WO2015091578A, EP2401375B1,

WO20 10097414A1, EP2773760B1, WO2013064584A1, EP3126501 Al, US10273484, WO2015144736A1, WO2016112963A1, WO2013097965A1, and WO2012038448A1, all of which are incorporated herein by reference.

[0096] These dsRNAs have been previously tested and screened for their capacity to agonize the TLR3 receptor and to activate TLR3 expressed by myeloid cells (macrophages and dendritic cells). Moreover, agonization of TLR3 by these dsRNAs induced the secretion of inflammatory cytokine and triggered the TLR3-dependent activation of inflammation and cell death in human or other mammal cancer cells. As such, in certain embodiments, the conjugates of the present technology, may also trigger TLR3-dependent activation of inflammation and cell death in human or other mammal cancer cells. In further embodiments, the conjugates of the present technology may trigger TLR3-dependent activation of inflammation and cell death in any cancer expressing the TLR3 receptor. Examples of such cancers include but are not limited to, breast, colon, ovarian, lung, brain and prostate cancers and melanoma.

[0097] In certain embodiments, the conjugate of the present technology comprises a dsRNA which serve as agonists of the TLR3 receptor which sense sequence is selected from: 5’ (1)10 - (U)50 - (1)10 3’

5’ (1)10 - (A)50 - (1)10 3’ 5’ (1)5 - (A)60 - (1)5 3’

5’ (1)15 - (A)40 - (1)15 3’

5’ (1)20 - (A)30 - (1)20 3’

5’ (1)25 - (A)20 - (1)25 3’

5’ (1)5 - (A)50 - (1)15 3’

5’ (1)13- (A)64 - (1)13 3’

5’ (1)10 - (A)70 - (1)10 3’

5’ (1)30 - (A)10 - (1)30 3’

5’ (A) 10 - (1)60 3’

5’ (A)60 - (1)10 3’

5’ (A)50 - (1)20 3’

5’ (A)20 - (1)50 3’ and 5’(A)35 - (1)35 3’.

[0098] In certain embodiments, the conjugate of the present technology comprises a dsRNA which serve as agonists of the TLR3 receptor which sense sequence is selected from:

5’ (1)10 - (U)50 - (1)10 3’

5’ (1)10 - (A)50 - (1)10 3’

5’ (1)5 - (A)60 - (1)5 3’

5’ (1)15 - (A)40 - (1)15 3’

5’ (1)20 - (A)30 - (1)20 3’

5’ (1)25 - (A)20 - (1)25 3’

5’ (1)5 - (A)50 - (1)15 3’

5’ (1)13 - (A)64 - (1)13 3’; and 5’ (1)10 - (A)70 - (1)10 3’.

[0099] In a particular embodiment, the conjugate of the present technology comprises a dsRNA which serve as agonists of the TLR3 receptor which sense sequence is selected among the group consisting of

5’ (1)10 - (A)50 - (1)10 3’; and 5’(A)35 - (1)35 3’.

[00100] In other embodiments, the conjugates of the present technology target the TLR3 receptor in the liver. Hepatocytes are known to express the TLR3 receptor. Activation of the TLR3 receptor in hepatocytes results in the secretion of IL-6, IP- 10 and type I interferon (IFN) following agonization (Luangsay, S. et al. Expression and functionality of Toll- and RIG-like receptors in HepaRG cells. J Hepatol 63, 1077-1085 and Lucifora J. & Bonnin M. etal, Direct antiviral properties of TLR ligands against HBV replication in immune-competent hepatocytes. Scientific Reports 8, 5390 (2018), both incorporated herein by reference).

[00101] In further embodiments, the conjugates of the present technology have anti -HBV effects in the liver. Activation of TLR3 with Poly (I:C)-HMW agonists has been previously shown to decrease total intracellular HBV DNA, and reduce HBeAg and HBsAg secretion in both HBV-infected dHepaRG cells and primary human hepatocytes (PHH) without significant toxicity to the cells (Lucifora J. & Bonnin M. et al, Direct antiviral properties of TLR ligands against HBV replication in immune-competent hepatocytes. Scientific Reports 8, 5390 (2018), incorporated herein by reference).

[00102] In some embodiments, the present technology demonstrates that conjugates comprising dsRNAs having sequences as described above, and serving as agonists for TLR3, reduce HBeAg secretion and have anti-HBV effects in HBV infected primary human hepatocytes.

[00103] In further embodiments, the conjugates of the present technology target the TLR3 receptor and activate immune cells. In general, the benefit of targeting TLR3 in various diseases is thought to ensue from both a direct effect on the target cells, as well as an indirect effect on immunity. Toll-like receptors are part of a large family of patter recognition receptors (PRR) which detect pathogen-associated molecular patters derived from viruses, bacteria, mycobacteria, fungi or parasites and generally activate downstream signaling events leading to specific gene expression programs and the secretion of interferons (IFN), inflammatory cytokines/chemokines, and other antimicrobial peptides, as part of the host innate immune response. Without being bound by theory, activation of the TRL3 receptor in the immune cells by the conjugates of the present technology is thought to results in an indirect anti-HBV effect, wherein the IFN and pro-inflammatory cytokines produced following TLR3 activation in the immune cells contributes in the mounting of the host innate immune response against HBV. Many IFNs and pro-inflammatory cytokines have been shown to have anti-HBV effect in hepatocytes. In particular, TLR7 and TLR9 ligands have been extensively used to induce endogenous IFNs and other cytokines and have been demonstrated to have anti-HBV benefits in in vivo models of the disease. In other embodiments, activation of the TRL3 receptor in the immune cells may also indirectly result in anti-cancer effects. iii) Carbohydrates

[00104] The conjugates of the present technology comprise at least one carbohydrate covalently linked to the dsRNA. In certain embodiments, the at least one carbohydrate is linked to the dsRNA through a linker. [00105] In certain embodiments, the carbohydrate enhances the distribution, targeting and/or lifetime of the dsRNA to which it is conjugated. In some embodiments, the carbohydrate of the present technology provides enhanced affinity for a selected target such as a molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Representative carbohydrates include the sugars (mono-, di-, tri- and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starches, glycogen, cellulose and polysaccharide gums. Further examples of carbohydrates include those carbohydrates that will not interfere with the activity of the dsRNA comprised in the conjugate of the present technology.

[00106] In certain embodiments, the carbohydrate in the conjugate of the present technology serves to target the dsRNA to a cell type. In such embodiments, the carbohydrates suitable for the conjugate of the present technology include any carbohydrate capable of targeting a specific cell surface receptor and include N-acetylgalactosamines (GalNAcs) and derivatives thereof, galactose, mannose, mannose-6p and clusters thereof, and the like. Advantageously, covalent linkage of the carbohydrate in the conjugate of the present technology overcomes the need for transfection agents, or additional conjugation to other lipophilic ligand groups to enhance delivery and cellular uptake. Furthermore, specific targeting of the conjugate of the present technology by covalent linkage of a carbohydrate to the dsRNA, advantageously decreases non-specific off-target effects.

[00107] In one embodiment, the carbohydrate comprised in the conjugate of the present technology is a monosaccharide, such as a GalNAcs or any derivative thereof. Advantageously, GalNAcs serve as Asialoglycoprotein receptor (ASGP - R) ligands. The ASGP-R is a high capacity receptor, which is highly abundant on hepatocytes and displays rapid internalization and turnover. Furthermore, the ASGP-R show a 50-fold higher affinity for GalNAc rather than D-Gal. Therefore, in such embodiments, GalNAcs serves to target the conjugate to the liver. [00108] In certain embodiments, the carbohydrate linked to the dsRNA may be represented by the chemical moiety of Formula (VI)

(VI) (CH)x - Ly - wherein

L is a linker as defined above,

CH is a carbohydrate as defined above, particularly GalNAc derivatives x is an integer selected from 1, 2, 3 and 4, and y is 0 or 1. [00109] In certain embodiments, the GalNAc derivatives may be attached through a monovalent, bivalent or trivalent branched linker. In other embodiments, the GalNAc derivative may be a triantennary GalNAc. It is established that multivalency of GalNAc is required to achieve nM affinity for the ASGP-R, while spacing among sugars is also crucial. A widely accepted model for ASGP-R binding proposes a triantennary scaffold with precise distances between the sugar moieties and the linker scaffold as the optimal ligand.

[00110] The GalNac derivatives and the chemical moiety of Formula (VI) comprising GalNac derivatives as carbohydrates suitable for the conjugate of the present technology, have been previously described in WO 2014/179620, WO 2015/006740, WO 2016/077321, WO 2016/055601, WO 2017/178656, WO 2018/044350 andUS10,233,448 (all incorporated herein by reference).

[00111] In other embodiments, the GalNAc derivative may be monovalent N-acetyl- galactosamine units sequentially conjugated to the dsRNA by non-nucleosidic linkers.

[00112] In another embodiment, the GalNAc derivative comprises three monovalent N- acetylgalactosamine units sequentially conjugated to the dsRNA by non-nucleosidic linkers. GalNAc derivatives having these configurations are also known in the art as (1+1+1) trivalent GalNAc. In such embodiments, the structure of the GalNAc derivative is as represented in one of formula A, Formula B or Formula C:

Formula A;

Formula C. [00113] In a particular embodiment, the GalNAc derivative is the derivative of Formula A.

[00114] In a particular embodiment, the GalNAc derivative is the derivative of Formula B.

[00115] In a particular embodiment, the GalNAc derivative is the derivative of Formula C.

[00116] Advantageously, synthesis of (1+1+1) trivalent GalNAc conjugated oligonucleotides compared to triantennary GalNAc conjugated oligonucleotides requires fewer steps but retains optimal valency, spatial orientation and distance between the sugar moieties for proper recognition by ASGPR.

[00117] In some embodiments of the conjugate of the present technology, the carbohydrate may be covalently linked to the 5’ or the 3’ end of the sense or antisense strand of the dsRNA. In certain implementations, the carbohydrate is covalently linked to the 3’ or the 5’ end of the antisense strand. In one embodiment, the carbohydrate is covalently linked to the 5’ end of the antisense strand.

[00118] In some embodiments of the conjugate of the present technology, the carbohydrate may be covalently linked to the 5’ or the 3’ end of the sense or antisense strand of the dsRNA as disclosed above, wherein the 5’ or 3’ end to which it is linked is a C. This means that in a particular embodiment, the dsRNA of the conjugate is a dsRNA of formula (I), (II), (III) and (V), wherein at least one of P, R, Y or Z is C. [00119] In certain embodiments, the conjugate of the present technology comprises a (1+1+1) trivalent GalNAc, particularly of Formula A, Formula B or Formula C, covalently linked to the 3’ end of antisense strand of the dsRNA. In other embodiments, the conjugate of the present technology comprises a (1+1+1) trivalent GalNAc covalently linked to the 5’ end of antisense strand of the dsRNA. Advantageously, the inventors have discovered that 5’ conjugated (1+1+1) trivalent GalNAc on the antisense strand of certain dsRNAs have the greatest potency in inducing an anti-HBV effect without toxicity compared to 3’ conjugated (1+1+1) trivalent GalNAc or the unconjugated dsRNA counterparts.

[00120] In certain embodiments, the conjugates of the present technology is a conjugate of Formula (VII)

(VII) SdsRNA

[(CH)x-Ly]z - ASdsRNA - [Ly-(CH)x]w wherein

SdsRNA is the sense sequence of the dsRNA defined above and particularly the dsRNA of one of formula (I), (II), (III), (IV) and (V),

ASdsRNA is the antisense sequence of the dsRNA defined above and particularly the dsRNA of one of formula (I) to (V),

L is a linker as defined above, and CH is a carbohydrate as defined above, x and y are defined above, and z and w are 0 or 1 with the proviso that when z + w = 1.

[00121] In certain embodiments, the conjugate of the present technology is a conjugate of Formula (VII) wherein z is 0.

[00122] In certain embodiments, the conjugate of the present technology is a conjugate of Formula (VII) wherein SdsRNA is selected among the group consisting of 5’ (1)10 - (U)50 - (1)10 3’

5’ (1)10 - (A)50 - (1)10 3’

5’ (1)5 - (A)60 - (1)5 3’

5’ (1)15 - (A)40 - (1)15 3’

5’ (1)20 - (A)30 - (1)20 3’

5’ (1)25 - (A)20 - (1)25 3’

5’ (1)5 - (A)50 - (1)15 3’

5’ (1)13— (A)64 - (1)13 3’

5’ (1)10 - (A)70 - (1)10 3’ 5’ (1)30 - (A)10 - (1)30 3’

5’ (A) 10 - (1)60 3’

5’ (A)60 - (1)10 3’

5’ (A)50 - (1)20 3’

5’ (A)20 - (1)50 3’ and 5’(A)35 - (1)35 3’.

[00123] In a particular embodiment, the conjugate of the present technology is a conjugate of Formula (VII) wherein SdsRNA is selected among the group consisting of 5’ (1)10 - (A)50 - (1)10 3’and 5’(A)35 - (1)35 3’.

[00124] In certain embodiments, the conjugates of the present technology is a conjugate of Formula (VIII)

(VIII) [P]a [A]b [R]c

[Y]a [U]b [Z]c - [Ly-(CH)x]w wherein P, R, Y, Z, a, b, and c have the same meaning as in Formula (II), and L, CH, w, x and y have the same meaning as in Formula (VII) above.

[00125] In certain embodiments, the conjugates of the present technology is a conjugate of Formula (VIII) wherein Z is C.

[00126] In certain embodiments, the conjugates of the present technology is a conjugate of Formula (VIII) wherein b is an integer selected from 30 to 60, particularly 35 to 55, more particularly 50; and a and c are equal and are an integer selected from 1 to 20, particularly 5 to 15, more particularly 10.

[00127] In certain embodiments, the conjugates of the present technology is a conjugate of Formula (IX)

(IX) [I] a [A]b [I]c

[C]a [U]b [C]c - [Ly-(CH)x]w wherein

L, CH, x, y and w are defined above, a is either 0 or equal to c b is an integer selected from 20 to 60, particularly 30 to 55, more particularly 35 to

50, and c is an integer selected from 1 to 60, particularly 5 to 50, more particularly 10 to 35. [00128] In other particular embodiments, the conjugates of the present technology is a conjugate of Formula (IX) disclosed above wherein b is an integer selected from 30 to 60, particularly 35 to 55, more particularly 50; and a and c are equal and are an integer selected from 1 to 20, particularly 5 to 15, more particularly 10.

[00129] In other particular embodiments, the conjugates of the present technology is a conjugate of Formula (IX) disclosed above wherein a is 0 and b and c are integers independently selected from 20 to 60, particularly 30 to 50, more particularly 25 to 40. In a particular embodiment b and c above are equal, particularly are 35.

[00130] In particular embodiments, the conjugates of the present technology is a conjugate of formulas (VII), (VIII), (IX) or (X) wherein the GalNAc derivative is selected among the derivatives of FormulaA, Formula B, Formula C, particularly the derivative of Formula C. [00131] Preparation of the conjugates comprises coupling the carbohydrate chemical moiety with the dsRNA.

[00132] Methods for such coupling are known, including methods disclosed in WO 2015/006740, WO 2016/077321, WO 2018/044350 andUS10,233,448 (all incorporated herein by reference).

[00133] In other embodiments, the conjugate of the present technology may be further linked to any one of more of an additional molecule such as an antibody, another protein or peptide, a lipid, additional sugars, another receptor ligand or nanoparticles. In such embodiments, the additional molecule may be covalently linked, or may be non-covalently associated with the conjugate. Examples of the additional molecules include those molecules which do not take part or interfere with the association of the conjugate to its target sites.

Pharmaceutical Compositions

[00134] In some embodiments, the conjugates of the present technology may be formulated into a pharmaceutically composition. The pharmaceutically compositions described herein comprise pharmaceutically acceptable carriers, adjuvants and/or vehicles. Pharmaceutically acceptable carriers, adjuvants and vehicles suitable for the composition of the present technology include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. [00135] The conjugates of the present technology are advantageously targeted to a specific site and/or receptor and are therefore suitable for systemic administration and confer limited off-target effects. As such, the pharmaceutical compositions of the present technology include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. In certain embodiments, the pharmaceutical composition is administered transdermally (e.g., using a transdermal patch or iontophoretic techniques). Other formulations may conveniently be presented in unit dosage form, e.g., tablets and sustained release capsules, and in liposomes, and may be prepared by any methods well known in the art of pharmacy. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA (17th ed. 1985), incorporated herein by reference. Such preparative methods include the step of bringing into association with the molecule to be administered ingredients such as the carrier that constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers, liposomes or finely divided solid carriers including nanoparticles, or both, and then if necessary, shaping the product.

[00136] In certain embodiments, the compound is administered orally. Compositions suitable for oral administration may be presented as discrete units such as capsules, sachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion, or packed in liposomes and as a bolus, etc. Soft gelatin capsules can be useful for containing such suspensions, which may beneficially increase the rate of compound absorption.

[00137] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets optionally may be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein. Methods of formulating such slow or controlled release compositions of pharmaceutically active ingredients, such as those herein and other compounds known in the art, are known in the art and described in several issued US Patents, some of which include, but are not limited to, US 4,369,172 and US 4,842,866, which are incorporated herein by refernce. Coatings can be used for delivery of compounds to the intestine (see, e.g., U.S. Patent Nos. 6,638,534, 5,217,720, and 6,569,457, 6,461,631, 6,528,080, 6,800,663, all incorporated herein by reference).

[00138] In the case of tablets for oral use, carriers that are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are administered orally, the active ingredient is combined with emulsifying and/or suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added. Surfactants such as sodium lauryl sulfate may be useful to enhance dissolution and absorption.

[00139] Compositions suitable for oral administration include lozenges comprising the ingredients in a flavored basis, usually sucrose and acacia or tragacanth; and pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia.

[00140] Compositions suitable for parenteral administration include aqueous and non- aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets. Such injection solutions may be in the form, for example, of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally- acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant.

[00141] The pharmaceutical compositions of the present technology may be administered in the form of suppositories for rectal or vaginal administration. These compositions can be prepared by mixing a compound with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components. Such materials include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.

[00142] Topical administration of the pharmaceutical compositions is especially useful when the desired treatment involves areas (including mucosa and mesothelial surfaces) or organs readily accessible by topical application. For application topically to the skin, the pharmaceutical composition will be formulated with a suitable ointment containing the active components suspended or dissolved in a carrier. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical composition can be formulated with a suitable lotion or cream containing the active compound suspended or dissolved in a carrier. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. Alternatively, the pharmaceutical composition can be formulated with a suitable gel. The pharmaceutical compositions of this invention may also be topically applied to the lower intestinal tract by rectal suppository formulation or in a suitable enema formulation. Topically-transdermal patches and iontophoretic administration are also described.

[00143] The pharmaceutical compositions may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art. Aerosol formulations that may be utilized in the methods of this invention also include those described in US 6,811,767, which is incorporated herein by reference.

[00144] In certain embodiments, the pharmaceutical composition may be administered to a tumour. In these embodiments, the composition may be applied on the surface, e.g. using an appropriate formulation such as a gel or a patch for prolonged contact with the tumor site, or in the tumour mass, e.g. using an implant, or injected into the tumour, e.g. using injectable compositions as described herein.

Methods of treatment

[00145] In certain embodiment, the conjugate of the present technology is suitable for use in the treatment of a liver disease. In certain implementation, the conjugate of the present technology is for use in the treatment of any one of more an HBV associated disease, or for use in the treatment of liver disorders characterized by unwanted cell proliferation or other liver disorders such as hematological disorders, metabolic disorders, and disorders characterized by inflammation of the liver.

[00146] In certain embodiment, the pharmaceutical composition of the present technology is suitable for use in the treatment of a liver disease. In certain implementation, the pharmaceutical composition is suitable for use in the treatment of any one of more an HBV associated disease.

[00147] Examples of HBV-associated diseases include, but are not limited to, hepatitis D virus infection, delta hepatitis, acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; liver fibrosis; end-stage liver disease; and hepatocellular carcinoma.

[00148] In other embodiments, the pharmaceutical composition of the present technology may also be suitable for use in the treatment of liver disorders characterized by unwanted cell proliferation.

[00149] A proliferation disorder of the liver can be, for example, a benign or malignant disorder, e.g., a cancer, e.g., a hepatocellular carcinoma (HCC), hepatic metastasis, or hepatoblastoma. In certain embodiments, the pharmaceutical composition of the present technology is suitable for use in the treatment of hepatocellular carcinoma including hepatocellular carcinomas having etiologies which do not involve the HBV virus, such as, chronic hepatitis C infection, cirrhosis, diabetes, non-alcoholic fatty liver disease, exposure to aflatoxins, and excessive alcohol consumption.

[00150] In other embodiments, the pharmaceutical composition of the present technology may be suitable for use in the treatment of other liver disorders such as hematological disorders, metabolic disorders, and disorders characterized by inflammation of the liver.

[00151] A hepatic hematology or inflammation disorder can be a disorder involving clotting factors, a complement-mediated inflammation or a fibrosis, for example. Metabolic diseases of the liver include dyslipidemias and irregularities in glucose regulation.

[00152] The present technology also provides for the use of a conjugate of the present technology is suitable for use in the treatment of a liver disease. In certain implementation, the conjugate of the present technology as disclosed herein for the preparation of a medicament for use in the treatment of any one of more an HBV associated disease, or for use in the treatment of liver disorders characterized by unwanted cell proliferation or other liver disorders such as hematological disorders, metabolic disorders, and disorders characterized by inflammation of the liver.

[00153] The present technology further provides for therapeutic methods. In some embodiments, the therapeutic methods of the present technology comprise administering to a subject having an HBV infection and/or HBV-associated disease, disorder, and/or condition, or prone to developing an HBV-associated disease, disorder, and/or condition a therapeutically effective amount of the conjugate or a pharmaceutical composition comprising the conjugate of the present technology. In other embodiments, the present technology provides methods of treating a subject having an HBV-associated disease including hepatitis D virus infection, delta hepatitis, acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; liver fibrosis; end- stage liver disease; hepatocellular carcinoma.

[00154] In other embodiments, the therapeutic methods of the present technology comprise administering to a subject having hepatocellular carcinoma, or prone to developing, hepatocellular carcinoma, a therapeutically effective amount of the conjugate or a pharmaceutical composition comprising the conjugate of the present technology. In further embodiments, the present technology provides methods of treating a subject having hepatocellular carcinoma including hepatocellular carcinomas having etiologies which do not involve the HBV virus, such as, chronic hepatitis C infection, cirrhosis, diabetes, non-alcoholic fatty liver disease, exposure to aflatoxins, and excessive alcohol consumption [00155] In some embodiments, the present technology provides methods for reducing levels of Hepatis B virus ccc DNA in a subject. In other embodiment, the present technology provides methods of reducing the level of HBV antigens such as HBsAg and/or HBeAg, in a subject. In a further embodiment, the present invention provides methods of reducing the viral load of HBV in a subject. In these embodiments the method comprises administering a therapeutically effective amount of the conjugate or the pharmaceutical composition of the present technology to a subject. Subjects that would benefit from a reduction and/or inhibition of Hepatis B virus ccc DNA, HBV antigens such as HBsAg, HBeAg and/or HBeAg, or viral load of HBV are those having an HBV infection and/or an HBV-associated disease or disorder as described herein.

[00156] In certain embodiments the conjugate or pharmaceutical composition of the present technology are administered to a subject having an HBV infection or an HBV associated disease such that the expression of HBV ccc DNA levels, HBV antigen levels and HBV viral load levels in a cells, tissue, blood or other tissue or fluid of the subject are reduced by at least about 0%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%,

41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%,

57%, 58%, 59%, 60%, 61%, 62%, 62%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,

73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,

89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% or more when the conjugate or pharmaceutical composition are administered to the subject.

[00157] In other embodiments administration of the conjugate or pharmaceutical composition of the present technology results in a reduction of the severity, signs, symptoms, and/or markers of HBV infection, HBV-associated diseases, or hepatocellular carcinoma. By "reduction" in this context is meant a statistically significant decrease in such level. The reduction can be, for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%.

[00158] Efficacy of treatment or prevention of disease can be assessed, for example by measuring disease progression, disease remission, symptom severity, reduction in pain, quality of life, dose of a medication required to sustain a treatment effect, level of a disease marker or any other measurable parameter appropriate for a given disease being treated or targeted for prevention. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. For example, efficacy of treatment of HBV may be assessed, by periodic monitoring of viral load and transaminase levels. Comparison of the later readings with the initial readings provide a physician an indication of whether the treatment is effective. A treatment or preventive effect is evident when there is a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated. As an example, a favorable change of at least 10% in a measurable parameter of disease, or at least 20%, 30%, 40%, 50% or more can be indicative of effective treatment. [00159] In certain embodiments, the conjugates or pharmaceutical compositions of the present technology may be administered in dosages sufficient to result in the uptake of the conjugate in the liver and to cause activation of the TLR3 receptor in liver cells. In other embodiments, the conjugates or pharmaceutical compositions of the present technology may be administered in dosages sufficient to result in the uptake of the conjugate in immune cells and to cause activation of the TLR3 receptor in immune cells. [00160] In general, a suitable dose of the pharmaceutical composition of the present technology will be in the range of about 0.001 to about 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of about 1 to 50 mg per kilogram body weight per day.

[00161] In certain embodiment, the pharmaceutical composition of the present technology may be administered to a subject as a weight-based dose. A "weight-based dose" (e.g., a dose in mg/kg) is a dose of the pharmaceutical composition that will change depending on the subject's weight. In another embodiment, the pharmaceutical composition of the present technology may be administered to a subject as a fixed dose. A "fixed dose" (e.g., a dose in mg) means that one dose of the pharmaceutical composition is used for all subjects regardless of any specific subject- related factors, such as weight.

[00162] In certain embodiments, the pharmaceutical compositions of the present technology may be administered as a single dose, multiple dose, or repeat dose regimen. A multi-dose regimen may include administration of a therapeutic amount of the pharmaceutical composition daily such as for two days, three days, four days, five days, six days, seven days, or longer. A repeat-dose regimen may include administration of a therapeutic amount the pharmaceutical composition on a regular basis, such as every other day, every third day, every fourth day, twice a week, once a week, every other week, or once a month.

[00163] The administration may be repeated, for example, on a regular basis, such as weekly, biweekly (i.e., every two weeks) for one month, two months, three months, four months or longer. After an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after administration weekly or biweekly for three months, administration can be repeated once per month, for six months or a year or longer.

[00164] The pharmaceutical composition can be administered once daily at maximum dosage, or can be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation. In that case, the conjugate contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the conjugate over a several day period. Sustained release formulations are well known in the art and are particularly useful for delivery of agents at a particular site, such as could be used with the conjugates of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose. [00165] In other embodiments, a single dose of the pharmaceutical compositions can be long lasting, such that subsequent doses are administered at not more than 3, 4, or 5-day intervals, or at not more than 1, 2, 3, or 4 week intervals. In some embodiments of the invention, a single dose of the pharmaceutical compositions of the invention is administered once per week. In other embodiments of the invention, a single dose of the pharmaceutical compositions of the invention is administered bi-monthly. In some embodiments of the invention, a single dose of the pharmaceutical compositions of the invention is administered once per month once every other month, or once quarterly (i.e., every three months).

[00166] The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the conjugates and pharmaceutical compositions encompassed by the present technology can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as known in the art.

[00167] In certain embodiments, the conjugate or the pharmaceutical compositions of the present technology may be administered with an additional therapeutic agent useful in treating HBV infections or HBV associated diseases. Examples of additional therapeutic agents useful in treating HBV infections or HBV associated diseases suitable for administration in combination with the conjugate or pharmaceutical composition of the present technology include but are not limited to, iRNA agents targeting the HBV genome, antiviral agents, reverse transcriptase inhibitors (e.g., Tenofovir disoproxil fumarate (TDF), Tenofovir alafenamide, Lamivudine, Adefovir dipivoxil, Entecavir (ETV), Telbivudine, and AGX-1009), immune stimulators (e.g., pegylated interferon alfa 2a (PEG-IFN-Cc2a), Interferon alfa-2b, a recombinant human interleukin-7, and a Toll-like receptor 7 (TLR7) agonist, therapeutic vaccines (e.g., GS-4774, DV-601, and TGI 050), viral entry inhibitors (e.g., Myrcludex), oligonucleotide that inhibits the secretion or release of HbsAg (e.g., REP 9AC), capsid inhibitors (e.g., Bay41-4109 and NVR-1221), cccDNA inhibitors (e.g., IHVR-25) The additional therapeutic agent may be administered at the same time and/or mixed in the same composition, or can be administered as part of a separate composition and/or at separate time by any methods known in the art.

[00168] In other embodiments, the conjugate or the pharmaceutical compositions of the present technology may be administered with an additional therapeutic agent useful in treating hepatocellular carcinoma. Examples of additional therapeutic agents useful in treating hepatocellular carcinoma suitable for administration in combination with the conjugate or pharmaceutical composition of the present technology include but are not limited to chemotherapeutic agents (e.g., tamoxifen, cisplatin, mitomycin, 5-fluorouracil, doxorubicin, sorafenib, octreotide, dacarbazine (DTIC), Cis-platinum, cimetidine, cyclophophamide), agents used in hormone therapy (e.g., anti-estrogen therapy, androgen deprivation therapy (ADT), luteinizing hormone-releasing hormone (LH-RH) agonists, aromatase inhibitors (AIs, such as anastrozole, exemestane, letrozole), estrogen receptor modulators (e.g., tamoxifen, raloxifene, toremifene)), or biological therapy), hematopoietic growth factors and cytokines (e.g. interleukins, such as IL-2 (including recombinant IL-II (“rIL2”) and canarypox IL-2), IL- 10, IL-12, and IL-18; interferons, such as interferon alfa-2a, interferon alfa-2b, interferon alfa- nl, interferon alfa-n3, interferon beta-I a, and interferon gamma-I b; GM-CF and GM-CSF; and EPO), monoclonal and polyclonal antibodies (e.g. trastuzumab (Herceptin®), rituximab (Rituxan®), bevacizumab (Avastin™), pertuzumab (Omnitarg™), tositumomab (Bexxar®), edrecolomab (Panorex®), and G250) as well as small molecule second active agents such as anti-cancer agents, antibiotics, immunosuppressive agents, steroids, and the like which are readily appreciated by the person skilled in the art.

[00169] The methods described herein may also be performed alone or in conjunction with other therapies, such as chemotherapy, radiation therapy, surgery, gene therapy, immunotherapy, chemoimmunotherapy, hepatic artery -based therapy, cryotherapy, ultrasound therapy, liver transplantation, local ablative therapy, radiofrequency ablation therapy, photodynamic therapy, and any other procedures used for treating HBV infection, HBV associated diseases, and hepatocellular carcinoma.

EXAMPLES

Example 1: Generation of Conjugates

[00170] Conjugates of the present technology were prepared with TLR3 agonists TL-532 and TL-533 as depicted in Table I. The dsRNAs were synthesized using standard phosphoramidite solid-phase synthesis technology as described in M. D. Matteucci et al, Tetrahedron Lett. 22, 1859- 1862 (1981), incorporated herein by reference. (1+1+1) trivalent GalNAcs were conjugated at the 3’ and/or 5’ ends of the antisense strand of the dsRNAs, whenever possible, according to known methods (Rajeev KG et al, Chembiochem 16, 903-908 (2015), incorporated herein by reference). Purity, length and carbohydrate conjugation were confirmed by gel electrophoresis. Briefly, 200 ng of material were loaded in a 6% native PAGE gel, and ran in TBElx buffer before revelation in 1 μg/mL of ethidium bromide. [00171] As seen on FIGs 1 A and IB, the unconjugated dsRNA controls on native acrylamide gel show a major clear band at about 70bp compared to the ladder, and a significant shift in the size of the 3’ and 5’ conjugates was seen on the gels compared to the unconjugated controls. Table 1 shows sequences and structure of the conjugates according to certain embodiments of the present technology.

[00172] The data in FIG. 1A is representative of three independent assays while the data in FIG. IB is representative of two independent assays.

Table 1: Sequences and structure of conjugates and unconjugated controls

Example 2: Assessing Anti-HBV Effect of Conjugates

[00173] To assess the anti-HBV activity of the conjugates of Example 1, primary human hepatocytes (PHH) were platted at a density of 250,000 cells in p48-well, infected with HBV at a multiplicity of infection of 100 viral genome equivalent (vge)/cell, and treated twice (on day 4 and day 7 post-infection) with the conjugates presented in Table 1 at the indicated concentrations. The end point of these experiments was on day 10 post-infection (i.e. 4 days after the second treatment). Treatment with Poly (I:C)-HMW (commercial Poly(I:C) High Molecular Weight with size ranging from 1.5-8 kbp) was used as a TLR3 -activated positive control. All dsRNAs were used without transfection reagent. Nucleoside analogue (NUC - tenofovir and lamivudine at a final concentration of 10 and 1 μM, respectively) was used as a positive control for reducing virions secretion. At the end of the treatment at day 10 post infection, supernatants were collected and HBeAg, HbsAg levels were assessed by ELISA (as described in Lucifora & Bonnin et al., Sci Reports, 2018, incorporated herein by reference, (Lucifora & Bonnin et al)). Newly secreted virions were measured by Q-PCR in the collected supernatants, as described in Lucifora & Bonnin et al. , with minor modifications. Briefly, 25 μl of supernatants were subjected to 100 μg/mL of RNAse and 100 μg/mL of DNAse degradation for 30 minutes at 37°C and heated for 5 minutes at 95°C before being diluted 4 times in sterile nuclease-free water. Q-PCR was then performed. Red neutral and sulforhodamine cell viability assays were performed as described in Lucifora & Bonnin et al. Levels of hlP10 and hIL-6 secretion were measured from supernatants collected 3 days after the first treatment, using ELISA kits as described by the manufacturer (BioLegend). FIGs. 2 to 9 demonstrate the results from these experiments which are expressed as mean ± SD. Data are the mean of three independent assays (FIGs. 2 to 7) or are the mean of two independent assays (FIG. 8). Unpaired Student’s t-test was used to determine significant differences (* = p<0.05; ** = p<0.01; *** = p<0.001; ns: not significant). Statistics are compared to Mock condition, unless otherwise stated.

[00174] As seen on FIGs. 2-4, treatment with (1+1+1) trivalent GalNAc conjugated TL-532 at the 3 ’end decreased all HBV parameters in HBV-infected PHH cells at similar or lower levels compared to treatment with the unconjugated TL-532 control used at the same concentration. Surprisingly, TL-532-5’ -GalNAc had the most potent anti-HBV effect compared to unconjugated TL-532 and TL-532-3 ’-GalNAc used at the same concentration, and resulted in a similar biological activity as TL-532 used at 2000 μg/mL when added at a concentration of 250 μg/mL, corresponding to nearly a 78% reduction in HBeAg secretion, nearly a 50% reduction in HBsAg secretion, and nearly an 75% reduction in new secreted virions compared to mock treated controls This suggests that 5’ conjugation of (1+1+1) trivalent GalNAcs on the antisense strand of dsRNAs may confer an advantage over 3’ conjugation and can increase the potency of interaction between the conjugates and hepatocytes and/or the activation of TLR3 in the endolysosomes of HBV-infected hepatocytes.

[00175] FIGs. 5 and 6 further demonstrate that the TL-532-5’-GalNac anti-HBV effects are not associated with cytotoxic effects in the HBV-infected PHH; as demonstrated respectively by Red Neutral and Sulforhodamine assays.

[00176] Treatment with TL-532-5 ’-GalNAc and TL-532-3 ’-GalNAc was further seen to be associated with Interferon-gamma inducible-protein- 10 ( hlP10) secretion (FIG. 7), without any noticeable effects on Interleukin 6 (hIL-6) secretion (FIG. 8), as compared to Poly (LC)-HMW which induces the secretion of both. The selective activation of hlP10 in these experiments suggest that TLR3 is engaged most likely through an IRF3/7 transduction signaling, but with a toxicity profile that should be less prominent than Poly(I:C)-HMW, due to the low/absence of hIL-6 secretion.

[00177] The effects of (1+1+1) trivalent GalNAc conjugated to TL-533 at the 5’end was also investigated. As seen in FIG. 9A, treatment with TL-533 5’ GalNac decreased all HBV parameters in HBV-infected PHH cells at similar levels compared to treatment with the unconjugated TL-533 when used at the same concentration, without induction of cytotoxicity. Treatment with TL-533 5’ GalNac was also associated with an increase in hlP10 secretion, without any noticeable effect on hIL-6 secretion (FIG. 9B). Data are the mean of at least two independent assays (FIGs. 9A and 9B). Unpaired Student’s t-test was used to determine significant differences (* = p<0.05; ** = p<0.01; *** = p<0.001; ns: not significant). Statistics are compared to Mock condition, unless otherwise stated.

Example 3: Assessing Potency of TL-532-3 ’-GalNAc and TL-532-5 ’-GalNAc in Inducing Apoptosis and Reducing Cell Viability in non-hepatocyte cells [00178] The further investigate if the conjugates of the present technology have potency in non-hepatocyte cell lines, Non Small Cell Lung Cancer (NSCLC) NCI-H292 WT cells were used.

[00179] As best seen in FIG. 10, contrary to the hepatoma cell line HepG2 which is known to express the ASGP-R protein (Kim et al ., Molecular Therapy, 2019, incorporate herein by reference), the NCI-H292 wt cells do not express this protein. Expression of the ASGP-R protein in these cells (or lack thereof) was determined by Flow Cytometry using an anti- hASGP-Rl antibody purchased from R&D systems (Catalogue number MAB050).

[00180] The ability of the 3’ and 5’ conjugates to induce apoptosis and decrease cell viability in the NSCLC NCI-H292 wt cells was then compared to that of the unconjugated control TL- 532. Briefly, 15,000 cells/well were plated in 100 ul of medium in p96-well plates (GREINER, USA, Cat#655098). 24 hours later, cells were treated with conjugated or unconjugated TL-532 without transfection reagent at 0, 1, 10 and 100 pg/ml for an extra 24 hours. Apoptosis was measured using the Real-Time Glo AnnexinV kit from Promega (France, Cat#JA1001). Cell viability was measured using an MTS assay kit (kit Cell Titer Aqueous solution, Promega). [00181] As seen in FIGs 11 and 12, no significant difference was observed between the 3’ and 5’ conjugates of TL-532 and the unconjugated control at any of the concentrations tested. These results demonstrate that the presence of GalNAc on the 3’ or 5’ end of TL-532 does not impact the induction of apoptosis and cell death in non-hepatocyte cell lines when ASGP-R is not expressed. These data further confirm that the potent anti-HBV effect observed with TL- 532-5 ’-GalNAc in the HBV-infected PHH in vitro model is dependent on both the presence of the 5’GalNac on TL-532 and on the high ASGP-R expression of in vitro hepatocytes as described in the literature (Huang et al., Bioconjug Chem, 2017, incorporated herein by reference).

[00182] The data in FIG. 10 are representative of one independent assay (N=l) while the data in FIGs. 11 and 12 represent the mean of three independent assays. Results are expressed as mean ± SD. Unpaired Student’s t-test was used to assess significant differences (* = p<0.05; ** = p<0.01; *** = p<0.001; ns: not significant). [00183] Variations and modifications will occur to those of skill in the art after reviewing this disclosure. The disclosed features may be implemented, in any combination and subcombinations (including multiple dependent combinations and subcombinations), with one or more other features described herein. The various features described or illustrated above, including any components thereof, may be combined or integrated in other systems. Moreover, certain features may be omitted or not implemented. Examples of changes, substitutions, and alterations are ascertainable by one skilled in the art and could be made without departing from the scope of the information disclosed herein.

[00184] All references cited in this specification, and their references, are incorporated by reference herein in their entirety where appropriate for teachings of additional or alternative details, features, and/or technical background.

[00185] While the disclosure has been particularly shown and described with reference to particular embodiments, it will be appreciated that variations of the above-disclosed and other features and functions, or alternatives thereof, may be desirably combined into many other different systems or applications. Also, that various presently unforeseen or unanticipated alternatives, modifications, variations or improvements therein may be subsequently made by those skilled in the art which are also intended to be encompassed by the following claims.

Claims

CLAIMS:

1. A conjugate, or pharmaceutically acceptable salt thereof, comprising a carbohydrate covalently linked to a double-stranded RNA, the double-stranded RNA having a sense strand and an antisense strand, wherein the sense strand comprises at least one block of poly A comprising at least 15 A, and the antisense strand comprises at least one complementary block poly U comprising at least 15 U, and wherein each of the sense and antisense strands have a length of between 50 and 200 nucleotides.

2. The conjugate of claim 1, wherein the sense strand comprises a sequence of formula

(I):

(I) [P]a [Q]b [R]c wherein:

R is at least one nucleotide selected from A, U, G, I and C; and combinations thereof.

3. The conjugate of claim 2, wherein the dsRNA comprises a sequence of formula (II):

(II) [P]a [A]b [R]c [Y]a [U]b [Z]c wherein Y and Z comprise the complementary nucleotides of P and R and are selected from A, U, G, I and C and combinations thereof.

4. The conjugate of claim 2 or 3, wherein P and R are independently selected from combinations of at least two different nucleotides, a block a single nucleotide, at least two blocks of different nucleotides, or a mixture of at least one block of a nucleotide and at least one other nucleotide.

5. The conjugate of claim 4, wherein P and R are a block of a single nucleotide.

6. The conjugate of any one of claims 2 to 5, wherein P and R are identical.

7. The conjugate of any one of claims 2 to 6, wherein P is I.

8. The conjugate of any one of claims 2 to 7, wherein a and c are equivalent.

9. The conjugate of any one of claims 2 to 8, wherein a+b+c is equivalent to 70.

10. The conjugate of any one of claims 2 to 9, wherein a=10, b=50 and c=10.

11. The conjugate of any one of claims 2 to 10, wherein Q is A, P is I, R is I, a is 10, b is 50, c is 10.

12. The conjugate of claim 11, wherein Y and Z are both C.

13. The conjugate of claim 2, wherein the dsRNA is of formula (III):

(III) [A]b [R]c [U]b [Z]c wherein Z comprises the complementary nucleotides of R selected from A, U, G, I and C and combinations thereof.

14. The conjugate of claim 13, wherein R is selected from random combinations of at least two different nucleotides, a block a single nucleotide, at least two blocks of different nucleotides, or a mixture of at least one block of a nucleotides and at least one other nucleotide.

15. The conjugate of claim 14, wherein R is a block of a single nucleotide.

16. The conjugate of claim 13 to 15, wherein R is I.

17. The conjugate of claim 13 to 16, wherein b+c is equivalent to 70.

18. The conjugate of claim 13 to 17, wherein b is 35 and c is 35.

19. The conjugate of claim 13 to 18, wherein R is I, b is 35 and c is 35 and Z is C.

20. The conjugate of any one of claims 1-19, wherein the double-stranded RNA is a TLR3 agonist.

21. The conjugate of any one of claims 1 to 20, wherein the carbohydrate is attached to one end of the double-stranded RNA.

22. The conjugate of any one of claims 1 to 21, wherein the carbohydrate is attached to the 3’ or the 5’ end of the anti-sense strand.

23. The conjugate of claim 22, wherein the carbohydrate is attached to the 5’ end of the anti-sense strand.

24. The conjugate of any one of claims 1 to 23, wherein the carbohydrate is a N-acetyl- galactosamine derivative.

25. The conjugate of any one of claims 1 to 24, wherein the carbohydrate comprises monovalent N-acetyl-galactosamine units sequentially conjugated to the dsRNA by non- nucleosidic linkers.

26. The conjugate of claim 24, wherein the carbohydrate comprises at least three monovalent N-acetylgalactosamine units sequentially conjugated to the dsRNA by non- nucleosidic linkers.

27. The conjugate of claim 26, wherein the carbohydrate structure is of Formula A, Formula B or Formula C:

Formula B; or

Formula C.

28. The conjugate of any one of claims 1 to 27, wherein the carbohydrate is an ASPG-R ligand.

29. The conjugate of any one of claims 1 to 28 for use in therapy.

30. A pharmaceutical composition comprising the conjugate of any one of claims 1 to 28, and one or more pharmaceutically acceptable vehicles, carriers or excipients.

31. The conjugate of any one of claims 1 to 28 or the pharmaceutical composition of claim 30, for use in the treatment of an HBV-associated disease.

32. The conjugate or the pharmaceutical composition for use of claim 31, wherein the HBV-associated disease is selected from the group consisting of hepatitis D virus infection, delta hepatitis, acute hepatitis B; acute fulminant hepatitis B; chronic hepatitis B; liver fibrosis; end-stage liver disease; and hepatocellular carcinoma.

33. The conjugate of any one of claims 1 to 28 or the pharmaceutical composition of claim

30, for use in the treatment of hepatocellular carcinoma.

34. A method of treating a subject having an HBV infection, comprising administering to the subject a therapeutically effective amount of the conjugate of any one of claims 1 to 28 or a pharmaceutical composition thereof.

35. A method of treating a subject having hepatocellular carcinoma, comprising administering to the subject a therapeutically effective amount of the conjugate of any one of claims 1 to 28 or a pharmaceutical composition thereof.