Compositions and methods for inhibiting alpha-1 antitrypsin gene expression

Abstract

The present invention relates to an RNA interference trigger that inhibits AAT gene expression via an RNA interference mechanism. The present invention also relates to a pharmaceutical composition comprising the AAT RNAi trigger and an excipient capable of improving delivery of the RNAi trigger to hepatocytes in vivo. Delivery of the AAT RNAi trigger in vivo to hepatocytes results in inhibition of AAT gene expression and treatment of alpha1-antitrypsin deficiencies and related diseases.

Description

Background Art

[0001] Alpha-1 antitrypsin deficiency is an inherited autosomal dominant genetic disorder that results in insufficient production of alpha-1-antitrypsin (A1AT), causing lung and liver disease and occurs with a frequency of approximately 1 in 1,500 to 3,500 individuals. Alpha-1 antitrypsin deficiency most commonly affects people of European ancestry worldwide.

[0002] Alpha-1 antitrypsin (α1-antitrypsin, α-1 proteinase inhibitor, A1AT or AAT) is a protease inhibitor belonging to the serpin superfamily. Normal AAT protein is synthesized by hepatocytes in the liver and secreted into the blood. Its physiological function is to inhibit neutrophil protease to protect host tissue from nonspecific damage during the inflammatory phase. The most important form of A1AT deficiency (AATD) is caused by Z mutation in clinical practice. The Z mutant allele (PiZ) tends to abnormally fold in the endoplasmic reticulum of hepatocytes via a single point mutation, resulting in intracellular retention. The loss of circulating protease activity makes the lungs susceptible to damage caused by neutrophil elastase, resulting in the development of emphysema. AAT enhancement treatment of AATD using purified human AAT every week obtains normal AAT plasma levels and prevents lung damage in affected individuals.

[0003] Although administration of purified AAT can alleviate lung damage caused by loss of endogenous secretory AAT, AATD patients are still susceptible to endoplasmic reticulum liver storage disease caused by accumulation of excessive abnormally folded AAT protein. 12% to 15% of AATD patients also develop liver disease, which may be severe or fatal, even in infancy. In AATD patients, intracellular accumulation of AAT protein in hepatocytes induces hepatocellular damage and death, as well as chronic liver damage. Clinical manifestations include chronic hepatitis, cirrhosis, hepatocellular carcinoma, elevated transaminases (transaminitis), cholestasis, fibrosis and even fulminant liver failure.

[0004] There is currently no specific treatment for preventing the onset of liver disease caused by AATD or slowing its progression. Because the liver damage caused by AATD occurs via a gain-of-function mechanism, inhibition or AAT gene expression will be applicable to preventing the accumulation of AAT protein in the liver, thereby providing a therapeutic treatment for AATD. It has been shown that double-stranded RNA molecules (dsRNA) and other RNAi triggers block gene expression with a highly conservative regulatory mechanism, which is called RNA interference (RNAi). The present invention provides AAT RNA interference (RNAi) triggers and compositions thereof for inhibiting the expression of the AAT gene in vivo. The present invention also provides methods for using AAT RNAi triggers to treat AATD and conditions and diseases caused by AATD (such as chronic hepatitis, cirrhosis, hepatocellular carcinoma and fulminant liver failure). SUMMARY OF THE INVENTION

[0006] The present invention provides alpha-1 antitrypsin (AAT) gene-specific RNA interference (RNAi) trigger molecules that can selectively and effectively reduce AAT expression. A method for therapeutically treating diseases associated with alpha-1 antitrypsin deficiency is provided using an AAT RNAi trigger. Such methods include administering an RNAi trigger targeting AAT to a human or animal.

[0007] In one embodiment, the present invention provides an RNAi trigger molecule for inhibiting the expression of human AAT gene. The RNAi trigger comprises at least two sequences that are partially, substantially or completely complementary to each other. In one embodiment, the two RNAi trigger sequences contain a sense strand comprising a first sequence and an antisense strand comprising a second sequence. In another embodiment, the two RNAi trigger sequences comprise two sense strands that jointly comprise a first sequence, and an antisense strand comprising a second sequence, wherein the sense strand and the antisense strand together form a partial meroduplex (Table 2 and Table 4). The AAT RNAi trigger sense strand comprises a sequence that has at least 90% identity with at least a portion of the AAT mRNA. Exemplary AAT RNAi trigger sense strands, antisense strands, sequence pairs, and partial duplexes are shown in Tables 1 to 5.

[0008] In one embodiment, the antisense strand comprises a nucleotide sequence complementary to a portion of the mRNA encoded by the AAT gene, and the length of the complementary region is optimally less than 30 nucleotides. In addition, preferably, the length (duplex length) of the RNAi trigger of the present invention described herein is in the range of about 16 to 30 nucleotides, particularly in the range of about 18 to 28 nucleotides. Particularly suitable for the context of the present invention is a duplex length of about 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides. The sense strand and the antisense strand may have the same length or they may have different lengths. For example, the sense strand and the antisense strand may have a length of 19, 20, 21, 22, 23 or 24 nucleotides. For example, the sense strand may have a length of 21 nucleotides, while the antisense strand may have a length of 23 nucleotides. A duplex segment of 19, 21, 22 or 23 nucleotides is optimal. The RNAi trigger inhibits the in vitro or in vivo expression of the AAT gene when delivered to a cell expressing the AAT gene.

[0009] The RNAi triggering molecules or pharmaceutical compositions described herein can be administered in a number of ways, depending on whether local or systemic treatment is desired and the area to be treated. Administration can be topical (e.g., by a transdermal patch), pulmonary, such as by inhalation or insufflation of a powder or aerosol, including by a nebulizer: intratracheal, intranasal, transdermal, and transdermal, oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular injection or infusion; subcutaneous, such as via an implanted device; or intracranial, such as intraparenchymal, intrathecal, or intraventricular administration.

[0010] The RNAi triggering molecules described herein can be delivered to target cells or tissues using any known oligonucleotide delivery technology known in the art. Nucleic acid delivery methods include, but are not limited to, encapsulation in liposomes, by iontophoresis or by incorporation of other carriers, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres, protein carriers or DPCs (WO 2000 / 053722, WO 2008 / 0022309, WO 2011 / 104169 and WO 2012 / 083185, each of which is incorporated herein by reference). In one embodiment, the AAT RNAi trigger is provided together with an in vivo delivery compound. Preferred in vivo delivery compounds include MLP delivery polymers.

[0011] In another preferred embodiment, the invention features a composition for delivering an AAT RNAi trigger to hepatocytes in vivo, comprising: an AAT RNAi trigger as described herein conjugated to a hydrophobic group containing at least 20 carbon atoms, such as cholesterol (RNA trigger-conjugate). BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Table 1. Core sequences of RNAi triggers targeting AAT mRNA.

[0013] Figure 2 Table 2. RNAi trigger sequences with 5' and 3' extensions. Capital letters represent ribonucleotides, lowercase letters "c", "g", "a", and "u" represent 2'O-methyl modified nucleotides, capital letters A, C, G, U followed by "f" represent 2'-fluoro nucleotides, "s" represents phosphorothioate, "dT" represents deoxythymidine, and (invdT) represents inverted deoxythymidine (3'-3'-linkage).

[0014] Figure 3 Table 3. Canonical AAT siRNA RNAi triggers. Capital letters indicate RNA nucleotides, lowercase letters "c", "g", "a", and "u" indicate 2'O-methyl modified nucleotides, capital letters A, C, G, U followed by "f" indicate 2'-fluoro nucleotides, "s" indicates phosphorothioate, "dT" indicates deoxythymidine, and (invdT) indicates inverted deoxythymidine (3'-3'-linked). T m is the melting temperature of the RNAi trigger.

[0015] Figure 4 Table 4. AAT partial duplex RNAi triggers. Capital letters represent RNA nucleotides, lowercase letters "c", "g", "a", and "u" represent 2'O-methyl modified nucleotides, capital letters A, C, G, U followed by "f" represent 2'-fluoro nucleotides, "s" represents phosphorothioate, "dT" represents deoxythymidine, and (invdT) represents inverted deoxythymidine (3'-3'-linked). T m is the melting temperature of the RNAi trigger.

[0016] Figure 5 Table 5. AAT UNA RNAi triggers. Capital letters represent RNA nucleotides, lowercase letters "c", "g", "a", and "u" represent 2'O-methyl modified nucleotides, capital letters A, C, G, and U followed by "f" represent 2'-fluoro nucleotides, and lowercase letters A, C, G, and U followed by " UNA The capital letters A, C, G, and U in the "" represent 2',3'-broken (unlocked) RNA nucleotide analogs, "s" represents phosphorothioate, "dT" represents deoxythymidine, and (invdT) represents inverted deoxythymidine (3'-3'-linked). T m is the melting temperature of the RNAi trigger.

[0017] Fig. 6A -E. List of MLP polymers suitable for in vivo delivery of the AAT RNAi triggers described herein.

[0018] Figure 7 . Graph depicting relative AAT expression in Hep3B cells in vitro using different concentrations of the indicated RNAi triggers.

[0019] Figure 8 . Graph depicting relative serum AAT levels in PiZ mice treated with saline or the AAT RNAi trigger of SEQ ID 52 / 63 (AD00370) and MLP delivery polymer (MLP).

[0020] Fig. 9 PAS-D staining to visualize Z-AAT accumulation in the liver. Liver sections from (A) PiZ mice sacrificed on study day 1; (B) PiZ mice receiving four biweekly intravenous doses of saline vehicle; (C) PiZ mice receiving four biweekly intravenous doses of 8 mg / kg Luc-RNAi trigger control + 8 mg / kg MLP delivery polymer; (D) PiZ mice receiving four biweekly intravenous (IV) doses of 8 mg / kg SEQ ID 52 / 63 and 8 mg / kg MLP delivery polymer.

[0021] Fig.10 Western blot analysis of soluble and insoluble fractions from liver of PiZ mice. Five-week-old mice received intravenous doses of saline, Luc-UNA (8 mg / kg SEQ ID 59 / 78 and 8 mg / kg MLP delivery polymer) or AAT-UNA (8 mg / kg SEQ ID 52 / 63 and 8 mg / kg MLP delivery polymer) once every two weeks for 8 weeks.

[0022] Fig.11 Western blot analysis of soluble and insoluble fractions from liver of PiZ mice. Five-week-old mice received four biweekly intravenous doses of saline, Luc-UNA (8 mg / kg SEQ ID 59 / 78 with 8 mg / kg MLP delivery polymer), or AAT-UNA (8 mg / kg SEQ ID 52 / 63 with 8 mg / kg MLP delivery polymer) for 8 weeks.

[0023] Fig.12. A bar graph showing globule size in six month old female PiZ mice that had received a single intravenous dose of saline, Luc-UNA RNAi trigger (8 mg / kg SEQ ID 59 / 78 with 8 mg / kg MLP delivery polymer), or AAT-UNA RNAi trigger (8 mg / kg SEQ ID 52 / 63 with 8 mg / kg MLP delivery polymer). Liver sections were cut, processed in formalin for histological observation, and stained with PAS-D for digital quantification of globule size and liver area covered by globules.

[0024] Fig.13 . A diagram showing AAT knockout after repeated administration of AAT-RNAi trigger and MLP delivery polymer in primates. Two monkeys were each given 2.0 mg / kg MLP delivery polymer (MLP delivery peptide) + 4.0 AAT-RNAi trigger SEQ ID 52 / 63 (AAT-UNA) or 3 mg / kg MLP delivery polymer (MLP delivery peptide) + 6 mg / kg RNAi trigger SEQID 52 / 63 (AAT-UNA). The first dose was given on day 1. The doses were all six weeks apart. DETAILED DESCRIPTION OF THE INVENTION

[0026] Appendix 1 to 5 relate to preferred molecules and sequences for forming AAT RNAi trigger molecules according to the present invention. The AAT RNAi trigger molecules described herein comprise one or two sense strands and an antisense strand, each of which contains a core sequence of about 18 nucleobases. The antisense strand core sequence is complementary to the nucleotide sequence (target sequence) present in AAT mRNA. The sense strand core sequence may have the same length as the antisense core sequence or it may have different lengths. The sense and antisense core sequences of the RNAi trigger anneal to form a complementary duplex region or double helix structure. Within the complementary duplex region, the sense strand core sequence is at least 90% complementary or 100% complementary to the antisense core sequence. For partially duplex (meroduplex) RNAi triggers, the sense strand core sequence is internally nicked and provides two sense strand sequences that hybridize together with the antisense strand core sequence. In addition, the sense strand and the antisense strand may independently contain an extension of 1 to 6 nucleobases at the 5' end of its core sequence, the 3' end of its core sequence, or the 5' end and 3' end of its core sequence. The antisense strand extension, if any, may be complementary or non-complementary to the corresponding nucleotides of the AAT mRNA or to any corresponding nucleotides of the sense strand. Similarly, the sense strand extension, if any, may be consistent or inconsistent with the corresponding nucleotides of the AAT mRNA or to any corresponding nucleotides of the antisense strand. When delivered to cells, the AAT RNAi triggers described herein "knock out" or inhibit the expression of the normal or Z mutant allele AAT gene.

[0027] The described AAT RNAi triggers and methods can be used to treat subjects suffering from diseases or conditions that would benefit from reduced or inhibited AAT expression. A therapeutically effective amount of any one or more of the described AAT RNAi triggers is administered to the subject. Treatment of subjects that would benefit from reduced and / or inhibited AAT gene expression includes therapeutic and / or prophylactic treatment. The subject can be a human, a patient, or a human patient. The described AAT RNAi trigger molecules can be used to provide methods for therapeutic treatment of diseases associated with mutant AAT expression. These methods include administering RNAi triggers targeting AAT to humans or animals.

[0028] AAT RNAi trigger sense and antisense strand core sequences are shown in Table 1. Table 2 provides illustrative examples of RNAi trigger sense and antisense strands with 5' or 3' extensions described herein. RNAi trigger sense and antisense strands with modified nucleotides are provided herein, and are specifically disclosed in Appendix 3 to 5, providing illustrative examples of modified RNAi trigger sense and antisense strands of the present invention. The relationship of the modified RNAi trigger strands shown in Tables 2 to 5 to the unmodified core sequences shown in Table 1 is represented by the core SEQ ID number. Modifications of these components of the RNAi triggers of the present invention are provided herein as examples of modifications and / or modification patterns.

[0029] RNAi triggers (also called dsRNAi triggers) inhibit gene expression through the biological process of RNA interference (RNAi). RNAi triggers include double-stranded RNA or RNA-like structures, which usually contain 15-50 base pairs, preferably 18-26 base pairs, and have a nucleobase sequence that is at least 90% complementary to the coding sequence in the target gene expressed in the cell. RNAi triggers include (but are not limited to): short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro RNA (miRNA), short hairpin RNA (shRNA) and dicer substrate (U.S. Patent Nos. 8,084,599, 8,349,809 and 8,513,207).

[0030] The RNAi triggers described herein are formed by annealing the antisense strand to one sense strand (for canonical siRNA RNAi triggers and UNA RNAi triggers) or two sense strands (for partial duplex RNAi triggers). In a preferred embodiment, the AAT RNAi trigger antisense strand comprises the nucleic acid sequence depicted in SEQ ID Nos: 1, 2, 3, 4, and 5. The corresponding AAT RNAi trigger sense strand comprises the nucleic acid sequence depicted in SEQ ID Nos: 8, 9, 10, 11, and 12. Thus, the AAT RNAi trigger molecule of the present invention may particularly comprise a sequence pair selected from the group consisting of the following SEQ ID pairs: 1 / 8, 2 / 9, 3 / 10, 4 / 11, and 5 / 12. Complementary pairs or partial duplexes (meroplexes) (RNAi triggers) are provided in Tables 1 to 5, as indicated by SEQ ID pairs or SEQ ID partial duplexes.

[0031] As described in detail below, the sense strand and antisense strand of RNAi trigger molecules described herein each include a core sequence and optionally, a 5' extension, a 3' extension, or a 5' extension and a 3' extension. As used herein, the extension at the 5' end or 3' end of the sense strand core sequence or the antisense strand core sequence includes 1 to 5 nucleotides. The extended nucleotides on the sense strand may or may not be complementary (base pairing) to the nucleotides (core sequence nucleotides or extended nucleotides) in the corresponding antisense strand. On the contrary, the extended nucleotides on the antisense strand may or may not be complementary (base pairing) to the nucleotides (core sequence nucleotides or extended nucleotides) in the corresponding sense strand.

[0032] In one embodiment, the AAT RNAi triggering molecule described herein comprises an antisense strand having a 3' extension of 1 to 5 nucleotides in length, preferably 1 to 2 nucleotides in length. In one embodiment, one or more of the antisense strand extension nucleotides comprises a uracil or thymidine nucleotide or a nucleotide complementary to the corresponding AAT mRNA sequence. In another embodiment, the antisense strand extension consists of dTdT or dTsdT, wherein dT represents a deoxythymidine nucleotide and sdT represents a deoxythymidine nucleotide with a 5' phosphorothioate.

[0033] In another preferred embodiment, the AAT RNAi triggering molecule described herein comprises a 3' extended sense strand having a length of 1 to 5 nucleotides, preferably 1 to 2 nucleotides. In one embodiment, one or more of the antisense strand extension nucleotides comprises adenosine, uracil or thymidine nucleotides, AT dinucleotides, or nucleotides corresponding to nucleotides in the AAT mRNA sequence. In a preferred embodiment, the 3' sense strand extension consists of Af (invdT), wherein Af represents 2'-fluoro adenosine nucleotides and invdT represents inverted (3'-3'-linked) deoxythymidine nucleotides.

[0034] In one embodiment, the AAT RNAi triggering molecule described herein comprises an antisense strand having a 5' extension of 1 to 5 nucleotides in length, preferably 1 to 2 nucleotides in length. In one embodiment, one or more of the antisense strand extension nucleotides comprises a uracil or thymidine nucleotide or a nucleotide complementary to the corresponding AAT mRNA sequence. In a preferred embodiment, the antisense strand extension consists of dT.

[0035] In another preferred embodiment, the AAT RNAi triggering molecule described herein comprises a 5' extended sense strand having a length of 1 to 5 nucleotides, preferably 1 to 3 nucleotides. In one embodiment, one or more of the sense strand extension nucleotides comprises a uracil or adenosine nucleotide or a nucleotide corresponding to a nucleotide in the AAT mRNA sequence. In a preferred embodiment, the sense strand extension consists of 5'UAU or 5'uAu, wherein u represents a 2'O-methyl-modified uridine nucleotide.

[0036] The RNAi triggering molecules described herein may contain 3' and / or 5' extensions independently on each sense strand and antisense strand. In one embodiment, both the sense strand and the antisense strand contain 3' and 5' extensions, each as described above. In one embodiment, one or more 3' extension nucleotides of one strand are base paired with one or more 5' extension nucleotides of another strand. In another embodiment, one or more 3' extension nucleotides of one strand are not base paired with one or more 5' extension nucleotides of another strand. The sense and antisense strands of the RNAi trigger may or may not contain the same number of nucleotide bases. The antisense and sense strands may form a duplex, wherein the 5' end has only a blunt end, the 3' end has only a blunt end, the 5' and 3' ends are both blunt ends, or the 5' and 3' ends are not blunt ends. In another embodiment, one or more of the nucleotides in the extension contain phosphorothioate, phosphorothioate, reverse deoxynucleotide (3' to 3' connection) nucleotides or are modified by ribonucleotides or deoxynucleotides.

[0037] In some embodiments, the sense and antisense strands of the RNAi triggers described herein contain different numbers of nucleotide bases. In some embodiments, the 5' end of the sense strand of the RNAi trigger described herein forms a blunt end with the 3' end of the antisense strand. In some embodiments, the 3' end of the sense strand of the RNAi trigger described herein forms a blunt end with the 5' end of the antisense strand. In some embodiments, both ends of the RNAi trigger described herein form blunt ends. In some embodiments, both ends of the RNAi trigger described herein are non-blunt ends. As used herein, a blunt end refers to the end of a double-stranded trigger molecule, wherein the terminal nucleotides of the two annealed chains are complementary (forming complementary base pairs). In some embodiments, the 5' end of the sense strand of the RNAi trigger described herein forms a frayed end with the 3' end of the antisense strand. In some embodiments, the 3' end of the sense strand of the RNAi trigger described herein forms a frayed end with the 5' end of the antisense strand. In some embodiments, both ends of the RNAi trigger described herein form frayed ends. In some embodiments, both ends of the RNAi trigger described herein are non-frayed ends. As used herein, an inverted end refers to the end of a double-stranded trigger molecule, wherein the terminal nucleotides of the two annealed chains are not complementary (i.e., form non-complementary base pairs). As used herein, an overhang is a segment of one or more unpaired nucleotides at the end of one chain of a double-stranded RNAi trigger molecule. The unpaired nucleotides may be on the sense strand or the antisense strand, thereby producing a 3' or 5' overhang. In some embodiments, the RNAi trigger molecule contains: a blunt end and an inverted end, a blunt end and a 5' overhang, a blunt end and a 3' overhang, an inverted end and a 5' overhang, an inverted end and a 3' overhang, two 5' overhangs, two 3' overhangs, or a 5' overhang and a 3' overhang.

[0038] In a preferred embodiment, the AAT RNAi triggering molecule of the present invention comprises a sequence pair selected from the group consisting of: SEQ ID NO: 15 / 23, 15 / 24, 16 / 28, 17 / 30, 18 / 31, 19 / 36 and 19 / 38. In another preferred embodiment, the AAT partial (duplex) RNAi triggering molecule of the present invention comprises a partial duplex selected from the group consisting of: SEQ ID NO: 15 / 25 / 41, 15 / 26 / 42, 15 / 27 / 43, 16 / 29 / 44, 18 / 32 / 45, 18 / 33 / 46, 18 / 34 / 47, 18 / 35 / 48 and 19 / 37 / 49.

[0039] RNAi triggers (also called dsRNAi triggers) inhibit gene expression through the biological process of RNA interference (RNAi). RNAi triggers include double-stranded RNA or RNA-like structures, which generally contain 15-50 base pairs and preferably 18-25 base pairs, and have a nucleobase sequence that is identical (completely complementary) or almost identical (substantially complementary) to the coding sequence in the target gene expressed in the cell. RNAi triggers include (but are not limited to): short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro RNA (miRNA), short hairpin RNA (shRNA), partial duplexes, dsRNA containing unlocked nucleic acid, and Dicer substrates (U.S. Patents 8,084,599, 8,349,809, and 8,513,207).

[0040] The AAT RNAi triggering molecules described herein may comprise naturally occurring nucleotides or may comprise at least one modified nucleotide or nucleotide mimetic. The sense and antisense strands of the RNAi triggers described herein may be synthesized and / or modified by methods well-established in the art.

[0041] Nucleobases (or nucleobases) are heterocyclic pyrimidine or purine compounds that are components of all nucleic acids and include adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U). Nucleosides are ribose or deoxyribose derivatives of certain pyrimidine or purine bases. They become glycosylamines or N-glycosides associated with nucleotides due to the lack of phosphorylation. They are also routinely included in nucleoside analogs in which the sugar group is attached to carbon rather than nitrogen ('C-nucleosides'). Nucleotides are compounds formally obtained by esterifying the 3' or 5' hydroxyl of a nucleoside with phosphoric acid. They are nucleic acid monomers.

[0042] As used herein, "G", "C", "A", "U", and "T" or "dT" each generally refers to a nucleobase, nucleoside, nucleotide, or nucleotide mimetic containing guanine, cytosine, adenine, uracil, and deoxythymidine as a base, respectively. Also, as used herein, the term "ribonucleotide" or "nucleotide" may also refer to a modified nucleotide or nucleotide mimetic, as further described below, or an alternative replacement moiety. Sequences comprising such replacement moieties are embodiments described herein.

[0043] For RNAi trigger molecules described herein, nucleosides or nucleotide bases can be linked by covalent internucleoside bonds containing phosphate (natural) or not containing phosphate (non-natural), i.e., RNAi trigger molecules can have natural or non-natural oligonucleotide backbones. In another embodiment, the RNAi trigger contains a non-standard (non-phosphate) bond between two nucleotide bases.

[0044] In a preferred embodiment, one or more nucleotides of the RNAi triggering molecule are modified nucleotides. In another embodiment, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% of the nucleotides are modified. Modified nucleotides include, but are not limited to, 2' modifications, 2'-O-methyl nucleotides (represented herein by lowercase 'n' in the nucleotide sequence), 2'-deoxy-2'-fluoro nucleotides (represented herein by Nf, also represented herein by 2'fluoro nucleotides), 2'-deoxy nucleotides (represented herein by dN), 2'-amino nucleotides, 2'-alkyl nucleotides, terminal 3' to 3' linkages, inverted deoxythymidine (represented herein by invdT), nucleotides containing 5'-phosphorothioate groups (represented herein by lowercase 's' before the nucleotide, as in sN), phosphorothioate bonds, phosphorodithioate groups, nucleotides containing non-natural bases, locked nucleotides, bridged nucleotides, peptide nucleic acids, 2',3'-broken nucleotide mimetics (unlocked nucleotides, represented herein by N UNA denoted), morpholino nucleotides and abasic nucleotides. It is not necessary that all positions in a given compound are uniformly modified. Rather, more than one modification may be incorporated into a single RNAi trigger compound or even a single nucleotide thereof. Ribose 2' modifications may be combined with modified nucleoside linkages.

[0045] Preferred modified internucleoside linkages or backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates (including 3'-alkylenephosphonic acid and chiral phosphonates), phosphinates, phosphoramidates (including 3'-aminophosphoramidates and aminoalkylphosphoramidates), thionophosphoramidates, thionoalkyl-phosphonates, thionoalkylphosphotriesters, and borophosphorates with normal 3'-5' linkages, their 2'-5' linkage analogs, and those with reverse polarity, wherein adjacent nucleoside unit pairs are 3'-5' linked to 5'-3' or 2'-5' linked to 5'-2'. Various salts, mixed salts, and acid-free forms are also included.

[0046] Preferred modified internucleoside linkages or backbones (i.e., oligonucleosides) that do not include phosphorus atoms have backbones formed by short-chain alkyl or cycloalkyl sugar-to-sugar linkages, mixed heteroatom and alkyl or cycloalkyl sugar-to-sugar linkages, or one or more short-chain heteroatom or heterocyclic sugar-to-sugar linkages. They include those with morpholinyl linkages (formed in part by the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formacetyl and thioformacetyl backbones; backbones containing methyleneformacetyl and thioformacetyl; backbones containing olefins; sulfamic acid ester backbones; methyleneimino and methylenehydrazine backbones; sulfonate and sulfonamide backbones; amide backbones; and other substances with mixed N, O, S, and CH2 components.

[0047] In another embodiment, the AAT RNAi triggering molecule described herein is a canonical siRNA with modified nucleotides. Exemplary sequences suitable for forming AAT canonical siRNA RNAi triggers with modified nucleotides are shown in Table 3. Exemplary AAT canonical siRNA RNAi triggers are the following SEQ ID pairs: 50 / 62, 50 / 63, 53 / 67, 54 / 69, 55 / 70, 56 / 75 and 56 / 77.

[0048] In another embodiment, the AAT RNAi trigger molecule described herein is a partial duplex with modified nucleotides. Exemplary sequences suitable for forming AAT partial duplex RNAi triggers with modified nucleotides are shown in Table 4. Exemplary AAT partial duplex RNAi triggers are the following SEQ ID partial duplexes: 50 / 64 / 81, 50 / 65 / 82, 50 / 66 / 83, 53 / 68 / 84, 55 / 71 / 85, 55 / 72 / 86, 55 / 73 / 87, 55 / 74 / 88, and 56 / 76 / 89.

[0049] The antisense strand of the AAT RNAi trigger preferably contains at least one "unlocking nucleotide" (UNA), AAT UNA RNAi trigger. UNA is an acyclic RNA mimetic, also known as 2',3'-broken-RNA, in which there is no C2'-C3' ribose bond. Due to the absence of ribose 2',3' bonds, UNA is flexible, thereby adjusting affinity and specificity. UNA shows a reduced binding affinity to the complementary strand, resulting in reduced thermal stability of the duplex. UNA can be located anywhere along the base chain of the RNAi trigger. The RNAi trigger described herein preferably contains a UNA located at position 6 or 7 (numbering includes a 5'dT nucleotide extension). Exemplary sequences suitable for forming AAT UNA RNAi triggers are shown in Table 5. Exemplary AAT UNA RNAi triggers are the following SEQ ID pairs: 51 / 63, 52 / 63, 57 / 77 and 58 / 77.

[0050] Modified nucleobases include other synthetic and natural nucleobases such as 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines (including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halo Uracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

[0051] In one embodiment, the AAT RNAi trigger described herein comprises a targeting moiety coupled to the RNAi trigger. We have found that coupling the RNAi trigger to a targeting moiety (wherein the targeting moiety comprises a hydrophobic group) or a galactose cluster facilitates the RNAi trigger to target the liver in vivo. The RNAi trigger-targeting moiety conjugate is formed by covalently linking the RNAi trigger to the targeting moiety. The targeting moiety can be linked to the 3' or 5' end of the sense strand or antisense strand of the RNAi trigger. Preferably, the targeting moiety is linked to the 5' end of the sense strand of the RNAi trigger.

[0052] In one embodiment, the targeting moiety is composed of a hydrophobic group. More specifically, the RNAi trigger targeting moiety is composed of a hydrophobic group with at least 20 carbon atoms. The hydrophobic group used as the targeting moiety is referred to herein as a hydrophobic targeting moiety. The hydrophobic targeting moiety is preferably a hydrocarbon containing only carbon and hydrogen atoms. However, it is allowed to maintain hydrophobic substitution or heteroatoms, such as fluorine. The hydrophobic group suitable for use as the targeting moiety can be selected from the group consisting of: alkyl, alkenyl, alkynyl, aryl, aralkyl, aralkenyl and aralkyne, each of which can be straight chain, branched or cyclic, cholesterol, cholesterol derivatives, sterols, steroids and steroid derivatives. Exemplary suitable hydrophobic groups can be selected from the group comprising: cholesterol, cholesterol derivatives, dicholesterol, tocopherol, ditocopherol, didecyl, didodecyl, didodecyl, didodecyl, didodecyl, didodecyl, isoprenoid and cholanamide.

[0053] In another embodiment, the targeting moiety comprises a galactose targeting moiety or a galactose cluster targeting moiety. As used herein, a galactose cluster comprises a molecule having two to four, typically three, terminal galactose derivatives. As used herein, the term galactose derivative includes galactose and derivatives with an affinity equal to or greater than galactose for the asialoglycoprotein receptor. Galactose or galactose clusters suitable for targeting oligonucleotides and other molecules to the liver in vivo are well known in the art.

[0054] Other terms commonly used in the art include triantennary galactose, trivalent galactose and galactose trimer. A preferred galactose derivative is N-acetyl-galactosamine (GalNAc). Other carbohydrates with affinity for asialoglycoprotein receptors may be selected from the list comprising galactose, galactosamine, N-formylgalactosamine, N-acetylgalactosamine, N-propionyl-galactosamine, N-n-butyrylgalactosamine and N-isobutyrylgalactosamine.

[0055] In one embodiment, the targeting moiety is coupled to the 5' end of the sense strand of the AAT RNAi trigger. In another preferred embodiment, the targeting moiety is coupled to the 5' end of the sense strand of the AAT RNAi trigger with a UAU extension. Preferably, the targeting moiety is a cholesterol-based derivative. Preferably, the UAU extension is a uAu extension. In yet another embodiment, the cholesterol-based derivative is connected to the 5' end of the sense strand of the AAT RNAi trigger via a linker.

[0056] Exemplary linkers include alkyl and PEG groups. A preferred PEG linker is a triethylene glycol linker.

[0057]

[0058] Chol-PEG targeting moiety (n=1-10)

[0059] Exemplary AAT RNAi triggers having cholesterol-based targeting moieties include: the following SEQ ID pairs or partial duplexes: 50 / 63, 56 / 77, 50 / 64 / 81, 50 / 65 / 82, 50 / 66 / 83, 53 / 68 / 84, 55 / 71 / 85, 55 / 72 / 86, 55 / 73 / 87, 55 / 74 / 88, 56 / 76 / 89, 51 / 63, 52 / 63, 57 / 77, and 58 / 77.

[0060] The RNAi triggering molecules described herein can be synthesized to have a reactive group, such as an amine group, at the 5' end. The reactive group can then be used to attach a targeting moiety using methods typical in the art.

[0061] As used herein, the term "sequence" refers to a chain of nucleotides described by the sequence referred to using standard nucleotide nomenclature. However, as detailed herein, the "chain constituting the sequence" may also include modifications, such as modified nucleotides and nucleotide mimetics.

[0062] As used herein, and unless otherwise specified, the term "complementary / complementarity" when used to describe a first nucleotide sequence (e.g., RNAi trigger sense strand or AATmRNA) relative to a second nucleotide sequence (e.g., RNAi trigger antisense strand), refers to the ability of an oligonucleotide or polynucleotide comprising a first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide comprising a second nucleotide sequence under certain conditions and form a duplex or double helix structure. Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs, and include natural or modified nucleotides or nucleotide mimetics, as long as the above requirements for their hybridization ability are met. Perfect or complete complementarity refers to the fact that all bases in the continuous sequence of the first polynucleotide will hybridize with the same number of bases in the continuous sequence of the second polynucleotide. The continuous sequence may include all or part of the first or second nucleotide sequence. Partial complementarity refers to the presence of one or more mismatched base pairs in the hybridization pair of the nuclear base sequence. As used herein, basic complementarity, when it comes to the RNAi triggers described herein, refers to the presence of 1 to 3 mismatched base pairs in the nuclear base sequence in the hybridization pair. With respect to base matching between the sense and antisense strands of an RNAi trigger or between the antisense strand of an RNAi trigger and the sequence of AAT mRNA, the terms "complementary", "completely complementary" and "substantially complementary" may be used herein.

[0063] We describe compositions and methods for inhibiting AAT expression in a cell, cell population, tissue or object, the method comprising: administering to the subject a therapeutically effective amount of an AAT RNAi trigger as described herein, thereby inhibiting AAT expression in the subject. Silencing, reducing, inhibiting, down-regulating or knocking out gene expression (as long as it refers to the AAT gene) means that when a cell, cell population, tissue or object transcribed by the AAT gene is treated with an AAT RNAi trigger as described herein, the expression of the ATT gene in the cell, cell population, tissue or object is reduced compared to a second cell, cell population, tissue or object not so treated, and the reduction can be measured by the level of RNA transcribed from the ATT gene in the cell, cell population, tissue or object or the level of a polypeptide, protein or protein subunit translated from mRNA.

[0064] Furthermore, the present invention relates to a method for inhibiting the expression of the AAT gene in a cell, tissue or organism, comprising the steps of: introducing an RNAi trigger as defined herein into the cell, tissue or organism; and maintaining the cell, tissue or organism for a time sufficient to achieve degradation of the mRNA transcript of AAT, thereby inhibiting the expression of AAT in a given cell.

[0065] In some embodiments, we describe a pharmaceutical composition comprising at least one of the AAT RNAi triggers described herein. These pharmaceutical compositions are particularly suitable for inhibiting AAT gene expression in cells, tissues or organisms. The pharmaceutical composition can be used to treat subjects suffering from diseases or conditions that will benefit from reduced or inhibited AAT expression. The pharmaceutical composition can be used to treat subjects at risk of developing diseases or conditions that will benefit from reduced or inhibited AAT expression. Diseases and / or conditions that will benefit from reduced or inhibited AAT expression can be selected from a list comprising: AATD, chronic hepatitis, cirrhosis, hepatocellular carcinoma and fulminant hepatic failure. Preferably, the subject is a mammal, preferably a human patient. In one embodiment, the method comprises administering to the mammal to be treated a composition comprising the AAT RNAi trigger molecule described herein. The above-mentioned pharmaceutical composition may also comprise one or more pharmaceutically acceptable excipients (including carriers, vehicles, diluents and / or delivery polymers).

[0066] In another embodiment, the present invention provides a method for treating, preventing or managing clinical manifestations associated with AATD (including AATD). The method comprises administering a therapeutically or prophylactically effective amount of one or more AAT RNAi triggers described herein to a subject in need of such treatment, prevention or management. Preferably, the subject is a mammal, most preferably a human patient. In one embodiment, the method comprises administering a composition comprising an AAT RNAi trigger molecule described herein to the mammal to be treated.

[0067] The terms "treat", "treatment" and similar terms in the context of the present invention refer to the alleviation or relief of symptoms associated with AATD.

[0068] The AAT RNAi triggers and methods described herein can be used to treat or prevent at least one symptom of a subject suffering from a disease or condition that would benefit from reduced or inhibited AAT expression. The subject is administered a therapeutically effective amount of any one or more of the RNAi triggers, thereby treating the symptom. The subject is administered a prophylactically effective amount of any one or more of the RNAi triggers, thereby preventing the at least one symptom.

[0069] In some embodiments, the gene expression level and / or mRNA level of AAT in a subject administered the AAT RNAi trigger is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% relative to a subject not receiving the AAT RNAi trigger. The gene expression level and / or mRNA level of a subject may be reduced in a cell, cell population, and / or tissue of a subject. In some embodiments, the AAT protein level of a subject administered with the AAT RNAi trigger is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% relative to a subject not receiving the AAT RNAi trigger. The protein level of a subject may be reduced in a cell, cell population, tissue, blood and / or other body fluid of the subject. The reduction in gene expression, mRNA or protein level can be assessed by any method known in the art. A reduction or decrease in AAT mRNA level and / or protein level is collectively referred to herein as AAT reduction or decrease or AAT expression inhibition or reduction.

[0070] When referring to RNAi triggers, "introducing into a cell" means that the RNAi trigger is functionally delivered into the cell. Functional delivery means that the RNAi trigger is delivered to the cell and has the expected biological activity, i.e., sequence-specific inhibition of gene expression. Many molecules (including RNAi trigger molecules) administered to the vascular system of mammals are usually cleared from the body by the liver. RNAi triggers are cleared by the liver, but RNAi triggers are degraded or otherwise processed to be removed from the body and wherein the RNAi trigger does not result in sequence-specific inhibition of gene expression, which is not considered functional delivery.

[0071] The route of administration is the path by which the RNAi trigger contacts the body. In general, methods of administering drugs and nucleic acids to treat mammals are well known in the art and can be applied to the administration of the compositions described herein. The compounds of the present invention can be administered via any suitable route, with formulations appropriately tailored for a specific route. Thus, the compounds of the present invention can be administered by injection, such as intravenous, intramuscular, intradermal, subcutaneous or intraperitoneal administration. Therefore, the present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient.

[0072] The AAT RNAi triggering molecules or compositions described herein can be delivered to cells, cell groups, tissues or objects using oligonucleotide delivery techniques known in the art. In general, any method known in the art suitable for delivering nucleic acid molecules (in vitro or in vivo) can be applied to the RNAi triggers of the present invention. For example, delivery can be achieved by topical administration (e.g., direct injection, implantation or topical administration), systemic administration, or subcutaneous, intravenous, oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intracerebral parenchyma and intrathecal), intramuscular, transdermal, airway (aerosol), nasal, rectal or topical (including buccal and sublingual) administration. In certain embodiments, the composition is administered by subcutaneous or intravenous infusion or injection.

[0073] RNAi triggers can be combined with lipids, nanoparticles, polymers, liposomes, micelles, DPCs, or other delivery systems available in the art. RNAi triggers can also be chemically coupled to targeting moieties, lipids (including but not limited to cholesterol and cholesterol-based derivatives), nanoparticles, polymers, liposomes, micelles, DPCs (WO 2000 / 053722, WO 2008 / 0022309, WO 2011 / 104169, and WO 2012 / 083185, each of which is incorporated herein by reference), or other delivery systems available in the art.

[0074] As used herein, a "pharmaceutical composition" comprises a pharmacologically effective amount of at least one RNAi trigger and a pharmaceutically acceptable carrier and optional excipients. One or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients (excipients) are substances that have been appropriately evaluated for safety and intentionally included in a drug delivery system in addition to active pharmaceutical ingredients (APIs, therapeutic products, such as AAT RNAi triggers). The excipient will not play or is not intended to play a therapeutic role at the expected dose. Excipients can be used to a) help process drug delivery systems during manufacturing, b) protect, support or enhance the stability, bioavailability or patient acceptability of APIs, c) help product identification, and / or d) enhance the overall safety, effectiveness, delivery of APIs during storage or use Any other attributes.

[0075] Excipients include, but are not limited to, absorption promoters, anti-adherents, defoamers, antioxidants, binders, binders, buffers, carriers, coatings, colorants, delivery enhancers, polydextrose, dextrose, diluents, disintegrants, emulsifiers, bulking agents, fillers, flavors, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, suspending agents, sustained-release matrices, sweeteners, thickeners, tonicity agents, carriers, hydrophobic agents, and wetting agents. Pharmaceutically acceptable excipients may or may not be inert substances.

[0076] The pharmaceutical composition may contain other additional components commonly found in pharmaceutical compositions. Pharmaceutically active substances may include, but are not limited to, antipruritic agents, astringents, local anesthetics or anti-inflammatory agents (e.g., antihistamines, diphenhydramine, etc.). It is also contemplated that cells, tissues or isolated organs expressing or containing RNAi triggers as defined herein can be used as "pharmaceutical compositions". As used herein, "pharmacologically effective amount", "therapeutically effective amount" or simply "effective amount" refers to the amount of RNAi triggers that produces the desired pharmacological, therapeutic or preventive results.

[0077] In one embodiment, the AAT RNAi trigger-targeting moiety conjugate is co-administered with the MLP delivery polymer (excipient). Co-administration refers to administering the AAT RNAi trigger and the delivery polymer to a mammal so that they are present in the mammal at the same time. The AAT RNAi trigger-targeting moiety conjugate and the delivery polymer can be administered simultaneously or they can be delivered sequentially. For simultaneous administration, they can be mixed before administration. For sequential administration, the AAT RNAi trigger-targeting moiety conjugate or the delivery polymer can be administered first.

[0078] In a preferred embodiment, the invention features a pharmaceutical composition for delivering an AAT RNAi trigger to hepatocytes in vivo, comprising: a) a conjugate of an AAT RNAi trigger and a hydrophobic group containing at least 20 carbon atoms, such as cholesterol (RNA trigger-conjugate), and b) an MLP delivery polymer. The MLP delivery polymer and the RNA trigger-conjugate are synthesized separately and can be provided in separate containers or a single container. In a preferred embodiment, the AAT RNAi trigger is not conjugated to the delivery polymer.

[0079] MLP delivery polymers

[0080] Melittin-like peptide MLP as used herein is a small amphipathic membrane active peptide comprising about 23 to about 32 amino acids derived from naturally occurring melittin, i.e., melittin, as described in WO 2012 / 083185. Naturally occurring melittin contains 26 amino acids and is predominantly hydrophobic at the amino terminus and predominantly hydrophilic (cationic) at the carboxyl terminus. The MLP described herein may be isolated from a biological source or it may be synthesized. Synthetic polymers are "artificially" formulated or manufactured by chemical methods and are not produced by naturally occurring biological processes. As used herein, MLP encompasses naturally occurring melittin peptides of the melittin family found in, for example, the venom of the following species: Apis florea, Apis mellifera, Apis cerana, Apis dorsata, Vespulamaculifrons, Vespa magnifica, Vespa velutina, Polistes sp. HQL-2001, and Polistes hebraeus. As used herein, MLP also encompasses synthetic peptides having amino acid sequences consistent with or similar to naturally occurring melittin peptides. Exemplary MLP amino acid sequences include those shown in FIG6. In addition to retaining amino acids with high membrane activity inherent to melittin, 1 to 8 amino acids may also be added to the amino or carboxyl terminus of the peptide. In particular, a cysteine ​​residue may be added to the amino or carboxyl terminus. Figure 1 The list in is not exhaustive, and other conservative amino acid substitutions can be easily envisioned. Synthetic MLPs may contain naturally occurring L-amino acids or enantiomeric D-amino acids (inverso). However, the MLP should contain essentially all L- or all D-amino acids, but may have amino acids with opposite stereocenters attached to the amino or carboxyl termini. The MLP amino acid sequence may also be reversed (reversed). A retro MLP may have L-amino acids or D-amino acids (retroinverso). In addition to shielding agents, the MLP may have modifying groups that enhance tissue targeting or aid in in vivo circulation, attached to the amino or carboxyl termini of the peptide. However, as used herein, MLPs do not include chains or polymers containing two or more MLP peptides covalently linked to each other or to another polymer or scaffold.

[0081] In one embodiment, the MLP comprises melittin from Apis mellifera (small or dwarf honey bee), Apis mellifera (western or European or large honey bee), Apis cerana (giant honey bee), Apis cerana (Eastern honey bee), or a derivative thereof. A preferred MLP comprises the following sequence: Xaa1-Xaa2-Xaa3-Ala-Xaa5-Leu-Xaa7-Val-Leu-Xaa 10 -Xaa 11 -Xaa 12 -Leu-Pro-Xaa 15 -Leu-Xaa 17 -Xaa 18 -Trp-Xaa 20 -Xaa 21 -Xaa 22 -X aa 23 -Xaa 24 -Xaa 25 -Xaa 26 ,in:

[0082] Xaa1 is leucine, D-leucine, isoleucine, norleucine, tyrosine, tryptophan, valine, alanine, dimethylglycine, glycine, histidine, phenylalanine or cysteine,

[0083] Xaa2 is isoleucine, leucine, norleucine or valine,

[0084] Xaa3 is glycine, leucine or valine,

[0085] Xaa5 is isoleucine, leucine, norleucine or valine,

[0086] Xaa7 is lysine, serine, asparagine, alanine, arginine or histidine,

[0087] XA 10 is alanine, threonine or leucine,

[0088] XA 11 is threonine or cysteine,

[0089] XA 12 is glycine, leucine or tryptophan,

[0090] XA 15 is threonine or alanine,

[0091] XA 17 isoleucine, leucine, norleucine or valine,

[0092] XA 18 is serine or cysteine,

[0093] XA 20 isoleucine, leucine, norleucine or valine,

[0094] XA 21 is lysine or alanine,

[0095] XA 22 Asparagine or arginine,

[0096] XA 23 is lysine or alanine,

[0097] XA 24 is arginine or lysine,

[0098] XA 25 is lysine, alanine or glutamine,

[0099] XA 26 is optional and, if present, is glutamine, cysteine, glutamine-NH2 or cysteine-NH2; and

[0100] And Xaa 21 , Xaa 23 Xaa 25 At least two of them are lysine.

[0101] In another embodiment, the MLP comprises the following sequence: Xaa1-Xaa2-Xaa3-Ala-Xaa5-Leu-Xaa7-Val-Leu-Xaa 10 -Xaa 11 -Xaa 12 -Leu-Pro-Xaa 15 -Le u-Xaa 17 -Ser-Trp-Xaa 20 -Lys-Xaa 22 -Lys-Arg-Lys-Xaa 26 ,in:

[0102] Xaa1 is leucine, D-leucine, norleucine or tyrosine,

[0103] Xaa2 is isoleucine, leucine, norleucine or valine,

[0104] Xaa3 is glycine, leucine or valine,

[0105] Xaa5 is isoleucine, valine, leucine or norleucine,

[0106] Xaa7 is lysine, serine, asparagine, alanine, arginine or histidine,

[0107] XA 10 is alanine, threonine or leucine,

[0108] XA 11 is threonine or cysteine,

[0109] XA 12 is glycine, leucine or tryptophan,

[0110] XA 15 is threonine or alanine,

[0111] XA 17 isoleucine, leucine or norleucine,

[0112] XA 20 isoleucine, leucine or norleucine,

[0113] XA 22 is asparagine or arginine, and

[0114] XA 26 For glutamine or cysteine.

[0115] In another embodiment, the MLP comprises the following sequence: Xaa1-Xaa2-Gly-Ala-Xaa5-Leu-Lys-Val-Leu-Ala-Xaa 11 -Gly-Leu-Pro-Thr-Leu-Xaa 17 -Ser-Trp-Xaa 20 -Lys-Xaa 22 -Lys-Arg-Lys-Xaa 26 ,in:

[0116] Xaa1, Xaa2, Xaa5, Xaa 17 Xaa 20 isoleucine, leucine or norleucine, Xaa 11 is threonine or cysteine,

[0117] XA 22 is asparagine or arginine, and

[0118] XA 26 For glutamine or cysteine.

[0119] In another embodiment, the MLP comprises: Leu-Ile-Gly-Ala-Ile-Leu-Lys-Val-Leu-Ale-Thr-Gly-Leu-Pro-Thr-Leu-Ile-Ser-Trp-Ile-Lys-Asn-Lys-Arg-Lys-Gln.

[0120] The MLP described herein is membrane active and therefore capable of destroying the plasma membrane or lysosome / endocytic membrane. As used herein, a membrane active peptide is a surface-active amphipathic peptide that is capable of inducing one or more of the following effects on a biological membrane: membrane changes or destruction that allow non-membrane permeable molecules to enter a cell or pass through the membrane, formation of holes in the membrane, membrane fission, or membrane destruction or dissolution. As used herein, a membrane or cell membrane comprises a lipid bilayer. Membrane changes or destruction can be functionally defined by the activity of the peptide in at least one of the following analyses: erythrocyte lysis (hemolysis), liposome leakage, liposome fusion, cell fusion, cell lysis, and intracellular release. Peptides that preferentially cause endosome or lysosome destruction on the plasma membrane are considered to have intracellular solubility. The effect of a membrane active peptide on a cell membrane can be transient. A membrane active peptide has an affinity for the membrane and can cause denaturation or deformation of the bilayer structure. Delivery of RNAi triggers to cells is mediated by MLPs disrupting or destabilizing the plasma membrane or the membrane of internal vesicles (such as endosomes or lysosomes), including forming pores in the membrane, or disrupting endosomal or lysosomal vesicles, thereby allowing the contents of the vesicles to be released into the cytoplasm.

[0121] The membrane activity of MLPs is reversibly masked to obtain MLP delivery polymers. Masking is achieved by reversibly attaching the masking agent to the primary amines of the MLPs.

[0122] The essential feature of the shielding agent is that, in aggregated form, it inhibits the membrane activity of the MLP and provides in vivo hepatocyte targeting. As used herein, if the modified MLP (MLP delivery polymer) does not show membrane activity and shows cell-specific (i.e., hepatocyte) in vivo targeting, the MLP is shielded. If the bond connecting the shielding agent to the peptide is broken, causing the amine on the MLP to recover, thereby restoring membrane activity, the MLP is reversibly shielded. The essential feature of the shielding agent is that the shielding agent is covalently bound to the MLP via a physiologically unstable reversible bond. By using a physiologically unstable reversible connection or bond, the shielding agent can be cleaved from the MLP in vivo, thereby deshielding the MLP and restoring the activity of the deshielded MLP. A sufficient number of shielding agents are attached to the MLP to achieve the desired degree of inactivation. The desired level of modification of the MLP by attaching a shielding agent is easily determined using an appropriate peptide activity assay. For example, if the MLP is membrane active in a given assay, a sufficient level of shielding agent is attached to the MLP to achieve the desired level of inhibition of membrane activity in that assay. Preferably, ≥80% or ≥90% of the primary amine groups on the MLP peptide population are modified, as determined by the amount of primary amines on the peptide in the absence of any shielding agent. It is also a preferred feature of the shielding agent that the shielding agent attached to the peptide reduces the positive charge of the polymer, thus forming a more neutral delivery polymer. It is desirable that the shielded peptide retains water solubility.

[0123] The MLP delivery polymer comprises an MLP reversibly modified by reaction of a primary amine on the peptide with a shielding agent containing an asialoglycoprotein receptor (ASGPr) ligand, wherein the reversible modification is physiologically unstable, as described in 2012 / 083185.

[0124] As used herein, the shielding agent comprises a preferably neutral (uncharged) compound having an ASGPr ligand and an amine reactive group, wherein the reaction of the amine reactive group with an amine on the peptide causes the ASGPr ligand to be attached to the peptide via a reversible physiologically unstable covalent bond. If cleavage of the modifying group restores the amine, the amine is reversibly modified. Preferred shielding agents containing ASGPr ligands have a neutral charge and comprise an ASGPr ligand, such as galactosamine or a galactosamine derivative having a disubstituted maleic anhydride amine reactive group. The membrane active polyamine may be coupled to the shielding agent in the presence of an excess of shielding agent. Excess shielding agent may be removed from the coupled delivery polymer prior to administration of the delivery polymer.

[0125] Galactose and galactose derivatives have been used to target molecules to hepatocytes in vivo by binding to asialoglycoprotein receptors (ASGPr) expressed on the surface of hepatocytes. As used herein, ASGPr ligands (or ASGPr ligands) include galactose and galactose derivatives with an affinity equal to or greater than galactose for ASGPr. The binding of the galactose targeting moiety to ASGPr facilitates cell-specific targeting of the delivery polymer to hepatocytes and endocytosis of the delivery polymer to hepatocytes. ASGPr ligands can be selected from a group comprising lactose, galactose, N-acetylgalactosamine (GalNAc), galactosamine, N-formylgalactosamine, N-acetyl-galactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine and N-isobutyryl-galactosamine (Lobst, ST and Drickamer, KJBC 1996, 271, 6686). ASGPr ligands can be monomeric (eg, having a single galactosamine) or polymeric (eg, having multiple galactosamines).

[0126] Preferred shielding agents include neutral hydrophilic disubstituted alkyl maleic anhydrides:

[0127]

[0128] Wherein R1 comprises an uncharged asialoglycoprotein receptor ligand. Preferably the alkyl group is methyl or ethyl. Examples of substituted alkyl maleic anhydrides consist of 2-propionic acid-3-alkyl maleic anhydride derivatives. Neutral hydrophilic 2-propionic acid-3-alkyl maleic anhydride derivatives are formed by connecting a neutral hydrophilic group to 2-propionic acid-3-alkyl maleic anhydride via a 2-propionic acid-3-alkyl maleic anhydride γ-carboxyl group.

[0129]

[0130] wherein R1 comprises a neutral ASGPr ligand and n = 0 or 1. In one embodiment, the ASGPr ligand is linked to the anhydride via a short PEG linker.

[0131] ASGPr ligands provide targeting function through affinity for ASGPr. Preferred ASGPr ligands contain sugars with affinity for ASGPr, including (but not limited to): galactose, N-acetyl-galactosamine and galactose derivatives. Galactose derivatives with affinity for ASGPr are well known in the art.

[0132] The present invention includes a conjugate delivery system consisting of:

[0133] NT and MLP-(LM) x ,

[0134] wherein N is an AAT RNAi trigger, T comprises a hydrophobic group having 20 or more carbon atoms, MLP is a melittin-like peptide as described herein, and M contains an ASGPr ligand as described herein covalently linked to MLP via a physiologically labile, reversible maleimide bond L. Cleavage of L restores unmodified amines on MLP. x is an integer greater than 1. More specifically, the value of x is greater than 80% and up to 100% of the number of primary amines on the MLP population. As used herein, MLP-(LM) x Delivery polymers for MLPs.

[0135] A sufficient percentage of the primary amines of the MLP are modified to inhibit the membrane activity of the peptide and provide hepatocyte targeting. Preferably, the value of x is greater than 80% and more preferably greater than 90% of the number of primary amines on the MLP population, as determined by the amount of amines on the MLP population in the absence of any shielding agent. It should be noted that a single MLP typically contains 3 to 5 primary amines (amino terminus if unmodified) and typically contains 2 to 4 lysine residues). In the unmodified state, the MLP is membrane active. However, the MLP delivery polymer MLP-(LM) x Not membrane active. Reversible modification of the primary amine of MLP by attachment of M reversibly inhibits or inactivates MLP membrane activity. Upon cleavage of the reversible bond L, the unmodified amine is restored, thereby restoring the MLP to its unmodified, membrane active state. MLP-(LM) x (i.e., an ASGPr-targeting reversibly shielded membrane-active polymer) and TN (i.e., an RNAi trigger-conjugate) are synthesized or manufactured separately. Neither T nor N is covalently linked directly or indirectly to MLP, L or M. For in vivo liver delivery of RNAi-trigger, electrostatic or hydrophobic association of RNAi trigger or RNAi-trigger-conjugate with shielding or deshielding polymer is required. The shielding polymer and RNAi-trigger conjugate can be provided in the same container or in separate containers. They can be combined, co-administered, or administered sequentially prior to administration.

[0136] In one aspect, we describe a pharmaceutical composition for inhibiting AAT gene expression, comprising an AAT RNAi trigger as described herein.

[0137] In one embodiment, the RNAi trigger is administered in an unbuffered solution. In one embodiment, the unbuffered solution is saline or water. In one embodiment, the RNAi trigger is administered with a buffered solution. In one embodiment, the buffered solution comprises acetate, citrate, prolamin, carbonate or phosphate or any combination thereof. In another embodiment, the buffered solution is phosphate buffered saline (PBS).

[0138] Administration of the AAT RNAi trigger according to the methods and uses described herein can reduce the severity, signs, symptoms and / or markers of a disease or disorder that would benefit from inhibition or reduction of AAT expression in a subject such as AATD. The efficacy of treatment or prevention of a disease can be assessed, for example, by measuring disease progression, disease remission, symptom severity, pain relief, quality of life, drug dosage required to maintain a therapeutic effect, disease marker levels, or any other suitable measurable parameter suitable for a given disease being treated or targeted for prevention.

[0139] As used herein, a "therapeutically effective amount" includes an amount of an AAT RNAi trigger or co-therapy that, when administered to a subject suffering from AATD, is sufficient to effectively treat the disease (e.g., by reducing, ameliorating, or maintaining existing disease or one or more symptoms). A "therapeutically effective amount" may vary depending on the AAT RNAi trigger, the co-therapy, how the trigger is administered, the disease and its severity and history, age, weight, family history, genetic makeup, type of prior or concomitant therapy (if any), and other individual characteristics of the subject to be treated.

[0140] As used herein, a "prophylactically effective amount" includes an amount of an AAT RNAi trigger or co-therapy sufficient to prevent or ameliorate a disease or one or more symptoms of a disease when administered to a subject who has AATD but has not (or currently) experienced or exhibited symptoms of the disease and / or is at risk of developing AATD. Amelioration of the disease includes slowing the course of the disease or reducing the severity of the disease that subsequently develops.

[0141] In one embodiment, the dosage may be: 0.0005, 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0. .725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0.95, 0.975, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 , 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.5, 11, 11.5, 12, 12 48,49,40,41,42,43,44,45,46,47,48,49 or 50 mg / kg. Values ​​between these values ​​are also intended to be part of the invention.

[0142] In another embodiment, the dosage may be: 0.1 to 50, 0.25 to 50, 0.5 to 50, 0.75 to 50, 1 to 50, 1.5 to 50, 2 to 50, 2.5 to 50, 3 to 50, 3.5 to 50, 4 to 50, 4.5 to 50, 5 to 50, 7.5 to 50, 10 to 50, 15 to 50, 20 to 50, 20 to 50, 25 to 50, 25 to 50, 30 to 50, 35 to 50, 40 to 50, or 45 to 50 mg / kg.

[0143] In another embodiment, the dosage may be: 0.1 to 45, 0.25 to 45, 0.5 to 45, 0.75 to 45, 1 to 45, 1.5 to 45, 2 to 45, 2.5 to 45, 3 to 45, 3.5 to 45, 4 to 45, 4.5 to 45, 5 to 45, 7.5 to 45, 10 to 45, 15 to 45, 20 to 45, 20 to 45, 25 to 45, 25 to 45, 30 to 45, 35 to 45, or 40 to 45 mg / kg.

[0144] In another embodiment, the dosage may be: 0.1 to 40, 0.25 to 40, 0.5 to 40, 0.75 to 40, 1 to 40, 1.5 to 40, 2 to 40, 2.5 to 40, 3 to 40, 3.5 to 40, 4 to 40, 4.5 to 40, 5 to 40, 7.5 to 40, 10 to 40, 15 to 40, 20 to 40, 20 to 40, 25 to 40, 25 to 40, 30 to 40, or 35 to 40 mg / kg.

[0145] In another embodiment, the dosage may be: 0.1 to 30, 0.25 to 30, 0.5 to 30, 0.75 to 30, 1 to 30, 1.5 to 30, 2 to 30, 2.5 to 30, 3 to 30, 3.5 to 30, 4 to 30, 4.5 to 30, 5 to 30, 7.5 to 30, 10 to 30, 15 to 30, 20 to 30, 20 to 30, 25 to 30 mg / kg.

[0146] In another embodiment, the dosage may be: 0.1 to 20, 0.25 to 20, 0.5 to 20, 0.75 to 20, 1 to 20, 1.5 to 20, 2 to 20, 2.5 to 20, 3 to 20, 3.5 to 20, 4 to 20, 4.5 to 20, 5 to 20, 7.5 to 20, 10 to 20, or 15 to 20 mg / kg.

[0147] In another embodiment, the dosage can be: 0.01 to 10, 0.05 to 10, 0.1 to 10, 0.2 to 10, 0.3 to 10, 0.4 to 10, 0.5 to 10, 1 to 10, 1.5 to 10, 2 to 10, 2.5 to 10, 3 to 10, 3.5 to 10, 4 to 10, 4.5 to 10, 5 to 10, 5.5 to 10, 6 to 10, 6.5 to 10, 7 to 10, 7.5 to 10, 8 to 10, 8.5 to 10, 9 to 10, or 9.5 to 10 mg / kg.

[0148] In another embodiment, the dosage may be: 0.01 to 5, 0.05 to 5, 0.1 to 5, 0.2 to 5, 0.3 to 5, 0.4 to 5, 0.5 to 5, 1 to 5, 1.5 to 5, 2 to 5, 2.5 to 5, 3 to 5, 3.5 to 5, 4 to 5, or 4.5 to 5 mg / kg.

[0149] In another embodiment, the dosage may be: 0.01 to 2.5, 0.05 to 2.5, 0.1 to 2.5, 0.2 to 2.5, 0.3 to 2.5, 0.4 to 2.5, 0.5 to 5, 1 to 2.5, 1.5 to 2.5, or 2 to 2.5 mg / kg.

[0150] In one embodiment, the AAT RNAi trigger is administered once. In another embodiment, the AAT RNAi trigger is administered repeatedly (i.e., a repeated dose regimen or a multiple dose regimen). For repeated administration, the AAT RNAi trigger may be administered to the subject once a day, every other day, once every three days, once every four days, once every five days, once every six days, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every four weeks, once every four (4) weeks to fourteen (14) weeks, twice a month, once a month, once every two months, once every three months or less, or once every four months or longer. Values ​​between the above values ​​are also part of the present invention. In another embodiment, the AAT trigger may be administered if necessary. In another embodiment, the initial treatment regimen may include repeated administration at initial time intervals and subsequent administration at a less frequent rate. For example, after one to six months of administration once a week or once every two weeks, administration may be performed once a month or less thereafter. The initial time interval may be a set number of administrations, a set time span, or until a determined AAT reduction is measured. For any dosing regimen, whether single or repeated, any of the above amounts may be used. For repeated administration, the same dose or different doses may be used for each administration.

[0151] The present invention also provides a cell comprising at least one RNAi trigger as described herein. The cell is preferably a mammalian cell, such as a human cell. In addition, tissues and / or non-human organisms comprising an RNAi trigger molecule as defined herein are embodiments of the present invention, whereby the non-human organism is particularly suitable for research purposes or as a research tool, for example in drug testing.

[0152] The embodiments and items of the present invention provided above are now illustrated in the following non-limiting examples. Example

[0153] Example 1. Identification of RNAi trigger sequences.

[0154] The selection method for identifying a lead UNA to target AAT begins with an in silico approach to identify conserved sequences in AAT gene variants. Initially, AAT cDNA sequences were screened for 17-nucleotide sequences with exact complementary sequences in 11 known human AAT variants. Sequences known to have manufacturing challenges, such as those with five (5) or more consecutive guanines or cytosines, and those predicted to have poor RNAi activity based on known siRNA parameters were eliminated. Sequences including single nucleotide polymorphisms (SNPs) and wherein the major allele frequency at positions 2 to 18 of the 19-mer sequence was greater than 0.2 were also eliminated. The sequences were then subjected to cross-species reactivity analysis to select candidates that would cross-react with cynomolgus monkey AAT. Computer simulation analysis yielded 840 19-mer sequences, and 939 17-mer sequences. The specificity of these sequences was then evaluated to avoid off-target effects against the human and cynomolgus monkey genomes. Sequences containing the conserved miRNA seed region at positions 2-7 of any siRNA chain containing off-target genes were eliminated. 47 candidate sequences were then selected for the generation of RNAi trigger molecules.

[0155] Example 2. Synthesis of RNAi triggers.

[0156] A) Synthesis. RNAi trigger molecules were synthesized according to the phosphoramidite technique on a solid phase for oligonucleotide synthesis. Depending on the scale, MerMade96E (Bioautomation) or MerMade12 (Bioautomation) were used. And for the reverse dT The synthesis was performed on a solid support made from Prime Synthesis, Inc., Aston, PA, USA. All 2'-modified RNA phosphoramidites and auxiliary reagents were purchased from Thermo Fisher Scientific, Milwaukee, WI, USA. Specifically, the following 2'-O-methyl phosphoramidites were used: (5'-O-dimethoxytrityl-N 6 -(Benzoyl)-2'-O-methyl-adenosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite, 5'-O-dimethoxy-trityl-N 4 -(acetyl)-2'-O-methyl-cytidine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite, (5'-O-dimethoxytrityl-N 2 -(isobutyryl)-2'-O-methyl-guanosine-3'-O-(2-cyano-ethyl-N,N-diisopropylamino)phosphoramidite and 5'-O-dimethoxy-trityl-2'-O-methyl-uridine-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite. 2'-Deoxy-2'-fluoro-phosphoramidite carries the same protecting group as 2'-O-methyl RNA amidite. 5'-(4,4'-dimethoxytrityl)-2',3'-sequenced-uridine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite were purchased from Link Technologies LTD (Scotland). All amides were dissolved in anhydrous acetonitrile (50 mM) and molecular sieves were added. To introduce TEG-cholesterol at the 5' end of the oligomer, 1-dimethoxytrityloxy-3-O-(N-cholesteryl-3-aminopropyl)-triethylene glycol-glyceryl-2-O-(2-cyanoethyl)-(N,N,-diisopropyl)-phosphoramidite from GRC (Glen Research, Sterling, VA, USA) was used. The 5'-modification was introduced without any modification of the synthesis cycle. 5-Benzylthio-1H-tetrazole (BTT, 250 mM in acetonitrile) was used as the activator solution. The ligation times were 10 min (RNA), 180 sec (cholesterol), 90 sec (2'OMe and UNA), and 60 sec (2'F and DNA). To introduce phosphorothioate linkages, a 100 mM solution of 3-phenyl 1,2,4-dithiazolin-5-one (POS, obtained from PolyOrg, Leominster, MA, USA) in anhydrous acetonitrile was used. See Tables 1-5 ( Figure 1-5 ).about

[0157]

[0158] (Chol-TEG)-RNA

[0159] n=1-10, preferably n=2

[0160] B. Cleavage and deprotection of support-bound oligomers. After completion of solid phase synthesis, the dried solid support was treated with a 1:1 volume of a 40 wt % methylamine solution in water and a 28 % ammonium hydroxide solution (Aldrich) at room temperature for 2 hours. The solution was evaporated and the solid residue was reconstituted in water (see below).

[0161] C. Purification. Crude cholesterol containing oligomers was purified by reverse phase HPLC using a Waters XBridge BEH300 C4 5u preparative column and a Shimadzu LC-8 system. Buffer A was 100mM TEAA (pH 7.5) and contained 5% acetonitrile, and buffer B was 100mM TEAA and contained 95% acetonitrile. A gradient of 45% B to 55% B was used over 25 minutes. UV traces were recorded at 260nm. The appropriate fractions were then run on a size exclusion HPLC using a GE Healthcare XK 16 / 40 column filled with Sephadex G-25 medium, using a running buffer of 100mM ammonium bicarbonate (pH 6.7) and 20% acetonitrile. Other crude oligomers were purified by anion exchange HPLC using a TKSgel SuperQ-5PW 13u column and a Shimadzu LC-8 system. Buffer A was 20 mM Tris, 5 mM EDTA (pH 9.0) and contained 20% acetonitrile, and buffer B was the same as buffer A with the addition of 1.5 M sodium chloride. A gradient of 32.5% B to 42.5% B was used over 25 minutes. UV traces were recorded at 260 nm. Appropriate fractions were pooled and then run on the size exclusion HPLC as described for the cholesterol-containing oligomers.

[0162] D. Annealing. Complementary strands were mixed to form RNAi triggers by combining equimolar RNA solutions (sense and antisense) in 0.2× PBS (phosphate buffered saline, 1×, Corning, Cellgro). This solution was placed in a 70°C thermomixer, heated to 95°C, kept at 95°C for 5 minutes, and slowly cooled to room temperature. Some RNAi triggers were lyophilized and stored at -15°C to -25°C. The duplex concentration was determined by measuring the absorbance of the solution in 0.2× PBS on a UV-Vis spectrometer. The absorbance of the solution at 260 nm was then multiplied by the conversion factor and the dilution factor to determine the duplex concentration. Unless otherwise stated, all conversion factors are 0.037 mg / (mL·cm). For some experiments, a conversion factor of 0.0502 mg / (mL·cm) was used.

[0163] Example 3. Melittin-like peptide (MLP) delivery polymer.

[0164] A) Synthesis of Melittin-like Peptides (MLPs). All MLPs were prepared using standard peptide synthesis techniques in the art. Suitable MLPs can be all L-amino acids, all D-amino acids (reverse configuration). Regardless of L or D form, the MLP sequence can be reversed (inverse).

[0165] B) Synthesis of CDM-NAG (N-acetylgalactosamine). To a solution of CDM (300 mg, 0.16 mmol) in 50 mL of dichloromethane were added oxalyl chloride (2 g, 10 weight equivalents) and dimethylformamide (5 μl). The reaction was allowed to proceed overnight, after which the excess oxalyl chloride and dichloromethane were removed by rotary evaporation to give CDM acid chloride. The acid chloride was dissolved in 1 mL of dichloromethane. To this solution was added 10 mL of dichloromethane containing 1.1 molar equivalents of (aminoethoxy)ethoxy-2-(acetylamino)-2-deoxy-β-D-pyranogalactoside (i.e., aminobisethoxy-ethyl NAG) and pyridine (200 μl, 1.5 equivalents). The solution was then stirred for 1.5 hours. The solvent was then removed, and the resulting solid was dissolved in 5 mL of water and purified using reverse phase HPLC with a 0.1% TFA water / acetonitrile gradient.

[0166]

[0167] R1 comprises a neutral ASGPr ligand. Preferably the shielding agent is uncharged.

[0168]

[0169] n is an integer from 1 to 10. As indicated above, a PEG spacer may be located between the anhydride group and the ASGPr ligand. Preferred PEG spacers contain 1-10 ethylene units. Alternatively, an alkyl spacer may be used between the anhydride and N-acetylgalactosamine.

[0170]

[0171] n is an integer of 0-6.

[0172] Other spacers or linkers may be used between the anhydride and N-acetylgalactosamine. However, hydrophilic, neutral (preferably uncharged) spacers or linkers are preferred.

[0173] C) Formation of MLP delivery polymer (i.e., shielding). MLP was reacted with a CDM-NAG shielding agent to obtain an MLP delivery polymer. The MLP component was first dissolved in an aqueous HEPES solution (sodium salt, GMP grade, about 430 mg / mL) to a final concentration of 8.5 mg / mL. The MLP solution was then cooled to 4°C, and the appearance was checked (a transparent to light yellow solution with no visible particles) and the concentration was detected by UV spectrophotometry. CDM-NAG was dissolved in water at 4°C to a final concentration of about 75 mg / mL. The solution appearance was checked (a transparent to light yellow solution with no visible particles) and the concentration was detected by UV spectrophotometry. The MLP in the solution was mixed with the CDM-NAG in the solution at a ratio of 5:1 (w / w) of CDM-NAG to MLP. The addition rate of the CDM-NAG solution was about 0.3 L per minute while stirring. After all the CDM-NAG solution had been added to the MLP solution, the mixture was stirred for 30 minutes. To stabilize the MLP delivery polymer, the pH was increased to 9.0 ± 0.2 by adding 1 M aqueous sodium hydroxide solution. The reaction of the disubstituted maleic anhydride shielding agent with the peptide yielded a compound having a structure represented by:

[0174]

[0175] Wherein R is MLP and R1 comprises an ASGPr ligand (eg, NAG).

[0176] A colorimetric trinitrobenzenesulfonic acid (TNBS) analysis of remaining free amines was used to determine that the MLPs were sufficiently shielded by CDM-NAG, with less than 10% of the total MLP amines remaining unmodified.

[0177] The MLP delivery polymer was purified by diafiltration against 10 mM, pH 9.2 carbonate buffer to remove excess CDM-NAG. Diafiltration method exchanges approximately 10 volumes of carbonate buffer per volume of shielded MLP reaction solution and is maintained at 2-8°C.

[0178] Components Quantity (nominal) MLP-EX1 acetate 30g / L <![CDATA[CDM-NAG a ]]> 25g / L Sodium carbonate 0.3g / L Sodium bicarbonate 0.6g / L water 1000g / L

[0179] a- Assuming five (5) CDM-NAG segments / MLP

[0180] The MLP delivery polymer was further formulated with polydextrose to 10% w / v and stored at 2° C. to 8° C. For some experiments, this solution was lyophilized prior to use.

[0181] D) Injection. The injection solution is formed by mixing the RNAi trigger and the MLP delivery polymer. The lyophilized MLP delivery polymer is dissolved in water and mixed with the RNAi trigger. The solution is then diluted with saline to the correct injection concentration.

[0182] Example 4. In vitro screening of siRNA.

[0183] Candidate sequences identified by computer simulation analysis (Example 1) were screened for chemically modified in vitro canonical siRNA screening. Forty-six of the potential AAT RNAi triggers identified by computer simulation were synthesized as canonical siRNAs and screened for in vitro efficacy. Human hepatocellular carcinoma cell line Hep3B cells were seeded in a 96-well format at approximately 10,000 cells / well. Each of the 46 siRNAs was transfected in triplicate at two concentrations, 1 nM and 10 nM, using Lipofectamine RNAiMax (Life Technologies, 0.3 μL / well). Twenty-four hours after transfection, cells were lysed and cDNA was generated (TaqMan Cells-to-CT Gene Expression Kit, Life Technologies). AAT gene knockdown was assessed by qRT-PCR and TaqMan chemistry-based assays (Life Technologies) for human AAT (Assay ID: Hs01097800_m1), normalized to the endogenous control human cyclophilin A (PPIA, 4326316E). Of the 46 siRNAs tested in vitro, five showed at least 80% knockdown of AAT. These were selected for further analysis. The same cells and transfection conditions were used to generate ten EC 50 curve, siRNA concentration ranged from 0.001-1 nM. In addition, each of the five most potent canonical siRNAs was redesigned and synthesized as a corresponding partial duplex RNAi trigger, a UNA RNAi trigger, and a locked nucleic acid (LNA) RNAi trigger. The results were compared by in vitro knockdown analysis, by two-point concentration analysis (at 1 nM and 0.1 nM) and by ten-point ED 50The most potent SEQ ID 50 / 62 targeted positions 1142-1160 in AAT mRNA and had an EC of 0.01 nM. 50 .

[0184] Serum Factor VII (F7) Activity Assay. Animal serum samples were prepared by collecting blood into microcentrifuge tubes. F7 activity in plasma was determined by a chromogenic method using the BIOPHEN VII kit (Hyphen BioMed / Aniara, Mason, OH) according to the manufacturer's recommendations. The absorbance of the colorimetric development was determined at 405 nm using a Tecan Safire2 microplate reader.

[0185] Example 5. In vivo analysis of RNAi trigger efficacy in the mouse AATD model.

[0186] To evaluate the in vivo efficacy of candidate RNAi triggers, a transgenic PiZ mouse model (PiZ mouse) was used. PiZ mice carry the human PiZ AAT mutant allele and model human AATD (Carlson et al., Journal of Clinical Investigation 1989). As noted above, AAT RNAi triggers were selected in silico for interaction with human and cynomolgus monkey AAT, but not with rat or mouse AAT.

[0187] Cholesterol-targeted RNAi triggers were delivered to PiZ mice using MLP delivery polymers. Each mouse received an intravenous (IV) injection of 200-250 μL of a solution to the tail vein containing 8 or 2 mg / kg RNAi triggers + 8 mg / kg MLP delivery polymers (1: 1 or 0.25: 1 w / w RNAi triggers: delivery polymers, respectively). By analyzing mouse serum using hAATELISA (Abcam), the human AAT protein (hAAT) content in serum was monitored until the hAAT expression level returned to baseline. For standardization, the AAT level of each animal at a certain time point was divided by the pre-treatment expression level of the animal (in this case, on the first day) to determine the expression rate "standardized for the first day". Then, for the saline control group, the expression of a specific time point was standardized by dividing the ratio of "standardized for the first day" of individual animals by the average "standardized for the first day" ratio of all mice in the saline control group. This standardizes the expression of each time point relative to the expression in the control group. The experimental error is given in the form of standard deviation.

[0188] mRNA quantification. RNA was isolated from PiZ mouse livers as follows. At the time of sacrifice, one to three liver slices were quick-frozen in a 1.5 mL microcentrifuge tube using liquid nitrogen. One liver slice from each mouse was transferred to 2 mL TRI Reagent RT (Molecular Research Center, Cincinnati, OH) in a 15 mL conical tube. Total RNA was isolated according to the manufacturer's recommended protocol. In brief, liver slices in TRI Reagent RT were treated with a tissue homogenizer for about 30 seconds. 1 mL of the homogenate was added to 50 μL of 4-bromoanisole, mixed, and the phases were separated by centrifugation. 0.25-0.5 mL of the aqueous phase was removed, precipitated with isopropanol, and centrifuged. The resulting precipitate was washed with 75% ethanol and suspended in 0.3-0.7 mL of nuclease-free water.

[0189] Total RNA (approximately 500 ng) was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Life Technologies, Grand Island, NY). The cDNA was then diluted 1:5 and multiplexed RT-qPCR was performed using 5' exonuclease chemistry with a commercially available FAM-labeled analyte for human α-1 antitrypsin (Assay ID Hs01097800_m1, Life Technologies), a VIC-labeled endogenous control analyte for mouse β-actin (Life Technologies), and VeriQuest Master Mix (Affymetrix, Santa Clara, CA). Comparative C T Gene expression data were analyzed using the method (Livak and Schmittgen, 2001).

[0190] Table 7. In vivo processes in PiZ mice.

[0191]

[0192] Example 6. Screening of AAT siRNA RNAi triggers and AAT knockdown time course.

[0193] Cholesterol-coupled canonical siRNA RNAi triggers were administered to PiZ mice as described above. Each mouse received a single intravenous (IV) dose of 2 mg / kg RNAi trigger and 8 mg / kg MLP delivery polymer. Human AAT protein levels in serum were monitored until 29 days. Knockout levels and duration of response are shown in Table 8. After administration of SEQ ID 50 / 63 and SEQID 56 / 77, hAAT serum protein levels were reduced by more than 95%.

[0194] Table 8. Serum hAAT protein levels in PiZ mice after administration of 2 mg / kg siRNA with 8 mg / kg MLP delivery polymer. AAT levels were normalized to day 1 and saline controls.

[0195]

[0196] Example 7. Screening of AAT partial duplex RNAi triggers and AAT knockout time course.

[0197] Cholesterol-coupled partial duplex RNAi triggers were administered to PiZ mice as described above. Each mouse received a single intravenous (IV) dose of 8 or 2 mg / kg RNAi trigger and 8 mg / kg MLP delivery polymer. Human AAT protein levels in serum were monitored until day 39. Knockout levels and duration of response are shown in Table 10.

[0198] Table 9. Serum FVII levels in PiZ mice following administration of 8 mg / kg partial duplex RNAi trigger with 8 mg / kg MLP delivery polymer. FVII levels were normalized to day 3 and saline controls.

[0199] treat FVII activity brine 1.00±0.06 SEQ ID 61 / 80 (FVII) 0.15±0.20 SEQ ID 50 / 64 / 81 1.18±0.17 SEQ ID 50 / 65 / 82 1.29±0.26 SEQ ID 50 / 66 / 83 1.12±0.47 SEQ ID 56 / 76 / 89 1.24±0.20 SEQ ID 55 / 72 / 86 1.33±0.09

[0200] Table 10. Serum hAAT protein levels in PiZ mice following administration of 8 mg / kg partial duplex RNAi trigger with 8 mg / kg MLP delivery polymer. AAT levels were normalized to day 1 and saline controls.

[0201]

[0202]

[0203] Example 8. In vivo screening of AAT UNA RNAi triggers and AAT knockout time course.

[0204] Cholesterol-conjugated UNA RNAi trigger was administered to PiZ mice as described above. Each mouse received a single intravenous (IV) dose of 8 or 2 mg / kg RNAi trigger with 8 mg / kg MLP delivery polymer. Human AAT protein levels in serum were monitored for 40 days.

[0205] Table 11. Serum hAAT protein levels in PiZ mice following administration of 8 or 2 mg / kg UNA RNAi trigger with 8 mg / kg MLP delivery polymer. AAT levels were normalized to day 1 and saline controls.

[0206]

[0207] a Standard siRNA control

[0208] hAAT serum protein levels were reduced by more than 95% after administration of canonical siRNA SEQ ID 50 / 63 and UNA SEQ ID 52 / 63. Maximum knockdown was observed 7 days after injection (day 8). With UNA SEQ ID 52 / 63, knockdown lasted for more than 21 days (day 22) maintaining more than 80% reduction. The knockdown duration of the UNA RNAi trigger was longer than that of the canonical chol-siRNA with the same sequence SEQ ID 50 / 63.

[0209] Example 9. Liver mRNA analysis.

[0210] AAT RNAi triggers were administered to PiZ mice as described above. Each mouse received a single intravenous (IV) dose of 6 mg / kg RNAi trigger and 6 mg / kg MLP delivery polymer. Liver hAAT mRNA production was measured on days 3 and 10. Reduced mRNA levels were associated with reduced serum hAAT protein levels, except that the mRNA reduction was a few days earlier than the protein reduction. In PiZ mice, liver hAAT mRNA production levels were measured on days 3 and 10 after a single dose of SEQ ID 52 / 63 and MLP delivery polymer. A sustained reduction in liver hAAT mRNA levels was observed, which was associated with the observed reduction in serum hAAT protein levels.

[0211] Table 12. Serum hAAT protein levels in PiZ mice following administration of 6 mg / kg SEQ ID 52 / 63 RNAi trigger or siLuc siRNA control with 6 mg / kg MLP delivery polymer. Serum hAAT levels were normalized to day 1 and saline control.

[0212]

[0213] Table 13. Liver hAAT mRNA levels in PiZ mice following administration of 6 mg / kg SEQ ID 52 / 63 RNAi trigger or siLuc siRNA control with 6 mg / kg MLP delivery polymer. AAT mRNA levels are expressed relative to mouse β-actin mRNA levels.

[0214]

[0215] Example 10. In vivo dose response of SEQ ID 52 / 63 RNAi trigger.

[0216] Different amounts of UNA SEQ ID 52 / 63 were administered to PiZ mice as described above. Each mouse received a single intravenous (IV) dose of SEQ ID 52 / 63 and 4 or 8 mg / kg MLP delivery polymer. Human AAT protein levels in serum were monitored for 35 days. The level of hAAT knockout was largely dose-dependent ( Figure 8 ).

[0217] Table 14. Serum hAAT levels in PiZ mice normalized to day 1 and saline control groups

[0218]

[0219] Example 11. In vivo dose response of SEQ ID 52 / 63 RNAi trigger.

[0220] Different amounts of UNA SEQ ID 52 / 63 were administered to PiZ mice as described above. Each mouse received a single intravenous (IV) dose of SEQ ID 52 / 63 and 2, 4 or 8 mg / kg MLP delivery polymer. Human AAT protein levels in serum were monitored for 36 days. Increasing UNA doses generally resulted in increased levels and duration of knockout at each amount of MLP delivery polymer excipient used.

[0221] Table 15. Serum hAAT protein levels in PiZ mice after administration of different doses of SEQ ID 52 / 63 UNA RNAi trigger with different doses of MLP delivery polymers. AAT levels were normalized to day 1 and saline controls.

[0222]

[0223] Example 12. Liver histology of PiZ transgenic mice treated with SEQ ID 52 / 63 RNAi trigger.

[0224] To further evaluate the efficacy of hAAT knockout in the liver, histological changes in liver samples of male PiZ mice after administration of SEQ ID 52 / 63 RNAi triggers and MLP delivery polymers were evaluated. UNASEQ ID 52 / 63 was administered to PiZ mice as described above. Each mouse received intravenous (IV) doses of 8 mg / kg SEQ ID 52 / 63 and 8 mg / kg MLP delivery polymers once every two weeks for 8 weeks. Mice were bled weekly to monitor hAAT levels in serum, and mice were sacrificed on day 57 after administration of SEQ ID 52 / 63 and MLP delivery polymers. Liver samples were collected, fixed in 10% neutral buffered formalin, and embedded in paraffin. Inflammatory infiltration was assessed by H&E staining. PiZ mice injected with 8 mg / kg SEQ ID52 / 63 and 8 mg / kg MLP delivery polymers once every two weeks for 8 weeks had normal morphology, no detectable inflammatory infiltrates, and very rare small Z-hAAT globules. PiZ mice injected with saline every two weeks had significant globules of accumulation and inflammatory infiltrates around damaged or dead hepatocytes. Z-hAAT aggregation was visualized by amylase-resistant periodic acid Schiff (PAS-D) staining of liver sections. Prior to PAS staining, glycogen was digested with amylase to stain positively for Z-AAT protein accumulations or "globules". PiZ mice receiving four biweekly intravenous (IV) doses of 8 mg / kg SEQ ID 52 / 63 and 8 mg / kg MLP delivery polymer over the course of 8 weeks showed a reduction in intracellular AAT globules compared to PiZ mice receiving saline or Luc UNA RNAi trigger 59 / 78 control injections (luciferase RNAi trigger: dTCfgAfaGfUUNAAfcUfcAfgCfgUfaAfgdTsdT, SEQ ID 59; (Chol-TEG)uAuCfuUfaCfgCfuGfaGfuAfcUfuCfgAf(invdT), SEQ ID 78). The number of globules, globule size, and area of ​​liver covered by globules were digitally quantified from liver samples stained with PAS-D. Compared with saline-injected controls, mice treated with AAT-UNA had 85% fewer globules, 85% smaller globules, and a 96% smaller area of ​​the liver covered by globules ( Fig.10 ).

[0225] Example 13. Analysis of soluble and insoluble Z-hAAT proteins in liver tissue of PiZ mice.

[0226] Homogenized liver tissue from PiZ mice treated with SEQ ID 52 / 63 RNAi effectors was further analyzed to determine whether both soluble Z-hAAT, which is expected to be mostly monomeric protein, and insoluble Z-hAAT polymers were effectively reduced. A modified Western blotting protocol was used to separate the soluble and insoluble Z-hAAT fractions under non-denaturing conditions as previously described (Mueller et al., Molecular Therapy 2012). PiZ mice given four biweekly intravenous (IV) doses of 8 mg / kg SEQ ID 52 / 63 and 8 mg / kg MLP delivery polymer for 8 weeks showed a 99% reduction in soluble Z-hAAT and a 79% reduction in insoluble Z-hAAT compared to PiZ mice given four biweekly intravenous (IV) doses of saline. Fig.11 ).

[0227] Table 16. Mean levels of soluble and insoluble Z-hAAT protein in liver lysates of PiZ mice normalized to saline injected mice

[0228]

[0229] Example 14. In vivo duration of the response induced by a single injection of SEQ ID 52 / 63 RNAi trigger in PiZ mice.

[0230] As described above, 6-month-old female PiZ mice were given a single intravenous dose of saline, 8 mg / kg Luc-UNA + 8 mg / kg MLP delivery polymer, or 8 mg / kg AAT RNAi trigger SEQ ID 52 / 63 + 8 mg / kg MLP delivery polymer. Human AAT protein levels in serum were monitored for 29 days. At the specified time, blood samples were collected and analyzed for hAAT by ELISA. Day 1 samples were collected before the trigger was given. For mRNA analysis, 3 to 4 mice were sacrificed on each of days 3, 8, 15, 22, and 29. For the sacrificed mice, a cardiac puncture (cardic stick) was performed for AAT ELISA serum separation (200 μl serum). Half of the left liver lobe was collected and flash frozen in liquid nitrogen for RNA isolation. The rest of the left lobe was embedded in a paraffin block for PAS-D staining with hematoxylin staining as counterstain. Serum hAAT levels in mice given AAT RNAi trigger SEQ ID 52 / 63 were reduced by 95% on day 8 and remained reduced until day 29, at which time the reduction was 79%. Mice given AAT RNAi trigger SEQ ID 52 / 63 were sacrificed on days 3, 8, 15, 22, or 29. Mice given saline or Luc-RNAi trigger (SEQ ID 59 / 78) were sacrificed on day 29. hAAT mRNA levels in the liver were determined by RT-qPCR. hAAT mRNA in mice given UNA SEQ ID 52 / 63 was reduced by 97% on day 3 and remained reduced until day 29, at which time the level was reduced by 56%. Liver samples stained with PAS-D were used to digitally quantify the size of the globules and the area of ​​the liver covered by the globules. Mice treated with AAT-UNA had 70% smaller globules on day 15 and 62% smaller globules on day 29. The area of ​​the liver covered by glomeruli was reduced by 83% on day 15 and by 72% on day 29 ( Fig.12 ).

[0231] Table 17. Serum hAAT levels in PiZ mice after one injection of saline, Luc-RNAi trigger SEQ ID 59 / 78 or AAT RNAi trigger SEQ ID 52 / 63.

[0232]

[0233]

[0234] Table 18. Relative hAAT mRNA levels in PiZ mice after one injection of saline, Luc-RNAi trigger SEQ ID 59 / 78 or AAT RNAi trigger SEQ ID 52 / 63.

[0235]

[0236] Example 15. Knockout of alpha-1 antitrypsin (AAT) in primates following delivery of AAT RNA trigger molecules via MLP delivery polymers.

[0237] MLP delivery polymers and RNAi triggers were prepared as described above and combined in a pharmaceutically acceptable buffer. On day 1, cynomolgus macaque (Macaca fascicularis) primates (male and female, 3 to 9 kg) were co-injected with MLP delivery polymers and AAT UNA RNAi trigger SEQ ID 52 / 63 at different dose combinations. The dose combinations injected were: 2.0 mg / kg MLP delivery polymer + 4.0 RNAi trigger (n = 3), 3 mg / kg MLP delivery polymer + 1.5 mg / kg RNAi trigger (n = 2), 3.0 mg / kg MLP delivery polymer + 3.0 mg / kg RNAi trigger (n = 3), 3.0 mg / kg MLP delivery polymer + 6.0 mg / kg RNAi trigger (n = 2), 6.0 mg / kg MLP delivery polymer + 12 mg / kg RNAi trigger (n = 3) (using a 0.050s conversion factor to determine RNAi trigger concentration) and 12 mg / kg MLP delivery polymer + 6.0 mg / kg RNAi trigger (n = 12). For each injection, MLP delivery polymer + RNAi trigger (2 ml / kg) was injected into the saphenous vein using a 22 to 25 gauge intravenous catheter. At designated time points, blood samples were drawn and analyzed for AAT and toxicity markers. Blood was collected from the femoral vein, and primates were fasted overnight before all blood collections. Blood tests for blood urea nitrogen (BUN), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and creatinine were performed on an automated chemical analyzer at Meriter Laboratories or BASi. AAT levels were determined according to manufacturer recommendations on Cobas Integra 400 (Roche Diagnostics). Significant knockout of AAT was observed at all dose combinations. No toxicity was observed at doses of 2 mg / kg, 3 mg / kg or 6 mg / kg MLP, but at 12 mg / kg MLP, liver enzymes (ALT and AST) and BUN and creatinine were increased after injection.

[0238] Table 19. AAT knockout percentage of NHP

[0239]

[0240] Table 20. Urea nitrogen (mg / dL)

[0241]

[0242] Table 21. Creatinine (mg / dL)

[0243]

[0244] Table 22. Alanine aminotransferase (U / L)

[0245]

[0246] Table 23. Aspartate aminotransferase (U / L)

[0247]

[0248] Example 16. Repeated administration.

[0249] Cynomolgus monkey primates were given five doses of RNAi trigger + MLP delivery polymer at six-week intervals. Each dose contained MLP delivery polymer and AAT-RNAi trigger SEQ ID 52 / 63 in a weight-to-weight ratio of 1:2 between MLP and RNAi trigger. The first injection was on day 1. The injected dose combinations were: 2.0 mg / kg MLP delivery polymer + 4.0 RNAi trigger (n=2) and 3 mg / kg MLP delivery polymer + 6 mg / kg RNAi trigger (n=2). Throughout the study, blood was collected at certain intervals, and the AAT level in serum was determined as described. Repeated dosing at six-week intervals reduced serum AAT levels by about 80-90% two to thirty weeks after the first treatment of primates with 3 mg / kg MLP delivery polymer + 6 mg / kg RNAi trigger. After the first treatment of primates with 2.0 mg / kg MLP delivery polymer + 4.0 RNAi trigger, serum AAT decreased by 80%, and decreased by 85% after the fourth treatment. The serum AAT level rebound measured six weeks after each treatment with 2.0 mg / kg MLP delivery polymer + 4.0 RNAi trigger was less than that of each other treatment ( Fig.13 ).

Claims

1. An RNA interference (RNAi) trigger molecule capable of inhibiting the expression of α-1 antitrypsin gene, wherein the RNAi trigger molecule comprises a sense sequence and an antisense sequence, wherein the antisense sequence comprises nucleotides 1-18 of SEQ ID No. 1, 2, 3, 4 or 5 in sequence. 2 . The RNAi triggering molecule of claim 1 , wherein the sense strand or the antisense strand further comprises a 3′ extension of 1 to 5 nucleotides in length.

3. The RNAi triggering molecule of claim 2, wherein the 3' extension of the antisense strand comprises dTdT or dTsdT. The RNAi triggering molecule of claim 2 , wherein the 3′ extension of the sense strand comprises Af(invdT). 5 . The RNAi triggering molecule of claim 1 , wherein the sense strand or the antisense strand further comprises a 5′ extension of 1 to 5 nucleotides in length. The RNAi triggering molecule of claim 5 , wherein the 5′ extension of the antisense strand comprises dT.

7. The RNAi triggering molecule of claim 5, wherein the 5' extension of the sense strand comprises UAU or uAu. The RNAi triggering molecule of claim 1 , wherein the targeting moiety is coupled to the 5′ end of the sense strand.

9. The RNAi triggering molecule of claim 8, wherein the targeting moiety comprises a cholesterol group.

10. The RNAi triggering molecule of claim 9, wherein the targeting moiety comprises a cholesterol-triethylene glycol group.

Images