Methods for treatment of alpha-1 antitrypsin deficiency (AATD)

Abstract

To provide methods for treatment of alpha-1 antitrypsin deficiency (AATD) in a human subject.SOLUTION: Described are methods for treating alpha-1 antitrypsin deficiency (AATD) in a human patient in need of treatment, using pharmaceutical compositions that include AAT RNAi agents. The pharmaceutical compositions disclosed herein that include AAT RNAi agents are administered to a human patient in need thereof for treating liver diseases associated with AAT deficiency such as chronic hepatitis, cirrhosis, increased risk of hepatocellular carcinoma, transaminitis, cholestasis, fibrosis, fulminant hepatic failure, and other liver-related diseases.SELECTED DRAWING: Figure 3

Description

Technical Field

[0001] Sequence Listing This application includes a sequence listing submitted in ASCII format, which is hereby incorporated by reference in its entirety into this specification. The ASCII manuscript is named 30674_WO1_SequenceListing.txt and is 6 kb in size.

[0002] Disclosed herein is a method for treating alpha-1 antitrypsin deficiency (AATD) in a human subject, including treating symptoms and diseases caused by AATD using a pharmaceutical composition comprising an RNA interference (RNAi) agent that inhibits the expression of the alpha-1 antitrypsin gene.

Background Art

[0003] Alpha-1 antitrypsin (AAT, α1-antitrypsin, or A1AT) is a protease inhibitor belonging to the serpin superfamily encoded by the SERPINA1 gene in humans. Normal AAT protein is a circulating glycoprotein protease inhibitor mainly synthesized by hepatocytes in the liver and secreted into the blood. The known physiological function of AAT is to inhibit neutrophil proteases, which helps protect host tissues from non-specific damage during inflammation.

[0004] Alpha-1 antitrypsin deficiency (AATD) is an autosomal co-dominant genetic disorder that results in low blood levels of AAT, causing juvenile lung disease in adults and liver disease in children and adults. The prevalence of AAT deficiency (AATD) ranges from about 1 in 1,500 to 5,000 people, and people of European descent are most affected.

[0005] The most clinically significant form of AATD is caused by the Z-variant. The Z-variant allele, through a single-point mutation, renders the mutant Z-AAT protein (the "Z-AAT protein") prone to misfolding and causes its intracellular retention in the hepatocyte endoplasmic reticulum (ER). Other, rarer mutations also result in misfolded and accumulated proteins in hepatocytes. The mutant Z-AAT protein monomers can accumulate into polymeric aggregates, sometimes called "globules". These polymeric globule masses exert pressure on the ER, causing a cycle of continuous hepatocyte damage and recovery, leading to fibrosis, cirrhosis, and an increased risk of hepatocellular carcinoma. Additionally, the absence of anti-protease activity in the blood leaves the lungs vulnerable to damage by neutrophil elastase, particularly in pulmonary inflammation, causing the development of respiratory complications such as emphysema or other lung diseases.

[0006] Individuals with the homozygous PiZZ genotype have a severe deficiency of functional AAT. Weekly use of AAT augmentation therapy with purified human AAT can help prevent lung damage in affected individuals. Currently available formulations include, for example, Prolastin®-C, Prolastin®, Glassia®, Aralast® NP, and Zemaira®. However, administration of purified AAT can improve lung damage caused by the absence or low levels of endogenously secreted AAT in some cases, and can also help prevent it in others, but AATD patients (with AAT mutations that result in polymer formation) remain vulnerable to endoplasmic reticulum liver storage disease caused by the deposition and accumulation of excessive misfolded AAT protein. The Z-AAT protein accumulated in the "globule" conformation of hepatocytes is a well-known histological feature of AATD liver disease and is thought to lead to the protein-toxic effects involved in the induction of liver injury, including hepatocyte injury, hepatocyte death, and chronic liver injury, in AATD patients (see, for example, D. Lindblad et al., Hepatology 2007, 46:1228-1235). Null / null patients who do not produce AAT develop severe lung disease but have been reported to have a normal liver morphology, providing evidence that it is the accumulation of mutant AAT, rather than the absence of plasma AAT, that leads to liver disease (Feldman, G. et al, The Ultrastructure of Hepatocytes in alpha-1 antitrypsin deficiency with genotype Pi_, Gut. 1975;16:796-799).

[0007] AATD predisposes children and adults to liver disease and adults to early-onset emphysema. AATD patients often develop liver disease, which can be severe or fatal even in infancy. Some AATD patients initially escape detection, but eventually fibrosis accumulates, causing clinically apparent liver disease. Clinical symptoms of liver injury include chronic hepatitis, cirrhosis, increased risk of hepatocellular carcinoma, hypertransaminasemia, cholestasis, fibrosis, and even fulminant liver failure.

[0008] The accumulation of Z-AAT protein globules in hepatocytes has been clearly recognized as a cause of progressive liver disease in AATD patients. Elimination of the mutant protein accumulation in hepatocytes may halt the progression of liver disease. Removal of mutant protein damage may also allow regression of existing fibrosis. Currently, there are no clinically approved treatments for the prevention of onset, delay of progression, or other treatment of liver disease caused by AATD.

[0009] RNAi agents have emerged as a promising means of treating AATD patients. The dosing method is an important matter to consider in the treatment of AATD with RNAi agents. A reduced dosing frequency is important for patients, leading to improved compliance, and a low dose may be beneficial for the overall safety profile of the drug. Therefore, a low-dose and low-frequency method for the treatment of AATD is needed. SUMMARY OF THE INVENTION

[0010] Disclosed herein is a method for treating a human subject in need of treatment for alpha-1 antitrypsin deficiency (AATD). In one embodiment, the method comprises administering to the human subject a pharmaceutical composition comprising the composition described in Table 2 (i.e., the AAT RNAi prodrug, also referred to herein as ADS-001) at a dose of about 5 mg to about 300 mg of the AAT RNAi prodrug, wherein the pharmaceutical composition is administered subcutaneously and there is at least about 1 month between administrations (i.e., administration at least once a month). In some embodiments, the pharmaceutical composition used in the methods disclosed herein comprises, consists of, or consists essentially of the formulated AAT RNAi prodrug described in Table 3 (also referred to herein as ADS-001-1).

[0011] Further disclosed herein is a method for treating a human subject in need of treatment for AATD, the method comprising administering to the human subject a pharmaceutical composition comprising the AAT RNAi prodrug described in Table 2 (i.e., ADS-001) at a dose of about 5 mg to about 200 mg, wherein the pharmaceutical composition is administered subcutaneously and there is at least about 1 month between dose administrations (i.e., administration once a month).

[0012] Also disclosed herein is a method for treating a human subject in need of treatment for AATD, the method comprising administering to the human subject a pharmaceutical composition comprising the AAT RNAi prodrug described in Table 2 (i.e., ADS-001) at a dose of about 5 mg to about 300 mg, wherein the pharmaceutical composition is administered subcutaneously and there is about 3 months between dose administrations (i.e., administration every 3 months).

[0013] Similarly disclosed herein is a method for treating a human subject in need of treatment for AATD, the method comprising administering to the human subject a pharmaceutical composition comprising the AAT RNAi prodrug described in Table 2 (i.e., ADS-001) at a dose of about 5 mg to about 200 mg, wherein the pharmaceutical composition is administered subcutaneously and there is about 3 months between dose administrations (i.e., administration every 3 months).

[0014] Disclosed herein is a method of treating a human subject in need of treatment for AATD, the method comprising administering to the human subject a pharmaceutical composition comprising the AAT RNAi drug substance (i.e., ADS-001) described in Table 2 at a dose of from about 5 mg to about 300 mg, wherein the pharmaceutical composition is administered subcutaneously, and following the first administration, a second administration is given at about 1 month later, and thereafter, for subsequent administrations, there is about 3 months between dose administrations.

[0015] Disclosed herein is a method of treating a human subject in need of treatment for AATD, the method comprising administering to the human subject a pharmaceutical composition comprising the AAT RNAi drug substance (i.e., ADS-001) described in Table 2 at a dose of from about 5 mg to about 200 mg, wherein the pharmaceutical composition is administered subcutaneously, and following the first administration, a second administration is given at about 1 month later, and thereafter, for subsequent administrations, there is about 3 months between dose administrations.

[0016] In some embodiments, the dose of the AAT RNAi drug substance administered per administration is from about 25 mg to about 200 mg. In some embodiments, the dose of the AAT RNAi drug substance administered per administration is from about 100 mg to about 200 mg. In some embodiments, the dose of the AAT RNAi drug substance administered per administration is about 100 mg. In some embodiments, the dose of the AAT RNAi drug substance administered per administration is about 200 mg. In some embodiments, the dose of the AAT RNAi drug substance administered per administration is 200 mg or less.

[0017] The treatment methods disclosed in this specification can slow down or halt the progression of liver diseases in human subjects with AATD, thereby enabling the repair of fibrotic tissue. By the methods disclosed in this specification, in some embodiments, it is possible to treat AATD liver diseases, including fibrosis, cirrhosis, increased risk of hepatocellular carcinoma, chronic hepatitis, hypertransaminasemia, cholestasis, acute liver failure, and other liver-related conditions and diseases caused by AATD.

[0018] Administering to a human subject a pharmaceutical composition comprising the AAT RNAi agent disclosed herein can inhibit the expression of the alpha-1 antitrypsin gene in the subject. In some embodiments, the subject is a human who has previously been diagnosed with having AATD.

[0019] Another aspect of the present invention provides the use of the AAT RNAi prodrug described in Table 2 for the treatment of a human subject in need of treatment for alpha-1 antitrypsin deficiency (AATD), the use comprising administering to the patient a pharmaceutical composition comprising the AAT RNAi prodrug described in Table 2 at a dose of about 5 mg to about 300 mg of the AAT RNAi prodrug, and the pharmaceutical composition is administered by subcutaneous injection once a month.

[0020] Another aspect of the present invention provides the use of the AAT RNAi prodrug described in Table 2 for the treatment of a human subject in need of treatment for alpha-1 antitrypsin deficiency (AATD), the use comprising administering to the patient a pharmaceutical composition comprising the AAT RNAi prodrug described in Table 2 at a dose of about 5 mg to about 300 mg of the AAT RNAi prodrug, and the pharmaceutical composition is administered by subcutaneous injection once every three months.

[0021] Other objects, features, aspects, and advantages of the present invention will become apparent from the following detailed description of the invention, the accompanying drawings, and the claims.

Brief Description of the Drawings

[0022]

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Mode for Carrying Out the Invention

[0023] RNAi agent The methods described herein include administration of a pharmaceutical composition to a human subject, and the pharmaceutical composition includes a composition comprising an RNA interference (RNAi) agent (referred to herein and in the art as an RNAi agent or RNAi trigger) capable of inhibiting the expression of the AAT gene. In some embodiments, the methods described herein include administration of a pharmaceutical composition to a human subject, and the pharmaceutical composition includes the AAT RNAi prodrug (also referred to as ADS-001) described in Table 2. Compositions suitable for use in the methods disclosed herein are composed of an RNAi agent that inhibits the expression of the AAT gene in a human subject, and a targeting moiety or targeting group. In some embodiments, the RNAi agent includes the nucleotide sequences shown in Tables 1A and 1B, and the sense strand of the RNAi agent is further linked or conjugated to a targeting group comprising three targeting N-acetyl-galactosamine moieties (see, e.g., Table B). An RNAi agent that inhibits the expression of the AAT gene in a human subject is referred to as an "AAT RNAi agent".

[0024] Generally, an AAT RNAi agent includes a sense strand (also called the passenger strand) and an antisense strand (also called the guide strand), which anneal to form a duplex. The AAT RNAi agents disclosed herein include RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecules that can specifically degrade the mRNA transcript of AAT messenger RNA (mRNA) or inhibit its translation. The AAT RNAi agents disclosed herein can act via the RNA interference mechanism (i.e., induce RNA interference through interaction with the RNA interference pathway mechanism (RNA-induced silencing complex or RISC) of mammalian cells), or via any alternative mechanism(s) or pathway(s). The term, AAT RNAi agent, as used herein, is generally thought to act mainly via the RNA interference mechanism, but the RNAi agents of the present disclosure are not bound or limited to any particular pathway or mechanism of action. RNAi agents generally consist of a sense strand and an antisense strand each 16 to 49 nucleotides in length and include, but are not limited to, short or small interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin-type RNA (shRNA), and dicer substrates.

[0025] The length of the sense strand of the AAT RNAi agent is usually 16 to 49 nucleotides in length, and the length of the antisense strand of the AAT RNAi agent is usually 18 to 49 nucleotides in length. In some embodiments, the sense strand and the antisense strand are independently 17 to 26 nucleotides in length. In some embodiments, the sense strand and the antisense strand are independently 21 to 26 nucleotides in length. In some embodiments, the sense strand and the antisense strand are independently 21 to 24 nucleotides in length. In some embodiments, the sense strand and / or the antisense strand are independently 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, both the sense strand and the antisense strand are 21 nucleotides in length. The sense strand and the antisense strand may be of the same length or different lengths. The sense strand and the antisense strand can also form overhang nucleotides at one or both ends of the AAT RNAi agent.

[0026] The AAT RNAi agent inhibits, silences, or knocks down the expression of the AAT gene. As used herein, the terms "silencing," "reducing," "inhibiting," "downregulating," or "knocking down" when referring to the expression of AAT mean that the expression of the gene, as measured by the level of RNA transcribed from the gene, or the level of polypeptide, protein, or protein subunit translated from the mRNA of the gene in a cell, cell population, tissue, organ, or subject in which the gene is transcribed, is reduced in a cell, cell population, tissue, organ, or subject treated with the RNAi agent as compared to a second cell, cell population, tissue, organ, or subject that has not been so treated. In some cases, the reduction in gene expression is measured by comparing the baseline level of AAT mRNA or AAT protein in a human subject before administration of a composition containing the AAT RNAi agent to the level of AAT mRNA or AAT protein after the treatment.

[0027] Inhibition, silencing, or knockdown of the AAT gene may be measured by any suitable assay or method known in the art. The non-limiting examples described herein, and the examples described in International Patent Application Publication No. WO2018 / 132432 (Patent Application No. PCT / US2018 / 013102), which is incorporated herein by reference in its entirety, show certain examples of assays suitable for measuring inhibition of AAT gene expression. A reference AAT mRNA gene transcript (SERPINA1) (referred to as transcript variant 1. GenBank NM_000295.4) from healthy humans can be found in SEQ ID NO: 1.

[0028] An AAT RNAi agent suitable for use in the methods disclosed herein can be covalently linked or conjugated to a targeting moiety that includes one or more N-acetyl-galactosamine moieties. In embodiments, an AAT RNAi agent suitable for use in the methods disclosed herein is covalently linked or conjugated to a targeting moiety that includes one or more N-acetyl-galactosamine moieties, thereby forming an AAT RNAi prodrug as set forth in Table 2. In some embodiments, the methods described herein include administering an AAT RNAi prodrug as set forth in Table 2. The AAT RNAi prodrugs set forth in Table 2 include AAT RNAi agents as set forth in Table 1A (antisense strand) and Table 1B (sense strand). The N-acetyl-galactosamine moiety facilitates targeting of the AAT RNAi agent to the asialoglycoprotein receptor (ASGPr) that is readily present on the surface of hepatocytes, which leads to internalization of the AAT RNAi agent by endocytosis or other means.

[0029] An AAT RNAi agent that may be suitable for use in the methods disclosed herein includes an antisense strand having a region complementary to at least a portion of AAT mRNA. AAT RNAi agents and AAT RNAi prodrugs suitable for use in the methods of the present disclosure are described in International Patent Application Publication No. WO2018 / 132432 (Patent Application No. PCT / US2018 / 013102), which is incorporated herein by reference in its entirety as described above.

[0030] As used herein, the terms "sequence" and "nucleotide sequence" mean the arrangement or order of nucleobases or nucleotides, and are represented by a series of characters using standard nomenclature. As used herein, the terms "nucleobase" and "nucleotide" have the same meaning as generally understood in the art.

[0031] As used herein, the term "complementary", when used to indicate a first nucleotide sequence (e.g., the antisense strand of an RNAi agent) in relation to a second nucleotide sequence (e.g., the sense strand of an RNAi agent or a target mRNA sequence), means that an oligonucleotide containing the first nucleotide sequence hybridizes (forms hydrogen bonds between base pairs) with an oligonucleotide containing the second nucleotide sequence under mammalian physiological conditions (or other appropriate conditions) and forms a double-stranded or double-helical structure under certain specific standard conditions. Those skilled in the art will be able to select a series of conditions optimal for hybridization tests. Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs, and include natural or modified nucleotides or nucleotide mimics to the extent that at least the above hybridization requirements are met. Sequence identity or complementarity is independent of modification. For example, as defined herein, a and Af are complementary to U (or T) and are identical to A in the determination of identity or complementarity.

[0032] As used herein, "fully complementary" or "sufficiently complementary" means that all (100%) of the bases in the continuous sequence of the first oligonucleotide hybridize with the same number of nucleotides in the continuous sequence of the second oligonucleotide. The continuous sequence may include all or part of the first or second nucleotide sequence.

[0033] As used herein, "partially complementary" means that in a hybridized pair of nucleotide sequences, at least 70%, but not all, of the bases in a continuous sequence of a first oligonucleotide hybridize to the same number of bases in a continuous sequence of a second polynucleotide.

[0034] As used herein, "substantially complementary" means that in a hybridized pair of nucleotide sequences, at least 85%, but not all, of the bases in a continuous sequence of a first oligonucleotide hybridize to the same number of bases in a continuous sequence of a second polynucleotide. As used herein, the terms "complementary", "fully complementary", "partially complementary", and "substantially complementary" are used with respect to nucleotide identity between the sense and antisense strands of an RNAi agent, or between the antisense strand of an RNAi agent and the sequence of AAT mRNA.

[0035] As used herein, the terms "substantially identical" or "substantial identity" as applied to a nucleic acid sequence mean that a nucleic acid sequence includes a sequence having at least about 85% or more sequence identity, such as at least 90%, at least 95%, or at least 99% identity, to a reference sequence. The percentage of sequence identity is determined by comparing two sequences that are optimally aligned over a comparison window. The percentage is calculated by measuring the number of positions at which the same nucleic acid base occurs in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions within the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. The invention disclosed herein encompasses nucleotide sequences that are substantially identical to those disclosed herein.

[0036] Modified Nucleotides and Modified Nucleoside Linkages The AAT RNAi agents disclosed herein may be composed of modified nucleotides, which in humans can maintain the activity of the RNAi agent while enhancing its serum stability and minimizing the possibility of activating interferon activity. As used herein, "modified nucleotide" refers to nucleotides other than ribonucleotides (2'-hydroxyl nucleotides). In some embodiments, at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) of the nucleotides are modified nucleotides. As used herein, modified nucleotides include any known modified nucleotides known in the art, including deoxyribonucleotides, nucleotide mimics, 2'-modified nucleotides, inverted nucleotides, nucleotides containing modified nucleobases, bridged nucleotides, peptide nucleic acids (PNA), 2',3'-seco nucleotide mimics (unlocked nucleic acid base analogs), locked nucleotides, 3'-O-methoxy (2' nucleoside internucleotide linkage) nucleotides, 2'-F-arabinonucleotides, 5'-Me,2'-fluoronucleotides, morpholinonucleotides, vinylphosphonate-containing nucleotides, and cyclopropylphosphonate-containing nucleotides, but are not limited thereto. In some embodiments, the modified nucleotides of the AAT RNAi agent are 2'-modified nucleotides (i.e., nucleotides having a group other than a hydroxyl group at the 2'-position of the five-membered sugar ring). Examples of 2'-modified nucleotides include 2'-O-methyl nucleotides, 2'-deoxy-2'-fluoronucleotides (generally simply referred to as 2'-fluoronucleotides), 2'-deoxynucleotides, 2'-methoxyethyl (2'-O-2-methoxyethyl) nucleotides, 2'-aminonucleotides, and 2'-alkyl nucleotides, but are not limited thereto. Additional 2'-modified nucleotides are known in the art. It is not necessary for all nucleotides of a given RNAi agent to be uniformly modified.Furthermore, if multiple modifications can be incorporated into a single AAT RNAi agent, they can even be incorporated into a single nucleotide. The sense and antisense strands of the AAT RNAi agent can be synthesized and / or modified by methods known in the art. Modifications of one nucleotide are independent of modifications of the other nucleotide.

[0037] In some embodiments, the nucleobases (often simply referred to as "bases") can be modified. Natural nucleobases commonly used in the art include the major purine bases adenine and guanine, and the major pyrimidine bases cytosine, thymine, and uracil. The nucleobases can be modified to include, but are not limited to, universal bases, hydrophobic bases, degenerate bases, size-expanded bases, and fluorinated bases. (See, e.g., Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008). The synthesis of such modified nucleobases (such as phosphoramidite compounds containing modified nucleobases) is known in the art.

[0038] Examples of modified nucleobases include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines (e.g., 2-aminopropyladenine, 5-propynyluracil, or 5-propynylcytosine), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, inosine, xanthine, hypoxanthine, 2-aminoadenine, 6-alkyl (e.g., 6-methyl, 6-ethyl, 6-isopropyl, or 6-n-butyl) derivatives of adenine and guanine, 2-alkyl (e.g., 2-methyl, 2-ethyl, 2-isopropyl, or 2-n-butyl) and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil, cytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-sulfhydryl, 8-thioalkyl, 8-hydroxy, and other 8-substituted adenines and guanines, 5-halo (e.g., 5-bromo), 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, and 3-deazaadenine.

[0039] In some embodiments, all or substantially all of the nucleotides of the AAT RNAi agent are modified nucleotides. As used herein, an RNAi agent in which substantially all of the nucleotides present are modified nucleotides is an RNAi agent in which four or fewer (i.e., 0, 1, 2, 3, or 4) nucleotides are ribonucleotides (i.e., unmodified) in both the sense and antisense strands. As used herein, a sense strand in which substantially all of the nucleotides present are modified nucleotides is a sense strand in which two or fewer (i.e., 0, 1, or 2) nucleotides are ribonucleotides. As used herein, an antisense strand in which substantially all of the nucleotides present are modified nucleotides is an antisense strand in which two or fewer (i.e., 0, 1, or 2) nucleotides are ribonucleotides.

[0040] In some embodiments, one or more nucleotides of the AAT RNAi agent are linked by non-standard linkages or backbones (i.e., modified internucleoside linkages or modified backbones). Modified internucleoside linkages or backbones include phosphorothioate groups, chiral phosphorothioates, thiophosphates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, alkylphosphonates (e.g., methylphosphonate or 3'-alkylene phosphonate), chiral phosphonates, phosphinates, phosphoramidates (e.g., 3'-aminophosphoramidate, aminoalkylphosphoramidate, or thionophosphoramidate), thionoalkyl-phosphonate, thionoalkylphosphotriester, morpholino linkages, boranophosphates having a normal 3'-5' linkage, 2'-5' linked analogs of boranophosphates, or boranophosphates having an inverted polarity in which adjacent pairs of nucleoside units are linked from 3'-5' to 5'-3' or from 2'-5' to 5'-2', but are not limited thereto. In some embodiments, the modified internucleoside linkage or backbone lacks a phosphorus atom. Modified internucleoside linkages lacking a phosphorus atom include short-chain alkyl or cycloalkyl intersugar linkages, heteroatom and alkyl or cycloalkyl mixed intersugar linkages, or one or more short-chain heteroatom or heterocyclic intersugar linkages, but are not limited thereto. In some embodiments, modified internucleoside backbones include siloxane backbones, sulfide backbones, sulfoxide backbones, sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate salt backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and other backbones having mixed components of N, O, S, and CH2, but are not limited thereto.

[0041] In some embodiments, the sense strand of the AAT RNAi agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages, the antisense strand of the AAT RNAi agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages, or both the sense and antisense strands can independently contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages. In some embodiments, the sense strand of the AAT RNAi agent can contain 1, 2, 3, or 4 phosphorothioate linkages, the antisense strand of the AAT RNAi agent can contain 1, 2, 3, or 4 phosphorothioate linkages, or both the sense and antisense strands can independently contain 1, 2, 3, or 4 phosphorothioate linkages.

[0042] In some embodiments, the sense strand of the AAT RNAi agent contains at least two phosphorothioate internucleoside linkages. In some embodiments, the at least two phosphorothioate internucleoside linkages are between nucleotides at positions 1-3 from the 3' end of the sense strand. In some embodiments, the at least two phosphorothioate internucleoside linkages are between nucleotides at positions 1-3, 2-4, 3-5, 4-6, 4-5, or 6-8 from the 5' end of the sense strand. In some embodiments, the phosphorothioate internucleoside linkages are used to link the terminal nucleotide of the sense strand to a capping residue present at the 5' end, 3' end, or both the 5' and 3' ends of the nucleotide sequence. In some embodiments, the phosphorothioate internucleoside linkages are used to link a target-directing group to the sense strand.

[0043] In some embodiments, the antisense strand of the AAT RNAi agent comprises three or four phosphorothioate internucleoside linkages. In some embodiments, the antisense strand of the AAT RNAi agent comprises three phosphorothioate internucleoside linkages. In some embodiments, the three phosphorothioate internucleoside linkages are between nucleotides at positions 1-3 from the 5' end of the antisense strand and between nucleotides at positions 19-21, 20-22, 21-23, 22-24, 23-25, or 24-26 from the 5' end. In some embodiments, the AAT RNAi agent comprises at least two phosphorothioate internucleoside linkages in the sense strand and three or four phosphorothioate internucleoside linkages in the antisense strand.

[0044] In some embodiments, the AAT RNAi agent comprises one or more modified nucleotides and one or more internucleoside linkages. In some embodiments, the 2'-modified nucleosides are combined with modified internucleoside linkages.

[0045] Capping residue or moiety In some embodiments, the sense strand may comprise one or more capping residues or moieties, which may also be referred to in the art as "caps", "terminal caps", or "capping residues". As used herein, a "capping residue" is a non-nucleotide compound or other moiety that can be incorporated at one or more ends of the nucleotide sequence of the RNAi agent disclosed herein. Capping residues can, in some cases, provide the RNAi agent with certain beneficial properties, such as protection from exonuclease degradation. In some embodiments, an inverted abasic residue (invAb) (also referred to in the art as an "inverted abasic site") is added as a capping residue (see Table A). (See, for example, F. Czauderna, Nucleic Acids Res., 2003, 31(11), 2705-16). Capping residues are generally known in the art and include, for example, inverted abasic residues and terminal C3H7 (propyl), C6H 13(hexyl), or C 12 H 25 contains a carbon chain such as (dodecyl) group. In some embodiments, the capping residue is present at the 5'-end, 3'-end, or both the 5'- and 3'-ends of the sense strand. In some embodiments, the 5'-end and / or 3'-end of the sense strand may contain two or more inverted abasic deoxyribose moieties as capping residues.

[0046] In some embodiments, one or more inverted abasic residues (invAb) are added to the 3'-end of the sense strand. In some embodiments, one or more inverted abasic residues (invAb) are added to the 5'-end of the sense strand. In some embodiments, one or more inverted abasic residues or inverted abasic sites are inserted between the target-directed ligand and the nucleotide sequence of the sense strand of the RNAi agent. In some embodiments, by including one or more inverted abasic residues or inverted abasic sites at or near the end(s) of the sense strand of the RNAi agent, it is possible to improve the activity of the RNAi agent or other desired properties.

[0047] In some embodiments, one or more inverted abasic residues (invAb) are added to the 5'-end of the sense strand. In some embodiments, one or more inverted abasic residues may be inserted between the target-directed ligand and the nucleotide sequence of the sense strand of the RNAi agent. The inverted abasic residue may be linked via a phosphate, phosphorothioate (e.g., as denoted herein as (invAb)s), or other internucleoside linkage. The chemical structures of the inverted abasic deoxyribose residues are shown in Table A below, as well as in the chemical structures shown in FIGS. 1A-1E and FIGS. 2A-2E.

[0048] [Table 1]

[0049] Target-directed moieties and target-directed groups The AAT RNAi agent can be conjugated to one or more non-nucleotide groups including, but not limited to, a targeting moiety or targeting group. The targeting moiety or targeting group can enhance the targeting or delivery of the RNAi agent. Examples of targeting moieties and targeting groups are known in the art. Specific examples of the targeting (NAG37)s group used in the AAT RNAi prodrug described in Table 2 herein containing three targeting N-acetyl-galactosamine moieties are shown in Table B. The targeting moiety or targeting group can be covalently attached to either the 3' and / or 5' end of either the sense strand and / or the antisense strand. In some embodiments, the AAT RNAi agent comprises a targeting group linked to the 3' and / or 5' end of the sense strand. In some embodiments, the targeting group is linked to the 5' end of the sense strand of the AAT RNAi agent. In some embodiments, the targeting group comprises, consists essentially of, or consists of the structure (NAG37)s and is linked to the 5' end of the sense strand of the AAT RNAi agent. The targeting group can be linked to the RNAi agent directly or indirectly via a linker / linkage group. In some embodiments, the targeting group is linked to the RNAi agent via a labile, cleavable, or reversible bond or linker. In some embodiments, the targeting group is linked to a reversed abasic residue at the 5' end of the sense strand.

[0050] The targeting group or targeting moiety can enhance the pharmacokinetic or biodistribution properties of the conjugate or RNAi agent to which they are attached, improving the cell-specific distribution and cellular uptake of the conjugate or RNAi agent. In some embodiments, the targeting group enhances the endocytosis of the RNAi agent. The targeting group can be monovalent, divalent, trivalent, tetravalent, or have a higher valence with respect to the target to which it is directed. Representative targeting groups include, but are not limited to, compounds having an affinity for cell surface molecules, cell receptor ligands, haptens, antibodies, monoclonal antibodies, antibody fragments, and antibody mimetics having an affinity for cell surface molecules.

[0051] In some embodiments, the target-directed group includes an asialoglycoprotein receptor ligand. In some embodiments, the asialoglycoprotein receptor ligand comprises or consists of one or more galactose derivatives. As used herein, the term galactose derivative includes both galactose and galactose derivatives having an affinity for the asialoglycoprotein receptor equal to or higher than that of galactose. Examples of galactose derivatives include, but are not limited to, galactose, galactosamine, N-formylgalactosamine, N-acetyl-galactosamine, N-propionyl-galactosamine, N-n-butanoyl-galactosamine, and N-iso-butanoyl-galactosamine (see, e.g., S. T. Iobst and K. Drickamer, J. B. C., 1996, 271, 6686). Galactose derivatives and clusters of galactose derivatives useful for targeting oligonucleotides and other molecules to the liver in vivo are known in the art (see, e.g., Baenziger and Fiete, 1980, Cell, 22, 611-620; Connolly et al., 1982, J. Biol. Chem., 257, 939-945).

[0052] Galactose derivatives have been used to target molecules to hepatocytes in vivo via binding to the asialoglycoprotein receptor expressed on the surface of hepatocytes. Binding of the asialoglycoprotein receptor ligand to the asialoglycoprotein receptor(s) promotes cell-specific targeting to hepatocytes and endocytosis of the molecule into hepatocytes. The asialoglycoprotein receptor ligand may be monomeric (e.g., having a single galactose derivative) or multimeric (e.g., having multiple galactose derivatives). The galactose derivative or “cluster” of galactose derivatives can be attached to the 3' or 5' end of the sense or antisense strand of the RNAi agent using methods known in the art.

[0053] In some embodiments, the target-directed group comprises a cluster of galactose derivatives. As used herein, a cluster of galactose derivatives includes molecules having 2 to 4 terminal galactose derivatives. The terminal galactose derivatives are attached to the molecule via their C-1 carbon. In some embodiments, the cluster of galactose derivatives is a trimer of galactose derivatives (also referred to as a branched trisaccharide galactose derivative or a trivalent galactose derivative). In some embodiments, the cluster of galactose derivatives comprises N-acetyl-galactosamine. In some embodiments, the cluster of galactose derivatives comprises 3 N-acetyl-galactosamines. In some embodiments, the cluster of galactose derivatives is a tetramer of galactose derivatives (also referred to as a branched tetrasaccharide galactose derivative or a tetravalent galactose derivative). In some embodiments, the cluster of galactose derivatives comprises 4 N-acetyl-galactosamines.

[0054] As used herein, a trimer of a galactose derivative comprises three galactose derivatives, each of which is linked to a central branching point. As used herein, a tetramer of a galactose derivative comprises four galactose derivatives, each of which is linked to a central branching point. The galactose derivative can be attached to the central branching point via the C-1 carbon of the sugar. In some embodiments, the galactose derivative is linked to the branching point via a linker or spacer. In some embodiments, the linker or spacer is a flexible hydrophilic spacer, such as, for example, a PEG group (see, e.g., U.S. Patent No. 5,885,968, Biessen et al. J. Med. Chem. 1995 Vol. 39 p. 1538-1546). The branching point can be any small molecule that enables the attachment of three galactose derivatives and further enables the attachment of the branching point to the RNAi agent. Examples of branching point groups are dilysine or diglutamic acid. The attachment of the branching point to the RNAi agent can be via a linker or spacer. In some embodiments, the linker or spacer is a flexible hydrophilic spacer, such as, but not limited to, a PEG spacer. In some embodiments, the linker comprises a rigid linker, such as, for example, a cyclic group. In some embodiments, the galactose derivative comprises or consists of N-acetyl-galactosamine. In some embodiments, the cluster of galactose derivatives is composed of a tetramer of galactose derivatives, which can be, for example, a tetramer of N-acetyl-galactosamine.

[0055] The preparation of a cluster of galactose derivatives containing a target-directed group, such as N-acetyl-galactosamine, is described, for example, in International Patent Application Publication No. WO2018 / 044350 (Patent Application No. PCT / US2017 / 021147) and International Patent Application Publication No. WO2007 / 156012 (Patent Application No. PCT / US2017 / 021175). The contents of both of these publications are hereby incorporated by reference in their entirety.

[0056] For example, the target-directed ligand conjugated to the AAT RNAi agent described in Tables 1A and 1B has the chemical structure (NAG37)s as shown in Table B below.

[0057]

Table 2

[0058] AAT RNAi agent and AAT RNAi drug substance (ADS-001) In some embodiments, the AAT RNAi agent used in the methods disclosed herein has the nucleotide sequence of the AAT RNAi drug substance (ADS-001) shown in Table 2. The nucleotide sequences of the AAT RNAi agents found in the AAT RNAi drug substance include the nucleotide sequence of the antisense strand described in Table 1A below, and the nucleotide sequence of the sense strand described in Table 1B below.

[0059]

Table 3

[0060]

Table 4

[0061] The following notations, used in Tables 1A, 1B, and 2 herein, are used to indicate modified nucleotides, target-directed groups, and linking groups: A, G, C, and U represent adenosine, cytidine, guanosine, or uridine, and a, c, g, and u represent 2'-O-methyladenosine, 2'-O-methylcytidine, 2'-O-methylguanosine, or 2'-O-methyluridine, respectively. Af, Cf, Gf, and Uf represent 2'-fluoroadenosine, 2'-fluorocytidine, 2'-fluoroguanosine, or 2'-fluorouridine, respectively. s represents a phosphorothioate bond, (invAb) represents an inverted deoxyribose residue lacking a base (see Table A), and (NAG37)s represents the structure shown in Table B above.

[0062] As will be readily understood by those skilled in the art, unless otherwise indicated (e.g., by phosphorothioate bond "s", etc.), when present in the chain, the monomers are linked to each other by 5'-3' phosphodiester bonds. As will be clearly understood by those skilled in the art, by including the phosphorothioate bonds shown in the modified nucleotide sequences disclosed herein, the phosphodiester bonds normally present in oligonucleotides are replaced. Further, it will be readily understood by those skilled in the art that the terminal nucleotide at the 3' end of a given oligonucleotide sequence normally has a hydroxyl (-OH) group at the 3' position of each monomer in place of the phosphate moiety in ex vivo. Further, in the embodiments disclosed herein, when each strand is viewed from 5'→3', the inverted abasic residue is inserted such that the 3' position of the deoxyribose is linked at the 3' end of the preceding monomer of each strand. Further, as will be readily understood and recognized by those skilled in the art, the chemical structure of the phosphorothioate shown herein normally exhibits an anion on its sulfur atom, but the present invention disclosed herein encompasses all phosphorothioate tautomers (e.g., when the sulfur atom has a double bond and the anion is on the oxygen atom). Unless otherwise clearly indicated herein, such understanding of those skilled in the art is used when describing the AAT RNAi agents and compositions containing the AAT RNAi agents disclosed herein.

[0063] Each sense strand and / or antisense strand may have any of the above-mentioned target-directed groups or linking groups conjugated to the 5' and / or 3' ends of the sequence, and other target-directed groups or linking groups.

[0064] The sequence of the antisense strand of the AAT RNAi agent is designed to target mRNA transcripts derived from both normal and mutant AAT genes, thereby silencing the translation of mutant Z-AAT protein using the RNA interference mechanism in human subjects with AATD.

[0065] In some embodiments, the methods disclosed herein use the AAT RNAi drug substance described in Table 2 below.

[0066]

Table 5

[0067] A schematic diagram of the AAT RNAi drug substance (ADS-001) is shown in Figure 3, and the representations of the complete chemical structures are shown in Figures 1A-1E (sodium salt form) and Figures 2A-2E (free acid form). In some embodiments, the AAT RNAi drug substance is prepared or provided as a salt, mixed salt, or free acid. In a preferred embodiment, the form is the sodium salt.

[0068] Pharmaceutical Compositions and Formulations AAT RNAi agents suitable for use in the methods disclosed herein can be formulated as pharmaceutical compositions or formulations for administration to human subjects. The pharmaceutical compositions can be used to treat subjects having a disease or disorder for which inhibition of AAT mRNA expression or reduction of AAT protein levels would be effective, e.g., human subjects having AATD. In some embodiments, the method comprises administering to a subject in need of treatment an AAT RNAi agent linked to a targeting group or targeting ligand described herein. In some embodiments, one or more pharmaceutically acceptable excipients (such as vehicles, carriers, diluents, and / or delivery polymers, etc.) are added to the pharmaceutical composition comprising the AAT RNAi agent, thereby forming a formulation suitable for in vivo delivery to a human subject.

[0069] A pharmaceutical composition comprising an AAT RNAi agent, when administered to a human subject using the methods disclosed herein, reduces the level of AAT mRNA in the subject.

[0070] In some embodiments, the pharmaceutical compositions described herein comprising an AAT RNAi agent are used to treat or manage clinical symptoms in subjects with AATD, such as increased risk of chronic hepatitis, cirrhosis, hepatocellular carcinoma, hypertransaminasemia, cholestasis, fibrosis, and even fulminant hepatic failure. In some embodiments, a therapeutically effective amount or prophylactically effective amount of one or more pharmaceutical compositions is administered to a subject in need of such treatment. In some embodiments, administration of any of the AAT RNAi agents of the present disclosure can be used to reduce the number, severity, and / or frequency of symptoms of a disease in a subject.

[0071] The pharmaceutical compositions described herein comprising an AAT RNAi agent can be used to treat at least one symptom in a subject having a disease or disorder in which reduction or inhibition of AAT mRNA expression would be effective. In some embodiments, the subject receives a therapeutically effective amount of administration of one or more pharmaceutical compositions comprising an AAT RNAi agent to treat the symptom. In other embodiments, the subject receives a prophylactically effective amount of administration of one or more AAT RNAi agents to prevent the at least one symptom.

[0072] The AAT RNAi agents disclosed herein are preparations appropriately adjusted for a particular route and can be administered via any suitable route. Accordingly, the pharmaceutical compositions described herein can be administered by injection, for example, intravenously or subcutaneously. In some embodiments, the pharmaceutical compositions described herein are administered via subcutaneous injection.

[0073] As used herein, a pharmaceutical composition or agent comprises a pharmacologically effective amount of at least one AAT RNAi agent and one or more pharmaceutically acceptable excipients. A pharmaceutically acceptable excipient (excipient) is a substance other than a pharmaceutical active ingredient (API, therapeutic agent, e.g., AAT RNAi agent) and is intentionally included in a drug delivery system. The excipient does not exert a therapeutic effect at the intended dosage and is not intended to do so. The excipient acts to a) assist in the processing of the drug delivery system during manufacture, b) protect, support, or enhance the stability, bioavailability, or patient acceptability of the API, c) assist in the identification of the formulation, and / or d) enhance any other attributes of the overall safety, efficacy, or delivery of the API during storage or use. A pharmaceutically acceptable excipient may or may not be an inert substance.

[0074] Excipients may include, but are not limited to, absorption promoters, antiadhesion agents, antifoaming agents, antioxidants, binders, buffers, carriers, coating agents, colorants, delivery promoters, delivery polymers, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavors, flow promoters, humectants, lubricants, oils, polymers, preservatives, physiological saline, salts, solvents, sugars, suspending agents, sustained release matrices, sweeteners, thickeners, isotonic agents, vehicles, water repellents, and wetting agents.

[0075] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (when water-soluble). For subcutaneous or intravenous administration, suitable carriers may include physiological saline, bacteriostatic water, Cremophor® ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). This should be stable under the conditions of manufacture and storage and should be protected against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium including water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

[0076] The sterile injectable solution can be prepared by placing the required amount of the active compound in a suitable solvent, together with one or a combination of the ingredients enumerated above as required, and then filtering and sterilizing. Generally, the dispersion is prepared by placing the active compound in a sterile medium that contains a basic dispersion medium and other ingredients required from those enumerated above.

[0077] In some embodiments, the pharmaceutical composition suitable for use in the methods disclosed herein comprises the ingredients specified in Formulation AAT RNAi drug substance shown in Table 3 below.

[0078] For ease of administration and uniformity of dosage, the AAT RNAi agent can be formulated in unit dosage form in the composition. The unit dosage form refers to physically discrete units suitable as a single dosage for a subject to be treated, and each unit contains a predetermined quantity of the active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. In some embodiments, the unit dosage is from about 5 mg to about 300 mg of the AAT RNAi drug substance. In some embodiments, the unit dosage is from about 25 mg to about 200 mg of the AAT RNAi drug substance. In some embodiments, the unit dosage is from about 100 mg to about 200 mg of the AAT RNAi drug substance. In some embodiments, the unit dosage is about 100 mg of the AAT RNAi drug substance. In some embodiments, the unit dosage is about 200 mg of the AAT RNAi drug substance.

[0079] The pharmaceutical composition can include other additional ingredients commonly found in pharmaceutical compositions. Such additional ingredients include, but are not limited to, antipruritics, astringents, local anesthetics, or anti-inflammatory agents (such as antihistamines, diphenhydramine, etc.).

[0080] As used herein, "pharmacologically effective amount", "therapeutically effective amount", or simply "effective amount" refers to an amount of the RNAi agent that produces a pharmacological, therapeutic, or prophylactic effect.

[0081] The pharmaceutically acceptable formulations described herein can be packaged in kits, containers, packs, or dispensers. The pharmaceutical compositions described herein can be packaged in prefilled syringes or vials.

[0082] Formulated AAT RNAi Prodrug In some embodiments, the AAT RNAi prodrug (ADS-001) shown in Table 2 is formulated with one or more pharmaceutically acceptable excipients to form a pharmaceutical composition suitable for administration to a human subject. In some embodiments, the AAT RNAi prodrug described in Table 2 is formulated at 230 mg / mL in aqueous sodium phosphate buffer (0.5 mM sodium dihydrogen phosphate, 0.5 mM disodium hydrogen phosphate) to form the formulated AAT RNAi prodrug (ADS-001-1) shown in Table 3.

[0083]

Table 6

[0084] The formulated AAT RNAi prodrug of Table 3 is prepared as a sterile formulation. In some embodiments, the formulated AAT RNAi prodrug is packaged in a container, such as a glass vial. In some embodiments, the formulated AAT RNAi prodrug is packaged in a glass vial having a volume of about 1.1 mL, and the volume desired for administration can be calculated based on the dosage level desired for administration.

[0085] In some embodiments, the formulated AAT RNAi prodrug described in Table 3 is administered to a human subject using the methods disclosed herein.

[0086] Human Subjects with AATD and Diagnosis of AATD The methods disclosed herein include treating a human subject in need of treatment for alpha-1 antitrypsin deficiency (AATD), including treating symptoms and diseases caused by AATD in the human subject using a pharmaceutical composition comprising the AAT RNAi prodrug described in Table 2. In some embodiments, the human subject is administered after being diagnosed with AATD. As described herein, AATD is a genetic disorder caused by mutations in gene transcripts that result in translation of mutant forms of the AAT protein that are prone to misfolding and lead to intracellular retention in hepatocytes. Various mutations in the SERPINA1 gene have been identified, but the PiZZ genotype, the most common and severe form of AATD, is caused by a single base pair substitution. In subjects with the PiZZ genotype, blood AAT levels are often reported to be less than 15% of healthy human levels. Often, patients are initially diagnosed with COPD, asthma, or other lung diseases without identification of the underlying disease. Over time, liver diseases, such as fibrosis and cirrhosis, can develop due to intracellular retention of misfolded (“Z-AAT”) protein and the inability to properly secrete the protein from hepatocytes. Pediatric patients typically present with clinical symptoms of liver disease, which can include asymptomatic chronic hepatitis, growth impairment, anorexia, or hepatomegaly and splenomegaly. AATD can be diagnosed and confirmed by standard genotyping of a subject's blood sample.

[0087] Administration and Inhibition of AAT Gene Expression Generally, the effective amount of the AAT RNAi agent ranges from about 0.1 to about 10 mg / kg body weight / day, for example, in the range of about 0.25 to about 5 mg / kg body weight / day. In some embodiments, the effective amount of the AAT RNAi agent ranges from about 0.5 to about 4 mg / kg body weight per dose. In some embodiments, the effective amount is a fixed dose. In some embodiments, a fixed dose of 5 mg to 300 mg of the AAT RNAi drug substance is the effective dose. In some embodiments, a fixed dose of 25 mg to 200 mg of the AAT RNAi drug substance is the effective dose. The dosage may depend on variable factors such as the overall age and health of the patient, the relative biological efficiency of the compound being delivered, the formulation of the drug, the presence and type of excipients in the formulation, and the route of administration. In some embodiments, a fixed dose of about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200, 220, 240, 260, or 280 mg to about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280 or 300 mg is the effective dose. In some embodiments, a fixed dose of about 25 mg, about 100 mg, or about 200 mg is the effective dose.

[0088] Also, it is understood that the initial dosage administered may, in some cases, be increased above the upper limit levels described above to rapidly achieve the desired blood or tissue levels, or the initial dosage may, in some cases, be made less than the optimal amount. For example, in some embodiments, an initial dose of about 25 mg to about 200 mg of the AAT RNAi drug substance is administered, followed by a second dose of about 25 to 200 mg of the AAT RNAi drug substance approximately 1 month later, and thereafter additional doses (similar concept to "maintenance doses") are administered once every 3 months (i.e., quarterly).

[0089] Regarding the treatment of a disease or the production of an agent or composition for the treatment of a disease, the pharmaceutical composition described herein comprising an AAT RNAi agent can be combined with an excipient or a second or other therapeutic agent including, but not limited to, a second RNAi agent, a small molecule drug, an antibody, an antibody fragment, a peptide, and / or an aptamer.

[0090] In some embodiments, the gene expression level and / or mRNA level of the AAT gene in a subject to which the described AAT RNAi agent is administered is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99%, or more than 99% lower compared to the subject before the administration of the AAT RNAi agent or a subject not receiving the AAT RNAi agent. The gene expression level and / or mRNA level in the subject is reduced in the cells, cell populations, and / or tissues of the subject.

[0091] In some embodiments, the level of the AAT protein in a subject administered the described AAT RNAi agent is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more than 99% lower compared to the subject before the administration of the AAT RNAi agent or a subject not receiving the AAT RNAi agent. The level of the protein in the subject is reduced in the cells, cell populations, tissues, blood, and / or other body fluids of the subject.

[0092] In some embodiments, the level of Z-AAT polymeric protein in a subject having AATD administered with the described AAT RNAi agent is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more than 99% lower than that in the subject before administration of the AAT RNAi agent or in a subject not receiving the AAT RNAi agent. In some embodiments, the level of Z-AAT polymeric protein in a subject administered with the described AAT RNAi agent is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more than 99% lower than that in the subject before administration of the AAT RNAi agent or in a subject not receiving the AAT RNAi agent.

[0093] The decrease in the expression of the AAT gene, the level of AAT mRNA, or the level of AAT protein can be evaluated and quantified by common methods known in the art. The examples disclosed herein are generally known methods for evaluating the inhibition of AAT gene expression and the decrease in the level of AAT protein. The decrease or reduction in the level of the AAT mRNA and / or the level of the protein (such as Z-AAT polymer and / or monomer, etc.) is collectively referred to herein as the decrease or reduction of AAT, or the inhibition or decrease in the expression of AAT.

[0094] As used herein, the terms "treating", "treatment", etc. mean a method or step performed to provide a reduction or alleviation in the number, severity, and / or frequency of one or more symptoms of a disease in a subject. As used herein, "treating" and "treatment" can include the prevention, management, prophylactic treatment, and / or inhibition of the number, severity, and / or frequency of one or more symptoms of a disease in a subject.

[0095] As used herein, the administration of "once a month" or "monthly" means every 28 days. As used herein, the administration of "every three months" or "every three months" means every 84 days. The term "about" when used in connection with once-a-month administration means once-a-month administration + / - 3 days. The term "about" when used in connection with administration every three months means administration every three months + / - 9 days.

[0096] As used herein, the phrase "introduce into a cell" when referring to an RNAi agent means functionally delivering the RNAi agent into the cell. The phrase "functional delivery" means delivering the RNAi agent into the cell in such a way that the RNAi agent has the expected biological activity, for example, sequence-specific inhibition of gene expression.

[0097] Unless otherwise specified, the use of the symbol

[0098]

Chemical formula

[0099] means that any group(s) can be linked thereto in accordance with the scope of the invention described herein.

[0100] As used herein, for each structure in which an asymmetric center is present and thus gives rise to enantiomers, diastereomers, or other stereoisomeric configurations, unless specifically specified as having a particular conformation in the structure, each structure disclosed herein is intended to represent all such possible isomers, including their optically pure forms and racemates. For example, the structures disclosed herein are intended to include not only single stereoisomers but also mixtures of diastereomers.

[0101] As used in the claims of this specification, the phrase "consisting of" excludes any element, step, or component not specified in the claim. As used in the claims of this specification, the phrase "consisting essentially of" limits the claim to those materials or steps and to other materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0102] One of ordinary skill in the art will readily understand and appreciate that the compounds and compositions disclosed herein may have a particular atom (e.g., an N, O, or S atom) in a protonated or deprotonated state depending on the environment in which the compound or composition is placed. Accordingly, the structures disclosed herein, as used herein, are intended to encompass the case where certain functional groups, such as OH, SH, or NH, etc., may be protonated or deprotonated. As will be readily understood by one of ordinary skill in the art, the disclosure herein is intended to include the compounds and compositions of the present disclosure regardless of the state of protonation based on the environment (e.g., pH, etc.).

[0103] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0104] The above-described embodiments and items are explained herein by the following non-limiting examples.

Example

[0105] Example 1. Synthesis and Formulation of AAT RNAi Prodrug (ADS-001) The AAT RNAi drug substance suitable for use in the methods disclosed herein can be synthesized using standard phosphoramidite techniques in solid-phase oligonucleotide synthesis known in the art. A commercially available oligonucleotide synthesizer (e.g., MerMade96E® (Bioautomation) or MerMade12® (Bioautomation)) may be used. Synthesis can be performed on a solid support made of controlled pore glass (CPG, 500 Å or 600 Å, obtained from Prime Synthesis, Aston, PA, USA). The monomer placed at the 3'-end of each strand can be coupled to this solid support as the starting point of synthesis. All RNAs, 2'-modified RNA phosphoramidites, and inverted deoxy base phosphoramidites are commercially available. Target-directed group-containing phosphoramidites suitable for addition to the 5'-end of the sense strand can be synthesized. Standard cleavage, deprotection, purification, and annealing steps known in the art can be used. Further descriptions regarding the synthesis of the AAT RNAi agent can be found, for example, in International Patent Application Publication Nos. WO2018 / 132432 (Application No. PCT / US2018 / 013102) and WO2018 / 044350 (PCT / US2017 / 021147), each of which is incorporated herein by reference in its entirety. The AAT RNAi drug substance can then be formulated by dissolving it in standard pharmaceutically acceptable excipients generally known in the art. For example, Table 3 shows the formulated AAT RNAi drug substance suitable for use in the methods disclosed herein.

[0106] Example 2. Phase I Clinical Trial of AAT RNAi Drug Substance (ADS-001) in Healthy Human Volunteers (NHV) A Phase 1, single- and multiple-dose escalation study was conducted to evaluate the safety, tolerability, pharmacokinetics, and effect on serum AAT levels of the AAT RNAi drug substance (ADS-001) in healthy human volunteers (NHV). The study population included healthy adult men and women aged 18 - 52 years with a BMI of 19.0 - 35.0 kg / m 2 2.

[0107] The NHV subjects were divided into a total of seven cohorts. Cohorts 1 to 4 were randomized to receive the AAT RNAi drug substance or placebo (Active Substance 4: Placebo 4) by subcutaneous injection as a single ascending dose of 35 mg (Cohort 1) and multiple ascending doses of 100 mg (Cohort 2), 200 mg (Cohort 3), and 300 mg (Cohort 4). Cohorts 1 to 4 were double-blind. Cohorts 2b, 3b, and 4b were open-label and consisted of four subjects each receiving a single dose of 100, 200, and 300 mg of the AAT RNAi drug substance. A total of 44 subjects completed the study. Figure 4 shows the final study design for the Phase I clinical trial. The study parameters are summarized in Table 4 below.

[0108] Table 4. Phase I Clinical Trial Parameters

Table 7-1

[0109]

Table 7-2

[0110]

Table 7-3

[0111] The results of the decrease in serum AAT in this study showed that administration of the AAT RNAi drug substance at doses of 35 - 300 mg significantly decreased serum AAT compared to placebo. Initially, a cohort at 400 mg of AAT RNAi drug substance per dose was proposed as part of this clinical trial protocol. However, considering the unexpected efficacy at doses of 35, 100, 200, and 300 mg, the 400 mg cohort was removed from the protocol of this study. In this Phase I study, serum AAT significantly decreased at doses of 35 mg, 100 mg, and 200 mg, and after multiple administrations at both 100 mg and 200 mg, the mean serum AAT decrease reached approximately 90%. Figures 5 - 11 report the serum AAT decrease in various cohorts in this Phase I study.

[0112] Surprisingly and unexpectedly, there was no clear dose - dependent response across all dose levels as significant (reaching approximately 90%) and similar knockdowns to the higher dose of 300 mg occurred at dose levels of 100 mg and 200 mg. The lowest dose of 35 mg was also quite active but was not as active as the 100 mg administered as a single dose and showed some degree of dose response.

[0113] The duration of the decrease in serum AAT (>58%) from a single administration of 35 mg was longer than initially expected, lasting up to 16 weeks after dose administration and then returning to baseline. For example, 34 weeks after a single administration of 35 mg, the serum AAT level of one subject returned to above 90 mg / dL, while the serum AAT level of the second subject remained at 40 mg / dL (60.4% decrease from baseline). There was no significant difference in the duration of response following a single administration of 100 mg - 300 mg of AAT RNAi drug substance, which returned to baseline between 8 - 16 weeks after a single administration.

[0114] Multiple administrations of the AAT RNAi drug substance generally maintain a substantial decrease in serum AAT over a longer period than a single administration. These data suggest that the second administration, received on day 29 (i.e., one month after the first administration), may further decrease or maintain the decrease in serum AAT levels, and subsequent administrations may be administered every 12 weeks (i.e., every three months) to maintain the maximum decrease in serum AAT.

[0115] In this Phase I trial, there were no deaths, serious adverse events (SAEs), or adverse events (AEs) evaluated as severe. Two subjects out of all subjects who received the AAT RNAi drug substance reported that the intensity of three AEs was moderate (upper respiratory tract infection, rhinorrhea, general chest pain). Three subjects out of all subjects who received placebo reported that the intensity of three AEs was moderate (gastroenteritis 2, musculoskeletal chest pain - left side). All other AEs were reported as mild. The majority of subjects reported AEs not related to the test treatment. One AE that occurred in a subject during AAT administration led to early termination of the treatment, but this subject was continued to be followed up in the trial. A total of 94 AEs were reported in 28 subjects who received at least a single - dose formulation of the AAT RNAi drug substance. 46 AEs were reported in 17 subjects who received placebo. There was no clear pattern of increase in the frequency or intensity of AEs with increasing dose.

[0116] In the entire cohort of the fully formulated AAT RNAi drug substance, six AEs at the injection site occurred in six subjects, and all of them occurred in subjects who received the drug. There were no injection - site AEs in placebo subjects. The reported injection - site reactions included intradermal hemorrhage, erythema, and pain at the injection site. These combined AEs at the injection site were reported in 21.4% of the subjects who received the formulated AAT RNAi drug substance. Six out of 50 injections of the formulated AAT RNAi drug substance, i.e., 12%, resulted in injection - site AEs. There were no injection - site AEs reported more than once in a single subject. All injection - site AEs were considered to be of mild intensity.

[0117] Other embodiments Although the present invention has been described in conjunction with the forms for carrying out the invention of the present invention, the foregoing description is intended to explain and not to limit the scope of the present invention, and the scope of the present invention should be understood to be defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. 1. A pharmaceutical composition for treating alpha-1 antitrypsin deficiency (AATD) in a human subject in need thereof, comprising an AAT RNAi drug substance, wherein the pharmaceutical composition is administered to the patient at a dose of about 100 mg to about 300 mg of the AAT RNAi drug substance; wherein the AAT RNAi drug substance comprises a sense strand having the structure of (NAG37)s(invAb)sagcguuuaGfGfCfauguuuaacas(invAb) (SEQ ID NO: 6) and an antisense strand having the structure of usGfsuUfaAfacaugCfcUfaAfaCfgCfsu (SEQ ID NO: 2), and the sense strand and the antisense strand are annealed to form a duplex; where a, c, g, and u represent 2'-O-methyl adenosine, 2'-O-methyl cytidine, 2'-O-methyl guanosine, and 2'-O-methyl uridine, respectively; Af, Cf, Gf, and Uf represent 2'-fluoro adenosine, 2'-fluoro cytidine, 2'-fluoro guanosine, and 2'-fluoro uridine, respectively; s represents a phosphorothioate bond; (invAb) represents an inverted abasic deoxyribose residue; and (NAG37)s represents the following structure: 【Chemical 1】 represents The pharmaceutical composition, wherein the pharmaceutical composition is administered by subcutaneous injection once every 84 days.

2. 1. A pharmaceutical composition for treating alpha-1 antitrypsin deficiency (AATD) in a human subject in need thereof, comprising an AAT RNAi drug substance, wherein the AAT RNAi drug substance comprises a sense strand having the structure of (NAG37)s(invAb)sagcguuuaGfGfCfauguuuaacas(invAb) (SEQ ID NO: 6) and an antisense strand having the structure of usGfsuUfaAfacaugCfcUfaAfaCfgCfsu (SEQ ID NO: 2), and the sense strand and the antisense strand are annealed to form a duplex; where a, c, g, and u represent 2'-O-methyl adenosine, 2'-O-methyl cytidine, 2'-O-methyl guanosine, and 2'-O-methyl uridine, respectively; Af, Cf, Gf, and Uf represent 2'-fluoro adenosine, 2'-fluoro cytidine, 2'-fluoro guanosine, and 2'-fluoro uridine, respectively; s represents a phosphorothioate bond; (invAb) represents an inverted abasic deoxyribose residue; and (NAG37)s represents the following structure: 【Chemistry 2】 represents an initial dose of the pharmaceutical composition is administered to the subject by subcutaneous injection at a dose of about 100 mg to about 300 mg of the AAT RNAi drug substance; a second dose of the pharmaceutical composition is administered to the subject by subcutaneous injection at a dose of about 100 mg to about 300 mg of the AAT RNAi drug substance about 28 days after the first dose; and A third dose of the pharmaceutical composition is administered to the subject by subcutaneous injection about 84 days after the second dose at a dose of about 100 mg to about 300 mg of the AAT RNAi drug substance.

3. 10. The pharmaceutical composition of claim 1, wherein the dose of the AAT RNAi drug substance is from about 100 mg to about 200 mg.

4. 10. The pharmaceutical composition of claim 1, wherein the dose of the AAT RNAi drug substance is from about 180 mg to about 240 mg.

5. 10. The pharmaceutical composition of claim 1, wherein the dose of the AAT RNAi drug substance is about 100 mg.

6. 10. The pharmaceutical composition of claim 1, wherein the dose of the AAT RNAi drug substance is about 200 mg.

7. 10. The pharmaceutical composition of claim 1, wherein the dose of the AAT RNAi drug substance is about 200 mg or less.

8. 3. The pharmaceutical composition of claim 2, wherein each dose of the AAT RNAi drug substance is about 100 mg to about 200 mg.

9. 3. The pharmaceutical composition of claim 2, wherein each dose of the AAT RNAi drug substance is from about 180 mg to about 240 mg.

10. 3. The pharmaceutical composition of claim 2, wherein each dose of the AAT RNAi drug substance is about 100 mg.

11. 3. The pharmaceutical composition of claim 2, wherein each dose of the AAT RNAi drug substance is about 200 mg.

12. 3. The pharmaceutical composition of claim 2, wherein each dose of the AAT RNAi drug substance is about 200 mg or less.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the condition or disease caused by AATD is a liver disease.

14. 14. The pharmaceutical composition of claim 13, wherein the liver disease is chronic hepatitis, cirrhosis, increased risk of hepatocellular carcinoma, hypertransaminasemia, cholestasis, fibrosis, or fulminant hepatic failure.

15. The pharmaceutical composition of any one of claims 1 to 14, wherein the subject is further administered an additional therapeutic agent for the treatment of AATD.

16. 16. The pharmaceutical composition of any one of claims 1 to 15, further comprising a therapeutic agent for the treatment of lung disorders, emphysema, or other lung diseases or disorders caused by a deficiency in endogenously secreted AAT protein.

17. 17. The pharmaceutical composition of claim 16, wherein the therapeutic agent comprises human AAT protein, purified human alpha-1 protease inhibitor, or recombinant AAT protein.

18. The pharmaceutical composition of any one of claims 1 to 17, wherein the pharmaceutical composition is packaged in a kit, container, pack, dispenser, pre-filled syringe, or vial.

19. 19. The pharmaceutical composition of any one of claims 1-18, wherein the pharmaceutical composition comprises, consists of, or consists essentially of a formulated AAT RNAi drug substance as set forth in Table 3.

20. 3. The pharmaceutical composition of claim 2, further comprising administering additional doses after administration of the third dose, wherein the additional doses are administered at intervals of about 84 days.

21. The pharmaceutical composition of any one of claims 1 to 20, wherein one or more doses of the pharmaceutical composition are administered by the subject.

22. The pharmaceutical composition of any one of claims 1 to 20, wherein administration of one or more doses of the pharmaceutical composition is performed by a medical professional.

23. 1. Use of an AAT RNAi drug substance for the manufacture of a medicament for treating alpha-1 antitrypsin deficiency (AATD) in a human subject in need thereof, comprising: wherein the AAT RNAi drug substance comprises a sense strand having the structure of (NAG37)s(invAb)sagcguuuaGfGfCfauguuuaacas(invAb) (SEQ ID NO: 6) and an antisense strand having the structure of usGfsuUfaAfacaugCfcUfaAfaCfgCfsu (SEQ ID NO: 2), and the sense strand and the antisense strand are annealed to form a duplex; where a, c, g, and u represent 2'-O-methyl adenosine, 2'-O-methyl cytidine, 2'-O-methyl guanosine, and 2'-O-methyl uridine, respectively; Af, Cf, Gf, and Uf represent 2'-fluoro adenosine, 2'-fluoro cytidine, 2'-fluoro guanosine, and 2'-fluoro uridine, respectively; s represents a phosphorothioate bond; (invAb) represents an inverted abasic deoxyribose residue; and (NAG37)s represents the following structure: 【Chemistry 3】 represents The treatment comprises administering the medicament to the subject by subcutaneous injection once every 84 days at a dose of about 100 mg to about 300 mg of the AAT RNAi drug substance.

24. 1. A pharmaceutical composition for treating alpha-1 antitrypsin deficiency (AATD) in a human subject in need thereof, comprising an AAT RNAi drug substance, wherein the AAT RNAi drug substance comprises a sense strand having the structure of (NAG37)s(invAb)sagcguuuaGfGfCfauguuuaacas(invAb) (SEQ ID NO: 6) and an antisense strand having the structure of usGfsuUfaAfacaugCfcUfaAfaCfgCfsu (SEQ ID NO: 2), and the sense strand and the antisense strand are annealed to form a duplex; where a, c, g, and u represent 2'-O-methyl adenosine, 2'-O-methyl cytidine, 2'-O-methyl guanosine, and 2'-O-methyl uridine, respectively; Af, Cf, Gf, and Uf represent 2'-fluoro adenosine, 2'-fluoro cytidine, 2'-fluoro guanosine, and 2'-fluoro uridine, respectively; s represents a phosphorothioate bond; (invAb) represents an inverted abasic deoxyribose residue; and (NAG37)s represents the following structure: 【Chemistry 4】 represents an initial dose of the pharmaceutical composition is administered to the subject by subcutaneous injection at a dose of about 200 mg of the AAT RNAi drug substance; a second dose of the pharmaceutical composition is administered to the subject by subcutaneous injection at a dose of about 200 mg of the AAT RNAi drug substance about 28 days after the first dose; and A third dose of the pharmaceutical composition is administered to the subject at a dose of about 200 mg of the AAT RNAi drug substance by subcutaneous injection about 84 days after the second dose.

25. The pharmaceutical composition of any one of claims 1 to 22 and 24, wherein the AAT RNAi drug substance is provided as a salt, mixed salt or free acid.

26. The pharmaceutical composition of claim 25, wherein the AAT RNAi drug substance is provided as a sodium salt.

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