RU2847618C2 - Methods of treating alpha-1-antitrypsin (aatd) deficiency - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to medicine, specifically to methods of treating alpha-1-antitrypsin deficiency (AATD) in a person in need thereof. Method of treating AATD in a human in need thereof comprises administering to a subject by subcutaneous injection of doses of a pharmaceutical composition comprising from about 100 mg to about 300 mg of an AAT RNA drug substance, wherein the pharmaceutical composition is administered once every 84 days. Wherein the RNAi AAT medicinal substance contains a sense chain comprising the structure (NAG37)s(invAb)sagcguuuaGfGfCfauguuuaacas(invAb) (SEQ ID NO: 6), and an antisense chain comprising the structure usGfsuUfaAfacaugCfcUfaAfaCfgCfsu (SEQ ID NO: 2), where the sense and anti-sense chains are hybridised to form a duplex, where a, c, g and u are 2'-O-methyladenosine, 2'-O-methylcytidine, 2'-O-methylguanosine and 2'-O-methyluridine, respectively; Af, Cf, Gf and Uf are 2'-fluoroadenosine, 2'-fluorocytidine, 2'-fluoroguanosine and 2'-fluorouridine, respectively; s is a phosphorothioate bond; (invAb) is an inverted deoxyribose residue with a remote base. Group of inventions also relates to embodiments of said method and use of said RNAi AAT drug for treating AATD in a person in need thereof.

EFFECT: invention provides the effective treatment with less frequent dosing of the therapeutic agent that leads to higher therapeutic adherence and improves a drug safety profile.

23 cl, 11 dwg, 4 tbl, 2 ex

Description

List of sequences

[1] This application contains a sequence listing, which has been provided in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy is named 30674_WO1_SequenceListing.txt and is 6 KB in size.

Field of technology

[2] Disclosed herein are methods for treating alpha-1 antitrypsin deficiency (AATD) in a human subject, including treating symptoms and diseases caused by AATD, using pharmaceutical compositions comprising RNA interference (RNAi) agents that inhibit the expression of the alpha-1 antitrypsin gene.

State of the art

[3] Alpha-1-antitrypsin (AAT, α1-antitrypsin, or A1AT) is a protease inhibitor belonging to the serpin superfamily and encoded in humans by the SERPINA1 gene. Normal AAT protein is a circulating glycoprotein protease inhibitor, primarily synthesized in the liver by hepatocytes and secreted into the blood. A known physiological function of AAT is the inhibition of neutrophil proteases, which provides protection of body tissues from nonspecific damage during periods of inflammation.

[4] Alpha-1 antitrypsin deficiency (AATD) is an autosomal codominant inherited disorder that results in decreased circulating levels of AAT and causes early-onset lung disease in adults and liver disease in children and adults. The prevalence of AATD is approximately 1 in 1,500–5,000 people and most commonly affects people of European descent.

[5] The most clinically significant form of AATD is caused by the Z mutation. The mutant Z allele, a single-point mutation, produces a mutant Z-form of the AAT protein (Z-AAT protein), which is prone to abnormal folding, leading to its intracellular retention in the endoplasmic reticulum (ER) of hepatocytes. Other, rarer mutations also lead to protein misfolding and accumulation in hepatocytes. Monomers of the mutant Z-AAT protein can assemble into polymeric aggregates, sometimes called “globules.” The polymeric globular masses cause ER stress and initiate a cycle of continuous damage and repair in the hepatocyte, leading to fibrosis, cirrhosis, and an increased risk of developing hepatocellular carcinoma. Furthermore, in the absence of antiprotease activity in the circulating blood, the lung becomes vulnerable to damage by neutrophil elastase, particularly in the setting of lung inflammation, leading to the development of pulmonary complications such as emphysema or other pulmonary disease.

[6] Individuals with the homozygous PiZZ genotype have a severe deficiency of functional AAT. Weekly administration of AAT replacement therapy with purified human AAT prevents lung damage in affected individuals. Such products currently available on the market include, for example, Prolastin®-C, Prolastin®, Glassia ^™ , Aralast® NP, and Zemaira®. However, although administration of purified AAT can ameliorate or prevent lung damage caused by the absence or low levels of endogenously secreted AAT, patients with AATD (with a polymer-forming AAT mutation) remain susceptible to endoplasmic reticulum storage disease (ERSD), caused by the deposition or accumulation of excess amounts of misfolded AAT protein. The accumulation of Z-AAT protein in a globular conformation in hepatocytes is a well-known histological characteristic of the liver disease AATD and is thought to lead to proteotoxic effects that induce liver injury, including liver cell damage and death, as well as chronic liver damage in individuals with AATD ( see, e.g. , D. Lindblad et al., Hepatology 2007, 46: 1228-1235). Patients with the null/null phenotype without AAT production have been reported to develop severe pulmonary disease while maintaining normal liver morphology, suggesting that it is the accumulation of mutant AAT, rather than a lack of circulating 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).

[7] AATD predisposes to the development of liver disease in children and adults, as well as to early-onset emphysema in adults. Patients with AATD frequently develop liver disease, which can be severe or fatal, even in childhood. Because some patients with AATD do not present with the disease at an early stage, fibrous tissue accumulates in the liver over time, leading to the development of clinically evident liver disease. Clinical manifestations of liver disease include chronic hepatitis, cirrhosis, increased risk of hepatocellular carcinoma, elevated transaminases, cholestasis, fibrosis, and even fulminant liver failure.

[8] It has been clearly established that accumulation of Z-AAT protein globules in hepatocytes is the cause of progressive liver disease in patients with AATD. Elimination of mutant protein accumulations from hepatocytes can halt the progression of liver disease. Elimination of the damaging effects of the mutant protein also promotes regression of already developed fibrosis. Currently, there is no clinically approved treatment to prevent disease onset, slow progression, or otherwise treat liver disease caused by AATD.

[9] RNAi agents have emerged as a promising treatment option for patients with AATD. Dosing strategies are an important aspect of treating AATD with RNAi agents. Less frequent dosing is preferred by patients and leads to increased compliance, and reduced dosing may be beneficial for the overall safety profile of the drug. Thus, there is a need for a treatment option for AATD with low, infrequent doses.

The essence of the invention

[10] Disclosed herein are methods for treating alpha-1 antitrypsin deficiency (AATD) in a human subject in need thereof. In one aspect, the methods comprise administering to the human subject a pharmaceutical composition that comprises the composition described in Table 2 (i.e., the AAT RNAi drug substance, also referred to herein as ADS-001) at a dose between about 5 mg and about 300 mg of the AAT RNAi drug substance, wherein the pharmaceutical composition is administered subcutaneously and there is a time interval of at least about one month between doses (i.e., at least monthly dosing). In some embodiments, the pharmaceutical composition used in the methods disclosed herein comprises, consists of, or consists essentially of a ready-to-use AAT RNAi drug substance as described in Table 3 (also referred to herein as ADS-001-1).

[11] Further described herein are methods of treating AATD in a human subject in need thereof, comprising administering to the human subject a pharmaceutical composition that comprises an AAT RNAi drug as described in Table 2 (i.e., ADS-001) at a dose between about 5 mg and about 200 mg, wherein the pharmaceutical composition is administered subcutaneously and there is a time interval of at least about one month between dose administrations (i.e., as monthly dosing).

[12] Further described herein are methods of treating AATD in a human subject in need thereof, comprising administering to the human subject a pharmaceutical composition that comprises an AAT RNAi drug as described in Table 2 (i.e., ADS-001) at a dose between about 5 mg and about 300 mg, wherein the pharmaceutical composition is administered subcutaneously and there is a time interval of about three months between doses (i.e., as quarterly dosing).

[13] Also described herein are methods of treating AATD in a human subject in need thereof, comprising administering to the human subject a pharmaceutical composition that comprises an AAT RNAi drug substance as described in Table 2 (i.e., ADS-001) at a dose between about 5 mg and about 200 mg, wherein the pharmaceutical composition is administered subcutaneously and there is a time interval of about three months between dose administrations (i.e., as quarterly dosing).

[14] Described herein are methods for treating AATD in a human subject in need thereof, comprising administering to the human subject a pharmaceutical composition that comprises an AAT RNAi drug as described in Table 2 (i.e., ADS-001) at a dose between about 5 mg and about 300 mg, wherein the pharmaceutical composition is administered subcutaneously, and wherein the initial dose is followed by a second dose about one month later, and then subsequent doses with a time interval of about three months between dose administrations.

[15] Described herein are methods for treating AATD in a human subject in need thereof, comprising administering to the human subject a pharmaceutical composition that comprises an AAT RNAi drug as described in Table 2 (i.e., ADS-001) at a dose between about 5 mg and about 200 mg, wherein the pharmaceutical composition is administered subcutaneously, and wherein the initial dose is followed by a second dose about one month later, and then subsequent doses with a time interval of about three months between dose administrations.

[16] In some embodiments, the dose of the AAT RNAi drug administered with each dose is between about 25 mg and about 200 mg. In some embodiments, the dose of the AAT RNAi drug administered with each dose is between about 100 mg and about 200 mg. In some embodiments, the dose of the AAT RNAi drug administered with each dose is about 100 mg. In some embodiments, the dose of the AAT RNAi drug administered with each dose is about 200 mg. In some embodiments, the dose of the AAT RNAi drug administered with each dose is no more than 200 mg.

[17] The treatment methods disclosed herein may slow or halt the progression of liver disease in a human subject with AATD, which may promote the restoration of fibrotic tissue. The methods disclosed herein may, in some embodiments, be used to treat AATD-associated liver diseases, including fibrosis, cirrhosis, increased risk of hepatocellular carcinoma, chronic hepatitis, elevated transaminases, cholestasis, fulminant liver failure, and other diseases and conditions associated with liver injury and caused by AATD.

[18] Pharmaceutical compositions comprising the AAT RNAi agents disclosed herein can be administered to a human subject to suppress the expression of the alpha-1 antitrypsin gene in the subject. In some embodiments, the subject is a human previously diagnosed with AATD.

[19] In another aspect of the invention, there is provided the use of the AAT RNAi drug substance described in Table 2 for the treatment of alpha-1-antitrypsin deficiency (AATD) in a human subject in need thereof, wherein the use comprises administering to the patient a pharmaceutical composition that comprises the AAT RNAi drug substance described in Table 2 at a dose of between about 5 mg and about 300 mg of the AAT RNAi drug substance, wherein the pharmaceutical composition is administered once per month by subcutaneous injection.

[20] In another aspect of the invention, there is provided the use of the AAT RNAi drug substance described in Table 2 for the treatment of alpha-1-antitrypsin deficiency (AATD) in a human subject in need thereof, wherein the use comprises administering to the patient a pharmaceutical composition that comprises the AAT RNAi drug substance described in Table 2 at a dose of between about 5 mg and about 300 mg of the AAT RNAi drug substance, wherein the pharmaceutical composition is administered once every three months by subcutaneous injection.

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

Brief description of graphic materials

[22] FIG. 1A-1E. Depiction of the chemical structure of the AAT RNAi drug described in Table 2 (referred to herein as ADS-001; i.e., the AAT RNAi agent conjugated to a tridentate N-acetylgalactosamine targeting group at the 5' end of the sense strand), presented in the form of the sodium salt.

[23] FIG. 2A-2E: Depiction of the chemical structure of the AAT RNAi drug described in Table 2, presented in the free acid form.

[24] FIG. 3. Schematic representation of the modified sense and antisense strands of the AAT RNAi drug described in Table 2 (referred to herein as ADS-001; i.e., the AAT RNAi agent conjugated to a tridentate N-acetylgalactosamine targeting group at the 5' end of the sense strand). The following abbreviations are used in FIG. 3: a, c, g, and u are 2'-O-methyl-modified nucleotides; Af, Cf, Gf, and Uf are 2'-fluoro- (also referred to in the art as 2'-deoxy-2'-fluoro) modified nucleotides; o is a phosphodiester linkage; s is a phosphate thioate linkage; invAb is an inverted residue or base-deleted subunit; and (NAG37)s is a tridentate N-acetylgalactosamine targeting ligand with the following chemical structure:

(presented in the form of sodium salt),

(presented in the form of free acid).

[25] FIG. 4. Design of the final Phase I study and dose escalation schedule for the Phase I clinical study described in Example 2.

[26] FIG. 5. Graph showing serum AAT levels in normal healthy human volunteers (NHV) who were administered placebo (all cohorts) or 35 mg of AAT RNAi drug (Cohort 1) from the Phase I clinical trial described in Example 2. As shown in FIG. 5-11, the term “active” refers to the AAT RNAi drug described in Table 2 (administered as a ready-to-use AAT RNAi drug as described in Table 3).

[27] FIG. 6. Graph showing serum AAT levels in NHVs administered placebo (all cohorts) or a single 100 mg dose of AAT RNAi drug (cohort 2b) from the phase I clinical trial described in Example 2.

[28] FIG. 7. Graph showing serum AAT levels in NHVs administered placebo (all cohorts) or a single 200 mg dose of AAT RNAi drug (cohort 3b) from the phase I clinical trial described in Example 2.

[29] FIG. 8. Graph showing serum AAT levels in NHVs administered placebo (all cohorts) or a single 300 mg dose of AAT RNAi drug (cohort 4b) from the phase I clinical trial described in Example 2.

[30] FIG. 9. Graph showing serum AAT levels in NHVs treated with placebo (all cohorts) or three 100 mg doses of AAT RNAi drug monthly (cohort 2) from the phase I clinical trial described in Example 2.

[31] FIG. 10. Graph showing serum AAT levels in NHVs treated with placebo (all cohorts) or three 200 mg doses of AAT RNAi drug monthly (cohort 3) from the phase I clinical trial described in Example 2.

[32] FIG. 11. Graph showing serum AAT levels in NHVs treated with placebo (all cohorts) or three 300 mg doses of AAT RNAi drug monthly (cohort 4) from the phase I clinical trial described in Example 2.

Detailed description

RNAi tools

[33] The methods described herein comprise administering to a human subject a pharmaceutical composition that comprises 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 an AAT gene. In some embodiments, the methods described herein comprise administering to a human subject a pharmaceutical composition that comprises the AAT RNAi therapeutic agent described in Table 2 (also referred to as ADS-001). Compositions suitable for use in the methods disclosed herein comprise an RNAi agent that inhibits the expression of an AAT gene in a human subject, as well as a targeting moiety or targeting group. In some embodiments, the RNAi agent comprises the nucleotide sequences set forth in Tables 1A and 1B, wherein the sense strand of the RNAi agent is further linked or conjugated to a targeting moiety comprising three N-acetylgalactosamine targeting 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."

[34] In general, the AAT RNAi agents comprise a sense strand (also referred to as the carrier strand) and an antisense strand (also referred to as the guide strand) that are hybridized to form a duplex. The AAT RNAi agents disclosed herein include an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule capable of reducing or inhibiting the translation of messenger RNA (mRNA) transcripts as AAT mRNA in a sequence-specific manner. The AAT RNAi agents disclosed herein may act through an RNA interference mechanism (i.e., by inducing RNA interference through interaction with the RNA interference pathway (RNA-inducible silencing complex or RISC) of mammalian cells) or any alternative mechanism(s) or pathway(s). Because AAT RNAi agents, as used herein, are believed to act primarily through RNA interference, the disclosed RNAi agents are not associated with or limited to any particular pathway or mechanism of action. RNAi agents typically consist of a sense strand and an antisense strand, each ranging in length from 16 to 49 nucleotides, and include, but are not limited to, short or small interfering RNAs (siRNAs), double-stranded RNAs (dsRNAs), microRNAs (miRNAs), short hairpin RNAs (shRNAs), and Dicer substrates.

[35] The sense strand of the AAT RNAi agent is typically 16 to 49 nucleotides in length, and the antisense strand of the AAT RNAi agent is typically 18 to 49 nucleotides in length. In some embodiments, the sense and antisense strands are each individually 17 to 26 nucleotides in length. In some embodiments, the sense and antisense strands are each individually 21 to 26 nucleotides in length. In some embodiments, the sense and antisense strands are each individually 21 to 24 nucleotides in length. In some embodiments, the sense and/or antisense strands are each individually 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 and antisense strands are 21 nucleotides long. The sense and antisense strands may be the same length or different lengths. The sense and antisense strands may also form overhanging nucleotides at one or both ends of the AAT RNAi agent.

[36] AAT RNAi agents inhibit, downregulate, or block the expression of the AAT gene. As used herein, the terms "suppress," "reduce," "inhibit," "downregulate," or "block" when used in relation to the expression of the AAT gene mean that the expression of the gene, as determined by the level of RNA transcribed from the gene or the level of the polypeptide, protein, or protein subunit translated from mRNA in a cell, group of cells, tissue, organ, or subject in which the gene is being transcribed, is decreased upon exposure of the cell, group of cells, tissue, organ, or subject to the RNAi agent, compared to a second cell, group of cells, tissue, organ, or subject without such exposure(s). In some cases, the reduction in gene expression is measured by comparing baseline levels of AAT mRNA or AAT protein in a human subject prior to administration of a composition containing an AAT RNAi agent with levels of AAT mRNA or AAT protein following administration of the drug.

[37] Inhibition, suppression, or blocking of AAT gene expression can be measured by any appropriate assay or method known in the art. The non-limiting examples set forth herein, as well as the examples set forth in International Patent Application Publication No. WO 2018/132432 (Patent Application No. PCT/US2018/013102), which is incorporated herein by reference in its entirety, provide some examples of appropriate assays for measuring inhibition of AAT gene expression. A reference gene transcript for AAT mRNA (SERPINA1) in normal humans (referred to as transcript variant 1; GenBank NM_000295.4) is set forth in SEQ ID NO:1.

[38] AAT RNAi agents suitable for use in the methods disclosed herein can be covalently linked or conjugated to a targeting moiety comprising one or more N-acetylgalactosamine moieties. In embodiments, AAT RNAi agents suitable for use in the methods disclosed herein are covalently linked or conjugated to a targeting moiety comprising one or more N-acetylgalactosamine moieties, thereby forming an AAT RNAi drug described in Table 2. In some embodiments, the methods described herein comprise administering the AAT RNAi drug described in Table 2. The AAT RNAi drug described in Table 2 includes the AAT RNAi agent shown in Table 1A (antisense strand) and Table 1B (sense strand). N-acetylgalactosamine moieties promote targeting of the AAT RNAi agent to asialoglycoprotein receptors (ASGPr) freely present on the surface of hepatocytes, resulting in uptake of the AAT RNAi agent by endocytosis or other means.

[39] AAT RNAi agents suitable for use in the methods disclosed herein comprise an antisense strand having a region complementary to at least a portion of AAT mRNA. AAT RNAi agents and AAT RNAi therapeutic agents suitable for use in the disclosed methods are described in International Patent Application Publication No. WO 2018/132432 (Patent Application No. PCT/US2018/013102), which, as previously noted, is incorporated herein by reference in its entirety.

[40] As used herein, the terms "sequence" and "nucleotide sequence" refer to a sequence or order of nucleotide bases or nucleotides described by a sequence of letters according to standard nomenclature. As used herein, the terms "nucleotide base" and "nucleotide" have the same meaning as commonly understood in the art.

[41] As used herein, the term "complementary" when used to describe a first nucleotide sequence (e.g., the antisense strand of an RNAi agent) with respect to a second nucleotide sequence (e.g., the sense strand of an RNAi agent or the targeting sequence of an mRNA) refers to the ability of an oligonucleotide comprising the first nucleotide sequence to hybridize (form base-pair hydrogen bonds under physiological conditions for mammals (or under other suitable conditions) and a duplex or double-stranded structure under certain standard conditions) with an oligonucleotide comprising the second nucleotide sequence. A person of ordinary skill in the art will be able to select a set of conditions most suitable for conducting a hybridization test. Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs, and also include natural or modified nucleotides or nucleotide mimics, at least to the extent that the above requirements for hybridization are met. The identity or complementarity of sequences is independent of modification. For example, when establishing identity or complementarity, a and Af, as defined herein, are complementary to U (or T) and identical to A.

[42] As used herein, "perfectly complementary" or "fully complementary" means that all (100%) bases in the contiguous sequence of the first oligonucleotide hybridize to the same number of nucleotides in the contiguous sequence of the second oligonucleotide. The contiguous sequence may comprise all or part of the first or second nucleotide sequence.

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

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

[45] As used herein, the term "substantially identical" or "substantial identity" with respect to a nucleotide sequence means that the nucleic acid sequence comprises a nucleic acid sequence that has at least about 85% sequence identity or more, such as at least 90%, at least 95%, or at least 99% sequence identity, compared to a reference sequence. The percentage sequence identity is determined by comparing two optimally aligned sequences over a comparison window. The percentage is calculated by determining the number of positions at which an identical nucleobase occurs in both sequences, thereby establishing the number of corresponding positions, and then dividing the number of corresponding positions by the total number of positions in the comparison window and multiplying the result by 100 to obtain the percentage sequence identity. The inventions disclosed herein include nucleotide sequences that are substantially identical to those disclosed herein.

Modified nucleotides and modified internucleoside linkages

[46] The AAT RNAi agents disclosed herein may contain modified nucleotides that are capable of maintaining the activity of the RNAi agent while simultaneously increasing serum stability and minimizing the potential for initiating interferon activity in humans. As used herein, a "modified nucleotide" is a nucleotide other than a ribonucleotide (2'-hydroxyl nucleotide). 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 modified nucleotides known in the art, including, but not limited to, deoxyribonucleotides, nucleotide mimics, 2'-modified nucleotides, inverted nucleotides, nucleotides containing modified nucleotide bases, bridged nucleotides, peptide nucleic acids (PNA), 2',3'seco nucleotide mimics (unlocked nucleobase analogs), locked nucleotides, 3'-O-methoxy (with a 2'-internucleoside linkage) nucleotides, 2'-F-arabino nucleotides, 5'-Me, 2'-fluoro nucleotides, morpholine nucleotides, nucleotides containing vinyl phosphonates, and nucleotides containing cyclopropyl phosphonates. In some embodiments, the modified nucleotides of the AAT RNAi agent are 2'-modified nucleotides (i.e., a nucleotide with a group other than a hydroxyl group at the 2' position of the five-membered sugar ring). 2'-modified nucleotides include, but are not limited to, 2'Omethyl nucleotides, 2'-deoxy-2'-fluoro nucleotides (commonly referred to simply as 2'-fluoro nucleotides), 2'-deoxy nucleotides, 2'-methoxyethyl (2'-O-2-methoxyethyl) nucleotides, 2'-amino nucleotides, and 2'-alkyl nucleotides. Additional 2'-modified nucleotides are known in the art. It is not necessary that all nucleotides in a particular RNAi agent be equally modified. Furthermore, more than one modification may be incorporated into a single AAT RNAi agent or even into a single nucleotide of the agent. The sense and antisense strands of the AAT RNAi agent can be synthesized and/or modified using methods known in the art. Modification of one nucleotide is independent of modification of another nucleotide.

[47] In some embodiments, a nucleotide base (often referred to simply as a "base") may be modified. According to common usage in the art, naturally occurring nucleotide bases include the primary purine bases adenine and guanine, and the primary pyrimidine bases cytosine, thymine, and uracil. A nucleotide base may be modified to include, among other things, universal bases, hydrophobic bases, mixed bases, enlarged 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 nucleotide bases (including phosphoramidite compounds containing modified nucleotide bases) is known in the art.

[48] Modified nucleotide bases include, for example, 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-hydroxyl and other 8-substituted adenines and guanines, 5-halogen (e.g., 5-bromo), 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine and 3-deazaadenine.

[49] 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 is an RNAi agent in which four or fewer (i.e., 0, 1, 2, 3, or 4) nucleotides are represented by ribonucleotides (i.e., unmodified) in both the sense strand and the antisense strand. As used herein, a sense strand in which substantially all of the nucleotides present are modified is a sense strand in which two or fewer (i.e., 0, 1, or 2) nucleotides are represented by ribonucleotides. In the context of this document, an antisense strand in which substantially all of the nucleotides present are modified is an antisense strand in which there are two or fewer (i.e., 0, 1, or 2) nucleotides represented by ribonucleotides.

[50] In some embodiments, one or more nucleotides of the AAT RNAi agent are linked by non-standard linkages or through the backbones of the molecules (i.e., modified internucleoside linkages or through modified backbones). Modified internucleoside linkages or backbones of molecules include, but are not limited to, phosphorothioate groups, chiral phosphorothioates, thiophosphates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, alkyl phosphonates (e.g., methyl phosphonates or 3′-alkylene phosphonates), chiral phosphonates, phosphinates, phosphoramidates (e.g., 3′-aminophosphoramidate, aminoalkyl phosphoramidates, or thionophosphoramidates), thionoalkyl phosphonates, thionoalkyl phosphotriesters, morpholine linkages, boranophosphates with normal 3′-5′ linkages, 2′-5′-linked boranophosphate analogs, or boranophosphates with inverted orientation in which adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. In some embodiments, the modified internucleoside linkage or backbone of the molecule lacks one phosphorus atom. Modified internucleoside linkages lacking one phosphorus atom include, but are not limited to, short-chain alkyl or cycloalkyl intersaccharide linkages, mixed heteroatom and alkyl or cycloalkyl intersaccharide linkages, or one or more short-chain heteroatom or heterocyclic intersaccharide linkages. In some embodiments, modified internucleoside backbones include, but are not limited to, siloxane backbones, sulfide backbones, sulfoxide backbones, sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimine and methylenehydrazine backbones, sulfonate and sulfonamide backbones, amide backbones, and other backbones with mixed N, O, S, and _CH2 components.

[51] In some embodiments, the sense strand of the AAT RNAi agent may comprise 1, 2, 3, 4, 5, or 6 phosphorothioate linkages, the antisense strand of the AAT RNAi agent may comprise 1, 2, 3, 4, 5, or 6 phosphorothioate linkages, or each of the sense and antisense strands may independently comprise 1, 2, 3, 4, 5, or 6 phosphorothioate linkages. In some embodiments, the sense strand of the AAT RNAi agent may comprise 1, 2, 3, or 4 phosphorothioate linkages, the antisense strand of the AAT RNAi agent may comprise 1, 2, 3, or 4 phosphorothioate linkages, or each of the sense and antisense strands may comprise 1, 2, 3, or 4 phosphorothioate linkages.

[52] In some embodiments, the sense strand of the AAT RNAi agent comprises 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, phosphorothioate internucleoside linkages are used to link terminal nucleotides in the sense strand to capping residues present at the 5' end, the 3' end, or both the 5' end and the 3' end of the nucleotide sequence. In some embodiments, phosphorothioate internucleoside linkages are used to link the targeting group to the sense strand.

[53] 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.

[54] In some embodiments, the AAT RNAi agent comprises one or more modified nucleotides and one or more modified internucleoside linkages. In some embodiments, a 2' modified nucleoside is combined with a modified internucleoside linkage.

Capping residues and fragments

[55] In some embodiments, the sense strand may include one or more capping residues or moieties, sometimes referred to in the art as a "cap,""terminalcap," or "capping residue." As used herein, a "capping residue" is a non-nucleotide compound or other moiety that may be included at one or more ends of the nucleotide sequence of the RNAi agent disclosed herein. A capping residue may, in some cases, impart certain beneficial properties to the RNAi agent, such as, for example, protection from exonuclease degradation. In some embodiments, inverted base-deleted (invAb) residues (also referred to in the art as "inverted base-deleted sites") are added as capping residues ( see Table A). (See, for example, F. Czauderna, Nucleic Acids Res., 2003, 31(11), 2705-16). Capping residues are widely known in the art and include, for _example , inverted base-depleted residues, as well as carbon chains such as _C3H7 (propyl), _{C6H13 (} hexyl), or _C12H25 (dodecyl ₎ end groups. In some embodiments, the capping residue is present at either the 5' end or the 3' end, or at 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 include more than one inverted base-depleted deoxyribose moiety as a capping residue.

[56] In some embodiments, one or more inverted base-deleted residues (invAb) are added to the 3' end of the sense strand. In some embodiments, one or more inverted base-deleted residues (invAb) are added to the 5' end of the sense strand. In some embodiments, one or more inverted base-deleted residues or inverted base-deleted regions are inserted between the targeting ligand and the nucleotide sequence of the sense strand of the RNAi agent. In some embodiments, the inclusion of one or more inverted base-deleted residues or inverted base-deleted regions in or near the terminal region or terminal regions of the sense strand of the RNAi agent enhances activity or improves other desired properties of the RNAi agent.

[57] 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 targeting ligand and the nucleotide sequence of the sense strand of the RNAi agent. The inverted abasic residues may be linked via phosphate, phosphorothioate (e.g., shown herein as (invAb)) or other internucleoside linkages. The chemical structures of inverted abasic deoxyribose residues are shown in Table A below, as well as in the chemical structures of Figs. 1A-1E, and Figs.

[58] Table A. Chemical structures of inverted (deoxyribose) with nitrogenous bases removed

When located within an oligonucleotide:

( invAb) When located within an oligonucleotide:

( invAb) When located at the 3' end of an oligonucleotide:

( invAb)

Targeting fragments and groups

[59] The AAT RNAi agent may be conjugated to one or more non-nucleotide moieties including, but not limited to, a targeting moiety or a targeting group. The targeting moiety or targeting group may enhance targeting or delivery of the RNAi agent. Examples of targeting moieties or targeting groups are known in the art. Table B provides specific examples of a targeting group (NAG37) used in the AAT RNAi drug substance described in Table 2 herein, which includes three N-acetylgalactosamine targeting moieties, is provided in Table B. The targeting moiety or targeting group may be covalently linked to the 3' end and/or 5' end of the sense strand and/or the antisense strand. In some embodiments, the AAT RNAi agent comprises a targeting moiety linked to the 3' and/or 5' end of the sense strand. In some embodiments, the targeting moiety is linked to the 5' end of the sense strand of the AAT RNAi agent. In some embodiments, the targeting moiety comprises, consists essentially of, or consists of the structure (NAG37) and is linked to the 5' end of the sense strand of the AAT RNAi agent. The targeting moiety may be linked to the RNAi agent directly or indirectly via a linker/linker group. In some embodiments, the targeting moiety is linked to the RNAi agent via a labile, cleavable, or reversible bond or linker. In some embodiments, the targeting moiety is linked to an inverted base-depleted residue at the 5' end of the sense strand.

[60] Targeting groups or targeting moieties may improve the pharmacokinetic or biodistribution properties of the conjugate or RNAi agent to which they are attached, resulting in improved cell-specific distribution and cell-specific uptake of the conjugate or RNAi agent. In some embodiments, the targeting group enhances endocytosis of the RNAi agent. The targeting group may be monovalent, divalent, trivalent, tetravalent, or have a higher valency for the target molecule to which it is directed. Representative targeting groups include, but are not limited to, compounds with affinity for cell surface molecules, cellular receptor ligands, haptens, antibodies, monoclonal antibodies, antibody fragments, and antibody mimics with affinity for cell surface molecules.

[61] In some embodiments, the targeting moiety comprises 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 that have an affinity for the asialoglycoprotein receptor equal to or greater than that of galactose. Galactose derivatives include, but are not limited to: galactose, galactosamine, N-formylgalactosamine, N-acetylgalactosamine, N-propionylgalactosamine, N-n-butanoylgalactosamine, and N-isobutanoylgalactosamine (see, e.g., S. T. Iobst and K. Drickamer, JBC, 1996, 271, 6686). Galactose derivatives and clusters of galactose derivatives useful for in vivo targeting of oligonucleotides and other molecules to the liver 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).

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

[63] In some embodiments, the targeting moiety comprises a galactose derivative cluster. As used herein, a galactose derivative cluster comprises a molecule having two to four terminal galactose derivatives. A terminal galactose derivative is attached to the molecule via its C-1 carbon atom. In some embodiments, the galactose derivative cluster is a galactose derivative trimer (also referred to as a triantennary galactose derivative or a trivalent galactose derivative). In some embodiments, the galactose derivative cluster comprises N-acetylgalactosamines. In some embodiments, the galactose derivative cluster comprises three N-acetylgalactosamines. In some embodiments, the galactose derivative cluster is a galactose derivative tetramer (also referred to as a tetraantennary galactose derivative or a tetravalent galactose derivative). In some embodiments, the galactose derivative cluster comprises four N-acetylgalactosamines.

[64] As used herein, a galactose derivative trimer comprises three galactose derivatives, each linked to a central branch point. As used herein, a galactose derivative tetramer comprises four galactose derivatives, each linked to a central branch point. The galactose derivatives may be linked to the central branch point via the C-1 carbon atoms of the saccharides. In some embodiments, the galactose derivatives are linked to the branch point via linkers or spacers. In some embodiments, the linker or spacer is a flexible hydrophilic spacer, such as 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 branch point may be any small molecule capable of attaching three galactose derivatives and may further attach the branch point to an RNAi agent. An example of a branch point moiety is dilysine or diglutamate. Attachment of the branch point to the RNAi agent may occur via a linker or spacer. In some embodiments, the linker or spacer comprises a flexible hydrophilic spacer, such as, but not limited to, a PEG spacer. In some embodiments, the linker comprises a rigid linker, such as a cyclic group. In some embodiments, the galactose derivative comprises or consists of N-acetylgalactosamine. In some embodiments, the galactose derivative cluster consists of a tetramer of the galactose derivative, which may be, for example, a tetramer of N-acetylgalactosamine.

[65] The preparation of targeting groups such as galactose derivative clusters containing N-acetylgalactosamine is described, for example, in International Patent Application Publication No. WO 2018/044350 (Patent Application No. PCT/US2017/021147) and International Patent Application Publication No. WO 2017/156012 (Patent Application No. PCT/US2017/021175), the contents of which are incorporated herein in their entirety by reference.

[66] For example, the targeting ligand conjugated to the AAT RNAi agent described in Tables 1A and 1B has the chemical structure (NAG37) as shown in Table B.

[67] Table B. Chemical structure (NAG37).

((NAG37), presented in the form of sodium salt)
((NAG37), presented in the form of free acid)

AAT RNAi agents and AAT RNAi drug (ADS-001)

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

[69] Table 1A. Sequence of the antisense strand of the RNAi agent AAT

SEQ ID NO. Antisense sequence (modified)
(5' → 3') SEQ ID NO. Underlying base sequence
(5' → 3') 2 usGfsuUfaAfacaugCfcUfaAfaCfgCfsu 3 UGUUAAACAUGCCUAAACGCU

[70] Table 1B. Nucleotide sequence of the sense strand of the AAT RNAi agent (shown as a modified version without the inverted residues with the nucleobases removed or the NAG targeting group present in the AAT RNAi drug substance)

SEQ ID NO. Semantic sequence (modified)
(5' → 3') SEQ ID NO. Underlying base sequence
(5' → 3') 4 agcguuuaGfGfCfauguuuaaca 5 AGCGUUUAGGCAUGUUUAACA

[71] In Tables 1A, 1B and 2 provided herein, the following symbols are used to designate modified nucleotides, targeting groups and linking groups: A, G, C and U represent adenosine, cytidine, guanosine or uridine; a, c, g and u represent 2′-O-methyladenosine, cytidine, guanosine or uridine, respectively; Af, Cf, Gf and Uf represent 2′-fluoroadenosine, cytidine, guanosine or uridine, respectively; s represents a phosphorothioate linkage; (invAb) represents an inverted deoxyribose residue with the nucleobases deleted ( see Table A); and (NAG37) represents the structure shown in Table B above.

[72] It will be readily apparent to one skilled in the art, unless otherwise indicated by a sequence (such as by a phosphorothioate "s" linkage) present in a strand, that the monomers are linked together via 5'-3' phosphodiester linkages. It will be readily apparent to one skilled in the art that the inclusion of a phosphorothioate linkage, as shown in the modified nucleotide sequences disclosed herein, replaces the phosphodiester linkage typically present in oligonucleotides. Furthermore, it will be readily apparent to one skilled in the art that the terminal nucleotide at the 3' end of a particular oligonucleotide sequence will typically have a hydroxyl (-OH) group at the corresponding 3' position of the particular monomer in place of the phosphate moiety ex vivo . Furthermore, for the embodiments disclosed herein, when considering the corresponding 5' strand? 3' inverted residues with deleted nucleobases are inserted such that the 3' position of the deoxyribose is linked to the 3' end of the preceding monomer of the corresponding strand. Moreover, it should be readily apparent to one skilled in the art that, although the phosphorothioate chemical structures depicted herein typically show the anion as located on the sulfur atom, the inventions disclosed herein encompass all phosphorothioate tautomers (in which, for example, the sulfur atom is double-bonded and the anion is located on the oxygen atom). Unless otherwise explicitly stated herein, one skilled in the art applies similar interpretations to describe the AAT RNAi agents and compositions that include the AAT RNAi agents disclosed herein.

[73] Each sense strand and/or antisense strand may have any of the above targeting groups or linking groups, as well as other targeting or linking groups conjugated to the 5′ end and/or 3′ end of the sequence.

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

[75] In some embodiments, the methods disclosed herein utilize an AAT RNAi drug listed in the following Table 2.

[76] Table 2. Drug substance for RNAi AAT (ADS-001)

Sense and antisense strands ( the sense and antisense strands are hybridized to form a duplex): Sense strand (modified sequence) (5' → 3'): (NAG37)s(invAb)sagcguuuaGfGfCfauguuuaacas(invAb) (SEQ ID
NO: 6) Antisense strand (modified sequence) (5' → 3'): usGfsuUfaAfacaugCfcUfaAfaCfgCfsu (SEQ ID
NO: 2) Chemical formula: C493H610F11N163Na43O312P43S6 (Na+ form) C493H653F11N163O312P43S6 (form H+) Molecular weight: 16,532.9 Yes (Na+ form) 15,587.6 Yes (form H+) Appearance: Powder from white to almost white

[77] A schematic representation of the RNAi-based AAT RNAi drug substance (ADS-001) is shown in Fig. 3, and the complete chemical structure images are shown in Figs. 1A-1E (in the sodium salt form) and Figs. 2A-2E (in the free acid form). In some embodiments, the AAT RNAi drug substance is prepared or provided as a salt, a mixed salt, or a free acid. In preferred embodiments, the form is a sodium salt.

Pharmaceutical compositions and formulations

[78] AAT RNAi agents suitable for use in the methods disclosed herein can be prepared as pharmaceutical compositions or formulations for administration to human subjects. The pharmaceutical compositions can be used to treat a subject with a disease or disorder that would be ameliorated by inhibiting AAT mRNA expression or by reducing AAT protein levels, such as a human subject with AATD. In some embodiments, the methods comprise administering an AAT RNAi agent that is linked to a targeting moiety or targeting ligand as described herein to a subject in need of treatment. In some embodiments, one or more pharmaceutically acceptable excipients (including fillers, carriers, diluents, and/or delivery polymers) are added to the pharmaceutical compositions that include the AAT RNAi agent, thereby creating a pharmaceutical formulation suitable for in vivo delivery to a human subject.

[79] Pharmaceutical compositions comprising an AAT RNAi agent, when administered to a human subject using the methods disclosed herein, reduce the level of AAT mRNA in the subject.

[80] In some embodiments, the disclosed pharmaceutical compositions comprising an AAT RNAi agent are used to treat or manage clinical manifestations in a subject with AATD, such as chronic hepatitis, cirrhosis, increased risk of hepatocellular carcinoma, elevated transaminases, cholestasis, fibrosis, and even fulminant liver failure. In some embodiments, a therapeutically 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 disclosed AAT RNAi agents can be used to reduce the number, severity, and/or frequency of disease symptoms in a subject.

[81] The disclosed pharmaceutical compositions comprising an AAT RNAi agent can be used to treat at least one symptom in a subject with a disease or disorder that would be improved by reducing or inhibiting the expression of AAT mRNA. In some embodiments, the subject is administered a therapeutically effective amount of one or more pharmaceutical compositions comprising an AAT RNAi agent, thereby treating the symptom. In other embodiments, the subject is administered a prophylactically effective amount of one or more AAT RNAi agents, thereby preventing the development of at least one symptom.

[82] The AAT RNAi agents disclosed herein can be administered by any suitable route in the form of a preparation suitably formulated for the particular route of administration. Thus, the pharmaceutical compositions described herein can be administered by injection, such as intravenously or subcutaneously. In some embodiments, the pharmaceutical compositions described herein are administered by subcutaneous injection.

[83] As used herein, a pharmaceutical composition or medicinal product comprises a pharmacologically effective amount of at least one of the AAT RNAi agents and one or more pharmacologically acceptable excipients. Pharmacologically acceptable excipients (excipients) are substances other than the active pharmaceutical ingredient (API, therapeutic product, e.g., AAT RNAi agent) that are intentionally included in a drug delivery system. Excipients do not exert or are not intended to exert a therapeutic effect at a given dose. The excipients function to a) facilitate processing of the drug delivery system during manufacture, b) maintain, support, or enhance the stability, bioavailability, or patient tolerability of the API, c) assist in product identification, and/or d) enhance any other characteristic 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.

[84] Excipients may include, but are not limited to: absorption enhancers, anti-adhesives, anti-foaming agents, antioxidants, binders, buffering agents, carriers, coating agents, colorants, delivery enhancers, delivery polymers, dextran, dextrose, diluents, disintegrating agents, emulsifiers, additives, fillers, flavors, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, suspending agents, sustained release matrices, sweeteners, thickeners, tonicity regulators, fillers, water repellents, and humectants.

[85] Pharmaceutical compositions suitable for injection use include sterile aqueous solutions (where water-soluble ones are required). For subcutaneous or intravenous administration, suitable carriers may include saline, bacteriostatic water, Cremophor® EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). They must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

[86] Sterile injection solutions can be prepared by incorporating the active compound in the required amount into an appropriate solvent with one or a combination of the above ingredients in the required volume, followed by filtered sterilization. Dispersions are usually prepared by incorporating the active compound into a sterile vehicle that contains the basic dispersion medium and the required other ingredients from those listed above.

[87] In some embodiments, a pharmaceutical composition suitable for use in the methods disclosed herein includes the components identified in the ready-to-use drug substance for RNAi AAT, as listed in Table 3 below.

[88] The ready-mixed AAT RNAi agents can be formulated as compositions in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form refers to physically discrete units suitable as unitary dosages for the subject to be treated, each unit containing a predetermined quantity of active compound calculated to provide the desired therapeutic effect in association with the required pharmaceutical carrier. In some embodiments, the dosage unit is between about 5 mg and about 300 mg of the AAT RNAi drug substance. In some embodiments, the dosage unit is between about 25 mg and about 200 mg of the AAT RNAi drug substance. In some embodiments, the dosage unit is between about 100 mg and about 200 mg of the AAT RNAi drug substance. In some embodiments, the dosage unit is about 100 mg of the AAT RNAi drug substance. In some embodiments, the dosage unit is about 200 mg of the RNAi AAT drug substance.

[89] The pharmaceutical composition may contain other additional components commonly found in pharmaceutical compositions. Such additional components include, but are not limited to: antipruritics, astringents, local anesthetics, or anti-inflammatory agents (e.g., antihistamine, diphenhydramine, etc.).

[90] As used herein, a “pharmacologically effective amount,” “therapeutically effective amount,” or simply “effective amount” means that amount of an RNAi agent necessary to provide a pharmacological, therapeutic, or prophylactic result.

[91] The pharmaceutically acceptable formulations described herein may be packaged in kits, containers, packs, or dispensers. The pharmaceutical compositions described herein may be packaged in pre-fillable syringes or vials.

Ready-to-use drug substance for RNAi AAT

[92] In some embodiments, the AAT RNAi drug substance as provided in Table 2 (ADS-001) 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 drug substance described in Table 2 is formulated as a 230 mg/mL dosage form in an aqueous sodium phosphate buffer solution (0.5 mM monobasic sodium phosphate, 0.5 mM dibasic sodium phosphate), creating a ready-to-use AAT RNAi drug substance (ADS-001-1) shown in Table 3:

[93] Table 3. Composition of the ready-to-use medicinal substance for RNAi AAT, per 1.0 ml

Component Function Quantity/
quality Concentration ADS-001 Active ingredient Own production 230 mg Monobasic sodium phosphate, monohydrate Suspending agent US Pharm., Eur. Pharm. 0.061 mg Sodium phosphate dibasic, anhydrous Suspending agent US Pharm., Eur. Pharm. 0.062 mg Water for injection (WFI) Carrier US Pharm., Eur. Pharm. 879.2 mg

[94] The ready-to-use AAT RNAi drug substance according to Table 3 is prepared as a sterile formulation. In some embodiments, the ready-to-use AAT RNAi drug substance is packaged in a container, such as a glass vial. In some embodiments, the ready-to-use AAT RNAi drug substance is packaged in a glass vial with a nominal volume of about 1.1 mL, wherein the required volume for administration can be calculated based on the amount of dose to be administered.

[95] In some embodiments, the ready-to-use RNAi drug substance for AAT listed in Table 3 is administered to a human subject using the methods disclosed herein.

Human subjects with AATD and AATD diagnosis

[96] The methods disclosed herein include treating alpha-1 antitrypsin deficiency (AATD) in a human subject in need thereof, including treating symptoms and diseases caused by AATD in the human subject, using pharmaceutical compositions comprising the AAT RNAi therapeutic agent described in Table 2. In some embodiments, the human subject is diagnosed with AATD prior to administration. As noted previously, AATD is an inherited disorder caused by mutations in a gene transcript that result in the translation of a mutant form of the AAT protein, causing some mutant forms to fold abnormally and become retained intracellularly within hepatocytes. While various mutations of the SERPINA1 gene have been identified, the most common and severe form of AATD, the PiZZ genotype, is caused by a single base pair substitution. There are frequent reports of individuals with the PiZZ genotype having circulating AAT levels less than 15% of those in healthy individuals. In many cases, patients are initially diagnosed with COPD, asthma, or another lung disease without identifying the underlying cause. Over time, liver disease, such as fibrosis or cirrhosis, may develop due to the intercellular retention of the misfolded protein (Z-AAT) and the inability of liver cells to properly secrete this protein. In pediatric patients, presentations are typically associated with clinical symptoms of liver disease, which may include asymptomatic chronic hepatitis, failure to thrive, malaise, or hepatomegaly and splenomegaly. A diagnosis of AATD can be made and confirmed using standard genotyping techniques on blood samples obtained from the individual.

Dosage and inhibition of AAT gene expression

[97] Typically, an effective amount of the AAT RNAi agent is in the range of about 0.1 to about 10 mg/kg body weight/day, such as about 0.25 to about 5 mg/kg body weight/day. In some embodiments, the effective amount of the AAT RNAi agent is in the range of 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 an effective dose. In some embodiments, a fixed dose of 25 mg to 200 mg of the AAT RNAi drug substance is an effective dose. The amount administered will likely depend on variables such as the overall age and health of the patient, the relative biological effectiveness of the compound being delivered, the formulation of the drug, the presence and types of excipients in the formulation, and the route of administration. In some embodiments, a fixed dose of from 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 an effective dose. In some embodiments, a fixed dose of about 25 mg, about 100 mg, or about 200 mg is an effective dose.

[98] It should also be understood that the administered initial dose may, in some cases, be increased to a level above the threshold in order to rapidly achieve the desired blood level or tissue level, or the initial dose may, in some cases, be less than optimal. For example, in some embodiments, an initial dose of about 25 mg to about 200 mg of the AAT RNAi drug was administered, followed approximately 1 month later by a second dose of about 25 mg to 200 mg of the AAT RNAi drug, and thereafter, additional doses (similar to "maintenance doses") were administered once every three months (i.e., quarterly).

[99] To treat a disease or to create a medicinal product or composition for treating a disease, the pharmaceutical compositions described herein, including an RNAi agent, can be combined with an excipient or with a second drug or a drug, including, but not limited to, a second or different RNAi agent, a small molecule drug, an antibody, an antibody fragment, a peptide and/or an aptamer.

[100] In some embodiments, the level of gene expression and/or the level of mRNA of the AAT gene in a subject administered with the described AAT RNAi agent is reduced by 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% compared to the subject before administration of the AAT RNAi agent or to a subject who has not received the AAT RNAi agent. The level of gene expression and/or mRNA level in the subject is reduced in a cell, a group of cells, and/or a tissue of the subject.

[101] In some embodiments, the level of AAT protein in a subject administered the described AAT RNAi agent 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%, 96%, 97%, 98%, 99%, or more than 99% compared to the subject before administration of the AAT RNAi agent or to a subject who has not received the AAT RNAi agent. The protein level in the subject is reduced in a cell, a group of cells, tissue, blood, and/or other fluid of the subject.

[102] In some embodiments, the level of the Z-AAT protein polymer in a subject with AATD who has been administered the described AAT RNAi agent 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%, 96%, 97%, 98%, 99%, or greater than 99% compared to the subject before administration of the AAT RNAi agent or to a subject who has not received the AAT RNAi agent. In some embodiments, the level of the Z-AAT protein polymer in a subject administered with the described AAT RNAi agent 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%, 96%, 97%, 98%, 99%, or more than 99% compared to the subject before administration of the AAT RNAi agent or to a subject who has not received the AAT RNAi agent.

[103] A reduction in the level of AAT gene expression, AAT mRNA, or AAT protein levels can be assessed and measured using conventional methods known in the art. The examples provided later in this document disclose conventional methods for assessing the degree of inhibition of AAT gene expression and a reduction in AAT protein levels. Both measures of a reduction or decrease in AAT mRNA levels and/or protein levels (including Z-AAT polymer and/or monomer) are referred to herein as a reduction or decrease in AAT or an inhibition or reduction in AAT expression.

[104] As used herein, the terms "treat," "treatment," and the like refer to methods or steps taken to provide for the alleviation or reduction of the number, severity, and/or frequency of one or more symptoms of a disease in a subject. As used herein, "treat" and "treating" may include prevention, control, prophylactic treatment, and/or suppression of the number, severity, and/or frequency of one or more symptoms of a disease in a subject.

[105] As used herein, "monthly dosing" or "monthly" administration means every 28 days. As used herein, "quarterly dosing" or "quarterly" administration means every 84 days. The term "about" when used with respect to monthly dosing means monthly dosing +/- 3 days. The term "about" when used with respect to quarterly dosing means quarterly dosing +/- 9 days.

[106] As used herein, the phrase "introduction into a cell" with respect to an RNAi agent means functional delivery of the RNAi agent into a cell. The phrase "functional delivery" means delivery of the RNAi agent into a cell in a manner that allows the RNAi agent to exhibit the desired biological activity, such as sequence-specific inhibition of gene expression.

[107] Unless otherwise stated, the use of the symbol in the context of this document means that any group or groups may be linked through it that are consistent with the scope of the inventions described herein.

[108] In the context of the present document, unless a structure is specifically identified as having a particular conformation, then for each structure in which asymmetric centers are present and which thus give rise to enantiomers, diastereomers, or other stereoisomeric configurations, each structure disclosed herein is intended to depict all possible isomers of such kind, including their optically pure and racemic forms. For example, the structures disclosed herein are intended to encompass mixtures of diastereomers as well as single stereoisomers.

[109] In the context of a claim of this document, the phrase "consisting of" excludes any element, step, or ingredient not specifically mentioned in the claim. When used in a claim, the phrase "consisting essentially of" limits the scope of the claim to the materials or steps mentioned, as well as those that do not materially affect the essential and novel characteristic(s) of the claimed invention.

[110] It should be readily apparent to one of ordinary skill in the art that the compounds and compositions disclosed herein may have certain atoms (e.g., N, O, or S atoms) in a protonated or deprotonated state depending on the environment in which the compound or composition is placed. Therefore, in the context of the present document, the structures disclosed herein give rise to certain functional groups, such as, for example, OH, SH, or NH, which may be protonated or deprotonated. The invention provided herein is intended to encompass the disclosed compounds and compositions regardless of their protonation state due to the environment (e.g., pH), as should be readily apparent to one of ordinary skill in the art.

[111] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art. While 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 literature references mentioned herein are herein incorporated by reference in their entirety. In case of conflict, the present description, including definitions, controls. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be exhaustive.

[112] The above embodiments and sections are demonstrated in the following non-limiting examples.

Examples

Example 1. Synthesis and composition of a drug substance for RNAi of AAT (ADS-001)

[113] The RNAi drug substance suitable for use in the methods disclosed herein can be synthesized using standard phosphoramidite solid-phase oligonucleotide synthesis technology known in the art. Commercially available oligonucleotide synthesizers (e.g., MerMade96E® (Bioautomation) or MerMade12® (Bioautomation)) can be used. Synthesis can be performed on a solid support made of glass with a defined pore size (CPG, 500 Å or 600 Å, purchased from Prime Synthesis, Aston, PA, USA). A monomer located at the 3' end of the appropriate strand can be attached to the solid support as a starting point for synthesis. All RNAs, 2'-modified RNA phosphoramidites, and base-depleted inverse phosphoramidites are commercially available. Phosphoramidites containing a targeting group that are suitable for addition to the 5' end of the sense strand can be synthesized. Standard cleavage, deprotection, purification, and hybridization steps known in the art can be used. Detailed descriptions of the synthesis of AAT RNAi agents can be found, for example, in International Patent Application Publication No. WO 2018/132432 (Application No. PCT/US2018/013102) and No. WO 2018/044350 (PCT/US2017/021147), each of which is incorporated herein by reference in its entirety. The AAT RNAi drug substance is then formulated into a dosage form by dissolution in standard pharmaceutically acceptable excipients generally known in the art. For example, Table 3 shows a ready-to-use drug substance for RNAi of AAT suitable for use in the methods described herein.

Example 2. Phase I clinical trial of an RNAi drug (ADS-001) in healthy volunteers (NHV).

[114] A phase 1, single- and multiple-dose, dose-escalation study was conducted to evaluate the safety, tolerability, pharmacokinetics, and effect of an AAT RNAi drug (ADS-001) on serum AAT levels in healthy volunteers (NHV). The study population included healthy adult men and women aged 18–52 years with a BMI between 19.0 and 35.0 kg/m².

[115] NHV subjects were divided into a total of seven cohorts. Cohorts 1-4 were randomized to receive AAT RNAi drug or placebo (4 active: 4 placebo) with single escalating doses of 35 mg (cohort 1) and multiple escalating doses of 100 mg (cohort 2), 200 mg (cohort 3), and 300 mg (cohort 4), administered as subcutaneous injections. Cohorts 1-4 were double-blind. Cohorts 2b, 3b, and 4b were open-label and consisted of 4 subjects receiving single doses of 100, 200, and 300 mg of AAT RNAi drug. 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 the following Table 4.

[116] Table 4. Phase I clinical trial parameters

Development phase Phase 1: Involving Humans for the First Time Objectives of the study Primary objectives:
To establish the proportion and frequency of adverse events possibly or probably related to treatment as a measure of safety and tolerability of the AAT RNAi medicinal product (ADS-001) using escalating single doses and escalating multiple doses in healthy volunteers (NHV).
Secondary objectives:
To study the pharmacokinetics of a drug for RNAi AAT after single and multiple administration in NHV.
To establish the degree of reduction of serum AAT in response to the use of a drug for AAT RNAi as a measure of drug activity.
research objectives:
To evaluate the effect of single doses of AAT RNAi drug on cytokines (cytokine panel A: interleukin-6 [IL-6], monocyte chemoattractant protein-1 [MCP-1], tumor necrosis factor alpha [TNF-alpha], interleukin-8 [IL-8], interleukin-1beta [IL-1beta], interferon alpha [IFN alpha], IL-10, IL-12 [p40], IL-12 [p70], macrophage inflammatory protein-1alpha [Mip-1alpha]) in NHV.
To evaluate the effect of single escalating doses of an AAT RNAi drug on complement factors Bb, CH50, C5a, C4a, and C3a in NHV.
Collect plasma samples from NHV for subsequent metabolite determination (results are reported as a separate report, not related to the study).
Collect urine samples from NHV for subsequent assessment of urinary excretion and determination of metabolites (results are reported as a separate report, not related to the study). Study design Cohorts 1-4: randomized, double-blind, placebo-controlled
Cohorts 2b, 3b, and 4b: open Study population The present study was conducted among NHV adult men and women aged 18–52 years with a BMI between 19.0 and 35.0 kg/m². Product under investigation AAT RNAi drug (ADS-001) (see Table 2) administered as a ready-to-use AAT RNAi drug (see Table 3) Dosage and frequency Cohort 1: randomized to receive AAT RNAi drug (ADS-001) or placebo (4 active:4 placebo) as a single 35 mg dose administered as a single subcutaneous injection.
Cohorts 2-4: randomized to receive three monthly (i.e., days 1, 29, and 57) doses of 100 mg (cohort 2), 200 mg (cohort 3), or 300 mg (cohort 4) of AAT RNAi drug or placebo (4 active: 4 placebo) by subcutaneous injection.
Cohorts 2b, 3b, and 4b: randomized to receive a single dose of 100 mg (cohort 2b), 200 mg (cohort 3b), or 300 mg (cohort 4b) of AAT RNAi drug (4 active), administered as a single subcutaneous injection. Reference composition Placebo (PBO): isotonic sodium chloride solution (0.9%) administered subcutaneously in the appropriate volume. Safety assessment criteria Safety was assessed by adverse events, serious adverse events, physical examination findings, vital signs (blood pressure, heart rate, temperature, and respiratory rate), resting ECG findings, clinical laboratory tests, concomitant medications/therapies, injection site reactions (IRS), reasons for discontinuation, and 90-day pregnancy follow-up after day 29 (cohort 1) and after day 113 (all other cohorts). Pharmacokinetic evaluation For pharmacokinetic analysis, blood samples were collected from each subject after dose 1 (cohort 1) and after doses 1 and 3 (cohorts 2, 3, and 4). Data analysis Screening, compliance, tolerability and safety data:
Safety data will be analyzed and results will be summarized for individual cohorts. The proportion and frequency of adverse events (AEs), serious adverse events (SAEs), treatment-emergent AEs, treatment-related SAEs, and AEs leading to treatment discontinuation will be summarized for each cohort by SOC, PT, and severity. Other safety parameters will be summarized at each scheduled time.
Pharmacokinetics (NHV subjects only):
Plasma concentrations of the drug components for RNAi AAT will be used to calculate the following PK parameters: maximum observed plasma concentration (Cmax), area under the concentration-time curve (AUC) from 0 to 24 hours (AUC0-24), AUC from time 0 to extrapolated to infinity (AUCinf), and terminal half-life (t½). Pharmacokinetic parameters will be determined using non-compartmental methods. Descriptive statistics of the PK parameters will include the mean, standard deviation (SD), coefficient of variation, median, minimum, and maximum values. PK results will be analyzed for dose proportionality and sex-related differences.

[117] The serum AAT reduction results obtained in the study showed that administration of the AAT RNAi drug at doses ranging from 35 to 300 mg resulted in robust reductions in serum AAT levels compared to placebo. The cohort was initially proposed as part of a clinical trial protocol with 400 mg of the AAT RNAi drug per dose. However, given the unanticipated activity at doses of 35, 100, 200, and 300 mg, the 400 mg cohort was removed from the study protocol. Doses of 35 mg, 100 mg, and 200 mg produced substantial reductions in serum AAT levels, with both 100 mg and 200 mg achieving approximately 90% of the mean serum AAT reduction after multiple dosing in the Phase I study. In Fig. 5-11 show the reduction in serum AAT levels in different cohorts in the phase I study.

[118] No clear dose-response was observed at all dose levels, as surprisingly and unexpectedly the 100 mg and 200 mg dose levels produced a significant reduction (reaching approximately 90%) similar to that of the 300 mg dose. While the lowest dose of 35 mg was still quite active, it was not as active as the 100 mg dose given as a single dose, indicating some degree of dose-response relationship.

[119] The duration of the reduction (>58%) in serum AAT levels with a single 35 mg dose was longer than initially expected, extending up to 16 weeks post-dose before returning to baseline. For example, thirty-four weeks after a single 35 mg dose, serum AAT levels in one subject had returned to levels above 90 mg/dL, while another subject's serum AAT levels remained at 40 mg/dL (60.4% below baseline). There was no significant difference in the duration of response across single doses of AAT RNAi drug ranging from 100 mg to 300 mg, with values beginning to return to baseline between 8 and 16 weeks after a single dose.

[120] Overall, multiple doses of the AAT RNAi drug produced robust serum AAT reductions for a longer period than a single dose. These data suggest that a second dose given on day 29 (i.e., one month after the initial dose) may further reduce serum AAT levels or maintain this reduction, and subsequent doses could be administered every 12 weeks (i.e., quarterly) to maintain maximally reduced serum AAT levels.

[121] There were no deaths, serious adverse events (SAEs), or adverse events (AEs) considered severe in intensity in the Phase I study. Among subjects receiving the AAT RNAi drug substance, three AEs of moderate intensity (upper respiratory tract infection, runny nose, generalized chest pain) were reported in two subjects. Among subjects receiving placebo, three AEs of moderate intensity were reported in three subjects (2 - gastroenteritis, left-sided musculoskeletal chest pain). All other AEs were mild. Most subjects reported AEs unrelated to study treatment. In one case, an AE in a subject receiving AAT led to early discontinuation of therapy, although the subject was continued to be followed during the study. Ninety-four AEs were reported in 28 subjects who received at least one dose of the ready-to-use AAT RNAi drug substance. Forty-six AEs were reported in 17 subjects receiving placebo. There was no apparent increase in the frequency or intensity of AEs with increasing dose.

[122] Six injection site AEs were observed in 6 subjects across all cohorts of AAT RNAi drug substance administration, all occurring in subjects receiving drug. No injection site AEs were observed in subjects receiving placebo. Injection site reactions that occurred included bruising, erythema, and pain at the injection site. These combined injection site AEs were reported by 21.4% of subjects receiving the AAT RNAi drug substance. Six out of 50, or 12%, of AAT RNAi drug substance injections resulted in the development of an injection site AE. Injection site AEs did not occur more than once in the same subject. All injection site AEs were rated as mild in intensity.

Other embodiments of the invention

[123] It should be understood that while the invention has been described along with the detailed description thereof, the foregoing description is intended to illustrate and not to limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are presented within the scope of the appended claims.

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SEQUENCE LIST

<110> ARROWHEAD PHARMACEUTICALS, INC.

<120> Treatment options for alpha-1 antitrypsin deficiency (AATD)

<130> 30674-WO1

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tttaaaggca aatgggagag accctttgaa gtcaaggaca ccgaggaaga ggacttccac 960

gtggaccagg tgaccaccgt gaaggtgcct atgatgaagc gtttaggcat gtttaacatc 1020

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gccatgtttt tagaggccat acccatgtct atcccccccg aggtcaagtt caacaaaccc 1440

tttgtcttct taatgattga acaaaatacc aagtctcccc tcttcatggg aaaagtggtg 1500

aatcccaccc aaaaataact gcctctcgct cctcaacccc tcccctccat ccctggcccc 1560

ctccctggat gacattaaag aagggttgag ctggtccctg cctgcatgtg actgtaaatc 1620

cctcccatgt tttctctgag tctccctttg cctgctgagg ctgtatgtgg gctccaggta 1680

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aggaaccaga taccagccat gaccccaggc tccaccaagc atcttcatgt ccccctgctc 1920

atcccccact cccccccacc cagagttgct catcctgcca gggctggctg tgcccacccc 1980

aaggctgccc tcctgggggc cccagaactg cctgatcgtg ccgtggccca gttttgtggc 2040

atctgcagca acacaagaga gaggacaatg tcctcctctt gacccgctgt cacctaacca 2100

gactcgggcc ctgcacctct caggcacttc tggaaaatga ctgaggcaga ttcttcctga 2160

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tctttgtgcc tgttgctgtc cctgcagtct tcagagggcc accgcagctc cagtgccacg 2580

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aaactaatct aggaatcact gtctaaccac tcactgtcat gaatgtgtac ttaaaggatg 2820

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<212> RNA

<213> Artificial sequence

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<223> Modified sequence of the antisense strand of the agent for

RNAi AAT

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<223> Modified sequence of the semantic thread of the means for

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agcguuuagg cauguuuaac a 21

<---

Claims (49)

1. A method for treating alpha-1 antitrypsin deficiency (AATD) in a human subject in need thereof, comprising administering to the subject by subcutaneous injection doses of a pharmaceutical composition comprising from about 100 mg to about 300 mg of an RNAi AAT drug substance, wherein the RNAi AAT drug substance comprises: a sense strand comprising the structure (NAG37)s(invAb)sagcguuuaGfGfCfauguuuaacas(invAb) (SEQ ID NO: 6) and an antisense strand comprising the structure usGfsuUfaAfacaugCfcUfaAfaCfgCfsu (SEQ ID NO: 2), wherein the sense and antisense strands are hybridized to form a duplex, where a, c, g, and u represent 2′-O-methyladenosine, 2′-O-methylcytidine, 2′-O-methylguanosine, and 2′-O-methyluridine, respectively; Af, Cf, Gf, and Uf represent 2′-fluoroadenosine, 2′-fluorocytidine, 2′-fluoroguanosine, and 2′-fluorouridine, respectively; s represents a phosphorothioate linkage; (invAb) represents an inverted deoxyribose residue with the base removed; and (NAG37)s represents the structure or wherein the pharmaceutical composition is administered once every 84 days. 2. A method for treating alpha-1 antitrypsin deficiency (AATD) in a human subject in need thereof, comprising administering to a subject by subcutaneous administration of doses of a pharmaceutical composition comprising from about 100 mg to about 300 mg of an RNAi AAT drug substance, wherein the RNAi AAT drug substance comprises a sense strand comprising the structure (NAG37)s(invAb)sagcguuuaGfGfCfauguuuaacas(invAb) (SEQ ID NO: 6) and an antisense strand comprising the structure usGfsuUfaAfacaugCfcUfaAfaCfgCfsu (SEQ ID NO: 2), wherein the sense and antisense strands are hybridized to form a duplex, where a, c, g, and u represent 2′-O-methyladenosine, 2′-O-methylcytidine, 2′-O-methylguanosine, and 2′-O-methyluridine, respectively; Af, Cf, Gf, and Uf represent 2′-fluoroadenosine, 2′-fluorocytidine, 2′-fluoroguanosine, and 2′-fluorouridine, respectively; s represents a phosphorothioate linkage; (invAb) represents an inverted deoxyribose residue with the base removed; and (NAG37)s represents the structure or where the introduction includes: a) administration of an initial dose of a pharmaceutical composition to the subject; b) administering to the subject a second dose of the pharmaceutical composition approximately 28 days after the initial dose; and c) administering to the subject a third dose of the pharmaceutical composition approximately 84 days after the second dose. 3. The method according to claim 1 or 2, wherein each dose of the pharmaceutical composition contains the medicinal substance RNAi AAT in an amount of about 100 mg. 4. The method according to claim 1 or 2, wherein each dose of the pharmaceutical composition contains the RNAi AAT medicinal substance in an amount of approximately 200 mg. 5. The method according to claim 1 or 2, wherein each dose of the pharmaceutical composition contains the RNAi AAT drug substance in an amount of approximately 200 mg or less. 6. The method according to any one of claims 1-5, wherein the condition or disease caused by AATD is a liver disease. 7. The method of claim 6, wherein the liver disease is chronic hepatitis, cirrhosis, increased risk of hepatocellular carcinoma, elevated transaminase activity, cholestasis, fibrosis, or fulminant liver failure. 8. The method of any one of claims 1-7, wherein the subject is further administered an additional drug for treating AATD. 9. The method of any one of claims 1-8, wherein the subject is further administered a drug for the treatment of lung injury, emphysema, or other lung diseases or disorders caused by a deficiency of endogenously secreted AAT protein. 10. The method of claim 9, wherein the additional medicinal product comprises human AAT protein, purified human alpha-1 protease inhibitor, or recombinant AAT protein. 11. The method according to any one of claims 1-10, wherein the pharmaceutical composition is packaged in a kit, container, package, dispenser, pre-fillable syringe or vials. 12. The method according to any one of claims 1-11, wherein the pharmaceutical composition comprises, consists of, or essentially consists of a ready-to-use AAT RNAi drug substance containing the AAT RNAi drug substance at a concentration of 230 mg/ml in an aqueous solution of sodium phosphate buffer with 0.5 mM monobasic sodium phosphate and 0.5 mM dibasic sodium phosphate. 13. The method of claim 2, further comprising administering additional doses after the third dose, wherein the additional doses are administered at intervals of approximately 84 days. 14. The method according to any one of claims 1-13, wherein the administration of one or more doses of the pharmaceutical composition is performed by a subject. 15. The method according to any one of claims 1-13, wherein the administration of one or more doses of the pharmaceutical composition is performed by a healthcare professional. 16. The method according to any one of claims 1-15, wherein the RNAi AAT medicinal substance is presented in the form of a salt, a mixed salt or a free acid. 17. The method according to any one of claims 1-16, wherein the RNAi AAT medicinal substance is presented in the form of a sodium salt. 18. Use of an RNAi AAT drug substance for the treatment of alpha-1 antitrypsin deficiency (AATD) in a human subject in need thereof, wherein the use comprises administering by subcutaneous administration to the subject doses of a pharmaceutical composition comprising from about 100 mg to about 300 mg of an RNAi AAT drug substance, wherein the RNAi AAT drug substance comprises: a sense strand comprising the structure (NAG37)s(invAb)sagcguuuaGfGfCfauguuuaacas(invAb) (SEQ ID NO: 6) and an antisense strand comprising the structure usGfsuUfaAfacaugCfcUfaAfaCfgCfsu (SEQ ID NO: 2), wherein the sense and antisense strands are hybridized to form a duplex, where a, c, g, and u represent 2′-O-methyladenosine, 2′-O-methylcytidine, 2′-O-methylguanosine, and 2′-O-methyluridine, respectively; Af, Cf, Gf, and Uf represent 2′-fluoroadenosine, 2′-fluorocytidine, 2′-fluoroguanosine, and 2′-fluorouridine, respectively; s represents a phosphorothioate linkage; (invAb) represents an inverted deoxyribose residue with the base removed; and (NAG37)s represents the structure or wherein the pharmaceutical composition is administered once every 84 days. 19. The use according to claim 18, wherein the medicinal substance RNAi AAT is presented in the form of a salt, a mixed salt or a free acid. 20. The use according to claim 18 or 19, wherein the medicinal substance RNAi AAT is presented in the form of a sodium salt. 21. A method for treating alpha-1 antitrypsin deficiency (AATD) in a human subject in need thereof, comprising administering to the subject via subcutaneous administration doses of a pharmaceutical composition comprising approximately 200 mg of an RNAi AAT drug substance, wherein the RNAi AAT drug substance comprises: a sense strand comprising the structure (NAG37)s(invAb)sagcguuuaGfGfCfauguuuaacas(invAb) (SEQ ID NO: 6) and an antisense strand comprising the structure usGfsuUfaAfacaugCfcUfaAfaCfgCfsu (SEQ ID NO: 2), wherein the sense and antisense strands are hybridized to form a duplex, where a, c, g, and u represent 2′-O-methyladenosine, 2′-O-methylcytidine, 2′-O-methylguanosine, and 2′-O-methyluridine, respectively; Af, Cf, Gf, and Uf represent 2′-fluoroadenosine, 2′-fluorocytidine, 2′-fluoroguanosine, and 2′-fluorouridine, respectively; s represents a phosphorothioate linkage; (invAb) represents an inverted deoxyribose residue with the base removed; and (NAG37)s represents the structure or where the introduction includes: a) administering to the subject an initial dose of the pharmaceutical composition; b) administering to the subject a second dose of the pharmaceutical composition approximately 28 days after the initial dose; and c) administering to the subject a third dose of the pharmaceutical composition approximately 84 days after the second dose. 22. The method according to claim 21, wherein the RNAi AAT medicinal substance is presented in the form of a salt, a mixed salt or a free acid. 23. The method according to claim 21 or 22, wherein the medicinal substance RNAi AAT is presented in the form of a sodium salt.