RNAi constructs and methods for inhibiting LPA expression

Abstract

The present invention relates to an RNAi construct for reducing the expression of the LPA gene, which encodes apolipoprotein(a), a component of lipoprotein(a) (Lp(a)) particles. Methods for using such RNAi constructs to treat or prevent cardiovascular diseases, such as coronary artery disease, peripheral artery disease, stroke, and myocardial infarction, and to reduce serum Lp(a) levels are also described.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 945,777, filed December 9, 2019, which is incorporated herein by reference in its entirety.

[0002] Description of electronically submitted text files

[0001] This application contains a Sequence Listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. A copy of the Sequence Listing in computer readable format, created on December 8, 2020, is titled A-2425-WO-PCT_ST25 and is 190 kilobytes in size.

[0003] The present invention relates to compositions and methods for modulating hepatic expression of the LPA gene, which encodes apolipoprotein(a) (apo(a)). In particular, the present invention relates to nucleic acid-based therapeutics for reducing LPA gene expression via RNA interference to reduce blood levels of lipoprotein(a) (Lp(a)) and treat or prevent cardiovascular disease, and methods of using such nucleic acid-based therapeutics. [Background technology]

[0004] Lp(a) is a low-density lipoprotein (LDL) composed of LDL particles and apo(a), a glycoprotein linked to apolipoprotein B of the LDL particles by disulfide bonds. Apo(a) is encoded by the LPA gene and is expressed almost exclusively in primates, including humans. Apo(a) exhibits homology to plasminogen and exists in various isoforms due to intragenic size polymorphism resulting from a variable number of kringle IV type 2 (KIV-2) domain repeats (see Kronenberg and Utermann, J. Intern. Med., Vol. 273:6-30, 2013). An inverse correlation has been observed between the size of apo(a) isoforms and the plasma concentration of Lp(a) particles (Sandholzer et al., Hum. Genet., Vol. 86;607-614, 1991).

[0005] Although the physiological function of Lp(a) is unknown, Lp(a) has been shown to play a pathogenic role in the development of atherosclerosis and thrombosis (Nordestgaard and Langsted, Lipid Res., Vol. 57:1953-75, 2016). Several genetic and observational studies have documented the association between Lp(a) levels and coronary artery disease, myocardial infarction, stroke, peripheral vascular disease, and aortic stenosis (Schmidt et al., J. Lipid Res., Vol. 57:1339-1359, 2016). This risk relationship is known to be continuous, with higher Lp(a) levels associated with proportionally greater risk. This association persists even after accounting for other lipid parameters (Emerging Risk Factors Collaboration, JAMA, Vol. 302:412-423, 2009).

[0006] High plasma Lp(a) concentrations are genetically determined, remain stable, and cannot be controlled by habitual changes (diet, exercise, or other environmental factors), nor are they effectively controlled by any of the currently available lipid-lowering medications. Currently, no approved therapies have been shown to reduce the risk of cardiovascular events by lowering Lp(a). Modest reductions in Lp(a) have been observed with proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitors, niacin, and mipomersen (Santos et al., Arterioscler. Thromb. Vasc. Biol., Vol. 35:689-699, 2015; Yeang et al., Curr. Opin. Lipidol., Vol. 26:169-178, 2015; and Landray et al., N. Engl. J. Med., Vol. 371:203-212, 2014). Although apheresis is effective in lowering Lp(a), it is currently only used in a few countries with limited availability (Julius, J. Cardiovasc. Dev. Dis., Vol. 5:27-37, 2018). Additionally, apheresis is a non-invasive, extremely expensive procedure requiring frequent office visits, making it unavailable as a long-term treatment for subjects requiring lifelong therapy (Khan et al., Eur. Heart J., Vol. 38:1561-1569, 2017; Roeseler et al., Arterioscler. Thromb. Vasc. Biol., Vol. 36:2019-2027, 2016; Leebmann et al., Circulation, Vol. 128:2567-2576, 2013; Safarova et al., Atheroscler. Suppl., Vol. 14:93-99, 2013).

[0007] Therefore, there is a need in the art for new agents that potently reduce elevated Lp(a) levels for extended periods of time to provide additional protection against cardiovascular disease. [Prior art documents]

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[0008]

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[0009] The present invention is based, in part, on the design and generation of RNAi constructs that target the LPA gene and reduce expression of the encoded apo(a) protein in liver cells. Sequence-specific inhibition of LPA gene expression is useful for treating or preventing conditions associated with elevated Lp(a) levels, such as cardiovascular disease. Thus, in one embodiment, the present invention provides an RNAi construct comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region having a sequence complementary to the LPA mRNA sequence. In certain embodiments, the antisense strand comprises or consists of a sequence selected from any of the antisense sequences listed in Table 1 or Table 2.

[0010] In some embodiments, the sense strand of an RNAi construct described herein comprises a sequence sufficiently complementary to that of the antisense strand to form a duplex region of about 15 to about 30 base pairs in length. In these and other embodiments, the sense and antisense strands are each independently about 19 to about 30 nucleotides in length. In some embodiments, the RNAi construct comprises one or two blunt ends. In other embodiments, the RNAi construct comprises one or two nucleotide overhangs. Such nucleotide overhangs may comprise one to six unpaired nucleotides and may be located at the 3'-end of the sense strand, the 3'-end of the antisense strand, or the 3'-ends of both the sense and antisense strands. In certain embodiments, the RNAi construct comprises two unpaired nucleotide overhangs at the 3'-end of the sense strand and the 3'-end of the antisense strand. In other embodiments, the RNAi construct comprises two unpaired nucleotide overhangs at the 3'-end of the antisense strand and blunt ends at the 3'-end of the sense strand / 5'-end of the antisense strand.

[0011] The RNAi constructs of the present invention may contain one or more modified nucleotides, including nucleotides with modifications to the ribose ring, nucleobase, or phosphodiester backbone. In some embodiments, the RNAi constructs contain one or more 2'-modified nucleotides. Such 2'-modified nucleotides may include 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides, 2'-O-alkyl-modified nucleotides, 2'-O-allyl-modified nucleotides, bicyclic nucleic acids (BNAs), deoxyribonucleotides, or combinations thereof. In a specific embodiment, the RNAi constructs contain one or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, or combinations thereof. In some embodiments, all nucleotides in the sense and antisense strands of the RNAi construct are modified nucleotides. The RNAi constructs of the present invention may incorporate abasic nucleotides as terminal nucleotides, for example, at the 3'-end, 5'-end, or both the 3'-end and 5'-end of the sense strand. In such embodiments, the abasic nucleotide may be in an inverted position, for example, linked to the adjacent nucleotide via a 3'-3' internucleotide linkage or a 5'-5' internucleotide linkage.

[0012] In some embodiments, the RNAi construct comprises at least one backbone modification, such as a modified internucleotide or internucleoside linkage. In certain embodiments, the RNAi construct described herein comprises at least one phosphorothioate internucleotide linkage. In certain embodiments, the phosphorothioate internucleotide linkage may be located at the 3'-end or 5'-end of the sense strand and / or the antisense strand.

[0013] In certain embodiments, the antisense and / or sense strands of an RNAi construct of the invention may comprise or consist of sequences derived from the antisense and sense sequences listed in Table 1 or Table 2. In certain embodiments, the RNAi construct may be any one of the duplex compounds listed in any one of Tables 1-15. In one embodiment, the RNAi construct is 4601, 4613, 4930, 4970, 6150, 6182, 6247, 8395, 8401, 10927, 11318, 11344, 11351, 11374, 11580, 17188, 17205, 18436, 18444, or 18446. In another embodiment, the RNAi construct is 4601, 4613, 10927, 11351, 11374, 11580, 18436, or 18444.

[0014] The RNAi construct may further comprise a ligand that facilitates delivery or uptake of the RNAi construct to specific tissues or cells, such as liver cells. In certain embodiments, the ligand targets delivery of the RNAi construct to liver cells. In these and other embodiments, the ligand may comprise galactose, galactosamine, or N-acetyl-galactosamine (GalNAc). In certain embodiments, the ligand comprises a multivalent galactose or multivalent GalNAc moiety, such as a trivalent or tetravalent galactose or GalNAc moiety. The ligand may be covalently attached to the 5' or 3' end of the sense strand of the RNAi construct, optionally via a linker. In certain embodiments, the ligand comprises a structure of Structure 1, as described herein. In one such embodiment, a ligand having this structure is covalently attached to the 5' end of the sense strand, optionally via a linker, such as an aminohexyl linker. In some embodiments, the RNAi construct comprises a ligand and a linker having the structure of any one of Formulas I-IX described herein. In certain embodiments, the RNAi construct comprises a ligand and a linker having the structure of Formula VII. In other embodiments, the RNAi construct comprises a ligand and a linker having the structure of Formula IV.

[0015] The present invention also provides pharmaceutical compositions comprising any of the RNAi constructs described herein and a pharmaceutically acceptable carrier, excipient, or diluent. Such pharmaceutical compositions are particularly useful for reducing LPA gene expression in cells (e.g., liver cells) of a patient in need thereof. Patients who may receive the pharmaceutical compositions of the present invention include patients with a history of myocardial infarction, patients diagnosed with or at risk for coronary artery disease or other forms of cardiovascular disease, and patients with elevated serum Lp(a) or plasma Lp(a) levels. Accordingly, the present invention includes methods for treating or preventing cardiovascular disease in a patient in need thereof by administering the RNAi constructs or pharmaceutical compositions described herein. In certain embodiments, the present invention provides methods for reducing Lp(a) levels in a patient in need thereof by administering the RNAi constructs or pharmaceutical compositions described herein.

[0016] The use of an LPA-targeting RNAi construct in any of the methods described herein, or for preparing a medicament for administration by the methods described herein, is specifically contemplated.For example, the present invention includes an LPA-targeting RNAi construct for use in a method for treating or preventing cardiovascular disease, including coronary artery disease, peripheral artery disease, myocardial infarction, or stroke, in a patient in need thereof.The present invention also includes an LPA-targeting RNAi construct for use in a method for reducing Lp(a) levels in a patient in need thereof.In some embodiments, the present invention provides an LPA-targeting RNAi construct for use in a method for reducing the risk of myocardial infarction in a patient in need thereof.

[0017] The present invention also encompasses the use of an LPA-targeting RNAi construct in the preparation of a medicament for treating or preventing cardiovascular disease, including coronary artery disease, peripheral artery disease, myocardial infarction, or stroke, in a patient in need thereof. In certain embodiments, the present invention provides the use of an LPA-targeting RNAi construct in the preparation of a medicament for reducing Lp(a) levels in a patient in need thereof. In certain other embodiments, the present invention provides the use of an LPA-targeting RNAi construct in the preparation of a medicament for reducing the risk of myocardial infarction in a patient in need thereof. [Brief explanation of the drawings]

[0018] [Figure 1-1] Figure 1 shows the nucleotide sequence of the transcript of human LPA (NCBI reference sequence NM_005577.4, SEQ ID NO: 1). The transcript sequence is shown as the complementary DNA (cDNA) sequence with uracil bases replaced by thymine bases. [Figure 1-2] Figure 1 shows the nucleotide sequence of the transcript of human LPA (NCBI reference sequence NM_005577.4, SEQ ID NO: 1). The transcript sequence is shown as the complementary DNA (cDNA) sequence with uracil bases replaced by thymine bases. [Figure 1-3] Figure 1 shows the nucleotide sequence of the transcript of human LPA (NCBI reference sequence NM_005577.4, SEQ ID NO: 1). The transcript sequence is shown as the complementary DNA (cDNA) sequence with uracil bases replaced by thymine bases. [Figure 1-4] Figure 1 shows the nucleotide sequence of the transcript of human LPA (NCBI reference sequence NM_005577.4, SEQ ID NO: 1). The transcript sequence is shown as the complementary DNA (cDNA) sequence with uracil bases replaced by thymine bases. [Figure 2] The percentage of residual serum Lp(a) is shown compared to pre-dose baseline levels in cynomolgus monkeys followed by a single subcutaneous injection of 2 mg / kg of the indicated LPA RNAi construct on day 1. [Figure 3]The percentage of residual serum Lp(a) is shown compared to pre-dose baseline levels in cynomolgus monkeys followed by a single subcutaneous injection of 2 mg / kg of the indicated LPA RNAi construct on day 1. [Figure 4] The percentage of residual serum Lp(a) is shown compared to pre-dose baseline levels in cynomolgus monkeys followed by a single subcutaneous injection of 2 mg / kg of the indicated LPA RNAi construct on day 1. DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention relates to compositions and methods for regulating the expression of the LPA gene in cells or mammals. In some embodiments, the compositions of the present invention comprise an RNAi construct that targets mRNA transcribed from the LPA gene encoding the apo(a) protein and reduces apo(a) expression in cells or mammals. Such RNAi constructs reduce serum levels of Lp(a) and are useful for treating or preventing various forms of cardiovascular disease, such as atherosclerosis, coronary artery disease, peripheral artery disease, and aortic stenosis, and for reducing the risk of myocardial infarction or stroke.

[0020] As used herein, the term "RNAi construct" refers to an agent comprising an RNA molecule that, when introduced into a cell, can downregulate the expression of a target gene (e.g., an LPA gene) via the RNA interference mechanism. RNA interference is a process in which a nucleic acid molecule induces the cleavage and degradation of a target RNA molecule (e.g., a messenger RNA or mRNA molecule) in a sequence-specific manner, for example, via the RNA-induced silencing complex (RISC) pathway. In some embodiments, an RNAi construct comprises a double-stranded RNA molecule comprising two antiparallel strands of consecutive nucleotides that are sufficiently complementary to each other to hybridize and form a duplex region. "Hybridizing" or "hybridization" typically refers to the pairing of complementary polynucleotides via hydrogen bonds (e.g., Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonds) between complementary bases in two polynucleotides. The strand containing a region having a sequence substantially complementary to a target sequence (e.g., a target mRNA) is referred to as the "antisense strand." "Sense strand" refers to the strand that includes a region that is substantially complementary to a region of the antisense strand. In some embodiments, the sense strand can include a region that has substantial sequence identity to a target sequence.

[0021] Double-stranded RNA molecules may contain chemical modifications to ribonucleotides, including modifications to the ribose sugar, base, or backbone components of ribonucleotides, such as those described herein or known in the art. Any such modifications as used in double-stranded RNA molecules (e.g., siRNA, shRNA, etc.) are encompassed by the term "double-stranded RNA" for purposes of this disclosure.

[0022] As used herein, a first sequence is "complementary" to a second sequence if, under certain conditions, such as physiological conditions, the polynucleotide comprising the first sequence can hybridize to the polynucleotide comprising the second sequence to form a duplex region. Other such conditions can include moderate or stringent hybridization conditions, which are known to those skilled in the art. A first sequence is considered to be fully complementary (100% complementary) to a second sequence if the polynucleotide comprising the first sequence base pairs with the polynucleotide comprising the second sequence without any mismatches along the entire length of one or both nucleotide sequences. A sequence is "substantially complementary" to a target sequence if the sequence is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary to the target sequence. The percent complementarity can be calculated by dividing the number of bases in a first sequence that are complementary to the bases at corresponding positions in a second sequence or target sequence by the total length of the first sequence. When two sequences hybridize, if there are 5, 4, 3, or 2 or fewer mismatches across a 30-base pair duplex region, the sequence can be said to be substantially complementary to the other sequence. Generally, if nucleotide overhangs, as defined herein, exist, the sequence of such overhangs is not considered when determining the degree of complementarity between two sequences. For example, a 21-nucleotide sense strand and a 21-nucleotide antisense strand that hybridize to form a 19-base pair duplex region with a 2-nucleotide overhang at the 3' end of each strand would be considered fully complementary as this term is used herein.

[0023] In some embodiments, the region of the antisense strand comprises a sequence that is substantially or completely complementary to a region of the target RNA sequence (e.g., LPA mRNA). In such embodiments, the sense strand may comprise a sequence that is completely complementary to the sequence of the antisense strand. In other such embodiments, the sense strand may comprise a sequence that is substantially complementary to the sequence of the antisense strand, for example, a sequence that has 1, 2, 3, 4, or 5 mismatches in the duplex region formed by the sense strand and the antisense strand. In certain embodiments, it is preferred that any mismatches occur within the terminal regions (e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5'-end and / or 3'-end of the strand). In one embodiment, any mismatches in the duplex region formed by the sense strand and the antisense strand occur within 6, 5, 4, 3, or 2 nucleotides of the 5'-end of the antisense strand.

[0024] In certain embodiments, the sense strand and antisense strand of double-stranded RNA can be hybridized to form a duplex region, but otherwise can be two separate molecules that are separate.This double-stranded RNA molecule formed by two separate strands is called "small interfering RNA" or "short interfering RNA" (siRNA).Therefore, in some embodiments, the RNAi construct of the present invention comprises siRNA.

[0025] In other embodiments, the sense and antisense strands that hybridize to form the duplex region may be part of a single RNA molecule, i.e., the sense and antisense strands are part of a self-complementary region of a single RNA molecule. In such cases, the single RNA molecule comprises a duplex region (also referred to as a stem region) and a loop region. The 3' end of the sense strand is connected to the 5' end of the antisense strand by a contiguous sequence of unpaired nucleotides, forming the loop region. The loop region is typically long enough to allow the RNA molecule to refold on itself so that the antisense strand can base pair with the sense strand to form the duplex or stem region. The loop region may contain about 3 to about 25, about 5 to about 15, or about 8 to about 12 unpaired nucleotides. Such RNA molecules having at least a partially self-complementary region are referred to as "short hairpin RNAs" (shRNAs). In certain embodiments, the RNAi constructs of the present invention comprise shRNAs. The length of the single, at least partially self-complementary RNA molecule can be from about 40 nucleotides to about 100 nucleotides, from about 45 nucleotides to about 85 nucleotides, or from about 50 nucleotides to about 60 nucleotides, including the duplex region and loop region, each having the lengths listed herein.

[0026] In some embodiments, the RNAi construct of the present invention comprises a sense strand and an antisense strand, and the antisense strand comprises a region having a sequence substantially or completely complementary to the messenger RNA (mRNA) sequence of LPA. As used herein, "LPA mRNA sequence" refers to any messenger RNA sequence, including allelic variants and splice variants, encoding apo(a) protein, including variants or isoforms of apo(a) protein from any species (e.g., non-human primates, humans). The LPA gene (also known as AK38, APOA, and LP) is a major component of low-density lipoprotein particles known as lipoprotein(a) or Lp(a). In humans, the LPA gene is found on chromosome 6 at locus 6q25.3-q26. The LPA gene is highly polymorphic due to differences between individuals in the copy number of the Kringle IV type 2 (KIV-2) domain, which can range from 2 to 40 or more copies of the gene alleles (see, e.g., Kronenberg and Utermann, J. Intern. Med., Vol. 273:6-30, 2013).

[0027] The LPA mRNA sequence also includes the transcript sequence expressed as its complementary DNA (cDNA) sequence. A cDNA sequence refers to the sequence of an mRNA transcript expressed as DNA bases (e.g., guanine, adenine, thymine, and cytosine) rather than RNA bases (e.g., guanine, adenine, uracil, and cytosine). Thus, the antisense strand of the RNAi construct of the present invention can include a region having a sequence that is substantially or completely complementary to the target LPA mRNA sequence or LPA cDNA sequence. The LPA mRNA or cDNA sequence may include, but is not limited to, any LPA mRNA or cDNA sequence selected from the NCBI reference sequence NM_005577.4 (human, FIG. 1, SEQ ID NO: 1), XM_015448520.1 (cynomolgus monkey), XM_028847001.1 (rhesus monkey), XM_024357489.1 (chimpanzee), and XM_031012244.1 (gorilla). In certain embodiments, the LPA mRNA sequence is the human transcript listed in the NCBI database as reference sequence NM_005577.4 (see FIG. 1, SEQ ID NO: 1).

[0028] The region of the antisense strand may be substantially or completely complementary to at least 15 contiguous nucleotides of the LPA mRNA sequence. In some embodiments, the target region of the LPA mRNA sequence to which the antisense strand is complementary may be in the range of about 15 to about 30 contiguous nucleotides, about 16 to about 28 contiguous nucleotides, about 18 to about 26 contiguous nucleotides, about 17 to about 24 contiguous nucleotides, about 19 to about 30 contiguous nucleotides, about 19 to about 25 contiguous nucleotides, about 19 to about 23 contiguous nucleotides, or about 19 to about 21 contiguous nucleotides. In certain embodiments, the region of the antisense strand that is substantially or completely complementary to the LPA mRNA sequence may, in some embodiments, comprise at least 15 contiguous nucleotides from an antisense sequence listed in Table 1 or Table 2. In other embodiments, the antisense sequence comprises at least 16, at least 17, at least 18, or at least 19 contiguous nucleotides from an antisense sequence listed in Table 1 or Table 2.

[0029] The sense strand of an RNAi construct typically contains a sequence sufficiently complementary to that of the antisense strand so that the two strands hybridize under physiological conditions to form a duplex region. A "duplex region" refers to a region of two complementary or substantially complementary polynucleotides that base-pair with each other through either Watson-Crick base pairing or other hydrogen-bonding interactions, creating a duplex between the two polynucleotides. The duplex region of an RNAi construct must be long enough to allow, for example, the Dicer enzyme and / or the RISC complex to associate with the RNAi construct, thereby allowing it to enter the RNA interference pathway. For example, in some embodiments, the duplex region is about 15 to about 30 base pairs in length. Other lengths of the duplex region within this range are also suitable, such as about 15 to about 28 base pairs, about 15 to about 26 base pairs, about 15 to about 24 base pairs, about 15 to about 22 base pairs, about 17 to about 28 base pairs, about 17 to about 26 base pairs, about 17 to about 24 base pairs, about 17 to about 23 base pairs, about 17 to about 21 base pairs, about 19 to about 25 base pairs, about 19 to about 23 base pairs, or about 19 to about 21 base pairs. In certain embodiments, the duplex region is about 17 to about 24 base pairs in length. In other embodiments, the duplex region is about 19 to about 21 base pairs in length. In one embodiment, the duplex region is about 19 base pairs in length. In another embodiment, the duplex region is about 21 base pairs in length.

[0030] In embodiments in which the sense and antisense strands are two separate molecules (e.g., the RNAi construct comprises an siRNA), the sense and antisense strands need not be the same length as the duplex region. For example, one or both strands may be longer than the duplex region and may have one or more unpaired nucleotides or mismatches adjacent to the duplex region. Thus, in some embodiments, the RNAi construct comprises at least one nucleotide overhang. As used herein, "nucleotide overhang" refers to an unpaired nucleotide at the end of a strand or a nucleotide that extends beyond the duplex region. Nucleotide overhangs are typically generated when the 3' end of one strand extends beyond the 5' end of the other strand, or when the 5' end of one strand extends beyond the 3' end of the other strand. The length of a nucleotide overhang is generally 1 to 6 nucleotides, 1 to 5 nucleotides, 1 to 4 nucleotides, 1 to 3 nucleotides, 2 to 6 nucleotides, 2 to 5 nucleotides, or 2 to 4 nucleotides. In some embodiments, a nucleotide overhang comprises 1, 2, 3, 4, 5, or 6 nucleotides. In one particular embodiment, the nucleotide overhang comprises 1 to 4 nucleotides. In certain embodiments, the nucleotide overhang comprises 2 nucleotides. In certain other embodiments, the nucleotide overhang comprises a single nucleotide.

[0031] The nucleotides in the overhang can be ribonucleotides or modified nucleotides as described herein. In some embodiments, the nucleotides in the overhang are 2'-modified nucleotides (e.g., 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides), deoxyribonucleotides, abasic nucleotides, inverted nucleotides (e.g., inverted abasic nucleotides, inverted deoxyribonucleotides), or combinations thereof. For example, in one embodiment, the nucleotides in the overhang are deoxyribonucleotides, e.g., deoxythymidine. In another embodiment, the nucleotides in the overhang are 2'-O-methyl-modified nucleotides, 2'-fluoro-modified nucleotides, 2'-methoxyethyl-modified nucleotides, or combinations thereof. In other embodiments, the overhang comprises a 5'-uridine-uridine-3' (5'-UU-3') dinucleotide. In such embodiments, the UU dinucleotide can comprise a ribonucleotide or a modified nucleotide, e.g., a 2'-modified nucleotide. In other embodiments, the overhang comprises a 5'-deoxythymidine-deoxythymidine-3' (5'-dTdT-3') dinucleotide. When a nucleotide overhang is present in the antisense strand, the nucleotides in the overhang may be complementary to the target gene sequence, may form a mismatch with the target gene sequence, or may comprise some other sequence (e.g., a polypyrimidine or polypurine sequence, such as UU, TT, AA, GG, etc.).

[0032] The nucleotide overhangs may be present at the 5'-end or 3'-end of one or both strands. For example, in one embodiment, the RNAi construct comprises nucleotide overhangs at the 5'-end and 3'-end of the antisense strand. In another embodiment, the RNAi construct comprises nucleotide overhangs at the 5'-end and 3'-end of the sense strand. In some embodiments, the RNAi construct comprises nucleotide overhangs at the 5'-end of the sense strand and the 5'-end of the antisense strand. In other embodiments, the RNAi construct comprises nucleotide overhangs at the 3'-end of the sense strand and the 3'-end of the antisense strand.

[0033] An RNAi construct may comprise a single nucleotide overhang at one end of the double-stranded RNA molecule and a blunt end at the other end. "Blunt end" means that the sense and antisense strands are perfectly base-paired at the ends of the molecule, with no unpaired nucleotides extending beyond the duplex region. In some embodiments, an RNAi construct comprises a nucleotide overhang at the 3' end of the sense strand and blunt ends at the 5' end of the sense strand and the 3' end of the antisense strand. In other embodiments, an RNAi construct comprises a nucleotide overhang at the 3' end of the antisense strand and blunt ends at the 5' end of the antisense strand and the 3' end of the sense strand. In certain embodiments, an RNAi construct comprises blunt ends at both ends of the double-stranded RNA molecule. In such embodiments, the sense and antisense strands have the same length, and the duplex region is the same length as the sense and antisense strands (i.e., the molecule is double-stranded throughout its entire length).

[0034] The sense strand and antisense strand of the RNAi constructs of the present invention can each independently be about 15 to about 30 nucleotides in length, about 19 to about 30 nucleotides in length, about 18 to about 28 nucleotides in length, about 19 to about 27 nucleotides in length, about 19 to about 25 nucleotides in length, about 19 to about 23 nucleotides in length, about 19 to about 21 nucleotides in length, about 21 to about 25 nucleotides in length, or about 21 to about 23 nucleotides in length. In certain embodiments, the sense strand and antisense strand are each independently about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 nucleotides in length. In some embodiments, the sense strand and antisense strand have the same length but form a duplex region that is shorter than the other strands, such that the RNAi construct has a two-nucleotide overhang. For example, in one embodiment, the RNAi construct comprises (i) a sense strand and an antisense strand each 21 nucleotides in length, (ii) a duplex region 19 base pairs in length, and (iii) a nucleotide overhang of two unpaired nucleotides at both the 3' end of the sense strand and the 3' end of the antisense strand. In another embodiment, the RNAi construct comprises (i) a sense strand and an antisense strand each 23 nucleotides in length, (ii) a duplex region 21 base pairs in length, and (iii) a nucleotide overhang of two unpaired nucleotides at both the 3' end of the sense strand and the 3' end of the antisense strand. In other embodiments, the sense strand and the antisense strand have the same length and form a duplex region along their entire length such that there are no nucleotide overhangs at either end of the double-stranded molecule. In one such embodiment, the RNAi construct is blunt-ended and comprises (i) a sense strand and an antisense strand each 21 nucleotides in length, and (ii) a duplex region 21 base pairs in length. In another such embodiment, the RNAi construct is blunt-ended and comprises (i) sense and antisense strands, each 23 nucleotides in length, and (ii) a duplex region 23 base pairs in length. In yet another such embodiment, the RNAi construct is blunt-ended and comprises (i) sense and antisense strands, each 19 nucleotides in length, and (ii) a duplex region 19 base pairs in length.

[0035] In other embodiments, the sense strand or antisense strand is longer than the other strand, and the two strands form a duplex region having a length equal to the length of the shorter strand, such that the RNAi construct includes at least one nucleotide overhang. For example, in one embodiment, the RNAi construct includes (i) a sense strand 19 nucleotides long, (ii) an antisense strand 21 nucleotides long, (iii) a duplex region 19 base pairs long, and (iv) a nucleotide overhang of two unpaired nucleotides at the 3' end of the antisense strand. In another embodiment, the RNAi construct includes (i) a sense strand 21 nucleotides long, (ii) an antisense strand 23 nucleotides long, (iii) a duplex region 21 base pairs long, and (iv) a nucleotide overhang of two unpaired nucleotides at the 3' end of the antisense strand.

[0036] The antisense strand of an RNAi construct of the present invention may comprise or consist of any one of the antisense sequences listed in Table 1 or Table 2, the sequence of nucleotides 1-19 of any of these antisense sequences, or the sequence of nucleotides 2-19 of any of these antisense sequences. Thus, in some embodiments, the antisense strand comprises or consists of a sequence selected from SEQ ID NOs: 134-241, 437-601, 611, or 617-619. In other embodiments, the antisense strand comprises or consists of the sequence of nucleotides 1-19 of any one of SEQ ID NOs: 134-241, 437-601, 611, or 617-619. In yet other embodiments, the antisense strand comprises or consists of the sequence of nucleotides 2-19 of any one of SEQ ID NOs: 134-241, 437-601, 611, or 617-619. In certain embodiments, the antisense strand comprises or consists of a sequence selected from SEQ ID NO:137, SEQ ID NO:145, SEQ ID NO:164, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:189, SEQ ID NO:194, SEQ ID NO:196, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:205, SEQ ID NO:216, SEQ ID NO:224, SEQ ID NO:225, SEQ ID NO:440, SEQ ID NO:448, SEQ ID NO:471, SEQ ID NO:492, SEQ ID NO:497, SEQ ID NO:499, SEQ ID NO:515, SEQ ID NO:525, SEQ ID NO:530, SEQ ID NO:536, SEQ ID NO:540, SEQ ID NO:546, SEQ ID NO:547, SEQ ID NO:550, SEQ ID NO:568, SEQ ID NO:576, or SEQ ID NO:577. In some embodiments, the antisense strand comprises or consists of a sequence selected from SEQ ID NO:145, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:194, SEQ ID NO:196, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:448, SEQ ID NO:492, SEQ ID NO:497, SEQ ID NO:525, SEQ ID NO:530, SEQ ID NO:536, or SEQ ID NO:540.

[0037] In these and other embodiments, the sense strand of an RNAi construct of the invention may comprise or consist of any one of the sense sequences listed in Table 1 or Table 2, the sequence of nucleotides 1-19 of any of these sense sequences, or the sequence of nucleotides 2-19 of any of these sense sequences. Thus, in some embodiments, the sense strand comprises or consists of a sequence selected from SEQ ID NOs: 2-133, 242-436, 610, or 612-616. In other embodiments, the sense strand comprises or consists of the sequence of nucleotides 1-19 of any one of SEQ ID NOs: 2-133, 242-436, 610, or 612-616. In still other embodiments, the sense strand comprises or consists of the sequence of nucleotides 2-19 of any one of SEQ ID NOs: 2-133, 242-436, 610, or 612-616. In certain embodiments, the sense strand comprises or consists of a sequence selected from SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:35, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:71, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:91, SEQ ID NO:106, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:245, SEQ ID NO:253, SEQ ID NO:282, SEQ ID NO:304, SEQ ID NO:307, SEQ ID NO:312, SEQ ID NO:314, SEQ ID NO:341, SEQ ID NO:350, SEQ ID NO:357, SEQ ID NO:362, SEQ ID NO:364, SEQ ID NO:370, SEQ ID NO:372, SEQ ID NO:377, SEQ ID NO:378, SEQ ID NO:404, SEQ ID NO:413, or SEQ ID NO:415. In certain other embodiments, the sense strand comprises or consists of a sequence selected from SEQ ID NO:13, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:106, SEQ ID NO:253, SEQ ID NO:304, SEQ ID NO:307, SEQ ID NO:312, SEQ ID NO:350, SEQ ID NO:357, SEQ ID NO:362, SEQ ID NO:370, or SEQ ID NO:404.

[0038] In certain embodiments of the present invention, the RNAi construct comprises (i) a sense strand comprising or consisting of a sequence selected from SEQ ID NOs: 2 to 133, 242 to 436, 610, or 612 to 616, and (ii) an antisense strand comprising or consisting of a sequence selected from SEQ ID NOs: 134 to 241, 437 to 601, 611, or 617 to 619. In some embodiments, the RNAi construct comprises: (i) a sequence selected from SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:35, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:71, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:91, SEQ ID NO:106, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:245, SEQ ID NO:253, SEQ ID NO:282, SEQ ID NO:304, SEQ ID NO:307, SEQ ID NO:312, SEQ ID NO:314, SEQ ID NO:341, SEQ ID NO:350, SEQ ID NO:357, SEQ ID NO:362, SEQ ID NO:364, SEQ ID NO:370, SEQ ID NO:372, SEQ ID NO:377, SEQ ID NO:378, SEQ ID NO:404, SEQ ID NO:413, or SEQ ID NO:415 and (ii) an antisense strand comprising or consisting of a sequence selected from SEQ ID NO:137, SEQ ID NO:145, SEQ ID NO:164, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:189, SEQ ID NO:194, SEQ ID NO:196, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:205, SEQ ID NO:216, SEQ ID NO:224, SEQ ID NO:225, SEQ ID NO:440, SEQ ID NO:448, SEQ ID NO:471, SEQ ID NO:492, SEQ ID NO:497, SEQ ID NO:499, SEQ ID NO:515, SEQ ID NO:525, SEQ ID NO:530, SEQ ID NO:536, SEQ ID NO:540, SEQ ID NO:546, SEQ ID NO:547, SEQ ID NO:550, SEQ ID NO:568, SEQ ID NO:576, or SEQ ID NO:577.In other embodiments, the RNAi construct comprises (i) a sense strand comprising or consisting of a sequence selected from SEQ ID NO: 13, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 106, SEQ ID NO: 253, SEQ ID NO: 304, SEQ ID NO: 307, SEQ ID NO: 312, SEQ ID NO: 350, SEQ ID NO: 357, SEQ ID NO: 362, SEQ ID NO: 370, or SEQ ID NO: 404, and (ii) an antisense strand comprising or consisting of a sequence selected from SEQ ID NO: 145, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 448, SEQ ID NO: 492, SEQ ID NO: 497, SEQ ID NO: 525, SEQ ID NO: 530, SEQ ID NO: 536, or SEQ ID NO: 540.

[0039] In certain embodiments, the RNAi construct of the present invention comprises: (i) a sense strand comprising or consisting of the sequence of SEQ ID NO: 13 and an antisense strand comprising or consisting of the sequence of SEQ ID NO: 145; (ii) a sense strand comprising or consisting of the sequence of SEQ ID NO: 35 and an antisense strand comprising or consisting of the sequence of SEQ ID NO: 164; (iii) a sense strand comprising or consisting of the sequence of SEQ ID NO: 53 and an antisense strand comprising or consisting of the sequence of SEQ ID NO: 177; (iv) a sense strand comprising or consisting of the sequence of SEQ ID NO: 91 and an antisense strand comprising or consisting of the sequence of SEQ ID NO: 205; (v) a sense strand comprising or consisting of the sequence of SEQ ID NO: 49 and an antisense strand comprising or consisting of the sequence of SEQ ID NO: 175; (vi) a sense strand comprising or consisting of the sequence of SEQ ID NO: 71 and an antisense strand comprising or consisting of the sequence of SEQ ID NO: 189; (vii) a sense strand comprising or consisting of the sequence of SEQ ID NO: 51 and an antisense strand comprising or consisting of the sequence of SEQ ID NO: 175; (viii) a sense strand comprising or consisting of the sequence of SEQ ID NO: 79 (ix) a sense strand comprising or consisting of the sequence of SEQ ID NO: 85 and an antisense strand comprising or consisting of the sequence of SEQ ID NO: 198; (x) a sense strand comprising or consisting of the sequence of SEQ ID NO: 106 and an antisense strand comprising or consisting of the sequence of SEQ ID NO: 216; (xi) a sense strand comprising or consisting of the sequence of SEQ ID NO: 83 and an antisense strand comprising or consisting of the sequence of SEQ ID NO: 200; (xii) a sense strand comprising or consisting of the sequence of SEQ ID NO: 78 and an antisense strand comprising or consisting of the sequence of SEQ ID NO: 196; (xiii) a sense strand comprising or consisting of the sequence of SEQ ID NO: 5 and an antisense strand comprising or consisting of the sequence of SEQ ID NO: 137; (xiv) a sense strand comprising or consisting of the sequence of SEQ ID NO: 117 and an antisense strand comprising or consisting of the sequence of SEQ ID NO: 225; (xv) a sense strand comprising or consisting of the sequence of SEQ ID NO: 115 and an antisense strand comprising or consisting of the sequence of SEQ ID NO: 224;(xvi) a sense strand comprising or consisting of the sequence of SEQ ID NO: 54 and an antisense strand comprising or consisting of the sequence of SEQ ID NO: 178, or (xvii) a sense strand comprising or consisting of the sequence of SEQ ID NO: 86 and an antisense strand comprising or consisting of the sequence of SEQ ID NO: 198.

[0040] In some embodiments, the RNAi construct of the present invention comprises: (i) a sense strand comprising or consisting of a sequence of modified nucleotides according to SEQ ID NO: 253 and an antisense strand comprising or consisting of a sequence of modified nucleotides according to SEQ ID NO: 448; (ii) a sense strand comprising or consisting of a sequence of modified nucleotides according to SEQ ID NO: 282 and an antisense strand comprising or consisting of a sequence of modified nucleotides according to SEQ ID NO: 471; (iii) a sense strand comprising or consisting of a sequence of modified nucleotides according to SEQ ID NO: 312 and an antisense strand comprising or consisting of a sequence of modified nucleotides according to SEQ ID NO: 497; (iv) a sense strand comprising or consisting of a sequence of modified nucleotides according to SEQ ID NO: 378 and an antisense strand comprising or consisting of a sequence of modified nucleotides according to SEQ ID NO: 547; (v) a sense strand comprising or consisting of a sequence of modified nucleotides according to SEQ ID NO: 304 and an antisense strand comprising or consisting of a sequence of modified nucleotides according to SEQ ID NO: 492; (vi) a sense strand comprising or consisting of a sequence of modified nucleotides according to SEQ ID NO: 341 (vii) a sense strand comprising or consisting of a sequence of modified nucleotides according to SEQ ID NO: 377 and an antisense strand comprising or consisting of a sequence of modified nucleotides according to SEQ ID NO: 550; (viii) a sense strand comprising or consisting of a sequence of modified nucleotides according to SEQ ID NO: 307 and an antisense strand comprising or consisting of a sequence of modified nucleotides according to SEQ ID NO: 492; (ix) a sense strand comprising or consisting of a sequence of modified nucleotides according to SEQ ID NO: 350 and an antisense strand comprising or consisting of a sequence of modified nucleotides according to SEQ ID NO: 525; (x) a sense strand comprising or consisting of a sequence of modified nucleotides according to SEQ ID NO: 362 and an antisense strand comprising or consisting of a sequence of modified nucleotides according to SEQ ID NO: 536; (xi) a sense strand comprising or consisting of a sequence of modified nucleotides according to SEQ ID NO: 404 and an antisense strand comprising or consisting of a sequence of modified nucleotides according to SEQ ID NO: 568.(xii) a sense strand comprising or consisting of a sequence of modified nucleotides according to SEQ ID NO: 370 and an antisense strand comprising or consisting of a sequence of modified nucleotides according to SEQ ID NO: 540; (xiii) a sense strand comprising or consisting of a sequence of modified nucleotides according to SEQ ID NO: 357 and an antisense strand comprising or consisting of a sequence of modified nucleotides according to SEQ ID NO: 530; (xiv) a sense strand comprising or consisting of a sequence of modified nucleotides according to SEQ ID NO: 245 and an antisense strand comprising or consisting of a sequence of modified nucleotides according to SEQ ID NO: 440; (xv) a sense strand comprising or consisting of a sequence of modified nucleotides according to SEQ ID NO: 415 and an antisense strand comprising or consisting of a sequence of modified nucleotides according to SEQ ID NO: 577; (xvi) a sense strand comprising or consisting of a sequence of modified nucleotides according to SEQ ID NO: 413 and an antisense strand (xvii) a sense strand comprising or consisting of a sequence of modified nucleotides according to SEQ ID NO: 314 and an antisense strand comprising or consisting of a sequence of modified nucleotides according to SEQ ID NO: 499; (xviii) a sense strand comprising or consisting of a sequence of modified nucleotides according to SEQ ID NO: 377 and an antisense strand comprising or consisting of a sequence of modified nucleotides according to SEQ ID NO: 546; (xix) a sense strand comprising or consisting of a sequence of modified nucleotides according to SEQ ID NO: 364 and an antisense strand comprising or consisting of a sequence of modified nucleotides according to SEQ ID NO: 536; or (xx) a sense strand comprising or consisting of a sequence of modified nucleotides according to SEQ ID NO: 372 and an antisense strand comprising or consisting of a sequence of modified nucleotides according to SEQ ID NO: 540.

[0041] An RNAi construct of the invention may be any one of the duplex compounds listed in Tables 1-15 (including the unmodified and / or modified nucleotide sequences of the compound). In some embodiments, the RNAi construct is any of the duplex compounds listed in Table 1. In other embodiments, the RNAi construct is any of the duplex compounds listed in Table 2 (including the unmodified and / or modified nucleotide sequences of the compound). In particular embodiments, the RNAi construct is 4601, 4613, 4930, 4970, 6150, 6182, 6247, 8395, 8401, 10927, 11318, 11344, 11351, 11374, 11580, 17188, 17205, 18436, 18444, or 18446. In one particular embodiment, the RNAi construct is 4601. In another specific embodiment, the RNAi construct is 4613. In another embodiment, the RNAi construct is 10927. In another embodiment, the RNAi construct is 11351. In another embodiment, the RNAi construct is 11374. In yet another embodiment, the RNAi construct is 11580. In yet another embodiment, the RNAi construct is 18436. In another embodiment, the RNAi construct is 18444.

[0042] The RNAi construct of the present invention may contain one or more modified nucleotides. "Modified nucleotide" refers to a nucleotide having one or more chemical modifications to the nucleoside, nucleobase, pentose ring, or phosphate group. As used herein, modified nucleotides do not include ribonucleotides containing adenosine monophosphate, guanosine monophosphate, uridine monophosphate, and cytidine monophosphate. However, the RNAi construct may contain a combination of modified nucleotides and ribonucleotides. Incorporation of modified nucleotides into one or both strands of a double-stranded RNA molecule can improve the in vivo stability of the RNA molecule, for example, by reducing the molecule's susceptibility to nucleases and other degradation processes. Incorporation of modified nucleotides can also enhance the efficacy of the RNAi construct for reducing the expression of a target gene.

[0043] In certain embodiments, modified nucleotides have modifications of the ribose sugar. These sugar modifications can include modifications at the 2' and / or 5' positions of the pentose ring and bicyclic sugar modifications. A 2'-modified nucleotide refers to a nucleotide having a pentose ring with a substituent at the 2' position other than OH. Such 2'-modifications include 2'-H (e.g., deoxyribonucleotides), 2'-O-alkyl (e.g., O-C1-C2). 10 Or O-C1~C 10 Modifications at the 5' position of the pentose ring include, but are not limited to, 5'-methyl (R or S), 5'-vinyl, and 5'-methoxy.

[0044] "Bicyclic sugar modification" refers to a modification of a pentose ring in which a bridge links two atoms of the ring to form a second ring, resulting in a bicyclic sugar structure. In some embodiments, a bicyclic sugar modification comprises a bridge between the 4' and 2' carbons of the pentose ring. Nucleotides comprising a sugar moiety having a bicyclic sugar modification are referred to herein as bicyclic nucleic acids or BNAs. Exemplary bicyclic sugar modifications include α-L-methyleneoxy (4'-CH2-O-2') bicyclic nucleic acids (BNAs); β-D-methyleneoxy (4'-CH2-O-2') BNAs (also referred to as locked nucleic acids or LNAs); ethyleneoxy (4'-(CH2)2-O-2') BNAs; aminooxy (4'-CH2-ON(R)-2') BNAs; oxyamino (4'-CH2-N(R)-O-2') BNAs; methyl(methyleneoxy) (4'-CH(CH3)-O-2' )BNA (also referred to as constrained ethyl or cEt); methylene-thio (4'-CH2-S-2')BNA; methylene-amino (4'-CH2-N(R)-2')BNA; methyl carbocyclic (4'-CH2-CH(CH3)-2')BNA; propylene carbocyclic (4'-(CH2)3-2')BNA; and methoxy(ethyleneoxy) (4'-CH(CHOMe)-O-2')BNA (also referred to as constrained MOE or cMOE). These and other sugar-modified nucleotides that can be incorporated into the RNAi constructs of the invention are described in U.S. Pat. No. 9,181,551, U.S. Patent Application Publication No. 2016 / 0122761, and Deleaviey and Damha, Chemistry and Biology, Vol. 19:937-954, 2012, all of which are incorporated by reference in their entireties.

[0045] In some embodiments, an RNAi construct comprises one or more 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, 2'-O-allyl modified nucleotides, bicyclic nucleic acids (BNAs), deoxyribonucleotides, or combinations thereof. In certain embodiments, an RNAi construct comprises one or more 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, or combinations thereof. In a particular embodiment, an RNAi construct comprises one or more 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, or combinations thereof.

[0046] Both the sense strand and the antisense strand of an RNAi construct may contain one or more modified nucleotides. For example, in some embodiments, the sense strand contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more modified nucleotides. In certain embodiments, all nucleotides in the sense strand are modified nucleotides. In some embodiments, the antisense strand contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more modified nucleotides. In other embodiments, all nucleotides in the antisense strand are modified nucleotides. In certain other embodiments, all nucleotides in the sense strand and all nucleotides in the antisense strand are modified nucleotides. In these and other embodiments, the modified nucleotides may be 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, or a combination thereof.

[0047] In certain embodiments, modified nucleotides incorporated into one or both strands of an RNAi construct of the invention have a nucleobase (also referred to herein as a "base") modification. A "modified nucleobase" or "modified base" refers to a base other than the naturally occurring purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases may be synthetic or naturally occurring modifications and include universal bases, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine (X), hypoxanthine (I), 2-aminoadenine, 6-methyladenine, 6-methylguanine, and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, ...azouracil, 2-azouracil, 2-azouracil, 2-azouracil, 2-azouracil, 2-azouracil, 2-azouracil, 2-azouracil, 2-azouracil, 2-azouracil, 2-azouracil, 2-azouracil, 2-azouracil, 2-azouracil, 2-azouracil, 2-azouracil, 2-azouracil, 2-azouracil, 2-azouracil, 2-azouracil, 2-azouracil, 2-azouracil, 2-azouracil, 2-azouracil, 2-azouracil, 2-azouracil, 2-azouracil, These include, but are not limited to, methyl, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine.

[0048] In some embodiments, the modified base is a universal base. "Universal base" refers to a base analog that indiscriminately base pairs with all natural bases in RNA and DNA without altering the double helix structure of the resulting double-stranded region. Universal bases are known to those skilled in the art and include, but are not limited to, inosine, C-phenyl, C-naphthyl and other aromatic derivatives, azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole.

[0049] Other suitable modified bases that can be incorporated into the RNAi constructs of the present invention include those described in Herdewijn, Antisense Nucleic Acid Drug Dev., Vol.10:297-310,2000, and Peacock et al., J.Org.Chem., Vol.76:7295-7300,2011, both of which are incorporated herein by reference in their entirety. Those skilled in the art will be well aware that guanine, cytosine, adenine, thymine and uracil may be substituted with other nucleobases, such as the above-mentioned modified nucleobases, without substantially changing the base pairing properties of polynucleotides containing nucleotides having such substituted nucleobases.

[0050] In some embodiments, the sense and antisense strands of an RNAi construct may contain one or more abasic nucleotides. An "abasic nucleotide" or "abasic nucleoside" is a nucleotide or nucleoside that lacks a nucleobase at the 1' position of the ribose sugar. In certain embodiments, an abasic nucleotide is incorporated at the end of the sense and / or antisense strand of an RNAi construct. In one embodiment, the sense strand contains an abasic nucleotide as the terminal nucleotide at its 3'-end, its 5'-end, or both its 3'-end and 5'-end. In another embodiment, the antisense strand contains an abasic nucleotide as the terminal nucleotide at its 3'-end, its 5'-end, or both its 3'-end and 5'-end. In such embodiments in which the abasic nucleotide is the terminal nucleotide, the terminal nucleotide may be an inverted nucleotide, i.e., linked to the adjacent nucleotide via a 3'-3' internucleotide bond (if on the 3'-end of the strand) or a 5'-5' internucleotide bond (if on the 5'-end of the strand) rather than a natural 3'-5' internucleotide bond. The abasic nucleotide may also include a sugar modification, such as any of the sugar modifications described above. In certain embodiments, the abasic nucleotide includes a 2'-modification, such as a 2'-fluoro modification, a 2'-O-methyl modification, or a 2'-H (deoxy) modification. In one embodiment, the abasic nucleotide includes a 2'-O-methyl modification. In another embodiment, the abasic nucleotide includes a 2'-H modification (i.e., a deoxyabasic nucleotide).

[0051] The RNAi constructs of the present invention may also contain one or more modified internucleotide linkages. As used herein, the term "modified internucleotide linkage" refers to an internucleotide linkage other than a natural 3'-5' phosphodiester linkage. In some embodiments, the modified internucleotide linkage is a phosphorus-containing internucleotide linkage, such as a phosphotriester, an aminoalkylphosphotriester, an alkylphosphonate (e.g., methylphosphonate, 3'-alkylenephosphonate), a phosphinate, a phosphoramidate (e.g., 3'-aminophosphoramidate and aminoalkylphosphoramidate), a phosphorothioate (P=S), a chiral phosphorothioate, a phosphorodithioate, a thionophosphoramidate, a thionoalkylphosphonate, a thionoalkylphosphotriester, and a boranophosphate. In one embodiment, the modified internucleotide linkage is a 2'-5' phosphodiester linkage. In other embodiments, the modified internucleotide linkage is a non-phosphorus-containing internucleotide linkage, and may therefore also be referred to as a modified internucleoside linkage. Such non-phosphorus-containing linkages include, but are not limited to, morpholino linkages (formed in part from the sugar portion of the nucleoside); siloxane linkages (—O—Si(H)—O—); sulfide, sulfoxide, and sulfone linkages; formacetyl and thioformacetyl linkages; alkene-containing backbones; sulfamate backbones; methylenemethylimino (—CH—N(CH)—O—CH—) and methylenehydrazino linkages; sulfonate and sulfonamide linkages; amide linkages; and others having mixed N, O, S, and CH component moieties. In one embodiment, the modified internucleoside linkage is a peptide-based linkage (e.g., aminoethylglycine) to generate peptide nucleic acids, or PNAs, as described in U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262.Other suitable modified internucleotide and internucleoside linkages that can be used in the RNAi constructs of the present invention are described in U.S. Pat. Nos. 6,693,187, 9,181,551, U.S. Patent Application Publication No. 2016 / 0122761, and Deleaviey and Damha, Chemistry and Biology, Vol. 19:937-954, 2012, all of which are incorporated by reference in their entireties.

[0052] In certain embodiments, an RNAi construct of the present invention comprises one or more phosphorothioate internucleotide linkages. The phosphorothioate internucleotide linkages may be present in the sense strand, the antisense strand, or both strands of the RNAi construct. For example, in some embodiments, the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, or more phosphorothioate internucleotide linkages. In other embodiments, the antisense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, or more phosphorothioate internucleotide linkages. In still other embodiments, both strands comprise 1, 2, 3, 4, 5, 6, 7, 8, or more phosphorothioate internucleotide linkages. The RNAi construct may comprise one or more phosphorothioate internucleotide linkages at the 3'-end, 5'-end, or both the 3'-end and 5'-end of the sense strand, the antisense strand, or both strands. For example, in certain embodiments, an RNAi construct comprises from about 1 to about 6 or more (e.g., about 1, 2, 3, 4, 5, 6 or more) consecutive phosphorothioate internucleotide linkages at the 3'-end of the sense strand, the antisense strand, or both strands. In other embodiments, an RNAi construct comprises from about 1 to about 6 or more (e.g., about 1, 2, 3, 4, 5, 6 or more) consecutive phosphorothioate internucleotide linkages at the 5'-end of the sense strand, the antisense strand, or both strands.

[0053] In some embodiments, the RNAi construct comprises a single phosphorothioate internucleotide linkage between the terminal nucleotides at the 3' end of the sense strand. In other embodiments, the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages between the terminal nucleotides at the 3' end of the sense strand. In one embodiment, the RNAi construct comprises a single phosphorothioate internucleotide linkage between the terminal nucleotides at the 3' end of the sense strand and a single phosphorothioate internucleotide linkage between the terminal nucleotides at the 3' end of the antisense strand. In another embodiment, the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages between the terminal nucleotides at the 3' end of the antisense strand (i.e., phosphorothioate internucleotide linkages at the first and second internucleotide linkages at the 3' end of the antisense strand). In another embodiment, the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages between the terminal nucleotides at both the 3' end and the 5' end of the antisense strand. In yet another embodiment, the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages between the terminal nucleotides at both the 3' and 5' ends of the antisense strand and two consecutive phosphorothioate internucleotide linkages at the 5' end of the sense strand. In yet another embodiment, the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages between the terminal nucleotides at both the 3' and 5' ends of the antisense strand and two consecutive phosphorothioate internucleotide linkages between the terminal nucleotides at the 3' end of the sense strand. In another embodiment, the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages between the terminal nucleotides at both the 3' and 5' ends of the antisense strand and two consecutive phosphorothioate internucleotide linkages between the terminal nucleotides at both the 3' and 5' ends of the sense strand (i.e., phosphorothioate internucleotide linkages at the first and second internucleotide linkages at both the 5' and 3' ends of the antisense strand, and phosphorothioate internucleotide linkages at the first and second internucleotide linkages at both the 5' and 3' ends of the sense strand).In yet another embodiment, the RNAi construct comprises two consecutive phosphorothioate internucleotide bonds between the terminal nucleotides at both the 3' and 5' ends of the antisense strand, and a single phosphorothioate internucleotide bond between the terminal nucleotides at the 3' end of the sense strand. In any of the embodiments in which one or both strands comprise one or more phosphorothioate internucleotide bonds, the remaining internucleotide bonds within the strand may be natural 3'-5' phosphodiester bonds. For example, in some embodiments, each internucleotide bond in the sense strand and the antisense strand is selected from phosphodiester and phosphorothioate, and at least one internucleotide bond is phosphorothioate.

[0054] In embodiments in which the RNAi construct comprises a nucleotide overhang, two or more of the unpaired nucleotides in the overhang may be linked by phosphorothioate internucleotide bonds. In certain embodiments, all of the unpaired nucleotides in the nucleotide overhang at the 3' end of the antisense strand and / or the sense strand are linked by phosphorothioate internucleotide bonds. In other embodiments, all of the unpaired nucleotides in the nucleotide overhang at the 5' end of the antisense strand and / or the sense strand are linked by phosphorothioate internucleotide bonds. In still other embodiments, all of the unpaired nucleotides in any of the nucleotide overhangs are linked by phosphorothioate internucleotide bonds.

[0055] In some embodiments of the RNAi constructs of the present invention, the 5'-end of the sense strand, the antisense strand, or both the antisense strand and the sense strand comprises a phosphate moiety. As used herein, the term "phosphate moiety" refers to a terminal phosphate group, including unmodified phosphate (-OP=O)(OH)OH) and modified phosphate. Modified phosphates include phosphates in which one or more of the O and OH groups are replaced with H, O, S, N(R), or alkyl (wherein R is H, an amino-protecting group, or unsubstituted or substituted alkyl). Exemplary phosphate moieties include, but are not limited to, 5'-monophosphate; 5'-diphosphate; 5'-triphosphate; 5'-guanosine cap (7-methylated or unmethylated); 5'-adenosine cap or any other modified or unmodified nucleotide cap structure; 5'-monothiophosphate (phosphorothioate); 5'-monodithiophosphate (phosphorodithioate); 5'-α-thiotriphosphate; 5'-γ-thiotriphosphate; 5'-phosphoramidate; 5'-vinyl phosphate; 5'-alkyl phosphonates (e.g., alkyl = methyl, ethyl, isopropyl, propyl, etc.); and 5'-alkyl ether phosphonates (e.g., alkyl ether = methoxymethyl, ethoxymethyl, etc.).

[0056] Modified nucleotides that can be incorporated into RNAi constructs of the present invention can have two or more chemical modifications described herein. For example, modified nucleotides can have a modification to the ribose sugar and a modification to the nucleobase. For example, modified nucleotides can include a 2' sugar modification (e.g., 2'-fluoro or 2'-O-methyl) and a modified base (e.g., 5-methylcytosine or pseudouracil). In other embodiments, modified nucleotides can include a sugar modification in combination with a modification to the 5' phosphate, resulting in a modified internucleotide or internucleoside linkage when the modified nucleotide is incorporated into a polynucleotide. For example, in some embodiments, modified nucleotides can include sugar modifications such as a 2'-fluoro modification, a 2'-O-methyl modification, or a bicyclic sugar modification, and a 5' phosphorothioate group. Thus, in some embodiments, one or both of the RNAi constructs of the present invention include a combination of a 2'-modified nucleotide or BNA and a phosphorothioate internucleotide linkage. In certain embodiments, both the sense and antisense strands of the RNAi constructs of the invention contain a combination of 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, and phosphorothioate internucleotide linkages. Exemplary RNAi constructs containing modified nucleotides and modified internucleotide linkages are shown in Table 2.

[0057] The RNAi constructs of the present invention can be easily produced using techniques known in the art, for example, by conventional solid-phase nucleic acid synthesis. The polynucleotides of the RNAi constructs can be assembled using standard nucleotide or nucleoside precursors (e.g., phosphoramidites) on a suitable nucleic acid synthesizer. Automated nucleic acid synthesizers are commercially available from several vendors, including the DNA / RNA synthesizer from Applied Biosystems (Foster City, CA), the MerMade synthesizer from BioAutomation (Irving, TX), and the OligoPilot synthesizer from GE Healthcare Life Sciences (Pittsburgh, PA). An exemplary method for synthesizing the RNAi constructs of the present invention is described in Example 1.

[0058] Oligonucleotides can be synthesized using phosphoramidite chemistry using a 2' silyl protecting group along with acid-labile dimethoxytrityl (DMT) at the 5' position of the ribonucleoside. Final deprotection conditions are known not to significantly degrade the RNA product. All syntheses can be performed on large, medium, or small scales in any automated or manual synthesizer. Synthesis can also be performed in multiwell plates, columns, or glass slides.

[0059] The 2'-O-silyl group can be removed by exposure to fluoride ions, which can include any source of fluoride ions, such as salts containing fluoride ions paired with inorganic counterions, such as cesium fluoride and potassium fluoride, or salts containing fluoride ions paired with organic counterions, such as tetraalkylammonium fluoride. In the deprotection reaction, a crown ether catalyst can be used in combination with an inorganic fluoride. Preferred fluoride ion sources are tetrabutylammonium fluoride or aminohydrofluoride (e.g., by mixing aqueous HF with triethylamine in a dipolar aprotic solvent, such as dimethylformamide).

[0060] The choice of protecting groups used for the phosphite triesters and phosphotriesters can alter the stability of the triesters to fluoride. Methyl-protecting the phosphotriester or phosphite triester can stabilize the bond to fluoride ions and improve process yields.

[0061] Because ribonucleosides have a reactive 2' hydroxyl substituent, it may be desirable to protect the reactive 2' position of RNA with a protecting group that is orthogonal to the 5'-O-dimethoxytrityl protecting group, e.g., a protecting group that is stable to acid treatment. Silyl protecting groups fulfill this requirement and can be easily removed in a final fluoride deprotection step, thereby minimizing RNA degradation.

[0062] A tetrazole catalyst can be used in standard phosphoramidite coupling reactions. Preferred catalysts include, for example, tetrazole, S-ethyl-tetrazole, benzylthiotetrazole, and p-nitrophenyltetrazole.

[0063] As can be appreciated by those of skill in the art, additional methods for synthesizing the RNAi constructs described herein will be apparent to those of skill in the art. Additionally, the various synthetic steps may be performed in an alternate order or sequence to arrive at the desired compound. Other synthetic chemical transformations, protecting groups (e.g., for hydroxyl, amino, etc. present on bases), and protecting group methodologies (protection and deprotection) useful in synthesizing the RNAi constructs described herein are known in the art, such as those described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.W. Greene and P.G.M. Buts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof. Custom synthesis of RNAi agents is also available from several commercial vendors, including Dharmacon, Inc. (Lafayette, CO), AxoLabs GmbH (Kulmbach, Germany), and Ambion, Inc. (Foster City, CA).

[0064] The RNAi construct of the present invention may include a ligand. As used herein, "ligand" refers to any compound or molecule that can directly or indirectly interact with another compound or molecule. The interaction of a ligand with another compound or molecule may elicit a biological response (e.g., trigger a signal transduction cascade, induce receptor-mediated endocytosis), or may simply be a physical association. A ligand can modify one or more properties of the double-stranded RNA molecule to which it is bound, such as the pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge, and / or clearance properties of the RNA molecule.

[0065] Ligands include serum proteins (e.g., human serum albumin, low-density lipoproteins, globulins), cholesterol moieties, vitamins (biotin, vitamin E, vitamin B 12), a folate moiety, a steroid, a bile acid (e.g., cholic acid), a fatty acid (e.g., palmitic acid, myristic acid), a carbohydrate (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid), a glycoside, a phospholipid, or an antibody or binding fragment thereof (e.g., an antibody or binding fragment that targets the RNAi construct to a specific cell type, such as the liver). Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinkers (e.g., psoralens, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules such as adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl groups, hexadecanol, methyl ... Examples of suitable oleic acid derivatives include glycerol, borneol, menthol, 1,3-propanediol, heptadecyl groups, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl or phenoxazine, peptides (e.g., antennapedia peptide, Tat peptide, RGD peptide), alkylating agents, polymers such as polyethylene glycol (PEG) (e.g., PEG-40K), polyamino acids, and polyamines (e.g., spermine, spermidine).

[0066] In certain embodiments, the ligand has endosomolytic properties. The endosomolytic ligand promotes lysis of endosomes and / or transport of the RNAi construct of the present invention or its components from endosomes to the cytoplasm of a cell. The endosomolytic ligand can be a polycationic peptide or peptidomimetic that exhibits pH-dependent membrane activity and fusogenicity. In one embodiment, the endosomolytic ligand adopts its active conformation at endosomal pH. An "active" conformation is one in which the endosomolytic ligand promotes lysis of endosomes and / or transport of the RNAi construct of the present invention or its components from endosomes to the cytoplasm of a cell. Exemplary endosomolytic ligands include GALA peptide (Subbarao et al., Biochemistry, Vol. 26:2964-2972, 1987), EALA peptide (Vogel et al., J. Am. Chern. Soc., Vol. 118:1581-1586, 1996), and derivatives thereof (Turk et al., Biochem. Biophys. Acta, Vol. 1559:56-68, 2002). In one embodiment, the endosomolytic component may contain a chemical group (e.g., an amino acid) that undergoes a change in charge or protonation in response to a change in pH. The endosomolytic component may be linear or branched.

[0067] In some embodiments, the ligand comprises a lipid or other hydrophobic molecule. In one embodiment, the ligand comprises a cholesterol moiety or other steroid. Cholesterol-conjugated oligonucleotides have been reported to be more active than their unconjugated counterparts (Manoharan, Antisense Nucleic Acid Drug Development, Vol. 12:103-228, 2002). Ligands comprising cholesterol moieties and other lipids for conjugation to nucleic acid molecules are also described in U.S. Pat. Nos. 7,851,615; 7,745,608; and 7,833,992, all of which are incorporated herein by reference in their entireties. In another embodiment, the ligand comprises a folate moiety. Polynucleotides conjugated to the folate moiety can be taken up into cells via receptor-mediated endocytosis. Such folate-polynucleotide conjugates are described in U.S. Pat. No. 8,188,247, incorporated herein by reference in its entirety.

[0068] The LPA gene is primarily expressed in the liver. Therefore, in certain embodiments, it is desirable to specifically deliver the RNAi construct of the present invention to liver cells. Thus, in certain embodiments, a ligand is used to target the specific delivery of the RNAi construct to liver cells (e.g., hepatocytes) using various means, as described in more detail below. In certain embodiments, the RNAi construct is targeted to liver cells by a ligand that binds to the surface-expressed asialoglycoprotein receptor (ASGR) or its components (e.g., ASGR1, ASGR2).

[0069] In some embodiments, the RNAi construct can be specifically targeted to the liver by using a ligand that binds to or interacts with a protein expressed on the surface of liver cells. For example, in certain embodiments, the ligand can comprise an antigen-binding protein (e.g., an antibody or a binding fragment thereof (e.g., Fab, scFv)) that specifically binds to a receptor expressed on liver cells, such as the asialoglycoprotein receptor and the LDL receptor. In a specific embodiment, the ligand comprises an antibody or a binding fragment thereof that specifically binds to ASGR1 and / or ASGR2. In another embodiment, the ligand comprises a Fab fragment of an antibody that specifically binds to ASGR1 and / or ASGR2. A "Fab fragment" is composed of one immunoglobulin light chain (i.e., the light chain variable region (VL) and constant region (CL)) and the CH1 region and variable region (VH) of one immunoglobulin heavy chain. In another embodiment, the ligand comprises a single-chain variable antibody fragment (scFv fragment) of an antibody that specifically binds to ASGR1 and / or ASGR2. An "scFv fragment" comprises the VH and VL regions of an antibody, these regions being present in a single polypeptide chain, and optionally including a peptide linker between the VH and VL regions that enables the Fv to form the desired structure for antigen binding. Exemplary antibodies and binding fragments thereof that specifically bind to ASGR1 and can be used as ligands for targeting the RNAi constructs of the invention to the liver are described in WO 2017 / 058944, the entire contents of which are incorporated herein by reference. Other antibodies or binding fragments thereof that specifically bind to ASGR1, LDL receptor, or other proteins expressed on the surface of the liver and suitable for use as ligands for the RNAi constructs of the invention are commercially available.

[0070] In certain embodiments, the ligand comprises a carbohydrate. "Carbohydrate" refers to a compound composed of one or more monosaccharide units (which may be linear, branched, or cyclic) having at least six carbon atoms, with an oxygen, nitrogen, or sulfur atom attached to each carbon atom. Carbohydrates include, but are not limited to, sugars (e.g., monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starch, glycogen, cellulose, and polysaccharide gums. In some embodiments, the carbohydrate incorporated into the ligand is a monosaccharide selected from pentose, hexose, or heptose, as well as disaccharides and trisaccharides containing such monosaccharide units. In other embodiments, the carbohydrate incorporated into the ligand is an amino sugar, such as galactosamine, glucosamine, N-acetylgalactosamine, and N-acetylglucosamine.

[0071] In some embodiments, the ligand comprises a hexose or hexosamine. The hexose may be selected from glucose, galactose, mannose, fucose, or fructose. The hexosamine may be selected from fructosamine, galactosamine, glucosamine, or mannosamine. In certain embodiments, the ligand comprises glucose, galactose, galactosamine, or glucosamine. In one embodiment, the ligand comprises glucose, glucosamine, or N-acetylglucosamine. In another embodiment, the ligand comprises galactose, galactosamine, or N-acetyl-galactosamine. In certain embodiments, the ligand comprises N-acetyl-galactosamine. Ligands comprising glucose, galactose, and N-acetyl-galactosamine (GalNAc) are particularly effective for targeting compounds to liver cells because such ligands bind to ASGR, which is expressed on the surface of liver cells. See, e.g., D'Souza and Devarajan, J. Control Release, Vol. 203:126-139, 2015. Examples of GalNAc- or galactose-containing ligands that can be incorporated into the RNAi constructs of the invention are described in U.S. Patent Nos. 7,491,805; 8,106,022; and 8,877,917; U.S. Patent Application Publication No. 20030130186; and WO 2013166155, all of which are incorporated by reference in their entireties.

[0072] In certain embodiments, the ligand comprises a multivalent carbohydrate moiety. As used herein, a "multivalent carbohydrate moiety" refers to a moiety containing two or more carbohydrate units that can independently bind or interact with other molecules. For example, a multivalent carbohydrate moiety contains two or more carbohydrate binding domains that can bind to two or more different molecules or to two or more different sites on the same molecule. The valency of the carbohydrate moiety indicates the number of individual binding domains within the carbohydrate moiety. For example, the terms "monovalent," "bivalent," "trivalent," and "tetravalent" with respect to a carbohydrate moiety refer to carbohydrate moieties having one, two, three, and four binding domains, respectively. A multivalent carbohydrate moiety can comprise a multivalent lactose moiety, a multivalent galactose moiety, a multivalent glucose moiety, a multivalent N-acetyl-galactosamine moiety, a multivalent N-acetyl-glucosamine moiety, a multivalent mannose moiety, or a multivalent fucose moiety. In some embodiments, the ligand comprises a multivalent galactose moiety. In other embodiments, the ligand comprises a multivalent N-acetyl-galactosamine moiety. In these and other embodiments, the multivalent carbohydrate moiety can be bivalent, trivalent, or tetravalent. In such embodiments, the polyvalent carbohydrate moiety can be biantennary or triantennary. In a specific embodiment, the polyvalent N-acetyl-galactosamine moiety is trivalent or tetravalent. In another specific embodiment, the polyvalent galactose moiety is trivalent or tetravalent. Exemplary trivalent or tetravalent GalNAc-containing ligands for incorporation into the RNAi constructs of the invention are detailed below.

[0073] The ligand may be directly or indirectly bound or conjugated to the RNA molecule of the RNAi construct. For example, in some embodiments, the ligand is directly covalently bound to the sense or antisense strand of the RNAi construct. In other embodiments, the ligand is covalently bound to the sense or antisense strand of the RNAi construct via a linker. The ligand may be bound to the nucleobase, sugar moiety, or internucleotide linkage of the polynucleotide (e.g., the sense or antisense strand) of the RNAi construct of the present invention. Conjugation or binding to a purine nucleobase or a derivative thereof can occur at any position, including endocyclic and exocyclic atoms. In certain embodiments, the 2-, 6-, 7-, or 8-position of the purine nucleobase is bound to the ligand. Conjugation or binding to a pyrimidine nucleobase or a derivative thereof can also occur at any position. In some embodiments, the 2-, 5-, and 6-positions of the pyrimidine nucleobase may be bound to the ligand. Conjugation or binding to the sugar moiety of the nucleotide can occur at any carbon atom. Exemplary carbon atoms of the sugar moiety that can be attached to a ligand include the 2', 3', and 5' carbon atoms. For example, in abasic nucleotides, the 1' position can also be attached to a ligand. Internucleotide linkages can also facilitate ligand attachment. In the case of phosphorus-containing linkages (e.g., phosphodiester, phosphorothioate, phosphorodithioate, phosphoramidate, etc.), the ligand can be attached directly to the phosphorus atom or to an O, N, or S atom attached to the phosphorus atom. In the case of amine- or amide-containing internucleoside linkages (e.g., PNA), the ligand can be attached to the nitrogen atom or adjacent carbon atom of the amine or amide.

[0074] In some embodiments, the ligand may be attached to the 3' or 5' end of either the sense strand or the antisense strand. In certain embodiments, the ligand is covalently attached to the 5' end of the sense strand. In such embodiments, the ligand is attached to the 5'-terminal nucleotide of the sense strand. In these and other embodiments, the ligand is attached at the 5' position of the 5'-terminal nucleotide of the sense strand. In embodiments where the inverted abasic nucleotide is the 5'-terminal nucleotide of the sense strand and is attached to the adjacent nucleotide via a 5'-5' internucleotide linkage, the ligand may be attached to the 3' position of the inverted abasic nucleotide. In other embodiments, the ligand is covalently attached to the 3' end of the sense strand. For example, in some embodiments, the ligand is attached to the 3'-terminal nucleotide of the sense strand. In certain such embodiments, the ligand is attached at the 3' position of the 3'-terminal nucleotide of the sense strand. In embodiments where the inverted abasic nucleotide is the 3'-terminal nucleotide of the sense strand and is attached to the adjacent nucleotide via a 3'-3' internucleotide linkage, the ligand may be attached to the 5' position of the inverted abasic nucleotide. In alternative embodiments, the ligand is attached near the 3'-end of the sense strand, but before one or more terminal nucleotides (i.e., before 1, 2, 3, or 4 terminal nucleotides). In some embodiments, the ligand is attached at the 2'-position of the sugar of the 3'-terminal nucleotide of the sense strand. In other embodiments, the ligand is attached at the 2'-position of the sugar of the 5'-terminal nucleotide of the sense strand.

[0075] In certain embodiments, the ligand is attached to the sense or antisense strand via a linker. A "linker" is an atom or group of atoms that covalently attaches the ligand to the polynucleotide component of an RNAi construct. Linkers can be about 1 to about 30 atoms in length, about 2 to about 28 atoms in length, about 3 to about 26 atoms in length, about 4 to about 24 atoms in length, about 6 to about 20 atoms in length, about 7 to about 20 atoms in length, about 8 to about 20 atoms in length, about 8 to about 18 atoms in length, about 10 to about 18 atoms in length, and about 12 to about 18 atoms in length. In some embodiments, the linker may comprise a bifunctional linking moiety, generally comprising an alkyl moiety bearing two functional groups. One of the functional groups is selected to bind to a compound of interest (e.g., the sense or antisense strand of an RNAi construct), and the other is selected to subsequently bind to any selected group, such as a ligand, as described herein. In certain embodiments, the linker comprises a chain structure or oligomer of repeating units, such as ethylene glycol units or amino acid units. Examples of functional groups commonly used in bifunctional linking moieties include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In some embodiments, bifunctional linking moieties include amino, hydroxyl, carboxylic acid, thiol, and unsaturation (e.g., double or triple bonds).

[0076] Linkers that may be used to attach a ligand to the sense or antisense strand of an RNAi construct of the invention include pyrrolidine, 8-amino-3,6-dioxaoctanoic acid, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, 6-aminohexanoic acid, substituted C1-C 10 Alkyl, substituted or unsubstituted C2-C 10 Alkenyl, or substituted or unsubstituted C2-C 10 Preferred substituents for such linkers include, but are not limited to, hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

[0077] In certain embodiments, the linker is cleavable. The cleavable linker is sufficiently stable outside the cell, but is cleaved upon entry into the target cell to release the two moieties held together by the linker. In some embodiments, the cleavable linker is cleaved at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold or more, or at least 100-fold faster in the target cell or under a first reference condition (e.g., which may be selected to mimic or represent intracellular conditions) than in the subject's blood or under a second reference condition (e.g., which may be selected to mimic or represent conditions found in blood or serum).

[0078] Cleavable linkers are sensitive to cleaving agents, such as pH, redox potential, or the presence of degradable molecules. Generally, cleaving agents are found to be more prevalent or at higher levels or activity inside cells than in serum or blood. Examples of such degrading agents include oxidizing or reducing agents, such as mercaptans, that are present inside cells and can degrade redox-cleavable linkers by reduction, and are selective for specific substrates or have no substrate specificity; esterases; agents that can generate endosomes or acidic environments, such as those that produce a pH of 5 or less; enzymes that can hydrolyze or degrade acid-cleavable linkers by acting as general acids, peptidases (which may be substrate-specific), and phosphatases.

[0079] The cleavable linker may contain a moiety that is sensitive to pH. While the pH of human serum is 7.4, the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. The pH of endosomes is more acidic, ranging from 5.5 to 6.0, and the pH of lysosomes is even more acidic, at about 5.0. Some linkers have a cleavable group that is cleaved at a preferred pH, thereby releasing the RNA molecule from the ligand to the interior of the cell or to a desired compartment of the cell.

[0080] The linker may contain a cleavable group that can be cleaved by a specific enzyme. The type of cleavable group incorporated into the linker may depend on the cell to be targeted. For example, a liver-targeting ligand may be linked to an RNA molecule via a linker containing an ester group. Because liver cells are rich in esterases, this linker will be cleaved more effectively in liver cells than in cell types that are not rich in esterases. Other types of cells that are rich in esterases include lung, renal cortex, and testicular cells. When targeting cells rich in peptidases, such as hepatocytes and synovial cells, a linker containing a peptide bond can be used.

[0081] In general, the suitability of a candidate cleavable linker can be evaluated by testing the ability of a degradative agent (or the conditions under which it is cleaved) to cleave the candidate linker. It may also be desirable to test the candidate cleavable linker for its ability to resist cleavage when contacted with blood or other non-target tissues. Thus, the relative susceptibility to cleavage can be determined between a first condition selected to exhibit cleavage in target cells and a second condition selected to exhibit cleavage in other tissues or bodily fluids, such as blood or serum. Evaluation can be performed in a cell-free system, cells, cell cultures, organ or tissue cultures, or in whole animals. It may be useful to perform initial evaluations in cell-free or culture conditions, followed by further confirmation by evaluation in whole animals. In some embodiments, a useful candidate linker is cleaved at least 2-fold, 4-fold, 10-fold, 20-fold, 50-fold, 70-fold, or 100-fold faster in cells (or under in vitro conditions selected to mimic intracellular conditions) than in blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0082] In other embodiments, a redox-cleavable linker is used. A redox-cleavable linker is cleaved upon reduction or oxidation. An example of a reductively cleavable group is a disulfide bond (-SS-). One or more methods described herein can be used to determine whether a candidate cleavable linker is a suitable "reductively cleavable linker," or suitable for use with, for example, a particular RNAi construct and a particular ligand. For example, a candidate linker can be evaluated by incubating it with dithiothreitol (DTT) or other reducing agents known in the art that mimic the cleavage rate observed in cells, e.g., target cells. Candidate linkers can also be evaluated under conditions selected to mimic blood or serum conditions. In certain embodiments, the candidate linker is cleaved at a rate of up to 10% in blood. In other embodiments, useful linker candidates are degraded at least 2, 4, 10, 20, 50, 70, or 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood (or under in vitro conditions selected to mimic extracellular conditions).

[0083] In yet other embodiments, the ligand is covalently attached to the sense or antisense strand of the RNAi construct using a phosphate-based cleavable linker that is cleaved by an agent that degrades or hydrolyzes the phosphate group. Examples of agents that hydrolyze phosphate groups within cells include enzymes such as intracellular phosphatases. Examples of phosphate-based cleavable groups are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, and -OP(S)(Rk)-S-, where Rk can be hydrogen or alkyl. Particular embodiments include -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O-, -SP(S)(H)-O-, -SP(O)(H)-S-, and -OP(S)(H)-S-. Another particular embodiment is -OP(O)(OH)-O-. These linker candidates can be evaluated using methods similar to those described above.

[0084] In other embodiments, the linker may include an acid-cleavable group, which is a group that is cleaved under acidic conditions. In some embodiments, the acid-cleavable group is cleaved in an acidic environment of about pH 6.5 or below (e.g., about 6.0, 5.5, 5.0 or below) or by an agent, such as an enzyme, that can act as a general acid. Within a cell, certain low-pH organelles, such as endosomes and lysosomes, can provide a cleavage environment for the acid-cleavable group. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, esters, and esters of amino acids. Acid-cleavable groups may have the general formula -C=NN-, C(O)O, or -OC(O). In a particular embodiment, the carbon bonded to the oxygen (alkoxy group) of the ester is an aryl group, a substituted alkyl group, or a tertiary alkyl group such as dimethyl, pentyl, or t-butyl. These candidates can be evaluated using methods similar to those described above.

[0085] In other embodiments, the linker may include an ester-based cleavable group that is cleaved by enzymes such as intracellular esterases and amidases. Examples of ester-based cleavable groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester cleavable groups have the general formula -C(O)O- or -OC(O)-. These linker candidates can be evaluated using methods similar to those described above.

[0086] In further embodiments, the linker may comprise a peptidic cleavable group that is cleaved by enzymes such as intracellular peptidases and proteases. Peptidic cleavable groups are peptide bonds formed between amino acids to give rise to oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptidic cleavable groups include amide groups (-C(O)NH-). Amide groups may be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond formed between amino acids to give rise to peptides and proteins. Peptidic cleavable groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to give rise to peptides and proteins. Peptidic cleavable linking groups have the general formula -NHCHR A C(O)NHCHR B C(O)—, where R A and R B are the side chains of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0087] Other types of linkers suitable for attaching a ligand to the sense or antisense strand of an RNAi construct of the invention are known in the art and include those described in U.S. Pat. Nos. 7,723,509; 8,017,762; 8,828,956; 8,877,917; and 9,181,551, all of which are incorporated herein by reference in their entireties.

[0088] In certain embodiments, the ligand covalently attached to the sense strand or antisense strand of an RNAi construct of the invention comprises a GalNAc moiety, e.g., a multivalent GalNAc moiety. In some embodiments, the multivalent GalNAc moiety is a trivalent GalNAc moiety and is attached to the 3'-end of the sense strand. In other embodiments, the multivalent GalNAc moiety is a trivalent GalNAc moiety and is attached to the 5'-end of the sense strand. In yet other embodiments, the multivalent GalNAc moiety is a tetravalent GalNAc moiety and is attached to the 3'-end of the sense strand. In yet other embodiments, the multivalent GalNAc moiety is a tetravalent GalNAc moiety and is attached to the 5'-end of the sense strand.

[0089] In certain embodiments, the RNAi construct of the present invention has the structure 1: [ka] The ligand includes a ligand having the structure: In a preferred embodiment, a ligand having this structure is covalently attached to the 5' end of the sense strand via a linker, such as those described herein. In one embodiment, the linker is an aminohexyl linker.

[0090] Exemplary trivalent and tetravalent GalNAc moieties and linkers that can be attached to the double-stranded RNA molecules of the RNAi constructs of the invention are shown in the following structural formulas I-IX, where "Ac" in the formulas listed herein represents an acetyl group.

[0091] In one embodiment, the RNAi construct comprises a ligand and a linker having the structure of Formula I below, where each n is independently 1 to 3, k is 1 to 3, m is 1 or 2, and j is 1 or 2, and the ligand is attached to the 3' end of the sense strand of a double-stranded RNA molecule (represented by a solid wavy line). [ka]

[0092] In another embodiment, the RNAi construct comprises a ligand and a linker having the structure of Formula II below, where each n is independently 1 to 3, k is 1 to 3, m is 1 or 2, and j is 1 or 2, and the ligand is attached to the 3' end of the sense strand of a double-stranded RNA molecule (represented by a solid wavy line). [ka]

[0093] In yet another embodiment, the RNAi construct comprises a ligand and a linker having the structure of Formula III below, wherein the ligand is attached to the 3' end of the sense strand of the double-stranded RNA molecule (represented by a solid wavy line). [ka]

[0094] In yet another embodiment, the RNAi construct comprises a ligand and a linker having the structure of Formula IV below, where the ligand is attached to the 3' end of the sense strand of the double-stranded RNA molecule (represented by a solid wavy line). [ka]

[0095] In certain embodiments, the RNAi construct comprises a ligand and a linker having the structure of Formula V below, where each n is independently 1 to 3 and k is 1 to 3, and the ligand is attached to the 5' end of the sense strand of a double-stranded RNA molecule (represented by a solid wavy line). [ka]

[0096] In other embodiments, the RNAi construct comprises a ligand and a linker having the structure of Formula VI below, where each n is independently 1 to 3 and k is 1 to 3, and the ligand is attached to the 5' end of the sense strand of a double-stranded RNA molecule (represented by a solid wavy line). [ka]

[0097] In one particular embodiment, the RNAi construct comprises a ligand and a linker having the structure of Formula VII below, where X=O or S, and the ligand is attached to the 5' end of the sense strand of a double-stranded RNA molecule (represented by a wavy line). [ka]

[0098] In some embodiments, the RNAi construct comprises a ligand and a linker having the structure of Formula VIII below, where each n is independently 1 to 3, and the ligand is attached to the 5' end of the sense strand of a double-stranded RNA molecule (represented by a solid wavy line). [ka]

[0099] In certain embodiments, the RNAi construct comprises a ligand and a linker having the structure of Formula IX below, where the ligand is attached to the 5' end of the sense strand of a double-stranded RNA molecule (represented by a solid wavy line). [ka]

[0100] To covalently attach the ligand and linker to the nucleic acid strand, phosphorothioate bonds can be substituted for the phosphodiester bonds shown in any one of Formulas I-IX.

[0101] The present invention also includes pharmaceutical compositions and formulations comprising the RNAi constructs described herein and pharmaceutically acceptable carriers, excipients, or diluents. Such compositions and formulations are useful for reducing the expression of the LPA gene in patients in need thereof. When clinical use is envisioned, pharmaceutical compositions and formulations are prepared in a form appropriate for the intended use. Generally, this involves preparing compositions that are substantially free of pyrogens as well as other impurities that may be harmful to humans or animals.

[0102] The phrases "pharmaceutically acceptable" or "pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to animals or humans. As used herein, "pharmaceutically acceptable carriers, excipients, or diluents" include solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are acceptable for use in formulating pharmaceuticals, such as pharmaceuticals suitable for administration to humans. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the RNAi construct of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the composition, provided that they do not inactivate the RNAi construct of the composition.

[0103] The composition and method for formulating a pharmaceutical composition depend on several criteria, including, but not limited to, the route of administration, the type and severity of the disease or injury being treated, or the dose to be administered. In some embodiments, the pharmaceutical composition is formulated based on the intended delivery route. For example, in certain embodiments, the pharmaceutical composition is formulated for parenteral delivery. Parenteral delivery forms include intravenous, intraarterial, subcutaneous, intrathecal, intraperitoneal, or intramuscular injection or infusion. In one embodiment, the pharmaceutical composition is formulated for intravenous delivery. In such embodiments, the pharmaceutical composition may include a lipid-based delivery vehicle. In another embodiment, the pharmaceutical composition is formulated for subcutaneous delivery. In such embodiments, the pharmaceutical composition may include a targeting ligand (e.g., a GalNAc-containing ligand or an antibody-containing ligand described herein).

[0104] In some embodiments, the pharmaceutical composition comprises an effective amount of an RNAi construct described herein. An "effective amount" is an amount sufficient to produce a beneficial or desired clinical result. In some embodiments, an effective amount is an amount sufficient to reduce expression of the LPA gene in a particular tissue or cell type (e.g., liver or hepatocytes) of a subject. An effective amount of an RNAi construct of the invention may be about 0.01 mg / kg body weight to about 100 mg / kg body weight and may be administered daily, weekly, monthly, or at longer intervals. Precisely determining what is considered an effective amount and effective frequency of administration can be based on several factors, including the patient's height, age, and general condition, the type of disorder being treated (e.g., myocardial infarction, coronary artery disease, peripheral artery disease, stroke), the particular RNAi construct used, and the route of administration.

[0105] The pharmaceutical compositions of the present invention can be administered via any common route as long as the target tissue is accessible via that route. Such routes include, but are not limited to, parenteral (e.g., subcutaneous, intramuscular, intraperitoneal, or intravenous), oral, nasal, buccal, intradermal, transdermal, and sublingual routes, or direct injection into liver tissue or delivery via the hepatic portal vein. In some embodiments, the pharmaceutical composition is administered parenterally. For example, in certain embodiments, the pharmaceutical composition is administered intravenously. In other embodiments, the pharmaceutical composition is administered subcutaneously.

[0106] Colloidal dispersion systems, such as oil-in-water emulsions, micelles, mixed micelles, and macromolecular complexes including liposomes, nanocapsules, microspheres, beads, and lipid-based systems, may be used as delivery vehicles for the RNAi constructs of the present invention. Commercially available lipid emulsions suitable for delivering the nucleic acids of the present invention include Intralipid® (Baxter International Inc.), Liposyn® (Abbott Pharmaceuticals), Liposyn® II (Hospira), Liposyn® III (Hospira), Nutrilipid (B. Braun Medical Inc.), and other similar lipid emulsions. A preferred colloidal system for use as an in vivo delivery vehicle is a liposome (i.e., an artificial membrane vesicle). The RNAi constructs of the present invention may be encapsulated within a liposome or may be complexed with a liposome, particularly a cationic liposome. Alternatively, the RNAi constructs of the present invention may be complexed with a lipid, particularly a cationic lipid. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylcholine (DMPC), and dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine), anionic (e.g., dimyristoylphosphatidylglycerol (DMPG)), and cationic (e.g., dioleoyltetramethylaminopropyl (DOTAP) and dioleoylphosphatidylethanolamine (DOTMA)). The preparation and use of such colloidal dispersion systems is well known in the art. Exemplary formulations are also disclosed in U.S. Pat. Nos. 5,981,505, 6,217,900; 6,383,512; 5,783,565; 7,202,227; 6,379,965; 6,127,170; 5,837,533; 6,747,014; and WO 03 / 093449.

[0107] In some embodiments, the RNAi constructs of the present invention are fully encapsulated within a lipid formulation, for example, to form SNALP or other nucleic acid-lipid particles. As used herein, the term "SNALP" refers to stable nucleic acid-lipid particles. SNALPs typically contain cationic lipids, non-cationic lipids, and lipids that prevent particle aggregation (e.g., PEG-lipid conjugates). SNALPs exhibit long circulatory life after intravenous injection and accumulate at distal sites (e.g., sites physically distant from the administration site), making them extremely useful for systemic administration. Nucleic acid-lipid particles typically have an average diameter of about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, or about 70 nm to about 90 nm and are substantially nontoxic. Additionally, nucleic acids present in nucleic acid-lipid particles are resistant to degradation by nucleases in aqueous solution. Nucleic acid-lipid particles and methods for their preparation are disclosed, for example, in U.S. Pat. Nos. 5,976,567, 5,981,501, 6,534,484, 6,586,410, 6,815,432, and WO 96 / 40964.

[0108] Pharmaceutical compositions suitable for injectable use include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Generally, these preparations are sterile and fluid to the extent that easy syringability exists. Preparations 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. Suitable solvents or dispersion media may contain, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained by the use of a coating, for example, lecithin, to maintain the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In many cases, it is preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of injectable compositions can be achieved by using agents delaying absorption, for example, aluminum monostearate and gelatin in the compositions.

[0109] Sterile injectable solution can be prepared by adding the active compound in an appropriate amount to a solvent together with any other desired ingredients (for example, as listed above), followed by filtration sterilization.Generally, dispersion is prepared by adding various sterilized active ingredients to a sterile vehicle containing, for example, a basic dispersion medium as listed above and other desired ingredients.For sterile powders for preparing sterile injectable solution, the preferred preparation method includes vacuum drying and freeze-drying technology, which can obtain powders of the active ingredient plus any additional desired ingredients from the previously sterile-filtered solution thereof.

[0110] The compositions of the present invention may generally be formulated in a neutral or salt form. Pharmaceutically acceptable salts include, for example, acid addition salts (formed with free amino groups) derived from inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids (e.g., acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). Salts formed with free carboxyl groups can also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxide) or organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine, etc.). In some embodiments, the RNAi constructs of the present invention are formulated as sodium salts.

[0111] For example, for parenteral administration in aqueous solution, the solution is generally suitably buffered and the liquid diluent is first rendered isotonic, for example, with sufficient saline or glucose. Such aqueous solutions may be used, for example, for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In light of the present disclosure, it is preferable to use a sterile aqueous medium as known to those skilled in the art. By way of illustration, a single dose may be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion fluid, or injected at the indicated infusion site (see, e.g., "Remington's Pharmaceutical Sciences," 15th Edition, pages 1035-1038 and 1570-1580). For human administration, preparations must meet sterility, pyrogenicity, general safety, and purity standards required by FDA standards. In certain embodiments, the pharmaceutical composition of the present invention comprises or consists of sterile saline and the RNAi construct described herein. In other embodiments, pharmaceutical compositions of the invention comprise or consist of an RNAi construct described herein and sterile water (e.g., water for injection, WFI). In yet other embodiments, pharmaceutical compositions of the invention comprise or consist of an RNAi construct described herein and phosphate buffered saline (PBS).

[0112] In some embodiments, the pharmaceutical composition of the present invention is packaged in or stored in an administration device.Devices for injectable formulations include, but are not limited to, injection ports, pre-filled syringes, automatic injection devices, injection pumps, on-body injectors, and injection pens.Devices for aerosolized formulations or powder formulations include, but are not limited to, inhalers, inhalers, inhalers, etc.Therefore, the present invention includes an administration device containing the pharmaceutical composition of the present invention for treating or preventing one or more of the disorders or diseases described herein.

[0113] The present invention provides methods for reducing or inhibiting LPA gene expression, and thus apo(a) protein production, in cells (e.g., liver cells) by contacting the cells with any one of the RNAi constructs described herein. The cells may be in vitro or in vivo. LPA gene expression can be assessed by measuring the amount or level of LPA mRNA, apo(a) protein, or another biomarker associated with LPA expression, such as serum levels of Lp(a). Reduction of LPA expression in cells or animals treated with an RNAi construct of the present invention can be determined compared to LPA expression in cells or animals not treated with an RNAi construct or treated with a control RNAi construct. For example, in some embodiments, the reduction of LPA expression is assessed by (a) measuring the amount or level of LPA mRNA in liver cells treated with an RNAi construct of the present invention, (b) measuring the amount or level of LPA mRNA in liver cells treated with a control RNAi construct (e.g., an RNAi construct targeting an RNA molecule not expressed in liver cells, or an RNAi construct having a nonsense or scrambled sequence), or in liver cells treated without the construct, and (c) comparing the LPA mRNA level measured from the cells treated in (a) with the LPA mRNA level measured from the control cells in (b). Prior to comparison, the LPA mRNA levels of the treated and control cells may be normalized to the RNA level of a control gene (e.g., 18S ribosomal RNA or a housekeeping gene). LPA mRNA levels can be measured by a variety of methods, including Northern blot analysis, nuclease protection assay, fluorescence in situ hybridization (FISH), reverse transcriptase (RT)-PCR, real-time RT-PCR, quantitative PCR, droplet digital PCR, etc.

[0114] In another embodiment, the reduction of LPA expression is assessed by (a) measuring the amount or level of apo(a) protein in liver cells treated with an RNAi construct of the present invention, (b) measuring the amount or level of apo(a) protein in liver cells treated with a control RNAi construct (e.g., an RNAi construct directed against an RNA molecule not expressed in liver cells, or an RNAi construct having a nonsense or scrambled sequence), or in liver cells treated without the construct, and (c) comparing the apo(a) protein level measured from the cells treated with (a) with the apo(a) protein level measured from the control cells in (b). Methods for measuring apo(a) protein levels are known to those skilled in the art and include Western blot, immunoassays (e.g., ELISA), and flow cytometry. Any method capable of measuring LPA mRNA or apo(a) protein can be used to evaluate the effectiveness of the RNAi construct of the present invention.

[0115] In some embodiments, the method for assessing LPA expression levels is performed in vitro in cells that naturally express the LPA gene (e.g., liver cells) or cells engineered to express the LPA gene. In certain embodiments, the method is performed in vitro in liver cells. Suitable liver cells include, but are not limited to, primary liver cells (e.g., human and non-human primate liver cells), HepAD38 cells, HuH-6 cells, HuH-7 cells, HuH-5-2 cells, BNLCL2 cells, Hep3B cells, or HepG2 cells. In one embodiment, the liver cells are HuH-7 cells. In another embodiment, the liver cells are human primary liver cells.

[0116] In other embodiments, the method for assessing LPA expression levels is performed in vivo. The RNAi construct and any control RNAi construct can be administered to an animal (e.g., a transgenic animal expressing the LPA gene or a non-human primate), and the LPA mRNA level or apo(a) protein level can be assessed in pancreatic tissue collected from the treated animal after treatment. Alternatively, or in addition, biomarkers or functional phenotypes associated with LPA expression can be assessed in treated animals. For example, apo(a) protein is the major component of Lp(a) present in serum or plasma. Therefore, serum or plasma levels of Lp(a) can be measured in animals treated with the RNAi constructs of the present invention to assess the functional effectiveness of reducing LPA expression. Exemplary methods for measuring serum or plasma Lp(a) levels are described in Examples 3 and 4.

[0117] In certain embodiments, the expression of LPA is reduced by at least 40%, at least 45%, or at least 50% in liver cells by the RNAi constructs of the present invention. In some embodiments, the expression of LPA is reduced by at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% in liver cells by the RNAi constructs of the present invention. In other embodiments, the expression of LPA is reduced by at least about 90% in liver cells by the RNAi constructs of the present invention, e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more. The percentage reduction in LPA expression can be measured by any of the methods described herein and other methods known in the art.

[0118] The present invention provides methods for reducing or inhibiting LPA gene expression, and thus apo(a) protein production, in patients in need thereof, and methods for treating or preventing conditions, diseases, or disorders associated with LPA expression or apo(a) activity. "Conditions, diseases, or disorders associated with LPA expression" refers to conditions, diseases, or disorders in which altered or elevated LPA expression levels are associated with an increased risk of developing the condition, disease, or disorder. Conditions, diseases, or disorders associated with LPA expression can also include conditions, diseases, or disorders resulting from abnormal changes in lipoprotein metabolism, such as changes resulting in abnormal or elevated levels of Lp(a), cholesterol, lipids, triglycerides, etc., or changes resulting in impaired clearance of these molecules. The apo(a) protein is the main component of Lp(a), and elevated Lp(a) levels are associated with increased risk of cardiovascular disease (see, e.g., Nordestgaard et al., Eur. Heart J., Vol. 31; 2844-2853, 2010; Kronenberg and Utermann, J. Intern. Med., Vol. 273: 6-30, 2013; Nordestgaard et al., J. Lipid Res., Vol. 57: 1953-1975, 2016; and Tsimikas, J. Am. Coll. Cardiol., Vol. 69: 692-711, 2017). Thus, in certain embodiments, the RNAi constructs of the present invention are particularly useful for treating or preventing cardiovascular disease (e.g., coronary artery disease and myocardial infarction) and reducing blood levels of Lp(a).

[0119] Conditions, diseases, and disorders associated with LPA expression that can be treated or prevented according to the methods of the present invention include, but are not limited to, cardiovascular diseases such as myocardial infarction, heart failure, stroke (ischemic and hemorrhagic), atherosclerosis, coronary artery disease, peripheral vascular disease (e.g., peripheral arterial disease), cerebrovascular disease, vulnerable plaque, and aortic stenosis; familial hypercholesterolemia; venous thrombosis; hypercholesterolemia; hyperlipidemia; and dyslipidemia.

[0120] In certain embodiments, the present invention provides a method for reducing LPA expression in a patient in need thereof, comprising administering any of the RNAi constructs described herein to the patient. As used herein, the term "patient" refers to a mammal, including a human, and can be used interchangeably with the term "subject." Preferably, the LPA expression level in the patient's liver cells is reduced after administration of the RNAi construct compared to the LPA expression level of the patient not receiving the RNAi construct, or compared to the LPA expression level of the patient before administration of the RNAi construct. In some embodiments, the LPA expression in the patient after administration of the RNAi construct of the present invention is reduced by at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The percent reduction in LPA expression can be measured by any of the methods described herein, as well as other methods known in the art. In certain embodiments, the percent reduction in LPA expression is determined by assessing Lp(a) levels in the patient's serum or plasma according to the methods described herein.

[0121] In some embodiments, a patient in need of reducing LPA expression is a patient at risk of suffering from myocardial infarction. A patient at risk of suffering from myocardial infarction may have a history of myocardial infarction (e.g., have previously suffered from myocardial infarction). A patient at risk of suffering from myocardial infarction may also have a family history of myocardial infarction or one or more risk factors for myocardial infarction. Such risk factors include, but are not limited to, high blood pressure, elevated levels of non-HDL cholesterol, elevated levels of triglycerides, diabetes, obesity, or an autoimmune disease (e.g., rheumatoid arthritis, lupus). In one embodiment, a patient at risk of suffering from myocardial infarction is a patient suffering from or diagnosed with coronary artery disease. The risk of myocardial infarction in these and other patients can be reduced by administering any of the RNAi constructs described herein to the patient. Accordingly, the present invention is directed to a method for reducing the risk of myocardial infarction in a patient in need thereof, comprising administering to the patient an RNAi construct described herein. In some embodiments, the present invention includes the use of any of the RNAi constructs described herein in the preparation of a medicament for reducing the risk of myocardial infarction in a patient in need thereof. In other embodiments, the present invention provides an LPA-targeting RNAi construct for use in a method for reducing the risk of myocardial infarction in a patient in need thereof.

[0122] In certain embodiments, a patient in need of reducing LPA expression has been diagnosed with or is at risk for cardiovascular disease. Accordingly, the present invention includes methods for treating or preventing cardiovascular disease in a patient in need thereof by administering any of the RNAi constructs of the present invention. In some embodiments, the present invention includes the use of any of the RNAi constructs described herein in the preparation of a medicament for treating or preventing cardiovascular disease in a patient in need thereof. In other embodiments, the present invention provides an LPA-targeting RNAi construct for use in a method for treating or preventing cardiovascular disease in a patient in need thereof. Cardiovascular disease includes, but is not limited to, myocardial infarction, heart failure, stroke (ischemic and hemorrhagic), atherosclerosis, coronary artery disease, peripheral vascular disease (e.g., peripheral artery disease), cerebrovascular disease, vulnerable plaque, and aortic stenosis. In some embodiments, the cardiovascular disease treated or prevented according to the methods of the present invention is coronary artery disease. In other embodiments, the cardiovascular disease treated or prevented according to the methods of the present invention is myocardial infarction. In still other embodiments, the cardiovascular disease treated or prevented according to the methods of the invention is stroke. In yet other embodiments, the cardiovascular disease treated or prevented according to the methods of the invention is peripheral artery disease. In certain embodiments, administration of an RNAi construct described herein reduces the risk of non-fatal myocardial infarction, fatal and non-fatal stroke, certain types of cardiac surgery (e.g., angioplasty, bypass), hospitalization for heart failure, chest pain in patients with cardiac disease, and / or cardiovascular events in patients with established cardiac disease (e.g., previous myocardial infarction, previous cardiac surgery, and / or chest pain with signs of arterial blockage). In some embodiments, administration of an RNAi construct described herein according to the methods of the invention can be used to reduce the risk of recurrent cardiovascular events.

[0123] In certain other embodiments, the patient in need of reduced LPA expression is a patient with elevated blood levels of Lp(a). Accordingly, in some embodiments, the present invention provides methods for reducing serum or plasma levels of Lp(a) in a patient in need thereof by administering any of the RNAi constructs described herein to the patient. In some embodiments, the present invention includes the use of any of the RNAi constructs described herein in the preparation of a medicament for reducing serum or plasma levels of Lp(a) in a patient in need thereof. In other embodiments, the present invention provides an LPA-targeting RNAi construct for use in a method for reducing serum or plasma levels of Lp(a) in a patient in need thereof. As described above, elevated blood levels of Lp(a) are associated with an increased risk of cardiovascular disease. In some embodiments, the level of Lp(a) in the serum or plasma of a patient after administration of the RNAi construct is reduced compared to the level of Lp(a) in the serum or plasma of the patient before administration of the RNAi construct, or compared to the level of Lp(a) in the serum or plasma of a patient not receiving the RNAi construct. In certain embodiments, after administration of an RNAi construct of the invention, the patient's serum or plasma Lp(a) level is reduced to about 150 nmol / L or less, about 125 nmol / L or less, about 100 nmol / L or less, about 75 nmol / L or less, about 70 nmol / L or less, about 65 nmol / L or less, about 60 nmol / L or less, about 55 nmol / L or less, about 50 nmol / L, about 45 nmol / L or less, about 40 nmol / L or less, about 35 nmol / L or less, or about 30 nmol / L or less. While there is a preference for Lp(a) levels measured in units of particle concentration (e.g., nmol / L) (see, e.g., Wilson et al., Journal of Clinical Lipidology, Vol. 13; 374-392, 2019), Lp(a) levels may also be calculated in units of mass concentration (e.g., mg / dL).In such embodiments, the RNAi constructs of the invention can reduce Lp(a) levels in a patient's serum or plasma to about 100 mg / dL or less, about 90 mg / dL or less, about 80 mg / dL or less, about 70 mg / dL or less, about 60 mg / dL or less, about 50 mg / dL or less, about 45 mg / dL or less, about 40 mg / dL or less, about 35 mg / dL or less, about 30 mg / dL or less, about 25 mg / dL or less, about 20 mg / dL or less, or about 15 mg / dL or less after administration. Lp(a) levels in plasma or serum samples can be measured using commercially available kits such as the Lp(a) ELISA assay kit from Mercodia AB (Uppsala, Sweden), the Lp(a) immunoturbidimetric assay from Randox Laboratories Ltd. (Crumlin, United Kingdom), or the Tina-quant® Lp(a) assay from F. Hoffmann-La Roche Ltd. (Basel, Switzerland), or using other methods known in the art, such as those described in Marcovina and Albers, J. Lipid Res., Vol. 57:526-537, 2016.

[0124] In some embodiments, patients treated according to the methods of the invention have elevated blood levels of Lp(a) (e.g., elevated serum or plasma levels of Lp(a)). Patients treated according to the methods of the invention may have blood Lp(a) levels of about 50 nmol / L or greater, about 55 nmol / L or greater, about 60 nmol / L or greater, about 65 nmol / L or greater, about 70 nmol / L or greater, about 75 nmol / L or greater, about 100 nmol / L or greater, about 125 nmol / L or greater, about 150 nmol / L or greater, about 175 nmol / L or greater, or about 200 nmol / L or greater. In certain embodiments, an RNAi construct of the invention is administered to a patient when the patient has a serum or plasma Lp(a) level of about 100 nmol / L or greater. In one embodiment, an RNAi construct of the invention is administered to a patient when the patient has a serum or plasma Lp(a) level of about 125 nmol / L or greater. In another embodiment, a patient is administered an RNAi construct of the invention if the patient has a serum or plasma Lp(a) level of about 150 nmol / L or greater. In embodiments in which blood Lp(a) levels are measured in mass concentration units, patients treated according to the methods of the invention may have blood Lp(a) levels of about 30 mg / dL or greater, about 35 mg / dL or greater, about 40 mg / dL or greater, about 45 mg / dL or greater, about 50 mg / dL or greater, about 55 mg / dL or greater, about 60 mg / dL or greater, about 65 mg / dL or greater, about 70 mg / dL or greater, about 75 mg / dL or greater, or about 100 mg / dL or greater. In one embodiment, a patient is administered an RNAi construct of the invention if the patient has a serum or plasma Lp(a) level of about 50 mg / dL or greater. In another embodiment, a patient is administered an RNAi construct of the invention if the patient has a serum or plasma Lp(a) level of about 70 mg / dL or greater.

[0125] In certain embodiments, patients treated according to the methods of the present invention are patients suffering from vulnerable plaques (also referred to as unstable plaques). Vulnerable plaques are accumulations of macrophages and lipids, primarily cholesterol, beneath the endothelial lining of the arterial wall. These vulnerable plaques can rupture, leading to the formation of a thrombus, which can potentially block blood flow through the artery and cause a myocardial infarction or stroke. Vulnerable plaques can be identified by methods known in the art, including, but not limited to, intravascular ultrasound and computed tomography (Sahara et al., European Heart Journal, Vol. 25; 2026-2033, 2004; Budhoff, J. Am. Coll. Cardiol., Vol. 48; 319-321, 2006; Hausleiter et al., J. Am. Coll. Cardiol., Vol. 48; 312-318, 2006).

[0126] The following examples, including the experiments conducted and results achieved, are offered for illustrative purposes only and are not to be construed as limiting the scope of the appended claims. [Example]

[0127] Example 1. Design and synthesis of RNAi constructs for LPA Bioinformatics analysis of the human LPA transcript identified candidate sequences for the design of therapeutic siRNA molecules targeting the human LPA gene, and the sequence is presented herein as SEQ ID NO: 1 (NCBI Reference SEQ ID NO: NM_005577.4; see Figure 1). The human LPA gene is highly polymorphic due to differences in the number of Kringle IV-2 (KIV-2) domain repeats among alleles of the gene between individuals. KIV-2 domain repeats can range from 2 to 43 copies among individuals. The transcript presented herein as SEQ ID NO: 1 is an allelic variant containing 15 copies of the KIV-2 domain. Sequences were analyzed using an in-house siRNA design algorithm and selected if they met certain criteria. Sequences were also evaluated for cross-reactivity with the LPA gene from cynomolgus monkeys (NCBI Reference SEQ ID NO: XM_015448520.1), sequence homology with other human gene sequences, seed sequence matches with human microRNA (miRNA) sequences to predict off-target effects, and overlap with known single nucleotide polymorphisms. Based on the results of the bioinformatics analysis, 465 sequences were selected, of which 320 sequences were prioritized for initial synthesis and in vitro testing.

[0128] RNAi constructs were synthesized using solid-phase phosphoramidite chemistry. Synthesis was performed on a MerMade 12 or MerMade 192X (Bioautomation) instrument. Various chemical modifications were incorporated into the molecules, including 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, abasic nucleotides, and phosphorothioate internucleotide linkages. RNAi constructs are generally formatted as 19-21 base pair duplexes when annealed with either no overhangs (double bluntmers) or one or two two-nucleotide overhangs at the 3' end of the antisense and / or sense strands. The sense strand of the RNAi construct was further conjugated to a trivalent N-acetyl-galactosamine (GalNAc) moiety, as described below.

[0129] material Acetonitrile (DNA synthesis grade, AXO152-2505, EMD) Capping Reagent A (80:10:10 (v / v / v) tetrahydrofuran / lutidine / acetic anhydride, BIO221 / 4000, EMD) Capping Reagent B (16% 1-methylimidazole / tetrahydrofuran, BIO345 / 4000, EMD) Activator solution (0.25 M 5-(ethylthio)-1H-tetrazole (ETT) in acetonitrile, BIO152 / 0960, EMD) Detritylation reagent (3% dichloroacetic acid in dichloromethane, BIO830 / 4000, EMD) Oxidizing reagent (0.02 M iodine in 70:20:10 (v / v / v) tetrahydrofuran / pyridine / water, BIO420 / 4000, EMD) Diethylamine solution (20% DEA in acetonitrile, NC0017-0505, EMD) Thiolating reagent (0.05 M 5-N-[(dimethylamino)methylene]amino-3H-1,2,4-dithiazole-3-thione (BIOSULII / 160K) in 40:60 (v / v) pyridine / acetonitrile) 5'-aminohexyl linker phosphoramidites of adenosine, guanosine, cytosine, and uridine, phosphorylated phosphoramidites, 2'-deoxythymidine phosphoramidite, and 2'-methoxy and 2'-fluorophosphoramidites (Thermo Fisher Scientific), 0.10 M in acetonitrile over approximately 10 mL of molecular sieves (3 Å, JT Baker). CPG support (high-load general-purpose support, 500A (BH5-3500-G1), 79.6μmol / g, 0.126g (10μmol)) Ammonium hydroxide (high concentration, JTBaker)

[0130] synthesis The reagent solution, phosphoramidite solution, and solvent were connected to the MerMade 12 instrument. A solid support was added to each column (4 mL SPE tubing with upper and lower frits), and the column was attached to the instrument. The column was washed twice with acetonitrile. The phosphoramidite and reagent solution lines were purged. Synthesis was initiated using Poseidon software. Synthesis was performed by repeated deprotection / coupling / oxidation / capping synthesis cycles. Specifically, a detritylation reagent was added to the solid support to remove the 5'-dimethoxytrityl (DMT) protecting group. The solid support was washed with acetonitrile. The phosphoramidite and activator solutions were added to the support, followed by incubation to allow the coupling of incoming nucleotides to the free 5'-hydroxyl group. The support was washed with acetonitrile. An oxidation or thiolation reagent was added to the support to convert the phosphite triester to a phosphate triester or phosphorothioate. Capping reagents A and B were added to the support to terminate any unreacted oligonucleotide chains. The support was washed with acetonitrile. After the final reaction cycle, the resin was washed with diethylamine solution to remove the 2-cyanoethyl protecting group, and the support was washed with acetonitrile and dried under vacuum.

[0131] GalNAc conjugation The sense strand was prepared for conjugation to a trivalent GalNAc moiety (structure shown in Formula VII below) using a 5'-aminohexyl linker. After automated synthesis, the column was removed from the instrument and transferred to a vacuum manifold in a hood. The 5'-monomethoxytrityl (MMT) protecting group was removed from the solid support by successive treatment with 2 mL aliquots of 1% trifluoroacetic acid (TFA) in dichloromethane (DCM) via vacuum filtration. Once no further orange / yellow color was observed in the eluate, the resin was washed with dichloromethane. The resin was then washed with 5 mL of 2% diisopropylethylamine in N,N-dimethylformamide (DMF). In a separate vial, a solution of GalNAc3-Lys2-Ahx (67 mg, 40 μmol) (the structure and synthesis of which are described below) in DMF (0.5 mL) was prepared using 1,1,3,3-tetramethyluronium tetrafluoroborate (TATU, 12.83 mg, 40 μmol) and diisopropylethylamine (DIEA) (13.9 μL, 80 μmol). The activated coupling solution was added to the resin, and the column was capped and incubated overnight at room temperature. The resin was washed with DMF and DCM and dried under vacuum.

[0132] Disconnect The synthesis columns were removed from the synthesizer or vacuum manifold. The solid support from each column was transferred to a 10 mL vial. 4 mL of concentrated ammonium hydroxide was added to the solid support. The bottles were tightly capped and the mixtures were heated at 55°C for 4 hours. The bottles were transferred to a freezer and allowed to cool for 20 minutes before being opened in a hood. The mixtures were filtered through 8 mL SPE tubes to remove the solid support. The vials and solid support were rinsed with 1 mL of 50:50 ethanol / water.

[0133] Analysis and Purification A portion of the combined filtrate was analyzed and purified by anion exchange chromatography. The pooled fractions were desalted by size exclusion chromatography and analyzed by ion-pair reversed-phase high-performance liquid chromatography-mass spectrometry (HPLC-MS). The pooled fractions were lyophilized to yield a white amorphous powder. Analytical Anion Exchange Chromatography (AEX): Column: Thermo DNAPac PA200RS (4.6 x 50 mm, 4 μm) Equipment: Agilent 1100 HPLC Buffer A: 20 mM sodium phosphate, 10% acetonitrile, pH 8.5 Buffer B: 20 ​​mM sodium phosphate, 10% acetonitrile, pH 8.5, 1 M sodium bromide Flow rate: 1 mL / min at 40°C Gradient: 20-65% B in 6.2 min Preparative Anion Exchange Chromatography (AEX): Column: Tosoh Corporation TSK Gel SuperQ-5PW, 21 x 150 mm, 13 μm Equipment: Agilent 1200 HPLC Buffer A: 20 mM sodium phosphate, 10% acetonitrile, pH 8.5 Buffer B: 20 ​​mM sodium phosphate, 10% acetonitrile, pH 8.5, 1 M sodium bromide Flow rate: 8mL / min Injection volume: 5mL Gradient: 35-55% B over 20 min Preparative Size Exclusion Chromatography (SEC): Column: GE Hi-Prep 26 / 10 Equipment: GE AKTA Pure Buffer: 20% ethanol in water Flow rate: 10mL / min Injection volume: 15 mL using a sample loading pump Ion-pair reversed phase (IP-RP) HPLC: Column: Water Xbridge BEH OST C18, 2.5 μm, 2.1 × 50 mm Equipment: Agilent 1100 HPLC Buffer A: 15.7 mM DIEA, 50 mM hexafluoroisopropanol (HFIP) in water Buffer B: 15.7 mM DIEA, 50 mM HFIP in 50:50 water / acetonitrile Flow rate: 0.5mL / min Gradient: 10-30% B over 6 min

[0134] annealing Small amounts of the sense and antisense strands were weighed into individual vials. siRNA reconstitution buffer (Qiagen) or phosphate-buffered saline (PBS) was added to the vials to a concentration of approximately 2 mM based on dry weight. Actual sample concentrations were measured using a NanoDrop One (ssDNA, extinction coefficient = 33 μg / OD260). The two strands were then mixed in an equimolar ratio, and the samples were heated in a 90°C incubator for 5 minutes and allowed to cool slowly to room temperature. Samples were analyzed by AEX. Duplexes were registered and subjected to in vitro and in vivo testing as described in more detail below.

[0135] Preparation of GalNAc3-Lys2-Ahx [ka] where X=O or S. The wavy line represents the point of binding to the 5'-terminal nucleotide of the sense strand of the RNAi construct.

[0136] To a 50 mL falcon tube was added Fmoc-Ahx-OH (1.13 g, 3.19 mmol) in DCM (30 mL), followed by DIEA (2.23 mL, 12.78 mmol). This solution was added to 2-Cl trityl chloride resin (3.03 g, 4.79 mmol) in a 50 mL centrifuge tube and placed on a shaker for 2 h. The solvent was drained, and the resin was washed with 17:2:1 DCM / MeOH / DIEA (2 x 30 mL), DCM (4 x 30 mL), and dried. The loading was determined to be 0.76 mmol / g by UV spectrophotometric detection at 290 nm.

[0137] 3 g of the charged 2-Cl trityl resin was suspended in 20% 4-methylpiperidine in DMF (20 mL), and the solvent was drained after 30 minutes. This process was repeated once more, and the resin was washed with DMF (30 mL x 3) and DCM (30 mL x 3).

[0138] To a solution of Fmoc-Lys(ivDde)-OH (3.45 g, 6 mmol) in DMF (20 mL) was added TATU (1.94 g, 6 mmol), followed by DIEA (1.83 mL, 10.5 mmol). The solution was then added to the deprotected resin, and the suspension was placed on a shaker overnight. The solvent was drained, and the resin was washed with DMF (30 mL x 3) and DCM (30 mL x 3).

[0139] The resin was treated with 20% 4-methylpiperidine in DMF (15 mL) and the solvent was drained after 10 min. This process was repeated once more, and the resin was washed with DMF (15 mL x 4) and DCM (15 mL x 4).

[0140] To a solution of Fmoc-Lys(Fmoc)-OH (3.54 g, 6 mmol) in DMF (20 mL) was added TATU (1.94 g, 6 mmol), followed by DIEA (1.83 mL, 10.5 mmol). The solution was then added to the deprotected resin, and the suspension was placed on a shaker overnight. The solvent was drained, and the resin was washed with DMF (30 mL x 3) and DCM (30 mL x 3).

[0141] The resin was treated with 5% hydrazine in DMF (20 mL) and the solvent was drained after 5 min. This process was repeated four more times, and the resin was washed with DMF (30 mL × 4) and DCM (30 mL × 4).

[0142] To a solution of 5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydro-2H-pyran-2-yl)oxy)pentanoic acid (4.47 g, 10 mmol) in DMF (40 mL) was added TATU (3.22 g, 10 mmol), and the solution was stirred for 5 min. To this solution was added DIEA (2.96 mL, 17 mmol), and then this mixture was added to the above resin. The suspension was kept at room temperature overnight, and the solvent was drained. The resin was washed with DMF (3 × 30 mL) and DCM (3 × 30 mL).

[0143] The resin was treated with 1% TFA in DCM (30 mL, containing 3% triisopropylsilane) and the solvent was drained after 5 min. This process was repeated three more times, and the combined filtrates were concentrated under reduced pressure. The residue was triturated with diethyl ether (50 mL), and the suspension was filtered and dried to give the crude product. The crude product was purified by reverse-phase chromatography, eluting with 0–20% MeCN in water. The combined fractions were lyophilized to give the product as a white solid.

[0144] Based on the activity in the in vitro cell-based assay described in Example 2 and the in vitro transgenic mouse study described in Example 3, 137 sequences targeting specific regions of the human LPA transcript were selected for structure-activity relationship (SAR) studies. Table 1 below lists the unmodified sense and antisense sequences of each molecule in the 137 sequence families. Table 1 also shows the nucleotide ranges targeted by siRNA molecules of each sequence family within the human LPA transcript (SEQ ID NO: 1). As mentioned above, because the human LPA gene contains KIV-2 domain repeats, siRNA molecules may have more than one target site within the transcript if the target site is within a conserved region in the KIV-2 domain or other KIV domains. For clarity, only the first target site within the transcript is shown.

[0145] Table 2 shows the sequences of the sense and antisense strands with chemical modifications in an exemplary duplex resulting from the SAR study. The nucleotide sequences are listed according to the following notation: a, u, g, and c = the corresponding 2'-O-methylribonucleotide; Af, Uf, Gf, and Cf = the corresponding 2'-deoxy-2'-fluoro ("2'-fluoro") ribonucleotide; Phos = the terminal nucleotide with a monophosphate group at its 5' end; and invAb = an inverted abasic nucleotide (i.e., an abasic nucleotide that is linked to an adjacent nucleotide via its 3'-substituent when located at the 3' end of the strand (3'-3' bond), or via its 5'-substituent when located at the 5' end of the strand (5'-5' internucleotide bond). The insertion of "s" in the sequence indicates that two adjacent nucleotides are linked by a phosphorothioate group (e.g., a phosphorothioate internucleotide bond). Unless otherwise indicated, all other nucleotides are linked by 3'-5' phosphodiester groups. [GalNAc3] represents the GalNAc moiety shown in Formula VII, which, when followed by an "s" in the [GalNAc3] designation, is covalently attached to the 5'-terminus of the sense strand via a phosphodiester or phosphorothioate bond.

[0146] [Table 1]

[0147] [Table 2]

[0148] [Table 3]

[0149] [Table 4]

[0150] [Table 5]

[0151] [Table 6]

[0152] [Table 7]

[0153] [Table 8]

[0154] [Table 9]

[0155] [Table 10]

[0156] [Table 11]

[0157] [Table 12]

[0158] [Table 13]

[0159] Example 2. In vitro evaluation of LPA RNAi constructs in cell-based assays Based on 320 distinct sequences initially prioritized by the bioinformatics analysis described in Example 1, 400 GalNAc-conjugated LPA siRNA molecules were evaluated at a single concentration (12 nM) for their ability to inhibit LPA mRNA synthesis in an in vitro primary human hepatocyte assay. Human primary hepatocytes (Xenotech / Sekisui donor lot number HC10-23) were thawed in OptiThaw medium (Xenotech catalog number K8000) according to the manufacturer's protocol. Cells were centrifuged, and after media aspiration, resuspended in OptiPlate Hepatocyte Medium (Xenotech catalog number K8200) and plated onto 96-well collagen-coated plates (Greiner catalog number 655950). After a 3-4 hour incubation period, the medium was removed and replaced with OptiCulture Hepatocyte Medium (Xenotech catalog number K8300). Three to five hours after adding OptiCulture medium, GalNAc-conjugated siRNA was delivered to cells by free uptake (without transfection reagent) either at a single point (12 nM) or in a dose-response format (0.2 μM–4 μM). Cells were incubated at 37°C and 5% CO2 for approximately 66–72 hours. RNA extraction was performed on either a Qiagen QIACube HT (9001793) instrument or a ThermoFisher KingFisher Flex (5400630) instrument. Using the Qiagen QIACube HT system, cells were lysed in Qiagen RLT buffer (79216) supplemented with 1% 2-mercaptoethanol (Sigma, M-3148), and the lysates were stored at -20°C. RNA was purified using the Qiagen QIACube HT kit (74171) on the Qiagen QIACube HT instrument according to the manufacturer's instructions. Samples were analyzed using a QIAxpert system (9002340). Cells were lysed using Lysis / Binding Concentrate (ThermoFisher Scientific AM8500) with a ThermoFisher KingFisher Flex system. Cell lysates were stored at -20°C, or in some cases, RNA extraction was performed immediately after cell lysis.RNA was purified using the ThermoFisher Scientific MagMAX™-96 Total RNA Isolation Kit (ThermoFisher Scientific AM1830) on a Flex instrument according to the manufacturer's instructions.

[0160] cDNA was synthesized from RNA samples using the Applied Biosystems High Capacity cDNA Reverse Transcription Kit (4368813). Reactions were assembled according to the manufacturer's instructions, with input RNA concentrations varied by sample. Reverse transcription was performed in a BioRad quadruplicate thermal cycler (model number PTC-0240G) using the following conditions: 25°C for 10 minutes, 37°C for 120 minutes, 85°C for 5 minutes, followed by an optional 4°C hold. Droplet digital PCR (ddPCR) was performed using a BioRad QX200 AutoDG droplet digital PCR system according to the manufacturer's instructions. Reactions were assembled in Eppendorf clear 96-well PCR plates (951020303) using BioRad ddPCR Supermix for probes (1863010), fluorescently labeled qPCR assays for LPA (IDT Hs.PT.58.1145110, ordered at a primer-to-probe ratio of 3.6:1, 45 nmoles each of forward and reverse primers, and 12.5 nmoles of 6-FAM / ZEN / IBFQ-labeled probe), and TATA box-binding protein (TBP) (IDT Hs.PT.53a.20105486, ordered at a primer-to-probe ratio of 3.6:1, 45 nmoles each of forward and reverse primers, and 12.5 nmoles of HEX / ZEN / IBFQ-labeled probe), and RNase-free water (Ambion, AM9937). Primer / probe sequences are listed below. Final primer / probe concentrations were 900 nM / 250 nM, respectively, and the concentration of input cDNA was varied between wells.

[0161] Droplets were generated using a BioRad Auto DG Droplet Generator (1864101) installed with the manufacturer's recommended consumables (BioRad DG32 Cartridge 1864108, BioRad Tip 1864121, Eppendorf Blue 96-well PCR Plate 951020362, BioRad Probe Droplet Generation Oil 1864110, and BioRad Droplet Plate Assembly). Droplets were amplified on a BioRad C1000 touch thermal cycler (1851197) using the following conditions: 95°C for 10 minutes for enzyme activation, 94°C for 30 seconds for denaturation, followed by 60°C for 1 minute for annealing / extension, 40 cycles with a ramp rate of 2°C / second, 98°C for 10 minutes for enzyme inactivation, and then an optional 4°C hold. Samples were then read on a BioRad QX200 droplet reader to measure the FAM / HEX signal, which correlates with the mRNA concentration of LPA or TBP, respectively. Data were analyzed using BioRad's QuantaSoft software package. Samples were gated by channel (fluorescent label) to measure the concentration per sample. Each sample was then expressed as the ratio of the concentration of the gene of interest (LPA) to the concentration of the housekeeping gene (TBP) to adjust for differences in sample loading. Data were then imported into Genedata Screener, and each test siRNA was normalized to the median of the neutral control wells (buffer only or control siRNA) and expressed as a percentage of control.

[0162] ddPCR assay sequence LPA: Primer 1: 5'-CAAAATGGAACATAAGGAAGTGGT-3' (SEQ ID NO: 602) Primer 2: 5'-GTGACAGTGGTGGAGTACG-3' (SEQ ID NO: 603) Probe: 5'- / 56-FAM / CATGGCTTT (SEQ ID NO: 604) / ZEN / GCTCAGGTGCTGC (SEQ ID NO: 605) / 3IABkFQ / -3' TBP: Primer 1: 5'-ATGACCCCCATCACTCCT-3' (SEQ ID NO: 606) Primer 2: 5'-TCAAGTTTACAACCAAGATTCACTG-3' (SEQ ID NO: 607) Probe: 5'- / 5HEX / AGCTGCGGT (SEQ ID NO: 608) / ZEN / ACAATCCCAGAACTC (SEQ ID NO: 609) / 3IABkFQ / -3'

[0163] Based on the results of the single-concentration assay, a subset of GalNAc-conjugated LPA siRNA molecules was selected for further testing in a 10-point dose-response format (0.2 μM to 4 μM) in a ddPCR assay in primary human hepatocytes. After a 72-hour incubation period of GalNAc-conjugated LPA siRNA molecules with hepatocytes, the ratio of LPA mRNA concentration to TBP mRNA concentration was measured. EC50 values ​​for each of the GalNAc-conjugated LPA siRNA molecules were calculated from the dose-response curve and are shown in Table 3 below, along with the maximal antagonist activity of each molecule, expressed as a percentage of remaining LPA mRNA (i.e., a percentage of the control).

[0164] [Table 14]

[0165] [Table 15]

[0166] [Table 16]

[0167] Some of the LPA siRNA molecules showed a maximal reduction of LPA mRNA levels by more than 85% compared to hepatocytes not treated with the siRNA molecules, with EC50 values ​​in the single-digit nanomolar range.

[0168] A subset of the more potent siRNA molecules from Table 3 was selected and further tested in a second in vitro assay using a dual-luciferase reporter system. In addition, the dual-luciferase reporter assay was used in a SAR study in combination with the transgenic mouse model described in Example 3. In this assay, the configuration of the siRNA molecules and the number and / or composition of chemical modifications (e.g., strand length and terminal nature) were varied to select sequence families that would optimize the magnitude and extent of inhibition of LPA gene expression.

[0169] A dual-luciferase reporter plasmid (pMIR0660) was constructed from the commercially available psiCHECK plasmid (Promega, Madison, WI), which contains the coding DNA sequences (CDS) for both Renilla luciferase and firefly luciferase. A portion of the CDS for human LPA, including KIV-3 to KIV-10, was cloned into this plasmid to generate a fusion between the Renilla luciferase CDS and the human LPA CDS. siRNA-mediated inhibition of LPA target sequence translation resulted in degradation of the fusion mRNA and reduced Renilla luciferase signal. Knockdown of the LPA gene was assessed by measuring Renilla luciferase levels normalized to the firefly luciferase levels constitutively expressed by the plasmid. Huh7 cells, a human hepatocellular carcinoma cell line, were plated in 96-well plates. After overnight incubation, cells were co-transfected with the dual reporter plasmid pMIR0660 and transfected with different concentrations of Lipofectamine according to the manufacturer's instructions. (商標) siRNA molecules were tested with the 2000 transfection reagent. An 8- to 11-point dose escalation protocol (0-10 nM) was performed in triplicate. After a second overnight incubation, dual luciferase activity was measured using an Envision luminometer (PerkinElmer, Waltham, MA). The EC50 values ​​and maximum antagonist activity (measured as the lowest ratio of Renilla luciferase levels to firefly luciferase levels) of each of the evaluated LPA siRNA molecules are reported in Table 4 below.

[0170] [Table 17]

[0171] [Table 18]

[0172] [Table 19]

[0173] [Table 20]

[0174] Example 3. In vivo efficacy of LPA RNAi constructs in transgenic mice expressing human apolipoproteins To evaluate the in vivo efficacy of the LPA RNAi construct, we used a double transgenic mouse model. There is no ortholog of the LPA gene in mice, and apo(a) (encoded by the LPA gene) is generally expressed only in primates. Transgenic mice expressing human apo(a) from a yeast artificial chromosome (YAC) containing the complete human LPA gene (Frazer et al., Nature Genetics, Vol. 9:424-431, 1995) were crossed with transgenic mice expressing human apoB-100 (Linton et al., J. Clin. Invest., Vol. 92:3029-3037, 1993). The resulting double transgenic mice express fully functional human Lp(a) particles, with baseline serum Lp(a) levels averaging approximately 50-60 mg / dL. Female double transgenic mice were randomized into different treatment groups for each study based on baseline Lp(a) serum levels, body weight, and age. Saline or LPA RNAi constructs were administered as a single subcutaneous injection at doses of 0.5 mg / kg, 1 mg / kg, or 2 mg / kg. Serum samples were collected before injection and then at weeks 1, 2, 3, 4, 6, 8, 10, and 12 after injection, or until serum Lp(a) levels returned to baseline levels. Serum Lp(a) concentrations were measured using an Lp(a) ELISA assay (catalog number 10-1106-01, Mercodia AB, Uppsala, Sweden). The percent change in Lp(a) levels for each animal at a particular time point was calculated based on the animal's baseline Lp(a) levels. The results of 11 separate studies of transgenic mice with different LPA RNAi constructs are shown in Tables 5–15 below. Data are expressed as the mean percent change from baseline for each treatment group (n=4 or 5 animals / group, except for studies 10 and 11 where n=6 animals / group).

[0175] [Table 21]

[0176] Table 22

[0177] Table 23

[0178] Table 24

[0179] Table 25

[0180] Table 26

[0181] Table 27

[0182] Table 28

[0183] Table 29

[0184] Table 30

[0185] Table 31

[0186] Most of the tested LPA RNAi constructs reduced serum Lp(a) levels by at least 50% two weeks after a single subcutaneous injection at a dose of 1 mg / kg or 2 mg / kg in transgenic animals. Some of the generated RNAi constructs sustained inhibition of Lp(a) serum levels up to week 4 after a single injection. For example, after a single injection of 1 mg / kg or 2 mg / kg of constructs 4601, 4613, 4930, 4970, 6150, 6182, 6247, 8395, 8401, 10927, 11318, 11344, 11351, 11374, 11580, 17188, 18436, 18444, and 18446, Lp(a) serum levels were still reduced by approximately 50% or more at week 4.

[0187] Example 4. In vitro efficacy of LPA RNAi constructs in non-human primates The efficacy of selected LPA RNAi constructs was evaluated in cynomolgus monkeys in three separate experiments. The RNAi constructs had sequences that cross-reacted with the sequence of the cynomolgus monkey LPA gene (NCBI reference sequence number XM_015448520.1). In the first experiment, cynomolgus monkeys (n=3 per treatment group) received a single subcutaneous injection of 2 mg / kg of LPA RNAi constructs 4601, 4613, or 4970. Blood samples were collected on day -1 (before administration) and on days 4, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 98, 105, 112, 119, 126, 133, and 140 after administration on day 1. Each sample was analyzed for Lp(a) serum levels using an Lp(a) ELISA assay (catalog number 10-1106-01, Mercodia AB, Uppsala, Sweden). Results from the first study are shown in Figure 2. Data are expressed as a percentage of remaining Lp(a) serum levels compared to pre-dose baseline. Constructs 4601 and 4613 suppressed serum Lp(a) levels by 80% or more compared to baseline levels for at least 6 weeks (e.g., by at least day 42).

[0188] In the second study, cynomolgus monkeys (n = 3 per treatment group) received a single subcutaneous injection of 2 mg / kg of LPA RNAi constructs 8401, 10927, 11318, 11344, or 11351. Blood samples were collected at the same time points as in the first study, and serum Lp(a) levels were analyzed as described above. The results of the second study are shown in Figure 3. Data are expressed as the percentage of remaining Lp(a) serum levels compared to pre-dose baseline. Notably, constructs 10927 and 11351 nearly completely suppressed Lp(a) serum levels by week 8. Significant reductions in serum Lp(a) levels were observed up to 112 days, nearly 4 months, after the single-dose injection. In contrast, constructs 8401 and 11344 produced a more moderate and transient reduction in serum Lp(a) levels. Construct 11318 suppressed serum Lp(a) to levels approximately 40% of baseline, and this reduced level was sustained for several weeks.

[0189] In the third study, cynomolgus monkeys (n=3 per treatment group) received a single subcutaneous injection of 2 mg / kg of LPA RNAi constructs 11374, 11580, 17205, 18444, or 18436. Blood samples were collected at the same time points and analyzed for serum Lp(a) levels as in the previous two studies described above. The results of the third study are shown in Figure 4. Data are expressed as the percentage of remaining Lp(a) serum levels compared to pre-dose baseline. Construct 11374 was the most effective of this group of molecules, suppressing Lp(a) serum levels to 20% of baseline levels for approximately 6 weeks after a single subcutaneous injection.

[0190] Example 5. Viscosity evaluation of LPA RNAi constructs The viscosity of the LPA RNAi construct 11374 in phosphate-buffered saline (PBS) was evaluated at different concentrations. Lyophilized 11374 was formulated in PBS to prepare a stock solution. The stock solution was diluted with PBS to prepare formulations of different 11374 constructs at concentrations ranging from 150 to 350 mg / mL. For comparison, the viscosity of the LPA RNAi construct AD03851 (described in WO 2017 / 059223) was also evaluated in parallel. The modified nucleotide sequence of AD03851 is listed below: Sense sequence: 5'-csagccccuUfAfUfuguuauacgs(invdA)-3' (SEQ ID NO: 620) Antisense sequence: 5'-usCfsgUfaUfaacaaUfaAfgGfgGfcsUfsg-3' (SEQ ID NO: 621) (where a, u, g, and c = the corresponding 2'-O-methylribonucleotides; Af, Uf, Gf, and Cf = the corresponding 2'-deoxy-2'-fluororibonucleotides; invdA = inverted deoxyadenosine nucleotide (i.e., 3'-3' linkage); and s = phosphorothioate internucleotide linkage). The 5' end of the sense strand was covalently linked via a phosphorothioate linkage to a trivalent GalNAc moiety (NAG25, the structure of which is described in WO 2017 / 059223).

[0191] To calculate the concentration of 11374 formulation, the absorbance of the samples was measured at 260 nm using an Agilent 8453 G1103A UV-Visible spectrophotometer. The measured extinction coefficient of AD03851 at 260 nm was 19.1 mL*mg -1 *cm -1 An approximate attenuation coefficient of 0.05 and a path length of 1 cm were used to calculate the concentration of the formulation using Beer's Law.

[0192] The viscosity of each formulation was measured using an Anton Paar MCR 302 cone-plate rheometer at 1000 s -1The viscosity measurements were taken at a shear rate of 100 s at 25° C. The viscosity measurements of the two LPA RNAi constructs at different concentrations in PBS are shown in Table 16 below.

[0193] [Table 32]

[0194] The LPA RNAi construct 11374 has a lower viscosity as a function of concentration compared to the reference RNAi construct AD03851, allowing for more concentrated formulations and reduced injection volumes.

[0195] All publications, patents, and patent applications discussed and cited herein are hereby incorporated by reference in their entirety. It is understood that the disclosed invention is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the appended claims.

[0196] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. An RNAi construct comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region having a sequence complementary to an mRNA sequence of LPA; (i) the sense strand comprises or consists of the sequence of SEQ ID NO: 79, and the antisense strand comprises or consists of the sequence of SEQ ID NO: 194; (ii) the sense strand comprises or consists of the sequence of SEQ ID NO: 80, and the antisense strand comprises or consists of the sequence of SEQ ID NO: 194; (iii) the sense strand comprises or consists of the sequence of SEQ ID NO: 78, and the antisense strand comprises or consists of the sequence of SEQ ID NO: 196; (iv) the sense strand comprises or consists of the sequence of SEQ ID NO: 85, and the antisense strand comprises or consists of the sequence of SEQ ID NO: 198; (v) the sense strand comprises or consists of the sequence of SEQ ID NO: 86, and the antisense strand comprises or consists of the sequence of SEQ ID NO: 198; or (vi) the sense strand comprises or consists of the sequence of SEQ ID NO: 83, and the antisense strand comprises or consists of the sequence of SEQ ID NO: 200; RNAi constructs.

2. The RNAi construct of claim 1 , wherein the RNAi construct comprises at least one modified nucleotide.

3. The RNAi construct of claim 2, wherein the modified nucleotide is a 2'-modified nucleotide.

4. 3. The RNAi construct of claim 2, wherein the modified nucleotide is a 2'-fluoro modified nucleotide, a 2'-O-methyl modified nucleotide, a 2'-O-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a 2'-O-allyl modified nucleotide, a bicyclic nucleic acid (BNA), a deoxyribonucleotide, or a combination thereof.

5. 3. The RNAi construct of claim 2, wherein all of the nucleotides in the sense strand and the antisense strand are modified nucleotides.

6. The RNAi construct of claim 5, wherein the modified nucleotides are 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, or a combination thereof.

7. The RNAi construct of any one of claims 1 to 6, wherein the sense strand comprises an abasic nucleotide as a terminal nucleotide at its 3' end, its 5' end, or both its 3' end and 5' end.

8. The RNAi construct of claim 7, wherein the abasic nucleotide is linked to an adjacent nucleotide via a 3'-3' internucleotide bond or a 5'-5' internucleotide bond.

9. The RNAi construct of any one of claims 1 to 8, wherein the sense strand, the antisense strand, or both the sense strand and the antisense strand comprise one or more phosphorothioate internucleotide linkages.

10. The RNAi construct of claim 9, wherein the antisense strand comprises two consecutive phosphorothioate internucleotide bonds between the terminal nucleotides at both the 3' and 5' ends.

11. 11. The RNAi construct of claim 9 or 10, wherein the sense strand comprises a single phosphorothioate internucleotide bond between the terminal nucleotides at the 3' end.

12. The RNAi construct of claim 9 or 10, wherein the sense strand comprises two consecutive phosphorothioate internucleotide bonds between the terminal nucleotides at the 3' end.

13. (i) the sense strand comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 350 and the antisense strand comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 525; (ii) the sense strand comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 354 and the antisense strand comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 525; (iii) the sense strand comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 351 and the antisense strand comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 530; (iv) the sense strand comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 357 and the antisense strand comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 530; (v) the sense strand comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 362 and the antisense strand comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 536; (vi) the sense strand comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 366 and the antisense strand comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 536; (vii) the sense strand comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 364 and the antisense strand comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 536; (viii) the sense strand comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 370 and the antisense strand comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 540, or (ix) the sense strand comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 372, and the antisense strand comprises or consists of a sequence of modified nucleotides according to SEQ ID NO: 540; The RNAi construct of claim 1.

14. The RNAi construct according to any one of claims 1 to 13, further comprising a ligand.

15. 15. The RNAi construct of claim 14, wherein the ligand comprises a cholesterol moiety, a vitamin, a steroid, a bile acid, a folate moiety, a fatty acid, a carbohydrate, a glycoside, or an antibody or antigen-binding fragment thereof.

16. The RNAi construct of claim 15, wherein the ligand comprises galactose, galactosamine, or N-acetyl-galactosamine.

17. 17. The RNAi construct of claim 16, wherein the ligand comprises a multivalent galactose moiety or a multivalent N-acetyl-galactosamine moiety.

18. 18. The RNAi construct of claim 17, wherein the multivalent galactose moiety or the multivalent N-acetyl-galactosamine moiety is trivalent or tetravalent.

19. The ligand has the following structure: 【Chemistry 1】 The RNAi construct of claim 14, comprising:

20. The RNAi construct of any one of claims 14 to 19, wherein the ligand is covalently bound to the sense strand via a linker.

21. 21. The RNAi construct of claim 20, wherein the ligand is covalently attached to the 5' end of the sense strand.

22. and further comprising a ligand covalently attached to the 5' end of the sense strand via a linker, wherein the ligand and linker have the structure of Formula VII: 【Chemistry 2】 The RNAi construct of any one of claims 1 to 13, having the formula: (wherein X = O or S).

23. A pharmaceutical composition comprising the RNAi construct of any one of claims 1 to 22 and a pharmaceutically acceptable carrier or excipient.

24. A pharmaceutical composition for reducing the expression of LPA in a patient, comprising the RNAi construct of any one of claims 1 to 22.

25. 25. The pharmaceutical composition of claim 24, wherein the patient is diagnosed with or at risk for cardiovascular disease.

26. 25. The pharmaceutical composition of claim 24, wherein the patient has a serum or plasma Lp(a) level of 100 nmol / L or greater.

27. 25. The pharmaceutical composition of claim 24, wherein the patient has a history of myocardial infarction.

28. A pharmaceutical composition for reducing serum or plasma Lp(a) levels in a patient, comprising the RNAi construct of any one of claims 1 to 22.

29. 29. The pharmaceutical composition of claim 28, wherein the patient is diagnosed with or at risk for cardiovascular disease.

30. 29. The pharmaceutical composition of claim 28, wherein the patient has a serum or plasma Lp(a) level of 100 nmol / L or greater.

31. A pharmaceutical composition for treating or preventing cardiovascular disease in a patient, comprising the RNAi construct of any one of claims 1 to 22.

32. 32. The pharmaceutical composition of claim 31, wherein the cardiovascular disease is coronary artery disease, peripheral artery disease, myocardial infarction, or stroke.

33. A pharmaceutical composition for reducing the risk of myocardial infarction in a patient, comprising the RNAi construct of any one of claims 1 to 22.

34. 34. The pharmaceutical composition of claim 33, wherein the patient has been diagnosed with coronary artery disease.

35. 34. The pharmaceutical composition of claim 33, wherein the patient has a serum or plasma Lp(a) level of 100 nmol / L or greater.

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