Rnai constructs for inhibiting SLC30a8 expression and methods of use thereof

Abstract

To provide RNAi constructs for reducing expression of the SLC30A8 gene, and to provide methods of using such RNAi constructs to treat or prevent diseases such as pre-diabetes or diabetes.SOLUTION: An RNAi construct comprising a sense strand and an antisense strand, where the antisense strand comprises a region having at least 15 contiguous nucleotides differing by no more than 3 nucleotides from a specific antisense sequence, and where the RNAi construct inhibits expression of zinc transporter 8 (SLC30A8).SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application No. 62 / 886,269, filed August 13, 2019, which is incorporated herein by reference in its entirety.

[0002] The present invention relates to compositions and methods for modulating pancreatic expression of zinc transporter 8 (SLC30A8 or ZnT8), particularly to nucleic acid-based therapeutics for reducing SLC30A8 expression via RNA interference and methods of using such nucleic acid-based therapeutics to treat or prevent diseases such as diabetes. [Background technology]

[0003] Approximately 80 million Americans are estimated to have prediabetes, defined by impaired glucose tolerance or elevated fasting glucose. Additionally, an estimated 21 million Americans have type 2 diabetes. Progressive beta cell failure is the primary cause of diabetes progression. Maintaining insulin-producing beta cell mass and function is critical for attenuating disease progression in all types of diabetes. Solute transporter family 30 member 8 (SLC30A8 or ZnT8), also known as zinc transporter 8, belongs to the cation diffusion facilitator protein (CDF) family. Two transcripts of human SLC30A8 encode isoform A (369 amino acids) and isoform B (319 amino acids). Isoform B differs from isoform A by an alternative start codon, resulting in the production of a shorter form of the protein that lacks the N-terminal 50 amino acids of isoform A. SLC30A8 is expressed almost exclusively in pancreatic islet beta cells, where it transports cytosolic zinc to insulin-secreting granules. Within the granules, Zn 2+SLC30A8 binds to insulin to form a crystalline hexamer. Recent genome-wide association studies (GWAS) have shown that a common variant (p.W325R) in SLC30A8, which exhibits higher zinc transporter activity, is associated with an increased risk of type 2 diabetes (T2D; Sladek R and Montpetit A (2007) Nature 445 881-885; Merriman C, and Fu D. (2016) JBC 29153:26950). Furthermore, multiple SLC30A8 loss-of-function (LOF) mutations discovered by deCODE et al. reduced the risk of T2D by approximately 65% ​​(Flannick J and Altshuler D. (2014) Nature Genetics 46:357; Flannick J and Boehnke M (2019) Nature 570:71-76). Thus, increased ZnT8 activity is associated with a higher risk of developing T2D, and decreased ZnT8 activity is associated with a reduced risk of T2D. Human genetic data and functional biochemical analyses strongly suggest that ZnT8 inhibition may be beneficial in preventing the progression of prediabetes to T2D.

[0004] Therapeutic gene silencing is an emerging technology that has shown promising results in preclinical and clinical studies. Small interfering RNA (siRNA) is a novel drug modality that blocks the production of disease-causing proteins. siRNAs can be designed to target the transcript of any gene, without being limited to so-called "druggable target classes."

[0005] Silencing SLC30A8 activity has been proposed to benefit individuals with prediabetes or diabetes. Therefore, novel therapeutic agents that target SLC30A8 function offer a novel approach to reducing SLC30A8 levels and treating diseases such as diabetes. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Sladek R and Montpetit A (2007) Nature 445 881-885 [Non-patent document 2] Merriman C,and Fu D.,(2016)JBC 29153:26950 [Non-patent document 3] Flannick J and Altshuler D.,(2014)Nature Genetics 46:357 [Non-patent document 4] Flannick J and Boehnke M (2019) Nature 570:71-76 Summary of the Invention [Means for solving the problem]

[0007] The present invention is based, in part, on the design and generation of RNAi constructs that target the SLC30A8 gene and reduce SLC30A8 expression in pancreatic cells, such as pancreatic islet beta cells. Sequence-specific inhibition of SLC30A8 expression is useful for treating or preventing conditions associated with SLC30A8 expression, such as prediabetes or diabetes. 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 SLC30A8 mRNA sequence. In certain embodiments, the antisense strand comprises a region having at least 15 contiguous nucleotides from an antisense sequence listed in Table 1.

[0008] 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 about 15 to about 30 nucleotides in length. In some embodiments, the RNAi construct comprises at least one blunt end. In other embodiments, the RNAi construct comprises at least one nucleotide overhang. Such a nucleotide overhang may comprise at least 1 to 6 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.

[0009] 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-allyl-modified nucleotides, bicyclic nucleic acids (BNAs), glycol nucleic acids (GNAs), inverted bases (e.g., inverted adenosines), 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.

[0010] In some embodiments, the RNAi construct comprises at least one backbone modification, such as a modified internucleotide or internucleoside bond. In certain embodiments, the RNAi construct described herein comprises at least one phosphorothioate internucleotide bond. In certain embodiments, the phosphorothioate internucleotide bond can be located at the 3' or 5' end of the sense strand and / or the antisense strand.

[0011] In some embodiments, the antisense and / or sense strands of the RNAi constructs of the present invention can comprise or consist of sequences from the antisense and sense sequences listed in Table 1. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention is directed to compositions and methods for modulating expression of the zinc transporter 8 (SLC30A8 or ZnT8) gene. In some embodiments, the gene can be in a subject, such as a cell or a mammal (e.g., a human). In some embodiments, the compositions of the invention comprise an RNAi construct that targets SLC30A8 mRNA and reduces SLC30A8 expression in the cell or mammal. Such RNAi constructs are useful for treating or preventing various forms of disease, such as, for example, prediabetes or diabetes.

[0013] RNA interference (RNAi) is a process in which exogenous RNA is introduced into cells to cause the specific degradation of mRNA encoding a target protein, thereby reducing protein expression. Advances in both RNAi technology and delivery to the pancreas, as well as the increasing success of other RNAi-based therapies, suggest that RNAi is a compelling means of treating diabetes by directly targeting SLC30A8. The inhibitory effects of these sequences were confirmed by screening in CHO transfected cells.

[0014] 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., SLC30A8) through 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 through hydrogen bonding (e.g., Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding) between complementary bases in the 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.

[0015] In some embodiments, the present invention is an RNAi molecule that targets SLC30A8. In some embodiments, the present invention is an RNAi molecule that contains any of the sequences listed in Table 1.

[0016] Double-stranded RNA molecules can 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. All modifications used in double-stranded RNA molecules (e.g., siRNA, shRNA, etc.) are encompassed by the term "double-stranded RNA" for purposes of this disclosure.

[0017] 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%, 99%, or 100% 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 or target sequence by the total length of the first sequence.When two sequences hybridize, even if there are 5, 4, 3, 2, or 1 mismatches or less across a 30-base pair duplex region, the sequence can still be said to be substantially complementary to another sequence.Generally, when nucleotide overhangs, as defined herein, exist, the sequence of such overhangs is not taken into account when determining the degree of complementarity between two sequences.For example, a 21-nucleotide sense strand and a 21-nucleotide antisense strand hybridize to form a 19-base pair duplex region with two nucleotide overhangs at the 3' end of each strand, and are considered to be fully complementary as the term is used herein.

[0018] In some embodiments, the region of the antisense strand comprises a sequence that is completely complementary to a region of the target RNA sequence (e.g., SLC30A8 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, any mismatches preferably occur within the terminal regions (e.g., within 6, 5, 4, 3, 2, or 1 nucleotide of the 5' 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, 2, or 1 nucleotide of the 5' end of the antisense strand.

[0019] 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 that is formed from 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.

[0020] When the two substantially complementary strands of dsRNA are composed of separate RNA molecules, these molecules do not need to be covalently linked, but can be.When the two strands are covalently linked between the 3'-end of one strand and the 5'-end of the other strand that form a duplex structure by means other than an uninterrupted chain of nucleotides, this linking structure is called a "linker".The RNA strands can have the same number of nucleotides or different numbers of nucleotides.The maximum number of base pairs in a duplex is the number of nucleotides of the shortest strand of dsRNA minus any overhangs present in the duplex.In addition to the duplex structure, RNAi can also include one or more nucleotide overhangs.

[0021] In other embodiments, the sense and antisense strands that hybridize to form a duplex region can be part of a single RNA molecule. That is, the sense and antisense strands can be 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 flanking sequence of unpaired nucleotides, forming the loop region. The loop region is typically long enough to allow the RNA molecule to fold back on itself so that the antisense strand can base pair with the sense strand to form the duplex or stem region. The loop region can contain about 3 to about 25, about 5 to about 15, or about 8 to about 12 unpaired nucleotides. Such RNA molecules with at least a partially self-complementary region are referred to as "short hairpin RNAs" (shRNAs). In some embodiments, the loop region can contain at least 1, 2, 3, 4, 5, 10, 20, or 25 unpaired nucleotides. In some embodiments, the loop region can have 10, 9, 8, 7, 6, 5, 4, 3, 2, or fewer unpaired nucleotides. In certain embodiments, the RNAi construct of the present invention comprises an shRNA. The length of the single, at least partially self-complementary RNA molecule can be about 35 to about 100 nucleotides, about 45 to about 85 nucleotides, or about 50 to about 60 nucleotides, and can include a duplex region and a loop region, each having a length as recited herein.

[0022] 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 SLC30A8. As used herein, "SLC30A8 mRNA sequence" refers to any messenger RNA sequence, including splice variants, encoding an SLC30A8 protein, including variants or isoforms of the SLC30A8 protein from any species (e.g., mouse, rat, non-human primate, human).

[0023] The SLC30A8 mRNA sequence also includes a 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 an RNAi construct of the present invention may comprise a region having a sequence substantially or completely complementary to the target SLC30A8 mRNA sequence or SLC30A8 cDNA sequence. Examples of SLC30A8 mRNA or cDNA sequences include, but are not limited to, any SLC30A8 mRNA or cDNA sequence that can be derived from human SLC30A8 (NM_173851.3; NM_001172814.2; NM_001172811.2; NM_001172813.2; or NM_001172815.2) or mouse SLC30A8 (NM_172816.4).

[0024] The region of the antisense strand may be substantially or completely complementary to at least 15 contiguous nucleotides of the SLC30A8 mRNA sequence. In some embodiments, the target region of the SLC30A8 mRNA sequence to which the antisense strand comprises a region of complementarity 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 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 comprising a sequence substantially or completely complementary to the SLC30A8 mRNA sequence may, in some embodiments, comprise at least 15 contiguous nucleotides from an antisense sequence listed in Table 1. 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. In some embodiments, the sense and / or antisense sequences comprise at least 15 nucleotides from a sequence listed in Table 1 with no more than 1, 2, or 3 nucleotide mismatches.

[0025] 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 within two complementary or substantially complementary polynucleotides that base-pair with each other through Watson-Crick base pairing or other hydrogen-bonding interactions to create a duplex between the two polynucleotides. The duplex region of an RNAi construct must be of sufficient length to enable the RNAi construct to enter the RNA interference pathway, for example, by engaging the Dicer enzyme and / or the RISC complex. 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 one embodiment, the duplex region is about 17 to about 24 base pairs in length. In another embodiment, the duplex region is about 19 to about 21 base pairs in length.

[0026] In some embodiments, the RNAi agents of the present invention contain a duplex region of about 24 to about 30 nucleotides that interacts with a target RNA sequence, e.g., an SLC30A8 target mRNA sequence, to induce cleavage of the target RNA. Without wishing to be bound by theory, long double-stranded RNA introduced into cells can be degraded into siRNAs by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a RNase III-like enzyme, processes dsRNA into short interfering RNAs of 19 to 23 base pairs with characteristic two-base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). Then, siRNA is incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to induce target recognition (Nykanen, et al., (2001) Cell 107:309). When it binds to the appropriate target mRNA, one or more endonucleases in RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev.15:188).

[0027] 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 can be longer than the duplex region and can have one or more unpaired nucleotides or mismatches flanking the duplex region. Thus, in some embodiments, the RNAi construct comprises at least one nucleotide overhang. As used herein, "nucleotide overhang" refers to one or more unpaired nucleotides that extend beyond the duplex region at the end of a strand. 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, the nucleotide overhang comprises 1, 2, 3, 4, 5, or 6 nucleotides. In a particular embodiment, the nucleotide overhang comprises 1 to 4 nucleotides. In certain embodiments, the nucleotide overhang comprises 2 nucleotides. The nucleotides in the overhang can be ribonucleotides, deoxyribonucleotides, or modified nucleotides as described herein. In some 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, such as a 2'-modified nucleotide. In other embodiments, the overhang comprises a 5'-deoxythymidine-deoxythymidine-3' (5'-dTdT-3') dinucleotide.

[0028] The nucleotide overhangs can be 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.

[0029] 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. "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).

[0030] The sense strand and the antisense strand can each independently be about 15 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 21 to about 25 nucleotides in length, or about 21 to about 23 nucleotides in length. In certain embodiments, the sense strand and the antisense strand are each 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 the antisense strand are the same length, but form a duplex region that is shorter than the strands, such that the RNAi construct has two nucleotide overhangs. For example, in one embodiment, the RNAi construct comprises (i) a sense strand and an antisense strand that are each 21 nucleotides in length, (ii) a duplex region that is 19 base pairs in length, and (iii) nucleotide overhangs 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) nucleotide overhangs 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) a sense strand and an antisense strand, each 23 nucleotides in length, and (ii) a duplex region 23 base pairs in length.

[0031] In other embodiments, the sense strand or antisense strand is longer than the other strand, so that the RNAi construct comprises at least one nucleotide overhang, and the two strands form a duplex region having a length equal to that of the shorter strand. For example, in one embodiment, the RNAi construct comprises (i) a sense strand that is 19 nucleotides long, (ii) an antisense strand that is 21 nucleotides long, (iii) a duplex region that is 19 base pairs long, and (iv) a single nucleotide overhang of two unpaired nucleotides at the 3' end of the antisense strand. In another embodiment, the RNAi construct comprises (i) a sense strand that is 21 nucleotides long, (ii) an antisense strand that is 23 nucleotides long, (iii) a duplex region that is 21 base pairs long, and (iv) a single nucleotide overhang of two unpaired nucleotides at the 3' end of the antisense strand.

[0032] The antisense strand of an RNAi construct of the present invention can comprise the sequence of any one of the antisense sequences listed in Table 1, or the sequence of nucleotides 1-19 or 1-21 of any of these antisense sequences.

[0033] Modified Nucleotides 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, and deoxyribonucleotides containing deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxythymidine monophosphate, and deoxycytidine monophosphate. However, the RNAi construct may contain a combination of modified nucleotides, ribonucleotides, and deoxyribonucleotides. 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.

[0034] 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. 2'-modified nucleotides refer to nucleotides having a pentose ring with a substituent at the 2' position other than H or OH. Such 2' modifications include, but are not limited to, 2'-O-alkyl (e.g., O-C1-C10 or O-C1-C10 substituted alkyl), 2'-O-allyl (O-CH2CH=CH2), 2'-C-allyl, 2'-fluoro, 2'-O-methyl (OCH3), 2'-O-methoxyethyl (O-(CH2)2OCH3), 2'-OCF3, 2'-O(CH2)2SCH3, 2'-O-aminoalkyl, 2'-amino (e.g., NH2), 2'-O-ethylamine, and 2'-azido. 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.

[0035] "Bicyclic sugar modification" refers to a modification of the pentose ring in which a bridge connects 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 called 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') methylene-thio (4'-CH-S-2') BNA; methylene-amino (4'-CH-N(R)-2') BNA; methyl carbocyclic (4'-CH-CH(CH)-2') BNA; propylene carbocyclic (4'-(CH)-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.

[0036] 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-allyl modified nucleotides, bicyclic nucleic acids (BNAs), 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.

[0037] 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.

[0038] In some embodiments, all pyrimidine nucleotides preceding an adenosine nucleotide in the sense strand, the antisense strand, or both strands are modified nucleotides. For example, when the sequence 5'-CA-3' or 5'-UA-3' appears in either strand, the cytidine and uridine nucleotides are modified nucleotides, preferably 2'-O-methyl modified nucleotides. In certain embodiments, all pyrimidine nucleotides in the sense strand are modified nucleotides (e.g., 2'-O-methyl modified nucleotides), and all 5' nucleotides of the sequence 5'-CA-3' or 5'-UA-3' present in the antisense strand are modified nucleotides (e.g., 2'-O-methyl modified nucleotides). In other embodiments, all nucleotides in the duplex region are modified nucleotides. In such embodiments, the modified nucleotides are preferably 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, or a combination thereof.

[0039] In embodiments in which the RNAi construct comprises a nucleotide overhang, the nucleotides in the overhang can be ribonucleotides, deoxyribonucleotides, or modified nucleotides. In one embodiment, the nucleotides in the overhang are deoxyribonucleotides, such as deoxythymidine. In another embodiment, the nucleotides in the overhang are modified nucleotides. For example, in some embodiments, the nucleotides in the overhang are 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-methoxyethyl modified nucleotides, or combinations thereof.

[0040] 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 the native 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, such as those 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. No. 6,693,187, 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.

[0041] In certain embodiments, an RNAi construct 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. An RNAi construct may comprise one or more phosphorothioate internucleotide linkages at the 3'-end, the 5'-end, or both the 3'-end and the 5'-end of the sense strand, the antisense strand, or both strands. For example, in certain embodiments, an RNAi construct comprises about one to about six 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 about one to about six 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. In one embodiment, an RNAi construct comprises a single phosphorothioate internucleotide linkage at the 3'-end of the sense strand and a single phosphorothioate internucleotide linkage at the 3'-end of the antisense strand. In another embodiment, an RNAi construct comprises two consecutive phosphorothioate internucleotide linkages 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 at both the 3' and 5' ends of the antisense strand.In yet another embodiment, the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages 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 at both the 3'- and 5'-ends of the antisense strand and two consecutive phosphorothioate internucleotide linkages 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 any of the embodiments in which one or both strands comprise one or more phosphorothioate internucleotide linkages, the remaining internucleotide linkages in the strands may be native 3'-5' phosphodiester linkages. For example, in some embodiments, each internucleotide linkage in the sense strand and the antisense strand is selected from phosphodiester and phosphorothioate, and at least one internucleotide linkage is phosphorothioate.

[0042] In embodiments where 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 3'-end nucleotide overhang 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 5'-end nucleotide overhang 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.

[0043] 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 "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 can be synthetic or naturally occurring modifications, such as the 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- These include, but are not limited to, azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thio, 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.

[0044] In some embodiments, the modified base is a universal base. "Universal base" refers to a base analog that indiscriminately forms base pairs with all of the naturally occurring bases in RNA and DNA without changing the resulting double-stranded region's double helix structure. 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.

[0045] 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.Chern., Vol.76:7295-7300, 2011, both of which are incorporated herein by reference in their entirety.Those skilled in the art will appreciate that guanine, cytosine, adenine, thymine and uracil can be substituted with other nucleobases, such as the modified nucleobases described above, without substantially changing the base pairing properties of polynucleotides containing nucleotides having such substituted nucleobases.

[0046] 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'-alpha-thiotriphosphate; 5'-gamma-thiotriphosphate; 5'-phosphoramidate; 5'-vinylphosphate; 5'-alkylphosphonates (e.g., alkyl = methyl, ethyl, isopropyl, propyl, etc.); and 5'-alkyl ether phosphonates (e.g., alkyl ether = methoxymethyl, ethoxymethyl, etc.).

[0047] Modified nucleotides that can be incorporated into RNAi constructs of the present invention can have multiple 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'-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, which results 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 a sugar modification, 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 strands of an RNAi construct of the present invention include a combination of 2'-modified nucleotides or BNAs and phosphorothioate internucleotide linkages. In certain embodiments, both the sense and antisense strands of the RNAi constructs of the present invention comprise a combination of 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, and phosphorothioate internucleotide linkages. Exemplary RNAi constructs comprising modified nucleotides and modified internucleotide linkages are shown in Table 2.

[0048] Function of RNAi constructs Preferably, the RNAi constructs of the present invention reduce or inhibit SLC30A8 expression in cells, particularly pancreatic islet beta cells. Thus, in one embodiment, the present invention provides a method for reducing SLC30A8 expression in cells by contacting the cells with any of the RNAi constructs described herein. The cells may be in vitro or in vivo. SLC30A8 expression can be assessed by measuring the amount or level of SLC30A8 mRNA, SLC30A8 protein, or another biomarker associated with SLC30A8 expression. The reduction in SLC30A8 expression in cells or animals treated with the RNAi constructs of the present invention can be determined compared to SLC30A8 expression in cells or animals not treated with the RNAi construct or treated with a control RNAi construct. For example, in some embodiments, reduced SLC30A8 expression is assessed by (a) measuring the amount or level of SLC30A8 mRNA in pancreatic cells treated with an RNAi construct of the present invention, (b) measuring the amount or level of SLC30A8 mRNA in pancreatic cells treated with a control RNAi construct (e.g., an RNAi agent targeting an RNA molecule not expressed in pancreatic cells or an RNAi construct having a nonsense or scrambled sequence) or not treated with the construct, and (c) comparing the SLC30A8 mRNA level measured from the treated cells in (a) with the SLC30A8 mRNA level measured from the control cells in (b). Prior to comparison, the SLC30A8 mRNA levels in the treated and control cells may be normalized to the RNA level of a control gene (e.g., 18S ribosomal RNA). SLC30A8 mRNA levels can be measured by a variety of methods, including Northern blot analysis, nuclease protection assay, fluorescent in situ hybridization (FISH), reverse transcriptase (RT)-PCR, real-time RT-PCR, quantitative PCR, and the like.

[0049] In another embodiment, the reduction of SLC30A8 expression is evaluated by (a) measuring the amount or level of SLC30A8 protein in pancreatic cells treated with an RNAi construct of the present invention, (b) measuring the amount or level of SLC30A8 protein in pancreatic cells treated with a control RNAi construct (e.g., an RNAi agent targeting an RNA molecule not expressed in pancreatic cells or an RNAi construct having a nonsense or scrambled sequence) or not treated with the construct, and (c) comparing the SLC30A8 protein level measured in the treated cells in (a) with the SLC30A8 protein level measured in the control cells in (b). Methods for measuring SLC30A8 protein level are known to those skilled in the art and include, for example, Western blot, immunoassay (e.g., ELISA), and flow cytometry. The effectiveness of the RNAi construct of the present invention can be evaluated using any method capable of measuring SLC30A8 mRNA or protein.

[0050] In some embodiments, the method for assessing the expression level of SLC30A8 is performed in vitro in cells that naturally express SLC30A8 (e.g., pancreatic cells) or cells engineered to express SLC30A8. In certain embodiments, the method is performed in vitro in pancreatic cells.

[0051] In another embodiment, the method for assessing the expression level of SLC30A8 is performed in vivo. The RNAi construct and any control RNAi construct can be administered to an animal (e.g., a rodent or non-human primate), and the mRNA or protein level of SLC30A8 can be assessed in pancreatic tissue harvested from the treated animal. Alternatively or additionally, biomarkers or functional phenotypes associated with SLC30A8 expression can be assessed in the treated animal.

[0052] In certain embodiments, the expression of SLC30A8 is reduced in pancreatic cells by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% by the RNAi construct of the present invention. In some embodiments, the expression of SLC30A8 is reduced in pancreatic cells by at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% by the RNAi construct of the present invention. In other embodiments, the expression of SLC30A8 is reduced in pancreatic cells by about 90% or more, for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more by the RNAi construct of the present invention. The percent reduction in SLC30A8 expression can be measured by any of the methods described herein and other methods known in the art.

[0053] In some embodiments, the efficacy of an RNAi construct of the present invention in inhibiting SLC30A8 expression in pancreatic cells is evaluated by calculating an IC50 value. An "IC50 value" is the dose / concentration required to achieve 50% inhibition of biological or biochemical function. The IC50 value of any particular substance or antagonist can be determined by constructing a dose-response curve and testing the effect of various concentrations of the substance or antagonist on expression levels or functional activity in any assay. The IC50 value of a given antagonist or substance can be calculated by determining the concentration required to inhibit half of the maximum biological response or native expression level. Thus, the IC50 value of any RNAi construct can be calculated by determining the concentration of the RNAi construct required to inhibit half of the native SLC30A8 expression level in pancreatic cells (e.g., the expression level of SLC30A8 in CHO-transfected cells or control pancreatic cells) in any assay, such as the immunoassay, RNA FISH assay, or droplet digital PCR assay described in the Examples. The RNAi constructs of the present invention can inhibit SLC30A8 expression in pancreatic cells with an IC50 of less than about 100 nM. For example, the RNAi constructs inhibit SLC30A8 expression in pancreatic cells with an IC50 of about 0.001 nM to about 100 nM, about 0.001 nM to about 20 nM, about 0.001 nM to about 10 nM, about 0.001 nM to about 5 nM, about 0.001 nM to about 1 nM, about 0.1 nM to about 10 nM, about 0.1 nM to about 5 nM, or about 0.1 nM to about 1 nM. In a specific embodiment, the RNAi constructs inhibit SLC30A8 expression in CHO transfected cells with an IC50 of about 1 nM to about 10 nM. In certain embodiments, the RNAi construct inhibits SLC30A8 expression in CHO transfected cells with an IC50 of about 0.1 nM to about 5 nM.

[0054] 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 a suitable nucleic acid synthesizer that utilizes standard nucleotide or nucleoside precursors (e.g., phosphoramidites). 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).

[0055] Oligonucleotides can be synthesized using phosphoramidite chemistry, using a 2' silyl protecting group with an 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 any automated or manual synthesizer, at large, medium, or small scales. Synthesis can also be performed in multiwell plates, columns, or glass slides.

[0056] 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, crown ether catalysts can be used in combination with inorganic fluorides. Preferred fluoride ion sources are tetrabutylammonium fluoride or amine hydrofluorides (for example, aqueous HF combined with triethylamine in a dipolar aprotic solvent, such as dimethylformamide).

[0057] The choice of protecting groups for use on the phosphite triesters and phosphotriesters can alter the stability of the triesters to fluoride. Methyl protection of the phosphotriester or phosphite triester can stabilize the bond to fluoride ions and improve process yields.

[0058] Because ribonucleosides have a reactive 2' hydroxyl substituent, it may be desirable to protect the reactive 2' position in the RNA with a protecting group that is orthogonal to the 5'-O-dimethoxytrityl protecting group, e.g., one that is stable to treatment with acid. Silyl protecting groups meet this requirement and can be easily removed in a final fluoride deprotection step, thereby minimizing RNA degradation.

[0059] Tetrazole catalysts can be used in standard phosphoramidite coupling reactions. Preferred catalysts include, for example, tetrazole, S-ethyl-tetrazole, benzylthiotetrazole, and p-nitrophenyltetrazole.

[0060] As can be understood by those skilled in the art, additional methods for synthesizing the RNAi constructs described herein will be apparent to those skilled in the art. In addition, various synthetic steps can be performed in an alternative sequence or order to obtain 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 & Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley & Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley & 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).

[0061] The RNAi construct of the present invention may contain 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 between a ligand and another compound or molecule may induce 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.

[0062] The ligand may comprise a serum protein (e.g., human serum albumin, low density lipoprotein, globulin), a cholesterol moiety, a vitamin (biotin, vitamin E, vitamin B12), 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 a pancreatic cell). Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinkers (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecyl). Examples of suitable oleic acid derivatives include glycerol, borneol, menthol, 1,3-propanediol, heptadecyl groups, 03-(oleoyl)lithocholic acid, 03-(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).

[0063] 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. The "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 their derivatives (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.

[0064] 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 a folate moiety can be taken up by cells via receptor-mediated endocytosis. Such folate-polynucleotide conjugates are described in U.S. Pat. No. 8,188,247, which is incorporated herein by reference in its entirety.

[0065] Given that SLC30A8 is expressed in pancreatic cells, in certain embodiments, it is desirable to specifically deliver an RNAi construct to those pancreatic cells. In some embodiments, the RNAi construct can be specifically targeted to the pancreas, particularly pancreatic islet beta cells, by using a ligand that binds to or interacts with a protein expressed on the surface of pancreatic cells. For example, in certain embodiments, the ligand can include 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 pancreatic cells.

[0066] The ligand can be directly or indirectly bound or conjugated to the RNA molecule of the RNAi construct. For example, in some embodiments, the ligand is covalently bound directly 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 can 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 occur at any position. In some embodiments, the 2-, 5-, and 6-positions of the pyrimidine nucleobase can 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. In abasic residues, the 1' position can also be attached to a ligand. Internucleotide linkages can also facilitate ligand attachment. In 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 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.

[0067] In certain embodiments, the ligand can be attached to the 3' or 5' end of the sense strand or the antisense strand. In certain embodiments, the ligand is covalently attached to the 5' end of the sense strand. 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 alternative embodiments, the ligand is attached near the 3' end of the sense strand but before one or more terminal nucleotides (i.e., before one, two, three, or four 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.

[0068] In certain embodiments, the ligand is attached to the sense strand 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 the 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 strand or antisense strand of the 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 unsaturated (e.g., double or triple bonds).

[0069] Linkers that can be used to attach a ligand to the sense or antisense strand in an RNAi construct of the invention include, but are not limited to, pyrrolidine, 8-amino-3,6-dioxaoctanoic acid, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, 6-aminohexanoic acid, substituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, or substituted or unsubstituted C2-C10 alkynyl. 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.

[0070] In certain embodiments, the linker is cleavable. A cleavable linker is one that is sufficiently stable outside the cell but is cleaved upon entry into the target cell to release the two moieties that the linker holds together. 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 (which, for example, can be selected to mimic or represent intracellular conditions) than in the subject's blood or under a second reference condition (which, for example, can be selected to mimic or represent conditions found in blood or serum).

[0071] Cleavable linkers are sensitive to cleaving agents, such as pH, redox potential, or the presence of degradative 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 degradative agents include redox agents that are selective for a specific substrate or lack substrate specificity (e.g., oxidases or reductases or reducing agents, such as mercaptans, that are present inside cells and can degrade redox-cleavable linkers by reduction); esterases; agents that can create endosomes or acidic environments, such as those that result in 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.

[0072] 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 cell interior or a desired compartment of the cell.

[0073] The linker may include a cleavable group that is cleavable by a specific enzyme. The type of cleavable group incorporated into the linker may depend on the cell to be targeted.

[0074] In general, the suitability of a candidate cleavable linker can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linker. It may also be desirable to test candidate cleavable linkers for their ability to resist cleavage when in blood or other non-target tissues. Thus, the relative susceptibility to cleavage can be determined between a first condition selected to be indicative of cleavage in target cells and a second condition selected to be indicative of cleavage in other tissues or biological fluids, such as blood or serum. Evaluation can be performed in a cell-free system, cells, cell cultures, organs, 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 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).

[0075] In other embodiments, a redox-cleavable linker is utilized. 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 incubation with dithiothreitol (DTT) or other reducing agents known in the art that mimic the cleavage rate that would be 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 some embodiments, useful linker candidates are degraded 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) compared to blood (or under in vitro conditions selected to mimic extracellular conditions).

[0076] In yet other embodiments, the phosphate-based cleavable linker is cleaved by an agent that degrades or hydrolyzes the phosphate group. An example of an agent that hydrolyzes phosphate groups within a cell is an enzyme such as an intracellular phosphatase. Examples of phosphate-based cleavable groups include -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-. 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-, -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.

[0077] In other embodiments, the linker may comprise 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 lower) or by an agent, such as an enzyme, that can act as a general acid. Within cells, certain low-pH organelles, such as endosomes and lysosomes, may 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 of the ester (alkoxy group) 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.

[0078] In other embodiments, the linker may contain an ester-based cleavable group, which is cleaved by enzymes such as esterases and amidases in cells. 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.

[0079] In further embodiments, the linker may comprise a peptide-based cleavable group, which is cleaved by enzymes such as intracellular peptidases and proteases. Peptide-based cleavable groups are peptide bonds formed between amino acids to give rise to oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include amide groups (—C(O)NH—). Amide groups can 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. Peptide-based cleavable groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to give rise to peptides and proteins, but do not include the entire amide functionality. Peptide-based cleavable linking groups have the general formula —NHCHRAC(O)NHCHRBC(O)—, where R and R are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0080] Other types of linkers suitable for attaching a ligand to the sense or antisense strand in the RNAi constructs of the present invention are known in the art and may 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.

[0081] In some embodiments, the RNAi constructs of the present invention can be delivered to cells or tissues of interest by administering a vector that encodes and controls the intracellular expression of the RNAi construct. A "vector" (also referred to herein as an "expression vector") is a composition of matter that can be used to deliver a nucleic acid of interest into a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides bound to ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, and retroviral vectors. A vector can replicate within a living cell, or it can be synthetically produced.

[0082] Generally, vectors for expressing the RNAi constructs of the present invention contain one or more promoters operably linked to a sequence encoding the RNAi construct. As used herein, the phrases "operably linked" or "under transcriptional control" mean that the promoter is in the correct position and orientation relative to a polynucleotide sequence to control initiation of transcription by RNA polymerase and expression of the polynucleotide sequence. A "promoter" refers to a sequence recognized by the cell's synthetic machinery or introduced synthetic machinery and required to initiate specific transcription of a gene sequence. Suitable promoters include, but are not limited to, RNA pol I, pol II, HI, or U6 RNA pol III, and viral promoters (e.g., the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, and the Rous sarcoma virus long terminal repeat). In some embodiments, the HI or U6 RNA pol III promoter is preferred. The promoter can be a tissue-specific or inducible promoter. Of particular interest is a pancreas-specific promoter.

[0083] In some embodiments in which the RNAi construct comprises an siRNA, the two separate strands (sense and antisense strands) can be expressed from a single vector or from two separate vectors. For example, in one embodiment, the sequence encoding the sense strand is operably linked to a promoter on a first vector, and the sequence encoding the antisense strand is operably linked to a promoter on a second vector. In such embodiments, the first and second vectors are simultaneously introduced into a target cell, for example, by infection or transfection, so that the sense and antisense strands are transcribed and hybridize to form an siRNA molecule in the cell. In another embodiment, the sense and antisense strands are transcribed from two separate promoters located in a single vector. In some such embodiments, the sequence encoding the sense strand is operably linked to a first promoter, and the sequence encoding the antisense strand is operably linked to a second promoter, and the first and second promoters are located in a single vector. In one embodiment, the vector comprises a first promoter operably linked to a sequence encoding an siRNA molecule and a second promoter operably linked in the opposite direction to the same sequence, such that transcription of the sequence from the first promoter results in synthesis of the sense strand of the siRNA molecule, and transcription of the sequence from the second promoter results in synthesis of the antisense strand of the siRNA molecule.

[0084] In other embodiments, where the RNAi construct comprises an shRNA, a sequence encoding a single, at least partially self-complementary RNA molecule is operably linked to a promoter that generates a single transcript. In some embodiments, the sequence encoding the shRNA comprises inverted repeats connected by a linker polynucleotide sequence to generate the stem and loop structure of the shRNA after transcription.

[0085] In some embodiments, the vector encoding the RNAi construct of the present invention is a viral vector.Various viral vector systems suitable for expressing the RNAi construct described herein include, but are not limited to, adenovirus vector, retrovirus vector (for example, lentivirus vector, Moloney murine leukemia virus), adeno-associated virus vector; herpes simplex virus vector; SV40 vector; polyomavirus vector; papillomavirus vector; picornavirus vector; and poxvirus vector (for example, vaccinia virus).In certain embodiments, the viral vector is a retrovirus vector (for example, lentivirus vector).

[0086] Various vectors suitable for use in the present invention, methods for inserting nucleic acid sequences encoding siRNA or shRNA molecules into the vector, and methods for delivering the vector to target cells are within the skill of one of ordinary skill in the art. For example, Dornburg, Gene Therap.,Vol.2:301-310,1995;Eglitis,Biotechniques,Vol.6:608-614,1988;Miller,HumGene Therap.,Vol.1:5-14,1990;Anderson,Nature,Vol.392:25-30,1998;Rubinson DA et al.,Nat. Genet.,Vol.33:401-406,2003;Brummelkamp et al.,Science,Vol.296:550-553,2002;Brummelkamp et al.,Cancer Cell,Vol.2:243-247,2002;Lee et al.,Nat Biotechnol,Vol.20:500-505,2002;Miyagishi et See, e.g., S. et al., Nat Biotechnol, Vol. 20:497-500, 2002; Paddison et al., GenesDev, Vol. 16:948-958, 2002; Paul et al., Nat Biotechnol, Vol. 20:505-508, 2002; Sui et al., Proc Natl Acad Sci USA, Vol. 99:5515-5520, 2002; and Yu et al., Proc Natl Acad Sci USA, Vol. 99:6047-6052, 2002, all of which are incorporated herein by reference in their entireties.

[0087] 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 SLC30A8 in subjects in need thereof. For clinical use, pharmaceutical compositions and formulations are prepared in a form suitable for the intended use. Generally, this will involve preparing compositions that are substantially free of pyrogens and other impurities that may be harmful to humans or animals.

[0088] 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 a drug, such as a drug suitable for human administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional media or agent is contemplated for use in therapeutic compositions, except insofar as it is incompatible with the RNAi construct of the present invention. Supplementary active ingredients may also be incorporated into the compositions, provided that they do not inactivate the vector or RNAi construct of the composition.

[0089] The compositions and methods for formulating pharmaceutical compositions depend on several criteria, including, but not limited to, the route of administration, the type and extent of the disease or disorder to be treated, or the dose to be administered. In some embodiments, the pharmaceutical composition is formulated based on the intended route of delivery. 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 an embodiment, the pharmaceutical composition may include a lipid-based delivery vehicle. In another embodiment, the pharmaceutical composition is formulated for subcutaneous delivery. In such an embodiment, the pharmaceutical composition may include a targeting ligand.

[0090] 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 SLC30A8 expression in pancreatic cells of a subject.

[0091] An effective amount of an RNAi construct of the present invention can be about 0.01 mg / kg to about 100 mg / kg body weight, about 0.05 mg / kg to about 75 mg / kg body weight, about 0.1 mg / kg to about 50 mg / kg body weight, about 1 mg / kg to about 30 mg / kg body weight, about 2.5 mg / kg to about 20 mg / kg body weight, or about 5 mg / kg to about 15 mg / kg body weight. In certain embodiments, an effective single dose of an RNAi construct of the present invention can be about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, or about 10 mg / kg. A pharmaceutical composition comprising an effective amount of an RNAi construct can be administered weekly, biweekly, monthly, quarterly, or semi-annually. The precise determination of what will be considered an effective dosage and frequency of administration can be based on several factors, including the size, age, and general condition of the patient, the type of disorder being treated (e.g., myocardial infarction, heart failure, coronary artery disease, hypercholesterolemia), the particular RNAi construct used, and the route of administration. Estimates of effective dosages and in vivo half-lives for any particular RNAi construct of the invention can be confirmed using conventional methods and / or testing in appropriate animal models.

[0092] 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. In some embodiments, the pharmaceutical compositions are administered parenterally. For example, in certain embodiments, the pharmaceutical compositions are administered intravenously. In other embodiments, the pharmaceutical compositions are administered subcutaneously.

[0093] Colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes, can be used as delivery vehicles for the RNAi constructs of the present invention or vectors encoding such constructs. Commercially available fat emulsions suitable for delivering nucleic acids of the present invention include Intralipid®, Liposyn®, Liposyn® II, Liposyn® III, Nutrilipid, and other similar fat 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 can be encapsulated within liposomes or complexed with liposomes, particularly cationic liposomes. Alternatively, the RNAi constructs of the present invention can be complexed with lipids, particularly cationic lipids. 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.

[0094] In some embodiments, the RNAi constructs of the present invention are fully encapsulated within a lipid formulation to form, for example, SPLPs, pSPLPs, SNALPs, or other nucleic acid-lipid particles. As used herein, the term "SNALP" refers to stable nucleic acid-lipid particles, including SPLPs. As used herein, the term "SPLP" refers to nucleic acid-lipid particles containing plasmid DNA encapsulated in lipid vesicles. SNALPs and SPLPs typically contain cationic lipids, non-cationic lipids, and lipids that prevent particle aggregation (e.g., PEG-lipid conjugates). SNALPs and SPLPs exhibit long circulatory lifetimes after intravenous injection and accumulate at distal sites (e.g., sites physically distant from the administration site), making them highly useful for systemic administration. SPLPs include "pSPLPs," which contain an encapsulated condensing agent-nucleic acid complex, as described in WO 00 / 03683. 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 non-toxic. Additionally, the nucleic acid present in the nucleic acid-lipid particles is 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.

[0095] Examples of 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, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action 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, such as sugars or sodium chloride. Prolonged absorption of injectable compositions can be achieved by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0096] Sterile injectable solutions 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, dispersions are prepared by adding various sterilized active ingredients to a sterile vehicle containing a basic dispersion medium and other desired ingredients, for example, as listed above.For sterile powders for preparing sterile injectable solutions, preferred preparation methods include vacuum drying and freeze-drying techniques, which produce powders of active ingredients and additional desired ingredients from the solution that has previously been sterile-filtered.

[0097] The compositions of the present invention can generally be formulated in neutral or salt form. Examples of pharmaceutically acceptable salts include 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 hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide) or organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine, etc.).

[0098] For parenteral administration in an aqueous solution, for example, the solution is usually suitably buffered and the liquid diluent first rendered isotonic, for example, with sufficient saline or glucose. Such aqueous solutions can be used, for example, for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. Particularly in light of the present disclosure, the use of sterile aqueous media, as known to those skilled in the art, is preferred. For example, a single dose can be dissolved in 1 ml of isotonic NaCl solution, added to 1000 ml of subcutaneous infusion fluid, and injected at the proposed injection site (see, e.g., "Remington's Pharmaceutical Sciences," 15th Edition, pages 1035-1038 and 1570-1580). For human administration, preparations should 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).

[0099] In some embodiments, the pharmaceutical composition of the present invention is packaged in or stored in an administration device.Devices for injection preparations include, but are not limited to, injection ports, pre-filled syringes, automatic injectors, injection pumps, wearable injectors, and injection pens.Devices for aerosolized preparations or powder preparations include, but are not limited to, inhalers, inhalers, inhalers, etc.Therefore, the present invention includes an administration device that comprises the pharmaceutical composition of the present invention for treating or preventing one or more of the disorders described herein.

[0100] Methods for inhibiting SLC30A8 expression The present invention also provides a method for inhibiting the expression of the SLC30A8 gene in a cell. The method includes inhibiting the expression of SLC30A8 in a cell by contacting the cell with an RNAi agent, e.g., a double-stranded RNAi agent, in an amount effective to inhibit the expression of SLC30A8 in the cell. Contacting the cell with the RNAi agent, e.g., a double-stranded RNAi agent, can be carried out in vitro or in vivo. Contacting the cell with the RNAi agent in vivo includes contacting a cell or a group of cells in a subject, e.g., a human subject, with the RNAi agent. A combination of in vitro and in vivo methods of contacting a cell is also possible.

[0101] The present invention provides methods for reducing or inhibiting the expression of SLC30A8 in a subject in need thereof, and methods for treating or preventing a pathology, disease, or disorder associated with SLC30A8 expression or activity. "Pathology, disease, or disorder associated with SLC30A8 expression" refers to a pathology, disease, or disorder in which an altered expression level of SLC30A8 or an increased expression level of SLC30A8 is associated with an increased risk of developing the pathology, disease, or disorder.

[0102] Contacting of cells can be direct or indirect, as described above. Additionally, contacting of cells can be via a targeting ligand, including ligands described herein or known in the art.

[0103] In one embodiment, contacting a cell with an RNAi includes "introducing" or "delivering RNAi to a cell" by facilitating or causing cellular uptake or absorption. Absorption or uptake of RNAi can occur by unassisted diffusive or active cellular processes or by auxiliary agents or devices. Introduction of RNAi into cells can be in vitro and / or in vivo. For example, for in vivo introduction, RNAi can be injected into a tissue site or administered systemically. In vitro introduction into cells includes methods known in the art, such as electroporation and lipofection. Additional methods are described herein below and / or known in the art.

[0104] As used herein, the term "inhibit" is used interchangeably with "reduce," "silencing," "downregulate," "suppress," and other similar terms, and includes all levels of inhibition.

[0105] The phrase "inhibiting the expression of SLC30A8" refers to the inhibition of expression of any SLC30A8 gene (such as a mouse SLC30A8 gene, rat SLC30A8 gene, monkey SLC30A8 gene, or human SLC30A8 gene) and variants or mutants of the SLC30A8 gene. Thus, the SLC30A8 gene may be a wild-type SLC30A8 gene, a mutant SLC30A8 gene (such as a mutant SLC30A8 gene that causes amyloid deposition), or a transgenic SLC30A8 gene in relation to a genetically engineered cell, cell group, or organism.

[0106] "Inhibiting the expression of the SLC30A8 gene" includes inhibition of any level of the SLC30A8 gene, for example, at least partial suppression of SLC30A8 gene expression. SLC30A8 gene expression can be assessed based on the level or change in the level of any variable associated with SLC30A8 gene expression, such as the SLC30A8 mRNA level, the SLC30A8 protein level, or the number or extent of amyloid deposits. This level can be assessed, for example, in individual cells or a group of cells comprising a sample derived from a subject.

[0107] Inhibition can be assessed by a decrease in the absolute or relative level of one or more variables associated with SLC30A8 expression compared to a control level, which can be any type of control level utilized in the art, such as a baseline level before administration or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (e.g., a buffer-only control or an inactive drug control). In some embodiments of the methods of the present invention, expression of the SLC30A8 gene is inhibited by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0108] Inhibition of SLC30A8 gene expression can be demonstrated by a decrease in the amount of mRNA expressed by a first cell or group of cells (such cells may be present, for example, in a sample derived from a subject) in which the SLC30A8 gene is transcribed and which have been treated (e.g., by contacting the cells with an RNAi agent of the present invention or by administering an RNAi agent of the present invention to a subject in which the cells are or were present) so that expression of the SLC30A8 gene is inhibited, compared to a second cell or group of cells (control cells) that are substantially identical to the first cell or group of cells but have not been similarly treated. In a preferred embodiment, inhibition is assessed by expressing the mRNA level in treated cells as a percentage of the mRNA level in subject cells using the following formula:

number

[0109] Alternatively, inhibition of SLC30A8 gene expression can be assessed in terms of a parameter functionally related to SLC30A8 gene expression, such as a reduction in SLC30A8 protein expression. SLC30A8 gene silencing can be determined by any assay known in the art in any cell that constitutively or by genome engineering expresses SLC30A8.

[0110] Inhibition of SLC30A8 protein expression can be evidenced by a decrease in the level of SLC30A8 protein expressed by a cell or group of cells (e.g., the level of the protein expressed in a sample derived from a subject). As explained above, for assessment of mRNA suppression, inhibition of protein expression levels in treated cells or group of cells can similarly be expressed as a percentage of the level of protein in control cells or group of cells.

[0111] Control cells or cell groups that can be used to evaluate the inhibition of SLC30A8 gene expression include cells or cell groups that have not yet been contacted with the RNAi agent of the present invention.For example, control cells or cell groups can be obtained from an individual subject (e.g., a human or animal subject) before the subject is treated with the RNAi agent.

[0112] The level of SLC30A8 mRNA expressed by a cell or group of cells or the level of circulating SLC30A8 mRNA can be determined using any method known in the art for assessing mRNA expression. In one embodiment, the expression level of SLC30A8 in a sample is determined by detecting a transcribed polynucleotide or a portion thereof, such as the mRNA of the SLC30A8 gene. RNA can be extracted from cells using RNA extraction techniques, including, for example, acid phenol / guanidine isothiocyanate extraction (RNAzol B; Biogenesis), the RNeasy RNA preparation kit (Qiagen), or PAXgene (PreAnalytix, Switzerland). Typical assay formats utilizing ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays (Melton et al., Nuc. Acids Res. 12:7035), Northern blotting, in situ hybridization, and microarray analysis. Circulating SLC30A8 mRNA can be detected using the methods described in PCT / US2012 / 043584, the entire contents of which are incorporated herein by reference.

[0113] In one embodiment, the level of SLC30A8 expression is determined using a nucleic acid probe. The term "probe" as used herein refers to any molecule that can selectively bind to a specific SLC30A8. Probes can be synthesized by those skilled in the art or derived from appropriate biological preparations. Probes can be specifically designed to be labeled. Examples of molecules that can be used as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0114] The isolated mRNA can be used in hybridization or amplification assays, including, but not limited to, Southern or Northern analysis, polymerase chain reaction (PCR) analysis, and probe arrays. One method for determining mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to SLC30A8 mRNA. In one embodiment, the mRNA is immobilized on a solid surface and contacted with the probe, for example, by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane such as nitrocellulose. In an alternative embodiment, the probe is immobilized on a solid surface and the mRNA is contacted with the probe, for example, in an Affymetrix gene chip array. Those skilled in the art can easily adapt known mRNA detection methods for use in determining SLC30A8 mRNA levels.

[0115] Alternative methods for determining the level of expression of SLC30A8 in a sample include, for example, RT-PCR (experimental embodiment described in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self-sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcription amplification systems (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-beta replicase (Lizardi et al. (1988) Bio / Technology 6:1197), rolling circle replication (Lizardi et al. (1988) Bio / Technology 6:1197), and the like.

[0013] This includes, for example, nucleic acid amplification of mRNA in a sample and / or reverse transcriptase (to prepare cDNA) using a method such as (U.S. Pat. No. 5,854,033, et al.) or any other nucleic acid amplification method, followed by detection of the amplified molecules using techniques well known to those skilled in the art. These detection schemes are useful for detecting nucleic acid molecules, particularly when they are present in very low numbers. In a specific embodiment of the present invention, the level of SLC30A8 expression is determined by quantitative fluorescent RT-PCR (i.e., TaqMan™ system). The expression level of SLC30A8 mRNA can be monitored using membrane blots (such as those used in hybridization analyses such as Northern, Southern, and dot) or microwells, sample tubes, gels, beads, or fibers (or any solid support containing bound nucleic acids). See U.S. Patent Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195, and 5,445,934, which are incorporated herein by reference. Determining the expression level of SLC30A8 can also involve the use of a nucleic acid probe in solution.

[0116] In preferred embodiments, the level of mRNA expression is assessed using branched DNA (bDNA) assays or real-time PCR (qPCR). The use of these methods is described and exemplified in the Examples presented herein.

[0117] The level of SLC30A8 protein expression can be determined using any method known in the art for measuring protein levels, including, for example, electrophoresis, capillary electrophoresis, high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), high-diffusion chromatography, liquid or gel precipitin reaction, absorption spectroscopy, colorimetric assay, spectrophotometric assay, flow cytometry, immunodiffusion (single or dual), immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, electrochemiluminescence assay, etc.

[0118] In some embodiments, the effectiveness of the methods of the invention can be monitored by detecting or monitoring a reduction in symptoms of SLC30A8 disease, such as reduced edematous swelling of the extremities, face, larynx, upper airway, abdomen, trunk, and genitals, prodrome, laryngeal edema, non-pruritic rash, nausea, vomiting, or abdominal pain. These symptoms can be assessed in vitro or in vivo using any method known in the art.

[0119] In some embodiments of the method of the present invention, an RNAi agent is administered to a subject so as to be delivered to a specific site within the subject. Inhibition of SLC30A8 expression can be assessed by measuring the level or change in the level of SLC30A8 mRNA or SLC30A8 protein in a sample derived from a body fluid or tissue from a specific site within the subject. In a preferred embodiment, the site is selected from the group consisting of the liver, choroid plexus, retina, and pancreas. The site can be a small unit or subgroup of cells from any one of the aforementioned sites. The site can also include cells expressing a specific type of receptor.

[0120] Methods for treating or preventing SLC30A8-related diseases The present invention provides therapeutic and preventive methods comprising administering a composition comprising an RNAi agent, a pharmaceutical composition comprising an RNAi agent, or a vector comprising an RNAi of the present invention to a subject suffering from or susceptible to an SLC30A8-related disease, disorder, and / or condition. Non-limiting examples of SLC30A8-related diseases include prediabetes and diabetes.

[0121] In certain embodiments, the present invention provides a method for reducing the expression of SLC30A8 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 expression level of SLC30A8 in pancreatic cells of the patient is reduced after administration of the RNAi construct compared to the expression level of SLC30A8 in the patient who has not been administered the RNAi construct.

[0122] The methods of the present invention are useful for treating subjects with an SLC30A8-related disease, such as subjects who would benefit from reduced SLC30A8 gene expression and / or SLC30A8 protein production. In one aspect, the present invention provides a method for reducing the expression level of the SLC30A8 gene in a subject with prediabetes or diabetes.

[0123] In another aspect, the present invention provides a method for treating a subject with prediabetes or diabetes. The treatment method (and use) of the present invention comprises administering to a subject, e.g., a human, a therapeutically effective amount of an RNAi agent of the present invention that targets the SLC30A8 gene, or a pharmaceutical composition comprising an RNAi agent of the present invention that targets the SLC30A8 gene, or a vector of the present invention that comprises an RNAi agent that targets the SLC30A8 gene.

[0124] In one aspect, the present invention provides a method for preventing at least one symptom in a subject with prediabetes or diabetes, comprising administering to the subject a therapeutically effective amount of an RNAi agent, e.g., a dsRNA, pharmaceutical composition, or vector, of the present invention, thereby preventing at least one symptom in the subject with a disorder that would benefit from reduced SLC30A8 gene expression.

[0125] In another aspect, the present invention provides use of a therapeutically effective amount of an RNAi agent of the present invention for treating a subject, e.g., a subject who would benefit from reduced and / or inhibited SLC30A8 gene expression. In a further aspect, the present invention provides use of an RNAi agent of the present invention that targets the SLC30A8 gene, e.g., a dsRNA or a pharmaceutical composition comprising an RNAi agent that targets the SLC30A8 gene, in the manufacture of a medicament for treating a subject, such as a subject with a disorder that would benefit from reduced SLC30A8 gene expression, e.g., an SLC30A8-associated disease, e.g., a subject who would benefit from reduced and / or inhibited SLC30A8 gene expression and / or SLC30A8 protein production.

[0126] In another aspect, the present invention provides use of an RNAi, e.g., a dsRNA, of the present invention for preventing at least one symptom in a subject suffering from a disorder that would benefit from reducing and / or inhibiting SLC30A8 gene expression and / or SLC30A8 protein production.

[0127] In a further aspect, the present invention provides the use of an RNAi agent of the present invention in the manufacture of a medicament for preventing at least one symptom in a subject suffering from a disorder that would benefit from reducing and / or inhibiting SLC30A8 gene expression and / or SLC30A8 protein production, such as an SLC30A8-associated disease.

[0128] In one embodiment, the RNAi agent targeting SLC30A8 is a dsRNA agent that, when administered to a subject, reduces expression of the SLC30A8 gene by at least about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, %, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 62%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least about 99% or more reduction in SLC30A8-associated disorder, such as prediabetes or diabetes.

[0129] The methods and uses of the present invention comprise administering a composition described herein so that expression of the target SLC30A8 gene is reduced for, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, or about 80 hours. In one embodiment, the expression of the target SLC30A8 gene is reduced for an extended period of time, for example, at least about 2, 3, 4, 5, 6, 7 days or more, for example, about 1 week, 2 weeks, 3 weeks, or about 4 weeks or more.

[0130] Administration of dsRNA according to the methods and uses of the present invention can result in a reduction in the severity, signs, symptoms, and / or markers of SLC30A8-related diseases, such as prediabetes or diabetes, in patients with such diseases or disorders. In this context, "reduction" refers to a statistically significant decrease in such levels. The reduction can be, for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%. The effectiveness of disease treatment or prevention can be assessed, for example, by measuring disease progression, disease remission, disease symptom severity, pain reduction, quality of life, the dose of a drug required to maintain the therapeutic effect, the level of a disease marker, or any other measurable parameter appropriate for the given disease being treated or targeted for prevention. It is well within the capabilities of one skilled in the art to monitor the effectiveness of treatment or prevention by measuring any one or any combination of such parameters. For example, the effectiveness of a treatment for prediabetes or diabetes can be assessed, for example, by regular monitoring of prediabetes or diabetes symptoms. A comparison of initial readings with subsequent readings provides a physician with an indication of whether the treatment is effective. It is well within the capabilities of one skilled in the art to monitor the effectiveness of treatment or prevention by measuring any one or any combination of such parameters. In relation to the administration of an RNAi targeting SLC30A8 or a pharmaceutical composition thereof, "effective against" an SLC30A8-related disease indicates that administration in a clinically relevant manner results in a beneficial effect in at least a statistically significant proportion of patients, such as improvement of symptoms, cure, reduction of disease, prolongation of life, improvement in quality of life, or other effect generally recognized as favorable by physicians familiar with the treatment of prediabetes or diabetes and / or SLC30A8-related diseases and related causes.

[0131] The therapeutic or preventive effect is evident when there is a statistically significant improvement in one or more parameters of the disease state, or when there is no worsening or progression of symptoms that would normally be expected.As an example, a favorable change of at least 10%, preferably at least 20%, 30%, 40%, 50% or more of the measurable parameters of the disease can indicate effective treatment.The effectiveness of a given RNAi drug or a formulation of this drug can also be determined using an experimental animal model for a given disease known in the art.When using an experimental animal model, the effectiveness of treatment is proven when a statistically significant reduction in markers or symptoms is observed.

[0132] The subjects included dsRNA at approximately 0.01 mg / kg, 0.02 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.15 mg / kg, 0.2 mg / kg, 0.25 mg / kg, 0.3 mg / kg, 0.35 mg / kg, 0.4 mg / kg, 0.45 mg / kg, 0.5 mg / kg, 0.55 mg / kg, 0.6 mg / kg, 0.65 mg / kg, 0.7 mg / kg, 0.75 mg / kg, 0.8 mg / kg, 0.85 mg / kg, 0.9 mg / kg, 0.95 mg / kg, 1.0 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.6 mg / kg, 1.7 mg / kg, 1.8 mg / kg, 1.9 mg / kg, 2.0 mg / kg, 2.1 mg / kg, 2.2 mg / kg, 2.3 mg / kg, 2.4 mg / kg, 2.5 mg / kg dsRNA, 2.6 mg / kg dsRNA, 2.7 mg / kg dsRNA, 2.8 mg / kg dsRNA, 2.9 mg / kg dsRNA, 3.0 mg / kg dsRNA, 3.1 mg / kg dsRNA, 3.2 mg / kg dsRNA, 3.3 mg / kg dsRNA, 3.4 mg / kg dsRNA, 3.5 mg / kg dsRNA, 3.6 mg / kg dsRNA, 3.7 mg / kg dsRNA, 3.8 mg / kg dsRNA, 3.9 mg / kg dsRNA, 4.0 mg / kg dsRNA, 4.1 mg / kg dsRNA, 4.2 mg / kg dsRNA, 4.3 mg / kg dsRNA, 4.4 mg / kg dsRNA, 4.5 mg / kg dsRNA, 4.6 mg / kg dsRNA, 4.7 mg / kg dsRNA, 4.8 mg / kg dsRNA, 4.9 mg / kg dsRNA, 5.0 mg / kg dsRNA, 5.1 mg / kg dsRNA, 5.2 mg / kg dsRNA, 5.3 mg / kg dsRNA, 5.4 mg / kg dsRNA, 5.5 mg / kg dsRNA, 5.6 mg / kg dsRNA, 5.7 mg / kg dsRNA, 5.8 mg / kg dsRNA, 5.9 mg / kg dsRNA, 6.0 mg / kg dsRNA, 6.1 mg / kg dsRNA, 6.2 mg / kg dsRNA, 6.3 mg / kg dsRNA, 6.4 mg / kg dsRNA, 6.5 mg / kg dsRNA, 6.6 mg / kg dsRNA, 6.7mg / kg dsRNA, 6.8mg / kg dsRNA, 6.9mg / kg dsRNA, 7.0mg / kg dsRNA, 7.1mg / kg dsRNA, 7.2mg / kg dsRNA, 7.3mg / kg dsRNA, 7.4mg / kg dsRNA, 7.5mg / kg dsRNA, 7.6mg / kg dsRNA, 7.7mg / kg dsRNA, 7.8mg / kg dsRNA, 7.9mg / kg dsRNA, 8.0mg / kg dsRNA, 8.1mg / kg dsRNA, 8.2mg / kg dsRNA, 8.3mg / kg dsRNA, 8.4mg / kg dsRNA, 8.5mg / kg dsRNA, 8.6mg / kg dsRNA, 8.7mg / kg dsRNA, 8.8mg / kg A therapeutic amount of RNAi such as dsRNA, 8.9 mg / kg dsRNA, 9.0 mg / kg dsRNA, 9.1 mg / kg dsRNA, 9.2 mg / kg dsRNA, 9.3 mg / kg dsRNA, 9.4 mg / kg dsRNA, 9.5 mg / kg dsRNA, 9.6 mg / kg dsRNA, 9.7 mg / kg dsRNA, 9.8 mg / kg dsRNA, 9.9 mg / kg dsRNA, 9.0 mg / kg dsRNA, 10 mg / kg dsRNA, 15 mg / kg dsRNA, 20 mg / kg dsRNA, 25 mg / kg dsRNA, 30 mg / kg dsRNA, 35 mg / kg dsRNA, 40 mg / kg dsRNA, 45 mg / kg dsRNA or about 50 mg / kg dsRNA may be administered. In one embodiment, a subject may be administered 0.5 mg / kg of dsRNA. Values ​​and ranges intermediate to these recited values ​​are also intended to be part of the invention.

[0133] Administration of RNAi can, for example, increase the presence of SLC30A8 protein levels in the patient's cells, tissues, blood, urine, or other compartment by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118 %, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least about 99% or more.

[0134] Prior to administration of the full dose of RNAi, the patient may be administered a smaller dose, such as a 5% infusion, and monitored for side effects, such as allergic reactions. In another example, the patient may be monitored for undesired immunostimulatory effects, such as elevated cytokine (e.g., TNF-alpha or INF-alpha) levels.

[0135] Due to the inhibitory effect on SLC30A8 expression, the composition according to the present invention or a pharmaceutical composition prepared therefrom can improve the quality of life.

[0136] The RNAi of the present invention can be administered in a "naked" form, in which the modified or unmodified RNAi agent is directly suspended in an aqueous or suitable buffer solution as a "free RNAi." The free RNAi is administered in the absence of a pharmaceutical composition. The free RNAi can be in a suitable buffer solution. The buffer solution can contain acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate-buffered saline (PBS). The pH and osmolality of the buffer solution containing the RNAi can be adjusted to be suitable for administration to a subject.

[0137] Alternatively, the RNAi of the present invention can be administered as a pharmaceutical composition, such as a dsRNA liposome formulation.

[0138] Subjects who would benefit from reducing and / or inhibiting SLC30A8 gene expression are those who have pre-diabetes or diabetes and / or an SLC30A8-related disease or disorder as described herein.

[0139] Treatment of subjects who would benefit from reducing and / or inhibiting SLC30A8 gene expression includes therapeutic and prophylactic treatment.

[0140] The present invention further provides methods and uses of RNAi agents or pharmaceutical compositions thereof for treating subjects who would benefit from reducing and / or inhibiting SLC30A8 gene expression, e.g., subjects with SLC30A8-associated diseases, in combination with other pharmaceutical agents and / or other therapies, e.g., known pharmaceutical agents and / or known therapies, e.g., those currently utilized to treat these disorders.

[0141] For example, in certain embodiments, an RNAi targeting the SLC30A8 gene is administered in combination with an agent useful for treating, e.g., an SLC30A8-associated disease, as described elsewhere herein. For example, additional therapeutic agents and therapies suitable for treating a subject who would benefit from reduced SLC30A8 expression, e.g., a subject with an SLC30A8-associated disease, include RNAi agents targeting different portions of the SLC30A8 gene, therapeutic agents and / or procedures for treating an SLC30A8-associated disease, or any combination of the foregoing.

[0142] In one embodiment, all of the nucleotides of the first and second sense strands and / or all of the nucleotides of the first and second antisense strands comprise a modification.

[0143] In one embodiment, at least one of the modified nucleotides is selected from the group consisting of a 3'-terminal deoxy-thymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxy modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide containing a non-natural base, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide containing a phosphorothioate group, a nucleotide containing a methylphosphonate group, a nucleotide containing a 5'-phosphate, and a nucleotide containing a 5'-phosphate mimic.

[0144] In certain embodiments, the first RNAi agent that targets the SLC30A8 gene is administered in combination with the second RNAi agent that targets a gene other than the SLC30A8 gene.The first RNAi agent that targets the SLC30A8 gene and the second RNAi agent that targets a gene other than the SLC30A8 gene can be administered as part of the same pharmaceutical composition.Alternatively, the first RNAi agent that targets the SLC30A8 gene and the second RNAi agent that targets a gene other than the SLC30A8 gene can be administered as part of different pharmaceutical compositions.

[0145] The RNAi agent and additional therapeutic agent and / or treatment may be administered simultaneously and / or in the same combination, e.g., parenterally, or the additional therapeutic agent may be administered as part of a separate composition or separately and / or by other methods known in the art or described herein.

[0146] The present invention also provides methods of using RNAi agents of the present invention and / or compositions containing RNAi agents of the present invention to reduce and / or inhibit SLC30A8 expression in cells. In another aspect, the present invention provides RNAi agents of the present invention and / or compositions comprising the RNAi agents of the present invention for use in reducing and / or inhibiting SLC30A8 gene expression in cells. In yet another aspect, the present invention provides use of RNAi agents of the present invention and / or compositions comprising the RNAi agents of the present invention for the manufacture of a medicament for reducing and / or inhibiting SLC30A8 gene expression in cells. In yet another aspect, the present invention provides RNAi agents of the present invention and / or compositions comprising the RNAi agents of the present invention for use in reducing and / or inhibiting SLC30A8 protein production in cells. In yet another aspect, the present invention provides use of RNAi agents of the present invention and / or compositions comprising the RNAi agents of the present invention for the manufacture of a medicament for reducing and / or inhibiting SLC30A8 protein production in cells. The methods and uses include contacting a cell with an RNAi of the present invention, e.g., dsRNA, and maintaining the cell for a period of time sufficient to obtain degradation of the mRNA transcript of the SLC30A8 gene, thereby inhibiting expression of the SLC30A8 gene or inhibiting SLC30A8 protein production in the cell.

[0147] The reduction in gene expression can be evaluated by any method known in the art. For example, the reduction in SLC30A8 expression can be determined by determining the mRNA expression level of SLC30A8 using a method conventional to those skilled in the art, such as Northern blotting or qRT-PCR, by determining the protein level of SLC30A8 using a method conventional to those skilled in the art, such as Western blotting, immunological techniques, flow cytometry, ELISA, etc., and / or by determining the biological activity of SLC30A8.

[0148] In the methods and uses of the present invention, the cells may be contacted in vitro or in vivo, i.e., the cells may be within a subject.

[0149] Cells suitable for treatment using the methods of the present invention can be any cells that express the SLC30A8 gene, such as cells from a subject with prediabetes or diabetes, or cells containing an expression vector containing the SLC30A8 gene or a portion of the SLC30A8 gene. Cells suitable for use in the methods and uses of the present invention can be mammalian cells, such as primate cells (human cells or non-human primate cells, such as monkey cells or chimpanzee cells), non-primate cells (such as cow cells, pig cells, camel cells, llama cells, horse cells, goat cells, rabbit cells, sheep cells, hamster cells, guinea pig cells, cat cells, dog cells, rat cells, mouse cells, lion cells, tiger cells, bear cells, or buffalo cells), avian cells (e.g., duck cells or goose cells), or whale cells. In one embodiment, the cells are human cells.

[0150] SLC30A8 gene expression is at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 1 It may be inhibited by 1%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or about 100%.

[0151] SLC30A8 protein production is at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124 The expression level may be inhibited by 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or about 100%.

[0152] The in vivo method and use of the present invention can include administering to a subject a composition containing an RNAi, wherein the RNAi comprises a nucleotide sequence complementary to at least a portion of the RNA transcript of the SLC30A8 gene of the mammal to be treated.When the organism to be treated is human, the composition can be administered by any means known in the art, including, but not limited to, subcutaneous, intravenous, oral, intraperitoneal or intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intramuscular, transdermal, airway (aerosol), nasal, parenteral routes including rectal, and topical (including buccal and sublingual) administration.In certain embodiments, the composition is administered by subcutaneous or intravenous infusion or injection.In one embodiment, the composition is administered by subcutaneous injection.

[0153] In some embodiments, administration is by depot injection. Depot injection can release RNAi consistently over a long period of time. Therefore, depot injection can reduce the frequency of administration required to achieve a desired effect, such as a desired SLC30A8 inhibition or therapeutic or preventive effect. Depot injection can also provide a more consistent serum concentration. Depot injection can include subcutaneous injection or intramuscular injection. In a preferred embodiment, depot injection is subcutaneous injection.

[0154] In some embodiments, administration is via a pump. The pump can be an external pump or a surgically implanted pump. In certain embodiments, the pump is an osmotic pump implanted subcutaneously. In other embodiments, the pump is an infusion pump. The infusion pump can be used for intravenous, subcutaneous, arterial, or epidural infusion. In a preferred embodiment, the infusion pump is a subcutaneous infusion pump. In other embodiments, the pump is a surgically implanted pump that delivers RNAi to a subject.

[0155] The mode of administration can be selected based on whether local or systemic treatment is desired and based on the area to be treated. The route and site of administration can be selected to enhance targeting.

[0156] In one aspect, the present invention also provides a method for inhibiting expression of the SLC30A8 gene in a mammal, for example, a human. The present invention also provides a composition comprising an RNAi, for example, a dsRNA, targeting the SLC30A8 gene in a mammalian cell for use in inhibiting expression of the SLC30A8 gene in a mammal. In another aspect, the present invention provides the use of an RNAi, for example, a dsRNA, targeting the SLC30A8 gene in a mammalian cell in the manufacture of a medicament for inhibiting expression of the SLC30A8 gene in a mammal.

[0157] These methods and uses include administering to a mammal, e.g., a human, a composition comprising an RNAi, e.g., dsRNA, that targets the SLC30A8 gene in the cells of the mammal, and maintaining the mammal for a period of time sufficient to result in degradation of mRNA transcripts of the SLC30A8 gene, thereby inhibiting expression of the SLC30A8 gene in the mammal.

[0158] The reduction in gene expression can be assessed in a peripheral blood sample from a subject administered RNAi by any method known in the art, for example, qRT-PCR as described herein. The reduction in protein production can be assessed by any method known in the art and as described herein, for example, ELISA or Western blotting. In one embodiment, a tissue sample serves as a tissue source for monitoring the reduction in expression of the SLC30A8 gene and / or protein. In another embodiment, a blood sample serves as a tissue source for monitoring the reduction in expression of the SLC30A8 gene and / or protein.

[0159] In one embodiment, verification of RISC-mediated cleavage of the target in vivo after administration of the RNAi agent is performed by performing 5'-RACE or a modification of protocols known in the art (Lasham A et al., (2010) Nucleic Acid Res., 38(3)p-el9) (Zimmermann et al. (2006) Nature 441:111-4).

[0160] It is understood that all ribonucleic acid sequences disclosed herein can be converted to deoxyribonucleic acid sequences by substituting thymine bases for uracil bases in the sequences. Similarly, all deoxyribonucleic acid sequences disclosed herein can be converted to ribonucleic acid sequences by substituting thymine bases for uracil bases in the sequences. Deoxyribonucleic acid sequences, ribonucleic acid sequences, and sequences containing mixtures of deoxyribonucleotides and ribonucleotides of all sequences disclosed herein are included in the present invention.

[0161] Additionally, any nucleic acid sequence disclosed herein may be modified by any combination of chemical modifications. Those skilled in the art will readily understand that, in certain cases, designations such as "RNA" or "DNA" to describe modified polynucleotides are arbitrary. For example, a polynucleotide containing nucleotides with 2'-OH substituents on the ribose sugar and thymine bases may be described as a DNA molecule with modified sugars (2'-OH for the native 2'-H in DNA) or an RNA molecule with modified bases (thymine (methylated uracil) for the native uracil in RNA).

[0162] Thus, the nucleic acid sequences provided herein, including but not limited to those in the Sequence Listing, are intended to encompass nucleic acids containing any combination of native or modified RNA and / or DNA, including but not limited to those with modified nucleic acid bases. As a further example, and not by way of limitation, a polynucleotide having the sequence "ATCGATCG" encompasses all polynucleotides having a sequence that includes RNA bases, whether modified or unmodified, such as those with the sequence "AUCGAUCG," as well as compounds including, but not limited to, those with some DNA bases and some RNA bases, such as "AUCGATCG" (where meC indicates a cytosine base containing a methyl group at the 5-position), and polynucleotides with other modified bases, such as "ATmeCGAUCG."

[0163] The following examples, including the experiments conducted and results achieved, are provided for illustrative purposes only and are not to be construed as limiting the scope of the appended claims.

[0164] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are incorporated by reference herein as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. However, the citation of a reference herein should not be construed as an admission that such reference is prior art to the present invention. To the extent that any definitions or terms provided in a reference incorporated by reference differ from the terms and discussion provided herein, the terms and definitions shall control.

[0165] equivalent The foregoing specification is considered sufficient to enable one skilled in the art to practice the invention. The foregoing description and examples detail certain preferred embodiments of the invention and set forth the best mode contemplated by the inventors. However, no matter how detailed the foregoing appears, it will be understood that the invention can be practiced in many ways and should be construed in accordance with the appended claims and any equivalents thereof.

[0166] The following examples, including the experiments conducted and results achieved, are provided for illustrative purposes only and should not be construed as limiting the scope of the invention. [Example]

[0167] Example 1: Efficacy of selected SLC30A8 siRNA molecules in an RNA FISH assay RNA FISH (fluorescence in situ hybridization) assays were performed to measure SLC30A8 mRNA knockdown by test siRNA. CHO cells stably overexpressing SLC30A8 (SLC30A8 / CHO, produced by Amgen) were cultured in F-12K medium (Mediatech) supplemented with 10% fetal bovine serum (FBS, Sigma) and 1% penicillin-streptomycin (PS, Corning). siRNA transfection was performed as follows: 1 μL of test siRNA and 4 μL of plain F-12K medium were added to a PDL-coated CellCarrier-384 Ultra assay plate (PerkinElmer) using a BioMek FX (Beckman Coulter). Next, 5 μL of Lipofectamine RNAiMAX (Thermo Fisher Scientific) prediluted in plain F-12K medium (0.05 μL of RNAiMAX in 5 μL of F-12K medium) was dispensed into the assay plate using a Multidrop Combi reagent dispenser (Thermo Fisher Scientific). After a 20-minute incubation of the siRNA / RNAiMAX mixture at room temperature (RT), 30 μL of SLC30A8 / CHO cells (1,500 cells per well) in F-12K medium supplemented with 10% FBS and 1% PS were added to the transfection complex using the Multidrop Combi reagent dispenser. The assay plate was incubated for 20 minutes at RT before being placed in an incubator. The cells were incubated at 37°C and 5% CO2 for 72 hours. The ViewRNA ISH cell assay was performed according to the manufacturer's protocol (Thermo Fisher Scientific) using an in-house assembled automated FISH assay platform for liquid handling. Briefly, cells were fixed in 4% formaldehyde (Thermo Fisher Scientific) for 15 min at RT, permeabilized with detergent for 3 min at RT, and then treated with protease solution for 10 min at RT.Incubation of the target-specific probe pair (Thermo Fisher Scientific) was performed for 3 hours, while incubation of the preamplifier, amplifier, and label probe (Thermo Fisher Scientific) was performed for 1 hour each. All hybridization steps were performed in a Cytomat 2 C-LIN automated incubator (Thermo Fisher Scientific) at 40°C. After the hybridization reaction, cells were stained with Hoechst and CellMask Blue (Thermo Fisher Scientific) for 30 minutes and then imaged using an Opera Phenix (PerkinElmer). Images were analyzed using a Columbus Image Data Storage and Analysis System (PerkinElmer) to obtain the average number of spots per cell. Spot counts were normalized using high (containing phosphate-buffered saline, Corning) and low (no target probe pair) control wells. Normalized values ​​were plotted against total siRNA concentration, and the data were fitted to a four-parameter sigmoidal model using Genedata Screener (Genedata) to obtain IC50 and maximum activity.

[0168] The results of the RNA FISH assay are shown in Table 1.

[0169] [Table 1]

[0170] [Table 2]

Claims

1. An RNAi construct comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region having at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from an antisense sequence listed in Table 1, and wherein the RNAi construct inhibits expression of zinc transporter 8 (SLC30A8).

2. The RNAi construct of claim 1 , wherein the antisense strand comprises a region complementary to an mRNA sequence of SLC30A8.

3. 3. The RNAi construct of claim 1 or 2, wherein the sense strand comprises a region having at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from an antisense sequence listed in Table 1.

4. The RNAi construct of any one of claims 1 to 3, wherein the sense strand comprises a sequence sufficiently complementary to a sequence of the antisense strand to form a duplex region of about 15 to about 30 base pairs in length.

5. The RNAi construct of claim 4, wherein the duplex region is about 17 to about 24 base pairs in length.

6. The RNAi construct of claim 4, wherein the duplex region is about 19 to about 21 base pairs in length.

7. The RNAi construct of claim 6 , wherein the duplex region is 19 base pairs in length.

8. The RNAi construct according to any one of claims 4 to 7, wherein the sense strand and the antisense strand are each about 15 to about 30 nucleotides in length.

9. The RNAi construct of claim 8, wherein the sense strand and the antisense strand are each about 19 to about 27 nucleotides in length.

10. The RNAi construct of claim 8, wherein the sense strand and the antisense strand are each about 21 to about 25 nucleotides in length.

11. The RNAi construct of claim 8, wherein the sense strand and the antisense strand are each about 21 to about 23 nucleotides in length.

12. The RNAi construct of any one of claims 1 to 11, comprising at least one blunt end.

13. 12. The RNAi construct of any one of claims 1 to 11, comprising at least one nucleotide overhang of 1 to 4 unpaired nucleotides.

14. The RNAi construct of claim 13 , wherein the nucleotide overhang has two unpaired nucleotides.

15. 15. The RNAi construct of claim 13 or 14, comprising a nucleotide overhang at the 3' end of the sense strand, the 3' end of the antisense strand, or the 3' end of both the sense strand and the antisense strand.

16. The RNAi construct of any one of claims 13 to 15, wherein the nucleotide overhang comprises a 5'-UU-3' dinucleotide or a 5'-dTdT-3' dinucleotide.

17. The RNAi construct of any one of claims 1 to 16, comprising at least one modified nucleotide.

18. The RNAi construct of claim 17, wherein the modified nucleotide is a 2'-modified nucleotide.

19. 18. The RNAi construct of claim 17, 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-allyl modified nucleotide, a bicyclic nucleic acid (BNA), a glycol nucleic acid, an inverted base, or a combination thereof.

20. 20. The RNAi construct of claim 19, wherein the modified nucleotides are 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-fluoro modified nucleotides, or a combination thereof.

21. 18. The RNAi construct of claim 17, wherein all of the nucleotides in the sense and antisense strands are modified nucleotides.

22. The RNAi construct of claim 21, wherein the modified nucleotides are 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, or a combination thereof.

23. 23. The RNAi construct of any one of claims 1 to 22, comprising at least one phosphorothioate internucleotide linkage.

24. 24. The RNAi construct of claim 23, comprising two consecutive phosphorothioate internucleotide linkages at the 3' end of the antisense strand.

25. The RNAi construct of claim 23, comprising two consecutive phosphorothioate internucleotide linkages 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.

26. 26. The RNAi construct of any one of claims 1 to 25, wherein the antisense strand comprises a sequence selected from the antisense sequences listed in Table 1.

27. 27. The RNAi construct of claim 26, wherein the sense strand comprises a sequence selected from the sense sequences listed in Table 1.

28. 28. The RNAi construct of any one of claims 1 to 27, which reduces the expression level of SLC30A8 in SLC30A8 CHO transfected cells or pancreatic cells after incubation with the RNAi construct compared to the expression level of SLC30A8 in SLC30A8 CHO transfected cells or pancreatic cells incubated with a control RNAi construct.

29. A pharmaceutical composition comprising the RNAi construct of any one of claims 1 to 28 and a pharmaceutically acceptable carrier, excipient, or diluent.

30. A method for reducing the expression of SLC30A8 in a patient in need thereof, comprising administering to the patient an RNAi construct according to any one of claims 1 to 28.

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