WO2020144583A1 - Cell-penetrating tripodal interfering rna - Google Patents

Abstract

A tripodal interfering RNA (tiRNA) polynucleotide complex, which can inhibit expression of multiple targets by RNA interference (RNAi), while having cell-penetrating capability, is provided. A tiRNA structure comprises three polynucleotides that are annealed together into a Y-shaped structure, thereby forming three small interfering RNAs (siRNAs). The tiRNA structure may induce silencing or reduction of expression of three RNA targets by RNAi simultaneously. A method of making a pharmaceutical composition for reducing expression of one or more target RNAs is also provided that involves testing multiple tiRNAs for the ability to reduce expression of target RNAs by RNAi, selecting a tiRNA from the multiple tiRNAs that inhibits expression of at least two of the target RNAs to a certain degree, and formulating the selected tiRNA as a pharmaceutical composition for delivery.

Description

CELL-PENETRATING TRIPODAL INTERFERING RNA

PRIORITY

This application claims priority to and benefit from U.S. Provisional Patent Application No. 62/789,817, filed January 8, 2019, the entire contents of which are incorporated herein by reference.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS- Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on January 7, 2020, is named OLX-013PC_ST25.txt and is 4,991 bytes in size.

BACKGROUND

RNA interference (RNAi) is a mechanism capable of inhibiting the expression of a gene in a highly specific and efficient manner, in which degradation of the target RNA is induced by introducing a double-stranded RNA, which comprises a sense strand having a sequence homologous to the target RNA and an antisense strand having a sequence complementary to the target RNA, into cells or the like, thereby inhibiting the expression of the target RNA.

These RNAi techniques have been expected to be effective for the treatment of many diseases, including cancer or viral infections. Many researchers reported that the replication of viruses was successfully inhibited using various RNAi techniques, such as small interfering RNA (siRNA) and short hairpin RNA (shRNA) techniques (Jacque J. M. et al., Nature, 418: 435, 2002; Novina C. D. et al., Nat. Med., 8:681 , 2002; Nishitsuji H. et al., Microbes Infect., 6:76, 2004). However, it was reported that, when the expression of a single target gene in a virus is inhibited by the RNAi mechanism, the virus can escape from RNAi-mediated inhibition of gene expression due to the substitution or deletion of a single nucleotide in the genome of the virus (Westerhout E. M. et al., Nucleic Acids Res., 33:796, 2005). One way to minimize the escape of viruses from RNAi-mediated inhibition of gene expression is to induce multiple RNAi mechanisms that target various regions in the viral genome.

Meanwhile, RNAi mechanisms have been used to develop drugs not only for inhibiting viral replication, but also for treating cancer. RNAi can induce cell cycle arrest and target genes essential for tumor survival, thereby inducing apoptosis in cancer cells. It was reported that the simultaneous inhibition of a plurality of genes induces strong apoptosis in cancer cells (Menendez J. A. et al., Proc. Natl. Acad. Sci. U.S.A., 101 :10715, 2004). Accordingly, the development of an efficient strategy for inhibiting the expression of a plurality of target genes using an RNAi mechanism has been required.

However, so far, multi-targeted RNAi mechanisms have been developed mainly based on shRN A expression systems (Konstantinova P. et al., Gene Ther., 13:1403, 2006; ter Brake O. et al., Mol. Ther., 14:883, 2006; Watanabe T. et al., Gene Ther., 13:883, 2006). Also, Khaled et al. developed RNA nanostructures for non- viral siRNA delivery, which comprise multiple siRNAs based on phi29 RNA backbones (Khaled A. et al., Nano Lett., 5:1797, 2005). However, such RNA backbone structures have an excessively long length so that they cannot be chemically synthesized, and thus the actual use thereof is limited. Accordingly, it has been required to develop new siRNA structures which can be applied to multiple siRNAs and, at the same time, can be chemically synthesized.

Another requirement for the siRNA technique is the efficient intracellular delivery of siRNA. It was reported that 21-base-pair siRNAs known in the prior art are unsuitable for binding to a cationic polymer such as PEI, unlike plasmid DNAs (Balcato-Bellemin A. L. et al., Proc. Natl. Acad. Sci. U.S.A., 104:16050, 2007).

Accordingly, compositions and methods for efficiently inhibiting expression of a plurality of target RNAs, including for therapy, are desired.

SUMMARY

In various aspects, the present invention provides a polynucleotide complex which can inhibit the expression of multiple targets by RNA interference (RNAi), while having cell-penetrating capability. In some aspects, the RNA complex comprises three polynucleotides (e.g., single-stranded polynucleotides) that are annealed or hybridized together into a Y- or tripod-shaped structure, thereby forming a “tripodal” interfering RNA comprising three small interfering RNAs (siRNAs). The three polynucleotides each comprise an antisense region for silencing a first target RNA by RNAi, and a sense region for silencing a second target RNA by RNAi. The RNA in accordance with the present invention is therefore referred to herein as a“tripodal interfering RNA" (tiRNA) or tiRNA structure. The tiRNA structure in accordance with the present disclosure may induce silencing or reduction of expression of three RNA targets by RNA interference simultaneously, wherein each of the three siRNAs can be directed towards a different RNA target.

In some embodiments, the three siRNAs forming the tiRNA have 3’ overhangs, and 5’ and 3’ ends of the three polynucleotides are selected to reduce expression of at least two of the target RNAs by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%. Additionally or alternatively, in some embodiments, at least two of the three 3’ ends are different by a length of the 3’ overhang and/or chemical modification. In some embodiments, one or more 3’ ends have a cell-targeting or cell-penetrating moiety. In some embodiments, the described tiRNA structures have improved cell-penetrating capability such that they can silence multiple targets without the aid of a separate intracellular delivery vehicle (e.g., a transfection reagent). The tiRNA structures in accordance with embodiments of the present disclosure can therefore be referred to as cell-penetrating tiRNAs.

In some aspects, the present invention provides a method of making a pharmaceutical composition for reducing expression of one or more target RNAs. The method includes providing a plurality of tiRNAs prepared in accordance with the present disclosure and testing the plurality of tiRNAs for the ability to reduce expression of the target RNAs. Each of the plurality of tiRNAs can be made by hybridizing three polynucleotides (e.g., RNAs or modified RNAs), which can be different from one another in various aspects, into the Y-shaped structure. For example, respective 3’ ends of the polynucleotides can be different by a length of the 3’ overhang and/or by at least one chemical modification, which can include a cell-targeting or cell-penetrating moiety. The multiple tiRNAs having different variations can be tested and one or more tiRNAs can be selected from the tested tiRNAs for formulating the selected tiRNA as a pharmaceutical composition. A tiRNA that inhibits expression of at least two of respective target RNAs by at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% can be selected for the pharmaceutical composition. In some embodiments, a tiRNA that inhibits expression of all three respective target RNAs by at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% can be selected for the pharmaceutical composition.

In some aspects, a tiRNA in accordance with the present disclosure has various chemical modifications. For example, in some embodiments, the 3’ end of at least one of the siRNAs can have at least one lipophilic moiety (e.g., cholesterol) or at least one non-lipophilic targeting moiety (e.g., N-acetylgalactosamine (GalNac)), at least one phosphorothioate (PS) or phosphorodithioate moiety, and/or any other nucleotide modification. In some embodiments, the 3’ ends of at least two of the three siRNAs of the tiRNA are different by a length of the 3’ overhangs. For example, the 3’ overhangs can be independently selected from a length of 2, 3, 4, or greater than 4 nucleotides (nt). In some embodiments, the hydroxyl group at the 2' position of the ribose of at least two nucleotides of at least one of the three polynucleotides is replaced by an -O-alkyl (e.g., 2'-OMe) group, a hydrogen atom, a fluorine atom, an—O-acyl group, or an amino group. In some embodiments, the at least two nucleotides with the 2'-0-alkyl (e.g., 2'-OMe) chemical modifications can be located at a junction of the Y-shaped structure, though the 2'-O-alkyl groups can be located at other portions of the tiRNA.

Other aspects and certain embodiments of the invention will be apparent from the following detailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates an example of a tiRNA structure in accordance with some embodiments of the present disclosure. The Y-shaped tiRNA structure is formed by hybridizing three single-stranded polynucleotide sequences of about 40 nt in length to form three siRNAs each having a 2-nt overhang at the 3' end thereof. Each of the three siRNAs (first, second, and third) includes an antisense strand having the respective target region or seed, and a sense strand complementary to the antisense strand and having the 2-nt 3' end overhang. As shown, each RNA strand has a first, antisense region for silencing a target RNA by RNAi, and a second, sense region for silencing a second target RNA by RNAi. The hydroxyl group at the 2' position of the ribose of at least two nucleotides of each of the three single-stranded polynucleotide sequences, at a junction of the Y-shaped structure, has been replaced by an -O-methyl (2'-OMe) group (shown by additional lines drawn adjacent to the lines representing polynucleotide strands).

FIG. 2 illustrates a tiRNA structure in accordance with some embodiments of the present disclosure. The Y- shaped tiRNA structure is formed by hybridizing three polynucleotides to form three siRNAs each having a 2-nt overhang, a cholesterol moiety conjugated to 3’ ends, and phosphorothioate (PS) linkages at the 3’ ends. Each of the three siRNAs (first, second, and third) includes an antisense strand having the respective target region or seed, and a sense strand complementary to the antisense strand and having the 2-nt 3' end overhang. Each of the siRNAs includes, at a junction of the Y-shaped structure, at least two 2'-OMe groups (shown by additional lines drawn adjacent to the lines representing polynucleotide strands).

FIG. 3 illustrates a tiRNA structure in accordance with some embodiments of the present disclosure. The Y- shaped tiRNA structure is formed by hybridizing three polynucleotides to form three siRNAs, wherein some siRNA components have a 4-nt overhang. Also, 3’ ends further comprise a cholesterol moiety and PS linkages. Each of the three siRNAs (first, second, and third) includes an antisense strand having the respective target region or seed, and a sense strand complementary to the antisense strand. Each of the siRNAs includes, at a junction of the Y-shaped structure, at least two 2'-OMe groups (shown by additional lines drawn adjacent to the lines representing polynucleotide strands).

FIG. 4 illustrates a tiRNA structure in accordance with some embodiments of the present disclosure. The Y- shaped tiRNA structure is formed by hybridizing three polynucleotides to form three siRNAs each having a 2-nt overhang, three PS linkages, and a N-acetylgalactosamine (GalNAc) ligand at the 3’ ends thereof. Each of the three siRNAs (first, second, and third) includes an antisense strand having the respective target region or seed, and a sense strand complementary to the antisense strand and having the 2-nt 3' end overhang. Each of the siRNAs includes, at a junction of the Y-shaped structure, at least two 2'-OMe groups (shown by additional lines drawn adjacent to the lines representing polynucleotide strands).

FIGS. 5A, 5B, and 5C illustrate three exemplary tiRNA structures each formed from three polynucleotides targeting Lamin A/C, DBP, and TIG3 mRNA. FIG. 5A shows a tiRNA structure having three siRNAs designed for targeting Lamin (L1), DBP (D1), and TIG3 (T1 ) mRNAs, each of the siRNAs including a 2-nt overhang at the 3' end thereof. FIG. 5B shows a tiRNA structure having three siRNAs designed for targeting Lamin (L2), DBP (D2), and TIG3 (T2) mRNAs, each of the siRNAs including a 2-nt overhang, a cholesterol moiety, and three PS linkages at the 3’ end thereof. FIG. 5C shows a third tiRNA structure having three siRNAs designed for targeting Lamin (L3), DBP (D3), and TIG3 (T3) mRNAs, each of the siRNAs including a 4-nt overhang, a cholesterol moiety, and four PS linkages at the 3’ end thereof. In FIGS. 5A, 5B, and 5C, each of the three siRNAs includes, at a junction of the Y-shaped structure, at least two 2'-OMe groups (shown by additional lines).

FIG. 6 illustrates results of testing of multiple tiRNA structures for their ability to reduce expression of respective three RNAs in a cell. The tiRNA structures were made using various combinations of polynucleotides selected from the nine polynucleotides forming the tiRNAs of FIGS. 5A to 5C. The x-axis shows the results in triplets of Lamin (Blue), DBP (Red), and TIG-3 (green), in this order; the y-axis shows relative RNA expression levels. The no-treatment (NT) control included 10 nM of each siRNA. The transfection controls included: (1) T1-D1-L1 tiRNA (made as shown in FIG. 5A) and a transfection reagent, (2) T2-D2-L2 tiRNA (made as shown in FIG. 5B) and a transfection reagent, and (3) T3-D3-L3 tiRNA (made as shown in FIG. 5C) and a transfection reagent. The passive uptake experiments included 27 various tiRNA structures without a transfection reagent, each formed of a different combination of T1 , D1 , L1 , T2, D2, L2, T3, D3, and L3, wherein each tiRNA structure includes one of T1 , T2, T3, one of D1 , D2, and D3, and one of L1 , L2, and L3. The tiRNAs were used at a concentration of 1 mM. DETAILED DESCRIPTION

The present invention is based, in part, on the design of small interfering RNAs (siRNA) complexes. An siRNA that induces RNA interference is a short double-stranded RNA capable of inhibiting expression of a target gene in a sequence-specific manner. For the development of effective therapeutic agents based on siRNAs, various problems associated with stability, silencing efficiency, immune responses, off-target effects and the like, are required to be solved, with the effective in vivo delivery being considered the most challenging to achieve. An siRNA typically cannot pass through the cell membrane, because it is highly negatively charged due to its phosphate backbone structure. In addition, because of its small size, the siRNA is quickly removed from blood, and thus it is difficult to deliver the siRNA to a target area in an amount sufficient for inducing RNAi.

Accordingly, the present invention provides siRNA structures with enhanced cell-penetrating capabilities. More specifically, the present invention provides a tripodal interfering RNA (tiRNA) in the form of a Y-shaped structure encompassing three siRNAs which can inhibit or reduce expression of three respective targets simultaneously. Moreover, the tiRNA in accordance with the present invention can include cell-targeting and/or cell penetrating moieties, and thus in some embodiments has enhanced intracellular penetrating ability such that it can be delivered into cells without the aid of a delivery vehicle.

In some embodiments, siRNA (small interfering RNA) refers to an RNA complex that mediates gene silencing in a sequence-specific manner. The RNA complex comprises an antisense strand and a sense strand. The antisense and sense strand can have one or more chemical modifications.

An antisense strand or region is a polynucleotide that is substantially or 100% complementary to a target nucleic acid sequence of interest. For example, an antisense strand or region may be complementary, in whole or in part, to a molecule of mRNA (messenger RNA), or an RNA sequence that is not mRNA (e.g., microRNA, piwiRNA, tRNA, rRNA, or hnRNA).

A sense strand or region is a polynucleotide that is complementary, in whole or in part, to the antisense strand or region. Generally, a sense strand has the same nucleotide sequence, in whole or in part, as a target nucleic acid, in which the polynucleotide is identical to a molecule of mRNA (messenger RNA), or an RNA sequence that is not mRNA (e.g., microRNA, piwiRNA, tRNA, rRNA, or hnRNA). In some aspects, a multiplex tiRNA structure is provided that is made of three polynucleotides hybridized or annealed together into a Y-shaped structure to form three siRNAs having 3’ overhangs. As used herein, the term“siRNA” (small interfering RNA) means a short double-stranded RNA that mediates gene silencing in a sequence-specific manner.

In some embodiments, in the tiRNA complex, each of the three polynucleotides forming the tiRNA forms an antisense region for silencing a first target RNA by RNAi and a sense region for silencing a different target RNA by RNAi. For example, a first polynucleotide may comprise an antisense region for silencing a first target RNA sequence by RNAi and a sense region for silencing a third target RNA sequence by RNAi; a second polynucleotide may comprise an antisense region for silencing the third target RNA sequence by RNAi and a sense strand for silencing a second target RNA sequence by RNAi; and a third polynucleotide may comprise an antisense region for silencing the second target RNA sequence by RNAi and a sense strand for silencing the first target RNA sequence by RNAi. The individual polynucleotides in various embodiments have lengths independently selected from 35 to 50 nucleotide, such as, e.g., from 38 to 45 nucleotide. For example, as shown in FIG. 1 , an RNA sequence forming the antisense region of the first siRNA, after the junction of the Y-structure, forms the sense region of the third siRNA. An RNA sequence forming the antisense region of the third siRNA, after the junction, forms the sense region of the second siRNA. And an RNA sequence forming the antisense region of the second siRNA after the junction forms the sense region of the first siRNA. Other variations of a structure of a tiRNA complex are possible as well. For example, in some embodiments, a polynucleotide in a tiRNA complex can form two antisense regions for targeting respective different RNAs. In some embodiments, a polynucleotide in a tiRNA complex can form two sense regions of respective different siRNAs. As another variation, in some embodiments, polynucleotides in a tiRNA complex can form the respective antisense and sense regions in different ways.

In some embodiments, the three polynucleotides are selected to target expression of three respective target RNAs by RNA interference, and each of the polynucleotides has a 3’ overhang. The 5’ and 3’ ends of the polynucleotides annealed into the siRNAs, which can be considered“arms” of the Y-shaped tiRNA, can vary in different ways. For example, the 3’ ends of the siRNAs can have different lengths and/or different types and number of chemical modifications. Other portions of the three siRNAs can also have one or more chemical modifications, including chemical modifications that are different among the three siRNAs. These can be selected to achieve silencing efficiency of each target. In some embodiments, at least two of the three 3’ ends of the respective three siRNAs are different by a length of the 3’ overhang and/or chemical modification. In some embodiments, a plurality of tiRNAs are made and tested for the ability to reduce expression of respective targets RNAs, e.g., in a cell line or in an animal model. Based on results of the testing, a tiRNA can be selected from the plurality of tiRNAs that inhibits expression of at least two of the target RNAs by at least 30%, at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, or by at least 75%. In some embodiments, a tiRNA can be selected from the plurality of tiRNAs that inhibits expression of all of the three target RNAs by at least 30%, at least 35%, by at least 40%, by at least 45%, or by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, or by at least 75%.

The target nucleic acid, not limited thereto, may be mRNA (messenger RNA) or small RNA such as microRNA(miRNA), or piRNA (piwi-interacting RNA). In some embodiments, the three target RNAs are mRNAs, non-coding RNAs, or combinations thereof. At least two of the three siRNAs of a tiRNA in accordance with some embodiments can target the same mRNA (e.g., different regions of the mRNA). In some embodiments, all three of the siRNAs target the same mRNA, which can be different regions of that mRNA. In some embodiments, at least two, or all three, of the siRNAs can target different regions within the same mRNA selected from the 5' untranslated region (5' UTR), the 3' untranslated region (3' UTR), and a coding region. In some embodiments, all three siRNAs target respective three different mRNAs and/or small RNAs (e.g., miRNAs). In some embodiments, at least one of the three siRNAs targets an miRNA. In some embodiments, the three targets are included in a common pathway, providing synergistic inhibition of the biological pathway. In some embodiments, the three target RNAs are viral RNA sequences. In some embodiments, the three target RNA sequences are involved in cell survival or can induce cell cycle arrest.

As mentioned above, in the described embodiments, the 3’ ends of the polynucleotides (which are the 3' ends of the siRNA sense strands) can have one or more chemical modifications, which can be different chemical modifications. Moreover, in the described embodiments, at least two of the three 3’ ends are different by a length of the 3’ overhang and/or chemical modification.

In some embodiments, at least one chemical modification of a 3’ end comprises at least one cell penetrating or cell targeting moiety, which can be at least one of a lipophilic moiety and a non-lipophilic moiety. Exemplary lipophilic moieties include cholesterol and cholesterol derivatives (including cholestenes, cholestanes, and cholestadienes), bile acids (such as cholic acid, deoxycholic acid and dehydrocholic acid), sterols, steroids, or other fat-soluble alcohol or thiol. Various lipophilic moieties can be conjugated at the 5' and/or 3' end of the polynucleotides, or along the polynucleotide backbone. In some embodiments, the lipophilic moieties can be conjugated directly or indirectly (e.g., through a linker) to the 3' end. In some embodiments, lipophilic moieties can be conjugated through a hydrocarbon moiety or other linker. In some embodiments, the lipophilic moiety can be an alcohol, such as a sterol, and, in such embodiments, the lipophilic moiety can be etherized with a hydrocarbon having a suitable terminal functional group.

In some embodiments, the lipophilic moiety is at least one cholesterol. In some embodiments, at least one of the three siRNAs comprises a cholesterol moiety conjugated at the 3’ end thereof. In some embodiments, at least one of the three siRNAs comprises one, two, or three cholesterols conjugated at the 3’ end thereof. In some embodiments, all three siRNAs each have at least one cholesterol conjugated at the 3’ end thereof. In some embodiments, all three siRNAs each have at least two cholesterols conjugated at the 3’ end thereof. In some embodiments, the at least one cholesterol moiety is attached to the 3' end of the sense strand of the siRNA.

In some embodiments, the non-lipophilic moiety comprises at least one N-acetylgalactosamine (GalNac) moiety. In some embodiments, the at least one GalNac moiety can be a multivalent (e.g., trivalent) GalNac moiety, and“a GalNac moiety” can include multivariate moieties. Multivalent GalNac moieties are described in U.S. Patent Application Publication No. 2011/0077386, which is hereby incorporated herein by reference in its entirety.

In some embodiments, at least one of the siRNAs comprises at least one GalNac moiety. In some embodiments, two or each of the three siRNAs comprises at least one GalNac moiety conjugated at the 3’ end thereof.

In some embodiments, at least two of the three siRNAs comprise respective different cell penetrating moieties. At least two of the three siRNAs can also differ by a number and/or type of cell penetrating moieties. For example, one of the siRNAs can have two cholesterol moieties, whereas another one of the siRNAs can have three cholesterol moieties. Additionally or alternatively, at least two of the three siRNAs can have respective cell penetrating moieties of different types. In some embodiments, all of the three siRNAs have respective different cell penetrating moieties. Moreover, in some embodiments, all of the three siRNAs have the same cell penetrating moieties. For example, all of the siRNA can have at least one or at least two cholesterol moieties at the 3’ end thereof. As another example, all of the siRNAs can have at least one GalNac moiety conjugated at the 3’ end thereof. In some embodiments, such chemical modifications comprising at least one GalNac moiety can facilitate delivery of the tiRNA to hepatocytes.

In some embodiments, the at least one chemical modification of at least one of the three 3’ ends of siRNAs comprises a modification in which the phosphate backbone of at least one nucleotide is replaced by any one or more of a phosphorothioate form (phosphorothioate (PS) moiety), phosphorodithioate form, alkylphosphonate form, phosphoroamidate form, and boranophosphate form. For example, in some embodiments, the chemical modification of at least one of the three 3’ ends comprises at least one PS or phosphorodithioate moiety. In some embodiments, the 3’ end of each of the three polynucleotides comprises at least one, at least two, at least three, or at least four PS or phosphorodithioate moieties. “Phosphorothioates” are a variant of normal DNA or RNA linkage in which one of the nonbridging oxygens is replaced by a sulfur.

Additionally, in some embodiments, the chemical modification may be obtained by replacing at least one nucleotide included in the tiRNA by any one of LNA (locked nucleic acid), UNA (unlocked nucleic acid), morpholino, and PNA (peptide nucleic acid).

In some embodiments, at least two of the three 3’ ends of the siRNAs are different by a number of PS or phosphorodithioate linkages. For example, in some embodiments, at least one of the three 3’ ends comprises at least three or four PS or phosphorodithioate linkages. In some embodiments, at least two of the three 3’ ends each has at least three or four PS or phosphorodithioate linkages. In some embodiments, each of the three 3’ ends has at least three or four PS or phosphorodithioate linkages. Also, in some embodiments, at least one of the three 3’ ends has three or four PS or phosphorodithioate linkages. In some embodiments, one, two, or all three of the 3’ ends can each have one, two, three, four, or more than four PS or phosphorodithioate linkages.

As mentioned above, in the described embodiments, each of the siRNAs has a 3’ overhang, and at least two of the three 3’ ends are different by lengths of their respective 3’ overhangs. The 3’ overhangs can be independently selected from various lengths. For example, in some embodiments, at least one of the three 3’ ends has a 3’ overhang of 2 nt in length. In some embodiments, at least one of the three 3’ ends has a 3’ overhang of 3 nt in length. In some embodiments, at least one of the three 3’ ends has a 3’ overhang of 4 nt in length. Furthermore, in some embodiments, at least one of the three 3’ ends of the siRNAs has a 3’ overhang of more than 4 nucleotides in length. In some embodiments, all of the three 3’ ends of the siRNAs have 3’ overhangs of different lengths. The 3’ overhangs of different lengths can be selected from the 3’ overhangs of 2, 3, 4, or greater than 4 nucleotides in length. In some embodiments, the 3’ overhangs of different lengths can be selected from 3’ overhangs of at least 4 nucleotides in length.

In addition to the one or more chemical modifications of the siRNAs 3’ ends comprising at least one cell- penetrating or cell-targeting moiety and/or at least one PS or phosphorodithioate moiety, another type of a chemical modification can additionally or alternatively be at least one 2' chemical modification. Such a modification can be obtained by replacing the hydroxyl group at position 2' of ribose of at least one nucleotide by any one of a hydrogen atom, a fluorine atom, an -O-alkyl group, an—O-acyl group and an amino group, but is not limited thereto. In order to increase the ability to deliver the tiRNA, the hydroxyl group may be replaced by any one of halogen (e.g.,— Br,—Cl, --F)— R,— R'OR,— SH,—SR,— N3 and—ON (R=alkyl, aryl, or alkylene).

In some embodiments, at least one of the siRNAs of a tiRNA can have at least one 2' chemical modification at a junction of the Y-shaped structure of the tiRNA. The 2' chemical modification can be one or more 2'-0- alkyl (e.g., 2'-OMe) nucleosides which carry a O-methyl group at the 2'-OH group of the ribose molecule. 2'- OMe-RNAs show the same (or similar) behavior as RNA, but are protected against nuclease degradation. Thus, in some embodiments, at least one of the three siRNAs comprises a plurality of 2’-0-modified (e.g., 2'- OMe) nucleosides (e.g., 2, 3, 4, 5, 6, or greater than 6 2'-0-modified nucleosides), which can be disposed at the junction of the Y-shaped structure, and/or at other portions of the siRNAs. In some embodiments, the 2'- O-modified nucleosides are positioned on both antisense and sense strands of each of the siRNAs. Thus, in some embodiments, the tiRNA comprises a plurality of 2'-0-modified nucleosides (e.g., 2, 3, 4, 5, 6, or greater than 6 2'-0-modified nucleosides, such as 2’-OMe nucleosides). For example, in some embodiments, at least six nucleotides of the tiRNA (with two nucleotides of each of the three siRNAs) forming the junction of the Ύ” of the tiRNA can each have a 2'-OMe group. In some embodiments, the tiRNA comprises 2'-0-methylated nucleosides that alternate with unmodified nucleosides.

In some embodiments, a 2'-0-modified (e.g., 2’-OMe) nucleoside is positioned at the 3' terminal region of the sense strand of a siRNA. In some embodiments, 3' terminal region of the sense strand comprises a plurality of 2'-0-modified nucleosides (e.g., 2, 3, 4, 5, 6, or greater than 6 2'-0-modified nucleosides, such as 2’-OMe). In some embodiments, the 3' terminal region of the sense strand comprises 2'-OMe nucleosides that alternate with unmodified nucleosides. In some embodiments, a tiRNA can have at least one of its siRNAs having the 3’ overhang of 2 nt in length, a cholesterol moiety conjugated to the 3’ end, and at least three PS moieties at the 3’ end thereof. At least one other of the siRNAs can have a 3’ overhang of 4 nucleotides in length, a cholesterol moiety conjugated to the 3’ end thereof, and at least four PS moieties at the 3’ end thereof.

In at least one embodiment, a tiRNA can have at least one of the siRNAs having a 3’ overhang of 2 nucleotides in length, a cholesterol moiety conjugated to the 3’ end thereof, and at least three PS moieties at the 3’ end thereof. At least one other of the siRNAs can have a 3’ overhang of 4 nt in length, a cholesterol moiety conjugated to the 3’ end thereof, and four PS moieties at the 3’ end thereof.

In at least one embodiment, a tiRNA can have a first siRNA, a second siRNA, and a third siRNA. The first siRNA can have a 3’ overhang of 2 nt in length and does not include a cholesterol or a PS moiety. The second siRNA can have a 3’ overhang of 2 nt in length, a cholesterol moiety conjugated to the 3’ end thereof, and at least three PS moieties at the 3’ end thereof. The third siRNA can have the 3’ overhang of 4 nt in length, a cholesterol moiety conjugated to the 3’ end thereof, and at least four PS moieties at the 3’ end thereof.

In some embodiments, the one or more chemical modifications of at least one of siRNAs of a tiRNA can be selected to reduce expression of each of the target RNAs by at least 25%, 30%, at least 35%, at least 40%, at least 45%, at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, or by at least 75%. In some embodiments, the one or more chemical modifications of at least one of siRNAs of a tiRNA can be selected to reduce expression of at least two of the target RNAs by at least 25%, 30%, at least 35%, at least 40%, at least 45%, at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, or by at least 75%.

FIG. 1 illustrates an example of a tiRNA in the form of a Y-shaped structure in accordance with some embodiments. As shown in FIG. 1 , the tiRNA is formed by annealing or hybridizing together of three polynucleotides (e.g., RNAs), into the Y-shaped structure. In this example, the three polynucleotides are selected for targeting expression of three respective target RNAs by RNA interference. The three targets can be three different target RNAs, or at least two of the targets can be different target RNAs. In some cases, at least two of the targets can be the same mRNA, and, in some embodiments, each of the targets can be a respective different region (e.g., the 5' untranslated region (5' UTR), the 3' untranslated region (3' UTR), or a coding region) of the same mRNA.

As shown in FIG. 1 , each of the three single-stranded RNAs can be about 40 nucleotides in length, including a 2-nt overhang. The three single-stranded RNAs are annealed together to form three siRNAs joined together at a junction of the Y-shaped structure. Each siRNA has a double-stranded region of 19 bp in length and the 2-nt overhang at the 3' end thereof.

FIG. 1 illustrates that the first polynucleotide in the tiRNA includes a first antisense region extending from the 5' end of the first polynucleotide, which targets a first target RNA by RNA interference. The first polynucleotide is about 40 nt in length including, from its 5' end, 19 nt that anneal to the third polynucleotide (to thereby form the first siRNA), and subsequent 19 nt that anneal to the second polynucleotide (to thereby form the third siRNA), plus the 2-nt overhang at the 3' end of the first polynucleotide. In this example, each of the second and third polynucleotide sequences is also about 40 nt in length, which are annealed to the other two polynucleotides, forming, together with the first polynucleotide, the three siRNAs.

In some embodiments, the tiRNA in accordance with embodiments of the present disclosure comprises one or more chemical modifications. For example, the chemical modification may be the one in which the hydroxyl group at the 2' position of the ribose of at least one nucleotide is replaced by any one of an—O-methyl (2' OMe) group, a hydrogen, a halogen group (e.g., fluorine), an—O-alkyl group, an—O-acyl group, or an amino group. In the example of FIG. 1 , the tiRNA includes at least six 2' OMe groups (shown with an additional line drawn adjacent to the line representing polynucleotide strands), introduced into the tiRNA such that at least two nucleotides at each duplex extending from the junction of the Y-structure has the modification. For example, at least two nucleotides of each of the first, second, and third RNA strands can have 2' OMe chemical modification, e.g., at least one nucleotide at each side of the junction the Y-shaped structure, as shown in FIG. 1. In some embodiments, each of the single-stranded RNAs hybridized into the tiRNA can have more than two 2' OMe groups - e.g., 3, 4, 5, 6, or greater than 6 2' OMe groups. Also, one or more 2' OMe groups can be disposed at other portions of the tiRNA, including portions that are farther away from the junction of the Y-shaped structure.

Furthermore, the tiRNA shown in FIG. 1 can have other 2' chemical modifications, including those in which the hydroxyl group at the 2' position of the ribose of 1 , 2, 3, 4, 5, 6, or greater than 6 nucleotides have been replaced by a hydrogen, a halogen such as fluorine, an— O-alkyl group, an—O-acyl group, or an amino group.

In some embodiments, a tiRNA in accordance with the present disclosure can be a variant of the tiRNA shown in FIG. 1. For example, a Y-shaped tiRNA can have various chemical modifications. At least one chemical modification can be, e.g., at least one cell-penetrating or cell-targeting moiety, which can be a lipophilic moiety (e.g., cholesterol moiety) and a non-lipophilic moiety (e.g., GalNac moiety). Additionally or alternatively, the chemical modification can be at least one PS or phosphorodithioate moiety at the 3’ end of each of the three polynucleotides, or any other type of a chemical modification. The tiRNA can be designed to target any three suitable targets. The siRNAs forming“arms” of the tiRNA can have various lengths, including lengths that differ from one another within the same tiRNA.

FIG. 2 illustrates an embodiment of a tiRNA having at least one cholesterol moiety conjugated at the 3’ end of at least one of the three polynucleotides, i.e., 3’ ends of the siRNAs formed by hybridizing the three polynucleotides together. In particular, in this example, as shown in FIG. 2, the tiRNA includes a cholesterol moiety conjugated at the 3’ end of each of the polynucleotides. As shown in FIG. 2, the tiRNA also comprises three PS moieties at the 3’ end of each of the siRNAs. Also, similar to the tiRNA of FIG. 1 , each of the polynucleotides of the tiRNA shown in FIG. 2 includes six 2' OMe nucleosides at the junction of the Y-shaped structure.

As mentioned above, a tiRNA in accordance with the present disclosure can have an overhang at the 3’ end of at least one of the three polynucleotides, and, optionally, all three polynucleotides, and which may be of independently selected lengths. The 3’ end overhang can have any suitable number of nucleotides (e.g., from 1 to 5 or from 2 to 4), including a different number of overhang nucleotides among at least two of the three polynucleotides. Thus, FIG. 3 illustrates an example of a tiRNA comprising a 4-nt overhang at the 3’ end of each of the three polynucleotides hybridized into the Y-shaped structure. In some embodiments, a tiRNA can have an independently selected 2, 3, 4, 5, 6, 7, 9, or 10-nt overhang at the 3’ end of each of the polynucleotides. The tiRNA of FIG. 3 also comprises a cholesterol moiety and four PS moieties at the 3’ end of each of the three polynucleotides. Also, similar to the tiRNAs of FIGS. 1 and 2, each of the polynucleotides of the tiRNA shown in FIG. 3 includes six 2' OMe nucleosides at the junction of the Y-shaped structure.

In some embodiments, a tiRNA in accordance with the present disclosure comprises at least three or at least four PS moieties at the 3’ end of at least one of the three RNA strands. In some embodiments, the tiRNA comprises at least five, at least six, at least seven, at least eight, at least nine, or at least ten PS or phosphorodithioate moieties at the 3’ end of at least one of the three polynucleotides.

As mentioned above, in a tiRNA in accordance with the present disclosure, a cell-penetrating or cell-targeting moiety conjugated to the 3’ end of at least one of the three polynucleotides hybridized into a Y-shaped structure, can be a non-lipophilic moiety. In some embodiments, the non-lipophilic moiety can be a GalNac moiety (e.g., multivalent or trivalent GalNac moiety) conjugated to the 3’ end of at least one of the three polynucleotides. FIG. 4 illustrates an example of a tiRNA comprising at least one GalNac moiety conjugated to the 3’ end of each of the three polynucleotides. As shown in FIG. 4, each of the 3’ ends also comprises three PS moieties, and each of the polynucleotides includes six 2' OMe nucleosides at the junction of the Y- shaped structure. The GalNac moiety can facilitate liver-specific delivery of the tiRNA.

In some embodiments, a method of preparing a tiRNA is provided, which comprises hybridizing three single stranded polynucleotides together. Each two of the three polynucleotides are base paired together such that the combination of three polynucleotides results in the formation of a Y- or tripod-shaped complex encompassing three arms in the form of double-stranded siRNAs. Each of the siRNAs can be designed to modulate expression of a respective target, such that the tiRNA targets expression of three targets. In each siRNA, an antisense sequence against a target mRNA is paired with a complementary sense sequence from another polynucleotide, forming siRNAs. Thus, the tiRNA structure is a combination of three different siRNAs held together in the Y-shaped structural format. The tiRNA structure is designed as a cell-penetrating tiRNA comprising at least one chemical modification, including at least one cell-penetrating moiety, and the tiRNA has the ability to penetrate a cellular membrane without the aid of a separate delivery vehicle. In some embodiments, the tiRNA is formulated as a pharmaceutical composition for delivery to a subject, without a cationic delivery vehicle.

The polynucleotides may be chemically or enzymatically synthesized molecules. In some embodiments, the antisense regions may be substantially complementary at the nucleotide sequence level to at least a portion of the target RNA, and in some embodiments are fully complementary to the target. In some embodiments, the antisense region has one or two mismatches with the target. For example, in some embodiment, an antisense region of each of the three RNA interference-inducing siRNAs of the tiRNA may be at least 90% or 100% complementary to the respective target RNA, e.g., to an mRNA sequence of a target gene or to the respective non-coding RNA. In some embodiments, the target gene may be an endogeneous gene or a transgene.

A plurality of tiRNA structures can be designed in accordance with embodiments of the present disclosure and tested, and a tiRNA exhibiting reduction of expression of at least two of the target RNAs by a certain degree (e.g., at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%) can be selected for formulation in a pharmaceutical composition. In the described embodiments, the tiRNA can silence or reduce expression of three genes simultaneously, and it can be delivered into cell in the absence of a separate intracellular delivery vehicle. Thus, the tiRNA can be a tool for targeting multiple disease genes with enhanced therapeutic efficacy.

In some embodiments, a method of making a pharmaceutical composition for reducing expression of one or more target RNAs is provided. The method includes providing a plurality tiRNAs, testing the plurality of tiRNAs for the ability to reduce expression of the target RNAs in a cell line or in an animal model, selecting a tiRNA from the plurality of tiRNAs that inhibits expression of the target RNAs by at least 50%, and formulating the selected tiRNA as a pharmaceutical composition for delivery. Each tiRNA in the plurality tiRNAs is a tiRNA according to any of the embodiments of the present disclosure, or a combination of any of the embodiments.

The pharmaceutical composition can further comprise a pharmaceutically acceptable carrier, excipient or diluent. The tiRNA may be included in the pharmaceutical composition in a pharmaceutically effective amount according to a disease, severity of the disease, a patient's age, gender, weight, health condition, a route of administration, a period of treatment, and/or other factors.

As used herein, the term “pharmaceutically acceptable” refers to a composition that is physiologically acceptable and does not cause gastric disorder, allergic reactions such as gastrointestinal disorder or vertigo, or similar reactions, when administered to humans. Examples of the carrier, excipient or diluent may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oils.

The pharmaceutical composition may additionally include fillers, anti-aggregating agents, lubricants, wetting agents, perfumes, emulsifiers and preservatives. Also, the pharmaceutical composition of the present disclosure may be formulated using a method well known in the art, such that it can provide a rapid, sustained, or delayed release of the active ingredient after administration to a subject (e.g., a mammal such as human, or a veterinary or zoological subject). The formulation may be in the form of an injectable solution, e.g., example an aqueous or non-aqueous sterile injection solution.

The pharmaceutical composition may be adapted for administration by any appropriate route, for example by the oral (including enteral, buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route. Such compositions may be prepared by any method known in the art of pharmacy, for example by admixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions. In some embodiments, a method of inhibiting or treating a disease or disorder is provided. The method comprises administering a cell-penetrating RNA interference-inducing tiRNA prepared in accordance with any of the embodiments of the present disclosure, or a combination of any of the embodiments.

In addition, the tiRNA in accordance with the present disclosure can be developed into therapeutic agents against localized diseases, and may be used together with various known cell-specific antibodies, aptamers, ligands or the like, and thus can be developed into therapeutic agents for gene regulation, which exhibit gene silencing effects only in a desired area.

In some embodiments, the disease or disorder comprises cancer. The cancer can be solid tumor or hematological cancer. Non-limiting examples of solid tumor include breast cancer, colon cancer, ovarian cancer, rectal cancer, pancreatic cancer, lung cancer, prostate cancer, gastric cancer, renal cell carcinoma, and liver cancer. Non-limiting examples of hematological cancer include lymphoma, leukemia, and myeloma.

In some embodiments, the cancer is selected from basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, cancer of the peritoneum, cervical cancer, choriocarcinoma, colon and rectum cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, cancer of the head and neck, gastric cancer (including gastrointestinal cancer), glioblastoma, hepatic carcinoma, hepatoma, intra-epithelial neoplasm, kidney or renal cancer, larynx cancer, leukemia, liver cancer, lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), melanoma, myeloma, neuroblastoma, oral cavity cancer (lip, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, cancer of the respiratory system, salivary gland carcinoma, sarcoma, skin cancer, squamous cell cancer, stomach cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, cancer of the urinary system, vulval cancer, lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate grade/follicular NHL, intermediate grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non- cleaved cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's Macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), Hairy cell leukemia, chronic myeloblastic leukemia, as well as other carcinomas and sarcomas, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs’ syndrome. In some embodiments, the disease or disorder comprises age-related macular degeneration (AMD), which can be wet AMD or dry AMD. In some embodiments, the tiRNA for treatment or prevention of AMD (wet or dry) can be administered by intravitreal injection.

In some embodiments, the disease or disorder comprises a viral infection.

In some embodiments, the disease or disorder that can be prevented and/or treated using a tiRNA in accordance with the present disclosure comprises a connective tissue growth factor (CTGF)-associated disease or disorder. The CTGF-associated disease or disorder can be keloid, hypertrophic scar, or fibrosis. These diseases have only limited treatment options. Hypertrophic scars and keloids can form as a result of wound healing (e.g., after an injury or infection) or as an autoimmune process. The keloid can be a burn keloid, posttraumatic keloids, and other types of abnormal proliferation of scar tissue. Fibrosis, keloid, and hypertrophic scars are characterized by increased expression of transforming growth factor (TFG)-beta. CTGF is a downstream mediator of TGF-beta activity that is associated with scarring and fibrosis.

Non-limiting examples of fibrosis include kidney fibrosis, retinal fibrosis, pulmonary fibrosis, hepatic fibrosis, systemic sclerosis, pachydermatosis, or dermatofibrosis.

In some embodiments, a tiRNA can be made such that it inhibits tyrosinase expression by a cell and can therefore be used for reducing melanin production and treating pigmentation-related disorders, including melasma and age spots. A pharmaceutical composition can be made including such tiRNAs, and the pharmaceutical composition can be used for inhibiting melanin production by a cell (e.g., a melanocyte) to thereby treat a pigmentation-related disorder.

In some embodiments, the tiRNA is administered (e.g., in a pharmaceutical composition) using a suitable invasive or non-invasive method. For example, in some embodiments, the tiRNA is administered by injection, e.g., intravitreal, intravenous, or another type of injection (e.g., intra-peritoneal, intramuscular, etc.). In some embodiments, the tiRNA is administered (e.g., in a pharmaceutical composition) intranasally (e.g., by intranasal inhalation). The tiRNA can be administered to a subject in need thereof. The present invention has both human medical and veterinary applications. Accordingly, the term“subject” refers to a human or a non-human mammal or other veterinary subject.

In the described embodiments, a tiRNA can be delivered into cells without the aid of a delivery vehicle. Accordingly, the tiRNA may have enhanced intracellular delivery capability. It should be appreciated, however, that, in some embodiments, the tiRNA can be complexed with a cationic cell delivery vehicle such as, e.g., a cationic polymer or a cationic lipid. For example, liposomes, such as, e.g., polyethylenimine (PEI) or Lipofectamine 2000 (Invitrogen), may be used as the cationic cell delivery vehicle, but it will be apparent to a person skilled in the art in view of this disclosure that any positively charged delivery agent may be used in embodiments in which a delivery vehicle is desired.

Definitions

As used herein,“a,”“an,” or“the” can mean one or more than one.

Further, the term“about” when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 10% of that referenced numeric indication. For example, the language “about 50” covers the range of 45 to 55.

An“effective amount,” when used in connection with medical uses is an amount that is effective for providing a measurable treatment, prevention, or reduction in the rate of pathogenesis of a disease of interest.

As referred to herein, all compositional percentages are by weight of the total composition, unless otherwise specified. As used herein, the word “include,” and its variants, is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the compositions and methods of this technology. Similarly, the terms“can” and“may” and their variants are intended to be non-limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present technology that do not contain those elements or features.

Although the open-ended term“comprising,” as a synonym of terms such as including, containing, or having, is used herein to describe and claim the invention, the present invention, or embodiments thereof, may alternatively be described using alternative terms such as“consisting of” or“consisting essentially of.”

As used herein, the words“preferred” and“preferably” refer to embodiments of the technology that afford certain benefits, under certain circumstances. Flowever, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the technology.

The amount of compositions described herein needed for achieving a therapeutic effect may be determined empirically in accordance with conventional procedures for the particular purpose. Generally, for administering therapeutic agents for therapeutic purposes, the therapeutic agents are given at a pharmacologically effective dose. A “pharmacologically effective amount,” “pharmacologically effective dose,”“therapeutically effective amount,” or“effective amount” refers to an amount sufficient to produce the desired physiological effect or amount capable of achieving the desired result, particularly for treating the disorder or disease. An effective amount as used herein would include an amount sufficient to, for example, delay the development of a symptom of the disorder or disease, alter the course of a symptom of the disorder or disease (e.g., slow the progression of a symptom of the disease), reduce or eliminate one or more symptoms or manifestations of the disorder or disease, and reverse a symptom of a disorder or disease. Therapeutic benefit also includes halting or slowing the progression of the underlying disease or disorder, regardless of whether improvement is realized.

Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to about 50% of the population) and the ED50 (the dose therapeutically effective in about 50% of the population). The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50. In some embodiments, compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from in vitro assays, including, for example, cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 as determined in cell culture, or in an appropriate animal model. Levels of the described compositions in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.

As used herein, “methods of treatment” are equally applicable to use of a composition for treating the diseases or disorders described herein and/or compositions for use and/or uses in the manufacture of a medicaments for treating the diseases or disorders described herein.

EXAMPLES

Hereinafter, the present invention will be described in further detail with reference to examples. It will be obvious to a person having ordinary skill in the art that these examples are illustrative purposes only and are not to be construed to limit the scope of the present invention. In addition, it will be apparent to those skilled in that art that various modifications and variations can be made without departing from the technical scope of the present invention. In the Examples below, Lamin A/C, DBP, and TIG3 genes are illustrated as target genes. It should be appreciated, however, that the inventive tiRNAs, pharmaceutical compositions, and methods of making thereof, relate to reducing expression or silencing of any target genes.

Example 1

Preparation of tiRNAs

The cell-penetrating tiRNAs (cp-tiRNAs) used in the described experiments were made by purchasing chemically synthesized RNA sequences from Dharmacon, Inc., and then hybridizing three RNA sequences according to the manufacturer's protocol. For each cp-tiRNA, three RNAs were hybridized together so as to form a Y-shaped structure encompassing three small interfering RNAs (siRNAs) having 3’ overhangs. In this example, 27 tiRNAs were created based on various combinations of nine RNAs, such that each tiRNA was constructed for targeting mRNAs of three different genes - Lamin A/C or Lamin (“L”), DBP (“D”) and TIG3 (“T”) mRNAs. FIGS. 5A to 5C illustrate the nine RNAs hybridized into three tiRNAs for targeting L, D, and T. In particular, FIG. 5A shows a tiRNA for targeting L1 , D1 , and T1 ; FIG. 5B shows a tiRNA for targeting L2, D2, and T2; and FIG. 5C shows a tiRNA for targeting L3, D3, and T3.

Table 1 below summarizes the properties of each of the nine RNA sequences shown in FIGS. 5A to 5C, including a length of a 3’ overhang, presence of at least one 2^'-OMe nucleoside, presence and number of a cholesterol moieties, and presence and number of PS moieties.

Table 1

The strands for constructing the tiRNAs were as follows:

where m: 2'-O-Methyl modification, chol: cholesterol, *: phosphorothioate linkage.

FIGS. 5A to 5C illustrate three tiRNA of 27 tiRNAs made in accordance with the present disclosure. The other tiRNAs were made using various combinations of the polynucleotides shown in FIGS. 5A-5C and described in Table 1.

Example 2

Measurement of Gene Silencing Ability of cp-tiRNAs

Each of the 27 cp-tiRNAs for targeting Lamin A/C, DBP, and TIG3 mRNAs, prepared as described in Example 1 , were introduced into FleLa cells (ATCC CCL-2) by passive uptake. The FleLa cells were cultured in Dulbecco's modified Eagle's medium (Hyclone), supplemented with 10% FBS (fetal bovine serum), in a 12- well plate. Before introduction of each complex, the cells were cultured in the antibiotic-free complete medium for 24 hours until a confluency of 80% was reached.

The siRNA mixture was used at a concentration of 10 nM of each siRNA, and the tiRNAs were used at a concentration of 1 mM. 6 hours after introduction of each tiRNA, the medium was replaced with a fresh medium, and the cells were harvested at 24 hours post-treatment. The levels of Lamin, DBP and TIG3 mRNAs were measured by quantitative real-time PCR (RT-PCR). Specifically, total RNA was extracted from the cell lysate using a Trireagent kit (Favorgen) and then used as a template for cDNA synthesis which was carried out using the Applied Biosystems high-capacity reverse transcription system according to the manufacturer's protocol. Then, the expression level of each of the target genes was measured using a step-one real-time PCR system (Applied Biosystems) according to the manufacturer's protocol using primer sequences shown below (in the 5'-3' order):

For transfection control, the tiRNAs made as shown in FIGS. 5A to 5C were transfected into HeLa cells (ATCC CCL-2) using Lipofectamine 2000, according to the same method as described above. 24 hours after introduction of each tiRNA, the mRNA level of each gene was measured by RT-PCR.

FIG. 6 illustrates results of the experiments, where the x-axis shows sets of three mRNAs being silenced in the experiments, where each of the triplets includes Lamin A/C mRNA (L, shown in blue), DBP mRNA (D, shown in red), and TIG-3 mRNA (T, shown in green), in this order; and the y-axis shows the relative target gene expression level.

As shown in FIG. 6, in the no-treatment (“NT”) control experiment including siRNA mixture (siLamin, siDBP, and siTIG3), no gene silencing activity was observed. In the transfection (control) experiment using a transfection reagent (Lipofectamine 2000), the tiRNA made as shown in FIG. 5A (T1-D1 -L1) exhibited the most potent gene silencing activity, and both the tiRNAs made as shown in FIGS. 5B (T2-D2-L2) and 5C (T3- D3-L3) caused the significant (greater than 50%) reduction of expression of Lamin and particularly TIG3.

Among 27 cp-tiRNAs introduced into the cells by passive uptake (i.e., in the absence of a separate intracellular delivery vehicle such as a transfection reagent), the tiRNAs demonstrated different degrees of reduction of expression of target genes. In FIG. 6, three of the multiple tiRNAs, shown with blocks, demonstrated particularly prominent reduction of expression of the target genes, measured in relative expression levels. Specifically, in these experiments, the T3-D3-L2, T2-D1 -L3, and T2-D2-L3 tiRNAs exhibited particularly notable expression reduction activity. The T2-D2-L3 tiRNA inhibited expression of each of the target RNAs by more than 70% (at least about 75%). The T3-D3-L2 tiRNA inhibited expression of each of the respective target RNAs by at least about 50%. The T2-D1-L3 tiRNAs inhibited expression of each of the respective target RNAs by at least about 50%.

EQUIVALENTS

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.

INCORPORATION BY REFERENCE

All patents and publications referenced herein are hereby incorporated by reference in their entireties.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections.

Claims

CLAIMS What is claimed is:

1. A tripodal interfering (tiRNA) having cell-penetrating capability, the tiRNA comprising three polynucleotides for targeting expression of three respective target RNAs by RNA interference, the three polynucleotides being hybridized together into a Y-shaped structure to form three small interfering RNAs (siRNAs) having 3’ overhangs, wherein 5’ and 3’ ends of the polynucleotides are selected to reduce expression of at least two of the target RNAs by at least 50%, and wherein at least two of the three 3’ ends are different by a length of the 3’ overhang and/or chemical modification.

2. The tiRNA of claim 1 , wherein the target RNAs are mRNAs.

3. The tiRNA of claim 2, wherein at least two of the siRNAs target the same mRNA, and wherein the at least two of the siRNAs optionally target different sequences within the same mRNA selected from the 5' untranslated region (5' UTR), the 3' untranslated region (3' UTR), and a coding region.

4. The tiRNA of claim 2, wherein all three siRNAs target the same mRNA, and wherein each of the three siRNAs optionally targets a respective different sequence within the same mRNA selected from the 5' untranslated region (5' UTR), the 3' untranslated region (3' UTR), and a coding region.

5. The tiRNA of claim 2, wherein all three siRNAs target respective three different mRNAs.

6. The tiRNA of claim 1 , wherein at least one of the three siRNAs targets a microRNA (miRNA).

7. The tiRNA of any one of claims 1 to 6, wherein at least one chemical modification of a 3’ end comprises at least one cell penetrating moiety.

8. The tiRNA of claim 7, wherein the at least one cell penetrating moiety is at least one of a lipophilic moiety and a non-lipophilic moiety.

9. The tiRNA of claim 8, wherein the lipophilic moiety is at least one cholesterol.

10. The tiRNA of claim 9, wherein at least one of the three siRNAs comprises a cholesterol moiety conjugated at the 3’ end thereof.

11. The tiRNA of any one of claims 7 to 10, wherein the non-lipophilic moiety comprises at least one N- acetylgalactosamine (GalNac) moiety.

12. The tiRNA of claim 11 , wherein the at least one GalNac moiety comprises a multivalent GalNac moiety.

13. The tiRNA of claims 11 or 12, wherein at least one of the three siRNAs comprises one GalNac moiety conjugated at the 3’ end thereof.

14. The tiRNA of any one of claims 7 to 13, wherein at least two of the three siRNAs comprise respective different cell penetrating moieties.

15. The tiRNA of any one of claims 7 to 14, wherein all of the three siRNAs comprise respective different cell penetrating moieties.

16. The tiRNA of any one of claims 1 to 15, wherein the chemical modification comprises at least one phosphorothioate (PS) or phosphorodithioate moiety.

17. The tiRNA of claim 16, wherein at least two of the three 3’ ends of the siRNAs are different by a number of PS or phosphorodithioate moieties.

18. The tiRNA of claims 16 or 17, wherein at least one of the three 3’ ends of the siRNAs comprises at least three or four PS or phosphorodithioate moieties.

19. The tiRNA of claims 16 or 17, wherein at least two of the three 3’ ends of the siRNAs each comprise at least three or four PS or phosphorodithioate moieties.

20. The tiRNA of claims 16 or 17, wherein each of the three 3’ ends of the siRNAs comprises at least three or four PS or phosphorodithioate moieties.

21. The tiRNA of any one of claims 1 to 20, wherein at least one of the three 3’ ends of the siRNAs has a 3’ overhang of 2 nucleotides in length.

22. The tiRNA of any one of claims 1 to 20, wherein at least one of the three 3’ ends of the siRNAs has a 3’ overhang of 3 nucleotides in length.

23. The tiRNA of any one of claims 1 to 20, wherein at least one of the three 3’ ends of the siRNAs has a 3’ overhang of 4 nucleotides in length.

24. The tiRNA of any one of claims 1 to 20, wherein at least one of the three 3’ ends of the siRNAs has a 3’ overhang of more than 4 nucleotides in length.

25. The tiRNA of any one of claims 21 to 24, wherein all of the three 3’ ends of the siRNAs have 3’ overhangs of different lengths.

26. The tiRNA of claim 25, wherein the 3’ overhangs of different lengths are selected from 3’ overhangs of 2 nucleotides in length, 3 nucleotides in length, and 4 nucleotides in length.

27. The tiRNA of claim 25, wherein the 3’ overhangs of different lengths include at least one 3’ overhang of at least 4 nucleotides in length.

28. The tiRNA of any one of claims 1 to 27, wherein the at least one chemical modification comprises at least one 2' chemical modification comprising any one of a hydrogen, a halogen, an -O-alkyl group, an— O-acyl group, and an amino group.

29. The tiRNA of claim 28, wherein the at least one 2' chemical modification comprises an—O-methyl

(2'-OMe) group.

30. The tiRNA of any one of claims 1 to 29, wherein at least one of the three siRNAs has at least two— O-methyl (2'-OMe) groups at a junction of the Y-shaped structure.

31. The tiRNA of claim 30, wherein at least one of the three siRNAs has at least four 2'-OMe groups at a junction of the Y-shaped structure.

32. The tiRNA of claim 31 , wherein at least one of the three siRNAs has at least six 2'-OMe groups at a junction of the Y-shaped structure.

33. The tiRNA of any one of claims 1 to 32, wherein:

at least one of the siRNAs has a 3’ overhang of 2 nucleotides in length, a cholesterol moiety conjugated to the 3’ end thereof, and at least three phosphorothioate (PS) moieties at the 3’ end thereof; and at least one other of the siRNAs has a 3’ overhang of 4 nucleotides in length, a cholesterol moiety conjugated to the 3’ end thereof, and at least four PS moieties at the 3’ end thereof.

34. The tiRNA of claim 33, wherein:

at least one of the siRNAs has a 3’ overhang of 2 nucleotides in length, a cholesterol moiety conjugated to the 3’ end thereof, and at least three PS moieties at the 3’ end thereof; and

at least one other of the siRNAs has a 3’ overhang of 4 nucleotides in length, a cholesterol moiety conjugated to the 3’ end thereof, and four PS moieties at the 3’ end thereof.

35. The tiRNA of any one of claims 1 to 34, wherein:

a first siRNA of the three siRNAs has a 3’ overhang of 2 nucleotides in length, and does not include a cholesterol or a PS moiety;

a second siRNA of the three siRNAs has a 3’ overhang of 2 nucleotides in length, a cholesterol moiety conjugated to the 3’ end thereof, and at least three phosphorothioate (PS) moieties at the 3’ end thereof; and

a third siRNA of the three siRNAs has a 3’ overhang of 4 nucleotides in length, a cholesterol moiety conjugated to the 3’ end thereof, and at least four PS moieties at the 3’ end thereof.

36. The tiRNA of any one of claims 1 to 35, wherein the 5’ and 3’ ends are selected to reduce expression of each of the target RNAs by at least 50%.

37. A method of making a pharmaceutical composition for reducing expression of one or more target RNAs, comprising:

providing a plurality of tripodal interfering RNAs (tiRNAs), each being a tiRNA of any one of claims 1 to 36;

testing the plurality of tiRNAs for the ability to reduce expression of the target RNAs in a cell line or in an animal model;

selecting a tiRNA from the plurality of tiRNAs that inhibits expression of at least two of the target RNAs by at least 50%; and

formulating the selected tiRNA as a pharmaceutical composition for delivery.

38. The method of claim 37, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient or diluent.

39. A method of inhibiting or treating a disease or disorder, the method comprising administering a cell- penetrating tripodal interfering RNA (tiRNA) of any one of claims 1 to 36 to a subject.

40. The method of claim 39, wherein the disease or disorder comprises cancer.

41. The method of claim 40, wherein the cancer is solid tumor or hematological cancer.

42. The method of claim 41 , wherein the solid tumor is breast cancer, colon cancer, ovarian cancer, rectal cancer, pancreatic cancer, lung cancer, prostate cancer, gastric cancer, renal cell carcinoma, and liver cancer.

43. The method of claim 41 , wherein the hematological cancer is lymphoma, leukemia, and myeloma.

44. The method of claim 39, wherein the disease or disorder comprises age-related macular degeneration (AMD).

45. The method of claim 44, wherein the AMD is wet AMD or dry AMD.

46. The method of claim 39, wherein the disease or disorder comprises a viral infection.

47. The method of claim 39, wherein the disease or disorder comprises a connective tissue growth factor (CTGF)-associated disease or disorder.

48. The method of claim 47, wherein the CTGF-associated disease or disorder comprises keloid.

49. The method of claim 47, wherein the CTGF-associated disease or disorder comprises a hypertrophic scar.

50. The method of claim 47, wherein the CTGF-associated disease or disorder is fibrosis.

51. The method of claim 50, wherein the fibrosis is kidney fibrosis, retinal fibrosis, pulmonary fibrosis, hepatic fibrosis, systemic sclerosis, pachydermatosis, or dermatofibrosis.

52. The method of any one of claims 39 to 51 , wherein the tiRNA is administered by injection.

53. The method of claim 52, wherein the injection is intravitreal injection.

54. The method of claim 52, wherein the injection is intravenous injection.

55. The method of any one of claims 39 to 51 , wherein the tiRNA is administered intranasally.

56. A method of inhibiting or treating a disease or disorder, the method comprising contacting a cell with a cell-penetrating tripodal interfering RNA (tiRNA) of any one of claims 1 to 36.