Oligonucleotide constructs with improved function and therapeutics

Abstract

Divalent RNA molecules containing chemically linked siRNA molecules are provided that inhibit the expression of TGFβ1 and cyclooxygenase-2 (Cox-2) genes. The divalent molecules may be used for treating a variety of cancers in a subject, particularly in cells that express Cox-2. Pharmaceutical compositions containing the linked siRNA molecules also are provided.

Description

Attorney Docket No.4690.0096I Oligonucleotide Constructs with Improved Function and Therapeutics Related Applications

[0001] This application claims the benefit of and priority to US Provisional Patent Application No.63 / 528,024, filed July 20, 2023, the contents of which are incorporated herein by reference in their entirety. Sequence Listing

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ST.26 (XML) format and is hereby incorporated by reference in its entirety. Said Sequence List, created on July 17, 2024, is named 4690_0096I_SL and is 144 kilobytes in size. Field

[0003] Oligonucleotide constructs comprising a linker between individual RNA molecules are provided that inhibit the expression of TGF-β1 and Cox-2 genes together with pharmaceutical compositions and methods of making and using them in therapeutic applications. Background

[0004] Over the past 25 years, 18 nucleic acid therapeutics have received approval for the treatment of various diseases. These therapeutics utilize a variety of mechanisms of action, including antisense oligonucleotides (ASOs), splice-switching oligonucleotides (SSOs), RNA interference (RNAi) such as siRNAs, and RNA aptamers that target specific proteins. (1, 2A, 3) Several diseases have been targeted by this new class of drugs, including homozygous familial hypercholesterolemia, spinal muscular atrophy, Duchenne muscular dystrophy, hereditary transthyretin-mediated amyloidosis, familial chylomicronemia syndrome, acute hepatic porphyria, and primary hyperoxaluria (1). Oligonucleotide drugs and mRNA vaccines approved through 2022 include: VITRAVENE®, MACUGEN®, KYNAMRO®, EXONDYS 51®, VYONDYS 53®, SPINRAZA®, HEPLISAV-B®, ONPATTRO®, TEGSEDI®, WAYLIVRA®, GIVLAARI® (8), OXLUMO®, VILTEPSO®, AMONDYS 45®, LEQVIO®, AMVUTTRA®, COMIRNATY® and SPIKEVAX®.

[0005] The development of these oligonucleotide drugs relied heavily on chemical modifications of DNA and RNA. The currently available oligonucleotide therapeutics incorporate a limited number of first- and second-generation modifications, such as 2'-fluoro- RNA, 2'-O-methyl RNA and phosphorothioates that have been in use for over 50 years. Two additional, notable chemical modifications are 2'-O-(2-methoxyethyl)-RNA (MOE) andAttorney Docket No.4690.0096I phosphorodiamidate morpholinos (PMO), which play a crucial role in enhancing target affinity, metabolic stability, and favorable pharmacokinetic and pharmacodynamic properties of oligonucleotides. (4, 5)

[0006] Significant progress has been made in lipid formulations and GalNAc conjugation techniques for delivering oligonucleotides into cells. Both have greatly improved the delivery efficiency and long-lasting gene silencing of modified oligonucleotides, particularly in hepatocytes. (2B, 5, 6, 7)

[0007] TGF-β is a cytokine belonging to the TGF-β family, which includes other growth factors like BMPs and GDFs. TGF-β is mainly secreted and stored in the extracellular matrix as a latent complex and requires activation to bind to its receptor and perform its biological functions. It plays a critical role in embryonic development and adult homeostasis through regulation of cell growth, differentiation, and apoptosis. It is produced by many types of cells including cancer cells and has been implicated in the development and progression of various types of cancer. Overexpression of TGF-β also has been linked to various metabolic disorders and dysfunctions, including immune dysfunction and fibrosis. (10)

[0008] TGF-β has a dual role in cancer, suppressing tumor growth by inhibiting cell proliferation, inducing apoptosis, and promoting tumor growth and metastasis by stimulating angiogenesis, cell migration, and invasion. (15) It suppresses tumors in premalignant cells, while it promotes tumor growth from carcinoma cells. In the early stages of tumorigenesis, TGF-β inhibits proliferation and induces apoptosis by promoting expression of cyclin- dependent kinase (CDK) inhibitors and inhibiting MYC expression. Thus, it prevents the development of cancer by modulating the cellular environment, in addition to other effects. However, tumor cells can escape the antitumor surveillance of TGF-β by accumulating mutations in TGF-β signaling cascades. For instance, mutations in genes that encode TGF-β ligands, receptors, and SMADs, such as SMAD4, TβR I, AVCR2A, BMPR2, SMAD2, and SMAD4, have been observed in various types of cancer, including pancreatic ductal adenocarcinoma, gastric cancer, colon cancer, and gastrointestinal cancer. These mutations promote the transition of premalignant cells to a more malignant phenotype.(13)

[0009] TGF-β can also promote tumor escape by facilitating an immunosuppressive microenvironment through both adaptive and innate immunity. TGF-β signaling can control adaptive immunity by promoting the expansion of immunosuppressive Treg cells and suppressing the regulatory CD4+T cell response. It can also control the function of effector T cells, natural killer (NK) cells, macrophages, antigen-presenting dendritic cells (DCs), and granulocytes. Dysregulation of NK cell homeostasis and augmented tumor cell metastasesAttorney Docket No.4690.0096I can be promoted by mutations of SMAD4. These actions on both adaptive immunity and innate immunity form a network of negative immune regulatory inputs.

[0010] Due to its critical role in various diseases, anti-TGF-β approaches have been introduced in treatments and clinical trials have verified the therapeutic effect of TGF-β- targeted drugs on tumors and fibrotic diseases. Given its complex role in cancer, there has been significant interest in targeting TGFβ-1 as a potential therapeutic strategy to treat cancer. Several approaches have been developed, including small molecule inhibitors, monoclonal antibodies, and gene therapies, with the goal of blocking TGFβ-1 signaling and thus inhibiting tumor growth and metastasis. Preclinical studies have shown promising results in various types of cancer, including breast, pancreatic, lung, and colorectal cancer. Carrying out clinical trials targeting TGFβ-1, however, has been a challenge because of the complex and context-dependent nature of TGFβ-1 activity in cancer. Nonetheless, several clinical trials are currently ongoing, and the results of these studies will provide further insights into the potential utility of TGFβ-1 inhibitors in cancer treatment.

[0011] Combination therapy using TGF-β targeting drugs with other antigens, such as OD- L1, is a popular treatment strategy. TGF-β-induced immune tolerance and inflammatory responses can be flexibly treated by ionizing radiation combined with hyperthermia and checkpoint inhibitor therapies. Researchers have explored the potential of combination therapy of TGF-β with other siRNAs, such as COX-2 siRNA, to improve the therapeutic effect of TGF-β-targeted therapy. Combination therapy of TGF-β siRNA and COX-2 siRNA has been found to exhibit a synergistic effect in the inhibition of cancer cell proliferation and migration. (14) Multi-Targeted siRNA Compositions

[0012] RNA interference (RNAi) is a process for specific degradation of RNA sequences, which offers a convenient and direct gene knockdown or silencing effect (2A, 7, 17, 18). In natural RNA interference, an RNase III / helicase protein, Dicer, cleaves double-stranded (ds) RNA into siRNA molecules of 19-23 nucleotides with 2-nucleotide overhangs at the 3' ends. These siRNAs become a part of a ribonuclease complex known as RNA-induced-silencing- complex (RISC). Within RISC, one strand of siRNA remains associated and acts as a guide to target complementary RNA sequences. A siRNA-directed endonuclease cleaves the target RNA, thereby disabling its function. Chemically synthesized 21-25-nucleotide siRNAs exhibit RNAi effects in mammalian cells, and the thermodynamic stability of siRNA hybridization, whether at the termini or internally, significantly influences the efficacy of the molecule (19, 20, 21).Attorney Docket No.4690.0096I

[0013] The unique advantage of siRNA enables the combination of multiple siRNA duplexes to target multiple disease-causing genes in a single treatment. This is possible because all siRNA duplexes share the same chemical composition, origin and manufacturing process (19, 21-28). However, accurately predicting the RNAi activity of candidate siRNA sequences targeting disease gene mRNA remains challenging. Consequently, individual candidate siRNA polynucleotide or oligonucleotide sequences need to be generated and tested in mammalian cell cultures to assess their effectiveness in interfering with the expression of the targeted gene(s). There is a need to develop potent siRNA duplexes specifically targeting the pro-inflammatory factor TGFβ-1, and the inflammation promoter COX-2. Further, there is a demand for the formulation of multi-targeted siRNA compositions. Accordingly, an improved therapeutic approach that includes these features is being sought for subjects with solid tumor diseases. Histidine-Lysine Polymer (HKP) carriers have been formulated with siRNA molecules into nanoparticles and have shown promise for in vivo siRNA delivery in various tissues, including tumors in ocular, brain, lung and joint tissues. (26-28).

[0014] Compared to the nanoparticle delivery formulation of mixture of two separate siRNAs targeting two different genes, the divalent, linked siRNA molecule approach provides a unique and convenient means of delivering siRNAs against at least two different gene targets at the same time to the same cell type, irrespective of whether the linker is cleaved intracellularly. Summary

[0015] What is provided is a divalent double stranded RNA molecule containing a first RNA molecule covalently linked to a second RNA molecule, where the first RNA molecule targets the expression of TGFβ-1 and the second RNA molecule targets expression of Cox-2. The covalent link may be, for example, an intracellularly cleavable disulfide linker. Alternatively, the covalent linker contains an ethylene glycol (PEG) moiety that is not intracellularly cleavable.

[0016] The divalent RNA molecule may contain chemically-linked RNA molecules that are double stranded siRNA molecules. Each of the chemically-linked siRNA molecules may, for example, contain one or more modified nucleotides selected from the group consisting of 2’- fluoro, 2’-O-methyl, phosphate, phosphorothioates, vinyl phosphonate, methyl vinyl phosphonate, MOE, phosphorodiamidate morpholinos (PMO), 2'-methoxyethoxy, 2'-fluoro, 2'-allyl, 2'-O-[2-(methylamino)-2-oxoethyl], 4'-thio, 4'-CH2-O-2'-bridge, 4'-(CH2)2-O-2'- bridge, 2'-LNA, 2'-amino and 2'-O--(N-methylcarbamate) ribonucleotide.Attorney Docket No.4690.0096I

[0017] In some embodiments, the linked RNA molecules are chemically linked through only their sense strands, or through only their antisense strands. The strands may be chemically linked at their 5’ ends, at their 3’ ends, or between a 5’ end of a first RNA molecule and a 3’ send of a second RNA molecule.

[0018] The divalent RNA molecule may contain strands selected from the group consisting of SEQ ID Nos.3-14 or 15-18. 13. Advantageously the divalent RNA molecule contains strands having the sequences of SEQ ID No.8 and / or SEQ ID No.9, and / or SEQ ID No.6 and / or SEQ ID No.4.

[0019] In some embodiments the antisense strand or strands are at least 80%, 90%, 95% or 99% complementary to the selected sense strands.

[0020] Further provided are compositions containing: the divalent RNA molecules as described above further containing a transfection enhancing agent, such as lipofectamine and / or a histidine-lysine copolymer.

[0021] Also provided are pharmaceutical compositions containing the divalent RNA molecule as described above and a pharmaceutically acceptable carrier, diluent, or excipient.

[0022] The pharmaceutical composition may contain a divalent siRNA molecule containing: two cleavably linked siRNA molecules selected from the group consisting of SEQ ID Nos.3- 14, and complementary antisense sequences selected from the group consisting of SEQ ID Nos.15-18, in which the composition also contains a histidine-lysine copolymer carrier, where the carrier is mixed with the divalent siRNA molecule to form nanoparticles. The nanoparticles may have an average diameter selected from the group consisting of 80-400nm, 100-300 nm, 130-250nm, and 150-200 nm. The histidine-lysine copolymer may have the formula (R)K(R)-K(R)-(R)KCX), where R=KHHHKHHHKHHHKHHHK (SEQ ID NO: 33), R=KHHHKHHHKHHHHKHHHK (SEQ ID NO: 34), X=C(O)NH2, K=lysine, and H=histidine.

[0023] Also provided are pharmaceutical compositions where the divalent siRNA molecule contains a first siRNA targeting the TGFβ-1 gene cleavably linked to a second siRNA targeting the Cox-2 gene, in which the link between the siRNA molecules is a disulfide bond.

[0024] Methods of treating a subject suffering from cancer using the pharmaceutical composition as described above are also provided, by administering to the subject a pharmacologically effective amount of the pharmaceutical composition, where administration of the composition results in the reduction in expression of at least one of TGFβ-1 or Cox-2 mRNAs in the subject. The reduction in mRNA expression may be at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%.Attorney Docket No.4690.0096I These methods of treatment may result in a reduction in cancer tumor volume of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%. The subject may be a mammal and, advantageously, is a human. Brief Description of the Drawings

[0025] FIG.1A shows the general structure of divalent siRNA molecules covalently linked through their sense strands. FIG.1B shows the chemical structure of the molecule, with the linker designed as either cleavable (L3=C6SSC6) or non-cleavable PEG (L4). The linkers may be tested to measure how each linker impacts the functionality of the siRNAs.

[0026] FIG.2A shows TGFβ1 gene expression in A549 cells 48 hours following transfection using serial dilutions of TGFβ1, TGFβ1-Cox2 hetero-dual, Cox2-TGFβ1 hetero-dual, or TGFβ1 homo-dual siRNAs. W = wild type; Wd = wild type di divalent siRNA. FIG.2B shows the effect of TGFβ1, TGFβ1-Cox2 hetero-dual, Cox2-TGFβ1 hetero-dual, and TGFβ1 homo-dual siRNAs on IC50values.

[0027] FIG.3 shows Cox2 gene expression in A549 cells 48 hours following transfection using serial dilutions of Cox2, TGFβ1-Cox2 hetero-dual, Cox2-TGFβ1 hetero-dual, or Cox2 homo-dual siRNAs.

[0028] FIG.4A shows TGFβ1 gene expression in HepG2 cells 48 hours following transfection using serial dilutions of TGFβ1, TGFβ1-Cox2 hetero-dual, Cox2-TGFβ1 hetero- dual, or TGFβ1 homo-dual siRNAs. FIG.4 B shows the effect of the four siRNA molecules on IC50values.

[0029] FIG.5 shows A549 cell viability after 72 hours following transfection using serial dilutions of TGFβ1 and Cox2 single and dual siRNAs.

[0030] FIG.6 shows HepG2 cell viability after 72 hours following transfection using serial dilutions of TGFβ1 and Cox2 single and dual siRNAs.

[0031] FIG.7 shows A549 cell viability after 72 hours following transfection using serial dilutions of hetero-dual siRNAs linked with a disulfide (L3) or PEG (L4) linker containing truncated siRNA or siRNA with 3’ UU overhangs on the antisense strand.

[0032] FIG.8 shows A549 cell viability after 72 hours following transfection using serial dilutions of Cox2 homo-dual siRNAs linked with a disulfide (L3) or PEG (L4) linker containing truncated siRNA or siRNA with 3’ UU overhangs on the antisense strand.

[0033] FIG.9 shows A549 cell viability after 72 hours following transfection using serial dilutions of mixed Cox2 and TGFβ1 homo-dual siRNA linked with a disulfide (L3) or PEGAttorney Docket No.4690.0096I (L4) linker containing truncated siRNA or siRNA with 3’ UU overhangs on the antisense strand.

[0034] FIG.10 shows PC3 cell viability after 72 hours following transfection using serial dilutions of TGFβ1 and Cox2 siRNAs, either combined as single siRNAs or linked by a disulfide linker.

[0035] FIG.11 shows PiaPaca-2 cell viability for 72 hours following transfection using serial dilutions of TGFβ1 and Cox2 siRNAs, either combined as single siRNAs or linked by a disulfide linker.

[0036] FIG.12 shows SCC-25 cell viability after 72 hours following transfection using serial dilutions of TGFβ1 and Cox2 siRNAs, either combined as single siRNAs or linked by a disulfide linker.

[0037] FIG.13 shows A549 cell viability after 72 hours following transfection using serial dilutions of disulfide-linked siRNAs targeting TGFβ1 or / and Cox2.

[0038] FIG.14 shows PC3 cell viability after 72 hours following transfection using serial dilutions of disulfide-linked siRNAs targeting TGFβ1 or / and Cox2.

[0039] FIG.15 shows WdCox2-L3 characterization by HPLC and mass spectrometry.

[0040] FIG.16 shows WdTGFβ1-L3 characterization by HPLC and mass spectrometry.

[0041] FIG.17 shows WdCox2-TGFβ1-L3 characterization by HPLC and mass spectrometry.

[0042] FIG.18 shows WdTGFβ1-Cox2-L3 characterization by HPLC and mass spectrometry. Detailed Description

[0043] Divalent RNA molecules containing chemically linked siRNA molecules are provided that inhibit the expression of the TGFβ1 and cyclooxygenase-2 (Cox-2) genes for treating a variety of cancers in a subject, particularly in cells that express Cox-2. Constructs are provided containing linked RNA molecules that target the TGFβ-1 and Cox-2 genes for therapeutic purposes; where the linkage occurs between sense strands or, optionally, antisense strands. The constructs can contain between two and four double stranded RNA molecules. In some embodiments the RNA molecules are siRNA molecules. In other embodiments, other forms of RNA molecules – shRNA, miRNA, etc. – may be used. Also provided are embodiments of the compositions for use in vitro or in vivo containing these constructs, and methods for their manufacture and use. Examples provided demonstrate a powerful combinatorial approach to modulating gene expression and treating various diseases due toAttorney Docket No.4690.0096I the combined delivery to, and, in certain embodiments, intracellular release of the RNA molecules in, the same cells at the same time.

[0044] The divalent siRNA molecules disclosed herein may be formulated for use in oncology, fibrosis, and other diseases, and offer a more comprehensive approach to disease treatment by simultaneously targeting multiple gene expressions or pathways. (16) As used in a composition intended for an in vitro or in vivo use, divalent siRNA molecules are designed to simultaneously target at least two disease-related genes or pathways and possibly several others, offering a potential solution for complex diseases with multiple genetic and other etiologies, such as cancer and inflammatory disorders, and viral infections. (26- 28) In some embodiments, by combining two or more siRNA molecules into a single formulation, enhanced therapeutic efficacy has been demonstrated, and a broadening of the scope of RNAi-based treatments is envisioned.

[0045] Use of divalent siRNA molecules formulated in nanoparticles, including embodiments where two or more double stranded (ds) siRNA molecules are cleavably linked through the sense or, optionally, antisense strands, represents an alternative approach to the delivery of at least two, three or four individual ds siRNA molecules in similar formulations.

[0046] In other embodiments the linked siRNA molecules remain linked intracellularly, in whole or part, and also exhibit similar enhanced effects. In one embodiment, a divalent siRNA contains two double stranded siRNA sequences in which the two siRNAs are covalently connected through their sense strands by a chemical linker that is cleavable once the molecules are inside the targeted cells.

[0047] In another embodiment the siRNA molecules are cleavably linked through their antisense strands, 3’ to 3’ ends. In some embodiments the ds siRNA molecules are linked through the joining of their sense strands, via linkage of the 5’ end of one siRNA to the 3’ end of the other siRNA.

[0048] In other embodiments the sense strands are linked through the 5’ end of one siRNA’s sense strand to the 5’ end of a second siRNA’s sense strand. In still other embodiments the siRNA molecules are covalently linked through their sense strands, 3’ to 3’ ends.

[0049] In other embodiments the siRNA molecules also may be linked through their antisense strands by their 3’ ends. In still other embodiments under some conditions, this configuration may result in a reduction in potency.

[0050] In still other embodiments the siRNAs (whether two, three or four, in series) remain linked intracellularly, and exert their enhanced effects. In other embodiments, certain of the links are retained intracellularly, while others are cleaved. For example, two siRNAs linkedAttorney Docket No.4690.0096I with an L4 PEG linker remain linked intracellularly and yet exhibit an enhanced effect to silence the targeted genes.

[0051] Similarly, in certain embodiments, three or four siRNAs linked with L3 cleavable linkages are released individually intracellularly. In other embodiments, a combination of L3 and L4 linkages permit the cleavage of the L3 links, while retaining the L4 links ion, intracellularly. For example, an L3 linkage could separate a single siRNA while three siRNAs remain linked, or the L3 linkage could separate two sets of two linked siRNAs. In still other embodiments, cleavable L3 linkages are evident between each siRNA. As noted, these linkages could be 5’ to 5’, 3’ to 3’, or 5’ to 3’ between any two siNRAs, regardless of whether L3 or L4 linkages are used. Both homo- divalent and multivalent antisense oligonucleotides have been shown to enhance efficacy. (17, 18) This provides an advantage to improved synergistic benefit in cancer treatment and other oligonucleotide drug development.

[0052] Both linked siRNA molecules may each have a role in targeting a specific disease- related gene or gene product involved in pathogenesis. Through careful design and selection, the siRNA molecules are chosen to exhibit complementary effects and collectively enhance the therapeutic outcome. In the disclosed embodiments, the linked siRNAs targeting TGFβ-1 and COX-2 genes provide a combination therapeutic approach for various types of cancer.

[0053] TGFβ-1 is a multifunctional cytokine that has been shown to play a complex role in cancer progression, with both tumor-suppressive and pro-tumorigenic effects. COX-2, on the other hand, is an enzyme that plays a role in inflammation and is upregulated in many types of cancer. COX-2 inhibitors have been studied as a cancer therapy due to their anti- inflammatory properties and potential to inhibit tumor growth. However, the use of COX-2 inhibitors alone has been limited by concerns about cardiovascular and gastrointestinal side effects. The combination of COX-2 inhibitors with TGFβ-1 inhibitors offers a solution to this problem by targeting multiple pathways involved in cancer progression.

[0054] Preclinical studies have shown that the combination of individual siRNA molecules targeting TGFβ-1 and COX-2 genes can enhance the antitumor effects of each agent alone in various types of cancer, including breast, lung, and colorectal cancer. Nucleic acids – siRNA, miRNA and mRNA

[0055] The nucleic acids used in some disclosed embodiments comprise siRNA, miRNA and / or mRNA molecules that target TGF-β1 and COX-2 genes of interest. Certain disclosed embodiments include at least one nucleic acid complex – e.g., linked siRNAs - in each composition. RNA molecules targeting TGF-β1 and COX-2 genes, their composition and methods of manufacture were disclosed in U.S. Patent No.9,642,873, incorporated byAttorney Docket No.4690.0096I reference herein. In some embodiments, the gene-targeting siRNA comprises at least one sense strand and an antisense strand, each containing a core sequence that is 19, 21, 23 or 25 nucleotides in length. The sense and antisense strands of siRNA typically anneal to form a duplex. Within the complementary duplex region, the sense strand core sequence is 100% complementary to the antisense core sequence. In some embodiments the siRNAs can be asymmetric where one strand is shorter than the other (typically by 2 bases e.g. a 21mer with a 23mer or a 19mer with a 21mer or a 23mer with a 25mer). The strands may be modified by inclusion of a dTdT overhang group on the 3’ end of selected strands. In some embodiments the antisense strands can be more than 50%, 60%, 70%, 80%, 90%, 95% or 99% complementary to the sense strands.

[0056] In some embodiments, double stranded siRNA may be unmodified or chemically modified at the 2’ position with 2’-OCH3, (or 2’-OMe) or by 2’-F, and / or at the 5’ position with -P(O)2=S, -P(S)2=O. Other chemical modifications, such as pegylation or lipid functionalization may be used to improve the overall stability and bioavailability of the RNAi. In some embodiments, siRNA duplexes are capable of targeting multiple genes with a single effector sequence.

[0057] In some embodiments in each of the RNA molecules, e.g., siRNA, one or more of the nucleotides in either or both the sense or the antisense strand can be a modified nucleotide. Modified nucleotides can improve stability and decrease immune stimulation by the siRNAs. The modified nucleotide may be, for example, a 2'-O-methyl, 2'-methoxyethoxy, 2'-fluoro, 2'- allyl, 2'-O-[2-(methylamino)-2-oxoethyl], 4'-thio, 4'-CH2-O-2'-bridge, 4'-(CH2)2-O-2'-bridge, 2'-LNA, 2'-amino or 2'-O--(N-methylcarbamate) ribonucleotide. In other embodiments, one or more of the phosphodiester linkages between the ribonucleotides may be modified to improve resistance to nuclease digestion. Suitable modifications are well-known in the art and may include the use of phosphorothioate and / or phosphorodithioate modified linkages. Design of a homo-divalent siRNA improves the potency in cancer treatment.

[0058] In some of the disclosed embodiments, the two siRNAs are covalently linked to form a divalent siRNA construct via a chemical linker through sense strands, which are subsequently annealed to complimentary antisense strand to form the dual (divalent) siRNA molecule. A series of homo (two of the same siRNA) divalent siRNA were designed and synthesized by solid phase oligonucleotide synthesizer, as known in the art and described in the examples below. In the disclosed embodiments, the homo-divalent anti- TGFβ1 or homo- divalent anti Cox2 siRNA, when covalently linked through their sense strands by a cleavable disulfide linker, C6-S-S-C6, significantly improved the in vitro potency of the combination inAttorney Docket No.4690.0096I cancer cell lines, particularly cell lines expressing Cox-2. This holds even if only the homo- divalent anti-TGFβ1 molecule is used. Table 1 provides the siRNA constructs with cleavable disulfide links as referenced by name or SEQ ID numbers elsewhere in this disclosure. Table 2 provides the siRNA constructs with PEG links, not cleavable intracellularly. Table 1: Synthesized siRNA Constructs with Cleavable Disulfide (C6SSC6) LinksAttorney Docket No.4690.0096I Table 2: Synthesized siRNA Constructs with Non-cleavable PEG Links

[0059] The W prefix in the Tables above refers to wild type; the Wd prefix refers to dual wild type, meaning two identified siRNA sequences are linked through the disulfide (L3) or PEG (L4) linkers. The linkers are discussed below. The siRNA sequences targeting TGFβ1, and those targeting Cox-2, without the linker, have been disclosed previously, see e.g., RE46,873, U.S. Pat. No.9,642,873, U.S.2020 / 392507 A1, and U.S.2021 / 324384 A1. Hetero-divalent siRNA improves the potency of dual target synergetic efficacy.

[0060] A series of linked siRNA sequences, each of which targets one of the two synergetic mRNA targets, were designed and fully chemically synthesized by methods known in the art. These also are shown in Table 1 (WdCox2-L3, WdTGFβ1-L3, WdCox2-TGFβ1-L3, WdTGFβ1-Cox2-L3, etc.). All of the divalent siRNA conjugates demonstrated improved potency in growth inhibition or reduced cell viability in multiple cancer cell lines, such as in A549 (Figure 5), in PC3 (Figure 10), and in SCC-25 (Figure 12).

[0061] In the hetero divalent siRNA construct of TGFβ1 and Cox2, both compounds, WdTGFβ1-WdCox2-L3 (SEQ ID No.9) and WdCox2-WdTGFβ1-L3 (SEQ ID No.8),Attorney Docket No.4690.0096I increased the cell death rate compared with the mixture of individual TGFβ1 and Cox2 siRNAs (IC50 was increased by 1.36 to 2.39-fold).

[0062] In the hetero divalent siRNA constructs, although they share the same composition, the orientation of the divalent molecule as WdCox2-WdTGFβ1-L3 (SEQ ID No.8) is slightly more potent than the differently oriented, WdTGFβ1-WdCox2-L3 (SEQ ID No.9). As the L3 linker is cleaved intracellularly, a portion of the link remains with each of the siRNAs. There is an effect the linker (L3) remnant that remains with the individual siRNAs after cleavage that makes the one or more of the siRNAs of SEQ ID No.8 more potent than the siRNAs of SEQ ID No.9, that is, the orientation of the molecules with respect to the location of the linker, L3, affects the potency of the cleaved siRNAs as can be seen in Figure 13. The mixture of WdTGFβ1-WdCox2-L3+WdTGFβ1-WdCox2-L3 demonstrated an IC50 of 0.0012 nM, which fell in between the two WdCox2-WdTGFβ1-L3 (IC50= 0.0007302) and WdTGFβ1-WdCox2-L3 with a IC50 =0.003011. The combination demonstrated greater potency than a mixture of two homo divalent siRNA constructs WdTGFβ1-L3+WdCox2-L3 (SEQ ID Nos.1-2). (Figures 13 and 14)

[0063] In comparison with the single siRNA mixture of TGFβ-1 or COX-2, the divalent siRNA conjugate of WdCox2-L3, or WdTGFβ1-L3 also significantly improved the potency of the single siRNA target by 2- to 4.76-fold, increasing IC50in the cell viability assay. (Figures 5, 10, and 12)

[0064] Cell selective inhibition was observed in vitro among those divalent siRNA constructs. When both mRNAs are highly expressed in a cancer cell line such as A549, PC3, or SCC-25, cancer cell growth was potently inhibited. In cell lines with lower COX-2 expression, for example, in HepG2 or MiaPaca-2 cells, however, the divalent constructs demonstrated lower potency. This is consistent with previous in vitro study results demonstrating the synergistic effects of the combination of separate anti-TGFβ-1 and anti- COX-2 siRNA molecules delivered in a nanoparticle formulation.

[0065] A non-cleavable reference linker, L4, was also designed and all the L4-linked divalent siRNA molecules were fully synthesized using a solid phase RNA synthesizer. These molecules are shown in Table 2. In comparison with the L3 cleavable linker, L4 linked divalent siRNAs against TGFβ-1 and COX-2, showed attenuated potency to kill cancer cells. Thus, although the divalent siRNA linked with the L4 linker also were successful, that is, the siRNAs both showed potency even when the siRNAs remained linked intracellularly, the fact that the divalent L3-linked siRNAs were more potent demonstrates the importance of the cleavage of the linker inside the cell, resulting in the release of the two siRNA moleculesAttorney Docket No.4690.0096I within the cellular compartment simultaneously, facilitating RISC loading of both target genes.

[0066] A 25mer siRNA of TGFβ-1 and COX-2, was designed and screened in vitro. When two additional overhangs were introduced in the 25mer siRNAs against TGFβ-1 or COX-2, there was a dramatic reduction of potency in cancer lines. This demonstrates the sensitivity of the siRNA to length and modifications on both strands, thus providing a guide for future designs of the longer siRNA in applications in oncology.

[0067] All the TGFβ-1 and COX-2 siRNA molecules are double stranded (ds) compounds having 2’-OH modifications, but without further chemical modification in the nanoparticle formulation study. For example, the double stranded siRNA can be anti - TGF-β1 or anti – COX-2 , and can be unmodified or chemically modified at the 2 ' position with , for example , 2’- OCH3, 2'-F, or 2’-O-MOE , or at the 5 ' position with P(O)2=S. Other chemical modifications are known in the art and can include, for example, pegylation functionalization to improve the overall stability and bioavailability of the RNAi. In specific embodiments, the double stranded siRNA may be duplexes consisting of 25 or 24 contiguous base pairs of any one or more of the duplexes shown in Tables 1 and 2. Chemical Linker

[0068] A chemical linker can be used to covalently link two oligonucleotide strands of siRNAs targeting TGF-β1 and COX-2 to produce the divalent molecules. Suitable linkers are well-known to one of ordinary skill in the art and include polymeric linkers, hydrocarbon linkers, nucleic acid linkers, and peptide linkers. The linker can join two sense strands, two antisense strands, one sense and one antisense strand, two sense strands and one antisense strand, two antisense strands and one sense strand, or two sense and two antisense strands.

[0069] The linker can be cleavable or non-cleavable, but advantageously is cleavable. In some embodiments the linker is cleavable due to the presence of a disulfide bond that is cleaved in vivo.

[0070] If the linker is a nucleic acid linker, it can contain one or more modified residues and can be 4-20 nucleotides long. If the linker is a peptide linker it can be 2-10 amino acids long and can include a protease cleavage site that is cleavable by a protease in vivo.

[0071] Advantageously, a cleavable linker contains an -OC6SSC6O-moiety as shown in Figure 1B, or a tetraethylene glycol linker as shown in Figure 1B.

[0072] The divalent siRNA molecule can be prepared through solid phase RNA synthesis or solution phase reactions. The cleavable linker, L3, (Figure 1B) was designed to be cleavableAttorney Docket No.4690.0096I by reductive GSH or enzyme conditions under intracellular conditions. Another linker, L4 (Figure 1B) was designed to link the two siRNAs, but is non-cleavable intracellularly.

[0073] In vitro experimental work reported here has validated the importance of not only the structure but the orientation of the linked molecules to the potency of the linked siRNAs. These data demonstrate the synergistic effects achieved by chemically linking the synthesized constructs against TGFβ-1 and COX-2, delivering them to targeted cells, and later, either cleaving the complex to individual RNA molecules within the cell (with the L3 link) or maintaining the L4 link. The combined administration of linked siRNA molecules results in a potent and selective inhibition of the disease-related gene expression, leading to a greater therapeutic impact compared to using either siRNA alone, even when formulated similarly with a carrier and administered in the same treatment. The synergistic action of the linked siRNA formulation ensures enhanced gene silencing efficacy, increased target specificity, and reduced off-target effects. Definitions

[0074] As used herein, "a" or "an" may mean one or more. As used herein, "another" may mean at least a second or more.

[0075] The term "amino acid" is inclusive of the 20 common amino acids, as well as "nonstandard amino acids," for example, D-amino acids and chemically (or biologically) produced derivatives of "common" amino acids, including for example, beta-amino acids.

[0076] A compound is "associated with" a second compound if the two compounds have formed a complex as a result of covalent or non-covalent interactions between the two compounds.

[0077] The term "copolymer" refers to a polymer that contains two or more types of units, regardless of the arrangement of units along the chain (random, alternating, block, graft), and regardless of its molecular structure (linear or branched). The term "histidine copolymer" means that the copolymer comprises histidine as one of its unit types. The term "transport polymer" means a polymer comprising the histidine copolymer of the disclosed embodiments. The term “branched histidine – lysine” polymer or copolymer (HKP) is a peptide consisting of histidine and lysine amino acids. In some embodiments, other amino acids may be included in the copolymer. By synthesizing multiple amino acids from a common lysine core, in some embodiments, the peptide may be branched. Such polymers are described in U.S. Patent. Nos.7,070,807, 7,163,695, and 7,772,201, which are incorporated herein by reference. Lipofectamine and other lipid- or amino acid-containing carriers mayAttorney Docket No.4690.0096I also be used for formulations of the disclosed embodiments of siRNA(s) for delivery as disclosed herein.

[0078] The term "branch" is inclusive of any monomer or linear polymer (including co- polymer) thereof, which is covalently attached at least one end to the side group of a branching monomer. A branch which itself comprises one or more branching monomers is referred to as a "non-terminal branch". A branch which does not comprise a branching monomer is referred to as a "terminal branch". A "terminal branch" may include for example, the final division of branching of histidine or lysine to the n-terminal amino acid of the branch. The terminal branch may include a non-histidine or lysine amino acid (e.g., a cysteine or other linking agent), which aids in conjugating a stabilizing agent (such as PEG or HPMA) and / or a targeting ligand. The term "branched polymer" is inclusive of any polymer comprising at least one backbone and at least one terminal branch. A branched polymer may further comprise one or more non-terminal branches.

[0079] The terms "HK peptide," "HK polymer," and "HK carrier" are intended to mean transport polymers, which comprise at least histidine and lysine residues, including the polymers encompassed by the disclosed embodiments. HKP and HKP(+H) both have a lysine backbone (three lysine residues) where the lysine side chain ε-amino groups and the N- terminus are coupled to [KH3]4K (SEQ ID NO: 33) (for HKP) or KH3KH4[KH3]2K (SEQ ID NO: 35) (for HKP(+H). Among the polymers with 4 branches, the repeating pattern of HHHK (SEQ ID NO: 36) (e.g., H3K4b) on the terminal branch appears to augment uptake of siRNA more effectively than the repeating patterns of HHK (e.g., H2K4b) or HK (e.g., HK4b). As a result, a similar pattern was adopted in constructing the highly branched H3K8b and found it to be highly effective for preparing carriers of siRNA. H3K8b has eight terminal branches and has a high percentage of histidine residues and a low percentage of lysine residues. Although the repeating pattern of HHK is present in H3K4b and H3K8b copolymers, N-terminal lysine residues were removed in the highly branched polymer, H3K8b. Reduction in the number of lysine residues in the terminal branches of H3K8b may lead to decreased binding of siRNA and increase the amount of siRNA in the cytoplasm compared to that in the nucleus. By adding a single lysine to each terminal branch of H3K8b (eight lysine residues total per polymer), the efficacy of the new polymer ((+K)H3K8b) in reducing the target mRNA was significantly impaired compared to that of H3K8b. A smaller polymer sequence (i.e., those not having the added lysine to each terminal branch) that accomplishes siRNA transport is advantageous in synthesizing polymers more readily. The idea that binding modulates siRNA release is consistent with the finding that a carrier peptideAttorney Docket No.4690.0096I with increased binding to siRNA is less effective as a carrier for siRNA. (Simeoni F, Morris M C, Heitz F, Divita G. Insight into the mechanism of the peptide-based gene delivery system MPG: implications for delivery of siRNA into mammalian cells. Nucleic Acids Res 2003; 31:2717-2724.). Nevertheless, the vast amount of HK carriers with varying abilities to bind nucleic acids were ineffective carriers of siRNA.

[0080] Non-limiting examples of HK polymers according to the present disclosed embodiments include, but are not limited to, one or more polymers such as HKP, HKP(+H), HKP, HKP(+H), H3K4b, H3K8b, and (-HHHK)H3K8b. Other modifications may be made by those skilled in the art within the scope of this disclosed embodiments. For example, ligands such as, e.g., peptides, aptamers, antibodies and carbohydrates such as hyaluronic acid (HA) targeting the CD44 receptor may be added to the polymer(s) within the scope of the present disclosed embodiments. In some embodiments the HK copolymer has the formula (R)K(R)-K(R)-(R)KCX), where R=KHHHKHHHKHHHKHHHK (SEQ ID NO: 33), R=KHHHKHHHKHHHHKHHHK (SEQ ID NO: 34), X=C(O)NH2, K=lysine, and H=histidine.

[0081] The term "lipid" is used as it is in the art and includes any chemical species having a hydrophobic and a hydrophilic portion. Hydrophilic characteristics typically derive from the presence of phosphato, carboxylic, sulfato, amino, sulfhydryl, nitro, and other like groups. Hydrophobicity may be conferred by cholesterol and derivatives thereof and by the inclusion of groups that include, but are not limited to, long chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic or heterocyclic group(s).

[0082] The term "non-cationic lipid" refers to any of a number of lipid species that exist either in an uncharged form a neutral zwitterionic form, or an anionic form at physiological pH. Such lipids include, for example diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cardiolipin, cerebrosides, DOPE, and cholesterol.

[0083] The term "cationic lipid" refers to any of a number of lipid species which carries a net positive charge at physiologic pH. Such lipids include, but are not limited to, DODAC, DOTMA, DDAB, DOSPER, DOSPA, DOTAP, DC-Chol and DMRIE. Additionally, a number of commercial preparations of cationic lipids are available which can be used in the disclosed embodiments. These include, for example, LIPOFECTIN.RTM. (Commercially available cationic liposomes comprising DOTMA and DOPE, from GIBCO / BRL, Grand Island, N.Y., USA); LIPOFECTAMINE.RTM. (Commercially available cationic liposomesAttorney Docket No.4690.0096I comprising DOSPA and DOPE, from GIBCO / BRL); and TRANSFECTAM.RTM. (Commercially available cationic liposomes comprising DOGS from Promega Corp., Madison, Wis., USA).

[0084] The term "peptide" is inclusive of both straight and branched amino acid chains, as well as cyclic amino acid chains, which comprise at least 2 amino acid residues. The terms "peptide" and "polypeptide" are used interchangeably herein.

[0085] A "pharmaceutical agent" includes any therapeutic agent useful in preventing, delaying or reducing the severity of the onset of a disease, or in reducing the severity of an ongoing disease, or in enhancing normal physiological functioning, as well as diagnostic agents, for example, a marker gene (GFP, luciferase). A "pharmaceutical agent" may consist of one or more therapeutic agents, one or more diagnostic agents, or a combination of one or more therapeutic and one or more diagnostic agents. As used herein, a "pharmaceutically acceptable" component (such as a salt, carrier, excipient or diluent) of a pharmaceutical agent delivery composition according to the present disclosed embodiments is a component which (1) is compatible with the other ingredients of the delivery composition in that it can be included in the delivery composition without eliminating the capacity of the composition to deliver the pharmaceutical agent; and (2) where the delivery composition is intended for therapeutic uses, is suitable for use with an animal (e.g., a human) without undue adverse side effects, such as toxicity, irritation, and allergic response. Side effects are "undue" when their risk outweighs the benefit provided by the pharmaceutical agent.

[0086] A "therapeutically effective amount" is an amount necessary to prevent, delay or reduce the severity of the onset of disease, or an amount necessary to arrest or reduce the severity of an ongoing disease, and also includes an amount necessary to enhance normal physiological functioning.

[0087] The term "siRNA" is used as it is in the art, and includes a duplex of RNA (19 to 25 bases or fewer in each strand) that targets mRNA. siRNA may be chemically or enzymatically synthesized. siRNA in accordance with the present disclosed embodiments may be incorporated and then activated in RISC.

[0088] The word "transfect" is broadly used herein to refer to introduction of an exogenous compound, such as a polynucleotide sequence, into a prokaryotic or eukaryotic cell; the term includes, without limitation, introduction of an exogenous nucleic acid into a cell, which may result in a permanent or temporary alteration of genotype in an immortal or non-immortal cell line.Attorney Docket No.4690.0096I

[0089] The term “ oligonucleotide ” refers to a chemically modified or unmodified nucleic acid molecule (RNA or DNA) having a length of fewer than 100 nucleotides ( for example fewer than 50 , fewer than 30 , or fewer than 25 nucleotides ) . It can be siRNA , microRNA , anti microRNA , microRNA mimics , dsRNA , SSRNA , aptamer , triplex forming oligonucleotides , aptamers . In one embodiment, the oligonucleotide is an RNAi agent . As used herein , an “ siRNA molecule ” or “ RNAi molecule ” is a duplex oligo nucleotide , that is a short , double - stranded polynucleotide , that interferes with the expression of a gene in a cell , after the molecule is introduced into the cell . For example, an siRNA molecule targets and binds to a complementary nucleotide sequence in a single stranded target RNA molecule . By convention, when an siRNA molecule is identified by a particular nucleotide sequence, the sequence refers to the sense strand of the duplex molecule . One or more of the ribonucleotides comprising the molecule can be chemically modified by techniques known in the art. In addition to being modified at the level of one or more of its individual nucleotides, the backbone of the oligonucleotide can be modified. Additional modifications include the use of small molecules ( e.g. sugar molecules ) , amino acids , peptides , cholesterol , and other large molecules for conjugation onto the siRNA molecule. Histidine-Lysine (HK) polypeptides

[0090] Effective means for transferring nucleic acids into target cells are important tools, both in the basic research setting and in clinical applications. A diverse array of nucleic acid carriers is currently required because the effectiveness of a particular carrier depends on the characteristics of the nucleic acid that is being transfected [Blakney et al., Biomacromolecules 2018, 19: 2870-2879. Goncalves et al., Mol Pharm 2016; 13: 3153-3163. Kauffman et al., Biomacromolecules 2018; 19: 3861-3873. Peng et al., Biomacromolecules 2019; 20: 3613-3626. Scholz et al., J Control Release 2012; 161: 554-565.]. Among various carriers, non-viral delivery systems have been developed and reported to be more advantageous than the viral delivery system in many aspects [Brito et al., Adv Genet.2015; 89: 179-233]. For example, the large molecular weight branched polyethylenimine (PEI, 25 kDa) is an excellent carrier for plasmid DNA but not for mRNA. However, by decreasing the molecular weight of PEI to 2 kDa, it becomes a more effective carrier of mRNA [Bettinger et al., Nucleic Acids Res 2001; 29: 3882-3891.].

[0091] The four-branched histidine-lysine (HK) peptide polymer H2K4b has been shown to be a good carrier of large molecular weight DNA plasmids [Leng et al., Nucleic Acids Res 2005; 33: e40.], but a poor carrier of relatively low molecular weight siRNA [Leng et al., J Gene Med 2005; 7: 977-986.]. Two histidine-rich peptides analogs of H2K4b, namely H3K4bAttorney Docket No.4690.0096I and H3K(+H)4b, were shown to be effective carriers of siRNA [Leng et al., J Gene Med 2005; 7: 977-986. Chou et al., Biomaterials 2014; 35: 846-855.], although H3K(+H)4b appeared to be modestly more effective [Leng et al., Mol Ther 2012; 20: 2282-2290.]. Moreover, the H3K(+H)4b carrier of siRNA induced cytokines to a significantly lesser degree in vitro and in vivo than H3K4b siRNA polyplexes [Leng et al., Mol Ther 2012; 20: 2282- 2290], which were already at very low levels. These polypeptides have a lysine backbone (three lysine residues) where the lysine side chain ε-amino groups and the N-terminus are coupled to various HK sequences. HK polypeptide carriers can be synthesized by methods that are well-known in the art including, for example, solid-phase peptide synthesis (SPPS).

[0092] It was found that such histidine-lysine peptide polymers (i.e., “HKP” or “HK polymers” or “HK copolymers”), in addition to their ability to package and carry siRNAs also were surprisingly effective as mRNA carriers, and that they can be used, alone or in combination with liposomes, to provide effective delivery of mRNA into target cells. Similar to PEI and other carriers, initial results suggested HK polymers differ in their ability to carry and release nucleic acids. However, because HK polymers can be reproducibly made on a peptide synthesizer, their amino acid sequence can be easily varied, thereby allowing fine control of the binding and release of siRNAs, miRNAs or mRNAs, as well as the stability of polyplexes containing the HK polymers and mRNA [Chou et al., Biomaterials 2014; 35: 846- 855. Midoux et al., Bioconjug Chem 1999; 10: 406-411. Henig et al., Journal of American Chemical Society 1999; 121: 5123-5126.]. When siRNA, miRNA, or mRNA molecules are mixed with one or more HKP carriers the components self-assemble into nanoparticles.

[0093] Methods well known in the art, including gel retardation assays, heparin displacement assays and flow cytometry can be performed to assess performance of different formulations containing HK polymer plus liposome in successfully delivering mRNA. Suitable methods are described in, for example, Gujrati et al., Mol. Pharmaceutics 11:2734−2744 (2014), Pärnaste et al., Mol Ther Nucleic Acids.7: 1–10 (2017).

[0094] Detection of nucleic acid uptake into cells can also be achieved using SmartFlare® technology (Millipore Sigma). These smart flares are beads that have a sequence attached that, when recognizing the RNA sequence in the cell, produce an increase in fluorescence that can be analyzed with a fluorescent microscope. siRNAs can reduce expression of a target gene while mRNA can increase it. miRNAs can either increase or decrease expression.

[0095] Other methods include measuring protein expressions from the nucleic acid, for example, an mRNA encoding luciferase can be used to measure the efficiency of transfection using methods that are well known in the art. See, for example, this was accomplished withAttorney Docket No.4690.0096I luciferase mRNA in a recent publication (He et al, J Gene Med.2021 Feb; 23(2):e3295) to demonstrate the efficacy of delivering mRNA using a HKP and liposome formulation. Synthesis of histidine-lysine copolymers

[0096] Synthesis of histidine-lysine copolymers is well known in the art (see e.g., US Pat. Nos.7,163,695, and 7, 772,201). Briefly, polypeptides may be prepared by any method known in the art for covalently linking any naturally occurring or synthetic amino acid to any naturally occurring or synthetic amino acid in a polypeptide chain which may have a side chain group able to react with the amino or carboxyl group on the amino acids so as to become covalently attached to the polypeptide chain.

[0097] For example, but not by way of limitation, branched polypeptides can be prepared as follows: (1) the amino acid to be branched from the main polypeptide chain can be prepared as an N-α-tert-butyloxycarbonyl (Boc) protected amino acid pentafluorophenyl (Opfp) ester and the residue within the main chain to which this branched amino acid will be attached can be an N-Fmoc-. α,γ-diaminobutyric acid; (2) the coupling of the Boc protected amino acid to diaminobutyric acid can be achieved by adding 5 grams of each precursor to a flask containing 150 ml DMF, along with 2.25 ml pyridine and 50 mg dimethylaminopyridine and allowing the solution to mix for 24 hours; (3) the polypeptide can then be extracted from the 150 ml coupling reaction by mixing the reaction with 400 ml dichloromethane (DCM) and 200 ml 0.12N HCl in a 1 liter separatory funnel, and allowing the phases to separate, saving the bottom aqueous layer and re-extracting the top layer two more times with 200 ml 0.12 N HCl; (4) the solution containing the polypeptide can be dehydrated by adding 2-5 grams magnesium sulfate, filtering out the magnesium sulfate, and evaporating the remaining solution to a volume of about 2-5 ml; (5) the dipolypeptide can then be precipitated by addition of ethyl acetate and then 2 volumes of hexanes and then collected by filtration and washed two times with cold hexanes; and (6) the resulting filtrate can be lyophilized to achieve a light powder form of the desired dipolypeptide. Branched polypeptides prepared by this method will have a substitution of diaminobutyric acid at the amino acid position which is branched. Branched polypeptides containing an amino acid or amino acid analog substitution other than diaminobutyric acid can be prepared analogously to the procedure described above, using the N-Fmoc coupled form of the amino acid or amino acid analog.

[0098] Polypeptides of the transport polymer can also be encoded by viral DNA and be expressed on the virus surface. Alternatively, histidine could be covalently linked to proteins through amide bonds with a water soluble dicarbodiimide.Attorney Docket No.4690.0096I

[0099] The HK transport polymer may also include a polypeptide--"synthetic monomer" copolymer. In these embodiments, the transport polymer backbone may comprise covalently linked segments of polypeptide and segments of synthetic monomer or synthetic polymer. The synthetic monomer or polymer may be biocompatible and / or biodegradable. Examples of synthetic monomers include ethylenically or acetylenically unsaturated monomers containing at least one reactive site for binding to the polypeptide. Suitable monomers as well as methods for preparing a polypeptide--"synthetic monomer" copolymer are described in U.S. Pat. No.4,511,478, for "Polymerizable compounds and methods for preparing synthetic polymers that integrally contain polypeptides," by Nowinski et al, which is herein incorporated by reference. Where the transport polymer comprises a branched polymer, synthetic monomer or polymer may be incorporated into the backbone(s) and / or branch(es). Furthermore, a backbone or branch may include a synthetic monomer or polymer. Finally, in this embodiment, the branching monomers may be branching amino acids or branching synthetic monomers. Branching synthetic monomers may include for example, ethylenically or acetylenically unsaturated monomers containing at least one substituent reactive side- group. Additionally, these side groups may consist of peptide (or non-peptide) sequences that are able to bind to select targets on cell membranes – providing the ability to specifically deliver siRNAs or other nucleotides to specific cell types within an organism.

[0100] Transport HK polymers in accordance with the present disclosed embodiments may be synthesized by methods known to those skilled in the art.

[0101] By way of non-limiting example, certain HK polymers discussed herein may be synthesized as follows. The Biopolymer Core Facility at the University of Maryland may be used to synthesize for example, the following HK polymers on a Ranin Voyager solid-phase synthesizer (PTI, Tucson, Ariz., USA): (1) H2K4b (83mer; molecular weight 11137 Da); (2) H3K4b (71mer; MW 9596 Da); (3) HK4b (79mer; MW 10896 Da); (4) H3K8b (163mer; MW 23218 Da); (5) H3K8b (166mer; MW 23564 Da); (6) (-HHHK)H3K8b (131mer; MW 18901 Da); (7) (-HHHK)H3K8b (134mer; MW 19243 Da); (8) ((K+) H3K8b (174mer; MW 24594 Da). The structures of certain branched polymers are shown in US Patent No. 7,772,201. The polymers with four branches (e.g. H3K4b, HK4b) may be synthesized by methods known in the art. The sequence of synthesis for highly branched polymers with eight terminal branches may be as follows: (1) RGD or other ligand (if present); (2) the 3-lysine core; (3) histidine-rich domain; (4) addition of a lysine; and (5) terminal branches. The RGD sequence may be initially synthesized by the instrument followed by three manual couplings with (fmoc)-Lys-(Dde)(the lysine core). The (Dde) protecting groups may be removed duringAttorney Docket No.4690.0096I the automatic deprotection cycle. To the lysine core, activated amino acids that comprise the histidine-rich domain may then be added sequentially by the instrument. A (fmoc)-Lys-(fmoc) amino acid was added to the histidine-rich domain and the fmoc protecting groups were then removed. To the α and ε amine groups of this lysine, activated amino acids of the terminal branches may then be added. The peptide is cleaved from the resin and precipitated by methods known in the art.

[0102] By way of non-limiting example, polymers of the disclosed embodiments may be analyzed as follows. Polymers may be first analyzed by high-performance liquid chromatography (HPLC; Beckman, Fullerton, Calif., USA) and might not be further purified if HPLC reveals that the purity of polymers is 95% or greater. The polymers may be purified on an HPLC column, for example with System Gold operating software, using a Dynamax 21-4.times.250 mm C-18 reversed phase preparative column with a binary solvent system. Detection may be at 214 nm. Further analyses of the polymers may be performed for example, using a Voyager matrix-assisted laser desorption ionization time-of-flight (MALDI- TOF) mass spectrometer (Applied Biosystems, Foster City, Calif., USA) and amino acid analysis (AAA Laboratory Service, Boring, Oreg., USA). Transfection agents such as, SuperFect (Qiagen, Valencia, Calif.), Oligofectamine (Invitrogen, Carlsbad, Calif.), Lipofectamine 2000 (Invitrogen), and Lipofectamine (Invitrogen) may be used according to the manufacturers' instructions. DOTAP liposomes may be prepared by methods known in the art. Formation of nanoparticles containing copolymer, and RNA

[0103] Nanoparticles advantageously are formed for administration to a subject. Various methods of nanoparticle formation are well known in the art. See, e.g., Babu et al., IEEE Trans Nanobioscience, 15: 849–863 (2016).

[0104] Nanoparticles may be formed using a microfluidic mixer system, in which one or more siRNA molecules are mixed with one or more HKP copolymers at a fixed flow rate. The flow rate can be varied to vary the size of the nanoparticles produced. Methods are described in Example 1 below.

[0105] Average nanoparticle diameter varies between about 80-400 nm, 100-300 nm, 130- 250nm, or 150-200 nm. Transfection

[0106] Branched carriers comprising histidine and lysine are useful for transfection of plasmids. (See Chen et al., “Branched co-polymers of histidine and lysine are efficient carriers of plasmids.” Nucleic Acids Res 2001; 29:1334-1340.) In these branched co-Attorney Docket No.4690.0096I polymers, the lysine and histidine component forms a complex with and partially neutralizes the negative charge of the plasmid DNA. In addition, the histidine component, with a pKa of about 6.0, buffers and aids in the release of plasmid DNA from endosomal vesicles. In general, linear HK peptides are ineffective for delivery of siRNA. In the present disclosed embodiments cover novel, highly branched HK polymers that are unexpectedly effective carriers of siRNA. The HK polymers of the present disclosed embodiments are advantageous, for example, in that they are less toxic and provide a more efficacious delivery of siRNA than other polymers.

[0107] The HK polymers of the present disclosed embodiments may be useful, for example, for in vitro delivery of siRNA to the interior of a cell. These polymers may, however, also have in vivo applications. These methods all include contacting a transfection complex with one or more cells to deliver the siRNA. The transfection complex includes at least one transport polymer and siRNA. The transport polymer includes histidine and lysine.

[0108] In general, a cell to be transfected includes, but is not limited to, any animal, plant or bacterial cell that is susceptible to intracellular delivery of siRNA using the transfection complex of the present disclosed embodiments either in vitro or in vivo. For example, suitable cellular targets include, without limitation, epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes, blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes, various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, and the like. In certain aspects, the cell is selected from the group consisting of lung cells, liver cells, endothelial cells, muscle cells, skin cells, hematopoietic stem cells and tumor cells.

[0109] According to certain embodiments, the cells include one or more cells selected from the group consisting of transformed, recombinant, malignant, and primary cell lines. By way of non-limiting example, cells according to the present disclosed embodiments may include one or more cells selected from SVR-bag4, MDA-MB-435, C6 and HUVEC (human umbilical endothelial vein) cell lines.

[0110] With plasmid-based therapy, nuclear import is important for transcription to occur and this appears to be a rate-limiting step in several cell lines. (Pollard et al.,”Polyethylenimine but not cationic lipids promotes transgene delivery to the nucleus in mammalian cells,” J Biol Chem 1998; 273:7507-7511; Zabner et al.,”Cellular and molecular barriers to gene transfer by a cationic lipid,” J Biol Chem 1995; 270:18997-19007.) Because nuclear import isAttorney Docket No.4690.0096I unnecessary for siRNA to degrade its target mRNA, it is believed that the polymers of the present disclosed embodiments will be effective as carriers of siRNA in most cell lines.

[0111] Methods of transfecting cells in accordance with the present disclosed embodiments may also include forming the transfection complex and allowing the transfection complex to stand for about 15 minutes to about 11 / 2 hours, or from about 15 to about 45 minutes at approximately room temperature before contacting the transfection complex with cells.

[0112] Transport polymers, that include histidine and lysine in accordance with the present disclosed embodiments include one or more HK carriers that are effective for transporting siRNA, including for example, polymers having between six and 10 terminal branches. According to certain embodiments, the transport polymer of the present disclosed embodiments includes eight terminal branches and a histidine-rich domain. According to certain embodiments, the transport polymer comprises a terminal branch having a sequence of -HHHKHHHKHHHKHHHKHHH (SEQ ID NO: 37)- or a version thereof. Non-limiting examples of transport polymers in accordance with the present disclosed embodiments include one or more polymers selected from H3K8b and structural analogs, such as H3K8b including one or more other ligand(s), (-HHHK)H3K8b, and the like.

[0113] Transport polymers of the present disclosed embodiments may optionally include one or more stabilizing agents. Suitable stabilizing agents would be apparent to those skilled in the art in view of this disclosure. Non limiting examples of stabilizing agents in accordance with the present disclosed embodiments include polyethyleneglycol (PEG) or hydroxypropylmethylacrylimide (HPMA).

[0114] Transport polymers of the present disclosed embodiments may optionally include one or more targeting ligands. Suitable targeting ligands would be apparent to those skilled in the art in view of this disclosure.

[0115] The disclosed embodiments are further directed to compositions, which include transfection complexes of the present disclosed embodiments. Such compositions may include for example, one or more intracellular delivery components in association with the HK polymer and / or the siRNA. The intracellular delivery component may include for example, a lipid (such as cationic lipids), a transition metal or other components that would be apparent to those skilled in the art.

[0116] In certain embodiments, transfection complex compositions include a transport polymer (which may act as an intracellular delivery component) and siRNA. In these embodiments the transport polymer may act as the intracellular delivery component withoutAttorney Docket No.4690.0096I need for additional delivery components, or may act in conjunction with other delivery components.

[0117] In other embodiments, the transfection complex compositions may include (i) the transport polymer, (ii) at least one intracellular delivery component in association with the transport polymer, and (iii) siRNA in association with the intracellular delivery component and / or the transport polymer. Methods of making these compositions may include combining (i) and (ii) for a time sufficient for the transport polymer and the siRNA to associate into a stable complex. Components (i), (ii) and (iii) may also be provided in a suitable carrier, such as a pharmaceutically acceptable carrier. In embodiments that include an intracellular delivery component other than the transport polymer, the transport polymer may interact with an intracellular delivery component, such as a liposome, through non-covalent or covalent interactions. The transport polymer may interact with siRNA through non-covalent or covalent interactions. Alternatively, the transport polymer need not interact directly with the siRNA, but rather, the transport polymer may react with an intracellular delivery component(s), which in turn interacts with the siRNA, in the context of the overall complex.

[0118] The present disclosed embodiments further include assays for determining an effective carrier of siRNA for transfection into cells. These assays include mixing siRNA with a transport polymer to form a transfection complex; contacting the transfection complex with one or more cells; and detecting the presence or absence of siRNA activity within the cells.

[0119] Intracellular delivery components of the presently disclosed embodiments comprise the transport polymer itself. Where intracellular delivery components other than the transport polymer are utilized, such delivery components may be viral or non-viral components. Suitable viral intracellular delivery components include, but are not limited to, retroviruses (e.g., murine leukemia virus, avian, lentivirus), adenoviruses and adeno-associated viruses, herpes simplex viruses, rhinovirus, Sendai virus, and Poxviruses. Suitable non-viral intracellular delivery components include, but are not limited to, lipids and various lipid- based substances, such as liposomes and micelles, as well as various polymers known in the art.

[0120] Suitable lipids include, but are not limited to, phosphoglycerides, sphingolipids, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidyleholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine,Attorney Docket No.4690.0096I distearoylphosphatidylcholine, dilinoleoylphosphatidylcholine, glycosphingolipid, amphipathic lipids. The lipids may be in the form of unilamellar or multilamellar liposomes.

[0121] The intracellular delivery component may include, but are not limited to, a cationic lipid. Many such cationic lipids are known in the art. A variety of cationic lipids have been made in which a diacylglycerol or cholesterol hydrophobic moiety is linked to a cationic headgroup by metabolically degradable ester bond, for example: 1,2-Bis(oleoyloxy)-3-(4-'- trimethylammonio)propane (DOTAP), 1,2-dioleoyl-3-(4'-trimethylammonio)butanoyl-sn- glycerol (DOTB), 1,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC) and cholesteryl (4'-trimethylammonio)butanoate (ChoTB). Other suitable lipids include, but are not limited to, cationic, non-pH sensitive lipids, such as: 1,2-dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide (DORI), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE), and 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DMRIE). Other non-pH-sensitive, cationic lipids include, but are not limited to,: O,O'-didodecyl-N-[p-(2-trimethylammonioethyloxy)benzoyl]-N,N,N-trimethylammonium chloride, Lipospermine, DC-Chol (3 beta [N-(N',N''-dimethylaminoethane) carbonyl]cholesterol), lipopoly(L-lysine), cationic multilamellar liposomes containing N- (alpha-trimethylamnmonioacetyl)-didodecyl-D-glutamate chloride (TMAG), TransfectACE.TM. (1:2.5 (w:w) ratio of DDAB which is dimethyl dioctadecylammonium bromide and DOPE) (Invitrogen) and lipofectAMINE.TM. (3:1 (w:w) ratio of DOSPA which is 2,3-dioleyloxy-N-[20([2,5-bis[(3-amino-propyl)amino]-1-oxypentyl]amino)et- hyl]-N,N- dimethyl-2,3-bis(9-octadecenylo-xy)-1-propanaminium trifluoroacetate and DOPE) (Invitrogen). Other suitable lipids are described in U.S. Pat. No.5,965,434, for "Amphipathic PH sensitive compounds and delivery systems for delivering biologically active compounds," by Wolff et al.

[0122] Cationic lipids that may be used in accordance with the presently disclosed embodiments comprise, but are not limited to, those that form liposomes in a physiologically compatible environment. Suitable cationic lipids include, but are not limited to cationic lipids selected from the group consisting of 1,2-dioleythyloxypropyl-3-trimethyl ammonium bromide; 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide; dimethyldioctadecyl ammonium bromide; 1,2-dioleoyl-3-(trimethylammonium)propane (DOTAP); 3.beta.N-(N',N'-dimethylaminoethane)carbamoyl]cholestero-1 (DC-cholesterol); 1,2 dioleolyl-sn-glycero-3-ethylphosphocholine; 1,2 dimyristoly-sn-glycero-3- ethylphosphocholine; [1-(2,3-diol-eyloxy)propyl]-N,N,N-trimethyl-ammonium chloride (DOTMA); 1,3-dioleoyloxy-2-(6carhoxys-permyl) propylamide (DOSPER); 2,3-dioleyloxy-Attorney Docket No.4690.0096I N-[2(spermine-carboxyamido)et-hyl]-N,N, dimethyl-1-propanamoniumtrifluoroacetate (DOSPA); and 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DMRIE).

[0123] Cationic lipids may be used with one or more helper lipids such as diloleoylphosphatidylethanolamine (DOPE) or cholesterol to enhance transfection. The molar percentages of these helper lipids in cationic liposomes are between about 5 and 50%. In addition, pegylated lipids, which can prolong the in vivo half-life of cationic liposomes, can be present in molar percentages of between about 0.05 and 0.5%.

[0124] Compositions in accordance with the disclosed embodiments may alternatively include one or more components to enhance transfection, to preserve reagents, or to enhance stability of the delivery complex. For example, in certain embodiments stabilizing compounds such as polyethylene glycol can be covalently attached to either the lipids or to the transport polymer.

[0125] Compositions of the disclosed embodiments may also suitably comprise various delivery-enhancing components known in the art. For example, the composition may comprise one or more compounds known to enter the nucleus or ligands subject to receptor- mediated endocytosis, and the like. For example, the ligand may comprise a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. Other examples of delivery-enhancing components include, but are not limited to, nuclear proteins, adenoviral particles, transferrin, surfactant-B, anti-thrombomodulin, intercalating agents, hemagglutinin, asialoglycoprotein, chloroquine, colchicine, integrin ligands, LDL receptor ligands, and viral proteins to maintain expression (e.g. integrase, LTR elements, rep proteins, oriP and EBNA-1 proteins) or viral components that interact with the cell surface proteins (e.g. ICAM, HA-1, MLV's gp70-phosphate transporter, and HIV's gp120-CD4). Delivery enhancing components can be covalently or non-covalently associated with the transport polymer, the intracellular delivery component, or the pharmaceutical agent. For instance, delivery to a tumor vasculature can be targeted by covalently attaching a -RGD- or - NGR- motif. This could be accomplished using a peptide synthesizer or by coupling to amino groups or carboxyl groups on the transport polymer with a water-soluble di-carbodiimide (e.g., 1-ethyl-3-(3-dimethyaminopropyl)carboiimide). Both of these methods are known to those familiar with the art.

[0126] Compositions of the present disclosed embodiments may suitably include a transition metal ion, such as a zinc ion. The presence of a transition metal in the complexes of the disclosed embodiments may enhance transfection efficiency.Attorney Docket No.4690.0096I Dosing and Administration

[0127] The pharmaceutical compositions described herein may be administered to subjects, which include mammals, including human subjects, by any mode of administration that is conventionally used to administer compositions. Thus, the compositions can be in the form of an aerosol, dispersion, solution, or suspension and can be formulated for inhalation, intramuscular, oral, sublingual, buccal, parenteral, nasal, subcutaneous, intradermal, or topical administration. The term parenteral as used herein includes percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, or intrathecal injection or infusion techniques and the like.

[0128] Composition embodiments for in vitro work may be used in amounts consistent with those used in the examples provided for the desired effect.

[0129] As used herein, an effective dose of a composition is the dose required to produce a protective immune response in the subject to whom the pharmaceutical composition is administered. A protective immune response in the present context is one that prevents or ameliorates a variety of diseases or disorders.

[0130] The composition may be administered one or more times. An initial measurement of an desired effect to the composition may be made by measuring one or more compounds in the circulation or tissue samples of the recipient subject. Methods of measuring a variety of compounds in this manner are also well known in the art, as is an appropriate dose effective in preventing or inhibiting the occurrence, or treating (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state.

[0131] The pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors that those skilled in the medical arts will recognize that, generally, an amount between 0.1 mg / kg and 100 mg / kg body weight / day of active ingredients is administered dependent upon potency of the formulated composition, between about 0.1 mg / kg and about 1.0 mg / kg, between about 1.0 mg / kg and about 2.0 mg / kg, from between about 2.0 mg / kg and 3.0 mg / kg, between about 3.0 and 5.0 mg / kg, between about 5 mg / kg and about 8 mg / kg, between about 8 mg / kg and about 15 mg / kg, between about 15 mg / kg and about 25 mg / kg, between about 25 mg / kg and about 35 mg / kg, between about 35 mg / kg and about 45 mg / kg, between about 45 mg / kg and about 55 mg / kg, between about 55 mg / kg and about 65 mg / kg, between about 65 mg / kg and about 75 mg / kg, between about 75 mg / kg and about 85 mg / kg,Attorney Docket No.4690.0096I between about 85 mg / kg and about 95 mg / kg, and between about 95 mg / kg and about 105 mg / kg.

[0132] However, their application has until recently been restricted by the instability and inefficient in vivo delivery of nucleic acids such as siRNA molecules. The methods described herein provide methods of making and using pharmaceutical compositions with a HK copolymer or other carrier in a nanoparticle delivery system.

[0133] Depending on the particular target RNA sequences and the dose of the nanoparticle composition delivered, partial or complete loss of function for the target RNAs may be observed. A reduction or loss of RNA levels or gene expression (either RNA expression or encoded polypeptide expression) in at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more of targeted cells is exemplary. Inhibition of target RNA levels or expression refers to the absence (or observable decrease) in the level of RNA or RNA- encoded protein. Specificity refers to the ability to inhibit the target RNA without manifest effects on other genes of the cell. The consequences of inhibition can be confirmed by examination of the outward properties of the cell or organism or by biochemical techniques such as RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring with a microarray, antibody binding, enzyme linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), other immunoassays, and fluorescence activated cell analysis (FACS). Inhibition of target RNA sequence(s) by the dsRNA agents of the disclosed embodiments also can be measured based upon the effect of administration of such dsRNA agents upon development / progression of a target RNA-associated disease or disorder, e.g., tumor formation, growth, metastasis, etc., either in vivo or in vitro. Treatment and / or reductions in tumor or cancer cell levels can include halting or reduction of growth of tumor or cancer cell levels or reductions of, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100%, and can also be measured in logarithmic terms, e.g., 10-fold, 100-fold, 1000-fold, 105-fold, 106-fold, or 107- fold reduction in cancer cell levels could be achieved via administration of the nanoparticle composition to cells, a tissue, or a subject. The subject may be a mammal, such as a human.

[0134] The methods described herein may be used in clinical applications of the siRNA include prophylactic and therapeutic compositions effective against various diseases, especially infectious diseases and oncological indications. Treatments

[0135] The present disclosed embodiments provide methods of treating diseases comprising using the divalent siRNA complexes and composition embodiments. In particular, methodsAttorney Docket No.4690.0096I are provided for treating a patient having a disease, by administering to the patient a therapeutically effective amount of a complex or composition of the present disclosed embodiments. Also encompassed are methods for treating a patient having a disease, by administering to the patient cells that have been transfected by the methods disclosed herein. Examples of genetic and / or non-neoplastic diseases potentially treatable with the complex, compositions, and methods include, but are not limited to the following: adenosine deaminase deficiency; purine nucleoside phosphorylase deficiency; chronic granulomatous disease with defective p47phox; sickle cell with HbS, β-thalassemia; Faconi's anemia; familial hypercholesterolemia; phenylketonuria; ornithine transcarbamylase deficiency; apolipoprotein E deficiency; hemophilia A and B; muscular dystrophy; cystic fibrosis; Parkinsons, retinitis pigmentosa, lysosomal storage disease (e.g., mucopolysaccharide type 1, Hunter, Hurler and Gaucher), diabetic retinopathy, human immunodeficiency virus disease virus infection, acquired anemia, cardiac and peripheral vascular disease, and arthritis. In some of these examples of diseases, the therapeutic gene may encode a replacement enzyme or protein of the genetic or acquired disease, an antisense or ribozyme molecule, a decoy molecule, or a suicide gene product. References: 1. Khvorova, A. & Watts, J. K. (2017) The chemical evolution of oligonucleotide therapies of clinical utility. Nat. Biotechnol.35:238–248. 2A. Martin Egli, Muthiah Manoharan. (2023) Chemistry, structure and function of approved oligonucleotide therapeutics, Nucleic Acids Research, 51:2529–2573. 2B. Ostergaard, M. E. et al., (2015) Efficient synthesis and biological evaluation of 5’-galnac conjugated antisense oligonucleotides. Bioconjug. Chem. , 26:1451–1455. 3. Lehman N. (2015) The RNA world: 4,000,000,050 years old. Life (Basel).; 5:1583–1586. 4. Eckstein F. (1966) Nucleoside phosphorothioates. J. Am. Chem. Soc.88:4292–4294. 5. Furukawa Y., Kobayashi K., Kanai Y., Honjo M. (1965) Synthesis of 2′-O-methyluridine, 2′-O-methylcytidine and their relating compounds. Chem.13:1273–1278 6. Bumcrot D., Manoharan M., Koteliansky V., Sah D.W.Y. (2006) RNAi therapeutics: a potential new class of pharmaceutical drugs. Nat. Chem. Biol.2:711–719. 7. Hu, B., Zhong, L., Weng, Y. et al., (2020) Therapeutic siRNA: state of the art. Sig Transduct Target Ther 5, 101. 8. Deleavey G.F., Damha M.J.. Designing chemically modified oligonucleotides for targeted gene silencing. Chem. Biol. (Oxford, U. K.).2012; 19:937–954. 9. Scott L.J. Givosiran: first approval. Drugs.2020; 80:335–339.Attorney Docket No.4690.0096I 10. Saito A, Horie M, Nagase T: TGF-beta Signaling in Lung Health and Disease. Int J Mol Sci 2018, 19. 11.Cowin, AJ et al., (2001) Expression of TGFbeta and its receptors in murine fetal and adult dermal wounds. Eur. J. Dermatol.11:424-431. 12. Peng, D., Fu, M., Wang, M. et al., (2022) Targeting TGF-β signal transduction for fibrosis and cancer therapy. Mol Cancer 21:104 13. David CJ, Massague J. (2018) Contextual determinants of TGFbeta action in development, immunity and cancer. Nat Rev Mol Cell Biol.19:419–35. 14. Liu S, Ren J, Ten Dijke P. (2021) Targeting TGFbeta signal transduction for cancer therapy. Signal Transduct Target Ther.;6:8. 15. Rifkin D, Sachan N, Singh K, Sauber E, Tellides G, Ramirez F: The role of LTBPs in TGF beta signaling. Dev Dyn 2021. 16. Zhou J., Zhao Y., Simonenko V., Xu J. J., Liu K., Wang D., Shi J., Zhong T., Zhang L., Zeng L., Huang B., Tang S., Lu A. Y., et al., (2017) Simultaneous silencing of TGF-β1 and COX-2 reduces human skin hypertrophic scar through activation of fibroblast apoptosis. Oncotarget 8: 80651-80665. 17. Alterman, J.F., Godinho, B.M.D.C., Hassler, M.R. et al., (2019) A divalent siRNA chemical scaffold for potent and sustained modulation of gene expression throughout the central nervous system. Nat Biotechnol. 37:884–894. 18. Vignesh N. Hariharan, Minwook Shin, Ching-Wen Chang, , and Anastasia Khvorova et al., (2023) Divalent siRNAs are bioavailable in the lung and efficiently block SARS-CoV-2 infection, PNAS 120 (11), e2219523120. 19. ManusM.T.and P.A.Sharp (2002), Gene silencing in mammals by small interfering RNAs. Nature Review, Genetics.3(10):737-747. 20. Lu, P.Y.et al.(2003) siRNA mediated antitumorigenesis for drug target validation and therapeutics. Current Opinion in Molecular Therapeutics.5(3):225-234. 21. Kim, B. et al., (2004) Inhibition of ocular angiogenesis by siRNA targeting vascular endothelial growth factor-path way genes; therapeutic strategy for herpetic stromal keratitis. Am. J. Pathol.165(6):2177-85. 22. Tuschl, Zamore, et al., (1999) Neutralising antibody to TGFB 1, 265 Genes & Dev. 13:3191-3197. 23. Elbashir, Lendeckel and Tuschl (2001). Genes & Dev.15: 188-200. 24. Kim, D H, J. J. Rossi et al.(2005) Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy. Nat Biotechnol. February; 23(2):222-6.Attorney Docket No.4690.0096I 25. Reynolds A, et al., Induction of the interferon response by siRNA is cell type- and duplex length-dependent. RNA.2006, 12(6):988-93. 26. Lu, P. Y. and M. Woodle (2005) Delivering siRNA in vivo for functional genomics and novel therapeutics. In RNA Interference Technology. Cambridge University Press. Pp.303- 317. 27. Lu, P. Y. et al., (2005) Modulation of angiogenesis with siRNA inhibitors for novel therapeutics. TRENDS in Molecular Medicine.11(3):104-13. 28. Lu P Y, Xie F, Woodle M C. (2005) In vivo application of RNA interference: from functional genomics to therapeutics. Adv Genet.54:117-42.

[0136] Ex vivo and in vivo gene therapy with siRNA could also be used to treat a variety of cancers. These siRNA applications include, without limitation: 1) reducing expression of growth factors, reducing proteins that augment the cell cycle (e.g., KRAS Raf-1, PI-3 kinase, MEK or mTOR), growth factor receptors (e.g., EGFR, Her-2), or proteins critical for supporting cells of the tumor (e.g., VEGF, VEGFR1-2 for tumor endothelial cells); 2) targeting or reducing expression of factors that are anti-apoptotic (e.g., BCL-2 or BCLXL); and 3) targeting proteins or enzymes that reduce immune activation toward tumor (PDL1, PD1 or CTLA4 among others).

[0137] The present disclosed embodiments also disclose a method of ex vivo gene therapy comprising: (i) removing a cell from a subject; (ii) delivering a nucleic acid (such as siRNA) to the interior of the cell by contacting the cell with a transfection complex or composition comprising such a transfection complex of the present disclosed embodiments; and (iii) administering the cell comprising the nucleic acid (e.g., siRNA) to the subject. In some embodiments used in vitro, lipofectamine is a carrier of choice.

[0138] Recombinant cells may be produced using the complexes of the present disclosed embodiments. Resulting recombinant cells can be delivered to a subject by various methods known in the art. In certain embodiments, the recombinant cells are injected, e.g., subcutaneously. In other embodiments, recombinant skin cells may be applied as a skin graft onto a patient. Recombinant blood cells (e.g., hematopoietic stem or progenitor cells) are preferably administered intravenously. The cells can also be encapsulated in a suitable vehicle and then implanted in the subject. The amount of cells administered depends on a variety of factors known in the art, for example, the desired effect, subject state, rate of expression of the chimeric polypeptides, etc., and can readily be determined by one skilled in the art.

[0139] All ranges and ratios disclosed here can and necessarily do describe all subranges and subratios therein for all purposes, and all such subranges and subratios also form part andAttorney Docket No.4690.0096I parcel of the disclosed embodiments. Any listed range or ratio can be easily recognized as sufficiently describing and enabling the same range or ratio being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range or ratio discussed herein can be readily broken down into a lower third, middle third and upper third, etc.

[0140] The embodiments disclosed of pharmaceutical formulations may be used alone or in combination with other treatments or components of treatments for other dermatological or nondermatological disorders.

[0141] The disclosed embodiments will be better understood by reference to the following examples which are intended for purposes of illustration and are not intended to be interpreted in any way to limit the scope of the appended claims. While specific embodiments and application of the disclosed embodiments have been illustrated and described, it is to be understood that the disclosed embodiments are not limited to the precise configuration and components disclosed herein. Various modifications, changes, and variations, which will be apparent to those skilled in the art may be made in the arrangement, operation, and details of the methods and systems of the embodiments disclosed herein, including those of the appended claims. Finally, various features of the disclosed embodiments herein may be combined to provide additional configurations which fall within the scope of the disclosed embodiments. The following examples are intended to illustrate the kinetic measures and the efficacy of inhibitory compounds tested, including those in the disclosed embodiments. Examples Materials and Methods Cell Culture

[0142] Human HepG2 cells hepatocyte carcinoma, ATCC-for mulated EMEM medium) and human A549 cells (lung carcinoma, ATCC-formulated F-12k medium) were purchased from the American Type Culture Collection. To make the complete growth medium fetal bovine serum was added to a final concentration of 10%. Cells were incubated at 37° C. in 5% CO2. siRNA Design

[0143] Blunt-ended, 25mer siRNA duplexes targeting TGFβ-1 and Cox-2 mRNA sequences were designed as known in the art and described elsewhere, and were linked as described herein, and shown in Tables 1-2. In some of the divalent siRNA complexes a two-nucleotide (UU) is added to the 3’ end of the strand as indicated. Each individual siRNA sequence is homologous for corresponding genes among human, mouse and pig.Attorney Docket No.4690.0096I Quantitative Real-Time PCR

[0144] 1x105HepG2 cells or 5x104A549 cells in 0.5 ml of appropriate growth medium per well were seeded into 24 well plates. 24 hours after seeding, cells were transfected with siRNA by mixing siRNA with 1.5 µl lipofectamine RNAi max (ThermoFisher) and 100 µl OptiMEM media (Gibco) per sample for 15 minutes, then adding the mixture to HepG2 cells. Cells were lysed and RNA was harvested 48 hours post-transfection using the RNeasy plus mini kit (Qiagen). Following RNA isolation, cDNA was synthesized using the Maxima First Strand Synthesis kit (ThermoFisher). 500-1000 ng of RNA was incubated with buffer and enzyme for at 25° C for 10 minutes, 50° C for 15 minutes, and 85° C for 5 minutes. qPCR was performed using TaqPath master mix (Applied Biosystems) and Taqman assays (Thermofisher) for human TGF-Β1 and Cox2. GAPDH was used as the endogenous control. The fluorescent dye VIC was used for endogenous control probes and FAM was used for experimental gene probes. A QuantStudio 5 real-time PCR machine (Thermofisher) and a program of 50° C for 2 minutes, 95° C for 10 minutes followed by 95° C for 15 minutes and 60° C for 1 minute repeated 40 times was used for PCR amplification. Relative expression was calculated from delta-delta Ct values. Cell Viability assay

[0145] 2.5x103HepG2 cells or 1x103A549 cells in 40 µl of appropriate growth medium per well were seeded into 384 well plates. 24 hours after seeding, cells were transfected with siRNA by mixing siRNA with 0.075 µl lipofectamine RNAi max (ThermoFisher) and 10 µl OptiMEM media (Gibco) per sample for 15 minutes, then adding the mixture to cells. 72 hours post-transfection, 25 µl CellTiter-Glo 2.0 was added to each well and incubated in the dark for 10 minutes. Bioluminescence was measured using a Cytation 5 plate reader (BioTek Inc). IC50and EC50calculations

[0146] IC50 and EC50 values were calculated in GraphPad Prism 9. To generate IC50 values, qPCR data was fitted to a sigmoidal four parameter logistic (4PL) curve. To generate EC50 values, cell viability data was fitted to an asymmetrical five parameter curve. Summary of LC-MS protocols

[0147] The identities of the final siRNA constructs were confirmed using ESI-MS profile analysis. The buffers utilized for LC-MS for Oligo’s were the same every time. Buffer A is composed of water, hexafluoro-2-propanol (HFIP), N, N-Diisopropylethylamine, and EDTA while Buffer B is composed of water, acetonitrile, hexafluoro-2-propanol, N, N- Diisopropylethylamine, and EDTA. The adopted method utilized a 260 nm 2.1 x 50 mmAttorney Docket No.4690.0096I Clarity 2.6u Oligo-XT Column, and gradient 5-25% B in 20min, 25-65% B in 1 min at 60° C. The two-buffer compositions are as follows: A=1%HFIPA / 0.1%DIEA and B=65%ACN / 0.075%HFIPA / 0.0375% DIEA. The data was analyzed by ThermoFisher LTQ Ion Trap in negative ion mode and all the samples are diluted to appropriate concentrations with nanopure water. The ESI-MS m / z results and the corresponding figures are listed in Table 3. Figures 15-18 show the representative characterization of siRNAs by HPLC chromatography and ESI-MS profiles. Table 3: ESI-MS analysis of the siRNA constructs.Example 1. General protocols of solid phase Oligonucleotide synthesis

[0148] Single siRNA strands were synthesized on ABI DNA / RNA synthesizer each at 10µmol scale of synthesis using either standard or Universal CPG support. The four phosphoramidites (U, A, C & G) used were all regular with 2’-O-tert-Butyldimethylsilyl group at 2’ position. The exocylic amino groups on the bases were protected with Acetyl, isobutyl and Benzyl groups for C, G & A respectively. Both CPG columns and the amiditesAttorney Docket No.4690.0096I were purchased from Glen Research Inc. Synthesis was achieved using a modified 10 µmol RNA protocol which involved 10 minutes coupling time for each base added with ETT as activator. Oxidation and deblock steps were both accomplished using 0.5M Iodine / water and 3% DCA / Dichloromethane solutions respectively; while Cap Mix A & B reagents were used for capping reaction. Synthesis of sense strands linked via a linker was achieved in two steps. In the first step, the first 25 nucleotides were coupled to the CPG with the 25th nucleotide added on DMT-ON mode. The second and the final step of synthesis involved addition of the linker, either disulfide or Spacer 9; and the last half of 25 nucleotides. The last nucleotide was added on DMT-ON mode to allow for efficient downstream purification of the final siRNA using Glen Pak RNA Cartridges. On the other hand, antisense strands were all synthesized in one step and DMT-ON mode. For all the siRNAs synthesized, stepwise coupling efficiencies were above 90% and the average overall yield was over 40 % based on the trityl maps. Example 2. CPG Cleavage and deprotection of the siRNAs

[0149] Upon completion of the synthesis, siRNAs were cleaved from the CPG support followed by deprotection of exocyclic amine and 2’-hydroxyl protecting groups. This was achieved following our standard operating protocols. The CPG in each 10 µmole column was split into two equal portions in 15mL vials. Each portion was incubated in 5mL of AMA cleavage solution for 2.5 hours in room 37lightly37re. This was followed by CPG filtration and the filtrate was concentrated to dryness using speed vacuum evaporator. The fluoride labile 2’-O-tert-Butyldimethylsilyl group was removed by reconstituting the crude solid residue in 300uL of dry DMSO.80uL of TEA were added to enhance the stability of DMT group under this deprotection condition. The mixture was thoroughly mixed by 37lightly warming it at 40C for 10 minutes. Triethylamine trihydrofluoride (TEA.3HF) solution (150µL) were then added into the DMSO solution, thoroughly mixed and the resultant solution mixture was incubated at 55°C for 2.5 hours. This was followed by cooling the reaction mixture at -20°C for 15 minutes and quenching the excess acidic HF reagent with quenching buffer (1.5mL) . The quenched solution (~2mL) was finally subjected to downstream purification using reverse phase-based Glen-Pak™ RNA purification cartridge (0.2-1mL) and following the recommended desalting protocols. Each RNA cartridge was conditioned using 1-2 mL of acetonitrile followed by 2mL of 2M TEAA. The oligo / buffer mixture solution was loaded to the cartridge. The cartridge was washed with 2mL of wash Solution (10% Acetonitrile: 90% 2M TEAA). This rinses away the failure sequences from the cartridge. Wash solution was rinsed by eluting the column with 2mL of deionized water.Attorney Docket No.4690.0096I Detritylation of the DMT group was achieved by rinsing the cartridge with 4 x lmL of 4% TFA. A clear pink / orange band was evident of full sequence with DMT-ON. The TFA acid was washed out flushing the cartridge with 3mL of deionized water. The full and purified siRNA was finally eluted with 1.5mL of 1M ammonium bicarbonate in 30% acetonitrile in water. The eluate was concentrated to dryness and reconstituted to 500µL for qualitative and quantitative analysis using HPLC and HRMS spectroscopic techniques. Example 3. Qualitative and quantitative analysis of the siRNAs Constructs

[0150] Qualitative analysis of the purified single siRNAs was performed using Reverse- Phase high pressure liquid chromatography (HPLC) on Agilent 1260 Infinity II Quaternary Pump LC systems with diode array detector, all operated by Agilent Open Lab processing software. For analytical and preparative purposes, Ion-pair, IPC18 advanceBio Oligonucleotide 4.6 x 150 mm and XBridge Oligonucleotide BEH C18 OBDTM Prep column 130A, 2.5 µm, 10 x 50 mm columns were used respectively. High resolution was achieved by gradually increasing the gradient of mobile phase B (90% Acetonitrile in 0.1M TEAA), 10-30% B in 20 minutes. Mobile phase A was 0.1M TEAA. The pH of both buffers was maintained at 7.0. The LC purity of all the single siRNAs was more than 95%. Pure single siRNAs were quantified using Agilent Cary UV-Vis Multicell Peltier and Thermo- Scientific NanoDrop™ One Microvolume UV-Vis Spectrophotometers. Pure single siRNAs were quantified and annealed following SOPs developed in our laboratory. Equimolar volumes of both sense and antisense strands were thoroughly mixed by vortex in 2mL vials and heated at 95oC for 5 minutes in a well-insulated heat block. The sample solution in the heat block was then allowed to gradually cool to room temperature taking 2hours. The solutions were finally cooled at 0-4°C for 30 minutes before further HPLC analysis to ascertain the quality of the duplexes. Table 1 below summarizes final duplex siRNA constructs synthesized for in vitro studies. The HPLC profiles depicting the purity of these constructs are illustrated in figures (1-24). Example 4. In vitro RT-PCR evaluation of TGF-β1 siRNAs in A549 cell line.

[0151] (2A.) TGF-Β1 expression in A549 cells treated with eleven 5x serial dilutions of TGF-Β1, TGF-Β1-Cox2 hetero-dual, Cox2-TGF-Β1 hetero-dual, or TGF-Β1 homo-dual siRNA for 48 hours. Final siRNA concentrations were 200, 40, 8, 1.6, .32, 0.64, 0.0128, 0.00256, 0.000512, 0.000102, 0.0000205 nM. TGF-Β1 expression was analyzed by qPCR. (2B.) IC50 values and other statistics were derived from data depicted in bar graphs. R2indicates strength of fitting to a sigmoidal four parameter logistic (4PL) curve. See Figure 2.Attorney Docket No.4690.0096I Example 5. Efficacy study of divalent TGF-Β1 and Cox-2 siRNAs by cell viability assay in A549 cell line.

[0152] Viability of 105A549 cells treated with ten 5x serial dilutions of TGF-Β1 and Cox2 single and dual siRNA for 72 hours. Final siRNA concentrations were 1000, 200, 40, 8, 1.6, .32, 0.64, 0.0128, 0.00256, 0.000512, 0.000102, 0.0000205 nM. Viability was determined by luminescence from Cell Titer Glo Assay 2.0. Relative viability was calculated by comparing signal to non-silencing (NS) control siRNA. R2indicates strength of fitting to an asymmetric five parameter curve. Example 6. In vitro RT-PCR evaluation of TGF-Β1 siRNAs in HepG2 cells.

[0153] TGF-Β1 expression in HepG2 cells treated with eleven 5x serial dilutions of TGF-Β1, TGF-Β1-Cox2 hetero-dual, Cox2-TGF-Β1 hetero-dual, or TGF-Β1 homo-dual siRNA for 48 hours. Final siRNA concentrations were 200, 40, 8, 1.6, .32, 0.64, 0.0128, 0.00256, 0.000512, 0.000102, 0.0000205 nM. TGF-Β1 expression was analyzed by qPCR. IC50 values and other statistics were derived from data depicted in bar graphs. R2indicates strength of fitting to a sigmoidal four parameter logistic (4PL) curve. See Figure 4a and 4b. Example 7. Efficacy study of TGF-Β1 and Cox-2 siRNAs by cell viability assay in PC3 cells.

[0154] Viability of PC3 cells treated with ten 5x serial dilutions of TGF-Β1 and Cox2 siRNA, either combined as single siRNAs or linked by a disulfide linker, for 72 hours. Viability was determined by luminescence from Cell Titer Glo Assay 2.0. Relative viability was calculated by comparing signal to non-silencing (NS) control siRNA. Final siRNA concentrations were 1000, 200, 40, 8, 1.6, .32, 0.64, 0.0128, 0.00256, 0.000512, 0.000102, 0.0000205 nM. R2indicates strength of fitting to an asymmetric five parameter curve. See Figure 10. Example 8. Efficacy study of TGF-Β1 and Cox-2 siRNAs by cell viability assay in MiaPaca-2 cells.

[0155] Viability of MiaPaca-2 cells treated with ten 5x serial dilutions of TGF-Β1 and Cox2 siRNA, either combined as single siRNAs or linked by a disulfide linker, for 72 hours. Viability was determined by luminescence from Cell Titer Glo Assay 2.0. Relative viability was calculated by comparing signal to non-silencing (NS) control siRNA. Final siRNA concentrations were 1000, 200, 40, 8, 1.6, .32, 0.64, 0.0128, 0.00256, 0.000512, 0.000102, 0.0000205 nM.. R2indicates strength of fitting to an asymmetric five parameter curve. See Figure 11.Attorney Docket No.4690.0096I Example 9. Efficacy study of TGF-Β1 and Cox-2 siRNAs by cell viability assay in SCC-25 cells.

[0156] Viability of SCC-25 cells treated with ten 5x serial dilutions of TGF-Β1 and Cox2 siRNA, either combined as single siRNAs or linked by a disulfide linker, for 72 hours. Viability was determined by luminescence from Cell Titer Glo Assay 2.0. Relative viability was calculated by comparing signal to non-silencing (NS) control siRNA. Final siRNA concentrations were 1000, 200, 40, 8, 1.6, .32, 0.64, 0.0128, 0.00256, 0.000512, 0.000102, 0.0000205 nM. R2indicates strength of fitting to an asymmetric five parameter curve. Example 10. Comparison study of divalent hetero- TGF-Β1 and Cox-2 siRNAs by cell viability assay in A549 cells.

[0157] A549 cells were transfected with disulfide-linked siRNAs targeting TGFβ1 or / and Cox2, followed by a 72-hour incubation. The siRNAs were diluted in a five-fold series, resulting in final concentrations of 1000, 200, 40, 8, 1.6, 0.32, 0.64, 0.0128, 0.00256, 0.000512, 0.000102, and 0.0000205 nM. Cell viability was determined by luminescence measurements using the Cell Tilter Glo Assay 2.0 (Promega) and BioTek Cytation reader (Agilent). The readings were normalized against Non-silence siRNA (Qiagen), and the data was fitted to the asymmetric (five-parameter) equation using GraphPad Prism. The bottom, EC50, and R2values were obtained from the best-fit analysis provided by the software. Example 11. Comparison study of divalent hetero- TGF-Β1 and Cox-2 siRNAs by cell viability assay in PC3 cells.

[0158] PC3 cells were transfected with disulfide-linked siRNAs targeting TGFβ1 or / and Cox2, followed by a 72-hour incubation. The siRNAs were diluted in a five-fold series, resulting in final concentrations of 1000, 200, 40, 8, 1.6, 0.32, 0.64, 0.0128, 0.00256, 0.000512, 0.000102, and 0.0000205 nM. Cell viability was determined by luminescence measurements using the Cell Tilter Glo Assay 2.0 (Promega) and BioTek Cytation reader (Agilent). The readings were normalized against Non-silence siRNA (Qiagen), and the data was fitted to the asymmetric (five-parameter) equation using GraphPad Prism. The bottom, EC50, and R2values were obtained from the best-fit analysis provided by the software.

Claims

Attorney Docket No.4690.0096I Claims 1. A divalent double stranded RNA molecule comprising a first RNA molecule covalently linked to a second RNA molecule, wherein said first RNA molecule targets the expression of TGFβ-1 and wherein said second RNA molecule targets expression of Cox-2.

2. The divalent RNA molecule of claim 1, wherein the covalent link is an intracellularly cleavable disulfide linker.

3. The divalent RNA molecule of claim 1, wherein the covalent link is an ethylene glycol (PEG) linker that is not intracellularly cleavable.

4. The divalent RNA molecule of any preceding claim, wherein the covalently linked RNA molecules are double stranded siRNA molecules.

5. The divalent RNA molecule of claim 4, wherein each of the covalently linked siRNA molecules comprises one or more modified nucleotides selected from the group consisting of 2’-fluoro, 2’-O-methyl, phosphate, phosphorothioates, vinyl phosphonate, methyl vinyl phosphonate, MOE, phosphorodiamidate morpholinos (PMO), 2'-methoxyethoxy, 2'-fluoro, 2'-allyl, 2'-O-[2-(methylamino)-2-oxoethyl], 4'-thio, 4'-CH2-O-2'-bridge, 4'-(CH2)2-O-2'- bridge, 2'-LNA, 2'-amino and 2'-O--(N-methylcarbamate) ribonucleotide.

6. The divalent RNA molecule of any preceding claim, wherein the linked RNA molecules are covalently linked through only their sense strands.

7. The divalent RNA molecule of any preceding claim, wherein the linked RNA molecules are covalently linked through only their antisense strands.

8. The divalent RNA molecule of claim 6 or 7, wherein the strands are covalently linked at their 5’ ends, at their 3’ ends, or with a 5’ end of a first RNA molecule to a 3’ end of a second RNA molecule.

9. The divalent RNA molecule of any preceding claim, comprising a single linker.

10. The divalent RNA molecule of any preceding claim, wherein the divalent molecule is selected from the group consisting of SEQ ID Nos.3-14.

11. The divalent RNA molecule of claim 10 further comprising antisense strands that are at least 80%, 90%, 95% or at least 99% complementary to the selected sense strands.

12. The divalent RNA molecule of claim 10 further comprising antisense strands selected from the group consisting of SEQ ID Nos.15-18.

13. The divalent RNA molecule of claim 10 comprising SEQ ID No.8 or SEQ ID No.

9.

14. The divalent RNA molecule of claim 10 comprises SEQ ID No.6 or SEQ ID No.

4.

15. A composition comprising: the divalent RNA molecule of any of claims 1-14, further comprising lipofectamine.Attorney Docket No.4690.0096I 16. A pharmaceutical composition comprising: the divalent RNA molecule of any of claims 1-14 and a pharmaceutically acceptable carrier, diluent, or excipient.

17. The composition of claim 16 further comprising a histidine-lysine copolymer.

18. A pharmaceutical composition comprising a divalent siRNA molecule comprising: two cleavably linked siRNA molecules selected from the group consisting of SEQ ID Nos.3- 14, and complementary antisense sequences selected from the group consisting of SEQ ID Nos.15- 18, wherein the composition further comprises a histidine-lysine copolymer carrier, and wherein the carrier is mixed with the divalent siRNA molecule to form nanoparticles.

19. The pharmaceutical composition of claim 18, wherein the nanoparticles have an average diameter selected from the group consisting of 80-400nm, 100-300 nm, 130-250nm, and 150-200 nm.

20. The pharmaceutical composition of claim 18 or 19, wherein the divalent siRNA molecule comprises a first siRNA targeting the TGFβ-1 gene cleavably linked to a second siRNA targeting the Cox-2 gene, wherein the link between the siRNA molecules is a disulfide bond.

21. The composition of any of claims 18-20, wherein the histidine-lysine copolymer has the formula (R)K(R)-K(R)-(R)KCX), where R=KHHHKHHHKHHHKHHHK (SEQ ID NO: 33), R=KHHHKHHHKHHHHKHHHK (SEQ ID NO: 34), X=C(O)NH2, K=lysine, and H=histidine.

22. A method of treating a subject with cancer the pharmaceutical composition of any of claims 18-21, comprising: administering to the subject an pharmacologically effective amount of the composition, wherein administration of the composition results in the reduction in expression of at least one of TGFβ-1 or Cox-2 genes in the subject.

23. The method of treating a subject with cancer of claim 22, wherein the reduction in gene expression of at least one of the TGFB-1 or the Cox-2 genes is selected from the group consisting of: at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, and at least 99%.

24. The method of treating a subject with cancer of claim 22 or claim 23, wherein the reduction in cancer tumor volume is selected from the group consisting of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, and 100%.

25. The method of any of claims 22-24, wherein the subject is a mammal.

26. The method or claim 25, wherein the subject is a human.

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