Compositions and methods for treating fibrosis

Abstract

Compositions and methods for reducing one or more symptoms associated with fibrosis are disclosed. The compositions include one or more agents that inhibit SUN2 expression or activity, directly, or indirectly. The composition includes one or more functional nucleic acids that inhibit the production or stability of SUN2, CTDNEP1, and / or NEP1R1. In some forms, the compositions include a dominant negative protein or peptide to disrupt SUN2 function, including dominant negative truncations of Sun or Nesprin proteins In some forms the composition is a gene editing composition targeting SUN2, CTDNEP1, and / or NEP1R1. The compositions can include nucleic acids and / or small molecule activators that increase expression of CK2. The compositions can be administered to a subject in need thereof to treat one or more conditions in which fibrosis is implicated.

Description

[0001] ATTORNEY DOCKET # YU 9154

[0002] COMPOSITIONS AND METHODS FOR TREATING FIBROSIS

[0003] CROSS REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of and priority to UU.S. Serial No. 63 / 730,404, filed December 10, 2024, the entire content of which is hereby incorporated herein by reference for all purposes in its entirety.

[0005] REFERENCE TO THE SEQUENCE LISTING

[0006] The Sequence Listing submitted as an XML file named “YU_9154_PCT_ST26”, created December 10, 2025, and having a size of 139,472 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.834(c)(1).

[0007] FIELD OF THE INVENTION

[0008] The disclosed invention is generally in the field of fibrosis and specifically in the area of compositions and methods of use thereof to treat fibrosis.

[0009] BACKGROUND OF THE INVENTION

[0010] Fibrosis is the replacement of functional tissue architecture with excess fibrous connective tissue, leading to a reduction in organ function and ultimately organ failure and death. Fibrosis can affect all tissues in the body and therefore is a ubiquitous problem that contributes massively to morbidity and mortality worldwide. While fibrosis is the common end-point for a wide range of diseases, the underlying etiologies and mechanisms can be either core or organ specific, and in the majority of cases remain ill-defined / idiopathic. There are only two approved anti-fibrotic therapies (Pirfenidone and Nintedanib) and both are licensed exclusively for the treatment of patients with mild- moderate Idiopathic Pulmonary Fibrosis (IFF). There is therefore an urgent unmet need to develop new anti-fibrotic therapies for use in other fibrotic diseases.

[0011] Fibrosis contributes to 45% of human deaths in the US and Europe, and there are few efficacious treatments that prevent or reverse existing fibrosis

[0012] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application.

[0013] BRIEF SUMMARY OF THE INVENTION

[0014] Compositions and methods for reducing one or more symptoms associated with fibrosis are disclosed. The compositions include one or more active agents, where the one or more active agents are agents that inhibit SUN2 expression or activity, directly, or indirectly. In some forms, the compositions are provided in a delivery system that binds to the unique markers present in

[0015] 45808517.1 1 ATTORNEY DOCKET # YU 9154 scar tissue, allowing the active agent(s) to concentrate at the site of fibrosis while minimizing damage to healthy tissue

[0016] In some forms, the active agent is a functional nucleic acid that inhibits the production or stability of SUN2, CTDNEP1, and / or NEP1 R1. In some forms, the active agent is a dominant negative protein or peptide to disrupt SUN2 function, including dominant negative truncations of Sun or Nesprin proteins

[0017] In some forms the active agent is a gene editing composition targeting SUN2, CTDNEP1, and / or NEP 1R1. The gene editing composition can be one that induces a single or double strand break at a SUN2, CTDNEP1, and / or NEP1R1 genetic locus in the subject and reduces expression thereof. An exemplary gene editing composition is a CRISPR / Cas system. The CRISPR / Cas system can include, for example, a single-guide RNA (sgRNA) that targets a SUN2, CTDNEP1, / or NEP1R1 and Cas nuclease or nickase.

[0018] In some forms, the active agent is a nucleic acid and / or a small molecule activator that increases expression of CK2, such as echinacoside ((Zeng, et al., Signal Transduct Target Ther, 2021;6(l):71 (doi: 10.1038 / s41392-020-00447-6.).

[0019] The disclosed compositions can be administered to a subject in need thereof to treat one or more conditions in which fibrosis is implicated. Exemplary conditions include, but are not limited to, NASH (Non-Alcoholic Steatohepatitis), keloids / hypertrophic scars, pulmonary fibrosis, for example, idiopathic pulmonary fibrosis, localized scleroderma, liver cirrhosis, systemic sclerosis and myelofibrosis.

[0020] The route of administration can be oral, parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), transdermal (either passively or using iontophoresis or electroporation), or transmucosal (nasal, vaginal, rectal, or sublingual) routes of administration or using bioerodible inserts and can be formulated in dosage forms appropriate for each route of administration.

[0021] Additional advantages of the disclosed method and compositions will be set forth in part in the description which follows, and in part will be understood from the description, or can be learned by practice of the disclosed method and compositions. The advantages of the disclosed method and compositions will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.

[0022] 45808517.1 2 ATTORNEY DOCKET # YU 9154

[0023] BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings illustrate several embodiments of the disclosed method and compositions and together with the description, serve to explain the principles of the disclosed method and compositions.

[0025] FIG. l is a schematic showing a kinase-phosphatase balance that regulates ER-associated degradation of SUN2 from the inner nuclear membrane (TNM) ((Krshnan, et al., (2022) eEife l l:e81573)).

[0026] FIG. 2A shows the expression of SUN2 in tissue sections of lung from a patient with idiopathic lung fibrosis and its colocalization in fibrotic regions (red).

[0027] FIG. 2B shows the expression of SUN2 in fibroblasts that have been plated on soft (3kPa) or stiff (500kPa) polydimethylsiloxane (PDMS) substrates.

[0028] FIG. 3A shows bleomycin induced lung fibrosis in WT and SUN2 KO mice illustrating a protection from fibrosis.

[0029] FIG. 3B shows bleomycin induced skin fibrosis in WT and SUN2 KO mice illustrating a protection from fibrosis.

[0030] DETAILED DESCRIPTION OF THE INVENTION

[0031] The disclosed methods and compositions are based at least on the identification of SUN2 as an important player in fibrosis. Data in the present application shows that reducing SUN2 protein expression is an effective therapeutic strategy for multi-tissue fibrosis. Loss of SUN2 does not impact normal tissue homeostasis.

[0032] The disclosed methods and compositions can be understood more readily by reference to the following detailed description of particular embodiments and the Example included therein and to the Figures and their previous and following description.

[0033] The disclosed compositions and methods are based at least on the identification of a new player in molecular pathways involved in mechanical signaling in fibrosis responses. Data in this application shows that intervening in this pathway can prevent fibrosis, opening up a new set of druggable targets.

[0034] It is to be understood that the disclosed method and compositions are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0035] 45808517.1 3 ATTORNEY DOCKET # YU 9154

[0036] I. DEFINITIONS

[0037] As used herein, the term “carrier” or “excipient” refers to an organic or inorganic ingredient, natural or synthetic inactive ingredient in a formulation, with which one or more active ingredients are combined.

[0038] As used herein, an “expression vector” is a vector that includes one or more expression control sequences.

[0039] As used herein, an “expression control sequence” is a DNA sequence that controls and regulates the transcription and / or translation of another DNA sequence.

[0040] "Effective amount" and “therapeutically effective amount,” used interchangeably, as applied to the nanoparticles, therapeutic agents, and pharmaceutical compositions described herein, mean the quantity necessary to render the desired therapeutic result. For example, an effective amount is a level effective to treat, cure, or alleviate the symptoms of a disease for which the composition and / or therapeutic agent, or pharmaceutical composition, is / are being administered.

[0041] As used herein, the term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequence that including coding sequences necessary for the production of a polypeptide, RNA (e.g., including, but not limited to, mRNA, tRNA and rRNA) or precursor. The polypeptide, RNA, or precursor can be encoded by a full-length coding sequence or by any portion thereof. The term also encompasses the coding region of a structural gene and the sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb on either end such that the gene corresponds to the length of the full-length mRNA. The term “gene” encompasses both cDNA and genomic forms of a gene, which may be made of DNA, or RNA. A genomic form or clone of a gene may contain the coding region interrupted with non-coding sequences termed “introns” or “intervening regions” or “intervening sequences.” Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or "spliced out" from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation.

[0042] As used herein, the term “inhibit” or other forms of the word such as “inhibiting” or “inhibition” means to hinder or restrain a particular characteristic, for example, to reduce, decrease or prevent, either partially or entirely. It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, “inhibits SUN2” means hindering or restraining the activity of the protein relative to a standard or a control.

[0043] 45808517.1 4 ATTORNEY DOCKET # YU 9154

[0044] “Inhibits SUN2” can also mean to hinder or restrain the synthesis or expression of the protein, or mRNA encoding the protein, relative to a standard or control.

[0045] As used herein, “mammal” includes both humans and non-humans and include but is not limited to humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

[0046] As used herein, the term “localization signal or sequence or domain or ligand” or “targeting signal or sequence or domain or ligand” are used interchangeably and refer to a signal that directs a molecule to a specific cell, tissue, organelle, or intracellular region. The signal can be polynucleotide, polypeptide, or carbohydrate moiety or can be an organic or inorganic compound sufficient to direct an attached molecule to a desired location.

[0047] As used herein, the term “microparticles” refers to particles having a diameter between one micron and 1000 microns, typically less than 400 microns, more typically less than 100 microns, most preferably for the uses described herein in the range of less than 10 microns in diameter. Microparticles include microcapsules and microspheres unless otherwise specified.

[0048] As used herein, the term “nanoparticles” refers to particles having a diameter of less than one micron, more typically between 50 and 1000 nanometers, preferably in the range of 100 to 300 nanometers.

[0049] As used herein, the term “operably linked” refers to a juxtaposition wherein the components are configured so as to perform their usual function. For example, control sequences or promoters operably linked to a coding sequence are capable of effecting the expression of the coding sequence, and an organelle localization sequence operably linked to protein will direct the linked protein to be localized at the specific organelle.

[0050] As used herein, the term “pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients.

[0051] As used herein, the term “pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water and emulsions such as an oil / water or water / oil emulsion, and various types of wetting agents.

[0052] As used herein, the terms “subject,” “individual,” and “patient” refer to any individual who is the target of treatment using the disclosed compositions. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human. The subjects can be symptomatic or asymptomatic. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered. A subject can include a control subject or a test subject.

[0053] 45808517.1 5 ATTORNEY DOCKET # YU 9154

[0054] As used herein, the term “treating” includes alleviating the symptoms associated with a specific disorder or condition and / or preventing or eliminating the symptoms.

[0055] As used herein, “transformed” and “transfected” encompass the introduction of a nucleic acid (e.g. a vector) into a cell by a number of techniques known in the art.

[0056] As used herein, a “vector” is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. The vectors described herein can be expression vectors.

[0057] II. COMPOSITIONS

[0058] The disclosed compositions include as active agents, one or more agents that directly or indirectly inhibit SUN2 (collectively, SUN2 inhibitors). SUN2 inhibitors are formulated in a pharmaceutically acceptable carrier for delivery to a site in a subject in need thereof.

[0059] In some forms, the composition includes one or more functional nucleic acids that inhibit SUN2, CTDNEP1, andlov NEP1R1. In some forms the composition is a gene editing composition targeting 5 UN2, CTDNEP1, and / or NEP1R1.

[0060] The gene editing composition can be one that induces a single or double strand break at a SUN2, CTDNEP1, and / or NEPlRl genetic locus in the subject and reduces expression thereof. An exemplary gene editing composition is a CRISPR / Cas system. The CRISPR / Cas system can include, for example, a single-guide RNA (sgRNA) that targets a SUN2, CTDNEP1, / or NEP1R1 and Cas nuclease or nickase.

[0061] In some forms, the compositions include nucleic acids and / or small molecule activators that increase expression of CK2.

[0062] In some forms, the compositions are configured for targeted delivery to the tissue / site in need thereof. In these forms the compositions are provided in a delivery system that binds to the unique markers present in scar tissue at the site, allowing the delivered active agent to concentrate at the site of fibrosis while minimizing damage to healthy tissue. Targeted expression at specific tissues can rely in some forms, on the use of tissue specific promoters, for example, cardiac or cardiomyocyte-specific promoters, hepatocyte-specific promoters, renal cellspecific promoters, lung cell-specific promoters, etc.

[0063] Exemplary cardiac- or cardiomyocyte- specific promoters include, but are not limited to troponin T r (cTnT), alpha myosin heavy chain (a-MHC) , myosin light chain (MLC2v) , atrial natriuretic factor (ANF) and sarcolipin (SLN) promoter. The promoters cTnT and a-MHC are only active in cardiomyocytes. ANF and SLN are only expressed in atrial contractile cardiomyocytes and MLC-2v is restricted to ventricular cardiomyocytes. SHoX2 is a promoter

[0064] 45808517.1 6 ATTORNEY DOCKET # YU 9154 potentially specific to non-contractile cardiomyocytes shaping the cardiac conduction system (CCS).

[0065] Exemplary renal-specific promoters include, but are not limited to sodium-glucose cotransporter- 2 (SGLT2) (proximal tubule), Na+-K+-Cl“ cotransporter-2 (NKCC2) (thick ascending limb), E-cadherin (distal tubule), and the entire nephron kidney-specific cadherin (KSPC), podocin (podocytes) and Kidney androgen-related protein (KAP) (androgen-responsive PCTs) and NPHS1 ((NPHS1 Adhesion Molecule, Nephrin) promoter.

[0066] Exemplary hepatocyte promoters include, but are not limited to the alpha- 1 antitrypsin (hAAT) promoter (often combined with enhancers like the alpha-1 microglobulin enhancer), al- antitrypsin promoter. Li et al (Gene Ther. 2009;16:43-51) have developed a small DNA fragment (347 bp) from the AAV chromosome 19 integration site that is capable of providing efficient and enhanced liver-specific transcription when used in recombinant AAV vectors.

[0067] Exemplary lung cell specific promoters include, but are not limited to, SP-B, SP-C (Surfactant Protein B / C), SP-A, SP-D, TTF-1 (Thyroid Transcription Factor 1), Cytokeratin 19 (KRT1 ), Carcinoembryonic Antigen (CEACAM5), and Gastrin-Releasing Peptide (GRP) promoters.

[0068] In some forms, expression vector used herein do not include a signal sequence.

[0069] A. SUN2 Inhibitors

[0070] The disclosed compositions include active agents, agents that inhibit SUN2. The active agent can inhibit SUN2 (a) directly, by inhibiting SUN2 gene expression or protein activity or (b) indirectly, by (i) modulating (inhibiting or increasing (as the case may be) the activity of a protein involved in SUN2 turnover.

[0071] 1. SUN2 Target Sequences

[0072] In some forms, the active agent inhibit SUN2 protein expression and / or activity.

[0073] Exemplary active include, but are not limited to, antisense, siRNA, miRNA, aptamers, ribozymes, triplex forming molecules, RNAi, external guide sequences specific for SUN2 or an mRNA transcribed therefrom, or a gene editing composition that targets SUN2 gene or a gene product thereof.

[0074] SAD1 / UNC84 domain protein-2 (SUN2) is a member of the SUN domain protein family, and a key component of linker of nucleoskeleton and cytoskeleton (LING) complex. The nuclear architecture functionally provides a framework for organizing and regulating diverse processes within cells. The LING complex is a nuclear envelope protein complex that mainly consists of SUN and nesprin proteins, connecting nuclear lamina and cytoskeletal filaments. LING complexes help to regulate the size and shape of the cell nucleus and also mechanically connect

[0075] 45808517.1 7 ATTORNEY DOCKET # YU 9154 the nucleus to the cytoskeleton and cell-matrix (integrin) adhesions. Several SUN proteins have been identified in several organisms, including Schizosaccharomyces pombe Sadi, Caenorhabditis elegans UNC-84 and SUN1, and five human SUN proteins. Human SUN proteins can be grouped into two subfamilies based on their intracellular localization: SUN1 and SUN2 are integral membrane components of the inner nuclear membrane (INM). SUN3 and the sperm-associated antigen 4 localize to endoplasmic reticulum and outer nuclear membrane (ONM). SUN proteins are conserved among all eukaryotes and characterized by a C-terminal 200 amino acid SUN domain.

[0076] WO 2019143300 discloses an approach within the context of Laminopathies, by providing Linker of Nucleoskeleton and Cytoskeleton (LINC) complex binding competitors (including dominant negative SUN1 or SUN2), thereby uncoupling the nucleus from its linkage to the cytoskeleton.

[0077] SUN2 undergoes regulated turnover. SUN2 levels at the INM are controlled by an ERAD-like mechanism. This process involves SUN2 ubiquitination by the SCFPTrCP ubiquitin ligase, membrane extraction by the p97 ATPase complex, and degradation by the proteasome. The opposing activities of Casein Kinase 2 (CK2) and CTDNEP1 / NEPR1 regulate SUN2 degradation. This kinase / phosphatase balance regulates SCFPTrCP binding to a non-canonical recognition motif in the SUN2 nucleoplasmic domain that is required for subsequent SUN2 ubiquitination (Krshnan, et al., (2022) eLife 11 :e81573) and proteasomal turnover. The kinase CK2 and the phosphatase CTDNEP1 act, respectively, as positive and negative regulators of SUN2 degradation by influencing the phosphorylation state of non-canonical binding sites for the SCFPTrCP ubiquitin ligase, primarily amino acids termed “Site 2’’ by Krshnan et al. Binding of SCFPTrCP to phosphorylated Site 2 results in SUN2 ubiquitination and the recruitment of the p97 ATPase complex, which facilitates SUN2 membrane extraction and delivery to the proteasome for degradation (FIG. 1, reproduced from Krshnan, et al., (2022) eLife 11 :e81573). The scheme illustrates the opposing effects of the kinase CK2 and the phosphatase CTDNEP1 (and its required cofactor, NEP1R1) on SUN2 degradation. Phosphorylation of SUN2 Site 2 promotes the binding of SCFPTrCP ubiquitin ligase and subsequent SUN2 ubiquitination, membrane extraction by the p97 ATPase complex and delivery to the proteasome for degradation. SUN2 is depicted as a monomer for simplicity however it functions as a homotrimer.

[0078] Exemplary target sequences for SUN2 are:

[0079] Human Sadi and UNC84 domain containing 2 (SUN2) (NCBI Gene ID: 25777; NCBI Ref.: NC_000022.11; 21265 bp) (SEQ ID NO: 1); Human SUN domain-containing protein 2

[0080] 45808517.1 8 ATTORNEY DOCKET # YU 9154 isoform 1 (NCB1 Reference: NP_001186508.1 ( (SEQ ID NO:2); Human SUN domaincontaining protein 2 isoform 2 (NCBI Reference: NP_001186509.1 (SEQ ID NO:3); Human SUN domain-containing protein 2 isoform 3 (NCBI Reference: NP_001381356.1) (SEQ ID NO:4); Human SUN domain-containing protein 2 isoform 4 (NCBI Reference: NP_001381359.1 (SEQ ID NO:5); Human SUN domain-containing protein 2 isoform 5 (NCBI Reference: NP_001381360.1 (SEQ ID NO: 6); Human SUN domain-containing protein 2 isoform 6 (NCBI Reference: NP_001381365.1 (SEQ ID NO:7); Human SUN domain-containing protein 2 isoform 7 (NCBI Reference: NP_001381367.1) (SEQ ID NO:8); Human SUN domain-containing protein 2 isoform 8 (NCBI Reference: NP_001381369.1 (SEQ ID NO: 9); Human SUN domaincontaining protein 2 isoform 9 (NCBI Reference: NP_001381371.1 ) (SEQ ID NO: 10); Human SUN domain-containing protein 2 isoform 10 (NCBI Reference: NP_001381372.1) (SEQ ID NO: 11); and Human SUN domain-containing protein 2 isoform 11 (NCBI Reference: NP_001381373.1 (SEQ ID NO: 12).

[0081] 2. CTDNEP1 Target Sequences

[0082] In some forms, the active agent is an agent that inhibit CTDNEP1 protein expression and / or activity, for example antisense, siRNA, miRNA, aptamers, ribozymes, triplex forming molecules, RNAi, external guide sequences specific for CTDNEP1 or an mRNA transcribed therefrom, or a gene editing composition that targets CTDNEP1 gene or a gene product thereof.

[0083] CTDNEP1 is a member of the C-terminal domain phosphatases (CTDPs), which is a subfamily of the haloacid dehalogenase (HAD) superfamily of magnesium-dependent phosphatases that share a characteristic Rossmann-like fold and catalytic mechanism involving a DxDx(V / T) active site motif.

[0084] The active agent can target human C-terminal domain Nuclear Envelope Phosphatase 1 (CTDNEP1) DNA Sequence (NCBI Gene ID: 23399; NCBI Ref.: NC_000017.11; 8388 bp) (SEQ ID NO: 13), the mRNA transcribed therefrom or the human CTD Nuclear envelope phosphatase 1 protein sequence (NCBI Reference: NP_001137247.1) (SEQ ID NO: 14).

[0085] 3. NEP1R1 Target Sequences

[0086] In some forms, the active agent is an agent that inhibits NEP1R1 protein expression and / or activity, antisense, siRNA, miRNA, aptamers, ribozymes, triplex forming molecules, RNAi, external guide sequences, specific for the NEP1R1 gene, or an mRNA transcribed therefrom or a gene editing composition that targets NEP1R1 gene or a gene product thereof.

[0087] Nuclear Envelope Phosphatase 1 Regulatory Subunit 1 (NEP1R1) is currently the best characterized binding partner of CTDNEP1. Biochemical evidence establishes NEP1R1 as an activating regulatory subunit for CTDNEP1, with NEP1R1 directly binding, stabilizing, and

[0088] 45808517.1 9 ATTORNEY DOCKET # YU 9154 enhancing the catalytic activity of CTDNEP1. Mechanistically, NEP1R1 acts as an activating regulatory subunit that directly binds and increases the phosphatase activity of CTDNEP1. Knockdown of NEP1R1 generates identical phenotypes to reported loss of CTDNEP1 in mammalian cells, establishing CTDNEP1-NEP1R1 as an evolutionarily conserved membrane protein phosphatase complex that restricts ER expansion. Gao et al., (PNAS 2024 Vol. 121 No. 22 e2321 167121 ).

[0089] For example, the active agent can target DNA Sequence (NCBI Gene ID: 255919; NCBI Ref: NC_000016.10; 11857 bp) (SEQ ID NO: 15), the mRNA transcribed therefrom, expression or levels of human nuclear envelope phosphatase-regulatory subunit 1 isoform 1 (NCBI Reference: NP_694993.2; 142 aa) (SEQ ID NO: 16); or human nuclear envelope phosphatase-regulatory subunit 1 isoform 2 (NCBI Reference: NP_001268718.1 ; 125 aa) (SEQ ID NO: 17).

[0090] 4. CK2 Target Sequences

[0091] In some forms, the active agent is an agent that activates Casein Kinase 2 (CK2) protein expression and / or activity.

[0092] CK2 is a constitutively active Ser / Thr protein kinase, which phosphorylates hundreds of substrates, controls several signaling pathways, and is implicated in a plethora of human diseases.

[0093] In some forms, expression of CK2 is increased at a site in need thereof, by administering to the site, of an isolated nucleic acid molecule, which includes an expression vector and a transgene, whereby the transgene is CK2 and is operably linked to a promoter, for example a tissue specific promoter, as described above.

[0094] In some embodiments, the expression vector is a virus expression vector.

[0095] In some embodiments, the virus expression vector is selected from the group consisting of Lentivirus, Adenovirus and Adeno-associated virus (AAV). Preferably the virus expression vector is adeno-associated virus (AAV).

[0096] In some embodiments, the AAV vector is selected from the group consisting of AAV9 (serotype 9), AAV1 (serotype 1), AAV6 (serotype 6), AAV8 (serotype 8), AAV2i8 and AAV9.45.

[0097] In some embodiments, the AAV vector is AAV9 (serotype 9).

[0098] For example, the active agent can upregulate / increase the expression of human casein kinase 2 alpha 1 (CSNK2A1; Gene ID: 1457; NCBI Ref.: NC_000020.11 ; 71293 bp) (SEQ ID NO:18)or the levels of Human Casein Kinase 2 subunit alpha isoform a (NCBI Reference:

[0099] 45808517.1 10 ATTORNEY DOCKET # YU 9154

[0100] NP_001349699.1) (SEQ ID NO: 19) or Human Casein Kinase 2 subunit alpha isoform b (NCB1 Reference: NP_808228.1) (SEQ ID NO:20).

[0101] 5. Functional Nucleic acid inhibitors

[0102] The inhibitor can be a functional nucleic acid. Protein expression and / or activity of a desired protein can be inhibited using a functional nucleic acid (herein, inhibiting NA), or vector encoding the same, which reduces expression of the desired protein i.e., SUN2, CTDNEP1 or NEP1R1. Functional nucleic acids (FNAs) refer to those nucleic acids whose functions are beyond the conventional genetic roles of nucleic acids. As discussed in more detail below, functional nucleic acid molecules can be divided into the following non- limiting categories: antisense molecules, siRNA, miRNA, aptamers, ribozymes, triplex forming molecules, RNAi, external guide sequences, and other gene editing compositions. The functional nucleic acid molecules can act as effectors, inhibitors, modulators, and stimulators of a specific activity possessed by a target molecule, or the functional nucleic acid molecules can possess a de novo activity independent of any other molecules.

[0103] Functional nucleic acid molecules can interact with any macromolecule, such as DNA, RNA, polypeptides, or carbohydrate chains. Thus, functional nucleic acids can interact with the mRNA or the genomic DNA of a target polypeptide, or they can interact with the polypeptide itself. Often functional nucleic acids are designed to interact with other nucleic acids based on sequence homology between the target molecule and the functional nucleic acid molecule. In other situations, the specific recognition between the functional nucleic acid molecule and the target molecule is not based on sequence homology between the functional nucleic acid molecule and the target molecule, but rather, is based on the formation of tertiary structure that allows specific recognition to take place.

[0104] Therefore, the compositions can include one or more functional nucleic acids designed to reduce expression of the SUN2, CTDNEP1, andlov NEP1R1 gene, or a gene product thereof. For example, the functional nucleic acid or polypeptide can be designed to target and reduce or inhibit expression or translation of SUN2, CTDNEP1, and / or NEP1R1 mRNA; or to reduce or inhibit expression, reduce activity, or increase degradation of SUN2, CTDNEP1, and / or NEP1R1 protein. In some embodiments, the composition includes a vector suitable for in vivo expression of the functional nucleic acid.

[0105] In some embodiments, a functional nucleic acid or polypeptide is designed to target a segment of the nucleic acid sequence encoding SUN2, CTDNEP1, and / or NEP1R1, or the complement thereof, or a genomic sequence corresponding therewith, or variants thereof having a nucleic acid sequence at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,

[0106] 45808517.1 11 ATTORNEY DOCKET # YU 9154

[0107] 80%. 81%. 82%. 83%. 84%. 85%. 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%, 97%, 98%, 99% identical to a sequence encoding SUN2, CTDNEP1, andlor NEP1R1.

[0108] In some embodiments, a functional nucleic acid or polypeptide is designed to target a segment of a the nucleic acid encoding the amino acid sequence of SUN2, CTDNEP1, and / or NEP1R1, or the complement thereof, or variants thereof having a nucleic acid sequence 65%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to a nucleic acid encoding the amino acid sequence of SUN2, CTDNEP1, and / or NEP1R1.

[0109] In some embodiments, the function nucleic acid hybridizes to the nucleic acid encoding SUN2, CTDNEP1, and / or NEP1R1, or a complement thereof, for example, under stringent conditions. In some embodiments, the functional nucleic acid hybridizes to a nucleic acid sequence that encodes the amino acid sequence of SUN2, CTDNEP1, and / or NEP1R1, or a complement thereof, for example, under stringent conditions. i. RNA Interference

[0110] In some embodiments, the functional nucleic acids induce gene silencing through RNA interference. Gene expression can also be effectively silenced in a highly specific manner through RNA interference (RNAi). This silencing was originally observed with the addition of double stranded RNA (dsRNA). Once dsRNA enters a cell, it is cleaved by an RNase III -like enzyme, Dicer, into double stranded small interfering RNAs (siRNA) 21-23 nucleotides in length that contains 2 nucleotide overhangs on the 3’ ends. In an ATP dependent step, the siRNAs become integrated into a multi-subunit protein complex, commonly known as the RNAi induced silencing complex (RISC), which guides the siRNAs to the target RNA. At some point the siRNA duplex unwinds, and it appears that the antisense strand remains bound to RISC and directs degradation of the complementary mRNA sequence by a combination of endo and exonucleases. However, the effect of iRNA or siRNA or their use is not limited to any type of mechanism.

[0111] Short Interfering RNA (siRNA) is a double- stranded RNA that can induce sequencespecific post-transcriptional gene silencing, thereby decreasing or even inhibiting gene expression. In one example, a siRNA triggers the specific degradation of homologous RNA molecules, such as mRNAs, within the region of sequence identity between both the siRNA and the target RNA. For example, WO 02 / 44321 discloses siRNAs capable of sequence-specific degradation of target mRNAs when base-paired with 3' overhanging ends, herein incorporated by reference for the method of making these siRNAs.

[0112] 45808517.1 12 ATTORNEY DOCKET # YU 9154

[0113] Sequence specific gene silencing can be achieved in mammalian cells using synthetic, short double-stranded RNAs that mimic the siRNAs produced by the enzyme dicer. siRNA can be chemically or in vz'Zro-synthesized or can be the result of short double-stranded hairpin-like RNAs (shRNAs) that are processed into siRNAs inside the cell. Synthetic siRNAs are generally designed using algorithms and a conventional DNA / RNA synthesizer. Suppliers include Ambion (Austin, Texas), ChemGenes (Ashland, Massachusetts), Dharmacon (Lafayette, Colorado), Glen Research (Sterling, Virginia), MWB Biotech (Esbersberg, Germany), Proligo (Boulder, Colorado), and Qiagen (Vento, The Netherlands). siRNA can also be synthesized in vitro using kits such as Ambion’s SILENCER® siRNA Construction Kit.

[0114] The production of siRNA from a vector is more commonly done through the transcription of a short hairpin RNAse (shRNAs). Kits for the production of vectors having shRNA arc available, such as, for example, Imgcncx’s GENESUPPRESSOR™ Construction Kits and Invitrogen’s BLOCK- IT™ inducible RNAi plasmid and lentivirus vectors.

[0115] In some embodiments, the functional nucleic acid is siRNA, shRNA, miRNA. In some embodiments, the composition includes a vector expressing the functional nucleic acid. Methods of making and using vectors for in vivo expression of functional nucleic acids such as antisense oligonucleotides, siRNA, shRNA, miRNA, EGSs, ribozymes, and aptamers are known in the art. ii. Antisense

[0116] SUN2, CTDNEP1 or NEP1R1 expression can be reduced using can be antisense molecules. Antisense molecules are designed to interact with a target nucleic acid molecule through either canonical or non-canonical base pairing. The interaction of the antisense molecule and the target molecule is designed to promote the destruction of the target molecule through, for example, RNAse H mediated RNA-DNA hybrid degradation. Alternatively, the antisense molecule is designed to interrupt a processing function that normally would take place on the target molecule, such as transcription or replication. Antisense molecules can be designed based on the sequence of the target molecule. There are numerous methods for optimization of antisense efficiency by finding the most accessible regions of the target molecule. Exemplary methods include in vitro selection experiments and DNA modification studies using DMS and DEPC. It is preferred that antisense molecules bind the target molecule with a dissociation constant (Ka) less than or equal to 10’6, ICT8, IO'10, or 10’12.

[0117] An “antisense” nucleic acid sequence (antisense oligonucleotide) can include a nucleotide sequence that is complementary to a “sense” nucleic acid encoding a protein, e.g., complementary to the TRF2. Antisense nucleic acid sequences and delivery methods are well known in the art. The antisense nucleic acid can be complementary to an entire coding strand of

[0118] 45808517.1 13 ATTORNEY DOCKET # YU 9154 a target sequence, or to only a portion thereof. An antisense oligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length.

[0119] An antisense nucleic acid can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. The antisense nucleic acid also can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).

[0120] Other examples of useful antisense oligonucleotides (AONs / ASOs) include an alpha- anomeric nucleic acid. An alpha-anomeric nucleic acid molecule forms specific double- stranded hybrids with complementary RNA in which, contrary to the usual beta-units, the strands run parallel to each other (Gaultier et al., Nucleic Acids. Res. 15:6625-6641 (1987)). The antisense nucleic acid molecule can also comprise a 2’-o-methylribonucleotide (Inoue et al. Nucleic Acids Res. 15:6131-6148 (1987)) or a chimeric RNA-DNA analogue (Inoue et al. FEBS Lett., 215:327-330 (1987)). iii. Triplex forming molecules

[0121] SUN2, CTDNEP1 or NEPIRlexpression can be reduced using triplex forming molecules. Triplex forming functional nucleic acid molecules are molecules that can interact with either double-stranded or single-stranded nucleic acid. When triplex molecules interact with a target region, a structure called a triplex is formed in which there are three strands of DNA forming a complex dependent on both Watson-Crick and Hoogsteen base-pairing. Triplex molecules are preferred because they can bind target regions with high affinity and specificity. It is preferred that the triplex forming molecules bind the target molecule with a Kd less than 10-6, 10-8, 10-10, or 10-12. iv. External guide sequences

[0122] SUN2, CTDNEP1 orNEPIRl RNA expression can be reduced using external guide sequences. External guide sequences (EGSs) are molecules that bind a target nucleic acid molecule forming a complex, which is recognized by Rnase P, which then cleaves the target molecule. EGSs can be designed to specifically target a RNA molecule of choice. RNAse P aids in processing transfer RNA (tRNA) within a cell. Bacterial RNAse P can be recruited to

[0123] 45808517.1 14 ATTORNEY DOCKET # YU 9154 cleave virtually any RNA sequence by using an EGS that causes the target RNA:EGS complex to mimic the natural tRNA substrate. Similarly, eukaryotic EGS / RNAse P-directed cleavage of RNA can be utilized to cleave desired targets within eukaryotic cells. Representative examples of how to make and use EGS molecules to facilitate cleavage of a variety of different target molecules are known in the art. v. Aptamers

[0124] The functional nucleic acids can be aptamers. Aptamers are molecules that interact with a target molecule, preferably in a specific way. Typically aptamers are small nucleic acids ranging from 15-50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G-quartets. Aptamers can bind small molecules, such as ATP and theophiline, as well as large molecules, such as reverse transcriptase and thrombin. Aptamers can bind very tightly with Kd’s from the target molecule of less than 10-12 M. It is preferred that the aptamers bind the target molecule with a Kd less thanlO-6, 10-8, 10-10, or 10-12. Aptamers can bind the target molecule with a very high degree of specificity. For example, aptamers have been isolated that have greater than a 10,000 fold difference in binding affinities between the target molecule and another molecule that differ at only a single position on the molecule. It is preferred that the aptamer have a Kd with the target molecule at least 10, 100, 1000, 10,000, or 100,000 fold lower than the Kd with a background binding molecule. It is preferred when doing the comparison for a molecule such as a polypeptide, that the background molecule be a different polypeptide. vi. Ribozymes

[0125] The functional nucleic acids can be ribozymes. Ribozymes are nucleic acid molecules that are capable of catalyzing a chemical reaction, either intramolecularly or intermolecularly. It is preferred that the ribozymes catalyze intermolecular reactions. There are a number of different types of ribozymes that catalyze nuclease or nucleic acid polymerase type reactions which are based on ribozymes found in natural systems, such as hammerhead ribozymes. There are also a number of ribozymes that are not found in natural systems, but which have been engineered to catalyze specific reactions de novo. Preferred ribozymes cleave RNA or DNA substrates, and more preferably cleave RNA substrates. Ribozymes typically cleave nucleic acid substrates through recognition and binding of the target substrate with subsequent cleavage. This recognition is often based mostly on canonical or non-canonical base pair interactions. This property makes ribozymes particularly good candidates for target specific cleavage of nucleic acids because recognition of the target substrate is based on the target substrates sequence.

[0126] 45808517.1 15 ATTORNEY DOCKET # YU 9154 vii. Other Gene Editing Compositions

[0127] In some embodiments the functional nucleic acids are gene editing compositions. Gene editing compositions can include nucleic acids that encode an element or elements that induce a single or a double strand break in the target cell’s genome, and optionally a polynucleotide. The compositions can be used, for example, to reduce or otherwise modify expression of SUN2, CTDNEP1 or NEPIRl. a. Strand Break Inducing Elements CRISPR / Cas

[0128] In some embodiments, the element that induces a single or a double strand break in the target cell’s genome is a CRISPR / Cas system. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is an acronym for DNA loci that contain multiple, short, direct repetitions of base sequences. The prokaryotic CRISPR / Cas system has been adapted for use as gene editing (silencing, enhancing or changing specific genes) for use in eukaryotes (sec, for example, Cong, Science, 15:339(6121):819-823 (2013) and Jinek, et al.. Science, 337(6096):816-21 (2012)). By transfecting a cell with the required elements including a Cas gene and specifically designed CRISPRs, the organism's genome can be cut and modified at any desired location. Methods of preparing compositions for use in genome editing using the CRISPR / Cas systems are described in detail in WO 2013 / 176772 and WO 2014 / 018423, which are specifically incorporated by reference herein in their entireties.

[0129] In general, “CRISPR system” refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g., tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), or other sequences and transcripts from a CRISPR locus. One or more tracr mate sequences operably linked to a guide sequence (e.g., direct repeat-spacer-direct repeat) can also be referred to as pre-crRNA (pre-CRISPR RNA) before processing or crRNA after processing by a nuclease.

[0130] In some embodiments, a tracrRNA and crRNA are linked and form a chimeric crRNA- tracrRNA hybrid where a mature crRNA is fused to a partial tracrRNA via a synthetic stem loop to mimic the natural crRNAlracrRNA duplex as described in Cong, Science, 15 :339(6121) :819- 823 (2013) and Jinek, et al., Science, 337(6096):816-21 (2012)). A single fused crRNA- tracrRNA construct can also be referred to as a guide RNA or gRNA (or single-guide RNA

[0131] 45808517.1 16 ATTORNEY DOCKET # YU 9154

[0132] (sgRNA)). Within an sgRNA, the crRNA portion can be identified as the ‘target sequence’ and the tracrRNA is often referred to as the ‘scaffold’.

[0133] In some embodiments, one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of a CRISPR system is derived from a particular organism including an endogenous CRISPR system, such as Streptococcus pyogenes.

[0134] In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system). In the context of formation of a CRISPR complex, “target sequence” refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. A target sequence can be any polynucleotide, such as DNA or RNA polynucleotides. In some embodiments, a target sequence is located in the nucleus or cytoplasm of a cell.

[0135] In the target nucleic acid, each protospacer is associated with a protospacer adjacent motif (PAM) whose recognition is specific to individual CRISPR systems. In the Streptococcus pyogenes CRISPR / Cas system, the PAM is the nucleotide sequence NGG. In the Streptococcus thermophiles CRISPR / Cas system, the PAM is the nucleotide sequence is NNAGAAW. The tracrRNA duplex directs Cas to the DNA target consisting of the protospacer and the requisite PAM via heteroduplex formation between the spacer region of the crRNA and the protospacer DNA.

[0136] Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (including a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence. All or a portion of the tracr sequence may also form part of a CRISPR complex, such as by hybridization to all or a portion of a tracr mate sequence that is operably linked to the guide sequence.

[0137] There are many resources available for helping practitioners determine suitable target sites once a desired DNA target sequence is identified. For example, numerous public resources, including a bioinformatically generated list of about 190,000 potential sgRNAs, targeting more than 40% of human exons, are available to aid practitioners in selecting target sites and designing the associate sgRNA to affect a nick or double strand break at the site. See also, crispr.u-psud.fr / , a tool designed to help scientists find CRISPR targeting sites in a wide range of species and generate the appropriate crRNA sequences.

[0138] 45808517.1 17 ATTORNEY DOCKET # YU 9154

[0139] In some embodiments, one or more vectors driving expression of one or more elements of a CRISPR system are introduced into a target cell such that expression of the elements of the CRISPR system direct formation of a CRISPR complex at one or more target sites.

[0140] For example, a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector. CRISPR system elements that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5' with respect to (“upstream” of) or 3' with respect to (“downstream” of) a second element. The coding sequence of one element can be located on the same or opposite strand of the coding sequence of a second clement, and oriented in the same or opposite direction. In some embodiments, a single promoter drives expression of a transcript encoding a CRISPR enzyme and one or more of the guide sequence, tracr mate sequence (optionally operably linked to the guide sequence), and a tracr sequence embedded within one or more intron sequences (e.g., each in a different intron, two or more in at least one intron, or all in a single intron). In some embodiments, the CRISPR enzyme, guide sequence, tracr mate sequence, and tracr sequence are operably linked to and expressed from the same promoter.

[0141] In some embodiments, a vector includes one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a “cloning site”). In some embodiments, one or more insertion sites (e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertion sites) are located upstream and / or downstream of one or more sequence elements of one or more vectors. In some embodiments, a vector includes an insertion site upstream of a tracr mate sequence, and optionally downstream of a regulatory element operably linked to the tracr mate sequence, such that following insertion of a guide sequence into the insertion site and upon expression the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence in a eukaryotic cell. In some embodiments, a vector includes two or more insertion sites, each insertion site being located between two tracr mate sequences so as to allow insertion of a guide sequence at each site. In such an arrangement, the two or more guide sequences can include two or more copies of a single guide sequence, two or more different guide sequences, or combinations of these. When multiple different guide sequences are used, a single expression construct may be used to target CRISPR activity to multiple different, corresponding target sequences within a cell. For example, a single vector can include about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 guide sequences. In some embodiments, about

[0142] 45808517.1 18 ATTORNEY DOCKET # YU 9154 or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, such guide-sequence-containing vectors may be provided, and optionally delivered to a cell.

[0143] In some embodiments, a vector includes a regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, such as a Cas protein.

[0144] Non-limiting examples of Cas proteins include Cask CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2. Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologues thereof, or modified versions thereof. In some embodiments, the unmodified CRISPR enzyme has DNA cleavage activity, such as Cas9. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands at the location of a target sequence, such as within the target sequence and / or within the complement of the target sequence. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence.

[0145] In some embodiments, a vector encodes a CRISPR enzyme that is mutated with respect to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence. For example, an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S'. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand). Other examples of mutations that render Cas9 a nickase include, without limitation, H840A, N854A, and N863A. As a further example, two or more catalytic domains of Cas9 (RuvC I, RuvC II, and RuvC III) can be mutated to produce a mutated Cas9 substantially lacking all DNA cleavage activity. In some embodiments, a D10A mutation is combined with one or more of H840A, N854A, or N863A mutations to produce a Cas9 enzyme substantially lacking all DNA cleavage activity. In some embodiments, a CRISPR enzyme is considered to substantially lack all DNA cleavage activity when the DNA cleavage activity of the mutated enzyme is less than about 25%, 10%, 5%>, 1%>, 0.1 %>, 0.01%, or lower with respect to its non-mutated form.

[0146] In some embodiments, an enzyme coding sequence encoding a CRISPR enzyme is codon optimized for expression in particular cells, such as eukaryotic cells. The eukaryotic cells can be those of or derived from a particular organism, such as a mammal, including but not limited to human, mouse, rat, rabbit, dog, or non-human primate. In general, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest

[0147] 45808517.1 19 ATTORNEY DOCKET # YU 9154 by replacing at least one codon (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules.

[0148] The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the “Codon Usage Database”, and these tables can be adapted in a number of ways. See Nakamura, et al., Nucl. Acids Res., 28:292 (2000). Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell, for example Gene Forge (Aptagen; Jacobus, PA), are also available. In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding a CRISPR enzyme correspond to the most frequently used codon for a particular amino acid.

[0149] In some embodiments, a vector encodes a CRISPR enzyme including one or more nuclear localization sequences (NLSs). When more than one NLS is present, each may be selected independently of the others, such that a single NLS may be present in more than one copy and / or in combination with one or more other NLSs present in one or more copies. In some embodiments, an NLS is considered near the N- or C-terminus when the nearest amino acid of the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptide chain from the N-or C-terminus.

[0150] In general, the one or more NLSs are of sufficient strength to drive accumulation of the CRISPR enzyme in a detectable amount in the nucleus of a eukaryotic cell. In general, strength of nuclear localization activity may derive from the number of NLSs in the CRISPR enzyme, the particular NLS(s) used, or a combination of these factors.

[0151] Detection of accumulation in the nucleus may be performed by any suitable technique. For example, a detectable marker may be fused to the CRISPR enzyme, such that location within a cell may be visualized, such as in combination with a means for detecting the location of the nucleus (e.g., a stain specific for the nucleus such as DAPI). Cell nuclei may also be isolated from cells, the contents of which may then be analyzed by any suitable process for detecting protein, such as immunohistochemistry, Western blot, or enzyme activity assay. Accumulation in

[0152] 45808517.1 20 ATTORNEY DOCKET # YU 9154 the nucleus may also be determined indirectly, such as by an assay for the effect of CR1SPR complex formation (e.g., assay for DNA cleavage or mutation at the target sequence, or assay for altered gene expression activity affected by CRISPR complex formation and / or CRISPR enzyme activity), as compared to a control no exposed to the CRISPR enzyme or complex, or exposed to a CRISPR enzyme lacking the one or more NLSs.

[0153] In some embodiments, one or more of the elements of CRISPR system are under the control of an inducible promoter, which can include inducible Cas, such as Cas9.

[0154] Cong, Science, 15 :339(6121 ):819- 823 (2013) reported heterologous expression of Cas9, tracrRNA, pre-crRNA (or Cas9 and sgRNA) can achieve targeted cleavage of mammalian chromosomes. Therefore, CRISPR system utilized in the methods disclosed herein can be encoded within a vector system which can include one or more vectors which can include a first regulatory clement operably linked to a CRISPR / Cas system chimeric RNA (chiRNA) polynucleotide sequence, wherein the polynucleotide sequence includes (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell, (b) a tracr mate sequence, and (c) a tracr sequence; and a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme which can optionally include at least one or more nuclear localization sequences. Elements (a), (b) and (c) can arranged in a 5' to 3 orientation, wherein components I and II are located on the same or different vectors of the system, wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, and wherein the CRISPR complex can include the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence, wherein the enzyme coding sequence encoding the CRISPR enzyme further encodes a heterologous functional domain. In some embodiment, one or more of the vectors encodes also encodes a suitable Cas enzyme, for example, Cas9. The different genetic elements can be under the control of the same or different promoters.

[0155] While the specifics can be varied in different engineered CRISPR systems, the overall methodology is similar. A practitioner interested in using CRISPR technology to target a DNA sequence (such as Brd.9, Ankibl, Cacngl, and Gtl3 (Cfap20)) can insert a short DNA fragment containing the target sequence into a guide RNA expression plasmid. The sgRNA expression plasmid contains the target sequence (about 20 nucleotides), a form of the tracrRNA sequence (the scaffold) as well as a suitable promoter and necessary elements for proper processing in eukaryotic cells. Such vectors are commercially available (see, for example, Addgene). Many of the systems rely on custom, complementary oligos that are annealed to form a double stranded

[0156] 45808517.1 21 ATTORNEY DOCKET # YU 9154

[0157] DNA and then cloned into the sgRNA expression plasmid. Co-expression of the sgRNA and the appropriate Cas enzyme from the same or separate plasmids in transfected cells results in a single or double strand break (depending of the activity of the Cas enzyme) at the desired target site.

[0158] The nuclease activity of the genome editing systems described herein cleave target DNA to produce single or double strand breaks in the target DNA. Double strand breaks can be repaired by the cell in one of two ways: non-homologous end joining, and homology- directed repair. In non-homologous end joining (NHEJ), the double-strand breaks are repaired by direct ligation of the break ends to one another. As such, no new nucleic acid material is inserted into the site, although some nucleic acid material may be lost, resulting in a deletion. In homology- directed repair, a donor polynucleotide with homology to the cleaved target DNA sequence is used as a template for repair of the cleaved target DNA sequence, resulting in the transfer of genetic infomiation from a donor polynucleotide to the target DNA. As such, new nucleic acid material can be inserted / copied into the site.

[0159] Therefore, in some embodiments, the genome editing composition optionally includes a donor polynucleotide. The modifications of the target DNA due to NHEJ and / or homology- directed repair can be used to induce gene correction, gene replacement, gene tagging, transgene insertion, nucleotide deletion, gene disruption, gene mutation, etc.

[0160] Accordingly, cleavage of DNA by the genome editing composition can be used to delete nucleic acid material from a target DNA sequence by cleaving the target DNA sequence and allowing the cell to repair the sequence in the absence of an exogenously provided donor polynucleotide. Thus, the subject methods can be used to knock out a gene (resulting in complete lack of transcription or altered transcription) or to knock in genetic material into a locus of choice in the target DNA.

[0161] Alternatively, if the genome editing composition includes a donor polynucleotide sequence that includes at least a segment with homology to the target DNA sequence, the methods can be used to add, i.e., insert or replace, nucleic acid material to a target DNA sequence (e.g., to “knock in” a nucleic acid that encodes for a protein, an siRNA, an miRNA, etc.), to add a tag (e.g., 6xHis, a fluorescent protein (e.g., a green fluorescent protein; a yellow fluorescent protein, etc.), hemagglutinin (HA), FLAG, etc.), to add a regulatory sequence to a gene (e.g., promoter, polyadenylation signal, internal ribosome entry sequence (IRES), 2A peptide, start codon, stop codon, splice signal, localization signal, etc.), to modify a nucleic acid sequence (e.g., introduce a mutation), and the like. As such, the compositions can be used to

[0162] 45808517.1 22 ATTORNEY DOCKET # YU 9154 modify DNA in a site- specific, i.e., “targeted”, way, for example gene knock-out, gene knock- in, gene editing, gene tagging, etc. as used in, for example, gene therapy.

[0163] In applications in which it is desirable to insert a polynucleotide sequence into a target DNA sequence, a polynucleotide including a donor sequence to be inserted is also provided to the cell. By a “donor sequence” or “donor polynucleotide” or “donor oligonucleotide” it is meant a nucleic acid sequence to be inserted at the cleavage site. The donor polynucleotide typically contains sufficient homology to a genomic sequence at the cleavage site, e.g., 70%, 80%, 85%, 90%, 95%, or 100% homology with the nucleotide sequences flanking the cleavage site, e.g., within about 50 bases or less of the cleavage site, e.g., within about 30 bases, within about 15 bases, within about 10 bases, within about 5 bases, or immediately flanking the cleavage site, to support homology-directed repair between it and the genomic sequence to which it bears homology. The donor sequence is typically not identical to the genomic sequence that it replaces. Rather, the donor sequence may contain at least one or more single base changes, insertions, deletions, inversions or rearrangements with respect to the genomic sequence, so long as sufficient homology is present to support homology-directed repair. In some embodiments, the donor sequence includes a non-homologous sequence flanked by two regions of homology, such that homology-directed repair between the target DNA region and the two flanking sequences results in insertion of the non-homologous sequence at the target region.

[0164] Donor sequences can also include a vector backbone containing sequences that are not homologous to the DNA region of interest and that are not intended for insertion into the DNA region of interest. Generally, the homologous region(s) of a donor sequence will have at least 50% sequence identity to a genomic sequence with which recombination is desired. In certain embodiments, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 99.9% sequence identity is present. Any value between 1% and 100% sequence identity can be present, depending upon the length of the donor polynucleotide.

[0165] The donor sequence can include certain sequence differences as compared to the genomic sequence, e.g., restriction sites, nucleotide polymorphisms, selectable markers (e.g., drug resistance genes, fluorescent proteins, enzymes etc.), etc., which can be used to assess for successful insertion of the donor sequence at the cleavage site or in some cases may be used for other purposes (e.g., to signify expression at the targeted genomic locus). In some cases, if located in a coding region, such nucleotide sequence differences will not change the amino acid sequence, or will make silent amino acid changes (i.e., changes which do not affect the structure or function of the protein). Alternatively, these sequences differences may include flanking

[0166] 45808517.1 23 ATTORNEY DOCKET # YU 9154 recombination sequences such as ELPs, loxP sequences, or the like, that can be activated at a later time for removal of the marker sequence.

[0167] The donor sequence can be a single-stranded DNA, single- stranded RNA, doublestranded DNA, or double-stranded RNA. It can be introduced into a cell in linear or circular form. If introduced in linear form, the ends of the donor sequence can be protected (e.g., from exonucleolytic degradation) by methods known to those of skill in the art. For example, one or more dideoxynucleotide residues are added to the 3' terminus of a linear molecule and / or self- complementary oligonucleotides are ligated to one or both ends. See, for example, Chang et al. Proc. Natl. Acad. Sci. USA 84:4959-4963 (1987); Nehls et al. Science 272:886-889 (1996). Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group(s) and the use of modified intemucleotide linkages such as, for example, phosphorothioates, phosphor amidates, and O-mcthyl ribose or deoxyribose residues.

[0168] As an alternative to protecting the termini of a linear donor sequence, additional lengths of sequence can be included outside of the regions of homology that can be degraded without impacting recombination. A donor sequence can be introduced into a cell as part of a vector molecule having additional sequences such as, for example, replication origins, promoters and genes encoding antibiotic resistance. b. Zinc Finger Nucleases

[0169] In some embodiments, the element that induces a single or a double strand break in the target cell’s genome is a nucleic acid construct or constructs encoding a zinc finger nucleases (ZFNs). ZFNs are typically fusion proteins that include a DNA-binding domain derived from a zinc-finger protein linked to a cleavage domain.

[0170] The most common cleavage domain is the Type IIS enzyme Fokl. Fokl catalyzes double-stranded cleavage of DNA, at 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other ( Kim et al. J. Biol. Chem. 269:31 ,978-31,982 (1994b)). One or more of these enzymes (or enzymatically functional fragments thereof) can be used as a source of cleavage domains.

[0171] The DNA-binding domain, which can, in principle, be designed to target any genomic location of interest, can be a tandem array of Cys2His2 zinc fingers, each of which generally recognizes three to four nucleotides in the target DNA sequence. The CyS2His2 domain has a general structure: Phe (sometimes Tyr)-Cys-(2 to 4 amino acids)-Cys-(3 amino acids)- Phe(sometimes Tyr)-(5 amino acids)-Leu-(2 amino acids)-His-(3 amino acids)-His. By linking together multiple fingers (the number varies: three to six fingers have been used per monomer in

[0172] 45808517.1 24 ATTORNEY DOCKET # YU 9154 published studies), ZEN pairs can be designed to bind to genomic sequences 18-36 nucleotides long.

[0173] Engineering methods include, but are not limited to, rational design and various types of empirical selection methods. Rational design includes, for example, using databases including triplet (or quadruplet) nucleotide sequences and individual zinc finger amino acid sequences, in which each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers which bind the particular triplet or quadruplet sequence. See, for example, U.S. Pat. Nos. 6, 140,081; 6,453,242; 6,534,261; 6,610,512; 6,746,838; 6,866,997; 7,067,617; U.S. Published Application Nos. 2002 / 0165356; 2004 / 0197892; 2007 / 0154989; 2007 / 0213269; and International Patent Application Publication Nos. WO 98 / 53059 and WO 2003 / 016496. c. Transcription Activator-Like Effector Nucleases

[0174] In some embodiments, the element that induces a single or a double strand break in the target cell’s genome is a nucleic acid construct or constructs encoding a transcription activatorlike effector nuclease (TALEN). TALENs have an overall architecture similar to that of ZFNs, with the main difference that the DNA-binding domain comes from TAL effector proteins, transcription factors from plant pathogenic bacteria. The DNA-binding domain of a TALEN is a tandem array of amino acid repeats, each about 34 residues long. The repeats are very similar to each other; typically they differ principally at two positions (amino acids 12 and 13, called the repeat variable diresidue, or RVD). Each RVD specifies preferential binding to one of the four possible nucleotides, meaning that each TALEN repeat binds to a single base pair, though the NN RVD is known to bind adenines in addition to guanine. TAL effector DNA binding is mechanistically less well understood than that of zinc-finger proteins, but their seemingly simpler code could prove very beneficial for engineered-nuclease design. TALENs also cleave as dimers, have relatively long target sequences (the shortest reported so far binds 13 nucleotides per monomer) and appear to have less stringent requirements than ZFNs for the length of the spacer between binding sites. Monomeric and dimeric TALENs can include more than 10, more than 14, more than 20, or more than 24 repeats.

[0175] Methods of engineering TAL to bind to specific nucleic acids are described in Cermak, et al, Nucl. Acids Res. 1-11 (2011). US Published Application No. 2011 / 0145940, which discloses TAL effectors and methods of using them to modify DNA. Miller et al. Nature Biotechnol 29: 143 (2011) reported making TALENs for site-specific nuclease architecture by linking TAL truncation variants to the catalytic domain of Fokl nuclease. The resulting TALENs were shown

[0176] 45808517.1 25 ATTORNEY DOCKET # YU 9154 to induce gene modification in immortalized human cells. General design principles for TALE binding domains can be found in, for example, WO 2011 / 072246.

[0177] Methods for delivering nucleic acid payloads are known in the art (reviewed in Paunovska, et al. Nat. Rev. Gen, 23:265-280 (2022).

[0178] 6. Nucleic acid Activators

[0179] In some forms, the disclosed compositions include a nucleic acid that increases Casein Kinase 2 (CK2) protein expression and / or activity.

[0180] Thus, in some forms, useful compositions include nucleic acids encoding Casein Kinase 2 (CK2) protein, RPA2, and / or RPA3, preferably, in a vector for delivery and expression in cells, for example, mammalian cells.

[0181] In preferred embodiments, the nucleic acid molecule is a messenger RNA (mRNA). As used herein, the term "messenger RNA" (mRNA) refers to any polynucleotide which encodes a polypeptide of interest and which is capable of being translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ or ex vivo.

[0182] Nucleic acids in vectors can be operably linked to one or more expression control sequences. For example, the control sequence can be incorporated into a genetic construct so that expression control sequences effectively control expression of a coding sequence of interest. Examples of expression control sequences include promoters, enhancers, and transcription terminating regions. A promoter is an expression control sequence composed of a region of a DNA molecule, typically within 100 nucleotides upstream of the point at which transcription starts (generally near the initiation site for RNA polymerase II). To bring a coding sequence under the control of a promoter, it is necessary to position the translation initiation site of the translational reading frame of the polypeptide between one and about fifty nucleotides downstream of the promoter. Hamann, et al., J. Biol. Eng., 13:7 (2019) demonstrated that gene expression in hBMSCs driven by cytomegalovirus (CMV) promoter, resulted in 10-fold higher transgene expression than transfection with plasmids containing elongation factor 1 a (EFla) or rous sarcoma virus (RSV) promoters.

[0183] Enhancers provide expression specificity in terms of time, location, and level. Unlike promoters, enhancers can function when located at various distances from the transcription site. An enhancer also can be located downstream from the transcription initiation site. A coding sequence is “operably linked’’ and “under the control” of expression control sequences in a cell when RNA polymerase is able to transcribe the coding sequence into mRNA, which then can be translated into the protein encoded by the coding sequence.

[0184] 45808517.1 26 ATTORNEY DOCKET # YU 9154

[0185] Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, tobacco mosaic vims, heipes viruses, cytomegalo vims, retroviruses, vaccinia vimses, adenovimses, and adeno-associated vimses. Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (La Jolla, CA), and Invitrogen Life Technologies (Carlsbad, CA). Recent transfection studies have investigated minicircle DNA (mcDNA), nucleic acids that are derived from pDNA by recombination that removes bacterial sequences.

[0186] The vectors including the nucleic acid of interest can be administered to subjects in need thereof resulting in transfection or transformation of the cells in the subject which in turn express the protein / peptide encoded by the nucleic acid.

[0187] B. Fibrotic Tissue targeting agents

[0188] The disclosed compositions can, in some forms, include delivery systems that target unique markers present in scar tissue, allowing the medication to concentrate at the site of fibrosis while minimizing damage to healthy tissue.

[0189] Hepatocyte targeting delivery system hepatocytes, which account for 60% of the total liver cells and 80% of the liver volume, are the primary cause of liver fibrosis. Asialoglycoprotein receptor (ASGPR) is the extracellular glycoprotein receptor located on the surface of hepatocytes, and the galactose is its specific ligand. Therefore, the delivery system modified by galactose can selectively target hepatocytes and improve delivery efficiency, which became a common hepatocyte-targeting ligand. Galactose-modified liposomes loading quercetin (QC) has been used to combat arsenic-induced hepatic fibrogenesis, confirming that galactosylated liposomal QC could improve drug accumulation in the hepatocytes and protect liver from fibrosis. HSCs are regarded as the main ECM-producing cells with the injured liver and thus drive the fibrogenic process, which play a critical role in the fibrogenesis of liver. Therefore, HSCs are well accepted to be the target cells of anti-fibrotic therapy. HSCs contain approximately 80% of the body's vitamin A (VA) with a gradual distribution in the liver lobules; retinol binding protein receptor (RBPR), cell REP (CRBP) and cell retinoic acid binding protein (CRABP) over-expressed on the surface of HSCs. Therefore, VA-based delivery carriers have been utilized for targeting system. Zhang et al. conjugated VA with low molecular weight polyethylenimine (PEI) to form nanoparticles by further combining with nucleotide (RcP) to allow the antisense oligonucleotide (ASO)-laden RcP carrier (RAP) direct into the HSCs (reviewed in Xing, et al., Asian J Pharm Sci. 2020 Jul 17; 16(1):47— 61). Cyclic Arg-Gly-Asp (RGD) peptide can specifically bind to the collagen VI receptors of HSCs. A nanosystem of poly

[0190] 45808517.1 27 ATTORNEY DOCKET # YU 9154

[0191] (ethylene glycol)-b-poly (epsilon-caprolactone) (PEG-b-PCE) modified with cyclic RGD peptides to target HSCs. The results demonstrated that the nanosystem could accumulate highly and inhibit the proliferation of HSCs. The mannose 6-phosphate / insulin-like growth factor II (M6P / IGF-II) receptor expression is increased on activated HSCs, particularly during fibrosis. The receptor has binding sites for IGF-II and M6P-containing ligands.

[0192] Pulmonary Targeting'. Polyplexes based on CXCR4-inhibiting poly(ethylenimine) derivative (PEI-C) can be used for targeted pulmonary delivery. (Ding, et al., Nanomedicine . 2018 Aug;14(6):1765-1776).

[0193] Renal targeting: Proximal tubular cells play an important role in the etiology of many renal diseases, such as the formation and development of interstitial inflammation and fibrosis. It is an effective strategy to deliver drags to the proximal tubules for the treatment of kidney disease. Low molecular weight chitosan (LMWC) has been used as a carrier for the site-specific delivery of prednisolone to kidney. This is mainly because aminoglycoside, a well-known ligand of megalin receptor, shares a similar glucosamine unit level with LMWC. Kidney-specific nanocomplexes from catechol-derived LMWC (HCA-Chi), metal ions and active drag molecules (Qiao, et al., (Biomaterials. 2014;35(25):7157-7171). Vimentin is involved in renal fibrosis; N- acetylglucosamine (GlcNAc) with PEI specifically interacts with vimentin-expressing cells (Singh, et al., I Nanosci Nanotechnol. 2014; 14( 11):8356- 8364.). Kidney fibroblast plays an important role in the fibrotic process, which maintains the homeostasis of interstitial matrix and adjacent tissue. When activated by cytokines, stress and other factors, it tends to myofibroblast phenotype and contributes to matrix deposition. Consequently, targeted delivery of drags to myofibroblasts is effective in suppressing the progress of interstitial renal failure. Conjugating an agent to be delivered to PDGFRP recognizing cyclic peptide C*SRNLIDC* (PPB) [PPB-PEG- IFN-g] provides renal myofibroblast targeting capacity (Poosti, et al., FASEB I.

[0194] 2015;29(3) : 1029-1042) (reviewed in Xing, et al., Asian J Pharm Sci. 2020 Jul 17; 16(1 ):47— 61)).

[0195] C. Delivery Vehicles

[0196] The disclosed active agents can be administered and taken up into the cells of a subject with or without the aid of a delivery vehicle. Appropriate delivery vehicles are known in the art and can be selected to suit the particular inhibitor. For example, if the compound is a nucleic acid or vector, the delivery vehicle can be a viral vector, for example a commercially available preparation, such as an adenovirus vector (Quantum Biotechnologies, Inc. (Laval, Quebec, Canada). The viral vector delivery can be via a viral system, such as a retroviral vector system which can package a recombinant retroviral genome (see e.g., Pastan et al., (1988) Proc. Natl. Acad. Sci. U.S.A. 85:4486; Miller et al., (1986) Mol. Cell. Biol. 6:2895). The recombinant

[0197] 45808517.1 28 ATTORNEY DOCKET # YU 9154 retrovirus can then be used to infect and thereby deliver to the infected cells nucleic acid encoding the compound inhibitor. The exact method of introducing the altered nucleic acid into mammalian cells is, of course, not limited to the use of retroviral vectors. Other techniques are widely available for this procedure including the use of adenoviral vectors (Mitani et al., Hum. Gene Ther. 5:941-948 (1994)), adeno-associated viral (AAV) vectors (Goodman et al., Blood 84:1492-1500 (1994)), lentiviral vectors (Naidini et al., Science 272:263-267 (1996)), pseudotyped retroviral vectors (Agrawal et al., Exper. Hematol. 24:738-747 (1996)).

[0198] Physical transduction techniques can also be used, such as liposome delivery and receptor-mediated and other endocytosis mechanisms (see, for example, Schwartzenberger et al., Blood 87:472-478 (1996)). For example, in some embodiments, the functional nucleic acid inhibitor is delivered via a liposome. Commercially available liposome preparations such as LIPOFECTIN, LIPOFECT AMINE (GIBCO-BRL, Inc., Gaithersburg, Md.), SUPERFECT (Qiagen, Inc. Hilden, Germany) TRANSFECT AM (Promega Biotec, Inc., Madison, Wis.), CELLFECTIN®, DMRIE-C, DMRIE, DOTAP, DOSPA, and DOSPER, and dendrimer compositions, particularly G5-G10 dendrimers, including dense star dendrimers, PAMAM dendrimers, grafted dendrimers, and dendrimers known as dendrigrafts and SUPERFECT®. as well as other liposomes developed according to procedures standard in the art are well known. In addition, the disclosed nucleic acid or vector can be delivered in vivo by electroporation, the technology for which is available from Genetronics, Inc. (San Diego, Calif.) as well as by means of a SONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson, Ariz.). This disclosed compositions and methods can be used in conjunction with any of these or other commonly used gene transfer methods.

[0199] In some embodiments, the delivery vehicle is incorporated into or encapsulated by a nanoparticle, microparticle, micelle, synthetic lipoprotein particle, or carbon nanotube. For example, the compositions can be incorporated into a vehicle such as polymeric microparticles which provide controlled release of the compound. In some embodiments, release of the drug(s) is controlled by diffusion of the compound out of the microparticles and / or degradation of the polymeric particles by hydrolysis and / or enzymatic degradation. Suitable polymers include ethylcellulose and other natural or synthetic cellulose derivatives. Polymers which are slowly soluble and form a gel in an aqueous environment, such as hydroxypropyl methylcellulose or polyethylene oxide may also be suitable as materials for drug containing microparticles. Other polymers include, but are not limited to, polyanhydrides, poly (ester anhydrides), polyhydroxy acids, such as polylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA),

[0200] 45808517.1 29 ATTORNEY DOCKET # YU 9154 poly-3 -hydroxybut rate (PHB) and copolymers thereof, poly-4-hydroxy butyrate (P4HB) and copolymers thereof, polycaprolactone and copolymers thereof, and combinations thereof.

[0201] D. Pharmaceutical Formulations

[0202] The SUN2 inhibitory agents described herein can be formulated for administration to a subject in need thereof.

[0203] The agents described herein can be formulated for enteral, parenteral, topical, or pulmonary administration. The agents can be combined with one or more pharmaceutically acceptable carriers and / or excipients that are considered safe and effective and may be administered to an individual without causing undesirable biological side effects or unwanted interactions. The carrier is all components present in the pharmaceutical formulation other than the active ingredient or ingredients.

[0204] 1. Parenteral Formulations

[0205] The compounds described herein can be formulated for parenteral administration.

[0206] For example, parenteral administration may include administration to a patient intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intravitreally, intratumorally, intramuscularly, subcutaneously, subconjunctivally, intravesicularly, intrapericardially, intraumbilically, by injection, and by infusion.

[0207] Parenteral formulations can be prepared as aqueous compositions using techniques known in the art. Typically, such compositions can be prepared as injectable formulations, for example, solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a reconstitution medium prior to injection; emulsions, such as water-in-oil (w / o) emulsions, oil-in-water (o / w) emulsions, and microemulsions thereof, liposomes, or emulsomes.

[0208] The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), oils, such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc.), and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and / or by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.

[0209] Solutions and dispersions of the active compounds as the free acid or base or pharmacologically acceptable salts thereof can be prepared in water or another solvent or dispersing medium suitably mixed with one or more pharmaceutically acceptable excipients

[0210] 45808517.1 30 ATTORNEY DOCKET # YU 9154 including, but not limited to, surfactants, dispersants, emulsifiers, pH modifying agents, viscosity modifying agents, and combination thereof.

[0211] Suitable surfactants may be anionic, cationic, amphoteric or nonionic surface- active agents. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions. Examples of anionic surfactants include sodium, potassium, ammonium of long chain alkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium bis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate. Cationic surfactants include, but are not limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzyl ammonium chloride, polyoxyethylene and coconut amine. Examples of nonionic surfactants include ethylene glycol monostcaratc, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG- 150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates, polyoxyethylene octylphenylether, PEG- 1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, Pol oxamer® 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide. Examples of amphoteric surfactants include sodium N-dodecyl-.beta.-alanine, sodium N-lauryl-.beta.- iminodipropionate, myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.

[0212] The formulation can contain a preservalive to prevent the growth of microorganisms. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. The formulation may also contain an antioxidant to prevent degradation of the active agent(s).

[0213] The formulation is typically buffered to a pH of 3-8 for parenteral administration upon reconstitution. Suitable buffers include, but are not limited to, phosphate buffers, acetate buffers, and citrate buffers.

[0214] Water-soluble polymers are often used in formulations for parenteral administration. Suitable water-soluble polymers include, but are not limited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, and polyethylene glycol.

[0215] Sterile injectable solutions can be prepared by incorporating the active compounds in the required amount in the appropriate solvent or dispersion medium with one or more of the excipients listed above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those listed above.

[0216] 45808517.1 31 ATTORNEY DOCKET # YU 9154

[0217] In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The powders can be prepared in such a manner that the particles are porous in nature, which can increase dissolution of the particles. Methods for making porous particles are well known in the art. i. Controlled Release Formulations

[0218] The parenteral formulations described herein can be formulated for controlled release including immediate release, delayed release, extended release, pulsatile release, and combinations thereof. a. Nano- and microparticles

[0219] For parenteral administration, the one or more compounds, and optional one or more additional active agents, can be incorporated into microparticles, nanoparticles, or combinations thereof that provide controlled release of the compounds and / or one or more additional active agents. In embodiments wherein the formulations contains two or more drugs, the drags can be formulated for the same type of controlled release (e.g., delayed, extended, immediate, or pulsatile) or the drags can be independently formulated for different types of release (e.g., immediate and delayed, immediate and extended, delayed and extended, delayed and pulsatile, etc.).

[0220] For example, the compounds and / or one or more additional active agents can be incorporated into polymeric microparticles, which provide controlled release of the drag(s). Release of the drag(s) is controlled by diffusion of the drag(s) out of the microparticles and / or degradation of the polymeric particles by hydrolysis and / or enzymatic degradation. Suitable polymers include ethylcellulose and other natural or synthetic cellulose derivatives.

[0221] Polymers, which are slowly soluble and form a gel in an aqueous environment, such as hydroxypropyl methylcellulose or polyethylene oxide, can also be suitable as materials for drug containing microparticles. Other polymers include, but are not limited to, polyanhydrides, poly(ester anhydrides), polyhydroxy acids, such as polylactide (PEA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA), poly-3 -hydroxybutyrate (PHB) and copolymers thereof, poly-4-hydroxybutyrate (P4HB) and copolymers thereof, polycaprolactone and copolymers thereof, and combinations thereof.

[0222] Alternatively, the drag(s) can be incorporated into microparticles prepared from materials which are insoluble in aqueous solution or slowly soluble in aqueous solution, but are capable of degrading within the GI tract by means including enzymatic degradation, surfactant action of

[0223] 45808517.1 32 ATTORNEY DOCKET # YU 9154 bile acids, and / or mechanical erosion. As used herein, the term “slowly soluble in water’’ refers to materials that are not dissolved in water within a period of 30 minutes. Preferred examples include fats, fatty substances, waxes, wax-like substances and mixtures thereof. Suitable fats and fatty substances include fatty alcohols (such as lauryl, myristyl stearyl, cetyl or cetostearyl alcohol), fatty acids and derivatives, including but not limited to fatty acid esters, fatty acid glycerides (mono-, di- and tri-glycerides), and hydrogenated fats. Specific examples include, but are not limited to hydrogenated vegetable oil, hydrogenated cottonseed oil, hydrogenated castor oil, hydrogenated oils available under the trade name Sterotex®, stearic acid, cocoa butter, and stearyl alcohol. Suitable waxes and wax-like materials include natural or synthetic waxes, hydrocarbons, and normal waxes. Specific examples of waxes include beeswax, glycowax, castor wax, carnauba wax, paraffins and candelilla wax. As used herein, a wax-like material is defined as any material, which is normally solid at room temperature and has a melting point of from about 30 to 300°C.

[0224] In some cases, it may be desirable to alter the rate of water penetration into the microparticles. To this end, rate-controlling (wicking) agents can be formulated along with the fats or waxes listed above. Examples of rate-controlling materials include certain starch derivatives (e.g., waxy maltodextrin and drum dried corn starch), cellulose derivatives (e.g., hydroxypropylmethyl-cellulose, hydroxypropylcellulose, methylcellulose, and carboxymethylcellulose), alginic acid, lactose and talc. Additionally, a pharmaceutically acceptable surfactant (for example, lecithin) may be added to facilitate the degradation of such microparticles.

[0225] Proteins, which are water insoluble, such as zein, can also be used as materials for the formation of drug containing microparticles. Additionally, proteins, polysaccharides and combinations thereof, which are water-soluble, can be formulated with drug into microparticles and subsequently cross-linked to form an insoluble network. For example, cyclodextrins can be complexed with individual drug molecules and subsequently cross-linked. b. Method of making Nano- and Microparticles

[0226] Encapsulation or incorporation of drug into carrier materials to produce drug-containing microparticles can be achieved through known pharmaceutical formulation techniques. In the case of formulation in fats, waxes or wax-like materials, the carrier material is typically heated above its melting temperature and the drug is added to form a mixture comprising drug particles suspended in the earner material, drug dissolved in the carrier material, or a mixture thereof. Microparticles can be subsequently formulated through several methods including, but not limited to, the processes of congealing, extrusion, spray chilling or aqueous dispersion. In a

[0227] 45808517.1 33 ATTORNEY DOCKET # YU 9154 preferred process, wax is heated above its melting temperature, drug is added, and the molten wax-drug mixture is congealed under constant stirring as the mixture cools. Alternatively, the molten wax-drug mixture can be extruded and spheronized to form pellets or beads. These processes are known in the art.

[0228] For some carrier materials it may be desirable to use a solvent evaporation technique to produce drug-containing microparticles. Tn this case drug and carrier material are co-dissolved in a mutual solvent and microparticles can subsequently be produced by several techniques including, but not limited to, forming an emulsion in water or other appropriate media, spray drying or by evaporating off the solvent from the bulk solution and milling the resulting material.

[0229] In some embodiments, drug in a particulate form is homogeneously dispersed in a waterinsoluble or slowly water soluble material. To minimize the size of the drug particles within the composition, the drug powder itself may be milled to generate fine particles prior to formulation. The process of jet milling, known in the pharmaceutical art, can be used for this purpose. In some embodiments drug in a particulate form is homogeneously dispersed in a wax or wax like substance by heating the wax or wax like substance above its melting point and adding the drug particles while stirring the mixture. In this case a pharmaceutically acceptable surfactant may be added to the mixture to facilitate the dispersion of the drug particles.

[0230] The particles can also be coated with one or more modified release coatings. Solid esters of fatty acids, which are hydrolyzed by lipases, can be spray coated onto microparticles or drag particles. Zein is an example of a naturally water-insoluble protein. It can be coated onto drag containing microparticles or drag particles by spray coating or by wet granulation techniques. In addition to naturally water-insoluble materials, some substrates of digestive enzymes can be treated with cross-linking procedures, resulting in the formation of non-soluble networks. Many methods of cross-linking proteins, initiated by both chemical and physical means, have been reported. One of the most common methods to obtain cross-linking is the use of chemical crosslinking agents. Examples of chemical cross-linking agents include aldehydes (gluteraldehyde and formaldehyde), epoxy compounds, carbodiimides, and genipin. In addition to these crosslinking agents, oxidized and native sugars have been used to cross-link gelatin. Cross-linking can also be accomplished using enzymatic means; for example, transglutaminase has been approved as a GRAS substance for cross-linking seafood products. Finally, cross-linking can be initiated by physical means such as thermal treatment, UV irradiation and gamma irradiation.

[0231] To produce a coating layer of cross-linked protein surrounding drag containing microparticles or drug particles, a water-soluble protein can be spray coated onto the microparticles and subsequently cross-linked by the one of the methods described above.

[0232] 45808517.1 34 ATTORNEY DOCKET # YU 9154

[0233] Alternatively, drug-containing microparticles can be microencapsulated within protein by coacervation-phase separation (for example, by the addition of salts) and subsequently crosslinked. Some suitable proteins for this puipose include gelatin, albumin, casein, and gluten.

[0234] Polysaccharides can also be cross-linked to form a water-insoluble network. For many polysaccharides, this can be accomplished by reaction with calcium salts or multivalent cations, which cross-link the main polymer chains. Pectin, alginate, dextran, amylose and guar gum are subject to cross-linking in the presence of multivalent cations. Complexes between oppositely charged polysaccharides can also be formed; pectin and chitosan, for example, can be complexed via electrostatic interactions. ii. Injectable / Implantable formulations

[0235] The compounds described herein can be incorporated into injectable / implantable solid or semi-solid implants, such as polymeric implants. In one embodiment, the compounds arc incorporated into a polymer that is a liquid or paste at room temperature, but upon contact with aqueous medium, such as physiological fluids, exhibits an increase in viscosity to form a semi-solid or solid material. Exemplary polymers include, but are not limited to, hydroxyalkanoic acid polyesters derived from the copolymerization of at least one unsaturated hydroxy fatty acid copolymerized with hydroxyalkanoic acids. The polymer can be melted, mixed with the active substance and cast or injection molded into a device. Such melt fabrication require polymers having a melting point that is below the temperature at which the substance to be delivered and polymer degrade or become reactive. The device can also be prepared by solvent casting where the polymer is dissolved in a solvent and the drug dissolved or dispersed in the polymer solution and the solvent is then evaporated. Solvent processes require that the polymer be soluble in organic solvents. Another method is compression molding of a mixed powder of the polymer and the drug or polymer particles loaded with the active agent.

[0236] Alternatively, the compounds can be incorporated into a polymer matrix and molded, compressed, or extruded into a device that is a solid at room temperature. For example, the compounds can be incorporated into a biodegradable polymer, such as polyanhydrides, polyhydroalkanoic acids (PHAs), PLA, PGA, PLGA, polycaprolactone, polyesters, polyamides, polyorthoesters, polyphosphazenes, proteins and polysaccharides such as collagen, hyaluronic acid, albumin and gelatin, and combinations thereof and compressed into solid device, such as disks, or extruded into a device, such as rods.

[0237] The release of the one or more compounds from the implant can be varied by selection of the polymer, the molecular weight of the polymer, and / or modification of the polymer to increase degradation, such as the formation of pores and / or incoiporation of hydrolyzable linkages. Methods

[0238] 45808517.1 35 ATTORNEY DOCKET # YU 9154 for modifying the properties of biodegradable polymers to vary the release profile of the compounds from the implant are well known in the art.

[0239] 2. Enteral Formulations

[0240] Suitable oral dosage forms include tablets, capsules, solutions, suspensions, syrups, and lozenges. Tablets can be made using compression or molding techniques well known in the art. Gelatin or non-gelatin capsules can prepared as hard or soft capsule shells, which can encapsulate liquid, solid, and semi-solid fill materials, using techniques well known in the art.

[0241] Formulations may be prepared using a pharmaceutically acceptable earner. As generally used herein “carrier” includes, but is not limited to, diluents, preservatives, binders, lubricants, disintegrators, swelling agents, fillers, stabilizers, and combinations thereof.

[0242] Carrier also includes all components of the coating composition, which may include plasticizers, pigments, colorants, stabilizing agents, and glidants.

[0243] Examples of suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and methacrylic resins that are commercially available under the trade name EUDRAGIT® (Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.

[0244] Additionally, the coating material may contain conventional carriers such as plasticizers, pigments, colorants, glidants, stabilization agents, pore formers and surfactants.

[0245] “Diluents”, also referred to as "fillers," are typically necessary to increase the bulk of a solid dosage form so that a practical size is provided for compression of tablets or formation of beads and granules. Suitable diluents include, but are not limited to, dicalcium phosphate dihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin, sodium chloride, dry starch, hydrolyzed starches, pregelatinized starch, silicone dioxide, titanium oxide, magnesium aluminum silicate and powdered sugar.

[0246] “Binders” are used to impart cohesive qualities to a solid dosage formulation, and thus ensure that a tablet or bead or granule remains intact after the formation of the dosage forms. Suitable binder materials include, but are not limited to, starch, pregelatinized starch, gelatin, sugars (including sucrose, glucose, dextrose, lactose and sorbitol), polyethylene glycol, waxes, natural and synthetic gums such as acacia, tragacanth, sodium alginate, cellulose, including hydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose, and veegum, and synthetic polymers such as acrylic acid and methacrylic acid copolymers, methacrylic acid

[0247] 45808517.1 36 ATTORNEY DOCKET # YU 9154 copolymers, methyl methacrylate copolymers, aminoalkyl methacrylate copolymers, polyacrylic acid / polymethacrylic acid and polyvinylpyrrolidone.

[0248] “Lubricants” are used to facilitate tablet manufacture. Examples of suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, glycerol behenate, polyethylene glycol, talc, and mineral oil.

[0249] “Disintegrants” are used to facilitate dosage form disintegration or "breakup" after administration, and generally include, but are not limited to, starch, sodium starch glycolate, sodium carboxymethyl starch, sodium carboxymethylcellulose, hydroxypropyl cellulose, pregelatinized starch, clays, cellulose, alginine, gums or cross linked polymers, such as crosslinked PVP (Polyplasdone® XL from GAF Chemical Corp).

[0250] “Stabilizers” are used to inhibit or retard drug decomposition reactions, which include, by way of example, oxidative reactions. Suitable stabilizers include, but arc not limited to, antioxidants, butylated hydroxytoluene (BHT); ascorbic acid, its salts and esters; Vitamin E, tocopherol and its salts; sulfites such as sodium metabisulphite; cysteine and its derivatives; citric acid; propyl gallate, and butylated hydroxyanisole (BHA).

[0251] Controlled Release Enteral Formulations

[0252] Oral dosage forms, such as capsules, tablets, solutions, and suspensions, can for formulated for controlled release. For example, the one or more compounds and optional one or more additional active agents can be formulated into nanoparticles, microparticles, and combinations thereof, and encapsulated in a soft or hard gelatin or non-gelatin capsule or dispersed in a dispersing medium to form an oral suspension or syrup. The particles can be formed of the drug and a controlled release polymer or matrix. Alternatively, the drug particles can be coated with one or more controlled release coatings prior to incorporation in to the finished dosage form.

[0253] In another embodiment, the one or more compounds and optional one or more additional active agents are dispersed in a matrix material, which gels or emulsifies upon contact with an aqueous medium, such as physiological fluids. In the case of gels, the matrix swells entrapping the active agents, which are released slowly over time by diffusion and / or degradation of the matrix material. Such matrices can be formulated as tablets or as fill materials for hard and soft capsules.

[0254] In still another embodiment, the one or more compounds, and optional one or more additional active agents are formulated into a sold oral dosage form, such as a tablet or capsule, and the solid dosage form is coated with one or more controlled release coatings, such as a

[0255] 45808517.1 37 ATTORNEY DOCKET # YU 9154 delayed release coatings or extended release coatings. The coating or coatings may also contain the compounds and / or additional active agents. a. Extended release dosage forms

[0256] The extended release formulations are generally prepared as diffusion or osmotic systems, which are known in the art. A diffusion system typically consists of two types of devices, a reservoir and a matrix, and is well known and described in the art. The matrix devices are generally prepared by compressing the drug with a slowly dissolving polymer carrier into a tablet form. The three major types of materials used in the preparation of matrix devices are insoluble plastics, hydrophilic polymers, and fatty compounds. Plastic matrices include, but are not limited to, methyl acrylate-methyl methacrylate, polyvinyl chloride, and polyethylene. Hydrophilic polymers include, but are not limited to, cellulosic polymers such as methyl and ethyl cellulose, hydroxyalkylcclluloscs such as hydroxypropyl-ccllulosc, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and Carbopol® 934, polyethylene oxides and mixtures thereof. Fatty compounds include, but are not limited to, various waxes such as carnauba wax and glyceryl tristearate and wax -type substances including hydrogenated castor oil or hydrogenated vegetable oil, or mixtures thereof.

[0257] In certain preferred embodiments, the plastic material is a pharmaceutically acceptable acrylic polymer, including but not limited to, acrylic acid and methacrylic acid copolymers, methyl methacrylate, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, aminoalkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid), methacrylic acid alkylamine copolymer poly(methyl methacrylate), poly(methacrylic acid)(anhydride), polymethacrylate, polyacrylamide, poly(methacrylic acid anhydride), and glycidyl methacrylate copolymers.

[0258] In certain preferred embodiments, the acrylic polymer is comprised of one or more ammonio methacrylate copolymers. Ammonio methacrylate copolymers are well known in the art, and are described in NF XVII as fully polymerized copolymers of acrylic and methacrylic acid esters with a low content of quaternary ammonium groups.

[0259] In one preferred embodiment, the acrylic polymer is an acrylic resin lacquer such as that which is commercially available from Rohm Pharma under the tradename EUDRAGIT t®. In further preferred embodiments, the acrylic polymer comprises a mixture of two acrylic resin lacquers commercially available from Rohm Pharma under the tradenames EUDRAGIT® RL30D and EUDRAGIT ® RS30D, respectively. EUDRAGIT® RL30D and EUDRAGIT ® RS30D are copolymers of acrylic and methacrylic esters with a low content of quaternary ammonium groups, the molar ratio of ammonium groups to the remaining neutral (meth)acrylic

[0260] 45808517.1 38 ATTORNEY DOCKET # YU 9154 esters being 1 :20 in EUDRAGIT ® RE30D and 1 :40 in EUDRAGIT® RS30D. The mean molecular weight is about 150,000. EUDRAGIT ® S-100 and EUDRAGIT ® L-100 are also preferred. The code designations RL (high permeability) and RS (low permeability) refer to the permeability properties of these agents. EUDRAGIT ® RL / RS mixtures are insoluble in water and in digestive fluids. However, multiparticulate systems formed to include the same are swellable and permeable in aqueous solutions and digestive fluids.

[0261] The polymers described above such as EUDRAGIT ® RL / RS may be mixed together in any desired ratio in order to ultimately obtain a sustained-release formulation having a desirable dissolution profile. Desirable sustained-release multiparticulate systems may be obtained, for instance, from 100% EUDRAGIT® RL, 50% EUDRAGIT® RL and 50% EUDRAGIT t® RS, and 10% EUDRAGIT® RL and 90% EUDRAGIT® RS. One skilled in the art will recognize that other acrylic polymers may also be used, such as, for example, EUDRAGIT® L.

[0262] Alternatively, extended release formulations can be prepared using osmotic systems or by applying a semi-permeable coating to the dosage form. In the latter case, the desired drug release profile can be achieved by combining low permeable and high permeable coating materials in suitable proportion.

[0263] The devices with different drug release mechanisms described above can be combined in a final dosage form comprising single or multiple units. Examples of multiple units include, but are not limited to, multilayer tablets and capsules containing tablets, beads, or granules An immediate release portion can be added to the extended release system by means of either applying an immediate release layer on top of the extended release core using a coating or compression process or in a multiple unit system such as a capsule containing extended and immediate release beads.

[0264] Extended release tablets containing hydrophilic polymers are prepared by techniques commonly known in the art such as direct compression, wet granulation, or dry granulation. Their formulations usually incorporate polymers, diluents, binders, and lubricants as well as the active pharmaceutical ingredient. The usual diluents include inert powdered substances such as starches, powdered cellulose, especially crystalline and microcrystalline cellulose, sugars such as fructose, mannitol and sucrose, grain flours and similar edible powders. Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts such as sodium chloride and powdered sugar. Powdered cellulose derivatives are also useful. Typical tablet binders include substances such as starch, gelatin and sugars such as lactose, fructose, and glucose. Natural and synthetic gums, including acacia, alginates, methylcellulose, and polyvinylpyrrolidone can also be used. Polyethylene glycol,

[0265] 45808517.1 39 ATTORNEY DOCKET # YU 9154 hydrophilic polymers, ethylcellulose and waxes can also serve as binders. A lubricant is necessary in a tablet formulation to prevent the tablet and punches from sticking in the die. The lubricant is chosen from such slippery solids as talc, magnesium and calcium stearate, stearic acid and hydrogenated vegetable oils.

[0266] Extended release tablets containing wax materials are generally prepared using methods known in the art such as a direct blend method, a congealing method, and an aqueous dispersion method. In the congealing method, the drug is mixed with a wax material and either spray- congealed or congealed and screened and processed. b. Delayed release dosage forms

[0267] Delayed release formulations can be created by coating a solid dosage form with a polymer film, which is insoluble in the acidic environment of the stomach, and soluble in the neutral environment of the small intestine.

[0268] The delayed release dosage units can be prepared, for example, by coating a drug or a drug-containing composition with a selected coating material. The drug-containing composition may be, e.g., a tablet for incorporation into a capsule, a tablet for use as an inner core in a "coated core" dosage form, or a plurality of drug-containing beads, particles or granules, for incorporation into either a tablet or capsule. Preferred coating materials include bioerodible, gradually hydrolyzable, gradually water-soluble, and / or enzymatically degradable polymers, and may be conventional "enteric" polymers. Enteric polymers, as will be appreciated by those skilled in the art, become soluble in the higher pH environment of the lower gastrointestinal tract or slowly erode as the dosage form passes through the gastrointestinal tract, while enzymatically degradable polymers are degraded by bacterial enzymes present in the lower gastrointestinal tract, particularly in the colon. Suitable coating materials for effecting delayed release include, but are not limited to, cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose acetate succinate, hydroxypropylmethyl cellulose phthalate, methylcellulose, ethyl cellulose, cellulose acetate, cellulose acetate phthalate, cellulose acetate trimellitate and carboxymethylcellulose sodium; acrylic acid polymers and copolymers, preferably formed from acrylic acid, methacrylic acid, methyl acrylate, ethyl acrylate, methyl methacrylate and / or ethyl methacrylate, and other methacrylic resins that are commercially available under the tradename Eudragit® (Rohm Pharma: Westerstadt, Germany), including EUDRAGIT® L30D-55 and L100-55 (soluble at pH 5.5 and above), EUDRAGIT® L-100 (soluble at pH 6.0 and above), EUDRAGIT® S (soluble at pH 7.0 and above, as a result of a higher degree of esterification), and EUDRAGITS® NE, RL and RS (water-insoluble polymers having different degrees of

[0269] 45808517.1 40 ATTORNEY DOCKET # YU 9154 permeability and expandability): vinyl polymers and copolymers such as polyvinyl pyrrolidone, vinyl acetate, vinylacetate phthalate, vinylacetate crotonic acid copolymer, and ethylene-vinyl acetate copolymer; enzymatically degradable polymers such as azo polymers, pectin, chitosan, amylose and guar gum; zein and shellac. Combinations of different coating materials may also be used. Multi-layer coatings using different polymers may also be applied.

[0270] The preferred coating weights for particular coating materials may be readily determined by those skilled in the art by evaluating individual release profiles for tablets, beads and granules prepared with different quantities of various coating materials. It is the combination of materials, method and form of application that produce the desired release characteristics, which one can determine only from the clinical studies.

[0271] The coating composition may include conventional additives, such as plasticizers, pigments, colorants, stabilizing agents, glidants, etc. A plasticizer is normally present to reduce the fragility of the coating, and will generally represent about 10 wt. % to 50 wt. % relative to the dry weight of the polymer. Examples of typical plasticizers include polyethylene glycol, propylene glycol, triacetin, dimethyl phthalate, diethyl phthalate, dibutyl phthalate, dibutyl sebacate, triethyl citrate, tributyl citrate, triethyl acetyl citrate, castor oil and acetylated monoglycerides. A stabilizing agent is preferably used to stabilize particles in the dispersion. Typical stabilizing agents are nonionic emulsifiers such as sorbitan esters, polysorbates and polyvinylpyrrolidone. Glidants are recommended to reduce sticking effects during film formation and drying, and will generally represent approximately 25 wt. % to 100 wt. % of the polymer weight in the coating solution. One effective glidant is talc. Other glidants such as magnesium stearate and glycerol monostearates may also be used. Pigments such as titanium dioxide may also be used. Small quantities of an anti-foaming agent, such as a silicone (e.g., simethicone), may also be added to the coating composition.

[0272] 3. Topical Formulations

[0273] Suitable dosage forms for topical administration include creams, ointments, salves, sprays, gels, lotions, emulsions, and transdermal patches. The formulation may be formulated for transmucosal, transepithelial, transendothelial, or transdermal administration. The compounds can also be formulated for intranasal delivery, pulmonary delivery, or inhalation. The compositions may further contain one or more chemical penetration enhancers, membrane permeability agents, membrane transport agents, emollients, surfactants, stabilizers, buffers, and combination thereof.

[0274] In certain embodiments, it may be desirable to provide continuous delivery of one or more compounds to a patient in need thereof. For topical applications, repeated application can

[0275] 45808517.1 41 ATTORNEY DOCKET # YU 9154 be done or a patch can be used to provide continuous administration of the compounds over an extended period of time

[0276] “Buffers” are used to control pH of a composition. Preferably, the buffers buffer the composition from a pH of about 4 to a pH of about 7.5, more preferably from a pH of about 4 to a pH of about 7, and most preferably from a pH of about 5 to a pH of about 7. In a preferred embodiment, the buffer is triethanolamine.

[0277] “Emollients” are an externally applied agent that softens or soothes skin and are generally known in the art and listed in compendia, such as the “Handbook of Pharmaceutical Excipients”, 4thEd., Pharmaceutical Press, 2003. These include, without limitation, almond oil, castor oil, ceratonia extract, cetostearoyl alcohol, cetyl alcohol, cetyl esters wax, cholesterol, cottonseed oil, cyclomethicone, ethylene glycol palmitostearate, glycerin, glycerin monostearate, glyceryl monoolcatc, isopropyl myristate, isopropyl palmitate, lanolin, lecithin, light mineral oil, medium-chain triglycerides, mineral oil and lanolin alcohols, petrolatum, petrolatum and lanolin alcohols, soybean oil, starch, stearyl alcohol, sunflower oil, xylitol and combinations thereof. In one embodiment, the emollients are ethylhexylstearate and ethylhexyl palmitate.

[0278] “Emulsifiers” are surface active substances which promote the suspension of one liquid in another and promote the formation of a stable mixture, or emulsion, of oil and water. Common emulsifiers are: metallic soaps, certain animal and vegetable oils, and various polar compounds. Suitable emulsifiers include acacia, anionic emulsifying wax, calcium stearate, carbomers, cetostearyl alcohol, cetyl alcohol, cholesterol, diethanolamine, ethylene glycol palmitostearate, glycerin monostearate, glyceryl monooleate, hydroxpropyl cellulose, hypromellose, lanolin, hydrous, lanolin alcohols, lecithin, medium-chain triglycerides, methylcellulose, mineral oil and lanolin alcohols, monobasic sodium phosphate, monoethanolamine, nonionic emulsifying wax, oleic acid, poloxamer, poloxamers, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, propylene glycol alginate, self-emulsifying glyceryl monostearate, sodium citrate dehydrate, sodium lauryl sulfate, sorbitan esters, stearic acid, sunflower oil, tragacanth, triethanolamine, xanthan gum and combinations thereof. In one embodiment, the emulsifier is glycerol stearate.

[0279] “Penetration enhancers” are known in the art and include, but are not limited to, fatty alcohols, fatty acid esters, fatty acids, fatty alcohol ethers, amino acids, phospholipids, lecithins, cholate salts, enzymes, amines and amides, complexing agents (liposomes, cyclodextrins, modified celluloses, and diimides), macrocyclics, such as macrocylic lactones, ketones, and anhydrides and cyclic ureas, surfactants, N-methyl pyrrolidones and derivatives thereof, DMSO

[0280] 45808517.1 42 ATTORNEY DOCKET # YU 9154 and related compounds, ionic compounds, azone and related compounds, and solvents, such as alcohols, ketones, amides, polyols (e.g., glycols). Examples of these classes are known in the art.

[0281] “Preservatives” can be used to prevent the growth of fungi and microorganisms. Suitable antifungal and antimicrobial agents include, but are not limited to, benzoic acid, butylparaben, ethyl paraben, methyl paraben, propylparaben, sodium benzoate, sodium propionate, benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, and thimerosal.

[0282] “Surfactants” are surface-active agents that lower surface tension and thereby increase the emulsifying, foaming, dispersing, spreading and wetting properties of a product. Suitable non-ionic surfactants include emulsifying wax, glyceryl monooleate, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polysorbate, sorbitan esters, benzyl alcohol, benzyl benzoate, cyclodextrins, glycerin monostearate, poloxamer, povidone and combinations thereof. In one embodiment, the non-ionic surfactant is stearyl alcohol. i. Emulsions

[0283] An emulsion is a preparation of one liquid distributed in small globules throughout the body of a second liquid. In particular embodiments, the non-miscible components of the emulsion include a lipophilic component and an aqueous component. The dispersed liquid is the discontinuous phase, and the dispersion medium is the continuous phase. When oil is the dispersed liquid and an aqueous solution is the continuous phase, it is known as an oil-in-water emulsion, whereas when water or aqueous solution is the dispersed phase and oil or oleaginous substance is the continuous phase, it is known as a water-in-oil emulsion. Either or both of the oil phase and the aqueous phase may contain one or more surfactants, emulsifiers, emulsion stabilizers, buffers, and other excipients. Preferred excipients include surfactants, especially non-ionic surfactants; emulsifying agents, especially emulsifying waxes; and liquid non-volatile non-aqueous materials, particularly glycols such as propylene glycol. The oil phase may contain other oily pharmaceutically approved excipients. For example, materials such as hydroxylated castor oil or sesame oil may be used in the oil phase as surfactants or emulsifiers.

[0284] The oil phase may consist at least in part of a propellant, such as an HFA propellant. Either or both of the oil phase and the aqueous phase may contain one or more surfactants, emulsifiers, emulsion stabilizers, buffers, and other excipients. Preferred excipients include surfactants, especially non-ionic surfactants; emulsifying agents, especially emulsifying waxes; and liquid non-volatile non-aqueous materials, particularly glycols such as propylene glycol. The oil phase may contain other oily pharmaceutically approved excipients. For example,

[0285] 45808517.1 43 ATTORNEY DOCKET # YU 9154 materials such as hydroxylated castor oil or sesame oil may be used in the oil phase as surfactants or emulsifiers.

[0286] A sub-set of emulsions are the self-emulsifying systems. These drug delivery systems are typically capsules (hard shell or soft shell) comprised of the drug dispersed or dissolved in a mixture of surfactant(s) and lipophilic liquids such as oils or other water immiscible liquids. When the capsule is exposed to an aqueous environment and the outer gelatin shell dissolves, contact between the aqueous medium and the capsule contents instantly generates very small emulsion droplets. These typically are in the size range of micelles or nanoparticles. No mixing force is required to generate the emulsion as is typically the case in emulsion formulation processes. ii. Lotions

[0287] A lotion can contain finely powdered substances that arc in soluble in the dispersion medium through the use of suspending agents and dispersing agents. Alternatively, lotions can have as the dispersed phase liquid substances that are immiscible with the vehicle and are usually dispersed by means of emulsifying agents or other suitable stabilizers. In one embodiment, the lotion is in the form of an emulsion having a viscosity of between 100 and 1000 centistokes. The fluidity of lotions permits rapid and uniform application over a wide surface area. Lotions are typically intended to dry on the skin leaving a thin coat of their medicinal components on the skin’s surface. iii. Creams

[0288] Creams may contain emulsifying agents and / or other stabilizing agents. In one embodiment, the formulation is in the form of a cream having a viscosity of greater than 1000 centistokes, typically in the range of 20,000-50,000 centistokes. Creams are often time preferred over ointments, as they are generally easier to spread and easier to remove.

[0289] The difference between a cream and a lotion is the viscosity, which is dependent on the amount / use of various oils and the percentage of water used to prepare the formulations. Creams are typically thicker than lotions, may have various uses and often one uses more varied oils / butters, depending upon the desired effect upon the skin. In a cream formulation, the waterbase percentage is about 60-75 % and the oil-base is about 20-30 % of the total, with the other percentages being the emulsifier agent, preservatives and additives for a total of 100 %. iv. Ointments

[0290] Examples of suitable ointment bases include hydrocarbon bases (e.g., petrolatum, white petrolatum, yellow ointment, and mineral oil); absorption bases (hydrophilic petrolatum, anhydrous lanolin, lanolin, and cold cream); water-removable bases (e.g., hydrophilic ointment),

[0291] 45808517.1 44 ATTORNEY DOCKET # YU 9154 and water-soluble bases (e.g., polyethylene glycol ointments). Pastes typically differ from ointments in that they contain a larger percentage of solids. Pastes are typically more absorptive and less greasy that ointments prepared with the same components. v. Gels

[0292] Gels are semisolid systems containing dispersions of small or large molecules in a liquid vehicle that is rendered semisolid by the action of a thickening agent or polymeric material dissolved or suspended in the liquid vehicle. The liquid may include a lipophilic component, an aqueous component or both. Some emulsions may be gels or otherwise include a gel component. Some gels, however, are not emulsions because they do not contain a homogenized blend of immiscible components. Suitable gelling agents include, but are not limited to, modified celluloses, such as hydroxypropyl cellulose and hydroxyethyl cellulose; Carbopol homopolymers and copolymers; and combinations thereof. Suitable solvents in the liquid vehicle include, but are not limited to, diglycol monoethyl ether; alklene glycols, such as propylene glycol; dimethyl isosorbide; alcohols, such as isopropyl alcohol and ethanol. The solvents are typically selected for their ability to dissolve the drug. Other additives, which improve the skin feel and / or emolliency of the formulation, may also be incorporated. Examples of such additives include, but are not limited, isopropyl myristate, ethyl acetate, C12-C15 alkyl benzoates, mineral oil, squalane, cyclomethicone, capric / caprylic triglycerides, and combinations thereof. vi. Foams

[0293] Foams consist of an emulsion in combination with a gaseous propellant. The gaseous propellant consists primarily of hydrofluoroalkanes (IIFAs). Suitable propellants include IIFAs such as 1,1,1,2-tetrafluoroethane (HFA 134a) and 1,1,1,2,3,3,3-heptafluoropropane (HFA 227), but mixtures and admixtures of these and other HFAs that are currently approved or may become approved for medical use are suitable. The propellants preferably are not hydrocarbon propellant gases, which can produce flammable or explosive vapors during spraying. Furthermore, the compositions preferably contain no volatile alcohols, which can produce flammable or explosive vapors during use.

[0294] 4. Pulmonary Formulations

[0295] In one embodiment, the compounds are formulated for pulmonary delivery, such as intranasal administration or oral inhalation.

[0296] The respiratory tract is the structure involved in the exchange of gases between the atmosphere and the blood stream. The lungs are branching structures ultimately ending with the alveoli where the exchange of gases occurs. The alveolar surface area is the largest in the respiratory system and is where drug absorption occurs. The alveoli are covered by a thin

[0297] 45808517.1 45 ATTORNEY DOCKET # YU 9154 epithelium without cilia or a mucus blanket and secrete surfactant phospholipids. The respiratory tract encompasses the upper airways, including the oropharynx and larynx, followed by the lower airways, which include the trachea followed by bifurcations into the bronchi and bronchioli. The upper and lower airways are called the conducting airways. The terminal bronchioli then divide into respiratory bronchiole, which then lead to the ultimate respiratory zone, the alveoli, or deep lung. The deep lung, or alveoli, is the primary target of inhaled therapeutic aerosols for systemic drug delivery.

[0298] Pulmonary administration of therapeutic compositions including low molecular weight drugs has been observed, for example, beta- androgenic antagonists to treat asthma. Other therapeutic agents that are active in the lungs have been administered systemically and targeted via pulmonary absorption. Nasal delivery is considered to be a promising technique for administration of therapeutics for the following reasons: the nose has a large surface area available for drug absorption due to the coverage of the epithelial surface by numerous microvilli, the sub epithelial layer is highly vascularized, the venous blood from the nose passes directly into the systemic circulation and therefore avoids the loss of drug by first-pass metabolism in the liver, it offers lower doses, more rapid attainment of therapeutic blood levels, quicker onset of pharmacological activity, fewer side effects, high total blood flow per cm3, porous endothelial basement membrane, and it is easily accessible.

[0299] Carriers for pulmonary formulations can be divided into those for dry powder formulations and for administration as solutions. Aerosols for the delivery of therapeutic agents to the respiratory tract are known in the art. Aerosols can be produced using standard techniques, such as ultrasonication or high-pressure treatment. For administration via the upper respiratory tract, the formulation can be formulated into a solution, e.g., water or isotonic saline, buffered or un-buffered, or as a suspension, for intranasal administration as drops or as a spray. Preferably, such solutions or suspensions are isotonic relative to nasal secretions and of about the same pH, ranging e.g., from about pH 4.0 to about pH 7.4 or, from pH 6.0 to pH 7.0. Buffers should be physiologically compatible and include, simply by way of example, phosphate buffers. For example, a representative nasal decongestant is described as being buffered to a pH of about 6.2. One skilled in the art can readily determine a suitable saline content and pH for an innocuous aqueous solution for nasal and / or upper respiratory administration.

[0300] Preferably, the aqueous solution is water, physiologically acceptable aqueous solutions containing salts and / or buffers, such as phosphate buffered saline (PBS), or any other aqueous solution acceptable for administration to an animal or human. Such solutions are well known to a person skilled in the art and include, but are not limited to, distilled water, de-ionized water,

[0301] 45808517.1 46 ATTORNEY DOCKET # YU 9154 pure or ultrapure water, saline, phosphate-buffered saline (PBS). Other suitable aqueous vehicles include, but are not limited to, Ringer's solution and isotonic sodium chloride. Aqueous suspensions may include suspending agents such as cellulose derivatives, sodium alginate, polyvinyl-pyrrolidone and gum tragacanth, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate.

[0302] Solvents that are low toxicity organic (i.e. nonaqueous) class 3 residual solvents, such as ethanol, acetone, ethyl acetate, tetrahydofuran, ethyl ether, and propanol may be used for the formulations. The solvent is selected based on its ability to readily aerosolize the formulation. The solvent should not detrimentally react with the compounds. An appropriate solvent should be used that dissolves the compounds or forms a suspension of the compounds. The solvent should be sufficiently volatile to enable formation of an aerosol of the solution or suspension. Additional solvents or aerosolizing agents, such as freons, can be added as desired to increase the volatility of the solution or suspension.

[0303] In one embodiment, compositions may contain minor amounts of polymers, surfactants, or other excipients well known to those of the art. In this context, "minor amounts" means no excipients are present that might affect or mediate uptake of the compounds in the lungs and that the excipients that are present are present in amount that do not adversely affect uptake of compounds in the lungs.

[0304] Dry lipid powders can be directly dispersed in ethanol because of their hydrophobic character. For lipids stored in organic solvents such as chloroform, the desired quantity of solution is placed in a vial, and the chloroform is evaporated under a stream of nitrogen to form a dry thin film on the surface of a glass vial. The film swells easily when reconstituted with ethanol. To fully disperse the lipid molecules in the organic solvent, the suspension is sonicated. Nonaqueous suspensions of lipids can also be prepared in absolute ethanol using a reusable PARI LC Jet-i- nebulizer (PARI Respiratory Equipment, Monterey, CA).

[0305] Dry powder formulations ("DPFs") with large particle size have improved flowability characteristics, such as less aggregation, easier aerosolization, and potentially less phagocytosis. Dry powder aerosols for inhalation therapy are generally produced with mean diameters primarily in the range of less than 5 microns, although a preferred range is between one and ten microns in aerodynamic diameter. Large "carrier" particles (containing no drug) have been codelivered with therapeutic aerosols to aid in achieving efficient aerosolization among other possible benefits.

[0306] Polymeric particles may be prepared using single and double emulsion solvent evaporation, spray drying, solvent extraction, solvent evaporation, phase separation, simple and

[0307] 45808517.1 47 ATTORNEY DOCKET # YU 9154 complex coacervation, interfacial polymerization, and other methods well known to those of ordinary skill in the art. Particles may be made using methods for making microspheres or microcapsules known in the art. The preferred methods of manufacture are by spray drying and freeze drying, which entails using a solution containing the surfactant, spraying to form droplets of the desired size, and removing the solvent.

[0308] The particles may be fabricated with the appropriate material, surface roughness, diameter and tap density for localized delivery to selected regions of the respiratory tract such as the deep lung or upper airways. For example, higher density or larger particles may be used for upper airway delivery. Similarly, a mixture of different sized particles, provided with the same or different EGS may be administered to target different regions of the lung in one administration.

[0309] Formulations for pulmonary delivery include unilamellar phospholipid vesicles, liposomes, or lipoprotein particles. Formulations and methods of making such formulations containing nucleic acid are well known to one of ordinary skill in the art. Liposomes are formed from commercially available phospholipids supplied by a variety of vendors including Avanti Polar Lipids, Inc. (Birmingham, Ala.). In one embodiment, the liposome can include a ligand molecule specific for a receptor on the surface of the target cell to direct the liposome to the target cell.

[0310] III. METHODS Of USING

[0311] The disclosed compositions can be administered to a subject in need thereof to treat one or more conditions in which fibrosis is implicated.

[0312] Fibrosis is a pathological process characterized by an excessive accumulation of extracellular matrix (ECM), such as collagens, fibronectin and hyaluronic acid, that occurs in chronic inflammation. Fibrosis can occur anywhere in the body, such as liver, lung, kidney, heart, blood vessel, eye, pancreas, skin, bone and so on. Further progress of fibrosis will lead to function decline and even failure and death in organs. Apart from organs damage, fibrosis also occurs on many chronic autoimmune diseases as a major pathological feature, including scleroderma, rheumatoid arthritis, Crohn's disease, ulcerative colitis, myelofibrosis and systemic lupus erythematosus. The early-stage of fibrosis can be treated and reversed to avoid serious health problem.

[0313] In some forms, the subject has been diagnosed with pulmonary fibrosis.

[0314] In some forms, the subject has been diagnosed with renal fibrosis.

[0315] In some forms, the subject has been diagnosed with liver fibrosis.

[0316] In some forms, the subject has been diagnosed with skin fibrosis.

[0317] 45808517.1 48 ATTORNEY DOCKET # YU 9154

[0318] In some forms, the subject has been diagnosed with cardiac fibrosis. In still some forms, the cardiac fibrosis is not caused one or more Lamin A mutations and / or the composition does not include a dominant negative SUN2 protein

[0319] Exemplary conditions include, but are not limited to NAS, keloids / hypertrophic scars, pulmonary fibrosis, for example, idiopathic pulmonary fibrosis, localized scleroderma, liver cirrhosis, systemic sclerosis and myelofibrosis.

[0320] The route of administration can be oral, parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), transdermal (either passively or using iontophoresis or electroporation), or transmucosal (nasal, vaginal, rectal, or sublingual) routes of administration or using bioerodible inserts and can be formulated in dosage forms appropriate for each route of administration.

[0321] In certain embodiments, the compositions arc administered locally, for example by injection or other application directly into or onto a site to be treated.

[0322] In some embodiments, the compositions are injected, topically applied. Typically, local administration causes an increased localized concentration of the compositions which is greater than that which can be achieved by systemic administration.

[0323] In some embodiments the route of administration is transdermal, for example, a transdermal patch or gel that is contacted with the skin of the subject. The composition can be administered directly or indirectly to adipose tissue and / or the target cells (e.g., WAT precursor cells).

[0324] The precise dosage will vary according to a variety of factors including but not limited to the inhibitor that is selected and subject-dependent variables (e.g., age, immune system health, clinical symptoms etc.).

[0325] The timing of the administration of the composition will also depend on the formulation and / or route of administration used. The compound may be administered once daily, but may also be administered two, three or four times daily, or every other day, or once or twice per week. For example, the subject can be administered one or more treatments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, days, weeks, or months apart.

[0326] In some embodiments, the compositions are formulated for extended release. For example, the formulation can be suitable for administration once daily or less. In some embodiments, the composition is only administered to the subject once every 24-48 hours.

[0327] In some embodiments, administration of the composition will be given as a long-term treatment regimen whereby pharmacokinetic steady state conditions will be reached.

[0328] 45808517.1 49 ATTORNEY DOCKET # YU 9154

[0329] A. Pulmonary Fibrosis

[0330] In some forms, the subject has been diagnosed with pulmonary fibrosis. Pulmonary fibrosis (PF) is a form of interstitial lung disease that causes scarring in the lungs. In pulmonary fibrosis, lung tissue becomes damaged and scarred. This thickened, stiff tissue makes it harder for the lungs to work properly. Pulmonary fibrosis worsens over time. Some people can stay stable for a long time, but the condition gets worse faster in others. As it gets worse, people become more and more short of breath. Idiopathic pulmonary fibrosis usually occurs in middle- aged and older adults. Some cases of PF are caused by autoimmune diseases like rheumatoid arthritis, scleroderma or Sjogren's syndrome. Certain viral infections and gastroesophageal reflux disease (GERD) are also risk factors for PF. GERD is a condition in which acid from your stomach backs up into your throat. Some people who have GERD may breathe in tiny drops of acid from their stomachs, which may injure the lungs. PF can be caused by exposure to hazardous materials. Examples include occupational exposures such as asbestos or silica. Some cases of PF are caused by breathing in bird or animal droppings. Radiation treatments and certain types of medications can cause PF. Cigarette smoking also increases a person's risk of developing PF. Sometimes pulmonary fibrosis is diagnosed in children and infants, but this is not common. Symptoms of pulmonary fibrosis may include: Shortness of breath, Dry cough, Extreme tiredness, Weight loss that's not intended, Aching muscles and joints, Widening and rounding of the tips of the fingers or toes, called clubbing.

[0331] For the treatment of diseases like IPF in the respiratory tract, drugs need to enter the deep airways.

[0332] B. Renal Fibrosis

[0333] In some forms, the subject has been diagnosed with renal fibrosis. Renal fibrosis is characterized by glomerular sclerosis and tubulointerstitial fibrosis, and is a dynamic process consisting of four phases: initiation, activation, execution and progression.

[0334] C. Liver Fibrosis

[0335] In some forms, the subject has been diagnosed with liver fibrosis. Liver fibrosis is the result of chronic liver damage caused by a variety of factors, including viral infection, drug toxicity, alcohol abuse, autoimmune disease, nonalcoholic fatty liver disease and dysplasia. The occurrence of liver fibrosis is a dynamic and continuous process, which means the continuous necrosis of hepatocytes and progressive accumulation of ECM. It reflects the imbalance of repair and scar formation of the liver when facing damage

[0033] . If left uncontrolled, it may develop into the end-stage liver disease or cirrhosis. And the loss of liver function, ascites, portal

[0336] 45808517.1 50 ATTORNEY DOCKET # YU 9154 hypertension, esophageal varices and increased risk of hepatic carcinoma are the most serious complications and are often fatal.

[0337] D. Skin Fibrosis

[0338] The hallmark features of skin fibrosis include increased collagen synthesis, increased deposition of extracellular matrix (ECM) proteins, and increased fibroblast proliferation . Skin fibrosis is a characteristic finding in several skin pathologies, including scleroderma and morphea, keloids and hypertrophic scars, chronic graft-versus-host disease, and gadolinium- induced nephrogenic systemic fibrosis. Fibrotic skin disorders lead to a significant decrease in patient lifespan and have a negative impact on quality of life .

[0339] Scleroderma is a fibrotic skin disorder characterized by thickened, hardened areas of skin. It affects approximately 100,000 people in the United States and has a median survival rate of 11 years. When scleroderma is accompanied by fibrosis in other organ systems, it is known as systemic sclerosis; when it is localized to the skin, it is known as morphea. Scleroderma and other fibrotic skin disorders are debilitating and affect the physical and functional abilities of patients. It is associated with low self-esteem, job discrimination, prejudicial societal reactions, isolation, depression, and other psychiatric comorbidities.

[0340] Keloids and hypertrophic scars are characterized by an overgrowth of fibrotic scar tissue that occurs secondary to defective wound healing. Keloids grow outside the original boundaries of the injury; while hypertrophic scars are contained to the original wound boundaries. These scars often have a functional, aesthetic, or psychosocial impact on patients.

[0341] Gadolinium-induced nephrogenic systemic fibrosis (NSF) is a fibrosing disorder occurring in patients with renal insufficiency who receive the contrast agent gadolinium prior to radiologic imaging. Fibrotic plaques located on the trunk and extremities characterize NSF.

[0342] Chronic graft-versus-host disease (cGVHD) is an autoimmune disease that occurs in bone marrow transplant recipients. cGVHD may involve a number of organ systems, mainly skin, gastrointestinal tract, lung, and liver.

[0343] E. Cardiac fibrosis

[0344] Fibrotic scars of the cardiac muscle most commonly occur after myocardial infarction; however, there are various other conditions promoting cardiac fibrosis such as hypertensive heart disease, diabetic hypertrophic cardiomyopathy and idiopathic dilated cardiomyopathy.

[0345] Cardiac fibrosis is a scarring event in the cardiac muscle that is characterized by an increased collagen type I deposition as well as cardiac fibroblast activation and differentiation into myofibroblasts.

[0346] 45808517.1 51 ATTORNEY DOCKET # YU 9154

[0347] These pathological changes lead to an increased matrix stiffness and lead to abnormalities in cardiac function. Three types of myocardial fibrosis have been identified: I) reactive interstitial fibrosis, II) infiltrative interstitial fibrosis and III) replacement fibrosis. Reactive interstitial fibrosis leads to a pressure overload and cardiomyopathies due to an increased ECM deposition without a significant loss of cardiomyocytes.

[0348] Infiltrative interstitial fibrosis is characterized by the glycolipid build up in different cells of the heart, which can be observed in patients with Fabry disease, a rare genetic disease that involves dysfunctional metabolism of sphingolipids.

[0349] Replacement fibrosis occurs after cardiac injury like myocardial infarction, where cardiac cells are damaged. Here, dead cells are replaced and a predominantly collagen type I-containing scar is formed

[0350] The disclosed compositions and methods can be further understood in view of the following non- limiting examples.

[0351] Examples

[0352] Analysis of SUN2 expression in idiopathic lung fibrosis:

[0353] Histological sections of lung patient with idiopathic lung fibrosis (e.g. FIG2) were stained with anti-SUN2 antibody [EPR6557] (Abeam abl24916) as well as staining for DNA (DAPI) and alpha smooth muscle actin (a-SMA) with antibody (Invitrogen; REF 14-9760-82).

[0354] Analysis of SUN2 expression on fibrotic substrates: Substrates were generated as described Mertz et al. PNAS, 110 (3) 842-847 (2012).

[0355] Human dermal fibroblasts were plated on fibronectin-coated silicone gel with Young’s modulus of 3 kPa or 500 kPa. SUN2 expression was analyzed using immunofluorescence with antibodies against SUN2 ([EPR6557] (Abeam abl 24916)). Immunofluorescence was performed as described Mertz et al. PNAS, 110 (3) 842-847 (2012).

[0356] Studies identified SUN2 as key regulator of mechano-sensing required for fibrosis. FIG. 2A shows SUN2 is upregulated in samples obtained from patients with idiopathic fibrosis of the lung. FIG. 2B shows that SUN2 is also upregulated in cell culture models of fibrosis. As shown in FIGs. 3A and 3B, inhibiting SUN2 protects multiple tissues from fibrosis.

[0357] Lung fibrosis induction in mice and analysis: Bleomycin administration, bronchealveolar lavage (BAL), and lung harvest were performed as previously reported (Sun et al Arthritis & Rheumatology 2016) in 7-9 week old WT or Sun2- / - mice. FFPE sections were stained with Masson’s trichrome to visualize collagen deposition. Images of the whole lung and subjected to a quantitative scoring system (modified Ashcroft Score) assigns a numerical value between 0

[0358] 45808517.1 52 ATTORNEY DOCKET # YU 9154 and 8 that corresponds to the extent of fibrosis in a histologic sample. Multiple regions of each individual sample were imaged and scored, then averaged. Histological analysis of each sample was performed by two individuals blinded to experimental groups.

[0359] Skin fibrosis induction in mice: Male mice between 6-8 weeks received daily subcutaneous injections of 300ug Bleo Sulfate (Enzo) for 11 days. Skin was isolated and histological analysis was performed as described (Schmidt and Horsley, Development 140(7): 1517-27.201 ).

[0360] It is understood that the disclosed method and compositions are not limited to the particular methodology, protocols, and reagents described as these can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0361] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the method and compositions described herein. Such equivalents are intended to be encompassed by the following claims.

[0362] 45808517.1 53

Claims

1. ATTORNEY DOCKET # YU 9154CLAIMSWe claim:

1. A method of reducing one or more symptoms associated with fibrosis in a subject in need thereof comprising administering to the subject an effective amount of (a) an inhibitor of a SUN2, CTDNEP1, and / or NEP1RJ gene or a gene product thereof and / or (b) an activator of CK2 or a gene product thereof to the subject, to reduce one or more symptoms of fibrosis.

2. The method of claim 2, wherein subject has been diagnosed with fibrosis.

3. The method of any one of claims 1-2 wherein the inhibitor is administered in an effective amount to decrease expression of SUN2, CTDNEP and / or NEPJR1.

4. The method of claim 3, wherein the inhibitor is an inhibitor of the SUN2 gene or a gene product thereof.

5. The method of claim 3, wherein the inhibitor is an inhibitor of CTDNEP1 or a gene product thereof.

6. The method of claim 3, wherein the inhibitor is an inhibitor of the NEP1R1 gene or a gene product thereof.

7. The method of any one of claims 1-6, wherein the inhibitor is antisense molecules, siRNA, miRNA, aptamers, ribozymes, triplex forming molecules, RNAi, external guide sequences, or a gene editing composition that targets the SUN2, CTDNEP1, and / or NEP1R1 gene or gene product thereof.

8. The method of any one of claims 1-6, wherein the inhibitor is a gene editing composition that induces a single or double strand break at SUN2, CTDNEP1, and / or NEP1R1 genetic locus in the subject and reduces expression thereof.

9. The method of claim 8, wherein the gene editing composition is a CRISPR / Cas system.

10. The method of claim 9, wherein the CRISPR / Cas system comprises a singleguide RNA (sgRNA) that targets the SUN2, CTDNEP1, and / or NEP1R1 and a Cas nuclease or nickase.

11. The method of any one of claims 1-10, wherein the inhibitor reduces expression or activity of a SUN2 gene and / or a gene product thereof.

12. The method of any one of claims 1-11, wherein the subject is a human subject.

13. The method of any one of claims 1-11, wherein the subject has been diagnosed with a condition selected from the group consisting of pulmonary fibrosis, renal fibrosis, liver fibrosis, skin fibrosis and / or cardiac fibrosis.

14. The method of claim 13, wherein the subject has pulmonary fibrosis45808517.1 54ATTORNEY DOCKET # YU 915415. The method of claim 13, wherein the subject has renal fibrosis16. The method of claim 13, wherein the subject has liver fibrosis.

17. The method of claim 13, wherein the subject has skin fibrosis.

18. The method of claim 13, wherein the subject has cardiac fibrosis19. A pharmaceutical composition comprising an effective amount of: (a) an inhibitor of a SUN2, CTDNEP1, and / or NEP1R1 gene or a gene product thereof and / or (b) an activator of CK2 or a gene product thereof, to reduce one or more symptoms associated with fibrosis following administration thereof to a subject.

20. The pharmaceutical composition of claim 19, wherein the inhibitor is a gene editing composition that induces a single or double strand break at the SUN2, CTDNEP1, and / or NEP1R1 genetic locus in the subject and reduces expression thereof.

21. The pharmaceutical composition of claim 20, wherein the gene editing composition is a CRISPR / Cas system.

22. The pharmaceutical composition of claim 21 , wherein the CRISPR / Cas system comprises a single-guide RNA (sgRNA) that targets the SUN2, CTDNEP1, and / or NEP1R1 and Cas nuclease or nickase.

23. The pharmaceutical composition of claim 22, wherein the CK2 activator is a small molecule activator or comprises a nucleic acid sequence encoding CK2, optionally, wherein the composition further comprises a tissue-targeting moiety.

24. The pharmaceutical composition of claim 23, wherein the inhibitor is an inhibitor of the SUN2, CTDNEP1, and / or NEP1R1 gene or gene product thereof.

25. The pharmaceutical composition of claim 24, wherein the inhibitor is selected from the group consisting of antisense molecules, siRNA, miRNA, aptamers, ribozymes, triplex forming molecules, RNAi, external guide sequences, and combinations thereof.

26. The pharmaceutical composition of any one of claims 19-25, wherein the SUN2, CTDNEP1, and / or NEP1R1 gene or a gene is a human gene or gene product.

27. The pharmaceutical composition of any one of claims 19-25, wherein the subject is a human subject, further comprising a tissue specific-targeting moiety.45808517.1 55

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