CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application Serial No.: 61/362,751 , entitled "TARGETED RECEPTOR-MEDIATED siRNA" filed on July 9, 2010, the entirety of which is hereby incorporated by reference.

TECHNICAL FIELD

The present disclosure is generally related to receptor ligand-siRNA conjugates and receptor-mediated delivery of the siRNA to a targeted cell.

BACKGROUND

Sjogren's Syndrome is common systemic autoimmune disease mainly affecting the salivary and lacrimal glands resulting in secretory hypofunction and dry mouth and dry eye, respectively, which adversely affects quality of life. Despite extensive studies into the mechanisms which contribute to the development or pathogenesis of Sjogren's Syndrome, the events that trigger disease onset in the target exocrine glands remain unknown.

Previous studies examining the salivary glands of the non-obese diabetic (NOD), and, more recently, the C57BL/6.NOD-AecMec2 mouse models of Sjogren's Syndrome, indicate alterations in the glandular environment even prior to disease onset, including apoptosis of acinar tissues and altered cell proliferation (Bulosan et a/. , (2008) Immunol. Cell Biol. 87: 81 - 90; Cha et a/. , (2004) Scand. J. Immunol. , 60: 552-565; Cha et al., (2001 ) Exp. Clin.

Immunogenet. 18: 143-160; Robinson er a/. , (1996) Clin. Immunol. Immunopathol. 79: 50-59; Cha et al. , (2004) Arthritis Rheum. 46: 1390-1398). Current therapeutic strategies for Sjogren's Syndrome mainly focus on palliative treatments to stimulate secretion or suppression of immune responses by corticosteroids. However, such treatments do not address the underlying causes of secretory dysfunction, one of which is the loss of acinar cells through apoptotic cell death.

RNA interference (RNAi) is the natural process occurring in most eukaryotic cells in which small double stranded RNA (dsRNA) molecules negatively regulate gene expression by causing the degradation or translation repression of specific mRNA targets (reviewed in Rana ef a/. , (2007) Nat. Rev. Mol. Cell Biol. 8: 23-36). One class of these small dsRNAs are small interfering RNA molecules (siRNA), which are 21 nucleotides long and bind specifically to their target mRNAs via complementary base-pair matching, leading to the cleavage of that mRNA by the RNA-induced silencing complex (RISC, Rana et al., (2007) Nat. Rev. Mol. Cell Biol. 8: 23-36).

Since the discovery of the RNAi pathway, there has been a surge in research towards developing siRNA-based therapeutics for otherwise "undruggable" targets. Two critical issues being considered in the development of siRNA therapies are preserving the efficacy and stability of the siRNA molecule in vivo and generating siRNA delivery systems. It has been determined that stability of siRNA in vivo can be achieved through various chemical modifications (Lopez-Fraga er a/. , (2009) BioDrugs, 23: 305-332).

siRNA delivery can be achieved by a variety of strategies including lipid-based formulations (Wu ef al. , (2009) AAPS J. 2009, 1 1 : 639-652), nanoparticles (Hart SL: Cell Biol. Toxicol. 26: 69-81 ), and magnetofection (Mykhaylyk er a/. , (2008) Curr. Opin. Mol. Ther. 10: 493-505). However, these strategies are nonspecific, and cell-type specific delivery is still the most challenging step blocking the progress of RNAi therapy in modern medicine. To target siRNA to specific cell or tissue types, specificity must be built into the delivery agents or expressed shRNAs. Some strategies for cell-type specific delivery include antibody targeting (Yu et al. ,. (2009) AAPS J. 1 1 : 195-203), cell-penetrating peptides (Endoh er a/., (2009) Adv. Drug Deliv. Rev. 61 : 704-709), chemical modifications (Wu et al., (2009) AAPS J. 1 1 : 639-652), and aptamers (Kim et al. , (2009) Trends Mol. Med. 15: 491 -500), but each of these strategies presents certain drawbacks such as cytotoxicity or immunogenicity.

SUMMARY

Briefly described, embodiments of this disclosure, among others, encompass compositions comprising molecular conjugates between a receptor ligand, most suitably a small molecule ligand, and an siRNA that when administered to an animal or human subject in need thereof, can modulate or relieve symptoms of Sjogren's Syndrome as well as inhibiting or reducing such cellular processes as apoptosis of glandular acinar cells that trigger or sustain Sjogren's Syndrome.

One aspect of the disclosure, therefore, encompasses embodiments of compositions comprising: a ligand characterized as having affinity for a surface receptor of a cell, an siRNA moiety wherein the ligand is linked to the siRNA moiety, and, optionally, a

pharmaceutically acceptable carrier.

In embodiments of this aspect of the disclosure, the cell can be an exocrine glandular cell.

In embodiments of this aspect of the disclosure, the ligand can be covalently conjugated to the siRNA moiety.

In embodiments of this aspect of the disclosure, the ligand can be linked to the siRNA moiety by a linker.

In embodiments of this aspect of the disclosure, the ligand can be characterized as having affinity for a muscarinic receptor.

In some embodiments of this aspect of the disclosure, the exocrine glandular cell can be a salivary or lacrimal cell susceptible to Sjogrens Syndrome.

In some embodiments of this aspect of the disclosure, the ligand characterized as having affinity for a muscarinic receptor can be a secretagogue. In embodiments of this aspect of the disclosure, the ligand is a muscarinic receptor agonist. In these embodiments of this aspect of the disclosure, the muscarinic receptor agonist can be selected from the group consisting of; carbachol, cevimeline, pilocarpine, methacholine, bethanechol, muscarine, and a muscarinic receptor subtype-specific agonist.

In some embodiments of this aspect of the disclosure, the siRNA moiety, when delivered to the salivary or lacrimal cell, or population of said cells, susceptible to Sjogrens Syndrome can increase the survivability of salivary or lacrimal acinar cells.

In other embodiments of this aspect of the disclosure, the siRNA moiety, when delivered to the salivary or lacrimal cell, or population of said cells, susceptible to Sjogrens Syndrome can reduce the level of apoptosis in said salivary or lacrimal cell, or population of said cells.

In embodiments of this aspect of the disclosure, the siRNA moiety, when delivered to the salivary or lacrimal cell, or population of said cells, susceptible to Sjogrens Syndrome can reduce the level of caspase-3 activity in said salivary or lacrimal cell, or population of said cells.

In embodiments of this aspect of the disclosure, the siRNA moiety can comprise a nucleotide sequence complementary to a region of an mRNA encoding a caspase, and wherein when delivered to the salivary or lacrimal cell, or population of said cells, reduces the level of caspase activity in said cell, or population of said cells.

In embodiments of this aspect of the disclosure, the siRNA moiety can comprise the nucleotide sequence 5'-AUAAAUUCAAGCUUGUCGG-3' (SEQ ID No.: 4) and, optionally, the complement thereof, and wherein said siRNA is characterized as reducing the level of caspase-3 activity when delivered to a salivary or lacrimal cell, or a population of said cells.

In embodiments of this aspect of the disclosure, the siRNA moiety, when delivered to the salivary or lacrimal cell or population of cells subject to Sjogrens Syndrome can modulate the expression of a gene thereby down-regulating a pro-inflammatory cytokine secretion and HLA class II expression, inhibiting a toll-like receptor (TLR)-mediated pathway, inhibiting the expression of a viral gene, or stimulating the differentiation of an acinar cell to a ductal cell.

Another aspect of the disclosure encompasses embodiments of a method for delivery of a molecular conjugate to a cell, comprising the steps of: (a) contacting with a cell or cells with a molecular conjugate comprising a ligand characterized as having affinity for a surface receptor of the cell, and an siRNA moiety linked to the ligand; and (b) maintaining the cell, or population of said cells, under conditions whereby the ligand specifically binds to a surface receptor of the cell or cells, whereupon the molecular conjugate enters the cell or cells by endocytosis, thereby delivering the siRNA moiety to the cytoplasm of the cell or cells. In embodiments of this aspect of the disclosure, the cell can be an exocrine glandular cell, or population of said cells.

In embodiments of this aspect of the disclosure, the surface receptor can be a muscarinic receptor. In some embodiments of this aspect of the disclosure, the exocrine glandular cell can be a cell of a salivary gland, a lacrimal gland, a tracheobronchial gland, a urodigestive gland, or a sweat gland. In some embodiments of this aspect of the disclosure, the exocrine glandular cell or population of said cells can be a salivary or lacrimal cell or population of said cells susceptible to Sjogrens Syndrome.

In embodiments of this aspect of the disclosure, the ligand can be a secretagogue or a muscarinic receptor agonist.

In embodiments of this aspect of the disclosure, the muscarinic receptor agonist can be selected from the group consisting of; carbachol, cevimeline, pilocarpine, methacholine, bethanechol, muscarine, and a muscarinic receptor subtype-specific agonist.

In some embodiments of this aspect of the disclosure, the siRNA moiety, when delivered to the salivary or lacrimal cell or population of cells susceptible to Sjogrens Syndrome, can increase the survivability of salivary or lacrimal acinar cells.

In some embodiments of this aspect of the disclosure, the siRNA moiety can reduce the level of apoptosis in said salivary or lacrimal cell or population of cells.

In some embodiments of this aspect of the disclosure, the siRNA moiety can comprise a nucleotide sequence complementary to a region of an mRNA encoding a caspase, and wherein when delivered to the salivary or lacrimal cell, or population of said cells, reduces the level of caspase activity in said cell, or population of said cells.

In embodiments of this aspect of the disclosure, the siRNA moiety comprises the nucleotide sequence 5 -AUAAAUUCAAGCUUGUCGG-3' (SEQ ID No.: 4) and, optionally, the complement thereof, and wherein said siRNA can be characterized as reducing the level of caspase-3 activity when delivered to a salivary or lacrimal cell, or a population of said cells.

In embodiments of this aspect of the disclosure, the method can further comprise administering the molecular conjugate to the subject animal or human, wherein the ligand increases secretion from the saliva gland or the lacrimal gland of said subject, and the siRNA reduces the level of apoptosis in said saliva gland or the lacrimal gland, thereby reducing the symptoms of Sjogrens Syndrome in said subject animal or human.

In embodiments of this aspect of the disclosure, the siRNA moiety, when delivered to the salivary or lacrimal cell or population of cells subject to Sjogrens Syndrome, can modulate the expression of a gene thereby down-regulating a pro-inflammatory cytokine secretion and HLA class II expression, inhibiting a toll-like receptor (TLR)-mediated pathway, inhibiting the expression of a viral gene, or stimulating the differentiation of an acinar cell to a ductal cell.

Another aspect of the disclosure provides an embodiment of a method of modulating the symptoms of Sjogrens Syndrome in a subject animal or human by administering to said subject a therapeutic dose of a pharmacologically acceptable composition comprising: (i) a ligand characterized as a secretagogue having affinity for a muscarinic receptor of an exocrine salivary or lacrimal glandular cell, or population of said cells, wherein the ligand is a muscarinic receptor agonist selected from the group consisting of; carbachol, cevimeline, pilocarpine, methacholine, bethanechol, muscarine, and a muscarinic receptor subtype- specific agonist; (ii) an siRNA moiety having the nucleotide sequence 5'-

AUAAAUUCAAGCUUGUCGG-3' (SEQ ID No.: 4) and is complementary to a region of an mRNA encoding caspase-3, wherein the ligand is linked to the siRNA moiety; and (iii) a pharmaceutically acceptable carrier, wherein the siRNA moiety reduces the level of caspase-3 activity in said salivary or lacrimal cell, thereby stimulating the secretion of the saliva gland or lacrimal gland and increasing the survivability of the acinar cell.

BRIEF DESCRIPTION OF THE DRAWINGS

Further aspects of the present disclosure will be more readily appreciated upon review of the detailed description of its various embodiments, described below, when taken in conjunction with the accompanying drawings.

Fig. 1 A illustrates a schematic diagram of a carbachol-siRNA conjugation for targeting caspase-3. The siRNA is directly conjugated to muscarinic receptor agonist, carbachol

Fig. 1 B schematically illustrates the two-step activation and conjugation of choline to 5'-amino-modified RNA. Activation of choline by reaction with 4-nitrophenyl chloroformate was followed by reaction with amino-modified RNA.

Figs. 2A and 2b illustrate graphs showing that the carbachol and the siRNA portions of conjugate retain function after conjugation.

Fig. 2A is a graph showing the level of caspase-3 expression when the conjugate was transfected into a human salivary gland cell line (HSG) at the indicated concentrations. Caspase-3 gene expression was analyzed by quantitative RT-PCR after 48 hours of incubation. Free siCaspase-3 treatment indicates" naked siRNA was added to the cells in the absence of transfecting reagent. Asterisks ( ^** ) indicate p<0.01 or ( ^* ) p<0.05 as determined by one-way ANOVA with Dunnett's multiple comparison test (n = 3 independent experiments). Bars represent mean with standard error.

Fig.2B is a graph showing the level of caspase-3 expression when the conjugate was added to HSG cells as indicated and intracellular calcium release was measured as fluorescence intensity, lonomycin is an ionophore that increases intracellular calcium release and was used as positive control. Bars represent mean with standard error (n = 3 independent experiments), and dashed line indicates level of background fluorescence.

Figs. 2C-2E shows a series of graphs illustrating calcium imaging of ParC5 cells treated with: Fig. 2C, 10ΌμΜ carbachol; Fig. 2D, a free siRNA control; and Fig. 2E, 8.7 μΜ conjugate. Each line represents calcium response in a single cell.

Fig. 3 is a series of digital images showing that conjugate treatment induced endocytosis similarly to carbachol. ParC5 cells transfected with YFP-tagged NBCel were treated with carbachol or conjugate and then co-stained with anti-EEA-1 endosomal marker. Co-localization of NBCel and EEA-1 indicate endocytosis was induced by carbachol and conjugate treatment. Images shown at 630x magnification; insets are enlarged to view co- localization.

Fig. 4 is a series of digital images showing that the conjugate was detected in HSG cell cytoplasm within 30 mins of incubation. HSG cells were treated with 5μΜ conjugate for the indicated times before being fixed and used for in situ hybridization with a FAM-labeled DNA oligonucleotide probe specific for the antisense strand of caspase-3 siRNA. Arrows indicate conjugate detected in cytoplasm of cells. Nuclei counterstained with DAPI. Images shown at 200x magnification.

Fig. 5 illustrates a graph showing that conjugate treatment reduced caspase-3 gene expression in HSG cells. HSG cells were treated with 8.71 μΜ conjugate or negative control conjugate and incubated for 48 hours. Caspase-3 gene expression was then analyzed by quantitative RT-PCR. Asterisks ( ^* ) indicate p<0.01 as determined by one-way ANOVA with Dunnett's multiple comparison test. Bars represent mean with standard error (n = 5).

Fig. 6 illustrates a graph showing that conjugate had no effect on M3R-negative cells. HeLa cells were treated with conjugate or transfected with caspase-3 siRNA, incubated for 72 hours, and caspase-3 gene expression was analyzed by qRT-PCR. Bars represent mean with standard error (n = 2).

Fig. 7 illustrates a graph showing that conjugate incubation did not induce interferon response. HSG cells were treated with conjugate or transfected with caspase-3 siRNA and incubated for 72 hours. qRT-PCR was used to analyze mRNA expression of interferon response genes OAS1 (white bars) and MX1 (black bars) which remained unchanged. Bars represent mean with standard error (n = 3).

Fig. 8 is a series of digital images showing that conjugate treatment reduced caspase-3 protein expression in HSG cells. HSG cells were treated with 8.71 μΜ conjugate and incubated for 72 hours. Caspase-3 protein expression was analyzed by

immunufluorescence using rabbit anti-caspase-3 antibodies and Alexa fluor 568 goat anti- rabbit IgG secondary antibodies. Cell nuclei were counterstained with DAPI, and caspase-3 siRNA or conjugate was detected by in situ hybridization. Images shown at 100x magnification.

Fig. 9 shows a graph illustrating the quantitation of caspase-3 protein levels measured using Image J image analysis software and normalized to untreated cells.

Asterisks ( ^* ) indicate p<0.05 as determined by student t test. Bars represent mean with standard error (n = 3 independent experiments).

Fig. 10 is a series of digital images showing Western blot analysis caspase-3 and GAPDH protein expression in lysates of cells treated as indicated.

Figs. 1 1 and 12 are a pair of graphs that illustrate that conjugate treatment prevented TNF-ot induced apoptosis in HSG cells. HSG cells were treated with 10 μΜ conjugate, 10 μΜ negative control conjugate, or transfected with caspase-3 siRNA and incubated for 96 hours. The cells were then treated with TNF-a (50ng/ml) and cycloheximide (Ι Ομς/ιηΙ) for eight hours and stained with Annexin-V and propidium iodide. The percent of early and late apoptotic cells after TNF -a/cycloheximide treatment was significantly reduced in caspase-3- transfected and conjugate-treated cells compared to cells treated with negative control conjugate (asterisks p<0.01 as determined by t test).

Fig. 1 shows a graph that illustrates that conjugate treatment prevented TNF-a induced apoptosis in HSG cells. Flow cytometry was used to assess early apoptotic cells (Annexin-V positive).

Fig. 12 shows a graph that illustrates that conjugate treatment prevented TNF-a induced apoptosis in HSG cells. Late apoptotic cells (Annexin-V/PI positive) in treated (black bars) and untreated (white bars) cells.

Fig. 13 is a graph showing caspase-3 gene expression measured by qRT-PCR after caspases-3 siRNA was added to the medium without transfection.

The drawings are described in greater detail in the description and examples below.

The details of some exemplary embodiments of the methods and systems of the present disclosure are set forth in the description below. Other features, objects, and advantages of the disclosure will be apparent to one of skill in the art upon examination of the following description, drawings, examples and claims. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be protected by the accompanying claims.

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, 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, since the scope of the present disclosure will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.

It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a support" includes a plurality of supports. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

As used herein, the following terms have the meanings ascribed to them unless specified otherwise. In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes," "including," and the like; "consisting essentially of or "consists essentially" or the like, when applied to methods and compositions encompassed by the present disclosure refers to compositions like those disclosed herein, but which may contain additional structural groups, composition components or method steps (or analogs or derivatives thereof as discussed above). Such additional structural groups, composition components or method steps, etc., however, do not materially affect the basic and novel characteristic (s) of the compositions or methods, compared to those of the corresponding compositions or methods disclosed herein. "Consisting essentially of or "consists essentially" or the like, when applied to methods and compositions encompassed by the present disclosure have the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

Prior to describing the various embodiments, the following definitions are provided and should be used unless otherwise indicated.

Definitions

In describing and claiming the disclosed subject matter, the following terminology will be used in accordance with the definitions set forth below.

The term "hybridization" as used herein refers to the process of association of two nucleic acid strands to form an anti-parallel duplex stabilized by means of hydrogen bonding between residues of the opposite nucleic acid strands.

The terms "hybridizing" and "binding" as used herein with respect to polynucleotides, are used interchangeably. The terms "hybridizing specifically to" and "specific hybridization" and "selectively hybridize to," as used herein refer to the binding, duplexing, or hybridizing of a nucleic acid molecule preferentially to a particular nucleotide sequence under stringent conditions. By "hybridization" or "hybridizing" as used herein are meant the formation of A-T and C-G base pairs between the nucleotide sequence of a fragment of a segment of a polynucleotide and a complementary nucleotide sequence of an oligonucleotide. By complementary is meant that at the locus of each A, C, G or T (or U in a ribonucleotide) in the fragment sequence, the oligonucleotide sequenced has a T, G, C or A, respectively. The hybridized fragment/oligonucleotide is called a "duplex." The term "binding' as used herein can also refer to the association of a ligand (a small molecule or such as, but not limited to, an aptamer, peptide or oligonucleotide ligand) that bonds to a target receptor by non-covalent interactions.

The term "target" as used herein refers to a cell or receptor thereof for which it is desired to modulate the bioactivity thereof.

As used herein, the term "oligonucleotide" refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction. A short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue.

Oligonucleotides may be chemically synthesized and may be used as primers or probes. Oligonucleotide means any nucleotide of more than 3 bases in length used to facilitate detection or identification of a target nucleic acid, including probes and primers.

The term "polynucleotide" as used herein refers to any polyribonucleotide or polydeoxribonucleotide that may be unmodified RNA or DNA or modified RNA or DNA. Thus, for instance, polynucleotides as used herein refers to, among others, single-and double-stranded DNA, DNA that is a mixture of single-and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. The terms "nucleic acid," "nucleic acid sequence," or "oligonucleotide" also encompass a polynucleotide as defined above. The terms "polynucleotide" and "oligonucleotide" also include DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein.

It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term polynucleotide as it is employed herein embraces such chemically, enzymatically, or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inter alia.

The terms "nucleotide" and a "nucleotide moiety" as used herein refer to a sub-unit of a nucleic acid (whether DNA or RNA or an analogue thereof) that may include, but is not limited to, a phosphate group, a sugar group and a nitrogen containing base, as well as analogs of such sub-units. Other groups (e.g., protecting groups) can be attached to the sugar group and nitrogen containing base group. The term "nucleoside" as used herein refers to a nucleic acid subunit including a sugar group and a nitrogen containing base. It should be noted that the term "nucleotide" is used herein to describe embodiments of the disclosure, but that one skilled in the art would understand that the term "nucleoside" and "nucleotide" are interchangable in most instances. One skilled in the art would have the understanding that additional modification to the nucleoside may be necessary, and one skilled in the art has such knowledge.

It will be appreciated that, as used herein, the terms "nucleotide" and "nucleoside" will include those moieties which contain not only the naturally occurring purine and pyrimidine bases, e.g. , adenine (A), thymine (T), cytosine (C), guanine (G), or uracil (U), but also modified purine and pyrimidine bases and other heterocyclic bases which have been modified (these moieties are sometimes referred to herein, collectively, as "purine and pyrimidine bases and analogs thereof"). Such modifications include, e.g., diaminopurine and its derivatives, inosine and its derivatives, alkylated purines or pyrimidines, acylated purines or pyrimidines thiolated purines or pyrimidines, and the like, or the addition of a protecting group such as acetyl, difluoroacetyl, trifluoroacetyl, isobutyryl, benzoyl, 9- fluorenylmethoxycarbonyl, phenoxyacetyl, dimethylformamidine, Ν,Ν-diphenyl carbamate, or the like. The purine or pyrimidine base may also be an analog of the foregoing; suitable analogs will be known to those skilled in the art and are described in the pertinent texts and literature. Common analogs include, but are not limited to, 1 -methyladenine, 2- methyladenine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentyladenine, Ν,Ν-dimethyladenine, 8-bromoadenine, 2-thiocytosine, 3-methylcytosine, 5-methylcytosine, 5-ethylcytosine, 4-acetylcytosine, 1 -methylguanine, 2-methylguanine, 7-methylguanine, 2,2- dimethylguanine, 8-bromoguanine, 8-chloroguanine, 8-aminoguanine, 8-methylguanine, 8- thioguanine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, 5-ethyluracil, 5- propyluracil, 5-methoxyuracil, 5-hydroxymethyluracil, 5-(carboxyhydroxymethyl)uracil, 5- (methylaminomethyl)uracil, 5-(carboxymethylaminomethyl)-uracil, 2-thiouracil, 5-methyl-2- thiouracil, 5-(2-bromovinyl)uracil, uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester, pseudouracil, 1 -methylpseudouracil, queosine, inosine, 1 -methylinosine,

hypoxanthine, xanthine, 2-aminopurine, 6-hydroxyaminopurine, 6-thiopurine, and 2,6- diaminopurine.

The terms "complementarity" or "complementary" as used herein refers to a sufficient number in the oligonucleotide of complementary base pairs in its sequence to interact specifically (hybridize) with the target nucleic acid sequence to be amplified or detected. As known to those skilled in the art, a very high degree of complementarity is needed for specificity and sensitivity involving hybridization, although it need not be 100%. Thus, for example, an oligonucleotide that is identical in nucleotide sequence to an oligonucleotide disclosed herein, except for one base change or substitution, may function equivalents to the disclosed oligonucleotides.

The term "cyclic polymerase-mediated reaction" as used herein refers to a biochemical reaction in which a template molecule or a population of template molecules is periodically and repeatedly copied to create a complementary template molecule or complementary template molecules, thereby increasing the number of the template molecules over time.

The term "fragment", of a molecule such as a protein or nucleic acid, as used herein refers to any portion of the amino acid or nucleotide genetic sequence.

The term "molecular conjugate" as used herein refers to an entity formed by the covalent linkage of at least two molecular moieties to form a single molecular structure. In the context of the present disclosure such a single molecular structure can comprise, but is not limited to, a receptor-specific ligand covalently attached to a siRNA moiety.

The term "cytokine" as used herein refers to any cytokine or growth factor that can induce the differentiation of a stem cell to a progenitor or precursor cell and/or induce the proliferation thereof. Suitable cytokines for use in the present invention include, but are not limited to, stem cell factor, interleukin-1 , interleukin-2, interleukin-3, interleukin-6, interleukin- 7, interleukin-15, interleukin-17, interleukin-18, interferon-a, interferon-β, interferon-γ, Flt3L, leukemia inhibitory factor, insulin-like growth factor, insulin, and the like. The term "cytokine" as used herein further refers to any natural cytokine or growth factor as isolated from an animal or human tissue, and any fragment or derivative thereof that retains biological activity of the original parent cytokine. The cytokine or growth factor may further be a recombinant cytokine or a growth factor such as, for example, recombinant insulin. The term "cytokine" as used herein further includes species-specific cytokines that while belonging to a structurally and functionally related group of cytokines, will have biological activity restricted to one animal species or group of taxonomically related species, or have reduced biological effect in other species.

The term "modify the level of gene expression" as used herein refers to generating a change, either a decrease or an increase in the amount of a transcriptional or translational product of a gene. The transcriptional product of a gene is herein intended to refer to a messenger RNA (mRNA) transcribed product of a gene and may be either a pre- or post- spliced mRNA. Alternatively, the term "modify the level of gene expression" may refer to a change in the amount of a protein, polypeptide or peptide generated by a cell as a consequence of interaction of an siRNA with the contents of a cell. For example, but not limiting, the amount of a polypeptide derived from a gene may be reduced if the

corresponding mRNA species is subject to degradation as a result of association with an siRNA introduced into the cell. The term "siRNA" as used herein refers to small inhibitory RNA duplexes that induce the RNA interference (RNAi) pathway. These molecules can vary in length (generally 18-30 base pairs) and contain varying degrees of complementarity to their target mRNA in the antisense strand. Some, but not all, siRNA have unpaired overhanging bases on the 5' or 3' end of the sense strand and/or the antisense strand. The term "siRNA" includes duplexes of two separate strands, as well as single strands that can form hairpin structures comprising a duplex region. siRNA may be divided into five (5) groups (non-functional, semi-functional, functional, highly functional, and hyper-functional) based on the level or degree of silencing that they induce in cultured cell lines. In this context, "non-functional siRNA" are defined as those siRNA that induce less than 50% (<50%) target silencing. "Semi-functional siRNA" induce 50-79% target silencing. "Functional siRNA" are molecules that induce 80-95% gene silencing. "Highly-functional siRNA" are molecules that induce greater than 95% gene silencing. "Hyperfunctional siRNA" are a special class of molecules. For purposes of this disclosure, hyperfunctional siRNA are defined as those molecules that: (1 ) induce greater than 95% silencing of a specific target when they are transfected at sub-nanomolar concentrations (i.e., less than one nanomolar); and/or (2) induce functional (or better) levels of silencing for greater than 96 hours. These relative functionalities (though not intended to be absolutes) may be used to compare siRNAs to a particular target for applications such as functional genomics, target identification and therapeutics.

siRNAs trigger host cell RNA degradation mechanisms in a sequence-specific manner. They may therefore be used to inactivate endogenous RNAs or pathogen RNA. It is preferred that there be not more than one mismatch (mismatches are defined as not including G:U pairs) in each double-stranded region, more preferably no mismatches, and most preferred that the double stranded region(s) be perfectly matched. Where the targeted molecule is variable, highly conserved regions should be targeted. A family of variants can be targeted provided they do not have more than one mismatch with one or other of the strands of the double-stranded region of the siRNA molecule. For longer siRNA molecules, several short duplexes may be joined, allowing targeting of multiple genes, which is preferred for targets with higher variablity. The siRNA duplexes may also be produced from longer RNA transcripts by splicing or self-cleaving means, for example by incorporating self- cleaving ribozymes between or flanking the duplex regions. siRNA molecules are easily formed from DNA molecules having an inverted repeat structure. Alternatively, siRNA duplexes may be formed from two RNA molecules with complementary regions. siRNA molecules with double-stranded regions of greater than 30 base pairs can be used if they are nuclear localized, e.g., if they are made without signals for cytoplasmic export such as polyadenylated sequences. The term "target" is used in a variety of different forms throughout this document and is defined by the context in which it is used. "Target mRNA" refers to a messenger RNA to which a given siRNA can be directed against. "Target sequence" and "target site" refer to a sequence within the mRNA to which the sense strand of an siRNA shows varying degrees of homology and the antisense strand exhibits varying degrees of complementarity. The phrase "siRNA target" can refer to the gene, mRNA, or protein against which an siRNA is directed. Similarly, "target silencing" can refer to the state of a gene, or the corresponding mRNA or protein.

The term "carbachol" as used herein (also known as carbamylcholine) refers to carbachol (a choline ester) and its derivatives that are capable of binding and stimulating acetylcholine receptors (e.g., muscarinic and nicotinic receptors).

The term "muscarinic receptor," as used herein without a prefix specifying the receptor subtype, refers to one or more of the five receptor subtypes M -Ms.

The term "modulating" as used herein means increasing or decreasing, e.g. activity, by a measurable amount. Compounds that modulate muscarinic activity by increasing the activity of the muscarinic receptors are called agonists. An agonist interacts with a muscarinic receptor to increase the ability of the receptor to transduce an intracellular signal in response to endogenous ligand binding. Conversely, an antagonist interacts with a muscarinic receptor to decrease the ability of the receptor to transduce an intracellular signal in response to endogenous ligand binding.

The term "modulator of the expression of a gene' as used herein refers to a molecule, including, but not limited to, an oligonucleotide such as an siRNA, that can interact with a genomic nucleotide sequence, an mRNA, or with a transcription factor whereby the expression of a selected gene is reduced, increased or eliminated when compared to the expression in the absence of the modulator. In this context, "gene expression"

encompasses formation an mRNA encoded by the gene, translocation of the mRNA from a cell nucleus to the translation system of the cell, or translation of the mRNA to a polypeptide product. Modulation of the gene expression by the modulator may be by mechanisms such as physically blocking transcription from the gene, the translation process from the mRNA, or by inducing such as degradation of the mRNA, such as by contact with an siRNA.

The term "exocrine gland" as used herein refers to glands that secrete their products (excluding hormones and other chemical messengers) into ducts (duct glands) which lead directly into the external environment. Typical exocrine glands include sweat glands, salivary (salivary) glands, tear (lacrimal) glands, mammary glands, tracheobronchial glands, stomach, liver, pancreas, and of the reproductive and urinary tracts. The term "secretogogue" as used herein refers to a substance that causes another substance to be secreted by a cell or tissue such as, but not limited to, an acrine cell of an exocrine gland.

The term "endocytosis" as used herein refers to the process by which cells absorb molecules (such as proteins) from outside the cell by engulfing them with their cell membrane. Large polar molecules cannot otherwise pass through the hydrophobic plasma or cell membrane.

The term "apoptosis" as used herein refer to molecular and morphological processes leading to controlled cellular self-destruction (see, e.g., Kerr er a/., (1972), Br. J. Cancer 26: 239-257). Apoptotic cell death can be induced by a variety of stimuli, such as ligation of cell surface receptors, starvation, growth factor/survival factor deprivation, heat shock, hypoxia, DNA damage, viral infection, and cytotoxic/chemotherapeutic agents. Apoptotic cells can be recognized by stereotypical morphological changes: the cell shrinks, shows deformation and looses contact to its neighboring cells. Its chromatin condenses, and finally the cell is fragmented into compact membrane-enclosed structures, called "apoptotic bodies" which contain cytosol, the condensed chromatin, and organelles. The apoptotic bodies are engulfed by macrophages and thus are removed from the tissue without causing an inflammatory response. This is in contrast to the necrotic mode of cell death in which case the cells suffer a major insult, resulting in loss of membrane integrity, swelling and disrupture of the cells. During necrosis, the cell contents are released uncontrolled into the cell's environment what results in damage of surrounding cells and a strong inflammatory response in the corresponding tissue. See, e.g., Tomei & Cope., eds., 1991 , Apoptosis: The Molecular Basis of Cell Death, Plainville, N.Y.: Cold Spring Harbor Laboratory Press; Isaacs J. T., (1993) Environ Health Perspect. 101 (suppl 5): 27-33, which is herein incorporated by reference in its entirety for all purposes. "Apoptosis" is characterized by certain cellular characteristics such as membrane blebbing, chromatin condensation and fragmentation, formation of apoptotic bodies and a positive "TUNEL" staining pattern. Degradation of genomic DNA during apoptosis results in formation of characteristic, nucleosome sized DNA fragments; this degradation produces a diagnostic (about) 180 bp laddering pattern when analyzed by gel electrophoresis. A later step in the apoptotic process is degradation of the plasma membrane, rendering apoptotic cells leaky to various dyes (e.g., trypan blue and propidium iodide). Accordingly, a variety of apoptosis assays are well known to one of skill in the art (e.g., DNA fragmentation assays, radioactive proliferation assays, DNA laddering assays for treated cells, fluorescence microscopy of 4'-6-Diamidino-2-phenylindole (DAPI) stained cells assays, and the like).

The term "increases the survivability of a salivary or a lacrimal cell or population of cells as used herein refers to the suppression or elimination of a process such as, but not limited to, apoptosis whereupon the cells have prolonged viability compared to untreated cells.

The term "caspase" as used herein refers to cysteine-aspartic proteases or cysteine- dependent aspartate-directed proteases. They are a family of cysteine proteases that play essential roles in apoptosis (programmed cell death), necrosis and inflammation.

Caspases are essential in cells for apoptosis, or programmed cell death, in development and most other stages of adult life. Some caspases are also required in the immune system for the maturation of cytokines.

Inflammatory caspases include human caspase-1 , caspase-4, caspase-5, and caspase-12, and murine caspase-1 , caspase-1 1 and caspase-12. These enzymes constitute a subgroup of caspases that are defined by their association with immune responses to microbial pathogens. Inflammatory caspases are essential regulators of inflammation that are activated by cellular sensors of danger signals, the inflammasomes, and subsequently convert pro-inflammatory cytokines into their mature active forms.

Caspase-1 is known to cleave the proforms of ILI -β, IL18, IL1 H4 (IL1 F7b), and IL33.

Caspase-4 has been shown to process proform of IL18 and IL1 H4 (IL1 F7b) inefficiently, and may also cleave caspase-3 into its active form. Caspase-5 has been reported to cleave caspase-3. Caspase-1 1 cleaves caspase-1 and caspase-3. The activation of some inflammatory caspases, such as caspase-1 and caspase-5, occurs upon assembly of an intracellular complex known as the inflammasome. This results in the cleavage and activation of the pro-inflammatory cytokines ΙΙ_1 -β and IL18.

Twelve caspases have been identified in humans. There are two types of apoptotic caspases: initiator (apical) caspases and effector (executioner) caspases. Initiator caspases (e.g. CASP2, CASP8, CASP9 and CASP10) cleave inactive pro-forms of effector caspases, thereby activating them. Effector caspases (CASP3, CASP6, and CASP7) in turn cleave other protein substrates within the cell, to trigger the apoptotic process. The initiation of this cascade reaction is regulated by caspase inhibitors.

The term "pharmaceutically acceptable" as used herein refers to a compound or combination of compounds that while biologically active will not damage the physiology of the recipient human or animal to the extent that the viability of the recipient is comprised. Preferably, the administered compound or combination of compounds will elicit, at most, a temporary detrimental effect on the health of the recipient human or animal is reduced.

The term "intravascularly" as used herein refers to a route of delivering a fluid, such as a pharmaceutically acceptable composition, to a blood vessel.

The term "dosage" as used herein refers to the amount of a composition of the present disclosure administered to an animal or human. The term "directly delivering" as used herein refers to delivering a pharmaceutical preparation into a mass of target cells or population of cells within a defined location within a subject human or animal, whereby the preparation is not delivered by administration into the circulatory system to be distributed throughout the body rather than specifically or mainly to the target tissue. It is expected that the administration may be by injection near the disease tissue, e.g., saliva gland, to minimize side effects, although another advantageous route is expected to be intravascularly, and most advantageously into a vessel leading into the area to be treated. The manner of administration may also be transdermal, intramuscular, topical, oral, ductal, subcutaneous, intracavity, or peristaltic. Regarding injection near the disease tissue, micro-pumps may be implanted in or near the disease tissue to administer the dose in a manner similar to insulin pumps.

Description

The present disclosure encompasses compositions and methods for the delivery of siRNA moieties to the intracellular environment. siRNA species can be linked to a ligand that has specificity for a cell-surface receptor of the targeted cell. When contacted with the cell, the conjugated ligand specifically binds to its associated receptor, whereupon the conjugate and the receptor are internalized by endocytosis. It is understood that upon delivery to the cytoplasm of the targeted cell the siRNA moiety may be, but not necessarily, detached from the ligand attachment for interaction with a region of a gene or other nucleic acid and thereby modulating the expression or activity of a gene. It is further contemplated that the compositions and methods of the present disclosure may incorporate any suitable ligand that can specifically recognize and interact with a cell surface receptor that upon binding to such a ligand is internalized by endocytosis. Preferably, but not necessarily, the ligand species incorporated into the compositions of the disclosure are small molecules unlikely to elicit an immune response during the period of exposure of a recipient subject human or animal to the compositions of the present disclosure. However, any suitable ligand may be used including, but not limited to, a small molecule, a peptide, an oligonucleotide, a known receptor ligand (natural or synthetic) wherein such a receptor ligand is an agonist or an antagonist, and the like.

The siRNA moiety of the compositions of the disclosure may be any siRNA species that it is desired to be delivered to be internalized by a cell for the purpose of modulating the expression of a gene as a nucleic acid species or as a protein or polypeptide product. The siRNA species selected to be linked to the receptor binding moiety and the moiety itself may be linked together by any means that will allow the ligand moiety to recognize and bind to a cell surface receptor of a targeted cell. It is contemplated that the attachment may be by a covalent bond between the siRNA and the ligand, through a non-covalent bond, and optionally with an intervening linker molecule situated between the siRNA and the ligand. In some embodiments of the disclosure, the bond between the siRNA and the ligand or linker may be cleaved to release the siRNA species within the interior of the cell. In other embodiments, the bond between the ligand and a linker may be cleaved after endocytosis, thereby releasing the siRNA attached to the linker (that optionally may also be cleaved to release the siRNA from the linker).

While not wishing to be bound by any one theory, conjugation of the ligand to the siRNA is preferably to the sense strand, and once in the cell, it will be the anti-sense strand that determines RNA interference. Accordingly, one mechanism for releasing the siRNA is for the antisense strand to dissociate from the complementary sense strand without a cleavage of the ligand from an oligoribonucleotide.

The ligand-conjugate strand may just remain as a non-functional entity in the cell while anti-sense strand determines RNA interference. Such a mechanism could explain why conjugation to a ligand, with or without an intervening linker, would not affect the desired function of siRNA.

By targeting a cell surface receptor of a particular cell type, the compositions of the disclosure may be adapted to target specific cell or tissue types to modulate a particular gene function. This may then, prevent or treat such as a disease by disrupting the underlying cause and/or relieve undesirable symptoms of the disease. An example, and not intended to be limiting is the relief of Sjogren's Syndrome.

Sjogren's Syndrome is characterized by xerophthalmia (excessive dryness of the eye) and xerostomia (excessive dryness of the mouth due to reduced flow of saliva) resulting from loss of secretory function due to immune cell infiltration in lacrimal and salivary glands. Current Sjogren's Syndrome therapeutic strategies employ secretagogues to induce secretion via muscarinic receptor stimulation. The present disclosure encompasses compositions utilizing ligands having high affinity for muscarinic type-3-receptor to deliver siRNA into cells via receptor-mediated endocytosis, thereby altering epithelial cell responses to external cues such as pro-inflammatory or death (apoptosis) signals. By the use of the muscarinic receptor-specific ligands that can also function as secretagogues, the compositions of the disclosure can simultaneously stimulate secretion. In some

embodiments of the disclosure, carbachol, synthesized with an active choline group, was conjugated directly to the terminal amine group of the sense strand of an siRNA targeting caspase-3. A human salivary gland cell line (HSG) was used to test the efficacy of this type of conjugate to deliver the siRNA to the interior cytoplasm of a cell.

Transfection of a conjugate according to the disclosure into cells resulted in at least 78% reduction in caspase-3 gene expression, confirming the retained function of siRNA after conjugation. External conjugate treatment of HSG cells resulted in intracellular calcium release and induction of endocytosis similar to carbachol-only stimulation, indicating that the carbachol portion of the conjugate retained function after conjugation.

Conjugate entry into cells was confirmed using a dye-labeled DNA oligonucleotide probe specific for the antisense strand of the caspase-3 siRNA. HSG cells treated with conjugate (without transfection) exhibited a 50% reduction in caspase-3 gene and protein expression indicating the conjugate structures of the disclosure are effective in delivering functional siRNA into cells via receptor-mediated endocytosis. Furthermore, TNF-a-induced apoptosis was reduced in conjugate treated cells. This therapeutic strategy using the conjugates of the present disclosure can be manipulated to siRNA-target different genes of interest while maintaining cell-type specificity, and has clinical applications in the treatment of Sjogren's Syndrome.

Accordingly, the present disclosure provides a vehicle that alters molecular signals in salivary epithelial cells using RNA interference (RNAi)-based strategies. A ligand for muscarinic type-3 receptor (M3R), i.e. carbachol, conjugated with small interfering RNAs (siRNAs), can deliver siRNA into a human salivary gland cell line (HSG) by receptor- mediated endocytosis, where it can silence gene expression by RNAi while simultaneously inducing secretion in Sjogren's Syndrome patients. This carbachol-siRNA conjugate is referred to herein as the conjugate. siRNA targeting caspase-3 in the conjugate was used to investigate if knockdown of caspase-3 can prevent cytokine-induced apoptosis of HSG cells, mimicking the in vivo environment of Sjogren's Syndrome salivary glands where the fluid- secreting acinar cells undergo apoptosis. Although one embodiment of the disclosure used carbachol, it is contemplated that the siRNA conjugation technique of the disclosure may also be useful conjugating an siRNA moiety to any FDA-approved muscarinic receptor agonist such as, and not limited to, cevimeline (EVOXAC.RTM). Thus RNAi therapy can be delivered to cells in a receptor-specific manner while simultaneously stimulating secretion in Sjogren's Syndrome patients.

It has now been determined that siRNA targeting caspase-3 conjugated to the muscarinic receptor agonist carbachol can be delivered to cells via receptor-mediated endocytosis, hence providing an approach for cell type-specific RNAi therapy in Sjogren's Syndrome. The data indicate that both the siRNA and carbachol portions of the conjugate retained their respective functions after the conjugation process (Figs. 2A, 2B, and 3), and conjugate entry into HSG cells was detectable using a FAM-labeled probe (Fig. 4).

Conjugate treatment of cells resulted in a 50% reduction in caspase-3 gene and protein expression (Figs. 5, 6A, and 6B), supporting that this siRNA-carbachol conjugate is successfully delivered into cells via MR receptor-mediated endocytosis.

This design offers advantages over current strategies, as well as some limitations. By avoiding relatively bulky protein complexes such as a biotin-streptavidin bridge to link the two components of the molecular conjugates of the disclosure, the overall size of the conjugates of the disclosure remain small and are unlikely to induce a significant immune response. Known limiting strategies that employ biotin-streptavidin linking of antibodies to siRNA make use of the specificity of antibody targeting. Once inside the cell, however, the siRNA often must be cleaved free from the antibody-biotin-streptavidin complex to be functional, and the large size of the complex increases the possibility of triggering an immune response.

Conjugating siRNA to various receptor ligands can be limited by the chemical structure and flexibility of those ligands. Ligands with more complex structures may be more difficult to modify to link an siRNA without disrupting the ligand's capacity to bind its target receptor. Accordingly, it is contemplated that the siRNA and the ligand components of the molecular conjugates may be linked by short linkers, the termini of which may be selected to react with the siRNA and the ligand. Such linkers may be, but are not intended to be limited to such moieties as a hexahistidine chain, a polyglycine, and the like.

The carbachol-siRNA conjugates according to the present disclosure are particularly suitable for the treatment of Sjogen's Syndrome. The dual nature of the molecular conjugates herein described, where the ligand may be selected as being a secretagogue that increases the release of secretions from saliva glands or from lacrimal glands, and the conjugated siRNA reduces such processes as apoptosis in the recipient target cells, will relieve the immediate symptoms of the disease and presents a reduction in the auto-immune process that ultimately can destroy the acinar cells of these glands.

Acinar cells show signs of apoptotic cell death even prior to disease onset in Sjogen's Syndrome-prone mouse model system (C57BL/6.NOD- \ec7Aec2). Accordingly, reversal of apoptotic cell death in the acinar cell population by knocking down caspase-3 while simultaneously stimulating fluid secretion with carbachol is an attractive therapeutic treatment of Sjogen's Syndrome. It is contemplated, however, that the molecular conjugates of the disclosure, allow that any gene of interest can be targeted by providing an alternative target sequence of the siRNA. For example, but not intended to be limiting, siRNAs may be selected that target molecular events in epithelial cells to down-regulate cytokine secretion, co-stimulatory molecules, or HLA class II expression for antigen presentation; improve mild cognitive impairment or fatigue in a subset of Sjogen's Syndrome patients by regulating secretion of pro-inflammatory cytokines from target tissues; targeting a macrophage-specific receptor to regulate caspase-1 , which is important for pro-inflammatory cytokine secretion that leads to epithelial cell death in the glands prior to disease onset; targeting viral antigens detected in the salivary glands to reduce immunogenicity; and differentiation ductal cells into acinar cells in situ in diseased glands by identifying master transcription factors, activators or repressors for differentiation. Early intervention is more effective and beneficial than late intervention with regards to maintaining saliva flow in Sjogren's Syndrome patients. Modulating host cell or innate immune response around the time of disease onset or while acinar cells are still viable in the diseased salivary glands would be as critical as regulating autoreactive immune cells to prevent severe secretory dysfunction. Pre-diagnostic markers for Sjogen's Syndrome, and Sjogren's Syndrome-disease specific diagnostic markers are subjects of research (see, for example, Cha et al. , (2004J Scand. J. Immunol. 60: 552-565; Cha et al. , (2001 ) Exp. Clin. Immunogenet. 18: 143-160; Brayer et al , (2001 ) Scand. J. Immunol. 54: 133-140; Cha et al. , (2002) Arthritis Rheum. 46: 1390-1398; Cha et al. , (2006) J. Rheumatol. 33: 296-306; Gao et al. , (2004) Arthritis Rheum. 50: 2615-2621 ; Gao ef al. , J. Autoimmun. 26: 90-103. Epub 2006; Nguyen et al. , (2006) Mol. Immunol. 43: 1332-1339; Nguyen er a/. , (2000) Arthritis Rheum. 43:2297-2306; Robinson et al. , (1998) Adv. Exp. Med. Biol. 438: 925-930; Robinson et al. , (1997) Proc. Natl. Acad. Sci. U. S. A. 94: 5767-5771 ; Robinson et al. , (1996) Clin. Immunol. Immunopathol. 79:.50-59, incorporated herein by reference in their entireties).

Pharmaceutical compositions comprising the molecular conjugate compositions of the present disclosure can be administered in dosages and by techniques well known to those skilled in the medical or veterinary arts, taking into consideration such factors as the age, sex, weight, species and condition of the particular patient, and the route of

administration. The route of administration can be via any route that delivers a safe and effective dose of a composition of the present disclosure to the desired target such as a saliva gland, a tear gland, and the like wherein secretion stimulation and prevention or treatment of cell loss is desirable. Pharmaceutical or therapeutic compositions can be administered alone, or can be co-administered or sequentially administered with other treatments or therapies. Forms of administration, including injectable administration, include, but are not limited to, intravenous, intraperitoneal, an intramuscular, an intrathecal, an intraarticular, an intrapulmonary, an intraperitoneal, a retroperitoneal, an intrapleural, a subcutaneous, a percutaneous, a transmucosal, an intranasal, an oral, a gastro-intestinal, ductal, and an intraocular route of administration of such as sterile solutions, suspensions or emulsions. A particularly advantageous route of delivery of the compositions of the disclosure to a gland is to directly introduce the composition into a blood vessel leading into the treatable area.

Pharmaceutical compositions may be administered in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, or the like. The compositions can contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, adjuvants, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard pharmaceutical texts, such as "Remmington's Pharmaceutical Science," 17th edition, 1985 may be consulted to prepare suitable preparations, without undue experimentation. The effective dosage and route of administration are determined by the therapeutic range and nature of the compound, and by known factors, such as the age, weight, and condition of the host, as well as LD ₅₀ and other screening procedures that are known and do not require undue experimentation. Dosages can generally range from a few hundred micrograms to a few grams administered as a bolus or over a sustained period as determined by the medical condition and need of a subject animal or human. The term "sustained" as used herein refers to any extended period ranging from several minutes to years.

Suitable dosage units for use in the methods of the present disclosure range from mg/kg body weight of the recipient subject to mg/kg. The therapeutic agent may be delivered to the recipient as a bolus or by a sustained (continuous or intermittent) delivery. Delivery of a dosage may be sustained over a period, which may be in the order of a few minutes to several days, weeks or months, or may be administer chronically for a period of years. In this regard, during the period of administration, each individual patient should be examined to see how they are reacting to the treatment of the present disclosure. For instance, the patient should be examined for the above noted possible adverse reactions.

In view of the above, the period of administration may be, but is not limited to, from about 1 day to about 1 week, about 1 week to 6 months, about 1 week to 3 months, about 2 weeks to 1 month, and about 2 to 3 weeks. If the period of administration is too long, the period of recovery between periods of administration is increased and adverse impacts on the patient's health are more likely.

The compositions of the present disclosure may comprise a pharmaceutical composition of the present disclosure and at least one pharmaceutically acceptable carrier or excipient. As used herein, the terms "pharmaceutically acceptable", "physiologically tolerable" and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a mammal without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like.

The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on formulation. Typically such compositions are prepared as injectables either as liquid solutions or suspensions, however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared. The preparation can also be emulsified.

The active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients are, for example, water, saline, 11 042170 dextrose, glycerol, ethanol or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance the effectiveness of the active ingredient.

Physiologically tolerable carriers are well known in the art. Exemplary of liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes.

Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions.

A therapeutic composition contains an amount of an active compound of the present disclosure, typically formulated to contain an amount of at least about 0.1 weight percent of active compound of the present disclosure per weight of total therapeutic composition. A weight percent is a ratio by weight of active compound to total composition. Thus, for example, about 0.1 weight percent is about 0.1 grams of active compound per 100 grams of total composition.

While not wishing to be bound by theory, the dosage is expected to depend upon factors such as period of administration, stage of disease tissue, e.g., tumor, endogenous factors, disease tissue, e.g., tumor, behavior, and the patient's individual physiology. For shorter periods of administration, higher dosages are generally used. For later stage disease tissue, e.g., tumors, the dosage should generally be higher. For example, if the tumor has metastasized, the dosage should generally be higher. Dosages will generally be higher for more resistant and/or aggressive disease tissue. The dosage should also be affected by the patient's individual physiology. For instance, if the individual is healthy, the dosage can be higher. Also, if the individual is tolerant to the composition of the present disclosure, the dosage should generally be higher. Conversely, if an individual has adverse reactions, the treatment method of the present disclosure may not be appropriate or the dosage should generally be reduced.

One aspect of the disclosure, therefore, encompasses embodiments of compositions comprising: a ligand characterized as having affinity for a surface receptor of a cell; an siRNA moiety, wherein the ligand is linked to the siRNA moiety; and, optionally, a

pharmaceutically acceptable carrier.

In embodiments of this aspect of the disclosure, the cell can be an exocrine glandular cell. In embodiments of this aspect of the disclosure, the ligand can be covalently conjugated to the siRNA moiety.

In embodiments of this aspect of the disclosure, the ligand can be linked to the siRNA moiety by a linker.

In embodiments of this aspect of the disclosure, the ligand can be characterized as having affinity for a muscarinic receptor.

In some embodiments of this aspect of the disclosure, the exocrine glandular cell can be a salivary or lacrimal cell susceptible to Sjogrens Syndrome.

In some embodiments of this aspect of the disclosure, the ligand characterized as having affinity for a muscarinic receptor can be a secretagogue.

In embodiments of this aspect of the disclosure, the ligand is a muscarinic receptor agonist. In these embodiments of this aspect of the disclosure, the muscarinic receptor agonist can be selected from the group consisting of; carbachol, cevimeline, pilocarpine, methacholine, bethanechoi, muscarine, and a muscarinic receptor subtype-specific agonist.

In some embodiments of this aspect of the disclosure, the siRNA moiety, when delivered to the salivary or lacrimal cell, or population of said cells, susceptible to Sjogrens Syndrome can increase the survivability of salivary or lacrimal acinar cells.

In other embodiments of this aspect of the disclosure, the siRNA moiety, when delivered to the salivary or lacrimal cell, or population of said cells, susceptible to Sjogrens Syndrome can reduce the level of apoptosis in said salivary or lacrimal cell, or population of said cells.

In embodiments of this aspect of the disclosure, the siRNA moiety, when delivered to the salivary or lacrimal cell, or population of said cells, susceptible to Sjogrens Syndrome can reduce the level of caspase-3 activity in said salivary or lacrimal cell, or population of said cells.

In embodiments of this aspect of the disclosure, the siRNA moiety can comprise a nucleotide sequence complementary to a region of an mRNA encoding a caspase, and wherein when delivered to the salivary or lacrimal cell, or population of said cells, reduces the level of caspase activity in said cell, or population of said cells.

In embodiments of this aspect of the disclosure, the siRNA moiety can comprise the nucleotide sequence S'-AUAAAUUCAAGCUUGUCGG-S' (SEQ ID No.: 4) and, optionally, the complement thereof, and wherein said siRNA is characterized as reducing the level of caspase-3 activity when delivered to a salivary or lacrimal cell, or a population of said cells.

In embodiments of this aspect of the disclosure, the siRNA moiety, when delivered to the salivary or lacrimal cell or population of cells subject to Sjogrens Syndrome can modulate the expression of a gene thereby down-regulating a pro-inflammatory cytokine secretion and HLA class II expression, inhibiting a toll-like receptor (TLR)-mediated pathway, inhibiting the expression of a viral gene, or stimulating the differentiation of an acinar cell.

Another aspect of the disclosure encompasses embodiments of a method for delivery of a molecular conjugate to a cell, comprising the steps of: (a) contacting with a cell or cells with a molecular conjugate comprising a ligand characterized as having affinity for a surface receptor of the cell, and an siRNA moiety linked to the ligand; and (b) maintaining the cell, or population of said cells, under conditions whereby the ligand specifically binds to a surface receptor of the cell or cells, whereupon the molecular conjugate enters the cell or cells by endocytosis, thereby delivering the siRNA moiety to the cytoplasm of the cell or cells.

In embodiments of this aspect of the disclosure, the cell can be an exocrine glandular cell, or population of said cells.

In embodiments of this aspect of the disclosure, the surface receptor can be a muscarinic receptor. In some embodiments of this aspect of the disclosure, the exocrine glandular cell can be a cell of a saliva gland, a lacrimal gland, a tracheobronchial giand, a digestive gland, or a sweat gland. In some embodiments of this aspect of the disclosure, the exocrine glandular cell or population of said cells can be a salivary or lacrimal cell or population of said cells susceptible to Sjogrens Syndrome.

In embodiments of this aspect of the disclosure, the ligand can be a secretagogue or a muscarinic receptor agonist.

In embodiments of this aspect of the disclosure, the muscarinic receptor agonist can be selected from the group consisting of; carbachol, cevimeline, pilocarpine, methacholine, bethanechol, muscarine, and a muscarinic receptor subtype-specific agonist.

In some embodiments of this aspect of the disclosure, the siRNA moiety, when delivered to the salivary or lacrimal cell or population of cells susceptible to Sjogrens Syndrome, can increase the survivability of salivary or lacrimal acinar cells.

In some embodiments of this aspect of the disclosure, the siRNA moiety can reduce the level of apoptosis in said salivary or lacrimal cell or population of cells.

In some embodiments of this aspect of the disclosure, the siRNA moiety can comprise a nucleotide sequence complementary to a region of an mRNA encoding a caspase, and wherein when delivered to the salivary or lacrimal cell, or population of said cells, reduces the level of caspase activity in said cell, or population of said cells.

In embodiments of this aspect of the disclosure, the siRNA moiety comprises the nucleotide sequence 5 -AUAAAUUCAAGCUUGUCGG-3' (SEQ ID No.: 4) and, optionally, the complement thereof, and wherein said siRNA can be characterized as reducing the level of caspase-3 activity when delivered to a salivary or lacrimal cell, or a population of said cells. In embodiments of this aspect of the disclosure, the method can further comprise administering the molecular conjugate to the subject animal or human, wherein the ligand increases secretion from the saliva gland or the lacrimal gland of said subject, and the siR A reduces the level of apoptosis in said saliva gland or the lacrimal gland, thereby reducing the symptoms of Sjogrens Syndrome in said subject animal or human.

In embodiments of this aspect of the disclosure, the siRNA moiety, when delivered to the salivary or lacrimal cell or population of cells subject to Sjogrens Syndrome, modulates the expression of a gene thereby down-regulating a cytokine secretion, down-regulating an HLA class II expression, regulating secretion of a pro-inflammatory cytokines, regulating caspase-1 expression, inhibiting the expression of a viral gene, or stimulating the differentiation of a ductal cell into an acinar cell.

Another aspect of the disclosure provides an embodiment of a method of modulating the symptoms of Sjogrens Syndrome in a subject animal or human by administering to said subject a therapeutic dose of a pharmacologically acceptable composition comprising: (i) a ligand characterized as a secretagogue having affinity for a muscarinic receptor of an exocrine salivary or lacrimal glandular cell, or population of said cells, wherein the ligand is a muscarinic receptor agonist selected from the group consisting of; carbachol, cevimeline, pilocarpine, bethanechol, and muscarine; (ii) an siRNA moiety having the nucleotide sequence 5'-AUAAAUUCAAGCUUGUCGG-3' (SEQ ID No.: 4) and is complementary to a region of an mRNA encoding caspase-3, wherein the ligand is linked to the siRNA moiety; and (iii) a pharmaceutically acceptable carrier, wherein the siRNA moiety reduces the level of caspase-3 activity in said salivary or lacrimal cell, thereby stimulating the secretion of the saliva gland or lacrimal gland and increasing the survivability of the acinar cell.

The specific examples below are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present disclosure to its fullest extent. All publications recited herein are hereby incorporated by reference in their entirety.

It should be emphasized that the embodiments of the present disclosure, particularly, any "preferred" embodiments, are merely possible examples of the implementations, merely set forth for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the above-described embodiment(s) of the disclosure without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure, and the present disclosure and protected by the following claims.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the compositions and compounds disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, eic), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

It should be noted that ratios, concentrations, amounts, and other numerical data may be expressed herein in a range format. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a concentration range of "about 0.1 % to about 5%" should be interpreted to include not only the explicitly recited concentration of about 0.1 wt% to about 5 wt%, but also include individual concentrations (e.g., 1 %, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5%, 1.1 %, 2.2%, 3.3%, and 4.4%) within the indicated range. The term "about" can include ±1 %, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, or ±10%, or more of the numerical value(s) being modified.

EXAMPLES

Example 1

Conjugation of carbachol with siRNA: The conjugate design aimed to utilize the specificity of the muscariinic receptor agonist carbachol to deliver siRNA into MR-expressing cells of the salivary glands via receptor-mediated endocytosis. Carbachol was synthesized with an active choline group which was linked to an siRNA targeting caspase-3 via a 5' amino group, as schematically shown in Fig. 1 B.

Carbachol-siRNA conjugation: Conjugate was synthesized by Solulink, Inc. (San Diego, CA). Carbachol was synthesized with an active choline group which was linked to the 5'-amino end on the sense strand of an siRNA targeting caspase-3 (targeting sequence 5'- CCGACAAGCUUGAAUUUAU-3' (SEQ ID NO. 1 )) or to a scrambled sequence 5'- GAUAUGUCAACUCAGUACU-3' (SEQ ID NO.: 2). Conjugate was synthesized four times and experimental variations between batches are minimal based on functional tests.

Activation of choline: To a solution of choline tosylate (13.8 mg; 50 pmol; SigmaAldrich, St. Louis, MO) in anhydrous DMF (250 pL) was added triethylamine (14 μΙ_; 100 pmol). A solution of 4-nitrophenyl chloroformate (10.1 mg; 50 pmol; SigmaAldrich) in anhydrous dichloromethane (100 μΙ_) was prepared and added directly to the choline/TEA/DMF solution. The reaction was incubated at room temperature for 2 hours. A sample was analyzed by electrospray mass spectrometry and the solution was used directly for modification of the amino-RNA sense strand if the major peak m/e = 269. Choline conjugation to amino-RNA sense strand: Both amino-modified sense and antisense strands were desalted into 100 mmol phosphate, 150 mM NaCI, pH 7.4 using 5K MWCO VIVASPIN.RTM diafiltration devices (SartoriusStedim, Purchase, NY). To the amino- modified sense strand (66.2 nmol; 28.2 μΙ_; 0.488 OD/μΙ) was added activated choline solution (9.76 μΙ_; 1 .3 pmol; 20 mol equivalents), vortexed and allowed to stand at room temperature for 1 h and overnight at 4 °C. The choline-modified RNA was isolated by desalting into nuclease-free water using a 5K MWCO VIVASPIN.RTM device. The product was analyzed by MALDI mass spectrometry: expected 6912; found m/e 6925 (starting amino-RNA m/e 6794 + choline-C=0 1 8).

Example 2

HSG cell culture: Cells were maintained in DMEM supplemented with 10% fetal calf serum, penicillin (100U/ml) and streptomycin (100pg/ml) (Life Techonologies, Burlington, Ontario, Canada). Cells were plated at medium density (2.5 x 10 ⁴ cells/cm ² ) in a 75cm ² flask and incubated at 37 °C with 5% C0 ₂ until confluent. Cells were harvest with 0.25%

trypsin/0.53mM EDTA (Hyclone), washed, and resuspended in new media. For conjugate treatment, HSG cells were seeded onto 8-chamber slides or 6-well plates in growth media and cultured overnight. The cells were then washed three times with Opti-MEM serum free media (Invitrogen, Carlsbad, CA). The cells were treated with conjugate diluted in OPTI- MEM. RTM to a final concentration of 3.97μΜ unless specified otherwise. Appropriate controls were included in each experiment. Negative control conjugate ("Neg Ctl

Conjugate") was synthesized using a scrambled siRNA sequence and carbachol. "Free siCaspase-3" indicates siRNA targeting caspase-3 was added to culture media in the absence of transfection reagent to serve as an additional control. "Carbachol only" indicates 100μΜ carbachol was added to the culture media. "siCaspase-3" or "transfected siCaspase- 3" indicates that the cells were transfected with 40nM siRNA targeting caspase-3 to serve as a positive control for caspase-3 knockdown.

Example 3

Transfection and qRT-PCR: HSG cells were transfected using LIPOFECTAMINE.RTM 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. siRNAs targeting human caspase-3 (target sequence 5'-CCGACAAGCUUGAAUUUAU-3' (SEQ ID No. 1 ) and human GAPDH (target sequence 5'-GGUCAUCCAUGACAACUUUGG-3' (SEQ ID No. 3)) were purchased from Dharmacon (Lafayette, CO) and Applied Biosystems (Carlsbad, CA), respectively, and transfected into cells at final concentrations of 40nM. Caspase-3 gene, OAS-1 , and MX-1 expression in HSG cells was analyzed after transfection or conjugate treatment using quantitative real time RT-PCR (qRT-PCR).

Total RNA was extracted using the MIRVANA.RTM miRNA Isolation kit (Ambion, Austin, TX) in accordance with the manufacturer's protocol. RNA concentrations were determined and 50ng of each RNA sample was used for qRT-PCR that was performed using the TAQMAN.RTM High-Capacity cDNA Reverse Transcription Kit, TAQMAN FAST.RTM PCR Master Mix, and TAQMAN.RTM mRNA assay primers (Applied Biosystems). All reactions were analyzed using StepOne Real-Time PCR System (Applied Biosystems). The levels of mRNA were normalized to 18S controls. The cycle threshold values, corresponding to the PCR cycle number at which fluorescence emission reaches a threshold above baseline emission, were determined and the relative mRNA expression was calculated using the AACt method (Livak er a/. , (2001 ) Methods, 25:402-408, incorporated herein by reference in its entirety).

Example 4

Calcium Release Assay: Untreated, conjugate treated and carbachol-treated HSG cells were monitored using FLUO-4 NW.RTM Calcium assay kit (Invitrogen) in accordance with the manufacturer's protocol. Briefly, 30,000 cells per well were cultured in a 96-well plate overnight in growth media. Before the assay, the growth media was removed and 100μΙ of dye-loading solution was added to each well. The cells were then incubated for 30 minutes at 37 °C, then at room temperature for 30 minutes. Carbachol or the carbachol-siRNA conjugate was added to the cells at the indicated concentrations, and emitted fluorescence (516nm) was measured using a fluorescent plate reader.

Example 5

Calcium Imaging: Parc5 cells were loaded with Fura 2-AM ( Regehr & Atluri (1995) Biophys. J. 68: 2156-2170). A 1 mM solution of Fura 2-AM was made in DMSO containing 20% pluronic acid. This was then diluted 100-fold in physiological solution. The dye was filled in a pipette and pressure injected for 30-45 min. Cells were incubated in 20 μΜ Fura 2-AM in 0.2% pluronic acid for an hour at room temperature and allowed to recover a further hour. Imaging was performed using a Zeiss Axioskop-2 upright microscope using a water- immersion 40x lens (numerical aperture, 0.8). Images were acquired using a cooled CCD camera (Cooke Sensicam; PCO, Kelheim, Germany). Excitation was achieved using a Sutter Instruments (Novato, CA) DG-4 fast wavelength switcher and standard Fura 2 excitation and emission filters were used (Chroma Technology, Brattleboro, VT). Images were acquired and processed using the SlideBook software (Intelligent Imaging Innovations, Denver, CO). Ratiometric data were acquired at 0.1-1 Hz. Analyzed data were graphed using the Microcal Origin software. Calcium signals were expressed as changes in the 340/380 ratio. Agonist was applied using pressure application via a patch pipette placed approximately 20 μΜ above the slice for 30 sees. Example 6

Endocytosis assay: ParC5 c(rat parotid acinar) cells were transfected with the Yellow Fluorescent Protein (YFP)-tagged NBCel construct (NBCe1-EYFP). 100 μΜ carbachol or 1 :100 diluted conjugate (8.71 μΜ) was added to serum-free culture media. Cells were then washed with PBS, fixed, permeabilized, and labeled with anti-Early Endosomal Antigen 1 (EEA1 ) primary antibodies followed by Alexa Fluor-Texas red secondary antibodies, washed and mounted with VECTASHIELD.RTM on slides for qualitative analysis through a confocal microscopy. Fluorescent images were taken using an Olympus Spinning Disk confocal microscope controlled by Slidebook 4.0.10.2. Confocal images were captured in z-stack intervals of 1 μηη using a 60x oil immersion objective (1 .45 A). Qualitative analysis was done to ascertain if co-localization occurred between NBCel -EYFP and Alexa Fluor-Texas red for EAA1 -endosomal marker.

Example 7

In situ hybridization: HSG cells were cultured on 8-chamber slides, treated with conjugate or transfected with caspase-3 or GAPDH siRNA, and incubated for the indicated times (30 minutes to 24 hours). The cells were then washed once with PBS, fixed in 4%

paraformaldehyde for 15 minutes at room temperature, permeabilized in 0.5% Triton X-100 for five minutes, and dehydrated with 70, 90, and 100% ethanol for one minute each. The FAM-labeled DNA oligonucleotide probe specific for the antisense strand of caspase-3 siRNA (Exiqon Woburn, MA) was diluted to 80nM in PBS and heated to 80 °C for two minutes before addition to the cells. Hybridization was performed at 47 °C for 45 minutes, and the cells were then washed and mounted in VECTASHIELD.RTM mounting medium containing DAPI (Vector Labs, Burlingame, CA). Images were taken with Zeiss Axiovert 200 M microscope and a Zeiss AxioCam MRm camera.

Example 8

Immunocytochemistry: HSG cells were cultured on 8-chamber slides, fixed in 4% paraformaldehyde for 10 minutes, and permeabilized in 0.5% Triton X-100 for 5 minutes. Caspase-3 protein was detected with rabbit anti-caspase-3 antibodies (Abeam, Cambridge, MA) used at a 1 :200 dilution. Secondary antibodies used were Alexa Fluor 568 goat anti- rabbit IgG (1 :400) from Molecular Probes (Carlsbad, CA). Slides were mounted using

Vectashield Mounting Medium containing DAPI (Vector Labs) and images were taken with Zeiss Axiovert 200 M microscope and a Zeiss AxioCam MRm camera. Image J image analysis software was used to measure the relative caspase-3 protein expression.

Example 9

Apoptosis assay: HSG cells were treated with TNF-a (50ng/ml, BD Biosciences, San Jose, CA) and cycloheximide (10 g/ml, Sigma, St. Louis, MO) for eight hours. Cycloheximide was required to sensitize the cells to TNF-a induced apoptosis (Cryns ef a/. , (1996) J. Biol. Chem. 271 : 31277-31282; Janicke et al. , (1998) J. Biol. Chem. , 273: 15540-15545). The cells were then washed once with PBS, briefly trypsinized, and pelleted. The cells were then stained with FITC-conjugated Annexin V (BD Biosciences) and propidium iodide (BD Biosciences) for 15 mins at room temeprature. The stained cells were analyzed by flow cytometry (FACS Calibur, BD Biosciences) to determine the percentage of early (annexin-V positive) and late (double positive) apoptotic cells.

Example 10

Statistical Analysis: Statistics were calculated using GraphPad Prism 4 software (GraphPad Software, La Jolla, CA). Student t tests or one-way ANOVA were used as indicated, with p<0.05 considered statistically significant.

Example 11

Carbachol and siRNA portions of conjugate retained function after conjugation: After conjugation, it was determined whether if the carbachol and siRNA portions of the conjugate retained function after the conjugation process. First, the conjugate was transfected into HSG cells in varying concentrations from about 100nM to about 10μΜ using

LIPOFECTAMINE.RTM 2000, incubated the cells for 48 hours, and analyzed caspase-3 gene expression by qRT-PCR. Cells were also transfected with a negative control conjugate containing a scrambled siRNA sequence.

Fig. 2A shows that transfected conjugate resulted in a significant decrease in the caspase-3 gene expression level compared to cells transfected with negative control conjugate ( ^* *p<0.01 , *p<0.05 as determined by one-way ANOVA with Dunnett's multiple comparison test). 100nM and 1 μΜ transfected conjugate resulted in 78% and 71 % reductions in caspase-3 gene expression, respectively, while 10μΜ transfected conjugate resulted in only a 38% reduction, as shown in Fig. 2A. Unconjugated caspase-3 siRNA transfected into cells in parallel gave a 53% reduction in caspase-3 gene expression, while siRNA and carbachol added to the cells individually had no effect on caspase-3 gene expression. These data indicate that the siRNA portion of the conjugate retained function after the conjugation process, and the conjugated carbachol did not influence siRNA efficacy on gene knockdown.

The carbachol portion of the conjugate was tested in a calcium release assay that quantitatively measured intracellular calcium release from HSG cells. Muscarinic receptor agonists such as carbachol, bind to receptors on the cell surface, initializing signal transduction that results in intracellular calcium release from the endoplasmic reticulum, ultimately leading to fluid secretion (Tobin et a/., (2009) J. Physiol. Pharmacol. 60: 3-21 ). Therefore, intracellular calcium release was monitored as a measure of carbachol function. HSG cells were treated with 1 -10ΌμΜ carbachol and 100ηΜ-87μΜ conjugate in parallel and, as shown in Fig. 2B, conjugate treatment resulted in similar levels of intracellular calcium release as carbachol treatment. To independently verify these results, calcium ratiometric analysis was performed using the rat parotid cell line, ParC5. Cells were treated with 100pM carbachol or 8.71 μΜ (1 :100 dilution) of conjugate or free siRNA control. Single cells stimulated with conjugate produced similar levels of calcium release as those stimulated with carbachol. These data indicate that the conjugation process did not alter the efficacy of the carbachol, as shown in Figs 2C-2E.

Another measure of carbachol function was its ability to induce muscarinic receptor- mediated endocytosis. To monitor carbachol-induced endocytosis with our conjugate, ParC5 cells were transfected with the Yellow Fluorescent Protein (YFP)-tagged NBCel construct (NBCel -EYFP), which is an electrogenic Na( ⁺ )-HCO( ₃ )( ^" ) co-transporter (NBCel ) known to be endocytosed upon cholinergic stimulation (Perry ef a/. , (2009) Am. J. Physiol. Cell Physiol. 297: C1409-1423), and then treated with 100 μΜ carbachol or 8.71 μΜ conjugate. Co-localization between NBCel -EYFP and EEA1 -endosomal marker indicated the induction of endocytosis. As shown in Fig.3, the merged images indicated that carbachol and the conjugate similarly stimulate endocytosis of basolateral NBCel in ParC5 cells, indicating that the carbachol portion of the conjugate is capable of inducing receptor- mediated endocytosis.

Example 12

Conjugate entry detected in HSG cells: When it was verified that both the carbachol and siRNA portions of the conjugate retained function after conjugation, it was determined whether the conjugate could enter cells through receptor-mediated endocytosis. A FA - labeled DNA oligonucleotide probe was designed to specifically bind the antisense strand of the caspase-3 siRNA, and in situ hybridization was used to visualize the entry of conjugate into HSG cells, as shown in Fig. 4.

Cells were treated with 5μΜ conjugate for 30 minutes to 24 hours or left untreated and fixed after each time point. As shown in Fig. 4, conjugate was detected in the cytoplasm (arrows) as soon as 30 minutes after treatment and up to 24 hours after treatment, although conjugate was detected in only about 30% of cells. This experiment was repeated six times with reproducible results; however, the percentage of cells which contained conjugate varied with target HSG cell batches. This may be accounted for by non-uniform muscarinic-3 receptor expression on HSG cells in response to subtle changes in culture condition overtime. This also reflects the importance of receptor density in the receptor-medicated endocytosis. Nonetheless, the data indicate that conjugate entry into cells was achieved. Example 13

Conjugate treatment results in caspase-3 gene and protein reduction: To determine if the conjugate was capable of effectively reducingcaspase-3 gene/protein expression after entry into the cells. Cells were treated with 8.71 μΜ conjugate for 4-6 hours in serum free media that was then replaced with growth media. After 48 hours of incubation, caspase-3 gene expression was analyzed by qRT-PCR. As shown in Fig. 5, conjugate-treated cells showed a 50% reduction in caspase-3 gene expression compared to cells treated with negative control conjugate ( ^* p<0.01 as determined by one-way ANOVA with Dunnett's multiple comparison test). After 72 hours of incubation, caspase-3 protein expression was analyzed by immunofluorescence.

Caspase-3 gene expression in MR-negative HeLa cells treated with 5μΜ conjugate was not affected (Fig. 6), indicating that the conjugate specifically targets MR-expressing cells. HSG cells were also treated with 50nM to 1 μΜ concentrations of free siCaspase-3 to demonstrate that siRNA alone is unable to enter the cells. As expected, free siCaspase-3 treated cells showed no reduction in caspase-3 gene expression. To ensure that the reduced caspase-3 gene/protein expression was not due to a non-specific interferon response, mR A expression of interferon response genes OAS1 and MX1 was monitored and shown to remain unchanged after conjugate treatment (Fig. 7). After 72 hours of incubation, caspase-3 protein expression was analyzed by immunofluorescence.

As shown in Fig. 8, caspase-3 staining was drastically reduced in conjugate-treated and caspase-3 siRNA-transfected cells compared to untreated cells. Transfected GAPDH siRNA, carbachol only, or siRNA only had no effect on caspase-3 protein levels. Caspase-3 siRNA was detected in transfected cells, but conjugate was minimally detected, indicating that the conjugate may be degraded after 72 hours. Quantitative analysis of capase-3 protein levels carried out using Image J software (Fig. 9) showed that caspase-3 protein levels were reduced by 50% in conjugate-treated cells, similarly to caspase-3 siRNA transfected cells.

Western blot was also performed to further demonstrate the reduction in caspase-3 protein levels after conjugate treatment. As shown in Fig. 10, cells treated with 5μΜ conjugate exhibited greater than 50% reduction in caspase-3 compared to mock transfected or cell treated with negative control conjugate.

Taken together, these data demonstrate that a carbachol-siRNA conjugate according to the present disclosure was effective in reducing caspase-3 gene and protein expression and reveal a novel strategy for RNAi therapies in conjugating siRNAs directly with receptor ligands having affinity for specific targeted receptors on the surface of cells.

Example 14 Conjugate treatment prevents TNF-a induced apoptosis of HSG cells: In Sjogren's syndrome, the presence of inflammatory cytokines in target tissues contributes to apoptosis of surrounding cells. Hence, our strategy is to prevent cytokine-induced apoptosis of acinar cells using the conjugate to knock down caspase-3 expression. HSG cells were treated with 5 μΜ conjugate or transfected with caspase-3 siRNA and incubated for 96 hours to allow for complete caspase-3 knockdown. The cells were then treated with TNF-a (50 ng/ml) and cycloheximide (10 pg/ml) for eight hours, and then stained with Annexin-V and propidium iodide and assessed by flow cytometry.

As shown in Fig. 1 1 , the percent of early apoptotic cells was significantly reduced in caspase-3-transfected and conjugate-treated cells after TNF-a/cycloheximide treatment

(p<0.001 ) indicating that the conjugate successfully prevents TNF-a induced apoptosis. The percent of late apoptotic cells (Annexin-V/PI positive) was reduced by 58% in caspase-3 transfected cells and by 25% in conjugate treated cells (Fig 12, p<0.01 compared to negative control conjugate-treated cells as determined by t test). Apoptosis in cells treated with negative control conjugate with a scrambled siRNA sequence was not significantly different from mock transfected cells. These data indicate the therapeutic potential of conjugate in preventing cytokine-induced apoptosis in salivary acinar cells and hence maintaining secretory function in Sjogren's syndrome patients.

Example 15

Free siCaspase-3 indicates siRNA targeting caspase-3 was added to culture media in the absence of transfection reagent to serve as an additional control. Cells were incubated with free siCaspase-3 for four hours, washed, and then incubated for 48 hours. Caspase-3 gene expression was then measured using qRT-PCR, as shown in Fig. 13.