# Patent

## Title
Compositions and methods for inhibiting expression of Nav1.8 gene

## Abstract

The invention relates to a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of the Nav1.8 gene (Nav1.8 gene), comprising an antisense strand having a nucleotide sequence which is less that 25 nucleotides in length and which is substantially complementary to at least a part of the Nav1.8 gene. The invention also relates to a pharmaceutical composition comprising the dsRNA together with a pharmaceutically acceptable carrier; methods for treating diseases caused by the expression of the Nav1.8 gene using the pharmaceutical composition; and methods for inhibiting the expression of the Nav1.8 gene gene in a cell.

## Background
RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No. 60/733,816, filed Nov. 4, 2005; U.S. Provisional Application No. 60/741,586, filed Dec. 2, 2005; U.S. Provisional Application No. 60/763,202, filed Jan. 26, 2006; U.S. Provisional Application No. 60/795,443, filed Apr. 27, 2006; and U.S. Provisional Application No. 60/849,364, filed Oct. 4, 2006. The contents of these prior applications are incorporated herein by reference in their entirety.


SEQUENCE LISTING
This application incorporates by reference the sequence listing saved as an ASCII text file on CD-ROM. The sequence listing saved on CD-ROM was created on Nov. 3, 2006, and is identified as “14174-127001.txt.” The file contains 217 KB of data. Three identical copies of the sequence listing have been submitted, including one “Computer-Readable Format” (CRF) and two “Official Copies” (Copy 1 and Copy 2).
FIELD OF THE INVENTION
This invention relates to double-stranded ribonucleic acid (dsRNA), and its use in mediating RNA interference to inhibit the expression of the Nav1.8 gene and the use of the dsRNA to treat pain.
BACKGROUND OF THE INVENTION
Neuropathic pain can be classified as peripheral and central neuropathic pain. Peripheral neuropathic pain is caused by injury or infection of peripheral sensory nerves, whereas central neuropathic pain is caused by damage to the CNS and/or the spinal cord. Both peripheral and central neuropathic pain can occur without obvious initial nerve damage.
A similar definition is given by the International Association for the Study of Pain (IASP, Seattle, Wash., USA): peripheral neuropathic pain is pain initiated or caused by a primary lesion or dysfunction in the peripheral nervous system. Central neuropathic pain is pain initiated or caused by a primary lesion or dysfunction in the central nervous system.
Peripheral lesions may be lesions of perpherial nerves, e.g. diabetic neuropathy, drug-inducted neuropathy, e.g. upon chemotherapy, lesions of nerve roots and posterior ganglia, e.g. post-herpetic neuralgia or nerve root avulsions, neuropathic cancer pain due to compression of peripheral nerves, nerve plexuses and nerve roots, etc. Central lesions may be lesions due to infarction, compressive tumors or abscesses, e.g. in the brainstem, or may be spinal cord lesions due to injury or operations (Jain K K, Emerging Drugs, 2000, 5:241-257; McQuay, 2002, European Journal of Pain 6 (Suppl. A): 11-18).
The above examples of peripheral and central neuropathic pain demonstrate that peripheral and central neuropathic pain are distinguished not only by the anatomical location of the lesion or dysfunction, but they also demonstrate that peripheral and central neuropathic pain can be distinguished by its mechanisms (McQuay, supra). Consequently, there is no clear relation between drug action mechanism and the effect in distinct pain conditions or for single drug classes and different pain conditions (Sindrup S H, Jensen T S, Pain 1999, 83:389-400).
Common analgesics like opioids and non-steroidal anti-inflammatory drugs (NSAIDs) improve only insufficiently chronic abnormal pain syndromes as peripheral and central neuropathic pain due to insufficient efficacy and/or dose-limiting side effects. In the search for alternative treatment regimes to produce satisfactory and sustained pain relief, corticosteroids, conduction blockade, glycerol, anti-convulsants, anti-arrhythmics, antidepressants, local anesthetics, topical agents such as capsaicin, gangliosides and electrostimulation have been tried, but only a subset of patients with neuropathic pain respond to such treatments and typically, significant pain remains even in these responders. The critical issue with current therapies is the therapeutic window; a particular treatment might have potential for efficacy but the patients are not ‘treated to effect’ because of limiting side effects upon dose escalation.
Central neuropathic pain is a form of neuropathic pain which is a particularly difficult form to be treated (Yezierski and Burchiel, 2002). Due to lesions in the spinothalamocortical pathways, ectopic neuronal discharges can occur in different neurons of the spinal cord and brain. Hyperexcitability in damaged areas of the central nervous system plays a major role in the development of central neuropathic pain. Patients with central neuropathic pain almost always have stimulus-independent pain. In addition, in the case of spinal cord injury, for example, stimulus-dependent pain may be present, usually because skin areas or viscera below the lesions are allodynic. Thus, partial spinal lesions may tend to produce pain to a greater extent than do complete lesions.
Other accepted forms of central neuropathic pain or diseases associated with central neuropathic pain exist. Examples include inflammatory CNS diseases such as multiple sclerosis, myelitis or syphilis, ischemia, hemorrhages or asteriovenous malformations (e.g. post-stroke neuropathic pain) located in the thalamus, spinothalamic pathway or thalamocortical projections, and syrnigomyelia (Koltzenburg, Pain 2002—An Updated Review: Refresher Course Syllabus; IASP Press, Seattle, 2002).
Na+-channels are central to the generation of action potentials in all excitable cells such as neurons and myocytes. As such they play key roles in a variety of disease states such as pain (See, Waxman, S. G., S. Dib-Hajj, et al. (1999) “Sodium channels and pain” Proc Natl Acad Sci USA 96(14): 7635-9 and Waxman, S. G., T. R. Cummins, et al. (2000) “Voltage-gated sodium channels and the molecular pathogenesis of pain: a review” J Rehabil Res Dev 37(5): 517-28). Three members of the gene family (NaV1.8, 1.9, 1.5) are resistant to block by the well-known Na channel blocker TTX, demonstrating subtype specificity within this gene family. Mutational analysis has identified glutamate 387 as a critical residue for TTX binding (See, Noda, M., H. Suzuki, et al. (1989) “A single point mutation confers tetrodotoxin and saxitoxin insensitivity on the sodium channel II” FEBS Lett 259(1): 213-6).
In general, voltage-gated sodium channels (NaVs) are responsible for initiating the rapid upstroke of action potentials in excitable tissue in nervous system, which transmit the electrical signals that compose and encode normal and aberrant pain sensations. Antagonists of NaV channels can attenuate these pain signals and are useful for treating a variety of pain conditions, including but not limited to acute, chronic, inflammatory, and neuropathic pain. Known NaV antagonists include TTX, lidocaine (See Mao, J. and L. L. Chen (2000) “Systemic lidocaine for neuropathic pain relief” Pain 87(1): 7-17.) bupivacaine, carbamazepine, mexilitene, phenytoin (See Jensen, T. S. (2002) “Anticonvulsants in neuropathic pain: rationale and clinical evidence” Eur J Pain 6 (Suppl A): 61-8), and lamotrigine (See Rozen, T. D. (2001) “Antiepileptic drugs in the management of cluster headache and trigeminal neuralgia” Headache 41 Suppl 1:S25-32 and Jensen, T. S. (2002). However, side effects include dizziness, somnolence, nausea and vomiting (See Tremont-Lukats, I. W., C. Megeff, and M. M. Backonja (2000) “Anticonvulsants for neuropathic pain syndromes: mechanisms of action and place in therapy” Drugs 60:1029-1052) that limit the utility of these known NaV antagonists for the treatment of pain. These side effects are thought to result at least in part from the block of multiple NaV subtypes. An agent that inhibits the NaV1.8 channel selectively would provide a much great therapeutic window than these non-selective, known NaV antagonists.
The detection and transmission of nociceptive stimuli is mediated by small sensory neurons. Immunohistochemical, in-situ hybridization and electrophysiology experiments have all shown that the sodium channel NaV1.8 is selectively localized to the small sensory neurons of the dorsal root ganglion and trigeminal ganglion (see Akopian, A. N., L. Sivilotti, et al. (1996) “A tetrodotoxin-resistant voltage-gated sodium channel expressed by sensory neurons” Nature 379(6562): 257-62.). After experimental nerve injury, immunohistochemical data demonstrated an accumulation of Nav1.8 at the site of nerve injury, concomitant with an upregulation in TTX-resistant sodium current, consistent with NaV1.8 as a mechanism underlying hyperalgesia (see Gold, M. S., D. Weinreich, et al. (2003) “Redistribution of Nav1.8 in uninjured axons enables neuropathic pain” J. Neurosci. 23:158-166). Attenuation of NaV1.8 expression with antisense oligodeoxynucleotides administered by intrathecal injection prevented experimental nerve-injury induced redistribution of Nav1.8 in the sciatic nerve and reversed neuropathic pain (tactile and thermal hyperalgesia), demonstrating a causal role of Nav1.8 in nerve-injury induced pain (see also Lai, J., M. S. Gold, et al. (2002) “Inhibition of neuropathic pain by decreased expression of the tetrodotoxin-resistant sodium channel, NaV1.8” Pain 95(1-2): 143-52, and Lai, J., J. C. Hunter, et al. (2000) “Blockade of neuropathic pain by antisense targeting of tetrodotoxin-resistant sodium channels in sensory neurons” Methods Enzymol 314: 201-13.). In inflammatory pain models, intrathecal administration of antisense oligodeoxynucleotides against NaV1.8 resulted in a significant reduction in PGE2-induced hyperalgesia (see Khasar, S. G., M. S. Gold, et al. (1998) “A tetrodotoxin-resistant sodium current mediates inflammatory pain in the rat” Neurosci Lett 256(1): 17-20) and in CFA (complete Freund's adjuvant)—induced hyperalgesia (Porreca, F., J. Lai, et al. (1999) “A comparison of the potential role of the tetrodotoxin-insensitive sodium channels, PN3/SNS and NaN/SNS2, in rat models of chronic pain” Proc. Nat'l. Acad. Sci. 96: 7640-7644). In addition, in a rat model of visceral pain, induced by intravesical infusion of acetic acid, bladder hyperactivity was reduced by intrathecal injection of antisense oligodeoxynucleotides against NaV1.8, showing that Nav1.8 contributes to the activation of sensory nerves in visceral pain (Yoshimura, N., S. Seki, et al. (2001) “The involvement of the tetrodotoxin-resistant sodium channel Nav1.8 (PN3/SNS) in a rat model of visceral pain” J. Neurosci. 21: 8690-8696).
Taken together, these data support a role for NaV1.8 in the detection and transmission of inflammatory and neuropathic pain.
Recently, double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). WO 99/32619 (Fire et al.) discloses the use of a dsRNA of at least 25 nucleotides in length to inhibit the expression of the Nav1.8 gene in C. elegans. dsRNA has also been shown to degrade target RNA in other organisms, including plants (see, e.g., WO 99/53050, Waterhouse et al.; and WO 99/61631, Heifetz et al0.), Drosophila (see, e.g., Yang, D., et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895, Limmer; and DE 101 00 586.5, Kreutzer et al.). This natural mechanism has now become the focus for the development of a new class of pharmaceutical agents for treating disorders that are caused by the aberrant or unwanted regulation of a gene.
Despite significant advances in the field of RNAi and advances in the treatment of pain, there remains a need for an agent that can selectively and efficiently silence the Nav1.8 gene using the cell's own RNAi machinery that has both high biological activity and in vivo stability, and that can effectively inhibit expression of a target Nav1.8 gene for use in treating pain.
SUMMARY OF THE INVENTION
The invention provides double-stranded ribonucleic acid (dsRNA), as well as compositions and methods for inhibiting the expression of the Nav1.8 gene in a cell or mammal using such dsRNA. The invention also provides compositions and methods for treating pathological conditions and diseases caused by the expression of the Nav1.8 gene, such as in the propagation of pain signals in neuropathic and inflammatory pain. The dsRNA of the invention comprises an RNA strand (the antisense strand) having a region which is less than 30 nucleotides in length and is substantially complementary to at least part of an mRNA transcript of the Nav1.8 gene.
In one embodiment, the invention provides double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of the Nav1.8 gene. The dsRNA comprises at least two sequences that are complementary to each other. The dsRNA comprises a sense strand comprising a first sequence and an antisense strand comprising a second sequence. The antisense strand comprises a nucleotide sequence which is substantially complementary to at least part of an mRNA encoding Nav1.8, and the region of complementarity is less than 30 nucleotides in length. The dsRNA, upon contacting with a cell expressing the Nav1.8, inhibits the expression of the Nav1.8 gene by at least 20%.
For example, the dsRNA molecules of the invention can be comprised of a first sequence of the dsRNA that is selected from the group consisting of the sense sequences of Tables 1, 4 and 6 and the second sequence is selected from the group consisting of the antisense sequences of Tables 1, 4 and 6. The dsRNA molecules of the invention can be comprised of naturally occurring nucleotides or can be comprised of at least one modified nucleotide, such as a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group. Alternatively, the modified nucleotide may be chosen from the group of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide. Preferably, the first sequence of said dsRNA is selected from the group consisting of the sense sequences of Tables 1, 4 and 6 and the second sequence is selected from the group consisting of the antisense sequences of Tables 1, 4 and 6.
In another embodiment, the invention provides a cell comprising one of the dsRNAs of the invention. The cell is preferably a mammalian cell, such as a human cell.
In another embodiment, the invention provides a pharmaceutical composition for inhibiting the expression of the Nav1.8 gene in an organism, comprising one or more of the dsRNA of the invention and a pharmaceutically acceptable carrier.
In another embodiment, the invention provides a method for inhibiting the expression of the Nav1.8 gene in a cell, comprising the following steps:

    
    
        (a) introducing into the cell a double-stranded ribonucleic acid (dsRNA), wherein the dsRNA comprises at least two sequences that are complementary to each other. The dsRNA comprises a sense strand comprising a first sequence and an antisense strand comprising a second sequence. The antisense strand comprises a region of complementarity which is substantially complementary to at least a part of an mRNA encoding Nav1.8, and wherein the region of complementarity is less than 30 nucleotides in length and wherein the dsRNA, upon contact with a cell expressing the Nav1.8, inhibits expression of the Nav1.8 gene by at least 20%; and
        (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the Nav1.8 gene, thereby inhibiting expression of the Nav1.8 gene in the cell.
    
    


In another embodiment, the invention provides methods for treating, preventing or managing pain comprising administering to a patient in need of such treatment, prevention or management a therapeutically or prophylactically effective amount of one or more of the dsRNAs of the invention.
In another embodiment, the invention provides vectors for inhibiting the expression of the Nav1.8 gene in a cell, comprising a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of one of the dsRNA of the invention.
In another embodiment, the invention provides a cell comprising a vector for inhibiting the expression of the Nav1.8 gene in a cell. The vector comprises a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of one of the dsRNA of the invention.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. In vitro activity of the dsRNAs provided in Table 1 against mRNA expression of transfected human Nav1.8 in Cos-7 cells.
FIG. 2. Lack of cross reactivity of Nav1.8 dsRNAs to endogenous Nav1.5 mRNA in SW620 cells in vitro.
FIG. 3. In vitro activity of the dsRNAs provided in Table 1 against endogenous Nav1.8 mRNA in primary cultures of rat dorsal root ganglion cells.
FIG. 4. Dose response of dsRNA AL-DP-6209 against endogenous Nav1.8 mRNA in primary cultures of dorsal root ganglion cells
FIG. 5. In vivo efficacy of dsRNA AL-DP-6209 with iFECT against complete Freund's adjuvant—induced tactile hyperalgesia in rats.
FIG. 6. In vivo efficacy of dsRNA AL-DP-4459 and AL-DP-4461 against complete Freund's adjuvant—induced tactile hyperalgesia in rats.
FIG. 7. In vivo efficacy of dsRNA AL-DP-6050 against complete Freund's adjuvant—induced tactile hyperalgesia in rats.
FIG. 8. In vivo efficacy of dsRNAs AL-DP-6050 and AL-DP-4459 against complete Freund's adjuvant—induced thermal hyperalgesia in rats.
FIG. 9. In vivo efficacy of dsRNA AL-DP-4459 against spinal nerve ligation—induced tactile and thermal hyperalgesia in rats.
FIG. 10. Stability of unconjugated dsRNAs AL-DP-6050, AL-DP-6209, AL-DP-6217, AL-DP-6218 and AL-DP-6219 in human cerebrospinal fluid at 37° C.
FIG. 11. Stability of cholesterol-conjugated dsRNA AL-DP-4459 in human cerebrospinal fluid at 37° C.
FIG. 12. Dose response of siRNA AL-DP-6050 and its cholesterol conjugate AL-DP-4459 against mRNA expression of endogenous Nav1.8 in primary cultures of rat dorsal root ganglion cells.
FIG. 13. In vivo efficacy of dsRNA AL-DP-6050 against spinal nerve ligation—induced thermal hyperalgesia in rats.
FIG. 14. In vivo efficacy of ND98-2.7 liposomal formulation of dsRNA AL-DP-6050 against spinal nerve ligation—induced thermal hyperalgesia in rats.
FIG. 15. Structure of ND98 lipid



DETAILED DESCRIPTION OF THE INVENTION
The invention provides double-stranded ribonucleic acid (dsRNA), as well as compositions and methods for inhibiting the expression of the Nav1.8 gene in a cell or mammal using the dsRNA. The invention also provides compositions and methods for treating pathological conditions and diseases in a mammal caused by the expression of the Nav1.8 gene using dsRNA. dsRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). The process occurs in a wide variety of organisms, including mammals and other vertebrates.
The dsRNA of the invention comprises an RNA strand (the antisense strand) having a region which is less than 30 nucleotides in length and which is substantially complementary to at least part of an mRNA transcript of the Nav1.8 gene. The use of these dsRNAs enables the targeted degradation of mRNAs of genes that are implicated in pain response in mammals. Using cell-based and animal assays, the present inventors have demonstrated that very low dosages of these dsRNA can specifically and efficiently mediate RNAi, resulting in significant inhibition of expression of the Nav1.8 gene. Thus, the methods and compositions of the invention comprising these dsRNAs are useful for treating pain.
The following detailed description discloses how to make and use the dsRNA and compositions containing dsRNA to inhibit the expression of a target Nav1.8 gene, as well as compositions and methods for treating diseases and disorders caused by the expression of Nav1.8, such as neuropathic and inflammatory pain. The pharmaceutical compositions of the invention comprise a dsRNA having an antisense strand comprising a region of complementarity which is less than 30 nucleotides in length and is substantially complementary to at least part of an RNA transcript of the Nav1.8 gene, together with a pharmaceutically acceptable carrier.
Accordingly, certain aspects of the invention provide pharmaceutical compositions comprising the dsRNA of the invention together with a pharmaceutically acceptable carrier, methods of using the compositions to inhibit expression of the Nav1.8 gene, and methods of using the pharmaceutical compositions to treat diseases caused by expression of the Nav1.8 gene.
I. Definitions
For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definition in this section shall prevail.
“G,” “C,” “A” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, and uracil as a base, respectively. However, it will be understood that the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety. The skilled person is well aware that guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of the invention by a nucleotide containing, for example, inosine. Sequences comprising such replacement moieties are embodiments of the invention.
By “Nav1.8” as used herein is meant, any Nav1.8 protein, peptide, or polypeptide associated with the development or maintenance of an ion channel. The terms “Nav1.8” also refer to nucleic acid sequences encoding any Nav1.8 protein, peptide, or polypeptide. For the Examples, the Nav1.8 mRNA sequences used were human (NM—006514), mouse (NM—009134), rat (NM—017247) and dog (NM001003203) mRNA sequences.
As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of the Nav1.8 gene, including mRNA that is a product of RNA processing of a primary transcription product.
As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.
As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.
This includes base-pairing of the oligonucleotide or polynucleotide comprising the first nucleotide sequence to the oligonucleotide or polynucleotide comprising the second nucleotide sequence over the entire length of the first and second nucleotide sequence. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they may form one or more, but preferably not more than 4, 3 or 2 mismatched base pairs upon hybridization, while retaining the ability to hybridize under the conditions most relevant to their ultimate application. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as “fully complementary” for the purposes of the invention.
“Complementary” sequences, as used herein, may also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled.
The terms “complementary”, “fully complementary” and “substantially complementary” herein may be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as will be understood from the context of their use.
As used herein, a polynucleotide which is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide which is substantially complementary to a contiguous portion of the mRNA of interest (e.g., encoding Nav1.8). For example, a polynucleotide is complementary to at least a part of a Nav1.8 mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding Nav1.8.
The term “double-stranded RNA” or “dsRNA”, as used herein, refers to a ribonucleic acid molecule, or complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary, as defined above, nucleic acid strands. The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′ end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop”. Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′ end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker”. The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA. In addition to the duplex structure, a dsRNA may comprise one or more nucleotide overhangs.
As used herein, a “nucleotide overhang” refers to the unpaired nucleotide or nucleotides that protrude from the duplex structure of a dsRNA when a 3′-end of one strand of the dsRNA extends beyond the 5′-end of the other strand, or vice versa. “Blunt” or “blunt end” means that there are no unpaired nucleotides at that end of the dsRNA, i.e., no nucleotide overhang. A “blunt ended” dsRNA is a dsRNA that is double-stranded over its entire length, i.e., no nucleotide overhang at either end of the molecule.
The term “antisense strand” refers to the strand of a dsRNA which includes a region that is substantially complementary to a target sequence. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches are most tolerated in the terminal regions and, if present, are preferably in a terminal region or regions, e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus.
The term “sense strand,” as used herein, refers to the strand of a dsRNA that includes a region that is substantially complementary to a region of the antisense strand.
“Introducing into a cell”, when referring to a dsRNA, means facilitating uptake or absorption into the cell, as is understood by those skilled in the art. Absorption or uptake of dsRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; a dsRNA may also be “introduced into a cell”, wherein the cell is part of a living organism. In such instance, introduction into the cell will include the delivery to the organism. For example, for in vivo delivery, dsRNA can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection.
The terms “silence” and “inhibit the expression of”, in as far as they refer to the Nav1.8 gene, herein refer to the at least partial suppression of the expression of the Nav1.8 gene, as manifested by a reduction of the amount of mRNA transcribed from the Nav1.8 gene which may be isolated from a first cell or group of cells in which the Nav1.8 gene is transcribed and which has or have been treated such that the expression of the Nav1.8 gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells). The degree of inhibition is usually expressed in terms of




  
    
      
        
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Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to Nav1.8 gene transcription, e.g. the amount of protein encoded by the Nav1.8 gene which is secreted by a cell, or the number of cells displaying a certain phenotype, e.g apoptosis. In principle, Nav1.8 gene silencing may be determined in any cell expressing the target, either constitutively or by genomic engineering, and by any appropriate assay. However, when a reference is needed in order to determine whether a given siRNA inhibits the expression of the Nav1.8 gene by a certain degree and therefore is encompassed by the instant invention, the assay provided in the Examples below shall serve as such reference.
For example, in certain instances, expression of the Nav1.8 gene is suppressed by at least about 20%, 25%, 35%, or 50% by administration of the double-stranded oligonucleotide of the invention. In a preferred embodiment, the Nav1.8 gene is suppressed by at least about 60%, 70%, or 80% by administration of the double-stranded oligonucleotide of the invention. In a more preferred embodiment, the Nav1.8 gene is suppressed by at least about 85%, 90%, or 95% by administration of the double-stranded oligonucleotide of the invention. In a most preferred embodiment, the Nav1.8 gene is suppressed by at least about 98%, 99% or more by administration of the double-stranded oligonucleotide of the invention.
The terms “treat”, “treatment”, and the like, refer to relief from or alleviation of the perception of pain, including the relief from or alleviation of the intensity and/or duration of a pain (e.g., burning sensation, tingling, electric-shock-like feelings, etc.) experienced by a subject in response to a given stimulus (e.g., pressure, tissue injury, cold temperature, etc.). Relief from or alleviation of the perception of pain can be any detectable decrease in the intensity or duration of pain. Treatment can occur in a subject (e.g., a human or companion animal) suffering from a pain condition or having one or more symptoms of a pain-related disorder that can be treated according to the present invention, or in an animal model of pain, such as the SNL rat model of neuropathic pain or CFA rat model of chronic pain described herein, or another animal model of pain. In the context of the present invention insofar as it relates to any of the other conditions recited herein below (other than pain), the terms “treat”, “treatment”, and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition.
As used herein, the phrases “therapeutically effective amount” and “prophylactically effective amount” refer to an amount that provides a therapeutic benefit in the treatment, prevention, or management of pain or an overt symptom of pain. The specific amount that is therapeutically effective can be readily determined by ordinary medical practitioner, and may vary depending on factors known in the art, such as, e.g. the type of pain, the patient's history and age, the stage of pain, and the administration of other anti-pain agents.
As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of a dsRNA and a pharmaceutically acceptable carrier. As used herein, “pharmacologically effective amount,” “therapeutically effective amount” or simply “effective amount” refers to that amount of an RNA effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 25% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 25% reduction in that parameter.
The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.
As used herein, a “transformed cell” is a cell into which a vector has been introduced from which a dsRNA molecule may be expressed.
II. Double-Stranded Ribonucleic Acid (dsRNA)
In one embodiment, the invention provides double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of the Nav1.8 gene in a cell or mammal, wherein the dsRNA comprises an antisense strand comprising a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of the Nav1.8 gene, and wherein the region of complementarity is less than 30 nucleotides in length and wherein said dsRNA, upon contact with a cell expressing said Nav1.8 gene, inhibits the expression of said Nav1.8 gene by at least 20%. The dsRNA comprises two RNA strands that are sufficiently complementary to hybridize to form a duplex structure. One strand of the dsRNA (the antisense strand) comprises a region of complementarity that is substantially complementary, and preferably fully complementary, to a target sequence, derived from the sequence of an mRNA formed during the expression of the Nav1.8 gene, the other strand (the sense strand) comprises a region which is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. Preferably, the duplex structure is between 15 and 30, more preferably between 18 and 25, yet more preferably between 19 and 24, and most preferably between 21 and 23 base pairs in length. Similarly, the region of complementarity to the target sequence is between 15 and 30, more preferably between 18 and 25, yet more preferably between 19 and 24, and most preferably between 21 and 23 nucleotides in length. The dsRNA of the invention may further comprise one or more single-stranded nucleotide overhang(s). The dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc. In a preferred embodiment, the Nav1.8 gene is the human Nav1.8 gene. In specific embodiments, the antisense strand of the dsRNA comprises the antisense sequences of Tables 1, 4 and 6 and the second sequence is selected from the group consisting of the sense sequences of Tables 1, 4 and 6.
In further embodiments, the dsRNA comprises at least one nucleotide sequence selected from the groups of sequences provided in Tables 1, 4 and 6. In other embodiments, the dsRNA comprises at least two sequences selected from this group, wherein one of the at least two sequences is complementary to another of the at least two sequences, and one of the at least two sequences is substantially complementary to a sequence of an mRNA generated in the expression of the Nav1.8 gene. Preferably, the dsRNA comprises two oligonucleotides, wherein one oligonucleotide is described by Tables 1, 4 and 6 and the second oligonucleotide is described by Tables 1, 4 and 6.
The skilled person is well aware that dsRNAs comprising a duplex structure of between 20 and 23, but specifically 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer dsRNAs can be effective as well. In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in Tables 1, 4 and 6, the dsRNAs of the invention can comprise at least one strand of a length of minimally 21 nt. It can be reasonably expected that shorter dsRNAs comprising one of the sequences of Tables 1, 4 and 6 minus only a few nucleotides on one or both ends may be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs comprising a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences of Tables 1, 4 and 6, and differing in their ability to inhibit the expression of the Nav1.8 gene in a FACS assay as described herein below by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated by the invention.
The dsRNA of the invention can contain one or more mismatches to the target sequence. In a preferred embodiment, the dsRNA of the invention contains more than 3 mismatches. If the antisense strand of the dsRNA contains mismatches to a target sequence, it is preferable that the area of mismatch not be located in the center of the region of complementarity. If the antisense strand of the dsRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to 5 nucleotides from either end, for example 5, 4, 3, 2, or 1 nucleotide from either the 5′ or 3′ end of the region of complementarity. For example, for a 23 nucleotide dsRNA strand which is complementary to a region of the Nav1.8 gene, the dsRNA preferably does not contain any mismatch within the central 13 nucleotides. The methods described within the invention can be used to determine whether a dsRNA containing a mismatch to a target sequence is effective in inhibiting the expression of the Nav1.8 gene. Consideration of the efficacy of dsRNAs with mismatches in inhibiting expression of the Nav1.8 gene is important, especially if the particular region of complementarity in the Nav1.8 gene is known to have polymorphic sequence variation within the population.
In one embodiment, at least one end of the dsRNA has a single-stranded nucleotide overhang of 1 to 4, preferably 1 or 2 nucleotides. dsRNAs having at least one nucleotide overhang have unexpectedly superior inhibitory properties than their blunt-ended counterparts. Moreover, the present inventors have discovered that the presence of only one nucleotide overhang strengthens the interference activity of the dsRNA, without affecting its overall stability. dsRNA having only one overhang has proven particularly stable and effective in vivo, as well as in a variety of cells, cell culture mediums, blood, and serum. Preferably, the single-stranded overhang is located at the 3′-terminal end of the antisense strand or, alternatively, at the 3′-terminal end of the sense strand. The dsRNA may also have a blunt end, preferably located at the 5′-end of the antisense strand. Such dsRNAs have improved stability and inhibitory activity, thus allowing administration at low dosages, i.e., less than 5 mg/kg body weight of the recipient per day. Preferably, the antisense strand of the dsRNA has a nucleotide overhang at the 3′-end, and the 5′-end is blunt. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.
In yet another embodiment, the dsRNA is chemically modified to enhance stability. The nucleic acids of the invention may be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry”, Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Chemical modifications may include, but are not limited to 2′ modifications, introduction of non-natural bases, covalent attachment to a ligand, and replacement of phosphate linkages with thiophosphate linkages. In this embodiment, the integrity of the duplex structure is strengthened by at least one, and preferably two, chemical linkages. Chemical linking may be achieved by any of a variety of well-known techniques, for example by introducing covalent, ionic or hydrogen bonds; hydrophobic interactions, van der Waals or stacking interactions; by means of metal-ion coordination, or through use of purine analogues. Preferably, the chemical groups that can be used to modify the dsRNA include, without limitation, methylene blue; bifunctional groups, preferably bis-(2-chloroethyl)amine; N-acetyl-N′-(p-glyoxylbenzoyl)cystamine; 4-thiouracil; and psoralen. In one preferred embodiment, the linker is a hexa-ethylene glycol linker. In this case, the dsRNA are produced by solid phase synthesis and the hexa-ethylene glycol linker is incorporated according to standard methods (e.g., Williams, D. J., and K. B. Hall, Biochem. (1996) 35:14665-14670). In a particular embodiment, the 5′-end of the antisense strand and the 3′-end of the sense strand are chemically linked via a hexaethylene glycol linker. In another embodiment, at least one nucleotide of the dsRNA comprises a phosphorothioate or phosphorodithioate groups. The chemical bond at the ends of the dsRNA is preferably formed by triple-helix bonds. Table 1 provides examples of modified RNAi agents of the invention.
In certain embodiments, a chemical bond may be formed by means of one or several bonding groups, wherein such bonding groups are preferably poly-(oxyphosphinicooxy-1,3-propandiol)- and/or polyethylene glycol chains. In other embodiments, a chemical bond may also be formed by means of purine analogs introduced into the double-stranded structure instead of purines. In further embodiments, a chemical bond may be formed by azabenzene units introduced into the double-stranded structure. In still further embodiments, a chemical bond may be formed by branched nucleotide analogs instead of nucleotides introduced into the double-stranded structure. In certain embodiments, a chemical bond may be induced by ultraviolet light.
In yet another embodiment, the nucleotides at one or both of the two single strands may be modified to prevent or inhibit the activation of cellular enzymes, such as, for example, without limitation, certain nucleases. Techniques for inhibiting the activation of cellular enzymes are known in the art including, but not limited to, 2′-amino modifications, 2′-fluoro modifications, 2′-alkyl modifications, uncharged backbone modifications, morpholino modifications, 2′-O-methyl modifications, and phosphoramidate (see, e.g., Wagner, Nat. Med. (1995) 1:1116-8). Thus, at least one 2′-hydroxyl group of the nucleotides on a dsRNA is replaced by a chemical group, preferably by a 2′-fluoro or a 2′-O-methyl group. Also, at least one nucleotide may be modified to form a locked nucleotide. Such locked nucleotide contains a methylene or ethylene bridge that connects the 2′-oxygen of ribose with the 4′-carbon of ribose. Oligonucleotides containing the locked nucleotide are described in Koshkin, A. A., et al., Tetrahedron (1998), 54: 3607-3630) and Obika, S. et al., Tetrahedron Lett. (1998), 39: 5401-5404). Introduction of a locked nucleotide into an oligonucleotide improves the affinity for complementary sequences and increases the melting temperature by several degrees (Braasch, D. A. and D. R. Corey, Chem. Biol. (2001), 8:1-7).
Conjugating a ligand to a dsRNA can enhance its cellular absorption. In certain instances, a hydrophobic ligand is conjugated to the dsRNA to facilitate direct permeation of the cellular membrane. Alternatively, the ligand conjugated to the dsRNA is a substrate for receptor-mediated endocytosis. These approaches have been used to facilitate cell permeation of antisense oligonucleotides. For example, cholesterol has been conjugated to various antisense oligonucleotides resulting in compounds that are substantially more active compared to their non-conjugated analogs. See M. Manoharan Antisense & Nucleic Acid Drug Development 2002, 12, 103. Other lipophilic compounds that have been conjugated to oligonucleotides include 1-pyrene butyric acid, 1,3-bis-O-(hexadecyl)glycerol, and menthol. One example of a ligand for receptor-mediated endocytosis is folic acid. Folic acid enters the cell by folate-receptor-mediated endocytosis. dsRNA compounds bearing folic acid would be efficiently transported into the cell via the folate-receptor-mediated endocytosis. Li and coworkers report that attachment of folic acid to the 3′-terminus of an oligonucleotide resulted in an 8-fold increase in cellular uptake of the oligonucleotide. Li, S.; Deshmukh, H. M.; Huang, L. Pharm. Res. 1998, 15, 1540. Other ligands that have been conjugated to oligonucleotides include polyethylene glycols, carbohydrate clusters, cross-linking agents, porphyrin conjugates, and delivery peptides.
In certain instances, conjugation of a cationic ligand to oligonucleotides often results in improved resistance to nucleases. Representative examples of cationic ligands are propylammonium and dimethylpropylammonium. Interestingly, antisense oligonucleotides were reported to retain their high binding affinity to mRNA when the cationic ligand was dispersed throughout the oligonucleotide. See M. Manoharan Antisense & Nucleic Acid Drug Development 2002, 12, 103 and references therein.
The ligand-conjugated dsRNA of the invention may be synthesized by the use of a dsRNA that bears a pendant reactive functionality, such as that derived from the attachment of a linking molecule onto the dsRNA. This reactive oligonucleotide may be reacted directly with commercially-available ligands, ligands that are synthesized bearing any of a variety of protecting groups, or ligands that have a linking moiety attached thereto. The methods of the invention facilitate the synthesis of ligand-conjugated dsRNA by the use of; in some preferred embodiments, nucleoside monomers that have been appropriately conjugated with ligands and that may further be attached to a solid-support material. Such ligand-nucleoside conjugates, optionally attached to a solid-support material, are prepared according to some preferred embodiments of the methods of the invention via reaction of a selected serum-binding ligand with a linking moiety located on the 5′ position of a nucleoside or oligonucleotide. In certain instances, an dsRNA bearing an aralkyl ligand attached to the 3′-terminus of the dsRNA is prepared by first covalently attaching a monomer building block to a controlled-pore-glass support via a long-chain aminoalkyl group. Then, nucleotides are bonded via standard solid-phase synthesis techniques to the monomer building-block bound to the solid support. The monomer building block may be a nucleoside or other organic compound that is compatible with solid-phase synthesis.
The dsRNA used in the conjugates of the invention may be conveniently and routinely made through the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is also known to use similar techniques to prepare other oligonucleotides, such as the phosphorothioates and alkylated derivatives.
Teachings regarding the synthesis of particular modified oligonucleotides may be found in the following U.S. patents: U.S. Pat. Nos. 5,138,045 and 5,218,105, drawn to polyamine conjugated oligonucleotides; U.S. Pat. No. 5,212,295, drawn to monomers for the preparation of oligonucleotides having chiral phosphorus linkages; U.S. Pat. Nos. 5,378,825 and 5,541,307, drawn to oligonucleotides having modified backbones; U.S. Pat. No. 5,386,023, drawn to backbone-modified oligonucleotides and the preparation thereof through reductive coupling; U.S. Pat. No. 5,457,191, drawn to modified nucleobases based on the 3-deazapurine ring system and methods of synthesis thereof; U.S. Pat. No. 5,459,255, drawn to modified nucleobases based on N-2 substituted purines; U.S. Pat. No. 5,521,302, drawn to processes for preparing oligonucleotides having chiral phosphorus linkages; U.S. Pat. No. 5,539,082, drawn to peptide nucleic acids; U.S. Pat. No. 5,554,746, drawn to oligonucleotides having β-lactam backbones; U.S. Pat. No. 5,571,902, drawn to methods and materials for the synthesis of oligonucleotides; U.S. Pat. No. 5,578,718, drawn to nucleosides having alkylthio groups, wherein such groups may be used as linkers to other moieties attached at any of a variety of positions of the nucleoside; U.S. Pat. Nos. 5,587,361 and 5,599,797, drawn to oligonucleotides having phosphorothioate linkages of high chiral purity; U.S. Pat. No. 5,506,351, drawn to processes for the preparation of 2′-O-alkyl guanosine and related compounds, including 2,6-diaminopurine compounds; U.S. Pat. No. 5,587,469, drawn to oligonucleotides having N-2 substituted purines; U.S. Pat. No. 5,587,470, drawn to oligonucleotides having 3-deazapurines; U.S. Pat. No. 5,223,168, and U.S. Pat. No. 5,608,046, both drawn to conjugated 4′-desmethyl nucleoside analogs; U.S. Pat. Nos. 5,602,240, and 5,610,289, drawn to backbone-modified oligonucleotide analogs; U.S. Pat. Nos. 6,262,241, and 5,459,255, drawn to, inter alia, methods of synthesizing 2′-fluoro-oligonucleotides.
In the ligand-conjugated dsRNA and ligand-molecule bearing sequence-specific linked nucleosides of the invention, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already bear the linking moiety, ligand-nucleotide or nucleoside-conjugate precursors that already bear the ligand molecule, or non-nucleoside ligand-bearing building blocks.
When using nucleotide-conjugate precursors that already bear a linking moiety, the synthesis of the sequence-specific linked nucleosides is typically completed, and the ligand molecule is then reacted with the linking moiety to form the ligand-conjugated oligonucleotide. Oligonucleotide conjugates bearing a variety of molecules such as steroids, vitamins, lipids and reporter molecules, has previously been described (see Manoharan et al., PCT Application WO 93/07883). In a preferred embodiment, the oligonucleotides or linked nucleosides of the invention are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates in addition to the standard phosphoramidites and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.
The incorporation of a 2′-O-methyl, 2′-O-ethyl, 2′-O-propyl, 2′-O-allyl, 2′-O-aminoalkyl or 2′-deoxy-2′-fluoro group in nucleosides of an oligonucleotide confers enhanced hybridization properties to the oligonucleotide. Further, oligonucleotides containing phosphorothioate backbones have enhanced nuclease stability. Thus, functionalized, linked nucleosides of the invention can be augmented to include either or both a phosphorothioate backbone or a 2′-O-methyl, 2′-O-ethyl, 2′-O-propyl, 2′-O-aminoalkyl, 2′-O-allyl or 2′-deoxy-2′-fluoro group.
In some preferred embodiments, functionalized nucleoside sequences of the invention possessing an amino group at the 5′-terminus are prepared using a DNA synthesizer, and then reacted with an active ester derivative of a selected ligand. Active ester derivatives are well known to those skilled in the art. Representative active esters include N-hydrosuccinimide esters, tetrafluorophenolic esters, pentafluorophenolic esters and pentachlorophenolic esters. The reaction of the amino group and the active ester produces an oligonucleotide in which the selected ligand is attached to the 5′-position through a linking group. The amino group at the 5′-terminus can be prepared utilizing a 5′-Amino-Modifier C6 reagent. In a preferred embodiment, ligand molecules may be conjugated to oligonucleotides at the 5′-position by the use of a ligand-nucleoside phosphoramidite wherein the ligand is linked to the 5′-hydroxy group directly or indirectly via a linker. Such ligand-nucleoside phosphoramidites are typically used at the end of an automated synthesis procedure to provide a ligand-conjugated oligonucleotide bearing the ligand at the 5′-terminus.
In one preferred embodiment of the methods of the invention, the preparation of ligand conjugated oligonucleotides commences with the selection of appropriate precursor molecules upon which to construct the ligand molecule. Typically, the precursor is an appropriately-protected derivative of the commonly-used nucleosides. For example, the synthetic precursors for the synthesis of the ligand-conjugated oligonucleotides of the invention include, but are not limited to, 2′-aminoalkoxy-5′-ODMT-nucleosides, 2′-6-aminoalkylamino-5′-ODMT-nucleosides, 5′-6-aminoalkoxy-2′-deoxy-nucleosides, 5′-6-aminoalkoxy-2-protected-nucleosides, 3′-6-aminoalkoxy-5′-ODMT-nucleosides, and 3′-aminoalkylamino-5′-ODMT-nucleosides that may be protected in the nucleobase portion of the molecule. Methods for the synthesis of such amino-linked protected nucleoside precursors are known to those of ordinary skill in the art.
In many cases, protecting groups are used during the preparation of the compounds of the invention. As used herein, the term “protected” means that the indicated moiety has a protecting group appended thereon. In some preferred embodiments of the invention, compounds contain one or more protecting groups. A wide variety of protecting groups can be employed in the methods of the invention. In general, protecting groups render chemical functionalities inert to specific reaction conditions, and can be appended to and removed from such functionalities in a molecule without substantially damaging the remainder of the molecule.
Representative hydroxyl protecting groups, for example, are disclosed by Beaucage et al. (Tetrahedron, 1992, 48:2223-2311). Further hydroxyl protecting groups, as well as other representative protecting groups, are disclosed in Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d ed., John Wiley & Sons, New York, 1991, and Oligonucleotides And Analogues A Practical Approach, Ekstein, F. Ed., IRL Press, N.Y, 1991.
Examples of hydroxyl protecting groups include, but are not limited to, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl, p,p′-dinitrobenzhydryl, p-nitrobenzyl, triphenylmethyl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, benzoylformate, acetate, chloroacetate, trichloroacetate, trifluoroacetate, pivaloate, benzoate, p-phenylbenzoate, 9-fluorenylmethyl carbonate, mesylate and tosylate.
Amino-protecting groups stable to acid treatment are selectively removed with base treatment, and are used to make reactive amino groups selectively available for substitution. Examples of such groups are the Fmoc (E. Atherton and R. C. Sheppard in The Peptides, S. Udenfriend, J. Meienhofer, Eds., Academic Press, Orlando, 1987, volume 9, p. 1) and various substituted sulfonylethyl carbamates exemplified by the Nsc group (Samukov et al., Tetrahedron Lett., 1994, 35:7821; Verhart and Tesser, Rec. Trav. Chim. Pays-Bas, 1987, 107:621).
Additional amino-protecting groups include, but are not limited to, carbamate protecting groups, such as 2-trimethylsilylethoxycarbonyl (Teoc), 1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), t-butoxycarbonyl (BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethyloxycarbonyl (Fmoc), and benzyloxycarbonyl (Cbz); amide protecting groups, such as formyl, acetyl, trihaloacetyl, benzoyl, and nitrophenylacetyl; sulfonamide protecting groups, such as 2-nitrobenzenesulfonyl; and imine and cyclic imide protecting groups, such as phthalimido and dithiasuccinoyl. Equivalents of these amino-protecting groups are also encompassed by the compounds and methods of the invention.
Many solid supports are commercially available and one of ordinary skill in the art can readily select a solid support to be used in the solid-phase synthesis steps. In certain embodiments, a universal support is used. A universal support allows for preparation of oligonucleotides having unusual or modified nucleotides located at the 3′-terminus of the oligonucleotide. Universal Support 500 and Universal Support II are universal supports that are commercially available from Glen Research, 22825 Davis Drive, Sterling, Va. For further details about universal supports see Scott et al., Innovations and Perspectives in solid-phase Synthesis, 3rd International Symposium, 1994, Ed. Roger Epton, Mayflower Worldwide, 115-124]; Azhayev, A. V. Tetrahedron 1999, 55, 787-800; and Azhayev and Antopolsky Tetrahedron 2001, 57, 4977-4986. In addition, it has been reported that the oligonucleotide can be cleaved from the universal support under milder reaction conditions when oligonucleotide is bonded to the solid support via a syn-1,2-acetoxyphosphate group which more readily undergoes basic hydrolysis. See Guzaev, A. I.; Manoharan, M. J. Am. Chem. Soc. 2003, 125, 2380.
The nucleosides are linked by phosphorus-containing or non-phosphorus-containing covalent intemucleoside linkages. For the purposes of identification, such conjugated nucleosides can be characterized as ligand-bearing nucleosides or ligand-nucleoside conjugates. The linked nucleosides having an aralkyl ligand conjugated to a nucleoside within their sequence will demonstrate enhanced dsRNA activity when compared to like dsRNA compounds that are not conjugated.
The aralkyl-ligand-conjugated oligonucleotides of the invention also include conjugates of oligonucleotides and linked nucleosides wherein the ligand is attached directly to the nucleoside or nucleotide without the intermediacy of a linker group. The ligand may preferably be attached, via linking groups, at a carboxyl, amino or oxo group of the ligand. Typical linking groups may be ester, amide or carbamate groups.
Specific examples of preferred modified oligonucleotides envisioned for use in the ligand-conjugated oligonucleotides of the invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined here, oligonucleotides having modified backbones or internucleoside linkages include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of the invention, modified oligonucleotides that do not have a phosphorus atom in their intersugar backbone can also be considered to be oligonucleosides.
Specific oligonucleotide chemical modifications are described below. It is not necessary for all positions in a given compound to be uniformly modified. Conversely, more than one modifications may be incorporated in a single dsRNA compound or even in a single nucleotide thereof.
Preferred modified internucleoside linkages or backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free-acid forms are also included.
Representative United States Patents relating to the preparation of the above phosphorus-atom-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; and 5,697,248, each of which is herein incorporated by reference.
Preferred modified intemucleoside linkages or backbones that do not include a phosphorus atom therein (i.e., oligonucleosides) have backbones that are formed by short chain alkyl or cycloalkyl intersugar linkages, mixed heteroatom and alkyl or cycloalkyl intersugar linkages, or one or more short chain heteroatomic or heterocyclic intersugar linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.
Representative United States patents relating to the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is herein incorporated by reference.
In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleoside units are replaced with novel groups. The nucleobase units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligonucleotide, an oligonucleotide mimetic, that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide-containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497.
Some preferred embodiments of the invention employ oligonucleotides with phosphorothioate linkages and oligonucleosides with heteroatom backbones, and in particular —CH2—NH—O—CH2—, —CH2—N(CH3)—O—CH2— [known as a methylene (methylimino) or MMI backbone], —CH2—O—N(CH3)—CH2—, —CH2—N(CH3)—N(CH3)—CH2—, and —O—N(CH3)—CH2—CH2— [wherein the native phosphodiester backbone is represented as —O—P—O—CH2—] of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.
The oligonucleotides employed in the ligand-conjugated oligonucleotides of the invention may additionally or alternatively comprise nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.
Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligonucleotides of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Id., pages 276-278) and are presently preferred base substitutions, even more particularly when combined with 2′-methoxyethyl sugar modifications.
Representative United States patents relating to the preparation of certain of the above-noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; and 5,808,027; all of which are hereby incorporated by reference.
In certain embodiments, the oligonucleotides employed in the ligand-conjugated oligonucleotides of the invention may additionally or alternatively comprise one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl, O-, S-, or N-alkenyl, or O, S- or N-alkynyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Particularly preferred are O[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2′ position: C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2 CH3, ONO2, NO2, N3, NH2, heterocycloalkyl heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy [2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE] (Martin et al., Helv. Chim. Acta, 1995, 78, 486), i.e., an alkoxyalkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2′-DMAOE, as described in U.S. Pat. No. 6,127,533, filed on Jan. 30, 1998, the contents of which are incorporated by reference.
Other preferred modifications include 2′-methoxy (2′-O—CH3), 2′-aminopropoxy (2′-OCH2CH2CH2NH2) and 2′-fluoro (2′-F). Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides.
As used herein, the term “sugar substituent group” or “2′-substituent group” includes groups attached to the 2′-position of the ribofuranosyl moiety with or without an oxygen atom. Sugar substituent groups include, but are not limited to, fluoro, O-alkyl, O-alkylamino, O-alkylalkoxy, protected O-alkylamino, O-alkylaminoalkyl, O-alkyl imidazole and polyethers of the formula (O-alkyl)m, wherein m is 1 to about 10. Preferred among these polyethers are linear and cyclic polyethylene glycols (PEGS), and (PEG)-containing groups, such as crown ethers and those which are disclosed by Ouchi et al. (Drug Design and Discovery 1992, 9:93); Ravasio et al. (J. Org. Chem. 1991, 56:4329); and Delgardo et. al. (Critical Reviews in Therapeutic Drug Carrier Systems 1992, 9:249), each of which is hereby incorporated by reference in its entirety. Further sugar modifications are disclosed by Cook (Anti-pain Drug Design, 1991, 6:585-607). Fluoro, O-alkyl, O-alkylamino, O-alkyl imidazole, O-alkylaminoalkyl, and alkyl amino substitution is described in U.S. Pat. No. 6,166,197, entitled “Oligomeric Compounds having Pyrimidine Nucleotide(s) with 2′ and 5′ Substitutions,” hereby incorporated by reference in its entirety.
Additional sugar substituent groups amenable to the invention include 2′-SR and 2′-NR2 groups, wherein each R is, independently, hydrogen, a protecting group or substituted or unsubstituted alkyl, alkenyl, or alkynyl. 2′-SR Nucleosides are disclosed in U.S. Pat. No. 5,670,633, hereby incorporated by reference in its entirety. The incorporation of 2′-SR monomer synthons is disclosed by Hamm et al. (J. Org. Chem., 1997, 62:3415-3420). 2′-NR nucleosides are disclosed by Goettingen, M., J. Org. Chem., 1996, 61, 6273-6281; and Polushin et al., Tetrahedron Lett., 1996, 37, 3227-3230. Further representative 2′-substituent groups amenable to the invention include those having one of formula I or II:




wherein,
E is C1-C10 alkyl, N(Q3)(Q4) or N═C(Q3)(Q4); each Q3 and Q4 is, independently, H, C1-C10 alkyl, dialkylaminoalkyl, a nitrogen protecting group, a tethered or untethered conjugate group, a linker to a solid support; or Q3 and Q4, together, form a nitrogen protecting group or a ring structure optionally including at least one additional heteroatom selected from N and O;
q1 is an integer from 1 to 10;
q2 is an integer from 1 to 10;
q3 is 0 or 1;
q4 is 0, 1 or2;
each Z1, Z2 and Z3 is, independently, C4-C7 cycloalkyl, C5-C14 aryl or C3-C15 heterocyclyl, wherein the heteroatom in said heterocyclyl group is selected from oxygen, nitrogen and sulfur;
Z4 is OM1, SM1, or N(M1)2; each M1 is, independently, H, C1-C8 alkyl, C1-C8 haloalkyl, C(═NH)N(H)M2, C(═O)N(H)M2 or OC(═O)N(H)M2; M2 is H or C1-C8 alkyl; and
Z5 is C1-C10 alkyl, C1-C10 haloalkyl, C2-C10 alkenyl, C2-C10 alkynyl, C6-C14 aryl, N(Q3)(Q4), OQ3, halo, SQ3 or CN.
Representative 2′-O-sugar substituent groups of formula 1 are disclosed in U.S. Pat. No. 6,172,209, entitled “Capped 2′-Oxyethoxy Oligonucleotides,” hereby incorporated by reference in its entirety. Representative cyclic 2′-O-sugar substituent groups of formula II are disclosed in U.S. Pat. No. 6,271,358, entitled “RNA Targeted 2′-Modified Oligonucleotides that are Conformationally Preorganized,” hereby incorporated by reference in its entirety.
Sugars having O-substitutions on the ribosyl ring are also amenable to the invention. Representative substitutions for ring O include, but are not limited to, S, CH2, CHF, and CF2. See, e.g., Secrist et al., Abstract 21, Program & Abstracts, Tenth International Roundtable, Nucleosides, Nucleotides and their Biological Applications, Park City, Utah, Sep. 16-20, 1992.
Oligonucleotides may also have sugar mimetics, such as cyclobutyl moieties, in place of the pentofuranosyl sugar. Representative United States patents relating to the preparation of such modified sugars include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,0531 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,700,920; and 5,859,221, all of which are hereby incorporated by reference.
Additional modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide. For example, one additional modification of the ligand-conjugated oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more additional non-ligand moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties, such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 111; Kabanov et al., FEBS Lett., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75, 49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990, 18, 3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923).
Representative United States patents relating to the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928; and 5,688,941, each of which is herein incorporated by reference.
The invention also includes compositions employing oligonucleotides that are substantially chirally pure with regard to particular positions within the oligonucleotides. Examples of substantially chirally pure oligonucleotides include, but are not limited to, those having phosphorothioate linkages that are at least 75% Sp or Rp (Cook et al., U.S. Pat. No. 5,587,361) and those having substantially chirally pure (Sp or Rp) alkylphosphonate, phosphoramidate or phosphotriester linkages (Cook, U.S. Pat. Nos. 5,212,295 and 5,521,302).
In certain instances, the oligonucleotide may be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to oligonucleotides in order to enhance the activity, cellular distribution or cellular uptake of the oligonucleotide, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such oligonucleotide conjugates have been listed above. Typical conjugation protocols involve the synthesis of oligonucleotides bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the oligonucleotide still bound to the solid support or following cleavage of the oligonucleotide in solution phase. Purification of the oligonucleotide conjugate by HPLC typically affords the pure conjugate.
Alternatively, the molecule being conjugated may be converted into a building block, such as a phosphoramidite, via an alcohol group present in the molecule or by attachment of a linker bearing an alcohol group that may be phosphitylated.
Importantly, each of these approaches may be used for the synthesis of ligand conjugated oligonucleotides. Aminolinked oligonucleotides may be coupled directly with ligand via the use of coupling reagents or following activation of the ligand as an NHS or pentfluorophenolate ester. Ligand phosphoramidites may be synthesized via the attachment of an aminohexanol linker to one of the carboxyl groups followed by phosphitylation of the terminal alcohol functionality. Other linkers, such as cysteamine, may also be utilized for conjugation to a chloroacetyl linker present on a synthesized oligonucleotide.
III. Pharmaceutical Compositions Comprising dsRNA
In one embodiment, the invention provides pharmaceutical compositions comprising a dsRNA, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical composition comprising the dsRNA is useful for treating a disease or disorder associated with the expression or activity of the Nav1.8 gene, such as neuropathic or inflammatory pain.
In another embodiment, the invention provides pharmaceutical compositions comprising at least two dsRNAs, designed to target different regions of the Nav1.8 gene, and a pharmaceutically acceptable carrier. In this embodiment, the individual dsRNAs are prepared as described in the preceding section, which is incorporated by reference herein. One dsRNA can have a nucleotide sequence which is substantially complementary to at least one part of the Nav1.8 gene; additional dsRNAs are prepared, each of which has a nucleotide sequence that is substantially complementary to different part of the Nav1.8 gene. The multiple dsRNAs may be combined in the same pharmaceutical composition, or formulated separately. If formulated individually, the compositions containing the separate dsRNAs may comprise the same or different carriers, and may be administered using the same or different routes of administration. Moreover, the pharmaceutical compositions comprising the individual dsRNAs may be administered substantially simultaneously, sequentially, or at preset intervals throughout the day or treatment period.
The pharmaceutical compositions of the invention are administered in dosages sufficient to inhibit expression of the Nav1.8 gene. The present inventors have found that, because of their improved efficiency, compositions comprising the dsRNA of the invention can be administered at surprisingly low dosages. A maximum dosage of 5 mg dsRNA per kilogram body weight of recipient per day is sufficient to inhibit or completely suppress expression of the Nav1.8 gene, or alleviate chronic pain.
In general, a suitable dose of dsRNA will be in the range of 0.01 to 5.0 milligrams per kilogram body weight of the recipient per day, preferably in the range of 0.1 to 200 micrograms per kilogram body weight per day, more preferably in the range of 0.1 to 100 micrograms per kilogram body weight per day, even more preferably in the range of 1.0 to 50 micrograms per kilogram body weight per day, and most preferably in the range of 1.0 to 25 micrograms per kilogram body weight per day. The pharmaceutical composition may be administered once daily, or the dsRNA may be administered as two, three, four, five, six or more sub-doses at appropriate intervals throughout the day or even using continuous infusion. In that case, the dsRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the dsRNA over a several day period. Sustained release formulations are well known in the art. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.
The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual dsRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.
Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as pain. Such models are used for in vivo testing of dsRNA, as well as for determining a therapeutically effective dose.
The pharmaceutical compositions encompassed by the invention may be administered by any means known in the art including, but not limited to oral or parenteral routes, including intravenous, intramuscular, intraperitoneal, epidural, intrathecal, intracerebroventricular, intraparenchymal (within the peripheral or central nervous system), subcutaneous, transdermal, intranasal, airway (aerosol), rectal, vaginal and topical (including buccal and sublingual) administration. In preferred embodiments, the pharmaceutical compositions are administered intrathecally by continuous infusion such as with a pump, or intrathecally by bolus injection. In other preferred embodiments, the pharmaceutical compositions are administered intravenously by continuous infusion such as with a pump, or intravenously by bolus injection.
For intrathecal, intracerebroventricular, intramuscular, intraparenchyrnal (within the peripheral or central nervous system), subcutaneous and intravenous use, the pharmaceutical compositions of the invention will generally be provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity. Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride. In a preferred embodiment, the carrier consists exclusively of an aqueous buffer. In this context, “exclusively” means no auxiliary agents or encapsulating substances are present which might affect or mediate uptake of dsRNA in the cells that express the Nav1.8 gene. Such substances include, for example, micellar structures, such as liposomes or capsids, as described below. Surprisingly, the present inventors have discovered that compositions containing only naked dsRNA and a physiologically acceptable solvent are taken-up by cells, where the dsRNA effectively inhibits expression of the Nav1.8 gene. Although microinjection, lipofection, viruses, viroids, capsids, capsoids, or other auxiliary agents are required to introduce dsRNA into cell cultures, surprisingly these methods and agents are not necessary for uptake of dsRNA in vivo. Aqueous suspensions according to the invention may include suspending agents such as cellulose derivatives, sodium alginate, polyvinyl-pyrrolidone and gum tragacanth, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate.
The pharmaceutical compositions useful according to the invention also include encapsulated formulations to protect the dsRNA against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811; PCT publication WO 91/06309; and European patent publication EP-A-43075, which are incorporated by reference herein.
Using the small interfering RNA vectors previously described, the invention also provides devices, systems, and methods for delivery of small interfering RNA to target locations in the nervous system and or the brain. The envisioned route of delivery is through the use of implanted, indwelling, intrathecal, intracerebroventricular or intraparenchymal catheters that provide a means for injecting small volumes of fluid containing the dsRNA of the invention directly into local nerves or local brain tissue, or into bodily fluids surrounding these tissues. The proximal end of these catheters may be connected to an implanted, intrathecal or intracerebral access port surgically affixed to the patient's body or cranium, or to an implanted drug pump located in the patient's torso.
Alternatively, implantable delivery devices, such as an implantable pump may be employed. Examples of the delivery devices within the scope of the invention include the Model 8506 investigational device (by Medtronic, Inc. of Minneapolis, Minn.), which can be implanted subcutaneously in the body or on the cranium, and provides an access port through which therapeutic agents may be delivered to the nerves or brain. Delivery occurs through a stereotactically implanted polyurethane catheter. Two models of catheters that can function with the Model 8506 access port include the Model 8770 ventricular catheter by Medtronic, Inc., for delivery to the intracerebral ventricles, which is disclosed in U.S. Pat. No. 6,093,180, incorporated herein by reference, and the IPAI catheter by Medtronic, Inc., for delivery to the brain tissue itself (i.e., intraparenchymal delivery), disclosed in U.S. Ser. Nos. 09/540,444 and 09/625,751, which are incorporated herein by reference. The latter catheter has multiple outlets on its distal end to deliver the therapeutic agent to multiple sites along the catheter path. In addition to the aforementioned device, the delivery of the small interfering RNA vectors in accordance with the invention can be accomplished with a wide variety of devices, including but not limited to U.S. Pat. Nos. 5,735,814, 5,814,014, and 6,042,579, all of which are incorporated herein by reference. Using the teachings of the invention and those of skill in the art will recognize that these and other devices and systems may be suitable for delivery of small interfering RNA vectors for the treatment of pain in accordance with the invention.
In one such embodiment, the method further comprises the steps of implanting a pump outside the body or brain, the pump coupled to a proximal end of the catheter, and operating the pump to deliver the predetermined dosage of the at least one small interfering RNA or small interfering RNA vector through the discharge portion of the catheter. A further embodiment comprises the further step of periodically refreshing a supply of the at least one small interfering RNA or small interfering RNA vector to the pump outside said body or brain.
Thus, the invention includes the delivery of small interfering RNA vectors using an implantable pump and catheter, like that taught in U.S. Pat. No. 5,735,814 and 6,042,579, and further using a sensor as part of the infusion system to regulate the amount of small interfering RNA vectors delivered to the nerves or brain, like that taught in U.S. Pat. No. 5,814,014. Other devices and systems can be used in accordance with the method of the invention, for example, the devices and systems disclosed in U.S. Ser. No. 09/872,698 (filed Jun. 1, 2001) and U.S. Ser. No. 09/864,646 (filed May 23, 2001), which are incorporated herein by reference.
Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit high therapeutic indices are preferred.
The data obtained from cell culture assays and animal studies can be used in formulation a range of dosage for use in humans. The dosage of compositions of the invention lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
In addition to their administration individually or as a plurality, as discussed above, the dsRNAs of the invention can be administered in combination with other known agents effective in treatment of pain. In any event, the administering physician can adjust the amount and timing of dsRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.
Methods for Treating Diseases Caused by Expression of the Nav1.8 Gene
In one embodiment, the invention provides a method for treating a subject having a pathological condition mediated by the expression of the Nav1.8 gene, such as neuropathic or inflammatory pain. In this embodiment, the dsRNA acts as a therapeutic agent for controlling the expression of the Nav1.8 protein. The method comprises administering a pharmaceutical composition of the invention to the patient (e.g., human), such that expression of the Nav1.8 gene is silenced. Because of their high specificity, the dsRNAs of the invention specifically target mRNAs of the Nav1.8 gene.
Pain
As used herein, the term “pain” is art recognized and includes a bodily sensation elicited by noxious chemical, mechanical, or thermal stimuli, in a subject, e.g., a mammal such as a human. The term “pain” includes chronic pain such as lower back pain; pain due to arthritis, e.g., osteoarthritis; joint pain, e.g., knee pain or carpal tunnel syndrome; myofascial pain, and neuropathic pain. The term “pain” further includes acute pain, such as pain associated with muscle strains and sprains; tooth pain; headaches; pain associated with surgery; and pain associated with various forms of tissue injury, e.g., inflammation, infection, and ischemia.
“Neuropathic pain” refers to pain caused by injury or disease of the central or peripheral nervous system. In contrast to the immediate (acute) pain caused by tissue injury, neuropathic pain can develop days or months after a traumatic injury. Neuropathic pain frequently is long lasting or chronic, and is not limited in duration to the period of tissue repair. Neuropathic pain can occur spontaneously, or as a result of stimulation that normally is not painful. Neuropathic pain is caused by aberrant somatosensory processing, and is associated with chronic sensory disturbances, including spontaneous pain, hyperalgesia (i.e., sensation of more pain than the stimulus would warrant) and allodynia (i.e., a condition in which ordinarily painless stimuli induce the experience of pain). Neuropathic pain includes, but is not limited to, pain caused by peripheral nerve trauma, viral infection, diabetes mellitus, chemotherapy, causalgia, plexus-avulsion, spinal cord injury, neuroma, limb amputation, vasculitis, nerve damage from surgery, nerve damage from chronic alcoholism, hypothyroidism, uremia, and vitamin deficiencies, among other causes. Neuropathic pain is one type of pain associated with cancer. Cancer pain can also be “nociceptive” or “mixed.”
“Chronic pain” can be defined as pain lasting longer than three months (Bonica, Semin. Anesth. 1986, 5:82-99), and may be characterized by unrelenting persistent pain that is not fully amenable to routine pain control methods. Chronic pain includes, but is not limited to, inflammatory pain, post-operative pain, cancer pain, osteoarthritis pain associated with metastatic cancer, chemotherapy-induced pain, trigeminal neuralgia, acute herpetic and post-herpetic neuralgia, diabetic neuropathy, pain due to arthritis, joint pain, myofascial pain, causalgia, brachial plexus avulsion, occipital neuralgia, reflex sympathetic dystrophy, fibromyalgia, gout, phantom limb pain, burn pain, pain associated with spinal cord injury, multiple sclerosis, reflex sympathetic dystrophy and lower back pain and other forms of neuralgia, neuropathic, and idiopathic pain syndromes.
“Nociceptive pain” is due to activation of pain-sensitive nerve fibers, either somatic or visceral. Nociceptive pain is generally a response to direct tissue damage. The initial trauma causes the release of several chemicals including bradykinin, serotonin, substance P, histamine, and prostaglandin. When somatic nerves are involved, the pain is typically experienced as an aching or pressure-like sensation.
In the phrase “pain and related disorders”, the term “related disorders” refers to disorders that either cause or are associated with pain, or have been shown to have similar mechanisms to pain. These disorders include addiction, seizure, stroke, ischemia, a neurodegenerative disorder, anxiety, depression, headache, asthma, rheumatic disease, osteoarthritis, retinopathy, inflammatory eye disorders, pruritis, ulcer, gastric lesions, uncontrollable urination, an inflammatory or unstable bladder disorder, inflammatory bowel disease, irritable bowel syndrome (IBS), irritable bowel disease (IBD), gastroesophageal reflux disease (GERD), functional dyspepsia, functional chest pain of presumed oesophageal origin, functional dysphagia, non-cardiac chest pain, symptomatic gastroesophageal disease, gastritis, aerophagia, functional constipation, functional diarrhea, burbulence, chronic functional abdominal pain, recurrent abdominal pain (RAP), functional abdominal bloating, functional biliary pain, functional incontinence, functional ano-rectal pain, chronic pelvic pain, pelvic floor dyssenergia, unspecified functional ano-rectal disorder, cholecystalgia, interstitial cystitis, dysmenorrhea, and dyspareunia.
The invention thus provides the use of an anti-Nav1.8 dsRNA administered to a human, particularly by intrathecal infusion or injection, or by intravenous infusion or injection, for the treatment of pain, including neuropathic and inflammatory pain.
The pharmaceutical compositions encompassed by the invention may be administered by any means known in the art including, but not limited to oral or parenteral routes, including intravenous, intramuscular, intraperitoneal, epidural, intrathecal, intracerebroventricular, intraparenchymal (within the peripheral or central nervous system), subcutaneous, transdermal, intranasal, airway (aerosol), nasal, rectal, vaginal and topical (including buccal and sublingual) administration, and epidural administration. In preferred embodiments, the pharmaceutical compositions are administered intrathecally by continuous infusion such as with a pump, or intrathecally by bolus injection. In other preferred embodiments, the pharmaceutical compositions are administered intravenously by continuous infusion such as with a pump, or intravenously by bolus injection.
Methods for Inhibiting Expression of the Nav1.8 Gene
In yet another aspect, the invention provides a method for inhibiting the expression of the Nav1.8 gene in a mammal. The method comprises administering a composition of the invention to the mammal such that expression of the target Nav1.8 gene is silenced. Because of their high specificity, the dsRNAs of the invention specifically target RNAs (primary or processed) of the target Nav1.8 gene. Compositions and methods for inhibiting the expression of these Nav1.8 genes using dsRNAs can be performed as described elsewhere herein.
In one embodiment, the method comprises administering a composition comprising a dsRNA, wherein the dsRNA comprises a nucleotide sequence which is complementary to at least a part of an RNA transcript of the Nav1.8 gene of the mammal to be treated. When the organism to be treated is a mammal such as a human, the composition may be administered by any means known in the art including, but not limited to oral or parenteral routes, including intravenous, intramuscular, intraperitoneal, epidural, intrathecal, intracerebroventricular, intraparenchymal (within the peripheral or central nervous system), subcutaneous, transdermal, intranasal, airway (aerosol), rectal, vaginal and topical (including buccal and sublingual) administration. In preferred embodiments, the compositions are administered by intrathecal infusion or injection, or by intravenous infusion or injection.
Unless otherwise defined, 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 invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
EXAMPLES
Example 1
Gene Walking of the Nav1.8 Gene
siRNAs were identified in a multi step sequence analysis process in order to design siRNAs targeting the Nav1.8 gene in 4 species of interest.
ClustalW multiple alignment function of BioEdit Sequence Alignment Editor (version 7.0.4.1) was used to generate a global alignment of human (NM—006514), mouse (NM—009134), rat (NM—017247) and dog (NM001003203) Nav1.8 mRNA sequences.
Conserved regions were identified by embedded sequence analysis function of the software. Conserved regions were defined as sequence stretches with a minimum length of 19 bases for all aligned sequences containing no internal gaps. Sequence positions of conserved regions were counted according to the human sequence.
The siRNA design web interface at Whitehead Institute for Biomedical Research (on the World Wide Web at jura.wi.mit.edu/siRNAext/) was used to identify all potential siRNAs targeting the conserved regions as well as their respective off-target hits to sequences in the human, mouse and rat RefSeq database. siRNAs satisfying the crossreactivity criteria were selected out of the candidates pool and subjected to the software embedded off-target analysis. For this, all selected siRNAs were analyzed in 3 rounds by the NCBI blast algorithm against the NCBI human, mouse and rat RefSeq database.
Blast results were downloaded and analyzed by a perl script in order to extract the identity of the best off-target hit for the antisense strain as well as the positions of occurring mismatches.
All siRNA candidates were ranked according to predicted properties. For this, different criteria were applied in order to identify siRNAs with the following properties:

    
    
        reactivity criterium: targeting human, mouse, rat and dog sequences
        specificity criteria: highly specific for human, at least specific for rat and mouse
    
    


The 2 siRNAs that satisfied the applied criteria were refered to as “multi-species targeting siRNAs”.
In order to identify more siRNAs a second round of siRNA identification steps were conducted correspondingly with additional regions that had been previously eliminated due to missing reactivity with dog sequences.
The resulting pool of 10 siRNAs matching the above mentioned criteria were refered to as ‘human/rat/mouse targeting siRNAs’.
A third round of the siRNA design process was conducted while disregarding cross-reactivity to mouse.
All candidate siRNAs were again extracted and ranked in 3 steps according to the following criteria:
Step 1:

    
    
        reactivity criterium: targeting human, rat and mouse sequences
        specificity criterium: highly specific for human and rat, moderately specific for mouse  4 siRNAs (added to pool of ‘human/rat/mouse targeting siRNAs’)
    
    


Step 2:

    
    
        reactivity criterium: targeting human and rat sequences
        sequence-embedded criterium: absense of stretches with more than 3 Gs in a row
        specificity criterium: highly specific for human  19 siRNAs
    
    


Step 3:

    
    
        reactivity criterium: targeting human and rat sequences
        specificity criterium: highly specific for rat, specific for human and favorable ddG value  1 siRNA
    
    


The pool resulting form steps 2 and 3 (20 siRNAs) were referred to as ‘human/rat targeting siRNAs’.
The in silico selected 36 siRNAs were synthesized (Table 1).
Additional sequence selections were performed using the above generalized methods except that cross reactivity between species was not used as a selection criterea: the sequence selection was based solely on the human Nav1.8 sequence. In addition, ranking was based on off-target scores based on the closest FASTA hit as was as the number of potential off target genes. These siRNAs are provided in Table 4.







TABLE 1







siRNAs specific for Nav1.8














SEQ

SEQ





ID
antisense strand sequence
ID


duplex name
sense strand sequence (5′-3′)
NO:
(5′-3′)
NO:















AL-DP-6042
cmcmcmggaumumumumaacmumacmacmcmTT
1
ggugumaguumaaaauccgggTT
2



 


AL-DP-6043
cmcmggaumumumumaacmumacmacmcmaTT
3
uggugumaguumaaaauccggTT
4


 


AL-DP-6044
gacmaacmcmcmggaumumumumaacmumTT
5
aguumaaaauccggguugucTT
6


 


AL-DP-6045
cmcmumumacmaacmcmagcmgcmaggaTT
7
uccugcgcugguugumaaggTT
8


 


AL-DP-6046
aacmcmcmggaumumumumaacmumacmaTT
9
ugumaguumaaaauccggguuTT
10


 


AL-DP-6047
umgumgcmaumgacmcmcmgaacmumgaTT
11
ucmaguucgggucmaugcmacmaTT
12


 


AL-DP-6048
acmaacmcmagcmgcmaggaumgumcmTT
13
gacmauccugcgcugguuguTT
14


 


AL-DP-6049
acmcmcmggaumumumumaacmumacmacmTT
15
gugumaguumaaaauccggguTT
16


 


AL-DP-6050
cmaumcmcmumaumgaacmcmaaumagcmTT
17
gcumauugguucmaumaggaugTT
18


 


AL-DP-6051
cmumumacmaacmcmagcmgcmaggaumTT
19
auccugcgcugguugumaagTT
20


 


AL-DP-6052
umumumaacmumacmacmcmagcmumumumgTT
21
cmaaagcuggugumaguumaaaTT
22


 


AL-DP-6053
gumgumgcmaumgacmcmcmgaacmumgTT
23
cmaguucgggucmaugcmacmacTT
24


 


AL-DP-6202
cmggaumumumumaacmumacmacmcmagTT
25
cuggugumaguumaaaauccgTT
26


 


AL-DP-6203
umacmaacmcmagcmgcmaggaumgumTT
27
acmauccugcgcugguugumaTT
28


 


AL-DP-6204
ggumcmumcmumgumgcmcmcmaumumgcmumTT
29
agcmaaugggcmacmagagaccTT
30


 


AL-DP-6205
cmcmcmaagumumcmumaumggumgagcmTT
31
gcucmaccmaumagaacuugggTT
32


 


AL-DP-6206
cmaagumumcmumaumggumgagcmumcmTT
33
gagcucmaccmaumagaacuugTT
34


 


AL-DP-6207
cmumacmagcmacmacmacmcmggacmaTT
35
uguccggugugugcugumagTT
36


 


AL-DP-6208
aagumumcmumaumggumgagcmumcmcmTT
37
ggagcucmaccmaumagaacuuTT
38


 


AL-DP-6209
umumumumgumcmumaaaumgagumumcmaTT
39
ugaacucmauuumagacmaaaaTT
40


 


AL-DP-6210
cmcmumcmumcmacmumgumumcmcmgcmcmumcmTT
41
gaggcggaacmagugagaggTT
42


 


AL-DP-6211
aacmcmagumumcmumumumgumggcmcmgTT
43
cggccmacmaaagaacugguuTT
44


 


AL-DP-6212
cmumcmacmumgumumcmcmgcmcmumcmaumgTT
45
cmaugaggcggaacmagugagTT
46


 


AL-DP-6213
cmcmaagumumcmumaumggumgagcmumTT
47
agcucmaccmaumagaacuuggTT
48


 


AL-DP-6214
cmagumumcmumumumgumggcmcmgumcmumTT
49
agacggccmacmaaagaacugTT
50


 


AL-DP-6215
ggcmumggcmaggumgcmgcmaagaTT
51
ucuugcgcmaccugccmagccTT
52


 


AL-DP-6216
cmumcmcmumcmumgagggcmagcmacmgTT
53
cgugcugcccucmagaggagTT
54


 


AL-DP-6217
gumcmumumcmacmumgumcmaumumumacmaTT
55
ugumaaaugacmagugaagacTT
56


 


AL-DP-6218
cmaacmcmagcmgcmaggaumgumcmumTT
57
agacmauccugcgcugguugTT
58


 


AL-DP-6219
agaagumaumcmumgaumcmumgggaTT
59
ucccmagaucmagaumacuucuTT
60


 


AL-DP-6220
umcmumacmagcmacmacmacmcmggacmTT
61
guccggugugugcugumagaTT
62


 


AL-DP-6221
agumumctnumaumggumgagcmumcmcmcmTT
63
gggagcucmaccmaumagaacuTT
64


 


AL-DP-6222
umcmumaumggumgagcmumcmcmcmagcmTT
65
gcugggagcucmaccmaumagaTT
66


 


AL-DP-6223
agumumcmumumumgumggcmcmgumcmumumTT
67
aagacggccmacmaaagaacuTT
68


 


AL-DP-6224
umcmacmumgumumcmcmgcmcmumcmaumgaTT
69
ucmaugaggcggaacmagugaTT
70


 


AL-DP-6225
ggaumumumumaacmumacmacmcmagcmTT
71
gcuggugumaguumaaaauccTT
72
















TABLE 4







Further siRNAs specific for Nav1.8


















Remaining








Luc




SEQ

SEQ
activity


duplex
Sense strand
ID
Antisense strand
ID
[% of


name
sequence (5′-3′)
NO:
sequence (5′-3′)
NO:
controls]
















AD-11159
ccgcuugcugcgcguauucTT
73
gaauacgcgcagcaagcggTT
74
 26 ± 2



 


AD-11160
cuugcuaccguaucguggaTT
75
uccacgauacgguagcaagTT
76
 52 ± 4


 


AD-11161
gacuucaucgcuaauccgaTT
77
ucggauuagcgaugaagucTT
78
 29 ± 4


 


AD-11162
uggauuuuagcgucauuacTT
79
guaaugacgcuaaaauccaTT
80
 70 ± 6


 


AD-11163
cgcuugcugcgcguauucaTT
81
ugaauacgcgcagcaagcgTT
82
 31 ± 5


 


AD-11164
caacuuccgucgcuuuacuTT
83
aguaaagcgacggaaguugTT
84
 24 ± 2


 


AD-11165
gucgcuuuacuccggagucTT
85
gacuccggaguaaagcgacTT
86
 20 ± 3


 


AD-11166
aaugaguucacguaccugaTT
87
ucagguacgugaacucauuTT
88
 17 ± 3


 


AD-11167
agacuugcuaccguaucguTT
89
acgauacgguagcaagucuTT
90
 38 ± 2


 


AD-11168
aguuuauuuauuacggucaTT
91
ugaccguaauaaauaaacuTT
92
 63 ± 7


 


AD-11169
aaaugaguucacguaccugTT
93
cagguacgugaacucauuuTT
94
 46 ± 4


 


AD-11170
ugucucggcauucgaugcaTT
95
ugcaucgaaugccgagacaTT
96
 72 ± 5


 


AD-11171
ucaaagcccuucgaacccuTT
97
aggguucgaagggcuuugaTT
98
 96 ± 4


 


AD-11172
gcgggcucuuucucgauuuTT
99
aaaucgagaaagagcccgcTT
100
 34 ± 4


 


AD-11173
ccuuguaccuuugucgauuTT
101
aaucgacaaagguacaaggTT
102
 17 ± 2


 


AD-11174
gugguucucuccauugcgaTT
103
ucgcaauggagagaaccacTT
104
 24 ± 3


 


AD-11175
caaaaggccuaucggagcuTT
105
agcuccgauaggccuuuugTT
106
 46 ± 3


 


AD-11176
aaaggccuaucggagcuauTT
107
auagcuccgauaggccuuuTT
108
 48 ± 5


 


AD-11177
ggugcaucaacuauaccgaTT
109
ucgguauaguugaugcaccTT
110
 21 ± 2


 


AD-11178
aacuaccguaacaaccgaaTT
111
uucgguuguuacgguaguuTT
112
 42 ± 8


 


AD-11179
ucgcuuuacuccggagucaTT
113
ugacuccggaguaaagcgaTT
114
 17 ± 1


 


AD-11180
gaaaacgccgggcuagucaTT
115
ugacuagcccggcguuuucTT
116
 36 ± 8


 


AD-11181
accgaaaaaauaucuccgcTT
117
gcggagauauuuuuuugguTT
118
105 ± 15


 


AD-11182
uucaucgcuaauccgacugTT
119
cagucggauuagcgaugaaTT
120
 87 ± 14


 


AD-11183
uccgucgcuuuacuccggaTT
121
uccggaguaaagcgacggaTT
122
 33 ± 3


 


AD-11184
gcuuuacuccggagucacuTT
123
agugacuccggaguaaagcTT
124
 14 ± 1


 


AD-11185
ggaaaacgccgggcuagucTT
125
gacuagcccggcguuuuccTT
126
 50 ± 5


 


AD-11186
acuaccguaacaaccgaaaTT
127
uuucgguuguuacgguaguTT
128
 41 ± 5


 


AD-11187
uggccaucguaccaaacagTT
129
cuguuugguacgauggccaTT
130
 85 ± 7


 


AD-11188
acuugcuaccguaucguggTT
131
ccacgauacgguagcaaguTT
132
109 ± 15


 


AD-11189
gauaagucucacagcgaagTT
133
cuucgcugugagacuuaucTT
134
 29 ± 4


 


AD-11190
auaagucucacagcgaagaTT
135
ucuucgcugugagacuuauTT
136
 34 ± 4


 


AD-11191
aagcccuucgaacccuucgTT
137
cgaaggguucgaagggcuuTT
138
 86 ± 10


 


AD-11192
agcccuucgaacccuucgcTT
139
gcgaaggguucgaagggcuTT
140
 98 ± 16


 


AD-11193
cccuucgaacccuucgcgcTT
141
gcgcgaaggguucgaagggTT
142
 64 ± 4


 


AD-11194
aacccaaucgaaauauacuTT
143
aguauauuucgauuggguuTT
144
 40 ± 4


 


AD-11195
cgcuuuacuccggagucacTT
145
gugacuccggaguaaagcgTT
146
 17 ± 2


 


AD-11196
gaccauuucccgguuuaguTT
147
acuaaaccgggaaauggucTT
148
 77 ± 4


 


AD-11197
cgguuuagugccacucgggTT
149
cccgaguggcacuaaaccgTT
150
 58 ± 6


 


AD-11198
cacagcaauagaucuccguTT
151
acggagaucuauugcugugTT
152
 34 ± 4


 


AD-11199
acuagggauugacacaaccTT
153
gguugugucaaucccuaguTT
154
 86 ± 3


 


AD-11200
cccacaauggaucaccuuuTT
155
aaaggugauccauugugggTT
156
 22 ± 0


 


AD-11201
ugucuuuucuaggccucgcTT
157
gcgaggccuagaaaagacaTT
158
 91 ± 10


 


AD-11202
agcugucgaugucucggcaTT
159
ugccgagacaucgacagcuTT
160
 42 ± 2


 


AD-11203
gucucggcauucgaugcagTT
161
cugcaucgaaugccgagacTT
162
 62 ± 5


 


AD-11204
ucaaaaucauugccuucgaTT
163
ucgaaggcaaugauuuugaTT
164
 42 ± 7


 


AD-11205
cccgcuggcacaugcacgaTT
165
ucgugcaugugccagcgggTT
166
 42 ± 4


 


AD-11206
ucauugucuuccguauccuTT
167
aggauacggaagacaaugaTT
168
 79 ± 11


 


AD-11207
uuggccaucguaccaaacaTT
169
uguuugguacgauggccaaTT
170
 59 ± 8


 


AD-11208
gcacgguggacugccuagaTT
171
ucuaggcaguccaccgugcTT
172
 95 ± 12


 


AD-11209
gcccuucgaacccuucgcgTT
173
cgcgaaggguucgaagggcTT
174
 48 ± 4


 


AD-11210
ugcgggcucuuucucgauuTT
175
aaucgagaaagagcccgcaTT
176
 35 ± 5


 


AD-11211
gaggugcaucaacuauaccTT
177
gguauaguugaugcaccucTT
178
 36 ± 4


 


AD-11212
caucaacuauaccgauggaTT
179
uccaucgguauaguugaugTT
180
 58 ± 4


 


AD-11213
aaaucauccuaugaaccaaTT
181
uugguucauaggaugauuuTT
182
 26 ± 2


 


AD-11214
aaggccuaucggagcuaugTT
183
cauagcuccgauaggccuuTT
184
 63 ± 4


 


AD-11215
uguacucccagacaaaucuTT
185
agauuugucugggagaacaTT
186
 40 ± 3


 


AD-11216
aggacaucuagcucaauacTT
187
guauugagcuagauguccuTT
188
 29 ± 2


 


AD-11217
ccgguuuagugccacucggTT
189
ccgaguggcacuaaaccggTT
190
 22 ± 2


 


AD-11218
caucgcuaauccgacugugTT
191
cacagucggauuagcgaugTT
192
 51 ± 2


 


AD-11219
aaacuaccguaacaaccgaTT
193
ucgguuguuacgguaguuuTT
194
 29 ± 3


 


AD-11220
aucgcuaauccgacuguguTT
195
acacagucggauuagcgauTT
196
 52 ± 1


 


AD-11221
cccgguuuagugccacucgTT
197
cgaguggcacuaaaccgggTT
198
 60 ± 1


 


AD-11222
uuuagcgucauuacccuggTT
199
ccaggguaaugacgcuaaaTT
200
105 ± 3


 


AD-11223
aauaagcgaggcacuucugTT
201
cagaagugccucgcuuauuTT
202
 66 ± 5


 


AD-11224
cuaccguaacaaccgaaaaTT
203
uuuucgguuguuacgguagTT
204
 25 ± 3


 


AD-11225
ucgcuaauccgacugugugTT
205
cacacagucggauuagcgaTT
206
 70 ± 5


 


AD-11226
ucccucgaaacuaacaacuTT
207
aguuguuaguuucgagggaTT
208
 24 ± 3


 


AD-11227
cuuccgucgcuuuacuccgTT
209
cggaguaaagcgacggaagTT
210
 31 ± 0


 


AD-11228
uuucccgguuuagugccacTT
211
guggcacuaaaccgggaaaTT
212
101 ± 2


 


AD-11229
gguuuagugccacucgggcTT
213
gcccgaguggcacuaaaccTT
214
 67 ± 1


 


AD-11230
acaacccggauuuuaacuaTT
215
uaguuaaaauccggguuguTT
216
 48 ± 3


 


AD-11231
cuagggauugacacaaccuTT
217
agguugugucaaucccuagTT
218
 39 ± 2


 


AD-11232
ugucgauugugaauaacaaTT
219
uuguuauucacaaucgacaTT
220
 25 ± 3


 


AD-11233
caucgugaccagacaagcuTT
221
agcuugucuggucacgaugTT
222
 47 ± 4


 


AD-11234
ccuaucggagcuaugugcuTT
223
agcacauagcuccgauaggTT
224
 58 ± 7


 


AD-11235
uuagcgucauuacccuggcTT
225
gccaggguaaugacgcuaaTT
226
 92 ± 7


 


AD-11236
agcaauagaucuccgugggTT
227
cccacggagaucuauugcuTT
228
 83 ± 4


 


AD-11237
aucgaaauauacugauccaTT
229
uggaucaguauauuucgauTT
230
 57 ± 3


 


AD-11238
ggaucccucgaaacuaacaTT
231
uguuaguuucgagggauccTT
232
 18 ± 1


 


AD-11239
acaccggacauuuauggugTT
233
caccauaaauguccgguguTT
234
 96 ± 2


 


AD-11240
guuuagugccacucgggccTT
235
ggcccgaguggcacuaaacTT
236
 95 ± 17


 


AD-11241
augacccgaacugaccuucTT
237
gaaggucaguucgggucauTT
238
 80 ± 7


 


AD-11242
auuuuagcgucauuacccuTT
239
aggguaaugacgcuaaaauTT
240
 70 ± 5


 


AD-11243
uuuuagcgucauuacccugTT
241
caggguaaugacgcuaaaaTT
242
 77 ± 8


 


AD-11244
gcaauagaucuccgugggaTT
243
ucccacggagaucuauugcTT
244
 23 ± 1


 


AD-11245
aaauaagcgaggcacuucuTT
245
agaagugccucgcuuauuuTT
246
 38 ± 3


 


AD-11246
auaagcgaggcacuucugaTT
247
ucagaagugccucgcuuauTT
248
 32 ± 2


 


AD-11247
ugauccuuacaaccagcgcTT
249
gcgcugguuguaaggaucaTT
250
 78 ± 2


 


AD-11248
uggccgagauaucucacucTT
251
gagugagauaucucggccaTT
252
 66 ± 1


 


AD-11249
caaccgccgcccacuagugTT
253
cacuagugggcggcgguugTT
254
 71 ± 4


 


AD-11250
uuagaugaaccuuuccgggTT
255
cccggaaagguucaucuaaTT
256
 90 ± 3


 


AD-11251
aaccuuuccgggcccaaagTT
257
cuuugggcccggaaagguuTT
258
 99 ± 7


 


AD-11252
auaaccuccguccuugaggTT
259
ccucaaggacggagguuauTT
260
105 ± 2


 


AD-11253
cuugugacggaucccuuugTT
261
caaagggauccgucacaagTT
262
 62 ± 5


 


AD-11254
ccuaccuucgaagccaugcTT
263
gcauggcuucgaagguaggTT
264
 84 ± 6


 


AD-11255
aaaucauugccuucgacccTT
265
gggucgaaggcaaugauuuTT
266
 92 ± 6


 


AD-11256
cauugccuucgacccauacTT
267
guaugggucgaaggcaaugTT
268
 42 ± 2


 


AD-11257
cuucgacccauacuauuauTT
269
auaauaguaugggucgaagTT
270
 35 ± 2


 


AD-11258
ucaucgcuaauccgacuguTT
271
acagucggauuagcgaugaTT
272
 76 ± 2


 


AD-11259
aauccgacugugugggucuTT
273
agacccacacagucggauuTT
274
 87 ± 5


 


AD-11260
agcacgguggacugccuagTT
275
cuaggcaguccaccgugcuTT
276
 75 ± 7


 


AD-11261
ggugcgcaagacuugcuacTT
277
guagcaagucuugcgcaccTT
278
 34 ± 3


 


AD-11262
aggugcaucaacuauaccgTT
279
cgguauaguugaugcaccuTT
280
 59 ± 5


 


AD-11263
aucaacuauaccgauggagTT
281
cuccaucgguauaguugauTT
282
113 ± 10


 


AD-11264
uugucgauugugaauaacaTT
283
uguuauucacaaucgacaaTT
284
 36 ± 5


 


AD-11265
aauggguuaccuugcacuuTT
285
aagugcaagguaacccauuTT
286
 70 ± 6


 


AD-11266
aggccuaucggagcuauguTT
287
acauagcuccgauaggccuTT
288
 72 ± 4


 


AD-11267
ggccuaucggagcuaugugTT
289
cacauagcuccgauaggccTT
290
 65 ± 7


 


AD-11268
uaucggagcuaugugcugcTT
291
gcagcacauagcuccgauaTT
292
110 ± 10


 


AD-11269
ccguccuaugagagugucaTT
293
ugacacucucauaggacggTT
294
 50 ± 3


 


AD-11270
ccauuggaucccucgaaacTT
295
guuucgagggauccaauggTT
296
 18 ± 3


 


AD-11271
cauuggaucccucgaaacuTT
297
aguuucgagggauccaaugTT
298
 23 ± 3


 


AD-11272
aucccucgaaacuaacaacTT
299
guuguuaguuucgagggauTT
300
 76 ± 5


 


AD-11273
gaaacuaacaacuuccgucTT
301
gacggaaguuguuaguuucTT
302
 21 ± 3


 


AD-11274
uuccgucgcuuuacuccggTT
303
ccggaguaaagcgacggaaTT
304
 87 ± 6


 


AD-11275
ccgucgcuuuacuccggagTT
305
cuccggaguaaagcgacggTT
306
 29 ± 2


 


AD-11276
ggaucuagauccguucuacTT
307
guagaacggaucuagauccTT
308
 22 ± 3


 


AD-11277
cacacaccggacauuuaugTT
309
cauaaauguccggugugugTT
310
 57 ± 2


 


AD-11278
cacaccggacauuuaugguTT
311
accauaaauguccggugugTT
312
101 ± 1


 


AD-11279
ggaccauuucccgguuuagTT
313
cuaaaccgggaaaugguccTT
314
 45 ± 4


 


AD-11280
accauuucccgguuuagugTT
315
cacuaaaccgggaaaugguTT
316
 89 ± 2


 


AD-11281
auuucccgguuuagugccaTT
317
uggcacuaaaccgggaaauTT
318
100 ± 4


 


AD-11282
aaccugaucagaagaacggTT
319
ccguucuucugaucagguuTT
320
 66 ± 5


 


AD-11283
ucaguuuauuuauuacgguTT
321
accguaauaaauaaacugaTT
322
 76 ± 4


 


AD-11284
gugcaugacccgaacugacTT
323
gucaguucgggucaugcacTT
324
 29 ± 1


 


AD-11285
augaguucacguaccugagTT
325
cucagguacgugaacucauTT
326
 37 ± 4


 


AD-11286
agcgucauuacccuggcauTT
327
augccaggguaaugacgcuTT
328
 31 ± 1


 


AD-11287
gcgucauuacccuggcauaTT
329
uaugccaggguaaugacgcTT
330
 16 ± 1


 


AD-11288
gucauuacccuggcauaugTT
331
cauaugccaggguaaugacTT
332
 21 ± 1


 


AD-11289
acagcaauagaucuccgugTT
333
cacggagaucuauugcuguTT
334
 64 ± 4


 


AD-11290
ggacauucagaguucuuagTT
335
cuaagaacucugaauguccTT
336
 26 ± 1


 


AD-11291
ucuacauaaauaagcgaggTT
337
ccucgcuuauuuauguagaTT
338
 88 ± 15


 


AD-11292
uaaauaagcgaggcacuucTT
339
gaagugccucgcuuauuuaTT
340
 68 ± 2


 


AD-11293
ugcccugaugguuauaucuTT
341
agauauaaccaucagggcaTT
342
 39 ± 3


 


AD-11294
caacccggauuuuaacuacTT
343
guaguuaaaauccggguugTT
344
 26 ± 2


 


AD-11295
gggaaaaucuauaugaucuTT
345
agaucauauagauuuucccTT
346
 15 ± 1


 


AD-11296
gcccucgagaugcuccggaTT
347
uccggagcaucucgagggcTT
348
 54 ± 9


 


AD-11297
agggauugacacaaccucuTT
349
agagguugugucaaucccuTT
350
 22 ± 1


 


AD-11298
gggauugacacaaccucucTT
351
gagagguugugucaaucccTT
352
 22 ± 2


 


AD-11299
cacaauggaucaccuuuaaTT
353
uuaaaggugauccauugugTT
354
 23 ± 0


 


AD-11300
ccgcucugauccuuacaacTT
355
guuguaaggaucagagcggTT
356
 34 ± 2


 


AD-11301
auccuuacaaccagcgcagTT
357
cugcgcugguuguaaggauTT
358
 90 ± 5


 


AD-11302
agcgcaggaugucuuuucuTT
359
agaaaagacauccugcgcuTT
360
 46 ± 3


 


AD-11303
cuuuucuaggccucgccucTT
361
gaggcgaggccuagaaaagTT
362
 94 ± 10


 


AD-11304
cuggaaaacgccgggcuagTT
363
cuagcccggcguuuuccagTT
364
 57 ± 2


 


AD-11305
aaacgccgggcuagucaugTT
365
caugacuagcccggcguuuTT
366
 95 ± 7


 


AD-11306
aacgccgggcuagucauggTT
367
ccaugacuagcccggcguuTT
368
101 ± 5


 


AD-11307
agaccacgaaagccaucggTT
369
ccgauggcuuucguggucuTT
370
 89 ± 6


 


AD-11308
cucccuagaagcccucuucTT
371
gaagagggcuucuagggagTT
372
 50 ± 3


 


AD-11309
agaugaacaccaaccgccgTT
373
cggcgguugguguucaucuTT
374
 62 ± 7


 


AD-11310
augaacaccaaccgccgccTT
375
ggcggcgguugguguucauTT
376
 99 ± 6


 


AD-11311
aaccgccgcccacuagugaTT
377
ucacuagugggcggcgguuTT
378
 72 ± 2


 


AD-11312
accgccgcccacuagugagTT
379
cucacuagugggcggcgguTT
380
 99 ± 13


 


AD-11313
gcugucgaugucucggcauTT
381
augccgagacaucgacagcTT
382
 67 ± 8


 


AD-11314
ucgaugucucggcauucgaTT
383
ucgaaugccgagacaucgaTT
384
 36 ± 2


 


AD-11315
ucucggcauucgaugcaggTT
385
ccugcaucgaaugccgagaTT
386
 99 ± 5


 


AD-11316
ggcauucgaugcaggacaaTT
387
uuguccugcaucgaaugccTT
388
 48 ± 5


 


AD-11317
gcauucgaugcaggacaaaTT
389
uuuguccugcaucgaaugcTT
390
 56 ± 5


 


AD-11318
cuuagaugaaccuuuccggTT
391
ccggaaagguucaucuaagTT
392
 47 ± 3


 


AD-11319
gaguguugucaguaucauaTT
393
uaugauacugacaacacucTT
394
 22 ± 2


 


AD-11320
caguaucauaaccuccgucTT
395
gacggagguuaugauacugTT
396
 27 ± 2


 


AD-11321
cucgaggagucugaacagaTT
397
ucuguucagacuccucgagTT
398
 32 ± 2


 


AD-11322
guaucugaucugggauugcTT
399
gcaaucccagaucagauacTT
400
 65 ± 3


 


AD-11323
agacaauucucuuugggcuTT
401
agcccaaagagaauugucuTT
402
 73 ± 2


 


AD-11324
caccuugugcaucguggugTT
403
caccacgaugcacaaggugTT
404
 47 ± 4


 


AD-11325
gcaugagcccuaccuucgaTT
405
ucgaagguagggcucaugcTT
406
 25 ± 1


 


AD-11326
uaccuucgaagccaugcucTT
407
gagcauggcuucgaagguaTT
408
 71 ± 2


 


AD-11327
caaaaucauugccuucgacTT
409
gucgaaggcaaugauuuugTT
410
 33 ± 3


 


AD-11328
aucauugccuucgacccauTT
411
augggucgaaggcaaugauTT
412
 38 ± 2


 


AD-11329
ucauugccuucgacccauaTT
413
uaugggucgaaggcaaugaTT
414
 29 ± 1


 


AD-11330
auugccuucgacccauacuTT
415
aguaugggucgaaggcaauTT
416
 77 ± 7


 


AD-11331
ucgacccauacuauuauuuTT
417
aaauaauaguaugggucgaTT
418
 55 ± 3


 


AD-11332
caucaucgucacugugaguTT
419
acucacagugacgaugaugTT
420
 31 ± 3


 


AD-11333
caucgucacugugagucugTT
421
cagacucacagugacgaugTT
422
 33 ± 4


 


AD-11334
ucgucacugugagucugcuTT
423
agcagacucacagugacgaTT
424
 44 ± 4


 


AD-11335
ggaaaacuaccguaacaacTT
425
guuguuacgguaguuuuccTT
426
 34 ± 1


 


AD-11336
uaccguaacaaccgaaaaaTT
427
uuuuucgguuguuacgguaTT
428
 21 ± 3


 


AD-11337
cguaacaaccgaaaaaauaTT
429
uauuuuuucgguuguuacgTT
430
 29 ± 2


 


AD-11338
aaaauccauaugccucaucTT
431
gaugaggcauauggauuuuTT
432
 87 ± 4


 


AD-11339
cuguucaucgcccugcuauTT
433
auagcagggcgaugaacagTT
434
 40 ± 2


 


AD-11340
uguucaucgcccugcuauuTT
435
aauagcagggcgaugaacaTT
436
 26 ± 1


 


AD-11341
cccugcuauugaacucuuuTT
437
aaagaguucaauagcagggTT
438
 29 ± 1


 


AD-11342
ggccaucguaccaaacaggTT
439
ccuguuugguacgauggccTT
440
 41 ± 2


 


AD-11343
ccaucguaccaaacaggcuTT
441
agccuguuugguacgauggTT
442
 47 ± 2


 


AD-11344
cagugacuucaucgcuaauTT
443
auuagcgaugaagucacugTT
444
 33 ± 2


 


AD-11345
cuucaucgcuaauccgacuTT
445
agucggauuagcgaugaagTT
446
 36 ± 2


 


AD-11346
uaauccgacugugugggucTT
447
gacccacacagucggauuaTT
448
 97 ± 3


 


AD-11347
gaaucugaucuugaugacuTT
449
agucaucaagaucagauucTT
450
 45 ± 2


 


AD-11348
aagaguccaugggauguggTT
451
ccacaucccauggacucuuTT
452
100 ± 2


 


AD-11349
gcaggugcgcaagacuugcTT
453
gcaagucuugcgcaccugcTT
454
 39 ± 3


 


AD-11350
gcgcaagacuugcuaccguTT
455
acgguagcaagucuugcgcTT
456
 29 ± 1


 


AD-11351
aagacuugcuaccguaucgTT
457
cgauacgguagcaagucuuTT
458
 82 ± 2


 


AD-11352
gacuugcuaccguaucgugTT
459
cacgauacgguagcaagucTT
460
 56 ± 2


 


AD-11353
cucauugugaauaucucacTT
461
gugagauauucacaaugagTT
462
 54 ± 3


 


AD-11354
ugauaagucucacagcgaaTT
463
uucgcugugagacuuaucaTT
464
 31 ± 1


 


AD-11355
aucaaagcccuucgaacccTT
465
ggguucgaagggcuuugauTT
466
 87 ± 8


 


AD-11356
aaagcccuucgaacccuucTT
467
gaaggguucgaagggcuuuTT
468
 83 ± 3


 


AD-11357
ccuucgaacccuucgcgcuTT
469
agcgcgaaggguucgaaggTT
470
 50 ± 1


 


AD-11358
cuucgaacccuucgcgcucTT
471
gagcgcgaaggguucgaagTT
472
 55 ± 4


 


AD-11359
ugcaucaacuauaccgaugTT
473
caucgguauaguugaugcaTT
474
 59 ± 7


 


AD-11360
cccuuguaccuuugucgauTT
475
aucgacaaagguacaagggTT
476
 24 ± 3


 


AD-11361
uuguaccuuugucgauuguTT
477
acaaucgacaaagguacaaTT
478
 39 ± 6


 


AD-11362
uuugauaauguugcaauggTT
479
ccauugcaacauuaucaaaTT
480
110 ± 14


 


AD-11363
gcaauggguuaccuugcacTT
481
gugcaagguaacccauugcTT
482
 93 ± 6


 


AD-11364
uggguuaccuugcacuucuTT
483
agaagugcaagguaacccaTT
484
 54 ± 11


 


AD-11365
gcuguugauucccgggaggTT
485
ccucccgggaaucaacagcTT
486
 36 ± 1


 


AD-11366
aucgugaccagacaagcuuTT
487
aagcuugucuggucacgauTT
488
 36 ± 5


 


AD-11367
cgucuucacaggcgaauguTT
489
acauucgccugugaagacgTT
490
 57 ± 2


 


AD-11368
ucacaggcgaaugugucauTT
491
augacacauucgccugugaTT
492
 76 ± 5


 


AD-11369
cacaggcgaaugugucaugTT
493
caugacacauucgccugugTT
494
 75 ± 4


 


AD-11370
caggcgaaugugucaugaaTT
495
uucaugacacauucgccugTT
496
 32 ± 5


 


AD-11371
aggcgaaugugucaugaagTT
497
cuucaugacacauucgccuTT
498
 48 ± 6


 


AD-11372
uguucgcuuugaggcaguaTT
499
uacugccucaaagcgaacaTT
500
 36 ± 4


 


AD-11373
gcaguacuacuucacaaauTT
501
auuugugaaguaguacugcTT
502
 27 ± 2


 


AD-11374
uccauugcgagccugauuuTT
503
aaaucaggcucgcaauggaTT
504
 24 ± 2


 


AD-11375
ccauugcgagccugauuuuTT
505
aaaaucaggcucgcaauggTT
506
 21 ± 3


 


AD-11376
aucgggcuguugcuauuccTT
507
ggaauagcaacagcccgauTT
508
 78 ± 11


 


AD-11377
aaaacccaaucgaaauauaTT
509
uauauuucgauuggguuuuTT
510
 33 ± 4


 


AD-11378
caaucgaaauauacugaucTT
511
gaucaguauauuucgauugTT
512
 66 ± 8


 


AD-11379
aaucgaaauauacugauccTT
513
ggaucaguauauuucgauuTT
514
128 ± 8


 


AD-11380
ucgaaauauacugauccagTT
515
cuggaucaguauauuucgaTT
516
119 ± 7


 


AD-11381
aaucauccuaugaaccaauTT
517
auugguucauaggaugauuTT
518
 39 ± 3


 


AD-11382
uaugaaccaauagcaaccaTT
519
ugguugcuauugguucauaTT
520
 61 ± 4


 


AD-11383
cacucuccgauggaagcaaTT
521
uugcuuccaucggagagugTT
522
 69 ± 5


 


AD-11384
cuaucggagcuaugugcugTT
523
cagcacauagcuccgauagTT
524
 95 ± 10


 


AD-11385
agcaaaugaaaauuguguaTT
525
uacacaauuuucauuugcuTT
526
 79 ± 6


 


AD-11386
uacucccagacaaaucugaTT
527
ucagauuugucugggaguaTT
528
 36 ± 4


 


AD-11387
agagugucacuagaggccuTT
529
aggccucuagugacacucuTT
530
 36 ± 1


 


AD-11388
ggccuuagugauagagucaTT
531
ugacucuaucacuaaggccTT
532
 35 ± 4


 


AD-11389
agugauagagucaacaugaTT
533
ucauguugacucuaucacuTT
534
 29 ± 5


 


AD-11390
gauagagucaacaugaggaTT
535
uccucauguugacucuaucTT
536
 32 ± 3


 


AD-11391
caucuagcucaauacaaaaTT
537
uuuuguauugagcuagaugTT
538
 23 ± 3


 


AD-11392
cuagcucaauacaaaaugaTT
539
ucauuuuguauugagcuagTT
540
 28 ± 5


 


AD-11393
aguauggagcugauugcccTT
541
gggcaaucagcuccauacuTT
542
108 ± 8









Example 2
Optimization of siRNAs by Chemical Modification
As has been experienced by those working in the antisense field, ribonucleic acids are often quickly degraded by a range of nucleases present in virtually all biological environments, e.g. endonucleases, exonucleases etc. This vulnerability may be circumvented by chemically modifying these oligonucleotides such that nucleases may no longer attack. Consequently, siRNAs in Table 1 represent chemically modified oligonucleotides; these chemically modified siRNAs were tested for inhibitory activity on Nav1.8 gene expression (Nav1.8 mRNA levels).
dsRNA Synthesis
Source of Reagents
Where the source of a reagent is not specifically given herein, such reagent may be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.
siRNA Synthesis
Single-stranded RNAs were produced by solid phase synthesis on a scale of 1 μmole using an Expedite 8909 synthesizer (Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass (CPG, 500 Å, Proligo Biochemie GmbH, Hamburg, Germany) as solid support. RNA and RNA containing 2′-O-methyl nucleotides were generated by solid phase synthesis employing the corresponding phosphoramidites and 2′-O-methyl phosphoramidites, respectively (Proligo Biochemie GmbH, Hamburg, Germany). These building blocks were incorporated at selected sites within the sequence of the oligoribonucleotide chain using standard nucleoside phosphoramidite chemistry such as described in Current protocols in nucleic acid chemistry, Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA. Phosphorothioate linkages were introduced by replacement of the iodine oxidizer solution with a solution of the Beaucage reagent (Chruachem Ltd, Glasgow, UK) in acetonitrile (1%). Further ancillary reagents were obtained from Mallinckrodt Baker (Griesheim, Germany).
Deprotection and purification of the crude oligoribonucleotides by anion exchange HPLC were carried out according to established procedures. Yields and concentrations were determined by UV absorption of a solution of the respective RNA at a wavelength of 260 nm using a spectral photometer (DU 640B, Beckman Coulter GmbH, Unterschleiβheim, Germany). Double stranded RNA was generated by mixing an equimolar solution of complementary strands in annealing buffer (20 mM sodium phosphate, pH 6.8; 100 mM sodium chloride), heated in a water bath at 85-90° C. for 3 minutes and cooled to room temperature over a period of 3-4 hours. The annealed RNA solution was stored at −20 ° C. until use.
For the synthesis of 3′-cholesterol-conjugated siRNAs (herein referred to as -Chol or -sChol, depending on whether the link to the cholesteryl group is effected via a phosphodiester or a phosporothioate diester group), an appropriately modified solid support was used for RNA synthesis. The modified solid support was prepared as follows:
Diethyl-2-azabutane-1,4-dicarboxylate AA




A 4.7 M aqueous solution of sodium hydroxide (50 mL) was added into a stirred, ice-cooled solution of ethyl glycinate hydrochloride (32.19 g, 0.23 mole) in water (50 mL). Then, ethyl acrylate (23.1 g, 0.23 mole) was added and the mixture was stirred at room temperature until completion of the reaction was ascertained by TLC. After 19 h the solution was partitioned with dichloromethane (3×100 mL). The organic layer was dried with anhydrous sodium sulfate, filtered and evaporated. The residue was distilled to afford AA (28.8 g, 61%).
3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl]-amino }-propionic acid ethyl ester AB




Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) was dissolved in dichloromethane (50 mL) and cooled with ice. Diisopropylcarbodiimde (3.25 g, 3.99 mL, 25.83 mmol) was added to the solution at 0° C. It was then followed by the addition of Diethyl-azabutane-1,4-dicarboxylate (5 g, 24.6 mmol) and dimethylamino pyridine (0.305 g, 2.5 mmol). The solution was brought to room temperature and stirred further for 6 h. Completion of the reaction was ascertained by TLC. The reaction mixture was concentrated under vacuum and ethyl acetate was added to precipitate diisopropyl urea. The suspension was filtered. The filtrate was washed with 5% aqueous hydrochloric acid, 5% sodium bicarbonate and water. The combined organic layer was dried over sodium sulfate and concentrated to give the crude product which was purified by column chromatography (50% EtOAC/Hexanes) to yield 11.87 g (88%) of AB.
3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethyl ester AC




3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoyl]-amino}-propionic acid ethyl ester AB (11.5 g, 21.3 mmol) was dissolved in 20% piperidine in dimethylformamide at 0° C. The solution was continued stirring for 1 h. The reaction mixture was concentrated under vacuum, water was added to the residue, and the product was extracted with ethyl acetate. The crude product was purified by conversion into its hydrochloride salt.
3-({6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}ethoxycarbonylmethyl-amino)-propionic acid ethyl ester AD




The hydrochloride salt of 3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethyl ester AC (4.7 g, 14.8 mmol) was taken up in dichloromethane. The suspension was cooled to 0° C. on ice. To the suspension diisopropylethylamine (3.87 g, 5.2 mL, 30 mmol) was added. To the resulting solution cholesteryl chloroformate (6.675 g, 14.8 mmol) was added. The reaction mixture was stirred overnight. The reaction mixture was diluted with dichloromethane and washed with 10% hydrochloric acid. The product was purified by flash chromatography (10.3 g, 92%).
1-{6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-4-oxo-pyrrolidine-3-carboxylic acid ethyl ester AE




Potassium t-butoxide (1.1 g, 9.8 mmol) was slurried in 30 mL of dry toluene. The mixture was cooled to 0° C. on ice and 5 g (6.6 mmol) of diester AD was added slowly with stirring within 20 mins. The temperature was kept below 5° C. during the addition. The stirring was continued for 30 mins at 0° C. and 1 mL of glacial acetic acid was added, immediately followed by 4 g of NaH2PO4.H2O in 40 mL of water The resultant mixture was extracted twice with 100 mL of dichloromethane each and the combined organic extracts were washed twice with 10 mL of phosphate buffer each, dried, and evaporated to dryness. The residue was dissolved in 60 mL of toluene, cooled to 0° C. and extracted with three 50 mL portions of cold pH 9.5 carbonate buffer. The aqueous extracts were adjusted to pH 3 with phosphoric acid, and extracted with five 40 mL portions of chloroform which were combined, dried and evaporated to dryness. The residue was purified by column chromatography using 25% ethylacetate/hexane to afford 1.9 g of b-ketoester (39%).
[6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamic acid 17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester AF




Methanol (2 mL) was added dropwise over a period of 1 h to a refluxing mixture of b-ketoester AE (1.5 g, 2.2 mmol) and sodium borohydride (0.226 g, 6 mmol) in tetrahydrofuran (10 mL). Stirring was continued at reflux temperature for 1 h. After cooling to room temperature, 1 N HCl (12.5 mL) was added, the mixture was extracted with ethylacetate (3×40 mL). The combined ethylacetate layer was dried over anhydrous sodium sulfate and concentrated under vacuum to yield the product which was purified by column chromatography (10% MeOH/CHCl3) (89%).
(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamic acid 17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester AG




Diol AF (1.25 gm 1.994 mmol) was dried by evaporating with pyridine (2×5 mL) in vacuo. Anhydrous pyridine (10 mL) and 4,4′-dimethoxytritylchloride (0.724 g, 2.13 mmol) were added with stirring. The reaction was carried out at room temperature overnight. The reaction was quenched by the addition of methanol. The reaction mixture was concentrated under vacuum and to the residue dichloromethane (50 mL) was added. The organic layer was washed with 1M aqueous sodium bicarbonate. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The residual pyridine was removed by evaporating with toluene. The crude product was purified by column chromatography (2% MeOH/Chloroform, Rf=0.5 in 5% MeOH/CHCl3) (1.75 g, 95%).
Succinic acid mono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl 2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl) ester AH




Compound AG (1.0 g, 1.05 mmol) was mixed with succinic anhydride (0.150 g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and dried in a vacuum at 40° C. overnight. The mixture was dissolved in anhydrous dichloroethane (3 mL), triethylamine (0.318 g, 0.440 mL, 3.15 mmol) was added and the solution was stirred at room temperature under argon atmosphere for 16 h. It was then diluted with dichloromethane (40 mL) and washed with ice cold aqueous citric acid (5 wt%, 30 mL) and water (2×20 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness. The residue was used as such for the next step.
Cholesterol derivatised CPG AI




Succinate AH (0.254 g, 0.242 mmol) was dissolved in a mixture of dichloromethane/acetonitrile (3:2, 3 mL). To that solution DMAP (0.0296 g, 0.242 mmol) in acetonitrile (1.25 mL), 2,2′-Dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) in acetonitrile/dichloroethane (3:1, 1.25 mL) were added successively. To the resulting solution triphenylphosphine (0.064 g, 0.242 mmol) in acetonitrile (0.6 ml) was added. The reaction mixture turned bright orange in color. The solution was agitated briefly using a wrist-action shaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (1.5 g, 61 mM) was added. The suspension was agitated for 2 h. The CPG was filtered through a sintered funnel and washed with acetonitrile, dichloromethane and ether successively. Unreacted amino groups were masked using acetic anhydride/pyridine. The achieved loading of the CPG was measured by taking UV measurement (37 mM/g).
The synthesis of siRNAs bearing a 5′-12-dodecanoic acid bisdecylamide group (herein referred to as “5′-C32-”) or a 5′-cholesteryl derivative group (herein referred to as “5′-Chol-”) was performed as described in WO 2004/065601, except that, for the cholesteryl derivative, the oxidation step was performed using the Beaucage reagent in order to introduce a phosphorothioate linkage at the 5′-end of the nucleic acid oligomer.
Nucleic acid sequences are represented below using standard nomenclature, and specifically the abbreviations of Table 2.







TABLE 2







Abbreviations of nucleotide monomers used in nucleic acid


sequence representation. It will be understood that these


monomers, when present in an oligonucleotide, are mutually


linked by 5′-3′-phosphodiester bonds.








Abbrevi-



ationa
Nucleotide(s)





A, a
2′-deoxy-adenosine-5′-phosphate, adenosine-5′-phosphate


C, c
2′-deoxy-cytidine-5′-phosphate, cytidine-5′-phosphate


G, g
2′-deoxy-guanosine-5′-phosphate, guanosine-5′-phosphate


T, t
2′-deoxy-thymidine-5′-phosphate, thymidine-5′-phosphate


U, u
2′-deoxy-uridine-5′-phosphate, uridine-5′-phosphate


N, n
any 2′-deoxy-nucleotide/nucleotide (G, A, C, or T, g, a, c



or u)


Am
2′-O-methyladenosine-5′-phosphate


Cm
2′-O-methylcytidine-5′-phosphate


Gm
2′-O-methylguanosine-5′-phosphate


Tm
2′-O-methyl-thymidine-5′-phosphate


Um
2′-O-methyluridine-5′-phosphate


Af
2′-fluoro-2′-deoxy-adenosine-5′-phosphate


Cf
2′-fluoro-2′-deoxy-cytidine-5′-phosphate


Gf
2′-fluoro-2′-deoxy-guanosine-5′-phosphate


Tf
2′-fluoro-2′-deoxy-thymidine-5′-phosphate


Uf
2′-fluoro-2′-deoxy-uridine-5′-phosphate


A, C, G,
underlined: nucleoside-5′-phosphorothioate


T, U, a,


c, g, t, u


am, cm, gm,
underlined: 2-O-methyl-nucleoside-5′-phosphoro-


tm, um
thioate





acapital letters represent 2′-deoxyribonucleotides (DNA), lower case letters represent ribonucleotides (RNA)






Single-Dose Screen of Nav1.8 siRNAs against mRNA Expression of Transfected Human Nav1.8 in Cos-7 Cells.
All Nav1.8 siRNAs in Table 1 and Table 4 were tested initially at a single dose of 100 nM (Table 1) or 50 nM (Table 4) for activity in reducing mRNA expression of transfected human Nav1.8 in Cos-7 cells. One day before transfection, Cos-7 cells (DSMZ, Braunschweig, Germany) were seeded at 1.5×104 cells/well on 96-well plates (Greiner Bio-One GmbH, Frickenhausen, Germany) in 100 μl of growth medium (Dulbecco's MEM, 10% fetal calf serum, 2 mM L-glutamine, 1.2 μg/ml sodium bicarbonate, 100 u penicillin/100 μg/ml streptomycin, all from Biochrom AG, Berlin, Germany). Four hours prior to siRNA transfection, 20 ng of plasmid/well (Table 1) or 50 ng/well (Table 4) were transfected with Lipofectamine2000 (Invitrogen) as described below for the siRNAs, with the plasmid diluted in Opti-MEM to a final volume of 12.5 μl/well, prepared as a mastermix for the whole plate.
siRNA transfections were performed in triplicate. For each well, 0.5 μl Lipofectamine2000 (Invitrogen GmbH, Karlsruhe, Germany) was mixed with 12 μl Opti-MEM (Invitrogen) and incubated for 15 min at room temperature. For an siRNA concentration of 100 nM in a transfection volume of 100 μl, 2 μl of a 5 μM siRNA were mixed with 10.5 μl Opti-MEM per well, combined with the Lipofectamine2000-Opti-MEM mixture and again incubated for 15 minutes at room temperature. During that incubation time, growth medium was removed from cells and replaced by 75 μl/well of fresh medium. In six wells,. growth medium was replaced by 100 μl of fresh medium and cells were lysed immediately by adding lysis mixture, as described below, in order to analyse the background value in the bDNA-assay caused by the Nav1.8-cDNA in the plasmid. siRNA-Lipofectamine2000-complexes were applied completely (25 μl each per well) to the cells and cells were incubated for 24 h at 37° C. and 5% CO2 in a humidified incubator (Heraeus GmbH, Hanau, Germany).
Cells were harvested by applying 50 μl of lysis mixture (from the QuantiGene bDNA-kit from Genospectra, Fremont, USA) to each well and were lysed at 53° C. for 30 min. Afterwards, 50 μl of the lysates were incubated with probesets specific to human Nav1.8 and human GAPDH (sequence of probesets see below) and processed according to the manufacturer's protocol for QuantiGene. Chemoluminescence was measured in a Victor2-Light (Perkin Elmer, Wiesbaden, Germanv) as RLUs (relative light units) and values obtained with the human Nav1.8 probeset were normalized to the respective human GAPDH (GAPDH sequence of Cercopithecus aethiops so far unknown) values for each well. Values obtained with cells lysed 4 h after plasmid transfection were subtracted from the values obtained with cells lysed 24 h after siRNA transfection. Values acquired with siRNAs directed against Nav1.8 were further normalized relative to the value obtained with an unrelated siRNA (directed against hepatitis C virus) which was set to 100%.
FIG. 1 provides the results from a representative experiment where siRNAs from Table 1 were tested at a single dose of 100 nM and Table 4 provides the results for additional siRNAs at a dose of 50 nM. Several siRNAs (in Tables 1 and 4) were effective at the dose tested in reducing Nav1.8 mRNA levels by at least 50% in COS-7 cells transfected with Nav1.8.
Dose-Response Curves for Selected siRNAs against mRNA Expression of Transfected Human Nav1.8 in Cos-7 Cells
Several effective siRNAs against Nav1.8 from the single dose screen (results in FIG. 1) were further characterized by dose response curves. For dose response curves, transfections were performed as for the single dose screen above, but with the following concentrations of siRNA (nM): 100, 33, 11, 3.7, 1.2, 0.4, 1, 0.14, 0.05, 0.015, 0.005 and mock (no siRNA). siRNAs were diluted with Opti-MEM to a final volume of 12.5 μl according to the above protocol.
Three independent dose response experiments were carried out to generate dose response curves (DRCs). The dose response curves were repeated, and a summary of the results are provided in Table 3.







TABLE 3







IC50 values for selected siRNAs targeting Nav1.8











IC50-values [nM]:
1st DRC screen
2nd DRC screen















AL-DP-6218
0.0068
0.021



AL-DP-6217
0.036
0.0021



AL-DP-6050
0.013
0.16



AL-DP-6209
0.060
0.012



AL-DP-6042
nd
0.016



AL-DP-6049
0.24
0.0010



AL-DP-6219
0.13
0.0069



AL-DP-6223
73140
22



AL-DP-6225
0.70
8.07










Specificity Testing of the Nav1.8 siRNAs against Nav1.5 MRNA
Nav1.5 is an NaV subtype that is closely related to Nav1.8, but has an important role in normal cardiac function. Therefore, it is important to confirm that siRNAs selective for Nav1.8 do not inhibit Nav1.5 expression. Several siRNAs were tested for specificity towards Nav1.8 by transfecting SW620 cells, which express endogenous human Nav1.5, with these siRNAs, and assessing Nav1.5 mRNA levels. A control unrelated siRNA (AL-DP-5002) was also transfected into SW620 cells. siRNAs targeting Nav1.8 were tested at the following doses: 1200 nM, 400 nM, 133.3 nM, 44.4 nM, 14.8 nM, 4.9 nM, 1.6 nM, and mock (without siRNA). One siRNA targeting Nav1.8 (AL-DP-6217) was tested at 1 nM, 10 nM, 100 nM and 1 uM. The expression of Nav1.5 mRNA, which encodes the protein with the highest homology to Nav1.8 in the Nav-family, was then quantified.
One day before transfection, SW620 cells (LCG Promochem, Wesel, Germany) were seeded at 1.5×104 cells/well on 96-well plates (Greiner Bio-One GmbH, Frickenhausen, Germany) in 100 μl of growth medium (Leibowitz L-15 Medium, 10% fetal calf serum, 2 mM L-glutamine, 100 u penicillin/100 μg/ml streptomycin, all from Biochrom AG, Berlin, Germany).
siRNA transfections were performed in triplicate. For each well, 0.6 μl Oligofectamine (Invitrogen GmbH, Karlsruhe, Germany) was mixed with 2.4 μl Opti-MEM (Invitrogen) and incubated for 10 min at room temperature. For an siRNA concentration of 1200 nM in 100 μl transfection volume, 5 μl of 24 μM siRNA was mixed with 12 μl Opti-MEM per well, combined with the Oligofectamine-Opti-MEM mixture and again incubated for 20 minutes at room temperature. During that incubation time, growth medium was removed from cells and replaced by 80 μl/well of fresh growth medium without serum. siRNA-Oligofectamine-complexes were applied completely (20 μl each per well) to the cells and cells were incubated for 24 h at 37° C. without CO2 in a humidified incubator (Heraeus GmbH, Hanau, Germany).
Cells were harvested by applying 50 μl of lysis mixture (content of the QuantiGene bDNA-kit from Genospectra, Fremont, USA) to each well and were lysed at 53° C. for 30 min. Afterwards, 50 μl of the lysates were incubated with probesets specific to human Nav1.5 and human GAPDH (sequence of probesets see below) and processed according to the manufacturer's protocol for QuantiGene. Chemoluminescence was measured in a Victor2-Light (Perkin Elmer, Wiesbaden, Germany) as RLUs (relative light units) and values obtained with the human Nav1.5 probeset were normalized to the respective human GAPDH values for each well.
An unrelated control siRNA (directed against hepatitis C virus) was used as a negative control.
FIG. 2 provides the results. At the concentrations tested, the selected siRNAs targeting Nav1.8, did not exhibit significant dose-dependent inhibition of Nav1.5 mRNA as compared with the unrelated control siRNA (AL-DP-5002), confirming the specificity of these Nav1.8 siRNAs for Nav1.8 over Nav1.5.
In addition, at concentrations up to 1 uM, AL-DP-6217 exhibited no significant inhibition of Nav1.5 mRNA (data not shown), confirming the specificity of this Nav1.8 siRNA for Nav1.8 over Nav1.5.








TABLE 5







bDNA Probesets for the quantitation of



human Nav1.8, Nav1.5 and GAPDH














SEQ






ID


FPL Name
Function
Sequence
NO:










Human Nav1.8 probeset:











hNa182001
CE
TTTGGAGGTTAAAGGTGATCCATTTTTTCTCTTGGAAAGAAAGT
543



 


hNa182002
CE
GGCGTTTTCCAGAGGCGAGTTTTTCTCTTGGAAAGAAAGT
544


 


hNa182003
CE
CCAGCAGCAGAGAGCCCCTTTTTCTCTTGGAAAGAAAGT
545


 


hNa182004
CE
CTGGGTTGAGGAAGAGGGCTTTTTTCTCTTGGAAAGAAAGT
546


 


hNa182005
CE
AACACTCATTGCCCTTTGGGTTTTTCTCTTGGAAAGAAAGT
547


 


hNa182006
CE
AAGGACGGAGGTTATGATACTGACTTTTTCTCTTGGAAAGAAAGT
548


 


hNa182007
CE
TGTTCAGACTCCTCGAGTTCCTCTTTTTCTCTTGGAAAGAAAGT
549


 


hNa182008
LE
CTATGCCTTCTCTCACTGGCATTTTTTTAGGCATAGGACCCGTGTCT
550


 


hNa182009
LE
CTGGTTGTAAGGATCAGAGCGGTTTTTAGGCATAGGACCCGTGTCT
551


 


hNa182010
LE
AACACACTGCCATGACTAGCCCTTTTTAGGCATAGGACCCGTGTCT
552


 


hNa182011
LE
GATGGCTTTCGTGGTCTCCATTTTTAGGCATAGGACCCGTGTCT
553


 


hNa182012
LE
CTGGCCAGCACCCCCACTTTTTAGGCATAGGACCCGTGTCT
554


 


hNa182013
LE
CATGCCTGGAGTCAGGGTTGTTTTTAGGCATAGGACCCGTGTCT
555


 


hNa182014
LE
AGACATCGACAGCTCCAGGGTTTTTAGGCATAGGACCCGTGTCT
556


 


hNa182015
LE
GTCCTGCATCGAATGCCGTTTTTAGGCATAGGACCCGTGTCT
557


 


hNa182016
LE
CAAGCAGGGTGGGCACTTCTTTTTAGGCATAGGACCCGTGTCT
558


 


hNa182017
BL
TGTGGGAGTGGAGAGAGGTTG
559


 


hNa182018
BL
CCCTCTGACACTCTTGGCTTTATT
560


 


hNa182019
BL
GGTGATTTGTTGTCTTCTGTGGAG
561


 


hNa182020
BL
GCCTAGAAAAGACATCCTGCG
562


 


hNa182021
BL
GCCAGGGGACCGGAAATGG
563


 


hNa182022
BL
CCCTCAGGGAGTGAGATATCTCG
564


 


hNa182023
BL
GGAAAGACTCCATCATCTGTGACT
565


 


hNa182024
BL
TCTAGGGAGGGGGCCTTG
566


 


hNa182025
BL
GCGGTTGGTGTTCATCTTCTC
567


 


hNa182026
BL
GCAAGCTCACTAGTGGGCG
568


 


hNa182027
BL
TCTGCTGACAAGAAAGTCTTCTTTT
569


 


hNa182028
BL
CCCGGAAAGGTTCATCTAAGTAT
570


 







Human GAPDH probeset:











hGAP001
CE
GAATTTGCCATGGGTGGAATTTTTTCTCTTGGAAAGAAAGT
571



 


hGAP002
CE
GGAGGGATCTCGCTCCTGGATTTTTCTCTTGGAAAGAAAGT
572


 


hGAP003
CE
CCCCAGCCTTCTCCATGGTTTTTTCTCTTGGAAAGAAAGT
573


 


hGAP004
CE
GCTCCCCCCTGCAAATGAGTTTTTCTCTTGGAAAGAAAGT
574


 


hGAP005
LE
AGCCTTGACGGTGCCATGTTTTTAGGCATAGGACCCGTGTCT
575


 


hGAP006
LE
GATGACAAGCTTCCCGTTCTCTTTTTAGGCATAGGACCCGTGTCT
576


 


hGAP007
LE
AGATGGTGATGGGATTTCCATTTTTTTAGGCATAGGACCCGTGTCT
577


 


hGAP008
LE
GCATCGCCCCACTTGATTTTTTTTTAGGCATAGGACCCGTGTCT
578


 


hGAP009
LE
CACGACGTACTCAGCGCCATTTTTAGGCATAGGACCCGTGTCT
579


 


hGAP010
LE
GGCAGAGATGATGACCCTTTTGTTTTTAGGCATAGGACCCGTGTCT
580


 


hGAP011
BL
GGTGAAGACGCCAGTGGACTC
581


 







Human Nav1.5 probeset:











hNa15001
CE
CGTTTTCTCCTCTTGCTTCTTCTCTTTTTCTCTTGGAAAGAAAGT
582



 


hNa15002
CE
GGGAGCCTGTCCTCCCCATTTTTCTCTTGGAAAGAAAGT
583


 


hNa15003
CE
TCGCCTGCGAAAGGTGAATTTTTCTCTTGGAAAGAAAGT
584


 


hNa15004
CE
CTCTCGCTCTCCCCCGCTTTTTCTCTTGGAAAGAAAGT
585


 


hNa15005
CE
CCGGGACTGGGCTGTCCTTTTTCTCTTGGAAAGAAAGT
586


 


hNa15006
CE
TTTTGCCATGGAGGGCGTTTTTTCTCTTGGAAAGAAAGT
587


 


hNa15007
LE
GGCTGAGATGATTCATTGCTCTGTTTTTAGGCATAGGACCCGTGTCT
588


 


bNa15008
LE
GCTGAGGCCACGGGTGATTTTTAGGCATAGGACCCGTGTCT
589


 


bNa15009
LE
AATGCTCCCGCGGCTGGTTTTTAGGCATAGGACCCGTGTCT
590


 


hNa15010
LE
CTGGGCACTGGTCCGGCTTTTTAGGCATAGGACCCGTGTCT
591


 


hNa15011
LE
GGCCAGGAGCCGAGGTTTTTTTAGGCATAGGACCCGTGTCT
592


 


bNa15012
LE
CCCAGTAATGAGACCACCCCATTTTTTAGGCATAGGACCCGTGTCT
593


 


hNa15013
LE
CCTCTGGGTCGCCTGCCTTTTTAGGCATAGGACCCGTGTCT
594


 


hNa15014
BL
CACTCCTCAGTTCCTGAAGACATC
595


 


hNa15015
BL
GGACCATCTTCTGAGTCAGACTTG
596


 


hNa15016
BL
AACGTGGCTTCATAGAAGTCCT
597


 


hNa15017
BL
AATCTGCTTCAGAACCCAGGTC
598


 


hNa15018
BL
TGTGCTGTTTTCATCATCTGCAA
599


 


hNa15019
BL
CCAGCAGTGATGTGTGGTGG
600


 


hNa15020
BL
GCAGGGGCCAGGGCA
601


 


hNa15021
BL
TGCAGTCCACAGTGCTGTTCT
602


 


hNa15022
BL
GGCTTCCTGGGGATGTGG
603









Single-Dose Screen of Nav1.8 siRNAs against mRNA Expression of Endogenous Nav1.8 in Primary Cultures of Rat Dorsal Root Ganglion Cells.
To confirm and extend the results obtained on mRNA expression of transfected human Nav1.8 in Cos-7 cells, Nav1.8 siRNAs from Table 1 were tested at a single dose of 200 nM for activity in reducing mRNA expression of endogenous Nav1.8 in primary cultures of rat dorsal root ganglion (DRG) cells.
DRG cells were isolated from Sprague-Dawley rats at postnatal day 3 to 6. DRGs were dissected and cells dissociated into single cells by incubation with 0.28 Wunsch units/ml Liberase Blendzyme (Roche) in S-MEM (Gibco) at 37° C. for 35 min. The cell suspension was pre-plated on tissue-culture plates to remove non-neuronal cells. Neurons were then plated onto tissue-culture BiocoatTM PDL Poly-D-Lysine/Laminin 96-well plates (BD Biosciences, Bedford Mass., USA) in F12-HAM's Medium containing glutamine (Invitrogen Gibco, Carlsbad Calif., USA) with 5% fetal bovine serum (FBS, heat inactivated) and 5% horse serum (heat inactivated) (both Invitrogen Gibco, Carlsbad Calif., USA) supplemented with 50 ng/ml mouse nerve growth factor 2.5S (NGF; Promega Corp., Madison Wis., USA) and kept at 37° C., 5% CO2 in a humidified incubator until transfection.
Nav1.8 siRNAs were screened in DRG cultures at 200 nM in duplicate using TransMessengerTM Transfection reagent (Qiagen GmbH, Hilden, Germany, cat. no. 301525) which is based on a lipid formulation, a specific RNA-condensing reagent (Enhancer RTM) and an RNA-condensing buffer (Buffer EC-RTM), keeping siRNA:Enhancer RTM ratio (μg:μl) constant at 1:2, and siRNA:TransMessengerTM ratio (μg:μl) constant at 1:12.
DRG neurons were transfected 24 h post-plating. For each well, 0.52 μl Enhancer RTM were first mixed with 13.68 μl Buffer EC-RTM. 0.8 μl of a 25 μM solution of AL-DP-5987 (0.26 μg) in annealing buffer (20 mM sodium phosphate, pH 6.8; 100 mM sodium chloride), or 0.8 μl of annealing buffer (siRNA-free control) were added and the mixture incubated for 5 min at RT. 3.12 μl TransMessengerTM Transfection Reagent were diluted with 6.88 μl Buffer EC-RTM, added to the mixture, and the mixture incubated for another 10 min at room temperature to allow transfection-complex formation. 75 μl serum free F12-HAM's Medium containing glutamine (Invitrogen Gibco, Carlsbad Calif., USA) supplemented with 50 ng/ml NGF 2.5S (Promega Corp., Madison Wis., USA) and 1:50 B27 supplement (Invitrogen Gibco, Carlsbad Calif., USA) were added to the transfection complexes and complete mixing achieved by gently pipetting up and down. The growth medium was removed from the DRG cells, and 90 μl of the above transfection complex mixture were added onto the cells. After 7 to 8 h of incubation at 37° C., 5% CO2 in a humidified incubator supernatant was removed from the cells, fresh F12-HAM's medium containing glutamine supplemented with 5% FBS, 5% horse serum (both Invitrogen Gibco, Carlsbad Calif., USA), 50 ng/ml mouse NGF 2.5S (Promega Corp., Madison Wis., USA) and 1:100 Penicillin/Streptomycin (Invitrogen Gibco, Carlsbad Calif., USA) was added, the cells were incubated for another 16 h at 37° C., 5% CO2 in a humidified incubator, and Nav1.8 mRNA was quantified.
Nav1.8 mRNA levels were measured using the QuantiGeneTM bDNA kit (Genospectra, Fremont, USA) according to manufacturer's protocol. Briefly, the supernatant was removed from the DRG cells, and the cells were lysed by addition of 150 μl of Lysis Working Reagent (1 volume of Lysis Mixture plus 2 volumes of medium) and incubation at 52° C. for 30min. 40 μl of the lysates were incubated at 52° C. for 40 min with the probe sets specific to rat Nav1.8 and mouse synuclein (SNCL). Chemoluminescence was read on a Victor2-LightTM (PerkinElmer Life And Analytical Sciences, Inc., Boston Mass., USA) as Relative Light Units (RLU). RLU for Nav1.8 were normalized to SNCL RLU for each well. Normalized Nav1.8/SNCL ratios were then compared to the siRNA-free control, which was set as 100%.
FIG. 3 provides the results. At 200 nM, at least 13 (indicated by ‘*’) of the Nav1.8 siRNAs tested showed at least 50% Nav1.8 mRNA knock down compared to the siRNA-free control (TransMessengerTM only; TM only), while an unrelated control siRNA against RhoA had no effect.
Dose Response of dsRNA AL-DP-6209 against MRNA Expression of Endogenous Nav1.8 in Primary Cultures of Rat Dorsal Root Ganglion Cells
One effective siRNA (AL-DP-6209) against Nav1.8 from the single dose screen in primary cultures of rat dorsal root ganglion cells was further characterized for dose dependence. For the dose response curve, experiments were performed as for the single dose screen in DRG cultures above, but with the following concentrations of siRNA: 175, 88, 44, 22, 11 and 5.5 nM. For all siRNA concentrations, siRNA:Enhancer R ratio (ug:ul) was kept constant at 1:2 and siRNA:TransMessenger ratio (ug/ul) was kept constant at 1:12 (FIG. 4).
FIG. 4 provides the result for the selected siRNA AL-DP-6209 from a dose response experiment. At 5.5 and 11 nM, AL-DP-6209 did not inhibit Nav1.8 mRNA expression relative to SNCL, whereas at 88 and 175 nM, AL-DP-6209 inhibited Nav1.8 mRNA expression relative to SNCL by >40%. Maximal inhibition of Nav1.8 mRNA expression relative to SNCL occurred in this experiment at 175 nM.
Intrathecal Bolus Administration of siRNAs against Nav1.8 with iFECT Prevents Inflammatory Pain
The effect of siRNAs against Nav1.8, formulated with iFECT, on complete Freund's adjuvant-induced tactile hypersensitivity was evaluated in rats (FIG. 5). Adult male Sprague-Dawley rats received an injection of CFA (150 uL) into the hindpaw on day 0. siRNAs against Nav1.8 were then administered by intrathecal bolus to the lumbar region of the spinal cord on days 1, 2 and 3; specifically, for each bolus injection, 2 ug of siRNA was complexed with iFECT transfection reagent (Neuromics, Minneapolis Minn., USA) at a ratio of 1:4 (w:v) in a total volume of 10 uL. Five groups of rats (with 5 rats per group) were treated with either siRNA (AL-DP-6049, AL-DP-6209, AL-DP-6217 or AL-DP-6218; Table 1), or PBS, in the presence of iFECT. Tactile hypersensitivity was expressed as tactile withdrawal thresholds which were measured by probing the hindpaw with 8 calibrated von Frey filaments (Stoelting, Wood Dale Ill., USA) (0.41 g to 15 g). Each filament was applied to the plantar surface of the paw. Withdrawal threshold was determined by sequentially increasing and decreasing the stimulus strength and calculated with a Dixon non-parametric test (see Dixon, W. J. (1980) “Efficient analysis of experimental observations” Annu Rev Pharmacol Toxicol 20:441-462; Chaplan, S. R., F. W. Bach, et al.(1994) “Quantitative assessment of tactile allodynia in the rat paw” J Neurosci Methods 53:55-63). Tactile thresholds were measured before CFA injection to assess baseline thresholds, and then on day 4 after CFA and treatment with test articles. In rats treated with PBS, tactile hypersensitivity was pronounced on day 4, as evidenced by reduced paw withdrawal threshold, as expected. In rats treated with AL-DP-6209, tactile thresholds were nearly normalized on day 4, demonstrating that the Nav1.8 siRNA, AL-DP-6209, is efficacious in vivo against inflammation-induced hyperalgesia. Treatment with the Nav1.8 siRNA, AL-DP-6217, resulted in the average tactile threshold trending towards baseline, with one of five rats demonstrating a normal tactile response. AL-DP-6049 and AL-DP-6218 did not significantly alter tactile thresholds compared to PBS treatment, in this experimental paradigm.
These results demonstrate that siRNAs targeting Nav1.8, formulated with transfection reagent and administered intrathecally, alleviate CFA-induced tactile hyperalgesia, and therefore represent a novel approach to providing effective treatment of clinical inflammatory pain.
Intrathecal Bolus Administration of siRNAs against Nav1.8 without Transfection Reagent Alleviates Inflammatory Pain
The effect of siRNAs against Nav1.8, formulated in phosphate buffered saline (PBS), on complete Freund's adjuvant (CFA)—induced tactile hypersensitivity was evaluated in rats (FIG. 6). Of 3 Nav1.8 siRNAs tested, 2 siRNAs were efficacious against CFA-induced tactile hypersensitivity: AL-DP-4461, an unconjugated siRNA with different chemical modifications but the same sequence as AL-DP-6050, and AL-DP-4459, a cholesterol-conjugated siRNA with the same chemical modifications and sequence as AL-DP-6050. With the dosing paradigms evaluated in this experiment, AL-DP-6980 (cholesterol-conjugated siRNA with the same chemical modifications and sequence as AL-DP-6209) was not efficacious against CFA-induced tactile hypersensitivity. The sequences of AL-DP-4461, AL-DP-4459 and AL-DP-6980 are shown in Table 6.







TABLE 6







Further modified siRNAs specific for Nav1.8














SEQ

SEQ



Duplex
sense strand
ID
anti-sense strand
ID


name
sequence (5′-3′)
NO:
sequence (5′-3′)
NO:





AL-DP-
cauccuaugaaccaauagceTeT
604
gcuauugguucauaggaugeceT
605



4461


 


AL-DP-
cmaumcmcmumaumgaacmcmaa
606
gcumauugguucmaumaggaugTT
607


4459
umagcmTT-sChol


 


AL-DP-
umumumumgumcmumaaaumgag
608
ugaacucmauuumagacmaaaaTT
609


6980
umumcmaTT-sChol





Note:


Prefix e represents 2′-O-(2-methoxyethyl) modified nucleotides






Adult male Sprague-Dawley rats received an injection of CFA into the hindpaw on day 0. Four groups of rats (with 5 rats per group) were treated starting on day 1 after CFA injection with either siRNA against Nav1.8 (AL-DP-4461, AL-DP-4459, or AL-DP-6980), or PBS. siRNAs against Nav1.8 (AL-DP-4461, AL-DP-4459 or AL-DP-6980) or PBS were administered by intrathecal bolus injections (10 uL per injection) to the lumbar level of the spinal cord twice per day (BID) on days 1, 2 and 3. Tactile hypersensitivity was measured as above, both before CFA injection to assess baseline thresholds, and then on day 4 after CFA and treatment with test articles. In rats treated with PBS, tactile hypersensitivity was pronounced on day 4, as evidenced by reduced paw withdrawal threshold, as expected. In rats treated with AL-DP-4461 by bolus BID (0.5 mg/bolus), tactile thresholds were moderately normalized on day 4, demonstrating that the Nav1.8 siRNA, AL-DP-4461, is moderately efficacious with this dosing paradigm in vivo against CFA-induced tactile hyperalgesia. Treatment with the Nav1.8 siRNA, AL-DP-4459, by bolus BID (0.15 mg/bolus), resulted in nearly complete normalization of tactile threshold, with all 5 rats demonstrating substantial recovery of tactile thresholds to more than 10.2 g by day 4. In contract, the Nav1.8 siRNA AL-DP-6980, by bolus BID (0.15 mg/bolus), did not affect tactile hypersensitivity, showing that not all cholesterol-conjugated siRNAs against a target are equally efficacious or potent, even if efficacious when transfected into cultured cells.
These results suggest that cholesterol conjugation enhances in vivo efficacy of siRNAs for neurological disorders, including chronic pain. Furthermore, these results demonstrate that siRNAs targeting Nav1.8, either with or without cholesterol-conjugation, formulated in saline and administered intrathecally by bolus injection, alleviate CFA-induced tactile hyperalgesia, and therefore represent a novel approach to providing effective treatment of clinical inflammatory pain.
Intrathecal Pump or Bolus Administration of siRNAs against Nav1.8 without Transfection Reagent Alleviates Inflammatory Pain
The effect of siRNAs against Nav1.8, formulated in phosphate buffered saline (PBS), on complete Freund's adjuvant (CFA)—induced tactile hypersensitivity was evaluated in rats after intrathecal pump infusion (FIG. 7, left) or intrathecal BID bolus injection (FIG. 7, right). Of 3 Nav1.8 siRNAs tested by continuous intrathecal pump infusion at 0.4 mg/day, 1 siRNA (AL-DP-6050, Table 1) was modestly efficacious against CFA-induced tactile hypersensitivity. AL-DP-6050, when tested by intrathecal BID bolus injection at 0.5 mg/bolus, was efficacious against CFA-induced tactile hypersensitivity.
For evaluating effects of siRNAs against NaV1.8 with continuous intrathecal pump infusion, adult male Sprague-Dawley rats received an injection of CFA into the hindpaw on day 0. Four groups of rats (with 5 rats per group) were treated starting on day 1 after CFA injection with either siRNA against Nav1.8 (AL-DP-6050, AL-DP-6218, or AL-DP-6219), or PBS. In all rats, test articles were intrathecally administered by continuous osmotic mini-pump infusion with an infusion rate of 0.5 uL/hour, beginning on day 1. siRNAs were infused at 0.4 mg/day. Tactile hypersensitivity was measured as above, both before CFA injection to assess baseline thresholds, and then on day 4 after CFA and treatment with test articles (FIG. 7, left). In rats treated with PBS, tactile hypersensitivity was pronounced on day 4, as evidenced by reduced paw withdrawal threshold, as expected. In rats treated with AL-DP-6050 by continuous intrathecal pump infusion (0.4 mg/day), tactile thresholds were modestly normalized on day 4, demonstrating that the Nav1.8 siRNA, AL-DP-6050, is modestly efficacious with this dosing paradigm in vivo against inflammatory pain.
For evaluating effects of siRNAs against NaV1.8 with BID intrathecal bolus injection, adult male Sprague-Dawley rats received an injection of CFA into the hindpaw on day 0. Two groups of rats (with 4 to 5 rats per group) were treated starting on day 1 after CFA injection with either siRNA against Nav1.8 (AL-DP-6050, 0.5 mg/bolus) or PBS. Tactile hypersensitivity was assessed as described above. Tactile thresholds were measured before CFA injection to assess baseline thresholds, and then on day 4 after CFA injection and treatment with test articles (FIG. 7, right). As expected, in rats treated with PBS, tactile hypersensitivity was pronounced on day 4, as evidenced by reduced paw withdrawal threshold. In rats treated with AL-DP-6050 by BID intrathecal bolus injection (0.5 mg/bolus), tactile thresholds were substantially normalized on day 4, demonstrating that the Nav1.8 siRNA, AL-DP-6050, is modestly efficacious with this dosing paradigm in vivo against inflammatory pain.
These results further demonstrate that siRNAs targeting Nav1.8 without cholesterol-conjugation, formulated in saline and administered intrathecally by either bolus injection or continuous pump infusion, alleviate CFA-induced tactile hyperalgesia, and therefore represent a novel approach to providing effective treatment of clinical inflammatory pain.
Intrathecal Bolus Administration of siRNAs against Nav1.8 without Transfection Reagent Alleviates Inflammatory Pain
The effect of siRNAs against Nav1.8, formulated in phosphate buffered saline (PBS), on complete Freund's adjuvant (CFA)—induced thermal hypersensitivity was evaluated in rats (FIG. 8). Both unconjugated dsRNA AL-DP-6050 (Table 1) and cholesterol-conjugated dsRNA AL-DP-4459 (Table 6) were efficacious against CFA-induced thermal hypersensitivity with the BID bolus intrathecal dosing paradigms evaluated.
Adult male Sprague-Dawley rats received an injection of CFA into the hindpaw on day 0. Three groups of rats (with 4 to 5 rats per group) were treated starting on day 1 after CFA injection with either siRNA against Nav1.8 (AL-DP-6050 or AL-DP-4459), or PBS. In all rats, test articles were administered intrathecally by BID bolus injection (10 uL per bolus) beginning on day 1. AL-DP-4459 was dosed at 0.15 mg/bolus whereas AL-DP-6050 was dosed at 0.5 mg/bolus. Thermal hypersensitivity was measured by assessing paw withdrawal latency to a noxious thermal stimulus as described by Hargreaves and colleagues (Hargreaves, K., R. Dubner, et al. (1988) “A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia” Pain 32:77-88). Latency to withdrawal of a hindpaw in response to noxious radiant heat was determined. A maximal cut-off of 40 sec prevented tissue damage.
Thermal responses were measured before CFA injection to assess baseline thresholds, and then on day 4 after CFA injection and treatment with test articles. As expected, in rats treated with PBS, thermal hypersensitivity was pronounced on day 4, as evidenced by reduced paw withdrawal latency. In rats treated with AL-DP-6050 (0.5 mg/bolus) or AL-DP-4459 (0.15 mg/bolus) by intrathecal BID bolus injection, thermal latencies were normalized on day 4, demonstrating that the unconjugated Nav1.8 siRNA, AL-DP-6050, and the cholesterol-conjugated Nav1.8 siRNA AL-DP-4459 are efficacious with this dosing paradigm in vivo against inflammatory pain.
These results demonstrate that siRNAs targeting Nav1.8, either with or without cholesterol-conjugation, formulated in saline and administered intrathecally by bolus injection, alleviate CFA-induced thermal hyperalgesia, in addition to tactile hyperalgesia (above), and therefore represent a novel approach to providing effective treatment of multiple types of hyperalgesia in clinical inflammatory pain.
Intrathecal Bolus Administration of Cholesterol-Conjugated siRNA against Nav1.8 without Transfection Reagent Alleviates Neuropathic Pain
The effect of AL-DP-4459 (Table 6) against Nav1.8, formulated in phosphate buffered saline (PBS), on spinal nerve ligation (SNL)-induced tactile and thermal hypersensitivity was evaluated in rats (FIG. 9). With the bolus intrathecal dosing paradigm evaluated (0.15 mg/bolus, BID), AL-DP-4459 was efficacious against SNL-induced tactile and thermal hypersensitivity.
Adult male Sprague-Dawley rats received unilateral ligation of the L5 and L6 spinal nerves on day 0 (SNL surgery). Three groups of rats (with 6 to 8 rats per group) were treated starting on day 3 after SNL surgery by intrathecal administration of either the siRNA AL-DP-4459 against Nav1.8, or PBS. In 2 of these groups, AL-DP-4459 or PBS was administered by intrathecal bolus injections (5 uL per injection) to the lumbar level of the spinal cord twice per day (BID) on post-SNL days 3 through 7. In one group of rats, AL-DP-4459 was intrathecally administered by continuous osmotic mini-pump infusion at 0.18 mg/day, with an infusion rate of 0.5 uL/hour, on post-SNL days 3 through 7. Tactile and thermal hypersensitivities were assessed as described above.
Tactile (FIG. 9, left) and thermal (FIG. 9, right) responses were measured before SNL surgery to assess baseline responses (BL), and on post-SNL day 3 before treatment with test articles to verify that tactile and thermal hyperalgesia had developed fully. In rats treated with PBS, tactile and thermal hypersensitivities were pronounced, as expected, on post-SNL days 3, 5 and 7 as evidenced by reduced paw withdrawal thresholds and reduced thermal latencies, respectively. The Nav1.8 siRNA AL-DP-4459, when intrathecally administered by continuous pump infusion at 0.18 mg/day, did not affect tactile or hypersensitivity significantly over the time-frame shown. In contrast, in rats treated with AL-DP-4459 by bolus BID (0.15 mg/bolus), tactile thresholds and thermal latencies were substantially normalized on post-SNL day 7 (day 5 of treatment), demonstrating that the Nav1.8 siRNA, AL-DP-4459, is efficacious in vivo against SNL-induced tactile and thermal hyperalgesia.
These results demonstrate that intrathecally administered cholesterol-conjugated siRNAs targeting Nav1.8, formulated in saline, are efficacious against experimental nerve injury-induced chronic pain in rats, and therefore, represent a novel approach to providing effective treatment of clinical neuropathic pain.
Unconjugated and Cholesterol-Conjugated siRNAs Targeting NaV1.8 are Stable in Human Cerebrospinal Fluid at 37° C.
To determine the stability in human cerebrospinal fluid (CSF) of unconjugated and cholesterol-conjugated siRNAs targeting NaV1.8, siRNA duplexes were incubated in human CSF for 48 hours at 37° C., and the single strands were measured by quantitative ion exchange chromatography. In this example, unconjugated siRNAs AL-DP-6050, AL-DP-6209, AL-DP-6217, AL-DP-6218 and AL-DP-6219 (Table 1), and cholesterol-conjugated siRNA AL-DP-4459 (Table 6) were evaluated. 30 μl of human CSF was mixed with 3μl of 50 μM siRNA (150 pmole/well) in a 96-well plate, sealed to avoid evaporation, and incubated for 1 to 48 hours at 37° C. Incubation in 30 μl PBS for 48 hours at 37° C. served as a control for nonspecific degradation. Reactions were stopped by the addition of 4 μl proteinase K (20 mg/ml) and 25μl of proteinase K buffer, and incubation of this mixture for 20 min at 42° C. Samples were spin filtered through a 0.2 μm 96-well filter plate at 3000 rpm for 20 min. Incubation wells were washed with 50 μl Millipore water twice and the combined washing solutions were spin filtered also. A 5 μl aliquot of 50 μM 40-mer RNA was added to each sample to act as an internal standard (IS) for normalization of volume changes in the filtration volume. Samples were analyzed by ion exchange HPLC under denaturing conditions.
1. HPLC System for Analysis of Cholesterol-Conjugated siRNA AL-DP-4459
Column: Dionex DNAPac PA200 (4×250 mm analytical column)
Temp.: 80° C. (denaturing conditions)
Flow: 1 ml/min
Injection: 50 ul
Detection: 260 (reference wavelength 600 nm)
HPLC Eluent A: 25 mM TRIS-HCl; 1 mM EDTA; 50% ACN; pH=8
HPLC Eluent B: 600 mM NaBr in A
Gradient table:












Time
% A
% B


















0.00
min
80
20


1.00
min
80
20


15.0
min
30
70


15.5
min
0
100


17.5
min
0
100


18.0
min
80
20


21.0
min
80
20









2. HPLC System for unconjugated siRNAs AL-DP-6050, 6209, 6217, 6218, 6219
Column: Dionex DNAPac PA200 (4×250 mm analytical column)
Temp.: 30° C. (denaturing conditions by pH=11)
Flow: 1 ml/min
Injection: 50 ul
Detection: 260 nm (reference wavelength 600 nm)
HPLC Eluent A: 20 mM Na3PO4 in 10% ACN; pH=11
HPLC Eluent B: 1 M NaBr in A
Gradient table:












Time
% A
% B


















0.00
min
75
25


1.00
min
75
25


21.0
min
40
60


21.5
min
0
100


23.0
min
0
100


24.5
min
75
25


28.0
min
75
25









Under the denaturing IEX-HPLC conditions, the duplexes eluted as two separated single strands. The internal standard eluted at higher retention times than the single strands of the duplex, and did not interfere with the analysis.
For every injection, the chromatograms were integrated automatically by the Dionex Chromeleon 6.60HPLC software, but were adjusted manually as necessary. All peak areas were corrected by the following equation to the internal standard (IS) peak:

CF(IS)=100/PeakArea(IS); Corrected PeakArea(s/as)=CF*PeakArea(a/as) 

The %-values for the remaining intact FLP at all time points are calculated by the following equation:

%-FLP(s/as)=(Corrected PeakArea(s/as)/Corrected PeakArea(s/as); t=0min)*100%

All values were normalized to the incubation at t=0 min.
The results for the unconjugated siRNAs are shown in FIG. 10. After 48 hours incubation at 37° C. in PBS (PBS-48), all five siRNAs were completely stable, with no loss detectable. After 48 hours incubation at 37° C. in human CSF, three (AL-DP-6050, AL-DP-6209 and AL-DP-6217) of the five siRNAs exhibited less than 20% loss, whereas two (AL-DP-6218 and AL-DP-6219) of the siRNAs exhibited greater than 50% loss. The results for the cholesterol-conjugated siRNA AL-DP-4459 are shown in FIG. 11. After 48 hours incubation at 37° C. in PBS (PBS-48), AL-DP-4459 was completely stable, with no loss detectable. When assessed for stability in human CSF, AL-DP-4459 was found to be stable at 37° C. for 48 hours, with less than 20% variation detected over this time period.
Unconjugated and Cholesterol-conjugated siRNAs Targeting NaV1.8 Reduce NaV1.8 mRNA in a Dose-dependent Manner in Primary Rat Sensory Neuronal Cultures
Another effective siRNA (AL-DP-6050, Table 1) against Nav1.8 from the single dose screen in primary cultures of rat dorsal root ganglion cells and its cholesterol conjugate AL-DP-4459 (Table 6) were further characterized for dose dependence. For the dose response curves, transfections were performed using Lipofectamine 2000 (see below) with the following concentrations of siRNA: 200, 80, 32, 12.8, 5.12, 2.05, 0.82 and 0.33 nM (FIG. 12).
DRG neurons were transfected 24 h post-plating. For each well, 0.4 μl LipofectamineTM 2000 (Invitrogen Corporation, Carlsbad, Calif.) was used and transfections were performed according to the manufacturer's protocol. Specifically, the siRNA: LipofectamineTM 2000 complexes were prepared as follows. The appropriate amount of siRNA was diluted in Opti-MEM I Reduced Serum Medium and mixed gently. The LipofectamineTM 2000 was vortexed before use; then for each well of a 96 well plate, 0.4 ul LipofectamineTM 2000 was diluted in 25 μl of Opti-MEM I Reduced Serum Medium, mixed gently and incubated for 5 minutes at room temperature. After the 5 minute incubation, 1 ul of the diluted siRNA was combined with the diluted LipofectamineTM 2000 (total volume, 26.4 μl). The complex was mixed gently and incubated for 20 minutes at room temperature to allow the siRNA: LipofectamineTM 2000 complexes to form. Then 100 μl of serum-free F12-HAM's Medium containing glutamine (Invitrogen Gibco, Carlsbad Calif., USA) supplemented with 50 ng/ml NGF 2.5S (Promega Corp., Madison Wis., USA) and 1:50 B27 supplement (Invitrogen Gibco, Carlsbad Calif., USA) was added to the transfection complexes, and mixing was achieved by gently pipetting up and down. The growth medium was removed from the DRG cells and 100 μl of the above transfection complex mixture was added to each well of a 96-well plate. After 20 h of incubation at 37° C., 5% CO2 in a humidified incubator, supernatant was removed from the cells, fresh F12-HAM's medium containing glutamine supplemented with 5% FBS, 5% horse serum (both Invitrogen Gibco, Carlsbad Calif., USA), 50 ng/ml mouse NGF 2.5S (Promega Corp., Madison Wis., USA) and 1:100 Penicillin/Streptomycin (Invitrogen Gibco, Carlsbad Calif., USA) was added. The cells were incubated for another 20-24 h at 37° C., 5% CO2 in a humidified incubator, and Nav1.8 mRNA was quantified by the bDNA assay, as described previously.
FIG. 12 provides the result for the selected siRNA AL-DP-6050 and its cholesterol conjugate AL-DP-4459 from a dose response experiment. AL-DP-6050 and its cholesterol conjugate AL-DP-4459 inhibited Nav1.8 mRNA expression relative to alpha-synuclein (SNCA) in a dose-dependent manner, with maximal inhibitions of  ̃40% at >30 nM siRNA, in this experiment.
Intrathecal Bolus Administration of Unconjugated siRNA against Nav1.8 without Transfection Reagent Alleviates Neuropathic Pain
The effect of AL-DP-6050 (Table 1) against Nav1.8, formulated in phosphate buffered saline (PBS), on spinal nerve ligation (SNL)—induced thermal hypersensitivity was evaluated in rats (FIG. 13). With the bolus intrathecal dosing paradigm evaluated (0.15 mg/bolus, BID), AL-DP-6050 was efficacious against SNL-induced thermal hypersensitivity.
Adult male Sprague-Dawley rats received unilateral ligation of the L5 and L6 spinal nerves on day 0 (SNL surgery). Two groups of rats (with 8 rats per group) were treated starting on day 3 after SNL surgery by intrathecal bolus injection with either the siRNA AL-DP-6050 against Nav1.8, or PBS. Intrathecal bolus injections (0.15 mg in 5 uL per injection) were administered to the lumbar level of the spinal cord twice per day (BID) on post-SNL days 3 through 7. Thermal hypersensitivity was assessed as described above.
Thermal responses were measured before SNL surgery to assess baseline responses (BL), and on post-SNL day 3 before treatment with test articles to verify that thermal hyperalgesia had developed fully. In rats treated with PBS, thermal hypersensitivity was pronounced, as expected, on post-SNL days 3, 5 and 7 as evidenced by reduced thermal latencies. In contrast, in rats treated with AL-DP-6050 by bolus BID (0.15 mg/bolus), thermal latencies were substantially normalized on post-SNL days 5 (day 3 of treatment) and 7 (day 5 of treatment), demonstrating that the Nav1.8 siRNA, AL-DP-6050, is efficacious in vivo against SNL-induced thermal hyperalgesia.
These results demonstrate that siRNAs targeting Nav1.8, formulated in saline and administered by intrathecal bolus injection, are efficacious against experimental nerve injury-induced chronic pain in rats, and therefore, represent a novel approach to providing effective treatment of clinical neuropathic pain.
Intrathecal Bolus Administration of ND98-2.7 Liposomal Formulation of siRNA against Nav1.8 Alleviates Neuropathic Pain
The effect of AL-DP-6050 (Table 1) against Nav1.8, formulated in ND98-2.7 (described below), and administered by intrathecal bolus injection, on spinal nerve ligation (SNL) -induced thermal hypersensitivity was evaluated in rats (FIG. 14). With the bolus intrathecal dosing paradigm evaluated (5 micrograms/bolus, daily), AL-DP-6050 was efficacious against SNL-induced thermal hypersensitivity.
Adult male Sprague-Dawley rats received unilateral ligation of the L5 and L6 spinal nerves on day 0 (SNL surgery). Two groups of rats (with 6 rats per group) were treated starting on day 3 after SNL surgery by intrathecal bolus injection with either the siRNA AL-DP-6050 against Nav1.8, formulated in ND98-2.7, or PBS. Intrathecal bolus injections (5 micrograms in 5 uL per injection) were administered to the lumbar level of the spinal cord daily on post-SNL days 3 through 5. Thermal hypersensitivity was assessed as described above.
Thermal responses were measured before SNL surgery to assess baseline responses (BL), and on post-SNL day 3 before treatment with test articles to verify that thermal hyperalgesia had developed fully. In rats treated with PBS, thermal hypersensitivity was pronounced, as expected, on post-SNL days 3, 5, 7 and 10 as evidenced by reduced thermal latencies. In contrast, in rats treated with AL-DP-6050 formulated in ND98-2.7 liposomes, by intrathecal bolus daily injection (5 micrograms/bolus), thermal latencies were substantially normalized on post-SNL day 5 (day 3 of treatment), demonstrating that the Nav1.8 siRNA, AL-DP-6050, formulated in ND98-2.7 liposomes and administered by intrathecal injection, is efficacious in vivo against SNL-induced thermal hyperalgesia. Moreover, the dose level of siRNA required for efficacy with the ND98-2.7 liposomal formulation (5 micrograms per day) was much lower than that observed for efficacy with the PBS formulation (300 micrograms per day).
These results demonstrate that siRNAs targeting Nav1.8, administered by intrathecal bolus injection and formulated in ND98-2.7 liposomes, are efficacious against experimental nerve injury-induced chronic pain in rats, and therefore, represent a novel approach to providing effective treatment of clinical neuropathic pain.
Intravenous Bolus and Continuous Pump Administration of ND98-2.7 Liposomal Formulation of siRNA against Nav1.8 Alleviates Neuropathic Pain
The effect of AL-DP-6050 (Table 1) against Nav1.8, formulated in ND98-2.7, and administered by intravenous bolus injection or intravenous continuous pump infusion, on spinal nerve ligation (SNL) -induced thermal hypersensitivity was evaluated in rats (FIG. 14). With the bolus intravenous dosing paradigm evaluated (daily 0.5 mg/bolus, equivalent to approximately 2 mg/kg/bolus), and with the continuous intravenous pump paradigm evaluated (0.24 mg/day, equivalent to approximately 1 mg/kg/day), AL-DP-6050, formulated in ND98-2.7 liposomes, was efficacious against SNL-induced thermal hypersensitivity.
Adult male Sprague-Dawley rats received unilateral ligation of the L5 and L6 spinal nerves on day 0 (SNL surgery). Two groups of rats (with 6 rats per group) were treated starting on day 3 after SNL surgery by intravenous bolus injection or intravenous continuous pump infusion with the siRNA AL-DP-6050 against Nav1.8, formulated in ND98-2.7. Intravenous bolus injections (0.5 mg in 1 mL per bolus) were administered daily on post-SNL days 3 through 5. Intravenous continuous pump infusion (10 uL/hour, equivalent to 0.24 mg/day) was administered on post-SNL days 3 through 10 via a cannula in the jugular vein. Thermal hypersensitivity was assessed as described above.
Thermal responses were measured before SNL surgery to assess baseline responses (BL), and on post-SNL day 3 before treatment with test articles to verify that thermal hyperalgesia had developed fully. Thermal hypersensitivity was pronounced, as expected, on post-SNL day 3, before treatment, as evidenced by reduced paw withdrawal latencies. In contrast, in rats treated with AL-DP-6050 formulated in ND98-2.7 liposomes, by intravenous bolus daily injection (0.5 mg/bolus), thermal latencies were substantially normalized on post-SNL day 5 (day 3 of treatment), and post-SNL day 7 (2 days after the last intravenous bolus treatment) demonstrating that the Nav1.8 siRNA, AL-DP-6050, formulated in ND98-2.7 liposomes and administered by intravenous bolus injection, is efficacious in vivo against SNL-induced thermal hyperalgesia. Furthermore, in rats treated with AL-DP-6050 formulated in ND98-2.7 liposomes, by intravenous continuous pump infusion (0.24 mg/day), thermal latencies were substantially normalized on post-SNL days 5 (day 3 of treatment), 7 (day 5 of treatment) and 10 (day 8 of treatment).
These results demonstrate that siRNAs targeting Nav1.8, formulated in ND98-2.7 liposomes, and administered by intravenous bolus injection or intravenous continuous pump infusion, are efficacious against experimental nerve injury-induced chronic pain in rats, and therefore, represent a novel approach to providing effective treatment of clinical neuropathic pain. Moreover, these results demonstrate that ND98-2.7 liposomal formulation of siRNA can provide effective delivery of siRNA to sensory neurons of the dorsal root ganglion, with systemic administration.
Formulation Procedure
The lipidoid ND98.4HCl (MW 1487), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) were used to prepare lipid-siRNA nanoparticles.
Stock solutions of each in ethanol were prepared: ND98 (FIG. 15, ND98 Isomer I), 133 mg/mL; Cholesterol, 25 mg/mL, PEG-Ceramide C16, 100 mg/mL. ND98, Cholesterol, and PEG-Ceramide C16 stock solutions were then combined in a 42:48:10 molar ratio. Combined lipid solution was mixed rapidly with aqueous siRNA (in sodium acetate pH 5) such that the final ethanol concentration was 35-45.% and the final sodium acetate concentration was 100-300 mM. Lipid-siRNA nanoparticles formed spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture was in some cases extruded through a polycarbonate membrane (100 nm cut-off) using a thermobarrel extruder (Lipex Extruder, Northern Lipids, Inc). In other cases, the extrusion step was omitted. Ethanol removal and simultaneous buffer exchange was accomplished by either dialysis or tangential flow filtration. Buffer was exchanged to phosphate buffered saline (PBS) pH 7.2.
Characterization of Formulations
Formulations prepared by either the standard or extrusion-free method are characterized in a similar manner. Formulations are first characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles are measured by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be 20-300 nm, and ideally, 40-100 nm in size. The particle size distribution should be unimodal. The total siRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated siRNA is incubated with the RNA-binding dye Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, 0.5% Triton-X100. The total siRNA in the formulation is determined by the signal from the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the “free” siRNA content (as measured by the signal in the absence of surfactant) from the total siRNA content. Percent entrapped siRNA is typically >85%.
dsRNA Expression Vectors
In another aspect of the invention, Nav1.8 specific dsRNA molecules that modulate Nav1.8 gene expression activity are expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be incorporated and inherited as a transgene integrated into the host genome. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).
The individual strands of a dsRNA can be transcribed by promoters on two separate expression vectors and co-transfected into a target cell. Alternatively each individual strand of the dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In a preferred embodiment, a dsRNA is expressed as an inverted repeat joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.
The recombinant dsRNA expression vectors are preferably DNA plasmids or viral vectors. dsRNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus (for a review, see Muzyczka, et al., Curr. Topics Micro. Immunol.(1992) 158:97-129)); adenovirus (see, for example, Berkner, et al., BioTechniques (1998) 6:616), Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld et al. (1992), Cell 68:143-155)); or alphavirus as well as others known in the art. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, in vitro and/or in vivo (see, e.g., Eglitis, et al., Science (1985) 230:1395-1398; Danos and Mulligan, Proc. Natl. Acad. Sci. USA (1998) 85:6460-6464; Wilson et al., 1988, Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al., 1990, Proc. Natl. Acad. Sci. USA 87:61416145; Huber et al., 1991, Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al., 1991, Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al., 1991, Science 254:1802-1805; van Beusechem. et al., 1992, Proc. Nad. Acad. Sci. USA 89:7640-19 ; Kay et al., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol. 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573). Recombinant retroviral vectors capable of transducing and expressing genes inserted into the genome of a cell can be produced by transfecting the recombinant retroviral genome into suitable packaging cell lines such as PA317 and Psi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone et al., 1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant adenoviral vectors can be used to infect a wide variety of cells and tissues in susceptible hosts (e.g., rat, hamster, dog, and chimpanzee) (Hsu et al., 1992, J. Infectious Disease, 166:769), and also have the advantage of not requiring mitotically active cells for infection.
The promoter driving dsRNA expression in either a DNA plasmid or viral vector of the invention may be a eukaryotic RNA polymerase I (e.g. ribosomal RNA promoter), RNA polymerase II (e.g. CMV early promoter or actin promoter or U1 snRNA promoter) or preferably RNA polymerase III promoter (e.g. U6 snRNA or 7SK RNA promoter) or a prokaryotic promoter, for example the T7 promoter, provided the expression plasmid also encodes T7 RNA polymerase required for transcription from a T7 promoter. The promoter can also direct transgene expression to the pancreas (see, e.g. the insulin regulatory sequence for pancreas (Bucchini et al., 1986, Proc. Natl. Acad. Sci. USA 83:2511-2515)).
In addition, expression of the transgene can be precisely regulated, for example, by using an inducible regulatory sequence and expression systems such as a regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems, suitable for the control of transgene expression in cells or in mammals include regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-D1-thiogalactopyranoside (EPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the dsRNA transgene.
Preferably, recombinant vectors capable of expressing dsRNA molecules are delivered as described below, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of dsRNA molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the dsRNAs bind to target RNA and modulate its function or expression. Delivery of dsRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.
dsRNA expression DNA plasmids are typically transfected into target cells as a complex with cationic lipid carriers (e.g. Oligofectamine) or non-cationic lipid-based carriers (e.g. Transit-TKOTM). Multiple lipid transfections for dsRNA-mediated knockdowns targeting different regions of a single Nav1.8 gene or multiple Nav1.8 genes over a period of a week or more are also contemplated by the invention. Successful introduction of the vectors of the invention into host cells can be monitored using various known methods. For example, transient transfection, can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection of ex vivo cells can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.
The Nav1.8 specific dsRNA molecules can also be inserted into vectors and used as gene therapy vectors for human patients. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

## Claims

1. A double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of a human Nav1.8 gene in a cell, wherein said dsRNA consists of a sense strand and an antisense strand that are complementary to each other, wherein said sense strand consists of SEQ ID NO:17 and said antisense strand consists of SEQ ID NO:18.

2. The dsRNA of claim 1, wherein said dsRNA, upon contact with a cell expressing said Nav1.8, inhibits expression of said Nav1.8 gene by at least 20%.

3. A cell comprising the dsRNA of claim 1.

4. A pharmaceutical composition for inhibiting the expression of the Nav1.8 gene in an organism, comprising a dsRNA and a pharmaceutically acceptable carrier, wherein the dsRNA consists of a sense strand and an antisense strand that are complementary to each other, wherein said sense strand consists of SEQ ID NO:17 and said antisense strand consists of SEQ ID NO:18.

5. The pharmaceutical composition of claim 4, wherein said dsRNA, upon contact with a cell expressing said Nav1.8 gene, inhibits expression of said Nav1.8 gene by at least 20%.

6. The pharmaceutical composition of claim 4, wherein said composition is formulated for administration selected from the group consisting of intrathecal infusion or injection, or intravenous infusion or injection.

7. A method for inhibiting the expression of the Nav1.8 gene in a cell, the method comprising:
(a) introducing into the cell a double-stranded ribonucleic acid (dsRNA), wherein the dsRNA consists of a sense strand and an antisense strand that are complementary to each other, wherein said sense strand consists of SEQ ID NO:17 and said antisense strand consists of SEQ ID NO:18; and
(b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the Nav1.8 gene, thereby inhibiting expression of the Nav1.8 gene in the cell.

8. The method of claim 7, wherein said dsRNA, upon contact with a cell expressing said Nav1.8, inhibits expression of said Nav1.8 gene by at least 20%.

9. A method of treating, preventing or managing pain comprising administering to a patient in need of such treatment, prevention or management a therapeutically or prophylactically effective amount of a dsRNA, wherein the dsRNA consists of a sense strand and an antisense strand that are complementary to each other wherein said sense strand consists of SEQ ID NO:17 and said antisense strand consists of SEQ ID NO:18.

10. The method of claim 9, wherein said dsRNA, upon contact with a cell expressing said Nav1.8, inhibits expression of said Nav1.8 gene by at least 20%.

11. The method of claim 8, wherein said pain is selected from the group consisting of neuropathic pain and inflammatory pain.