COMPOSITIONS AND METHODS FOR INHIBITING EXPRESSION OF

Nayl.8 GENE

Related Applications

This application claims the benefit of U.S. Provisional Application No. 60/733,816, filed November 4, 2005; U.S. Provisional Application No.

60/741,586, filed December 2, 2005; U.S. Provisional Application No. 60/763,202, filed January 26, 2006; U.S. Provisional Application No. 60/795,443, filed April 27, 2006; and U.S. Provisional Application No.60/849,364, filed October 4, 2006. The contents of these prior applications are incorporated herein by reference in their entirety.

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 Navl.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 maybe 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, antiarrhythmics, 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 syringomyelia (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" ProcNatl 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: areview" J Rehabil Res Dev 37(5): 517-28). Three members of the gene family (NaVl.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 glutaraate 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 NaV 1.8 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 NaVl.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 Navl.8 at the site of nerve injury, concomitant with an upregulation in TTX- resistant sodium current, consistent with NaVl.8 as a mechanism underlying hyperalgesia (see Gold, M. S., D. Weinreich, et al. (2003) "Redistribution of Navl.8 in uninjured axons enables neuropathic pain" J. Neurosci. 23: 158-166). Attenuation of NaVl.8 expression with antisense oligodeoxynucleotides administered by intrathecal injection prevented experimental nerve-injury induced redistribution of Navl .8 in the sciatic nerve and reversed neuropathic pain (tactile and thermal hyperalgesia), demonstrating a causal role of Navl.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, NaVl, 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.). hi inflammatory pain models, intrathecal administration of antisense oligodeoxynucleotides against NaV1.8 resulted in a significant reduction in PGE ₂ -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 NaVl.8, showing that Navl.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 Navl .8 (PN3/SNS) in a rat model of visceral pain" J. Neurosci. 21 : 8690-8696).

Taken together, these data support a role for NaVl.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 Navl .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 al.), Drosophila (see, e.g., Yang, D., et al., Curr. Biol. (2000) 10:1191- 120O) ₅ 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 Navl.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 Navl.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 Navl ,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 Navl, 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 Navl .8 gene.

In one embodiment, the invention provides double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of the Navl .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 Navl .8, and the region of complementarity is less than 30 nucleotides in length. The dsRNA, upon contacting with a cell expressing the Navl .8, inhibits the expression of the Navl .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 ^l -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 Navl .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 Navl.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 Navl .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 Navl.8, inhibits expression of the Navl .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 Nav 1 , 8 gene, thereby inhibiting expression of the Navl .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 Navl .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 Navl .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 Navl .8 in Cos-7 cells.

FIG. 2. Lack of cross reactivity of Navl.8 dsRNAs to endogenous Navl.5 mRNA in SW620 cells in vitro.

FIG. 3. In vitro activity of the dsRNAs provided in Table 1 against endogenous Navl .8 mRNA in primary cultures of rat dorsal root ganglion cells.

FIG. 4. Dose response of dsRNA AL-DP-6209 against endogenous Navl .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 ⁰ 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 Navl .8 in primary cultures of rat dorsal root ganglion cells.

FIG. 13. 7« 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 Navl ,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 Navl .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 Navl .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 Navl.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 Navl.8 gene, as well as compositions and methods for treating diseases and disorders caused by the expression of Navl .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 Navl .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 Navl.8 gene, and methods of using the pharmaceutical compositions to treat diseases caused by expression of the Navl .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 "Navl.8" as used herein is meant, any Navl.8 protein, peptide, or polypeptide associated with the development or maintenance of an ion channel. The terms "Navl.8" also refer to nucleic acid sequences encoding any Navl.8 protein, peptide, or polypeptide. For the Examples, the Navl .8 mRNA sequences used were human (NM_006514), mouse (NM_009134), rat (NM_017247) and dog (NMOO 1003203) 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

Navl.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, 5O ⁰ C or 7O ⁰ 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. i

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 Navl .8). For example, a polynucleotide is complementary to at least a part of a Navl .8 mRNA if the sequence is substantially complementary to a non- interrupted portion of an mRNA encoding Navl .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 Navl.8 gene, herein refer to the at least partial suppression of the expression of the Navl.8 gene, as manifested by a reduction of the amount of mRNA transcribed from the Navl.8 gene which maybe isolated from a first cell or group of cells in which the Navl .8 gene is transcribed and which has or have been treated such that the expression of the Navl .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

(mRNA in control cells) - (mRNA in treated cells) .

(mRNA in control cells)

Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to Navl.8 gene transcription, e.g. the amount of protein encoded by the Navl.8 gene which is secreted by a cell, or the number of cells

displaying a certain phenotype, e.g apoptosis. In principle, Navl .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 Navl .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 Navl.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 Navl .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 Navl.8 gene is suppressed by at least about 85%, 90%, or 95% by administration of the double-stranded oligonucleotide of the invention. Li a most preferred embodiment, the Navl .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)

hi one embodiment, the invention provides double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of the Navl .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 Navl .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 Navl .8 gene, inhibits the expression of said Navl .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 Navl .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 Navl .8 gene is the human Navl .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 Navl .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 Navl.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 no 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 Navl .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 Navl .8 gene. Consideration of the efficacy of dsRNAs with mismatches in inhibiting expression of the Navl.8 gene is important, especially if the particular region of complementarity in the Navl .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, NY, 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-l,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.

hi 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'-<9-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 peπneation 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, l,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.

hi 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 aralkyϊ 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, CA). 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 ^? -fiuoro-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 ^f -O-methyl, 2'-0-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'-0-ethyl, 2'-O-propyl, 2'-O- aminoalkyl, 2'-O-allyl or 2'-deoxy-2 ^l -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-Modifϊer 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, l-(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.l) 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, Virginia. 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 5yn-l,2-acetoxyphosphate group which more readily undergoes basic hydrolysis. See Guzaev, A. L; Manoharan, M. J Am. Chem. Soc. 2003, 125, 2380.

The nucleosides are linked by phosphorus-containing or non-phosphorus- containing covalent internucleoside 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 noraial 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 internucleoside 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 CH ₂ 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). hi 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 -CH ₂ -NH-O-CH ₂ --, ~CH ₂ -N(CH ₃ )-O~CH ₂ - [known as a methylene (methylimino) or MMI backbone], -CH ₂ ~O~N(CH ₃ )~CH2 --, -CH ₂ -N(CH ₃ )- N(CHs)-CH ₂ -, and -O~N(CH ₃ )~CH ₂ -CH ₂ - [wherein the native phosphodiester backbone is represented as --0-P-O-CH ₂ -] 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-ρropyl 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. L, 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 O-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 ⁰ 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 T 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 C ₁ to C ₁₀ alkyl or C ₂ to C ₁₀ alkenyl and alkynyl. Particularly preferred are O[(CH ₂ ) _n O] _m CH ₃ , O(CH ₂ ) _n OCH ₃ , O(CH ₂ ) _n NH ₂ , O(CH ₂ ) _n CH ₃ , O(CH ₂ ) _n ONH ₂ , and O(CH ₂ ) _n ON[(CH ₂ ) _n CH ₃ )] ₂ , where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2' position: C ₁ to C ₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH ₃ , OCN, Cl, Br, CN, CF ₃ , OCF ₃ , SOCH ₃ , SO ₂ CH ₃ , ONO ₂ , NO ₂ , N ₃ , NH ₂ , 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— CH ₂ CH ₂ OCH ₃ , also known as 2'-O-(2-methoxyethyl) or 2'-MOE] (Martin et al, HeIv. Chim. Acta, 1995, 78, 486), i.e., an alkoxyalkoxy group. A further preferred modification includes 2'-dimethylaminooxyethoxy, i.e., a O(CH ₂ ) ₂ ON(CH ₃ ) 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'-0-CH ₃ ), T- aminopropoxy (2'-OCH ₂ CH ₂ CH ₂ NH ₂ ) 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 T-S ¹ 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, fiuoro, O- alkyl, O-alkylamino, O-alkylalkoxy, protected 0-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). Fiuoro, O-alkyl, O-alkylamino, O-alkyl imidazole, O- alkylaminoalkyl, and alkyl amino substitution is described in U.S. Patent 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'-NR ₂ 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:

Z ₅ ) q4

I II

wherein, E is C ₁ -C ₁₀ alkyl, N(Q ₃ )(Q ₄ ) or N=C (Q ₃ )(Q ₄ ); each Q ₃ and Q ₄ is, independently, H, C ₁ -C ₁₀ alkyl, dialkylaminoalkyl, a nitrogen protecting group, a tethered or untethered conjugate group, a linker to a solid support; or Q ₃ and Q ₄ , together, form a nitrogen protecting group or a ring structure optionally including at least one additional heteroatom selected from N and O;

qi is an integer from 1 to 10;

q ₂ is an integer from 1 to 10;

q ₃ is 0 or 1;

q ₄ is 0, 1 or 2;

each Z ₁ , Z ₂ and Z ₃ is, independently, C ₄ -C ₇ cycloalkyl, C ₅ -C ₁₄ aryl or C ₃ -C ₁₅ heterocyclyl, wherein the heteroatom in said heterocyclyl group is selected from oxygen, nitrogen and sulfur;

Z ₄ is OM ₁ , SM ₁ , OrN(MO ₂ ; each M ₁ is, independently, H ₃ C ₁ -C ₈ alkyl, C ₁ -C ₈ haloalkyl, C(=NH)N(H)M ₂ , C(=O)N(H)M ₂ or OC(=O)N(H)M ₂ ; M ₂ is H or C ₁ -C ₈ alkyl; and

Z ₅ is C ₁ -Ci ₀ alkyl, C ₁ -Cj ₀ haloalkyl, C ₂ -C ₁₀ alkenyl, C ₂ -C ₁₀ alkynyl, C ₆ -C ₁₄ aryl, N(Q ₃ )(Q ₄ ), OQ ₃ , halo, SQ ₃ or CN.

Representative 2'-O-sugar substituent groups of formula I 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. Patent 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, CH ₂ , CHF, and CF ₂ . See, e.g., Secrist et al., Abstract 21, Program & Abstracts, Tenth International Roimdtable, 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. Sd. 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. NY. Acad. Sd., 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.,

EMBOJ., 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 l,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. Sd. 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. NY. Acad. Sd., 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 l,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 maybe 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 Navl .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 Navl .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 Navl .8 gene; additional dsRNAs are prepared, each of which has a nucleotide sequence that is substantially complementary to different part of the Navl .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 Navl .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 Navl .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, intraparencliymal (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, intraparenchymal (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 Navl.8 gene. Such substances include, for example, mi cellar 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 Navl.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. Patent 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 IPAl 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. Nos. 09/872,698 (filed Jun. 1, 2001) and 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.

hi 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 Nayl.8 gene

In one embodiment, the invention provides a method for treating a subject having a pathological condition mediated by the expression of the Navl .8 gene, such as neuropathic or inflammatory pain. In this embodiment, the dsRNA acts as a

therapeutic agent for controlling the expression of the Navl .8 protein. The method comprises administering a pharmaceutical composition of the invention to the patient (e.g., human), such that expression of the Navl.8 gene is silenced. Because of their high specificity, the dsRNAs of the invention specifically target mRNAs of the Navl .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, hi 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-iVαv/.<§ 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 Nayl.8 gene

In yet another aspect, the invention provides a method for inhibiting the expression of the Navl .8 gene in a mammal. The method comprises administering a composition of the invention to the mammal such that expression of the target Navl .8 gene is silenced. Because of their high specificity, the dsRNAs of the invention specifically target RNAs (primary or processed) of the target Navl .8 gene.

Compositions and methods for inhibiting the expression of these Navl.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 Navl .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 Nayl.8 gene

siRNAs were identified in a multi step sequence analysis process in order to design siRNAs targeting the Navl .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) Navl.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 (http://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 refered 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 Navl.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 forNavl.8

Ui Ul

Table 4: Further siRNAs specific for Navl .8

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Example 2: Optimization of siRNAs bv 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 Navl.8 gene expression (Navl.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, 5OθA, 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 '-(9-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, NY, 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 - 9O ⁰ C for 3 minutes and cooled to room temperature over a period of 3 - 4 hours. The annealed RNA solution was stored at -20 ⁰ C until use.

For the synthesis of 3 '-cholesterol-conjugated siRNAs (herein referred to as - Choi 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 x 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-ylmethoxycarbony l-amino)- hexanoyl] -amino} -propionic acid ethyl ester AB

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 ⁰ C. It was then followed by the addition of Diethyl-azabutane-l,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]-propion ic acid ethyl ester AC

AC

3-{Ethoxycarbonylniethyl-[6-(9H-fluoren-9-ylmethoxycarbon ylamino)- 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-(l ,5-Dimethyl-hexyl)- 10, 13-dimethyl- 2,3,4,7,8,9,10,1 ^π^SJ^lSjlό^T-tetradecahydro-lH-cyclopentafaJphenanthren-S - yloxycarbonylaminoj-hexanoyl} ethoxycarbonylmethyl-amino)-propionic acid ethyl ester AD

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%).

l-{6-[17-(l,5-Dimemyl-hexyl)-10,13-dimethyl- 2,3,4,7,8,9,10,1 l,12,13,14,15,16,17-tetradecahydro-lH-cyclopenta[a] phenanthren-3- yloxycarbonylamino]-hexanoyl}-4-oxo-pyrrolidine-3-carboxylic acid ethyl ester AE

AE

Potassium t-butoxide (1.1 g, 9.8 mmol) was slurried in 30 mL of dry toluene. The mixture was cooled to 0 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 OfNaH ₂ PO ₄ -H ₂ O 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-l-yl)-6-oxo-hexy l]-carbamic acid 17-(l,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,l 1,12,13,14,15,16,17- tetradecahydro-lH-cyclopentafajphenanthren-S-yl ester AF

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 x 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/CHCl ₃ ) (89%).

(6- { 3 - [Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl] -4-hydroxy-pyrrolidin-

1 -yl}-6-oxo-hexyl)-carbamic acid 17-(l ,5-dimethyl-hexyl)-l 0, 13-dimethyl- 2,3,4,7,8,9, 10, 11 , 12, 13, 14, 15, 16, 17-tetradecahydro- lH-cyclopenta[a]phenanthren-3-yl ester AG

AG

Diol AF (1.25 gm 1.994 mmol) was dried by evaporating with pyridine (2 x 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 IM 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/CHCls) (1.75 g, 95%).

Succinic acid mono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-l-{6- [17-(l,5-dimethyl-hexyl)-10,13-dimethyl 2,3,4,7,8,9,10,11,12,13,14,15,16,17- tetradecahydro-lH cyclopentafaJphenanthren-S-yloxycarbonylaminoJ-hexanoyl}- pyrrolidin-3-yl) ester AH

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 niL), 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 X 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

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.

"capital letters represent 2'-deoxyribonucleotides (DNA), lower case letters represent ribonucleotides (RNA)

Single-dose screen of Nayl.8 siRNAs against mRNA expression of transfected human Nayl.8 in Cos-7 cells.

All Navl.8 siRNAs in Table 1 and Table 4 were tested initially at a single dose of 100 riM (Table 1) or 5OnM (Table 4) for activity in reducing mRNA expression of transfected human Navl .'8 in Cos-7 cells. One day before transfection, Cos-7 cells (DSMZ, Braunschweig, Germany) were seeded at 1.5 x 10 ⁴ 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, 10Ou penicillin / 100 μg/ml streptomycin, all from Biochrom AG, Berlin, Germany). Four hours prior to siRNA transfection, 20ng of plasmid/well (Table 1) or 50ng/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 Navl .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 ⁰ C and 5 % CO ₂ 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 Navl.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, Germany) as RLUs (relative light units) and values obtained with the human Navl.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 Navl .8 were further normalized relative to the value obtained with an unrelated siRNA (directed against hepatitis C virus) which was set to 100%.

Figure 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 5OnM. Several siRNAs (in Tables 1 and 4) were effective at at the dose tested in reducing Navl .8 mRNA levels by at least 50% in COS-7 cells transfected with Navl .8.

Dose-response curves for selected siRNAs against mRNA expression of transfected human Nayl.8 in Cos-7 cells

Several effective siRNAs against Navl.8 from the single dose screen (results in Figure 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 Navl.8

Specificity testing of the Nayl.8 siRNAs against Nayl.5 mRNA

Navl .5 is an NaV subtype that is closely related to Navl .8, but has an important role in normal cardiac function. Therefore, it is important to confirm that siRNAs selective for Navl .8 do not inhibit Navl .5 expression. Several siRNAs were tested for specificity towards Navl .8 by transfecting SW620 cells, which express endogenous human Navl .5, with these siRNAs, and assessing Navl .5 mRNA levels. A control unrelated siRNA (AL-DP-5002) was also transfected into SW620 cells. siRNAs targeting Navl .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 Navl.8 (AL-DP-6217) was tested at 1 nM, 10 nM, 100 nM and 1 uM. The expression of Navl .5 mRNA, which encodes the protein with the highest homology to Navl .8 in the Nav-family, was then quantified.

One day before transfection, SW620 cells (LCG Promochem, Wesel,

Germany) were seeded at 1.5 x 10 ⁴ 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, lOOu 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 CO ₂ 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 Navl.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 Navl .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.

Figure 2 provides the results. At the concentrations tested, the selected siRNAs targeting Navl .8, did not exhibit significant dose-dependent inhibition of Navl.5 mRNA as compared with the unrelated control siRNA (AL-DP-5002), confirming the specificity of these Navl .8 siRNAs for Navl .8 over Navl .5.

In addition, at concentrations up to 1 uM, AL-DP-6217 exhibited no significant inhibition of Navl .5 mRNA (data not shown), confirming the specificity of this Navl .8 siRNA for Navl .8 over Navl .5.

Table 5: bDNA Probesets for the quantitation of human Navl.8, Navl.5 and GAPDH

Human Navl .8 probeset:

SEQ ID

FPL Name Function Sequence NO: hNal 82001 CE TTTGGAGGTTAAAGGTGATCCATTTTTTCTCTTGGAAAGAAAGT 543 hNal 82002 CE GGCGTTTTCCAGAGGCGAGTTTTTCTCTTGGAAAGAAAGT 544

SEQ ID

FPL Name Function Sequence NO: hNal 82003 CE CCAGCAGCAGAGAGCCCCTTTTTCTCTTGGAAAGAAAGT 545 hNal 82004 CE CTGGGTTGAGGAAGAGGGCTTTTTTCTCTTGGAAAGAAAGT 546 hNal 82005 CE AACACTCATTGCCCTTTGGGTTTTTCTCTTGGAAAGAAAGT 547 hNal82006 CE AAGGACGGAGGTTATGATACTGACTTTTTCTCTTGGAAAGAAAGT 548 hNal 82007 CE TGTTCAGACTCCTCGAGTTCCTCTTTTTCTCTTGGAAAGAAAGT 549 hNal 82008 LE CTATGCCTTCTCTCACTGGCATTTTTTTAGGCATAGGACCCGTGTCT 550 hNal 82009 LE CTGGTTGTAAGGATCAGAGCGGTTTTTAGGCATAGGACCCGTGTCT 551 hNal82010 LE AACACACTGCCATGACTAGCCCTTTTTAGGCATAGGACCCGTGTCT 552 hNal82011 LE GATGGCTTTCGTGGTCTCCATTTTTAGGCATAGGACCCGTGTCT 553 hNal82012 LE CTGGCCAGCACCCCCACTTTTTAGGCATAGGACCCGTGTCT 554 hNal82013 LE CATGCCTGGAGTCAGGGTTGTTTTTAGGCATAGGACCCGTGTCT 555 hNal82014 LE AGACATCGACAGCTCCAGGGTTTTTAGGCATAGGACCCGTGTCT 556 hNal82015 LE GTCCTGCATCGAATGCCGTTTTTAGGCATAGGACCCGTGTCT 557 hNal82016 LE CAAGCAGGGTGGGCACTTCTTTTTAGGCATAGGACCCGTGTCT 558 hNal82017 BL TGTGGGAGTGGAGAGAGGTTG 559

IiNaI 82018 BL CCCTCTGACACTCTTGGCTTTATT 560 hNal 82019 BL GGTGATTTGTTGTCTTCTGTGGAG 561 hNal 82020 BL GCCTAGAAAAGACATCCTGCG - 562 hNal 82021 BL GCCAGGGGACCGGAAATGG 563 hNal 82022 BL CCCTCAGGGAGTGAGATATCTCG 564 hNal 82023 BL GGAAAGACTCCATCATCTGTGACT 565 hNal 82024 BL TCTAGGGAGGGGGCCTTG 566 hNal 82025 BL GCGGTTGGTGTTCATCTTCTC 567 hNal 82026 BL GCAAGCTCACTAGTGGGCG 568 hNal 82027 BL TCTGCTGACAAGAAAGTCTTCTTTT 569 hNal 82028 BL CCCGGAAAGGTTCATCTAAGTAT 570

Human GAPDH probeset:

SEQ ID

FPL Name Function Sequence NO: hGAPOOl CE GAATTTGCCATGGGTGGAATTTTTTCTCTTGGAAAGAAAGT 571 hGAP002 CE GGAGGGATCTCGCTCCTGGATTTTTCTCTTGGAAAGAAAGT 572 hGAP003 CE CCCCAGCCTTCTCCATGGTTTTTTCTCTTGGAAAGAAAGT 573 hGAP004 CE GCTCCCCCCTGCAAATGAGTTTTTCTCTTGGAAAGAAAGT 574 hGAP005 LE AGCCTTGACGGTGCCATGTTTTTAGGCATAGGACCCGTGTCT 575 hGAPOOδ LE GATGACAAGCTTCCCGTTCTCTTTTTAGGCATAGGACCCGTGTCT 576 hGAP007 LE AGATGGTGATGGGATTTCCATTTTTTTAGGCATAGGACCCGTGTCT 577 hGAP008 LE GCATCGCCCCACTTGATTTTTTTTTAGGCATAGGACCCGTGTCT 578 hGAP009 LE CACGACGTACTCAGCGCCATTTTTAGGCATAGGACCCGTGTCT 579 hGAPOlO LE GGCAGAGATGATGACCCTTTTGTTTTTAGGCATAGGACCCGTGTCT 580

SEQ ID

FPL Name Function Sequence NO: hGAPOl l BL GGTGAAGACGCCAGTGGACTC 581

Human Navl .5 probeset:

SEQ ID

FPL Name Function Sequence NO: hNal5001 CE CGTTTTCTCCTCTTGCTTCTTCTCTTTTTCTCTTGGAAAGAAAGT 582 hNal5002 CE GGGAGCCTGTCCTCCCCATTTTTCTCTTGGAAAGAAAGT 583 hNal5003 CE TCGCCTGCGAAAGGTGAATTTTTCTCTTGGAAAGAAAGT 584 hNal5004 CE CTCTCGCTCTCCCCCGCTTTTTCTCTTGGAAAGAAAGT 585 hNal5005 CE CCGGGACTGGGCTGTCCTTTTTCTCTTGGAAAGAAAGT 586 hNal5006 CE TTTTGCCATGGAGGGCGTTTTTTCTCTTGGAAAGAAAGT 587 hNal5007 LE GGCTGAGATGATTCATTGCTCTGTTTTTAGGCATAGGACCCGTGTCT 588 hNal5008 LE GCTGAGGCCACGGGTGATTTTTAGGCATAGGACCCGTGTCT 589 hNal5009 LE AATGCTCCCGCGGCTGGTTTTTAGGCATAGGACCCGTGTCT 590 hNal5010 LE CTGGGCACTGGTCCGGCTTTTTAGGCATAGGACCCGTGTCT 591 hNal5011 LE GGCCAGGAGCCGAGGTTTTTTTAGGCATAGGACCCGTGTCT 592 hNal5012 LE CCCAGTAATGAGACCACCCCATTTTTTAGGCATAGGACCCGTGTCT 593 hNal5013 LE CCTCTGGGTCGCCTGCCTTTTTAGGCATAGGACCCGTGTCT 594 hNal5014 BL CACTCCTCAGTTCCTGAAGACATC 595 hNal5015 BL GGACCATCTTCTGAGTCAGACTTG 596 hNal5016 BL AACGTGGCTTCATAGAAGTCCT 597 hNal5017 BL AATCTGCTTCAGAACCCAGGTC 598 hNal5018 BL TGTGCTGTTTTCATCATCTGCAA 599 hNal5019 BL CCAGCAGTGATGTGTGGTGG 600 hNal5020 BL GCAGGGGCCAGGGCA 601 hNal5021 BL TGCAGTCCACAGTGCTGTTCT 602 hNal5022 BL GGCTTCCTGGGGATGTGG 603

Single-dose screen of Nayl.8 siRNAs against mRNA expression of endogenous Nayl.8 in primary cultures of rat dorsal root ganglion cells.

To confirm and extend the results obtained on mRNA expression of transfected human Navl.8 in Cos-7 cells, Navl.8 siRNAs from Table 1 were tested at a single dose of 200 nM for activity in reducing mRNA expression of endogenous Navl.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 ⁰ 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 Biocoat™ PDL PoIy-D-

Lysine/Laminin 96-well plates (BD Biosciences, Bedford MA, USA) in F12-HAM's Medium containing glutamine (Invitrogen Gibco, Carlsbad CA, USA) with 5% fetal bovine serum (FBS, heat inactivated) and 5% horse serum (heat inactivated) (both Invitrogen Gibco, Carlsbad CA, USA) supplemented with 50 ng/ml mouse nerve growth factor 2.5S (NGF; Promega Corp., Madison WI, USA) and kept at 37°C, 5%CO ₂ in a humidified incubator until transfection.

Navl.8 siRNAs were screened in DRG cultures at 200 nM in duplicate using TransMessenger™ Transfection reagent (Qiagen GmbH, Hilden, Germany, cat. no. 301525) which is based on a lipid formulation, a specific RNA-condensing reagent (Enhancer R™) and an RNA-condensing buffer (Buffer EC-R™), keeping siRNA: Enhancer R™ ratio (μg:μl) constant at 1:2, and siRNA:TransMessenger™ ratio (μg:μl) constant at 1 :12.

DRG neurons were transfected 24h post-plating. For each well, 0.52 μl Enhancer R™ were first mixed with 13.68 μl Buffer EC-R™. 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 TransMesssenger™ Transfection Reagent were diluted with 6.88 μl Buffer EC-R™, 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 CA, USA) supplemented with 50 ng/ml NGF 2.5S (Promega Corp., Madison WI, USA) and 1:50 B27 supplement (Invitrogen Gibco, Carlsbad CA, 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 ⁰ C, 5%CO ₂ 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 CA, USA), 50 ng/ml mouse NGF 2.5S (Promega Corp., Madison WI, USA) and 1:100 Penicillin/Streptomycin (Invitrogen Gibco, Carlsbad CA, USA) was added, the cells were incubated for another 16 h at 37°C, 5%CO ₂ in a humidified incubator, and Navl.8 mRNA was quantified.

Navl .8 mRNA levels were measured using the QuantiGene™ 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 Navl .8 and mouse synuclein

(SNCL). Chemoluminescence was read on a Victor ² -Light™ (PerkinElmer Life And Analytical Sciences, Inc., Boston MA, USA) as Relative Light Units (RLU). RLU for Navl .8 were normalized to SNCL RLU for each well. Normalized Navl .8/SNCL ratios were then compared to the siRNA-free control, which was set as 100%.

Figure 3 provides the results. At 200 nM, at least 13 (indicated by '*') of the

Navl .8 siRNAs tested showed at least 50% Navl .8 mRNA knock down compared to the siRNA-free control (TransMessenger™ 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 Nayl.8 in primary cultures of rat dorsal root ganglion cells

One effective siRNA (AL-DP-6209) against Navl .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 (Figure 4).

Figure 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 Navl .8 mRNA expression relative to SNCL, whereas at 88 and 175 nM, AL-DP-6209 inhibited Navl .8 mRNA expression relative to SNCL by >40%. Maximal inhibition of Navl .8 mRNA expression relative to SNCL occurred in this experiment at 175 nM.

Intrathecal bolus administration of siRNAs against Nayl.8 with iFECT prevents inflammatory pain

The effect of siRNAs against Navl.8, formulated with iFECT, on complete Freund's adjuvant-induced tactile hypersensitivity was evaluated in rats (Figure 5). Adult male Sprague-Dawley rats received an injection of CFA (150 uL) into the hindpaw on day 0. siRNAs against Navl.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 MN, 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 IL, 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, WJ. (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 Navl.8 siRNA, AL-DP-6209, is efficacious in vivo against inflammation-induced hyperalgesia. Treatment with the Navl.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 Navl .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 Nayl.8 without transfection reagent alleviates inflammatory pain

The effect of siRNAs against Navl .8, formulated in phosphate buffered saline

(PBS), on complete Freund's adjuvant (CFA) -induced tactile hypersensitivity was evaluated in rats (Figure 6). Of 3 Navl.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 Navl.8

SEQ SEQ

Duplex sense strand ID anti-sense strand ID name sequence (5 '-3') NO: sequence (5 "-3") NO:

AL-DP-

4461 cauccuaugaaccaauagceTeT 604 gcuauugguucauaggaugeceT 605

AL-DP- cmaumcmcmumaumgaacmcmaa

4459 umagcmTT-sChol 606 gcumauugguucmaumaggaugTT 607

AL-DP- umumumumgumcmumaaaumgag

6980 umumcmaTT-sCho1 608 ugaacucmauuumagacmaaaaTT 609

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 Navl.8 (AL-DP-4461, AL-DP- 4459, or AL-DP-6980), or PBS. siRNAs against Navl.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 Navl .8 siRNA, AL-DP-4461, is moderately efficacious with this dosing paradigm in vivo against CFA-induced tactile hyperalgesia. Treatment with the Navl .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 Navl .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 Navl .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 Nayl.8 without transfection reagent alleviates inflammatory pain

The effect of siRNAs against Navl.8, formulated in phosphate buffered saline (PBS), on complete Freund's adjuvant (CFA) -induced tactile hypersensitivity was evaluated in rats after intrathecal pump infusion (Figure 7, left) or intrathecal BID bolus injection (Figure 7, right). Of 3 Navl.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 NaV 1.8 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 Navl.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 (Figure 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 Navl .8 siRNA, AL-DP- 6050, is modestly efficacious with this dosing paradigm in vivo against inflammatory pain.

For evaluating effects of siRNAs against NaV 1.8 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 Navl.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 (Figure 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 Navl .8 siRNA, AL-DP- 6050, is modestly efficacious with this dosing paradigm in vivo against inflammatory pain.

These results further demonstrate that siRNAs targeting Navl.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 Nayl.8 without transfection reagent alleviates inflammatory pain

The effect of siRNAs against Navl.8, formulated in phosphate buffered saline (PBS), on complete Freund's adjuvant (CFA) -induced thermal hypersensitivity was evaluated in rats (Figure 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 Navl.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

Navl.8 siRNA, AL-DP-6050, and the cholesterol-conjugated Navl.8 siRNA AL-DP- 4459 are efficacious with this dosing paradigm in vivo against inflammatory pain.

These results demonstrate that siRNAs targeting Navl.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 Nayl.8 without transfection reagent alleviates neuropathic pain

The effect of AL-DP-4459 (Table 6) against Navl .8, formulated in phosphate buffered saline (PBS), on spinal nerve ligation (SNL) -induced tactile and thermal hypersensitivity was evaluated in rats (Figure 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 Navl .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 (Figure 9, left) and thermal (Figure 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 Navl.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 Navl.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 Navl.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 ⁰ C

To determine the stability in human cerebrospinal fluid (CSF) of unconjugated and cholesterol-conjugated siRNAs targeting NaV 1.8, 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. 30μl of human CSF was mixed with 3μl of 50μM siRNA (150pmole/well) in a 96-well plate, sealed to avoid evaporation, and incubated for 1 to 48 hours at 37 ⁰ C. Incubation in 30μl PBS for 48 hours at 37 ⁰ C served as a control for nonspecific degradation.

Reactions were stopped by the addition of 4μl proteinase K (20mg/ml) and 25 μl of proteinase K buffer, and incubation of this mixture for 20min at 42° C. Samples were

spin filtered through a 0.2μm 96-well filter plate at 3000 rpm for 20min. Incubation wells were washed with 50μl Millipore water twice and the combined washing solutions were spin filtered also. A 5μl aliquot of 50μM 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 (4x250 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:

2. HPLC System for unconjugated siRNAs AL-DP-6050, 6209, 6217, 6218, 6219

Column: Dionex DNAPac PA200 (4x250 mm analytical column)

Temp.: 3O ⁰ C (denaturing conditions by pH=l 1)

Flow: 1 ml/min

Injection: 50 ul

Detection: 260 nm (reference wavelength 600 ran)

HPLC Eluent A: 2OmM Na ₃ PO ₄ in 10% ACN; pH=l 1

HPLC Eluent B: 1 M NaBr in A

Gradient table:

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 6.60 HPLC 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=omi _n ) * 100%

All values were normalized to the incubation at t=0 min.

The results for the unconjugated siRNAs are shown in Figure 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 Figure 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 ⁰ C for 48 hours, with less than 20% variation detected over this time period.

Unconjugated and cholesterol-conjugated siRNAs targeting NaV1.8 reduce Na Vl.8 mRNA in a dose-dependent manner in primary rat sensory neuronal cultures

Another effective siRNA (AL-DP-6050, Table 1) against Navl.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 (Figure 12).

DRG neurons were transfected 24h post-plating. For each well, 0.4 μl Lipofectamine™ 2000 (Invitrogen Corporation, Carlsbad, CA) was used and transfections were performed according to the manufacturer's protocol. Specifically,

the siRNA: Lipofectamine™ 2000 complexes were prepared as follows. The appropriate amount of siRNA was diluted in Opti-MEM I Reduced Serum Medium and mixed gently. The Lipofectamine™ 2000 was vortexed before use; then for each well of a 96 well plate, 0.4 ul Lipofectamine™ 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 Lipofectamine™ 2000 (total volume, 26.4 μl). The complex was mixed gently and incubated for 20 minutes at room temperature to allow the siRNA: Lipofectamine™ 2000 complexes to form. Then 100 μl of serum-free F12-HAM's Medium containing glutamine (Invitrogen Gibco, Carlsbad CA, USA) supplemented with 50 ng/ml NGF 2.5S (Promega Corp., Madison WI, USA) and 1 :50 B27 supplement (Invitrogen Gibco, Carlsbad CA, 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 2Oh of incubation at 37 ⁰ C, 5%CO ₂ 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 CA, USA), 50 ng/ml mouse NGF 2.5S (Promega Corp., Madison WI, USA) and 1:100 Penicillin/Streptomycin (Invitrogen Gibco, Carlsbad CA, USA) was added. The cells were incubated for another 20-24h at 37 ⁰ C, 5%CO ₂ in a humidified incubator, and Navl.8 mRNA was quantified by the bDNA assay, as described previously.

Figure 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 Navl .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 Nayl.8 without transfection reagent alleviates neuropathic pain

The effect of AL-DP-6050 (Table 1) against Navl.8, formulated in phosphate buffered saline (PBS), on spinal nerve ligation (SNL) -induced thermal hypersensitivity was evaluated in rats (Figure 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 Navl .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. Li 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 Navl.8 siRNA, AL-DP-6050, is efficacious in vivo against SNL-induced thermal hyperalgesia.

These results demonstrate that siRNAs targeting Navl.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 Nayl.8 alleviates neuropathic pain

The effect of AL-DP-6050 (Table 1) against Navl .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 (Figure 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 Navl.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, hi 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 Navl.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 Navl .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 Nayl.8 alleviates neuropathic pain

The effect of AL-DP-6050 (Table 1 ) against Navl .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 (Figure 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 Navl .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 Navl.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 Navl .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 ND984HC1 (MW 1487), Cholesterol (Sigma-Aldrich), and PEG- Ceramide Cl 6 (Avanti Polar Lipids) were used to prepare lipid-siRNA nanoparticles.

Stock solutions of each in ethanol were prepared: ND98 (Figure 15, ND98

Isomer I), 133 mg/mL; Cholesterol, 25 mg/mL, PEG-Ceramide C 16, 100 mg/mL. ND98, Cholesterol, and PEG-Ceramide Cl 6 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 ran 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, Navl.8 specific dsRNA molecules that modulate Navl .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, US 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. ScL 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. hi 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. Sd.

USA (1998) 85:6460-6464; Wilson et al., 1988, Proc. Natl. Acad. Sci. USA 85:3014- 3018; Aπnentano 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. Patent No. 4,868,116; U.S. Patent 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 Ul 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)).

hi 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-Dl - 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-TKO™). Multiple lipid transfections for dsRNA-mediated knockdowns targeting different regions of a single Navl.8 gene or multiple Navl.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 Navl.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. Patent 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.