Chemically Modified Oligonucleotides

Abstract

The present disclosure relates in some aspects to chemically modified double-stranded nucleic acid molecules directed against a gene encoding T cell immunoreceptor with Ig and ITIM domains (TIGIT).

Description

[Technical field]

[0001] Related Applications This application claims the benefit under 35 USC § 119(e) of U.S. Provisional Application No. 63 / 229,438, filed August 4, 2021, entitled “CHEMICALLY MODIFIED OLIGONUCLEOTIDES,” the entire disclosure of which is incorporated herein by reference in its entirety.

[0002] Reference to electronic sequence listing The contents of the electronic sequence listing (R065970058WO00-SEQ-JXV.xml; size: 103,856 bytes; and creation date: August 4, 2022) are incorporated herein by reference in their entirety.

[0003] Field In some aspects, the present disclosure relates to chemically modified double-stranded nucleic acid molecules directed against a gene encoding T cell immunoreceptor with Ig and ITIM domains (TIGIT). [Background technology]

[0004] background The physiological function of the immune system is to recognize and eliminate neoplastic cells. Thus, an aspect of tumor progression is the development of immune resistance mechanisms. Once developed, these resistance mechanisms not only prevent the innate immune system from affecting tumor growth, but also limit the effectiveness of immunotherapeutic approaches against cancer. Immune resistance mechanisms include immune inhibitory pathways, often referred to as immune checkpoints. Immune inhibitory pathways play a particularly important role in the interaction between tumor cells and CD8+ cytotoxic T lymphocytes, including adoptive cell transfer (ACT) therapeutic agents.

[0005] Various methods of adoptive cell transfer (ACT) involve ex vivo treatment of cells harvested from a patient sample, such as blood or tumor material. Common steps involved in the preparation of cell-based treatments are isolation of cells from a primary source (e.g., peripheral blood), gene editing (e.g., engineering of chimeric antigen receptor (CAR) T cells or engineered T cell receptor (TCR) cells), activation, and expansion.

[0006] During ex vivo processing, cells undergo certain phenotypic changes that may affect their therapeutic properties, such as, among others, their trafficking to tumors, their in vivo proliferation capacity and life span, and their efficacy in an immunosuppressive environment. For example, T cell differentiation and maturation states typically progress through the following sequence of subtypes: naive (TN)-stem cell memory (TSCM)-central memory (TCM)-effector memory (TEM)-terminally differentiated effector T cells (TEFF). It has been observed that the phenotypic and functional attributes of early memory T cells (TSCM / TCM) among CD8+ T cells demonstrate superior in vivo expansion, persistence, and antitumor efficacy than more differentiated effector cells (e.g., TEM, TEFF, etc.).

[0007] Cancer immunotherapy has become increasingly important in clinical practice. Immunotherapies designed to induce or amplify immune responses can be classified as activating immunotherapies, whereas immunotherapies that reduce or suppress immune responses can be classified as suppressing immunotherapies. One activating immunotherapeutic strategy to counteract cancer immune resistance mechanisms is to inhibit immune checkpoints (e.g., by using checkpoint-targeting monoclonal antibodies) to stimulate or maintain the host immune response.

[0008] However, there are many drawbacks to using cancer immunotherapeutics in combination with checkpoint inhibitors. For example, blocking immune checkpoints can lead to the destruction of immune self-tolerance, thereby inducing a novel syndrome of autoimmune / autoinflammatory side effects, which have been termed "immune-related adverse events". In addition, the toxicity profile of checkpoint inhibitors is reportedly different from that reported for other classes of tumor agents, and they can also induce inflammatory events in multiple organ systems, including the skin, gastrointestinal tract, endocrine, lung, liver, eye, and nervous system. Summary of the Invention

[0009] overview The present disclosure provides, in some aspects, a chemically modified double-stranded nucleic acid molecule directed against a gene encoding a T cell immunoreceptor with Ig and ITIM domains (TIGIT), wherein the chemically modified double-stranded nucleic acid molecule comprises at least 12 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1-6 and 27.

[0010] In some embodiments, the chemically modified double-stranded nucleic acid molecule comprises a sequence selected from SEQ ID NOs: 1-6 and 27. Another aspect of the present disclosure provides a chemically modified double-stranded nucleic acid molecule directed against a gene encoding a T cell immunoreceptor with Ig and ITIM domains (TIGIT), wherein the chemically modified double-stranded nucleic acid molecule is directed against a sequence comprising at least 12 contiguous nucleotides of SEQ ID NO:28. In some embodiments, the chemically modified double-stranded nucleic acid molecule is directed against SEQ ID NO:28.

[0011] In some embodiments, the chemically modified double-stranded nucleic acid molecule comprises INTASYL™. In some embodiments, the chemically modified double-stranded nucleic acid molecule is hydrophobically modified. In some embodiments, the chemically modified double-stranded nucleic acid molecule is linked to one or more hydrophobic conjugates. In some embodiments, the hydrophobic conjugate is cholesterol. In some aspects, the chemically modified double-stranded nucleic acid molecule comprises at least one 2'-O-methyl modification and / or at least one 2'-O-fluoro modification, and at least one phosphorothioate modification.

[0012] In some embodiments, the chemically modified double-stranded nucleic acid molecule comprises a sequence selected from TIGIT22 (SEQ ID NOs: 1-2), TIGIT23 (SEQ ID NOs: 3-4), and TIGIT24 (SEQ ID NOs: 5-6). In some embodiments, the chemically modified double-stranded nucleic acid molecule comprises a sense strand having the sequence set forth in SEQ ID NO:1 (TIGIT22 sense strand) and / or an antisense strand having the sequence set forth in SEQ ID NO:2 (TIGIT22 antisense strand).

[0013] In some embodiments, the chemically modified double-stranded nucleic acid molecule comprises a sense strand having the sequence set forth in SEQ ID NO:3 (TIGIT23 sense strand) and / or an antisense strand having the sequence set forth in SEQ ID NO:4 (TIGIT23 antisense strand). In some embodiments, the chemically modified double-stranded nucleic acid molecule comprises a sense strand having the sequence set forth in SEQ ID NO:5 (TIGIT24 sense strand) and / or an antisense strand having the sequence set forth in SEQ ID NO:6 (TIGIT24 antisense strand).

[0014] In some aspects, the present disclosure provides a composition comprising a chemically modified double-stranded nucleic acid molecule described herein and a pharma- ceutically acceptable excipient. An aspect of the present disclosure provides an immunogenic composition comprising a host cell comprising a chemically modified double-stranded nucleic acid molecule comprising at least 12 contiguous nucleotides of SEQ ID NOs: 1-6 and 27, wherein the host cell is selected from the group consisting of a T cell, an antigen presenting cell (APC), a dendritic cell (DC), a stem cell (SC), an induced pluripotent stem cell (iPSC), and a stem cell memory T cell. In some embodiments, the chemically modified double-stranded nucleic acid molecule comprises a sequence selected from SEQ ID NOs: 1-6 and 27. An aspect of the present disclosure provides an immunogenic composition comprising a host cell comprising a chemically modified double-stranded nucleic acid molecule directed against a gene encoding a T cell immunoreceptor with Ig and ITIM domains (TIGIT), wherein the chemically modified double-stranded nucleic acid molecule is directed against a sequence comprising at least 12 contiguous nucleotides of SEQ ID NO:28, and wherein the host cell is selected from the group consisting of a T cell, an antigen presenting cell (APC), a dendritic cell (DC), a stem cell (SC), an induced pluripotent stem cell (iPSC), and a stem cell memory T cell.

[0015] In some embodiments, the chemically modified double-stranded nucleic acid molecule is directed against SEQ ID NO:28. In some embodiments, the chemically modified double-stranded nucleic acid molecule comprises INTASYL™. In some embodiments, the chemically modified double-stranded nucleic acid molecule is hydrophobically modified. In some embodiments, the chemically modified double-stranded nucleic acid molecule is linked to one or more hydrophobic conjugates. In some embodiments, the hydrophobic conjugate is cholesterol. In some aspects, the chemically modified double-stranded nucleic acid molecule comprises at least one 2'-O-methyl modification and / or at least one 2'-O-fluoro modification, and at least one phosphorothioate modification.

[0016] In some embodiments, the chemically modified double-stranded nucleic acid molecule comprises INTASYL™. In some embodiments, the chemically modified double-stranded nucleic acid molecule is hydrophobically modified. In some embodiments, the chemically modified double-stranded nucleic acid molecule is linked to one or more hydrophobic conjugates. In some embodiments, the hydrophobic conjugate is cholesterol. In some aspects, the chemically modified double-stranded nucleic acid molecule comprises at least one 2'-O-methyl modification and / or at least one 2'-O-fluoro modification, and at least one phosphorothioate modification.

[0017] In some embodiments, the host cell is a T cell. In some embodiments, the T cell comprises one or more transgenes expressing a high affinity T cell receptor (TCR) and / or a chimeric antibody receptor (CAR). In some embodiments, the host cell is derived from a healthy donor.

[0018] In some embodiments, the chemically modified double-stranded nucleic acid molecule induces at least 50% inhibition of TIGIT in a host cell. In another aspect, the present disclosure provides a method of producing an immunogenic composition, the method comprising introducing into a host cell one or more chemically modified double-stranded nucleic acid molecules, at least one of which comprises at least 12 consecutive nucleotides of SEQ ID NOs: 1-6 and 27, wherein the one or more chemically modified double-stranded nucleic acid molecules target T cell immunoreceptor with Ig and ITIM domains (TIGIT), thereby producing a host cell, and wherein the host cell is selected from the group consisting of a T cell, an antigen presenting cell (APC), a dendritic cell (DC), a stem cell (SC), an induced pluripotent stem cell (iPSC), and a stem cell memory T cell.

[0019] In some embodiments, at least one of the one or more chemically modified double-stranded nucleic acid molecules comprises a sequence selected from SEQ ID NOs: 1-6 and 27. Another aspect of the present disclosure provides a method of producing an immunogenic composition, the method comprising introducing into a host cell one or more chemically modified double-stranded nucleic acid molecules, at least one of which is directed against a sequence comprising at least 12 consecutive nucleotides of SEQ ID NO: 28, where the one or more chemically modified double-stranded nucleic acid molecules target T cell immunoreceptor with Ig and ITIM domains (TIGIT), thereby producing a host cell, and where the host cell is selected from the group consisting of a T cell, an antigen presenting cell (APC), a dendritic cell (DC), a stem cell (SC), an induced pluripotent stem cell (iPSC), and a stem cell memory T cell.

[0020] In some embodiments, at least one of the one or more chemically modified double-stranded nucleic acid molecules is directed against SEQ ID NO:28. Aspects of the present disclosure relate to methods of producing an immunogenic composition, comprising introducing a chemically modified double-stranded nucleic acid molecule described herein into a host cell, wherein the host cell is selected from the group consisting of a T cell, an antigen presenting cell (APC), a dendritic cell (DC), a stem cell (SC), an induced pluripotent stem cell (iPSC), and a stem cell memory T cell. In some embodiments, the cells are T cells. In some embodiments, the T cells contain one or more transgenes expressing a high affinity T cell receptor (TCR) and / or a chimeric antibody receptor (CAR). In some embodiments, the cells are derived from a healthy donor.

[0021] In some embodiments, the method provided herein further comprises resuspending the host cells in a wash medium that does not contain one or more chemically modified double-stranded nucleic acid molecules for 24 hours to 6 days. In some embodiments, the host cells are resuspended in the wash medium for 24 hours to 96 hours. In some embodiments, the host cells are resuspended in the wash medium for 48 hours. In some embodiments, the host cells are resuspended in the wash medium for 96 hours.

[0022] In some embodiments, the host cells are not resuspended in a wash medium that does not contain the one or more chemically modified double-stranded nucleic acid molecules. Aspects of the disclosure relate to methods for treating a subject suffering from a proliferative or infectious disease, the methods comprising administering to the subject a composition or an immunogenic composition described herein.

[0023] In some embodiments, the proliferative disease is cancer. Each of the limitations of the present invention may encompass various aspects of the present invention. Thus, each of the limitations of the present invention involving any one element or combination of elements is considered to be included in each aspect of the present invention. The present invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The present invention is capable of other embodiments and of being practiced or carried out in various ways.

[0024] Also, the phraseology and terminology used herein are for purposes of description and should not be regarded as limiting. The use of "including," "comprising," "having," "containing," "involving," and variations thereof herein is meant to encompass the items listed thereafter, and equivalents thereof, as well as additional items. [Brief description of the drawings]

[0025] BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings are not intended to be drawn to scale. In the drawings, identical or nearly identical components that are illustrated in various figures are represented by like numerals. For purposes of clarity, not every component may be labeled in every figure.

[0026] [Figure 1]FIG. 1 provides an overview of the experimental setup utilized to test the ability of INTASYL™ compounds targeting TIGIT to enhance NK cell activity. [Diagram 2] Figure 2 shows tumor cell killing by NK cells treated with the TIGIT-targeting INTASYL™ compound. TIGIT mRNA levels are shown on the left and specific lysis percentages on the right.

[0027] [Diagram 3] Figure 3 shows tumor cell killing by NK cells treated with the TIGIT-targeting INTASYL™ compound after a 96 hour washout period. Expression of TIGIT surface protein is shown on the left and percent specific lysis on the right. UTC = untransfected control, NTC = non-targeted control. [Figure 4] Figure 4 shows tumor cell killing by NK cells treated with the TIGIT-targeting INTASYL™ compound after a 6-day washout period. TIGIT surface protein expression is shown on the left and specific lysis percentage on the right. UTC = untransfected control, NTC = non-targeted control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Detailed Description In some aspects, the present disclosure relates to chemically modified double-stranded nucleic acid molecules (e.g., INTASYL™) that target Ig and T cell immune receptors with ITIM domains. The chemically modified double-stranded nucleic acid molecules disclosed herein can be used for immunotherapy.

[0029] INTASYL™ technology is particularly suitable for regulating genes of interest in host cells (e.g., T cells) for immunotherapy. Some advantages of INTASYL™ include: (i) INTASYL™ was developed in a short period of time and can silence virtually any target, including "non-druggable" targets, e.g., those that are difficult to inhibit with small molecules, e.g., transcription factors; (ii) compared to alternative ex vivo siRNA transfection techniques (e.g., lipid-mediated transfection or electroporation), INTASYL™ can transfect various cell types, including T cells, with high transfection efficiency while retaining high cell viability; (iii) when added to cell culture medium during the initial expansion phase, INTASYL™ compounds provide transient silencing of the target of interest for 8-10 division cycles; (iv) INTASYL™ can be used in combination to simultaneously silence multiple targets, thus providing considerable flexibility for use in various types of cell treatment protocols. Described herein are INTASYL™ compounds that are directed against TIGIT.

[0030] As used herein, "nucleic acid molecule" includes, but is not limited to, INTASYL™, sd-rxRNA, rxRNAori, oligonucleotide, ASO, siRNA, shRNA, miRNA, ncRNA, cp-lasiRNA, aiRNA, single-stranded nucleic acid molecule, double-stranded nucleic acid molecule, RNA and DNA. In some embodiments, the nucleic acid molecule is a chemically modified nucleic acid molecule, such as a chemically modified oligonucleotide. In some embodiments, the nucleic acid molecule is double-stranded. In some embodiments, the chemically modified double-stranded nucleic acid molecule described herein is an INTASYL™ (also known as sd-rxRNA) molecule.

[0031] INTASYL(TM)(sd-rxRNA) molecule Aspects of the present invention relate to INTASYL™ molecules that target TIGIT. In some embodiments, the INTASYL™ molecules described herein comprise, consist of, target, or are directed against the sequences set forth in Table 1 or fragments thereof.

[0032] As used herein, "sd-rxRNA" or "sd-rxRNA molecule" or "INTASYL™" or "INTASYL™ molecule" or "INTASYL™ compound" refers to any of the compounds disclosed in U.S. Patent No. 8,796,443, entitled "REDUCED SIZE SELF-DELIVERING RNAI COMPOUNDS," granted on August 5, 2014, U.S. Patent No. 9,080,171, entitled "REDUCED SIZE SELF-DELIVERING RNAI COMPOUNDS," granted on July 14, 2015, U.S. Patent No. 9,175,289, entitled "REDUCED SIZE SELF-DELIVERING RNAI COMPOUNDS," granted on November 3, 2015, U.S. Patent No. 9,175,289, entitled "REDUCED SIZE SELF-DELIVERING RNAI COMPOUNDS," granted on March 26, 2019, and ... No. 10,240,149 entitled "REDUCED SIZE SELF-DELIVERING RNAI COMPOUNDS", granted on September 15, 2020; U.S. Patent No. 10,774,330 entitled "REDUCED SIZE SELF-DELIVERING RNAI COMPOUNDS", granted on September 14, 2021; U.S. Patent No. 11,118,178 entitled "REDUCED SIZE SELF-DELIVERING RNAI COMPOUNDS", filed on September 22, 2009 (Application No. PCT Publication No. WO2010 / 033247) entitled "REDUCED SIZE SELF-DELIVERING RNAI COMPOUNDS" (Application No. PCT / US2009 / 005247); and U.S. Patent No. 10,240,149 entitled "REDUCED SIZE SELF-DELIVERING RNAI COMPOUNDS", granted on September 15, 2020; U.S. Patent No. 10,240,149 entitled "REDUCED SIZE SELF-DELIVERING RNAI COMPOUNDS", granted on September 15, 2020; "INTASYL™" refers to self-delivering RNA molecules such as those described in PCT Publication No. WO2011 / 119852 (Application No. PCT / US2011 / 029824) entitled "INTASYL™ COMPOUNDS," which are incorporated herein by reference. ナノ(also referred to as INTASYL™) is an isolated asymmetric double-stranded nucleic acid molecule comprising a guide strand with a minimum length of 16 nucleotides and a passenger strand with a length of 8-18 nucleotides, where the double-stranded nucleic acid molecule has a double-stranded region and a single-stranded region, where the single-stranded region has a length of 4-12 nucleotides and has at least three nucleotide backbone modifications. In a preferred embodiment, the double-stranded nucleic acid molecule has one end that is blunt or contains one or two nucleotide overhangs. INTASYL™ molecules can be optimized through chemical modifications, in some instances through the attachment of hydrophobic conjugates. Each of the above referenced patents and publications is incorporated herein by reference in their entirety.

[0033] In some embodiments, INTASYL™ comprises an isolated double-stranded nucleic acid molecule comprising a guide strand and a passenger strand, wherein the region of the molecule that is double-stranded is 8-15 nucleotides in length, wherein the guide strand contains a single-stranded region 4-12 nucleotides in length, wherein the single-stranded region of the guide strand contains 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 phosphorothioate modifications, and wherein at least 40% of the nucleotides of the double-stranded nucleic acid are modified.

[0034] In some embodiments, INTASYL™ comprises an isolated double-stranded nucleic acid molecule comprising a guide strand and a passenger strand, wherein the region of the molecule that is double-stranded is 8-15 nucleotides in length, wherein the guide strand contains a single-stranded region that is 4-12 nucleotides in length, wherein the single-stranded region of the guide strand contains 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 phosphorothioate modifications, and wherein at least 40% of the nucleotides of the double-stranded nucleic acid are modified, and wherein one strand is conjugated to cholesterol at the 3' or 5' end of the strand. In some embodiments, the passenger strand is conjugated to cholesterol at the 5' or 3' end of the strand.

[0035] In some embodiments, INTASYL™ comprises an isolated double-stranded nucleic acid molecule comprising a guide strand and a passenger strand, wherein the region of the molecule that is double-stranded is 8-15 nucleotides in length, wherein the guide strand contains a single-stranded region 4-12 nucleotides in length, wherein the single-stranded region of the guide strand contains 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 phosphorothioate modifications, and wherein at least 40% of the nucleotides of the double-stranded nucleic acid are modified, and wherein one strand (e.g., the passenger strand) contains a hydrophobic moiety conjugated at the 3' end of the passenger strand, wherein the hydrophobic moiety is cholesterol.

[0036] Nucleic acid molecules related to the present disclosure are referred to herein as isolated double-stranded or duplex nucleic acids, chemically modified double-stranded or duplex nucleic acids, oligonucleotides, polynucleotides, nanomolecules, nanoRNAs, sd-rxRNAs, and the like, of the present invention. ナノ , sd-rxRNA, INTASYL™ or RNA molecules.

[0037] Compared with traditional siRNA, INTASYL ™ molecules are taken up by cells much more effectively.These molecules are highly efficient in silencing target genes, and offer great advantages over previously described RNAi molecules, including high activity in the presence of serum, efficient self-delivery, compatibility with various linkers, and reduced or complete absence of chemical modifications associated with toxicity.

[0038] In contrast to single-stranded polynucleotides, duplex polynucleotides have traditionally been difficult to deliver to cells because they have a rigid structure and multiple negative charges, making their membrane transport difficult.However, INTASYL™ molecules, although partially double-stranded, are recognized as single-stranded in vivo, and therefore can be delivered efficiently across cell membranes.As a result, polynucleotides of the present invention can be self-delivered in many cases.Thus, polynucleotides of the present invention can be formulated in a manner similar to conventional RNAi agents, or delivered alone (or with non-delivery carriers) to cells or subjects, allowing for self-delivery.In one embodiment of the present invention, a self-delivering asymmetric double-stranded RNA molecule is provided, in which one part of the molecule resembles a conventional RNA duplex, and a second part of the molecule is single-stranded.

[0039] In some aspects, the oligonucleotide of the present invention has an asymmetric structure, comprising a double-stranded region and a single-stranded region of 5 nucleotides or longer, and a specific chemical modification pattern, and is conjugated to lipophilic or hydrophobic molecules.In some embodiments, this class of RNAi-like compounds has excellent efficacy in vitro and in vivo.It is believed that the reduction in size of the strong duplex region, combined with the phosphorothioate modification applied to the single-stranded region, contributes to the excellent efficacy observed.

[0040] In some embodiments, the RNAi compounds of the present invention comprise asymmetric compounds that include a duplex region of 8-15 bases in length (required for efficient RISC entry) and a single-stranded region of 4-12 nucleotides in length. In some embodiments, the duplex region is 13 or 14 nucleotides in length, and in some embodiments, the single-stranded region is 6-7 nucleotides in length. The single-stranded region of the RNAi compound (e.g., INTASYL™ molecule) also comprises 2-12 phosphorothioate internucleotide linkages (referred to as phosphorothioate modifications). In some embodiments, the single-stranded region comprises 6-8 phosphorothioate internucleotide linkages. Additionally, the RNAi compounds of the present invention also comprise unique chemical modification patterns that provide stability and are compatible with RISC entry. In some embodiments, the combination of these elements results in unexpected properties that are highly useful for delivery of RNAi reagents in vitro and in vivo.

[0041] The chemical modification pattern that provides stability and is compatible with RISC entry includes modifications to the sense or passenger strand as well as the antisense or guide strand. As an example, the passenger strand can be modified with any chemical entity that ensures stability and does not interfere with activity. Such modifications include backbone modifications such as 2' ribo modifications (O-methyl, 2'F, 2 deoxy, etc.) and phosphorothioate modifications. In some embodiments, the chemical modification pattern in the passenger strand includes O-methyl modifications of C and U nucleotides in the passenger strand. Alternatively, the passenger strand may be fully O-methyl modified.

[0042] The guide strand may also be modified by any chemical modification that ensures stability without interfering with RISC entry in some embodiments. In some embodiments, the chemical modification pattern in the guide strand includes a majority of C and U nucleotides that are 2'F modified and phosphorylated at the 5' end. In some embodiments, the chemical modification pattern in the guide strand includes 2'O methyl modifications of C / U at positions 1 and 11-18 and chemical phosphorylation at the 5' end. In some embodiments, the chemical modification pattern in the guide strand includes 2'O methyl modifications of C / U at positions 1 and 11-18 and chemical phosphorylation at the 5' end as well as 2'F modifications of C / U at positions 2-10. In some embodiments, the passenger strand and / or guide strand contain at least one 5-methyl C or U modification.

[0043] In some embodiments, at least 30% of the nucleotides in the sd-rxRNA (e.g., INTASYL™ compounds) are modified. For example, at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 102%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 109%, 109%, 109%, 109%, 109%, 1 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% are modified. In some embodiments, 100% of the nucleotides in the INTASYL™ compound are modified.

[0044] The above-mentioned chemical modification patterns of the oligonucleotide of the present invention show good tolerance and improve the efficacy of asymmetric RNAi compounds.In some embodiments, the elimination of any of the described components (guide strand stabilization, phosphorothioate extension, sense strand stabilization, hydrophobic conjugate, and / or targeting ligand) or the increase in size can result in suboptimal efficacy, and in some cases, complete loss of efficacy.The combination of elements leads to the development of compounds that are fully active even after passive delivery to cells.

[0045] INTASYL™ can be further improved in some instances by using novel types of chemistries to improve the hydrophobicity of the compounds. For example, one chemistry involves the use of hydrophobic base modifications. Any base at any position may be modified as long as the modification results in an increase in the partition coefficient of the base. The preferred positions for modification chemistry are pyrimidine positions 4 and 5. The major advantages of these positions are (a) ease of synthesis, and (b) no interference with base pairing and A form helix formation, which is essential for RISC complex loading and target recognition. In some embodiments, INTASYL™ compounds are used in which multiple deoxyuridines are present without interfering with the overall compound potency. In addition, major improvements in tissue distribution and cellular uptake can be obtained by modifying the structure of the hydrophobic conjugate. In some embodiments, the structure of the sterol is modified to change (increase / decrease) the C17 attached chain. This type of modification results in greatly increased cellular uptake in vivo and improved tissue uptake prosperities.

[0046] In some embodiments, the chemically modified double-stranded nucleic acid molecule is a hydrophobically modified siRNA antisense hybrid molecule comprising a double-stranded region of about 13-22 base pairs with or without a 3'-overhang on each of the sense and antisense strands, and a 3' single-stranded tail on the antisense strand of about 2-9 nucleotides. In some embodiments, the chemically modified double-stranded nucleic acid molecule contains at least one 2'-O-methyl modification, at least one 2'-fluoro modification, and at least one phosphorothioate modification, and at least one hydrophobic modification selected from sterol, cholesterol, vitamin D, naphthyl, isobutyl, benzyl, indole, tryptophan, phenyl, etc., hydrophobic modifier. In some embodiments, the chemically modified double-stranded nucleic acid molecule comprises a plurality of such modifications.

[0047] In some embodiments, the chemically modified double-stranded nucleic acid molecule targets the gene encoding TIGIT. As used herein, "TIGIT" refers to a T cell immunoreceptor with Ig and ITIM domains, which is an immunoreceptor that downregulates T cell-mediated immunity, for example, by increasing the production of interleukin 10 (IL-10) via the CD226 / TIGIT-PVR pathway. In some embodiments, TIGIT is encoded by a nucleic acid sequence represented by NCBI reference sequence number NM_173799.3. Non-limiting examples of TIGIT sequences that may be targeted by the chemically modified double-stranded nucleic acid molecules of the present disclosure are listed in Tables 1-2. In some embodiments, the chemically modified double-stranded nucleic acid molecule, such as INTASYL™, targets any one of SEQ ID NOs: 28-48, or a portion thereof.

[0048] In some embodiments, a chemically modified double-stranded nucleic acid molecule, such as INTASYL™, comprises at least 12 contiguous nucleotides of a sequence in Table 1 or 2. In some embodiments, a chemically modified double-stranded nucleic acid molecule, such as INTASYL™, comprises at least one sequence of a sequence in Table 1 or 2. In some embodiments, a chemically modified double-stranded nucleic acid molecule, such as INTASYL™, comprises at least 12 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1-48. In some embodiments, a chemically modified double-stranded nucleic acid molecule comprises at least 12 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1-27. In some embodiments, a chemically modified double-stranded nucleic acid molecule comprises a sequence selected from SEQ ID NOs: 1-48. In some embodiments, a chemically modified double-stranded nucleic acid molecule comprises a sense strand having a sequence set forth in SEQ ID NO: 1, and / or an antisense strand having a sequence set forth in SEQ ID NO: 2. In some embodiments, a chemically modified double-stranded nucleic acid molecule comprises a sense strand having a sequence set forth in SEQ ID NO: 3, and / or an antisense strand having a sequence set forth in SEQ ID NO: 4. In some embodiments, the chemically modified double-stranded nucleic acid molecule comprises a sense strand having the sequence set forth in SEQ ID NO:5 and / or an antisense strand having the sequence set forth in SEQ ID NO:6.

[0049] Thus, aspects of the present invention relate to isolated double-stranded nucleic acid molecules comprising a guide (antisense) strand and a passenger (sense) strand. The term "double-stranded" as used herein refers to one or more nucleic acid molecules in which at least a portion of the nucleomonomers are complementary and hydrogen bonded to form a double-stranded region. In some embodiments, the length of the guide strand ranges from 16 to 29 nucleotides in length. In some embodiments, the guide strand is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 nucleotides in length. The guide strand has complementarity to a target gene. Complementarity between the guide strand and the target gene can exist over any portion of the guide strand. Complementarity as used herein can be perfect or less perfect, so long as the guide strand is sufficiently complementary to mediate RNAi to the target. In some embodiments, complementarity refers to less than 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2% or 1% mismatch between guide strand and target. Full complementarity refers to 100% complementarity. In some embodiments, siRNAs with insertions, deletions and single point mutations compared to the target sequence have also been found to be effective for inhibition. In addition, not all sites of siRNAs contribute equally to target recognition. Mismatches in the center of siRNA are the most important and essentially abolish the cleavage of target RNA. For antisense strand, mismatches upstream of the center or upstream of the cleavage site are tolerant but significantly reduce the cleavage of target RNA. For antisense strand, mismatches in the center or downstream of the cleavage site, preferably located near the 3' end of the antisense strand, for example 1, 2, 3, 4, 5 or 6 nucleotides from the 3' end of the antisense strand, are tolerant and only slightly reduce the cleavage of target RNA.

[0050] Without wishing to be bound by any particular theory, in some embodiments of the double-stranded nucleic acid molecules described herein, the guide strand is at least 16 nucleotides in length and anchors the Argonaute protein in RISC. In some embodiments, when the guide strand loads into RISC, it has a distinct seed region, and cleavage of the target mRNA occurs over positions 10-11 of the guide strand. In some embodiments, the 5' end of the guide strand is phosphorylated or can be phosphorylated. The nucleic acid molecules described herein may also be referred to as minimum trigger RNAs.

[0051] In some embodiments of the double-stranded nucleic acid molecules described herein, the length of the passenger strand ranges from 8 to 15 nucleotides in length. In some embodiments of the double-stranded nucleic acid molecules described herein, the length of the passenger strand ranges from 8 to 16 nucleotides in length. In some embodiments, the passenger strand is 8, 9, 10, 11, 12, 13, 14, 15 or 16 nucleotides in length. The passenger strand has complementarity to the guide strand. Complementarity between the passenger strand and the guide strand may exist over any portion of the passenger or guide strand. In some embodiments, there is 100% complementarity between the guide strand and the passenger strand within the double-stranded region of the molecule.

[0052] Aspects of the invention relate to double-stranded nucleic acid molecules having a minimal double-stranded region. In some embodiments, the double-stranded region of the molecule ranges from 8 to 15 nucleotides in length. In some embodiments, the double-stranded region of the molecule ranges from 8 to 16 nucleotides in length. In some embodiments, the double-stranded region of the molecule is 8, 9, 10, 11, 12, 13, 14, 15 or 16 nucleotides in length. In some embodiments, the double-stranded region is 13 or 14 nucleotides in length. In some embodiments, the region of the molecule that is double-stranded is 13 to 22 nucleotides in length. In some embodiments, the region of the molecule that is double-stranded is 16, 17, 18, 19, 20, 21 or 22 nucleotides in length.

[0053] There may be 100% complementarity between the guide strand and the passenger strand, or there may be one or more mismatches between the guide strand and the passenger strand. In some embodiments, at one end of the double-stranded molecule, the molecule is blunt-ended or has a one-nucleotide overhang. The single-stranded region of the molecule is, in some embodiments, 4-12 nucleotides long. For example, the single-stranded region may be 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides long. However, in some embodiments, the single-stranded region may also be less than 4 nucleotides long or more than 12 nucleotides long. In some embodiments, the single-stranded region is at least 6 or at least 7 nucleotides long. In some embodiments, the single-stranded region is 2-9 nucleotides long, including 2 or 3 nucleotides long.

[0054] The RNAi constructs related to the present invention can have a thermodynamic stability (ΔG) of less than -13 kcal / mol. In some embodiments, the thermodynamic stability (ΔG) is less than -20 kcal / mol. In some embodiments, there is a loss of potency when (ΔG) is less than -21 kcal / mol. In some embodiments, (ΔG) values ​​higher than -13 kcal / mol are compatible with aspects of the present invention. Without wishing to be bound by any theory, in some embodiments, molecules with relatively high (ΔG) values ​​may be active at relatively high concentrations, while molecules with relatively low (ΔG) values ​​may be active at relatively low concentrations. In some embodiments, (ΔG) values ​​may be higher than -9 kcal / mol. The gene silencing effect mediated by the RNAi constructs related to the present invention with minimal double-stranded regions cannot be predicted, since molecules with nearly identical designs but lower thermodynamic stability have been shown to be inactive (Rana et al. 2004).

[0055] Without wishing to be bound by any theory, the results described herein suggest that 8-10 bp stretches of dsRNA or dsDNA will be structurally recognized by protein components of RISC or cofactors of RISC. Furthermore, there is a free energy requirement for a triggering compound that can be sensed by the protein components and / or be stable enough to interact with such components and thus be loaded into the Argonaute protein. If permissible thermodynamics exist and there is a double-stranded portion that is preferably at least 8 nucleotides, the duplex will be recognized and loaded into the RNAi machinery.

[0056] In some embodiments, thermodynamic stability is increased through the use of LNA base. In some embodiments, additional chemical modifications are introduced. Some non-limiting examples of chemical modifications include 5'phosphate, 5'phosphonate, 5'vinylphosphonate, 2'-O-methyl, 2'-O-ethyl, 2'-fluoro, ribothymidine, C-5 propynyl-dC (pdC) and C-5 propynyl-dU (pdU); C-5 propynyl-C (pC) and C-5 propynyl-U (pU); 5-methylC, 5-methylU, 5-methyldC, 5-methyldU methoxy, (2,6-diaminopurine), 5'-dimethoxytrityl-N4-ethyl-2'-deoxycytidine and MGB (minor groove binder). It should be understood that more than one chemical modification can be combined in the same molecule.

[0057] In some embodiments, the molecules related to the present disclosure are optimized for increased potency and / or reduced toxicity. For example, the length of the nucleotides of the guide and / or passenger strands and / or the number of phosphorothioate modifications in the guide and / or passenger strands affect the potency of the RNA molecule in some aspects, while replacing 2'-fluoro (2'F) modifications with 2'-O-methyl (2'OMe) modifications affects the toxicity of the molecule in some aspects. Specifically, reducing the 2'F content of the molecule is expected to reduce the toxicity of the molecule. Furthermore, the number of phosphorothioate modifications in the RNA molecule can affect the efficiency of the uptake of the molecule into cells, for example, the passive uptake of the molecule into cells. Preferred embodiments of the molecules described herein are characterized by the absence of 2'F modifications and still comparable potency in cellular uptake and tissue penetration. Such molecules represent a significant improvement over the prior art, such as the molecules described by Accell and Wolfrum, which are heavily modified with the extensive use of 2'F.

[0058] In some embodiments, the guide strand is approximately 18-20 nucleotides in length and has approximately 2-14 phosphate modifications. For example, the guide strand may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more than 14 phosphate modified nucleotides. The guide strand may contain one or more modifications that increase stability without interfering with RISC entry. The phosphate modified nucleotides, such as phosphorothioate modified nucleotides, may be at the 3' end, at the 5' end, or may be spread throughout the guide strand. In some embodiments, the 3'-terminal 10 nucleotides of the guide strand contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphorothioate modified nucleotides. The guide strand may also contain 2'F and / or 2'OMe modifications, which may be located throughout the molecule. In some embodiments, the nucleotide at position 1 of the guide strand (the nucleotide at the 5'-most position of the guide strand) is 2'OMe-modified and / or phosphorylated and / or contains vinyl phosphonate. C and U nucleotides in the guide strand can be 2'F-modified. For example, C and U nucleotides at positions 2-10 of a 20 nucleotide guide strand (or corresponding positions in a different length strand) can be 2'F-modified. C and U nucleotides in the guide strand can also be 2'OMe-modified. For example, C and U nucleotides at positions 11-18 of a 19 nucleotide guide strand (or corresponding positions in a different length strand) can be 2'OMe-modified. In some embodiments, the nucleotide at the 3'-most end of the guide strand is unmodified. In some embodiments, the majority of the C and U in the guide strand are 2'F-modified and the 5'-end of the guide strand is phosphorylated. In other embodiments, C or U at positions 1 and 11-18 are 2'OMe-modified and the 5'-end of the guide strand is phosphorylated. In another embodiment, the C or U at positions 1 and 11-18 are 2'OMe modified, the 5' end of the guide strand is phosphorylated, and the C or U at positions 2-10 are 2'F modified.

[0059] In some aspects, the passenger strand is approximately 11-14 nucleotides in length. The passenger strand may contain modifications that increase stability. One or more nucleotides in the passenger strand may be 2'OMe modified. In some embodiments, one or more C and / or U nucleotides in the passenger strand are 2'OMe modified, or all of the C and U nucleotides in the passenger strand are 2'OMe modified. In some embodiments, all nucleotides in the passenger strand are 2'OMe modified. One or more nucleotides on the passenger strand may also be phosphate modified, such as phosphorothioate modified. The passenger strand may also contain 2' ribo, 2' F and 2 deoxy modifications, or any combination of the above. Chemical modification patterns in both the guide strand and the passenger strand may be well tolerated, and combinations of chemical modifications may result in increased potency and self-delivery of the RNA molecule.

[0060] Aspects of the present invention relate to RNAi constructs that have relatively long single-stranded regions relative to double-stranded regions when compared to molecules previously used for RNAi. The single-stranded regions of the molecules may be modified to facilitate cellular uptake or gene silencing. In some embodiments, phosphorothioate modifications of the single-stranded regions affect cellular uptake and / or gene silencing. The phosphorothioate modified regions of the guide strand may include nucleotides in both the single-stranded and double-stranded regions of the molecule. In some embodiments, the single-stranded region includes 2-12 phosphorothioate modifications. For example, the single-stranded region may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 phosphorothioate modifications. In some examples, the single-stranded region includes 6-8 phosphorothioate modifications.

[0061] Molecules related to the present invention are also designed for cellular uptake. In the RNA molecules described herein, the guide strand and / or passenger strand may be attached to a conjugate. In some embodiments, the conjugate is hydrophobic. The hydrophobic conjugate may be a small molecule with a partition coefficient higher than 10. The conjugate may be a sterol-type molecule, such as cholesterol, or a molecule with a polycarbon chain of increased length attached to C17, and the presence of the conjugate may affect the ability of the RNA molecule to be taken up by cells with or without a lipid transfection reagent. The conjugate may be attached to the passenger strand or the guide strand through a hydrophobic linker. In some embodiments, the hydrophobic linker is 5-12C in length and / or is hydroxypyrrolidine-based. In some embodiments, the hydrophobic conjugate is attached to the passenger strand, and a CU residue in either the passenger strand and / or the guide strand is modified. In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the CU residues of the passenger strand and / or guide strand are modified. In some aspects, the molecules related to the present invention are self-delivering (sd). As used herein, "self-delivery" refers to the ability of a molecule to be delivered to a cell without the need for additional delivery vehicles such as transfection reagents.

[0062] The molecules related to the present invention are designed for targeted delivery to the liver. In the RNA molecules described herein, the guide strand and / or passenger strand can be attached to a conjugate. In some embodiments, the conjugate is a targeting ligand. The targeting ligand conjugate can be a sugar, such as N-acetylgalactosamine (GalNac) moiety and its derivatives. In some embodiments, the RNA molecule can contain one, two, three, four, five or more GalNac moieties. The targeting ligand conjugate(s) can be attached to the passenger strand or guide strand via a linker, or can be incorporated into the passenger strand or guide strand, for example, as a phosphoramidite.

[0063] Aspects of the invention relate to selecting molecules for use in RNAi. In some embodiments, molecules having a double-stranded region of 8-15 nucleotides may be selected for use in RNAi. In some embodiments, molecules are selected based on their thermodynamic stability (ΔG). In some embodiments, molecules having a (ΔG) of less than -13kkal / mol will be selected. For example, the (ΔG) value may be less than -13, -14, -15, -16, -17, -18, -19, -21, -22 or -22kkal / mol. In other embodiments, the (ΔG) value may be greater than -13kkal / mol. For example, the (ΔG) value may be greater than -12, -11, -10, -9, -8, -7 or -7kkal / mol. It should be understood that ΔG may be calculated using any method known in the art. In some embodiments, ΔG is calculated using Mfold, available through the Mfold internet site (mfold.bioinfo.rpi.edu / cgi-bin / rna-for m1.cgi). Methods for calculating ΔG are described in the following references, which are incorporated by reference therefrom: Zuker, M. (2003) Nucleic Acids Res., 31(13):3406-15; Mathews, DH, Sabina, J., Zuker, M. and Turner, DH (1999) J. Mol. Biol. 288:911-940; Mathews, DH, Disney, MD, Childs, JL, Schroeder, SJ, Zuker, M., and Turner, DH (2004) Proc. Natl. Acad. Sci. 101:7287-7292; Duan, S., Mathews, DH, and Turner, DH (2006) Biochemistry 45:9819-9832; Wuchty, S., Fontana, W., Hofacker, IL, and Schuster, P. (1999) Biopolymers 49:145-165.

[0064] In some embodiments, the polynucleotide contains a 5' and / or 3' terminal overhang. The number and / or sequence of nucleotide overhangs at one end of the polynucleotide may be the same or different from the other end of the polynucleotide. In some embodiments, one or more of the overhanging nucleotides may contain a chemical modification(s), such as a phosphorothioate or 2'-OMe modification.

[0065] In some embodiments, the polynucleotide is unmodified. In other embodiments, at least one nucleotide is modified. In further embodiments, the modification comprises a 2'-H or 2'-modified ribose sugar at the second nucleotide from the 5' end of the guide sequence. The "second nucleotide" is defined as the second nucleotide from the 5' end of the polynucleotide.

[0066] As used herein, "2' modified ribose sugar" includes ribose sugars that do not have a 2'-OH group. "2' modified ribose sugar" does not include 2'-deoxyribose (as found in unmodified standard DNA nucleotides). For example, the 2' modified ribose sugar may be a 2'-O-alkyl nucleotide, a 2'-deoxy-2'-fluoro nucleotide, a 2'-deoxy nucleotide, or a combination thereof.

[0067] In certain embodiments, the 2' modified nucleotide is a pyrimidine nucleotide (eg, C / U). Examples of 2'-O-alkyl nucleotides include 2'-O-methyl nucleotides or 2'-O-allyl nucleotides.

[0068] In certain embodiments, the sd-rxRNA polynucleotides of the present invention having the above-mentioned 5' end modifications exhibit significantly (e.g., at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more) less "off-target" gene silencing when compared to similar constructs lacking the specified 5' end modifications, thereby greatly improving the overall specificity of the RNAi reagent or therapy.

[0069] As used herein, "off-target" gene silencing refers to unintended gene silencing due, for example, to spurious sequence homology between an antisense (guide) sequence and an unintended target mRNA sequence. In accordance with this aspect of the invention, certain guide strand modifications further increase nuclease stability and / or reduce interferon induction without significantly reducing RNAi activity (or without reducing RNAi activity at all).

[0070] Certain combinations of modifications may provide additional unexpected advantages, manifested in part by enhanced ability to inhibit expression of a target gene, enhanced serum stability, and / or increased target specificity. In certain embodiments, the guide strand comprises a 2'-O-methyl modified nucleotide at the second nucleotide at the 5' end of the guide strand and no other modified nucleotides.

[0071] In another aspect, the chemically modified double-stranded nucleic acid molecule structure of the present invention mediates sequence-dependent gene silencing by the microRNA mechanism. As used herein, the term "microRNA" ("miRNA"), also referred to in the art as "small temporal RNA" ("stRNA"), refers to small (10-50 nucleotide) RNAs that are genetically encoded (e.g., by viral, mammalian or plant genomes) and capable of directing or mediating RNA silencing. "miRNA disorder" shall refer to a disease or disorder characterized by aberrant expression or activity of miRNA.

[0072] MicroRNA is involved in downregulating target genes in important pathways such as development or cancer in mice, nematodes and mammals. Gene silencing through microRNA mechanism is achieved by specific but still imperfect base pairing between miRNA and its target messenger RNA (mRNA). Various mechanisms may be used in microRNA-mediated downregulation of target mRNA expression.

[0073] miRNAs are approximately 22-nucleotide non-coding RNAs that can regulate gene expression at the post-transcriptional or post-translational level throughout plant and animal development. One common feature of miRNAs is that they are excised from approximately 70-nucleotide precursor RNA stem-loops called pre-miRNAs, presumably by the RNase III enzyme Dicer or its homologues. Naturally occurring miRNAs are expressed by endogenous genes in vivo and processed from hairpin or stem-loop precursors (pre-miRNAs or pri-miRNAs) by Dicer or other RNAses. miRNAs can exist transiently as double-stranded duplexes in vivo, but only one strand is incorporated into the RISC complex to direct gene silencing.

[0074] In some embodiments, chemically modified double-stranded nucleic acid molecule compound versions are described that are effective in cellular uptake and inhibiting miRNA activity.Compounds are essentially similar to RISC-invasive versions, but the chemical modification pattern of the larger strand blocks cleavage and acts as an effective inhibitor of RISC action.For example, compounds may be fully or mostly O-methyl modified with phosphorothioate content as described above.For these types of compounds, in some embodiments, 5' phosphorylation is not required.The presence of double-stranded regions is preferred, as it promotes cellular uptake and efficient RISC loading.

[0075] Another pathway that uses small RNAs as sequence-specific regulators is the RNA interference (RNAi) pathway, which is an evolutionarily conserved response to the presence of double-stranded RNA (dsRNA) in cells. dsRNA is cleaved by Dicer into ~20 base pair (bp) duplex small interfering RNAs (siRNAs). These small RNAs are assembled into a multiprotein effector complex called the RNA-induced silencing complex (RISC). The siRNAs then guide the cleavage of target mRNAs with perfect complementarity.

[0076] Some aspects of biogenesis, protein complexes, and function are shared between the siRNA and miRNA pathways. Single-stranded polynucleotides may mimic dsRNA in the siRNA mechanism or mimic microRNA in the miRNA mechanism.

[0077] In certain embodiments, a modified RNAi construct may have improved stability in serum and / or cerebrospinal fluid compared to an unmodified RNAi construct having the same sequence.

[0078] In some embodiments, the structure of the RNAi construct does not induce an interferon response in primary cells, such as primary mammalian cells, including primary cells from humans, mice and other rodents, and other non-human mammals. In some embodiments, the RNAi construct may also be used to inhibit the expression of target genes in invertebrate organisms.

[0079] To further increase the in vivo stability of the subject constructs, the 3' end of the structure may be blocked by a protecting group(s). Protecting groups such as inverted nucleotides, inverted abasic moieties, or amino-terminal modified nucleotides may be used. Inverted nucleotides may include inverted deoxynucleotides. Inverted abasic moieties may include inverted deoxy abasic moieties, such as 3',3' linked or 5',5' linked deoxy abasic moieties.

[0080] The RNAi construct of the present invention can inhibit the synthesis of any target protein encoded by the target gene(s).The present invention includes a method for inhibiting the expression of target gene in cells, either in vitro or in vivo.Therefore, the RNAi construct of the present invention is useful for treating patients with diseases characterized by overexpression of target gene.

[0081] The target gene may be endogenous to the cell or exogenous (e.g., introduced into the cell by a virus or using recombinant DNA technology). Such methods may include introducing RNA into the cell in an amount sufficient to inhibit expression of the target gene. For example, such an RNA molecule may have a guide strand that is complementary to the nucleotide sequence of the target gene, such that the composition inhibits expression of the target gene.

[0082] The invention also relates to vectors expressing the nucleic acids of the invention, and to cells containing such vectors or nucleic acids. The cells can be mammalian cells, such as human cells, in vivo or in culture. The present invention further relates to compositions comprising a subject RNAi construct and a pharma- ceutically acceptable carrier or diluent. The methods may be performed in vitro, ex vivo, or in vivo, for example, in cultured mammalian cells, such as human cells in culture.

[0083] The target cell (eg, a mammalian cell) may be contacted in the presence of a delivery reagent, such as a lipid (eg, a cationic lipid) or a liposome. Another aspect of the invention provides a method for inhibiting expression of a target gene in a mammalian cell comprising contacting the mammalian cell with a vector expressing an RNAi construct of interest.

[0084] In one aspect of the invention, a longer duplex polynucleotide is provided, comprising a first polynucleotide ranging in size from about 16 to about 30 nucleotides and a second polynucleotide ranging in size from about 26 to about 46 nucleotides, where the first polynucleotide (antisense strand) is complementary to both the second polynucleotide (sense strand) and the target gene, and both polynucleotides form a duplex, where the first polynucleotide contains a single-stranded region longer than 6 bases in length, is modified with an alternative chemical modification pattern, and / or contains a conjugate moiety to facilitate cellular delivery. In this embodiment, about 40 to about 90% of the nucleotides of the passenger strand, about 40 to about 90% of the nucleotides of the guide strand, and about 40 to about 90% of the nucleotides of the single-stranded region of the first polynucleotide are chemically modified nucleotides.

[0085] In one embodiment, the chemically modified nucleotide in the polynucleotide duplex may be any chemically modified nucleotide known in the art, such as those discussed in detail above. In a particular embodiment, the chemically modified nucleotide is selected from the group consisting of 2'F modified nucleotide, 2'-O-methyl modified and 2'deoxy nucleotide. In another particular embodiment, the chemically modified nucleotide results from the "hydrophobic modification" of the nucleotide base. In another particular embodiment, the chemically modified nucleotide is phosphorothioate. In yet another particular embodiment, the chemically modified nucleotide is a combination of phosphorothioate, 2'-O-methyl, 2'deoxy, hydrophobic modification and phosphorothioate. If these groups of modifications refer to modifications of the ribose ring, backbone and nucleotide, it is also feasible that some modified nucleotides have a combination of all three types of modifications.

[0086] In another embodiment, the chemical modifications are not the same across the various regions of the duplex. In a particular embodiment, the first polynucleotide (passenger strand) has multiple and various chemical modifications at various positions. For this polynucleotide, up to 90% of the nucleotides may be chemically modified and / or may have mismatches introduced.

[0087] In another embodiment, chemical modifications of the first or second polynucleotide include, but are not limited to, modifications of uridine and cytosine at the 5' position (4-pyridyl, 2-pyridyl, indolyl, phenyl (CHOH); tryptophanyl (CHN)CHCH(NH)CO), isobutyl, butyl, aminobenzyl; phenyl; naphthyl, etc.), where the chemical modification may alter the base pairing ability of the nucleotide. For the guide strand, an important feature of this aspect of the invention is the location and sequence of the chemical modification relative to the 5' end of the antisense. For example, chemical phosphorylation of the 5' end of the guide strand is usually beneficial for efficacy. O-methyl modifications in the seed region of the sense strand (positions 2-7 relative to the 5' end) are generally not well tolerated, whereas 2'F and deoxy are well tolerated. The middle portion of the guide strand and the 3' end of the guide strand are more tolerant of the type of chemical modification applied. Deoxy modifications are not tolerated at the 3' end of the guide strand.

[0088] A unique feature of this aspect of the invention involves the use of hydrophobic modifications to the bases. In one embodiment, the hydrophobic modifications are preferably located near the 5' end of the guide strand, in other embodiments they are located in the middle of the guide strand, in other embodiments they are located at the 3' end of the guide strand, and in yet other embodiments they are distributed throughout the entire length of the polynucleotide. The same type of pattern is applicable to the passenger strand of the duplex. The other part of the molecule is a single-stranded region, predicted to range from 7 to 40 nucleotides.

[0089] In one embodiment, the single-stranded region of the first polynucleotide contains modifications selected from the group consisting of 40%-90% hydrophobic base modifications, 40%-90% phosphorothioates, 40%-90% modifications of the ribose moiety, and any combination of the foregoing. Because the efficiency of loading of the guide strand (first polynucleotide) into the RISC complex may vary for heavily modified polynucleotides, in one embodiment, the duplex polynucleotide contains a mismatch between nucleotides 9, 11, 12, 13, or 14 on the guide strand (first polynucleotide) and the opposite nucleotide on the sense strand (second polynucleotide) to facilitate efficient guide strand loading.

[0090] Duplex Features The double-stranded oligonucleotide of the present invention may be formed by two separate, complementary nucleic acid strands. Duplex formation may occur either inside or outside the cell containing the target gene.

[0091] The term "duplex" as used herein includes a region of a double-stranded nucleic acid molecule or molecules that is hydrogen-bonded to a complementary sequence. The double-stranded oligonucleotide of the present invention may include a nucleotide sequence that is sense to a target gene and a complementary sequence that is antisense to the target gene. The sense and antisense nucleotide sequences correspond to the target gene sequence, and are, for example, identical to the target gene sequence or sufficiently identical (e.g., about at least about 98% identical, 96% identical, 94%, 90% identical, 85% identical or 80% identical) to cause inhibition of the target gene.

[0092] In some embodiments, the double-stranded oligonucleotide of the present invention is double-stranded over its entire length, i.e., it does not have any single-stranded sequence protruding at either end of the molecule, i.e., it is blunt-ended.In other embodiments, the individual nucleic acid molecules may be of different lengths.In other words, the double-stranded oligonucleotide of the present invention is not double-stranded over its entire length.For example, when two separate nucleic acid molecules are used, one of the molecules, for example the first molecule that contains the antisense sequence, may be longer than the second molecule that hybridizes to it (making a portion of the molecule single-stranded).Similarly, when a single nucleic acid molecule is used, a portion of either end of the molecule may remain single-stranded.

[0093] In one embodiment, the double-stranded oligonucleotide of the present invention contains mismatches and / or loops or bulges, but is double-stranded over at least about 70% of the length of the oligonucleotide. In another embodiment, the double-stranded oligonucleotide of the present invention is double-stranded over at least about 80% of the length of the oligonucleotide. In another embodiment, the double-stranded oligonucleotide of the present invention is double-stranded over at least about 90%-95% of the length of the oligonucleotide. In another embodiment, the double-stranded oligonucleotide of the present invention is double-stranded over at least about 96%-98% of the length of the oligonucleotide. In some embodiments, the double-stranded oligonucleotide of the present invention contains at least or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mismatches.

[0094] qualification The nucleotides of the invention may be modified at various positions, including the sugar moiety, the phosphodiester linkage, and / or the base.

[0095] In some embodiments, the base moiety of the nucleoside may be modified. For example, the pyrimidine base may be modified at the 2, 3, 4, 5, and / or 6 positions of the pyrimidine ring. In some embodiments, the exocyclic amine of the cytosine may be modified. The purine base may also be modified. For example, the purine base may be modified at the 1, 2, 3, 6, 7, or 8 positions. In some embodiments, the exocyclic amine of the adenine may be modified. In some cases, the nitrogen atom of the ring of the base moiety may be replaced with another atom, such as carbon. The modification to the base moiety may be any suitable modification. Examples of modifications are known to those skilled in the art. In some embodiments, the modification of the base includes alkylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocycles.

[0096] In some embodiments, the pyrimidine may be modified at the 5-position. For example, the 5-position of the pyrimidine may be modified with an alkyl group, an alkynyl group, an alkenyl group, an acyl group, or a substituted derivative thereof. In other examples, the 5-position of the pyrimidine may be modified with a hydroxyl group or an alkoxyl group, or a substituted derivative thereof. Also, the N of the pyrimidine may be modified with an alkyl group, an alkynyl group, an alkenyl group, an acyl group, or a substituted derivative thereof. 4 In yet another example, the pyrimidine 5-6 bond may be saturated, the nitrogen atom in the pyrimidine ring may be replaced by a carbon atom, and / or the O 2 Or O 4 The atom may be substituted with a sulfur atom. It should be understood that other modifications are also possible.

[0097] In another example, the N of a purine 7 Position and / or N 2 and / or N 3 The third ring may be modified with an alkyl group or a substituted derivative thereof. In a further example, the third ring may be fused to a purine bicyclic ring system and / or the nitrogen atom in the purine ring system may be replaced with a carbon atom. It should be understood that other modifications are also possible. Non-limiting examples of pyrimidines modified at the 5-position are disclosed in U.S. Pat. No. 5,591,843, U.S. Pat. No. 7,205,297, U.S. Pat. No. 6,432,963 and U.S. Pat. No. 6,020,483; 4 Non-limiting examples of pyrimidines modified at the 8-position are disclosed in U.S. Pat. No. 5,580,731; non-limiting examples of purines modified at the 8-position are disclosed in U.S. Pat. No. 6,355,787 and U.S. Pat. No. 5,580,972; 6 Non-limiting examples of purines modified at the A position are disclosed in U.S. Pat. No. 4,853,386, U.S. Pat. No. 5,789,416, and U.S. Pat. No. 7,041,824; and non-limiting examples of purines modified at the 2 position are disclosed in U.S. Pat. No. 4,201,860 and U.S. Pat. No. 5,587,469; all of which are incorporated herein by reference.

[0098] Non-limiting examples of modified bases include N 4 ,N 4 -Ethanocytosine, 7-Deazaxanthosine, 7-Deazaguanosine, 8-Oxo-N 6 -methyladenine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N 6 -Isopentenyl-adenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N 6 -Methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, 5-methoxyuracil, 2-methylthio-N 6-isopentenyladenine, pseudouracil, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, 2-thiocytosine and 2,6-diaminopurine. In some embodiments, the base moiety may be a heterocyclic base other than a purine or pyrimidine. The heterocyclic base may be optionally modified and / or substituted.

[0099] Sugar moieties include naturally occurring unmodified sugars, e.g., monosaccharides (pentoses, e.g., ribose, deoxyribose), modified sugars and sugar analogs. In general, possible modifications of nucleomonomers, particularly those of the sugar moiety, include, for example, replacement of one or more hydroxyl groups with halogens, heteroatoms, aliphatic groups, or functionalization of hydroxyl groups as ethers, amines, thiols, etc.

[0100] A useful group of modified nucleomonomers are 2'-O-methyl nucleotides. Such 2'-O-methyl nucleotides may be referred to as "methylated", and the corresponding nucleotides are made from unmethylated nucleotides by alkylation or directly from methylated nucleotide reagents. Modified nucleomonomers may be used in combination with unmodified nucleomonomers. For example, the oligonucleotides of the present invention may contain both methylated and unmethylated nucleomonomers.

[0101] Some exemplary modified nucleomonomers include ribonucleotides with sugar or backbone modifications. Modified ribonucleotides may contain non-naturally occurring bases (instead of naturally occurring bases), such as uridine or cytidine modified at the 5' position, e.g., 5'-(2-amino)propyluridine and 5'-bromouridine; adenosine and guanosine modified at the 8 position, e.g., 8-bromoguanosine; deazanucleotides, e.g., 7-deaza-adenosine; and N-alkylated nucleotides, e.g., N6-methyladenosine. Sugar-modified ribonucleotides may also have a 2'-OH group replaced with H, alkoxy (or OR), R or alkyl, halogen, SH, SR, amino (e.g., NH2, NHR, NR2), or CN group, where R is lower alkyl, alkenyl, or alkynyl.

[0102] Modified ribonucleotides may also have a phosphodiester group attached to an adjacent ribonucleotide replaced by a modified group, such as a phosphorothioate group. More generally, the various nucleotide modifications may be combined.

[0103] The antisense (guide) strand may be substantially identical to at least a portion of the target gene(s), but the sequence does not need to be completely identical, at least in terms of base pairing properties, to be useful, for example to inhibit the phenotypic expression of the target gene.In general, higher homology can be used to compensate for the use of shorter antisense genes.In some cases, the antisense strand will generally be substantially identical (in antisense direction) to the target gene. The use of 2'-O-methyl modified RNA can also be beneficial in situations where it is desirable to minimize cellular stress responses. RNA with 2'-O-methyl nucleomonomers cannot be recognized by cellular mechanisms that are thought to recognize unmodified RNA. 2'-O-methylated or partially 2'-O-methylated RNA can avoid the interferon response to double-stranded nucleic acids while maintaining target RNA inhibition. This can be useful in both short RNAi (e.g. siRNA) sequences that induce interferon response, and longer RNAi sequences that can induce interferon response, for example, to avoid interferon or other cellular stress responses.

[0104] In general, modified sugars include D-ribose, 2'-O-alkyl (including 2'-O-methyl and 2'-O-ethyl), i.e., 2'-alkoxy, 2'-amino, 2'-S-alkyl, 2'-halo (including 2'-fluoro), 2'-methoxyethoxy, 2'-allyloxy (-OCH2CH=CH2), 2'-propargyl, 2'-propyl, ethynyl, ethenyl, propenyl, and cyano. In one embodiment, the sugar moiety may be a hexose and may be incorporated into an oligonucleotide as described (Augustyns, K., et al., Nucl. Acids. Res. 18:4711 (1992)). Exemplary nucleomonomers may be found, for example, in U.S. Patent No. 5,849,902, which is incorporated herein by reference. Specific functional group definitions and chemical terminology are described in more detail below. For the purposes of the present invention, chemical elements are identified according to the CAS version of the Periodic Table of the Elements, inside cover of the Handbook of Chemistry and Physics, 75th Ed., and specific functional groups are generally defined as described therein. Furthermore, general principles of organic chemistry and specific functional moieties and reactivities are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito:1999, the entire contents of which are incorporated herein by reference.

[0105] Certain compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, racemic mixtures thereof, and other mixtures thereof, as being within the scope of the present invention. Additional asymmetric carbon atoms may be present in a substituent, such as an alkyl group. All such isomers and mixtures thereof are intended to be included in the present invention.

[0106] Isomeric mixtures containing any of a variety of isomeric ratios may be utilized in accordance with the present invention. For example, when only two isomers are combined, mixtures containing isomeric ratios of 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0 are all contemplated by the present invention. Those skilled in the art will readily appreciate that similar ratios are contemplated for more complex isomeric mixtures.

[0107] By way of example, if a particular enantiomer of a compound of the invention is desired, it may be prepared by asymmetric synthesis or by derivatization with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary is cleaved to provide the pure desired enantiomer. Alternatively, if the molecule contains a basic functional group such as amino or an acidic functional group such as carboxyl, a diastereomeric salt may be formed with a suitable optically active acid or base, and the diastereomers thus formed may then be resolved by fractional crystallization or chromatographic means well known in the art, followed by recovery of the pure enantiomer.

[0108] In certain embodiments, the oligonucleotides of the invention include 3' and 5' termini (except for circular oligonucleotides). In one embodiment, the 3' and 5' termini of an oligonucleotide can be substantially protected from nucleases, for example, by modifying the 3' or 5' linkage (e.g., U.S. Pat. No. 5,849,902 and WO 98 / 13526). For example, an oligonucleotide can be made resistant by including a "blocking group." As used herein, the term "blocking group" refers to a substituent (e.g., other than an OH group) that can be attached to an oligonucleotide or nucleomonomer as either a protecting group for synthesis or as a linking group (e.g., FITC, propyl (CH2-CH2-CH3), glycol (-O-CH2-CH2-O-) phosphate (PO3 2- ), hydrogen phosphonate or phosphoramidite). "Blocking groups" also include "terminal blocking groups" or "exonuclease blocking groups," which protect the 3' and 5' termini of oligonucleotides, including modified nucleotides and non-nucleotide exonuclease resistant structures.

[0109] Exemplary terminal blocking groups include cap structures (e.g., 7-methylguanosine cap), inverted nucleomonomers, such as those with 3'-3' or 5'-5' terminal inversions (see, e.g., Ortiagao et al. 1992. Antisense Res. Dev. 2:129), methylphosphonates, phosphoramidites, non-nucleotide groups (e.g., non-nucleotide linkers, amino linkers, conjugates), and the like. The 3'-terminal nucleomonomers may include modified sugar moieties. The 3'-terminal nucleomonomers include a 3'-O that may be optionally replaced with a blocking group that prevents 3'-exonuclease degradation of the oligonucleotide. For example, the 3'-hydroxyl may be esterified to the nucleotide via a 3'→3' internucleotide linkage. For example, the alkyloxy radical may be methoxy, ethoxy, or isopropoxy, preferably ethoxy. Optionally, the 3'→3' linked nucleotide at the 3' end may be linked by an alternative linkage. To reduce nuclease degradation, the 5'-most 3'→5' linkage can be a modified linkage, such as a phosphorothioate or P-alkyloxyphosphotriester linkage. Preferably, the two 5'-most 3'→5' linkages are modified linkages. Optionally, the 5'-terminal hydroxyl moiety can be esterified with a phosphorus-containing moiety, such as phosphate, phosphorothioate, or P-ethoxyphosphate.

[0110] Those skilled in the art will understand that synthetic methods utilize various protecting groups as described herein. The term "protecting group" as used herein means that a specific functional moiety, such as O, S or N, is temporarily blocked so that reaction can be selectively carried out at another reactive site in a multifunctional compound. In some embodiments, the protecting group reacts selectively with good yield to give a protected substrate that is stable to the planned reaction; the protecting group should be selectively removable with good yield by a reagent that is easily available and preferably non-toxic and does not attack other functional groups; the protecting group forms an easily separable derivative (more preferably, without the generation of a new stereocenter); and the protecting group has minimal additional functionality to avoid having additional reactive sites.

[0111] As detailed herein, oxygen, sulfur, nitrogen and carbon protecting groups may be utilized. Hydroxyl protecting groups include: methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl ( SEMOR), tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,

[0112] 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxide, diphenylmethyl, p,p'-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, α-naphthalene phthalic acid diphenylmethyl, p-methoxyphenyl diphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4'-bromophenacyloxyphenyl)diphenylmethyl, 4,4',4''-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4',4''-tris(levulinoyloxyphenyl)methyl, 4,4',4''-tris(benzoyloxyphenyl)methyl, 3-(imidazol-1-yl)bis(4',4''-dimethoxyphenyl)methyl, 1,1-bis(4-methoxyphenyl)-1'-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl, 1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxide,

[0113] Trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate, Acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate (levulinoyl dithioacetal), pivaloate, adamantate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate),

[0114] Alkyl methyl carbonate, 9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenylphosphonio)ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate, alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-1-naphthyl carbonate, methyl dithiocarbonate,

[0115] 2-Iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate, 2,6-dichloro-4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodipheny alkyl acetate, isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate, o-(methoxycarbonyl)benzoate, α-naphthoate, nitrate, alkyl N,N,N',N'-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate and tosylate (Ts).

[0116] For protecting 1,2- or 1,3-diols, protecting groups include: methylene acetal, ethylidene acetal, 1-t-butylethylidene ketal, 1-phenylethylidene ketal, (4-methoxyphenyl)ethylidene acetal, 2,2,2-trichloroethylidene acetal, acetonide, cyclopentylidene ketal, cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal, p-methoxybenzylidene acetal, 2,4-dimethoxybenzylidene ketal, 3,4-dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal, methoxymethylene acetal, ethoxymethylene acetal, dimethyoxymethylene acetal, methyl ... xymethylene ortho esters, 1-methoxyethylidene ortho esters, 1-ethoxyethylidene ortho esters, 1,2-dimethoxyethylidene ortho esters, α-methoxybenzylidene ortho esters, 1-(N,N-dimethylamino)ethylidene derivatives, α-(N,N'-dimethylamino)benzylidene derivatives, 2-oxacyclopentylidene ortho esters, di-t-butylsilylene groups (DTBS), 1,3-(1,1,3,3-tetraisopropyldisiloxanylidene) derivatives (TIPDS), tetra-t-butoxydisiloxane-1,3-diylidene derivatives (TBDS), cyclic carbonates, cyclic boronates, ethyl boronates and phenyl boronates.

[0117] Amino protecting groups include: methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluorenylmethyl carbamate, 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyi)]methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)- 1-Methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2'- and 4'-pyridyl)ethyl carbamate (Pyoc),

[0118] 2-(N,N-dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropyl allyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, Carbamates, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitrobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate,

[0119] 2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenylcarbamate (Mtpc), 2,4-dimethylthiophenylcarbamate (Bmpc), 2-phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl carbamate , 5-benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methyl carbamate, phenothiazinyl-(10)-carbonyl derivatives, N'-p-toluenesulfonylaminocarbonyl derivatives, N'-phenylaminothiocarbonyl derivatives,

[0120] t-Amyl carbamate, S-benzylthiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxycarbonylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzyl carbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, carbamate, 2-iodoethyl carbamate, isobornyl carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p'-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate, 1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate,

[0121] Phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate, 2,4,6-trimethylbenzyl carbamate, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivatives,

[0122] Benzamide, p-phenylbenzamide, o-nitrophenylacetamide, o-nitrophenoxyacetamide, acetoacetamide, (N'-dithiobenzyloxycarbonylamino)acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide, 2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutane amide, 3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine derivatives, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),

[0123] 5-Substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-ones, 5-Substituted 1,3-dibenzyl-1,3,5-triazacyclohexan-2-ones, 1-Substituted 3,5-dinitro-4-pyridones, N-Methylamines, N-Allylamines, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-pyrrolin-3-yl)amine, Quaternary ammonium salts, N-Benzylamines, N-Di(4-methoxyphenyl)methylamine, N-5-Dibenzosuberylamine, N-Triphenylmethylamine (Tr), N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-Phenylfluorenylamine (PhF), N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), N-2-picolylamino N'-oxide, N-1,1-dimethylthiomethyleneamine, N-benzylideneamine, Np-methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine, N-(N',N'-dimethylaminomethylene)amine, N,N'-isopropylidenediamine, Np-nitrobenzylideneamine, N-salicylideneamine, N-5-chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,

[0124] N-Borane derivatives, N-diphenylborinic acid derivatives, N-[phenyl(pentacarbonylchromium- or tungsten)carbonyl]amine, N-copper chelates, N-zinc chelates, N-nitroamines, N-nitrosamines, amine N-oxides, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidates, diphenyl phosphoramidates, benzenesulfenamides, o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, 3-nitropyridine sulfenamide (Npys),

[0125] p-Toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6-trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts ), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4',8'-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide and phenacylsulfonamide.

[0126] Exemplary protecting groups are detailed herein. However, the present invention is not intended to be limited to these protecting groups, but rather, various additional equivalent protecting groups can be easily identified using the above criteria and utilized in the methods of the present invention. In addition, various protecting groups are described in Protective Groups in Organic Synthesis, Third Ed. Greene, TW and Wuts, PG, Eds., John Wiley & Sons, New York: 1999, the entire contents of which are incorporated herein by reference.

[0127] It is understood that compounds may be substituted with any number of substituents or functional moieties as described herein. In general, the term "substituted" and the substituents contained in the formulas of this invention, whether preceded by the term "optionally" or not, refer to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent. When more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituents may be the same or different at each position. The term "substituted" as used herein is intended to include all permissible substituents of organic compounds. In a broad sense, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Heteroatoms, such as nitrogen, may have hydrogen substituents and / or any permissible substituents of organic compounds described herein that satisfy the valence of the heteroatom. Moreover, this invention is not intended to be limited in any manner by the permissible substituents of organic compounds. Combinations of substituents and variables envisioned by the present invention are preferably those that result in the formation of stable compounds useful, for example, in the treatment of infectious or proliferative diseases. The term "stable" preferably, as used herein, refers to compounds that have sufficient stability to permit manufacture, maintain compound integrity for a sufficient period of time to be detected, and preferably be useful for the purposes detailed herein.

[0128] The term "aliphatic" as used herein includes both saturated and unsaturated, straight-chain (i.e. unbranched), branched, acyclic, cyclic or polycyclic aliphatic hydrocarbons, optionally substituted with one or more functional groups. As will be understood by those skilled in the art, "aliphatic" as used herein is intended to include, but is not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl and cycloalkynyl moieties. Thus, the term "alkyl" as used herein includes straight-chain, branched and cyclic alkyl groups. A similar convention applies to other general terms, such as "alkenyl", "alkynyl", etc. Moreover, the terms "alkyl", "alkenyl", "alkynyl", etc. as used herein include both substituted and unsubstituted groups. In certain embodiments, "lower alkyl" as used herein is used to refer to alkyl groups (cyclic, acyclic, substituted, unsubstituted, branched or unbranched) having 1 to 6 carbon atoms.

[0129] In certain embodiments, the alkyl, alkenyl, and alkynyl groups employed in the present invention contain 1-20 aliphatic carbon atoms. In certain embodiments, the alkyl, alkenyl, and alkynyl groups employed in the present invention contain 1-10 aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the present invention contain 1-8 aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the present invention contain 1-6 aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the present invention contain 1-4 aliphatic carbon atoms.

[0130] Thus, exemplary aliphatic groups include, but are not limited to, for example, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, -CH2-cyclopropyl, vinyl, allyl, n-butyl, sec-butyl, isobutyl, tert-butyl, cyclobutyl, -CH2-cyclobutyl, n-pentyl, sec-pentyl, isopentyl, tert-pentyl, cyclopentyl, -CH2-cyclopentyl, n-hexyl, sec-hexyl, cyclohexyl, -CH2-cyclohexyl moieties, which may in turn bear one or more substituents. Alkenyl groups include, but are not limited to, for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like. Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like.

[0131] Some examples of substituents for the above aliphatic (and other) moieties of the compounds of the invention include, but are not limited to, the following: aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; -F; -Cl; -Br; -I; -OH; -NO2; -CN; -CF3; -CH2CF3; -CHCl2; -CH2OH; -CH2CH2OH; -CH2NH2; -CH2SO2CH3; -C(O)R x ;-CO2(R x );-CON(R x )2;-OC(O)R x ;-OCO2R x ;-OCON(R x )2;-N(R x )2;-S(O)2R x ;-NR x (CO)R xwherein each occurrence of Rx independently includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, where any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and where any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted. Additional examples of generally applicable substituents are illustrated by the specific embodiments described herein.

[0132] The term "heteroaliphatic" as used herein refers to an aliphatic moiety that contains one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms, for example, in place of carbon atoms. Heteroaliphatic moieties can be branched or unbranched, cyclic or acyclic, and can include saturated and unsaturated heterocycles, such as morpholino, pyrrolidinyl, and the like. In some embodiments, heteroaliphatic moieties are substituted by independent replacement of one or more hydrogen atoms thereon with one or more moieties, including, but not limited to, the following: aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; -F; -Cl; -Br; -I; -OH; -NO2; -CN; -CF3; -CH2CF3; -CHCl2; -CH2OH; -CH2CH2OH; -CH2NH2; -CH2SO2CH3; -C(O)R x ;-CO2(R x );-CON(R x )2;-OC(O)R x ;-OCO2R x ;-OCON(R x )2;-N(R x )2;-S(O)2R x ;-NR x (CO)R x ;

[0133] Here, R xEach occurrence independently includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, where any of the aliphatic, heteroaliphatic, arylalkyl, or heteroarylalkyl substituents described above and herein can be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and where any of the aryl or heteroaryl substituents described above and herein can be substituted or unsubstituted. Additional examples of generally applicable substituents are illustrated by specific embodiments described herein. The terms "halo" and "halogen" as used herein refer to an atom selected from fluorine, chlorine, bromine, and iodine.

[0134] The term "alkyl" includes saturated aliphatic groups, including straight chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.), branched chain alkyl groups (isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups (cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In certain embodiments, a straight chain or branched chain alkyl group has 6 or fewer carbon atoms in its backbone (e.g., C1-C6 for straight chain, C3-C6 for branched chain), more preferably 4 or fewer carbon atoms. Similarly, preferred cycloalkyls have 3-8 carbon atoms in their ring structure, more preferably 5 or 6 carbons in the ring structure. The term C1-C6 includes alkyl groups containing 1-6 carbon atoms.

[0135] Moreover, unless otherwise specified, the term alkyl includes both "unsubstituted alkyls" and "substituted alkyls," the latter of which refers to alkyl moieties having independently selected substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents include, for example, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonate, phosphinate, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonate, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Cycloalkyl may be further substituted, e.g., with the substituents described above. An "alkylaryl" or an "arylalkyl" moiety is an alkyl substituted with an aryl (e.g., phenylmethyl (benzyl)). The term "alkyl" also includes the side chains of natural or unnatural amino acids. The term "n-alkyl" refers to a straight-chain (i.e., unbranched), unsubstituted alkyl group.

[0136] The term "alkenyl" includes unsaturated aliphatic groups similar in length to the alkyls described above and which may be substituted, but which contain at least one double bond. For example, the term "alkenyl" includes straight chain alkenyl groups (e.g., ethylenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, etc.), branched chain alkenyl groups, cycloalkenyl (alicyclic) groups (cyclopropenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl), alkyl- or alkenyl-substituted cycloalkenyl groups, and cycloalkyl- or cycloalkenyl-substituted alkenyl groups. In some embodiments, straight chain or branched chain alkenyl groups have 6 or fewer carbon atoms in their backbone (e.g., C2-C6 for straight chain, C3-C6 for branched chain). Likewise, cycloalkenyl groups may have from 3-8 carbon atoms in their ring structure, and more preferably have 5 or 6 carbons in the ring structure. The term C2-C6 includes alkenyl groups containing from 2-6 carbon atoms.

[0137] Moreover, unless otherwise specified, the term alkenyl includes both "unsubstituted alkenyls" and "substituted alkenyls," the latter of which refers to alkenyl moieties having independently selected substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents include, for example, alkyl groups, alkynyl groups, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonate, phosphinate, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonate, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

[0138] The term "alkynyl" includes unsaturated aliphatic groups similar in length and possible substitution to the alkyls described above, but containing at least one triple bond. For example, the term "alkynyl" includes straight chain alkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, etc.), branched chain alkynyl groups, and cycloalkyl or cycloalkenyl substituted alkynyl groups. In certain embodiments, a straight chain or branched chain alkynyl group has 6 or fewer carbon atoms in its backbone (e.g., C2-C6 for straight chain, C3-C6 for branched chain). The term C2-C6 includes alkynyl groups containing 2-6 carbon atoms.

[0139] Moreover, unless otherwise specified, the term alkynyl includes both "unsubstituted alkynyls" and "substituted alkynyls," the latter of which refers to alkynyl moieties having independently selected substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents include, for example, alkyl groups, alkynyl groups, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonate, phosphinate, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonate, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

[0140] Unless the number of carbons is otherwise specified, "lower alkyl" as used herein means an alkyl group, as defined above, having from 1 to 5 carbon atoms in its backbone structure. "Lower alkenyl" and "lower alkynyl", for example, have chain lengths of from 2 to 5 carbon atoms.

[0141] The term "alkoxy" includes substituted and unsubstituted alkyl, alkenyl, and alkynyl groups covalently linked to an oxygen atom. Examples of alkoxy groups include methoxy, ethoxy, isopropyloxy, propoxy, butoxy, and pentoxy groups. Examples of substituted alkoxy groups include halogenated alkoxy groups. Alkoxy groups include alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonate, phosphinate, cyano, amino(alkylamino, dialkylamino, arylamino, diarylamino, and alkylaryl). The halogen substituted alkoxy groups may be substituted with independently selected groups such as arylamino, acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulffydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfmyl, sulfonate, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or aromatic or heteroaromatic moieties. Examples of halogen substituted alkoxy groups include, but are not limited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy, trichloromethoxy, and the like.

[0142] The term "heteroatom" includes atoms of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur and phosphorus. The term "hydroxy" or "hydroxyl" includes groups with an --OH or --O-- (with an appropriate counterion). The term "halogen" includes fluorine, bromine, chlorine, iodine, etc. The term "perhalogenated" generally refers to a moiety where all hydrogens have been replaced by halogen atoms.

[0143] The term "substituted" includes independently selected substituents that may be placed on the moiety and that enable the molecule to perform its intended function. Examples of substituents are alkyl, alkenyl, alkynyl, aryl, (CR'R'') 0~3 NR'R'', (CR'R'') 0~3 CN, NO2, halogen, (CR'R'') 0~3 C(halogen)3, (CR'R'') 0~3 CH(Halogen)2, (CR'R'') 0~3 CH2(Halogen), (CR'R'') 0~3 CONR'R'', (CR'R'') 0~3 S(O) 1~2 NR'R'', (CR'R'') 0~3 CHO, (CR'R'') 0~3 O(CR'R'') 0~3 H, (CR'R'') 0~3 S(O) 0~2 R', (CR'R'') 0~3 O(CR'R'') 0~3 H, (CR'R'') 0~3 COR', (CR'R'') 0~3 CO2R', or (CR'R'') 0~3 an OR' group; where each R' and R'' is each independently a hydrogen, a C1-C5 alkyl, a C2-C5 alkenyl, a C2-C5 alkynyl or an aryl group, or R' and R'' taken together are a benzylidene group or a -(CH2)2O(CH2)2- group.

[0144] The term "amine" or "amino" includes compounds or moieties in which a nitrogen atom is covalently bonded to at least one carbon or heteroatom. The term "alkylamino" includes groups and compounds in which the nitrogen is bonded to at least one additional alkyl group. The term "dialkylamino" includes groups in which the nitrogen atom is bonded to at least two additional alkyl groups. The term "ether" includes compounds or moieties which contain an oxygen bonded to two different carbon atoms or heteroatoms. For example, this term includes "alkoxyalkyl," which refers to an alkyl, alkenyl, or alkynyl group covalently bonded to an oxygen atom which is covalently bonded to another alkyl group.

[0145] The terms "polynucleotide", "nucleotide sequence", "nucleic acid", "nucleic acid molecule", "nucleic acid sequence" and "oligonucleotide" refer to a polymer of two or more nucleotides. A polynucleotide can be DNA, RNA or derivatives or modified versions thereof. A polynucleotide can be single-stranded or double-stranded. A polynucleotide can be modified at the base moiety, sugar moiety or phosphate backbone to improve, for example, the stability of the molecule, its hybridization parameters, etc. A polynucleotide can contain a modified base moiety selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine,

[0146] Inosine, N6-isopentenyl adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosyl eosin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methy Ruthio-N6-isopentenyladenine, wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine.

[0147] Polynucleotides may contain modified sugar moieties (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, 2'-O-methylcytidine, arabinose, and hexose) and / or modified phosphate moieties (e.g., phosphorothioate and 5'-N-phosphoramidite linkages). Nucleotide sequences typically carry genetic information, including the information used by cellular machinery to make proteins and enzymes. These terms include double- or single-stranded genomic and cDNA, RNA, any synthetic and engineered polynucleotides, and both sense and antisense polynucleotides. This includes single- and double-stranded molecules, i.e., DNA-DNA, DNA-RNA, and RNA-RNA hybrids, as well as "protein nucleic acids" (PNAs) formed by conjugating bases to an amino acid backbone.

[0148] The term "targeting moiety" or "targeting ligand" includes, but is not limited to, N-acetylglucosamine or N-acetylgalactosamine (GalNac). The term "base" includes the known purine and pyrimidine heterocyclic bases, deazapurines and their analogs (including heterocyclic substituted analogs, e.g., aminoethoxyphenoxazine), derivatives (e.g., 1-alkyl-, 1-alkenyl-, heteroaromatic-, and 1-alkynyl derivatives) and tautomers thereof. Examples of purines are adenine, guanine, inosine, diaminopurine, and xanthine and their analogs (e.g., 8-oxo-N 6 -methyladenine or 7-diazaxanthine), and derivatives thereof. Pyrimidines include, for example, thymine, uracil and cytosine, and analogs thereof (e.g., 5-methylcytosine, 5-methyluracil, 5-(1-propynyl)uracil, 5-(1-propynyl)cytosine, and 4,4-ethanocytosine). Other examples of suitable bases include non-purinyl and non-pyrimidinyl bases, such as 2-aminopyridine and triazines.

[0149] In some embodiments, the nucleomonomers of the oligonucleotide of the present invention are RNA nucleotides. In another preferred embodiment, the nucleomonomers of the oligonucleotide of the present invention are modified RNA nucleotides. Thus, the oligonucleotide contains modified RNA nucleotides. The term "nucleoside" includes a base covalently linked to a sugar moiety, preferably ribose or deoxyribose. Examples of preferred nucleosides include ribonucleosides and deoxyribonucleosides. Nucleosides also include a base linked to an amino acid or amino acid analog that may contain a free carboxyl group, a free amino group, or a protecting group. Suitable protecting groups are well known in the art (see PGM Wuts and T.W. Greene, Protective Groups in Organic Synthesis, 2nd ed., Wiley-Interscience, New York, 1999). The term "nucleotide" includes nucleosides that further include a phosphate group or a phosphate analog.

[0150] The nucleic acid molecule may be linked to a hydrophobic moiety for targeting and / or delivery of the molecule to a cell. In some embodiments, the hydrophobic moiety is linked to the nucleic acid molecule via a linker. In some embodiments, the linkage is via a non-covalent interaction. In other embodiments, the linkage is via a covalent bond. The nucleic acid molecule may be linked to a targeting ligand moiety for targeting and / or delivery of the molecule to a cell. In some embodiments, the targeting ligand moiety is linked to the nucleic acid molecule as a phosphoramidite or alternatively via a linker. In some embodiments, the linkage is via a non-covalent interaction. In other embodiments, the linkage is via a covalent bond. Any linker known in the art may be used to link the nucleic acid to the hydrophobic moiety. Linkers known in the art are described in published International PCT applications WO92 / 03464, WO95 / 23162, WO2008 / 021157, WO2009 / 021157, WO2009 / 134487, WO2009 / 126933, U.S. Patent Application Publication No. 2005 / 0107325, U.S. Patent No. 5,414,077, U.S. Patent No. 5,419,966, U.S. Patent No. 5,512,667, U.S. Patent No. 5,646,126, and U.S. Patent No. 5,652,359, which are incorporated herein by reference.

[0151] The linker may be as simple as a covalent bond to a polyatomic linker. The linker may be cyclic or acyclic. The linker may be optionally substituted. In some embodiments, the linker is capable of being cleaved from the nucleic acid. In some embodiments, the linker is capable of being hydrolyzed under physiological conditions. In some embodiments, the linker is capable of being cleaved by an enzyme (e.g., an esterase or a phosphodiesterase). In some embodiments, the linker includes a spacer element that separates the nucleic acid from the hydrophobic moiety. The spacer element may include 1-30 carbon or heteroatoms.

[0152] In some embodiments, the linker and / or spacer element comprises a protonatable functional group. Such a protonatable functional group may facilitate endosomal escape of the nucleic acid molecule. The protonatable functional group may also assist in the delivery of the nucleic acid to the cell, for example, by neutralizing the overall charge of the molecule. In other embodiments, the linker and / or spacer element is biologically inert (i.e., does not impart biological activity or function to the resulting nucleic acid molecule).

[0153] In some embodiments, the nucleic acid molecule having the targeting moiety and / or linker, and the hydrophobic moiety, is of a formula described herein. [ka] During the ceremony, X is N or CH; A is a bond; a substituted or unsubstituted, cyclic or acyclic, branched or unbranched aliphatic; or a substituted or unsubstituted, cyclic or acyclic, branched or unbranched heteroaliphatic; R 1 is a hydrophobic moiety; R 2 is hydrogen; an oxygen protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; and R 3 is a nucleic acid, It is expressed as:

[0154] In some embodiments, the molecule has the formula: [ka] It is expressed as: In some embodiments, the molecule has the formula: [ka] It is expressed as:

[0155] In some embodiments, the molecule has the formula: [ka] It is expressed as: In some embodiments, the molecule has the formula: [ka] It is expressed as:

[0156] In some embodiments, X is N. In some embodiments, X is CH. In some embodiments, A is a bond. In some embodiments, A is a substituted or unsubstituted, cyclic or acyclic, branched or unbranched aliphatic. In some embodiments, A is an acyclic, substituted or unsubstituted, branched or unbranched aliphatic. In some embodiments, A is an acyclic, substituted, branched or unbranched aliphatic. In some embodiments, A is an acyclic, substituted, unbranched aliphatic. In some embodiments, A is an acyclic, substituted, unbranched alkyl. In some embodiments, A is an acyclic, substituted, unbranched C 1~20 In some embodiments, A is an acyclic, substituted, unbranched C alkyl. 1~12 In some embodiments, A is an acyclic, substituted, unbranched C alkyl. 1~10 In some embodiments, A is an acyclic, substituted, unbranched C alkyl. 1~8 In some embodiments, A is an acyclic, substituted, unbranched C alkyl. 1~6 In some embodiments, A is an alkyl. In some embodiments, A is a substituted or unsubstituted, cyclic or acyclic, branched or unbranched heteroaliphatic. In some embodiments, A is an acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic. In some embodiments, A is an acyclic, substituted, branched or unbranched heteroaliphatic. In some embodiments, A is an acyclic, substituted, branched or unbranched heteroaliphatic. In some embodiments, A is an acyclic, substituted, unbranched heteroaliphatic.

[0157] In some embodiments, A has the formula: [ka] It is expressed as:

[0158] In some embodiments, A has the formula: [ka] It is expressed as one of the following.

[0159] In some embodiments, A has the formula: [ka] It is expressed as one of the following.

[0160] In some embodiments, A has the formula: [ka] It is expressed as one of the following.

[0161] In some embodiments, A has the formula: [ka] It is expressed as: In some embodiments, A has the formula: [ka] It is expressed as:

[0162] In some embodiments, A has the formula: [ka] During the ceremony, each occurrence of R is independently the side chain of a natural or unnatural amino acid; and n is an integer between 1 and 20, inclusive; It is expressed as:

[0163] In some embodiments, A has the formula: [ka] It is expressed as: In some embodiments, each occurrence of R is independently the side chain of a natural amino acid. In some embodiments, n is an integer from 1 to 15, inclusive. In some embodiments, n is an integer from 1 to 10, inclusive. In some embodiments, n is an integer from 1 to 5, inclusive.

[0164] In some embodiments, A has the formula: [ka] where n is an integer from 1 to 20, inclusive; It is expressed as:

[0165] In some embodiments, A has the formula: [ka] It is expressed as: In some embodiments, n is an integer from 1 to 15, inclusive. In some embodiments, n is an integer from 1 to 10, inclusive. In some embodiments, n is an integer from 1 to 5, inclusive.

[0166] In some embodiments, A has the formula: [ka] where n is an integer from 1 to 20, inclusive; It is expressed as:

[0167] In some embodiments, A has the formula: [ka] It is expressed as: In some embodiments, n is an integer from 1 to 15, inclusive. In some embodiments, n is an integer from 1 to 10, inclusive. In some embodiments, n is an integer from 1 to 5, inclusive.

[0168] In some embodiments, the molecule has the formula: [ka] In the formula, X, R 1 , R 2 and R 3 is as defined herein; and A' is substituted or unsubstituted, cyclic or acyclic, branched or unbranched aliphatic; or substituted or unsubstituted, cyclic or acyclic, branched or unbranched heteroaliphatic; It is expressed as:

[0169] In some embodiments, A' has the formula: [ka] It is expressed as one of the following.

[0170] In some embodiments, A has the formula: [ka] It is expressed as one of the following.

[0171] In some embodiments, A has the formula: [ka] It is expressed as one of the following.

[0172] In some embodiments, A has the formula: [ka] It is expressed as:

[0173] In some embodiments, A has the formula: [ka] It is expressed as: In one embodiment, R 1 is a steroid. In some embodiments, R1 is cholesterol. In some embodiments, R 1 is a lipophilic vitamin. In some embodiments, R 1 is vitamin A. In some embodiments, R 1 is vitamin E.

[0174] In one embodiment, R 1 is the expression: [ka] In the formula, R A is a substituted or unsubstituted, cyclic or acyclic, branched or unbranched aliphatic; or is a substituted or unsubstituted, cyclic or acyclic, branched or unbranched heteroaliphatic; It is expressed as:

[0175] In one embodiment, R 1 is the expression: [ka] It is expressed as:

[0176] In one embodiment, R 1 is the expression: [ka] It is expressed as:

[0177] In one embodiment, R 1 is the expression: [ka] It is expressed as:

[0178] In one embodiment, R 1 is the expression: [ka] It is expressed as:

[0179] In one embodiment, R 1 is the expression: [ka] It is expressed as:

[0180] In one embodiment, the nucleic acid molecule has the formula: [ka] During the ceremony, X is N or CH; A is a bond; a substituted or unsubstituted, cyclic or acyclic, branched or unbranched aliphatic; or a substituted or unsubstituted, cyclic or acyclic, branched or unbranched heteroaliphatic; R 1 is a hydrophobic moiety; R 2 is hydrogen; an oxygen protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; and R 3 is a nucleic acid, It is expressed as:

[0181] In one embodiment, the nucleic acid molecule has the formula: [ka] During the ceremony, X is N or CH; A is a bond; a substituted or unsubstituted, cyclic or acyclic, branched or unbranched aliphatic; or a substituted or unsubstituted, cyclic or acyclic, branched or unbranched heteroaliphatic; R 1 is a hydrophobic moiety; R 2is hydrogen; an oxygen protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; and R 3 is a nucleic acid, It is expressed as:

[0182] In one embodiment, the nucleic acid molecule has the formula: [ka] During the ceremony, X is N or CH; A is a bond; a substituted or unsubstituted, cyclic or acyclic, branched or unbranched aliphatic; or a substituted or unsubstituted, cyclic or acyclic, branched or unbranched heteroaliphatic; R 1 is a hydrophobic moiety; R 2 is hydrogen; an oxygen protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; and R 3 is a nucleic acid, It is expressed as:

[0183] In one embodiment, the nucleic acid molecule has the formula: [ka] It is expressed as:

[0184] In one embodiment, the nucleic acid molecule has the formula: [ka] It is expressed as:

[0185] In one embodiment, the nucleic acid molecule has the formula: [ka] In the formula, R 3 is a nucleic acid, It is expressed as:

[0186] In one embodiment, the nucleic acid molecule has the formula: [ka] In the formula, R 3 is a nucleic acid; and n is an integer between 1 and 20, inclusive. It is expressed as:

[0187] In one embodiment, the nucleic acid molecule has the formula: [ka] It is expressed as:

[0188] In one embodiment, the nucleic acid molecule has the formula: [ka] It is expressed as:

[0189] In one embodiment, the nucleic acid molecule has the formula: [ka] It is expressed as:

[0190] In one embodiment, the nucleic acid molecule has the formula: [ka] It is expressed as:

[0191] In one embodiment, the nucleic acid molecule has the formula: [ka] It is expressed as:

[0192] As used herein, the term "linkage" refers to a naturally occurring, unmodified phosphodiester moiety (-O-(PO 2- )-O-). The term "substitute linkage" as used herein includes any analog or derivative of a native phosphodiester group that covalently bonds adjacent nucleomonomers. Substitute linkages include phosphodiester analogs such as phosphorothioate, phosphorodithioate, and P-ethyoxyphosphodiester, P-ethoxyphosphodiester, P-alkyloxyphosphodiester, methylphosphonate, and non-phosphorous containing linkages such as acetals and amides. Such substitute linkages are known in the art (see, for example, Bjergarde et al. 1991. Nucleic Acids Res. 19:5843; Caruthers et al. 1991. Nucleosides Nucleotides. 10:47). In some embodiments, non-hydrolyzable linkages such as phosphorothioate linkages are preferred.

[0193] In some embodiments, the oligonucleotide of the present invention comprises hydrophobically modified nucleotides or "hydrophobic modifications". As used herein, "hydrophobic modifications" refers to bases that have been modified such that (1) the overall hydrophobicity of the base is significantly increased, and / or (2) the base is still able to form similar normal Watson-Crick interactions. Some non-limiting examples of base modifications include uridine and cytidine modifications at the 5th position, such as phenyl, 4-pyridyl, 2-pyridyl, indolyl, and isobutyl, phenyl (CHOH); tryptophanyl (CHN)CHCH(NH)CO), isobutyl, butyl, aminobenzyl; phenyl; and naphthyl. Other types of conjugates that can be attached to the ends (3' or 5' ends), loop regions, or any other portion of the chemically modified double-stranded nucleic acid molecule include sterols, sterol-type molecules, peptides, small molecules, proteins, etc. In some embodiments, a chemically modified double-stranded nucleic acid molecule such as sd-rxRNA (INTASYL™) may contain more than one conjugate (of the same or different chemical nature). In some embodiments, the conjugate is cholesterol. Alternatively, in some embodiments, the conjugate is GalNac.

[0194] In some embodiments, the first nucleotide to the 5' end of the guide strand has a 2'-O-methyl modification, where optionally the 2'-O-methyl modification is a 5P-2'O-methyl U modification, or a 5' vinylphosphonate 2'-O-methyl U modification. Another method for increasing target gene specificity or reducing off-target silencing effects is to introduce a 2' modification, such as a 2'-O-methyl modification, at a position corresponding to the second nucleotide at the 5' end of the guide sequence. The antisense (guide) sequences of the present invention can be "chimeric oligonucleotides" that contain RNA-like and DNA-like regions.

[0195] The term "RNase H activating region" includes a region of an oligonucleotide, e.g., a chimeric oligonucleotide, that is capable of recruiting RNase H, which cleaves a target RNA strand to which the oligonucleotide binds. Typically, an RNase H activating region comprises a minimal core of DNA or DNA-like nucleomonomers (of at least about 3-5, typically about 3-12, more typically about 5-12, more preferably about 5-10 consecutive nucleomonomers) (see, e.g., U.S. Pat. No. 5,849,902). Preferably, the RNase H activating region comprises about 9 consecutive deoxyribose-containing nucleomonomers.

[0196] The term "non-activating region" includes a region of an antisense sequence, such as a chimeric oligonucleotide, that does not recruit or activate RNase H. Preferably, the non-activating region does not contain phosphorothioate DNA. The oligonucleotides of the present invention include at least one non-activating region. In one embodiment, the non-activating region can be nuclease stable or can provide specificity for a target by being complementary to the target and forming hydrogen bonds with a target nucleic acid molecule bound by the oligonucleotide.

[0197] In one embodiment, at least a portion of the contiguous polynucleotides are linked by substituted linkages, such as phosphorothioate linkages. In some embodiments, most or all of the nucleotides beyond the guide sequence are linked by phosphorothioate linkages (whether 2' modified or not). Such constructs tend to have improved pharmacokinetics due to their higher affinity for serum proteins. Phosphorothioate linkages in the non-guide sequence portion of a polynucleotide generally do not interfere with the activity of the guide strand once it is loaded into RISC. In some embodiments, a high level of phosphorothioate modification can result in improved delivery. In some embodiments, the guide and / or passenger strands are fully phosphorothioated.

[0198] The antisense (guide) sequence of the present invention may comprise a "morpholino oligonucleotide". Morpholino oligonucleotides are non-ionic and function by an RNase H-independent mechanism. The four genetic bases (adenine, cytosine, guanine and thymine / uracil) of morpholino oligonucleotides are linked to a six-membered morpholine ring. Morpholino oligonucleotides are made by linking four different subunit types, for example, by non-ionic phosphorodiamidate intersubunit linkages. Morpholino oligonucleotides have many advantages, including: complete resistance to nuclease (Antisense & Nucl.Acid Drug Dev.1996.6:267); predictable targeting (Biochemica Biophysica Acta.1999.1489:141); reliable activity in cells (Antisense & Nucl.Acid Drug Dev.1997.7:63); excellent sequence specificity (Antisense & Nucl.Acid Drug Dev.1997.7:151); minimal non-antisense activity (Biochemica Biophysica Acta.1999.1489:141); and easy osmotic or scraping delivery (Antisense & Nucl.Acid Drug Dev.1997.7:291). Morpholino oligonucleotides are also preferred due to their non-toxicity at high doses. A discussion of the preparation of morpholino oligonucleotides can be found in Antisence & Nucl. Acid Drug Dev. 1997.7:187.

[0199] The chemical modifications described herein are believed to facilitate the loading of single-stranded polynucleotides into RISC.Single-stranded polynucleotides have been shown to be active in loading into RISC and inducing gene silencing.However, the level of activity for single-stranded polynucleotides is believed to be 2-4 orders of magnitude lower when compared to duplex polynucleotides.

[0200] The present invention provides a description of chemical modification patterns that (a) significantly increase the stability of single-stranded polynucleotides, (b) facilitate efficient loading of polynucleotides into RISC complexes, and (c) improve cellular uptake of single-stranded nucleotides. The chemical modification patterns may include combinations of ribose modifications, backbone modifications, hydrophobic nucleoside modifications, and conjugate-type modifications. Additionally, in some embodiments, the 5' end of the single polynucleotide may be chemically phosphorylated.

[0201] In yet another aspect, the present invention provides a description of chemical modification patterns that improve the function of RISC inhibitory polynucleotides. Single-stranded polynucleotides have been shown to inhibit the activity of preloaded RISC complexes through a substrate competition mechanism. For these types of molecules, commonly referred to as antagomers, activity usually requires high concentrations and in vivo delivery is less effective. The present invention provides a description of chemical modification patterns that (a) significantly increase the stability of single-stranded polynucleotides, (b) promote efficient recognition of polynucleotides as substrates by RISC, and / or (c) improve the uptake of single-stranded nucleotides by cells. Chemical modification patterns may include a combination of ribose modifications, backbone modifications, hydrophobic nucleoside modifications, and conjugate-type modifications.

[0202] The modifications provided by the present invention are applicable to all polynucleotides, including single-stranded RISC invasive polynucleotides, single-stranded RISC inhibitory polynucleotides, conventional duplexed polynucleotides of various lengths (15-40 bp), asymmetric duplexed polynucleotides, etc. The polynucleotides may be modified with various chemical modification patterns, including 5'-end modifications, ribose modifications, backbone modifications, and hydrophobic nucleoside modifications.

[0203] synthesis The oligonucleotides of the present invention can be synthesized by any method known in the art, for example, using enzymatic synthesis and / or chemical synthesis. Oligonucleotides can be synthesized in vitro (for example, using enzymatic synthesis and chemical synthesis) or in vivo (using recombinant DNA techniques well known in the art).

[0204] In some embodiments, chemical synthesis is used for modified polynucleotides. Chemical synthesis of linear oligonucleotides is well known in the art and can be achieved by solution or solid phase techniques. Preferably, synthesis is by solid phase method. Oligonucleotides can be made by any of several different synthetic procedures, including phosphoramidite, phosphite triester, H-phosphonate and phosphotriester methods, typically by automated synthesis methods.

[0205] Protocols for the synthesis of oligonucleotides are well known in the art and include, for example, U.S. Pat. No. 5,830,653; WO 98 / 13526; Stec et al. 1984. J. Am. Chem. Soc. 106:6077; Stec et al. 1985. J. Org. Chem. 50:3908; Stec et al. J. Chromatog. 1985. 326:263; LaPlanche et al. 1986. Nucl. Acid. Res. 1986. 14:9081; Fasman GD, 1989. Practical Handbook of Biochemistry and Molecular Biology. 1989. CRC Press, Boca Raton, Fla.; Lamone. 1993. Biochem. Soc. Trans. 21:1; U.S. Patent No. 5,013,830; U.S. Patent No. 5,214,135; U.S. Patent No. 5,525,719; Kawasaki et al. 1993. J. Med. Chem. 36:831; WO 92 / 03568; U.S. Patent No. 5,276,019; and U.S. Patent No. 5,264,423.

[0206] The synthetic method selected may depend on the length of the desired oligonucleotide, and such a selection is within the skill of a person skilled in the art. For example, phosphoramidite and phosphite triester methods can generate oligonucleotides with 175 or more nucleotides, while H-phosphonate methods work well for oligonucleotides with less than 100 nucleotides. When modified bases are incorporated into the oligonucleotide, and especially when modified phosphodiester linkages are used, synthetic procedures may be changed according to known procedures as necessary. In this regard, Uhlmann et al. (1990, Chemical Reviews 90:543-584) provide references and outline procedures for making oligonucleotides with modified bases and modified phosphodiester linkages. Other exemplary methods for making oligonucleotides are taught in Sonveaux. 1994. "Protecting Groups in Oligonucleotide Synthesis"; Agrawal. Methods in Molecular Biology 26:1. Exemplary synthesis methods are also taught in "Oligonucleotide Synthesis - A Practical Approach" (Gait, MJIR L Press at Oxford University Press. 1984). Moreover, linear oligonucleotides of defined sequence, including some sequences with modified nucleotides, are readily available from several commercial sources.

[0207] Oligonucleotides may be purified by polyacrylamide gel electrophoresis or by any of a number of chromatographic methods, including gel chromatography and high pressure liquid chromatography. To confirm the nucleotide sequence, particularly the unmodified nucleotide sequence, the oligonucleotides may be subjected to DNA sequencing by any of the known procedures, including Maxam-Gilbert sequencing, Sanger sequencing, capillary electrophoretic sequencing, wandering spot sequencing procedures, or by using selective chemical degradation of oligonucleotides bound to Hybond paper. The sequence of short oligonucleotides may also be analyzed by laser desorption mass spectrometry or fast atom bombardment (McNeal, et al., 1982, J. Am. Chem. Soc. 104:976; Viari, et al., 1987, Biomed. Environ. Mass Spectrom. 14:83; Grotjahn et al., 1982, Nuc. Acid Res. 10:4671). Sequencing methods are also available for RNA oligonucleotides.

[0208] The quality of the synthesized oligonucleotides can be confirmed by testing the oligonucleotides by capillary electrophoresis and denaturing strong anion HPLC (SAX-HPLC), for example, using the method of Bergot and Egan. 1992. J. Chrom. 599:35.

[0209] Other exemplary synthesis techniques are well known in the art (see, e.g., Sambrook et al., Molecular Cloning: a Laboratory Manual, Second Edition (1989); DNA Cloning, Vol umes I and II (DN Glover Ed. 1985); Oligonucleotide Synthesis (MJ Gait Ed, 1984); Nucleic Acid Hybridisation (BD Hames and SJ Higgins eds. 1984); A Practical Guide to Molecular Cloning (1984); or the series Methods in Enzymology (Academic Press, Inc.)).

[0210] In some embodiments, the target RNAi construct or at least a part thereof is transcribed from the expression vector that codes the target construct.For this purpose, any vector recognized in the art may be used.The transcribed RNAi construct may be isolated and purified before desired modification (such as replacing unmodified sense strand with modified one) is carried out.

[0211] Delivery / Carrier While not wishing to be bound by any particular theory, the inventors believe that the particular pattern of modifications on the passenger and guide strands of the double-stranded nucleic acid molecules described herein (e.g., INTASYL™) facilitates entry of the guide strand into the nucleus, where the guide strand mediates gene silencing (e.g., silencing of a target gene such as TIGIT). Without wishing to be bound by any theory, several potential mechanisms of action could explain this activity. For example, in some embodiments, the guide strand (e.g., antisense strand) of a nucleic acid molecule (e.g., INTASYL™) can dissociate from the passenger strand and enter the nucleus as a single strand. Once inside the nucleus, the single-stranded guide strand binds to RNAse H or another ribonuclease and cleaves the target (e.g., TIGIT) ("antisense mechanism of action"). In some embodiments, the guide strand (e.g., antisense strand) of a nucleic acid molecule (e.g., INTASYL™) can bind to an Argonaute (Ago) protein in the cytoplasm or outside the nucleus to form a loaded Ago complex. This loaded Ago complex can migrate to the nucleus and then cleave the target (e.g., TIGIT). In some embodiments, both strands (e.g., a duplex) of a nucleic acid molecule (e.g., INTASYL™) can enter the nucleus and the guide strand can bind to RNAse H, an Ago protein, or another ribonuclease, and cleave the target (e.g., TIGIT).

[0212] Those skilled in the art will appreciate that the sense strand of the double-stranded molecules described herein (e.g., the sense strand of INTASYL™) is not limited to delivery of the guide strand of the double-stranded nucleic acid molecules described herein. Rather, in some embodiments, the passenger strand described herein is attached (e.g., covalently, non-covalently, conjugated, hybridized via a region of complementarity, etc.) to another molecule (e.g., an antisense oligonucleotide, ASO) for the purpose of targeting the same molecule to the nucleus of a cell. In some embodiments, the molecule attached to the sense strand described herein is a synthetic antisense oligonucleotide (ASO). In some embodiments, the sense strand attached to the antisense oligonucleotide is 8-15 nucleotides in length, chemically modified, and includes a hydrophobic conjugate.

[0213] While not wishing to be bound by any particular theory, the ASO may be attached to the complementary passenger strand by hydrogen bonding. Thus, in some aspects, the disclosure provides a method of delivering a nucleic acid molecule to a cell, comprising administering to the cell an isolated nucleic acid molecule, wherein the isolated nucleic acid comprises a sense strand that is complementary to an antisense oligonucleotide (ASO), wherein the sense strand is 8-15 nucleotides in length and comprises at least two phosphorothioate modifications, wherein at least 50% of the pyrimidines in the sense strand are modified, and wherein the molecule comprises a hydrophobic conjugate.

[0214] Cellular uptake of oligonucleotides The oligonucleotides and oligonucleotide compositions are contacted (i.e., contacted or herein referred to as administered or delivered) with and incorporated into one or more cells or cell lysates. The term "cell" includes prokaryotic and eukaryotic cells, preferably vertebrate cells, more preferably mammalian cells. In some embodiments, the oligonucleotide compositions of the present invention are contacted with bacterial cells. In some embodiments, the oligonucleotide compositions of the present invention are contacted with eukaryotic cells (e.g., plant cells, mammalian cells, arthropod cells such as insect cells). In some embodiments, the oligonucleotide compositions of the present invention are contacted with stem cells. In some embodiments, the oligonucleotide compositions of the present invention are contacted with liver cells, such as hepatocytes, and incorporated via receptor-mediated uptake. In some embodiments, the oligonucleotide compositions of the present invention are contacted with human cells (e.g., neutrophils, T cells, B cells, monocytes / macrophages, and combinations thereof) or human cell lines (e.g., T cell lines, such as Jurkat cells).

[0215] The oligonucleotide composition of the present invention may be contacted with cells in vitro, for example in a test tube or culture dish (with or without being introduced into a subject), or in vivo, for example in a subject, such as a mammalian subject, or ex vivo. In some embodiments, the oligonucleotide is administered locally or through electroporation. Oligonucleotides are taken up into cells at a slow rate by endocytosis, but endocytosed oligonucleotides are generally sequestered and not available for hybridization to, for example, target nucleic acid molecules. In one embodiment, uptake by cells can be facilitated by electroporation or calcium phosphate precipitation. However, these procedures are only useful in in vitro or ex vivo embodiments, are not convenient, and in some cases are associated with cytotoxicity.

[0216] In another embodiment, delivery of oligonucleotides into cells can be enhanced by suitable, art-recognized methods including calcium phosphate, DMSO, glycerol or dextran, electroporation, or by transfection, e.g., using cationic, anionic or neutral lipid compositions or liposomes, using methods known in the art (see, e.g., WO90 / 14074; WO91 / 16024; WO91 / 17424; U.S. Patent No. 4,897,355; Bergan et al. 1993. Nucleic Acid Research. 21:3567). Enhanced delivery of oligonucleotides can also be mediated through the use of vectors (see, e.g., Shi, Y. 2003. Trends Genet 2003 Jan. 19:9; Reichhart JM et al. Genesis. 2002. 34(1-2):1604, Yu et al. 2002. Proc. Natl. Acad Sci. USA 99:6047; Sui et al. 2002. Proc. Natl. Acad Sci. USA 99:5515), viruses, polyamines or polycation conjugates using compounds such as polylysine, protamine or Ni, N12-bis(ethyl)spermine (see, e.g., Bartzatt, R. et al. 1989. Biotechnol. Appl. Biochem. 11:133; Wagner E. et al. 1992. Proc. Natl. Acad. Sci. 88:4255).

[0217] The protocol used for uptake of oligonucleotides will depend on a number of factors, the most important of which is the type of cells used. Other factors that are important in uptake include, but are not limited to, the nature and concentration of the oligonucleotide, the confluence of the cells, the type of culture in which the cells are placed (e.g., suspension culture or plate culture), and the type of medium in which the cells are cultured.

[0218] Immunogenic compositions and methods for producing same In some embodiments, the chemically modified double-stranded nucleic acid molecules described herein are useful for producing immunogenic compositions.As used herein, "immunogenic composition" refers to a composition that comprises a host cell that comprises the chemically modified nucleic acid molecules described herein, and optionally one or more pharma-ceutically acceptable excipients or carriers.Without wishing to be bound by any particular theory, the immunogenic composition as described by the present disclosure is characterized by the reduction (e.g., inhibition) of the expression or activity of one or more immune checkpoint proteins (e.g., TIGIT), and thus, in some embodiments, is useful for stimulating the immune response of a subject.

[0219] As used herein, a "host cell" is a cell into which one or more chemically modified double-stranded nucleic acid molecules are introduced. Typically, the host cell is a mammalian cell, such as a human cell, a mouse cell, a rat cell, a pig cell, etc. However, in some embodiments, the host cell is a non-mammalian cell, such as a prokaryotic cell (e.g., a bacterial cell), a yeast cell, an insect cell, etc. In general, the host cell is derived from a donor, such as a healthy donor (e.g., the cell into which the chemically modified double-stranded nucleic acid is introduced is taken from a donor, such as a healthy donor). For example, the cell may be isolated from a biological sample, such as bone marrow or blood, obtained from a donor, such as a healthy donor. As used herein, a "healthy donor" refers to a subject that does not have or is not suspected of having a proliferative disorder or an infectious disease (e.g., a bacterial infection, a viral infection, or a parasitic infection). However, in some embodiments, the host cell is derived from a subject that has (or is suspected of having) a proliferative disorder or an infectious disease, for example, in the context of autologous cell therapy.

[0220] In some embodiments, the cell (e.g., a host cell) is an immune cell, such as a T cell, a B cell, a dendritic cell (DC), a granulocyte, a macrophage, etc. In some embodiments, the cell (e.g., a host cell) is a cell that can differentiate into an immune cell, such as a stem cell (SC) or an induced pluripotent stem cell (iPSC). In some embodiments, the cell (e.g., a host cell) is a stem cell memory T cell, e.g., as described in Gattinoni et al. (2017) Nature Medicine 23;18-27, incorporated by reference therein.

[0221] In some embodiments, the cell (e.g., a host cell) is a T cell, such as a killer T cell, a helper T cell, or a regulatory T cell. In some embodiments, the T cell is a killer T cell (e.g., a CD8+ T cell). In some embodiments, the T cell is a helper T cell (e.g., a CD4+ T cell). In some embodiments, the T cell is an activated T cell (e.g., a T cell in which a peptide antigen is presented by an MHC class II molecule on an antigen-presenting cell). In some embodiments, the T cells comprise one or more transgenes expressing a high affinity T cell receptor (TCR) and / or a chimeric antibody receptor (CAR).

[0222] In some aspects, the present disclosure relates to the discovery that introducing one or more chemically modified double-stranded nucleic acid molecules of the present disclosure into a cell (e.g., an immune cell obtained from a donor) to produce a host cell results in a significant decrease in expression or activity of an immune checkpoint protein (e.g., TIGIT) in the host cell. In some embodiments, the host cell is characterized by about 5% to about 50% decreased expression of the immune checkpoint protein compared to a cell (e.g., an immune cell of the same cell type) that does not contain the chemically modified double-stranded nucleic acid molecule. In some embodiments, the host cells are characterized by expression of an immune checkpoint protein that is reduced by more than 50% (e.g., 51%, 52%, 53%, 54%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or any percentage between 51% and 100%) compared to cells (e.g., immune cells of the same cell type) that do not contain the chemically modified double-stranded nucleic acid molecule (e.g., immune cells of a subject having or suspected of having a proliferative or infectious disease).

[0223] In some embodiments, the immunogenic compositions described by the present disclosure include a plurality of host cells. In some embodiments, the plurality of host cells is about 10,000 host cells (e.g., 10,000 host cells / kg of subject), about 50,000 host cells (e.g., 50,000 host cells / kg of subject), about 100,000 host cells (e.g., 100,000 host cells / kg of subject), about 250,000 host cells (e.g., 250,000 host cells / kg of subject), about 500,000 host cells (e.g., 500,000 host cells / kg of subject), about 1x10 6 Host cells (e.g., about 1x10 6 host cells / kg of subject), approximately 5x10 6 Host cells (e.g., 5x10 6 host cells / kg of subject), approximately 1x10 7 Host cells (e.g., 1x10 7 host cells / kg of subject), approximately 1x10 8 Host cells (e.g., 1x10 8host cells / kg of subject), approximately 1x10 9 Host cells (e.g., 1x10 9 kg of host cells / subject), or 1x10 9 More than 1x10 host cells (e.g., 9 In some embodiments, the number of host cells is greater than about 1x10 5 and 1x10 14 Between the host cell (for example, 1x10 5 and 1x10 14 (between 10 and 15 kg of host cells / kg of subject).

[0224] In some aspects, the present disclosure provides a method for producing the immunogenic composition described in the present disclosure.In some embodiments, the method includes introducing one or more chemically modified double-stranded nucleic acid molecules into a cell, wherein the one or more chemically modified double-stranded nucleic acid molecules target TIGIT, thereby producing a host cell. The method of producing the immunogenic composition (e.g., producing a host cell) may be performed in vitro, ex vivo, or in vivo, e.g., in mammalian cells in culture, such as human cells in culture. In some embodiments, the target cells (e.g., cells obtained from a donor) may be contacted in the presence of a delivery reagent, such as a lipid (e.g., a cationic lipid) or a liposome, to facilitate entry of the chemically modified double-stranded nucleic acid molecule into the cell, as described in more detail elsewhere in this disclosure.

[0225] In some embodiments, the method of producing an immunogenic composition (e.g., producing a host cell) may further include subjecting the host cell (e.g., the cell containing one or more chemically modified double-stranded nucleic acid molecules) to a washing period. As used herein, "washing period" refers to a period during which the host cell is suspended in a washing medium (a medium that does not contain one or more chemically modified double-stranded nucleic acid molecules). In some embodiments, the washing period is 24 hours to 6 days (e.g., 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, 120 hours, 136 hours, or 144 hours). In some embodiments, the washing period is 24 hours. In some embodiments, the washing period is 48 hours. In some embodiments, the washing period is 96 hours. In some embodiments, the washing period is 6 days. In some embodiments, the method of producing an immunogenic composition (e.g., producing a host cell) does not include subjecting the host cell (e.g., a cell comprising one or more chemically modified double-stranded nucleic acid molecules) to a washing period.

[0226] Carriers and Complexing Agents The present disclosure further relates to a composition comprising the RNAi constructs described herein and a pharma- ceutically acceptable carrier or diluent. In some aspects, the present disclosure relates to an immunogenic composition comprising the RNAi constructs described herein and a pharma- ceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes suitable solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. The use of such media and agents for pharmaceutical active substances is well known in the art. Any conventional media or agent can be used in the therapeutic composition, except in the case where it is incompatible with the active ingredient. Auxiliary active ingredients can also be incorporated into the composition.

[0227] For example, in some embodiments, oligonucleotides may be incorporated into liposomes, or liposomes modified with polyethylene glycol or mixed with cationic lipids, for parenteral administration. The incorporation of additional substances into liposomes, such as antibodies reactive to membrane proteins found on specific target cells, can help target oligonucleotides to specific cell types (e.g., immune cells such as NK cells). The encapsulating agent traps the oligonucleotide in the vesicle. In another embodiment of the present invention, the oligonucleotide may be associated with a carrier or vehicle, such as a liposome or micelle, but it will be understood by those skilled in the art that other carriers can be used. Liposomes are vesicles that are composed of a lipid bilayer with a structure similar to that of biological membranes. Such carriers are used to promote cellular uptake, or to target the oligonucleotide, or to improve the pharmacokinetics or toxicological properties of the oligonucleotide.

[0228] For example, the oligonucleotide of the present invention may also be encapsulated and administered in liposomes, which are pharmaceutical compositions in which active ingredient is contained in a dispersed form or in a body that is composed of an aqueous-concentrated layer attached to a lipid layer.Depending on solubility, oligonucleotides may be present in both the aqueous layer and the lipid layer, or in what is generally called liposomal suspension.The hydrophobic layer generally, but not necessarily, contains phospholipids such as lecithin and sphingomyelin, steroids such as cholesterol, more or less ionic surfactants such as diacetyl phosphate, stearylamine or phosphatidic acid, or other materials of hydrophobic nature.The diameter of liposomes generally ranges from approximately 15 nm to about 5 microns.

[0229] The use of liposomes as drug delivery vehicles offers several advantages. Liposomes increase intracellular stability, increase uptake efficiency, and improve biological activity. Liposomes are hollow spherical vesicles that are composed of lipids arranged in a manner similar to the lipids that compose cell membranes. They have an internal aqueous space that encapsulates water-soluble compounds, and range in size from 0.05 to several microns in diameter. Several studies have shown that liposomes can deliver nucleic acids to cells, and that the nucleic acids remain biologically active. For example, lipid delivery vehicles originally designed as research tools, such as lipofectin or LIPOFECTAMINE™ 2000, can deliver intact nucleic acid molecules to cells. Specific advantages of using liposomes include: they are non-toxic and biodegradable in composition; they exhibit long circulatory half-lives; and recognition molecules can be easily attached to their surface for targeting to tissues. Finally, the cost-effective production of liposome-based pharmaceuticals, either in liquid suspensions or lyophilized products, demonstrates the viability of this technology as an acceptable drug delivery system.

[0230] In some aspects, the formulations related to the present invention may be selected for the class of naturally occurring or chemically synthesized or modified saturated and unsaturated fatty acid residues.Fatty acids may be present in the form of triglycerides, diglycerides or individual fatty acids.In another embodiment, the use of well-validated mixtures of fatty acids and / or lipid emulsions currently used in pharmacology for parenteral nutrition may be utilized. Liposome-based formulations are widely used for the delivery of oligonucleotides. However, most of the commercially available lipid or liposome formulations contain at least one positively charged lipid (e.g., cationic lipid). The presence of this positively charged lipid is considered essential to obtain high levels of oligonucleotide loading and to enhance the membrane fusion properties of liposomes. Several methods have been implemented and published to identify functional positively charged lipid compounds. However, commercially available liposome formulations containing cationic lipids are characterized by high levels of toxicity. With limited therapeutic index in vivo, it has been revealed that liposome formulations containing positively charged lipids are associated with toxicity (e.g., increased liver enzymes) at concentrations only slightly higher than those required to achieve RNA silencing.

[0231] The nucleic acids related to the present invention can be hydrophobically modified and can be included in neutral nanotransporters. Further description of neutral nanotransporters is incorporated by reference from PCT application PCT / US2009 / 005251, entitled "Neutral Nanotransporters", filed on September 22, 2009. Such particles allow quantitative incorporation of oligonucleotides into uncharged lipid mixtures. The lack of toxicity levels of cationic lipids in such neutral nanotransporter compositions is an important property.

[0232] As demonstrated in PCT / US2009 / 005251, oligonucleotides can be efficiently incorporated into lipid mixtures that do not have cationic lipids, and such compositions can effectively deliver therapeutic oligonucleotides to cells in a functional manner.For example, when lipid mixtures are composed of phosphatidylcholine-based fatty acids and sterols such as cholesterol, high levels of activity have been observed.As an example, one preferred formulation of neutral lipid mixtures is composed of at least 20% DOPC or DSPC and at least 20% sterols such as cholesterol.It has been shown that lipid to oligonucleotide ratios as low as 1:5 are sufficient to obtain complete encapsulation of oligonucleotides in uncharged formulations.

[0233] The neutral nanotransporter composition allows for the efficient loading of oligonucleotides into neutral lipid formulations. The composition includes oligonucleotides that are modified in such a way that the hydrophobicity of the molecule is increased (e.g., hydrophobic molecules are attached (covalently or non-covalently) to the oligonucleotide terminal or non-terminal nucleotide, base, sugar or backbone), and the modified oligonucleotides are mixed with neutral lipid formulations (e.g., containing at least 25% cholesterol and 25% DOPC or its analogs). Cargo molecules such as other lipids can also be included in the composition. This composition allows for the efficient encapsulation of oligonucleotides in neutral lipid particles when part of the formulation is constructed in the oligonucleotide itself.

[0234] In some aspects, stable particles ranging in size from 50 to 140 nm can be formed by complexing hydrophobic oligonucleotides with the preferred formulations, which by themselves typically do not form small particles, but rather form agglomerates that are converted to stable 50-120 nm particles by addition of hydrophobically modified oligonucleotides. In some embodiments, the neutral nanotransporter composition comprises a hydrophobically modified polynucleotide, a neutral lipid mixture, and optionally a cargo molecule. As used herein, a "hydrophobically modified polynucleotide" is a polynucleotide of the present invention (e.g., sd-rxRNA) that has at least one modification that makes the polynucleotide more hydrophobic than before the modification of the polynucleotide. The modification may be achieved by attaching (covalently or non-covalently) a hydrophobic molecule to the polynucleotide. In some examples, the hydrophobic molecule is or includes a lipophilic group.

[0235] The term "lipophilic group" refers to a group that has an affinity for lipids that is greater than its affinity for water. Examples of lipophilic groups include, but are not limited to, cholesterol, cholesteryl or modified cholesteryl residues, adamantine, dihydrotesterone (dihyd), and the like. drotesterone), long chain alkyl, long chain alkenyl, long chain alkynyl, oleyl-lithocholic acid, cholenic acid, oleoyl-cholenic acid, palmitic acid, heptadecylic acid, myristic acid, bile acid, cholic acid or taurocholic acid, deoxycholic acid, oleyllithocholic acid, oleoylcholenic acid, glycolipids, phospholipids, sphingolipids, isoprenoids such as steroids, vitamins such as vitamin E, fatty acids either saturated or unsaturated, fatty acid esters such as triglycerides, pyrenes, porphyrins, texaphyrin, adamantane, acridines, biotin, coumarin, fluorescein, rhodamine, Texas-Red, digoxigenin, dimethoxytrityl, t-butyldimethylsilyl, t-butyldiphenylsilyl, cyanine dyes (e.g., Cy3 or Cy5), Hoechst 33258 dyes, psoralen or ibuprofen. The cholesterol moiety may be reduced (eg, as in cholestane) or substituted (eg, by halogen). A combination of different lipophilic groups in one molecule is also possible.

[0236] The hydrophobic molecule may be attached at various positions of the polynucleotide. As mentioned above, the hydrophobic molecule may be linked to the terminal residue of the polynucleotide, such as the 3' or 5' end of the polynucleotide. Alternatively, it may be linked to an internal nucleotide or a nucleotide on a branch of the polynucleotide. The hydrophobic molecule may be attached to the 2' position of the nucleotide, for example. The hydrophobic molecule may also be linked to the heterocyclic base, sugar or backbone of the nucleotide of the polynucleotide.

[0237] The hydrophobic molecule may be linked to the polynucleotide by a linker moiety. Optionally, the linker moiety is a non-nucleotidic linker moiety. The non-nucleotidic linker is, for example, an abasic residue (d spacer), an oligoethylene glycol such as triethylene glycol (spacer 9) or hexaethylene glycol (spacer 18), or an alkanediol such as butanediol. The spacer units are preferably linked by phosphodiester or phosphorothioate bonds. The linker units may occur only once in the molecule or may be incorporated several times, for example, via phosphodiester, phosphorothioate, methylphosphonate or amine linkages.

[0238] A typical conjugation protocol involves the synthesis of a polynucleotide carrying an amino linker at one or more positions of the sequence, however, a linker is not required. The amino group is then reacted with the molecule to be conjugated using an appropriate coupling or activation reagent. The conjugation reaction may be carried out with the polynucleotide still attached to a solid support or following cleavage of the polynucleotide in solution phase. Purification of the modified polynucleotide by HPLC typically results in a pure material.

[0239] In some embodiments, the hydrophobic molecule is a sterol-type conjugate, a phytosterol-conjugate, a cholesterol-conjugate, a sterol-type conjugate of varying side chain length, a fatty acid conjugate, any other hydrophobic group conjugate, and / or a hydrophobic modification of an internal nucleoside that provides sufficient hydrophobicity for incorporation into a micelle.

[0240] For the purposes of the present invention, the term "sterol" or steroid alcohols refers to a subgroup of steroids that have a hydroxyl group at the 3-position of the A ring. These are amphipathic lipids synthesized from acetyl-coenzyme A via the HMG-CoA reductase pathway. The overall molecule is quite flat. The hydroxyl group on the A ring is polar. The remainder of the aliphatic chain is non-polar. Sterols are usually considered to have an 8-carbon chain at the 17-position.

[0241] For purposes of the present invention, the term "sterol-type molecules" refers to steroid alcohols, which are structurally similar to sterols. The main differences are the ring structure and the number of carbons in the side chain attached at the 21st position. For purposes of the present invention, the term "phytosterols" (also referred to as plant sterols) is a group of steroidal alcohols that are phytochemicals that occur naturally in plants. Over 200 phytosterols are known.

[0242] For the purposes of the present invention, the term "sterol side chain" refers to the chemical composition of the side chain attached at the 17-position of a sterol-type molecule. In the standard definition, sterols are limited to a four-ring structure carrying an eight-carbon chain at the 17-position. In the present invention, sterol-type molecules with longer and shorter side chains than conventional ones are described. The side chains may be branched or contain a double backbone. Thus, sterols useful in the present invention include, for example, cholesterol, as well as unique sterols with side chains longer than 2-7 or 9 carbons attached at position 17. In some embodiments, the length of the polycarbon tail varies between 5-9 carbons. Such conjugates may have significantly better in vivo efficacy, especially in delivery to the liver. These types of molecules are expected to work at 5-9 fold lower concentrations than conventional cholesterol-conjugated oligonucleotides.

[0243] Alternatively, the polynucleotide may be bound to a protein, peptide, or positively charged chemical that acts as a hydrophobic molecule.The protein may be selected from the group consisting of protamine, dsRNA binding domain, and arginine-rich peptide.Exemplary positively charged chemicals include spermine, spermidine, cadaverine, and putrescine. In another embodiment, hydrophobic molecule conjugates may demonstrate even greater potency when combined with specific chemical modification patterns of polynucleotides, including but not limited to hydrophobic modifications, phosphorothioate modifications, and 2' ribo modifications (as described in detail herein).

[0244] In another embodiment, the sterol type molecule can be a naturally occurring phytosterol.The polycarbon chain can be longer than 9, linear, branched, and / or contain double bonds.Some phytosterols containing polynucleotide conjugates can be significantly more potent and active in delivering polynucleotide to various tissues.Some phytosterols can demonstrate tissue selectivity, and can therefore be used as a means to deliver RNAi specifically to certain tissues.

[0245] The hydrophobically modified polynucleotide is mixed with a neutral fatty acid mixture to form a micelle. The neutral fatty acid mixture is a mixture of lipids that have a net neutral or slight net negative charge at or near physiological pH that can form micelles with the hydrophobically modified polynucleotide. For the purposes of the present invention, the term "micelle" refers to a small nanoparticle formed by a mixture of uncharged fatty acids and phospholipids. The neutral fatty acid mixture may contain cationic lipids, so long as they are present in an amount that does not cause toxicity. In some embodiments, the neutral fatty acid mixture does not contain cationic lipids. A mixture that does not contain cationic lipids is one in which less than 1%, preferably 0%, of the total lipids are cationic lipids. The term "cationic lipid" includes lipids and synthetic lipids that have a net positive charge at or near physiological pH. The term "anionic lipid" includes lipids and synthetic lipids that have a net negative charge at or near physiological pH.

[0246] Neutral lipids bind to the oligonucleotides of the invention through strong, but non-covalent, attractive forces (e.g., electrostatic, van der Waals, pi-stacking, etc. interactions). Neutral lipid mixture may comprise a preparation selected from the class of naturally occurring or chemically synthesized or modified saturated and unsaturated fatty acid residues.Fatty acids may be present in the form of triglycerides, diglycerides or individual fatty acids.In another embodiment, well-defined mixtures of fatty acids and / or lipid emulsions currently used in pharmacology for parenteral nutrition may be utilized.

[0247] The neutral fatty acid mixture is preferably a mixture of choline-based fatty acids and sterols. Choline-based fatty acids include, for example, synthetic phosphocholine derivatives such as DDPC, DLPC, DMPC, DPPC, DSPC, DOPC, POPC and DEPC. DOPC (compound registry number 4235-95-4) is dioleoylphosphatidylcholine (also known as dielaidoylphosphatidylcholine, dioleoyl-PC, dioleoylphosphocholine, dioleoyl-sn-glycero-3-phosphocholine, dioleylphosphatidylcholine). DSPC (compound registry number 816-94-4) is distearoylphosphatidylcholine (also known as 1,2-distearoyl-sn-glycero-3-phosphocholine).

[0248] The sterol in the neutral fatty acid mixture may be, for example, cholesterol. The neutral fatty acid mixture may be entirely composed of choline-based fatty acids and sterols, or it may optionally include a cargo molecule. For example, the neutral fatty acid mixture may have at least 20% or 25% fatty acids and 20% or 25% sterols.

[0249] For the purposes of the present invention, the term "fatty acid" refers to the conventional description of fatty acids. They may exist as individual entities or in the form of diglycerides and triglycerides. For the purposes of the present invention, the term "lipid emulsion" refers to a safe lipid formulation given intravenously to subjects who are unable to obtain sufficient lipids from their diet. It is an emulsion of soybean oil (or other naturally occurring oils) and egg phospholipids. Lipid emulsions have been used for the formulation of some insoluble anesthetic agents. In the present disclosure, lipid emulsions may be part of commercial formulations such as Intralipid, Liposyn, Nutrilipid, modified commercial formulations enriched with specific fatty acids, or entirely new formulated combinations of fatty acids and phospholipids.

[0250] In one embodiment, the cells contacted with the oligonucleotide composition of the present invention are contacted with a mixture comprising an oligonucleotide and a mixture comprising a lipid, such as one of the lipids or lipid compositions described above, for about 12 hours to about 24 hours. In another embodiment, the cells contacted with the oligonucleotide composition are contacted with a mixture comprising an oligonucleotide and a mixture comprising a lipid, such as one of the lipids or lipid compositions described above, for about 1 to about 5 days. In one embodiment, the cells are contacted with a mixture comprising a lipid and an oligonucleotide for as long as about 3 days to about 30 days. In another embodiment, the mixture comprising a lipid is left in contact with the cells for at least about 5 to about 20 days. In another embodiment, the mixture comprising a lipid is left in contact with the cells for at least about 7 to about 15 days.

[0251] 50%-60% of the formulation may optionally be any other lipid or molecule. Such lipid or molecule is referred to herein as cargo lipid or cargo molecule. Cargo molecules include, but are not limited to, intralipids, small molecules, fusogenic peptides or lipids, or other small molecules may be added to alter cellular uptake, endosomal release, or tissue distribution properties. The ability to tolerate cargo molecules is important for modifying the properties of these particles, if such properties are desired. As an example, the presence of some tissue-specific metabolites may significantly alter the tissue distribution profile. For example, the use of intralipid-type formulations enriched with shorter or longer lipid chains with various saturation levels will affect the tissue distribution profile (and their loading) of these types of formulations.

[0252] An example of a cargo lipid useful according to the present invention is a membrane fusogenic lipid. For example, the zwitterionic lipid DOPE (compound registry number 4004-5-1, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) is a preferred cargo lipid. Intralipid may be composed of the following composition: 1000 mL contains: 90 g refined soybean oil, 12 g refined egg phospholipids, 22 g anhydrous glycerol, water for injection (sufficient for 1000 mL). The pH is adjusted to about pH 8 with sodium hydroxide. Energy content / L: 4.6 MJ (190 kcal). Osmolality (approximate): 300 mOsm / kg water. In another embodiment, the lipid emulsion is Liposyn, which contains 5% safflower oil, 5% soybean oil, up to 1.2% egg phospholipids added as an emulsifier, and 2.5% glycerin in water for injection. It may also contain sodium hydroxide for pH adjustment. pH 8.0 (6.0-9.0). Liposyn has an osmolality of 276 mOsmol / liter (actual value).

[0253] Variations in cargo lipid identity, amount and ratio affect the cellular uptake and tissue distribution characteristics of these compounds.For example, the length and saturation level of lipid tail affect different uptake in liver, lung, fat and cardiomyocytes.Addition of special hydrophobic molecules such as vitamins or different forms of sterols can favor distribution to special tissues involved in the metabolism of certain compounds.In some embodiments, vitamin A or E is used.Complexes are formed at different oligonucleotide concentrations, with higher concentrations favoring more efficient complex formation.

[0254] In another embodiment, the lipid emulsion is based on a mixture of lipids. Such lipids may include natural compounds, chemically synthesized compounds, purified fatty acids, or any other lipids. In yet another embodiment, the composition of the lipid emulsion is completely artificial. In a particular embodiment, the lipid emulsion is more than 70% linoleic acid. In yet another particular embodiment, the lipid emulsion is at least 1% cardiolipin. Linoleic acid (LA) is an unsaturated omega-6 fatty acid. It is a colorless liquid consisting of a carboxylic acid with an 18-carbon chain and two cis double bonds. In yet another embodiment of the invention, alteration of the composition of lipid emulsions is used as a means to alter the tissue distribution of hydrophobically modified polynucleotides. This methodology results in specific delivery of polynucleotides to specific tissues.

[0255] In another embodiment, the lipid emulsion of the cargo molecule contains more than 70% linoleic acid (C 18 H 32 O2) and / or cardiolipin. Lipid emulsions such as intralipid have been previously used as a delivery formulation for some water-insoluble drugs, such as Propofol (reformulated as Diprivan). The unique features of the present invention include (a) the concept of combining modified polynucleotides with one or more hydrophobic compound(s) so that they can be incorporated into lipid micelles, and (b) mixing them with lipid emulsions to provide a reversible carrier. After injection into the bloodstream, micelles typically bind to albumin, HDL, LDL and other serum proteins. This binding is reversible, and the lipids are eventually absorbed by cells. The polynucleotides incorporated as part of the micelles will then be delivered near the surface of the cells. Cellular uptake can then occur through a variety of mechanisms, including but not limited to sterol-type delivery.

[0256] The complexing agent binds to the oligonucleotide of the present invention by strong but non-covalent attractive forces (e.g., electrostatic, van der Waals, pi-stacking, etc. interactions). In one embodiment, the oligonucleotide of the present invention can be complexed with a complexing agent to increase cellular uptake of the oligonucleotide. Examples of complexing agents include cationic lipids. Cationic lipids can be used to deliver oligonucleotides to cells. However, as noted above, formulations that do not include cationic lipids are preferred in some embodiments.

[0257] The term "cationic lipid" includes lipids and synthetic lipids with both polar and non-polar domains that can be positively charged at or near physiological pH and bind to polyanions such as nucleic acids to facilitate delivery of the nucleic acid into cells. In general, cationic lipids include saturated and unsaturated alkyl and alicyclic ethers and amine esters, amides, or derivatives thereof. The linear and branched alkyl and alkenyl groups of the cationic lipids can contain, for example, 1 to about 25 carbon atoms. Preferred linear or branched alkyl or alkene groups have 6 or more carbon atoms. Alicyclic groups include cholesterol and other steroid groups. Cationic lipids include, for example, Cl. - , Br - , I - , F - They can be prepared with a variety of counterions (anions), including acetate, trifluoroacetate, sulfate, nitrite, and nitrate.

[0258] Examples of cationic lipids include polyethyleneimine, polyamidoamine (PAMAM) starburst dendrimers, Lipofectin (a combination of DOTMA and DOPE), Lipofectase, LIPOFECTAMINE™ (e.g., LIPOFECTAMINE™ 2000), DOPE, Cytofectin (Gilead Sciences, Foster City, Calif.), and Eufectins (JBL, San Luis Obispo, Calif.).

[0259] Exemplary cationic liposomes can be prepared from N-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA), N-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP), 3β-[N-(N′,N′-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol), 2,3,-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide; and dimethyldioctadecylammonium bromide (DDAB). For example, the cationic lipid N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA) was found to increase the antisense effect of phosphorothioate oligonucleotides by 1000-fold (Vlassov et al., 1994, Biochimica et Biophysica Acta 1197:95-108). The oligonucleotides may also be complexed, for example, with poly(L-lysine) or avidin, with or without lipids in the mixture, for example, steryl-poly(L-lysine).

[0260] Cationic lipids have been used in the art to deliver oligonucleotides to cells (see, for example, U.S. Patent Nos. 5,855,910; 5,851,548; 5,830,430; 5,780,053; 5,767,099; Lewis et al. 1996. Proc. Natl. Acad. Sci. USA 93:3176; Hope et al. 1998. Molecular Membrane Biology 15:1). Other lipid compositions that can be used to facilitate the uptake of the present oligonucleotides can be used in combination with the claimed method. In addition to those listed above, other lipid compositions are also known in the art, including those taught in, for example, U.S. Patent Nos. 4,235,871; 4,501,728; 4,837,028; 4,737,323. In one embodiment, the lipid composition can further comprise an agent, such as a viral protein, to enhance lipid-mediated transfection of oligonucleotides (Kamata, et al., 1994. Nucl. Acids. Res. 22:536). In another embodiment, the oligonucleotide is contacted with cells as part of a composition comprising an oligonucleotide, a peptide and a lipid, for example as taught in U.S. Pat. No. 5,736,392. Improved lipids that are serum-resistant have also been described (Lewis, et al., 1996. Proc. Natl. Acad. Sci. 93:3176). Cationic lipids and other complexing agents act to increase the number of oligonucleotides that are transported into cells through endocytosis.

[0261] In another embodiment, N-substituted glycine oligonucleotides (peptoids) can be used to improve the uptake of oligonucleotides. Peptoids have been used to create cationic lipid-like compounds for transfection (Murphy, et al., 1998. Proc. Natl. Acad. Sci. 95:1517). Peptoids can be synthesized using standard methods (e.g. RN, et al. 1992. J. Am. Chem. Soc. 114:10646; Zuckermann, RN, et al. 1992. Int. J. Peptide Protein Res. 40:497). Liptoids, which are a combination of cationic lipids and peptoids, can also be used to improve the uptake of oligonucleotides of interest (Hunag, et al., 1998. Chemistry and Biology. 5:345). Liptoids can be synthesized by producing a peptoid oligonucleotide and coupling the amino-terminal submonomer to a lipid through its amino group (Hunag, et al., 1998. Chemistry and Biology. 5:345).

[0262] It is known in the art that positively charged amino acids can be used to make highly active cationic lipids (Lewis et al. 1996. Proc. Natl. Acad. Sci. USA 93:3176). In one embodiment, the composition for delivering the oligonucleotide of the present invention comprises a number of arginine, lysine, histidine or ornithine residues linked to lipophilic moieties (see, for example, U.S. Patent No. 5,777,153).

[0263] In another embodiment, the composition for delivering oligonucleotides of the invention comprises a peptide having about 1 to about 4 basic residues. These basic residues can be located, for example, at the amino terminus, C-terminus or internal region of the peptide. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine (also considered to be non-polar), asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Besides the basic amino acids, the majority or all of the other residues of the peptide may be selected from non-basic amino acids, e.g., from amino acids other than lysine, arginine or histidine. Preferably, neutral amino acids with long neutral side chains are predominately used.

[0264] In one embodiment, the composition for delivering the oligonucleotide of the present invention comprises a natural or synthetic polypeptide having one or more gamma carboxyglutamic acid residues or γ-Gla residues.These gamma carboxyglutamic acid residues allow the polypeptides to bind to each other and to membrane surfaces.In other words, the polypeptide having a series of γ-Gla can be used as a universal delivery modality to help RNAi constructs to stick to whatever membrane they come into contact with.This can delay the clearance of RNAi constructs from bloodstream and enhance their chances of homing to targets.

[0265] Gamma carboxyglutamic acid residues may be present in naturally occurring proteins (e.g., prothrombin has 10 γ-Gla residues). Alternatively, they may be introduced into purified, recombinantly produced, or chemically synthesized polypeptides by carboxylation, e.g., using vitamin K-dependent carboxylase. Gamma carboxyglutamic acid residues may be contiguous or non-contiguous, and the total number and location of such gamma carboxyglutamic acid residues in a polypeptide may be controlled / tweaked to achieve different levels of "stickiness" of the polynucleotide.

[0266] In one embodiment, the cells contacted with the oligonucleotide composition of the present invention are contacted with a mixture comprising an oligonucleotide and a mixture comprising a lipid, such as one of the lipids or lipid compositions described above, for about 12 hours to about 24 hours. In another embodiment, the cells contacted with the oligonucleotide composition are contacted with a mixture comprising an oligonucleotide and a mixture comprising a lipid, such as one of the lipids or lipid compositions described above, for about 1 to about 5 days. In one embodiment, the cells are contacted with a mixture comprising a lipid and an oligonucleotide for as long as about 3 days to about 30 days. In another embodiment, the mixture comprising a lipid is left in contact with the cells for at least about 5 to about 20 days. In another embodiment, the mixture comprising a lipid is left in contact with the cells for at least about 7 to about 15 days.

[0267] For example, in one embodiment, the oligonucleotide composition may be contacted with cells for an extended incubation period as described herein in the presence of a lipid such as cytofectin CS or GSV (available from Glen Research; Sterling, Va.), GS3815, GS2888.

[0268] In one embodiment, incubation of the cells with a mixture comprising lipid and oligonucleotide composition does not reduce the viability of the cells. Preferably, after the transfection period, the cells are substantially viable. In one embodiment, after the transfection, the cells are at least about 70% to at least about 100% viable. In another embodiment, the cells are at least about 80% to at least about 95% viable. In yet another embodiment, the cells are at least about 85% to at least about 90% viable. In one embodiment, the oligonucleotide is modified by attaching a peptide sequence that transports the oligonucleotide into a cell, referred to herein as a "transport peptide." In one embodiment, a composition comprises an oligonucleotide that is complementary to a target nucleic acid molecule encoding a protein and a covalently attached transport peptide.

[0269] The term "transport peptide" includes amino acid sequences that facilitate the transport of oligonucleotides into cells.Exemplary peptides that facilitate the transport of the moiety to which they are linked into cells are known in the art, and include, for example, HIV TAT transcription factor, lactoferrin, herpes VP22 protein, and fibroblast growth factor 2 (Pooga et al. 1998. Nature Biotechnology. 16:857; and Derossi et al. 1998. Trends in Cell Biology. 8:84; Elliott and O'Hare. 1997. Cell 88:223). The oligonucleotides can be attached to the transport peptide using known techniques (e.g., Prochiantz, A. 1996. Curr. Opin. Neurobiol. 6:629; Derossi et al. 1998. Trends Cell Biol. 8:84; Troy et al. 1996. J. Neurosci. 16:253; Vives et al. 1997. J. Biol. Chem. 272:16010). For example, in one embodiment, an oligonucleotide bearing an activated thiol group is linked through its thiol group to a cysteine ​​present in a transport peptide (e.g., to a cysteine ​​present in the β-turn between the second and third helices of the Antennapedia homeodomain, as taught in, for example, Derossi et al. 1998. Trends Cell Biol. 8:84; Prochiantz. 1996. Current Opinion in Neurobiol. 6:629; Allinquant et al. 1995. J Cell Biol. 128:919). In another embodiment, a Boc-Cys-(Npys)OH group can be coupled to a transport peptide, such that an oligonucleotide bearing the last (N-terminal) amino acid and an SH group can be coupled to the peptide (Troy et al. 1996. J. Neurosci. 16:253).

[0270] In one embodiment, a linking group can be attached to the nucleomonomer and the transport peptide can be covalently attached to the linker. In one embodiment, the linker can function both as a binding site for the transport peptide and can provide stability against nucleases. Examples of suitable linkers include substituted or unsubstituted C1-C 20 Alkyl chain, C2-C 20 Alkenyl chain, C2-C 20They include alkynyl chains, peptides, and heteroatoms (e.g., S, O, NH, etc.). Other exemplary linkers include bifunctional crosslinkers such as sulfosuccinimidyl-4-(maleimidophenyl)-butyrate (SMPB) (see, e.g., Smith et al. Bioch em J 1991.276:417-2).

[0271] In one embodiment, the oligonucleotides of the invention are synthesized as molecular conjugates that utilize receptor-mediated endocytosis mechanisms for delivery of genes into cells (see, e.g., Bunnell et al. 1992. Somatic Cell and Molecular Genetics. 18:559 and references cited therein). Other carriers for delivery of RNAi reagents in vitro and / or in vivo are known in the art and can be used to deliver the RNAi construct of interest (e.g., to a host cell, such as a T cell) (see, for example, U.S. Patent Application Publication Nos. 20080152661, 20080112916, 20080107694, 20080038296, 20070231392, 20060240093, 20060178327, 20060008910, 20050265957, 20050064595, to name a few). See, e.g., Nos. 20050042227, 20050037496, 20050026286, 20040162235, 20040072785, 20040063654, 20030157030, WO2008 / 036825, WO04 / 065601, and AU2004206255B2, all of which are incorporated by reference.

[0272] Treatment method In some aspects, the present disclosure provides methods of treating a proliferative or infectious disease by administering an immunogenic composition described herein to a subject (e.g., a subject having or suspected of having a proliferative or infectious disease). Without wishing to be bound by any particular theory, the immunogenic compositions described herein are, in some embodiments, useful for stimulating the immune system of a subject having a particular proliferative or infectious disease characterized by decreased expression of an immune checkpoint protein, and thus, an increased expression of an immune checkpoint protein.

[0273] As used herein, "proliferative disease" refers to diseases and disorders characterized by excessive cell proliferation and cell-matrix turnover, including cancer, atherosclerosis, rheumatoid arthritis, psoriasis, idiopathic pulmonary fibrosis, scleroderma, cirrhosis of the liver, etc. Examples of cancer include, but are not limited to, small cell lung cancer, colon cancer, breast cancer, lung cancer, prostate cancer, ovarian cancer, pancreatic cancer, melanoma, hematological malignancies such as chronic myeloid leukemia (CML), etc. Examples of subjects include mammals, such as humans and other primates; cows, pigs, horses, and farming (agricultural) animals; dogs, cats, and other domesticated pets; mice, rats, and transgenic non-human animals.

[0274] In some embodiments, the immunogenic compositions described in this disclosure are administered to a subject by adoptive cell transfer (ACT) therapy. Examples of ACT modalities include, but are not limited to, autologous cell therapy (e.g., the subject's own cells are removed, genetically engineered, and returned to the subject), and allogeneic cell therapy (e.g., cells are removed from a donor, genetically engineered, and placed in a recipient). In some embodiments, ACT is performed with a human cell line (e.g., Jurkat cells). In some embodiments, the cells utilized in the ACT therapy method may be genetically engineered to express a chimeric antigen receptor (CAR), which is an engineered cell receptor that displays specificity for a target antigen based on a selected antibody portion. Consequently, in some embodiments, CAR T cells (e.g., CART) may be transfected with chemically modified double-stranded nucleic acid using the methods described herein for the purpose of ACT therapy. Other immune cells, such as neutrophils, B cells, monocytes / macrophages, and combinations thereof, may be used in addition to or instead of T cells in the ACT therapy disclosed herein.

[0275] For in vivo application, the formulations of the present invention can be administered to patients in various forms adapted to the chosen route of administration (e.g., parenterally, orally or intraperitoneally). Preferred parenteral administration includes administration by the following routes: intravenous; intramuscular; intratumoral; interstitially; intraarterial; subcutaneous; intraocular; intrasynovial; transepithelial, including transdermal; pulmonary via inhalation; ophthalmic; sublingual and buccal; topical, including ophthalmic; transdermal; ocular; rectal; and intranasal inhalation via insufflation.

[0276] Pharmaceutical preparations for parenteral administration include aqueous solutions of active compounds in water-soluble or water-dispersible form. In addition, suspensions of active compounds as suitable oily injection suspensions may also be administered. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran, and optionally, the suspension may also contain a stabilizer. The oligonucleotides of the present invention may be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the oligonucleotides may be formulated in solid form and redissolved or suspended immediately before use. Lyophilized forms are also included in the present invention.

[0277] Drug delivery vehicles can be selected, for example, for in vitro administration, for systemic administration. These vehicles can be designed to act as delayed release reservoirs or to deliver their contents directly to target cells. The advantage of using some direct delivery drug vehicles is that multiple molecules are delivered per ingestion. Such vehicles have been shown to extend the circulatory half-life of drugs that would otherwise be rapidly cleared from the bloodstream. Some examples of such specialized drug delivery vehicles that fall into this category are liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres.

[0278] The administration of an active amount of the oligonucleotide of the present invention is defined as an effective amount, in the dosage and time required to achieve the desired result.For example, the active amount of oligonucleotide may vary according to factors such as the type of cell, the oligonucleotide used, and for in vivo use, the disease state, the age, sex and weight of the individual, and the ability of the oligonucleotide to elicit the desired response in the individual.The establishment of a therapeutic level of oligonucleotide in cells depends on the rate of uptake and the rate of efflux or degradation.Reducing the degree of degradation extends the intracellular half-life of the oligonucleotide.Therefore, chemically modified oligonucleotides, such as those with phosphate backbone modifications, may require different dosages.

[0279] The exact dosage of the immunogenic composition and the number of doses administered will depend on the data generated experimentally and in clinical trials.Several factors, such as the desired effect, delivery vehicle, disease symptoms and route of administration, will affect the dosage.The dosage can be easily determined by those skilled in the art and formulated into the subject pharmaceutical composition.Preferably, the duration of treatment will cover at least the entire course of the disease symptoms.

[0280] Dosage regimen may be adjusted to provide optimal therapeutic response.For example, immunogenic composition may be repeatedly administered, for example, several doses may be administered daily, or the dose may be proportionally reduced according to the requirements of the therapeutic situation.No matter whether chemically modified double-stranded nucleic acid molecule or immunogenic composition is administered to cell or to subject, those skilled in the art may easily determine the appropriate dose and administration schedule thereof for the subject. Administration of the immunogenic composition, such as through intradermal injection or subcutaneous delivery, can be optimized through testing of dosing regimens. In some embodiments, a single administration is sufficient. To further extend the effect of the administered immunogenic composition, the composition can be administered in a sustained release formulation or device, as is well known to those skilled in the art.

[0281] In other embodiments, the chemically modified double-stranded nucleic acid molecule or immunogenic composition is administered multiple times.In some examples, it is administered daily, twice a week, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months or less than once every 6 months.In some examples, it is administered multiple times per day, week, month and / or year.For example, it can be administered approximately every hour, every 2 hours, every 3 hours, every 4 hours, every 5 hours, every 6 hours, every 7 hours, every 8 hours, every 9 hours, every 10 hours, every 12 hours or more than every 12 hours.It can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 times per day.

[0282] Aspects of the invention relate to administering an immunogenic composition to a subject. In some instances, the subject is a patient and administering the immunogenic composition involves administering the composition in a doctor's office. In some embodiments, more than one immunogenic composition is administered simultaneously. For example, a composition comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 different compositions may be administered. In some embodiments, the composition comprises two or three different immunogenic compositions.

[0283] Self-delivered RNAi immunotherapeutics As described in U.S. Patent No. 10,934,550, the entire contents of which are incorporated herein by reference, immunotherapeutic agents have been produced by treating cells with specific INTASYL™ agents designed to target and knock down specific genes involved in immune suppression mechanisms. Several cells and cell lines have been successfully treated with INTASYL™ compounds and shown to knock down at least 70% of targeted gene expression in certain human cells. These studies demonstrated the utility of these immunogenic agents in suppressing target gene expression in cells that are normally highly resistant to transfection and suggested that the agents could reduce target cell expression in any cell type.

[0284] For purposes of the present invention, ranges may be expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. Furthermore, for purposes of the present invention, the occurrence of the term "a" or "an" refers to one or more of the occurrences; for example, "a protein" or "a nucleic acid molecule" refers to one or more of these compounds or at least one compound. Thus, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein. It should also be noted that the terms "comprising," "including," and "having" can be used interchangeably. Furthermore, a compound "selected from the group consisting of" refers to one or more of the compounds in the list that follows, including mixtures (i.e., combinations) of two or more compounds.

[0285] According to the present invention, an isolated or biologically pure protein or nucleic acid molecule is a compound that is removed from its natural environment.Therefore, "isolated" and "biologically pure" do not necessarily reflect the degree to which a compound has been purified.An isolated compound of the present invention can be obtained from its natural source, can be produced using molecular biology techniques, or can be produced by chemical synthesis. The compositions and methods described herein are further illustrated by the following examples, which should not be construed as further limiting in any way. The entire contents of all references cited throughout this application (including literature references, issued patents, published patent applications, and co-pending patent applications) are hereby expressly incorporated by reference.

[0286] example Example 1: Self-delivered RNAi immunotherapeutic The gene encoding TIGIT (NCBI GenBank Accession No. NM_173799) was analyzed using a proprietary algorithm to identify preferred INTASYL™ target sequences and target regions. (Table 1) The sequences of Tigit1 through Tigit20 have been previously disclosed in U.S. Patent Publication No. 2020 / 0215113, and are incorporated by reference therein, the entire patent publication of which is incorporated by reference herein. Tigit21 is identified herein as an additional preferred INTASYL™ target sequence.

[0287] Table 1 TIGIT target sequences and gene regions [Table 1-1] [Table 1-2]

[0288] Example 2: Chemical Modifications and Structures of INTASYL™ Compounds Evaluated INTASYL™ compounds were designed and synthesized to assess their ability to reduce gene expression levels in the liver following systemic administration. Non-limiting examples of INTASYL™ sequences are shown in Table 2. Table 2. TIGIT INTASYL™ Sequences (Passenger Strand / Sense Strand; Guide Strand / Antisense Strand)

[0289] [Table 2] sign A=Adenosine G=guanosine U = uridine C=Cytodine m = 2'-O-methyl nucleotide f=2' fluoro nucleotide Y=5-methyluridine X=5-methylcytosine *= phosphorothioate linkage .=phosphodiester linkage TEG-Chl = cholesterol-TEG-glyceryl P=5' inorganic phosphate VP-5' vinylphosphonate

[0290] Example 3: Silencing of TIGIT protein in KHYG-1 cells by flow cytometry KHYG1 cells were cultured in RPMI+10%FBS+100IU / mL rhIL-2 (Peprotech). Cells were harvested for transfection at a density of 0.3e6 cells / mL in culture medium. Cells were added directly to 24-well plates containing INTASYL™ compounds at final concentrations of 2 μM, 1 μM, 0.5 μM, and 0.25 μM. Plates were gently rocked to ensure complete mixing of cells and compounds. Transfections were performed twice for each condition. Cells were kept at 37° C. and 5% CO2 for the duration of the experiment. After 96 hours, cells were harvested and surface TIGIT protein was analyzed by flow cytometry.

[0291] For flow cytometry analysis, cells were collected in 96-well V-bottom plates and stained at 4°C according to the manufacturer's instructions. Briefly, 30 μL of cell suspension for each condition was pelleted in a 96-well V-bottom plate. Cells were resuspended in cell staining buffer containing TIGIT-APC antibody (R&D Systems) and incubated on ice for 1 hour. Cells were washed twice with cell staining buffer and resuspended to a final volume of 80 μL. Cells were analyzed using a NovoCyte flow cytometer (ACEA) and analyzed using NovoExpress software (ACEA). 25 μL of cell suspension was collected for each sample. As shown in FIG. 1, expression of TIGIT was downregulated in KHYG-1 cells after incubation with INTASYL™-Tigit (UTC represents the untreated control).

[0292] Example 4: Enhanced killing of tumor cells by natural killer cells treated with TIGIT KHYG1 natural killer (NK) cells were cultured with TIGIT-22 INTASYL™ compound for 72 hours. After 72 hours, cells were pelleted and resuspended in fresh medium in the absence of INTASYL™ compound for an additional 48 hours. KHYG1 cells were then analyzed for TIGIT mRNA levels (Figure 2, left graph), as well as their tumor-killing activity (K562 cells) was determined using the DELFIA cytotoxicity assay (at an E:T ratio of 5:1) (Figure 2, right graph). KHYG1 cells treated with TIGIT-22 INTASYL™ compound demonstrated enhanced tumor cell activity compared to controls.

[0293] KHYG1 NK cells were cultured with TIGIT22 INTASYL™ compound for 48 hours. After 48 hours, cells were pelleted and resuspended in fresh medium in the absence of INTASYL™ compound for a washing period (96 hours or 6 days). After the washing period, KHYG1 cells treated with TIGIT22 INTASYL™ compound were analyzed for surface TIGIT protein levels (Figures 3 and 4, left graphs) and their tumor-killing activity (K562 cells) was determined using the DELFIA cytotoxicity assay (E:T ratio of 5:1) (Figures 3 and 4, right graphs). KHYG1 cells treated with TIGIT22 INTASYL™ compound demonstrated enhanced tumor cell-killing activity compared to controls.

[0294] Equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. All references, including patent documents, are incorporated by reference in their entirety.

Claims

1. A chemically modified double-stranded nucleic acid molecule directed against a gene encoding a T-cell immunoreceptor with Ig and ITIM domains (TIGIT), wherein the chemically modified double-stranded nucleic acid molecule comprises at least 12 consecutive nucleotides of a sequence selected from SEQ ID NOs: 1-4 and 27.

2. (i) The chemically modified double-stranded nucleic acid molecule comprises a sequence selected from SEQ ID NOs: 1 to 4 and 27. (ii) the chemically modified double-stranded nucleic acid molecule is hydrophobically modified; (iii) the chemically modified double-stranded nucleic acid molecule is linked to one or more hydrophobic conjugates; (iv) the chemically modified double-stranded nucleic acid molecule is linked to one or more hydrophobic conjugates, and the one or more hydrophobic conjugates is cholesterol; (v) the chemically modified double-stranded nucleic acid molecule comprises at least one 2'-O-methyl modification and / or at least one 2'-O-fluoro modification, and at least one phosphorothioate modification; and / or (vi) The chemically modified double-stranded nucleic acid molecule according to claim 1, wherein the chemically modified double-stranded nucleic acid molecule comprises a sequence selected from TIGIT22 (SEQ ID NOs: 1-2) and TIGIT23 (SEQ ID NOs: 3-4).

3. The chemically modified double-stranded nucleic acid molecule of claim 1 or 2, wherein the chemically modified double-stranded nucleic acid molecule comprises a sense strand (TIGIT22 sense strand) having the sequence set forth in SEQ ID NO: 1 and / or an antisense strand (TIGIT22 antisense strand) having the sequence set forth in SEQ ID NO:

2.

4. The chemically modified double-stranded nucleic acid molecule of claim 1 or 2, wherein the chemically modified double-stranded nucleic acid molecule comprises a sense strand (TIGIT23 sense strand) having the sequence set forth in SEQ ID NO: 3 and / or an antisense strand (TIGIT23 antisense strand) having the sequence set forth in SEQ ID NO:

4.

5. A composition comprising the chemically modified double-stranded nucleic acid molecule of claim 1 or 2 and a pharmaceutically acceptable excipient.

6. An immunogenic composition comprising a host cell comprising a chemically modified double-stranded nucleic acid molecule comprising at least 12 consecutive nucleotides of any one of SEQ ID NOs: 1-4 and 27, wherein the host cell is selected from the group consisting of a T cell, an antigen-presenting cell (APC), a dendritic cell (DC), a stem cell (SC), an induced pluripotent stem cell (iPSC), a natural killer cell, a macrophage, a neutrophil, and a stem cell memory T cell. (i) The chemically modified double-stranded nucleic acid molecule comprises a sequence selected from SEQ ID NOs: 1 to 4 and 27. (ii) the chemically modified double-stranded nucleic acid molecule is hydrophobically modified; (iii) the chemically modified double-stranded nucleic acid molecule is linked to one or more hydrophobic conjugates; (iv) the chemically modified double-stranded nucleic acid molecule comprises at least one 2'-O-methyl modification and / or at least one 2'-O-fluoro modification, and at least one phosphorothioate modification; and / or (v) The immunogenic composition of claim 6, wherein the chemically modified double-stranded nucleic acid molecule induces at least 50% inhibition of TIGIT in host cells. (i) the host cell is a T cell or a natural killer cell; (ii) the host cell is a T cell, and the T cell contains one or more transgenes expressing a high affinity T cell receptor (TCR) and / or a chimeric antibody receptor (CAR); (iii) the host cell is a natural killer cell, and the natural killer cell comprises one or more transgenes expressing a high affinity T cell receptor (TCR) and / or a chimeric antibody receptor (CAR); and / or (iv) The immunogenic composition of claim 6 or 7, wherein the host cells are derived from a healthy donor or a subject having or suspected of having a proliferative disorder or an infectious disease.

9. 1. A method for producing an immunogenic composition, the method comprising introducing one or more chemically modified double-stranded nucleic acid molecules into a host cell, at least one of the molecules comprising at least 12 consecutive nucleotides of any one of SEQ ID NOs: 1-4 and 27, wherein the one or more chemically modified double-stranded nucleic acid molecules target T cell immunoreceptor with Ig and ITIM domains (TIGIT), and wherein the host cell is selected from the group consisting of T cells, antigen-presenting cells (APCs), dendritic cells (DCs), stem cells (SCs), induced pluripotent stem cells (iPSCs), natural killer cells, and stem cell memory T cells.

10. A method for producing an immunogenic composition, the method comprising introducing the chemically modified double-stranded nucleic acid molecule of claim 1 or 2 into a host cell, wherein the host cell is selected from the group consisting of T cells, antigen-presenting cells (APCs), dendritic cells (DCs), stem cells (SCs), induced pluripotent stem cells (iPSCs), natural killer cells, and stem cell memory T cells.

11. The method described in claim 9, wherein the host cells are derived from a healthy donor.

12. 12. The method of claim 9 or 11, further comprising resuspending the host cells in a wash medium that does not contain the one or more chemically modified double-stranded nucleic acid molecules for 24 hours to 6 days, 24 hours to 96 hours, 48 hours, or 96 hours, or wherein the host cells are not resuspended in a wash medium that does not contain the one or more chemically modified double-stranded nucleic acid molecules.

13. A composition as described in claim 5 for treating a proliferative disease in a subject, wherein the proliferative disease is cancer.

14. An immunogenic composition as described in claim 6 or 7 for treating a proliferative disease in a subject, wherein the proliferative disease is cancer.

15. A method for adoptive cell transfer (ACT), comprising: The method comprises contacting cells collected from a patient or a healthy donor ex vivo with the chemically modified double-stranded nucleic acid molecule of claim 1 or 2.

16. The method described in claim 15, wherein the cells recovered from the patient or healthy donor are immune cells, and the immune cells are selected from one or more of the group consisting of neutrophils, B cells, T cells, monocytes, natural killer cells, and macrophages.

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