Chemically modified oligonucleotides targeting bromodomain-containing protein 4 (BRD4) for immunotherapy

Abstract

In some aspects, the present disclosure relates to methods and compositions for producing immunomodulatory compositions. In some embodiments, the present disclosure provides host cells that have been treated ex vivo with one or more oligonucleotide agents that can control and / or reduce the differentiation of host cells. In some embodiments, the compositions and methods described by the present disclosure are useful as immunogenicity modulators for treating cancer.

Description

[Technical Field]

[0001] Related applications This application claims, under 35 U.S.C. § 119(e), the benefit as of the filing date of U.S. Provisional Application No. 62 / 932,813, filed on 8 November 2019, entitled "CHEMICALLY MODILIED OLIGONUCLEOTIDES TARGETING BROMODOMAIN CONTAINING PROTEIN 4 (BRD4) FOR IMMUNOTHERAPY," the full disclosure thereof is incorporated herein by reference in its entirety.

[0002] field In several respects, this disclosure relates to immunomodulatory compositions and methods for preparing immunomodulatory compositions, including the use of oligonucleotides to modulate bromodomain-containing protein 4 (BRD4), a gene target involved in transcription and epigenetic regulation, for improving a population or subset of therapeutic immune cells. This disclosure further relates to methods for using immunomodulatory compositions for the treatment of cytoproliferative disorders or infectious diseases, including cancer and autoimmune disorders. [Background technology]

[0003] background The physiological function of the immune system is to recognize and eliminate neoplastic cells. Therefore, 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 influencing tumor growth but also limit the effectiveness of immunotherapeutic approaches to cancer. Immune resistance mechanisms include immunosuppressive pathways, often referred to as immune checkpoints. Immunosuppressive pathways play a particularly important role in the interaction between tumor cells and CD8+ cytotoxic T lymphocytes and include adoptive cell transplantation (ACT) therapies.

[0004] Various methods of adoptive cell transfer (ACT) involve ex vivo treatment of cells recovered from a patient's 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.

[0005] During ex vivo processing, cells undergo certain phenotypic changes that can affect their therapeutic properties, among other things, such as transport to the tumor, in vivo proliferative capacity and lifespan, and their effectiveness in an immunosuppressive environment. For example, the state of T cell differentiation and maturation typically proceeds through the following sequence of subtypes: naive (T CM ) - stem cell memory (T SCM ) - central memory (T CM ) - effector memory (T EM ) - terminally differentiated effector T cells (T​​​​​​​​​​​​​​​​​​​​​​The present disclosure relates to compositions and methods for controlling the T cell differentiation process during the production of immunomodulatory compositions to enhance the level of a desired subtype of T cells. The present disclosure is based in part on immunomodulatory (e.g., immunogenic) compositions comprising host cells containing oligonucleotide molecules that target genes related to signaling / transcription factors, epigenetic, metabolic and co-inhibitory / negative regulatory targets, and methods for producing such compositions. In some aspects, the present disclosure provides chemically modified oligonucleotide molecules for use in methods for producing immunomodulatory compositions. In some embodiments, the methods and compositions described herein are useful for the production of immunomodulatory compositions and for treating subjects with proliferative or infectious diseases.

[0007] Consequently, in several respects, this disclosure provides chemically modified double-stranded nucleic acid molecules that target (for example, directed towards genes encoding) bromodomain-containing protein 4 (BRD4), which is a member of the bromodomain and extraterminal (BET) families.

[0008] In some embodiments, the chemically modified double-stranded nucleic acid molecule is directed to a sequence containing at least 12 consecutive nucleotides of a sequence selected from the sequences in Table 1. In some embodiments, the chemically modified double-stranded nucleic acid molecule is a self-delivery RNA (e.g., INTASYL®; also referred to herein as sd-rxRNA). In some embodiments, the chemically modified double-stranded nucleic acid molecule (e.g., INTASYL®) contains, consists of, targets, or is directed to sequences or fragments thereof listed in Table 1 or 2.

[0009] In some respects, chemically modified double-stranded nucleic acid molecules include at least one 2'-O-methyl modification and / or at least one 2'-O-fluoro modification, and at least one phosphorothioate modification. In some aspects, this disclosure provides INTASYL® compounds directed to the gene encoding BRD4. In some embodiments, the INTASYL® compound (sd-rxRNA) comprises at least 12 consecutive nucleotides of a sequence selected from the sequences in Table 2.

[0010] In some embodiments, the INTASYL® compound is hydrophobically modified. In some embodiments, the INTASYL® compound is linked to one or more hydrophobic conjugates. In some embodiments, the hydrophobic conjugate is cholesterol. In some embodiments, the chemically modified double-stranded nucleic acid molecules or INTASYL® compounds described herein include or consist of sequences represented by a BRD4-20 sense or antisense strand, a BRD4-21 sense or antisense strand, or a BRD4-22 sense or antisense strand.

[0011] In some embodiments, the chemically modified double-stranded nucleic acid molecules or INTASYL® compounds described herein include, or consist of, a sense strand having a sequence represented by the BRD4-20 sense strand and / or an antisense strand having a sequence represented by the BRD4-20 antisense strand. In some embodiments, the chemically modified double-stranded nucleic acid molecules or INTASYL® compounds described herein include, or consist of, a sense strand having a sequence represented by the BRD4-21 sense strand and / or an antisense strand having a sequence represented by the BRD4-21 antisense strand. In some embodiments, the chemically modified double-stranded nucleic acid molecules or INTASYL® compounds described herein include, or consist of, a sense strand having a sequence represented by the BRD4-22 sense strand and / or an antisense strand having a sequence represented by the BRD4-22 antisense strand.

[0012] In some respects, this disclosure provides compositions comprising the chemically modified double-stranded nucleic acid molecules or INTASYL® described herein and pharmaceutically acceptable excipients. In some embodiments, the compositions described herein include a chemically modified double-stranded nucleic acid molecule or an INTASYL® compound directed toward BRD4. In some embodiments, the chemically modified double-stranded nucleic acid molecule or INTASYL® compound directed toward BRD4 includes at least 12 consecutive nucleotides of a sequence selected from Table 2.

[0013] In some aspects, this disclosure provides immunomodulatory compositions comprising host cells (e.g., immune cells such as T cells or NK cells) and the host cells being treated ex vivo with chemically modified double-stranded nucleic acid molecules to control and / or reduce the level of differentiation of the host cells (e.g., T cells) to enable the production of a specific immune cell population (e.g., a population enriched for a particular T cell subtype) for administration in humans. In some embodiments, the immunomodulatory composition comprises a plurality of host cells enriched for a particular cell type (e.g., a T cell subtype). For example, in some embodiments, the immunomodulatory composition comprises T SCM or T CM The cells contain at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% (for example, comprehensively, any percentage between 50% and 100%) of T cells of a specific T cell subtype.

[0014] In some embodiments, the immunomodulatory composition comprises a host cell containing a chemically modified double-stranded nucleic acid molecule as described in the specification (e.g., a chemically modified double-stranded nucleic acid molecule or INTASYL® directed to a gene encoding BRD4). In some embodiments, the chemically modified double-stranded nucleic acid molecule or INTASYL® compound is directed to a sequence comprising at least 12 consecutive nucleotides of a sequence selected from the sequences in Table 1. In some embodiments, the chemically modified double-stranded nucleic acid molecule (e.g., INTASYL®) contains, consists of, targets, or is directed to the sequences or fragments thereof listed in Tables 1 and 2. In some embodiments, the host cell contains a chemically modified double-stranded nucleic acid molecule directed toward BRD4. In some embodiments, the chemically modified double-stranded nucleic acid molecule directed toward BRD4 contains at least 12 consecutive nucleotides of a sequence selected from Table 2.

[0015] In some embodiments, the host cells are selected from the group consisting of T cells, NK cells, antigen-presenting cells (APCs), dendritic cells (DCs), stem cells (SCs), induced pluripotent stem cells (iPSCs), stem cell memory T cells, and cytokine-induced killer cells (CIKs). In some embodiments, the host cells are T cells. In some embodiments, the T cells are CD8+ T cells. In some embodiments, the T cells are T cells after introduction of a chemically modified double-stranded nucleic acid molecule or an INTASYL® compound. SCM or T CM They differentiate into specific T cell subtypes, such as T cells.

[0016] In some embodiments, T cells contain one or more transgenes that express high-affinity T cell receptors (TCRs) and / or chimeric antigen receptors (CARs). In some embodiments, the host cells are derived from a healthy donor. In some aspects, this disclosure provides a method for producing an immunomodulatory composition, comprising introducing one or more chemically modified double-stranded nucleic acid molecules or INTASYL® compounds described herein into cells. In some embodiments, the chemically modified double-stranded nucleic acid molecules or sd-rxRNA are introduced into cells ex vivo.

[0017] In some embodiments of the methods described herein, the cells are T cells, NK cells, antigen-presenting cells (APCs), dendritic cells (DCs), stem cells (SCs), induced pluripotent stem cells (iPSCs), stem cell memory T cells, and cytokine-induced killer cells (CIKs). In some embodiments, T cells are CD8+ T cells. In some embodiments, T cells are T cells after the introduction of a chemically modified double-stranded nucleic acid molecule or sd-rxRNA. SCM or T CM They differentiate into specific T cell subtypes, such as T cells. In some embodiments, the T cells contain one or more transgenes that express high-affinity T cell receptors (TCRs) and / or chimeric antigen receptors (CARs). In some embodiments, the cells are derived from a healthy donor.

[0018] In some aspects, this disclosure provides a method for treating a subject suffering from a proliferative disorder or an infectious disorder, the method comprising administering an immunomodulatory composition described herein to the subject. In some embodiments, the proliferative disorder is cancer. In some embodiments, the infectious disorder is a pathogenic infection such as a viral infection, a bacterial infection, or a parasitic infection.

[0019] Each limitation of the present invention may encompass various aspects of the present invention. Therefore, each limitation of the present invention involving any one element or combination of elements may be considered to be included in each aspect of the present invention. In its application, the present invention is not limited to the details of interpretation and arrangement of components described in the following description or illustrated in the drawings. Other aspects of the present invention are possible and can be practiced or implemented in a variety of ways. Furthermore, the terminology and terminology used in this book are for illustrative purposes only and should not be considered limiting. The use of “including,” “comprising,” “having,” “containing,” “involving,” and their variations herein means that they encompass the items listed thereafter, their equivalents, and any additional items. [Brief explanation of the drawing]

[0020] A brief explanation of the drawing. The attached drawings are not intended to be drawn to a fixed scale. In the drawings, identical or nearly identical components illustrated in various figures are represented by similar numbers. For the sake of clarity, not all components can be labeled in all drawings.

[0021] [Figure 1] Figure 1 shows the two-point dose response of BRD4-targeting chemically modified INTASYL™ molecules for mRNA silencing in A549 cells. [Figure 2] Figure 2 shows the dose-response curves of chemically modified INTASYL® molecules targeting BRD4 in human primary T cells. For each chemically modified INTASYL® molecule, the tested concentrations were 2 μM, 1 μM, 0.25 μM, 0.125 μM, and 0.06 μM, from left to right. [Figure 3] Figure 3 shows the percentage of BRD4-negative cells at different time points after or after treatment with BRD4-20, untargeted control (NTC; negative control), or JQ1 (positive control).

[0022] [Figure 4]Figures 4A-4B show the test protocol (Figure 4A) and the percentage of CCR7+ / CD62L+ cells after treatment with no treatment (UNT), non-targeted control (NTC), BRD4-20, and positive control (JQ1) (Figure 4B). [Figure 5] Figure 5 shows the concentrations of interferon-γ (IFN-γ) in melanoma-derived tumor-infiltrating lymphocytes (TILs) co-incubated with human melanoma after untreated (UNT), non-targeted control (NTC; negative control), and BRD4-20 or JQ1 (positive control).

[0023] [Figure 6] Figures 6A and 6B show the results of flow cytometry analysis of TILs at day 12 of the National Cancer Institute's Rapid Expansion Protocol (REP). Figure 6A shows the raw data, and Figure 6B shows the quantified data. Results were obtained after treatment with no treatment (UNT), non-targeted control (NTC; negative control), BRD4-20, or JQ1 (positive control).

[0024] [Figure 7] Figure 7 shows the tumor volume over time in Hepa 1-6 tumor-bearing mice measured after treatment with PBS, non-targeted control (NTC), BRD4-20 (0.5 mg / dose), BRD4-20 (2 mg / dose), or JQ1 (positive control). [Figure 8] Figure 8 shows the percentage of CD45+ TIL measured after the treatments shown in the graph in Hepa 1-6 tumor-carrying mice. [Figure 9] Figures 9A and 9B show the tumor volume during the experiment. Figure 9A shows the mean tumor volume over time, and Figure 9B shows the tumor volume AUC after the indicated treatment. [Modes for carrying out the invention]

[0025] Detailed explanation In several respects, this disclosure relates to compositions and methods for immunotherapy. This disclosure is in part to chemically modified double-stranded nucleic acid molecules (e.g., INTASYL®) that target genes associated with controlling the differentiation process of T cells and / or regulating T cell expression or activity, such as BRD4. INTASYL® technology specifically controls the differentiation process of cells, including T cells, and the desired subtype (T SCM / T CM It is suitable for producing therapeutic cells rich in ). Some of the advantages of INTASYL® include: (i) INTASYL® can be developed in a short period of time and can silence virtually any target, including “non-drugable” targets, such as those that are difficult to inhibit with small molecules, such as transcription factors; (ii) Compared with 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 maintaining high cell viability; (iii) When added to cell culture medium in the initial expansion stage, INTASYL® compounds provide transient silencing of the target of interest during 8-10 mitotic cycles, allowing the silencing effect to disappear in the final population of cells by the time of their reinjection into the patient; (iv) INTASYL® can be used in combination to silence multiple targets simultaneously, thus providing considerable flexibility for use in various types of cell processing protocols.

[0026] INTASYL® compounds targeted at specific targets involved in T cell differentiation, and the beneficial effects of such INTASYL® on the T cell phenotype or after ex vivo expansion, are described herein. Screening methods that may be used to identify INTASYL® compounds suitable for specific cell production protocols are also presented.

[0027] As used herein, “nucleic acid molecule” includes, but is not limited to, INTASYL® compounds, sd-rxRNA, rxRNAori, oligonucleotides, ASO, siRNA, shRNA, miRNA, ncRNA, cp-lasiRNA, aiRNA, single-stranded nucleic acid molecules, double-stranded nucleic acid molecules, 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 INTASYL® (also known as sd-rxRNA). ) It is a molecule.

[0028] INTASYL™ compound (sd-rxRNA) molecule Aspects of the present invention relate to INTASYL® molecules that target genes associated with controlling the T cell differentiation process and / or regulating T cell expression or activity, such as BRD4. In some embodiments, this disclosure provides INTASYL® molecules that target the BRD4 gene. In some embodiments, the INTASYL® molecules described herein include, consist of, target, or are directed to the sequences or fragments thereof listed in Table 2.

[0029] As used herein, "sd-rxRNA" or "sd-rxRNA molecule" or "INTASYL®" or "INTASYL® molecule" or "INTASYL compound" refers to U.S. Patent No. 8,796,443, entitled "REDUCED SIZE SELF-DELIVERING RNAI COMPOUNDS," granted on August 5, 2014. This refers to self-delivering RNA molecules, such as those described in U.S. Patent No. 9,175,289, entitled "REDUCED SIZE SELF-DELIVERING RNAI COMPOUNDS," granted on November 3, 2015; U.S. Patent No. 10,774,330, entitled "REDUCED SIZE SELF-DELIVERING RNAI COMPOUNDS," granted on September 15, 2020; and PCT Publication No. WO2010 / 033247 (Application No. PCT / US2009 / 005247), entitled "REDUCED SIZE SELF-DELIVERING RNAI COMPOUNDS," filed on September 22, 2009, and incorporated herein by reference. In short, INTASYL (trademark) (sd-rxRNA ナノ INTASYL® (also known as INTASYL) is an isolated asymmetric double-stranded nucleic acid molecule comprising a guide strand of a minimum length of 16 nucleotides and a passenger strand of 8–18 nucleotides in length, wherein the double-stranded nucleic acid molecule has a double-stranded region and a single-stranded region, the single-stranded region having a length of 4–12 nucleotides and having at least three nucleotide backbone modifications. In a preferred embodiment, the double-stranded nucleic acid molecule has a blunt single end or contains one or two nucleotide protrusions. INTASYL® molecules can be optimized through chemical modification, and in some examples through the attachment of hydrophobic conjugates. The above-referenced patents and publications are incorporated herein by reference in their entirety.

[0030] In some embodiments, INTASYL™ comprises an isolated double-stranded nucleic acid molecule comprising a guide strand and a passenger strand, wherein the double-stranded region of the molecule is 8 to 15 nucleotides long, wherein the guide strand contains a single-stranded region of 4 to 12 nucleotides long, 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. The nucleic acid molecules of the present invention, as used herein, include isolated double-stranded or duplex nucleic acids, oligonucleotides or polynucleotides, nanomolecules, nanoRNAs, and sd-rxRNAs. ナノ It is also called sd-rxRNA, INTASYL (trademark), or RNA molecule.

[0031] INTASYL™ molecules are taken up by cells far more effectively than conventional siRNAs. These molecules are highly efficient in silencing target genes and offer significant advantages over previously described RNAi molecules, including high activity in the presence of serum, efficient self-delivery, compatibility with diverse linkers, and reduced or complete absence of toxicity-related chemical modifications.

[0032] In contrast to single-stranded polynucleotides, duplex polynucleotides have traditionally been difficult to deliver to cells because their rigid structure and numerous negative charges make membrane transport difficult. However, INTASYL™ molecules, despite being partially double-stranded, are recognized as single-stranded in vivo and can therefore be efficiently delivered across the cell membrane. As a result, the polynucleotides of the present invention are self-deliverable in many cases. Thus, the polynucleotides of the present invention may be formulated in a manner similar to conventional RNAi agents, or delivered to cells or targets alone (or with a non-delivery carrier), enabling self-delivery. In one embodiment of the present invention, a self-delivering asymmetric double-stranded RNA molecule is provided, in which a portion of the molecule is similar to a conventional RNA duplex and the second portion of the molecule is single-stranded.

[0033] The oligonucleotides of the present invention, in several aspects, have an asymmetric structure comprising a double-stranded region and a single-stranded region of 5 nucleotides or longer, combined with a specific chemical modification pattern, and are conjugated to lipophilic or hydrophobic molecules. In some embodiments, this class of RNAi-like compounds exhibits excellent efficacy in vitro and in vivo. The reduction in the size of the robust duplex region, when combined with phosphorothioate modifications applied to the single-stranded region, is thought to contribute to the observed excellent efficacy.

[0034] In some embodiments, the RNAi compounds of the present invention comprise an asymmetric compound comprising an 8-15 nucleotide duplex region (required for efficient RISC entry) and a 4-12 nucleotide single-stranded region. In some embodiments, the duplex region is 13 or 14 nucleotides long, and in some embodiments, the single-stranded region is 6-7 nucleotides long. The single-stranded region of the RNAi compound (e.g., the INTASYL™ molecule) also comprises 2-12 phosphorothioate internucleotide junctions (referred to as phosphorothioate modifications). In some embodiments, the single-stranded region comprises 6-8 phosphorothioate internucleotide junctions. Furthermore, the RNAi compounds of the present invention also comprise a unique chemical modification pattern that provides stability and is compatible with RISC entry. In some embodiments, the combination of these elements results in an unexpected property that is highly useful for the delivery of RNAi reagents in vitro and in vivo.

[0035] Chemical modification patterns that provide stability and are suitable for RISC entry include modifications to the sense or passenger strand as well as the antisense or guide strand. For example, the passenger strand may be modified by any chemical entity that ensures stability and does not interfere with activity. Such modifications include skeletal 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 within the passenger strand. Alternatively, the passenger strand may be entirely O-methyl modified.

[0036] The guide chain may also be modified in some embodiments by any chemical modification that ensures stability without interfering with RISC entry. In some embodiments, the chemical modification pattern in the guide chain includes a majority of C and U nucleotides being 2'F modified and the 5' end being phosphorylated. In some embodiments, the chemical modification pattern in the guide chain includes 2'O methyl modification 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 chain includes 2'O methyl modification of C / U at positions 1 and 11-18 and chemical phosphorylation at the 5' end, as well as 2'F modification of C / U at positions 2-10. In some embodiments, the passenger chain and / or guide chain contain at least one 5-methyl C or U modification.

[0037] In some embodiments, at least 30% of the nucleotides in 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%, or 99% are modified. In some embodiments, 100% of the nucleotides in the INTASYL® compound are modified.

[0038] The above chemical modification patterns of oligonucleotides of the present invention exhibit good resistance and improve the potency of asymmetric RNAi compounds. In some embodiments, the exclusion of any of the described components (guide chain stabilization, phosphorothioate elongation, sense chain stabilization, and hydrophobic conjugate), or in some examples, an increase in size, results in suboptimal potency and, in some examples, complete loss of potency. Combinations of components lead to the development of compounds that remain sufficiently active even after passive delivery to cells such as HeLa cells or T cells.

[0039] INTASYL® can be further improved in several instances by enhancing the hydrophobicity of the compound using novel types of chemistries. For example, one chemistry relates to the use of hydrophobic base modification. Any base at any position may be modified insofar as the modification results in an increase in the base partition coefficient. Preferred positions for the chemistry of modification are positions 4 and 5 of the pyrimidine. The main advantages of these positions are (a) ease of synthesis and (b) no interference with base pairing and A-form helix formation, which are 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 potency of the compound. 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 alter (increase / decrease) the C17 attached chain. This type of modification leads to a significant increase in cellular uptake in vivo and improved tissue uptake success rates (prosperities).

[0040] 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 to 22 base pairs, with or without 3'-overhangs on the sense and antisense strands, and a 3' single-stranded tail of about 2 to 9 nucleotides on the antisense strand. 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, as well as at least one hydrophobic modification selected from sterols, cholesterol, vitamin D, naphthyl, isobutyl, benzyl, indole, tryptophan, phenyl, etc., and a hydrophobic modifier. In some embodiments, the chemically modified double-stranded nucleic acid molecule contains multiple such modifications.

[0041] In some respects, this disclosure relates to chemically modified double-stranded nucleic acid molecules that target genes encoding targets related to cell differentiation (e.g., T cell differentiation), such as signaling / transcription target, epigenetic target, and metabolic and co-inhibitory / negative regulatory target. Examples of epigenetic proteins include, but are not limited to, BRD4. In some embodiments, the chemically modified double-stranded nucleic acid target targets the gene encoding BRD4.

[0042] As used herein, “BRD4” (also known as CAP, MCAP, HUNK1, and HUNKI) refers to bromodomain-containing protein 4 or bromodomain-containing 4, which is a member of the bromodomain and extra-terminus (BET) family of transcriptional and epigenetic regulators that play a role during cancer development. BRD4 contains two bromodomains that recognize acetylated lysine residues on DNA histone tails. As a chromatin regulatory protein, BRD4 binds to acetylated histones and is involved in the transmission of epigenetic memory across cell division and transcriptional regulation. Specifically, once the protein binds, it remains with acetylated chromatin throughout the entire cell cycle, providing epigenetic memory to post-mitotic G1 gene transcription by preserving higher-order chromatin structure (Wang et al. (2012) J. Biol. Chem. 287:10738-10752). BRD4 promotes gene transcription during the initiation and elongation steps by recruiting the positive transcription elongation factor P-TEFb (Yang et al. (2005) Mol Cell. 19(4):535-45). Due to its role in regulating the transcriptional elongation of genes involved in the cell cycle and apoptosis, such as c-Myc and BCL2, BRD4 has been suggested to be involved in cancer (Jung et al. (2015) Epigenomics, 7(3):487-501). In some embodiments, BRD4 is encoded by a nucleic acid sequence represented by NCBI reference sequence number NM_058243.2. Non-limiting examples of BRD4 sequences that may be targeted by the chemically modified double-stranded nucleic acid molecules of this disclosure are listed in Table 2.

[0043] In some embodiments, the chemically modified double-stranded nucleic acid molecule contains at least 12 nucleotides from the sequences in Table 2. In some embodiments, the chemically modified double-stranded nucleic acid molecule contains at least one sequence from Table 2 (for example, a sense strand or an antisense strand containing a sequence listed in any one of Table 2). In some embodiments, the chemically modified double-stranded nucleic acid molecule (for example, INTASYL®) contains, consists of, targets, or is directed towards the sequences or fragments thereof listed in Table 2.

[0044] In some embodiments, a chemically modified double-stranded nucleic acid molecule (e.g., INTASYL®) comprises a sense strand having the sequence described in the BRD4-20 sense strand and / or an antisense strand having the sequence described in the BRD4-20 antisense strand. In some embodiments, a chemically modified double-stranded nucleic acid molecule (e.g., INTASYL®) comprises a sense strand having the sequence described in the BRD4-21 sense strand and / or an antisense strand having the sequence described in the BRD4-21 antisense strand. In some embodiments, a chemically modified double-stranded nucleic acid molecule (e.g., INTASYL®) comprises a sense strand having the sequence described in the BRD4-22 sense strand and / or an antisense strand having the sequence described in the BRD4-22 antisense strand.

[0045] In some embodiments, the dsRNAs formulated according to the present invention are rxRNAori. rxRNAori refers to a class of RNA molecules described and incorporated by reference in PCT publication number WO2009 / 102427 (application number PCT / US2009 / 000852), filed on 11 February 2009, titled "MODIFIED RNAI POLYNUCLEOTIDES AND USES THEREOF," and U.S. Patent Publication number 2011 / 0039914, filed on 1 November 2010, also titled "MODIFIED RNAI POLYNUCLEOTIDES AND USES THEREOF."

[0046] In some embodiments, the rxRNAori molecule comprises a 12-35 nucleotide double-stranded RNA (dsRNA) construct for inhibiting the expression of a target gene, which comprises a sense strand having 5' and 3' ends, where the sense strand is highly modified with 2'-modified ribose sugar, and where 3-6 nucleotides in the middle of the sense strand are not modified with 2'-modified ribose sugar, and an antisense strand having 5' and 3' ends, which hybridizes with the sense strand and the mRNA of the target gene, where the dsRNA inhibits the expression of the target gene in a sequence-dependent manner.

[0047] The rxRNAori may contain any of the modifications described herein. In some embodiments, at least 30% of the nucleotides in the rxRNAori 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%, 6% of the nucleotides in the rxRNAori are modified. 2%, 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 sd-rxRNA are modified. In some embodiments, only the passenger strand of the rxRNAori contains modifications.

[0048] Accordingly, aspects of the present invention relate to isolated double-stranded nucleic acid molecules comprising a guide (antisense) strand and a passenger (sense) strand. As used herein, the term “double-stranded” 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 is in the range of 16 to 29 nucleotides. In some embodiments, the guide strand is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 nucleotides long. The guide strand is complementary to the target gene. The complementarity between the guide strand and the target gene can exist across any portion of the guide strand. As used herein, the complementarity may be complete or less complete, insofar as the guide strand is sufficiently complementary to mediate RNAi to the target. In some embodiments, complementarity refers to a mismatch of less than 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% between the guide strand and the target. Perfect 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. Furthermore, not all sites on the siRNA contribute equally to target recognition. Mismatches at the center of the siRNA are most important and essentially neutralize the cleavage of the target RNA. With respect to the antisense strand, mismatches upstream of the center or upstream of the cleavage site exhibit resistance but significantly reduce the cleavage of the target RNA. With respect to the antisense strand, mismatches downstream of the center or cleavage site, preferably near the 3' end, for example, located 1, 2, 3, 4, 5, or 6 nucleotides from the 3' end of the antisense strand, exhibit resistance and only slightly reduce the cleavage of the target RNA.

[0049] While we do not wish to be constrained by any particular theory, in some embodiments of the double-stranded nucleic acid molecules described herein, the guide strand is at least 16 nucleotides long and anchors the Argonaut protein in RISC. In some embodiments, when the guide strand is loaded 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 or can be phosphorylated. The nucleic acid molecules described herein may also be referred to as minimum trigger RNAs.

[0050] In some embodiments of the double-stranded nucleic acid molecules described herein, the length of the passenger strand is in the range of 8 to 15 nucleotides. In some embodiments, the passenger strand is 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides long. The passenger strand is complementary to the guide strand. The complementarity between the passenger strand and the guide strand may exist across either the passenger or the guide strand. In some embodiments, 100% complementarity exists between the guide strand and the passenger strand within the double-stranded region of the molecule.

[0051] An aspect of the present invention relates to a double-stranded nucleic acid molecule having a minimum double-stranded region. In some embodiments, the double-stranded region of the molecule is in the range of 8 to 15 nucleotides in length. In some embodiments, the double-stranded region of the molecule is 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length. In some embodiments, the double-stranded region is 13 or 14 nucleotides in length. In some embodiments, the double-stranded region of the molecule is 13 to 22 nucleotides in length. In some embodiments, the double-stranded region of the molecule is 16, 17, 18, 19, 20, 21, or 22 nucleotides in length.

[0052] 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 either blunt-ended or has a 1-nucleotide protrusion. In some embodiments, the single-stranded region of the molecule is 4 to 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 longer 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 to 9 nucleotides long, including 2 or 3 nucleotides long.

[0053] The RNAi constructs related to the present invention may 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, loss of efficacy occurs when (ΔG) is less than -21 kcal / mol. In some embodiments, a (ΔG) value higher than -13 kcal / mol is compatible with aspects of the present invention. While we do not wish to be constrained by any theory, in some embodiments, molecules with relatively high (ΔG) values ​​may become active at relatively high concentrations, while molecules with relatively low (ΔG) values ​​may become active at relatively low concentrations. In some embodiments, the (ΔG) value may be higher than -9 kcal / mol. The gene silencing effect mediated by the RNAi constructs related to the present invention having a minimum double-stranded region is unpredictable, because molecules with nearly identical design but lower thermodynamic stability have been shown to be inactive (Rana et al. 2004).

[0054] While we do not wish to be constrained by any theory, the results described herein suggest that an 8-10 bp elongation of dsRNA or dsDNA would be structurally recognized by RISC protein components or RISC cofactors. Furthermore, there exists 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 loaded into the Argonaut protein. If acceptable thermodynamics exist and a double-stranded portion, preferably of at least 8 nucleotides, is present, the duplex will be recognized and loaded into the RNAi mechanism.

[0055] In some embodiments, thermodynamic stability is increased through the use of LNA bases. In some embodiments, additional chemical modifications are introduced. Several non-limiting examples of chemical modifications include 5'-phosphonate, 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-methyl C, 5-methyl U, 5-methyl dC, 5-methyl dU methoxy, (2,6-diaminopurine), 5'-dimethoxytrityl-N4-ethyl-2'-deoxycytidine, and MGB (a secondary groove binder). It should be understood that more than one chemical modification may be combined within the same molecule.

[0056] The molecules related to the present invention are optimized for increased potency and / or reduced toxicity. For example, the length of nucleotides in 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 several aspects, while replacing 2'-fluoro (2'F) modifications with 2'-O-methyl (2'OMe) modifications affects the toxicity of the molecule in several 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 may affect the efficiency of the molecule's uptake into cells, for example, passive uptake into cells. Preferred embodiments of the molecules described herein are characterized by the absence of 2'F modifications while still exhibiting equivalent potency in cellular uptake and tissue penetration. Such molecules represent a significant improvement over prior art such as the molecules described by Accell and Wolfrum, which are heavily modified by the extensive use of 2'F.

[0057] In some embodiments, the guide chain is approximately 18–20 nucleotides long and has approximately 2–14 phosphate modifications. For example, the guide chain may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more phosphate-modified nucleotides. The guide chain may contain one or more modifications that increase stability without interfering with RISC entry. Phosphate-modified nucleotides, such as phosphorothioate-modified nucleotides, may be located at the 3' end, the 5' end, or spread throughout the entire guide chain. In some embodiments, the 10 nucleotides at the 3' end of the guide chain contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 phosphorothioate-modified nucleotides. The guide chain 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 chain (the nucleotide at the furthest 5' position of the guide chain) is 2'OMe modified and / or phosphorylated and / or contains vinyl phosphonate. C and U nucleotides in the guide chain may be 2'F modified. For example, C and U nucleotides at positions 2–10 (or corresponding positions in chains of different lengths) of a 20-nucleotide guide chain may be 2'F modified. C and U nucleotides in the guide chain may also be 2'OMe modified. For example, C and U nucleotides at positions 11–18 (or corresponding positions in chains of different lengths) of a 19-nucleotide guide chain may be 2'OMe modified. In some embodiments, the nucleotide at the furthest 3' end of the guide chain is unmodified. In one embodiment, most of the C and U in the guide chain are 2'F modified, and the 5' end of the guide chain is phosphorylated. In other embodiments, C or U at position 1, and at positions 11–18, are 2'OMe modified, and the 5' end of the guide chain is phosphorylated. In another embodiment, the carbon or utrients at positions 1 and 11–18 are 2'OMe modified, the 5' end of the guide chain is phosphorylated, and the carbon or utrients at positions 2–10 are 2'F modified.

[0058] In some aspects, the passenger strand is approximately 11–14 nucleotides long. 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 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 modification. The passenger strand may also contain 2'ribo, 2'F and 2'deoxy modifications, or any combination thereof. 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.

[0059] Aspects of the present invention relate to RNAi constructs having a single-stranded region that is relatively longer than the double-stranded region when compared to molecules previously used for RNAi. The single-stranded region of the molecule may be modified to promote cellular uptake or gene silencing. In some embodiments, phosphorothioate modifications of the single-stranded region affect cellular uptake and / or gene silencing. The phosphorothioate-modified region of the guide strand may contain nucleotides within both the single-stranded and double-stranded regions of the molecule. In some embodiments, the single-stranded region contains 2 to 12 phosphorothioate modifications. For example, the single-stranded region may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 phosphorothioate modifications. In some examples, the single-stranded region contains 6 to 8 phosphorothioate modifications.

[0060] 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 greater than 10. The conjugate may be a sterol-type molecule such as cholesterol, or a molecule having an increased length polycarbonate chain 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 the presence of a lipid transfection reagent. The conjugate may be attached to the passenger strand or guide strand via a hydrophobic linker. In some embodiments, the hydrophobic linker is 5–12C long and / or hydroxypyrrolidine-based. In some embodiments, the hydrophobic conjugate is attached to the passenger strand, and a CU residue on either the passenger strand and / or 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 chain and / or guide chain are modified. In some aspects, the molecules relating to the present invention are self-delivery (sd). As used herein, “self-delivery” refers to the ability of a molecule to be delivered to cells without requiring an additional delivery vehicle, such as a transfection reagent.

[0061] An aspect of the present invention relates to the selection of molecules for use in RNAi. In some embodiments, molecules having a double-stranded region of 8 to 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 -13 kcal / mol will be selected. For example, the (ΔG) value may be less than -13, -14, -15, -16, -17, -18, -19, -21, -22, or -22 kcal / mol. In other embodiments, the (ΔG) value may be higher than -13 kcal / mol. For example, the (ΔG) value may be higher than -12, -11, -10, -9, -8, -7, or -7 kcal / mol. It should be understood that ΔG can be calculated using any method known in the art. In some embodiments, ΔG is calculated using Mfold, which is available through the Mfold internet site (mfold.bioinfo.rpi.edu / cgi-bin / rna-form1.cgi). The method for calculating ΔG is described in the following references and incorporated by reference: Zuker, M. (2003) Nucleic Acid 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.

[0062] In one embodiment, the polynucleotide contains 5' and / or 3' terminal overhangs. The number and / or sequence of nucleotide overhangs at one end of the polynucleotide may be the same as or different from that at the other end. In one embodiment, one or more of the overhanging nucleotides may contain chemical modifications such as phosphorothioates or 2'-OMe modifications. In one embodiment, the polynucleotide is unmodified. In another embodiment, at least one nucleotide is modified. In a further embodiment, the modification includes 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.

[0063] As used herein, "2'-modified ribose sugar" includes ribose sugars that do not have a 2'-OH group. "2'-modified ribose sugar" does not contain 2'-deoxyribose (found in unmodified reference DNA nucleotides). For example, a 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. In one embodiment, the 2'-modified nucleotide is a pyrimidine nucleotide (e.g., C / U). Examples of 2'-O-alkyl nucleotides include 2'-O-methyl nucleotides or 2'-O-allyl nucleotides.

[0064] In one embodiment, the sd-rxRNA polynucleotides of the present invention having the aforementioned 5' terminal modification exhibit significantly lower "off-target" gene silencing (e.g., at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more) compared to similar constructs without the identified 5' terminal modification, thereby greatly improving the overall specificity of RNAi reagents or treatments. As used herein, “off-target” gene silencing refers to unintended gene silencing resulting, for example, from false sequence homology between an antisense (guide) sequence and an unintended target mRNA sequence.

[0065] According to this aspect of the present invention, certain guide chain modifications further increase nuclease stability and / or reduce interferon induction without significantly reducing (or not reducing at all) RNAi activity. Certain combinations of modifications may yield further unexpected benefits, which may be partially expressed by enhanced ability to inhibit target gene expression, enhanced serum stability, and / or increased target specificity. In one embodiment, the guide chain contains a 2'-O-methyl modified nucleotide at the second nucleotide at the 5' end of the guide chain, but does not contain any other modified nucleotides.

[0066] In other aspects, the chemically modified double-stranded nucleic acid molecular structures of the present invention mediate sequence-dependent gene silencing via the microRNA mechanism. The term “microRNA” (“miRNA”) as used herein is also referred to in the art as “small temporal RNA” (“stRNA”), and refers to small (10-50 nucleotides) RNAs genetically encoded (e.g., by viruses, mammalian or plant genomes) that can direct or mediate RNA silencing. “miRNA disorder” should refer to a disease or disorder characterized by abnormal expression or activity of miRNAs.

[0067] MicroRNAs are involved in downregulating target genes in important pathways such as development and cancer in mice, nematodes, and mammals. Gene silencing via microRNA mechanisms is achieved through specific but still incomplete base pairing between miRNA and its target messenger RNA (mRNA). A variety of mechanisms may be employed in the microRNA-mediated downregulation of target mRNA expression.

[0068] miRNAs are non-coding RNAs of approximately 22 nucleotides that can regulate gene expression at post-transcriptional or post-translational levels throughout plant and animal development. One common characteristic of miRNAs is that they are cleaved from a precursor RNA stem-loop of approximately 70 nucleotides called pre-miRNA, possibly by a type III RNase enzyme such as Dicer or its homolog. Naturally occurring miRNAs are expressed in vivo by endogenous genes and are processed from hairpin or stem-loop precursors (pre-miRNA or pri-miRNA) by Dicer or other RNAses. miRNAs can exist transiently in vivo as double-stranded duplexes, but only one strand is incorporated into the RISC complex to direct gene silencing.

[0069] In some embodiments, versions of chemically modified double-stranded nucleic acid molecular compounds effective in inhibiting cellular uptake and miRNA activity are described. The compounds are essentially similar to the RISC-invading versions, but the large-chain chemical modification pattern blocks cleavage and acts as an effective inhibitor of RISC activity. For example, the compounds may be fully or mostly O-methyl modified with the phosphorothioate-containing compounds described earlier. For these types of compounds, 5' phosphorylation is not required in some embodiments. The presence of the double-stranded region is preferred because it promotes cellular uptake and efficient RISC loading.

[0070] 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. dsRNAs are cleaved by a 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 a target mRNA with perfect complementarity.

[0071] Several 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 microRNA in the miRNA mechanism. In one embodiment, a modified RNAi construct may have improved stability in serum and / or cerebrospinal fluid compared to an unmodified RNAi construct having the same sequence.

[0072] In one embodiment, the structure of the RNAi construct does not induce an interferon response in primary cells, such as mammalian primary cells, including primary cells from humans, mice, and other rodents, as well as other non-human mammals. In another embodiment, the RNAi construct may also be used to inhibit the expression of target genes in invertebrates.

[0073] To further increase the in vivo stability of the target construct, the 3' end of the structure may be blocked by a protecting group(s). Protecting groups such as inverted nucleotides, inverted debasing moieties, or amino-terminated nucleotides may be used. Inverted nucleotides may include inverted deoxynucleotides. Inverted debasing moieties may include inverted deoxybasing moieties such as 3',3'-linked or 5',5'-linked deoxybasing moieties.

[0074] The RNAi construct of the present invention can inhibit the synthesis of any target protein encoded by a target gene(s). The present invention includes a method for inhibiting the expression of a 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 the overexpression of a target gene.

[0075] The target gene may be endogenous or exogenous to the cell (e.g., introduced into the cell by a virus or using recombinant DNA technology). Such a method may involve introducing RNA into the cell in an amount sufficient to inhibit the expression of the target gene. For example, such RNA molecule may have a guide strand complementary to the nucleotide sequence of the target gene so that the composition inhibits the expression of the target gene. The present invention also relates to vectors expressing the nucleic acids of the present invention, and to cells containing such vectors or nucleic acids. The cells may be mammalian cells, such as human cells, in vivo or in culture.

[0076] The present invention further relates to a composition comprising a target RNAi construct and a pharmaceutically acceptable carrier or diluent. The method may be performed in vitro, ex vivo, or in vivo, for example, in cultured mammalian cells such as human cells in culture. Target cells (e.g., mammalian cells) may be brought into contact with a delivery reagent such as a lipid (e.g., a cationic lipid) or liposomes. Another aspect of the present invention provides a method for inhibiting the expression of a target gene in mammalian cells, comprising contacting mammalian cells with a vector expressing a target RNAi construct.

[0077] In one aspect of the present invention, a longer duplex polynucleotide is provided, comprising a first polynucleotide having a size in the range of about 16 to about 30 nucleotides and a second polynucleotide having a size in the range of about 26 to about 46 nucleotides, wherein the first polynucleotide (antisense strand) is complementary to both the second polynucleotide (sense strand) and the target gene, and both polynucleotides form a duplex, wherein the first polynucleotide contains a single-stranded region longer than 6 bases, is modified by a different chemical modification pattern, and / or contains a conjugate portion to facilitate cell delivery. In this embodiment, about 40 to about 90% of the nucleotides in the passenger strand, about 40 to about 90% of the nucleotides in the guide strand, and about 40 to about 90% of the nucleotides in the single-stranded region of the first polynucleotide are chemically modified nucleotides.

[0078] In one embodiment, the chemically modified nucleotides in a polynucleotide duplex may be any chemically modified nucleotides known in the art, such as those discussed in detail above. In a particular embodiment, the chemically modified nucleotides are selected from the group consisting of 2'F modified nucleotides, 2'-O-methyl modified nucleotides, and 2'-deoxy nucleotides. In another particular embodiment, the chemically modified nucleotides result from "hydrophobic modifications" of the nucleotide base. In yet another particular embodiment, the chemically modified nucleotides are phosphorothioates. In yet another particular embodiment, the chemically modified nucleotides are combinations of phosphorothioates, 2'-O-methyl, 2'-deoxy, hydrophobic modifications, and phosphorothioates. If the modifications in these groups refer to modifications of the ribose ring, backbone, and nucleotide, it is also feasible for some modified nucleotides to have combinations of all three types of modifications.

[0079] In another embodiment, the chemical modifications are not identical across diverse regions of the duplex. In a particular embodiment, the first polynucleotide (passenger chain) has numerous diverse chemical modifications at diverse sites. Up to 90% of this polynucleotide may be chemically modified and / or may have introduced mismatches.

[0080] In another embodiment, the chemical modification of the first or second polynucleotide includes, but is not limited to, modifications of uridine and cytosine at the 5' position (4-pyridyl, 2-pyridyl, indolyl, phenyl (C6H5OH); tryptophanyl (C8H6N)CH2CH(NH2)CO), isobutyl, butyl, aminobenzyl; phenyl; naphthyl, etc.), where the chemical modification may alter the base-pairing ability of the nucleotide. Regarding the guide strand, a key feature of this aspect of the invention is the location and sequence of chemical modifications relative to the 5' end of the antisense. For example, chemical phosphorylation of the 5' end of the guide strand is usually beneficial for potency. O-methyl modifications in the seed region of the sense strand (positions 2-7 relative to the 5' end) generally do not show good tolerance, while 2'F and deoxy modifications do. 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 do not show tolerance at the 3' end of the guide strand.

[0081] A unique feature of this aspect of the present invention is the use of hydrophobic modifications to the bases. In one embodiment, the hydrophobic modifications are preferably located near the 5' end of the guide chain; in another embodiment, they are localized in the middle of the guide chain; in yet another embodiment, they are localized at the 3' end of the guide chain; and in yet another embodiment, they are distributed throughout the entire length of the polynucleotide. The same type of pattern is applicable to the passenger chain of a duplex.

[0082] The other part of the molecule is a single-stranded region. This single-stranded region is predicted to range from 7 to 40 nucleotides. In one embodiment, the single-stranded region of the first polynucleotide contains modifications selected from the group consisting of 40% to 90% hydrophobic base modifications, 40% to 90% phosphorothioate modifications, 40% to 90% ribose moiety modifications, and any combination thereof. Since the efficiency of loading the guide chain (first polynucleotide) into the RISC complex may vary for heavily modified polynucleotides, in one embodiment, to facilitate efficient guide chain loading, the duplex polynucleotide contains a mismatch between nucleotides 9, 11, 12, 13, or 14 on the guide chain (first polynucleotide) and the opposite nucleotide on the sense chain (second polynucleotide). More detailed aspects of the present invention are described in the following sections.

[0083] Features of duplex The double-stranded oligonucleotide of the present invention may be formed from two separate, complementary nucleic acid strands. Duplex formation may occur either inside or outside the cell containing the target gene. As used herein, the term “duplex” includes a region of one or more double-stranded nucleic acid molecules that are hydrogen-bonded to a complementary sequence. The double-stranded oligonucleotides of the present invention may include a nucleotide sequence that is sense to the 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 to cause inhibition of the target gene (e.g., nearly at least about 98% identical, 96% identical, 94%, 90% identical, 85% identical, or 80% identical).

[0084] In one embodiment, the double-stranded oligonucleotide of the present invention is double-stranded along its entire length, that is, it does not have any protruding single-stranded sequences at any end of the molecule, i.e., it is blunt-ended. In another embodiment, individual nucleic acid molecules may be of different lengths. In other words, the double-stranded oligonucleotide of the present invention is not double-stranded along its entire length. For example, when two separate nucleic acid molecules are used, one of the molecules, for example, the first molecule containing the antisense sequence, may be longer than the second molecule that hybridizes to it (part of the molecule being single-stranded). Similarly, when a single nucleic acid molecule is used, any portion of the molecule at either end may remain single-stranded.

[0085] In one embodiment, the double-stranded oligonucleotide of the present invention contains mismatches and / or loops or bulges, but is double-stranded for at least about 70% of the oligonucleotide's length. In another embodiment, the double-stranded oligonucleotide of the present invention is double-stranded for at least about 80% of the oligonucleotide's length. In another embodiment, the double-stranded oligonucleotide of the present invention is double-stranded for at least about 90% to 95% of the oligonucleotide's length. In another embodiment, the double-stranded oligonucleotide of the present invention is double-stranded for at least about 96% to 98% of the oligonucleotide's length. In one embodiment, 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.

[0086] qualification The nucleotides of the present invention may be modified at various positions, including sugar moieties, phosphodiester linkages, and / or bases. In some embodiments, the base moiety of the nucleoside may be modified. For example, a pyrimidine base may be modified at positions 2, 3, 4, 5, and / or 6 of the pyrimidine ring. In some embodiments, the extracyclic amine of cytosine may be modified. Purine bases may also be modified. For example, a purine base may be modified at positions 1, 2, 3, 6, 7, or 8. In some embodiments, the extracyclic amine of 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 of the base moiety may be any preferred 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.

[0087] In some embodiments, the pyrimidine may be modified at position 5. For example, position 5 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, position 5 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 4 The position may be alkylated. In further examples, the pyrimidine 5-6 bond may be saturated, the nitrogen atom in the pyrimidine ring may be substituted with a carbon atom, and / or O 2 or O 4 The atom may be substituted with a sulfur atom. It should be understood that other modifications are also possible.

[0088] In other examples, the N of pudding 7 Position and / or N 2 and / or N 3 The position may be modified with an alkyl group or its substituted derivative. In further examples, the third ring may be condensed into a purine dicyclic system, and / or the nitrogen atom in the purine ring system may be substituted with a carbon atom. It should be understood that other modifications are also possible.

[0089] Non-limiting examples of pyrimidines modified at position 5 are disclosed in U.S. Patents 5,591,843, 7,205,297, 6,432,963 and 6,020,483; N 4 Non-limiting examples of pyrimidines modified at position 8 are disclosed in U.S. Patent No. 5,580,731; non-limiting examples of purines modified at position 8 are disclosed in U.S. Patent Nos. 6,355,787 and 5,580,972; N 6 Non-limiting examples of puddings modified with 1 are disclosed in U.S. Patents No. 4,853,386, No. 5,789,416 and No. 7,041,824; non-limiting examples of puddings modified with 2 are disclosed in U.S. Patents No. 4,201,860 and No. 5,587,469; all of these are incorporated herein by reference.

[0090] Non-restrictive examples of modified bases include N 4 ,N 4 -Ethanocytosine, 7-Deazaxanthosine, 7-Deazaguanosine, 8-Oxo-N 6 -Methyladenine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N 6 -Isopentenyl-adenine, 1-methyladenine, 1-methylpsoidouracil, 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-The bases include isopentenyl adenine, pseudouracil, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, 2-thiocytosine, and 2,6-diaminopurine. In some embodiments, the base portion may be a heterocyclic base other than a purine or pyrimidine. The heterocyclic base may be optionally modified and / or substituted.

[0091] The sugar moiety includes naturally occurring unmodified sugars, such as monosaccharides (pentoses, e.g., ribose, deoxyribose), modified sugars, and sugar analogs. Generally, possible modifications of the nucleomonomone, particularly those of the sugar moiety, include, for example, the substitution of one or more hydroxyl groups with halogens, heteroatoms, or aliphatic groups, or the functionalization of hydroxyl groups with ethers, amines, thiols, etc.

[0092] A particularly useful group of modified nucleomonomers are 2'-O-methyl nucleotides. Such 2'-O-methyl nucleotides may also be referred to as "methylated," and the corresponding nucleotides are produced from unmethylated nucleotides by alkylation or directly from methylation 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.

[0093] Some exemplary modified nucleomonomons include ribonucleotides with modified sugars or backbones. Modified ribonucleotides may contain bases that do not exist naturally (in place 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 (NH2, NHR, NR2, etc.), or CN group, where R is a lower alkyl, alkenyl, or alkynyl group.

[0094] Modified ribonucleotides may also have phosphodiester groups linked to adjacent ribonucleotides, which are replaced by modifying groups, such as phosphorothioate groups. More generally, a variety of nucleotide modifications may be combined. The antisense (guide) strand may be substantially identical with respect to at least a portion of the target gene(s), but the sequence does not need to be exactly identical, for example, to inhibit the phenotypic expression of the target gene, as long as it is useful, at least in relation to base-pairing properties. Generally, 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 with respect to the target gene (in the antisense direction).

[0095] The use of 2'-O-methyl-modified RNA may also be beneficial in situations where minimizing the cellular stress response is desirable. RNA with a 2'-O-methyl nucleomonomer cannot be recognized by cellular mechanisms thought to recognize unmodified RNA. 2'-O-methylated or partially 2'-O-methylated RNA may evade the interferon response to double-stranded nucleic acids while maintaining target RNA inhibition. This may be useful, for example, in both short RNAi (e.g., siRNA) sequences that induce an interferon response and longer RNAi sequences that can induce an interferon response, in order to evade interferon or other cellular stress responses.

[0096] Overall, the 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 as described (Augustyns, K., et al., Nucl. Acids. Res. 18:4711 (1992)) and may be incorporated into oligonucleotides. Exemplary nucleomonomers can be found, for example, in U.S. Patent No. 5,849,902, which is incorporated herein by reference.

[0097] Specific definitions of functional groups and chemical terms are described in further detail below. For the purposes of this invention, chemical elements are identified according to the periodic table on the inside cover of the CAS version of Handbook of Chemistry and Physics, 75th Ed., and specific functional groups are generally defined as described therein. Furthermore, general principles of organic chemistry, as well as specific functional parts and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito: 1999, the entirety of which is incorporated herein by reference.

[0098] Some compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention takes into account 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 chiral carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are intended to be included in the present invention.

[0099] Isomer mixtures containing any of the various isomer ratios may be used in accordance with the present invention. For example, when only two isomers are combined, mixtures containing isomer 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 intended in the present invention. Those skilled in the art will readily understand that similar ratios are intended for more complex isomer mixtures.

[0100] For example, if a specific enantiomer of the compound of the present invention is desired, it may be prepared by asymmetric synthesis or by derivatization with a chiral auxiliary group, and the resulting diastereomer mixture is separated and the auxiliary group 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 diastereomer salt is formed with a suitable optically active acid or base, and the diastereomer thus formed is then separated by fractional crystallization or chromatographic means known in the art, and subsequently the pure enantiomer is recovered.

[0101] In one embodiment, the oligonucleotides of the present invention include 3' and 5' termini (except for cyclic oligonucleotides). In one embodiment, the 3' and 5' termini of an oligonucleotide can be substantially protected from nucleases by modifying, for example, the 3' or 5' bond (e.g., U.S. Patent No. 5,849,902 and WO98 / 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 nucleo monomer as either a protecting group or a bonding group for synthesis (e.g., FITC, propyl (CH2-CH2-CH3), glycol (-O-CH2-CH2-O-), phosphate (PO3)). 2- ), hydrogen phosphonate or phosphoramidite). The “blocking group” also includes “terminal blocking groups” or “exonuclease blocking groups,” which protect the 3' and 5' ends of oligonucleotides, including modified nucleotides and non-nucleotide exonuclease-resistant structures.

[0102] Exemplary terminal blocking groups include cap structures (e.g., 7-methylguanosine cap), inverted nucleomonomers, e.g., those with 3'-3' or 5'-5' terminal inversions (see, e.g., Ortiagao et al. 1992. Antisense Res. Dev. 2:129), methylphosphonates, phosphoramidites, and non-nucleotide groups (e.g., non-nucleotide linkers, aminolinkers, conjugates). The 3'-terminal nucleomonomer may contain a modified sugar moiety. The 3'-terminal nucleomonomer may contain a 3'-O that can be optionally substituted with a blocking group that prevents 3'-exonuclease degradation of the oligonucleotide. For example, a 3'-hydroxyl group can be esterified to a nucleotide via a 3'→3' nucleotide linkage. For example, the alkyloxy radical may be methoxy, ethoxy, or isopropoxy, preferably ethoxy. Optionally, the 3'→3' linked nucleotide at the 3' terminus may be linked by alternative linkages. To reduce nuclease degradation, the most 5' 3'→5' linkage can be a modified linkage, such as a phosphorothioate or p-alkyloxyphosphotriester linkage. Preferably, the two most 5' 3'→5' linkages are modified linkages. Optionally, the 5'-terminal hydroxyl moiety can be esterified with a phosphorus-containing moiety, such as a phosphate, phosphorothioate, or p-ethoxyphosphate.

[0103] Those skilled in the art will understand that the synthesis method utilizes various protecting groups, as described herein. As used herein, the term “protecting group” means that a particular functional group, e.g., O, S, or N, is temporarily blocked, allowing the reaction to proceed selectively at another reaction site in a polyfunctional compound. In some embodiments, the protecting group reacts selectively in good yield to provide a protected substrate that is stable for the planned reaction; the protecting group should be selectively removable in good yield with readily available, preferably non-toxic, reagents that do not attack other functional groups; the protecting group forms a readily separable derivative (more preferably without the formation of new stereocenters); and the protecting group has minimal additional functionality to avoid having further reaction sites.

[0104] As detailed herein, oxygen, sulfur, nitrogen, and carbon protecting groups may be used. Hydroxyl protecting groups include: methyl, methoxymethyl (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-methoxypiperidine-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,

[0105] 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-dibenzosberyl, triphenylmethyl, α-na Phthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, 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-(imidazole-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,

[0106] 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 (rebrinate), 4,4-(ethylenedithio)pentanoate (rebrinoyldithioacetal), pivaloate, adamantate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (methitoate),

[0107] Alkylmethylcarbonate, 9-Fluorenylmethylcarbonate (Fmoc), Alkylethylcarbonate, Alkyl 2,2,2-Trichloroethylcarbonate (Troc), 2-(Trimethylsilyl)ethylcarbonate (TMSEC), 2-(Phenylsulfonyl)ethylcarbonate (Psec), 2-(Triphenylphosphonio)ethylcarbonate (Peoc), Alkylisobutylcarbonate, Alkylvinylcarbonate, Alkylallylcarbonate, Alkylp-nitrophenylcarbonate, Alkylbenzylcarbonate, Alkylp-methoxybenzylcarbonate, Alkyl3,4-Dimethoxybenzylcarbonate, Alkylo-nitrobenzylcarbonate, Alkylp-nitrobenzylcarbonate, AlkylS-benzylthiocarbonate, 4-Ethoxy-1-Naphthylcarbonate, Methyldithiocarbonate,

[0108] 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, chlorodiphen Luacetate, isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate, o-(methoxycarbonyl)benzoate, α-naphthoate, nitrate, alkyl N,N,N',N'-tetramethylphosphodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinthiole, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate and tosylate (Ts).

[0109] To protect 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, dimethoxymethylene orthoester, 1-methoxyethylidene orthoester, 1-ethoxyethylidene orthoester, 1,2-dimethoxyethylidene orthoester, α-methoxybenzylidene orthoester, 1-(N,N-dimethylamino Ethylidene derivatives, α-(N,N'-dimethylamino)benzylidene derivatives, 2-oxacyclopentylidene orthoesters, 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.

[0110] The amino protecting groups include: methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluorenylmethyl carbamate, and 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxantyl)]methyl carbamate (DBD-Tm). oc), 4-methoxyphenacylcarbamate (Phenoc), 2,2,2-trichloroethylcarbamate (Troc), 2-trimethylsilylethylcarbamate (Teoc), 2-phenylethylcarbamate (hZ), 1-(1-adamantyl)-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2-haloethylcarbamate, 1,1-dimethyl-2,2-dibromoethylcarbamate (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), 2-(N,N-dicyclohexylcarboxamide)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, alkyl dithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),

[0111] p-nitrobenzylcarbamate, p-bromobenzylcarbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzylcarbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethylcarbamate, diphenylmethylcarbamate, 2-methylthioethylcarbamate, 2-methylsulfonylethylcarbamate, 2-(p-toluenesulfonyl)ethylcarbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenylcarbamate (Mtpc), 2,4-dimethylthiophenylcarbamate (Bmpc), 2-phosphonioethylcarbamate (Peoc), 2-triphenylphosphonioisopropylcarbamate Maat (Ppoc), 1,1-dimethyl-2-cyanoethylcarbamate, m-chloro-p-acyloxybenzylcarbamate, p-(dihydroxyboryl)benzylcarbamate, 5-benzisoxazolylmethylcarbamate, 2-(trifluoromethyl)-6-chromonylmethylcarbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzylcarbamate, o-nitrobenzylcarbamate, 3,4-dimethoxy-6-nitrobenzylcarbamate, phenyl(o-nitrophenyl)methylcarbamate, phenothiazinyl-(10)-carbonyl derivatives, N'-p-toluenesulfonylaminocarbonyl derivatives, N'-phenylaminothiocarbonyl derivatives,

[0112] t-Amylcarbamate, S-Benzylthiocarbamate, p-Cyanobenzylcarbamate, Cyclobutylcarbamate, Cyclohexylcarbamate, Cyclopentylcarbamate, Cyclopropylmethylcarbamate, p-Decyloxybenzylcarbamate, 2,2-Dimethoxycarbonylvinylcarbamate, o-(N,N-Dimethylcarboxamide)benzylcarbamate, 1,1-Dimethyl-3-(N,N-Dimethylcarboxamide)propylcarbamate, 1,1-Dimethylpropynylcarbamate, Di(2-Pyridyl)methylcarbamate, 2-Furanylmethylcarbamate, 2-Iodoethylcarbamate, Isobornylcarbamate, Isobutylcarbamate, Isonicotinyl Carbamate, p-(p'-methoxyphenylazo)benzylcarbamate, 1-methylcyclobutylcarbamate, 1-methylcyclohexylcarbamate, 1-methyl-1-cyclopropylmethylcarbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethylcarbamate, 1-methyl-1-(p-phenylazophenyl)ethylcarbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethylcarbamate, phenylcarbamate, p-(phenylazo)benzylcarbamate, 2,4,6-tri-t-butylphenylcarbamate, 4-(trimethylammonium)benzylcarbamate, 2,4,6-trimethylbenzylcarbamate,

[0113] Formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl derivatives, benzamide, p-phenylbenzamide, o-nitrophenylacetamide, o-nitrophenoxyacetamide, acetamide, (N'-dithiobenzyloxycarbonylamino)acetamide, 3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide, 2-methyl-2-(o-phenylazofenoxy)propanamide D, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine derivative, 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), 5-substituted 1,3-dimethyl-1,3,5-triazacyclohexane-2-one, 5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexane-2-one, 1-substituted 3,5-dinitro-4-pyridone,

[0114] N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1-isopropyl-4-nitro-2-oxo-3-pyrroline-3-yl)amine, quaternary ammonium salt, N-benzylamine, N-di(4-methoxyphenyl)methylamine, N-5-dibenzosperylamine, 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-benzylidene Min, 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-cyclohexyllideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine, N-borane derivatives, N-diphenylboric acid derivatives, N-[phenyl(pentacarbonylchromium- or tungsten)carbonyl]amine, N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosamine, amine N-oxide,

[0115] Diphenylphosphinamide (Dpp), Dimethylthiophosphinamide (Mpt), Diphenylthiophosphinamide (Ppt), Dialkylphosphorumidates, Dibenzylphosphorumidates, Diphenylphosphorumidates, Benzensulfenamide, o-Nitrobenzenesulfenamide (Nps), 2,4-Dinitrobenzenesulfenamide, Pentachlorobenzenesulfenamide, 2-Nitro-4-methoxybenzenesulfenamide, Triphenylmethylsulfenamide, 3-Nitropyridinesulfenamide (Npys), p-Toluenesulfonamide (Ts), Benzensulfonamide, 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.

[0116] Exemplary protecting groups are described in detail herein. However, the present invention is not intended to be limited to these protecting groups, but rather various additional equivalent protecting groups can be readily 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.

[0117] As described herein, it is understood that compounds may be substituted with any number of substituents or functional moieties. In general, whether preceded by the term “arbitrarily,” the term “substituted” and the substituents included in the formulas of this invention refer to the replacement of a hydrogen radical in a given structure with a particular substituent radical. If more than one position in any given structure may be substituted with more than one substituent selected from a particular group, the substituents may be identical or different at each position. As used herein, the term “substituted” is intended to include all acceptable substituents of an organic compound. In a broad view, acceptable substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of an organic compound. Heteroatoms such as nitrogen may have any acceptable substituent of the organic compounds described herein that satisfy the valence of the hydrogen substituent and / or the heteroatom. Furthermore, the present invention is not intended to be limited in any way by the acceptable substituents of an organic compound. The combinations of substituents and variables envisioned in the present invention preferably result in the formation of stable compounds useful for treating, for example, infectious diseases or proliferative disorders. The term “stable” preferably, as used herein, means a compound that is stable enough to enable production, maintains the integrity of the compound for a sufficient time to be detectable, and preferably is useful for a sufficient time for the purposes detailed herein.

[0118] As used herein, the term “aliphatic” includes both saturated and unsaturated linear (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, as used herein, the term “alkyl” includes linear, branched, and cyclic alkyl groups. Similar conventions apply to other general terms, such as “alkenyl,” “alkynyl,” etc. Furthermore, as used herein, the terms “alkyl,” “alkenyl,” “alkynyl,” etc., encompass both substituted and unsubstituted groups. In some embodiments, as used herein, “lower alkyl” is used to refer to alkyl groups (cyclic, acyclic, substituted, unsubstituted, branched, or unbranched) having 1 to 6 carbon atoms.

[0119] In one embodiment, the alkyl, alkenyl, and alkynyl groups used in the present invention contain 1 to 20 aliphatic carbon atoms. In another embodiment, the alkyl, alkenyl, and alkynyl groups used in the present invention contain 1 to 10 aliphatic carbon atoms. In yet another embodiment, the alkyl, alkenyl, and alkynyl groups used in the present invention contain 1 to 8 aliphatic carbon atoms. In yet another embodiment, the alkyl, alkenyl, and alkynyl groups used in the present invention contain 1 to 6 aliphatic carbon atoms. In yet another embodiment, the alkyl, alkenyl, and alkynyl groups used in the present invention contain 1 to 4 aliphatic carbon atoms. Therefore, the aliphatic groups exemplified are not limited to these, but include, 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 moiety, etc., which may have one or more substituents. The alkenyl groups are not limited to these, but include, for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, etc. Typical alkynyl groups are not limited to these, but include, for example, ethinyl, 2-propynyl (propargyl), 1-propynyl, etc.

[0120] Some examples of substituents on the aliphatic (and other) moieties of the compounds of the present invention, but not limited to these, include: 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(Rx )2;-OC(O)R x ;-OCO2R x ;-OCON(R x )2;-N(R x )2;-S(O)2R x ;-NR x (CO)R x Herein, each appearance of Rx independently includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl or heteroarylalkyl substituents, 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. Examples of generally applicable additions of substituents are illustrated by the specific embodiments described herein.

[0121] As used herein, the term "heteroaliphatic" refers to an aliphatic moiety containing, for example, one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms instead of carbon atoms. Heteroaliphatic moieties may be branched or unbranched, cyclic or acyclic, and may include saturated and unsaturated heterocycles such as morpholino and pyrrolidinyl. In some embodiments, a heteroaliphatic moiety is substituted by the independent substitution of one or more hydrogen atoms thereon by one or more moieties, including but not limited to: 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 ; Here R x Each of the appearances independently includes, but is not limited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl substituents, 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 any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted. Examples of generally applicable additions of substituents are illustrated by the specific embodiments described herein. As used herein, the terms "halo" and "halogen" refer to an atom selected from fluorine, chlorine, bromine, and iodine.

[0122] The term "alkyl" includes saturated aliphatic groups, which include linear alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.), branched alkyl groups (e.g., isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups (cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In some embodiments, linear or branched alkyl groups have 6 or fewer carbon atoms in their skeleton (e.g., C1-C6 for linear groups, C3-C6 for branched groups), more preferably 4 or fewer carbon atoms. Similarly, preferred cycloalkyl groups have 3-8 carbon atoms in their ring structure, more preferably 5 or 6 carbon atoms in their ring structure. The term C1-C6 includes alkyl groups containing 1-6 carbon atoms.

[0123] Furthermore, unless otherwise specified, the term alkyl includes both "unsubstituted alkyl" and "substituted alkyl," the latter referring to an alkyl moiety having independently selected substituents that replace hydrogens on one or more carbons of a hydrocarbon skeleton. 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, sulfonamide, nitro, trifluoromethyl, cyano, azide, heterocyclyl, alkylaryl, or aromatic or heteroaromatic moieties. Cycloalkyls may be further substituted, for example, by the substituents mentioned above. The "alkylaryl" or "arylalkyl" moiety is an alkyl group substituted with an aryl group (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.

[0124] The term "alkenyl" includes unsaturated aliphatic groups that are similar in length to the alkyl groups described above and are substituted for them, but which contain at least one double bond. For example, the term "alkenyl" includes linear alkenyl groups (e.g., etylene, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, etc.), branched 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, linear or branched alkenyl groups have six or fewer carbon atoms in their skeleton (e.g., C2-C6 for linear groups, C3-C6 for branched groups). Similarly, a cycloalkenyl group may have 3 to 8 carbon atoms in its ring structure, more preferably 5 or 6 carbon atoms. The term C2-C6 includes alkenyl groups containing 2 to 6 carbon atoms.

[0125] Furthermore, unless otherwise specified, the term alkenyl includes both "unsubstituted alkenyls" and "substituted alkenyls," the latter referring to an alkenyl moiety having independently selected substituents that replace hydrogens on one or more carbons of a hydrocarbon skeleton. Such substituents include, for example, alkyl groups, alkynyl groups, halogens, hydroxyls, alkylcarbonyloxys, arylcarbonyloxys, alkoxycarbonyloxys, carboxylates, alkylcarbonyls, arylcarbonyls, alkoxycarbonyls, aminocarbonyls, alkylaminocarbonyls, dialkylaminocarbonyls, alkylthiocarbonyls, alkoxyls, phosphates, phosphonates, phosphinates, cyanos, aminos (including alkylaminos, dialkylaminos, arylaminos, diarylaminos, and alkylarylaminos), acylaminos (including alkylcarbonylaminos, arylcarbonylaminos, carbamoyls, and ureidos), amidinos, iminos, sulfhydryls, alkylthios, arylthios, thiocarboxylates, sulfates, alkylsulfinyls, sulfonates, sulfamoyls, sulfonamides, nitros, trifluoromethyls, cyanos, azides, heterocyclyls, alkylaryls, or aromatic or heteroaromatic moieties.

[0126] The term "alkynyl" includes unsaturated aliphatic groups that are similar in length to the alkyl groups described above and are substituted for them, but which contain at least one triple bond. For example, the term "alkynyl" includes linear alkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octinyl, noninyl, desynyl, etc.), branched alkynyl groups, and cycloalkyl or cycloalkenyl-substituted alkynyl groups. In some embodiments, linear or branched alkynyl groups have six or fewer carbon atoms in their skeleton (e.g., C2-C6 for linear groups, C3-C6 for branched groups). The term C2-C6 includes alkynyl groups containing two to six carbon atoms.

[0127] Furthermore, unless otherwise specified, the term alkynyl includes both "unsubstituted alkynyls" and "substituted alkynyls," the latter referring to an alkynyl moiety having independently selected substituents that replace hydrogens on one or more carbons of a hydrocarbon skeleton. Such substituents include, for example, alkyl groups, alkynyl groups, halogens, hydroxyls, alkylcarbonyloxys, arylcarbonyloxys, alkoxycarbonyloxys, carboxylates, alkylcarbonyls, arylcarbonyls, alkoxycarbonyls, aminocarbonyls, alkylaminocarbonyls, dialkylaminocarbonyls, alkylthiocarbonyls, alkoxyls, phosphates, phosphonates, phosphinates, cyanos, aminos (including alkylaminos, dialkylaminos, arylaminos, diarylaminos, and alkylarylaminos), acylaminos (including alkylcarbonylaminos, arylcarbonylaminos, carbamoyls, and ureidos), amidinos, iminos, sulfhydryls, alkylthios, arylthios, thiocarboxylates, sulfates, alkylsulfinyls, sulfonates, sulfamoyls, sulfonamides, nitros, trifluoromethyls, cyanos, azides, heterocyclyls, alkylaryls, or aromatic or heteroaromatic moieties.

[0128] Unless otherwise specified, “lower alkyl” means an alkyl group having 1 to 5 carbon atoms in its skeletal structure, as defined above, as used herein. “Lower alkenyl” and “lower alkynyl” have, for example, a chain length of 2 to 5 carbon atoms. The term "alkoxy" includes substituted and unsubstituted alkyl, alkenyl, and alkynyl groups covalently bonded 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 alkylali) They may be substituted with independently selected groups such as halogen-substituted alkoxy groups (including arylaminos), acylaminos (including alkylcarbonylaminos, arylcarbonylaminos, carbamoyls, and ureidos), amidinos, iminos, sulffydryls, alkylthios, arylthios, thiocarboxylates, sulfates, alkylsulfumyls, sulfonates, sulfamoyls, sulfonamides, nitros, trifluoromethyls, cyanos, azides, heterocyclyls, alkylaryls, or aromatic or heteroaromatic moieties. Examples of halogen-substituted alkoxy groups include, but are not limited to, fluoromethoxys, difluoromethoxys, trifluoromethoxys, chloromethoxys, dichloromethoxys, and trichloromethoxys.

[0129] 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" refers to a group that has -OH or -O- (with the appropriate counterion). The term "halogen" includes elements such as fluorine, bromine, chlorine, and iodine. The term "perhalogenation" generally refers to a region where all hydrogen atoms are replaced by halogen atoms.

[0130] The term "substituted" includes an independently selected substituent that can be placed in the part and enables 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 OR' group; where each R' and R'' independently comprises hydrogen, a C1-C5 alkyl, a C2-C5 alkenyl, a C2-C5 alkynyl, or aryl group, or together R' and R'' comprises a benzylidene group or a -(CH2)2O(CH2)2- group.

[0131] The terms "amine" or "amino" include 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 nitrogen is bonded to at least one further alkyl group. The term "dialkylamino" includes groups in which a nitrogen atom is bonded to at least two additional alkyl groups. The term "ether" includes a compound or moiety containing oxygen bonded to two different carbon atoms or heteroatoms. For example, the term includes "alkoxyalkyl," which refers to an alkyl, alkenyl, or alkynyl group covalently bonded to an oxygen atom that is covalently bonded to another alkyl group.

[0132] The terms "polynucleotide," "nucleotide sequence," "nucleic acid," "nucleic acid molecule," "nucleic acid sequence," and "oligonucleotide" refer to polymers of two or more nucleotides. Polynucleotides may be DNA, RNA, or derivatives or modified versions thereof. Polynucleotides may be single-stranded or double-stranded. Polynucleotides may be modified in the base moiety, sugar moiety, or phosphate backbone to improve, for example, molecular stability or their hybridization parameters. Polynucleotides may contain modified base moieties 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, inosine, N 6-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-methylthio -N6-isopentenyl adenine, wybutoxosine, pseudouracil, queosin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetate methyl ester, uracil-5-oxyacetate, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Polynucleotides may also 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-phosphoamidite bonds). Nucleotide sequences typically contain genetic information, including information used by cellular mechanisms to make proteins and enzymes. These terms encompass double-stranded or single-stranded genomes and cDNA, RNA, any synthetic and genetically engineered polynucleotides, and both sense and antisense polynucleotides. This includes single-stranded and double-stranded molecules, namely DNA-DNA, DNA-RNA, and RNA-RNA hybrids, as well as "protein nucleic acids" (PNAs) formed by conjugating bases to an amino acid backbone.

[0133] The term "base" includes 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. Examples of purines include adenine, guanine, inosine, diaminopurine, and xanthine and their analogs (e.g., 8-oxo-N6-methyladenine or 7-diazaxanthine) and derivatives. Pyrimidines include, for example, thymine, uracil, and cytosine and their analogs (e.g., 5-methylcytosine, 5-methyluracil, 5-(1-propynyl)uracil, 5-(1-propynyl)cytosine, and 4,4-ethanocytosine). Other suitable examples of bases include non-purinyl and non-pyrimidinyl bases such as 2-aminopyridines and triazines.

[0134] In a preferred embodiment, the nucleomonomer of the oligonucleotide of the present invention is an RNA nucleotide. In another preferred embodiment, the nucleomonomer of the oligonucleotide of the present invention is a modified RNA nucleotide. Thus, the oligonucleotide contains a modified RNA nucleotide. The term “nucleoside” includes a base covalently bonded to a sugar moiety, preferably ribose or deoxyribose. Examples of preferred nucleosides include ribonucleosides and deoxyribonucleosides. Nucleosides also include bases bonded to amino acids or amino acid analogs, which 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 TW Greene, “Protective Groups in Organic Synthesis,” 2nd edition, Wiley-Interscience, New York, 1999). The term "nucleotide" includes nucleosides that further contain a phosphate group or a phosphate analog.

[0135] Nucleic acid molecules may be bound to a hydrophobic moiety for targeting and / or delivery of the molecule to cells. In one embodiment, the hydrophobic moiety is bound to the nucleic acid molecule via a linker. In one embodiment, the binding is via a non-covalent interaction. In another embodiment, the binding is via a covalent bond. Any linker known in the art may be used to bind 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 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. 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 may be cleaved from nucleic acids. In one embodiment, the linker can be hydrolyzed under physiological conditions. In another embodiment, the linker can be cleaved by an enzyme (e.g., an esterase or phosphodiesterase). In one embodiment, the linker includes a spacer element that separates the nucleic acid from the hydrophobic moiety. The spacer element may contain 1 to 30 carbon or heteroatoms. In one embodiment, the linker and / or spacer element includes a protonable functional group. Such a protonable functional group may facilitate the endosomal escape of the nucleic acid molecule. The protonable 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 another embodiment, the linker and / or spacer element is biologically inert (i.e., does not confer biological activity or function to the resulting nucleic acid molecule).

[0136] In one embodiment, the nucleic acid molecule having a linker and a hydrophobic moiety is of the formula described herein. In one embodiment, the nucleic acid molecule is of the formula: [ka] During the ceremony, X is either N or CH; A is a conjugated; substituted or unsubstituted, cyclic or acyclic, branched or unbranched aliphatic; or a substituted or unsubstituted, cyclic or acyclic, branched or unbranched heteroaliphatic; R 1 This is the hydrophobic part; R 2 are hydrogen; oxygen protecting groups; 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 It is a nucleic acid. It is represented as follows.

[0137] In one aspect, the numerator is given by the formula: [ka] It is represented as follows.

[0138] In one aspect, the numerator is given by the formula: [ka] It is represented as follows.

[0139] In one aspect, the numerator is given by the formula: [ka] It is represented as follows.

[0140] In one aspect, the numerator is given by the formula: [ka] It is represented as follows.

[0141] In one embodiment, X is N. In another embodiment, X is CH. In one embodiment, A is a bond. In one embodiment, A is a substituted or unsubstituted, cyclic or acyclic, branched or unbranched aliphatic. In one embodiment, A is an acyclic, substituted or unsubstituted, branched or unbranched aliphatic. In one embodiment, A is an acyclic, substituted, branched or unbranched aliphatic. In one embodiment, A is an acyclic, substituted, unbranched aliphatic. In one embodiment, A is an acyclic, substituted, unbranched alkyl. In one embodiment, A is an acyclic, substituted, unbranched C 1~20 It is alkyl. In one embodiment, A is acyclic, substituted, unbranched C 1~12 It is alkyl. In one embodiment, A is acyclic, substituted, unbranched C 1~10 It is alkyl. In one embodiment, A is acyclic, substituted, unbranched C 1~8 It is alkyl. In one embodiment, A is acyclic, substituted, unbranched C 1~6 It is 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, unbranched heteroaliphatic.

[0142] In one way, A is given by the formula: [ka] It is represented as follows.

[0143] In one way, A is given by the formula: [ka] It is represented by one of the following.

[0144] In one way, A is given by the formula: [ka] It is represented by one of the following.

[0145] In one way, A is given by the formula: [ka] It is represented by one of the following.

[0146] In one way, A is given by the formula: [ka] It is represented as follows.

[0147] In one way, A is given by the formula: [ka] It is represented as follows.

[0148] In one way, A is given by the formula: [ka] During the ceremony, Each appearance of R is independently a side chain of a natural or unnatural amino acid; and n is an integer from 1 to 20 (including the boundary). It is represented as follows.

[0149] In one way, A is given by the formula: [ka] It is represented as follows.

[0150] In one embodiment, each occurrence of R is independently a side chain of a native amino acid. In one embodiment, n is an integer from 1 to 15 (inclusive of the boundary). In one embodiment, n is an integer from 1 to 10 (inclusive of the boundary). In one embodiment, n is an integer from 1 to 5 (inclusive of the boundary).

[0151] In one way, A is given by the formula: [ka] In the formula, n is an integer from 1 to 20 (including the boundary). It is expressed as follows: In one embodiment, A is given by the formula: [ka] It is represented as follows.

[0152] In one embodiment, n is an integer from 1 to 15 (including the boundary). In another embodiment, n is an integer from 1 to 10 (including the boundary). In another embodiment, n is an integer from 1 to 5 (including the boundary).

[0153] In one way, A is given by the formula: [ka] In the formula, n is an integer from 1 to 20 (including the boundary). It is represented as follows.

[0154] In one way, A is given by the formula: [ka] It is represented as follows.

[0155] In one embodiment, n is an integer from 1 to 15 (including the boundary). In another embodiment, n is an integer from 1 to 10 (including the boundary). In another embodiment, n is an integer from 1 to 5 (including the boundary).

[0156] In one aspect, the numerator is given by the formula: [ka] In the formula, X, R 1 , R 2 and R 3 This is as defined herein; and A' is a substituted or unsubstituted, cyclic or acyclic, branched or unbranched aliphatic; or a substituted or unsubstituted, cyclic or acyclic, branched or unbranched heteroaliphatic. It is represented as follows.

[0157] In one aspect, A' is given by the formula: [ka] It is represented by one of the following.

[0158] In one way, A is given by the formula: [ka] It is represented by one of the following.

[0159] In one way, A is given by the formula: [ka] It is represented by one of the following.

[0160] In one way, A is given by the formula: [ka] It is represented as follows.

[0161] In one embodiment, A is of the formula:

Chem.

[0162] In one embodiment, R 1 is a steroid. In one embodiment, R 1 is cholesterol. In one embodiment, R 1 is a lipophilic vitamin. In one embodiment, R 1 is vitamin A. In one embodiment, R 1 is vitamin E.

[0163] <​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ is represented by

[0167] In one embodiment, R 1 is of the formula:

Chem.

[0168] In one embodiment, R 1 is of the formula:

Chem.

[0169] In one embodiment, the nucleic acid molecule is of the formula:

Chem.

[0170] In one embodiment, the nucleic acid molecule is of the formula:

Chem.

[0171] In one embodiment, nucleic acid molecules are given by formula: [ka] During the ceremony, X is either N or CH; A is a conjugated; substituted or unsubstituted, cyclic or acyclic, branched or unbranched aliphatic; or a substituted or unsubstituted, cyclic or acyclic, branched or unbranched heteroaliphatic; R 1 This is the hydrophobic part; R 2 are hydrogen; oxygen protecting groups; 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 It is a nucleic acid. It is represented as follows.

[0172] In certain embodiments, the nucleic acid molecule has the formula: [Chemical formula] as represented by:

[0173] In certain embodiments, the nucleic acid molecule has the formula: [Chemical formula] as represented by:

[0174] In certain embodiments, the nucleic acid molecule has the formula: [Chemical formula] wherein R 3 is a nucleic acid, as represented by:

[0175] In certain embodiments, the nucleic acid molecule has the formula: [Chemical formula] wherein R 3 is a nucleic acid; and n is an integer from 1 to 20 (including the boundaries).<0C00986>as represented by:

[0176] In certain embodiments, the nucleic acid molecule has the formula: [Chemical formula] as represented by:

[0177] " In certain embodiments, the nucleic acid molecule has the formula: [Chemical formula] as represented by:

[0178] In certain embodiments, the nucleic acid molecule has the formula: [ka] It is represented as follows.

[0179] In one embodiment, nucleic acid molecules are given by formula: [ka] It is represented as follows.

[0180] In one embodiment, nucleic acid molecules are given by formula: [ka] It is represented as follows.

[0181] As used herein, the term "linkage" refers to an unmodified phosphodiester moiety (-O-(PO) that is covalently bonded to an adjacent nucleomonomer in nature. 2- This includes )-O-). The term “substitute linkage” as used herein includes any analog or derivative of a native phosphodiester group that is covalently bonded to an adjacent nucleomonomer. Substitute linkages include phosphodiester analogs, e.g., phosphorothioates, phosphorodithioates and P-ethyoxyphosphodiesters, P-ethoxyphosphodiesters, P-alkyloxyphosphodiesters, methylphosphonates and phosphorus-free linkages, e.g., acetals and amides. Such substitute linkages are known in the art (e.g., 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.

[0182] In some embodiments, the oligonucleotides of the present invention include hydrophobically modified nucleotides or “hydrophobic modifications.” As used herein, “hydrophobic modifications” refer to bases modified such that (1) the overall hydrophobicity of the base is significantly increased, and / or (2) the base can still form interactions similar to the normal Watson-Crick interaction. Some non-limiting examples of base modifications include uridine and cytidine modifications at the 5th position, e.g., phenyl, 4-pyridyl, 2-pyridyl, indolyl, and isobutyl, phenyl(C6H5OH); tryptophanyl(C8H6N)CH2CH(NH2)CO), isobutyl, butyl, aminobenzyl; phenyl; and naphthyl.

[0183] Other types of conjugates that can attach to the terminal (3' or 5' end), loop region, or any other part of a chemically modified double-stranded nucleic acid molecule include sterols, sterol-type molecules, peptides, small molecules, proteins, and the like. 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 properties). In some embodiments, the conjugate is cholesterol.

[0184] In some embodiments, the first nucleotide relative 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' vinyl phosphonate 2'-O-methyl U modification. Another method for increasing target gene specificity or reducing off-target silencing 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) sequence of the present invention may be a "chimeric oligonucleotide" containing RNA-like and DNA-like regions.

[0185] The term “RNase H activating region” includes a region of an oligonucleotide, such as a chimeric oligonucleotide, that can recruit RNase H to cleave a target RNA strand to which the oligonucleotide binds. Typically, the RNase H activating region includes a minimum core of DNA or DNA-like nucleomonomers (at least about 3 to 5, typically about 3 to 12, more typically about 5 to 12, more preferably about 5 to 10, consecutive nucleomonomers) (see, for example, U.S. Patent No. 5,849,902). Preferably, the RNase H activating region includes about 9 consecutive deoxyribose-containing nucleomonomers.

[0186] The term "inactivation region" includes antisense sequences, such as regions of a chimeric oligonucleotide, that do not recruit or activate RNase H. Preferably, the inactivation region does not contain phosphorothioate DNA. The oligonucleotide of the present invention comprises at least one inactivation region. In one embodiment, the inactivation region may be stable to a nuclease or may provide specificity to a target by being complementary to the target and forming hydrogen bonds with the target nucleic acid molecule bound by the oligonucleotide. In one embodiment, at least a portion of a continuous polynucleotide is linked by a substitution linkage, such as a phosphorothioate linkage.

[0187] In some embodiments, most or all nucleotides beyond the guide sequence are linked (whether 2' modified or not) by phosphorothioate ligatures. Such constructs tend to exhibit improved pharmacokinetics due to their higher affinity for serum proteins. Phosphorothioate ligatures in the non-guide sequence portion of polynucleotides generally do not interfere with the activity of the guide chain once it has been loaded into RISC. In some embodiments, high levels of phosphorothioate modification can result in improved delivery. In some embodiments, the guide and / or passenger chains are fully phosphorothioate-modified.

[0188] The antisense (guide) sequence of the present invention may include a "morpholino oligonucleotide." Morpholino oligonucleotides are nonionic and function by an RNase H-independent mechanism. The four gene bases of a morpholino oligonucleotide (adenine, cytosine, guanine, and thymine / uracil) are linked to a six-membered morpholine ring. Morpholino oligonucleotides are created by linking four different subunit types, for example, by nonionic phosphorodiamidate subunit ligators. Morpholino oligonucleotides offer numerous advantages, including: complete resistance to nucleases (Antisence & Nucl. Acid Drug Dev. 1996. 6:267); predictable targeting (Biochemica Biophysica Acta. 1999. 1489:141); reliable activity in cells (Antisence & Nucl. Acid Drug Dev. 1997. 7:63); excellent sequence specificity (Antisence & Nucl. Acid Drug Dev. 1997. 7:151); minimal non-antisense activity (Biochemica Biophysica Acta. 1999. 1489:141); and convenient delivery by osmotic pressure or scraping (Antisence & 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.

[0189] The chemical modifications described herein are thought to promote 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 thought to be two to four orders of magnitude lower compared to duplex polynucleotides. The present invention provides a description of chemical modification patterns that (a) significantly increase the stability of single-stranded polynucleotides, (b) promote efficient loading of polynucleotides into RISC complexes, and (c) improve cellular uptake of single-stranded polynucleotides. Chemical modification patterns may include combinations of ribose modification, skeletal modification, hydrophobic nucleoside modification, and conjugate modification. In addition, in some embodiments, the 5' end of a single polynucleotide may be chemically phosphorylated.

[0190] In yet another embodiment, 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 pre-loaded RISC complexes through substrate competition mechanisms. For these types of molecules, commonly referred to as antagonists, activity is usually required at high concentrations, and in vivo delivery is not very effective. The present invention provides a description of chemical modification patterns that (a) significantly increase the stability of single-stranded polynucleotides, (b) facilitate efficient recognition of polynucleotides as substrates by RISC, and / or (c) improve the uptake of single-stranded polynucleotides by cells. Chemical modification patterns may include combinations of ribose modifications, skeletal modifications, hydrophobic nucleoside modifications, and conjugate modifications.

[0191] The modifications provided by the present invention are applicable to all polynucleotides. This includes single-stranded RISC-penetrating polynucleotides, single-stranded RISC-inhibiting polynucleotides, conventional duplexed polynucleotides of various lengths (15-40 bp), and asymmetric duplexed polynucleotides. Polynucleotides may be modified by a variety of chemical modification patterns, including 5'-end modifications, ribose modifications, skeletal modifications, and hydrophobic nucleoside modifications.

[0192] synthesis The oligonucleotides of the present invention can be synthesized by any method known in the art, for example, by enzymatic synthesis and / or chemical synthesis. The oligonucleotides can be synthesized in vitro (for example, by enzymatic synthesis and chemical synthesis) or in vivo (by recombinant DNA techniques well known in the art). In some embodiments, chemical synthesis is used for modified polynucleotides. The chemical synthesis of linear oligonucleotides is well known in the art and can be achieved by solution or solid-phase techniques. Preferably, the synthesis is by solid-phase methods. Oligonucleotides may be produced by any of several different synthetic procedures, typically by automated methods, including phosphoramidite, phosphite triester, H-phosphonate, and phosphotryester methods.

[0193] The synthesis protocols for oligonucleotides are well known in the art, for example, U.S. Patent No. 5,830,653; WO98 / 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. It can be found in 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; WO92 / 03568; U.S. Patent No. 5,276,019; and U.S. Patent No. 5,264,423.

[0194] The choice of synthesis method may depend on the desired oligonucleotide length, and such choice is within the scope of the art. For example, the phosphoramidite and phosphite triester methods can produce oligonucleotides having 175 or more nucleotides, while the H-phosphonate method works well for oligonucleotides with fewer than 100 nucleotides. When modified bases are incorporated into the oligonucleotide, and especially when modified phosphodiester linkages are used, the synthesis procedure can be modified as needed according to known procedures. In this regard, Uhlmann et al. (1990, Chemical Reviews 90:543-584) provide references and outline procedures for preparing oligonucleotides having modified bases and modified phosphodiester linkages. Other exemplary methods for preparing 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, MJ IRL Press at Oxford University Press, 1984). Furthermore, linear oligonucleotides of specified sequences, including several sequences with modified nucleotides, are readily available from several commercial sources.

[0195] 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, oligonucleotides may be subjected to DNA sequencing by any known procedure, including Maxam-Gilbert sequencing, Sanger sequencing, capillary electrophoresis sequencing, wandering spot sequencing, or by selective chemical degradation of oligonucleotides bound to Hybond paper. The sequences of short oligonucleotides can also be analyzed by laser desorption mass spectrometry or fast atomic 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.

[0196] The quality of synthesized oligonucleotides can be confirmed by testing them using, for example, the method described in Bergot and Egan. 1992. J. Chrom. 599:35, by capillary electrophoresis and denaturing strong anion HPLC (SAX-HPLC). Other exemplary synthesis techniques are well known in the art (see, for example, Sambrook et al., Molecular Cloning: a Laboratory Manual, Second Edition (1989); DNA Cloning, Volumes I and II (DN Glover Ed. 1985); Oligonucleotide Synthesis (MJ Gait Ed. 1984); Nucleic Acid Hybridization (BD Hames and SJ Higgins eds. 1984); A Practical Guide to Molecular Cloning (1984); or the series Methods in Enzymology (Academic Press, Inc.)).

[0197] In one embodiment, the target RNAi construct or at least a portion thereof is transcribed from an expression vector encoding 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 any desired modifications (such as replacing the unmodified sense strand with a modified one) are made.

[0198] Delivery / Carrier While not wishing to be bound by any particular theory, the inventors believe that certain patterns of modifications on the passenger and guide strands of the double-stranded nucleic acid molecules described herein (e.g., INTASYL™) facilitate the entry of the guide strand into the nucleus, and that the guide strand mediates gene silencing (e.g., silencing of target genes such as BRD4).

[0199] While we do not wish to be constrained by any theory, several potential mechanisms of action may 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 in the nucleus, the single-stranded guide strand binds to RNAse H or another ribonuclease and cleaves the target (e.g., BRD4) ("antisense mechanism"). In some embodiments, the guide strand (e.g., antisense strand) of a nucleic acid molecule (e.g., INTASYL™) can bind to an Argonaut (Ago) protein in the cytoplasm or outside the nucleus to form a loaded Ago complex. This loaded Ago complex can then migrate to the nucleus and subsequently cleave the target (e.g., BRD4). In some embodiments, both strands (e.g., 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 to cleave a target (e.g., BRD4).

[0200] Those skilled in the art will understand that the sense strand of the double-stranded molecule described herein (e.g., the sense strand of INTASYL®) is not limited to the delivery of the guide strand of the double-stranded nucleic acid molecule described herein. Rather, in some embodiments, the passenger strand described herein is bound to another molecule (e.g., an antisense oligonucleotide, ASO) for the purpose of targeting the cell nucleus (e.g., by covalent, non-covalent, conjugation, hybridization via a complementary region, etc.). In some embodiments, the molecule bound to the sense strand described herein is a synthetic antisense oligonucleotide (ASO). In some embodiments, the sense strand bound to the antisense oligonucleotide is 8 to 15 nucleotides long, chemically modified, and includes a hydrophobic conjugate.

[0201] While we do not wish to be constrained by any particular theory, ASOs can be bound to a complementary passenger chain by hydrogen bonds. In some respects, this disclosure provides a method for delivering a nucleic acid molecule to a cell, comprising administering an isolated nucleic acid molecule to a cell, wherein the isolated nucleic acid comprises a sense chain complementary to an antisense oligonucleotide (ASO), wherein the sense chain is 8-15 nucleotides long and comprises at least two phosphorothioate modifications, wherein at least 50% of the pyrimidines in the sense chain are modified, wherein the molecule comprises a hydrophobic conjugate.

[0202] Cellular uptake of oligonucleotides Oligonucleotides and oligonucleotide compositions are brought into contact with (i.e., contacted with, or referred to herein as administered or delivered to) one or more cells or cell lysates and taken up therein. 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 brought into contact with bacterial cells. In some embodiments, the oligonucleotide compositions of the present invention are brought into contact 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 brought into contact with stem cells. In some embodiments, the oligonucleotide compositions of the present invention are brought into contact with immune cells such as T cells (e.g., CD8+ T cells). In some embodiments, T cells are T SCM or T CM These are cells. In a preferred embodiment, the oligonucleotide composition of the present invention is brought into contact with human cells.

[0203] The oligonucleotide compositions of the present invention may be brought into contact with cells in vitro, for example, in a test tube or culture dish (with or without introduction into the subject), or in vivo, for example, in a subject such as a mammalian subject, or ex vivo. In some embodiments, the oligonucleotides are administered topically or via electroporation. Oligonucleotides are taken up by cells at a slow rate by endocytosis, but endocytosized oligonucleotides are generally isolated and not available for hybridization to target nucleic acid molecules, for example. In one embodiment, cellular uptake can be facilitated by electroporation or calcium phosphate precipitation. However, these procedures are useful only in in vitro or ex vivo embodiments, are not convenient, and in some cases are associated with cytotoxicity.

[0204] In another embodiment, the delivery of oligonucleotides into cells may be enhanced by preferred methods recognized in the art, including calcium phosphate, DMSO, glycerol, or dextran, electroporation, or by transfection, using, for example, 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 oligonucleotide delivery can also be achieved using 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 polylysine, protamine or polycation conjugates using compounds such as 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. It can also be mediated by the use of (see 88:4255).

[0205] In one embodiment, the chemically modified double-stranded nucleic acid molecules of the present invention may be delivered using a variety of beta-glucan-containing particles, referred to as GeRP (glucan-encapsulated RNA-loaded particles), which are described in U.S. Provisional Application No. 61 / 310,611, filed March 4, 2010, titled “Formulations and Methods for Targeted Delivery to Phagocytic Cells,” and incorporated by reference. Such particles are also described in U.S. Patent Publications US2005 / 0281781 A1 and US2010 / 0040656, and PCT Publications WO2006 / 007372 and WO2007 / 050643, and incorporated by reference. The chemically modified double-stranded nucleic acid molecules may be hydrophobically modified and optionally bound to lipids and / or amphiphilic peptides. In one embodiment, the beta-glucan particles are derived from yeast. In one embodiment, the payload trapping molecule is a polymer with a molecular weight of at least about 1,000 Da, 10,000 Da, 50,000 Da, 100 kDa, 500 kDa, etc. Preferred polymers include (without limitation) cationic polymers, chitosan, or PEI (polyethyleneimine).

[0206] Glucan particles may be derived from insoluble components of fungal cell walls, such as yeast cell walls. In some embodiments, the yeast is baker's yeast. Yeast-derived glucan molecules may include one or more β-(1,3)-glucans, β-(1,6)-glucans, mannans, and chitins. In some embodiments, the glucan particles contain hollow yeast cell walls, thereby allowing the particles to maintain a cell-like three-dimensional structure, within which they can complex with or encapsulate molecules such as RNA molecules. Some advantages associated with the use of yeast cell wall particles include the availability of their components, their biodegradable nature, and their ability to target phagocytic cells.

[0207] In some embodiments, glucan particles may be prepared by extracting insoluble components from cell walls, for example, by extracting baker's yeast (Fleischmann's) with 1 M NaOH / pH 4.0, H2O, followed by washing and drying. Methods for preparing yeast cell wall particles are discussed in the following literature and incorporated by reference therefrom: U.S. Patents 4,810,646, 4,992,540, 5,082,936, 5,028,703, 5,032,401, 5,322,841, 5,401,727, and 5,504. U.S. Patent Publications No. 079, No. 5,607,677, No. 5,968,811, No. 6,242,594, No. 6,444,448, No. 6,476,003, U.S. Patent Publications 2003 / 0216346, 2004 / 0014715, and 2010 / 0040656, and PCT Publication WO02 / 12348.

[0208] Protocols for preparing glucan particles are also described in the following literature and are incorporated by reference: Soto and Ostroff (2008), "Characterization of multilayered nanoparticles encapsulated in yeast cell wall particles for DNA delivery"; Bioconjug Chem 19(4):840-8; Soto and Ostroff (2007), "Oral Macrophage Mediated Gene Delivery System," Nanotech, Volume 2, Chapter 5 ("Drug Delivery"), pages 378-381; and Li et al. (2007), "Yeast glucan particles activate murine resident macrophages to secrete proinflammatory cytokines via MyD88-and Syk kinase-dependent pathways." Clinical Immunology 124(2):170-181.

[0209] Glucan-containing particles, such as yeast cell wall particles, can also be commercially available. Some non-limiting examples include: Nutricell MOS 55 from Biorigin (Sao Paolo, Brazil), SAF-Mannan (SAF Agri, Minneapolis, Minn.), Nutrex (Sensient Technologies, Milwaukee, Wis.), alkali-extracted particles, e.g., those manufactured by Nutricepts (Nutricepts Inc., Burnsville, Minn.) and ASA Biotech, acid-extracted WGP particles and organic solvent-extracted particles, e.g., Adjuvax™ from Alpha-beta Technology, Inc. (Worcester, Mass.) and microparticle glucans from Novogen (Stamford, Conn.).

[0210] Glucan particles, such as yeast cell wall particles, can have varying levels of purity depending on the manufacturing and / or extraction method. In some cases, particles are extracted using alkaline, acid, or organic solvent extraction to remove intracellular components and / or the outer mannoprotein layer of the cell wall. Such protocols can produce particles with a glucan content (w / w) ranging from 50 to 90%. In some cases, low-purity particles, i.e., particles with a low glucan w / w content, may be preferred, while in other embodiments, high-purity particles, i.e., particles with a high glucan w / w content, may be preferred.

[0211] Glucan particles, such as yeast cell wall particles, may have a natural lipid content. For example, particles may contain lipids exceeding 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or 20% w / w. In some embodiments, the presence of natural lipids may facilitate the complexation or capture of RNA molecules.

[0212] Glucan-containing particles typically have a diameter of 2 to 4 microns, but particles with a diameter of less than 2 microns or greater than 4 microns also fit aspects of the present invention. The RNA molecule(s) to be delivered is complexed with a glucan particle or "captured" within its shell. The particle shell or RNA component may be labeled for visualization, as described and incorporated by reference in Soto and Ostroff (2008) Bioconjug Chem 19:840. The method for loading GeRP is discussed further below.

[0213] The protocol used for oligonucleotide uptake will depend on numerous factors, but the most important is the type of cells used. Other important factors in uptake include, but are not limited to, the properties and concentration of the oligonucleotide, cell confluence, 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.

[0214] Immunomodulatory composition and method for producing the same In some embodiments, the chemically modified double-stranded nucleic acid molecules described herein (e.g., INTASYL® molecules) are useful for the production of specific cell subtypes or T cell subtypes for immunomodulatory compositions. When used herein, “immunomodulatory composition” is a composition comprising host cells containing the chemically modified nucleic acid molecules described herein and / or host cells treated with the chemically modified nucleic acid molecules described herein. The immunomodulatory composition may optionally further comprise one or more pharmaceutically acceptable excipients or carriers. Without intending to be bound by any specific theory, the immunomodulatory compositions described herein are useful for the production of specific cell subtypes (e.g., T SCM or T CMCharacterized by populations of immune cells (e.g., T cells, NK cells, antigen-presenting cells (APCs), dendritic cells (DCs), stem cells (SCs), induced pluripotent stem cells (iPSCs), etc.) that have been engineered to have enriched populations of T cell subtypes such as T cells, and therefore, in some embodiments, are useful for modulating (e.g., stimulating or inhibiting) the immune response of a target.

[0215] As used herein, “host cell” refers to a cell into which one or more chemically modified double-stranded nucleic acid molecules have been introduced. Typically, host cells are mammalian cells, such as human cells, mouse cells, rat cells, pig cells, etc. However, in some embodiments, host cells are non-mammalian cells, such as prokaryotic cells (e.g., bacterial cells), yeast cells, insect cells, etc. Generally, host cells are derived from a donor, such as a healthy donor (e.g., cells into which chemically modified double-stranded nucleic acids are introduced are taken from a donor, such as a healthy donor). For example, cells 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, “healthy donor” refers to a subject that does not have, or is not suspected of having, a proliferative disorder or infectious disease (e.g., a bacterial infection, a viral infection, or a parasitic infection). However, in some embodiments, host cells are derived from a subject that has (or is suspected of having) a proliferative disorder or infectious disease, for example, in the context of autologous cell therapy.

[0216] In some embodiments, the cells (e.g., host cells) are immune cells, such as T cells, B cells, dendritic cells (DCs), granulocytes, natural killer cells, macrophages, etc. In some embodiments, the cells (e.g., host cells) are cells that can differentiate into immune cells such as stem cells (SCs) or induced pluripotent stem cells (iPSCs). In some embodiments, the cells (e.g., host cells) are stem cell memory T cells, as described, for example, in Gattinoni et al. (2017) Nature Medicine 23;18-27, which are incorporated by reference thereto. In some embodiments, the cells (e.g., host cells) are T cells such as killer T cells, helper T cells, regulatory T cells, or tumor-infiltrating lymphocytes (TILs). In some embodiments, the T cells are killer T cells (e.g., CD8+ T cells). In some embodiments, the T cells are helper T cells (e.g., CD4+ T cells). In some embodiments, the T cells are activated T cells (e.g., T cells on which a peptide antigen is presented by an MHC class II molecule on an antigen-presenting cell). In some embodiments, T cells contain one or more transgenes that express a high-affinity T cell receptor (TCR) and / or a chimeric antibody receptor (CAR).

[0217] In some aspects, this disclosure relates to the finding that introducing one or more chemically modified double-stranded nucleic acid molecules of this disclosure (e.g., one or more INTASYL® molecules) into cells (e.g., immune cells obtained from a donor) to produce host cells is characterized in the host cells by a significant decrease in the expression or activity of one or more signaling / transcription factors, epigenetic, metabolic and / or co-inhibitory / negative regulatory proteins (e.g., BRD4, etc.). In some embodiments, the host cells are characterized by approximately 5% to approximately 50% reduced expression of immune checkpoint proteins compared to cells without chemically modified double-stranded nucleic acid molecules (e.g., immune cells of the same cell type). In some embodiments, host cells are characterized by a greater than 50% reduction in the expression of molecularly related targets (e.g., signaling molecules, kinases / phosphatases, transcription factors, epigenetic modulators, metabolic and regulatory targets) compared to cells that do not contain chemically modified double-stranded nucleic acid molecules (e.g., immune cells of the same cell type) (e.g., immune cells of a subject having or suspected of having a proliferative or infectious disease).

[0218] In some embodiments, the immunomodulatory compositions described herein include a plurality of host cells. In some embodiments, the plurality of host cells may be approximately 10,000 host cells per kilogram, approximately 50,000 host cells per kilogram, approximately 100,000 host cells per kilogram, approximately 250,000 host cells per kilogram, approximately 500,000 host cells per kilogram, or approximately 1 x 10¹⁶ host cells per kilogram. 6 Host cells, approximately 5 x 10⁶ per kilogram 6 Host cells, approximately 1 x 10⁶ per kilogram 7 Host cells, approximately 1 x 10⁶ per kilogram 8 Host cells, approximately 1 x 10⁶ per kilogram 9 Host cells, or 1 x 10⁶ per kilogram 9 The host cell population is larger than the host cell population. In some aspects, multiple host cells are approximately 1 x 10⁶ per kilogram. 5 ~1x10 14 It is a host cell.

[0219] In some aspects, this disclosure provides a method for producing the immunomodulatory compositions described herein. In some embodiments, the method comprises introducing one or more chemically modified double-stranded nucleic acid molecules (e.g., INTASYL®) into cells, wherein one or more chemically modified double-stranded nucleic acid molecules target BRD4, thereby targeting a specific cell subtype or T cell subtype (e.g., T SCM or T CM It produces host cells containing ).

[0220] Methods for producing immunomodulatory compositions (e.g., host cells or populations of host cells) may be carried out in vitro, ex vivo, or in vivo in mammalian cells in culture, such as human cells in culture. In some embodiments, target cells (e.g., cells obtained from a donor) may be contacted in the presence of delivery reagents such as lipids (e.g., cationic lipids) or liposomes to facilitate the entry of chemically modified double-stranded nucleic acid molecules into the cells, as described in further detail elsewhere in this disclosure.

[0221] Carrier and composite agent This disclosure further relates to compositions comprising the RNAi construct described herein and a pharmaceutically acceptable carrier or diluent. In some aspects, this disclosure relates to immunomodulatory compositions comprising the RNAi construct described herein and a pharmaceutically acceptable carrier.

[0222] As used herein, “pharmaceutically acceptable carrier” includes suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption retarders, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional media or agent may be used in a therapeutic composition unless it is incompatible with the active ingredient. Auxiliary active ingredients may also be incorporated into the composition.

[0223] 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 the liposomes, such as antibodies reactive to membrane proteins found in specific target cells, may help target the oligonucleotides to specific cell types (e.g., immune cells such as T cells).

[0224] The encapsulating agent captures the oligonucleotide within the vesicle. In another embodiment of the present invention, the oligonucleotide may be bound to a carrier or vehicle, such as a liposome or micelle, but other carriers may be used, as will be understood by those skilled in the art. Liposomes are vesicles consisting of a lipid bilayer having a structure similar to that of a biological membrane. Such carriers are used to promote cellular uptake, to target the oligonucleotide, or to improve the pharmacokinetic or toxicological properties of the oligonucleotide.

[0225] For example, the oligonucleotides of the present invention may also be administered encapsulated in liposomes, which are pharmaceutical compositions in which the active ingredient is dispersed therein or contained in small bodies comprising an aqueous concentrated layer adhered to a lipid layer. Depending on their solubility, the oligonucleotides may be present in both the aqueous and lipid layers, or in what is generally called a liposomic 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 phosphatidylic acid, or other materials with hydrophobic properties. The diameter of liposomes generally ranges from approximately 15 nm to about 5 microns.

[0226] The use of liposomes as drug delivery vehicles offers several advantages. Liposomes increase intracellular stability, enhance uptake efficiency, and improve biological activity. Liposomes are hollow, spherical vesicles composed of lipids arranged in a manner similar to the lipids that make up the cell membrane. They have an internal aqueous space for encapsulating 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.

[0227] The specific advantages of using liposomes include: they are non-toxic and biodegradable in composition; they exhibit a long cyclic half-life; and the recognition molecules can easily adhere to their surfaces for targeting of tissues. Finally, the cost-effective production of liposome-based pharmaceuticals, whether in liquid suspension or lyophilized form, demonstrates the viability of this technology as an acceptable drug delivery system.

[0228] In some aspects, formulations relating to the present invention may be selected from classes of saturated and unsaturated fatty acid residues, whether naturally occurring, chemically synthesized, or modified. Fatty acids may exist in the form of triglycerides, diglycerides, or individual fatty acids. In another embodiment, the use of well-established mixtures of fatty acid and / or lipid emulsions currently used in pharmacology for parenteral nutrition may be utilized.

[0229] Liposome-based formulations are widely used for oligonucleotide delivery. However, most 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 for obtaining high levels of oligonucleotide loading and for enhancing the membrane fusion properties of liposomes. Several methods have been implemented and published for identifying functionally positively charged lipid compounds. However, commercially available liposome formulations containing cationic lipids are characterized by high levels of toxicity. Limited therapeutic in vivo indices have 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.

[0230] Nucleic acids related to the present invention may be hydrophobically modified and incorporated into neutral nanotransporters. Further description of neutral nanotransporters is incorporated by reference from PCT application PCT / US2009 / 005251, titled "Neutral Nanotransporters," filed September 22, 2009, and U.S. Patent Publication U.S.2011 / 0237522, titled "Neutral Nanotransporters," published September 29, 2011. Such particles enable the quantitative incorporation of oligonucleotides into uncharged lipid mixtures. An important characteristic of such neutral nanotransporter compositions is that cationic lipids are not at toxic levels.

[0231] As demonstrated in PCT / US2009 / 005251, oligonucleotides can be efficiently incorporated into lipid mixtures that do not contain cationic lipids, and such compositions can effectively deliver therapeutic oligonucleotides to cells in a functional manner. For example, high levels of activity have been observed when the lipid mixture consists of phosphatidylcholine-based fatty acids and sterols such as cholesterol. As an example, one preferred formulation of a neutral lipid mixture consists of at least 20% DOPC or DSPC and at least 20% sterols such as cholesterol. It has been shown that even a low lipid-to-oligonucleotide ratio of 1:5 is sufficient to obtain complete encapsulation of oligonucleotides in uncharged formulations.

[0232] The neutral nanotransporter composition enables the efficient loading of oligonucleotides into neutral lipid formulations. The composition comprises oligonucleotides modified in a manner that increases the hydrophobicity of the molecules (e.g., hydrophobic molecules are attached (covalently or noncovalently) to the hydrophobic molecules on oligonucleotide-terminated or non-terminative nucleotides, bases, sugars, or backbones), and the modified oligonucleotides are mixed with a neutral lipid formulation (e.g., containing at least 25% cholesterol and 25% DOPC or its analogues). Cargo molecules, such as other lipids, may also be included in the composition. This composition enables the efficient encapsulation of oligonucleotides in neutral lipid particles when a portion of the formulation is constructed within the oligonucleotides themselves.

[0233] In some respects, stable particles in the size range of 50 to 140 nm can be formed by complexing hydrophobic oligonucleotides with a preferred formulation. The formulation itself typically does not form small particles, but rather aggregates, which are converted into stable 50–120 nm particles by the addition of hydrophobic modified oligonucleotides.

[0234] In some embodiments, the neutral nanotransporter composition comprises a hydrophobic modified polynucleotide, a neutral lipid mixture, and optionally a cargo molecule. As used herein, “hydrophobic modified polynucleotide” refers to the polynucleotide of the present invention (e.g., sd-rxRNA) having at least one modification that makes the polynucleotide more hydrophobic than before the modification. The modification may be achieved by attaching (covalently or noncovalently) a hydrophobic molecule to the polynucleotide. In some examples, the hydrophobic molecule is or contains a lipophilic group.

[0235] The term "lipophilic group" refers to a group that has a higher affinity for lipids than for water. Examples of lipophilic groups are not limited to these, but include cholesterol, cholesteryl or modified cholesteryl residues, adamantine, dihydrotesterone, long-chain alkyl groups, long-chain alkenyl groups, long-chain alkynyl groups, oleyl-lithocholic acid, collentic acid, oleoyl-colenic acid, palmitic acid, heptadecylic acid, myristic acid, bile acids, cholic acid or taurocholic acid, deoxycholic acid, oleyl-lithocholic acid, oleoyl-colenic acid, glycolipids, phospholipids, and sulfites. This material contains ingolipids, isoprenoids such as steroids, vitamins such as vitamin E, saturated or unsaturated fatty acids, fatty acid esters such as triglycerides, pyrenes, porphyrins, texaphyllin, adamantane, acridines, biotin, coumarin, fluorescein, rhodamine, Texas-Red, digoxigenin, dimethoxytrityl, t-butyldimethylsilyl, t-butyldiphenylsilyl, cyanine pigments (e.g., Cy3 or Cy5), Hoechst 33258 pigment, psoralen, or ibuprofen. The cholesterol portion may be reduced (e.g., as in cholestane) or substituted (e.g., by halogens). Combinations of different lipophilic groups in a single molecule are also possible.

[0236] Hydrophobic molecules may be attached at various positions on the polynucleotide. As described above, hydrophobic molecules may be ligated to terminal residues of the polynucleotide, such as the 3' or 5' end. Alternatively, they may be ligated to nucleotides inside the polynucleotide or to nucleotides on branches. Hydrophobic molecules may be attached, for example, to the 2' position of a nucleotide. Hydrophobic molecules may also be ligated to heterocyclic bases, sugars, or backbones of the nucleotides in the polynucleotide.

[0237] Hydrophobic molecules may be linked to polynucleotides by linker moieties. Optionally, the linker moieties are non-nucleotide linker moieties. Non-nucleotide linkers include, for example, debasic residues (d-spacers), oligoethylene glycols such as triethylene glycol (spacer 9) or hexaethylene glycol (spacer 18), or alkanediols such as butanediol. The spacer units are preferably linked by phosphodiester or phosphorothioate bonds. The linker units may appear only once in the molecule, or they may be incorporated several times, for example, via phosphodiesters, phosphorothioate, methylphosphonate, or amine linkages.

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

[0239] In some embodiments, the hydrophobic molecules are sterol conjugates, phytosterol conjugates, cholesterol conjugates, sterol conjugates with altered side chain lengths, fatty acid conjugates, any other hydrophobic group conjugates, and / or hydrophobic modifications of internal nucleosides, which provide sufficient hydrophobicity for incorporation into micelles.

[0240] For the purposes of this invention, the term "sterol" or steroid alcohols refers to a subgroup of steroids having a hydroxyl group at position 3 of the A ring. These are amphiphilic lipids synthesized from acetyl-coenzyme A via the HMG-CoA reductase pathway. The overall molecule is extremely flattened. The hydroxyl group on the A ring is polar. The remainder of the aliphatic chain is nonpolar. Typically, sterols are considered to have an 8-carbon chain at position 17. For the purposes of this invention, the term "sterol-type molecule" refers to steroid alcohols, which are structurally similar to sterols. The main differences are the ring structure and the number of carbon atoms in the side chain bonded at position 21.

[0241] For the purposes of this invention, the term "phytosterol" (also referred to as plant sterol) refers to a group of steroid alcohols, which are naturally occurring phytochemicals in plants. More than 200 types of phytosterols are known. For the purposes of this invention, the term "sterol side chain" refers to the chemical composition of the side chain attached at position 17 of a sterol-type molecule. In the standard definition, sterols are limited to a tetracyclic structure having an 8-carbon chain at position 17. In this invention, sterol-type molecules having longer and shorter side chains than conventional ones are described. The side chain may be branched or may contain a double skeleton.

[0242] Therefore, sterols useful in the present invention include, for example, cholesterol, as well as unique sterols having a side chain longer than 2 to 7 or 9 carbons attached at the 17th position. In some embodiments, the length of the polycarbonate tail varies between 5 and 9 carbons. Such conjugates may have significantly better in vivo efficacy, particularly in delivery to the liver. These types of molecules are expected to work at concentrations 5 to 9 times lower than conventional cholesterol-conjugated oligonucleotides.

[0243] Alternatively, the polynucleotide may be bound to a protein, peptide, or a positively charged chemical substance that functions as a hydrophobic molecule. The protein may be selected from the group consisting of protamines, dsRNA-binding domains, and arginine-rich peptides. Exemplary positively charged chemical substances include spermine, spermidine, cadaverine, and putrescine.

[0244] In another embodiment, hydrophobic molecular 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).

[0245] In another embodiment, the sterol-type molecule may be a naturally occurring phytosterol. The polycarbonate chain may be longer than nine units, linear, branched, and / or contain double bonds. Some phytosterols containing polynucleotide conjugates may be significantly more potent and active in the delivery of polynucleotides to diverse tissues. Since some phytosterols can demonstrate tissue selectivity, they may be used as a means for the specific delivery of RNAi to particular tissues.

[0246] Hydrophobic modified polynucleotides are mixed with a neutral fatty acid mixture to form micelles. The neutral fatty acid mixture is a mixture of lipids that are net neutral or slightly net negatively charged at or near the physiological pH in which micelles can be formed with the hydrophobic modified polynucleotides. For the purposes of this invention, the term “micelle” refers to small nanoparticles formed from a mixture of uncharged fatty acids and phospholipids. The neutral fatty acid mixture may contain cationic lipids, insofar as they are present in amounts that do 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 the physiological pH. The term “anionic lipid” includes lipids and synthetic lipids that have a net negative charge at or near the physiological pH. Neutral lipids bind to the oligonucleotides of the present invention by strong but non-covalent attractive forces (e.g., electrostatic, van der Waals, pi-stacking, etc.).

[0247] The neutral lipid mixture may comprise formulations selected from naturally occurring, chemically synthesized, or modified classes of saturated and unsaturated fatty acid residues. Fatty acids may exist in the form of triglycerides, diglycerides, or individual fatty acids. In another embodiment, well-established mixtures and / or lipid emulsions of fatty acids currently used for parenteral nutrition in pharmacology may be utilized.

[0248] 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 registration number 4235-95-4) is dioleoylphosphatidylcholine (also known as dielydoylphosphatidylcholine, dioleoyl-PC, dioleoylphosphocholine, dioleoyl-sn-glycero-3-phosphocholine, and dioleylphosphatidylcholine). DSPC (compound registration number 816-94-4) is distearoylphosphatidylcholine (also known as 1,2-distearoyl-sn-glycero-3-phosphocholine).

[0249] The sterols in the neutral fatty acid mixture may, for example, be cholesterol. The neutral fatty acid mixture may consist entirely of choline-based fatty acids and sterols, or it may optionally contain cargo molecules. For example, the neutral fatty acid mixture may have at least 20% or 25% fatty acids and 20% or 25% sterols.

[0250] For the purposes of this invention, the term “fatty acid” refers to the conventional description of fatty acids. These may exist as individual entities or in the form of diglycerides and triglycerides. For the purposes of this invention, the term “lipid emulsion” refers to a safe lipid formulation administered intravenously to subjects who cannot obtain sufficient lipids from their diet. This is an emulsion of soybean oil (or other naturally occurring oils) and egg phospholipids. Lipid emulsions have been used for formulations of several insoluble anesthetics. In this disclosure, lipid emulsions may be part of commercially available formulations such as Intralipid, Liposyn, and Nutrilipid, modified commercially available formulations with concentrated specific fatty acids, or entirely novel combinations of fatty acids and phospholipids.

[0251] In one embodiment, cells to be contacted with the oligonucleotide composition of the present invention are contacted with a mixture containing the oligonucleotide and a mixture containing lipids, such as the lipids or lipid compositions described above, for about 12 to about 24 hours. In another embodiment, cells to be contacted with the oligonucleotide composition are contacted with a mixture containing the oligonucleotide and a mixture containing lipids, such as the lipids or lipid compositions described above, for about 1 to about 5 days. In one embodiment, cells are contacted with a mixture containing lipids and oligonucleotides for about 3 to about 30 days. In another embodiment, the lipid-containing mixture is kept in contact with cells for at least about 5 to about 20 days. In another embodiment, the lipid-containing mixture is kept in contact with cells for at least about 7 to about 15 days.

[0252] 50%–60% of the formulation may optionally consist of any other lipid or molecule. Such lipids or molecules are referred to herein as cargo lipids or cargo molecules. Cargo molecules include, but are not limited to, intralipids, small molecules, membrane fusion peptides or lipids, or other small molecules may be added to alter cellular uptake, endosomal release or tissue distribution properties. Where such properties are desirable, the ability to tolerate cargo molecules is important for modifying the properties of these particles. For example, the presence of certain tissue-specific metabolites can significantly alter the tissue distribution profile. For instance, the use of intralipid formulations enriched with shorter or longer lipid chains with diverse saturation levels affects the tissue distribution profile (and their loading) of these types of formulations.

[0253] Examples of useful cargo lipids according to the present invention are membrane fusion lipids. For example, the zwitterionic lipid DOPE (compound registration number 4004-5-1, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) is a preferred cargo lipid. Intralipid may consist of the following composition: 1000 mL contains: 90 g of refined soybean oil, 12 g of refined egg phospholipid, 22 g of anhydrous glycerol, and a sufficient amount of water for injection (enough for 1000 mL). The pH is adjusted to approximately pH 8 with sodium hydroxide. Energy content / L: 4.6 MJ (190 kcal). Osmotic pressure (approximately): 300 mOsm / 1 kg of water. In another embodiment, the lipid emulsion is liposyn, containing 5% safflower oil, 5% soybean oil, up to 1.2% egg phospholipid added as an emulsifier, and 2.5% glycerol in water for injection. This may also contain sodium hydroxide for pH adjustment. pH 8.0 (6.0~9.0). Liposyn has an osmotic pressure of 276 mOsmol / liter (measured value).

[0254] Variations in the identity, quantity, and ratio of cargo lipids influence the characteristics of cellular uptake and tissue distribution of these compounds. For example, the length and saturation level of the lipid tail affect different uptake into liver, lung, adipose, and cardiomyocytes. The addition of special hydrophobic molecules, such as vitamins or different forms of sterols, can favor the distribution of specific compounds to the tissues involved in their metabolism. In some embodiments, vitamins A or E are used. Complexes are formed at different oligonucleotide concentrations, with higher concentrations favoring more efficient complex formation.

[0255] 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 lipid. In yet another embodiment, the composition of the lipid emulsion is entirely 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 having an 18-carbon chain and two cis double bonds.

[0256] In yet another embodiment of the present invention, modification of the composition of a lipid emulsion is used as a means to alter the tissue distribution of hydrophobic modified polynucleotides. This methodology results in the specific delivery of polynucleotides to specific tissues. In another embodiment, the lipid emulsion of cargo molecules contains more than 70% linoleic acid (C 18 H 32 Contains O2) and / or cardiolipin.

[0257] Lipid emulsions, such as intralipid, have long been used as delivery formulations for several water-insoluble drugs (such as propofol (reformulated as Diprivan)). The unique features of the present invention include (a) the concept of combining a modified polynucleotide with one or more hydrophobic compounds so that it can be incorporated into lipid micelles, and (b) mixing this with a lipid emulsion 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 ultimately absorbed by the cells. The polynucleotide incorporated as part of the micelle will then be delivered near the cell surface. Subsequently, cellular uptake may occur through a variety of mechanisms, including but not limited to sterol-type delivery.

[0258] 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.). In one embodiment, the oligonucleotide of the present invention may be complexed with a complexing agent to increase the uptake of the oligonucleotide by cells. Examples of complexing agents include cationic lipids. Cationic lipids may be used to deliver the oligonucleotide to cells. However, as described above, formulations that do not contain cationic lipids are preferred in some embodiments.

[0259] The term "cationic lipid" includes lipids and synthetic lipids having both polar and nonpolar domains, which can be positively charged at or near physiological pH and which bind to polyanions such as nucleic acids to facilitate the delivery of nucleic acids into cells. Generally, cationic lipids include saturated and unsaturated alkyls, as well as alicyclic ethers and amine esters, amides, or derivatives thereof. The linear and branched alkyl and alkenyl groups of cationic lipids may 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 - It can be prepared with a variety of counterions (anions), including acetates, trifluoroacetic acid, sulfates, nitrites, and nitrates.

[0260] Examples of cationic lipids include polyethyleneimines, 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.). Exemplary cationic liposomes can be produced from N-[1-(2,3-diorheoroxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA), N-[1-(2,3-diorheoroxy)-propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP), 3β-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol), 2,3,-dioleyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propaneaminium 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 times (Vlassov et al., 1994, Biochimica et Biophysica Acta 1197:95-108). Oligonucleotides may also be complexed with, for example, poly(L-lysine) or avidin, and the lipid may or may not be included in this mixture, for example, steryl-poly(L-lysine).

[0261] 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 oligonucleotides may be used in combination with the claimed method. In addition to those listed above, other lipid compositions, including those taught in, for example, U.S. Patent Nos. 4,235,871; 4,501,728; 4,837,028; and 4,737,323, are also known in the art.

[0262] In one embodiment, the lipid composition may further contain agents, such as viral proteins, to enhance the lipid-mediated transfection of oligonucleotides (Kamata, et al., 1994. Nucl. Acids. Res. 22:536). In another embodiment, oligonucleotides are brought into contact with cells as part of a composition comprising oligonucleotides, peptides, and lipids, for example, as taught in U.S. Patent No. 5,736,392. Improved lipids that are serum-tolerant 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 delivered into the cell via endocytosis.

[0263] In another embodiment, N-substituted glycine oligonucleotides (peptoids) may be used to improve the uptake of oligonucleotides. Peptoids have been used to prepare 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., Zuckermann, 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 combinations of cationic lipids and peptoids, may also be used to improve the uptake of the target oligonucleotide (Hunag, et al., 1998. Chemistry and Biology. 5:345). Lipoids can be synthesized by producing peptoid oligonucleotides and coupling the amino-terminant submonomer to lipids via their amino groups (Hunag, et al., 1998. Chemistry and Biology. 5:345).

[0264] It is known in the art that positively charged amino acids can be used to produce highly active cationic lipids (Lewis et al. 1996. Proc. Natl. Acad. Sci. US.A. 93:3176). In one embodiment, the composition for delivering oligonucleotides of the present invention comprises a number of arginine, lysine, histidine, or ornithine residues bound to a lipophilic moiety (see, for example, U.S. Patent No. 5,777,153).

[0265] In another embodiment, a composition for delivering oligonucleotides of the present invention comprises a peptide having about 1 to about 4 basic residues. These basic residues may be located, for example, at the amino terminus, C terminus, or in the interior region of the peptide. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine (which can also be considered nonpolar), asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar 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). In addition to basic amino acids, the majority or all of the other residues of the peptide may be selected from non-basic amino acids, such as lysine, arginine, or histidine. Preferably, neutral amino acids with long neutral side chains are predominantly used.

[0266] In one embodiment, the composition for delivering oligonucleotides 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 enable the polypeptides to bind to each other and to membrane surfaces. In other words, a series of γ-Gla-containing polypeptides can be used as a versatile delivery modality to help RNAi constructs adhere to whatever membrane they come into contact with. This can at least delay the clearance of RNAi constructs from the bloodstream and enhance their chances of homing to their targets.

[0267] Gamma-carboxyglutamic acid residues may be present in naturally occurring proteins (e.g., prothrombin has 10 γ-Gla residues). Alternatively, they can be introduced into purified, recombinant, or chemically synthesized polypeptides by carboxylation, for example, using vitamin K-dependent carboxylase. The gamma-carboxyglutamic acid residues may be continuous or discontinuous, and the total number and position of such residues in the polypeptide can be controlled / tuned to achieve different levels of "stickiness" of the polynucleotide.

[0268] In one embodiment, cells to be contacted with the oligonucleotide composition of the present invention are contacted with a mixture containing oligonucleotides and a mixture containing lipids, such as one of the above-mentioned lipids or lipid compositions, for about 12 to about 24 hours. In another embodiment, cells to be contacted with the oligonucleotide composition are contacted with a mixture containing oligonucleotides and a mixture containing lipids, such as one of the above-mentioned lipids or lipid compositions, for about 1 to about 5 days. In one embodiment, cells are contacted with a mixture containing lipids and oligonucleotides for a period of about 3 to about 30 days. In another embodiment, the lipid-containing mixture is left in contact with cells for at least about 5 to about 20 days. In another embodiment, the lipid-containing mixture is left in contact with cells for at least about 7 to about 15 days.

[0269] For example, in one embodiment, the oligonucleotide composition may be exposed to cells for a long incubation period as described herein, in the presence of lipids such as cytofectin CS or GSV (available from Glen Research; Sterling, Va.), GS3815, or GS2888. In one embodiment, incubation of cells with a mixture containing lipid and oligonucleotide compositions does not reduce the viability of the cells. Preferably, after the transfection period, the cells are substantially viable. In one embodiment, after transfection, the cells are viable by at least about 70% to at least about 100%. In another embodiment, the cells are viable by at least about 80% to at least about 95%. In yet another embodiment, the cells are viable by at least about 85% to at least about 90%.

[0270] In one embodiment, the oligonucleotide is modified by attaching a peptide sequence, referred herein as a “transport peptide,” which transports the oligonucleotide into a cell. In one embodiment, the composition comprises an oligonucleotide complementary to a protein-coding target nucleic acid molecule and a transport peptide covalently attached thereto.

[0271] The term "transport peptide" refers to an amino acid sequence that facilitates the transport of oligonucleotides into cells. Exemplary peptides that facilitate the transport of the portion 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).

[0272] Oligonucleotides can be attached to transport peptides 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 possessing an activated thiol group can be coupled via that thiol group to cysteine ​​present in the transport peptide (for example, to cysteine ​​present in the β-turn between the second and third helices of the Antennapedia homeodomain, as taught in 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, the Boc-Cys-(Npys)OH group can be coupled to the transport peptide so that an oligonucleotide possessing the last (N-terminal) amino acid and SH group can be coupled to the peptide (Troy et al. 1996. J. Neurosci. 16:253).

[0273] In one embodiment, a linking group may be attached to a nucleomonomone, and the transport peptide may be covalently attached to the linker. In one embodiment, the linker may function both as a binding site for the transport peptide and as something that can provide stability to the nuclease. Examples of suitable linkers include substituted or unsubstituted C1-C1 20 Alkyl chain, C2~C 20 Alkenyl chain, C2~C 20These include alkynyl chains, peptides, and heteroatoms (e.g., S, O, NH, etc.). Other exemplary linkers include bifunctional crosslinking agents such as sulfosuccinimidyl-4-(maleimidophenyl)-butyric acid (SMPB) (see, e.g., Smith et al. Biochem J 1991.276: 417-2). In one embodiment, the oligonucleotides of the present invention are synthesized as molecular conjugates that utilize receptor-mediated endocytosis mechanisms for the delivery of genes into cells (see, for example, Bunnell et al. 1992. Somatic Cell and Molecular Genetics. 18:559 and the references cited herein).

[0274] Other carriers for the delivery of RNAi reagents in vitro and / or in vivo are known in the art and may be used to deliver the RNAi construct of interest (for example, to host cells such as T cells). For example, a few examples include U.S. Patent Application Publications 20080152661, 20080112916, 20080107694, 20080038296, 20070231392, 20060240093, 20060178327, 20060008910, 20050265957, and 20050064595. See issues 20050042227, 20050037496, 20050026286, 20040162235, 20040072785, 20040063654, 20030157030, WO2008 / 036825, WO04 / 065601 and AU2004206255B2 (all incorporated by reference).

[0275] Treatment method In some aspects, this disclosure provides a method for treating proliferative disorders or infectious disorders by administering an immunomodulatory composition described herein (e.g., an immunomodulatory composition comprising one or more host cells of a specific cell subtype or T cell subtype) to a subject (e.g., a subject having or suspected of having a proliferative disorder or infectious disorder). In some embodiments, the immunomodulatory compositions described herein are characterized as a population of immune cells (e.g., T cells, NK cells, antigen-presenting cells (APCs), dendritic cells (DCs), stem cells (SCs), induced pluripotent stem cells (iPSCs), etc.) having reduced (e.g., inhibited) expression or activity of one or more genes (e.g., BRD4, etc.) relating to the control of the T cell differentiation process.

[0276] As used herein, “proliferative disorders” 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, and so on. Examples of cancer include, but are not limited to, any other disease or disorder characterized by uncontrolled cell proliferation such as neoplasms, malignancies, metastases, or cancer. In some embodiments, cancer is primary cancer. In some embodiments, cancer is metastatic cancer.

[0277] Examples of cancer include: biliary tract cancer; bladder cancer; brain cancer including glioblastoma and medulloblastoma; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; hematological neoplasms including acute lymphoblastic and myeloid leukemia; multiple myeloma; AIDS-related leukemia and adult T-cell leukemia / lymphoma; carcinoma in situ including Bowen's disease and Paget's disease; liver cancer; lung cancer; lymphoma including Hodgkin's disease and lymphocytic lymphoma; neuroblastoma; This includes oral cancers, including squamous cell carcinoma; ovarian cancers, including those arising from epithelial cells, stromal cells, germ cells, and mesenchymal cells; pancreatic cancer; prostate cancer; rectal cancer; sarcomas, including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma; skin cancers, including melanoma, Kaposi's sarcoma, basal cell carcinoma, and squamous cell carcinoma; testicular cancers, including germ cell tumors such as seminomas, nonseminomas, and teratomas; tumors with a high tumor mutational burden; choriocarcinoma; stromal tumors and germ cell tumors; thyroid cancers, including thyroid adenocarcinoma and medullary carcinoma; and renal cancers, including adenocarcinoma and Wilms' tumor. In some embodiments, cancers are selected from the group consisting of hematological malignancies such as small cell lung cancer, colon cancer, breast cancer, lung cancer, prostate cancer, ovarian cancer, pancreatic cancer, melanoma, chronic myeloid leukemia, etc. In some embodiments, the subject has one type of cancer. In some embodiments, the subjects have more than one type of cancer (e.g., two, three, four, five, or more types). In some embodiments, the cancers include small cell lung cancer, colon cancer, breast cancer, lung cancer, prostate cancer, ovarian cancer, pancreatic cancer, melanoma, or hematological malignancies such as chronic myeloid leukemia (CML).

[0278] As used herein, the term “infectious disease” refers to diseases and disorders caused by pathogens that result in infection of the subject. Examples of human pathogens include, but are not limited to, certain bacteria (e.g., certain strains of E. coli, Salmonella, etc.), viruses (e.g., HIV, HCV, influenza, etc.), parasites (e.g., protozoa, helminths, amoebas, etc.), yeasts (e.g., certain Candida species, etc.), and fungi (e.g., certain Aspergillus species). Examples of animals covered include mammals, such as humans and other primates; cattle, pigs, horses, and farming (agricultural) animals; dogs, cats, and other domesticated pets; mice, rats, and transgenic non-human animals.

[0279] In some embodiments, the immunomodulatory compositions described herein are administered to a subject by adoptive cell transplantation (ACT) therapy. Examples of ACT modalities, but not limited to, include autologous cell therapy (e.g., cells of the subject themselves are taken, genetically engineered, and returned to the subject), tumor-infiltrating lymphocytes (TILs), and allogeneic cell therapy (e.g., cells are taken from a donor, genetically engineered, and placed in the recipient). In some embodiments, cells used in ACT therapy may be genetically engineered to express chimeric antigen receptors (CARs), which are engineered T cell receptors that display specificity to a target antigen based on a selective antibody portion. Consequently, in some embodiments, CAR T cells (e.g., CARTs) may be transfected with chemically modified double-stranded nucleic acids using the methods described herein for the purpose of ACT therapy.

[0280] With regard to in vivo application, the formulations of the present invention can be administered to patients in various forms adapted to the selected route of administration (e.g., parenteral, oral, or intraperitoneal). Preferred parenteral administrations include administration by the following routes: intravenous; intramuscular; intratumorally; interstitially; intraarterial; subcutaneous; intraocular; intrasynovial; transepithelial, including percutaneous; pulmonary inhalation; ophthalmic; sublingual and oral; topical, including ophthalmic; percutaneous; ocular; rectal; and nasal inhalation via air insufflation.

[0281] Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compound in a water-soluble or water-dispersible form. In addition, suspensions of the active compound 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. The aqueous injection suspension may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran, and optionally the suspension may also contain stabilizers. The oligonucleotides of the present invention may be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hanks' solution or Ringer's solution. Furthermore, the oligonucleotides may be formulated in solid form and redissolved or suspended immediately before use. Lyophilized forms are also included in the present invention.

[0282] Drug delivery vehicles can be selected for purposes such as in vitro administration or systemic administration. These vehicles may be designed to function as delayed-release reservoirs or to deliver their contents directly to target cells. One advantage of using some direct-delivery drug vehicles is that multiple molecules are delivered with each uptake. Such vehicles have been shown to extend the circulating 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 include liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres.

[0283] The active amount of oligonucleotides in this invention is defined as the effective amount in terms of the dosage and time required to achieve the desired result. For example, the active amount of oligonucleotides may vary depending on factors such as cell type, the oligonucleotide used, and, for in vivo use, disease state, age, sex and weight of the individual, and the ability of the oligonucleotide to elicit a desired response in the individual. Establishing therapeutic levels of oligonucleotides 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 modifications to the phosphate backbone, may require different doses.

[0284] The precise dosage and frequency of administration of an immunomodulatory composition will depend on data generated experimentally and in clinical trials. Several factors, such as the desired effect, delivery vehicle, disease symptoms, and route of administration, will influence the dosage. The dosage can be readily determined by those skilled in the art and formulated into the pharmaceutical composition of interest. Preferably, the duration of treatment will extend at least to the entire course of the disease symptoms.

[0285] The drug regimen may be modified to provide a targeted therapeutic response. For example, the immunomodulatory composition may be administered repeatedly, for example, several doses daily, or the dose may be proportionally reduced according to the requirements of the treatment situation. Whether the chemically modified double-stranded nucleic acid molecule or immunomodulatory composition is administered to cells or to a subject, a person skilled in the art will be able to readily determine the appropriate dose and administration schedule for the subject.

[0286] The administration of immunomodulatory compositions, such as by intradermal injection or subcutaneous delivery, can be improved through testing of the drug regimen. In some embodiments, a single dose is sufficient. To further extend the effect of the administered immunomodulatory composition, as is well known to those skilled in the art, the composition may be administered in a sustained-release formulation or device.

[0287] In other embodiments, chemically modified double-stranded nucleic acid molecules or immunomodulatory compositions are administered multiple times. In some examples, this is administered daily, twice a week, weekly, every two weeks, every three weeks, monthly, every two months, every three months, every four months, every five months, every six months, or less than once every six months. In some examples, this is administered multiple times per day, week, month and / or year. For example, this can be administered approximately every hour, every two hours, every three hours, every four hours, every five hours, every six hours, every seven hours, every eight hours, every nine hours, every ten hours, every twelve hours, or at intervals longer than twelve hours. This can be administered one, two, three, four, five, six, seven, eight, nine, ten, or more than ten times per day.

[0288] An aspect of the present invention relates to the administration of immunomodulatory compositions to a subject. In some examples, the subject is a patient, and the administration of the immunomodulatory composition involves administering the composition in a hospital setting. In some embodiments, more than one immunomodulatory composition is administered simultaneously. For example, a composition containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different compositions may be administered. In some embodiments, the composition contains two or three different immunomodulatory compositions.

[0289] Self-delivering RNAi immunotherapy agents As described in U.S. Patent Publication No. US 2016 / 0304873, the entire contents of which are incorporated herein by reference, immunotherapeutic agents were produced by treating cells with specific INTASYL® agents designed to target and knock down specific genes involved in immunosuppressive mechanisms. Several cells and cell lines have been successfully treated with INTASYL® compounds and shown to knock down at least 70% of the targeted gene expression in certain human cells. These studies demonstrated the usefulness of these immunomodulatory agents in suppressing the expression of target genes in cells that are normally highly resistant to transfection, suggesting that these agents can reduce the expression of target cells in any cell type.

[0290] For the purposes of the present invention, a range may be expressed herein as “about” one particular value to and / or “about” another particular value. Where such a range is expressed, another aspect includes from one particular value to and / or other particular values. Similarly, where the use of the antecedent “about” expresses a value as an approximation, it will be understood that a particular value forms another aspect. It will be further understood that the endpoints of each range are important together, both in relation to and independently of the other endpoints.

[0291] Furthermore, for the purposes of this invention, the term "a" or "an" refers to the presence of one or more such compounds; for example, "a protein" or "a nucleic acid molecule" refers to one or more such compounds or at least one such 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 compounds in the following list that include mixtures (i.e., combinations) of two or more compounds.

[0292] According to the present invention, isolated or biologically pure protein or nucleic acid molecules are compounds extracted from their natural environment. Therefore, "isolated" and "biologically pure" do not necessarily reflect the degree to which the compound has been purified. The isolated compounds of the present invention can be obtained from their natural sources, produced using molecular biological techniques, or produced by chemical synthesis.

[0293] The compositions and methods described herein are further illustrated by the following examples, but these should not be construed as further limiting. The entire contents of all references cited throughout this application (including references, granted patents, published patent applications and concurrently pending patent applications) are expressly incorporated herein by reference. [Examples]

[0294] example Example 1: Identification of BRD4-targeted INTASYL(trademark) sequences To identify preferred INTASYL® sequences that target the BRD4 sequence and target region, the BRD4 gene was analyzed using a proprietary algorithm. Non-restrictive examples of BRD4 targeting sequences and / or INTASYL® sequences are shown in Tables 1 and 2.

[0295] Example 2: Two dose-response studies of chemically modified INTASYL™ molecules targeting BRD4 in A549 cells. A549 cells were obtained from ATCC and cultured in F12K medium containing 10% fetal bovine serum and 1% Pen / Strep. Cells were plated into 96 wells 24 hours prior to transfection. Chemically modified INTASYL® molecules targeting BRD4 were prepared by diluting INTASYL® molecules to 0.2–2 μM in serum-free Accell medium (wells), and INTASYL® containing the medium was divided into cells (100 μl / well in a 96-well plate).

[0296] 72 hours after administration, cells were lysed, and mRNA levels were determined by a Quantigene branched DNA assay using gene-specific probes, according to the manufacturer's protocol. Data were normalized to housekeeping genes (PPIBs) and graphed against untargeted controls. Error bars represent the standard deviation from the biological triple mean. The results shown in Figure 1 demonstrate significant silencing of BRD4-targeted INTASYL® molecules BRD4-11, BRD4-20, BRD4-21, BRD4-22, and BRD4-23 delivered to A549 cells, and that 2 μM of INTASYL® molecules inhibited gene expression by more than 60–70%.

[0297] Example 3: Five dose-response curves for the INTASYL™ molecule targeting BRD4 in T cells. Primary human T cells were obtained from AllCells (CA) and cultured in Immunocult medium containing 10% fetal bovine serum (Gibco) and 1000 IU / mL IL2. Cells were activated with anti-CD3 / CD28 Dynabeads (Gibco, 11131) at least 4 days prior to transfection, according to the manufacturer's instructions. INTASYL® molecules targeting BRD4 were prepared by individually diluting the compound to 0.12–4 μM in serum-free RPMI per sample (well) and divided into 50 μl / well portions in 96-well plates. Cells were prepared to 1,000,000 cells / ml in Immunocult medium containing 5% FBS and 2000 U / ml IL2 and seeded at 50 μl / well in 96-well plates containing pre-diluted INTASYL® molecules. After 72 hours, transfected cells were lysed with 50 μl of lysis mixture and 3 μl of protease K per well. Cells were lysed at 37°C for 30 minutes. mRNA levels were determined by branched DNA assay according to the manufacturer's protocol. The results shown in Figure 2 demonstrate dose-dependent silencing of BRD4-targeted INTASYL® molecules in T cells, with over 70-80% of gene expression inhibited by 2 μM INTASYL® molecules BRD4-20 and 21.

[0298] Example 4: Ex vivo treatment of tumor-infiltrating lymphocytes (TILs) with BRD-4 targeted INTASYL® compound. CD8+ T cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy human volunteers by negative selection. These cells were then expanded using the National Cancer Institute's Rapid Expansion Protocol (REP). During the REP, cells were treated with or left untreated with either BRD4-20, an untargeted control (NTC), or JQ1 (positive control). Compound additions are outlined in Figure 4A. The percentage of BRD4-negative cells was determined on days 0, 8, 12, and 14. On day 14 of the REP, cells were harvested and analyzed for BRD4 protein and differentiation marker levels by flow cell cytometry. Treatment of CD8+ T cells with BRD4-20 (2 μM) resulted in an increase in the population of BRD4-negative CD8+ T cells (demonstrating BRD4 protein reduction) (Figure 3) and an increase in the frequency of CD8+ T cells with stem cell-like memory phenotype (CCR7+ / CD62L+) compared to the control (Figure 4B).

[0299] Furthermore, a subset of CD8+ T cells treated as described above was used in co-culture with the malignant melanoma cell line A375 to determine the functional recognition of tumor cells. Treatment with BRD4-20 during REP resulted in CD8+ T cells with enhanced recognition of tumor cells, as demonstrated by increased levels of INFγ production (Figure 5). Treatment with BRD4-20 during REP also affects stem cell memory T cells (T SCMIt was found that this resulted in differentiation into ). Figures 6A-6B show the flow cytometry results on REP day 12, which show that cells treated with BRD4-20 had decreased CD45RA+CD62L+ staining and increased CD45RA+CCR7+ staining compared to other treatment groups.

[0300] Example 5: High-dose intratumoral injection of BRD4-20 results in inhibition of tumor growth in vivo. Hepa 1-6 tumor-bearing mice (female C57BL / 6Crl mice subcutaneously injected with mouse hepatocellular carcinoma) were treated intratumorally with INTASYL® (BRD4-20), a BRD4-targeting agent, at two doses: 0.5 mg / tumor and 2 mg / tumor, on days 1, 4, 7, 10, and 14. JQ1, a nonspecific inhibitor of bromodomain protein, was used as a positive control. An untargeted control (NTC) was used as a negative control. Longitudinal mean tumor volume (mm) was measured. 3 The tumor growth rate (TILs) was recorded and plotted throughout the study period (Figure 7). Intratumoral injection of BRD4-20 was found to inhibit tumor growth at both dose levels. Mice were sacrificed 14 days after the last dose, and tumors were excised. TILs were isolated and analyzed for the CD45+ population by flow cytometry. As shown in Figure 8, treatment with BRD4-20 increased CD45+ TILs in the tumor microenvironment (TME) at both dose levels.

[0301] Example 6. The dose-response of BRD4-20 in Hepa 1-6 tumor-carrying mice results in inhibition of tumor growth in vivo. Hepa 1-6 tumor-bearing mice were administered intratumoral doses of INTASYL® (BRD4-20), which targets BRD4 at increased dose levels, on days 1, 3, 7, 10, and 14 (0.02 mg to 0.5 mg per injection). The tumor volume target for initiating treatment was 150 mm. 3 The satellite group (n=6) was sacrificed on day 12 for TME analysis. The research schedule is shown in Table 3.

[0302] [Table 1]

[0303] Non-targeted controls (NTCs) were used as negative controls. Longitudinal mean tumor volume (mm) 3 The tumor volume AUC was recorded (Figure 9A) and calculated using trapezoidal transformation (Figure 9B). Statistical significance was assessed by one-way ANOVA and Tukey's multiple comparison post-hoc study. Intratumoral administration of BRD4-20 resulted in dose-dependent inhibition of tumor growth.

[0304] [Table 2-1] [Table 2-2]

[0305] [Table 3-1] [Table 3-2]

[0306] Equivalents Those skilled in the art will be able to understand or recognize numerous equivalents to specific embodiments of the invention described herein by conventional experimentation alone. Such equivalents are intended to be encompassed by the following claims. All references, including patent documents disclosed herein, are incorporated by reference in their entirety.

Claims

1. A chemically modified double-stranded nucleic acid molecule, comprising a sense strand and an antisense strand, directed toward a gene encoding BRD4, wherein the chemically modified double-stranded nucleic acid molecule comprises a sense strand consisting of the sequence defined by Sequence ID No. 87 (fG.mA.fU.mG.fU.mG.fU.mU.fC.mG.fA.mA.fA*mU*fA.TEG-Chl) and an antisense strand consisting of the sequence defined by Sequence ID No. 88 (P.mA.fA.mU.fU.mU.fC.mG.fA.mA.fC.mA.fC.mA.fU*mC*fC*mU*fG*mG*fA), Here, the chemically modified double-stranded nucleic acid molecule is characterized in that the 3' end of the sense strand of the chemically modified double-stranded nucleic acid molecule is linked to the cholesterol portion.

2. An asymmetric double-stranded ribonucleic acid (dsRNA) including a sense strand and an antisense strand, directed to the gene encoding BRD4, The dsRNA includes a sense strand consisting of the sequence defined by SEQ ID NO: 87 (fG.mA.fU.mG.fU.mG.fU.mU.fC.mG.fA.mA.fA*mU*fA.TEG-Chl), and an antisense strand consisting of the sequence defined by SEQ ID NO: 88 (P.mU.fA.mU.fU.mU.fC.mG.fA.mA.fC.mA.fC.mA.fU*mC*fC*mU*fG*mG*fA), The dsRNA wherein the 3' end of the sense strand of the dsRNA is ligated to the cholesterol portion.

3. A composition comprising a chemically modified double-stranded nucleic acid molecule according to claim 1 or a dsRNA according to claim 2, and a pharmaceutically acceptable excipient.

4. An immunomodulatory composition comprising host cells, wherein the host cells are ex vivo treated with the chemically modified double-stranded nucleic acid molecule described in claim 1, or the dsRNA described in claim 2, or the composition described in claim 3, to control and / or reduce the level of differentiation of the host cells, thereby enabling the production of a specific population of immune cells for administration in humans.

5. The host cells are selected from the following groups: T cells, tumor-infiltrating lymphocytes (TILs), NK cells, antigen-presenting cells (APCs), dendritic cells (DCs), stem cells (SCs), induced pluripotent stem cells (iPSCs), stem cell memory T cells, tumor cells, and cytokine-induced killer cells (CIKs). Alternatively, the host cell is a T cell, the T cell is a CD8+ T cell, and / or the T cell contains one or more transgenes that express high-affinity T cell receptor (TCR) and / or chimeric antigen receptor (CAR), and / or the immunomodulatory composition according to claim 4, wherein the host cells are derived from a healthy donor.

6. A method for producing an immunomodulatory composition, comprising introducing a chemically modified double-stranded nucleic acid molecule according to claim 1 or a dsRNA according to claim 2 into a cell in vitro or ex vivo.

7. The method according to claim 6, wherein the cells are T cells, NK cells, antigen-presenting cells (APCs), dendritic cells (DCs), stem cells (SCs), induced pluripotent stem cells (iPSCs), stem cell memory T cells, or cytokine-induced killer cells (CIKs), and / or the cells are derived from a healthy donor.

8. The method according to claim 7, wherein the cell is a T cell, the T cell is a CD8+ T cell, and / or the cell is a T cell, the T cell comprises one or more transgenes expressing a high-affinity T cell receptor (TCR) and / or a chimeric antigen receptor (CAR).

9. The immunomodulatory composition according to claim 4 or 5 for use in a method for treating a proliferative disorder or an infectious disorder.

10. (a) The proliferative disorder is cancer, (b) The infectious disease is caused by a pathogen, or (c) The infectious disease is a pathogenic infection, and the pathogenic infection is a bacterial infection, a viral infection, or a parasitic infection, and / or (d) The immunomodulatory composition according to claim 9, wherein the chemically modified double-stranded nucleic acid molecule according to claim 1 or the dsRNA according to claim 2 is to be administered via intratumoral injection.

11. A chemically modified double-stranded nucleic acid molecule according to claim 1, or dsRNA according to claim 2, or a composition according to claim 3, for use in a method for treating proliferative or infectious diseases.

12. A chemically modified double-stranded nucleic acid molecule, dsRNA, or composition according to claim 11, intended for administration via intratumor injection.

13. T cells after introduction of the chemically modified double-stranded nucleic acid molecule described in claim 1 or the dsRNA described in claim 2, ＳＣＭ or T ＣＭ It differentiates into, and / or here, the immunomodulatory composition is T ＳＣＭ or T ＣＭ The immunomodulatory composition according to claim 5, comprising at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% cells.

14. T cells after introduction of the chemically modified double-stranded nucleic acid molecule described in claim 1 or the dsRNA described in claim 2, ＳＣＭ or T ＣＭ It differentiates into, and / or here, the immunomodulatory composition is T ＳＣＭ or T ＣＭ The method according to claim 8, comprising at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% cells.

Images