P-ethoxy nucleic acids for BCL2 inhibition

Abstract

Provided herein are improved delivery systems for oligonucleotides, said delivery system comprising a liposome that comprises neutral phospholipids and a P-ethoxy oligonucleotide, which targets a BCL2-encoding polynucleotide. Methods of treating patients with said delivery systems are also provided.

Description

<DP=1>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²DESCRIPTION²P-ETHOXY NUCLEIC ACIDS FOR BCL2 INHIBITION²100011 The present application claims the priority benefit of United States ²provisional ²application number 62 / 487,302, filed April 19, 2017, the entire contents of ²which is ²incorporated herein by reference.²BACKGROUND OF THE INVENTION²1. Field of the Invention²100021 The present invention relates generally to the field of medicine. More ²particularly, it concerns liposomal formulations of P-ethoxy oligonucleotides ²that hybridize ²to a BCL2 polynucleotide gene product and methods of making and using such ²formulations ²in medicine, even more particularly in the treatment of cancers that have high ²expression or ²increased activity of the BCL2 gene.²2. Description of Related Art²100031 Bc1-2 is a protein that is involved in regulating apoptosis or ²programmed cell ²death. Over-expression of Bc1-2 prevents the induction of apoptosis in ²response to cellular ²insults such as treatment with chemotherapeutic agents. Bc1-2 is over-²expressed in more than ²90% of follicular B-cell non-Hodgkins lymphoma due to a chromosomal ²rearrangement and ²is the key factor in the initiation of this malignancy. Bc1-2 is also ²overexpressed in a wide ²variety of solid tumors (it is estimated to be over-expressed in 40 percent of ²cancers). For ²example, Bc1-2 overexpression has been associated with the progression of ²prostate cancer ²from hormone dependence to hormone independence and may contribute to the ²relative drug ²resistant phenotype typically observed in hormone independent prostate cancer.²SUMMARY OF THE INVENTION²100041 Provided herein are compositions and methods that induce growth ²inhibition ²and / or apoptosis in a wide range of cancer cells controlled by Bc12. Bc12 ²expression is ²targeted by a non-toxic nuclease resistant oligonucleotide that targets Bc12-²encoding ²polynucleotides in combination with a neutral liposome that prevents Bc12 ²protein ²expression, thus eliminating the pool of available Bc12 protein.²1²<DP=2>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²10005j In one embodiment, compositions are provided comprising a population of ²²oligonucleotides that hybridize to a BCL2 polynucleotide gene product. In some ²aspects, the ²oligonucleotides of the population are composed of nucleoside molecules linked ²together ²through phosphate backbone linkages, wherein at least one of the phosphate ²backbone ²linkages in each oligonucleotide is a P-ethoxy backbone linkage, and wherein ²no more than ²80% of the phosphate backbone linkages in each oligonucleotide are P-ethoxy ²backbone ²linkages. In some aspects, at least one of the phosphate backbone linkages in ²each ²oligonucleotide is a phosphodiester backbone linkage. In some aspects, the ²oligonucleotides ²of the population comprise a sequence according to any one of SEQ ID NOs: 1-3. ²In some ²aspects, the oligonucleotides of the population comprise a sequence according ²to SEQ ID ²NO: 1. In one aspect, the oligonucleotides of the population comprise a ²sequence according ²to SEQ ID NO: 1 and the phosphate backbone linkages at least between ²nucleotides 5 and 6, ²between nucleotides 7 and 8, between nucleotides 9 and 10, between nucleotides ²11 and 12, ²and between nucleotides 14 and 15 of the oligonucleotides of the population ²are ²phosphodiester backbone linkages. In some aspects, the oligonucleotides of the ²population ²comprise a sequence according to SEQ ID NO: 2. In one aspect, the ²oligonucleotides of the ²population comprise a sequence according to SEQ ID NO: 2 and the phosphate ²backbone ²linkages at least between nucleotides 5 and 6, between nucleotides 7 and 8, ²and between ²nucleotides 9 and 10 of the oligonucleotides of the population are ²phosphodiester backbone ²linkages. In some aspects, the oligonucleotides of the population comprise a ²sequence ²according to SEQ ID NO: 3. In various aspects, the oligonucleotides of the ²population inhibit ²the expression of Bc12. In some aspects, the composition is lyophilized.²100061 In some aspects, 10% to 80% of the phosphate backbone linkages are P-²ethoxy backbone linkages; 20% to 80% of the phosphate backbone linkages are P-²ethoxy ²backbone linkages; 30% to 80% of the phosphate backbone linkages are P-ethoxy ²backbone ²linkages; 40% to 80% of the phosphate backbone linkages are P-ethoxy backbone ²linkages; ²50% to 80% of the phosphate backbone linkages are P-ethoxy backbone linkages; ²or 60% to ²70% of the phosphate backbone linkages are P-ethoxy backbone linkages, or any ²range ²derivable therein. In some aspects, 20% to 90% of the phosphate backbone ²linkages are ²phosphodiester backbone linkages; 20% to 80% of the phosphate backbone ²linkages are ²phosphodiester backbone linkages; 20% to 70% of the phosphate backbone ²linkages are ²phosphodiester backbone linkages; 20% to 60% of the phosphate backbone ²linkages are ²phosphodiester backbone linkages; 20% to 50% of the phosphate backbone ²linkages are²2²<DP=3>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²phosphodiester backbone linkages; or 30 A to 400 o of the phosphate backbone ²linkages are ²phosphodiester backbone linkages, or any range derivable therein. In various ²aspects, at least ²500, 1000, 15%, 2000, 2500, 30%, 350, 40%, 4500, 50%, 5500, 60%, 65%, 60%, ²65%, 70%, ²7500, 800 0, 850 0, 900 0, or 950 0, or any value therein, of the phosphate ²backbone linkages are ²P-ethoxy backbone linkages. In various aspects, at most 5%, 1000, 15%, 20%, ²25%, 30%, ²350, 40%, 450, 50%, 550, 60%, 65%, 70%, 750, 80%, 85%, 90%, or 95%, or any ²value ²therein, of the phosphate backbone linkages are phosphodiester backbone ²linkages. In certain ²aspects, the phosphodiester backbone linkages are distributed throughout the ²oligonucleotides. As such, the oligonucleotides are not chimeric molecules. In ²some aspects, ²the oligonucleotides do not comprise a phosphorothioate backbone linkage.²10007j In some aspects, the oligonucleotides of the population have a size ²ranging ²from 7 to 30 nucleotides. In certain aspects, the oligonucleotides of the ²population have a ²size ranging from 12 to 25 nucleotides. In various aspects, the ²oligonucleotides of the ²population have a size of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, ²17, 18, 19, 20, 21, 22, ²23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. The size range may be an ²average size of the ²oligonucleotides in the population.²100081 In some aspects, the oligonucleotides of the population have an average ²size of ²7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, ²27, 28, 29, or 30 ²nucleotides, wherein no more than 5, 6, 7, 8, 8, 9, 10, 11, 11, 12, 13, 14, ²15, 15, 16, 17, 18, ²19, 20, 20, 21, 22, 23, or 24, respectively, of the phosphate backbone ²linkages in each ²oligonucleotide is a P-ethoxy backbone linkage. In some aspects, the ²oligonucleotides of the ²population have an average size of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, ²18, 19, 20, 21, 22, ²23, 24, 25, 26, 27, 28, 29, or 30 nucleotides and at least 2, 2, 2, 2, 3, 3, ²3, 3, 4, 4, 4, 4, 4, 5, 5, ²5, 5, 5, 5, 6, 6, 6, 6, or 6, respectively, of the phosphate backbone linkages ²in each²oligonucleotide is a phosphodiester backbone linkage. By ²way of example, the²oligonucleotides of the population may have an average size of 18 nucleotides, ²wherein no ²more than 14 of the phosphate backbone linkages in each oligonucleotide is a P-²ethoxy ²backbone linkage; the oligonucleotides of the population may have an average ²size of 20 ²nucleotides, wherein no more than 16 of the phosphate backbone linkages in ²each ²oligonucleotide is a P-ethoxy backbone linkage; the oligonucleotides of the ²population may ²have an average size of 25 nucleotides, wherein no more than 20 of the ²phosphate backbone ²linkages in each oligonucleotide is a P-ethoxy backbone linkage; or the ²oligonucleotides of²3²<DP=4>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²the population may have an average size of 30 nucleotides, wherein no more ²than 24 of the ²phosphate backbone linkages in each oligonucleotide is a P-ethoxy backbone ²linkage.²100091 In some aspects, the population of oligonucleotides comprises a single ²species ²of oligonucleotides. In other aspects, the population of oligonucleotides ²comprises at least ²two species of oligonucleotides. A single species of oligonucleotide may have ²the same ²nucleotide sequence but either have or lack P-ethoxy linkages in different ²positions within the ²molecule. As such, the population may be homogeneous as to the nucleotide ²sequence and ²heterogeneous as to the distribution of phosphodiester backbone linkages among ²the ²oligonucleotides of the population. In addition, the population may be ²heterogeneous as to ²the number of P-ethoxy backbone linkages and phosphodiester backbone linkages ²among the ²oligonucleotides of the population. As a non-limiting example, a first portion ²of the ²oligonucleotides of the population may have 70% P-ethoxy linkages and 30% ²phosphodiester ²linkages while a second portion of the oligonucleotides of the population may ²have 60% P-²ethoxy linkages and 40% phosphodiester linkages. In some aspects, the ²population of ²oligonucleotides comprises anti sense oligonucleotides, short interfering RNAs ²(siRNAs), ²microRNAs (miRNAs), or piwiRNAs (piRNAs).²

[0010] In various aspects, the composition further comprises phospholipids. In ²some ²aspects, the phospholipids and oligonucleotides are present at a molar ratio ²of from about 5:1²to about 100:1. In some aspects, the oligonucleotides and phospholipids ²form an ²oligonucleotide-lipid complex, such as, for example, a liposome complex. In ²some aspects, ²at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, ²²89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the liposomes are ²less ²than 5 microns in diameter. In some aspects, at least 75%, 76%, 77%, 78%, 79%, ²80%, 81%, ²82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, ²97%, ²98%, or 99% of the liposomes are less than 4 microns in diameter. In some ²aspects, the ²population of oligonucleotides are incorporated in the population of ²liposomes.²[00111 In some aspects, the phospholipids are uncharged or have a neutral ²charge at ²physiologic pH. In some aspects, the phospholipids are neutral phospholipids. ²In certain ²aspects, the neutral phospholipids are phosphatidylcholines. In certain ²aspects, the neutral ²phospholipids are dioleoylphosphatidyl choline. In some aspects, the ²phospholipids are ²essentially free of cholesterol.²4²<DP=5>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²10012j In one embodiment, pharmaceutical compositions are provided comprising ²a ²composition of oligonucleotides and phospholipids of the present embodiments ²and a ²pharmaceutically acceptable carrier. In some aspects, the composition further ²comprises a ²chemotherapeutic agent.²100131 In one embodiment, methods are provided for delivering a ²therapeutically ²effective amount of an oligonucleotide to a cell comprising contacting the ²cell with a ²pharmaceutical composition of the present embodiments. In some aspects, the ²method is a ²method of treating hyperplasia, cancer, an autoimmune disease, or an ²infectious disease.²100141 In one embodiment, methods are provided for treating a subject with ²cancer, ²an autoimmune disease, or an infectious disease comprising administering to ²the subject a ²therapeutically effective amount of a pharmaceutical composition of the ²present ²embodiments. In some aspects, the subject is a human. In some aspects, the ²cancer is a ²bladder, blood, lymphoma, pancreas, bone, bone marrow, brain, breast, colon, ²esophagus, ²stomach, head and neck, kidney, liver, lung, prostate, skin, testis, tongue, ²ovary, or uterine ²cancer. In some aspects, the lymphoma is germinal center B-cell-like diffuse ²large B cell ²lymphoma, activated B-cell-like subtype diffuse large B cell lymphoma, mantle ²cell ²lymphoma, or Burkitt lymphoma. In some aspects, the leukemia is myeloid ²leukemia. In ²some aspects, the autoimmune disease is Lupus erythematosis, ²Spondyloarthropathy, ²Sjogren's disease, Crohn's disease, diabetes mellitus, multiple sclerosis, or ²rheumatoid ²arthritis. In some aspects, the infectious disease is a bacterial infection, ²fungal infection, viral²infection, or parasitic infection. In some aspects, the composition is ²administering ²subcutaneously, intravenously, or intraperitoneally. In some aspects, the ²method further ²comprises administering at least a second anticancer therapy to the subject. ²In some aspects, ²the second anticancer therapy is a surgical therapy, chemotherapy, radiation ²therapy, ²cryotherapy, hormone therapy, immunotherapy, or cytokine therapy. In some ²aspects, the ²immunotherapy is a checkpoint blockade therapy. In some aspects, ²administration of the ²composition reduces expression of Bc12 protein in the patient.²100151 An oligonucleotide includes an antisense nucleic acid molecule that ²specifically hybridizes to a nucleic acid molecule encoding a target protein ²or regulating the ²expression of the target protein. "Specific hybridization" means that the ²antisense nucleic ²acid molecule hybridizes to the targeted nucleic acid molecule and regulates ²its expression. ²Preferably, "specific hybridization" also means that no other genes or ²transcripts are affected.²<DP=6>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²An oligonucleotide can be a single-stranded nucleic acid and may comprise 7, ²8, 9, 10, 11, ²12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or ²more nucleobases. ²In particular aspects the oligonucleotide can comprise 15 to 30, 19 to 25, 20 ²to 23, or 21 ²contiguous nucleobases. In certain embodiments, the oligonucleotide inhibits ²the translation ²of a gene that promotes growth of a cancerous or pre-cancerous or hyperplastic ²mammalian ²cell (e.g., a human cell). An oligonucleotide may induce apoptosis in the ²cell, and / or inhibit ²the translation of an oncogene or other target gene. In certain embodiments, ²the ²oligonucleotide component comprises a single species of oligonucleotide. In ²other ²embodiments, the oligonucleotide component comprises a 2, 3, 4 or more species ²of ²oligonucleotide that target 1, 2, 3, 4, or more genes. The composition may ²further comprise a ²chemotherapeutic or other anti-cancer agent, which may or may not be ²incorporated in a lipid ²component or liposome of the invention. In further embodiments, the ²oligonucleotide ²component is incorporated within the liposome or lipid component.²100161 "Entrap," "encapsulate," and "incorporate" refer to the lipid or ²liposome ²forming an impediment to free diffusion into solution by an association with ²or around an ²agent of interest, e.g., a liposome may encapsulate an agent within a lipid ²layer or within an ²aqueous compartment inside or between lipid layers. In certain embodiments, ²the ²composition is comprised in a pharmaceutically acceptable carrier. The ²pharmaceutically ²acceptable carrier may be formulated for administration to a human subject or ²patient.²10017j In certain embodiments, the lipid component has an essentially neutral ²charge ²because it comprises a neutral phospholipid or a net neutral charge. In ²certain aspects a ²neutral phospholipid may be a phosphatidylcholine, such as DOPC, egg ²phosphatidylcholine ²("EPC"), dilauroylphosphatidylcholine ("DLPC"), dimyristoylphosphatidylcholine ²²("DMPC"), dipalmitoylphosphatidylcholine ("DPPC"), ²distearoylphosphatidylcholine ²("DSPC"), dilinoleoylphosphatidylcholine, 1,2-diarachidoyl-sn-glycero-3-²phosphocholine ²("DAPC"), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine ("DEPC"), 1-myristoy1-2-²²palmitoyl phosphatidylcholine ("WPC"), 1-palmitoy1-2-myristoyl ²phosphatidylcholine ²("PMPC"), 1-palmitoy1-2-stearoyl phosphatidylcholine ("PSPC"), 1-stearoy1-2-²palmitoyl ²phosphatidylcholine ("SPPC"), 1-palmitoy1-2-oleoyl phosphatidylcholine ²("POPC"), 1-²oleoy1-2-palmitoyl phosphatidylcholine ("OPPC"), or lysophosphatidylcholine. ²In other ²aspects the neutral phospholipid can be a phosphatidylethanolamine, such as ²dioleoylphosphatidylethanolamine ("DOPE"), distearoylphophatidylethanolamine ²("DSPE"),²6²<DP=7>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²dimyristoyl phosphatidylethanolamine ("DMPE"), dipalmitoyl ²phosphatidylethanolamine²("DPPE"), palmitoyloleoyl phosphatidylethanolamine ("POPE"), or²lysophosphatidylethanolamine. In certain embodiments, the phospholipid ²component can ²comprise 1, 2, 3, 4, 5, 6, 7, 8, or more kinds or types of neutral ²phospholipid. In other ²embodiments, a phospholipid component can comprise 2, 3, 4, 5, 6 or more kinds ²or type of ²neutral phospholipids.²100181 In certain embodiments, a lipid component can have an essentially ²neutral ²charge because it comprises a positively charged lipid and a negatively ²charged lipid. The ²lipid component may further comprise a neutrally charged lipid(s) or ²phospholipid(s). The ²positively charged lipid may be a positively charged phospholipid. The ²negatively charged ²lipid may be a negatively charged phospholipid. The negatively charged ²phospholipid may be ²a phosphatidylserine, such as dimyristoyl phosphatidylserine ("DMPS"), ²dipalmitoyl ²phosphatidylserine ("DPPS"), or brain phosphatidylserine ("BPS"). The ²negatively charged ²phospholipid may be a phosphatidylglycerol, such as ²dilauroylphosphatidylglycerol ²("DLPG"), dimyristoylphosphatidylglycerol ("DWG"), ²dipalmitoylphosphatidylglycerol ²("DPPG"), distearoylphosphatidylglycerol ("DSPG"), or ²dioleoylphosphatidylglycerol ²("DOPG"). In certain embodiments, the composition further comprises ²cholesterol or ²polyethyleneglycol (PEG). In other embodiments, the composition is essentially ²free of ²cholesterol. In certain embodiments, a phospholipid is a naturally-occurring ²phospholipid. In ²other embodiments, a phospholipid is a synthetic phospholipid.²100191 Liposomes can be made of one or more phospholipids, as long as the ²lipid ²material is substantially uncharged. It is important that the composition be ²substantially free ²of anionic and cationic phospholipids and cholesterol. Suitable phospholipids ²include ²phosphatidylcholines and others that are well known to persons that are ²skilled in this field.²100201 Another aspect of the present invention involves methods for delivering ²²oligonucleotide to a cell comprising contacting the cell with a neutral lipid ²composition of the ²invention. The methods will provide an inventive composition in an effective ²amount. An ²effective amount is an amount of therapeutic component that attenuates, slows, ²reduces or ²eliminates a cell, condition, or disease state in a subject. The cell may be ²comprised in a ²subject or patient, such as a human. The method may further comprise a method ²of treating ²cancer or other hyperplastic condition. The cancer may have originated in the ²bladder, blood, ²bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, ²head, kidney, liver,²7²<DP=8>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²lymph node, lung, nasopharynx, neck, prostate, skin, stomach, testis, tongue, ²ovary, or uterus. ²In certain embodiments, the method further comprises a method of treating a ²non-cancerous ²disease or hyperplastic condition. The cell may be a pre-cancerous or a ²cancerous cell. In ²certain embodiments, the compositions and methods inhibit the growth of the ²cell, induce ²apoptosis in the cell, and / or inhibit the translation of an oncogene. The ²oligonucleotide may ²inhibit the translation of a gene that is overexpressed in the cancerous cell.²100211 In certain embodiments, the methods of the invention further comprise ²administering an additional therapy to the subject. The additional therapy may ²comprise ²administering a chemotherapeutic (e.g., paclitaxel or docetaxel), a surgery, a ²radiation ²therapy, and / or a gene therapy. In certain aspects the chemotherapy is ²docetaxel, paclitaxel, ²cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, ²cyclophosphamide, ²camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, ²dactinomycin, ²daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), ²tamoxifen, ²raloxifene, estrogen receptor binding agents, taxol, gemcitabien, navelbine, ²farnesyl-protein ²tansferase inhibitors, transplatinum, 5-fluorouracil, vincristin, vinblastin, ²methotrexate, or ²combinations thereof. In certain embodiments the chemotherapy is a taxane such ²as docetaxal ²or paclitaxel. The chemotherapy can be delivered before, during, after, or ²combinations ²thereof relative to a neutral lipid composition of the invention. A ²chemotherapy can be ²delivered within 0, 1, 5, 10, 12, 20, 24, 30, 48, or 72 hours or more of the ²neutral lipid ²composition. The neutral lipid composition, the second anti-cancer therapy, or ²both the ²neutral lipid composition and the anti-cancer therapy can be administered ²intratumorally, ²intravenously, intraperitoneally, subcutaneously, orally or by various ²combinations thereof.²100221 It is contemplated that any embodiment discussed in this specification ²can be ²implemented with respect to any method or composition of the invention, and ²vice versa. ²Furthermore, compositions of the invention can be used to achieve the methods ²of the ²invention.²[00231 As used herein, "essentially free," in terms of a specified component, ²is used ²herein to mean that none of the specified component has been purposefully ²formulated into a ²composition and / or is present only as a contaminant or in trace amounts. The ²total amount of ²the specified component resulting from any unintended contamination of a ²composition is ²therefore well below 0.05%, preferably below 0.01%. Most preferred is a ²composition in²8²<DP=9>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²which no amount of the specified component can be detected with standard ²analytical ²methods.²100241 As used herein the specification, "a" or "an" may mean one or more. As ²used ²herein in the claim(s), when used in conjunction with the word "comprising," ²the words "a" ²or "an" may mean one or more than one.²100251 The use of the term "or" in the claims is used to mean "and / or" unless ²explicitly indicated to refer to alternatives only or the alternatives are ²mutually exclusive, ²although the disclosure supports a definition that refers to only alternatives ²and "and / or." As ²used herein "another" may mean at least a second or more.²100261 Throughout this application, the term "about" is used to indicate that ²a value ²includes the inherent variation of error for the device, the method being ²employed to ²determine the value, or the variation that exists among the study subjects.²100271 Other objects, features and advantages of the present invention will ²become ²apparent from the following detailed description. It should be understood, ²however, that the ²detailed description and the specific examples, while indicating preferred ²embodiments of the ²invention, are given by way of illustration only, since various changes and ²modifications ²within the spirit and scope of the invention will become apparent to those ²skilled in the art ²from this detailed description.²BRIEF DESCRIPTION OF THE DRAWINGS²100281 The following drawings form part of the present specification and are ²included ²to further demonstrate certain aspects of the present invention. The invention ²may be better ²understood by reference to one or more of these drawings in combination with ²the detailed ²description of specific embodiments presented herein.²100291 FIG. 1 ¨ Liposomal BCL2 antisense does not inhibit the viability of ²normal ²peripheral blood mononuclear cells. The ability of liposomal BCL2 antisense to ²inhibit the ²viability of normal peripheral blood mononuclear cells was tested by ²incubating liposomal ²BCL2 antisense corresponding to one of SEQ ID NO: 1 with the cells for four ²days.²100301 FIGs. 2A-J ¨ Liposomal BCL2 antisense inhibits the viability of ²germinal ²center B-cell-like subtype diffuse large B cell lymphoma cells. The ability of ²liposomal²9²<DP=10>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²BCL2 antisense to inhibit the growth of germinal center B-cell-like subtype ²diffuse large B ²cell lymphoma cells was tested in ten human germinal center B-cell-like ²subtype diffuse large ²B cell lymphoma cell lines: DOHH-2 (FIG. 2A), SU-DHL-4 (FIG. 2B), SU-DHL-6 ²(FIG. ²2C), SU-DHL-10 (FIG. 2D), OCI-LY-18 (FIG. 2E), OCI-LY-19 (FIG. 2F), WSU-DLCL2 ²(FIG. 2G), RL (FIG. 2H), OCI-LY-1 (FIG. 21), and OCI-LY-7 (FIG. 2J).²10031j FIGs. 3A-C ¨ Liposomal BCL2 antisense inhibits the viability of ²activated B-²cell-like subtype diffuse large B cell lymphoma cells. The ability of ²liposomal BCL2 ²antisense to inhibit the growth of activated B-cell-like subtype diffuse large ²B cell lymphoma ²cells was tested in three human activated B-cell-like subtype diffuse large B ²cell lymphoma ²cell lines: SU-DHL-2 (FIG. 3A), U-2932 (FIG. 3B), and RI-1 (FIG. 3C).²100321 FIGs. 4A-B ¨ Liposomal BCL2 antisense inhibits the viability of ²lymphoma ²cells. The ability of liposomal BCL2 antisense to inhibit the growth of ²lymphoma cells was ²tested in two human lymphoma cell lines: GRANTA-519 (mantle cell lymphoma; ²FIG. 4A) ²and Ramos (Burkitt lymphoma; FIG. 4B).²100331 FIGs. 5A-C ¨ Liposomal BCL2 antisense inhibits the viability of myeloid ²²leukemia cells. The ability of liposomal BCL2 antisense to inhibit the growth ²myeloid ²leukemia cells was tested in three human myeloid leukemia cell lines: K562 ²(FIG. 5A), MV-²4-11 (FIG. 5B), and Kasumi-1 (FIG. 5C).²DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS²100341 Bc1-2 is a potential therapeutic target for cancer, such as, for ²example, ²aggressive NHL. To inhibit the expression of Bc1-2 protein, the present ²invention provides ²compositions and methods for delivery of an anti- BCL2 oligonucleotide (e.g., ²an inhibitor of ²gene expression) to a cell via a lipid composition, in certain aspects a lipid ²composition with ²a net charge of about zero, i.e., a neutral lipid composition, which allows it ²to be delivered ²systemically via intravenous infusion. These methods may be effectively used ²to treat a ²cancer.²I. Lipids and Liposomes²100351 "Liposomes" is used herein to mean lipid-containing vesicles having a ²lipid ²bilayer, as well as other lipid carrier particles that can entrap or ²incorporate antisense²<DP=11>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²oligonucleotides. As such, liposome is a generic term encompassing a variety ²of unilamellar, ²multilamellar, and multivesicular lipid vehicles formed by the generation of ²enclosed lipid ²bilayers or aggregates. In addition, liposomes may have an undefined lamellar ²structure. ²Liposomes may be characterized as having vesicular structures with a ²phospholipid bilayer ²membrane and an inner aqueous medium. Multilamellar liposomes have multiple ²lipid layers ²separated by aqueous medium. They form spontaneously when phospholipids are ²suspended ²in an excess of aqueous solution. The lipid components undergo self-²rearrangement before ²the formation of closed structures and entrap water and dissolved solutes ²between the lipid ²bilayers (Ghosh and Bachhawat, 1991). However, the present invention also ²encompasses ²compositions that have different structures in solution than the normal ²vesicular structure. For ²example, the lipids may assume a micellar structure or merely exist as non-²uniform ²aggregates of lipid molecules.²100361 Liposomes are a form of nanoparticles that are carriers for delivering ²a variety ²of drugs into a diseased tissue. Optimal liposome size depends on the target ²tissue. In tumor ²tissue, the vasculature is discontinuous, and pore sizes vary from 100 to 780 ²nm (Siwak et at., ²2002). By comparison, pore size in normal vascular endothelium is <2 nm in ²most tissues, ²and 6 nm in post-capillary venules. Negatively charged liposomes are thought ²to be more ²rapidly removed from circulation than neutral or positively charged liposomes; ²however, ²recent studies have indicated that the type of negatively charged lipid ²affects the rate of ²liposome uptake by the reticulo-endothelial system (RES). For example, ²liposomes ²containing negatively charged lipids that are not sterically shielded ²(phosphatidylserine, ²phosphatidic acid, and phosphatidylglycerol) are cleared more rapidly than ²neutral liposomes. ²Interestingly, cationic liposomes (1,2-dioleoy1-3-trimethylammonium-propane ²[DOTAI]) ²and cationic-liposome-DNA complexes are more avidly bound and internalized by ²endothelial cells of angiogenic blood vessels via endocytosis than anionic, ²neutral, or ²sterically stabilized neutral liposomes (Thurston et at., 1998; Krasnici et ²at., 2003). Cationic ²liposomes may not be ideal delivery vehicles for tumor cells because surface ²interactions ²with the tumor cells create an electrostatically derived binding-site barrier ²effect, inhibiting ²further association of the delivery systems with tumor spheroids (Kostarelos ²et at., 2004). ²However, neutral liposomes appear to have better intratumoral penetration. ²Toxicity with ²specific liposomal preparations has also been a concern. Cationic liposomes ²elicit dose-²dependent toxicity and pulmonary inflammation by promoting release of reactive ²oxygen ²intermediates, and this effect is more pronounced with multivalent cationic ²liposomes than²11²<DP=12>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²monovalent cationic liposomes, such as DOTAP (Dokka et at., 2000). Neutral and ²negative ²liposomes do not appear to exhibit lung toxicity (Guitierrez-Puente et at., ²1999). Cationic ²liposomes, while efficiently taking up nucleic acids, have had limited success ²for in vivo gene ²down-regulation, perhaps because of their stable intracellular nature and ²resultant failure to ²release nucleic acid contents. Lipids with neutral charge or lipid ²compositions with a ²neutralized charge, e.g., 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), are ²used herein ²because of the neutral properties and success in delivering antisense ²oligonucleotides in vivo.²100371 The present invention provides methods and compositions for associating ²an ²oligonucleotide, such as an antisense oligonucleotide, with a lipid and / or ²liposome. The ²oligonucleotide may be incorporated in the aqueous interior of a liposome, ²interspersed ²within the lipid bilayer of a liposome, attached to a liposome via a linking ²molecule that is ²associated with both the liposome and the oligonucleotide, entrapped in a ²liposome, ²complexed with a liposome, dispersed in a solution containing a lipid, mixed ²with a lipid, ²combined with a lipid, contained as a suspension in a lipid, contained or ²complexed with a ²micelle, or otherwise associated with a lipid. The liposome or ²liposome / oligonucleotide-²associated compositions provided herein are not limited to any particular ²structure in ²solution. For example, they may be present in a bilayer structure, as ²micelles, or with a ²"collapsed" structure. They may also simply be interspersed in a solution, ²possibly forming ²aggregates that are not uniform in either size or shape.²A. Lipids²100381 Lipids are fatty substances that may be naturally occurring or ²synthetic. For ²example, lipids include the fatty droplets that naturally occur in the ²cytoplasm as well as the ²class of compounds that are well known to those of skill in the art that ²contain long-chain ²aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, ²amines, amino ²alcohols, and aldehydes. An example is the lipid 1,2-dioleoyl-sn-glycero-3-²phosphocholine ²(DOPC).²100391 Lipid compositions of the present invention may comprise phospholipids. ²In ²certain embodiments, a single kind or type of phospholipid may be used in the ²creation of ²lipid compositions, such as liposomes. In other embodiments, more than one ²kind or type of ²phospholipid may be used.²12²<DP=13>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²10040j Phospholipids include glycerophospholipids and certain sphingolipids. ²Phospholipids include, but are not limited to, dioleoylphosphatidylycholine ²("DOPC"), egg²phosphatidylcholine ("EPC"), dilauryloylphosphatidylcholine ²("DLPC"),²dimyristoylphosphatidylcholine ("DMPC"), dipalmitoylphosphatidylcholine ²("DPPC"), ²distearoylphosphatidylcholine ("DSPC"), dilinoleoylphosphatidylcholine, 1,2-²diarachidoyl-²sn-glycero-3-phosphocholine ("DAPC"), 1,2-dieicosenoyl-sn-glycero-3-²phosphocholine ²("DEPC"), 1-myristoy1-2-palmitoyl phosphatidylcholine ("MPPC"), 1-palmitoy1-2-²myristoyl ²phosphatidylcholine ("PMPC"), 1-palmitoy1-2-stearoyl phosphatidylcholine ²("PSPC"), 1-²stearoy1-2-palmitoyl phosphatidylcholine ("SPPC"), palmitoyloeoyl ²phosphatidylcholine²("POPC"), 1-oleoy1-2-palmitoyl phosphatidylcholine ²("OPPC"),²dilauryloylphosphatidylglycerol ("DLPG"), dimyristoylphosphatidylglycerol ²("DMPG"), ²dipalmitoylphosphatidylglycerol ("DPPG"), distearoylphosphatidylglycerol ²("DSPG"), ²dioleoylphosphatidylglycerol ("DOPG"), dimyristoyl phosphatidic acid ("DMPA"), ²²dipalmitoyl phosphatidic acid ("DPPA"), distearoyl phosphatidic acid ("D ²SPA"), dioleoyl ²phosphatidic acid ("DOPA"), dimyristoyl phosphatidylethanolamine ("DMPE"), ²dipalmitoyl²phosphatidylethanolamine ("DPPE"), di ²stearoylphophatidylethanolamine ("D SPE"),²dioleoylphosphatidylethanolamine ("DOPE"), palmitoyloeoyl ²phosphatidyletlianolamine ²("POPE"), dimyristoyl phosphatidylserine ("DMPS"), dipalmitoyl ²phosphatidylserine ²("DPPS"), brain phosphatidylserine ("BPS"), distearoyl sphingomyelin ("DSSP"), ²brain ²sphingomyelin ("B SP"), dipalmitoyl sphingomyelin ("DP SP"), ²lysophosphatidylcholine, and ²lysophosphatidylethanolamine.²100411 Phospholipids include, for example, ²phosphatidylcholines,²phosphatidylglycerols, and phosphatidylethanolamines; because ²phosphatidylethanolamines ²and phosphatidylcholines are non-charged under physiological conditions (i.e., ²at about pH ²7), these compounds may be particularly useful for generating neutral ²liposomes. In certain ²embodiments, the phospholipid DOPC is used to produce non-charged liposomes or ²lipid ²compositions. In certain embodiments, a lipid that is not a phospholipid ²(e.g., a cholesterol) ²can also be used²10042 Phospholipids may be from natural or synthetic sources. However, ²phospholipids from natural sources, such as egg or soybean ²phosphatidylcholine, brain ²phosphatidic acid, brain or plant phosphatidylinositol, heart cardiolipin, and ²plant or bacterial ²phosphatidylethanolamine, are not used in certain embodiments as the primary ²phosphatide²13²<DP=14>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²(i.e., constituting 50% or more of the total phosphatide composition) because ²this may result ²in instability and leakiness of the resulting liposomes.²B. Neutral Liposomes²10043j "Neutral liposomes or lipid composition" or "non-charged liposomes or ²lipid ²composition," as used herein, are defined as liposomes or lipid compositions ²having one or ²more lipids that yield an essentially-neutral net charge (substantially non-²charged). In certain ²embodiments, neutral liposomes or lipid compositions may include mostly lipids ²and / or ²phospholipids that are themselves neutral. In certain embodiments, amphipathic ²lipids may be ²incorporated into or used to generate neutral liposomes or lipid compositions. ²For example, a ²neutral liposome may be generated by combining positively and negatively ²charged lipids so ²that those charges substantially cancel one another, thereby yielding an ²essentially-neutral net ²charge. By "essentially neutral" or "essentially non-charged," it is meant ²that few, if any, ²lipids within a given population (e.g., a population of liposomes) include a ²charge that is not ²canceled by an opposite charge of another component (e.g., fewer than 10% of ²components ²include a non-canceled charge, more preferably fewer than 5%, and most ²preferably fewer ²than 1%). In certain embodiments of the present invention, a composition may ²be prepared ²wherein the lipid component of the composition is essentially neutral but is ²not in the form of ²liposomes.²10044j The size of the liposomes varies depending on the method of synthesis. ²A ²liposome suspended in an aqueous solution is generally in the shape of a ²spherical vesicle, ²and may have one or more concentric layers of lipid bilayer molecules. Each ²layer consists of ²a parallel array of molecules represented by the formula XY, wherein X is a ²hydrophilic ²moiety and Y is a hydrophobic moiety. In aqueous suspension, the concentric ²layers are ²arranged such that the hydrophilic moieties tend to remain in contact with an ²aqueous phase ²and the hydrophobic regions tend to self-associate. For example, when aqueous ²phases are ²present within the liposome, the lipid molecules may form a bilayer, known as ²a lamella, of ²the arrangement XY-YX. Aggregates of lipids may form when the hydrophilic and ²hydrophobic parts of more than one lipid molecule become associated with each ²other. The ²size and shape of these aggregates will depend upon many different variables, ²such as the ²nature of the solvent and the presence of other compounds in the solution.²100451 Liposomes within the scope of the present invention can be prepared in ²accordance with known laboratory techniques, such as, for example, the method ²of Bangham²14²<DP=15>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²et at. (1965), the contents of which are incorporated herein by reference; the ²method of ²Gregoriadis (1979), the contents of which are incorporated herein by ²reference; the method of ²Deamer and Uster (1983), the contents of which are incorporated by reference; ²and the ²reverse-phase evaporation method as described by Szoka and Papahadjopoulos ²(1978). The ²aforementioned methods differ in their respective abilities to entrap aqueous ²material and ²their respective aqueous space-to-lipid ratios.²100461 In certain embodiments, a neutral liposome may be used to deliver an ²oligonucleotide, such as an antisense oligonucleotide. The neutral liposome ²may contain a ²single species of oligonucleotide directed to the suppression of translation ²of a single gene, or ²the neutral liposome may contain multiple species of oligonucleotides that are ²directed to the ²suppression of translation of multiple genes. Further, the neutral liposome ²may also contain a ²chemotherapeutic in addition to the oligonucleotide; thus, in certain ²embodiments, a ²chemotherapeutic and an oligonucleotide may be delivered to a cell (e.g., a ²cancerous cell in ²a human subject) in the same or separate compositions.²100471 Dried lipids or lyophilized liposomes may be dehydrated and ²reconstituted at ²an appropriate concentration with a suitable solvent (e.g., DPBS or Hepes ²buffer). The ²mixture may then be vigorously shaken in a vortex mixer. The liposomes may be ²resuspended at an appropriate total phospholipid concentration (e.g., about 10-²200 mM). ²Unencapsulated oligonucleotide may be removed by centrifugation at 29,000 g ²and the ²liposomal pellets washed. Alternatively, the unencapsulated oligonucleotides ²may be ²removed by dialyzing against an excess of solvent. The amount of ²oligonucleotide ²encapsulated can be determined in accordance with standard methods.²Inhibition of Gene Expression²100481 An inhibitory oligonucleotide can inhibit the transcription or ²translation of a ²gene in a cell. An oligonucleotide may be from 5 to 50 or more nucleotides ²long, and in ²certain embodiments from 7 to 30 nucleotides long. In certain embodiments, the ²²oligonucleotide maybe 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, ²22, 23, 24, 25, ²26, 27, 28, 29, or 30 nucleotides long. The oligonucleotide may comprise a ²nucleic acid ²and / or a nucleic acid analog. Typically, an inhibitory oligonucleotide will ²inhibit the ²translation of a single gene within a cell; however, in certain embodiments, ²an inhibitory ²oligonucleotide may inhibit the translation of more than one gene within a ²cell.²<DP=16>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²10049j Within an oligonucleotide, the components of the oligonucleotide need ²not be ²of the same type or homogenous throughout (e.g., an oligonucleotide may ²comprise a ²nucleotide and a nucleic acid or nucleotide analog). In certain embodiments of ²the present ²invention, the oligonucleotide may comprise only a single nucleic acid or ²nucleic acid analog. ²The inhibitory oligonucleotide may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, ²15, 16, 17, 18, ²19, 20, 25, 30 or more contiguous nucleobases, including all ranges ²therebetween, that ²hybridize with a complementary nucleic acid to form a double-stranded ²structure.²III. Nucleic Acids²100501 The present invention provides methods and compositions for the ²delivery of ²an oligonucleotide via neutral liposomes. Because an oligonucleotide is ²composed of a ²nucleic acid, methods relating to nucleic acids (e.g., production of a nucleic ²acid, ²modification of a nucleic acid, etc.) may also be used with regard to an ²oligonucleotide.²10051j The term "nucleic acid" is well known in the art. A "nucleic acid" as ²used ²herein generally refers to a molecule (i.e., a strand) of DNA, RNA, or a ²derivative or analog ²thereof, comprising a nucleobase. These definitions refer to a single-stranded ²or double-²stranded nucleic acid. Double-stranded nucleic acids may be formed by fully ²complementary ²binding; however, in some embodiments, a double-stranded nucleic acid may be ²formed by ²partial or substantial complementary binding. As used herein, a single-²stranded nucleic acid ²may be denoted by the prefix "ss" and a double-stranded nucleic acid by the ²prefix "ds."²A. Nucleobases²10052j As used herein a "nucleobase" refers to a heterocyclic base, such as, ²for ²example, a naturally occurring nucleobase (i.e., an A, T, G, C or U) found in ²at least one ²naturally occurring nucleic acid (i.e., DNA and RNA), and naturally or non-²naturally ²occurring derivative(s) and analogs of such a nucleobase. A nucleobase ²generally can form ²one or more hydrogen bonds (i.e., "anneal" or "hybridize") with at least one ²naturally ²occurring nucleobase in a manner that may substitute for naturally occurring ²nucleobase ²pairing (e.g., the hydrogen bonding between A and T, G and C, and A and U). A ²nucleobase ²may be comprised in a nucleoside or nucleotide, using any chemical or natural ²synthesis ²method described herein or known to one of ordinary skill in the art.²16²<DP=17>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²10053j "Purine" and / or "pyrimidine" nucleobase(s) encompass naturally ²occurring ²purine and / or pyrimidine nucleobases and also derivative(s) and analog(s) ²thereof, including ²but not limited to, a purine or pyrimidine substituted by one or more of an ²alkyl, ²carboxyalkyl, amino, hydroxyl, halogen (i.e., fluoro, chloro, bromo, or iodo), ²thiol, or ²alkylthiol moiety. Preferred alkyl (e.g., alkyl, caboxyalkyl, etc.) moieties ²comprise of from ²about 1, about 2, about 3, about 4, about 5, to about 6 carbon atoms. Other ²non-limiting ²examples of a purine or pyrimidine include a deazapurine, a 2,6-diaminopurine, ²a 5-²fluorouracil, a xanthine, a hypoxanthine, a 8-bromoguanine, a 8-chloroguanine, ²a ²bromothyline, a 8-aminoguanine, a 8-hydroxyguanine, a 8-methylguanine, a 8-²thioguanine, ²an azaguanine, a 2-aminopurine, a 5-ethylcytosine, a 5-methylcyosine, a 5-²bromouracil, a 5-²ethyluracil, a 5-iodouracil, a 5-chlorouracil, a 5-propyluracil, a thiouracil, ²a 2-methyladenine, ²a methylthioadenine, a N,N-diemethyladenine, an azaadenines, a 8-bromoadenine, ²a 8-²hydroxyadenine, a 6-hydroxyaminopurine, a 6-thiopurine, a 4-(6-²aminohexyl / cytosine), and ²the like. Purine and pyrimidine derivatives or analogs include, but are not ²limited to²(abbreviation / modified base description): ac4 ²c / 4-acetyl cyti dine, Mam5 s2u / 5-²methoxy ami nomethyl -2-thi ouri dine, Chm5u / 5-(carboxyhydroxylmethyl) uri ²dine, Man²q / Beta, D-mannosylqueosine, Cm / 21-0-methylcyti dine, ²Mcm5 s2u / 5-²methoxy c arb onylm ethyl -2-thi ouri dine, ²Cmnm5s2u / 5-carboxymethylamino-methy1-2-²thioridine, Mcm5u / 5 -methoxycarb onylmethyluri dine, ²Cmnm5u / 5-²carboxymethylaminomethyluridine, Mo5u / 5-methoxyuridine, D / Dihydrouridine, ²Ms2i6a, 2-²methylthio-N6-isopentenyladenosine, Fm / 21-0-methylpseudouridine, Ms2t6a / N-((9-²beta-D-²rib ofuranosy1-2-m ethylthi opurine-6-yl)c arb amoyl)threonine, Gal ² q / B eta,D-²galactosylqueosine, ²Mt6a / N-((9-b eta-D-rib ofuranosylpurine-6-y1)N-m ethyl-²carb am oyl)threonine, Gm / 21-0-methylguanosine, Mv / Uridine-5-oxyacetic acid ²methylester,²I / Inosine, o5u / Uridine-5-oxyacetic acid ²(v), I6a / N6-i sopentenyladenosine,²Osyw / Wybutoxosine, mla / l-methyladenosine, P / Pseudouridine, mlf / l-²methylpseudouridine, ²Q / Queosine, ml g / 1 -methylguanosine, s2c / 2-thiocytidine, mlI / 1 -²methylinosine, s2t / 5-methy1-²2-thiouridine, m22g / 2,2-dimethylguanosine, s2u / 2-thiouridine, m2a / 2-²methyladenosine, ²s4u / 4-thi ouri dine, m2g / 2-m ethylguanosine, T / 5 -m ethyluri dine, m3 c / 3 -²methyl cyti dine, t6a / N-²((9-b eta-D-rib ofuranosylpurine-6-yl)carb am oyl)threonine, m5 c / 5 -m ethyl ²cyti dine, Tm / 21-0-²methy1-5-methyluridine, m6a / N6-methyladenosine, Um / 21-0-methyluridine, m7g / 7-²methylguanosine, Yw / Wybutosine, Mam5u / 5-methylaminomethyluridine, or X / 3-(3-²amino-²3 -carb oxypropyl)uridine, (acp3)u.²17²<DP=18>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²B. Nucleosides²100541 As used herein, a "nucleoside" refers to an individual chemical unit ²comprising a nucleobase covalently attached to a nucleobase linker moiety. A ²non-limiting ²example of a "nucleobase linker moiety" is a sugar comprising 5-carbon atoms ²(i.e., a "5-²carbon sugar"), including but not limited to a deoxyribose, a ribose, an ²arabinose, or a ²derivative or an analog of a 5-carbon sugar. Non-limiting examples of a ²derivative or an ²analog of a 5-carbon sugar include a 2'-fluoro-2'-deoxyribose or a carbocyclic ²sugar where a ²carbon is substituted for an oxygen atom, in the sugar ring. As used herein, a ²"moiety" ²generally refers to a smaller chemical or molecular component of a larger ²chemical or ²molecular structure.²100551 Different types of covalent attachment(s) of a nucleobase to a ²nucleobase ²linker moiety are known in the art. By way of non-limiting example, a ²nucleoside comprising ²a purine (i.e., A or G) or a 7-deazapurine nucleobase typically comprises a ²covalent ²attachment of the 9 position of the purine or 7-deazapurine to a 1 '-position ²of a 5-carbon ²sugar. In another non-limiting example, a nucleoside comprising a pyrimidine ²nucleobase ²(i.e., C, T, or U) typically comprises a covalent attachment of the 1 position ²of the pyrimidine ²to a l'-position of a 5-carbon sugar (Kornberg and Baker, 1992).²C. Nucleotides²100561 As used herein, a "nucleotide" refers to a nucleoside further ²comprising a ²"backbone linkage." A backbone linkage generally covalently attaches a ²nucleotide to ²another molecule comprising a nucleotide, or to another nucleotide to form a ²nucleic acid. ²The "backbone linkage" in naturally occurring nucleotides typically comprises ²a phosphate ²moiety (e.g., a phosphodiester backbone linkage), which is covalently attached ²to a 5-carbon ²sugar. The attachment of the backbone moiety typically occurs at either the 3'-² or 5'-position ²of the 5-carbon sugar. However, other types of attachments are known in the ²art, particularly ²when a nucleotide comprises derivatives or analogs of a naturally occurring 5-²carbon sugar or ²phosphate moiety.²D. Nucleic Acid Analogs²100571 A nucleic acid may comprise, or be composed entirely of, a derivative ²or ²analog of a nucleobase, a nucleobase linker moiety, and / or backbone linkage ²that may be ²present in a naturally occurring nucleic acid. As used herein a "derivative" ²refers to a²18²<DP=19>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²chemically modified or altered form of a naturally occurring molecule, while ²the terms ²"mimic" or "analog" refer to a molecule that may or may not structurally ²resemble a naturally ²occurring molecule or moiety, but possesses similar functions. Nucleobase, ²nucleoside, and ²nucleotide analogs or derivatives are well known in the art.²100581 Non-limiting examples of nucleosides, nucleotides, or nucleic acids ²comprising 5-carbon sugar and / or backbone linkage derivatives or analogs, ²include those in ²U.S. Pat. No. 5,681,947 which describes oligonucleotides comprising purine ²derivatives that ²form triple helixes with and / or prevent expression of dsDNA; U.S. Pat. Nos. ²5,652,099 and ²5,763,167 which describe nucleic acids incorporating fluorescent analogs of ²nucleosides ²found in DNA or RNA, particularly for use as fluorescent nucleic acids probes; ²U.S. Pat. No. ²5,614,617 which describes oligonucleotide analogs with substitutions on ²pyrimidine rings ²that possess enhanced nuclease stability; U.S. Pat. Nos. 5,670,663, 5,872,232 ²and 5,859,221 ²which describe oligonucleotide analogs with modified 5-carbon sugars (i.e., ²modified 2'-²deoxyfuranosyl moieties) used in nucleic acid detection; U.S. Pat. No. ²5,446,137 which ²describes oligonucleotides comprising at least one 5-carbon sugar moiety ²substituted at the 4' ²position with a substituent other than hydrogen that can be used in ²hybridization assays; U.S. ²Pat. No. 5,886,165 which describes oligonucleotides with both ²deoxyribonucleotides with 3'-²5' backbone linkages and ribonucleotides with 2'-5' backbone linkages; U.S. ²Pat. No. ²5,714,606 which describes a modified backbone linkage wherein a 3'-position ²oxygen of the ²backbone linkage is replaced by a carbon to enhance the nuclease resistance of ²nucleic acids; ²U.S. Pat. No. 5,672,697 which describes oligonucleotides containing one or ²more 5' ²methylene phosphonate backbone linkages that enhance nuclease resistance; U.S. ²Pat. Nos. ²5,466,786 and 5,792,847 which describe the linkage of a substituent moiety ²that may ²comprise a drug or label to the 2' carbon of an oligonucleotide to provide ²enhanced nuclease ²stability and ability to deliver drugs or detection moieties; U.S. Pat. No. ²5,223,618 which ²describes oligonucleotide analogs with a 2 or 3 carbon backbone linkage ²attaching the 4' ²position and 3' position of adjacent 5-carbon sugar moiety to enhanced ²cellular uptake, ²resistance to nucleases, and hybridization to target RNA; U.S. Pat. No. ²5,470,967 which ²describes oligonucleotides comprising at least one sulfamate or sulfamide ²backbone linkage ²that are useful as nucleic acid hybridization probes; U.S. Pat. Nos. ²5,378,825, 5,777,092, ²5,623,070, 5,610,289 and 5,602,240 which describe oligonucleotides with a ²three or four ²atom backbone linkage moiety replacing the phosphodiester backbone linkage ²used for ²improved nuclease resistance, cellular uptake, and regulating RNA expression; ²U.S. Pat. No.²19²<DP=20>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²5,858,988 which describes hydrophobic carrier agent attached to the 2'-0 ²position of ²oligonucleotides to enhance their membrane permeability and stability; U.S. ²Pat. No. ²5,214,136 which describes oligonucleotides conjugated to anthraquinone at the ²5' terminus ²that possess enhanced hybridization to DNA or RNA; enhanced stability to ²nucleases; U.S. ²Pat. No. 5,700,922 which describes PNA-DNA-PNA chimeras wherein the DNA ²comprises ²2'-deoxy-erythro-pentofaranosyl nucleotides for enhanced nuclease resistance, ²binding ²affinity, and ability to activate RNase H; U.S. Pat. No. 5,708,154 which ²describes RNA ²linked to a DNA to form a DNA-RNA hybrid; U.S. Pat. No. 5,908,845 which ²describes ²polyether nucleic acids wherein one or more nucleobases are linked to chiral ²carbon atoms in ²a polyether backbone; U.S. Pat. Nos. 5,786,461, 5,891,625, 5,786,461, ²5,773,571, 5,766,855, ²5,736,336, 5,719,262, 5,714,331, 5,539,082, and WO 92 / 20702 which describe ²peptide ²nucleic acids (PNA or peptide-based nucleic acid analog; or PENAM) that ²generally ²comprise one or more nucleotides or nucleosides that comprise a nucleobase ²moiety, a ²nucleobase linker moiety that is not a 5-carbon sugar (e.g., aza nitrogen ²atoms, amido and / or ²ureido tethers), and / or a backbone linkage that is not a phosphate backbone ²linkage (e.g., ²aminoethylglycine, polyamide, polyethyl, polythioamide, polysulfinamide, or ²polysulfonamide backbone linkage); and U.S. Pat. No. 5,855,911 which describes ²the ²hydrophobic, nuclease resistant P-ethoxy backbone linkage.²10059j Other modifications and uses of nucleic acid analogs are known in the ²art, and ²it is anticipated that these techniques and types of nucleic acid analogs may ²be used with the ²present invention.²E. Preparation of Nucleic Acids²10060! A nucleic acid may be made by any technique known to one of ordinary ²skill ²in the art, such as chemical synthesis, enzymatic production or biological ²production. Non-²limiting examples of a synthetic nucleic acid (e.g., a synthetic ²oligonucleotide) include a ²nucleic acid made by in vitro chemical synthesis using phosphotriester, ²phosphite, or ²phosphoramidite chemistry and solid phase techniques, such as described in EP ²266,032, ²incorporated herein by reference, or by deoxynucleoside H-phosphonate ²intermediates as ²described by Froehler et at. (1986) and U.S. Pat. No. 5,705,629, each ²incorporated herein by ²reference. In the methods of the present invention, one or more species of ²oligonucleotide ²may be used. Various mechanisms of oligonucleotide synthesis have been ²disclosed in, for²<DP=21>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²example, U.S. Pat. Nos. 4,659,774, 4,816,571, 5,141,813, 5,264,566, 4,959,463, ²5,428,148, ²5,554,744, 5,574,146, 5,602,244, each of which is incorporated herein by ²reference.²F. Purification of Nucleic Acids²10061 j A nucleic acid may be purified on polyacrylamide gels, cesium chloride ²²centrifugation gradients, or by any other means known to one of ordinary skill ²in the art (see ²for example, Sambrook et at. (2001), incorporated herein by reference).²10621 In certain embodiments, the present invention concerns a nucleic acid ²that is ²an isolated nucleic acid. As used herein, the term "isolated nucleic acid" ²refers to a nucleic ²acid molecule (e.g., an RNA or DNA molecule) that has been isolated free of, ²or is otherwise ²free of, the bulk of the total genomic and transcribed nucleic acids of one or ²more cells. In ²certain embodiments, "isolated nucleic acid" refers to a nucleic acid that has ²been isolated ²free of, or is otherwise free of, the bulk of cellular components or in vitro ²reaction ²components, such as, for example, macromolecules, such as lipids or proteins, ²small ²biological molecules, and the like.²G. Hybridization²100631 As used herein, "hybridization," "hybridize(s)," or "capable of ²hybridizing" is ²understood to mean the forming of a double or triple stranded molecule or a ²molecule with ²partial double or triple stranded nature. The term "anneal" as used herein is ²synonymous with ²"hybridize."²10064j As used herein "stringent condition(s)" or "high stringency" are those ²conditions that allow hybridization between or within one or more nucleic acid ²strand(s) ²containing complementary sequence(s), but precludes hybridization of random ²sequences. ²Stringent conditions tolerate little, if any, mismatch between a nucleic acid ²and a target ²strand. Such conditions are well known to those of ordinary skill in the art, ²and are preferred ²for applications requiring high selectivity.²100651 Stringent conditions may comprise low salt and / or high temperature ²conditions, such as provided by about 0.02 M to about 0.15 M NaCl at ²temperatures of about ²50 C to about 70 C. It is understood that the temperature and ionic strength ²of a desired ²stringency are determined in part by the length of the particular nucleic ²acid(s), the length and ²nucleobase content of the target sequence(s), the charge composition of the ²nucleic acid(s),²21²<DP=22>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²and to the presence or concentration of formamide, tetramethylammonium ²chloride, or other ²solvent(s) in a hybridization mixture.²100661 It is also understood that these ranges, compositions and conditions ²for ²hybridization are mentioned by way of non-limiting examples only, and that the ²desired ²stringency for a particular hybridization reaction is often determined ²empirically by ²comparison to one or more positive or negative controls. Depending on the ²application ²envisioned it is preferred to employ varying conditions of hybridization to ²achieve varying ²degrees of selectivity of a nucleic acid towards a target sequence. In a non-²limiting example, ²identification or isolation of a related target nucleic acid that does not ²hybridize to a nucleic ²acid under stringent conditions may be achieved by hybridization at low ²temperature and / or ²high ionic strength. Such conditions are termed "low stringency" or "low ²stringency ²conditions," and non-limiting examples of low stringency include hybridization ²performed at ²about 0.15 M to about 0.9 M NaCl at a temperature range of about 20 C to about ²50 C. Of ²course, it is within the skill of one in the art to further modify the low or ²high stringency ²conditions to suit a particular application.²IV. Method of Manufacturing Liposomal P-ethoxy Antisense Drug Product²10067j Antisense oligonucleotides (oligos) complementary to specific regions ²of a ²target mRNA have been used to inhibit the expression of endogenous genes. When ²the ²antisense oligonucleotides bind to a target mRNA, a DNA-RNA hybrid is formed. ²This ²hybrid formation inhibits the translation of the mRNA and, thus, the ²expression of the ²encoded protein. If the protein is essential for the survival of the cell, the ²inhibition of its ²expression may lead to cell death. Therefore, antisense oligonucleotides can ²be useful tools in ²anticancer and antiviral therapies.²100681 The main obstacles in using antisense oligonucleotides to inhibit gene ²expression are cellular instability, low cellular uptake, and poor ²intercellular delivery. Natural ²phosphodiesters are not resistant to nuclease hydrolysis; thus high ²concentrations of antisense ²oligonucleotides are needed before any inhibitory effect is observed. Modified ²²phosphodiester analogs, such as P-ethoxy, have been made to overcome this ²nuclease ²hydrolysis problem, but they have not provided a satisfactory solution to the ²problem.²100691 The cellular uptake of antisense oligonucleotides is low. To solve this ²²problem, physical techniques, such as calcium-phosphate precipitation, DEAE-²dextran²22²<DP=23>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²mediation, or electroporation, have been used to increase the cellular uptake ²of ²oligonucleotides. These techniques are difficult to reproduce and are ²inapplicable in vivo. ²Cationic lipids, such as Lipofectin, have also been used to deliver ²oligonucleotides. An ²electrostatic interaction is formed between the cationic lipids and the ²negatively charged ²oligonucleotides, which results in a complex that is then taken up by the ²target cells. Since ²these cationic lipids do not protect the oligonucleotides from nuclease ²digestion and are ²harmful to the cell membrane, they are only useful in delivering the nuclease-²resistant ²phosphorothioates, but not the nuclease-cleavable phosphodiesters.²100701 Another modified phosphodiester analog that has been prepared is P-²ethoxy. ²The P-ethoxy antisense backbone does not have an adverse effect on bleeding ²and ²complement activation, which are some of the toxicities that have been ²reported for other ²antisense analogs. The modifications of P-ethoxy oligonucleotides are made in ²the phosphate ²backbone so that the modification will not interfere with the binding of these ²oligonucleotides ²to a target mRNA. P-ethoxy oligonucleotides are made by adding an ethyl group ²to the non-²bridging oxygen atom of the phosphate backbone, thus rendering these ²oligonucleotides ²uncharged compounds. In spite of their resistance to nucleases, the cellular ²uptake and ²intracellular delivery of P-ethoxy oligonucleotides is poor because upon ²internalization, these ²oligonucleotides remain sequestered inside the endosomal / lysosomal vacuoles, ²impeding ²their access to target mRNA.²A. P-ethoxy antisense drug product²100711 The liposomal P-ethoxy antisense drug product is composed of two cGMP ²products, both of which have a FDA-required Certificate of Analysis with FDA-²approved ²release criteria. The raw materials, solvents, and final drug product are ²described herein. ²When manufactured, the drug product is a lyophilized crystal or powder of ²amber or white ²color that comprises the following materials: oligonucleotide (e.g., P-ethoxy ²antisense drug ²substance), neutral lipids (e.g., DOPC), and surfactant (e.g., polysorbate ²20). In preparation ²for administration to a patient, normal saline is added to the vial, at which ²time liposomes are ²formed with the P-ethoxy antisense incorporated into the interior.²B. P-ethoxy antisense drug substance²100721 Specific physical properties (e.g., solubility and hydrophobicity, ²which then ²affect drug product solubility in saline, incorporation of oligo into ²liposomes, and liposome²23²<DP=24>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²particle size) of the finished product can be defined using a pre-determined P-²ethoxy and ²phosphodiester amidite raw material mix during production of the P-ethoxy ²antisense drug²substance. While loss of the P-ethoxy backbone group randomly occurs during ²²oligonucleotide manufacturing resulting in phosphodiester bonds at those ²linkages, that loss ²may not generate the preferred ratio of P-ethoxy : phosphodiester backbone ²linkage within ²the oligonucleotide. In this case, the mix of P-ethoxy and phosphodiester ²amidite raw ²material supplements the expected value of P-ethoxy backbone deletions, thus ²generating an ²oligonucleotide with the desired ratio. Increasing the number of P-ethoxy ²molecules in the ²backbone of the oligonucleotide causes the molecule to be more hydrophobic ²(which results ²in larger liposome particles; Table 1), less polar, and less soluble (Table ²2). Methods of ²testing the charge-neutral, hydrophobic P-ethoxy drug substance include mass ²spectrometry ²to determine the distribution of oligonucleotide lengths and assays to ²determine the solubility ²of drug substance, which for practical purposes for solubility is a visual ²inspection of the drug ²product reconstituted in saline. As the oligonucleotide becomes less soluble ²due to a greater ²number of P-ethoxy backbone linkages the reconstituted solution becomes whiter ²until ²particulates form as hydrophobicity becomes too high.²100731 Formulation must use a particle size, wherein the 90% value is less ²than 5000 ²nm in size and is soluble, which is a function of the nucleotide composition. ²By way of ²example, if an oligonucleotide is 18-20 nucleotides in length, then at least ²five of the ²phosphate backbone linkages should be phosphodiester backbone linkages. This ²is supported ²by the Experiments 7-10 below in Table 1, which provides data from 18mer ²oligonucleotides.. Wherein if an oligonucleotide is 25 nucleotides in length, ²then at least six ²of the phosphate backbone linkages should be phosphodiester backbone linkages.²Table 1. Liposome Particle Size Variability with Antisense Backbone ²Composition²Post-Manufacturing Particle Size ²Characteristics: ²Backbone Ethyl Deletion Cumulative Distribution Function²Experiment Engineered Principal Composite 90% 50% ²300 nm²Antisense Peakd Deletione Value Value ²Value (%)²Backbone (nm) ** (nm)²1 3 amidite -6 -5.67 2130 911 15.30²substitution²2 3 amidite -6 -5.67 2420 1004 15.50²substitution²3 3 amidite -6 -6.12 3682 943 15.50²substitution²4 3 amidite -7 -6.66 3805 978 14.60²24²<DP=25>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²substitution² 100% P- -5 -5.66 3924 976 16.00²ethoxy²6 2 amidite -5 -5.32 4387 1888 11.60²substitution²7a 100%P- -4 -4.22 5057 1131 17.70²ethoxy²8 100% P- -4 -4.52 5659 1359 10.00²ethoxy²9b 100% P- -4 -4.38 7571 1909 2.60²ethoxy²10' 100% P- -4 -4.38 7994 1653 14.40²ethoxy²** Drug product release criteria is for 90% of the liposome particles to be ²less than or equal ²to 5000 nm.²a. This lot was discarded due to poor solubility; specifically, antisense ²particles in the ²reconstituted solution.²b. This lot had lower DMSO and tBA volume with 2 mg antisense in a 20 mL vial, ²which ²added an additional component to liposome enlargement.²c. This lot was not released because it failed the particle size release spec.²d. The principal peak represents the most common number of p-ethoxy deletions ²in the ²oligonucleotides of the population.²e. The composite deletion represents the average number of p-ethoxy deletions ²in the ²population of oligonucleotides.²Table 2. Liposome Particle Solubility with Antisense Backbone Composition² Post-Manufacturing Drug Solubility²Backbone Ethyl Deletion²Experiment Engineered Principal Composite Visual Solubility²Antisense Peak ²Deletion Observation Assessment²Backbone **²1 3 amidite -6 -5.67 skim milk good²substitution solution²2 3 amidite -6 -5.67 skim milk good²substitution solution²3 3 amidite -6 -6.12 skim milk good²substitution solution²4 3 amidite -7 -6.66 skim milk good²substitution solution²5 100% P- -5 -5.66 skim milk good²ethoxy solution²6 2 amidite -5 -5.32 skim milk good²substitution solution²7 100% P- -4 -4.52 white pass²ethoxy solution²8b 100% P- -4 -4.38 white pass²ethoxy solution²9' 100% P- -4 -4.38 white pass²<DP=26>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²ethoxy solution²10a 100% P- -4 -4.22 white fail²ethoxy solution²particles²** If the drug product sample has particles the lot will be rejected²a. This lot was discarded due to poor solubility; specifically, antisense ²particles in the ²reconstituted solution.²b. This lot had lower DMSO and tBA volume with 2 mg antisense in a 20 mL vial, ²which ²added an additional component to liposome enlargement.²c. This lot was not released because it failed the particle size release spec.²C. ²Formulation, filtration, and lyophilization of liposomal P-ethoxy antisense²drug product²100741 One gram (1 g) of pE oligos is dissolved in DMSO at a ratio of 10 mg ²oligonucleotide per 1 mL DMSO. Next, DOPC is added to tert-butyl alcohol at a ²ratio of 1 g ²DOPC per 1719 mL of tert-butyl alcohol. The oligo and DOPC are combined and ²mixed at a ²ratio of 1 g oligonucleotide per 2.67 g DOPC. Then, 20 mL of a 0.835% (v / v) ²solution of ²polysorbate 20 is added to the mixture resulting in a final concentration of ²0.039 mg / mL. ²The solution is passed through a sterile filter prior to dispensing into glass ²vials for ²lyophilization.²100751 The effect of the surfactant on liposome particle size was determined ²by ²titrating the amount of surfactant (Table 3). In the absence of polysorbate ²20, only 2.8% of ²the particles had a diameter of 300 nm or less. In the presence of lx ²polysorbate 20, 12.5% ²of the particles had a diameter of 300 nm or less. With the addition of 3x-10x ²polysorbate 20, ²around 20% of the particles had a diameter of 300 nm or less. Thus an increase ²in surfactant ²from lx to 3x results in a decrease in particle size.²Table 3. Liposome Particle Size Variability with Surfactant²Particle Size Characteristics:²Cumulative Distribution Function²Experiment Amount of Surfactant 50% Value 90% Value ** 300 nm ²Value²1 Ox 5301 nm ²10719 nm 2.8%²2 lx 1053 nm ²4054 nm 12.5%²3 3x 785 nm 2926 nm ²19.1%²4 5x 721 nm 2691 nm ²21.9%² 10x 734 nm 2937 nm 21.4%²** Drug product release criteria is for 90% of the liposome particles to be ²less than or equal ²to 5000 nm.²26²<DP=27>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²D. Preparation of liposomal P-ethoxy antisense drug product for ²administration²100761 The lyophilized preparation was hydrated with normal saline (0.9% / 10 mM ²²NaCl) at a final oligo concentration of 10-5000 p.M. The liposomal-P-ethoxy ²oligos were ²mixed by hand shaking.²E. Methods of Testing Liposomal P-ethoxy Antisense Drug Product²100771 Visual Inspection of Manufactured Drug Product: After manufacturing, a ²sample vial containing drug product is selected and visually inspected. The ²absence of liquid ²is mandatory, and then amber crystals at the bottom of the vial are ²acceptable, and increasing ²in acceptance to a white, flocculated powder or appearance, the best result. ²The white ²appearance indicates a better drying process, with a high surface area to mass ²ratio, which is ²very conducive to reconstitution for use.²100781 Visual Inspection of Reconstituted Drug Ready for Patient IV: Normal ²saline ²is added to a vial containing the manufactured Liposomal P-ethoxy Antisense ²Drug Product ²and shaken to reconstitute into a solution with the drug crystal or powder ²completely ²dissolved. Three main observations are made: 1) that the crystal or powder is ²completely ²dissolved, 2) there are no white clumps of undissolved material, and 3) the ²appearance is a ²milky white or skim milk appearance. The bluer the appearance of the ²reconstituted liquid, ²the better, as this signals a smaller liposome particle size that reflects ²light in the blue ²spectrum.²100791 Mass Spectrometry: Mass spectrometry (mass spec) is used to display the ²²profile of the various masses in a sample. When P-ethoxy antisense material is ²produced, a ²mass spec is run on the sample. The result shows peaks of material present on ²a grid that has ²increasing mass on the "x" axis to the right, and relative mass abundance on ²the "y" axis ²increasing upward. The profile from a sample is analyzed to determine the ²relative quantity ²of P-ethoxy backbones in the P-ethoxy sample, recognizing that the profile of ²peaks ²represents (starting farthest to the right), full length material with all ²backbones comprised of ²the P-ethoxy linkage, the next peak moving left a full length with one ²backbone with a P-²ethoxy deletion (and therefore, the ethyl being knocked off and the result ²being a normal ²phosphodiester backbone linkage), and continuing. The mass spec pattern ²shifted to the right ²represents a P-ethoxy sample having more P-ethoxy backbones, and therefore ²having the ²properties of being more hydrophobic and less soluble; and likewise, shifted ²to the left having²27²<DP=28>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²the opposite effects. Inspection of the mass spec chart of a sample also can ²be used to ²determine if filtration during manufacturing produces any adverse effects on ²oligonucleotide ²composition present in the filtered drug product.²100801 UV Testing: Ultraviolent light testing is used to determine the mass of ²²oligonucleotide present in a sample. Oligonucleotides absorb light in the 260 ²nanometer ²range. As a result, UV testing of the finished reconstituted drug product has ²come to be used ²as a method in determining the quantity of oligonucleotide drug substance in a ²vial of drug ²product. In terms of manufacturing development and innovations, UV testing was ²used to ²determine if there were problems experienced during filtration in ²manufacturing or poor ²solubility of the P-ethoxy antisense drug substance, resulting in less ²oligonucleotide in ²solution and therefore a lower UV reading. The method will be validated and ²likely become ²part of the final product release testing.²100811 Liposome Particle Size: A vial of finished drug product is ²reconstituted and ²tested for liposome particle size. The result is often a roughly normal ²distribution, having a ²central point, tails and average values or a roughly normal distribution of ²the majority of the ²particles and smaller, secondary peaks of the smaller liposomes particles ²resulting from ²second-order particle formation effects. It is important that liposome ²particles not be too ²large, as they may create adverse effects in patients (for example, create ²blood flow problems ²in smaller blood vessels in the lungs). As a result, the drug product release ²criteria include ²that particle size testing show that 90% of liposomes be 5 microns or less in ²size. In addition, ²smaller liposomes are preferred because they will have better uptake into ²cells, and secondly, ²smaller liposomes can penetrate vascular pores, thereby allowing the liposomes ²to penetrate ²inside tumors, increasing treatment effectiveness of a Liposomal P-ethoxy ²Antisense Drug ²Product.²V. Methods of Treatment²100821 Certain aspects of the present invention provide an ²oligonucleotide¨lipid ²complex (e.g., an oligonucleotide incorporated into a non-charged liposome) ²for treating ²diseases, such as cancer, autoimmune disease, or infectious disease. ²Particularly, the ²oligonucleotide may have a sequence that allows for base pairing with a human ²nucleotide ²sequence and thus may inhibit the expression of a protein encoded by the human ²nucleotide ²sequence.²28²<DP=29>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²10083j "Treatment" and "treating" refer to administration or ²application of a ²therapeutic agent to a subject or performance of a procedure or modality on a ²subject for the ²purpose of obtaining a therapeutic benefit of a disease or health-related ²condition. For ²example, a treatment may include administration of a pharmaceutically ²effective amount of ²an oligonucleotide¨lipid complex.²10084j "Subject" and "patient" refer to either a human or non-human, ²such as ²primates, mammals, and vertebrates. In particular embodiments, the subject is ²a human.²100851 The term "therapeutic benefit" or "therapeutically effective" ²as used ²throughout this application refers to anything that promotes or enhances the ²well-being of the ²subject with respect to the medical treatment of this condition. This ²includes, but is not ²limited to, a reduction in the frequency or severity of the signs or symptoms ²of a disease. For ²example, treatment of cancer may involve, for example, a reduction in the size ²of a tumor, a ²reduction in the invasiveness of a tumor, reduction in the growth rate of the ²cancer, or ²prevention of metastasis. Treatment of cancer may also refer to prolonging ²survival of a ²subject with cancer. Treatment of an autoimmune disease may involve, for ²example, ²reducing the expression of a self-antigen against which there is an undesired ²immune ²response, inducing tolerance of a self-antigen against which there is an ²undesired immune ²response, or inhibiting the immune response towards the self-antigen. ²Treatment of an ²infectious disease may involve, for example, eliminate the infectious agent, ²reduce the level ²of the infectious agent, or maintain the level of the infectious agent at a ²certain level.²10086j Tumors for which the present treatment methods are useful include any ²malignant cell type, such as those found in a solid tumor, a hematological ²tumor, metastatic ²cancer, or non-metastatic cancer. Exemplary solid tumors can include, but are ²not limited to, ²a tumor of an organ selected from the group consisting of pancreas, colon, ²cecum, esophagus, ²gastrointestine, gum, liver, skin, stomach, testis, tongue, uterus, stomach, ²brain, head, neck, ²ovary, kidney, larynx, sarcoma, bone, lung, bladder, melanoma, prostate, and ²breast. ²Exemplary hematological tumors include tumors of the bone marrow, T or B cell ²malignancies, leukemias, lymphomas, such as, for example, diffuse large B-cell ²lymphoma, ²blastomas, myelomas, and the like. Further examples of cancers that may be ²treated using the ²methods provided herein include, but are not limited to, carcinoma, lymphoma, ²blastoma, ²sarcoma, leukemia, squamous cell cancer, lung cancer (including small-cell ²lung cancer, non-²small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of ²the lung),²29²<DP=30>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer ²(including ²gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic ²cancer, glioblastoma, ²cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, ²colon cancer, ²colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, ²kidney or ²renal cancer, prostate cancer, vulval cancer, thyroid cancer, various types of ²head and neck ²cancer, melanoma, superficial spreading melanoma, lentigo malignant melanoma, ²acral ²lentiginous melanomas, nodular melanomas, as well as B-cell lymphoma ²(including low ²grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; ²intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade ²immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved ²cell ²NHL; bulky disease NHL; diffuse large B-cell lymphoma; mantle cell lymphoma; ²AIDS-²related lymphoma; and Waldenstrom's macroglobulinemia), chronic lymphocytic ²leukemia ²(CLL), acute lymphoblastic leukemia (ALL), Hairy cell leukemia, multiple ²myeloma, acute ²myeloid leukemia (AML) and chronic my el obla sti c leukemia.²10087j The cancer may specifically be of the following histological type, ²though it is ²not limited to these: neoplasm, malignant; carcinoma; carcinoma, ²undifferentiated; giant and ²spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous ²cell carcinoma; ²lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; ²transitional cell ²carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, ²malignant; ²cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular ²carcinoma and ²cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; ²adenocarcinoma ²in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid ²carcinoma; carcinoid ²tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary ²adenocarcinoma; ²chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil ²²carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular ²adenocarcinoma; ²papillary and follicular adenocarcinoma; nonencapsulating sclerosing ²carcinoma; adrenal ²cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine ²adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; ²mucoepidermoid ²carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous ²cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; ²signet ring ²cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular ²carcinoma; ²inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; ²adenosquamous ²carcinoma; adenocarcinoma w / squamous metaplasia; thymoma, malignant; ovarian ²stromal²<DP=31>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; ²androblastoma, ²malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell ²tumor, malignant; ²paraganglioma, malignant; extra-mammary paraganglioma, malignant; ²pheochromocytoma; ²glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial ²spreading ²melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell ²melanoma; blue ²nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; ²myxosarcoma; ²liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; ²alveolar ²rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed ²tumor; ²nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; ²brenner ²tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, ²malignant; ²dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, ²malignant; ²choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; ²hemangioendothelioma, ²malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; ²²osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, ²malignant; ²mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; ²odontogenic ²tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ²ameloblastic ²fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; ²astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; ²glioblastoma; ²oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar ²sarcoma; ²ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic ²tumor; ²meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular ²cell tumor, ²malignant; malignant lymphoma; hodgkin's disease; hodgkin's; paragranuloma; ²malignant ²lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; ²malignant ²lymphoma, follicular; mycosis fungoides; other specified non-hodgkin's ²lymphomas; ²malignant histiocytosis; multiple myeloma; mast cell sarcoma; ²immunoproliferative small ²intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; ²erythroleukemia; ²lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; ²eosinophilic ²leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; ²myeloid ²sarcoma; and hairy cell leukemia.²100881 Autoimmune diseases for which the present treatment methods are useful ²include, without limitation, spondyloarthropathy, ankylosing spondylitis, ²psoriatic arthritis, ²reactive arthritis, enteropathic arthritis, diabetes mellitus, celiac disease, ²autoimmune thyroid ²disease, autoimmune liver disease, Addison's disease, transplant rejection, ²graft vs. host²31²<DP=32>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²disease, host vs. graft disease, ulcerative colitis, Crohn's disease, ²irritable bowel disease, ²inflammatory bowel disease, rheumatoid arthritis, juvenile rheumatoid ²arthritis, familial ²Mediterranean fever, amyotrophic lateral sclerosis, Sjogren's syndrome, early ²arthritis, viral ²arthritis, multiple sclerosis, or psoriasis. The diagnosis and treatment of ²these diseases are ²well documented in the literature.²10089j Infectious diseases for which the present treatment methods are useful ²include, ²without limitation, bacterial infections, viral infections, fungal infections, ²and parasitic ²infections. Exemplary viral infections include hepatitis B virus, hepatitis C ²virus, human ²immunodeficiency virus 1, human immunodeficiency virus 2, human papilloma ²virus, herpes ²simplex virus 1, herpes simplex virus 2, herpes zoster, varicella zoster, ²coxsackievirus A16, ²cytomegalovirus, ebola virus, enterovirus, Epstein-Barr virus, hanta virus, ²hendra virus, viral ²meningitis, respiratory syncytial virus, rotavirus, west nile virus, ²adenovirus, and influenza ²virus infections. Exemplary bacterial infections include Chlamydia ²trachomatis, Listeria ²monocytogenes, Helicobacter pylori, Escherichia coil, Borelia burgdorferi, ²Legionella ²pneumophilia, Mycobacteria sps (e.g., M tuberculosis, M avium, M intracellular ²e, M ²kansaii, M gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria ²meningitides, Streptococcus pyogenes (Group A Streptococcus), Streptococcus ²agalactiae ²(Group B Streptococcus), Streptococcus (viridans group), Streptococcus ²faecalis, ²Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus pneumoniae, ²pathogenic ²Campylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillus ²anthracis, ²Corynebacterium diphtheriae, corynebacterium sp., Erysipelothrix ²rhusiopathiae, ²Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, ²Klebsiella pneumoniae, ²Pasturella multocida, Bacteroides sp., Fusobacterium nucleatum, ²Streptobacillus ²moniliformis, Treponema pallid / urn, Treponema pertenue, Leptospira, ²Rickettsia, ²Actinomyces israelli, Shigella sps (e.g., S.flexneri, S. sonnei, S. ²dysenteriae), and Salmonella ²spp infections. Exemplary fungal infections include Candida albicans, Candida ²glabrata, ²Aspergillus fumigatus, Aspergillus terreus, Cryptococcus neoformans, ²Histoplasma ²capsulatum, Coccidioides immitis, Blastomyces dermatitidis, and Chlamydia ²irachomatis ²infections.²100901 The oligonucleotide¨lipid complex may be used herein as an antitumor, ²antiviral, antibacterial, antifungal, antiparasite, or anti-autoimmune agent ²in a variety of ²modalities. In a particular embodiment, the invention contemplates methods of ²using an²32²<DP=33>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²oligonucleotide¨lipid complex comprises contacting a population of diseased ²cells with a ²therapeutically effective amount of an oligonucleotide¨lipid complex for a ²time period ²sufficient to inhibit or reverse disease.²100911 In one embodiment, the contacting in vivo is accomplished by ²administering, ²by intravenous, intraperitoneal, subcutaneous, or intratumoral injection, a ²therapeutically ²effective amount of a physiologically tolerable composition comprising an ²oligonucleotide¨²lipid complex of this invention to a patient. The oligonucleotide¨lipid ²complex can be ²administered parenterally by injection or by gradual infusion over time.²100921 Therapeutic compositions comprising oligonucleotide¨lipid complex are ²conventionally administered intravenously or subcutaneously, such as by ²injection of a unit ²dose, for example. The term "unit dose" when used in reference to a ²therapeutic composition ²refers to physically discrete units suitable as unitary dosage for the ²subject, each unit ²containing a predetermined quantity of active material calculated to produce ²the desired ²therapeutic effect in association with the required diluent, i.e., carrier, or ²vehicle.²100931 The compositions are administered in a manner compatible with ²the ²dosage formulation, and in a therapeutically effective amount. The quantity to ²be ²administered depends on the subject to be treated, capacity of the subject's ²system to utilize ²the active ingredient, and degree of therapeutic effect desired. Precise ²amounts of active ²ingredient required to be administered depend on the judgment of the ²practitioner and are ²peculiar to each individual. However, suitable dosage ranges for systemic ²application are ²disclosed herein and depend on the route of administration. Suitable regimes ²for initial and ²booster administration are also contemplated and are typified by an initial ²administration ²followed by repeated doses at one or more hour intervals by a subsequent ²injection or other ²administration. Exemplary multiple administrations are described herein and ²are particularly ²preferred to maintain continuously high serum and tissue levels of ²polypeptide. ²Alternatively, continuous intravenous infusion sufficient to maintain ²concentrations in the ²blood in the ranges specified for in vivo therapies are contemplated.²100941 It is contemplated that an oligonucleotide of the invention can be ²administered ²systemically or locally to treat disease, such as to inhibit tumor cell growth ²or to kill cancer ²cells in cancer patients with locally advanced or metastatic cancers. They can ²be ²administered intravenously, intrathecally, subcutaneously, and / or ²intraperitoneally. They can²33²<DP=34>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²be administered alone or in combination with anti-proliferative drugs. In one ²embodiment, ²they are administered to reduce the cancer load in the patient prior to ²surgery or other ²procedures. Alternatively, they can be administered after surgery to ensure ²that any ²remaining cancer (e.g., cancer that the surgery failed to eliminate) does not ²survive.²100951 A therapeutically effective amount of an oligonucleotide is a ²predetermined ²amount calculated to achieve the desired effect, i.e., to inhibit the ²expression of a target ²protein. Thus, the dosage ranges for the administration of oligonucleotides of ²the invention ²are those large enough to produce the desired effect. The dosage should not be ²so large as to ²cause adverse side effects, such as hyperviscosity syndromes, pulmonary edema, ²congestive ²heart failure, neurological effects, and the like. Generally, the dosage will ²vary with age of, ²condition of, sex of, and extent of the disease in the patient and can be ²determined by one of ²skill in the art. The dosage can be adjusted by the individual physician in ²the event of any ²complication.²100961 A composition of the present invention is preferably administered to a ²patient ²parenterally, for example by intravenous, intraarterial, intramuscular, ²intralymphatic, ²intraperitoneal, subcutaneous, intrapleural, or intrathecal injection, or may ²be used ex vivo. ²Preferred dosages are between 5-25 mg / kg. The administration is preferably ²repeated on a ²timed schedule until the cancer disappears or regresses, and may be in ²conjunction with other ²forms of therapy.²VI. Pharmaceutical Preparations²100971 A pharmaceutical composition comprising the liposomes will usually ²include ²a sterile, pharmaceutically acceptable carrier or diluent, such as dextrose or ²saline solution.²100981 Where clinical application of non-charged lipid component (e.g., in the ²form ²of a liposome) containing an oligonucleotide is undertaken, it will generally ²be beneficial to ²prepare the lipid complex as a pharmaceutical composition appropriate for the ²intended ²application. This will typically entail preparing a pharmaceutical composition ²that is ²essentially free of pyrogens, as well as any other impurities that could be ²harmful to humans ²or animals. One may also employ appropriate buffers to render the complex ²stable and allow ²for uptake by target cells.²34²<DP=35>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²10099j The phrases "pharmaceutical or pharmacologically acceptable" refers to ²molecular entities and compositions that do not produce an adverse, allergic ²or other ²untoward reaction when administered to an animal, such as a human, as ²appropriate. The ²preparation of a pharmaceutical composition that contains at least one non-²charged lipid ²component comprising an oligonucleotide or additional active ingredient will ²be known to ²those of skill in the art in light of the present disclosure, as exemplified ²by Remington: The ²Science and Practice of Pharmacy, 21st, 2005, incorporated herein by ²reference. Moreover, ²for animal (e.g., human) administration, it will be understood that ²preparations should meet ²sterility, pyrogenicity, general safety and purity standards as required by ²FDA Office of ²Biological Standards.²1001001 As used herein, "pharmaceutically acceptable carrier" ²includes any and ²all solvents, dispersion media, coatings, surfactants, antioxidants, ²preservatives (e.g., ²antibacterial agents, antifungal agents), isotonic agents, absorption delaying ²agents, salts, ²preservatives, drugs, drug stabilizers, gels, binders, excipients, ²disintegration agents, ²lubricants, sweetening agents, flavoring agents, dyes, such like materials and ²combinations ²thereof, as would be known to one of ordinary skill in the art. A ²pharmaceutically acceptable ²carrier is preferably formulated for administration to a human, although in ²certain ²embodiments it may be desirable to use a pharmaceutically acceptable carrier ²that is ²formulated for administration to a non-human animal but which would not be ²acceptable ²(e.g., due to governmental regulations) for administration to a human. Except ²insofar as any ²conventional carrier is incompatible with the active ingredient, its use in ²the therapeutic or ²pharmaceutical compositions is contemplated.²1001011 The actual dosage amount of a composition of the present ²invention ²administered to a patient or subject can be determined by physical and ²physiological factors ²such as body weight, severity of condition, the type of disease being treated, ²previous or ²concurrent therapeutic interventions, idiopathy of the patient and on the ²route of ²administration. The practitioner responsible for administration will, in any ²event, determine ²the concentration of active ingredient(s) in a composition and appropriate ²dose(s) for the ²individual subject.²1001021 In certain embodiments, pharmaceutical compositions may ²comprise, ²for example, at least about 0.1% of an active compound. In other embodiments, ²the an active ²compound may comprise between about 2% to about 75% of the weight of the unit, ²or²<DP=36>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²between about 25% to about 60%, for example, and any range derivable therein. ²In other non-²limiting examples, a dose may also comprise from about 1 microgram / kg / body ²weight, about ² microgram / kg / body weight, about 10 microgram / kg / body weight, about 50 ²microgram / kg / body weight, about 100 microgram / kg / body weight, about 200 ²microgram / kg / body weight, about 350 microgram / kg / body weight, about 500 ²microgram / kg / body weight, about 1 milligram / kg / body weight, about 5 ²milligram / kg / body ²weight, about 10 milligram / kg / body weight, about 50 milligram / kg / body weight, ²about 100 ²milligram / kg / body weight, about 200 milligram / kg / body weight, about 350 ²milligram / kg / body weight, about 500 milligram / kg / body weight, to about 1000 ²mg / kg / body ²weight or more per administration, and any range derivable therein. In non-²limiting examples ²of a derivable range from the numbers listed herein, a range of about 5 ²[tg / kg / body weight to ²about 1000 mg / kg / body weight, about 5 microgram / kg / body weight to about 500 ²milligram / kg / body weight, etc., can be administered.²1001031 An oligonucleotide of the present embodiments may be ²administered ²in a dose of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, ²80, 90, 100 or more jig ²of nucleic acid per dose. Each dose may be in a volume of 1, 10, 50, 100, 200, ²500, 1000 or ²more pi or ml.²1001041 Solutions of therapeutic compositions can be prepared in ²water ²suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions ²also can be ²prepared in glycerol, liquid polyethylene glycols, mixtures thereof and in ²oils. Under ordinary ²conditions of storage and use, these preparations contain a preservative to ²prevent the growth ²of microorganisms.²

[00105] The therapeutic compositions of the present invention are ²advantageously administered in the form of injectable compositions either as ²liquid solutions ²or suspensions; solid forms suitable for solution in, or suspension in, liquid ²prior to injection ²may also be prepared. These preparations also may be emulsified. A typical ²composition for ²such purpose comprises a pharmaceutically acceptable carrier. For instance, ²the composition ²may contain 10 mg, 25 mg, 50 mg or up to about 100 mg of human serum albumin ²per ²milliliter of phosphate buffered saline. Other pharmaceutically acceptable ²carriers include ²aqueous solutions, non-toxic excipients, including salts, preservatives, ²buffers and the like.²36²<DP=37>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²1001061 Examples of non-aqueous solvents are propylene glycol, ²polyethylene ²glycol, vegetable oil and injectable organic esters such as ethyloleate. ²Aqueous carriers ²include water, alcoholic / aqueous solutions, saline solutions, parenteral ²vehicles such as ²sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid ²and nutrient ²replenishers. Preservatives include antimicrobial agents, anti-oxidants, ²chelating agents and ²inert gases. The pH and exact concentration of the various components the ²pharmaceutical ²composition are adjusted according to well known parameters.²1901071 The therapeutic compositions of the present invention may ²include ²classic pharmaceutical preparations. Administration of therapeutic ²compositions according to ²the present invention will be via any common route so long as the target ²tissue is available ²via that route. This includes oral, nasal, buccal, rectal, vaginal or topical. ²Topical ²administration may be particularly advantageous for the treatment of skin ²cancers, to prevent ²chemotherapy-induced alopecia or other dermal hyperproliferative disorder. ²Alternatively, ²administration may be by orthotopic, intradermal, subcutaneous, intramuscular, ²²intraperitoneal or intravenous injection. Such compositions would normally be ²administered ²as pharmaceutically acceptable compositions that include physiologically ²acceptable carriers, ²buffers or other excipients. For treatment of conditions of the lungs, aerosol ²delivery can be ²used. Volume of the aerosol is between about 0.01 ml and 0.5 ml.²1001081 An effective amount of the therapeutic composition is ²determined ²based on the intended goal. The term "unit dose" or "dosage" refers to ²physically discrete ²units suitable for use in a subject, each unit containing a predetermined-²quantity of the ²therapeutic composition calculated to produce the desired responses discussed ²above in ²association with its administration, i.e., the appropriate route and treatment ²regimen. The ²quantity to be administered, both according to number of treatments and unit ²dose, depends ²on the protection or effect desired.²1001991 Precise amounts of the therapeutic composition also depend ²on the ²judgment of the practitioner and are peculiar to each individual. Factors ²affecting the dose ²include the physical and clinical state of the patient, the route of ²administration, the intended ²goal of treatment (e.g., alleviation of symptoms versus cure) and the potency, ²stability and ²toxicity of the particular therapeutic substance.²37²<DP=38>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²VII. Combination Treatments²1001191 In certain embodiments, the compositions and methods of the ²present ²invention involve an inhibitory oligonucleotide, or oligonucleotide capable of ²expressing an ²inhibitor of gene expression, in combination with a second or additional ²therapy. The ²methods and compositions including combination therapies enhance the ²therapeutic or ²protective effect, and / or increase the therapeutic effect of another anti-²cancer or anti-²hyperproliferative therapy. Therapeutic and prophylactic methods and ²compositions can be ²provided in a combined amount effective to achieve the desired effect, such as ²the killing of a ²cancer cell and / or the inhibition of cellular hyperproliferation. This process ²may involve ²contacting the cells with both an inhibitor of gene expression and a second ²therapy. A tissue, ²tumor, or cell can be contacted with one or more compositions or ²pharmacological ²formulation(s) including one or more of the agents (i.e., inhibitor of gene ²expression or an ²anti-cancer agent), or by contacting the tissue, tumor, and / or cell with two ²or more distinct ²compositions or formulations, wherein one composition provides 1) an ²inhibitory ²oligonucleotide; 2) an anti-cancer agent, or 3) both an inhibitory ²oligonucleotide and an anti-²cancer agent. Also, it is contemplated that such a combination therapy can be ²used in ²conjunction with a chemotherapy, radiotherapy, surgical therapy, or ²immunotherapy.²1901111 An inhibitory oligonucleotide may be administered before, ²during, ²after or in various combinations relative to an anti-cancer treatment. The ²administrations may ²be in intervals ranging from concurrently to minutes to days to weeks. In ²embodiments where ²the inhibitory oligonucleotide is provided to a patient separately from an ²anti-cancer agent, ²one would generally ensure that a significant period of time did not expire ²between the time ²of each delivery, such that the two compounds would still be able to exert an ²advantageously ²combined effect on the patient. In such instances, it is contemplated that one ²may provide a ²patient with the inhibitory oligonucleotide therapy and the anti-cancer ²therapy within about ²12 to 24 or 72 h of each other and, more preferably, within about 6-12 h of ²each other. In ²some situations it may be desirable to extend the time period for treatment ²significantly ²where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 ²or 8) lapse between ²respective administrations.²1001121 In certain embodiments, a course of treatment will last 1, ²2, 3, 4, 5, 6,²7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, ²27, 28, 29, 30, 31, 32,²33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, ²52, 53, 54, 55, 56, 57,²38²<DP=39>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, ²77, 78, 79, 80, 81, 82, ²83, 84, 85, 86, 87, 88, 89, 90 days or more. It is contemplated that one agent ²may be given on ²day 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, ²22, 23, 24, 25, 26, 27, ²28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, ²47, 48, 49, 50, 51, 52, ²53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, ²72, 73, 74, 75, 76, 77, ²78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, and / or 90, any combination ²thereof, and another ²agent is given on day 1,2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, ²18, 19, 20, 21, 22, ²23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, ²42, 43, 44, 45, 46, 47, ²48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, ²67, 68, 69, 70, 71, 72, ²73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, and / or 90, ²or any combination ²thereof Within a single day (24-hour period), the patient may be given one or ²multiple ²administrations of the agent(s). Moreover, after a course of treatment, it is ²contemplated that ²there is a period of time at which no anti-cancer treatment is administered. ²This time period ²may last 1, 2, 3, 4, 5, 6, 7 days, and / or 1, 2, 3, 4, 5 weeks, and / or 1, 2, 3, ²4, 5, 6, 7, 8, 9, 10, ²11, 12 months or more, depending on the condition of the patient, such as ²their prognosis, ²strength, health, etc.²1901131 Various combinations may be employed. For the example below ²an ²inhibitory oligonucleotide therapy is "A" and an anti-cancer therapy is "B":²A / B / A B / A / B B / B / A A / A / B A / B / B B / A / A A / BBB ²B / A / B / B BBB / A B / B / A / B A / A / B / B A / B / A / B A / B / B / A ²B / B / A / A B / A / B / A B / A / A / B A / A / A / B B / A / A / A A / B / A / A ²A / A / B / A²1001141 Administration of any compound or therapy of the present ²invention to ²a patient will follow general protocols for the administration of such ²compounds, taking into ²account the toxicity, if any, of the agents. Therefore, in some embodiments ²there is a step of ²monitoring toxicity that is attributable to combination therapy. It is ²expected that the ²treatment cycles would be repeated as necessary. It also is contemplated that ²various standard ²therapies, as well as surgical intervention, may be applied in combination ²with the described ²therapy.²1001151 In specific aspects, it is contemplated that a standard ²therapy will ²include chemotherapy, radiotherapy, immunotherapy, surgical therapy or gene ²therapy and²39²<DP=40>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²may be employed in combination with the inhibitor of gene expression therapy, ²anticancer ²therapy, or both the inhibitor of gene expression therapy and the anti-cancer ²therapy, as ²described herein.²A. Chemotherapy²1001161 A wide variety of chemotherapeutic agents may be used in ²accordance ²with the present embodiments. The term "chemotherapy" refers to the use of ²drugs to treat ²cancer. A "chemotherapeutic agent" is used to connote a compound or ²composition that is ²administered in the treatment of cancer. These agents or drugs are categorized ²by their mode ²of activity within a cell, for example, whether and at what stage they affect ²the cell cycle. ²Alternatively, an agent may be characterized based on its ability to directly ²cross-link DNA, ²to intercalate into DNA, or to induce chromosomal and mitotic aberrations by ²affecting ²nucleic acid synthesis.²1001171 Examples of chemotherapeutic agents include alkylating ²agents, such ²as thiotepa and cyclosphosphamide; alkyl sulfonates, such as busulfan, ²improsulfan, and ²piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and ²uredopa; ²ethylenimines and methylamelamines, including altretamine, ²triethylenemelamine, ²trietylenephosphoramide, triethiylenethiophosphoramide, and ²trimethylolomelamine; ²acetogenins (especially bullatacin and bullatacinone); a camptothecin ²(including the synthetic ²analogue topotecan); bryostatin; callystatin; CC-1065 (including its ²adozelesin, carzelesin ²and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 ²and ²cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, ²KW-2189 and ²CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen ²mustards, such ²as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, ²mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, ²phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosureas, ²such as ²carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ²ranimnustine; antibiotics, ²such as the enediyne antibiotics (e.g., calicheamicin, especially ²calicheamicin gammalI and ²calicheamicin omegaIl); dynemicin, including dynemicin A; bisphosphonates, ²such as ²clodronate; an esperamicin; as well as neocarzinostatin chromophore and ²related ²chromoprotein enediyne anti obi oti c chromophores, acl acinomy sins, ²actinomycin, ²authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, ²carzinophilin, ²chromomycini s, dactinomycin, daunorubicin, detorubicin, 6-di azo-5-oxo-L-norl ²eucine,²<DP=41>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-²pyrrolino-²doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, ²marcellomycin, ²mitomycins, such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycins, ²²peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, ²streptozocin, ²tubercidin, ubenimex, zinostatin, and zorubicin; anti-metabolites, such as ²methotrexate and 5-²fluorouracil (5-FU); folic acid analogues, such as denopterin, pteropterin, ²and trimetrexate; ²purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, and ²thioguanine; ²pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, ²cytarabine, ²dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens, such ²as calusterone, ²dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti-²adrenals, such ²as mitotane and trilostane; folic acid replenisher, such as frolinic acid; ²aceglatone; ²aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; ²bestrabucil; ²bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; ²elliptinium ²acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; ²lonidainine; ²maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; ²²mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; ²podophyllinic ²acid; 2-ethylhydrazide; procarbazine; P SKpolysacchari de complex; razoxane; ²rhizoxin; ²si zofi ran; spirogermanium; tenuazonic acid; triaziquone; 2,2,2" -tri chl ²orotri ethyl amine; ²trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); ²urethan; ²vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; ²gacytosine; ²arabinoside ("Ara-C"); cyclophosphamide; taxoids, e.g., paclitaxel and ²docetaxel ²gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, ²such as ²cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-²16); ifosfamide; ²mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; ²daunomycin; ²aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase ²inhibitor RFS ²2000; difluorometlhylornithine (DMF0); retinoids, such as retinoic acid; ²capecitabine; ²carboplatin, procarbazine,plicomycin, gemcitabien, navelbine, farnesyl-protein ²tansferase ²inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or ²derivatives of any ²of the above.²B. Radiotherapy²1001181 Other factors that cause DNA damage and have been used ²extensively ²include what are commonly known as y-rays, X-rays, and / or the directed ²delivery of ²radioisotopes to tumor cells. Other forms of DNA damaging factors are also ²contemplated²41²<DP=42>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²such as microwaves, proton beam irradiation (U.S. Pat. Nos. 5,760,395 and ²4,870,287) and ²UV-irradiation. It is most likely that all of these factors affect a broad ²range of damage on ²DNA, on the precursors of DNA, on the replication and repair of DNA, and on ²the assembly ²and maintenance of chromosomes. Dosage ranges for X-rays range from daily ²doses of 50 to ²200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of ²2000 to 6000 ²roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-²life of the ²isotope, the strength and type of radiation emitted, and the uptake by the ²neoplastic cells.²1901191 The terms "contacted" and "exposed," when applied to a cell, ²are used ²herein to describe the process by which a therapeutic construct and a ²chemotherapeutic or ²radiotherapeutic agent are delivered to a target cell or are placed in direct ²juxtaposition with ²the target cell. To achieve cell killing, for example, both agents are ²delivered to a cell in a ²combined amount effective to kill the cell or prevent it from dividing.²C. Immunotherapy²1001201 In the context of cancer treatment, immunotherapeutics, ²generally, rely ²on the use of immune effector cells and molecules to target and destroy cancer ²cells. ²Trastuzumab (HerceptinTM) is such an example. The immune effector may be, for ²example, ²an antibody specific for some marker on the surface of a tumor cell. The ²antibody alone may ²serve as an effector of therapy or it may recruit other cells to actually ²affect cell killing. The ²antibody also may be conjugated to a drug or toxin (chemotherapeutic, ²radionuclide, ricin A ²chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting ²agent. Alternatively, ²the effector may be a lymphocyte carrying a surface molecule that interacts, ²either directly or ²indirectly, with a tumor cell target. Various effector cells include cytotoxic ²T cells and NK ²cells. The combination of therapeutic modalities, i.e., direct cytotoxic ²activity and inhibition ²or reduction of ErbB2 would provide therapeutic benefit in the treatment of ²ErbB2 ²overexpressing cancers.²1001211 Another immunotherapy could also be used as part of a ²combined ²therapy with gen silencing therapy discussed above. In one aspect of ²immunotherapy, the ²tumor cell must bear some marker that is amenable to targeting, i.e., is not ²present on the ²majority of other cells. Many tumor markers exist and any of these may be ²suitable for ²targeting in the context of the present invention. Common tumor markers ²include ²carcinoembryonic antigen, prostate specific antigen, urinary tumor associated ²antigen, fetal ²antigen, tyrosinase (p9'7), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, ²MucB,²42²<DP=43>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²PLAP, estrogen receptor, laminin receptor, erb B and p155. An alternative ²aspect of ²immunotherapy is to combine anticancer effects with immune stimulatory ²effects. Immune ²stimulating molecules also exist including: cytokines such as IL-2, IL-4, IL-²12, GM-CSF, ²gamma-IFN, chemokines such as MIP-1, MCP-1, IL-8 and growth factors such as ²FLT3 ²ligand. Combining immune stimulating molecules, either as proteins or using ²gene delivery in ²combination with a tumor suppressor has been shown to enhance anti-tumor ²effects. ²Moreover, antibodies against any of these compounds can be used to target the ²anti-cancer ²agents discussed herein.²1001221 Examples of immunotherapies currently under investigation or ²in use ²are immune adjuvants e.g., Mycobacterium bovis, Plasmodium falciparum, ²dinitrochlorobenzene and aromatic compounds (U.S. Pat. Nos. 5,801,005 and ²5,739,169; Hui ²and Hashimoto, 1998; Christodoulides et al., 1998), cytokine therapy, e.g., ²interferons a, f3 ²and y; IL-1, GM-CSF and TNF (Bukowski et al., 1998; Davidson et al., 1998; ²Hellstrand et ²al., 1998) gene therapy, e.g., TNF, IL-1, IL-2, p53 (Qin et al., 1998; Austin-²Ward and ²Villaseca, 1998; U.S. Pat. Nos. 5,830,880 and 5,846,945) and monoclonal ²antibodies, e.g., ²anti-ganglioside GM2, anti-HER-2, anti-p185 (Pietras et al., 1998; Hanibuchi ²et al., 1998; ²U.S. Pat. No. 5,824,311). It is contemplated that one or more anti-cancer ²therapies may be ²employed with the gene silencing therapies described herein.²1001231 In active immunotherapy, an antigenic peptide, polypeptide ²or protein, ²or an autologous or allogenic tumor cell composition or "vaccine" is ²administered, generally ²with a distinct bacterial adjuvant (Ravindranath and Morton, 1991; Morton et ²al., 1992; ²Mitchell et al., 1990; Mitchell et al., 1993).²

[00124] In adoptive immunotherapy, the patient's circulating ²lymphocytes, or ²tumor infiltrated lymphocytes, are isolated in vitro, activated by lymphokines ²such as IL-2 or ²transduced with genes for tumor necrosis, and readministered (Rosenberg et ²al., 1988; 1989).²1001251 In some embodiments, the immunotherapy may be an immune ²checkpoint inhibitor. Immune checkpoints either turn up a signal (e.g., co-²stimulatory ²molecules) or turn down a signal. Inhibitory immune checkpoints that may be ²targeted by ²immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also ²known ²as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T-lymphocyte-²associated ²protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), ²killer-cell²43²<DP=44>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²immunoglobulin (KIR), lymphocyte activation gene-3 (LAG3), programmed death 1 ²(PD-1), ²T-cell immunoglobulin domain and mucin domain 3 (TIM-3) and V-domain Ig ²suppressor of ²T cell activation (VISTA). In particular, the immune checkpoint inhibitors ²target the PD-1 ²axis and / or CTLA-4.²1001261 The immune checkpoint inhibitors may be drugs such as small ²molecules, recombinant forms of ligand or receptors, or, in particular, are ²antibodies, such as ²human antibodies (e.g., International Patent Publication W02015016718; ²Pardo11, Nat Rev ²Cancer, 12(4): 252-64, 2012; both incorporated herein by reference). Known ²inhibitors of the ²immune checkpoint proteins or analogs thereof may be used, in particular ²chimerized, ²humanized or human forms of antibodies may be used. As the skilled person will ²know, ²alternative and / or equivalent names may be in use for certain antibodies ²mentioned in the ²present disclosure. Such alternative and / or equivalent names are ²interchangeable in the ²context of the present disclosure. For example, it is known that lambrolizumab ²is also known ²under the alternative and equivalent names MK-3475 and pembrolizumab.²1001271 In some embodiments, the PD-1 binding antagonist is a ²molecule that ²inhibits the binding of PD-1 to its ligand binding partners. In a specific ²aspect, the PD-1 ²ligand binding partners are PDL1 and / or PDL2. In another embodiment, a PDL1 ²binding ²antagonist is a molecule that inhibits the binding of PDL1 to its binding ²partners. In a specific ²aspect, PDL1 binding partners are PD-1 and / or B7-1. In another embodiment, the ²PDL2 ²binding antagonist is a molecule that inhibits the binding of PDL2 to its ²binding partners. In a ²specific aspect, a PDL2 binding partner is PD-1. The antagonist may be an ²antibody, an ²antigen binding fragment thereof, an immunoadhesin, a fusion protein, or ²oligopeptide. ²Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, ²and 8,008,449, ²all incorporated herein by reference. Other PD-1 axis antagonists for use in ²the methods ²provided herein are known in the art such as described in U.S. Patent ²Publication Nos. ²20140294898, 2014022021, and 20110008369, all incorporated herein by ²reference.²1001281 In some embodiments, the PD-1 binding antagonist is an anti-²PD-1 ²antibody (e.g., a human antibody, a humanized antibody, or a chimeric ²antibody). In some ²embodiments, the anti-PD-1 antibody is selected from the group consisting of ²nivolumab, ²pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is ²an ²immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 ²binding portion ²of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an ²immunoglobulin²44²<DP=45>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²sequence). In some embodiments, the PD-1 binding antagonist is AMP- 224. ²Nivolumab, also ²known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO , is an anti-²PD-1 antibody described in W02006 / 121168. Pembrolizumab, also known as MK-²3475, ²Merck 3475, lambrolizumab, KEYTRUIDA , and SCH-900475, is an anti-PD-1 ²antibody ²described in W02009 / 114335. CT-011, also known as hBAT or hBAT-1, is an anti-²PD-1 ²antibody described in W02009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-²Fc ²fusion soluble receptor described in W02010 / 027827 and W02011 / 066342.²1901291 Another immune checkpoint that can be targeted in the ²methods ²provided herein is the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), ²also known as ²CD152. The complete cDNA sequence of human CTLA-4 has the Genbank accession ²number L15006. CTLA-4 is found on the surface of T cells and acts as an "off' ²switch when ²bound to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a ²member of ²the immunoglobulin superfamily that is expressed on the surface of Helper T ²cells and ²transmits an inhibitory signal to T cells. CTLA4 is similar to the T-cell co-²stimulatory ²protein, CD28, and both molecules bind to CD80 and CD86, also called B7-1 and ²B7-2 ²respectively, on antigen-presenting cells. CTLA4 transmits an inhibitory ²signal to T cells, ²whereas CD28 transmits a stimulatory signal. Intracellular CTLA4 is also found ²in regulatory ²T cells and may be important to their function. T cell activation through the ²T cell receptor ²and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for ²B7 molecules.²1001301 In some embodiments, the immune checkpoint inhibitor is an ²anti-²CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric ²antibody), an ²antigen binding fragment thereof, an immunoadhesin, a fusion protein, or ²oligopeptide.²1001311 Anti-human-CTLA-4 antibodies (or VH and / or VL domains ²derived ²therefrom) suitable for use in the present methods can be generated using ²methods well ²known in the art. Alternatively, art recognized anti-CTLA-4 antibodies can be ²used. For ²example, the anti-CTLA-4 antibodies disclosed in: US Patent No. 8,119,129, WO ²01 / 14424, ²WO 98 / 42752; WO 00 / 37504 (CP675,206, also known as tremelimumab; formerly ²ticilimumab), U.S. Patent No. 6,207,156; Hurwitz et at. (1998) Proc Natl Acad ²Sci USA ²95(17): 10067-10071; Camacho et at. (2004) J Clin Oncology 22(145): Abstract ²No. 2505 ²(antibody CP-675206); and Mokyr et at. (1998) Cancer Res 58:5301-5304 can be ²used in the ²methods disclosed herein. The teachings of each of the aforementioned ²publications are ²hereby incorporated by reference. Antibodies that compete with any of these ²art-recognized²<DP=46>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²antibodies for binding to CTLA-4 also can be used. For example, a humanized ²CTLA-4 ²antibody is described in International Patent Application No. W02001014424, ²W02000037504, and U.S. Patent No. 8,017,114; all incorporated herein by ²reference.²1001321 An exemplary anti-CTLA-4 antibody is ipilimumab (also known ²as ²10D1, MDX- 010, MDX- 101, and Yervoyg) or antigen binding fragments and ²variants ²thereof (see, e.g., WO 01 / 14424). In other embodiments, the antibody comprises ²the heavy ²and light chain CDRs or VRs of ipilimumab. Accordingly, in one embodiment, the ²antibody ²comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab, and ²the ²CDR1, CDR2 and CDR3 domains of the VL region of ipilimumab. In another ²embodiment, ²the antibody competes for binding with and / or binds to the same epitope on ²CTLA-4 as the ²above-mentioned antibodies. In another embodiment, the antibody has at least ²about 90% ²variable region amino acid sequence identity with the above-mentioned ²antibodies (e.g., at ²least about 90%, 95%, or 99% variable region identity with ipilimumab).²1001331 Other molecules for modulating CTLA-4 include CTLA-4 ligands ²and ²receptors such as described in U.S. Patent Nos. 5844905, 5885796 and ²International Patent ²Application Nos. W01995001994 and W01998042752; all incorporated herein by ²reference, ²and immunoadhesins such as described in U.S. Patent No. 8329867, incorporated ²herein by ²reference.²1001341 In some embodiment, the immune therapy could be adoptive ²immunotherapy, which involves the transfer of autologous antigen-specific T ²cells generated ²ex vivo. The T cells used for adoptive immunotherapy can be generated either ²by expansion ²of antigen-specific T cells or redirection of T cells through genetic ²engineering (Park, ²Rosenberg et al. 2011). Isolation and transfer of tumor specific T cells has ²been shown to be ²successful in treating melanoma. Novel specificities in T cells have been ²successfully ²generated through the genetic transfer of transgenic T cell receptors or ²chimeric antigen ²receptors (CARs) (Jena, Dotti et al. 2010). CARs are synthetic receptors ²consisting of a ²targeting moiety that is associated with one or more signaling domains in a ²single fusion ²molecule. In general, the binding moiety of a CAR consists of an antigen-²binding domain of ²a single-chain antibody (scFv), comprising the light and variable fragments of ²a monoclonal ²antibody joined by a flexible linker. Binding moieties based on receptor or ²ligand domains ²have also been used successfully. The signaling domains for first generation ²CARs are ²derived from the cytoplasmic region of the CD3zeta or the Fc receptor gamma ²chains. CARs²46²<DP=47>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²have successfully allowed T cells to be redirected against antigens expressed ²at the surface of ²tumor cells from various malignancies including lymphomas and solid tumors ²(Jena, Dotti et ²al. 2010).²I001351 In one embodiment, the present application provides for a ²combination ²therapy for the treatment of cancer wherein the combination therapy comprises ²adoptive T-²cell therapy and a checkpoint inhibitor. In one aspect, the adoptive T-cell ²therapy comprises ²autologous and / or allogenic T cells. In another aspect, the autologous and / or ²allogenic T cells ²are targeted against tumor antigens.²D. Surgery²1001361 Approximately 60% of persons with cancer will undergo ²surgery of ²some type, which includes preventative, diagnostic or staging, curative, and ²palliative ²surgery. Curative surgery is a cancer treatment that may be used in ²conjunction with other ²therapies, such as the treatment of the present invention, chemotherapy, ²radiotherapy, ²hormonal therapy, gene therapy, immunotherapy and / or alternative therapies.²1004371 Curative surgery includes resection in which all or part of ²cancerous ²tissue is physically removed, excised, and / or destroyed. Tumor resection ²refers to physical ²removal of at least part of a tumor. In addition to tumor resection, treatment ²by surgery ²includes laser surgery, cryosurgery, electrosurgery, and microscopically ²controlled surgery ²(Mohs' surgery). It is further contemplated that the present invention may be ²used in ²conjunction with removal of superficial cancers, precancers, or incidental ²amounts of normal ²tissue.²1001381 Upon excision of part or all of cancerous cells, tissue, or ²tumor, a ²cavity may be formed in the body. Treatment may be accomplished by perfusion, ²direct ²injection or local application of the area with an additional anti-cancer ²therapy. Such ²treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or ²every 1, 2, 3, 4, ²and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These ²treatments may be of ²varying dosages as well.²E. Other Agents²1004391 It is contemplated that other agents may be used in ²combination with ²certain aspects of the present embodiments to improve the therapeutic efficacy ²of treatment.²47²<DP=48>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²These additional agents include agents that affect the upregulation of cell ²surface receptors ²and GAP junctions, cytostatic and differentiation agents, inhibitors of cell ²adhesion, agents ²that increase the sensitivity of the hyperproliferative cells to apoptotic ²inducers, or other ²biological agents. Increases in intercellular signaling by elevating the ²number of GAP ²junctions would increase the anti-hyperproliferative effects on the ²neighboring ²hyperproliferative cell population. In other embodiments, cytostatic or ²differentiation agents ²can be used in combination with certain aspects of the present embodiments to ²improve the²anti-hyperproliferative efficacy of the treatments. ²Inhibitors of cell adhesion are²contemplated to improve the efficacy of the present embodiments. Examples of ²cell adhesion ²inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is ²further ²contemplated that other agents that increase the sensitivity of a ²hyperproliferative cell to ²apoptosis, such as the antibody c225, could be used in combination with ²certain aspects of the ²present embodiments to improve the treatment efficacy.²VIII. Kits and Diagnostics²1001401 In ²various aspects of the invention, a kit is envisioned containing²therapeutic agents and / or other therapeutic and delivery agents. In some ²embodiments, the ²present invention contemplates a kit for preparing and / or administering a ²therapy of the ²invention. The kit may comprise reagents capable of use in administering an ²active or ²effective agent(s) of the invention. Reagents of the kit may include at least ²one inhibitor of ²gene expression (e.g., a BCL2 oligonucleotide), one or more lipid component, ²one or more ²anti-cancer component of a combination therapy, as well as reagents to ²prepare, formulate, ²and / or administer the components of the invention or perform one or more steps ²of the ²inventive methods.²1001411 In ²some embodiments, the kit may also comprise a suitable container²means, which is a container that will not react with components of the kit, ²such as an ²eppendorf tube, an assay plate, a syringe, a bottle, or a tube. The container ²may be made from ²sterilizable materials such as plastic or glass.²1001421 The ²kit may further include an instruction sheet that outlines the²procedural steps of the methods, and will follow substantially the same ²procedures as ²described herein or are known to those of ordinary skill.²48²<DP=49>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²IX. Examples²1001431 The following examples are included to demonstrate preferred ²²embodiments of the invention. It should be appreciated by those of skill in ²the art that the ²techniques disclosed in the examples which follow represent techniques ²discovered by the ²inventor to function well in the practice of the invention, and thus can be ²considered to ²constitute preferred modes for its practice. However, those of skill in the ²art should, in light ²of the present disclosure, appreciate that many changes can be made in the ²specific ²embodiments which are disclosed and still obtain a like or similar result ²without departing ²from the spirit and scope of the invention.²Example 1 ¨ BCL2-targeted P-ethoxy oligonucleotides²1001441 Oligonucleotides targeting BCL2 were designed for use in a ²liposomal ²BCL2 antisense drug product to inhibit the expression of Bc12. The contiguous ²cDNA ²sequence of BCL2 variant alpha is provided in SEQ ID NO: 4 and the protein ²sequence of ²BCL2 variant alpha is provided in SEQ ID NO: 5. The contiguous cDNA sequence ²of BCL2 ²variant beta is provided in SEQ ID NO: 6 and the protein sequence of BCL2 ²variant beta is ²provided in SEQ ID NO: 7. The sequence of each of the oligonucleotides is ²provided in ²Table 4.²Table 4. BCL2 antisense sequences²Antisense name Sequence SEQ ID NO:²20 base' 5'- CAG CGT GCG CCA TCC TTC CC -3' 1²18 base' 5'- GCG TGC GCC ATC CTT CCC -3' 2²7 base 5'- TCC TTC C -3' 3²a Underlining indicates the location of a known phosphodiester backbone ²linkage²1001451 The liposomal BCL2 antisense drug product was manufactured ²according to the methods described herein. Mass spectrometry testing for the ²20 base ²oligonucleotide showed that over 80% of the oligonucleotide drug substance had ²between ²five and seven phosphodiester backbone linkages and that over 98% of the ²oligonucleotide ²drug substance had between five and eight phosphodiester backbone linkages. ²Particle ²testing for the 20 base oligonucleotide showed that 90% of the liposomes had a ²particle²49²<DP=50>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²diameter of 2462 nm or less, 50% of the liposomes had a particle diameter of ²607 nm or less, ²and about 28% of the liposomes had a particle diameter of 300 nm or less.²Example 2 ¨ Inhibition of normal and cancer cell viability by liposomal BCL2 ²antisense²100146] The ability of liposomal BCL2 antisense to inhibit the ²viability of ²normal peripheral blood mononuclear cells (PBMCs) was tested. Liposomal BCL2 ²antisense ²corresponding to one of SEQ ID NO: 1 with 3x detergent, SEQ ID NO: 2 with lx ²detergent, ²SEQ ID NO: 2 with 3x detergent, and SEQ ID NO: 3 with lx detergent was ²incubated with ²the cells for four days. As the data in FIG. 1 show, incubation with liposomal ²BCL2 ²antisense did not reduce the cell viability of normal PBMCs.²100147] The ability of liposomal BCL2 antisense to inhibit the ²viability of ²germinal center B-cell-like subtype diffuse large B cell lymphoma was tested ²in ten cell lines: ²DOHH-2, SU-DHL-4, SU-DHL-6, SU-DHL-10, OCI-LY-18, OCI-LY-19, WSU-DLCL2, ²RL, OCI-LY-1, and OCI-LY-7. Liposomal BCL2 antisense corresponding to one of ²SEQ ID ²NO: 1 with 3x detergent, SEQ ID NO: 2 with lx detergent, SEQ ID NO: 2 with 3x ²detergent, ²and SEQ ID NO: 3 with lx detergent was incubated with each cell line for four ²days. ²Cytotoxicity was assessed using a sulforhodamine B cytotoxicity assay. As the ²data in FIGs. ²2A-J show, incubation with liposomal BCL2 antisense induced 50% inhibition in ²six of ten ²germinal center B-cell-like subtype diffuse large B cell lymphoma cell lines.²1001481 The ability of liposomal BCL2 antisense to inhibit the ²viability of ²activated B-cell-like subtype diffuse large B cell lymphoma was tested in ²three cell lines: ²SU-DHL-2, U-2932, and RI-1. Liposomal BCL2 antisense corresponding to one of ²SEQ ID ²NO: 1 with 3x detergent, SEQ ID NO: 2 with lx detergent, SEQ ID NO: 2 with 3x ²detergent, ²and SEQ ID NO: 3 with lx detergent was incubated with each cell line for four ²days. ²Cytotoxicity was assessed using a sulforhodamine B cytotoxicity assay. As the ²data in FIGs. ²3A-C show, incubation with liposomal BCL2 antisense induced 50% inhibition in ²all three ²activated B-cell-like subtype diffuse large B cell lymphoma cell lines.²1001491 The ability of liposomal BCL2 antisense to inhibit the ²viability of ²lymphoma cells was tested in two cell lines: GRANTA-519 (mantle cell lymphoma) ²and ²Ramos (Burkitt lymphoma). Liposomal BCL2 antisense corresponding to one of SEQ ²ID ²NO: 1 with 3x detergent, SEQ ID NO: 2 with lx detergent, SEQ ID NO: 2 with 3x ²detergent, ²and SEQ ID NO: 3 with lx detergent was incubated with each cell line for four ²days.²<DP=51>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²Cytotoxicity was assessed using a sulforhodamine B cytotoxicity assay. As the ²data in FIGs. ²4A-B show, incubation with liposomal BCL2 antisense induced 50% inhibition in ²both cell ²lines.²1001501 The ²ability of liposomal BCL2 antisense to inhibit the viability of²myeloid leukemia cells was tested in three cell lines: K562, MV-4-11, and ²Kasumi-1. ²Liposomal BCL2 antisense corresponding to one of SEQ ID NO: 1 with 3x ²detergent, SEQ ²ID NO: 2 with lx detergent, SEQ ID NO: 2 with 3x detergent, and SEQ ID NO: 3 ²with lx ²detergent was incubated with each cell line for four days. Cytotoxicity was ²assessed using a ²sulforhodamine B cytotoxicity assay. As the data in FIGs. 5A-C show, ²incubation with ²liposomal BCL2 antisense induced 50% inhibition in all three myeloid leukemia ²cell lines.²* * *²100151 All ²of the methods disclosed and claimed herein can be made and executed²without undue experimentation in light of the present disclosure. While the ²compositions and ²methods of this invention have been described in terms of preferred ²embodiments, it will be ²apparent to those of skill in the art that variations may be applied to the ²methods and in the ²steps or in the sequence of steps of the method described herein without ²departing from the ²concept, spirit and scope of the invention. More specifically, it will be ²apparent that certain ²agents which are both chemically and physiologically related may be ²substituted for the ²agents described herein while the same or similar results would be achieved. ²All such similar ²substitutes and modifications apparent to those skilled in the art are deemed ²to be within the ²spirit, scope and concept of the invention as defined by the appended claims.²51²<DP=52>²CA 03058486 2019-09-27²WO 2018 / 195253 PCT / US2018 / 028267²REFERENCES²The following references, to the extent that they provide exemplary procedural ²or ²other details supplementary to those set forth herein, are specifically ²incorporated herein by ²reference.²U.S. Pat. No. 4,659,774 ²U.S. Pat. No. 4,816,571 ²U.S. Pat. No. 4,870,287 ²U.S. Pat. No. 4,959,463 ²U.S. Pat. No. 5,141,813 ²U.S. Pat. No. 5,214,136 ²U.S. Pat. No. 5,223,618 ²U.S. Pat. No. 5,264,566 ²U.S. Pat. No. 5,378,825 ²U.S. Pat. No. 5,428,148 ²U.S. Pat. No. 5,446,137 ²U.S. Pat. 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Claims

<DP=1>²WHAT IS CLAIMED IS:²1. A composition comprising a population of oligonucleotides, wherein the ²oligonucleotides hybridize to a BCL2 polynucleotide gene product, wherein ²oligonucleotides ²of the population are composed of nucleoside molecules linked together through ²phosphate ²backbone linkages, wherein at least one of the phosphate backbone linkages in ²each ²oligonucleotide is a P-ethoxy backbone linkage, and wherein no more than 80% ²of the ²phosphate backbone linkages in each oligonucleotide are P-ethoxy backbone ²linkages.²2. The composition of claim 1, wherein oligonucleotides of the population ²comprise a sequence according to any one of SEQ ID NOs: 1-3.²3. The composition of claim 2, wherein oligonucleotides of the population ²comprise a sequence according to SEQ ID NO: 1.²4. The composition of claim 3, wherein the phosphate backbone linkages at ²least ²between nucleotides 5 and 6, between nucleotides 7 and 8, between nucleotides ²9 and 10, ²between nucleotides 11 and 12, and between nucleotides 14 and 15 of the ²oligonucleotides of ²the population are phosphodiester backbone linkages.²5. The composition of claim 2, wherein oligonucleotides of the population ²comprise a sequence according to SEQ ID NO: 2.²6. The composition of claim 2, wherein the phosphate backbone linkages at ²least ²between nucleotides 5 and 6, between nucleotides 7 and 8, and between ²nucleotides 9 and 10 ²of the oligonucleotides of the population are phosphodiester backbone ²linkages.²7. The composition of claim 2, wherein oligonucleotides of the population ²comprise a sequence according to SEQ ID NO: 3.²8. The composition of claim 1, wherein 50% to 80% of the phosphate backbone ²²linkages are P-ethoxy backbone linkages.²9. The composition of claim 8, wherein 60% to 75% of the phosphate backbone ²²linkages are P-ethoxy backbone linkages.²58²<DP=2>²10. The composition of claim 1, wherein 20% to 50% of the phosphate ²backbone ²linkages are phosphodiester backbone linkages.²11. The composition of claim 10, wherein 25% to 40% of the phosphate ²backbone ²linkages are phosphodiester backbone linkages.²12. The composition of claim 1, wherein the phosphodiester backbone ²linkages ²are distributed throughout each oligonucleotide.²13. The composition of claim 1, wherein the phosphodiester backbone ²linkages ²are not clustered within a portion of each oligonucleotide.²14. The composition of claim 1, wherein the population of oligonucleotides ²is ²heterogeneous as to the number of P-ethoxy backbone linkages and ²phosphodiester backbone ²linkages present in the oligonucleotides of the population.²15. The composition of claim 1, wherein the oligonucleotides of the ²population ²have a size ranging from 18 to 30 nucleotides.²16. The composition of claim 15, wherein the oligonucleotides of the ²population ²have an average size of 18 nucleotides, wherein no more than 14 of the ²phosphate backbone ²linkages in each oligonucleotide is a P-ethoxy backbone linkage.²17. The composition of claim 15, wherein the oligonucleotides of the ²population ²have an average size of 20 nucleotides, wherein no more than 16 of the ²phosphate backbone ²linkages in each oligonucleotide is a P-ethoxy backbone linkage.²18. The composition of claim 15, wherein the oligonucleotides of the ²population ²have an average size of 25 nucleotides, wherein no more than 20 of the ²phosphate backbone ²linkages in each oligonucleotide is a P-ethoxy backbone linkage.²19. The composition of claim 15, wherein the oligonucleotides of the ²population ²have an average size of 30 nucleotides, wherein no more than 24 of the ²phosphate backbone ²linkages in each oligonucleotide is a P-ethoxy backbone linkage.²20. The composition of claim 1, wherein the population of oligonucleotides ²comprises a single species of oligonucleotides.²59²<DP=3>²21. The composition of claim 1, wherein the population of oligonucleotides ²comprises at least two species of oligonucleotides.²22. The composition of claim 1, wherein the population of oligonucleotides ²is ²heterogeneous as to the distribution of phosphodiester backbone linkages among ²the ²oligonucleotides of the population.²23. The composition of claim 1, wherein the oligonucleotides of the ²population ²inhibit the expression of Bcl2 protein.²24. The composition of claim 1, further comprising phospholipids and ²wherein the ²oligonucleotides and phospholipids form an oligonucleotide¨lipid complex.²25. The composition of claim 24, wherein the phospholipids are uncharged or ²²have a neutral charge at physiologic pH.²26. The composition of claim 25, wherein the phospholipids are neutral ²phospholipids.²27. The composition of claim 26, wherein the neutral phospholipids are ²phosphatidylcholines.²28. The composition of claim 26, wherein the neutral phospholipids are ²dioleoylphosphatidyl choline.²29. The composition of claim 24, wherein the phospholipids are essentially ²free of ²cholesterol.²30. The composition of claim 24, wherein the phospholipids and ²oligonucleotides ²are present at a molar ratio of from about 5:1 to about 100:1.²31. The composition of claim 24, wherein the oligonucleotide¨lipid complex ²is ²further defined as a population of liposomes.²32. The composition of claim 31, wherein at least 90% of the liposomes are ²less ²than 5 microns in diameter.²33. The composition of claim 31, wherein the population of oligonucleotides ²is ²incorporated in the population of liposomes.²<DP=4>²34. The composition of claim 1, wherein the composition is lyophilized.²35. A method for reducing the expression level of Bcl2 protein in a cell ²comprising contacting the cell with a composition of claim 1.²36. A pharmaceutical composition comprising a composition according to ²claim ²24 and a pharmaceutically acceptable carrier.²37. The composition of claim 36, further comprising a chemotherapeutic ²agent.²38. A method for delivering a therapeutically effective amount of an ²oligonucleotide to a cell comprising contacting the cell with a pharmaceutical ²composition of ²claim 36.²39. The method of claim 38, wherein the method is a method of treating ²hyperplasia, cancer, autoimmune disease, or infectious disease.²40. A method of treating a subject with a cancer comprising administering ²to the ²subject a therapeutically effective amount of a pharmaceutical composition of ²claim 36.²41. The method of claim 40, wherein the subject is a human.²42. The method of claim 40, wherein the cancer is a non-small cell lung ²cancer, ²pancreatic adenocarcinoma, breast cancer, prostate cancer, melanoma, colon ²cancer, ²leukemia, lymphoma, glioblastoma, osteosarcoma, oral cavity cancer, ovarian ²cancer, uterine ²cancer, bone cancer, brain cancer, prostate cancer, kidney cancer, stomach ²cancer, esophageal ²cancer, rectal cancer, bladder cancer, testicular cancer, or liver cancer.²43. The method of claim 42, wherein the lymphoma is germinal center B-cell-²like ²diffuse large B cell lymphoma.²44. The method of claim 42, wherein the lymphoma is activated B-cell-like ²subtype diffuse large B cell lymphoma.²45. The method of claim 42, wherein the lymphoma is mantle cell lymphoma.²46. The method of claim 42, wherein the lymphoma is Burkitt lymphoma.²47. The method of claim 42, wherein the leukemia is myeloid leukemia.²61²<DP=5>²48. The method of claim 40, wherein the composition is administered ²subcutaneously, intravenously, or intraperitoneally.²49. The method of claim 40, further comprising administering at least a ²second ²anticancer therapy to the subject.²50. The method of claim 49, wherein the second anticancer therapy is a ²surgical ²therapy, chemotherapy, radiation therapy, cryotherapy, hormone therapy, ²immunotherapy, ²anti-viral therapy, immune suppression therapy, anti-bacterial therapy, anti-²parasite therapy, ²anti-fungal therapy, or cytokine therapy.²51. The method of claim 40, wherein administration of the composition ²reduces ²expression of Bcl2 protein in the patient.²62²