Nucleoside-functionalized nanocarriers for delivery of nucleic acids

Abstract

A self-assembling composition includes molecules of a first polymer, molecules of a positively charged second polymer, and a nucleic add. First pendant groups conjugated to pendant amine group of the first polymer include a hydrophilic polymer. Second pendant groups conjugated to the pendant amine groups include a hydrophobic group. Third pendant groups conjugated to the pendant amine groups include a group selected from a nucleoside and a nucleoside analog. Fourth pendant groups conjugated to the second polymer via pendant amine groups thereof include the hydrophilic polymer. Fifth pendant groups are conjugated to the amine groups of the second polymer include the hydrophobic group. The second polymer also includes repeat units wherein the pendant amine groups are positively charged in vivo.

Description

Atty. Docket No.: 24-018PCT NUCLEOSIDE-FUNCTIONALIZED NANOCARRIERS FOR DELIVERY OF NUCLEIC ACIDS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Patent Application Serial No.63 / 689,987, filed September 3, 2024, the disclosure of which is incorporated herein by reference. STATEMENT OF GOVERNMENT INTEREST

[0002] This invention was made with government support under grant numbers CA278608 and CA270623 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND ART

[0003] The following information is provided to assist the reader in understanding technologies disclosed below and the environment in which such technologies may typically be used. The terms used herein are not intended to be limited to any particular narrow interpretation unless clearly stated otherwise in this document. References set forth herein may facilitate understanding of the technologies or the background thereof. The disclosure of all references cited herein are incorporated by reference.

[0004] Immunotherapy, such as immune checkpoint blockade (ICB), represents a promising approach for various type of cancers. However, only a small number of cancer patients benefit from ICB. Strategies aimed at enhancing ICB effectiveness include facilitating the infiltration of cytotoxic T lymphocytes (CTLs) and boosting CTL activation at the different stages of immune response. Despite these advances, relatively few therapeutic strategies have been explored that target the mechanisms of resistance after CTLs and NK cells are recruited and activated. Granzyme B (GzmB), a potent cytotoxic molecule secreted by activated CTLs or NK cells, triggers tumor cell apoptosis by cleaving and activating caspases-3 and -8. The gene expression as well as the activity of GzmB is regulated by various mechanisms at transcriptional and post-transcriptional levels. Overexpression of SERPINB9 (SPB9), a potent endogenous inhibitor of GzmB, represents another important mechanism of cancer resistance to immunotherapy.

[0005] SPB9, first defined in 1995, is a member of the largest and most widely distributed superfamily of proteinase inhibitors known as serpins, which specifically inhibit serine proteases. SPB9 offers cytoprotective benefits against self-inflicted damage caused by GzmBAtty. Docket No.: 24-018PCT in various immune cells, including cytotoxic T cells, dendritic cells and neutrophils. This protection allows immune cells to deploy GzmB to eliminate target cells without falling prey to their own cytolytic mechanisms. However, it has been established that SPB9 also protects tumor cells from GzmB mediated destruction, regardless of whether GzmB originates from cytotoxic lymphocytes or is self-produced by tumor cells. In addition, SPB9 has been observed to shield immunosuppressive cells and tumor-associated fibroblasts (TAF) from GzmB induced destruction. Several studies have reported that SPB9 expression is markedly elevated in several types of cancers, suggesting that its upregulation could be a crucial mechanism of resistance to immunotherapies, including ICB. Therefore, selective inhibition of SPB9 activity in tumor cells and immunosuppressive immune cells while preserving its function in CTLs, shall represent an attractive and effective strategy to boost ICB as well as other immunotherapies. So far, the specific role of SPB9 in modulating the response of cancer cells to chemotherapeutic agents remains poorly understood.

[0006] Chemotherapies may also induce immune suppressive mechanisms. Gemcitabine (GEM) is one of the front-line chemotherapeutic agents for pancreatic cancer (PCa). Recent studies show that GEM is capable of boosting antitumor immune response through various mechanisms. However, several immune suppressive mechanisms are also induced following GEM treatments, which limits the overall therapeutic outcome. SUMMARY OF THE INVENTION

[0007] In one aspect, a composition includes a plurality of molecules of a first polymer. Each molecule of the first polymer includes a first backbone including repeat units including a pendant amine group. A plurality of first pendant groups including a hydrophilic polymer is conjugated to the first backbone via the pendant amine groups thereof. A plurality of second pendant groups including a hydrophobic group is conjugated to the first backbone via the pendant amine groups thereof. Further, a plurality of third pendant groups including a group selected from a nucleoside and a nucleoside analog is conjugated to the first backbone via the pendant amine groups thereof. The composition also includes a plurality of molecules of a second polymer. Each molecule of the second polymer is positively charged and includes a second backbone including the repeat units. A plurality of fourth pendant groups including the hydrophilic polymer is conjugated to the second backbone via the pendant amine groups thereof. A plurality of fifth pendant groups including the hydrophobic group is conjugated to the second backbone via the amine groups thereof. The second polymer also includes a plurality of repeat units wherein the pendant amine groups are groups that are positively charged in vivo. The composition further includes molecules of a nucleic acid. The pluralityAtty. Docket No.: 24-018PCT of molecules of the first polymer, the pluralities of molecules of the second polymer, and the molecules of the nucleic acid assemble into nanostructures in an aqueous medium.

[0008] The pendant amine groups that are positively charged in vivo may, for example, have the formula -NHX, wherein X is selected from the group consisting of :In a number of embodiments, X is H.

[0009] The hydrophobic group may, for example, be a lipid, a poly(methyl acrylate), a polyethylene, a polystyrene, a polyisobutane, a polyester, a polypeptide, and vorinostat. In a number of embodiments, the hydrophobic group is a lipid. In a number of embodiments, the lipid is selected from the group consisting of an oleic acid, stearic acid, palmitic acid, palmitoleic acid, myristic acid, lauric acid, decanoic acid, cholesterol, Vitamin A, Vitamin D, Vitamin E, ceramides, and Farnesyl Thiosalicylic Acid

[0010] In a number of embodiments, each of the first polymer and the second polymer is prepared from a precursor polymer including a backbone including one of the repeat units selected from the group consisting of:Atty. Docket No.: 24-018PCT,wherein R is selected from the group consisting of :Atty. Docket No.: 24-018PCT.

[0011] The second polymer may, for example, be formed by reacting the precursor polymer with a plurality of first molecules including the hydrophilic polymer and a functional group reactive with primary amine groups of the precursor polymer and with a plurality of second molecules including the hydrophobic group and a functional group reactive with primary amine groups of the precursor polymer in a predetermined ratio of the first molecules to the second molecules and in a predetermined molar amount such that primary amine groups of the precursor polymer remain unreacted. The first polymer may, for example, be formed by reaction of a portion of the second polymer with a plurality of molecules including the nucleoside or nucleoside analog and a function group reactive with primary amine groups of the second polymer.

[0012] The hydrophilic polymer may, for example, be selected from the group consisting of: a polyalkylene oxide, a polyvinylalcohol, a polyacrylic acid, a polyacrylamide, a polyoxazoline, a polysaccharide, and a polypeptide. In a number of embodiments, the hydrophilic polymer is a polyalkylene oxide. The polyalkylene oxide may, for example, be a polyethylene glycol. In a number of embodiments, the hydrophilic polymer has a molecular weight in the range from 100 Da to 5 kDa.

[0013] The nucleoside analog may but need not be a chemotherapeutic agent. In a number of embodiments, the nucleoside analog is selected from the group consisting of an analog of cytidine, an analog of adenine, and an analog of pyrimidine. In a number of embodiments, the nucleoside analog is selected from the group consisting of an analog of a pyrimidine nucleoside and an analog of a purine nucleoside. In a number of embodiments, the nucleoside analog is selected from the group consisting of gemcitabine, azacitidine, cytarabine, decitabine, RX-3117, trifluridine, 5-fluorouracil, capecitabine, tegafur, carmofur, floxuridine, DMT-OMe-rC(Ac), uridine, 2 -eeoxy-2 -fluoroadenosine, tezacitabine,Atty. Docket No.: 24-018PCT LY2334737, 2'-deoxy-2'-fluoro-5-iodouridine, 1-(2-deoxy-2-fluoro-beta-D- arabinofuranosyl)uracil, tipiracil, idoxuridine, zidovudine, eniluracil, sorivudine, cytidine, thymidine; adenosine, 8-chloroadenosine, clofarabine, n6-benzyladenosine, clofarabine, fludarabine phosphate, cladribine, fludarabine, nelarabine, pentostatin, ganciclovir, forodesine hydrochloride, viltolarsen, cordycepin, vidarabine, didanosine, tenofovir, adefovir, 6-thioguanine, mercaptopurine, azathioprine, forodesine, ganciclovir, acyclovir, valacyclovir, penciclovir, famciclovir, ribavirin and tiazofurin, guanosine. In a number of embodiments, the nucleoside analog is a gemcitabine.

[0014] The nucleic acid may be selected from the group of mRNA and siRNA. The nucleic acid may, for example, be siRNA. In a number of embodiments, the siRNA is siSPB9, siNEBL, siiRhom1,siCD200, siCT, or siEGFR.

[0015] The nucleoside analog, particularly when it is a therapeutically active agent, may be conjugated to the pendant amine group via a linking group including a moiety that is labile in vivo. The moiety that is labile in vivo may include at least one of an ester bond, an orthoester bond, a thioether-ester bond, an anhydride bond, an amid bond, a carbonate bond, a disulfide bond, a hydrazone bond, a cic-acotinyl bond, an acetal bond, a carboxydimethyl maleate bond, an imine bond, an oxime bond, a silyl ether bond, a ketal bond, a thioketal bond and a protease cleavable peptide. In a number of embodiments, the moiety that is labile in vivo includes at least one of an ester bond and a disulfide bond. In a number of embodiments, the moiety that is labile in vivo includes a disulfide bond.

[0016] In a number of embodiments, the first polymer has the formula:and the second polymer has the formula:wherein R1 is a group including a group selected from the group of the nucleoside and the nucleoside analog, R2 is a group including the hydrophilic polymer, R3 is a group including theAtty. Docket No.: 24-018PCT hydrophobic group, and x is in the range of 30 to 100%, y is in the range of 1 to 50%, and z is in the range of 1 to 20%, and x1 is in the range of 30 to 100%, y1 is in the range of 1 to 50%, and z1 is in the range of 3 to 20%. X may, for example, be selected from the group consisting of:. In a number of embodiment, X is H.

[0017] In a number of embodiments, the nucleic acid is siRNA and the mass ratio of first polymer to siRNA is within the range of 10:1 to 100:1, or in the range of 20:1 to 80:1. The nucleic acid may be siRNA and the N:P ratio of the composition may be in the range of 2:1 to 15:1.

[0018] The nanostructures may be micelles. In a number of embodiments, the nanostructures have an average diameter in the range of 30 to 200 nm, or in the range of 30 to 100 nm.

[0019] In a number of embodiment, the composition further includes a therapeutic agent loaded into the nanostructure during self-assembly thereof (for example, by mixing the therapeutic agent with the components of the composition in the aqueous medium). The therapeutic agent may, for example, be selected from the group consisting of a hydrophobic therapeutic agent and a hydrophilic therapeutic agent, wherein the hydrophilic therapeutic agent is conjugated with a lipid before being loaded into the nanostructure during self- assembly thereof. In a number of embodiments, the therapeutic agent is a chemotherapy agent. The therapeutic agent may be a small molecule therapeutic compound. For example,Atty. Docket No.: 24-018PCTthe therapeutic agent may have a molecular weight below 1 kDa. In a number of embodiments, the therapeutic agent is selected from the group consisting of paclitaxel, doxorubicin, docetaxel, gefitinib, imatinib, dasatinib, curcumin, camptothecin, etoposide, edelfosine, vincristine, temsirolimus, carmustine, cisplatin, oxaliplatin, or a chemotherapeutically active derivative thereof.

[0020] In another aspect, a method of delivering a nucleic acid to a patient includesadministering a plurality of nanostructures compositions as set forth herein.

[0021] The therapeutic agent may also be an immunotherapeutic agent. The therapeuticagent may, for example, include an anti-PD-1 antibody.

[0022] In another aspect, a polymer is prepared from a precursor polymer including one ofrepeat units selected from the group consisting of: ,, , , ,Atty. Docket No.: 24-018PCTwherein R is selected from the group consisting of:,

[0023] The precursor polymer is reacted with a plurality of first molecules including ahydrophilic polymer and a functional group reactive with primary amine groups of the precursor polymer and with a plurality of second molecules including a hydrophobic group and a functional group reactive with primary amine groups of the precursor polymer in aAtty. Docket No.: 24-018PCTpredetermined ratio of the first molecules to the second molecules and in a predetermined molar amount such that primary amine groups of the precursor polymer remain unreacted.

[0024] In another aspect, a polymer is prepared from a precursor polymer including one ofrepeat units selected from the group consisting of:Atty. Docket No.: 24-018PCTwherein R is selected from the group consisting of :, wherein the precursor polymer is reacted with a first plurality of molecules including a hydrophilic polymer and a functional group reactive with primary amine groups of the precursor polymer, with a second plurality of molecules including a hydrophobic group and a functional group reactive with primary amine groups of the precursor polymer, and with a third plurality of molecules including a group selected from the group consisting of a nucleoside and a nucleoside analog and a functional group reactive with primary amine groups of the polymer.

[0025] In a further aspect, a method of providing treatment to a patient, includesadministering a therapeutic agent, wherein the therapeutic agent causes upregulation of SPB9, and administering siSPB9. The therapeutic agent may, for example, be a chemotherapeutic agent. In a number of embodiments, the therapeutic agent is a nucleoside or a nucleoside analog.Atty. Docket No.: 24-018PCT

[0026] The therapeutic agent and siSPB9 may be co-administered via nanostructures. The nanostructures may, for example, be formed by mixing in an aqueous medium components including the following: (i) a plurality of molecules of a first polymer, each molecule of the first polymer including a first backbone including repeat units including a pendant amine group, a plurality of first pendant groups including a hydrophilic polymer being conjugated to the first backbone via the pendant amine groups thereof, a plurality of second pendant groups including a hydrophobic group being conjugated to the first backbone via the pendant amine groups thereof, and a plurality of third pendant groups including a group selected from the group consisting of a nucleoside and a nucleoside analog being conjugated to the first backbone via the pendant amine groups thereof, (ii) a plurality of molecules of a second polymer, each molecule of the second polymer being positively charged and including a second backbone including the repeat units, a plurality of fourth pendant groups including the hydrophilic polymer being conjugated to the second backbone via the pendant amine groups thereof, a plurality of fifth pendant groups including the hydrophobic group being conjugated to the second backbone via the amine groups thereof, and a plurality of repeat units wherein the pendant amine groups are groups that are positively charged in vivo, and (iii) molecules of siSPB9. The plurality of molecules of the first polymer, the pluralities of molecules of the second polymer, and the molecules of siSPB9 assemble into the nanostructures in the aqueous medium.

[0027] The present devices, systems, and methods, along with the attributes and attendant advantages thereof, will best be appreciated and understood in view of the following detailed description taken in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0028] FIG.1A illustrates schematically the formation of a nanostructure or nanocarrier delivery system with a representative embodiment of a PPO / PPOGEM / siRNE (POEM / siRNA) system hereof.

[0029] FIG.1B illustrates tissue distribution (H, heart; S, spleen; Lu, lung; Li, liver; K, kidney; T, tumor; T / Li, tumor / liver; T / K, tumor / kidney; T / Ti tumor / tissues) of POEM / siRNA using different PPO polymers.

[0030] FIG.2A illustrates embodiments of synthetic route for synthesis of PEG1K-COOH, synthesis of PPO polymers, and synthesis of PPOGEM polymers.

[0031] FIG.2B illustrates structures of PSugar and PCytidine control polymers for use in the studies hereof.Atty. Docket No.: 24-018PCT

[0032] FIG.2C illustrates a generalized formula for representative PPO, PPOGEM, PSugar, and PCytidine polymers of the studies hereof.

[0033] FIG.2D illustrates Size and ZP of PPOGEM / PPO / siRNA (POEM / siRNA) at a ratio of 50 / 5 / 1, and Psugar / PPO / siRNA at a ratio of 40 / 5 / 1. n = 3 independent samples.

[0034] FIG.2E illustrates studies of cumulative release of gemcitabine at 72h or 12h from POEM / siRNA NPs under different conditions, wherein n = 3 independent samples, data are presented as mean ± s.e.m, and statistical analysis was performed by one-way ANOVA with Tukey’s post hoc test for comparison.

[0035] FIG.2F illustrates a gel-retardation assay of free siRNA or POEM / siRNA after incubation with RNAse.

[0036] FIG.3A illustrates quantified radiant efficiency of ex vivo imaging of KPC-C2 subcutaneous tumor and major organs (H, heart; Li, liver; S, spleen; Lu, lung; K, kidney; T, tumor) at 24 h following intravenous administration of free siSPB9-cy5.5 or various POEM / siSPB9-cy5.5 NPs, wherein n = 3 independent samples, data are presented as mean ± s.e.m, and statistical analysis was performed by two-way ANOVA with Tukey’s post hoc test for comparison.

[0037] FIG.3B illustrates quantified radiant efficiency of ex vivo imaging of KPC-C2 orthotopic pancreatic tumor and major organs at 24 h following intravenous administration of free siSPB9-cy5.5 or POEM / siSPB9-cy5.5 NPs at a ratio of 50 / 5 / 1, wherein n = 3, independent samples data are presented as mean ± s.e.m, and statistical analysis was performed by two-way ANOVA with Tukey’s post hoc test for comparison.

[0038] FIG.3C illustrates percentage of injected dose and tissue concentration of siSPB9 in different organs following i.v. injection of free siSPB9 or POEM / siSPB9 at a dose of 1 mg / kg for siRNA, wherein n = 3 independent samples, independent samples data are presented as mean ± s.e.m, and statistical analysis was performed by two-way ANOVA with Tukey’s post hoc test for comparison.

[0039] FIG.3D illustrates plasma concentrations of siSPB9 over time after i.v. injection of siSPB9 or POEM / siSPB9 at a dose of 1 mg / kg for siRNA, wherein n = 3 independent samples, independent samples data are presented as mean ± s.e.m, and statistical analysis was performed by two-way ANOVA with Tukey’s post hoc test for comparison.

[0040] FIG.3E illustrates Table 1 in which pharmacokinetic parameters of siSPB9 were analyzed by a one-compartmental model. t1 / 2: half-life; AUC : the area under the plasmaconcentration versus time curve; AUMC : the area under the plasma concentration versusthe first moment curve (AUMC); CL: clearance; Vd: volume of distribution.Atty. Docket No.: 24-018PCT

[0041] FIG.4A illustrates a quantification of luminous intensity of whole body imaging of tumors of KPC-C2-Luc tumor-bearing C57BL / 6 mice receiving intravenous administration of POEM / siLuc NPs once every 3 days. siCT refers to non-targeting siRNA as a control, wherein n = 5 mice, data are presented as mean ± s.e.m, and statistical analysis was performed by a two-way ANOVA with Tukey’s post hoc test for comparison.

[0042] FIG.4B illustrates the mRNA expression levels of SPB9 in KPC-C2 tumors following various treatments three times. n = 5 mice, data are presented as mean ± s.e.m., and statistical analysis was performed by one-way ANOVA with Tukey’s post hoc test for comparison.

[0043] FIG.4C illustrates changes of average tumor volume in KPC-C2 WT tumor (subcutaneous)-bearing mice receiving various treatments, wherein n = 5 mice, data are presented as mean ± s.e.m., and statistical analysis was performed by a two-way ANOVA with Tukey’s post hoc test for comparison.

[0044] FIG.4D illustrates changes of individual tumor volume in KPC-C2 WT tumor (subcutaneous)-bearing mice receiving various treatments, wherein n = 5 mice, data are presented as mean ± s.e.m., and statistical analysis was performed by two-way ANOVA with Tukey’s post hoc test for comparison.

[0045] FIG.4E illustrates schematically a scheme for the establishment of pancreatic orthotopic model and treatments.

[0046] FIG.4F illustrates luminous intensity of tumors from whole-body imaging of KPC-C2- Luc tumor (orthotopic)-bearing mice following different treatments, wherein n = 6 mice, data are presented as mean ± s.e.m., and statistical analysis was performed by two-way ANOVA with Tukey’s post hoc test for comparison.

[0047] FIG.4G illustrates survival rate of KPC-C2-Luc tumor (orthotopic)-bearing mice receiving various treatments, wherein n = 6 mice, and statistical analysis was performed by log rank test for comparison.

[0048] FIG.5A illustrates changes of average tumor volumes and individual tumor volume in mice bearing KPC-C2 gemcitabine resistant (GEMR) subcutaneous tumor receiving various treatments, wherein n = 5 mice, data are presented as mean ± s.e.m., and statistical analysis was performed by two-way ANOVA with Tukey’s post hoc test for comparison.

[0049] FIG.5B illustrates tumor weights and images of KPC-C2 GEMR tumor-bearing mice receiving different treatments, wherein n = 5 mice, data are presented as mean ± s.e.m., and statistical analysis was performed by one-way ANOVA with Tukey’s post hoc test for comparisonAtty. Docket No.: 24-018PCT

[0050] FIG.5C illustrates changes of average tumor volumes and individual tumor volume in KPC-C2-GEMR tumor (subcutaneous)-bearing mice receiving PBS, anti-PD1, POEM / siSPB9 and combination treatment, wherein n = 5 mice, data are presented as mean ± s.e.m., and statistical analysis was performed by two-way ANOVA with Tukey’s post hoc test for comparison.

[0051] FIG.6A illustrates tumor growth curves of 3LL tumor-bearing mice receiving various treatments, wherein the mice received treatments once every 3 days for 5 times at 3 and 1 mg / kg for AZA and siRNA (siNebl), respectively, wherein n =5, and Nebl is an oncogene that is upregulated in various types of cancer including lung cancer and contributes to oncogenesis and tumor progression.

[0052] FIG.6B illustrates shows the tumor growth curves from another therapeutic study based on the 5-fluorouracil nucleoside formulation, wherein the curves depict the growth of CT26 subcutaneous tumors in mice, which received different treatments every 3 days for a total of 5 injections (N = 5).

[0053] FIG.7A illustrates Table 2 setting forth antibodies for Western blot and flow cytometry.

[0054] FIG.7B illustrates Table 3 setting forth sequences of siRNA.

[0055] FIG.7C illustrates Table 4 setting forth sequences of primers for qRT-PCR.

[0056] FIG.7D illustrates Table 5 wetting for sequences of the SERPINB9 promoter region and three putative ATF3 binding motifs.

[0057] FIG.7E illustrates Table 6 setting forth sequences of primers for siSerpinb9 quantification by qRT-PCR. DESCRIPTION

[0058] It will be readily understood that the components of the embodiments, as generally described and illustrated in the figures herein, may be arranged and designed in a wide variety of different configurations in addition to the described representative embodiments. Thus, the following more detailed description of the representative embodiments, as illustrated in the figures, is not intended to limit the scope of the embodiments, as claimed, but is merely illustrative of representative embodiments.

[0059] Reference throughout this specification to “one embodiment” or “an embodiment” (or the like) means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearance of the phrases “in one embodiment” or “in an embodiment” or the like inAtty. Docket No.: 24-018PCT various places throughout this specification are not necessarily all referring to the same embodiment.

[0060] Furthermore, described features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. In the following description, numerous specific details are provided to give a thorough understanding of embodiments. One skilled in the relevant art will recognize, however, that the various embodiments can be practiced without one or more of the specific details, or with other methods, components, materials, et cetera. In other instances, well known structures, materials, or operations are not shown or described in detail to avoid obfuscation.

[0061] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, reference to “a primary amine group” includes a plurality of such primary amine groups and equivalents thereof known to those skilled in the art, and so forth, and reference to “the primary amine group” is a reference to one or more such primary amine groups and equivalents thereof known to those skilled in the art, and so forth. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value, as well as intermediate ranges, are incorporated into the specification as if individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contraindicated by the text.

[0062] As used herein, the term “polymer” refers to a chemical compound that is made of a plurality of small molecules or monomers that are arranged in a repeating structure to form a larger molecule. Thus, a polymer is a compound having multiple repeat units (or monomer units) and includes the term “oligomer,” which is a polymer that has only a few repeat units. The term “copolymer” refers to a polymer including two or more dissimilar repeat units (including terpolymers - comprising three dissimilar repeat units - etc.). Polymers may occur naturally or be formed synthetically. The use of the term “polymer” encompasses homopolymers (having a single repeat unit) as well as copolymers. The term “copolymer” is used herein to include any polymer having two or more different monomers. Copolymers may, for example, include alternating copolymers, periodic copolymers, statistical copolymers, random copolymers, block copolymers, graft copolymers etc.

[0063] As used herein, the term “pendant” refers to a group or moiety attached to a backbone chain of a long molecule such as a polymer as described above. Pendant group may be either (1) short chain or low molecular weight groups or (2) long chain or highAtty. Docket No.: 24-018PCT molecular groups such as polymers. Pendant groups are sometime referred to as side groups. Long chain pendant groups or high molecular weight pendant groups are sometimes referred to as “pendant chains” or “side chains.”

[0064] As used herein, the term “approximately” when used in connection with a value means within 5%, within 2%, or within 1% of the value unless otherwise indicated herein or otherwise clearly contraindicated by the text. As used herein the term “and / or” means one of or both of an entity. Thus, A and / or B means A or B, or both A and B.

[0065] Although SPB9 (an endogenous inhibitor of granzyme B or GmzB) is known to be upregulated in immunotherapy, until the present studies, its upregulation had not been demonstrated in connection with other therapies, including cancer chemotherapy. In studies hereof, it was shown chemotherapeutic agents such as GEM also induces SPB9 expression in tumor cells or tissues in vitro and in vivo, indicating another feedback mechanism that negatively regulates the antitumor immune response. The mechanism by which GEM induces the SPB9 gene expression was studied at transcriptional level, and the role of GzmB / SPB9 axis in regulating the chemosensitivity of pancreatic cancer or PCa cells to GEM was evaluated. Studies hereof have demonstrated that levels of GmzB and SPB9 are each increased in connection with, for example, nucleoside analog chemotherapy. The studies hereof further demonstrated that tumor cells became much more sensitive to chemotherapy through SPB9 inhibition / knock down. Polymer nanocarriers hereof such as a representative POEM (PPO / PPOGEM) nanocarrier were developed and evaluated for codelivery of nucleosides such as GEM and nucleic acids such as siSPB9 to overcome both chemotherapy and immune resistance.

[0066] As described above, GEM is a front-line treatment for PCa but shows limited efficacy. To study the mechanisms of resistance in PCa, RNA sequencing was conducted to analyze changes in the gene expression profiles of murine wild type (WT, KPC-C2 (clone KPC 6694c2) and GEM resistant (KPC C2GEMR) PDAC cell lines. Cutoff values for both log2(fold change) and log10(p-value) were established at 2. Twenty-two (22) genes were shown to be upregulated in GEMR cells including 3 genes (Atf3, Gadd45a, and Asns) that were previously shown to be involved in gemcitabine resistance. The expression of Serpinb9b gene was most significantly induced among the 22 upregulated genes. Upregulation of Serpinb9b was further confirmed by qRT-PCR in 3 murine GEMR cell lines (KPC-C2, KPC-C5 (clone KPC 6914c5), and Panc02). Serpinb9b is a murine orthologue of human SERPINB9 (SPB9), although humans do not have the SERPINB9 subtype SERPINB9b. Several bioinformatic and experimental studies suggest that SPB9 might be implicated in immunotherapy resistance inAtty. Docket No.: 24-018PCT both animal models and in cancer patients, but its role in chemoresistance had not yet been explored before the present studies. Therefore, SPB9 was selected as a primary target to elucidate its potential involvement in chemoresistance. To bridge the findings with clinical applications, the expression of Serpinb9 in the murine GEMR cell lines was further assessed via qRT-PCR. The upregulation of the orthologue was also observed in the murine GEMR cell lines.

[0067] To further explore the clinical implications of SPB9, its expression across various cancer types in The Cancer Genome Atlas Program (TCGA) database were studied. SPB9 overexpression was not only seen in pancreatic ductal adenocarcinoma (PDAC), but also found in diffuse large B-cell lymphoma (DLBC), kidney renal clear cell carcinoma (KIRC), skin cutaneous melanoma (SKCM), and stomach adenocarcinoma (STAD). Elevated levels of SPB9 expression were negatively correlated with the clinical prognosis of cancer patients across all cancer types (PANCAN) including PDAC patients.

[0068] It was found that GEM treatment induced the expression of SPB9 mRNA in a dose- dependent manner in KPC-C2, KPC-C5 and Panc02 cell lines. Upregulation of SPB9 mRNA by GEM was also observed in human PCa cell lines (PANC-1, MIA PaCa-2, Panc 10.05 and Panc 02.03) in both time- and dose-dependent manners. Induction of SPB9 by GEM was further confirmed at the protein level in several murine (C2, C5 and Panc02) and human pancreatic cancer cell lines (PANC-1, MIA PaCa-2, Panc 10.05 and Panc 02.03). In addition to GEM, doxorubicin (DOX) and cisplatin (CDDP) induced SPB9 mRNA expression in several murine PCa cell lines tested (C2, C5 and Panc02).

[0069] The above results indicate that chemotherapy drugs induce SPB9 gene expression, likely at transcriptional level. RNA-seq data was reanalyzed to identify candidate transcription factors whose expression levels were also significantly induced. Similar to the cutoff value established earlier, cutoff values for both log2(fold change) and log10(p-value) were set at 2. ATF3, a transcription factor critical to stress responses, was identified as the most significantly upregulated gene. The gene set ATF3_Q6 includes those genes with at least one ATF3 binding motif, within a 4 kb region centered on their transcription starting sites. See Gene Set Enrichment Analysis, Human Gene Sets: ATF3_Q6. This gene set was also significantly upregulated). The findings were validated by qRT-PCR in GEMR PCa cell lines. A transient treatment with GEM for 24 or 48 hours induced the expression of ATF3 at both mRNA and protein levels. The positive correlation of the expression levels of SPB9 and ATF3 was further shown in tumor samples of PAAD patients from TCGA database and a referenced study described in Balagurunathan Y, et al., Gene expression profiling-based identification ofAtty. Docket No.: 24-018PCT cell-surface targets for developing multimeric ligands in pancreatic cancer, Mol Cancer Ther 7(9): 3071-3080 (2008). A similar correlation was also seen in tumors from a cohort of Pan- Cancer patients from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) program and TCGA database. Studies hereof demonstrated that siATF3 could achieve over 60% knockdown efficiency. Knockdown of ATF3 via siRNA significantly attenuated the GEM-mediated induction of SPB9 in both murine (KPC-C5) and human (MIA PaCa-2) PDAC cells at both mRNA and protein levels. ATF3 KD had no impact on GEM-induced expression of murine orthologue Serpinb9b, however.

[0070] The above data indicated that ATF3 plays a role in GEM-mediated induction of SPB9 gene expression. Three (3) putative ATF3-binding sites (-303-311 (ATF3-1), -390-397 (ATF3- 2), and -1503-1510 (ATF3-3)) upstream of human SPB9 gene were identified based on an ATF3 chromatin immunoprecipitation sequencing (ChIP-Seq) and the UCSC Genome Browser. A DNA fragment spanning all 3 putative binding sites was generated by PCR and cloned into the pGL3 vector. In addition, 3 other reporters were constructed by cloning a 5- times repeat of each of the 3 putative ATF3 binding sequences, respectively. The transcriptional activity of each cloned sequence was examined by luciferase assay using MIA PaCa-2, PANC-1, and Panc 02.03 cell lines. GEM induced the expression of luciferase reporter in a dose-dependent manner in cells transfected with the plasmid containing all 3 putative binding sequences as well as the one with 5x repeat of ATF3-3 sequence. In contrast, minimal transcriptional activity was shown for ATF3-1 and ATF3-2 sequences. In addition, deletion of ATF3-3 from the full-length sequence completely abolished its transcriptional activity. Taking together, the data indicates that ATF3 is a key transcriptional factor that drives the GEM-induced SPB9 gene expression through binding to ATF3-3 in SPB9 gene promoter.

[0071] The GzmB-SPB9 axis is importantly involved in the escape of cancer cells from immune surveillance as well as poor response to immunotherapy. However, its role in chemotherapy resistance remains unexplored. In studies of the role of GzmB in GEM- mediated tumor killing, KPC-C2 SPB9 KO cell line were first generated using the CRISPR / Cas9 system. Western blot analysis confirmed the disappearance of SPB9 protein, both the monomeric form and the SPB9-GzmB complex. Notably, the levels of monomeric GzmB were increased in SPB9 KO cells, demonstrating that GzmB is dissociated from its complex as a result of the depletion of SPB9. SPB9 KO cells showed a cell viability that was similar to that of WT cells as evaluated by MTT assay. However, SPB9 KO cells showed significant retardation in tumor formation in mice with tumor volumes and weights of about 1 / 6 ofAtty. Docket No.: 24-018PCT those of WT tumors. The proportion of Annexin V+tumor cells was also significantly increased in the KO group.

[0072] KO of SPB9 significantly improved the tumor immune microenvironment as evident from increases in the infiltration of CD45+immune cells. The numbers of both total CD8+T cells and IFN + T cells (both CD4+ and CD8+) were also increased in the SPB9-deficienttumors. In addition, there was a notable increase in the recruitment of dendritic cells in SPB9-deficient tumors. Those changes were accompanied by a substantial reduction in myeloid-derived suppressor cells (MDSCs). SPB9 KO further facilitated a reversal from the predominance of M2-like macrophages, suggesting a reprogramming of the tumor- associated macrophage phenotype.

[0073] SPB9 KO drastically increased the responsiveness of both WT and GEMR KPC-C2 cells to GEM. Similarly, SPB9 KD with siRNA (siSPB9) also enhanced the sensitivity of both WT and GEMR murine PCa cell lines (KPC-C2, KPC-C5 and Panc02) to GEM treatment. Similar results were seen in human PCa cell lines (Panc 02.03 and MIA PaCa-2). The proportion of Annexin V+cells was significantly increased when GEM was combined with siSPB9 or SPB9 inhibitor 3034, suggesting a strong synergistic effect between GEM treatment and SPB9 KD or inhibition. In line with in vitro results, GEM-based nanoparticles (NPs) effectively halted the tumor progression in all five SPB9 KO tumors.

[0074] To gain mechanistic insights, the expression of GzmB following GEM treatment at both transcriptional and translational levels was studied through PCR, Western blot, and flow cytometry. Both the expression levels of GzmB and the abundance of GzmB+cells were increased by GEM treatment across several cell lines (KPC-C2 WT, KPC-C2 SPB9 KO, KPC-C5, Panc02, PANC-1, Panc 02.03, Panc 10.05 and MIA PaCa-2). Specifically, GEM induced the expression of GzmB in SPB9 KO cells at concentrations as low as 2 ng / mL. In addition, the activity of GzmB was also induced in murine KPC C2 cells and human PANC-1 cells. KD of GzmB in SPB9 KO cells using siRNA significantly attenuated the effects of GEM treatment. A similar effect was observed with the pharmacological inhibition of GzmB: treatment with the GzmB inhibitor 368050 led to decreased apoptosis. Development of GEM resistance was associated with marked increases in SPB9 expression levels along with decreases in GzmB expression. The data indicates that SPB9 KD or inhibition in tumor cells may benefit increased sensitivity to GEM-based chemotherapy in addition to its benefits in improving antitumor immune response.

[0075] The above and other studies indicate that SPB9 represents a promising therapeutic target for cancer, particularly in combination with other treatments such as chemotherapy.Atty. Docket No.: 24-018PCT As discussed further below, it was chosen to develop a representative therapy based on combination of GEM with siSPB9 instead of a small molecule inhibitor of SPB9. Various strategies for siRNA delivery, ranging from synthetic NPs to extracellular vesicles, have been studied. Current lipid nanoparticle (LNP) systems have shown substantial efficacy in siRNA delivery to hepatocytes through i.v. administration. However, such systems have shown limited success in targeting to extrahepatic organs or tissues including solid tumors. Polymer-based delivery systems have been explored as an alternative to LNPs due to their potential to enhance tumor targeting through various surface modifications and functionalization strategies. Nevertheless, such polymeric systems often fall short in achieving the desired amounts of accumulation in tumors and / or knockdown efficiency. A PMBOP-CP-based polymeric nanocarrier that is highly effective in selective codelivery of siRNA and lipophilic / hydrophobic drugs to tumors is, for example, discussed in PCT International Patent Application Publication No.2023 / 136868.

[0076] In various attempts to reduce the effects of SPB9 in connection with immunotherapy, a number of small molecules SPB9-inhibiting drugs have been developed. However, small-molecule SPB9 inhibitors may be readily taken up by various immune cells and diminish the cytoprotective benefits against self-inflicted damage provided by SPB9 in such immune cells. The present inventors hypothesized that using siRNA instead of small molecules to inhibit SPB9 can improve therapeutic treatment in which SPB9 is upregulated in a more targeted manner (that is, without significant negative effect on immune cells). In the case of a chemotherapeutic drug, for example, it is desirable to deliver siRNA to inhibit SPB9 (or other targets) in a tumor specific manner.

[0077] Delivery of nucleic acids such as siRNA and mRNA had, for example, previously relied on the use of nanocarriers with cationic components such as cationic lipids, cationic polymers, hybrid cationic molecules etc. Excess amounts of cationic molecules are often needed, which contributes to the toxicity of nanocarriers. In addition, codelivery of other small molecule drugs with nucleic acids carriers has been challenging, especially for codelivery of highly hydrophilic drugs such as nucleosides-based drugs.

[0078] Nucleosides include nucleobase (sometimes referred to a nitrogenous base, and which includes a nitrogen containing organic ring structure) and a five-carbon sugar (that is, ribose or 2'-deoxyribose, or a modified version thereof). A nucleoside analog is a structurally modified version of a natural nucleoside. In general, a nucleoside analog is a synthetic mimic of a natural nucleoside that includes the majority of the canonical structure of the natural nucleoside, but includes modifications which alter biological activity or impart otherAtty. Docket No.: 24-018PCT properties. A nucleoside analog thus retains the canonical nucleoside framework, and includes a nucleobase (that is, purine, pyrimidine, or a modified version thereof through substitutions, deletions, or additions within the purine or the pyrimidine ring system) which is covalently linked to a sugar moiety (that is, -deoxyribose, or a modified version thereof).

[0079] The present inventors have discovered that amphiphilic polymers which can self- assemble into nanostructures and include covalently incorporated nucleoside or nucleoside analog pendant groups provide very good carriers for nucleic acids including siRNA. In that regard, and without limitation to any mechanism, it is hypothesized that nucleosides and nucleoside analogs, which (as described above) include a sugar molecule linked to a nitrogen- -interactions, via hydrophobic interactions, and via hydrogen bonding. Such interactions may occur regardless of the structure of the nucleoside or nucleoside analog. In addition to formation of stable and small nanostructures (for example, micelles) for delivery of nucleic acid, the conjugation of therapeutic nucleoside / nucleoside analogs into amphiphilic polymers hereof provides an efficient delivery path for hydrophilic nucleoside / nucleoside analogs while protecting such nucleoside / nucleoside analogs from, enzymatic (for example, deaminase) attack and destruction during systemic delivery.

[0080] In a number of embodiments hereof, a composition or nanocarrier composition includes a first polymer, which is an amphiphilic polymer. The first polymer includes at least one group selected from the group of a nucleoside and a nucleoside analog. The composition further includes a second polymer which is a positively charged in vivo (that is, which includes cationic groups in vivo) and which is an amphiphilic polymer. The second polymer does not include nucleoside or nucleoside analog groups. The first polymer and the second polymer self-assemble into nanostructures in an aqueous medium. An individual nanostructure hereof include molecules of both of the first polymer and the second polymer. A nucleic acid may be added to the nanostructures and may be retained on the interface thereof. The first polymer may be approximately neutral in charge, while the second polymer includes a positive charge for charge-charge interaction with the nucleic acids. Interactions of the nucleoside groups and / or nucleoside analog groups of the first polymer with the nucleic acid molecule(s) stabilize the nanostructures and enable the formation of nanostructures having relatively small average diameter.

[0081] While nanostructures may be formed from a single amphiphilic polymer which, for example, includes pendant hydrophilic polymer groups, pendant hydrophobic groups, pendant groups including a group selected from the group consisting of a nucleoside or a nucleoside analog, as well as pendant cationic groups, a number of advantages are providedAtty. Docket No.: 24-018PCT by the two-polymer compositions hereof. In that regard, it is significantly more difficult to synthesize a single-polymer composition as describe above. Multiple steps (for example, requiring 4 or more steps) are required and separation or purification may be difficult. In the two-polymer system or composition hereof, fewer steps are required and separation may be easier (for example, achieved via dialysis and precipitation). Moreover use of two polymers in forming the nanostructures hereof facilitates modification or adjustment of various ratios and other variables in the nanostructure compositions (for example, the ratio of nucleoside:hydrophilic group:hydrophobic group and the nitrogen to phosphorous ratio or N:P ratio). Moreover, it is easier to adjust the amount of nucleoside / nucleoside analog introduced as well as the positive charge introduced in the case of a two-polymer system.

[0082] Each of a first polymer and a second polymer of compositions hereof are biocompatible and may, for example, have a molecular weight (Mw) of no greater than 30kDa to facilitate removal through kidney clearance. In a number of embodiments, the Mw of the first polymer and the second polymer is between 3kDa and 30kDa. In a number of embodiments, the degree of polymerization of the polymer backbone of the first polymer differs from the degree of polymerization of the polymer backbone of the second polymer by less than 30%, 20% or 10%. The first polymer and the second polymer can have a different number of pendant hydrophilic polymer groups and hydrophobic groups (as well as a different ratio thereof).

[0083] In a number of embodiments, the composition includes a plurality of molecules of a first polymer, wherein each molecule of the first polymer includes a first backbone formed of repeat units including a pendant amine group. A plurality of first pendant groups include a hydrophilic polymer and which are conjugated to the first backbone via the pendant amine groups thereof. The first polymer also includes a plurality of second pendant groups which include a hydrophobic group and which conjugated to the first backbone via the pendant amine groups thereof. The first polymer further includes a plurality of third pendant groups which includes a group selected from the group of a nucleoside or a nucleoside analog which is conjugated to the first backbone via the pendant amine groups thereof. The composition further includes a plurality of molecules of a second polymer. Each molecule of the second polymer is positively charged and includes a second backbone comprising the repeat units. A plurality of fourth pendant groups including the hydrophilic polymer are conjugated to the second backbone via the pendant amine groups thereof. A plurality of fifth pendant groups including the hydrophobic group are conjugated to the second backbone via the amine groups thereof. The second polymer further includes a plurality of repeat units wherein theAtty. Docket No.: 24-018PCT pendant amine groups thereof are groups that are positively charged in vivo. The pendant amine groups that are positively charged in vivo may, for example, have the formula -NHX, wherein X is selected from the croup consisting of :.

[0084] The plurality of molecules of the first polymer and the pluralities of molecules of the second polymer assemble into nanostructures in an aqueous medium. Molecules of a nucleic acid are added to the nanostructures and become associated therewith. In a number of embodiments, the pendant amine groups of the second polymer are primary amine groups.

[0085] The hydrophobic group may, for example, be a lipid, a poly(methyl acrylate), a polyethylene, a polystyrene, a polyisobutane, a polyester, a polypeptide oleic acid, stearic acid, palmitic acid, palmitoleic acid, myristic acid, lauric acid, cholesterol, Vitamin A, Vitamin D, Vitamin E, ceramides, Farnesyl Thiosalicylic Acid and vorinostat. In a number of embodiments, the hydrophobic group is a lipid.

[0086] Polymers hereof may be formed via monomers corresponding to the repeat units wherein the monomers are functionalized with the pendant groups described above before polymerization. Alternatively, in a number of embodiments, the first polymer and the second polymer are prepared from a precursor polymer including a backbone having repeat units including a pendant primary amine group. In a number of embodiments, the repeat units of the backbone are the same (that is, a homopolymer backbone). The precursor polymer is then reacted with molecules including a group reactive with the primary amine to attach the pendant groups described above. The first polymer and the second polymer may, for example, be prepared from a precursor polymer comprising a backbone comprising one of the repeat units selected from the group consisting of:Atty. Docket No.: 24-018PCTO O H N O NH2,NH2,O O OO O O,NH2,NH2, and O O N R NH2 , , ,, , , , , andAtty. Docket No.: 24-018PCTwherein R is selected from the group consisting of : ,.

[0087] The second polymer may, for example, be formed by reacting the precursor polymerwith a plurality of first molecules comprising the hydrophilic polymer and a functional group reactive with primary amine groups of the precursor polymer and with a plurality of second molecules comprising the hydrophobic group and a functional group reactive with primary amine groups of the precursor polymer in a predetermined ratio of the first molecules to the second molecules and in a predetermined molar amount such that primary amine groups of the precursor polymer remain unreacted. The first polymer may, for example, be formed by reaction of a portion of the second polymer with a plurality of molecules comprising the nucleoside or nucleoside analog and a function group reactive with primary amine groups of the second polymer.

[0088] The hydrophilic polymer may, for example, be selected from the group consisting of:a polyalkylene oxide, a polyvinylalcohol, a polyacrylic acid, a polyacrylamide, a polyoxazoline, apolysaccharide, and a polypeptide. In a number of embodiments, the hydrophilic polymeris a polyalkylene oxide. The hydrophilic polymer may, for example, be a polyethylene glycol.Atty. Docket No.: 24-018PCT The hydrophilic polymer may, for example, have a molecular weight in the range from 100 Da to 5 kDa.

[0089] The nucleoside or nucleoside analog hereof need not be biologically active or therapeutic. In embodiments in which a nucleoside or nucleoside analog is not biologically active or therapeutic, the nanostructures hereof may be used to deliver a nucleic acid (for example, siRNA or mRNA) and / or a therapeutic agent loaded into the nanostructures (for example, a small-molecule drug or therapeutic agent). In a number of embodiments, the third pendant group of the first polymer includes a therapeutic nucleoside analog. The nucleoside analog may, for example, be a chemotherapeutic agent. The nucleoside analog may, for example, be selected from the group consisting of an analog of cytidine, an analog of adenine, and an analog of pyrimidine. In a number of embodiments, the nucleoside analog is selected from the group consisting of gemcitabine, azacitidine, cytarabine, decitabine, RX-3117, trifluridine, 5-fluorouracil, capecitabine, tegafur, carmofur, floxuridine, DMT-OMe-rC(Ac), uridine, 2 -eeoxy-2 -fluoroadenosine, tezacitabine, LY2334737, 2'-deoxy-2'-fluoro-5-iodouridine, 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)uracil, tipiracil, idoxuridine, zidovudine, eniluracil, sorivudine, cytidine, thymidine; adenosine, 8- chloroadenosine, clofarabine, n6-benzyladenosine, clofarabine, fludarabine phosphate, cladribine, fludarabine, nelarabine, pentostatin, ganciclovir, forodesine hydrochloride, viltolarsen, cordycepin, vidarabine, didanosine, tenofovir, adefovir, 6-thioguanine, mercaptopurine, azathioprine, forodesine, ganciclovir, acyclovir, valacyclovir, penciclovir, famciclovir, ribavirin and tiazofurin, guanosine. In a number of embodiments, the nucleoside analog is a gemcitabine.

[0090] The nucleic acid may, for example, be selected from the group of mRNA and siRNA. In a number of embodiments, the nucleic acid is siRNA. In a number of embodiments, the siRNA is siSPB9, siNEBL, siXkr8, siiRhom1, siCD200, siCT, or siEGFR.

[0091] In a number of embodiments, a nucleoside analog, which is a therapeutic agent, is conjugated to the pendant amine group via a linking group including a moiety that is labile in vivo. The moiety that is labile in vivo may, for example, include at least one of an ester bond, an orthoester bond, a thioether-ester bond, an anhydride bond, an amid bond, a carbonate bond, a disulfide bond, a hydrazone bond, a cic-acotinyl bond, an acetal bond, a carboxydimethyl maleate bond, an imine bond, an oxime bond, a silyl ether bond, a ketal bond, a thioketal bond and a protease cleavable peptide. In a number of embodiments, the moiety that is labile in vivo includes at least one of an ester bond and a disulfide bond. In a number of embodiments, the moiety that is labile in vivo includes a disulfide bond.Atty. Docket No.: 24-018PCT

[0092] In a number of embodiments, the second polymer has the formula:and the first polymer has the formula:wherein R1 is a group comprising a group selected from the group of the nucleoside and the nucleoside analog, R2 is a group comprising the hydrophilic polymer, R3 is a group comprising the hydrophobic group, and x is in the range of 30 to 100%, y is in the range of 1 to 50%, and z is in the range of 1 to 20%, and x1 is in the range of 30 to 100%, y1 is in the range of 1 to 50%, and z1 is in the range of 3 to 20%. X may, for example, be selected from the group consisting of:

[0093] In a number of embodiments wherein the nucleic acid is siRNA, the mass ratio of first polymer to siRNA is within the range of 10:1 to 100:1, and optionally in the range of 20:1 to 80:1. In a number of embodiments wherein the nucleic acid is siRNA, the N:P ratio of the composition is in the range of 2:1 to 15:1.Atty. Docket No.: 24-018PCT

[0094] The nanostructures may, for example, be micelles. The nanostructures may, for example, have an average diameter in the range of 30 to 200, or in the range of 30 to 100.

[0095] The composition hereof may further include a therapeutic agent loaded into the nanostructure during self-assembly thereof. The therapeutic agent may, for example, be selected from the group consisting of a hydrophobic therapeutic agent and a hydrophilic therapeutic agent, wherein the hydrophilic therapeutic agent is conjugated with a lipid before being loaded into the nanostructure during self-assembly thereof. The therapeutic agent may, for example, be a chemotherapy agent. The therapeutic agent may be a small molecule therapeutic compound. For example, the therapeutic agent may have a molecular weight below 1 kDa. Representative example, of suitable therapeutic agents include paclitaxel, doxorubicin, docetaxel, gefitinib, imatinib, dasatinib, curcumin, camptothecin, etoposide, edelfosine, vincristine, temsirolimus, carmustine, cisplatin, oxaliplatin, or a chemotherapeutically active derivative thereof. In the case of hydrophilic cisplatin of oxaliplatin, the cisplatin or oxaliplatin would be conjugated with a lipid before loading.

[0096] In a number of studies hereof, a representative, simple and small-sized (~40 nm) platform was developed that is highly effective in selective codelivery of nucleic acids such as siRNA and various hydrophilic nucleoside-based drugs as demonstrated by a representative two-polymer ‘POEM’ nanocarrier in codelivery of siSBP9 and GEM. The representative POEM nanocarrier includes the polyalkylene oxide polyethylene glycol (PEG) and lipid-derivatized polylysine (PEG-PLL-Oleic acid, PPO, which is positively charged) and lipid-derivatized, GEM-conjugated polylysine (PEG-PLL-OA-GEM, PPOGEM, which is nearly neutral in charge). Such components form a compact nanoassembly PPO / PPOGEM (sometimes referred to herein as POEM) that efficiently loads nucleic acids such as siRNA into NPs (see FIG.1A). Design of this nanocarrier was based on the following rationales: 1) conjugation of GEM to a polymer enhances its delivery and protects it from degradation by deaminase; 2) lipid-derivatization of both polymers aids in forming compact micelles and facilitate endosomal release; 3) the electrostatic interactions between PPO and siRNA initiate the formation of POEM / siRNA complexes; 4) POEM / siRNA complexes may be further -s and hydrogen bonding between the built-in GEM and siRNA(see FIG.1A). The multiple modes of interaction reduce the nitrogen / phosphate (N / P) ratio needed to form stable siRNA NPs, which decreases toxicity. In addition, this helps to reduce the surface positive charges, which shall decrease the non-selective uptake by lung and liver; 5) The synthesis of polymers such as PPO and PPOGEM is simple and can be readily scaled up. In addition, POEM / siRNA NPs can be prepared via a one-step of mixing followed byAtty. Docket No.: 24-018PCT removal of solvents via dialysis. Systematic in vitro characterizations were conducted to optimize the ratio of the 3 components (PPO, PPOGEM, and siRNA) with a goal to identify top candidates that are small in sizes (<100 nm) and close to neutral in surface charges. Such optimization studies are readily performed with other polymer and nucleic acid combinations hereof. The in vivo tumor targeting efficiency of the top candidates was then evaluated with near infrared fluorescence imaging using both subcutaneous and orthotopic tumor models.

[0097] A small library of PPO cationic polymers that varied in the ratios of PEG / OA (y / z) and the amounts of cationic amino acids (histidine, lysine, and arginine) introduced was first synthesized (see FIG.1B). Polymers with unfavorable biophysical properties (low solubility and / or size over 400 nm) were triaged for further evaluations. Candidate PPO polymers selected from the initial screening were then mixed with PPOGEM and siRNA at an “empirical” ratio of 100 / 20 / 1 (w / w for PPOGEM / siRNA (100 / 1) and N / P for PPO / siRNA (20 / 1)) to form POEM / siRNA. Ex vivo near-infrared fluorescence imaging (NIRFI) was used to evaluate the Cy5.5-siRNA NPs with respect to their tumor-targeting efficiency. As depicted in FIG.3B, a PPO with a PEG / OA ratio of 5:2 and without further amino acid modification formed the most effective (among the compositions tested) siRNA NPs with PPOGEM in terms of the amounts of Cy5.5 signals in the tumor tissues, as well as the ratios of the signals in tumors over those in other normal tissues / organs. That PPO was selected for further optimization of POEM / siRNA via extensive characterizations of the biophysical properties and in vivo distribution of 18 NPs that vary in PPOGEM / PPO / siRNA ratio.

[0098] Gel retardation assay revealed that siRNA could be effectively loaded into PPO polymer at a N / P ratio as low as 5 / 1. However, PPO alone cannot form stable complexes with siRNA. Interestingly, incorporation of PPOGEM polymer into the PPO / siRNA system resulted in NPs with a single-peaked size distribution, indicating that PPOGEM helps stabilize the system. Judging from gel retardation assay, siRNA was well loaded into POEM NPs at an N / P ratio of 5 / 1, 10 / 1 and 20 / 1, respectively. Increasing the amounts of PPOGEM led to a gradual decrease of zeta potential likely as a result of the shielding effect from PPOGEM. Notably, at an N / P ratio of 5 / 1 and a PPOGEM / siRNA mass ratio of 50 / 1, the POEM / siRNA system demonstrated the most desirable biophysical properties with a size of approximately 45.6 nm and a zeta potential of around 9.3 mV (NPs with zeta potential in a range of -10 ~ +10 are considered close to neutral).

[0099] As an initial step to understand the interaction of siRNA with POEM carrier, a control polymer with only sugar ring conjugated to the PLL backbone (Psugar) was synthesized toAtty. Docket No.: 24-018PCT elucidate a role of GEM aromatic ring in stabilizing the siRNA NPs. Representative embodiments of a synthetic scheme for PPO an POGEM are illustrated in FIG.2A, and the structures of PSugar and PCytadine (discussed below) are illustrated in FIG.2B. FIG.2C illustrates a general structure for the PPO, PPOGEM, PSugar, and PCytadine polymers of the studies hereof. FIG.2D shows that replacement of PPOGEM with Psugar led to formation of much larger-sized NPs (~160 nm compared to ~40 nm for PPOGEM) with higher zeta potential (~18 mV compared to ~9 mV for PPOGEM). Moreover, increasing the amounts of Psugar led to further increases in the sizes of the resulting NPs that eventually form precipitates due to poor stability. In addition, the zeta potential of the NPs could not be reduced to below 10 mV at all PPO / Psugar ratios examined. The above data highlight the significant role of GEM in formulating the POEM NP system.

[0100] To further characterize the interactions between POEM and siRNA and the behavior of siRNA within the POEM carrier, all-atom molecular dynamics (MD) simulations were conducted. Considering that the system is very dynamic, the radius of gyration (RoG) was used to monitor the conformational variation of POEM / siRNA. The RoG measures how far the components of an object are spread out from its center of mass. A smaller RoG value indicates that the mass of the object is more tightly clustered around the center, indicating a denser and more compact structure. The RoG of POEM system was compared to that of the Psugar system. During the simulation, after the systems reached a plateau, it was obvious that the POEM system (~54.87 Å) exhibits lower RoG values than the control polymer, Psugar (~55.87 Å). This can be further explained by a representative conformation of POEM / siRNA, wherein siRNA effectively interacts with PPOGEM through both T- -and hydrogen bonding interactions. During the simulation, the whole NP system was constructed based on a molar ratio of PPOGEM (or Psugar), PPO and siRNA of 33: 9: 1, thus, the simulation accurately represents the composition of the NPs. However, in the real-world scenario, NPs are composed of numerous copies of the simulation system which can be considered as a unit. While the RoG difference between the POEM and Psugar systems is only around 1 Å in the simulation, this small difference at the unit level can lead to a significant variation in the overall size of the assembled NPs. Consequently, the POEM system (~40 nm) and the Psugar system (~160 nm) exhibit a substantial difference in their final dimensions.

[0101] POEM / siRNA exhibited a lower critical micelle concentration of 12 ng / mL compared to the Psugar system. The morphology of POEM / siRNA at a ratio of 50 / 5 / 1 was assessedAtty. Docket No.: 24-018PCT using cryo-electron microscopy (cryo-EM), revealing spherical particles with a relatively uniform diameter.

[0102] The GEM release profile of POEM / siRNA NPs was evaluated using dialysis method. As shown in FIG.2E, minimal GEM release was observed from the NPs in PBS (pH 7.4) with or without 10% of FBS or murine serum. However, a rapid release of GEM (over 30%) occurred at 12 h in the presence of 10 mM GSH (glutathione) due to the cleavage of built-in disulfide bond (-SS-), a concentration that is commonly found in tumor cells (FIG.2D). In addition, POEM demonstrated a sustained release of GEM over a period of 72 h followed. The above data indicate that GEM is well protected prior to delivery to tumor tissues and achieves sustained release upon intracellular delivery to tumor cells and exposure to high concentrations of GSH. The gel retardation study of FIG.2F shows that siRNA formulated in the POEM NPs was well protected from RNase-mediated degradation.

[0103] The in vitro gene knockdown efficiency of POEM NPs was evaluated using KPC-C2- Luc and Panc02-Luc cell lines. POEM NPs were more effective than the commercial siRNA transfection reagent RNAiMAX (available from Thermo Fisher Scientific of Waltham, MA US) in mediating the siLuc transfection and silencing luciferase transgene expression in both cell lines.

[0104] The in vivo distribution of POEM NPs was first evaluated by IVIS imaging 24 hours after i.v. injection into KPC-C2 tumor (subcutaneous)-bearing mice. The NPs were prepared with an N / P ratio of 5:1. While lower N / P ratios are possible, an N / P ratio of 5:1 represented a minimum ratio required to effectively load siRNA at a desirable loading capacity for studies hereof. Additionally, the biodistribution of the POEM system at different amounts of PPOGEM was investigated to examine how changes in size and zeta potential affect their in vivo behavior. Cy5.5 signals were predominantly accumulated in the kidney and were barely detectable in tumors following injection of free siRNA-cy5.5 (see FIG.3A). Positively charged PPO / siRNA-cy5.5 complexes were primarily found in lungs (Lu) and kidneys (K), suggesting the initial aggregation in blood followed by gradual release of siRNA from the complexes. Incorporating PPOGEM at a ratio of 25 / 5 / 1 led to improved accumulation at the tumor site, with significant amounts of signals also observed in the liver (Li) and lungs, likely due to size reduction (~50 nm) but still insufficient shielding of surface positive charges (~20 mV) by PPOGEM. Increasing the PPOGEM ratio from 25 / 5 / 1 to 50 / 5 / 1 resulted in a gradual increase of the Cy5.5 signal in tumors and a decrease of signal in liver due to improvements in both size reduction and shieling of surface positive charge (~50 nm, ~10 mV) (Fig.4a). Further increase in the amount of PPOGEM led to reduced tumor signals due to the larger size of theAtty. Docket No.: 24-018PCT NPs (~86.5 nm). Subsequent studies were conducted with POEM / siRNA prepared at a PPOGEM / PPO / siRNA ratio of 50 / 5 / 1.Similar results were obtained in Panc02 subcutaneous model. Effective tumor targeting of POEM / siRNA NPs was also demonstrated in PCa KPC-C2 and Panc02 orthotopic (o.t.) models (see FIG.3B). However, more signals were seen in liver in o.t. models compared to subcutaneous models. PCa o.t. model is known to have a high propensity of developing liver metastasis.

[0105] The tissue distribution and pharmacokinetics of siSPB9 via qRT-PCR was further quantitatively analyzed. Free siRNA was found in all organs, with a notably higher concentration observed in the spleen (S). See FIG.3C. In contrast, siSPB9 was largely concentrated in tumors with approximately 6% of the injected dose localized in tumors at 24 hours following i.v. injection of POEM / siRNA (FIG.3C). The concentration of siSPB9 in tumors was significantly higher than that in the liver (~ 3-fold) (FIG.3C). There were some discrepancies between the imaging and qRT-PCR data, which was likely due to the limited sensitivity of whole-body and ex vivo imaging as reported in literature. In addition, qRT-PCR detects intact siRNA while imaging detects all fluorescence signals. FIG.3D shows the kinetic changes in the concentrations of siSPB9 in blood after injection of free siSPB9 or POEM siSPB9 NPs into KPC-C2 tumor-bearing mice. It is apparent that POEM siSPB9 persisted in the circulation for a significantly longer time compared with free siSPB9. Encapsulation of siSPB9 within POEM substantially enhanced its half-life (t1 / 2) and area under the curve (AUC), while significantly reducing its volume of distribution (Vd) and clearance (CL) compared to the free form of siSPB9 (Table 1 of FIG.3E). The extended circulation time of POEM / siSPB9 NPs was also confirmed by fluorescence measurement of serum samples. The long circulation time of POEM / siRNA is likely attributed to the excellent stability of the NPs in blood, which contributes to the effective tumor targeting.

[0106] The effective tumor accumulation of POEM NPs is likely to benefit from a passive targeting through enhanced permeation and retention (EPR) effect due to their relatively small sizes (40~50 nm). To further elucidate a potential role of active transport in tumor targeting by POEM NPs, proteomics study was conducted to investigate the protein corona formed around POEM NPs, which is regarded as an important factor in influencing the in vivo behaviors thereof, including tissue distribution. Preliminary data indicated that fibronectin, which can contribute to NPs’ tumor targeting and penetration, is involved in the formation of the NPs protein corona. A transcytosis study revealed that blocking fibronectin receptor ITGA5 with its antibody significantly inhibited the transcytosis of the NPs. In lineAtty. Docket No.: 24-018PCT with this observation, cellular uptake experiments confirmed that both anti-ITGA5 and chlorpromazine were able to block the uptake of POEM NPs.

[0107] In studies of co-delivery of siSPB9 and GEM, the knockdown efficiency of siRNA delivered by the POEM system was first evaluated. A luciferase-expressing tumor cell line (KPC-C2-Luc) was used to establish a subcutaneous tumor model and the gene knockdown efficiency was examined by following changes in luminescent intensity following treatment with POEM NPs loaded with siLuc. A non-targeting siRNA was included as a control (siCT). FIG.4A shows that repeated injections of siLuc NPs led to a gradual reduction in luciferase activity in KPC-C2-Luc tumor-bearing mice as assessed by whole-body bioluminescence imaging. FIG.4B shows that POEM / siSPB9 NPs effectively suppressed both basal and GEM- induced SPB9 mRNA levels in vivo.

[0108] The findings discussed above have shown that targeting SPB9 can directly sensitize PCa to GEM-based treatment in addition to enhancing antitumor immune response. In addition, a POEM nanocarrier capable of codelivery of GEM and siSPB9 has been developed. FIGS.4C and 4D show the therapeutic efficacy of POEM / siSPB9 NPs in a KPC-C2 subcutaneous tumor model. A pharmacologically “inert” control nanocarrier, POC (PPO / PCytidine; see FIG.2B), was also developed as a control NP to load siSPB9 via replacing gemcitabine in PPOGEM with cytidine. POC shows similar size and zeta potential compared to POEM. In addition, POC and POEM show similar interactions with siRNA as demonstrated by comparable radiuses in molecular dynamic simulation. Tumor-bearing mice received various treatments when the tumors reached ~50 mm3. Both free GEM and POC / siSPB9 showed modest antitumor activity while POEM / siCT was more effective than free GEM. Combination of POC / siSPB9 with GEM led to a significantly improved antitumor activity (FIG. 4C), suggesting that POC / siSPB9, as a simple and stand-alone siRNA therapy, can be readily combined with current standard of care such as gemcitabine and other treatments. It is also apparent that POEM loaded with siSPB9 showed a further improvement in overall antitumor activity compared with GEM+POC / siSPB9, including stabilization of tumor growth in 2 mice (FIG.4D), clearly demonstrating the therapeutic benefit of codelivery of gemcitabine and siSPB9 using POEM NPs.

[0109] There were no significant changes in body weights following the different treatments. In addition, the treatments did not adversely affect liver and kidney functions, as indicated by minimal alterations in serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine. Furthermore, histological examination revealed no notable changes in major organs including heart (H) , liver (Li), spleen (S), lungAtty. Docket No.: 24-018PCT (Lu), and kidney (K) in all groups. Such findings underscore the excellent safety profile of co- delivery of siSPB9 and GEM using POEM NPs, at dosages that have shown significant therapeutic efficacy.

[0110] We further evaluated the therapeutic potential of POEM / siSPB9 in KPC-C2 o.t. model. Luciferase-expressing KPC-C2 cells (KPC-C2-luc) were orthotopically transplanted into the pancreas of mice. Tumor-bearing mice received different treatments starting on day 5 post-inoculation of tumor cells (FIG.4E). FIG.4F displays the changes in luminescence intensities of tumor areas over time, prior to any mortality in each group. POC / siSPB9 and POEM / siCT were both effective in controlling tumor growth, with the POEM / siSPB9 group demonstrating superior anti-tumor effects (FIG.4F). The enhanced inhibition of tumor growth by POEM / siSPB9 was translated into a significant prolongation of survival time (FIG. 4G). Among all treatments evaluated, POEM / siSPB9 led to the longest survival.

[0111] GEM resistance presents a significant challenge in clinical treatment. To further assess the effectiveness of POEM / siSPB9 NPs in combating GEM-resistant tumors, therapeutic efficacy thereof in KPC-C2 GEMR model was evaluated. Similar to the study in wild-type model, mice received various treatments 5 days after tumor inoculation (FIGS.5A and 5B). POEM / siCT showed a level of antitumor activity that is comparable to POC / siSPB9, supporting the notion that SPB9 is a viable therapeutic target in the treatment of GEM- resistant pancreatic cancer. However, GEM and siSPB9 combination was less effective in controlling the resistant tumor compared to what was shown earlier in WT tumor model (FIGS.5A and 5B). These results prompted further investigation of the tumor immune microenvironment in the GEMR model as discussed below.

[0112] Flow cytometry analysis demonstrated that all the treatments resulted in increased infiltration of CD45+immune cells. Formulations loaded with siSPB9, particularly POEM / siSPB9, led to the polarization of M1-like macrophages (CD80+) and an increase in the M1 / M2 ratio (CD80+ / CD163+). In addition, treatments with POEM / siCT and POEM / siSPB9 significantly increased the numbers of CD8+T cells, along with GzmB+CD8+T cells, especially in the POEM / siSPB9 group. Activated CD8+T cells (CD69+) were also increased. Moreover, there were significant increases of -secreted CD4+and CD8+T cells, which are indicative of an enhanced immune response against the tumor. Meanwhile, regulatory T cells, which typically suppress immune responses and promote immune tolerance, were downregulated within the TME. Overall, the combination of GEM and siSPB9 using POEM NPs fostered an immunoactive tumor microenvironment, contributing to the inhibition of proliferation andAtty. Docket No.: 24-018PCT killing of tumor cells as was evident from decreased numbers of Ki67+and increased numbers of Annexin V+tumor cells.

[0113] Despite the favorable changes in various immune cell subsets as described above, treatment with GEM or siSPB9 significantly upregulated PD-1 expression in CD4+and CD8+T cells. PD-1 is a protein that inhibits the immune system's ability to attack cancer cells. This observation led to exploration the potential of combining anti-PD-1 antibody with treatments hereof to enhance the overall therapeutic efficacy. As demonstrated in FIG.5C, monotherapy with anti-PD-1 exhibited limited antitumor efficacy. However, the combination of anti-PD-1 with POEM / siSPB9 significantly enhanced the therapeutic outcome. Tumor growth was effectively suppressed following the 1sttreatment, and by day 20, four out of five tumors were completely regressed upon gross examination. Moreover, all treatments were well tolerated, as indicated by minimal changes in body weights. These results suggest that the combination of POEM / siSPB9 with anti-PD-1 treatment represents a promising regimen towards GEM resistant pancreatic cancer.

[0114] As described above, as an endogenous inhibitor of GzmB, SPB9 has been shown to be significantly involved in immune homeostasis including viral infection, autoimmune diseases and tumor immune escape. Inhibition of SPB9 in tumor cells has been proposed as a strategy for improving cancer immunotherapy. In the studies hereof, the understanding of SPB9 was extending by showing that GzmB / SPB9 axis is also involved in regulating the sensitivity of representative PCa cells to the representative chemotherapy agent GEM through a non-immunological mechanism. The studies hereof demonstrated that GEM induces the gene expression of both SPB9 and GzmB. SPB9 KO or KD led to sensitization of PCa cells to GEM in a GzmB-dependent manner. Interestingly, GEM resistance is associated with increased expression levels of SPB9 and decreased expression of GzmB. Data of the present studied indicates that KD of SPB9 can directly sensitize PCa to GEM treatment in addition to improving the antitumor immune response.

[0115] Nanocarriers formed from two-polymer systems hereof (such as POEM) represents an effective nanocarrier in codelivery of nucleosides and nucleoside analogs (for example, GEM) and nucleic acids (for example, siRNA, mRNA etc.). The aromatic of nucleosides and nucleoside analog (pyrimidine in GEM) structure is important in stabilizing the siRNA NPs - sugar ring are also important in promoting the interaction with a nucleic acid such as siRNA, albeit less important compared to pyrimidine motif of GEM. The use of two individual polymers (for example, PPO and PPOGEM) with cationic (for example, -NH2) and nucleosidesAtty. Docket No.: 24-018PCT and nucleoside analogs (for example, GEM) motif, respectively will provide the flexibility of adjusting their ratio in formulation optimization. In addition, a very similar structure of the two polymers will facilitate their mixing to form compact and stable micelles. Although this study focuses on evaluating combination of GEM and siRNA, this strategy can be applied to codelivery of siRNA with other nucleoside and nucleoside analogue-based drugs such as AZA. In addition, other drugs such as cisplatin can be readily incorporated into POEM NPs following lipid-derivatization. Therefore, the nanocarrier systems hereof may be tailor- designed to develop precision medicines to suit different combination therapies.

[0116] Representative studies of another representative example of a formulation hereof is illustrated in FIG.6A. In that regard, FIG.6A illustrates tumor growth curves of 3LL tumor- bearing mice receiving various treatments, wherein the mice received treatments once every 3 days for 5 times at 3 and 1 mg / kg for azacitidine or AZA and siRNA (siNebl), respectively. N =5. Nebl is an oncogene that is upregulated in various types of cancer including lung cancer and contributes to oncogenesis and tumor progression. The PPO-AZA polymer is similar in structure to PPOGEM other than GEM is replaced by azacitidine (AZA).

[0117] FIG.6B illustrates a further representative study of a formulation hereof. FIG.6 B illustrates tumor growth curves from a therapeutic study based on the 5-fluorouracil nucleoside formulation. The curves depict the growth of CT26 subcutaneous tumors in mice, which received different treatments every 3 days for a total of 5 injections (N = 5). POF nanoparticles of the study included two PPO polymers as described herein. The first PPO polymer included pendant groups of 5-fluoro- -deoxyuridine, which is a nucleoside analogand an active metabolite of 5-fluorouracil. The second PPO polymer did not contain nucleoside analog pendant groups. POF is used as a mixture of the two polymers described above. In FIG.6B, Pt represents oxaliplatin used in the system via loading thereof into the nanostructures. CD200 is the oncogene investigated in this study. siCT and siCD200 refer to siRNAs with control and CD200- the CD200 antibody, which is used for comparison with the effect of siCD200 delivered by POF.

[0118] Recently, protein corona (PC), a layer of proteins absorbed on the surface of NPs upon exposure to biological fluids, has been increasingly recognized to be important in defining the in vivo behaviors of the NPs especially the tissue tropism. Much of this understanding has been derived from studies on lipid nanoparticles (LNPs) used for targeted delivery to hepatocytes. It has recently reported that fibronectin was preferentially enriched in ultrasmall-sized PAZA NPs (~15 nm) but not the large-sized counterpart without AZA motifAtty. Docket No.: 24-018PCT (~150 nm), which contributes significantly to both tumor accumulation and deep penetration of PAZA NPs. Luo Z, et al., In Situ Formation of Fibronectin-Enriched Protein Corona on Epigenetic Nanocarrier for Enhanced Synthetic Lethal Therapy, Adv Sci (Weinh) e2307940 (2024). Fibronectin was also one of the proteins that were enriched in the protein corona of POEM NPs. The fact that fibronectin is involved in facilitating cellular uptake and transcytosis of POEM NPs by tumor cells indicates that this protein, and possibly other proteins as well, may also play a role in enhancing the accumulation and penetration of POEM NPs. EPR shall also contribute to the tumor targeting by POEM NPs as well, especially considering their compact sizes (40~50 nm). The combination of EPR-mediated passive targeting and fibronectin-mediated active targeting contributes significantly to the effective tumor accumulation of POEM NPs in both subcutaneous and orthotopic models: around 6% of injected siRNA formulated in POEM NPs was found in the tumor tissues at 24 h, and the siRNA concentrations in tumors were 3~4 folds higher than those in liver. More mechanistic studies on tumor targeting may, for example, be used to achieve further improved tumor delivery systems.

[0119] Studies hereof demonstrated that codelivery of siSPB9 with GEM led to significant antitumor activity in both WT and GEMR models along with improved tumor immune microenvironment. The enhanced antitumor activity with that representative combination therapy likely stems from multiple synergistic mechanisms. Firstly, GEM contributes directly to tumor cell death and also induces the production of GzmB in the tumor cells. Concurrently, SPB9 KD sensitizes tumor cells to both the direct pharmacological effect of GEM and the apoptosis induced by immune cells. Additionally, this combination therapy leads to a more robust antitumor immune response, creating a hostile environment for cancer progression. Combination of anti-PD1 antibody with this regimen leads to further improvement in the overall therapeutic effect.

[0120] Once again, there have been efforts to develop small molecule inhibitors of SPB9 such as 3034 due to the ease in clinical application of such small molecule inhibitors.3034 has been shown to inhibit tumor growth in several murine tumor models. Codelivery of 3034 with DOX via NPs has also been shown to improve the overall therapeutic efficacy in a murine breast cancer model. A concern with the small molecule inhibitor, however, is the potential detrimental effect on T cells and NK cells via blocking the activity of SPB9 inside these cells. Despite the likely improved delivery of a SPB9 inhibitor to tumors using NPs, it is likely that T cells will also be exposed to the small molecule compound released inside tumor tissues. Systemic use of a SPB9 inhibitor may have more concerns on its impact on theAtty. Docket No.: 24-018PCT immune homeostasis in normal organs and tissues. On the other hand, T cells are notoriously known to be hard to transfect. Thus, uptake of siSPB9 NPs by T cells may cause minimal impact on the SPB9 levels inside these immune cells. In that regard, the representative POEM NPs hereof exhibited minimal uptake by T cells compared to tumor cells, both in vitro and in vivo following systemic administration. This low uptake of POEM NPs by T cells is likely due to their limited interactions with T cells, in contrast to other immune cell types such as macrophages. The efficient cellular uptake by TAFs indicates that the representative POEM NPs hereof may serve as a powerful tool to deliver therapies to these cells directly and disrupt the TAFs-mediated pro-tumor processes. It is also interesting to note that POEM NPs were effectively taken by tumor endothelial cells as well. I has recently been reported that transcytosis across endothelial cells contributes significantly to the accumulation of various types of NPs. A role of the active trans-endothelial transport in tumor targeting by POEM NPs requires more study in the future. In a study, it was demonstrated that codelivery of siSPB9 and GEM using POEM NPs was more effective in inhibiting the tumor growth than POEM NPs-mediated codelivery of 3034 and GEM.

[0121] Experimental Methods

[0122] Ethics statement. All animals were housed under pathogen free conditions according to AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care) guidelines. All animal-related experiments were performed in full compliance with institutional guidelines and approved by the Animal Use and Care Administrative Advisory Committee at the University of Pittsburgh under protocol number 21099779. The maximum tumor volume permitted by the IACUC guidelines was 2,000 mm³. Throughout the study, tumor size and animal health were monitored daily, and the maximum allowed tumor burden was not exceeded.

[0123] Reagents. Gemcitabine was purchased from LC Laboratories (MA, USA). Poly- -L-lysine HCl (MW 3500-4500 Da) was purchased from BIOSYNTH (KY, USA). Oleic acid, poly(ethylene glycol) methacrylate (average Mn = 950, PEG950), 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide (MTT), Dulbecco's Modified Eagle's Medium (DMEM), trypsin-EDTA solution, D-Luciferin were purchased from Sigma-Aldrich (MO, USA). RPMI- 1640 medium, fetal bovine serum (FBS) and penicillin-streptomycin solution were purchased from Invitrogen (NY, USA). GlutaMAX (100×) was purchased from Thermo Fisher Scientific (MA, USA). Antibodies for Western blot and flow cytometry were listed in Table 2 of FIG.7A.

[0124] Cell lines and animals. All cell lines were purchased from certified vendors (ATCC) and used at low passage. Authentication was performed by the suppliers using standard STRAtty. Docket No.: 24-018PCT profiling. Routine mycoplasma testing was conducted using PCR-based assays to confirm the absence of contamination throughout the study. Panc02, PANC-1, MIA PaCa-2, Panc 02.03 and Panc 10.05 cell lines were obtained from ATCC (Manassas, VA). KPC-C2 and KPC-C5 cell lines were kindly gifted by Dr. Wen Xie (University of Pittsburgh). KPC-C2, KPC-C5 and Panc02 gemcitabine resistant cell lines were generated according to published protocols. Shah, A.N., et al., Development and characterization of gemcitabine-resistant pancreatic tumor cells, Ann Surg Oncol, 14(12): 3629-3637 (2007). SERPINB9 KO KPC-C2, SERPINB9 KO KPC-C2 GEMR, Panc02-Luc and KPC-C2-Luc cells were produced by following lentiviral / retroviral infection protocol as detailed in lentiviral infection section below. Panc02 and PANC-1 were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin / streptomycin (100 U / ml). KPC-C2, SERPINB9 KO KPC-C2 and KPC-C5 were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 1% GlutaMAX (100×) and penicillin / streptomycin (100 U / ml). MIA PaCa-2 was cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 2.5% horse serum and penicillin / streptomycin (100 U / ml). Panc 02.03 and Panc 10.05 were cultured in RPMI-1640 supplemented with 15% fetal bovine serum (FBS), 10 Units / ml human recombinant insulin and penicillin / streptomycin (100 U / ml). Gemcitabine resistant cell lines were cultured in the full medium with gemcitabine (260 ng / mL). All cells were cultured at 37 °C in a humidified atmosphere with 5% CO2.

[0125] Female C57BL / 6 mice aged between 4-6 or 8-10 weeks were purchased from The Jackson Laboratory (ME, USA). Mice were housed at an ambient temperature of 22 °C (range: 22-24 °C) and a humidity of 45%, with a 14 / 10 day / night cycle (lights on at 6:00, off at 20:00), and allowed access to food ad libitum.

[0126] Vector, RNA interference, and lentiviral infection. Edit-R Mouse Serpinb9 mCMV- EGFP All-in-one Lentiviral sgRNA was purchased from Horizon Discovery Ltd. (Cambridge, UK). psPAX2 and pMD2.G were provided by Dr. Da Yang (University of Pittsburgh). pLentipuro3 / TO / V5-GW / EGFP-Firefly Luciferase Plasmid was purchased from Addgene (MA, USA).

[0127] Murine and human SERPINB9 siRNA (siSPB9), murine and human ATF3 siRNA (siATF3) and control siRNA (siCT) were purchased from Sigma-Aldrich (MO, USA). Murine granzyme B siRNA (siGzmB) was purchased from Thermo Fisher Scientific (MA, USA). SiSPB9- cy5.5 was synthesized by Sigma-Aldrich. Cy5.5 was introduced to siSPB9 via phosphate linkage using phosphoramidite chemistry. The sequences of siSPB9, siSPB9-cy5.5, siATF3 and siCT are shown in Table 3 of FIG.7B.Atty. Docket No.: 24-018PCT

[0128] SERPINB9 KO cell lines were generated by using CRISPR technology. Cells were infected with the lentivirus packaged by Serpinb9-All-in-one lentiviral sgRNA-CRISPR-Cas9 plasmid encoding EGFP. The successfully knocked out cells were selected by cell sorting of EGFP+population followed by single clone culture and further confirmed through Western blot analysis for the lack of SERPINB9 proteins.

[0129] SERPINB9 control vector cell line was generated by using a control lentiviral vector with Cas9 coding sequence but without the specific guiding sequences (Lenti CRISPR plasmid without sgRNA sequence).

[0130] The luciferase-expressing cell lines were generated according to a published protocol. Brennan, T.V., et al., Generation of Luciferase-expressing Tumor Cell Lines, Bio Protoc, 8(8) (2018). Cells were infected with the lentivirus packaged from pLentipuro3 / TO / V5-GW / EGFP-Firefly Luciferase plasmid encoding EGFP. The luciferase- expressing cells were selected by cell sorting of the EGFP+population, followed by single clone culture, and further confirmed through flow cytometry analysis for EGFP expression.

[0131] RNA sequencing analysis. KPC-C2 gemcitabine resistant cells and wide type cells were harvested for RNA sequencing (RNA-seq), which was performed by the Health Sciences Sequencing Core at Children’s Hospital, University of Pittsburgh. Raw sequence data was analyzed by HISAT-Stringtie workflow as described in previously published protocol57, 58, 59to generate transcript level gene expression. Pertea M., et al., ,Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown, Nature Protocols, 11(9): 1650-1667 (2016); Wang Y., et al., Systematic identification of non-coding pharmacogenomic landscape in cancer, Nature communications, 9(1): 1-15 (2018); and Wan, Z., et al., Targeting metabotropic glutamate receptor 4 for cancer immunotherapy. Science Advances, 7(50): eabj4226. The Gene Set Enrichment Analysis (GSEA) was further performed based on the gene lists ranked by the log2FC between two groups. Yuan F., et al., Mirovascular permeability and interstitial penetration of sterically stabilized (stealth) liposomes in a human tumor xenograft, Cancer Research 54(13): 3352-3356 (1994).

[0132] qRT-PCR. To examine the mRNA level of different genes from RNA-seq results, murine pancreatic cancer cell lines (Panc02, KPC-C2, and KPC-C5; WT vs GEMR) were collected and subjected to qRT-PCR as detailed below. To examine the effect of GEM on the mRNA level of GranzymeB, SERPINB9 and ATF3, different pancreatic cancer cells were treated with various concentrations of GEM. Cells were collected 24 or 48 hours later and subjected to qRT-PCR as detailed below. cDNA was generated from the purified RNA extracted from the indicated cultured cells using High-Capacity cDNA Reverse TranscriptionAtty. Docket No.: 24-018PCT Kit (Thermo Fisher Scientific, MA, USA) according to the manufacturer’s instructions. qRT- PCR was performed using Power SYBR Green PCR Master Mix (Thermo Fisher Scientific, MA, USA) on a 7900HT Fast Realtime PCR System. Relative target mRNA levels were analyzed using delta-delta-Ct calculations. The primers are shown in Table 4 of FIG.7C.

[0133] Western blot assay. The total protein was extracted from the indicated cells by using radioimmunoprecipitation assay (RIPA) lysis buffer (Thermo Fisher Scientific, MA, USA) through gently shaking on ice for 30 min. After centrifugation at 12,500 g for 10 min, the supernatants were collected, and the concentrations of proteins were determined using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, MA, USA). The protein samples were mixed with 5× SDS loading buffer, denatured at 98OC for 10 min, loaded onto 10% SDS- polyacrylamide gel electrophoresis (PAGE) gel for electrophoresis, followed by transferring to PVDF membrane. The membranes were then blocked in 3% BSA dissolved in phosphate buffer containing 0.05% Tween-20 (PBST) for 1h at room temperature. Afterwards, the membranes were incubated with primary antibody in diluted buffer (3% BSA in PBST) with gentle agitation overnight at 4OC. After washing with PBST for three times, the membranes were subsequently incubated with horseradish peroxidase-conjugated secondary antibody at room temperature for 1 hour. The membranes were then washed three times with PBST before being incubated with Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, MA, USA). Signal was visualized by films with the AX700LE film processor (Alphatek) or iBright™ FL1500 Imaging System.

[0134] Granzyme B activity assay. Granzyme B activity was measured using the GranToxiLux PLUS kit. Cells subjected to different treatments were incubated with the Granzyme B substrate for 1 hour at 37OC. The cleaved Granzyme B substrate (GranToxiLux) exhibited an excitation peak at 488 nm and an emission peak at 520 nm. GranToxiLux fluorescence was measured by flow cytometry, and cell populations positive for GranToxiLux were considered to have active Granzyme B.

[0135] Construction of luciferase reporter assay plasmid. The UCSC genome browser identified a 1601 bp promoter region of human SERPINB9 gene, revealing 3 putative ATF3- binding motifs. The specific sequence of the SERPINB9 promoter region is shown in Table 5 of FIG.7D. Three plasmids each with a 5× repeated sequences of one of the three motifs (Supplementary Table 4) were constructed, respectively by using pGL3-Basic (Promega, CA, USA) as the backbone. The three DNA fragments with different 5 x repeated sequences were synthesized by AZENTA (MA, USA) with added restriction enzyme sites. NheI (Thermo Fisher Scientific, MA, USA) and HindIII (Thermo Fisher Scientific, MA, USA) restriction enzymesAtty. Docket No.: 24-018PCT were used to digest the pGL3-Basic plasmid and the synthesized fragments. The 3 DNA fragments were individually ligated with the backbone by using T4 DNA ligase (Invitrogen, s (Thermo Fisher Scientific, MA, USA). After plasmid extraction, the concentration was determined by NanoDrop, and the sequence was confirmed by Sanger sequence.

[0136] Similarly, a full length promoter sequence containing all three binding motifs was synthesized by Twist Bioscience (CA, USA) and cloned into pGL3-Basic. The mutated plasmid was constructed through site-directed mutagenesis. In brief, a pair of primers for mutagenesis were designed and synthesized (IDT, Coralville, IA, USA). This pair of primers was designed to include the front and back sequence of each binding motif but without the motif sequence. Then the PCR reaction with this pair of primers was performed using the previously generated full-length plasmid as a template. The PCR product was treated with Dpn1 enzyme to remove the template. Then, the PCR product was used for the tion was determined by NanoDrop. The Sanger sequencing confirmed the sequence.

[0137] Luciferase Reporter Assay. PANC-1, MIA PaCa-2 and Panc 02.03 cells were transfected with each of the plasmids described above using Lipofectamine 3000 (Invitrogen, MA, USA) for 48 hours. Subsequently, the cells were treated with gemcitabine for an additional 48 hours. Cell lysis was performed using the lysis buffer from the Pierce Firefly Luciferase Glow Assay Kit (Thermo Fisher Scientific, MA, USA). D-Luciferin was added to the cell lysate, and bioluminescence was detected using a luminometer, according to the manufacturer's instructions.

[0138] In vitro cytotoxicity assay. Cytotoxicity was evaluated by MTT assay with indicated cell lines. Cells were seeded in 96-well plates (attached 96 well tissue culture plate (CELLTREAT, MA)) at a density of 2×103received various treatments including GEM alone, GEM+siCT or GEM+siSPB9 combination at various GEM and siRNA concentrations for 48 hours. For the combination treatment, cells were transfected with siRNA using Lipofectamine RNAiMAX (Invitrogen, MA, USA) for 48 hours followed by drug treatment for another 48 hours. MTT assay was then performed on the cultured cancer cells. The absorbances of each well were measured at 590 nm. The cell viability was determined via the following formula: (ODtreated- ODblank) / (ODcontrol-ODblank) × 100 %.

[0139] Cell apoptosis study. Cells were digested following different treatments for 24 h and stained with Zombie NIR and BV421 anti-mouse Annexin V or FITC anti-mouse Annexin V forAtty. Docket No.: 24-018PCT cells.

[0140] Synthesis and characterization of PEG1K-COOH. A solution of PEG1K(5 g, 5 mmol) in chloroform (15 mL) was added to a dry flask containing succinic anhydride (2.5 g, 25 mmol). Subsequently, a solution of DMAP (3.05 g, 25 mmol) in 10 mL of chloroform was introduced into the reaction mixture. The mixture was then refluxed at 60 °C for 24 hours. After the reaction, the solvent was reduced to about half of the original volume using rotary evaporation. The product was recrystallized from cold ether, filtered, and the precipitate was collected. The synthesis route for PEG1K-COOH is presented in FIG.2A.

[0141] Synthesis and characterization of PPO polymer library. To a stirred solution of PEG1K- COOH and oleic acid (OA) in 30 mL DMSO, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added. The mixture was stirred for 2 hours, after which Poly- -L-lysine HCl (PLL) and triethylamine (TEA) were added. Twelve hours later,the reaction mixture was purified by dialysis using a dialysis membrane (MWCO 3500 Da) in a DMSO / H2O (4:1) mixture for 24 hours, followed by water for an additional 24 hours. The product (PEG-PLL-Oleic acid, PPO) was lyophilized and stored at 4OC. The synthesis route and NMR data are demonstrated in FIG.2A.

[0142] To synthesize PPO polymers conjugated with various amino acids, tert- butyloxycarbonyl (BOC)-protected amino acids (histidine, lysine, or arginine) were dissolved in DMSO. The activation of the amino acid carboxyl groups was achieved by adding 1-ethyl- 3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The reaction mixture was stirred for 2 hours to promote conjugation. Next, the non-amino acid- decorated PPO polymer and triethylamine (TEA) were introduced, and the mixture was stirred for an additional 12 hours. The reaction product was purified by dialysis using a dialysis membrane (MWCO 3500 Da). Dialysis was performed in a DMSO / H2O (4:1) mixture for 24 hours, followed by 24 hours in pure water. The final product was lyophilized and stored at 4OC.

[0143] Synthesis and characterization of PPOGEM polymer.4,4'-Dithiodibutyric acid (2.5 g, 10 mmol) and 10 mL of acyl chloride were combined in a 50 mL flask and refluxed at 65OC for 4 hours. After completion, the excess acyl chloride was evaporated, and the inner anhydride was obtained without further purification. Subsequently, PPO (500 mg), TEA (0.9 mL, 6.42 mmol), and 30 mL DMSO were added to the flask. The mixture was stirred and reacted at 50°C for 24 hours. Purification was conducted by dialysis using a dialysis membrane (MWCO 3500 Da) with a DMSO / H2O mixture (4:1) for 24 h, followed by water forAtty. Docket No.: 24-018PCT another 24 h. The compound PLL(COOH)-OA-PEG was then lyophilized and stored at 4OC. NMR results confirmed synthesis of PLL(COOH)-OA-PEG.

[0144] For the subsequent reaction, PLL(COOH)-OA-PEG (300 mg), hydroxybenzotriazole (HOBt) (60 mg, 0.44 mmol), EDC (150 mg, 0.98 mmol), gemcitabine (560 mg, 2.1 mmol), and TEA (0.45 mL, 3.2 mmol) were dissolved in 30 mL of DMSO. The mixture was then reacted at 50°C for 72 hours. Following the reaction, the product underwent purification by dialysis using a dialysis membrane (MWCO 3500 Da) with a DMSO / H2O mixture (4:1) for 24 hours and then water for another 24 hours. The final product was lyophilized and stored at 4OC. The synthesis routes are illustrated in FIG.2A. The content of gemcitabine in the polymer was determined by UV absorbance.

[0145] Synthesis and characterization of Psugar and PCytidine polymers. The control polymers Psugar and PCytidine were synthesized using a method similar to that for the PPOGEM polymer. PLL(COOH)-OA-PEG (300 mg), hydroxybenzotriazole (HOBt) (60 mg, 0.44 mmol), EDC (150 mg, 0.98 mmol), D-Glucosamine sulfate (600 mg, 2.1 mmol), and TEA (0.45 mL, 3.2 mmol) were dissolved in 30 mL of DMSO to synthesize Psugar polymer. PLL(COOH)- OA-PEG (300 mg), hydroxybenzotriazole (HOBt) (60 mg, 0.44 mmol), EDC (150 mg, 0.98 mmol), cytidine (520 mg, 2.1 mmol), and TEA (0.45 mL, 3.2 mmol) were dissolved in 30 mL of DMSO to obtain PCytidine polymer. Both reactions were stirred at 50 OC for 72 hours. Thesubsequent dialysis and lyophilization processes followed the methods described above. The final compound structures are demonstrated in FIG.2B. The conjugation rates of cytidine and glucosamine were determined by UV absorbance.

[0146] Fabrication and physicochemical characterization of nanoparticles. To prepare the POEM / siRNA or Psugar / PPO / siRNA NPs, Psugar, PPOGEM and PPO were dissolved in DMSO, separately (40 mg / mL). SiRNA was dissolved in deionized water at a concentration of 25 different centrifuged using an Amicon Ultra centrifugal filter device at 15,000 g for 15 minutes. Deionized water was added, and the centrifugation was repeated at 15,000 g for 15 minutes to remove residual DMSO. The final nanoparticles in the remaining liquid in the tube were collected for subsequent studies.

[0147] The formation of stable nanoparticle complexes was confirmed using gel-retardation assay. The particle size was measured via Zetasizer (Malvern Panalytical, UK) from three batches of formulation. The critical micelle concentration (CMC) of POEM / siRNA and Psugar / PPO / siRNA were determined through measuring the light scattering intensity. ToAtty. Docket No.: 24-018PCT assess the resistance of POEM / siRNA NPs against nuclease-mediated degradation, the NPs were incubated with RNase (50 U / mL) (NEB, MA, USA) at 37OC for one hour. Subsequently, the NPs were disrupted by 0.05% SDS, and the integrity of siRNA was evaluated by electrophoresis. Free siRNA served as a control.

[0148] Molecular simulation systems. The chemical structures of PPO, PPOGEM, Psugar and PCytidine are all composed of different numbers of three residue types, A, B and C as illustrated in FIG.2C. For PPOGEM, Psugar and PCytidine, the residue composition is x=24 for A, y=3 for B, z=3 for C, while for PPO, the residue numbers of A, B, and C are present in a ratio of 23:5:2. The residue topologies of all residues were generated using the Antechamber software. Wang J., et al., Automatic atom type and bond type perception in molecular mechanical calculations. J Mol Graph Model, 25(2): 247-260 (2006). Specifically, even though the numbers of different residues in the polymer have been determined, the precise arrangement of these residues within the polymeric chain remains unknown. To maximize the separation of the same type of residues, the 1D sequence of PPOGEM, Psugar and PCytidine is depicted as the follows: A-A-A-A-A-A-A-A-B-A-A-A-A-A-A-A-A-B-A-A-A-A-A-A-A-A- B-C-C-C, while the sequence of PPO is: A-A-A-A-A-B-A-A-A-A-A-B-A-A-A-A-B-A-A-A-A-A-B-A-A- A-A-B-C-C. Next, we utilized the NAB program in the Amber Tools to generate the siRNA structure based on the human siRNA sequence listed in Table 3. Finally, POEM nanoparticle system and the control nanoparticle (Psugar / PPO / siRNA, PCytidine / PPO / siRNA) were constructed with PPOGEM (Psugar or PCytidine), PPO, and siRNA in a ratio of 33:9:1. To construct the topology of the simulation system, the GAFF2 force field was utilized to model lipids, while the OL3 and OL15 force fields were used to model RNA and DNA, respectively. Wang J., et al., Development and testing of a general amber force field, J Comput Chem, 25(9): 1157-1174 (2004); Wang, J., et al., Development and testing of a general amber force field, J Comput Chem, 25(9): 1157-1174 (2004).

[0149] Molecular dynamics simulations. The micelle-like structure of POEM loaded with siRNA was formed through a series of molecular dynamics (MD) simulations using the PMEMD.cuda program in the AMBER 22 software package. Salomon-Ferrer, R., et al., Routine Microsecond Molecular Dynamics Simulations with AMBER on GPUs.2. Explicit Solvent Particle Mesh Ewald, J Chem Theory Comput, 9(9): 3878-3888 (2013); Gotz, A.W., et al., Routine Microsecond Molecular Dynamics Simulations with AMBER on GPUs.1. Generalized Born, J Chem Theory Comput, 8(5): 1542-1555 (2012). Initial system relaxation was achieved by a 1000-step conjugated-gradient energy minimization on the entire POEM / siRNA structure. Next, constant-pressure MD simulation was performed forAtty. Docket No.: 24-018PCT approximately 3.5 nanoseconds (ns), facilitating system shrinking and stabilization, with an objective of achieving a density close to 1. Subsequently, a two-stage MD simulation protocol was applied to the shrunk system, with a 600 ns MD simulation to continue to stabilize the system in stage one. Upon the completion of stage one, the last snapshot was solvated in a rectangular water box filled in with TIP3P water and 0.15 M Na+ and Cl ions.The whole system was neutralized and the simulation box has a dimension of 220 220 220Å. Then a 300 ns MD simulation was conducted with period boundary condition enforced in the second stage. During the whole simulation phase, position restraints were applied to the siRNA with a force constant of 100 kcal / mol / Å2to prevent its distortion but the other atoms were allowed to move freely. The snapshot with the smallest root-mean-square deviation (RMSD) from the average structure of snapshots collected after the system reached equilibrium (the last 150 ns) was selected as the representative conformation. A similar protocol was applied for the PCytidine / siRNA and Psugar / siRNA system.

[0150] CryoEM methods. Samples were initially examined using negative-stain electron -discharged continuous carbon copper grid and stained with 1% uranyl acetate solution. The grids were then inserted into a Tecnai TF20 electron microscope (Thermo Fisher Scientific, MA, USA), equipped with a field emission gun and images captured using an XF416 CMOS camera (TVIPS GmbH, Gilching, Germany) to assess nanoparticle uniformity and concentration.

[0151] For cryo- -flat CF-2 / 1-3CU-T grid (Protochips, NC, USA) that had been glow discharged at 25mA for 30 seconds using an Emitech KX100 glow discharger. The grids were then processed in a Thermo Fisher Vitrobot Mk 4, set to a relative humidity of 95%. They were blotted for 3 seconds with a force setting of 4 and subsequently plunged into a 40 / 60 mixture of liquid ethane / propane that was precooled in a liquid nitrogen bath. The grids were then placed onto a Gatan 9103-grid cryoholder (Gatan, Inc, CA, USA) and inserted into the TF20 aperture. Cryo-electron micrographs were collected at a nominal magnification of 150,000× on the TVIPS XF416 CMOS camera, with a pixel size of 0.74 Å at the sample. Low dose methods were employed to minimize electron beam damage, and images were acquired using TVIPS EMplified software in movie mode to correct for drift.

[0152] Release kinetics of gemcitabine. The kinetics of gemcitabine release from POEM nanoparticles under different conditions was performed by dialysis method. In brief, 2 mL ofAtty. Docket No.: 24-018PCT POEM nanoparticles (PPOGEM / PPO / siSPB9; 5 mg / 0.5 mg / 0.1 mg) PBS solution under different conditions (10% FBS, 10% murine serum, 10 mM GSH and 100 mM GSH) were placed in a dialysis bag (MWCO 3.5 kDa) and immersed in 40 mL of 0.1 M PBS solution containing 0.5% (w / v) Tween 80. The experiment was performed in an incubation shaker at 37ºC with gentle shaking mL medium outside the dialysis bag were withdrawn while same amount of fresh dialysis solution was added for replenishment. The concentration of gemcitabine was measured by HPLC-UV according to a previously published method. Sun, J., et al., High Loading of Hydrophobic and Hydrophilic Agents via Small Immunostimulatory Carrier for Enhanced Tumor Penetration and Combinational Therapy, Theranostics, 10(3): 1136-1150 (2020).

[0153] In vitro knock down efficiency. To test the in vitro knockdown efficiency of POEM NPs, KPC-C2-Luc and Panc02-Luc cells were treated with different formulations of POEM NPs with varying ratios of PPOGEM and PPO. After 48 hours treatment, cell lysates were collected and analyzed by measuring the bioluminescent intensity. Commercial Lipofectamine RNAiMAX was used as a control.

[0154] Cellular uptake. For the cellular uptake study, KPC C2 WT cells were pretreated with chlorpromazine, anti-ITGA5, or IgG for 1 hour, in medium depleted of FBS. The cells were then incubated with POEM / siRNA / DiD dye NPs for 1 hour. After incubation, the cells were digested, and the fluorescence intensity of DiD dye was measured.

[0155] Transcytosis study. KPC C2 cells were seeded into the upper chamber of a transwell and cultured for 3 days until a confluent monolayer was formed. Prior to adding POEM / siRNA / DiD NPs to the upper chamber, the cells were pretreated with anti-ITGA5 or IgG for 1 hour. After 8 hours of incubation, the medium in the lower chamber was collected, followed by measurement of its fluorescence intensity.

[0156] Murine tumor models. To establish the subcutaneous tumor models, 5×105Panc02, KPC-C2, KPC-C2 SERPINB9 KO, KPC-C2 control vector cells or KPC-C2 GEMR cells were inoculated into the right flank of C57BL / 6 mice (4-6 weeks). The Panc02, KPC-C2 or KPC-C2 GEMR tumor-bearing mice received different treatments when the tumor size reached the indicated volume. Tumor growth in KPC-C2 SERPINB9 KO or KPC-C2 control vector tumor- bearing mice was monitored for 20 days, followed by analysis of tumor-infiltrating immune cells as detailed in “Analysis of tumor-infiltrating immune cells” section.

[0157] The pancreatic orthotopic tumor models were established according to a previous publication. Cai, X., et al., Inhibition of the SLC35B2-TPST2 Axis of Tyrosine Sulfation Attenuates the Growth and Metastasis of Pancreatic Ductal Adenocarcinom, Cell MolAtty. Docket No.: 24-018PCT Gastroenterol Hepatol, 16(3): 473-495 (2023). Briefly, anesthetized C57BL / 6 mice (8-10 weeks) were shaved, and the surgical area on the left flank was disinfected. The pancreas was exposed through a small incision (~1 cm) on the left flank. Fifty (50) μL Panc02-Luc or KPC-C2-Luc single cell suspension (1×105per mouse) was injected cell into the tail of the pancreas using 28-gauge hypodermic needles. The needle was slowly removed after the injection of cell suspension. After injection of tumor cells, the mice were maintained at the heating pad and observed until complete recovery. The development of pancreatic cancer was monitored by bioluminescence radiance intensity after the mice were injected with D- Luciferin (GoldBio, 150 mg / kg) intraperitoneally.

[0158] Whole-body NIRF imaging and ex vivo imaging. For tissue biodistribution study, mice bearing subcutaneous (~300 mm3) or orthotopic (~10 days after inoculation) pancreatic tumors were intravenously injected with free siSPB9-cy5.5 and POEM / siSPB9-cy5.5 NPs with different PPOGEM / siRNA ratios, respectively. The mice were sacrificed at 24 hours after injection. Tumor and major organs including heart, liver, spleen, lung, and kidney were collected and imaged by IVIS 200 system (Perkin Elmer, MA, USA) at a constant 1 s exposure time with excitation at 679 nm and emission at 702 nm. For the study of PK in blood, blood was collected in EDTA-containing tubes at 10 min, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 48 h and 72 h timepoints and plasma samples were prepared by centrifugation at 21,000 g for 10 min and imaged by the IVIS 200 system.

[0159] Blood pharmacokinetics and Biodistribution of siSPB9. Groups of 3 C57BL / 6 mice (4- 6 weeks) bearing subcutaneous KPC-C2 tumors (~300 mm3) received tail vein injection of POEM / siSPB9-cy5.5 NPs or free siSPB9-cy5.5 at a dose of 1 mg / kg for siRNA. At different timepoints (10 min, 0.5 h, 1 h, 4 h, 24 h and 48 h) post injection, blood was collected in the tubes containing EDTA. The amount of siRNA in the plasma was quantified by qRT-PCR as detailed below. The PK parameters were obtained by fitting the blood siRNA concentration versus time using a one-compartment model. Hearts, livers, spleens, lungs, kidneys and tumors were collected at 24 h and homogenized. SiRNA in the tissues was quantified through qRT-PCR.

[0160] SiSPB9 quantification by qRT-PCR. The siSPB9 was measured by qRT-PCR as previously published. Chen, Y., et al., Targeting Xkr8 via nanoparticle-mediated in situ co- delivery of siRNA and chemotherapy drugs for cancer immunochemotherapy, Nat Nanotechnol, 18(2): 193-204 (2023). Briefly, the plasma, major organs and tumors collected were homogenized in . was added to the homogenized tissues. After 5 min of incubation, the samples were centrifuged at 21,000 g for 15 min. TheAtty. Docket No.: 24-018PCT upper aqueous phase was used for qRT-PCR. A standard curve was generated by spiking a known amount of siSPB9-cy5.5 (0- / mL) in the tissues obtained from non-treatedcontrol animals and used to calculate the amount of siRNA in the samples. A SuperScript III reverse transcription kit (Invitrogen, NY, USA) was used to convert siRNA into cDNA. For reverse transcription, 0.1 M , RNAse OUT (Invitrogen, 96-well plate. GS primer and template were premixed, heated at 85 °C for 2 min, snap-chilled on ice, and RT premix was added. The 10 mixture was incubated at 50 °C for 30 min, 85 °C for 5 min, cooled to r.t., and diluted 10- nscription, quadruplicate measurements of-well optical PCR plate using a 7900 HT PCR instrument (Applied Biosystems, MA, USA). SYBR green PCR mix Biosystems, MA, USA - The primer sequences are shown in Table 6 of FIG.7E.

[0161] In vitro T cell uptake. Spleen and KPC-C2 tumor tissues were harvested from C57BL / 6 mice, followed by the preparation of single-cell suspensions. CD4+and CD8+T cellswere isolated using CD4 / CD8 MicroBeads (Miltenyi Biotec, North Rhine-Westphalia, Germany). Cells were cultured in RPMI-1640 medium and stimulated by anti-CD3 (Invitrogen, NY, USA) and anti-CD28 (Invitrogen, NY, USA). Subsequently, the cells were treated with PBS, free siSPB9-cy5.5, POEM / siSPB9-cy5.5, or Lipofectamine RNAiMAX / siSPB9- cy5.5 as a control for 4 hours. Cellular uptake of siRNA was examined by flow cytometry.

[0162] Analysis of in vivo cellular uptake. KPC-C2 tumor-bearing mice were treated with POEM / siSPB9-cy5.5 NPs intravenously. Tumors were collected and prepared into single cell suspensions, which were then stained for antibody (PE-Cyanine7 anti-mouse CD45, BUV737 anti-mouse CD4, BV480 anti-mouse CD8, eF405 anti-mouse CD31, FITC anti-mouse CD140a, BV711 anti-mouse Gr1 and PE anti-mouse CD11b; dilution: 1 / 200 for antibody) for flow cytometry analysis. Chen, Y., et al., An immunostimulatory dual-functional nanocarrier that improves cancer immunochemotherapy, Nat Commun, 7: 13443 (2016).

[0163] In vivo gene knockdown. POEM NPs loaded with luciferase siRNA (siLuc) or control siRNA (siCT) were intravenously injected into KPC-C2-Luc tumor-bearing mice at a dose of 1 mg / kg. The efficiency of gene knockdown was measured three times by whole body bioluminescence imaging on the 2nd day following the 1st, 2nd, and 3rd injection of the NPsAtty. Docket No.: 24-018PCT once every 3 days, respectively. The exposure time was set at 60 s for every experiment. Mice were anesthetized according to protocol prior to imaging.

[0164] In vivo SPB9 knockdown. When the tumor volume reached ~50 mm3, KPC-C2 tumor- bearing mice were randomly assigned to different groups (n = 5). They were intravenously administered with PBS, PPO / PCytidine / siSPB9 (POC / siSPB9) NPs, POEM / siCT NPs or POEM / siSPB9 NPs three times at an interval of 5 days (on day 5, 10 and 15). The doses for PPOGEM and siRNA were 50 mg / kg and 1 mg / kg, respectively. Twenty-four hours after the final injection, tumors were collected and subjected to qRT-PCR of SPB9 expression as detailed above.

[0165] Therapeutic treatment. Murine pancreatic cancer (KPC-C2 and KPC-C2 GEMR) models were established for in vivo antitumor efficacy study. When the tumor volume reached ~ 50 mm3, mice were treated with PBS, POC / siSPB9, POEM / siCT, and POEM / siSPB9 respectively (n = 5), every three days for a total of 5 times (PCytidine: 50 mg / kg; PPOGEM: 50 mg / kg; siSPB9: 1 mg / kg) through intravenous injection. Tumor volumes and mouse body weights were monitored every three days following the initiation of the treatment. The tumor volumes (V) were calculated by the formula: (Length × Width2) / 2. After completing the in vivo experiment, tumor tissues and major organs were harvested for histochemical staining. In addition, blood sample was collected, and plasma was isolated after centrifugation at 21,000 g for 10 min. Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and creatinine levels in plasma were measured as indicators of hepatic and renal function.

[0166] For orthotopic pancreatic cancer model, the KPC-C2-Luc tumor-bearing mice were treated intravenously with PBS, POC / siSPB9, POEM / siCT, and POEM / siSPB9 respectively, at dosage described above. The treatments were conducted every three days for a total of five times. The whole-body tumor burden was monitored and quantified by measuring the luminous intensity using LivingImagging 4.1.0 software.

[0167] To test the therapeutic effect of the combinational therapy of POEM / siSPB9 NPs with anti-PD-1, the treatment was started when the KPC-C2 GEMR tumors reached ~50 mm3in size. Mice were treated with anti-PD-1 (clone RMP1-14, Bio X Cell) alone, POEM / siSPB9 NPs alone or the combination once every 5 days three times. Anti-PD-1 was administered at 10 mg / kg, while POEM / siSPB9 NPs were given intravenously at dosage described above. Mice were followed until death or were killed if the tumor size reached 2,000 mm3, the maximal tumor size permitted by the Animal Use and Care Administrative Advisory Committee at the University of Pittsburgh.Atty. Docket No.: 24-018PCT

[0168] Histopathological analysis. Tumors and major organs including heart, liver, spleen, lung and kidney were excised and fixed in 10% formalin at the end of the in vivo therapy study, followed by embedment in paraffin. The paraffin embedded samples were sectioned then processed for hematoxylin and eosin staining (H&E) for histological evaluation according to published protocols. Huang, H., et al., A novel immunochemotherapy based on targeting of cyclooxygenase and induction of immunogenic cell death, Biomaterials, 270: 120708 (2021). The images were observed under a BZ-X710 Fluorescence Microscope (Keyence, Itasca, IL, USA).

[0169] Analysis of tumor-infiltrating immune cells. Flow cytometry was performed with LSRII (BD Biosciences) and Aurora (Cytek Biosciences) instruments and analyzed by FlowJo (BD Biosciences). Spleens and tumors were collected 1 day after the last treatment. Single- cell suspensions were prepared as previously described. Briefly, tumors were dissected and transferred into RPMI-1640 medium. Tumors were disrupted mechanically using scissors, digested with a mixture of deoxyribonuclease I (0.3 mg / mL, Sigma-Aldrich) and TL Liberase (0.25 mg / mL, Roche) in serum-free RPMI-1640 at 37 °C for 30 min, and dispersed through a was performed using a Zombie NIR Fixable Viability Kit (BioLegend, dilution: 1 / 1,000) at 4OCfor 30 min in PBS. Surface staining was performed at 4OC for 30 min in FACS staining buffer (1× PBS / 5% FBS / 0.5% sodium azide) containing designated antibody cocktails (PerCP anti- mouse CD45 antibody, Brilliant Violet 737 anti-mouse CD4 antibody, Brilliant Violet 480 anti- mouse CD8 antibody, Brilliant Violet 615 anti-mouse PD-1 antibody, APC anti-mouse CD11b antibody, Brilliant Violet 510 anti-mouse Gr-1 antibody, APC / Cyanine7 anti-mouse F4 / 80 antibody, Pacific Blue anti-mouse MHC II antibody, Brilliant Violet 737 anti-mouse CD80 antibody, PE anti-mouse CD163 antibody, FITC anti-mouse CD69; dilution: 1 / 200 for all antibodies). Cells were fixed and permeabilized using the BD Cytofix / Cytoperm kit, following the manufacturer’s instructions. For intracellular cytokine staining (PE-Cyanine7 anti-mouse IFN- and eF450 anti-mouse GzmB antibody; dilution: 1 / 200 for antibody), cellswere stimulated with phorbol 12-myristate-13-acetate (100 ng / mL) and ionomycin (500 ng / mL) for 6 hours in the presence of Monensin. Cells were fixed / permeabilized using the BD Cytofix / Cytoperm kit before cell staining.

[0170] Statistics and reproducibility. Statistical analysis was performed with two-tailed Student’s t-test for comparison between two groups, one-way analysis of variance (ANOVA) for comparison between multiple groups, and log-rank (Mantel-Cox) test for survival analysisAtty. Docket No.: 24-018PCT as indicated in figure legend. Results were considered statistically significant if P < 0.05. Prism 10.1.0 (GraphPad Software) was used for data analysis and graph plotting. All experiments were repeated at least three times with similar results. No data were excluded from the analyses.

[0171] The foregoing description and accompanying drawings set forth a number of representative embodiments at the present time. Various modifications, additions and alternative designs will, of course, become apparent to those skilled in the art in light of the foregoing teachings without departing from the scope hereof, which is indicated by the following claims rather than by the foregoing description. All changes and variations that fall within the meaning and range of equivalency of the claims are to be embraced within their scope.

Claims

Atty. Docket No.: 24-018PCT WHAT IS CLAIMED IS:

1. A composition, comprising: a plurality of molecules of a first polymer, each molecule of the first polymer comprising a first backbone comprising repeat units comprising a pendant amine group, a plurality of first pendant groups comprising a hydrophilic polymer being conjugated to the first backbone via the pendant amine groups thereof, a plurality of second pendant groups comprising a hydrophobic group being conjugated to the first backbone via the pendant amine groups thereof, and a plurality of third pendant groups comprising a group selected from a nucleoside and a nucleoside analog being conjugated to the first backbone via the pendant amine groups thereof, a plurality of molecules of a second polymer, each molecule of the second polymer being positively charged and comprising a second backbone comprising the repeat units, a plurality of fourth pendant groups comprising the hydrophilic polymer being conjugated to the second backbone via the pendant amine groups thereof, a plurality of fifth pendant groups comprising the hydrophobic group being conjugated to the second backbone via the amine groups thereof, and a plurality of repeat units wherein the pendant amine groups are groups that are positively charged in vivo, and molecules of a nucleic acid, wherein the plurality of molecules of the first polymer, the pluralities of molecules of the second polymer, and the molecules of the nucleic acid assemble into nanostructures in an aqueous medium.

2. The composition of claim 1 wherein the pendant amine groups that are positively charged in vivo have the formula -NHX, wherein X is selected from the group consisting of :Atty. Docket No.: 24-018PCT3. The composition of claim 2 wherein X is H.

4. The composition of claim 1 wherein the hydrophobic group is selected from the group consisting of a lipid, a poly(methyl acrylate), a polyethylene, a polystyrene, a polyisobutane, a polyester, a polypeptide, and vorinostat.

5. The composition of claim 4 wherein the hydrophobic group is a lipid, wherein the lipid is optionally selected from the group consisting of an oleic acid, stearic acid, palmitic acid, palmitoleic acid, myristic acid, lauric acid, decanoic acid, cholesterol, Vitamin A, Vitamin D, Vitamin E, ceramides, and Farnesyl Thiosalicylic Acid.

6. The composition of any one of claims 1 through 5 wherein each of the first polymer and the second polymer is prepared from a precursor polymer comprising a backbone comprising one of the repeat units selected from the group consisting of:Atty. Docket No.: 24-018PCTAtty. Docket No.: 24-018PCT.

7. The composition of claim 6 wherein the second polymer is formed by reacting the precursor polymer with a plurality of first molecules comprising the hydrophilic polymer and a functional group reactive with primary amine groups of the precursor polymer and with a plurality of second molecules comprising the hydrophobic group and a functional group reactive with primary amine groups of the precursor polymer in a predetermined ratio of the first molecules to the second molecules and in a predetermined molar amount such that primary amine groups of the precursor polymer remain unreacted, and wherein the first polymer is formed by reaction of a portion of the second polymer with a plurality of molecules comprising the nucleoside or nucleoside analog and a functional group reactive with primary amine groups of the second polymer.

8. The composition of claim 1 wherein the hydrophilic polymer is selected from the group consisting of: a polyalkylene oxide, a polyvinylalcohol, a polyacrylic acid, a polyacrylamide, a polyoxazoline, a polysaccharide, and a polypeptide.

9. The composition of claim 8 wherein the hydrophilic polymer is a polyalkylene oxide.

10. The composition of claim 9 wherein the hydrophilic polymer is a polyethylene glycol.

11. The composition of claim 1 wherein the hydrophilic polymer has a molecular weight in the range from 100 Da to 5 kDa.

12. The composition of claim 1 wherein the nucleoside analog is a chemotherapeutic agent.

13. The composition of claim 1 wherein nucleoside analog is selected from the group consisting of an analog of cytidine, an analog of adenine, and an analog of pyrimidine.

14. The composition of claim 1 wherein the nucleoside analog is selected from the group consisting of an analog of a pyrimidine nucleoside and an analog of a purine nucleoside.

15. The composition of claim 1 wherein the nucleoside analog is selected from the group consisting of gemcitabine, azacitidine, cytarabine, decitabine, RX-3117, trifluridine, 5- fluorouracil, capecitabine, tegafur, carmofur, floxuridine, DMT-OMe-rC(Ac), uridine, 2 -eeoxy-2 -fluoroadenosine, tezacitabine, LY2334737, 2'-deoxy-2'-fluoro-5-iodouridine, 1-(2-Atty. Docket No.: 24-018PCT deoxy-2-fluoro-beta-D-arabinofuranosyl)uracil, tipiracil, idoxuridine, zidovudine, eniluracil, sorivudine, cytidine, thymidine; adenosine, 8-chloroadenosine, clofarabine, n6- benzyladenosine, clofarabine, fludarabine phosphate, cladribine, fludarabine, nelarabine, pentostatin, ganciclovir, forodesine hydrochloride, viltolarsen, cordycepin, vidarabine, didanosine, tenofovir, adefovir, 6-thioguanine, mercaptopurine, azathioprine, forodesine, ganciclovir, acyclovir, valacyclovir, penciclovir, famciclovir, ribavirin and tiazofurin, guanosine.

16. The composition of claim 1 wherein the nucleic acid is selected from the group of mRNA and siRNA.

17. The composition of claim 16 wherein the nucleic acid is siRNA.

18. The composition of claim 17 wherein the siRNA is siSPB9, siNEBL, siiRhom1, siCD200, siCT, or siEGFR.

19. The composition of claim 1 wherein the nucleoside analog is a gemcitabine.

20. The composition of claim 1 wherein the nucleoside analog is conjugated to the pendant amine group via a linking group comprising a moiety that is labile in vivo.

21. The composition of claim 20 wherein the moiety that is labile in vivo comprises at least one of an ester bond, an orthoester bond, a thioether-ester bond, an anhydride bond, an amid bond, a carbonate bond, a disulfide bond, a hydrazone bond, a cic-acotinyl bond, an acetal bond, a carboxydimethyl maleate bond, an imine bond, an oxime bond, a silyl ether bond, a ketal bond, a thioketal bond and a protease cleavable peptide.

22. The composition of claim 21 wherein the moiety that is labile in vivo comprises at least one of an ester bond and a disulfide bond.

23. The composition of claim 21 wherein the moiety that is labile in vivo comprises a disulfide bond.

24. The composition of claim 1 the first polymer has the formula:and the second polymer has the formula:Atty. Docket No.: 24-018PCTwherein R1is a group comprising a group selected from the group of the nucleoside and the nucleoside analog, R2is a group comprising the hydrophilic polymer, R3is a group comprising the hydrophobic group, and x is in the range of 30 to 100%, y is in the range of 1 to 50%, and z is in the range of 1 to 20%, and x1is in the range of 30 to 100%, y1is in the range of 1 to 50%, and z1is in the range of 3 to 20%, wherein X is selected from the croup consisting of:.

25. The composition of claim 24 wherein X is H.

26. The composition of claim 24 wherein the nucleic acid is siRNA and the mass ratio of first polymer to siRNA is within the range of 10:1 to 100:1, and optionally in the range of 20:1 to 80:

1.

27. The composition of claim 24 wherein the nucleic acid is siRNA and the N:P ratio of the composition is in the range of 2:1 to 15:

1.

28. The composition of claim 1 wherein the nanostructures are micelles.

29. The composition of claim 28 wherein the nanostructures have an average diameter in the range of 30 to 200 nm.

30. The composition of claim 28 wherein the nanostructures have an average diameter in the range of 30 to 100 nm.Atty. Docket No.: 24-018PCT31. The composition of claim 1 further comprising a therapeutic agent loaded into thenanostructures during self-assembly thereof.

32. The composition of claim 31 wherein the therapeutic agent is selected from the groupconsisting of a hydrophobic therapeutic agent and a hydrophilic therapeutic agent, wherein the hydrophilic therapeutic agent is conjugated with a lipid before being loaded into the nanostructures during self-assembly thereof.

33. The composition of claim 32 wherein the therapeutic agent is a chemotherapy agent.

34. The composition of claim 31 wherein the therapeutic agent is a small molecule therapeuticcompound.

35. The composition of claim 34 wherein the therapeutic agent has a molecular weight below 1kDa.

36. The composition of claim 32 wherein the therapeutic agent is selected from the groupconsisting of paclitaxel, doxorubicin, docetaxel, gefitinib, imatinib, dasatinib, curcumin, camptothecin, etoposide, edelfosine, vincristine, temsirolimus, carmustine, cisplatin, oxaliplatin, or a chemotherapeutically active derivative thereof.

37. The composition of claim 31 wherein the therapeutic agent is an immunotherapeutic agent.

38. The composition of claim 37 wherein the therapeutic agent is an anti-PD-1 antibody.

39. A polymer prepared from a precursor polymer comprising one of repeat units selected fromthe group consisting of:Atty. Docket No.: 24-018PCTwherein R is selected from the group consisting of:,Atty. Docket No.: 24-018PCTwherein the precursor polymer is reacted with a plurality of first molecules comprising a hydrophilic polymer and a functional group reactive with primary amine groups of the precursor polymer and with a plurality of second molecules comprising a hydrophobic group and a functional group reactive with primary amine groups of the precursor polymer in a predetermined ratio of the first molecules to the second molecules and in a predetermined molar amount such that primary amine groups of the precursor polymer remain unreacted.

40. A polymer prepared from a precursor polymer comprising one of repeat units selected fromthe group consisting of:Atty. Docket No.: 24-018PCTwherein R is selected from the group consisting of : ,, wherein the precursor polymer is reacted with a first plurality of molecules comprising a hydrophilic polymer and a functional group reactive with primary amine groups of the precursor polymer, with a second plurality of molecules comprising a hydrophobic group and a functional group reactive with primary amine groups of the precursor polymer, and with a third plurality of molecules comprising a group selected from the group consisting ofAtty. Docket No.: 24-018PCT a nucleoside and a nucleoside analog and a functional group reactive with primary amine groups of the polymer.

41. A method of delivering, a nucleic acid to a patient, comprising, administering a plurality of nanostructures compositions as set forth in claim 1.

42. A method of providing treatment to a patient, comprising: administering a therapeutic agent, wherein the therapeutic agent causes upregulation of SPB9, and administering siSPB9.

43. The method of claim 42 wherein the therapeutic agent is a chemotherapeutic agent.

44. The method of claim 42 wherein the therapeutic agent is a nucleoside or a nucleoside analog.

45. The method of claim 42 wherein the therapeutic agent and SPB9 are co-administered via nanostructures, formed by mixing in an aqueous medium: a plurality of molecules of a first polymer, each molecule of the first polymer comprising a first backbone comprising repeat units comprising a pendant amine group, a plurality of first pendant groups comprising a hydrophilic polymer being conjugated to the first backbone via the pendant amine groups thereof, a plurality of second pendant groups comprising a hydrophobic group being conjugated to the first backbone via the pendant amine groups thereof, and a plurality of third pendant groups comprising a group selected from the group consisting of a nucleoside and a nucleoside analog being conjugated to the first backbone via the pendant amine groups thereof, a plurality of molecules of a second polymer, each molecule of the second polymer being positively charged and comprising a second backbone comprising the repeat units, a plurality of fourth pendant groups comprising the hydrophilic polymer being conjugated to the second backbone via the pendant amine groups thereof, a plurality of fifth pendant groups comprising the hydrophobic group being conjugated to the second backbone via the amine groups thereof, and a plurality of repeat units wherein the pendant amine groups are groups that are positively charged in vivo, and molecules of siSPB9, wherein the plurality of molecules of the first polymer, the pluralities of molecules of the second polymer, and the molecules of siSPB9 assemble into the nanostructures in the aqueous medium.

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