Lipid nanoparticle delivery for reducing PD-l1 expression in cancer

Abstract

The disclosure provides materials and methods of inducing an immune response to a tumor. The method comprises administering to a subject in need thereof a liposome nanoparticle comprising (I) nucleic acides that inhibit the expression of PD-L1 and (II) tumor RNA.

Description

Docket No. 32917 / 70821LIPID NANOPARTICLE DELIVERY FOR REDUCING PD-L1 EXPRESSION IN CANCERFIELD

[0001] The disclosure relates to lipid nanoparticles comprising an inhibitor of PD-L1 expression and methods of use.GOVERNMENT SUPPORT CLAUSE

[0002] This invention was made with government support under Grant No. R37 CA251978, awarded by the National Institutes of Health. The government has certain rights in the invention.CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 736691 , filed December 20, 2024, the disclosure of which is hereby incorporated by reference in its entirety.

[0004] The following applications also are incorporated by reference: International Patent Application No. PCT / US20 / 42606, filed July 17, 2020; and International Patent Application No. PCT / US21 / 16925, filed February 5, 2021 .INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0005] A sequence listing, which is a part of the present disclosure, is submitted concurrently with the specification as an XML file. The name of the electronic file containing the sequence listing is “70821_SeqListing.xml”, which was created on December 16, 2025 and is 15,304 bytes in size. The subject matter of the sequence listing is incorporated herein in its entirety by reference.BACKGROUND

[0006] Due to severe and non-specific deleterious effects of radiation and chemotherapy, targeted therapies capable of selectively killing tumor cells in patients are essential. Tumorspecific immunotherapy can be harnessed to eradicate tumors with exquisite precision and without collateral damage to normal tissue. Immunotherapy relies on the cytotoxic potential of activated T cells, which scavenge to recognize and reject tumor associated or specific antigens (TAAs or TSAs). Unlike most drug agents, activated T cells can traverse the blood brain barrier (BBB) via integrin (i.e., LFA-1 , VLA-4) binding of ICAMs / VCAMs.

[0007] T cells can be endogenously activated using cancer vaccines; but, in a randomized phase III trial for patients with primary GBM, peptide vaccines targeting the tumor specific EGFRVIII surface antigen failed to mediate enhanced survival benefits over control vaccines. The EGFRVIII vaccine’s failure to mediate anti-tumor efficacy highlights theDocket No. 32917 / 70821 challenge of therapeutic cancer vaccines. While prophylactic cancer vaccines work to prevent malignancies (e.g., HPV vaccine to prevent cervical cancer), the vaccines require several boosts over months to years to confer protection in immune-replete patients. Furthermore, therapeutic cancer vaccines must induce immunologic response much more rapidly against malignancies (e.g., GBM) that are rapidly evolving. An added challenge with brain cancers, in particular, are that the cancers are highly invasive and heterogeneous tumors associated with profound systemic / intratumoral suppression that can stymie a nascent immunotherapeutic response.

[0008] There is a need for alternative immunotherapy options to trigger immune responses against cancer, particularly brain cancers.SUMMARY

[0009] The disclosure provides a method of inducing an immune response to a tumor, the method comprising administering to a subject in need thereof a liposome nanoparticle comprising (i) nucleic acids that inhibit the expression of PD-L1 and (ii) tumor RNA. In various aspects, liposome nanoparticle comprises one or more cationic lipids (e.g., 1 ,2- Dioleoyl-3-trimethylammoniumpropane (DOTAP)), one or more neutral lipids (e.g., dioleoylphosphatidylethanolamine (DOPE), disteroylphosphatidyl choline (DSPC), or a combination thereof), and one or more PEG-modified lipids (e.g., PEG-distearoyl glycerol). For instance, the liposome nanoparticle may comprises about 0.5% to about 15% on a molar basis of the neutral lipid and / or from about 0.5% to about 20% on a molar basis of PEG- modified lipid. Optionally, the liposome nanoparticle comprises a sterol (e.g., cholesterol), such as sterol in an amount of from about 5% to about 50% on a molar basis. Optionally, the liposome nanoparticle comprises a folated lipid (e.g., distearoylphosphatidyl ethanolamine (DSPE)-PEG(2000)-folate, dipalmitoylphosphatidyl ethanolamine (DPPE)- PEG(2000)-folate, cholesterol PEG folate, folated stearylamine, or a combination thereof). In various aspects, the liposome nanoparticle comprises disteroylphosphatidyl choline (DSPC), 1 ,2-Dioleoyl-3-trimethylammoniumpropane (DOTAP), cholesterol, dioleoylphosphatidylethanolamine (DOPE), and PEG-distearoyl glycerol (PEG-DMG), optionally about 2.5-100 w% DOTAP, about 0.5-18 w% DSPC, about 1 -34 w% cholesterol, about 2-80 w% DOPE, and about 0.5-8 w% PEG-DMG, such as about 2.62-100 w% DOTAP, about 0.59-7 w% DSPC, about 1 .12-34 w% cholesterol, about 2.5-76 w% DOPE, and about 0.07-6 w% PEG-DMG. In various aspects, the liposome nanoparticle further comprises about 0.5-15 w% folated lipid, such as about 0.6-13 w% folated lipid. In various aspects, the liposome nanoparticle comprises an n / p ratio for lipid to RNA of about 4.5:1 .Docket No. 32917 / 70821

[0010] Also provided is a liposome nanoparticle as described herein. For instance, the disclosure provides a liposome nanoparticle comprising disteroylphosphatidyl choline (DSPC), 1 ,2-Dioleoyl-3-trimethylammoniumpropane (DOTAP), cholesterol, dioleoylphosphatidylethanolamine (DOPE), and PEG-distearoyl glycerol (PEG-DMG), and further comprising (i) nucleic acids that inhibit the expression of PD-L1 and (ii) tumor RNA. The liposome nanoparticle may comprise about 2.5-100 w% DOTAP, about 0.5-18 w% DSPC, about 1-34 w% cholesterol, about 2-80 w% DOPE, and about 0.5-8 w% PEG-DMG, such as about 2.62-100 w% DOTAP, about 0.59-7 w% DSPC, about 1 .12-34 w% cholesterol, about 2.5-76 w% DOPE, and about 0.07-6 w% PEG-DMG. In various aspects, the liposome nanoparticle further comprises a folated lipid, such as about 0.5-15 w% folated lipid (e.g., about 0.6-13 w% folated lipid). The liposome nanoparticle may comprise an n / p ratio for lipid to RNA of about 4.5:1 , although this is not required.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIGs. 1 A and 1 B are bar graphs illustrating the results of PD-L1 knockdown studies using anti-PD-L1 siRNA complexed with formulation F8 lipid nanoparticles (LNPs) in mouse derived dendritic cells (DC2.4). LNPs complexed with PD-L1 inhibitory nucleic acids demonstrated reduced PD-L1 expression as quantified by flow cytometry compared to scrambled siRNA and PD-L1 siRNA alone. Put another way, LNPs complexed with PD-L1 inhibitory nucleic acids inhibited PD-L1 expression to a greater extent than scrambled siRNA and PD-L1 siRNA which was not present in the LNPs of the disclosure. Geometric mean of PD-L1 is provided on the y-axis. Material tested is indicated on the x-axis (scrambled siRNA, PD-L1 siRNA, or F8 LNPs comprising PD-L1 siRNA).

[0012] FIG. 2 is a graph illustrating results of a study characterizing luciferase (mLuc) RNA complexed with LNPs. The x-axis identifies the LNPs tested (mLuc F7 = formula 7; mLuc F8f (EIM); and mLuc F8f (NM)). The bars represent, from left to right, polydispersity index (PDI), surface zeta potential (mV), and particle size (nm).

[0013] FIG. 3 is a graph illustrating features of LNPs of the disclosure as determined by dynamic light scattering (DLS). Hydrodynamic diameter (nm) is provided on the y-axis. The x-axis identifies the LNPs tested (kLuc TTRNA F8f = formula 8, total tumor RNA + PD-L1 siRNA; and co-F8f). The bars represent, from left to right, polydispersity index (PDI), surface zeta potential (mV), and particle size (nm).DETAILED DESCRIPTION

[0014] The disclosure provides, in various aspects, a method of inducing or improving an immune response to a tumor. The method comprises administering to a subject in need thereof a liposome nanoparticle (LNP) comprising (i) nucleic acids that inhibit the expressionDocket No. 32917 / 70821 of PD-L1 and (ii) tumor mRNA. The LNP formulations described herein complexed with anti- PD-L1 nucleic acids (e.g., siRNA) and tumor RNA (e.g., tumor antigen mRNA) reduces PD- L1 expression in tumor associated immune cells and enhance a subject’s immunological response against the tumor, resulting in tumor volume reduction and improved survival outcomes. The disclosure also provides materials and methods for treating cancer, such as a brain cancer, e.g., medulloblastoma or glioblastoma, in a human subject in need thereof. The method comprises administering to the subject a composition comprising a liposomal nanoparticle comprising nucleic acid molecules that inhibit the expression of PD-L1 (e.g., RNA-based inhibitor of PD-L1 , such as siRNA that targets PD-L1 such that translation is inhibited and reduced amounts of PD-L1 transcription factor are produced) in combination in tumor RNA (e.g., comprising tumor specific antigen mRNA).

[0015] The nanoparticles of the disclosure comprise a lipid and nucleic acids (such as siRNA and / or mRNA). As used herein the term “nanoparticle” refers to a particle that is less than about 1000 nm in diameter. In exemplary aspects, the nanoparticle has a diameter within the nanometer range. In exemplary aspects, the nanoparticle has a diameter between about 50 nm to about 500 nm, e.g., about 50 nm to about 450 nm, about 50 nm to about 400 nm, about 50 nm to about 350 nm, about 50 nm to about 300 nm, about 50 nm to about 250 nm, about 50 nm to about 200 nm, about 50 nm to about 150 nm, about 50 nm to about 100 nm, about 100 nm to about 500 nm, about 150 nm to about 500 nm, about 200 nm to about 500 nm, about 250 nm to about 500 nm, about 300 nm to about 500 nm, about 350 nm to about 500 nm, or about 400 nm to about 500 nm. In exemplary aspects, the nanoparticle has a diameter between about 50 nm to about 300 nm, e.g., about 100 nm to about 250 nm, about 110 nm ± 5 nm, about 115 nm ± 5 nm, about 120 nm ± 5 nm, about 125 nm ± 5 nm, about 130 nm ± 5 nm, about 135 nm ± 5 nm, about 140 nm ± 5 nm, about145 nm ± 5 nm, about 150 nm ± 5 nm, about 155 nm ± 5 nm, about 160 nm ± 5 nm, about165 nm ± 5 nm, about 170 nm ± 5 nm, about 175 nm ± 5 nm, about 180 nm ± 5 nm, about190 nm ± 5 nm, about 200 nm ± 5 nm, about 210 nm ± 5 nm, about 220 nm ± 5 nm, about230 nm ± 5 nm, about 240 nm ± 5 nm, about 250 nm ± 5 nm, about 260 nm ± 5 nm, about270 nm ± 5 nm, about 280 nm ± 5 nm, about 290 nm ± 5 nm, or about 300 nm ± 5 nm. In exemplary aspects, the nanoparticle is about 50 nm to about 250 nm in diameter. In some aspects, the nanoparticle is about 70 nm to about 200 nm in diameter. In exemplary aspects, the nanoparticle is present in a composition (e.g., pharmaceutical composition) comprising a heterogeneous mixture of nanoparticles ranging in diameter, e.g., about 50 nm to about 500 nm or about 50 nm to about 250 nm in diameter. Optionally, the pharmaceutical composition comprises a heterogeneous mixture of nanoparticles ranging from about 150 nm to about 250 nm in diameter.Docket No. 32917 / 70821

[0016] In exemplary instances, the nanoparticle is characterized by a zeta potential of about +20 mV to about +60 mV or about +40 mV to about +60 mV, e.g., about +40 mV to about +55 mV, about +40 mV to about +50 mV, about +40 mV to about +50 mV, about +40 mV to about +45 mV, about +45 mV to about +60 mV, about +50 mV to about +60 mV, about +55 mV to about +60 mV. In exemplary aspects, the nanoparticle has a zeta potential of about +45 mV to about +55 mV. The nanoparticle in various instances, has a zeta potential of about +50 mV. In various aspects, the zeta potential is greater than +30 mV or +35 mV. In other aspects, the zeta potential is about +20 mV to about +30 mV.

[0017] In exemplary aspects, the nanoparticles comprise one or more cationic lipids. In some embodiments, the cationic lipid is a low molecular weight cationic lipid such as those described in U.S. Patent Application Publication No. 20130090372, the contents of which are herein incorporated by reference in their entirety. The cationic lipid in exemplary instances is a cationic fatty acid, a cationic glycerolipid, a cationic glycerophospholipid, a cationic sphingolipid, a cationic sterol lipid, a cationic prenol lipid, a cationic saccharolipid, or a cationic polyketide. In exemplary aspects, the cationic lipid comprises two fatty acyl chains, each chain of which is independently saturated or unsaturated. In exemplary instances, the cationic lipid is DOTAP (1 ,2-dioleoyl-3-trimethylammonium-propane), or a derivative thereof. In exemplary instances, the cationic lipid is DOTMA (1 ,2-di-O-octadecenyl-3- trimethylammonium propane), or a derivative thereof. In exemplary aspects, the nanoparticle comprises 2.5-100 w% (weight percent) (e.g., 2.62-100 w%) of cationic lipid. For instance, the nanoparticle may comprise from about 5 w% to about 50 w%, from about 10 w% to about 40 w%, from about 20 w% to about 45 w%, from about 30 w% to about 45 w%, or from about 35 w% to about 40 w% cationic lipid, such as DOTAP.

[0018] In some aspects, the nanoparticle comprises one or more neutral lipids. Examples of neutral lipids include, but are not limited to, distearoyl phosphatidyl choline (DSPC), 1 - palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), dipalmitoylphosphatidylcholine (DPPC), Dioleoyl phosphatidyl ethanolamine (DOPE), and sphingomyelin (SM). In various aspects, the nanoparticle comprises disteroylphosphatidyl choline (DSPC), or a derivative thereof. In exemplary aspects, the lipid is dioleoylphosphatidylethanolamine (DOPE), or a derivative thereof. In various aspects, the LNP comprises from about 0.5% to about 15% on a molar basis of the neutral lipid e.g., from about 3 to about 12%, from about 5 to about 10% or about 15%, about 10%, or about 7.5% on a molar basis. In some aspects, the LNP comprises from about 10% to about 40% on a molar basis of the neutral lipid or from about 50% to about 60% on a molar basis of the neutral lipid. For instance, the LNP may comprise about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60% on a molar basis of the neutral lipid.Docket No. 32917 / 70821

[0019] In some embodiments, the nanoparticle comprises one or more sterols. For instance, the nanoparticle optionally includes from about 5% to about 50% on a molar basis of the sterol (e.g., about 15 to about 45%, about 20 to about 40%, about 40%, about 38.5%, about 35%, or about 31% on a molar basis). An exemplary sterol is cholesterol.

[0020] In some embodiments, the nanoparticle comprises one or more PEG-modified lipids. For instance, the nanoparticle may comprise from about 0.5% to about 20% on a molar basis of the PEG or PEG-modified lipid (e.g., about 0.5 to about 10%, about 0.5 to about 5%, about 1 .5%, about 0.5%, about 1 .5%, about 3.5%, or about 5% on a molar basis). In some embodiments, the PEG or PEG modified lipid comprises a PEG molecule of an average molecular weight of 2,000 Da. In other embodiments, the PEG or PEG modified lipid comprises a PEG molecule of an average molecular weight of less than 2,000, for example around 1 ,500 Da, around 1 ,000 Da, or around 500 Da. Examples of PEG-modified lipids include, but are not limited to, PEG-distearoyl glycerol (PEG-DMG, e.g., DMG- PEG2000 (also referred herein as PEG-014 or C14-PEG), PEG-cDMA, further discussed in Reyes et al., J. Controlled Release, 107, 276-287 (2005), the contents of which is herein incorporated by reference in its entirety).

[0021] In various aspects, the formulation comprises one or more folated lipids. Folate- conjugated lipids may be generated via any suitable method. For instance, folated lipid may be synthesized by reacting the carboxylic group of folic acid to a complementarily reactive lipid or cholesterol. Folated lipids bind with folate receptors on target cells prompting invagination and formation of an endosome. Examples of folated lipids include, but are not limited to, distearoylphosphatidyl ethanolamine (DSPE)-PEG(2000)-folate, dipalmitoylphosphatidyl ethanolamine (DPPE)-PEG(2000)-folate, cholesterol PEG folate, and folated stearylamine. Production of folated lipids is further described in, e.g., Kumar et al., Formulation Strategies for Folate-Targeted Liposomes and Their Biomedical Applications. Pharmaceutics. 2019 Aug 2;11 (8):381 .

[0022] The nanoparticles in some aspects are composed of multiple lipid components (e.g., three to six lipid components, such as three to five lipid components) in addition to the nucleic acid molecules. In exemplary aspects, the liposome comprises disteroylphosphatidyl choline (DSPC), DOTAP, sterol (e.g., cholesterol), dioleoylphosphatidylethanolamine (DOPE), and / or PEG-DMG. In various aspects, the nanoparticle comprises DOTAP, DSPC, cholesterol, DOPE, and PEG-DMG. In exemplary aspects, the nanoparticle comprises 2.5- 100 w% (e.g., 2.62-100 w%) DOTAP, 0.5-18 w% (e.g., 0.59-7 w%) DSPC, 1-34 w% (e.g., 1 .12-34 w%) cholesterol, 2-80 w% (e.g., 2.5-76 w%) DOPE, 0.5-8 w% (e.g., 0.07-6 w%) PEG-DMG, and / or about 0.5-15 w% (e.g., 0.6-13 w%) folated lipid. In exemplary aspects, the n / p ratio for lipid to RNA ranges between about 4.5:1 , about 6:1 , about 9:1 and aboutDocket No. 32917 / 7082115:1 . “N / p ratio” refers to the molar ratio of positively charged amine groups (N) from the cationic lipid to negatively charged phosphate groups (P) from the nucleic acid (DNA / RNA) cargo.

[0023] In exemplary embodiments, the nanoparticle comprises a surface and an interior comprising at least two nucleic acid layers, optionally, formed around a core, which may comprise nucleic acid or may not comprise nucleic acid. In exemplary instances, each nucleic acid layer is positioned between a lipid layer, e.g., a cationic lipid layer. In exemplary aspects, the nanoparticles are multilamellar comprising alternating layers of nucleic acid and lipid. In exemplary embodiments, the nanoparticle comprises at least three nucleic acid layers, each of which is positioned between a cationic lipid bilayer. In exemplary aspects, the nanoparticle comprises at least four or five nucleic acid layers, each of which is positioned between a cationic lipid bilayer. In exemplary aspects, the nanoparticle comprises at least more than five (e.g., 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleic acid layers, each of which is positioned between a cationic lipid bilayer. As used herein the term “cationic lipid bilayer” is meant a lipid bilayer comprising, consisting essentially of, or consisting of a cationic lipid or a mixture thereof. Suitable cationic lipids are described herein. As used herein the term “nucleic acid layer” is meant a layer of the presently disclosed nanoparticle comprising, consisting essentially of, or consisting of a nucleic acid, e.g., RNA.

[0024] The unique structure of multilamellar nanoparticles results in mechanistic differences in how the multilamellar nanoparticles (ML-NPs) exert a biological effect. Previously described RNA-based nanoparticles exert their effect, at least in part, through the toll-like receptor 7 (TLR7) pathway. Surprisingly, the multilamellar nanoparticles of the instant disclosure mediate efficacy independent of TLR7. While not wishing to be bound to any particular theory, intracellular pathogen recognition receptors (PRRs), such as MDA-5, appear more relevant to biological activity of the multi-lamellar nanoparticles than TLRs. This likely allows ML RNA-NPs to stimulate multiple intracellular PRRs (e.g., RIG-I, MDA-5) as opposed to singular TLRs (e.g., TLR7 in the endosome) culminating in greater release of type I interferons and induction of more potent innate immunity.

[0025] The liposome nanoparticle (LNP) of the disclosure comprises nucleic acids that inhibit the expression of PD-L1 . Programmed death-ligand 1 (PD-L1 ; also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1)) is a transmembrane protein that functions to suppress the immune system in, e.g., pregnancy, tissue allografts, and autoimmune disease. Binding of PD-L1 to its receptor PD-1 transmits an inhibitory signal that reduces the proliferation and function of T cells and can induce apoptosis. PD-L1 is expressed not only by immune cells, but also in various tumor cell lines and cancer tissue.Docket No. 32917 / 70821See, e.g., Latchman et al, Nature Immunology, 2001 , Vol. 2, No. 3, pp.261 -267. Human PD- L1 amino acid sequence is known in the art. See, e,g.., Uniprot Q9NZQ7-1 . The nucleic acid sequence of human PD-L1 is provided as GenBank Accession No. AF177937.1 (SEQ ID NO: 1).

[0026] In various aspects of the disclosure, the nucleic acids that inhibit the expression of PD-L1 are RNA, such as antisense RNA (i.e. , RNA complementary to a gene product encoding messenger RNA (mRNA) with which it hybridizes and blocks translation). Optionally, the RNA is siRNA, shRNA, miRNA, or any combination thereof. An inhibitor nucleic acid (e.g., RNA) can be single stranded or double stranded).

[0027] The antisense nucleic acid can be one which mediates RNA interference (RNAi). RNAi is a ubiquitous mechanism of gene regulation in plants and animals in which target mRNAs are degraded in a sequence-specific manner (Sharp, Genes Dev., 15, 485-490 (2001); Hutvagner et al., Curr. Opin. Genet. Dev, 12, 225-232 (2002); Fire et al. Nature, 391 , 806-811 (1998); Zamore et al. Cell, 101 , 25-33 (2000)). The natural RNA degradation process is initiated by the dsRNA-specific endonuclease Dicer, which promotes cleavage of long dsRNA precursors into double-stranded fragments between 21 and 25 nucleotides long, termed small interfering RNA (siRNA; also known as short interfering RNA) (Zamore, et al. Cell. 101 , 25-33 (2000); Elbashir et al. Genes Dev, 15, 188-200 (2001); Hammond et al. Nature, 404, 293-296 (2000); Bernstein et al. Nature, 409, 363-366 (2001)). siRNAs are incorporated into a large protein complex that recognizes and cleaves target mRNAs (Nykanen et al. Cell, 107, 309-321 (2001)). It has been reported that introduction of dsRNA into mammalian cells does not result in efficient Dicer-mediated generation of siRNA and therefore does not induce RNAi (Caplen et al. Gene 252, 95-105 (2000); Ui-Tei et al, FEBS Lett, 479, 79-82 (2000)). The requirement for Dicer in maturation of siRNAs in cells can be bypassed by introducing synthetic 21 -nucleotide siRNA duplexes, which inhibit expression of transfected and endogenous genes in a variety of mammalian cells (Elbashir et al. Nature, 411 : 494-498 (2001)).

[0028] In various aspects, the nucleic acid molecule is a short interfering RNA (siRNA). A siRNA molecule is a duplex comprising a sense strand and complementary antisense strand, the antisense strand having sufficient complementary to PD-L1 mRNA to mediate RNA interference. In this respect, the siRNA interferes with PD-L1 expression by, e.g, degrading mRNA after transcription, thereby preventing translation of PD-L1 . siRNA molecules are typically about 10-50 or more nucleotides in length (e.g, from about 15-30, e.g, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides (or nucleotide analogs) in each strand, wherein one of the strands is sufficiently complementary to a target region of a PD-L1 nucleic acid sequence to reduce translation). In various aspects, theDocket No. 32917 / 70821 strands of the duplex comprise at least 1 , 2, or 3 bases at the end of the strands which do not align (i.e., overhang) when strands are duplexed.

[0029] siRNA molecules have sufficient complementarity with target sequence within PD- L1 such that the siRNA mediates RNA interference (RNAi). For instance, the sense strand of the siRNA comprises a sequence sufficiently identical to a portion of the target sequence within PD-L1 (e.g., greater than 80% identity, such as 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% identity, between the sense strand of the siRNA duplex and the target PD-L1 RNA sequence). The sense strand of the siRNA duplex may comprise 4, 3, 2, 1 , or 0 mismatched nucleotide(s) with a target region of PD-L1 mRNA, or the siRNA sequence may comprise small insertions or deletions (e.g., 1 or 2 nucleotides), or comprise substitutions or insertions while still mediating RNAi. siRNAs can be designed by using any method known in the art, such as, for example, the method described in U.S. Patent No. 12,077,755. See also Shah et al., “sIR: siRNA Information Resource, a web-based tool for siRNA sequence design and analysis and an open access siRNA database.” BMC Bioinformatics, vol. 8, 31 May 2007, p. 178. Exemplary siRNA targeting PD-L1 comprises the nucleic acid sequences of SEQ ID NO: 2 (CCCACAUAAAAAACAGUUGTT) or SEQ ID NO:-3 (CAACUGUUUUUUAUGUGGGTT). See also, e.g., Kwak et al, ACS Nano, 11 , 10135- 10146 (2017), incorporated herein by reference. Another exemplary sequence targeting PD- L1 comprises the nucleic acid sequence of SEQ ID NO: 4 (ACG GGC CAC ATC AAC TCA TTG ATA GAC AAT GCG TCC ACT GCC CGT). See, e.g., Lai et al, Mol Ther Nuc Acids, 5, e397 (2016), incorporated herein by reference. Other examples of siRNA targeting PD-L1 include nucleic acids comprising the nucleic acid sequence of SEQ ID NOs: 5-6 and 7-8.

[0030] In alternative aspects, the inhibitory RNA molecule is a short hairpin RNA (shRNA) molecule specific for inhibiting the expression of PD-L1 . The term "shRNA" refers to a molecule of about 20 or more base pairs in which a single-stranded RNA partially contains a palindromic base sequence and forms a double-strand structure therein (i.e. a hairpin structure). An shRNA can be an siRNA (or siRNA analog) which is folded into a hairpin structure. shRNAs typically comprise about 45 to about 60 nucleotides, including the approximately 21 nucleotide antisense and sense portions of the hairpin, optional overhangs on the non-loop side of about 2 to about 6 nucleotides long, and the loop portion that can be, e.g., about 3 to 10 nucleotides long. The shRNA can be chemically synthesized.Alternatively, the shRNA can be produced by linking sense and antisense strands of a DNA sequence in reverse directions and synthesizing RNA in vitro with T7 RNA polymerase using the DNA as a template.Docket No. 32917 / 70821

[0031] In exemplary aspects, the PD-L1 inhibitory nucleic acid is a microRNA (miRNA). “microRNA” refers to a small (e.g., 15-22 nucleotides), non-coding RNA molecule which base pairs with mRNA molecules to silence gene expression via translational repression or target degradation. microRNA and the therapeutic potential thereof are described in the art. See, e.g., Mulligan, MicroRNA: Expression, Detection, and Therapeutic Strategies, Nova Science Publishers, Inc., Hauppauge, NY, 2011 ; Bader and Lammers, “The Therapeutic Potential of microRNAs” Innovations in Pharmaceutical Technology, pages 52-55 (March 2011 ).

[0032] The LNPs further comprise tumor RNA (e.g., transfer RNA (tRNA), ribosomal RNA (rRNA), messenger RNA (mRNA), or combinations thereof). The term “tumor RNA” is meant to reference RNA from any cancer cell type. In various aspects, tumor RNA is directly isolated from a tumor, but this is not required. The sequence of the “tumor RNA” reflects the sequence of RNA found in a tumor, such as the particular type of tumor present in a subject or the particular tumor present in a subject. In various aspects, the RNA is total RNA isolated from a tumor cell or a cancer cell. The tumor RNA may be derived from the subject being treated (autologous) or may be derived from other sources (allogeneic). Methods of obtaining total tumor RNA is known in the art. In various aspects, the nanoparticle comprises a mixture of RNA which is RNA isolated or derived from a tumor of a human, optionally, a malignant brain tumor, optionally, a glioblastoma, medulloblastoma, diffuse intrinsic pontine glioma, or a peripheral tumor with metastatic infiltration into the central nervous system. In various aspects, the RNA comprises a sequence encoding a poly(A) tail so that the in vitro transcribed RNA molecule comprises a poly(A) tail at the 3’ end.

[0033] In exemplary instances, the tumor RNA molecules are mRNA. In various aspects, mRNA is in vitro transcribed mRNA. In various instances, the mRNA molecules are produced by in vitro transcription (IVT). Suitable techniques of carrying out IVT are known in the art. Optionally, the in vitro transcription template is cDNA made from RNA extracted from a tumor cell. Some or all mRNAs, in exemplary aspects, encode a protein. Optionally, at least a portion of the mRNA encodes a tumor antigen. In exemplary aspects, the tumor antigen is an antigen derived from a viral protein, an antigen derived from point mutations, or an antigen encoded by a cancer-germline gene. In exemplary aspects, the tumor antigen is pp65, p53, KRAS, NRAS, MAGEA, MAGEB, MAGEC, BAGE, GAGE, LAGE / NY-ES01 , SSX, tyrosinase, gp100 / pmel17, Melan-A / MART-1 , gp75 / TRP1 , TRP2, CEA, RAGE-1 , HER2 / NEU, WT1.

[0034] The disclosure also contemplates LNPs comprising non-tumor RNA, such as RNA encoding proteins that are not specific to tumor cells. For instance, the protein may be a cytokine or a co-stimulatory molecule. In this regard, a co-stimulatory molecule, a cytokine,Docket No. 32917 / 70821 a growth factor, a hematopoietic factor, and a lymphokine, including, e.g., cytokines and growth factors that are effective in inhibiting tumor metastasis, and cytokines or growth factors that have been shown to have an antiproliferative effect on at least one cell population, are contemplated. Such cytokines, lymphokines, growth factors, or other hematopoietic factors include, but are not limited to: M-CSF, GM-CSF, TNF, IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 , IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IFN, TNFa, TNF1 , TNF2, G-CSF, Meg-CSF, GM-CSF, thrombopoietin, stem cell factor, and erythropoietin. Additional growth factors for use herein include angiogenin, bone morphogenic protein-1 , bone morphogenic protein-2, bone morphogenic protein-3, bone morphogenic protein-4, bone morphogenic protein-5, bone morphogenic protein-6, bone morphogenic protein-7, bone morphogenic protein-8, bone morphogenic protein-9, bone morphogenic protein-10, bone morphogenic protein-11 , bone morphogenic protein-12, bone morphogenic protein-13, bone morphogenic protein-14, bone morphogenic protein-15, bone morphogenic protein receptor I A, bone morphogenic protein receptor IB, brain derived neurotrophic factor, ciliary neurotrophic factor, ciliary neurotrophic factor receptor a, cytokine-induced neutrophil chemotactic factor 1 , cytokine- induced neutrophil, chemotactic factor 2 a, cytokine-induced neutrophil chemotactic factor 2 b, b endothelial cell growth factor, endothelin 1 , epithelial-derived neutrophil attractant, glial cell line-derived neurotrophic factor receptor a 1 , glial cell line-derived neurotrophic factor receptor a 2, growth related protein, growth related protein a, growth related protein b, growth related protein g, heparin binding epidermal growth factor, hepatocyte growth factor, hepatocyte growth factor receptor, insulin-like growth factor I, insulin-like growth factor receptor, insulinlike growth factor II, insulin-like growth factor binding protein, keratinocyte growth factor, leukemia inhibitory factor, leukemia inhibitory factor receptor a, nerve growth factor nerve growth factor receptor, neurotrophin-3, neurotrophin-4, pre-B cell growth stimulating factor, stem cell factor, stem cell factor receptor, transforming growth factor a, transforming growth factor b, transforming growth factor b1 , transforming growth factor b1 .2, transforming growth factor b2, transforming growth factor b3, transforming growth factor b5, latent transforming growth factor b1 , transforming growth factor b binding protein I, transforming growth factor b binding protein II, transforming growth factor b binding protein III, tumor necrosis factor receptor type I, tumor necrosis factor receptor type II, urokinase-type plasminogen activator receptor, and chimeric proteins and biologically or immunologically active fragments thereof. In exemplary aspects, the co-stimulatory molecule is selected from the group consisting of CD80 and CD86.

[0035] In some aspects, the protein is not expressed by a tumor cell or by a human. In some aspects, the protein is non-specific relative to a tumor or cancer. In exemplaryDocket No. 32917 / 70821 instances, the protein is not related to a tumor antigen or cancer antigen. For example, the non-specific protein may be green fluorescence protein (GFP) or ovalbumin (OVA). In various aspects, the protein is a viral antigen or bacterial antigen, such as viral or bacterial antigens not associated with tumorigenesis.

[0036] The subject is a mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits, mammals from the order Carnivora, including Felines (cats) and Canines (dogs), mammals from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). In some aspects, the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). In some aspects, the mammal is a human. In some aspects, the human is an adult aged 18 years or older. In some aspects, the human is a child aged 17 years or less.

[0037] The disclosure provides, in various aspects, a method of inducing or improving an immune response to a tumor. Increased host immune response may be determined in any of a number of ways. For example, administration of the LNP increase the number of cytotoxic T cells in a tumor and / or enhance cytotoxic T cell activity. For example, in various embodiments, the method may increase perforin, IFN-gamma, and / or granzyme production by cytotoxic T cells and increase cytolytic activity. Further, the method described herein may enhance T cell survival, promote T cell longevity, and / or restrict loss of replicative potential. Methods of measuring T cell activity and immune responses are known in the art. T cell activity can be measured by, for example, a cytotoxicity assay, such as those described in Fu et al., PLoS ONE 5(7): e11867 (2010). Other T cell activity assays are described in Bercovici et al. , Clin Diagn Lab Immunol. 7(6): 859-864 (2000). Methods of measuring immune responses are also described in e.g., Macatangay et al., Clin Vaccine Immunol 17(9): 1452-1459 (2010), and Clay et al., Clin Cancer Res.7(5):1127-35 (2001). In various aspects, the method of the disclosure enhances cytotoxic T cell mediated killing of cancer cells within the tumor.

[0038] In various aspects, the disclosure provides a method of treating cancer in subject or treating a tumor in a subject in needed thereof. The tumor or cancer treatable by the materials and methods disclosed herein may be any cancer, e.g., any malignant growth or tumor caused by abnormal and uncontrolled cell division that may spread to other parts of the body through the lymphatic system or the blood stream (although this is not required). In exemplary aspects, the cancer is located across the blood brain barrier and / or the subject has cancer (i.e. , a tumor) located in the brain. In some aspects, the cancer is a glioma, a low-grade glioma or a high-grade glioma, specifically a grade III astrocytoma or a glioblastoma. In various aspects, the cancer is glioblastoma and the subject is an adult,Docket No. 32917 / 70821 although pediatric patients also are contemplated. In various aspects, the cancer is medulloblastoma. Optionally, the subject in need thereof is suffering from (or at risk of suffering from) Grade 3 (Group 3) medulloblastoma. See, e.g., Kool et al., “Molecular subgroups of medulloblastoma: an international meta-analysis of transcriptome, genetic aberrations, and clinical data of WNT, SHH, Group 3, and Group 4 medulloblastomas.” Acta Neuropathol. 2012; 123:473-484.

[0039] The cancer in some aspects is one selected from the group consisting of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer (e.g., glioma), breast cancer (e.g., triple negative breast cancer), cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the head, neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal cancer (e.g., gastrointestinal carcinoid tumor), Hodgkin lymphoma, endometrial or hepatocellular carcinoma, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer (e.g., non-small cell lung cancer, bronchioloalveolar carcinoma), malignant mesothelioma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer (e.g., renal cell carcinoma (ROC)), small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, ureter cancer, and urinary bladder cancer. In particular aspects, the cancer is selected from the group consisting of head and neck, ovarian, cervical, bladder and oesophageal cancers, pancreatic, gastrointestinal cancer, gastric, breast, endometrial and colorectal cancers, hepatocellular carcinoma, glioblastoma, bladder, and lung cancer (e.g., non-small cell lung cancer (NSCLC), bronchioloalveolar carcinoma). In various aspects, the subject has a solid tumor.

[0040] As used herein, the term “treat,” as well as words related thereto, do not necessarily imply 100% or complete treatment (i.e., complete remission or eradication of the disease). The methods of treating a disease of the present disclosure can provide any amount or any level of treatment. For example, a therapeutic response optionally refers to one or more of the following improvements in the disease: (1) a reduction in the number of neoplastic cells; (2) an increase in neoplastic cell death; (3) inhibition of neoplastic cell survival; (4) inhibition (i.e., slowing to some extent, preferably halting) of tumor growth or appearance of new lesions; (5) an increased patient survival rate; and / or (6) some relief from one or more symptoms associated with the disease or condition (e.g., pain, weight loss, weakness or fatigue, anemia, or bleeding). Disease state is monitored by, e.g., clinicalDocket No. 32917 / 70821 examination, X-ray, computerized tomography (CT, such as spiral CT), magnetic resonance imaging (MRI), positron emission tomography (PET), ultrasound, endoscopy and laparoscopy, tumor marker levels (e.g., carcinoembryonic antigen (CEA)), cytology, histology, tumor biopsy sampling, and / or counting of tumor cells in circulation. The treatment provided by the presently disclosed method may delay the onset or reoccurrence / relapse of the disease being prophylactical ly treated. The prophylactic treatment encompasses reducing the risk of the disease being treated. In exemplary aspects, the method reduces the risk of the disease or relapse by 2-fold, 5-fold, 10-fold, 20- fold, 50-fold, 100-fold, or more.

[0041] In various aspects, response to treatment is evaluated by measuring parameters of one or more target lesions over time. For glioma, for instance, response determination may be based on a comparison of an area [W (longest diameter of the target lesion) x T (transverse measurement, perpendicular to W)] between the baseline assessment and after treatment. A complete response is characterized by the disappearance of lesions. A partial response is characterized by at least a 50% decrease in the size of target lesions. A subject exhibiting "stable disease" exhibits neither sufficient shrinkage to qualify for complete response or partial response nor sufficient increase to qualify for progressive disease, characterized by at least a 25% increase in the sum of the size of target lesions. The disclosure contemplates improvement of any of these parameters, and preferably improvement sufficient to achieve at least a partial response. For example, in various aspects, the subject achieves at least a 10% reduction, at least a 20% reduction, at least a 30% reduction (e.g., at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, at least a 70% reduction, at least an 80% reduction, or at least a 90% reduction) in the area of target lesions (compared to baseline before treatment) or demonstrates a complete response. Alternatively or in addition, the subject experiences progression-free survival for at least six months (e.g., at least nine months) after cessation of treatment, optionally experiencing progression-free survival for 12 months or longer (e.g., 18 months or longer or 24 months or longer) after cessation of treatment. The method of the disclosure may also improve the stage or grade of the cancer.

[0042] The nanoparticle is typically provided in the form of a pharmaceutical composition comprising a plurality of nanoparticles according to the present disclosure and a pharmaceutically acceptable carrier, diluent, or excipient and intended for administration to a human. In exemplary aspects, the composition is a sterile composition. In various aspects, the composition comprises about 108-1010nanoparticles per mL to about 1015nanoparticles per mL, optionally about 1012nanoparticles ± 10% per mL.Docket No. 32917 / 70821

[0043] In exemplary aspects, the composition of the present disclosure may comprise additional components other than the nanoparticle. The composition, in various aspects, comprises any pharmaceutically acceptable ingredient, including, for example, acidifying agents, additives, adsorbents, aerosol propellants, air displacement agents, alkalizing agents, anticaking agents, anticoagulants, antimicrobial preservatives, antioxidants, antiseptics, bases, binders, buffering agents, chelating agents, coating agents, coloring agents, desiccants, detergents, diluents, disinfectants, disintegrants, dispersing agents, dissolution enhancing agents, dyes, emollients, emulsifying agents, emulsion stabilizers, fillers, film forming agents, flavor enhancers, flavoring agents, flow enhancers, gelling agents, granulating agents, humectants, lubricants, mucoadhesives, ointment bases, ointments, oleaginous vehicles, organic bases, pastille bases, pigments, plasticizers, polishing agents, preservatives, sequestering agents, skin penetrants, solubilizing agents, solvents, stabilizing agents, suppository bases, surface active agents, surfactants, suspending agents, sweetening agents, therapeutic agents, thickening agents, tonicity agents, toxicity agents, viscosity-increasing agents, water-absorbing agents, water-miscible cosolvents, water softeners, or wetting agents. See, e.g., the Handbook of Pharmaceutical Excipients, Third Edition, A. H. Kibbe (Pharmaceutical Press, London, UK, 2000), which is incorporated by reference in its entirety. Remington’s Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980), which is incorporated by reference in its entirety.

[0044] The composition of the present disclosure can be suitable for administration by any acceptable route, including parenteral and subcutaneous routes. Exemplary routes include, but are not limited to, intravenous, intradermal, intranasal, intrathecal, intracranial, intramuscular, intraperitoneal, intranodal and intrasplenic routes of administration, for example. In exemplary aspects, the composition is suitable for systemic administration. In exemplary aspects, the composition is suitable for intravenous administration. In exemplary aspects, the composition is suitable for intramuscular administration. In exemplary aspects, the composition is suitable for intra-nasal, intrathecal, and intracranial administrations.

[0045] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0046] The use of the terms “a” and “an” and “the” and similar referents in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” andDocket No. 32917 / 70821“containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. If aspects of the invention are described as "comprising" a feature, embodiments also are contemplated "consisting of" or "consisting essentially of" the feature.

[0047] 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 and each endpoint, unless otherwise indicated herein, and each separate value and endpoint is incorporated into the specification as if it were individually recited herein. Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about" as that term would be interpreted by the person skilled in the relevant art.

[0048] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.EXAMPLES

[0049] The example provided herein relates to production and characterization of lipid nanoparticle (LNP) formulations complexed with anti-PD-L1 nucleic acids (here, siRNA) and tumor antigen specific mRNA. Formulations F7 and F8f are described. The LNPs were characterized for particle size, polydispersity indices, zeta potential, and particle concentrations using Malvern’s Zetasizer, and NanoSight’s NTA systems. LNPs were screened for transfection efficiency in DC2.4 cells, using GFP mRNA as a fluorescent probe. LNPs will be screened for transfection efficiency against additional cell types (e.g., SMA 560, and Kluc cells). Formulations are complexed with GFP mRNA and anti-PD-L1 si- RNA / antigen mRNA using Nanoassemblr microfluidic equipment (Cytiva, Precision NanoSystems). The formulations are characterized for visual inspection, pH, particle size, zeta potential, polydispersity indices (PDI) , and particle concentration using Malvern’s Zetasizer, and NanoSight NTA. Additionally, RNA encapsulation efficiency is performed quantitatively using ribogreen assay and qualitatively using 1 % agarose gel electrophoresis. Furthermore, LNP formulations are evaluated in mouse model of glioblastoma (GBM) for efficacy and survival outcome benefits.Docket No. 32917 / 70821

[0050] PD-L1 knockdown studies using anti-PD-L1 siRNA complexed with formulation F8 in mouse derived dendritic cells (DC2.4) demonstrated reduced PD-L1 expression post treatment compared to scrambled siRNA and PDL1 siRNA alone, as determined by flow cytometry (FIG. 1 A and 1 B). The results demonstrate that anti-PD-L1 inhibitory nucleic acids (here, siRNA) complexed LNPs efficiency inhibit PD-L1 expression in a clinically relevant cell line model.

[0051] Biodistribution studies demonstrated brain localization for LNP formulations F7, and F8f following a single i.v. injection into healthy mice. Combining anti-PD-L1 nucleic acids (e.g., siRNA) and antigenic mRNA with the LNP platform described herein will potentiate enhanced immunological responses near the target tumor site and provide maximum efficacy with minimum off-target toxicities, thereby enhance the overall survival outcomes. LNP formulations F7, and F8f localize to the brain, providing a means to deliver anti-PD-L1 nucleic acids and tumor RNA the brain to achieve a synergistic improvement of an immune stimulatory response.

[0052] Preparation of Formulation F7 LNPs

[0053] Formulation 7 LNPs were generated using an ethanol injection solvent evaporation method. Initially, DOTAP (Lot#890890P-500MG-R-179), distearoyl phosphatidyl choline (DSPC, Avanti, Lot#850365P-500MG-D-178), cholesterol (700100P-1 OOmg, Avanti polar lipids, lot#700100P-100MG-B-036), dioleoyl phosphatidyl ethanolamine (DOPE, Avanti, lot#850725P-500MG-B-445), and dimyristoyl glycerol (DMG) PEG 2000 (DMG-PEG2000, Avanti, lot #880151 P-1 G-F-024) were dissolved in ethanol (2 mL, molecular biology grade BP2818-500, Fisher bioreagents, Lot #224553). For DOTAP weight measurements, powder vial of DOTAP, 100 mg, was dissolved in 2 mL ethanol, resulting in 25 mg / mL solution. DOTAP solution (1 .572 mL) was added to the weighed lipids in a 100 mL glass beaker (Bomex®). For DOPE weight measurements, 4 mL ethanol was added to the vial containing 25 mg DOPE powder. Ethanolic solution (3.013 mL) was used and added to the lipid mixture. An additional 1 .3 mL ethanol was added to the beaker containing lipids followed by bath sonication and warming the lipids ethanolic solution to 50 °C for 3 minutes to completely dissolve lipids. The glass syringe (Fortuna®, Optima®, 10 mL, Lot# F00037) was then filled with ethanolic solution of lipids. Aqueous phase containing 30 mL UltraPure distilled water (Invitrogen, 500 mL, Lot# 242907) contained in a 100 mL glass beaker (Bomex®) was warmed to 50 °C under water bath equipped with hot plate stirrer (Thermo, XLD-T 100S) maintained at the stirring rate of 1000 rpm. Ethanolic solution was then added dropwise to the aqueous solution under stirring and maintained at 50 °C under flume hood. Both the beaker and glass syringe were further rinsed with additional 1 mL molecular gradeDocket No. 32917 / 70821 ethanol, warmed to 50 °C for 3 minutes, and added dropwise to the aqueous solution maintained at 50 °C. The hydroalcoholic solution was stirred at 50 °C for a further 2 hours under hood for ethanol removal. The final volume of the liposomal suspension obtained was approximately 15 mL. The liposomal formulation was dialyzed using dialysis membrane (SnakeSkin™ Dialysis Tubing, 3.5k MWCO, Ref:88242, Lot #YA353160) against distilled water for two washes every three hours (kept under shaking (Thermo Fisher, 90 rpm and 37 °C in beaker for each wash)), and a third wash was performed against PBS and dialyzed overnight. Dialyzed formulation was then homogenized using microfluidizer 110 S for 15 mins. Microfluidized liposomes were evaluated for their size distribution, polydispersity indices (PDI), and zeta potential measurements. The formulation was complexed with luciferase mRNA (mLuc) and evaluated for percent encapsulation efficiency by electrophoresis (FIG. 2) and for percent cell transfection by fluorescence microscopy and flow cytometry. Pre-clinical in vivo imaging (I VIS) also was performed.

[0054] TABLE 1 : Formulation F7 lipid composition

[0055] Preparation of Formulation F8 LNPs

[0056] Formulation 8 LNPs were generated using an ethanol injection solvent evaporation microfluidization method. Initially, DOTAP (Avanti Polar lipids, Lot#890890P 500MG L179), distearoyl phosphatidyl choline (DSPC, Avanti, Lot#850365P 500MG-D-178), cholesterol (700100P-1 OOmg, Avanti polar lipids, lot#700100P-100MG-B-036), dioleoyl phosphatidyl ethanolamine (DOPE, Avanti polar lipids, lot #850725P-500MG-B-445), and dimyristoyl glycerol (DMG) PEG 2000 (DMG PEG2000, Avanti, lot #880151 P-1 G-F-024) were dissolved in ethanol (3 mL, molecular biology grade BP2818-500, Fisher bioreagents, Lot #224553). For dissolving cholesterol, an additional 1 .5 mL heptane was added to the Eppendorf tube containing cholesterol, followed by warming the solution to 50 °C for 3 minutes to completely dissolve cholesterol. Solubilized cholesterol was then added to the beaker containing other lipids and warmed to 50 °C. A glass syringe (Fortuna®, Optima®, 10 mL, Lot# F00037) wasDocket No. 32917 / 70821 then filled with ethanolic solution of lipids. Aqueous phase containing 30 mL UltraPure distilled water (Invitrogen, 500 mL, Lot# 242907) contained in a 100 mL glass beaker (Bomex®) was warmed to 50 °C under water bath equipped with a hot plate stirrer (Thermo, XLD-T100S), which was maintained at a stirring rate of 1000 rpm. Ethanolic solution was then added dropwise to the aqueous solution under stirring and maintained at 50 °C under flume hood. Both the beaker and glass syringe were further rinsed with additional 1 mL molecular grade ethanol, warmed to 50 °C for 3 minutes, and added dropwise to the aqueous solution maintained at 50 °C. Hydroalcoholic solution was stirred at 50 °C for further two hours under hood for ethanol removal. The final volume of the liposomal suspension obtained was approximately 15 mL. The resulting liposomal formulation was dialyzed using dialysis membrane (SnakeSkin™ Dialysis Tubing, 3.5k MWCO, Ref:88242, Lot #YA353160) against distilled water for two washes every three hours (kept under shaking (Thermo Fisher, 90 rpm and 37 °C in beaker for each wash)), and a third wash was performed against PBS and dialyzed overnight. Dialyzed formulation was then homogenized using microfluidizer 110 S for 15 mins. Microfluidized formulation was further heated to 50 °C for 1 hour to reduce the volume down to 10 mL. The formulation was evaluated for LNP size distribution, polydispersity indices (PDI) , and zeta potential. The formulation was then complexed with PD-L1 siRNA (Table 3) and evaluated for anti-PDL1 knockdown efficiency on DC2.4 cells (FIG. 1 A and 1 B)

[0057] TABLE 2: Formulation F8 lipid composition

[0058] TABLE 3: Anti-PDL1 siRNA complexation with F8 LNPsDocket No. 32917 / 70821

[0059] Preparation of formulation mLuc F8f (EIM) using microfluidic mixing

[0060] Initially, DOTAP (Avanti Polar lipids, Lot#890890P-500MG B 180), distearoyl phosphatidyl choline (DSPC, Avanti, Lot#850365P-500MG R-178), cholesterol (700100P- 100mg, Avanti polar lipids, lot#700100P-100MG-B-036), dioleoyl phosphatidyl ethanolamine (50.22 mg, DOPE, Avanti polar lipids, lot #850725P-200MG-C-446), and dimyristoyl glycerol (DMG) PEG 2000 (DMG-PEG2000, Avanti, lot #880151 P-1 G-F-024) were dissolved in ethanol (3 mL, molecular biology grade BP2818-500, Fisher bioreagents, Lot #224553). Targeted lipid (folated, 12.5 mg) was dissolved in heptane (2 mL), sonicated and warmed to 50 °C, and then added to the lipid mixture. For dissolving cholesterol, an additional 1 .5 mL heptane was added to the Eppendorf tube containing cholesterol followed by warming the solution to 50 °C for 3 minutes to completely dissolve cholesterol. Solubilized cholesterol was then added to the beaker containing other lipids and warmed to 50 °C. A glass syringe (Fortuna®, Optima®, 10 mL, Lot# F00037) was then filled with ethanolic solution of lipids. Aqueous phase containing 30 mL UltraPure distilled water (Invitrogen, 500 mL, Lot# 242907) contained in a 100 mL glass beaker (Bomex®) was warmed to 50 °C under water bath equipped with hot plate stirrer (Thermo, XLD-T100S) maintained at the stirring rate of 1000 rpm. Ethanolic solution was then added dropwise to the aqueous solution under stirring and maintained at 50 °C under flume hood. Both the beaker and glass syringe were further rinsed with additional 1 mL molecular grade ethanol, warmed to 50 °C for 3 minutes, and added dropwise to the aqueous solution maintained at 50 °C. The hydroalcoholic solution was stirred at 50 °C for further 2 hours under hood for ethanol removal. The final volume of the liposomal suspension obtained was approximately 15 mL. The liposomal formulation was dialyzed using dialysis membrane (Slide-A-Lyzer™ G3 Dialysis Cassettes, 3.5k MWCO, 15 mL, Ref: A52967, Lot #FP2868359) against PBS overnight (kept under stirring (Thermo Fisher, 1000 rpm and room temperature in beaker). The dialyzed formulation was homogenized using microfluidizer 110 S (Microfluidics™) for 10 mins. Microfluidized formulation was made up to 20 mL with PBS. Microfluidized liposomes were evaluated for their size distribution, polydispersity indices (PDI), and zeta potential measurements (FIG. 2). Formulations will be complexed with Luc mRNA and evaluated for encapsulation efficiency by electrophoresis and biodistribution profile using I VIS bioluminescence imaging.Docket No. 32917 / 70821

[0061] TABLE 4: Formulation F8f (EIM) lipid composition

[0062] Preparation of formulation mLuc F8f NM using microfluidic mixing

[0063] Initially, lipid mixture in ethanol was made. Briefly, DOTAP (Lot#890890P 500MG- B-180), distearoyl phosphatidyl choline (DSPC, Avanti, Lot#850365P-500MG-D-178), cholesterol (Avanti, lot#700107P-100MG-G 112), dioleoyl phosphatidyl ethanolamine (DOPE, Avanti, lot #850725P 200MG-C-446), dimyristoyl glycerol (DMG) PEG 2000 (DMG- PEG2000, Avanti, lot #880151 P-1 G-F-024), and targeted lipid were dissolved in ethanol heptane mixture (v / v). The targeted (folated) lipid was initially dissolved in heptane 2 mL, sonicated, and warmed to 50 °C. All lipids were weighed according to Table 5 and dissolved in ethanol in a glass vial and made up to 5 mL with ethanol serving as the lipid mixture. One mL ethanolic lipid mixture was used to achieve an n / p ratio of 4.5 between DOTAP (n) and mRNA (p), and 13.09 w / w ratio of DOTAP to mRNA. After completing the lipid mixture, Luc mRNA (125 ug) was dissolved in 3 mL of citrate buffer (60 mM) contained in a 10 mL falcon tube and kept under ice. The aqueous and lipid mixture contained in the organic solvent was microfluidized at a total flow rate of 12:1 and aqueous to organic phase ratio of 3:1 . Liposomal Luc mRNA LNPs obtained using the Nanoassemblr microfluidizer were dialyzed using dialysis cassettes (Thermo Scientific™ Slide-A-Lyzer™ G3 Dialysis Cassettes, 3.5K MWCO, 3 mL, Catalog number: A52966) against distilled PBS for two washes every three hours (kept in a beaker and magnetic bead rotation (Thermo Fisher plate, 500 rpm and refrigeration in beaker for each washes)), and third wash was performed overnight under refrigeration condition. The formulation Luc mRNA F8 LNPs were then concentrated using Amicon filters (30kDa cut off) under centrifugation at 800 g, 2 hours down (4°C, acceleration and deacceleration set to 4) to 1 mL to attain a 25 ug / mL concentration of Luc mRNA in F8 LNP formulation. The formulation was bath sonicated for 3 minutes under ice bath.Microfluidized-sonicated liposomes were evaluated for their size distribution, polydispersity indices (PDI), and zeta potential measurements using Zetasizer and NanoSight’s NTA analysis (FIG. 2).Docket No. 32917 / 70821

[0064] TABLE 5: Formulation mLuc F8f NM lipid composition

[0065] Preparation of formulation co-F8f LNPs

[0066] This paragraph describes preparation F8f LNPs comprising anti-PD-L1 siRNA plus GI261 total tumor RNA (TTRNA) using NanoAssemblr™ Ignite™ microfluidizer. Initially, lipid mixture in ethanol was made. Briefly, DOTAP (Lot#890890P 500MG C 180), distearoyl phosphatidyl choline (DSPC, Avanti, Lot#850365P 500MG D-178), cholesterol (Avanti, lot#700107P-100MG-G-112), dioleoyl phosphatidyl ethanolamine (DOPE, Avanti, lot #850725P-200MG-C-446), dimyristoyl glycerol (DMG) PEG 2000 (DMG-PEG2000, Avanti, lot #880151 P-1 G-F-024), and targeted lipid were dissolved in ethanol heptane mixture (v / v). The targeted lipid was initially dissolved in heptane 2 mL, sonicated, and warmed to 50 °C. 400 uL of the targeted lipid in heptane was then introduced into the lipid phase dissolved in ethanol according to Table 6. All lipids were weighed according to Table 6 and dissolved in ethanol in a glass vial and made up to 1 mL with ethanol serving as the lipid mixture to achieve an n / p ratio of 4.5 between DOTAP (n) and siRNA (p), and 13.09 w / w ratio of DOTAP to siRNA. After completing the lipid mixture, anti-PD-L1 siRNA (240 ug) and GL261 (glioma cell line) TTRNA (200 ug) were dissolved in 3 mL of citrate buffer (60 mM) contained in a 5 mL sterile Eppendorf tube and kept under ice. The aqueous and lipid mixture contained in the organic solvent was microfluidized at a total flow rate of 12:1 and aqueous to organic phase ratio of 3:1 . The F8f NM LNPs were then concentrated using Amicon filters (30kDa cut off) under centrifugation at 1000 g, 1 hour down (4 °C, acceleration and deacceleration set to 4) to 1 mL to attain a 240 and 200 ug / mL concentration of anti-PD-L1 siRNA and GL261 TTRNA, respectively, in the F8f LNP formulation. Liposomal siRNA LNPs were then dialyzed using dialysis cassettes (Thermo Scientific™ Slide-A-Lyzer™ G3 Dialysis Cassettes, 3.5K MWCO, 15 mL, Catalog number: A52967) against distilled PBS twice (kept in a beaker and magnetic bead rotation (Thermo Fisher plate, 1000 rpm)) and refrigeration in beaker for each wash) each for the duration of 12 hours. Microfluidized liposomes were evaluated for their size distribution, polydispersity indices (PDI) , and zeta potential measurements using Zetasizer and NanoSight’s NTA analysis (FIG. 3).Docket No. 32917 / 70821

[0067] TABLE 6: Formulation co-F8f lipid composition

[0068] Kluc derived TTRNA F8f LNPs (kTTRNA F8f_02)

[0069] Initially, lipid mixture in ethanol was made. Briefly, DOTAP (Lot#890890P-500MG- B 180), distearoyl phosphatidyl choline (DSPC, Avanti, Lot#850365P-500MG-D-178), cholesterol (Avanti, lot#700107P-100MG-H-036), dioleoyl phosphatidyl ethanolamine (DOPE, Avanti, lot #850725P-200MG-C-446), dimyristoyl glycerol (DMG) PEG 2000 (DMG- PEG2000, Avanti, lot #880151 P-1 G-F-024), and targeted lipid (6 mg) were dissolved in heptane mixture (400 uL). The targeted lipid was initially dissolved in heptane, sonicated, and warmed to 50 °C. All lipids were weighed according to Table 7 and dissolved in ethanol in a glass vial and made up to 2 mL with ethanol serving as the lipid mixture. After completing the lipid mixture, Kluc tumor (high-grade glioma tumor cell line) derived TTRNA (750 ug) was dissolved in 9 mL of citrate buffer (60 mM) contained in a 15 mL of sterile tube and kept under ice. The aqueous and lipid mixture contained in the organic solvent was microfluidized at a total flow rate of 12:1 and aqueous to organic phase ratio of 9:3. The F8f LNPs were then concentrated using Amicon filters (30 kDa cut off) under centrifugation at 1000 g, 1 hour down (4°C, acceleration and deacceleration set to 4) to 6 mL to attain a 125 ug / mL concentration of Kluc TTRNA in F8 LNP formulation. A dose of 12.5 ug / 100 uL will be injected into the tail of implanted mice. Liposomal TTRNA F8f LNPs were then dialyzed using dialysis cassettes (Thermo Scientific™ Slide-A-Lyzer™ G3 Dialysis Cassettes, 3.5K MWCO, 3 mL, Catalog number: A52967) against distilled PBS twice (kept in a beaker and magnetic bead rotation (Thermo Fisher plate, 1000 rpm) and refrigeration in beaker for each washes) each for the duration of 4 hours, and a third wash was performed overnight.Microfluidized liposomes were then evaluated for their size distribution, polydispersity indices (PDI), and zeta potential measurements using Zetasizer and NanoSight’s NTA analysis (FIG. 3).Docket No. 32917 / 70821

[0070] TABLE 7: Formulation composition of kTTRNA F8f_02 LNPs

[0071] Summary

[0072] The luciferase mRNA (mLuc) complexed F7 and F8 LNPs prepared using the ethanol injection solvent evaporation - microfluidization (EIM) demonstrated positive surface zeta potential values (45-50 mV), and particle size and PDI values between 166-236 nm and 0.35-0.43 respectively (Figure 2). Formulation F8f prepared using the microfluidic process demonstrated relatively lower particle size (86.3 ± 44.0 nm), PDI (0.03 ± 0.04), and zeta potential values (27.58 ± 0.967 mV) (Figure 2). Similarly, formulation F8f complexed with Kluc derived TTRNA (kTTRNA F8f_02) demonstrated lower particle size distribution (NTA measurement, 204.7 ± 76.0 nm), polydispersity (0.1812 ± 0.02), and a positive surface zeta potential values (47.37 ± 0.851 mV) (Figure 3). Oo-F8f LNPs loaded with anti-PD-L1 siRNA and TTRNA showed relatively higher particle size distribution (308 ± 99.4 nm) by NTA measurement, a positive surface zeta potential (25.96 ± 0.454 mV), and a polydispersity of 0.336 ± 0.045 (Figure 3).

Claims

Docket No. 32917 / 70821What is claimed is:1 . A method of inducing an immune response to a tumor, the method comprising administering to a subject in need thereof a liposome nanoparticle comprising (i) nucleic acids that inhibit the expression of PD-L1 and (ii) tumor RNA.

2. The method of claim 1 , wherein the liposome nanoparticle comprises one or more cationic lipids, one or more neutral lipids, and one or more PEG-modified lipids.

3. The method of claim 2, wherein the cationic lipid is 1 ,2-Dioleoyl-3- trimethylammoniumpropane (DOTAP).

4. The method of claim 2 or claim 3, wherein the liposome nanoparticle comprises about 0.5% to about 15% on a molar basis of the neutral lipid.

5. The method of any one of claims 2-4, wherein the neutral lipid is dioleoylphosphatidylethanolamine (DOPE), disteroylphosphatidyl choline (DSPC), or a combination thereof.

6. The method of any one of claims 2-5, wherein the liposome nanoparticle comprises from about 0.5% to about 20% on a molar basis of PEG-modified lipid.

7. The method of any one of claims 2-6, wherein the PEG-modified lipid is PEG- distearoyl glycerol.

8. The method of any one of claims 1 -7, wherein the liposome nanoparticle comprises a sterol.

9. The method of claim 8, wherein the liposome nanoparticle comprises from about 5% to about 50% on a molar basis of the sterol.

10. The method of any one of claims 1 -9, wherein the liposome nanoparticle comprises a folated lipid.11 . The method of claim 10, wherein the folated lipid is distearoylphosphatidyl ethanolamine (DSPE)-PEG(2000)-folate, dipalmitoylphosphatidyl ethanolamine (DPPE)- PEG(2000)-folate, cholesterol PEG folate, folated stearylamine, or a combination thereof.

12. The method of claim 1 , wherein the liposome nanoparticle comprises disteroylphosphatidyl choline (DSPC), 1 ,2-Dioleoyl-3-trimethylammoniumpropane (DOTAP), cholesterol, dioleoylphosphatidylethanolamine (DOPE), and PEG-distearoyl glycerol (PEG- DMG).

13. The method of claim 12, wherein the liposome nanoparticle comprises 2.5- 100 w% DOTAP, 0.5-18 w% DSPC, 1 -34 w% cholesterol, 2-80 w% DOPE, and 0.5-8 w% PEG-DMG.Docket No. 32917 / 7082114. The method of claim 12, wherein the liposome nanoparticle comprises 2.62- 100 w% DOTAP, 0.59-7 w% DSPC, 1 .12-34 w% cholesterol, 2.5-76 w% DOPE, and 0.07-6 w% PEG-DMG.

15. The method of any one of claims 12-14, wherein the liposome nanoparticle further comprises about 0.5-15 w% folated lipid.

16. The method of claim 15, wherein the liposome nanoparticle further comprises about 0.6-13 w% folated lipid.

17. The method of any one of claims 1 -16, wherein the liposome nanoparticle comprises an n / p ratio for lipid to RNA of about 4.5:1 .

18. A liposome nanoparticle comprising disteroylphosphatidyl choline (DSPC), 1 ,2-Dioleoyl-3-trimethylammoniumpropane (DOTAP), cholesterol, dioleoylphosphatidylethanolamine (DOPE), and PEG-distearoyl glycerol (PEG-DMG), and further comprising (i) nucleic acids that inhibit the expression of PD-L1 and (ii) tumor RNA.

19. The liposome nanoparticle of claim 18, wherein the liposome nanoparticle comprises 2.5-100 w% DOTAP, 0.5-18 w% DSPC, 1-34 w% cholesterol, 2-80 w% DOPE, and 0.5-8 w% PEG-DMG.

20. The liposome nanoparticle of claim 18, wherein the liposome nanoparticle comprises 2.62-100 w% DOTAP, 0.59-7 w% DSPC, 1 .12-34 w% cholesterol, 2.5-76 w% DOPE, and 0.07-6 w% PEG-DMG.21 . The liposome nanoparticle of any one of claims 18-20, further comprising a folated lipid.

22. The liposome nanoparticle of claim 21 , comprising about 0.5-15 w% folated lipid.

22. The liposome nanoparticle of claim 21 , comprising about 0.6-13 w% folated lipid.

23. The liposome nanoparticle of any one of claims 18-22 wherein the liposome nanoparticle comprises an n / p ratio for lipid to RNA of about 4.5:1 .

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