Lipid-nanoparticle delivery of sirna to treat brain tumors

Abstract

The disclosure provides a liposome nanoparticle, a pharmaceutical composition comprising liposome nanoparticles, and a method of treating brain cancer. In some aspects, the liposome nanoparticle comprising a folate bearing lipid and nucleic acid molecules (e.g., siRNA or shRNA) that reduce the expression of H2.0 Like Homeobox (HLX). The liposome nanoparticle optionally comprises a diameter of about 50 nm to about 400 nm. In some aspects, the pharmaceutical composition comprises the liposome nanoparticles and a pharmaceutically acceptable carrier, diluent, or excipient. In some aspects, the method of treating brain cancer comprises administering to a subject in need thereof the liposome nanoparticle.

Description

Docket No. 32917 / 70713LIPID-NANOPARTICLE DELIVERY OF SIRNA TO TREAT BRAIN TUMORSFIELD

[0001] The disclosure relates to lipid nanoparticles comprising an HLX inhibitor and methods of use.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 696,591 filed on September 19, 2024, the disclosure of which are fully incorporated herein by reference.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0003] A Sequence Listing, which is part of the present disclosure, is submitted concurrently with the specification. The name of the file containing the Sequence Listing is “70713P_SeqListing.xml”. The Sequence Listing was created on August 25, 2025 and is 6,322 bytes in size. The subject matter of the Sequence Listing is incorporated by reference herein in its entirety.GOVERNMENT SUPPORT CLAUSE

[0004] This invention was made with government support under Grant Number R37 CA251978, awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0005] Medulloblastoma (MB) is the most common malignant pediatric brain tumor.Molecular profiling studies have subcategorized MB into four main molecular subgroups: wingless pathway (WNT) activated (associated with mutations in CTNNB1 and APC), sonic hedgehog pathway (SHH) activated (associated with mutations in PATCH1 , TP53 + / -), group 3 (G3), and group 4 (G4). While the main common driver pathways for G3 and G4 MB have not been identified, these groups are characterized by c-Myc and MYCN signatures, respectively. Among the four subgroups, G3 is the most aggressive MB with a 45%-60% five-year survival rate, while other subgroups (WNT >90%, 6 SHH 60%-80%,7 and group 4 75%-80%5) have more favorable prognoses.

[0006] The standard treatment for MB includes surgical resection, radiotherapy, and chemotherapy. Although craniospinal irradiation after surgery improves long-term outcomes, radio-resistance development hampers therapeutic efficacy and survival.

[0007] Even with advanced molecular classification, robust candidate therapeutic targets for the different subgroups, especially G3 and G4 MB, have yet to be identified.Docket No. 32917 / 70713SUMMARY

[0008] Disclosed is a liposome nanoparticle comprising a folate bearing lipid and nucleic acid molecules (e.g., siRNA or shRNA) that reduce the expression of H2.0 Like Homeobox (HLX). In some aspects, the liposome nanoparticle comprises a diameter of about 50 nm to about 400 nm.

[0009] In some aspects, the liposome nanoparticle further comprises DOTAP, DSPC, cholesterol, DOPE, and / or PEG-DMG (e.g., DOTAP, DSPC, cholesterol, DOPE, and PEG- DMG). In some aspects, the liposome nanoparticle comprises (a) about 34-100 w% DOTAP; and / or (b) about 7-12 w% DSPC; and / or (c) about 14-22 w% cholesterol; and / or (d) about 8- 34 w% DOPE; and / or (e) about 0.9-6 w% PEG-DMG; and / or (f) about 5-10 w% folate bearing lipid. In some aspects, the liposome nanoparticle comprises about 8.3 w% folate bearing lipid.

[0010] Optionally, the liposome nanoparticle comprises a positively charged surface and an interior comprising (i) a core and (ii) at least two nucleic acid layers comprising the siRNA or shRNA, wherein each nucleic acid layer is positioned between a cationic lipid bilayer.

[0011] In some aspects, the liposome nanoparticle comprises siRNA, and the siRNA comprises a sense strand comprising a nucleic acid sequence of SEQ ID NO:2 and / or the siRNA comprises an antisense strand complementary to HLX mRNA and comprises a nucleic acid sequence of SEQ ID NO: 3.

[0012] In some aspects, the liposome nanoparticle is prepared by mixing the nucleic acid molecules (e.g., siRNA or shRNA) and the cationic lipid at a nucleic acid molecule:cationic lipid ratio of about 1 to 4.5, about 1 to 9. In some aspects, the liposome nanoparticle comprises a diameter of about 50 nm to about 200 nm.

[0013] Also disclosed is a pharmaceutical composition comprising a plurality of liposome nanoparticles and a pharmaceutically acceptable carrier, diluent, or excipient. In some aspects, the pharmaceutically acceptable carrier, diluent or excipient is selected from the group consisting of acidifying agents, antiseptics, binders, disintegrants, stabilizing agents, and preservatives.

[0014] Also disclosed is a method of treating brain cancer in a subject in need thereof, the method comprising administering to the subject a liposome nanoparticle comprising nucleic acid molecules that reduce the expression of H2.0 Like Homeobox (HLX). In some aspects, the brain cancer is a glioma or a medulloblastoma. In some aspects, the glioma is selected from the group consisting of astrocytoma, low-grade glioma, high-grade glioma, and glioblastoma.Docket No. 32917 / 70713

[0015] In some aspects, the medulloblastoma is a Grade 3 medulloblastoma, and / or the astrocytoma is a Grade III astrocytoma.

[0016] In some aspects, the subject is an adult greater than 18 years old or the subject is less than 18 years old.

[0017] In some aspects, the liposome nanoparticle is administered to the subject parenterally or subcutaneously and / or the liposome nanoparticle is administered to the subject systemically.

[0018] In some aspects, the liposome nanoparticle is administered in a sterile pharmaceutical composition comprising about 1010 liposome nanoparticles per mL to about 1015 liposome nanoparticles per mL.

[0019] It should be understood that, while various embodiments in the specification are presented using “comprising” language, under various circumstances, a related embodiment may also be described using “consisting of” or “consisting essentially of” language. The disclosure contemplates embodiments described as “comprising” a feature to include embodiments which “consist of” or “consist essentially of” the feature. The term “a” or “an” refers to one or more. For example, “a bispecific antibody construct” is understood to represent one or more bispecific antibody constructs. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein. The term “or” should be understood to encompass items in the alternative or together, unless context unambiguously requires otherwise.

[0020] It should also be understood that when describing a range of values, the disclosure contemplates individual values found within the range. For example, “about 5-10 w% folate bearing lipid,” could be, but is not limited to, 5.0%, 5.5%, 6.2%, 8.3%, etc., and any value in between such values. In any of the ranges described herein, the endpoints of the range are included in the range. However, the description also contemplates the same ranges in which the lower and / or the higher endpoint is excluded. When the term “about” is used, it means the recited number plus or minus 5%, 10%, or more of that recited number. The actual variation intended is determinable from the context.

[0021] Additional features and variations of the invention will be apparent to those skilled in the art from the entirety of this application, including the figures and detailed description, and all such features are intended as aspects of the invention. Likewise, features of the invention described herein can be re-combined into additional embodiments that also are intended as aspects of the invention, irrespective of whether the combination of features is specified as an aspect or embodiment of the invention. The entire document is intended to be related as a unified disclosure, and it should be understood that all combinations ofDocket No. 32917 / 70713 features described herein (even if described in separate sections) are contemplated, even if the combination of features is not found together in the same sentence, or paragraph, or section of this document. Also, only such limitations which are described herein as critical to the invention should be viewed as such; variations of the invention lacking limitations which have not been described herein as critical are intended as aspects of the invention.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIGs. 1 A and 1 B are bar graphs illustrating that long non-coding RNA HLX-2-7 (lnc-HLX-2-7) is highly increased in both Group 3 MB cell lines (FIG. 1 A, left graph) and patient derived xenograft samples (FIG. 1 B, right graph) compared to other groups. Foldchange relative to cerebellum is provided on the y-axis. Cell lines / xenografts are indicated on the x-axis.

[0023] FIGs. 2A-2B are graphs illustrating the results of lnc-HLX-2-7 knock down studies. As described in Example 1 , a CRISPR / Cas9 system to knock-down lnc-HLX-2-7 in D425 and MED211 cells. FIG. 2A demonstrates that the CRISPR / Cas9 system was effective in knocking down lnc-HLX-2-7; fold change compared to controls was significant. Cell viability assays were performed and the results are illustrated in FIG. 2B. Number of living cells is provided on the y-axis; days post knock down is provided on the x-axis. Control samples are noted as circles while lnc-HLX-2-7 knock down samples are noted as squares. In both cell lines, lnc-HLX2-7 knock down was associated with a statistically significant growth reduction.

[0024] FIGs. 3A and 3B are graphs illustrating the results of intracranial studies. Intracranial MB xenografts were established in NOD-SCID mice using control cells (CTRL) and lnc-HLX-2-7 KD cells containing a luciferase reporter. Weekly analysis by I VIS imaging revealed significantly reduced tumor growth in mice transplanted with lnc-HLX-2-7 KD cells compared to control cells. Total photon bioluminescense (photons / seconds) is provided on the y-axis while days post-implantation is provided on the x-axis. Control cells are noted as circles while knock cells are represented by squares. Results of studies using D425 cells are provided in FIG. 2A, and results of studies using MED211 cells are provided in FIG. 2B.

[0025] FIGs. 4A and 4B are graphs illustrating results of an HLX functional study. FIG. 4A illustrates expression levels of HLX in patient subgroups WNT, SHH, G3, G4, and normal subjects. FIG. 4B is a line graph illustrating overall survival in patients classified as “HLX high” (bottom line) and “HLX low” (top line). Overall survival of HLX high MB patients is reduced.

[0026] FIGs. 5A-5D illustrate results from a knock down study of HLX to confirm its functional role. FIG. 5A is a bar graph illustrating reduction of HLX following knock down in D425 and MED5211 cell lines. Reduced HLX mediated by short hairpin RNA is confirmed inDocket No. 32917 / 70713FIG. 5B. FIG. 5C is line graphs demonstrating that knock down of HLX reduces tumor cell growth. The number of living cells is provided on the y-axis and time is provided on the x- axis. In both D425 cells and MED211 cells, knock down of HLX significantly reduced viable cells. FIG. 5D includes line graphs illustrating the effect of HLX knock down on tumor growth after implantation. Total photon bioluminescense (photons / seconds) is provided on the y- axis while days post-implantation is provided on the x-axis. For both D425 cells and MED211 cells, knock down of HLX resulted in reduced growth.

[0027] FIG. 6 is an illustration of a study using a liposome nanoparticle comprising siRNA targeting HLX (si-HLX).

[0028] FIG. 7 is an illustration of accumulation of LNP-si-HLX described in Example 1 in brain tumors. Packaging of the siRNA into the nanoparticle of the disclosure significantly increased accumulation in the brain compared to other parts of the body.

[0029] FIGs. 8A and 8B illustrate data from additional preclinical in vivo studies described in Example 1 . D425 cells were transplanted into mice and established for 14 days. LNP-si- CTRL (control) or LNP-si-HLX (siRNA targeting HLX) was administered every three days at a dose of 25 ug of siRNA, as illustrated in FIG. 8A. FIG. 8B is a line graph comparing total photon bioluminescense (photons / seconds; y-axis) and days after the first administration (x- axis). Tumor size became much smaller in LNP-si-HLX treated mice.DETAILED DESCRIPTION

[0030] The disclosure is based, at least in part, on the identification of a transcription factor, HLX (H2.0 Like Homeobox), upregulated in G3 MB tumors and assessment as a therapeutic target. It was determined that HLX is highly and specifically expressed in G3 MB patients, cell lines, and PDXs. Further it is demonstrated herein that liposome nanoparticles comprising nucleic acid molecules that inhibit HLX are effective in promoting tumor cell death and shrinking brain tumors (e.g., medulloblastoma, such as G3 medulloblastoma) in vivo. Indeed, the results demonstrate preclinical efficacy and safety for the nanoparticles of the disclosure in the treatment of brain cancer, including medulloblastoma, such as G3 medulloblastoma. The disclosure provides materials and methods for treating a brain cancer, such as 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 reduce the expression of HLX (e.g., RNA-based inhibitor of HLX, such as siRNA that targets HLX such that translation is inhibited and reduced amounts of HLX transcription factor are produced).

[0031] The liposome nanoparticles of the disclosure comprise a lipid and nucleic acids (such as siRNA or shRNA). In some aspects, the liposome nanoparticle comprises a folateDocket No. 32917 / 70713 bearing lipid and nucleic acids (e.g., siRNA or shRNA) that reduce the expression of H2.0 Like Homeobox (HLX). Optionally, the liposome nanoparticle comprises a diameter of about 50 nm to about 400 nm.

[0032] 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, about130 nm ± 5 nm, about 135 nm ± 5 nm, about 140 nm ± 5 nm, about 145 nm ± 5 nm, about150 nm ± 5 nm, about 155 nm ± 5 nm, about 160 nm ± 5 nm, about 165 nm ± 5 nm, about170 nm ± 5 nm, about 175 nm ± 5 nm, about 180 nm ± 5 nm, about 190 nm ± 5 nm, about200 nm ± 5 nm, about 210 nm ± 5 nm, about 220 nm ± 5 nm, about 230 nm ± 5 nm, about240 nm ± 5 nm, about 250 nm ± 5 nm, about 260 nm ± 5 nm, about 270 nm ± 5 nm, about280 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, a pharmaceutical composition comprises a heterogenous mixture of liposome nanoparticles comprising diameter ranging from about 50 nm to about 400 nm. Optionally, the pharmaceutical composition comprises a heterogeneous mixture of nanoparticles ranging from about 70 nm to about 200 nm in diameter.

[0033] In exemplary aspects, the nanoparticles comprise a cationic lipid or an anionic lipid. In various aspects, the nanoparticle comprises a folated lipid or a folate bearing lipid (“folated lipid” and “folate bearing lipid” are used interchangeably herein). In some aspects, a folated lipid or a folate bearing lipid comprises a lipid that has been conjugated with a folate or folate derivative including folic acid. Folate-conjugated lipids (e.g., folated lipid or folate bearing lipid) may be generated via any suitable method, including the method described in Example 2. For instance, folated lipid may be synthesized by reacting the carboxylic groupDocket No. 32917 / 70713 of folic acid to a complementarily reactive lipid or cholesterol. Non-limiting 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, although other folate bearing lipids are contemplated. In some aspects, the nanoparticle comprises about 1 -20 w% (weight percent) of folated lipid, about 5-15 w% of folated lipid, or about 5-10 w% of folated lipid. In some aspects, the nanoparticle comprises about 1 w% of folated lipid, about 2 w% of folated lipid, about 3 w% of folated lipid, about 4 w% of folated lipid, about 5% w% of folated lipid, about 6 w% of folated lipid, about 7 w% of folated lipid, about 8 w% of folated lipid, about 8.3 w% of folated lipid, about 9 w% of folated lipid, about 10 w% of folated lipid, about 11 w% of folated lipid, about 12 w% of folated lipid, about 13 w% of folated lipid, about 14 w% of folated lipid, about 15 w% of folated lipid, about 16 w% of folated lipid, about 17 w% of folated lipid, about 18 w% of folated lipid, about 19 w% of folated lipid, or about 20 w% of folated lipid.

[0034] In exemplary aspects, the nanoparticle comprises a surface and an interior comprising (i) a core and (ii) at least two nucleic acid layers, optionally, more than two nucleic acid layers. 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 lipid bilayer (e.g., cationic lipid bilayer). In exemplary aspects, the nanoparticle comprises at least four or five nucleic acid layers, each of which is positioned between a lipid bilayer (e.g., 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.

[0035] In various aspects, the presently disclosed nanoparticle comprises a positively- charged surface. In some instances, the positively-charged surface comprises a lipid layer, e.g., a cationic lipid layer. In various aspects, the outermost layer of the nanoparticle comprises a cationic lipid bilayer. Optionally, the cationic lipid bilayer comprises, consists essentially of, or consists of DOTAP. In various instances, the surface comprises a plurality of hydrophilic moieties of the cationic lipid of the cationic lipid bilayer. In some aspects, theDocket No. 32917 / 70713 core comprises a cationic lipid bilayer. In various instances, the core lacks nucleic acids, optionally, the core comprises less than about 0.5 wt% nucleic acid.

[0036] In exemplary instances, the nanoparticle is characterized by a zeta potential of 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. The zeta potential is one parameter which distinguishes the nanoparticles of the present disclosure and those described in Sayour et al., Oncoimmunology 6(1): e1256527 (2016).

[0037] In various aspects, the presently disclosed nanoparticles comprise a negatively- charged surface and an interior comprising (i) a core and (ii) at least two nucleic acid (e.g., RNA) layers, wherein each nucleic acid (e.g., RNA) layer is positioned between an anionic lipid bilayer. In some instances, the negatively-charged surface comprises a lipid layer, e.g., anionic lipid layer. In various aspects, the nanoparticle has a negative zeta potential. In some aspects, the nanoparticles comprising a negatively charged surface may be generated by mixing RNA molecules and lipid nanoparticles at ratios to oversaturate lipid nanoparticles with negative charge.

[0038] In some instances, the nanoparticle is characterized by a negative zeta potential. For instance, the nanoparticle, in various aspects, is characterized by a zeta potential of about -10 mV to about -70 mV, -10 mV to about -60 mV, -10 mV to about -50 mV, e.g., about -10 mV to about -45 mV, about -10 mV to about -40 mV, about -10 mV to about -35 mV, about -10 mV to about -30 mV, about -15 mV to about -50 mV, about -15 mV to about -45 mV, about -15 mV to about -30 mV, about -20 mV to about -60 mV, about -20 mV to about - 50 mV, about -20 mV to about -45 mV, about -20 mV to about -40 mV, about -20 mV to about -35 mV, about -20 mV to about -30 mV, about -30 mV to about -60 mV, about -40 mV to about -50 mV, about -50 mV to about -60 mV, or about -25 mV to about -30 mV. In exemplary aspects, the nanoparticle has a zeta potential of about -20 mV to about -30 mV. In exemplary instances, the nanoparticle is characterized by a zeta potential of about -40 mV to about -65 mV, e.g., about -45 mV to about -60 mV, about -50 mV to about -60 mV, or about -50 mV to about -58 mV.

[0039] In some embodiments, the nanoparticle comprises cationic lipid, and at least one 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 byDocket No. 32917 / 70713 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-0-octadecenyl-3-trimethylammonium propane) or a derivative thereof.

[0040] In some aspects, the nanoparticle 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. Examples of neutral lipids include, but are not limited to, DSPC, POPC, DPPC, DOPE, and 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 nanoparticle does not comprise a neutral lipid. In some embodiments, the formulation 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.

[0041] In some embodiments, the formulation includes 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) (also referred herein as PEG-C14 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).

[0042] 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 34- 100 w% DOTAP, 7-12 w% DSPC, 14-22 w% cholesterol, 8-34 w% DOPE, 0.9-6 w% PEG-Docket No. 32917 / 70713DMG, and / or about 5-10 w% folate bearing lipid (including about 8.3 w% folate bearing lipid). In exemplary aspects, the nanoparticle comprises 34-100 w% DOTAP, 7-12 w% DSPC, 14- 22 w% cholesterol, 8-34 w% DOPE, 0.9-6 w% PEG-DMG, and about 5-10 w% of folate bearing lipid. In some aspects, the nanoparticle comprises about 34-40 w% DOTAP, about 7-10 w% DSPC, about 14-20 w% cholesterol, about 28-24 w% DOPE, about 0.9-3 w% PEG- DMG, and / or about 5-10 w% folate bearing lipid. In some aspects, the nanoparticle comprises about 34-36 w% DOTAP, about 7-9 w% DSPC, about 14-16 w% cholesterol, about 30-34 w% DOPE, about 0.9-2 w% DMG-PEG and / or about 7-9 w% folated lipid. For instance, the nanoparticle may comprise 34-100 w% DOTAP, 7-12 w% DSPC, 14-22 w% cholesterol, 8-34 w% DOPE, 0.9-6 w% PEG-DMG, and / or about 8.3 w% folated lipid.

[0043] A representative nanoparticle comprises DOTAP, DSPC, cholesterol, DOPE, DMG-PEG, and folate bearing lipid with an n / p ratio of 4.5 between DOTAP (n) and nucleic acid (e.g., siRNA) (p). For example, the nanoparticle may comprise about 34 w% DOTAP, about 7.8 w% of DSPC, about 14.8 w% of cholesterol, about 33.3 w% of DOPE, about 0.9 w% of DMG-PEG, and about 8.3 w% of folated lipid.

[0044] A representative nanoparticle comprises DOTAP, DSPC, cholesterol, DOPE, and DMG-PEG with a n / p ration of 9 between DOTAP (n) and nucleic acid (e.g., siRNA) (p). For example, the nanoparticle may comprise about 52.09 w% DOTAP, about 11 .76 w% of DSPC, about 22.2 w% cholesterol, about 8.3% DOPE, and about 5.6 w% of DMG-PEG.

[0045] 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 HLX mRNA (including sufficient complementarity to the nucleic acid sequence of SEQ ID NO: 1) to mediate RNA interference. In this respect, the siRNA interferes with HLX expression by, e.g., degrading mRNA after transcription, thereby preventing translation of the transcription factor. 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 in each strand, wherein one of the strands is sufficiently complementary to a target region of HLX to reduce translation). In various aspects, the 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.

[0046] siRNA molecules have sufficient complementarity with target sequence within HLX 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 HLX (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%Docket No. 32917 / 70713 identity, between the sense strand of the siRNA duplex and the target HLX 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 HLX 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 HLX comprises the nucleic acid sequences of rCrCrCrUrUrArArArCrUrCrGrArArCrCrCrArArUrU (SEQ ID NO: 2) and rUrUrGrGrGrUrUrCrGrArGrUrUrUrArArGrGrGrArC (SEQ ID NO: 3). In some aspects, the siRNA comprises a sense strand comprising the nucleic acid sequence of SEQ ID NO: 2. In some aspects, the siRNA comprises an antisense strand complementary to HLX mRNA and comprising a nucleic acid sequence of SEQ ID NO: 3.

[0047] In some aspects, the nucleic acid molecule is a short hairpin RNA (shRNA), specific for inhibiting the expression of HLX. 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. In some aspects, the shRNA may have sufficient complementarity with a target sequence within HLX. In some aspects, the shRNA may comprise the nucleic acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3 or may have complementarity to the nucleic acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3.

[0048] In some aspects, the liposome nanoparticles may comprise nucleic acid molecules (e.g., antisense nucleic acid molecules, siRNA, or shRNA) that targets a protein of an immune checkpoint pathway to mediate reduced expression of the protein. In various aspects, the protein of the immune checkpoint pathway is CTLA-4, PD-1 , PD-L1 , PD-L2, B7- H3, B7-H4, TIGIT, LAG3, CD112 TIM3, BTLA, or co-stimulatory receptor: ICOS, 0X40, 41 BB, or GITR. The protein of the immune-checkpoint pathway in certain instances is CTLA4, PD-1 , PD-L1 , B7-H3, B7H4, or TIM3. Immune checkpoint signaling pathways are reviewed in Pardoll, Nature Rev Cancer 12(4): 252-264 (2012).

[0049] In some aspects, the liposome nanoparticles may be prepared by mixing the nucleic acid molecules and the lipid(s) at different nucleic acid molecule: lipid ratios. ForDocket No. 32917 / 70713 example, in some aspects, the nucleic acid molecules (e.g., siRNA or shRNA) within the liposome nanoparticle are present at a nucleic acid molecule: cationic lipid (or anionic lipid) ratio of about 0.5 to about 2 (nucleic acid) to about 0.5 to about 20 (lipid). In some aspects, the nucleic acid molecules are present at a nucleic acid molecule: cationic lipid (or anionic lipid) ratio of about 1 to about 1 to about 1 to about 20. In some aspects, the nucleic acid molecules are present at a nucleic acid molecule: cationic lipid (or anionic lipid) ratio of about 1 :1 , about 1 :2, about 1 :3, about 1 :4, about 1 :4.5, about 1 :5, about 1 :6, about 1 :7, about 1 :8, about 1 :9, about 1 :10, about 1 :11 , about 1 :12, about 1 :13, about 1 :14, about 1 :15, about 1 :16, about 1 :17, about 1 :18, about 1 :19, or about 1 :20.

[0050] In some aspects, the liposome nanoparticle is prepared by mixing the nucleic acid molecules (e.g., siRNA or shRNA) and the lipids (e.g., cationic, neutral, or anionic) at a w / w ratio of about 1 :5 to about 1 :30. For example, in some aspects, the nucleic acid molecules are mixed with the lipids at a w / w ratio of about 1 :5, about 1 :6, about 1 :7, about 1 :8, about 1 :9, about 1 :10, about 1 :11 , about 1 :12, about 1 :13, about 1 :13.09, about 1 :14, about 1 :15, about 1 :16 about 1 :17, about 1 :18, about 1 :19, about 1 :20, about 1 :21 , about 1 :22, about 1 :23, about 1 :24, about 1 :25, about 1 :26, about 1 :26.19, about 1 :27, about 1 :28, about 1 :29, or about 1 :30.

[0051] As used herein, the term “nucleic acid molecule: cationic lipid ratio” is meant a mass ratio, where the mass of the nucleic acid molecule is relative to the mass of the cationic lipid (or anionic lipid, if anionic lipid is used instead of cationic lipid). Also, in exemplary aspects, the term “nucleic acid molecule: cationic lipid ratio” is meant the ratio of the mass of the nucleic acid molecule, e.g., siRNA or shRNA, added to the liposomes comprising cationic lipids (or anionic lipid, if anionic lipid is used instead of cationic lipid) during the process of manufacturing the nanoparticles of the present disclosure. In exemplary aspects, the nanoparticle comprises about 15 pg nucleic acid (e.g., siRNA or shRNA) molecules per 15 pg lipid mixture.

[0052] Also disclosed herein are methods of treating cancer comprising administering to a subject in need thereof a liposome nanoparticle of the disclosure. In some aspects, 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.Docket No. 32917 / 70713

[0053] The 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, 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.

[0054] 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., clinical 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 prophylactically 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.

[0055] 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 afterDocket No. 32917 / 70713 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.

[0056] 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 1010nanoparticles per mL to about 1015nanoparticles per mL, optionally about 1012nanoparticles ± 10% per mL.

[0057] 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, tonicityDocket No. 32917 / 70713 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.

[0058] The composition of the present disclosure can be suitable for administration by any acceptable route, including parenteral and subcutaneous routes. Other routes include intravenous, intradermal, intramuscular, intraperitoneal, intranodal and intrasplenic, for example. In exemplary aspects, the composition is suitable for systemic (e.g., intravenous) administration.

[0059] All of the references cited herein, including patents, patent applications, literature publications, and the like, are hereby incorporated in their entireties by reference.

[0060] While this invention has been described with an emphasis upon preferred embodiments, it will be obvious to those of ordinary skill in the art that variations of the preferred materials and methods may be used and that it is intended that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the invention as defined by the following claims.EXAMPLES

[0061] Example 1

[0062] To identify MB group-specific long non-coding RNAs (IncRNAs), 175 RNA-seq data was obtained from patients representing all four MB subgroups from the ICGC (International Cancer Genome Consortium) database, and a machine learning algorithm was applied to classify them. A group of IncRNAs was identified that is highly expressed and statistically significant in G3 MB patients compared to other groups. A small group of IncRNA (lnc-HLX-1 , lnc-HLX-2-7, lnc-HLX-5, and lnc-HLX-6) was determined to be the most significantly increased in G3 MB compared to other groups. Q-PCR further confirmed that lnc-HLX-2-7 is highly upregulated in both G3 MB cell lines and patient derived xenograft samples compared to other groups. See FIGs. 1 A and 1 B.

[0063] The CRISPR / Cas9 system was utilized to knock-down lnc-HLX-2-7 to confirm its oncogenic role. For this study, the G3 MB cell lines D425 and MED211 were utilized.Stable lnc-HLX-2-7-knock down cells (lnc-HLX-2-7 KD) were generated. In both cell lines,Docket No. 32917 / 70713 lnc-HLX2-7 knock down showed a statistically significant growth reduction. See FIGs. 2A and 2B.

[0064] Next, intracranial MB xenografts were established in NOD-SCID mice using control (CTRL) and lnc-HLX-2-7 KD cells containing a luciferase reporter. Weekly analysis by I VIS® imaging revealed significantly reduced tumor growth in mice transplanted with lnc-HLX-2-7 KD cells compared to control cells. The same results were achieved with a xenograft using another cell line. The results establish that lnc-HLX-2-7 has an oncogenic role in vivo. See FIGs. 3A and 3B.

[0065] Functional analysis of HLX (H2.0 Like Homeobox), the host coding gene for Inc- HLX2-7, was performed using samples from MB patients. HLX was observed to be highly upregulated in Group 3 MB patients, and high HLX expression was correlated with poor survival of MB patients. See FIGs. 4A and 4B. An HLX knock down (KD) experiment was performed to confirm its functional role. Stable HLX knock down significantly decreased the growth of G3 MB cells in vitro and in vivo. These results further establish that HLX contributes to G3 MB cell proliferation and tumor growth. See FIGs. 5A-5D.

[0066] Additional studies were performed to characterize the effect of an inhibitor of HLX (siRNA) packaged into a liposome nanoparticle (LNP-si-HLX) as described in the instant disclosure. See FIG. 6. In this regard, double-stranded si-HLX complex lipid nanoparticles (LNPs) were administered to immunocompromised mice with medulloblastomas, alone and in combination with standard-of-care regimens. D425 cells were transplanted into NOD / SCID mice for this MB model. The model is complemented by using a syngeneic, immune- competent G3 MB model, in which normal stem cells are isolated from pups infected with retroviruses encoding Myc and dominant-negative Trp53, with or without siHLX and transplanted into the cerebella of immune-competent adult mice. After 21 days, LNP-si-HLX accumulation in mouse brain tumor was observed, and anti-tumor efficacy was studied. The particles generated comprise HLX siRNA (Integrated DNA technologies™) packaged into cationic lipid pockets, which are encapsulated into phospholipid nanoparticles coated in PEG and folate. FIG. 7 shows that LNP-si-HLX specifically accumulated and retained in mice brain tumors. Additionally, in vivo implantation experiments were conducted as described in FIG. 8A. LNP-si-NC negative control (NC / CTRL; scrambled RNA (Integrated DNA technologies™)) or LNP-si-HLX was administered and antitumor effect was evaluated. As illustrated in FIG. 8B, a drastic reduction in tumor size was achieved in LNP-si-HLX treated mice.

[0067] To summarize, the results from the study establish HLX as a therapeutic target for MB, and that HLX inhibitors such as shRNA and siRNA achieve a therapeutic effect in vivo.Docket No. 32917 / 70713The effect of HLX on tumor growth in vivo was determined using intracranial MB xenografts in NOD-SCID mice. D425 Med and MED211 cells with control sh-RNA (sh-NC) and D425 Med and MED211 with sh-HLX (sh-HLX) cells were pre-infected with a lentivirus containing a luciferase reporter. Weekly evaluation of tumor growth by bioluminescence imaging revealed significantly smaller tumors in mice transplanted with HLX KD D425 Med and MED211 cells compared to mice transplanted with control cells. On day 28, tumors were harvested, cut into sections, and then subjected to Ki67 and TUNEL staining. Ki67 analysis showed reduced cell proliferation in HLX KD D425 Med cell-transplanted mice. TUNEL analysis found that HLX depletion induced a significantly higher percentage of TUNEL- positive cells than compared to mice transplanted with control cells. Kaplan-Meier plots demonstrated that the group transplanted with HLX KD D425 Med and MED211 cells had significantly prolonged survival compared to the control. The studies confirm that HLX drives G3 MB growth and is a target for treating G3 MBs. HLX is also highly expressed in other brain tumors (for example, GBM). As such, HLX is a therapeutic target for brain tumors other than medulloblastomas.

[0068] Example 2

[0069] This example describes folate targeted lipid synthesis or generation of folate bearing lipids.

[0070] An amidation reaction was carried out between stearylamine (SN, octadecylamine, cat# 8410290250, Sigma-Aldrich™) amino group and folic acid (FA, cat# F8798-5G, Sigma- Aldrich™) carboxylic group at equimolar ratio using coupling agent, N-(3- Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDAC, 03449-1 G, Sigma- Aldrich™) as a catalyst. Initially, FA (88 mg, 0.4 mM) was dissolved in 2 mL of 10 N NaOH introduced into a 250 mL RBF followed by drop-wise addition of solution of EDAC (44 mg, 0.2 mM) in 1 mL of water under constant stirring at approximately 300 rpm on a magnet stirrer (Isotemp, Fisher Scientific™) at room temperature under dark for one hour. Following this, SN in equimolar quantity (53.8 mg, 0.2 mM) dissolved in 5 mL of ethanol was added drop-wise and the mixture was kept under constant stirring under dark conditions for 48 hours. The final product was dialyzed against distilled water overnight to remove unreacted folic acid. Product was rinsed with ethanol under filtration assembly to remove excess of unreacted SN, and allowed to dry to obtain the powdered folated lipid which was then stored at -20 °C until further use.

[0071] Example s

[0072] This example describes production of anti-HLX siRNA F1 DOTAP LNP formulation.Docket No. 32917 / 70713

[0073] Briefly, cationic lipid DOTAP (2.5 mg, Lot#890890P-500MG-B-180, Avanti Polar Lipids, Inc, part of Croda International Pic) was dissolved in ethanol (1 mL, molecular biology grade BP2818-500, Lot #235726, Fisher BioReagents™). For DOTAP weight measurements, powder vial of DOTAP, 100 mg was dissolved in 4 mL ethanol, resulting in 25 mg / mL solution. All lipids were weighed according to Table 1 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 9 between DOTAP (n) and siRNA (p), and 26.19 w / w ratio of DOTAP to siRNA.

[0074] Anti-HLX siRNA (15 nmoles, 200 ug, Integrated DNA Technologies™) was dissolved in 3 mL of citrate buffer (60 mM) contained in a 5 mL of Eppendorf tube and kept under ice. Anti-HLX stock solution (119.3 nmoles, 1 .5902 mg) was initially dissolved in 1 mL of acetate buffer, and sub-aliquoted to into 8 Eppendorfs of 125 uL, each containing 200 ug of anti-HLX siRNA.

[0075] Formulation of anti-HLX siRNA LNPs obtained using the Nanoassemblr™ Ignite™ microfluidizer (Precision Nanosystems, now part of Cytiva) was dialyzed using dialysis cassettes (Thermo Scientific™ Slide-A-Lyzer™ G3 Dialysis Cassettes, 30K MWCO, Catalog number: A52967) against distilled PBS for two washes every three hours (kept under stirring (Thermo Fisher) at 1000 rpm under refrigeration in a beaker for each washes), and third wash was performed against PBS and dialyzed overnight.

[0076] Anti-HLX siRNA F1 LNPs were then concentrated using Amicon® Ultra Centrifugal filters (30kDa cut off, Lot# 00000273863, cat# UFC903024, MilliPore Sigma) under centrifugation at 800 g, 1 hour (4 °C, acceleration and deacceleration set to 4) down to 1 mL to attain a 200 ug / mL concentration of siRNA in F1 LNP formulation.

[0077] Microfluidized liposomes were evaluated for their size distribution, polydispersity indices (PDI), and zeta potential measurements using Malvern Panalytical™ Zetasizer and NanoSight's NTA analysis, followed by qualitative siRNA encapsulation efficiency by agarose gel electrophoresis.

[0078] TABLE 1

[0079] Example 4

[0080] This example describes production of Alexa HLX siRNA F8 formulation.

[0081] Briefly, DOTAP (13.0975 mg, Lot#890890P-500MG-B-180, Avanti Polar Lipids,Inc, part of Croda International Pic), distearoyl phosphatidyl choline (DSPC, Avanti, 0.59 mg,Docket No. 32917 / 70713Lot#850365P-500MG-D-178), cholesterol (1.12 mg, lot#700107P-100MG-G-112, Avanti Polar Lipids), dioleoyl phosphatidyl ethanolamine (12.555 mg, DOPE, lot #850725P- 200MG-C-446, Avanti Polar Lipids) dimyristoyl glycerol (DMG) PEG 2000 (DMG-PEG2000, 0.071 mg, lot #880151 P-1 G-F-024, Avanti Polar Lipids), and folate targeted lipid (0.625 mg) were dissolved in ethanol heptane mixture (v / v). Targeted lipid (i.e., folate bearing lipid) was initially dissolved in heptane, sonicated and warmed to 50 °C. A 200 uL of the targeted lipid in heptane was then introduced into the lipid phase dissolved in ethanol according to the table below. Complete solubilization is desired; if any component lipid did not solubilize, bath sonication was followed for 5 minutes and then lipids were warmed to 50 °C. For DOTAP weight measurements, powder vial of DOTAP, 100 mg was dissolved in 4 mL ethanol, resulting in 25 mg / mL solution. For DOPE weight measurements, 4 mL ethanol was added to the vial containing 100 mg DOPE powder resulting in 25 mg / mL solution. All lipids were weighed according to the Table 2 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.

[0082] After completing the lipid mixture, Alexa HLX siRNA (200 ug, Integrated DNA Technologies™) was dissolved in 3 mL of citrate buffer (60 mM) contained in a 5 mL of sterile Eppendorf tube and kept under ice.

[0083] The aqueous and lipid mixture contained in the organic solvent was microfluidized (Ignite™, Nanoassemblr™, Precision Nanosystems, now part of Cytiva) at a total flow rate of 12:1 and aqueous to organic phase ratio of 3:2.

[0084] Liposomal Alexa HLX siRNA LNPs obtained using the Nanoassemblr™ microfluidizer was dialyzed using dialysis cassettes (Thermo Scientific™ Slide-A-Lyzer™ G3 Dialysis Cassettes, 3.5K MWCO, 15 mL, catalog number: A52967) against distilled PBS for two washes every three hours (kept in a beaker and magnetic bead rotation (Thermo Fisher plate, 1000 rpm) and 2 - 8 °C refrigeration in beaker for each wash), and third wash was performed overnight.

[0085] Alexa HLX siRNA F8 LNPs were concentrated using Amicon® filters (30kDa cut off) under centrifugation at 800g, 2 hours down (4 °C, acceleration and deacceleration set to 4) to 0.4 mL to attain a 200 ug / mL concentration of Alexa HLX siRNA in F8 LNP formulation.

[0086] Microfluidized liposomes were evaluated for their size distribution, PDI, and zeta potential measurements using Malvern Panalytical™ Zetasizer and NanoSight’s NTA analyses, followed by qualitative alexa anti-HLX siRNA’s encapsulation efficiency by agarose gel electrophoresis.

[0087] TABLE 2Docket No. 32917 / 70713

[0088] Example 5

[0089] This example describes preparation of formulation 5.

[0090] Briefly, DOTAP (5.239 mg, Lot#890890P-500MG-N-179, Avanti Polar Lipids, Inc, part of Croda International Pic), distearoyl phosphatidyl choline (DSPC, 1.1825 mg, Lot#850365P-500MG-D-178, Avanti Polar Lipids, Inc), cholesterol (2.2329 mg, lot#700107P- 100MG-A-010, Avanti Polar Lipids, Inc), dioleoyl phosphatidyl ethanolamine (0.837 mg, DOPE, Avanti, lot #850725P-25MG-E-443), dimyristoyl glycerol (DMG) PEG 2000 (DMG- PEG2000, 0.5646 mg, lot #880151 P-1 G-F-024, Avanti Polar Lipids, Inc) were dissolved in ethanol (1 mL, molecular biology grade BP2818-500, Fisher BioReagents™, Lot #235726). Complete solubilization is desired; if any of the component lipid did not solubilize, bath sonication was performed for 5 minutes and lipids mixture was warmed to 50 °C. All lipids were weighed according to the Table 3 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 9 between DOTAP (n) and siRNA (p), and 26.19 w / w ratio of DOTAP to siRNA.

[0091] After completing the lipid mixture, anti-HLX siRNA (15 nmoles, 200 ug, Integrated DNA Technologies™) was dissolved in 3 mL of acetate buffer (3 mM, pH 5.2) contained in a 5 mL Eppendorf tube and kept under ice.

[0092] The aqueous and lipid mixture contained in the organic solvent was microfluidized (Ignite™, Nanoassemblr™, Precision Nanosystems, now part of Cytiva) at a total flow rate of 12:1 and aqueous to organic phase ratio of 3:1 .

[0093] Liposomal anti-HLX siRNA LNP formulation obtained using the Nanoassemblr™ microfluidizer was dialyzed using dialysis cassettes (Thermo Scientific™ Slide-A-Lyzer™ G3 Dialysis Cassettes, 3.5K MWCO, Catalog number: A52967) against PBS for two washes every three hours (kept under shaking (Thermo Fisher, 90 rpm) and room temperature inDocket No. 32917 / 70713 beaker for each washes), and third wash was performed against PBS and dialyzed overnight.

[0094] Anti-HLX siRNA F5 LNPs were then concentrated using Amicon® filters (30 kDa cut off) under centrifugation at 1000 g, 3 hours down to 1 mL to attain a 200 ug / mL concentration of siRNA loaded in F5 LNP formulation.

[0095] Microfluidized liposomes were evaluated for their size distribution, PDI, and zeta potential measurements using Malvern Panalytical™ Zetasizer and NanoSight’s NTA analyses, followed by qualitative alexa anti-HLX siRNA’s encapsulation efficiency by agarose gel electrophoresis.

[0096] TABLE 3

[0097] Example 6

[0098] This example describes preparation process for anti-HLX siRNA.

[0099] Briefly, DOTAP (13.0975 mg, Lot#890890P-500MG-B-180, Avanti Polar Lipids, Inc, part of Croda International Pic), distearoyl phosphatidyl choline (DSPC, 0.59 mg, Lot#850365P-500MG-D-178, Avanti Polar Lipids, Inc.), cholesterol (1.12 mg, lot#700107P- 100MG-G-112, Avanti Polar Lipids, Inc.), dioleoyl phosphatidyl ethanolamine (12.555 mg, DOPE, lot #850725P-200MG-C-446, Avanti Polar Lipids, Inc.), dimyristoyl glycerol (DMG) PEG 2000 (DMG-PEG2000, Avanti, 0.071 mg, lot #880151 P-1 G-F-024, Avanti Polar Lipids, Inc.), and folate targeted lipid (3.125 mg) were dissolved in ethanol heptane mixture (v / v). Targeted (i.e., folated) lipid (6.25 mg) was initially dissolved in heptanesonicated and warmed to 50 °C. A 500 uL of the targeted lipid in heptane was then introduced into the lipid phase dissolved in ethanol according to the table below. Complete solubilization is desired; if any of the component lipid did not solubilize, bath sonication was performed for 5 minutes and lipids mixture was warmed to 50 °C. All lipids were weighed according to Table 4 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.Docket No. 32917 / 70713

[0100] Anti-HLX siRNA (75 nmoles, 1000 ug, Integrated DNA Technologies™) was dissolved in 6 mL of citrate buffer (60 mM) contained in a 10 mL of falcon tube and kept under ice.

[0101] 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 6:4.

[0102] Liposomal Anti-HLX siRNA LNPs obtained using the Nanoassemblr microfluidizer was dialyzed using dialysis cassettes (Thermo Scientific™ Slide-A-Lyzer™ G3 Dialysis Cassettes, 3.5K MWCO, 15 mL, Catalog number: A52967) against distilled PBS for two washes every three hours (kept in a beaker and magnetic bead rotation (Thermo Fisher plate, 1000 rpm) and refrigeration in beaker for each washes), and third wash was performed overnight.

[0103] Anti-HLX siRNA F8 LNPs were then concentrated using Amicon® filters (30 kDa cut off) under centrifugation at 800 g, 1 hour down (4 °C, acceleration and deacceleration set to 4) to 5 mL to attain a 200 ug / mL concentration of Anti-HLX siRNA in F8 LNP formulation.

[0104] Microfluidized liposomes were evaluated for their size distribution, PDI, and zeta potential measurements using Malvern Panalytical™ Zetasizer and NanoSight’s NTA analyses, followed by qualitative alexa anti-HLX siRNA’s encapsulation efficiency by agarose gel electrophoresis.

[0105] TABLE 4

[0106] Example 7

[0107] This example describes production of NC siRNA F8f.

[0108] Briefly, DOTAP (13.0975 mg, Lot#890890P-500MG-B-180, Avanti Polar Lipids,Inc, part of Croda International Pic.), distearoyl phosphatidyl choline (DSPC, 0.59 mg,Docket No. 32917 / 70713Lot#850365P-500MG-D-178, Avanti Polar Lipids, Inc.), cholesterol (1.12 mg, lot#700107P- 100MG-G-112, Avanti Polar Lipids, Inc.), dioleoyl phosphatidyl ethanolamine (12.555 mg, DOPE, lot #850725P-200MG-C-446, Avanti Polar Lipids, Inc.) dimyristoyl glycerol (DMG) PEG 2000 (DMG-PEG2000, 0.071 mg, lot #880151 P-1 G-F-024, Avanti Polar Lipids, Inc.), and folate targeted lipid (3.125 mg) were dissolved in ethanol heptane mixture (v / v). Targeted lipid (3.125 mg) was initially dissolved in heptane, sonicated and warmed to 50 °C. A 500 uL of the targeted lipid in heptane was then introduced into the lipid phase dissolved in ethanol according to the Table 5 below. If any of the component lipid did not solubilize, bath sonication was performed for 5 minutes and lipids mixture was warmed to 50 °C. All lipids were weighed according to the Table 5 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.

[0109] NC siRNA (~75 nmoles, 1000 ug) was dissolved in 6 mL of citrate buffer (60 mM) contained in a 10 mL of falcon tube and kept under ice.

[0110] 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 6:4.

[0111] Liposomal NC siRNA LNPs thus obtained using the Nanoassemblr™ microfluidizer was dialyzed using dialysis cassettes (Thermo Scientific™ Slide-A-Lyzer™ G3 Dialysis Cassettes, 3.5K MWCO, 15 mL, Catalog number: A52967) against distilled PBS for two washes every three hours (kept in a beaker and magnetic bead rotation (Thermo Fisher™ plate, 1000 rpm) and refrigeration in beaker for each washes), and third wash was performed overnight.

[0112] An NC siRNA F8f LNPs were then concentrated using Amicon® filters (30kDa cut off) under centrifugation at 800g, 1 hour (4 °C, acceleration and deacceleration set to 4) down to 5 mL to attain a 200 ug / mL concentration of NC siRNA in F8 LNP formulation.

[0113] Microfluidized liposomes were evaluated for their size distribution, PDI, and zeta potential measurements using Malvern Panalytical™ Zetasizer and NanoSight’s NTA analyses, followed by qualitative alexa anti-HLX siRNA’s encapsulation efficiency by agarose gel electrophoresis.

[0114] TABLE 5Docket No. 32917 / 70713

Claims

Docket No. 32917 / 70713What is claimed is:1 . A liposome nanoparticle comprising a folate bearing lipid and siRNA or shRNA that reduce the expression of H2.0 Like Homeobox (HLX), and wherein the liposome nanoparticle comprises a diameter of about 50 nm to about 400 nm.

2. The liposome nanoparticle of claim 1 , wherein the liposome nanoparticle comprises DOTAP, DSPC, cholesterol, DOPE, and PEG-DMG.

3. The liposome nanoparticle of any of claims 1-2, wherein the liposome nanoparticle comprises(a) about 34-100 w% DOTAP; and / or(b) about 7-12 w% DSPC; and / or(c) about 14-22 w% cholesterol; and / or(d) about 8-34 w% DOPE; and / or(e) about 0.9-6 w% PEG-DMG; and / or(f) about 5-10 w% folate bearing lipid.

4. The liposome nanoparticle of claim 3, wherein the liposome nanoparticle comprises about 8.3 w% folate bearing lipid.

5. The liposome nanoparticle of any of claims 1-4, wherein the liposome nanoparticle comprises a positively charged surface and an interior comprising (i) a core and (ii) at least two nucleic acid layers comprising the siRNA or shRNA, wherein each nucleic acid layer is positioned between a cationic lipid bilayer.

6. The liposome nanoparticle of any of claims 1-5, wherein the siRNA comprises a sense strand comprising a nucleic acid sequence of SEQ ID NO:2.

7. The liposome nanoparticle of any of claims 1-5, wherein the siRNA comprises an antisense strand complementary to HLX mRNA and comprising a nucleic acid sequence of SEQ ID NO: 3.

8. The liposome nanoparticle of any of claims 1-7, wherein the liposome nanoparticle is prepared by mixing the siRNA or shRNA and the cationic lipid at a nucleic acid molecule:cationic lipid ratio of about 1 to 4.5, or about 1 to 9.

9. The liposome nanoparticle of any of claims 1-8, wherein the liposome nanoparticle comprises a diameter of about 50 nm to about 200 nm.Docket No. 32917 / 7071310. A pharmaceutical composition comprising a plurality of liposome nanoparticles according to any of claims 1-9 and a pharmaceutically acceptable carrier, diluent, or excipient.11 . The pharmaceutical composition of claim 10, wherein the pharmaceutically acceptable carrier, diluent or excipient is selected from the group consisting of: acidifying agents, antiseptics, binders, disintegrants, stabilizing agents, and preservatives.

12. A method of treating brain cancer in a subject in need thereof, the method comprising administering to the subject a liposome nanoparticle of any of claims 1-9.

13. The method of claim 12, wherein the brain cancer is a glioma or a medulloblastoma.

14. The method of any of claim 13, wherein the glioma is selected from the group consisting of astrocytoma, low-grade glioma, high-grade glioma, and glioblastoma.

15. The method of claim 13, wherein the medulloblastoma is a Grade 3 medulloblastoma.

16. The method of claim 14, wherein the astrocytoma is a Grade III astrocytoma.

17. The method of any of claims 12-16, wherein the subject is an adult greater than 18 years old.

18. The method of any of claims 12-16, wherein the subject is less than 18 years old.

19. The method of any of claims 12-18, wherein the liposome nanoparticle is administered to the subject parenterally or subcutaneously.

20. The method of any of claims 12-19, wherein the liposome nanoparticle is administered to the subject systemically.21 . The method of any of claims 12-20, wherein the liposome nanoparticle is administered in a sterile pharmaceutical composition comprising about 1010liposome nanoparticles per mL to about 1015liposome nanoparticles per mL.

Images