Method for treating miRNA-based cancer using tumor-navigating peptides

Abstract

A nanomedicine platform comprising the use of a blood-brain permeable tumor navigation probe containing a peptide p28 covalently bound to an antisense miRNA. The probe localized to human pediatric glioblastoma tumors in the brain of mice. Upon entering the cells, the probe significantly inhibited tumor cell survival by silencing the oncogenic miRNA miR-20a. Notably, systemic administration of the probe enabled complete regression of the primary tumor and a significant extension of the overall survival period without any apparent adverse effects.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 365,417, filed on May 27, 2022, the entire disclosure of which is incorporated herein by reference.

[0002] Description of Research Funded by the Federal Government This invention was made with government support under Federal Grant No. R21CA252370 awarded by the NIH / NCI R21. The government has certain rights in this invention.

[0003] Reference to the Sequence Listing This application is electronically filed in ASCII format and includes a sequence listing that is incorporated herein by reference in its entirety. The name of the ASCII copy created on May 26, 2023 is 46466 - 59.xml and its size is 61440 bytes.

Background Art

[0004] Glioblastoma is the most common primary malignant brain tumor. However, glioblastoma is less common in children than in adults, and pediatric glioblastoma (pGBM) remains a devastating disease with significant morbidity and mortality. The median survival of pGBM is 13 - 73 months, and the 5-year survival rate is less than 20%. pGBM treatment currently mainly includes gross total resection followed by local irradiation with additional chemotherapy. Despite these treatments, pGBM remains incurable. The main challenges in the development of new therapeutic agents are: i) the limitation of transport across the blood-brain barrier (BBB), ii) insufficient intracellular penetration, and iii) tumor heterogeneity. Furthermore, clinical trials in children with glioblastoma are often based on regimens that do not account for the differences in tumor biology between children and adults. In recent years, our understanding of the origin and biological characteristics of pediatric brain tumors has improved considerably through genomic and epigenomic molecular profiling. These results indicate that abnormal expression of microRNA (miRNA) has significant implications in cancer progression.

[0005] miRNAs are small (18 - 25 nucleotides) non-coding RNA molecules. Unlike small interfering RNAs (siRNAs), a single miRNA can regulate multiple target genes and thereby simultaneously regulate multiple signaling pathways, including proliferation, angiogenesis, and differentiation.

[0006] Reducing the overexpression of oncogenic miRNAs using antisense-miRNAs is a promising strategy as it selectively targets and silences both "druggable" and "undruggable" genes. However, the effective and safe delivery of antisense miRNAs to target tissues remains a major challenge for miRNA-based therapies. To develop an effective delivery system for miRNAs, biocompatible non-viral-based miRNA delivery agents such as cell-penetrating peptides (CPPs), lipids, and extracellular vesicle carriers are being explored. Among these delivery systems, CPPs are a promising strategy for improving intracellular delivery and are thought to be versatile for enhancing the permeation ability of cells or tissues when combined with other delivery vehicles. More importantly, the presence of the BBB limits the penetration of vehicles to the tumor site and reduces the delivery and thus the efficiency of miRNA-based drugs for targeting pGBM. Therefore, the development of effective carriers that can overcome these challenges is necessary, and new miRNA-based therapeutic approaches remain unmet medical needs in the absence of alternative therapeutic approaches for pGBM.

[0007] Therapeutic approaches for suppressing or restoring the expression of disease-related miRNAs, such as virus-based miRNA and anti-miRNA oligonucleotide delivery systems, have been developed. However, the safe and efficient delivery of miRNAs to target tissues remains a significant challenge for miRNA-based therapies. To overcome this challenge, biocompatible non-viral-based miRNA delivery with reduced toxicity, such as cell-penetrating peptides (CPPs), lipids, and extracellular vesicle carriers, is being explored. Among these delivery systems, CPPs are considered a promising strategy for improving intracellular delivery due to the simplicity of chemical and biological synthesis, efficient tissue penetration due to their small hydrodynamic size, and versatility in enhancing the permeation ability of cells or tissues when combined with other delivery vehicles.

[0008] Furthermore, the complexity of the brain and the presence of the physiological blood-brain barrier (BBB) limit the penetration of most molecules into the brain / tumor site, reducing the efficiency of oligonucleotide drugs. Therefore, a systematic design of vectors that can overcome these delivery challenges is necessary for the successful targeted delivery of miRNAs to brain tumor sites. Summary of the Invention

[0009] The inventors have previously developed a CPP, p28, derived from Pseudomonas aeruginosa azurin that preferentially invades cancer cells and inhibits their growth. P28 (NSC745104) has been clinically tested in two Phase I clinical trials as a single therapeutic agent, and since p28 was well tolerated without obvious adverse effects, toxicity, or immunogenicity in pediatric patients with recurrent and refractory central nervous system tumors (NCI and Pediatric Brain Tumor Consortium) and adult patients with advanced solid tumors, it received FDA orphan drug designation for the treatment of pediatric high-grade glioma and rare pediatric disease designation for the treatment of diffuse pontine glioma. The two Phase I clinical trials demonstrated the safety and non-toxicity characteristics of p28, but its efficacy was moderate. To improve efficacy, the inventors used p28 as a carrier molecule because it has great potential for an effective and safe tumor-targeting approach.

[0010] To target miR-20a, which is specifically and highly overexpressed in pGBM compared to aGBM and normal brain cells, the inventors designed a novel and unique agent, AmiR20-p28, by covalently linking the BBB-permeable tumor-navigating CPP p28 and antisense miR-20a. After systemic administration, AmiR20-p28 crosses the BBB, preferentially accumulates in pGBM tumors, and efficiently inhibits oncogenic miR-20a expression. Further experiments in a pGBM mouse model generated using drug-resistant cancer stem cells showed that AmiR20-p28 decreased tumor burden and significantly extended overall survival without obvious adverse effects. The inventors' findings develop a nanomedicine platform that includes the use of a BBB-permeable tumor-navigating peptide p28 linked to an antisense miRNA. It provides a widely applicable strategy for cancer treatment with potential for clinical translation.

[0011] Other methods, features, and / or advantages will be apparent or will become apparent to those of ordinary skill in the art upon examination of the following drawings and detailed description. Such additional methods, features, and advantages are all included within this description and are intended to be protected by the accompanying claims.

[0012] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the invention will be obtained from the following detailed description, which illustrates exemplary embodiments in which the principles of the invention are utilized, and from the accompanying drawings.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0032] pGBM cannot be histologically distinguished from adult glioblastoma (aGBM), but there are significant differences at the molecular level. Analysis of the miRNA expression profile in aGBM showed significant changes in the expression of a group of miRNAs overexpressed in aGBM, including miR-21. In contrast, the expression of miRNAs in the miR-17-92 cluster (miR-17, miR-18a, miR-19a, miR-19b-1, miR-20a, and miR-92a-1) was shown to be significantly increased in pGBM compared to aGBM and healthy individuals.

[0033] Pediatric glioblastoma (pGBM) remains a devastating disease, in part due to the lack of a standard curative chemotherapy regimen, low cellular permeability, lack of tumor targeting, and limitations in drug delivery across the blood-brain barrier (BBB). Therefore, the inventors developed a nanomedicine platform that includes the use of a BBB-permeable tumor-navigating peptide p28 covalently linked to antisense miRNA20a (i.e., AmiR20-p28). Here, the inventors show that AmiR20-p28 crosses the BBB and preferentially localizes to human pGBM tumors in the brains of mice. Upon entering cells, AmiR20-p28 significantly inhibits pGBM cell survival by silencing the oncogenic miRNA miR-20a, which alters multiple signaling pathways. Notably, systemic administration of AmiR20-p28 enabled complete regression of early tumors and a significant increase in overall survival without obvious adverse effects in orthotopic xenograft mice. Thus, the development of miRNA-based platforms containing p28 represents a translatable strategy for the treatment of pGBM.

[0034] Definition As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0035] As used herein, the term "cell" includes both the singular and plural of this term. The terms "isolated", "purified", or "biologically pure" refer to a substance that substantially or essentially does not contain the components normally associated with a substance as it is found in its natural state. Terms such as "heterologous DNA", "heterologous nucleic acid sequence", or "exogenous" as used herein refer to a nucleic acid sequence that is (a) foreign to a given host microorganism (i.e., not naturally found), (b) the sequence is naturally found in a given host microorganism but in an unnatural (e.g., greater than expected) amount, or (c) the nucleic acid sequence contains two or more sub-sequences that are not normally found in the same relationship to each other, at least one of which is true.

[0036] The terms "peptide" and "polypeptide" are used interchangeably herein and refer to a compound composed of a chain of amino acid residues linked by peptide bonds. The "active portion" of a polypeptide means a peptide that is less than the full-length polypeptide but retains measurable biological activity and biological detectability.

[0037] As used herein, the term "tumor" refers to any neoplastic growth, proliferation, or mass, whether benign or malignant (cancerous), primary site lesion or metastasis.

[0038] As used herein, a "therapeutically effective amount" refers to an amount of a composition that reduces (to some extent, as determined by one of ordinary skill in the art) one or more symptoms of a disease or condition in a mammal. Further, a "therapeutically effective amount" of a composition means an amount that partially or completely returns to normal a physiological or biochemical parameter associated with or causative of a disease or condition. A clinician in the art can determine a therapeutically effective amount of a composition for treating or preventing a particular disease state or disorder when administered, for example, intravenously, subcutaneously, intraperitoneally, orally, or by inhalation. The exact amount of the composition required to be therapeutically effective depends on many factors in addition to many patient-specific considerations, for example, the specific activity of the active agent, the delivery device used, the physical properties of the agent, the purpose of administration, etc. However, determination of a therapeutically effective amount is within the skill of one of ordinary skill in the art by understanding the disclosure described herein.

[0039] As used herein, "treating", "treatment", and "treat", and the like, refer to any action that provides a benefit to a patient at risk of or suffering from a disease, including improvement of a condition by reduction or suppression of at least one symptom, delay of disease progression, prevention or delay of disease onset, and the like. Treatment also includes partially or completely destroying unwanted proliferating cells while minimizing the destructive effect on normal cells. A subject at risk is a subject determined to have a higher than average risk of developing cancer, which can be determined, for example, by family history or detection of a gene that causes a predisposition to cancer.

[0040] As used herein, the term "subject" refers to mammalian species, including but not limited to primates such as monkeys and humans, equids (e.g., horses), canids (e.g., dogs), felids, various domestic animals (e.g., ungulates such as swine, pigs, goats, sheep), and animals kept as pets and in zoos.

[0041] Where the methods and steps described herein indicate particular events occurring in a particular order, one of ordinary skill in the art will recognize that the order of the particular steps can be changed and that such changes are in accordance with variations of the invention. Further, the particular steps may be performed simultaneously in a parallel process, if possible, or sequentially.

[0042] The meanings of the abbreviations are as follows. As is clear from the usage, "C" means Celsius or degrees Celsius, "s" means seconds, "min" means minutes, "h", "hr" or "hrs" means hours, "psi" means pounds per square inch, "nm" means nanometers, "d" means days, "μL", "uL" or "ul" means microliters, "mL" means milliliters, "L" means liters, "mm" means millimeters, "nm" means nanometers, "mM" means millimoles, "μM" or "uM" means micromoles, "M" means moles, "mmol" means millimoles, "μmol" or "uMol" means micromoles, "g" means grams, "μg" or "ug" means micrograms, "ng" means nanograms, "PCR" means polymerase chain reaction, "kDa" means kilodaltons, "g" means the gravitational constant, "bp" means base pairs, "kbp" means kilobase pairs, "%w / v" means weight / volume percent, "%v / v" means volume / volume percent, "rpm" means revolutions per minute, "HPLC" means high performance liquid chromatography, and "GC" means gas chromatography.

[0043] It is understood that "aurA", "aurB", and "azurin" can each refer to any molecule having a peptide sequence that is substantially similar, either wholly or in part, to SEQ ID NO: 1, 2, or 67, respectively. Any polypeptide that includes SEQ ID NO: 1, 2, or 67 in part and is approximately 24 to 32 nucleotides in length can be considered 96% identical to p28 (SEQ ID NO: 64) and is recognized to be usable as a probe in the methods described herein. That is, if a single amino acid in the aurA, aurB, or azurin partial sequence that is p28, which is approximately 28 amino acids in length (e.g., ±3 amino acids), is altered by substitution with a different amino acid, the new sequence is 96% identical to SEQ ID NO: 1, 2, or 67. Similarly, when a single amino acid is added to the p28 sequence, a sequence that is 96% identical to SEQ ID NO: 1, 2, or 64 is obtained. SEQ ID NOs: 65 and 66 are exemplary sequences for explaining the substantial similarity described herein. The modified cupredoxin-derived peptide can include X 1 SX 2 AADX 3 X 4 X 5 VVX 6 DX 7 X 8 ASGLDKDYLKPDX 9 (SEQ ID NO: 65), where X 1 is L or acetylated L, X 2 is T or W, X 3 is M, L, or V, X 4 is Q or W, X 5 is G or A, X 6 is T or W, X 7 is G, T, or W, X 8 is M, L, or V, X 9 is amidated D. In other embodiments, the modified cupredoxin-derived peptide includes X 1 SX 2 AADX 3 X 4 X 5 VVX 6 DX 7 X 8 ASGLDKDYLKPDX 9It can consist of (SEQ ID NO: 66), where X 1 is L or acetylated L, and X 2 is T or W, and X 3 is M, L or V, and X 4 is Q or W, and X 5 is G or A, and X 6 is T or W, and X 7 is G, T or W, and X 8 is M, L, or V, and X 9 is amidated D.

[0044] Summary Large-scale dataset analysis revealed molecular-level significant differences between pGBM and normal brain tissues, such as miRNA expression. In particular, miRNA20 is upregulated in pGBM and functions as an oncogenic miRNA. Therefore, targeting specific oncogenic miRNAs in pGBM is an attractive approach for developing new miRNA-based drugs for pGBM. Reducing the level of oncogenic miRNAs in pGBM by using anti-sense miRNAs is a promising approach, but i) transport to the brain by crossing the blood-brain barrier (BBB), and ii) tumor-targeted delivery of anti-miRNAs are currently major challenges for moving forward to the clinic. In this study, the inventors overcome the current limitations by using the cell-penetrating peptide p28 as a carrier molecule. The inventors demonstrated that the redox protein azurin secreted by the opportunistic pathogen Pseudomonas aeruginosa preferentially invades human cancer cells and induces apoptosis. The inventors further confirmed that a fragment of azurin, p28, crosses the BBB and preferentially invades human pGBM cells, SJ-GBM2 and CHLA-200. Furthermore, p28 has no obvious toxicity or immunogenicity in clinical trials of pediatric CNS patients. Therefore, p28 is likely to be an ideal carrier for pGBM-targeted delivery. Here, the inventors created a unique drug (i.e., AmiR20-p28) by covalently linking p28 and anti-sense miRNA20. AmiR20-p28 exposure showed preferential penetration and a clear dose-dependent anti-proliferation / apoptosis effect on pGBM cell lines, but not on human astrocytes used as non-malignant controls. In contrast, anti-sense miRNA20 alone without conjugation with p28 showed little effect at any concentration tested in these cancer cells. Such an apoptosis effect induced by AmiR20-p28 was associated with a significant decrease in the level of endogenous oncogenic miRNA20 in pGBM.

[0045] Furthermore, it is suggested that the p53 and ERK signaling pathways are altered by AmiR20-p28, indicating that AmiR20-p28 can regulate multiple pathways simultaneously. Notably, treatment with AmiR20-p28 at the early stage of pGBM tumor development enables complete regression and dramatically extends survival in an orthotopic xenograft mouse model with drug-resistant cancer stem pGBM cells. PCR analysis showed preferential accumulation of AmiR20-p28 in a given tumor in the mouse brain. Based on these findings, the development of p28 as a carrier provides a novel strategy for the tumor-targeted delivery of miRNAs. It can overcome the current limitations in developing miRNA-based drugs and positively impact the therapeutic outcome (e.g., morbidity, quality of life) of pGBM.

[0046] In some embodiments, compositions are described that include an antisense microRNA covalently linked to a cell-permeable peptide. The cell-permeable peptide can include p28, can include p28 as part of a larger peptide, can include only p28, can include a peptide having significant homology to p28, can include a peptide having a single amino acid variant derived from p28, or can include a peptide having 90% sequence identity to p28. The antisense microRNA is antisense miR20, antisense miR21, or antisense miR21a, or an antisense microRNA corresponding to any suitable microRNA target. The antisense microRNA covalently linked to the cell-permeable peptide can be in any form desirable for pharmaceutical use. The covalent bond between the microRNA and p28 can occur via a maleimide-thiol reaction. In some embodiments, the covalent bond between the microRNA and p28 can occur between hexynyl-antisense microRNA and azido-p28 via click chemistry conjugation.

[0047] In some embodiments, methods for inhibiting tumor cell proliferation are described. Briefly, systemic administration of a composition comprising any of the compositions according to any aspect of the present invention to tumor cells results in inhibition of tumor cell proliferation.

[0048] In some embodiments, methods of targeting antisense microRNA to tumor cells are described. Briefly, systemic administration of a composition comprising any of the compositions according to any aspect of the present invention to tumor cells allows the antisense miRNA to target tumor cells and enter the tumor cells. In some embodiments, the cells are in vivo cells. In some embodiments, an antisense microRNA covalently bound to a cell-permeable peptide forms a complex that crosses the blood-brain barrier.

[0049] In some embodiments, methods of reducing endogenous microRNA in tumor cells are described. Briefly, systemic administration of any of the compositions according to any aspect of the present invention to tumor cells results in a decrease in endogenous miRNA within the tumor cells. In some embodiments, the tumor cells are in vivo cells.

[0050] Example Example 1: miR-20a is more highly expressed in pGBM than in aGBM and normal astrocytes The reported gene expression profiles of the miR-17-92 cluster and their roles in tumorigenic signaling in gliomas prompted the inventors to further investigate the role of this cluster in pGBM. Furthermore, miRNA profiling by the GSE42657 dataset demonstrated that miR-17-92 cluster genes were abnormally expressed in pGBM. In particular, miR-20a, a member of the miR-17-92 cluster, was significantly and highly expressed in pGBM (Figure 1A). Therefore, the inventors further examined miR-17-92 cluster genes differentially expressed in pGBM cell lines (SJ-GBM2 and CHLA-200), aGBM cell line (LN-229), and normal human astrocytes. qPCR analysis showed that these genes were differentially expressed between pHGG and aHGG and were on average 2.5-fold higher in SJ-GBM2 and CHLA-200 cells compared to LN-229 cells (Figure 1B). The results showed that miR-20a was the most highly expressed miRNA among miR-17-92 gene cluster members in pGBM cell lines and that miR-20a was abnormally expressed in pGBM compared to aGBM and normal astrocytes (Figure 2A). This result was also observed in pediatric patients with high-grade gliomas (pHGG) (Figure 2B). Furthermore, miR-20a expression in pGBM cells was the highest among the other solid tumors tested (aGBM, breast cancer, ovarian cancer, pancreatic cancer, colon cancer, prostate cancer, and melanoma cells, Figure 2C). These results indicate a distinct difference in the abnormal upregulation of miR-20a in the pediatric population with GBM, suggesting that this molecule may be a specific and attractive therapeutic target for pGBM.

[0051] Example 2: The conjugate complex consisting of p28 and antisense miR-20a (AmiR20-p28) preferentially penetrates cancer cells Our previous findings showed that CPP, p28 preferentially enters various human cancer cells over non-cancerous cells. As shown in Figure 2D, p28 preferentially permeated into SJ-GBM2 cells and CHLA-200 cells compared to normal astrocytes (high magnification images in Figure 2D, low magnification images in Figure 3A). To utilize the ability of p28 to specifically target tumor cells, we combined the tumor targeting ability of p28, a CPP, and the anti-cancer activity of anti-sense miR-20a by covalently conjugating p28 and anti-sense miR-20a (AmiR20-p28) to create a new agent that efficiently delivers anti-sense miR-20a and the target miR-20a to pGBM. Maleimide-anti-sense miR-20a and Cys-p28 were conjugated by a disulfide bond (Figure 3B). First, the cellular invasion of AmiR20-p28 was evaluated using the same set of cell lines. Confocal images showed preferential invasion of AmiR20-p28 in pGBM cells compared to normal human astrocytes (Figure 2E). The intracellular invasion of AmiR20-p28 was also quantitatively confirmed by the detection of anti-sense miR-20a. qPCR analysis showed preferential and dose-dependent penetration of AmiR20-p28 into pGBM and aGBM cells (Figure 2F). The final products after qPCR analysis of SJ-GBM2 cells, CHLA-200 cells, LN-229 cells, and normal human astrocytes treated with AmiR20-p28 were subsequently visualized on an agarose gel. This assay confirmed the cellular invasion of AmiR20-p28 in pGBM and aGBM cells, but little signal was detected in normal human astrocytes (Figure 2G). These results indicate that p28, a tumor-targeting CPP, can efficiently deliver the cargo molecule, anti-sense miR-20a, in a dose-dependent manner.

[0052] Example 3: AmiR20-p28 downregulates oncogenic miR-20a and inhibits cell proliferation To determine whether AmiR20-p28 captures miR-20a and reduces endogenous miR-20a levels, the inventors measured miR-20a expression levels in pGBM cells, aGBM cells, and normal human astrocytes. The results of qPCR showed that miR-20a expression levels were significantly decreased by 94%, 96%, and 84% in SJ-GBM2, CHLA-200, and LN-229 cells, respectively. miR-20a levels in normal human astrocytes did not change significantly, presumably due to the preferential invasion characteristics of AmiR20-p28 (Figure 4A). In contrast, two components of AmiR20-p28, the antisense miR-20a oligonucleotide (AmiR20) alone and p28, when administered alone, did not have a significant inhibitory effect in any of the tested cell lines (Figure 4A). This result suggests that significant miR-20a silencing in GBM was due to the effect of antisense miR-20a delivered by p28.

[0053] Next, the inventors examined whether silencing of miR-20a by AmiR20-p28 promotes an inhibitory effect on cancer cell viability in vitro. The Cell Counting Kit-8 (CCK-8) viability assay showed that AmiR20-p28 significantly decreased the viability of pGBM cells (SJ-GBM2 and CHLA-200) (Figure 4B). In contrast, the effect of AmiR20-p28 on cell viability was not as great in aGBM (LN-229) cells (compared to pGBM) and was negligible in normal human astrocytes (Figure 4B). These results are correlated with the degree to which miR-20a was silenced in the cells. Here too, neither p28 nor AmiR20 alone had an inhibitory effect on cell viability. This finding is consistent with reports that p28 alone shows an inhibitory effect on cancer cell viability at a higher dose than that tested in GBM cells in this study (50 μM). Notably, the low dose of 10 nM AmiR20-p28 decreased cell viability by approximately 45% and 50% in SJ-GBM2 and CHLA-200 cells, respectively. The IC 50 values of AmiR20-p28 in SJ-GBM2, CHLA-200, and LN-229 cells were 8 nM, 11 nM, and 170 nM, respectively. To determine that the inhibitory effect on cell viability was due to the chemical linker, the inventors prepared AmiR20-p28 by different chemical conjugation methods by click chemistry (Figure 3C). Considering that a similar inhibitory effect on cell viability was obtained in the same set of cell lines using AmiR20-p28 prepared by click chemistry in the set of cell lines tested (Figure 5A), such an effect was not due to the linker used in the conjugation process.

[0054] Furthermore, the inventors performed flow cytometry analysis to detect apoptotic cells using annexin V / propidium iodide (PI) staining. Among pGBM cells exposed to 10 nM AmiR20-p28 for 24 hours, SJ-GBM2 cells showed a significant 16% increase in apoptotic cells, and the number of apoptotic cells significantly increased by 14% in CHLA-200 cells, but did not increase in the control groups (NT, p28 alone, AmiR20 alone) (FIGS. 4C and 5B). In contrast, apoptosis after AmiR20-p28 treatment of aGBM and astrocytes was 6.7% and 0.5%, respectively. As shown in FIGS. 2A-2C, oncogenic miR-20a is abnormally expressed in GBM, particularly pGBM. As expected, AmiR20-p28 treatment had little effect on the viability of MDA-MB231, SKOV3, Mia-Paca2, Colon 205, and DU145 cells, suggesting that AmiR20-p28 specifically affects pGBM cells in which miR-20a is abnormally expressed (FIGS. 2A-2C and 5C). These results demonstrate that AmiR20-p28 preferentially penetrates GBM cells, silences miR-20a, thereby inhibiting the viability of cancer cells.

[0055] Example 4: AmiR20-p28 targets oncogenic signaling pathways in pGBM After it was revealed that AmiR20-p28 silences miR-20a and reduces cell viability in pGBM cells, the inventors aimed to clarify the mechanism by which this occurs. To elucidate the signaling pathway changed by AmiR20-p28, the inventors determined the expression levels of downstream targets of miR-20a. Genes related to the EGFR, p53, and TGF pathways were studied because of their important roles in the tumorigenic signaling network, and miR-20a was suggested to regulate these genes. qPCR analysis showed that the expression levels of the EGFR, Ras, MEK1, ERK1, c-Fos, c-Jun, β-catenin, TCF-1, and Lef1 genes significantly decreased after treatment with AmiR20-p28, but not with p28 alone, AmiR20 alone, or PBS (NT), whereas a strong increase in the expression of p53, p21, Bax, and E2F was observed in SJ-GBM2 and CHLA-200 cells (Figure 4D). In LN-229 cells, AmiR20-p28 significantly increased the expression of p53, p21, Bax, and E2F and decreased the EGFR expression level, but no significant effect on gene expression levels was observed in normal human astrocytes (Figures 6A and 6B). There is a significant difference between pGBM and aGBM in the degree to which these genes are regulated (Figures 4D and 6A). Genes of the TGF and Wnt pathways, such as β-catenin and TCF1, were significantly downregulated by AmiR20-p28 in pGBM, but not in aGBM LN229 (Figures 4D and 6A). This difference between pGBM and aGBM may be due to the fact that the miR-20a expression level in pGBM is significantly higher than that in aGBM, being one order of magnitude higher than that in pGBM in LN-229 cells. The IC 50which is reflected. To determine whether AmiR20-p28 similarly affects the expression of these genes at the protein level, the inventors evaluated the protein levels of the major downstream targets of miR-20a. Consistent with the qPCR results (Figure 4D), Western blot analysis showed that the levels of ERK and EGFR decreased, while the levels of p53, p21, and Bax increased upon AmiR20-p28 treatment compared to the controls (Figure 4E). These results suggest that AmiR20-p28 can reduce oncogenic miR-20a expression and simultaneously alter multiple signaling pathways by targeting and silencing both "druggable" genes such as RAS and "undruggable" genes in pGBM (Figure 6C).

[0056] Example 5: AmiR20-p28 exhibits cytotoxic activity against tumor spheroids with characteristics of glioblastoma stem cells Pediatric brain tumors generally recur due to the presence of tumor heterogeneity, which is often associated with drug resistance, and the self-renewal ability of glioblastoma stem cells (GSCs). To determine drug sensitivity, pGBM cells were cultured in the presence of temozolomide (TMZ), a chemotherapeutic agent for aGBM. Cell viability assays showed that SJ-GBM2 cells had significantly lower sensitivity to TMZ than CHLA-200 (IC 50 = ~0.6 mM) (Figure 7A). Furthermore, SJ-GBM2 cells were positively stained for CD133, the most common surface marker of GSCs (Figure 7B).

[0057] Generally, monolayer cell cultures are used to evaluate the efficacy of lysed drugs that are readily accessible in cell culture media. In contrast, natural tumors contain densely packed cells surrounded by other cells. To better mimic the complex tumor structure, the inventors generated tumor spheroids using SJ-GBM2 cells. To test the penetration ability of AmiR20-p28, the inventors first examined its uptake in SJ-GBM2 tumor spheroids. Confocal images showed that AmiR20-p28 penetrated the tumor spheroids (Figure 7C). After confirming that AmiR20-p28 penetrated the tumor spheroids, the inventors investigated the possibility that AmiR20-p28 might affect the complex structure of the tumor in tumor spheroids. The structure of SJ-GBM2 spheroids exposed to AmiR20-p28 began to disintegrate at 24 hours, and this disintegration increased at 48 hours (Figure 7D), suggesting that AmiR20-p28 can penetrate 3D cultures and disrupt the GSC spheroid structure. These in vitro results suggest that AmiR20-p28, if applied by a treatment method in pGBM, may overcome the GSC characteristics of pGBM tumors.

[0058] Example 6: AmiR20-p28 inhibits orthotopic glioblastoma tumor growth To determine the therapeutic efficacy and tumor inhibitory ability of AmiR20-p28 in vivo, the inventors established an intracranial orthotopic xenograft mouse model by injecting SJ-GBM2 cells and tested tumor development. The SJ-GBM2 tumor was prominent in T2-weighted magnetic resonance (MR) images, and histological examination also showed tumor-positive regions in the coronal section of the brain (Figure 8A). After systemic intravenous (i.v.) administration of indocyanine green (ICG) dye conjugated to p28 to the mice, near-infrared (NIR) fluorescence images of the brain showed that p28 preferentially localized to the tumor lesions (Figure 8A and Figure 9). The ability of the vehicle to cross the BBB is a major challenge for gene delivery. Here, the inventors used a 3D human BBB assay kit in vitro to test the ability of AmiR20-p28 to cross the BBB. The BBB permeability of AmiR20-p28 and AmiR20 in two directions, from apical to basolateral (A-B) and from basolateral to apical (B-A), was measured. Quantitative analysis using qPCR showed that AmiR20-p28 crossed the BBB at a significant rate (Figure 8B). In contrast, AmiR20 was not detected, suggesting that the ability of AmiR20-p28 to cross the BBB is due to the p28 motif. Since the high influx (from blood to brain) and low efflux (from brain to blood) of AmiR20-p28 are desirable for drug accumulation in the brain, the inventors determined the influx and efflux rates of AmiR20-p28. The influx Papp (A-B) and efflux Papp (B-A) rates were 4.39×10 -6 and 0.95×10 -6 cm / sec, respectively (Figure 8B). The influx transport rate of p28 into the brain was higher than the efflux transport rate, suggesting that AmiR20-p28 is an ideal BBB-permeable agent.

[0059] After determining that AmiR20-p28 could successfully cross the BBB in vitro, the inventors aimed to examine the therapeutic effect of AmiR20-p28 in vivo. They transplanted SJ-GBM2 cells genetically engineered to express luciferase (SJ-GBM2-luc cells) into mice and monitored luciferase signaling via bioluminescence intensity imaging. After i.v. systemic treatment with 0.1, 1, or 5 mg / kg of AmiR20-p28, there was a significant inhibition of tumor growth compared to mice treated with negative control (PBS) or the lowest dose (0.1 mg / kg) of AmiR20-p28 (Figures 8C and 10). After 4 weeks of treatment consisting of 12 injections per mouse administered three times a week, the groups receiving AmiR20-p28 at 1 and 5 mg / kg were further divided into two subgroups. One group continued to receive treatment (i.v. three times a week), and the other group stopped treatment to examine whether the tumors recurred. Throughout an additional experimental period of approximately two months, neither the 1 mg / kg dose group nor the 5 mg / kg dose group showed signs of tumor recurrence (Figure 8C).

[0060] Subsequently, another series of experiments was conducted to verify and evaluate the efficacy of AmiR20-p28 in the regulation of tumor growth at various stages / sizes. Intravenous (i.v.) treatment with AmiR20-p28 at the minimum effective dose (1 mg / kg) was initiated at three different time points: 10 days (early stage), 20 days (mid-stage), and 40 days (late stage) after cell injection into mice (Figure 8D, upper panel). Consistent with the previous experimental results (Figure 8C), mice treated with AmiR20-p28 at the early stage of tumor development showed complete tumor suppression (Figures 8D, 8E, 11A - 11B). Mice treated with AmiR20-p28 at the mid-stage of tumor development showed continuous suppression of tumor volume (Figures 8D, 11A). Despite tumor progression being observed in late-stage animals, 80% of the mice in this group survived until day 49, unlike the mice in the control groups (PBS and AmiR20) (Figures 8D - 8F, and 11A). Mice were either euthanized due to disease or sacrificed based on humane endpoint criteria, including abnormal behavior (e.g., paralysis) (Figure 12). Based on the results of toxicity tests [since miRNAs are known to be metabolized in the liver, hepatotoxicity assays (Figures 13A - 13B), and since AmiR20-p28 is injected i.v., hemolysis assays (Figure 13C)], and histological examination of major organs (Figure 13D), AmiR20-p28 induced an antitumor effect without obvious harmful effects. The overall survival period in the AmiR20-p28 treatment group was associated with their antitumor effect. The overall survival rates were 100% (early stage), 80% (mid-stage), and 40% (late stage), which were different from those of the control groups administered PBS or AmiR20 (Figure 8F).

[0061] To evaluate whether systemically delivered AmiR20-p28 can localize to tumor lesions in vivo, the inventors measured AmiR20-p28 in normal brain tissue and tumor tissue. Mice with SJ-GBM2 tumors were injected with 1 mg / kg of AmiR20-p28 for 30 minutes or 120 minutes. Brain samples were isolated and separated into normal brain tissue and detectable tumor tissue by bioluminescence imaging and quantitative human-specific Alu sequencing (huAlu) (since the Alu repeat DNA sequence is specific to human cells, human-derived SJ-GBM2 cell tumors transplanted into mice can be identified). (Figures 14A - 14B). Tumor tissue and normal brain tissue were subjected to qPCR to detect AmiR20-p28. Even 30 minutes after injection, substantially higher levels of AmiR20-p28 were detected in tumor tissue than in normal brain tissue (Figure 8G). These results indicated that AmiR20-p28 is a BBB-permeable agent with a high influx rate / low efflux rate and is an effective targeting agent for pGBM in a preclinical setting.

[0062] To determine whether the tumor localization of AmiR20-p28 has a biological effect on miR-20a levels, after tumors regressed due to long-term exposure to AmiR20-p28, mice were treated three times a week for 1 week with PBS or 1 mg / kg of AmiR20-p28. Brain samples were isolated from PBS-treated mice and AmiR20-p28-treated mice and separated into normal brain tissue and tumor tissue. A significant decrease in miR-20a levels was detected in tumor tissue excised from AmiR20-p28-treated mice compared to that from PBS-treated mice, which remained at a similar level to that of the original SJ-GBM2 cells (Figure 2C) (Figure 8H). Collectively, these results confirmed the in vivo efficacy and tumor specificity of AmiR20-p28 in inhibiting oncogenic miR-20a.

[0063] One of the most recent emerging areas for cancer diagnosis and prognosis is the development of non-invasive circulating serum biomarkers. To examine whether tumor burden correlates with circulating oncogenic genes, we measured miR-20a in human serum. Evaluation of circulating miR-20a levels showed that they were significantly higher in pHGG patients than in healthy volunteers (Figure 8I). This result indicates that serum miR-20a may function as a pGBM biomarker. To further examine serum miR-20a levels in the in vivo animal model (Figure 8D), we analyzed miR-20a levels in serum samples from the PBS-treated group and the AmiR20-p28-treated group (animals in the experiment shown in Figure 8D) and compared them to those from serum samples of tumor-free control mice. As expected, qPCR analysis demonstrated that miR-20a levels were significantly higher in the PBS-treated group than in the tumor-free group (Figure 8J), which was consistent with the results of Figure 8I. More importantly, serum levels of miR20a correlated with tumor burden (Figure 8J and Figure 15). Serum miR-20a levels in the initial group did not differ significantly from the basal levels in tumor-free mice (P = 0.78), and circulating miR-20a levels can be used as a biomarker to monitor tumor response (Figure 8J). The lack of an increase in serum miR-20a levels indicated that tumors in these mice had completely disappeared in the initial treatment group. Overall, systemically administered AmiR20-p28 crossed the BBB and preferentially accumulated in orthotopic xenograft tumors. Without causing obvious adverse effects, AmiR20-p28 treatment inhibited tumor growth, which was associated with overall survival and could be monitored by circulating miR-20a levels as a serum biomarker.

[0064] Example 7: AmiR20-p28 inhibits tumor growth in orthotopic glioblastoma To determine the therapeutic efficacy and inhibitory ability of AmiR20-p28 in vivo, an intracranial orthotopic xenograft mouse model was utilized for systemic delivery of anti-miR20. SJ-GBM2-luc cells genetically engineered to express luciferase were transplanted into control group mice and AmiR20-p28-treated mice, and the luciferase signal was monitored by bioluminescence intensity imaging. After systemic treatment with AmiR20-p28 (1 mg / kg), there was a significant suppression in tumor growth compared to mice treated with PBS as a negative control and 0.1 mg / kg AmiR20-p28 (Figure 17A). After i.v. injection of AmiR20-p28, the luciferase activity obtained from AmiR20-p28-treated mice was significantly lower than that of the PBS control group, indicating that repeated treatment with AmiR20-p28 decreased the rate of glioblastoma growth. To evaluate the efficacy of AmiR20-p28 in controlling the size of tumor growth, mice were treated i.v. with 1 mg / kg AmiR20-p28 at three different time points: 10 days (early stage), 21 days (mid-stage), and 42 days (late stage) after cell injection. As observed from the previous experiment, mice treated with AmiR20-p28 at the early stage of tumor development, 10 days after cell injection, showed complete suppression of the tumor (Figure 17B). Furthermore, AmiR20-p28 injected into mice with larger tumor masses developed 21 days after cell injection showed continuous suppression of tumor growth (Figure 17B). Mice treated with AmiR20-p28 at the late stage (42 days after cell injection) showed tumor progression (Figure 17B). These antitumor effects of AmiR20-p28 were associated with their overall survival period. Treatment with AmiR20-p28 resulted in an increase in overall survival rate, including 100% (early stage), 80% (mid-stage), and 60% (late stage) survival probabilities, compared to the control groups (PBS and Amir20 alone) (Figure 17C). Furthermore, the inventors detected AmiR20-p28 uptake in the xenografted tumor tissue. Mice with SJGBM2 tumors were injected with 1 mg / kg AmiR20-p28 for 30 minutes or 120 minutes. After treatment, the tumor region and normal brain tissue region were subjected to real-time PCR. The results showed that higher levels of AmiR20 were detected in the tumor region (Figure 17D).To further confirm the uptake of AmiR20-p28 in tumors, AmiR20-p28 labeled with Alexa fluor 568 was injected into mice bearing SJGBM2. Confocal images of brain sections showed a significant contrast of Alexa fluor signal at the tumor site compared to normal brain regions (Figure 17E). These results suggest that systemic administration of AmiR20-p28 preferentially accumulates in a given tumor in the mouse brain and that AmiR20-p28 treatment enables control of tumor growth associated with an increase in overall survival.

[0065] Example 8: Effect of AmiR21-p28 on various solid tumors The cell-permeable peptide p28 was chemically conjugated to anti-sense miR21 (e.g., AmiR21-p28). miR21 is known to be an oncogenic miRNA and is highly expressed in various types of cancer cells.

[0066] In contrast to miR20, adult glioblastoma (aGBM) LN229 showed the highest level of miR21 compared to pGBM (Figure 18A). Exposure of pGBM and aGBM to 10 nM AmiR21-p28 significantly decreased endogenous miR21 (Figure 18B - Figure 18D), but not in human normal astrocytes (Figure 18E). p28 alone and anti-sense miR21 (anti-miR21) alone did not induce a significant decrease in miR21 in any of the cell lines tested. Changes in the gene expression profiles induced by AmiR21-p28 are different from those induced by AmiR20-p28 (e.g., PTEN), suggesting that the target genes of miR21 are different from those of miR20 (Figure 19).

[0067] The inventors have developed a novel miRNA delivery system using the cell-penetrating peptide p28, which can deliver drugs across the BBB. AmiR20-p28 showed preferential penetration and cytotoxicity in pGBM cells. Importantly, the inventors found that AmiR20-p28 targets multiple signaling pathways simultaneously to inhibit pGBM growth and improves the overall survival by silencing oncogenic miR20.

[0068] Despite extensive research to understand the molecular profiles of gliogenesis in pediatric patients, current knowledge remains insufficient to identify targets for developing therapeutic agents. In recent years, numerous evidences have shown that microRNAs (miRNAs) play important roles in the molecular and cellular mechanisms of tumorigenesis in various cancers, including pediatric glioblastoma multiforme (pGBM), acting as either oncogenes or tumor suppressors. Furthermore, miRNAs can simultaneously alter the expression of multiple target genes, often disrupting entire signaling networks. Gene expression profiling studies have shown that the most upregulated miRNAs in pediatric samples, compared to adult populations and normal brain tissues, include miR15a, miR17, miR18a, miR19a, miR19b, miR20, miR27a, miR-100, miR-106a, miR-195, and miR497. Increasing evidence indicates that miR20 plays an important role as an oncogene in the development of a wide range of malignancies. In addition, miR20 plays an important role in glioblastoma stem cell (GSC) invasion. In addition to their small size and broad impact on biological processes, these findings make miRNAs attractive therapeutic agents for pGBM. Another important factor in gene regulation is small interfering RNA (siRNA), which has been studied in recent years as a new class of therapeutic agents for treating a wide range of disorders, including cancer. Clinical trials of siRNA- and miRNA-based drugs have already been initiated. siRNAs and miRNAs share the similarity of consisting of short double-stranded RNAs that both exert gene silencing, but while siRNAs are highly specific for one mRNA target, miRNAs can simultaneously alter the expression of multiple target genes, often disrupting entire signaling networks and resulting in efficient changes in the activity of target cells. Therefore, the inventors' research has been advanced by targeting miR20 against pGBM. However, the broad functionality of miRNAs can also make it difficult to control off-target effects and toxicity.Furthermore, the clinical application of miRNA as a therapeutic agent is hampered by delivery vector constraints, such as low stability, low transfection efficiency, and carrier-related toxicity arising from off-target effects. Another major barrier in the development of new drugs for CNS disorders is the delivery of therapeutic agents across the BBB. The BBB represents a complex structure in the brain that controls molecular transport, maintains homeostasis, and excludes toxins by selectively transporting nutrients and other molecules across gap junctions. Among recent advances in the development of novel delivery systems, the transport of therapeutic molecules into the CNS via CPP-based delivery systems appears more promising due to their low cytotoxicity and high efficiency in transporting macromolecules across cell membranes (Silva S et al., 2019). In this study, we use the p28 delivery system, which has been shown to cross the BBB in vitro, and its application in human clinical trials, which provides indirect evidence of p28 crossing the BBB. Based on this previous knowledge, we conjugated anti-miR20 to p28 to inhibit miR20.

[0069] The conjugate complex of AmiR20-p28 showed preferential penetration in the pGBM cell line and suppressed miR20. This suppression of miR20 was associated with a significant decrease in cell viability in the pGBM cell line compared to astrocytes. One of the important features of gliomas is the impairment of the natural programmed cell death mechanism. These findings indicate that AmiR20-p28 is tumor cell-specific. p28 itself shows significant cytotoxicity against human carcinomas, such as glioblastoma, melanoma, breast, prostate, and colorectal. However, this effect of p28 in AmiR20-p28 treatment can be excluded as shown by the absence of cytotoxicity in glioblastoma cells when treated with a relatively low dose of 10 nM p28 alone. Furthermore, clinical findings reported in two Phase I clinical trials of p28 (NCS745104) as a single agent in patients with progressive solid tumors and pediatric patients showed no adverse effects, toxicity, or immunogenicity, highlighting the safety of p28 as a carrier agent. Glioma stem cells (GSCs) are now known to be one of the reasons for the high level of heterogeneity in pediatric tumors associated with a high recurrence rate. GSCs can perform both self-renewal and differentiation as a means of tumor regrowth by producing more GSCs. GSCs in brain tumors have been shown to be resistant to aggressive radiotherapy and are hardly affected by standard chemotherapy. These aspects of GSCs create an urgent need to find therapeutic agents that target these cells to reduce recurrence. In the HGG stem cell population, CD133 is considered a stem cell-like cell surface marker and is widely used to identify putative stem cells. SJ-GBM2 shows a higher CD133 expression level than astrocytes, suggesting the spheroid formation ability shown by SJ-GBM2. The data of the present inventors reveal that AmiR20-p28 can penetrate into spheroids and target spheroids.

[0070] To understand the mechanism of AmiR20-p28, the expression analysis of pGBM cells treated with AmiR20-p28 was determined. Along with the p53 axis, AmiR20-p28 regulated the expression level of E2F. Dysregulation of EGFR leads to enhanced tumorigenicity and is well-established in adult glioblastoma, which shows resistance to both radiotherapy and chemotherapy, and is considered less important in pGBM. In this study, the inventors observed significant downregulation of the EGFR pathway in multiple downstream signaling cascades, EGFR, Ras, MEK, ERK, ELK1, and c-fos. Although there is no universal expression in all pediatric brain tumors, overexpression of EGFR is actually more frequently observed in pGBM by datasets that vary from 10% to 80%.

[0071] In the SJ-GBM2 pGBM orthotopic mouse model, complete regression was observed with initial treatment by AmiR20-p28. The relative tumor volume in the human pediatric brain was estimated to be about 40 cm based on the initial mouse brain tumor, 3 suggesting that AmiR20-p28 could be theoretically effective in pediatric patients with tumors the size of a golf ball (about 4 cm). Therefore, AmiR20-p28 treatment could be realistically effective in initial pGBM patients and as adjuvant therapy in patients with relatively large tumors even after surgery.

[0072] Furthermore, the identification and development of new biomarkers to improve early cancer detection and drug efficacy monitoring are clinically important. For biomarkers, liquid biopsy is important because it is a less invasive and safer approach compared to conventional biopsy for diagnosis. Based on the preclinical evaluation of the inventors, the level of miR-20a was significantly higher in serum from pHGG patients, and a similar association was found in in vivo mouse experiments.

[0073] In summary, the inventors highlight a new approach for miRNA-based therapy for pGBM that utilizes p28 as a delivery platform. p28 promotes the permeation of AmiR20 across the BBB, enables preferential intracellular delivery, results in suppression of endogenous miR20, regulation of multiple target proteins, inhibition of tumor growth, and extension of overall survival. Further systemic or neurological toxicity, pharmacokinetic, and biodistribution studies are to determine the off-target potential or initiate the design of early clinical trials. Overall, the results support p28-mediated delivery for miRNA-based approaches for cancer therapy.

[0074] Method Cell line and cell culture

[0075] SJ-GBM2 (female, 50 months, progressive disease, post-chemotherapy, histone 3 variant H3.3: wild-type, TP53: R273C) and CHLA-200 cells (male, 144 months, treated with chemotherapy and radiation for multiple relapses, histone 3 variant H3.3: wild-type, TP53: wild-type) were obtained from the Children’s Oncology Group Cell Culture Repository (Lubbock, TX). Normal human astrocytes (NHA, CC-2565) were obtained from Lonza (Bend, OR). Human cancer cells (LN-229, MDA-MB231, SKOV3, MIA PaCa-2, Colon 205 and DU145 cells) were purchased from the American Type Culture Collection (Manassas, VA). SJ-GBM2 cells and CHLA-200 cells were maintained in IMDM supplemented with 20% fetal bovine serum (FBS), 100 units / ml penicillin, 100 μg / ml streptomycin and 1% ITS. LN-229, MDA-MB231, SKOV3, MIA PaCa-2, Colon 205 and DU145 cells were maintained in MEME supplemented with 10% FBS, 100 units / ml penicillin and 100 μg / ml streptomycin. Astrocytes were maintained in an astrocyte growth medium bullet kit (CC-3186). All cells were cultured at 37 °C in a humidified chamber containing 5% CO 2 2. Melanoma (Mel-2) was developed in our laboratory as described previously.

[0076] Preparation of AmiR20-p28

[0077] miRNA-peptide conjugation was performed based on the thiol-maleimide reaction. Antisense-miR-20a with a 5'-terminal maleimide group modification was obtained from Integrated DNA Technologies (Coralville, IA). All RNA bases had 2'-O-methyl modifications. The sequence was 5'-maleimide CUA CCU GCA CUA UAA GCA CUU UA-3' (SEQ ID NO: 3). Cys-p28 (CLSTA ADMQG VVTDG MASGL DKDYL KPDD (SEQ ID NO: 4)) was p28 with one cysteine group added to the N-terminus (CS Bio, CA). The maleimide-conjugated antisense miR-20a was first deprotected by a retro-Diels-Alder reaction by suspending 80 nmol of the lyophilized oligonucleotide in 2 ml of anhydrous toluene under absolute dry conditions at 90 °C for 4 hours, and then evaporated to obtain a white residue consisting of the active maleimide moiety conjugated to the antisense miRNA. The prepared form of this oligonucleotide was then reacted with 5 equivalents of thiol (400 nmol) containing Cys-p28 in phosphate-buffered saline (PBS) at room temperature for 1 hour while maintaining the final pH at 6.5 - 7.5. Subsequently, the conjugation reaction was confirmed by mass spectrometry (Bruker MALDI TOF Microfelx, Germany). A widely used negative control, miRNA (5'-maleimide CAGUACUUUUGUGUAGUACAA-3' (SEQ ID NO: 5)), was obtained from Integrated DNA Technologies and conjugated with p28 as described above.

[0078]

[0001] AmiR20-p28 was prepared using click chemistry conjugation between hexynyl-antisense miR20a and azido-p28. Hexynyl-antisense miR20a (5’-hexynyl-CUACCUGCACUAUAAGCACUUUA (SEQ ID NO: 6)) was purchased from Integrated DNA Technologies. Azido-p28 was obtained from Biomatik. 200 μl of 1 mM hexynyl-antisense miR20a in 500 μl of D.W. was prepared in a pressure-resistant vial such that the final concentration would be 200 μM after adding solutions in subsequent steps. 100 μl of pH 7.0, 2 M triethylammonium acetate buffer (TEA) was added to the solution, followed by 20 μl of DMSO. 30 μl of 10 mM azido-p28 was added to the solution and vortexed. Then, 100 μl of 5 mM ascorbic acid solution (Alfa Aesar, #A15613.18) was added to the mixture, and this was degassed for 30 seconds by bubbling argon. Next, 0.5 mM Cu-TBTA complex (Lumiprobe, #21050) was added to the mixture and degassed. The reaction was incubated overnight with gentle stirring at room temperature. The conjugate was precipitated with acetone, at least 4 volumes of the mixture, held at -20 °C for 20 minutes, and centrifuged at 10,000 r.p.m. for 10 minutes at 4 °C. The pellet was washed again with acetone and dried at room temperature. Finally, the pellet was reconstituted in PBS and stored at -80 °C. Subsequently, the conjugation reaction was confirmed by mass spectrometry.

[0079]

[0002] Quantitative real-time PCR assay The microRNA enriched fraction was extracted using the miRNeasy Mini Kit (Qiagen, catalog #217004) according to the manufacturer's instructions. miRNA expression was analyzed using stem-loop real-time qPCR. The inventors designed miR-20a stem-loop RT primers and amplification primers according to the previously described method 79. cDNA was synthesized from the extracted RNA using the unique stem-loop RT primers. The sequence of the stem-loop primer for miR-20a was 5’-GTCGTATCCAGTGCAGGGTCCGAGGTCGGCAATTGCACTGGATACGACCTACCT-3’ (SEQ ID NO: 7). The reverse transcriptase reaction contained the following reagents: 10 ng of RNA sample, 60 nM of stem-loop RT primer, 1× RT buffer, 0.25 mM dNTP, and 4 U / μl of reverse transcriptase. The reaction was carried out by incubation at 16 °C for 30 minutes, 42 °C for 30 minutes, and 85 °C for 5 minutes. Real-time PCR (7300 Real time PCR Systems, Applied Biosystems, CA) was performed using a specific forward primer for miR-20a (5’-GTAAAGTGCTTATAGTGCAG-3’ (SEQ ID NO: 8)) and a universal reverse primer (5’-GTGCAGGGTC CGAGGT-3’ (SEQ ID NO: 9)). The 20 μl PCR mixture contained 1× SYBR Premix (Invitrogen), 2 μl of the RT product, and 10 nM each of the forward and reverse primers. In a 96-well plate, the reaction was carried out by incubation at 95 °C for 30 seconds, followed by 35 cycles of 95 °C for 30 seconds and 60 °C for 30 seconds. U6 was used as an internal control to normalize the levels of the target miRNA. The results were verified by performing PCR using M13-modified forward and reverse primers specific to the stem-loop sequence. The PCR products were visualized on a 2% agarose gel and the DNA sequences were confirmed.Human tumor and serum samples were obtained from patients who signed an informed consent, and approval from the institutional ethics committee was obtained at the participating institutions of the Pediatric Brain Tumor Consortium (PBTC-041).

[0080]

[0004] p28 / AmiR20-p28 labeling p28 or AmiR20-p28 was dissolved in PBS mixed with Alexa Fluor 568 dye (Invitrogen) at a peptide / dye ratio of 1:2 at 37°C. Sodium bicarbonate (pH 8.5) was added and incubated at 4°C with continuous stirring. Alexa Fluor 568-labeled p28 and AmiR20-p28 were dialyzed against cold PBS using Slide-A-Lyzer Dialysis Cassettes (Pierce Biotechnology). Similarly, p28 was labeled with ICG as described above.

[0081]

[0006] In vitro BBB assay The permeability experiment was conducted using a 3D Human Blood Brain Barrier Model kit (Alphabioregen, catalog #EP010). The BBB kit was activated according to the manufacturer's instructions. Briefly, the BBB model was thawed using the BBB growth medium (#BBB-GM001) and incubated overnight with fresh BBB growth medium. The model was incubated with Endo-Neuro-Pharmaceuticals medium (#NMBBB001) for 3 days. AmiR20-p28 was prepared in assay buffer (Dulbecco's PBS containing CaCl2 and MgCl4, 100 mM HEPES, pH 7.0, 25 mM D-glucose). The permeability of AmiR20-p28 in two directions, from apical to basolateral (A-B) and from basolateral to apical (B-A), was measured. The apical side represented blood, and the basolateral side represented brain tissue. For transport, the insert from the BBB well was transferred to another well containing pre-warmed assay buffer. Depending on the direction, the medium in the donor compartment of the BBB model was replaced with AmiR20-p28. The plate was incubated at 37 °C for 30 minutes. Samples were then collected from the donor and acceptor compartments and analyzed by real-time PCR. The concentration of AmiR20-p28 in the compartments was calculated based on the standard curve developed using 1, 10, 100, and 1,000 nM of AmiR20-p28 and its relative Ct value obtained from real-time PCR. The apparent permeability (Papp) coefficient in cm / s was calculated according to the formula, Papp=(dC / dT×V) / (A×C 0 ). In the formula, dC / dt is the change in concentration (μg / sec) in the basal chamber, V is the volume of the basal chamber, A is the membrane surface area, and C 0 is the initial concentration in the apical chamber at 0 minutes. The magnitude of efflux was estimated using the efflux ratio (ER) defined as the ratio of Papp(B-A) / Papp(A-B).

[0082]

[0008] Cell viability assay Cell viability was evaluated by CCK-8 assay (Dojindo, Japan). Cells were seeded at a density of 4,000 cells / well in 100 μl of growth medium in a 96-well plate and grown overnight. The cells were then exposed to anti-sense miR-20a alone, p28 alone, or negative control or AmiR20-p28 over a range of concentrations and incubated for 24 hours. According to the manufacturer's instructions, 10 μl of CCK-8 solution was added to each well, followed by incubation at 37 °C for 1 hour. The absorbance at 450 nm was measured by a multiplate reader. Cell viability was calculated as follows. Cell viability (%) = Abs (sample) - Abs (blank) / Abs (untreated) - Abs (blank) × 100

[0083]

[0011] Apoptosis assay Cells were seeded in a 10 cm dish and cultured overnight. The cells were then treated with 10 nM anti-sense miR-20a alone, p28 alone, or AmiR20-p28 for 24 hours. The cells were washed with 1×PBS, detached with TrypleE (Invitrogen), and collected by centrifugation. The cells were washed and resuspended in 1× annexin binding buffer (Life Technologies, catalog #V13242), and the cell density was maintained at 10 6 cells / ml. Subsequently, 5 μl of annexin-V was added to 100 μl of cell suspension, and the mixture was incubated at room temperature for 15 minutes. The cells were washed, incubated on ice with 5 μl of PI for an additional 15 minutes, washed, and resuspended in 200 μl of buffer. Untreated cells were used as a control. After washing, the cells were subjected to flow cytometry (Gallios flow cytometer) and analyzed with Kaluza software to obtain the percentages of dead cells (upper left quadrant), late apoptotic cells (upper right quadrant), early apoptotic cells (lower right quadrant), and viable cells (lower left quadrant).

[0084]

[0013] Western blot analysis RIPA buffer (Cell Signaling Technology, #9806S) was used according to the manufacturer's protocol to prepare whole cell lysates. Total protein was quantified with Bradford reagent (Bio-Rad), and the proteins were subjected to one-dimensional gel electrophoresis on a NuPAGE system and then transferred to a nitrocellulose membrane (Bio-Rad). The nitrocellulose membrane was incubated in blocking buffer (Thermo Scientific, #37535) for 2 hours at room temperature and then blotted overnight at 4°C with each primary antibody (1:1000 anti-p53, Santa Cruz SC-17846; 1:1000 anti-p21, Santa Cruz SC-397; 1:1000 anti-ERK, Santa Cruz SC-94; 1:1000 anti-Bax, Santa Cruz SC-20067) and anti-EGFR (1:1000; Invitrogen MA5-13070). The membrane was washed and incubated with an HRP-conjugated secondary antibody. Each band was visualized using Pierce SuperSignal West Pico Chemiluminescent substrate (Thermo Fisher Scientific). Anti-actin (1:1000, Santa Cruz SC-1616) was used as an internal loading control.

[0085]

[0015] Intracranial tumor transplantation All animal experiments were conducted in accordance with the use of animals in research and approved by the IACUC. Chemotransfection method (FuGENE HD, Promega) in the presence of G418 antibiotic (GoldBio) was used to generate SJ-GBM2 cells stably expressing the luciferase gene (pGL4.51[luc2 / CMV / Neo], Promega). Four- to five-week-old athymic mice were purchased from Jackson Laboratories. The SJ-GBM2-luc cell line was maintained at 70% confluence and their luminescence activity was checked. Cells were 10 6Cells were resuspended in PBS at a final concentration of 3 μl / cell. After anesthesia, a midline incision extending from bregma to lambda suture was made on the cranial crown. A 3-mm hole was drilled in the skull at a position 2 mm posterior to bregma and 2 mm outside the coronal suture. A 26-gauge Hamilton syringe was inserted into the hole, and 10 6 cells (3 μl) were slowly injected. The injection site was covered with sterile bone wax and sutured. As the first set of experiments, mice were randomly divided into experimental groups, and a dose-setting test was performed using AmiR20-p28 at 0.1 and 1 mg / kg. AmiR20-p28 was administered i.v. from the tail vein three times a week every other day (200 μl / injection). As the second set of experiments, mice were randomly divided into groups of PBS or AmiR20-p28 at 1 and 5 mg / kg (N = 5). After 4 weeks of treatment (a total of 12 injections / mouse, three times a week, i.v.), to examine whether tumors recurred over an additional experimental period of about 2 months, the AmiR20-p28 groups at 1 and 5 mg / kg were further divided into two subgroups, one group received continuous treatment (i.v. three times a week), while the other group received no treatment. As the third set of experiments, AmiR20-p28 at 1 mg / kg was administered i.v. three times a week at three different tumor growth stages [10 days (early), 20 days (mid), 40 days (late) after cell injection]. Mice were sacrificed based on the following humane endpoints, such as >20% weight loss, presence of labored breathing, abnormal behavior (e.g., paralysis), and loss of walking ability.

[0086]

[0017] In vivo bioluminescence imaging D-Luciferin potassium salt (GoldBio, #LUCK-100) was injected intraperitoneally into mice at 150 mg / kg. Mice were anesthetized in a chamber containing isoflurane / oxygen and placed for bioluminescence imaging using a Spectral Lago X imaging system (Accela, Czech Republic). A series of images were acquired over a period up to 30 minutes after D-luciferin injection time. Aura software (version 3.2) was used to calculate the region of interest (ROI) and integrate the total bioluminescence signal in each ROI. The radiance in the ROI (photons / sec / cm2 Based on the (), the data was analyzed. For each animal in the experimental group, the radiance values were plotted to create a tumor growth curve. The overall survival period was also evaluated based on their survival periods (the mice either died due to the disease or were sacrificed based on the humane endpoint criteria). For X-ray imaging, anesthetized mice were placed and images were taken using the Spectral Lago X imaging system. The Aura software was used to combine the images taken with the X-ray and bioluminescence systems.

[0087]

[0019] Confocal microscopy SJ-GBM2 cells, CHLA-200 cells, and astrocytes (2×10 4 cells / well) were cultured on glass slides placed in 24-well plates. After overnight culture, the cells were washed with PBS and incubated with 20 μM Alexa Fluor 568-labeled p28 peptide in medium (5% FBS, without phenol red) at 37 °C for 2 hours. The cells were fixed with 2% formalin for 10 minutes. After washing, slides were prepared and mounted using Vectashield containing DAPI (Vector Laboratories, catalog number 94010). The slides were examined by an LSM 710 confocal laser scanning microscope (Zeiss). The images were processed using ZEN software (ZEISS ZEN Lite). For GSC markers, the slides were first incubated with Alexa Fluor 568-labeled AmiR20-p28 at 37 °C for 2 hours. The cells were fixed with 2% formalin for 10 minutes. After washing, the slides were incubated with blocking buffer at room temperature for 1 hour, followed by sequential staining with anti-CD133 antibody (1:500, R&D Systems FAB11331G). The slides were washed and mounted using Vectashield containing DAPI. The slides were examined by an LSM 710 confocal laser scanning microscope.

[0088]

[0021] Histological analysis The collected samples were fixed with buffered 3.7% formalin (Anatech) for 24 hours. After fixation, the formalin was replaced with 70% ethanol. The samples were paraffin-embedded, and the blocks were cut into 4-μm-thick sections and mounted on slides for H&E staining. The H&E-stained slides were analyzed by a pathologist who was blinded to the experimental groups.

[0089]

[0023] Blood collection Before sacrifice, whole blood samples (about 500 μl) were collected into tubes by cardiac puncture. To perform cardiac puncture, the mice were deeply anesthetized under isoflurane, and a 21-gauge needle was inserted into the heart. The mice were euthanized immediately after cardiac puncture. After whole blood collection, the samples were allowed to clot by standing at room temperature for 15 minutes. The blood clots were then removed by centrifugation at 2,000 × g for 10 minutes at 4°C. Human serum samples from healthy volunteers were obtained from Discovery Life Sciences (Huntsville, AL). Serum from pediatric patients with GBM (ID#16) and malignant glioma (ID#17) were from our previous studies. Circulating miRNAs in the serum were extracted using the NucleoSpin miRNA extraction kit (Takara, #740971) according to the manufacturer's instructions. miRNA expression was analyzed using stem-loop real-time qPCR.

[0090]

[0025] Statistical analysis GraphPad Prism 9.0 was used for statistical analysis of the data. The two-sided Student's t-test was used for single comparisons, and group differences were evaluated using ANOVA. Survival assays were analyzed using the Kaplan-Meier test. The data are presented as mean ± SEM, and all experiments were performed in triplicate unless otherwise specified.

[0091]

[0027] Differential miRNA expression analysis The dataset analyzed in this study is available in the GSE42657 repository. The data was processed with a significant cutoff at a p-value of less than 0.05. The miRNA expression dataset includes 5 grade IV pGBM (age range 4 - 15 years) and 2 control tissues (age range 21 - 22 years).

[0092]

[0029] MR imaging Brain MR images were recorded by a 9.4T MRI system (Agilent, Santa Clara, CA) as described previously for 80. Briefly, mice were anesthetized with isoflurane / oxygen, temperature was maintained, and respiration was monitored throughout the whole scan. T2-weighted MR images were acquired using a fast spin echo sequence with the following acquisition parameters: TR / TE 2050 / 8 ms, echo train length 8, matrix 128×128, FOV 19.2mm×19.2mm, slice thickness 1mm. Images were visualized using MirocDicom (ver.2022.1).

[0093]

[0031] Tumor spheroid formation SJ-GBM2 spheroids were formed by self-aggregation of cells at the bottom of non-adhesive round-bottom 96-well plates (Thermo Scientific, #174925 96U). A suspension of 10,000 cells in 100 μl volume of medium was pipetted into individual wells and allowed to form cell-cell bonds over 3 days.

[0094]

[0033] NIR fluorescence imaging Mice with tumors in the brain were injected with 0.5 mg / kg of ICG-labeled p28. After 24 hours, the brains were scanned by an Odyssey imaging system (Li-cor, NE). Specific NIR signals at 800 nm were recorded.

[0095]

[0035] Toxicity assay Hemolysis assay: The potential hemolytic activity of AmiR20-p28 was determined as described above. Briefly, human whole blood samples were centrifuged at 1,000×g for 10 min, the pellets were washed with PBS and HKR buffer (pH 7.4), resuspended in HKR buffer, and 10 μl was transferred to a tube containing 190 μl of AmiR20-p28. 0.1% Triton X-100 detergent was used as a control for disrupting the RBC membrane. After 30 min, the tubes were centrifuged and the absorbance (540 nm) of the supernatant was recorded. Hemoglobin release in the presence of Triton X-100 was defined as 100%.

[0096]

[0037] Analysis of liver enzymes Alanine transaminase (ALT) activity was measured using an ALT assay kit (Sigma, #MAK052) and serum aspartate transaminase (AST) activity was measured using an AST assay kit (Sigma, #MAK055) according to the manufacturer's instructions. Briefly, serum samples at different dilutions were reacted with ALT or AST reaction mixture, plated in a 96-well plate, and subsequently read at A450. Final calculations were made by comparing the sample readings with those of the standard curves (pyruvate standards for ALT and glutamate standards for AST).

[0097]

[0039] Estimation of tumor size in human brain 10 out of 96 wells 2 , 10 3 , 10 4 , 10 5 , 10 6 and 10 7The density of SJ-GBM2-luc cells per well was maintained by trypsinization and cell counting, followed by incubation with 5× cell lysis buffer (Promega, catalog number #E153A) for 5 minutes. The cells were then subjected to 100 μl of D-luciferin potassium salt at a final concentration of 150 μg / ml, and bioluminescence was observed using a Spectral Lago X imaging system. Aura software was used to calculate the region of interest (ROI), integrate the total bioluminescence signal in each ROI, and generate a standard curve for ROIs with known concentrations of cell numbers. The cell number was measured for the desired luminescence signal from the ROI and converted to relative tumor size in the pediatric brain. The calculations were performed assuming that a tumor of 1 cm 3 contains 109 cells, the average size of the whole brain of the mouse is 0.58 cm 3 and the average size of the pediatric brain is 1,312 cm 3 is.

[0098]

[0041] Detection of cancer cells in tissues by Alu sequencing Quantitative analysis using human-specific Alu sequences (hAlu) was performed on samples taken from isolated brain tissues. Detection of human-derived tumors transplanted into mice can be identified by Alu sequences specific to human cells. Genomic DNA was extracted from the collected tissues using the DNeasy Blood&Tissue kit (Qiagen). Real-time PCR was performed using primers specific for hAlu and mGAPDH (5 ng of genomic DNA, 0.5 μM of each primer, and PowerUp SYBR Green Master Mix; Life Technologies, USA). Each reaction was carried out at a final volume of 10 μl, with 2 minutes at 50 °C and 2 minutes at 95 °C, followed by 30 cycles of 30 seconds at 95 °C, 30 seconds at 63 °C, and 30 seconds at 72 °C. The amounts of hAlu and mGAPDH DNA in the tissues were calculated by comparison with a standard curve, and the calculated amount of hAlu DNA was normalized against the relative amount of mGAPDH. The results were verified by performing PCR using M13-modified forward and reverse primers specific for hAlu. The PCR products were visualized on a 2% agarose gel and confirmed by DNA sequencing. Actin was used as a loading control by using primers that recognize actin in both mice and humans (521 nt - 575 nt).

[0099]

[0043] List of primers

Table 1-1

Table 1-2

[0100]

[0044] Preferred embodiments of the present invention have been shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Without departing from the present invention, numerous variations, modifications, and substitutions will occur to those skilled in the art. It is to be understood that various alternative forms of the embodiments of the present invention described herein may be used in practicing the present invention. The following claims define the scope of the present invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. A composition comprising an antisense microRNA covalently bound to a cell-permeable peptide, wherein the cell-permeable peptide contains p28.

2. The composition according to claim 1, wherein the antisense microRNA is antisense miR20, antisense miR21, or antisense miR21a.

3. The composition according to claim 1, wherein the covalent bond is formed between the maleimide-antisense microRNA and the cysteine ​​residue of p28 by a maleimide-thiol reaction.

4. The composition according to claim 1, wherein a covalent bond is formed between hexinyl-antisense microRNA and azide-p28.

5. The composition according to claim 1, wherein the cell-permeable peptide contains at least 90% sequence identity with p28.

6. A composition for use in a method for inhibiting tumor cell proliferation: However, the method comprises systemically administering the composition, which contains an antisense microRNA covalently bound to a cell-permeable peptide, to the tumor cells, wherein the cell-permeable peptide contains p28; and The antisense microRNA is antisense miR20, antisense miR21, or antisense miR21a; or The covalent bond is formed by a maleimide-thiol reaction between the maleimide-antisense microRNA and the cysteine ​​residue of p28, or the covalent bond is formed between the hexynyl-antisense microRNA and azide-p28; or The cell-permeable peptide contains at least 90% sequence identity with p28.

7. A composition for use in a method for targeting tumor cells with microRNA: However, the method comprises administering the composition, which contains an antisense microRNA covalently bound to a cell-permeable peptide, to the tumor cells, wherein the cell-permeable peptide contains p28; and The antisense microRNA is antisense miR20, antisense miR21, or antisense miR21a.

8. The composition according to claim 7, which is as follows: The covalent bond is formed between the maleimide-antisense microRNA and the cysteine ​​residue of p28 by a maleimide-thiol reaction, or the covalent bond is formed between the hexynyl-antisense microRNA and azide-p28, The cell-permeable peptide contains at least 90% sequence identity with p28, or The aforementioned cells are in vivo cells, or The antisense microRNA covalently bound to the cell-permeable peptide forms a complex that crosses the blood-brain barrier.

9. A composition for use in a method for reducing endogenous microRNAs in tumor cells: However, the method comprises systemically administering the composition containing an antisense microRNA covalently bound to a cell-permeable peptide to the tumor cells, wherein the cell-permeable peptide contains p28, and The antisense microRNA is antisense miR20, antisense miR21, or antisense miR21a.

10. Methods for evaluating the pGBM tumor status in subjects, including the following: To provide a diagram showing the correlation between miRNA concentration and the presence and size of tumors; To obtain a liquid biopsy sample from the aforementioned subject; To measure the miR20 concentration in the aforementioned liquid biopsy sample; To compare the miR20 concentration in the liquid biopsy sample with the figure; Here, the miR20 concentration indicates the presence and size of the tumor, and provides an evaluation of the pGBM tumor status. The step of measuring miR20 includes the use of an antisense microRNA covalently bound to a cell-permeable peptide and to a detectable label, wherein the cell-permeable peptide comprises p28.

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