Uses of circular RNA encoding PTEN protein and composition thereof in combination with immune checkpoint inhibitor in treatment of tumor

Abstract

The present invention provides uses of a circular RNA encoding PTEN and a composition thereof. The present invention further relates to use of a composition of the described circular RNA in combination with an immune checkpoint inhibitor in the treatment of a tumor. In vivo and in vitro pharmacodynamic experiments prove that the circular RNA encoding PTEN and the composition thereof can effectively inhibit tumor growth, and can achieve better efficacy when used in combination with the immune checkpoint inhibitor.

Description

Application of circular RNA encoding PTEN protein and its combination with immune checkpoint inhibitors in tumor therapy Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of a circular RNA encoding PTEN protein and its composition, and more particularly to the application of a composition of a circular RNA encoding PTEN protein combined with an immune checkpoint inhibitor in tumor treatment. Background Technology

[0002] PTEN (phosphatase and tensin homolog deleted on chromosome 10), a homologous gene for phosphatase and tensin deleted on human chromosome 10, is a key tumor suppressor gene. Its encoded protein plays a crucial role in various biological processes. The core function of PTEN is its ability to produce phosphatase activity, specifically removing the phosphate group from phosphatidylinositol-3,4,5-triphosphate (PIP3), converting it to phosphatidylinositol-4,5-diphosphate (PIP2). PIP3 is a key messenger in the PI3K (phosphatidylinositol 3-kinase) pathway, which is essential for cell growth, proliferation, and survival. Therefore, PTEN can antagonize PI3K activity by reducing PIP3 levels, thereby regulating downstream signaling pathways and influencing several key biological processes such as cell cycle, apoptosis, and migration. Under normal functional conditions, PTEN regulates the PI3K / AKT signaling pathway, preventing excessive cell proliferation and growth. However, when the PTEN gene mutates or the protein is inactivated, it leads to an abnormally high level of PIP3, which in turn activates the PI3K / AKT signaling pathway and its downstream effector molecules, causing uncontrolled cell growth and division, thereby increasing the risk of cancer. In fact, PTEN mutations and inactivation have been widely observed in various human cancers, including pancreatic cancer, breast cancer, prostate cancer, liver cancer, and colorectal cancer. Furthermore, germline mutations in the PTEN gene can lead to PTEN hamartoma syndrome, a group of hereditary diseases characterized by multiple benign tumors and cancer susceptibility, posing a significant health threat to patients. Therefore, targeted therapy strategies against PTEN have enormous potential application in cancers and diseases with PTEN dysfunction.

[0003] RNA protein supplementation therapy is a treatment based on RNA technology that temporarily provides protein-coding information without altering cellular DNA, thereby guiding cells to produce missing or underfunctional proteins. This is used to treat diseases caused by protein dysfunction due to gene mutations or deletions. Compared to messenger RNA (mRNA), which is more widely studied, circular RNA (circular RNA) is a novel RNA molecule that can express proteins while possessing advantages such as a longer half-life, more persistent protein expression, and greater stability. Therefore, by directly delivering circular RNA encoding PTEN protein into tumor cells to restore or enhance PTEN protein expression, it can inhibit tumor cell proliferation, induce apoptosis, and reverse the immunosuppressive microenvironment, thus exerting an anti-tumor effect and providing a new approach to cancer treatment. This method also offers a novel strategy for diseases caused by germline mutations in the PTEN tumor suppressor gene.

[0004] Currently, the main treatments for tumors include surgery, radiotherapy and chemotherapy, targeted therapy, and immunotherapy. Immunotherapy primarily targets programmed death receptor 1 (PD-1) / PD-L1 and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), inhibiting tumor growth by enhancing or activating the function of immune cells to kill tumor cells. While immunotherapy has shown better therapeutic effects than traditional treatments, the clinical response rate is not high. Based on our previous research, circular RNA encoding PTEN protein combined with immune checkpoint inhibitors can effectively activate anti-tumor immune responses, inhibit tumor growth, and prolong the survival time of animals. Therefore, circular RNA encoding PTEN protein combined with immune checkpoint inhibitors could serve as a novel treatment option for tumors. Summary of the Invention

[0005] The purpose of this invention is to provide an optimized circular RNA molecule encoding PTEN protein, which can express PTEN protein in vitro and in vivo, regulate the PI3K / AKT signaling pathway, and prevent excessive cell proliferation and growth, thereby having a therapeutic effect on tumors. Simultaneously, it provides a new treatment strategy for diseases caused by germline mutations in the PTEN tumor suppressor gene. Furthermore, the circular RNA molecule encoding PTEN protein in this invention can be combined with immune checkpoint inhibitors to effectively activate anti-tumor immune responses, improve the therapeutic effect of drugs, and provide a new method for tumor treatment.

[0006] In a first aspect, the present invention provides a circular RNA molecule encoding a PTEN protein, said circular RNA molecule encoding any one of the following proteins:

[0007] (1) Wild-type PTEN protein, wherein the amino acid sequence of the PTEN protein is shown in SEQ ID NO: 1;

[0008] (2) A mutant protein that has more than 80% identity with the wild-type PTEN protein shown in SEQ ID NO: 1 and has the same function.

[0009] In some embodiments of the present invention, the mutant protein has 85%, 90%, 95% or more of the same identity as the wild-type PTEN protein shown in SEQ ID NO: 1.

[0010] In some embodiments of the present invention, the mutant protein has 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the wild-type PTEN protein shown in SEQ ID NO: 1.

[0011] In some embodiments of the present invention, the sequence of the circular RNA encoding the PTEN protein includes any of the following:

[0012] (1) The circular RNA molecule or fragment thereof shown in SEQ ID NO: 2;

[0013] (2) A degenerate or complementary sequence of SEQ ID NO: 2;

[0014] (3) has more than 80% identity with (1) or (2) and has a circular RNA molecule that encodes the same functional protein.

[0015] In some embodiments of the present invention, the circular RNA molecule described in (3) has 85%, 90%, 95% or more of the same identity as (1) or (2).

[0016] In some embodiments of the present invention, the circular RNA molecule described in (3) has the same identity as (1) or (2) 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0017] In some embodiments of the present invention, the circular RNA molecule includes an IRES sequence and an open reading frame (ORF).

[0018] In some embodiments of the present invention, the circular RNA molecule includes the sequence shown in SEQ ID NO: 3 or 4.

[0019] In some embodiments of the present invention, the circular RNA molecule has a loop structure with the ends connected.

[0020] In some embodiments of the present invention, the transcribed linear RNA can be self-splicing into a circular form by type I intron ribozymes or directly intramolecularly ligated into a circular form by T4 RNA ligase catalysis.

[0021] In some embodiments of the present invention, the transcribed linear RNA is self-spliced ​​into a circular form by a type I intron ribozyme, the sequence of which is shown in SEQ ID NO: 3.

[0022] In some embodiments of the present invention, the transcribed linear RNA is circularized using T4 RNA ligase, and its sequence is shown in SEQ ID NO: 4.

[0023] In some embodiments of the present invention, the circular RNA molecule is an open reading frame (ORF) encoding the wild-type PTEN protein (SEQ ID NO: 1) or its homolog, fragment, or variant.

[0024] In some embodiments of the invention, the open reading frame (ORF) encoding variants of the wild-type PTEN protein has at least 80%, 85%, 90%, 95%, or 98% sequence identity with the wild-type protein and has the same function.

[0025] In some embodiments of the present invention, the identity is 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0026] In some embodiments of the present invention, the circular RNA molecule is a degenerate or complementary sequence encoding the above-mentioned protein sequence, or an mRNA sequence having at least 80%, 85%, 90%, 95% or 98% identity with it, or a sequence encoding a protein with the same function.

[0027] In some embodiments of the present invention, the identity is 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0028] In some embodiments of the present invention, the open reading frame (ORF) further includes encoding a polypeptide or protein fused with the target protein.

[0029] In some embodiments of the present invention, the polypeptide or protein is used for the expression, transport, detection or tracing of the target protein.

[0030] In some embodiments of the present invention, the polypeptide or protein includes a tag protein and / or a protein localization sequence.

[0031] In some embodiments of the present invention, the tag protein includes any one or a combination of at least two of the following: Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, or SUMO tag.

[0032] In some embodiments of the present invention, the tag protein is an HA tag, the nucleotide sequence of which is shown in SEQ ID NO: 11.

[0033] In some embodiments of the present invention, the circular RNA molecule comprises or consists of sequences as shown in any one of SEQ ID NO: 3-4.

[0034] In some embodiments of the invention, one or more nucleotides in the circular RNA molecule may be modified. For example, one or more nucleotides (e.g., all nucleotides) in the circular RNA molecule may each be independently replaced with a naturally occurring nucleotide analog or a synthetically produced nucleotide analog.

[0035] In some embodiments of the present invention, naturally occurring nucleotide analogues include pseudouridine, 2-thiouridine, 5-methyluridine, 5-methylcytidine, and N6-methyladenosine.

[0036] In some embodiments of the present invention, the artificially synthesized nucleotide analogs include N1-methylpseuuridine and 5-ethynyluridine. In some embodiments of the present invention, the artificially synthesized nucleotide analogs include α-adenosine triphosphate, α-cytidine triphosphate, α-guanosine triphosphate, α-uridine triphosphate, α-pseuuridine triphosphate, and N1-methylpseuuridine triphosphate.

[0037] A second aspect of the invention provides a DNA molecule encoding the circular RNA molecule described in the first aspect of the invention. In some embodiments of the invention, the DNA molecule can be used to prepare the circular RNA of the invention via in vitro transcription.

[0038] In some embodiments of the present invention, the DNA molecule comprises or consists of sequences as shown in any one of SEQ ID NO: 5-6.

[0039] A third aspect of the present invention provides a recombinant plasmid comprising the DNA molecule described in the second aspect of the present invention.

[0040] In some embodiments of the present invention, the backbone of the recombinant plasmid is the pCDNA3.1 plasmid. Of course, those skilled in the art can reasonably select other conventional empty vector plasmids in the art as backbones for the transfer of the target gene, including but not limited to pCDNA3.1, according to the needs of use.

[0041] In some embodiments of the present invention, the recombinant plasmid further comprises a start sequence, a T7 promoter, and a resistance gene.

[0042] In some embodiments of the present invention, the starting sequence is pUC Ori, which contains the nucleotide sequence shown in SEQ ID NO: 7.

[0043] In some embodiments of the present invention, the T7 promoter comprises a nucleotide sequence as shown in SEQ ID NO: 8.

[0044] In some embodiments of the present invention, the resistance gene is an ampicillin resistance gene and / or a neomycin resistance gene.

[0045] In some embodiments of the present invention, the recombinant plasmid comprises, or is composed of, the nucleotide sequences shown in SEQ ID NO: 9-10.

[0046] In some embodiments of the present invention, SEQ ID NO: 9 is a recombinant plasmid pCDNA3.1-circPTEN containing a protein-coding sequence, which is self-circulated using a type I intron ribozyme.

[0047] In some embodiments of the present invention, SEQ ID NO: 10 is a recombinant plasmid containing a protein-coding sequence circularized using T4 RNA ligase.

[0048] A fourth aspect of the present invention provides a circular RNA-carrier particle composition comprising a circular RNA molecule encoding the PTEN protein as described in the first aspect of the present invention, and a carrier material encapsulating the circular RNA molecule.

[0049] In some embodiments of the present invention, the carrier material may be selected from protein, polypeptide nanoparticles (PNP), lipid nanoparticles (LNP), polymer materials or inorganic nanoparticles.

[0050] In some embodiments of the present invention, the carrier material is LNP.

[0051] In some embodiments of the present invention, the LNP may comprise one or more of ionized lipids, polyethylene glycol-modified lipids, cholesterol and its derivatives, or phospholipids. For example, the LNP may comprise any one of ionized lipids, polyethylene glycol-modified lipids, cholesterol and its derivatives, or phospholipids, wherein any two, any three, or a combination of all four.

[0052] A fifth aspect of the present invention provides a method for treating tumors, the method comprising administering to a subject an effective amount of any one or a combination of at least two of the circular RNA and circular RNA-carrier particles of the present invention.

[0053] In this invention, the term "treatment" as used refers to a reduction in the risk of acquiring or developing a disease or condition, i.e., preventing at least one clinical symptom of the disease from developing in a subject susceptible to the disease or unexposed to the pathogen before the onset of the disease. For example, treatment may include: (i) preventing the disease, disorder, and / or condition in a patient who may be susceptible to the disease, disorder, and / or condition but has not yet been diagnosed with the disease, disorder, and / or condition; (ii) suppressing the disease, disorder, and / or condition, i.e., preventing its development; or (iii) alleviating the disease, disorder, and / or condition, i.e., causing the remission of the disease, disorder, and / or condition.

[0054] In this invention, the term "effective amount" refers to the amount of a compound sufficient to achieve such treatment or prevention when administered to a subject for the treatment or prevention of a disease. "Effective amount" can vary depending on the compound, the disease and its severity, and the age, weight, etc., of the subject being treated. "Therapeutic effective amount" refers to an effective amount for therapeutic treatment. "Prophylactic effective amount" refers to an effective amount for preventative treatment.

[0055] In some embodiments of the invention, the therapeutically effective amount is an effective amount for tumor treatment. The therapeutically effective amount is determined by a diagnostic participant, such as someone skilled in the art, using known techniques and observations in similar circumstances. In determining the therapeutically effective amount or dosage, the diagnostic participant should consider many factors, including but not limited to: animal species, weight, age, health status, disease acquired, severity of disease, individual response, specific compound used, specific composition used, route of administration, biocompatibility of the formulation, duration of treatment, excretion rate of the specific compound used, selected dose range, drugs used in combination with or concurrently with the specific compound used, and similar factors well known in the medical field, and other relevant circumstances.

[0056] In some embodiments of the present invention, the effective therapeutic dose is 0.0001-300 mg / kg body weight per day.

[0057] In some embodiments of the present invention, the therapeutically effective dose is 0.01-200 mg / kg body weight.

[0058] In this invention, the total daily dose can be a single dose or an equal portion. If necessary, the effective daily dose can be divided into multiple doses for administration. Therefore, a single-dose composition can contain such an amount or an approximation thereof that constitutes the daily dose.

[0059] Of course, it should be understood that the effective therapeutic dose in this invention can be reasonably adjusted based on the subject's health status, disease progression, species origin, and other information, and can be converted into equal or approximate doses between different species based on common knowledge in the field.

[0060] In this invention, the term "administration" as used refers to the physical introduction of a drug agent into a subject using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral administration routes, such as by injection or infusion.

[0061] In this invention, the terms "subject," "individual," and "patient" are well-known in the art and are used interchangeably herein to refer to any subject requiring treatment, particularly a mammalian subject. Examples include, but are not limited to, humans and other primates, including non-human primates such as chimpanzees and other ape and monkey species. Non-human mammals may be, but are not limited to, mice, rats, guinea pigs, hamsters, pigs, dogs, sheep, monkeys, rabbits, cats, cattle, or horses. The terms individual, subject, and patient do not inherently indicate a specific age, sex, race, etc.

[0062] In some embodiments of the present invention, the disease includes tumors, such as pancreatic cancer, breast cancer, glioma, thyroid cancer, liver cancer, kidney cancer, melanoma, or lymphoma.

[0063] A sixth aspect of the present invention provides the use of at least one of the circular RNA molecule encoding PTEN protein described in the first aspect, the DNA molecule described in the second aspect, the recombinant plasmid described in the third aspect, or the circular RNA-vector particle described in the fourth aspect in the preparation of a medicament.

[0064] In some embodiments of the present invention, the drug is an anti-tumor drug.

[0065] In some embodiments of the present invention, the tumor includes solid tumors and / or non-solid tumors.

[0066] In some embodiments of the present invention, the tumor includes any one of pancreatic cancer, breast cancer, glioma, thyroid cancer, liver cancer, kidney cancer, melanoma, or lymphoma.

[0067] In some embodiments of the present invention, the drug further contains a second active ingredient.

[0068] In some embodiments of the present invention, the second active ingredient includes an immune checkpoint inhibitor;

[0069] In some embodiments of the present invention, the immune checkpoint inhibitor includes at least one of anti-PD1 antibody, anti-PD-L1 antibody, or anti-CTLA4 antibody.

[0070] In some embodiments of the present invention, the medicament further contains pharmaceutically acceptable excipients.

[0071] In some embodiments of the present invention, the pharmaceutically acceptable excipients include, but are not limited to, at least one of fillers, binders, disintegrants, lubricants, etc.

[0072] Specifically, based on the beneficial effects of the circular RNA molecule described in this invention, these beneficial effects are ultimately manifested in the drug. More specifically, the drug contains any mass percentage of the aforementioned circular RNA molecule ranging from 1.00% to 99.00%, further, the drug contains any mass percentage of the aforementioned circular RNA molecule ranging from 5.00% to 95.00%, and even further, the drug contains any mass percentage of the aforementioned circular RNA molecule ranging from 10.00% to 90.00%.

[0073] A seventh aspect of the present invention provides a method for treating tumors in combination with an immune checkpoint inhibitor, the method comprising administering to a subject an effective amount of any one or a combination of at least two of the circular RNA and circular RNA-carrier particles of the present invention, simultaneously or sequentially administering an immune checkpoint inhibitor.

[0074] In some embodiments of the present invention, the immune checkpoint inhibitor includes at least one antibody from the group consisting of anti-PD1 antibody, anti-PD-L1 antibody, and anti-CTLA4 antibody.

[0075] In some embodiments of the present invention, the tumor includes any one of pancreatic cancer, breast cancer, glioma, thyroid cancer, liver cancer, kidney cancer, melanoma, or lymphoma.

[0076] An eighth aspect of the present invention provides a method for treating diseases caused by germline mutations in the PTEN tumor suppressor gene, the method comprising administering to a subject an effective amount of any one or a combination of at least two of the circular RNA of the present invention and circular RNA-carrier particles.

[0077] In embodiments of the method of the present invention, diseases caused by germline mutations in the PTEN tumor suppressor gene include, but are not limited to, Cowden syndrome, Bannayan-Riley-Ruvalcaba (BRRS) syndrome, Proteus syndrome, megalocerebellar-capillary malformation syndrome, or cerebellar dysplasia gangliocytoma.

[0078] A ninth aspect of the present invention provides the use of at least one of the circular RNA molecule encoding PTEN protein described in the first aspect, the DNA molecule described in the second aspect, the recombinant plasmid described in the third aspect, or the circular RNA-vector particle described in the fourth aspect in the preparation of a medicament for treating diseases caused by germline mutations of the PTEN tumor suppressor gene or related disease conditions.

[0079] In some embodiments of the present invention, the disease includes PTEN hamartoma syndrome.

[0080] In some embodiments of the present invention, the PTEN hamartoma syndrome includes any one of Cowden syndrome, Bannayan-Riley-Ruvalcaba (BRRS) syndrome, Proteus syndrome, megalocerebellar-capillary malformation syndrome, or cerebellar dysplasia gangliocytoma.

[0081] In some embodiments of the present invention, disease-related conditions caused by germline mutations in the PTEN tumor suppressor gene include neurodevelopmental disorders, behavioral and cognitive deficits, skin and mucous membrane involvement, thyroid abnormalities, vascular involvement, gastrointestinal involvement, fibrocystic breast disease, uterine fibroids, and endometrial hyperplasia.

[0082] Compared with the prior art, the present invention has the following beneficial effects:

[0083] This invention provides an application of circular RNA encoding PTEN protein and its composition thereof, and also provides a method for combining circular RNA encoding PTEN protein and its composition with immune checkpoint inhibitors. This invention increases the stability of RNA molecules and prolongs their half-life by producing circular RNA encoding PTEN protein, enabling efficient and long-term expression of PTEN protein in vitro and in vivo. This invention also provides a DNA sequence and recombinant plasmid for preparing circular RNA encoding PTEN protein. The circular RNA-vector particles provided by this invention can express PTEN protein in vitro and in vivo, supplementing or restoring the function of PTEN protein, thereby achieving the purpose of treating tumors and diseases caused by PTEN germline mutations. Attached Figure Description

[0084] Figure 1 shows the electrophoresis results of the mRNA and circular RNA encoding the PTEN protein obtained by plasmid transcription.

[0085] Figure 2 shows the results of Western blot analysis of PTEN protein expression by mRNA and circular RNA encoding PTEN protein at different time points.

[0086] Figure 3 shows the results of Panc-02 cell apoptosis induced by PTEN protein mRNA and circular RNA.

[0087] Figure 4 shows the cell growth curves of Panc-02 cells after treatment with mRNA and circular RNA encoding PTEN protein.

[0088] Figures 5A-5C show the biosafety assessment of LNP delivery of mRNA and circular RNA encoding PTEN protein. Figure 5A shows the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CREA) in mice of each treatment group; Figure 5B shows the serum levels of inflammatory factors interleukin-2 (IL-2), interleukin-6 (IL-6), immunoglobulin G (IgG), and tumor necrosis factor-α (TNF-α) in each group; Figure 5C shows the H&E staining of tissue sections.

[0089] Figures 6A-6D show the tumor-suppressing effects of mRNA and circular RNA encoding PTEN, as well as the combination with aPD-1, in an orthotopic pancreatic cancer tumor model (Figure 6A), the comparison of body weight among groups (Figure 6C), the results of mouse survival (Figure 6D), and the results of comparing the effects of each experimental group on the spleen of mice (Figure 6B).

[0090] Figure 7 shows an image of an orthotopic pancreatic cancer tumor in a mouse.

[0091] Figures 8A-8F show the activation of anti-tumor immune responses in patients with orthotopic pancreatic cancer by LNP delivery of mRNA and circular RNA encoding PTEN protein. Figure 8A shows flow cytometry plots of CD4+CD8 in each group; Figure 8B shows flow cytometry plots of CD206+F4 / 80; Figure 8C shows flow cytometry plots of CD80+F4 / 80; Figure 8D shows flow cytometry plots of CD8 in each group. + Figure 8E shows a comparison of the proportion of T cells in each group; Figure 8F shows a comparison of the proportion of CD206M2 cells in each group. Detailed Implementation

[0092] The present invention will now be described in detail with reference to specific embodiments. The given embodiments are merely illustrative and are not intended to limit the scope of the invention. The specific implementation methods disclosed herein can be more readily understood by referring to the following detailed description in conjunction with the accompanying drawings, which constitute a part of this disclosure.

[0093] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. If specifically stated otherwise, the materials, reagents, etc., used in the following embodiments are commercially available.

[0094] Abbreviations and Definitions

[0095] IRES sequence: internal ribosome entry site sequence; IVT: in vitro transcription; TECP: tricarboxyethylphosphine; DOPE: dioleoylphosphatidylethanolamine; DMG-PEG 2000: 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol 2000; LNP: lipid nanoparticles; TAM cells: tumor-associated macrophages;

[0096] Example 1: Synthesis of circular RNA encoding PTEN protein

[0097] 1. Synthesis of circular RNA encoding PTEN protein

[0098] 1.1 Sequence design encoding PTEN protein

[0099] A DNA coding sequence was designed based on the wild-type PTEN sequence. An IRES sequence was added to the DNA coding sequence and inserted into the pCDNA3.1 plasmid via homologous recombination to construct the recombinant plasmid pCDNA3.1-circPTEN (SEQ ID NO: 9), which contains the protein coding sequence and is self-circulated using a type I intron ribozyme. A recombinant plasmid containing the protein coding sequence, circularized with T4 RNA ligase, was obtained using the same method; its sequence is SEQ ID NO: 10.

[0100] Sequencing confirmed the accuracy of the constructed plasmid sequence.

[0101] 1.2 Sequence optimization of the PTEN protein

[0102] By optimizing the nucleotide sequence encoding the PTEN protein obtained from the above steps, its yield during transcription, its intracellular stability, and its translation efficiency can be improved.

[0103] The specific method is as follows:

[0104] Utilize publicly available RNA sequence optimization platforms, such as LinearDesign sequence design algorithm and GenSmart. TMCodon optimization, tools such as VectorBuilder and Java Codon Adaptation Tool, are used to optimize the coding sequences of RNA. Strategies employed include using host-preferred codons and increasing CG content.

[0105] In this embodiment, two optimized sequences are provided, as shown in SEQ ID NO: 12 and 13.

[0106] 2. Preparation of circular RNA

[0107] The constructed plasmid was transformed into *E. coli* for amplification. *E. coli* containing the plasmid were collected, and the plasmid was harvested and purified. The purified plasmid was linearized by cutting it with restriction endonucleases. In vitro transcription was performed using T7 RNA polymerase with the linearized plasmid as a template. In this embodiment, a conventional co-transcription system (as shown in Table 1) was used for transcription.

[0108] Table 1. Co-transcription system (20 μL system)

[0109] After incubating the above system at 37°C for 2-4 hours, linear RNA (pre-circPTEN) was obtained.

[0110] The same in vitro transcription method was used to obtain mRNA encoding PTEN (mPTEN) and mRNA with randomly shuffled PTEN sequences (scrPTEN), which were used as the control group for circPTEN in the experiment.

[0111] Linear RNA was circularized to obtain circular RNA (circPTEN) encoding the PTEN protein. The circularization reaction system used is as follows:

[0112] Table 2 Cyclization reaction system (20 μL system)

[0113] Following the cyclization reaction, the linear RNA in the RNase R digestion system (Kaikai Biotechnology, RNR-EE001) was used to identify, enrich, and purify the circular RNA. The specific digestion time and amount of RNase R were determined according to the manufacturer's instructions.

[0114] The pCDNA3.1-circPTEN plasmid was transcribed in vitro to obtain the circular RNA molecule circPTEN, as shown in SEQ ID NO: 3. circPTEN was translated into PTEN protein at the cellular and animal levels. The quality of circPTEN was assessed using agarose gel electrophoresis and capillary electrophoresis.

[0115] The results are shown in Figure 1.

[0116] The agarose gel electrophoresis results in Figure 1 show that the PTEN mRNA obtained from transcription and the pre-circPTEN are both single bands without degradation, while the circulated circPTEN has a smaller molecular weight than the pre-circPTEN and contains byproduct bands.

[0117] Example 2: Preparation of lipid nanoparticles of circular RNA

[0118] Lipid nanoparticles containing circular RNA were prepared using the circular RNA synthesized in Example 1. In this example, the mass ratio of mRNA to LNP was fixed at 5.9%.

[0119] The specific steps are as follows: Cationic lipids (obtained from the prior Chinese application CN202410591611.1), dioleoylphosphatidylethanolamine (DOPE), cholesterol, and 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG 2000) were dissolved in ethanol at a molar ratio of 45:30:23.5:1.5 to obtain a lipid mixture. This lipid mixture was rapidly mixed with mRNA dissolved in 50 mM sodium acetate buffer (pH 5.0) at a volume ratio of 1:3. The lipid mixture and mRNA were then mixed using a microfluidic mixer to obtain lipid nanoparticles. Ethanol was removed by dialysis, followed by ultrafiltration concentration and filtration through a 0.22 μm filter to obtain LNP-mRNA nanoparticles, which were stored at 4°C for use. Unloaded LNPs were used as a blank control. The particle size, polydispersity index (PDI), and encapsulation efficiency data of the obtained formulation are shown in Table 3.

[0120] Table 3 Parameters of lipid nanoparticles

[0121] Example 3: Cellular Level Evaluation

[0122] In this embodiment, the lipid nanoparticles obtained in the above embodiments were used to conduct an effect evaluation experiment at the cellular level.

[0123] 1. Cell culture:

[0124] Mouse pancreatic cancer cells Panc-02 (Beijing Solarbio Science & Technology Co., Ltd.) were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% antibiotics at 37°C in a cell culture incubator containing 5% CO2.

[0125] 2. Cell transfection:

[0126] One day before transfection, Panc-02 cells were digested with trypsin, counted, and seeded into culture plates. 24 hours after plating, lipid nanoparticle-encapsulated LNP-mPTEN and LNP-circPTEN were diluted with culture medium and added to the cell culture plates for transfection. The culture plates were then incubated at 37°C with 5% CO2. Based on the experimental objectives, appropriate detection methods were selected to measure the corresponding indicators.

[0127] 3. Western blot for protein immunoblotting

[0128] Panc-02 cells were collected at three different time points: 24h, 48h, and 72h. Cells were washed twice with pre-chilled PBS, and cell lysis buffer was added to lyse the cells to obtain total cellular protein. The total protein content was then determined using the Pierce BCA protein assay (Thermo Fisher Scientific). Samples were separated by 12% SDS-PAGE gel, transferred to PVDF membranes (Sigma-Aldrich), and blocked with 5% skim milk powder dissolved in TBST. The PVDF membranes were incubated overnight at 4°C with antibodies in the following ratios: anti-PTEN (1:1000, Abclonal, A19104) and anti-GAPDH (1:50000, Abclonal, A19056). The next day, the PVDF membranes were washed five times in TBST for 5 minutes each time. The membranes were then incubated with horseradish peroxidase (HRP)-labeled secondary antibody, reacted with ECL substrate (Pierce), and developed.

[0129] The results are shown in Figure 2.

[0130] The Western blot results in Figure 2 show that both LNP-encapsulated mPTEN and circPTEN can translate and express PTEN protein in cells. Furthermore, comparing the protein expression efficiency of mPTEN and circPTEN reveals that circPTEN maintains PTEN protein expression for a longer period, exhibiting a longer half-life. 72 hours after transfection, the protein expression level of mPTEN significantly decreased, while circPTEN maintained a relatively high level of protein expression.

[0131] 4. Flow cytometry detection of apoptosis

[0132] Panc-02 cells were seeded in 12-well plates and cultured for 24 hours. Then, LNP-mPTEN and LNP-circPTEN diluted in 1640 medium were added for transfection. The amount of lipid nanoparticles added was determined by a concentration of 2 μg / mL for both mPTEN and circPTEN. After 48 hours of incubation, flow cytometry was performed. Cells were first digested and centrifuged to collect them, then resuspended in Binding Buffer, stained with Annexin V and PI apoptosis dye, and then analyzed for apoptosis using flow cytometry.

[0133] The results are shown in Figure 3.

[0134] Flow cytometry analysis revealed that LNP-encapsulated circPTEN induced more significant apoptosis than mPTEN (80.4% vs 72.8%), demonstrating stronger application advantages (Figure 3).

[0135] 5. Cell proliferation detection

[0136] Panc-02 cell suspensions were prepared and seeded in 96-well plates, then cultured overnight at 37°C with 5% CO2. Cells were transfected with cell culture medium containing LNP empty vector, LNP-scrPTEN, LNP-mPTEN, and LNP-circPTEN, with RNA concentrations of 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, and 4 μg / mL, respectively. After transfection, cells were incubated for 48 h. The cell-killing effects of mPTEN and circPTEN were assessed using the CCK8 assay according to the kit instructions.

[0137] The results are shown in Figure 4.

[0138] Figure 4 shows that compared with the LNP empty vector group and LNP-scrPTEN, LNP-mPTEN and LNP-circPTEN significantly reduced cell proliferation, and circPTEN was more effective than mPTEN.

[0139] Example 4: In vivo biosafety evaluation

[0140] In this embodiment, 4-6 week old female C57BL / 6J mice (purchased from Guangdong Yaokang Biotechnology Co., Ltd.) were selected as experimental mice and were raised under standardized conditions in a specific pathogen-free (SPF) grade animal facility.

[0141] The in vivo administration regimen for the experimental group was as follows: LNP nanolipid particles were prepared according to the method described in the above embodiments to deliver mPTEN and circPTEN, and were injected into normal C57BL / 6J mice via different administration routes (intravenous IV and intraperitoneal IP).

[0142] The experiment consisted of six groups: PBS (blank), scrPTEN (IP), mPTEN (IV), mPTEN (IP), circPTEN (IV), and circPTEN (IP). Mice were administered the drugs every three days for a total of five doses. Mice were sacrificed on day 15 post-administration, and serum and organ tissues were collected. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CREA) were analyzed using a biochemical analyzer. Serum levels of inflammatory factors, such as IL-2, IL-6, IgG, and TNF-α, were measured using an ELISA kit. Furthermore, major organ tissues were extracted, prepared into paraffin sections, and stained with hematoxylin and eosin (H&E) to observe their histological morphology.

[0143] The results are shown in Figures 5A-5C.

[0144] As shown in Figure 5A, the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CREA) in all treatment groups remained within the normal range compared to the PBS group, indicating that the mice maintained normal liver and kidney function. Meanwhile, Figure 5B shows that the serum levels of inflammatory factors interleukin-2 (IL-2), interleukin-6 (IL-6), immunoglobulin G (IgG), and tumor necrosis factor-α (TNF-α) in all groups showed no significant changes. Hematologic and epithelial eosinophil (H&E) results of tissue sections showed normal morphology of the major organs (heart, liver, spleen, lung, and kidney), indicating good in vivo biocompatibility in all experimental groups (Figure 5C).

[0145] The above data indicate that LNP-mPTEN and LNP-circPTEN formulations administered via tail vein (IV) or intraperitoneal injection (IP) have good in vivo biocompatibility and do not cause liver or kidney dysfunction or inflammation.

[0146] Example 5: In vivo evaluation - mouse tumor experiment

[0147] In this embodiment, 4-6 week old female C57BL / 6J mice (purchased from Guangdong Yaokang Biotechnology Co., Ltd.) were selected as experimental mice and were raised under standardized conditions in a specific pathogen-free (SPF) grade animal facility.

[0148] The method for constructing the mouse orthotopic pancreatic cancer model in the experimental group was as follows: Panc-02 mouse pancreatic cancer cells in the logarithmic growth phase were collected and prepared into a cell suspension using matrix gel. After anesthetizing C57BL / 6J female mice with sodium pentobarbital solution, a 1cm longitudinal incision was made along the left upper quadrant of the abdomen near the rectum using sterile surgical instruments in a laminar flow hood to carefully expose the pancreas. The Panc-02 cell suspension was then injected into the pancreatic tissue. The abdominal wound was sutured to ensure a tight seal and prevent infection and adhesion of abdominal organs. The mice were properly housed, and their recovery was closely monitored, with regular observation of their condition. Tumor growth in the mice was also checked periodically.

[0149] The experiment consisted of seven groups: PBS (blank), LNP-scrPTEN, aPD-1 (positive group), LNP-scrPTEN+aPD-1, LNP-mPTEN, LNP-circPTEN, and LNP-circPTEN+aPD-1. Orthotopic pancreatic cancer tumor treatment was administered: treatment began three days after tumor inoculation in the experimental groups. The specific treatment regimen was as follows: LNP-encapsulated RNA was injected intraperitoneally into mice, with the first administration defined as day 1 of treatment. The combination group received an intraperitoneal injection of aPD-1 monoclonal antibody (Amycin, BE0146) on day 2 after LNP administration. Administered every three days for a total of five administrations. The dosage of mPTEN and circPTEN was 1.5 mg / kg body weight, and the dosage of aPD-1 was 5 mg / kg body weight. After treatment, tumor tissue and spleen tissue were photographed and weighed. During the treatment period, mouse weight was measured every two days, and weight changes were recorded. The physiological state of the mice was continuously observed, and their survival time was recorded.

[0150] The results are shown in Figures 6A-6D.

[0151] Figure 6A shows that LNP-delivered mPTEN and circPTEN significantly inhibited the growth of pancreatic cancer in situ, with circPTEN exhibiting better tumor-suppressive effects than mPTEN. Statistical analysis revealed a significant difference in tumor-suppressive effect between circPTEN and mPTEN, and a significant difference between the circPTEN group and the group receiving aPD-1 immunoblockade therapy. These results indicate that circPTEN has a better anti-tumor effect than mPTEN and can effectively improve tumor responsiveness to aPD-1 immunoblockade therapy. Therefore, circPTEN combined with aPD-1 immunoblockade therapy has significant clinical application potential in cancer treatment.

[0152] Meanwhile, there was no significant difference in body weight among the different treatment groups, and the LNP-circPTEN carrier particle composition showed no significant side effects (Figure 6C). The spleen weight of mice in the LNP-circPTEN combined with aPD-1 group was normal, with no significant difference compared to the PBS group, demonstrating the good biocompatibility of LNP-circPTEN combined with aPD-1 therapy and indicating that circPTEN has lower immunogenicity than mPTEN (Figure 6B). Figure 6D shows that circPTEN+aPD-1 and circPTEN can significantly prolong the survival time of experimental animals, consistent with the tumor growth inhibition effect.

[0153] As can be seen, the circular RNA molecule circPTEN encoding PTEN protein in this embodiment of the invention can efficiently express PTEN protein in mice. LNP-circPTEN has a good inhibitory effect on tumors and effectively improves the tumor's response to aPD-1 immunotherapy. LNP-circPTEN combined with aPD-1 immunotherapy showed a highly efficient tumor-suppressive effect and significantly prolonged the survival time of experimental animals.

[0154] Example 6: Activation of anti-tumor immune response

[0155] After the orthotopic pancreatic cancer animal efficacy experiment in the above embodiments, tumor tissues were removed from the mice in each group. The tumor tissues were homogenized into single-cell suspensions using a tissue homogenizer. Flow cytometry antibodies containing Brilliant Violet 421 anti-CD45, FITC anti-CD3, APC anti-CD4, and PE anti-CD8 were added to detect CD8 infiltration in the tumor tissues. + T cell content. The content of infiltrating M2 TAM cells in tumor tissue was detected by flow cytometry using Brilliant Violet 421 anti-CD45, PE-Cy7 anti-CD11b, FITC anti-F4 / 80, and APC anti-CD206. The content of infiltrating M1 macrophages in tumor tissue was detected by flow cytometry using Brilliant Violet 421 anti-CD45, PE-Cy7 anti-CD11b, FITC anti-F4 / 80, and APC anti-CD80.

[0156] The results are shown in Figures 8A-8F.

[0157] Figures 8A-8F show that CD8 is lacking in pancreatic cancer tumor tissue. + T-cell infiltration was only 6.49% in the PBS experimental group. After LNP delivery of circPTEN and mPTEN, CD8...+ The proportion of T cells increased to 38.51% and 27.04%, effectively activating anti-tumor effector T cells. LNP-circPTEN combined with aPD-1 therapy achieved the best effector T cell activation effect, reaching 47.89%. Simultaneously, LNP-circPTEN combined with aPD-1 therapy significantly reduced the content of M2 TAMs infiltrating tumor tissue, effectively reversing the immunosuppressive environment. The number of anti-tumor M1 macrophages in tumor tissue was significantly increased after treatment with LNP-circPTEN and LNP-circPTEN combined with aPD-1 therapy. These results demonstrate that LNP-delivered ircPTEN combined with aPD-1 therapy has a good anti-tumor immune activation effect.

[0158] The preferred embodiments of the present invention have been described in detail above. However, the detailed description of the specific embodiments is intended to illustrate, rather than limit, the scope of the invention, which is defined by the scope of the appended claims. Furthermore, different embodiments of the present invention can be combined in any way without departing from the core spirit of the invention, and such combinations should also be considered part of the invention. To avoid repetition, all possible combinations will not be described in detail here.

[0159] While the invention has been described with reference to specific embodiments, those skilled in the art will understand that various modifications can be made and equivalents can be used without departing from the spirit and scope of the invention. Various modifications can be made to suit specific circumstances, materials, compositions, methods, and steps to conform to the purpose and spirit of the invention. All such modifications are intended to be within the scope of the claims.

Claims

1. A circular RNA molecule encoding a PTEN protein, characterized in that, The circular RNA molecule encodes any one of the following proteins: (1) a wild-type PTEN protein, the amino acid sequence of which is shown in SEQ ID NO: 1; (2) a mutant protein having more than 80% identity with the wild-type PTEN protein shown in SEQ ID NO: 1 and having the same function.

2. The circRNA molecule encoding PTEN according to claim 1, characterized in that, The sequence of the circular RNA encoding the PTEN protein includes any one of the following: (1) a circular RNA molecule shown in SEQ ID NO: 2 or a fragment thereof; (2) a degenerate sequence or a complementary sequence of the sequence shown in SEQ ID NO: 2; (3) a circular RNA molecule having more than 80% identity with (1) or (2) and encoding a protein having the same function.

3. The circRNA molecule encoding PTEN according to any one of claims 1-2, characterized in that, The circular RNA molecule comprises an IRES sequence and an open reading frame (ORF); the circular RNA molecule comprises a sequence shown in SEQ ID NO: 3 or 4.

4. The circRNA molecule encoding PTEN according to any one of claims 1 to 3, characterized in that, The circular RNA molecule has a circular structure with a head-to-tail connection.

5. A DNA molecule, characterized in that, The DNA molecule encodes the circular RNA molecule encoding PTEN according to any one of claims 1-4; Preferably, the DNA molecule comprises a nucleotide sequence shown in SEQ ID NO: 5-6.

6. A recombinant plasmid, characterized in that, The recombinant plasmid comprises the DNA molecule according to claim 5; Preferably, the backbone of the recombinant plasmid is a pCDNA3.1 plasmid; Preferably, the recombinant plasmid further comprises a start sequence, a T7 promoter and a resistance gene; Preferably, the start sequence comprises a pUC Ori comprising a nucleotide sequence shown in SEQ ID NO: 7; Preferably, the T7 promoter comprises a nucleotide sequence shown in SEQ ID NO: 8; Preferably, the resistance gene is an ampicillin resistance gene and / or a neomycin resistance gene; Preferably, the recombinant plasmid comprises a nucleotide sequence shown in SEQ ID NO: 9-10.

7. An mRNA-carrier particle, characterized in that, The circular RNA-carrier particle comprises the circular RNA molecule encoding the PTEN protein according to any one of claims 1-4 and a carrier material encapsulating the circular RNA molecule; Preferably, the carrier material comprises a protein, a polypeptide nanoparticle (PNP), a lipid nanoparticle (LNP), a polymer material or an inorganic nanoparticle; Preferably, the carrier material is a lipid nanoparticle (LNP); Preferably, the LNP comprises one or more of an ionizable lipid, a pegylated lipid, cholesterol or a derivative thereof, a phospholipid.

8. A medicament for treating a tumor, characterized by comprising the compound according to claim 1. The tumor treatment drug comprises at least one of the circular RNA molecule encoding the PTEN protein according to any one of claims 1-4, the DNA molecule according to claim 5, the recombinant plasmid according to claim 6 or the circular RNA-carrier particle according to claim 7; Preferably, the tumor treatment drug further comprises an immune checkpoint inhibitor; Preferably, the immune checkpoint inhibitor comprises at least one of an anti-PD1 antibody, an anti-PD-L1 antibody and an anti-CTLA4 antibody.

9. Use of at least one of the circular RNA molecule encoding PTEN protein according to any one of claims 1-4, the DNA molecule according to claim 5, the recombinant plasmid according to claim 6 or the circular RNA-carrier particle according to claim 7 in the preparation of a medicament. Preferably, the medicament is an anti-tumor medicament. Preferably, the tumor comprises pancreatic cancer, breast cancer, brain glioma, thyroid cancer, liver cancer, kidney cancer, melanoma and lymphoma. Preferably, the medicament further comprises a second active ingredient. Preferably, the second active ingredient is an immune checkpoint inhibitor. Preferably, the medicament further comprises a pharmaceutically acceptable excipient.

10. Use of at least one of the circular RNA encoding PTEN according to any one of claims 1-4, the DNA according to claim 5, the recombinant plasmid according to claim 6 or the circular RNA-carrier particle according to claim 7 in the preparation of a medicament for treating a disease caused by germline mutation of PTEN tumor suppressor gene or a related disease condition thereof. Preferably, the disease caused by germline mutation of PTEN tumor suppressor gene comprises PTEN hamartoma syndrome. Preferably, the PTEN hamartoma syndrome comprises Cowden syndrome, Bannayan-Riley-Ruvalcaba (BRRS) syndrome, Proteus syndrome and megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome or lissencephaly-polydactyly-hydrocephalus syndrome. Preferably, the related disease condition of the disease caused by germline mutation of PTEN tumor suppressor gene comprises neurodevelopmental disorder, behavioral cognitive deficits, cutaneous mucosal involvement, thyroid abnormalities, vascular involvement, gastrointestinal involvement, fibrocystic breast disease, uterine fibroids and endometrial hyperplasia.

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