Universal mRNA-lnp vaccine for a population and method of making same

Abstract

The present application relates to a kind of mRNA-LNP vaccine for a class of crowd and its preparation method.Specifically, the present application relates to a method for constructing a set of human de novo origin new genes, the gene set constructed by the method, part of the coding sequence or full-length coding sequence of any gene in the gene set, the use of molecule for detecting or regulating the expression of the gene in the preparation of drug for treating tumor or diagnostic reagent for diagnosing tumor, polynucleotide molecule encoding the protein expressed by any gene in the gene set, antigenic fragment thereof or variant of protein or antigenic fragment, liposome complex containing the polynucleotide molecule, the use of the liposome complex in the preparation of vaccine for treating and / or preventing tumor, and the targeted molecule for regulating the expression of any gene selected from the gene set.

Description

Technical Field

[0001] This invention pertains to the medical field. Specifically, the invention relates to a method for constructing a gene set, the gene set constructed by the method, the use of a partial or full-length coding sequence of any gene in the gene set, a molecule for detecting or regulating the expression of the gene in the preparation of a drug for treating tumors or a diagnostic reagent for diagnosing tumors, a polynucleotide molecule encoding a protein, an antigenic fragment thereof, or a variant of the protein or antigenic fragment expressed by any gene in the gene set, a liposome complex containing the polynucleotide molecule, the use of the liposome complex in the preparation of a vaccine for treating and / or preventing tumors, and a targeting molecule for regulating the expression of any gene selected from the gene set. Background Technology

[0002] Cancer is one of the major health challenges worldwide. According to data released by the International Agency for Research on Cancer (IARC) of the World Health Organization, approximately 20 million new cancer cases and 9.7 million cancer deaths were reported globally in 2022. 1 In China, the incidence and mortality rates of cancer continue to rise. Data from the National Cancer Center shows that in 2022, China saw 4.82 million new cancer cases and 2.57 million cancer deaths, with annual medical expenses for malignant tumors exceeding 220 billion RMB. To effectively alleviate the growing burden of cancer, my country has clearly identified promoting comprehensive risk factor control, cancer screening, early diagnosis and treatment, and standardization of treatment protocols as key strategies for effectively reducing the increasing cancer burden before 2030. 2 .

[0003] Currently, traditional cancer treatments mainly include surgery, radiotherapy, and chemotherapy, but these methods all have limitations, such as significant side effects and the tendency to develop drug resistance. Therefore, developing safer and more effective early cancer prevention or treatment strategies to reduce the cancer burden has become a major health problem urgently needing to be addressed. Existing immunotherapies, such as DNA vaccines, anti-PD-1, and monoclonal antibodies, while showing some efficacy, are expensive, have complex manufacturing processes, and carry numerous risks. For example, DNA vaccines may induce mutations in the body, and immune checkpoint inhibitors such as anti-PD-1 may cause activated T cells to attack normal tissues, increase autoantibodies and cytokines, thereby inducing autoimmune inflammation and producing a series of immune-related adverse reactions. Furthermore, monoclonal antibody treatment has a long treatment cycle, and patients are prone to developing drug resistance. In addition, tumors to which the immune system responds weakly or cannot effectively attack are called "cold tumors." They typically exhibit low levels of immune cell infiltration, a lack of good angiogenesis within the tumor, and low expression of immune-related genes. Cold tumors often have a high level of immunosuppressive microenvironment, meaning that the tumor can evade immune surveillance and continue to grow and spread. For example, cold tumors are relatively common in cancer types such as pancreatic cancer and prostate cancer. Due to the aforementioned characteristics, these tumors have poor responsiveness to traditional immunotherapy, resulting in relatively poor patient prognosis and extremely high treatment difficulty. How to transform cold tumors into hot tumors to improve their sensitivity to PD-L1 therapy is a hot topic in cold tumor treatment. Therefore, developing more effective strategies to improve the sensitivity of cold tumors to immunotherapy is an important research direction in the current field of cancer treatment.

[0004] mRNA tumor vaccines are nucleic acid vaccines designed to target tumor antigens. These tumor antigens are typically expressed by or specific to tumor cells and, after release, are presented to immune cells such as dendritic cells, macrophages, and B cells. Subsequently, T cells exert their killing effect by recognizing specific antigens in tumor tissue. However, the expression level of these tumor antigens is low, failing to activate a strong immune response. mRNA tumor vaccines, on the other hand, are prepared and injected into the body based on a modified and assembled mRNA encoding tumor-associated antigens or tumor-specific antigens. After being phagocytosed by antigen-presenting cells, the vaccine relies on the cell's protein synthesis system to translate specific proteins, which serve as "targets," inducing an immune response against these "targets" and thus specifically attacking tumor cells. However, naked mRNA is extremely unstable, large in size, hydrophilic, and negatively charged. Early mRNA molecules faced limitations due to instability and low expression efficiency. Unmodified mRNA can induce high immunogenicity, activating innate immune responses through Toll-like receptors (TLR3, TLR7, TLR8, and TLR9). Subsequently, incorporating modified nucleosides (such as m5C, m6A, m5U, s2U, and pseudouracil) into the mRNA formulation significantly reduced their immunogenicity, and completely replacing uracil with pseudouracil improved mRNA stability and translation efficiency. 3 Modified mRNA vaccines offer numerous advantages. Compared to DNA-based treatments, mRNA vaccines do not integrate into the host cell genome, eliminating the risk of mutations. Furthermore, the transient nature of mRNA expression ensures that antigen stimulation is temporary, effectively reducing the likelihood of chronic inflammation or autoimmune responses. In general, mRNA vaccines have no significant toxic side effects and can be synthesized in vitro, with simple and rapid preparation processes. By activating both humoral and cellular immunity simultaneously through host protein synthesis, mRNA vaccines can stimulate a high immune response rate, making them ideal anti-tumor vaccines. Current research on mRNA cancer vaccines focuses on eliminating the immunogenicity and instability of mRNA, developing efficient and targeted mRNA delivery systems, and antigen identification for mRNA vaccines. Currently, biocompatible lipid nanoparticles (LNPs) have been validated as effective carriers for in vivo mRNA delivery, used in various preventative and therapeutic vaccine strategies. LNPs demonstrate superiority over other carriers in mRNA delivery, and future research requires optimizing passive and active strategies to improve the performance and safety of LNPs in mRNA and drug delivery.

[0005] With the application of two COVID-19 vaccines, the prevalence of mRNA vaccines has increased significantly. In the field of cancer treatment, research on mRNA vaccines has also made significant progress. Since 2020, mRNA vaccines have received widespread attention in the field of personalized cancer treatment. Currently, anti-tumor mRNA vaccine strategies are mainly divided into two categories:

[0006] I. mRNA vaccines encoding viral antigens. These vaccines are suitable for virus-related malignancies, such as those caused by human papillomavirus (HPV), hepatitis B virus (HBV), Epstein-Barr virus (EBV), and human immunodeficiency virus (HIV), which are closely associated with the development of malignancies such as cervical cancer, liver cancer, and nasopharyngeal carcinoma. These mRNA vaccines offer a therapeutic strategy for these tumors, different from traditional preventative strategies, by stimulating the body's immune system to recognize and attack cells expressing these viral antigens. For example, a recent study showed that a messenger RNA-HPV therapeutic vaccine (mHTV) exhibited strong immunogenicity and anti-tumor effects in mice and non-human primates, making mHTV a promising therapeutic and preventative vaccine for HPV-related diseases. 4 .

[0007] II. mRNA Vaccines Encoding Tumor Antigens. These vaccines achieve immunotherapeutic effects by encoding tumor-associated antigens (TAAs) expressed higher in tumors than in normal tissues, tumor-specific antigens (TSAs) expressed or significantly overexpressed, and by targeting immune cells in the tumor microenvironment and encoding immunomodulatory factors. In recent years, mRNA vaccines encoding tumor antigens have developed rapidly, with some already entering clinical trials. For example, Autogene cevumeran (BNT122) is a personalized cancer treatment based on mRNA vaccine technology. It utilizes mRNA technology to express endogenous tumor antigens within human cells, thereby activating specific anti-tumor immunity to attack cancer cells. This vaccine, in combination with the anti-PD-L1 antibody atezolizumab and chemotherapy, has shown positive results in a Phase I clinical trial in patients with locally advanced or metastatic solid tumors. 5Furthermore, mRNA-4157 encodes 34 antigens targeting unique mutations in the tumor DNA of individual patients and is currently undergoing clinical trials in melanoma, non-small cell lung cancer, and other solid tumors. Testing showed that in clinical trials of mRNA-4157 in combination with pembrolizumab (an anti-PD-1 drug manufactured by Merck) for patients with head and neck squamous cell carcinoma, the overall response rate was 50% and the disease control rate was 90%. Two phase IIb clinical trials combining mRNA-4157 and pembrolizumab for high-risk melanoma patients reduced the risk of recurrence or death by 44% and 49%, respectively, indicating greater benefits with longer treatment duration. Currently, the combination therapy of mRNA-4157 and pembrolizumab is the first mRNA cancer vaccine to enter a phase III clinical trial (NCT05933577). 6 .

[0008] Despite the immense potential of mRNA vaccines in anti-tumor immunotherapy, their practical application faces numerous challenges. The scarcity of highly effective and specific tumor neoantigens, stemming from the diverse and highly heterogeneous mutations in tumor cells, has become a major bottleneck in their application. 7 This challenge is particularly prominent in the development of prophylactic mRNA vaccines targeting multiple tumor types and covering a broad population. Finding universally applicable neoantigen epitopes to reduce the time and financial costs of personalized tumor neoantigen epitope screening, thereby enabling more timely treatment for patients, is crucial for the further development of neoantigen mRNA vaccine anti-tumor therapy. Furthermore, designing LNP structures to exclude potential inflammatory responses, improve mRNA stability and delivery efficiency, reduce immunogenicity, and ensure long-term safety and efficacy are also urgent issues to be addressed. These challenges highlight the importance of developing more effective mRNA vaccines to overcome existing bottlenecks, improve treatment outcomes, and bring new hope to cancer patients.

[0009] To address these challenges, researchers have begun exploring optimization strategies, including improving vector sequences, antigen combinations, and delivery systems, to enhance the immunogenicity and safety of vaccines. For example, using novel delivery mechanisms and improved synthesis methods can further enhance the expression efficiency and biological stability of self-amplified mRNA.

[0010] In this invention, the mRNA vaccine antigen used is derived from a carefully selected class of de novo novel genes encoding human-specific proteins, namely human-specific de novo genes. It is noteworthy that these genes typically exist as non-coding regions in other species, but have acquired protein-coding capabilities in humans, theoretically closely related to novel human-specific phenotypes and diseases. However, due to their novelty, these genes are characterized by small open reading frames (ORFs), low expression levels, and narrow expression profiles. 8,9 Furthermore, they tend to be expressed in repetitive sequences. 10,11 This increases the difficulty of its accurate identification, resulting in a lack of sufficient and reliable in vivo expression and functional evidence. 12,13 Therefore, these special genes have often been overlooked in previous studies. Furthermore, due to the lack of homologous proteins, accurate identification is difficult, resulting in a lagging understanding of the function and disease impact of these new genes, and a lack of experimental evidence, leading to their long-term neglect in gene and functional research. 14 Nevertheless, some studies have revealed that these new genes can maintain the activity of spermatogonia in the testes. 15,16 Furthermore, it is crucial for maintaining the stability of the neural stem cell pool during brain development. 10,17 These functions suggest their potential role in tumorigenesis and development, and some case studies have already associated these genes with tumor development and prognosis. 18-24 In summary, human-specific de novo genes are closely related to tumorigenesis and development. Due to their unique origin, young gene age, and insufficient expression studies, these genes are often overlooked in proto-oncogene screening using existing technologies, mistakenly treated as non-coding genes, and belong to a group of genes ignored in current proto-oncogene screening techniques. Some proteins translated from these genes have extremely low expression levels or very narrow expression profiles in normal adults, expressed only in a few tissues (brain and testes) or during development. However, in tumors, the sudden high expression of these genes makes them a type of natural tumor-specific antigen. Given the special properties of this type of antigen, the vaccine of this invention exhibits the excellent characteristic of specifically killing tumor cells without affecting normal cells, a conclusion verified in the embodiments of this invention. Furthermore, as a universal anti-tumor antigen, this type of antigen has a far wider application scope than personalized antigens, and is expected to become a key breakthrough point for anti-tumor vaccines. In addition, this invention also optimized the lipid nanoparticle (LNP) delivery process and LNP structure, significantly improving the efficacy and safety of the vaccine. Summary of the Invention

[0011] In a first aspect, the present invention provides a method for constructing a gene set, the method comprising:

[0012] (a) Collect de novo genes specific to the human family and eliminate erroneous or duplicated candidate genes by determining their gene and transcript structure, thereby obtaining a candidate gene set;

[0013] Optionally (b) perform a linear alignment of the entire mammalian genome to exclude candidate genes that have coding capabilities in orthologous regions of the ancestral genome;

[0014] Optionally (c) homology analysis is performed to exclude candidate genes that may have a duplication origin; and

[0015] Optionally (d) a transcriptional profiling analysis was performed to screen for candidate genes with evidence of in vivo protein expression.

[0016] In some embodiments, collecting human-specific de novo genes includes integrating publicly reported human-specific de novo genes. In some embodiments, the human-specific de novo genes refer to human-specific newborn human genes. In some embodiments, the step of determining gene and transcript structure can be performed by sequence similarity search, for example using GENCODE. In some embodiments, step (a) involves manually verifying the coding sequence regions of candidate genes and the structure of transcripts to exclude erroneous or duplicated candidate genes from the collected de novo genes.

[0017] In some embodiments, mammalian genome alignment can be performed by whole-genome alignment of 20 or more, 40 or more, 60 or more, 80 or more, 100 or more, 110 or more, or 120 or more mammals. In some embodiments, the mammals of the present invention are selected from the mammals listed in Table 1. In some embodiments, the genomes used for mammalian genome alignment in the method of the present invention are selected from the genomes listed in Table 1. In some embodiments, mammalian genome alignment may include whole-genome alignment of representative primate species. In some embodiments, step (b) includes assessing the coding potential of each candidate gene in a common ancestor on a phylogenetic tree. In some embodiments, if the start site of the reconstructed ancestral ORF is precisely aligned with the candidate gene or aligned within a hexanucleotide frame downstream of the translation start site, and the assumed ORF length of the ancestral sequence reaches 70% of the candidate gene, then the orthologous region in the ancestral genome is considered to be coding. In some implementations, step (b) further includes excluding cases of start codon deletion, frameshift, or stop codon gain in outgroup species based on the primate whole-genome alignment results, in order to further confirm that the discovery of these candidate genes (or orphan proteins) represents a recent genetic origin event in humans, rather than an ancestral ORF lost in an outgroup.

[0018] In some embodiments, the homology analysis in step (c) is performed through a homology search in humans and representative outgroups to exclude candidate genes with a possible origin of replication. In some embodiments, the representative outgroups are selected from: *Pan troglodytes*, *Pan paniscus*, *Gorilla gorilla*, *Pongo abelii*, *Nomascus leucogenys*, *Macaca mulatta*, *Macaca fascicularis*, *Callithrix jacchus*, *Otolemurgarnettii*, *Oryctolagus cuniculus*, *Mus musculus*, *Canis lupus familiaris*, *Loxodontaafricana*, *Monodelphis domestica*, or combinations thereof. In some embodiments, the homology search requires an E value less than 10. -4 Less than 10⁻ 5 Less than 10⁻ 6 or less than 10 -7The consistency is greater than 30%, 40%, 50%, or 60%, and / or the query coverage is greater than 40%, 50%, or 60%. In some implementations, in step (c), candidate genes with multiple homologs mapped to different genomic locations, transcripts, or proteins are excluded during the homology search.

[0019] In some embodiments, the transcriptomic profiling in step (d) includes studying transcriptome data, ribosome imprinting (Ribo-seq) datasets, and / or mass spectrometry (MS) data, preferably with comprehensive coverage of different human tissues and cell lines. In some embodiments, step (d) utilizes representative regions unique to newly generated gene transcripts for quantification. In some embodiments, defining a representative region involves: identifying and merging exons of all transcripts containing complete coding sequences (CDS); excluding regions that overlap with exons of all cis-antense genes; and removing fragments shorter than 100 base pairs (bp). In some embodiments, step (d) screens for gene sets with at least one, two, or three independent studies providing translational evidence supporting their ability to translate into human-specific proteins.

[0020] In the method of the present invention, steps (b), (c) and / or (d) can be performed in any order.

[0021] In some embodiments, the method of the first aspect further includes:

[0022] (e) Further screening for genes that are expressed at low levels or with a narrow expression profile in normal adult tissues and / or are specifically highly expressed in tumors.

[0023] In some embodiments, step (e) includes screening for genes with a transcriptional level (TPM) ≤0.5 (low expression in all normal tissues) or spatial expression specificity ≥0.75 (narrow expression profile) in at least 20, at least 23, at least 25, or at least 27 human tissues. The human tissues are selected, for example, from the cerebral cortex, frontal cortex, amygdala, hippocampus, hypothalamus, cerebellum, cerebellar hemispheres, esophageal mucosa, left ventricle, thyroid gland, breast, lung, stomach, pancreas, liver, renal cortex, sigmoid colon, transverse colon, bladder, skin, whole blood, ovary, cervical adventitia, cervical endometrium, uterus, prostate, testis, or combinations thereof. In some embodiments, high specific expression in tumors refers to a fold change >1.5 between the quartile of the TPM value in one or more tumors and the corresponding normal sample.

[0024] In some implementations, the genes specifically highly expressed in tumors that were screened are as follows (the cancer types specifically highly expressed by the gene are in parentheses): RNF32-DT (LUAD, LUSC, KIRP, COAD, SKCM, OV, CESC), TIPARP-AS1 (GBM, LUSC, LIHC, KIRC, KIRP, BLCA, OV, PRAD), LINC01547 (LUAD, OV), TYMSOS (HNSC, THCA, BRCA, LUAD, LUSC, STAD, LIHC, KIRP, BLCA, SKCM, OV, CESC), PAPRN-AS1 (TGCT), ODC1-DT (KIRC, KIRP), ADORA2A-AS1 (KIRC), ZNF843 (LGG, KIRP), AATBC (HNSC, COAD, BLCA, UCEC), MOCS 2-DT (LUAD, LUSC, KIRC, KIRP, COAD, SKCM), SPTSSB (BRCA, LUAD, LUSC, PAAD, OV), MYEOV (LUAD, LUSC, STAD, PAAD, KIRC, COAD, BLCA, CESC), TENM3-AS1 (HNSC, KIRP, SKCM) , MYCNOS (LGG), LINC00632 (TGCT), ELFN1-AS1 (ESCA, STAD, PAAD, COAD, BLCA, SKCM, OV, CESC, UCEC, PRAD), LINC02188 (PAAD, KIRC, KIRP), SMIM45 (LUSC, SKCM, OV, CESC).

[0025] In some embodiments, the genes specifically highly expressed in tumors that were screened are as follows (the cancer types specifically highly expressed by the gene are in parentheses): SERP1 (LGG, KIRC, KIRP); YIF1B (HNSC, BLCA, SKCM, OV); MYEOV (LUAD, LUSC, STAD, PAAD, KIRC, COAD, BLCA, CESC); ZNF843 (LGG, KIRP); TYMSOS (HNSC, THCA, BRCA, LUAD, LUSC, STAD, LIHC, KIRP, BLCA, SKCM, OV, CESC); TENM3-AS1 (HNSC, KIRP, SKCM); ADORA2A-AS1 (KIRC); S PEN-AS1 (KIPR); RNF32-DT (LUAD, LUSC, KIRP, COAD, SKCM, OV, CESC); LINC01547 (LUAD, OV); SPTSSB (BRCA, LUAD, LUSC, PAAD, OV); LINC0 0632 (TGCT); SMIM45 (LUSC, SKCM, OV, CESC); AATBC (HNSC, COAD, BLCA, UCEC); EXOC3-AS1 (LUSC, KIRC, KIRP); ENSG00000224376 (HNSC, KI RP); MYCNOS (LGG); ELFN1-AS1 (ESCA, STAD, PAAD, COAD, BLCA, SKCM, OV, CESC, UCEC, PRAD); TIPARP-AS1 (GBM, LUSC, LIHC, KIRC, KIRP, BL CA, OV, PRAD); MOCS2-DT (LUAD, LUSC, KIRC, KIRP, COAD, SKCM); ODC1-DT (KIRC, KIRP); PAPLN-AS1 (TGCT); LINCO2188 (PAAD, KIRC, KIRP).

[0026] In a second aspect, the present invention relates to gene sets constructed by the method of the first aspect.

[0027] In some embodiments, the gene set of the second aspect comprises any one or more genes selected from the following: KCNK10, SERP1, ENSG00000145063, SCP2D1-AS1, YIF1B, MYEOV, RPP38-DT, ZNF843, TYMSOS, TENM3-AS1, ADORA2A-AS1, EIF3J-DT, SPEN-AS1, CHRM2, RNF32-DT, LINC01547, PAX8-AS1, LINC00523, SPTSSB, C20orf203, LINC01565, LINC00632, TDRG1, LINC02397, ENSG00000205414, SMIM4 5. AATBC, EXOC3-AS1, ENSG00000224376, NTM-AS1, MIR137HG, SATB2-AS1, HDAC2-AS2, LINC02370, ENSG00000233290, MYCNOS, ELFN1-AS1, ENSG00000243004, TIPA RP-AS1, UBR5-DT, MOCS2-DT, DNAH10OS, PBOV1, ENSG00000255384, ZNF252P-AS1, CLLU1, ODC1-DT, PAPLN-AS1, LINC02188, GTSCR1, ENSG00000279491, LINC00602.

[0028] In some embodiments, the gene set of the second aspect comprises any one or more genes selected from the following: RNF32-DT, TIPARP-AS1, LINC0547, TYMSOS, PAPLN-AS1, ODC1-DT, ADORA2A-AS1, ZNF843, AATBC, MOCS2-DT, SPTSSB, MYEOV, TENM3-AS1, MYCNOS, LINC00632, ELFN1-AS1, LINC02188, SMIM45.

[0029] In some embodiments, the gene set of the second aspect comprises one or more genes selected from the following: SERP1, YIF1B, MYEOV, ZNF843, TYMSOS, TENM3-AS1, ADORA2A-AS1, SPEN-AS1, RNF32-DT, LINC01547, SPTSSB, LINC00632, SMIM45, AATBC, EXOC3-AS1, ENSG00000224376, MYCNOS, ELFN1-AS1, TIPARP-AS1, MOCS2-DT, ODC1-DT, PAPLN-AS1, and LINC02188.

[0030] Thirdly, the present invention relates to the use of partial or full-length coding sequences of genes selected from the set of the second aspect, and molecules for detecting or regulating the expression of genes selected from the set of the second aspect in the preparation of medicaments for treating tumors or diagnostic reagents for diagnosing tumors.

[0031] In some embodiments, the partial coding sequence encodes an antigenic fragment of the selected gene or a variant thereof. In some embodiments, the antigenic fragment or variant thereof can be used to generate antibodies against tumors.

[0032] In some embodiments, the partial or full-length coding sequence encodes a polypeptide comprising an amino acid sequence shown in any one of SEQ ID NO:1-420 and SEQ ID NO:423-787, or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% identity with an amino acid sequence shown in any one of SEQ ID NO:1-420 and SEQ ID NO:423-787. In some embodiments, the full-length coding sequence comprises the sequence shown in any one of SEQ ID NO:947-965. In some embodiments, the full-length coding sequence has at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% identity with the sequence shown in any one of SEQ ID NO:947-965, and encodes the exact same amino acid sequence encoded by the sequence shown in any one of SEQ ID NO:947-965. In some embodiments, the full-length coding sequence is a codon-optimized sequence of the sequence shown in any one of SEQ ID NO:947-965.

[0033] Fourthly, the present invention relates to a polynucleotide molecule encoding a protein, an antigenic fragment thereof, or a variant of a protein or antigenic fragment expressed by a gene selected from the following: KCNK10, SERP1, ENSG00000145063, SCP2D1-AS1, YIF1B, MYEOV, RPP38-DT, ZNF843, TYMSOS, TENM3-AS1, ADORA2A-AS1, EIF3J-DT, SPEN-AS1, CHRM2, RNF32-DT, LINC01547, PAX8-AS1, LINC00523, SPTSSB, C20orf203, LINC01565, LINC00632, TDRG1, LINC02397, ENSG00 000205414, SMIM45, AATBC, EXOC3-AS1, ENSG00000224376, NTM-AS1, MIR137HG, SATB2-AS1, HDAC2-AS2, LINC02370, ENSG00000233290, MYCNOS, ELFN1-AS1, ENSG00000243 004, TIPARP-AS1, UBR5-DT, MOCS2-DT, DNAH10OS, PBOV1, ENSG00000255384, ZNF252P-AS1, CLLU1, ODC1-DT, PAPLN-AS1, LINC02188, GTSCR1, ENSG00000279491, LINC00602.

[0034] In some embodiments, the polynucleotide molecule encodes a protein, an antigenic fragment thereof, or a variant of a protein or antigenic fragment expressed by a gene selected from the following: RNF32-DT, TIPARP-AS1, LINC0547, TYMSOS, PAPLN-AS1, ODC1-DT, ADORA2A-AS1, ZNF843, AATBC, MOCS2-DT, SPTSSB, MYEOV, TENM3-AS1, MYCNOS, LINC00632, ELFN1-AS1, LINC02188, SMIM45.

[0035] In some embodiments, the polynucleotide molecule encodes a polypeptide comprising an amino acid sequence represented by any one of SEQ ID NO:1-420 and SEQ ID NO:423-787, or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% identity with an amino acid sequence represented by any one of SEQ ID NO:1-420 and SEQ ID NO:423-787. In some embodiments, the polynucleotide molecule encodes a polypeptide comprising an amino acid sequence represented by any one of SEQ ID NO:9, SEQ ID NO:37, SEQ ID NO:130, SEQ ID NO:129, SEQ ID NO:191, and SEQ ID NO:418-420, or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% identity with an amino acid sequence represented by any one of SEQ ID NO:9, SEQ ID NO:37, SEQ ID NO:130, SEQ ID NO:129, SEQ ID NO:191, and SEQ ID NO:418-420.

[0036] In some embodiments, the polynucleotide molecule is an mRNA molecule. In some embodiments, the polynucleotide molecule comprises a 5' cap structure, preferably with a cap1 modification at the 5' end. In some embodiments, at least 50%, at least 70%, at least 90%, at least 95%, or 100% of the uridine (U) in the polynucleotide molecule is replaced with pseudouridine (Ψ).

[0037] In some embodiments, the polynucleotide molecule comprises the sequence shown in SEQ ID NO:788 or SEQ ID NO:789, or a polynucleotide sequence having at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% identity with the sequence shown in SEQ ID NO:788 or SEQ ID NO:789. In some embodiments, the polynucleotide molecule comprises a sequence encoding any tag that facilitates detection, separation, and / or purification, such as a Flag tag, like the sequence shown in SEQ ID NO:790, or a polynucleotide sequence having at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% identity with the sequence shown in SEQ ID NO:790.

[0038] Fifthly, the present invention relates to a liposome complex comprising a polynucleotide molecule as described in the fourth aspect, and lipid nanoparticles encapsulating the polynucleotide molecule in a lipid shell, the lipid nanoparticles comprising protonable cationic lipids, phospholipids, steroids and / or PEG lipids.

[0039] In some embodiments, the liposome complex comprises about 40 mol% to about 60 mol% of protonable cationic lipids, about 5 mol% to about 20 mol% of phospholipids, about 30 mol% to about 50 mol% of cholesterol, and / or about 0.1 mol% to about 5 mol% of PEG lipids. In some embodiments, the liposome complex comprises SM-102, DSPC, cholesterol, and / or DMG-PEG2k, preferably in a ratio of about 50 / about 10 / about 38.5 / about 1.5 of SM-102 / DSPC / cholesterol / DMG-PEG2k in the liposome complex.

[0040] Sixthly, the present invention relates to a targeting molecule for regulating the expression of genes selected from the following: KCNK10, SERP1, ENSG00000145063, SCP2D1-AS1, YIF1B, MYEOV, RPP38-DT, ZNF843, TYMSOS, TENM3-AS1, ADORA2A-AS1, EIF3J-DT, SPEN-AS1, CHRM2, RNF32-DT, LINC01547, PAX8-AS1, LINC00523, SPTSSB, C20orf203, LINC01565, LINC00632, TDRG1, LINC02397, ENSG00000205414, SMIM45, A ATBC, EXOC3-AS1, ENSG00000224376, NTM-AS1, MIR137HG, SATB2-AS1, HDAC2-AS2, LINC02370, ENSG00000233290, MYCNOS, ELFN1-AS1, ENSG00000243004, TIPAR P-AS1, UBR5-DT, MOCS2-DT, DNAH10OS, PBOV1, ENSG00000255384, ZNF252P-AS1, CLLU1, ODC1-DT, PAPLN-AS1, LINC02188, GTSCR1, ENSG00000279491, LINC00602.

[0041] In some embodiments, the targeting molecule regulates the expression of the following genes: SERP1, YIF1B, MYEOV, ZNF843, TYMSOS, TENM3-AS1, ADORA2A-AS1, SPEN-AS1, RNF32-DT, LINC01547, SPTSSB, LINC00632, SMIM45, AATBC, EXOC3-AS1, ENSG00000224376, MYCNOS, ELFN1-AS1, TIPARP-AS1, MOCS2-DT, ODC1-DT, PAPLN-AS1, and LINC02188.

[0042] In some embodiments, the targeting molecule regulates the expression of the following genes: ELFN1-AS1, ODC1-DT, SPTSSB, SMIM45, AATBC, MYEOV, TENM3-AS1, LINC01547, ENSG00000224376, TIPARP-AS1, MOCS2-DT, LINC00632, PAPLN-AS1, EXOC3-AS1, PAX8-AS1, RNF32-DT, and TDRG1. ADORA2A-AS1, CHRM2, ENSG00000145063, C20orf203, YIF1B, DNAH10OS, SATB2-AS1, SERP1, LINC00523, ENSG00000255384, ENSG00000233290, CLLU1, LINC02397, KCNK10, ENSG00000279491, SCP2D1-AS1 or LINC02370.

[0043] In some embodiments, the targeting molecule regulates the expression of the following genes: ELFN1-AS1, ODC1-DT, SPTSSB, SMIM45, AATBC, MYEOV, TENM3-AS1, LINC01547, ENSG00000224376, TIPARP-AS1, MOCS2-DT, LINC00632, PAPLN-AS1, EXOC3-AS1, PAX8-AS1, RNF32-DT, TDRG1, ADORA2A-AS1, CHRM2, ENSG00000145063, C20orf203, YIF1B, DNAH10OS, SATB2-AS1, SERP1, LINC00523, ENSG00000255384, ENSG00000233290, or CLLU1.

[0044] In some embodiments, the targeting molecule regulates the expression of the following genes: ODC1-DT, ELFN1-AS1, AATBC, SPTSSB, SMIM45, TIPARP-AS1, LINC01547, MOCS2-DT, MYEOV, TENM3-AS1, PAPLN-AS1, LINC00632, RNF32-DT, or ADORA2A-AS1.

[0045] In some implementations, the targeting molecule reduces the expression of the target gene by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%.

[0046] In some embodiments, the targeting molecule is a guide RNA, preferably sgRNA. In some embodiments, the targeting molecule is siRNA. In some embodiments, the targeting molecule comprises a targeting region shown in any one of SEQ ID NO:791-946 or SEQ ID NO:966-1031, or a targeting region having at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% identity with the sequence shown in any one of SEQ ID NO:791-946 or SEQ ID NO:966-1031.

[0047] In some embodiments, the antisense strand of the siRNA includes a target region shown in any one of SEQ ID NO:966-1031 or a target region having at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% identity with the sequence shown in any one of SEQ ID NO:966-1031. In some embodiments, the antisense strand of the siRNA also includes a 3' overhang (e.g., "TT").

[0048] In some embodiments, the antisense strand of the siRNA comprises a complementary sequence to a consecutive segment of at least 15 nt, 16 nt, 17 nt, 18 nt, or 19 nt from any of the sequences shown in SEQ ID NO: 947-965. In some embodiments, the complementary sequence includes not only perfectly complementary sequences but also substantially complementary sequences, meaning sequences that can hybridize with the consecutive segments under stringent conditions known in the art. In some embodiments, the antisense strand of the siRNA does not hybridize with non-target regions under stringent conditions. In some embodiments, the antisense strand of the siRNA has a low off-target risk.

[0049] In another aspect, the present invention relates to a method for diagnosing, preventing or treating cancer, the method comprising administering to a subject in need a polynucleotide molecule of the fourth aspect, a liposome complex of the fifth aspect, or a targeting molecule of the sixth aspect.

[0050] In another aspect, the present invention relates to the use of a fourth aspect of a polynucleotide molecule in the preparation of a vaccine for the treatment and / or prevention of tumors.

[0051] In some embodiments, the tumor of the present invention includes, but is not limited to:

[0052] Respiratory system tumors: lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), bronchogenic carcinoma, small cell lung cancer, non-small cell lung cancer;

[0053] Digestive system tumors: gastric cancer (e.g., gastric adenocarcinoma (STAD)), pancreatic cancer (e.g., pancreatic adenocarcinoma (PAAD)), colon cancer (e.g., colonic adenocarcinoma (COAD)), rectal cancer, bile duct cancer, gallbladder cancer, esophageal cancer (ESCA, e.g., esophageal adenocarcinoma), hepatocellular carcinoma (LIHC), appendix cancer;

[0054] Urogenital tumors: bladder cancer (e.g., bladder urothelial carcinoma (BLCA)), clear cell renal cell carcinoma (KIRC), papillary renal cell carcinoma (KIRP), prostate cancer (PRAD), testicular germ cell tumor (TGCT), ovarian cancer, endometrial cancer (UCEC), and cervical squamous cell carcinoma (CESC).

[0055] Head and neck tumors: squamous cell carcinoma of the head and neck (HNSC), thyroid carcinoma (THCA), nasopharyngeal carcinoma, laryngeal carcinoma;

[0056] Nervous system tumors: low-grade glioma (LGG), glioblastoma multiforme (GBM), medulloblastoma, glioma (such as astrocytoma, oligodendroglioma);

[0057] Skin and soft tissue tumors: skin melanoma (SKCM), basal cell carcinoma, squamous cell carcinoma, soft tissue sarcoma (such as liposarcoma, chondrosarcoma), angiosarcoma (such as Kaposi's sarcoma), osteosarcoma;

[0058] Hematologic malignancies: leukemia (such as acute myeloid leukemia), lymphoma (such as diffuse large B-cell lymphoma), multiple myeloma;

[0059] Breast and gynecological tumors: breast cancer (such as adenocarcinoma of the breast, invasive breast cancer (BRCA)), ovarian cancer (such as ovarian serous cystadenocarcinoma (OV)), vulvar cancer.

[0060] In some embodiments, exemplary tumors of the present invention include, but are not limited to: colon cancer, lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), gastric adenocarcinoma (STAD), pancreatic adenocarcinoma (PAAD), clear cell renal carcinoma (KIRC), colonic adenocarcinoma (COAD), bladder urothelial carcinoma (BLCA), cervical squamous cell carcinoma (CESC), low-grade glioma (LGG), renal papillary cell carcinoma (KIRP), head and neck squamous cell carcinoma (HNSC), thyroid cancer (THCA), invasive breast cancer (BRCA), hepatocellular carcinoma (LIHC), cutaneous melanoma (SKCM), ovarian serous cystadenocarcinoma (OV), endometrial cancer (UCEC), esophageal cancer (ESCA), prostate cancer (PRAD), glioblastoma multiforme (GBM), and testicular germ cell tumor (TGCT).

[0061] In summary, this invention targets a group of genes that are overlooked in existing proto-oncogene screening techniques. These humanoid-specific de novo genes, which maintain cell stemness, are naturally occurring proto-oncogenes. The antigen set screened by the method of this invention has broad spectrum and is an ideal antigen candidate for developing universally applicable vaccines.

[0062] This invention pioneers a method based on a human-specific de novo gene set. Through this invention and other studies, it has been demonstrated that this set contains many naturally occurring proto-oncogenes, which are often overlooked in general gene screening due to their neogenic nature. Previous research has not explored the application of these genes as anti-tumor mRNA vaccines. This invention, through meticulous screening, first constructs a human-specific de novo gene set containing 52 newly generated human genes. Based on this, further screening of expression levels yields a set of 18 proto-oncogene antigens with high population universality and specificity, opening a new pathway for tumor vaccine development. Analysis of gene expression levels in numerous patient cases from TCGA demonstrates that these human-specific de novo genes are widely expressed in most cancer patients, indicating that these genes, as neoantigens, also possess broad-spectrum characteristics. In summary, this screening method not only overcomes the limitations of traditional tumor vaccines in antigen selection but also ensures that the vaccine antigen specifically targets tumor cells without harming normal tissue cells, thus achieving significant improvements in safety, efficacy, and broad-spectrum efficacy.

[0063] Furthermore, this invention has successfully developed a specific mRNA vaccine delivery system (LNP) targeting these novel proto-oncogenes. This technological innovation not only optimizes vaccine delivery efficiency but also ensures stable expression of the vaccine antigen in vivo and effective induction of an immune response. More importantly, this invention selected ELFN1-AS1 and TYMSOS antigens as examples. First, the effectiveness of the mRNA delivery system was verified through physicochemical properties. Then, experiments were conducted in humanized mouse tumor models, showing both single-agent vaccination and combined vaccination with PD1-laIL2 immunotherapy. Vaccination with the mRNA vaccine significantly reduced tumor size in the mouse models, and combined vaccination with PD1-laIL2 immunotherapy resulted in a more significant therapeutic effect. This further confirms the effectiveness of the mRNA vaccine delivery system. The experimental results not only demonstrate that the vaccine can elicit a strong specific immune response but also directly prove its ability to inhibit tumor growth in live animal models, laying a solid foundation for subsequent clinical trials. The results of these two examples are also expected to be extended to a population-wide set of 18 genes, validating the correctness and future potential of the initial selection of 52 human-specific de novo genes.

[0064] It should be noted that the methods and technologies proposed in this invention have broad applicability, not limited to specific tumor types or populations, and provide important technical support and theoretical basis for the future development of precision tumor vaccines based on individual genomic information. With the continuous advancement of gene sequencing technology and in-depth research in tumor immunology, the antigen screening method and mRNA vaccine delivery system constructed in this invention are expected to play a more important role in future tumor immunotherapy, bringing safer and more effective treatment options to cancer patients.

[0065] In summary, this invention has not only achieved a significant breakthrough in scientific theory, but also demonstrated great application potential in technical practice, making important contributions to the development of tumor vaccines and the field of tumor immunotherapy. Attached Figure Description

[0066] Figure 1 This demonstrates the process for identifying newly emerging protein-coding genes in the human family.

[0067] Figure 2This section shows the expression profiles and translational evidence of human-specific de novo genes at different tissue developmental stages. A. Expression profiles of 27 human-specific and 25 human-specific de novo genes at different developmental stages in various tissues. The heatmap above shows the relative expression levels of 52 genes in various tissues and three developmental stages. The dotted heatmap below shows the amount of ORF translational evidence detected from all data (All evidence), including re-analyzed and annotated Ribo-seq data, in-house or public mass spectrometry data (In-house, PeptideAtlas, and MassIVE), and Western Blot evidence (WB). B. Number of P-site reads near start and stop codons in de novo gene ORFs, showing a clear trinucleotide periodicity. C. Heatmap of the proportion of P-site reads near start and stop codons in typical protein-coding gene ORFs from different datasets.

[0068] Figure 3 This section illustrates the tissue and spatiotemporal expression specificity of human de novo genes. A. Correlation between tissue and spatiotemporal specificity of human de novo genes. B. Comparison of tissue and spatiotemporal expression specificity of human de novo genes, housekeeping genes, and randomly selected genes from the whole genome.

[0069] Figure 4 The spatiotemporal tissue expression profiles of human de novo genes in six organ types are shown. A. Relative spatial expression profiles of human de novo genes in 27 tissues; brain and testis tissues are highlighted with a gray background. Gray bars represent the expression differences between denovo and housekeeping genes. B. Time specificity of de novo genes in six organs; different colored dots represent the median spatial specificity for different gene types (blue, de novo genes; gray, genome-wide background genes; red, housekeeping genes). C. Spatial expression profiles of de novo genes in six organs, identified as major expression trends in categories 0-6. c1-c2 are late-expressed genes, and c4-c6 are early-expressed genes. D. Number of time-dynamically expressed genes (TDGs) classified into categories 0-6 in the six organs.

[0070] Figure 5 This diagram illustrates the spatiotemporal expression specificity of human de novo genes and their expression expansion in tumor tissues. The heatmap shows the expression of de novo genes in normal tissues and the significant fold change in their upper quartile expression values ​​between tumor and normal tissues. The left-hand bar chart shows the number of genes expressed in normal tissues and the number of significantly upregulated genes in tumors.

[0071] Figure 6 This figure illustrates the expression of novel, specific proto-oncogenes in multiple tissues during the development of a population-wide tumor vaccine. The broken line represents the expression level of the de novo gene in normal tissues, while the scatter plots represent the expression levels of the de novo gene in different tumor types calculated using TCGA data. Different tissue origins are distinguished by different colors.

[0072] Figure 7 The inhibitory effect of siRNA on tumor cell proliferation was demonstrated.

[0073] Figure 8 This demonstrates how CRISPR-Cas9 screening proves that de novo genes affect the proliferation of different cancer cell lines. A. CRISPR-Cas9 screening library design strategy. B. A heatmap of CRISPR-Cas9 screening results shows 4 tumor-related genes (top section) and 14 feasible de novo genes with / without expression amplification (middle and bottom sections, respectively; expression amplification is determined by...). Figure 5 (Definition) Log-2 fold change in sequencing read counts from day P0 to day P10 in tumors; statistical significance of the log2 Fold Change for each sgRNA count is indicated by an asterisk. *|L2FC(counts)|>1; **|L2FC(counts)|>1.5. C. Proportion of de novo genes significantly associated with poor prognosis in various tumor types. D. Kaplan-Meier survival analysis of ELFN1-AS1 expression in patients with colorectal adenocarcinoma (COAD) (Cox proportional hazards model for prognostic significance, P < 0.01).

[0074] Figure 9 This diagram illustrates a strategy for preparing mRNA vaccines using the human-specific de novo genes ELFN1-AS1 and TYMSOS as tumor neoantigens. A schematic diagram of mRNA-LNP vaccines targeting the ELFN1-AS1 and TYMSOS genes is shown. LNPs consist of SM-102 / DSPC / cholesterol / DMG-PEG2K (50 / 10 / 38.5 / 1.5) to deliver either ELFN1-AS1-3×Flag mRNA or TYMSOS-3×Flag mRNA. The mRNA sequences include 5' and 3' untranslated regions (UTRs), the coding sequences for ELFN1-AS1 and TYMSOS, a 3×Flag tag, and a poly-A tail. The lengths of the two mRNAs are labeled in the diagram. The 5' end of the mRNA is modified to Cap1, and all uridine (U) is replaced with pseudouridine (Ψ).

[0075] Figure 10Physicochemical properties of LNP-mRNA and in vitro translational evidence are presented. A. Physicochemical properties of mRNA-LNPs, including particle size, polydispersity index (PD index), encapsulation efficiency (EE), and zeta potential (mV). Data are presented as mean ± standard error (3 biologically independent samples). B. 293T cells were transfected with 0.5 μg / ml mRNA-ELFN1-AS1 and mRNA-TYMSOS, and then collected for Western blotting analysis.

[0076] Figure 11 This diagram illustrates how vaccination with an LNP-mRNA vaccine based on the de novo gene induces the maturation and activation of dendritic cells in vivo. A. Schematic diagram of the vaccination protocol for humanized mice and the sampling of spleen and peripheral blood. Each treatment had three independent biological replicates. B. Flow cytometry analysis of CD11c in the spleen of mice vaccinated with LNP-empty, LNP-ELFN1-AS1, and LNP-TYMSOS vaccines. + CD80 in cells + CD86 + and CD83 + C. Quantitative comparison of flow cytometry analysis. (3 biologically independent samples; one-sided t-test, *P<0.05, ***P < 0.001, ns represents statistically insignificant).

[0077] Figure 12 This study demonstrates that vaccination with an LNP-mRNA vaccine based on the de novo gene can induce specific T cell activation. A. Flow cytometry analysis was used to detect CD137 in the spleen of mice vaccinated with LNP-empty, LNP-ELFN1-AS1, and LNP-TYMSOS vaccines. + CD8 + Histogram of T cell proportions. Blue represents the LNP-control group, yellow represents the LNP-ELFN1-AS1 group, and beige represents the LNP-TYMSOS group. B. Quantitative comparison of flow cytometry analysis (3 biologically independent samples; one-sided t-test, **P<0.01, ****P < 0.0001, ns indicates statistically insignificant).

[0078] Figure 13This study demonstrates the predicted specific affinity of epitope peptides for MHC-I as detected by T2 cell epitope peptide binding assays. A. Flow cytometry analysis was used to detect the expression level of HLA-A2 molecules on the cell surface of T2 cells after stimulation with epitope peptides designed for ELFN1-AS1 and TYMSOS. Epitope peptide CDC73-Q254E-1-NIFAILESV (SEQ ID NO:421) served as a positive control, KRAS(A11)-G12C-VGACGVGK (SEQ ID NO:422) served as a negative control, and DMSO stimulation of unstimulated T2 cells served as a background control. B. Fluorescence index (FI) of HLA-A2 molecule expression levels on the surface of T2 cells stimulated by epitope peptides in each group. FI = (mean fluorescence of PE-HLA-A2 in experimental group T2 – mean fluorescence of PE-HLA-A2 in negative control group T2) / (mean fluorescence of PE-HLA-A2 in negative control group T2), FI > 1.0 is the standard for high affinity peptides.

[0079] Figure 14 This diagram illustrates the IFN-γ ELISpot assay used to validate the specific immune response of peripheral blood T cells after LNP-mRNA vaccination. A. Schematic diagram of the ELISpot assay procedure. PBMCs from mice vaccinated with LNP-mRNA were isolated and co-incubated with the corresponding predicted epitope peptides for 24 hours. The ELISpot assay detected a positive IFN-γ response. B. ELISpot results: Red shading represents the results of LNP-ELFN1-AS1 vaccine vaccination with the corresponding epitope peptide, and blue shading represents the results of LNP-TYMSOS vaccine vaccination with the corresponding epitope peptide; NC is the negative control (DMSO treatment without epitope peptide), and PC is the positive control (anti-CD3 treatment). C. ELISpot count results after standardization. The results of each group were compared with the corresponding group of mice vaccinated with LNP-control. Each point represents an independent mouse biological replicate (one-sided t-test, *P<0.05).

[0080] Figure 15 This paper demonstrates that vaccination with an LNP-mRNA vaccine based on the de novo gene can inhibit early tumor development. A. Schematic diagram of humanized mouse vaccination and subcutaneous tumor inoculation regimens. B. Subcutaneous tumor growth curves after vaccine prophylaxis (6 biologically independent samples, one-sided t-test, *P<0.05).

[0081] Figure 16This paper demonstrates that vaccination with a de novo gene-based LNP-mRNA vaccine combined with PD1-laIL2 immunotherapy can effectively inhibit tumor development. A. Schematic diagram of humanized mouse vaccination, anti-PD1-laIL2 immunotherapy treatment, and subcutaneous tumor inoculation regimen. B. Subcutaneous tumor growth curves after vaccine administration (6 or 7 biologically independent samples, one-sided t-test, *P<0.05, **P<0.01). Detailed Implementation

[0082] definition

[0083] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0084] As used herein, the terms “about” or “approximately” mean within an acceptable margin of error for a particular value as determined by one of ordinary skill in the art, depending in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can refer to any value within 20%, preferably within 10%, more preferably within 5%, and even more preferably within 1%, of a given value. Alternatively, particularly for biological systems or processes, “about” can refer to any value within an order of magnitude of a given value, preferably within 5 times, and more preferably within 2 times.

[0085] As used herein, the terms “comprising,” “including,” or “having” mean that any of the listed elements must be included, and other elements may optionally be included. The term “consisting of” means that all unlisted elements are excluded. The terms “comprising,” “including,” or “having” cover “consisting of” or “substantially consisting of”.

[0086] As used herein, the term "identity" refers to the relationship between two or more polypeptide or nucleotide sequences determined by sequence comparison. Sequence identity is determined by comparing the sequences after a given sequence has been optimally aligned with a reference sequence to produce the highest degree of sequence similarity, such as by matching strings of these sequences. Sequence identity can be readily calculated using known methods, such as BLAST. Those skilled in the art can use computer programs well-known in the art (e.g., DNASTAR™ software) to determine which amino acid residues can be replaced, inserted, or deleted without eliminating biological activity. For example, functional variants of the invention may include conserved substitutions. Conserved substitutions replace amino acids with other amino acids having similar chemical structures, similar chemical properties, or similar side chain volumes. The introduced amino acids may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge as the amino acids they replace. Exemplary conserved substitutions are well known in the art (see, for example, WO 97 / 09433, page 10, published March 13, 1997; Lehninger, Biochemistry, second edition; Worth Publishers, Inc. NY:NY (1975), pp. 71–77; Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA (1990), page 8). It is well known that conserved substitutions between amino acids of similar properties generally do not affect the activity of the peptide sequence.

[0087] As used herein, the term "de novo gene" refers to a newly emerging gene derived from a non-genetic sequence of an ancestor. As used herein, the term "Hominidae" is a family of primates in biological taxonomy, including Homo sapiens and all extinct humans and all chimpanzees. In some cases, the "Hominidae de novo gene" or "Hominidae-specific de novo gene" of this invention refers to a newly emerging protein-coding gene within the Hominidae family.

[0088] As used in this article, "MHC" refers to the major histocompatibility complex. The human MHC is called HLA (human leukocyte antigen). Endogenous synthetic antigens in the cytoplasm of all cells are presented to CD8+ T cells in the form of peptides that bind to MHC I molecules.

[0089] As used in this article, the term “cap1 modification” refers to mRNA having a 7-methylguanosine modification at the 5' end, and simultaneously being methylated at the 2'-O position of the first nucleotide.

[0090] As used in this article, pseudouridine (ψ) is an isomer of uridine (U). Unlike uridine, where uracil is linked to ribose via a carbon-nitrogen bond (C1-N1), in pseudouridine, uracil and ribose are linked via a carbon-carbon bond (C1-C5).

[0091] As used herein, “guide RNA (gRNA)” is an RNA containing a genome-specific CRISPR-RNA (called 'crRNA'). “Single guide RNA (sgRNA)” refers to a chimeric RNA containing crRNA and a trans-activating crRNA (called 'tracrRNA') attached to its 3' end. sgRNA typically contains a specific targeting region and a universal Cas nuclease recruitment sequence. tracrRNA sequences recruiting various Cas nucleases are well known in the art and are available from a variety of commercially available sgRNA expression plasmids.

[0092] As used herein, siRNA (Small interfering RNA) is a single-stranded double-stranded RNA molecule of 19-25 nucleotides in length that specifically inhibits the expression of target genes through RNA interference (RNAi) mechanisms. siRNA guides mRNA degradation or translational repression through complementary pairing of its antisense strand with the target gene mRNA. The siRNA of this invention may contain a 3' dangling at the 3' end of its sense and / or antisense strands, for example, 2-4 unpaired deoxynucleotides such as "TT".

[0093] As used herein, the term "antigenic fragment" refers to a protein fragment capable of evoking an immune response. The term "variant" refers to a polypeptide that has one or more amino acid modifications compared to a protein or protein fragment derived from the wild type and still retains its biological function. In some embodiments, the "antigenic fragment" or "variant of the antigenic fragment" of the present invention has 5 to 15 amino acids, preferably 8 to 11 amino acids, more preferably 9 or 10 amino acids.

[0094] lipid nanoparticles

[0095] As used herein, "lipid nanoparticles" or "LNP" refers to particles having a nanoscale size (e.g., 1-1,000 nm) comprising one or more lipids. In some embodiments, the lipid nanoparticles of the present invention comprise protonable cationic lipids, phospholipids, steroids, and / or PEG lipids.

[0096] As used herein, "protonable cationic lipid" refers to a lipid that is protonated and becomes positively charged under specific conditions, such as an acidic environment. Protonable cationic lipids can be included in the lipid nanoparticles in varying amounts. For example, based on total lipids, the lipid nanoparticles may comprise about 10 mol% to about 100 mol%, such as about 20 mol% to about 70 mol%, about 40 mol% to about 60 mol%, such as about 50 mol% of protonable cationic lipids. The protonable cationic lipids of the present invention can be any cationic lipid suitable for use in lipid nanoparticles. In some embodiments, the protonable cationic lipids of the present invention comprise (heptadecane-9-yl-8-((2-hydroxyethyl)(6-keto-6-(undecapoxy)hexyl)amino)octanoate) (SM-102), (6Z,9Z,28Z,31Z)-heptadecane-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA), (4-hydroxybutyl)azanidinediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), or combinations thereof.

[0097] In this invention, examples of "phospholipids" include, but are not limited to, 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine (DSPE), 1,2-dimyristoyl-sn-glycerol-phosphate choline (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-diundecanoyl-sn-glycerol-phosphate choline (DUPC), and any mixture thereof. In some embodiments, the molar fraction of phospholipids, based on total lipids, is from about 0 mol% to about 40 mol%, for example, about 1 mol%, about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 15 mol%, about 20 mol%, about 25 mol%, about 30 mol%, about 35 mol%, or about 40 mol%.

[0098] In this invention, examples of "steroids" include, but are not limited to, cholesterol and its derivatives. In some embodiments, the molar fraction of steroids, based on total lipids, is from about 0 mol% to about 70 mol%, for example, about 5 mol%, about 10 mol%, about 15 mol%, about 20 mol%, about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, or about 50 mol%, for example, about 38.5 mol%.

[0099] As used herein, the term "PEG lipid" refers to any PEG-modified lipid, examples of which include, but are not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, etc. In some embodiments, the PEG lipid is 1,2-dimyristoyl-sn-glycerol-methoxy(polyethylene glycol) MW 2000 (DMG-PEG2000). In some embodiments, the PEG modification has a molecular weight of about 100 to about 15,000. In some embodiments, the molecular weight of the PEG-modified material is from about 100, 200, 400, 500, 600, 800, 1,000, 1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,500 to about 15,000. The PEG lipids may be included in the lipid nanoparticles in different amounts. For example, based on total lipids, the lipid nanoparticles may include about 0 mol% to about 10 mol%, such as about 0.01 mol% to about 10 mol%, about 0.1 mol% to about 5 mol%, about 1 mol% to about 2 mol%, such as about 1.5 mol% of PEG lipids.

[0100] In this invention, the mass ratio of total lipids to nucleic acids in the liposome complex is 1-100:1, preferably 10-80:1, more preferably 10-60:1, for example 10:1, 20:1, 30:1, 40:1, 50:1, 60:1.

[0101] Example

[0102] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. However, those skilled in the art will understand that the embodiments described below are some embodiments of the present invention, but not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention.

[0103] Example 1: Method for constructing a novel set of specific proto-oncogene antigens for the development of a universally applicable tumor vaccine for a population

[0104] First, in order to discover tumor neoantigens applicable to a broad population, the inventors focused on human-specific de novo genes. These genes underwent a rapid evolutionary process from their origin to the formation of their functions, quickly transforming into crucial factors playing key roles in life regulation. The regulatory role of these de novo genes in maintaining progenitor cell stemness... 17,25 Furthermore, de novo genes have been reported to be specifically expressed in immune-immune-exempt tissues (brain and testes) and during development, and they play a role in maintaining the stability of the neural stem cell pool during brain development. 10,17 At the same time, it maintains the activity of spermatocytes in the testes. 26,27 This suggests that these newly generated de novo genes can maintain the stem cell state and are naturally occurring proto-oncogenes. Some case studies have already associated these genes with tumor development and prognosis. 18-24 Due to their inherent instability, these newly formed genes may play the role of proto-oncogenes in the occurrence and development of tumors, thereby inducing cancer. However, due to their nascent nature, these genes are characterized by small open reading frames (ORFs), low expression levels, and narrow expression profiles. 8,9 Furthermore, they tend to appear in repetitive sequences. 10,11 This increases the difficulty of its accurate identification, resulting in a lack of sufficient and reliable in vivo expression and functional evidence. 12,13 Therefore, these special genes have often been overlooked in previous studies. Furthermore, due to the lack of homologous proteins, accurate identification is difficult, resulting in a lagging understanding of the function and disease impact of these new genes. Lacking experimental evidence, they have long been classified as non-coding RNAs in gene and functional research, thus remaining largely ignored. 14 .

[0105] This invention integrates expression level information from 5,271 clinical patients in the TCGA database, validating the broad-spectrum expression of these genes in cancer patients. Compared to personalized antigens, broad-spectrum universal antigens significantly improve time efficiency and preparation costs, and have a wider range of applications. Therefore, human-specific de novo genes hold promise as an ideal antigen candidate for developing population-wide anti-tumor vaccines, despite previous omissions.

[0106] In short, this invention first systematically screened newly emerging human protein-coding genes. After obtaining a high-confidence set of human-specific protein-coding genes, the inventors further conducted in-depth screening based on gene expression levels and expression stages, aiming to identify genes expressed in specific tissues and developmental stages, but for which no corresponding antibodies exist in normal adults. These genes were ultimately identified as a set of novel specific proto-oncogene antigens for the development of universal tumor vaccines.

[0107] Specifically, in order to identify the newly emerging de novo gene set in the human family, this invention delves into their origin, evolutionary path, and gene expression patterns, and conducts a comprehensive analysis within the context of the latest genome. Figure 1 First, this invention integrates all reported de novo genes from thirteen publicly published papers. 9,10,28-38 These original studies typically used genome-wide alignments and protein annotations from a few species to identify de novo genes, and due to the limited and incomplete data available at the time, these gene sets likely contained numerous errors. Building upon this initial set, the inventors used GENCODE (Version 43) for sequence similarity searches, manually verifying the coding sequence regions and transcript structures of these genes. After removing duplicate genes and manually verifying genomic locations, the inventors constructed a list of 100 candidate de novo genes.

[0108] In further evaluation, the inventors utilized whole-genome alignment information from 120 mammals (Table 1) to thoroughly assess whether these ORFs originated from non-coding regions, supplemented by whole-genome alignment information from representative primate species to determine the precise emergence time of the ORFs. At this evaluation stage, more stringent criteria were introduced to ensure that the ancestral corresponding regions had absolutely no coding potential, and proteins encoded entirely by these regions were excluded. To this end, the inventors used PHAST (v1.4) to extract high-quality orthologous sequences, retained homologous sequences from other mammals requiring alignment coverage exceeding 95% with human homologous sequences, and reconstructed the ancestral sequences using PRANK (v170427). For each candidate gene, the inventors evaluated its coding potential across all common ancestors on a phylogenetic tree. Specifically, if the reconstructed ancestral ORF start site precisely aligned with the candidate gene or aligned within a hexanucleotide frame downstream of the translation start site, and the assumed ORF length of the ancestral sequence reached 70% of the candidate gene, then the orthologous region in that ancestral genome was considered to have coding capacity. Only those candidate genes (84 in total) that were completely lacking in coding potential in all the more ancient ancestors before the first appearance of coding potential were preserved.

[0109] Table 1: Names of 120 mammal species and their genomes

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[0113] Based on the aforementioned whole-genome alignment information, the inventors further determined the evolutionary age of the ORFs encoded by these candidate genes. Specifically, if the assumed ORF length encoded by an outgroup species exceeds 70% of that of the candidate species and has significant sequence similarity to the ORF encoded by the candidate species (Hominidae), then the ORF is considered to exist in that outgroup species. The age of these candidate genes was then determined by assessing the presence or absence of complete ORFs in outgroup species along the phylogenetic tree, retaining only candidate genes for young ORFs newly formed in the ape (Hominidae) lineage. Using the latest primate whole-genome alignment results, the inventors carefully examined the deletion of start codons, frameshifts, or gain of stop codons in outgroup species to further confirm that the discovery of these candidate genes (or orphan proteins) represents a recent genetic origin event in humans, rather than ancestral ORFs lost in outgroups, resulting in a further screening of 78 candidate genes.

[0114] Finally, to rule out the possibility that these genes originated from replication, the inventors used BLASTn to perform a homology search on the human genome (hg38) and annotated transcripts (GENCODE Version 43), requiring an E value of less than 10⁻. 6 Consistency greater than 50% and query coverage greater than 50%. Furthermore, BLASTp was used to perform homology searches on the annotated proteomes (Ensembl) of 14 representative outgroups (Pan troglodytes, Pan paniscus, Gorilla gorilla, Pongo abelii, Nomascus leucogenys, Macaca mulatta, Macaca fascicularis, Callithrix jacchus, Otolemur garnettii, Oryctolaguscuniculus, Mus musculus, Canis lupus familiaris, Loxodonta africana, and Monodlphis domesticica), requiring an E value less than 10⁻. 5The consistency was greater than 40%, and the query coverage was greater than 50%. Candidate genes with multiple homologs that map to different genomic locations, transcripts, or proteins were excluded, resulting in 72 candidate genes of de novo origin.

[0115] To further investigate the expression of these newly emerging candidate de novo genes in human tissues, this invention collected and analyzed 1300 sets of data from the Genotype-Tissue Expression (GTEx V8) project and 300 sets from Cardoso-Moreira et al. 39 The study's transcriptome data also integrated 262 published ribosomal imprinting datasets (Ribo-seq) and 100 million mass spectrometry datasets. This data comprehensively covers different human tissues and cell lines (…). Figure 1 and 2 A).

[0116] To reduce the risk of false positives from naturally occurring antisense transcripts, the inventors used representative regions unique to newly emerging gene transcripts for quantification when analyzing transcriptome data for each de novo gene, ensuring the accuracy and reliability of transcriptional quantification. The method for defining representative regions involves the following steps: First, identifying and merging exons of all transcripts containing complete coding sequences (CDS). Next, excluding regions that overlap with exons of all cis-antisense genes to avoid inaccurate quantification of expression levels. Furthermore, fragments shorter than 100 base pairs (bp) are removed, as these short fragments may not provide accurate expression data. Finally, these screened and merged regions are used as the basis for gene expression level analysis. Based on the aforementioned extensive transcriptome, translatome, and proteome data, this invention ultimately identified 52 newly emerging de novo genes in humans. The novo gene, and at least three independent studies provide translational evidence supporting its ability to translate into human-specific proteins: KCNK10, SERP1, ENSG00000145063, SCP2D1-AS1, YIF1B, MYEOV, RPP38-DT, ZNF843, TYMSOS, TENM3-AS1, ADORA2A-AS1, EIF3J-DT, SPEN-AS1, CHRM2, RNF32-DT, LINC01547, PAX8-AS1, LINC00523, SPTSSB, C20orf203, LINC01565, LINC00632, TDRG1, LINC02397, ENSG00000205414, SM IM45, AATBC, EXOC3-AS1, ENSG00000224376, NTM-AS1, MIR137HG, SATB2-AS1, HDAC2-AS2, LINC02370, ENSG00000233290, MYCNOS, ELFN1-AS1, ENSG00000243004, TI PARP-AS1, UBR5-DT, MOCS2-DT, DNAH10OS, PBOV1, ENSG00000255384, ZNF252P-AS1, CLLU1, ODC1-DT, PAPLN-AS1, LINC02188, GTSCR1, ENSG00000279491, LINC00602. These genes include 25 de novo genes specific to humans (Homo sapiens) and 27 de novo genes specific to the Hominidae family. Figure 2 A). Furthermore, ribosomal footprint analysis within these gene ORFs revealed a clear trinucleotide periodicity (A). Figure 2B) further demonstrates that these de novo genes can be translated in vivo and possess the characteristics of protein-coding genes. Figure 2 C). Most of these 52 human-specific de novo genes are defined as non-coding genes in the latest authoritative international database Ensembl (Table 2). Some patent literature has also only focused on the use of these genes as non-protein-coding genes, such as CN115029442A, CN118685514A, CN114344322A, and CN113293210A. Existing technologies have limited research on these genes, or treat them only as non-protein-coding genes, demonstrating the inadequacy of previous research. When using existing technologies for proto-oncogene screening, these potential protein-coding genes are easily overlooked, thus proving the special nature and advantages of the de novo gene set identified in this invention. Their basic information and encoded protein sequences are shown in Table 3.

[0117] Table 2: Definitions of 52 human-specific de novo genes in the latest authoritative international database Ensembl. Data source: Ensembl database version 113. Gene type abbreviations: protein_coding, lncRNA (long non-coding RNA), TEC (To be experimentally confirmed).

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[0119]

[0120] Table 3: Basic information on 52 de novo genes and their encoded protein sequences.

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[0127] Since gene expression profiles can typically map to their corresponding biological functions, this invention identified the transcriptomic characteristics of these de novo genes in 27 human tissues and multiple developmental stages, and quantified the temporal and spatial specificity of each gene. Based on the median transcriptional level (TPM value) of de novo genes in different tissues or developmental stages, the Tau index was used to calculate their temporal specificity index in each organ and their spatial specificity index in the 27 human tissues. The Tau equation is a statistical formula for assessing the temporal specificity of gene expression, as follows:

[0128]

[0129] For the Tau value calculation of each de novo gene, TPM i TPM represents the median TPM value in each organ or tissue. max This represents the maximum TPM value in each organ or tissue. A pseudo-value of 0.01 is added to avoid zero-value calculations. N represents the number of organ or tissue types. When analyzing time-specific expression, the TPM value at each time point is used and the same equation is applied for calculation.

[0130] The results showed that there were significant temporal and spatial correlations among different de novo genes (Pearson correlation coefficient R = 0.702, P < 2.2 × 10⁻⁶). 16 () Figure 3 A), and compared with housekeeping genes and randomly selected genes from the whole genome, de novo genes showed significantly higher spatiotemporal and tissue specificity. Figure 3 B), which is consistent with previous literature results. 9,40,41 This finding further confirms the uniqueness and complexity of the de novo gene's expression patterns. De novo genes exhibit specific expression preferences in different tissue types. Specifically, de novo genes are more likely to be expressed in testicular and brain tissues, and their expression duration in these tissues is narrower, meaning their expression in these tissues is more concentrated and specific. Figure 4 (A and B). This discovery reveals that de novo genes may play an important role in specific tissues and developmental stages.

[0131] To gain a deeper understanding of the expression dynamics of these de novo genes at different developmental stages, this invention further identified temporal dynamic genes (TDGs) whose expression changes significantly at different developmental stages. Based on the preferential expression characteristics of de novo genes in the tissue at early or late developmental stages, the inventors divided them into two groups: early-expressing genes and late-expressing genes. Figure 4C). The results showed that most de novo genes tended to be expressed late in testicular tissue, while de novo genes tended to be expressed early in hindbrain, liver, and kidney tissue. Figure 4 D). This finding suggests that the expression patterns of de novo genes are closely related to the developmental stage and specific function of tissues, and that they may play different biological roles in different developmental stages and tissues.

[0132] Based on the above findings, it can be concluded that the spatiotemporally specific expression pattern of human-specific de novo genes not only elucidates their crucial role in regulating spermatogonial apoptosis in the testes, as previously reported in the literature, but also... 15,16 Furthermore, it is crucial for maintaining the stability of the neural stem cell pool during early brain development, a point demonstrated in the literature. 10,17 The specific expression of these genes in different tissues and developmental stages may provide humans with unique advantages in areas such as reproduction and nervous system development, which may have played a key role in human evolution.

[0133] Notably, the potential roles of these de novo genes in stem cell maintenance and anti-spermatogonia apoptosis have prompted the inventors to delve deeper into their possible functions in tumorigenesis. To explore this possibility, this invention utilized 5,278 transcriptome datasets from 22 tumor tissues in the TCGA database, compiled from clinical patients, for in-depth analysis. The results showed that, compared to low expression levels in normal tissues (except the brain and testes), these de novo genes generally exhibited a trend of upregulated expression in their respective tumors. Figure 5 In particular, the tissue expression profiles of 23 de novo genes were significantly upregulated in tumor samples, and the tissue expression profiles of 18 de novo genes were significantly expanded in tumor samples. Figure 5 Some of these genes are even expressed only in tumor tissue types.

[0134] These results indicate that these newly emerging de novo genes are not only universally upregulated in tumor tissues but also exhibit a spatiotemporally and tissue-wide expression pattern. This suggests that while these genes are strictly restricted in normal tissues due to certain mechanisms, this restriction is lifted during tumorigenesis, leading to their specific upregulation. This expression pattern suggests that they may play a role in tumorigenesis by reprogramming malignant proliferating cells and maintaining cellular plasticity, similar to carcinoembryonic genes widely used in early cancer screening. 42Therefore, transcriptome data from 5,278 clinical patients confirmed the broad-spectrum upregulation of these de novo genes, suggesting their potential to specifically target adult tumor tissues without attacking normal tissues in mRNA vaccine therapy, providing new targets and approaches for cancer treatment.

[0135] Based on expression characteristics, the inventors have screened a gene set for universally applicable siRNA delivery for tumor treatment in a specific population, and a method for designing siRNA from the corresponding gene's CDS region. This set requires that the genes be upregulated in certain tumors, with the expectation that expression interference will inhibit tumor tissue proliferation. By requiring that the fold change of the gene's expression level in one or more tumors at the quartile of the TPM value compared to the corresponding normal sample is >1.5, the corresponding gene's CDS sequence constitutes the universally applicable siRNA delivery for tumor treatment in this invention. The gene set for universally applicable siRNA therapy for tumors obtained in this invention includes: SERP1, YIF1B, MYEOV, ZNF843, TYMSOS, TENM3-AS1, ADORA2A-AS1, SPEN-AS1, RNF32-DT, LINC01547, SPTSSB, LINC00632, SMIM45, AATBC, EXOC3-AS1, ENSG00000224376, MYCNOS, ELFN1-AS1, TIPARP-AS1, MOCS2-DT, ODC1-DT, PAPLN-AS1, and LINC02188.

[0136] The specific upregulated genes and corresponding cancer types are shown in Table 3S-1.3:

[0137] Table 3S-1.3 Tumor Upregulated Genes and Corresponding Cancer Types

[0138]

[0139]

[0140] The specific upregulated gene CDS sequences are shown in Table 3S-1.4:

[0141] Table 3S-1.4 CDS sequences of tumor-upregulated genes

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[0143]

[0144]

[0145]

[0146] Note: The transcribed mRNA sequence replaces T in this table with uracil (U).

[0147] Finally, by combining expression and translation characteristics, the inventors screened a new set of specific proto-oncogene antigens for the development of a population-wide tumor vaccine. This set requires that the genes not only be specifically upregulated in certain tumors but also have low expression levels or narrow expression profiles in 27 normal adult tissues to ensure that the neoantigens can specifically target tumors without killing normal tissues. By requiring a transcriptional level TPM ≤ 0.5 (low expression in all normal tissues) or spatial specificity ≥ 0.75 (narrow expression profile) in all normal tissues, the inventors obtained 18 human-specific de novo genes that are lowly expressed or expressed in very few of the 27 normal adult tissues, but significantly highly expressed in certain tumors (the fold change of the TPM value of the gene in one or more tumors compared to the corresponding normal sample is > 1.5). Their protein-coding sequences constitute the new set of specific proto-oncogene antigens for the development of a population-wide tumor vaccine in this invention. Figure 6 The novel specific proto-oncogene set obtained by this invention for developing a universal tumor vaccine for the population is as follows: RNF32-DT, TIPARP-AS1, LINC0547, TYMSOS, PAPLN-AS1, ODC1-DT, ADORA2A-AS1, ZNF843, AATBC, MOCS2-DT, SPTSSB, MYEOV, TENM3-AS1, MYCNOS, LINC00632, ELFN1-AS1, LINC02188, and SMIM45.

[0148] Specific highly expressed genes and their corresponding cancer types are shown in Table 3S-1:

[0149] RNF32-DT (LUAD, LUSC, KIRP, COAD, SKCM, OV, CESC), LINC01547 (LUAD, OV), PAPLN-AS1 (TGCT), ODC1-DT (KIRC, KIRP), AATBC (HNSC, COAD, BLCA, UCEC), MOCS2-DT (LUAD , LUSC, KIRC, KIRP, COAD, SKCM), SPTSSB (BRCA, LUAD, LUSC, PAAD, OV), MYCNOS (LGG), LINC00632 (TGCT), LINC02188 (PAAD, KIRC, KIRP), SMIM45 (LUSC, SKCM, OV, CESC).

[0150] Table 3S-1 Tumor-specific highly expressed genes and corresponding cancer types

[0151]

[0152]

[0153] Example 2: The effect of genes in the collection of the present invention on the proliferation of different cancer cell lines and the prognosis of cancer patients.

[0154] To verify the potential application of siRNA targeting de novo genes in young human tumor therapy, this invention designed and implemented a series of experiments, specifically including siRNA design and synthesis, cell culture and transfection, cell proliferation detection, and result analysis. First, validation was performed on 19 genes from a set of 23 tumor-upregulated genes (corresponding CDS sequences are shown in Table 3S-1.4), and three sets of specific siRNA sequences were designed. siRNA is a double-stranded RNA molecule, 21 nucleotides in length, that can specifically degrade the mRNA of target genes through RNA interference (RNAi) mechanisms, thereby inhibiting gene expression. Each siRNA sequence underwent rigorous bioinformatics screening to ensure it targets a specific region of the target gene (such as the CDS region or downstream of the start codon) and avoids cross-reactions with non-target genes. The siRNA was synthesized by Berry Genomics using chemical synthesis methods, and its purity and concentration were verified through quality control. To improve knockdown efficiency, three siRNA sequences designed for each gene were used in combination, with non-targeting siRNAs serving as negative controls. siRNAs that have been reported to interfere with tumor cell proliferation, such as those for the MYC, Bcl2, and Plk1 genes, were used as positive controls. The specific target regions of the siRNAs and their corresponding 19 genes are shown in Table 3S-1.5.

[0155] Table 3S-1.5 siRNA sequences used in RNAi experiments

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[0157]

[0158]

[0159] Next, five representative tumor cell lines were selected for the experiment, including human cervical cancer cells (HeLa), glioblastoma cells (U87), renal cell carcinoma cells (OS-RC-2), colon adenocarcinoma cells (SW480), and undifferentiated thyroid cancer cells (Cal62). These cell lines all exhibited high proliferative capacity and well-defined tumor characteristics, making them suitable for siRNA function validation experiments. All cell lines were cultured in DMEM medium (containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin) at 37°C in a 5% CO2 incubator. Cells were digested with 0.25% trypsin during passage to ensure optimal cell condition. When the cell density reached 60%, transfection was performed using Lipofectamine RNAiMAX (Invitrogen). siRNA and liposomes were mixed at a 1:1 volume ratio in Opti-MEM medium and incubated at room temperature for 15 minutes to form the siRNA-liposome complex. 50 μL of the complex was added to each well, resulting in a final siRNA concentration of 20 nM. Six hours after transfection, the medium was replaced with complete culture medium, and cultured for another 24 hours to ensure gene knockdown.

[0160] To evaluate the inhibitory effect of siRNA on tumor cell proliferation, the CCK-8 assay was used. Transfected cells were seeded into 96-well plates at a density of 2 × 10³ / well, with six biological replicates per group to ensure data reliability. CCK-8 reagent (Solarbio) was added at 0, 24, 48, 72, and 96 hours, and the cells were incubated at 37°C for 2 hours. The absorbance (OD value) at 450 nm was measured using a microplate reader. Using the non-targeted siRNA group as a baseline, the relative proliferation rate of the experimental groups at 96 hours was calculated (formula: experimental group OD / control group OD × 100%), and statistical analysis was performed using a two-way ANOVA. The mRNA level of the target gene was detected by qPCR. The results showed that the mRNA level of the target gene was significantly reduced after siRNA transfection, with a knockdown efficiency >70% (P < 0.01), indicating the effectiveness of the siRNA design. There was no significant difference in cell proliferation between the non-targeted siRNA group and the untransfected group (P > 0.05), indicating that the inhibitory effect of siRNA is targeted and specific.

[0161] Figure 7 A shows that siRNA interference experiments demonstrate that de novo genes affect the proliferation of different cancer cell lines. The heatmap of siRNA screening results displays three known genes affecting tumor cell proliferation and 19 upregulated denovo genes in tumors (upregulation is caused by…). Figure 5(Definition) CCK-8 reagent (Solarbio) was added at 0, 24, 48, 72, and 96 hours after siRNA transfection, and the cells were incubated at 37°C for 2 hours. The absorbance (OD value) at 450 nm was measured using a microplate reader. The log2 fold change of each siRNA group was obtained by taking the logarithm of the fold change in the relative proliferation rate of the non-targeted siRNA group at 96 hours. Significance was determined by two-way ANOVA of the experimental group and the non-targeted siRNA group: * P < 0.5; ** P < 0.01; *** P < 0.001. Figure 7 B showed that after knocking down two representative genes, ADORA2A-AS1 and ELFN1-AS1, in five types of tumor cells from 0 to 96 hours, the CCK-8 cell proliferation curves showed that the interference of their gene expression had a significant inhibitory effect on cell proliferation.

[0162] This embodiment successfully inhibited the proliferation of various tumor cells by targeting newly formed genes in young humans with siRNA, verifying the important role of these genes in tumorigenesis and development. Experimental results show that siRNA technology can efficiently and specifically knock down target genes, providing a concrete implementation plan for anti-cancer therapy based on RNA interference technology. Furthermore, the innovative design of the siRNA sequence and the application of multiple gene combinations in this embodiment provide strong support for subsequent claims. By further optimizing the siRNA delivery system and expanding the scope of cell line validation, this invention is expected to provide new strategies and tools for tumor treatment.

[0163] Furthermore, inspired by the upregulation and spatiotemporal expansion of novel gene expression in tumors, this invention, based on the 18 novel specific proto-oncogene sets obtained from the invention for the development of universal tumor vaccines for the population, simultaneously conducted CRISPR / Cas9 screening experiments targeting 14 of these genes (ODC1-DT, ELFN1-AS1, AATBC, SPTSSB, SMIM45, TIPARP-AS1, LINC01547, MOCS2-DT, MYEOV, TENM3-AS1, PAPLN-AS1, LINC00632, RNF32-DT, ADORA2A-AS1), analyzed their effects on the proliferation of multiple cancer cell lines, and used 3 recognized proto-oncogenes (PIK3CA, KMT2D, KRAS) and 1 tumor suppressor gene (PTEN) as positive controls.

[0164] This library contains 144 single-stranded guide RNAs (sgRNAs), with 4-5 sgRNAs per gene. It includes 20 non-targeting sgRNAs as negative controls (nc) and 16 sgRNAs targeting known oncogenes as positive controls. Specific sgRNA targeting sequences are shown in Table 3S-2. The inventors tested the effects of these sgRNAs on the growth of multiple cancer cell lines, including U87, SW480, HeLa, Cal62, and OS-RC-2. The abundance of each sgRNA was compared immediately after infection and after ten cell passages. Figure 8 A). A significant reduction in sgRNAs targeting oncogenes validated the effectiveness of the screening experiment, and disruption of 10 out of 14 de novo genes (72%, ODC1-DT, ELFN1-AS1, AATBC, SPTSSB, SMIM45, TIPARP-AS1, MOCS2-DT, MYEOV, PAPLN-AS1, RNF32-DT) significantly inhibited the proliferation of at least one tumor cell line. Figure 8 (B) emphasizes their direct regulatory role in tumor growth.

[0165] Table 3S-2: CRISPR-Cas9 screening of corresponding sgRNA sequences

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[0168]

[0169]

[0170]

[0171] Note: T in this sequence represents uracil in RNA.

[0172] Based on these findings, this invention further explored the potential of de novo genes as biomarkers for clinical prognosis in tumors. This invention downloaded and reanalyzed datasets of clinical and associated gene expression from 5,111 sets of the TCGA public database. Prognostic significance was assessed using Kaplan-Meier analysis and Cox proportional hazards models, grouped by median de novo gene expression. Results showed that high expression of 83.3% of these genes was associated with poor prognosis in at least one cancer type. Figure 8 C). For example, ELFN1-AS1 expression was significantly associated with the prognosis of colon adenocarcinoma (P=0.011; Figure 8D). In summary, the novel specific antigen set for the development of universally applicable tumor vaccines obtained in this invention and the human-specific de novo gene set have similar roles and mechanisms in tumorigenesis and development. This also suggests that young human de novo genes are candidate targets for anti-tumor drugs.

[0173] Example 3: Presentation of de novo gene-encoded protein by tumor cells

[0174] To further verify whether tumor cells would present peptides encoded by de novo genes and thus aid in subsequent tumor vaccine development, this invention utilized the NetMHCpan-4.1 computational tool to predict the 9-10 amino acid epitope peptides of these 18 genes bound to different MHC-IA / B alleles. This tool, trained on millions of datasets obtained through binding affinity (BA) and eluted ligand (EL) mass spectrometry measurements using an artificial neural network model, can predict the probability of a specific peptide of a protein (Table 4, Peptide) being bound and presented by MHC molecules and output the identified binding core (Table 4, Core). Half-inhibitory concentration (IC50) and percentile rank (Table 3) are indicators of a peptide's ability to bind to MHC molecules (Allele) (Table 3). Peptides with IC50 < 500 nM or Percentile Rank < 2.0 are considered capable of binding to MHC-I molecules, while peptides with IC50 < 50 nM or Percentile Rank < 0.5 are considered to have strong binding ability to MHC-I molecules. This invention provides a peptide (Table 4) that is most likely to bind to each de novo gene and each MHC-I A / B allele (requiring IC50 < 500), demonstrating that they can be presented to the cell surface by tumor cells and subsequently specifically recognized and killed by CD8+ T cells, confirming their potential as a universal tumor vaccine.

[0175] Table 4: 18 de novo genes and their peptides that are most likely to bind to each MHC-I A / B allele (IC50 < 500).

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[0194] The novel set of specific proto-oncogenes developed in this invention for the development of a universally applicable tumor vaccine provides new ideas and methods for tumor vaccine research and development. By utilizing these genes with unique expression patterns and potential tumor relevance as antigens, the inventors hope to develop more effective and safer tumor vaccines, providing new means for the treatment and prevention of tumors.

[0195] Example 4: Specific mRNA vaccine delivery system (LNP) targeting novel proto-oncogenes

[0196] This invention also provides a specific mRNA vaccine delivery system (LNP) targeting novel proto-oncogenes, comprising a liposome complex of non-replicating nucleic acids, the liposome complex including nucleic acid molecules and cationic lipid nanoparticles encapsulating the nucleic acid molecules in a lipid shell, the cationic lipid nanoparticles comprising protonable cationic lipids, structural lipids, auxiliary lipids, and surfactants. The LNP delivery vector (SM-102 LNP) developed by Moderna has achieved great success in the development of COVID-19 vaccines. In this embodiment, the inventors used SM-102 LNP as a delivery system to test the feasibility of using novel proto-oncogenes as vaccines. Those skilled in the art will expect that any other LNP delivery vector can be used to achieve the same or similar effects.

[0197] This embodiment focuses on two de novo genes with unique expression patterns—ELFN1-AS1 and TYMSOS—which are specifically expressed in early developmental stages and reactivated in tumor tissue. Based on this discovery, the inventors designed and prepared mRNA vaccines targeting these two genes. Figure 9 The sequences used in the design of the mRNA vaccine are shown in Table 4S below.

[0198] Table 4. Sequences used in the design of S mRNA vaccines

[0199]

[0200] In the process of creating the mRNA vaccine, the inventors constructed an advanced vaccine delivery system and modified the mRNA molecule to enhance its stability and reduce immunogenicity. Specifically, they added pseudouracil (ψ) and a Cap1 structure to the mRNA. These modifications help reduce mRNA degradation within cells and lower the risk of it being recognized by the immune system, thereby improving the vaccine's effectiveness. Figure 9 ).

[0201] Subsequently, an mRNA vaccine encapsulated with SM-102 LNP was prepared. This formulation has been proven effective in clinical applications for vaccines against COVID-19 and SARS-CoV-2. 7,43,44 In short, the lipid mixture was dissolved in ethanol at a molar ratio of 50 / 10 / 38.5 / 1.5 (SM102 / DSPC / cholesterol / DMG-PEG2K), while the mRNA was dissolved in 10 mM citrate buffer (pH 4.0). The mixtures were then combined in a microfluidic system at a volume ratio of 3:1 (aqueous phase: ethanol phase), resulting in a final lipid-to-mRNA weight ratio of 20:1. The mRNA-LNPs were divided into three groups: LNP control group, LNP-ELFN1-AS1 group, and LNP-TYMSOS group.

[0202] In designing the mRNA sequences, the inventors inserted a 3×Flag tag downstream of the coding sequence (CDS). This design enabled them to detect the translational expression of ELFN1-AS1 and TYMSOS proteins in cells using Western blotting. Experimental results showed that both mRNAs were successfully translated in in vitro cell lines, producing proteins tagged with the 3×Flag (see...). Figure 10 B).

[0203] To verify the physicochemical properties of mRNA-LNPs, the inventors performed detailed characterization. The results showed that the prepared mRNA-LNPs exhibited high homogeneity, with a polydispersity index (PDI) of less than 0.15 and similar diameters. Furthermore, the inventors determined their encapsulation efficiency and Zeta potential to ensure their stability and effectiveness (see [link to study]). Figure 10 A).

[0204] In summary, the mRNA vaccines targeting ELFN1-AS1 and TYMSOS designed in this invention exhibit superior physicochemical properties and in vitro translation capabilities, providing a feasible strategy and method for developing universal tumor prevention or treatment vaccines. This achievement is expected to bring new breakthroughs and progress to the field of tumor vaccine research and development.

[0205] Example 5: Efficacy of a specific mRNA vaccine targeting a novel proto-oncogene in humanized mice.

[0206] To verify the effectiveness of the two mRNA vaccines obtained in the above embodiments, this invention used a humanized mouse model (huHSC-NKG-ProF strain) to simulate the complexity of the human immune system, thereby evaluating the immunogenicity and therapeutic efficacy of these two mRNA vaccines. Specifically, this humanized mouse model, achieved by transplanting human hematopoietic stem cells into C-NKG mice, showed that after 10 weeks, the proportion of humanized T cells in peripheral blood mononuclear cells reached 30% to 60%. Furthermore, various types of human myeloid cells, such as dendritic cells, could be detected in the spleen of the humanized mice, making this mouse strain highly suitable for evaluating tumor vaccines.

[0207] Subsequently, humanized mice were vaccinated with LNP-control, LNP-ELFN1-AS1, and LNP-TYTMSOS vaccines, once a week for a total of four doses (30ug per mouse per dose). Figure 11 A). Following vaccination, the inventors assessed the immunogenicity of each group of humanized mice. Because mature dendritic cells express specific co-stimulatory molecules while presenting antigens, this promotes CD8... +T-cell immune response activation is crucial; therefore, this invention first assessed the maturation and activation of dendritic cells in the spleen of mice vaccinated with mRNA vaccines using flow cytometry. The results showed that, compared to mice vaccinated with the LNP-control vaccine, mice vaccinated with both LNP-ELFN1-AS1 and LNP-TYTMSOS vaccines exhibited increased expression of CD83, a marker of mature dendritic cells, as well as the T-cell activation helper molecules CD80 and CD86, in the spleen. Figure 11 (B and C). This indicates that human dendritic cells in humanized mice were effectively activated after vaccination with the vaccine designed in this invention. The above results demonstrate that these two mRNA vaccines can promote the maturation of dendritic cells and endow them with phenotypic characteristics that stimulate cellular immune responses.

[0208] Following antigen processing, activated dendritic cells migrate to secondary lymphoid organs and interact with T cells, which are then activated upon antigen presentation and co-stimulatory signals. Results showed that CD8+ expressing CD137 was observed in the spleen of mice vaccinated with LNP-ELFN1-AS1 or LNP-TYTMSOS vaccines. + An increased proportion of T cells demonstrates CD8+ specific antigen response. + T cells are also activated. Figure 12 (A and B).

[0209] To further verify whether there is an antigen-specific T-cell response to these two mRNA vaccines in humanized mice vaccinated with these two LNP-mRNA vaccines, this invention selected three peptides with the top three IC50 rankings based on the previous calculation and prediction results of their binding ability to MHC-I A / B for synthesis and subsequent experiments (Table 5).

[0210] Table 5. Affinities of epitope peptides predicted by ELFN1-AS1 and TYMSOS to bind to MHC-I (HLA-A*02:01).

[0211]

[0212] Table 5 lists the affinities of peptides predicted by ELFN1-AS1 and TYMSOS to bind to MHC-I (HLA-A*02:01). The table lists the gene name, peptide name, length, peptide sequence, IC50 value, and percentile rank.

[0213] Subsequently, this invention verified the binding of peptides to HLA through T2 cell epitope peptide binding experiments. T2 cell lines lack antigen peptide transporters (TAPs) and therefore cannot effectively present endogenous antigens, leading to unstable expression and rapid degradation of HLA-A2 molecules on their surface. However, when exogenous antigen peptides bind to HLA-A2 molecules, the stability of HLA-A2 expression is enhanced. The stronger the binding strength between the antigen peptide and HLA-A2 molecules, the less HLA-A2 molecules are degraded on the T2 cell surface, and the higher the expression level. Therefore, the increased expression level of HLA-A2 molecules on the T2 cell surface can serve as an indicator of the binding affinity between exogenous epitope peptides and HLA-A2 molecules. The results showed that the epitope peptides ELFN1-AS1-p2, TYMSOS-p1, TYMSOS-p2, and TYMSOS-p3 have a high affinity for HLA. Therefore, the researchers used these peptides in the subsequent IFN-γ ELISpot experiment to induce T cell responses. Figure 13 (A and B).

[0214] After identifying the HLA-affinity peptide, this invention isolated peripheral blood mononuclear cells (PBMCs) from humanized mice one week after administering four doses of the mRNA vaccine, and co-incubated the cells with the corresponding epitope peptide. The IFN-γ Elispot assay showed that a significant increase in the number of IFN-γ T cells was detected in the peripheral blood of humanized mice vaccinated with the LNP-mRNA vaccine after stimulation with the corresponding epitope peptide. However, no specifically responding T cells were detected in humanized mice vaccinated with the control LNP-control vaccine or those not stimulated with the corresponding peptide. This indicates that ELFN1-AS1 and TYMSOS, as neoantigens, are not present in the autoantigen repertoire of humanized mice with a human immune system, and that both LNP-mRNA vaccines, LNP-ELFN1-AS1 and LNP-TYMSOS, can specifically induce an IFN-γ T cell response. Figure 14 ).

[0215] In summary, based on the specific activation of dendritic cells and T cells in vivo and the antigen-specific CD8 in vitro... +The T-test results demonstrate that humanized mice vaccinated with both LNP-ELFN1-AS1 and LNP-TYMSOS mRNA vaccines elicited specific immune activation responses. This verifies that LNP-mRNA vaccines designed based on the de novo gene can induce in vivo immune responses and specific immune memory, suggesting that these LNP-mRNA vaccines may possess the potential for anti-tumor immune responses.

[0216] To elucidate the role of this LNP-mRNA vaccine in anti-tumor immunity, humanized mice were further vaccinated weekly for a total of three weeks. SW480 colon cancer cells were then subcutaneously implanted into the mice to test the efficacy of the mRNA vaccine in preventing tumor growth. Figure 15 A). Consistent with the results of in vitro T-cell specific immune response detection, this invention found that both LNP-ELFN1-AS1 and LNP-TYMSOS can effectively inhibit the progression of tumors in the early stages ( Figure 15 B).

[0217] Colorectal cancer is a highly complex disease, and in its clinical treatment, immune checkpoint inhibitor therapies targeting PD-1 (such as Pembrolizumab and Nivolumab), PD-L1 (such as Durvalumab and Atezolizumab), and CTLA-4 (such as Ipilimumab) have shown promising clinical efficacy. 45,46 However, the response rate of PD-1 / PDL-1 therapy is relatively low, and many patients may not benefit from PD-1 / PDL-1 immunotherapy. Therefore, the development of multiple combination therapies has always been a focus of clinical research in colorectal cancer. Based on the inhibitory effect of mRNA vaccines in the early stages of tumor growth described above, this invention further combines mRNA vaccine prevention with PD1-1aIL2 immunotherapy to observe the effect in tumor treatment. Figure 16 A), the results showed that LNP-ELFN1-AS1 and LNP-TYMSOS combined with PD1-laIL2 achieved significant inhibitory effects on tumor growth ( Figure 16 B).

[0218] In summary, these studies demonstrate that the mRNA vaccine of this invention can induce a specific immune response and inhibit tumor growth. The de novo gene set screened in this invention, combined with its overall specific origin, low or narrow expression profile in normal tissues, tumor-specific upregulation, direct relationship with tumor growth, and prognostic association with clinical patients, reasonably suggests the potential of other de novo genes in the set as neoantigens in anti-tumor therapy, providing strong support for the development of universally applicable tumor mRNA vaccines.

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Claims

1. A method for constructing a gene set, the method comprising: (a) Collect de novo genes specific to the human family and eliminate erroneous or duplicated candidate genes by determining their gene and transcript structure, thereby obtaining a candidate gene set; Optionally (b) perform a linear alignment of the entire mammalian genome to exclude candidate genes that have coding capabilities in orthologous regions of the ancestral genome; Optionally (c) homology analysis is performed to exclude candidate genes that may have a duplication origin; as well as Optionally (d) a transcriptional profiling analysis was performed to screen for candidate genes with evidence of in vivo protein expression.

2. The method of claim 1, wherein step (b) comprises assessing the coding potential of each candidate gene in a common ancestor on a phylogenetic tree, wherein if the start site of the reconstructed ancestral ORF is precisely aligned with the candidate gene or aligned within a hexanucleotide frame downstream of the start translation site, and the assumed ORF length of the ancestral sequence reaches 70% of that of the candidate gene, then the orthologous region in the ancestral genome is considered to be coding.

3. The method according to claim 1 or 2, wherein the homology analysis in step (c) is performed by a homology search in humans and representative outgroups to exclude candidate genes with the possibility of replication origin, preferably requiring an E value of less than 10 for the homology search. -4 Less than 10⁻ 5 Less than 10⁻ 6 or less than 10 -7 Consistency greater than 30%, 40%, 50% or 60%, and / or query coverage greater than 40%, 50% or 60%.

4. The method according to any one of claims 1 to 3, wherein the transcriptional profiling analysis in step (d) includes studying transcriptome data, ribosomal imprinting (Ribo-seq) datasets and / or mass spectrometry (MS) data.

5. The method according to any one of claims 1 to 4, wherein the method further comprises: (e) Further screening for genes that are expressed at low levels or with a narrow expression profile in normal adult tissues and / or are specifically highly expressed in tumors.

6. The method of claim 5, wherein step (e) comprises screening for genes with a transcriptional level TPM ≤ 0.5 (low expression in all normal tissues) or spatial expression specificity ≥ 0.75 (narrow expression profile) in at least 20, at least 23, at least 25 or at least 27 human tissues.

7. The method according to claim 5 or 6, wherein step (e) includes screening for genes that are specifically highly expressed in tumors, preferably the fold change of the expression level of the gene in one or more tumors at the quartile of the TPM value compared with the corresponding normal sample is >1.

5.

8. A gene set constructed by the method according to any one of claims 1-7, preferably the gene set comprising one or more genes selected from the following: KCNK10, SERP1, ENSG00000145063, SCP2D1-AS1, YIF1B, MYEOV, RPP38-DT, ZNF843, TYMSOS, TENM3-AS1, ADORA2A-AS1, EIF3J-DT, SPEN-AS1, CHRM2, RNF32-DT, LINC01547, PAX8-AS1, LINC00523, SPTSSB, C20orf203, LINC01565, LI NC00632, TDRG1, LINC02397, ENSG00000205414, SMIM45, AATBC, EXOC3-AS1, ENSG00000224376, NTM-AS1, MIR137HG, SATB2-AS1, HDAC2-AS2, LINC0 2370, ENSG00000233290, MYCNOS, ELFN1-AS1, ENSG00000243004, TIPARP-AS1, UBR5-DT, MOCS2-DT, DNAH10OS, PBOV1, ENSG00000255384, ZNF252P- AS1, CLLU1, ODC1-DT, PAPRN-AS1, LINC02188, GTSCR1, ENSG00000279491, LINC00602; more preferably, the gene set includes any one or more genes selected from the following: RNF32-DT, TIPARP-AS1, LINC0547, TYMSOS, PAPRN-AS1, ODC1-DT, ADORA2A-AS1, ZNF843, AATBC, MOCS2-DT, SPTSSB, MYEOV, TENM3-AS1, MYCNOS, LINC00632, ELFN1-AS1, LINC02 188, SMIM45, and more preferably the gene set comprises any one or more genes selected from the following: SERP1, YIF1B, MYEOV, ZNF843, TYMSOS, TENM3-AS1, ADORA2A-AS1, SPEN-AS1, RNF32-DT, LINC01547, SPTSSB, LINC00632, SMIM45, AATBC, EXOC3-AS1, ENSG00000224376, MYCNOS, ELFN1-AS1, TIPARP-AS1, MOCS2-DT, ODC1-DT, PAPLN-AS1, LINC02188.

9. Use of a partial or full-length coding sequence of any gene in the gene set according to claim 8, or a molecule for detecting or regulating the expression of said gene, in the preparation of a medicament for treating tumors or a diagnostic reagent for diagnosing tumors.

10. A polynucleotide molecule encoding a protein, an antigenic fragment thereof, or a variant of a protein or antigenic fragment expressed by a gene selected from the following: KCNK10, SERP1, ENSG00000145063, SCP2D1-AS1, YIF1B, MYEOV, RPP38-DT, ZNF843, TYMSOS, TENM3-AS1, ADORA2A-AS1, EIF3J-DT, SPEN-AS1, CHRM2, RNF32-DT, LI NC01547, PAX8-AS1, LINC00523, SPTSSB, C20orf203, LINC01565, LINC00632, TDRG1, LINC02397, ENSG00000205414 , SMIM45, AATBC, EXOC3-AS1, ENSG00000224376, NTM-AS1, MIR137HG, SATB2-AS1, HDAC2-AS2, LINC02370, ENSG00000 233290, MYCNOS, ELFN1-AS1, ENSG00000243004, TIPARP-AS1, UBR5-DT, MOCS2-DT, DNAH10OS, PBOV1, ENSG00000255384, ZNF252P-AS1, CLLU1, ODC1-DT, PAPRN-AS1, LINC02188, GTSCR1, ENSG00000279491, LINC00602; preferably, the polynucleotide molecule encodes Proteins, antigenic fragments, or variants of proteins or antigenic fragments expressed by the following genes are selected: RNF32-DT, TIPARP-AS1, LINC0547, TYMSOS, PAPLN-AS1, ODC1-DT, ADORA2A-AS1, ZNF843, AATBC, MOCS2-DT, SPTSSB, MYEOV, TENM3-AS1, MYCNOS, LINC00632, ELFN1-AS1, LINC02188, SMIM45.

11. The polynucleotide molecule of claim 10, encoding a polypeptide comprising an amino acid sequence represented by any one of SEQ ID NO:1-420 and SEQ ID NO:423-787 or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% identity with any one of SEQ ID NO:1-420 and SEQ ID NO:423-787, preferably the polynucleotide molecule encoding a polypeptide comprising an amino acid sequence represented by any one of SEQ ID NO:9, SEQ ID NO:37, SEQ ID NO:130, SEQ ID NO:129, SEQ ID NO:191, and SEQ ID NO:418-420 or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% identity with any one of SEQ ID NO:9, SEQ ID NO:37, SEQ ID NO:130, SEQ ID NO:129, SEQ ID NO:191, and SEQ ID NO:418-420.

12. The polynucleotide molecule according to claim 10 or 11, wherein the polynucleotide molecule is an mRNA molecule, preferably the polynucleotide molecule contains a 5' cap structure, more preferably contains a cap1 modification at the 5' end, and / or at least 50%, at least 70%, at least 90%, at least 95% or 100% of uridine (U) in the polynucleotide molecule is replaced with pseudouridine (Ψ).

13. A liposome complex comprising a polynucleotide molecule according to any one of claims 10 to 12, and lipid nanoparticles encapsulating the polynucleotide molecule in a lipid shell, said lipid nanoparticles comprising protonable cationic lipids, phospholipids, steroids, and / or PEG lipids.

14. Use of the liposome complex according to claim 13 in the preparation of vaccines for the treatment and / or prevention of tumors.

15. A targeting molecule that regulates the expression of the following genes: KCNK10, SERP1, ENSG00000145063, SCP2D1-AS1, YIF1B, MYEOV, RPP38-DT, ZNF843, TYMSOS, TENM3-AS1, ADORA2A-AS1, EIF3J-DT, SPEN-AS1, CHRM2, RNF32-DT, LINC01547, PAX8-AS1, LINC00523, SPTSSB, C20orf203, LINC01565, LINC00632, TDRG1, LINC02397 , ENSG00000205414, SMIM45, AATBC, EXOC3-AS1, ENSG00000224376, NTM-AS1, MIR137HG, SATB2-AS1, HDAC2-AS2, LINC02370, ENSG0000023329 0.MYCNOS, ELFN1-AS1, ENSG00000243004, TIPARP-AS1, UBR5-DT, MOCS2-DT, DNAH10OS, PBOV1, ENSG00000255384, ZNF252P-AS1, CLLU1, ODC1- DT, PAPRN-AS1, LINC02188, GTSCR1, ENSG00000279491, LINC00602, preferably the targeted molecular regulation is selected from the following gene expression: ODC1-DT, ELFN1-AS1, AATBC, SPTSSB, SMIM45, TIPARP-AS1, LINC01547, MOCS2-DT, MYEOV, TENM3-AS1, PAPRN-AS1, LINC00632, RNF32-DT, ADORA2A-AS1, and even more preferably the targeted molecular regulation is selected from the following gene expression: SERP1, YIF 1B, MYEOV, ZNF843, TYMSOS, TENM3-AS1, ADORA2A-AS1, SPEN-AS1, RNF32-DT, LINC01547, SPTSSB, LINC00632, SMIM45, AATBC, EXOC3-AS1, ENSG00000224376, MYCNOS, ELFN1-AS1, TIPARP-AS1, MOCS2-DT, ODC1-DT, PAPLN-AS1, LINC02188, more preferably the target molecule is a guide RNA, such as sgRNA, or the target molecule is siRNA.

16. The targeting molecule of claim 15, wherein the targeting molecule comprises a targeting region shown in any one of SEQ ID NO:791-946 or SEQ ID NO:966-1031 or a targeting region having at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% identity with the sequence shown in any one of SEQ ID NO:791-946 or SEQ ID NO:966-1031.

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