Polynucleotides that upregulate telomerase and uses and methods of treatment

Abstract

The present invention provides a composition comprising one or more polynucleotides that upregulate telomerase expression, wherein the telomerase comprises a protein component having reverse transcriptase activity encoded by a telomerase reverse transcriptase (TERT) gene and a non-coding RNA encoded by a telomerase RNA component (TERC) gene. In one embodiment, the one or more polynucleotides of the present invention upregulate telomerase by downregulating antisense RNA that regulates telomerase genes. In one embodiment, the one or more polynucleotides comprise siRNA or shRNA. In one embodiment, the one or more polynucleotides of the present invention are delivered by nanoparticles (e.g., exosomes) that encapsulate the one or more polynucleotides. The present invention also provides a method of treating a disease or disorder associated with telomerase deficiency using the one or more polynucleotides described in any embodiment of the present invention.

Description

[0001] Reference to merge into sequence list XML

[0002] This specification provides a sequence listing XML file entitled "Telomerase Upregulating Polynucleotide Sequence Listing.xml", created on January 15, 2025, with a size of 132 KB. The contents of this sequence listing XML file are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention provides a composition comprising one or more polynucleotides that upregulate telomerase and a method thereof. Background Technology

[0004] Telomerase is a ribonucleoprotein polymerase that maintains telomere ends by adding telomeric repeat sequences (TTAGGG). Telomerase comprises a protein component with reverse transcriptase activity encoded by the telomerase reverse transcriptase (TERT) gene and a non-coding RNA encoded by the telomerase RNA component (TERC) gene, which serves as a template for telomeric repeat sequences. Telomerase expression is associated with cellular aging, as it is typically suppressed in postnatal somatic cells, leading to gradual telomere shortening. Dysregulation of telomerase expression in somatic cells may be associated with tumorigenesis. Mouse studies have shown that de novo synthesis of telomeric repeat sequences may occur at doublet breaks, indicating that telomerase also participates in chromosome repair. Furthermore, increased telomerase expression has been found to be associated with cellular senescence and aging, and overexpression has been shown to improve idiopathic pulmonary fibrosis (IPF) (https: / / doi.org / 10.1083 / jcb.202002120). Therefore, there is a need to upregulate telomerase expression to treat lung diseases and potentially reverse fibrosis. Summary of the Invention

[0005] A pharmaceutical composition comprising a therapeutically effective amount of one or more polynucleotides that upregulate telomerase, wherein the nucleotide sequence of each of the one or more polynucleotides that upregulate telomerase is identical to that of SEQ ID NO. 26, SEQ ID NO. 29, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 17, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 18, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. The similarity of 39 or its combinations is at least about 80%, about 85%, about 90%, about 95%, or about 100%.

[0006] A method for increasing the expression of TERT, TERC, or a combination thereof in a subject, comprising administering to the subject a therapeutically effective amount of one or more polynucleotides that upregulate telomerase, as described in any embodiment of the present invention.

[0007] A method for treating a subject with telomerase-related disease includes the step of increasing telomerase expression in the subject. In one embodiment, the step of increasing telomerase expression in a subject of the present invention includes administering a therapeutically effective amount of one or more telomerase-upregulating polynucleotides as described in any embodiment of the present invention to the subject. Attached Figure Description

[0008] Figure 1 Examples of lncRNAs associated with the TERC locus are shown, along with the preferred siRNA target sites for inhibiting TERC-related lncRNAs.

[0009] Figure 2 Examples of lncRNAs associated with the TERT locus are shown, and the preferred siRNA target site for repressing the TERT-associated lncRNA ENST00000666708.1 is shown, where ENST00000666708.1 is the transcript of ENSG00000287486.

[0010] Figure 3Examples show siRNAs targeting human TERC-related lncRNAs and schematic diagrams for screening human TERC-related lncRNAs.

[0011] Figure 4 This paper illustrates the screening of siRNAs targeting TERC-related lncRNAs. TERC expression was assessed in HEK293 cells transfected with 100 nM siRNA. RNA was collected 72 hours post-transfection, and TERC expression was determined by qRT-PCR. The p-values ​​of the triple-treatment cultures are shown as standard deviation and paired T-tests (two-tailed si2 and one-tailed si10).

[0012] Figure 5 The effects of various siRNAs on TERC expression are illustrated. HEK293 cells were harvested 72 hours after transfection with 100 nM siRNA, and TERC expression was measured by qRT-PCR. The p-values ​​of the triple-treatment culture are shown as standard deviation and paired T-test.

[0013] Figure 6 Examples of preferred siRNAs for TERC activation are shown. A variety of siRNAs targeting TERC-associated antisense RNAs are screened. Preferred siRNAs and TERC F1 / R1 or TERC F2 / R2 amplicons used in qRT-PCR evaluation are displayed relative to the TERC gene.

[0014] Figure 7 Example of the target site of siRNA in 3' TERT-associated antisense lncRNA (ENSG00000287486).

[0015] Figure 8 This illustrates siRNA-mediated activation of TERT in HEK293 cells. HEK293 cells were transfected with 100 nM L2K, and TERT mRNA expression was assessed after 72 hours.

[0016] Figure 9 This illustrates siRNA-mediated activation of TERT in HEK293 cells. HEK293 cells were collected 72 hours after transfection with the specified siRNA (12-well plate). RNA was extracted using the RNeasy kit and subjected to DNase treatment and qPCR primer amplification: β-actin, TERT F1, R1 (Table 1).

[0017] Figure 10 Example of combined TERT mRNA expression in cells treated with E666-si4-1 and si4-2. Standard deviation shows the mean of triple-repeated treatment of HEK cells.

[0018] Figure 11 An example vector map of the dual-expression shRNA EV production vector is shown. The vector pRP[Exp]-{pshTERC10&13_Dual}-CMV>Puro is co-transfected with shRNA packaging and enhancement vectors from the RNex system (patent applications 63 / 598499 and 63 / 607,552) and cells are subjected to multiplex drug screening.

[0019] Figure 12 This illustration depicts a Transwell assay used to assess the packaging and translocation efficacy of therapeutic RNAs using the RNex system. Producer cells are transfected with different combinations of plastids, reprogramming them to produce exosomes (EVs) that encapsulate therapeutic RNAs targeting various diseases. EV-asRNA, EV-shRNA, or EV-mRNA from the reprogrammed producer cells can penetrate a 0.4 µm membrane and be taken up by recipient cells. Recipient cells can be transduced cell lines stably expressing reporter genes, HEK293 cells directly transfected with various reporter plastids containing therapeutic RNA target sites fused with various reporter transgenes such as RLuc and GFP, or patient-derived organoid cells, allowing for further evaluation of their impact on target disease-related genes.

[0020] Figure 13 Example usage Figure 11 TERC activation was performed using a dual-expression shRNA EV production vector (shTERC-2+shTERC-10). The drug-selected production cell line 2 was co-cultured with the recipient HEK293 cells in Transwell. Figure 12 Recipient cells were collected after co-culturing for 4 days (96 hours), with n = 3 replicates per experiment, and specific targets were evaluated by qRT-PCR. Detailed Implementation

[0021] In this specification and the following claims, unless the context clearly indicates otherwise, the singular forms “a”, “an”, and “the” also include plural indicators. Thus, for example, “an ingredient” also includes a mixture of ingredients, “an active pharmaceutical ingredient” also includes more than one active pharmaceutical ingredient, and so on.

[0022] In this specification, the term “about” as a modifier of quantity means ±5%, ±10%, ±15%, or ±20% of that quantity, including the quantity itself.

[0023] In this specification, the term "nucleic acid" means nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and their polymers, which may be single-stranded, double-stranded, or multi-stranded, or complementary strands thereof. The terms "polynucleotide," "oligonucleotide," "oligomer," or similar terms, in their usual and customary sense, mean a linear sequence of nucleotides. The term "nucleotide," in its usual and customary sense, means a single unit of a polynucleotide, i.e., a monomer. Nucleotides may be ribonucleotides, deoxyribonucleotides, or modified versions thereof. Examples of nucleic acids herein include single-stranded and double-stranded DNA, single-stranded and double-stranded RNA, and hybrid molecules having mixtures of single-stranded and double-stranded DNA and RNA. Examples of nucleic acids here include various RNAs (e.g., antisense RNA, messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), guide RNA, Dicer substrate RNA, Dicer substrate siRNA (dsiRNA) (dsiRNA is cleaved by the RNase I endonuclease Dicer into a 21- to 23-base-pair double strand with two 3'-overhead ends), and various types of DNA, such as genomic DNA, plasmid DNA, and minicircle DNA. DNA and fragments thereof. In the context of nucleic acid terminology, the term "duplex" generally and conventionally refers to a double-stranded structure. Nucleic acids can be linear or branched. For example, nucleic acids can be linear chains of nucleotides, or they can be branched, such as nucleic acids containing one or more nucleotide arms or branches. Optionally, branched nucleic acids can be further branched to form higher-order structures such as dendrimers. In one embodiment, the nucleotide sequence is represented by the symbol ATCG (adenine (A), cytosine (C), guanine (G), and thymine (T)) to represent a DNA molecule, and by the symbol AUCG (adenine (A), cytosine (C), guanine (G), and uracil (U)) to represent an RNA molecule. In one embodiment, the symbols T and U are used interchangeably to represent DNA and RNA molecules prepared according to the nucleotide sequence, respectively.

[0024] In this specification, the terms "polypeptide," "peptide," and "protein" generally refer to polymers of amino acid residues. In this specification, the terms also apply to amino acid polymers comprising one or more amino acid chemical analogs or modified derivatives, which may correspond to naturally occurring amino acids or be non-natural amino acids. In this specification, the term "protein" generally refers to a polymer of amino acids bonded together by peptide bonds to form a polypeptide, and the chain length of the polypeptide is sufficient to produce tertiary and / or quaternary structures. In one embodiment, the "polypeptide," "peptide," or "protein" of the present invention is prepared from a plastid encoding the "polypeptide," "peptide," or "protein." Therefore, the “polypeptide”, “peptide” or “protein” of the present invention further comprises a nucleotide sequence encoding a genetic code that can be converted into the “polypeptide”, “peptide” or “protein”, the genetic code being such as, but not limited to, the standard genetic code.

[0025] In this specification, the terms "sequence identity" and "%identity" refer to a value calculated by comparing two best-aligned sequences within a comparison window, wherein, for best alignment of the two sequences, a portion of the sequence within the comparison window may include additions or deletions compared to a reference sequence. The calculation method involves determining the number of positions in the two sequences where the same amino acid residues occur to generate a matching position number, then dividing the matching position number by the total number of positions within the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Unless otherwise specified, the comparison window is the entire length of the sequence in question.

[0026] In this specification, the term "% similarity" is calculated in the same way as "% identity," with the following exceptions: hydrophobic residues alanine (Ala), valine (Val), phenylalanine (Phe), proline (Pro), leucine (Leu), isoleucine (Ile), tryptophan (Trp), methionine (Met), and cysteine ​​(Cys) are considered similar; basic residues lysine (Lys), arginine (Arg), and histidine (His) are considered similar; acidic residues glutamic acid (Glu) and aspartic acid (Asp) are considered similar; and hydrophilic and uncharged residues glutamic acid (Gln), aspartate (Asn), serine (Ser), threonine (Thr), and tyrosine (Tyr) are considered similar. In this context, the remaining natural amino acid glycine (Gly) is not considered similar to any other amino acid.

[0027] In this specification, the terms “subject,” “individual,” or “patient” are used interchangeably and refer to a vertebrate, preferably a mammal, and more preferably a human. Mammals include, but are not limited to, rodents, apes, humans, farm animals, sporting animals, and pets.

[0028] In this specification, the terms "effective amount" or "therapeutically effective amount" refer to the amount of a drug, compound, pharmacologically active dosage form, or pharmaceutical composition administered, which is sufficient to achieve the desired result. The specific required dose varies from subject to subject and depends on factors such as the subject's species, age, general condition, disease severity, specific active dosage form, method of administration, and desired outcome. In some embodiments of the invention, "therapeutically effective amount" refers to an amount of a drug, compound, pharmacologically active dosage form, or pharmaceutical composition sufficient to inhibit or reverse the progression of any of the diseases described herein, which may occur in a subject or biological sample (e.g., cells). In some embodiments, the inhibition of disease progression is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100%. In some embodiments, the drug, compound, pharmacologically active dosage form, or pharmaceutical composition inhibits disease progression by at least about 25%, at least about 50%, at least about 75%, at least about 90%, or at least about 100%. In some embodiments of the invention, "therapeuticly effective amount" means an amount of drug, compound, pharmacologically active dosage form, or pharmaceutical composition sufficient to induce disease reversal. In some embodiments, disease reversal is about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 100%, or any value or range within the foregoing ranges.

[0029] In this specification, the term "target" or "targeting" polynucleotide refers to the direct or indirect regulation of the expression level, biological function, or a combination thereof of the polynucleotide. Indirect regulation of the polynucleotide includes indirectly regulating the expression level, biological function, or a combination thereof by regulating one or more antisense RNAs that regulate the targeted polynucleotide. In one embodiment, in the case of direct regulation, downregulating the polynucleotide may include disrupting or degrading the targeted or regulated polynucleotide. In one embodiment, in the case of indirect regulation, downregulating the polynucleotide may include disrupting or degrading one or more antisense RNAs that regulate the targeted polynucleotide. In one embodiment, the disruption or degradation uses small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozyme, deoxyribozyme, aptamer, or a combination thereof to target or regulate the polynucleotide.

[0030] This invention provides one or more polynucleotides that upregulate telomerase and pharmaceutical compositions thereof. In one embodiment, the one or more polynucleotides of this invention upregulate telomerase by regulating the expression levels of TERT, TERC, or combinations thereof. In one embodiment, regulating the expression levels of TERT, TERC, or combinations thereof includes regulating the expression levels of antisense regulatory factors of TERT, TERC, or combinations thereof. In one embodiment, the one or more polynucleotides of this invention that target TERT, TERC, or combinations thereof for upregulating telomerase increase or de-inhibit the expression levels of TERT, TERC, or combinations thereof by inhibiting the expression levels of antisense RNA regulatory factors of TERT, TERC, or combinations thereof. In one embodiment, the antisense RNA regulatory factor of TERT, TERC, or combinations thereof is antisense RNA targeting the promoter of TERT, TERC, or combinations thereof. In one embodiment, the nucleotide sequence of the antisense RNA regulator of TERT, TERC, or a combination thereof is at least about 80%, about 85%, about 90%, about 95%, or about 100% identical to the following nucleotide sequences of the subject: AW207347 (SEQ ID NO. 1), AI825849 (SEQ ID NO. 2), AW136367 (SEQ ID NO. 3), AI380754 (SEQ ID NO. 4), AW293800 (SEQ ID NO. 5), BM695101 (SEQ ID NO. 6), CV370609 (SEQ ID NO. 7), MG677549 (SEQ ID NO. 8), BF802688 (SEQ ID NO. 9), AA748707 (SEQ ID NO. 10), AA811084 (SEQ ID NO. 11), AI824948 (SEQ ID NO. 12), AW270031 (SEQ ID NO. 10), CV370609 (SEQ ID NO. 11), MG677549 (SEQ ID NO. 12), BF802688 (SEQ ID NO. 13), AW270031 (SEQ ID NO. 11), CV370609 (SEQ ID NO. 11), MG677549 (SEQ ID NO. 12 ...CV370609 (SEQ ID NO. 11), MG677549 (SEQ ID NO. 12), CV370609 (SEQ ID NO. 11), MG677549 (SEQ ID NO. 12), CV370609 (SEQ ID NO The SEQ ID NO. 13), AW276315 (SEQ ID NO. 14), DB345416 (SEQ ID NO. 15) (which is stored in the NCBI Nucleotide database (https: / / www.ncbi.nlm.nih.gov / nuccore / )), or ENST00000666708.1 (SEQ ID NO. 16) (which is stored in the Ensembl database (https: / / asia.ensembl.org / index.html)) are mentioned. In one embodiment, the subject comprises human cells.

[0031] SEQ ID NO. 1 (AW207347): TTTTTTTTTTTTTTTTTCAGGTTTGGGGGTTCACAAGCCCCCATTGCCGGCGAGGGGTGACGGATGCGCACGATCGGCGTTCCCCCCACCAACAGGAAAGCGAACTGCATGTGTGAGCCGAGTCCTGGGTGCACGTCCCACAGCTCAGGGAATCGCGCCGCGCGCGGGGACTCGCTCCGTTCCTCTTCCTGCGGCCTGAAAGGCCTGAACCTCGCCCTCGCCCCCGAGAGACCCGCGGCTGACAGAGCCCAACTCTTCGCGGTGGCAGTGGGTGCCTCCGGAGAAGCCCCGGGCCGACCGCGGGCTCCAGGCGGGGTTCGGGGGCTGGGCAGGCGACCCGCCGCAGGTCCCCGGGAGGGGCGAACGGGCCAGCAGCTGACATTTTTTG SEQ ID NO. 2(AI825849): AGGTTTGGGGGTTCACAAGCCCCCATTGCCGGCGAGGGGTGACGGATGCTCACGATCGGCGTTCCCCCCACCAACAGGAAAGCGAACTGCATGTGTGAGCCGAGTCCTGGGTGCACGTCCCACAGCTCAGGGAATCGCGCCGCGCGCGGGGACTCGCTCCGTTCCTCTTCCTGCGGCCTGAAAGGCCTGAACCTCGCCCTCGCCCCCGAGAGACCCGCGGCTGACAGAGCCCAACTCTTCGCGGTGGCAGTGGGTGCCTCCGGAGAAGCCCCGGGCCGACCGCGGCCTCCAGGCGGNGTTCGGNGGCTGGGCAGGCGACCCGCCGCAGGTCCCCGGGAGGGGCGAACGGGCCAGCAGCTGACATTTTTTGTTTGCTCTAGAATGAACGGTGGAAGGCGGCAGGCCGAGGCTTTTCCGCCCGCTGAAAGTCAGCGAGAAAAACA SEQ ID NO. 3(AW136367): TTTTTTTTTTTTTTTTTACTGCATGTGTGAGCCGAGTCCTGGGTGCACGTCCCACAGCTCAGGGAATCGCGCCGCGCGCGGGGACTCGCTCCGTTCCTCTTCCTGCGGCCTGAAAGGCCTGAACCTCGCCCTCGCCCCCGAGAGACCCGCGGCTGACAGAGCCCAACTCTTCGCGGTGGCAGTGGGTGCCTCCGGAGAAGCCCCGGGCCGACCGCGGCCTCCAGGCGGGGTTCGGGGGCTGGGCAGGCGACCCGCCGCAGGTCCCCGGGAGGGGCGAACGGGCCAGCAGCTGACATTTTTTGTTTGCTCTAGAATGAACGGTGGAAGGCGGCAGGCCGAGGCTTTTCCGCCCGCTGAAAGTCAGCGAGAAAAACAGCGCGCGGGGAGCAAAAGCACGGCGCCCCTCGTGCCG SEQ ID NO. 4(AI380754): TTTTCTTCTGCATGTGTGAGCCGAGTCCTGGGTGCACGTCCCACAGCTCAGGGAATCGCGCCGCGCGCGGGGACTCGCTCCGTTCCTCTTCCTGCGGCCTGAAAGGCCTGAACCTCGCCCTCGCCCCCGAGAGACCCGCGGCTGACAGAGCCCAACTCTTCGCGGTGGCAGTGGGTGCCTCCGGAGAAGCCCCGGGCCGACCGCGGCCTCCAGGCGGGGTTCGGGGGCTGGGCAGGCGACCCGCCGCAGGTCCCCGGGAGGGGCGAACGGGCCAGCAGCTGACATTTTTTGTTTGCTCTAGAATGAACGGTGGAAGGCGGCAGGCCGAGGCTTTTCCGCCCGCTGAAAGTCAGCGAGAAAAACAGCGCGCGGGGAG SEQ ID NO. 5(AW293800): TTTTTTCCTTTTTTTCTGCATGTGTGAGCCGAGTCCTGGGTGCACGTCCCACAGCTCAGGGAATCGCGCCGCGCGCGGGGACTCGCTCCGTTCCTCTTCCTGCGGCCTGAAAGGCCTGAACCTCGCCCTCGCCCCCGAGAGACCCGCGGCTGACAGAGCCCAACTCTTCGCGGTGGCAGTGGGTGCCTCCGGAGAAGCCCCGGGCCGACCGCGGCCTCCAGGCGGGGTTCGGGGGCTGGGCAGGCGACCCGCCGCAGGTCCCCGGGAGGGGCGAACGGGCCAGCAGCTGACATTTTTTGTTTGCTCTAGAATGAACGGTGGAAGGCGGCAGGCCGAGGCTTTTCCGCCCGCTGAAAGTCAGCGAGAAAAACAGCGCGCGGGGAGCAAAAGCACGGCGCCTACGCCCTTCTCAGTTAGGGGT SEQ ID NO. 6(BM695101): TGACCATTAAAGGAACACAATTTCCAATGTTCATTTAGATCTTCTAATTAAATATTCATTAAATGTTAAATGATCTCTCAAAAAAAAATGACTGTTCTCCCACACCCCGTTGAGGGGACTGGTCGAGATCTACCTTGGGAGAAGCAAAAACCTCAACAAAATCTGCAGAGCAGGAACTAAGTTGTAATACAACCATAAAAGGCAACAAAAAGCGGAAGACGGGAGAACCCACGCAGGAACGGCTCCAGGCAACCCCGGCTCACTGCCCATTCATTTTGGCCGACTTTGGAGGTGCCTTCACGTCTCCTGCCAATTTGCAGCACACTGGCCCAGTCAGTCAGGTTTGGGGGCTC SEQ ID NO. 7(CV370609): TTTTCCGCCCGCTGAAAGTCAGCGAGAAAAACAGCGCGCGGGAAGCAAAAGCACGGTGCCTACGCACTTCTCAGTTAGGGTTAGACAAAAAATGGNCACCAACCCTCCCAGGGCCAC SEQ ID NO. 8(MG677549): SEQ ID NO. 9(BF802688): GATTAGAGGATCCCAGTAGTCGGACGTGTAGTGAATGAGATGATGGTCTCACACCATCACCCAGACCAGGTTATGCACACAGGGCGGTGGTCAGAAGGATTGGCAGGACGTTGATATACGATGACATCAGGTTGTCTGACGAAGGCAGGATTCATGATAAGTACTCTGACGCTGTCCGCATCCTCTCAGGTTCACGCATGTGTGCTGCAGCTCCCATTTCATCAGCAAGTTTGGAAGAACCCCACATTTTTCCTGCGCGTCATCTCTGACACGGCCTCCCTCTGCTACTCCATCCTGCGGTCCTGAGCTTAACAGCTTCTACTTTCTGTTCTTTCTGTGTTGTGGAAATTTCACCTCCCAGGGCGGGTGCCAGGCTCGCAGTGGAGCTGGACATACGTCCTTCCTCAGGCAGAAGGAACTGGAAGGATTGCAGAGAACAGGAGGGGCGGCTCAGAGGGACGCAGTCTTGGGGTGAAGAAACAGCCCCTCCTCAGAAGTTGGCTTGGGCCACACGCGGTC SEQ ID NO. 10(AA748707): TTTTTTTCAAAACTGAAAAACTCATATATTCAGTATTTTACTCCCACAGCACCTCCCCCCAATTTGACCCACAGGGACCCCCATCCAGGTGCAGGGTCCTCGCCTGTGTACAGGGCACACCTTTGGTCACTCCAAATTCCCAGAGCTCCCAGGGTCCTTCTCAGGGTCTCCACCTGGATGGTGGGCCCCGGCCCCCAAACCACGGCACGGCGAGGGGTGAACAATGGCGAATCTGGGGATGGACTATTCCTATGTGGGGAGTGGAAGCCGGGCTCCTGGTGAGGAAAAGCTGGCCCTGGGGTGGAGCCGAGCGCA SEQ ID NO. 11(AA811084): TTTTTTTTTTTTTTTTTTCAAAACTGAAAAACTCATATATTCAGTATTTTACTCCCACAGCACCTCCCCCCAATTTGACCCACAGGACCCCCATCCAGGTGCAGGGTCCTCGCCTGTGTACAGGGCACACCTTTGGTCACTCCAAATTCCCAGAGCTCCCAGGGTCCTTCTCAGGGTCTCCACCTGGATGGTTGGGGTGGAAGGCAAAGGAGGGCAGGGCGAGGGGTGAACAATGGCGAATCTGGGGATGGACTATTCCTATGTGGGGAGTGGAAGCCGGGCTCCTGGTGAGGAAAAGCTGGCCCTGGGGTGGAGCCGAGCGCCACCTGTGGGGAAGTGA SEQ ID NO. 12(AI824948): TTTTTTTTTTTTTTTCAAAACTGAAAAACTCATATATTCAGTATTTTACTCCCACAGCACCTCCCCCCAATTTGACCCACAGGGACCCCCATCCAGGTGCAGGGTCCTCGCCTGTGTACAGGGCACACCTTTGGTCACTCCAAATTCC SEQ ID NO. 13(AW270031): TTCAAAACTGAAAAACTCATATATTCAGTATTTTACTCCCACAGCACCTCCCCCCAATTTGACCCACAGGGACCCCCATCCAGGTGCAGGGTCCTCGCCTGTGTACAGGGCACACCTTTGGTCACTCCAAATTCCCAGAGCTCCCAGGGTCCTTCTCAGGGTCTCCACCTGGATGGTGGGGGTGGAAGGCAAAGGAGGGCAGGGCGAGGGGTGAACAATGGCGAATCTGGGGATGGACTATTCCTATGTGGGGAGTGGAAGCCGGGCTCCTGGTGAGGAAAAGCTGGCCCTGGGGTGGAGCCGAGCGCCAGCCTGTGGNGAAGTGAAGACGGCAGGTGTGCTGGACACTCAGCCCTTGGCTGGACACTCGCTCAGGCCTCAGCCGGACACTCAGCCTTCAACCGGACATGCAGGCCTCGGCCAAACACTCACTCAGGCCTCAGACTCCCAGCGGTGCGGGCCTGGGTG SEQ ID NO. 14(AW276315): TCAAAACTGAAAAACTCATATATTCAGTATTTTACTCCCACAGCACCTCCCCCCAATTTGACCCACAGGGACCCCCATCCAGGTGCAGGGTCCTCGCCTGTGTACAGGGCACACCTTTGGTCACTCCAAATTCCCAGAGCTCCCAGGGTCCTTCTCAGGGTCTCCACCTGGATGGTGGGGGTGGAAGGCAAAGGAGGGCAGGGCGAGGGGTGAACAATGGCGAATCTGGGGATGGACTATTCCTATGTGGGGAGTGGAAGCCGGGCTCCTGGTGAGGAAAAGCTGGCCCTGGGGTGGAGCCGAGCGCCAGCCTGTGGGGAAGTGAAGACGGCAGGTGTGCTGGACACTCAGCCCTTGGCTGGACACTCGCTCAGGCCTCAGCCGGACACTCAGCCTTCAGCCGGACATGCAGGCCT SEQ ID NO. 15(DB345416): CAAAACTGAAAAACTCATATATTCAGTATTTTACTCCCACAGCACCTCCCCCCAATTTGACCCACAGGGACCCCCATCCAGGTGCAGGGTCCTCGCCTGTGTACAGGGCACACCTTTGGTCACTCCAAATTCCCAGAGCTCCCAGGGTCCTTCTCAGGGTCTCCACCTGGATGGTGGGGGTGGAAGGCAAAGGAGGGCAGGGCGAGGGGTGAACAATGGCGAATCTGGGGATGGACTATTCCTATGTGGGGAGTGGAAGCCGGGCTCCTGGTGAGGAAAAGCTGGCCCTGGGGTGGAGCCGAGCGCCAGCCTGTGGGGAAGTGAAGACGGCAGGTGTGCTGGACACTCAGCCCTTGGCTGGACACTCGCTCAGGCCTCAGCCGGACACTCAGCCTTCAGCCGGACATGCAGGCCTCGGCCAAACACTCACTCAGGCCTCANACTCCCAGCGGTGCGGGCCTGGGTGTGGGCCGCCCCTCCCTCCCTGGGACGTANAGCCCGGCGTGACAGGGCTGCTGGTGTCTGCTCTCGGCCTGGCT SEQ ID NO. 16(ENST00000666708.1): In one embodiment, the polynucleotide length of the upregulating telomerase of the present invention is from about 10 to about 100 nucleotides (nt), for example, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 nt, and includes any length or length range falling within these numerical ranges. In one embodiment, the polynucleotide length of the upregulating telomerase of the present invention is from about 15 to about 55 nucleotides, from about 20 to about 30 nucleotides, or from about 21 to about 23 nucleotides. In one embodiment, the polynucleotide of the upregulating telomerase of the present invention may be double-stranded or single-stranded. In one embodiment, the polynucleotide of the upregulating telomerase of the present invention may be blunt-ended or include overhanging ends. In one embodiment, the polynucleotide of the upregulating telomerase of the present invention may be chemically synthesized or recombinant produced. In one embodiment, one strand of the polynucleotide that upregulates telomerase of the present invention comprises a nucleotide sequence that is sufficiently complementary to an antisense RNA regulator of TERT, TERC, or a combination thereof, such that the polynucleotide can guide the cleavage of the antisense RNA regulator through RNA interference.

[0032] In one embodiment, one or more polynucleotides of the present invention that upregulate telomerase comprise small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozyme, deoxyribozyme, aptamer, or a combination thereof. In one embodiment, one or more polynucleotides of the present invention that upregulate telomerase comprise siRNA. In one embodiment, the nucleotide sequence of the polynucleotide that upregulates telomerase with the siRNA of the present invention is at least about 80%, about 85%, about 90%, about 95%, or about 100% identical to SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, or SEQ ID NO. 39. In one embodiment, the polynucleotide that upregulates telomerase with siRNA of the present invention contains a 3' deoxythymidine dinucleotide (dTdT) overhang to increase resistance to nucleases.

[0033] In one embodiment, one or more polynucleotides that upregulate telomerase of the present invention comprise a first siRNA and a second siRNA, wherein the nucleotide sequence of the first siRNA is at least about 80%, about 85%, about 90%, about 95%, or about 100% identical to SEQ ID NO. 26, and the nucleotide sequence of the second siRNA is at least about 80%, about 85%, about 90%, about 95%, or about 100% identical to SEQ ID NO. 29.

[0034] SEQ ID NO. 17 (siasTERC1): GCCUACGCCCUUCUCAGUUAG

[0035] SEQ ID NO. 18 (siasTERC2): GGCCGAGGCUUUUCCGCCCGC

[0036] SEQ ID NO. 19(siasTERC3):AGAGCCCAACUCUCCGCGGUG

[0037] SEQ ID NO. 20(siasTERC4):GCAGCUGACAUUUUUUGUUUG

[0038] SEQ ID NO. 21(siasTERC5):CCGCCGCAGGUCCCCGGGAGG

[0039] SEQ ID NO. 22(siasTERC6):AUCGGGCUUCCCCCCACCAAC

[0040] SEQ ID NO. 23(siasTERC7):GCCUUCACGUCUCCUGCCAAU

[0041] SEQ ID NO. 24(siasTERC8):GGUUCCAAGCCCCCAUUGCC

[0042] SEQ ID NO. 25(siasTERC9):AUUUUGGCCGACUUUGGAGGU

[0043] SEQ ID NO. 26(siasTERC10):CUGCCCAUUCAUUUUGGCCGA

[0044] SEQ ID NO. 27(siasTERC11):UUAGCUUAUUUUCUUAAAGGA

[0045] SEQ ID NO. 28(siasTERC12):GUUAAAUGAUCUCUCAAAAAA

[0046] SEQ ID NO. 29(siasTERC13):UCAUUUAGAUCUUCUAAUUAA

[0047] SEQ ID NO. 30(siasTERC14):AAGGUCUAAUUUUUCAAAAAA

[0048] SEQ ID NO. 31(siasTERC15):UCUUUUCCGUCUUUCAUUAUG

[0049] SEQ ID NO. 32(MG-si1):GAUGAUGUCUUAGUCAUCACAGUGU

[0050] SEQ ID NO. 33(MG-si2):GGAAAAUUUGUUCCAACAAAAGUUA

[0051] SEQ ID NO. 34(MG-si3):GAGGCCAACAUCUGGUCACAUCCCG

[0052] SEQ ID NO. 35(E666-si1):CCAGCAGGUACUUAUCAUGAAUCCU

[0053] SEQ ID NO. 36(E666-si2):GUACACUGCGUUUCUGUGCACCAUC

[0054] SEQ ID NO. 37(E666-si3):CCUGUUCUCUGCAAUCCUUCCAGUU

[0055] SEQ ID NO. 38(E666-si4-1):GGCACCAGAGUGCAUUUACGA

[0056] SEQ ID NO. 39(E666-si4-2):UUCUCUGAAGCAGCAGCAAUA

[0057] In one embodiment, the one or more telomerase-upregulating polynucleotides of the present invention comprise short hairpin RNA (shRNA). In one embodiment, each of the one or more shRNA-upregulating telomerase polynucleotides of the present invention comprises a first nucleotide sequence and a second nucleotide sequence, wherein the second nucleotide sequence and the first nucleotide sequence are inversely complementary. In one embodiment, the nucleotide sequence of the first nucleotide sequence of each of the one or more shRNAs upregulating telomerases of the present invention is at least about 80%, about 85%, about 90%, about 95%, or about 100% identical to SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, or SEQ ID NO. 39. In one embodiment, the shRNA of the present invention further comprises a loop structure connecting a first nucleotide sequence and a second nucleotide sequence. In one embodiment, the loop structure of the upregulated telomerase shRNA of the present invention comprises a nucleotide sequence of about 2 to 9 base pairs (bp), for example, about 2, 3, 4, 5, 6, 7, 8, or 9 base pairs. In one embodiment, the nucleotide sequence of the loop structure of the upregulated telomerase shRNA of the present invention has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 43, or SEQ ID NO. 44. In one embodiment, the loop structure can be cleaved in the RNA interference pathway.

[0058] SEQ ID NO. 40: UUGC

[0059] SEQ ID NO. 41: CCUGACCCA

[0060] SEQ ID NO. 42: AAGCACA

[0061] SEQ ID NO. 43: UGUGCUU

[0062] SEQ ID NO. 44: UUG

[0063] In one embodiment, one or more telomerase-upregulating polynucleotides of the present invention comprise a first short hairpin RNA (shRNA) and a second short hairpin RNA (shRNA), wherein the nucleotide sequence of the first nucleotide sequence of the first shRNA is at least about 80%, about 85%, about 90%, about 95%, or about 100% identical to SEQ ID NO. 45, and the nucleotide sequence of the first nucleotide sequence of the second shRNA is at least about 80%, about 85%, about 90%, about 95%, or about 100% identical to SEQ ID NO. 46.

[0064] SEQ ID NO. 45: TCGGCCAAAATGAATGGGCAGTTGCCTGCCATTCATTTTGGCCGA SEQ ID NO. 46: TTAATTAGAAGATCTAAATGAGCAATCATTTAGATCTTCTAATTAA In one embodiment, the polynucleotide upregulating telomerase in any embodiment of the present invention further comprises N-acetylgalactosamine (GalNAc), wherein the one or more polynucleotides upregulating telomerase of the present invention bind to GalNAc. In one embodiment, the small interfering RNA (siRNA) upregulating telomerase in any embodiment of the present invention further comprises GalNAc, wherein the one or more siRNAs upregulating telomerase of the present invention bind to GalNAc. In one embodiment, the short hairpin RNA (shRNA) upregulating telomerase in any embodiment of the present invention further comprises GalNAc, wherein the one or more shRNAs upregulating telomerase of the present invention bind to GalNAc.

[0065] In one embodiment, the telomerase-upregulating polynucleotide of any embodiment of the present invention upregulates the telomerase activity of a subject by at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 500%, about 750%, or about 1000%. In one embodiment, the telomerase-upregulating polynucleotide of any embodiment of the present invention restores the telomerase activity of a subject to at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the normal telomerase activity of the subject in a healthy state. In one embodiment, the telomerase-upregulating polynucleotide of any embodiment of the present invention restores the telomerase activity of a subject to at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the normal telomerase activity of a healthy human. In one embodiment, the telomerase-upregulating polynucleotide of any embodiment of the present invention restores the telomerase activity of a subject to at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the normal telomerase activity of a healthy human with the same biostatistical characteristics (e.g., age, sex, height, weight, or combinations thereof) as the subject.

[0066] In one embodiment, the polynucleotide that upregulates telomerase in any embodiment of the present invention upregulates the expression of TERT, TERC, or a combination thereof in the subject by at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 500%, about 750%, or about 1000%. In one embodiment, the telomerase-upregulating polynucleotide of any embodiment of the present invention restores the expression of TERT, TERC, or a combination thereof in a subject to at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the normal expression levels of TERT, TERC, or a combination thereof in a healthy human being. In one embodiment, the telomerase-upregulating polynucleotide of any embodiment of the present invention restores the expression of TERT, TERC, or a combination thereof in a subject to at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the normal expression levels of TERT, TERC, or a combination thereof in a healthy human being. In one embodiment, the polynucleotide that upregulates telomerase in any embodiment of the present invention restores the expression of TERT, TERC, or a combination thereof in the subject to at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the normal expression levels of TERT, TERC, or a combination thereof in an average healthy human with the same biostatistical characteristics (such as age, sex, height, weight, etc. or combinations thereof) as the subject.

[0067] The present invention also provides a pharmaceutical composition comprising nanoparticles encapsulating one or more polynucleotides that upregulate telomerase, as described in any embodiment of the present invention. In one embodiment, the nanoparticles of the present invention comprise chemical nanoparticles, such as, but not limited to, lipid nanoparticles, polymer nanoparticles, lipid-polymer hybrid nanoparticles, and biological nanoparticles, such as, but not limited to, liposomes, exosomes, viruses, or virus-like particles.

[0068] In one embodiment, the nanoparticles comprise exosomes. In one embodiment, the exosomes encapsulate one or more fusion proteins and one or more cargo RNAs. In one embodiment, the cargo RNA comprises pack RNA and a packing domain, wherein the pack RNA comprises one or more telomerase-upregulating polynucleotides as described in any embodiment of the invention, and wherein the packing domain is capable of binding to the packing protein of the fusion protein. In one embodiment, the fusion protein comprises an exosome-associated transmembrane protein fused to the packing protein. In one embodiment, the exosome-associated transmembrane protein comprises CD9, CD37, CD53, CD63, CD68, CD81, CD82, LAMP-1, LAMP-2A, LAMP-2B, LAMP-2C, lactadherin, or PTGFRN. In one embodiment, the packing protein comprises U1a protein. In one embodiment, the packaging protein includes an RNA-binding protein capable of binding to the packaging domain, thereby enabling the cargo RNA to bind to the fusion protein via the RNA-binding protein and the packaging domain.

[0069] In one embodiment, the nanoparticles comprise exosomes prepared by an exosome-based packaging and delivery system. In one embodiment, the system comprises a low-immunogenic exosome-based packaging and delivery system. In one embodiment, the exosome-based RNA packaging and delivery system comprises exosome-producing cells, cargo RNA plasmids, and one or more fusion protein bodies, wherein the cargo RNA plasmids encode packaging RNA, the packaging RNA comprising one or more telomerase-upregulating polynucleotides as described in any embodiment of the invention, and a packaging domain capable of binding to a packaging protein of the fusion protein, the fusion protein being encoded by one or more fusion protein bodies. In one embodiment, the fusion protein expressed by the exosome-producing cells according to the fusion protein body comprises an exosome-associated transmembrane protein fused to the packaging protein. In one embodiment, the exosome-associated transmembrane protein includes CD9, CD37, CD53, CD63, CD68, CD81, CD82, LAMP-1, LAMP-2A, LAMP-2B, LAMP-2C, lactobacin, or PTGFRN. In one embodiment, the packaging protein includes an RNA-binding protein. In one embodiment, the packaging protein is endogenous to the subject. Various embodiments of recombinant fusion proteins comprising exosome-associated transmembrane proteins and packaging proteins are disclosed in PCT application No. PCT / US2021 / 026892, filed April 12, 2021, the contents of which are incorporated herein by reference in their entirety. In one embodiment, the packaging protein includes the U1a protein.

[0070] In one embodiment, the one or more fusion proteins comprise the CD63-U1a protein. In one embodiment, the nucleotide sequence encoding the plasmid of the CD63-U1a fusion protein of the present invention has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with SEQ ID NO. 47. In one embodiment, the amino acid sequence of the CD63-U1a fusion protein has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity with SEQ ID NO. 48. In one embodiment, the one or more fusion proteins comprise the CD81-U1a protein. In one embodiment, the nucleotide sequence encoding the plasmid of the CD81-U1a fusion protein of the present invention has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with SEQ ID NO. 49. In one embodiment, the amino acid sequence of the CD81-U1a fusion protein has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 50. In one embodiment, the one or more fusion proteins comprise PTGFRN-U1a protein. In one embodiment, the nucleotide sequence encoding the plastid of the PTGFRN-U1a fusion protein of the present invention has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity to SEQ ID NO. 51. In one embodiment, the amino acid sequence of the PTGFRN-U1a fusion protein has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 52.

[0071] SEQ ID NO. 47 (nt CD63-U1a): The uppercase sequence represents CD63, the lowercase sequence represents the connector sequence, and the bold sequence represents U1 small nucleus ribonucleoprotein polypeptide A (U1 snRNP A).

[0072] SEQ ID NO. 48 (aa CD63-U1a): MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVFRDKVMSEFNNNFRQQMENYPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCI NVTVGCGINFNEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGIVFACCLVKSIRSGYEVMefggggsMAVPETRPNHTIYINNLNEKIKKDELKKSLYAIFSQFGQILDILVSRSLKMRGQAFVIFKEVSSATNALRSMQGFPFYDKPMRIQYAKTDSDIIAKMK The uppercase sequence represents CD63, the lowercase sequence represents the connector sequence, and the bold sequence represents U1 small nucleus ribonucleoprotein polypeptide A (U1 snRNP A).

[0073] SEQ ID NO. 49 (nt CD81-U1a): The uppercase sequence represents CD81, the lowercase sequence represents the connector sequence, and the bold sequence represents U1 small nucleus ribonucleoprotein polypeptide A (U1 snRNP A).

[0074] SEQ ID NO. 50 (aa CD81-U1a): MSGLRSRAQASGVEGCTKCIKYLLFVFNFVFWLAGGVILGVALWLRHDPQTTNLLYLELGDKPAPNTFYVGIYILIAVGAVMMFVGFLGCYGAIQESQCLLGTFFTCLVILFACEVAAGIWGFVNKDQIAKDVKQFYDQALQQAVVDDDANNAKAVVKTFHETLDCCGSSTLTAL TTSVLKNNLCPSGSNIISNLFKEDCHQKIDDLFSGKLYLIGIAAIVVAVIMIFEMILSMVLCCGIRNSSVYEFGGGGSMAVPETRPNHTIYINNLNEKIKKDELKKSLYAIFSQFGQILDILVSRSLKMRGQAFVIFKEVSSATNALRSMQGFPFYDKPMRIQYAKTDSDIIAKMK SEQ ID NO. 51 (nt PTGFRN-U1a): The uppercase sequence represents PTGFRN, the lowercase sequence represents the connector sequence, and the bold sequence represents U1 small nucleus ribonucleoprotein polypeptide A (U1 snRNP A).

[0075] SEQ ID NO. 52 (aa PRGFRN-U1a): MGRLASRPLLLALLSLALCRGRVVRVPTATLVRVVGTELVIPCNVSDYDGPSEQNFDWSFSSLGSSFVELASTWEVGFPAQLYQERLQRGEILLRRTANDAVELHIKNVQPSDQGHYKCSTPSTDATVQGNYEDTVQVKVLADSLHVGPSARPPPSLSLREGEPFELRCTAASASPLHTHLALLWEVHRGPARRSVLALTHEGRFHPGLGYEQRYHSGDVRLDTVGSDAYRLSVSRALSADQGSYRCIVSEWIAEQGNWQEIQEKAVEVATVVIQPSVLRAAVPKNVSVAEGKELDLTCNITTDRADDVRPEVTWSFSRMPDSTLPGSRVLARLDRDSLVHSSPHVALSHVDARSYHLLVRDVSKENSGYYYCHVSLWAPGHNRSWHKVAEAVSSPAGVGVTWLEPDYQVYLNASKVPGFADDPTELACRVVDTKSGEANVRFTVSWYYRMNRRSDNVVTSELLAVMDGDWTLKYGERSKQRAQDGDFIFSKEHTDTFNFRIQRTTEEDRGNYYCVVSAWTKQRNNSWVKSKDVFSKPVNIFWALEDSVLVVKARQPKPFFAAGNTFEMTCKVSSKNIKSPRYSVLIMAEKPVGDLSSPNETKYIISLDQDSVVKLENWTDASRVDGVVLEKVQEDEFRYRMYQTQVSDAGLYRCMVTAWSPVRGSLWREAATSLSNPIEIDFQTSGPIFNASVHSDTPSVIRGDLIKLFCIITVEGAALDPDDMAFDVSWFAVHSFGLDKAPVLLSSLDRKGIVTTSRRDWKSDLSLERVSVLEFLLQVHGSEDQDFGNYYCSVTPWVKSPTGSWQKEAEIHSKPVFITVKMDVLNAFKYPLLIGVGLSTVIGLLSCLIGYCSSHWCCKKEVQETRRERRRLMSMEMDEFGGGGSMAVPETRPNHTIYINNLNEKIKKDELKKSLYAIFSQFGQILDILVSRSLKMRGQAFVIFKEVSSATNALRSMQGFPFYDKPMRIQYAKTDSDIIAKMK In one embodiment, the cargo RNA comprises packaging RNA and a packaging domain, wherein the packaging RNA comprises one or more telomerase-upregulating polynucleotides as described in any embodiment of the present invention, and the packaging domain is capable of binding to the packaging protein of the fusion protein. In one embodiment, the packaging domain comprises an UR domain or an L2 domain. In one embodiment, the nucleotide sequence of the UR domain is at least about 80%, about 85%, about 90%, about 95%, or about 100% identical to SEQ ID NO. 53, and the nucleotide sequence of the L2 domain is at least about 80%, about 85%, about 90%, about 95%, or about 100% identical to SEQ ID NO. 54.

[0076] In one embodiment, the packaging domain further comprises a stabilizing domain. In one embodiment, the stabilizing domain comprises an OH domain and / or a MorrisMotif domain, wherein the nucleotide sequence of the OH domain is at least about 80%, about 85%, about 90%, about 95%, or about 100% identical to SEQ ID NO. 55, and the nucleotide sequence of the MorrisMotif domain is at least about 80%, about 85%, about 90%, about 95%, or about 100% identical to SEQ ID NO. 56.

[0077] SEQ ID NO. 53 (UR): AATCATTGCACTCCGGATT

[0078] SEQ ID NO. 54 (L2): AATCATTGCACTCCGGATTT

[0079] SEQ ID NO. 55 (OH): CTGCAGATATCCAGCACAGTGGC

[0080] SEQ ID NO. 56 (MorrisMotif): GCGCAGCGCGCAGCGC

[0081] In one embodiment, the cargo RNA further comprises a SIRLOIN (SINE-derived nuclear RNALOcalizatIoN) nuclear localization sequence, wherein the SIRLOIN is located upstream of the UR sequence or L2 sequence of the cargo RNA. In one embodiment, the nucleotide sequence of the SIRLOIN has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with SEQ ID NO. 57.

[0082] SEQ ID NO. 57 (SIRLOIN): CGCCTCCCGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGA In one embodiment, the exosome-based packaging and delivery system further includes an Ago2 (argonaute2) encoding plasmid. In one embodiment, the Ago2 includes an S387A mutant. Overexpression of Ago2 or its S387A mutant can improve the efficiency of cargo RNA being encapsulated into exosomes in exosome-producing cells.

[0083] In one embodiment, the pharmaceutical composition comprising nanoparticles encapsulating one or more polynucleotides of the present invention that upregulate telomerase further comprises a nanoparticle carrier release enhancer. In one embodiment, the nanoparticle carrier release enhancer enhances the release of the carrier by at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, or at least about 25-fold compared to the same composition without the release enhancer. In one embodiment, the nanoparticle carrier release enhancer comprises a modified myoferlin protein. In one embodiment, the modified myoferlin protein comprises C2F, C2G, a transmembrane domain, or a combination thereof. In one embodiment, the modified myoferlin protein is composed of C2F, C2G, a transmembrane domain, or a combination thereof. In one embodiment, the amino acid sequence of the C2F domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 58. In one embodiment, the amino acid sequence of the C2G domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 59. In one embodiment, the amino acid sequence of the transmembrane domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 60.

[0084] SEQ ID NO. 58 (C2F): QFRELPDSVPQECTVRIYIVRGLELQPQDNGLCDPYIKITLGKKVIEDRDHYIPNTLNPVFGRMYELSCYLPQEKDLKISVYDYDTFTRDEKVGETIIDLENRFLSRFGSHCGIPEEYCVSGV SEQ ID NO. 59 (C2G): PFNITPRKAKKYYLRVIIWNTKDVILDEKSITGEEMSDIYVKGWVPGNEENKQKTDVHYRSLDGEGNFNWRFVFPFDYLPAEQLCIVAKKEHFWSIDQTEFRIPPRLIIQIWDNDKFSLDDYLGFLELDLRH SEQ ID NO. 60 (transmembrane domain): PDLKAMPLKAKTASLFEQKSMKGWWPCYAEKDGARVMAGKVEMTLEILNEKEADERPAGKGRDEPNMNPKLDLPNRPETSFLWFTNPCKTMKFIVWRRFKWVIIGLLFLLLILLLFVAV In one embodiment, the modified muscle Fehling protein comprises C2A, FerA, FerB, DysFN, a transmembrane domain, or a combination thereof. In one embodiment, the modified muscle Fehling protein consists of C2A, FerA, FerB, DysFN, and a transmembrane domain. In one embodiment, the amino acid sequence of the C2A domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 61. In one embodiment, the amino acid sequence of the FerA domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 62. In one embodiment, the amino acid sequence of FerB has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 63. In one embodiment, the DysFN domain comprises DysFN-1, DysFN-2, or a combination thereof. In one embodiment, the amino acid sequence of DysFN-1 is at least about 80%, about 85%, about 90%, about 95%, or about 100% identical or similar to SEQ ID NO. 64. In one embodiment, the amino acid sequence of DysFN-2 is at least about 80%, about 85%, about 90%, about 95%, or about 100% identical or similar to SEQ ID NO. 65. In one embodiment, the amino acid sequence of the transmembrane domain is at least about 80%, about 85%, about 90%, about 95%, or about 100% identical or similar to SEQ ID NO. 60.

[0085] SEQ ID NO. 61 (C2A): MLRVIVESASNIPKTKFGKPDPIVSVIFKDEKKKTKKVDNELNPVWNEILEFDLRGIPLDFSSSLGIIVKDFETIGQNKLIGTATVALKDLTGDQSRSLPYKLISLLNERGQDTGATIDLVIGYDPPSAPHPNDLS SEQ ID NO. 62 (FerA): LQTNIEALKSGIQGKIPANQLAELWLKLIDEVIEDTRYTLPLTEGKANVTVLDTQIRK SEQ ID NO. 63 (FerB): WLDKLMQLTEEPQNSMPDIIIWMIRGEKRLAYARIPAHQVLYSTSGENASGKYCGKTQTIFLKYPQEKNNGP SEQ ID NO. 64 (DysFN-1): AVEKKFNSFAEGTFTVFAEMYENQALMFGKWGTSGLVGRHKFSDVTGKIKLKREFFLP SEQ ID NO. 65 (DysFN-2): DPERSLLTEADAGHTEFTDEVYQNESRYPGGDWKPAEDTYTDANGDKAASPSELTCP In one embodiment, the modified myofibril protein of any embodiment of the present invention further comprises C2B, C2C, C2D, C2E domains or combinations thereof. In one embodiment, the amino acid sequence of the C2B domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 66. In one embodiment, the amino acid sequence of the C2C domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 67. In one embodiment, the amino acid sequence of C2D has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 68. In one embodiment, the amino acid sequence of C2E has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 69.

[0086] SEQ ID NO. 66 (C2B): PQDFQIRVRVIEGRQLSGNNIRPVVKVHVCGQTHRTRIKRGNNPFFDELFFYNVNMTPSELMDEIISIRVYNSHSLRADCLMGEFKIDVGFVYDEPGHAVMRKWLLLNDP SEQ ID NO. 67(C2C): TFLLKIYRAEDIPQMDDAFSQTVKEIFGGNADKKNLVDPFVEVSFAGKKVCTNIIEKNANPEWNQVVNLQIKFPSVCEKIKLTIYDWDRLTKNDVVGTTYLHLSKIAASGGEVEDFSSSGTGAASYTVNTGETEVGFVPTFGPCYLNLYGSPREYTGFPDPYDE SEQ ID NO. 68(C2D): TPIVSCNFDRVYIYHLRCYVYQARNLLALDKDSFSDPYAHICFLHRSKTTEIIHSTLNPTWDQTIIFDEVEIYGEPQTVLQNPPKVIMELFDNDQVGKDEFLGRSIFSPVVKLNSEMDITPKLLWHPVMNGDKA SEQ ID NO. 69(C2E): RNMKNFQMASITSPSLVVECGGERVESVVIKNLKKTPNFPSSVLFMKVFLPKEELYMPPLVIKVIDHRQFGRKPVVG In one embodiment, the modified myofibril protein of the present invention does not contain a C2A domain, wherein the amino acid sequence of the C2A domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 61. In one embodiment, the modified myofibril protein of the present invention does not contain a C2B domain, wherein the amino acid sequence of the C2B domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 66. In one embodiment, the modified myofibril protein of the present invention does not contain a C2C domain, wherein the amino acid sequence of the C2C domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 67. In one embodiment, the modified myofibril protein of the present invention does not contain a C2D domain, wherein the amino acid sequence of the C2D domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 68. In one embodiment, the modified myofibril protein of the present invention does not contain a C2E domain, wherein the amino acid sequence of the C2E domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 69. In one embodiment, the modified myofibril protein of the present invention does not contain a C2F domain, wherein the amino acid sequence of the C2F domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 58. In one embodiment, the modified myofeline protein of the present invention does not contain a C2G domain, wherein the amino acid sequence of the C2G domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity with SEQ ID NO. 59. In one embodiment, the modified myofeline protein of the present invention does not contain a FerA domain, wherein the amino acid sequence of the FerA domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity with SEQ ID NO. 62. In one embodiment, the modified myofeline protein of the present invention does not contain a FerB domain, wherein the amino acid sequence of the FerB domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity with SEQ ID NO. 63. In one embodiment, the modified myofibrin protein of the present invention does not contain a DysFN-1 domain, wherein the amino acid sequence of the DysFN-1 domain is at least about 80%, about 85%, about 90%, about 95%, or about 100% identical or similar to SEQ ID NO. 64.In one embodiment, the modified myofibril protein of the present invention does not contain a DysFN-2 domain, wherein the amino acid sequence of the DysFN-2 domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 65. In one embodiment, the modified myofibril protein of the present invention does not contain a transmembrane domain, wherein the amino acid sequence of the transmembrane domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 60.

[0087] In one embodiment, the amino acid sequence of the modified myofibrin of the present invention is at least about 80%, about 85%, about 90%, about 95%, or about 100% identical or similar to that of SEQ ID NO. 70.

[0088] SEQ ID NO. 70 (C2F-C2G): MVPAPPRQFRELPDSVPQECTVRIYIVRGLELQPQDNGLCDPYIKITLGKKVIEDRDHYIPNTLNPVFGRMYELSCYLPQEKDLKISVYDYDTFTRDEKVGETIIDLENRFLSRFGSHCGIPEEYCVSGVNTW RDQLRPTQLLQNVARFKGFPQPILSEDGSRIRYGGRDYSLDEFEANKILHQHLGAPEERLALHILRTQGLVPEHVETRTLHSTFQPNISQGKLQMWVDVFPKSLGPPGPPFNITPRKAKKYYLRVIIWNTKDVI LDEKSITGEEMSDIYVKGWVPGNEENKQKTDVHYRSLDGEGNFNWRFVFPFDYLPAEQLCIVAKKEHFWSIDQTEFRIPPRLIIQIWDNDKFSLDDYLGFLELDLRHTIIPAKSPEKCRLDMIPDLKAMNPLKA KTASLFEQKSMKGWWPCYAEKDGARVMAGKVEMTLEILNEKEADERPAGKGRDEPNMNPKLDLPNRPETSFLWFTNPCKTMKFIVWRRFKWVIIGLLFLLLILLLFVAVLLYSLPNYLSMKIVKPNVYPYDVPDYA This invention also provides a protein-coding polynucleotide of modified myofibril protein, which encodes the protein of the modified myofibril protein in any embodiment of this invention. In one embodiment, the protein-coding polynucleotide of the modified myofibril protein encodes a C2A domain, a C2B domain, a C2C domain, a C2D domain, a C2E domain, a C2F domain, a C2G domain, a FerA domain, a FerB domain, a DysFN-1 domain, a DysFN-2 domain, a transmembrane domain, or a combination thereof. In one embodiment, the C2A domain is encoded by a polynucleotide, wherein the nucleotide sequence of the polynucleotide has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with SEQ ID NO. 71. In one embodiment, the C2B domain is encoded by a polynucleotide, wherein the nucleotide sequence of the polynucleotide has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with SEQ ID NO. 72. In one embodiment, the C2C domain is encoded by a polynucleotide, wherein the nucleotide sequence of the polynucleotide is at least about 80%, about 85%, about 90%, about 95%, or about 100% identical to SEQ ID NO. 73. In one embodiment, the C2D domain is encoded by a polynucleotide, wherein the nucleotide sequence of the polynucleotide is at least about 80%, about 85%, about 90%, about 95%, or about 100% identical to SEQ ID NO. 74. In one embodiment, the C2E domain is encoded by a polynucleotide, wherein the nucleotide sequence of the polynucleotide is at least about 80%, about 85%, about 90%, about 95%, or about 100% identical to SEQ ID NO. 75. In one embodiment, the C2F domain is encoded by a polynucleotide, wherein the nucleotide sequence of the polynucleotide is at least about 80%, about 85%, about 90%, about 95%, or about 100% identical to SEQ ID NO. 76. In one embodiment, the C2G domain is encoded by a polynucleotide, wherein the nucleotide sequence of the polynucleotide is at least about 80%, about 85%, about 90%, about 95%, or about 100% identical to SEQ ID NO. 77. In one embodiment, the FerA domain is encoded by a polynucleotide, wherein the nucleotide sequence of the polynucleotide is at least about 80%, about 85%, about 90%, about 95%, or about 100% identical to SEQ ID NO. 78. In one embodiment, the FerB domain is encoded by a polynucleotide, wherein the nucleotide sequence of the polynucleotide is at least about 80%, about 85%, about 90%, about 95%, or about 100% identical to SEQ ID NO. 79.In one embodiment, the DysFN-1 domain is encoded by a polynucleotide, wherein the nucleotide sequence of the polynucleotide is at least about 80%, about 85%, about 90%, about 95%, or about 100% identical to SEQ ID NO. 80. In one embodiment, the DysFN-2 domain is encoded by a polynucleotide, wherein the nucleotide sequence of the polynucleotide is at least about 80%, about 85%, about 90%, about 95%, or about 100% identical to SEQ ID NO. 81. In one embodiment, a transmembrane domain is encoded by a polynucleotide, wherein the nucleotide sequence of the polynucleotide is at least about 80%, about 85%, about 90%, about 95%, or about 100% identical to SEQ ID NO. 82.

[0089] SEQ ID NO. 71 (nt C2A): ATGCTGCGAGTGATTGTGGAATCTGCCAGCAATATCCCTAAAACGAAATTTGGCAAGCCGGATCCTATTGTTTCTGTCATTTTTAAGGATGAGAAAAAAGAAAACAAAGAAAGTTGATAATGAATTGAACCCTGTCTGGAATGAGATTTTGGAGTTTGACTTGAGGGGTATACCACTGGACTTTTCATCTTCCCTTGGGATTATT GTGAAAGATTTTGAGACAATTGGACAAAATAAATTAATTGGCACGGCGACTGTAGCCCTGAAGGACCTGACTGGTGACCAGAGCAGATCCCTGCCGTACAAGCTGATCTCCCTGCTAAATGAAAGAGGGCAAGATACTGGGGCCACCATTGACTTGGTGATCGGCTATGATCCGCCTTCTGCTCCACATCCAAATGACCTGAGC SEQ ID NO. 72 (nt C2B): CCACAGGACTTCCAGATCCGCGTCCGAGTGATTGAGGGCCGACAGTTAAGTGGCAACAACATAAGGCCTGTGGTCAAAGTTCACGTCTGTGGCCAGACACACCGAACAAGAATCAAGAGAGGAAACAACCCTTTTTTTGATGAGTTGTTTTTCTACAATGTCAAC ATGACCCCTTCTGAATTGATGGATGAGATCATCAGCATCCGGGTTTATAATTCTCACTCTCTGCGGGCAGATTGTCTGATGGGGGAATTTAAGATTGATGTTGGATTTGTTTATGATGAACCTGGCCATGCTGTCATGAGAAAGTGGCTTCTTCTCAATGACCCG SEQ ID NO. 73 (nt C2C): ACCTTCTTGCTGAAAATCTACCGAGCTGAGGACATCCCCCAGATGGATGATGCCTTCTCACAGACAGTAAAGGAAATATTTGGAGGCAATGCAGATAAGAAAAATCTCGTGGATCCTTTTGTAGAAGTTTCCTTTGCTGGAAGAAGGTTTGTACAAACATAATTGAGAAGAATGCAAACCCAGAGGTGGAATCAGGTCGTCAATCTTCAGATCAAGTTTCCTTCAGTGTGTGAAAAAATAAAACTA ACAATATATGACTGGGACCGTCTTACTAAATGATGTAGTTGGAACAACATATCTACACCTCTCTAAAATTGCTGCCTCTGGTGGGGAAGTGGAAGATTTCTCATCTTCGGGAACTGGGGCTGCATCATATACAGTAAACACAGGAGAAACAGAGGTAGGCTTTGTTCCAACGTTTGGACCTTGTTACCTGAATCTTTATGGAAGCCCCAGAGAGGTACACGGGATTCCCAGACCCCTATGATGAG SEQ ID NO. 74 (nt C2D): ACCCCCATTGTTTCCTGCAATTTTGACAGAGTCTACATCTACCATCTGCGCTGCTATGTCTATCAAGCCAGAAACCTCTTGGCTTTAGATAAGGATAGCTTTTCAGATCCATATGCTCATATCTGTTTCCTCCATCGGAGCAAAACCACTGAGATCATCCATTCAACCCTGAATCCCACGTGGGACCAAACAATTATATTCGATGAAGTTGAAATCTATGGGGAACCCCAAACAGTTCTACAGAATCCACCCAAAGTTATCATGGAACTTTTTGACAATGACCAAGTGGGCAAAGATGAATTTTTAGGACGAAGCATTTTCTCTCCTGTGGTGAAACTGAACTCAGAAATGGACATCACACCCAAACTTCTCTGGCACCCAGTAATGAATGGAGACAAAGCC SEQ ID NO. 75(nt C2E): AGAAATATGAAAAACTTCCAGATGGCTTCTATCACATCCCCCAGTCTTGTTGTGGAGTGTGGAGGAGAAAGGGTGGAATCGGTGGTGATCAAAAACCTTAAGAAGACACCCAACTTTCCAAGTTCTGTTCTCTTCATGAAAGTGTTCTTGCCCAAGGAGGAATTGTACATGCCCCCACTGGTGATCAAGGTCATCGACCACAGGCAGTTTGGGCGGAAGCCTGTCGTCGGC SEQ ID NO. 76(nt C2F): CAGTTTCGGGAATTACCTGACAGCGTCCCACAGGAATGCACGGTTAGGATTTACATTGTTCGAGGCTTAGAGCTCCAGCCCCAGGACAACAATGGCCTGTGTGACCCTTACATAAAAATAACACTGGGCAAAAAAGTCATTGAAGACCGAGATCACTACATTCCCAACACTCTCAACCCAGTCTTTGGCAGGATGTACGAACTGAGCTGCTACTTACCTCAAGAAAAAGACCTGAAAATTTCTGTCTATGATTATGACACCTTTACCCGGGATGAAAAAGTAGGAGAGACAATTATTGATCTGGAAAACCGATTCCTTTCCCGCTTTGGGTCCCACTGCGGCATACCAGAGGAGTACTGTGTTTCTGGAGTC SEQ ID NO. 77(nt C2G): CCTTTCAACATCACACCCCGGAAAGCCAAGAAATACTACCTGCGTGTGATCATCTGGAACACCAAGGATGTTATCTTGGATGAGAAAAGCATCACAGGAGAGGAAATGAGTGACATCTACGTCAAAGGCTGGGTTCCTGGCAATGAAGAAAACAAACAGAAAACAGATGTCCATTACAGATCTTTGGATGGTGAAGGGAATTTTAACTGGCGATTTGTTTTCCCGTTTGACTACCTTCCAGCCGAACAACTCTGTATCGTTGCGAAAAAAGAGCATTTCTGGAGTATTGACCAAACGGAATTTCGAATCCCACCCAGGCTGATCATTCAGATATGGGACAATGACAAGTTTTCTCTGGATGACTACTTGGGTTTCCTAGAACTTGACTTGCGTCAC SEQ ID NO. 78(nt FerA): CTGCAAACAAATATAGAAGCTCTAAAATCAGGGATACAAGGTAAAATTCCTGCAAACCAGCTGGCTGAATTGTGGCTGAAGCTGATAGATGAAGTTATAGAAGACACGAGATACACGTTGCCTCTCACAGAAGGAAAAGCCAACGTCACAGTTCTCGATACTCAGATCCGAAAG SEQ ID NO. 79(nt FerB): TGGCTTGATAAATTAATGCAGCTGACTGAAGAGCCACAGAACAGCATGCCTGACATCATCATCTGGATGATCCGGGGAGAGAAGAGACTGGCCTATGCACGAATTCCCGCACATCAGGTCTTGTACTCCACCAGTGGTGAGAATGCATCTGGAAAATACTGTGGGAAAACCCAAACCATCTTTCTGAAGTATCCACAGGAGAAAAACAACGGGCCA SEQ ID NO. 80(nt DysFN-1): GCTGTGGAGAAGAAGTTTAACAGCTTCGCAGAAGGAACTTTCACCGTCTTTGCTGAAATGTATGAAAATCAAGCTCTCATGTTTGGAAAATGGGGTACTTCTGGATTAGTAGGACGTCATAAGTTTTCTGATGTCACAGGAAAAATAAAACTCAAGAGGGAATTTTTTCTGCCT SEQ ID NO. 81(nt DysFN-2): GATCCTGAAAGAAGCTTGCTGACTGAGGCAGATGCAGGTCACACGGAGTTCACTGATGAAGTCTACCAGAACGAGAGCCGCTACCCCGGGGGCGACTGGAAGCCGGCCGAGGACACCTACACGGATGCGAACGGCGATAAAGCAGCATCACCCAGCGAGTTGACTTGTCCT SEQ ID NO. 82(nt TM): CCGGACCTCAAAGCCATGAACCCCCTTAAAGCCAAGACAGCCTCCCTCTTTGAGCAGAAGTCCATGAAAGGATGGTGGCCATGCTACGCAGAGAAAGATGGCGCCCGCGTAATGGCTGGGAAAGTGGAGATGACATTGGAAATCCTCAACGAGAAGGAGGCCGACGAGAGGCCAGCCG GGAAGGGGCGGGACGAACCCAACATGAACCCCAAGCTGGACTTACCAAATCGACCAGAAACCTCCTTCCTTGGTTCACCAACCCATGCAAGACCATGAAGTTCATCGTGTGGCGCCGCTTTAAGTGGGTCATCGGCTTGCTGTTCCTGCTTATCCTGCTGCTCTTCGTGGCCGTG In one embodiment, the modified myofibril protein of the present invention further comprises a connexin 43 protein, wherein the dual protein comprising the modified myofibril protein and the connexin 43 protein can be used to significantly improve the efficacy and delivery efficiency of RNA or nucleic acid cargoes. In one embodiment, the connexin 43 protein comprises the S368A mutation. In one embodiment, the amino acid sequence of the connexin 43 protein of the present invention has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 83.

[0090] SEQ ID NO. 83 (connector protein 43): MATTMGDWSALGKLLDKVQAYSTAGGKVWLSVLFIFRILLLGTAVESAWGDEQSAFRCNTQQPGCENVCYDKSFPISHVRFWVLQIIFVSVPTLLY LAHVFYVMRKEEKLNKKEEELKVAQTDGVNVDMHLKQIEIKKFKYGIEEHGKVKMRGGLLRTYIISILFKSIFEVAFLLIQWYIYGFSLSAVYTCKR DPCPHQVDCFLSRPTEKTIFIIFMLVVSLVSLALNIIELFYVFFKGVKDRVKGKSDPYHATSGALSPAKDCGSQKYAYFNGCSSPTAPLSPMSPPGYKLVTGDRNNSSCRNYNKQASEQNWANYSAEQNRMGQAGSTISNSHAQPFDFPDDNQNSKKLAAGHELQPLAIVDQRPSSRAASRASSRPRPDDLEI The present invention further provides a dual protein polynucleotide encoding a dual protein comprising a modified myofelin protein of the present invention fused with an S368A mutant of a protein of linker protein 43, wherein the nucleotide sequence of the dual protein polynucleotide is at least about 80%, about 85%, about 90%, about 95%, or about 100% identical to SEQ ID NO. 84.

[0091] SEQ ID NO. 84: In one embodiment, various components include the fusion protein of the present invention or any variant thereof, the Ago2 protein of the present invention or any variant thereof, the modified myofiber ferring protein of the present invention or any variant thereof, and the linker protein 43 of the present invention or any variant thereof. These components may be integrated or encapsulated in all possible combinations into nanoparticles encapsulating one or more telomerase-upregulating polynucleotides of the present invention to enhance the delivery and uptake of the nanoparticles in a subject. In one embodiment, the nanoparticles encapsulating one or more telomerase-upregulating polynucleotides of the present invention further include the fusion protein of the present invention or any variant thereof. In one embodiment, the nanoparticles encapsulating one or more telomerase-upregulating polynucleotides of the present invention further include the Ago2 protein of the present invention or any variant thereof. In one embodiment, the nanoparticles encapsulating one or more telomerase-upregulating polynucleotides of the present invention further include the modified myofiber ferring protein of the present invention or any variant thereof. In one embodiment, the nanoparticles encapsulating one or more telomerase-upregulating polynucleotides of the present invention further include the linker protein 43 of the present invention or any variant thereof. In one embodiment, the nanoparticles encapsulating one or more telomerase-upregulating polynucleotides of the present invention do not include the fusion protein of the present invention or any variant thereof. In one embodiment, the nanoparticles encapsulating one or more telomerase-upregulating polynucleotides of the present invention do not contain the Ago2 protein or any variant thereof of the present invention. In one embodiment, the nanoparticles encapsulating one or more telomerase-upregulating polynucleotides of the present invention do not contain the modified myofiber-feline protein or any variant thereof of the present invention. In one embodiment, the nanoparticles encapsulating one or more telomerase-upregulating polynucleotides of the present invention do not contain the linker protein 43 or any variant thereof of the present invention. In one embodiment, when the nanoparticles contain exosomes of one or more telomerase-upregulating polynucleotides of the present invention, and a fusion protein containing a transmembrane protein associated with the exosome fused to the packaging protein of the present invention, the one or more telomerase-upregulating polynucleotides are fused to the packaging domain of the present invention or any variant thereof. In one embodiment, the packaging domain of the present invention or any variant thereof contains a UR domain, an L2 domain, an OH domain, a MorrisMotif domain, a nuclear localization sequence SIRLOIN, or a combination thereof.

[0092] In one embodiment, nanoparticles encapsulating polynucleotides that upregulate telomerase in any embodiment of the present invention upregulate the telomerase activity of a subject by at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 500%, about 750%, or about 1000%. In one embodiment, nanoparticles encapsulating polynucleotides that upregulate telomerase in any embodiment of the present invention restore the telomerase activity of a subject to at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the normal telomerase activity of the subject when healthy. In one embodiment, nanoparticles encapsulating polynucleotides that upregulate telomerase in any embodiment of the present invention restore the telomerase activity of a subject to at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the normal telomerase activity of an average healthy human. In one embodiment, nanoparticles encapsulating polynucleotides that upregulate telomerase in any embodiment of the present invention restore the telomerase activity of a subject to at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the normal telomerase activity of an average healthy human with the same biostatistical characteristics (e.g., age, sex, height, weight, etc. or combinations thereof) as the subject.

[0093] In one embodiment, nanoparticles of the polynucleotides that encapsulate the upregulating telomerase of any embodiment in any embodiment of the present invention upregulate the expression of TERT, TERC, or a combination thereof in a subject by at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 500%, about 750%, or about 1000%. In one embodiment, nanoparticles encapsulating the polynucleotides that upregulate telomerase in any embodiment of the present invention restore the expression of TERT, TERC, or a combination thereof in a subject to at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the normal expression levels of TERT, TERC, or a combination thereof in a healthy subject. In one embodiment, nanoparticles encapsulating the polynucleotides that upregulate telomerase in any embodiment of the present invention restore the expression of TERT, TERC, or a combination thereof in a subject to at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the normal expression levels of TERT, TERC, or a combination thereof in an average healthy human. In one embodiment, nanoparticles encapsulating the polynucleotides that upregulate telomerase in any embodiment of the present invention restore the expression of TERT, TERC, or a combination thereof in a subject to at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the normal expression levels of TERT, TERC, or a combination thereof in an average healthy human with the same biostatistical characteristics (e.g., age, sex, height, weight, etc. or combinations thereof) as the subject.

[0094] This invention provides a method for increasing the expression of TERT, TERC, or a combination thereof in a subject, comprising administering to the subject a therapeutically effective amount of one or more polynucleotides of the invention that upregulate telomerase. In one embodiment, the subject has been diagnosed with a telomerase-related disease. In one embodiment, the telomerase-related disease includes telomere disease, aging-related disease, cellular aging-related disease, or a combination thereof. In one embodiment, the telomerase-related disease includes chronic lung disease associated with TERT or TERC, such as, but not limited to, chronic obstructive pulmonary disease, lung cancer, or idiopathic pulmonary fibrosis; chronic liver disease associated with TERT or TERC, such as, but not limited to, liver fibrosis or cirrhosis; or bone marrow failure (BMF) associated with TERT or TERC, such as, but not limited to, congenital dyskeratosis (DC) or aplastic anemia.

[0095] The present invention also provides a method for treating telomerase-related diseases in a subject, comprising the step of increasing telomerase expression in the subject. In one embodiment, the step of increasing telomerase expression in the subject comprises increasing the expression of TERT, TERC, or a combination thereof in the subject. In one embodiment, the step of increasing the expression of TERT, TERC, or a combination thereof comprises administering to the subject a therapeutically effective amount of one or more telomerase-upregulating polynucleotides of any embodiment of the present invention. In one embodiment, the step of increasing telomerase expression comprises administering to the subject nanoparticles of one or more telomerase-upregulating polynucleotides encapsulated in any embodiment of the present invention.

[0096] In one embodiment, telomerase-related diseases include telomere disorders, aging-related diseases, cellular aging-related diseases, or combinations thereof. In one embodiment, telomerase-related diseases include chronic lung diseases associated with TERT or TERC, such as, but not limited to, chronic obstructive pulmonary disease, lung cancer, or idiopathic pulmonary fibrosis; chronic liver diseases associated with TERT or TERC, such as, but not limited to, liver fibrosis or cirrhosis; or bone marrow failure (BMF) associated with TERT or TERC, such as, but not limited to, congenital dyskeratosis (DC) or aplastic anemia.

[0097] In one embodiment, a method of treating a subject with telomerase-related disease includes the step of administering a therapeutically effective amount of a pharmaceutical composition of one or more telomerase-upregulating polynucleotides of the present invention. In another embodiment, a method of treating telomerase-related disease includes the step of administering a therapeutically effective amount of a pharmaceutical composition of any embodiment of the present invention comprising nanoparticles encapsulating one or more telomerase-upregulating polynucleotides. The pharmaceutical compositions of the present invention can be administered in different ways depending on whether local or systemic treatment is desired, or depending on the area of ​​treatment. Administration can be local (including ophthalmic, vaginal, rectal, intranasal, and transdermal), oral, or parenteral. Parenteral administration can include intravenous infusion, subcutaneous injection, intraperitoneal injection, intramuscular injection, intrathecal administration, or intraventricular administration. The route and site of administration can be selected to enhance the delivery of agents containing site-specific targeting molecules or to target specific locations. For example, intravenous injection can be used to target hepatocytes.

[0098] The pharmaceutical compositions of the present invention can be administered in the form of exosomes according to any embodiment of the present invention. In this specification, the term "exosome" refers to a small vesicle (with a diameter between 20 and 300 nm, preferably 40 and 200 nm) derived from cells, having a membrane structure to enclose its internal space, and which can be formed directly by budding from the cell's plasma membrane or by fusion of late endosomes with the plasma membrane. The exosomes comprise lipids or fatty acids and polypeptides, and further comprise the inhibitory nucleic acids described in this specification as goods. The exosomes can be derived from production cells and can be isolated from the production cells according to their size, density, biochemical parameters, or combinations thereof. Exosomes can be directly loaded with exogenous nucleic acids or drugs by electroporation, lipid transfection, ultrasonic treatment, or contact with calcium chloride. Furthermore, purified exosomes can also be loaded by methods such as electroporation in vitro (exvivo) methods.

[0099] The exosomes of this invention can produce cells cultured in vitro or bodily fluids from a subject. When producing cells cultured in vitro using exosomes, various production cells can be used, such as HEK293 cells, Chinese hamster ovary (CHO) cells, or mesenchymal stem cells (MSCs).

[0100] The pharmaceutical compositions of the present invention can also be formulated by integrating the inhibitory nucleic acid described herein into adenovirus or adeno-associated virus (AAV), or formulated as cell-penetrating peptides, lentiviral vectors, polymers, dendritic polymers, or prepared as siRNA bioconjugates, such as the GalNAc–siRNA conjugate delivery platform.

[0101] In one embodiment, if exosomes or vectors are used as delivery vehicles for siRNA, the candidate siRNA is delivered in the form of shRNA. Both siRNA and shRNA can target and inhibit viruses, and they are functionally equivalent. When the candidate siRNA is delivered in the form of shRNA, it originates from the cellular system and is packaged into exosomes or vectors (AAV or lentiviral vectors) as described above.

[0102] In one embodiment, the shRNA may be provided to an expression cassette containing a promoter sequentially linked to the siRNA described herein. In various embodiments, the promoter is a polII or polIII promoter, such as the U6 promoter (e.g., the mouse U6 promoter) or the H1 promoter. In various embodiments, the expression cassette further contains a marker gene. In various embodiments, the promoter is a polII promoter. In various embodiments, the activator is a tissue-specific activator. In various embodiments, the promoter is an inducible promoter. In various embodiments, the promoter is a polIII promoter. In various embodiments, the promoter is a U6 or H1 promoter.

[0103] In addition, a vector containing the expression cassette described herein is also provided. Examples of suitable vectors include adenovirus, lentivirus, adeno-associated viral (AAV), poliovirus, herpes simplex virus (HSV), or murine Maloney-based viral vectors. In one embodiment, the vector is an adeno-associated viral (AAV) vector.

[0104] In one embodiment, the shRNA molecule comprises paired RNA sequences and a loop structure located between the paired RNA sequences to form a hairpin structure. The length of the loop structure is variable. In some embodiments, the length of the loop structure is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In some embodiments, the length of the loop structure is 18 nucleotides. The hairpin structure may also include 3' and / or 5' overhanging ends. In some embodiments, the overhanging ends are 3' and / or 5' overhanging ends, and their length is 0, 1, 2, 3, 4, or 5 nucleotides. The nucleotide sequence of the loop structure region can vary, for example, it may be (5'-GCAA-3'), (5'-GCGC-3'), or (5'-TTGC-3'), or other sequences well known to those skilled in the art.

[0105] The pharmaceutical compositions described in this specification can be administered in doses sufficient to inhibit the expression of a target gene or the biological activity of a non-translated target sequence (e.g., a regulatory sequence) in the treated cells, tissues, or organism. The specific dosage of the inhibitory nucleic acid will depend on a variety of factors, such as the route of administration, the physiological characteristics of the subject (including health status), etc. For example, the appropriate dosage of the pharmaceutical compositions containing inhibitory nucleic acids described in this specification can be influenced by a variety of factors, including but not limited to the physiological characteristics of the subject (e.g., age, weight, sex), the progression of a specific coronavirus infection (i.e., pathological state), and other factors readily identifiable by one of ordinary skill in the art. In determining the appropriate dosage, various general factors may be considered, such as those described in *Remington's Pharmaceutical Sciences* (1990), edited by Gennaro et al., Mack Publishing Co., Easton, Pennsylvania, USA; and *Goodman and Gilman's: The Pharmacological Bases of Therapeutics* (1990), edited by Gilman et al., Pergamon Press. The non-limiting examples of applicable doses of the inhibitory nucleic acids described in this specification include dose ranges of 0.01 to 200 mg / kg body weight per day, 1 to 50 mg / kg body weight per day, 1 to 40 mg / kg body weight per day, 1 to 30 mg / kg body weight per day, 1 to 20 mg / kg body weight per day, 1 to 10 mg / kg body weight per day, 1 to 5 mg / kg body weight per day, 1 to 3 mg / kg body weight per day, 1 to 2 mg / kg body weight per day, 0.1 to 1 mg / kg body weight per day, and 0.1 to 0.9 mg / kg body weight per day. The dosage ranges are as follows: 0.1 to 0.8 mg per kilogram of body weight per day, 0.1 to 0.7 mg per kilogram of body weight per day, 0.1 to 0.6 mg per kilogram of body weight per day, 0.1 to 0.5 mg per kilogram of body weight per day, 0.1 to 0.4 mg per kilogram of body weight per day, 0.1 to 0.3 mg per kilogram of body weight per day, 0.1 to 0.2 mg per kilogram of body weight per day, 0.01 to 0.1 mg per kilogram of body weight per day, 0.01 to 0.05 mg per kilogram of body weight per day, 0.01 to 0.02 mg per kilogram of body weight per day, and 0.005 to 0.01 mg per kilogram of body weight per day.

[0106] Those skilled in the art can determine, through routine experiments, the effective and non-toxic dosage of the pharmaceutical composition and / or inhibitory nucleic acid described in this specification for use in a single dose or a series of doses to achieve the desired therapeutic effect.

[0107] Generally, in therapeutic applications, the treatment will continue during the infection, disease state, or symptom. Furthermore, those skilled in the art will understand that the optimal dosage and intervals for individual doses will depend on the nature and severity of the infection, disease state, or symptom being treated, the dosage form, route of administration, and site of application, as well as the characteristics of the individual being treated. Such optimal conditions can also be determined using conventional techniques.

[0108] The pharmaceutical compositions described in this specification are suitable for multiple administrations in many cases. For example, they may be administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, or more. The administration interval may be from about one week to about 12 weeks. In some embodiments, the administration interval may be from about one week to about four weeks. When a subject is repeatedly exposed to a specific pathogen targeted by the pharmaceutical compositions described in this specification, a strategy of periodic re-administration may be applicable.

[0109] Those with general knowledge in the relevant technical field can also determine the optimal treatment course through routine treatment decision tests.

[0110] Techniques applicable to the introduction of the inhibitory nucleic acids described in this specification into cells, tissues, and organisms include various carrier systems, vectors, and reagents. Non-limiting examples include lipid nanoparticles (LNPs), micelles, nucleic acid-lipid particles, lipid complexes, liposomes, nucleic acid polymers, single chemical entity conjugates, virions, virus-like particles (VLPs), or mixtures thereof.

[0111] The pharmaceutical compositions of the present invention can be administered by any suitable manner, such as intravenous, buccal, parenteral, intranasal, oral, sublingual, or topical administration. Therefore, administration methods may include topical, pulmonary (e.g., by inhalation or spraying of aerosols or powders, including the use of a nebulizer), intranasal, intratracheal, epidermal, percutaneous, oral, or parenteral administration. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; or intracranial (e.g., intraparenchymal, subarachnoid, or intraventricular) administration.

[0112] In one embodiment, the pharmaceutical composition is suitable for intranasal administration.

[0113] In one embodiment, the pharmaceutical composition of the present invention can be formulated as a direct-acting nasal spray. In one embodiment, the nasal spray can be self-administered by a subject in a medical setting.

[0114] It should be understood that the above general description and detailed description of the present invention are merely illustrative and explanatory, and should not be construed as limiting the scope of the patent applications of the present invention. Generally, the terminology used in this specification should not be construed as limiting the technology to the specific embodiments disclosed in the specification, unless these terms are explicitly defined in the foregoing detailed description. Therefore, the actual scope of the technology covers the disclosed embodiments and all equivalent implementations and applications.

[0115] example

[0116] Materials and Methods

[0117] siRNA was transfected into cells at a final concentration of 100 nM using Lipofectamine 2000 (L2K). 72 or 96 hours post-transfection, cellular RNA was collected using the Qiagen RNeasy kit and treated with DNase. The resulting RNA was converted to cDNA using a Thermo Fisher reverse transcription kit. Subsequently, using primers listed in Table 1, the differences in gene expression relative to β-actin were assessed by quantitative PCR.

[0118] Table 1. PCR primers used for RNA detection

[0119] result

[0120] Example 1: siRNA activating TERC

[0121] To achieve the goal of activating the TERT / TERC pathway, we investigated the TERC and TERT loci and observed several annotated antisense long non-coding RNAs (lncRNAs), which may be TERC ( Figure 1 ) or TERT ( Figure 2 Endogenous regulatory factors of TERC and TERT expression. Numerous reports in the literature indicate that antisense lncRNAs can control gene expression through various mechanisms, including blocking miRNA targeting protein-coding genes and targeting epigenetic regulatory proteins at specific loci within the gene body. Therefore, we hypothesize that using siRNA to target lncRNAs associated with TERC and TERT may be a feasible method to de-repress TERC and TERT, thereby activating TERC / TERT. To confirm the effectiveness of inhibiting various lncRNAs associated with the TERC locus (… Figure 1 To first activate TERC's ability, we developed several siRNAs (Table 2) to target these hypothesized regulatory lncRNAs. Figure 3These siRNAs target TERC-associated antisense RNAs as a means of "de-repressing" and activating TERC expression. siRNAs were screened using HEK293 cells and analyzed by qRT-PCR (…). Figure 4 We analyzed the effects of these siRNAs on TERC expression. Notably, we found that siRNAs (numbered 2, 10, and 13) increased TERC expression. Figure 4 and Figure 5 ), of which siasTERC2 targets EST CV370609, siasTERC10 targets EST AW207347, AI825849, AW136367, AI380754, and AW293800, while siasTERC13 targets EST BM695101 ( Figure 6 ).

[0122] Table 2. siRNAs targeting TERC-related antisense lncRNAs

[0123] Example 2: siRNA activating TERT

[0124] Next, we evaluated the TERT locus and identified candidate lncRNAs that are antisense to the TERT gene. Figure 2 To confirm the role of unblocking the inhibition of various lncRNAs associated with the TERT locus (). Figure 2 To activate TERT's ability, we designed several siRNAs that target these putative regulatory lncRNAs (Table 3). Figure 7 These siRNAs target TERT-associated antisense RNAs as a means of de-repressing and activating TERT expression. HEK293 cells were treated with siRNAs, and the effects of these siRNAs on TERT expression were determined by qRT-PCR. Figures 8 to 9 Notably, we discovered two siRNAs, E666-si14-1 and E666-si14-2, both of which target the regulatory lncRNA (ENST00000666708.1) and can enhance TERT expression. Figure 10 Overall, these data suggest that targeting regulatory lncRNAs of TERC and TERT with siRNA is a means of activating TERC / TERT expression.

[0125] Example 3: Exosome delivery of TERT / TERC siRNA

[0126] Recently, we modified HEK293 cells to produce stable shRNA exosomes. We modified HEK293 cells to produce shRNA exosomes (shEVs) that target and activate TERC by co-transfection with a dual shRNA expression vector and the RNex packaging system (Table 4). Figure 11 Next, we screened these transfected cells with drugs to produce stable shRNA-producing cells, and then used the Transwell assay method (…). Figure 12 The effects of exosomes produced by the cells on TERC activation were evaluated. Our plastid system comprises packaging plastid p24 and enhancing plastid p54 (Table 4), which is functionally required in all formulations to ultimately enhance shRNA packaging and endosome release. This effect can be assessed by shRNA knockdown and activation of the target gene in recipient cell lines. Figure 13 Stable cell lines containing dual-expressing shRNAs (sh10 and sh13) were established. These shRNAs target TERC regulatory lncRNAs, and the results were verified by qRT-PCR using TERC-specific primers (Table 1). The results showed that they could activate TERC expression in recipient cells. Figure 13 Furthermore, these shRNAs are designed with a 7 bp hairpin loop structure, but can also be used with loop structures of 4 bp, 5 bp, 6 bp, 8 bp, or 9 bp. shRNA 10 targets AW207347, AI825849, AW136367, AI380754, and AW293800, while shRNA 13 targets BM695101. These data indicate that shRNAs targeting regulatory lncRNAs controlling TERC expression can be packaged and delivered to recipient cells via exosomes.

[0127] Table 3. siRNAs targeting TERT-related antisense lncRNAs

[0128] Table 4 lists the plastids required to convert cell lines into shRNA-EV production systems. The plastids were co-transfected at equimolar ratios and screened for drugs to produce stable shRNA-EV production cells.

[0129] Those skilled in the art will understand that modifications can be made to the above examples without departing from their broad inventive concept. Therefore, this invention is not limited to the specific examples disclosed, but is intended to cover modifications consistent with the spirit and scope of the invention, as defined by the appended claims.

Claims

1. A pharmaceutical composition comprising one or more polynucleotides that upregulate telomerase, wherein, The nucleotide sequence of each of the one or more polynucleotides that upregulate telomerase is at least about 80%, about 85%, about 90%, about 95%, or about 100% identical to SEQ ID NO. 26, SEQ ID NO. 29, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 17, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 18, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 39, or combinations thereof.

2. The pharmaceutical composition according to claim 1, wherein, The one or more polynucleotides that upregulate telomerase upregulate the expression levels of TERT, TERC, or combinations thereof by regulating the expression levels of one or more antisense regulators of TERT, TERC, or combinations thereof.

3. The pharmaceutical composition according to claim 2, wherein, The nucleotide sequences of the antisense RNA regulatory factors of TERT, TERC, or combinations thereof are identical to those of AW207347 (SEQ ID NO. 1), AI825849 (SEQ ID NO. 2), AW136367 (SEQ ID NO. 3), AI380754 (SEQ ID NO. 4), AW293800 (SEQ ID NO. 5), BM695101 (SEQ ID NO. 6), CV370609 (SEQ ID NO. 7), MG677549 (SEQ ID NO. 8), BF802688 (SEQ ID NO. 9), AA748707 (SEQ ID NO. 10), AA811084 (SEQ ID NO. 11), AI824948 (SEQ ID NO. 12), AW270031 (SEQ ID NO. 13), AW276315 (SEQ ID NO. 14), and DB345416 (SEQ ID NO. 1). The similarity to 15) or ENST00000666708.1 (SEQ ID NO. 16) is at least about 80%, about 85%, about 90%, about 95%, or about 100%.

4. The pharmaceutical composition according to claim 1, wherein, The one or more polynucleotides that upregulate telomerase include small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozyme, deoxyribozyme, aptamer, or a combination thereof.

5. The pharmaceutical composition according to claim 1, wherein, The one or more telomerase-upregulating polynucleotides comprise one or more first telomerase-upregulating polynucleotides and one or more second telomerase-upregulating polynucleotides, wherein the nucleotide sequence of the one or more first telomerase-upregulating polynucleotides is at least about 80%, about 85%, about 90%, about 95%, or about 100% identical to the nucleotide sequence of SEQ ID NO. 26, and the nucleotide sequence of the one or more second telomerase-upregulating polynucleotides is at least about 80%, about 85%, about 90%, about 95%, or about 100% identical to the nucleotide sequence of SEQ ID NO.

29.

6. The pharmaceutical composition according to claim 1, further comprising nanoparticles, wherein, The nanoparticles encapsulate one or more polynucleotides that upregulate telomerase.

7. The pharmaceutical composition according to claim 6, wherein, The nanoparticles include lipid nanoparticles, polymer nanoparticles, lipid-polymer hybrid nanoparticles, liposomes, exosomes, or viral or virus-like particles.

8. The pharmaceutical composition according to claim 7, further comprising a fusion protein and a packaging domain, wherein, The nanoparticles comprise exosomes, wherein one or more telomerase-upregulating polynucleotides are fused to the packaging domain, wherein the fusion protein comprises an exosome-associated transmembrane protein fused to the packaging protein, wherein the packaging domain comprises an UR domain, an L2 domain, or a combination thereof, and wherein the packaging domain is capable of binding to the packaging protein.

9. The pharmaceutical composition according to claim 8, wherein, The one or more polynucleotides that upregulate telomerase are further fused to the nuclear localization sequence SIRLOIN.

10. The pharmaceutical composition according to claim 8, wherein, The fusion protein comprises CD63-U1a, CD81-U1a, PTGFRN-U1a, or a combination thereof.

11. The pharmaceutical composition according to claim 8, wherein, The exosomes further contain the Ago2 protein or its S387A mutant.

12. The pharmaceutical composition according to claim 8, wherein, The exosomes further contain proteins that have been modified with myofibrin.

13. The pharmaceutical composition according to claim 12, wherein, The modified myofibrin protein comprises the C2F, C2G, or transmembrane domains of the myofibrin protein, or a combination thereof.

14. The pharmaceutical composition according to claim 12, wherein, The modified myofibrin protein further includes a protein of linker protein 43 or its S368A mutant.

15. The pharmaceutical composition according to claim 8, wherein, The exosomes are prepared using an exosome-based packaging and delivery system comprising one or more cargo RNA-encoding plasmids and one or more fusion protein-encoding plasmids, wherein each of the one or more cargo RNA-encoding plasmids encodes one or more polynucleotides upregulated by telomerase fused to the packaging domain, and wherein each of the one or more fusion protein-encoding plasmids encodes the fusion protein, the fusion protein comprising a transmembrane protein associated with the exosome fused to the packaging protein.

16. The pharmaceutical composition according to claim 15, wherein, The exosome-based packaging and delivery system further comprises plasmids encoding a modified myofelin protein, a protein of linker protein 43 or its S368A mutant, or a combination thereof.

17. A method for increasing the expression of TERT, TERC, or a combination thereof in a subject, comprising the step of administering a therapeutically effective amount of the pharmaceutical composition of claim 1 to the subject.

18. The method according to claim 17, wherein, The subjects suffered from telomerase-related diseases, including telomere disease, aging-related diseases, cellular senescence-related diseases, or combinations thereof.

19. The method according to claim 18, wherein, The telomerase-related diseases include chronic lung diseases associated with TERT or TERC, including chronic obstructive pulmonary disease, lung cancer, or idiopathic pulmonary fibrosis; chronic liver diseases associated with TERT or TERC, including liver fibrosis or cirrhosis; or bone marrow failure (BMF) associated with TERT or TERC, including congenital dyskeratosis (DC) or aplastic anemia.

20. A method of treating a subject with telomerase-related disease, comprising administering a therapeutically effective amount of the pharmaceutical composition according to claim 1 to the subject.

21. The method according to claim 20, wherein, The telomerase-related diseases include telomere disease, aging-related diseases, cellular senescence-related diseases, or combinations thereof.

22. The method according to claim 20, wherein, The telomerase-related diseases include chronic lung diseases associated with TERT or TERC, including chronic obstructive pulmonary disease, lung cancer, or idiopathic pulmonary fibrosis; chronic liver diseases associated with TERT or TERC, including liver fibrosis or cirrhosis; or bone marrow failure (BMF) associated with TERT or TERC, including congenital dyskeratosis (DC) or aplastic anemia.

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