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CN-121975759-A - Mitochondrial targeting recombinant extracellular vesicle delivery system and application thereof

CN121975759ACN 121975759 ACN121975759 ACN 121975759ACN-121975759-A

Abstract

The present disclosure provides a mitochondrial targeting catalase or variant thereof, a nucleic acid sequence encoding the mitochondrial targeting catalase or variant thereof, a vector comprising the nucleic acid sequence, a delivery system comprising the mitochondrial targeting catalase or variant thereof and/or the nucleic acid sequence or vector, a pharmaceutical or cosmetic composition comprising the mitochondrial targeting catalase or variant thereof or the nucleic acid sequence or vector or delivery system. The present disclosure also provides a method of manufacturing the delivery system and uses thereof.

Inventors

  • LI MING
  • CHEN YIYOU

Assignees

  • 艾思曼生物科技有限公司

Dates

Publication Date
20260505
Application Date
20260202
Priority Date
20250311

Claims (20)

  1. 1. A catalase or variant thereof, characterized in that the catalase or variant thereof has at least one biological activity of scavenging Reactive Oxygen Species (ROS), reducing oxidative stress, and/or improving visible signs and/or biomarkers of skin photoaging.
  2. 2. The catalase or variant thereof according to claim 1, wherein the catalase or variant thereof is se:Sup>A wild-type catalase or se:Sup>A modified catalase, wherein the modified catalase is selected from at least one of (i) se:Sup>A fusion protein comprising (se:Sup>A) se:Sup>A wild-type catalase or variant thereof, and (b) other functional proteins or polypeptides having specific biological functions, optionally the functional proteins or polypeptides having tissue targeting activity, more optionally having mitochondrial targeting activity, most optionally the fusion protein is obtained based on deletion of the C-terminal amino acid residue "K-se:Sup>A-N-L" of catalase and addition of an N-terminal mitochondrial targeting sequence.
  3. 3. The catalase or variant thereof according to claim 1 or 2, wherein the catalase or variant thereof is selected from at least one of the group consisting of: (i) A mitochondrial targeting catalase having the amino acid sequence shown in SEQ ID No. 2, or a mitochondrial targeting catalase having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to SEQ ID No. 2.
  4. 4. A nucleic acid sequence encoding the catalase or variant thereof according to any one of claims 1-3.
  5. 5. A vector comprising the nucleic acid sequence of claim 4.
  6. 6. A delivery system, the delivery system comprising: (i) A catalase or a variant thereof according to claim 1 to 3, and/or, (Ii) A nucleic acid sequence according to claim 4 or a vector according to claim 5; (iii) A delivery vehicle, wherein the delivery vehicle has the function of delivering catalase or a variant thereof into a specific tissue or cell.
  7. 7. The delivery system of claim 6, wherein the delivery vehicle is selected from at least one of a liposome, an Extracellular Vesicle (EV), and a virus-like particle (VLP).
  8. 8. The delivery system of claim 6, wherein the delivery vehicle is selected from at least one of a generic extracellular vesicle or a modified extracellular vesicle, Alternatively, the extracellular vesicles are universal exosomes or modified exosomes.
  9. 9. The delivery system of claim 8, wherein the extracellular vesicles are harvested from production cells; Alternatively, the extracellular vesicles are exosomes, and the exosomes may be modified to enhance production, exposure duration, tissue-specific targeting, or endosomal escape; More optionally, the exosomes comprise (i) peptides and/or proteins comprising GPI-anchored signal sequences, (ii) peptide/antibody fragment modifications, and/or (iii) other protein modifications.
  10. 10. The delivery system of claim 9, wherein the producer cells overexpress at least one protein or protein fragment selected from the group consisting of CD46, CD52, CD55, CD58, and CD 59.
  11. 11. The delivery system of claim 9, wherein the producer cell is a non-human mammalian cell line or a human cell line.
  12. 12. The delivery system of claim 9, wherein the producer cell is selected from a HEK 293F cell line, a HEK 293T cell line, a stem cell line, or any combination thereof.
  13. 13. A pharmaceutical composition, characterized in that, the pharmaceutical composition comprises: (i) A catalase or a variant thereof according to claim 1 to 3, or, (Ii) The nucleic acid sequence according to claim 4 or the vector according to claim 5, or, (Iii) The delivery system of any one of claims 6-12, and (Iv) Pharmaceutically acceptable excipients.
  14. 14. A cosmetic composition, characterized in that it comprises: (i) A catalase or a variant thereof according to claim 1 to 3, or, (Ii) The nucleic acid sequence according to claim 4 or the vector according to claim 5, or, (Iii) The delivery system of any one of claims 6-12, and (Iv) Acceptable excipients.
  15. 15. A method of manufacturing a delivery system according to any one of claims 6 to 12, wherein the delivery system is formed and secreted/released into a culture medium by production cells transfected with the vector according to claim 5 and produced intracellularly, and isolated and purified.
  16. 16. The method of manufacturing according to claim 15, wherein the producer cell is selected from the group consisting of HEK 293F cell line, HEK 293T cell line, stem cell line, or any combination thereof.
  17. 17. A method of manufacturing a pharmaceutical composition according to claim 13 or a cosmetic composition according to claim 14, wherein the pharmaceutical composition or the cosmetic composition uses a catalase or variant thereof according to any one of claims 1 to 3, or a nucleic acid sequence according to claim 4, or a vector according to claim 5, or a delivery system according to any one of claims 6 to 12.
  18. 18. A method of using the pharmaceutical composition of claim 13 or the cosmetic composition of claim 14, comprising administering to a subject in need thereof an effective amount of the catalase of any one of claims 1 to 3 or variant thereof, or the nucleic acid sequence of claim 4, or the vector of claim 5, or the delivery system of any one of claims 6 to 12.
  19. 19. Use of a catalase or variant thereof according to any of claims 1-3, a nucleic acid sequence according to claim 4 or a vector according to claim 5, or a delivery system according to any of claims 6-12 for the preparation of a pharmaceutical or cosmetic composition for scavenging active oxygen, alleviating oxidative stress and/or improving visible signs and/or biomarkers of skin photoaging.
  20. 20. The use according to claim 19, wherein the pharmaceutical or cosmetic composition is for improving photoaged skin conditions.

Description

Mitochondrial targeting recombinant extracellular vesicle delivery system and application thereof Cross reference The present application claims priority from PCT application No. PCT/CN2025/081856 filed on day 3 and 11 of 2025. The content of the prior PCT application is considered part of the present disclosure and is incorporated herein in its entirety. Technical Field The present disclosure relates to the field of biological medicine, and in particular to a mitochondria-targeted recombinant extracellular vesicle delivery system and uses thereof. Background Skin aging and related cellular aging are caused by exogenous factors (e.g., ultraviolet irradiation, smoking, pollution) and endogenous factors (e.g., time, genetic factors, hormones). Ultraviolet (UV) radiation is the strongest external driving factor for age-related changes in skin (i.e. "photoaging"). Facial aging is about 80% attributable to photoaging. Skin photoaging is clinically manifested mainly as photoelastic tissue degeneration, wrinkles and pigmentation. Ultraviolet radiation triggers the overproduction of Reactive Oxygen Species (ROS) in the epidermis and dermis of the skin, mitochondria being the primary site of ROS production in cells. ROS negatively affect skin cells, including keratinocytes and fibroblasts, by inducing oxidative damage and promoting inflammatory responses. These effects can lead to decreased cell viability, accelerated collagen degradation, and up-regulation of pro-inflammatory cytokines. Various features of skin aging are associated with inflammation. Elevated ROS levels are typically observed during polarization of M0 macrophages to pro-inflammatory M1 macrophages. At the same time, excessive ROS may further promote M1 polarization. Cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) are markers of M1 macrophages, whose levels are found to be upregulated in photoaged skin. The therapeutic use of small molecules is undesirable because they do not effectively scavenge intracellular reactive oxygen species. The reaction speed of antioxidant enzyme and these oxidants is thousands to millions times faster than that of small molecules, and is the main antioxidant defense mechanism. Catalase, as the most active antioxidant enzyme in nature and in the human body, plays a vital role in relieving oxidative stress by effectively scavenging ROS. The ability of catalase to decompose hydrogen peroxide into water and oxygen significantly reduces oxidative damage caused by ultraviolet radiation. This enzymatic activity not only protects keratinocytes and fibroblasts from ROS-induced cell damage, but also helps to preserve the integrity and elasticity of the skin. Catalase acts as an effective strategy for improving visible signs of photoaging, such as wrinkles and pigmentation. Extracellular Vesicles (EVs) are lipid bilayer nanoparticles that are released from all cell types into the extracellular space. EVs naturally transport the necessary cellular components (e.g., proteins or active enzymes, lipids, nucleic acids such as mRNA, micro-RNA) for intercellular communication. Thus, EVs hold great promise in delivering active ingredients, payloads, or therapeutic agents to target cells or within cells. As a natural secretion product of cells, EVs have numerous advantages as drug delivery systems/vectors compared to synthetic drug delivery vectors (e.g., liposomes, lipid nanoparticles, and viral vectors). EVs are naturally derived from cells, making them inherently biocompatible and less prone to eliciting immune responses than synthetic vectors. This property is critical to reducing potential side effects in therapeutic applications, EVs have inherent tissue targeting and the ability to cross biological barriers, EVs exhibit extremely high stability in biological fluids, which helps to extend circulation time in extracellular matrix and blood. EVs have been widely validated in scientific research and clinical applications, and their effectiveness and safety have been demonstrated in the field of medical cosmetology. Potential therapeutic applications include anti-aging, anti-pigmentation, wound healing, hair regrowth, and the like. However, the active ingredients in natural stem cell derived EVs are very limited. Catalase has a short half-life after entering the body due to rapid proteolytic degradation and immunogenicity. Thus, encapsulation of catalase in extracellular vesicles enhances its stability, extends its effective half-life, allows for better penetration into target tissues and cells, and provides for efficient scavenging of reactive oxygen species therein. Currently, a variety of methods have been developed for loading therapeutic agents into EVs, including sonication, electroporation, and passive incubation. However, these conventional methods involve multiple manufacturing steps, including catalase protein expression, EV preparation, and loading. They suffer from disadvantages such as rela