WO2023155880A1 - Methods for membrane repair - Google Patents

Methods for membrane repair Download PDF

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Publication number
WO2023155880A1
WO2023155880A1 PCT/CN2023/076779 CN2023076779W WO2023155880A1 WO 2023155880 A1 WO2023155880 A1 WO 2023155880A1 CN 2023076779 W CN2023076779 W CN 2023076779W WO 2023155880 A1 WO2023155880 A1 WO 2023155880A1
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Prior art keywords
cell
tspan4
membrane
agent
damage
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PCT/CN2023/076779
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English (en)
French (fr)
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Li Yu
Yuwei Huang
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Tsinghua University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]

Definitions

  • the plasma membrane is a barrier surrounding the cell.
  • Various types of damage which may be physical, chemical or biological in nature, can cause loss of integrity of the cell membrane, which will inevitably lead to the demise of the cell if not repaired.
  • Several different mechanisms including exocytosis, endocytosis, SYX-2-EFF-1 repair machinery and ESCRT machinery, have been shown to play essential roles in membrane repair 1-8 .
  • ESCRT-mediated membrane repair ESCRT components are recruited to the site of damage and pinch off the damaged membrane to facilitate repair in a calcium-dependent manner 9 .
  • ESCRT-mediated membrane repair plays important roles in maintaining cell viability following diverse types of damage, including damage caused by laser, detergents, necrosis and pyroptosis 10-12 .
  • ESCRT is capable of repairing small patches of membrane damage around 100 nm; however, it is less clear how larger wounds are repaired.
  • Tetraspanin 4 is a transmembrane protein which contains 4 transmembrane domains. It is a member of the Tetraspanin family. Tetraspanin family members organize membranes into cholesterol-enriched membrane domains named Tetraspanin-enriched membrane microdomains (TEMs) 13-16 , and Tetraspanins are known to affect the properties of membrane 15, 17-19 . TEMs can assemble into micrometer-sized Tetraspanin-enriched membrane macrodomains (TEMAs) , which are required for formation of migrasomes 20 . It is unknown whether assembly of TEMAs can have other functions besides mediating migrasome formation.
  • TEMs micrometer-sized Tetraspanin-enriched membrane macrodomains
  • the present disclosure provides a method for regulating membrane repair and/or a membrane repair related biological process, comprising regulating the amount and/or function of TSPAN4, which can help cells mitigate damage caused by laser, detergent, pyroptosis and natural killer cells.
  • TEMAs Tetraspanin-enriched macrodomains
  • Tspan4 plays an important role in membrane repair by mediating assembly of a Tetraspanin-enriched macrodomain, which seals off the damage site to prevent it expanding, and thus facilitates membrane repair (Fig. 4j) .
  • This mechanism which could be viewed as a parallel and possibly indirect mechanism, maintains membrane integrity upon damage in synergy with the ESCRT machinery and possibly other mechanisms which directly repair wounds smaller than 100 nm.
  • the present disclosure provides a method for regulating membrane repair and/or a membrane repair related biological process, comprising regulating the amount and/or function of TSPAN4.
  • said membrane repair related biological process comprises wound healing and/or cell damage regulation.
  • said cell damage comprises cell death
  • said cell death comprises apoptosis, necrosis, and/or pyroptosis.
  • said cell damage is caused by a physical, a chemical and/or a biological factor.
  • said cell damage is caused by laser, a detergent, a perforin, a photon, and/or a killer cell.
  • said killer cell comprises a Natural Killer (NK) cell.
  • NK Natural Killer
  • said membrane has a damaged region with a diameter of at least about 100 nm.
  • said method comprises regulating the amount and/or function of the TSPAN4 in the membrane to be repaired.
  • said membrane is derived from a biological material and/or is an artificial membrane.
  • said method comprises regulating the amount and/or function of the TSPAN4 expressed or contained by said cell.
  • TSPAN4 is human TSPAN4.
  • TSPAN4 has an amino acid sequence as set forth in any one of SEQ ID NO: 1-2.
  • membrane repair comprises inhibition of membrane damage expansion and/or promotion of membrane healing.
  • said method increases said membrane repair, mitigates said cell damage, promotes said wound healing, and/or protects the cell from said cell damage.
  • said method comprises increasing the amount and/or function of said TSPAN4.
  • said method comprises overexpressing a TSPAN4 protein, a functional fragment thereof, a functional variant thereof, and/or a nucleic acid molecule encoding one or more of them.
  • said method comprises increasing the amount and/or function of said TSPAN4 in said cell.
  • said method comprises promoting:
  • said method further comprises increasing the amount and/or function of a second agent capable of increasing membrane repair, mitigating cell damage, promoting wound healing, and/or protecting the cell from cell damage.
  • said second agent comprises one or more components of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery, TSPAN5, cholesterol, and/or an agent capable of increasing the amount and/or function thereof.
  • ESCRT Endosomal Sorting Complex Required for Transport
  • said one or more components of the ESCRT comprises Chmp4b, Vps4a, and/or Chmp3.
  • said method inhibits said membrane repair, inhibits said wound healing and/or promotes said cell damage.
  • said method comprises decreasing the amount and/or function of said TSPAN4.
  • said method comprises decreasing the amount and/or function of said TSPAN4 in said cell.
  • said method comprises knocking down or knocking out the expression of a gene encoding for tetraspanin 4.
  • said method comprises inhibiting:
  • said method comprises administering a TSPAN4 inhibitor.
  • said method further comprises decreasing the amount and/or function of a second agent capable of increasing membrane repair, mitigating cell damage, promoting wound healing, and/or protecting the cell from cell damage.
  • said second agent comprises one or more components of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery, TSPAN5, cholesterol, and/or an agent capable of increasing the amount and/or function thereof.
  • ESCRT Endosomal Sorting Complex Required for Transport
  • said one or more components of the ESCRT comprises Chmp4b, Vps4a, and/or Chmp3.
  • said method further comprises administering an additional agent capable of inhibiting membrane repair and/or causing cell damage.
  • said additional agent comprises a physical, a chemical and/or a biological agent.
  • said additional agent comprises laser, a detergent, a perforin, a photon, and/or a killer cell.
  • said killer cell comprises a Natural Killer (NK) cell.
  • NK Natural Killer
  • said method is an in vivo method.
  • said method is an in vitro method or an ex vivo method.
  • the present disclosure provides an agent capable of regulating the amount and/or function of TSPAN4, for use in regulating membrane repair and/or a membrane repair related biological process.
  • said membrane repair related biological process comprises wound healing and/or cell damage regulation.
  • said cell damage comprises cell death
  • said cell death comprises apoptosis, necrosis, and/or pyroptosis.
  • said cell damage is caused by a physical, a chemical and/or a biological factor.
  • said cell damage is caused by laser, a detergent, a perforin, a photon, and/or a killer cell.
  • said killer cell comprises a Natural Killer (NK) cell.
  • NK Natural Killer
  • said membrane has a damaged region with a diameter of at least about 100 nm.
  • said membrane is derived from a biological material and/or is an artificial membrane.
  • said membrane is cell membrane
  • said agent is capable of regulating the amount and/or function of the TSPAN4 expressed or contained by said cell.
  • TSPAN4 is human TSPAN4.
  • TSPAN4 has an amino acid sequence as set forth in any one of SEQ ID NO: 1-2.
  • membrane repair comprises inhibition of membrane damage expansion and/or promotion of membrane healing.
  • said agent for use in increasing said membrane repair, mitigating said cell damage, promoting said wound healing and/or protecting the cell from said cell damage.
  • said agent is capable of increasing the amount and/or function of said TSPAN4.
  • said agent is capable of resulting in an overexpression of a TSPAN4 protein, a functional fragment thereof, a functional variant thereof, and/or a nucleic acid molecule encoding one or more of them.
  • said agent comprises a TSPAN4 protein, a functional fragment thereof, a functional variant thereof, and/or a nucleic acid molecule encoding one or more of them.
  • said membrane is cell membrane
  • said agent is capable of increasing the amount and/or function of said TSPAN4 in said cell.
  • said agent is capable of promoting:
  • said agent for use in combination with a second agent capable of increasing membrane repair, mitigating cell damage, and/or protecting the cell from cell damage.
  • said second agent comprises one or more components of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery, TSPAN5, cholesterol and/or an agent capable of increasing the amount and/or function thereof.
  • ESCRT Endosomal Sorting Complex Required for Transport
  • said one or more components of the ESCRT comprise Chmp4b, Vps4a, and/or Chmp3.
  • said agent for use in inhibiting said membrane repair, inhibiting said wound healing and/or promotes said cell damage for use in inhibiting said membrane repair, inhibiting said wound healing and/or promotes said cell damage.
  • said agent is capable of decreasing the amount and/or function of said TSPAN4.
  • said membrane is cell membrane
  • said agent is capable of decreasing the amount and/or function of said TSPAN4 in said cell.
  • said agent is capable of knocking down or knocking out the expression of a gene encoding for tetraspanin 4.
  • said agent is capable of inhibiting:
  • said agent comprises a TSPAN4 inhibitor.
  • said agent for use in combination with a second agent capable of decreasing the amount and/or function of an agent capable of increasing membrane repair, mitigating cell damage, promoting wound healing, and/or protecting the cell from cell damage.
  • said second agent is capable of decreasing the amount and/or function of: one or more components of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery, TSPAN5, cholesterol, and/or an agent capable of increasing the amount and/or function thereof.
  • ESCRT Endosomal Sorting Complex Required for Transport
  • said one or more components of the ESCRT comprises Chmp4b, Vps4a, and/or Chmp3.
  • said agent for use in combination with an additional agent capable of inhibiting membrane repair and/or causing cell damage.
  • said additional agent comprises a physical, a chemical and/or a biological agent.
  • said additional agent comprises laser, a detergent, a perforin, a photon, and/or a killer cell.
  • said killer cell comprises a Natural Killer (NK) cell.
  • NK Natural Killer
  • the present disclosure provides an engineered cell with altered ability for regulating membrane repair and/or a membrane repair related biological process comparing to an unmodified corresponding cell, said engineered cell has been modified to alter the amount and/or function of TSPAN4 therein.
  • said membrane repair related biological process comprises wound healing and/or cell damage regulation.
  • said cell damage comprises cell death
  • said cell death comprises apoptosis, necrosis, and/or pyroptosis.
  • said cell damage is caused by a physical, a chemical and/or a biological factor.
  • said cell damage is caused by laser, a detergent, a perforin, a photon, and/or a killer cell.
  • said killer cell comprises a Natural Killer (NK) cell.
  • NK Natural Killer
  • said membrane has a damaged region with a diameter of at least about 100 nm.
  • TSPAN4 is human TSPAN4.
  • TSPAN4 has an amino acid sequence as set forth in any one of SEQ ID NO: 1-2.
  • membrane repair comprises inhibition of membrane damage expansion and/or promotion of membrane healing.
  • said engineered cell has increased ability for membrane repair, mitigating cell damage, promoting wound healing and/or protecting the cell from cell damage.
  • said engineered cell has been modified to increase the amount and/or function of said TSPAN4 therein.
  • said engineered cell overexpresses a TSPAN4 protein, a functional fragment thereof, a functional variant thereof, and/or a nucleic acid molecule encoding one or more of them.
  • said engineered cell has been modified to promote:
  • said engineered cell has been further modified to increase the amount and/or function of a second agent capable of increasing membrane repair, mitigating cell damage, promoting wound healing and/or protecting the cell from cell damage.
  • said second agent comprises one or more components of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery, TSPAN5, cholesterol and/or an agent capable of increasing the amount and/or function thereof.
  • ESCRT Endosomal Sorting Complex Required for Transport
  • said one or more components of the ESCRT comprises Chmp4b, Vps4a, and/or Chmp3.
  • said engineered cell has decreased ability for membrane repair and/or increase ability for promoting cell damage.
  • said engineered cell comprises decreased amount and/or function of said TSPAN4 comparing to an unmodified corresponding cell.
  • said engineered cell has been modified to inhibit:
  • said engineered cell has been treated with a TSPAN4 inhibitor.
  • said engineered cell has been further modified to decrease the amount and/or function of a second agent capable of increasing membrane repair, mitigating cell damage, promoting wound healing and/or protecting the cell from cell damage.
  • said second agent comprises one or more components of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery, TSPAN5, cholesterol and/or an agent capable of increasing the amount and/or function thereof.
  • ESCRT Endosomal Sorting Complex Required for Transport
  • said one or more components of the ESCRT comprises Chmp4b, Vps4a, and/or Chmp3.
  • said engineered cell has been further treated with an additional agent capable of inhibiting membrane repair and/or causing cell damage.
  • said additional agent comprises a physical, a chemical and/or a biological agent.
  • said additional agent comprises laser, a detergent, a perforin, a photon, and/or a killer cell.
  • said killer cell comprises a Natural Killer (NK) cell.
  • NK Natural Killer
  • the present disclosure provides a use of the agent according to the present disclosure and/or the engineered cell according to the present disclosure in the preparation of a regulator for said membrane repair and/or said membrane repair related biological process.
  • the present disclosure provides a composition, comprising the agent according to the present disclosure, and/or the engineered cell according to the present disclosure.
  • said composition is a pharmaceutical composition and optionally comprises a pharmaceutically acceptable excipient.
  • the present disclosure provides a kit, comprising the agent according to the present disclosure, the engineered cell according to the present disclosure, and/or the composition according to the present disclosure.
  • said kit further comprising said second agent.
  • said second agent is comprised in a separate container or compartment.
  • FIG. 1A illustrates the photo-damage assay.
  • FIG. 1B illustrates the time-lapse imaging of NRK cells overexpressing Tspan4-GFP were subjected to photo-damage treatment as in FIG. 1A.
  • Time-lapse imaging was performed by confocal microscopy. Scale bar, 5 ⁇ m.
  • FIG. 1C illustrates how cells were treated with detergent by a micro-pipette needle.
  • FIG. 1D illustrates the time-lapse imaging of NRK cells overexpressing Tspan4-GFP were treated with detergent by a micro-pipette glass needle as in FIG. 1C.
  • Time-lapse imaging was performed by confocal microscopy. Green, Tspan4; red, PI; yellow, merge. Scale bar, 10 ⁇ m.
  • FIG. 1E illustrates YTS NK cells and MGC803 cells expressing Tspan4-BFP were co-cultured and imaged by confocal microscopy.
  • YTS cells were labeled by CD56-APC. Yellow, BFP; cyan, APC. Scale bar, 10 ⁇ m.
  • FIG. 1F illustrates the time-lapse images of NRK Tspan4-GFP cells subjected to photo-damage treatment.
  • PI sodium iodide
  • Scale bar 10 ⁇ m.
  • FIG. 2A illustrates Chmp4b-GFP was co-expressed with Tspan4-mCherry in NRK cells in the first panel; in the last two panels, Vps4a-mCherry and Chmp3-mCherry were expressed in NRK Tspan4-GFP cells. Imaging was conducted by confocal microscopy after photo-damage. Green, GFP; red, mCherry; yellow, merge. Scale bar, 5 ⁇ m.
  • FIG. 2B illustrates NRK cells co-expressing Tspan4-GFP and Chmp4b-mCherry were photo-damaged, then time-lapse images were collected by confocal microscopy. Scale bar, 10 ⁇ m.
  • FIG. 2D illustrates the localization of Tspan4-GFP and Chmp4b-mCherry was analyzed by SIM after photo-damage. Green, Tspan4; red, Chmp4b-mCherry. White arrows, overlapping sites. Scale bar, 5 ⁇ m; zoom in, 1 ⁇ m.
  • FIG. 2E illustrates the localization of Tspan4 and Vps4a at damage sites was analyzed by confocal microscopy after photo-damage in cells co-expressing Tspan4-GFP and Vps4a-mCherry or Vps4a E228Q-mCherry. Scale bar, 10 ⁇ m.
  • FIG. 2F illustrates the images of Tspan4-GFP at the damage site were collected from WT NRK cells or Chmp4b-KO NRK cells after photo-damage treatment. Scale bar, 10 ⁇ m
  • FIG. 2G illustrates the method for analyzing the area positive for Chmp4b-mCherry signals on the bottom of the cell.
  • the upper panel shows the cell at the time of photo-damage; the lower panel shows the cell at subsequent time points.
  • FIG. 2H illustrates Chmp4b-mCherry was expressed in NRK cells overexpressing Tspan4 (NRK Tspan4-OE) , NRK WT cells, and NRK cells with knockout of Tspan4 (NRK Tspan4-KO cells) .
  • the cells were photo-damaged then monitored by time-lapse imaging. Scale bar, 10 ⁇ m.
  • FIG. 3A illustrates Integrin ⁇ 5-mCherry, CD63-mCherry and CD151-mCherry in Tspan4-GFP NRK cells after photo-damage. Scale bar, 5 ⁇ m
  • FIG. 3B illustrates Tspan4-GFP NRK cells under different cholesterol depletion conditions, including 10%full cholesterol medium (FBS) , cholesterol depletion medium (LPDS) and cholesterol depletion medium with 30 ⁇ M Pravastatin, were observed by confocal microscopy before and after photo-damage. Scale bar, 10 ⁇ m.
  • FBS 10%full cholesterol medium
  • LPDS cholesterol depletion medium
  • Pravastatin cholesterol depletion medium with 30 ⁇ M Pravastatin
  • FIG. 3D illustrates two-photon fluorescence microscopy analysis of laurdan-labeled Tspan4-mCherry NRK cells before and after photo-damage. Intensity images of Channel 1 (400-460 nm) , Channel 2 (470-500 nm) and the corresponding GP images are shown. Scale bar, 10 ⁇ m.
  • FIG. 3F illustrates Tspan4-GFP NRK cells were mock treated or exposed to Latrunculin A (500 nM) or Latrunculin A (500 nM) + nocodazole (10 ⁇ M) . Cells were photo-damaged, then observed by confocal microscopy. Scale bar, 10 ⁇ m.
  • FIG. 3G illustrates the stages of the in vitro collapse assay.
  • FIG. 3H illustrates Tspan4-OE GPMVs were used in the in vitro collapse assay. After fixation, immunofluorescence imaging was conducted by confocal microscopy. Yellow, anti-GFP; gray, Rho-PE; cyan, anti-Na/K ATPase. Scale bar, 5 ⁇ m.
  • FIG. 3I illustrates the tatistical analysis of fluorescence enrichment on the edge of damage membrane relative to the non-damaged membrane following the same method as in FIG8B.
  • FIG. 3J illustrates the statistical analysis of the percentage of ruptured pancake membrane structures per random view.
  • FIG. 3K illustrates GPMVs with or without Tspan4-GFP were placed in a micropipette system and then treated by foscholine-12 detergent. Time-lapse imaging was conducted by confocal microscopy. Green, Tspan4; red, rhodamine-PE; gray, DIC. Scale bar, 5 ⁇ m.
  • FIG. 3L illustrates the quantification of the lifetime of detergent-treated GPMVs with or without Tspan4-GFP as in k.
  • N 39 from 3 independent experiments. Mean ⁇ s.e.m., unpaired t-test.
  • FIG. 3M illustrates proteo-liposomes embedded with Tspan4 or not were observed by cryo-electron microscopy after routine cryo-EM sample preparation. Scale bar, 100 nm.
  • FIG. 3O illustrates the reconstructed cryo-electron tomographic Z-stack images of partially opened structures in Tspan4 proteo-liposomes. Yellow arrow, partially opened site. Scale bar, 10 nm.
  • FIG. 4A illustrates images of NRK Tspan4-OE, NRK WT and NRK Tspan4-KO cells after photo-damage. PI influx was used to indicate cell death. Green, Tspan4-GFP; red, PI. Scale bar, 10 ⁇ m.
  • FIG. 4C illustrates heat map images of PI entry signals in NRK, NRK Tspan4-KO and NRK Tspan4/Tspan5 double-knockout cells after photo-damage treatment.
  • Time-lapse imaging was conducted by confocal microscopy. Scale bar, 5 ⁇ m.
  • FIG. 4E illustrates statistical analysis of cell viability in each group (NRK Tspan4-OE, NRK WT, NRK Tspan4-KO, NRK Tspan4/Tspan5 double-knockout cells) after detergent treatment. Mean ⁇ s.e.m., summary of 6 or 8 independent experiments. Unpaired t-test was used.
  • FIG. 4F illustrates diagram of the experiment to assess the effect of Tspan4 on cell viability after LPS electroporation.
  • FIG. 4G illustrates statistical analysis of cell viability in each group (NRK Tspan4-OE, NRK WT and NRK Tspan4-KO) after PBS or LPS electroporation treatment. Mean ⁇ s.e.m., summary of 8 independent experiments. Unpaired t-test was used.
  • FIG. 4H illustrates diagram of the NK killing assay.
  • NK cells YTS
  • cancer cells MGC803
  • FIG. 4I illustrates statistical analysis of the viability of Tspan4-OE, WT and Tspan4-KO cells in the NK cell killing assay. Mean ⁇ s.e.m., summary of 4 independent experiments. Unpaired t-test was used.
  • FIG. 4J illustrates model illustrating the function of Tspan4 in repairing membrane damage.
  • FIG. 5A illustrates L929 Tspan4-mCherry and MGC803 Tspan4-GFP cells were subjected to photo-damage treatment and Tspan4 distribution at the damage site was observed by confocal microscopy. Scale bar, 5 ⁇ m.
  • FIG. 5B illustrates NRK cells co-expressing Tspan4-GFP and Na/K ATPase-mCherry were photo-damaged, then time-lapse images were collected by confocal microscopy. Scale bar, 5 ⁇ m.
  • FIG. 5C illustrates after cells were photo-damaged, PI was added into the system at different time points (0, 1, 2 and 2.5 min) and the PI intensity was traced. Representative images were showed. Green, Tspan4-GFP; red, PI. Scale bar, 5 ⁇ m.
  • FIG. 6A illustrates NRK cells co-expressing Tspan4-GFP and Chmp4b-mCherry were subjected to detergent (25 ⁇ M digitonin) treatment.
  • Z-projection images were collected by 3D-SIM. Yellow, Tspan4; cyan, Chmp4b-mCherry. Scale bar, 5 ⁇ m.
  • FIG. 6B illustrates Tspan4-GFP over-expressing cells treated with DMSO (as control) or 10 ⁇ M BAPTA-AM were subjected to photo-damage. Time-lapse images were collected by confocal microscopy. Scale bar, 5 ⁇ m.
  • FIG. 6C illustrates localization of Tspan4 and Chmp3 at the damage site was analyzed by confocal microscopy after photo-damage in cells co-expressing Tspan4-GFP and Chmp3-mCherry or Chmp3 1-179-mCherry. Scale bar, 10 ⁇ m.
  • FIG. 6D illustrates verification of the NRK Chmp4b knockout cell line by PCR.
  • FIG. 7A-7C illustrate Tspan4-GFP NRK cells under different cholesterol depletion conditions, including 10%full cholesterol medium (FBS) , cholesterol depletion medium (LPDS) and cholesterol depletion medium with 30 ⁇ M Pravastatin, were observed by confocal microscopy before and after photo-damage. PI influx was used to indicate cell death. Scale bar, 5 ⁇ m.
  • FBS 10%full cholesterol medium
  • LPDS cholesterol depletion medium
  • Pravastatin cholesterol depletion medium with 30 ⁇ M Pravastatin
  • FIG. 8A illustrates time-lapse images of Tspan4-GFP GPMVs or GFP-CAAX (as control) GPMVs in the in vitro collapse assay. Green, GFP; red, Rho-PE; yellow, merge. Scale bar, 5 ⁇ m.
  • FIG. 8B illustrates the method used to measure fluorescence enrichment.
  • FIG. 8C illustrates images were collected by confocal microscopy after Tspan4-OE GPMVs, NRK GPMVs and NRK Tspan4-KO GPMVs were used in the in vitro collapse assay.
  • Scale bar 10 ⁇ m;zoom in, 2 ⁇ m.
  • FIG. 8D illustrates time-lapse imaging of rupture events in Tspan4-GFP GPMVs after detergent treatment. Yellow arrow, rupture site. Scale bar, 5 ⁇ m.
  • FIG. 9A illustrates cell death was analyzed by PI permeability in NRK Tspan4-OE, NRK WT and NRK Tspan4-KO cells after detergent treatment. Cells were imaged by confocal microscopy. Scale bar, 10 ⁇ m
  • FIG. 9C illustrates verification of the Tspan4-KO Tspan5 knockout cell line by PCR.
  • FIG. 9D illustrates Tspan4-OE (Tspan4-overexpression) , WT, and Tspan4/Tspan5-KO cells pre-stained by FM1-43 (5 ⁇ M) were subjected to photo-damage treatment and the FM1-43 signal was monitored. Time-lapse images were collected by confocal microscopy and displayed as heat-map images. Scale bar, 10 ⁇ m.
  • FIG. 9F illustrates statistical analysis of cell viability in each group (NRK Tspan4-OE, NRK WT, NRK Tspan4-KO, NRK Tspan4/Tspan5 double-knockout cells) after PBS or LPS electroporation treatment. Mean ⁇ s.e.m., summary of 4 independent experiments. Unpaired t-test was used.
  • FIG. 10A illustrates killing of MGC803 Tspan4-BFP cells by YTS NK cells was observed by confocal microscopy. Cell death was indicated by PI influx. Yellow, BFP; cyan, CD56-APC; red, PI. Scale bar, 10 ⁇ m.
  • FIG. 10B illustrates Killing of MGC803 MGC803 WT cells by YTS NK cells was observed by confocal microscopy. Cell death was indicated by PI influx. Yellow, BFP; cyan, CD56-APC; red, PI. Scale bar, 10 ⁇ m.
  • FIG. 10C illustrates Killing of MGC803 MGC803 Tspan4-KO cells by YTS NK cells was observed by confocal microscopy. Cell death was indicated by PI influx. Yellow, BFP; cyan, CD56-APC; red, PI. Scale bar, 10 ⁇ m.
  • TSPAN4 Tetraspanin 4
  • TSPAN4 generally refers to a TSPAN4 gene and/or a protein that is encoded by the TSPAN4 gene.
  • the NCBI Entrez Gene for TSPAN4 may be 7106.
  • the UniProtKB/Swiss-Prot number for Tetraspanin 4 may be O14817.
  • Tetraspanin 4 may encompass various isoforms of the Tetraspanin 4, the naturally-occurring allelic and processed forms thereof.
  • TSPAN4 or a fragment thereof coupled to, for example, a tag (e.g., a histidine tag) , mouse or human Fc, or a signal sequence.
  • TSPAN4 comprises functional variants and/or fragments thereof, it also comprises orthologue and homologs thereof.
  • TSPAN4 encompasses the TSPAN4 gene or protein from any species, such as human, or a non-human animal, e.g., dog, mouse, rat, pig, monkey (e.g., Rhesus monkey) , cow, cat, chicken, zebrafish, etc.
  • apoptosis generally refers to the process of programmed cell death, with its accompanying cellular morphological changes and loss of cell viability. It may include caspase-dependent cell death, which is characterized by any of the following properties: cell shrinkage, nuclear condensation, DNA fragmentation or membrane blebbing. For example, it may be a form of cell death that prevents immune activation. Apoptotic cells may have a particular microscopic appearance. The cell activates proteins called caspases that are normally dormant. These caspases dismantle the cell from within. The apoptotic cell breaks into small packages that can be engulfed by other cells. This prevents the cell contents leaking out of the dying cell and allows the components to be recycled.
  • cell damage generally refers to a variety of changes of stress that a cell suffers due to external and/or internal environmental changes. Amongst other causes, this can be due to physical, chemical, infectious, biological, nutritional or immunological factors. Cell damage can be reversible or irreversible. Depending on the extent of injury, the cellular response may be adaptive and where possible, homeostasis is restored. Cell death occurs when the severity of the injury exceeds the cell's ability to repair itself. Cell death is relative to both the length of exposure to a harmful stimulus and the severity of the damage caused.
  • cell death generally refers to an event of a biological cell ceasing to carry out its functions. This may be the result of the natural process of old cells dying and being replaced by new ones, or may result from such factors as disease, localized injury, or the death of the organism of which the cells are part. Apoptosis or Type I cell-death, and autophagy or Type II cell-death are both forms of programmed cell death, while necrosis is a non-physiological process that occurs as a result of infection or injury. Cell death is an important process in the body as it promotes the removal of unwanted cells. Failure of cells to die, or cells dying when they shouldn’ t, can lead to or exacerbate many diseases.
  • apoptosis apoptosis, necrosis, necroptosis and pyroptosis. Some occur by an organized, ‘programmed’ process. Some cell death processes leave no trace of the dead cell, whereas others activate the immune system with substances from the dead cell.
  • Chmp3 generally refers to a protein that sorts transmembrane proteins into lysosomes/vacuoles via the multivesicular body (MVB) pathway.
  • This protein along with other soluble coiled-coil containing proteins, forms part of the ESCRT-III protein complex that binds to the endosomal membrane and recruits additional cofactors for protein sorting into the MVB.
  • the protein encoded by human Chmp3 has the accession number of Q9Y3E7 in UniProtKB/Swiss-Prot.
  • Chmp3 or a fragment thereof coupled to, for example, a tag (e.g., a histidine tag) , mouse or human Fc, or a signal sequence.
  • Chmp3 comprises functional variants and/or fragments thereof, it also comprises orthologue and homologs thereof.
  • Chmp3 encompasses the Chmp3 gene or protein from any species, such as human, or a non-human animal, e.g., dog, mouse, rat, pig, monkey (e.g., Rhesus monkey) , cow, cat, chicken, zebrafish, etc.
  • Charged Multivesicular Body Protein 4B or “Chmp4b” generally refers to a member of the chromatin-modifying protein/charged multivesicular body protein (CHMP) protein family.
  • CHMP chromatin-modifying protein/charged multivesicular body protein
  • the protein is part of the endosomal sorting complex required for transport (ESCRT) complex III (ESCRT-III) , which functions in the sorting of endocytosed cell-surface receptors into multivesicular endosomes.
  • ESCRT-III complex III
  • the ESCRT machinery also functions in the final abscission stage of cytokinesis and in the budding of enveloped viruses such as HIV-1.
  • the three proteins of the CHMP4 subfamily interact with programmed cell death 6 interacting protein (PDCD6IP, also known as ALIX) , which also functions in the ESCRT pathway.
  • PDCD6IP programmed cell death 6 interacting protein
  • the CHMP4 proteins assemble into membrane-attached 5-nm filaments that form circular scaffolds and promote or stabilize outward budding. These polymers are proposed to help generate the luminal vesicles of multivesicular bodies.
  • the protein encoded by human Chmp4b has the accession number of Q9H444 in UniProtKB/Swiss-Prot.
  • the term also encompasses Chmp4b or a fragment thereof coupled to, for example, a tag (e.g., a histidine tag) , mouse or human Fc, or a signal sequence.
  • Chmp4b comprises functional variants and/or fragments thereof, it also comprises orthologue and homologs thereof.
  • Chmp4b encompasses the Chmp4b gene or protein from any species, such as human, or a non-human animal, e.g., dog, mouse, rat, pig, monkey (e.g., Rhesus monkey) , cow, cat, chicken, zebrafish, etc.
  • the term “dominant-negative” generally refers to a mutant or variant protein, or the gene encoding the mutant or variant protein, that substantially prevents a corresponding protein having wild-type function from performing the wild-type function.
  • it may refer to a gene or gene variant thereof that encodes a gene product that antagonizes the gene product of a wildtype gene.
  • ESCRT Endosomal Sorting Complex Required for Transport
  • ESCRT generally refers to the ESCRT machinery, which typically is made up of cytosolic protein complexes, known as ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III.
  • the first two complexes function mainly in protein sorting and in recruitment of ESCRT-III, together with Bro1-domain containing proteins.
  • the ESCRT-III complex coordinates the membrane-severing function.
  • the ESCRT machinery plays a vital role in a number of cellular processes including multivesicular body (MVB) biogenesis, cellular abscission, and viral budding.
  • MVB multivesicular body
  • the ESCRT machinery is recruited to sites of action by subfunction-specific targeting modules. These factors include ESCRT-0 (MVE formation) , CEP55 (cytokinesis) , and Gag (virus budding) , that are able to associate with ESCRT components and Bro1-domain proteins.
  • ESCRT-0 MVE formation
  • CEP55 cytokinesis
  • Gag virus budding
  • ESCRT-mediated membrane repair ESCRT components are recruited to the site of damage and pinch off the damaged membrane to facilitate repair.
  • ESCRT-mediated membrane repair plays important roles in maintaining cell viability following diverse types of damage, including damage caused by laser, detergents, necrosis and pyroptosis.
  • ESCRT is capable of repairing small patches of membrane damage around 100 nm.
  • ESCRT components comprise Chmp4b, Vps4a, and Chmp3.
  • engineered generally refers to one or more alterations of a nucleic acid, e.g., the nucleic acid within an organism's genome, of a polypeptide, or of other components.
  • engineered can refer to alterations, additions, and/or deletions of the genes, polypeptides or other components.
  • engineered cell generally refers to a modified cell of human or non-human origin.
  • an engineered cell can refer to a cell with an added, deleted and/or altered gene, polypeptide or other components.
  • ex vivo method generally refers to a method with substantially all steps performed outside of an organism (e.g., an animal or a human body) .
  • an ex vivo method may be performed in or on a tissue from an organism in an external environment with minimal alteration of natural conditions. Tissues may be removed in many ways, including in part, as whole organs, or as larger organ systems.
  • the samples to be tested may have been extracted from the organism. For example, using living cells or tissue from the same organism may also be considered to be ex vivo.
  • One widely performed ex vivo study is the chick chorioallantoic membrane (CAM) assay. In this assay, angiogenesis is promoted on the CAM membrane of a chicken embryo outside the organism (chicken) .
  • CAM chick chorioallantoic membrane
  • in vitro method generally refers to a method performed with microorganisms, cells, or biological molecules outside their normal biological context.
  • an in vitro method may be performed in labware such as test tubes, flasks, Petri dishes, and microtiter plates.
  • In vitro methods may be performed using components of an organism that have been isolated from their usual biological surroundings. For example, microorganisms or cells can be studied in culture media, and proteins can be examined in solutions.
  • the term “in vivo method” generally refers to a method wherein the effects of various biological entities are tested on whole, living organisms or cells, usually animals, including humans, and plants, as opposed to a tissue extract or dead organism.
  • the in vivo method may be performed in a whole organism, rather than in isolated cells thereof.
  • the term "functional fragment” generally refers to a fragment having a partial region of a full-length protein or nucleic acid, but retaining or partially retaining the biological activity or function of the full-length protein or nucleic acid.
  • the term "functional variant” generally refers to a nucleic acid molecule, or a polypeptide having similar amino acid or nucleic acid sequences as the parent sequence and retain one or more properties of the parent sequence.
  • the term “knock down” generally refers to a measurable reduction in the expression of a target mRNA or the corresponding protein in a genetically modified cell or organism as compared to the expression of the target mRNA or the corresponding protein in a counterpart control cell or organism that does not contain the genetic modification to reduce expression.
  • a target mRNA or the corresponding protein in a genetically modified cell or organism as compared to the expression of the target mRNA or the corresponding protein in a counterpart control cell or organism that does not contain the genetic modification to reduce expression.
  • RNA-mediated inhibition techniques e.g., siRNA, shRNA, microRNA, antisense RNA, or other RNA-mediated inhibition techniques, to knock down a target polynucleotide sequence.
  • the term “knock out” generally includes deleting all or a portion of the target polynucleotide sequence in a way that interferes with the function of the target polynucleotide sequence.
  • a knock-out can be achieved by altering a target polynucleotide sequence by inducing a deletion in the target polynucleotide sequence in a functional domain of the target polynucleotide sequence.
  • CRISPR/Cas systems e.g., ZFN, TALEN, TgAgo
  • membrane damage expansion generally refers to enlargement of the membrane damage site such as diameter enlargement, or even irreparable flying membrane damage such as membrane lysis.
  • membrane repair or “membrane healing” generally refers to an active or passive resealing of membrane disruptions to maintain homeostasis and/or prevent cell death or progression of membrane damage.
  • Membrane repair may include cellular membrane repair and non-cellular membrane repair. Cell membrane repair may involve mechanisms from various cellular functions, including vesicle trafficking, exocytosis, and endocytosis, to mend the broken or damaged membrane.
  • the term “pharmaceutically acceptable excipient” generally refers to any material, which is inert in the sense that it substantially does not have a therapeutic and/or prophylactic effect per se. Such an excipient is added with the purpose of making it possible to obtain a pharmaceutical composition having acceptable technical properties.
  • pyroptosis generally refers to an inflammatory form of programmed cell death. It occurs most frequently upon infection with intracellular pathogens and forms part of the immune system's antimicrobial response. In this process, immune cells recognize foreign danger signals within themselves after ingesting a pathogen, which causes the immune cells to release pro-inflammatory cytokines capable of bringing in and activating innate and adaptive immune cells, swell, burst and die.
  • the term pyroptosis is coined from the Greek word pyro, meaning fire or fever, and ptosis, meaning falling, which reflects its inflammatory nature and highlights that it is distinct from other forms of programmed cell death.
  • Pyroptosis requires the function of the enzyme caspase-1, which is activated by an inflammasome. Pyroptosis is characterized by cell swelling, the rapid loss of cell membrane integrity, and the release of pro-inflammatory cytokines and intracellular contents. In contrast to apoptosis, which is primarily anti-inflammatory, pyroptosis is associated with strong pro-inflammatory activity due to the processing and release of IL-1 ⁇ and IL-18.
  • TEMAs tetraspanin-enriched macrodomains
  • TEMAs generally refers to the micrometer-scaled macrodomains assembled by many small TEMs (tetraspanin-enriched microdomain) .
  • TEMs generally refers to the tetraspanin-and cholesterol-enriched microdomains on membranes.
  • TEMs are about 100 nm in size, and are highly enriched with a set of tetraspanins such as TSPAN4 and so-called raft lipids such as cholesterol.
  • tetraspanin generally refers to a membrane protein, which is also known as the transmembrane 4 superfamily (TM4SF) protein, and may have four transmembrane alpha-helices and two extracellular domains.
  • TM4SF transmembrane 4 superfamily
  • tetraspanin may encompass various isoforms of the tetraspanin, as well as the naturally-occurring allelic and processed forms thereof.
  • TSPAN5 Tetraspanin 5
  • TSPAN5 generally refers to a TSPAN5 gene and/or a protein that is encoded by the TSPAN5 gene.
  • the NCBI Entrez Gene for TSPAN5 may be 10098.
  • the term “Tetraspanin 5” may encompass various isoforms of the Tetraspanin 5, the naturally-occurring allelic and processed forms thereof.
  • the term also encompasses TSPAN5 or a fragment thereof coupled to, for example, a tag (e.g., a histidine tag) , mouse or human Fc, or a signal sequence.
  • TSPAN5 comprises functional variants and/or fragments thereof, it also comprises orthologue and homologs thereof.
  • TSPAN5 encompasses the TSPAN5 gene or protein from any species, such as human, or a non-human animal, e.g., dog, mouse, rat, pig, monkey (e.g., Rhesus monkey) , cow, cat, chicken, zebrafish, etc.
  • a non-human animal e.g., dog, mouse, rat, pig, monkey (e.g., Rhesus monkey) , cow, cat, chicken, zebrafish, etc.
  • composition also encompasses “is” , “has” and “consist of” .
  • a composition comprising X and Y may be understood to encompass a composition that comprises at least X and Y. It shall also be understood to disclose a composition that only comprises X and Y (i.e., a composition consisting of X and Y) .
  • the term "about” or “approximately” as used herein is understood to be within normal tolerances in the art, e.g., within 2 standard deviations of the mean. About may be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01%of the stated value. Unless obvious from the context, all values provided herein are approximately modified by the term.
  • the present disclosure provides a method for regulating membrane repair and/or a membrane repair related biological process, comprising regulating the amount and/or function of TSPAN4.
  • said membrane repair related biological process comprises wound healing and/or cell damage regulation.
  • said cell damage comprises cell death
  • said cell death comprises apoptosis, necrosis, and/or pyroptosis.
  • the present disclosure provides a method for membrane repair, wound healing and/or cell damage regulation, comprising regulating the amount and/or function of TSPAN4, wherein said cell damage may comprise cell death, wherein said cell death may comprise apoptosis, necrosis, and/or pyroptosis.
  • said cell damage is caused by a physical, a chemical and/or a biological factor.
  • said cell damage is caused by laser, a detergent, a perforin, a photon, and/or a killer cell.
  • the present disclosure provides a method for regulating membrane repair, wound healing and/or cell damage regulation, comprising regulating the amount and/or function of TSPAN4, wherein said cell damage may be caused by laser, a detergent, a perforin, a photon, and/or a killer cell.
  • said killer cell comprises a Natural Killer (NK) cell.
  • NK Natural Killer
  • the present disclosure provides a method for regulating membrane repair, wound healing and/or cell damage regulation, comprising regulating the amount and/or function of TSPAN4, wherein said cell damage may be caused by NK cells.
  • said membrane has a damaged region with a diameter of at least about 100 nm.
  • said membrane may have a damaged region with a diameter of about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm, about 150 nm or larger.
  • the present disclosure provides a method for regulating membrane repair, comprising regulating the amount and/or function of TSPAN4, wherein said membrane may have a damaged region with a diameter of at least about 100 nm, wherein said damaged region may be caused by a physical, a chemical and/or a biological factor such as laser, a detergent, a perforin, a photon, and/or a killer cell.
  • a physical, a chemical and/or a biological factor such as laser, a detergent, a perforin, a photon, and/or a killer cell.
  • said method comprises regulating the amount and/or function of the TSPAN4 in the membrane to be repaired.
  • said method comprises regulating the amount and/or function of a TPSAN4 in the membrane to be repaired.
  • said membrane is derived from a biological material and/or is an artificial membrane.
  • said membrane may include cell membranes, membranes of tissues/organ, extracellular vesicle membrane, and/or membranes of cellular substructures (e.g., membranes of organelles) .
  • said membrane may also include naturally occurring as well as modified membranes, e.g., said membrane may be natural cell membrane, e.g., said membrane may also be modified cell membrane.
  • the present disclosure provides a method for regulating membrane repair and/or a membrane repair related biological process, comprising regulating the amount and/or function of TSPAN4, wherein said membrane may be natural cell membrane or modified cell membrane, and said membrane may have a damaged region with a diameter of at least about 100 nm, wherein said damaged region may be caused by a physical, a chemical and/or a biological factor such as laser, a detergent, a perforin, a photon, and/or a killer cell.
  • a method for regulating membrane repair and/or a membrane repair related biological process comprising regulating the amount and/or function of TSPAN4, wherein said membrane may be natural cell membrane or modified cell membrane, and said membrane may have a damaged region with a diameter of at least about 100 nm, wherein said damaged region may be caused by a physical, a chemical and/or a biological factor such as laser, a detergent, a perforin, a photon, and/or a killer cell.
  • the present disclosure provides a method for regulating membrane repair and/or a membrane repair related biological process, comprising regulating the amount and/or function of TSPAN4, wherein said membrane may be natural extracellular vesicle membrane or modified extracellular vesicle membrane, and said membrane may have a damaged region with a diameter of at least about 100 nm, wherein said damaged region may be caused by a physical, a chemical and/or a biological factor such as laser, a detergent, a perforin, a photon, and/or a killer cell.
  • artificial membrane is generally used to refer to synthetic membranes, including membrane structures that are present or absent in nature.
  • said membrane may be an artificial cell membrane, or an artificial extracellular vesicle membrane.
  • the present disclosure provides a method for regulating membrane repair and/or a membrane repair related biological process, comprising regulating the amount and/or function of TSPAN4, wherein said membrane may be artificial cell membrane or artificial extracellular vesicle membrane.
  • said method comprises regulating the amount and/or function of the TSPAN4 expressed or contained by said cell.
  • membrane repair comprises inhibition of membrane damage expansion and/or promotion of membrane healing.
  • said method increases said membrane repair, mitigates said cell damage, promotes said wound healing, and/or protects the cell from said cell damage.
  • said method comprises increasing the amount and/or function of said TSPAN4.
  • the present disclosure provides a method for increasing said membrane repair, mitigating said cell damage, promoting said wound healing, and/or protecting the cell from said cell damage, comprising increasing the amount and/or function of TSPAN4.
  • said method comprises overexpressing a TSPAN4 protein, a functional fragment thereof, a functional variant thereof, and/or a nucleic acid molecule encoding one or more of them.
  • the present disclosure provides a method for increasing said membrane repair, mitigating said cell damage, promoting said wound healing, and/or protecting the cell from said cell damage, comprising overexpressing a TSPAN4 protein, a functional fragment thereof, a functional variant thereof, and/or a nucleic acid molecule encoding one or more of them.
  • said method comprises increasing the amount and/or function of said TSPAN4 in said cell.
  • the present disclosure provides a method for increasing said cell membrane repair, mitigating said cell damage, and/or protecting the cell from said cell damage, comprising overexpressing a TSPAN4 protein, a functional fragment thereof, a functional variant thereof, and/or a nucleic acid molecule encoding one or more of them in said cell.
  • said method comprises promoting:
  • the present disclosure provides a method for increasing membrane repair and/or a membrane repair related biological process, comprising promoting:
  • the present disclosure provides a method for increasing said membrane repair, mitigating said cell damage, promoting said wound healing, and/or protecting the cell from said cell damage, comprising promoting:
  • TPSAN4 may be in the membrane to be repaired and/or in said cell.
  • said method further comprises increasing the amount and/or function of a second agent capable of increasing membrane repair, mitigating cell damage, promoting wound healing, and/or protecting the cell from cell damage.
  • the present disclosure provides a method for increasing membrane repair and/or a membrane repair related biological process, comprising increasing the amount and/or function of TSPAN4 and increasing the amount and/or function of a second agent capable of increasing membrane repair, mitigating cell damage, promoting wound healing, and/or protecting the cell from cell damage.
  • said second agent comprises one or more components of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery, TSPAN5, cholesterol, and/or an agent capable of increasing the amount and/or function thereof.
  • ESCRT Endosomal Sorting Complex Required for Transport
  • said one or more components of the ESCRT comprises Chmp4b, Vps4a, Chmp3, a functional fragment thereof, a functional variant thereof, and/or a nucleic acid molecule encoding one or more of them.
  • Chmp4b is rat Chmp4b.
  • Chmp4 is human Chmp4.
  • Chmp4 has an amino acid sequence as set forth in SEQ ID NO: 4 or an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99%of identity with an amino acid sequence as set forth in SEQ ID NO: 4.
  • Vps4a is rat Vps4a.
  • Vps4a is human Vps4a.
  • Vps4a has an amino acid sequence as set forth in SEQ ID NO: 3 or an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99%of identity with an amino acid sequence as set forth in SEQ ID NO: 3.
  • Chmp3 is rat Chmp3.
  • Chmp3 is human Chmp3.
  • Chmp3 has an amino acid sequence as set forth in SEQ ID NO: 5 or an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99%of identity with an amino acid sequence as set forth in SEQ ID NO: 5.
  • the present disclosure provides a method for increasing membrane repair and/or a membrane repair related biological process, comprising increasing the amount and/or function of TSPAN4 and increasing the amount and/or function of a second agent capable of increasing membrane repair, mitigating cell damage, promoting wound healing, and/or protecting the cell from cell damage;
  • TSPAN4 may comprise overexpressing a TSPAN4 protein, a functional fragment thereof, a functional variant thereof, and/or a nucleic acid molecule encoding one or more of them;
  • said second agent may comprise one or more components of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery (such as Chmp4b, Vps4a, and/or Chmp3) , TSPAN5, cholesterol, and/or an agent capable of increasing the amount and/or function thereof.
  • ESCRT Endosomal Sorting Complex Required for Transport
  • said method inhibits said membrane repair, inhibits said wound healing and/or promotes said cell damage.
  • said method comprises decreasing the amount and/or function of said TSPAN4.
  • the present disclosure provides a method for decreasing membrane repair and/or a membrane repair related biological process, comprising decreasing the amount and/or function of TSPAN4.
  • the present disclosure provides a method for inhibiting membrane repair, inhibiting said wound healing and/or promoting said cell damage, comprising decreasing the amount and/or function of TSPAN4.
  • said method comprises decreasing the amount and/or function of said TSPAN4 in said cell.
  • the present disclosure provides a method for inhibiting cell membrane repair, and/or promoting said cell damage, comprising decreasing the amount and/or function of TSPAN4 in said cell.
  • said method comprises knocking down or knocking out the expression of a gene encoding for tetraspanin 4.
  • the present disclosure provides a method for inhibiting cell membrane repair, and/or promoting said cell damage, comprising knocking down or knocking out the expression of a gene encoding for tetraspanin 4 in said cell.
  • said method comprises inhibiting:
  • the present disclosure provides a method for inhibiting cell membrane repair, and/or promoting said cell damage, comprising comprises inhibiting:
  • said method comprises administering a TSPAN4 inhibitor.
  • said method further comprises decreasing the amount and/or function of a second agent capable of increasing membrane repair, mitigating cell damage, promoting wound healing, and/or protecting the cell from cell damage.
  • said second agent comprises one or more components of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery, TSPAN5, cholesterol, and/or an agent capable of increasing the amount and/or function thereof.
  • ESCRT Endosomal Sorting Complex Required for Transport
  • said one or more components of the ESCRT comprises Chmp4b, Vps4a, and/or Chmp3.
  • the present disclosure provides a method for decreasing membrane repair and/or a membrane repair related biological process, comprising administering a TSPAN4 inhibitor and decreasing the amount and/or function of a second agent capable of increasing membrane repair, mitigating cell damage, promoting wound healing, and/or protecting the cell from cell damage;
  • said second agent may comprise one or more components of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery (such as Chmp4b, Vps4a, and/or Chmp3) , TSPAN5, cholesterol, and/or an agent capable of increasing the amount and/or function thereof.
  • ESCRT Endosomal Sorting Complex Required for Transport
  • said method further comprises administering an additional agent capable of inhibiting membrane repair and/or causing cell damage.
  • said additional agent comprises a physical, a chemical and/or a biological agent.
  • said additional agent comprises laser, a detergent, a perforin, a photon, and/or a killer cell.
  • said killer cell comprises a Natural Killer (NK) cell.
  • NK Natural Killer
  • the present disclosure provides a method for decreasing membrane repair and/or a membrane repair related biological process, comprising administering a TSPAN4 inhibitor and administering an additional agent capable of inhibiting membrane repair and/or causing cell damage;
  • said additional agent comprises laser, a detergent, a perforin, a photon, and/or a killer cell such as NK cells.
  • the present disclosure provides a method for decreasing membrane repair and/or a membrane repair related biological process, comprising administering a TSPAN4 inhibitor, decreasing the amount and/or function of a second agent, and administering an additional agent capable of inhibiting membrane repair and/or causing cell damage;
  • said second agent may comprise one or more components of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery (such as Chmp4b, Vps4a, and/or Chmp3) , TSPAN5, cholesterol, and/or an agent capable of increasing the amount and/or function thereof;
  • ESCRT Endosomal Sorting Complex Required for Transport
  • said additional agent may comprise laser, a detergent, a perforin, a photon, and/or a killer cell.
  • said method is an in vivo method.
  • the present disclosure provides an in vivo method for regulating membrane repair and/or a membrane repair related biological process, comprising regulating the amount and/or function of TSPAN4.
  • said method is an in vitro method or an ex vivo method.
  • the present disclosure provides an in in vitro method or an ex vivo method for regulating membrane repair and/or a membrane repair related biological process, comprising regulating the amount and/or function of TSPAN4.
  • the present disclosure provides an agent capable of regulating the amount and/or function of TSPAN4, for use in regulating membrane repair and/or a membrane repair related biological process.
  • said membrane repair related biological process comprises wound healing and/or cell damage regulation.
  • said cell damage comprises cell death
  • said cell death comprises apoptosis, necrosis, and/or pyroptosis.
  • the present disclosure provides an agent capable of regulating the amount and/or function of TSPAN4, for use in regulating membrane repair, wound healing and/or cell damage regulation.
  • said cell damage is caused by a physical, a chemical and/or a biological factor.
  • said cell damage is caused by laser, a detergent, a perforin, a photon, and/or a killer cell.
  • said killer cell comprises a Natural Killer (NK) cell.
  • NK Natural Killer
  • the present disclosure provides an agent capable of regulating the amount and/or function of TSPAN4, for use in regulating membrane repair, wound healing and/or cell damage regulation, wherein said cell damage may be caused by a physical, a chemical and/or a biological factor.
  • said membrane has a damaged region with a diameter of at least about 100 nm.
  • said membrane may have a damaged region with a diameter of about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm, about 150 nm or larger.
  • the present disclosure provides an agent capable of regulating the amount and/or function of TSPAN4, for use in regulating membrane repair, wherein said membrane damage may have a damaged region with a diameter of at least about 100 nm.
  • the present disclosure provides an agent capable of regulating the amount and/or function of TSPAN4 in the membrane, for use in regulating membrane repair and/or a membrane repair related biological process.
  • said membrane is derived from a biological material and/or is an artificial membrane.
  • said membrane may include cell membranes, membranes of tissues/organ, extracellular vesicle membrane, and/or membranes of cellular substructures (e.g., membranes of organelles) .
  • said membrane may also include naturally occurring as well as modified membranes, e.g., said membrane may be natural cell membrane, e.g., said membrane may also be modified cell membrane.
  • the present disclosure provides an agent capable of regulating the amount and/or function of TSPAN4, for use in regulating membrane repair and/or a membrane repair related biological process, wherein said membrane may be natural cell membrane or modified cell membrane, and said membrane may have a damaged region with a diameter of at least about 100 nm, wherein said damaged region may be caused by a physical, a chemical and/or a biological factor such as laser, a detergent, a perforin, a photon, and/or a killer cell.
  • a physical, a chemical and/or a biological factor such as laser, a detergent, a perforin, a photon, and/or a killer cell.
  • the present disclosure provides an agent capable of regulating the amount and/or function of TSPAN4, for use in regulating membrane repair and/or a membrane repair related biological process, wherein said membrane may be natural extracellular vesicle membrane or modified extracellular vesicle membrane, and said membrane may have a damaged region with a diameter of at least about 100 nm, wherein said damaged region may be caused by a physical, a chemical and/or a biological factor such as laser, a detergent, a perforin, a photon, and/or a killer cell.
  • a physical, a chemical and/or a biological factor such as laser, a detergent, a perforin, a photon, and/or a killer cell.
  • artificial membrane is generally used to refer to synthetic membranes, including membrane structures that are present or absent in nature.
  • said membrane may be an artificial cell membrane, or an artificial extracellular vesicle membrane.
  • the present disclosure provides an agent capable of regulating the amount and/or function of TSPAN4, for use in regulating membrane repair and/or a membrane repair related biological process, wherein said membrane may be artificial cell membrane or artificial extracellular vesicle membrane.
  • said membrane is cell membrane
  • said agent is capable of regulating the amount and/or function of the TSPAN4 expressed or contained by said cell.
  • membrane repair comprises inhibition of membrane damage expansion and/or promotion of membrane healing.
  • said agent for use in increasing said membrane repair, mitigating said cell damage, promoting said wound healing and/or protecting the cell from said cell damage.
  • said agent is capable of increasing the amount and/or function of said TSPAN4.
  • the present disclosure provides an agent capable of increasing the amount and/or function of TSPAN4, for use in increasing said membrane repair, mitigating said cell damage, promoting said wound healing and/or protecting the cell from said cell damage.
  • the present disclosure provides an agent capable of increasing the amount and/or function of TSPAN4 expressed or contained by said cell, for use in increasing said membrane repair, mitigating said cell damage, promoting said wound healing and/or protecting the cell from said cell damage.
  • said agent is capable of resulting in an overexpression of a TSPAN4 protein, a functional fragment thereof, a functional variant thereof, and/or a nucleic acid molecule encoding one or more of them.
  • the present disclosure provides an agent capable of resulting in an overexpression of a TSPAN4 protein, a functional fragment thereof, a functional variant thereof, and/or a nucleic acid molecule encoding one or more of them, for use in increasing said membrane repair, mitigating said cell damage, promoting said wound healing and/or protecting the cell from said cell damage.
  • said agent comprises a TSPAN4 protein, a functional fragment thereof, a functional variant thereof, and/or a nucleic acid molecule encoding one or more of them.
  • the present disclosure provides an agent comprising a TSPAN4 protein, a functional fragment thereof, a functional variant thereof, and/or a nucleic acid molecule encoding one or more of them, for use in increasing said membrane repair, mitigating said cell damage, promoting said wound healing and/or protecting the cell from said cell damage.
  • said membrane is cell membrane
  • said agent is capable of increasing the amount and/or function of said TSPAN4 in said cell.
  • the present disclosure provides an agent capable of increasing the amount and/or function of said TSPAN4 in said cell, for use in increasing cell membrane repair, mitigating said cell damage, promoting said wound healing and/or protecting the cell from said cell damage.
  • said agent is capable of promoting:
  • the present disclosure provides an agent for use in increasing cell membrane repair, mitigating said cell damage, promoting said wound healing and/or protecting the cell from said cell damage, wherein said agent is capable of promoting:
  • TPSAN4 is in the membrane and/or said TSPAN4 is in said cell.
  • said agent for use in combination with a second agent capable of increasing membrane repair, mitigating cell damage, and/or protecting the cell from cell damage.
  • said second agent comprises one or more components of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery, TSPAN5, cholesterol and/or an agent capable of increasing the amount and/or function thereof.
  • ESCRT Endosomal Sorting Complex Required for Transport
  • said one or more components of the ESCRT comprise Chmp4b, Vps4a, and/or Chmp3.
  • Chmp4b is rat Chmp4b.
  • Chmp4 is human Chmp4.
  • Chmp4 has an amino acid sequence as set forth in SEQ ID NO: 4 or an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99%of identity with an amino acid sequence as set forth in SEQ ID NO: 4.
  • Vps4a is rat Vps4a.
  • Vps4a is human Vps4a.
  • Vps4a has an amino acid sequence as set forth in SEQ ID NO: 3 or an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99%of identity with an amino acid sequence as set forth in SEQ ID NO: 3.
  • Chmp3 is rat Chmp3.
  • Chmp3 is human Chmp3.
  • Chmp3 has an amino acid sequence as set forth in SEQ ID NO: 5 or an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99%of identity with an amino acid sequence as set forth in SEQ ID NO: 5.
  • the present disclosure provides an agent capable of increasing the amount and/or function of said TSPAN4 in said cell, for use in increasing cell membrane repair, mitigating said cell damage, and/or protecting the cell from said cell damage, wherein said agent for use in combination with a second agent, wherein said second agent comprises one or more components of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery (such as Chmp4b, Vps4a, and/or Chmp3) , TSPAN5, cholesterol, and/or an agent capable of increasing the amount and/or function thereof.
  • ESCRT Endosomal Sorting Complex Required for Transport
  • said agent for use in inhibiting said membrane repair, inhibiting said wound healing and/or promotes said cell damage for use in inhibiting said membrane repair, inhibiting said wound healing and/or promotes said cell damage.
  • said agent is capable of decreasing the amount and/or function of said TSPAN4.
  • said membrane is cell membrane
  • said agent is capable of decreasing the amount and/or function of said TSPAN4 in said cell.
  • the present disclosure provides an agent capable of decreasing the amount and/or function of said TSPAN4 in said cell, for use in inhibiting said membrane repair, inhibiting said wound healing and/or promotes said cell damage.
  • said agent is capable of knocking down or knocking out the expression of a gene encoding for tetraspanin 4.
  • the present disclosure provides an agent capable of knocking down or knocking out the expression of a gene encoding for tetraspanin 4 in said cell, for use in inhibiting said membrane repair, inhibiting said wound healing and/or promotes said cell damage.
  • said agent is capable of inhibiting:
  • the present disclosure provides an agent for use in inhibiting said membrane repair, inhibiting said wound healing and/or promotes said cell damage, wherein said agent may be capable of inhibiting:
  • said agent comprises a TSPAN4 inhibitor.
  • said agent for use in combination with a second agent capable of decreasing the amount and/or function of an agent capable of increasing membrane repair, mitigating cell damage, promoting wound healing, and/or protecting the cell from cell damage.
  • said second agent is capable of decreasing the amount and/or function of: one or more components of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery, TSPAN5, cholesterol, and/or an agent capable of increasing the amount and/or function thereof.
  • ESCRT Endosomal Sorting Complex Required for Transport
  • said one or more components of the ESCRT comprises Chmp4b, Vps4a, and/or Chmp3.
  • the present disclosure provides an agent for use in inhibiting said membrane repair, inhibiting said wound healing and/or promotes said cell damage, said agent for use in combination with a second agent comprising one or more components of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery (such as Chmp4b, Vps4a, and/or Chmp3) , TSPAN5, cholesterol, and/or an agent capable of increasing the amount and/or function thereof.
  • ESCRT Endosomal Sorting Complex Required for Transport
  • said agent for use in combination with an additional agent capable of inhibiting membrane repair and/or causing cell damage.
  • said additional agent comprises a physical, a chemical and/or a biological agent.
  • said additional agent comprises laser, a detergent, a perforin, a photon, and/or a killer cell.
  • said killer cell comprises a Natural Killer (NK) cell.
  • NK Natural Killer
  • the present disclosure provides an agent for use in inhibiting said membrane repair, inhibiting said wound healing and/or promotes said cell damage, said agent for use in combination with an additional agent capable of inhibiting membrane repair and/or causing cell damage;
  • said additional agent may comprise laser, a detergent, a perforin, a photon, and/or a killer cell such as NK cells.
  • an agent may be a small molecule compound, an antibody, a nucleic acid molecule, a polypeptide, or fragments thereof.
  • the agent may comprise one or more active components, present in a single molecule or as separate molecules.
  • the agent may be provided in a non-active form and be converted into an active form in vitro or in vivo before, during or after administration.
  • the agent may be a pharmaceutical agent or an agent for non-pharmaceutical use.
  • the agent may exert the desired functions directly or indirectly via the function of additional agents, compositions or cells.
  • the present disclosure provides an engineered cell with altered ability for regulating membrane repair and/or a membrane repair related biological process comparing to an unmodified corresponding cell, said engineered cell has been modified to alter the amount and/or function of TSPAN4 therein.
  • a “corresponding unmodified cell” or “unmodified corresponding cell” generally refers to a cell that has not been modified to alter the amount and/or function of the sphingomyelin therein, while with all the other features substantially the same as the engineered cell.
  • the corresponding unmodified cell is a wildtype cell (e.g., of the same cell type as the engineered cell) .
  • the corresponding unmodified cell may comprise one or more modifications, but the modification may be for other purposes.
  • a cell may be modified by any approach applicable for the purpose of the present disclosure.
  • the modification may be a genetic modification.
  • the modification may comprise treating the cell with one or more agent causing the desired change or effect.
  • the modification may be temporary, transient or may be stable or permanent.
  • the engineered cell may be a progeny of a parent cell that has been modified.
  • said membrane repair related biological process comprises wound healing and/or cell damage regulation.
  • said cell damage comprises cell death
  • said cell death comprises apoptosis, necrosis, and/or pyroptosis.
  • the present disclosure provides an engineered cell with altered ability for regulating membrane repair, wound healing and/or cell damage regulation comparing to an unmodified corresponding cell, said engineered cell has been modified to alter the amount and/or function of TSPAN4 therein, wherein said cell damage may comprise cell death, wherein said cell death may comprise apoptosis, necrosis, and/or pyroptosis.
  • said cell damage is caused by a physical, a chemical and/or a biological factor.
  • said cell damage is caused by laser, a detergent, a perforin, a photon, and/or a killer cell.
  • said killer cell comprises a Natural Killer (NK) cell.
  • NK Natural Killer
  • the present disclosure provides an engineered cell with altered ability for regulating membrane repair, wound healing and/or cell damage regulation comparing to an unmodified corresponding cell, said engineered cell may have been modified to alter the amount and/or function of TSPAN4 therein, wherein said cell damage may be caused by a physical, a chemical and/or a biological factor such as laser, a detergent, a perforin, a photon, and/or a killer cell.
  • the present disclosure provides an engineered cell with altered ability for regulating membrane repair, wound healing and/or cell damage regulation comparing to an unmodified corresponding cell, said engineered cell may have been modified to alter the amount and/or function of TSPAN4 therein, wherein said cell damage may be caused by NK cells.
  • said membrane has a damaged region with a diameter of at least about 100 nm.
  • said membrane may have a damaged region with a diameter of about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm, about 150 nm or larger.
  • said engineered cell has been modified to increase the amount and/or function of the TSPAN4 in the membrane.
  • the present disclosure provides an engineered cell with altered ability for regulating membrane repair and/or a membrane repair related biological process comparing to an unmodified corresponding cell, said engineered cell may have been modified to increase the amount and/or function of the TSPAN4 in the membrane.
  • membrane repair comprises inhibition of membrane damage expansion and/or promotion of membrane healing.
  • said engineered cell has increased ability for membrane repair, mitigating cell damage, promoting wound healing and/or protecting the cell from cell damage.
  • said engineered cell has been modified to increase the amount and/or function of said TSPAN4 therein.
  • said engineered cell overexpresses a TSPAN4 protein, a functional fragment thereof, a functional variant thereof, and/or a nucleic acid molecule encoding one or more of them.
  • said engineered cell has been modified to promote:
  • the present disclosure provides an engineered cell with altered ability for regulating membrane repair and/or a membrane repair related biological process comparing to an unmodified corresponding cell, said engineered cell may have been modified to promote:
  • said engineered cell has been further modified to increase the amount and/or function of a second agent capable of increasing membrane repair, mitigating cell damage, promoting wound healing and/or protecting the cell from cell damage.
  • the present disclosure provides an engineered cell with altered ability for regulating membrane repair and/or a membrane repair related biological process comparing to an unmodified corresponding cell, said engineered cell may have been modified to increase the amount and/or function of the TSPAN4 and increase the amount and/or function of a second agent capable of increasing membrane repair, mitigating cell damage, promoting wound healing and/or protecting the cell from cell damage.
  • said second agent comprises one or more components of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery, TSPAN5, cholesterol and/or an agent capable of increasing the amount and/or function thereof.
  • ESCRT Endosomal Sorting Complex Required for Transport
  • said one or more components of the ESCRT comprises Chmp4b, Vps4a, Chmp3, a functional fragment thereof, a functional variant thereof, and/or a nucleic acid molecule encoding one or more of them.
  • the present disclosure provides an engineered cell with altered ability for regulating membrane repair and/or a membrane repair related biological process comparing to an unmodified corresponding cell, said engineered cell may have been modified to increase the amount and/or function of the TSPAN4 and increase the amount and/or function of a second agent, wherein said second agent may comprise one or more components of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery, TSPAN5, cholesterol and/or an agent capable of increasing the amount and/or function thereof.
  • ESCRT Endosomal Sorting Complex Required for Transport
  • said engineered cell has decreased ability for membrane repair and/or increase ability for promoting cell damage.
  • said engineered cell comprises decreased amount and/or function of said TSPAN4 comparing to an unmodified corresponding cell.
  • said engineered cell has been modified to inhibit:
  • said engineered cell has been treated with a TSPAN4 inhibitor.
  • said engineered cell has been further modified to decrease the amount and/or function of a second agent capable of increasing membrane repair, mitigating cell damage, promoting wound healing and/or protecting the cell from cell damage.
  • said second agent comprises one or more components of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery, TSPAN5, cholesterol and/or an agent capable of increasing the amount and/or function thereof.
  • ESCRT Endosomal Sorting Complex Required for Transport
  • said one or more components of the ESCRT comprises Chmp4b, Vps4a, Chmp3, a functional fragment thereof, a functional variant thereof, and/or a nucleic acid molecule encoding one or more of them.
  • said engineered cell has been further treated with an additional agent capable of inhibiting membrane repair and/or causing cell damage.
  • said additional agent comprises a physical, a chemical and/or a biological agent.
  • said additional agent comprises laser, a detergent, a perforin, a photon, and/or a killer cell.
  • said killer cell comprises a Natural Killer (NK) cell.
  • NK Natural Killer
  • the present disclosure provides an engineered cell with altered ability for regulating membrane repair and/or a membrane repair related biological process comparing to an unmodified corresponding cell, said engineered cell may have been modified to decrease the amount and/or function of the TSPAN4 and treated with an additional agent capable of inhibiting membrane repair and/or causing cell damage.
  • the present disclosure provides an engineered cell with altered ability for regulating membrane repair and/or a membrane repair related biological process comparing to an unmodified corresponding cell, said engineered cell may have been modified to decrease the amount and/or function of the TSPAN4 and treated with an additional agent, wherein said additional agent may comprise laser, a detergent, a perforin, a photon, and/or a killer cell such as NK cells.
  • additional agent may comprise laser, a detergent, a perforin, a photon, and/or a killer cell such as NK cells.
  • the present disclosure provides a method for preparing an engineered cell with altered ability for regulating membrane repair and/or a membrane repair related biological process comparing to an unmodified corresponding cell, comprising modifying said cell to alter the amount and/or function of TSPAN4 therein.
  • the present disclosure provides a use of an agent modifying said cell to alter the amount and/or function of TSPAN4 therein, in the preparation of an engineered cell with altered ability for regulating membrane repair and/or a membrane repair related biological process comparing to an unmodified corresponding cell.
  • the present disclosure provides a use of the agent according to the present disclosure and/or the engineered cell according to the present disclosure in the preparation of a regulator for said membrane repair and/or said membrane repair related biological process.
  • the present disclosure provides a composition, comprising the agent according to the present disclosure, and/or the engineered cell according to the present disclosure.
  • composition of the present disclosure may be a pharmaceutical composition.
  • the pharmaceutical composition may comprise a pharmaceutically acceptable excipient.
  • the composition may comprise an effective amount of the agent of the present disclosure.
  • the effective amount may be an amount of the agent that when administered alone or in combination with another agent to a cell, tissue, or subject is effective to achieve the desired effect (e.g., regulating migrasome formation and/or in regulating a migrasome-mediated biological process) .
  • compositions may further include pharmaceutically acceptable materials, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, i.e., carriers.
  • a liquid or solid filler such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, i.e., carriers.
  • carriers are involved in transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier should be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject.
  • the formulation and delivery methods will generally be adapted according to the site and the disease to be treated.
  • Exemplary formulations include, but are not limited to, those suitable for parenteral administration, e.g., intravenous, intra-arterial, intramuscular, or subcutaneous administration
  • parenteral administration e.g., intravenous, intra-arterial, intramuscular, or subcutaneous administration
  • the dosage of the agents of the disclosure will vary according to the extent and severity of the need for regulation, the activity of the administered composition, the general health of the subject, and other considerations well known to the skilled artisan.
  • compositions suitable for administration Such compositions typically comprise the agent and a pharmaceutically acceptable carrier. Supplementary active compounds can also be incorporated into the compositions.
  • the agents described herein are delivered locally. Localized delivery allows for the delivery of the agent non-systemically, for example, to the site of regulation in need.
  • the kit of the present disclosure may comprise the agent, the engineered cell, and/or the composition according to the present disclosure.
  • the agent, the engineered cell, and/or the composition may be comprised in suitable packaging, and written material that can include instructions for use, discussion of experimental studies (such as clinical studies) , listing of side effects, and the like.
  • kits may also include information, such as scientific literature references, package insert materials, experimental results (such as clinical trial results) , and/or summaries of these and the like, which indicate or establish the activities and/or advantages of the agent, the engineered cell and/or the composition, and/or which describe dosing, administration, side effects, drug interactions, or other information useful to the users (such as health care provider or consumers) .
  • Such information may be based on the results of various studies, for example, studies using experimental animals involving in vivo models and studies based on human clinical trials.
  • the kit may further contain an additional agent.
  • the agent, engineered cell and/or the composition of the present invention and the additional agent may be provided as separate compositions in separate containers within the kit.
  • the agent, the engineered cell and/or the composition of the present disclosure and the additional agent are provided as a single composition within a container in the kit.
  • Suitable packaging and additional articles for use e.g., measuring cup for liquid preparations, foil wrapping to minimize exposure to air, and the like
  • Kits described herein can be provided, marketed and/or promoted to users (such as health providers) , including scientists, physicians, nurses, pharmacists, formulary officials, and the like. Kits may also, in some embodiments, be marketed directly to the consumer.
  • TSPAN4 means functional TSPAN4 in its normal role in membrane repair, unless otherwise specified.
  • TSPAN4 includes variant polypeptides that are functional.
  • TSPAN4 is used to refer to a TSPAN4 polypeptide (or protein) alone, a TSPAN4 polypeptide fused to additional polypeptides, and a TSPAN4 polypeptide associated with one or more additional polypeptides as long as the TSPAN4 protein exhibits a TSPAN4 function/activity.
  • TSPAN4 functions/activities include, but are not limited to, an ability to mediate formation of TEMs, an ability to mediate formation of TEMs, or an ability to form a migrasome.
  • the TSPAN4 can be the human, porcine, canine, rat, or murine TSPAN4. Alternatively spliced transcript variants encoding different isoforms have been identified.
  • the full-length polypeptide and polynucleotide sequences are known, as are many functional fragments, mutants and modified versions.
  • TSPAN4 include, e.g., full-length TSPAN4, and/or TSPAN4 polypeptide with a full or partial deletion of the non-four hydrophobic domains.
  • TSPAN4 is rat TSPAN4.
  • TSPAN4 is human TSPAN4.
  • TSPAN4 may have an amino acid sequence as set forth in any one of SEQ ID NO: 1-2 or an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99%of identity with an amino acid sequence as set forth in any one of SEQ ID NO: 1-2.
  • the present disclosure provides a method for regulating membrane repair and/or a membrane repair related biological process, comprising regulating the amount and/or function of human TSPAN4, wherein said human TSPAN4 may have an amino acid sequence as set forth in SEQ ID NO: 2 or an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99%of identity with an amino acid sequence as set forth in SEQ ID NO: 2.
  • the present disclosure provides an agent capable of regulating the amount and/or function of human TSPAN4, for use in regulating membrane repair and/or a membrane repair related biological process, wherein said human TSPAN4 may have an amino acid sequence as set forth in SEQ ID NO: 2 or an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99%of identity with an amino acid sequence as set forth in SEQ ID NO: 2.
  • the present application provides a method for regulating the amount and/or function of a TPSAN4.
  • increasing the amount and/or function of TPSAN4 may comprise providing TPSAN4, overexpressing TPSAN4, and/or activating TPSAN4.
  • increasing the amount and/or function of TPSAN4 may comprise introducing TPSAN4 and/or a gene encoding for TPSAN4.
  • increasing the amount and/or function of TPSAN4 may comprise promoting the interaction between the TPSAN4 and other ingredients of TEMs (such as cholesterol, other tetraspanins and/or Integrin- ⁇ 5) .
  • decreasing the amount and/or function of TPSAN4 may comprise knocking out the expression of a gene encoding for TPSAN4, knocking down the expression of a gene encoding for TPSAN4, and/or administering an agent capable of inhibiting the function of TPSAN4.
  • decreasing the amount and/or function of TPSAN4 may comprise introducing CRISPR Cas9 system and/or miRNA targeting TPSAN4.
  • decreasing the amount and/or function of TPSAN4 may comprise inhibiting the interaction between the TPSAN4 and other ingredients of TEMs (such as cholesterol, other tetraspanins and/or Integrin- ⁇ 5) .
  • decreasing the amount and/or function of TPSAN4 may comprise binding to the TPSAN4.
  • Increasing the expression of the TSPAN4 may comprise overexpressing the TSPAN4.
  • the overexpression may be achieved either by introducing an exogenous protein or an exogenous nucleic acid molecule encoding the protein, or by causing increased expression of the endogenous protein or the endogenous gene encoding for said protein.
  • such overexpression may be caused by a mutation in the regulatory region of a gene encoding for the protein.
  • the overexpression may be achieved by changing the function of one or more components of the transcriptional and/or translational machinery.
  • the TSPAN4 protein may comprise an amino acid sequence as set forth in any one of SEQ ID NO: 1-2.
  • Knocking out the TSPAN4 refers to a genetic process in which the TSPAN4 encoding gene is made inoperative ( “knocked out” ) .
  • the TSPAN4 encoding gene When the TSPAN4 encoding gene is knocked out, it may comprise heterozygous knock out or homozygous knock out. In the heterozygous knock out, only one of two gene copies (alleles) is knocked out, in the homozygous knock out, both copies are knocked out.
  • Knockouts may be accomplished through a variety of techniques. In some cases, the knockouts may be naturally occurring mutations that are screened out or identified (e.g., by DNA sequencing or other methods) .
  • the knockouts are generated by homologous recombination.
  • it may involve creating a nucleic acid (e.g., DNA) construct containing the desired mutation.
  • the construct may also comprise a drug resistance marker in place of the desired knockout gene.
  • the construct may further contain a minimum length (e.g., 2kb or above) of homology to the target sequence.
  • the construct may be delivered to target cells (for example, through microinjection, electroporation or other methods, such as transfection, using a virus or a non-virus system) . This method then relies on the cell’s own repair mechanisms to recombine the nucleic acid construct into the existing DNA (e.g., the genome of the cell) .
  • the drug selection marker on the construct may be used to select for cells in which the recombination event has occurred.
  • diploid organisms which contain two alleles for most genes, and may as well contain several related genes that collaborate in the same role, additional rounds of transformation and selection may be performed until every targeted gene is knocked out. Selective breeding may be required to produce homozygous knockout animals.
  • the knockouts are generated using site-specific nucleases.
  • Various methods may be used to precisely target a DNA sequence in order to introduce a double-stranded break. Once this occurs, the cell’s repair mechanisms will attempt to repair this double stranded break, often through non-homologous end joining (NHEJ) , which involves directly ligating the two cut ends together. This may be done imperfectly, therefore sometimes causing insertions or deletions of base pairs, which cause frameshift mutations. These mutations can render the gene in which they occur nonfunctional, thus creating a knockout of that gene.
  • NHEJ non-homologous end joining
  • a zinc-finger nuclease may be used to generate such knockouts.
  • Zinc-finger nucleases comprise DNA binding domains that can precisely target a DNA sequence. Each zinc finger can recognize codons of a desired DNA sequence, and therefore can be modularly assembled to bind to a particular sequence. These binding domains are coupled with a restriction endonuclease that can cause a double stranded break (DSB) in the DNA. Repair processes may introduce mutations that destroy functionality of the gene.
  • DSB double stranded break
  • TALENs Transcription activator-like effector nucleases
  • TALENs contain a DNA binding domain and a nuclease that can cleave DNA.
  • the DNA binding region may comprise amino acid repeats that each recognize a single base pair of the desired targeted DNA sequence. If this cleavage is targeted to a gene coding region, and NHEJ-mediated repair introduces insertions and deletions, a frameshift mutation often results, thus disrupting function of the gene.
  • CRISPR Clustered regularly interspaced short palindromic repeats
  • the CRISPR/Cas9 method is a method for genome editing that contains a guide RNA complexed with a Cas9 protein.
  • the guide RNA can be engineered to match a desired DNA sequence through simple complementary base pairing.
  • the coupled Cas9 may cause a double stranded break in the DNA. Following the same principle as zinc-fingers and TALENs, the attempts to repair these double stranded breaks often result in frameshift mutations that result in a nonfunctional gene.
  • the knockout may also comprise a conditional gene knockout.
  • a conditional gene knockout allows gene deletion in a tissue or cell when certain conditions are fulfilled, for example, in a tissue specific manner. It may be achieved by introducing short sequences called loxP sites around the gene. These sequences will be introduced into the germ-line via the same mechanism as a knock-out. This germ-line can then be crossed to another germline containing Cre-recombinase which is a viral enzyme that can recognize these sequences, recombines them and deletes the gene flanked by these sites.
  • Knocking down the TSPAN4 refers to a process by which the expression of the TSPAN4 encoding gene is reduced. The reduction can occur either through genetic modification or by treatment with a reagent such as a short DNA or RNA oligonucleotide that has a sequence complementary to either gene or an mRNA transcript.
  • a reagent such as a short DNA or RNA oligonucleotide that has a sequence complementary to either gene or an mRNA transcript.
  • the knocking down may be through a genetic modification or may be transient. If a DNA of an organism or cell is genetically modified, the resulting organism or cell may be referred to as a “knockdown organism” or a “knockdown cell” . If the change in gene expression is caused by an oligonucleotide binding to an mRNA or temporarily binding to a gene, this leads to a temporary change in gene expression that does not modify the chromosomal DNA, and the result may be referred to as a “transient knockdown” .
  • Binding can occur either through the blocking of transcription (in the case of gene-binding) , the degradation of the mRNA transcript (e.g. by small interfering RNA (siRNA) ) or RNase-H dependent antisense, or through the blocking of either mRNA translation, pre-mRNA splicing sites, or nuclease cleavage sites used for maturation of other functional RNAs, including miRNA (e.g. by morpholino oligos or other RNase-H independent antisense) .
  • siRNA small interfering RNA
  • RNA interference is a means of silencing genes by way of mRNA degradation. Gene knockdown by this method is achieved by introducing small double-stranded interfering RNAs (siRNA) into the cytoplasm. Small interfering RNAs can originate from inside the cell or can be exogenously introduced into the cell. Once introduced into the cell, exogenous siRNAs are processed by the RNA-induced silencing complex (RISC) .
  • RISC RNA-induced silencing complex
  • the siRNA is complementary to the target mRNA to be silenced, and the RISC uses the siRNA as a template for locating the target mRNA. After the RISC localizes to the target mRNA, the RNA is cleaved by a ribonuclease.
  • a shRNA is used for knocking down the TSPAN4.
  • the shRNA may be introduced into the cell via a viral construct.
  • the viral construct is a lentiviral construct.
  • Increasing the function of the TSPAN4 may comprise introducing a mutation to the TSPAN4 protein thereby increasing one or more function thereof. In some cases, increasing the function of the TSPAN4 may be achieved by increasing or decreasing a factor capable of regulating the function of the TSPAN4 and thereby indirectly increasing the function of the TSPAN4. In some cases, increasing the function of the TSPAN4 comprises administering an activator of the TSPAN4.
  • Inhibiting or decreasing the function of the TSPAN4 may comprise introducing a mutation to the TSPAN4 protein or introducing a mutated TSPAN4 protein (e.g., a dominant-negative TSPAN4) , thereby compromising the function of the TSPAN4.
  • decreasing the function of the TSPAN4 may be achieved by increasing or decreasing a factor capable of regulating the function of the TSPAN4 and thereby indirectly inhibiting or decreasing the function of the TSPAN4.
  • decreasing the function of the TSPAN4 comprises administering to the cell an inhibitor of the TSPAN4.
  • Membrane repair and/or a membrane repair related biological process may be monitored and/or determined by observation, e.g. using microscopy, such as scanning electron microscope (SEM) and/or transmission electron microscope (TEM) .
  • microscopy such as scanning electron microscope (SEM) and/or transmission electron microscope (TEM) .
  • a membrane repair related biological process may include all biological processes related to membrane repair, such as wound healing and/or cell damage regulation, as long as that involves membrane damage repair.
  • a membrane repair related biological process may also include a part of the membrane repair mechanism such as the TSPAN4 mediated formation of TEMs, the TSPAN4 mediated formation of TEMAs, and/or the recruitment of said TSPAN4 towards the site of the damaged membrane.
  • the membrane repair may comprise inhibition of membrane damage expansion and/or promotion of membrane healing.
  • Membrane repair may comprise multiple mechanisms, including exocytosis, endocytosis, SYX-2-EFF-1 repair machinery and ESCRT machinery.
  • said membrane repair and/or a membrane repair related biological process may comprise TSPAN4 and/or TSPAN5 mediated membrane repair.
  • the TSPAN4 forms TEMs and is recruited to the membrane damage in a TEMs form and can form TEMAs for promoting membrane damage repair.
  • TEMAs are in the liquid-ordered phase and form a rigid ring around the damaged site, which restricting the spreading of the damage and prevents membrane disintegration, thus facilitating membrane repair by other mechanisms.
  • the TSPAN4 content of the edge of the site of the damaged membrane can be about 0.1%higher, 1%higher, 10%higher, 20%higher, 30%higher, 40%higher, 50%higher, 60%higher, 70%higher, 80%higher, 90%higher, 95%higher, 99%higher, 100%higher, 2 times higher, 3 times higher, 4 times higher, 5 times higher, 10 times higher, 50 times higher, 100 times higher, 1000 times higher, or 10000 times higher than that of an undamaged site.
  • TSPAN4 mediated formation of TEMs can comprise TSPAN4 organize membranes into cholesterol-enriched membrane domains, which named Tetraspanin-enriched membrane microdomains (TEMs) .
  • TEMs may recruit a large array of membrane proteins.
  • TEM-enriched molecules may comprise other tetraspanins and Integrin- ⁇ 5.
  • TSPAN4 mediated formation of TEMs may comprise TSPAN4 organize membranes into cholesterol-enriched membrane domains, which named Tetraspanin-enriched membrane microdomains (TEMs) .
  • TEMs may assemble into micrometer-sized Tetraspanin-enriched membrane macrodomains (TEMAs) .
  • TEMAs may be in the liquid-ordered phase and form a rigid ring around the damaged site, which may restrict the spreading of the damage and prevent membrane disintegration, thus facilitating repair by other mechanisms.
  • a second agent includes any active agent known in the art to be useful in increasing membrane repair, mitigating cell damage, promoting wound healing, and/or protecting the cell from cell damage.
  • active agent can comprise a protein, antibody, peptide, nucleotide, small molecule drug, etc. that provides a beneficial effect to an animal patient when administered to said patient. Synthetically produced, naturally derived or recombinantly produced fractions are also included in this term. Active agents may be analogues, derivatives, agonists, antagonists, enantiomers or pharmaceutically acceptable salts of biologically active agents.
  • a second agent may be a molecule that is functionally alternative to TSPAN4, eg, the second agent may be other tetraspanin family proteins, such asTSPAN5.
  • a second agent may be a molecule that promotes the formation of TEMS from the TSPAN4, promotes the formation of TMAS from TEMS, and/or promotes the migration of TSAN4 towards the site of membrane damage.
  • said second agent may be cholesterol.
  • a second agent can include an active agent for membrane repair mediated by several different mechanisms, including exocytosis, endocytosis, SYX-2-EFF-1 repair machinery and ESCRT machinery.
  • a second agent can promote membrane repair by regulating exocytosis.
  • a second agent can promote membrane repair by regulating endocytosis.
  • a second agent can promote membrane repair by SYX-2-EFF-1 repair machinery.
  • a second agent can promote membrane repair by ESCRT machinery.
  • a second agent can comprise one or more components of the ESCRT machinery.
  • the second agent can be Chmp4b, Vps4a, and Chmp3 or functional fragments thereof.
  • one or more second agents may be administered, for example the second agent comprises TSPAN5 and cholesterol. Also, for example, the second agent comprises TSPAN5 and chmp4b. Also, for example, the second agent TSPAN5, cholesterol, chmp4b, Vps4a, and Chmp3.
  • the method comprises administering to the cell a first agent and a second agent, the first agent for promoting the amount and function of TSPAN4.
  • the second agent and the first agent are administered sequentially. In some embodiments, the second agent and the first agent are administered simultaneously. In some embodiments, the second agent and the first agent are administered concurrently.
  • the first agent or the second agent may be administered using a variety of formulations known in the art.
  • the first agent is a nucleic acid molecule encoding TSPAN4 and the second agent is a nucleic acid molecule encoding TSPAN5, Chmp4b, Vps4a, and/or Chmp3.
  • the first agent is a nucleic acid molecule encoding TSPAN4 and the second agent is cholesterol.
  • inhibitor generally refers to a compound/substance or composition that is capable of completely or partially preventing or reducing the physiological function of one or more specific biomolecules (e.g., proteins, polypeptides, lipopolysaccharides, glycoproteins, ribonucleoprotein complexes, etc. ) .
  • Said reduction in the physiological function of one or more specific proteins may comprise a reduction in the activity of the protein itself (e.g., the ability to bind to other molecules, etc. ) or a reduction in the amount of the protein itself present.
  • Suitable inhibitor molecules may include antagonist antibodies or antibody fragments, fragments or derivatives of small molecules, peptides, antisense oligonucleotides, small organic molecules, etc.
  • said inhibitor is capable of blocking the activation of cellular signaling pathways.
  • TSPAN4 inhibitor relates to a compound that targets, reduces or inhibits TSPAN4, which may directly or indirectly target one or more TSPAN4, can inhibit said TSPAN4 at the transcriptional level, at the translational level and/or at the level of post-translational modification of the protein, can inhibit the amount or expression of said TSPAN4, and can also inhibit the activity of said TSPAN4.
  • said TSPAN4 inhibitor can reduce or knock down the expression level of TSPAN4 by substances such as siRNA, shRNA, CRISPER/cas systems, etc.
  • said TSPAN4 inhibitor can inhibit the function of TSPAN4, such as inhibiting the formation of TEMS mediated by TSPAN4, or the migration of TPAN4 to sites of membrane damage.
  • said TSPAN4 inhibitor may comprise a dominant negative TSPAN4 mutant, which may substantially prevent a wild-type TSPAN4 from performing the wild-type function.
  • Standard abbreviations may be used, e.g., pl, picoliter (s) ; s or sec, second (s) ; min, minute (s) ; h or hr, hour (s) ; aa, amino acid (s) ; nt, nucleotide (s) ; i. m., intramuscular (ly) ; i. p., intraperitoneal (ly) ; s. c., subcutaneous (ly) ; r. t., room temperature; and the like.
  • Adherent cells including Normal Rat Kidney (NRK) , MGC803 and L929 cells and their derivatives, were cultured at 37°C and 5%CO 2 in DMEM supplemented with 10%serum and 1%penicillin-streptomycin.
  • YTS cells were cultured at 37°C and 5%CO 2 in RPIM-1640 supplemented with 10%serum and 1%penicillin-streptomycin.
  • Chmp4b KO cells The Chmp4b gene in NRK cells was deleted using a modified PX458 plasmid (kindly provided by Dr. Wei Guo from Zhejiang University) that contains two guide RNAs coupled with Cas9 nuclease.
  • the sgRNA sequences used for CRISPR/Cas9 were 5’-GAGGATCGCGCACTGCATGC-3’ (sgRNA#1, SEQ ID NO: 6) and 5’-GTACCAATCAACAACAGGAC-3’ (sgRNA#2, SEQ ID NO: 7) . 72 hours after transfection, the cells were seeded into 96-well plates by fluorescence-activated cell sorter (FACS) selection of EGFP signal.
  • FACS fluorescence-activated cell sorter
  • test primer sequences were 5’-AGCCCGAAAACTCAGACT-3’ (forward, SEQ ID NO: 8) and 5’-TGGATATTGTAAGCACTCTCA-3’ (reverse, SEQ ID NO: 9) .
  • Tspan4/Tspan5 double KO cells The Tspan5 gene was deleted in previously generated Tspan4-KO NRK cells, following the same method described above.
  • the Tspan5-KO gRNA sequences used for CRISPR/Cas9 were 5’-GCTCAGCCGCGCGGACCGAG-3’ (sgRNA#1, SEQ ID NO: 10) and 5’-GGCAGAGCGTGGTGTCATAA-3’ (sgRNA#2, SEQ ID NO: 11) .
  • the test primer sequences were 5’-CTACCCTGGCTCCTCTAAAT-3’ (forward, SEQ ID NO: 12) and 5’- TGCAAGGAGATACACGCTGA-3’ (reverse, SEQ ID NO: 13) .
  • Tspan4-KO gRNA sequences used for CRISPR/Cas9 were 5’-GATGGGGCGTCCGGGAGCCA-3’ (sgRNA#1, SEQ ID NO: 14) and 5’-GCGCACGTGCTCACAAGACC-3’ (sgRNA#2, SEQ ID NO: 15) for NRK cells.
  • sgRNA#1, SEQ ID NO: 14 5’-GCGCACGTGCTCACAAGACC-3’
  • sgRNA#2 SEQ ID NO: 15
  • NRK cells and their derivatives 1/3 of a 6 cm-dish of cells was transfected with 2 ⁇ g DNA via Amaxa nucleofection using solution T and program X-001, and then grown for 15 h for protein expression.
  • a 3.5 cm-dish of cells was transfected with 4 ⁇ g DNA using a Lipofectamine-3000 transfection kit (Invitrogen) and then grown for 15-18 h for protein expression.
  • micropipette glass needle Cells cultured on a 24-mm glass cover slip were put onto a micropipette system and the glass needle was positioned close to the target area on a cell. The cell was injected with 0.02%TritonX-100 or mock, and time-lapse imaging was begun simultaneously.
  • 5 ⁇ M laurdan dye was applied in fresh medium to cells for 30 min for pre-staining at 37°C.
  • the cells were first imaged by an 800 nm laser using the two-photon microscopy mode, and then quickly photo-damaged in confocal microscopy mode. Next, damaged cells were time-lapse imaged by two-photon microscopy for 3 min.
  • GP values were calibrated and calculated as described 21 , 22 and presented as a statistical plot. Images from channel 1 and channel 2 were calibrated and analyzed as before for presentation in GP value mode.
  • GPMVs Preparation of GPMVs
  • 1 ml GPMV buffer containing freshly added 2 mM DTT, 25 mM formaldehyde and 10 ⁇ g/ml PE-Rhodamine (Avanti) was applied to the cells at 37°C for 1 h, with shaking at 70 rpm/min. Finally, GPMVs were prepared in the 1 ml suspension.
  • In-vitro micro-pipette treatment GPMVs were placed in a micropipette system, and the glass needle was set close to the target GPMV. 0.5%foscholine-12 or mock was injected into the GMPV, and time-lapse imaging was started simultaneously.
  • a glass-bottom chamber for confocal imaging was glow-discharged using a plasma cleaning device (PDC-32G, Harrick Plasma) , with 2.5 min vacuum treatment and 60 s exposure to high level ionizing radiation.
  • the chamber was placed on the imaging adapter and a 3- ⁇ l sample of GPMVs was applied onto the center of the base of the chamber.
  • the lid was added and the chamber/samples were allowed to stand for 3 min at room temperature.
  • Time-lapse images (512 ⁇ 512 pixels) were then acquired for 10-15 min. For statistical analysis, images were collected randomly about 20 min after the samples were applied to the chamber.
  • Tspan4-GFP protein was purified and inserted into liposomes to form proteo-liposomes as described before 20 .
  • the tomographic data from the vitrified specimen was collected on an FEI Titan Krios 300 kv TEM equipped with a Cs corrector, an energy filter and a K2 Summit direct electron detector camera (Gatan) . Images were acquired at a magnification of 64,000 ⁇ (pixel size at the specimen level) and -1.5 ⁇ m defocus, with a Volta phase plate inserted to generate a phase shift of ⁇ 0.5 ⁇ . Tilt serials were collected by using SerialEM software 32 at every 3° from -42°to 42°, with a total dose of The structures were then reconstructed in IMOD 33 and visualized in Chimera 34 .
  • LPDS lipoprotein-deficient serum
  • LPDS was prepared from fetal bovine serum (FBS) as described before 35, 36 .
  • FBS fetal bovine serum
  • Tspan4-GFP-expressing NRK cells were cultured in DMEM medium supplemented with 10%FBS, LPDS, or LPDS with 30 ⁇ M Pravastatin (TargetMol, T0672) for 60 hours as described in our previous work 20 Then the photo-damage assay was conducted on cells from each group one by one. The total cellular cholesterol levels of each group were determined using the Red cholesterol assay kit (Invitrogen, A12216) according to the manufacturer’s instructions.
  • NRK cells and their derivatives 3 ⁇ 10 5 cells were transfected with PBS (mock) or 0.5 ⁇ g LPS dissolved in PBS via Amaxa nucleofection using solution T and program X-001, and then seeded into 96-well plates at a density of 1 ⁇ 10 3 per well. 8 h after transfection, cell viability was analyzed using a CellTiter-Glo luminescent cell viability kit.
  • 1.5 ⁇ 10 5 of YTS cells and 1.5 ⁇ 2 ⁇ 10 4 of MGC803 cells or their derivatives were co-cultured, or cultured separately as control, in 100 ⁇ l cell medium at 37°C and 5%CO 2 for 3 h in 1.5 ml ventilate EP tubes.
  • the cells were gently mixed every 30 min during the 3-h period.
  • the cells were centrifuged at 2000 rpm, then stained for CD56-APC at 4°C for 30 min. After one wash in PBS, the cells were stained by AnnexinV-FITC and PI for further flow cytometry analyze.
  • ROIs regions of interest
  • NIS-analysis software For analysis of damage site intensity in photo-damage assays ROIs (regions of interest) were firstly designated by NIS-analysis software. The damaged area was set as ROI 1 and a randomly selected non-damaged area was set as ROI 2. After tracing the ROIs frame by frame during the whole imaging period, the absolute fluorescence intensity of all ROIs was measured. The intensity of the ROI 1s was calibrated by their paired ROI 2s and normalized as a percentage for presentation in plots.
  • Chmp4b-positive area and fluorescence intensity The area covered by Chmp4b-mCherry signals was circled by the shortest connection between the outermost Chmp4b-mCherry punctate signals. The cell total substrate area was manually quantified in the DIC channel image. Based on these measurements frame by frame, the percentage of the area covered by Chmp4b-mCherry signal was calculated and the total fluorescence intensity of mCherry was measured.
  • PI Propidium Iodide
  • BAPTA-AM (Selleck S7534) was added into cell culture system to the final concentration of 10 ⁇ M for 30min at 37°C. Then the cells were subjected to the photo-damage assay.
  • FM1-43 (ThermoFisher F35355) was added into cell culture system to the final concentration of 5 ⁇ g/mL for 15min at 37°C. Then the cells were subjected to the photo-damage assay.
  • Tspan4-GFP first appeared as a series of puncta around the damaged area; eventually, Tspan4-GFP formed a ring-like structure surrounding the damage site (FIG. 1B) .
  • the recruitment of Tspan4-GFP appeared to be specific, as membrane proteins such as Na/K ATPase were not recruited to the damage site (FIG. 5B) .
  • Other forms of damage including localized addition of detergent (FIG. 1C and FIG. 1D) and damage caused by natural killer cells (FIG.
  • 2.1 Tspan4 can protect cells from membrane damage
  • Tspan4 significantly reduced the uptake of PI in cells undergoing photo-damage (FIG. 4A and FIG. 4B) , as well as in detergent-induced membrane damage (FIG. 9A and FIG. 9B) , while knockout of Tspan4 had a relatively minor effect on enhancing PI entry. This suggested that Tspan4 played a role in protecting cells from photo-damage-induced and detergent-induced cell death.
  • RNA-seq indicated that beside Tspan4, Tspan5, 7, 3 and 31 also have relatively high expression levels in NRK cells, which raises the possibility that functional redundancy of Tetraspanins may explain the minor effect of Tspan4 knockout.
  • Tspan4/Tspan5 double-knockout cells showed significantly increased PI entry.
  • Tspan4/Tspan5 double-knockout cells had a higher FM 1-43 signal, indicating that there was more damaged membrane on these cells.
  • Tspan4/Tspan5 double-knockout cells showed a marked enhancement of detergent-induced viability loss (FIG. 4C-4E and FIG. 9C-9E) .
  • Physiological stimuli such as pyroptosis have been shown to cause massive membrane damage, which can be countered by the ESCRT-mediated membrane repair mechanism. It was found that overexpressing Tspan4-GFP protected cells from pyroptosis induced by LPS transfection 24 (FIG. 4F) . Conversely, knockout of Tspan4 sensitizes cells to LPS transfection-induced pyroptosis, and simultaneous knockout of Tspan5 and Tspan4 further exacerbated the sensitivity (FIG. 4G and FIG. 9F) . These results suggested that Tetraspanin-mediated membrane repair played an important role in maintaining cell viability during pyroptosis.
  • NK cells kill their target cells by perforin-mediated membrane disruption and by apoptosis 25-31 .
  • the recruitment of Tspan4-BFP to the contact sites between NK cells and their target cells was observed (FIG. 1E) . It was found that indeed overexpression of Tspan4 protected target cells from NK-mediated killing, while knockout of Tspan4 sensitized target cells to NK-mediated cell death. Thus, Tspan4 protected target cells from NK cells (FIG. 4H-4I and FIG. 10A-10C) .
  • Tspan4 is recruited to damage sites in the form of Tetraspanin-enriched microdomains (TEMs) . It was found that all these molecules were recruited to damage sites (FIG. 3A) . Besides proteins, cholesterol is an essential component of TEMs, and formation of TEMAs is dependent on cholesterol. It was found that cholesterol depletion blocked assembly of the TEMA ring, and significantly increased the PI influx (FIG. 3B-3C and FIG. 7A-7D) . Put together, these data suggested that Tspan4-GFP was indeed recruited to the damage site in the form of TEMs. Once the TEMs reached the damage site, they assembled into a ⁇ m-scale TEMA. Tspan4 may promote membrane repair by forming a rigid ring which surrounds the damaged area and restricts its further expansion.
  • Membrane microdomains like Tetraspanin-enriched domains tend to be in a liquid-ordered phase because of their high cholesterol concentration, which results in high membrane rigidity.
  • phase dye laurdan were used.
  • Generalized polarization (GP) of laurdan changes only with phase state; thus, laurdan has been widely used to identify phase states in membranes 21, 22 . It was found that laurdan dye staining revealed a ring-like structure which encloses the damage site with a GP value around 0.4; thus, the TEMA enclosing the damage site was in the lipid ordered phase (FIG. 3D and FIG. 3E) .
  • Tspan4-GFP was localized on the plasma membrane and on intra-cellular vesicles. That fact that the recruitment of Tspan4 was not dependent on the cytoskeleton (FIG. 3F) suggested that Tspan4 was recruited from the plasma membrane.
  • Tspan4-GFP partially co-localized with various ESCRT components including Chmp4b, Vps4a, and Chmp3 (FIG. 2A) .
  • Tspan4-GFP formed a relatively smooth ring which closely surrounds the damage site, while ESCRT components formed puncta around the damage site, with the highest density on the Tspan4-GFP-positive area (FIG. 2B) .
  • Time-lapse imaging revealed that recruitment of Tspan4-GFP occurred almost simultaneously with recruitment of the ESCRT component Chmp4b. After 5 minutes, the Chmp4b signal became weaker, while the Tspan4-GFP signal kept rising slowly even after the disappearance of Chmp4b (FIG. 2C) .
  • Tspan4-GFP overexpression of Tspan4-GFP dramatically reduced the area around the damage site that was positive for Chmp4b puncta.
  • Chmp4b puncta were restricted to an area immediately surrounding the Tspan4-GFP signal, while in control cells, Chmp4b puncta quickly expanded from the damage site and occupy a much larger area (FIG. 2G-2I) .
  • Knockout of Tspan4 only slightly exacerbated the spreading of Chmp4b puncta, which suggested that other Tetraspanins may compensate for the role of Tspan4.
  • overexpressing Tspan4-GFP markedly reduced the amount of Chmp4b recruited to the damage site, while knockout of Tspan4 only marginally enhanced the recruitment of Chmp4b (FIG. 2H and FIG. 2J) .
  • Example 6 the membrane repair process can be reconstituted in vitro
  • GPMVs giant plasma membrane vesicles
  • NRK cells with or without Tspan4-GFP expression
  • GPMVs contacted the hydrophilic surface, they became flatter, and formed an increasingly thin pancake-shaped double-membrane structure. As the “pancake” gets larger and thinner, the upper layer of membrane ruptures at its center, eventually forming a single-membrane bilayer attached to the supporting surface.
  • Example 7 GPMVs from Tspan4 expressing cells were more resistant to detergent
  • Tspan4-GFP-embedded proteoliposomes showed an unusual morphology under cryo-EM. Control liposomes, with no Tspan4, were all intact in cryo-EM images. In contrast, in Tspan4-GFP-embedded proteoliposomes, some were ruptured yet still retain vesicular morphology by cryo-electron tomography were observed (FIG. 3M-3O) . Close to the sites of rupture, disheveled membrane fragments were observed, which were likely the expelled fragments of damaged membrane.

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