[go: up one dir, main page]

WO2025191080A1 - Procédés et compositions - Google Patents

Procédés et compositions

Info

Publication number
WO2025191080A1
WO2025191080A1 PCT/EP2025/056903 EP2025056903W WO2025191080A1 WO 2025191080 A1 WO2025191080 A1 WO 2025191080A1 EP 2025056903 W EP2025056903 W EP 2025056903W WO 2025191080 A1 WO2025191080 A1 WO 2025191080A1
Authority
WO
WIPO (PCT)
Prior art keywords
rbcnps
protein
contain
rbcnp
payload
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2025/056903
Other languages
English (en)
Inventor
Sujaan DAS
Luca BOLONDI
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Crane Biosciences Ug
Original Assignee
Crane Biosciences Ug
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Crane Biosciences Ug filed Critical Crane Biosciences Ug
Publication of WO2025191080A1 publication Critical patent/WO2025191080A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0641—Erythrocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle

Definitions

  • the present invention relates to red blood cell-derived nanoparticles (RBCNPs), uses thereof and methods of production.
  • RBCNPs red blood cell-derived nanoparticles
  • LNPs lipid nanoparticles
  • Extracellular vesicles are small, membrane-bound particles ubiquitously released by several cell types into various bodily fluids, including but not limited to blood, urine, and saliva. Functionally, they are integral to numerous biological mechanisms, encompassing intercellular communication, coagulation, and immune regulation.
  • the present invention introduces a new type of RBCEV-like particle, named red blood cell-derived nanoparticle (RBCNP), that can be easily manufactured at scale using filtration-based methods.
  • RBCNP red blood cell-derived nanoparticle
  • the present invention provides an ultrascalable method for red blood cell-derived nanoparticle (RBCNP) production.
  • RBCNPs generated are useful for in vivo and in vitro delivery of payloads, since they do not cause immune reactions and are naturally taken up by cells, thereby acting as a superior replacement to current gene transduction methods.
  • the RBCNPs of the present invention are derived from readily available red blood cells, eliminating complex, costly, and time-consuming cell culture processes.
  • the RBCNPs lack nuclei and immune- triggering surface markers (e.g. MHC l/MHC II) minimizing immune recognition and clearance, whilst possessing several native RBC membrane properties that ensure low toxicity and excellent in vivo stability.
  • the invention provides a red blood cell-derived nanoparticle (RBCNP) containing: a. At least one protein from Table A; b. At least one protein from Tables A and B; c. At least one protein from Tables A, B, and C; or d. At least one protein from Tables A, B, C, and D.
  • the invention provides a red blood cell-derived nanoparticle (RBCNP) wherein said RBCNP does not contain at least one of the following proteins: a. ALIX (ALG-2-interacting protein X); b. TSG101 (Tumor susceptibility gene 101 protein); c. Synexin; and/or d. Any protein from Table F
  • the invention provides a red blood cell-derived nanoparticle (RBCNP) wherein said RBCNP does not contain TSG101 .
  • the invention provides a method of producing a plurality of red blood cell-derived nanoparticles (RBCNPs) wherein the method comprises at least one filtration step.
  • RBCNPs red blood cell-derived nanoparticles
  • the invention provides RBCNPs obtained by a method disclosed herein.
  • the invention provides RBCNPs loaded with a payload obtained by a method disclosed herein.
  • the invention provides a solution comprising a plurality of RBCNPs as disclosed herein.
  • the invention provides a composition comprising a plurality of RBCNPs as disclosed herein.
  • the invention provides a method of delivering a releasable payload to one or more tissues selected from: brain, kidney, adrenal glands, liver, lung, ovaries, spleen, lymph nodes and thymus, comprising administering the RBCNPs as described herein to a subject.
  • FIG. 1 Schematic method of red blood cell-derived nanoparticle (RBCNP) production.
  • the schematic shows a blood bag, separation of the red blood cells, red blood cell vesiculation, and a series of filtration steps to purify RBCNPs.
  • Figure 2 Particle size and concentration measurement.
  • RBCNPs exhibit a size distribution with a peak at approximately 135-140 nm.
  • the observed yield for 200 mL of red blood cells is approximately 1 x 10 14 - 2 x 10 14 .
  • FIG. 3 RBCNP stability. RBCNPs were kept for 25 days at temperature conditions of -20°C, 4 °C and room temperature (RT). Minimal losses (10%) in particle numbers were observed even after 25 days.
  • FIG. 4 Functional delivery of siRNA to human cell lines.
  • RBCNPs were loaded with siRNA cargo against the enzyme GAPDH using the ExoFect kit (System Biosciences). Loaded RBCNPs were incubated with A59 cells, and GAPDH mRNA expression was measured relative to beta actin expression using qPCR after 2 days (relative mRNA expression).
  • the graph shows the relative GAPDH mRNA expression for untreated cells, cells treated with 10 x 10 10 RBCNPs with 2.5 pL siRNA and cells treated with 10 x 10 10 RBCNPs with 5 pL siRNA.
  • Figure 5 Functional delivery of Cre recombinase mRNA to human cell lines.
  • Cre mRNA was successfully delivered to the cells, Cre mRNA was translated to Cre recombinase protein, which then caused RFP expression. Fluorescence imaging showed RFP expression in >85% cells.
  • Figure 6 Functional delivery of Cy2/labelled GFP mRNA to human cell lines.
  • Cy2/labelled eGFP mRNA was packaged into RBCNPs using the ExoFect kit. Loaded RBCNPs were incubated with HeLa cells cultured in complete medium (DMEM), and GFP expression was observed within 1 day.
  • Figure 7 Functional delivery of GFP plasmid DNA to human cell lines.
  • Plasmid DNA for GFP expression was packaged into RBCNPs using the Exofect kit. Loaded RBCNPs were incubated with HeLa cells cultured in complete medium (DMEM), and GFP expression was observed within 1 day.
  • Figure 8 Functional delivery of Cas9 mRNA to human cell lines.
  • Cas9 mRNA together with a guide RNA against GFP were loaded into RBCNPs, and delivered to human cells stably expressing GFP (HEK293 N1 cell line). Fluorescence imaging showed 5 to 10 % of cells displayed a loss of GFP expression in the Cas9 group compared to only ⁇ 1 % in the control group. On day 3 post-treatment, many groups of cells were observed without GFP expression in the Cas9 treated wells (demonstrated by the white arrows).
  • the RBCNPs were tolerated by both groups of mice, with no changes in body weight when compared to control (saline solution) 9A; 9B).
  • the RBCNPs were tolerated by both groups of mice, with no changes in cumulative feed consumption when compared to control (saline solution) (10A; 10B).
  • the RBCNPs were tolerated by both groups of mice and the inflammatory cytokine profiles of the mice were measured. No increase in inflammatory cytokine IFN-y was observed when compared to control (saline solution) (1 1 A; 11 B).
  • the RBCNPs were tolerated by both groups of mice and the inflammatory cytokine profiles of the mice were measured. No increase in inflammatory cytokine TNF-a was observed when compared to control (saline solution) (12A; 12B).
  • the RBCNPs were tolerated by both groups of mice and the locomotor activity of the mice were measured. No significant difference in locomotor activity was observed when compared to control (saline solution) (13A; 13B).
  • All mice were sacrificed, and the organs collected and imaged fluorometrically.
  • a strong fluorescent signal was seen in thymus and ovaries of the treated mice, pointing to the accumulation of RBCNPs in the above organs. Smaller signals were detected in spleen, heart, brain, and intestine. (14)
  • FIG 15 Further storage data Further storage experiments were carried out on RBCNPs loaded with Cre mRNA after storage (postloaded RBCNPs) and RBCNPs loaded before storage (pre-loaded RBCNPs). RBCNPs were stored at different storage conditions for 8 weeks (post-loaded RBCNPs) or 36 hours (pre-loaded RBCNPs). Loaded RBCNPs were incubated with a Cre reporter cell line (HEK293 LoxP switch GFP/RFP) and imaged for GFP and RFP fluorescence. To compare outcomes, the “Switching factor” (SF) was defined as the mean intensity of the Red channel, divided by the mean intensity of the Green channel.
  • Figure 15A shows results from the post-loaded RBCNPs and Figure 15B shows results from pre-loaded RBCNPs.
  • Figure 16 Functional delivery of plasmid DNA to dendritic cells.
  • Dendritic cells in culture derived from mouse bone marrow induced pluripotent stem cells (IPSCs) were incubated with 50 ul of the plasmid pmaxgfp (1 mg/mL) showed no GFP expression after 3 days, whereas RBCNPs loaded with pmaxGFP using the Exofect kit efficiently delivered plasmid DNA to cells, which exhibited robust GFP expression (lower panel) within the same time frame.
  • IPCs mouse bone marrow induced pluripotent stem cells
  • Figure 17 Functional delivery of small molecules to human cell lines.
  • RBCNPs (3.3uL) loaded with Cre mRNA (1 ug) were incubated in PBS, incomplete RPMI medium, or complete RPMI medium (which contains FBS and therefore proteases and RNAses) for 7 days at 4 C. These preparations were then overlayed onto the cell line HEK293 loxP GFP RFP (acquired from GenTarget). These cells can switch from GFP to RFP expression upon successful delivery of Cre recombinase, which was robustly observed after 3 days irrespective of the medium of storage. Naked Cre mRNA was used a as a control where no switching was observed.
  • This study investigated the biodistribution of mRNA-loaded RBCNPs in a single-dose imaging study using Ai9 transgenic mice.
  • the study was divided into two groups: Group 1 , consisting of control mice (3 males and 3 females), and Group 2, consisting of mice treated with 130 pL of RBCNP solution (3 males and 3 females).
  • the dosed solution contained 15 pg (range: 10-20 pg) of Cre mRNA and 1000ug (range: 750-1500 pg) of RBCNP per mouse.
  • Mice were anesthetized with isoflurane for imaging, which was performed using the IVIS system at 72, 168 and 192 hours post-dose.
  • tissue samples were collected from several organs, including the adrenal gland, brain, heart, kidneys, liver, lungs, lymph nodes, ovaries, spleen, testis, thymus and bone marrow (representative IVIS image shown on top right panel). The observed fluorescence was recorded and data from a representative mouse is shown in the bottom right panel.
  • IHC analysis revealed tdTomato staining exclusively in treated mice, with no staining observed in control tissues. The organ and cell distribution of tdTomato expression were scored and reported by a trained pathologist (lower panel).
  • IHC analysis immunolabeling positivity (%) by tissue was determined. >75% immunolabeling positivity was found in the brain, kidney, adrenal glands and liver.
  • RBNCPs produced by a method disclosed herein have a different proteomic composition compared to RBCEVs known in the prior art.
  • the invention therefore provides a red blood cell-derived nanoparticle (RBCNP) containing: a. At least one protein from Table A; b. At least one protein from Tables A and B; c. At least one protein from Tables A, B, and C; or d. At least one protein from Tables A, B, C, and D.
  • RBCNP red blood cell-derived nanoparticle
  • the RBCNP contains: a. At least W proteins from Table A; b. At least X proteins from Table A and B; c. At least Y proteins from Table A, B, and C; or d. At least Z proteins from Table A, B, C, and D; where W is between 1 and 103, X is between 1 and 141 , Y is between 1 and 209, and Z is between 1 and 375.
  • W is therefore from 1-103, for example 1-100, 2-90, 3-80, 4-70, 5-60, 6-70, 7-60, 8-50, 9-40, 10-30 or 20-25, for example W ean be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 101 , 102 or 103.
  • X is therefore from 1-141 , for example 1-140, 2-130, 3-120, 4-110, 5-100, 6-90, 7-80, 8-70, 7-60, 8-50, 9-40, 10-30 or 15-20, for example Y can be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140 or 141.
  • Y is therefore from 1-209, for example 1-200, 2-190, 3-180, 4-170, 5-160, 6-150, 7-140, 8-130, 7-120, 8-110, 9-100, 10-90, 15-80, 20-70, 30-60 or 40-50, for example Y can be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 206, 207, 208 or 209.
  • Z is therefore from 1-375, for example 1-375, 2-370, 3-360, 4-350, 5-340, 6-330, 7-320, 8-310, 7-300, 8-290, 9-280, 10-270, 15-260, 20-250, 30-240, 40-230, 50-220, 60-210, 70-200, 80-190, 90-180, 100- 170, 110-160, 120-150 or 130-140, for example Y can be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15,
  • the proteins from any of Tables A, B, C and D can be present in any combination.
  • the proteins from any of Table G, H and I represent a subset of the proteins present in Table A.
  • the proteins from any of Table G, H and I can be present in any combination.
  • the at least one protein from Table A is Apolipoprotein A-L In some embodiments, the at least one protein from Table A is Apolipoprotein A-l and albumin. Both Apolipoprotein A-l and albumin are proteins present in Table H.
  • RBCNPs of the invention do not contain at least one of the following proteins: a. ALIX (ALG-2-interacting protein X); b. TSG101 (Tumor susceptibility gene 101 protein); c. Synexin; and/or d. At least one protein from Table F.
  • the invention provides a red blood cell-derived nanoparticle (RBCNP) wherein said RBCNP does not contain at least one of the following proteins: a. ALIX (ALG-2-interacting protein X); b. TSG101 (Tumor susceptibility gene 101 protein); c. Synexin; and/or d. Any protein from Table F
  • RBCNPs of the invention do not contain TSG101. Accordingly, in a third aspect, the invention provides a red blood cell-derived nanoparticle (RBCNP) wherein said RBCNP does not contain TSG101.
  • RBCNP red blood cell-derived nanoparticle
  • TSG101 Tumor Susceptibility Gene 101
  • ESCRT endosomal sorting complex required for transport
  • Vps vacuolar protein sorting
  • TSG101 has been shown to play a role in immune modulation.
  • TSG101-containing vesicles influence immune responses by modulating antigen-presenting cells, such as dendritic cells (DCs), leading to alterations in cytokine secretion and immune activation (Papayannakos et al., 2021).
  • DCs dendritic cells
  • TSG101 -containing vesicles have been associated with both upregulation and downregulation of inflammatory processes by regulating MMP-9 expression (Sai et al., 2015). Given these immunoregulatory effects and the inherent variability associated with TSG101 -containing vesicles, developing vesicles that are free of TSG101 could help mitigate unintended immune activation or suppression and reduce therapeutic outcome variability, making them more reliable and predictable for clinical applications. It is therefore desirable to obtain red blood cell-derived vesicles and solutions of red blood cell-derived vesicles that are substantially free of TSG101 to prevent unintended immune regulation.
  • RBCEVs of the prior art contain TSG101 , likely due to immature RBCs (reticulocytes, a normal component of human blood) expressing TSG101 .
  • the RBCNPs of the present invention do not express TSG101 , despite starting with blood that was acquired from the Bavarian Red Cross blood banks. It would be expected that TSG101 would be present in RBCNPs and/or RBCNP preparations using packed RBCs from a blood bank however there is no TSG101 present in the final preparations of the present invention. We hypothesise this is a result of the manufacturing method of the present invention. Without intending to be bound by theory, this may be a result of:
  • the RBCNPs contain at least one protein from Table A, and do not contain any protein from Table F. In some embodiments, the RBCNPs contain at least one protein from Table A, and do not contain ALIX. In some embodiments, the RBCNPs contain at least one protein from Table A, and do not contain TSG101. In some embodiments, the RBCNPs contain at least one protein from Table A, and do not contain Synexin.
  • the RBCNPs contain at least one protein from Table A, and do not contain ALIX and do not contain TSG101 . In some embodiments, the RBCNPs contain at least one protein from Table A, and do not contain ALIX and do not contain Synexin. In some embodiments, the RBCNPs contain at least one protein from Table A, and do not contain Synexin and do not contain any protein from Table F. In some embodiments, the RBCNPs contain at least one protein from Table A, and do not contain ALIX and do not contain any protein from Table F. In some embodiments, the RBCNPs contain at least one protein from Table A, and do not contain ALIX, do not contain TSG101 and do not contain Synexin.
  • the RBCNPs contain at least one protein from Table A, and do not contain ALIX, do not contain TSG101 and do not contain any protein from Table F. In some embodiments, the RBCNPs contain at least one protein from Table A, and do not contain ALIX, do not contain Synexin and do not contain any protein from Table F. In some embodiments, the RBCNPs contain at least one protein from Table A, and do not contain ALIX, do not contain TSG101 , do not contain Synexin and do not contain any protein from Table F. In some embodiments, the RBCNPs contain at least one protein from Table A, and do not contain TSG101 and do not contain Synexin.
  • the RBCNPs contain at least one protein from Table A, and do not contain TSG101 and do not contain any protein from Table F. In some embodiments, the RBCNPs contain at least one protein from Table A, and do not contain TSG101 , do not contain Synexin and do not contain any protein from Table F.
  • the RBCNPs do not contain ALIX and contain at least one protein from Table G, Table H and/or Table I.
  • the RBCNPs do not contain TSG101 and contain at least one protein from Table G, Table H and/or Table I.
  • the RBCNPs do not contain Synexin and contain at least one protein from Table G, Table H and/or Table I.
  • the RBCNPs do not contain ALIX and TSG101 and contain at least one protein from Table G, Table H and/or Table I.
  • the RBCNPs do not contain ALIX and Synexin and contain at least one protein from Table G, Table H and/or Table I.
  • the RBCNPs do not contain ALIX, TSG101 and Synexin, and contain at least one protein from Table G, Table H and/or Table I. In some embodiments, the RBCNPs do not contain TSG101 and Synexin and contain at least one protein from Table G, Table H and/or Table I.
  • the at least one protein from Table H is Apolipoprotein A-l. In some embodiments, the at least one protein from Table H is Apolipoprotein A-l and albumin.
  • the RBCNPs do not contain ALIX and contain Apolipoprotein A-l. In some embodiments, the RBCNPs do not contain TSG101 and contain Apolipoprotein A-L In some embodiments, the RBCNPs do not contain Synexin and contain Apolipoprotein A-L In some embodiments, the RBCNPs do not contain ALIX and TSG101 and contain Apolipoprotein A-l. In some embodiments, the RBCNPs do not contain ALIX and Synexin and contain Apolipoprotein A-L In some embodiments, the RBCNPs do not contain ALIX, TSG101 and Synexin, and contain Apolipoprotein A-l. In some embodiments, the RBCNPs do not contain TSG101 and Synexin and contain Apolipoprotein A-l. In some embodiments, the RBCNPs do not contain TSG101 and Synexin and contain Apolipoprotein A-l. In some embodiments, the RBCNPs do not contain TSG101 and Synexin and
  • the RBCNPs do not contain ALIX and contain Apolipoprotein A-l and albumin. In some embodiments, the RBCNPs do not contain TSG101 and contain Apolipoprotein A-l and albumin. In some embodiments, the RBCNPs do not contain Synexin and contain Apolipoprotein A-l and albumin. In some embodiments, the RBCNPs do not contain ALIX and TSG101 and contain Apolipoprotein A-l and albumin. In some embodiments, the RBCNPs do not contain ALIX and Synexin and contain Apolipoprotein A-l and albumin.
  • the RBCNPs do not contain ALIX, TSG101 and Synexin, and contain Apolipoprotein A-l and albumin. In some embodiments, the RBCNPs do not contain TSG101 and Synexin and contain Apolipoprotein A-l and albumin.
  • the RBCNP does not contain 1-69 proteins from Table F, for example it does not contain 1- 68, 2-67, 3-66, 4-65, 5-64, 6-63, 7-62, 8-61 , 9-60, 10-55, 11-50, 12-45, 13-40, 14-35, 15-30, 16-25 or 17-20 proteins from Table F, for example it does not contain 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 61 , 62, 63, 64, 65, 66, 67, 68 or 69 proteins from Table F.
  • the at least one protein from Table H is Apolipoprotein A-l. In some embodiments, the at least one protein from Table H is Apolipoprotein A-l and albumin.
  • the RBCNPs do not contain any protein from Table F and contain Apolipoprotein A-l. In some embodiments, the RBCNPs do not contain any protein from Table F and ALIX, and contain Apolipoprotein A-l. In some embodiments, the RBCNPs do not contain any protein from Table F and TSG101 , and contain Apolipoprotein A-L In some embodiments, the RBCNPs do not contain any protein from Table F and Synexin and contain Apolipoprotein A-l. In some embodiments, the RBCNPs do not contain any protein from Table F, ALIX, TSG101 , Synexin, and contain Apolipoprotein A-l.
  • the RBCNPs do not contain any protein from Table F and contain Apolipoprotein A-l and albumin. In some embodiments, the RBCNPs do not contain any protein from Table F and ALIX and contain Apolipoprotein A-l and albumin. In some embodiments, the RBCNPs do not contain any protein from Table F and TSG101 and contain Apolipoprotein A-l and albumin. In some embodiments, the RBCNPs do not contain any protein from Table F and Synexin and contain Apolipoprotein A-l and albumin. In some embodiments, the RBCNPs do not contain any protein from Table F, ALIX, TSG101 , Synexin and contain Apolipoprotein A-l and albumin.
  • the presence or absence of proteins on the surface of the RBCNPs can be detected using any suitable method, for example mass spectrometry or Western blotting.
  • the invention provides a method of producing a plurality of red blood cell-derived nanoparticles (RBCNPs) wherein the method comprises at least one filtration step.
  • RBCNPs red blood cell-derived nanoparticles
  • nanoparticle refers to a particle of matter ranging in size between 1 to 500 nanometers (nm) in diameter. In the case of RBCNPs the diameter is typically between 50 and 200 nm in diameter.
  • extracellular vesicle or “EV” refers to small, membrane-enclosed structures naturally released by cells into their extracellular environment throughout the cellular lifecycle. These vesicles contain various biomolecules, including proteins, lipids, and nucleic acids such as RNA and DNA. The release of extracellular vesicles is a fundamental aspect of cellular communication, allowing cells to exchange information with other cells in their vicinity or at distant locations.
  • red blood cell-derived extracellular vesicles refers to extracellular vesicles that are naturally released from red blood cells (erythrocytes) into the extracellular environment. Erythrocytes are the most abundant type of blood cells and primarily function to transport oxygen from the lungs to various tissues and organs throughout the body. RBCEVs are a subtype of extracellular vesicles and can include both exosomes and microvesicles that are released by red blood cells. RBCEVs can be isolated from human or animal blood. The contents of RBCEVs may vary, and their release can be influenced by various physiological and pathological conditions.
  • red blood cell-derived nanoparticles or “RBCNP” refers to RBCEV-like particles that are distinct from RBCEVs as a result of their manufacturing technique and composition.
  • the at least one filtration step is a plurality of filtration steps.
  • the plurality of filtration steps comprises using filters of varying sizes.
  • the plurality of filtration steps comprises using filters of the same size.
  • the at least one filtration step comprises use of a filter of no greater than 0.8 pm.
  • the at least one filtration step comprises use of a filter of no greater than 0.5 pm.
  • the plurality of filtration steps comprises use of a filter of no greater than 0.5 pm for the final filtration step.
  • the filter can be no greater than 0.45 pm.
  • the filter can be no greater than 0.4 pm.
  • the filter can be no greater than 0.35 pm.
  • the filter can be no greater than 0.3 pm, for example a 0.3 pm filter.
  • the filter can be no greater than 0.25 pm.
  • the filter can be no greater than 0.2 pm.
  • a vacuum pump can be used for filtration.
  • Ultrafiltration can be used for filtration.
  • the method further comprises subjecting a resulting filtrate to ultrafiltration.
  • ultrafiltration is centrifugal ultrafiltration.
  • the ultrafiltration is performed using a 100 nm polyethersulfone (PES) membrane.
  • PES polyethersulfone
  • ultrafiltration refers to a filtration (i.e. separation) process that uses a semipermeable membrane to selectively remove particles and solutes from a liquid based on their size and molecular weight. Filtration is achieved by applying a pressure gradient across the membrane, driving the liquid through the membrane pores.
  • ultrafiltration is “centrifugal ultrafiltration” which refers to a technique that combines the principles of ultrafiltration with the use of centrifugal force to separate and concentrate substances in a liquid sample. As the sample spins, the centrifugal force drives liquid and particles and molecules of desired sizes through the filter membrane, while particles and molecules of an undesired size are retained on the membrane surface. This allows for the separation and concentration of the desired components in the liquid.
  • the yield of RBCNPs generated by the method of the present invention can be between 1 x 10 14 to 5 x 10 14 per 200 mL of packed RBCs. In some embodiments, the observed yield of RBCNPs can be 1 x 10 14 to 3 x 10 14 per 200 mL of packed RBCs. In some embodiments, the observed yield of RBCNPs can be 1 x 10 14 to 2 x 10 14 per 200 mL of packed RBCs.
  • packed RBCs refers to red blood cells commonly used in transfusions.
  • pRBCs are derived from whole blood donations, where the blood is processed to remove most of the plasma, platelets, and leukocytes (white blood cells), leaving primarily red blood cells (RBCs). This concentration process increases the proportion of RBCs in the final product.
  • the RBCNPs obtained by a method disclosed herein can be obtained by subjecting red blood cells (RBCs) to vesiculation or extrusion.
  • RBCs red blood cells
  • vesiculation is induced by incubating RBCs with calcium ionophore.
  • the calcium ionophore is added at a final concentration of 1 pM to 10 mM.
  • the calcium ionophore is added at a final concentration of 1 pM to 100pM.
  • the calcium ionophore is added at a final concentration of 5pM to 20
  • the calcium ionophore is added at a final concentration of 10 pM.
  • vesiculation is induced by incubating RBCs with calcium ionophore at a final concentration of 5 pM for 1 hour at 37°C.
  • vesiculation refers to the process of forming or producing vesicles. Vesicles are small, membrane-bound sacs or bubbles that can be found in cells or formed from cellular structures. Vesiculation often involves the budding or release of vesicles from cellular membranes.
  • exusion refers to the process of passing RBC membranes through a needle (23 gauge or other sizes) to create a population of nanoparticles.
  • the method can further comprise the addition of CaCI 2 .
  • the CaCI 2 is added at a final concentration of 0.1 mM to 100 mM. In some embodiments the CaCI 2 is added at a final concentration of 0.1 mM to 10 mM. In some embodiments the CaCI 2 is added at a final concentration of 0.5 mM to 2 mM. In some embodiments CaCI 2 is added at a final concentration of 1 mM. In some embodiments, the CaCI 2 is added sequentially to or simultaneously with the calcium ionophore. In some embodiments, the CaCI 2 is added simultaneously with the calcium ionophore.
  • the RBCNPs are derived or obtained from human or mammalian RBCs.
  • the RBCNPs and/or RBCs are obtained from hematopoietic stem cells in vitro.
  • the RBCNPs and/or RBCs are obtained from human induced pluripotent stem cells (hiPSCs) in vitro.
  • the human or mammalian RBCs can be isolated, derived or obtained from blood.
  • iPSCs Induced pluripotent stem cells
  • iPSCs refers to iPSCs, in particular human iPSCs (hiPSCs) which express at least one marker selected from the list consisting of POU Class 5 Homeobox 1 (POU5F1') (also known as OCT4), OCT3, Nanog homeobox (NANOG) and SRY-box transcription factor 2 (SOX2).
  • POU5F1' also known as OCT4
  • OCT3 Nanog homeobox
  • SOX2 SRY-box transcription factor 2
  • the iPSCs may be genetically modified.
  • HSCs Hematopoietic stem cells
  • myeloid-lineage and lymphoid-lineage cells HSCs
  • HSCs differentiate into common myeloid progenitors and megakaryocyte-erythroid progenitors, then sequentially differentiate into unipotent progenitors restricted to the erythroid lineage, ultimately resulting in reticulocytes that mature into RBCs.
  • RBCs can be produced via erythroid differentiation of HSCs.
  • the blood used in the present invention can be stored for at least 7 days. For example, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, or 13 days.
  • the blood used in the present invention can be stored for at least 14 days. For example, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, or 20 days.
  • the blood used in the present invention can be stored for at least 21 days.
  • the blood used in the present invention can be “expired blood”.
  • the term “expired blood” refers broadly to blood that has reached its expiration date. Blood is carefully screened, processed and stored to ensure safety. Blood products have expiration dates, and after this date, they are not used for transfusion.
  • Blood is typically stored at 4°C and is generally considered “expired” if it is over 21 days old. Therefore, the maximum period of blood storage for transfusion is typically 21 days at 4°C.
  • the 30-minute rule states that red blood cell (RBC) units left out of controlled temperature storage for more than 30 minutes should not be returned to storage for reissue; the 4-hour rule states that transfusion of RBC units should be completed within 4 hours of their removal from controlled temperature storage.
  • RBC red blood cell
  • the invention provides RBCNPs obtained by a method disclosed herein.
  • the RBCNPs obtained by a method disclosed herein, such as the method of the fourth aspect, have the characteristics set out above in relation to a RBCNP of the first, second or third aspect.
  • the invention provides RBCNPs obtained by a method of the invention, wherein said RBCNPs contain: a. At least one protein from Table A; b. At least one protein from Tables A and B; c. At least one protein from Tables A, B, and C; or d. At least one protein from Tables A, B, C, and D.
  • the invention provides RBCNPs obtained by a method of the invention, wherein said RBCNPs do not contain TSG101 .
  • the RBCNPs of the fifth aspect are a plurality of RBCNPs.
  • the RBCNPs have a particle size distribution peak between 50-200 nm.
  • the RBCNPs have a particle size distribution peak between 135-140 nm.
  • the RBCNPs have a particle size distribution peak between 135-145 nm.
  • the particle size distribution can be determined by any suitable method, for example Nanoparticle Tracking Analysis (NTA), Laser Diffraction (LD), Dynamic Light Scattering (DLS), Dynamic Image Analysis (DIA) or Sieve Analysis.
  • the particle size distribution peak is determined by Nanoparticle Tracking Analysis (NTA).
  • the RBCNPs can comprise a releasable payload.
  • the method of the fourth aspect can further comprise loading the RBCNPs with a payload.
  • the method of the fourth aspect further comprises incubating the payload and RBCNPs under sufficient conditions for the RBCNPs to be loaded with said payload.
  • the loading can be performed in vitro or ex vivo. Typically loading is achieved by means of transfection, electroporation or hydropo ration.
  • the term “payload” refers to a material or functional component carried by a delivery system.
  • a RBCNP may be loaded with a therapeutic gene as its payload that upon delivery of the payload to a target cell, means the cell can express the therapeutic gene, perhaps to provide a therapeutic effect. Payload can be used interchangeably with the term “cargo”.
  • the payload is typically releasable, which means that on delivery of the RBCNP to its intended location in the body, it releases the payload. This is typically achieved in two steps, uptake of the RBCNP into a cell (including potentially membrane fusion and receptor-mediated endocytosis), and release of the cargo in the cytoplasm (typically via endosomal escape). Typically, as the RBCNP is taken up by the cell, it is engulfed in an exosome. The exosome then breaks (“endosomal escape”) and the cargo is released into the cytoplasm.
  • the payload is a polynucleotide, metabolite, small molecule, protein, enzyme, CRISPR/Cas editing system or any combination thereof.
  • the payload is selected from the group of RNA, DNA, plasmids, antisense oligonucleotides (ASO) and combinations thereof.
  • the RNA is selected from the group of mRNA, siRNA, miRNA, circRNA, gRNA and combinations thereof.
  • the payload is Cas9 mRNA and a guide RNA; or Cas9 and gRNA plasmids.
  • the payload size can be up to 14kb (kilobases). In some embodiments the payload size is up to 12kb, for example up to 1 1.2kb. In some embodiments, the payload size can be up to 6 kb. In some embodiments, the payload size is 0.5-2kb, for example 1.4 kb. In some embodiments the payload size is 4-6kb, for example 4.5-5kb, for example, 4.5kb, for example 4.7kb.
  • the payload size can be quantified by the number of nucleotides, for example up to 30 nucleotides, for example 29, 28, 27, 26, 25, 24, 23, 22, 21 , 20 nucleotides. In some embodiments, the payload comprises 20 to 25 nucleotides. In some embodiments, the payload comprises 21 to 23 nucleotides. This is a typical length of an siRNA payload.
  • the RBCNPs are stable at room temperature (approximately 20°C) for up to 2 months.
  • RBCNPs can be stored at 4°C for up to 2 months and at -20°C or lower temperatures, for example -80°C, for several months, for example up to 12 months, for example 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months.
  • RBCNPs loaded with a payload such as an RNA are typically stable at room temperature for up to 36 hours, for example 24 hours, 12 hours, 8 hours or 4 hours.
  • the invention provides a solution comprising a plurality of RBCNPs as disclosed herein.
  • the RBCNPs may be the RBCNPs of the first, second, third, fifth or sixth aspect.
  • solution refers to a liquid preparation comprising the RBCNPs of the present invention present, for example resuspended, in a liquid.
  • the liquid is PBS.
  • the liquid may also be a pharmaceutically acceptable carrier or diluent.
  • the solution does not contain TSG101 (Tumor susceptibility gene 101 protein).
  • the invention provides a method of delivering a releasable payload to one or more tissues selected from: brain, kidney, adrenal glands, liver, lung, ovaries, spleen, lymph nodes and thymus, comprising administering the RBCNPs as described herein to a subject.
  • the RBCNPs may be the RBCNPs of the first, second, third, fifth or sixth aspect, wherein the RBCNPs are loaded with a payload.
  • the RBCNPs may be delivered in a solution according to the seventh aspect or in a composition according to an eighth aspect.
  • the subject is typically a human.
  • the invention provides the RBCNPs, solution comprising RBCNPs or composition comprising RBCNPs of the present invention for use in a method of delivering a releasable payload to one or more tissues selected from: brain, kidney, adrenal glands, liver, lung, ovaries, spleen, lymph nodes and thymus, comprising administering the RBCNPs as described herein to a subject.
  • the invention provides the use of the RBCNPs, solution comprising RBCNPs or composition comprising RBCNPs of the present invention to deliver a releasable payload to one or more tissues selected from: brain, kidney, adrenal glands, liver, lung, ovaries, spleen, lymph nodes and thymus in a subject.
  • the RBCNPs may be the RBCNPs of the first, second, third, fifth or sixth aspect, wherein the RBCNPs are loaded with a payload.
  • the RBCNPs may be delivered in a solution according to the seventh aspect or in a composition according to an eighth aspect.
  • the releasable payload is delivered to the brain.
  • the RBCNPS are administered locally or systemically.
  • the RBCNPS are administered by direct injection.
  • the RBCNPs are administered parenterally or enterally.
  • the RBCNPs are administered intracathecally, intravenously, intramuscularly, transdermally, intradermally, intra-articularly, extra-articularly, intraarterially, intracranially, subcutaneously, intraorbitally, intraventricularly, intraspinally, or intraperitoneally.
  • the RBCNPs are administered intravenously or subcutaneously.
  • Immune evasion refers to strategies employed by, for example, pathogens, cancer calls or other entities, to avoid detection or attack by the immune system.
  • the term “about” in the context of concentration, size, length of time, or other stated values refers to a value that is similar to the stated reference value.
  • the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 1 1 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %), or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
  • the terms “improve,” “increase” or “reduce,” or grammatical equivalents indicate values that are relative to a reference (e.g., baseline) measurement, such as a measurement taken under comparable conditions (e.g., in the same individual prior to initiation of treatment described herein, or a measurement in a control individual (or multiple control individuals) in the absence of treatment) described herein.
  • a reference e.g., baseline
  • comparable conditions e.g., in the same individual prior to initiation of treatment described herein, or a measurement in a control individual (or multiple control individuals) in the absence of treatment
  • the term "subject”, “individual”, or “patient” refers to any organism upon which embodiments of the invention may be used or administered, e.g. for experimental, diagnostic, prophylactic, and/or therapeutic purposes. These terms include mammals, such as humans and both domestic animals such as laboratory animals and household pets (e.g., cats, dogs, swine, cattle, sheep, goats, horses, rabbits), and non-domestic animals such as wildlife and the like.
  • pharmaceutically acceptable carrier or diluent means any substance suitable for use in administering to an animal.
  • a pharmaceutically acceptable carrier or diluent is sterile saline.
  • such sterile saline is pharmaceutical grade saline.
  • target cell or target tissue refers to any cell, tissue, or organism.
  • O negative (O -ve) blood (1 unit, 400mL) was acquired from the Bavarian Red Cross. The blood was tested for the absence of HIV, Hepatitis B, and other blood-borne infections. Red blood cells (RBCs) were separated and washed with RPMI medium and stored in the fridge in 20 mL aliquots (in 50 mL falcon tubes) for 1-2 weeks. Wherein each bag of blood yields approximately 10-12 falcon tubes. Calcium ionophore (A23187, Sigma Aldrich) was stored as a 10 mM stock solution in DMSO.
  • Each 20mL RBC aliquot was washed 1x with PBS, resuspended in PBS to a total volume of 50 mL, and Calcium ionophore was added at a final concentration of 10 pM. CaCL was also optionally added at a final concentration of 1 mM, to enhance the release of nanoparticles from the RBCs.
  • the falcon tubes were then incubated at 37°C for 48 hours with constant shaking, followed by centrifugation at 4500 rpm (basic swing rotor benchtop centrifuge) for 30 minutes.
  • Figure 1 includes a schematic representation of RBCNP production.
  • the manufacturing cost of the novel process is significantly lower ( ⁇ 1000x) than reported by other groups (as described in US2019/054192A1). This is predominantly due to a reduction of ⁇ 1000x in the amount of Calcium Ionophore (one of the main cost drivers).
  • Previous studies have used calcium ionophore at a concentration of 10mM, worth >20,000 EUR for every donation bag of blood needed to process.
  • ultracentrifugation for which the machinery is expensive and non-scalable, is used in previous studies, whereas this invention uses a series of filtration steps which are readily scalable.
  • the present invention uses a unique combination of chemical and physical processes, which allows for a reduction of calcium ionophore use by 1000x (10 pM), elimination of ultracentrifugation, and improved yields at lower costs.
  • RBCNPs Functional delivery was tested by incubation of loaded RBCNPs with suitable cell lines (including HeLa cell line, SC018 LoxP switch red green cell line, etc). Between 10 5 to 10 10 RBCNPs loaded with a suitable cargo were incubated with cells growing in 5mm x 5mm coverslips in DMEM complete medium, at 37°C, 5% CO2. Cells were then imaged 2-3 days later using a 20x objective on a Leica DMi8 widefield microscope with a DF C9000 GTC camera. Suitable lasers and filter sets were used for fluorescence excitation/emission of GFP/RFP.
  • suitable cell lines including HeLa cell line, SC018 LoxP switch red green cell line, etc.
  • RBCNPs were loaded with the various kinds of polynucleotide cargo, including but not limited to siRNA, mRNA, DNA and small molecules using the Exo-FectTM Exosome Transfection Kit (Catalog #EXFT10A- 1) (System Biosciences) according to the manufacturer’s instructions.
  • a pool of siRNA against the enzyme GAPDH was acquired from siTools GMBH and loaded into RBCNPs according to instructions in the ExoFect kit. Loaded RBCNPs were incubated with A549 cells, and GAPDH mRNA was measured relative to beta actin expression using qPCR after 2 days. Very high functional knockdown of GAPDH mRNA (-80%) was observed (see Figure 4).
  • Cre reporter cell line HEK293 LoxP switch GFP/RFP, SC018 from GenTarget. Cre mRNA was successfully delivered to the cells, since a colour switch from GFP to RFP was observed in >85% of cells (see Fig 5).
  • a colour switch implies successful recombination between loxP sites in the cell line genome enabling expression of RFP in live cells. Successful recombination between loxP sites is exclusively caused by Cre protein, which comes from the translation of successfully delivered Cre mRNA (see Figure 5).
  • Cy2/labelled GFP mRNA was also loaded into RBCNPs and delivered to HeLa cells, demonstrating GFP expression within 1 day ( Figure 6) and similarly, plasmid DNA for GFP expression was loaded into RBCNPs and delivered to HeLa cells, GFP expression was functionally delivered (see Figure 7).
  • Cas9 mRNA together with a guide RNA against GFP were loaded into RBCNPs, and delivered to human cells stably expressing GFP (HEK293 N1 cell line). It was expected to observe populations of cells without GFP expression, due to the Cas9+GFPgRNA causing a double strand break at the gfp genetic locus, which then would be repaired by NHEJ mechanisms to yield a gfp mutant locus. 5-10% of cells lost GFP expression in the Cas9 ground compared to ⁇ 1 % in the control group (see Figure 8). On day 3 post-treatment, many groups of cells were observed without GFP expression in the Cas9 treated wells (demonstrated by the white arrow of Figure 8).
  • Dendritic cells in culture derived from mouse bone marrow induced pluripotent stem cells were incubated with 50 pl of the plasmid pmaxgfp (1 mg/mL) and showed no GFP expression after 3 days (see Figure 16), whereas RBCNPs loaded with pmaxGFP using the Exofect kit efficiently delivered plasmid DNA to cells, which exhibited robust GFP expression (lower panel) within the same time frame.
  • IPCs mouse bone marrow induced pluripotent stem cells
  • the cell-non-permeable actin filament binding drug Phalloidin (1 pg) was loaded into RBCNPs and overlayed on HELA cells in vitro (2cm dish). Delivery of Phalloidin was observed in >70% of cells.
  • the fluorescently labelled anti-cancer drug Paclitaxel (1 pg) was loaded into RBCNPs and overlayed on HELA cells in vitro (2cm dish). Delivery of fluorescent Paclitaxel was observed in 50-70% of cells.
  • RBCNPs loaded with 5 pg FITC-dextran was successfully delivered to HFF cells in culture, but not FITC dextran alone. See Figure 17.
  • SC018 HEK293 switch LoxP cells
  • RBCNP formulations (1 , 2, and 3
  • a control naked Cre mRNA
  • Cre mRNA remained protected for 7 days at 4°C by RBCNPs, regardless of the presence or absence of protease-containing enzymes, ensuring stability in various storage conditions, see Figure 18.
  • G1 control
  • G2 treated
  • Both G1 and G2 were made up of five female and five male BALB/c mice. Mice from G1 were injected with saline solution into the tail vein at a 5mL/kg dose. Injections were repeated every 48 hours for a total of 5 injections.
  • mice from G2 were injected with RBCNP solution into the tail vein at a 1 .25mL/kg dose. Injections were repeated every 48 hours for a total of 5 injections. Each mouse received approximately 10 A 12 RBCNPs over the course of the study.
  • locomotor activity was measured via an actophotometer to highlight any depressant or stimulant effects on the central nervous system. Results are shown in Figure 13.
  • mice were humanely sacrificed, and organs were collected for histopathological investigation and organ weight measurement.
  • mice Immediately after the second injection, all mice were sacrificed, and the organs collected and imaged fluorometrically. When compared to control, a strong fluorescent signal was seen in thymus and ovaries of the treated mice, pointing to the accumulation of RBCNPs in the above organs. Smaller signals were detected in spleen, heart, brain, and intestine. Results are shown in Figure 14.
  • TdTomato staining was conducted using a rabbit polyclonal antibody and visualized with DAB chromogen and hematoxylin counterstaining. IHC analysis revealed tdTomato staining exclusively in treated mice, with no staining observed in control tissues.
  • CREmRNA-RBCNP treated animal Strong, diffuse immunolabeling was observed in endothelial cells across all tissues. Moderate to strong cytoplasmic labeling was detected in the majority of cell types in tissues where labelling was present.
  • CNS Cells neurons in the cerebral cortex, Cerebellar Purkinje layer, Choroid plexus epithelium
  • Renal and Pulmonary Cells renal tubular cells (proximal and distal), Pulmonary type II pneumocytes, Bronchiolar epithelial cells (weak labeling)
  • Cell type SC018 LoxP switch cells (as described in Example 4).
  • Cargo type CRE mRNA, 2.5 ug per well Procedure:
  • SF switching factor
  • Cell type SC018 LoxP switch cells (as described in Example 4).
  • Cargo type CRE mRNA, 2.5 ug per well
  • RBCNPs were manufactured using the disclosed filtration-based method (described in Example 1).
  • Loaded RBCNPs were stored at different storage conditions for 36hrs. Storage conditions were -80C, 4C.
  • SF switching factor
  • pre-loading RBCNPs with mRNA caused a decrease in switching factor when compared to post-loading (previous experiment). This decrease is more pronounced when loaded RBCNPs are stored at 4C vs at -80C. The decrease is likely attributed to degradation of RNA during the storage period.
  • RBCNPs were manufactured using the method described in Example 1 from 4 independent RBC preparations.
  • Table F represents RBCEV-specific proteins (not present on RBCNPs of the invention). The tables were obtained by comparing the proteins present in RBCNPs with proteins present in RBCEVs (Pham et al., 2021).
  • Table A RBCNPs-specific proteins present in 100% of analyzed samples
  • Table B RBCNPs-specific proteins present in 75% of analyzed samples
  • Table F RBCEV-specific proteins (not present on RBCNPs of the invention). Table obtained from supplemental information to Pham et al., 2021
  • Chiangjong et al 2021 lists Synexin a major protein marker of RBCEVs. It is not present in the RBCNP samples.
  • Pham et al 2021 includes a mass-spectrometry-based list of the proteomic composition of RBCEVs. While some proteins are shared between RBCEVs and RBCNPs, most of the proteins we found are unique to RBCNPs (86%).
  • LC MS/MS was performed on RBCNPs manufactured using the method described in Example 1.
  • RBCNPs were assessed from 4 independent preparations. Proteins uniquely identified across all 4 RBCNPs preparations but not identified in previous reports (e.g. Pham et al., 2021), which are advantageous for maintenance of red blood cell (RBC) membrane structure and endothelial tissue binding, crossing the blood-brain barrier (BBB), and suppression of immune clearance suppression are included in Tables G-l below. Accession numbers included in the table below are obtained from UniProtKB.
  • Table H Improved delivery to target cells and blood-brain-barrier crossing
  • Table I Calpain Calpstatin system
  • TSG101 a tumor susceptibility gene, bidirectionally modulates cell invasion through regulating MMP-9 mRNA expression.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Cell Biology (AREA)
  • Medicinal Preparation (AREA)

Abstract

La présente invention concerne des nanoparticules dérivées de globules rouges (RBCNP), leurs utilisations et des procédés de production. Les nanoparticules dérivées de globules rouges de la présente invention sont utiles pour l'administration in vivo et in vitro de charges utiles.
PCT/EP2025/056903 2024-03-13 2025-03-13 Procédés et compositions Pending WO2025191080A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB2403647.7 2024-03-13
GBGB2403647.7A GB202403647D0 (en) 2024-03-13 2024-03-13 Method and compositions

Publications (1)

Publication Number Publication Date
WO2025191080A1 true WO2025191080A1 (fr) 2025-09-18

Family

ID=90730766

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2025/056903 Pending WO2025191080A1 (fr) 2024-03-13 2025-03-13 Procédés et compositions

Country Status (2)

Country Link
GB (1) GB202403647D0 (fr)
WO (1) WO2025191080A1 (fr)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA3056085A1 (fr) * 2017-02-10 2018-08-16 Universite Laval Vesicules extracellulaires derivees d'erythrocytes et proteines associees a de telles vesicules en tant que biomarqueurs de la maladie de parkinson
US20190054192A1 (en) 2017-08-16 2019-02-21 City University Of Hong Kong ISOLATION OF EXTRACELLULAR VESICLES (EVs) FROM RED BLOOD CELLS FOR GENE THERAPY
US20200316129A1 (en) * 2016-05-25 2020-10-08 The Johns Hopkins University Engineered anucleate cellular and extracellular vesicles as a novel biologics delivery platform
WO2021145821A1 (fr) * 2020-01-13 2021-07-22 Carmine Therapeutics Pte. Ltd. Vésicules extracellulaires de globules rouges chargées d'acide nucléique
WO2021194425A1 (fr) 2020-03-26 2021-09-30 National University Of Singapore Procédé d'administration d'acide nucléique à des cellules immunitaires à l'aide de rbcev
WO2022211740A1 (fr) 2021-03-31 2022-10-06 Carmine Therapeutics Pte. Ltd. Vésicules extracellulaires chargées avec au moins deux acides nucléiques différents

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200316129A1 (en) * 2016-05-25 2020-10-08 The Johns Hopkins University Engineered anucleate cellular and extracellular vesicles as a novel biologics delivery platform
CA3056085A1 (fr) * 2017-02-10 2018-08-16 Universite Laval Vesicules extracellulaires derivees d'erythrocytes et proteines associees a de telles vesicules en tant que biomarqueurs de la maladie de parkinson
US20190054192A1 (en) 2017-08-16 2019-02-21 City University Of Hong Kong ISOLATION OF EXTRACELLULAR VESICLES (EVs) FROM RED BLOOD CELLS FOR GENE THERAPY
WO2021145821A1 (fr) * 2020-01-13 2021-07-22 Carmine Therapeutics Pte. Ltd. Vésicules extracellulaires de globules rouges chargées d'acide nucléique
WO2021194425A1 (fr) 2020-03-26 2021-09-30 National University Of Singapore Procédé d'administration d'acide nucléique à des cellules immunitaires à l'aide de rbcev
WO2022211740A1 (fr) 2021-03-31 2022-10-06 Carmine Therapeutics Pte. Ltd. Vésicules extracellulaires chargées avec au moins deux acides nucléiques différents

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
CHIANGJONG ET AL.: "Red blood cell extracellular vesicle-based drug delivery: challenges and opportunities", FRONTIERS IN MEDICINE (LAUSANNE, vol. 8, 2021, pages 761362
LAUR�N EVA ET AL: "Phospholipid composition of packed red blood cells and that of extracellular vesicles show a high resemblance and stability during storage", BIOCHIMICA ET BIOPHYSICA ACTA (BBA) - MOLECULAR AND CELL BIOLOGY OF LIPIDS, vol. 1863, no. 1, 1 January 2018 (2018-01-01), pages 1 - 8, XP085263671, ISSN: 1388-1981, DOI: 10.1016/J.BBALIP.2017.09.012 *
PAPAYANNAKOS, C.J ET AL.: "Extracellular vesicles produced by primary human keratinocytes in response to TLR agonists induce stimulus-specific responses in antigen-presenting cells", CELLULAR SIGNALLING, 2021
PHAM ET AL.: "Covalent conjugation of extracellular vesicles with peptides and nanobodies for targeted therapeutic delivery", JOURNAL OF EXTRACELLULAR VESICLES, vol. 10, 2021, XP055948785, DOI: 10.1002/jev2.12057
PHAM TIN CHANH ET AL: "Covalent conjugation of extracellular vesicles with peptides and nanobodies for targeted therapeutic delivery", JOURNAL OF EXTRACELLULAR VESICLES, vol. 10, no. 4, 1 February 2021 (2021-02-01), UK, XP055948785, ISSN: 2001-3078, Retrieved from the Internet <URL:https://onlinelibrary.wiley.com/doi/full-xml/10.1002/jev2.12057> DOI: 10.1002/jev2.12057 *
SAI, X. ET AL.: "a tumor susceptibility gene, bidirectionally modulates cell invasion through regulating MMP-9 mRNA expression", BMC CANCER, vol. 15, 2015
USMAN ET AL.: "Efficient RNA drug delivery using red blood cell extracellular vesicles", NATURE COMMUNICATIONS, vol. 9, no. 2359, 2018
WAQAS MUHAMMAD USMAN ET AL: "Efficient RNA drug delivery using red blood cell extracellular vesicles", NATURE COMMUNICATIONS, VOL. 9, N. 1, ART. 2359, 15 June 2018 (2018-06-15), pages 1 - 15, XP055694577, Retrieved from the Internet <URL:http://www.nature.com/articles/s41467-018-04791-8> [retrieved on 20200513], DOI: 10.1038/s41467-018-04791-8 *

Also Published As

Publication number Publication date
GB202403647D0 (en) 2024-04-24

Similar Documents

Publication Publication Date Title
de Abreu et al. Native and bioengineered extracellular vesicles for cardiovascular therapeutics
JP7746319B2 (ja) 筋ジストロフィーの処置における心筋球由来細胞およびこのような細胞によって分泌されたエキソソーム
Hu et al. Platelet membrane and stem cell exosome hybrids enhance cellular uptake and targeting to heart injury
Maumus et al. Mesenchymal stem cell-derived extracellular vesicles: opportunities and challenges for clinical translation
Johnson et al. From mesenchymal stromal cells to engineered extracellular vesicles: a new therapeutic paradigm
Kim et al. Delivery of high mobility group box-1 siRNA using brain-targeting exosomes for ischemic stroke therapy
Zou et al. Lipid-mediated delivery of RNA is more efficient than delivery of DNA in non-dividing cells
You et al. Vitamin A-coupled stem cell-derived extracellular vesicles regulate the fibrotic cascade by targeting activated hepatic stellate cells in vivo
WO2021174738A1 (fr) Nanoparticule bionique recouverte d&#39;une membrane de cellules souches mésenchymateuses avec surface surexprimant la molécule pd-l1, sa préparation et son application
Liu et al. Therapeutic applications of extracellular vesicles for myocardial repair
Pan et al. Small extracellular vesicles: a novel drug delivery system for neurodegenerative disorders
AU2021272903A1 (en) Compositions and methods related to megakaryocyte-derived extracellular vesicles
Hecker Non-viral, lipid-mediated DNA and mRNA gene therapy of the central nervous system (CNS): chemical-based transfection
Hecker Nonviral, cationic lipid-mediated delivery of mRNA
Wu et al. Treg-derived extracellular vesicles: roles in diseases and theranostics
CA3195213A1 (fr) Vesicules de membrane cellulaire et utilisations associees
Lee et al. Heterogeneous subcellular origin of exosome-mimetic nanovesicles engineered from cells
WO2017070392A1 (fr) Appareil et procédés de production de tissus acellulaires pour la régénération d&#39;organes
WO2025191080A1 (fr) Procédés et compositions
Kim et al. TRITC-loaded PLGA nanoparticles as drug delivery carriers in mouse oocytes and embryos
Phelps et al. Extracellular Vesicles for the Treatment of Alzheimer's Disease: A Systematic Review
US20250195684A1 (en) Delivery methods using platelets
WO2025091119A1 (fr) Administration d&#39;agents thérapeutiques à base de cellules sanguines
US20240216532A1 (en) Compositions and methods related to megakaryocyte-derived extracellular vesicles for fanconi anemia
CN112675149B (zh) 负载环孢菌素a的脑靶向递药系统的制备方法和应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 25712563

Country of ref document: EP

Kind code of ref document: A1