Methods for enhanced production and isolation of cell-derived vesicles

Patent metadata

Field	Value
Publication number	US-2019008902-A1
Application number	US-201616067586-A
Country	US
Kind code	A1
Filing date	Dec 30, 2016
Priority date	Dec 30, 2015
Publication date	Jan 10, 2019
Grant date	—

Abstract

This disclosure relates to populations and compositions of purified cell-derived vesicles and uses thereof. One aspect of the disclosure relates to methods for purifying the cell-derived vesicles.

First claim

1 . A highly purified population of cell-derived vesicles prepared by culturing stem cells producing the cell-derived vesicles under conditions of hypoxia and low serum conditions, optionally wherein the cell-derived vesicles comprise exosomes and/or microvesicles.

2 . (canceled)

3 . The purified population of claim 1 , wherein the cell-derived vesicles are isolated from one or more stem cells of the group of adult stem cells, embryonic stem cells, embryonic-like stem cells, neural stem cells, mesenchymal stem cells, or induced pluripotent stem cells.

4 . (canceled)

5 . The purified population of claim 1 , wherein the cell-derived vesicles of the population further comprise at least one exogenous nucleic acid and/or at least one exogenous protein, optionally wherein the population of cell-derived vesicles do not comprise exogenous VEGFR and/or VEGF.

6 . The purified population of claim 5 , wherein the exogenous nucleic acid encodes a micro RNA (miRNA), optionally wherein the miRNA is selected from the group consisting of miR-150, miR-126, miR-132, miR-296, and let-7.

7 . (canceled)

8 . The purified population of claim 5 , wherein the exogenous protein is one or more of platelet derived growth factor receptor (PDGFR), Collagen, Type 1, Alpha 2 (COL1A2), Collagen, Type VI, Alpha 3 (COL6A3), EGF-like repeats- and discoidin i-like domains-containing protein 3 (EDIL3), epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), fibronectin (FN1), Milk fat globule-EGF factor 8 (MFGE8), lectin, galactoside-binding, soluble, 3 binding protein (LGALS3BP), nuclear factor-kappaB (NFκB), or transferrin (TF).

9 . (canceled)

10 . The purified population of claim 1 , wherein the cell-derived vesicles of the population comprise one or more of miR-126, miR-132, miR-150, miR-210, miR-214, miR-296, and miR-424.

11 - 16 . (canceled)

17 . The purified population of claim 1 , wherein the cell-derived vesicles of the population comprise one or more of 3,6-anhydro-D-galactose, 4-aminobutyric acid, 5′-deoxy-5′-methylthioadenosine, 5-methoxytryptamine, s-adenosylmethionine, s-adenosylhomocysteine, adipic acid, aminomalonate, arabinose, aspartic acid, beta-alanine, cholesterol, citric acid, creatinine, cysteine, cytidine-5-monophosphate, erythritol, fructose, fumaric acid, galacturonic acid, glucose, glucose-1-phosphate, glucose-6-phosphate, glutamine, glyceric acid, glycerol-alpha-phosphate, glycine, guanosine, hexitol, hexuronic acid, inosine, isohexonic acid, isomaltose, lactamide, lactic acid, lactose, leucine, levoglucosan, maleimide, malic acid, maltotriose, mannose, methanolphosphate, methionine, N-acetylaspartic acid, N-acetyl-D-galactosamine, nicotinamide, N-methylalanine, oxoproline, pantothenic acid, pentadecanoic acid, phenol, putrescine, pyruvic acid, ribitol, ribose, sorbitol, squalene, succinic acid, threitol, threonic acid, threonine, thymine, trans-4-hydroxyproline, trehalose, urea, uridine, valine, and xylitol.

18 - 26 . (canceled)

27 . The purified population of claim 1 , wherein the cell-derived vesicles of the population comprise one or more of Ceramide (d32:1), Ceramide (d33:1), Ceramide (d34:0), Ceramide (d34:1), Ceramide (d34:2), Ceramide (d34:2), Ceramide (d36:1), Ceramide (d38:1), Ceramide (d39:1), Ceramide (d40:0), Ceramide (d40:1), Ceramide (d40:2), Ceramide (d41:1), Ceramide (d42:1), Ceramide (d42:2) B, Ceramide (d44:1), Fatty Acid (20:4), Fatty Acid (22:0), Fatty Acid (22:6), Fatty Acid (24:0), Fatty Acid (24:1), glucosylceramides (d40:1), glucosylceramides (d41:1), glucosylceramides (d42:1), glucosylceramides (d42:2), Lysophosphatidylcholines (16:0), Lysophosphatidylcholines (18:0) A, Lysophosphatidylcholines (18:1), lysophosphatidylethanolamine (20:4), Phosphatidylcholines (32:1), Phosphatidylcholines (33:1), Phosphatidylcholines (34:0), Phosphatidylcholines (34:1), Phosphatidylcholines (34:2), Phosphatidylcholines (35:2), Phosphatidylcholines (36:1), Phosphatidylcholines (36:2), Phosphatidylcholines (36:3), Phosphatidylcholines (38:2), Phosphatidylcholines (38:3), Phosphatidylcholines (38:5), Phosphatidylcholines (38:6), Phosphatidylcholines (40:5), Phosphatidylcholines (40:6), Phosphatidylcholines (40:7), Phosphatidylcholines (p-34:0), Phosphatidylcholines (o-34:1), Phosphatidylethanolamines (34:1), Phosphatidylethanolamines (34:2), Phosphatidylethanolamines (36:3), Phosphatidylethanolamines (36:4), Phosphatidylethanolamines (38:4), B Phosphatidylethanolamines (38:6), Phosphatidylethanolamines (p-34:1), Phosphatidylethanolamines (o-34:2), Phosphatidylethanolamines (p-36:1), Phosphatidylethanolamines (o-36:2), Phosphatidylethanolamines (p-36:4), Phosphatidylethanolamines (o-36:5), Phosphatidylethanolamines (p-38:4), Phosphatidylethanolamines (o-38:5), Phosphatidylethanolamines (p-38:5), Phosphatidylethanolamines (o-38:6), Phosphatidylethanolamines (p-38:6), Phosphatidylethanolamines (o-38:7), Phosphatidylethanolamines (p-40:4), Phosphatidylethanolamines (o-40:5), Phosphatidylethanolamines (p-40:5), Phosphatidylethanolamines (o-40:6), Phosphatidylethanolamines (p-40:6), Phosphatidylethanolamines (o-40:7), Phosphatidylethanolamines (p-40:7), Phosphatidylethanolamines (o-40:8), Sphingomyelin (d30:1), Sphingomyelin (d32:0), Sphingomyelin (d32:2), Sphingomyelin (d33:1), Sphingomyelin (d34:0), Sphingomyelin (d36:1), Sphingomyelin (d36:2), Sphingomyelin (d38:1), Sphingomyelin (d40:1), Sphingomyelin (d40:2), Sphingomyelin (d41:1), Sphingomyelin (d41:2), Sphingomyelin (d42:2), and B Sphingomyelin (d42:3).

28 - 36 . (canceled)

37 . The purified population of claim 1 , wherein the cell-derived vesicles of the population comprise one or more of CD9, HSPA8, PDCD6IP, GAPDH, ACTB, ANXA2, CD63, SDCBP, ENO1, HSP90AA1, TSG101, PKM, LDHA, EEF1A1, YWHAZ, PGK1, EEF2, ALDOA, ANXA5, FASN, YWHAE, CLTC, CD81, ALB, VCP, TPI1, PPIA, MSN, CFL1, PRDX1, PFN1, RAP1B, ITGB1, HSPA5, SLC3A2, GNB2, ATP1A1, WHAQ, FLOT1, FLNA, CLIC1, CDC42, CCT2, A2M, YWHAG, RAC1, LGALS3BP, HSPA1A, GNAI2, ANXA1, RHOA, MFGE8, PRDX2, GDI2, EHD4, ACTN4, YWHAB, RAB7A, LDHB, GNAS, TFRC, RAB5C, ANXA6, ANXA11, KPNB1, EZR, ANXA4, ACLY, TUBA1C, RAB14, HIST2H4A, GNB1, UBA1, THBS1, RAN, RAB5A, PTGFRN, CCT5, CCT3, BSG, RAB5B, RAB1A, LAMP2, ITGA6, GSN, FN1, YWHAH, TKT, TCP1, STOM, SLC16A1, and RAB8A proteins.

38 - 46 . (canceled)

47 . The purified population of claim 1 , wherein the cell-derived vesicles of the population comprise one or more of FN1, EDIL3, TF, ITGB1, VCAN, ANXA2, MFGE8, TGB1, TGFB2, TGFBR1, TGBFR2, TGFBI, TGFBRAP1, BASP1, COL1, COL6, GAPDH, ITGA3, FBN1, ITGAV, ITGB5, NOTCH2, SDCBP, HSPA2, HSPA8, NT5E, MRGPRF, RTN4, NEFM, INA, NRP1, HSPA9, FBN1, BSG, PRPH, FBLN1, PARP4, FLNA, YBX1, EVA1B, ADAM10, HSPG2, MCAM, POSTN, GNB2, GNB1, ANPEP, ADAM9, ATP1A1, CSPG4, EHD2, PXDN, SERPINE2, CAV1, PKM, GNB4, NPTN, CCT2, LGALS3BP, and MVP proteins.

48 - 56 . (canceled)

57 . The purified population of claim 1 , wherein the cell-derived vesicles of the population comprise one or more of FBLN2, TIMP1, NID1, IGFBP3, LTBP1, DUSP3, ITGAV, LAMA5, COL1A1, NOTCH2, NRG1, ERBB2, COL4A2, LDLR, TSB, MMP2, TIMP2, TPI1, ACVR1B, INHBA, EGFR, APH1A, NCSTN, TGFB2, SPARC, TGFB1, F2, SERPINE1, SDC4, SDC3, ACAN, IFI16, MMP14, PLAT, COL18A1, NOTCH3, DSP, PKP4, SERPINE2, SRGN, NRP2, EPHA2, ITGA5, NRP1, PLAU, SERPINB6, CLEC3B, CD47, SDC1, PSMA7, ENG, S100A13, TIMP3, TMED10, TGFBI, CTGF, DCN, ITGB3, PDGFRA, JAG1, TGFBR2, PLAUR, PDGFRB, FYN, THY1, HSPG2, TENC1, TGFBR1, PLXNA1, LRP1, STAT1, CXCL12, VCAN, MET, FN1, CD36, STAT3, THBS1, FGFR1, GRB14, FGB, API5, HAPLN1, RECK, LAMC1, CYR61, GPC1

Assignees

• Univ California

Inventors

• Anderson Johnathon D
• Nolta Jan A
• Bauer Gerhard
• Fury Brian

Classifications

• C07K14/71

  for growth factors; for growth regulators · CPC title

• C12M47/12

  Purification (C12M47/04 takes precedence) · CPC title

• A61P25/00

  Drugs for disorders of the nervous system · CPC title

• C12M47/06

  Hydrolysis; Cell lysis; Extraction of intracellular or cell wall material (lysis of microorganisms C12N1/06; extracting or separating nucleic acids from biological samples C12N15/1003) · CPC title

• A61K9/19

  lyophilised {, i.e. freeze-dried, solutions or dispersions (lyophilised products with subsequent particle size reduction A61K9/14; granules or pellets made by lyphilisation A61K9/1682; solid oral dosage forms made by lyophilisation A61K9/2095; lyophilisation additives A61K47/00)} · CPC title