WO2020077030A1 - Aggf1 et cellules sensibilisées par aggf1 pour le traitement de maladies et d'états pathologiques - Google Patents

Aggf1 et cellules sensibilisées par aggf1 pour le traitement de maladies et d'états pathologiques Download PDF

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WO2020077030A1
WO2020077030A1 PCT/US2019/055513 US2019055513W WO2020077030A1 WO 2020077030 A1 WO2020077030 A1 WO 2020077030A1 US 2019055513 W US2019055513 W US 2019055513W WO 2020077030 A1 WO2020077030 A1 WO 2020077030A1
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aggf1
peptide
seq
composition
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Qing Wang
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Cleveland Clinic Foundation
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Cleveland Clinic Foundation
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    • 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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/515—Angiogenesic factors; Angiogenin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/44—Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
    • 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
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1891—Angiogenesic factors; Angiogenin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • compositions, systems, kits, and methods for treating a subject with a disease or condition characterized by abnormal glucose metabolism (e.g., diabetes) or ischemic vascular disease (e.g., PAD or CAD) by administering a composition comprising AGGF1 peptides, or an expression vector encoding said peptides, or AGGF1 -primed endothelial progenitor cells, to a subject.
  • a composition comprising AGGF1 peptides, or an expression vector encoding said peptides, or AGGF1 -primed endothelial progenitor cells, to a subject.
  • the AGGF1 peptide is full- length AGGF1, or a biologically active fragment or mutant thereof.
  • the AGGF1 peptides or expression vectors are administered around the same time insulin is administered to the subject.
  • Diabetes mellitus is a common and heterogeneous disease with high morbidity and mortality worldwide. An estimated 422 million adults are affected with diabetes based on the WHO’s most recent 2016 data.
  • the prevalence of diabetes, and in particular, of type 2 diabetes (T2DM) increases rapidly each year due to lifestyle habits and increased rates of aging, inactivity, and obesity, and poses an important challenge to human health.
  • T2DM type 2 diabetes
  • the treatment of diabetes remains suboptimal in a significant proportion of patients.
  • >50% of T2DM patients have poor glycemic control and 18% develop severe complications.
  • Population HbAlc concentrations failed to decline as much as expected due to poor medication compliance and other causes. To address this grand challenge, much is needed to understand the pathophysiology of diabetes and to develop new innovative therapies. In particular, long-lasting treatment can dramatically reduce poor medication compliance.
  • compositions, systems, kits, and methods for treating a subject with a disease or condition characterized by abnormal glucose metabolism (e.g., diabetes) or ischemic vascular disease (e.g., PAD or CAD) by administering a composition comprising AGGF1 peptides, or an expression vector encoding said peptides, or AGGF1 -primed endothelial progenitor cells, to a subject.
  • a composition comprising AGGF1 peptides, or an expression vector encoding said peptides, or AGGF1 -primed endothelial progenitor cells, to a subject.
  • the AGGF1 peptide is full- length AGGF1, or a biologically active fragment or mutant thereof.
  • the AGGF1 peptides or expression vectors are administered around the same time insulin is administered to the subject.
  • provided herein are methods of treating a subject with a disease or condition characterized by abnormal glucose metabolism comprising:
  • the methods further comprise administering insulin to the subject.
  • the insulin is administered at about the same time as the composition.
  • kits for treating a subject comprising: administering a composition to a subject, wherein the subject has at least one of the following: a disease or condition characterized by abnormal glucose metabolism, ischemic vascular disease, pulmonary hypertension, cardiac hypertrophy, and heart failure: wherein said composition comprises: i) an AGGF1 peptide, and/or ii) an expression vector encoding said AGGF1 peptide.
  • a cardiovascular disease or condition characterized comprising: administering a composition comprising: i) an AGGF1 peptide that is no longer than 600 amino acids (e.g., 100-600 or 100-300 or 100-150 amino acids in length), or ii) an expression vector encoding the AGGF1 peptide, to the subject with the disease or condition.
  • the disease or condition is selected from the group consisting of: coronary artery disease, myocardial infarction, cardiac hypertrophy, heart failure, peripheral artery disease, restenosis and in-stent thrombosis after angioplasty and stenting, and pulmonary arterial hypertension.
  • pulmonary arterial hypertension comprising: administering a composition comprising: i) an AGGF1 peptide, or ii) an expression vector encoding the AGGF1 peptide, to the subject with the PAH.
  • the subject in any of the methods herein is a human and: A) the composition comprises said AGGF1 peptide, and wherein the dosage is selected from the group consisting of: 0.1-15 mg/kg (e.g., 0.1 mg/kg ... 4.0 mg/kg ... 8.0 mg/k ... 12.0 mg/kg ... and 15 mg/kg), 0.5-10 mg/kg, and 1-7 mg/kg; or B) said composition comprises said expression vector encoding said AGGF1 peptide, and wherein said dosage is about 1.0 x 10 11 - 5.0 x 10 13 (e.g., 1.0 x 10 11 ... 1.0 x 10 12 ... or 5.0 x 10 13 ).
  • kits comprising: a) a blood glucose monitoring device or article of manufacture; and b) a composition comprising: i) an AGGF1 peptide, or ii) an expression vector encoding the AGGF1 peptide.
  • the glucose monitoring device or article of manufacture is selected from the group consisting of: a blood glucose test strip, a reader for reading the glucose test strip, FreeStyle Libre, GlucoTrack, Eversense, GlucoWise, NovioSense, GlucoSense, and a smart contact lens.
  • kits comprising: a) a composition comprising: i) an AGGF1 peptide, or ii) an expression vector encoding the AGGF1 peptide; and b) an implantable pump containing the composition, wherein the implantable pump is configured to deliver an amount of the composition into the bloodstream of a subject.
  • compositions comprising: a) an AGGF1 peptide, or an expression vector encoding the AGGF1 peptide; and b) insulin peptide, or an expression vector encoding the insulin peptide.
  • kits for treating a subject comprising: administering a composition to a subject, wherein the subject has a disease or condition characterized by abnormal glucose metabolism and/or ischemic vascular disease, and wherein the composition comprises an AGGFl-primed endothelial progenitor cell.
  • the disease or condition is selected from the group consisting of peripheral artery disease (PAD), coronary artery disease (CAD), coronary heart disease (CHD), and myocardial infarction (MI).
  • the AGGFl-primed endothelial progenitor cell comprises an endothelial progenitor cell (EPC) from the subject, wherein the EPC has been exposed to AGGF1 peptides ex vivo.
  • the AGGFl- primed endothelial progenitor cell comprises an endothelial progenitor cell (EPC) from a relative of the subject, wherein the EPC has been exposed to AGGF1 peptides ex vivo.
  • the AGGFl-primed endothelial progenitor cell comprises an endothelial progenitor cell (EPC) from a non-relative of the subject, wherein the EPC has been exposed to AGGF1 peptides ex vivo.
  • EPC endothelial progenitor cell
  • compositions comprising: an AGGFl- primed endothelial progenitor cell.
  • the composition further comprises cell growth media.
  • the AGGFl-primed endothelial progenitor cell comprises an endothelial progenitor cell (EPC) from a subject with a disease or condition characterized by abnormal glucose metabolism and/or ischemic vascular disease, wherein the EPC has been exposed to AGGF1 peptides ex vivo.
  • the compositions further comprise a physiological tolerable liquid.
  • kits and systems comprising: a) an endothelial progenitor cell (EPC), and b) a composition comprising: i) an AGGF1 peptide, and/or ii) an expression vector encoding the AGGF1 peptide.
  • EPC endothelial progenitor cell
  • the AGGF1 peptide comprises full length human AGGF1 shown in SEQ ID NO: 1.
  • the AGGF1 peptide has at least 97% sequence identity with SEQ ID NO: 1 (e.g., 97 ... 98 .. 99 .. 99.5% identify).
  • the AGGF1 peptide is selected from any of SEQ ID Nos:2-l6 or i) a peptide comprising the amino acid sequence from 574 to 614 of SEQ ID NO:l, ii) a peptide comprising the amino acid sequence from 574 to 624 of SEQ ID NO:l, or iii) a peptide comprising the amino acid sequence from 604 to 714 of SEQ ID NO:l.
  • the AGGF1 peptide has at least 97% sequence identity (e.g., 97 ... 98 .. 99 ..
  • the subject is a human.
  • the AGGF1 peptide consists of, consists essentially of, or comprises QRDDAPASVH (SEQ ID NO: 18).
  • an expression vector encoding SEQ ID NO: 18 is employed.
  • the sequence encoding SEQ ID NO: 18 is CAAAGAGAUGAUG
  • CUCCUGCAUCUGUUCAU (SEQ ID NO:25).
  • the expression vectors herein comprise SEQ ID NO:25.
  • AGGF1 regulates glucose metabolism and insulin resistance.
  • A Body weight of AGGFl +/_ KO mice and littermate WT mice fed a standard chow diet or a high fat diet (HFD).
  • B Fasting glucose levels in AGGFl +/_ KO mice and littermate WT mice fed HFD.
  • C Fasting insulin levels in AGGFl +/ KO mice and littermate WT mice fed HFD.
  • D Glucose tolerance tests in AGGFl +/_ KO mice and littermate WT mice fed HFD.
  • E Insulin tolerance tests in AGGFl +/ KO mice and littermate WT mice fed HFD. *P ⁇ 0.01,
  • Fig. 3 Therapeutic effects of chronic AGGF1 protein therapy on lowering fasting glucose levels.
  • 24-week-old male HFD-induced diabetic mice were treated by intraperitoneal injection of 0.25 mg/kg AGGF1 protein or PBS control every 2 days for 36 days (chronic treatment).
  • A Fasting serum glucose levels.
  • AGGF1 protein therapy is insulin-dependent.
  • A Therapeutic effects of insulin alone (1.00 ug/kg body weight) and in combination with AGGF1 (0.25 mg/kg body weight) in 24-week-old male HFD-induced diabetic mice.
  • B No therapeutic effect for AGGF1 protein injection on fasting glucose levels in 16-week-old male diabetic mice induced by streptozotocin (STZ) for 4 weeks.
  • Figure 5a shows the amino acid sequence of full-length human AGGF1 (714 amino acid residues) (SEQ ID NO:l).
  • Figure 5b shows the amino acid sequence of AGGF1-N1 (696 amino acids; 19 amino acid deletion from the N-terminus of AGGF1) (SEQ ID NO:2).
  • Figure 5c shows the amino acid sequence of AGGF1-N2 (629 amino acids; 86 amino acid deletion from the N-terminus of AGGF1) (SEQ ID NOG).
  • Figure 6a shows the amino acid sequence of AGGF1-N3 (550 amino acids; 165 amino acid deletion from the N-terminus of AGGF1) (SEQ ID NO:4).
  • Figure 6b shows the amino acid sequence of AGGF1-N4 (496 amino acids; 219 amino acid deletion from the N- terminus of AGGF1) (SEQ ID NOG).
  • Figure 6c shows the amino acid sequence of AGGF1- N5 (431 amino acids; 284 amino acid deletion from the N-terminus of AGGF1) (SEQ ID NO:6).
  • Figure 6d shows the amino acid sequence of AGGF1-N6 (366 amino acids; 349 amino acid deletion from the N-terminus of AGGF1) (SEQ ID NO:7).
  • Figure 7a shows the amino acid sequence of AGGF1-N7 (302 amino acids; 413 amino acid deletion from the N-terminus of AGGF1) (SEQ ID NOG).
  • Figure 7b shows the amino acid sequence of AGGF1-N8 (244 amino acids; 472 amino acid deletion from the N- terminus of AGGF1) (SEQ ID NO:9).
  • Figure 7c shows the amino acid sequence of AGGF1- N9 (193 amino acids; 522 amino acid deletion from the N-terminus of AGGF1) (SEQ ID NO: 10).
  • Figure 7d show the amino acid sequence of AGGF1-N10 (141 amino acids; 574 amino acid deletion from the N-terminus of AGGF1) (SEQ ID NO: 11).
  • Figure 7e shows the amino acid sequence of AGGF1-N101 (131 amino acids; 584 amino acid deletion from the N-terminus of AGGF1) (SEQ ID NO: 12).
  • Figure 7f shows the amino acid sequence of AGGF1-N102 (121 amino acids; 594 amino acid deletion from the N-terminus of AGGF1) (SEQ ID NO: 13).
  • Figure 8a shows the amino acid sequence of AGGF1-N103 (110 amino acids; 604 amino acid deletion from the N-terminus of AGGF1) (SEQ ID NO: 14).
  • Figure 8b shows the amino acid sequence of AGGF1-C1 (664 amino acids; 50 amino acid deletion from the C- terminus of AGGF1) (SEQ ID NO: 15).
  • Figure 8c shows the amino acid sequence of AGGF1-C2 (614 amino acids; 100 amino acid deletion from the C-terminus of AGGF1) (SEQ ID NO: 16).
  • Figure 9 shows the full length human AGGF1 cDNA (SEQ ID NO: 17), with arrows showing truncation mutants.
  • FIG 10 shows a diagram showing the full length AGGF1 (WT) and its serial N- terminal and C-terminal deletion mutant proteins.
  • Figure 11 shows results of purification of mutant AGGF1 proteins with serial N- and C-terminal deletions.
  • Figure 12 shows results of AGGF1 -endothelial cell adhesion (binding) assays for serial N- and C-terminal AGGF1 deletion mutants.
  • Figure 13 shows results of Endothelial cell migration assays for serial N- and C- terminal AGGF1 deletion mutants.
  • Figure 14 shows results of angiogenesis assays for serial N- and C-terminal AGGF1 deletion mutants (capillary endothelial tube formation assays).
  • FIG. 15 Micro-deletion analysis identified the short mutant form of AGGF1 with full functions of the full-length AGGF1 protein.
  • A A diagram showing the full length AGGF1 (WT) and serial N-terminal deletion mutant proteins.
  • B Endothelial cell adhesion assay with AGGF1 and its N-terminal deletion mutants. Coating buffer (CB) with and without BSA was used as a negative control. *P ⁇ 0.05.
  • C Endothelial cell migration assay with AGGF1 and its N-terminal deletion mutants. BSA was used as a negative control. *P ⁇ 0.05.
  • D Endothelial tube formation assay AGGF1 and its N-terminal deletion mutants. BSA was used as a negative control (left panel).
  • FIG. 16 Aggfl +/ KO mice spontaneously developed PAH.
  • Male Agg[] 0,‘o/+ KO mice (16-20 weeks) showed much higher mPAP (mean pulmonary arterial pressure) (A, B), total pulmonary resistance (TPR) (C), and right ventricular hypertrophy compared to WT mice (D).
  • mPAP mean pulmonary arterial pressure
  • TPR total pulmonary resistance
  • D right ventricular hypertrophy compared to WT mice
  • FIG. 17 Aggfl +/ KO causes vascular remodeling.
  • A Representative H&E of pulmonary arteries.
  • B Mean medial wall thickness.
  • C Resistance pulmonary arterial muscularization.
  • lung sections (5 pm) were co-immunostained with an a-smooth muscle actin antibody (a-SMA, VSMC marker) and a CD31 antibody (EC maker). Vessels were classified as fully (100%), partially, or non-muscularized (0%) vessels based on the percentage of CD31 signal surrounded by a-SMA signal.
  • Aggfl is required for essential functions of EPCs in capillary tube formation, proliferation, transendothelial migration, and migration as in a diabetic mouse model ( db/db mice).
  • EPCs were isolated from bone marrow of WT, heterozygous Aggfl +/ KO mice, db/db mice, and Aggfl +/ db/db mice and characterized.
  • Aggfl is required for essential functions of EPCs in capillary tube formation, proliferation, transendothelial migration, and migration as in a diabetic mouse model ( db/db mice).
  • EPCs were isolated from bone marrow of WT, heterozygous Aggfl +/ KO mice, db/db mice, and Aggfl +/ db/db mice and characterized.
  • haploinsufficiency inhibits angiogensis mediated by EPCs.
  • B Images from (A) were analyzed, quantified and plotted.
  • C Aggfl haploinsufficiency inhibits EPC proliferation.
  • FIG. 20 AGGF1 dramatically improves essential functions of EPCs impaired by HG.
  • A Western blot analysis showing increased AGGF1 expression in EPCs.
  • B AGGF1 treatment reversed the impairment of angiogenic function of EPCs by HG.
  • C Images from (B) were analyzed, quantified and plotted.
  • D AGGF1 treatment reversed the reduced cell proliferation of EPCs by HG.
  • E AGGF1 treatment reversed the HG-impaired
  • FIG. 21 AGGF1 protein treatment robustly potentiates the therapeutic effects of EPCs on peripheral vascular complications in a hindlimb ischemia model in db/db mice.
  • a hindlimb ischemia model was created in db/db mice.
  • C Therapeutic effects of AGGF1 -pretreated EPCs on ambulatory impairment compared with EPCs without AGGF1 pretreatment.
  • D Effects of AGGF1 -pretreated EPCs on the density of CD3l-positive capillary vessels compared with EPCs without AGGF1 pretreatment.
  • FIG. 22 AGGF1 regulates the nuclear accumulation of Nrf2.
  • A Western blot analysis for the effect of AGGF1, HG and HG+AGGF1 on the expression levels of nuclear Nrf2 (n-Nrf2) and its downstream signaling molecules, including HQ-l, NQO-l, and CAT. GAPDH was used as loading control.
  • FIG. 23 AGGF1 activates AKT-Fyn-Nrf2 signaling in EPCs.
  • A-B Western blot analysis showing the effect of siRNA for A KT (siAKT) (A) and wortmannin (B) on AGGF1- activated phosphorylation of AKT and nuclear accumulation of Fyn (n-Fyn).
  • FIG. 24 Knockdown of AKT expression, wortmannin treatment, and knockdown of Nrf2 expression attenuate the protective effects of AGGF1 on EPCs.
  • A Western blot analysis for AGGF1 in EPCs.
  • B-E Effects of AKT siRNA on AGGF1 -mediated rescue of HG-impaired cell proliferation (B), tube formation (C), transendothelial migration (D), and cell migration (E) by EPCs.
  • G-J Effects of wortmannin on AGGF1 -mediated rescue of HG-impaired cell proliferation (G), tube formation (H), transendothelial migration (I), and cell migration (J) by EPCs.
  • FIG. 25 Isolation and characterization of bone marrow derived EPCs from mice.
  • Bone marrow mononuclear cells (MNCs) were washed out from the femurs and tibias of mice and isolated by density gradient centrifugation with histopaque-l083.
  • MNCs were plated on gelatin-coated culture dishes and maintained in endothelial growth factor- supplemented media (EGM-2 bullet kit). Ten days after maintenance in endothelial-specific media, non-adherent MNCs were removed and the remaining adherent cells were subjected to immunostaining to verify their EPC identity.
  • EMM-2 bullet kit Endothelial growth factor- supplemented media
  • FIG. 26 Aggfl haploinsufficiency significantly reduces the number of bone marrow mononuclear cells (MNCs).
  • MNCs were washed out from the femurs and tibias of WT and Aggfl +/ KO mice and purified by density gradient centrifugation with histopaque-l083.
  • MNCs were plated into gelatin-coated culture dishes and maintained in endothelial growth factor-supplemented media (EGM-2 bullet kit for ten days). Non-adherent MNCs were removed and the remaining adherent cells were subjected to immunostaining to verify their EPC identity.
  • FIG. 27 The expression level of the AGGF1 protein is increased in EPCs isolated from db/db mice or mice treated with HFD.
  • A Western blot analysis showing increased AGGF1 expression in isolated EPCs from db/db mice.
  • FIG. 28 Increased glucose levels in mice fed with a high fat diet (HFD).
  • HFD high fat diet
  • C57BL/6J mice were fed with a high fat diet alone to induce T2DM.
  • Mice fed with a chow diet were sued as controls.
  • blood glucose levels were measured using a glucometer, and then a hind-limb ischemia model was established with the mice.
  • FIG. 30 Aggfl is required for essential functions of EPCs in capillary tube formation, proliferation, transendothelial migration, and migration as in a HFD-induced diabetic mouse model.
  • EPCs were isolated from bone marrow of wild-type (WT) and heterozygous Aggfl +/ knockout (KO) mice fed with and without a high glucose diet (HFD), and characterized.
  • WT wild-type
  • KO heterozygous Aggfl +/ knockout mice fed with and without a high glucose diet
  • HFD high glucose diet
  • Aggfl haploinsufficiency inhibits angiogensis mediated by EPCs. Angiogenic function of EPCs was assessed by a matrigel-based capillary tube formation.
  • B Images from (A) were analyzed, quantified and plotted.
  • C Aggfl haploinsufficiency inhibits EPC proliferation.
  • EPCs Proliferation of EPCs was examined using the CCK-8 kit.
  • D Aggfl haploinsufficiency inhibits transendothelial migration of EPCs in transwell assays.
  • HUVECs about lxlO 4 cells per well
  • EPCs were harvested, resuspended in basal culture medium (EBM-2, 0.5% BSA), and added to the upper chamber of the transwell plate (0.2 ml). Images were captured 24 hours after EPCs plating with an inverted Nikon Eclipse Ti microscope.
  • E Images from (D) were quantified and plotted.
  • F Aggfl haploinsufficiency inhibits EPC migration in wound scratch migration assays.
  • EPCs monolayers in 6-well cell culture plates were wounded with a scratch by a 200 pl pipette tip and examined 24 hours after wounding. Images were captured with an inverted Nikon Eclipse Ti microscope.
  • FIG 32 AGGF1 protein treatment robustly potentiates the therapeutic effects of EPCs on angiogenesis and homing in a hindlimb ischemia model in db/db mice.
  • FIG. 33 AGGF1 protein treatment robustly potentiates the therapeutic effects of EPCs on peripheral vascular complications in a hindlimb ischemia model.
  • a hindlimb ischemia model was created in mice. Two days after the surgery, the mice were injected with EPCs, AGGFl-pretrearted EPCs ( ⁇ lxl0 6 ), and control PBS via the tail vein. The therapeutic effects were evaluated using a Vevo 2100 High- Resolution Micro-Ultrasound System before the ischemic surgery and 7, 14 and 28 days after ischemia.
  • A Transplantation of AGGF1- pretreated EPCs dramatically improved blood perfusion compared with EPCs without AGGF1 pretreatment.
  • Scale bar l00 pm.
  • DCFH-DA Dichlorodihydrofluorescein diacetate
  • FIG. 36 AGGF1 increases the level of nuclear Nrf2 in EPCs.
  • FIG. 37 AGGF1 does not affect the expression levels of Nrf2 and KEAP-l in EPCs.
  • EPCs were cultured in media with or without high glucose (HG, 30 mM) as well as a combination of high glucose and AGGF1 protein.
  • A Total RNA was extracted with Trizol reagent and used for real-time RT-PCR analysis for the level of Nrf2 mRNA.
  • B Western blot analysis with an anti-Nrf2 antibody.
  • FIG 38 Western blot analysis for nuclear accumulation of nNrf2 in EPCs treated with Aggfl siRNA and Fyn siRNA under the normal condition (Control) and under a high glucose condition (HG). GAPDH was used as loading control.
  • FIG 39 A schematic diagram showing the hypothetical molecular signaling pathway by which AGGF1 protein therapy potentiates the therapeutic effects of EPCs on diabetes-induced vascular complications. It is noted that the present invention is not limited to this particular mechanism, and an understanding of the mechanism is not necessary to practice the inventin.
  • AGGF1 activates AKT signaling, which inhibits Fyn-mediated export and degradation of nuclear Nrf2. Nrf2 binds to the promoter and regulatory region and activate the transcription of CAT, H(2-l, and NQO-1, which function as antioxidants to block generation of ROS, reducing oxidative stress and increasing function of EPCs. Overall, AGGF1 functions as an antioxidant regulator to protect the function of EPCs, and becomes a potential therapeutic target for improving the ischemia-reparative capacity of EPCs transplantation in DM.
  • Figure 40 shows the duration of the glucose-lowering effect of injection of a single dose of AGGF1 protein is more than 10 times longer than insulin in db/db diabetic mice.
  • A Experimental design of the 48-hour AGGF1 treatment assay in Example 4. The time points for AGGF1 injection, glucose measurement, fasting period, and eating period are shown.
  • the term“or” is an inclusive“or” operator and is equivalent to the term“and/or” unless the context clearly dictates otherwise.
  • the meaning of“a”,“an”, and“the” include plural references.
  • the meaning of“in” includes“in” and“on.”
  • the terms“subject” and“patient” refer to any animal, such as a mammal like a dog, cat, bird, livestock, and preferably a human (e.g., a human with a disease such as diabetes, cardiovascular disease, or related conditions).
  • the term“administration” refers to the act of giving a drug, prodrug, or other agent, or therapeutic treatment to a subject.
  • exemplary routes of administration to the human body can be through the mouth (oral), skin (transdermal, topical), nose (nasal), lungs (inhalant), oral mucosa (buccal), inhaled, an implanted pump activation, by injection (e.g., intravenously, subcutaneously, intratumorally, intraocular, intraperitoneally, etc.), and the like.
  • compositions, systems, kits, and methods for treating a subject with a disease or condition characterized by abnormal glucose metabolism (e.g., diabetes) or ischemic vascular disease (e.g., PAD or CAD) by administering a composition comprising AGGF1 peptides, or an expression vector encoding said peptides, or AGGF1 -primed endothelial progenitor cells, to a subject.
  • a composition comprising AGGF1 peptides, or an expression vector encoding said peptides, or AGGF1 -primed endothelial progenitor cells, to a subject.
  • the AGGF1 peptide is full- length AGGF1, or a biologically active fragment or mutant thereof.
  • the AGGF1 peptides or expression vectors are administered around the same time insulin is administered to the subject.
  • AGGF1 protein and its derivatives can effectively lower glucose levels.
  • therapies with AGGF1 and AGGF1 derivatives have a longer therapeutic effect, and do not cause hypoglycemia.
  • work conducted during development of embodiments herein demonstrated AGGF1 treatment has a much longer therapeutic effect with a peak effect at 18-24 hours and lasting for >42 hours.
  • AGGF1 peptides or expression vector encoding AGGF1
  • insulin are administered at about the same time (e.g., within 1-25 minutes of each other).
  • Combined treatment with both insulin and AGGF1 can quickly decrease the fasting glucose level within 30 minutes, but also significantly extend the effect of insulin. Therefore, the therapy with combined insulin and AGGF1 can have a sustained lowering effect on glucose levels for a much longer time than insulin alone.
  • patients typically require insulin injections 3 times per day. Even the long-acting insulin such as insulin glargine (Lantus, Sanofi) requires injection once a day.
  • the combined AGGF1 and insulin treatment reduces the frequency of injection to once per >2 days.
  • the various embodiments described herein can enable more patients with type 1 and type 2 diabetes mellitus to reach better glucose targets by reducing the frequency of injections of drugs and enhancing medication compliance, and maintain a better quality of life.
  • AGGF1 is an angiogenic protein that has been proposed previously for use in treating cardiovascular diseases. In work conducted during the development of embodiments herein, it was shown that AGGF1 has activities similar to insulin. In AGGF1 KO mice, fasting glucose levels were significantly elevated when mice were fed a high fat diet and were insulin resistant. In diabetic mice, AGGF1 administration significantly lowered fasting glucose levels. In streptozotocin mice, the effects of AGGF1 seemed to depend on and potentiate insulins activities.
  • AGGF1 therapy e.g., protein or expression vector encoding protein
  • benefits of AGGF1 therapy include, but are not limited to, AGGF1 peptides regulate glucose levels more precisely than insulin, no evidence of hypoglycemia, longer therapeutic window, peak glucose lowering at 18-24 hours, lasting for >42 hrs.
  • AGGF1 peptide and expression vectors encoding AGGF1 can be used to better control glucose levels over extended periods of time compared with insulin alone.
  • the AGGF1 gene encodes a 714 amino acid angiogenic factor essential for embryonic development.
  • KO heterozygous AGGF1 knockout mice develop diabetes with an increased fasting glucose level, impaired glucose tolerance, and insulin resistance, and that AGGF1 protein therapy dramatically reduces the fasting glucose level in high fat diet (HFD)-induced diabetic mice.
  • HFD high fat diet
  • AGGF1 treatment has a much longer therapeutic effect with a peak effect at 18-24 hours and lasting for >42 hours. It was found that AGGF1 protein therapy of one injection per two days maintained the low glucose level constantly.
  • long term expression can allow a subject to receive a single administration without further treatment of for a longer period of time (e.g., 2 months).
  • diabetic patients are treated with AGGF1 therapy who also have coronary artery disease, at risk for heart attack, or heart failure, and receive a benefit for both conditions.
  • compositions according to the present disclosure comprises or consists of a pharmaceutically acceptable carrier, diluent, or excipient (including combinations thereof).
  • a pharmaceutically acceptable carrier e.g., a pharmaceutically acceptable carrier, diluent, or excipient (including combinations thereof).
  • Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington’s Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).
  • the choice of pharmaceutical carrier, excipient, or diluent is selected with regard to the intended route of administration and standard pharmaceutical practice.
  • pharmaceutical comprise as, or in addition to, the carrier, excipient, or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), and/or solubilizing agent(s).
  • This pharmaceutical composition will desirably be provided in a sterile form. It may be provided in unit dosage form and will generally be provided in a sealed container. A plurality of unit dosage forms may be provided.
  • composition inside of pumps that are surgical embedded within a patient such that the AGGF1 peptide or expression vector can be injected (e.g., in a manner similar to insulin) to the subject’s blood stream when needed (see, e.g., Shah et ak, Int J Pharm Investig. 2016 Jan-Mar; 6(1): 1-9, herein incorporated by reference).
  • compositions within the scope of the present technology may include one or more of the following: preserving agents, solubilizing agents, stabilizing agents, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, odorants, and/or salts.
  • Compounds of the present technology may themselves be provided in the form of a pharmaceutically acceptable salt.
  • embodiments may comprise buffers, coating agents, antioxidants, suspending agents, adjuvants, excipients, and/or diluents. Examples of preservatives include sodium benzoate, sorbic acid, and esters of p-hydroxybenzoic acid.
  • compositions of the present technology may also contain other therapeutically active agents in addition to compounds of the present technology (e.g., insulin). Where two or more therapeutic agents are used they may be administered separately (e.g., at different times and/or via different routes) and therefore do not always need to be present in a single composition. Thus, combination therapy is within the scope of the present technology.
  • compositions herein contain an expression vector encoding the AGGF1 peptide.
  • the expression vectors is a plasmid, an adenovirus-associated viruses, or other viruses.
  • the expression vectors are delivered by nano-technologies and/or targeted delivery systems.
  • the routes for administration include, but are not limited to, one or more of: oral (e.g. as a tablet, capsule, or as an ingestable solution), implantable pump, topical, mucosal (e.g. as a nasal spray or aerosol for inhalation), nasal, parenteral (e.g. by an injectable form), gastrointestinal, intraspinal, intraperitoneal, intramuscular, intravenous, intrauterine, intraocular, intradermal, intracranial, intratracheal, intravaginal,
  • AGGF1 peptides and expression vectors described herein is administered parenterally, then examples of such administration include one or more of: intravenously, intra-arterially, intraperitoneally, intrathecally, intraventricularly, intraurethrally,
  • compositions adapted for oral administration are provided as capsules or tablets; as powders or granules; as solutions, food product, syrups or suspensions (in aqueous or non-aqueous liquids); as edible foams or whips; or as emulsions.
  • Tablets or hard gelatin capsules may comprise lactose, maize starch or derivatives thereof, stearic acid or salts thereof.
  • Soft gelatin capsules may comprise vegetable oils, waxes, fats, semi-solid, or liquid polyols etc.
  • Solutions and syrups may comprise water, polyols and sugars.
  • oils e.g., vegetable oils
  • An active agent intended for oral administration may be coated with or admixed with a material that delays disintegration and/or absorption of the active agent in the gastrointestinal tract (e.g., glyceryl monostearate or glyceryl distearate may be used).
  • a material that delays disintegration and/or absorption of the active agent in the gastrointestinal tract e.g., glyceryl monostearate or glyceryl distearate may be used.
  • the AGGF1 peptides described herein may be applied topically in the form of a gel, hydrogel, lotion, solution, cream, ointment or dusting powder.
  • the agent of the present technology may also be dermally or transdermally administered, for example, by the use of a skin patch.
  • the agent of the present technology can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene
  • polyoxypropylene compound emulsifying wax and water.
  • it can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
  • AGGF1 peptides are administered parenterally, then examples of such administration include one or more of: intravenously, intra-arterially, intraperitoneally, intrathecally, intraventricularly, intraurethrally, intrasternally, intracranially, intramuscularly or subcutaneously administering the agent; and/or by using infusion techniques.
  • the dosage is between 0.1 and 5 mg per kilogram of the subject (e.g., 0.1 ... 0.8 ... 1.5 ... 2.5 ... 4.0 ... 5.0 mg per kg).
  • the agent and/or the pharmaceutical composition of the present technology may be administered in accordance with a regimen of from 1 to 10 times per day, such as once or twice per day, or only once every other day or every third day.
  • the daily dosage level of the agent may be in single or divided doses.
  • AGGF1 may be regarded as the“the 2 nd insulin” for treatment of diabetes. Compared to typical insulin treatment, which has a fast-acting effect in 30 minutes but lasts about 3 hours, AGGF1 treatment, in this Example, has a longer therapeutic effect with a peak effect at 18-24 hours and lasting for >42 hours. AAV-AGGF1 gene therapy can make its therapeutic effect last even for months.
  • GTT glucose tolerance test
  • AGGF1 protein (0.25 mg/kg body weight) was injected into HFD-induced hyperglycemic mice. Surprisingly, injection of AGGF1 protein dramatically reduced the fasting glucose level in HFD hyperglycemic mice compared with control PBS buffer (Fig. 2). The maximal glucose lowering effect of AGGF1 was achieved at 18-24 hours after injection, and the sustained effect can be observed 42 hours after the injection (Fig. 2). Chronic AGGF1 treatment of HFD-induced hyperglycemic mice every 2 days for 36 days maintained the significantly low fasting glucose level for the entire treatment period (Fig. 3).
  • HFD-induced hyperglycemic mice were administered with insulin alone (1 ug/kg body weight) or combination of insulin (1 ug/kg body weight) and AGGF1 protein (0.25 mg/kg body weight) by intraperitoneal injection.
  • Insulin treatment quickly decreased the fasting glucose level within 30 minutes and the effect of insulin was sustained for only 150 minutes.
  • AGGF1 protein treatment significantly extended the effect of insulin (Fig. 4A).
  • AGGF1 protein therapy is dependent on insulin.
  • STZ streptozotocin
  • Fig. 4B the glucose-lowering effect of AGGF1 was severely blunted when insulin secretion was inhibited by somatostatin
  • Fig. 4C the glucose-lowering effect of AGGF1 protein therapy lowers blood glucose levels in an insulin-dependent manner.
  • the interaction domain between AGGF1 and endothelial cells is localized between amino acids 574 and 624.
  • endothelial cell adhesion assays were performed with human umbilical vascular endothelial cells (HUVECs) for full-length AGGF1 (WT) and serial N-terminal deletion mutant proteins. Significantly stronger binding (i.e. high fluorescence) was observed with WT-AGGF1 and deletion mutant proteins Nl to N10 than deletion mutant proteins Nl 1 and N12 ( Figure 12, left panel). These results suggest that the potential interaction domain of AGGF1 is between amino acid 574 and 624.
  • Endothelial cell adhesion assays were also performed with WT-AGGF1 and serial C- terminal deletion mutant proteins of AGGF1. Significantly higher binding (i.e. fluorescence) was observed with WT-AGGF1 and deletion mutants Cl and C2 than deletion mutants C3 to C13 ( Figure 12, right panel). These results suggest that the potential interaction domain of AGGF1 with endothelial cells is between amino acid 564 and 614 ( Figure 10). Together, these results narrow the AGGFl-endothelail cell interaction domain to a 40-amino acid region (amino acids 574-614) at the C-terminus of AGGF1.
  • AGGF1 responsible for endothelial cell migration is localized between amino acids 574 and 624.
  • the cellular mechanism of angiogenesis involves endothelial cell motility.
  • Endothelial cell migration assays were performed with WT-AGGF1 and serial N- and C- terminal deletion mutant AGGF1 proteins. Significantly more HUVECs migrated into the ‘wounded area’ after 24 hours in presence of WT-AGGF1 and deletion mutant proteins Nl to N10 than deletion mutant proteins Nll and N12 ( Figure 13). Significantly more HUVECs migration was also observed with deletion mutant proteins Cl and C2 than deletion mutant proteins C3 to C13 ( Figure 13). These endothelial cell migration assays demonstrate that the 40 amino acid AGGF1 binding domain (amino acids 574-614) is also responsible for inducing endothelial cell migration.
  • AGGF1 responsible for angiogenesis is localized between amino acids 574 and 624.
  • Angiogenesis assays were performed using the capillary endothelial tube formation assays.
  • a significantly higher number of‘enclosed circles’ i.e. angiogenesis
  • were formed by HUVECs after 16 hours in presence of WT-AGGF1 and deletion mutant proteins Nl to N10 than deletion mutant proteins Nll and N12 Figure 14
  • a significantly higher number of‘enclosed circles’ were formed by HUVECs in presence of WT-AGGF1 and deletion mutant proteins Cl and C2 than deletion mutant proteins C3 to C13 ( Figure 14).
  • These endothelial tube formation assays demonstrate that the 40 amino acid AGGF1 binding domain (amino acids 574-614) is responsible for angiogenesis.
  • a short mutant AGGF1 protein with amino acids 604-714 has full functions of full- length AGGF1 in endothelial cell proliferation, migration, and angiogenesis
  • AGGFl604-7i4 has full AGGF1 functions, we can utilize it for treatment of, for example, cardiac and vascular diseases, and the other diseases listed herein. Because AGGFl604-7i4has only 110 amino acid residues, it can be synthesized in vitro and used for clinical treatment of cardiac and vascular diseases. AGGFl 6 04-7i4 can be further optimized by many different modifications to increase its stability and functional activities in treatment of diseases.
  • AGGF U04-714 can be further modified by removing more amino acids to identify the shortest form of AGGF1, but with full activities, which can be used for treatment of diseases (e.g., using the assays and procedures described in this Example).
  • AGGFl604-7i4 can be further modified by adding some amino acids to increase its stability and activities.
  • AGGFl604-7i4 can be further modified by adding some amino acids (e.g. mimotope) or by conjugating carriers for its targeted delivery to specific organs, tissues and cells for treatment of specific diseases.
  • AGGFI604-714 can be further modified by synthesizing as a circular form (cyclotide) to increase its stability for disease treatment.
  • Pulmonary arterial hypertension is characterized by an increased mean pulmonary arterial pressure (mPAP) of >25 mmHg, which is caused by functional and structural remodeling of pulmonary arteries, and in turn increases the burden on the right ventricle and eventually leads to right ventricular failure and premature death 5 ’ 6 .
  • the pathogenesis of PAH is complex and involves pulmonary endothelial dysfunction, proliferation of vascular smooth muscle cells (VSMCs), inflammation, fibrosis, as well as abnormal thickening and contraction of pulmonary arteries, thrombus formation, and genetic abnormalities, leading to incremental increases of pulmonary vascular resistance, increased right ventricular loading, and eventual right ventricular failure and death. 7 11 Accurate and early diagnosis of PAH is seriously lacking because PAH symptoms appear late.
  • Aggfl +/ KO mice develop spontaneous PAH.
  • AGGF1 protein therapy can block PAH.
  • RVSP right ventricular systolic pressure
  • Figure 18 AGGF1 protein therapy also reduced RV hypertrophy (RV/LV+S) compared with IgG control ( Figure 18) although additional mice are needed to achieve statistical significance.
  • AGGF1 protein therapy for PAH has several advantages.
  • other VSMC proliferation blocking agents proteasome inhibitors bortezomib BTZ and carfizomib CFZ
  • VSMC proliferation blocking agents have off-target effects (causing cardiac apoptosis in RV and LV).
  • existing therapy for PAH such as calcium channel blockers causes hypotension, but AGGF1 treatment does not affect blood pressure under a normal physiological condition.
  • Galie et ak Eur Heart J. 2016;37:67-119. 6. Galie et ak; 2015 ESC/ERS Guidelines for the Diagnosis and Treatment of Pulmonary Hypertension. Rev Esp Cardiol (Engl Ed). 20l6;69:l77.
  • EPCs endothelial progenitor cells
  • Aggfl +/ mice showed impairment of proliferation, migration, angiogenesis, and transendothelial migration as in hyperglycemic mice fed with a high fat diet (HFD) or db/db mice.
  • HFD high fat diet
  • db/db mice mice fed with a high fat diet
  • the number of EPCs from Aggfl +/ mice was significantly reduced.
  • AGGF1 protein can fully reverse damaging effects of hyperglycemia on EPCs.
  • transplantation of AGGF1 -primed EPCs successfully restores blood flow, and blocks tissue necrosis and ambulatory impairment in HFD-induced hyperglycemic mice or db/db mice with diabetic hind-limb ischemia.
  • AGGF1 activates AKT and reduces nuclear localization of Fyn, which increases the nuclear level of Nrf2 and expression of anti-oxidative genes, and inhibits ROS generation.
  • DM Diabetes mellitus
  • cardiovascular disease cardiovascular disease and diabetic peripheral arterial disease (PAD)
  • PAD diabetic peripheral arterial disease
  • EPCs endothelial progenitor cells
  • EPCs are progenitors of endothelial cells (ECs) and have the potential to proliferate, migrate, home into the disrupted endothelium and differentiate into ECs to maintain endothelium integrity, restore endothelial dysfunction, promote neovascularization and repair damaged vessels (11; 13-15) .
  • Cell-based therapy based on implantation of EPCs has emerged as a potential therapy for myocardial ischemia, brain ischemia and pulmonary embolism (16; 17).
  • the efficacy of a therapy using EPC implantation for diabetic vascular complications is uncertain (18).
  • autologous EPCs transplantation may not work efficiently because of the impaired function of such EPCs in DM by hyperglycemia (9; 11; 18).
  • Wild-type (WT) C57BL/6J mice and db/db mice (Jackson Lab) were used in the study.
  • Aggfl+/- KO mice with exons 2-11 deleted were described previously (20). Because homozygous Aggfl-/- KO mice die before E8.5, heterozygous Aggfl+/- KO mice were studied.
  • a high fat diet (HFD) -induced mouse model for type 2 diabetes mellitus (T2DM) was previously described (25-27).
  • EPCs were isolated from the bone marrow of WT, HFD-induced diabetic mice, and db/db mice, and cultured as described (11; 31). Mice were anesthetized with chloral hydrate (3%, m/v) via intraperitoneal injection. Bone marrow mononuclear cells (MNCs) were washed out from the femurs and tibias of mice with phosphate buffer saline (PBS), and then purified by density gradient centrifugation (2000 r/min) with histopaque-l083 (Sigma- Aldrich, St. Louis, MO) for 30 min at room temperature with a horizontal rotor centrifuge (Anke, LCJ-2B, Shanghai, China).
  • PBS phosphate buffer saline
  • the volume ratio of single cell suspension and histopaque-l083 separation liquid was 2:1.
  • the cells were then washed twice using PBS, resuspended gently, plated in 0.1% (m/v) gelatin-coated cell culture dishes, and maintained in endothelial growth factor-supplemented media (EGM-2 bullet kit; Lonza, Switzerland) with 10% fetal bovine serum (FBS).
  • EGM-2 bullet kit Lonza, Switzerland
  • FBS fetal bovine serum
  • Cells were cultured at 37°C with 5% C02 in a humidified water jacket incubator (Thermo Fisher Scientific, MA, USA).
  • the EGM-2 medium was replaced every 3 days after first plating, and cellular morphology was monitored every day.
  • MNCs Characterization of MNCs was performed as described (11; 31). Seven days after maintenance in endothelial-specific media and the removal of non-adherent MNCs, the remaining cells were subjected to immunostaining analysis. Cells were plated on gelatin- coated slide (ibidi) for one day, and then incubated with 10 pg/mL of acetylated Dil lipoprotein from human plasma (Dil-Ac-LDL, Thermo Fisher Scientific, MA, USA) at 37°C for 4 hours.
  • the cells were washed 3 times with PBS, and incubated with 10 pg/mL of fluorescein isothiocyanate-labeled ulex europaeus lectin-l (Sigma-Aldrich, St. Louis, MO, USA) for 1 hour at 37°C.
  • the cells were rinsed 3 times with PBS again, and visualized under a confocal microscopy.
  • MNCs were also characterized by flow cytometry analysis.
  • Cells were incubated with 5% BSA (Sigma-Aldrich, St. Louis, MO) for 15 minutes for to block non-specific binding, and then stained with anti-mouse CD34-phycoerythrin (PE), CD31-PE, CD45-PE or CD144- PE (BD Biosciences, San Jose, CA, USA) at room temperature for 1 hour, respectively.
  • BSA Sigma-Aldrich, St. Louis, MO
  • PE anti-mouse CD34-phycoerythrin
  • CD31-PE CD45-PE
  • CD144- PE BD Biosciences, San Jose, CA, USA
  • the same fluorescein-labeled isotype IgG served as negative control.
  • Cells were analyzed using a Beckman CytoFLEX, and data were analyzed using FlowJo software version 10 (TreeStar, Ashland, OR, USA). Immunostaining for CD31
  • muscle samples were fixed overnight in paraformaldehyde (4%), embedded in paraffin and sectioned. Sections were deparaffinized, dehydrated and rehydrated after being sectioned. After microwave antigen retrieval, endogenous peroxidase blocking and normal goat serum blocking in equilibration buffer, sections were subject to
  • AGGF1 protein was purified as described previously (21; 33). EPCs isolated from mice were treated with purified AGGF1 protein (0.5 pg/ml) or negative control elution buffer for purification at 37°C for l2h. Two days after the diabetic hind-limb ischemia surgery, AGGFl-primed EPCs (-1x106) were injected into mice via the tail vein. Blood flow in both legs was measured in mice anesthetized with chloral hydrate (3%, m/v) using a Vevo 2100 High- Resolution Microultrasound System (Visualsonics Inc) immediately before the ischemic surgery and at time-points of 7, 14 and 28 days after ischemia.
  • Visualsonics Inc Vevo 2100 High- Resolution Microultrasound System
  • Vs peak systolic velocity
  • Vd minimal end diastolic flow velocity
  • Va temporal average velocity per cardiac cycle
  • Human embryonic kidney 293 cells (HEK293) were transfected with a lentiviral vector with EGFP, the packaging plasmid psPAX2 and the envelope plasmid pMD2.G using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Viral supernatants were produced from the transfected HEK293 as described (11). HEK293 cells were maintained at 37°C in the high-glucose Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% (V/V) FBS (Gibico Life Technologies, New York, USA) in a humidified water jacket incubator with 5% C02. The supernatant was harvested 72 hours after transfection, filtered through 0.45-pm polyvinylidene fluoride filters, and stored at -80°C until use.
  • DMEM Modified Eagle’s medium
  • EPCs were infected with the GFP-lentiviruses overnight at a multiplicity of infection of 50 with 2.5 pg/mL polybrene supplemented, and the culture medium was replaced with the fresh growth medium 24 hours after infection. After transfection for 72 hours, the infection efficiency was determined by GFP expression under a Nikon EclipseTi micorscopy (Nikon, Japan).
  • siRNAs for Nrf2 and AKT negative control siRNAs were chemically synthesized by Ribobio.
  • the control siRNA is the silencer scrambled negative control siRNA (siNC) having no significant homology to any known gene sequences from the mouse genome.
  • siRNAs are as follows: Nrf2 sense: 5’-CGAGAAGUGUUUGACUUUATT-3’ (SEQ ID NO:l9), Nrf2 antisense: 5’-UAAAGUCAAACA-CUUCUCGTT-3’ (SEQ ID NO:20); AKT sense: 5'-UGCCCUUCUACAACCAGGATT-3' (SEQ ID NO:2l), AKT antisense: 5’-UCCUGGUUGUAGAAGGGCATT-3’ (SEQ ID NO:22); siNC sense: 5’- UUCUC-CGAACGUGUCACGUTT-3’ (SEQ ID NO:23), and siNC antisense: 5’- ACGU G AC AC- GUUCGG AG AATT- 3’ (SEQ ID NO:24). EPCs were transfected with siRNA in the Opti-MEM medium overnight using transfection reagent (Santa Cruz).
  • EPCs were seeded into 96-well cell culture plates, and treated with AGGF1 (0.5 pg/mL) or a solution with high D-glucose (30 mM, Sigma, St. Louis, MO, control, L-glucose). Then, 10 pl/well of CCK-8 solution was added, and the plates were incubated for 1-4 hours in dark. Cell proliferation was determined by the measurement of absorbance at 450 nm using a Molecular Devices VERSA max microplate reader as described (39; 40).
  • TEM transendothelial migration
  • EPCs Human umbilical vein endothelial cells (HUVECs) (about lxlO 4 cells per well) were cultured in the upper chamber of a 24-transwell insert (8.0 pm pores; BD Falcon, San Jose, CA, USA) to confluency. Monolayer confluency was confirmed under an inverted fluorescence microscopy before each experiment.
  • EPCs were treated with AGGF1 vs. elution buffer, a high glucose solution vs. control, and siRNAs, and cultured for 24 hours. The EPCs were harvested and resuspended in the basal culture medium (EBM-2, 0.5% BSA).
  • EPC suspension ( ⁇ 0.2 mL) was added to the upper chamber of Transwell, and 0.5 ml of EBM-2 medium was added to the lower chamber.
  • Cells were cultured for 24 hours at 37°C, and EPCs that migrated from the upper chamber to the lower chamber of Transwell were visualized. Images were captured at 40x magnification under an inverted Nikon Eclipse Ti microscope (Nikon, Japan). The number of cells was counted and analyzed.
  • EPCs in cell culture dishes were trypsinized and resuspended in growth media and allowed to grow to a confluent monolayer in 6-well plates. EPCs were starved for 6 hours by replacing the regular growth media with serum-free growth media. EPC monolayers were then wounded with a 200 m ⁇ pipette tip gently through the cell sheet to remove some cells and make a wound. The growth medium was aspirated out and wells washed twice with PBS to remove cell debris. Media supplemented with AGGF1 (0.5 pg/ml) were added to the wells in a total volume of 1 ml of serum-free growth media. Cells were cultured for 24 hours by incubating the plate at 37°C and 5% C02. Images were captured at 40X magnification under an inverted Nikon Eclipse Ti microscope (Nikon, Japan). The movement of cells into the scraped area was measured.
  • Protein extracts were prepared from cultured EPCs with Western-IP lysis buffer (Beyotime, Beijing, China) supplemented with a proteinase inhibitor cocktail (Roche, Basel, Switzerland), separated by 12% SDS-PAGE, and transferred to polyvinylidene-fluoride membranes.
  • the membranes were blocked with skim milk and incubated with appropriate primary antibodies overnight at 4°C with gentle shaking. The membranes were then incubated with appropriate secondary antibodies for 2 hours at room temperature after standard TBST washing procedures.
  • Nrf2 The primary antibodies against Nrf2 (1:1000 dilution), heme oxygenase-l (HQ-l, 1:2000), NAD(P)H dehydrogenase quinone 1 (NQO-l, 1:1000), catalase (1:1000), and GAPDH (1:5000) were purchased from ProteinTech (Wuhan, China).
  • Ser473; 1:1000 were purchased from Cell Signaling Technology (Danvers, MA, USA). Images from Western blot analysis were developed using a ChemiDoc XRS (Bio-Rad Laboratories, Richmond, CA, USA) with the SuperSignal West Pico Chemiluminescent Substrate (Pierce Chemical Co., Rockford, IL, USA), and further analyzed with a Gel-Pro analyzer. GAPDH was used as the loading control.
  • RNA samples were isolated from EPCs using Trizol reagent (TaKaRa Biotech, Japan), and reversed transcribed into cDNA with M-MLV reverse transcriptase (Promega, Madison, wi) according to the manufacturer’s instruction.
  • DNase I Thermo Fisher
  • Quantitative real-time RT-PCR analysis was performed in triplicate with a 10 pl reaction system using the FastStart Universal SYBR Green Master (Roche, Basel, Switzerland) and a 7900 HT Fast Real-Time PCR System (ABI, US).
  • the PCR profile was 94°C for 5 min, and 40 cycles of 94°C for 10 s and 60°C for 15 s. Fold differences between samples were assessed with a comparative cycle time (Ct) method.
  • Ct comparative cycle time
  • GAPDH served as an internal standard. The data were analyzed using 2-AACt relative expression quantity as reported (41).
  • MNCs bone marrow-derived mononuclear cells
  • WT wild type
  • Aggfl +/- KO mice The isolated MNCs appeared to be centrally rounded EPCs with a peripheral spindle shape four days after culture (Fig. 25 A). The cells appeared to be spindle-shaped adherent EPCs and some showed cobblestone-like morphology at day 8 (Fig. 25A).
  • the isolated EPCs were confirmed by a Dil-Ac-LDL endocytosis assay (red fluorescence) and a UEA-l binding assay (green fluorescence).
  • the isolated EPCs were able to endocytose Ac-LDL and bind UEA-l (Fig. 25B), suggesting that they are endothelial lineage cells with characteristics of EPCs as previously described (32;
  • Fig. 19D-E neovascularization in a transwell assay
  • Fig. 19F-G reduced migration in a scratch- wound assay
  • WT EPCs isolated form wild type mice
  • Aggfl +/- showed similar effects as db/db mice (Fig. 19A-G).
  • Aggfl +/- db/db double KO mice showed exacerbated effects compared with Aggfl +/- KO mice (Fig. 19A-
  • AGGF1 is an angiogenic factor that can be secreted outside of cells (21), therefore, we treated EPCs with the purified human AGGF1 protein in culture to determine the effects of AGGF1 on functions of EPCs (Fig. 20A). All the defects by high glucose on EPC functions were blocked by AGGF1 treatment (Fig. 20B-H).
  • Fig. 20A All the defects by high glucose on EPC functions were blocked by AGGF1 treatment (Fig. 20B-H).
  • Fig. 20B-H We analyzed the expression levels of VEGF, VEGFR2 and CXCR4 mRNA in EPCs treated with or without AGGF1 in combination with or without high glucose.
  • AGGF1 did not affect the expression levels of VEGF, VEGFR2 and CXCR4 in EPCs (Fig. 31).
  • AGGF1 Boosts EPC-Mediated Angiogenesis and Blood Perfusion in T2MD
  • AGGF1 In a T2DM mouse model (db/db mice), AGGF1 was found to greatly boost the therapeutic effects of cell therapy with EPC implantation on vascular complications under ischemia in vivo. As AGGF1 can reverse the damaging effects of T2DM on EPCs, we hypothesized that AGGFl-primed EPCs can serve as a successful treatment tool for vascular complications in T2MD in vivo.
  • AGGFl-primed EPCs were significantly more effective than elution-buff er- pretreated EPCs in increasing blood flow in diabetic mice with ischemia (Fig. 21A).
  • the rate of tissue necrosis was significantly decreased by AGGFl-primed EPCs compared with elution-buffer-pretreated control EPCs (Fig. 21B).
  • Similar significant inhibitory effect on ambulatory impairment was detected for AGGFl-primed EPCs (Fig. 21C).
  • Immunostaining showed that the density of CD3l-postiive vessels was significantly higher for AGGFl- primed EPCs than for elution-buffer-pretreated control EPCs at day 28 after transplantation (Fig. 21D and Fig. 32A).
  • the therapeutic effect of AGGFl-primed EPCs may be related to the possibility that AGGF1 increases the homing of transplanted EPCs to the ischemic areas.
  • AGGF1 increases the homing of transplanted EPCs to the ischemic areas.
  • AGGF1 can activate PI3K and AKT signaling in endothelial cells and in zebrafish (19; 20).
  • PI3K and AKT were shown to regulate phosphorylation and nuclear localization of Fyn (19; 20).
  • Fyn was shown to phosphorylate Nrf2 at Y568, resulting in nuclear export and degradation of Nrf2 (19; 20). Therefore, we hypothesized that AGGF1 regulates ROS generation and EPC functions via an AGGF1- AKT-Fyn-Nrf2 signaling pathway.
  • EPCs showed a significantly reduced level of phosphorylation of AKT, however, the effect was reversed by AGGF1 treatment (Fig. 23A).
  • Nrf2 is a transcription factor important to cellular defense against oxidative stress. Nrf2 needs to be translocated into the nucleus to execute its function, and its nuclear localization is regulated by AKT-Fyn signaling. AGGF1 activates AKT, which leads to de-phosphorylation of Fyn, resulting in reduced translocation of Fyn into the nucleus and decreased nuclear Fyn (n-Fyn).
  • n-Fyn leads to decreased phosphorylation of Nrf2 and inhibits the nuclear export, ubiquitination and degradation of Nrf2, increasing the expression levels of nuclear Nrf2 and downstream cytoprotective genes such as NQO-l and CAT (Fig. 22).
  • Oxidative stress was considered to be a critical factor accounting for EPC dysfunction in T2DM (44; 45).
  • Our results suggest that AGGF1 protects EPCs partly by inhibiting hyperglycemia-induced ROS generation through a novel anti-oxidative stress signaling pathway (Fig. 39).
  • Endothelial dysfunction is a major problem in T2DM, and lack of endothelial regeneration and impaired angiogenesis by EPCs are responsible for the vascular abnormalities in DM (10; 11).
  • DM vascular abnormalities
  • the data in this Example establish AGGF1 as an important molecule to repair diabetic EPC dysfunction induced by hyperglycemia or dyslipidemia in db/db mice and HFD-induced hyperglycemia mice (Fig. 21, Fig.32 and Fig. 33).
  • An AGGFl-based EPC therapy for diabetic ischemia may have additional benefits for DM patients who are often affected with CAD/MI, heart failure, peripheral vascular disease, and other conditions. Furthermore, DM can also cause many other diseases including diabetic cardiomyopathy, end-stage renal disease (ESRD) and vascular aging, which are all associated with abnormal angiogenesis and vascular dysfunction. Based on our data, it is likely that AGGF1 -primed EPC therapy may serve as a new strategy not only for treating DM-associated vascular abnormalities, and those with vascular abnormalities without DM, but also for many other diabetes mellitus complications such as diabetic cardiomyopathy, ESRD and vascular aging.
  • ESRD end-stage renal disease
  • AGGF1 protein therapy reduces plasma glucose levels in db/db diabetic mice.
  • AGGF1 protein therapy is effective in lowering fasting glucose levels in HFD- induced diabetic mice.
  • the db/db mice are a well-established murine model for diabetes.
  • the overall study design is shown in Figure 40A.
  • Figure 40B compared to IgG control, AGGF1 significantly lowers the plasma glucose levels for more than 24 hours. The effect was lost at the time point of 30 hours.
  • AGGF1 The therapeutic effects of AGGF1 is more than 10 times longer than insulin treatment.
  • U.K. prospective diabetes study 16 Overview of 6 years' therapy of type II diabetes: a progressive disease. U.K. Prospective Diabetes Study Group. Diabetes.

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  • Heart & Thoracic Surgery (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biotechnology (AREA)
  • Toxicology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des compositions, des systèmes, des kits et des méthodes pour traiter un sujet souffrant d'une maladie ou d'un état pathologique caractérisé par un métabolisme anormal du glucose (par exemple, le diabète) ou une maladie vasculaire ischémique (par exemple, PAD ou CAD), par l'administration d'une composition comprenant des peptides AGGF1, ou un vecteur d'expression codant pour lesdits peptides, ou des cellules progénitrices endothéliales sensibilisées par AGGF1, à un sujet. Dans certains modes de réalisation, le peptide AGGF1 est un AGGF1 pleine longueur, ou un fragment biologiquement actif ou un mutant de celui-ci. Dans certains modes de réalisation, les peptides AGGF1 ou les vecteurs d'expression sont administrés à peu près au même moment où l'insuline est administrée au sujet.
PCT/US2019/055513 2018-10-11 2019-10-10 Aggf1 et cellules sensibilisées par aggf1 pour le traitement de maladies et d'états pathologiques Ceased WO2020077030A1 (fr)

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US201862744488P 2018-10-11 2018-10-11
US62/744,488 2018-10-11
US201962842283P 2019-05-02 2019-05-02
US62/842,283 2019-05-02

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WO2020077030A1 true WO2020077030A1 (fr) 2020-04-16

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