US20170096682A1 - Aav vectors for vascular gene therapy in coronary heart disease and peripheral ischaemia - Google Patents

Aav vectors for vascular gene therapy in coronary heart disease and peripheral ischaemia Download PDF

Info

Publication number
US20170096682A1
US20170096682A1 US15/303,823 US201515303823A US2017096682A1 US 20170096682 A1 US20170096682 A1 US 20170096682A1 US 201515303823 A US201515303823 A US 201515303823A US 2017096682 A1 US2017096682 A1 US 2017096682A1
Authority
US
United States
Prior art keywords
mrtf
raav
aav vector
mammal
human
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.)
Abandoned
Application number
US15/303,823
Other languages
English (en)
Inventor
Christian Kupatt
Rabea Hinkel
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.)
BIOTECH GmbH
Original Assignee
Individual
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 Individual filed Critical Individual
Publication of US20170096682A1 publication Critical patent/US20170096682A1/en
Assigned to BIOTECH GMBH reassignment BIOTECH GMBH ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BERMPOHL, ANDREAS, DR.
Abandoned legal-status Critical Current

Links

Images

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
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • 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
    • A61K48/005—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 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the invention is the field of gene therapy.
  • the invention is directed to providing gene therapy for coronary heart disease and peripheral ischemia in mammals.
  • coronary heart disease remains the most common cause of death, in spite of improved treatments such as revascularization of an occluded coronary vessel (Lloyd-Jones et al., Circulation 2010, 121:e46-e215).
  • myocardial ischemia may occur through a slow, chronic occlusion of a coronary vessel, which can progress to heart insufficiency and even to cardiac failure (Suero et al., J Am Coll Cardiol 2001, 38:409-14).
  • Chronic ischemic disease of the heart or peripheral muscle is presently treated using surgical or interventional measures in order to revascularize constricted or occluded vascular networks.
  • drug therapy following the re-opening of an occluded vessel, and thus event-free survival of patients has been greatly improved in the last years, a number of patients still develop heart insufficiency (Levy et al., N Engl J Med 2002, 347:1397-402).
  • conventional therapeutic strategies become exhausted and clinical benefit is then expected from adjuvant neovascularization therapies (angiogenesis/arteriogenesis).
  • angiogenesis (collateral growth), a substantial element of improvement in flow-through, did not prolong walking time in patients afflicted with limb ischemia when supporting GM-CSF treatment was applied without induction of microvessel growth and stabilization (van Royen et al., Circulation 2005, 112:1040-6).
  • adaptive collateralization (Schierling et al., J Vasc Res 2009, 46:365-374) occurred when a proangiogenic factor like VEGF-A was combined with the maturation factors PDGF-B (Kupatt et al., J Am Coll Cardiol 2010, 56:414-22) or angiopoietin-1 (Smith et al., J Am Coll Cardiol 2012, 59:1320-8).
  • Event-free survival of patients might be improved significantly using gene therapy in cases of angiogenesis, arteriogenesis, in addition to improved heart function. However, for these purposes, it is necessary to select the correct gene therapy vector and target cells.
  • the present invention advantageously solves these problems through the use of AAV vectors in vascular gene therapy strategies against coronary heart disease.
  • the invention relates to an adeno-associated viral vector (AAV vector) comprising a gene encoding a myocardin-related transcription factor A (MRTF-A).
  • AAV vector adeno-associated viral vector
  • MRTF-A myocardin-related transcription factor A
  • the invention relates to an adeno-associated viral vector (AAV vector) comprising a gene encoding a thymosin ⁇ 4 (T ⁇ 4).
  • AAV vector adeno-associated viral vector
  • the AAV vector can be an AAV2/9 or an AAV vector pseudotyped with envelope proteins of AAV9, preferably AAV2.9, AAV1.9, or AAV6.9.
  • the AAV vector comprises a gene encoding an MRTF-A.
  • the invention relates to an adeno-associated viral vector (AAV vector) comprising a gene encoding a myocardin-related transcription factor A (MRTF-A), and a second gene encoding a thymosin ⁇ 4 (T ⁇ 4) and/or a third gene encoding an MRTF-A.
  • AAV vector adeno-associated viral vector comprising a gene encoding a myocardin-related transcription factor A (MRTF-A), and a second gene encoding a thymosin ⁇ 4 (T ⁇ 4) and/or a third gene encoding an MRTF-A.
  • the first gene is under the control of a cardio-specific promoter. In one embodiment, the first gene is under the control of a CMV promoter, an MRC2 promoter, a MyoD promoter, or a troponin promoter.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an AAV vector of the invention and a pharmaceutically acceptable carrier.
  • the invention further relates to an AAV vector of the invention or a pharmaceutical composition of the invention for use as a medicament.
  • the AAV vector of the invention or the pharmaceutical composition of the invention is for use in the treatment of coronary heart disease or peripheral ischemia in a mammal, preferably in a human, a mouse, a rabbit, or a pig.
  • the coronary heart disease can be an acute heart attack, myocardial ischemia, stable angina pectoris, and/or hibernating myocardium.
  • the mammal is a human No-Option-Patient.
  • FIG. 1 Angiogenesis induced by MRTF activation and translocation into the nucleus via CCN1 and CCN2 activation
  • HMECs human microvascular endothelial cells
  • Overexpression of T ⁇ 4 showed similar effects if no MRTF shRNA was co-administered or a T ⁇ 4 mutant (T ⁇ 4m) lacking the G actin binding motif KLKKTET was used (scale bar: 200 ⁇ m).
  • T ⁇ 4 transfection of myocytic HL-1 cells enabled translocation of MRTF-A (green fluorescence) into the nucleus (blue fluorescence), an effect which was absent if the T ⁇ 4m construct without the G actin binding site was used (scale bar: 20 ⁇ m).
  • FIG. 1 h and FIG. 1 i Tubus maturation, evaluated as pericyte recruiting (PC, green fluorescence) on endothelial rings (EC rings, red fluorescence, scale bar: 200 ⁇ m), was induced by MRTF-A and T ⁇ 4.
  • PC pericyte recruiting
  • EC rings red fluorescence, scale bar: 200 ⁇ m
  • FIG. 2 The T ⁇ 4-MRTF-A signaling cascade induces angiogenesis in vitro
  • FIG. 2 a T ⁇ 4 transfection of cardiomyocytic HL-1 cells enables the translocation of MRTF into the nucleus, an effect lacking when a T ⁇ 4m construct without acting binding site was used.
  • FIG. 2 b Analysis of the MRTF-A protein level in the nucleus by Western blot showed an elevated MRTF-A protein level after T ⁇ 4 overexpression in HL-1 cells. T ⁇ 4m did not increase the MRTF-A level.
  • FIG. 2 c and FIG. 2 d qRT-PCR shows that CCN1/2 shRNA prevented accumulation of CCN1/2 transcripts after T ⁇ 4 expression.
  • FIG. 2 f rAAV.T ⁇ 4-transduced cardiomyocytic HL-1 cells induced angiogenesis (tubus formation) in endothelial cells co-cultured with HL-1 cells, if no MRTF shRNA was co-transduced, whereas rAAV.T ⁇ 4m had no effect (scale bar: 200 ⁇ m).
  • FIG. 2 g and FIG. 2 h MRTF-A mRNA expression levels (g) and MRTF-A protein level (h) were not influenced by T ⁇ 4 overexpression, but were significantly elevated after MRTF-A transfection.
  • FIG. 2 i FIG.
  • FIG. 2 j Tubus formation after T ⁇ 4 release from rAAV.T ⁇ 4-transduced HL-1 cells was disrupted by CCN1 shRNA (scale bar: 200 ⁇ m).
  • FIG. 3 Importance of MRTF signaling for neovascularization in vivo
  • FIG. 3 a qRT-PCR analysis showed an increase in MRTF-A in the ischemic hind limb transduced with rAAV.MRTF-A.
  • FIG. 3 b rAAV.MRTF-A induced MRTF/SRF target genes CCN1 and CCN2 in vivo.
  • FIG. 3 c and FIG. 3 d rAAV.MRTF-A transduction increased the capillary/muscle fiber ratio (c/mf) in a manner similar to MRTF activator T ⁇ 4.
  • FIG. 3 e and FIG. 3 f Functionally, transduction with rAAV.MRTF-A and -T ⁇ 4, but not rAAV.T ⁇ 4m or rAAV.T ⁇ 4+MRTF-shRNA, improved the perfusion of the hind limb on day 3 and day 7.
  • FIG. 3 e and FIG. 3 f Functionally, transduction with rAAV.MRTF-A and -T ⁇ 4, but not rAAV.T ⁇ 4m or rAAV.T ⁇ 4+MRTF-shRNA, improved the perfusion of the hind limb on day 3 and day 7.
  • FIG. 3 h Perfusion increased by rAAV.T ⁇ 4 was suppressed in MRTF-A/B ⁇ / ⁇ Vi mice.
  • FIG. 4 MRTF-A induced vessel growth in mouse hind limb ischemia
  • FIG. 4 a Protocol for mouse hind limb ischemia. Intramuscular (i.m.) rAAV administration was performed on day ⁇ 14 and the femoral artery was ligated on day 0. Subsequent laser Doppler flowthrough measurements (LDF) were performed on days 0, 3, and 7.
  • FIG. 4 b i.m. injection of rAAV.Cre induced homogenous muscle transduction, shown by a change of Tomato fluorescence (red) to GFP fluorescence (green) in Tomato reporter gene mice.
  • FIG. 4 c i.m. injection of rAAV.LacZ (3 ⁇ 10 12 virus particles) led to a homogenous transduction (blue staining) of the targeted hind limb, but not of the opposite one.
  • FIG. 4 d qRT-PCR detection of T ⁇ 4 in the rAAV.T ⁇ 4-transduced ischemic hind limbs, but not in the rAAV.LacZ-transduced hind limbs.
  • FIG. 4 e HPLC analysis showed an increase of T ⁇ 4 protein concentration in the rAAV.T ⁇ 4-transduced ischemic hind limbs.
  • FIG. 4 g T ⁇ 4-induced maturation of capillaries (pericyte investment, NG2 staining) was suppressed in MRTF-A/B ⁇ / ⁇ Vi hind limbs.
  • FIG. 4 g T ⁇ 4-induced maturation of capillaries (pericyte investment, NG2 staining) was suppressed in MRTF-A/B ⁇ / ⁇ Vi hind limbs.
  • FIG. 5 T ⁇ 4/MRTF-A-induced microvessel maturation: essential role for collateral growth and improved perfusion
  • FIG. 5 a HPLC analysis showed a significant increase of T ⁇ 4 protein after rAAV.T ⁇ 4 transduction of ischemic rabbit hind limbs, whereas rabbit-specific T ⁇ 4-Ala remained unchanged.
  • FIG. 5 b , FIG. 5 c and FIG. 5 d rAAV.MRTF-A or rAAV.T ⁇ 4 administration increased capillary density (PECAM-1 staining) and pericyte investment (NG2 staining, scale bar: 50 ⁇ m), both of which were abolished by co-application of angiopoietin 2 (rAAV.Ang2).
  • FIG. 5 f Angiographies of ischemic hind limbs on day 35 showed an increased collateral formation in rAAV.MRTF-A- and rAAV.T ⁇ 4-treated animals (arrows show site of excision of the femoral artery). Co-application of rAAV.Ang2 abolished this effect.
  • FIG. 6 T ⁇ 4-MRTF-A-induced vessel growth in rabbits
  • FIG. 6 a Protocol of a model for rabbit hind limb ischemia (femoral artery excision).
  • FIG. 6 b P ⁇ -galactosidase staining 5 weeks after i.m. injection of rAAV.LacZ into the rabbit hind limb.
  • FIG. 6 f T ⁇ 4 overexpression only in the lower limb (rAAV.T ⁇ 4 LL) increased capillary density (PECAM-1 staining) in the lower limb, whereas T ⁇ 4 transduction only in the upper limb (rAAV.T ⁇ 4 UL) did not influence the capillarization in the lower limb (scale bar: 50 ⁇ m).
  • FIG. 6 g and FIG. 6 h Collateralization was increased in the rAAV.T ⁇ 4 UL group, and to an even greater extent in the rAAV.T ⁇ 4 LL group, whereas perfusion FIG. 6 i was increased only in the rAAV.T ⁇ 4 LL group and not in the rAAV.T14 UL group.
  • FIG. 6 g and FIG. 6 h Collateralization was increased in the rAAV.T ⁇ 4 UL group, and to an even greater extent in the rAAV.T ⁇ 4 LL group, whereas perfusion FIG. 6 i was increased only in the rAAV.
  • FIG. 7 MRTF-A improves collateral formation and perfusion in hibernating myocardium of pigs
  • FIG. 7 a - FIG. 7 c In hibernating pig myocardium (see FIG. 8 a ), rAAV.MRTF-A transduction and ubiquitous overexpression of T ⁇ 4 (T ⁇ 4tg, see FIG. 9 ) induced capillary sprouting (PECAM-1 staining, scale bar: 50 ⁇ m) and pericyte investment (NG2 staining).
  • FIG. 7 d and FIG. 7 e Moreover, collateral growth was detected in rAAV.MRTF-A-transduced hearts, similarly to T ⁇ 4tg hearts.
  • FIG. 7 f The regional flow reserve, obtained by fast atrial stimulation (130 beats per minute), was increased in rAAV-MRTF-A-transduced and T ⁇ 4-transgenic hearts.
  • FIG. 7 g Regional myocardium function, measured by subendocardial segment shortening at rest and under atrial stimulation (130 and 150 beats per minute), showed improved functional reserve either by rAAV.MRTF-A transduction or in T ⁇ 4tg hearts.
  • FIG. 7 h The ejection fraction, a parameter of global myocardium function, was improved in rAAV.MRTF-A-transduced animals on day 56, compared with day 28. Constitutively overexpressing animals (T ⁇ 4tg), however, showed no loss of function on day 28.
  • FIG. 7 g Regional myocardium function, measured by subendocardial segment shortening at rest and under atrial stimulation (130 and 150 beats per minute), showed improved functional reserve either by rAAV.MRTF-A transduction or in T ⁇ 4tg hearts.
  • FIG. 7 h The ejection fraction, a parameter of global myocardium function, was improved in rAAV.MRTF-A-transduced animals on day 56, compared
  • MRTF-A or T ⁇ 4 transduction induces an increased amount of MRTF-A not bound to G actin that interacts with SRF upon translocation into the nucleus and induces e.g. CCN1 and CCN2 as target genes.
  • FIG. 8 Functional efficiency of the T ⁇ 4-MRTF-A axis in chronically ischemic pig hearts
  • FIG. 8 a Protocol of the pig model for hibernating myocardium.
  • FIG. 8 b RT-PCR-detection of MRTF-A and T ⁇ 4 in control pigs compared with rAAV.T ⁇ 4 and T ⁇ 4-transgenic (T ⁇ 4tg) pig hearts.
  • FIG. 7 c Examples of LacZ staining (blue) after rAAV.LacZ retroinfusion (5 ⁇ 10 12 virus particles) into the pig heart.
  • FIG. 7 d Before treatment (on day 28), retention analysis of fluorescent microbeads at rest showed a reduced blood flow in the ischemic area of rAAV.LacZ und rAAV.MRTF-A hearts, but not of T ⁇ 4tg hearts, similarly to the flow reserve FIG. 7 e at fast heart rate (130 bpm).
  • FIG. 7 f 4 weeks after treatment (on day 56), the regional myocardial blood flow in rAAV.MRTF-A und T ⁇ 4tg animals improved.
  • FIG. 7 g Furthermore, the Rentrop score showed an increased collateralization on day 56 in rAAV.MRTF-A-transduced or T ⁇ 4tg hearts.
  • FIG. 7 h Examples of MRT analysis on day 56 for control (left) and rAAV.MRTF-A-treated pig hearts.
  • FIG. 7 i The left ventricular end-diastolic pressure (LVEDP) increased in ischemic hearts from day 28 to day 56 if MRTF-A was not overexpressed.
  • FIG. 9 Production of T ⁇ 4-transgenic pigs
  • Fibroblasts of donor pigs were isolated and cultured.
  • pCMV-T ⁇ 4 was transfected by electroporation and the cells were cultured for 14 days. After detection of stable transfection of T ⁇ 4, a somatic nuclear transfer into pig oocytes was performed. Offspring were analyzed for T ⁇ 4 expression and fibroblasts of T ⁇ 4-expressing animals were cultured and subsequently used for a second somatic nuclear transfer. After genotyping, animals of this generation were used for the pig model of chronic ischemia.
  • FIG. 10 MRTFs are necessary for T ⁇ 4-induced cardioprotection
  • FIG. 10 a and FIG. 10 b rAAV.T ⁇ 4 induced capillary growth (PECAM-1 staining) and FIG. 10 c pericyte investment (NG2 staining, scale bar 50 ⁇ m), unless co-administration of rAAV.MRTF-shRNA prevented both processes.
  • FIG. 10 d and FIG. 10 e Collateral growth was detected in rAAV.T ⁇ 4-transduced animals, but not after co-administration of rAAV.MRTF-shRNA.
  • FIG. 10 f Rentrop scores showed increased collateralization after rAAV.T ⁇ 4 transduction, except in the case of co-administration of MRTF-A shRNA.
  • FIG. 10 f Rentrop scores showed increased collateralization after rAAV.T ⁇ 4 transduction, except in the case of co-administration of MRTF-A shRNA.
  • FIG. 10 g Regional myocardial blood flow at flow reserve (atrial stimulation 130/min) improved in rAAV.T ⁇ 4-treated animals, but not in rAAV.T ⁇ 4+MRTF-shRNA hearts.
  • FIG. 10 h Analysis of the ejection fraction showed improved systolic myocardium function in rAAV.T ⁇ 4-transduced animals (day 56), as compared with day 28 (day of transduction). No improvement of the ejection fraction was observed in rAAV.T ⁇ 4+MRTF-shRNA-treated hearts.
  • FIG. 11 Production and cardial phenotyping of INS C94Y -transgenic pigs (diabetes mellitus type I)
  • FIG. 11 a Process of producing the INS C94Y -transgenic pigs.
  • FIG. 11 b Blood glucose levels of wild type and diabetic pigs.
  • FIG. 11 c Fluorescence staining of endothelial cells (PECAM-1-positive cells, red) and pericytes (NG-2-positive cells, green).
  • FIG. 11 d Number of endothelial cells in the myocardium of wild type and diabetic pigs.
  • FIG. 11 e Left ventricular end-diastolic pressure in animals with diabetes mellitus type I and wild type animals.
  • FIG. 12 Characterization of the chronically ischemic myocardium model with cardiovascular risk factors
  • FIG. 12 a Protocol of the pig model for hibernating myocardium with diabetes mellitus type I or hypercholesterolemia.
  • FIG. 12 b Blood glucose concentration of the specific groups of animals over the duration of the experiment: control wild type; wild type treated with rAAV.T ⁇ 4; control with diabetes; diabetes treated with rAAV.T ⁇ 4.
  • FIG. 12 c and FIG. 12 d Serum trigylceride and cholesterol levels in animals with hypercholesterolemia (fat rich diet) and normal diet.
  • FIG. 13 Influence of rAAV.T ⁇ 4 application on angio- and arteriogenesis in animals with diabetes mellitus type I
  • FIG. 13 a Fluorescence staining of endothelial cells (PECAM-1-positive cells, red) and pericytes (NG-2-positive cells, green) in hibernating pig myocardium of diabetic and wild type animals.
  • FIG. 13 b and FIG. 13 c Number of endothelial cells and pericytes.
  • FIG. 13 d Number of collaterals formed.
  • FIG. 14 Functional efficiency of rAAV.T ⁇ 4 application in animals with diabetes mellitus type I
  • FIG. 14 a and FIG. 14 b Left ventricular end-diastolic pressure on days 28 and 56 and its change between these time points.
  • FIG. 14 c and FIG. 14 d Ejection fraction on days 28 and 56 and its change between these time points.
  • FIG. 15 Influence of elevated cholesterol levels on T ⁇ 4-mediated angio- and arteriogenesis
  • FIG. 16 Functional efficiency of rAAV.T ⁇ 4 application in animals with hypercholesterolemia
  • FIG. 16 a and FIG. 16 b Left ventricular end-diastolic pressure on days 28 and 56 and its change between these time points in hypercholesterolemic control and rAAV-T ⁇ 4-treated animals.
  • FIG. 16 c and FIG. 16 d Ejection fraction on days 28 and 56 and its change between these time points in hypercholesterolemic control and rAAV-T ⁇ 4-treated animals.
  • FIG. 16 e Regional myocardium function, measured as subendocardial segment shortening at rest and with increased heart rate (130 and 150 beats per minute).
  • FIG. 17 rAAV.T ⁇ 4 and rAAV.MRTF-A pretreatment in a mouse model of sepsis
  • FIG. 17 a Protocol of the sepsis tests in mice.
  • FIG. 17 b Scoring scheme for the assessment of sepsis symptoms in mice and determination of the stop criteria.
  • FIG. 17 c Peripheral arterial blood pressure values after 12 and 24 hours in animals with sepsis treated with different rAAV.
  • FIG. 17 d Symptom scores of the animals with sepsis in the treatment groups.
  • FIG. 17 e Cumulated survival after LPS-induced sepsis.
  • FIG. 18 Role of MRTF-A and T ⁇ 4 in vascular integrity during sepsis
  • FIG. 18 a and FIG. 18 b Histologic analyses of the endothelial cells (PECAM-1-positive cells) and the pericytes (NG-2-positive cells) in the hearts and the peripheral musculature of mice with sepsis.
  • FIG. 18 c and FIG. 18 d Exemplary images and quantitative analysis of a permeability measurement by means of fluorescently labeled high molecular dextran 6 hours after induction of sepsis.
  • a key feature of MRTF-A activation is translocation into the nucleus after decrease of G actin levels and export from the nucleus when the amount of G actin increases (Miralles et al., Cell 2003, 113:329-42; Vartiainen et al., Science 2007, 316:1749-52).
  • Enforced expression of MRTF-A or T ⁇ 4, a peptide activating MRTF-A by G actin binding FIG. 1 a - FIG. 1 i ), initiates an orchestrated micro- and macrovascular growth response in the case of chronic ischemia of peripheral ( FIG. 3 a - FIG. 3 l , FIG. 5 a - FIG. 5 g ) and heart muscle cells ( FIG.
  • MRTF-A-SRF signaling provides myofilaments is of particular interest, since a loss of the actin cytoskeleton is a hallmark of hibernating myocardium caused by chronic coronary hypo-perfusion (Bito et al., Circ Res 2007, 100:229-37).
  • MRTF-A is located at the interface of myocyte and vascular regeneration in hibernating myocardium.
  • T ⁇ 4 the most abundant G actin-binding peptide of the cytosol, can influence vascular growth by endothelial migration and sprouting (Grant et al., J Cell Sci 1995, 108:3685-94: Smart et al., Nature 2007, 445:177-82).
  • a substantial role of MRTF-A in T ⁇ 4 signaling has been shown in vitro and in vivo, since MRTF-A shRNA could suppress endothelial migration and sprouting ( FIG. 1 b , FIG. 1 d ) and micro- and macrovascular growth ( FIG. 3 d , FIG. 3 f ) and functional improvement of the heart ( FIG. 10 ).
  • MRTF-A endothelium-specific deficiency in MRTFs caused incomplete formation of the primary vascular plexus in the developing retina (Weinl et al., J Clin Invest 2013, 123:2193-206).
  • MRF the main target of MRTF-A
  • VEGF-A leads to the growth of immature and unstable capillaries (Dor et al., EMBO J. 2002, 21:1939-47), in contrast to T ⁇ 4-MRTF-A, thus indicating a difference in the signaling mechanisms for these two vascular growth factors.
  • the invention comprises in a first embodiment an adeno-associated viral vector (AAV vector) comprising a first gene encoding a myocardin-related transcription factor A (MRTF-A).
  • AAV vectors herein are particles displaying the envelope of an adeno-associated virus while comprising in their interior a single-stranded DNA encoding a gene of interest.
  • the gene of interest can be introduced into a target cell by infection of the target cell with an AAV vector.
  • the MRTF-A can be derived from a human, a mouse, a rabbit, a pig, or any other mammal.
  • an AAV vector comprising envelope proteins, in particular the cap protein, of AAV9.
  • AAV9 shows heart muscle tropism and thus provides for homogenous and stable expression in the heart muscle of a plurality of species.
  • an AAV vector pseudotyped with AAV9 may also be used.
  • a vector is meant comprising envelope proteins of AAV9, but otherwise expressing proteins of another strain and also containing genomic elements, for example internal terminal repeats (ITRs), from the other strain.
  • ITRs internal terminal repeats
  • AAV2.9 is an AAV2 vector pseudotyped with envelope proteins of AAV9.
  • AAV2.9, AAV1.9, and AAV6.9 are suitable as pseudotyped vectors.
  • an AAV vector with skeletal muscle tropism may also be used, in particular for the treatment of peripheral ischemia.
  • Examples are AAV6, AAV1, AAV9, or vectors pseudotyped with these strains.
  • the vector of the invention can further comprise additional expressible genes, e.g. an expression cassette for a thymosin ⁇ 4 (T ⁇ 4) or an MRTF-B.
  • T ⁇ 4 can be derived from a human, a mouse, a rabbit, a pig, or any other mammal.
  • the MRTF-B can be derived from a human, a mouse, a rabbit, a pig, or any other mammal. Expression of these genes in the heart muscle also supports therapeutic neovascularization in myocardial ischemia.
  • the MRTF-A gene in the vector of the invention is preferably under the control of a cardio-specific promoter, i.e. a promoter enabling expression mainly in the heart muscle.
  • a cardio-specific promoter i.e. a promoter enabling expression mainly in the heart muscle.
  • Exemplary cardio-specific promoters are the MLC2 promoter, the ⁇ myosin heavy chain promoter ( ⁇ -MHC promoter) and the troponin I promoter (TnI promoter).
  • ⁇ -MHC promoter ⁇ myosin heavy chain promoter
  • TnI promoter troponin I promoter
  • other constitutive or inducible promoters may be used, e.g. a CMV promoter or a MyoD promoter.
  • the MRTF-A gene can also be under the control of several promoters.
  • AAV vectors for the transfer of specific genes of interest are known in the state of the art (see e.g. Bell et al., J Clin Invest 2011, 121:2427-35).
  • One method consists in the triple transfection of a suitable producer cell line, e.g. U293, and subsequent purification by cesium chloride gradient, as described in the section “Materials and methods” below.
  • the producer cells are transfected with three vectors: A first vector encodes the gene of interest, flanked by corresponding packaging signals; a second vector encodes the necessary AAV proteins, in particular rep and cap; and a third vector provides the adenoviral helper functions without which no AAV particle production is possible.
  • the invention relates also to a pharmaceutical composition
  • a pharmaceutical composition comprising a vector of the invention and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition can be destined for every administration known in the art. Compositions for intravenous or intramuscular injection are preferred.
  • the pharmaceutical composition can additionally comprise salts, buffers, stabilizers, coloring agents, thickeners, flavors, etc.
  • the invention also relates to the AAV vector described herein or the pharmaceutical composition of the invention for use as a medicament.
  • such use can occur in a mammal for treatment of coronary heart diseases or peripheral ischemia.
  • Preferred mammals are human, pig, rabbit and mouse.
  • coronary heart disease means a disease of the coronary vessels of the heart.
  • the coronary heart disease can be myocardial ischemia, acute heart attack (myocardial infarction), stable angina pectoris and/or hibernating myocardium, but also cardiac arrhythmia and/or heart insufficiency.
  • myocardial ischemia is an insufficient perfusion or a complete loss of perfusion of a tissue or organ outside of the heart, while “myocardial ischemia” affects the heart muscle itself.
  • the vectors of the invention are particularly suitable for the treatment of “no option” patients. In such patients, all interventional and surgical therapeutic options are exhausted. Generally, slowing the progression of the disease by drug therapy is attempted. This however targets lipid reduction and platelet inhibition, but not neovascularization. Therapeutic neovascularization can overcome this hurdle, if molecular signaling pathways leading to balanced neovascularization are used.
  • MRTF-A and also T ⁇ 4 are two molecules that induce this type of balanced neovascularization (capillaries, microvascular maturation, and collateral formation) in ischemic tissue with concomitant lack of unwanted side effects.
  • vectors of the invention are particularly suitable for the treatment of subjects bearing additional cardiovascular risk factors.
  • risk factors include diabetes mellitus, in particular diabetes mellitus type I or type II.
  • the risk factor may also be an elevated concentration of cholesterol in the blood (hypercholesterolemia) that can be caused by a diet characterized as fat-rich.
  • the elevated cholesterol concentration can be elevated LDL cholesterol concentration or elevated HDL cholesterol concentration.
  • Example 1 Induction of Hallmarks of Angiogenesis by MRTF-A In Vitro
  • FIG. 1 a and FIG. 1 d We have found ( FIG. 1 a and FIG. 1 d ) that MRTF-A induced hallmarks of angiogenesis, i.e. migration and tubus formation of cultured human microvascular endothelial cells, to a comparable degree as T ⁇ 4.
  • the pro-angiogenic effect of MRTF-A was found to be dependent on the G actin binding motif of T ⁇ 4, since mutation of this domain and abolition of G actin binding eliminated the effect of T ⁇ 4 on vascular growth, similar to an shRNA shown to disrupt transcription of MRTF-A and -B (MRTF shRNA; Leitner et al., J Cell Sci 2011, 124:4318-31). Consistent therewith, T ⁇ 4 increased MRTF-A translocation into the nucleus ( FIG.
  • FIGS. 1 e , FIG. 2 a and FIG. 2 b similar to the transcription of an MRTF/SRF-dependent reporter gene containing three SRF binding sites of the c-fos promoter (p3DA.Luc, FIG. 1 f ; Geneste et al., J Cell Biol 2002, 157:831-8).
  • MRTF-A and T ⁇ 4 induced expression of genes involved in microvascular growth, in particular CCN1, mediating angiogenesis (Hanna et al., J. Biol. Chem. 2009, 284:23125-36), and CCN2, which is relevant for the attraction of 10T/2 pericyte-like cells ( FIGS.
  • rAAV recombinant AAV vectors
  • FIG. 3 a tissue concentration of target proteins in the treated limb
  • transcript levels of downstream mediators CCN1 and CCN2 FIG. 3 b , FIG. 4 d and FIG. 4 f
  • rAAV.MRTF-A induced capillary growth ( FIG. 3 c and FIG. 3 d ) and increased perfusion on day 7 ( FIG. 3 e and FIG. 3 ).
  • T ⁇ 4 had a similar effect on vascular growth and function ( FIG. 2 c - FIG. 2 f ), unless the G actin binding motif was missing (T ⁇ 4m) or an rAAV.MRTF-shRNA was co-administered.
  • This vector encodes an shRNA directed against both MRTF-A and MRTF-B and having the sequence 5′-GAUCCCCGCAUGGAGCUGGUGGAGAAGAAUUC AAGAGAUUCUUCUCCACCAGCUCCAUGUUUUUGGAAA-3′ (SEQ ID NO: 1).
  • T ⁇ 4-induced vascular growth rAAV.Cre was administered to Mrtfa ⁇ / ⁇ -Mrtf flox/flox hind limbs to generate MRTF-A/B double insufficiency.
  • T ⁇ 4 was not capable of stimulating capillary growth ( FIG. 3 g ) and pericyte recruitment ( FIG. 4 g and FIG. 4 h ) and of improving perfusion ( FIG. 3 h , FIG. 4 i ) on day 7 after induction of ischemia.
  • hind limbs did not show T ⁇ 4-mediated increase of capillaries ( FIG. 3 i and FIG.
  • FIG. 6 a The mutual dependence of microvascular growth and arteriogenesis for the mediation of regeneration of flowthrough was studied in a rabbit model of ischemic hind limbs ( FIG. 6 a ), which is compatible with topical separation of the microvascular growth area (lower limb) and the collateralization area (upper limb).
  • Regional transduction of ischemic calf muscle with MRTF-A or T ⁇ 4 led to functional neovascularization, including CD31 + capillary sprouting ( FIG. 5 b and FIG. 5 c ), NG2 + pericyte investment ( FIG. 5 b and FIG. 5 d ) and collateral growth ( FIG. 5 e and FIG. 5 f ).
  • MRTF activation via T ⁇ 4 transduction of the hip region did not increase perfusion, whereas limiting MRTF-A activation via T ⁇ 4 to the calf region was sufficient to significantly stimulate micro- and macrovascular growth and perfusion ( FIG. 6 e - FIG. 6 i ).
  • Detachment of microvascular pericytes by enforced angiopoietin 2 expression abolished T ⁇ 4-mediated collateralization and flowthrough improvement ( FIG. 5 e and FIG. 5 g ).
  • Example 4 Treatment of Hibernating Myocardium in the Pig with AAV-Based MRTF-A Gene Therapy
  • Collateral growth and perfusion under fast heart rate were still impaired on day 28, i.e. before LacZ and MRTF-A transduction ( FIG. 8 c - FIG. 8 f ), but improved on day 56, i.e. 4 weeks after MRTF-A transduction, but not after LacZ transduction ( FIGS. 7 d - f ).
  • Increased collateral perfusion ( FIG. 8 g ) generated an improved functional reserve of the ischemic area at fast heart rate (130 and 150 beats per minute, FIG. 7 g ).
  • FIG. 7 h an improved ejection fraction as a marker of global systolic function
  • FIG. 8 i a drop of the left ventricular end-diastolic pressure
  • Transgenic pigs that ubiquitously and constitutively express T ⁇ 4 showed similar capillary growth and maturation ( FIG. 7 a - FIG. 7 c ).
  • the blood flow reserve in the ischemic area was increased ( FIG. 7 f ) and the functional reserve in the ischemic region ( FIG. 7 g ) or the entire heart ( FIG. 7 h ) demonstrated an increase similar to rAAV.MRTF-A-treated hearts.
  • T ⁇ 4tg animals did not experience a significant loss of perfusion or myocardial function at rest or at fast heart rate ( FIG. 7 g , FIG. 8 d , FIG. 8 g , FIG. 8 i ).
  • FIG. 10 a - FIG. 10 f The overall gain in global ( FIG. 10 h , examples in FIG. 10 i ) and regional myocardium function ( FIG. 10 j ) was abolished when T ⁇ 4 transduction was combined with MRTF-A inhibition by a suitable shRNA.
  • MRTFs stimulate the growth and maturation of microvessels as well as an increased collateral blood flow after arterial occlusion in hind limb and coronary networks.
  • MRTF translocation downstream of thymosin ⁇ 4 co-activates SRF and induces CCN1/CCN2, thereby leading to increased angiogenesis and recruitment of vascular smooth muscle cells and formation of functional vessels that can carry collateral flow ( FIG. 7 i ).
  • transgenic pigs bearing the C94Y mutation in the insulin gene is shown in FIG. 11 .
  • This mutation is also depicted in Renner et al., Diabetes 2013, 62:1505-1511.
  • the C94Y mutation leads to misfolding of the insulin protein in the ⁇ cells of the pancreas and an accumulation of the misfolded insulin in the endoplasmic reticulum (ER).
  • ER stress leads to ⁇ cell apoptosis and thereby eventually to diabetes mellitus type I.
  • an INS C94Y expression vector was introduced into pig fibroblasts by means of nucleotransfection. After selection of the fibroblasts, a first round of somatic nucleus transfer into oocytes was performed. Subsequently, the offspring were analyzed by Southern blot and the animals with elevated blood glucose levels and delayed growth were used for renewed cloning (see Renner et al. 2013). These animals were then used for subsequent testing at 3-4 months of age.
  • FIG. 11 c and FIG. 11 d Analyses of the heart tissue for endothelial cells (PECAM-1-positive cells, red) and for pericytes (NG-2-positive cells, green) revealed a marked reduction of the endothelial cell and pericyte number even without additional stress.
  • FIG. 12 shows further effects of diabetes mellitus Type I or a fat-rich diet on the myocardium of pigs.
  • FIG. 12 illustrates the experimental protocol of the pig model for hibernating myocardium with diabetes type I or hypercholesterolemia.
  • the INS C94Y -transgenic animals with diabetes mellitus type I (labeled as control tg and rAAV.T ⁇ 4 tg) showed elevated blood glucose levels for the entire assay period ( FIG. 12 b ).
  • rAAV.T ⁇ 4 transduction induces capillary sprouting (PECAM-1 staining, red) and pericyte recruitment (NG-2 staining, green) in both groups (wild type and diabetes); FIG. 13 a - FIG. 13 c .
  • considerable collateral growth was induced by overexpression of T ⁇ 4 via rAAV ( FIG. 13 d ), and a considerably better filling of the distal blood vessel could be measured by means of the Rentrop score ( FIG. 13 e ).
  • the left ventricular end-diastolic pressure a parameter of global myocardium function, which showed an increase in the control animals of both groups from day 28 to day 56, was considerably reduced in the animals with rAAV.T ⁇ 4 transduction ( FIG. 14 a and FIG. 14 b ).
  • the left ventricular end-diastolic pressure a parameter of global myocardium function, which was increasing in the control animals from day 28 to day 56, was considerably reduced in the animals with rAAV.T ⁇ 4 transduction ( FIG. 16 a and FIG. 16 b ).
  • the ejection fraction a further parameter of global myocardium function, showed a further decrease of values from day 28 to day 56 in control animals, whereas the value after T ⁇ 4 overexpression improved considerably ( FIG. 16 c and FIG. 16 d ).
  • Example 7 Role of MRTF-A and T ⁇ 4 in Vascular Integrity of Mice with Sepsis
  • FIG. 17 a illustrates the protocol of the sepsis experiments conducted in mice. Sepsis was induced 14 days after rAAV treatment (rAAV.MRTF-A or rAAV.T ⁇ 4) by injection of LPS. At seven time points after sepsis induction (12 h, 24 h, 36 h, 48 h, 72 h, 96 h, 120 h), an assessment of the symptoms was carried out by means of the table shown in FIG. 17 b . The transduction of MRTF-A or T ⁇ 4 via rAAV before induction of sepsis leads to increased peripheral arterial blood pressure values after 12 and 24 hours ( FIG. 17 c ).
  • FIG. 18 a and FIG. 18 b Histological analyses of endothelial cells (PECAM-1-positive cells) and pericytes (NG-2-positive cells) showed an elevated cell number in the heart and the peripheral muscles of animals treated with T ⁇ 4 ( FIG. 18 a and FIG. 18 b ) compared to control animals transduced with rAAV.LacZ.
  • Exemplary images and a quantitative analysis of a permeability measurement by means of fluorescently labeled high molecular dextran 6 hours after sepsis induction are shown in FIG. 18 c and FIG. 18 d .
  • Contrast agent Solutrast 370 was supplied by Byk Gulden (Konstanz).
  • Recombinant vectors rAAV.MRTF-A, rAAV.T ⁇ 4, r.AAV.T ⁇ 4m, rAAV.LacZ, rAAV.Cre, and rAAV. MRTF-shRNA were produced by means of triple transfection of U293 cells.
  • rAAV.MRTF-A this was the plasmid pAAV-CMV-mMRTF-A (SEQ ID NO:16).
  • a plasmid encoding human MRTF-A may also be used, e.g.
  • pAAV-CMV-hMRTF-A (SEQ ID NO: 17).
  • a second plasmid provided AAV2 rep and AAV9 cap in trans (Bish et al., Hum. Gene Ther. 2008, 19:1359-68), while a third plasmid (delta F6) supplemented adenoviral helper functions.
  • Cells were harvested 48 hours later and vectors purified using a cesium chloride gradient as described previously (Lehrke et al., Cell Metab 2005, 1:297-308). Viral titers were measured by real time PCR versus the polyA tail of the bGH of the vector (see primer sequences in Table 1).
  • Trans and helper plasmids were supplied by courtesy of James M. Wilson, University of Pennsylvania.
  • SatisFection TPP AG, Trasadingen, Switzerland
  • HMECs human microvascular endothelial cells
  • bEnd.3 murine endothelial cells
  • HL-1 myocytic cell line HL-1 according to the manufacturer's instructions.
  • 100 ⁇ l serum- and antibiotic-free DMEM medium were mixed with 3 ⁇ l of SatisFection transfection reagent.
  • HMECs were transfected with pcDNA, MRTF-A, T ⁇ 4 ⁇ MRTF-shRNA, T ⁇ 4m (lacking the G actin binding motif KLKKTET; Bednarek et al., J. Biol. Chem. 2008, 283:1534-44), or T ⁇ 4 ⁇ CCN1-shRNA.
  • Cells (8000 cells per well) were seeded on Matrigel (BD MatrigelTM Basement Membrane Matrix, BD Biosciences, San Jose, USA) in basal endothelium growth media with a supplement of 5% fetal calf serum and images were made after 18 h. The number of rings in the low power field was quantified.
  • HL-1 cells were transduced with r.AAV.T ⁇ 4 ⁇ CCN1-shRNA, rAAV.MRTF-shRNA, or rAAV.T ⁇ 4m (1 ⁇ 10 6 AAV6 particles per cell).
  • HL-1 and HMECs embedded in Matrigel were physically separated by a semipermeable membrane. After 18 h, the HL-1 cells were removed and ring formation in the low power field was quantified.
  • CH3/10T1/2 pericyte cell attraction to murine endothelial cells was tested after transfection of the endothelial compartment with pcDNA, MRTF-A, or T ⁇ 4 ⁇ CCN2-shRNA by means of SatisFection (Agilent, Boblingen). Endothelial cells were stained with DiD (red, Vybrant®, Life Technologies) and seeded on Matrigel (12.000 cells per well). After 6 h, pericyte-like cells stained with DiO (Vybrant®, Life Technologies) (2,000 cells per well) were added and migration to the tubi was allowed for 2 h. The co-culturing images were made by means of confocal laser microscopy (Carl Zeiss, Jena).
  • HMECs were transfected as above with the indicated transgenes. 60,000 cells were grown to confluence in wells with a strip-like insert (ibidi GmbH, Planegg). After 48 h, the nuclei were stained with Syto62. Then cells were fixed with 2% PFA, permeabilized, and incubated with an anti-MRTF-A antibody (Santa Cruz Biotech, Santa Cruz, USA) and a secondary antibody (Alexa 488-coupled, Invitrogen, Düsseldorf). Images were made by means of confocal laser microscopy (Carl Zeiss, Jena) and the mean fluorescence intensity of the area of 100 nuclei, identified with Syto62, were automatically evaluated using the LS5 image browser.
  • T ⁇ 4 Detection of T ⁇ 4 was performed as described earlier (Huff et al., Ann. N. Y. Acad. Sci. 2007, 1112:451-7). Here, tissue samples were disrupted by adding 4 M perchloric acid with 1% thiodiethanol up to a final concentration of 0.4 M. Mixtures were homogenized, incubated for 30 min at 4° C. and centrifuged for 10 min at 20,000 g. The supernatant was analyzed using reverse phase chromatography. In rabbits, endogenous and exogenous T ⁇ 4 were distinguished by detection of the rabbit-specific T ⁇ 4-Ala.
  • Pellets of cells were obtained and lysed, further purified by centrifugation for 10 min at 4° C. and 13.000 rpm and used for the determination of firefly luciferase activity and Renilla luciferase activity. The ratio of firefly/ Renilla luciferase activity was calculated.
  • RT-PCR Real time PCR
  • SYBR Green dye iQ SYBR Green Supermix, Bio-Rad, Munchen
  • iQ cycler Bio-Rad, Minchen
  • the primers are listed in Table 1. Expression levels were normalized to GAPDH and shown as multiples of the pcDNA control situation.
  • the comparative 2 DDCt method was performed as described earlier (Pfosser et al., Cardiovasc Res 2005, 65: 728-36).
  • MRTF-A protein For the analysis of whole MRTF-A protein, cell culture and tissue samples were homogenized in 1 ml lysis buffer containing 20 mM Tris, 1 mM EDTA, 140 mM NaCl, 1% Nonidet P-40 (NP-40), 0.005 mg/ml leupeptin, 0.01 mg/ml aprotinin, 1 mM PMSF, pH7.5. 60 ⁇ g whole protein extract were separated by polyacrylamide gel electrophoresis with 10% sodium dodecyl sulfate (SDS-PAGE).
  • SDS-PAGE sodium dodecyl sulfate
  • the proteins were electrotransferred to a PVDF membrane (Millipore, Billerica, USA), blocked with 5% fat-free milk in PBS buffer containing 0.1% Tween 20 and incubated overnight at 4° C. with primary antibodies against MRTF-A (C-19; Santa Cruz Biotech, Santa Cruz, USA). After washing, the membrane was incubated with a secondary antibody (donkey anti-goat IgG, HRP-conjugated; Santa Cruz Biotech, Santa Cruz, USA) and developed with a chemiluminescence reagent (ECL; GE Healthcare, Buckinghamshire, England).
  • ⁇ -tubulin (6A204; Santa Cruz Biotech, Santa Cruz, USA) or, for the nucleus fraction, lamin B1 (ZL-5; Santa Cruz Biotech, Santa Cruz, USA) was used.
  • 3 ⁇ 10 12 AAV9 virus particles were administered intramuscularly to the right leg as described (Qin et al., PLoS ONE 2013, 8:e61831). On day 0, the left leg underwent mock surgery, whereas in the right leg the femoral artery was ligated.
  • tissue samples were digested as previously described (Thein et al., Comput. Methods Programs Biomed. 2000, 61:11-21; Kupaxtt et al., J Am Coll Cardiol 2010, 56:414-22). Fluorescence analysis was carried out with a Tecan Saphire 2 microtiter plate reader at the emission wavelengths 680 nm, 638 nm, 598 nm, 545 nm, 515 nm, 468 nm, and 424 nm, depending on the fluorescent dye employed. Calculations were carried out as described previously (Lebherz et al., Endothelium 2003, 10:257-65).
  • the global myocardial function (LVEDP) was examined by a Millar pressure tip catheter (Sonometrics, Ontario, Canada). An angiogram of the left ventricle for global myocardial function was performed on day 28 and day 56. The ejection fraction was obtained by planimetry of the end-systolic and end-diastolic angiogram images (Image J 1.43u, National Institute of Health, USA).
  • the analysis of regional myocardial blood flow was performed on day 28 (before rAAV treatment) and day 56 (28 days after AAV treatment) by means of fluorescent microbeads (Molecular Probes®).
  • the microbeads (15 ⁇ m, 5 ⁇ 10 6 particles per injection) were injected into the left ventricle with a pigtail catheter. Blood flow measurements were carried out at rest and at elevated heart rate (130 bpm).
  • the fluorescence content was analyzed by means of a Tecan Sapphire 2 microtiter plate reader and a calculation of the regional myocardial blood flow was performed, either as ml/g tissue absolute or as the ratio to the non-ischemic region at rest (% non-ischemic blood flow; Kupatt et al., J Am Coil Cardiol 2010, 56:414-22).
  • Tissue samples of the ischemic and non-ischemic area were examined for capillary density (PECAM-1-positive cells, red) and pericyte investment (NG-2-positive cells, green). Staining of capillaries was carried out with an anti-CD31 antibody (SC1506, Santa Cruz Biotech, Santa Cruz, USA) and a rhodamine-labeled secondary antibody, while vascular maturation was quantified by pericyte co-staining (anti-NG2-antibody AB5320, Millipore, Billerica, USA). Images of the ischemic and non-ischemic region were made with high power field magnification (40 times), and 5 independent images per region (ischemic and non-ischemic) and animal were quantified.
  • control mice, rabbits and pigs were treated with rAAV.LacZ.
  • Cryostatic sections of the LacZ-transduced animals were prepared and stained for ⁇ -galactosidase (blue staining).
  • RT-PCR for the several transgenes was carried out using the primers described in Table 1 and analyzed as described above.
  • mice homozygously expressing mT/mG express loxP sites on both sides of a membrane-directed tdTomato (mT) and a membrane-directed eGFP (Muzumdar et al., Genesis 2007, 45:593-605). Cre expression via rAAV.Cre for the determination of virus transduction efficiency deleted mT (red fluorescence) in the cells and enabled eGFP expression (green fluorescence) in the same cells ( FIG. 4 b ).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Virology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
US15/303,823 2014-04-14 2015-04-13 Aav vectors for vascular gene therapy in coronary heart disease and peripheral ischaemia Abandoned US20170096682A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10201207153.4 2014-04-14
DE102014207153.4A DE102014207153A1 (de) 2014-04-14 2014-04-14 AAV-Vektoren für vaskuläre Gentherapie in koronarer Herzerkrankung und peripherer Ischämie
PCT/EP2015/057987 WO2015158667A2 (de) 2014-04-14 2015-04-13 Aav-vektoren für vaskuläre gentherapie in koronarer herzerkrankung und peripherer ischämie

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2015/057987 A-371-Of-International WO2015158667A2 (de) 2014-04-14 2015-04-13 Aav-vektoren für vaskuläre gentherapie in koronarer herzerkrankung und peripherer ischämie

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US17/720,369 Continuation US20240167053A1 (en) 2014-04-14 2022-04-14 Aav vectors for vascular gene therapy in coronary heart disease and peripheral ischemia

Publications (1)

Publication Number Publication Date
US20170096682A1 true US20170096682A1 (en) 2017-04-06

Family

ID=52991710

Family Applications (2)

Application Number Title Priority Date Filing Date
US15/303,823 Abandoned US20170096682A1 (en) 2014-04-14 2015-04-13 Aav vectors for vascular gene therapy in coronary heart disease and peripheral ischaemia
US17/720,369 Pending US20240167053A1 (en) 2014-04-14 2022-04-14 Aav vectors for vascular gene therapy in coronary heart disease and peripheral ischemia

Family Applications After (1)

Application Number Title Priority Date Filing Date
US17/720,369 Pending US20240167053A1 (en) 2014-04-14 2022-04-14 Aav vectors for vascular gene therapy in coronary heart disease and peripheral ischemia

Country Status (5)

Country Link
US (2) US20170096682A1 (de)
EP (1) EP3132041B1 (de)
DE (1) DE102014207153A1 (de)
ES (1) ES2791877T3 (de)
WO (1) WO2015158667A2 (de)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4034661B1 (de) 2020-04-08 2023-09-06 Georg-August-Universitaet Goettingen Stiftung Oeffentlichen Rechts, Universitaetsmedizin All-in-one aav-vektoren zur behandlung von coronavirus-induzierten erkrankungen

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140271574A1 (en) * 2004-11-22 2014-09-18 The Arizona Board Of Regents On Behalf Of The University Of Arizona Methods and compositions for treating congestive heart failure

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140271574A1 (en) * 2004-11-22 2014-09-18 The Arizona Board Of Regents On Behalf Of The University Of Arizona Methods and compositions for treating congestive heart failure

Also Published As

Publication number Publication date
WO2015158667A2 (de) 2015-10-22
EP3132041A2 (de) 2017-02-22
DE102014207153A1 (de) 2015-10-15
US20240167053A1 (en) 2024-05-23
EP3132041B1 (de) 2020-03-18
ES2791877T3 (es) 2020-11-06
WO2015158667A3 (de) 2016-02-25

Similar Documents

Publication Publication Date Title
Hinkel et al. MRTF-A controls vessel growth and maturation by increasing the expression of CCN1 and CCN2
Mearini et al. Mybpc3 gene therapy for neonatal cardiomyopathy enables long-term disease prevention in mice
Singh et al. BRCA1 is a novel target to improve endothelial dysfunction and retard atherosclerosis
ES2288993T3 (es) Distribucion de factores angiogenicos mediada por virus adenoasociados.
JP6726291B2 (ja) 心筋線維化の処置のための医薬組成物
US20200308582A1 (en) Kcnk3-based gene therapy of cardiac arrhythmia
US20240167053A1 (en) Aav vectors for vascular gene therapy in coronary heart disease and peripheral ischemia
US20050158281A1 (en) Compositions and methods for systemic nucleic acid sequence delivery
Hu et al. Effect of combined VEGF165/SDF-1 gene therapy on vascular remodeling and blood perfusion in cerebral ischemia
JP2002515065A (ja) 脈管形成トランスジーンのインビボ送達により心不全および心室再構成を処置するための技術および組成物
EP1663317A1 (de) Gentransfer zum verbessern von zellentherapie
US8680064B2 (en) ShRNA gene therapy for treatment of ischemic heart disease
US11268107B2 (en) SMAD7 gene delivery into muscle cells
JP2021176848A (ja) 治療用コネキシン４５阻害剤
US11235073B2 (en) Method for treating or preventing heart failure
US20190328836A1 (en) Methods for treating ischemic injury
US20240026354A1 (en) Suppressing hippo signaling in the stem cell niche promotes skeletal muscle regeneration
WO2006123644A1 (ja) 低酸素ストレス応答に関与するInt6タンパク質、およびその利用
Pun-Garcia The role of the beta3-adrenergic receptor in animal models of cardiac injury
Sasipong Targeted overexpression of relaxin receptor 1 (RXFP1) with chronic administration of relaxin as a novel inotropic approach for heart failure treatment
US8496928B2 (en) Method for preventing and treating cardiovascular diseases with BRCA1
WO2008033304A2 (en) Compositions for promoting non-leaky collateral vascularization
Heineke et al. Cardiomyocyte GATA4 functions as a stress-responsive regulator of angiogenesis in the murine heart.
Bish Myostatin regulation and function in the heart

Legal Events

Date Code Title Description
AS Assignment

Owner name: BIOTECH GMBH, GERMANY

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:BERMPOHL, ANDREAS, DR.;REEL/FRAME:042386/0178

Effective date: 20170516

STPP Information on status: patent application and granting procedure in general

Free format text: FINAL REJECTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER

STPP Information on status: patent application and granting procedure in general

Free format text: FINAL REJECTION COUNTED, NOT YET MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: FINAL REJECTION MAILED

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION