US20170096682A1

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

Info

Publication number: US20170096682A1
Application number: US15/303,823
Authority: US
Inventors: Christian Kupatt; Rabea Hinkel
Original assignee: Individual
Current assignee: BIOTECH GmbH
Priority date: 2014-04-14
Filing date: 2015-04-13
Publication date: 2017-04-06
Also published as: DE102014207153A1; WO2015158667A2; EP3132041A2; EP3132041B1; WO2015158667A3; ES2791877T3

Abstract

The invention relates to the provision of a gene therapy for coronary heart disease and peripheral ischemia in mammals. One embodiment is an adeno-associated viral vector (AAV vector) comprising a first gene encoding a myocardin-related transcription factor A (MRTF-A). The invention further also relates to a pharmaceutical composition comprising an AAV vector of the invention and a pharmaceutically acceptable carrier. Methods for preparing the vector of the invention are also disclosed.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase entry under 35 U.S.C. §371 of International Patent Application PCT/EP2015/057987, filed Apr. 13, 2015, published as International Patent Publication WO 2015/158667 on Oct. 22, 2015, which claims the benefit of German Patent Application DE 10 2014 207 153.4, filed on Apr. 14, 2014; a, the contents of all are hereby incorporated by reference.

FIELD OF THE INVENTION

The invention is the field of gene therapy. In particular, the invention is directed to providing gene therapy for coronary heart disease and peripheral ischemia in mammals.

BACKGROUND OF THE INVENTION

In industrialized countries, 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). Besides the manifestation of coronary heart disease as an acute heart attack, 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. Although 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). In a growing population of patients, conventional therapeutic strategies become exhausted and clinical benefit is then expected from adjuvant neovascularization therapies (angiogenesis/arteriogenesis).
Previous pre-clinical (Kupatt et al., J Am Coll Cardiol 2010, 56:414-22) and clinical studies (Rissanen and Ylä-Herttuala, Mol Ther 2007, 15:1233-47) failed to reveal any increase in perfusion, if angiogenesis (capillary growth) was reinforced in the absence of microvessel maturation, i.e. recruiting of pericytes and smooth muscle cells (Jain, Nat Med 2003, 9:685-693; Potente et al., Cell 2011, 146:873-887). Furthermore, 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). In contrast, 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). On the other hand, inhibition of NF-κB signaling, hampering VEGF-A and PDGF-B expression led to a hyper-branched and immature collateral network (Tirziu et al., Circulation 2012, 126:2589-600). Consequently, an increase in stable and regulated microvessels is necessary for a successful induction of functional neovascularization.
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.

SUMMARY OF THE INVENTION

In one embodiment, the invention relates to an adeno-associated viral vector (AAV vector) comprising a gene encoding a myocardin-related transcription factor A (MRTF-A).
In another embodiment, the invention relates to an adeno-associated viral vector (AAV vector) comprising a gene encoding a thymosin β4 (Tβ4).
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.
In one embodiment, the AAV vector comprises a gene encoding an MRTF-A.
In one embodiment, 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 β4 (Tβ4) and/or a third gene encoding an MRTF-A.
In one embodiment, 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.
Furthermore, the invention also relates to 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. In one embodiment, 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.
In one embodiment, the mammal is a human No-Option-Patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Angiogenesis induced by MRTF activation and translocation into the nucleus via CCN1 and CCN2 activation

FIG. 1a and FIG. 1b , MRTF-A transfection increased endothelial cell migration in a wound scratch assay in vitro (bordered area=uncovered area); and FIG. 1c and FIG. 1d tubus formation of human microvascular endothelial cells (HMECs) in vitro (lpf=low power field). Overexpression of Tβ4 showed similar effects if no MRTF shRNA was co-administered or a Tβ4 mutant (Tβ4m) lacking the G actin binding motif KLKKTET was used (scale bar: 200 μm). FIG. 1e Tβ4 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 Tβ4m construct without the G actin binding site was used (scale bar: 20 μm). FIG. If Tβ4 transfection of HL-1 cells induced an MRTF-SRF-sensitive luciferase reporter (comprising three copies of the SRF binding site c-fos=p3DA.Luc, see Posern et al., Mol. Biol. Cell 2002, 13:4167-78), in contrast to transfection with the Tβ4 mutant. FIG. 5g Tβ4-induced tubus formation was abolished in the case of shRNA co-transfection of the MRTF-SRF target gene CCN1 (Cyr61) (scale bar: 200 μm). FIG. 1h and FIG. 1i Tubus maturation, evaluated as pericyte recruiting (PC, green fluorescence) on endothelial rings (EC rings, red fluorescence, scale bar: 200 μm), was induced by MRTF-A and Tβ4. Co-transfection of shRNA against the MRTF target gene CCN2 (CTGF) abolished the Tβ4 effect (mean±standard deviation, n=5, * p<0.05, ** p<0.001).

FIG. 2: The Tβ4-MRTF-A signaling cascade induces angiogenesis in vitro

FIG. 2a Tβ4 transfection of cardiomyocytic HL-1 cells enables the translocation of MRTF into the nucleus, an effect lacking when a Tβ4m construct without acting binding site was used. FIG. 2b Analysis of the MRTF-A protein level in the nucleus by Western blot showed an elevated MRTF-A protein level after Tβ4 overexpression in HL-1 cells. Tβ4m did not increase the MRTF-A level. FIG. 2c and FIG. 2d qRT-PCR shows that CCN1/2 shRNA prevented accumulation of CCN1/2 transcripts after Tβ4 expression. FIG. 2e and FIG. 2f rAAV.Tβ4-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.Tβ4m had no effect (scale bar: 200 μm). FIG. 2g and FIG. 2h MRTF-A mRNA expression levels (g) and MRTF-A protein level (h) were not influenced by Tβ4 overexpression, but were significantly elevated after MRTF-A transfection. FIG. 2i , FIG. 2j Tubus formation after Tβ4 release from rAAV.Tβ4-transduced HL-1 cells was disrupted by CCN1 shRNA (scale bar: 200 μm). FIG. 2k and FIG. 2l Co-transfection of rAAV.Tβ4 and CCN2 shRNA did not influence Tβ4-induced tubus formation. Furthermore, CCN1 shRNA did not influence recruitment of pericyte-like cells (mean±standard deviation, n=5, * p<0.05, ** p<0.001).

FIG. 3: Importance of MRTF signaling for neovascularization in vivo

FIG. 3a qRT-PCR analysis showed an increase in MRTF-A in the ischemic hind limb transduced with rAAV.MRTF-A. FIG. 3b rAAV.MRTF-A induced MRTF/SRF target genes CCN1 and CCN2 in vivo. FIG. 3c and FIG. 3d rAAV.MRTF-A transduction increased the capillary/muscle fiber ratio (c/mf) in a manner similar to MRTF activator Tβ4. rAAV.Tβ4m, a mutant without the G actin binding domain, or co-application of Tβ4 and rAAV.MRTF-A-shRNA, had no effect (PECAM-1 staining, scale bar 100 μm). FIG. 3e and FIG. 3f Functionally, transduction with rAAV.MRTF-A and -Tβ4, but not rAAV.Tβ4m or rAAV.Tβ4+MRTF-shRNA, improved the perfusion of the hind limb on day 3 and day 7. FIG. 3g After rAAV.Cre vector-induced MRTF-B deletion in MRTF-A-deficient mice (Mrtfa^−/−/b^flox/flox+rAAV.Cre=MRTF-A/B^−/−Vi), Tβ4 transduction could not induce angiogenesis, in contrast to Mrtfa^+/−/b^flox/floxmice (=MRTF-A/B^+/−). FIG. 3h Perfusion increased by rAAV.Tβ4 was suppressed in MRTF-A/B^−/−Vi mice. FIG. 3i and FIG. 3j In CCN1^−/−Vi mice (=CCN1^flox/flox+rAAV.Cre), both the increase of the capillary/muscle fiber ratio (PECAM-1 staining, scale bar 100 μm) and the increase of hind limb perfusion FIG. 3k and FIG. 3l were suppressed (mean±standard deviation, n=5, * p<0.05, ** p<0.001).

FIG. 4: MRTF-A induced vessel growth in mouse hind limb ischemia

FIG. 4a 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. 4b 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. 4c i.m. injection of rAAV.LacZ (3×10¹²virus particles) led to a homogenous transduction (blue staining) of the targeted hind limb, but not of the opposite one. FIG. 4d qRT-PCR detection of Tβ4 in the rAAV.Tβ4-transduced ischemic hind limbs, but not in the rAAV.LacZ-transduced hind limbs. FIG. 4e HPLC analysis showed an increase of Tβ4 protein concentration in the rAAV.Tβ4-transduced ischemic hind limbs. FIG. 4f rAAV.Tβ4 induced MRTF target genes CCN1 and CCN2 in vivo. FIG. 4g Tβ4-induced maturation of capillaries (pericyte investment, NG2 staining) was suppressed in MRTF-A/B^−/−Vi hind limbs. FIG. 4h and FIG. 4i In both MRTF-A^−/−/B^flox/floxmice (MRTF-A knockout) and MRTF-A^+/−/B^−/− Vi mice (MRTF-B knockout) rAAV.Tβ4 transduction induced an increase of capillary density (h) and perfusion (i). However, the rAAV.Tβ4 effect was largest in wild type mice (MRTF-A^+/−/B^flox/flox). Furthermore, there was no significant difference between MRTF-A and MRTF-B knockout mice (mean±standard deviation, n=4, * p<0.05 vs. control).

FIG. 5: Tβ4/MRTF-A-induced microvessel maturation: essential role for collateral growth and improved perfusion

FIG. 5a HPLC analysis showed a significant increase of Tβ4 protein after rAAV.Tβ4 transduction of ischemic rabbit hind limbs, whereas rabbit-specific Tβ4-Ala remained unchanged. FIG. 5b , FIG. 5c and FIG. 5d rAAV.MRTF-A or rAAV.Tβ4 administration increased capillary density (PECAM-1 staining) and pericyte investment (NG2 staining, scale bar: 50 μm), both of which were abolished by co-application of angiopoietin 2 (rAAV.Ang2). FIG. 5e and FIG. 5f Angiographies of ischemic hind limbs on day 35 showed an increased collateral formation in rAAV.MRTF-A- and rAAV.Tβ4-treated animals (arrows show site of excision of the femoral artery). Co-application of rAAV.Ang2 abolished this effect. FIG. 5g rAAV.MRTF-A and rAAV.Tβ4 induced an increase of perfusion in ischemic hind limbs, unless rAAV.Ang2 or L-NAME, which inhibits nitrogen oxide formation, were co-applied (mean±standard deviation, n=5, * p<0.05, ** p<0.001).

FIG. 6: Tβ4-MRTF-A-induced vessel growth in rabbits

FIG. 6a Protocol of a model for rabbit hind limb ischemia (femoral artery excision). FIG. 6b Pβ-galactosidase staining 5 weeks after i.m. injection of rAAV.LacZ into the rabbit hind limb. FIG. 6c and FIG. 6d qRT-PCR of Tβ4 (c) and angiopoietin 2 (d), normalized to GAPDH, in control and treated animals (n=3). FIG. 6e and FIG. 6f Tβ4 overexpression only in the lower limb (rAAV.Tβ4 LL) increased capillary density (PECAM-1 staining) in the lower limb, whereas Tβ4 transduction only in the upper limb (rAAV.Tβ4 UL) did not influence the capillarization in the lower limb (scale bar: 50 μm). FIG. 6g and FIG. 6h Collateralization was increased in the rAAV.Tβ4 UL group, and to an even greater extent in the rAAV.Tβ4 LL group, whereas perfusion FIG. 6i was increased only in the rAAV.Tβ4 LL group and not in the rAAV.T14 UL group. FIG. 6j Compared to rAAV.Tβ4-transduced rabbits, the co-administration of L-NAME did not decrease the NG2/PECAM-1 ratio, indicating robust and balanced microvessel growth. In contrast, L-NAME or rAAV.Ang2 treatment alone was not able to increase the NG2/PECAM-1 ratio as strongly as rAAV.Tβ4. FIG. 6k and FIG. 6l Tβ4-dependent increase of collateralization and perfusion was significantly reduced if L-NAME was co-administered (mean±standard deviation, n=5, ** p<0.01).

FIG. 7: MRTF-A improves collateral formation and perfusion in hibernating myocardium of pigs

FIG. 7a -FIG. 7c In hibernating pig myocardium (see FIG. 8a ), rAAV.MRTF-A transduction and ubiquitous overexpression of Tβ4 (Tβ4tg, see FIG. 9) induced capillary sprouting (PECAM-1 staining, scale bar: 50 μm) and pericyte investment (NG2 staining). FIG. 7d and FIG. 7e Moreover, collateral growth was detected in rAAV.MRTF-A-transduced hearts, similarly to Tβ4tg hearts. FIG. 7f The regional flow reserve, obtained by fast atrial stimulation (130 beats per minute), was increased in rAAV-MRTF-A-transduced and Tβ4-transgenic hearts. FIG. 7g 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 Tβ4tg hearts. FIG. 7h 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 (Tβ4tg), however, showed no loss of function on day 28. FIG. 7i Mechanisms of MRTF-mediated therapeutic neovascularization: MRTF-A or Tβ4 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. CCN1 enables capillary growth (angiogenesis), whereas CCN2 increases pericyte investment (vascular maturation). Together, these mechanisms induce collateral growth in a nitrogen oxide-dependent manner, leading to therapeutic neovascularization (mean±standard deviation, n=5, * p<0.05, ** p<0.001).

FIG. 8: Functional efficiency of the Tβ4-MRTF-A axis in chronically ischemic pig hearts

FIG. 8a Protocol of the pig model for hibernating myocardium. FIG. 8b RT-PCR-detection of MRTF-A and Tβ4 in control pigs compared with rAAV.Tβ4 and Tβ4-transgenic (Tβ4tg) pig hearts. FIG. 7c Examples of LacZ staining (blue) after rAAV.LacZ retroinfusion (5×10¹²virus particles) into the pig heart. FIG. 7d 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 Tβ4tg hearts, similarly to the flow reserve FIG. 7e at fast heart rate (130 bpm). FIG. 7f 4 weeks after treatment (on day 56), the regional myocardial blood flow in rAAV.MRTF-A und Tβ4tg animals improved. FIG. 7g Furthermore, the Rentrop score showed an increased collateralization on day 56 in rAAV.MRTF-A-transduced or Tβ4tg hearts. FIG. 7h Examples of MRT analysis on day 56 for control (left) and rAAV.MRTF-A-treated pig hearts. FIG. 7i The left ventricular end-diastolic pressure (LVEDP) increased in ischemic hearts from day 28 to day 56 if MRTF-A was not overexpressed. Tβ4tg constitutively overexpressing Tβ4 showed no change from day 28 to day 56 (mean±standard deviation, n=5, * p<0.05, ** p<0.001).

FIG. 9: Production of Tβ4-transgenic pigs

Fibroblasts of donor pigs were isolated and cultured. pCMV-Tβ4 was transfected by electroporation and the cells were cultured for 14 days. After detection of stable transfection of Tβ4, a somatic nuclear transfer into pig oocytes was performed. Offspring were analyzed for Tβ4 expression and fibroblasts of Tβ4-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 Tβ4-induced cardioprotection

FIG. 10a and FIG. 10b rAAV.Tβ4 induced capillary growth (PECAM-1 staining) and FIG. 10c pericyte investment (NG2 staining, scale bar 50 μm), unless co-administration of rAAV.MRTF-shRNA prevented both processes. FIG. 10d and FIG. 10e Collateral growth was detected in rAAV.Tβ4-transduced animals, but not after co-administration of rAAV.MRTF-shRNA. FIG. 10f Rentrop scores showed increased collateralization after rAAV.Tβ4 transduction, except in the case of co-administration of MRTF-A shRNA. FIG. 10g Regional myocardial blood flow at flow reserve (atrial stimulation 130/min) improved in rAAV.Tβ4-treated animals, but not in rAAV.Tβ4+MRTF-shRNA hearts. FIG. 10h Analysis of the ejection fraction showed improved systolic myocardium function in rAAV.Tβ4-transduced animals (day 56), as compared with day 28 (day of transduction). No improvement of the ejection fraction was observed in rAAV.Tβ4+MRTF-shRNA-treated hearts. FIG. 10i MRT images of rAAV.Tβ4-transduced hearts without (left) or with (right) rAAV.MRTF-shRNA co-administration. FIG. 10j Regional myocardium function, measured by subendocardial segment shortening at rest and at atrial stimulation (130 and 150 bpm) shows increased functional reserve after rAAV.Tβ4 but not rAAV.Tβ4+MRTF-shRNA transduction (mean±standard deviation, n=5, * p<0.05, ** p<0.001).

FIG. 11: Production and cardial phenotyping of INS^C94Y-transgenic pigs (diabetes mellitus type I)

FIG. 11a Process of producing the INS^C94Y-transgenic pigs. FIG. 11b Blood glucose levels of wild type and diabetic pigs. FIG. 11c Fluorescence staining of endothelial cells (PECAM-1-positive cells, red) and pericytes (NG-2-positive cells, green). FIG. 11d Number of endothelial cells in the myocardium of wild type and diabetic pigs. FIG. 11e 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. 12a Protocol of the pig model for hibernating myocardium with diabetes mellitus type I or hypercholesterolemia. FIG. 12b Blood glucose concentration of the specific groups of animals over the duration of the experiment: control wild type; wild type treated with rAAV.Tβ4; control with diabetes; diabetes treated with rAAV.Tβ4. FIG. 12 c and FIG. 12d Serum trigylceride and cholesterol levels in animals with hypercholesterolemia (fat rich diet) and normal diet.

FIG. 13: Influence of rAAV.Tβ4 application on angio- and arteriogenesis in animals with diabetes mellitus type I

FIG. 13a 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. 13b and FIG. 13c Number of endothelial cells and pericytes. FIG. 13d Number of collaterals formed. FIG. 13e Rentrop score.

FIG. 14: Functional efficiency of rAAV.Tβ4 application in animals with diabetes mellitus type I

FIG. 14a and FIG. 14b Left ventricular end-diastolic pressure on

days

28 and 56 and its change between these time points. FIG. 14c and FIG. 14d Ejection fraction on

days

28 and 56 and its change between these time points.

FIG. 15: Influence of elevated cholesterol levels on Tβ4-mediated angio- and arteriogenesis

Number of FIG. 15a endothelial cells, FIG. 15b collaterals, and FIG. 15c Rentrop score in the ischemic area of hypercholesterolemic control and rAAV-Tβ4-treated animals.

FIG. 16: Functional efficiency of rAAV.Tβ4 application in animals with hypercholesterolemia

FIG. 16a and FIG. 16b Left ventricular end-diastolic pressure on

days

28 and 56 and its change between these time points in hypercholesterolemic control and rAAV-Tβ4-treated animals. FIG. 16c and FIG. 16d Ejection fraction on

days

28 and 56 and its change between these time points in hypercholesterolemic control and rAAV-Tβ4-treated animals. FIG. 16e Regional myocardium function, measured as subendocardial segment shortening at rest and with increased heart rate (130 and 150 beats per minute).

FIG. 17: rAAV.Tβ4 and rAAV.MRTF-A pretreatment in a mouse model of sepsis

FIG. 17a Protocol of the sepsis tests in mice. FIG. 17b Scoring scheme for the assessment of sepsis symptoms in mice and determination of the stop criteria. FIG. 17c Peripheral arterial blood pressure values after 12 and 24 hours in animals with sepsis treated with different rAAV. FIG. 17d Symptom scores of the animals with sepsis in the treatment groups. FIG. 17e Cumulated survival after LPS-induced sepsis.

FIG. 18: Role of MRTF-A and Tβ4 in vascular integrity during sepsis

FIG. 18a and FIG. 18b 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. 18c and FIG. 18d Exemplary images and quantitative analysis of a permeability measurement by means of fluorescently labeled high molecular dextran 6 hours after induction of sepsis.

DETAILED DESCRIPTION OF THE INVENTION

In our experiments (see Examples), we have found that the combination of a long-acting vector and the overexpression of an effective vasoactive growth factor represents a therapeutic option for patients with chronic ischemic diseases of skeletal or heart muscle tissue. The combination of an adeno-associated vector and thymosin β4 (Tβ4) or MRTF-A transgene, respectively, leads to robust therapeutic vessel reformation in three species (mouse, rabbit, and pig). This therapeutic neovascularization in turn leads to a notably improved perfusion in the models of peripheral arterial obstruction disease and chronic myocardial ischemia. In the model of chronic ischemic cardiomyopathy in pigs it leads additionally to increased heart function. This specific effect can be achieved even in large animals with additional cardiovascular risk factors (elevated sugar or lipid levels).
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 Tβ4, a peptide activating MRTF-A by G actin binding (FIG. 1a -FIG. 1i ), initiates an orchestrated micro- and macrovascular growth response in the case of chronic ischemia of peripheral (FIG. 3a -FIG. 3l , FIG. 5a -FIG. 5g ) and heart muscle cells (FIG. 7a -FIG. 7i ). Consistent with these observations, chronic dysfunction of hibernating pig myocardium was resolved both by direct MRTF-A activation and MRTF-A activation via Tβ4 (FIG. 7). The idea that 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). Thus, MRTF-A is located at the interface of myocyte and vascular regeneration in hibernating myocardium. Tβ4, 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 Tβ4 signaling has been shown in vitro and in vivo, since MRTF-A shRNA could suppress endothelial migration and sprouting (FIG. 1b , FIG. 1d ) and micro- and macrovascular growth (FIG. 3d , FIG. 3f ) and functional improvement of the heart (FIG. 10). Correspondingly, 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). Furthermore, MRF, the main target of MRTF-A, has recently been identified as essential for the behavior of apical cells in sprouting angiogenesis after VEGF-A stimulation (Franco et al., Development 2013, 2321-33: Andoh et al., J Biochem 2006, 140:483-9). Nevertheless, VEGF-A leads to the growth of immature and unstable capillaries (Dor et al., EMBO J. 2002, 21:1939-47), in contrast to Tβ4-MRTF-A, thus indicating a difference in the signaling mechanisms for these two vascular growth factors.
Collectively, our data demonstrate that activation of Tβ4-MRTF via CCN1/CCN2 augments collateral blood flow in the ischemic heart and hind limb via induction of CCN1/CCN2. At the cellular level this response involves endothelial sprouting via CCN-1 (CYR61) and maturation, i.e. pericyte investment, via CCN2 (CTGF), resulting in a stable and functional vascular network that can carry collateral blood flow and improve conductance. Pericyte investment is crucial here, since Ang-2, by virtue of disrupting pericyte investment (Ziegler et al., J Clin Invest 2013, 123:3436-45), abolished the positive effects exerted by Tβ4-MRTF signaling (FIG. 3). This finding supports a central role of vessel maturation and balanced vessel growth and paves the way for new therapeutic avenues towards functional neovascularization.
Therefore, 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.
Particularly preferred is the use of 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. However, an AAV vector pseudotyped with AAV9 may also be used. By this 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. For example, AAV2.9 is an AAV2 vector pseudotyped with envelope proteins of AAV9. For the present invention, AAV2.9, AAV1.9, and AAV6.9 are suitable as pseudotyped vectors. By using a heart muscle-tropic vector, it is ensured that expression of MRTF-A occurs in the heart muscle, where it can initiate therapeutic neovascularization.
Alternatively, 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 β4 (Tβ4) or an MRTF-B. The Tβ4 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. Exemplary cardio-specific promoters are the MLC2 promoter, the α myosin heavy chain promoter (α-MHC promoter) and the troponin I promoter (TnI promoter). However, 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.
Methods for the production of 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. Here, 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.
In a further embodiment, the invention relates also to 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. In particular, such use can occur in a mammal for treatment of coronary heart diseases or peripheral ischemia. Preferred mammals are human, pig, rabbit and mouse.
The term “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. “Peripheral 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 Tβ4 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.
Furthermore, vectors of the invention are particularly suitable for the treatment of subjects bearing additional cardiovascular risk factors. Such 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.

EXAMPLES

Example 1: Induction of Hallmarks of Angiogenesis by MRTF-A In Vitro

We have found (FIG. 1a and FIG. 1d ) that MRTF-A induced hallmarks of angiogenesis, i.e. migration and tubus formation of cultured human microvascular endothelial cells, to a comparable degree as Tβ4. The pro-angiogenic effect of MRTF-A was found to be dependent on the G actin binding motif of Tβ4, since mutation of this domain and abolition of G actin binding eliminated the effect of Tβ4 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, Tβ4 increased MRTF-A translocation into the nucleus (FIG. 1e , FIG. 2a and FIG. 2b ), similar to the transcription of an MRTF/SRF-dependent reporter gene containing three SRF binding sites of the c-fos promoter (p3DA.Luc, FIG. 1f ; Geneste et al., J Cell Biol 2002, 157:831-8). Both MRTF-A and Tβ4 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. 2c-g ; Hall-Glenn et al., PLoS ONE 2012, 7:e30562). We observed that Tβ4 transfection did not affect the MRTF-A content (FIG. 2h ), in contrast to MRTF-A transfection. Consistently with CCN1/2 being downstream of MRTFs and relevant for vessel formation, disruption by CCN1 shRNA prevented Tβ4-induced tubus formation (FIG. 1g ), whereas CCN2 shRNA suspended the attachment of a murine pericyte-like cell line (C3H/10T1/2) to endothelial tubi in vitro (FIG. 1i and FIG. 1j ).

Example 2: Treatment of Hind Limb Ischemia in the Mouse with AAV-Based MRTF-A Gene Therapy

In order to further demonstrate the relevance of MRTF-A signaling in vivo, we employed a mouse model with hind limb ischemia. Intramuscular injection of recombinant AAV vectors (rAAV, FIG. 4a and FIG. 4c ) increased tissue concentration of target proteins in the treated limb (FIG. 3a ) and transcript levels of downstream mediators CCN1 and CCN2 (FIG. 3b , FIG. 4d and FIG. 4f ). Consistent therewith, rAAV.MRTF-A induced capillary growth (FIG. 3c and FIG. 3d ) and increased perfusion on day 7 (FIG. 3e and FIG. 3). As an upstream activator, Tβ4 had a similar effect on vascular growth and function (FIG. 2c -FIG. 2f ), unless the G actin binding motif was missing (Tβ4m) 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). In order to further examine the relevance of Tβ4-induced vascular growth, rAAV.Cre was administered to Mrtfa^−/−-Mrtf^flox/floxhind limbs to generate MRTF-A/B double insufficiency. In Cre-induced MRTF-A/B knockout mice, Tβ4 was not capable of stimulating capillary growth (FIG. 3g ) and pericyte recruitment (FIG. 4g and FIG. 4h ) and of improving perfusion (FIG. 3h , FIG. 4i ) on day 7 after induction of ischemia. Similarly, hind limbs did not show Tβ4-mediated increase of capillaries (FIG. 3i and FIG. 3j ) or perfusion (FIG. 3k and FIG. 3l ) on day 7, if rAAV.Cre was administered to CCN1^flox/floxmice. Consequently, MRTF-A transduction or MRTF-A activation via Tβ4-mediated G actin sequestration stimulates transcription of CCN1 to mediate functional vascular regeneration.

Example 3: Treatment of Hind Limb Ischemia in the Rabbit with AAV-Based MRTF-A Gene Therapy

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. 6a ), 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 Tβ4 (FIG. 5a , FIG. 6b and FIG. 6d ) led to functional neovascularization, including CD31⁺ capillary sprouting (FIG. 5b and FIG. 5c ), NG2⁺ pericyte investment (FIG. 5b and FIG. 5d ) and collateral growth (FIG. 5e and FIG. 5f ). In particular, MRTF activation via Tβ4 transduction of the hip region, though capable of inducing moderate collateral growth, did not increase perfusion, whereas limiting MRTF-A activation via Tβ4 to the calf region was sufficient to significantly stimulate micro- and macrovascular growth and perfusion (FIG. 6e -FIG. 6i ). Detachment of microvascular pericytes by enforced angiopoietin 2 expression (FIG. 5b and FIG. 5d ) abolished Tβ4-mediated collateralization and flowthrough improvement (FIG. 5e and FIG. 5g ). Furthermore, blocking of flow-induced vasodilation by oral administration of L-NAME, a non-selective nitrogen oxide synthase inhibitor, did not affect capillary growth and maturation (FIG. 6j ), but prevented formation of collaterals and increased perfusion (FIG. 6k and FIG. 6l ). Thus nitrogen oxide, subsequent to microvascular growth and maturation, appears to mediate collateral growth. This observation is supplemented by the finding that direct Tβ4 injection into the area of collateral growth (upper limb) did not improve perfusion to the same degree as distant injection of rAAV.Tβ4 into the lower limb, the area of microvascular growth (FIG. 6e -FIG. 6i ). These findings indicate that microvascular maturation and nitrogen oxide signaling are processes that must take place in the sequence of MRTF-A-mediated vascular growth to achieve a functional neovascularization.

Example 4: Treatment of Hibernating Myocardium in the Pig with AAV-Based MRTF-A Gene Therapy

Although both peripheral and coronary arteries perfuse muscle tissue, permanent contraction activity is a unique feature of the heart muscle, which requires a continuous oxygen supply. A chronic drop in oxygen supply changes the cellular composition of living cardiomyocytes in the ischemic area, leading to a regional loss of contraction force called hibernating myocardium (Heusch and Schulz, J Mol Cell Cardiol 1996, 28:2359-72; Nagueh et al., Circulation 1999, 100:490-6). Within cardiomyocytes, hallmarks of hibernating myocardium are reduced myofilament (Bito et al., Circ Res 2007, 100:229-37) and mitochondria content and increased glycogen content (St. Louis et al., Ann Thoracic Surg 2000, 69:1351-7). We examined the potential of MRTF-A to resolve dysfunction in hibernating myocardium induced by percutaneous implantation of a reduction stent in pig hearts (Kupat et al., J Am Coll Cardiol 2007, 49:1575-84) leading to a gradual occlusion of the ramus circumflexus (RCx, FIG. 8a ). On day 28, following rAAV.MRTF-A administration into the ischemic area that significantly increased MRTF-A content in the tissue (FIG. 8b ), we detected a significantly higher degree of capillary density and pericyte investment (FIG. 7a -FIG. 7c ). Collateral growth and perfusion under fast heart rate (130/min) were still impaired on day 28, i.e. before LacZ and MRTF-A transduction (FIG. 8c -FIG. 8f ), but improved on day 56, i.e. 4 weeks after MRTF-A transduction, but not after LacZ transduction (FIGS. 7d-f ).
Increased collateral perfusion (FIG. 8g ) generated an improved functional reserve of the ischemic area at fast heart rate (130 and 150 beats per minute, FIG. 7g ). At the same time, we observed an improved ejection fraction as a marker of global systolic function (FIG. 7 h) and a drop of the left ventricular end-diastolic pressure (FIG. 8i ), a predictive marker of the beginning of heart failure.
Transgenic pigs that ubiquitously and constitutively express Tβ4 (FIG. 9) showed similar capillary growth and maturation (FIG. 7a -FIG. 7c ). On day 56, the blood flow reserve in the ischemic area was increased (FIG. 7f ) and the functional reserve in the ischemic region (FIG. 7g ) or the entire heart (FIG. 7h ) demonstrated an increase similar to rAAV.MRTF-A-treated hearts. In particular, due to the constitutive Tβ4 overexpression from day 0 to day 28, Tβ4tg animals did not experience a significant loss of perfusion or myocardial function at rest or at fast heart rate (FIG. 7g , FIG. 8d , FIG. 8g , FIG. 8i ).
Furthermore, rAAV.Tβ4-induced micro- and macrovascular growth and subsequent increases in the perfusion reserve were suppressed when inhibiting MRTF-A shRNA was co-administered (FIG. 10a -FIG. 10f ). The overall gain in global (FIG. 10h , examples in FIG. 10i ) and regional myocardium function (FIG. 10j ) was abolished when Tβ4 transduction was combined with MRTF-A inhibition by a suitable shRNA.
We therefore demonstrate, using a combined genetic and physiologic approach in each of a mouse, rabbit and pig model, that 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. Mechanistically, we show that MRTF translocation downstream of thymosin β4 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. 7i ).

Example 5: Treatment of Hibernating Myocardium in Diabetic Pigs with AAV-Based Tβ4 Gene Therapy

Generation and Cardial Phenotyping of INS^C94Y-Transgenic Pigs (Diabetes Mellitus Type I)
The generation of transgenic pigs bearing the C94Y mutation in the insulin gene (INS^C94Y) 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 β cells of the pancreas and an accumulation of the misfolded insulin in the endoplasmic reticulum (ER). ER stress leads to β cell apoptosis and thereby eventually to diabetes mellitus type I.
First, an INS^C94Yexpression 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.
Once insulin treatment was stopped, the animals showed a markedly elevated blood glucose level (FIG. 11c and FIG. 11d ). 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. The analysis of the left ventricular end-diastolic pressure shows a significant increase in animals with diabetes mellitus type I as a sign of reduced global heart function already at an early stage (FIG. 11e ; mean±standard deviation; n=4, * p<0.05, **p<0.001).
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. Compared with the control groups (wt±rAAV.Tβ4), the INS^C94Y-transgenic animals with diabetes mellitus type I (labeled as control tg and rAAV.Tβ4 tg) showed elevated blood glucose levels for the entire assay period (FIG. 12b ). However, no difference appeared between the rAAV.Tβ4-treated group and the control group either for the wild type group or for the transgenic animals (FIG. 12c and FIG. 12d ). An influence of Tβ4 or MRTF-A on the blood glucose level is not to be expected. In the animals with hypercholesterolemia, considerably elevated triglyceride and cholesterol levels appeared in the serum after 9 weeks of ingesting a fat-rich diet (mean±standard deviation; n=4, **p<0.001).
Effect of rAAV.Tβ4 Application in Animals with Diabetes Mellitus Type I
In hibernating pig myocardium, rAAV.Tβ4 transduction induces capillary sprouting (PECAM-1 staining, red) and pericyte recruitment (NG-2 staining, green) in both groups (wild type and diabetes); FIG. 13a -FIG. 13c . Moreover, considerable collateral growth was induced by overexpression of Tβ4 via rAAV (FIG. 13d ), and a considerably better filling of the distal blood vessel could be measured by means of the Rentrop score (FIG. 13e ). The effect could also be measured in both groups: wild type and diabetes (mean±standard deviation; n=4, * p<0.05, **p<0.001).
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.Tβ4 transduction (FIG. 14a and FIG. 14b ). 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 Tβ4 overexpression considerably improved in both groups (wild type and diabetes) (FIG. 14c and FIG. 14d ; mean±standard deviation; n=4, * p<0.05, **p<0.001).

Example 6: Effects of Tβ4 Gene Therapy on the Myocardium of Pigs with Hypercholesterolemia

In control animals receiving 9 weeks of a fat-rich feeding, a considerable reduction of capillaries (PECAM-1-positive cells) appeared in the ischemic area (FIG. 15a ). With rAAV.Tβ4 application, the capillaries (PECAM-1-positive cells) in the ischemic area could be considerably increased. With rAAV.Tβ4 transduction, the collateral growth could be increased even in animals having elevated cholesterol levels (FIG. 15b ). This also led to a better filling of the distal vessel section in the ischemic area, as shown by the Rentrop score (FIG. 15c ; mean±standard deviation; n=4, * p<0.05, **p<0.001).
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.Tβ4 transduction (FIG. 16a and FIG. 16b ). 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 Tβ4 overexpression improved considerably (FIG. 16c and FIG. 16d ). The regional myocardium function, measured as segment shortening at rest and under increased heart rate (130 and 150 beats per minute) showed an improved functional reserve in animals with rAAV.Tβ4 therapy (FIG. 16e ; mean±standard deviation; n=4, * p<0.05, **p<0.001).

Example 7: Role of MRTF-A and Tβ4 in Vascular Integrity of Mice with Sepsis

FIG. 17a illustrates the protocol of the sepsis experiments conducted in mice. Sepsis was induced 14 days after rAAV treatment (rAAV.MRTF-A or rAAV.Tβ4) 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. 17b . The transduction of MRTF-A or Tβ4 via rAAV before induction of sepsis leads to increased peripheral arterial blood pressure values after 12 and 24 hours (FIG. 17c ). In the course of up to 36 hours after sepsis, the rAAV.Tβ4- and rAAV.MRTF-A-treated animals show considerably lower symptom scores as compared with the rAAV.LacZ-treated control animals (FIG. 17d ). The cumulative survival after LPS-induced sepsis is considerably improved by overexpression of Tβ4 and MRTF-A (FIG. 17e ; mean±standard deviation; n=7-15, * p<0.05, **p<0.001).
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 Tβ4 (FIG. 18a and FIG. 18b ) 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. 18c and FIG. 18d . Here a considerably reduced leakage of the indicator was observed after Tβ4 overexpression compared to the lacZ-transduced control animals (mean±standard deviation; n=4, * p<0.05, **p<0.001).
Materials and Methods
The experiments described in the examples were performed using the techniques described in the following.
Reagents
All cell culture media and chemicals were purchased from SIGMA (Deisenhofen), if not indicated to the contrary. Contrast agent Solutrast 370 was supplied by Byk Gulden (Konstanz).
Adeno-Associated Viral Vectors
Recombinant vectors rAAV.MRTF-A, rAAV.Tβ4, r.AAV.Tβ4m, rAAV.LacZ, rAAV.Cre, and rAAV. MRTF-shRNA were produced by means of triple transfection of U293 cells. One plasmid encoded the transgene under control of a CMV promoter flanked by cis-acting internal terminal repeats of AAV2. In the case of rAAV.MRTF-A, this was the plasmid pAAV-CMV-mMRTF-A (SEQ ID NO:16). However, 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.
Cell Culture
SatisFection (TPP AG, Trasadingen, Switzerland) was used for the transfection of human microvascular endothelial cells (HMECs), murine endothelial cells (bEnd.3), and the myocytic cell line HL-1 according to the manufacturer's instructions. 100 μl serum- and antibiotic-free DMEM medium were mixed with 3 μl of SatisFection transfection reagent.
In Vitro Tubus Formation and Co-Culturing Experiments
For the Matrigel experiments, HMECs were transfected with pcDNA, MRTF-A, Tβ4±MRTF-shRNA, Tβ4m (lacking the G actin binding motif KLKKTET; Bednarek et al., J. Biol. Chem. 2008, 283:1534-44), or Tβ4±CCN1-shRNA. Cells (8000 cells per well) were seeded on Matrigel (BD Matrigel™ 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.
In co-culturing experiments, HL-1 cells were transduced with r.AAV.Tβ4±CCN1-shRNA, rAAV.MRTF-shRNA, or rAAV.Tβ4m (1×10⁶AAV6 particles per cell). HL-1 and HMECs embedded in Matrigel (8,000 per well) 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 (bEnd.3) was tested after transfection of the endothelial compartment with pcDNA, MRTF-A, or Tβ4±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).
Migration Assay
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, Karlsruhe). 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.
HPLC Analysis
Detection of Tβ4 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 Tβ4 were distinguished by detection of the rabbit-specific Tβ4-Ala.
Luciferase Assay
To determine MRTF-dependent luciferase activity, HMECs and HL-1 cells were transfected with p3DA.Luc (=a construct of a synthetic promoter having three copies of the c-fos SRF binding site and a Xenopus type 5 actin TATA box plus a transcription start site inserted in pGL3; Posern et al., Mol. Biol. Cell 2002, 13; 4167-78), an SRF reporter gene, and 930 ng of pcDNA, Tβ4 or Tβ4m. Comparable transfection efficiencies were ensured by co-transfection of 50 ng ptkRL (Renilla luciferase reporter). 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.
RNA Modulation and Detection
Real time PCR (RT-PCR) was conducted with SYBR Green dye (iQ SYBR Green Supermix, Bio-Rad, Munchen) and measured on an 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).
Western Blot Analysis of MRTF-A
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 μg whole protein extract were separated by polyacrylamide gel electrophoresis with 10% sodium dodecyl sulfate (SDS-PAGE). After electrophoresis, 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). For analysis of the MRTF-A protein content in the nucleus or the cytosol, respectively, a separation with the Ne-Per® reagents for cytoplasmic and nucleus extraction (Thermo Scientific, Rockford, USA) was conducted according to the manufacturer's guidelines. Then a Western blot analysis was carried out as described above. As a control protein, either α-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.
Animal Experiments
Animal care and all experimental procedures were carried out under strict adherence to the German and NIH animal guidelines and have been approved by the Animal Protection Commission of the Government of Upper Bavaria (AZ 55.2-1-54-2531-26/09, 130/08, 140/07). All animal experiments were conducted at the Walter Brendel Center for Experimental Medicine in Munich.
Mouse Hind Limb Ischemia
Unilateral hind limb ischemia of the right leg was performed in male C56Bl mice of the same age (Charles River, Sulzfeld) and in MRTF-A^+/−/B^flox/flox, MRTF-A^−/−/B^flox/flox, MRTF-A^+/−/B^−/−Vi (=MRTF-A-^+/−/B^flox/flox+3×10¹²rAAV.cre), MRTF-A^−/−/B^−/−Vi (=MRTF-A-^−/−/B^flox/flox+3×10¹²rAAV.cre) (Weinl et al., J. Clin. Invest. 2013, 123:2193-226) and CCN1^−/−Vi mice (=Cyr61^flox/flox+3×10¹²rAAV.Cre; produced in the laboratory of Ralf Adams at the Max Planck Institute for Molecular Biomedicine in Münster) as previously described (Limbourg et al., Nat. Protocols 2009, 4:1737-48). Before induction of ischemia (day −14), 3×10¹²AAV9 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. The measurements of post-ischemic blood flow recovery were conducted by means of laser Doppler flowthrough cytometry (Moor Instruments, Devon, England). Measurements were made directly before and after surgery, on day 3, and on day 7. The results are given as the ratio of right leg to left leg including subtraction of the background tissue value. RT-PCR and HPLC analysis were carried out on day 5 after induction of ischemia; tissue was collected from treated and non-treated legs. Analyses of capillary density and vascular maturation were carried out on day 7 in all groups by means of PECAM-1 (sc1506, Santa Cruz Biotech, Santa Cruz, USA) and NG2 staining (in MRTF-A^+/−/B^flox/floxmice; Chemicon, Nürnberg) in frozen tissue samples of the M. gastrocnemius and M. adductor.
Rabbit Hind Limb Ischemia
On day 0, the complete femoral artery of the right leg in New Zealand rabbits was removed (Pfosser et al., Cardiovasc. Res. 2005, 65:728-736) and rAAV administration (5×10¹²virus particles) was performed by means of intramuscular injection into the right hind limb as indicated. On day 7 and day 35, angiography was performed by injection of contrast agent (Solutrast 370, Byk Gulden, Konstanz) into the ischemic leg with an automatic injector (Harvard Apparatus, Freiburg). Furthermore, fluorescent microbeads (15 μm, Molecular Probes®, Life Technologies, Carlsbad, USA) were used for blood flow measurements in ischemic and non-ischemic tissue. For blood flow analysis, 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). Analysis of capillary density and vascular maturation was carried out by means of PECAM-1 (sc1506, Santa Cruz Biotech, Santa Cruz, USA) and NG2 staining (in MRTF-A^+/−/B^flox/floxmice; Chemicon, Nürnberg) in frozen tissue samples of the ischemic and non-ischemic leg.
Chronic Myocardial Ischemia in Pigs
Pigs were anesthetized and treated as described previously (von Degenfeld et al., J. Am. Coil. Cardiol. 2003, 42:1120-8). To this end, a reduction stent coated with a PTFE membrane was implanted in the proximal RCx, leading to 75% reduction of blood flow. Correct localization of the stent and permissibility of the distal vessel were ensured by the injection of contrast agent. On day 28, the baseline measurements for global myocardium function (left ventricular end-diastolic pressure=LVEDP, ejection fraction=EF) and myocardial perfusion (fluorescent microbeads, 15 μm, Molecular Probes®) were conducted. Then selective pressure-regulated retroinfusion into the large cardiac vein draining the RCx-perfused myocardium was carried out for κ×10¹²virus particles of rAAV.MRTF-A and rAAV.Tβ4±rAAV.MRTFA-shRNA. On day 56, the measurements of global myocardium function and blood flow were repeated and the regional myocardium function of the ischemic and non-ischemic area were determined (at rest and under fast heart stimulation, 130 and 150 bpm). Post mortem angiography was carried out for the calculation of the collateral value and analysis by Rentrop score (0=no filling, 1=side branch filling; 2=partial main vessel filling; 3=complete main vessel filling). Tissue was collected for the analysis of regional myocardial blood flow and immunohistology.
Global Myocardial Function
On day 28 and day 56, 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).
Regional Myocardial Function
On day 56 after induction of ischemia, sternotomy was performed and ultrasound crystals were placed subendocardially in a standardized manner in the non-ischemic area (LAD control region) and the ischemic area (Cx perfused region). Subendocardial segment shortening (SES, Sonometrics, Ontario, Canada) was examined at rest and under elevated heart rate (functional reserve, rate 130 and 150) and evaluated off-line depending on ECG.
Regional Myocardial Blood Flow
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 μm, 5×10⁶particles 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).
Histology
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.
rAAV Transduction Efficiency
For the evaluation of the rAAv transduction efficiency, control mice, rabbits and pigs were treated with rAAV.LacZ. Cryostatic sections of the LacZ-transduced animals were prepared and stained for β-galactosidase (blue staining). Furthermore, RT-PCR for the several transgenes was carried out using the primers described in Table 1 and analyzed as described above.
Tomato Reporter Gene Mice
These mice homozygously expressing mT/mG (Jackson Laboratory, Bar Harbor, USA) 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. 4b ).
Statistical Methods
The results are shown as means±standard deviation. Statistical analyses were performed using one-way variance analysis (ANOVA). Every time a significant effect was found (p<0.05), we conducted multiple comparative tests between groups with the Student Newman Keul method (IBM SPSS 19.0; IBM, Chicago, USA). Differences between groups were regarded as significant at p<0.05.

TABLE 1

Primer sequences used for PCR:

BGH forward	5′-TCT AGT TGC CAG CCA TCT GTT GT-3′	SEQ ID NO: 2
BGH reverse	5′-TGG GAG TGG CAC CTT CCA-3′	SEQ ID NO: 3

GAPDH forward	5′-AAT TCA ACG GCA CAG TCA AG-3′	SEQ ID NO: 4
GAPDH reverse	5′-ATG GTG GTG AAG ACA CCA GT-3′	SEQ ID NO: 5

Tβ4 forward	5′-TCA TCG ATA TGT CTG ACA AAC-3′	SEQ ID NO: 6
Tβ4 reverse	5′-CAG CTT GCT TCT CTT GTT CAA-3′	SEQ ID NO: 7

MRTF-A forward	5′-AAT CCA TGG GTC GAC GGT ATC GAT-3′	SEQ ID NO: 8
MRTF-A reverse	5′-ATA CCA TGG TCA GGC ACC GGG CTT-3′	SEQ ID NO: 9

CCN1 (CYR61) forward	5′-GCT AAA CAA CTC AAC GAG GA-3′	SEQ ID NO: 10
CCN1 (CYR61) reverse	5′-GGC TGC AAC TGC GCT CCT CTG-3′	SEQ ID NO: 11

CCN2 (CTGF) forward	5′-CCC TAG CTG CCT ACC GAC T-3′	SEQ ID NO: 12
CCN2 (CTGF) reverse	5′-CAT TCC ACA GGT CTT AGA ACA GG-3′	SEQ ID NO: 13

Ang2 forward	5′-TCG AAT ACG ATG ACT CGG TG-3′	SEQ ID NO: 14
Ang2 reverse	5′-GTT TGT CCC TAT TTC TAT C-3′	SEQ ID NO: 15

Claims

1. An adeno-associated viral vector (AAV vector) comprising a gene encoding a neovasoactive growth factor, wherein the neovasoactive growth factor is a myocardin-related transcription factor A (MRTF-A) or thymosin β4 (Tβ4), or a combination thereof.

2. The AAV vector according to claim 1, wherein the AAV vector is an AAV9 vector or an AAV vector pseudotyped with AAV9 envelope proteins selected from AAV2.9, AAV1.9 and AAV6.9.

3. The AAV vector according to claim 1, further comprising a gene encoding an MRTF-B.

4. The AAV vector according to claim 1, wherein the MRTF-A gene is under the control of a cardio-specific promoter.

5. The AAV vector according to claim 4, wherein the cardio-specific promoter is a CMV promoter, an MRC2 promoter, a MyoD promoter, or a troponin promoter.

6. A pharmaceutical composition comprising an AAV vector of claim 1 and a pharmaceutically acceptable carrier.

7. The AAV vector according to claim 1 formulated for treating coronary heart disease or chronic ischemic diseases in a mammal, wherein the AAV vector is present in an amount effective to enhance MRTF-A activation.

8. A method of treating coronary heart disease or peripheral ischemia in a mammal by administering the pharmaceutical composition of claim 6.

9. The method according to claim 8, wherein the coronary heart disease is acute heart attack, myocardial ischemia, stable angina pectoris and/or hibernating myocardium.

10. (canceled)

11. The method according to claim 8, wherein the mammal is suffering from diabetes mellitus or hypercholesterolemia.

12. The method of claim 8, wherein the mammal is a human, a mouse, a rabbit, or a pig.

13. The method according to claim 12, wherein the human is a human no option patient.

14. The AAV vector according to claim 7, wherein the coronary heart disease is acute heart attack, myocardial ischemia, stable angina pectoris and/or hibernating myocardium.

15. The AAV vector according to claim 7, wherein the mammal is a human, a mouse, a rabbit, or a pig.

16. The method according to claim 15, wherein the human is a human no option patient.

17. The AAV vector according to claim 7, wherein the mammal is suffering from diabetes mellitus or hypercholesterolemia.

18. A method for therapeutic vessel reformation and increasing vessel profusion comprising administering the AAV vector of claim 1 to a mammal in an amount sufficient to enhance MRTF-A activation.

19. The method according to claim 18, wherein the mammal is a human, a mouse, a rabbit, or a pig.

20. The method according to claim 18, wherein the mammal suffers from coronary heart disease, chronic ischemic diseases, diabetes or hypercholesterolemia.