KR102578015B1 - An AAV vector containing a promoter specifically expressed in cerebrovascular endothelial cells and coated with a nano-peptide for targeting cerebrovascular endothelial cells, and its use for cerebrovascular endothelial cell-specific gene delivery - Google Patents

Abstract

The present invention relates to an AAV vector containing a promoter specifically expressed in cerebrovascular endothelial cells and coated with a nano-peptide targeting cerebrovascular endothelial cells, and a technology for using the same for cerebrovascular endothelial cell-specific gene delivery.

Description

AAV vector containing a promoter specifically expressed in cerebrovascular endothelial cells and coated with nano-peptide for targeting cerebrovascular endothelial cells and its use for cerebrovascular endothelial cell-specific gene delivery {An AAV vector containing a promoter specifically expressed in cerebrovascular endothelial cells and coated with a nano-peptide for targeting cerebrovascular endothelial cells, and its use for cerebrovascular endothelial cell-specific gene delivery}

The examples below relate to an AAV vector containing a promoter specifically expressed in cerebrovascular endothelial cells and coated with a nano-peptide targeting cerebrovascular endothelial cells, and technology for using the same for cerebrovascular endothelial cell-specific gene delivery.

Adeno-associated virus (AAV) is the preferred vector for preclinical and clinical applications and possesses a wide range of assisting, suppressing, and gene editing transgenes. Cells from various organs such as liver, lung, etc. have been targets of natural tropism of AAV serotypes, with myotropic AAV being mainly used to target the heart in various experiments, especially serotypes 8 or 9 in animal models. Preferred for gene editing. On the other hand, in the case of vascular cells, AAV is rarely used in vascular gene therapy because the transduction efficiency is low.

In this regard, several approaches to retarget AAV to endothelial cells have been reported for AAV2 and AAV9, although peptide insertion ultimately resulted in transduction of endothelial cells of large vessels (aorta, vena cava) in vivo, but not of microvessels. It was confirmed that this was not the case in the compartment. Other peptide variants (PPS, BR1) have been applied to target brain microvascular endothelial cells in vivo, but lack expression in other vascular beds such as heart or skeletal muscle.

Meanwhile, cardiovascular diseases are the number one cause of death worldwide and the number is increasing every year. In particular, arteriosclerosis is a representative cardiovascular disease that occurs when aged arteries, whose elasticity has decreased due to causes such as high blood pressure, hyperlipidemia, smoking, diabetes, obesity, lack of exercise, and stress, narrow with blood clots. Depending on the area, if it occurs in the cerebral blood vessels and carotid arteries, it causes cerebral infarction (stroke), and if it occurs in the coronary arteries of the heart, it causes arteriosclerotic heart disease (angina, myocardial infarction). There is a high need for efficient treatment methods for these cardiovascular diseases.

Korean Patent Publication No. 10-2022-0011616

Examples relate to a recombinant adeno-associated virus in which the surface of a viral capsid is coated with nanoparticles composed of a peptide that specifically binds to cardiovascular endothelial cells.

To achieve the above object, a recombinant adeno-associated virus is provided in which the surface of a viral capsid is coated with nanoparticles composed of a peptide that specifically binds to endothelial cells.

Additionally, a method for producing the recombinant adeno-associated virus is provided.

Additionally, a composition for preventing or treating atherosclerosis comprising the recombinant adeno-associated virus is provided.

Examples show that nanoparticles composed of a peptide that specifically binds to cardiovascular endothelial cells on the surface can target drugs to blood vessels and vascular endothelial cells through a recombinant adeno-associated virus coated on the surface of a viral capsid, and leukocytes in the blood vessel wall. It can prevent blood vessel occlusion by preventing sticking, and by removing the eNOS gene, it can contract muscles and control blood pressure.

However, the effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below.

Figures 1 and 2 are diagrams showing the G2 ^CYS coating of AAV9 and its results.
Figure 3 shows the control of AAV9-Cre transduction in vivo by G2 ^CNN coating in mTmG mice.
Figure 4 shows the results of endothelial transduction of mTmG pigs using AAV9-Cre.
Figure 5 relates to the process and results of endothelial S1FG delivery through endothelial retargeting AAV9.
Figure 6 shows that Annexin A1 retargeted to the endothelium inhibits leukocyte recruitment in a mouse model of chronic atherosclerosis.
Figure 7 shows blood pressure changes caused by Cas9-mediated eNOS deletion using endothelial retargeted AAV9.
Figures 8 to 10 show the structures of pSEW (Template), eNOSp-GFP (human eNOS-Promotor in SEW), and eNOSp-TK (human eNOS-Promotor in SETKW8 (basiert auf SEW)), respectively (eNOS: human eNOS) -Promotor; 1600 bp).

Hereinafter, embodiments will be described in detail with reference to the attached drawings. However, various changes can be made to the embodiments, so the scope of the patent application is not limited or limited by these embodiments. It should be understood that all changes, equivalents, or substitutes for the embodiments are included in the scope of rights.

Specific structural or functional descriptions of the embodiments are disclosed for illustrative purposes only and may be modified and implemented in various forms. Accordingly, the embodiments are not limited to the specific disclosed form, and the scope of the present specification includes changes, equivalents, or substitutes included in the technical spirit.

Terms such as first or second may be used to describe various components, but these terms should be interpreted only for the purpose of distinguishing one component from another component. For example, a first component may be named a second component, and similarly, the second component may also be named a first component.

When a component is referred to as being “connected” to another component, it should be understood that it may be directly connected or connected to the other component, but that other components may exist in between.

The terms used in the examples are for descriptive purposes only and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, terms such as “comprise” or “have” are intended to designate the presence of features, numbers, steps, operations, components, parts, or combinations thereof described in the specification, but are not intended to indicate the presence of one or more other features. It should be understood that this does not exclude in advance the possibility of the existence or addition of elements, numbers, steps, operations, components, parts, or combinations thereof.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by a person of ordinary skill in the technical field to which the embodiments belong. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and unless explicitly defined in the present application, should not be interpreted in an ideal or excessively formal sense. No.

The advantages and features of the present invention and methods for achieving them will become clear by referring to the embodiments described in detail below along with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below and will be implemented in various different forms. The present embodiments only serve to ensure that the disclosure of the present invention is complete and that common knowledge in the technical field to which the present invention pertains is not limited. It is provided to fully inform those who have the scope of the invention, and the present invention is only defined by the scope of the claims.

In the embodiments of the present invention, unless otherwise defined, all terms used herein, including technical or scientific terms, are the same as those commonly understood by a person of ordinary skill in the technical field to which the present invention pertains. It has meaning. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and unless clearly defined in the embodiments of the present invention, have an ideal or excessively formal meaning. It is not interpreted as

The shapes, sizes, proportions, angles, numbers, etc. disclosed in the drawings for explaining embodiments of the present invention are illustrative, and the present invention is not limited to the matters shown. Additionally, in describing the present invention, if it is determined that a detailed description of related known technologies may unnecessarily obscure the gist of the present invention, the detailed description will be omitted. When 'includes', 'has', 'consists of', etc. mentioned in this specification are used, other parts may be added unless 'only' is used. When a component is expressed in the singular, the plural is included unless specifically stated otherwise.

When interpreting a component, it is interpreted to include the margin of error even if there is no separate explicit description.

The size and thickness of each component shown in the drawings are shown for convenience of explanation, and the present invention is not necessarily limited to the size and thickness of the components shown.

Each feature of the various embodiments of the present invention can be partially or fully combined or combined with each other, and as can be fully understood by those skilled in the art, various technical interconnections and operations are possible, and each embodiment may be implemented independently of each other. It may be possible to conduct them together due to a related relationship.

One aspect of the present invention provides a recombinant adeno-associated virus in which the surface of a viral capsid is coated with nanoparticles composed of a peptide that specifically binds to cardiovascular endothelial cells.

In one embodiment, the nanoparticle may be a G2 dendrimer.

In one embodiment, the peptide that specifically binds to cardiovascular endothelial cells is CNN (CNNSGMRN) or SLR (SLRSPPS), has a positive zeta potential of 4.3 to 4.5 mV on the surface of the virus, and skeletal muscle endothelial cells. and a plasmid encoding the anti-inflammatory protein Annexin A1 (Anxa1), which has enhanced targeting to cardiovascular endothelial cells and contains in its genome the transgene S1FG, which consists of SDF-1 as a chemokine head, a fractalkine stem, and a GPI anchor. Including, it may be a recombinant adeno-associated virus that increases adhesion to white blood cells and prevents the development of vascular occlusion and arteriosclerosis caused by white blood cells.

More specifically, in the present invention, the expression levels of Ly6C+, Ly6G+, and CD11b+ were confirmed by staining leukocytes that cause arteriosclerosis, thereby confirming the effect of preventing leukocytes from adhering to blood vessel walls.

In one embodiment, the virus contains a gene encoding an aritificial adhesion molecule (S1FG; complex of [SDF-1] - [Mucin domain] - [GPI anchor]) in its genome, and expresses the molecule to induce testes ( Drugs can be delivered specifically to the venules of the cremaster.

In one embodiment, the peptide that specifically binds to cardiovascular endothelial cells is CNN (CNNSGMRN) or SLR (SLRSPPS), has a positive zeta potential of 4.3 to 4.5 mV on the surface of the virus, and skeletal muscle endothelial cells. and enhanced targeting to cardiovascular endothelial cells, containing the plasmid of Figure 9 in the viral genome, and targeting between exons 6 and 10 of the endothelial nitric oxide synthase (eNOS) gene in Cas9-transgenic (Cas9-tg) mice. It may be a recombinant adeno-associated virus that encodes AAV9-gRNA, which excises and can increase muscle contraction and arterial pressure. Regulating systemic blood pressure is a very important function in health, and the present invention uses vascular endothelial cell-targeted Cas9-AAV gene therapy (CRISPR/Cas9 method to remove exons (6 to 10) of the eNOS gene to remove muscle tissue. It was confirmed that blood pressure can be controlled by contracting.

In one embodiment, the recombinant adeno-associated virus may be transduced with one or more vectors of FIGS. 8 to 10.

In one embodiment, the recombinant adeno-associated virus may be pseudotype rAAV2/9, and the molar ratio of G2 dendrimer and NHSPEG-OPSS linker dissolved in DMSO may be 1:4.

One aspect of the present invention is an amine-terminated diaminobutane core PAMAM G2 dendrimer, an NHSPEG-OPSS linker dissolved in DMSO, and an amino acid sequence of CNN (CNNSGMRN) or SLR (SLRSPPS) that specifically binds to cardiovascular endothelial cells. Incubating the peptides together; After the above steps, loading on a cation exchange column and a salt gradient of 0.6 to ³ m NaCl, 20 Fractionation step; After the above steps, filtering the product with a centrifugal filter device; and a dendrimer coating step of adding the adeno-associated virus particles after the above step, mixing them by gently aspirating them with a pipette tip, and incubating at room temperature for 30 minutes. More specifically, the manufacturing method may include the manufacturing steps described in the examples below.

One aspect of the present invention provides a composition for preventing or treating atherosclerosis, comprising the above-described recombinant adeno-associated virus as an active ingredient.

Atherosclerosis is a representative cardiovascular disease that occurs when aged arteries, whose elasticity has decreased due to causes such as high blood pressure, hyperlipidemia, smoking, diabetes, obesity, lack of exercise, and stress, narrow with thrombosis. Depending on the site of occurrence, the brain If it occurs in blood vessels or carotid arteries, it can cause cerebral infarction (stroke), and if it occurs in the coronary arteries of the heart, it can cause arteriosclerotic heart disease (angina pectoris, myocardial infarction), which is a fatal disease that causes necrosis and death of each part of the body due to lack of blood supply.

In the present invention, it is a gene therapy designed to regenerate necrotic myocardial tissue (myocardium) by specifically targeting endothelial cells of coronary arteries to induce angiogenesis, targeting adeno-associated virus ( AAV (Adeno-associated virus) vector was used.

Hereinafter, the configuration of the present invention and its effects will be described in more detail through specific examples and comparative examples. However, these examples are for illustrating the present invention in more detail, and the scope of the present invention is not limited to these examples.

1. Experimental method

Virus creation:

Recombinant AAV9 (pseudotype rAAV2/9) was generated using the triple transfection method. Briefly, transfection of HEK293T cells (CRL-3216, ATCC) consisted of two plasmids: one plasmid encoding the transgene flanked by cis-acting AAV2 inverted terminal repeats (ITRs), the AAV2 rep and the AAV9 cap in trans. This was performed using the second plasmid, while the third plasmid (delta F6, Puresyn) was complemented with adenoviral helper function using polyethyleneimine (PEI-Max, Polysciences Inc.). The titer of AAV was determined via quantitative real-time polymerase chain reaction (qRT-PCR) against the ITR region.

Cell culture and peptide selection:

Human microvascular endothelial cells (HMEC-1, CRL-3243, ATCC) or HUVEC (Promocell) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) and 10% fetal calf serum (FCS) supplemented with endothelium. Cell Growth Medium 2 (Promocell) Incubate 2 x 10 ¹¹ plaque-forming units of the CX7C M13KE phage library (New England Biolabs) at 1 x 10 cells per tube of HMEC-1 or ^HUVEC cells in 1 mL of 1% bovine serum albumin (BSA). /DMEM and incubated for 120 min at 4°C with gentle rolling.

After washing, bound phage was eluted with 1 mL of 0.2 m glycine-HCl. 1 mL of selected phage eluate was incubated with 20 mL of E. coli ER2738 bacterial suspension (A600 nm 0.5) (New England Biolabs,). The recovered phages were centrifuged at 4°C/13,000rpm/20 minutes. The supernatant was then transferred to a new tube containing 1/6 volume of 20% PEG8000/2.5 m NaCl and incubated overnight at 4°C. Phages were then pelleted at 4°C/13,000 rpm/15 min and the supernatant was discarded. The phage pellet was thoroughly resuspended in 1 mL of TBS. 10 μL of phage eluate was incubated with 300 μL of E. coli ER2738. A single blue positive plaque was selected and amplified with 2 mL of E. coli ER2738. Each selected phage DNA was amplified by direct PCR using KAPA HiFi DNA polymerase (Peqlab). Ten phage clone sequences were selected by sequencing analysis with M13 phage primer sequences: forward 5′- TTA TTC GCA ATT CCT TTA GTG G -3′, reverse 5′-CCC TCA TAG TTA GCG TAA CG -3′. PCR products were then isolated and sequenced (Eurofins). Among the seven various sequences, CNNSGMRN was selected after alignment analysis by the Clustal W program.

Conjugate synthesis :

Amine-terminated diaminobutane core PAMAM G2 dendrimer was purchased from Dendritic Nanotechnologies and Andrews ChemServices. Conjugation of PAMAM G2 dendrimer, NHSPEG-OPSS linker, and endothelial cell-specific transduction peptide (CNN or SLR) was performed. Briefly, 1 μmol of G2 was incubated with 4 μmol of NHS-PEG2kDa-OPSS (Rapp Polymere) dissolved in dimethyl sulfoxide (DMSO) and loaded onto a cation exchange column (Macro-Prep High S; BioRad, Germany) at 0.6–3 m. Fractionated in a salt gradient of NaCl, 20 × 10 ^-3 m HEPES (=4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid), pH 7.4) solution. Afterwards, the product was filtered with a centrifugal filter device (Amicon Ultra 3K, Merck) and the G2 content of the conjugate was determined by TNBS (=trinitrobenzene sulfonic acid) analysis.

Dendrimer coating:

Complexes of AAV and G2 PAMAM were formed by diluting the indicated amounts of PAMAM dendrimer in Opti-MEM I (Thermo Fisher). Virus particles were added to the diluted PAMAM solution, immediately mixed by gentle aspiration with a pipette tip, and allowed to incubate for 30 minutes at room temperature before further use.

Zeta potential:

1 × 10 ¹² vgs AAV in 100 μL phosphate-buffered saline (PBS) was incubated in Hepes-buffered glucose (HBG, 20 × 10 ^-3 m HEPES, 5% w/v glucose, pH = 7.4) for 30 min at 25°C. Then, it was further diluted to 0.8 mL in a 1:1 mixture of PBS/HBG to be measured on zetasizer Nano ZS (Malvern Pananalytical). Samples were run in transparent disposable zeta cells at 25 °C using the chosen dispersant of water (0.8872 cP viscosity) and set to automatic voltage (always reaching 50.3 V) for up to 100 runs with a cooling time of 60 s between collections. Run it twice. The results were analyzed using Zetasizer Software v7.13 (Malvern), and only those extracted with good result quality according to the software were analyzed.

TEM:

AAV was coated with PAMAM conjugate as described above and processed according to the prior art. Briefly, 2 μL samples were pipetted onto formvar-coated 300 mesh copper grids, left to evaporate for 6 h, then coated dropwise with 20 μL 1% phosphorus tungstic acid solution for 3 min, excess liquid drained and left to dry overnight. Samples were imaged on a Libra 120 system (Zeiss,) using a Sharp:eye camera (TRS) and an in-column camera Morada G2 (11 MP) with a 120 kV operating filter and LaB6 filament.

Mouse Model:

Animal care and all experimental procedures were performed in accordance with the Bavarian Animal Care and Use Committee (ROB-55.2-2532.Vet_02-18-99, etc.). Rosa26 mTmG (007576, The Jackson Laboratory) and Rosa26-LSL-Cas9 (024857, The Jackson Laboratory) mice were provided by Ralf Adams (Max Planck Institute for molecular biomedicine, Muenster, Germany) and Roland Rad (Klinikum Rechts der Isar, Munich, Germany), respectively. Germany), provided by Oliver Sohnlein (Institute for Cardiovascular Prevention, Ludwig-Maximilians-University, Munich, Germany). ApoE-/- mice were used on a C57Bl6 background and fed with 21% fat and 0.15% cholesterol (ssniff, Soest) for 4 weeks. All AAV in vivo experiments were performed with tail vein injections of 2.5 × 10 ¹² vgs.

AAV injection into mTmG reporter pigs:

Transduction of mTmG reporter pigs was performed according to conventional techniques. Briefly, pigs (n = 2) were anesthetized, two 6F sheaths were inserted into the carotid artery and external jugular vein, and the left anterior descending artery was occluded with a wire balloon. AAV (1 × 10 ¹⁴ vgs) was injected through the lumen of this balloon while occluding the anterior interventricular vein with a Swan Ganz catheter. Another virus (1 × 10 ¹⁴ vgs) was also injected intramuscularly into the biceps femoris. Animals were sacrificed after 3 weeks, hearts were excised, sampled in a systematic manner, and flash frozen in isopentane for further investigation. An area of the biceps femoris muscle centered around the intramuscular injection site was also collected for flow cytometric analysis.

Fluorescence microscopy:

Microscopy was performed using a Leica THUNDER Imager Tissue (Leica Microsystems,) equipped with a 63XxNA1.40 oil immersion objective. Optical zoom was used where applicable. The SOLA light engine (Lumencor) was used for fluorescence excitation. Acquisition was performed with 20482 pixels. Image processing was performed using Leica LAS

Antibodies used for immunofluorescence staining were as follows: EGFP (CAB4211, Thermo Fisher), murine CD31 (BM4086, Origene), porcine CD31 (LCI-4, Bio-Rad), and CRISPR-Cas9 (7A9-3A3, Novus Biologicals). ), AAV9 intact capsid (ADK9, Origene).

Highly cross-adsorbed Alexa Fluor conjugated secondary antibodies were used for fluorescent labeling (A-11034, A48265, A-11029, A-11032, Thermo Fisher). GFP/CD31 double-positive cells in immunofluorescence images were counted in 10 sections using ImageJ.

Flow cytometry analysis of transduction:

Tissue samples were digested as follows: 10 mg of murine post-ischemic heart slices were digested with collagenase II (1 mg mL−1; C6885, Sigma) and DNase I (50 μg mL−1; D4263, Sigma) for 1 h. Porcine biceps femoris samples were cut into 50 mg pieces and digested with 1 mg mL ^-1 papain (P3375, Sigma) for 30 min and then digested with 1 mg mL ^-1 collagenase/dispase (Roche) for 30 min. The cell suspension was pressed through a cell strainer (70 μm) and centrifuged at 300 xg. Red blood cells were washed twice, lysed with ammonium potassium buffer, and stained with trypan blue for mouse cells and LIVE/DEAD (Thermo Fisher) for porcine cells. Murine cells were stained with a specific antibody against CD31 (BM4086, Origene) for 15 hours. Secondary antibody staining (A48265, Thermo Fisher) was performed. Porcine cells were fixed with 4% w/v paraformaldehyde, permeabilized with Triton A48255, Thermo Fisher) was performed. Fluorescence measurements were performed on LSRFortessa (BD Biosciences).

In Vivo Cremaster Microscopy :

AAV9 S1FG-injected C57Bl6 mice were anesthetized and the cremaster muscle was exposed on the microscope stage. Morphological parameters such as vein diameter, segment length, and leukocyte turnover rate were evaluated. The number of adherent cells per mm2 was determined using an intravital microscope (Olympus BX51WI microscope, immersion objective ×20, 0.95 numerical aperture). All scenes were recorded using a charge-coupled device (CCD) camera (model CF8/1, Kappa) and virtual dubbing software for offline analysis. During the entire observation, the cremaster muscle was replaced with temperature-controlled (35°C) bicarbonate-buffered saline solution. The range of observed venules behind capillaries was confirmed to be 20 to 40 μm.

Atherosclerosis Trial:

Eight-week-old Apoe−/− mice were intravenously injected with non-specific AAV carrying Annexin A1 (2.5 × 10 ¹² vgs), endothelium-specific AAV carrying Annexin A1 (2.5 × 10 ¹² vgs), or vehicle (100 μL, NaCl). administered. Mice were fed a high-fat diet containing 21% fat and 0.15% cholesterol (ssniff, Soest) for 4 weeks. All animal procedures were performed in accordance with national guidelines for animal welfare and were approved by the Bavarian Animal Care and Use Committee.

Intravital microscopy of the carotid artery:

The mouse was placed supine and the right jugular vein was cannulated with a catheter for antibody injection. Phycoerythrin (PE)-conjugated antibody to Ly6G (1 μg, clone 1A8, 127608, BioLegend), fluorescein (FITC)-conjugated antibody to Ly6C (1 μg, HK1.1, 128022, BioLegend), and PE-conjugated Intravital microscopy was performed after injection of antibody CD11b (1 μg, clone M1/70, 101208, BioLegend). Leukocyte-endothelial interactions along the carotid artery were visualized using an Olympus BX51 microscope equipped with a Hamamatsu 9100-02 electron multiplication CCD camera and a 10 × saline immersion objective. A 30-second video was collected offline and analyzed. One video was analyzed per mouse and video analysis was performed in a blinded manner.

Fluorescence-activated cell sorting (FACS):

The aorta was digested with 1.25 mg mL ^-1 Liberase (Roche) and incubated at 37°C for 1 h. The following antibodies were used to stain single cell suspensions: CD45-eFluor450 (30-F11, 48-0451-82, Thermo Fisher), CD31-APC (MEC13.3, 102510, BioLegend), Zombie Aqua Fixable Viability Kit ( 423102), Bio Legend). Cells were washed with Hanks Balanced Salt Solution, sorted directly with FACSAria III (BD) in extraction buffer from the PicoPure RNAIsolation kit (Thermo Fisher), and incubated for 30 min at 42°C. RNA was extracted using the same kit according to the manufacturer's instructions.

RNaseq analysis:

Library preparation for bulk sequencing of poly(A)-RNA was performed as described in the prior art. Briefly, barcode cDNA for each sample was generated with Maxima RT polymerase (Thermo Fisher) using oligo-dT primers containing the barcode, unique molecular identifier (UMI), and adapter. The 5'-end of the cDNA was extended by template switch oligo (TSO) and the full-length cDNA was amplified with primers binding to the TSO site and adapter. cDNA was fragmented using the NEB UltraII FS kit (New England Biolabs). After end repair and A-tailing, TruSeq adapters were ligated and the 3' end fragment was finally amplified using primers with Illumina P5 and P7 overhangs. Compared to Parek et al. (2016), the P5 and P7 sites were exchanged to allow sequencing of cDNA in read1 and barcode and UMI in read2 to achieve better cluster recognition. The library was sequenced on a NextSeq 500 (Illumina) with 63 cycles for cDNA in read1 and 16 cycles for barcode and UMI in read2. Data were processed using the published Drop-seq pipeline (v1.0) to generate sample- and gene-specific UMI tables. Principal component analysis and heatmap generation were performed by the R package Pheatmap.

Cas9-mediated eNOS deletion:

A candidate single guide RNA targeting murine eNOS was cloned into spCas9 plasmid (px459 #62988, Addgene) and subjected to polyethyleneimine transduction (PEI-MAX, Polysciences Inc) by T7 endonuclease assay (New England Biolabs). Efficiency of double-strand break induction was evaluated in Neuro2A cells (CCL-131, ATCC). Two sgRNAs, 5'-GCG AGG GGA CCC CGC CAA CG-3' and 5'-CAA TCC AGG CCC AAT CGG CA-3', were reverse-directed onto a self-complementary AAV transgene plasmid with the opposite pEndo.Cre cassette. Cloned and AAV9 virions were generated as described above (AAV9.pEndo.Cre.sgRNAeNOS). Adult LSL-Cas9 mice (8 weeks old) underwent initial noninvasive blood pressure measurements using the CODA 2 Noninvasive Blood Pressure Measurement System (Kent Scientific). After baseline blood pressure measurement, 2.5 × 10 ¹² vgs of uncoated AAV9.pEndo.Cre or G2CNN coated AAV9.pEndo.Cre.sgRNAeNOS was injected via tail vein injection. Noninvasive blood pressure measurements were repeated after 4 and 8 weeks. Eight weeks after AAV injection, mice underwent left ventricular pressure volume loop recordings. For this purpose, mice were anesthetized by intraperitoneal injection of MMF (Midazolam 5 mg kg-1 body weight, Medetomidin 0.5 mg kg-1 body weight, and Fentanyl 0.05 mg kg-1 body weight). After complete sedation, a pressure volume catheter (Scisense P/V Catheter, 1.2F, 3.5mm electrode gap, Transonic) was inserted into the left ventricle through the right carotid artery. After blood pressure was stabilized, 30 P/V loops were analyzed using LabChart 8 software. Cardiac and gastrocnemius muscles were snap frozen in isopentane. Cryo-sections were stained with CRISPR-Cas9 antibody (7A9-3A3, Novus Biologicals) and CD31 antibody (BM4086, Origen). DNA extraction was performed by column purification (Nucleospin Tissue XS, Macherey-Nagel). PCR of the edited region was performed by Q5 polymerase (New England Biolabs) using primers 5′ GAG GCA ATC TTC GGT GAG TGA CCC T 3′ and 5′ AAG GGG AGG AGC ATG GAA GAA AGC A 3′. The proportion of excised relative to wild-type amplicon was determined by quantifying band intensity on agarose gel by ImageJ.

Statistical analysis:

Data were expressed as mean ± SEM (standard error of the mean). To compare the means of two groups, an unpaired 2-tailed Student's T-test was used, whereas for comparisons of multiple groups, 1-way ANOVA (analysis of variance) with Dunnett's post hoc test was applied. Statistical significance was defined as p<0.05. *p<0.05, **p<0.01, ***p<0.001,****p<0.0001.

2.Experiment results

2.1 AAV9 G2 ^CYS Coating promotes target cell entry without changing orientation

The PEGylated G2-conjugate (G2 ^CYS ) (hereafter G2CYS) was readily bound to the negatively charged surface of the AAV9 capsid and its size was significantly increased by the particle diameter estimated from the respective transmission electron microscopy (TEM) images. (32.97 ± 2.24 nm, n = 153 for naked AAV9 capsids vs. 35.01 nm ± 2.74, 450 ng per 2.5 × 10 ¹² viral genomes, n = 69 for G2CYS-coated capsids = vgs, p < 0.00001). G2CYS conjugate coating changed the zeta potential from negative to positive surface charge values (−9.9 ± 1.18 mV for AAV9 vs. 4.4 ± 1.19 mV for G2CYS AAV9, p < 0.00001. For reference, the positively charged The surface was sufficient to dose-dependently increase AAV9-transduction of human endothelial cells and non-endothelial cells (HUVEC and HEK293T cells, respectively) in vitro (Figures 1 and 2).

Subsequently, in mTmG mice containing a floxed tdTomato-transgene and a floxed stop-codon preceding the enhanced green fluorescent protein (EGFP) transgene at the Rosa26 locus, AAV9-Cre transduction efficacy detected as a red-to-green transition was observed. The effect of G2CYS coating was investigated. Cleavage of both floxed alleles via Cre-nuclease provides a fluorescence switch from red to green in vivo.

In particular, upon systemic application of 2.5 × 10 ¹² vgs, skeletal myocyte and cardiomyocyte conversion rates of 47.3% ± 3.6% and 51.3% ± 5.9% were obtained, respectively, confirming the myopic nature of AAV9. However, clear endothelial AAV9-Cre transduction could not be detected, potentially masked by the fluorescence of neighboring cardiomyocytes. Therefore, we switched to the endoglin promoter (pEndo) using the CMV-promoter (cytomegalovirus, pCMV), which limits Cre-expression to endothelial cells. Eliminating muscle transduction using AAV9.pEndo.Cre resulted in only minimal endothelial AAV9 transduction in skeletal muscle and heart with or without G2CYS coating (Figure 3B).

2.2. G2 of AAV9-Cre ^CNN Coating enables expression of Stop-Loxed EGFP in vitro and in vivo

EP was added to overcome the tropism stability of G2CYS. After three rounds of biopanning in cultured endothelial cells using the M13-CX7C phage library, we obtained the CNNSGMRN peptide (CNN) linked to G2-PAMAM via OPN (G2 ^CNN ) (hereafter, G2CNN), which was then linked to native AAV9. Compared to G2Cys, the size increased similarly. (Figures 1 and 2). After systemic injection of G2CNN AAV9 carrying p.Endo.Cre into mTmG mice, we found that microvascular endothelial transport in skeletal muscle (Figure 3 C) and heart was significantly increased. The Cre-mediated redto-green fluorescence switch depended on EPCNN, an antibody against the hyaluronan receptor for endocytosis (Stab2), which is partially homologous to the CNN-motif and ruled out endothelial transduction (Figure 3D,E).

Quantification of Cre transduced PECAM-1 (platelet and endothelial cell adhesion molecule 1, CD31) positive endothelial cells amounted to 32.9% ± 4.1% in skeletal muscle and 42.7% ± 2.1% in heart. These results were confirmed by flow cytometry analysis of PECAM-1-positive EGFP-expressing cells after isolating cells from whole hearts. Here, the addition of G2CNN increased the transduction of PECAM-1 positive endothelial cells from 5.1% ± 2% to 27.3% ± 7.2% in mice.

To investigate whether the high selectivity of the CNN motif itself or the molecular composition of the G2-OPN sandwich linked to an endothelial-tropic peptide is essential for the observed endothelial targeting, we used an alternative EP, SLRSPPS (=SLR). This peptide has been described to transduce large blood vessels when expressed directly from amino acid residue A589 of the AAV9 capsid, but does not provide for microvascular transduction. SLR linked to G2 similarly enhanced AAV9.pEndo.Cre transduction of microvascular endothelial cells in muscle tissue unless the antibody prevented binding of G2SLR AAV9 to its cognate receptor FGFR3.

Next, whether G2CNN AAV9 encoding for pEndo.Cre can switch endothelial cells from native fluorescent red (tdTomato) to fluorescent green (EGFP) when applied topically to mTmG pigs co-containing the floxed td-Tomato allele. evaluated. In contrast to AAV9.pCMV.Cre, which was unable to activate EGFP expression in porcine heart or skeletal muscle endothelium, G2CNN AAV9.pEndo.Cre readily transduced the microvascular endothelium (PECAM-1+) of both peripheral and cardiac tissues ( Figure 4). Local injection of G2CNN-coated pEndo.Cre carrier vector is sufficient to transduce 15.8% ± 0.9% of m.quadriceps and 32.7% ± 2.8% of cardiac muscle capillary endothelium, whereas equivalent doses of wild-type AAV9 induce this effect. Did not do it. This was supported by flow cytometry analysis of PECAM-1 positive EGFP expressing cells in digested hindlimb muscle samples (10.9% ± 3.4% vs. 1.8% ± 0.4%, p < 0.05).

No toxicity was identified during systemic application of AAV9 with G2CNN modification.

2.3. AAV-mediated expression of S1FG, an artificial adhesion molecule for increased leukocyte adhesion.

The expression of adhesion molecules responsible for attracting flowing blood cells to the endothelium is tightly controlled by microvascular endothelial cells, which cannot be replaced by subendothelial or parenchymal cells. Therefore, we tested the hypothesis that AAV9, encoding an adhesion molecule, would only recruit leukocytes if altered by endothelial retargeting modifications. To assess the ability of G2CNN AAV9 to recruit leukocytes to the unstimulated microvascular endothelium, an artificial adhesive consisting of a SDF1 (= stromal cell-derived factor 1) chemokine head, a fractalkine stem, and a GPI (= glycosidolphosphatidylinositol) anchor S1FG was constructed with a molecular transgene (Figure 5A). Using the cremaster model of intravital microscopy to analyze adherent leukocytes (Figure 5 B,C), we showed that unmodified AAV9.pCMV.S1FG did not increase leukocyte adhesion compared to the control (AAV9.pCMV.LacZ). found. However, G2CNN AAV9.pEndo.S1FG increased adherent leukocytes by 4.6-fold (Figure 5 C,D). Of note, simultaneous application of AMD3100, which interferes with the binding of SDF-1 to its receptor CXCR4 on circulating leukocytes, completely abrogated this effect (Figure 5 D).

2.4. Preventing endothelial activation in a chronic atherosclerosis model.

AAV vectors are known to have long expression intervals, at least in muscle cells. To test whether this function could provide long-term quelling of chronic inflammatory conditions such as atherosclerosis, we used a murine model of atherosclerosis: apoE-/- mice fed a high-fat diet characterized by aortic valve formation. (Figure 6A). G2CNN AAV9.pEndo, encoding the anti-inflammatory protein Annexin A1 (Anxa1), was utilized to prevent leukocyte recruitment to the aortic endothelium, a hallmark of plaque formation in hyperlipidemic mice. Upon systemic administration of G2CNNAAV9.pEndo-Anxa1, we found a strong increase in Anxa1 transgene expression compared to uncoated AAV9.pEndo-Anxa1. Two months after systemic application of 2.5 × 10 ¹² vgs of G2CNN AAV9 or wild-type AAV9 encoding Anxa1, carotid arteries were analyzed for leukocyte recruitment. Among CD11b+-leukocytes recruited to the carotid artery wall of hyperlipidemic mice when G2CNN AAV9.pEndo.Anxa1 was applied, significantly lower amounts of Ly6C+-monocytes and Ly6G+-neutrophils compared to EGFP-control vector or wild-type AAV9.pCMV.Anxa1. was detected. (Figure 6 B,C). Bulk RNA sequencing of isolated aortic endothelial cells showed cells from G2CNN AAV9.pEndo.Anxa1 transduced animals separated into distinct clusters from those isolated from EGFP-control vector transduced ones.

2.5. AAV affects blood pressure regulation through Cas9-mediated endothelial nitric oxide synthase ablation.

Systemic blood pressure regulation is a complex, multilayered, and overlapping phenomenon due to its important functions. Nonetheless, endothelial nitric oxide synthase (eNOS) plays a central role in vasodilation of microcirculatory arterioles by releasing gaseous auto-nitric oxide that relaxes the adjacent smooth muscle cell cytoskeleton (Figure 7 A) and maintains blood pressure in the physiological range. .

Here, we investigated in Cas9-transgenic (Cas9-tg) mice whether G2CNN AAV9-gRNA-mediated knockout of endothelial NOS could increase systemic blood pressure ( Figure 7 A). We found that G2CNN AAV9-gRNA, designed to excise between exons 6 and 10 in Cas9-tg mice, was sufficient to increase mean arterial pressure by 23.7 ± 2.3 mmHg 2 months after systemic transduction in non-invasive measurements (compared to 9.4 in control animals). ± 4.7 mmHg vs.). Similar results were obtained for diastolic and systolic blood pressure (Figure 7 B-D). At 2 months, cardiac catheterization was performed to confirm the increase in blood pressure in the G2CNN AAV9.gRNAeN°S group compared to the mock-transduced control group (Figure 7 E). In the endothelial cells of these animals, where abundant Cas9 expression was observed (Figure 7 F), editing of eNOS was obtained in 20.7% ± 1.6% of the eNOS alleles in CD31+ endothelial cells (Figure 7 G).

In addition to cardiac and muscle endothelium, G2CNN-coated AAV9 virus transduced a variety of tissue-specific CD31+ endothelial cells, including aorta, liver, diaphragm, spleen, and brain. However, the amount of microvascular delivery in skeletal muscle and cardiac tissue raised the possibility that the original tropism would steer the vector primarily toward the endothelium of the target tissue since these tissues are within the tropism of the AAV9 serotype. Muscle fiber transduction of the concomitant G2CNN-vector was characterized. Additionally, analysis of fluorescently labeled vector capsids suggested entry through the abluminal plasmalemma, fitting the concept of intact proximal cells in situ for G2CNN AAV9, which is attracted to neighboring endothelial cells by virtue of its EP.

Therefore, coating AAV capsids with G2-EP results in retargeting to endothelial cells, and this result can be used in vascular disease models, e.g., plaque targeting such as atherosclerosis mice, inherited vascular diseases such as Marfan syndrome or aortic aneurysm. Targeting the aorta of mice with treatment) may suggest the possibility of broader AAV application. Additionally, endothelial transduction for microvascular growth and maturation can be achieved through endothelial retargeting AAV.

Claims (3)

A composition for the prevention or treatment of atherosclerosis, comprising as an active ingredient a recombinant adeno-associated virus coated on the surface of a viral capsid with nanoparticles composed of a peptide that specifically binds to cardiovascular endothelial cells,
In the recombinant adeno-associated virus, the nanoparticle is a G2 dendrimer; The peptide that specifically binds to the cardiovascular endothelial cells is CNN (CNNSGMRN) or SLR (SLRSPPS); The zeta potential of the virus surface has a positive value of 4.3 to 4.5 mV; Enhanced targeting toward skeletal muscle endothelial cells and cardiovascular endothelial cells; The genome contains genes encoding the S1FG complex, which consists of SDF-1 as a chemokine head, a fractalkine stem, and a GPI anchor; Containing the plasmid of Figure 9 in the viral genome; and a plasmid encoding the anti-inflammatory protein Annexin A1 (Anxa1), which prevents the development of vascular occlusion and arteriosclerosis by leukocytes by increasing adhesion to leukocytes. Composition for.


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