CN113774088A - Gene delivery composition and application of sildenafil in improving target gene expression - Google Patents

Abstract

The invention relates to a gene delivery composition, a medicament containing the gene delivery composition, application of sildenafil in improving the expression of a target gene mediated by a recombinant AAV vector, and application of sildenafil in reducing a neutralizing antibody of the AAV vector. The use of sildenafil can improve the safety and effectiveness of AAV vectors in gene therapy.

Description

Gene delivery composition and application of sildenafil in improving target gene expression

Technical Field

The present disclosure relates to gene delivery compositions, medicaments comprising the gene delivery compositions, the use of sildenafil for increasing recombinant AAV vector-mediated expression of a gene of interest, and the use of sildenafil for reducing neutralizing antibodies to AAV vectors.

Background

Adeno-associated virus (AAV) technology is one of the important technologies widely used in the field of gene therapy. AAV has the advantages of low pathogenicity, high efficiency, long-term gene expression, easy gene operation, etc. However, AAV vectors also have certain limitations in their applications. In the treatment of diseases such as Duchenne Muscular Dystrophy (DMD) using gene therapy, high doses of AAV vectors are sometimes administered by systemic injection through the circulatory system to achieve therapeutic efficacy. In addition, the use of AAV9 vectors for the treatment of brain diseases also requires high doses to cross the blood-brain barrier, thereby infecting target tissues and delivering genes expressing normal proteins. However, clinical data show that high doses of AAV vectors can elicit a severe immune response, leading to unwanted side effects. High doses of AAV vectors can cause a toxic response in the liver on the one hand and reduce the efficacy of gene therapy due to the production of neutralizing antibodies on the other hand.

Therefore, it is desirable to increase protein expression of AAV-mediated genes, so that reduction of viral vector doses can be achieved without significantly reducing expression of the gene of interest, thereby alleviating adverse reactions in gene therapy due to high doses of AAV vectors.

Disclosure of Invention

The present inventors have surprisingly found that sildenafil, a phosphodiesterase type 5 inhibitor, not only increases the expression of a gene of interest mediated by a recombinant AAV vector, but also decreases neutralizing antibodies to the AAV vector.

Accordingly, in a first aspect, the present disclosure provides a gene delivery composition comprising sildenafil and a recombinant adeno-associated virus (AAV) vector comprising an AAV capsid protein and a gene of interest.

In one embodiment, the gene of interest encodes a therapeutic protein or a non-therapeutic protein.

In a preferred embodiment, the therapeutic protein comprises a protein having an effect in treating a disease in a subject, the disease comprising: cancer, autoimmune diseases, diseases caused by exposure to infectious agents, and genetic diseases.

In a preferred embodiment, the non-therapeutic protein comprises a protein having a beneficial effect in the subject other than treating a disease, for example a protein having nutritional or cosmetic efficacy.

In one embodiment, the AAV capsid protein is a native AAV capsid protein or an artificially engineered AAV capsid protein. In a preferred embodiment, the AAV is selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-DJ8, AAV-DJ9, AAVrh8, AAVrh8R and AAVrh10, more preferably AAV 9.

In one embodiment, the gene delivery composition comprises 1 × 10⁹To 1X 10¹⁵vg of a recombinant AAV vector and 5 μ g to 100mg of sildenafil. In a preferred embodiment, the gene delivery composition comprises 1 × 10¹²To 2X 10¹³vg of a recombinant AAV vector and 1mg to 50mg of sildenafil.

In a second aspect, the present disclosure provides an isolated host cell transfected or transduced with a gene delivery composition as described in the first aspect.

In a third aspect, the present disclosure provides a medicament comprising a gene delivery composition according to the first aspect and an excipient.

In one embodiment, the excipient comprises one or more of a polyol, a sugar, a buffer, a preservative, and an inorganic salt.

In one embodiment, the drug is in the form of an injection such as an injection solution and a sterile powder for injection

In one embodiment, the drug is administered by a systemic route or a local route, such as intravenous administration, intramuscular administration, subcutaneous administration, oral administration, local contact, intraperitoneal administration, and intralesional administration.

In a fourth aspect, the present disclosure provides the use of sildenafil in increasing the expression of a gene of interest mediated by a recombinant AAV vector, wherein the recombinant AAV vector comprises an AAV capsid protein and a gene of interest; the AAV capsid protein is a natural AAV capsid protein or an artificially modified AAV capsid protein, and the target gene encodes a therapeutic protein or a non-therapeutic protein.

In a fifth aspect, the present disclosure provides a method of increasing recombinant AAV vector-mediated expression of a gene of interest, comprising: administering to a subject in need thereof a recombinant AAV vector comprising a gene of interest; and, administering to the subject sildenafil, the gene of interest encoding a therapeutic or non-therapeutic protein.

In a preferred embodiment, 1 x 10 is administered to the subject¹¹To 2X 10¹⁴vg/kg of recombinant AAV vector. In a more preferred embodiment, 1 × 10 is administered to the subject¹²To 2X 10¹⁴vg/kg, preferably 5X 10¹²To 2X 10¹⁴vg/kg, more preferably 5X 10¹²To 5X 10¹³vg/kg, more preferably 1X 10¹³vg/kg to 5X 10¹³vg/kg, particularly preferably 3X 10¹³vg/kg to 5X 10¹³vg/kg of recombinant AAV vector.

In a preferred embodiment, 5 to 50mg/kg of sildenafil is administered to the subject. In a more preferred embodiment, 10 to 50mg/kg, preferably 20 to 50mg/kg, more preferably 30 to 50mg/kg, particularly preferably 40 to 50mg/kg of sildenafil is administered to the subject.

In a sixth aspect, the present disclosure provides the use of sildenafil in reducing neutralizing antibodies to AAV vectors.

In a preferred embodiment, the AAV vector is a recombinant AAV vector.

In one embodiment, the recombinant AAV vector comprises a gene of interest encoding a therapeutic protein or a non-therapeutic protein.

In a seventh aspect, the present disclosure provides a method of reducing neutralizing antibodies to an AAV vector, comprising: administering an AAV vector to a subject in need thereof; and, administering sildenafil to the subject.

In a preferred embodiment, the AAV vector is a recombinant AAV vector.

In one embodiment, the recombinant AAV vector comprises a gene of interest encoding a therapeutic protein or a non-therapeutic protein.

In a preferred embodiment, 1 x 10 is administered to the subject¹¹To 2X 10¹⁴vg/kg of recombinant AAV vector. In a more preferred embodiment, 1 × 10 is administered to the subject¹²To 2X 10¹⁴vg/kg, preferably 5X 10¹²To 2X 10¹⁴vg/kg, more preferably 5X 10¹²To 5X 10¹³vg/kg, more preferably 1X 10¹³vg/kg to 5X 10¹³vg/kg, particularly preferably 3X 10¹³vg/kg to 5X 10¹³vg/kg of recombinant AAV vector.

In a preferred embodiment, 5 to 50mg/kg of sildenafil is administered to the subject. In a more preferred embodiment, 10 to 50mg/kg, preferably 20 to 50mg/kg, more preferably 30 to 50mg/kg, particularly preferably 40 to 50mg/kg of sildenafil is administered to the subject.

The gene delivery composition improves the tissue infection efficiency of the AAV vector and the protein expression of the target gene by combining sildenafil, thereby reducing the use dosage of the AAV vector without obviously influencing the curative effect and realizing the safe and effective application of the AAV vector in gene therapy.

More advantageously, the gene delivery composition of the present invention reduces neutralizing antibodies of AAV vectors by combining sildenafil, thereby reducing clearance of AAV vectors in a subject, further increasing infection efficiency of AAV vectors.

In addition, the present disclosure demonstrates that sildenafil in combination with AAV vectors has no significant toxic side effects.

Drawings

FIG. 1 shows different doses (5, 10, 50mg/kg) of sildenafil against injection of AAV9-Gluc virus (1X 10)¹³vg/kg) in the serum of mice (n-3).

FIG. 2A shows the tissue section GFP immunofluorescence results for sildenafil (10mg/kg) against AAV9-GFP virus (5X 10)¹³vg/kg) effect of GFP expression in brain (cortex, hippocampus), liver, heart, kidney, muscle tissue 3 weeks after C57 infection. 10 Xlens shots with a 50 μm scale.

Fig. 2B shows the results of quantitative analysis of GFP protein fluorescence in each tissue (mean ± sd, n ═ 3).

FIG. 3A shows the expression levels of GFP protein in the cortex, muscle, liver, heart and kidney of each group of mice.

FIG. 3B shows the results of quantitative analysis of GFP protein expression in the cortex, muscle, liver, heart and kidney of each group of mice.

FIG. 4A shows fluorescence imaging plots for different treatment groups.

Figure 4B shows qPCR results for different treatment groups. (average ± sd, n ═ 3).

FIG. 5A shows the Treg cell marker CD25 for the different treatment groups⁺、FoxP3⁺And (4) flow chart.

Figure 5B shows a graph of flow analysis of Treg cells for different treatment groups.

FIG. 5C shows the B cell marker CD19 for different treatment groups⁺、B220⁺And (4) flow chart.

Fig. 5D shows a B-cell flow analysis plot for the different treatment groups.

Fig. 5E shows that sildenafil (10mg/kg) reduced the expression of the inflammatory factors IL1, IL6, TNF α in mouse spleen tissue (mean ± sd, n ═ 6).

Figure 5F shows that sildenafil (10mg/kg) reduces neutralizing antibodies (mean ± sd, n ═ 3) produced following systemic administration of mouse AAV virus.

Figure 6A shows the effect of sildenafil at different doses (5, 10, 50mg/kg) on mouse body weight (mean ± sd, n ═ 3).

Fig. 6B shows the effect of sildenafil (10mg/kg) on mouse liver markers (ALT, AST) (mean ± sd, n ═ 3).

Figure 6C shows the results of the CCK8 toxicity of sildenafil on huvec cells.

FIG. 7A shows immunofluorescence results for dystrophin in tissue sections. And respectively shooting by 10x and 20x times of mirrors. The scales are 50 μm and 20 μm, respectively.

Fig. 7B shows the results of fluorescent quantitation of dystrophin in each tissue (mean ± sd, n ═ 3).

FIG. 8A shows the expression levels of dystrophin in the leg muscles (quadriceps brachialis, gastrocnemius, tibialis anterior) and heart tissues of the mice in each group.

FIG. 8B shows the results of quantitative analysis of dystrophin expression in the leg muscles (quadriceps brachialis, gastrocnemius, tibialis anterior) and heart tissues of each group of mice.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") in this context.

As used herein, the terms "patient" and "subject" are used interchangeably and in their conventional sense to refer to an organism that has or is susceptible to a disorder that can be prevented or treated by administration of a medicament of the present disclosure, and include humans and non-human animals (e.g., rodents or other mammals).

In one embodiment, the subject is a non-human animal (e.g., chimpanzees and other apes and monkey species; farm animals such as cows, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats, and guinea pigs; birds, including poultry, pheasants, and game birds such as chickens, turkeys, and other chickens, ducks, geese, etc.). In one embodiment, the subject is a mammal. In one embodiment, the subject is a human.

Herein, the term "treating" a condition, disease or disorder includes: (1) inhibiting the condition, disease or disorder, i.e., arresting, reducing or delaying the development of the disease or its recurrence or the development of at least one clinical or subclinical symptom thereof; or (2) ameliorating the disease, i.e., causing regression of at least one of the conditions, diseases or disorders or clinical or subclinical symptoms thereof.

Herein, the term "preventing" a condition, disease or disorder includes: preventing, delaying or reducing the incidence and/or likelihood of the occurrence of at least one clinical or subclinical symptom of a condition, disease or disorder developing in a subject who may be suffering from or susceptible to the condition, disease or disorder but who has not experienced or exhibited clinical or subclinical symptoms of the condition, disease or disorder.

Herein, the term "topical administration" or "topical route" refers to an administration having a local effect.

As used herein, the terms "transduction," "transfection," and "infection" refer to the process of delivering an exogenous nucleic acid (gene of interest) into a host cell and producing its RNA and protein products, which includes the use of recombinant viruses to introduce exogenous polynucleotides into the host cell.

As used herein, the term "gene delivery" refers to the introduction of an exogenous polynucleotide into a cell for gene delivery, including targeting, binding, uptake, transport, replicon integration, and expression.

As used herein, the term "gene expression" or "expression" refers to the process by which a gene is transcribed, translated, and post-translationally modified to produce the RNA or protein product of the gene.

As used herein, "AAV virus" or "AAV viral particle" or "rAAV vector" refers to a viral particle made up of at least one AAV capsid protein and a polynucleotide. If the viral particle includes a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is often referred to as a recombinant AAV vector or rAAV.

Sildenafil (Sildenafil), also known as Sildenafil, is an oral drug commonly used to treat Erectile Dysfunction (ED).

In some embodiments, the recombinant AAV vector is produced by three plasmid (plasmid 1: cis-element plasmid; plasmid 2: AAV Rep/Cap plasmid; plasmid 3: helper plasmid) transfection of HEK293 cells. In one embodiment, AAV particles are harvested from the culture medium and lysate of HEK293 cells. Purification methods such as affinity chromatography, ion exchange chromatography, cesium chloride and iodixanol gradient ultracentrifugation.

In one embodiment, to produce recombinant AAV vectors, three plasmid transfections of HEK293 cells were performed as follows: plasmid 1: a cis-element plasmid having an ITR; plasmid 2: an AAV Rep/Cap plasmid having a capsid protein coding sequence; plasmid 3: a helper plasmid having an adenoviral component, which is capable of facilitating replication, assembly and packaging of AAV virions. In one embodiment, AAV particles produced by HEK293 cells are purified by affinity chromatography and iodixanol density gradient ultracentrifugation.

In one embodiment, the AAV capsid protein may be any AAV serotype capsid protein, including native AAV capsid proteins (e.g., capsid proteins of native AAV types 1-11, avian AAV, bovine AAV, canine AAV, equine AAV and ovine AAV) and other artificially engineered AAV capsid proteins (e.g., capsid proteins of artificially engineered AAV types 1-11, avian AAV, bovine AAV, canine AAV, equine AAV and ovine AAV). The genomic sequences, ITR sequences, Rep and Cap proteins of different AAV serotypes are known in the art. These sequences can be found in the literature or in public databases, such as the GenBank database.

One skilled in the art can use standard methods known to produce recombinant and synthetic polypeptides or proteins thereof, design nucleic acid sequences, produce transformed cells, construct recombinant AAV vectors, engineer capsid proteins, package vectors expressing AAV Rep and/or Cap sequences, and transiently or stably transfect packaging cells. These techniques are known to those skilled in the art. See, e.g., MOLECULAR CLONING (MOLECULAR CLONING): a LABORATORY Manual (A Laboratory Manual), second edition, (Cold spring harbor, N.Y., 1989).

In one embodiment, the gene delivery compositions of the present disclosure are used to conduct studies, such as determining the effect of expression of a gene of interest on cell viability and/or function.

In one embodiment, the gene delivery composition of the present disclosure is used as a medicament. When used as a medicament, the gene delivery compositions of the present disclosure may be used to treat various types of diseases, including cancer, such as lung cancer, liver cancer, kidney cancer, thyroid cancer, prostate cancer, kidney cancer, breast cancer, colorectal cancer, cervical cancer, leukemia, lymphoma, melanoma, and glioblastoma, depending on the gene of interest involved; autoimmune diseases such as celiac disease, type 1 diabetes, diffuse toxic goiter, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis and systemic lupus erythematosus; diseases caused by exposure to infectious agents; and genetic diseases.

In one embodiment, the gene delivery composition of the present disclosure is prepared as a unit dose of a drug. In some embodiments, the unit dose comprises 1 × 10 of recombinant AAV vector⁹To 1X 10¹⁵Vector genome (vg) and 5. mu.g to 100mg of sildenafil, e.g. 1X 10¹⁰、1×10¹¹、1×10¹²、1×10¹³Or 1X 10¹⁴vg of recombinant AAV vector and 50 μ g, 100 μ g, 500 μ g, 1mg, 50mg, or 100mg of sildenafil. In a preferred embodiment, the unit dose comprises 1 × 10¹²To 2X 10¹³vg of a recombinant AAV vector and 1mg to 50mg of sildenafil.

In some embodiments, the gene delivery compositions of the present disclosure can be used to transduce various types of cells in vitro to produce stable cell lines expressing protein products, which can then be introduced in vivo for therapeutic or non-therapeutic purposes (e.g., cosmetic, weight management such as weight loss and muscle enhancement, and nutritional fortification). Types of cells include, but are not limited to, endothelial cells, myoblasts, fibroblasts, astrocytes, muller cells, oligodendrocytes, microglia, rod and cone cells, neurons, hematopoietic stem cells, monocytes, granulocytes, lymphocytes, osteoclasts, and macrophages. The principles and techniques for introducing or transplanting cells into a subject are known to those skilled in the art.

In one embodiment, the AAV vector can be loaded with an exogenous polynucleotide for delivery of the gene of interest into a target cell. Thus, AAV vectors can be used to deliver genes of interest to cells in vitro or in vivo.

In one embodiment, the gene of interest delivered by the AAV vector encodes a polypeptide that acts as a reporter (i.e., a reporter protein). The reporter protein is used to indicate cells successfully infected with AAV. These reporter proteins include, but are not limited to, Green Fluorescent Protein (GFP), β -galactosidase, alkaline phosphatase, luciferase, and chloramphenicol acetyltransferase.

In one embodiment, the gene of interest delivered to the target cell by the AAV vector encodes a protein for therapeutic use, either a natural protein or a synthetic protein.

In one embodiment, the gene of interest delivered to the target cell by the AAV vector encodes a protein for non-therapeutic use, which is a natural protein or a synthetic protein.

In one embodiment, the gene delivery composition of the present disclosure is formulated into a pharmaceutical formulation (e.g., injection such as injection solution and sterile powder for injection, tablet, capsule, powder, eye drop) for administration to a human or other mammal. The pharmaceutical preparation may further comprise other ingredients, such as pharmaceutical excipients, water-soluble or organic solvents (e.g. water, glycerol, ethanol, methanol, isopropanol, chloroform, phenol or polyethylene glycol), salts (e.g. sodium chloride, potassium chloride, phosphate, acetate, bicarbonate, Tris-HCl and Tris-acetate), dissolution retarding agents (e.g. paraffin), surfactants, antimicrobial agents, liposomes, lipid complexes, immunosuppressants (e.g. cortisone, prednisone, cyclosporine), microspheres of non-steroidal anti-inflammatory drugs (NSAIDs, e.g. aspirin, ibuprofen, paracetamol), rigid matrices, semi-solid carriers, nanospheres or nanoparticles. In addition, the pharmaceutical formulations can be delivered in single or multiple doses by inhalation, systemic or local (e.g., intravenous, intramuscular, subcutaneous, oral, contact, intraperitoneal, and intralesional) administration. The pharmaceutical formulation may be contained in a container, package, or dispenser (e.g., syringe) with instructions for administration.

In one embodiment, a host cell is transfected with a gene delivery composition of the present disclosure. In one embodiment, the host cell can be any host cell that can produce rAAV virions, including but not limited to, for example, mammalian cells (e.g., HEK-293 cells), insect cells (e.g., SF9 cells), microorganisms, and yeast.

In one embodiment, the excipient may be a solid, liquid, semi-solid, or gas (in the case of an aerosol composition). The excipient comprises more than one of polyalcohol, sugar, buffer, antiseptic and inorganic salt.

In one embodiment, the cell is contacted with the recombinant AAV vector and sildenafil. In one embodiment, the cells are contacted with 1X 10¹¹To 2X 10¹⁴vg of the recombinant AAV vector and 5 to 50 μ g/ml of sildenafil. In a preferred embodiment, the cells are contacted with 1X 10¹²To 2X 10¹⁴vg、5×10¹²To 2X 10¹⁴vg、5×10¹²To 5X 10¹³vg or 3X 10¹³To 5X 10¹³vg of a recombinant AAV vector and 10 to 50 μ g/ml, 20 to 50 μ g/ml, 30 to 50 μ g/ml, or 40 to 50 μ g/ml sildenafil. In some embodiments, the contacting occurs in vitro. In some embodiments, the contacting occurs in vivo, i.e., the recombinant AAV vector and sildenafil are administered to a subject. In one embodiment, 1 × 10 will be used¹¹To 2X 10¹⁴vg/kg of recombinant AAV vector and 5 to 50mg/kg of sildenafil are administered to the subject. In a preferred embodiment, 1 × 10 is added¹²To 2X 10¹⁴vg/kg, preferably 5X 10¹²To 2X 10¹⁴vg/kg, more preferably 5X 10¹²To 5X 10¹³vg/kg, more preferably 1X 10¹³vg/kg to 5X 10¹³vg/kg, particularly preferably 3X 10¹³vg/kg to 5X 10¹³Weight of vg/kgGroup AAV vectors and 10 to 50mg/kg, preferably 20 to 50mg/kg, preferably 30 to 50mg/kg, particularly preferably 40 to 50mg/kg of sildenafil are administered to the subject.

In one embodiment, co-administration of sildenafil and the recombinant AAV vector results in enhanced expression of the protein of interest by the cell in vivo or in vitro, under the same conditions, as compared to a control group administered with the recombinant AAV vector alone. In a preferred embodiment, the cell is a mammalian cell. In a more preferred embodiment, the mammalian cell is a human cell. In a preferred embodiment, the co-administration of sildenafil and the recombinant AAV vector results in an increase in the expression of the protein of interest of the cell in vivo or in vitro, e.g., by at least about 1.2-fold, preferably 1.5-fold, more preferably 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, or 20-fold, compared to a control group administered the recombinant AAV vector alone.

In one embodiment, the amount of expression of the protein of interest can be measured, for example, by immunoassay.

In one embodiment, co-administration of sildenafil and the recombinant AAV vector results in a decrease in neutralizing antibodies of the AAV vector produced by the cell in vivo or in vitro, under the same conditions, as compared to a control group administered with the recombinant AAV vector alone. In a preferred embodiment, the cell is a mammalian cell. In a more preferred embodiment, the mammalian cell is a human cell. In a preferred embodiment, the co-administration of sildenafil and the recombinant AAV vector results in a reduction of neutralizing antibodies of the AAV vector produced by the cells in vivo or in vitro, e.g., by at least about 1.2 fold, preferably by at least 1.5 fold, more preferably by at least 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, as compared to a control group administered the recombinant AAV vector alone.

In one embodiment, neutralizing antibody titers can be measured by, for example, ELISA assays.

The present disclosure is described in further detail below with reference to the accompanying drawings and examples. The following examples are merely illustrative of the present disclosure and are not intended to limit the scope of the present disclosure. The experimental procedures, in which the specific conditions are not indicated in the examples, are carried out according to the conventional conditions known in the art or according to the conditions recommended by the manufacturer.

Examples

Example 1 sildenafil increases AAV vector-mediated expression of Gluc (secreted luciferase)

Normal C57 female mice were injected with varying concentrations of sildenafil (5, 10, 50mg/kg) and AAV9-Gluc virus (1X 10)¹³vg/kg), or injection of AAV9-Gluc virus alone (1X 10)¹³vg/kg). The control group was injected with physiological saline only. The expression of Gluc in serum was measured 1 week, 2 weeks, 3 weeks, 4 weeks after injection.

The results showed that, as shown in fig. 1, the group injected with sildenafil (5, 10, 50mg/kg) + AAV9-Gluc virus increased the expression of Gluc in the serum of mice relative to the group injected with AAV9-Gluc virus alone, and the expression amount increased with the increase in the dose of sildenafil (fig. 1). In the group injected with only AAV9-Gluc virus, the expression of Gluc in mouse serum reached a peak at week 3 and began to decline at week 4. The group injected with sildenafil (10, 50mg/kg) + AAV9-Gluc virus still showed an upward trend at week 4. The above results indicate that sildenafil can increase the expression of AAV9-Gluc in mice.

Example 2 sildenafil enhances AAV-mediated expression of GFP in mouse tissues

To investigate the effect of sildenafil on infection after systemic administration of AAV in various tissues of mice, sildenafil was selected at a dose of 10mg/kg, and after intraperitoneal injection of C57 mice for 1 week, AAV9-GFP (5X 10) was administered into tail vein¹³vg/kg) virus. After 3 weeks, brain (cortex, hippocampus), liver, heart, kidney, and muscle tissues were perfused and cryosectioned, each 10 μm thick. After 0.3% Triton X-100 permeabilization and 5% BSA blocking, primary antibody (anti-GFP) was incubated overnight at 4 ℃. Next, a secondary antibody with a fluorophore (absorption wavelength 488nm) corresponding to the primary antigen was incubated with DAPI at room temperature for 2 hours. Finally, imaging was performed using the Leica Thunder system and quantitative fluorescence analysis of GFP was performed under consistent imaging parameters without overexposure.

FIGS. 2A and 2B show the effect of sildenafil on GFP expression in various tissues of AAV9-GFP virus-infected mice. The results showed that, as shown in fig. 2A, fluorescence intensity of GFP was increased in all of cortex, hippocampus, liver, heart, kidney and muscle tissues in brain sections of the sildenafil-treated mouse group compared to the mouse group injected with AAV9-GFP alone, where statistical analysis of fluorescence of cortex, hippocampus, kidney and muscle was p <0.05 (fig. 2B).

In addition, the protein was extracted from homogenates of different tissues of each group of mice for immunoblotting. FIG. 3 shows sildenafil (10mg/kg) against AAV9-GFP virus (5X 10)¹³vg/kg) effect of GFP expression in tissues 3 weeks after infection of C57 mice. The results showed that the expression of GFP protein was increased in the cortical, muscle, liver, heart and kidney tissues in the sildenafil-treated mouse group (fig. 3A), where statistical analysis of muscle, liver and kidney fluorescence p<0.05 (fig. 3B).

The results show that sildenafil can improve the infection efficiency of AAV9-GFP virus in various tissues of mice and increase the expression of a target gene GFP.

Example 3 sildenafil increases tissue sensitivity to AAV by enhancing vesicle-mediated endocytosis and macropinocytosis Dyeing efficiency

Transport of AAV viruses in vivo is known to be achieved primarily through endocytosis, and involves three major pathways, namely clathrin-mediated endocytosis, vesicle-mediated endocytosis, and macropinocytosis.

In order to deeply explore the action mechanism of sildenafil for increasing AAV tissue infection efficiency, the inventors constructed a transwell vascular model in vitro. 293 cells were seeded in the lower chamber and an in vitro vascular model was treated with sildenafil in combination with three pathway inhibitors. Inhibitors of three pathways, clathrin-mediated endocytosis, vesicle-mediated endocytosis, and macropinocytosis, are Filipin (Filipin), Amiloride (amioride), and Chlorpromazine (Chlorpromazine), respectively.

Using a mixture containing sildenafil (50. mu.g/ml), Filipin (5. mu.g/ml) + sildenafil (50. mu.g/ml), Amiloride (50. mu.M) + sildenafilCells were treated with medium other than (50. mu.g/ml), Chloromazine (10. mu.g/ml) + sildenafil (50. mu.g/ml) for 12 hours, respectively, and then changed to contain 1X 10¹¹Serum-free ECM medium of vg AAV9-GFP virus was cultured for an additional 6 hours, after which the upper chamber was removed and the lower medium was replaced and culture continued for an additional 72 hours.

Fluorescence microscopy showed that the GFP fluorescence of 293 cells of the sildenafil-only treated group was significantly stronger than that of the untreated group (fig. 4A). When the Filipin or amioride inhibitor is added, the fluorescence intensity is obviously reduced to be similar to that of a sildenafil untreated group. While the Chroprazine inhibitor had no significant effect on fluorescence intensity (FIG. 4A). This indicates that the Filipin and Amiloride inhibitors can antagonize the enhancement of sildenafil infection ability to AAV tissues, while Chloromazine has no obvious effect on the enhancement of sildenafil.

Next, 293 cells of total mRNA extraction for qPCR detection. The results showed that the transcription level of GFP was higher in 293 cells of the sildenafil-only treated group than in the untreated group. When Filipin or amioride inhibitors were added, GFP transcript levels were significantly reduced to a level similar to that of the sildenafil untreated group. While the Chlorlormazine inhibitor had no significant effect on the level of GFP transcription, this result was consistent with the fluorescence imaging results (FIG. 4B).

The above results indicate that sildenafil enhances the infection efficiency of AAV9 to tissues by enhancing vesicle-mediated endocytosis and macropinocytosis, and increases the distribution of AAV9 in each tissue.

Example 4 sildenafil alleviates immune responses by AAV and reduces AAV neutralizing antibodies

To investigate the effect of sildenafil on the immune response elicited by AAV, the inventors analyzed subpopulations of T cells and B cells in the spleen using flow cytometry. Wherein CD25⁺FoxP3⁺Labelling regulatory T cells (Tregs), B220⁺CD19⁺Labeling the B cells.

The results showed that mice injected with AAV9 alone had a decrease in the number of Treg cells in the spleen (fig. 5A, fig. 5B) and an increase in the number of B cells (fig. 5C, fig. 5D) compared to the blank control group, indicating that injection of AAV activated an immune response. And, the number of Treg cells in the spleen of mice in the sildenafil-treated group (10mg/kg) was significantly increased and the number of B cells was significantly decreased (p <0.05) compared to the group injected with AAV9 alone, and the number of Treg cells and B cells in the spleen of mice in the sildenafil group was not significantly different from that in the blank control group (p > 0.05). The above results indicate that sildenafil can reduce immune responses by AAV.

In addition, mRNA from spleens of each group of mice was extracted and the level of inflammation was detected by qPCR. The results showed that the transcription levels of the inflammatory factors IL1, IL6, TNF α in the spleen of the mice in the sildenafil-treated group (10mg/kg) were significantly lower than those in the AAV9 virus-only injection group (fig. 5E), indicating that sildenafil has a certain anti-inflammatory effect.

Finally, neutralizing antibody experimental studies found that sildenafil treated groups significantly reduced the production of neutralizing antibodies to mouse AAV9 after 2 and 3 weeks of AAV virus injection (fig. 5F).

It can be seen that sildenafil can effectively suppress the immune response caused by AAV9, reduce inflammatory response and reduce production of AAV9 neutralizing antibodies by modulating tregs and reducing B cell numbers. Thus, sildenafil can reduce clearance of AAV9 in mice, increasing its infection efficiency.

Example 5 evaluation of safety of sildenafil in combination with AAV vectors

The toxicity of sildenafil to CCK8 of huvec cells was first investigated. The CCK8 results showed that sildenafil had no significant toxic effect on huvec cells (fig. 6C).

Next, the safety of sildenafil was evaluated. The results show that sildenafil at different doses (5, 10, 50mg/kg) in combination with AAV9 did not affect the body weight of the mice (fig. 6A), sildenafil in combination with AAV9 did not affect the liver function markers of the mice, and ALT and AST levels were not significantly different from the blank group (fig. 6B), indicating that sildenafil is safer for use in combination with AAV viruses.

Example 6 sildenafil increases AAV vector-mediated dystrophin expression

Further validation of the effects of sildenafil on DMD Gene model mice. Selecting 10mg/kg sildenafil, injecting DMD mouse intraperitoneally for 1 week, and injecting AAV 9-dystropin (3 × 10)¹³vg/kg) virus. After 3 weeks, heart tissue and leg muscles, including gastrocnemius muscle (GAS), quadriceps muscle (QF), and Tibialis Anterior (TA) tissue frozen sections were perfused to 10 μm thick. After 0.3% Triton X-100 permeabilization and 5% BSA blocking, primary antibody (Anti-Dystrophin) was incubated overnight at 4 ℃. Then, a secondary antibody with a fluorophore (absorption wavelength 555nm) corresponding to the primary antigen was incubated with DAPI at room temperature for 2 hours. Finally, imaging was performed using the Leica Thunder system and quantitative fluorescent analysis of dystrophin (dystropin) was performed with consistent imaging parameters and without overexposure.

The results show that, as shown in fig. 7, the fluorescence intensity of dystrophin in leg muscles of mice of the sildenafil-treated group was enhanced compared to those of the AAV 9-dysthiophin-only injected group, with a significant increase in GAS and QF (p < 0.05). The same results were observed in mouse myocardial tissue sections.

In addition, the protein was extracted from homogenates of different tissues of each group of mice for immunoblotting. The results show that sildenafil treatment significantly increased dystrophin expression in gastrocnemius, quadriceps, tibialis anterior and cardiac muscles with p <0.05 (figure 8). The above results indicate that sildenafil can effectively increase the expression of dystrophin mediated by AAV vectors.

Furthermore, in addition to AAV9 demonstrated in the above examples, it is reasonable to expect based on the present disclosure that sildenafil also has varying degrees of enhancement of the expression of genes of interest of other AAV serotypes, and can also reduce neutralizing antibodies of other AAV serotypes to some extent.

While the present disclosure has been shown and described with reference to certain preferred embodiments thereof, it will be understood by those skilled in the art that the foregoing is a more detailed description of the disclosure than is possible with reference to the specific embodiments, and that no limitation to the specific embodiments of the disclosure is intended. Various changes in form and detail, including simple deductions or substitutions, may be made by those skilled in the art without departing from the spirit and scope of the present disclosure.

Claims (11)

1. A gene delivery composition comprising:

sildenafil, and

a recombinant adeno-associated virus (AAV) vector,

wherein the recombinant AAV vector comprises an AAV capsid protein and a gene of interest.

2. The gene delivery composition of claim 1, wherein the gene of interest encodes a therapeutic protein or a non-therapeutic protein, the therapeutic protein comprising a protein that has the effect of treating a disease in a subject, the disease comprising: cancer, autoimmune diseases, diseases caused by exposure to infectious agents, and genetic diseases; the non-therapeutic protein includes proteins that have a beneficial effect in a subject other than treating a disease, such as proteins that have nutritional or cosmetic efficacy.

3. The gene delivery composition according to claim 1 or 2, wherein the AAV capsid protein is a native AAV capsid protein or an artificially engineered AAV capsid protein; preferably, the AAV is selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-DJ8, AAV-DJ9, AAVrh8, AAVrh8R and AAVrh10, more preferably AAV 9.

4. The gene delivery composition of any one of claims 1 to 3, wherein the gene delivery composition comprises 1 x 10⁹To 1X 10¹⁵vg of a recombinant AAV vector and 5. mu.g to 100mg of sildenafil, preferably 1X 10¹²To 2X 10¹³vg of a recombinant AAV vector and 1mg to 50mg of sildenafil.

5. An isolated host cell transfected or transduced with the gene delivery composition of any one of claims 1 to 4.

6. A medicament comprising the gene delivery composition of any one of claims 1 to 4 and an excipient comprising one or more of a polyol, a sugar, a buffer, a preservative, and an inorganic salt; preferably, the drug is in the form of an injection such as an injection solution and a sterile powder for injection.

7. The medicament of claim 6, wherein the medicament is administered by a systemic route or a local route, such as intravenous administration, intramuscular administration, subcutaneous administration, oral administration, local contact, intraperitoneal administration, and intralesional administration.

8. Use of sildenafil for increasing the expression of a target gene mediated by a recombinant AAV vector, wherein the recombinant AAV vector comprises an AAV capsid protein and a target gene; the AAV capsid protein is a natural AAV capsid protein or an artificially modified AAV capsid protein, and the target gene encodes a therapeutic protein or a non-therapeutic protein.

9. A method of increasing recombinant AAV vector-mediated expression of a gene of interest, comprising: administering to a subject in need thereof a recombinant AAV vector comprising a gene of interest; and, administering sildenafil to the subject,

the gene of interest encodes a therapeutic protein or a non-therapeutic protein,

preferably, 1 x 10 is administered to the subject¹¹To 2X 10¹⁴vg/kg of recombinant AAV vector; preferably, 5 to 50mg/kg of sildenafil is administered to the subject.

10. Use of sildenafil in reducing neutralizing antibodies to an AAV vector, preferably the AAV vector is a recombinant AAV vector, more preferably the recombinant AAV vector comprises a gene of interest encoding a therapeutic protein or a non-therapeutic protein.

11. A method of reducing neutralizing antibodies to an AAV vector, comprising: administering an AAV vector to a subject in need thereof; and, administering sildenafil to the subject, preferably the AAV vector is a recombinant AAV vector, more preferably the recombinant AAV vector comprises a gene of interest encoding a therapeutic protein or a non-therapeutic protein,

preferably, 1 x 10 is administered to the subject¹¹To 2X 10¹⁴vg/kg of recombinant AAV vector; preferably, 5 to 50mg/kg of sildenafil is administered to the subject.

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