CN112852881A

CN112852881A - Method for enhancing transduction efficiency of adeno-associated virus in central nervous system by using cell-penetrating peptide

Info

Publication number: CN112852881A
Application number: CN202110173370.5A
Authority: CN
Inventors: 罗光佐; 韩雪飞; 孟缘
Original assignee: Nanjing Best Bio Technology Co ltd
Current assignee: Nanjing Best Bio Technology Co ltd
Priority date: 2021-02-09
Filing date: 2021-02-09
Publication date: 2021-05-28
Anticipated expiration: 2041-02-09
Also published as: CN112852881B

Abstract

The present invention provides a method for enhancing transduction efficiency of adeno-associated virus type 9 using Cell Penetrating Peptides (CPPs), which can be used to enhance the ability of vectors to deliver transgenes to the Central Nervous System (CNS) of a patient. By applying CPPs and recombinant adeno-associated virus 9 (rAAV 9), the efficiency of rAAV9 passing through a Blood Brain Barrier (BBB) can be improved, the expression of transgenes in a central nervous system is greatly improved, excessive immune response can be avoided, and the clinical prospect of delivering nucleic acid from a medicinal preparation to cells of the brain and/or the central nervous system is greatly expanded.

Description

Method for enhancing transduction efficiency of adeno-associated virus in central nervous system by using cell-penetrating peptide

Technical Field

The invention relates to a method for promoting the transduction efficiency of a viral vector, in particular to a method for promoting the gene expression of AAV9 in a central nervous system by using cell-penetrating peptide, belonging to the technical field of genetic engineering.

Background

Recombinant adeno-associated virus (rAAV) has been widely used in gene therapy for decades and has been widely used for eye diseases, cancer, vascular diseases, etc., but its use in the treatment of Central Nervous System (CNS) diseases is severely limited due to its very low efficiency in delivering rAAV vectors through the blood-brain barrier (BBB).

The blood-brain barrier is a highly selective osmotic barrier that separates circulating blood from the brain extracellular fluid of the central nervous system and prevents most drugs from passing from the blood into the brain. It is formed by capillary endothelial cells. Astrocytes are the major type of glial cells in the mammalian brain and are essential for the formation of the blood brain barrier. They provide a variety of support functions for partner neurons in the central nervous system, such as neuronal guidance during development and life-long nutritional and metabolic support.

There have been many studies aimed at improving the transduction efficiency of AAV vectors in the brain, but progress in this regard has remained very limited to date. Some preclinical studies have shown that engineered AAV vectors show positive results, but further studies have found that these AAV vectors may not enhance transduction efficiency in large animals and humans. It is not clear whether mutations on the viral capsid affect its productivity and cause any safety hazards, and AAV engineering is time consuming and expensive. Therefore, there is a need to find an effective and economical strategy to improve AAV transduction efficiency in the CNS.

Cell-penetrating peptides (CPPs) have been reported to enhance transduction of AAV2 and AAV8 in vivo and in vitro, and no cytotoxicity of CPPs was detected in vivo. Past studies have known that THR peptides can increase BBB crossing ability and transduction efficiency of AAV8 in the brain, but the number of AAV8 virions found in the liver is much greater (about 175 fold) compared to the brain following systemic injection of AAV 8/polypeptide, i.e. AAV8 is a highly hepatropic vector (Zhang X, He T, Chai Z, Samulski RJ, and Li c. biomaterials, 2018; 176: 71-83), which indicates that AAV8 is not an ideal AAV vector for delivering target genes to the brain. Although CPPs can enhance AAV-directed transduction, it is unknown whether CPPs can alter the tropism of the vector.

LAH4 is an antibacterial peptide which strongly interacts with phospholipid membrane and can be adsorbed on the membrane, mediates the transport of functional β -galactosidase into cells, has high nucleic acid transfection efficiency and cell penetration activity, and related derivatives have been developed to enhance viral transduction for gene therapy. Leptin30 is a 30 amino acid peptide derived from the endogenous hormone Leptin, used as a brain targeting ligand, and specific ligand-receptor binding mediated endocytosis is considered as one of the possible strategies to overcome the blood brain barrier. ApoE is a secreted lipid transporter in the peripheral and Central Nervous System (CNS), and has also been studied as a blood brain barrier receptor targeting agent.

Disclosure of Invention

The main objective of the present invention is to provide a method for enhancing transduction efficiency of adeno-associated virus type 9 by using cell-penetrating peptide, so as to overcome the deficiencies in the prior art.

In order to achieve the purpose, the invention provides the following technical scheme:

the present invention provides a method of delivering a transgene to the central nervous system, the method comprising administering: (i) a cell-penetrating peptide selected from LAH4, LEPTIN30, APOE or a combination thereof; (ii) recombinant adeno-associated virus vectors.

Preferably, the recombinant adeno-associated viral vector is adeno-associated virus type 9 or any mutant thereof.

More preferably, the above-mentioned adeno-associated virus type 9 comprises the chicken β -actin promoter.

Preferably, the LAH4 comprises SEQ ID NO: 1.

Preferably, the aforementioned APOE comprises SEQ ID NO: 2.

Preferably, the aforementioned LEPTIN30 comprises SEQ ID NO: 3.

Preferably, the above-mentioned adeno-associated virus type 9 and cell-penetrating peptide form a complex.

Preferably, the recombinant adeno-associated viral vector is capable of crossing the blood-brain barrier.

Preferably, the above transgene is capable of being expressed in human brain endothelial cells and glial cells.

In another embodiment, the present invention also provides a composition comprising the cell-penetrating peptide described above and a recombinant adeno-associated viral vector.

Preferably, the composition further comprises a pharmaceutically acceptable carrier.

The invention also provides the use of the aforementioned composition for treating a central nervous system disorder in a patient.

In the present invention, it was found that several cell-penetrating peptides (CPPs) can significantly enhance the in vitro transduction efficiency of adeno-associated virus type 9 (AAV serotype 9, AAV 9), a promising AAV vector for the treatment of CNS disorders. In particular embodiments, the peptide LAH4 that enhances transduction efficiency most, and the enhancement of AAV9 transduction efficiency by LAH4 peptide is dose dependent, which is dependent on binding of AAV9 capsid to the peptide. In addition, LAH4 peptide was also shown to increase AAV9 transduction within the CNS after systemic administration.

The CPP provided by the invention can directly interact with AAV9, and enhances the ability of AAV vector to cross BBB, thereby further inducing higher expression of genes in brain, and being beneficial to improving the application of AAV gene transfer vector in the treatment of central nervous system diseases.

As described above, the enhancement of the efficiency of viral transduction mediated by CPPs is caused by the direct interaction between AAV particles and CPPs, and thus, the purified AAV-CPP complex will have higher efficiency, better safety and lower immune response than the unpurified mixture.

Compared with the prior art, the invention has the beneficial effects that:

the method for promoting the AAV-mediated expression capacity of the exogenous gene in the central nervous system by using the cell-penetrating peptide combines the cell-penetrating peptide with AAV-mediated gene therapy by using the characteristics of the cell-penetrating peptide, carries the AAV to reach regions which are difficult to penetrate, and improves the gene expression efficiency. The invention can improve the efficiency of rAAV9 passing through the Blood Brain Barrier (BBB), greatly improve the expression of transgene in the central nervous system, and simultaneously avoid excessive immunoreaction, and has important significance for promoting the development of the fields of gene therapy, cell engineering and the like.

Definition of

Recombinant AAV:

the term "recombinant AAV" refers to an AAV or an artificially produced AAV that has been isolated from its native environment (e.g., from a host cell, tissue, or subject), and the recombinant AAV is produced using recombinant methods. The recombinant aav (rAAV) preferably has tissue-specific targeting capabilities such that a transgene of the rAAV will be specifically delivered to one or more predetermined tissues.

Transfection:

the term "transfection" refers to the uptake of foreign DNA by a cell, which is "transfected" when the foreign DNA is introduced into the cell membrane. A number of transfection techniques are well known in the art (see, e.g., Graham et al (1973) Virology, 52:456, Sambrook et al (1989) Molecular Cloning, a laboratory, Cold Spring Harbor Laboratories, New York, Davis et al (1986) Basic methods in Molecular Biology, Elsevier, and Chu et al (1981) Gene 13: 197) and can be used to introduce one or more foreign nucleic acids, such as nucleotide integration vectors and other nucleic acid molecules, into a suitable host cell.

Carrier:

the term "vector" includes any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc., which is capable of replication when combined with appropriate control elements and which permits transfer of gene sequences between cells. Thus, the term includes cloning and expression vectors as well as viral vectors. In some embodiments, useful vectors are considered to be those in which the nucleic acid fragment to be transcribed is under the transcriptional control of a promoter. The term "promoter" refers to a DNA sequence recognized by the synthetic machinery of the cell or introduced synthetic machinery that is required to initiate specific transcription of a gene. The term "expression vector" means any type of genetic construct containing a nucleic acid in which part or all of the nucleic acid coding sequence can be transcribed. In some embodiments, expression includes transcription of a nucleic acid, e.g., production of a biologically active polypeptide product or inhibitory RNA (e.g., shRNA, miRNA) from a transcribed gene.

Recombinant AAV vectors:

a "recombinant AAV (raav) vector" as described herein typically consists of at least a transgene (e.g., encoding GFP) and its regulatory sequences, and 5 'and 3' AAV Inverted Terminal Repeats (ITRs). This recombinant AAV vector is packaged as a capsid protein and delivered to selected target cells. The nucleic acid coding sequence is operably linked to regulatory components in a manner that allows for transcription, translation, and/or expression of the transgene in cells of the target tissue. The term "operably linked" refers to the promoter being in the correct position relative to the nucleic acid to control RNA polymerase initiation and gene expression.

Drawings

FIG. 1: schematic representation of the present invention.

FIGS. 2A-2B: the effect of CPPs used in the present invention on AAV9 transduction into HEK293T cells. Figure 2A shows that peptides of LAH4, LEPTIN30, and APOE increased transduction of HEK293T cells by AAV 9; FIG. 2B is a statistics of the number of GFP positive cells in FIG. 2A.

FIGS. 3A-3B: the effect of CPPs used in the present invention on AAV9 transduction into EC and HA cells. Fig. 3A shows that peptides of LAH4, LEPTIN30, and APOE increased transduction of EC cells, HA cells by AAV 9; FIG. 3B is a statistics of the number of GFP positive cells in FIG. 3A.

FIGS. 4A-4C: effect of CPPs used in the present invention on target gene expression in HEK293T, EC and HA cells. FIG. 4A shows that peptides of LAH4, LEPTIN30, and APOE increased the level of GFP mRNA in cells; FIG. 4B shows that LAH4, LEPTIN30, APOE increased the level of GFP protein in the cells; FIG. 4C is a graph quantifying the level of GFP protein in FIG. 4B.

FIG. 5: co-localization of LAH4 peptide and AAV9 particle in cells results.

FIGS. 6A-6H: transduction efficiency of AAV9-CPP complexes with EC cells at different incubation times. Figure 6A shows the effect of incubation time on the transduction efficiency of the complexes; FIG. 6B shows total RNA isolated from the cells of FIG. 6A and the mRNA level of GFP assessed by quantitative polymerase chain reaction; FIG. 6C shows GFP protein levels of the cells in FIG. 6A; FIG. 6D is a graph of the quantification of GFP protein levels in FIG. 6C; FIG. 6E shows the effect of incubation temperature on transduction efficiency of complexes; FIG. 6F shows total RNA isolated from the cells of FIG. 6E and the mRNA level of GFP assessed by quantitative polymerase chain reaction; FIG. 6G shows GFP protein levels of the cells in FIG. 6E; FIG. 6H is a graph quantifying GFP protein levels in FIG. 6G.

FIGS. 7A-7B: LAH4 facilitated in vitro testing results of AAV9 across the BBB. FIG. 7A shows the change in viral titer over time in EC cells of AAV9 and AAV9-LAH4 (20 μ M) treated groups; FIG. 7B shows the change in viral titer in EC cells over time after treatment with different concentrations of AAV9-LAH 4.

FIGS. 8A-8D: after AAV9 vector and AAV9-LAH4 complex were injected into mice, respectively, the result of GFP expression in mouse liver and brain tissue was obtained. FIG. 8A shows the expression of both vectors and controls in hippocampus, cerebellum, cortex, liver; FIG. 8B is a statistical chart of FIG. 8A; FIG. 8C shows the expression amount of GFP in genomic DNA in the brain and liver; FIG. 8D shows the expression amount of GFP in mRNA in brain and liver.

FIGS. 9A-9E: AAV9-LAH4 complex in mice in immune response. FIG. 9A shows mRNA levels of IL-1 β and TNF- α in the liver and brain; figure 9B shows levels of CD4 and CD8 in the mouse brain; FIG. 9C is a graph quantifying the expression levels of CD4 and CD8 in FIG. 9B; figure 9D shows levels of CD4 and CD8 in mouse liver; fig. 9E is a graph quantifying the expression levels of CD4 and CD8 in fig. 9D.

FIGS. 10A-10D: the effect of different inhibitors on viral transduction of cells infected with AAV9 or AAV9-LAH4 was tested. FIG. 10A shows the levels of GFP protein in infected cells after preincubation with chlorpromazine (CPZ, 15 nmol/L) and Amiloride (Amilolide, 1 mmol/L); FIG. 10B is a graph of quantifying the level of GFP protein in FIG. 10A; FIG. 10C shows the levels of GFP protein in cells that were re-infected after pre-incubation with Erythrina cristagalli lectin (ECL, 50 ug/mL); FIG. 10D is a graph quantifying GFP protein levels in FIG. 10C.

Detailed Description

The present invention will be further described with reference to the following embodiments and drawings, and the present invention is not limited to the following embodiments. It is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. It is intended that all such alterations and advantages be included in the invention, which occur to those skilled in the art, be considered as within the spirit and scope of the inventive concept, and that all such modifications and advantages be considered as within the scope of the appended claims and any equivalents thereof. In the description and claims of the present invention, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. As used herein, the expressions "cell" and "cell line" are used interchangeably and all such designations include progeny thereof, it being understood that not all progeny are precisely the same DNA content, including mutant progeny that have the same function or biological activity as screened for in the originally transformed cell, due to deliberate or inadvertent mutation. The experimental procedures in the following examples, in which specific conditions are not specified, are all common knowledge and general knowledge of those skilled in the art, or conditions recommended by the manufacturer. All materials and reagents used in the examples are commercially available products unless otherwise specified.

Cell lines

HEK293T (a derivative of human embryonic kidney cell HEK 293) was cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Hyclone, Northbrook, Ill., USA) containing 10% (v/v) heat-inactivated Fetal Bovine Serum (FBS) (Sera Pro SA).

Human brain microvascular endothelial cells (hCMEC/D3), purchased from Sigma-Aldrich (PN SCC 066), were cultured in endothelial basal medium (EBM-2) (Lonza, Basel, Switzerland) containing 1% lipid concentrate, 1% HEPES, 0.055% cortisol, 0.002% bFGF, 0.00055% vitamin c.

Human Astrocytes (HAs), purchased from scientific cell research laboratory (carlsbad, usa), were cultured in astrocyte medium (scientific cell research laboratory, carlsbad, california, usa).

The cell lines were cultured at 37 ℃ in 5% CO₂Cultured in an incubator.

Polypeptides

All polypeptides used were synthesized in GenScript (Nanjing, China), 95% pure, and dissolved in 10% dimethyl sulfoxide in Dulbecco's Phosphate Buffered Saline (DPBS) to make 10 mM stock. The amino acid sequences of the LAH4, ApoE and Leptin30 peptides are KKALLALHHLAHLALHLALALALKKAC (SEQ ID NO: 1), LRKLRKRLLLRKLRKRLL (SEQ ID NO: 2) and YQQILTSMPSRNVIQISNDLENLRDLLHVL (SEQ ID NO: 3), respectively.

Plasmids

AAV9 rep/cap plasmid, pAAV/CBA-GFP and pAD helper plasmid for recombinant AAV9 vector production were obtained from Addgene (Cambridge, Mass.).

Western blot analysis

Total protein in cells or animal tissues was isolated using RIPA buffer (Shanghai Biyum Biotechnology institute, Shanghai, China). Protein quantification equal amounts of protein samples were separated by SDS-PAGE using a Wanleibo BCA protein detection kit (PN WLA004, Shenyang, China), and then electrophoretically transferred onto PVDF membrane. To avoid non-specific binding, the membrane was blocked with 5% skim milk for 45 minutes at room temperature, and then incubated with anti-GFP antibody (1: 100,000 dilution, PN 66002-1-lg, Proteintech, Wuhan, China) for 2 hours at room temperature. After washing, the membrane was incubated with HRP-conjugated secondary antibody at room temperature for 2 hours (1: 5,000 dilution, PN SA00001-1, wuhan protein technologies, ltd.). After 3 washes with Tris buffered saline containing Tween 20 (TBST), immunoblots were visualized by Enhanced Chemiluminescence (ECL) kit (PN 180-.

Real-time PCR analysis

Total RNA was isolated from cells and tissues using TRIzol reagent (PN DP424, TIANGEN, Beijing, China). The concentration and mass of RNA were then determined using a NanoPhotometer N50 touch (Implen). Total RNA was reverse transcribed to cDNA using Takara PrimeScript RT Master Mix (RR 036A). Quantitative PCR was performed on the LightCycler 96 real-time system (Roche) using primer pair 5'-GCACAAGCTGGAGTACAACTA-3' (SEQ ID NO: 4) and 5'-TGTTGTGGCGGATCTTGAA-3' (SEQ ID NO: 5) specific for the GFP transgene. The specific primer pairs for the GAPDH gene were 5'-GAAGGTGAAGGTCGGAGTC-3' (SEQ ID NO: 6) and 5'-GAAGATGGTGATGGGATTTC-3' (SEQ ID NO: 7) using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an endogenous control. Relative quantification (2- Δ Ct) was used to calculate relative expression values.

Immunohistochemical analysis

AAV9（3×10¹¹Pellet/mouse) was preincubated with 0.001mM LAH4 peptide at 37 ℃ for 2 hours. As shown in fig. 1, AAV9 or AAV9-CPP complex was injected via tail vein into C57BL/6 mice, and 21 days after injection, the brain and liver of the mice were excised, fixed, frozen, cryosectioned, and mounted on glass slides (Thermo, Cryotome E). Frozen sections were washed with cold PBS, fixed with 4% paraformaldehyde in antigen recovery buffer (100 × EDTA buffer, pH 8.0), slides were blocked with 3% BSA, then washed with PBS, then incubated with rabbit anti-GFP (Proteitech 50430-2-AP), then incubated with the antibody, and counterstained with 4', 6-diamino-2-phenylindole (DAPI) for 10 minutes (Keygen BioTECH KGA 215). Fluorescence images were obtained from a Nikon (Nikon DS-U3) eclipsec1 microscope with regions of interest randomly selected at 200 Xmagnification.

Example 1: production of recombinant AAV9 viral vectors

Recombinant AAV9 whole particles with GFP expression driven by the chicken β -actin (CBA) promoter were produced using triple transfection in HEK293T cells. Briefly, the transgenic plasmid pAAV/CBA-GFP, the AAV helper plasmid containing the Rep and Cap genes (AAV 9 Rep/Cap), and the adenovirus helper plasmid pAD were co-transfected into HEK293T cells. HEK293T cells were harvested and lysed 72 hours post transfection, and the supernatant was subjected to cesium chloride gradient ultracentrifugation.

Example 2: in vitro transduction efficiency assay

To test the effect of CPPs on AAV9 transduction, cells were plated at 3 × 10 per well at least 6 hours prior to AAV9 transduction⁵The density of individual cells was seeded in 24-well plates, then CPPs (peptides of LAH4, LEPTIN30 and APOE) at different concentrations (final concentration of 0.5 μ M or 20 μ M) were incubated with AAV9 vector encoding green fluorescent protein (AAV 9/GFP) with an MOI of 1,000, respectively, and then infection of HEK293T cells was examined. As shown in fig. 2A and 2B, peptides of LAH4, LEPTIN30 and APOE induced a dose-dependent increase in viral transduction in HEK293T cells. As expected, no fluorescence was found in cells without AAV9 transduction (NC); in cells incubated with AAV9 vector alone, fluorescence in the cells was very weak, but when AAV9 vector was incubated with peptides of 5 μ M LAH4, LEPTIN30, or APOE and HEK293 cells were then transduced, the level of fluorescence increased significantly. When the concentration of CPPs was increased to 20 μ M, the group fluorescence levels of added CPPs (LAH 4, LEPTIN30, and APOE peptide) were increased by 550%, 230%, and 189%, respectively, compared to cells transduced with AAV only, with LAH4 being the best transduction efficiency. These results indicate that CPPs can effectively improve transduction of AAV9 into HEK293T cells.

To examine the role of CPPs in AAV9 transduction into human brain endothelial cells (hCMEC/D3, EC for short) and glial cells (HA), cells were plated at 3X 10/well at least 6 hours prior to AAV9 transduction⁵The density of individual cells was seeded in 24-well plates, then CPPs (peptides of LAH4, LEPTIN30 or APOE) at different concentrations (final concentration of 0.5. mu.M or 20. mu.M) were incubated with AAV9/GFP, respectively, at an MOI of 1,000, and then the infection status of EC and HA cells was examined. As shown in fig. 3A and 3B, peptides of LAH4, LEPTIN30 and APOE induced dose-dependent enhancement of viral transduction in EC and HA cells, similar to that in HEK293T cells. As expected, no fluorescence was found in cells without AAV9 transduction (NC); in cells incubated with AAV9 vector alone, the fluorescence in the cells was very weak; however, when AAV9 vector was incubated with 5 μ M peptide of LAH4, LEPTIN30, or APOE and then transduced EC or HA cells, the fluorescence level increased significantly. With cells transduced with AAV aloneIn contrast to the cells, when the concentration of CPPs was increased to 20 μ M, the fluorescence levels of EC cells were increased by 350%, 230% and 139% and the fluorescence levels of HA cells were increased by 250%, 130% and 119% in the groups to which CPPs (LAH 4, LEPTIN30 and APOE peptide) were added, respectively. LAH4 is the most effective polypeptide to enhance viral transduction in EC and HA cells, similar to HEK293T, compared to LEPTIN30 and APOE. Based on these data, we concluded that CPPs can effectively improve transduction of AAV9 to EC and HA cells.

Example 3: target gene expression delivered by AAV9 vector

Since CPP of LAH4, LEPTIN30 and APOE enhanced transduction of AAV9/GFP in HEK293, EC and HA cells, it was speculated that expression of the GFP gene should be increased accordingly. To validate this hypothesis, total RNA was purified from the cells of fig. 2A and 3A and subjected to real-time reverse transcription polymerase chain reaction (RT-qPCR). As shown in fig. 4A, peptides of LAH4, LEPTIN30 and APOE induced a dose-dependent increase of GFP mRNA in HEK293T, EC and HA cells. In cells incubated with AAV9 vector alone, GFP mRNA levels in the cells were very low; however, mRNA levels were significantly increased after AAV9 vector was incubated with 5 μ M peptides of LAH4, LEPTIN30, or APOE, followed by transduction of HEK293T, EC, or HA cells. When the concentration of CPP was increased to 20 μ M, mRNA levels in EC cells increased 350%, 230% and 139%, respectively, and mRNA levels in HA cells increased 250%, 130% and 119%, respectively, compared to cells transduced with AAV only, with LAH4 being the best effect of the three polypeptides.

In addition, GFP protein levels were also examined by immunoblotting using rabbit anti-GFP antibodies. As shown in fig. 4B and 4C, peptides of LAH4, LEPTIN30 and APOE induced dose-dependent increases in GFP protein levels in HEK293T, EC and HA cells. In cells incubated with AAV9 vector alone, GFP protein levels in cells were very low, but protein levels were significantly increased after AAV9 vector was incubated with 5 μ M peptides of LAH4, LEPTIN30, or APOE, followed by transduction of HEK293T, EC, or HA cells. When the concentration of CPPs was increased to 20 μ M, the protein levels in EC cells were increased by 350%, 230%, and 139%, respectively, and the protein levels in HA cells were increased by 250%, 130%, and 119%, respectively, compared to cells transduced with AAV vector alone, with the best of the three polypeptides remaining LAH 4.

Example 4: co-localization of LAH4 peptide and AAV9 vector particles in cells

To determine whether the enhanced viral transduction efficiency mediated by CPPs was the result of direct interaction of AAV9 vector particles with CPPs, empty capsid AAV9 particles that were not transgenic were preincubated with 5 μ M Fluorescein Isothiocyanate (FITC) -labeled LAH4 peptide at 37 ℃ for 1 hour, then the mixture was incubated with EC and HA cells, respectively, 3 hours later, the cells were collected and fixed, and then photographed by fluorescence confocal microscopy. The AAV9 was immunostained with intact AAV particle-specific antibodies, and the results are shown in fig. 5, where most of AAV9 co-localized with Fluorescein Isothiocyanate (FITC) -labeled LAH4 peptide, demonstrating the formation of AAV9-LAH4 complex, which confirms that the enhanced viral transduction mediated by CPPs is due to direct interaction of AAV9 particles with CPPs.

Example 5: optimization of AAV9-LAH4 complex formation

5.1 determination of optimal action time:

to optimize the experimental conditions for the production of the AAV9-CPP complex with the strongest transduction efficiency, we first investigated the AAV9 vector (1X 10) encoding Green Fluorescent Protein (GFP)⁴) Incubate with 5 μ M LAH4 peptide for 0, 60 and 120 minutes, respectively. AAV9-CPP complexes were incubated with EC cells for 24 hours for transduction, and the cells were then examined by fluorescence microscopy. As shown in fig. 6A, LAH4 peptide induced a time-dependent increase in fluorescence levels in EC cells; if AAV9 vector and LAH4 peptide were not incubated (time 0), fluorescence in the cells was very weak, whereas after 2 hours of incubation of AAV9 vector with LAH4 peptide, the fluorescence level increased significantly.

Total RNA and proteins were extracted from the cells of FIG. 6A, and GFP mRNA levels and protein levels were detected by RT-qPCR and Western blot, respectively. As shown in fig. 6B-6D, LAH4 peptide induced a time-dependent increase in GFP mRNA and protein levels in EC cells, with results similar to fluorescence levels, and after 2 hours of incubation, GFP mRNA and protein levels increased by about 250% on average over the unincubated (time 0).

5.2 determination of optimum action temperature:

past studies have indicated that CPPs interact with AAV particles through covalent bonds, which is an energy-dependent process (Liu et al, 2014). To confirm whether the formation of AAV9-CPP complex is energy-dependent, transduction efficiencies of AAV9-LAH4 complex formed at 4 ℃, 25 ℃ and 37 ℃ were tested, respectively. AAV9-CPP complexes were first incubated with EC cells for 24 hours for transduction, and the cells were then examined by fluorescence microscopy. As shown in FIG. 6E, the LAH4 peptide induced a temperature-dependent increase in fluorescence levels in EC cells, which were very weak at 4 ℃ and increased significantly when AAV9 vector was incubated with the LAH4 peptide at 25 ℃ and 37 ℃.

Total RNA and proteins were extracted from the cells of FIG. 6E, and GFP mRNA levels and protein levels were detected by RT-qPCR and Western blot, respectively. As shown in fig. 6F-6H, LAH4 peptide also induced a temperature-dependent increase in GFP mRNA and protein levels in EC cells, with similar results to fluorescence levels, with GFP mRNA and protein levels 120% and 50% higher at 37 ℃ than at 4 ℃, respectively.

Taken together, 120 min of reaction at 37 ℃ resulted in AAV9-CPP complex with maximal transduction potential.

Example 6: LAH4 increases AAV9 crossing the BBB

EC cells were cultured in monolayers and incubated with either AAV9 or AAV9-LAH4 complex (final concentration 20 μ M), respectively, and the culture fluid in the substrate chamber was collected at different time points and analyzed for viral titer by qPCR, all treatment groups were repeated three times (P <0.05, P <0.01, compared to cells treated with AAV9 alone). As shown in figure 7A, viral titers were significantly increased in cells treated with AAV9-LAH4 and exhibited a time-dependent increase compared to AAV9 alone.

EC cells were cultured in monolayers and incubated with different concentrations of AAV9-LAH4, respectively, and the culture fluid in the substrate chamber was collected at different time points and analyzed for viral titer by qPCR, all treatments were repeated three times (. P <0.05,. P <0.01, compared to cells treated with 20 μ M LAH 4). As shown in FIG. 7B, the virus titers of the experimental groups treated at 30. mu.M, 40. mu.M and 50. mu.M were significantly increased compared to the group treated at 20. mu.M.

Example 7: delivery of target Gene expression in vivo by AAV9-LAH4 Complex

The Blood Brain Barrier (BBB) consists of cerebral microvascular Endothelial Cells (ECs) that control the process of substance flow into and out of the brain. Since CPPs can enhance transduction of AAV9 vectors to EC and HA cells, it is speculated that CPPs can also enhance transduction of AAV9 vectors across the BBB in the CNS. The present invention injects Phosphate Buffered Saline (PBS), AAV9 vector and AAV9-LAH4 complex into different groups of mice by Intravenous (IV) injection, and after two weeks, the GFP fluorescence in liver and brain tissues of the mice is examined by immunohistochemistry. As shown in fig. 8A and 8B, the mice injected with the PBS group had weak background fluorescence in both brain and liver, whereas the mice injected with AAV9 vector had slightly elevated fluorescence levels in both brain and liver; GFP fluorescence was increased by about 100% in both brain and liver in mice injected with AAV9-LAH4 complex compared to mice injected with AAV9 vector alone.

To confirm the results, mRNA levels and DNA levels in brain and liver were also examined by RT-qPCR. As shown in fig. 8C, GFP DNA copy number was increased by about 270% and 400% in mice injected with AAV9-LAH4 complex, respectively, compared to mice injected with AAV9 alone; as shown in fig. 8D, GFP mRNA levels were increased by about 70% and 340% in mice injected with AAV9-LAH4 complex, respectively, compared to mice injected with AAV 9. In summary, LAH4 peptide enhances expression of target genes in brain and liver delivered by AAV9 by systemic administration in vivo.

Example 8: LAH4 peptides reduce immune responses to AAV9 transduction in the brain

Systemic administration of AAV generally leads to an immune response, which leads to a decrease in the efficiency of gene therapy and increased safety concerns for gene therapy, and to demonstrate whether the immune response increases with higher transduction of AAV9 in the brain, mice injected with AAV9-CPP complex were examined for immune response profiles, two inflammatory factors were examined by RT-qPCR: mRNA levels of interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha). As shown in FIG. 9A, the mRNA levels of IL-1 β and TNF- α in the liver of mice injected with AAV9-LAH4 were approximately 130% and 140% of the liver of mice injected with AAV9 alone, respectively, demonstrating that LAH4 peptide increases the immune response of the liver; however, the mRNA levels of IL-1 β and TNF- α in the brain of mice injected with AAV9-LAH4 were approximately 55% and 60% of those of mice injected with AAV9, respectively, demonstrating that LAH4 peptide may reduce the immune response transduced by AAV9 in the brain. This result was further confirmed by the reduced levels of CD4 and CD8 in the brain of mice injected with AAV9-LAH4, and by the reduced levels of CD4 and CD8 in the liver of mice injected with AAV9-LAH4, as shown in figures 9B and 9C, compared to mice injected with AAV9 alone.

Example 9: mechanism for AAV9-CPP complex entry into cells

Clathrin, caveolin and macroendocytic pathways are the main pathways for virus, protein and nanoparticle endocytosis, and in order to evaluate the effects of the three endocytic pathways in the entry of AAV9-CPP complex into cells, drug inhibition experiments were performed to test the effects of different inhibitors on AAV9 or AAV9-LAH4 infected cells and virus transduction. Selected inhibitors include: chlorpromazine (CPZ), Amiloride (Amiloride) which can block clathrin-mediated endocytosis, and Ertythrina Cristagalli Lectin (ECL) which inhibits entry of AAV9 and AAV9-LAH4 into EC cells by preventing induction of membrane ruffles, and Amiloride which inhibits AAV9 from its receptor by inhibiting macrocyclic cytidase lectin.

First, EC cells were pretreated with different inhibitors, respectively, followed by infection of inhibitor-treated and untreated cells with AAV9 or AAV9-LAH4 complex alone (final concentration 5 μ M), total protein extraction and determination of GFP expression levels of EC cells by Western blot, and with β -actin as control, all treatments were repeated three times (P <0.05, P < 0.01). FIGS. 10A and 10B show the results of preincubation of EC cells with chlorpromazine (15 nmol/L) or amiloride (1 mmol/L) for 30 min at 37 deg.C, respectively, as shown, amiloride did not inhibit transduction of AAV9 in EC cells, chlorpromazine did, and amiloride and chlorpromazine both significantly inhibited the transduction efficiency of AAV9-LAH4 into EC cells, showing that the endocytosis of AAV9-LAH4 plays an important role in this process; fig. 10C and 10D are results of preincubation of EC cells with ECL (50 ug/mL) at 4 ℃ for 15 minutes, as shown, ECL significantly inhibited AAV9 and AAV9-LAH4 from entering EC cells, showing that AAV9 receptor also plays an important role in AAV9-CPP entry into cells.

All documents referred to herein are incorporated by reference in their entirety. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the above teachings of the present invention, and such equivalent modifications also fall within the scope of the appended claims.

Sequence listing

<110> Nanjing Beisiao Biotechnology Ltd

<120> method for enhancing transduction efficiency of adeno-associated virus in central nervous system using cell-penetrating peptide

<141> 2021-01-27

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Claims

1. A method of delivering a transgene to the central nervous system, comprising administering:

(i) a cell-penetrating peptide selected from LAH4, LEPTIN30, APOE, or a combination thereof;

(ii) a recombinant adeno-associated viral vector which is adeno-associated virus type 9 or any mutant thereof.

2. The method of claim 1, wherein the LAH4 comprises SEQ ID NO: 1.

3. The method of claim 1, wherein the APOE comprises the amino acid sequence of SEQ ID NO: 2.

4. The method of claim 1, wherein said LEPTIN30 comprises the amino acid sequence of SEQ ID NO: 3.

5. The method of claim 1, wherein the adeno-associated virus type 9 comprises the chicken β -actin promoter.

6. The method of claim 1, wherein the adeno-associated virus type 9 and the cell-penetrating peptide form a complex.

7. The method of claim 1, wherein the recombinant adeno-associated viral vector is capable of crossing the blood-brain barrier.

8. The method of claim 1, wherein the transgene is capable of being expressed in human brain endothelial cells and glial cells.

9. A composition comprising the cell-penetrating peptide of claim 1 and a recombinant adeno-associated viral vector.