CN118743768A - A platelet-AAV packaging complex and its preparation method and application - Google Patents

Abstract

The present invention relates to the field of biomedicine, and in particular to a platelet-AAV packaging complex and its preparation method and application. The present invention provides a method for preparing a platelet-AAV packaging complex, comprising the steps of mixing platelets and adeno-associated viruses, performing oscillation treatment, and obtaining the platelet-AAV packaging complex. The present invention packages AAV with platelets, and utilizes the homing characteristics of platelets themselves to target damaged sites in the body. For example, in injuries such as myocardial ischemia or myocardial infarction, platelets carry the AAV packaged therein and will actively enrich to the damaged site, further improving the targeting of AAV gene therapy delivery. At the same time, packaging AAV with platelets and delivering it can protect AAV from attacks by the immune system in the body.

Description

A platelet-AAV packaging complex and its preparation method and application

Technical Field

The present invention relates to the field of biomedicine, and in particular to a platelet-AAV packaging complex and a preparation method and application thereof.

Background Art

Adeno-associated virus (AAV) belongs to the Parvoviridae family. It has no envelope and consists of a single-stranded DNA genome wrapped in an icosahedral capsid protein with a diameter of about 26 nm. According to the different compositions of AAV capsid proteins, AAV is divided into different serotypes, which have different tropisms for different organs and tissues. For example, recombinant AAV2/9 has high tropism for the nervous system, myocardium, lungs and other tissues. When used for gene therapy, artificial recombination technology can be used to construct AAV (recombinant adeno-associated virus) carrying exogenous genes with therapeutic effects. After that, this AAV product can be injected into the human body, and human cells can be transfected by AAV to transport the therapeutic gene fragments into the patient's cells to exert a therapeutic effect. When constructing AAV, its capsid protein can also be mutated and modified to change its tropism for different tissues or reduce immunogenicity; or AAV can be packaged in artificial or natural carriers (such as vesicles) to improve safety or efficacy.

At present, there are still two major problems in the delivery of gene therapy with AAV: insufficient targeting and insufficient effectiveness. There are two main reasons for this. First, after AAV enters the body, it is inevitable that off-target effects will occur, that is, infection of non-target organs. In particular, AAV has a natural enrichment in the liver, and the liver toxicity it causes can lead to serious adverse reactions. If the treatment itself is targeted at the liver, a smaller dose of AAV can complete the delivery of gene therapy, but if it is targeted at other organs and tissues, a larger dose of AAV is required, which increases the burden on the liver. Second, natural AAV (wild-type AAV) is widely present in the natural environment. Studies have found that among the general population over the age of 2, a considerable proportion of people have already developed antibodies against AAV capsid protein due to exposure to wild-type AAV in their lives. The presence of this antibody will cause the antibodies in the body to attack the AAV vector when receiving gene therapy using AAV as a vector in the future, thereby weakening the treatment effect, or more seriously, causing a strong immune response and resulting in serious adverse consequences. Moreover, if the patient has already received gene therapy delivered by AAV, the anti-AAV antibodies produced in the body will interfere with subsequent treatments that may be needed. It can be seen that when using AAV to deliver gene therapy, the human immune system is very likely to attack foreign AAV, thereby weakening the effectiveness of AAV-delivered gene therapy. If the AAV dose is increased to enhance the therapeutic effect, it will bring greater safety issues.

Current approaches to addressing the above difficulties include: a) selecting patients with low levels of anti-AAV antibodies from the outset to administer AAV gene therapy, but due to the widespread presence of anti-AAV antibodies in the population, this will exclude many patients who need treatment; b) using immunosuppressants to suppress the patient's immune system, thereby protecting the introduced AAV vector, but suppressing the immune system will bring other risks, such as infection; c) repeatedly performing plasma exchange to reduce the level of anti-AAV antibodies in the body, but this method requires multiple plasma exchanges and cannot completely eliminate high levels of anti-AAV antibodies.

This shows that there is an urgent need to develop more targeted delivery therapies to reduce the viral dose required for treatment and improve efficacy.

Summary of the invention

The purpose of the present invention is to provide a platelet-AAV packaging complex and its preparation method and application to solve the problems existing in the above-mentioned prior art. The present invention uses platelets to package AAV, thereby effectively protecting AAV from the attack of the body's immune system, thereby reducing the viral dose required for treatment and improving the efficacy.

To achieve the above object, the present invention provides the following solutions:

The present invention provides a method for preparing a platelet-AAV packaging complex, comprising the following steps:

The platelets and adeno-associated virus are mixed and then shaken to obtain the platelet-AAV packaging complex.

Preferably, the ratio of the number of platelets to adeno-associated virus is 1:(50-100).

Preferably, the shaking treatment is carried out at a temperature of 37° C. for 1-2 h.

The present invention provides a platelet-AAV packaging complex prepared by the above-mentioned preparation method.

The present invention provides the use of the platelet-AAV packaging complex in preparing a drug delivery system.

The present invention provides a drug delivery system, wherein the carrier of the drug delivery system is the platelet-AAV packaging complex.

The present invention provides application of the above-mentioned drug delivery system in preparing drugs.

Preferably, the drug comprises a drug for treating heart disease.

Preferably, the heart disease includes myocardial ischemia or myocardial infarction.

The present invention discloses the following technical effects:

The present invention obtains a platelet-AAV packaging complex by packaging AAV with platelets, and utilizes the platelet's own homing characteristics to target the damaged site in the body. For example, in injuries such as myocardial ischemia or myocardial infarction, the platelets carry the AAV packaged therein and will actively enrich to the damaged site, further improving the targeting of AAV gene therapy delivery, reducing the AAV dose required for gene therapy, and reducing toxicity. At the same time, packaging AAV with platelets and delivering it can protect AAV from attack by the body's immune system.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

Figure 1 is a stratified diagram of whole blood after centrifugation;

FIG2 is an electron micrograph of the recombinant adeno-associated virus prepared in Example 1 and naked AAV without platelet packaging; (a) is an electron micrograph of the recombinant adeno-associated virus prepared in Example 1; (b) is a partial enlarged view of (a); (c) is an electron micrograph of naked AAV without platelet packaging;

FIG3 is a schematic diagram of the synthesis of platelets as carriers for packaging recombinant adeno-associated viruses;

FIG4 is an electron micrograph of the recombinant adeno-associated virus prepared in Example 2; the arrow points to the portion of the AAV that has been swallowed;

FIG5 is an electron micrograph of the recombinant adeno-associated virus prepared in Comparative Example 1;

FIG6 is a fluorescence imaging diagram.

DETAILED DESCRIPTION

Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and/or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

Platelets are cell fragments derived from megakaryocytes and play an important role in hemostasis, thrombosis, atherosclerosis, angiogenesis, tumor growth and metastasis. The mechanism by which platelets can play a role in these processes depends on the receptors on the surface of platelets, which enable platelets to recognize and adhere to sites where vascular integrity is impaired; on the other hand, it depends on the unique α granules, dense granules and open canalicular system (OCS) of platelets, which can store a variety of active substances and release them after platelets are activated to mediate corresponding functions. Recent studies have found that platelets can not only store substances derived from megakaryocytes or synthesized by themselves, but also use their unique OCS to "swallow" certain foreign substances from the outside world, including proteins, bacteria, and viruses, which can be released after platelets are activated. These characteristics enable us to develop new drug delivery systems around platelets.

Sources of related substances in the examples:

ACD anticoagulant: manufacturer is Sigma-Aldrich (Sigma-Aldrich (Shanghai) Trading Co., Ltd.), brand number C3821;

Prostaglandin E1 (PGE1): manufacturer is MedChemExpress (Shanghai Haoyuan Pharmaceutical Co., Ltd. (agent)), brand number is HY-B0131;

Tyrode’s buffer: manufacturer is Wuhan Pronocell Life Science Co., Ltd., brand number is PB180341;

PBS buffer: The manufacturer is Wuhan Saiweier Biotechnology Co., Ltd., brand number G4200;

HEPES buffer solution: the manufacturer is Wuhan Saiweier Biotechnology Co., Ltd., the brand is G4210-100mL;

Bovine serum albumin (BSA): manufacturer: MedChemExpress (Shanghai Haoyuan Pharmaceutical Co., Ltd. (agent)), brand: HY-D0842;

Glucose: The manufacturer is MedChemExpress (Shanghai Haoyuan Pharmaceutical Co., Ltd. (agent)), the brand number is HY-B0389.

Example 1

A method for preparing a recombinant adeno-associated virus packaged with platelets as carriers, the schematic diagram of which is shown in FIG3 (in the schematic diagram, it can be seen that platelets engulf AAV into OCS to form a platelet-AAV packaging complex), and the specific steps are as follows:

Obtain human platelets or extract platelets from whole blood as follows:

Collect the whole blood in a test tube containing 1/10 volume of ACD anticoagulant, mix gently, and then centrifuge to separate it into 3 layers, as shown in Figure 1. The centrifugation conditions have a large variable range. At room temperature, such as from 800×g for 5min to 100×g for 20min, different centrifugation conditions can produce stable and good separation effects. In this embodiment, 800×g for 5min is selected. Then carefully absorb the top platelet-rich plasma (PRP) layer liquid and transfer it to a new test tube. When absorbing PRP, avoid absorbing the lower 2 layers of liquid. Add HEPES buffer solution to the absorbed PRP to make the final concentration of HEPES 1.9mM (the final concentration of HEPES can achieve the same effect between 1-30mM), add PGE1, and make the final concentration of PGE1 1μM to prevent premature activation of platelets. After gentle mixing, centrifuge at room temperature for 20min at 800×g, discard the supernatant, and the white precipitate at the bottom is the platelet precipitate. Gently resuspend the platelet pellet with Tyrode’s buffer, and add glucose and BSA to make the final concentration of glucose 5mM and BSA 3mg/mL. Then, PGE1 can be added to make the final concentration of PGE1 1μM to prevent platelet activation. Thus, a platelet suspension is obtained.

The AAV to be packaged is added to the platelet suspension, and the ratio of platelets to AAV is 1:50; the same effect can be achieved when the ratio of platelets to AAV is within the range of 1:50-1:100.

Gently shake the platelet suspension containing AAV at 37°C for 2 hours (the same effect can be achieved within 1-2 hours) to obtain platelet-packaged AAV, that is, recombinant adeno-associated virus packaged with platelets as carriers, also known as platelet-AAV packaging complex. The platelet-packaged AAV should be used as soon as possible.

Then, the platelet-packaged AAV and the naked AAV without platelet packaging were subjected to conventional resin embedding and fixation to prepare ultrathin sections. The images under a transmission electron microscope are shown in Figure 2. It can be seen that after the platelets engulf the AAV, a platelet-AAV packaging complex is formed (a and b); the image under a transmission electron microscope of the naked AAV without platelet packaging loaded on a copper mesh after negative staining is shown in Figure 2 (c).

Example 2

A method for preparing a recombinant adeno-associated virus packaged with platelets as carriers, wherein the specific steps are the same as those in Example 1, except that the ratio of platelets to AAV is 1:100.

The platelet-packaged AAV was then routinely embedded and fixed with resin to produce ultrathin sections. The image under a transmission electron microscope is shown in Figure 4 , which shows that the platelets were able to successfully engulf the AAV to form a platelet-AAV packaging complex.

Comparative Example 1

Obtain human platelets, or extract platelets from whole blood according to the steps in Example 1.

The AAV to be packaged is added to the platelet suspension in a ratio of platelets to AAV = 1:10.

The platelet suspension containing AAV was gently shaken at 37°C for 2 hours, and then the sample was routinely embedded in resin and fixed. The ultrathin sections were made under a transmission electron microscope as shown in Figure 5. No AAV particles were found in the platelets. Because AAV is a very small virus with a diameter of only about 26-30nm, when the ratio of platelets to AAV is too low, the contact between the two is reduced when the platelets and AAV are shaken in a mixed liquid, thereby reducing the efficiency of platelets in capturing and engulfing AAV, so that AAV particles cannot be detected in most platelets.

Experimental Example 3 Targeted Detection

Experimental animals: 30 C57BL6 mice were randomly divided into 3 groups. The average body weight of each group was 20 g. There was no statistical difference in body weight between and within groups.

AAV used in the experiment: Commercial AAV expressing EGFP fluorescent protein, purchased from Heyuan Biotechnology (Shanghai) Co., Ltd.

Group setup and treatment: Set up 3 groups of mice as follows:

(1) Sham operation + platelet-AAV group: After gas inhalation anesthesia, mice were intubated and connected to a ventilator to maintain anesthesia. After thoracotomy, no cardiac treatment was performed and the chest was sutured immediately. After surgery, the platelet-AAV complex formed by platelet-packaged AAV was injected into the tail vein of mice at an injection volume of 2.5×10 ¹¹ vg AAV/mouse.

(2) IR+AAV group: After anesthesia as described in the sham operation+platelet-AAV group, the mice were subjected to ischemia-reperfusion (IR) injury surgery after thoracotomy. The left anterior descending branch of the coronary artery was ligated with a slipknot for 40 minutes and then released to simulate the condition of cardiac ischemia-reperfusion injury. After the operation, AAV without special packaging was injected into the tail vein of mice at an injection volume of 2.5×10 ¹¹ vg AAV/mouse.

(3) IR+platelet-AAV group: As described for the IR+AAV group, mice underwent IR surgery. After surgery, platelet-AAV complexes formed by AAV packaged with platelets (platelet-AAV complexes prepared in Example 1) were injected through the tail vein of mice, with an injection volume equivalent to 2.5×10 ¹¹ vgAAV/mouse (equivalent means that the corresponding amount of solution containing 2.5×10 ¹¹ vgAAV is calculated based on the ratio of platelet to AAV number of 1:50 in Example 1).

Four weeks after the operation, mice in each group were killed, and the hearts, livers, kidneys and spleens were obtained for fluorescence imaging detection. The results are shown in Figure 6. Compared with the sham operation + platelet-AAV group and the IR + AAV group, the IR + platelet-AAV group showed obvious fluorescence reaction at the IR injury site of the heart, indicating that platelets have played the role of enrichment at the injury site. Therefore, after heart injury, platelet-packaged AAV can better target the heart compared with the existing technology.

Experimental Example 4: Platelet-AAV gene overexpression therapy improves myocardial ischemia-reperfusion injury

Preparation of platelet-packaged AAV:

Obtain human platelets, or extract platelets from whole blood according to the steps in Example 1.

The AAV encoding the overexpressed SERCA2a gene to be packaged (disclosed in "Extracellular Vesicle-Encapsulated Adeno-Associated Viruses for Therapeutic Gene Delivery to the Heart" (Li X, La Salvia S, Liang Y, et al. Extracellular Vesicle-Encapsulated Adeno-Associated Viruses for Therapeutic Gene Delivery to the Heart. Circulation. 2023; 148(5): 405-425. doi: 10.1161/CIRCULATIONAHA.122.063759)) is added to the platelet suspension, and the number ratio of platelets to AAV encoding the overexpressed SERCA2a gene is 1:50; the same effect can be achieved when the number ratio of platelets to AAV is in the range of 1:50-1:100.

The platelet suspension containing AAV is gently shaken at 37°C for 2 hours (the same effect can be achieved within the range of 1-2 hours) to obtain platelet-packaged AAV.

Experimental animals: 16 C57BL6 mice were randomly divided into 4 groups. The average body weight of each group was 20 g. There was no statistical difference in body weight between and within groups.

Group setup and treatment: Set up 4 groups of mice as follows:

(1): Control group without IR injury.

(2): IR injury group.

(3): The conventional SERCA2a overexpression AAV treatment group after IR injury, i.e., direct injection of AAV encoding the overexpressed SERCA2a gene.

(4): Platelet-SERCA2a overexpression AAV treatment group after IR injury, i.e., the platelet-packaged AAV constructed above was injected.

The mice in the IR injury group, the common SERCA2a overexpression AAV treatment group after IR injury, and the platelet-SERCA2a overexpression AAV treatment group after IR injury were given gas inhalation anesthesia, and the mice were intubated and connected to a ventilator to maintain anesthesia. After thoracotomy, ischemia-reperfusion (IR) injury surgery was performed, and the left anterior descending branch of the coronary artery was ligated with a slipknot for 40 minutes and then released to simulate the condition of cardiac ischemia-reperfusion injury.

After surgery, mice in the SERCA2a overexpression treatment group after IR injury and the platelet-SERCA2a overexpression AAV treatment group after IR injury were injected with unpackaged therapeutic AAV or platelet-packaged therapeutic AAV (platelet-AAV complex) prepared in this experimental example through the tail vein, with the same dose of 2.5×10 ¹¹ vgAAV/mouse.

After 4 weeks, the mice were subjected to echocardiography for cardiac function test. The results are shown in Table 1 below. It can be seen that the platelet-AAV prepared in this experimental example has a better cardiac function protection effect than ordinary AAV.

Table 1 Transthoracic echocardiography

Note: EF is ejection fraction; FS is shortening fraction; CO is cardiac output; LVIDd is left ventricular internal diameter at end diastole; *p<0.05 compared with the control group without IR injury; #p<0.05 compared with the IR surgical injury group; ##p<0.05 compared with the group treated with common SERCA2a overexpression AAV after IR injury.

The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims (9)

1. A method of preparing a platelet-AAV packaging complex comprising the steps of:

Mixing the platelet and the adeno-associated virus, and then carrying out oscillation treatment to obtain the platelet-AAV packaging complex.

2. The method of claim 1, wherein the ratio of the number of platelets to adeno-associated virus is 1: (50-100).

3. The method according to claim 1, wherein the temperature of the shaking treatment is 37 ℃ for 1-2 hours.

4. A platelet-AAV packaging complex prepared by the method of any one of claims 1-3.

5. Use of the platelet-AAV packaging complex of claim 4 in the preparation of a drug delivery system.

6. A drug delivery system, wherein the carrier of the drug delivery system is the platelet-AAV packaging complex of claim 4.

7. Use of the drug delivery system of claim 6 for the manufacture of a medicament.

8. The use according to claim 7, wherein the medicament comprises a medicament for treating a heart disease.

9. The use according to claim 8, wherein the heart disease comprises myocardial ischemia or myocardial infarction.