HK1208046B - A novel drug delivery system based on jcv-vlp - Google Patents

Description

A novel drug delivery system based on JCV-virus-like particles

Technical Field

The present invention relates to the use of human polyomavirus-like virus-like particles (VLPs) as a drug delivery system.

Technical Background

A huge challenge in the field of modern medicine is drug delivery. Drug delivery to selected site of action (for example, the microchamber of selected organ, tissue, cell type or cell, etc.) is called drug targeting. Even if systemic medication is used, it is possible to increase the drug concentration of this specific site by drug targeting. In addition, relative to common systemic medication, other parts of health are not or only exposed to the drug to a lower degree. This causes the risk of adverse side effects to be reduced and allows higher doses of medicine. Further, extremely toxic drugs (for example, cytotoxic agents for cancer treatment) may be used for the mankind for the first time because their toxic side effects are minimized by drug delivery systems. In some cases, because the drug is protected by the delivery method, the other advantage of drug targeting is to prevent the premature inactivation (metabolism), undesirable absorption, excretion or undesirable modification of the drug.

Drug delivery to the central nervous system (CNS), especially the brain, is a significant challenge, as the active ingredient must first cross the blood-brain barrier and then reach the target cells.

The blood-brain barrier is formed by the capillary endothelium. These endothelial cells are tightly connected to each other by so-called tight junctions, preventing substances above a certain molecular weight from entering the central nervous system. The blood-brain barrier thus provides an effective protective barrier. While ensuring the supply of nutrients to the central nervous system, it also enables the removal of metabolic products from the system.

Transporting hydrophilic substances across the blood-brain barrier is a major concern. Nearly 95% of all in vitro effective drug candidates are unable to cross the blood-brain barrier at pharmacologically active concentrations. Therefore, in order to achieve sufficient pharmacological concentrations of the drug in the central nervous system, the treatment of many central nervous system diseases, such as brain tumors or central nervous system infections, requires high plasma concentrations of the drug. This involves the risk of adverse side effects. Therefore, in pharmaceutical research, ways to improve the transport of drugs (especially hydrophilic substances) across the blood-brain barrier into the central nervous system are sought.

Some approaches use lipophilic particles that allow receptor-mediated transport across the blood-brain barrier. For this purpose, for example, nanoparticle delivery systems have been developed. Nanoparticles are typically composed of polymers and range in size from 10 to 1000 nanometers. They are often surface-modified.

These nanoparticles often face the problem of being expelled from the central nervous system by efflux pumps expressed in the blood-brain barrier. Furthermore, nanoparticles have been shown to affect the integrity of the blood-brain barrier (Rempe et al., Biochem Bioph Res Comm 2011, 406(1): 64-69). Consequently, the protective function of the blood-brain barrier is put at risk, including the risk of unwanted or infectious substances entering the central nervous system and causing side effects. This significant disadvantage is crucial for the clinical application of nanoparticles as drug delivery systems.

Other possible drug delivery systems are under discussion. For example, it is known that a pseudocapsid derived from the mouse polyomavirus VP1 protein bound to DNA encoding β-galactosidase can be used to deliver the DNA to the brain after intravenous administration, resulting in the expression of β-galactosidase in the brain (Krauzewicz et al., Gene Therapy 2000, 7, 1094-1102; see also WO 98/48841 A1). However, these findings do not provide a full understanding of the suitability of virus-like particles, especially those derived from human polyomavirus and loaded with cargo, as drug delivery systems for the central nervous system.

It is therefore an object of the present invention to provide a drug delivery system that allows drug transport to cells of the central nervous system, ie, drug delivery across the blood-brain barrier.

SUMMARY OF THE INVENTION

The inventors discovered that in a blood-brain barrier model based on primary porcine endothelial cells, virus-like particles (VLPs) derived from human JC virus, loaded with nucleic acid encoding a reporter protein, were able to cross the BBB and deliver the nucleic acid to cells of the central nervous system, where it was expressed. This model is believed to represent a physiologically intact BBB in vivo. Thus, it was demonstrated that VLPs, not just active substances alone, can cross the BBB and enter the central nervous system. Therefore, VLPs derived from human polyomavirus and loaded with a cargo are suitable as drug delivery systems for entering the central nervous system, particularly the brain.

The inventors have further discovered that the passage of drug-loaded virus-like particles does not require, nor does it result in, a loss of blood-brain barrier integrity or increased permeability. Thus, virus-like particles do not substantially affect the integrity of the blood-brain barrier. Therefore, virus-like particles can be used as a drug delivery system for access to the central nervous system without the significant risk of adverse side effects caused by unwanted or infectious agents entering the central nervous system.

According to the present invention, virus-like particles derived from human polyomavirus and loaded with a cargo are used to cross the blood-brain barrier together with the cargo. Importantly, passage of the virus-like particles through the blood-brain barrier enables the virus-like particles to demonstrate their ability to target specific cell populations in the brain, i.e., deliver the cargo to the target cells. In the context of the present invention, the delivery includes delivery to the target cells, delivery to the interior of the target cells, or delivery into the target cells. Thus, according to the present invention, functional virus-like particles and the cargo are passed through the blood-brain barrier together.

The cargo, preferably the active substance, is preferably completely encapsulated within the capsid of the virus-like particle. However, this does not exclude that the virus-like particle may additionally be bound to the active substance, for example, by binding the active substance to the outer structure of the capsid. Some molecules of the active substance may not even be completely encapsulated within the capsid. However, at least a portion of the active substance molecules are completely encapsulated within the capsid.

Therefore, according to the present invention, a composition of virus-like particles from human polyomavirus and an active substance is provided for use as a drug delivery system, wherein at least 1%, at least 2%, at least 3%, at least 5%, at least 7%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, more preferably at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% of the total amount of the active substance (load) is encapsulated within the capsid of the virus-like particle.

In the most preferred embodiment of the present invention, the virus-like particles of the composition are non-aggregating. Non-aggregating means that the virus-like particles are able to form dispersed particles when suspended in water.

Therefore, one aspect of the present invention provides a drug delivery system for the central nervous system that allows for the co-transport of virus-like particles and active substances. Transport can be active or passive. Interestingly, the inventors have further discovered that virus-like particles are not or substantially not cleared from the central nervous system by efflux transporters of the blood-brain barrier.

The virus-like particles of the present invention do not require surface treatment or the use of adjuvants to effectively cross the blood-brain barrier and enter the central nervous system. Therefore, in one aspect of the present invention, the drug delivery system has a microparticle structure and does not require any further surface treatment or modification, nor the use of adjuvants. The drug delivery system is preferably acellular.

As described above, according to the present invention, virus-like particles are used for drug delivery into the central nervous system, particularly into the brain. This delivery into the brain enables drugs to be targeted to specific cells, for example, by utilizing the natural chemotaxis of human polyomaviruses (particularly JC virus).

Detailed Description of the Invention

Virus-like particles (VLPs) derived from human polyomaviruses, in particular JC virus, are used according to the present invention for drug delivery, preferably containing at least one VP1. The VLP is preferably composed of a capsid constructed from VP1 assembled into a pentamer structure. Preferably, the VLP is composed of several VP1s, in particular several VP1 pentamers, especially 72 VP1 pentamers. However, the VLP may optionally contain other molecules inserted into the capsid. The structural molecules assembled into the VLP may be identical to the native polyomavirus proteins or may be modified to optimize the characteristics of the VLP.

Further, VLP according to the present invention also contains a loaded cargo. In a particularly preferred embodiment of the present invention, the major part of the total cargo amount is fully encapsulated in the capsid. In order to describe that the cargo molecule is fully encapsulated by the VLP, the term "loaded" is used. Therefore, "loaded VLP" refers to a VLP with a fully encapsulated cargo.

The loading material can be any molecule or composition contained in the space surrounded by the capsid. Preferably, the loading material is a cytotoxic agent, a detectable agent such as a radionuclide, a protein, a peptide or a nucleic acid, in particular selected from the group consisting of nucleic acids encoding a desired protein such as mRNA, cDNA, a plasmid or a vector, inhibitory nucleic acids such as siRNA or miRNA, and nucleic acids with catalytic activity such as ribozymes. The loading material is sometimes referred to as "active substance", "drug substance" or "active ingredient" hereinafter, and unless otherwise expressly indicated, these terms are used as synonyms.

In some embodiments, VLP is prepared by combining the components of the present invention with those of ordinary skill in the art. For example, VLP can be assembled into VLP by adding a plurality of different components, for example ^VP1 , ^VP2 , VP3 or their combination and a plurality of different loading materials ...

The term "human polyomavirus" refers to the human polyomavirus family, including JC virus, BK and SV40. In a particularly preferred embodiment, the human polyomavirus is JC virus.

According to the present invention, "VP1" or "viral protein 1" refers to a protein that is identical to or derived from the native VP1 of JC virus having the amino acid sequence set forth in SEQ ID NO: 1. A protein derived from the native VP1 of JC virus preferably has at least 60%, more preferably at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% amino acid sequence homology or identity with the amino acid sequence set forth in SEQ ID NO: 1 over a sequence of at least 100 consecutive amino acids, preferably at least 150, at least 200, at least 250, or at least 300 consecutive amino acids. More preferably, the amino acid homology or identity can be calculated over the entire length of the native JC virus VP1. The terms "VP1 derived from the native VP1 of JC virus" and "VP1 derived from JC virus" specifically also include VP1 that is identical to the native VP1 of JC virus.

According to the present invention, the term "VP1" also includes fragments and derivatives of natural VP1 that can be assembled into VLPs. Preferably, the fragments and derivatives of VP1 comprise at least amino acids 32 to 316 of the amino acid sequence of SEQ ID NO: 1, or derivatives thereof that have at least 60%, more preferably at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% homology or identity with the amino acid sequence at amino acid positions 32 to 316 of SEQ ID NO: 1 over a sequence of at least 100 consecutive amino acids, preferably at least 150, at least 200, at least 250, or at least 300 consecutive amino acids, and preferably over the entire sequence.

According to the present invention, VP1 may also include a heterologous nuclear localization signal (NLS). Preferably, the NLS is introduced before or within the N-terminus of VP1, particularly within the first 30, first 25, first 20, first 15 or first 10 amino acids of VP1. For example, NLSs described in WO 2009/036933 (e.g., page 10, lines 4 to 13 and Figure 4A) or Shishido-Hara et al. (Shishido-Hara, Y., Hara, Y., Larson, T., Yasui, K., Nagashima, K. & Stoner, G.L. Analysis of Capsid Formation of Human Polyomavirus JC (Tokyo-1 Strain) by a Eukaryotic Expression System: Splicing of Late RNAs, Translation and Nuclear Transport of Major Capsid Protein VP1, and Capsid Assembly. Journal of Virology 74, 1840-1853 (2000).

According to one embodiment, the amino acid sequence shown in SEQ ID NO: 5 is introduced into the N-terminal portion of VP1, in particular, between the 9th and 10th amino acids shown in SEQ ID NO: 1.

According to the present invention, "VP2" or "viral protein 2" refers to a protein that is identical to or derived from the native VP2 of JC virus having the amino acid sequence set forth in SEQ ID NO: 3. A protein derived from the native VP2 of JC virus preferably has at least 60%, more preferably at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% amino acid sequence homology or identity with the amino acid sequence set forth in SEQ ID NO: 3 over a sequence of at least 100 consecutive amino acids, preferably at least 150, at least 200, at least 250, or at least 300 consecutive amino acids. More preferably, the amino acid homology or identity is calculated over the entire length of the native JC virus VP2. The terms "VP2 derived from the native VP2 of JC virus" and "VP2 derived from JC virus" specifically also include VP2 that is identical to the native VP2 of JC virus.

According to the present invention, the term "VP2" also includes fragments and derivatives of natural VP2 that can be assembled into VLPs together with VP1. Preferably, the VP2 fragment comprises at least amino acids 214 to 318 of the amino acid sequence of SEQ ID NO: 3, or a derivative thereof having at least 60%, more preferably at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% homology or identity with the amino acid sequence at amino acid positions 214 to 318 of SEQ ID NO: 3 over a sequence of at least 100 consecutive amino acids, preferably at least 150, at least 200, at least 250, or at least 300 consecutive amino acids, preferably over the entire sequence.

According to the present invention, a "peptide" can be composed of any number of amino acids of any type, preferably naturally occurring amino acids, which are preferably connected by peptide bonds. In particular, the peptide contains at least 3 amino acids, preferably at least 5, at least 7, at least 9, at least 12 or at least 15 amino acids. Furthermore, there is no upper limit on the length of the peptide. However, the peptides of the present invention preferably do not exceed 500, preferably 300, 250, 200, 150 or 120 amino acids in length.

As used herein, the expression "comprising," in addition to its literal meaning, also includes, and specifically refers to, the semantics of "consisting essentially of" and "consisting of." Thus, the expression "comprising" refers to both embodiments in which the subject matter specifically comprising the listed elements does not contain other elements, and embodiments in which the subject matter specifically comprising the listed elements can and/or does contain other elements. Similarly, the expression "having" should be understood as meaning "comprising," also including, and specifically referring to, the semantics of "consisting essentially of" and "consisting of."

Application method

The VLPs of the present invention can be administered by various routes. Particularly preferred are dosage forms that allow systemic effects of the active substance. Most preferred are oral or parenteral administration, especially intravenous administration.

Preparation method

Preparation of virus-like particles

In another aspect, the present invention provides a method for preparing virus-like particles according to the present invention. The method comprises, in particular, the following steps:

(a) providing viral protein VP1 derived from JC virus;

(b) optionally providing viral proteins VP2 and/or VP3 derived from JC virus, preferably VP2, and mixing VP1 and VP2 (and/or VP3);

(c) allowing VP1 and optionally VP2 (and/or VP3) to assemble into virus-like particles.

Preferably, the method further comprises the steps of providing a loading material and mixing VP1 and optionally VP2 and/or VP3 with the loading material. Preferably, VP1 and VP2 are mixed.

When VLPs are assembled, the cargo is preferably encapsulated within the interior of the VLP. Preferably, at least one VLP carries the cargo, and more preferably, at least 1%, at least 2%, at least 3%, at least 5%, at least 7%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% or 100% of the assembled VLPs carry the cargo.

Assembly of the virus-like particles preferably occurs in solution, more preferably in an aqueous solution. Enabling VLP assembly preferably includes adjusting the Ca ²⁺ ion concentration in the solution to a level at which VLP assembly can occur. The Ca ²⁺ ion concentration is particularly in the range of 0.1 mM to 5 mM, preferably from 0.2 mM to 3 mM, more preferably from 0.5 mM to 2 mM or 0.8 mM to 1.2 mM, and most preferably about 1 mM. Further, allowing VLP assembly to occur under oxidizing conditions, particularly in the absence of significant concentrations of reducing agents such as DTT, DTE, or mercaptoethanol.

Providing viral protein and allowing VLP to assemble can be carried out simultaneously.Especially, supply viral protein under the condition that VLP assembling can take place.In a preferred embodiment, providing viral protein and VLP assembling to carry out in vivo.Especially, VLP is assembled when expressing host cell inside of viral protein or cracking or destroying host cell.

For example, assembly of VLPs may be performed as described in EP 0 862 633, the disclosure of which is incorporated herein by reference.

Delivery Method

The present invention further provides a method for delivering a substance or composition to a target cell in the central nervous system using the virus-like particles of the present invention. The method preferably comprises the following steps:

(a) providing a virus-like particle of the present invention, which comprises a substance or composition as a cargo; and

(b) administering the virus-like particle into a living body, preferably into a human body.

The target cell may be a natural target cell of the JC virus.

The active substance or composition can be of any type or nature. In a preferred embodiment, the active substance is a nucleic acid, particularly a nucleic acid encoding a protein or peptide, or an inhibitory nucleic acid such as siRNA or miRNA. In another preferred embodiment, the active substance is a protein or peptide. In yet another embodiment of the present invention, the active substance is a small molecule, particularly a negatively charged small molecule. The active substance can also be a mixture of various active substances.

The active substance is preferably a cytotoxic agent.

Furthermore, the present invention generally relates to VLPs that can be used as drug delivery systems for the treatment and diagnosis of neurological, neuronal or neurodegenerative diseases such as in particular multiple sclerosis, Parkinson's disease or Alzheimer's disease.

In preferred embodiments, the VLPs are transported inside, to, or into oligodendrocytes.

Example

1. VP1-VLPs in an in vitro blood-brain barrier (BBB) model

This in vitro experiment demonstrated the ability of VLPs to cross the blood-brain barrier in a model system that matches the organization and properties of the human blood-brain barrier. In this model system, primary porcine cerebral vascular endothelial cells (PBCEC) that can form a blood-brain barrier in vitro were used (Angelow S, Zeni P and Galla HJ "Usefulness and limitation of primary cultured porine horoid plexus epithel cells as an in vitro model to study drug transport at the blood-CSF barrier", Adv Drug Discovery 2004; 56(12): 1859-73).

PBCECs were prepared and cultured as described in Rempe et al., BBRC, 2011: Transport of Poly-(n-butylcyano-acrylate) nanoparticles across the blood-brain-barrier in vitro and their influence on barrier integrity.

The effect of VLPs containing cargo on a Transwell filtration system was investigated using relative transendothelial electrical resistance (TEER) measurements (Rempe et al., 2011). To establish quantitative evidence of delivery, we used plasmid DNA as cargo. VP1 virus-like particles did not affect the integrity of the blood-brain barrier under any circumstances and at any concentration.

In order to confirm that the cargo is transported through the blood-brain barrier in vitro, plasmid DNA is loaded into VLP according to Goldmann et al., 1999 (Journal of Virology:Molecular cloning and expression of major structural protein VP1 of the human polyoma virus:formation of virus like particles useful for immunological and therapeutic studies and measured quantitatively by specific qPCR). The copy number of the plasmid DNA on the apical and basal sides of the blood-brain barrier in the quantitative in vitro model is measured. The apical side is the location where the VLP is added (inside the blood vessel cavity in vivo), and the basal side refers to the brain. The quantitative evidence of the basal layer plasmid DNA represents that the molecules mediated by the VLP pass through the brain endothelial cells and the cargo are delivered through the blood-brain barrier.

2. In vivo reporter gene delivery and expression

A reporter gene delivery experiment was designed to demonstrate the ability of JC VP1 virus-like particles to deliver substances into cells and tissues in vivo. In this experiment, reporter gene expression is one of the best methods to demonstrate not only delivery but also the functionality of the delivered substance (Hoffman, R.M., 2005: THE MULTIPLE USES OF FLUORESCENT PROTEINS TO VISUALIZE CANCER IN VIVO. Nat. Rev. Cancer; 5(10): 796-806).

Material

The VP1-VLP probes of different types are used to detect the ability that external loading and sending reporter plasmid enter Cos 7 (green monkey kidney cell) cell, and reason is that this cell line is known to be by JCV VLP transduction.The VP1-VLP probes of 9 salt precipitations and 15 chromatography-purified VP1-VLP probes are arranged.External transduction experiment repeats 5 times.In this experiment, the ability of the maximum luminescent signal of the fluorescein substrate that detects and sends is used for experiment in vivo.

Salt-precipitated VP1-VLPs were precipitated overnight and dialyzed for 24 hours against Standard Buffer (150 mM NaCl, 10 mM Tris-HCl, pH 7.5). Reporter gene plasmid loading was accomplished by chemical isolation and recombination as described by Goldmann et al., 1999, Journal of Virology: Molecular cloning and expression of major structural protein VP1 of the human polyomavirus: formation of virus-like particles useful for immunological and therapeutic studies.

Experimental design

VLPs were injected intravenously into the tail vein of immunocompetent BALB/c mice under isoflurane anesthesia.

The animals were grouped as follows:

-5 μg salt-precipitated VLPs (4 animals)

-50 μg salt-precipitated VLPs (4 animals)

-5 μg chromatography-purified VLPs (4 animals)

-DNA control (reporter gene plasmid only) (3 animals)

-VLP control (VP1-VLP capsid only) (3 animals)

Bioluminescence was measured 12 minutes after intraperitoneal injection of luciferin substrate on days 2, 4, 7, 14, and 22. The results for each group were combined to calculate the mean and standard deviation. Means were analyzed by two-way ANOVA with a Holm-Sidak test as a post hoc test.

The results are shown in Figures 3 and 4.

3. Transduction efficiency of Cos7 cells (African green monkey kidney cells) using JC VP1-VLP loaded with luciferase plasmid and JC VP1-VLP mixed with luciferase plasmid

1. 18 hours before transduction, plate Cos7 cells into 24-well plates.

2. VP1-VLPs were separated with DTT and EGTA, mixed with luciferase plasmid, and dialyzed against rebinding buffer at 4°C overnight.

3. The next day, the loaded VP1-VLPs were removed from the dialysis tube.

4. VP1-VLP mixed with luciferase plasmid was prepared according to Krauzewicz (Gene Therapy (2000) 7, 1094-1102).

a. The mixing ratio of VLP and luciferase plasmid was 30:1 (w/w).

b. Incubate the mixture at room temperature for 15 minutes.

c. Dilute the mixture with DMEM cell culture medium and pipette it onto Cos7 cells in a 24-well plate.

5. Pipette the luciferase plasmid-loaded VP1-VLPs into Cos 7 cells. Perform the same operation for the luciferase plasmid-mixed VP1-VLPs.

6. After 72 hours, cells were lysed and luciferase activity was measured in groups of three using Promega's Luciferase Assay.

The results of independent transduction experiments are shown in Figure 5 .

4. Immunohistochemical detection of luciferase and VP1 proteins in mouse brains after intravenous administration of luciferase plasmid-loaded VLPs

As described above, detection of plasmid DNA (by quantitative PCR) or luciferase activity demonstrated that JC VP1-VLPs are capable of delivering substances into cells and tissues in vivo. Immunohistochemical detection of luciferase protein and VP1 protein in the brain further supported these experimental results.

Brain tissue was isolated from immunocompetent BALB/c mice (treated as described above) injected intravenously with VLPs, fixed in PFA and embedded in paraffin as described in J. Jankowski et al., The Journal of comparative Neurology 472: 87-99, 2004: "Engrailed-2 negatively regulates the onset if prenatal Purkinje Cell differentiation". Thin sections of 7 to 10 microns were prepared and mounted on Histobond plus slides. The sections were rehydrated to inactivate endogenous peroxidase and make the sections permeable. The blocking step was performed in a 3% bovine serum albumin solution. Luciferase protein or VP1 protein was detected with monoclonal antibodies, respectively. To increase signal intensity, the TSA fluorescence enhancement system (TSA ^™ Plus Fluorescein System, Perkin Elmer) was used.

Results: As shown in the right figure of Figure 6B, immunohistochemical analysis using anti-VP1 antibodies was able to detect VP1 protein in cells of the central nervous system. Brain sections showed scattered spots of irregular sizes, indicating the cellular localization of VP1 in the brain parenchyma. Using anti-luciferase antibodies, luciferase protein was also able to be detected in cells of the central nervous system. In accordance with the results of VP1 protein, luciferase-derived staining of brain sections also showed scattered spots of irregular sizes, indicating the cellular localization of luciferase in the brain parenchyma.

Discussion: The presentation of luciferase and VP1 protein in brain cells represents further support for the basic concept of the invention, as it demonstrates that the VLPs, and not just the active substances alone, pass the blood-brain barrier and enter the central nervous system.

5. Colocalization analysis showed the presence of VP1 protein in oligodendrocytes

Based on the above-mentioned detection of VP1 protein in cells within the central nervous system, a colocalization experiment was performed to identify the target cells. For this purpose, the detected VP1 protein colocalized with Olig 2, a marker that is specifically localized to oligodendrocytes (B. Menn et al., "Origin of oligodentrocytes in the subventricular zone of the adult brain", The Journal of Neuroscience, 2006, 26(30): 7907-7918).

Result: as shown in the lower left figure of Figure 8, several irregular spots can be detected in brain slices by using the Olig2 marker, which can also be stained with DAPI by nuclear staining (upper left figure of Figure 8) in each instance, thus indicating oligodendrocytes. As shown in the lower right figure of Figure 8, each of the oligodendrocytes is also VP1 protein positive. Olig2 and VP1 positive cells are marked with white arrows. Therefore, VP1 protein is located in the oligodendrocytes of the central nervous system, which indicates that these cells are infected by VLPs administered intravenously.

Discussion: The detection of VP1 protein in brain oligodendrocytes is consistent with the natural tropism of JCV virus. As a result, the VLPs claimed in the present invention are particularly suitable for treating diseases related to oligodendrocytes such as multiple sclerosis.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: A, B - Changes in relative transendothelial electrical resistance after addition of VP1-VLPs at concentrations ranging from 5*10 ⁸ to 2*10 ¹² particles per well (A and B) (n=3).

Figure 2: In vitro model, DNA-loaded VP1-VLPs were measured by quantitative PCR using specific primers to cross the blood-brain barrier. Added VLPs - the number of VLPs added to the apical side of the blood-brain barrier model, Detected VLPs - the number of plasmids detected on the apical and basolateral sides of the blood-brain barrier model (n=3).

Figure 3: Bioluminescent signals of mouse bodies after intravenous administration of: 5 μg salt-precipitated VLPs; 50 μg salt-precipitated VLPs; 5 μg chromatography-purified VLPs; DNA control (reporter gene plasmid only); VLP control (VP1-VLP capsid only). For example, ventral and dorsal images of 1 mouse per group.

Figure 4: Bioluminescent signal in the head of mice after intravenous administration of: 5 μg salt-precipitated VLPs; 50 μg salt-precipitated VLPs; 5 μg chromatography-purified VLPs; DNA control (reporter gene plasmid only). The mean value of each group was subtracted from the VLP control (as background signal).

Figure 5: A, B: Cos7 cells were transduced with VP1-VLPs loaded with luciferase plasmid DNA. RLU—relative light units; 25 μg loaded VLPs—25 μg JC VP1-VLPs loaded with luciferase plasmid; 50 μg loaded VLPs—50 μg JC VP1-VLPs loaded with luciferase plasmid; 50 μg VLP mix—50 μg JC VP1-VLPs mixed with luciferase plasmid; DNA control—luciferase plasmid control.

Figure 6: Immunohistochemical analysis of brain sections. The left column shows DAPI staining of cell nuclei, and the right column shows fluorescently labeled proteins: A - negative control; B - FITC staining of VP1 protein; C - FITC staining of luciferase protein.

Figure 7: Negative control for colocalization of VP1 protein with the oligodendrocyte marker Olig2. The upper left panel shows nuclear staining using the dye DAPI. The upper right panel shows the signal after fluorescence detection without the anti-VP1 antibody. The lower left panel shows the signal after fluorescence detection without the anti-Olig2 antibody. The lower right panel represents a fusion image of the two control stainings.

Figure 8: Colocalization of VP1 protein with the oligodendrocyte marker Olig2. The upper left panel shows nucleus staining with the dye DAPI. The upper right panel shows the localization of VP1 protein (FITC-fluorescence). The lower left panel shows staining with an anti-Olig2 antibody (TRITC-fluorescence). The lower right panel represents a fusion image of Olig2-marker and VP1 protein staining.

Claims (20)

1. Use of a drug-containing, virus-like particle (VLP) derived from human polyomavirus in the preparation of a drug delivery system for treating or diagnosing central nervous system diseases in living humans, wherein the VLP comprises at least one VP1 of human polyomavirus. 2. The use according to claim 1, wherein the VLP is derived from the JC virus. 3. The use according to claim 1 or 2, wherein the VLP, together with the drug encapsulated in the VLP, crosses the blood-brain barrier and enters the central nervous system. 4. The use according to claim 1, wherein the VLP containing the drug crosses the physiologically intact blood-brain barrier. 5. The use according to claim 1, wherein the central nervous system disease is a neurological, neuronal, or neurodegenerative disease. 6. The use according to claim 1, wherein the VLP is composed of the VP1 protein of the JC virus. 7. The use according to claim 1, wherein the VLP is administered via a route that allows the drug to produce a systemic effect. 8. The use according to claim 1, wherein the VLP is prepared for intravenous administration. 9. The use according to claim 1, wherein the VLP comprises VP1 and/or VP2, wherein VP1 comprises an amino acid sequence whose full length is at least 80% identical to the amino acid sequence shown in SEQ ID NO:1, and/or wherein VP2 comprises an amino acid sequence whose full length is at least 80% identical to the amino acid sequence shown in SEQ ID NO:3. 10. The use according to claim 1, wherein the drug is selected from the group consisting of cytotoxic agents, detectable reagents, proteins, peptides, and nucleic acids. 11. The use according to claim 10, wherein the detectable reagent is a radionuclide. 12. The use according to claim 10, wherein the nucleic acid encodes the desired protein and is mRNA, cDNA, plasmid, or vector nucleic acid. 13. The use according to claim 10, wherein the nucleic acid is a repressive nucleic acid and is siRNA or miRNA. 14. The use according to claim 10, wherein the nucleic acid is a catalytically active nucleic acid and is a ribozyme. 15. The use according to claim 1, wherein the VLP is loaded with a drug. 16. The use according to claim 1, wherein at least 1% of the total amount of pharmaceutical substance is completely encapsulated in the coating of the VLP. 17. The use according to claim 16, wherein at least 15% of the total amount of pharmaceutical substance is completely encapsulated in the coating of the VLP. 18. The use according to claim 16, wherein at least 50% of the total amount of pharmaceutical substance is completely encapsulated in the coating of the VLP. 19. The use according to claim 16, wherein at least 95% of the total amount of pharmaceutical substance is completely encapsulated in the coating of the VLP. 20. The use according to claim 16, wherein the VLP is non-aggregated.