WO2018132409A1

WO2018132409A1 - Treatment of cystic fibrosis via microvesicle-mediated cftr replacement

Info

Publication number: WO2018132409A1
Application number: PCT/US2018/013070
Authority: WO
Inventors: Jess G. Thoene
Original assignee: Thoene Jess G
Priority date: 2017-01-13
Filing date: 2018-01-10
Publication date: 2018-07-19

Abstract

A method for treating cystic fibrosis, including administering to a subject an effective amount of a microvesicle containing a cystic fibrosis transmembrane conductance regulator (CFTR) protein. A method for replacing a defective CFTR protein in a subject, including administering an effective amount of a microvesicle containing a CFTR protein. The microvesicle is produced by infecting Spodoptera cell with baculovirus containing a gene encoding a CFTR protein.

Description

TREATMENT OF CYSTIC FIBROSIS VIA MICRO VESICLE-MEDIATED CFTR

REPLACEMENT

TECHNICAL FIELD

The present invention relates to a method for providing a plasma membrane protein to a subject, in particular a method for treating cystic fibrosis by delivering a cystic fibrosis transmembrane conductance regulator (CFTR) protein to the subject via microvesicle.

BACKGROUND

Cystic fibrosis (CF) is a lethal genetic disease caused by a mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The human CFTR gene is an about 25,000 bp-long gene located in the chromosome 7 and encodes a membrane protein (i.e., CFTR protein) having about 1,480 amino acids. The CFTR protein is a member of the ATP -binding cassette (ABC) transporter family and functions as a chloride ion channel at cell membranes. There are various mutations in the CFTR gene that can cause defects in the CFTR protein, including but not limited to AF508, G542X, G55 ID, and N1303K.

Among them, the most common mutation is AF508, that is, a deletion of phenylalanine at position 508, and accounts for about 70% of the entire CF patients and about 90% of the CF patients in the United States.

If the CFTR protein is defective or malfunctioning due to a mutation to the CFTR gene, the chloride ion channel does not freely transfer ions through the membrane. As a result, viscous mucus accumulates in organs including lungs, pancreas, gut, and testes, and the accumulated mucus may cause blockages, infection, and inflammation of the organs. Many CF patients suffer from respiratory failure due to blockages of airways in lung, or infection and inflammation of various organs.

Ivacaftor (KALYDECO®) has been used to treat CF. Ivacaftor is a first drug approved by U.S. Food and Drug Administration (FDA) to treat CF. However, this drug works only for CF patients having certain mutations in the CFTR gene, and is not be effective for patients having the most common AF508 mutation. In addition, ivacaftor can cause severe side effects such as abdominal pain, headache, diarrhea, rash, dizziness, and nasal congestion, and the treatment with ivacaftor is very expensive as it costs about $300,000 a year for a patient. There is no cure for CF patients other than those having the specific mutations. The majority of CF treatment focuses on alleviating symptoms by administering antibiotics, anti-inflammatory agents, mucus viscosity modulators, and nutrient supplements.

There have been attempts to provide gene therapies to CF patients, however successful deliver of a functional CFTR protein has not been accomplished. Cloning of functional transmembrane proteins such as CFTR is generally believed to be impossible due to irreversible aggregation of proteins when they are removed from their lipid membrane environment.

U.S. Patent No. 9,023,798 (incorporated herein by reference in its entirety) describes treatment of cystinosis involving administration of a cystinosin replacement factor.

However, a successful replacement therapy for CF has not been reported.

One aspect of the present invention relates to treatment of CF. In one embodiment, the present invention relates to a replacement therapy for CF. In one embodiment, the present invention relates to a treatment of CF by administering a vesicle containing a CFTR protein. In one embodiment, the present invention relates to a method for treating CF by replacing a deficient CFTR protein with a functional CFTR protein via endobronchial instillation. In one aspect, the method may be performed by administering a vesicle containing a CFTR protein to a patient suffering from CF.

Another aspect of the present invention relates to delivering a plasma membrane protein, including but not limited to a CFTR protein, in a subject.

Still another aspect of the present invention relates to treatment of diseases or symptoms involving a plasma membrane protein, including but not limited to diabetes mellitus and hypertension.

SUMMARY

A first aspect of the present invention relates to a method for treating cystic fibrosis, in which an effective amount of a microvesicle containing a recombinant cystic fibrosis transmembrane conductance regulator protein is administered to a subject. The

microvesicle is produced by infecting a Spodoptera cell with baculovirus containing a gene encoding a cystic fibrosis transmembrane conductance regulator protein. In one

embodiment, the cystic fibrosis transmembrane conductance regulator protein is a wild-type human cystic fibrosis transmembrane conductance regulator protein. In one embodiment, the recombinant cystic fibrosis transmembrane conductance regulator protein is

encapsulated within the microvesicle.

A second aspect of the present invention relates to a method for replacing a defective cystic fibrosis transmembrane conductance regulator protein in a subject. In this method, an effective amount of a microvesicle containing a cystic fibrosis transmembrane conductance regulator protein is administered to the subject. The microvesicle is produced by infecting a Spodoptera cell with baculovirus containing a gene encoding a cystic fibrosis transmembrane conductance regulator protein.

A third aspect of the present invention relates to a method for delivering a plasma membrane protein to a human subject. In this method, an effective amount of a

microvesicle containing a plasma membrane protein is administered to the human subject. The microvesicle is produced by infecting a Spodoptera cell with baculovirus containing a gene encoding the plasma membrane protein. In one embodiment, the plasma membrane protein is involved in diabetes mellitus or hypertension.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows eGFP-tagged CFTR protein in cultured human fibroblasts after incubation with microvesicles containing the GFP -tagged CFTR protein. DETAILED DESCRIPTION

The present invention relates to a method for replacing a plasma membrane protein in a subject. In one embodiment, the present invention relates to a method for treating diseases involving one or more defective plasma membrane proteins. In one embodiment, the present invention relates to a method for treating cystic fibrosis (CF), for example a method for treating CF by replacing one or more proteins including a cystic fibrosis transmembrane conductance regulator (CFTR) protein in the CF patient. In another embodiment, the present invention relates to a method for treating a disease other than CF, for example a method for treating a disease by replacing one or more defective plasma membrane proteins in the patient.

A first embodiment of the present invention relates to a method for treating CF. The method may be a replacement therapy for CF. The method may be performed by administering a vesicle including a suitable protein to a patient suffering from CF.

According to one aspect, the vesicle may be an extracellular vesicle including a recombinant protein. The vesicle may be a microvesicle that is capable of being administered to a subject, preferably a mammalian subject, more preferably a human subject, without substantially producing adverse reactions, e.g., toxic, allergic, or immunological reactions.

In one embodiment, the vesicle may be a microvesicle derived from cells. The cells may be a plant cell, a bacterial cell, a yeast cell, an insect cell, and/or a mammalian cell, preferably an insect cell. Insect cell lines derived from Bombyx such as B. mori, Mamestra such as M. brassicae, Spodoptera such as S. frugiperda, Trichoplusia such as T. ni,

Drosophila cell such as D. melanogaster, and the like, may be used. Among them,

Spodoptera cells, in particular S. frugiperda cells, in particular Sf21 cells and Sf9 cells, in particular Sf9 cells are preferably used in the present invention. In a preferred embodiment, the vesicle is derived from Sf9 cells.

According to one aspect, the vesicle includes a biomolecule, in particular a protein and/or a peptide. As used herein, the term "protein" refers to compounds comprising amino acids joined via peptide bonds and may be used interchangeably with the term "polypeptide." In one aspect, the protein included in the vesicle is a plasma membrane protein, such as a protein forming an ion channel at a cell membrane, or an enzyme regulating an ion channel at a cell membrane. The peptide may be a part of a plasma membrane protein or a part of a protein forming an ion channel at a cell membrane, or may be a part of an enzyme regulating an ion channel at a cell membrane. Preferably, the vesicle includes a protein, for example a plasma membrane protein, in particular a cystic fibrosis transmembrane conductance regulator (CFTR) protein. Preferably, the vesicle includes a human CFTR protein, in particular the wild-type human CFTR protein.

In one aspect of the present invention, the vesicle is produced by transfecting or infecting a cell with a vector including a nucleotide or a gene encoding a desired protein or a peptide. For example, the vesicle is produced by a cell transfected or infected with a vector including a nucleotide or a gene encoding a desired protein or a peptide.

As used herein, the term "transfection," "transfecting," or "transfected" refer to the introduction of foreign DNA into cells. Transfection may be accomplished by a variety of means known to the art including calcium phosphate-DNA co-precipitation, DEAE-dextran- mediated transfection, polybrene-mediated transfection, glass beads, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, viral infection, biolistics (i.e., particle bombardment) and the like. Baculovirus may infect Spodoptera cells without the need for transfection. As used herein, the term "vector" refers to nucleic acid molecules that transfer DNA segment(s) from one cell to another. The term "vehicle" is sometimes used interchangeably with "vector." The vector may be a plasmid vector or a viral vector. Preferably, the vector is a viral vector, such as a baculovirus (BV), such as an Autographa californica baculovirus.

As used herein, the term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises coding sequences necessary for the production of an RNA, or a polypeptide or its precursor (e.g., proinsulin). A functional polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence as long as the desired activity or functional properties of the polypeptide are retained. The term "portion" when used in reference to a gene refers to fragments of that gene. The fragments may range in size from a few nucleotides to the entire gene sequence minus one nucleotide.

The term "gene" also encompasses the coding regions of a structural gene and includes sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb on either end such that the gene corresponds to the length of the full- length mRNA. The sequences which are located 5' of the coding region and which are present on the mRNA are referred to as 5' non-translated sequences. The sequences which are located 3' or downstream of the coding region and which are present on the mRNA are referred to as 3' non-translated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed "introns" or "intervening regions" or

"intervening sequences." Introns are segments of a gene which are transcribed into nuclear RNA (mRNA). Introns may contain regulatory elements such as enhancers. Introns are removed or "spliced out" from the nuclear or primary transcript. Introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

In addition to containing introns, genomic forms of a gene may also include sequences located on both the 5' and 3' end of the sequences which are present on the RNA transcript. These sequences are referred to as "flanking" sequences or regions. These flanking sequences are located 5' or 3' to the non-translated sequences present on the mRNA transcript. The 5' flanking region may contain regulatory sequences such as promoters and enhancers which control or influence the transcription of the gene. The 3' flanking region may contain sequences which direct the termination of transcription, posttranscriptional cleavage and polyadenylation. In one embodiment, the vector is modified to include a gene encoding a CFTR protein, in particular a human CFTR protein, preferably the wild-type human CFTR protein. In some preferred embodiments, BV is modified to include a gene encoding a CFTR protein (e.g., human CFTR protein, wild-type human CFTR protein, etc.), and an insect cell is transfected or infected with the modified BV. In some preferred embodiments, BV is modified to include a gene encoding a human CFTR protein (e.g., wild-type human CFTR protein), and a Spodoptera cell is infected with the modified BV. In some preferred embodiments, a BV is modified to include a gene encoding a human CFTR protein (e.g., wild-type human CFTR protein), and a Sf9 cell is infected with the modified BV.

For example, a CFTR gene (e.g. wild-type, mutated, truncated, etc.) is cloned into a suitable vector, and the vector is transformed or transfected into a cell. In some

embodiments, cells containing the vector are grown in a suitable media under conditions such that a CFTR protein is expressed (e.g. overexpressed).

In some embodiments, a CFTR gene is cloned into a vector by the method described in U.S. Patent No. 9,023,798, which is incorporated herein by reference.

In one aspect of the present invention, the gene included in the vector may be expressed in a vesicle such as a microvesicle. In some embodiments, a cell is transfected or infected with a vector including a gene encoding a plasma membrane protein. In some preferred embodiments, an insect cell is transfected or infected with a viral vector including a gene encoding a CFTR protein, and the CFTR protein is expressed in a vesicle produced by the insect cell. In some preferred embodiments, a Spodoptera cell is infected with BV including a gene encoding a CFTR protein, and the CFTR protein is expressed in a vesicle produced by the Spodoptera cell. In some preferred embodiments, a Sf9 cell is infected with BV including the human CFTR gene, and the human CFTR protein is expressed in a microvesicle produced by the Sf9 cell.

In some embodiments, the transfected cells (e.g. Sf9 cells) generate vesicles containing a plasma membrane protein, such as a protein capable of forming an ion channel at a cell membrane. In some embodiments, the transfected cells generate microvesicles containing a CFTR protein. The CFTR protein may be encapsulated within the

microvesicles.

In some embodiments, vesicles (e.g. microvesicles encapsulating a CFTR protein and/or a plasma membrane protein) generated from cells are purified and/or isolated from other cellular and/or media components. In some embodiments, vesicles (e.g. microvesicles encapsulating a CFTR protein and/or a plasma membrane protein) are purified and/or isolated from some or all other cellular and/or media components by standard methodologies known to those in the art, not limited to: dialysis, centrifugation,

chromatography, gel electrophoresis, filtration, etc. In one aspect of the present invention, the encapsulated contents (e.g. a CFTR protein and/or a plasma membrane protein) of vesicles may be co-purified with the vesicles, and a CFTR protein and/or a plasma membrane protein is not purified from vesicles before use and/or administration. However, a CFTR protein and/or a plasma membrane protein may be purified and/or isolated from vesicles.

As used herein the term "purified" refers to molecules that are removed from their natural environment, isolated or separated. As used herein, the term "purified" or "to purify" also refers to the removal of contaminants from a sample. The removal of contaminating proteins results in an increase in the percent of molecules of interest in the sample. In another example, recombinant proteins or polypeptides are expressed in plant, bacterial, yeast, insect, or mammalian host cells and the proteins or polypeptides are purified by the removal of host cell proteins; the percent of recombinant polypeptides is thereby increased in the sample.

In some embodiments, cells, media, vesicles, and or other factors are purified and/or isolated by suitable means. In some embodiments, the media used to culture cells is collected, e.g., by centrifugation, filtration, and the like. In some embodiments, material secreted by cells are collected with the media. In some embodiments, the media is separated into fractions (e.g. by chromatography, by centrifugation, by ultracentrifugation, by filtration, by affinity, etc.). In some embodiments, one or more elements, vesicles, compositions, compounds, proteins, etc. are isolated from the media. In some embodiments, the media is purified away from one or more contaminants.

The term "sample" is used in its broadest sense including biological and

environmental samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include but are not limited to blood products, such as plasma, serum, etc. Environmental samples include but are not limited to environmental material such as surface matter, soil, water, and industrial samples.

In some embodiments, vesicles (e.g. microvesicles encapsulating a CFTR protein and/or a plasma membrane) are about 50-100 nm in diameter. In some embodiments, vesicles (e.g. microvesicles encapsulating a CFTR protein and/or a plasma membrane protein) are small enough to pass through a 0.22 μπι sterilization filter. In some embodiments, microvesicles are freeze-thaw resistant. In some embodiments, microvesicles provide a transmembrane delivery system for a CFTR protein and/or another plasma membrane protein, such as a protein capable of forming an ion channel at a cell membrane.

In some embodiments, the present invention provides therapy for a subject suffering from CF. In some embodiments, the present invention provides replacement therapy for a subject suffering from CF. In some embodiments, replacement therapy provides one or more replacement factors which perform one or more functions of the missing and/or deficient CFTR.

In one embodiment, the present invention relates to a method for treating CF by replacing a deficient CFTR membrane protein with a functional CFTR protein via endobronchial instillation. In one embodiment, the method is performed by administering a vesicle including a CFTR protein to a patient suffering from CF.

According to one aspect of the present invention, a microvesicle is administered to a subject. In some embodiments, administration of the microvesicle provides a therapy for CF. In some embodiments, the microvesicle contains an active CFTR protein. In some embodiments, the microvesicle contains a protein capable of forming an ion channel, in particular a protein capable ofi rming a chloride ion channel, at a cell membrane in the subject. In some embodiments, the present invention provides administering an isolated microvesicle containing CFTR as a therapy for CF.

As used herein, the term "administration" or "administer" refers to the act of giving a drug, prodrug, or other agent, or therapeutic treatment to a subject (e.g., a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs). When the subject is a human, exemplary routes of administration to the human body can be through the eyes (ophthalmic), mouth (oral), skin (transdermal), nose (nasal), lungs (inhalant), oral mucosa (buccal), ear, rectal, by injection (e.g., intravenously, subcutaneously, intratumorally, intraperitoneally, etc.), and the like. In some embodiments, the administration may be parenteral ,

intraperitoneal, and/or intraspinal fluid administration.

In preferred embodiments of the present invention, the microvesicles are bronchially administered to the subject through lungs. The bronchial administration may be performed by using, e.g., an inhaler.

The subject may be a mammalian subject, preferably a human subject. The subject may have a mutation in the CFTR gene, and may have a deficient, malfunctional, or nonfunctional CFTR protein. The subject may or may not exhibit symptoms of CF. The subject may be suffering from CF. In some embodiments, a subject is deficient in the endogenous production of one or more plasma membrane protein (e.g. CFTR). In some embodiments, a subject produces defective protein, and is thereby deficient in normal/active/functioning protein. In some embodiments, a properly expressed protein is rendered defective through the action of another factor or factors, or by an unknown mechanism or pathway. In some embodiments, a deficiency of protein (e.g. deficiency of CFTR) results in a disease, condition, and or disorder (e.g. CF) in the protein deficiency subject.

In some embodiments, treatment of a disease or disorder (e.g. CF) is performed by replacing the defective protein (e.g. CFTR) with normal/active/functioning protein (e.g. exogenously expressed CFTR), a variant of the protein, or another factor (e.g. related or unrelated). In some embodiments, replacement of, supplementing of, or compensation for the protein for which a subject is deficient provides therapy (e.g. curative, palliative) for the related condition (e.g. symptom reduction). In some embodiments, the protein provided in replacement therapy is the protein for which the subject is deficient (e.g. CFTR). In some embodiments, the protein provided in replacement therapy is not the protein for which the subject is deficient. In some embodiments, the protein provided in replacement therapy is related to, a product of, and/or a by-product of expression of the protein for which the subject is deficient. In some embodiments, the protein provided in replacement therapy is a variant, mutant, or truncated version of the protein for which the subject is deficient (e.g. CFTR).

A variant or a mutant of a protein may have an amino acid sequence having, e.g. 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more identity as compared to an amino acid sequence of the

corresponding wild type protein, including all ranges and subranges therebetween. A truncated version of a protein may have an amino acid length of, e.g., 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more as compared to the length of the corresponding wild type protein, including all ranges and subranges therebetween.

For example, when the protein is a human CFTR protein having the amino acid sequence of SEQ ID NO: 1 (AAC 13657.1, available at National Center for Biotechnology Information (NCBI) website, on January 13, 2017, which is incorporated herein by reference), the variant or a mutant of the human CFTR protein may have an amino acid sequence having, e.g., 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more identity to the amino acid sequence of SEQ ID NO: 1.

As used herein, "amino acid sequence" refers to an amino acid sequence of a protein or peptide molecule. An "amino acid sequence" can be deduced from the nucleic acid sequence encoding the protein. However, terms such as "polypeptide" or "protein" are not meant to limit the amino acid sequence to the deduced amino acid sequence, but include post-translational modifications of the deduced amino acid sequences, such as amino acid deletions, additions, and modifications such as glycolsylations and addition of lipid moieties.

In some embodiments, a protein generated by the cells may provide a replacement or supplement for the subject. In some embodiments, the protein for which the subject is deficient is generated by the cells expressing the protein for which the subject is deficient, and the generated protein provides a replacement or supplement for the subject. In some embodiments, one or more proteins are purified and/or isolated from the liquid media. In some embodiments, one or more proteins are purified and/or isolated along with a plurality of other compounds and compositions from the liquid media. In some embodiments, a mixture including the desired factor or factors and additional components from the liquid media provide a replacement and/or supplement for a protein deficiency. In some embodiments, the proteins may be encapsulated in a vesicle generated by the cells.

The microvesicle administered to a subject may be in a pharmaceutically or pharmacologically acceptable carrier. That is, a suspension including the microvesicle and a pharmaceutically or pharmacologically acceptable carrier may be administered to a subject. Here, the terms "pharmaceutically acceptable" or "pharmacologically acceptable" refer to compositions that do not substantially produce adverse reactions, e.g., toxic, allergic, or immunological reactions, when administered to a subject.

The term "pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers including, but not limited to, phosphate buffered saline solution, water, emulsions (e.g., such as an oil/water or water/oil emulsions), and various types of wetting agents, any and all solvents, dispersion media, coatings, sodium lauryl sulfate, isotonic and absorption delaying agents, disintigrants (e.g., potato starch or sodium starch glycolate), and the like. The compositions also can include stabilizers and preservatives. Examples of carriers include but are not limited to, stabilizers and adjuvants.

The amount of the modified vesicle administered to the subject is not limited, as long as the effective amount is administered to the subject. Here, the term "effective amount" refers to the amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route. For example, when the subject has a deficient CFTR protein, the effective amount of the modified vesicle administered to the subject may be the amount sufficient to deliver a microvesicle containing the CFTR protein to the subject. The recombinant product is preferably a CFTR protein, more preferably the human CFTR protein.

In one embodiment of the present invention, the amount of the microvesicle administered to a subject in one dose is from about 0.1 ml to about 20 ml, preferably from about 0.5 ml to about 10 ml, more preferably from about 1 ml to 5 ml, including all ranges and subranges therebetween.

When a suspension including the microvesicle is administered to a subject, the concentration of the microvesicle in the suspension is from about 1 x 10⁵ to about 1 x 10¹⁵ per ml, preferably from about 1 ^χ 10⁹ to about 1 ^χ 10¹² per ml, more preferably from about 1 ^χ 10¹⁰ to 3 x 10¹¹ per ml, particularly preferably about 0.1 x 10¹¹ per ml, including all ranges and subranges therebetween.

In one embodiment of the present invention, the microvesicles are administered to a subject once a month, once a week, once a day, twice a day, three times a day, or four times a day, for a period of five days, one week, ten days, two weeks, three weeks, one month, or lifelong.

The amount and concentration of the microvesicles administered to a subject and/or the frequency of the administration described above are exemplary, and not limited to those amounts, concentrations, and frequencies. The amount, concentration, and frequency may vary depending on the diseases and/or symptoms from which the subject is suffering, or severeness of the diseases and/or symptoms.

In some embodiments, microvesivles including (e.g. encapsulating) a CFTR protein and/or a protein capable of forming an ion channel at a cell membrane are administered to a subject to treat and/or prevent a disease, condition or disorder (e.g. CF).

Another embodiment of the present invention relates to a method for treating diseases or symptoms other than CF.

The diseases or symptoms other than CF may be diseases or symptoms involving one or more defective plasma membrane proteins. Such diseases or symptoms include but are not limited to diabetes mellitus, hypertension, various hormonal disturbances, and defects of cell signaling that can lead to either growth failure or neoplasia. The method may be a replacement therapy for such diseases or symptoms. For example, the replacement therapy may be performed by replacing one or more defective plasma membrane proteins in a patient suffering from diseases or symptoms involving one or more defective plasma membrane receptor proteins. The method may be performed by administering a vesicle containing a suitable protein to a patient suffering from such diseases or symptoms. In some preferred embodiments, the method may be performed by administering a microvesicle containing a protein which is defective or deficient in the patient. A microvesicle containing such a protein may be produced by the method similar to the above-described method for producing a microvesicle containing CFTR.

The methods of the present invention may be used to treat any cell types. Cells may be in vitro, in culture, ex vivo, or in vivo. In some embodiments, the systems and methods of the present invention may be used in research, clinical, or diagnostic applications.

EXAMPLES

A human CFTR encoding gene was cloned into a viral vector, Baculovirus

(Autographa calif or nica), and then the Baculovirous bearing the human CFTR gene was transfected in the insect host cells, Spodoptera. The human CFTR protein was tagged with an e-GFP marker.

After lytic phase infection of Spodoptera cells, a microvesicle solution including 1.3 x 10¹¹ microvesicles/ml was prepared from a conditioned media. Cells from normal human fibroblasts line GM00010 were incubated for 96 hours in Ham's F12 tissue culture media supplemented with the microvesicle solution.

After fixation and sectioning, the e-GFP labeled CFTR protein was visualized by confocal immunofluoroscopy. The results are shown in Fig. 1. As shown in Fig. 1, green fluorescence is seen in the cultured human fibroblasts, indicating that the CFTR protein was successfully delivered to the recipient cultured fibroblasts.

Various modification and variation of the above-described method of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims. REFERENCES

The following references are incorporated herein by reference in their entireties:

1. Cant et al., CFTR structure and cystic fibrosis, The International Journal of Biochemistry & Cell Biology, 52 (2014) 15-25.

2. Edelman et al., Cystic fibrosis, a multi- systemic mucosal disease: 25 years after the discovery of CFTR, Editorial, The International Journal of Biochemistry & Cell Biology, 52 (2014) 2-4.

3. Larsen et al., Clusters of CI^" channels in CFTR-expressing Sf9 cells switch

spontaneously between slow and fast gating modes, Pflugers Archiv, 1996 Jul;432(3):528- 37.

4. Thoene et al., In vitro correction of disorders of lysosomal transport by microvesicles derived from baculovirus-infected Spodoptera cells, Molecular Genetics and Metabolism 109 (2013) 77-85.

Claims

1. A method of treating cystic fibrosis, comprising:

administering to a subject in need thereof an effective amount of microvesicles comprising a cystic fibrosis transmembrane conductance regulator protein.

2. The method of claim 1, wherein the microvesicles are produced by infecting a Spodoptera cell with baculovirus containing a gene encoding a cystic fibrosis

transmembrane conductance regulator protein.

3. The method of claim 1, wherein the cystic fibrosis transmembrane conductance regulator protein is a wild-type human cystic fibrosis transmembrane conductance regulator protein.

4. The method of claim 1, wherein the cystic fibrosis transmembrane conductance regulator protein is encapsulated in the microvesicles.

5. The method of claim 1, wherein the Spodoptera cell is a Spodoptera frugiperda cell.

6. The method of claim 5, wherein the Spodoptera frugiperda cell is a Sf9 cell.

7. The method of claim 1, wherein the subject has a cystic fibrosis transmembrane conductance regulator protein having a deletion of an amino acid at a position

corresponding to position 508 of the wild type human cystic fibrosis transmembrane conductance regulator protein.

8. The method of claim 1, wherein the microvesicles are bronchially administered to the subject.

9. The method of claim 1, wherein the administering of the microvesicle comprises administering a suspension comprising the microvesicles and a pharmaceutically acceptable carrier, and the suspension includes 1 ^χ 10⁵ to 1 ^χ 10¹⁵ of the microvesicles per ml.

10. The method of claim 9, wherein the microvesicles are administered to the subject from once to four times per day.

11. The method of claim 9, wherein 0.1 ml to 20 ml of the suspension is administered to the subject.

12. A method for replacing a defective cystic fibrosis transmembrane conductance regulator protein in a subject, the method comprising:

administering to the subject an effective amount of microvesicles comprising a cystic fibrosis transmembrane conductance regulator protein.

13. The method of claim 12, wherein the microvesicles are produced by infecting a Spodoptera cell with baculovirus containing a gene encoding a cystic fibrosis

transmembrane conductance regulator protein.

14. The method of claim 12, wherein the cystic fibrosis transmembrane conductance regulator protein is a wild-type human cystic fibrosis transmembrane conductance regulator protein.

15. The method of claim 12, wherein the cystic fibrosis transmembrane conductance regulator protein is encapsulated in the microvesicles.

16. The method of claim 12, wherein the Spodoptera cell is a Spodoptera frugiperda cell.

17. The method of claim 16, wherein the Spodoptera frugiperda cell is a Sf9 cell.

18. The method of claim 12, wherein the subject is suffering from cystic fibrosis.

19. The method of claim 12, wherein the microvesicles are administered to the subject such that the cystic fibrosis transmembrane conductance regulator protein is delivered to the subject.

20. The method of claim 12, wherein the microvesicles are bronchially administered to the subject.

21. The method of claim 12, wherein the administering of the microvesicles comprises administering a suspension comprising the microvesicles and a pharmaceutically acceptable carrier, and the suspension includes 1 ^χ 10⁵ to 1 ^χ 10¹⁵ of the microvesicles per ml.

22. The method of claim 21, wherein the microvesicles are administered to the subject from once to four times per day.

23. The method of claim 21, wherein 0.1 ml to 20 ml of the suspension is administered to the subj ect.

24. A method for delivering a plasma membrane protein in a human subject,

comprising:

administering to the human subject an effective amount of microvesicles comprising a plasma membrane protein.

25. The method of claim 24, wherein the microvesicles are produced by infecting a Spodoptera cell with baculovirus comprising a gene encoding the plasma membrane protein.

26. The method of claim 24, wherein the Spodoptera cell is a Spodoptera frugiperda cell.

27. The method of claim 24, wherein the Spodoptera frugiperda cell is a Sf9 cell.

28. The method of claim 24, wherein the plasma membrane protein is involved in diseases selected from the group consisting of diabetes mellitus and hypertension.

29. The method of claim 24, wherein the human subject is suffering from diseases selected from the group consisting of diabetes mellitus and hypertension.