Method for preparing liposomes

Abstract

The present invention relates to liposomes, methods for producing liposomes, and methods for loading cell-derived liposomes with cargo molecules. The invention extends to such liposomes themselves and their use as cellular delivery systems for the delivery of biologically and therapeutically active payload molecules, such as small molecules, RNAi molecules (e.g., siRNA), bioactive proteins, genome editing tools (e.g., Cas9), and drugs to cells to treat various disorders. Liposomes can also be used in a range of diagnostic and therapeutic applications. The invention also extends to pharmaceutical compositions comprising such liposomes, including populations of extracellular vesicles (EVs), exosomes, and fusion proteins. [Selection diagram] Figure 7

Description

Technical Field

[0001] The present invention relates to liposomes, and in particular, but not limited to, methods for generating liposomes and methods for loading cargo molecules into cell-derived liposomes. The present invention extends to the use of these liposomes themselves and small molecules, RNAi molecules (e.g., siRNA), antisense oligonucleotides (ASO), bioactive proteins, genome editing tools (such as nucleases like Cas9), and as cell transport systems for the stealth transport of biological and therapeutically active payload molecules such as drugs to cells for treating various disorders. These liposomes can also be used in the range of diagnostic and therapeutic applications. The present invention extends to pharmaceutical compositions and fusion proteins, such as the liposomes containing a population of extracellular vesicles (EV).

Background Art

[0002] Synthetic, nanoscale, advanced drug transport systems have been demonstrated for the targeted intracellular transport and controlled release of both small molecules and high molecular weight therapeutic agents [1]. They non-exhaustively incorporate nanoparticle systems, liposomes, hydrogels, emulsions, micelles, and soluble polymer-based drug delivery technologies [1]. In recent years, the potential of using biological systems that recombine cryptic protein domains that have evolved to perform specific tasks (i.e., facilitate the transport of therapeutic agents) has attracted attention [2]. Examples of these achievements include the use of protein toxin-derived materials to navigate the intracellular membrane system to achieve the targeted transport of large, membrane-impermeable molecules such as antisense oligonucleotides (ASO) [2]. This is useful because the bioavailability of these reagents (ASO) is limited in the absence of a means to cross the intracellular compartment barrier [2].

[0003] Proteins such as anthrax toxin (Atx) have evolved to disrupt the intracellular membrane system and gain access to the cytosol. This occurs via reverse fusion events between the inner boundary membranes of multivesicular endosomes (MVE) / multivesicular bodies (MVB) within intraluminal vesicles (ILV) and during the apoptosis-linked gene 2-interacting protein X (ALIX)-dependent process [3 and 4].

[0004] The PA oligomer (pore) component of Atx has been reported in the literature to be a cation-selective pore involved in the transfer of edema factor (EF) and lethal factor (LF) from the lumen of MVE / MVB to the lumen of ILV [6]. Furthermore, both EF and LF need to undergo a molten globule transition (i.e., unfolding) to pass through the PA63n oligomer [7]. Additionally, it is known that CAS9 does not pass through the PA pore [8].

[0005] Furthermore, there is only indirect evidence in response to the situation where pore switching occurs during the transport of siRNA or ASO [2], which is because (especially when high concentrations of PA83 protein are used) there is always a possibility that switching occurs across the inner boundary membrane of the MVE / MVB [3], or that the inner boundary membrane of the MVE / MVB has ruptured.

[0006] When the catalytic subunit of LF (i.e., domains II-IV) is removed from LF, the resulting non-toxic LF truncation (LFn) has been shown to help promote the movement of a fused selective cargo across the PA pore into the cytosol [2]. The use of PA83 and LFn-GAL4 for transporting ASO, or the use of PA83::LFn-PKR for transporting siRNA into the cytosol of cells has been previously reported [2].

[0007] Nucleocytosolic transport of nucleic acids (and proteins) via PA83:LFn-GAL4 or PA83::LFn-PKR is not ideal for several reasons. First, systematic use of this system, i.e., after i.v. administration, exposes the cargo and drug delivery system to the body's defenses. Since the components of this transport technology are known to be immunogenic (i.e., PA83 and LFn)

[11] , the plasma residence time of these constructs may be limited if repeated dosing is required. Second, there is also the possibility that the protein transport system or its cargo may be disrupted during transport to target cells after systemic administration. Furthermore, since the expression of the receptor involved in the internalization of PA83 has been shown to be almost ubiquitous, there is little room for targeted transport to specific cell populations

[12] . Finally, consideration should also be given to the possibility of PK-PD limitations caused by the charge of polyanions such as ASOs, i.e., the rapid removal from the plasma pool by cells of the reticuloendothelial system

[13] .

[0008] Wild-type LF has been reported in both ILVs and liposomes (e.g., exosomes), and considering that ILVs are known to be secreted from cells as liposomes after the release of calcium stored in the ER [5], the inventors inferred that recombinant LF could also be trapped or loaded into liposomes. Furthermore, the use of ionophores (e.g., ionomycin) releases ER calcium as needed, triggering the exocytosis of ILVs as liposomes. As a result, ionomycin was used to temporarily capture ILVs containing cargo from cells previously treated with an Atx-derived delivery system. Next, liposomes loaded with the cargo secreted into the cell culture medium were isolated thereby.

[0009] The present invention seeks to address one or more problems inherent in the prior art.

[0010] The inventors have developed a new methodology for loading luminal vesicles (or liposomes), which can be harvested as exosomes containing membrane-impermeable (therapeutic) cargo materials. This strategy confers "stealth" qualities to the cargo confined within the lumen of the vesicles, protects the cargo from the immune system, and protects the cargo from destruction by enzymes associated with serum or other body fluids. The liposomes described herein can protect luminal contents from enzymatic destruction and immune responses and are often referred to as a naturally occurring paracrine transport system that protects antigenic or enzymatically labile substances during transport. They also have the ability to target cells or tissues.

SUMMARY OF THE INVENTION

[0011] Accordingly, in a first aspect of the present invention, there is provided a method for preparing liposomes, the method comprising contacting at least one cell with the following ([ i ) and ([ ii ): ( i ) a pore-forming protein, or a pore-forming domain or variant or fragment thereof; ( ii ) a shuttle protein, which may be attached to a bioactive payload molecule, wherein the pore-forming protein, or a pore-forming domain or variant or fragment thereof, creates pores through the phospholipid bilayer of at least one of the cells, and the shuttle protein interacts with the pore-forming protein, or a pore-forming domain or variant or fragment thereof, thereby being internalized into the cell, thereby producing liposomes, wherein the shuttle protein may be loaded with a bioactive payload molecule.

[0012] Advantageously, the inventors have demonstrated that the method of the first aspect enables the efficient generation of liposomes (also referred to herein as "exosomes") that include an inner lumen surrounded or encapsulated by an outer lipid bilayer. FIG. 7 shows one embodiment of the method of the present invention. As shown in FIG. 1, the inventors have found that a labeled shuttle protein, preferably a fluorophore covalently attached to said shuttle protein, most preferably Texas Red-labeled LFn-PKR, can bind to intraluminal vesicles within a structure delimited by a CD63-positive membrane in the presence of the pore-forming protein PA83. Furthermore, these intraluminal vesicles can be isolated as shown in FIGS. 2 and 5. In some embodiments, said liposomes can be successfully loaded with a bioactive payload molecule or cargo molecule and then taken up by target cells (e.g., the cells of a patient suffering from a particular condition). Said bioactive payload molecule or cargo molecule can produce an effective biological or therapeutic result (see, e.g., FIG. 6). Thus, the inventors envision therapeutic applications of the liposomes. The inventors have also shown that a bioactive payload molecule can bind to the shuttle protein, and FIG. 3 provides clear evidence that said isolated liposomes contain a bioactive payload molecule labeled with said fluorophore, Texas Red. In FIG. 3, said bioactive payload molecule is the nuclease protein Cas9, and in FIG. 4, said payload molecule is a small molecule such as TexasRed.

[0013] The method of the present invention can be carried out in vitro, in vivo, or ex vivo. In a preferred embodiment, the method further comprises isolating said liposomes from said cells. Said shuttle protein may not be bound to a bioactive payload molecule. However, in a preferred embodiment, said shuttle protein is attached to a bioactive payload molecule either covalently or non-covalently.

[0014] The inventors have also demonstrated that, as shown in FIG. 6, the pharmacologically active siRNA can be successfully loaded into the liposomes using the pore-forming protein and the shuttle protein. Using the method of the present invention, biologically active cargo compounds can be loaded into and transported by liposomes, and the liposomes can be isolated and used to transfer the cargo from one cell population to another. Therefore, considering these data, it is clear that the liposomes produced by the method of the first aspect can be used to treat a wide range of disorders depending on which bioactive payload molecule is being carried.

[0015] According to a second aspect, there is provided a liposome obtained or obtainable by the method of the first aspect.

[0016] According to a third aspect, there is provided a liposome comprising a phospholipid bilayer surrounding a lumen, a pore-forming protein, or a pore-forming domain or a variant or fragment thereof, and a shuttle protein.

[0017] It is also contemplated that the liposomes of the present invention can be used in the treatment, prevention, or amelioration of a disease, or in diagnosis.

[0018] Therefore, according to a fourth aspect, there is provided a liposome according to either the second or third aspect for use in therapy or diagnosis.

[0019] According to a fifth aspect, there is provided a liposome according to either the second or third aspect for use in the treatment, prevention, or amelioration of a disease.

[0020] According to a sixth aspect, there is provided a method of treating, preventing, or ameliorating a disease in a subject, the method comprising administering to the subject in need of such treatment a therapeutically effective amount of a liposome according to either the second or third aspect.

[0021] Specifically, the inventors assume that the liposome may be useful for the treatment of FMO5-regulated obesity or male pattern alopecia. Thus, in one preferred embodiment, the disease to be treated is obesity, more preferably FMO5-regulated obesity. In other preferred embodiments, the disease to be treated is regulated by prostaglandin D2. The prostaglandin D2-regulated disease may be selected from the group consisting of male pattern alopecia (AGA); acne; seborrheic dermatitis; and prostate cancer.

[0022] In other preferred embodiments, the liposome may be useful in the treatment as a prophylactic agent. The liposome may be used for the treatment of dengue fever (or dengue virus disease), Ebola virus disease, acquired immunodeficiency syndrome (human immunodeficiency virus), Stat3-responsive cancer, P53-deficient cancer, virus-mediated cervical cancer (i.e., human papillomavirus), familial hypercholesterolemia, Duchenne muscular dystrophy, spinal muscular atrophy, Crohn's disease, and various inflammatory diseases, particularly intestinal diseases involved in the overexpression of intracellular adhesion molecule-1 (ICAM-1), but is not limited thereto.

[0023] According to a seventh aspect, a method for diagnosing a disease of a subject is provided, the method comprising obtaining a biological sample from the test subject and using the cells in the sample to generate liposomes loaded with a diagnostic compound using the method of the first aspect.

[0024] In one embodiment, this comprises loading the liposome with a seranostic compound that can report the presence or absence of the disease through the interaction between the cargo of the liposome having the disease marker and the disease cells.

[0025] In other embodiments, and as shown in Example 8, liposomes not derived from patient cells (e.g., liposomes derived from cultured mesenchymal stem cells) are loaded with seranostic compounds. In yet other embodiments, liposomes derived from cell lines rather than patients are used as therapeutic agents by loading them with other cargo. In one embodiment, the disease can be treated with liposomes not derived from the patient. In one embodiment, the liposomes not derived from the patient are derived from non-patient cells. In other embodiments, the liposomes not derived from the patient are derived from stem cells. Next, the obtained liposomes can be administered to the patient without immunologically adverse effects, i.e., as a "stealth" therapy.

[0026] According to an eighth aspect, there is provided a kit comprising a liposome according to either the second or third aspect, and instructions for use.

[0027] The liposome may comprise vesicles that can be extracellular vesicles (EVs) or intracellular vesicles, or intraluminal vesicles (ILVs). Most preferably, the liposome comprises exosomes. Although the lipid structures referred to herein are mainly extracellular, it will be understood by those skilled in the art that the present invention also extends to substantially intracellular lipid bilayer structures such as lysosomes, endosomes, and other intracellular lipid bilayer structures of both eukaryotes and prokaryotes, and vesicles therein. It will also be understood that the present invention extends to artificial lipid bilayer structures such as artificial vesicles, artificial liposomes, and other artificial lipid bilayer structures.

[0028] In one embodiment, the liposome has an average diameter between 10 nm and 500 nm. The dimensions of the liposome can be measured, for example, using small-angle neutron scattering [2]. In a preferred embodiment, the liposome has an average diameter between 20 nm and 400 nm. In a more preferred embodiment, the liposome has an average diameter between 30 nm and 300 nm. In an even more preferred embodiment, the liposome has an average diameter between 40 nm and 200 nm. In an even more preferred embodiment, the liposome has an average diameter between 50 nm and 150 nm. In the most preferred embodiment, the liposome has an average diameter between 60 nm and 120 nm.

[0029] In a preferred embodiment, the liposome comprises a phospholipid bilayer having a pore-forming protein, or its pore-forming domain or variant or fragment within the phospholipid bilayer. The pore-forming protein, or its pore-forming domain or variant or fragment, may extend completely across the width of the phospholipid bilayer or may only partially extend across the width of the phospholipid bilayer. The pore-forming protein, or pore-forming domain or its variant or fragment, can extend into the lumen of the cell and / or it can extend into the extracellular space of the cell. The pore-forming protein, or its pore-forming domain or variant or fragment, may extend only across the phospholipid bilayer and not into the lumen of the cell and / or the extracellular space of the cell.

[0030] Preferably, the cells used in the method of the first aspect include biological cells. Preferably, the cells include mammalian cells, most preferably human cells. Most preferably, the cells include cells obtained from the subject being treated. For example, the cells can be abnormal cells obtained from the subject, such as those collected from a biopsy. Alternatively, the cells include cells obtained from a stem cell line. The stem cell line can be a mesenchymal cell line.

[0031] In one non-limiting example, healthy cells can be collected from a target tissue and grown in culture before being used to generate liposomes (e.g., exosomes) loaded with a therapeutic compound appropriate for treating the clinical condition in question. This minimizes the likelihood that the liposomes will be recognized as "non-self" by the body. There is also the possibility of treating the liposomes to remove residual antigenic substances that may remain from the process of loading the therapeutic agent. In one embodiment, the cells collected may include healthy cells. In an alternative non-limiting example, the cells collected may not include healthy cells.

[0032] Preferably, the pore-forming protein, or its pore-forming domain or variant or fragment, comprises or is derived from non-toxic proteins. In one embodiment, the pore-forming protein, or its pore-forming domain or variant or fragment, is lysin.

[0033] In a preferred embodiment, the pore-forming protein, or its pore-forming domain or variant or fragment, is derived from Bacillus anthracis. In a preferred embodiment, the pore-forming protein is the Bacillus anthracis pathogenicity factor protective antigen (PA). In one embodiment, the pore-forming protein is Bacillus anthracis PA83. In one embodiment, the Bacillus anthracis PA83 has the amino acid sequence provided herein as SEQ ID NO: 1 as follows: MRGSHHHHHHGSEVKQENRLLNESESSSQGLLGYYFSDLNFQAPMVVTSSTTGDLSIPSSELENIPSENQYFQSAIWSGFIKVKKSDEYTFATSADNHVTMWVDDQEVINKASNSNKIRLEKGRLYQIKIQYQRENPTEKGLDFKLYWTDSQNKKEVISSDNLQLPELKQKSSNSRKKRSTSAGPTVPDRDNDGIPDSLEVEGYTVDVKNKRTFLSPWISNIHEKKGLTKYKSSPEKWSTASDPYSDFEKVTGRIDKNVSPEARHPLVAAYPIVHVDMENIILSKNEDQSTQNTDSQTRTISKNTSTSRTHTSEVHGNAEVHASFFDIGGSVSAGFSNSNSSTVAIDHSLSLAGERTWAETMGLNTADTARLNANIRYVNTGTAPIYNVLPTTSLVLGKNQTLATIKAKENQLSQILAPNNYYPSKNLAPIALNAQDDFSSTPITMNYNQFLELEKTKQLRLDTDQVYGNIATYNFENGRVRVDTGSNWSEVLPQIQETTARIIFNGKDLNLVERRIAAVNPSDPLETTKPDMTLKEALKIAFGFNEPNGNLQYQGKDITEFDFNFDQQTSQNIKNQLAELNATNIYTVLDKIKLNAKMNILIRDKRFHYDRNNIAVGADESVVKEAHREVINSSTEGLLLNIDKDIRKILSGYIVEIEDTEGLKEVINDRYDMLNISSLRQDGKTFIDFKKYNDKLPLYISNPNYKVNVYAVTKENTIINPSENGDTSTNGIKKILIFSKKGYEIG [SEQ ID NO: 1] Therefore, preferably, the pore-forming protein, or its pore-forming domain or variant or fragment, comprises, or consists essentially of, the amino acid sequence set forth in SEQ ID NO: 1, or a variant or fragment thereof.

[0034] In one preferred embodiment, the Bacillus anthracis PA83 comprises a PA83 variant (referred to herein as "MRSG-6His-PA83") having the amino acid sequence provided herein as SEQ ID NO: 2 as follows: MRGSHHHHHHGSEVKQENRLLNESESSSQGLLGYYFSDLNFQAPMVVTSSTTGDLSIPSSELENIPSENQYFQSAIWSGFIKVKKSDEYTFATSADNHVTMWVDDQEVINKASNSNKIRLEKGRLYQIKIQYQRENPTEKGLDFKLYWTDSQNKKEVISSDNLQLPELKQKSSNSRKKRSTSAGPTVPDRDNDGIPDSLEVEGYTVDVKNKRTFLSPWISNIHEKKGLTKYKSSPEKWSTASDPYSDFEKVTGRIDKNVSPEARHPLVAAYPIVHVDMENIILSKNEDQSTQNTDSQTRTISKNTSTSRTHTSEVHGNAEVHASFFDIGGSVSAGFSNSNSSTVAIDHSLSLAGERTWAETMGLNTADTARLNANIRYVNTGTAPIYNVLPTTSLVLGKNQTLATIKAKENQLSQILAPNNYYPSKNLAPIALNAQDDFSSTPITMNYNQFLELEKTKQLRLDTDQVYGNIATYNFENGRVRVDTGSNWSEVLPQIQETTARIIFNGKDLNLVERRIAAVNPSDPLETTKPDMTLKEALKIAFGFNEPNGNLQYQGKDITEFDFNFDQQTSQNIKNQLAELNATNIYTVLDKIKLNAKMNILIRDKRFHYDRNNIAVGADESVVKEAHREVINSSTEGLLLNIDKDIRKILSGYIVEIEDTEGLKEVINDRYDMLNISSLRQDGKTFIDFKKYNDKLPLYISNPNYKVNVYAVTKENTIINPSENGDTSTNGIKKILIFSKKGYEIG [SEQ ID NO: 2] Therefore, preferably, the pore-forming protein, or its pore-forming domain or variant or fragment, comprises, or consists essentially of, the amino acid sequence set forth in SEQ ID NO: 2, or a variant or fragment thereof.

[0035] Those skilled in the art will understand that the PA83 variant according to SEQ ID NO: 2 includes an N-terminal tagged variant, whereby the tag includes MRSG-6H and has the amino acid sequence provided as SEQ ID NO: 3 herein as follows: MRGSHHHHHH[SEQ ID NO: 3] An alternative protein tag containing 6His is provided as SEQ ID NO: 4 herein as follows: HHHHHH[SEQ ID NO: 4] Those skilled in the art will understand that the MRSG-6His and 6-His tags described in SEQ ID NO: 3 and SEQ ID NO: 4 can be added to the N-terminus or C-terminus of any of the proteins described herein, and such disclosure is understood to protect both tagged and untagged protein variants.

[0036] It will be understood that the pore-forming protein may include a single execution domain or subunit that can be formed into an oligomer such as Bacillus anthracis PA63. Thus, in other preferred embodiments, the pore-forming protein is Bacillus anthracis PA63. In one embodiment, the Bacillus anthracis PA63 has the amino acid sequence provided as SEQ ID NO: 5 herein as follows: STSAGPTVPDRDNDGIPDSLEVEGYTVDVKNKRTFLSPWISNIHEKKGLTKYKSSPEKWSTASDPYSDFEKVTGRIDKNVSPEARHPLVAAYPIVHVDMENIILSKNEDQSTQNTDSQTRTISKNTSTSRTHTSEVHGNAEVHASFFDIGGSVSAGFSNSNSSTVAIDHSLSLAGERTWAETMGLNTADTARLNANIRYVNTGTAPIYNVLPTTSLVLGKNQTLATIKAKENQLSQILAPNNYYPSKNLAPIALNAQDDFSSTPITMNYNQFLELEKTKQLRLDTDQVYGNIATYNFENGRVRVDTGSNWSEVLPQIQETTARIIFNGKDLNLVERRIAAVNPSDPLETTKPDMTLKEALKIAFGFNEPNGNLQYQGKDITEFDFNFDQQTSQNIKNQLAELNATNIYTVLDKIKLNAKMNILIRDKRFHYDRNNIAVGADESVVKEAHREVINSSTEGLLLNIDKDIRKILSGYIVEIEDTEGLKEVINDRYDMLNISSLRQDGKTFIDFKKYNDKLPLYISNPNYKVNVYAVTKENTIINPSENGDTSTNGIKKILIFSKKGYEIG [SEQ ID NO: 5] Therefore, preferably, the pore-forming protein, or its pore-forming domain or variant or fragment, comprises, or consists essentially of, the amino acid sequence set forth in SEQ ID NO: 5, or a variant or fragment thereof.

[0037] In some cases, it will also be understood that the pore-forming protein, or its pore-forming domain or variant or fragment, may include a fragment of the pore-forming protein. For example, the pore-forming protein can be truncated or digested to leave only the pore-forming fragment, which can be, for example, by an enzyme. Thus, in one embodiment, the pore-forming protein is a fragment of anthrax PA83, whereby the extracellular domain has been removed by an enzyme. In another embodiment, the pore-forming protein is a fragment of anthrax PA63, whereby the extracellular domain has been removed by an enzyme. It will be understood by those skilled in the art that both of these protein fragments have the amino acid sequence provided herein as SEQ ID NO: 6 as follows: VHVDMENIILSKNEDQSTQNTDSQTRTISKNTSTSRTHTSEVHGNAEVHASFFDIGGSVSAGFSNSNSSTVAIDHSLSLAGERTWAETMGLNTADTARLNANIRYVNT[SEQ ID NO: 6] Thus, preferably, the pore-forming protein, or its pore-forming domain or variant or fragment, comprises or consists essentially of the amino acid sequence set forth in SEQ ID NO: 6, or a variant or fragment thereof.

[0038]

[0039] In a further embodiment, the pore-forming protein is the anthrax PA83 D512K variant and has the amino acid sequence provided herein as SEQ ID NO: 7 as follows: MRGSHHHHHHGSEVKQENRLLNESESSSQGLLGYYFSDLNFQAPMVVTSSTTGDLSIPSSELENIPSENQYFQSAIWSGFIKVKKSDEYTFATSADNHVTMWVDDQEVINKASNSNKIRLEKGRLYQIKIQYQRENPTEKGLDFKLYWTDSQNKKEVISSDNLQLPELKQKSSNSRKKRSTSAGPTVPDRDNDGIPDSLEVEGYTVDVKNKRTFLSPWISNIHEKKGLTKYKSSPEKWSTASDPYSDFEKVTGRIDKNVSPEARHPLVAAYPIVHVDMENIILSKNEDQSTQNTDSQTRTISKNTSTSRTHTSEVHGNAEVHASFFDIGGSVSAGFSNSNSSTVAIDHSLSLAGERTWAETMGLNTADTARLNANIRYVNTGTAPIYNVLPTTSLVLGKNQTLATIKAKENQLSQILAPNNYYPSKNLAPIALNAQDDFSSTPITMNYNQFLELEKTKQLRLDTDQVYGNIATYNFENGRVRVDTGSNWSEVLPQIQETTARIIFNGKDLNLVERRIAAVNPSKPLETTKPDMTLKEALKIAFGFNEPNGNLQYQGKDITEFDFNFDQQTSQNIKNQLAELNATNIYTVLDKIKLNAKMNILIRDKRFHYDRNNIAVGADESVVKEAHREVINSSTEGLLLNIDKDIRKILSGYIVEIEDTEGLKEVINDRYDMLNISSLRQDGKTFIDFKKYNDKLPLYISNPNYKVNVYAVTKENTIINPSENGDTSTNGIKKILIFSKKGYEIG [SEQ ID NO: 7] Therefore, preferably, the pore-forming protein, or its pore-forming domain or variant or fragment, comprises, or consists essentially of, the amino acid sequence set forth in SEQ ID NO: 7, or a variant or fragment thereof.

[0040] In yet another embodiment, the pore-forming protein is the Bacillus anthracis PA83 K245G;R252N

[16] variant and has the amino acid sequence provided herein as SEQ ID NO: 8 as follows: MRGSHHHHHHGSEVKQENRLLNESESSSQGLLGYYFSDLNFQAPMVVTSSTTGDLSIPSSELENIPSENQYFQSAIWSGFIKVKKSDEYTFATSADNHVTMWVDDQEVINKASNSNKIRLEKGRLYQIKIQYQRENPTEKGLDFKLYWTDSQNKKEVISSDNLQLPELKQKSSNSRKKRSTSAGPTVPDRDNDGIPDSLEVEGYTVDVKNKRTFLSPWISNIHEKKGLTKYKSSPEKWSTASDPYSDFEKVTGRIDGNVSPEANHPLVAAYPIVHVDMENIILSKNEDQSTQNTDSQTRTISKNTSTSRTHTSEVHGNAEVHASFFDIGGSVSAGFSNSNSSTVAIDHSLSLAGERTWAETMGLNTADTARLNANIRYVNTGTAPIYNVLPTTSLVLGKNQTLATIKAKENQLSQILAPNNYYPSKNLAPIALNAQDDFSSTPITMNYNQFLELEKTKQLRLDTDQVYGNIATYNFENGRVRVDTGSNWSEVLPQIQETTARIIFNGKDLNLVERRIAAVNPSDPLETTKPDMTLKEALKIAFGFNEPNGNLQYQGKDITEFDFNFDQQTSQNIKNQLAELNATNIYTVLDKIKLNAKMNILIRDKRFHYDRNNIAVGADESVVKEAHREVINSSTEGLLLNIDKDIRKILSGYIVEIEDTEGLKEVINDRYDMLNISSLRQDGKTFIDFKKYNDKLPLYISNPNYKVNVYAVTKENTIINPSENGDTSTNGIKKILIFSKKGYEIG [SEQ ID NO: 8] Therefore, preferably, the pore-forming protein, or its pore-forming domain or variant or fragment, comprises, or consists essentially of, the amino acid sequence set forth in SEQ ID NO: 8, or a variant or fragment thereof.

[0041] In yet other embodiments, the pore-forming protein is the Bacillus anthracis PA83 K245N;R252S

[16] mutant and has the amino acid sequence provided herein as SEQ ID NO:9 as follows: MRGSHHHHHHGSEVKQENRLLNESESSSQGLLGYYFSDLNFQAPMVVTSSTTGDLSIPSSELENIPSENQYFQSAIWSGFIKVKKSDEYTFATSADNHVTMWVDDQEVINKASNSNKIRLEKGRLYQIKIQYQRENPTEKGLDFKLYWTDSQNKKEVISSDNLQLPELKQKSSNSRKKRSTSAGPTVPDRDNDGIPDSLEVEGYTVDVKNKRTFLSPWISNIHEKKGLTKYKSSPEKWSTASDPYSDFEKVTGRIDNNVSPEASHPLVAAYPIVHVDMENIILSKNEDQSTQNTDSQTRTISKNTSTSRTHTSEVHGNAEVHASFFDIGGSVSAGFSNSNSSTVAIDHSLSLAGERTWAETMGLNTADTARLNANIRYVNTGTAPIYNVLPTTSLVLGKNQTLATIKAKENQLSQILAPNNYYPSKNLAPIALNAQDDFSSTPITMNYNQFLELEKTKQLRLDTDQVYGNIATYNFENGRVRVDTGSNWSEVLPQIQETTARIIFNGKDLNLVERRIAAVNPSDPLETTKPDMTLKEALKIAFGFNEPNGNLQYQGKDITEFDFNFDQQTSQNIKNQLAELNATNIYTVLDKIKLNAKMNILIRDKRFHYDRNNIAVGADESVVKEAHREVINSSTEGLLLNIDKDIRKILSGYIVEIEDTEGLKEVINDRYDMLNISSLRQDGKTFIDFKKYNDKLPLYISNPNYKVNVYAVTKENTIINPSENGDTSTNGIKKILIFSKKGYEIG. [SEQ ID NO:9] Thus, preferably, the pore-forming protein, or its pore-forming domain or mutant or fragment, comprises, or consists essentially of, the amino acid sequence set forth in SEQ ID NO:9, or a mutant or fragment thereof.

[0042] In other embodiments, the pore-forming protein comprises a Bacillus anthracis PA83-HL hybrid molecule having the amino acid sequence provided herein as SEQ ID NO: 10, which incorporates the transmembrane domain of hemolysin

[17] instead of the PA63 transmembrane domain: GSEVKQENRLLNESESSSQGLLGYYFSDLNFQAPMVVTSSTTGDLSIPSSELENIPSENQYFQSAIWSGFIKVKKSDEYTFATSADNHVTMWVDDQEVINKASNSNKIRLEKGRLYQIKIQYQRENPTEKGLDFKLYWTDSQNKKEVISSDNLQLPELKQKSSNSRKKRSTSAGPTVPDRDNDGIPDSLEVEGYTVDVKNKRTFLSPWISNIHEKKGLTKYKSSPEKWSTASDPYSDFEKVTGRIDKNVSPEARHPLVAAYPIVHVDMENIILSKNEDQSTQNTDSQTRTISKNTSKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYAIDHSLSLAGERTWAETMGLNTADTARLNANIRYVNTGTAPIYNVLPTTSLVLGKNQTLATIKAKENQLSQILAPNNYYPSKNLAPIALNAQDDFSSTPITMNYNQFLELEKTKQLRLDTDQVYGNIATYNFENGRVRVDTGSNWSEVLPQIQETTARIIFNGKDLNLVERRIAAVNPSDPLETTKPDMTLKEALKIAFGFNEPNGNLQYQGKDITEFDFNFDQQTSQNIKNQLAELNATNIYTVLDKIKLNAKMNILIRDKRFHYDRNNIAVGADESVVKEAHREVINSSTEGLLLNIDKDIRKILSGYIVEIEDTEGLKEVINDRYDMLNISSLRQDGKTFIDFKKYNDKLPLYISNPNYKVNVYAVTKENTIINPSENGDTSTNGIKKILIFSKKGYEIG [SEQ ID NO: 10] Thus, preferably, the pore-forming protein, or its pore-forming domain or variant or fragment, comprises or consists essentially of the amino acid sequence set forth in SEQ ID NO: 10, or a variant or fragment thereof.

[0043] The above embodiments represent examples of pore-forming proteins and it will be understood by those skilled in the art that they are in no way limiting or exclusive. It will be understood that other fragments, mutants, or variants of the pore-forming proteins are also within the scope of the present invention.

[0044] Other examples of preferred pore-forming proteins include the PA83 or PA63 components of Atx, mutants of PA83 or PA63 such as the described octamer-forming mutants

[16] , or PA hybrids such as the described PA-α hemolysin hybrids, or non-Atx pore-forming proteins modified to mediate translocation across lipid bilayers such as recombinant streptolysin O (SLO) or α-hemolysin.

[0045] The term "shuttle protein" can refer to any protein or peptide configured to facilitate transport through the pores of a preformed protein. In a preferred embodiment, the shuttle protein is configured to facilitate the transport of a bioactive payload molecule through the pores. Thus, the shuttle protein is preferably an excipient that can pass through the inner boundary membrane of the endosome through the pores and carry the payload or cargo together. The payload can be covalently or non-covalently bound to the shuttle protein. Figure 7 shows the interaction between the shuttle protein carrying the payload and the pore-forming protein.

[0046] Preferably, the shuttle protein comprises an attenuated toxin protein. In particular, the shuttle protein can be a lethal factor (LF) or an edema factor (EF) derived from Bacillus anthracis. In one embodiment, the lethal factor domain I (LFn) has the amino acid sequence provided herein as SEQ ID NO: 11 as follows: MERNKTQEEHLKEIMKHIVKIEVKGEEAVKKEAAEKLLEKVPSDVLEMYKAIGGKIYIVDGDITKHISLEALSEDKKKIKDIYGKDALLHEHYVYAKEGYEPVLVIQSSEDYVENTEKALNVYYEIGKILSRDILSKINQPYQKFLDVLNTIKNASDSDGQDLLFTNQLKEHPTDFSVEFLEQNSNEVQEVFAKAFAYYIEPQHRDVLQLYAPEAFNYMDKFNEQEINLS[SEQ ID NO: 11] Therefore, preferably, the shuttle protein comprises the amino acid sequence set forth in SEQ ID NO: 11, or a variant or fragment thereof, or consists essentially of them.

[0047] In a preferred embodiment, the shuttle protein also comprises a linker protein. Preferably, in the attenuated toxin, at least one toxin domain, for example, one or more toxicity domains II-IV of the Bacillus anthracis lethal factor protein toxin, is replaced by the linker protein. In a more preferred embodiment, the linker protein comprises a nucleic acid binding domain. For example, the nucleic acid binding domain can be Saccharomyces cerevisiae GAL4 (fused with LFn) having the amino acid sequence provided herein as SEQ ID NO: 12 as follows: MGKPIPNPLLGLDSTMERNKTQEEHLKEIMKHIVKIEVKGEEAVKKEAAEKLLEKVPSDVLEMYKAIGGKIYIVDGDITKHISLEALSEDKKKIKDIYGKDALLHEHYVYAKEGYEPVLVIQSSEDYVENTEKALNVYYEIGKILSRDILSKINQPYQKFLDVLNTIKNASDSDGQDLLFTNQLKEHPTDFSVEFLEQNSNEVQEVFAKAFAYYIEPQHRDVLQLYAPEAFNYMDKFNEQEINLSMKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKDAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVSHHHHHH [SEQ ID NO:12] Therefore, preferably, the shuttle protein comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:12, or a variant or fragment thereof.

[0048] However, the inventors have found that LFn-GAL4 can be difficult to use because it has a tendency to aggregate non-specifically in an unregulated manner. The use of LFn-protein kinase R (PKR) for binding to ASO (essentially DNA-based rather than RNA-based) is novel and reported herein for the first time. It should be noted that LFn-PKR has not been shown to promote the translocation of plasmid DNA into the cytosol in the same manner as LFn-GAL4 [9 and 10]. Therefore, the inventors have also developed a novel and improved construct by replacing GAL4 in the above construct with PKR.

[0049] The inventors believe that PKR forms a linker protein that is more stable than GAL4 and binds to both the RNA and the double-stranded portion of the ASO. Thus, in a preferred embodiment, the linker protein comprises a protein kinase R or a fragment, variant, or mutant thereof having the amino acid sequence provided herein as SEQ ID NO: 13 as follows: MGKPIPNPLLGLDSTMERNKTQEEHLKEIMKHIVKIEVKGEEAVKKEAAEKLLEKVPSDVLEMYKAIGGKIYIVDGDITKHISLEALSEDKKKIKDIYGKDALLHEHYVYAKEGYEPVLVIQSSEDYVENTEKALNVYYEIGKILSRDILSKINQPYQKFLDVLNTIKNASDSDGQDLLFTNQLKEHPTDFSVEFLEQNSNEVQEVFAKAFAYYIEPQHRDVLQLYAPEAFNYMDKFNEQEINLSMAGDLSAGFFMEELNTYRQKQGVVLKYQELPNSGPPHDRRFTFQVIIDGREFPEGEGRSKKEAKNAAAKLAVEILNKEHHHHHH [SEQ ID NO: 13] Thus, preferably, the shuttle protein comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 13, or a variant or fragment thereof.

[0050] The inventors consider this to be an important aspect of the present invention.

[0051] Thus, according to a ninth aspect of the present invention, there is provided a shuttle protein comprising a detoxified toxin protein attached to a protein kinase R (PKR).

[0052] Preferably, the shuttle protein according to the ninth aspect comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 11, or a fragment or variant thereof.

[0053] In the most preferred embodiment, the shuttle protein is bound to a bioactive payload molecule. In some embodiments, the binding may include a covalent bond. In alternative embodiments, the binding may include a non-covalent bond. In other words, the payload may be bound to the shuttle protein by a covalent or non-covalent bond. The bioactive payload molecule may be a therapeutically active molecule that is active within the cytosol, within the nucleus, within intracellular structures such as organelles or vesicles or vacuoles, within the cell surface lipid membrane, or within the cell surface lipid membrane or intracellular lipid membranes, but is not limited thereto. The bioactive payload molecule may be active per se, but is not limited thereto, or may be inactive until activated intracellularly. The bioactive payload molecule may also, but is not limited to, be degraded intracellularly to form active or inactive components. The bioactive molecule may be a small molecule, a protein, an RNA molecule or fragment, or a DNA construct, but is not limited thereto. The molecular weight of the bioactive compound may be between 1 Da and 10 MDa.

[0054] The inventors have found that the liposome can effectively load small molecules that are preferably therapeutically active. Thus, in one embodiment, the bioactive payload molecule includes a small molecule. The molecular weight of the small molecule may be from 1 to 900 Da. Alternatively, the molecular weight of the small molecule may be from 100 to 800 Da, from 200 to 700 Da, from 300 to 600 Da, or from 400 to 500 Da. The small molecule may be an agent or drug having agonist or antagonist properties, but is not limited thereto, or may be a dye or a fluorescent molecule.

[0055] The inventors have also found that the liposomes can effectively load large molecules such as therapeutically active or bioactive proteins. Thus, in an alternative embodiment, the bioactive payload molecule comprises a large molecule such as a protein or an enzyme. In a preferred embodiment, the protein comprises an enzyme or a fragment thereof. In a further embodiment, the protein comprises an antibody or an antigen-binding fragment thereof, preferably a monoclonal antibody or an antigen-binding fragment thereof, or an antibody mimetic or an aptamer. In one embodiment, the bioactive payload molecule comprises a Fab or a vNAR.

[0056] In other preferred embodiments, the bioactive payload molecule comprises a diphtheria toxin A (DTA) chain conjugated to LFn having the amino acid sequence provided herein as SEQ ID NO: 15 as follows: MGSSHHHHHHSSGLVPRGSHMAGGHGDVGMHVKEKEKNKDENKRKDEERNKTQEEHLKEIMKHIVKIEVKGEEAVKKEAAEKLLEKVPSDVLEMYKAIGGKIYIVDGDITKHISLEALSEDKKKIKDIYGKDALLHEHYVYAKEGYEPVLVIQSSEDYVENTEKALNVYYEIGKILSRDILSKINQPYQKFLDVLNTIKNASDSDGQDLLFTNQLKEHPTDFSVEFLEQNSNEVQEVFAKAFAYYIEPQHRDVLQLYAPEAFNYMDKFNEQEINLSAMGSSHHHHHHSSGLVPRGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKGFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNR[SEQ ID NO: 15] Therefore, preferably, the bioactive payload molecule comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 15, or a variant or fragment thereof.

[0057] In one embodiment, the bioactive payload molecule is encoded by a nucleic acid sequence provided herein as SEQ ID NO: 16 as follows: Therefore, preferably, the bioactive payload molecule is substantially encoded by a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 16, or a variant or fragment thereof. The use of DTA as a payload is particularly useful for the treatment of cancers such as cervical cancer.

[0058] The inventors have further discovered that liposomes can be loaded with diagnostic agents such as dyes and fluorescent molecules, such as Texas Red (Example 4). Thus, in other embodiments, the bioactive molecule is a diagnostic label. The bioactive molecule may comprise a dye or fluorescent molecule at the time of diagnosis. The bioactive molecule may comprise a protein (e.g., GFP), or a small molecule (e.g., Texas Red) at the time of diagnosis.

[0059] In some embodiments, the bioactive payload molecule or component may also be useful in theranostics (i.e., combined therapeutic and diagnostic applications).

[0060] The bioactive payload molecule may comprise nucleotides, which can be DNA or RNA.

[0061] The inventors have discovered that antisense oligonucleotides (ASOs) can be effectively loaded into the liposomes (such as exosomes). Thus, in one embodiment, the bioactive payload molecule comprises an ASO.

[0062] In one embodiment, the ASO may comprise the anti-tandem dimer tomato ASO sequence provided herein as SEQ ID NO: 17 as follows: ZZE OZE ZOO FOE ZFE ZFE ZFE GCA TGC CGG CAT CAG AGC AGC CGG CAT [SEQ ID NO: 17] In other embodiments, the ASO may comprise the anti-tandem dimer tomato ASO sequence provided herein as SEQ ID NO: 18 as follows: ZZE OZE ZOO FOE ZFE ZFE ZFE GCA TGC CGG CTG CTC TGA TGC CGG CAT [SEQ ID NO:18] Therefore, preferably, the ASO comprises, or consists essentially of, the nucleic acid sequence set forth in SEQ ID NO: 17 or 18, or a variant or fragment thereof. The table in Example 10 illustrates the phosphorothioate code described above.

[0063] The inventors have also found that the liposomes (such as exosomes) can effectively load RNA molecules. Thus, in one embodiment, the bioactive payload molecule comprises RNA. Preferably, the bioactive payload molecule comprises mRNA, miRNA, guide RNA (for use in genome editing), or snRNA. Most preferably, the bioactive payload molecule comprises siRNA.

[0064] In other embodiments, the bioactive payload molecule comprises DNA such as a plasmid.

[0065] As shown in Example 3, the inventors have shown a method for successfully delivering gene editing nucleases such as Cas9 for use in gene editing methods using the liposomes of the present invention. As described above, the inventors have demonstrated that the genome editing nuclease, Cas9, can be encapsulated within the liposomes of the present invention and thus the liposomes can be used in genome editing techniques. Also shown (see Examples 4, 5 and 6), both an RNA analog and an RNA binding protein (LFn-PKR) can be trapped or loaded within the liposomes (e.g., exosomes). As a result, transport of the RNA or RNA analog is possible. This means that both trapping or loading of Cas9 and RNA within the liposomes is shown. Since Cas9 requires a guide RNA (gRNA) for target sequence specificity, both of these aspects are important for the utility of Cas9.

[0066] Accordingly, according to the tenth aspect, liposomes according to either the second or third aspect are provided for use in genome editing techniques.

[0067] According to the eleventh aspect, there is provided a genome editing method comprising: (i) loading a guide RNA into a liposome according to the second or third aspect; and / or (ii) using a nuclease or a gene construct encoding a nuclease, and the liposome loaded in gene editing therapy.

[0068] It will be understood that the genome editing method can be carried out in vitro, in vivo, or ex vivo.

[0069] Accordingly, in one embodiment, the bioactive payload molecule comprises a genome editing tool such as a nuclease. In a preferred embodiment, the bioactive payload molecule comprises Cas9 or Cpf1 or TALEN or zinc finger nuclease. Accordingly, the bioactive payload molecule can be used for transcriptional interference or transcriptional activation in a patient's cells.

[0070] In one embodiment, the genome editing method may comprise loading a construct encoding a nuclease such as Cas9 into a liposome according to the second or third aspect. The construct can be a plasmid or an expression vector containing a nucleic acid sequence encoding a nuclease. In a preferred embodiment, the plasmid encodes Cas9.

[0071] According to other embodiments, there is provided a genome editing method comprising loading a guide RNA targeting a gene sequence to be edited into a liposome according to the second or third aspect.

[0072] In one preferred embodiment, the bioactive molecule comprises Cas9 and binds to LFn having the amino acid sequence provided herein as SEQ ID NO: 14 as follows: Therefore, preferably, the bioactive molecule comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 14, or a variant or fragment thereof.

[0073] The liposomes and the bioactive payload molecules encapsulated therein can be used as a single-agent therapy for treating, ameliorating, or preventing disorders (e.g., FMO5-regulated obesity, prostaglandin-D2, androgenetic alopecia, acne, rosacea, prostate cancer, dengue fever, Ebola virus disease, acquired immunodeficiency syndrome, Stat3-responsive cancer, P53-deficient cancer, virus-mediated cervical cancer, familial hypercholesterolemia, Duchenne muscular dystrophy, spinal muscular atrophy, Crohn's disease, and various inflammatory diseases), or can be used as a nuclease for genome editing (e.g., Cas9). Alternatively, the liposomes according to the present invention can be used as an adjunct to, or in combination with, known therapies for treating, ameliorating, or preventing a disorder or symptoms of a disorder.

[0074] The liposomes according to the present invention can be combined into compositions having several different forms, depending in particular on the way in which the composition is used. Thus, for example, the composition can be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micellar solution, transdermal patch, liposome suspension, or any other suitable form that can be administered to a human or animal in need of treatment. It will be understood that the excipients of the medicament according to the present invention should be well tolerated by the subject to which it is administered.

[0075] The liposomes according to the present invention can also be incorporated into sustained release or delayed release devices. Such devices can be inserted, for example, above or below the skin, and the drug can be released over several hours, days, weeks, or months. The device can be placed at least adjacent to the treatment site. Such devices can be particularly advantageous when long-term treatment with the liposomes is required and usually requires frequent administration (e.g., at least daily injections).

[0076] The drug of the liposomes can be administered to a subject by directly injecting it into the bloodstream, a nerve, or a site in need of treatment. The injection can be intravenous (bolus or infusion) or subcutaneous (bolus or infusion), intradermal (bolus or infusion), intrathecal (bolus or infusion), or an injection into the cerebrospinal fluid (CSF) via epidural or spinal tap (bolus or infusion).

[0077] The amount of liposomes required is determined by its bioactive payload molecule encapsulated inside, as well as its biological activity and bioavailability, which will be understood to depend on the mode of administration, the physicochemical properties of the liposomes and the bioactive payload molecule encapsulated inside, and whether it is used in monotherapy or combination therapy. The frequency of administration will also be affected by the half-life of the payload molecule within the subject being treated as well as the half-life of the target molecule (i.e., the protein). The optimal dosage to be administered can be determined by those skilled in the art and will vary depending on the specific liposomes and specific bioactive payload molecule used, the strength of the pharmaceutical composition, the mode of administration, and the progression of the disorder or condition being treated. Additional factors depending on the specific subject being treated, such as the age, weight, gender, diet, and time of administration of the subject, may require adjustment of the dosage.

[0078] Generally, the daily dose of the payload molecule according to the present invention can be between 0.001 μg / kg body weight and 10 mg / kg body weight, or between 0.01 μg / kg body weight and 1 mg / kg body weight. Depending on the liposomes and bioactive payload molecules used, it is used to treat, improve, or prevent a specific disorder or symptoms of a specific disorder.

[0079] The liposomes can be administered before, during, or after the onset of the disorder or symptoms being treated. The daily dose can be given as a single administration (e.g., a single injection or inhalation of a nasal spray per day). Alternatively, the liposomes may require more than two administrations per day.

[0080] As an example, the liposomes can be administered as a daily dose of two times (or depending on the severity of the disorder being treated) between 0.07 μg and 700 mg (assuming a body weight of 70 kg). The patient being treated can administer the first dose when waking up and then the second dose in the evening (in the case of a two-dose regimen), or thereafter at 3- or 4-hour intervals. Alternatively, a sustained-release device can be used to provide the patient with the optimal dose of the liposomes according to the present invention without the need to administer repeated doses.

[0081] Known procedures such as those conventionally employed in the pharmaceutical industry (e.g., in vivo experiments, clinical trials, etc.) are used to form a specific formulation of the liposomes according to the present invention, an exact treatment regimen (such as the daily dose and frequency of administration of the agent), or the amount of the bioactive payload molecule encapsulated within the liposomes. The inventors believe they are the first to propose the loading of liposomes by the method described herein.

[0082] According to a thirteenth aspect, there is provided a pharmaceutical composition comprising the liposomes according to the second or third aspect and a pharmaceutically acceptable excipient.

[0083] According to the 14th aspect, a method for preparing a pharmaceutical composition according to the 13th aspect is provided, which method includes contacting the liposome according to either the 2nd or 3rd aspect with a pharmaceutically acceptable excipient.

[0084] The "subject" can be a vertebrate, a mammal, or a domestic animal. Thus, the compositions and agents according to the present invention can be used to treat any mammal, for example, a domestic animal (such as a horse), a pet, or can be used in other veterinary applications. However, most preferably, the subject is a human.

[0085] The "therapeutically effective amount" of the liposome and the bioactive payload molecule encapsulated therein is any amount that is the aforementioned amount required to treat a disorder or the symptoms of a disorder when administered to a subject.

[0086] For example, the therapeutically effective amount of the liposome and the bioactive payload molecule encapsulated therein can be from about 0.01 mg to about 800 mg, preferably from about 0.01 mg to about 500 mg. The amount of the encapsulated liposome and the bioactive payload molecule is preferably an amount from about 0.1 mg to about 250 mg, most preferably from about 0.1 mg to about 20 mg.

[0087] The "pharmaceutically acceptable excipient" referred to herein is any known compound or combination of known compounds that are known to those skilled in the art to be useful in formulating a pharmaceutical composition.

[0088] In one embodiment, the pharmaceutically acceptable excipient can be solid, and the composition can be in the form of a powder or a tablet. Solid pharmaceutically acceptable excipients can include one or more substances that can also act as flavoring agents, lubricants, solubilizing agents, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet disintegrants. The excipient can also be a encapsulating material. The pharmaceutically acceptable excipient can also be configured for controlled release in the body, such as in the stomach, blood, or other internal organs and structures, by using appropriate encapsulation, such as enteric encapsulation. In the powder, the excipient is a finely divided solid mixed with the finely divided active agent according to the present invention. In the tablet, the active agent (e.g., the encapsulated liposomes and bioactive payload molecules according to the present invention) can be mixed with an excipient having the necessary compression properties in an appropriate ratio and compressed into a desired shape and size. The powder and the tablet preferably contain up to 99% of the active agent. Suitable solid excipients include, for example, calcium phosphate, magnesium stearate, talc, sugar, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting wax, and ion exchange resins. In other embodiments, the pharmaceutical excipient can be a gel, and the composition can be in the form of a cream or the like.

[0089] However, the pharmaceutical excipient can be a liquid, and the pharmaceutical composition is in the form of a solution. The liquid excipient is used in the preparation of solutions, suspensions, emulsions, syrups, elixirs, and pressurized compositions. The liposomes according to the present invention can be dissolved or suspended in a pharmaceutically acceptable liquid excipient such as water, an organic solvent, a mixture of both, or a pharmaceutically acceptable oil. The liquid excipient can contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers, or osmotic pressure regulators. Examples of liquid excipients suitable for oral and parenteral administration include water (partially containing additives such as those described above, for example, a sodium carboxymethylcellulose solution), alcohols (including monohydric and polyhydric alcohols such as glycols) and their derivatives, and oils (for example, fractionated coconut oil and peanut oil). In the case of parenteral administration, the excipient can be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid excipients are useful in compositions in sterile liquid form for parenteral administration. The liquid excipient for pressurized compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.

[0090] Liquid pharmaceutical compositions that are sterile solutions or suspensions can be utilized, for example, by intramuscular, intrathecal, epidural, intraperitoneal, intravenous, and particularly subcutaneous injection. The liposomes can be prepared as a sterile solid composition that can be dissolved or suspended at the time of administration using sterile water, physiological saline, or other suitable sterile injection media.

[0091] The liposomes of the present invention can be orally administered in the form of a sterile solution or suspension containing other solutes or suspending agents (e.g., physiological saline or glucose sufficient to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monolaurate, polysorbate 80 (oleic acid ester of sorbitol and its anhydride copolymerized with ethylene oxide), etc. The liposomes according to the present invention can also be orally administered in either the form of a liquid or solid composition. Compositions suitable for oral administration include solid forms such as pills, capsules, granules, tablets, and powders, as well as liquid forms such as solutions, syrups, elixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions. Alternatively, the liposomes can be administered rectally, for example via an enema.

[0092] It will be understood that the present invention extends to any nucleic acid or peptide or variant, derivative or analog substantially comprising an amino acid or nucleic acid sequence of any of the sequences referred to herein, including variants or fragments thereof. The terms "substantially amino acid / nucleotide / peptide sequence", "variant" and "fragment" can be sequences having at least 40% sequence identity with any one of the amino acid / nucleotide / peptide sequences of any of the sequences referred to herein, for example, sequences having 40% identity with the sequences identified as SEQ ID NOs: 1 to 14.

[0093] Amino acid / polynucleotide / polypeptide sequences having more than 65%, more preferably more than 70%, even more preferably more than 75%, even more preferably more than 80% sequence identity to any of the referenced sequences are also contemplated. Preferably, said amino acid / polynucleotide / polypeptide sequence has at least 85% identity, more preferably at least 90% identity, even more preferably at least 92% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, and most preferably at least 99% identity to any of the sequences referred to herein.

[0094] One of ordinary skill in the art will understand how to calculate the percentage of identity between two amino acid / polynucleotide / polypeptide sequences. To calculate the percentage of identity between two amino acid / polynucleotide / polypeptide sequences, it is first necessary to prepare an alignment of the two sequences and then calculate the sequence identity value. The percentage of identity between said two sequences can take different values depending on: (i) the method used to align the sequences, for example, ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison; and, (ii) the parameters used in the alignment method (such as local alignment and global alignment), the pair score matrix used (such as BLOSUM62, PAM250, Gonnet, etc.), the gap penalty, for example, the functional form and the constant.

[0095] After alignment, there are many different ways to calculate the percentage of identity between two arrays. For example, the number of IDs can be classified as follows: (i) the length of the shortest sequence; (ii) the length of the alignment; (iii) the average length of the sequences; (iv) the number of positions without gaps; or (v) the number of equivalent positions excluding overhangs. Furthermore, it will be understood that the percentage of identity also strongly depends on the length. Thus, the shorter the pair of arrays, the higher the sequence identity that can be expected to occur by chance.

[0096] Thus, it will be understood that the accurate alignment of protein or DNA sequences is a complex process. The popular multiple alignment program ClustalW (Thompson et al., 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et al., 1997, Nucleic Acids Research, 24, 4876-4882) is a preferred method for generating multiple alignments of proteins or DNAs according to the present invention. The appropriate parameters for ClustalW are as follows: For DNA alignment: gap open penalty = 15.0, gap extension penalty = 6.66, and matrix = identity.

[0097] For protein alignment: gap open penalty = 10.0, gap extension penalty = 0.2, and matrix = Gonnet. For DNA and protein alignment: ENDGAP = -1, and GAPDIST = 4. Those skilled in the art will notice that they may need to change these and other parameters for optimal sequence alignment.

[0098] Preferably, the percentage identity between two amino acid / polynucleotide / polypeptide sequences can be calculated from an alignment such as (N / T)*100, where N is the number of positions at which the sequences share identical residues, and T is the total number of positions compared, which includes gaps and may or may not include overhangs. Preferably, overhangs are included in the calculation. Thus, the most preferred method for calculating the percentage identity between two sequences includes the following: (i) preparing a sequence alignment using the ClustalW program with an appropriate set of parameters, such as as described above; and, (ii) inserting the values of N and T into the following formula: - sequence identity = (N / T)*100.

[0099] Alternative methods for identifying similar sequences will be known to those of skill in the art. For example, substantially similar nucleotide sequences would be encoded by sequences that hybridize to the DNA sequences or their complements under stringent conditions. By stringent conditions, the inventors mean hybridizing the nucleotides to DNA or RNA bound to a filter in about 3-fold sodium chloride / sodium citrate (SSC) at about 45° C., followed by washing at least once with about 0.2-fold SSC / 0.1% SDS at about 20-65° C. Alternatively, substantially similar polypeptides can differ by, for example, at least one but less than 5, 10, 20, 50 or 100 amino acids from the sequences shown in SEQ ID NOs: 1-14.

[0100] Due to the degeneracy of the genetic code, it is clear that any of the nucleic acid sequences described herein can be modified or altered without substantially affecting the sequence of the protein encoded thereby, to provide functional variants thereof. Suitable nucleotide variants have sequences that are altered by substitution of different codons that encode the same amino acid within the sequence, thus resulting in silent (synonymous) changes. Other suitable variants have homologous nucleotide sequences, but include all or part of the sequence, and the sequence is altered by substitution of different codons that encode amino acids having side chains with biophysical properties similar to the amino acid it replaces, to produce conservative changes. For example, small non-polar hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large non-polar hydrophobic amino acids include phenylalanine, tryptophan, and tyrosine. The polar neutral amino acids include serine, threonine, cysteine, asparagine, and glutamine. The positively charged (basic) amino acids include lysine, arginine, and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Thus, it is understood which amino acids can be replaced with amino acids having similar biophysical properties, and those skilled in the art will know the nucleotide sequences encoding these amino acids.

[0101] All features described herein (including the appended claims, abstract and drawings), and / or all steps of any method or process thus disclosed, may be combined in any combination with any of the above aspects, except combinations in which at least some of said features and / or steps are mutually exclusive.

[0102] For a better understanding of the present invention and to show how embodiments of the present invention may be carried out, reference is made here, by way of example, to the accompanying drawings.

Brief Description of the Drawings

[0103]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Mode for Carrying Out the Invention

[0104] Examples The present inventors have developed a novel method for generating liposomes (e.g., exosomes), and a new cell transport system comprising these liposomes for the stealth transport of biologically and therapeutically active payload molecules such as small molecules, antisense oligonucleotides (ASO), RNA molecules (e.g., siRNA), bioactive proteins, genome editing tools (e.g., cas9), and drugs to cells for treating various disorders.

[0105] Referring to Fig. 7, a schematic diagram summarizing the four stages (described below) in which the liposomes of the present invention are generated is shown.

[0106] 1. The pore-forming protein (e.g., PA83) oligomerizes in the cell membrane to form a pore, and the shuttle protein (e.g., LFn or PKR) which may interact with a bioactive payload molecule (e.g., siRNA or Cas9 or ASO, etc.) interacts with the pore-forming protein and is taken into the cell by endocytosis, thereby forming an endocytic vesicle.

[0107] 2. In the endosomal sorting complex required for transport (ESCRT) mechanism, the endocytic vesicle loaded with the pore-forming protein and the shuttle protein which may be bound to the bioactive payload molecule is transported to the multivesicular body (MVB), where the endocytic vesicle forms intraluminal vesicles (ILVs).

[0108] 3. During the apoptosis-linked gene 2 interacting protein X (ALIX)-dependent process, the intracellular membrane system is disrupted by an inverse fusion event between the ILV within the MVB and the inner boundary membrane of the MVB (this usually causes fusion with lysosomes and destruction of the ILV), accessing the cytosol.

[0109] 4. Next, the MVB containing the ILV loaded with the pore-forming protein and the shuttle protein to which the bioactive payload molecule may be attached is released from the liposome / exosome containing the pore-forming protein and the shuttle protein to which the bioactive payload molecule may be attached.

[0110] Materials and Methods General Chemicals, Fluorescent Probes and Reagents General experimental reagents were obtained from Sigma Aldrich (Dorset, UK) unless otherwise specified. Texas Red-N-hydroxysuccinimide ester (TxR-SE) was obtained from Invitrogen (Paisley, UK). Dulbecco's minimum essential medium, Eagle's minimum essential medium, non-essential amino acids, penicillin, streptomycin, and glutamine solution were all from Gibco (ThermoFisher Scientific, Paisley UK), Blasticidin solution was from Invitrogen (Paisley UK), and ionomycin was from Sigma Aldrich (Dorset, UK). Mouse monoclonal anti-CD63 was from AbCam and monoclonal anti-Lamp2 was from DHSB (University of Iowa, IA, USA). Alexaflour 488-labeled goat anti-mouse antibody was obtained from Invitrogen (Paisley UK). Goat anti-Texas Red monoclonal antibody and HRP-labeled donkey anti-goat antibody were obtained from VectorLabs. The exoEasy Maxi kit (20) (catalog number: 76064) was from QIAgen and the Total exosome Isolation reagent (from cell culture medium) (catalog number: 4478359) (PEG solution) was from Invitrogen (Paisley UK). StealthRNAi TM siRNAGFP Reporter Control (catalog number: 12935145) was obtained from Invitrogen (Paisley UK) and provided as a 20 μM solution.

[0111] Exosome-free media Bovine liposomes / exosomes in FCS were sedimented at 180,000 x g for 18 hours at 4°C. The supernatant was collected, added to serum-free but complete medium (MEM), and filter-sterilized (0.2 μm filter, Sartorus) under negative pressure.

[0112] Cell culture The cultivation and subculture of HeLa (ATCC: CCL2) and HEK293 (AMSBIO: SCOO8) cells were performed according to the supplier's instructions. Cells for microscopy were seeded onto sterile cover slips at a density of 1×105 cells / well. Fixation, antibody hybridization, and detection were performed as previously described [2]. CD63 immunostained cells were fixed with cold methanol.

[0113] Protein production, isolation, and concentration The DNA sequences encoding protein PA83 (based on GenBank accession numbers AAF86457 and AAT98414) have been previously described [2]. LFn-PKR was synthesized by BioBasic Inc. (Ontario, Canada) using GenBank accession numbers AAY15237 (for LFn) and NM_002759 (for PKR). The open reading frame encoding LFn-PKR was subcloned into the bacterial expression cassette pET151 / D (Invitrogen, Paisley, UK) as previously described in [2] and PCT / GB2014 / 051918. Plasmids encoding LFn-Staphylococcus aureus (Sa) Cas9 and GST-PA63 were synthesized by Invitrogen using the pET151 bacterial expression system as the parental plasmid. The GST sequence used was from pGEX3x, and the SaCAS9 sequence was codon-optimized from Genbank accession number CCK74173.1 (i.e., SEQ ID NO: 14). The addition of a V5 epitope tag and a 6x histidine affinity tag allowed for immunodetection and affinity purification of the protein from bacterial lysates.

[0114] LFn-PKR and PA83 were concentrated from the E. coli culture supernatant at a yield of approximately 2 mg / L (LFn-SaCas9 was approximately 0.5 mg / L). Chemically competent E. coli BL21*DE3pLys (Invitrogen, Paisley, UK) was transformed with 10 ng of plasmid, cultured overnight in 2xYT containing 200 μg / mL ampicillin (Sigma, Dorset, UK), and grown for 3 h at 37 °C and 200 rpm in 1000 mL of 2xYT. Subsequently, isopropylthio-β-galactoside (IPTG) (Sigma, Dorset, UK) was added to a final concentration of 1 mM and incubated for an additional 3 h. Bacterial pellets prepared by centrifugation (6000 xg for 6 min at 4 °C) were lysed using a French press (Thermo Scientific, Paisley, UK) set at 15,000 psi. The lysate was clarified (20,000 xg for 20 min at 4 °C), and the supernatant was passed over a 6x histidine affinity chromatography column (Talon(R) resin; Clontech, Saint-Germain-en-Laye, France). 6xHis-tagged proteins were eluted in 1 mL fractions using 150 mM imidazole in PBS (Sigma, Dorset, UK). Protein fractions were analyzed for purity and concentration, pooled, dialyzed thoroughly against PBS, and finally filter sterilized (0.2 μm filter, Sartorus). The final protein preparation was evaluated by SDS-PAGE and subjected to Coomassie staining (to determine purity) and Western blot analysis using the described antibodies.

[0115] Probe synthesis and characterization LFn-PKR-TxR and LFn-SaCas9-TxR were prepared using the method described previously

[14] .

[0116] Briefly, TxR-SE (5 mg) was dissolved in DMSO (5 mL). 100 μL of the TxR solution was added to approximately 5 mg of recombinant protein in 2.5 mL of PBS and placed in the dark at 25 °C for 1 hour. The product was purified using a PD-10 column (GE Healthcare, Chalfont St Giles, UK) and PBS as the eluent, and 0.5 mL fractions were collected. Next, the fraction with the highest optical density was selected and pooled to obtain either the LFn-PKR-TxR or LFn-SaCas9-TxR conjugate. The fluorescent conjugate was then filter sterilized (0.2 μm filter, Sartorus) and frozen at -80 °C.

[0117] Cell culture for exosome loading Cells were seeded in a 175 cm2 TC-treated dish and grown under standard incubation conditions, i.e., at 37 °C in 5% (v / v) CO2. At 90% confluence, the cell monolayer was washed three times with PBS and then exosomes were loaded.

[0118] Loading of LFn-PKR-TxR or LFn-SaCas9-TxR into liposomes / exosomes Cells were incubated with PA83 (50 μg / mL) and LFn-PKR-TxR (50 μg / mL) or LFn-SaCas9-TxR (50 μg / mL) in 3 mL of serum-free DMEM at 37 °C for 1 hour. After 1 hour, 5 mL of exosome-free DMEM containing 10% (v / v) FCS was added to a final volume of 8 mL and the plate was incubated at 37 °C for 3 hours.

[0119] Loading of LFn-PKR::siRNA into liposomes / exosomes LFn-PKR (50 μg / mL) was incubated with GFP siRNA (50 nM) in serum-free DMEM for 5 minutes before adding PA83 (50 μg / mL). Next, the mixture was added to the cell monolayer and the cells were incubated at 37 °C for 1 hour. After 1 hour, 5 mL of exosome-free DMEM containing 10% (v / v) FCS was added to a final volume of 8 mL, and the plates were incubated at 37 °C for 3 hours.

[0120] Isolation of Liposomes / Exosomes Ionomycin (50 μM) was added to the medium and incubated for 30 minutes under standard conditions. Next, the medium was collected and centrifuged at 1,500 x g for 2 minutes at 4 °C to pellet cell debris. The resulting supernatant was filter-sterilized (0.8 μm, Sartorous) before freezing or exosome isolation. Exosome isolation was performed using any of the following three methods.

[0121] 1. Differential Centrifugation This was modified from

[15] . Briefly, the frozen-filtered conditioned medium was thawed on ice and exposed to 10,000 x g for 30 minutes to precipitate EVs. Next, the supernatant was exposed to 110,000 x g for 70 minutes at 4 °C, and the pellet was collected in 1 mL of PBS. Next, the resuspended pellet was subjected to a second precipitation at 100,000 x g for 70 minutes at 4 °C. Next, the resulting pellet was suspended in 1000 μL of exosome-free medium, filtered through a 0.2 μm filter (Sartorus), and stored frozen at -20 °C until needed.

[0122] 2. Separation by Polyethylene Glycol Precipitation Briefly, the volume of clarified and filtered cell culture medium was estimated and 0.5 volume of isolation reagent was added to this. This preparation was left at 4 °C overnight. Next, the mixture was centrifuged at 10,000 x g for 1 hour at 4 °C. Next, the resulting pellet was suspended in a final volume of 1000 μL of exosome-free medium and stored frozen at -20 °C until needed.

[0123] 3. Separation by membrane adsorption This was carried out using the QIAgenexoEasy kit according to the manufacturer's specifications. Briefly, 8 mL of XPB buffer was mixed with the cell culture reagent, and an additional step was added to remove the elution (XE) buffer from the exosome preparation after separation. This was achieved by centrifuging the eluate at 110,000 x g for 70 minutes at 4 °C. Next, the obtained pellet was suspended in 1000 μL of exosome-free medium and stored frozen at -20 °C until needed.

[0124] Quantification of proteins The bicinchoninic acid assay (BCA) assay was performed according to the specifications of the bicinchoninic acid kit (BCA-1) (Sigma Aldrich, Dorset UK) to measure the protein concentration of the final exosome sample before storage. Additionally, a μlite (BioDrop Inc.) device was used to measure the DNA and protein concentrations at OD 260 and OD 280 respectively, according to the manufacturer's recommended protocol.

[0125] Microscopy Microscopic visualization of liposomes / exosomes was performed by mixing equal amounts of exosome preparations with equal amounts of cell mask reagent incorporating the Cy5 fluorophore (catalog number C10046; Invitrogen, Paisley, UK). Thereby, imaging of liposomes / exosomes under fluorescence was performed using an LSM880 laser scanning confocal microscope equipped with an Airyscan unit (Carl Zeiss Ltd, Germany). The super-resolution function of the Airyscan unit enabled the resolution of liposomes / exosomes. In the case of liposomes / exosomes loaded with proteins labeled with Texas Red, the liposomes / exosomes were directly visualized using the super-resolution function of the Airyscan unit of the LSM880 (Carl Zeiss Ltd, Germany). In both cases, a Plan-Apochromat 63x / 1.40 numerical aperture oil DICf / ELYRA objective lens was used. Immunostaining was performed on either paraformaldehyde-fixed or cold (20 °C) methanol-fixed cells grown on coverslips as described above.

[0126] Assay of siRNA activity To evaluate the transport of siRNA, a control siRNA specific for GFP was purchased. This was directed against a stably expressed transgene expressed in HEK293 (SCOO8) cells and used as a standard for reporting gene activity. HEK293 cells overexpressing GFP fused in-frame to beta-galactosidase, an enzyme involved in the hydrolysis of x-gal from a colorless precursor to an insoluble blue compound, were detected spectrophotometrically at 620 nm. As a result, GFP siRNA activity could be monitored by measuring X-gal conversion via beta-galactosidase. Finally, beta-galactosidase activity was expressed as a percentage of the untreated control after normalization to protein concentration (OD 620 )).

[0127] Cell culture for gene regulation assays Activity was evaluated using a 6-well plate. Cells were seeded at 5×105 cells / well (HEK293, AMSBIO) and incubated at 37 °C with 5% (v / v) CO 2 for 24 h before treatment.

[0128] Administration of liposomes / exosomes Cells were treated with 200 μL of exosome preparation diluted in 2 mL of complete medium. This preparation was filter sterilized (0.2 μm filter, Sartorus) and then incubated with the cells for the desired times (24 h, 48 h, 72 h).

[0129] Analysis of gene regulation The medium was discarded and the cell monolayer was carefully washed three times with chilled PBS, after which 500 μL of RIPA buffer (R0278-50ML, Sigma Aldrich) was added to each well. After a 15-min incubation period on ice, the cell lysate from each well was aspirated 10 times and decanted into labeled Eppendorf tubes. After centrifugation at 21,000 x g for 10 min at 4 °C, the supernatant was transferred to a new Eppendorf tube and the pellet was discarded. Next, 10 μL of the lysate was added to 100 μL of 2% BCA reagent in a 96-well plate and incubated at 37 °C. The remaining 400 μL of the supernatant was mixed with 12 μl of X-gal (50 mM in DMSO) (R0404, ThermoFisher) and transferred to a 96-well plate at 100 μL / well. X-Gal conversion was assayed over time at 620 nm using a spectrophotometer set at 37 °C (readings were taken every 15 min for 5 h).

[0130] Western blotting and TCA precipitation Western blotting and immunodetection were performed using a Mini-PROTEAN Tetra cell apparatus (BioRad) according to the manufacturer's instructions. For protein separation, a 10% (w / v) acrylamide gel was used and run at 200 V for 60 minutes. Transfer to a nitrocellulose membrane was performed at 400 mM for 60 minutes. Blocking was carried out for 45 minutes using a 5% (w / v) non-fat dry milk solution in PBS containing 0.1% (v / v) Tween 20 reagent. Antibody hybridization was performed at 37 °C for 60 minutes with 3 mL under shaking conditions using the antibody diluent proposed by the manufacturer. Detection of the HRP-labeled secondary antibody was performed using an enhanced ECL reagent (Pierce, ThermoFisher Scientific) according to the manufacturer's instructions. Gels and blots were calibrated by running a broad range of prestained protein markers (Invitrogen). TCA precipitation of exosome proteins was performed by adding 0.6 volumes of TCA to the exosome preparation. This was then incubated at 4 °C for 30 minutes. Next, the preparation was sedimented at 21,000 x g for 10 minutes at 4 °C and the pellet was washed twice with acetone, also at 4 °C. The resulting pellet was dissolved in Laemmli buffer and Western immunoblotting was performed, probing with either an anti-LAMP2 specific primary antibody (DHSB, University of Iowa, Iowa, USA) under non-reducing conditions; or a Texas Red specific primary antibody (Vectorlabs) using the diluent proposed by the manufacturer.

[0131] Example 1 - Liposomes are taken up by cells As shown in Fig. 1, the cargo labeled with Texas red (i.e., LFn-PKR labeled with Texas Red) localizes to the luminal structures within HeLa cells. Since these luminal structures are positive for the exosome immune marker CD63 (also known as LAMP3), this luminal signal may be present within multivesicular endosomes, i.e., late endosomes. This indicates that LFn-PKR labeled with Texas red can preferentially label the luminal inner membrane within MVE / MVB 3 hours after being added to the cells when added to the cells with PA83.

[0132] Example 2 - Liposomes are of a size similar to physiological exosomes Fig. 2 shows the inventors' discovery that liposomes / exosomes isolated from Hela conditioned medium, stained with Cy5-Cell Mask, and visualized using an Airyscan detector are of approximately the appropriate size (60 - 120 nm) for exosomes. Note that the limit of resolution of this system is 120 nM in the x-y plane. Liposomes / exosomes isolated from the exoEasy kit were also subject to immunoblotting analysis using LAMP2 as a probe, and as predicted, the bands were seen at approximately the appropriate molecular weight within the exosome preparation. This means that not only are the liposomes / exosomes isolated using the exoEasy kit of approximately the correct size, but they also contain exosome immune markers that were adequately characterized as predicted.

[0133] Example 3 - Cas9 can be loaded into liposomes The liposomes / exosomes are effectively loaded with Staphylococcus aureus Cas9, that is, SaCAS9. In Figure 3, the red signal from LFn-SaCAS9 can be clearly seen in the exosome preparation from Hela cells exposed to LFn-SaCAS9 labeled with PA83 and TexasRed. Control liposomes / exosomes Rwith: without PA83, without cargo, or non-translocated cargo (BSA-Texas Red) did not generate a red signal even when incubated with a cell mask to confirm the focal plane (Figure 3B).

[0134] Example 4 - Small molecules can be loaded into liposomes It has been further shown that liposomes / exosomes can effectively load small molecules. In Figure 4, it is shown that the small molecule Texas Red is incorporated into the exosome preparation from Hela cells exposed to LFn-PKR or PA83 labeled with PA83 and TexasRed after 3 hours. BSA labeled with TexasRed was used as a negative control. Here, LFn-PKR labeled with Texas Red can be easily detected within the cell mask positive population of liposomes / exosomes, but BSA labeled with Texas Red cannot be detected.

[0135] Example 5 - The loaded protein is present in isolated liposomes Figure 5 shows the results of TCA-precipitated liposomes / exosomes prepared with either PA83 and Texas Red-labeled LFn-SaCAS9, Texas Red-labeled LFn-PKR, or Texas Red-labeled BSA. Texas Red is clearly visible in the pellet from preparations of LFn-SaCAS9 labeled with PA83 Texas Red and LFn-PKR labeled with PA83 Texas Red. Texas Red cannot be readily detected in the "untreated" or "treated" controls of PA83 and BSA-Texas Red. Similarly, after immunoblotting and detection using a Texas Red-specific primary antibody, Texas Red labeling a protein of the predicted molecular weight was detected from the same TCA precipitate as before.

[0136] Example 6 - Liposomes loaded with siRNA are effective in protein downregulation Figure 6 shows the biological activity of liposomes / exosomes isolated from cell culture media using the exoEasy kit and fractionation centrifugation. Here, a decrease in beta-galactosidase activity per unit cell protein was recorded, showing the following: 1) siRNA in liposomes / exosomes and 2) the biological activity of liposomes / exosomes loaded with siRNA, i.e., their ability to transport siRNA into the cytosol of a second population of cells.

[0137] Example 7 - Treatment of Zika virus infection As a theoretical example, liposomes generated by the method of the first aspect are used to treat a patient infected with Zika virus. First, a biopsy is performed on the patient and many of the patient's cells are isolated. Next, liposomes are generated from the cells using the method of the first aspect and loaded with anti-Zika virus siRNA. Next, the liposomes containing anti-Zika virus siRNA are administered to the patient and taken up by the target cells via endocytosis. Thus, the anti-Zika virus siRNA is present in the patient's cells and the ability of Zika virus to proliferate in the patient's body is inhibited.

[0138] Example 8 - Treatment of Obesity As another theoretical example, liposomes generated by the method of the first aspect are used to treat patients suffering from FMO5-regulated obesity. In this example, the liposomes are derived from cultured mesenchymal stem cells using the method of the first aspect and loaded with anti-FMO5 siRNA. Next, the liposomes containing anti-FMO5 siRNA are administered to the patient and taken up by the patient's cells via endocytosis. Thus, the anti-FMO5 siRNA is present in the patient's cells, the FMO5 enzyme is downregulated, and the patient no longer exhibits obesity-related symptoms.

[0139] Discussion The inventors used PA83::LFn fusions to load materials into liposomes / exosomes, including low molecular weight covalent conjugates (i.e., Texas Red), LFn-PKR::siRNA, Texas Red conjugated to LFn-PKR, LFn-Gal4::eGFP-Rab5, and Texas Red conjugated to LFn-SaCAS9. Subsequently, the data presented herein support the new idea that LFn fusion proteins can transport selective cargos (not only LF or EF) into a biologically derived stealth delivery system called exosomes. Herein, a methodology for achieving exosome loading without overexpression of the cargo or disruption of the exosomes is first disclosed, and evidence supporting this conclusion is discussed.

[0140] Figure 1 shows that LFn-PKR labeled with Texas Red can bind to luminal vesicles within CD63-positive membrane-bound structures in the presence of PA83. These data support the hypothesis that LFn-PKR uses a similar cytosolic trafficking pathway as wild-type LF [3]. Figure 2 verifies the isolation of liposomes / exosomes through both microscopic examination (bladder size measurement) and immunoblotting detection of the exosome marker LAMP2 within the isolated population of liposomes / exosomes using the QIAgen exoEasy kit. This data indicates that the liposomes / exosomes were actually isolated. Figure 3 shows evidence that the isolated liposomes / exosomes contain a cargo protein labeled with the fluorophore Texas Red. In this case, the cargo protein is LFn-SaCAS9, which has previously been reported to have a low likelihood of functioning as a PA translocase substrate. Here, LFn-SaCas9 is recorded within the isolated population of liposomes / exosomes. Figure 4 (panel a) is a replication of this methodology and shows reproducibility only when using a different cargo protein: Texas Red-labeled LFn-PKR. This further demonstrates the ability of this system to move selected small molecules covalently bound to a PA pore substrate, such as an LFn fusion protein, into the population of liposomes / exosomes. Figure 4 (panel B) functions as a negative control showing that: 1) BSA-labeled - Texas Red does not function as a PA translocase substrate, 2) the recorded signal is specific to Texas Red and does not result from either bleed-through autofluorescence from the Cy5 cell mask channel.

[0141] Figure 5 shows that the Texas Red signal from isolated liposomes / exosomes can be precipitated using trichloroacetic acid (TCA) (i.e., bound to proteins), and that after Western analysis, it was of the predicted molecular weight. This indicates that there is no damage to the protein and that the Texas Red fluorophore remains bound even after PA pore translocation. Figure 6 shows that siRNA can be loaded into liposomes / exosomes using PA and LFn-PKR, and that these liposomes / exosomes can be isolated by the exoEasy kit or by fractionation centrifugation. It also shows that the liposomes / exosomes are active, have recipient cell fusion ability, and can transport pharmacologically active siRNA. This is a proof of concept, i.e., demonstration that cargos can be loaded and transported into liposomes / exosomes using the described exosome loading method, and that liposomes / exosomes can be isolated and biologically active cargos transferred from one cell population to another. This supports the idea that this methodology can be used to load drugs into exosomes derived from ex vivo expanded patient cells to facilitate tertiary targeting and stealth transport of embodied precision medicine such as siRNA, gene editing proteins, gRNA, shRNA, miRNA, genes, and therapeutic proteins.

[0142] In attempts to optimize the loading of substances into liposomes / exosomes, PA63 (generated as PA63-TEV recognition site-GST), PA83, PA83 D512K, PA83 G to N, and PA83 N to S

[16] were also investigated for their ability to load liposomes / exosomes. Finally, a PA83 hybrid molecule incorporating the transmembrane domain of hemolysin

[17] instead of the PA63 transmembrane domain was encoded in a bacterial expression cassette (pET151) and constructed to investigate the role of the PA83 transmembrane assembly with respect to the rate-limiting ratchet phenomenon of Brownian motion during pore transport of the cargo [6].

[0143] Example 9 The inventors loaded exosomes with the conditional lethal cargo LFn-diphtheria toxin A chain (DTA) using PA83 as described above, except for the plasmid used, i.e., the plasmid was from Addgene (pET-15b LFn-DTA, Addgene number 11075) (https: / / www.addgene.org / 11075 / ).

[0144] Loading of exosomes Exosomes were loaded by incubating LFnDTA-sequence number 15 (protein) and sequence number 16 (DNA)-with HeLa cells at a concentration of 10 μg / ml, and 50 μg / ml of sequence number 2 (PA83) in a humid atmosphere containing 5% (v / v) CO 2 for 4 hours at 37°C. Thereafter, the cells were washed with PBS and incubated with 5 μM ionomycin (Sigma Chemical catalog number I9657-1MG) in serum-free medium. Next, exosomes were immediately isolated from the conditioned medium using fractionation centrifugation as described above.

[0145] Trypsin digestion To half of the exosome preparation, 5 μl of cell culture trypsin / EDTA (TE) buffer (ThermoFisher Scientific catalog number 25200056) was added to the exosomes and the volume was adjusted to 100 μl with PBS. To the other half of the preparation, 100 μl of PBS was added. Next, the exosome preparation was incubated for 60 minutes under conditions that have already been demonstrated to be sufficient to digest 5 μg of LFnDTA, which greatly exceeds the amount of LFnDTA contained in the exosome preparation. Next, the exosomes were added to a culture of HeLa cells containing a trypsin control (which has been found to be non-toxic), and cell viability was assayed 24 hours later. The results were expressed as DTA activity (%) normalized to the untreated control (LFnDTA-containing exosomes killed approximately 45% of the cells).

[0146] Results Referring to Figure 8, exosomes initially loaded with cargo (i.e., LFn-diphtheria toxin A chain (DTA)-SEQ ID NO: 15 and SEQ ID NO: 16) using PA83 (i.e., SEQ ID NO: 2) were then characterized to show that they were protected from the activity of an external enzyme (i.e., trypsin). These data indicate that LFnDTA, which can be used for the treatment of cancers such as cervical cancer, can be successfully loaded into exosomes.

[0147] Example 10 In this example, the inventors characterized the size of exosomes and extracellular vesicles (EVs) isolated using differential centrifugation by means of dynamic light scattering AKA photon correlation spectroscopy.

[0148] Exosome Loading Exosomes from HeLa cells were loaded with phosphorothioate-phosphodiester hybrid antisense oligonucleotides (ASOs) pre-hybridized to tandem dimer tomato at a concentration of 200 pMol / ml (total ASO)-SEQ ID NO: 17 and SEQ ID NO: 18. The following table describes the phosphorothioate code (obtained from the Thermofisher website).

[0149] [Table 1]

[0150] These ASOs were loaded into exosomes using 50 μg / ml LFnPKR and 50 μg / ml SEQ ID NO: 2 (PA83) or 50 μg / ml PA forced octamer mutant. This mixture was incubated with HeLa cells in serum-free medium at 37 °C for 4 h in a humidified atmosphere containing 5% (v / v) CO 2 and then the medium was removed, the cells were washed with PBS, and then incubated with 5 μM ionomycin in serum-free medium for 30 min.

[0151] Isolation of Exosomes The conditioned medium was first removed by centrifugation at 1.5 kxg for 2 minutes and filtered through a 0.8 micron filter. The flow-through was centrifuged at 10,000 xg for 30 minutes at 4 °C. Finally, the exosomes were isolated by sedimentation at 110,000 xg for 70 minutes at 4 °C, resuspended in PBS, and resedimented at 110,000 xg for 70 minutes at 4 °C. The exosomes were stored at 4 °C until then. When used for cell culture, the exosomes were diluted with the required amount of serum-free medium and filtered again through a 0.8 μ filter before incubating with the cells.

[0152] Results Referring to Figure 9, it was shown that when loading 200 pMol / ml anti-tandem dimer Tomato (TdTom) antisense oligonucleotide (ASO) using 50 μg / ml PA83 (SEQ ID NO: 2) and 50 μg / ml LFnPKR (SEQ ID NO: 13), the size of the exosomes was predicted.

[0153] Referring to Figure 10, it was also shown that when loading 200 pMol / ml anti-TdTom ASO using the forced octamer PA83 mutants, namely 25 μg / ml PA83 D512K (SEQ ID NO: 7) and 25 μg / ml PA83 K245G;R252N (SEQ ID NO: 8) with 50 μg / ml LFnPKR (SEQ ID NO: 13), the exosomes had the predicted size. These data indicate that the generated membrane fractions are of the predicted size of exosomes and EVs.

[0154] Example 11 The inventors tested whether exosomes loaded with active ASO retain their pharmacological activity.

[0155] Loading of Exosomes HeLa cells were loaded with exosomes by incubating them with 200 pMol / ml anti-TdTom ASO (SEQ ID NO: 17 and SEQ ID NO: 18) and 50 μg / ml heptamer (PA83 - SEQ ID NO: 2), or a forced octamer PA83 mutant (i.e., 25 μg / ml - SEQ ID NO: 7 (PA83 D512K) and 25 μg / ml SEQ ID NO: 8 (PA83 K245G;R252N)) and 50 μg / ml SEQ ID NO: 13 (LFnPKR). After separation, these ASO-loaded exosomes showed antisense activity against mRNA that bicistronically expresses a tandem dimer tomato that encodes GFP-fused β-galactosidase and is overexpressed in HEK293 cells (ASMBIO catalog number SC008). HEK cells (approximately 1x106 per well) were treated as follows: 57 μg [total protein measured at OD 280 of heptamer::ASO exosome prep or 42 μg [total protein measured at OD 280 of octamer::ASO.

[0156] Results Referring to Figure 11, exosomes loaded with ASO have been shown to have pharmacological activity. These data show that ASO can be loaded into exosomes using this methodology and that exosomes are (i) fusogenic; (ii) contain ASO; (iii) the ASO is active; and, (iv) ASO can be transported using exosomes. These data also show that exosomes can be loaded using mutants of PA.

[0157] Example 12 Exosomes were loaded by resuspending 50 nM stealth reporter anti-GFP siRNA (Invitrogen catalog number 12935-145), 50 μg / ml PA83 (SEQ ID NO: 2), and 50 μg / ml LFnPKR (SEQ ID NO: 13) in serum-free medium. Exosomes were isolated as before, namely, after a 4-hour incubation with the protein::siRNA mixture and a 30-minute incubation with 5 μM ionomycin in serum-free medium. All incubations were performed at 37° C., and cells were washed with PBS during incubation as before. Exosomes were isolated using the Qiagen exoEasy kit (catalog number 76064). Exosomes were washed with PBS and pelleted as before (i.e., 110,000 xg for 70 minutes at 4° C.) and then resuspended in 500 μl of PBS. Exosomes were diluted using complete medium and added to HEK293 cells stably expressing GFP fusion beta-galactosidase and tandem dimer tomato (ASMBIO catalog number SC008). Next, beta-galactosidase activity in cell lysates was assayed by measuring X-gal conversion at OD 620 over time and normalizing the conversion to the total cell lysate protein concentration.

[0158] Referring to FIG. 12, it can be appreciated that anti-GFP siRNA loaded into HeLa-derived exosomes using PA83 and LFnPKR has pharmacological activity. These data, similar to the ASO of Example 11, show that siRNA retains its activity in exosomes.

[0159] Summary Advantages of the liposome aspects and embodiments described herein are directed to addressing one or more problems inherent in the prior art by isolating ILVs loaded with Atx-related cargo as liposomes (e.g., exosomes) prior to ILV reverse fusion. Further addressed is the use of transmembrane sequences that perform the same function as Atx PA63 but include recombinant transmembrane sequences.

[0160] Liposomes / exosomes are well - known as natural paracrine transport systems that can protect the luminal contents from enzymatic destruction and immune responses, protecting antigenic or enzymatically labile substances during transport. Wild - type LF has been reported in both ILVs and liposomes (e.g., exosomes), and considering that it has been demonstrated that ILVs are secreted from cells as liposomes (e.g., exosomes) after the release of calcium stored in the ER [5], the inventors inferred that recombinant LF could also be trapped or loaded into liposomes (e.g., exosomes). Furthermore, the use of ionophores (e.g., ionomycin) suppresses the release of ER calcium upon demand and causes the exocytosis of ILVs as liposomes (e.g., exosomes). As a result, ionomycin was used to transiently capture ILVs containing cargo from cells previously treated with an Atx - derived delivery system. This enabled the isolation of exosomes loaded with cargo secreted into the cell culture medium. Although the Atx components protective antigen (PA)83 or PA63, lethal factor (LF), and edema factor (EF) have been reported to localize in liposomes / exosomes [4], the loading of related molecules (i.e., LFn - GAL4, LFn - PKR, LFn - PKR - Texas Red, siRNA, CAS9 or Cas9 - Texas Red) into liposomes / exosomes using pore - forming recombinant proteins has not been reported so far.

[0161] [References] [1]Tyagi P., Subramony J.A. (2018) Nanotherapeutics in oral and parenteral drug delivery: Key learnings and future outlooks as we think small. J. Control Release. 272:159 - 168. [2]S. C. Richardson, S. C. Winistorfer, V. Poupon, J. P. Luzio, R. C. Piper. (2004) Mammalian late vacuole protein sorting orthologues participate in early endosomal fusion and interact with the cytoskeleton, Mol Biol Cell. 15, 1197-1210. [3]Abrami, L., Lindsay, M., Parton, R. G., Leppla, S. H., & van der Goot, F. G. (2004). Membrane insertion of anthrax protective antigen and cytoplasmic delivery of lethal factor occur at different stages of the endocytic pathway. The Journal of Cell Biology, 166(5), 645-651. [4]Abrami, L., Brandi, L., Moayeri, M., Brown, M. J., Krantz, B. A., Leppla, S. H., & van der Goot, F. G. (2013). Hijacking Multivesicular Bodies Enables Long-Term and Exosome-Mediated Long-Distance Action of Anthrax Toxin, 5(4), 986-996. [5]Kuznetsov G., Brostrom M. A., and Brostrom C. (1992) Demonstration of a Calcium Requirement for Secretory Protein Processing and Export. The Journal of Biological Chemistry. 267(6); 3932-3939. [6]Blaustein, R. O., Koehler, T. M., Collier, R. J., and Finkelstein, A. Pr℃. Natl. Acad. Sci. USA 86, 2209-2213 (1989). [7]B.A. Krantz, et al., Acid-induced unfolding of the amino-terminal domains of the lethal and edema factors of anthrax toxin, J. Mol. Biol. 344 (3) (2004) 739-756. [8]Auger A., Park M., NitschkeF., MinassianL. M., BeilhartzG. L., Minassian B. A., and Melnyk R. A. (2015) Efficient Delivery of Structurally Diverse Protein Cargo into Mammalian Cells by a Bacterial Toxin. Mol. Pharmaceutics,, 12 (8), pp 2962-2971 [9]Dyer P.D. (2013) Development of a Protein-Based Antisense Delivery Platform Modelled on Anthrax Toxin. PhD Thesis, University of Greenwich.

[10] Gaur, R., Gupta, P., Goyal, A., Wels, W. & Singh, Y. Delivery of nucleic acid into mammalian cells by anthrax toxin. Bi℃hem. Biophys. Res. Commun. 297, 1121-1127 (2002).

[11] Baillie, L W., Huwar, T. B., Moore, S., Mellado-Sanchez, G., Rodriguez, L., Neeson, B. N., et al. (2010). An anthrax subunit vaccine candidate based on protective regions of Bacillus anthracis protective antigen and lethal factor. Vaccine, 28(41), 6740-6748.

[12] Khandia R., Bhatia S., Chanu K. V., Sood R. and Dhama K. (2014). Anthrax Toxin Receptors, Functions and their Possible Use in Therapeutics: A Review. Asian Journal of Animal and Veterinary Advances, 9: 599-609.

[13] Guo S. & Huang L. (2011) Nanoparticles Escaping RES and Endosome: Challenges for siRNA Delivery for Cancer Therapy. Journal of Nanomaterials. Article ID 742895. DoI: 10.1155 / 2011 / 742895

[14] S. C. Richardson, S. C. Winistorfer, V. Poupon, J. P. Luzio, R. C. Piper. Mammalian late vacuole protein sorting orthologues participate in early endosomal fusion and interact with the cytoskeleton, Mol Biol Cell. (2004) 15, 1197-1210.

[15] Willms E., Johansson H. J., Mager I., Lee Y., et al., (2015) Cells Release Subpopulations of Exosomes with Distinct Molecular and Biological Properties. Scientific Reports 6: 22519.

[16] Phillips D.D., Fattah R. J., Crown D., et al. (2013) Engineering Anthrax Toxin Variants That Exclusively Form ℃tamers and Their Application to Targeting Tumors. J. Biol. Chem., 288: 9058-9065.

[17] Karginov, V.A., Nestorovich E.M., Schmidtmann, F. Robinson T. M., Yohannes, A., Fahmi N. E., Bezrukov S. M.,, and Hecht S. M. (2007) Inhibition of S. aureus α-Hemolysin and B. anthracis Lethal Toxin by β-Cyclodextrin Derivatives. Bioorg Med Chem.; 15(16): 5424-5431.

[18] Feld G.K., Brown,M. J. & Krantz B. A. (2012) Ratcheting up protein transl℃ation with anthrax toxin. PROTEIN SCIENCE. 21:606-624.

Claims

**Claim 1** A method for preparing exosomes, comprising at least one cell, the following ( ｉ ), and ( ｉｉ ): ( ｉ ) a pore-forming protein, or a pore-forming domain thereof; ( ｉｉ ) A shuttle protein attached to a bioactive payload molecule, comprising contacting with, wherein the pore-forming protein, or its pore-forming domain, creates pores through the phospholipid bilayer of at least one of said cells, and the shuttle protein interacts with the pore-forming protein, or its pore-forming domain, is internalized into said cell to produce exosomes loaded with a bioactive payload molecule, wherein a bioactive payload molecule is attached to the shuttle protein, and the molecular weight of the bioactive payload molecule is between 1 Da and 10 MDa, the method further comprising isolating the exosomes from the cells, the pore-forming protein, or its pore-forming domain, comprises Bacillus anthracis pathogenic factor protective antigen (PA) 83 or Bacillus anthracis PA63, the shuttle protein comprises a lethal factor (LF) derived from Bacillus anthracis, the method. **Claim 2** the exosomes having an average diameter between 10 nm and 500 nm, or between 20 nm and 400 nm, or between 30 nm and 300 nm, or between 40 nm and 200 nm, or between 50 nm and 150 nm, or between 60 nm and 120 nm, and / or the method according to claim 1, wherein the cells comprise biological cells which may be mammalian or human cells. **Claim 3** the method according to claim 1 or 2, wherein the pore-forming protein, or its pore-forming domain, comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1, 2, 5-10, or consists essentially thereof. **Claim 4** the shuttle protein is configured to facilitate transport of the bioactive payload molecule through the pores of the pore-forming protein, the method according to any one of claims 1-3, wherein the shuttle protein comprises the amino acid sequence set forth in SEQ ID NO: 11, or consists essentially thereof. **Claim 5** The shuttle protein is configured to facilitate transport of the bioactive payload molecule through the pores of the pore-forming protein, the method according to any one of claims 1-3, wherein the shuttle protein comprises a linker protein. **Claim 6** The shuttle protein comprises an attenuated toxin protein, The method according to claim 5, wherein in the attenuated toxin, at least one toxin domain, which is one or more of the toxicity domains II-IV of the anthrax lethal factor protein toxin, is replaced by the linker protein.

7. The method according to claim 5 or 6, wherein the linker protein contains a nucleic acid binding domain.

8. The method according to claim 7, wherein the nucleic acid binding domain is Saccharomyces cerevisiae GAL4.

9. The method according to any one of claims 1 to 3, wherein the shuttle protein contains the amino acid sequence set forth in SEQ ID NO: 12 or 13, or consists essentially of them.

10. The bioactive payload molecule is a therapeutically active molecule that is active within the cell cytosol, within the nucleus, within intracellular structures such as organelles or vesicles or vacuoles, within the cell surface lipid membrane, or within the intracellular lipid membrane, and / or The bioactive payload molecule is: (i) a small molecule, protein, nucleotide, DNA or DNA construct, plasmid, RNA or RNA construct, mRNA, miRNA, guide RNA, snRNA, siRNA, antisense oligonucleotide (ASO), or (ii) a macromolecule such as a protein or enzyme, nuclease, etc. The method according to any one of claims 1 to 4.

11. A method for manufacturing a pharmaceutical composition, the method comprising a preparation step of preparing the exosome according to any one of claims 1 to 10, and the pharmaceutical composition comprising the exosome prepared by the preparation step and a pharmaceutically acceptable excipient.

12. The method according to claim 11, wherein the pharmaceutical composition is for use in treatment or diagnosis.

13. The method according to claim 11, wherein the pharmaceutical composition is for use in the treatment, prevention, or amelioration of a disease.

14. The disease is obesity, more preferably FMO5-regulated obesity, or male pattern alopecia (AGA); acne; seborrheic dermatitis; and a prostaglandin D2-regulated disease selected from the group consisting of prostate cancer, or The method according to claim 13, wherein the exosome is used for treating, preventing, or improving Zika fever (or Zika virus disease), Ebola virus disease, acquired immunodeficiency syndrome (human immunodeficiency virus), Stat3-responsive cancer, P53-deficient cancer, virus-mediated cervical cancer (i.e., human papillomavirus), familial hypercholesterolemia, Duchenne muscular dystrophy, spinal muscular atrophy, Crohn's disease, or an inflammatory disease, particularly an intestinal inflammatory disease associated with overexpression of intracellular adhesion molecule-1 (ICAM-1).

15. The cell used for producing the exosome is obtained from the subject to be treated, A healthy cell is obtained from the stem cell line of the subject or from the target tissue of the subject, and is grown in culture or obtained from the subject before being used for producing an exosome loaded with a therapeutic compound suitable for treating the clinical condition in question. The method according to any one of claims 11 to 13, wherein the cell obtained from the patient biopsy does not contain a healthy cell which may also be present.

16. The method according to claim 11, wherein the pharmaceutical composition is used in genome editing technology.

17. The exosome is loaded with (i) a guide RNA and / or (ii) a nuclease or a gene construct encoding a nuclease, the pharmaceutical composition is used in gene editing therapy, and the nuclease comprises Cas9 or Cpf1, or TALEN or zinc finger nuclease. The method according to claim 11.

Images