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Title:
A METHOD FOR SELECTIVE LOADING OF PROTEINS INTO EXOSOMES AND PRODUCTS THEREOF
Document Type and Number:
WIPO Patent Application WO/2020/170129
Kind Code:
A1
Abstract:
The present invention relates to a method to selectively load molecules into exosomes in living cells. The method is based on the addition of a selected sequence of amino acids acting as targeting-peptide to a bioactive molecule.The present invention allows the expression and loading of proteins of virtually any kind irrespective of size, charge and conformation into exosomes even during exosome biogenesis. Therefore, it is possible to integrate DNA sequences of the fusion protein into the cells genome thus creating a stable cell line that can be clonally expanded and even stored for future use and to express, at the same time, multiple proteins targeted for exosomes in the same cell line, creating, in a single exosome isolation step, a multi-targeting exosome pack, allowing for more complex therapeutic approaches.The loaded exosomes can be used in therapies based in exosome engineering and delivery to selected cells or tissues in the organism.

Inventors:
DE CARVALHO PEREIRA PAULO (PT)
OLIVEIRA FERREIRA JOÃO VASCO (PT)
ROSA SOARES ANA MARGARIDA (PT)
Application Number:
PCT/IB2020/051341
Publication Date:
August 27, 2020
Filing Date:
February 18, 2020
Export Citation:
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Assignee:
INST DE BIOLOGIA EXPERIMENTAL E TECNOLOGICA (IBET) (PT)
International Classes:
A61K9/127; A61K9/00; A61K38/00; C12N15/11
Domestic Patent References:
WO2020049158A12020-03-12
Foreign References:
US20130065267A12013-03-14
Other References:
RANJIT SAHU ET AL: "Microautophagy of Cytosolic Proteins by Late Endosomes", DEVELOPMENTAL CELL, CELL PRESS, US, vol. 20, no. 1, 4 December 2010 (2010-12-04), pages 131 - 139, XP028390382, ISSN: 1534-5807, [retrieved on 20101210], DOI: 10.1016/J.DEVCEL.2010.12.003
HIROSHI KOGA ET AL: "A photoconvertible fluorescent reporter to track chaperone-mediated autophagy", NATURE COMMUNICATIONS, vol. 2, 1 January 2011 (2011-01-01), pages 386 - 386, XP055257010, ISSN: 2041-1723, DOI: 10.1038/ncomms1393
JING XU ET AL: "The interplay between exosomes and autophagy - partners in crime", JOURNAL OF CELL SCIENCE, vol. 131, no. 15, 1 August 2018 (2018-08-01), Cambridge, XP055698653, ISSN: 0021-9533, DOI: 10.1242/jcs.215210
CHUNYING LIU ET AL: "Design strategies and application progress of therapeutic exosomes", THERANOSTICS, vol. 9, no. 4, 1 January 2019 (2019-01-01), AU, pages 1015 - 1028, XP055698379, ISSN: 1838-7640, DOI: 10.7150/thno.30853
ANA MARGARIDA ROSA SOARES: "RUN: A new mechanism for selective protein loading into exosomes", NOVA MEDICAL SCHOOL - FACULDADE DE CIÊNCIAS MÉDICAS, 1 December 2019 (2019-12-01), XP055698659, Retrieved from the Internet [retrieved on 20200526]
FERREIRA, J.V. ET AL.: "STUB1/CHIP is required for HIF1A degradation by chaperone-mediated autophagy", AUTOPHAGY, vol. 9, no. 9, 2013, pages 1349 - 66
FERREIRA, J.V. ET AL.: "K63 linked ubiquitin chain formation is a signal for HIF1A degradation by Chaperone-Mediated Autophagy", SCI REP, vol. 5, 2015, pages 10210
SAMBROOK, J.D.W. RUSSELLJ. SAMBROOK: "The condensed protocols from Molecular cloning : a laboratory manual", 2006, COLD SPRING HARBOR LABORATORY PRESS, pages: 800
BALBAS, P.A. LORENCE: "Methods in Molecular Biology", vol. 267, 2004, HUMANA PRESS, article "Recombinant Gene Expression: Reviews and Protocols"
LOBB, R.J. ET AL.: "Optimized exosome isolation protocol for cell culture supernatant and human plasma", J EXTRACELL VESICLES, vol. 4, 2015, pages 27031, XP055279331, DOI: 10.3402/jev.v4.27031
CATARINO, S. ET AL.: "Ubiquitin-mediated internalization of connexin43 is independent of the canonical endocytic tyrosine-sorting signal", BIOCHEM J, vol. 437, no. 2, 2011, pages 255 - 67
YIM, N. ET AL.: "Exosome engineering for efficient intracellular delivery of soluble proteins using optically reversible protein-protein interaction module", NAT COMMUN, vol. 7, 2016, pages 12277
Attorney, Agent or Firm:
OLIVEIRA LOURENÇO, Nuno Miguel (PT)
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Claims:
CLAIMS

1. A method for selectively loading molecules into exosomes in living cells comprising the following steps:

a) Providing selected nucleic acid sequences;

b) Adding the coded targeting-peptides resulting from the nucleic acid sequences of (a) to a target bioactive protein molecule to form a target-peptide;

c) Loading the desired molecules with the target-peptide of (b) into exosomes present in living cells, where the target peptides are one or more penta-peptide sequences comprising L-stereoisomers or D-stereoisomers , or a mixture of:

- A "Q" or an "N" as the first or last amino acid in the sequence ;

- Up to two of the hydrophobic amino acids "I", "F", "L" or "V";

- Up to two of the positive amino acids "R" or "K"; and

- One negative charged amino acid, either "E" or "D".

2. A method according to claim 1, wherein the nucleic acid sequences of step (a) are selected from one or more of the following :

SEQ. ID.1 : GTTAAAAAAGACCAAGCAGAACCACTACACCGAAAATTCGAA

CGACAA;

SEQ. ID.2 : GTTAAAAAAGACCAAGCAGAACCACTACACCGAAATGAATTC

AAGTTG;

SEQ. ID.3 : GTTAAAAAAGACCAAGCAGAACCACTACACCGAGATCGCCTG

CTTCAGGCAGAACCACTACACCGAGCTGACTACAGCATT ;

SEQ. ID.4 : AAATTCGAACGACAAGCAGAACCACTACACCGACATGGTGTG

ACAACAGCAGAACCACTACACCGAGTTAAAAAAGACCAA.

3. A method according to any of the claims 1 or 2, wherein the targeting-peptides of (b) are the following:

SEQ. ID. 5: VKKDQAEPLHRKFERQ;

SEQ. ID. 6: VKKDQAEPLHRNEFKL ;

SEQ. ID. 7: VKKDQAEPLHRDRLLQAEPLHRADYS I ;

SEQ. ID. 8: KFERQAEPLHRHGVTTAEPLHRVKKDQ .

4. A method according to any of the claims 1 to 3, wherein the targeting-peptides have a degree of similarity to the targeting-peptides as described in claim 3, of more than 50%, preferable more than 60%, more preferable more than 70%, even more preferable more than 80%, preferably more than 90%, in any of the levels defined by their sequence, structure and/or function implying or not implying homology .

5. A method according to any of the claims 1 to 4, wherein the targeting-peptides further includes targeting-peptides preceded or followed by any number of other amino acids.

6. A method according to any of the claims 1 to 5, wherein the targeting-peptides further include targeting-peptides separated by any number of other amino acids or amino acid linkers either before, after or in between said targeting- peptides .

7. A method according to claim 1 wherein step (b) is performed by fusing a signal peptide as described in any of the claims 1 to 6 to the cDNA sequence of a target bioactive protein molecule or by providing recombinant proteins that include said targeting-peptide and to be incorporated into any cells for further loading into exosomes.

8. A targeting-peptide coded by one or more of the following nucleic acid sequences:

SEQ. ID.1 : GTTAAAAAAGACCAAGCAGAACCACTACACCGAAAATTCGAA

CGACAA

SEQ. ID.2 : GTTAAAAAAGACCAAGCAGAACCACTACACCGAAATGAATTC

AAGTTG;

SEQ. ID.3 : GTTAAAAAAGACCAAGCAGAACCACTACACCGAGATCGCCTG

CTTCAGGCAGAACCACTACACCGAGCTGACTACAGCATT ;

SEQ. ID.4 : AAATTCGAACGACAAGCAGAACCACTACACCGACATGGTGTG

ACAACAGCAGAACCACTACACCGAGTTAAAAAAGACCAA

9. A targeting-peptide according to claim 8, comprising one or more penta-peptide sequences of L-stereoisomers or D- stereoisomers , or a mixture of:

- A "Q" or an "N" as the first or last amino acid in the sequence ;

- Up to two of the hydrophobic amino acids "I", "F", "L" or "V";

- Up to two of the positive amino acids "R" or "K"; and

- One negative charged amino acid, either "E" or "D".

10. A targeting-peptide according to any of the claims 8 or 9 comprising one or more of the following sequences:

SEQ. ID. 5: VKKDQAEPLHRKFERQ;

SEQ. ID. 6: VKKDQAEPLHRNEFKL ;

SEQ. ID. 7: VKKDQAEPLHRDRLLQAEPLHRADYS I ;

SEQ. ID. 8: KFERQAEPLHRHGVTTAEPLHRVKKDQ .

11. A targeting-peptide according to any of the claims 8 to 10 comprising a degree of similarity to the targeting- peptides of claim 8, of more than 50%, preferable more than 60%, more preferable more than 70%, even more preferable more than 80%, preferably more than 90%, in any of the levels defined by their sequence, structure and/or function implying or not implying homology.

12. A targeting-peptide according to any of the claims 8 to 11 comprising targeting-peptides preceded or followed by any number of other amino acids.

13. A targeting-peptide according to any of the claims 8 to 12 comprising targeting-peptides separated by any number of other amino acids or amino acid linkers either before, after or in between said targeting-peptides.

14. A bioactive protein molecule comprising a targeting- peptide as described in any of the claims 8 to 13.

15. An exosome comprising a bioactive protein molecule as described in claim 14.

16. An exosome composition comprising exosomes as described in claim 15.

17. An exosome composition according to claim 16 comprising exosomes expressing fused molecules with pharmaceutical and/or cosmetic drugs.

18. The use of a signal peptide as described in any of the claims 8 to 13 to bind to any target protein/peptide, thus allowing to target a protein/peptide to the inside of the exosome .

19. The use of a bioactive protein molecule as described in claim 14 for targeting a protein/peptide to the inside of the exosome.

20. The use of an exosome as described in claim 15 to produce compositions for delivering selected molecules to human, animal or other living organisms.

21. The use of an exosome composition according to claim 16 for delivering bioactive protein molecule into living cells, tissues and/or organs.

22. The use of an exosome composition according to claim 17 for delivering pharmaceutical and/or cosmetic drugs into living cells, tissues and/or organs.

Description:
DESCRIPTION

A METHOD FOR SELECTIVE LOADING OF PROTEINS INTO EXOSOMES AND

PRODUCTS THEREOF

TECHNICAL DOMAIN OF THE INVENTION

The present invention relates to a method to selectively load molecules into exosomes in living cells. The method comprises developing selected amino acids into sequences that act as targeting-peptides and adding it to a bioactive protein molecule. The addition of the peptide to the bioactive protein acts as a signal for loading into exosomes. These peptides can be used to design therapies based in exosome engineering for delivery to selected cells or tissues in the organism.

The present invention uses genetic engineering tools to provide a system for the stable expression of proteins, or any peptide, fused to the targeting-peptide. The fusion protein is loaded into the exosomes and can subsequently be delivered to selected cells or tissues in the organism. This provides a novel therapeutic approach, for example, by providing an alternative means to deliver currently undruggable proteins.

BACKGROUND OF THE INVENTION

Exosomes are small extracellular vesicles of endosomal origin and are secreted by virtually any cell type. Exosomes are carriers of bioactive information between cells and tissues in the form of proteins, DNA and RNA. They circulate in the blood stream and can potentially reach all tissues and organs. Exosomes offer exciting features for therapeutic delivery, including biocompatibility, stability in the circulation, biological barrier permeability, low immunogenicity, and low toxicity .

The cargo of exosomes is determined inside the cell, when the membrane of endosomes invaginates to form small intraluminal vesicles (ILVs) . When endosomes fuse with the plasma membrane they release their ILV cargo into the extracellular space. The secreted ILVs are thereafter referred to as exosomes.

Exosomes are widely regarded as a very promising tool for the treatment of human diseases. The contents of exosomes depend on the contents of the cell itself. Therefore, the main challenge in the field has been the integration and /or loading of selected cargo into these nanoparticles.

Initial cell-engineering approaches focused in the use of specific cell types or specific stimuli upon which cells would produce exosomes with the desired cargo.

More recent advances include sophisticated methodologies, intended to manipulate exosomal content in a more precise and systematic way. The loading of bioactive molecules to exosomes is achieved by the following methods: i) Incubation of drugs with exosomes: Exosomes are simply incubated with drugs. The drugs passively diffuse into the lumen of the exosomes. The drug loading efficiency depends on the hydrophobicity of the drug molecules. There is also a successful case of loading a tetramer protein of 250 kDa into exosomes with PBS buffer at room temperature for 18 h. The main drawback to this method is its low loading capacity; ii ) Incubation of drugs with cells that produce exosomes: The donor cells are treated with a drug. The drug passively diffuses into the cell. The cells then secrete exosomes loaded with the drug. The drug loading efficiency depends on the hydrophobicity of the drug molecules;

iii) Sonication : Exosomes isolated from cell culture medium are mixed with drugs or proteins and subsequently sonicated by using a homogenizer probe. The drawback is that the mechanical shear force from the sonicator probe compromises the membrane integrity of the exosomes. iv) Extrusion : Exosomes are mixed with a drug, and the mixture is loaded into a syringe-based lipid extruder with 100-400 nm porous membranes under a controlled temperature. The process causes alteration of the exosomal membrane and in some cases induces cytotoxicity;

v) Freeze and thaw cycles: In this procedure, drugs are incubated with exosomes at room temperature for a fixed amount of time. Subsequently the mixture is rapidly frozen at -80°C or in liquid nitrogen and thawed at room temperature. However, the method can induce aggregation of the exosomes, thus resulting in a broad size distribution of the drug-loaded exosomes. The drug loading capacity of the freeze/thaw method is generally low;

vi) Electroporation : This technique creates small pores in the exosome membrane through application of an electrical field to exosomes suspended in a conductive solution. The electrical current disturbs the phospholipid bilayer of the exosomes, thus resulting in the formation of temporary pores. Drug, nucleotides or proteins can subsequently diffuse into the interior of the exosomes via the pores. However, electroporation may cause RNA aggregation and exosome instability, thereby resulting in a low loading capacity and poor exosome quality;

vii) Incubation with membrane permeabilizer : Uses surfactant molecules, such as detergents, that can form complexes with cholesterol in cell membranes and generate pores, thus leading to an increase in membrane permeabilization. However, surfactant molecules are usually haemolytic. In addition, the concentrations used for drug loading have to be low and the exosomes must be purified after the procedure;

viii) Click chemistry method for direct conjugation: Chemical methods can also be used to directly attach molecules to the surfaces of exosomes via covalent bonds. Copper catalyzed azide alkyne cycloaddition, known as click chemistry can be used for bioconjugation of small molecules and macromolecules. Since molecules are attached to the exterior of the exosome they are amenable to degradation or other forms of chemical modification that can impair a molecule's mode of action and delivery to target cells;

ix) Transfection : For the loading of RNAs and DNAs, researchers have used commercially available polymer molecules capable of translocating genetic material across lipid membranes. The obvious disadvantages of this methodology include the costs of buying or producing the polymers and that it cannot be applied to proteins ;

x) Endogenous passive loading by overexpression of proteins : Cells overexpress the proteins of interest that are passively integrated into exosomes. This method displays low yield of biomolecule loading and does not provide specificity.

In general, all of the above techniques show limited loading efficiency, particularly with respect to protein loading. The vast majority of these approaches have additional drawbacks such as having to build, isolate and purify the genetic and protein material to load into exosomes ex vivo, using in vitro protocols that are expensive, time consuming and often inefficient .

Moreover, proteins, unlike chemical drugs or nucleic acids, have a very low capacity to passively penetrate cellular membranes .

In addition, proteins are easily modified by the physical and chemical environment, thus further limiting the applicability of in vitro models of incubating isolated proteins with exosomes while maintaining the biological activity of the loaded proteins.

All these methods have been largely unsuccessful in efficiently loading high molecular weight proteins and/or protein complexes into exosomes.

Traditional approaches used by companies for the loading of cargo (including some proteins) into exosomes are based on the above techniques or approaches.

Presently, efforts are being directed towards making the engineering of exosomes less expensive, less time consuming, more efficient while increasing the repertoire of proteins that can be loaded into exosomes. A preferred approach would be to engineer the cells so that they produce and load desired proteins into exosomes.

More recent and sophisticated approaches for exosome engineering include: i) Expression of transmembrane proteins: Since many proteins present in the membrane of exosomes are present in the plasma membrane and/or in the membrane of vesicles of the endocytic pathway, one strategy has been the expression of transmembrane proteins of interest that undergo endocytic transport. Some of these proteins are used to increase the tropism of an exosome for a particular cell type or organ, or to improve the uptake or transfer of relevant exosomal content into the receiving cells, other proteins are chelators of the circulating ligands. Alternatively, fusion chimeras consisting of a transmembrane protein domain and an extra-exosomal protein domain capable of binding bioactive molecules, such as a drug or other active compounds including proteins, can be expressed in cells for exosomal membrane loading. In this case, the loading of the bioactive cargo still happens after exosomal isolation but does not require the use of polymers nor electroporation. On the negative side, the binding of the molecules, including proteins, to the external side of the exosomal membrane leaves them amenable to chemical alterations capable of inactivating the bioactive molecules .

ii ) Exosomal membrane-anchored protein (XPACK) and Exosomes for protein loading using optogenetically reversible protein-protein interaction (EXPLOR) : proteins are expressed as a fusion protein with a transmembrane domain attached to a protein located at the inner layer of the exosomal membrane. These chimeras are integrated with exosomes along natural exosome biogenesis. In the case of EXPLOR the attachment of the protein of interest is dependent of blue light through a photo specific binding protein. This photoactivable protein is present in the protein of interest and on the transmembrane chimera, such that proteins are loaded into exosomes under blue light and are separated from the membrane portion of the construction when blue light is interrupted, releasing the protein inside the exosome. With XPACK the proteins of interest are irreversible fused to the transmembrane domain and therefore remain at exosomal membrane, limiting the therapeutic potential of proteins introduced in exosomes by this process, specifically in the case of soluble proteins. With EXPLOR the drawback is the need of blue light specific equipment to load the proteins into exosomes and the addition of a large tag to the protein of interest, that might irreversibly modify the protein function;

iii) Exosome packing of nucleic acids using pre-mir-451:

Describes a method for preparing exosomes packaged with a nucleic acid of interest such as a gene silencing nucleic acid, a nucleic acid of interest, or a precursor. The method includes introducing into an exosome- producing cell a nucleic acid construct comprising the nucleic acid sequence of interest incorporated in a pre- miR-451 structural mimic, and allowing the cell to produce exosomes. This method only allows for the targeting of RNA molecules, thus being restricted in what concerns to therapeutics delivery compositions. Based on the current state of the art, loading of proteins in exosomes is still an area where research has provided limited information as to the molecular mechanisms and key players involved in targeting proteins to exosomes. Thus, the technological approaches for improved loading of proteins is to a large extent based in empirical evidence and resorts to general technology to introduce molecules in nanoparticles. Because the mechanisms are not fully understood the efficiency of the majority of processes to load proteins remains quite low .

The present invention proposes an exosome-based delivery system that relies in cell engineering and protein expression allowing for the loading of proteins or peptides of any kind irrespective of size, charge and conformation, into exosomes. This will provide a means to deliver such bioactive molecules to any selected cell or tissue.

Moreover, the present invention also allows for the loading of any molecule/compound able of binding to a protein/peptide, into exosomes during their biogenesis. This invention, does not require any type of further protocol such as exposure to an electric or magnetic field, as well as, specific temperatures or light being applied to the exosomes. Proteins are produced and loaded in their physiological cell environment which further ensures maintenance of biological properties.

SUMMARY OF THE INVENTION

The present invention relates to a method to selectively load molecules into exosomes in living cells and products thereof. The method comprises developing selected amino acids into sequences that act as targeting-peptides and adding it to a bioactive protein molecule. The fusion of the peptide with the bioactive protein acts as a signal for loading into exosomes. These targeting peptides can be used to design therapies based in exosome engineering for delivery to selected cells or tissues in the organism.

Therefore, in a first embodiment, the present invention relates to a method for selectively load molecules into exosomes in living cells according to claim 1.

In a second embodiment, the present invention relates to targeting-peptides according to claim 8.

These targeting-peptides are useful to bind to any target protein/peptide, thus allowing to target a protein/peptide to the inside of the exosome.

In a third embodiment the present invention relates to fused molecules comprising the above-mentioned targeting-peptides according to claim 14.

These fused molecules are useful for targeting a protein/peptide to the inside of the exosome.

A fourth embodiment of the present invention is related to exosomes loaded with the said fused molecules according to claim 15.

These exosomes are useful to produce compositions for delivering selected molecules to human, animal or other living organisms . A fifth embodiment of the present invention relates to exosome composition comprising exosomes expressing fused molecules according to claim 16.

In another embodiment the present invention related to exosome compositions comprising exosomes expressing fused molecules for pharmaceutical and/or cosmetic drugs according to claim 17.

In another embodiment, the present invention relates to the use of a fused bioactive protein molecule for loading selected molecules, cell and/or tissues into exosomes according to claim 19.

The method of the present invention does not require further manipulation of exosomes after their isolation for cargo loading depending only on the fusion of a small peptide to the protein of interest, preserving the function of the protein.

In another embodiment, the present invention is related to exosome compositions for delivering selected molecules, cell and/or tissues to the human and animal organisms according to claim 20.

In general, the present invention decreases the costs of exosome protein loading since it is possible to integrate the DNA sequence of the fusion protein into the cells genome thus creating a stable cell line that can be clonally expanded and even stored for future use.

Also, it is possible to express, at the same time, multiple proteins targeted for exosomes in the same cell line, creating in a single exosome isolation step a multitargeting exosome pack, allowing for more complex therapeutic approaches.

DESCRIPTION OF THE FIGURES

Figure 1. This figure shows one preferred embodiment of the present invention, where the targeting-peptide, hereafter referred to as ExoSignal, loads the mCherry protein into exosomes .

Human cells were transduced with lentiviral vectors expressing either mCherry (exogenous non-human protein) or mCherry fused to the targeting-peptide (ExoSignal) . Subsequently exosomes were isolated from the media supernatant of cultured cells. Cells and exosome extracts were separated in and SDS-PAGE gel and transferred to a nitrocellulose membrane. Membranes were blotted with antibodies raised against mCherry and the exosomal markers Flotillinl and CD63. It is possible to observe that mCherry fused to the ExoSignal is 150 times enriched in isolated exosomes when compared to mCherry with no exosignal.

Figure 2. Quantification of the amount of unmodified mCherry or mCherry fused to the ExoSignal present in exosomes released by a retinal epithelial cell line in culture.

DESCRIPTION OF THE INVENTION

This invention describes a method for the loading of proteins into exosomes. The method includes the creation of a peptide from a combination of amino acids, arranged in a particular order, to create a targeting-peptide for targeting proteins into exosomes.

This signal is subsequently added to any peptide or protein intended to be loaded into exosomes. By using simple genetic tools, the targeting-peptide can be fused to the cDNA sequence of a target protein and subsequently expressed in cells or, alternatively, using protein loading tools, recombinant proteins that include said targeting-peptide can be incorporated into any cells for their loading into exosomes.

Therefore, the present invention also related to selected targeting-peptides, to fusion proteins comprising said targeting-peptides and to exosomes comprising said fusion proteins .

This invention is applicable to the loading of any known proteins into exosomes. This invention is further applicable to the loading of any designed peptides into exosomes. This invention is further applicable to the loading in exosomes of any DNA, RNA, peptide, molecule or pharmaceutical composition able to bind any peptide tagged with the particular selected targeting-peptide of the invention.

The present invention is also related to compositions comprising the exosomes loaded with the relevant fusion proteins and therefore, it is further applicable to produce the said compositions to be used to deliver molecules to cells or tissues for the treatment of humans or animals. These compositions are also useful to produce cosmetic products.

In a first step it is provided or selected a DNA sequence that encodes for a known protein, a portion of a protein or any other designed peptide, which is intended to be added to a targeting-peptide such as for example the ones presented in Table 1.

Table 1. Some of the most relevant DNA sequences and coded targeting-peptides .

DNA sequence:

SEQ . ID .1 : GTTAAAAAAGACCAAGCAGAACCACTACACCGAAAATTCGAACGACAA; SEQ . ID .2 : GTTAAAAAAGACCAAGCAGAACCACTACACCGAAATGAATTCAAGTTG; SEQ . ID .3 : GTTAAAAAAGACCAAGCAGAACCACTACACCGAGATCGCCTGCTTCAGGC

AGAACCACTACACCGAGCTGACTACAGCATT ;

SEQ . ID .4 : AAATTCGAACGACAAGCAGAACCACTACACCGACATGGTGTGACAACAG

CAGAACCACTACACCGAGTTAAAAAAGACCAA.

Table 2. Some of the most relevant coded targeting-peptides

- SEQ. ID. 5: VKKDQAEPLHRKFERQ;

- SEQ. ID. 6: VKKDQAEPLHRNEFKL ;

- SEQ. ID. 7: VKKDQAEPLHRDRLLQAEPLHRADYS I ;

- SEQ. ID. 8: KFERQAEPLHRHGVTTAEPLHRVKKDQ .

In the scope of the present invention amino acids are described and listed according to the IUPAC nomenclature and abbreviations, as in Table 3.

Table 3.

Targeting-peptides according to the present invention are one or more penta-peptide sequences consisting of L-stereoisomers or D-stereoisomers, or a mixture of both of:

- A "Q" or an "N" as the first or last amino acid in the sequence ;

- Up to two of the hydrophobic amino acids "I", "F", "L" or

"V" ;

- Up to two of the positive amino acids "R" or "K";

- One negative charged amino acid, either "E" or "D";

These sequences follow the rules for their recognition by the Hsc70 molecular chaperone [1, 2] . One of the many roles of this chaperone is to participate in the degradation of proteins containing these sequences in the lysosome by a process known as Chaperone-Mediated Autophagy.

In the scope of the present invention, it was surprisingly observed that these sequences play an important role in the way proteins are selectively loaded into exosomes. If a protein possesses such a sequence or, in the case that no such sequence is present, the addition of one or more of those sequences to the protein is sufficient to target those proteins to exosomes.

Targeting-peptides sharing a degree of conservation or similarity between those described above are also comprised in the scope of the present invention.

In the sense of the present invention "conservation" or "similarity" in amino acid sequences means, where an amino acid at a specific position has been substituted with a different one that has functionally equivalent physicochemical properties .

Therefore, preferred targeting-peptides have a degree of similarity of more than 50%, preferable more than 60%, more preferable more than 70%, even more preferable more than 80%, preferably more than 90%, in any of the levels defined by their sequence, structure and/or function implying or not implying homology to the above-described peptides are within the scope of the invention.

In another embodiment, the invention also comprises targeting- peptides having any of the above mentioned targeting-peptides and/or their similar targeting-peptides preceded or followed by any number of other amino acids. In another embodiment, the invention also comprises targeting- peptides having any of the above mentioned targeting-peptides and/or their similar targeting-peptides separated by any number of other amino acids or amino acid linkers either before, after or in between the targeting-peptides.

In the scope of the present invention amino acid linkers are amino acid sequences created in nature or designed to separate multiple domains of a protein, such that the different domains do not interact with each other.

The most preferred targeting-peptides according to the present invention are:

- SEQ. ID. 5: VKKDQAEPLHRKFERQ;

- SEQ. ID. 6: VKKDQAEPLHRNEFKL ;

- SEQ. ID. 7: VKKDQAEPLHRDRLLQAEPLHRADYS I ;

- SEQ. ID. 8: KFERQAEPLHRHGVTTAEPLHRVKKDQ .

The selected sequence or combination of sequences is then cloned into a plasmid for the expression of the construct in a cell such as disclosed by Sambrook et al . [3] or for the production of recombinant protein [4] using any other existing techniques .

The selection of ExoSignal sequences may be optimized for each protein. Different variations of the ExoSignal sequences may result in different degrees of exosomal loading. The preferred embodiment shows 150x enrichment for mCherry but specific variations and combinations of sequences can increase loading for other proteins.

Cells can then be infected according to known genetic engineering techniques, such as by using viral vectors, transfected by using transfection reagents, or electroporated with the plasmid that contains the construct. Electroporation can also be used to transfer the fusion protein produced in vitro into cells. Cells are grown and allowed to produce exosomes. Subsequently exosomes are isolated from the cultured media using any method available, for example the one described by Richard J. Lobb et al . [5] depending on the subsequent intended use for the exosomes.

The design and development of targeting peptides allows the efficient and cost effective targeting of selected proteins to exosomes, in any cell type, with great potential for therapies based in exosome engineering and delivery to selected cells or tissues in the organism.

DESCRIPTION OF A PREFERRED EMBODIMENT OF THE INVENTION

Signal peptides are created following the set of rules established for said purpose. For any given protein the design of the signal peptide can be adapted, thus there are no fixed formulations as in EXPLOR and XPAC, in terms of size and composition to achieve maximum loading capability with minimum disruption of protein activity. Preferable exosignal peptides herein disclosed are the ones having at least one of the sequences selected from: SEQ. ID. 1, SEQ. ID. 2, SEQ. ID. 3, or SEQ. ID. 4, as disclosed before.

In a preferred embodiment, a sequence consisting of PA-mCherry fused to the ExoSignal, with a peptide sequence of the invention is synthesized into a suitable vector/carrier such as a plasmid. The sequence is then flanked by Spel/Hindlll cloning sites to achieve a final protein sequence, which is variable depending on the peptide sequence used as exosignal.

Subsequently the sequence is cloned into a suitable DNA system, such as the ones described by Catarino et al . [6] using an adequate system of restriction enzymes followed by the incubation with the clonase, for the transfer of the PA- mCherry-ExoSignal to a suitable plasmid.

Next, competent E. coli are transformed with a suitable amount of the reaction. Transformed bacteria are plated on culture medium, such as LB, containing the appropriate antibiotic to select for expression clones.

Eukaryotic cells, at 80% confluency, are transfected with mCherry-ExoSignal and accessory plasmids, harvested and centrifuged to remove cells debris. The supernatant , containing viral particles, is then used to infect cells.

For cell infection, cells are plated in culture media and lentiviral particles mix is used to infect cells. Then, after the addition of the cell culture media cells are allowed to reach confluency. At this point cells are incubated with a suitable antibiotic for the selection of expressing cells.

Clones expressing the highest levels of the desired Exosignal are screened and selected.

For exosome isolation the cell media supernatant is subjected to differential centrifugation at low temperatures, for example around 4°C, starting with a centrifugation for example at 300 to 350g for 10 to 15 min, followed by a centrifugation at 16500 to 17000g, for 20 to 25 min. Subsequently the larger particles can be removed by filtration. A final centrifugation at 100000 to 120000g for 70 to 90 min is used to pellet the exosomes .

EXAMPLES

Example 1. Creation of a signal peptide

The sequence consisting of PA-mCherry fused to the exosignal, with the peptide sequence SEQ. ID. 1 VKKDQAEPLHRKFERQ was synthesized into a pUC57 plasmid (GeneCust, Luxembourg) .

The sequence was flanked by Spel/Hindlll cloning sites as follows SEQ. ID. 9: actagtATGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATTAAGGAGTTCATGCGC T TCAAGGTGCACATGGAGGGGTCCGTGAACGGCCACGTGTTCGAGATCGAGGGCGAGGGCG A GGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGCGGCCCCCT G CCCCTCACCTGGGACATCCTGAGCCCTCAGTTCATGTACGGCTCCAATGCCTACGTGAAG C ACCCCGCCGACATCCCCGACTACTTTAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGC G CGTGATGAAATTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGA C GGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTG A TGCAGAAGAAGACCATGGGCTGGGAGGCCCTCTCCGAGCGGATGTACCCCGAGGACGGCG C CCTGAAGGGCGAGGTCAAGCCCAGAGTGAAGCTGAAGGACGGCGGCCACTACGACGCTGA G GTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAAC C GCAAGCTGGACATCACCAGCCACAACGAGGACTACACCATCGTGGAGCAGTACGAGAGAG C CGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGAGATCTGCTAGCGTTAA A AAAGACCAAGCAGAACCACTACACCGAAAATTCGAACGACAATGaagc11

The Final Protein sequence was SEQ. ID. 10:

MVSKGEEDNMAI IKEEMRFKVHMEGSVNGHVFEIEGEGEGRPYEGTQTAKLKVTKGGPLPL TWDILSPQEMYGSNAYVKHPADIPDYFKLSFPEGFKWERVMKFEDGGWTVTQDSSLQDGE FIYKVKLRGTNFPSDGPVMQKKTMGWEALSERMYPEDGALKGEVKPRVKLKDGGHYDAEV K TTYKAKKPVQLPGAYNVNRKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKRSASVKK D

QAEPLHRKFERQ

Subsequently the sequenced was cloned into a pcDNA-ENTR-BP [ 6 ] using the Nhel/Hindlll enzymes followed by the incubation with LR clonase for the transfer of the PA-mCherry-ExoSignal to the pLenti6 plasmid (Gateway, ThermoFisher ) .

In brief, it was mixed:

50-100 ng of PA-mCherry-ExoSignal-pENTR :

100-150 ng/ml pLenti6

LR Clonase™ II enzyme mix 3.0 ml

Final volume 15 ml

The mix was incubated at 25°C for 2 hours. Subsequently we added 2 ml of proteinase K solution followed by another incubation at 37°C for 10 minutes. 50 ml of competent E. coli were transformed with 1 ml of the reaction. Transformed bacteria were plated on LB containing the appropriate antibiotic to select for expression clones.

After pLenti 6-PA-mCherry-Exosignal 293 cells at 80% confluency were transfected with pLenti 6-PA-mCherry-Exosignal , as well as, psPAX2 (Addgene, 12260) and pMD2.G (Addgene, 12259) . 48h to 72h after transfection 293 cells incubation media was harvested and centrifuged at 1000-2000g to remove cells debris. The supernatant, containing the lentiviral particles, was used to infect cells.

For cell infection, 25000 to 50000 cells were plated in a 6- well plate. 1:1 ration of cell culture media and lentiviral particles mix, supplemented with 5-10 ug/ml of polybrene was use to infect cells. 20-28h after 1 volume of cell culture media was added and cells were allowed to reach confluency. At this point cells were incubated with puromycin for the selection of expressing cells.

100-200 infected cells were plated in 150 mm cell culture dishes until small colonies arise (7 to 15 days) . Individual colonies were removed from the cell culture dishes using sterile filter paper circles soaked with trypsin for 3 to 5 min at 37 °C. Several filter papers with the cells were plated in a 48-well plate until confluency at which time cells were trypsinized and plated in a 60 to 100 mm cell culture dish. Clones expressing the highest levels of pLenti 6-PA-mCherry- Exosignal were selected using a confocal microscope.

Clones expressing pLenti 6-PA-mCherry-Exosignal were platted in 3 to 4 100mm dishes (18 to 24 million cells) and incubated for 48 to 72 hours in cell culture media supplemented with ExoFree 10% Fetal Bovine Serum (FBS) . In brief, to obtain ExoFree FBS the serum was centrifuged at at 100000 to 120000g for 14 to 18 hours in a SW32Ti Sorvall rotor.

Example 2. Exosome isolation

For exosome isolation the cell media supernatant was subjected to differential centrifugation at 4°C, starting with a centrifugation at 300 to 350g, for 10 to 15 min followed by a centrifugation at 16500 to 17000g, for 20 to 25 min. To remove larger particles, the supernatant was filtered with a 0.22pm filter unit, after which it was ultra-centrifuged at at 100000 to 120000g, for 70 to 90 min. The resulting pellet was washed once with PBS and ultra-centrifuged at at 100000 to 120000g, for 70 to 90 min. After ultracentrifugation, exosomes were re suspended in PBS . Example 3. Composition comprising exosomes loaded with mCherry

Human cells (ARPE-19 cells) were transduced /incubated with lentiviral particles containing the PA-mCherry-Exosignal expressing either PA-mCherry (exogenous non-human protein) or PA-mCherry fused to the Exosignal SEQ. ID. 1: VKKDQAEPLHRKFERQ as described in Example 1.

Subsequently exosomes were isolated from the media supernatant of cultured cells as described in Example 2 by differential centrifugation at 4°C, starting with a centrifugation at 300 to 350g, for 10 min followed by a centrifugation at 16500 to 17000g, for 20 min. To remove larger particles, the supernatant was filtered with a 0.22pm filter unit, after which it was ultra-centrifuged at 120000g, for 70 min. The resulting pellet was washed with PBS, and after ultracentrifugation, exosomes were re-suspended in PBS.

Cells and exosome extracts were separated in and SDS-PAGE gel. Samples were loaded in the gel under an electric current of 70 to 140 volts. Proteins were transferred to a nitrocellulose membrane by an electric current of 90 to 100 volts for lh 15 min to lh 30 min.

Membranes were blocked in 5% low-fat dry milk in Tris-Buffer Saline solution with tween for 30 min to 1 h blotted with antibodies raised against mCherry and the exosomal markers Flotillinl and CD63.

For protein detection the membranes were incubated with a secondary antibody coupled to horseradish peroxidase (HRP) for 1 hour, the chemiluminescence it is then obtained by adding the luminol substrate. The luminescence signal was acquired with an appropriate equipment (Chemidoc touch, Biorad; USA) . Protein bands were quantified using ImageJ software.

By adding the Exosignal to mCherry we were able to enrich 150x, the amount of mcherry inside exosomes, when compared to mCherry Wild-type .

In the same way mCherry loading with XPACK is system is 30x and with EXPLOR is 120x [7] .

References

1. Ferreira, J.V., et al . , STUBl/CHIP is required for HIF1A degradation by chaperone-mediated autophagy. Autophagy, 2013. 9(9) : p. 1349-66.

2. Ferreira, J.V., et al . , K63 linked ubiquitin chain formation is a signal for HIF1A degradation by Chaperone-Mediated Autophagy. Sci Rep, 2015. 5: p. 10210.

3. Sambrook, J., D.W. Russell, and J. Sambrook, The condensed protocols from Molecular cloning : a laboratory manual. 2006, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, v, 800 p.

4. Baibas, P. and A. Lorence, Recombinant Gene Expression: Reviews and Protocols. Methods in Molecular Biology. Vol . 267. 2004: Humana Press.

5. Lobb, R.J., et al . , Optimized exosome isolation protocol for cell culture supernatant and human plasma. J Extracell Vesicles, 2015. 4: p.

27031.

6. Catarino, S., et al . , Ubiquitin-mediated internalization of connexin43 is independent of the canonical endocytic tyrosine-sorting signal. Biochem J, 2011. 437(2): p. 255-67.

7. Yim, N., et al . , Exosome engineering for efficient intracellular delivery of soluble proteins using optically reversible protein-protein interaction module. Nat Commun, 2016. 7: p. 12277.