Patent Application
20240238441
The present disclosure relates generally to recombinant polypeptides. More particularly, the present disclosure relates to extracellular-vesicle targeted recombinant polypeptides.
Extracellular vesicles (EVs) are lipid bilayer-delimited particles that are naturally released from a cell. Exosomes are 40-150 nm small extracellular vesicles (EVs) of endocytic origin involved in intercellular communication that transfer bioactive cargo, for example lipids, proteins, microRNAs, and mRNAs, to distal cells.
Because of their ability to function as an intercellular transfer system, EVs have been studied for use as potential vehicles delivery of therapeutic molecules. In addition, certain EVs also possess inherent therapeutic characteristics.
In order to understand how EVs can be used for therapeutic purposes, it is important to understand the processes by which they are formed and how they function in health and disease.
It would desirable to have additional ways of targeting molecules of interest to EVs.
It is an object of the present disclosure to obviate or mitigate at least one disadvantage of previous approaches.
In a first aspect, the present disclosure provides an extracellular vesicle (EV) comprising: coat protein complex 1 (COPI), and a recombinant EV-directed polypeptide comprising: a cargo polypeptide, and an extracellular vesicle signal peptide (ESP) comprising a coatomer binding motif (CBM), wherein the cargo polypeptide is tethered to an external surface of the EV via the coatomer binding motif.
In another aspect, there is provided a recombinant extracellular vesicle (EV)-directed polypeptide comprising: a cargo polypeptide, and an extracellular vesicle signal peptide (ESP) comprising a coatomer binding motif (CBM).
In another aspect, there is provided a nucleic acid molecule encoding the recombinant EV-directed polypeptide as described herein.
In another aspect, there is provided a viral particle comprising the nucleic acid as described herein.
In another aspect, there is provided a recombinant host cell comprising the nucleic acid as described herein.
In another aspect, there is provided a composition comprising the EV as described herein, the nucleic acid as described herein, or the viral particle as described herein; together with an excipient diluent, or carrier.
In another aspect, there is provided a use of the EV as described herein for delivery of the cargo polypeptide to a cell.
In another aspect, there is provided a use of the EV as described herein for preparation of a composition for delivery of the cargo polypeptide to a cell.
In another aspect, there is provided the EV as described herein for use in delivery of a cargo polypeptide to a cell.
In another aspect, there is provided a method of delivering a cargo polypeptide to a cell comprising contacting the cell with the EV as described herein.
In one aspect, there is provided a recombinant skeletal muscle-directed extracellular vesicle (EV) comprising coat protein complex 1 (COPI), a skeletal muscle targeting moiety comprising a Wnt family polypeptide, or polypeptide at least 90% identical thereto, and a payload for delivery to skeletal muscle.
In one aspect, there is provided a method for delivering a payload to skeletal muscle comprising contacting a cell with the recombinant skeletal muscle-directed EV as defined herein.
In one aspect, there is provided a use the recombinant skeletal muscle-directed EV as defined herein for delivery of the payload to skeletal muscle.
In one aspect, there is provided the recombinant skeletal muscle-directed EV as defined herein for use in delivery of the payload to skeletal muscle.
In one aspect, there is provided a recombinant Wnt protein comprising an extracellular vesicle signal peptide (ESP) sequence comprising one or more coatomer binding motifs (CBMs), wherein at least one of the one or more CBMs is mutated relative to a corresponding wild-type sequence to form a mutated CBM that reduces or abrogates extracellular vesicle-targeting activity of the ESP sequence relative to the corresponding wild-type sequence.
In one aspect, there is provided a recombinant nucleic acid encoding the recombinant Wnt protein as defined herein.
In one aspect, there is provided a vector comprising the recombinant nucleic acid as defined herein.
In one aspect, there is provided a host cell comprising the recombinant nucleic acid as defined here, or the vector as defined herein.
In one aspect, there is provided a use of the recombinant nucleic acid as defined here, or the host cell defined here, for production of the recombinant Wnt protein as defined herein, wherein the recombinant Wnt protein is free of extracellular vesicles.
In one aspect, there is provided a method for producing the recombinant Wnt protein as defined herein comprising introducing the recombinant nucleic acid as defined herein to a cell, and culturing the cell to produce the recombinant Wnt protein, wherein the recombinant Wnt protein is free of extracellular vesicles.
In one aspect, there is provided a method for producing the recombinant Wnt protein as defined herein comprising culturing the host cell as defined herein to produce the recombinant Wnt protein, wherein the recombinant Wnt protein is free of extracellular vesicles.
Other aspects and features of the present disclosure will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments in conjunction with the accompanying figures.
Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures.
Generally, the present disclosure is based on the surprising finding described herein that Wnt7a, and apparently other Wnt family members, are trafficked to extracellular vesicles (EVs) via interactions with coat protein complex 1 (COPI) and/or its components. COPI has not previously been associated with EVs or EV trafficking. Extracellular vesicle signal peptides (ESPs), each comprising at least one key Coatomer binding motif (CBM), are described, and these are shown to mediate EV trafficking of Wnt family members. The ESPs may be used to target other cargo polypeptides for display on EVs, thereby lending themselves to generation of recombinant EV-directed polypeptides or EVs comprising such recombinant polypeptides.
In one aspect, there is provided an extracellular vesicle (EV) comprising:
By “extracellular vesicle” (EV) is meant cell-derived membranous structures, including exosomes and microvesicles, and apoptotic bodies. These extracellular vesicles generally are categorized based on their size, specific markers, cellular origin and biogenesis processes. Exosomes are 40-150 nm vesicles of endosomal-origin released from the cell upon fusion of a multivesicular body (MVB) membrane with the plasma membrane. Exosomes are produced by every cell type and their release can be induced by a variety of stimuli, including stress, hypoxia, cell death, and infection. Classical microvesicles (also known as microparticles) are 100 nm-1 μm vesicles released from the cell by shedding of the plasma membrane. Cancer cells can also secrete larger microvesicles (>1 μm) called oncosomes, which only differ from classical microvesicles in regard to their size. Like exosomes, microvesicle release can be induced by stress and viral infection, and their contents are heterogeneous. Apoptotic bodies are large EVs that are released from apoptotic cells by blebbing and range in size from 200 nm to 5 μm. These phosphatidylserine- and Annexin V-coated EVs contain cytoplasmic contents from the dying cell. Traditionally, EVs that pelleted at 100,000 g were referred to as exosomes, but in fact this pellet contains a combination of microvesicles and exosomes. It is now known that separation of different types of vesicles (microvesicles, apoptotic bodies, exosomes, etc.) is possible using proper pre-clarification processes, such as Tangential Flow Filtration, used herein. Though their biogenesis pathways are distinct, exosomes and microvesicles have many similarities and are difficult to distinguish from one another once released from the cell. Recently, the International Society for Extracellular Vesicles suggested the term Small EVs (sEVs) should be used for particles less than 200 nm in size, while the term Large EVs (IEVs) should be used for particles greater than 200 nm.
In one embodiment, the EV is an exosome.
By “coat protein complex 1” or “COPI” is meant the coatomer protein complex that coats certain membrane-bound vesicles. Two types of coatomers are known. COPII is involved in anterograde transport from ER to the cis-Golgi. COPI is conventionally known to be involved in retrograde transport from trans-Golgi network to cis-Golgi network and endoplasmic reticulum. However, here it has been shown that COPI is also associated with EVs. COPI consists of seven core subunits α-COP, β′-COP, ε-COP, β-COP, δ-COP, γ-COP and ζ-COP. A cytoplasmic heptamer of these subunits, termed coatomer, is recruited to the membrane bilayer to form a COPI coat. Coatomer becomes stably membrane associated through interaction with activated Arf1. Stable association of coatomer leads to polymerization. Localized recruitment and activation of Arf1 and/or coat polymerization leads to localizes stress on the membrane, leading to vesicle scission. While COPI is known to dissociate from vesicles, residual COPI remains on the surface of vesicles.
As use herein, an “extracellular vesicle signal peptide” or “ESP” is a signal sequence containing a CBM, and which mediates EVs secretion of the cargo protein.
A “coatomer binding motif” or “CBM” as used herein, is the specific amino acid residues within and ESP that mediates interaction with COPI or one its subunits.
In one embodiment, the ESP is for binding to a α-COP (COPα or COPA), β′-COP (COP β2), or γ-COP (COPγ) subunit of the COPI. In one embodiment, the ESP is for binding to α-COP or β′-COP.
In one embodiment, the CBM comprises a two- or three-amino acid motif comprising two positively charged amino acids residues. In one embodiment, the two- or three-amino acid motif comprises KR, KK, KxK, or RR, wherein x is any amino acid. In one embodiment, the two- or three-amino acid motif comprises RR.
In one embodiment, the CBM is located in the EV-directed polypeptide: in an unstructured loop of the cargo polypeptide, in an unstructured tail that is positioned C-terminally with respect to the cargo polypeptide, or in an unstructured leader sequence that is positioned at N-terminally with respect to the cargo polypeptide, wherein the EV-directed polypeptide lacks a signal peptide.
In one embodiment, the ESP is at least 10 amino acids in length. In one embodiment, the ESP is at least 11 amino acids in length. In one embodiment, the ESP is at least 12 amino acids in length. In one embodiment, the ESP is at least 13 amino acids in length. In one embodiment, the ESP is at least 14 amino acids in length. In one embodiment, the ESP is at least 15 amino acids in length. In one embodiment, the ESP is at least 16 amino acids in length. In one embodiment, the ESP is at least 17 amino acids in length. In one embodiment, the ESP is at least 18 amino acids in length. In one embodiment, the ESP is from 18 to 34 amino acids in length.
In one embodiment, the ESP is an ESP from a protein in the Wnt family. In one embodiment, the ESP is at least 80% identical to an ESP from a protein in the Wnt family. In one embodiment, the ESP is at least 90% identical to an ESP from a protein in the Wnt family. In one embodiment, the ESP is at least 95% identical to an ESP from a protein in the Wnt family. In one embodiment, the ESP is at least 98% identical to an ESP from a protein in the Wnt family. In one embodiment, the protein in the Wnt family is human Wnt2, Wnt2b, Wnt4, Wnt5b, Wnt7a, Wnt8a, Wnt10a, Wnt10b, Wnt11, or Wnt16.
Table 1 sets forth sequence information for these Wnt family members and their respective ESPs and CBMs.
Table 2 provides sequences of the proteins listed in Table 1, with ESPs underlined and CBMs bolded.
VRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQ
FLPSAEAELIFLEESPDYCTCNSSLGIYGTEGRECLQNSHNTSRWERRSCGRLCTECGLQ
PRRLSGELVYFEKSPDFCERDPTMGSPGTRGRACNKTSRLLDGCGSLCCGRGHNVLRQTR
In one embodiment, the ESP comprises a sequence from a Wnt family member listed in Table 1 or 2 and comprises the respective CBM identified in Table 1 or 2. In one embodiment, the ESP comprises a sequence that is 80% identical to a sequence of a Wnt family member listed in Table 1 or 2 and comprises the respective CBM identified in Table 1 or 2. In one embodiment, the ESP comprises a sequence that is 90% identical to a sequence of a a Wnt family member listed in Table 1 or 2 and comprises the respective CBM identified in Table 1 or 2. In one embodiment, the ESP comprises a sequence that is 95% identical to a sequence of a Wnt family member listed in Table 1 or 2 and comprises the respective CBM identified in Table 1 or 2. In one embodiment, the ESP comprises a sequence that is 99% identical to a sequence of a Wnt family member listed in Table 1 or 2 and comprises the respective CBM identified in Table 1 or 2.
In one embodiment, the ESP comprises an ESP selected from the group consisting of those ESPs depicted in Table 1 and 2. In one embodiment, the ESP is 80% identical to an ESP selected from the group consisting of those ESPs depicted in Table 1 and 2 and comprises the respective CBM. In one embodiment, the ESP is 90% identical to an ESP selected from the group consisting of those ESPs depicted in Table 1 and 2 and comprises the respective CBM. In one embodiment, the ESP is 95% identical to an ESP selected from the group consisting of those ESPs depicted in Table 1 and 2 and comprises the respective CBM. In one embodiment, the ESP is 98% identical to an ESP selected from the group consisting of those ESPs depicted in Table 1 and 2 and comprises the respective CBM.
In one embodiment, the ESP is PNKKLASPRITFKPKRRV; a sequence at least 80% identical thereto that retains at least KK, KR, or RR; a sequence at least 90% identical thereto that retains at least KK, KR, or RR; or a sequence at least 95% identical thereto that retains at least KK, KR, or RR.
In one embodiment, the ESP is from a Wnt family member from a non-human species. Wnt family members in other species are identifiable, for example, by homology-based sequence searching using human Wnt family member sequences as query sequences. ESPs and CBMs can be located in non-human Wnt family members by sequence alignment. In one embodiment, the ESP is at least 80% identical to an ESP from a non-human Wnt homologue. In one embodiment, the ESP is at least 90% identical to an ESP from a non-human Wnt homologue. In one embodiment, the ESP is at least 95% identical to an ESP from a non-human Wnt homologue. The non-human Wnt homologue may be, for example, one of those Wnt7a homologues depicted in Table 3.
Mus
Musculus
KPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDL
Rattus
norvegicus
KPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDL
Canis
lupus
familiaris
KPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDL
Bos
Taurus
KPLSYRKPMDTELVYIEKSPSYCEEDPATGSVGTQGRACNKTAPQASGCDL
Macaca
mulatta
KPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDL
Gallus
gallus
KPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRMCNKTAQQSNGCDL
Xenopus
tropicalis
KIKNSYRKPMDTDLVYIEKSPNYCEEDPMTGSVGTQGRLCNKTAQHTSSCD
Danio
rerio
KPYSYRKPMDTDLVYIEKSPNYCEADPVTGSMGTQGRICNKTAQHTNGCDL
In one embodiment, the ESP is for binding to y-COP.
In one embodiment, the CBM comprises FFxxBB, wherein x is any amino acid and B is a basic amino acid.
In one embodiment, the cargo protein is a therapeutic polypeptide.
By “therapeutic polypeptide” is meant any polypeptide for which delivery is desired to achieve a therapeutic end, such as disease treatment or prophylaxis.
In one embodiment the therapeutic protein comprises an antibody or an antigen-binding fragment thereof, an enzyme, a cytotoxic protein, an antigen, a receptor-binding molecule, or a protein that is deficient in disease state.
In one aspect, there is provided a recombinant extracellular vesicle (EV)-directed polypeptide comprising:
In one embodiment, the EV-directed polypeptide is an exosome-directed polypeptide.
In one embodiment, the ESP is for binding to a α-COP, β′-COP, or γ-COP of coat protein complex 1 (COPI).
In one embodiment, the ESP is for binding to α-COP or β′-COP.
In one embodiment, the CBM comprises a two- or three-amino acid motif comprising two positively charged amino acids residues. In one embodiment, the two- or three-amino acid motif comprises KR, KK, KxK, RK, or RR, wherein x is any amino acid. In one embodiment, the two- or three-amino acid motif comprises RR. In one embodiment, the two- or three-amino acid motif comprises KRK. In one embodiment, the two- or three-amino acid motif comprises KxK, wherein x is any amino acid. In one embodiment, the CBM comprises a four-amino acid motif comprising at least two positively charged amino acid residues. In one embodiment, the CBM comprises a four-amino acid motif comprising at least three positively charged amino acid residues. In one embodiment, the four-amino acid motif comprises KxKK.
In one embodiment, the CBM is located in the EV-directed polypeptide: in an unstructured loop of the cargo polypeptide, in an unstructured tail that is positioned C-terminally with respect to the cargo polypeptide, or in an unstructured leader sequence that is positioned at N-terminally with respect to the cargo polypeptide, wherein the EV-directed polypeptide lacks a signal peptide.
In one embodiment, the ESP is at least 10 amino acids in length. In one embodiment, the ESP is at least 11 amino acids in length. In one embodiment, the ESP is at least 12 amino acids in length. In one embodiment, the ESP is at least 13 amino acids in length. In one embodiment, the ESP is at least 14 amino acids in length. In one embodiment, the ESP is at least 15 amino acids in length. In one embodiment, the ESP is at least 16 amino acids in length. In one embodiment, the ESP is at least 17 amino acids in length. In one embodiment, the ESP is at least 18 amino acids in length. In one embodiment, the ESP is from 18 to 34 amino acids in length.
In one embodiment, the ESP is an ESP from a protein in the Wnt family. In one embodiment, the ESP is at least 80% identical to an ESP from a protein in the Wnt family. In one embodiment, the ESP is at least 90% identical to an ESP from a protein in the Wnt family. In one embodiment, the ESP is at least 95% identical to an ESP from a protein in the Wnt family. In one embodiment, the ESP is at least 98% identical to an ESP from a protein in the Wnt family. In one embodiment, the protein in the Wnt family is human Wnt2, Wnt2b, Wnt4, Wnt5b, Wnt7a, Wnt8a, Wnt10a, Wnt10b, Wnt11, or Wnt16.
In one embodiment, the ESP comprises a sequence from a Wnt family member listed in Table 1 or 2 and comprises the respective CBM identified in Table 1 or 2. In one embodiment, the ESP comprises a sequence that is 80% identical to a sequence of a Wnt family member listed in Table 1 or 2 and comprises the respective CBM identified in Table 1 or 2 In one embodiment, the ESP comprises a sequence that is 90% identical to a sequence of a a Wnt family member listed in Table 1 or 2 and comprises the respective CBM identified in Table 1 or 2. In one embodiment, the ESP comprises a sequence that is 95% identical to a sequence of a Wnt family member listed in Table 1 or 2 and comprises the respective CBM identified in Table 1 or 2. In one embodiment, the ESP comprises a sequence that is 99% identical to a sequence of a Wnt family member listed in Table 1 or 2 and comprises the respective CBM identified in Table 1 or 2.
In one embodiment, the coatomer binding motif is for binding to γ-COP.
In one embodiment, the coatomer binding motif comprises FFxxBB, wherein x is any amino acid and B is a basic amino acid.
In one embodiment, the cargo protein is a therapeutic protein.
In one embodiment, the therapeutic protein comprises an antibody or an antigen-binding fragment thereof, an enzyme, a cytotoxic protein, an antigen, a receptor-binding molecule, or a protein that is deficient in disease state.
In one aspect, there is provided a nucleic acid molecule encoding the recombinant EV-directed polypeptide as defined herein.
In one aspect, there is provided a viral particle comprising the nucleic acid as defined herein.
In one aspect, there is provided a recombinant host cell comprising the nucleic acid as defined herein.
In one aspect, a composition comprising the EV as defined herein the nucleic acid as defined herein, or the viral particle as defined herein; together with an excipient diluent, or carrier.
In one aspect, there is provided a use of the EV as defined herein for delivery of the cargo polypeptide to a cell.
In one aspect, there is provided a use of the EV as defined herein for preparation of a composition for delivery of the cargo polypeptide to a cell.
In one aspect, there is provided the EV as defined herein for use in delivery of the cargo polypeptide to a cell.
In one aspect, there is provided a method of delivering a cargo polypeptide to a cell comprising contacting the cell with the EV as defined herein.
The skeletal muscle targeting activity of EV-bound Wnts may, in some embodiments, allow for targeting recombinant EVs comprising a payload to skeletal muscle cells.
In one aspect, there is provided a recombinant skeletal muscle-directed extracellular vesicle (EV) comprising coat protein complex 1 (COPI), a skeletal muscle targeting moiety comprising a Wnt family polypeptide, or polypeptide at least 90% identical thereto, and a payload for delivery to skeletal muscle.
The payload may be any molecule intended for delivery to skeletal muscle. The payload may be a small molecule, such as a small molecule drug, therapeutic agent, or cytotoxic agent. The payload may comprise a nucleic acid. The payload may comprise a payload polypeptide.
In one embodiment, there is provided a recombinant skeletal muscle-directed extracellular vesicle (EV) comprising coat protein complex 1 (COPI), a skeletal muscle targeting moiety comprising a Wnt family polypeptide, or polypeptide at least 90% identical thereto, and a payload polypeptide for delivery to skeletal muscle.
The “payload polypeptide” may be any molecule that it is desirably to deliver to the cells of skeletal muscle. The payload polypeptide may, for example, be an enzyme, a therapeutic polypeptide, a cytotoxic polypeptide, or a fluorescent protein.
In one embodiment, the skeletal muscle targeting moiety comprises the Wnt family member.
In one embodiment, the Wnt family member is human Wnt2, Wnt2b, Wnt4, Wnt5b, Wnt7a, Wnt8a, Wnt10a, Wnt10b, Wnt11, or Wnt16.
In one embodiment, the Wnt family member is human Wnt7a.
The polypeptide defined by percent identity to the Wnt family may be at least 80% identical to the Wnt family member. The polypeptide defined by percent identity may be at least 85% identical to the Wnt family member. The polypeptide defined by percent identity may be at least 90% identical to the Wnt family member. The polypeptide defined by percent identity may be at least 95% identical to the Wnt family member. The polypeptide defined by percent identity may be at least 98% identical to the Wnt family member. The polypeptide defined by percent identity may be at least 99% identical to the Wnt family member. In each case, alignment may be calculated across the full length of the full length sequence of the Wnt family member. The polypeptide defined by percent identity may retain substantially the same skeletal muscle targeting activity as the Wnt family member.
In one embodiment, the payload polypeptide is a free polypeptide within the EV. In these embodiments, the payload polypeptide is not linked or connected to the skeletal muscle targeting moiety.
In one embodiment, wherein the payload polypeptide is linked to the skeletal muscle targeting moiety.
In one aspect, there is provided a method for delivering a payload to skeletal muscle comprising contacting a cell with the recombinant skeletal muscle-directed EV as defined herein. The payload may be a payload polypeptide.
In one aspect, there is provided a use the recombinant skeletal muscle-directed EV as defined herein for delivery of the payload to skeletal muscle. The payload may be a payload polypeptide.
In one aspect, there is provided the recombinant skeletal muscle-directed EV as defined herein for use in delivery of the payload to skeletal muscle. The payload may be a payload polypeptide.
The identification of ESPs and CBMs within Wnts may, in some embodiments, allow recombinant Wnts to be produced that less apt to be secreted in EVs than their wild type counterparts.
In one aspect, there is provided a recombinant Wnt protein comprising an extracellular vesicle signal peptide (ESP) sequence comprising one or more coatomer binding motifs (CBMs), wherein at least one of the one or more CBMs is mutated relative to a corresponding wild-type sequence to form a mutated CBM that reduces or abrogates extracellular vesicle-targeting activity of the ESP sequence relative to the corresponding wild-type sequence.
My “mutated” is meant an amino acid sequence change relative to the same position of the corresponding wild-type sequence. Mutations may be amino acid sequences changes, deletions, insertions, or a combination thereof.
Where a “corresponding wild-type sequence” is referred to in these embodiments, it will be understood that this refers to the sequence of the parent molecule from which the recombinant Wnt protein is derived. For sample, corresponding wild-type sequences may be obtained from GenBank reference sequences. Alignments maybe generated with well-known tools.
In one embodiment, each of the one or more CBMs is mutated relative to the corresponding wild-type sequence to form mutated CBMs that reduce or abrogate extracellular vesicle-targeting activity of the ESP sequence relative to the corresponding wild-type sequence.
By “reduce” in this context is meant that the recombinant Wnt protein exhibits a reduction in secretion in EVs relative to the corresponding wild-type protein (with a corresponding increase in the fraction of free protein produced).
In one embodiment, the recombinant Wnt protein may be secreted as more than 50% free protein. In one embodiment, the recombinant Wnt protein may be secreted as more than 60% free protein. In one embodiment, the recombinant Wnt protein may be secreted as more than 70% free protein. In one embodiment, the recombinant Wnt protein may be secreted as more than 75% free protein. In one embodiment, the recombinant Wnt protein may be secreted as more than 80% free protein.
By “abrogate” or “disrupt” in this context is meant that the EV-targeting activity is substantially removed. This term must be understood in technical context, however. For example, deletion and replacement of the entirety of the ESP (see Example 2) of Wnt7a resulted in 86.7% of protein being in the free fraction, and result may vary depending on the particular Wnt.
In one embodiment, the recombinant Wnt protein may be secreted as more than 85% free protein. In one embodiment, the recombinant Wnt protein may be secreted as more than 90% free protein. In one embodiment, the recombinant Wnt protein may be secreted as more than 95% free protein.
Likewise, where it is mentioned that the recombinant Wnt is “free of EVs” it will be understood that the recombinant Wnt is secreted in a free form in a greater proportion than its corresponding wild type sequence.
In one embodiment, the mutated CBM(s) comprise(s) an amino acid substitution, deletion, and/or insertion relative to the corresponding wild-type sequence.
In one embodiment, the one or more CBMs each independently comprises a two- or three-amino acid motif comprising KR, KK, KxK, RK, or RR, wherein x is any amino acid.
In some embodiments, the mutations result a sequence change in at least one K or R in the CBM to a different amino acid. In some embodiments, the mutations result a sequence change in at least one K or R to a neutral or negatively charged amino acid. In some embodiments, more than one K and/or R residues of the CBM are mutated. It is also envisaged that the sequences could be scrambled. The sequences could be deleted and/or replaced with a non-natural sequence. Combinations of mutations are also envisaged. The effects of mutations may be tested with assays similar to those described herein.
In one embodiment, the ESP may be at least partly deleted. In one embodiment, the entirety of the ESP may be deleted. In one embodiment, the entirety of the ESP may be deleted and replaced with a different amino acid sequence. In one embodiment, the different amino acid sequence comprises a linker. In one embodiment, the linker comprises GSGS.
In the embodiments below, where the the recombinant Wnt protein is described as “comprising” a particular sequence, it will be understood that this definition accommodates the inclusion of the mutation(s) to the CBM(s) to reduce or abrogate EV-targeting activity.
In one embodiment, the recombinant Wnt protein comprises an amino acid sequence of Wnt2 (GenBank Accession No. P09544) or an amino acid sequence at least 80% identical thereto, and wherein the mutated CBM is located at amino acid positions corresponding to 261-262 of Wnt2. The amino acid sequence defined by percent identity may be at least 85% identical thereto. The amino acid sequence defined by percent identity may be at least 90% identical thereto. The amino acid sequence defined by percent identity may be at least 95% identical thereto. The amino acid sequence defined by percent identity may be at least 98% identical thereto. The amino acid sequence defined by percent identity may be at least 99% identical thereto. In one embodiment, the recombinant Wnt protein comprises an amino acid sequence of Wnt2 (GenBank Accession No. P09544). In one embodiment thereof, the ESP is deleted. In one embodiment thereof, the ESP is replaced with a linker. In one embodiment thereof, the linker comprises GSGS.
In one embodiment, the recombinant Wnt protein comprises an amino acid sequence of Wnt2b (GenBank Accession No. Q93097) or an amino acid sequence at least 80% identical thereto, and wherein the mutated CBM is located at amino acid positions corresponding to 292-293 of Wnt2b. The amino acid sequence defined by percent identity may be at least 85% identical thereto. The amino acid sequence defined by percent identity may be at least 90% identical thereto. The amino acid sequence defined by percent identity may be at least 95% identical thereto. The amino acid sequence defined by percent identity may be at least 98% identical thereto. The amino acid sequence defined by percent identity may be at least 99% identical thereto. In one embodiment, the recombinant Wnt protein comprises an amino acid sequence of Wnt2b (GenBank Accession No. Q93097). In one embodiment thereof, the ESP is deleted. In one embodiment thereof, the ESP is replaced with a linker. In one embodiment thereof, the linker comprises GSGS.
In one embodiment, the recombinant Wnt protein comprises an amino acid sequence of Wnt4 (GenBank Accession No. P56705) or an amino acid sequence at least 80% identical thereto, and wherein the mutated CBM is located at amino acid positions corresponding to 247-248 of Wnt4. The amino acid sequence defined by percent identity may be at least 85% identical thereto. The amino acid sequence defined by percent identity may be at least 90% identical thereto. The amino acid sequence defined by percent identity may be at least 95% identical thereto. The amino acid sequence defined by percent identity may be at least 98% identical thereto. The amino acid sequence defined by percent identity may be at least 99% identical thereto. In one embodiment, the recombinant Wnt protein comprises an amino acid sequence of Wnt4 (GenBank Accession No. P56705). In one embodiment thereof, the ESP is deleted. In one embodiment thereof, the ESP is replaced with a linker. In one embodiment thereof, the linker comprises GSGS.
In one embodiment, the recombinant Wnt protein comprises an amino acid sequence of Wnt5 (GenBank Accession No. 81029) or an amino acid sequence at least 80% identical thereto, and wherein the mutated CBM is located at amino acid positions corresponding to 259-260 of Wnt5. The amino acid sequence defined by percent identity may be at least 85% identical thereto. The amino acid sequence defined by percent identity may be at least 90% identical thereto. The amino acid sequence defined by percent identity may be at least 95% identical thereto. The amino acid sequence defined by percent identity may be at least 98% identical thereto. The amino acid sequence defined by percent identity may be at least 99% identical thereto. In one embodiment, the recombinant Wnt protein comprises an amino acid sequence of Wnt5 (GenBank Accession No. 81029). In one embodiment thereof, the ESP is deleted. In one embodiment thereof, the ESP is replaced with a linker. In one embodiment thereof, the linker comprises GSGS.
In one embodiment, the recombinant Wnt protein comprises an amino acid sequence of Wnt 7a (GenBank Accession No. 000755) or an amino acid sequence at least 80% identical thereto, and wherein the mutated CBM is located at amino acid positions corresponding to one or more of 247-248 and 253-256 of Wnt7a. The amino acid sequence defined by percent identity may be at least 85% identical thereto. The amino acid sequence defined by percent identity may be at least 90% identical thereto. The amino acid sequence defined by percent identity may be at least 95% identical thereto. The amino acid sequence defined by percent identity may be at least 98% identical thereto. The amino acid sequence defined by percent identity may be at least 99% identical thereto. In one embodiment, the recombinant Wnt protein comprises an amino acid sequence of Wnt 7a (GenBank Accession No. 000755). In one embodiment thereof, the ESP is deleted. In one embodiment thereof, the ESP is replaced with a linker. In one embodiment thereof, the linker comprises GSGS.
In one embodiment, the recombinant Wnt protein comprises an amino acid sequence of Wnt8a (GenBank Accession No. Q9H1J5) or an amino acid sequence at least 80% identical thereto, and wherein the mutated CBM is located at amino acid positions corresponding to 222-223 of Wnt8a. The amino acid sequence defined by percent identity may be at least 85% identical thereto. The amino acid sequence defined by percent identity may be at least 90% identical thereto. The amino acid sequence defined by percent identity may be at least 95% identical thereto. The amino acid sequence defined by percent identity may be at least 98% identical thereto. The amino acid sequence defined by percent identity may be at least 99% identical thereto. In one embodiment, the recombinant Wnt protein comprises an amino acid sequence of Wnt8a (GenBank Accession No. Q9H1J5). In one embodiment thereof, the ESP is deleted. In one embodiment thereof, the ESP is replaced with a linker. In one embodiment thereof, the linker comprises GSGS.
In one embodiment, the recombinant Wnt protein comprises an amino acid sequence of Wnt10a (GenBank Accession No. Q9GZT5) or an amino acid sequence at least 80% identical thereto, and wherein the mutated CBM is located at amino acid positions corresponding to 328-329 of Wnt10a. The amino acid sequence defined by percent identity may be at least 85% identical thereto. The amino acid sequence defined by percent identity may be at least 90% identical thereto. The amino acid sequence defined by percent identity may be at least 95% identical thereto. The amino acid sequence defined by percent identity may be at least 98% identical thereto. The amino acid sequence defined by percent identity may be at least 99% identical thereto. In one embodiment, the recombinant Wnt protein comprises an amino acid sequence of Wnt10a (GenBank Accession No. Q9GZT5). In one embodiment thereof, the ESP is deleted. In one embodiment thereof, the ESP is replaced with a linker. In one embodiment thereof, the linker comprises GSGS.
In one embodiment, the recombinant Wnt protein comprises an amino acid sequence of Wnt10b (GenBank Accession No. 000744) or an amino acid sequence at least 80% identical thereto, and wherein the mutated CBM is located at amino acid positions corresponding to 302-303 of Wnt10b. The amino acid sequence defined by percent identity may be at least 85% identical thereto. The amino acid sequence defined by percent identity may be at least 90% identical thereto. The amino acid sequence defined by percent identity may be at least 95% identical thereto. The amino acid sequence defined by percent identity may be at least 98% identical thereto. The amino acid sequence defined by percent identity may be at least 99% identical thereto. In one embodiment, the recombinant Wnt protein comprises an amino acid sequence of Wnt10b (GenBank Accession No. 000744). In one embodiment thereof, the ESP is deleted. In one embodiment thereof, the ESP is replaced with a linker. In one embodiment thereof, the linker comprises GSGS.
In one embodiment, the recombinant Wnt protein comprises an amino acid sequence of Wnt11 (GenBank Accession No. 096014) or an amino acid sequence at least 80% identical thereto, and wherein the mutated CBM is located at amino acid positions corresponding to 255-256 of Wnt11. The amino acid sequence defined by percent identity may be at least 85% identical thereto. The amino acid sequence defined by percent identity may be at least 90% identical thereto. The amino acid sequence defined by percent identity may be at least 95% identical thereto. The amino acid sequence defined by percent identity may be at least 98% identical thereto. The amino acid sequence defined by percent identity may be at least 99% identical thereto. In one embodiment, the recombinant Wnt protein comprises an amino acid sequence of Wnt11 (GenBank Accession No. 96014). In one embodiment thereof, the ESP is deleted. In one embodiment thereof, the ESP is replaced with a linker. In one embodiment thereof, the linker comprises GSGS.
In one embodiment, the recombinant Wnt protein comprises an amino acid sequence of Wnt16 (GenBank Accession No.) or an amino acid sequence at least 80% identical thereto, and wherein the mutated CBM is located at amino acid positions corresponding to one or more of 264-265, 265-266, 268-269, 269-270, and 275-276 of Wnt16. The amino acid sequence defined by percent identity may be at least 85% identical thereto. The amino acid sequence defined by percent identity may be at least 90% identical thereto. The amino acid sequence defined by percent identity may be at least 95% identical thereto. The amino acid sequence defined by percent identity may be at least 98% identical thereto. The amino acid sequence defined by percent identity may be at least 99% identical thereto. In one embodiment, the recombinant Wnt protein comprises an amino acid sequence of Wnt16 (GenBank Accession No.). In one embodiment thereof, the ESP is deleted. In one embodiment thereof, the ESP is replaced with a linker. In one embodiment thereof, the linker comprises GSGS.
In one aspect, there is provided a recombinant polypeptide comprising the recombinant Wnt protein as defined herein.
In one embodiment, there is provided a composition comprising the recombinant Wnt protein as defined herein, together with an acceptable excipient, diluent, or carrier.
In one aspect, there is provided a recombinant nucleic acid encoding the recombinant Wnt protein as defined herein.
In one embodiment, the recombinant nucleic acid comprises DNA or RNA.
In one aspect, there is provided a vector comprising the recombinant nucleic acid as defined herein.
In one aspect, there is provided a host cell comprising the recombinant nucleic acid as defined here, or the vector as defined herein.
In one aspect, there is provided a use of the recombinant nucleic acid as defined here, or the host cell defined here, for production of the recombinant Wnt protein as defined herein, wherein the recombinant Wnt protein is free of extracellular vesicles. In one embodiment, there is proportionally more recombinant Wnt protein produced as free protein compared to the corresponding wild-type sequence. Accordingly, there is proportionally less recombinant Wnt protein produced as EV-bound protein compared to the corresponding wild-type sequence.
In one aspect, there is provided a method for producing the recombinant Wnt protein as defined herein comprising introducing the recombinant nucleic acid as defined herein to a cell, and culturing the cell to produce the recombinant Wnt protein, wherein the recombinant Wnt protein is free of extracellular vesicles. In one embodiment, there is proportionally more recombinant Wnt protein produced as free protein compared to the corresponding wild-type sequence. Accordingly, there is proportionally less recombinant Wnt protein produced as EV-bound protein compared to the corresponding wild-type sequence.
In one aspect, there is provided a method for producing the recombinant Wnt protein as defined herein comprising culturing the host cell as defined herein to produce the recombinant Wnt protein, wherein the recombinant Wnt protein is free of extracellular vesicles. In one embodiment, there is proportionally more recombinant Wnt protein produced as free protein compared to the corresponding wild-type sequence. Accordingly, there is proportionally less recombinant Wnt protein produced as EV-bound protein compared to the corresponding wild-type sequence.
Wnt proteins are a secreted family of hydrophobic glycoproteins that regulate important developmental processes. Here the molecular mechanisms that enables long-range Wnt signaling via exosomes, a class of secreted extracellular vesicles, is investigated. It is discovered that Wnt7a is secreted at high levels on exosomes following muscle injury to stimulate regeneration. Structure-function analysis identified the signal sequence in Wnt7a, the Extracellular Vesicle Signal Peptide, which directs exosomal secretion, and revealed that palmitoylation is not required. This peptide forms a heretofore unknown functional association with Coatomer proteins through a positively charged motif to direct trafficking of Wnt to exosomes. The positively charged motif and mechanism are conserved among Wnts. These studies identify a signal peptide that traffics cargo to the surface of exosomes and elucidates the mechanism that facilitate long-range Wnt signaling. The signal peptide can be used in recombinant polypeptide constructs to target other cargo molecules to exosomes.
Exosomes are 40-150 nm small EVs of endocytic origin involved in intercellular communication that transfer bioactive cargo, for example lipids, proteins, microRNAs, and mRNAs, to distal cells. Exosomes have been used in therapeutic applications.
Wnt proteins are an evolutionary conserved family of secreted glycoproteins that govern essential developmental, growth, and regenerative processes, as well as being involved in pathological conditions like cancer. Wnt signaling plays multiple roles in regulating stem cell function, including proliferation, cell polarity and symmetric division, motility, and fate specification. Despite their relative insolubility due to the palmitoylation required for specific Frizzled receptor binding, Wnt proteins actively participate in long-range paracrine signaling between Wnt-producing cells and distal recipient cells. Several mechanisms have been proposed to mediate long-range intercellular Wnt signaling including transfer of Wnt proteins via lipoproteins, cell extensions called cytonemes, association with soluble Wnt-binding proteins, or via a class of extracellular vesicles (EVs) termed exosomes.
In vitro studies have shown different Wnt proteins are secreted on the surface of exosomes, and that exosomal-Wnts are capable of eliciting appropriate signaling in target cells. Moreover, examples have been noted where up to 40% of Wnt proteins are secreted on exosomes. Considerable in vivo evidence derived from studies in Caenorhabditis and Drosophila, support the importance of EVs for long-range Wnt signaling. To date, long-range Wnt signaling mediated by exosomes has not been documented in vivo in mammals.
Following acute injury in adult skeletal muscle, Wnt7a is highly upregulated where it positively stimulates regenerative myogenesis by acting at multiple levels. Wnt7a/Fzd7 signaling via the planar-cell-polarity pathway stimulates symmetric muscle stem cell expansion and cell motility. Wnt7a/Fzd7 signaling via the AKT/mTOR pathway in myofibers stimulates anabolic growth and hypertrophy. Consequently, intramuscular injection of Wnt7a protein significantly ameliorates disease progression in mdx mice, a mouse model for Duchenne Muscular Dystrophy (DMD). Together, these findings indicate that Wnt7a is a promising candidate therapy for DMD. However, systemic delivery of Wnt7a via the circulation has remained a challenge because of the high hydrophobicity conferred by the conserved palmitoylation.
It has been found that Wnt7a is secreted at high levels on exosomes following muscle injury. Structure function analysis was performed and a novel specific signal sequence in Wnt7a was identified that was termed the Extracellular Vesicle Signal Peptide (ESP), which comprises a positively charged motif, which mediates Wnt7a-EVs secretion. Linking of ESP sequence to other cargo resulted in secretion on EVs. Furthermore, it was found that analogous ESP sequences are found in other Wnts that are secreted on EVs. Using Bio-ID, Coatomer proteins were identified as necessary for binding the ESP to traffic Wnt7a to the exterior of EVs. Finally, modeling and mutagenesis confirmed that the interaction occurs between the positively charged motif in the ESP and COPα and COPβ2.
The Wnt family of proteins generally, and Wnt7a specifically, were selected as a model to study exosome trafficking in the hope of elucidating general principles of wider application.
Cell culture. HEK293T cells were obtained from ATCC (CRL-3216) and verified to be free from mycoplasma contamination using the MycoSensor PCR Assay Kit (Agilent Technologies). Cells were cultured as in DMEM (Lonza) supplemented with 10% FBS, 100 U/mL penicillin, 100 U/mL streptomycin and maintained at 37° C. in a humidified incubator equilibrated with 5% CO2. Primary myoblasts were purified from C57BL/10 mice by magnetic cell separation (MACS) as previously described by Sincennes et al. Primary myoblasts were cultured on collagen-coated dishes with HAM F12-X, 10% FBS, 100 U/mL penicillin, 100 U/mL streptomycin and maintained at 37° C. in a humidified incubator equilibrated with 5% CO2. For differentiation, myoblasts were grown up to 80% confluence and growth media was replaced with differentiation medium [HAM F12-X: DMEM (1:1), 2% HS, 100 U/mL penicillin, and 100 U/mL streptomycin] for 4 days unless otherwise stated. During differentiation serums were treated to be free of extracellular vesicles prior to assays.
Mice and animal care. All experimental protocols for mice used in this study were approved in accordance with the guidelines of the Canadian Council on Animal Care. Food and water were administered ad libitum. For muscle regeneration experiments, eight week-old male mice were used, an F2 cross between the offspring of Myf5-Cre mice and Wnt7afl/fl mice in a C57BL/6 genetic background. Muscle regeneration was assessed four days following cardiotoxin injury as previously described with the following modifications. Mice were anesthetized with isoflurane and CTX injection was performed on a single injection into the TA (50 μL, 10 μM) and muscle regeneration assessed after 96 h.
Pre-embedding immunogold labeling for tissue TEM. Tibialis Anterior muscles from 8-week-old C57BL/6 mice were processed 96 h after cardiotoxin injury. Briefly, specimens were fixed in Karnovsky's fixative for 2 weeks. After fixation all segments were subsequently washed with 0.1M sodium cacodylate, 0.1% sodium borohydride, permeabilized with 0.1% triton X-100 and blocked with 10% donkey serum+0.6% fish gelatin. TA samples were incubated with Wnt7a antibody. After 48 h incubation, segments were rinsed thoroughly with PBS and incubated overnight with the secondary antibody Ultra small (0.8 nm) Gold conjugated (EMS) in blocking buffer at RT. Later, samples were rinsed with 0.1M sodium cacodylate and post-fixed with 2% glutaraldehyde in 0.1 M sodium cacodylate. Pre-embedding enhancement was realized with silver enhancement kit (AURION R-Gent SE-EM, EMS) according to the manufacturer's instructions. After enhancement, samples were secondly post-fixed with 1% osmium tetroxide in 0.1 M sodium cacodylate buffer. Then, samples were dehydrated in increasing concentration of ethanol and infiltrated in Spurr resin. Ultrathin transversal sections (80 nm) were collected onto 200-mesh copper grids and counterstained with 2% aqueous uranyl acetate and with Reynold's lead citrate. Finally, specimens were observed under a transmission electron microscope (Hitachi 7100, Gatan digital camera). For the analysis, approximately 50 immunoelectron micrographs were examined per muscle at different magnifications.
Pre-embedding immunogold labeling for cells and EVs TEM. Fixed HEK293T cells/exosome pellets were treated separately with 0.1% sodium borohydride in PBS. After a permeabilization step pellets were blocked in blocking buffer (10% donkey serum+0.6% gelatin from cold water fish skin in PBS) for 2 h. Cell/exosome pellets were incubated with the primary antibody for 48 h. Pellets were incubated overnight with the secondary antibody (Jackson ImmunoResearch). Immunogold-labelled cells and EVs were fixed with 2% glutaraldehyde in 0.1 M sodium cacodylate buffer and enhancement was performed with a silver enhancement kit on the immunogold-labelled cells. All samples were post-fixed with 1% osmium tetroxide in 0.1 M sodium cacodylate buffer. Specimens were dehydrated and embedded in resin and polymerized overnight at 70° C. Immunogold-labelled exosome ultrathin sections were observed by transmission electron microscopy at 100 000× and 150 000×.
Conditioned media production for EVs. Equal numbers of HEK293T cells were seeded and the different plasmids were transfected with linear polyethylenimine (Polysciences), accordingly to manufacturer's instructions. After 48 h of secretion in DMEM and 10% FBS exosome-depleted, conditioned media was collected for exosomal isolation. For tissue EVs a protocol has been standardized to obtained conditioned media from muscles explants. Briefly, both hind limbs of mice were injured with cardiotoxin (90 μL per leg, 10 μM). Four days later, injured muscles were harvested and cultured as explants on an exosome-depleted FBS pre-coated dish with high-glucose DMEM (Gibco) and maintained at 37° C. in a humidified incubator equilibrated with 5% CO2. After 48 h conditioned media was collected for exosomal isolation.
EVs isolation. Conditioned media (20 mL) was clarified by sequential centrifugation (300 g at 4° C. for 10 min; 2500 g at 4° C. for 10 min and 20,000 g at 4° C. for 20 min). Supernatant was transferred to Flexboy bag (Sartorius) and subjected to tangential flow filtration (TFF) under sterile conditions. Briefly, a KrosFlo Research 2i TFF system (Spectrum Laboratories) coupled to a MidGee Hoop ultrafiltration hollow fiber cartridge (GE Healthcare) 500-KDa MWCO was used. Transmembrane pressure was automatically adjusted at 3 PSI and a shear rate at 3000 s−1. Sample was concentrated up to 10 mL and then subjected to continuous diafiltration. Finally, sample was concentrated at 5 mL and recovered from the cartridge. Lastly, EVs were pellet down after spinning on an ultra bench centrifuge for 30 min at 100,000 g at 4° C.
Immunoblot analysis. Immunoblot analysis was performed as described previously with the following modifications. The lysates from EVs were not clarified by centrifugation. The immunoblot transferring was performed onto PVDF membranes. All antibodies and dilutions are provided in Table 4.
Immunohistochemistry. TA muscle cryosections were rehydrated using PBS, and then fixed with 2% PFA in PBS at room temperature. After washing with PBS, permeabilization with a solution of 0.1% Triton and 0.1 M glycine in PBS was applied for 10 min at room temperature. Mouse on mouse blocking reagent was used at a dilution of 1:40 in blocking solution of 10% goat serum, 1% bovine serum albumin (BSA) and 0.1% Tween 20 in PBS for one hour at room temperature. Primary antibodies were incubated overnight. Nuclei were counterstained with DAPI before mounting in Permafluor.
Hypertrophy assay. Myoblasts were differentiated for 4 days along with EVs stimulation at 10 μg/mL (based on total extracellular vesicle protein quantification after lysis) or recombinant Wnt7a protein at 100 ng/ml. Myotubes were fixed with 4% PFA. Permeabilization and blocking solution consisting of 0.3 M glycine, 1% BSA and 0.1% Tween in PBS was added for 90 mins. p-MHC primary antibody was incubated overnight. Nuclei were counterstained with DAPI before mounting in Permafluor. FIJI software was used to analyze myotube diameter. Ten blind images were acquired per well at 20×. The 50 largest myotubes from each well were included in the analysis.
Construction of Wnt7a mutants. Wnt7a was originated from a pcDNA3-hWnt7a-HA plasmid. Wnt10a and Wnt16 originate from pcDNA-hWnt10a-V5 (Addgene 35939) or pcDNA-hWnt16-V5 (Addgene 35942) plasmid respectively. Wnt10b used herein was a gift from Marian Waterman, David Virshup and Xi He from theplasmid kit (Addgene kit #1000000022). Mutation and truncation were generated by overlap extension PCR with specially designed primers. BamHI and EcoRI restriction sites were included in primers. OE-PCR products and pcDNA3-HA vector were digested with BamHI and EcoRI and ligated with Takara ligase Solution. All constructs were verified by sequencing. All primers and coding sequences sources are provided in Table 5.
RASRNKRPTFLKIKKPLSYRKPMDTDLVYI
PTFLKIKKPLSYRKPMDTDLVYIEKSPNYC
VRASRNKRPYPYDVPDYA
VRASRNKRPTFLKIKKPLSYRKPMDTDLVY
YPYDVPDYA
PTFLKIKKPLSYRKPMDTDLVYIEKSPNYC
YPYDVPDYA
TFLKIKKPLSYRKPMDTDLVYIEKSPNYCE
GSGSLSYRKPMDTDLVYIEKSPNYCEEDPV
YPYDVPDYA
TFLKIKKPLSYRKPMDTDLVYIEKSPNYCE
PVRASRNKRPTFLKIKKPLSYRKPMDTDLV
VRASRNKRPTFLKIKKPLSYRKPMDTDLVY
PTFLKIKKPNARTLMNLHNNEAGRKILEEN
KPLSYRKPMDTDLVYIEKSPNYCEEDPVTG
PTFLKIKKPNARTLMNLHNNEAGRKILEEN
PVRASRNKRPTFLKIKKPMNRKARRCLGHL
NKKLASPRITFKPKRRVLSYRKPMDTDLVY
VRASRNARPTFLKIKKPLSYRKPMDTDLVY
VRASRNKRPTFLAIKKPLSYRKPMDTDLVY
VRASRNKRPTFLKIAKPLSYRKPMDTDLVY
VRASRNKRPTFLKIKAPLSYRKPMDTDLVY
In-silico homology modeling of Wnt7a. The homology model of human Wnt7a was constructed through its sequence annealing over the resolved structure of Wnt3 protein (PDB 6AHY) with FoldX BuildModel command (Centre for Genomic Regulation, http://foldxsuite.crg.eu/command/BuildModel). The annealing of the sequence resulted in no energetic conflicts enlighting that the folding captured by the crystal represents a stable configuration of proteins within the Wnt family. GSGS linker length was chosen in order to replace ESP. In order to affect folding, the distance criteria with respect to the terminal residues of the ESP were taken into account (afterwards confirmed experimentally,
In-silico determination of the ESP region. The in-silico determination of the ESP region was performed through the free energy measurement of folding of the Wnt7a model (ΔGwt) versus the free energy resulting of the truncation of windows of 15 aa (ΔGtruncated) along the whole sequence. Those regions not contributing to the protein folding present a very negative variation energy (ΔΔGtruncated_WT<<0). The N-terminal region is not structured since the mapping of the Wnt folding domain (PFAM (pfam http://pfam.xfam.org/) PF00110) starts in position 41, the C-terminal region presents also low energies being a folded region not in close contact with the rest of the protein. Besides the terminal regions, the only sequence window presenting very low energy was selected as ESP (afterwards confirmed experimentally) since it is not important for the folding, is highly variable along the Wnt family, evincing that its sequence codifies for functional behavior.
Modeling of the ESP-loop swapping. All the unstructured regions within the Wnt7a generated model that were surrounded by secondary structured regions where evaluated in terms of end-to-end distances and torsional angles to establish their ability to room the ESP region though a sequence swap. Using ModelX (Centre for Genomic Regulation http://modelx.crg.es/) fragment replacement the ESP was inserted using as anchoring terminal aminoacids GLU171 and ASN 175. Energies of the replaced model was measured then with the FoldX force field and no energetic conflicts or clashes where found, demonstrating that the sequence swapping was supported by the structure.
Uptake assays. HEK293T cells were transfected with pcDNA3_HALO and pcDNA3_HALO-EBP plasmids that were generated from a Pax7-HALO plasmid (Epoch Life Science) using PEI, as aforementioned. EVs from transfected cells were isolated as previously described and added to fresh seeded HEK293T for 15 min. After, stimulated cells were labeled with HaloTag® Ligands for Super Resolution Microscopy-Janelia 549 (Promega) accordingly to manufacturer's instructions. Cells were then fixed in 2% PFA for 5 min and washed three times with PBS. Lastly, cells were analyzed by image cytometry in the Amnis ImageStream X platform to verify the location of the fluorescence inside the cell. The flourescence detected by the Amnis ImageStream was excited using 561 nm laser and detected by the 580-30 emission filter channel.
BioID assay. Stable primary myoblast cell lines expressing BioID2 BioID2-ESP and Wnt7a-BioID2 fusion proteins were generated using the mycBioID2-pBABE-puro vector (Addgene Plasmid). Myoblasts were grown in 15 cm culture dishes at subconfluency and incubated with biotin (Sigma-Aldrich: dissolved in DMSO) at a final concentration of 50 μM for 18 h. Plates were scraped in ice cold PBS, spun at 20817 g for 5 min to concentrate cell pellet, then resuspended in RIPA lysis buffer containing protease inhibitor cocktail. Cells were incubated on ice for 30 min, and then spun down 20817 g at 4° C. for 20 min. Supernatant was transferred to new low retention Eppendorf tube, and protein concentration was quantified using Bradford reagent and spectrometry. Magnetic streptavidin beads (New England Biolabs) were used to precipitate the biotinylated protein fraction. Streptavidin beads were washed twice in RIPA lysis buffer and subsequently added to protein lysates for overnight incubation at 4° C. rotating. The following day, beads were sequentially washed with RIPA buffer, 1 M KCl, 0.1 M Na2CO3, 2 M urea in 10 mM Tris-HCl (pH 8), and a final RIPA buffer wash. Biotinylated proteins were then eluted from beads by boiling for 10 min in 25 ul 6× Laemmli buffer containing 20 mM DTT and 2 mM biotin. Supernatant was loaded into precast gradient gel (4-15% Mini-PROTEAN® TGX Stain-Free™ Protein Gel) and run for 30 min at 100V. Colloidal blue dye (Thermofisher) was applied for 3 h, then rinsed in miliQ water while shaking overnight. The entire protein containing lane for each condition was then cut out and stored in 1% acetic acid. Samples were then transferred for further processing as described below.
Proteomic analysis. Proteins were digested in-gel using trypsin (Promega) according to the method of Shevchenko. Peptide extracts were concentrated by Vacufuge (Eppendorf) and purified by ZipTip (Sigma-Millipore). LC-MS/MS was performed using a Dionex Ultimate 3000 RLSC nano HPLC (Thermo Scientific) and Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific). MASCOT software version 2.6.2 (Matrix Science) was used to infer peptide and protein identities from the mass spectra. The observed spectra were matched against sequences from SwissProt (version 2020-01) and also against an in-house database of common contaminants. The results were exported to Scaffold (Proteome Software) for further validation and viewing. Enrichment heatmap was generated by computing the log 2 of the fold enrichment of each condition versus its control. Gene Ontology term enrichment analysis was performed over the “cellular component” branch using ClueGO plugin on Cytoscape software.
Proximity ligation assay (PLA). Fixed myotubes were permeabilized (0.1% Triton X-100, 0.1 M Glycine, PBS) for 10 min and blocked with Duolink Blocking Solution (Sigma) for 3 h. Incubation with primary antibodies diluted in Duolink Blocking Solution (Sigma) was performed overnight at 4° C. PLA reactions were subsequently performed using Duolink PLA probes for goat-mouse and goat-rabbit and Duolink In Situ Detection Reagents Red (Sigma) following the manufacturer's protocol. Myotubes were counterstained with GM130 to visualize the Golgi Apparatus. After the final wash, cells were mounted with VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories). For analysis Z-stack images of myotubes were acquired on an epifluorescent microscope equipped with a motorized stage (Zeiss AxioObserver Z1) with a step size of 0.2 μm to span the cell (25 slices in total) and images were deconvoluted using Zen Software (Zeiss). 3D sum intensity Z-projection was performed with ImageJ software.
Immunoprecipitation. Wnt7a-HA was overexpressed in HEK293T cells using Lipofectamine 2000 (Life Technologies) according to the manufacturer's instructions. Cells and EVs were isolated two days post-transfection and lysed in immunoprecipitation lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 2 mM MgCl2, 0.5 mM EDTA, 0.5% Triton X-100, and protease inhibitors) for 30 min on ice. Lysates from cells were cleared by centrifugation and were incubated with either HA (Benthyl) or COPβ2 (Cusabio) antibodies-Dynabeads Protein G (Thermo Fisher) overnight at 4° C., accordingly to the manufacturer's instructions. Beads were washed 4 times with lysis buffer and eluted with Laemmli buffer. Immunoprecipitates were resolved by SDS-PAGE and analyzed by immunoblot with the indicated antibodies.
SIRNA silencing. siRNA transfections were performed on HEK293T cells at 16 h post-culture using Lipofectamine RNAiMAX (Life Technologies) according to the manufacturer's instructions. siRNAs for COPα and COPβ2 were purchased from Dharmacon and used at a final concentration of 10 nM and 20 nM respectively. The following day cells were transfected with Wnt7a as aforementioned.
COPα-COPβ2 binding energy determination. Departing from the determined structure of Copβ2 binding to dilysine motif KxK (PDB 4J77) other binding motifs candidates described in literature were modeled by annealing them onto the crystallized binding peptide using FoldX BuildModel command. Positively charged motifs (KR, KK, RR) demonstrated a high compatibility with the binding pocket, presenting stronger binding energies than the crystallized motifs, while the truncation of these positive residues with alanine make the interactions weaker. Results were extrapolated to Copa subunit since it presents an identical folding than the β2 subunit and a sequence conserved binding pocket (PDB 5NZR).
Statistical analysis. Experiments were performed with a minimum of three biological replicates and results are presented as the mean±SEM. Student's t-test were performed to assess the statistical significance of two-tailed analysis. For multiple comparisons ANOVA test was employed and TUKEY test for post-hoc analysis. P-values are indicated as *p≤0.05, ** p≤0.01, *** p≤0.001, and P-values <0.05 were considered to be statistically significant.
Wnt7a expression is highly upregulated in newly differentiating myofibers following acute injury of skeletal muscle. Examination of muscle cryosections 96 h following cardiotoxin injury by Immunogold Electron Transmission Microscopy (iTEM) labeling revealed extensive secretion of Wnt7a on the surface of exosomes (
To eliminate contamination with free secreted proteins that is typically found when using ultracentrifugation to concentrate exosomal fractions, tangential flow filtration (TFF) was employed, which allows independent purification of both freely secreted Wnt7a and Wnt7a bound to EVs (see
To establish whether the bioactivity of Wnt7a-EVs is due to the Wnt7a cargo, EVs were isolated from regenerating muscle from mice with a functional Wnt7a gene (Myf5+/+:Wnt7afl/fl), or from mice where Wnt7a is specifically deleted in muscle (Myf5Cre/+: Wnt7afl/fl) (
Several groups have asserted the importance of palmitoylation for Wnt secretion and activation. Therefore, Wnt7a secretion was tested following mutation of the two conserved palmitoylation sites, cysteine 73 and serine 206. These sites have been previously shown to be critical for Wnt3a secretion. Notably, it was observed that secretion of Wnt7a on EVs was entirely unaffected by mutation of the palmitoylation sites (
Therefore, structure-function analysis was performed of Wnt7a to map the regions required for localization to EVs. A series of N-terminal and C-terminal deletions of Wnt7a-HA was constructed (
Wnt7a secretion on EVs was not impaired upon deletion of the 68aa following the SP (Wnt7a_Δ32-99), and the last 48aa (Wnt7a_Δ301-349) (
To identify the region that mediates the targeting of Wnt7a to EVs, Wnt7a was 3D-modeled based on XWnt8a structure (
Replacement of the 17 aa sequence between position 240 and 257 with the linker domain GSGS (Wnt7a_ΔESP*GSGS) resulted in a loss of Wnt7a targeting to EVs, with a corresponding increase in secretion as free protein, and with no effect on total Wnt7a protein expression (
It was next investigated whether the ESP is sufficient to target a different protein for secretion on EVs. First, the ESP was added to a truncated Wnt7a that was previously found to not localize to EVs (Wnt7a_Δ213-349) (
Insertion of the linker GSG or ESP between position 171 and 175 into full length Wnt7a-HA (Wnt7a-FL) had no effect on secretion on EVs (
It was next contemplated whether fusing the ESP to a non-Wnt protein would confer the ability to be secreted on EVs. Therefore, the ESP was fused to the HALO tag, a 297-residue peptide derived from a bacterial enzyme designed to covalently bind to a fluorescent ligands (
To investigate the molecular basis whereby the ESP mediates the secretion of Wnt7a on EVs, BioID analysis was performed to identify potential binding proteins. Myc-tagged BirA was used—a highly efficient proximity dependent biotin ligase—that tags proteins interacting with the constructs, even if the interaction is transient (
GO-term analysis of the common ESP and Wnt7a interacting proteins strongly supports the hypothesis that Wnt7a is secreted via the COPI vesicle pathway (
Proximity Ligation Assays between Wnt7a and COPα or COPβ2 confirmed the interaction of Wnt7a with these coatomers, displaying a different pattern of interaction at the Golgi versus the cell membrane respectively (
To directly assess the role of COPβ2 and COPα in mediating secretion of Wnt7a on EVs, Wnt7a-HA secreting HEK293T cells were transfected with siRNA to knock down COPβ2 or COPα (
The KR Motif within the ESP is Required for Binding to the Coatomer Complex
The presence of Coatomer proteins on EVs has been previously noted. Moreover, COPα and COPβ2 have been shown to bind with the positively charged motifs (KKxx, KxKxx, and in the case of β′-COP also RKxx) present in interacting proteins. Therefore, the role played by the positively charged motif present in the ESP was evaluated to mediate secretion of Wnt7a on EVs was evaluated.
First, a Wnt7a mutant was tested in which the ESP sequence was scrambled whilst maintaining the positively charged motifs (
In silico 3D modeling of the Wnt7a interaction with COPβ2 suggests a stable interaction through hydrogen bonds and hydrophobic interactions with the positively charged Wnt7a motif with three different residues of COPβ2. Due to the presence of an identical binding pocket in COPα subunit (
To empirically test the structural model, single point mutations were performed of the lysines residues to alanine across the ESP domain. Only the disruption of KR was found to impair the secretion of Wnt7a on EVs. Indeed, mutation of K256 that disrupts both positively charged motifs, KIK and KK, did not affect secretion of Wnt7a-EVs (
The ESP region corresponds with a linking peptide that connects the N- and C-terminal domains with a high variable length and sequence among the 19 human Wnt proteins (
To test the ability of candidate ESPs from different Wnts to mediate secretion on EVs, the ESP of Wnt7a was replaced with either the ESP from Wnt10a, containing only the RR motif, or the ESP from Wnt16, that contains both motifs RR and KR. Both the Wnt10a and the Wnt16 ESP were compatible for efficient secretion on EVs (
Here, the structural mechanism that targets Wnt proteins to the surface of EVs has been elucidated. A new role for COPI vesicles as mediators of Wnt secretion on EVs has been identified. It has been discovered that COPα and COPβ2 interact through their N-terminal β-propeller domains with a positively charged KR motif found in a loop within Wnt7a that has been termed ESP. This interaction mediates the targeting of Wnt7a to EVs, thus facilitating long-range signaling by Wnt7a. This dual requirement is interpreted as reflecting the changing interactions along the secretion pathway: first, the interaction with COPα in the cytosolic membrane of the Golgi Apparatus; and second, the interaction with COPβ2 in the cellular membrane. Interestingly, it was found that when COPβ2 is knocked down, COPα is also down regulated in EVs. This suggests that both proteins are required for the proper formation of the COPI vesicle and secretion of Wnt7a-EVs is abolished in the absence of either component.
Several groups have shown that Wnt secretion requires an interaction with Evi, a chaperone transmembrane protein that facilitates the secretion to the membrane. Moreover, it has been shown that Evi interacts with COPI vesicles to mediate the recycling of Evi and thus promote Wnt secretion. This data would suggest that Evi could also act as the linker be-tween Coatomer and Wnt facilitating the transfer to the membrane. However, recently it was confirmed that the interaction of Wnt to Evi it is through palmitoylation of Wnt. However, it was observed that palmitoylation was dispensable for Wnt7a secretion on EVs, ruling out the possibility of Evi mediating the Coatomer-dependent Wnt-EV secretion mechanism. Accordingly, no interaction of Wnt7a with Evi was detected by Bio-ID. The experiments suggest an alternative Coatomer-dependent mechanism for Wnt secretion on EVs, where COPI vesicles mediate intracellular trafficking of Wnt7a from the Golgi apparatus surface to the cellular membrane. Indeed, the results indicate that upon mutation of the ESP, secretion as a free protein is enhanced to the detriment of Wnt-EV secretion, thus confirming that both modes of secretion function independently. Exosomal secretion mechanism would compensate for the inability of free Wnt to signal long-range and provide for fast distal-range diffusion after acute muscle injury. This data reinforces the concept of independent co-existing secretion pathways and the ability of the cell to switch from one to another based on cellular homeostasis.
The N-terminal SP has long been understood to be required for extracellular protein secretion. Also, it has been assumed that proteins targeted for exosomal secretion are endocytosed directly from the cell membrane before being subsequently transferred back to the Multivesicular Body (MVB). Importantly, it has been found that the SP is not required for secretion of Wnt7a on EVs. Therefore, this data suggests that Wnt7a trafficking onto EVs occurs inside of the cell and not after being secreted as a free protein, which is later endocytosed. Since Wnt proteins have been described to be secreted through the classical ER-Golgi pathway it is unlikely that Wnt located in the luminal side of the Golgi could interact with the cytosolic Coatomer proteins. Therefore, a novel mechanism is suggested, wherein proteins would bypass the classical pathway ER-Golgi pathway (
The BioID data has shown an enrichment on Sec63, a chaperone that facilitates targeting of proteins bearing a SP into the Sec61 channel at the ER. This finding is consistent with the notion that Wnt proteins are translated in the cytosol and translocated to the ER with the assistance of Sec63. The fraction of Wnt remaining in the cytosol, however, would be available for direct cytosolic interaction with COPα at the Golgi. This new role for COPI vesicles is reinforced by the lack of any retrograde signal within the EBP sequence, that could relate this interaction with a retrograde mechanism. Also, the results showed that neither palmitoylation nor the SP is required for Wnt-EV secretion. Indeed, other Wnt proteins, such as WntD, have been previously shown to be secreted without palmitoylation. Furthermore, it has been shown that Wnt7a is fully bioactive upon secretion on EVs, as several authors have previously shown for other Wnts. Therefore maturation through the ER-Golgi classical pathway seems to be dispensable for specific Wnt-EV secretion and bioactivity.
Studies have previously identified the involvement of COPI vesicles in endosome trafficking. In Drosophila, knockdown of COPα or COPβ2 results in adult flies that display notched wings, suggesting an essential role for COPI vesicles in Wg secretion. Protein secretion pathways have been described that function independently of the classical ER-GA pathway. Therefore, the data is consistent with the assertion that Wnt7a secretion on EVs is occurring via a Coatomer-dependent leaderless secretion pathway rather than the classical ER-GA pathway. However, the mechanism that specifically regulates COPI vesicles cargo into MVB for protein-EV secretion needs further investigation.
The same linker region that forms the ESP has been implicated in other Wnt7a functions. The Reck receptor binds Wnt7a through the same region that encodes the ESP to form a signalosome that induces canonical Wnt7a-Fzd signaling. Moreover, Wnt7a similarly binds the canonical Frizzled co-receptor LRP6 through the ESP sequence. Together these findings suggest that this unstructured loop acts as an intrinsically disordered protein, to coordinate different functions possibly regulated by combinatorial posttranslational modifications. Notably, Reck was not detected in the BioID assays, and Reck expression by immunoblot analysis were not detected. Together, these data reinforce the notion that multiple mechanisms act on Wnt-signaling in different cell types to enforce distinct signaling outcomes.
It has been found that equivalent ESP sequences are conserved in several Wnts to mediate secretion on EVs. Further, it has been found that the mechanism of action is conserved through interaction with non-canonical charged amino acid motifs such us RR. It has been shown that Wnt secretion on EVs can be abrogated by mutating a single amino acid within the ESP without disrupting other types of secretion.
A novel role for COPI vesicles has been defined, which involves Wnt-Coatomer protein binding to target Wnt proteins for EVs secretion. The sequence requirements of ESPs and their coatomer binding motifs (CBMs) have been defined and it has been shown that a similar mechanism is involved in EVs secretion of multiple Wnts. These experiments suggest that systemic delivery of Wnt7a loaded on exosomes represents a potential therapy for neuromuscular diseases such as DMD. Moreover, the use of ESPs and/or CMBs to direct the display of other cargo proteins on the surface of exosomes opens the door for multiple therapeutic applications involving targeting of recombinant cargo proteins to EVs. In particular the unexpected involvement of COPI in Wnt trafficking to EVs suggest that other known CBMs, such as KR, KK, KxK (which bind to α-COP and/or β′-COP) will also be useful in this regard, as well as the motif FFxxBB (which binds to γ-COP). The significance of RR as a CBM, as described herein, also appears to be new. It is expected that these discoveries will serve as a basis for recombinant delivery systems.
As illustrated in
Extracellular Vesicles Signal Peptide (ESP) Deletion (Replacement with Linker) Increases Extracellular Secretion of Free Wnt7a Protein
As illustrated in
The disruption in the above cases was deletion of the ESP and replacement with a GSGS linker. However, it is clear that there would be other ways to reduce or disrupt ESP activity to achieve a similar increase in free protein, such as by mutation of one or more key residues in the CBM(s) of a given Wnt, or by making other deletions in the ESP. These approaches should be useful to generate free Wnts, including for therapeutic applications.
All references referred to herein are incorporated by reference in their entireties.
In the preceding description, for purposes of explanation, numerous details are set forth in order to provide a thorough understanding of the embodiments. However, it will be apparent to one skilled in the art that these specific details are not required. In other instances, well-known electrical structures and circuits are shown in block diagram form in order not to obscure the understanding. For example, specific details are not provided as to whether the embodiments described herein are implemented as a software routine, hardware circuit, firmware, or a combination thereof.
The above-described embodiments are intended to be examples only. Alterations, modifications and variations can be effected to the particular embodiments by those of skill in the art. The scope of the claims should not be limited by the particular embodiments set forth herein, but should be construed in a manner consistent with the specification as a whole.
This application claims priority from U.S. Provisional Application No. 63/183,902 entitled “EXTRACELLULAR VESICLE-DIRECTED POLYPEPTIDE TAG”, which was filed May 4, 2021, which is hereby incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2022/050689 | 5/3/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63183902 | May 2021 | US |
