Patent Application
20200140493
The Sequence Listing submitted Jan. 22, 2020 as a text file named “21101_0379U2_Sequence_Listing.txt,” created on Jan. 10, 2020, and having a size of 138,564 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).
Viruses have traditionally been categorized as either “enveloped” or “non-enveloped”, but in recent years this distinction has become blurred with the identification of “quasi-enveloped” viruses. In the conventional view, enveloped viruses contain single capsids surrounded by a membrane that they acquire, together with transmembrane envelope glycoproteins, as they bud from producer cells (
Disclosed herein are modified capsid proteins comprising a capsid forming protein, a membrane binding element and an ESCRT-recruiting element, wherein at least one of the membrane binding element and the ESCRT-recruiting element is heterologous to the capsid forming protein.
Disclosed herein are capsids comprising a plurality of the disclosed modified capsid proteins.
Disclosed herein are multimeric assemblies comprising a plurality of any one of the disclosed capsids within a membrane.
Disclosed herein are modified non-enveloped viruses comprising a capsid wherein the capsid comprises a plurality of modified capsid proteins, wherein the plurality of modified capsid proteins comprise a capsid forming protein, a membrane binding element and an ESCRT-recruiting element, wherein at least one of the membrane binding element and the ESCRT-recruiting element is heterologous to the modified capsid protein, wherein the capsid forming protein is a capsid forming protein of a non-enveloped virus.
Additional advantages of the disclosed method and compositions will be set forth in part in the description which follows, and in part will be understood from the description, or may be learned by practice of the disclosed method and compositions. The advantages of the disclosed method and compositions will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the disclosed method and compositions and together with the description, serve to explain the principles of the disclosed method and compositions.
The disclosed method and compositions may be understood more readily by reference to the following detailed description of particular embodiments and the Example included therein and to the Figures and their previous and following description.
It is to be understood that the disclosed method and compositions are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed method and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, is this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.
It is understood that the disclosed method and compositions are not limited to the particular methodology, protocols, and reagents described as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “a modified capsid protein” includes a plurality of such proteins, reference to “the modified capsid protein” is a reference to one or more modified capsid proteins and equivalents thereof known to those skilled in the art, and so forth.
A “capsid forming protein” is a structural protein which is part of the complex forming a capsid. As used herein, a “capsid forming protein” refers to an amino acid sequence capable of forming a capsid. For example, a capsid forming protein can be an amino acid sequence responsible for the self-interaction between capsid proteins. In some aspects, a capsid forming protein can be an alpha protein or VP2. For example, a capsid forming protein can be an FHV Alpha protein or AAV VP2 protein.
As used herein, “a capsid” is the protein shell of a virus. The capsid comprises one or more proteins referred to as capsid proteins or protomers. The capsid can enclose the viral genome or simply enclose nucleic acids, proteins, or small molecules for delivery. In some aspects, the capsid can have an icosahedral structure. In some aspects the capsid can be helical or prolate.
As used herein, “a capsid protein” is a protein that when assembled with other capsid proteins forms a capsid. A capsid protein can also be known as a capsid subunit or substructure because it is a smaller element of the capsid. In some aspects, a capsid protein is referred to as a protomer.
As used herein, “a modified capsid protein” is a capsid protein that is not naturally occurring or native to that particular capsid protein. A modified capsid protein can be a capsid protein with an amino acid substitution or an amino acid that has been altered from the amino acid sequence found in nature. A modified capsid protein can comprise sequences from other non-capsid proteins or from other non-heterologous capsid proteins. For example, a modified capsid protein can comprise a capsid forming protein, a membrane binding element and an ESCRT-recruiting element, wherein at least one of the membrane binding element and the ESCRT-recruiting element is heterologous to the capsid forming protein.
“Optional” or “optionally” means that the subsequently described event, circumstance, or material may or may not occur or be present, and that the description includes instances where the event, circumstance, or material occurs or is present and instances where it does not occur or is not present.
Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, also specifically contemplated and considered disclosed is the range from the one particular value and/or to the other particular value unless the context specifically indicates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another, specifically contemplated embodiment that should be considered disclosed unless the context specifically indicates otherwise. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint unless the context specifically indicates otherwise. Finally, it should be understood that all of the individual values and sub-ranges of values contained within an explicitly disclosed range are also specifically contemplated and should be considered disclosed unless the context specifically indicates otherwise. The foregoing applies regardless of whether in particular cases some or all of these embodiments are explicitly disclosed.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed method and compositions belong. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present method and compositions, the particularly useful methods, devices, and materials are as described. Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. No admission is made that any reference constitutes prior art. The discussion of references states what their authors assert, and applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of publications are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.
Throughout the description and claims of this specification, the word “comprise” and variations of the word, such as “comprising” and “comprises,” means “including but not limited to,” and is not intended to exclude, for example, other additives, components, integers or steps. In particular, in methods stated as comprising one or more steps or operations it is specifically contemplated that each step comprises what is listed (unless that step includes a limiting term such as “consisting of”), meaning that each step is not intended to exclude, for example, other additives, components, integers or steps that are not listed in the step.
The term “sequence of interest” or “gene of interest” can mean a nucleic acid sequence (e.g., a therapeutic gene), that is partly or entirely heterologous, i.e., foreign, to a cell into which it is introduced.
The term “sequence of interest” or “gene of interest” can also mean a nucleic acid sequence, that is partly or entirely complementary to an endogenous gene of the cell into which it is introduced. For example, the sequence of interest can be micro RNA (miRNA), short hairpin RNA (shRNA), or short interfering RNA (siRNA).
A “sequence of interest” or “gene of interest” can also include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of a selected nucleic acid. A “protein of interest” means a peptide or polypeptide sequence (e.g., a therapeutic protein), that is expressed from a sequence of interest or gene of interest.
The term “operatively linked to” refers to the functional relationship of a nucleic acid with another nucleic acid sequence. Promoters, enhancers, transcriptional and translational stop sites, and other signal sequences are examples of nucleic acid sequences operatively linked to other sequences. For example, operative linkage of DNA to a transcriptional control element refers to the physical and functional relationship between the DNA and promoter such that the transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA.
Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed then “less than or equal to 10” as well as “greater than or equal to 10” is also disclosed. It is also understood that throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point “15” are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15.
Disclosed are modified capsid proteins comprising a capsid forming protein, a membrane binding element and an ESCRT-recruiting element, wherein at least one of the membrane binding element and the ESCRT-recruiting element is heterologous to the capsid forming protein.
In some aspects, the capsid forming protein can be a capsid forming protein of an enveloped virus. Examples of enveloped virus include but are not limited to Herpesviridae, Poxviridae, Coronaviridae, Retroviridae, Orthomyxoviridae and Hepadnaviridae. In some aspects, the capsid forming protein can be a capsid forming protein of a non-enveloped virus. Examples of non-enveloped virus include but are not limited to Adenoviridae, Polyomaviridae, Papillomaviridae, Rudiviridae, Clavaviridae, Parvoviridae, Bimaviridae, Reovirideae, Totiviridae, Picomavirideae, Comoviridae, Bromoviridae, Hepeviridae, and Nodaviridae.
In some aspects, the membrane binding element can be a membrane binding element of an enveloped virus. In some aspects, the ESCRT-recruiting element can be an ESCRT-recruiting element of an enveloped virus. In some aspects, the membrane binding element can be membrane binding element of a non-enveloped virus. In some aspects, the ESCRT-recruiting element can be an ESCRT-recruiting element of a non-enveloped virus.
In some aspects, the membrane binding element can be a non-viral membrane binding protein. For example, the membrane binding element can be a cellular membrane binding protein. In some aspects, the ESCRT-recruiting element can be a non-viral membrane binding protein. In some aspects, the membrane binding element can comprise an amino acid sequence derived from a viral or non-viral membrane binding protein sequence.
In some aspects, the membrane binding element can be the membrane binding PH element from phospholipase C. In some aspects, the ESCRT-recruiting element can be the p6Gag polypeptide from human immunodeficiency virus.
In some aspects, the membrane binding element and/or the ESCRT-recruiting element does not occur in any naturally occurring protein (i.e. is non-naturally occurring). In some aspects, the membrane binding element and/or the ESCRT-recruiting element are synthetic. For example, the membrane binding element and/or the ESCRT-recruiting element can comprise an amino acid sequence derived from a viral or non-viral membrane binding protein sequence.
In some aspects, the membrane binding element and the ESCRT-recruiting element can be a membrane binding element and an ESCRT-recruiting element of the same virus. In some aspects, the membrane binding element and the ESCRT-recruiting element can be a membrane binding element and an ESCRT-recruiting element of different viruses.
In some aspects, the membrane binding element and ESCRT-recruiting element can be located within any region of the modified capsid protein that allows the capsid protein to retain its function and do not inhibit assembly of a capsid. For example, in some aspects the membrane binding element and ESCRT-recruiting element can be located within at least one exposed surface loop of the modified capsid proteins. In some aspects, the membrane binding element and ESCRT-recruiting element can be located within the same exposed surface loop of the modified capsid protein. In some aspects, the membrane binding element and ESCRT-recruiting element can be located within different exposed surface loops of the modified capsid protein. In some aspects, the membrane-binding element and ESCRT-recruiting element can be located at either the N or C terminus of the modified capsid protein. In some aspects, the membrane-binding element can be located at either the N or C terminus of the modified capsid protein and the ESCRT-recruiting element can be located at the opposite terminus of the modified capsid protein from the membrane-binding element.
In some aspects, the disclosed modified capsid proteins can further comprise a desired cargo, sequence of interest, gene of interest, packaging moiety, or a targeting moiety.
1. Membrane Binding Element
The membrane binding element, also referred to as an “M domain,” can be any suitable polypeptide that is capable of binding to a lipid bilayer via any suitable mechanism, including but not limited to non-covalently interacting with the lipid bilayer membrane. In some aspects, such interactions can include but are not limited to interacting via specific binding pockets with the polar head groups of lipid molecules in the lipid bilayer, interacting electrostatically with charged polar head groups, interacting non-covalently with the hydrophobic interior of the lipid bilayer, or by harboring a chemical modification (non-limiting examples can be fatty acid or acylation modifications such as myristoylation) that interacts non-covalently with the lipid bilayer. A given membrane binding element can employ one or more mechanisms of interaction with a lipid bilayer. As described herein, the multimeric assembly described herein can comprise one or more membrane binding elements. In some aspects, each modified capsid protein in a multimeric assembly comprises one or more membrane binding elements. In other aspects, some fraction (30%, 40%, 50%, 60%, 70%, 80%, 90%, or more) of the plurality of modified capsid proteins comprise one or more membrane binding elements. In some aspects, each capsid protein in the plurality of modified capsid proteins comprises one or more membrane binding elements. In some aspects, one or more membrane binding elements is required per multimeric assembly as described herein in order to drive association of the multimeric assembly with the lipid bilayer via any suitable mechanism.
The membrane binding elements present in a resulting modified capsid protein, capsid, multimeric assembly, or modified non-enveloped virus can all be the same, all different, or some the same and some different.
In various embodiments, the one or more membrane binding elements can comprise or consist of a polypeptide having an acylation motif, including but not limited to N-terminal myristoylation motifs (including but not limited to MGXXXT/S (SEQ ID NO: 1) motif and non-limiting example sequences below), palmitoylation motifs (including but not limited to non-limiting example sequences below), famesylation motifs, and geranylgeranylation motifs (Resh M (1999) Fatty acylation of proteins: new insights into membrane targeting of myristoylated and palmitoylated proteins. Biochim. Biophys. Acta 1451: 1-16; Resh M (2013) Covalent lipid modifications of proteins. Curr. Biol. 23:R431-5); a polar headgroup-binding domain (including but not limited to non-limiting example sequences 100-106 in the attached appendices and the domains defined in: Stahelin R V (2009) Lipid binding domains: more than simple lipid effectors. J. Lipid Res. 50:S299-304); or transmembrane protein domains (the latter preferably when the multimeric assembly is enveloped by a lipid bilayer). In various further embodiments, the M domain may comprise envelope proteins of enveloped viruses, membrane protein transporters, membrane protein channels, B-cell receptors, T-cell receptors, transmembrane antigens of human pathogens, growth factors receptors, G-protein coupled receptors (GPCRs), or complement regulatory proteins including but not limited to CD55 and CD59.
In further embodiments, the membrane binding element may comprise or consist of one or more of the following peptides:
Further exemplary membrane binding elements can comprise or consist of one or more of the peptides that follow (Resh M (1999) Biochim. Biophys. Acta 1451: 1-16; Resh M (2013) Curr. Biol. 23:R431-5; Stahelin RV (2009) J. Lipid Res. 50:S299-304).
In some aspects, the membrane binding element can comprise the membrane binding myristoylated and palmitoylated element of Lyn kinase.
i. N-Terminal Membrane Binding Elements
The following peptides can be at the N terminus of the polypeptide in which they appear in order to function as a membrane binding element.
Any amino acid sequence conforming to the consensus motif (M)GXXX(S/T) (SEQ ID NO: 1), where the membrane binding element is in the initiator methionine at the N terminus of the polypeptide sequence.
ii. C-Terminal Membrane Binding Elements
The following peptides can be at the C terminus of the polypeptide which they appear in order to function as a membrane binding element.
iii. Polar Headgroup Binding Domains that Function as Membrane Binding Elements
The following peptides are non-limiting examples of polar headgroup-binding domains that can function as membrane binding elements. These domains can appear anywhere in the polypeptides of the invention consistent with proper folding and multimerization of the multimeric assembly.
Based on the disclosure herein, it is well within the level of those of skill in the art to identify membrane binding elements suitable for use in producing the modified capsid proteins, capsids, multimeric assemblies, and modified non-enveloped viruses disclosed herein. In one embodiment, a suitable membrane binding element can be identified as follows: As described throughout, a membrane binding element for use in the present disclosure can be any suitable polypeptide element that is capable of binding to a lipid bilayer via any suitable mechanism, including but not limited to non-covalently interacting with the lipid bilayer membrane. As will be known to those of skill in the art, a membrane binding element can be demonstrated to perform the function of membrane binding using a variety of standard assays. Many in vitro assays exist for assaying whether or not a polypeptide interacts with lipid membranes and for evaluating the characteristics of the interaction, such as the nature of the interaction (e.g., electrostatic or hydrophobic), the strength of the interaction, and whether the interaction deforms or remodels the membrane. Such assays include but are not limited to vesicle sedimentation assays, vesicle co-flotation assays, isothermal titration calorimetry, measuring changes in intrinsic or extrinsic protein or lipid fluorescence, fluorescence anisotropy, and membrane morphology analysis by electron microscopy or fluorescence microscopy (Zhao H, Lappalainen P (2012) A simple guide to biochemical approaches for analyzing lipid-protein interactions. Mol. Biol. Cell 23:2823-30). In addition, membrane binding element—dependent localization of proteins to membranes in cells can also be used as an assay for the interaction of a membrane binding element with membranes, and can yield information about the specificity of a given M domain for particular membranes, membrane subdomains, or lipids (Zacharias D A, Violin J D, Newton A C, Tsien R Y (2002) Partitioning of lipid-modified GFPs into membrane microdomains in live cells. Science 296:913-916; Lemmon M A (2008) Membrane recognition by phospholipid-binding domains. Nat. Rev. Mol. Cell. Biol. 9:99-111). Whether in vitro or in cells, either an isolated membrane binding element or a membrane binding element linked via genetic fusion or another method to a carrier protein that facilitates observation (for example, green fluorescent protein) can be used to evaluate the ability of the membrane binding element to interact with lipid membranes.
2. ESCRT-Recruiting Element
The ESCRT-recruiting element, also referred to as an “L domain,” can be used in any suitable polypeptide that is capable of effecting membrane scission by recruiting the ESCRT machinery to the site of budding by binding to one or more ESCRT or ESCRT-associated proteins directly or indirectly via any suitable mechanism, including but not limited to non-covalently or covalently. Preferably, the ESCRT-recruiting element interacts with proteins known to recruit the ESCRT machinery to sites of budding in vivo, such as Tsg101, ALIX, or the Nedd4 family of ubiquitin E3 ligases (McDonald B, Martin-Serrano J (2009) No strings attached: the ESCRT machinery in viral budding and cytokinesis. J. Cell Sci. 122:2167-77; Votteler J, Sundquist W I (2013) Virus budding and the ESCRT pathway. Cell Host & Microbe 14:232-41). Most preferably, the ESCRT-recruiting element interacts with the human, murine, or other mammalian forms of these proteins. Each protein subunit in a multimeric assembly contains one or more L domains. The ESCRT-recruiting elements present in a resulting multimeric assembly may all be the same, all different, or some the same and some different.
In various embodiments, the one or more ESCRT-recruiting element can comprise or consist of a linear amino acid sequence motif selected from the group consisting of P(T/S)AP (SEQ ID NO: 152), ΦYX0/2(P/Φ)X0/3íI/I), PPXY (SEQ ID NO: 153), and overlapping combinations thereof (Bieniasz P D (2006) Late budding domains and host proteins in enveloped virus release. Virology 344:55-63; Votteler J, Sundquist W I (2013) Virus budding and the ESCRT pathway. Cell Host & Microbe 14:232-41), where Φ denotes a hydrophobic residue, X can be any amino acid, and numbered subscripts indicate amino acid spacers of varying lengths. Such overlapping combinations include, but are not limited to P(T/S)APPXY (SEQ ID NO: 155), P(T/S)APYP(X)nL (SEQ ID NO: 156), PPXYP(T/S)AP (SEQ ID NO: 157), PPXYYP(X)nL (SEQ ID NO: 158), YP(X)nLPPXY (SEQ ID NO: 159), and YP(X)nLPPXY (SEQ ID NO: 160).
Further exemplary ESCRT-recruiting element can comprise or consist of one or more of the peptides that follow:
As will be understood by those of skill in the art, the ESCRT-recruiting element can include additional sequences, beyond those directly responsible for recruiting the ESCRT machinery, as appropriate for an intended use, so long as the ESCRT-recruitment motifs are not buried in the peptide core in such a way as to render them inaccessible for binding their interaction partners. In various further embodiments, the ESCRT-recruiting element can comprise or consist of the following peptides that include one or more ESCRT-recruitment motifs plus additional residues (ESCRT-recruitment motifs noted by underlined text):
PTAPPEYGGS;
PTAPGGS;
PPEYGGS;
YPLTSLGGS;
YPDLGGS;
FPIVGGS;
Further exemplary ESCRT-recruiting elements can comprise or consist of one or more of the following polypeptides:
Based on the disclosure herein, it is well within the level of those of skill in the art to identify ESCRT-recruiting element suitable for use in producing the modified capsid proteins, multimeric assemblies, and modified non-enveloped viruses disclosed herein. As described herein, aESCRT-recruiting element for use in the present invention can be any suitable polypeptide domain that is capable of effecting membrane scission and release of an enveloped multimeric assembly from a cell by recruiting the ESCRT machinery to the site of budding by binding to one or more ESCRT proteins directly or indirectly via any suitable mechanism, including but not limited to non-covalently or covalently. As will be known to those with skill in the art, the ability of an ESCRT-recruiting element to recruit the ESCRT machinery and effect membrane scission and release of an enveloped multimeric assembly can be assessed using budding assays. In the budding assay, a candidate ESCRT-recruiting element is genetically fused to a viral structural protein that has been rendered defective in budding by mutation or deletion of its late domain, and the ability of the candidate ESCRT-recruiting element to restore budding of virus-like particles is evaluated by analyzing the culture supernatant for the presence of the viral structural protein using standard techniques such as SDS-PAGE and Western blotting (Parent L J, Bennett R P, Craven R C, Nelle T D, Krishna N K, Bowzard J B, Wilson C B, Puffer B A, Montelaro R C, Wills J W (1995) Positionally independent and exchangeable late budding functions of the Rous Sarcoma Virus and Human Immunodeficiency Virus Gag proteins. J. Virol. 69:5455-5460). Any viral structural protein that is known to be defective in budding can be used in the budding assay, including but not limited to budding-defective versions of HIV-1 Gag, RSV Gag, MuMoLV Gag, SV5 M, Ebola VP40 and other structural proteins from different families of enveloped viruses including retroviruses, filoviruses, rhabdoviruses, arenaviruses, and paramyxoviruses. In addition, as described below, the multimeric assemblies of the invention can be used to test the ability of an L domain to effect membrane scission and release of an enveloped multimeric assembly in a similar manner. The ESCRT-recruiting element of a modified capsid protein can be replaced with a candidate ESCRT-recruiting element, and the ability of the resulting construct to be released from cells can be determined by analyzing the culture supernatant for the presence of the protein subunits of the multimeric assembly using standard techniques such as SDS-PAGE and Western blotting. Finally, as will be known to those with skill in the art, the ability of an ESCRT-recruiting element to bind to one or more ESCRT proteins directly or indirectly can be assessed using a variety of biochemical, biophysical, and cell biological techniques including but not limited to co-immunoprecipitation, pull-down assays, isothermal titration calorimetry, biosensor binding assays, NMR spectroscopy, and X-ray crystallography.
3. Cargo
In some aspects, the disclosed modified capsid proteins can further comprise a desired cargo. The desired cargo can be, but is not limited to, a nucleic acid (e.g. a sequence of interest or a gene of interest), protein (e.g. a peptide of interest or a protein of interest), a ribonucleoprotein complex, a small molecule, or any combination thereof.
In some aspects, a desired cargo can be anything of interest that can be enclosed within a capsid or found on the surface of a capsid. In some aspects, a desired cargo can be linked to or interact with a packaging moiety disclosed herein and thus recruited to the multimeric assembly, including but not limited to therapeutics, diagnostics, antigens, adjuvants, imaging agents, dyes, radioisotopes, etc. In some aspects, if the desired cargo is a protein or polypeptide, the cargo can be expressed as a genetic fusion with the membrane binding element, or the ESCRT-recruiting element in order to directly incorporate the desired cargo into a modified capsid protein or multimeric assembly without the use of a distinct packaging moiety. In various embodiments, the desired cargo can be selected from the group consisting of, but not limited to, proteins, nucleic acids, lipids, small organic compounds or combinations thereof.
In some aspects, the cargo can be a detectable label.
In some aspects, the desired cargo comprises a polynucleotide with a nucleic acid sequence which is a recognition sequence known to bind to the corresponding packaging moieties described herein. In some aspects, the polynucleotide with a nucleic acid sequence which is a recognition sequence known to bind to a packaging moiety can be
In some aspects, the capsid protein is translated from a nucleic acid sequence that itself is replicated by the viral enzymes together with the nucleic acid sequence encoding the cargo. In this case, both the polynucleotide sequences encoding the capsid as well as the cargo contain recognition sequences for the viral replication enzymes. In some aspects, the nucleic acid sequence to bind a viral replication enzyme can be:
In some aspects, the desired cargo comprises a nucleic acid comprising one or more of the disclosed recognition sequences. Examples can be found in International Application Publication No WO 2016/138525, herein incorporated by reference in its entirety.
4. Packaging Moiety
In a further embodiment, the capsids, multimeric assemblies, or modified non-enveloped viruses of any embodiment or combination of embodiments herein can further comprise a packaging moiety. As used herein, a “packaging moiety” can be any moiety capable of interacting with a desired “cargo”, with the effect of recruiting the cargo to the capsid, multimeric assembly, or modified non-enveloped virus. The interaction between the packaging moiety and the cargo can be any type of interaction, covalent or non-covalent, that results in effective interaction with and recruitment to the capsid, multimeric assembly, or modified non-enveloped virus. As will be apparent to those of skill in the art, the ability to widely modify surface amino acid residues without disruption of the protein structure permits many types of modifications to endow the resulting self-assembled multimers with a variety of functions. In one non-limiting example, at least one of the modified capsid proteins can be modified, such as by introduction of various cysteine residues or non-canonical amino acids at defined positions to facilitate linkage to one or more cargo of interest. In another non-limiting example, the modified capsid protein can be modified to comprise as a genetic fusion a polypeptide domain or sequence known to interact with a desired cargo covalently or non-covalently. In one embodiment, a non-canonical amino acid can be incorporated recombinantly using amber codon suppression (see L. Wang, A. Brock, B. Herberich, P. G. Schultz, Science 292, 498 (2001)). In another embodiment, the packaging moiety comprises the polypeptide sequence:
wherein the packaging moiety polypeptide is expressed as a genetic fusion with the M domain or the L domain. This sequence is the p6 domain of HIV Gag, which is known to interact with the HIV protein Vpr via a non-covalent protein-protein interaction (Cavrois M, et al. (2002) Nat. Biotech. 20: 1151-4). For example, by including SEQ ID NO: 186 in a multimeric assembly of the invention, any polypeptide sequence or other molecule that is fused, tethered, or otherwise connected to the Vpr sequence can be packaged into the multimeric assembly.
In some aspects, the packaging moiety can be bound or fused to the membrane binding element, the ESCRT-recruiting element, or the capsid-forming protein.
Additional packaging moieties can comprise or consist of one or more of the following peptides expressed as a genetic fusion with the membrane binding element, the ESCRT-recruiting element, or the capsid-forming protein, each of which binds to corresponding recognition sequences present in a nucleic acid cargo of interest, resulting in recruitment of the nucleic acid cargo of interest to the multimeric assembly.
5. Additional Moieties
In some aspects, the disclosed capsids, multimeric assemblies, and modified non-enveloped viruses further comprise one or more transmembrane protein or membrane-anchored protein embedded in the lipid bilayer. This embodiment can be used to add additional functionality of any desired type to the capsids, multimeric assemblies, and modified non-enveloped viruses. In this embodiment, the transmembrane protein or membrane-anchored protein can be one not present as part of the capsid or modified capsid proteins, in that they are added to the assembly or virus during or after envelopment of the multimeric assembly and modified non-enveloped virus by the lipid bilayer and do not necessarily interact with the protein subunits either covalently or non-covalently. Any suitable transmembrane protein or membrane-anchored protein can be added that provides any desired additional functionality to the assembly, in terms of cell targeting, the display of transmembrane or membrane-anchored antigen for vaccines, or other desired use. In one non-limiting example, the transmembrane protein or membrane-anchored protein embedded in the lipid bilayer comprises a viral envelope protein that enables the enveloped multimeric assembly, capsid or modified non-enveloped virus to enter cells via receptor-mediated endocytosis and/or mediates fusion of the lipid bilayer of the enveloped multimeric assembly, capsid or modified non-enveloped virus with cellular membranes. In the study of enveloped viruses, the practice of incorporating a foreign viral envelope protein in the membrane of an enveloped virus is referred to as “pseudotyping.” By co-expressing the foreign viral envelope protein with the viral or virus-like particle proteins, the foreign viral envelope protein becomes embedded in the membrane bilayer of the cells, and is therefore incorporated into the membrane envelope of the budding virions or virus-like particles. As the inventors have shown below, viral envelope proteins (in one embodiment, the G protein of Vesicular Stomatitis Virus) can be incorporated in the membrane envelopes of the enveloped multimeric assemblies, capsids or modified non-enveloped viruses of the disclosure in a similar manner. In various non-limiting embodiments, additional classes of membrane proteins can be incorporated into the membrane envelopes of the multimeric assemblies, capsid or modified non-enveloped viruses of the disclosure. In various non-limiting embodiments, the transmembrane or membrane-anchored protein is selected from the group consisting of the envelope proteins of enveloped viruses, membrane protein transporters, membrane protein channels, B cell receptors, T cell receptors, transmembrane antigens of human pathogens, growth factors receptors, G-protein coupled receptors (GPCRs), complement regulatory proteins including but not limited to CD55 and CD59, or processed versions thereof.
In specific embodiments, the one or more transmembrane protein or membrane-anchored protein embedded in the lipid bilayer comprise one or more of the following polypeptides, or a processed version thereof. As will be understood by those of skill in the art, the polypeptide sequences provided are full-length protein precursors, which are cleaved or otherwise processed (i.e., “processed”) to generate the final envelope protein embedded in the lipid bilayer.
In some aspects, any known targeting moiety can be used. A targeting moiety can be any nucleic acid or amino acid sequence that targets a particular cell surface protein or intracellular target.
As used herein, the term “targeting moiety” refers to any moiety that specifically binds a selected target. The targeting moiety can be, for example, a polysaccharide, a peptide, peptide ligand, an aptamer, an antibody or fragment thereof, a single chain variable fragment (scFv) of an antibody, or a Fab′ fragment, or a nanobody. Targeting moieties can also include other forms of an antibody as disclosed in Rissiek et al.; “Nanobodies as modulators of inflammation: potential applications for acute brain injury,” Front. Cell. Neurosci., 21 Oct. 2014; and Cuesta et al.; “Multivalent antibodies: when design surpasses evolution;” Trends in Biotechnology; Vol. 28, Issue 7, pp. 355-362, July 2010. The cited references are incorporated herein by reference in their entirety.
As used herein, a “targeting moiety” can be specific to a recognition molecule on the surface of a cell or a population of cells, such as, for example B cells or T cells. In an aspect of the disclosed compositions and methods, a targeting moiety can include, but is not limited to: a monoclonal antibody, a polyclonal antibody, full-length antibody, a chimeric antibody, Fab′, Fab, F(ab)2, F(ab′)2, a single domain antibody, Fv, a single chain Fv (scFv), a minibody, a diabody, a triabody, hybrid fragments, a phage display antibody, a ribosome display antibody, an oligonucleotide, a modified oligonucleotide, a peptide, a peptide ligand, a hormone, a growth factor, a cytokine, a saccharide or polysaccharide, and an aptamer.
As used herein, “aptamers” refer to molecules that interact with a target molecule, preferably in a specific way. Typically, aptamers are small nucleic acids ranging from 15-50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G-quartets. Aptamers can bind small molecules and large molecules. Aptamers can bind very tightly with Kd's from the target molecule of less than 10−12 M. Aptamers can bind the target molecule with a very high degree of specificity. Aptamers are known to the art and representative examples of how to make and use aptamers to bind a variety of different target molecules can be found in the following non-limiting list of U.S. Pat. Nos. 5,476,766, 5,503,978, 5,631,146, 5,731,424, 5,780,228, 5,792,613, 5,795,721, 5,846,713, 5,858,660, 5,861,254, 5,864,026, 5,869,641, 5,958,691, 6,001,988, 6,011,020, 6,013,443, 6,020,130, 6,028,186, 6,030,776, and 6,051,698. In an aspect, the aptamer can be synthetic, nonimmunogenic antibody mimics. In an aspect, the aptamer can be a DNA aptamer. In an aspect, the DNA aptamer can be anti-PD-1 aptamer.
As used herein, the term “contacting” refers to bringing a disclosed composition, compound, conjugate or protein together with an intended target (such as, e.g., a cell or population of cells, a receptor, an antigen, or other biological entity) in such a manner that the disclosed composition, compound, conjugate or fusion protein can affect the activity of the intended target (e.g., receptor, transcription factor, cell, population of cells, etc.), either directly (i.e., by interacting with the target itself), or indirectly (i.e., by interacting with another molecule, co-factor, factor, or protein on which the activity of the target is dependent). In an aspect, a disclosed composition or fusion protein can be contacted with a cell or population of cells, such as, for example, one or more lymphocytes (e.g., T cells and/or B cells).
Disclosed are capsids comprising a plurality of the modified capsid proteins disclosed herein. Thus, for example, disclosed are capsids comprising a plurality of modified capsid proteins, wherein the modified capsid proteins comprise a capsid forming protein, a membrane binding element and an ESCRT-recruiting element, wherein at least one of the membrane binding element and the ESCRT-recruiting element is heterologous to the capsid forming protein.
In some aspects, the modified capsid proteins are derived from a non-envelope virus. In other words, the modified capsid proteins can comprise a capsid forming protein, a membrane binding element and an ESCRT-recruiting element, wherein at least one of the membrane binding element and the ESCRT-recruiting element is heterologous to the capsid forming protein and wherein the capsid forming protein can be a capsid forming protein of a non-enveloped virus. If the capsid forming protein is from a non-enveloped virus then the capsid can be referred to as a non-enveloped capsid.
In some aspects, the plurality of modified capsid proteins interact with each other forming the capsid.
In some aspects, the membrane binding element and ESCRT-recruiting element can be located within a region of the plurality of modified capsid proteins that does not disrupt the ability of the capsid to form.
In some aspects, the disclosed capsids can further comprise a packaging moiety as described herein. The packaging moiety can be present on one or more of the modified capsid proteins. In some aspects, the packaging moiety is bound to a desired cargo. For example, the packaging moiety can be bound to a desired cargo with a covalent or non-covalent bond. In some aspects, the packaging moiety can be a modified amino acid within one or more of the modified capsid proteins. In some aspects, the packaging moiety can be a polypeptide.
In some aspects, the disclosed capsids further comprise a desired cargo as described herein.
In some aspects, the disclosed capsids can further comprise a membrane surrounding the capsid. The membrane can be a lipid bilayer. Thus, the presence of a membrane surrounding a capsid comprised of modified capsid proteins, wherein the modified capsid proteins comprise capsid forming proteins of a non-enveloped virus can convert a non-enveloped capsid to an enveloped capsid.
Disclosed are multimeric assemblies comprising a one or more of the capsids disclosed herein within a membrane. Disclosed are multimeric assemblies comprising a two or more of the capsids disclosed herein within a membrane. Thus, for example, disclosed are multimeric assemblies comprising a plurality of capsids, wherein the capsids comprise a plurality of modified capsid proteins, wherein the modified capsid proteins comprise a capsid forming protein, a membrane binding element and an ESCRT-recruiting element, wherein at least one of the membrane binding element and the ESCRT-recruiting element is heterologous to the capsid forming protein.
In some aspects, the membrane binding element and ESCRT-recruiting element can be located within a region of the plurality of modified capsid proteins that does not disrupt the ability of the multimeric assembly to form.
As disclosed herein, the membrane can be a lipid bilayer. As used herein, the membrane can also be referred to as an envelope.
In some aspects, the multimeric assembly further comprises a packaging moiety. In some aspects, the packaging moiety can be bound to a desired cargo. In some aspects, the packaging moiety is bound to a desired cargo with a covalent or non-covalent bond. In some aspects, the packaging moiety can be a modified amino acid within one or more of the modified capsid proteins. In some aspects, the packaging moiety can be a polypeptide. In some aspects, the desired cargo can be a nucleic acid, protein, or small molecule. The packaging moiety and desired cargo can be any of those disclosed herein.
Disclosed herein are modified non-enveloped viruses comprising one or more of the capsids disclosed herein. Thus, for example, disclosed are modified non-enveloped viruses comprising a capsid wherein the capsid comprises a plurality of modified capsid proteins, wherein the plurality of modified capsid proteins comprise a membrane binding element and an ESCRT-recruiting element, wherein at least one of the membrane binding element and the ESCRT-recruiting element is heterologous to the modified capsid protein, wherein the capsid forming protein is a capsid forming protein of a non-enveloped virus.
In some aspects, the membrane binding element and ESCRT-recruiting element can be located within any region of the modified capsid protein that they retain their function and do not inhibit assembly of a capsid. For example, in some aspects the membrane binding element and ESCRT-recruiting element can be located within at least one exposed surface loop of the modified capsid proteins. In some aspects, the membrane binding element and ESCRT-recruiting element can be located within the same exposed surface loop of the modified capsid protein. In some aspects, the membrane binding element and ESCRT-recruiting element can be located within different exposed surface loops of the modified capsid protein. In some aspects, the membrane-binding element and ESCRT-recruiting element can be located at either the N or C terminus of the modified capsid protein. In some aspects, the membrane-binding element can be located at either the N or C terminus of the modified capsid protein and the ESCRT-recruiting element can be located at the opposite terminus of the modified capsid protein from the membrane-binding element.
In some aspects, the modified non-enveloped virus can be, but is not limited to, Adenoviridae, Polyomaviridae, Papillomaviridae, Rudiviridae, Clavaviridae, Parvoviridae, Bimaviridae, Reovirideae, Totiviridae, Picomavirideae, Comovirinae, Bromoviridae, Hepeviridae, and Nodaviridae. Thus, the capsid forming protein can be a capsid forming protein of, but is not limited to, Adenoviridae, Polyomaviridae, Papillomaviridae, Rudiviridae, Clavaviridae, Parvoviridae, Bimaviridae, Reovirideae, Totiviridae, Picomavirideae, Comovirinae, Bromoviridae, Hepeviridae, or Nodaviridae.
In some aspects, the modified non-enveloped virus can be a Flock House Virus or Nodamura Virus. In some aspects, one or both of the membrane binding element and ESCRT-recruiting element are located at position 206 of the alpha protein of Flock House Virus. In some aspects, one or both of the membrane binding element and ESCRT-recruiting element are located at position 264 of the alpha protein of Flock House Virus. In some aspects, one or both of the membrane binding element and ESCRT-recruiting element can be located at position 199 of the alpha protein of Nodamura Virus. In some aspects, one or both of the membrane binding element and ESCRT-recruiting element can be located at position 259 of the alpha protein of NoV. In some aspects, membrane binding and ESCRT recruiting elements can be located at the N terminus of the VP2 protein of AAV. In some aspects, membrane binding and ESCRT recruiting elements can be located at the truncated N terminus of the VP2 protein of AAV.
In some aspects, the membrane binding element can be any of the membrane binding elements described herein. For example, the membrane binding element can be the membrane binding PH element from phospholipase C.
In some aspects, the ESCRT-recruiting element can be any of the ESCRT-recruiting elements described herein. For example, the ESCRT-recruiting element can be the p6Gag polypeptide from human immunodeficiency virus.
In some aspects, the disclosed modified non-enveloped viruses can further comprise a membrane surrounding the capsid. In some aspects, the membrane can be a lipid bilayer. In some aspects, the membrane can also be referred to as an envelope. Thus, the presence of the membrane surrounding the disclosed modified non-enveloped viruses can convert the modified non-enveloped viruses to enveloped viruses and can be referred to as quasi-enveloped viruses.
In some aspects, the membrane can comprise any of the targeting moieties described herein.
In some aspects, the disclosed modified non-enveloped viruses can be replication defective.
In some aspects, the disclosed modified non-enveloped viruses can comprise a desired cargo. The desired cargo can be any of those disclosed herein. In some aspects, the desired cargo can be the viral genome. In some aspects, the viral genome can be modified.
Disclosed are nucleic acid sequences that encode any of the disclosed modified capsid proteins.
Disclosed are constructs comprising the nucleic acid sequence that encodes any of the modified capsid proteins disclosed herein.
Disclosed are constructs comprising a modified viral genome wherein the modified viral genome comprises a nucleic acid sequence capable of encoding any of the disclosed modified capsid proteins. In some aspects, the modified viral genome can be a modified Flock House Virus or Nodamuravirus genome. In some aspects, the modified viral genome can be a modified AAV genome.
In some aspects, a modified viral genome is a viral genome that renders the resulting virus replication defective.
Disclosed are methods of producing a vector comprising transfecting a cell with a packaging plasmid comprising a sequence of interest; and any of the constructs disclosed herein. In some aspects, the sequence of interest can be a therapeutic agent or a detectable agent.
Disclosed are methods of treating a subject with any of the disclosed capsids, any of the disclosed multimeric assemblies, any of the disclosed modified non-enveloped virus, or any of the disclosed vectors produced by the methods disclosed herein.
In some aspects, the sequence of interest encodes a protein that is non-functional or absent in the subject. In some aspects, the sequence of interest can be a therapeutic agent.
The materials described above as well as other materials can be packaged together in any suitable combination as a kit useful for performing, or aiding in the performance of, the disclosed method. It is useful if the kit components in a given kit are designed and adapted for use together in the disclosed method. For example disclosed are kits for producing the disclosed modified capsid proteins, capsids, multimeric assemblies or modified non-enveloped viruses, the kit comprising a membrane binding element and an ESCRT-recruiting element. The kits also can contain lipids, desired cargo, or targeting moieties.
One goal of the current disclosure is to generate a virus-based vector system in which capsids from non-enveloped viruses are modified so that they efficiently bud from cells inside membrane enclosed vesicles, thereby converting non-enveloped viruses into quasi-enveloped viruses. Three modular activities are necessary: membrane binding, self-assembly, and the ability to recruit ESCRT machinery to catalyze the membrane fission reactions required for vesicle release.
Quasi-enveloped viral capsids were generated by modifying the capsid-forming Alpha proteins from two non-enveloped nodaviruses: Flock House (FHV) and Nodamuraviruses (NOV). The membrane-binding PH domain from phospholipase C (PH-PLC) and the ESCRT-recruiting p6 sequence from HIV-1 Gag were inserted within exposed surface loops of the capsid-forming Alpha protein (
Quasi-enveloped viral capsids were generated by modifying the VP2 protein in the capsid of Adeno-associated virus. The membrane-binding 13 N-terminal amino acids of Lyn kinase, which include a myristoylation and a palmitoylation motif, and the ESCRT-recruiting p6 sequence from HIV-1 Gag, were inserted at the N terminus of the VP2 protein (
1. Plasmid Design and Construction
The polypeptide sequences of the ESCRT-recruiting HIV-1 p6Gag domain and the membrane-binding PH domain were inserted into exposed surface loops of the Alpha proteins (at amino acid positions 264 and 206 of FHV Alpha, and at equivalent sites of NoV Alpha, see
The polypeptide sequences of the ESCRT-recruiting HIV-1 p6Gag domain and the membrane-binding 13 N-terminal residues from Lyn Kinase that induce myristoyation and palmitoylation of the protein were inserted into the N terminus of AAV VP2. To uncouple expression of VP2 from VP1 and VP3, start codons for VP1 and VP2 were modified in the plasmid pXR2 by site-directed mutagenesis (see
2. Mammalian Cell Culture
All experiments were carried out in human embryonic kidney (HEK) 293T cells obtained from American Type Culture Collection (ATCC) and cultured at 37° C. and 5% CO2 in D-MEM media (ThermoFisher) containing 10% FBS, penicillin (100 U/ml) and streptomycin (0.1 mg/ml). Cells were tested for mycoplasma contamination every 3 months using the MycoAlert™ Mycoplasma Detection Kit (Lonza).
3. Viron Release Assays
To assay release of the designed FHV and NoV constructs, 8×105 293T cells were seeded in 6-well-plates 24 h prior to transfection. Cells were transfected with 2 μg of plasmid DNA expressing the FHV and NoV constructs (
4. Vesicle Purification
Released vesicles used in biochemical and cryo-EM studies were purified from culture supernatants of 293T cells (T225 flask, 6×106 cells seeded per flask) following transient transfection with plasmids encoding eFHV and eNoV (30 rtg/flask) using the PEI method. Transfected cells were incubated overnight and the media was replaced after 5 h with exosome production media (D-MEM supplemented with 10% FBS, depleted of contaminating extracellular particles by centrifugation overnight at 100,000×g at 4° C., and subsequently filtered through a 0.22 μm filter). Cells were grown for 48 h and extracellular vesicles were purified by a series of filtration and centrifugation steps. Briefly, cell debris was removed by centrifugation of the supernatant at 1,000×g for 5 min followed by filtration through a 0.22 μm filter (EMD Millipore). Vesicles were collected by centrifugation at 100,000×g in an SW32Ti (BeckmanCoulter) at 4° C. for 1 h. Pellets were resuspended in PBS and pooled in a single tube (SW41 rotor, BeckmanCoulter). PBS was added to fill the tube and vesicles were collected by centrifugation at 100,000×g at 4° C. for 1 h. Pellets were resuspended in 1 ml of PBS and concentrated by centrifugation at 100,000×g at 4° C. for 1 h in an OptimaMAX-E (BeckmanCoulter) benchtop ultracentrifuge using a TLS-55 rotor. FHV and NOV release levels were quantified by western blotting as described above.
5. Protease Protection Assays
To test for membrane envelopment, purified vesicles were resuspended and incubated under three different conditions, 10 l each: (i) untreated sample (
6. Cryo-EM Tomographic Imaging of Vesicles
To prepare samples for cryo-EM tomography, 3 μl of purified vesicles in PBS were mixed with 3 l of BSA-coated gold fiducials (10 nm size, Electron Microscopy Sciences). 3.5 l samples of the suspension were applied to glow-discharged R2/2 holey carbon coated EM grids (Quantifoil), within the environmental chamber of a Vitrobot Mark IV (FEI) maintained at 4° C., 80% relative humidity. Excess liquid was blotted from the grids for 1.5 s (blot force 20, 1 blot) with filter paper (Whatman), before plunge freezing in liquid ethane. Cryo-grids were placed in a Gatan 626 cryoholder (Gatan) and imaged in a 200 kV Tecnai TF20 microscope (FEI) equipped with a K2 summit direct electron detector (Gatan). Tilt series were recorded bidirectionally starting from 0° to ±˜600 with a 30 step size at a magnification of 22,500× and a defocus of ˜8 μm (total dose per specimen ˜300 e-/Å2), using the low-dose mode in SerialEM. Tomograms were generated using the IMOD software package. Image stacks were aligned and binned by 4 within IMOD. Aligned image stacks were Fourier filtered (cutoff 0.25, sigma 0.08) and tomographic reconstructions were performed using the simultaneous reconstruction technique (SIRT). Noise reduction was performed with the non-linear anisotropic diffusion (NAD) method in IMOD, using a K value of 0.04 with 12 iterations.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the method and compositions described herein. Such equivalents are intended to be encompassed by the following claims.
This application claims the benefit of U.S. Application No. 62/751,457, filed on Oct. 26, 2018. The content of this earlier filed application is hereby incorporated by reference herein in its entirety.
This invention was made with government support under grant no. GM082545 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62751457 | Oct 2018 | US |
