WO2025080793A1 - Multi-domain polypeptide and methods for targeted neuronal delivery of cargo - Google Patents

Abstract

A multi-domain polypeptide is provided herein that includes a synapse targeting domain, a cleavage domain, and an entry domain. The multi-domain polypeptide can be fused to cargo, such as a therapeutic agent or research tool, to form a complex to deliver the cargo to a target cell. Vectors, host cells, and pharmaceutical compositions comprising the multi-domain polypeptide and/or complex are also provided herein.

Description

MULTI-DOMAIN POLYPEPTIDE AND METHODS FOR TARGETED NEURONAL

DELIVERY OF CARGO

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 63/543,550 filed on 11 October 2023, the entire content of which is hereby incorporated by reference.

GOVERNMENT SUPPORT STATEMENT

[0002] This invention was made with government support under R00 MH110665 and RF1 MH126706 awarded by the National Institute of Mental Health. The government may have certain rights in the invention.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] This application contains references to nucleic acid sequences and/or amino acid sequences which have been submitted concurrently herewith as the sequence listing .xml file entitled “ST26_SL_8_October_2024.xml”, file size 119,519 Bytes (B), created on 8 October 2024. The aforementioned sequence listing is hereby incorporated by reference in its entirety.

FIELD

[0004] This disclosure generally relates to a multi-domain polypeptide including a synapse targeting domain, a cleavage domain, and an entry domain, and methods for using said multidomain polypeptide for targeted neuronal delivery of cargo.

BACKGROUND

[0005] This section provides background information related to the present disclosure which is not necessarily prior art.

[0006] Protein tags are peptide sequences that can be genetically fused to a recombinant protein. Protein tags may be used for a variety of functions, such as for purification, detection, solubilization, localization, and protease protection. For example, a green fluorescent protein (GFP) may be used for detection. Protein tags may be used to deliver cargo, such as a drug, into cells. One method for protein delivery into cells involves using a cell-penetrating peptide (CPP), which can deliver protein cargoes into cells through a non-invasive route.

[0007] Alternative to protein tags, pathogenic agents have been used to deliver cargo via neurons. However, such methods suffer from lack of specificity (e.g., wheat germ agglutinin (WGA)) and/or from toxicity (e.g., HSV-1 H129). There is a lack of safe and specific tools for delivering cargo via neurons for targeted therapies for neurological disorders. Thus, there is a need in the art for new and improved methods for delivering cargo via the neural circuit.

SUMMARY

[0008] This section provides a general summary of the disclosure and is not a comprehensive disclosure of its full scope or all of its features.

[0009] In certain aspects, the present disclosure provides a multi-domain polypeptide including one or more synapse targeting domain; one or more cleavage domain; and one or more entry domain, wherein each domain is covalently linked. In some embodiments, the multi-domain polypeptide may additionally include one or more localization domain.

[0010] Also provided is a multi-domain polypeptide including a means for targeting a neuron synapse, a means for cleavage of one or more protein domains, and a means for entering one or more target cell. In some embodiments, the multi-domain polypeptide may additionally include a means for localization to one or more target cell.

[0011] In some embodiments, the multi-domain polypeptide may further include one or more linker, linking at least two of the domains together.

[0012] In some embodiments, the multi-domain polypeptide may be used to deliver one or more cargo to one or more target cell. Thus, also provided herein is a complex including the multidomain polypeptide and one or more cargo, such as a therapeutic agent or research tool.

[0013] Additionally, a polynucleotide molecule encoding the multi-domain polypeptide, a vector including the multi-domain polypeptide, a host cell including the multi-domain polypeptide, and a pharmaceutical composition including the multi-domain polypeptide are provided herein.

[0014] In some aspects, the present disclosure provides a method for preparing the multidomain polypeptide that includes concatenating the synapse targeting, cleavage, and entry domains.

[0015] Additionally, the present disclosure provides a method of delivering cargo to one or more target cell in a subject, including administering the multi-domain polypeptide to a subject.

[0016] Further areas of applicability will become apparent from the description provided herein. The description and specific examples in this summary are intended for purposes of illustration only and are not intended to limit the scope of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0018] The drawings described herein are for illustrative purposes only of selected embodiments and not all possible implementations and are not intended to limit the scope of the present disclosure.

[0019] FIG. 1 includes laser scanning confocal microscopy images showing RFP+ (red) basal pontine neuron axons and GFP+ (green) cerebellar granule cells in adeno-associated virus (AAV) Control Cre, AAV Tagged Cre #1, and AAV Tagged Cre #2.

[0020] FIG. 2 includes laser scanning confocal microscopy images showing RFP+ (red) basal pontine neuron axons and GFP+ (green) cerebellar granule cells in AAV Control Flp, AAV Tagged Flp , and AAV Tagged Flp (Inset).

[0021] FIG. 3A-FIG. 3E includes laser scanning confocal microscopy images showing SST Cre mice have RFP+ labelling of starter SST interneurons in the hilar region of the dentate gyrus and GFP+ labeling in canonical anterograde targets including granule cells and hilar mossy cells (FIG. 3A), VIP Cre mice have labeling of RFP+ dentate granule neurons and anterograde GFP+ hilar mossy cells, absent canonically retrograde perforant path labelling (FIG. 3B), SST Cre mice have RFP+ cerebellar Golgi cell starter cell labeling and GFP+ canonically anterograde granule and Golgi cells, absent retrograde GFP + mossy fibers (FIG. 3C), Kit Cre mice have RFP+ cerebellar cortex Molecular Layer Interneurons and GFP+ canonically anterograde Purkinje cells, without GFP+ retrograde granule cells (FIG. 3D), and DocklO Cre mice show RFP+ labeling of starter dentate gyrus granule cells, and canonical anterograde GFP+ CA3 pyramidal neurons, absent retrograde GFP+ Entorhinal Cortex neurons (FIG. 3E). Cell nuclei per se are illustrated by DAPI labeling (shown in blue), to illustrate the region of tissue in which retrograde neurons would have been labeled GFP if they were present.

[0022] FIG. 4 includes laser scanning confocal microscopy images showing GFP+ and RFP+ labeling in Retrovirus Tagged Cre and Retrovirus Control Cre CA3 neurons and dentate gyrus (DG).

[0023] FIG. 5 is a schematic showing the concept of tags (e.g., a multi-domain polypeptide described herein) for retrograde and anterograde access to specific cell types. [0024] FIG. 6 is a schematic representing an example tag-Cre AAV (e.g., a multi-domain polypeptide described herein) with examples components and an example control-Cre AAV. WPRE stands for Woodchuck Post-transcriptional Regulatory Element and ITR stands for Inverted Terminal Repeat(s), both of which are AAV components.

[0025] FIG. 7 is a schematic indicating each component of AAV Tagged Cre #1, AAV Tagged Cre #2, and AAV Control Cre.

[0026] FIG. 8 is a schematic indicating each component of AAV Tagged Flp and AAV Control Flp.

[0027] FIG. 9 is a schematic indicating each component of Retrovirus Tagged Cre and Retrovirus Control Cre.

DETAILED DESCRIPTION

A. Introduction

[0028] Due to the lack of safe and specific tools to achieve cargo delivery via the neuronal circuit, the use of a multi-domain protein tag to achieve such function was investigated. Although preferential action in specific cell types is known, it was discovered that the claimed multi-domain polypeptide, having a synapse targeting domain, a cleavage domain, and an entry domain, can achieve neuronal delivery of cargo to specific target cell types and tissues.

[0029] Synapse targeting domains, cleavage domains, and entry domains are individually known in the art, however, there have been no previous attempts to combine them. There especially have been no previous attempts to combine them to release cargo in a desired way via neurons.

[0030] Example embodiments are provided so that this disclosure will be thorough and will fully convey the scope to those who are skilled in the art. Numerous specific details are set forth such as examples of specific compositions, components, and methods, to provide a thorough understanding of embodiments of the present disclosure. It will be apparent to those skilled in the art that specific details need not be employed, that example embodiments may be embodied in many different forms and that neither should be construed to limit the scope of the disclosure. In some example embodiments, well-known processes, well-known device structures, and well- known technologies are not described in detail.

B. Definitions [0031] The terminology used herein is for the purpose of describing particular example embodiments only and is not intended to be limiting. As used herein, the singular forms “a,” “an,” and “the” may be intended to include the plural forms as well, unless the context clearly indicates otherwise. The terms “comprises,” “comprising,” “including,” and “having,” are inclusive and therefore specify the presence of stated features, elements, compositions, steps, integers, operations, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. Although the open-ended term “comprising,” is to be understood as a non-restrictive term used to describe and claim various embodiments set forth herein, in certain aspects, the term may alternatively be understood to instead be a more limiting and restrictive term, such as “consisting of’ or “consisting essentially of.” Thus, for any given embodiment reciting compositions, materials, components, elements, features, integers, operations, and/or process steps, the present disclosure also specifically includes embodiments consisting of, or consisting essentially of, such recited compositions, materials, components, elements, features, integers, operations, and/or process steps. In the case of “consisting of,” the alternative embodiment excludes any additional compositions, materials, components, elements, features, integers, operations, and/or process steps, while in the case of “consisting essentially of,” any additional compositions, materials, components, elements, features, integers, operations, and/or process steps that materially affect the basic and novel characteristics are excluded from such an embodiment, but any compositions, materials, components, elements, features, integers, operations, and/or process steps that do not materially affect the basic and novel characteristics can be included in the embodiment.

[0032] Any method steps, processes, and operations described herein are not to be construed as necessarily requiring their performance in the particular order discussed or illustrated, unless specifically identified as an order of performance. It is also to be understood that additional or alternative steps may be employed, unless otherwise indicated.

[0033] The use of the term "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." As such, the terms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a compound" may refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or greater numbers of compounds.

[0034] The use of the term "at least one" will be understood to include one as well as any quantity more than one, including but not limited to, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" may extend up to 100 or 1000 or more, depending on the term to which it is attached; in addition, the quantities of 100/1000 are not to be considered limiting, as higher limits may also produce satisfactory results. In addition, the use of the term "at least one of X, Y, and Z" will be understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z. The use of ordinal number terminology (i.e., "first," "second," "third," "fourth," etc.) is solely for the purpose of differentiating between two or more items and is not meant to imply any sequence or order or importance to one item over another or any order of addition, for example.

[0035] The use of the term "or" in the claims is used to mean an inclusive "and/or" unless explicitly indicated to refer to alternatives only or unless the alternatives are mutually exclusive. For example, a condition "A or B" is satisfied by any of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0036] As used herein, any reference to "one embodiment," "an embodiment," "some embodiments," "one example," "for example," or "an example" means that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. The appearance of the phrase "in some embodiments" or "one example" in various places in the specification is not necessarily all referring to the same embodiment, for example. Further, all references to one or more embodiments or examples are to be construed as non-limiting to the claims.

[0037] Throughout this disclosure, the term "about" is used to indicate that a value includes the inherent variation of error for a composition/apparatus/device, the method being employed to determine the value, or the variation that exists among the study subjects. For example, but not by way of limitation, when the term "about" is utilized, the designated value may vary by plus or minus twenty percent, or fifteen percent, or twelve percent, or eleven percent, or ten percent, or nine percent, or eight percent, or seven percent, or six percent, or five percent, or four percent, or three percent, or two percent, or one percent from the specified value, as such variations are appropriate to perform the disclosed methods and as understood by persons having ordinary skill in the art. Particularly in reference to a given quantity, number or percentage, “about” is meant to encompass deviations of plus or minus ten percent (± 10). For example, about 5% encompasses any value between 4.5% to 5.5%, such as 4.5, 4.6, 4.7, 4.8, 4.9, 5, 4.1, 5.2, 5.3, 5.4, or 5.5. Accordingly, unless otherwise indicated, the numerical parameters set forth in this specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter. [0038] The term "or combinations thereof" as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0039] As will be understood by one skilled in the art, for any and all purpose, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Furthermore, as will be understood by one skilled in the art, a range includes each individual member.

[0040] The terms “peptide,” “polypeptide,” or “protein” refer to peptide, oligopeptide, oligomer, or protein including at least two amino acids joined to each other, for example, by a normal peptide bond. The term “recombinant polypeptide” refers to a polypeptide that is produced by recombinant DNA techniques, wherein generally DNA encoding the expressed protein or RNA is inserted into a suitable expression vector that is in turn used to transform a host cell to produce the polypeptide or RNA.

[0041] The term “domain” as used herein refers to an element of a polypeptide and/or protein including a sequence of nucleotides and/or amino acids of any length. Domains may have certain functions and/or structures in a protein.

[0042] The term “concatenate” or “concatenating” as used herein is defined as linking together in a chain or series. For example, the domains of the multi-domain polypeptide may be concatenated.

[0043] The term "covalent linkage" (also referred to as a covalent bond), as used herein refers to a chemical bond that involves the sharing of electron pairs between atoms. A covalent linkage involves a stable balance of attractive and repulsive forces between atoms when they share electrons. For many molecules, the sharing of electrons allows each atom to attain the equivalent of a full outer shell, corresponding to a stable electronic configuration. Covalent bonding includes many kinds of interactions, including for example a-bonding, 7t-bonding, metal-to-metal bonding, agostic interactions, and three-center two-electron bonds. Accordingly, the complex according to the present invention may also be referred to as "compound", in particular it may be referred to as "molecule". [0044] The term “axonal transport” as used herein refers to the bidirectional ATP-dependent process neurons use to shuttle diverse substances along axon microtubules. Axonal transport can be in the anterograde (i.e., orthograde) direction or the retrograde direction. Anterograde transport moves materials and signals from the cell body (i.e., soma) towards the synapses at the ends of axons (or cell membrane) and is kinesin-driven. Retrograde transport carries used materials and signals from the synapse back to the cell body for recycling or further signaling and is dynein- driven.

[0045] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. In particular, this disclosure utilizes routine techniques in the field of targeted delivery of biologically active molecules.

C. Multi-Domain Polypeptide

[0046] Provided herein is a multi-domain polypeptide that includes a synapse targeting domain, a cleavage domain, and an entry domain. The multi-domain polypeptide may optionally include a localization domain. Such domains may each be covalently linked.

[0047] Also provided herein is a multi-domain polypeptide including a means for targeting a neuron synapse, a means for cleavage of one or more protein domains, and a means for entering one or more target cell. The multi-domain polypeptide may optionally include a means for localization to one or more target cell. In some embodiments, the means for targeting a neuron synapse may be via the synapse targeting domain described herein. In some embodiments, the means for cleavage of one or more protein domains may be via the cleavage domain described herein. In some embodiments, the means for entering one or more target cell may be via the entry domain described herein. In some embodiments, the means for localizing to one or more target cell may be via the localization domain described herein.

[0048] A domain may refer to a nucleotide sequence or an amino acid sequence that provides a structure or function. Disclosed herein is a polypeptide including multiple domains, each of which may provide one or more function or structure. In some embodiments, each of the domains may work together to achieve a desired function and/or structure. In certain embodiments, the multi-domain polypeptide may function as a targeting protein (e.g., protein “tag”) that delivers one or more cargo to a target cell type or tissue. In some embodiments, one or more domains may fold independently of the rest of the multi-domain polypeptide. Domains with specific structures and/or functions may be known in the art and may be present in one or more type of protein. In some embodiments, domains with similar functions may have similar sequences and/or structures. Alternatively, in some embodiments, domains with similar functions may not have similar sequences and/or structures.

[0049] In some embodiments, the multi-domain polypeptide may produce a protein or a system of proteins. In some embodiments, such proteins or system of proteins may confer, to cargo that it is fused to, the delivery of said cargo from one population of cells to another. In some embodiments, such action is conferred by a combination of domains that accomplish targeting a neuron synapse, a cleaving of one or more protein domains, entering one or more target cell, and, optionally, localizing to one or more target cell.

Synapse targeting domain

[0050] The synapse targeting domain may include an amino acid sequence, or be encoded by a nucleotide sequence, that achieves localization to the subcellular domain of neurons where chemical neurotransmission takes place. The synapse may be pre- or post-synaptic. Specifically, in some embodiments, the sequence may achieve localization to the extracellular aspect of the synapse, including the extracellular domain of transmembrane proteins at the synapse or the lumen of neuronal secretory vesicles. In some embodiments, the multi-domain polypeptide may include more than one synapse targeting domain. For example, the multi-domain polypeptide may include 1, 2, 3, 4, or 5, synapse targeting domains. In some embodiments, when there is more than one synapse targeting domain, the multiple domains may be referred to as a first synapse targeting domain, second synapse targeting domain, third synapse targeting domain, etc. In some embodiments, the synapse targeting domain may be referred to as a synapse localization domain.

[0051] In some embodiments, the synapse targeting domain may include one or more ion channel sequence, neurotransmitter sequence, neuromodulator receptor sequence, neurotransmission-involved protein sequence, neuronal cell adhesion molecule sequence, interacting protein sequence, synthetic binding protein sequence, or a combination thereof.

[0052] As referred to herein, a neurotransmission-involved protein sequence may be any sequence that is involved in neurotransmission. In some embodiments, the neurotransmission- involved protein may be a synaptic vesicle protein or an axon terminal or active zone resident protein. In some embodiments, the synaptic vesicle protein may be a VAMP2/synaptobrevin, synapsin, synaptophysin, syntaxin, synaptotagmin, synaptogyrin, SCAMP, SV2, RAB, or vesicular neurotransmitter transporter or synthase. In some embodiments, the vesicular neurotransmitter transporter or synthase may be VGLUT1-3, VGAT, VEAT, VNUT, VMAT, or VAChT. In some embodiments, the axon terminal or active zone resident protein may be a neurexin/neuroligin family protein or a Shisa family protein. In certain embodiments, the synapse targeting domain may be a Presynaptic mGrasp Component or VAMP2. In some embodiments, one synapse targeting domain may be made up of two or more parts. In other words, there may be another domain and/or sequence between two or more parts of the same synapse targeting domain. In some embodiments, the two or more parts of the one synapse targeting domain may work together to achieve a function. For example, one part may be the signal peptide for the second part. Thus, in some embodiments, multiple parts of the same synapse targeting domain may be referred to as synapse targeting domain (part 1), synapse targeting domain (part 2), synapse targeting domain (part 3), etc.

[0053] In some embodiments, an interacting protein sequence encodes a protein, such as an adaptor protein, that interacts with one or more of any of the cargos described herein.

[0054] In some embodiments, the synthetic binding protein sequence may encode a protein that binds one or more of any of the cargos described herein. For example, in some embodiments, the synthetic binding protein may be a monobody, single domain antibody (such as camelid), or fibronectin intrabody.

[0055] In some embodiments, the synapse targeting domain may be encoded by a nucleotide sequence at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1, 15, 25, or a combination thereof. In some embodiments, the synapse targeting domain may be present in the multi-domain polypeptide as two separate parts (i.e., the parts may have one or more nucleotide between them). In certain embodiments, the synapse targeting domain may be encoded by SEQ ID NO: 1 and SEQ ID NO: 15. In certain embodiments, the synapse targeting domain may be encoded by SEQ ID NO: 25.

[0056] In some embodiments, the synapse targeting domain may include an amino acid sequence at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2, 16, 26, or a combination thereof. In some embodiments, the synapse targeting domain may be present in the multi-domain polypeptide as two separate parts (i.e., the parts may have one or more amino acid between them). In certain embodiments, the synapse targeting domain may include SEQ ID NO: 2 and SEQ ID NO: 16. In certain embodiments, the synapse targeting domain may include SEQ ID NO: 26.

Cleavage domain

[0057] A cleavage domain may include an amino acid sequence, or be encoded by a nucleotide sequence, that achieves liberation and/or separation of at least two domains in the multi-domain polypeptide. In some embodiments, the cleavage domain achieves liberation and/or separation of other domain components from the synapse targeting domain. This can be achieved, for example, by recognition of an endogenous enzyme (e.g., a protease) such that the other concatenated domains (i.e., entry domain and, optionally, localization domain) and the cargo to which they are fused may become liberated from the synapse targeting domain. Thus, the cargo may be rendered soluble/diffusable and/or released. In some embodiments, the multi-domain polypeptide may include more than one cleavage domain. For example, the multi-domain polypeptide may include 1, 2, 3, 4, or 5, cleavage domains. In some embodiments, when there is more than one cleavage domain, the multiple domains may be referred to as a first cleavage domain, second cleavage domain, third cleavage domain, etc. In some embodiments, a cleavage domain may be referred to as a cleavage site.

[0058] In some embodiments, the cleavage domain may include one or more inherent cleavage sites, endogenous cleavage enzyme sequence, exogenous cleavage enzyme sequence, or a combination thereof.

[0059] As referred to herein, one or more inherent cleavage site may be included in any part of the multi-domain polypeptide sequence (e.g., in any of the domains and/or additional components). In some embodiments, the inherent cleavage site may function by spontaneous action of the sequence. In some embodiments, the inherent cleavage site may be a disulfide linker.

[0060] In some embodiments, one cleavage domain may be made up of two or more parts. In other words, there may be another domain and/or sequence between two or more parts of the same cleavage domain. In some embodiments, the two or more parts of the one cleavage domain may work together to achieve a function. Thus, in some embodiments, multiple parts of the same cleavage domain may be referred to as cleavage domain (part 1), cleavage domain (part 2), cleavage domain (part 3), etc.

[0061] In some embodiments, the endogenous cleavage enzyme may be a protease, such as a secretase, sheddase, or convertase. In some embodiments, a protease may be any enzyme which breaks down peptide and/or proteins. In some embodiments, a secretase may cleave an amyloid precursor protein. In some embodiments a secretase may be capable of “snipping” pieces off of a protein that is embedded in the cell membrane. In some embodiments, the secretase may function in the generation of beta-amyloid. In some embodiments, a sheddase may be a membrane-bound enzyme that cleaves extracellular portions of transmembrane proteins and may release soluble ectodomains from the cell surface. In some embodiments, a sheddase may be in the ADAM or aspartic acid (BACE) protein families, which includes enzymes that can activate a transmembrane protein if it is a receptor or cut off the part of the transmembrane protein which has already bound an agonist. In some embodiments, this may allow the agonist to go and stimulate a receptor on another cell. Thus, in some embodiments, sheddases may demultiply the yield of agonists. In some embodiments, a convertase may be in endocrine and/or neuroendocrine tissues. In some embodiments, convertases may process prohormones and proneuropeptides into bioactive peptides and may comprise a collection of enzymes from all classes of proteolytic enzymes.

[0062] In some embodiments, the endogenous cleavage enzyme may be a Bacel secretase, gamma secretase, ADAM metalloproteases, site 1 protease, matrix metalloproteases, urokinase, cathepsin, furin and other convertases, extracellular matrix proteases, neuropsin, or neurotrypsin.

[0063] In certain embodiments, the exogenous cleavage enzyme may be a Tobacco Etch Virus protease (TEVp) in conjunction with one or more of its protease recognition sites. Thus, in some embodiments, there may be two cleavage domains. For example, a first cleavage domain may include a TEVp and a second cleavage domain may include its protease recognition site (or vice versa). In some embodiments, the TEVp may be self-cleaving or non-self-cleaving (such as uTEV). In some embodiments, the protease recognition site may be a high affinity site (e.g., ENLYFQS (SEQ ID NO: 86) or a low affinity site (e.g., ENLYFQM (SEQ ID NO: 8)). In some embodiments, the exogenous cleavage enzyme may be a protein splicing system, such as those in the extein/intein family.

[0064] In certain embodiments, the cleavage domain may include a Tobacco Etch Virus (TEV) protease recognition/cut site and/or a TEV protease.

[0065] In some embodiments, the cleavage domain may be encoded by a nucleotide sequence at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7, 11, 45, 71, 85, or a combination thereof. In some embodiments, the multi-domain polypeptide may include two cleavage domains. Thus, in some embodiments, the cleavage domains may be encoded by SEQ ID NO: 7 and SEQ ID NO: 11. In certain embodiments, the cleavage domain may be encoded by SEQ ID NO: 45. In certain embodiments, the cleavage domain may be encoded by SEQ ID NO: 71. In certain embodiments, the cleavage domain may be encoded by SEQ ID NO: 85.

[0066] In some embodiments, the cleavage domain may include an amino acid sequence at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8, 12, 46, 72, 86, or a combination thereof. In some embodiments, the multi- domain polypeptide may include two cleavage domains. Thus, in some embodiments, the cleavage domains may include SEQ ID NO: 8 and SEQ ID NO: 12. In certain embodiments, the cleavage domain may include SEQ ID NO: 46. In certain embodiments, the cleavage domain may include SEQ ID NO: 72. In certain embodiments, the cleavage domain may include SEQ ID NO: 86.

Entry domain

[0067] An entry domain may include an amino acid sequence, or be encoded by a nucleotide sequence, that achieves entry into one or more cells or one or more specific classes of cells, for example, by endocytosis, membrane translocation, phagocytosis, pinocytosis, vesicle recycling, or other mechanisms of cell entry. In certain embodiments, the endocytosis or vesicle recycling may include neurotransmitter release. In some embodiments, the multi-domain polypeptide may include more than one entry domain. For example, the multi-domain polypeptide may include 1, 2, 3, 4, or 5, entry domains. In some embodiments, when there is more than one entry domain, the multiple domains may be referred to as a first entry domain, second entry domain, third entry domain, etc. In some embodiment, the entry domain may be referred to as a cell entry domain.

[0068] In some embodiments, the entry domain may be able to transport different types of cargo across a plasma membrane, and, thus, facilitate cellular uptake of various cargoes (from nanosized particles to small chemical molecules and large fragments of DNA). Thus, in some embodiments, the entry domain aids in cellular internalization. Cellular internalization of the cargo molecule linked to the entry domain generally means transport of the cargo molecule across the plasma membrane and thus entry of the cargo molecule into the cell. In some embodiments, the cargo molecule can then be released in the cytoplasm, directed to an intracellular organelle, or further presented at the cell surface.

[0069] In some embodiments, the entry domain may allow cargo to enter one or more cell types. In other words, the entry domain may include a cell-type specific affinity sequence. Thus, in some embodiments, the entry domain may be referred to as a cell-type specific affinity domain. In other embodiments, the multi-domain polypeptide may include an entry domain and a cell-type specific affinity domain. A cell-type specific affinity domain may include an amino acid sequence, or be encoded by a nucleotide sequence, that achieves binding to, and which may or may not also intrinsically perform as an entry domain for, a specific type of cell (e.g., target cell) based upon affinity to cell surface exposed molecules expressed by that cell type to which the cargo is targeted for delivery to.

[0070] In some embodiments, the entry domain may allow cargo to enter most or all cell types. In some embodiments, the entry domain may allow cargo to enter to enter at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 cell types. Examples of target cell types are described in Sec. F herein.

[0071] In some embodiments, the entry domain may include one or more receptor binding domain, secretion signal sequence, cell penetrating peptide (CPP) sequence, non-CPP entry sequence, viral protein sequence, glycan or proteoglycan binding sequence, extracellular matrix (ECM) component sequence, or a combination thereof. In some embodiments, the secretion signal may be ala ptenL or AAV NLS/escape. In some embodiments, the CPP may be TAT or R9.

[0072] In certain embodiments, the entry domain may include a CPP. CPPs may have an amino acid composition that either contains a high relative abundance of positively charged amino acids, such as lysine or arginine, or have a sequence that contains an alternating pattern of polar/charged amino acids and non-polar, hydrophobic amino acids. CPPs may be of different sizes, amino acid sequences, and charges, however, most CPPs have a common characteristic that is the ability to translocate the plasma membrane and facilitate the delivery of various molecular cargoes to the cytoplasm or to an organelle of a cell. Example CPP entry mechanisms include direct penetration in the membrane, endocytosis-mediated entry, and translocation through the formation of a transitory structure.

[0073] In some embodiments, the non-CPP may be a zinc finger nuclease (e.g., ZFN.3 or ZiF) or A22p3. In some embodiments, the viral protein sequence may be derived from the rabies virus G-protein or HSV1 (e.g., strain H129 glycoprotein D). In some embodiments, the rabies virus G- protein may have a predicted retrograde efficacy. In some embodiments, the glycan or proteoglycan binding sequence or the ECM component sequence may be a carbohydrate binding protein sequence, such as a lectin binding protein (e.g., Wheat Germ Agglutin). In some embodiments, the ECM component sequence may be a hyaluronan, tenascin, or integrin.

[0074] In certain embodiments, the entry domain may be an R9 cell penetrating peptide. In some embodiments, the entry domain may be a Poly-Histidine and TAT cell penetrating peptide. In some embodiments, one entry domain may be made up of two or more parts. In other words, there may be another domain and/or sequence between two or more parts of the same entry domain. In some embodiments, the two or more parts of the one entry domain may work together to achieve a function. Thus, in some embodiments, multiple parts of the same entry domain may be referred to as entry domain (part 1), entry domain (part 2), entry domain (part 3), etc.

[0075] In some embodiments, the entry domain may be encoded by a nucleotide sequence at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3, 47, 51, or a combination thereof. In some embodiments, the entry domain may be present in the multi-domain polypeptide as two separate parts (i.e., the parts may have one or more nucleotide between them). In certain embodiments, the entry domain may be encoded by SEQ ID NO: 47 and SEQ ID NO: 51. In some embodiments, the entry domain may be encoded by SEQ ID NO: 3.

[0076] In some embodiments, the entry domain may include an amino acid sequence at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4, 48, 52, or a combination thereof. In some embodiments, the entry domain may be present in the multi-domain polypeptide as two separate parts (i.e., the parts may have one or more amino acid between them). In certain embodiments, the entry domain may include SEQ ID NO: 48 and SEQ ID NO: 52. In some embodiments, the entry domain may include SEQ ID NO: 4.

Optional Localization domain

[0077] A localization domain may include an amino acid sequence, or be encoded by a nucleotide sequence, that achieves, for targeted cargo, localization to and/or desired action at a specific aspect of the target cell, such as to a specific subcellular domain, organelle, or to a specific protein. In some embodiments, the localization domain may be referred to as a target peptide. In some embodiments, the multi-domain polypeptide may include more than one localization domain. For example, the multi-domain polypeptide may include 1, 2, 3, 4, or 5, localization domains. In some embodiments, when there is more than one localization domain, the multiple domains may be referred to as a first localization domain, second localization domain, third localization domain, etc. In some embodiments, the localization domain may be referred to as a sub-cellular localization domain.

[0078] In some embodiments, the localization domain may include one or more nuclear localization signal (NLS) sequence, organelle trafficking sequence, specific protein affinity sequence, spatiotemporal control sequence, or a combination thereof.

[0079] In some embodiments, the NLS may include DNA or RNA binding sequences. In some embodiments, the NLS may be from endogenous proteins, such as Myc, heterogeneous nuclear ribonucleoproteins (hnRNPs), or MECP2. In some embodiments, the NLS may be from an exogenous protein, such as SV40 large T antigen, HIV TAT, Nipah Virus W protein, Zika Virus NS5, or adeno-associated virus (AAV) (inclusive or exclusive of its lysosomal escape motif).

[0080] In some embodiments, the organelle trafficking sequence may be an endoplasmic reticulum trafficking sequence (e.g. FFAT), a Golgi targeting sequence (e.g., CENP-R altORF), mitochondrial targeting sequence/signal, lysosome targeting sequence, or peroxisome targeting sequence. In some embodiments, the specific protein affinity sequence may be an interacting domain or nanobody.

[0081] In some embodiments, a spatiotemporal control sequence may permit spatiotemporal control and/or regulation of the subcellular localization. In some embodiments, the spatiotemporal control sequence may be used for stabilizing and/or activating a protein. In some embodiments, the spatiotemporal control sequence may be used for temporal and/or spatial control. In some embodiments, the spatiotemporal control sequence may be used for destabilizing a protein. In some embodiments, the spatiotemporal control sequence may be an optogenetic regulator, such as Cry2/CIBN). In some embodiments, Cry2/CIBN may be used for optical control of intracellular signaling, lipid metabolism, gene expression, and/or organelle transport, and may function robustly in a variety of organisms without requiring an exogenous cofactor. In some embodiments, the spatiotemporal control sequence may include Cre/Lox, FLP, or TET-On/Off. In some embodiments, site-specific recombinases may be used to regulate gene expression. Thus, in some embodiments, the Cre (cyclization recombination) and/or FLP (flipase) enzymes may be used to either activate or inactivate genes in a precise spatiotemporal manner. In some embodiments, Cre- Lox recombination may be used as a site-specific recombinase technology, used to carry out deletions, insertions, translocations and/or inversions at specific DNA sites. In some embodiments, Cre-Lox may allow the DNA modification to be targeted to a specific cell type or be triggered by a specific external stimulus. In some embodiments, Cre may be referred to as Cre recombinase. In some embodiments, the tetracycline-controlled Tet-Off and Tet-On gene expression systems may be used to silence gene expression by administration of tetracycline (Tc) or tetracycline-derivatives like doxycycline (dox), whereas the Tet-On system may allow activation of gene expression by dox.

[0082] In some embodiments, one localization domain may be made up of two or more parts. In other words, there may be another domain and/or sequence between two or more parts of the same localization domain. In some embodiments, the two or more parts of the one localization domain may work together to achieve a function. Thus, in some embodiments, multiple parts of the same localization domain may be referred to as localization domain (part 1), localization domain (part 2), localization domain (part 3), etc.

[0083] In certain embodiments, the localization domain may be a nuclear localization signal/sequence (NLS). [0084] In some embodiments, inclusion of a localization domain may be useful for improving the targeted action of biological products, such as antibodies or Fc fusions; genome, epigenetic, or transcript modifying proteins (i.e., CRISPR/Cas enzymes); therapeutic peptide, protein, or conjugates thereof, such as neuroactive peptides, hormones, growth factors, etc.

[0085] In some embodiments, the localization domain may be encoded by a nucleotide sequence at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 33, 53, 61, or a combination thereof. In some embodiments, the localization domain may be encoded by SEQ ID NO: 33. In some embodiments, the localization domain may be encoded by SEQ ID NO: 53. In some embodiments, the localization domain may be encoded by SEQ ID NO: 61.

[0086] In some embodiments, the localization domain may include an amino acid sequence at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 34, 54, 62, or a combination thereof. In some embodiments, the localization domain may include SEQ ID NO: 34. In some embodiments, the localization domain may include SEQ ID NO: 54. In some embodiments, the localization domain may include SEQ ID NO: 62.

Additional optional multi-domain polypeptide components

[0087] In some embodiments, the multi-domain polypeptide may include one or more additional components (i.e., one or more additional amino acids). Thus, in some embodiments, the nucleotide sequence encoding the multi-domain polypeptide may include one or more additional components (i.e., one or more additional nucleotide). In some embodiments, the additional component may be an additional domain. In some embodiments, the additional component may positively impact correct biosynthesis and trafficking of the multi-domain polypeptide. In some embodiments, the multi-domain polypeptide may include more than one additional component. For example, the multi-domain polypeptide may include 1, 2, 3, 4, or 5, 6, 7, 8, 9, or 10 additional components. In some embodiments, one additional component may be made up of two or more parts. In other words, there may be another domain and/or sequence between two or more parts of the same additional component. In some embodiments, the two or more parts of the one additional component may work together to achieve a function.

[0088] In some embodiments, an additional component may be an epitope tag. Epitope tags are known in the art and commercially available, such as V5 and HA (available from Thermo Fischer Scientific), and FLAG® or 3x-FLAG® (available from Sigma- Aldrich). In certain embodiments, the multi-domain polypeptide includes the epitope tag HA. In some embodiments, an additional component, such as an epitope tag, may be used to track the spread and/or location of one or more parts of the multi-domain peptide. For example, in some embodiments, where a multi-domain polypeptide is cleaved once, an epitope tag may be used to track one or both parts of the cleaved multi-domain polypeptide. Thus, in some embodiments, the multi-domain polypeptide may include more than one epitope tag to track each of the multi-domain polypeptide parts after cleavage. In other words, if the multi-domain polypeptide is cleaved into two parts (part 1 and part 2), one epitope tag may be attached to part 1 and another epitope tag may be attached to part 2. In some embodiments, the multi-domain polypeptide may be cleaved more than once. Thus, in some embodiments, one or more epitope tag may be attached to one or more of all parts of the cleaved multi-domain polypeptide. In some embodiments, if the multi-domain polypeptide is cleaved to separate the synaptic targeting domain from the rest of the multi-domain polypeptide, one or more epitope may be included with the synaptic targeting domain portion and/or in the portion containing the rest of the polypeptide. In some embodiments, when the synaptic targeting domain is cleaved from the rest of the multi-domain polypeptide, the rest of the multi-domain polypeptide may be referred to as the “liberated portion.”

[0089] In some embodiments, an additional component may be a fluorescent protein or a non- fluorescent (i.e., dark) version of a fluorescent protein. In some embodiments, the fluorescent or non-fluorescent protein may positively impact correct biosynthesis and trafficking of the multidomain polypeptide.

[0090] In certain embodiments, the fluorescent protein may be green fluorescent protein (GFP). In certain embodiments, the fluorescent protein may be red fluorescent protein (RFP).

[0091] In some embodiments, the fluorescent protein may be encoded by a nucleotide sequence at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 17, 39, 67, or a combination thereof. In certain embodiments, the fluorescent protein may be encoded by SEQ ID NO: 17. In certain embodiments, the fluorescent protein may be encoded by SEQ ID NO: 39. In certain embodiments, the fluorescent protein may be encoded by SEQ ID NO: 67.

[0092] In some embodiments, the fluorescent protein may include an amino acid sequence at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 18, 40, 68, or a combination thereof. In certain embodiments, the fluorescent protein may include SEQ ID NO: 18. In certain embodiments, the fluorescent protein may include SEQ ID NO: 40. In certain embodiments, the fluorescent protein may include SEQ ID NO: 68.

[0093] In some embodiments, an additional component may be a 2A sequence. 2A peptides are a class of 18-22 amino acids long, which can induce ribosomal skipping during translation of a protein in a biological cell. In some embodiments, the 2A sequence is F2A (derived from foot- and-mouth disease virus).

[0094] In some embodiments, the 2A sequence may be encoded by a nucleotide sequence at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 41, 69, or a combination thereof. In certain embodiments, the 2A sequence may be encoded by SEQ ID NO: 41. In certain embodiments, the 2A sequence may be encoded by SEQ ID NO: 69.

[0095] In some embodiments, the 2A sequence may include an amino acid sequence at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 42, 70, or a combination thereof. In certain embodiments, the 2A sequence may include SEQ ID NO: 42. In certain embodiments, the 2A sequence may include SEQ ID NO: 70.

[0096] In some embodiments, an additional component may be an intervening sequence. An intervening sequence may also be referred to as an intron. Thus, in some embodiments, intervening sequences may include a nucleotide sequence that is not expressed. In some embodiments, the intervening sequence may not influence the function of the multi-domain polypeptide or one or more of its domains. In some embodiments, intervening sequences are added to aid in combining one or more of the multi-domain polypeptide domains.

[0097] In some embodiments, the intervening sequence may be encoded by a nucleotide sequence at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 49. In certain embodiments, the intervening sequence may be encoded by SEQ ID NO: 49. In some embodiments, the intervening sequence may be encoded by a nucleotide sequence at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to ATGGTG. Thus, in some embodiments the intervening sequence may be encoded by ATGGTG. In some embodiments, the intervening sequence may be encoded by a nucleotide sequence at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to AGTAC. Thus, in some embodiments the intervening sequence may be encoded by AGTAC.

[0098] In some embodiments, the intervening sequence may include an amino acid sequence at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 50. In certain embodiments, the intervening sequence may include an amino acid sequence at least 85% identical to SEQ ID NO: 50. In some embodiments, the intervening sequence may include an amino acid sequence at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to MV. Thus, in some embodiments the intervening sequence may include MV. In some embodiments, the intervening sequence may include an amino acid sequence at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to ST. Thus, in some embodiments the intervening sequence may be ST.

Covalently linked

[0099] In some embodiments, one or more of the multi-domain polypeptide domains may be covalently linked. In other words, linkage between at least two of the domains of the multi-domain polypeptide is a covalent linkage. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the domains of the multi-domain polypeptide are covalently linked. In some embodiments, all of the domains are covalently lined. Thus, a linear molecule may be formed.

[00100] In some embodiments, one or more domain may be covalently linked by chemical coupling in any suitable manner known in the art, such as cross-linking methods. However, many known chemical cross-linking methods are non-specific, i.e., they do not direct the point of coupling to any particular site on the domains. Thus, the use of non-specific cross-linking agents may attack functional sites or sterically block active sites, rendering the fused domains of the multi-domain polypeptide biologically inactive. It is referred to the knowledge of the skilled artisan to block potentially reactive groups by using appropriate protecting groups. Alternatively, the use of oxime and hydrazone ligation techniques, which are chemo-selective entities that can be applied for the cross-linking of one or more domains, may be employed.

[00101] In some embodiments, the multi-domain polypeptide may include one or more linker (also referred to as a spacer). In some embodiments, a linker may provide flexibility, maintain proper spacing, and/or prevent clashes between functional units in a protein, such as the multidomain protein described herein. Conventional linkers can be determined for connecting domains as described herein by those skilled in the art. Linkers may be included between one or more of the multi-domain polypeptide domains. It is customary to change the composition and/or length of the linker based on therapeutic and/or experimental needs. In some embodiments, instead of a linker, two domains may be directly appended.

[00102] In some embodiments, a linker may provide further functionalities in addition to linking of the domains. For example, the linker may be cleavable. Thus, in some embodiments, the linker may be naturally cleavable inside the target cell, e.g. by enzymatic cleavage. In some embodiments, such further functionalities do not include any immunological functionalities.

[00103] In some embodiments, the multi-domain polypeptide may include at least one, two, three, four, five, six, seven, eight, nine, or ten linkers. In some embodiments, the linker may connect one or more of the domains together. For instance, the linker may link the synapse targeting domain to the cleavage domain, synapse targeting domain to the entry domain, synapse targeting domain to the localization domain, the cleavage domain to the entry domain, the cleavage domain to the localization domain, or the entry domain to the localization domain. In certain embodiments, the linker may be between the synapse targeting domain and the cleavage domain.

[00104] In some embodiments, the linker may be a flexible linker, rigid linker, cleavable linker, or a combination thereof. In a specific embodiment, the linker may be a flexible linker. In some embodiments, a linker may consist of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

[00105] In some embodiments, the linker may be encoded by a nucleotide sequence at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 9, 13, 27, 43, or a combination thereof. In some embodiments, the multi-domain polypeptide may include two linkers. Thus, in some embodiments, the linkers may be encoded by SEQ ID NO: 9 and SEQ ID NO: 13. In some embodiments, the linkers may be encoded by SEQ ID NO: 13 and SEQ ID NO: 27. In some embodiments, the linker may be encoded by a nucleotide sequence at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to GGC. Thus, in some embodiments, the linker may include GGC. In some embodiments, the linkers may be encoded by SEQ ID NO: 43 and GGC. [00106] In some embodiments, the linker may include an amino acid sequence at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10, 14, 28, 44, or a combination thereof. In some embodiments, the multi-domain polypeptide may include two linkers. Thus, in some embodiments, the linkers may include SEQ ID NO: 10 and SEQ ID NO: 14. In some embodiments, the linkers may include SEQ ID NO: 14 and SEQ ID NO: 28. In some embodiments, the linker may include an amino acid sequence at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to G. Thus, in some embodiments, the linker may include G. In some embodiments, the linkers may include SEQ ID NO; 44 and G.

Multi-Domain Polypeptide Arrangement

[00107] Those skilled in the art will understand the disclosed domains may be arranged in the multi-domain polypeptide in any order based on desired function, structure, etc. of the multidomain polypeptide.

[00108] The 5’ end of DNA sequences corresponds with the N terminus of the protein, while the 3’ end of DNA sequences corresponds with the C terminus of the protein. In some embodiments, it is conventional to present DNA sequences from 5’ to 3’ and/or protein sequences from N terminus to C terminus (when reading from left to right).

[00109] In some embodiments, the domain exposed to the extracellular/luminal environment could be on the N terminal, on the C terminal, or in an internal domain/loop. In some embodiments, if the domain is in an internal domain/loop, the multi-domain polypeptide may require cleavage at both ends of the domain. If cleavage at both ends of a domain is required, this may be achieved, for example, by including at least 2 cleavage domains or by utilizing intein/extein type protein splicing.

[00110] In some embodiments, the synapse targeting domain, cleavage domain, and entry domain may be arranged from N terminus to C terminus direction. In some embodiments, the synapse targeting domain, cleavage domain, and entry domain may be arranged from C terminus to N terminus direction. In some embodiments, the synapse targeting domain, cleavage domain, entry domain, and localization domain may be arranged from N terminus to C terminus direction. In some embodiments, the synapse targeting domain, cleavage domain, entry domain, and localization domain may be arranged from C terminus to N terminus direction. In some embodiments, the cleavage domain may separate the synapse targeting domain from the other domains (i.e., from the entry and/or localization domain).

[00111] In certain embodiments, the multi-domain polypeptide may be arranged from N terminus to C terminus in one of the following exemplary orders:

Example 1: Synapse targeting domain (part 1) - entry domain - cargo - first cleavage domain - first linker - second cleavage domain - second linker - synapse targeting domain (part 2) - fluorescent protein

Example 2: Synapse targeting domain - first linker - first cleavage domain - second linker

- second cleavage domain - entry domain - cargo

Example 3: Fluorescent protein - T2A sequence - synaptic targeting domain - first linker

- cleavage domain - second linker - entry domain (part 1) - intervening sequence - entry domain (part 2) - localization domain - cargo

Example 4: Cargo - localization domain - entry domain (part 1) - intervening sequence - entry domain (part 2) - first linker - cleavage domain - second linker - synaptic targeting domain - T2A sequence - fluorescent protein

Example 5: Fluorescent protein - synapse targeting domain (part 1) - first linker - first cleavage domain - second linker - second cleavage domain - cargo - entry domain - synapse targeting domain (part 2)

Example 6: Cargo - entry domain - first cleavage domain - first linker - second cleavage domain - second linker - synapse targeting domain

Example 7: Cargo - entry domain - localization domain - cleavage domain - synapse targeting domain

Example 8: Cargo - cleavage domain - localization domain - synapse targeting domain - entry domain

Example 9: Localization domain - cargo - cleavage domain - entry domain- synapse targeting domain

Example 10: Entry domain - synapse targeting domain - cargo - cleavage domain - localization domain

Whole multi-domain polypeptide sequences (includes all domains/linkers)

[00112] In some embodiments, the multi-domain polypeptide may be encoded by a nucleotide sequence at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 21, 23, 31, 59, 77, or a combination thereof. Thus, in some embodiments, the multi-domain polypeptide may be in two parts (e.g., the cargo may be located between two parts). Thus, in some embodiments, the multi-domain polypeptide may be encoded by SEQ ID NO: 21 and SEQ ID NO: 23. In some embodiments, the multi-domain polypeptide may be encoded by SEQ ID NO: 31. In certain embodiments, the multi-domain polypeptide may be encoded by SEQ ID NO: 59. In certain embodiments, the multi-domain polypeptide may be encoded by SEQ ID NO: 77.

[00113] In some embodiments, the multi-domain polypeptide may include an amino acid sequence at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 22, 24, 32, 60, 78, or a combination thereof. Thus, in some embodiments, the multi-domain polypeptide may be in two parts (e.g., the cargo may be located between two parts). Thus, in some embodiments, the multi-domain polypeptide may include SEQ ID NO: 22 and SEQ ID NO: 24. In some embodiments, the multi-domain polypeptide may include SEQ ID NO: 32. In certain embodiments, the multi-domain polypeptide may include SEQ ID NO: 60. In certain embodiments, the multi-domain polypeptide may include SEQ ID NO: 78.

D. Method for Preparing the Multi-Domain Polypeptide

[00114] Also disclosed is a method for preparing the multi-domain polypeptide described herein. The method includes concatenating the synapse targeting, cleavage, and entry domains of the multi-domain polypeptide. The method may also include concatenating the synapse targeting, cleavage, entry, and localization domains of the multi-domain polypeptide. In other words, the method may also include concatenating the localization domain with the synapse targeting, cleavage, and entry domains. In some embodiments, the domains may be concatenated using one or more linker, as described herein.

[00115] In some embodiments, the domains may be arranged in any order. As mentioned herein, the synapse targeting domain, cleavage domain, and entry domain may be arranged from N terminus to C terminus direction. In some embodiments, the synapse targeting domain, cleavage domain, and entry domain may be arranged from C terminus to N terminus direction. In some embodiments, the synapse targeting domain, cleavage domain, entry domain, and localization domain may be arranged from N terminus to C terminus direction. In some embodiments, the synapse targeting domain, cleavage domain, entry domain, and localization domain may be arranged from C terminus to N terminus direction. In some embodiments, the cleavage domain may separate the synapse targeting domain from the other domains (i.e., from the entry and/or localization domain).

[00116] In some embodiments, multi-domain polypeptide may be prepared via recombinant DNA techniques, such as via a vector and/or plasmid. In some embodiments, the multi-domain polypeptide may be prepared using restriction enzyme and ligation, recombinase mediated cassette exchange, CRISPR/Cas type generation of cut sites to mediate targeted insertions from a donor, editing cassette or plasmid, targeted gene manipulation, or a combination thereof. For example, in certain embodiments, synthetic double stranded DNA blocks and Gibson/NEB HiFi assembly methods may be used to generate a plasmid including the multi-domain polypeptide. In some embodiments, gene editing technologies may be used to place the multi-domain polypeptide or portion of a multi-domain polypeptide into an endogenous gene. For example, in some embodiments, an endogenous synapse targeting domain gene could be used to which other domains and cargo may be appended through gene editing. In some embodiments, an endogenous gene could be used as the cargo and the multi-domain polypeptide may be appended.

[00117] In some embodiments, the vector/plasmid may be derived from AAV. Thus, in some embodiments, the vector/plasmid may include elements of AAV genomes. For example, the vector/plasmid may include Woodchuck Post-transcriptional Regulatory Element (WPRE/WRE). WPRE is present in most viral vectors, as it may help with the efficiency of transcription/translation of the encoded sequence. In some embodiment, WPRE may be omitted to give more space for other desired elements or omitted to reduce levels. In some embodiments, the plasmid/vector may include Inverted Terminal Repeat(s) (ITRs). In some embodiments, whatever is in between ITRs is what the vector will encode. In some embodiments, the vector/plasmid may be derived from a retrovirus.

E. Multi-Domain Polypeptide + Cargo Complex

[00118] Also disclosed is a complex including the multi-domain polypeptide as described herein and one or more cargo. In some embodiments, the complex may include at least one, two, three, four, five, six, seven, eight, nine, or ten cargos.

Cargo

[00119] Cargo may be any biologically active molecule, such as a peptide or protein, that one may choose to deliver to a target cell or target tissue for any reason, such as for research/experimentation use and/or therapeutic use. In some embodiments, cargo may be an enzyme. [00120] Cargo may be functionally bound, fused to, linked, or otherwise endowed with affinity to the multi-domain polypeptide. In some embodiments, the interaction between cargo and multidomain polypeptide may be achieved by direct genetic (DNA or RNA) sequence encoding, protein expression, or chemical synthesis. In some embodiments, the interaction between cargo and multidomain polypeptide may be achieved by indirect and/or orthogonal approaches such as protein splicing systems, SNAP-tag®, CLIP-tag™, CLICK chemistry systems, or affinity-based interaction. In some embodiments, the cargo may be linked to the multi-domain polypeptide with a linker as described herein. See, FIG. 6 which represents an example arrangement of multidomain polypeptide (i.e., tag) components bound to cargo.

[00121] In some embodiments, the cargo may be located 5’ (i.e., at the N terminus) of the multidomain polypeptide. Alternatively, in some embodiments, the cargo may be located 3’ (i.e., at the C terminus) of the multi-domain polypeptide. In some embodiments, the cargo may be located between two domains of the multi-domain polypeptide. For example, in specific embodiments, the cargo may be located between the synapse targeting domain and the rest of the multi-domain polypeptide.

[00122] In some embodiments, the cargo may include one or more therapeutic agent. In some embodiments, the therapeutic agent may be investigational or approved by a government agency, such as the United States Food and Drug Administration (FDA). In some embodiments, the cargo may include one or more research tool. In some embodiments, the therapeutic agent and/or research tool may include one or more gene sequence modifier, gene expression modifier, antibody, Fc-fusion, peptide, protein, recombinase, Tet-off transactivator (tTA), peroxidase, biotin ligase, agonist, antagonist, toxin, or a combination thereof.

[00123] In some embodiments, the gene sequence modifier or gene expression modifier may be a CRISPR/Cas enzyme (e.g., Cas9, Casl2a, HiFi Cas9, EbCasl2a, EnGen® SpRY Cas9 system, Transposon-associated RNA-guided nuclease (TnpB protein), Fanzor, etc.), TALEN, ZFN, Epigenetic modifier (e.g., CHARM), RNA, siRNA, aptamer, morpholinos as through affinity granted by RNA/DNA binding proteins.

[00124] In some embodiments, the cargo may include or have fused/linked to it a reporter, such as green or red fluorescent protein. Any reporter may work in such embodiments to determine where the cargo is located. For example, in experimental aspects, a researcher may be able to determine if the cargo was delivered to the desired target cell or target tissue.

[00125] In some embodiments, the peptide or protein may be a conjugate of a peptide or protein. In some embodiments, the peptide or protein may be a growth factor (such as brain- derived neurotrophic factor (BDNF)), therapeutic protein (such as ELEVIDYS (delandistrogene moxeparvovec-rokl)), peptide neurotransmitter, antagonist derivative, peptide toxin, or/or an FDA approved peptide.

[00126] In some embodiments, the recombinase may be Cre, Flp, Dre, vCre, or a derivative thereof, or a recombinase able to regulate, such as regulation by light (e.g., iCreV) and by drug (e.g., Cre-ERT2). In certain embodiments, the recombination is Cre. In certain other embodiments, the recombinase is Flp.

[00127] In some embodiments, the cargo may be encoded by a nucleotide sequence at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 5, 55, 73, 79, or a combination thereof. In some embodiments, the cargo may be encoded by SEQ ID NO: 5. In certain embodiments, the cargo may be encoded by SEQ ID NO: 55. In certain embodiments, the cargo may be encoded by SEQ ID NO: 73. In certain embodiments, the cargo may be encoded by SEQ ID NO: 79.

[00128] In some embodiments, the cargo may include an amino acid sequence at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6, 56, 74, 80, or a combination thereof. In some embodiments, the cargo may include SEQ ID NO: 6. In certain embodiments, the cargo may include SEQ ID NO: 56. In certain embodiments, the cargo may include SEQ ID NO: 74. In certain embodiments, the cargo may include SEQ ID NO: 80.

[00129] Thus, in some embodiments, the multi-domain polypeptide as described herein may be referred to as a therapeutic multi-domain polypeptide or a research multi-domain polypeptide.

Complex Arrangement

[00130] In some embodiments, the cargo may be covalently linked to the multi-domain polypeptide. In some embodiments, the cargo may be covalently linked to the N terminal or the C terminal of the multi-domain polypeptide. In certain embodiments, the cargo may be covalently linked to the N terminal of the multi-domain polypeptide.

[00131] In some embodiments, the cleavage domain may separate the synapse targeting domain from the other domains (i.e., from the entry and/or localization domain). Thus, in some embodiments, after cleavage, the other domains (i.e., the entry and/or localization domain) may remain contiguous with the cargo. [00132] In some embodiments, the cargo is located N terminal to the multi-domain polypeptide. In some embodiments, the cargo is located C terminal to the multi-domain polypeptide.

[00133] It should be appreciated that any domain or domain component may be located anywhere in relation to other domains/components to achieve a desired experimental and/or therapeutic result.

F. Vectors and Host Cells

[00134] Also disclosed is a polynucleotide molecule encoding the multi-domain polypeptide and/or complex as described herein. Additionally, a vector including the polynucleotide molecule is provided.

[00135] In some embodiments, the polynucleotide sequence encoding the multi-domain polypeptide and/or complex is provided to a host cell by way of a recombinant vector, which comprises a promoter operably linked to the polynucleotide sequence. In certain embodiments, the promoter may be a developmentally-regulated, an organelle- specific, a tissue-specific, an inducible, a constitutive, or a cell- specific promoter.

[00136] As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid, i.e., a polynucleotide sequence, to which it has been linked. One type of useful vector is an episome (i.e., a nucleic acid capable of extra-chromosomal replication). Useful vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as “expression vectors.” In general, expression vectors of utility in recombinant DNA techniques are often in the form of “plasmids,” which refer generally to circular double stranded DNA loops that, in their vector form, are not bound to the chromosome. The terms “plasmid” and “vector” are used interchangeably herein, inasmuch as a plasmid is the most commonly used form of vector. However, also included are such other forms of expression vectors that serve equivalent functions and that become known in the art subsequently hereto.

[00137] Vectors can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. [00138] For stable transformation of host cells, it is known that, depending upon the expression vector and transformation technique used, only a small fraction of cells will take-up and replicate the expression vector. In order to identify and select these transformants, a gene that encodes a selectable marker (e.g., resistance to an antibiotic) can be introduced into the host cells along with the nucleotide sequence encoding the multi-domain polypeptide. Selectable markers include those that confer resistance to drugs such as, but not limited to, ampicillin, kanamycin, chloramphenicol, or tetracycline. Nucleic acids encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a multi-domain polypeptide described herein or can be introduced on a separate vector. Cells stably transformed with the introduced nucleic acid can be identified by growth in the presence of an appropriate selection drug.

[00139] Thus, also provided herein is a host cell that includes and may express the multidomain polypeptide, complex, polynucleotide, and/or vector described herein.

[00140] In some embodiments, the host cell may be a bacterial cell, a cyanobacterium, a green- sulfur bacterium, a green non-sulfur bacterium, a purple sulfur bacterium, a purple non-sulfur bacterium, an extremophile, a yeast, algae, a fungus, an engineered organism thereof, or a synthetic organism.

[00141] In further exemplary embodiments, the host cell may be a mammalian cell, plant cell, insect cell, yeast cell, fungus cell, filamentous fungi cell, an algal cell, a cyanobacterial cell, or a bacterial cell. In certain embodiments, the host cell may be from the genus Escherichia, Bacillus, Pseudomonas, Lactobacillus, Rhodococcus, Synechococcus, Synechoystis, Pseudomonas, Aspergillus, Trichoderma, Neurospora, Fusarium, Humicola, Rhizomucor, Kluyveromyces, Pichia, Mucor, Myceliophtora, Penicillium, Phanerochaete, Pleurotus, Trametes, Chrysosporium, Saccharomyces, Stenotrophamonas, Schizosaccharomyces, Yarrowia, or Streptomyces. In certain embodiments, the host cell may be E. coli.

[00142] In some embodiments, the multi-domain polypeptide, complex, vector, and/or host cell may be used to deliver one or more cargo to one or more target cell. Thus, in some embodiments, expression of the multi-domain polypeptide may confer delivery of one or more cargo to one or more target cell. Thus, in some embodiments, expression of the multi-domain polypeptide in a host cell may confer delivery of one or more cargo to one or more target cell. G. Pharmaceutical Composition

[00143] Also provided is a pharmaceutical composition including the multi-domain polypeptide, polynucleotide encoding the multi-domain polypeptide, complex including the multidomain polypeptide, and/or vector including the multi-domain polypeptide as described herein, and a pharmaceutically acceptable carrier.

[00144] In some embodiments, the pharmaceutical composition may be useful, for example, in the prevention and/or treatment of a disease and/or a disorder including cancer, a hematological disorder, an infectious disease, an autoimmunity disorder, a transplant rejection, a chronic pain disorder or other nerve disorder, or a combination thereof. Examples of disease and/or disorder types are described in Sec. I herein.

Pharmaceutically acceptable carrier

[00145] In some embodiments, the pharmaceutical composition may include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 pharmaceutically acceptable carriers. In certain embodiments, the pharmaceutical composition may include at least one pharmaceutically acceptable carrier.

[00146] In some embodiments, it may be beneficial to include one or more excipients in a pharmaceutical composition. One of skill in the art would appreciate that the choice of any one excipient may influence the choice of any other excipient. For example, the choice of a particular excipient may preclude the use of one or more additional excipients because the combination of excipients would produce undesirable effects. One of skill in the art would be able to determine empirically which excipients, if any, to include in the formulations or compositions disclosed herein. Excipients may include, but are not limited to, co-solvents, solubilizing agents, buffers, pH adjusting agents, bulking agents, surfactants, encapsulating agents, tonicity-adjusting agents, stabilizing agents, protectants, and viscosity modifiers. In some embodiments, it may be beneficial to include a pharmaceutically acceptable carrier.

[00147] Also provided herein is a vaccine including the multi-domain polypeptide, polynucleotide encoding the multi-domain polypeptide, complex including the multi-domain polypeptide, and/or vector including the multi-domain polypeptide as described herein and/or a pharmaceutical composition as described herein. H. Method of Delivering Cargo to a Target Cell

[00148] A method of delivering one or more cargo to one or more target cell in a subject is also provided herein. The method includes administering the multi-domain polypeptide, polynucleotide encoding the multi-domain polypeptide, complex including the multi-domain polypeptide, and/or vector including the multi-domain polypeptide as described herein to the subject.

[00149] Thus, the multi-domain polypeptide may be referred to as a multi-domain targeting polypeptide and/or protein.

[00150] As described herein, the cargo may be one or more therapeutic agent and/or research tool.

Target cell

[00151] In some embodiments, the target cell may be any endogenous cell. In some embodiments, the target cell may be any endogenous healthy cell. In some embodiments, the target cell may be an endogenous unhealthy and/or abnormal cell, such as a cancerous or non-cancerous tumor cell or a pathogenic (e.g., infected, degenerating, etc.) cell.

[00152] In some embodiments, the target cell may be a nervous system cell, such as a neuron or a non-neuronal cell in synaptic proximity. In some embodiments, for example, the neuron may include those of the neurotransmitter classes: excitatory (i.e., glutamatergic), inhibitory (i.e., GABAergic, glycinergic), and modulatory (e.g., acetylcholine, dopamine, serotonin, histamine, norepinephrine, nitric oxide). In some embodiments, the non-neuronal cell in synaptic proximity may be a vascular cell, glial cell (e.g., astrocyte or satellite cell, ensheathing cell, Schwann cell, oligodendrocyte), or immune cell (nervous system resident cell such as microglia, or infiltrating immune cell such as monocyte, neutrophil, T cell, or macrophage).

[00153] In some embodiments, the target cell may be a cell outside of the nervous system, such as a muscle or specific organ cell. In some embodiments, the muscle cell may be a cardiac, skeletal, or smooth muscle cell. In some embodiments, the organ cell may include an eye, heart, gut, bladder, urinary sphincter, gut sphincter, sex organ (arousal, erection, ejaculation, etc.), salivary gland, nasal gland, lacrimal gland, exocrine gland, or sensory organ cell. In some embodiments, the sensory organ may be for vision, audition, olfaction, gustation, proprioception, nociception, thermoception, exteroception, interoception. [00154] In some embodiments, the target cell may be an exogenously derived desirable cell, such as from an autologous or allogenic transplant. In some embodiments, the target cell may be a pathogen, such as a virus, bacteria, fungi, parasite, or pathogenic protein, such as a prion.

[00155] In some embodiments, a target cell may refer to a population of target cells, such as a population of cells that make up a certain kind of tissue. Thus, one or more target cell may also refer to one or more target tissue.

Subject

[00156] “Subject,” ‘ ‘individual,” and “patient” interchangeably refer to a mammal, for example, a human or a non-human primate, but also domesticated mammals (e.g., canine or feline), laboratory mammals (e.g., mouse, rat, rabbit, hamster, guinea pig), and agricultural mammals (e.g., equine, bovine, porcine, ovine). In some embodiments, the subject may be human (e.g., adult male, adult female, adolescent male, adolescent female, male child, female child). Alternatively, in some embodiments, the subject may be a non-human animal. For example, in some embodiments, the non-human animal may be a canine or a feline. In certain embodiments, the subject can be under the care of a physician or other health worker. In certain embodiments the subject may not be under the care of a physician or other health worker. In some embodiments, a subject may be suffering from any disease and/or disorder.

[00157] In some embodiments, the multi-domain polypeptide’s action may be non-toxic in a subject.

Cargo Delivery

[00158] In some embodiments, the cargo is delivered to a target cell or tissue via one or more neuron. In some embodiments, the neurons are connected in circuits. For example, the complex including the multi-domain polypeptide and cargo may start in one cell type or tissue and be delivered to another cell type or tissue (e.g., target cell type or tissue) via multiple neurons connected in circuits. In other words, the one or more cells (e.g., one or more neurons) may be synaptically coupled. Synapses are made by neurons, thus, two neurons may be synaptically coupled or a neuron and a non-neuronal cell may be synaptically coupled.

[00159] In some embodiments, each domain included in the multi-domain polypeptide may provide one or more function, ultimately allowing cargo to be released in a target cell type or tissue in a desired way. Thus, in some embodiments, the synapse targeting domain, cleavage domain, entry domain, and, optionally, the localization domain of the multi-domain polypeptide each may function either simultaneously or in any order to deliver the cargo to the target cell or tissue.

[00160] In some embodiments, the cargo may be delivered via axonal transport. For example, in some embodiments, the cargo may be delivered by anterograde or retrograde transport. In certain embodiments, the cargo may be delivered by anterograde transport. In some embodiments, for anterograde delivery, the multi-domain polypeptide accomplishes subcellular localization to the lumen of neuropeptide or neurotransmitter vesicles, or the extracellular aspect of axon terminals. In other embodiments, the cargo may be delivered by retrograde transport. In some embodiments, for retrograde delivery, the multi-domain polypeptide accomplishes subcellular localization to the lumen of somato/dendritic vesicles and/or the extracellular aspect of dendrites or of dendritic spines (at/near postsynaptic densities). See, FIG. 5 which shows the concept of tags (e.g., a multi-domain polypeptide as described herein) for retrograde and anterograde access to specific cell types.

[00161] In some embodiments, the multi-domain polypeptide and/or cargo may be efficacious over a period of time. For example, the multi-domain polypeptide and/or cargo may maintain one or more of its domain function(s) and/or its overall function of delivering cargo to a target cell/tissue for at least 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 12 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, or 20 years. In some embodiments, the multi-domain polypeptide and/or cargo may maintain one or more of its domain function(s) for the entire lifespan of an organism. In some embodiments, the cargo may be a crystallin. Thus, in some embodiments, the multi-domain polypeptide may maintain one or more of its domain function(s) and/or its overall function of delivering cargo to a target cell/tissue in vivo (e.g., in a subject) or in vitro. I. Method of Treating and/or Preventing a Disease/Disorder

[00162] Additionally, a method of treating and/or preventing a disease or disorder in a subject in need thereof is provided herein. The method includes administering the multi-domain polypeptide, polynucleotide encoding the multi-domain polypeptide, complex including the multidomain polypeptide and cargo, and/or vector including the multi-domain polypeptide as described herein to a subject.

[00163] In some embodiments, the method may include administering to a subject an effective amount of a multi-domain polypeptide, multi-domain polypeptide-cargo complex, a pharmaceutical composition, and/or a vaccine as described herein. In some embodiments, the method may include administering to a subject a combination of a means for targeting a neuron synapse, a means for cleavage of one or more protein domains, a means for entering one or more target cell, and optionally, a means for localization to one or more target cell, and a pharmaceutically acceptable carrier.

[00164] In some embodiments, administration may be in an “effective amount” or a “therapeutically effective amount” (as the case may be), this being sufficient to show benefit to the individual/subject. The actual amount administered, and rate and time-course of administration, may depend on the nature and severity of what is being treated.

[00165] The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or concurrently with an existing therapeutic agent and once or multiple times.

[00166] Administration of the pharmaceutical composition can be systemic, mucosal and/or proximal to the location of a target site (e.g., near a tumor). Suitable routes of administration will be apparent to those of skill in the art, depending on the type of condition to be prevented or treated, the therapeutically active agent used, and/or the target cell population or tissue. Various acceptable methods of administration include, but are not limited to, intravenous administration, intraperitoneal administration, intramuscular administration, intranodal administration, intracoronary administration, intraarterial administration (e.g., into a carotid artery), subcutaneous administration, retroorbital administration, transdermal delivery, intratracheal administration, subcutaneous administration, intraarticular administration, intraventricular administration, inhalation (e.g., aerosol), intracranial, intraspinal, intraocular, aural, intranasal, oral, pulmonary administration, impregnation of a catheter, and direct injection into a tissue. In one aspect, routes of administration include: intravenous, intraperitoneal, subcutaneous, intradermal, intranodal, intramuscular, transdermal, inhaled, intranasal, oral, intraocular, intraarticular, intracranial, and intraspinal. Parenteral delivery can include intradermal, intramuscular, intraperitoneal, intrapleural, intrapulmonary, intravenous, subcutaneous, atrial catheter and venal catheter routes. Aural delivery can include ear drops, intranasal delivery can include nose drops or intranasal injection, and intraocular delivery can include eye drops. Aerosol (inhalation) delivery can also be performed using methods standard in the art. Other routes of administration that modulate mucosal immunity are useful in the treatment of viral infections. Such routes include bronchial, intradermal, intramuscular, intranasal, other inhalatory, rectal, subcutaneous, topical, transdermal, vaginal, and urethral routes.

[00167] A suitable amount of the pharmaceutical composition to be administered can be determined by routine experiments with animal models. Such models include, without implying any limitation, rabbit, sheep, mouse, rat, dog and non-human primate models. Example unit dose forms for injection include sterile solutions of water, physiological saline or mixtures thereof. The pH of such solutions should be adjusted to about 7.4. Suitable carriers for injection include hydrogels, devices for controlled or delayed release, polylactic acid and collagen matrices. For injection, the pharmaceutical composition may be provided, for example, in a pre-filled syringe Suitable pharmaceutically acceptable carriers for topical application include those which are suitable for use in lotions, creams, gels and the like. If the inventive composition, in particular the inventive vaccine, is to be administered orally, tablets, capsules and the like may be used as a unit dose form. The pharmaceutically acceptable carriers for the preparation of unit dose forms which can be used for oral administration are well known in the prior art. The choice thereof will depend on secondary considerations such as taste, costs and storability, which are not critical for the purposes of the present invention, and can be made without difficulty by a person skilled in the art.

[00168] In some embodiments, “treat” and “treatment” refer to an approach for obtaining beneficial or desired results, including clinical results. In some embodiments, the subject in need of treatment may include a subject diagnosed as having, or suspected to have, a disease and/or disorder. In a particular embodiment, treatment may include administering a multi-domain polypeptide-cargo complex or pharmaceutical composition as described herein to a subject having, diagnosed as having, or suspected of having a disease and/or disorder. In some embodiments, the subject may be asymptomatic. [00169] In some embodiments, prevention includes treatment of a disease/disorder that causes the clinical symptoms of the disease/disorder not to develop or progress, or reduce the occurrence of.

Example diseases/disorders

[00170] In some embodiments, the disease or disorder may be cancer, a hematological disorder, infectious disease, autoimmunity disorder or a transplant rejection.

[00171] In some embodiments, the cancer may be pancreatic cancer, prostate cancer, breast cancer, bladder cancer, endometrial cancer, cholangiocarcinoma, ovarian cancer, kidney cancer, renal cell carcinoma, leukemia, liver cancer, intrahepatic bile duct cancer, lymphoma (e.g., nonHodgkin lymphoma), thyroid cancer (e.g., papillary thyroid cancer), cervical cancer, gallbladder cancer, gastric cancer, skin cancer, bronchus cancer, lung cancer, colon cancer, rectal cancer, melanoma, multiple myeloma, urothelial carcinoma, osteosarcoma, head and neck cancers, colorectal cancer, hepatocellular carcinoma, glioma, medulloblastoma, testicular cancer, or a combination thereof.

[00172] In some embodiments, the hematological disorder may be a sickle cell disease, thalassemia, methemoglobinemia, anemia (e.g., iron-deficiency anemia, megaloblastic anemia, hemolytic anemia, aplastic anemia), HIV, myelodysplastic syndrome, myelofibrosis, neutropenia, polycythemia vera, erythrocytosis, leukocytosis, thrombocytosis, clotting protein disorder (e.g., hemophilia), platelet disorder, hematological malignancy (e.g., lymphoma, myeloma, leukemia), hemochromatosis, asplenia, hypersplenism, or a combination thereof.

[00173] In some embodiments, the infectious disease may be a bacterial infection (e.g., actinomycosis, brucellosis, cholera, epididymitis, melioidosis, pneumonia, syphilis, tuberculosis), fungal infection (e.g., aspergillosis, candidiasis, histoplasmosis, urethritis), viral infection (e.g., bird flu, chikungunya fever, Ebola, measles, pox disease, rubella, Zika fever), protozoan infection (avian malaria, coccidiosis, toxoplasmosis, trypanosomiasis), or a combination thereof.

[00174] In some embodiments, the autoimmune disorder may be lupus, rheumatoid arthritis, Crohn’s disease, ulcerative colitis, Psoriasis, Type 1 diabetes, or a combination thereof.

[00175] In some embodiments, the transplant rejection may be a hyperacute, acute, or chronic rejection. In some embodiments, the transplant rejection may be a kidney transplant rejection, liver transplant rejection, lung transplant rejection, or a combination thereof. In some embodiments, the disease or disorder may be a blood transfusion reaction. [00176] In some embodiment, the disease or disorder may be a chronic pain disorder or other nerve, neuronal, and/or nervous system disorder. In some embodiments, the disease or disorder may be a neurogenic disorder, neurodevelopmental disorder, epilepsy, neurodegenerative disease, or muscular disease. In some embodiments, the disease or disorder may be seizure disorder, Alzheimer’s or other dementias, Parkinson’s, Huntington’s, multiple sclerosis, stroke, migraine or other headache, Bell’ s palsy or other palsy, spinal cord or other nervous inject, or a combination thereof. In some embodiments, the disease/disorder may be a muscular disorder, such as a skeletal muscle disorder or myotonia. In some embodiments, the disease/disorder may be a smooth muscle disorder, such as gastrointestinal dysmotility, overactive bladder, unstable bladder, inability to empty and/or fill the bladder, urinary or fecal incontinence, or hypertension. In some embodiments, the disease or disorder may be a cardiac muscle disorder, such as tachycardia, bradycardia, arrythmias, or cardiomyopathies. In some embodiments, the disease or disorder may be disorders of the peripheral, autonomic, somatic, or enteric nervous system. A non-limiting list of autonomic dysfunction includes heart rate, arousal, and urination dysfunction.

EXAMPLES

[00177] The following examples are merely illustrative, and do not limit this disclosure in any way.

EXAMPLE 1: Adeno Associated Viral Vectors to Express Tagged Cre

[00178] The goal of this study was to determine if a rationally designed multi-domain polypeptide (also referred to herein as a tag) could confer trans-neuronal delivery of cargo (e.g., biologically active molecules) to which it was appended.

[00179] Serotype 1 Adeno Associated Viral (AAV) vector particles were prepared that utilized the CAG promoter to express either Cre recombinase (AAV Control Cre) or experimental AAV Tagged Cre #1 or AAV Tagged Cre #2. The AAV Control Cre construct paavCAG-iCre (Addgene Plasmid #51904) was previously existing and publicly available. The whole AAV Control Cre nucleotide sequence is SEQ ID NO: 35, which encodes amino acid sequence SEQ ID NO: 36. The whole AAV Control Cre sequence consists of an intervening sequence (atggtg, which encodes MV), a localization domain (SEQ ID NO: 33, which encodes SEQ ID NO: 34), and Cre (SEQ ID NO: 5, which encodes SEQ ID NO: 6). Refer to FIG. 7 that includes a schematic of the AAV Control Cre construct.

[00180] The AAV Tagged Cre #1 was prepared using synthetic double stranded DNA blocks and Gibson/NEB HiFi assembly methods to yield a plasmid wherein Cre (SEQ ID NO: 5, which encodes amino acid sequence SEQ ID NO: 6) was fused to a multi-domain polypeptide consisting of an entry domain (R9 cell penetrating peptide) (SEQ ID NO: 3, which encodes SEQ ID NO: 4), a first cleavage domain (Tobacco Etch Virus (TEV) protease recognition/cut site) (SEQ ID NO: 7, which encodes SEQ ID NO: 8), a first linker (SEQ ID NO: 9, which encodes SEQ ID NO: 10), a second cleavage domain (TEV protease) (SEQ ID NO: 11, which encodes SEQ ID NO: 12), a second linker (SEQ ID NO: 13, which encodes SEQ ID NO: 14), a fluorescent protein marker (mCerulean) (SEQ ID NO: 17, which encodes SEQ ID NO: 18) and a synapse targeting domain (Presynaptic mGrasp Component derived from paavCAG-pre-mGRASP-mCerulean (Addgene Plasmid #34910)) (SEQ ID NO: 1 and SEQ ID NO: 15, which encode SEQ ID NO: 2 and SEQ ID NO: 16, respectively). The synapse targeting domain is made up of two parts, located apart from each other. The first part, consisting of SEQ ID NO: 1, is the signal peptide for the second part, consisting of SEQ ID NO: 15). The multi-domain polypeptide “tag” comprises two parts, since the cargo (Cre) is located on the inside of the polypeptide sequence. Thus, one part consists of SEQ ID NO: 21 and the other part consists of SEQ ID NO: 23, which encode SEQ ID NO: 22 and SEQ ID NO: 24, respectively. Thus, the whole AAV Tagged Cre #1 sequence is SEQ ID NO: 19, which encodes SEQ ID NO: 20. Refer to FIG. 7 that includes a schematic of the AAV Tagged Cre #1 construct.

[00181] The AAV Tagged Cre #2 was prepared using synthetic double stranded DNA blocks and Gibson/NEB HiFi assembly methods to yield a plasmid wherein Cre (SEQ ID NO: 5, which encodes SEQ ID NO: 6) was fused to a multi-domain polypeptide consisting of a synapse targeting domain (VAMP2 obtained from VAMP2-SNAP (Addgene Plasmid #105288)) (SEQ ID NO: 25, which encodes SEQ ID NO: 26), a first linker (SEQ ID NO: 27, which encodes SEQ ID NO: 28), a first cleavage domain (TEV protease) (SEQ ID NO: 7, which encodes SEQ ID NO: 8), a second linker (SEQ ID NO: 13, which encodes SEQ ID NO: 14), a second cleavage domain (TEV protease recognition/cut site) (SEQ ID NO: 11, which encodes SEQ ID NO: 12), and an entry domain (R9 Cell penetrating peptide) (SEQ ID NO: 3, which encodes SEQ ID NO: 4). Thus, the multi-domain polypeptide “tag” consists of SEQ ID NO: 31, which encodes SEQ ID NO: 32. The whole AAV Tagged Cre #2 sequence is SEQ ID NO: 29, which encodes SEQ ID NO: 30. Refer to FIG. 7 that includes a schematic of the AAV Tagged Cre #2 construct. The constructs were validated by whole-plasmid sequencing.

[00182] Y oung adult mice of two to four months of age were utilized that harbored a fluorescent Cre reporter allele (eg Cre-on TdTomato Red Fluorescent Protein (RFP) from the “Ail4” strain B6. Cg-Gt(ROSA)26Sor^tnil4(CAG'^tdTomato)Hze/J). Mice underwent isoflurane based anesthesia and lidocaine/ketoprofen analgesia for a stereotaxic injection of 200 nanoliters of AAV Control Cre, AAV Tagged Cre #1, or AAV Tagged Cre #2 into the left basal pontine nucleus. Mice also underwent injection into the right cerebellar cortex of a serotype 8 Cre-reporter AAV (CAG-Flex- rev-3xGFP).

[00183] Histology. After an incubation period of several weeks, animals were terminally anesthetized with a tribromoethanol (Avertin) solution and subjected to transcardial perfusion with a 4% sucrose containing saline, and then with a 4% sucrose, 4% formaldehyde saline solution. After an overnight post fixation period, brains were sectioned by vibratome between 50 and 100 microns thick. In some cases, standard methods were used for fluorescent immunohistochemistry for the fluorescent proteins under evaluation and/or used DAPI as a counterstain for nuclei. Slides were mounted with an antifade solution (e.g., ProLong Glass). Slides were evaluated by laser scanning confocal microscopy.

[00184] Delivery of the AAV Control Cre, AAV Experimental Cre #1 , and AAV Experimental Cre #2 vectors was validated by the presence of RFP+ (shown in red) basal pontine neuron axons (mossy fibers) in the right cerebellar cortex. Potential evidence of transneuronal spread of the AAV Tagged Cre #1 and Cre #2 constructs was assayed by evaluating the presence of fluorescent, GFP+ (shown in green) cerebellar granule cell labeling, as cerebellar granule cells are anterograde/postsynaptic to basal pontine neuron mossy fibers.

[00185] The results suggest that the presence of at least two different designs of tags is associated with the emergent property of transneuronal delivery and enzymatic activity of Cre recombinase when delivered by AAVs. AAV Control Cre acts in a cell-autonomous fashion, rendering the pontine neurons mossy fiber axons fluorescent without appreciable anterograde action. AAV Tagged Cre #1 and AAV Tagged Cre #2 gave apparent labeling not only in pontine mossy fibers, but also in postsynaptic cerebellar granule cells (FIG. 1).

EXAMPLE 2: Adeno Associated Viral Vectors to Express Tagged Flp

[00186] The inventor sought to determine whether the multi-domain polypeptide tags may also confer transneuronal delivery /action to biological molecules besides Cre recombinase. Thus, AAV vectors were designed to express Flp recombinase (AAV Control Flp) or Flp recombinase fused to a candidate Tag (AAV Tagged Flp). Both constructs were of a Cre-on design.

[00187] A previously existing, publicly available AAV vector was used as starting material to generate both AAV Control Flp and AAV Tagged Flp. Namely, pAAV-hSynl-Flex-mRuby2- GSG-P2A-GCaMP6s-WPRE-pA (Addgene Plasmid #68720), where in the P2A-GCAMP6s portion was replaced by either T2A-Flp (untagged) or by T2A-Tagged Flp using Gibson assembly/NEB HiFi methods. The AAV Control Flp sequence is SEQ ID NO: 63, which encodes SEQ ID NO: 64. The AAV Control FLP sequence consisted of a fluorescent protein (SEQ ID NO: 39, which encodes SEQ ID NO: 40), T2A sequence (SEQ ID NO: 41, which encodes SEQ ID NO: 42), localization domain (SEQ ID NO: 61, which encodes SEQ ID NO: 62), and FlpO (SEQ ID NO: 55, which encodes SEQ ID NO: 56). Refer to FIG. 8 that includes a schematic of the AAV Control Flp construct.

[00188] The AAV Tagged Flp consisted of a fluorescent protein (mRuby2) (SEQ ID NO: 39, which encodes SEQ ID NO: 40), T2A sequence (SEQ ID NO: 41, which encodes SEQ ID NO: 42), synapse targeting domain (VAMP2) (SEQ ID NO: 25, which encodes SEQ ID NO: 26), a first linker (SEQ ID NO: 43, which encodes SEQ ID NO: 44), a cleavage domain (SEQ ID NO: 45, which encodes SEQ ID NO: 46), a second linker (ggc, which encodes G), an entry domain, consisting of two parts (Poly-Histidine and TAT Cell penetrating peptide, obtained from pTriEx- HTNC (Addgene Plasmid #13763)) (part 1: SEQ ID NO: 47, which encodes SEQ ID NO: 48; part 2: SEQ ID NO: 51, which encodes SEQ ID NO: 52), an intervening sequence (SEQ ID NO: 49, which encodes SEQ ID NO: 50), and a localization domain (Nuclear Localization Sequence) (SEQ ID NO: 53, which encodes SEQ ID NO: 54) fused to Flp (SEQ ID NO: 55, which encodes SEQ ID NO: 56). The multi-domain polypeptide “tag” consists of SEQ ID NO: 59, which encodes SEQ ID NO: 60). Thus, the whole AAV Tagged Flp sequence is SEQ ID NO: 57, which encodes SEQ ID NO: 58. Refer to FIG. 8 that includes a schematic of the AAV Tagged Flp construct. From these viral plasmids, viral particles were packaged of the AAV1 serotype.

[00189] The mice used in this study expressed a Slcl7a7 Cre transgene to achieve Cre expression in the glutamatergic projection neurons of the basal pontine nucleus. The mice also harbored a Flp-On enhanced Green Fluorescent Protein (GFP) reporter allele Gt(ROSA)26Sor^{tml 2(CAG}'^EGFP>Fsh/Mmjax) and/or a Flp-On fluorescent reporter virus (e.g., AAV CAG-FRT-rev-3xGFP). Mice underwent isoflurane based anesthesia and lidocaine/ketoprofen analgesia for a stereotaxic injection of 200 nanoliters of AAV Control Flp or AAV Tagged Flp into the left basal pontine nucleus.

[00190] After 8 weeks, mice underwent Histology as described in EXAMPLE 1. Successful delivery of the Cre-on AAV Control Flp or of the AAV Tagged Flp vectors was validated by the presence of RFP+ basal pontine neuron axons (mossy fibers) in the right cerebellar cortex. Potential evidence of transneuronal spread of the AAV Tagged Flp construct was assayed by evaluating the presence of fluorescent cerebellar granule cell labeling, as cerebellar granule cells are anterograde/postsynaptic to basal pontine neuron mossy fibers.

[00191] GFP+ granule cells in the Tagged Flp vs Control Flp condition suggest that the Tag confers transneuronal delivery. Furthermore the presence of the GFP (trasnsgenic Flp activity reporter), in the absence of RFP (AAV mediated mRuby2), suggests that it is the transneuronal action conferred by the polypeptide Tag, and not transneuronal trafficking of AAV itself, which is responsible for the anterograde action observed. AAV Control Flp acts in a cell-autonomous fashion, rendering the pontine neurons mossy fiber axons fluorescent without appreciable anterograde action (FIG. 2A). In the AAV Tagged Flp condition, there are multiple labeled mossy fibers, but there are also GFP+/RFP- granule cells (FIG. 2B). In an inset of (FIG. 2B), there are readily identifiable individual postsynaptic anterograde labeled granule cells, which express the GFP+ Flp reporter, but which do not express the mRuby2 fluorescent protein (red fluorescent protein, i.e., RFP) (FIG. 2C).

EXAMPLE 3: Adeno Associated Viral Vectors to Express Tagged Flp in Diverse Neurons [00192] The inventor sought to determine whether Tagged cargo delivery was feasible across multiple neuronal populations. The Cre-on AAV Tagged Flp particles from EXAMPLE 2 were used, and these particles were injected into target brain regions of different strains of mice that expressed Cre in discrete specific starter populations. All mice also expressed thee Flp-On enhanced Green Fluorescent Protein (GFP) reporter allele. The Cre driver mouse strains utilized are shown in TABLE 1.

TABLE 1: Cre driver mouse strains

[00193] Mice underwent isoflurane based anesthesia and lidocaine/ketoprofen analgesia for a stereotaxic injection of 200 nanoliters to 1 microliter of AAV Tagged Flp serotype 1 into designated brain regions.

[00194] After 2-4 weeks, mice underwent Histology as described in EXAMPLE 1. Successful delivery of the Cre-on AAV Tagged Flp vectors was validated by the presence of RFP protein positive starter population neurons. Potential evidence of transneuronal spread of the AAV Tagged Flp construct was assayed by evaluating the presence of GFP labeling outside of the starter population.

[00195] The results indicate that the Cre-On AAV mediated expression of Tagged Flp enables the transneuronal trafficking and activity of Flp in the anterograde cell population. The absence of RFP in the anterograde cell population suggests that it is the designed transneuronal spread of the polypeptide Tagged Flp cargo, and not of the AAV viral vector itself, responsible for biologic action. SST Cre mice have RFP+ labelling of starter SST interneurons in the hilar region of the dentate gyrus and GFP+ labeling in canonical anterograde targets including granule cells and hilar mossy cells (FIG. 3A). VIP Cre mice have labeling of RFP+ dentate granule neurons and anterograde GFP+ hilar mossy cells, absent canonically retrograde perforant path labelling (FIG. 3B). SST Cre mice have RFP+ cerebellar Golgi cell starter cell labeling and GFP+ canonically anterograde granule and Golgi cells, absent retrograde GFP + mossy fibers (FIG. 3C). Kit Cre mice have RFP+ cerebellar cortex Molecular Layer Interneurons and GFP+ canonically anterograde Purkinje cells, without GFP+ retrograde granule cells (FIG. 3D). DocklO Cre mice show RFP+ labeling of starter dentate gyrus granule cells, and canonical anterograde GFP+ CA3 pyramidal neurons, absent retrograde GFP+ Entorhinal Cortex neurons (FIG. 3E). Cell nuclei per se are illustrated by DAPI labeling (shown in blue), to illustrate the region of tissue in which retrograde neurons would have been labeled GFP if they were present.

EXAMPLE 4: Gamma-Retroviral Vectors to Express Tagged Cre

[00196] The inventor sought to determine whether tagged cargoes could undergo transneuronal delivery when expressed via mechanisms beyond AAVs. Thus, the action of Control Cre vs Tagged Cre constructs was compared as delivered by replication defective gamma-retroviral particles.

[00197] Control Cre Retrovirus was generated from a previously existing publicly available plasmid, pRubiG-T2A-Cre (Addgene Plasmid #66693), which expressed GFP separately from Cre recombinase via the T2A motif. The Retrovirus Control sequence is SEQ ID NO: 81, which encodes SEQ ID NO: 82. The Retrovirus Control sequence consists of a fluorescent protein (SEQ ID NO: 67, which encodes SEQ ID NO: 68), T2A (SEQ ID NO: 69, which encodes SEQ ID NO: 70), an intervening sequence (agtac, which encodes ST), and Cre (SEQ ID NO: 79, which encodes SEQ ID NO: 80). Refer to FIG. 9 that includes a schematic of the Retrovirus Control Cre construct. [00198] The design of the Tagged Cre Retrovirus was similar, and it also placed a Tag N- terminal to Cre, which was accomplished through Gibson/NEB HiFi assembly. The Tag design consisted of a fluorescent protein (SEQ ID NO: 67, which encodes SEQ ID NO: 68), T2A (SEQ ID NO: 69, which encodes SEQ ID NO: 70), a synapse targeting domain (VAMP2) (SEQ ID NO: 25, which encodes SEQ ID NO: 26), a first linker (SEQ ID NO: 43, which encodes SEQ ID NO: 44), a cleavage domain (SEQ ID NO: 71, which encodes SEQ ID NO: 72), a second linker (ggc, which encodes G), an entry domain, consisting of two parts (Poly-Histidine and TAT Cell penetrating peptide) (part 1: SEQ ID NO: 47, which encodes SEQ ID NO: 48; part 2: SEQ ID NO: 51, which encodes SEQ ID NO: 52), an intervening sequence (SEQ ID NO: 49, which encodes SEQ ID NO: 50), and a localization domain (Nuclear Localization Sequence) (SEQ ID NO: 53, which encodes SEQ ID NO: 54), fused to Cre recombinase (SEQ ID NO: 73, which encodes SEQ ID NO: 74). The sequences for (VAMP2) and for (Poly-Histidine Tag, TAT, and NLS) were from previously existing publicly available plasmids. The multi-domain polypeptide “tag” comprises SEQ ID NO: 77, which encodes SEQ ID NO: 78. Thus, the whole Retrovirus Tagged Cre sequence comprises SEQ ID NO: 75, which encodes SEQ ID NO: 76. Refer to FIG. 9 that includes a schematic of the Retrovirus Tagged Cre construct. These retroviral plasmids were packaged into replication defective retroviral particles pseudotyped with the VSV-G protein.

[00199] Seven day old mice of the Ai9 (B6.Cg-Gt(ROSA)26So/^m9fCAG-^fdr°^mato)Hz7J) strain underwent isoflurane based anesthesia and lidocaine/ketoprofen analgesia for a stereotaxic injection of 2 microliter Control Cre Retrovirus or Tagged Cre Retrovirus into the hippocampal formation, targeting the dentate gyrus, to infect postnatally generated dentate gyrus granule cells. [00200] After 2.5 weeks, mice underwent Histology as described in EXAMPLE 1. Successful delivery of the Retrovirus Control Cre was assayed by both the presence of GFP, and by the induction of the RFP in dentate gyrus granule cells. The extent of transneuronal Cre activity was then determined by the induction of RFP in other neuronal populations.

[00201] The results indicate that expression of the Retrovirus Tagged Cre leads to transneuronal Cre activity in the anterograde CA3 cell population, as indicated by RFP induction. The absence of GFP expression in the CA3 population indicates that is the transneuronal delivery of the polypeptide tagged Cre, and not of the retrovirus itself, that is responsible for biologic action. Retrovirus Control Cre leads to the presence of GFP, and the robust induction of the RFP expression in dentate gyrus granule cells, without Cre activity in other neuronal cell populations. In contrast, while Retrovirus Tagged Cre leads to the presence of GFP, and the robust induction of the RFP expression in dentate gyrus granule cells, it also leads to Cre activity /RFP expression in the CA3, without CA3 GFP (FIG. 4).

TABLE 2: Sequence Listing

Claims

WHAT IS CLAIMED IS:

1. A multi-domain polypeptide comprising: one or more synapse targeting domain; one or more cleavage domain; and one or more entry domain, wherein each domain is covalently linked.

2. The multi-domain polypeptide of claim 1, wherein the multi-domain polypeptide further comprises one or more localization domain.

3. The multi-domain polypeptide of claim 1 or claim 2, further comprising one or more linker, such as a flexible linker, rigid linker, cleavable linker, or a combination thereof, connecting at least two of the domains.

4. The multi-domain polypeptide of any one of the previous claims, wherein the one or more synapse targeting domain comprises one or more ion channel sequence, neurotransmitter sequence, neuromodulator receptor sequence, neurotransmission-involved protein sequence, neuronal cell adhesion molecule sequence, interacting protein sequence, synthetic binding protein sequence, or a combination thereof.

5. The multi-domain polypeptide of any one of the previous claims, wherein the one or more cleavage domain comprises one or more inherent cleavage site, endogenous cleavage enzyme sequence, exogenous cleavage enzyme sequence, or a combination thereof.

6. The multi-domain polypeptide of any one of the previous claims, wherein the one or more entry domain comprises one or more secretion signal sequence, cell penetrating peptide (CPP) sequence, non-CPP entry sequence, viral protein sequence, glycan or proteoglycan binding sequence, extracellular matrix (ECM) component sequence, or a combination thereof.

7. The multi-domain polypeptide of any one of claims 2-6, wherein the one or more localization domain comprises one or more nuclear localization signal sequence, organelle trafficking sequence, specific protein affinity sequence, spatiotemporal control sequence, or a combination thereof.

8. The multi-domain polypeptide of any one of the previous claims, wherein the one or more synapse targeting domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 2, 16, 26, or a combination thereof.

9. The multi-domain polypeptide of any one of the previous claims, wherein the one or more cleavage domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 8, 12, 46, 72, 86, or a combination thereof.

10. The multi-domain polypeptide of any one of the previous claims, wherein the one or more entry domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 4, 48, 52, or a combination thereof.

11. The multi-domain polypeptide of any one claims 2-10, wherein the one or more localization domain comprises an amino acid sequence at least 85% identical to SEQ ID NO: 34, 54, 62, or a combination thereof.

12. The multi-domain polypeptide of any one of the previous claims, wherein the multidomain polypeptide comprises an amino acid sequence at least 85% identical to SEQ ID NO: 32, 60, or 78.

13. The multi-domain polypeptide of any one of claims 1-12, for use in delivering one or more cargo to one or more target cell.

14. A complex comprising the multi-domain polypeptide of any one of the previous claims covalently linked to one or more cargo.

15. The complex of claim 14, wherein the cargo is covalently linked to the N-terminal or C- terminal of the multi-domain polypeptide.

16. The complex of claim 14 or 15, wherein the cargo comprises one or more therapeutic agent and/or research tool.

17. The complex of claim 16, wherein the therapeutic agent and/or research tool comprises one or more gene sequence or gene expression modifier, antibody, Fc-fusion, peptide, protein, recombinase, Tet-off transactivator (tTA), peroxidase, biotin ligase, agonist, antagonist, toxin, or a combination thereof.

18. The complex of any one of claims 14-17, for use in delivering the cargo to one or more target cell.

19. A polynucleotide molecule encoding the multi-domain polypeptide of any one of claims 1-13.

20. A vector comprising the polynucleotide molecule of claim 19.

21. A host cell comprising the multi-domain polypeptide of any one of claims 1-13, the complex of any one of claims 14-18, the polynucleotide molecule of claim 19, or the vector of claim 20.

22. The host cell of claim 21, wherein the host cell is a bacterial cell, a cyanobacterium, a green-sulfur bacterium, a green non-sulfur bacterium, a purple sulfur bacterium, a purple non-sulfur bacterium, an extremophile, a yeast, algae, a fungus, an engineered organism thereof, or a synthetic organism.

23. The host cell of claim 21 or claim 22, wherein expression of the multi-domain polypeptide confers delivery of one or more cargo to one or more target cell.

24. The host cell of claim 23, wherein the target cell is a nervous system cell, such as a neuron or non-neuronal cell in synaptic proximity.

25. The host cell of claim 23, wherein the target cell is a cell outside of the nervous system, such as a muscle, heart, or lung cell.

26. A pharmaceutical composition comprising the multi-domain polypeptide of any one of claims 1-13, the complex of any one of claims 14-18, the polynucleotide molecule of claim 19, or the vector of claim 20 and a pharmaceutically acceptable carrier.

27. A method for preparing the multi-domain polypeptide of any one of claims 1-13, comprising concatenating the synapse targeting, cleavage, and entry domains.

28. The method of claim 27, further comprising concatenating the localization domain with the synapse targeting, cleavage, and entry domains.

29. The method of claim 27 or 28, wherein the domains are concatenated using one or more linker.

30. A method of delivering cargo to one or more target cell in a subject, comprising administering the multi-domain polypeptide of claims 1-13, the complex of any one of claims 14-18, the polynucleotide molecule of claim 19, or the vector of claim 20 to the subject.

31. The method of claim 30, wherein the cargo comprises one or more therapeutic agent and/or research tool.

32. The method of claim 31, wherein the therapeutic agent and/or research tool comprises one or more gene sequence modifier, gene expression modifier, antibody, Fc-fusion, peptide, protein, recombinase, Tet-off transactivator (tTA), peroxidase, biotin ligase, agonist, antagonist, toxin, or a combination thereof.

33. The method of any one of claims 30-32, wherein the target cell is a nervous system cell, such as a neuron or non-neuronal cell in synaptic proximity.

34. The method of any one of claims 30-32, wherein the target cell is a cell outside of the nervous system, such as a muscle, heart, or lung cell.

35. The method of any one of claims 30-34, wherein the cargo is delivered via axonal transport, such as anterograde or retrograde transport.

36. A complex comprising: a means for targeting a neuron synapse; a means for cleavage of one or more protein domains; a means for entering one or more target cell; and one or more cargo.

37. The complex of claim 36, further comprising a means for localization to one or more target cell.