WO2025165878A1 - Methods of delivering therapeutic agents to target tissues and organs - Google Patents

Abstract

The disclosure relates generally to compositions and methods for delivery of an agent, e.g., a therapeutic agent, such as a nucleic acid, to a target tissue or organ. More particularly, the disclosure relates to the use of plant viral capsid proteins, for example, protein complexes comprising plant viral capsid proteins, for delivering therapeutic agents, such as nucleic acids, to target tissues and organs.

Description

METHODS OF DELIVERING THERAPEUTIC AGENTS TO

TARGET TISSUES AND ORGANS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63/626,413, filed January 29, 2024, which is hereby incorporated by reference in its entirety for all purposes.

FIELD OF THE INVENTION

[0002] The disclosure relates generally to compositions and methods for delivery of an agent, e.g., a therapeutic agent, such as a nucleic acid, to a target tissue or organ. More particularly, the disclosure relates to the use of plant viral capsid proteins, for example, protein complexes comprising plant viral capsid proteins, for delivering therapeutic agents, such as nucleic acids, to target tissues and organs.

BACKGROUND

[0003] Targeted delivery’ of therapeutic agents to a specific tissue and/or organ (e.g.. a diseased tissue or organ) is desirable as it can improve therapeutic efficacy and reduce off- target and/or toxic effects. However, the development of therapeutic modalities that are capable of effectively delivering therapeutic agents, such as nucleic acids, to a particular cell or tissue type is an ongoing challenge in the field. For example, delivery of therapeutic nucleic acids can be particularly challenging, in part due to their high molecular weight and strong negative charge, which prevents passive diffusion across cellular membranes. Further, nucleic acids are sensitive to degradation by nucleases.

[0004] Current strategies for delivery of therapeutic agents include the use of human viruses (e.g., adenoviruses) and nanoparticles (e.g., lipid nanoparticles (LNPs)), which may be guided to a particular tissue by local administration and combining with antibodies. However, therapeutic viral vector infection can activate a wide variety of immune responses both humoral and cellular, which may increase the risk of acute toxicity among other adverse effects. Pro-inflammatory concerns have also been raised with nanoparticle delivery modalities, such as LNPs. [0005] Despite existing delivery methods, there is a need in the art for compositions and methods to deliver therapeutic agents, such as nucleic acids, to specific tissues and organs.

SUMMARY OF THE INVENTION

[0006] The present disclosure is based, in part, on the discovery that agents, e.g., therapeutic agents, such as nucleic acids, can be delivered to specific tissues and/or organs using a complex comprising a plant viral capsid protein, or a fragment thereof.

[0007] Accordingly, in one aspect, the disclosure relates to a method of delivering a therapeutic agent to a specific target tissue or organ with an therapeutic composition in a patient in need thereof. The method includes orally or intravenously administering to the patient a therapeutically effective amount of a therapeutic composition comprising a complex comprising a plant viral capsid protein (e.g., an isolated plant viral capsid protein) or fragment thereof and a therapeutic agent, and a pharmaceutically acceptable excipient, thereby delivering the therapeutic agent to the patient's specific target tissue or organ.

[0008] In another aspect, the disclosure relates to a method of delivering a nucleic acid to a specific target tissue or organ with a therapeutic composition in a patient in need thereof. The method includes administering to the patient a therapeutically effective amount of a therapeutic composition comprising a complex comprising a plant viral capsid protein (e g., an isolated plant viral capsid protein) or fragment thereof and a nucleic acid, and a pharmaceutically acceptable excipient, thereby delivering the nucleic acid to the patient’s specific target tissue or organ, wherein the nucleic acid can be expressed in the specific target tissue or organ. In certain embodiments, where a nucleic acid is a non-coding RNA, the nucleic acid is not expressed.

[0009] In another aspect, the disclosure relates to a method of delivering a therapeutic agent to a specific target tissue or organ with a therapeutic composition in a patient in need thereof. The method includes administering to the patient a therapeutically effective amount of a therapeutic composition comprising a complex comprising a plant viral capsid protein (e g., an isolated plant viral capsid protein) or fragment thereof and a therapeutic agent, and a pharmaceutically acceptable excipient, thereby delivering the therapeutic agent to the patient’s specific target tissue or organ, wherein the specific target tissue is selected from the group consisting of adipose, adrenal, artery, blood, brain, breast, cervix, colon, esophagus, heart, kidney, liver, lung, muscle, nerve, ovary, pancreas, pituitary, prostate, salivary gland, small intestine, spleen, stomach, testis, thyroid, and uterus.

[0010] In another aspect, the disclosure relates to a method of delivering a therapeutic agent to a specific target tissue or organ with a therapeutic composition in a patient in need thereof. The method includes administering to the patient a therapeutically effective amount of a therapeutic composition comprising a complex comprising a plant viral capsid protein (<?.g., an isolated plant viral capsid protein) or fragment thereof and a therapeutic agent, and a pharmaceutically acceptable excipient, wherein the viral capsid protein or fragment thereof is a Bromoviridae, Alphaflexiviridae, Virgaviridae, Potyviridae, Solemoviridae, Closteroviridae, or Betaflexiviridae capsid protein or fragment thereof

[0011] In certain embodiments, the plant viral capsid protein or fragment thereof is an Alfamovirus, Anulavirus, Bromovirus, Cucumovirus, liarvirus, or Oleavirus capsid protein or fragment thereof. In certain embodiments, the plant viral capsid protein or fragment thereof is an alfalfa mosaic virus capsid protein or fragment thereof, a brome mosaic virus capsid protein or fragment thereof, a lilac leaf chlorosis virus capsid protein or fragment thereof, an apple necrotic mosaic virus capsid protein or fragment thereof, or a cowpea chlorotic mottle virus capsid protein or fragment thereof.

[0012] In certain embodiments, the plant viral capsid protein or fragment thereof is an Allexivirus, Botrexvirus, Lolavirus, Platypuvirus, Potexvirus, or Sclerodarnavirus capsid protein or fragment thereof. In certain embodiments, the plant viral capsid protein or fragment thereof is a clover yellow mosaic virus capsid protein or fragment thereof, a pepino mosaic virus capsid protein or fragment thereof, or a white clover mosaic virus capsid protein or fragment thereof.

[0013] In certain embodiments, the plant viral capsid protein or fragment thereof is a Furovirus, Goravirus, Hordeivirus, Pecluvirus, Pomovirus, Tobanwvirus. or Tobravirus viral capsid protein or fragment thereof. In certain embodiments, the plant viral capsid protein or fragment thereof is a cucumber green mottle mosaic virus capsid protein or fragment thereof, a tobacco mild green mosaic virus capsid protein or fragment thereof, a tobacco mosaic virus capsid protein or fragment thereof, a tomato mosaic virus capsid protein or fragment thereof, or a tropical soda apple mosaic virus capsid protein or fragment thereof. [0014] In certain embodiments, the plant viral capsid protein or fragment thereof is an Arepavirus, Bevemovirus, Brambyvirus, Bymovirus. Celavirus, Ipomovirus, Macluravirus , Poacevirus. Potyvirus, Roymovirus, Rymovirus, or Tritimovirus capsid protein or fragment thereof. In certain embodiments, the plant viral capsid protein or fragment thereof is a pepper mild mottle virus capsid protein or fragment thereof or a potato virus Y capsid protein or fragment thereof.

[0015] In certain embodiments, the plant viral capsid protein or fragment thereof is an Enamovirus, Polemovirus, Polerovirus, or Sobemovirus capsid protein or fragment thereof. In certain embodiments, the plant viral capsid protein or fragment thereof is a poinsettia latent virus capsid protein or fragment thereof.

[0016] In certain embodiments, the plant viral capsid protein or fragment thereof is a Carlavirus, Foveavirus, Robigovirus, Capillovirus , Chordovirus, Citrivirus, Divavirus. Prunevirus, Ravavirus. Tepovirus, Trichovirus, Vitivirus, or Wamavirus capsid protein or fragment thereof. In certain embodiments, the plant viral capsid protein or fragment thereof is an apple stem pitting virus capsid protein or fragment thereof.

[0017] In certain embodiments, the plant viral capsid protein or fragment thereof is a Ampelovirus, Closterovirus, Crinivirus, or Velarivirus capsid protein or fragment thereof. In certain embodiments, the plant viral capsid protein or fragment thereof is a citrus tristeza virus capsid protein or fragment thereof.

[0018] In certain embodiments, the plant viral capsid protein or fragment thereof is a modified plant viral capsid protein. In certain embodiments, the plant viral capsid protein or fragment thereof is naturally occurring.

[0019] In certain embodiments, an amino acid sequence of the plant viral capsid protein or fragment thereof is at least 90% identical to a naturally occurring plant viral capsid protein or fragment thereof.

[0020] In certain embodiments, the fragment of the plant viral capsid protein comprises at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, or at least 450 consecutive (i.e., contiguous) amino acids present in a corresponding full- length plant viral capsid protein. [0021] In certain embodiments, the therapeutic agent is a non-naturally occurring nucleic acid. In certain embodiments, the therapeutic agent is a heterologous nucleic acid. In certain embodiments, the non-naturally occurring or heterologous nucleic acid is a DNA or an RNA.

[0022] In certain embodiments, the non-naturally occurring or heterologous nucleic acid is expressed in the specific target tissue or organ. In certain embodiments, the specific target tissue is selected from the group consisting of adipose, adrenal, artery, blood, brain, breast, cervix, colon, esophagus, heart, kidney, liver, lung, muscle, nerve, ovary, pancreas, pituitary, prostate, salivary' gland, small intestine, spleen, stomach, testis, thyroid, and uterus tissue.

[0023] In another aspect, the disclosure relates to a composition comprising a plant viral capsid protein (e.g., an isolated plant viral capsid protein) or fragment thereof and a heterologous therapeutic agent, and wherein the viral capsid protein or fragment thereof is a Bromoviridae, Alphaflexiviridae, Virgaviridae, Potyviridae. Solemoviridae, or Betaflexiviridcie capsid protein or fragment thereof.

[0024] In certain embodiments, the heterologous therapeutic agent is a heterologous nucleic acid. In certain embodiments, the heterologous nucleic acid is a DNA or an RNA. In certain embodiments, the heterologous nucleic acid is an RNA.

[0025] In certain embodiments, an amino acid sequence of the plant viral capsid protein or fragment thereof is at least 90% identical to a naturally occurring plant viral capsid protein or fragment thereof.

[0026] In certain embodiments, the fragment of the plant viral capsid protein comprises at least 50, at least 100, at least 150, at least 200. at least 250, at least 300, at least 350, at least 400, or at least 450 consecutive amino acids present in a corresponding full-length plant viral capsid protein.

[0027] In certain embodiments, the plant viral capsid protein or fragment thereof is an Alfamovirus, Anulavirus. Bromovirus, Cucumovirus, Ilarvirus, or Oleavirus capsid protein or fragment thereof. In certain embodiments, the plant viral capsid protein or fragment thereof is an alfalfa mosaic vims capsid protein or fragment thereof, a brome mosaic virus capsid protein or fragment thereof, a lilac leaf chlorosis virus capsid protein or fragment thereof, an apple necrotic mosaic virus capsid protein or fragment thereof, or a cowpea chlorotic mottle vims capsid protein or fragment thereof. [0028] In certain embodiments, the plant viral capsid protein or fragment thereof is an Allexivirus. Botrexvirus, Lolavirus, Platypuvirus, Potexvirus, or Sclerodarnavirus plant viral capsid protein or fragment thereof. In certain embodiments, the plant viral capsid protein or fragment thereof is a clover yellow mosaic virus capsid protein or fragment thereof, a pepino mosaic virus capsid protein or fragment thereof, or a white clover mosaic virus capsid protein or fragment thereof.

[0029] In certain embodiments, the plant viral capsid protein or fragment thereof is a Furovirus, Goravirus, Hordeivirus, Pecluvirus, Pomovirus, Tobamovirus, or Tobravirus capsid protein or fragment thereof. In certain embodiments, the plant viral capsid protein or fragment thereof is a cucumber green mottle mosaic virus capsid protein or fragment thereof, a tobacco mild green mosaic virus capsid protein or fragment thereof, a tobacco mosaic virus capsid protein or fragment thereof, a tomato mosaic virus capsid protein or fragment thereof, or a tropical soda apple mosaic virus capsid protein or fragment thereof.

[0030] In certain embodiments, the plant viral capsid protein or fragment thereof is an Arepavirus, Bevemovirus, Brambyvirus, Bymovirus, Celavirus, Ipomovirus, Macluravirus , Poacevirus, Potyvirus, Roymovirus, Rymovirus, or Tritimovirus capsid protein or fragment thereof. In certain embodiments, the plant viral capsid protein or fragment thereof is a pepper mild mottle virus capsid protein or fragment thereof or a potato virus Y capsid protein or fragment thereof.

[0031] In certain embodiments, the plant viral capsid protein or fragment thereof is an Enamovirus, Polemovirus. Polerovirus, or Sobemovirus plant viral capsid protein or fragment thereof In certain embodiments, the plant viral capsid protein or fragment thereof is a poinsettia latent vims capsid protein or fragment thereof.

[0032] In certain embodiments, the plant viral capsid protein or fragment thereof is a Carlavirus, Foveavirus, Robigovirus, Capillovirus, Chordovirus, Citrivirus, Divavirus, Prunevirus, Ravavirus, Tepovirus, Trichovirus, Vitivirus, or Wamavirus plant viral capsid protein or fragment thereof. In certain embodiments, the plant viral capsid protein or fragment thereof is an apple stem pitting vims capsid protein or fragment thereof.

[0033] In another aspect, the disclosure relates to a pharmaceutical composition comprising any of the foregoing compositions and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is suitable for oral delivery or intravenous delivery. [0034] In another aspect, the disclosure relates to a nucleic acid or nucleic acids encoding any of the foregoing compositions.

[0035] In another aspect, the disclosure relates to a host cell comprising any of the foregoing compositions or nucleic acids. In certain embodiments, the host cell is a plant cell.

[0036] In another aspect, the disclosure relates to a method of delivering a therapeutic agent to a specific target tissue or organ in a patient. The method includes administering to the patient a therapeutically effective amount of a therapeutic composition comprising a complex comprising an Alfamovirus capsid protein or a fragment thereof and the therapeutic agent; and, optionally, a pharmaceutically acceptable excipient, thereby delivering the therapeutic agent to the patient’s specific target tissue or organ, wherein the specific target tissue or organ is selected from brain, prostate, esophagus, small intestine, breast, salivary gland, nerve, thyroid, adrenal, adipose, lung, muscle, heart, colon, artery, blood, liver, stomach, testis, pancreas, and ovary.

[0037] In certain embodiments, the Alfamovirus capsid protein or fragment thereof is an alfalfa mosaic virus capsid protein or fragment thereof.

[0038] In certain embodiments, the therapeutic agent is a heterologous nucleic acid. In certain embodiments, the heterologous nucleic acid is DNA or RNA.

[0039] In certain embodiments, an amino acid sequence of the Alfamovirus capsid protein or fragment thereof is at least 90% identical to a naturally occurring A Ifamovirus capsid protein or fragment thereof.

[0040] In certain embodiments, the fragment of the Alfamovirus capsid protein comprises at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, or at least 450 consecutive amino acids present in a corresponding full-length Alfamovirus capsid protein.

[0041] In another aspect, the disclosure relates to a method of delivering a therapeutic agent to a pituitary tissue in a patient. The method includes administering to the patient a therapeutically effective amount of a therapeutic composition comprising a complex comprising an Foveavirus capsid protein or a fragment thereof and the therapeutic agent; and, optionally, a pharmaceutically acceptable excipient, thereby delivering the therapeutic agent to the patient's pituitary tissue.

[0042] In certain embodiments, the Foveavirus capsid protein or a fragment thereof is an apple stem pitting virus capsid protein or fragment thereof.

[0043] In certain embodiments, the therapeutic agent is a heterologous nucleic acid. In certain embodiments, the heterologous nucleic acid is DNA or RNA.

[0044] In certain embodiments, an amino acid sequence of the Foveavirus capsid protein or fragment thereof is at least 90% identical to a naturally occurring Foveavirus capsid protein or fragment thereof.

[0045] In certain embodiments, the fragment of the Foveavirus capsid protein comprises at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, or at least 450 consecutive amino acids present in a corresponding full-length Foveavirus capsid protein.

[0046] In another aspect, the disclosure relates to a method of delivering a therapeutic agent to a specific tissue or organ in a patient. The method includes administering to the patient a therapeutically effective amount of a therapeutic composition comprising a complex comprising a Bromovirus capsid protein or a fragment thereof and the therapeutic agent; and, optionally, a pharmaceutically acceptable excipient, thereby delivering the therapeutic agent to the patient’s ovary, prostate, brain, breast, adipose, nerve, artery, small intestine, thyroid, blood, colon, lung, liver, muscle, esophagus, adrenal, or uterus tissue.

[0047] In certain embodiments, the Bromovirus capsid protein or a fragment thereof is a brome mosaic virus capsid protein or fragment thereof, a lilac leaf chlorosis virus capsid protein or fragment thereof, or a cowpea chlorotic mottle virus capsid protein or fragment thereof.

[0048] In certain embodiments, the therapeutic agent is a heterologous nucleic acid. In certain embodiments, the heterologous nucleic acid is DNA or RNA.

[0049] In certain embodiments, an amino acid sequence of the Bromovirus capsid protein or fragment thereof is at least 90% identical to a naturally occurring Bromovirus capsid protein or fragment thereof. [0050] In certain embodiments, the fragment of the Bromovirus capsid protein comprises at least 50, at least 100. at least 150, at least 200. at least 250, at least 300, at least 350, at least 400, or at least 450 consecutive amino acids present in a corresponding full-length Bromovirus capsid protein.

[0051] In another aspect, the disclosure relates to a method of delivering a therapeutic agent to a specific tissue or organ in a patient. The method includes administering to the patient a therapeutically effective amount of a therapeutic composition comprising a complex comprising a Potexvirus capsid protein or a fragment thereof and the therapeutic agent; and, optionally, a pharmaceutically acceptable excipient, thereby delivering the therapeutic agent to the patient's artery, blood, brain, esophagus, adipose, stomach, breast, adrenal, thyroid, cervix, small intestine, spleen, colon, testis, ovary, nerve, muscle, heart.

[0052] In certain embodiments, the Potexvirus capsid protein or fragment thereof is a clover yellow mosaic virus capsid protein or fragment thereof, a white clover mosaic virus capsid protein or fragment thereof, or a pepino mosaic virus capsid protein or fragment thereof.

[0053] In certain embodiments, the therapeutic agent is a heterologous nucleic acid. In certain embodiments, the heterologous nucleic acid is DNA or RNA.

[0054] In certain embodiments, an amino acid sequence of the Potexvirus capsid protein or fragment thereof is at least 90% identical to a naturally occurring Potexvirus capsid protein or fragment thereof.

[0055] In certain embodiments, the fragment of the Potexvirus capsid protein comprises at least 50, at least 100, at least 150, at least 200. at least 250, at least 300, at least 350, at least 400, or at least 450 consecutive amino acids present in a corresponding full-length Potexvirus capsid protein.

[0056] In another aspect, the disclosure relates to a method of delivering a therapeutic agent to a specific tissue or organ in a patient. The method includes administering to the patient a therapeutically effective amount of a therapeutic composition comprising a complex comprising a Tobamovirus capsid protein or a fragment thereof and the therapeutic agent; and, optionally, a pharmaceutically acceptable excipient, thereby delivering the therapeutic agent to the patient’s specific tissue or organ, wherein the specific tissue or organ is artery. brain, adipose, lung, blood, nerve, breast, esophagus, muscle, thyroid, testis, kidney, pituitary', prostate, liver, stomach, adrenal, heart, uterus, colon, small intestine, ovary, pancreas, spleen.

[0057] In certain embodiments, the Tobamovirus capsid protein or fragment thereof is a tobacco mild green mosaic virus capsid protein or fragment thereof, a tobacco mosaic virus capsid protein or fragment thereof, a tomato mosaic virus capsid protein or fragment thereof, a tropical soda apple mosaic virus capsid protein or fragment thereof, a pepper mild mottle virus capsid protein or fragment thereof, or a cucumber green mottle mosaic virus capsid protein or fragment thereof.

[0058] In certain embodiments, the therapeutic agent is a heterologous nucleic acid. In certain embodiments, the heterologous nucleic acid is DNA or RNA.

[0059] In certain embodiments, an amino acid sequence of the Tobamovirus capsid protein or fragment thereof is at least 90% identical to a naturally occurring Tobamovirus capsid protein or fragment thereof.

[0060] In certain embodiments, the fragment of the Tobamovirus capsid protein comprises at least 50, at least 100. at least 150, at least 200. at least 250, at least 300, at least 350, at least 400, or at least 450 consecutive amino acids present in a corresponding full-length Tobamovirus capsid protein.

[0061] In another aspect, the disclosure relates to a method of delivering a therapeutic agent to a heart tissue in a patient. The method includes administering to the patient a therapeutically effective amount of a therapeutic composition comprising a complex comprising a Polemovirus capsid protein or a fragment thereof and the therapeutic agent; and, optionally, a pharmaceutically acceptable excipient, thereby delivering the therapeutic agent to the patient’s heart tissue.

[0062] In certain embodiments, the Polemovirus capsid protein or fragment thereof is a poinsettia latent virus capsid protein or fragment thereof.

[0063] In certain embodiments, the therapeutic agent is a heterologous nucleic acid. In certain embodiments, the heterologous nucleic acid is DNA or RNA. [0064] In certain embodiments, an amino acid sequence of the Polemovirus capsid protein or fragment thereof is at least 90% identical to a naturally occurring Polemovirus capsid protein or fragment thereof.

[0065] In certain embodiments, the fragment of the Polemovirus capsid protein comprises at least 50, at least 100. at least 150, at least 200. at least 250, at least 300, at least 350, at least 400, or at least 450 consecutive amino acids present in a corresponding full-length Polemovirus capsid protein.

[0066] In another aspect, the disclosure relates to a method of delivering a therapeutic agent to an artery (e.g.. a coronary artery) tissue in a patient. The method includes administering to the patient a therapeutically effective amount of a therapeutic composition comprising a complex comprising a Potyvirus capsid protein or a fragment thereof and the therapeutic agent; and, optionally, a pharmaceutically acceptable excipient, thereby delivering the therapeutic agent to the patient’s artery (e.g., coronary) tissue.

[0067] In certain embodiments, the Potyvirus capsid protein or fragment thereof is a potato virus Y virus capsid protein or fragment thereof.

[0068] In certain embodiments, the therapeutic agent is a nucleic acid. In certain embodiments, the nucleic acid is DNA or RNA.

[0069] In certain embodiments, an amino acid sequence of the Potyvirus capsid protein or fragment thereof is at least 90% identical to a naturally occurring Potyvirus capsid protein or fragment thereof.

[0070] In certain embodiments, the fragment of the Potyvirus capsid protein comprises at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, or at least 450 consecutive amino acids present in a corresponding full-length Potyvirus capsid protein.

[0071] In another aspect, the disclosure relates to a method of delivering a therapeutic agent to a specific organ or tissue in a patient. The method includes administering to the patient a therapeutically effective amount of a therapeutic composition comprising a complex comprising a Closterovirus capsid protein or a fragment thereof and the therapeutic agent; and, optionally, a pharmaceutically acceptable excipient; thereby delivering the therapeutic agent to the patient’s specific tissue or organ, wherein the specific tissue or organ is adipose, breast, artery, or nen e tissue.

[0072] In certain embodiments, the Closterovirus capsid protein or fragment thereof is a citrus tristeza virus capsid protein or fragment thereof.

[0073] In certain embodiments, the therapeutic agent is a heterologous nucleic acid. In certain embodiments, the heterologous nucleic acid is DNA or RNA.

[0074] In certain embodiments, an amino acid sequence of the Closterovirus capsid protein or fragment thereof is at least 90% identical to a naturally occurring Closterovirus capsid protein or fragment thereof.

[0075] In certain embodiments, the fragment of the Closterovirus capsid protein comprises at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, or at least 450 consecutive amino acids present in a corresponding full-length Closterovirus capsid protein.

[0076] In certain embodiments, the therapeutic composition is orally administered to the patient.

[0077] In certain embodiments, the therapeutic composition is intravenously administered to the patient.

[0078] These and other aspects and features of the invention are described in the following detailed description and claims.

DETAILED DESCRIPTION

[0079] The present disclosure provides, among other things, plant viral capsid proteins (e g., any plant viral capsid protein described herein), or fragments thereof, which assemble into protein complexes (e.g., particles) comprising said plant viral capsid proteins, or fragments thereof. The complexes comprising the plant viral capsid proteins or fragments thereof are capable of delivering, or directing the delivery of, an agent (e.g., a therapeutic agent) to a specific cell or tissue and/or organ. In many embodiments, the present disclosure provides a protein complex (e.g., a particle) comprising a plant viral capsid protein, or a fragment thereof, and an agent, where the protein complex delivers the agent to a specific tissue and/or organ based at least in part by the tropism of the protein complex and/or the plant viral capsid protein. Accordingly, the present disclosure provides, among other things, compositions and methods for the targeted delivery of an agent, such as a therapeutic agent, to a specific tissue and/or organ in a patient with a disease or disorder.

[0080] The protein complex of the present disclosure can be or comprise a naturally occurring plant virus capsid or an engineered plant virus capsid (e.g., a modified plant virus capsid). In some embodiments, a protein complex of the present disclosure is or comprises a virus-like nanoparticle (VLP), e.g., a provided plant viral capsid is used to form a VLP.

[0081] The present disclosure provides compositions, including pharmaceutical compositions, comprising a plant viral capsid (e.g, any plant viral capsid provided herein) and an agent (e.g, a therapeutic agent, or any other agent described herein). The present disclosure further provides compositions, including pharmaceutical compositions, comprising a protein complex comprising a plant viral capsid protein, or a fragment thereof, and an agent, such as a therapeutic agent.

[0082] The present disclosure additionally provides methods of making a composition comprising a protein complex comprising a plant viral capsid protein, or a fragment thereof, and an agent, e.g., a therapeutic agent.

[0083] The present disclosure provides methods of screening targeting specificity of a protein complex comprising plant viral capsid protein, or a fragment thereof, (e.g, a complex comprising a plant viral capsid protein) and a therapeutic agent.

[0084] The present disclosure also provides methods of using a protein complex comprising a plant viral capsid protein, or a fragment thereof, and a therapeutic agent to treat a disease or disorder in a patient or subject. As used herein, the term “therapeutic agent” is also intended to include a prophylactic agent, such as a vaccine.

I. Definitions

[0085] The methods and compositions described herein can be used alone or in combination with other therapeutic agents and/or modalities. The term administered “in combination,” as used herein, is understood to mean that two (or more) different treatments are delivered to the patient during the course of the patient’s affliction with the disorder, such that the effects of the treatments on the patient overlap at a point in time. In certain embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery.” In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In certain embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In certain embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery' can be such that an effect of the first treatment delivered is still detectable when the second is delivered.

[0086] Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps.

[0087] In the application, where an element or component is said to be included in and/or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components.

[0088] Further, it should be understood that elements and/or features of a composition or a method described herein can be combined in a variety of ways without departing from the spirit and scope of the present invention, whether explicit or implicit herein. For example, where reference is made to a particular compound, that compound can be used in various embodiments of compositions of the present invention and/or in methods of the present invention, unless otherwise understood from the context. In other words, within this application, embodiments have been described and depicted in a way that enables a clear and concise application to be written and drawn, but it is intended and will be appreciated that embodiments may be variously combined or separated without parting from the present teachings and invention(s). For example, it will be appreciated that all features described and depicted herein can be applicable to all aspects of the invention(s) described and depicted herein.

[0089] It should be understood that the expression "at least one of includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use. The expression “and/or” in connection with three or more recited objects should be understood to have the same meaning unless otherwise understood from the context.

[0090] The use of the term “include,” “includes,” “including,” “have,” “has,” “having,” “contain,” “contains,” or “containing,” including grammatical equivalents thereof, should be understood generally as open-ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0091] Where the use of the term “about” is before a quantitative value, the present invention also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term “about” refers to a ± 10% variation from the nominal value unless otherwise indicated or inferred.

[0092] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the present invention remain operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0093] The use of any and all examples, or exemplary language herein, for example, “such as” or “including,” is intended merely to illustrate better the present invention and does not pose a limitation on the scope of the invention unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the present invention.

II. Plant Viral Capsids

[0094] The present disclosure provides, among other things, plant viral capsid proteins, or fragments thereof that are capable of directing the delivery⁷ of an agent (e.g., a therapeutic agent) to a specific tissue and/or organ. For example, the plant viral capsid proteins can be isolated or recombinant plant viral capsid proteins, including those assembled into protein complexes, such as capsids, virus-like particles (VLPs), etc.). [0095] The present disclosure encompasses the recognition that plant viruses have specific tissue tropism in tissues and organs (e.g, human tissues and organs). The structure of a plant virus is typically given by its coat of proteins (e.g, one or more types of plant viral capsid proteins, etc.), which surround the viral genome. Without wishing to be bound by theory, it is understood that the viral coat or viral capsid and/or the one or more viral capsid proteins therein, are, at least in part, responsible for observed tissue and/or organ tropism for certain viruses. It is further understood that one or more other proteins (non-capsid proteins) or other molecules associated with a viral capsid or viral capsid protein may, in part, be responsible for observed tissue and/or organ tropism for certain viruses. The present disclosure further encompasses the recognition that a capsid protein (CP) or fragment of a capsid protein from such plant viruses can be isolated, optionally modified, and assembled into a protein complex (e.g, a capsid, or engineered capsid) around an agent (or assembled to otherwise associate with an agent, e.g, on the internal or external surface of the complex), such as a nucleic acid, and administered to a patient, thereby to deliver the therapeutic agent to the specific tissue and/or organ in the patient. Accordingly, the present disclosure relates, in part, to the modification, production, and administration of plant viral capsid proteins (e.g, in the form of protein complexes, such as capsids, VLPs, etc.) carrying a therapeutic agent.

[0096] A plant viral capsid protein used in accordance with the present disclosure can be a naturally occurring plant viral capsid protein or a non-naturally occurring plant virus capsid protein. In some embodiments, a plant viral capsid protein comprises a naturally occurring plant viral capsid protein. In some embodiments, a plant viral capsid protein comprises a naturally occurnng plant viral capsid protein from a family or genus described in TABLE 1. In some embodiments, a plant viral capsid protein comprises a modified plant virus capsid protein. In some embodiments, a modified plant virus capsid protein can be, for example, a plant virus capsid protein having one or more modifications (e.g.. amino acid substitutions, deletions, or insertions) as compared to a naturally occurring plant viral capsid protein (e.g, a naturally occurring capsid protein from a family or genus described in TABLE 1). In certain embodiments, the plant virus capsid protein is from a rod virus, flexuous/filamentous virus, a spherical virus, icosahedral virus, helical virus, filamentous virus, etc., or a virus having a complex viral structure (e.g., oval or brick-shaped).

[0097] In some embodiments, a modified plant virus capsid protein comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more modifications relative to a naturally occurring or wild-type plant viral capsid protein. In some embodiments, a modification comprises an insertion, deletion, or substitution of at least one amino acid residue relative to a naturally occurring or wild-type plant viral capsid protein. In some embodiments, a modification comprises a substitution of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more amino acid residues relative to a naturally occurring or wild-type plant viral capsid protein. In some embodiments, at least one amino acid residue of a modified plant viral capsid protein has been substituted with an unnatural amino acid that allows for conjugation of an agent of interest (e.g. , a therapeutic agent, or any other agent described herein). In some embodiments, a modification comprises fusing or operably linking a viral capsid protein to an agent of interest (e.g., a therapeutic agent, or any other agent described herein), e.g.. to form a fusion protein.

[0098] In some embodiments, a plant viral capsid protein used in accordance with the present disclosure can be aBromoviridae, Alphaflexiviridae, Virgaviridae, Potyviridae, Solemoviridae, Closteroviridae, or Betaflexiviridae capsid protein. In certain embodiments, the plant viral capsid protein is an Alfamovirus, Anulavirus, Bromovirus, Cucumovirus, liarvirus, or Oleavirus capsid protein. In certain embodiments, the plant viral capsid protein is an Allexivirus, Botrexvirus, Lolavirus. Platypuvirus, Potexvirus, or Sclerodarnavirus capsid protein. In certain embodiments, the plant viral capsid protein is a Furovirus, Goravirus, Hordeivirus, Pecluvirus, Pomovirus, Tobamovirus, or Tobravirus capsid protein. In certain embodiments, the plant viral capsid protein is an Arepavirus, Bevemovirus, Brambyvirus. Bymovirus, Celavirus, Ipomovirus, Macluravirus. Poacevirus, Potyvirus, Roymovirus, Rymovirus. or Tritimovirus capsid protein. In certain embodiments, the plant viral capsid protein is an Enamovirus, Polemovirus, Polerovirus, or Sobemovirus capsid protein. In certain embodiments, the plant viral capsid protein is a Carlavirus, Foveavirus, Robigovirus, Capillovirus, Chordovirus, Cilrivirus, Divavirus, Prunevirus, Ravavirus, Tepovirus, Trichovirus, Vitivirus, or Wamavirus capsid protein. In certain embodiments, the plant viral capsid protein is an alfalfa mosaic virus (AMV) capsid protein, a brome mosaic virus (BMV) capsid protein, a lilac leaf chlorosis virus (LLCV) capsid protein, an apple necrotic mosaic virus capsid protein, a cowpea chlorotic mottle virus (CCMV) capsid protein, clover yellow mosaic virus (C1YMV) capsid protein, a pepino mosaic virus (PepMV) capsid protein, a white clover mosaic virus (WCMV) capsid protein, cucumber green mottle mosaic virus (CGMMV) capsid protein, a tobacco mild green mosaic virus (TMGMV) capsid protein, a tobacco mosaic virus (TMV) capsid protein, a tomato mosaic virus (ToMV) capsid protein, a tropical soda apple mosaic virus (TSAMV) capsid protein, pepper mild mottle virus (PMMoV) capsid protein, a potato virus Y (PVY) capsid protein, poinsettia latent virus (PnLV) capsid protein, an apple stem pitting virus (ASPV) plant viral capsid protein, or a citrus tristeza virus capsid protein.

[0099] In certain embodiments, the plant viral capsid protein is selected from a plant viral capsid protein provided in TABLE 1. In certain embodiments, the plant viral capsid protein is a modified capsid protein having one or more modifications as compared to a plant viral capsid protein provided in TABLE 1. In certain embodiments, the plant viral capsid protein is used to deliver an agent as described herein to one or more specific tissues provided in TABLE 1 under the heading “Contemplated Tissue Tropism”.

[00100]

TABLE 1: Exemplary Plant Viral Capsid Proteins

[00101] As used herein, the term “viral capsid protein” also includes a modified viral capsid protein that retains, for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the activity of the corresponding full-length, naturally occurring viral capsid protein. The activity of a viral capsid protein may be assayed by any method known in the art, including, for example, by measuring the ability of the viral capsid protein to assemble in a complex with a therapeutic agent and/or to deliver a therapeutic agent to a specific tissue or organ, as described in the Examples herein.

[00102] In certain embodiments, a viral capsid protein is a modified viral capsid protein comprising an amino acid sequence having at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a naturally occurring viral capsid protein sequence. In certain embodiments, a modified viral capsid protein comprises an amino acid sequence having at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 75%, at least 76%, at least

77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least

84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least

91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least

98%, or at least 99% sequence identity to any one of SEQ ID NOs: 1-25. In certain embodiments, a modified viral capsid protein comprises an amino acid substitution relative to a naturally occurring viral capsid protein sequence provided herein. For example, in certain embodiments, a modified viral capsid protein comprises 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, or up to 10 amino acid substitutions relative to a naturally occurring viral capsid protein sequence (e.g., a naturally occurring viral capsid protein sequence provided herein, such as any one of SEQ ID NOs: 1-25).

[00103] In certain embodiments, the modified viral capsid protein comprises a conservative substitution relative to a naturally occurring viral capsid protein sequence (e.g, a naturally occurring viral capsid protein sequence provided herein). For example, in certain embodiments, the modified viral capsid protein comprises 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, or up to 10 conservative substitutions relative to a viral capsid protein sequence provided herein. As used herein, the term “conservative substitution” refers to a substitution with a structurally similar amino acid. For example, conservative substitutions may include those within the following groups: Ser and Cys; Leu, He, and Vai; Glu and Asp; Lys, Arg, and His; Phe, Tyr, and Trp; and Gin, Asn, Glu, Asp. Conservative substitutions may also be defined by the BLAST (Basic Local Alignment Search Tool) algorithm, the BLOSUM substitution matrix (e.g., BLOSUM 62 matrix), or the PAM substitution^ matrix (e.g., the PAM 250 matrix). In certain embodiments, the viral capsid protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative substitutions.

[00104] As used herein, the phrase “percent identity” and “% identity” refers to the extent to which two sequences e.g., two polypeptides or two nucleic acids have the same respective amino acid or nucleotide at the same positions in an alignment. As used herein, “percent identity” between a polypeptide sequence and a reference sequence is defined as the percentage of amino acid residues in the polypeptide sequence that are identical to the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Similarly, “percent identity” between a nucleic acid sequence and a reference sequence is defined as the percentage of nucleotides in the nucleic acid sequence that are identical to the nucleotides in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity (e.g., nucleic acid sequence identity or amino acid sequence identity) can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST (Basic Local Alignment Search Tool), BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, or MUSCLE software. For a discussion of basic issues in searching sequence databases see Altschul et al., (1994) Nature Genetics 6: 119-129, which is fully incorporated by reference herein. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[00105] The alignment algorithms above may take into account a scoring matrix to calculate an alignment score (see Chao et al., Biomolecules (2022) 12(4): 546). For example, for an amino acid sequence at least 85 amino acids in length, the scoring matrix recommended by the BLAST algorithm is BLOSUM-62. The BLOSUM-62 scoring matrix assigns positive, zero, or negative scores between each pair or standard amino acid residues (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA (1992) 89, 10915-19 at FIG. 2). A positive score between two amino acid residues indicates that substitution of these amino acid residues for each other is conservative. As used herein, “similarity” between a subject amino acid sequence and a reference amino acid sequence refers to the percentage of amino acid residues in the subject amino acid sequence that are identical or have a conservative substitution according to the BLOSUM-62 scoring matrix, relative to the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum sequence alignment score.

[00106] As used herein, the term “fragment” of a viral capsid protein refers to functional fragment of a viral capsid protein (i.e., a polypeptide comprising an amino acid sequence of at least a portion of a viral capsid protein) that retains, for example, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the activity of the corresponding full-length, viral capsid protein. The activity of a viral capsid protein may be assayed by any method known in the art, including, for example, by measuring the ability of the viral capsid protein to assemble in a complex with a therapeutic agent and/or to deliver a therapeutic agent to a specific tissue or organ, as described in the Examples herein.

[00107] In certain embodiments, the viral capsid protein fragment comprises at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, or at least 450 consecutive amino acids present in a viral capsid protein provided herein (e.g., any one of SEQ ID NOs: 1-25). In certain embodiments, the viral capsid protein fragment comprises at least 50 consecutive amino acids of any one of SEQ ID NOs: 1-25. In certain embodiments, the viral capsid protein fragment comprises at least 100 consecutive amino acids of any one of SEQ ID NOs: 1-25. In certain embodiments, the viral capsid protein fragment comprises at least 150 consecutive amino acids of any one of SEQ ID NOs: 1-25. In certain embodiments, the viral capsid protein fragment comprises at least 200 consecutive amino acids of any one of SEQ ID NOs: 1-25. In certain embodiments, the viral capsid protein fragment comprises at least 250 consecutive amino acids of any one of SEQ ID NOs: 1-25. In certain embodiments, the viral capsid protein fragment comprises at least 300 consecutive amino acids of any one of SEQ ID NOs: 1-25. In certain embodiments, the viral capsid protein fragment comprises at least 350 consecutive amino acids of any one of SEQ ID NOs: 1-25. In certain embodiments, the viral capsid protein fragment comprises at least 400 consecutive amino acids of any one of SEQ ID NOs: 1-25. In certain embodiments, the viral capsid protein fragment comprises at least 450 consecutive amino acids of any one of SEQ ID NOs: 1-25.

[00108] In some embodiments, the viral capsid protein fragment comprises a truncation at the N-terminus, the C-terminus, and/or at least one internal site relative to a naturally occurring or wild-type viral capsid protein. In some embodiments, the viral capsid protein fragment comprises a truncation at the N-terminus relative to a naturally occurring or wild-type viral capsid protein. In some embodiments, the viral capsid protein fragment comprises a truncation at the C-terminus relative to a naturally occurring or wild-type viral capsid protein. In some embodiments, the viral capsid protein fragment comprises a truncation at at least one internal site relative to a naturally occurring or wild-type viral capsid protein. In certain embodiments, the functional fragment comprises a truncation of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids as compared to a viral capsid protein provided herein (e.g., a naturally occurring viral capsid protein, such as one of SEQ ID NOs: 1-25).

[00109] In some embodiments, a plant viral capsid protein as described herein may be assembled into a protein complex in the presence of an agent (e.g, any agent described herein) such that the agent becomes associated with the protein complex produced. In some embodiments, the agent is associated at an internal or external surface of a protein complex, e.g, via intermolecular forces. In some embodiments, the agent is associated with an internal or external surface of a protein complex via a covalent interaction (e.g., chemical conjugation). In some embodiments, an agent is fused, or linked, to a plant viral capsid protein (e.g., by any method described herein) and thereby becomes part of a protein complex when the plant viral capsid protein is incorporated.

[00110] In some embodiments, a plant viral capsid protein, or fragment thereof, of the present disclosure comprises an epitope tag. In some embodiments, a plant viral capsid protein, or fragment thereof, of the present disclosure comprises an epitope tag at the N-terminus. a plant viral capsid protein, or fragment thereof, of the present disclosure comprises an epitope tag at the C-terminus. In some embodiments, an epitope tag is also a reporter polypeptide. In some embodiments, an epitope tag is selected from the group consisting of: c-Myc, human influenza hemagglutinin (HA), FLAG, 3xFLAG, 6xHis (SEQ ID NO: 26), glutathione-S-transferase (GST), DYKDDDDK (SEQ ID NO: 27), maltose binding protein (MBP), GFP, RFP, mCherry, or variants or combinations thereof. In some embodiments, an epitope tag is a His tag. In some embodiments, a plant viral capsid protein, or fragment thereof, comprises an epitope tag, and the plant viral capsid protein, or fragment thereof, and epitope tag are linked by a linker (e.g., a cleavable linker, or any linker described herein).

[00111] As described herein, in some embodiments of the present disclosure, a plant viral capsid protein, or fragment thereof, may comprise one or more linkers or linker domains. For example, in some embodiments, a plant viral capsid protein may be linked to an epitope tag with a linker. In some embodiments, a plant viral capsid protein may be linked to an agent with a linker. In some embodiments, a linker is a peptide linker e.g., a short amino acid sequence). In some embodiments, a linker is a chemical linker (e.g., facilitated by conjugation of a bioconjugation handle and a crosslinking agent as is the case in various click chemistries).

[00112] In some embodiments, a linker comprises a glycine-serine linker sequence. In some embodiments, a linker comprises one or more glycine residues and/or one or more serine residues. In some embodiments, a linker may comprise repeating linker sequences, or combinations of linker sequences.

[00113] In some embodiments, a linker comprises at least about 2 amino acids, at least about 3 amino acids, at least about 4 amino acids, at least about 5 amino acids, at least about 6 amino acids, at least about 7 amino acids, at least about 8 amino acids, at least about 9 amino acids, at least about 10 amino acids, or at least about 15 amino acids. In some embodiments, a linker comprises about 2 amino acids, about 3 amino acids, about 4 amino acids, about 5 amino acids, about 6 amino acids, about 7 amino acids, about 8 amino acids, about 9 amino acids, about 10 amino acids, or about 15 amino acids.

[00114] In some embodiments, a linker is a cleavable linker. In some embodiments, a linker is an enzyme-cleavable linker. In some embodiments, a cleavable linker comprises a protease cleavage site. In some embodiments, a protease cleavage site is cleaved by tobacco etch virus (TEV). In some embodiments, a linker is a chemically labile linker. In some embodiments, a chemically labile linker is an acid-cleavable linker, or a reducible or disulfide linker. III. Production of Plant Viral Capsids Comprising a Therapeutic Agent

A. Production of Plant Viral Capsids

[00115] Provided plant viral capsid proteins, or fragments thereof (e.g., any plant viral capsid protein, or fragment thereof, described herein) can be made or obtained using any suitable method known in the field. To prepare plant viral capsid proteins, or fragments thereof, for a cell-free capsid assembly (e.g., formation of an engineered viral protein complex) viral capsid proteins can be obtained by expression and purification. Plant viral capsid proteins can be obtained generating the proteins in planta, e.g., by infecting a plant cell such as leaf tissue, and purifying the viral capsid protein. Purification or isolation of a plant viral capsid protein that is made or obtained using any of the methods described herein can be carried out according to standard techniques, e.g., exemplary protein purification protocols are described in Example 1 herein.

[00116] Capsid proteins are either expressed in soluble form, which is normally favorable for capsid assembly, or as aggregates forming inclusion bodies (e.g., within E. coli cells). To increase the soluble protein yield during expression, different factors such as the choice of the host cell, expression vector, codon usage, expression temperature, induction condition and medium can be optimized. It will be understood by a skilled artisan that the choice host cell system (or cell-free system) for capsid protein expression may alter one or more steps in a process for making, isolating or purifying, or otherwise obtaining a plant viral capsid protein for use in accordance with the present disclosure.

[00117] In some embodiments, epitope tags may be fused to viral capsid protein termini at the genetic level, as described herein, to aid protein folding and/or purification. Taking into account that the additional tag may interfere with capsid formation, in some embodiments, a protease cleavage site can be added and used prior to assembly. If proteins tend to form inclusion bodies (e.g., in E. coli) owing to incorrect folding, this expression strategy may be exploited with a strong promoter to produce high yields per dry mass and ease initial purification. The inclusion bodies can be easily separated from the cell debris and solubilized using known techniques.

[00118] While E. coli is a common host for expressing viral capsid proteins, other favorable hosts include mammalian systems, such as CHO cells, the yeast P. pastoris and insect cells, such as Sf9 cells, in combination with baculovirus vectors and yeast. These organisms are amenable to scale up and offer the advantage of eukaryotic post-translational protein modification. Cell-free protein synthesis systems have also been used to produce viral capsid proteins in defined transcription/translation reactions. This method allows for a direct control of capsid protein expression and protein complex (e.g., protein complexes, such as VLPs, etc.) assembly conditions and enables production of toxic and insoluble proteins.

[00119] Accordingly, a plant viral capsid protein, or fragment thereof, provided by the present disclosure may be expressed in any suitable host cell protein expression system. In some embodiments, a host cell is a prokaryotic cell. In some embodiments, a host cell is a bacterial cell. In some embodiments, a host cell is an E. coll cell. In some embodiments, a host cell is a eukaryotic cell. In some embodiments, a host cell is mammalian cell. In some embodiments, a host cell is a yeast cell. In some embodiments, a host cell is P. pastoris. In some embodiments, a host cell is an insect cell.

[00120] In some embodiments, a plant viral capsid protein of the present disclosure is expressed in a cell-free system, such as a rabbit reticulocyte cell-free system, HeLa cell-free system, or CHO cell-free system.

[00121] In some embodiments, a plant viral capsid protein used in accordance with the present disclosure is obtained (e.g., isolated or substantially purified) from a plant or plant cell infected with a virus comprising the plant viral capsid protein. For example, plant cells (e.g., leaf tissue) are infected with a virus and the capsid virus protein is purified using standard techniques (e.g., Bruckman and Steinmetz (2014) Methods Mol Biol. 1108: 173-85). Isolated viral particles can be disassembled using standard protocols ( .g., Lam et al. (2016) Sci Rep. (2016) 6:23803) and capsid proteins purified using methods known in the art, for example, as described in Example 1. In some embodiments, a plant viral capsid protein is obtained from a plant or plant cell infected with a virus species described in TABLE 1.

B. Production of Agents (e.g., Therapeutic Agents)

[00122] In some embodiments of the present disclosure, an agent of interest (e.g., a therapeutic agent) is associated with a plant viral capsid protein, or fragment thereof, or a protein complex comprising said plant viral capsid protein, or fragment thereof. For example, in some embodiments, an agent may be associated with a plant viral capsid protein, or fragment thereof via intermolecular interactions. In some embodiments, an agent may be packaged within or on the surface of a protein complex formed by a plant viral capsid protein. In some embodiments, an agent may be fused, or linked, to a plant viral capsid, e.g., as described herein. In some embodiments, the agent is a heterologous agent, i.e., the agent does not naturally occur with the plant viral capsid protein.

[00123] In some embodiments, an agent is a nucleic acid (e.g., DNA, RNA, etc.), a polypeptide or a small molecule or any combination thereof ( .g., a nucleic acid and a polypeptide, a nucleic acid and a small molecule, a polypeptide and a small molecule, or a nucleic acid, polypeptide and small molecule). In some embodiments, an agent is a nucleic acid. In some embodiments, an agent is a polypeptide. In some embodiments, an agent is a small molecule. In some embodiments, an agent is or comprises a bioactive moiety. In some embodiments, an agent is a therapeutic agent. In some embodiments, a therapeutic agent is a therapeutic polypeptide. In some embodiments, a therapeutic polypeptide comprises a cytokine, a hormone, an antibody, an affibody, a nanobody, an enzyme, a bioactive peptide, or derivatives or functional fragments thereof.

[00124] In some embodiments, the nucleic acid is expressed in a specific target tissue or organ. As used herein, “express”, “expressed”, “expression”, and so on, is used to indicate that the nucleic acid is transcribed and optionally translated in the specific target tissue or organ. For example, if the nucleic acid is DNA encoding a non-coding RNA, expression of such a nucleic acid includes only transcription into RNA. If the nucleic acid is DNA encoding an mRNA, expression of such a nucleic acid may include both transcription into mRNA and translation of the mRNA into protein. In certain embodiments, where a nucleic acid is a non-coding RNA, the nucleic acid may be delivered to a specific target tissue or organ but is not transcribed or translated. [00125] In some embodiments, the nucleic acid is an mRNA. In some embodiments, the nucleic acid is a non-coding RNA, such as an siRNA or siRNA precursor, tRNA, rRNA, microRNA (miRNA) or miRNA precursor, miRNA or siRNA cleavage blocker, decoy, or sponge, short hairpin RNA (shRNA), aptamer, or ribozyme. In some embodiments, the nucleic acid is a DNA, e.g., comprising one or more coding sequences for a polypeptide or bioactive nucleic acid (e.g., a therapeutic polypeptide, or therapeutic nucleic acid, such as an miRNA or miRNA precursor, mRNA, siRNA or siRNA precursor, etc.). In some embodiments, the nucleic acid is an mRNA encoding a reporter polypeptide (e.g., any reporter polypeptide described herein). In some embodiments, the nucleic acid is an miRNA or miRNA precursor. In some embodiments, an agent is an siRNA or siRNA precursor. In some embodiments, an agent is a circular RNA. In some embodiments, an agent (e.g., a therapeutic agent) is a therapeutic nucleic acid. In some embodiments, a therapeutic nucleic acid is an mRNA encoding a therapeutic polypeptide (e.g., a polypeptide for protein replacement therapy, or any therapeutic polypeptide described herein).

[00126] A nucleic acid agent (e.g., an mRNA) can be engineered to (1) improve interactions with a capsid protein, for example, to improve assembly into a complex and/or (2) improve transcription and/or translation in a cell (e.g., a mammalian cell). In certain embodiments, plant virus genetic elements are introduced into the mRNA construct to modulate its interaction with the viral capsid protein, for example, to enhance packaging and complex assembly. In certain embodiments, a regulatory element, such as an untranslated region (UTR) is incorporated to modulate stability and/or translation efficiency. Nucleic acid agents can be synthesized or produced recombinantly using known techniques, for example, those described in Example 2 herein.

[00127] Polypeptide agents described herein may be made using any known technique, e.g., a wide variety of expression vectors can be employed for expressing a nucleic acid molecule encoding a polypeptide agent of the present disclosure including, without limitation, viral expression vectors, prokaryotic expression vectors, eukaryotic expression vectors (e.g., yeast expression vectors), insect expression vectors, mammalian expression vectors, and cell-free extract expression vectors. Expression vectors may include polynucleotides encoding protein tags, or epitope tags, to aid in isolation, purification or selection (e.g., His tags, hemagglutinin tags, fluorescent protein tags, bioluminescent tags, nuclear localization tags, or any other tag described herein). As described herein, coding sequences for such protein tags can be fused to the coding sequences for provided polypeptides (e.g., viral capsid proteins, or fragments thereof) or can be included in a separate expression cassette. Non-limiting examples of expression vectors, along with well-established reagents and conditions for making and using an expression construct from such expression vectors are readily available from commercial vendors that include, without limitation, BD Biosciences-Clontech, Palo Alto, Calif; BD Biosciences Pharmingen, San Diego, Calif.; Invitrogen, Inc, Carlsbad, Calif.; EMD Biosciences-Novagen, Madison, Wis.; QIAGEN, Inc., Valencia, Calif; and Stratagene, La Jolla, Calif. The selection, making and use of an appropriate expression vector are routine procedures well within the scope of one skilled in the art and from the teachings herein.

[00128] In some embodiments, an agent is a reporter molecule. In some embodiments, a reporter molecule is a reporter polypeptide. In some embodiments, a reporter polypeptide is selected from the group consisting of a luciferase (e.g., Gaussia luciferase (Glue), nanoluc, etc.), a fluorescent polypeptide (e.g., GFP, RFP, YFP, e/c.), horseradish peroxidase (HRP), betaglucosidase, and variants or combinations thereof.

[00129] In some embodiments, an agent is a small molecule, such as a small molecule reporter or therapeutic small molecule.

[00130] As discussed herein, a plant viral capsid protein, or a fragment thereof, may be associated with an agent (e.g., a therapeutic agent, or any other agent described herein), e.g., a plant viral capsid protein, or a fragment thereof, may be associated with an agent via intermolecular forces, being linked by being expressed as a fusion protein or through chemical linkage, or otherwise. A plant viral capsid protein, or fragment thereof, may be associated with an agent directly (e.g., where a capsid protein and an agent are in direct contact or linked directly to each other) or indirectly (e.g., where a capsid protein and an agent are brought into proximity through a linker or other molecule).

[00131] A plant viral capsid protein, or fragment thereof, may be a fusion protein or polypeptide, e.g., a fusion polypeptide comprising a plant viral capsid protein, or fragment thereof, fused or linked to an agent (e.g., any agent described herein). For example, in some embodiments, a plant viral capsid protein, or fragment thereof, and an agent are expressed as a fusion protein. For example, both plant viral capsid protein and agent nucleic acid coding sequences can be directly linked or linked by a nucleic acid linker sequence, and upon expression in a suitable expression system, a viral capsid-agent fusion protein is made. In some embodiments, a plant viral capsid protein is linked to an agent using chemical conjugation (e.g., via click chemistry). In some embodiments, an agent is linked to the N-terminus of a plant viral capsid protein. In some embodiments, an agent is linked to the C-terminus of a plant viral capsid protein. In some embodiments, an agent is linked to an internal site of a plant viral capsid protein.

C. Assembly of Plant Viral Capsids Comprising an Agent or Therapeutic Agent

[00132] Capsid self-assembly is driven by Brownian motion and interactions between subunits as well as subunits and other viral or non-viral components to minimize free energy in higher structures. Without wishing to be bound by theory, capsid assembly is generally proposed to occur by an initial nucleation phase, in which a capsid oligomer nucleus is formed from the viral capsid proteins, followed by a growth phase, in which building blocks (e.g., capsid protein monomers, and/or capsid protein oligomers) are added to the capsid oligomer nucleus until a complete protein complex is formed. In certain embodiments, an agent (e.g., a therapeutic agent, such as a therapeutic nucleic acid) is packaged within, or generally associated with, a protein complex comprising a plant viral capsid protein. A protein complex comprising a plant viral capsid protein and an agent, such as a therapeutic nucleic acid, can be assembled using any known techniques, e.g., exemplary protocols for packaging various agents in capsid assemblies can be found in Azizgolshani et al. (2013) Virology 441 12-17, Lam and Steinmetz (2019) Biomater Sci. 7(8):3138-3142, de Ruiter et al. (2019) Journal of Controlled Release 307:342- 354, and Lu et al. (2012) Journal of General Virology 93: 1120- 1126, the entire contents of each of which are hereby incorporated by reference for all purposes. An assembled plant viral capsid protein complex comprising an agent is also referred to herein as a “multimodal vector (MMV)”.

[00133] In some embodiments, a plant viral capsid protein and an agent are assembled into a protein complex by combining both plant viral capsid protein and agent in a suitable buffered solution in a mass ratio of about 1 : 1, 1.5:1, 2: 1, 2.5:1, 3:1, 3.5: 1, 4: 1, 4.5: 1, 5: 1, 5.5:1, 6: 1, 6.5: 1, 7: 1, 7.5: 1, 8: 1, 8.5: 1, 9:1, 9.5:1, or 10: 1. In some embodiments, a plant viral capsid protein and an agent are combined in a mass ratio of about 4: 1. In some embodiments, a plant viral capsid protein and an agent are combined in a mass ratio of about 5: 1. In some embodiments, a plant viral capsid protein and an agent are combined in a mass ratio of about 6: 1 . Tn some embodiments, a plant viral capsid protein and an agent are combined in a mass ratio of about 7:1. In some embodiments, a plant viral capsid protein and an agent are combined in a mass ratio of about 8: 1.

[00134] In some embodiments, a plant viral capsid protein and a nucleic acid (e.g., a therapeutic nucleic acid, or any other nucleic acid described herein) are assembled into a protein complex by combining both plant viral capsid protein and nucleic acid in a suitable buffered solution in a mass ratio of about 1 : 1, 1.5: 1, 2: 1, 2.5:1, 3:1, 3.5:1, 4: 1, 4.5:1, 5: 1, 5.5:1, 6: 1, 6.5: 1, 7: 1, 7.5: 1, 8: 1, 8.5: 1, 9:1, 9.5: 1, or 10: 1. In some embodiments, a plant viral capsid protein and a nucleic acid are combined in a mass ratio of about 4: 1 . In some embodiments, a plant viral capsid protein and a nucleic acid are combined in a mass ratio of about 5:1. In some embodiments, a plant viral capsid protein and a nucleic acid are combined in a mass ratio of about 6:1. In some embodiments, a plant viral capsid protein and a nucleic acid are combined in a mass ratio of about 7: 1. In some embodiments, a plant viral capsid protein and a nucleic acid are combined in a mass ratio of about 8:1.

[00135] In some embodiments, a plant viral capsid protein and an RNA (e.g., a therapeutic RNA, or any other RNA described herein) are assembled into a protein complex by combining both plant viral capsid protein and RNA in a suitable buffered solution in a mass ratio of about 1 : 1, 1.5: 1, 2: 1, 2.5: 1 , 3: 1, 3.5:1 , 4: 1, 4.5: 1, 5: 1, 5.5: 1, 6:1, 6.5: 1, 7: 1 , 7.5: 1, 8: 1 , 8.5: 1, 9: 1 , 9.5: 1, or 10:1. In some embodiments, a plant viral capsid protein and an RNA are combined in a mass ratio of about 4: 1. In some embodiments, a plant viral capsid protein and an RNA are combined in a mass ratio of about 5:1. In some embodiments, a plant viral capsid protein and an RNA are combined in a mass ratio of about 6: 1. In some embodiments, a plant viral capsid protein and an RNA are combined in a mass ratio of about 7:1. In some embodiments, a plant viral capsid protein and an RNA are combined in a mass ratio of about 8: 1.

IV. Methods of Administration

[00136] Actual doses of provided compositions and pharmaceutical compositions to be administered will vary depending upon the age, weight, and general condition of the patient as well as the severity of the condition being treated, the judgment of the health care professional, and particular cells and compositions being administered. Therapeutically effective amounts can be determined by one of ordinary skill in the art, and will be adjusted to the particular requirements of each particular case. Generally, a therapeutically effective amount for the use of a protein complex (e.g., an engineered capsid, such as a VLP, etc.) is measured in parti cles/kg. Typically, a dose will include from about 1 x 10⁹ to about 2 x 10¹⁴ particles/kg of the protein complexes. A therapeutically effective dose can be determined experimentally by repeated administration of increasing amounts of the composition in order to determine which amount produces a clinically desired endpoint.

[00137] Administration can be in a single bolus dose, or can be in two or more doses, in the same day, or one or more days apart. The amount of composition administered will depend on the potency of said composition, and/or route of administration.

[00138] Administration is not limited to any particular route, but rather can refer to any route accepted as appropriate by the medical community. For example, the present disclosure contemplates routes of administering that include, but are not limited to, oral (PO), intravenous (IV), intramuscular (IM), intra-arterial, intramedullary, intrathecal, subcutaneous (SQ), intraventricular, transdermal, interdermal, intradermal, rectal (PR), vaginal, intraperitoneal (IP), intragastric (IG), topical and/or transdermal (e.g., by lotions, creams, powders, ointments, liniments, gels, drops, etc.), mucosal, intranasal, buccal, enteral, vitreal, and/or sublingual administration; by intratracheal instillation, bronchial instillation, and/or inhalation; as an oral spray, nasal spray, and/or aerosol, and/or through a portal vein catheter; and/or combinations of any of the foregoing. In some embodiments of the present invention, administration is oral, intragastric, mucosal, intranasal, buccal, enteral, and/or sublingual. In some embodiments of the present disclosure, administration is oral. In some embodiments, administration is intravenous.

[00139] The methods and compositions described herein can be used alone or in combination with other therapeutic agents and/or modalities. The term administered “in combination,” as used herein, is understood to mean that two (or more) different treatments are delivered to a patient during the course of said patient’s affliction with a disease or disorder, such that the effects of each treatment on said patient overlap at a point in time. In some embodiments, delivery of one treatment is still occurring when delivery of a second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery.” In some embodiments, delivery of one treatment ends before delivery of another treatment begins. In some embodiments of either case, a treatment is more effective because of combined administration.

[00140] For example, a second treatment may be more effective, e.g., an equivalent effect is seen with less of said second treatment, or a second treatment may reduce symptoms to a greater extent, than would be seen if said second treatment were administered in the absence of a first treatment, or the analogous situation is seen with a provided first treatment. In certain embodiments, delivery is such that reduction in one or more symptoms, or other parameters related to a disease or disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of a first treatment delivered is still detectable when a second is delivered.

A. Administration of Agents to Specific Cells and/or Tissues

[00141] The present disclosure relates to, among other things, the discovery that plant virus capsid proteins, and fragments thereof, when administered to a patient (e.g., a human patient), are delivered to specific cell and/or tissue types. Accordingly, in some embodiments, the disclosure relates to methods and compositions for delivering a therapeutic agent to a specific target tissue or organ in a patient in need thereof. Provided methods, in some embodiments, include administering to the patient in need thereof a therapeutically effective amount of a composition or pharmaceutical composition comprising a protein complex comprising a plant viral capsid protein (e.g., an isolated plant viral capsid protein), or fragment thereof, and a therapeutic agent, thereby delivering the therapeutic agent to the patient’s specific target tissue or organ, wherein the specific target tissue is selected from the group consisting of adipose, adrenal, artery, blood, brain, breast, cervix, colon, esophagus, heart, kidney, liver, lung, muscle, nerve, ovary, pancreas, pituitary, prostate, salivary gland, small intestine, spleen, stomach, testis, thyroid, and uterus tissue. In some embodiments, in a method of the present disclosure a provided composition or pharmaceutical composition is administered as an orally or intravenously.

[00142] In some embodiments, the present disclosure provides a method of delivering a therapeutic agent to a specific target tissue or organ in a patient, the method comprising administering to the patient a therapeutically effective amount of a provided composition or pharmaceutical composition comprising: a complex comprising an Alfamovirus capsid protein (e.g., an alfalfa mosaic virus capsid protein) or a fragment thereof and the therapeutic agent, thereby delivering the therapeutic agent to the patient’s specific target tissue or organ. In some embodiments, the specific target tissue or organ is selected from brain, prostate, esophagus, small intestine, breast, salivary gland, nerve, thyroid, adrenal, adipose, lung, muscle, heart, colon, artery, blood, liver, stomach, testis, pancreas, and ovary. For example, the brain tissue may be selected from hippocampus, basal ganglia (e.g., caudate, putamen, nucleus accumbens), cortex (e.g., frontal cortex, anterior cingulate), cerebellar hemisphere, cervical spinal cord, substantia nigra, hypothalamus, amygdala, and cerebellum. The esophagus tissue may be selected from gastroesophageal junction and muscularis. The small intestine tissue may be terminal ileum. The breast tissue may be mammary tissue. The nerve tissue may by tibial nerve tissue. The adrenal tissue may be gland tissue. The adipose tissue may be subcutaneous or visceral omentum tissue. The artery tissue may be tibial tissue or coronary tissue. The colon tissue may be sigmoid tissue.

[00143] In some embodiments, the present disclosure provides a method of delivering a therapeutic agent to a pituitary tissue in a patient, the method comprising administering to the patient a therapeutically effective amount of a provided composition or pharmaceutical composition comprising: a complex comprising an Foveavirus capsid protein (e.g., an apple stem pitting virus capsid protein) or a fragment thereof and the therapeutic agent, thereby delivering the therapeutic agent to the patient’s pituitary tissue.

[00144] In some embodiments, the present disclosure provides a method of delivering a therapeutic agent to a specific tissue or organ in a patient, the method comprising administering to the patient a therapeutically effective amount of a provided composition or pharmaceutical composition comprising: a complex comprising a. Bromovirus capsid protein (e.g., a brome mosaic virus capsid protein, a lilac leaf chlorosis virus capsid protein, an apple necrotic mosaic virus capsid protein, or a cowpea chlorotic mottle virus capsid protein) or a fragment thereof and the therapeutic agent, thereby delivering the therapeutic agent to the patient’s ovary, prostate, brain, breast, adipose, nerve, artery, small intestine, thyroid, blood, colon, lung, liver, muscle, esophagus, adrenal, or uterus tissue. For example, the brain tissue may be hypothalamus or cortex tissue. The breast tissue may be mammary tissue. The adipose tissue may be subcutaneous adipose tissue. The nerve tissue may be tibial tissue. The artery tissue may be tibial tissue. The small intestine tissue may be terminal ileum tissue. The colon tissue may be sigmoid tissue.

[00145] In some embodiments, the present disclosure provides a method of delivering a therapeutic agent to a specific tissue or organ in a patient, the method comprising administering to the patient a therapeutically effective amount of a provided composition or pharmaceutical composition comprising: a complex comprising a Potexvirus capsid protein (e.g., a clover yellow mosaic virus capsid protein, a white clover mosaic virus capsid protein, or a pepino mosaic virus capsid protein) or a fragment thereof and the therapeutic agent, thereby delivering the therapeutic agent to the patient’s artery, blood, brain, esophagus, adipose, stomach, breast, adrenal, thyroid, cervix, small intestine, spleen, colon, testis, ovary, nerve, muscle, heart. For example, the artery tissue may be coronary, aorta, or tibial tissue. The brain tissue may be cervical spinal cord, frontal cortex, or cerebellum tissue. The adipose tissue may be visceral omentum or subcutaneous tissue. The breast tissue may be mammary tissue. The adrenal tissue may be gland tissue. The esophagus tissue may be muscularis tissue. The cervix tissue may be endocervix tissue. The small intestine tissue may be terminal ileum tissue. The colon tissue may be transverse colon tissue. The nerve tissue may be tibial nerve tissue.

[00146] In some embodiments, the present disclosure provides a method of delivering a therapeutic agent to a specific tissue or organ in a patient, the method comprising administering to the patient a therapeutically effective amount of a provided composition or pharmaceutical composition comprising: a complex comprising a Tobamovirus capsid protein (e.g., tobacco mild green mosaic virus capsid protein, a tobacco mosaic virus capsid protein, a tomato mosaic virus capsid protein, a tropical soda apple mosaic virus capsid protein, a pepper mild mottle virus capsid protein, or a cucumber green mottle mosaic virus capsid protein) or a fragment thereof and the therapeutic agent, thereby delivering the therapeutic agent to the patient’s specific tissue or organ. In some embodiments, the specific tissue or organ is artery, brain, adipose, lung, blood, nerve, breast, esophagus, muscle, thyroid, testis, kidney, pituitary, prostate, liver, stomach, adrenal, heart, uterus, colon, small intestine, ovary, pancreas, spleen. . For example, the adipose tissue may be subcutaneous or visceral omentum tissue. The artery tissue may be coronary, tibial, or aorta tissue. The brain tissue may be substantia nigra, cortex (e.g., frontal cortex, anterior cingulate cortex), cervical spinal cord, basal ganglia (e.g., caudate, putamen, accumbens), cerebellar hemisphere; amygdala, hippocampus, cerebellum, cortex, or hypothalamus. The nerve tissue may be tibial tissue. The breast tissue may be mammary tissue. The esophagus tissue may be muscularis or gastroesophageal junction tissue. The kidney tissue may be cortex tissue. The adrenal tissue may be cortex or gland tissue. The colon may be transverse or sigmoid tissue. The small intestine tissue may be terminal ileum tissue.

[00147] In some embodiments, the present disclosure provides a method of delivering a therapeutic agent to a heart tissue in a patient, the method comprising administering to the patient a therapeutically effective amount of a provided composition or pharmaceutical composition comprising: a complex comprising a Polemovirus capsid protein (e.g., a poinsettia latent virus capsid protein) or a fragment thereof and the therapeutic agent, thereby delivering the therapeutic agent to the patient’s heart tissue.

[00148] In some embodiments, the present disclosure provides a method of delivering a therapeutic agent to an artery tissue in a patient, the method comprising administering to the patient a therapeutically effective amount of a provided composition or pharmaceutical composition comprising: a complex comprising a Potyvirns capsid protein (e.g., a potato virus Y virus capsid protein) or a fragment thereof and the therapeutic agent, thereby delivering the therapeutic agent to the patient’s artery tissue (e.g., coronary artery tissue).

[00149] In some embodiments, the present disclosure provides method of delivering a therapeutic agent to a specific organ or tissue in a patient, the method comprising administering to the patient a therapeutically effective amount of a provided composition or pharmaceutical comprising: a complex comprising a Closter ovirus capsid protein e.g., a citrus tristeza virus capsid protein) or a fragment thereof and the therapeutic agent, thereby delivering the therapeutic agent to the patient’s specific tissue or organ, wherein the specific tissue or organ is adipose, breast, artery, or nerve tissue.

[00150] The present disclosure also provides methods of screening targeting specificity of a protein complex comprising plant viral capsid protein, or a fragment thereof, (e.g., a complex comprising a plant viral capsid protein) and a therapeutic agent. In certain embodiments, the method can include administering a complex comprising a plant viral capsid protein to an animal (e.g., a mammal) and testing different tissue types for the presence of a capsid protein sequence. In certain embodiments, RNA is extracted from tissue from the animal and the RNA is amplified (e.g., using RT-PCR) and identified. Tn certain embodiments, the capsid protein sequence is identified using sequencing.

[00151] In certain embodiments, a complex comprising a plant viral capsid protein and a unique RNA sequence can be assembled and tested for targeting specificity. In this embodiment, the unique RNA sequence serves as a molecular barcode for its identification. In this embodiment, RNA extracted from each tissue can be processed for RNA sequencing. Then, the barcode RNA can be amplified to serve as template for a sequencing library preparation (e.g., using an Illumina sequencing library preparation). This method can be performed using multiple different complexes, wherein each complex comprises a unique RNA sequence.

V. Compositions and Pharmaceutical Compositions

[00152] Compositions comprising a plant viral capsid protein (e.g., a complex comprising a plant viral capsid protein and an agent) can be used to infect plant cells, and infected plant cells can be orally administered to a patient in need thereof. In certain embodiments, a plant is infected with a plant viral capsid protein (e.g., a complex comprising a plant viral capsid protein and an agent) and the infected plant is orally administered to a patient in need thereof.

[00153] Alternatively, or in addition, a composition comprising a plant viral capsid protein (e.g., a complex comprising a plant viral capsid protein and an agent) can be formulated into a pharmaceutical composition. One or more pharmaceutically acceptable excipients or carriers can be included in the composition for proper formulation. Suitable formulations for use in the present disclosure are found in Adeboye Adejare, Remington: The Science and Practice of Pharmacy (23rd ed. 2020).

[00154] In certain embodiments, a pharmaceutical composition may contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta- cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; saltforming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and/or pharmaceutical adjuvants (see, Adeboye Adejare, Remington: The Science and Practice of Pharmacy (23rd ed. 2020)).

[00155] Pharmaceutical compositions containing a provided plant viral capsid protein, or fragment thereof (e.g., in the form of a protein complex, such as a VLP, etc.) can be presented in a dosage unit form and can be prepared by any suitable method. A pharmaceutical composition should be formulated to be compatible with its intended route of administration. Examples of routes of administration are intravenous, intradermal, inhalation, transdermal, topical, transmucosal, intrathecal and rectal administration. Formulation components suitable for parenteral administration include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose.

[00156] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). The carrier should be stable under the conditions of manufacture and storage, and should be preserved against microorganisms. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. An intravenous drug delivery formulation of the present disclosure may be contained in a bag, a pen, or a syringe. In certain embodiments, the bag may be connected to a channel including a tube and/or a needle.

[00157] Depending upon the circumstances, the formulation can be a liquid formulation. An aqueous formulation can be prepared including the viral capsid protein or protein complex in a pH-buffered solution. The pH of the liquid formulation may be set by addition of a pharmaceutically acceptable acid and/or base. In certain embodiments, the pharmaceutically acceptable acid may be hydrochloric acid. In certain embodiments, the base may be sodium hydroxide. In certain embodiments, a salt or buffer components may be added in an amount of 10 mM to 200 mM. The salts and/or buffers are pharmaceutically acceptable and are derived from various known acids (inorganic and organic) with “base forming” metals or amines. In certain embodiments, the buffer may be phosphate buffer. In certain embodiments, the buffer may be glycinate, carbonate, citrate buffers, in which case, sodium, potassium or ammonium ions can serve as counterion. Intravenous formulations can be diluted with 0.9% sodium chloride solution before administration. In certain embodiments, the diluted drug product for injection is isotonic and suitable for administration by intravenous infusion.

[00158] Alternatively, the formulation can be a lyophilized formulation including the plant viral capsid protein or protein complex and a lyoprotectant. The lyoprotectant may be sugar, e.g., di saccharides. In certain embodiments, the lyoprotectant may be sucrose or maltose. The lyophilized formulation may also include one or more of a buffering agent, a surfactant, a bulking agent, and/or a preservative. The amount of sucrose or maltose useful for stabilization of the lyophilized drug product may be in a weight ratio of at least 1 :2 protein to sucrose or maltose. In certain embodiments, the protein to sucrose or maltose weight ratio may be of from 1 :2 to 1:5. Before lyophilization, the pH of the solution containing the protein of the present disclosure may be adjusted between 6 to 8. In certain embodiments, the pH range for the lyophilized drug product may be from 7 to 8. In certain embodiments, a “bulking agent” may be added. A “bulking agent” is a compound which adds mass to a lyophilized mixture and contributes to the physical structure of the lyophilized cake (e.g, facilitates the production of an essentially uniform lyophilized cake which maintains an open pore structure). Illustrative bulking agents include mannitol, glycine, polyethylene glycol and sorbitol. The lyophilized formulations of the present disclosure may contain such bulking agents. [00159] In certain embodiments, the lyophilized drug product may be constituted with an aqueous carrier. The aqueous carrier of interest herein is one which is pharmaceutically acceptable (e.g., safe and non-toxic for administration to a human) and is useful for the preparation of a liquid formulation, after lyophilization. Illustrative diluents include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate-buffered saline), sterile saline solution, Ringer’s solution or dextrose solution. In certain embodiments, the lyophilized protein product of the instant disclosure is constituted to about 4.5 mL water for injection and diluted with 0.9% saline solution (sodium chloride solution).

[00160] Pharmaceutical formulations preferably are sterile. Sterilization can be accomplished by any suitable method, e.g., filtration through sterile filtration membranes. Where the composition is lyophilized, filter sterilization can be conducted prior to or following lyophilization and reconstitution. The resulting aqueous solutions may be packaged for use as-is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the preparations typically will be between 3 and 11, more preferably between 5 and 9 or between 6 and 8, and most preferably between 7 and 8, such as 7 to 7.5. The resulting compositions in solid form may be packaged in multiple single dose units, each containing a fixed amount of the above-mentioned agent or agents. The composition in solid form can also be packaged in a container for a flexible quantity.

[00161] Depending upon the circumstances, the resulting pharmaceutical composition is formulated as a liquid formulation in either a USP / Ph Eur type I 50R vial closed with a rubber stopper and sealed with an aluminum crimp seal closure. The stopper may be made of elastomer complying with USP and Ph Eur. In certain embodiments, the liquid formulation may be diluted with 0.9% saline solution prior to use.

[00162] A preservative may be optionally added to the formulations to reduce bacterial action. The addition of a preservative may, for example, facilitate the production of a multi-use (multiple-dose) formulation.

[00163] In certain embodiments, a pharmaceutical composition may contain a sustained- or controlled-delivery formulation. Techniques for formulating sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. Sustained-release preparations may include, e.g., porous polymeric microparticles or semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained release matrices may include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl -L-glutamate, poly (2- hydroxyethyl-inethacrylate), ethylene vinyl acetate, or poly-D(-)-3 -hydroxybutyric acid. Sustained release compositions may also include liposomes that can be prepared by any of several methods known in the art.

[00164] The compositions described herein may be administered locally or systemically. Administration will generally be parenteral administration. In certain embodiments, the pharmaceutical composition is administered subcutaneously and in other embodiments the pharmaceutical composition is administered intravenously. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.

[00165] It will be understood by a skilled artisan that the amount of a provided pharmaceutical composition administered will depend on variables such as the type and extent of disease or indication to be treated, the overall health of the patient, the in vivo potency of the viral capsid composition, the pharmaceutical formulation, and the route of administration. The initial dosage can be increased beyond the upper level in order to rapidly achieve the desired blood-level or tissue-level. Alternatively, the initial dosage can be smaller than the optimum, and the daily dosage may be progressively increased during the course of treatment. Human dosage can be optimized, e.g., in a conventional Phase I dose escalation study. Dosing frequency can vary, depending on factors such as route of administration, dosage amount, serum half-life of the antibody, fusion protein, and/or antibody conjugate, and the disease being treated.

[00166] Exemplary dosing frequencies are at least once per day, at least once per week, at least once every two weeks, or at least once every month.

VI. Methods of Treatment

[00167] Once produced, the viral capsid proteins, protein complexes, and pharmaceutical compositions described herein can used in a variety of therapies and therapeutic methods. For example, the viral capsid proteins, protein complexes, and pharmaceutical compositions described herein can be used in a method of treating a patient with a disease or disorder. Provided methods comprises administering to the patient a therapeutically effective amount of a viral capsid protein or protein complex of the present disclosure (e.g., any viral capsid protein or protein complex described herein) or a pharmaceutical composition containing such viral capsid protein or protein complex. An administered viral capsid protein can be selected from any capsid protein described in TABLE 1, and assembled in a protein complex to be associated with any agent described herein (e.g., a therapeutic agent, such as a therapeutic nucleic acid).

[00168J Throughout the description, where apparatus, devices, and systems are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are apparatus, devices, and systems of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps.

[00169] Practice of the invention will be more fully understood from the foregoing examples, which are presented herein for illustrative purposes only, and should not be construed as limiting the invention in any way.

EXAMPLES

[00170] The following Examples are merely illustrative and are not intended to limit the scope or content of the disclosure in any way.

[00171] The following examples describe the production, engineering, and testing of engineered viral protein complexes using “multimodal vectors (MMVs),” as disclosed herein. It is contemplated that assembled engineered complexes comprising MMVs can be used to deliver therapeutic payloads or agents to specific tissues and organs, e.g., in the treatment of a disease or disorder.

Example 1: Production and isolation of viral capsid proteins

[00172] Viral capsid proteins (CP) can be made or obtained using different methods depending on the multimodal vector (MMV). For example, provided CPs can be expressed recombinantly in either E. coli or yeast P. pastoris), or they can be purified from live variants isolated from infected plants. A list of exemplary MMV candidates, contemplated expression systems, and CP sequences is provided in TABLE 1. Recombinant expression of CP in E. coli

[00173] An exemplary MMV that can be expressed in E. coli is cowpea chlorotic mottle virus (CCMV). A CCMV CP cDNA is synthetized as a gene block and cloned into the expression vector pET302/NT-His (Invitrogen #K6302-03) via Gibson Assembly. The pET302/CCMV-CP construct is transformed into the E. coli strain One Shot BL21 (D3E) (Life Technologies), and colonies are selected through antibiotic resistance and verified by sequencing.

[00174] To produce the CCMV CP protein, 2 m of an overnight pET302N/CCMV-CP culture is transferred to 100 mL of TB broth (Life Technologies) with 100 mg/mL carbenicillin and grown at 37 °C until OD600 reached 0.6-0.8. Protein expression is induced using 0.5 mM IPTG and the culture is allowed to grow, for example, at 20 °C for 18 hours. The cultures are then harvested by centrifugation at 4,200x g for 30 minutes at 4 °C. The cell pellet is stored at - 80 °C or used directly for protein production. For protein production, the cell pellet is resuspended in a buffer containing 50 mM Tris pH 7.5, 500 mM NaCl, 5 mM imidazole, 1 mM EDTA, and 2 mM TCEP before adding lysozyme (Thermo Scientific) to 1 mg/mL and HALT Protease Inhibitor Cocktail (Thermo Scientific). Resuspended cells are lysed using a Dounce homogenizer (Coming) and the lysate is centrifuged at 4,000x g for 30 minutes at 4 °C. The resulting cell supernatant is added to 1 mL HisPur Cobalt Resin (Thermo Scientific) and allowed to batch bind for 1 hour at 4 °C. His-tagged CCMV CPs are collected 1 mL fractions using affinity purification and elution with a buffer containing 50 mM Tris pH 7.5, 500 mMNaCl, 400 mM imidazole, 2 mM TCEP. Fractions containing protein are pooled and buffer exchanged using Zeba Desalting columns (Thermo Scientific) into a protein buffer containing 20mM Tris pH 7.2, IM NaCl ImM EDTA 2mM TCEP. Fractions are stored at 4 °C. Purified protein is analyzed by SDS-PAGE on a 4-12% Bis-Tris gel in MES buffer (Thermo Scientific). To determine protein quantity, the extinction coefficient is determined using the amino acid sequence of the CP. The absorbance is measured at 280 nm and calculated based on the measurement done in triplicate.

[00175] Other MMV candidates from the same family, such as brome mosaic virus (BMV), lilac leaf chlorosis virus (LLCV) and alfalfa mosaic virus (AMV), are produced using a similar procedure. Recombinant expression of CP in P. pastoris

[00176] Some MMVs, such as tobacco mosaic virus (TMV) and related viruses, are expressed in P. pastoris following procedures similar to those used in previous studies (Brumfield, et al. (2004) J Gen Virol. 85(Pt 4): 1049-1053 and Zhu, et al. (2020) Protein Expr Pnrif. 174: 105679). In this Example, TMV CP cDNA is cloned into a plasmid vector such as pPINKa-HC and expressed using the PichiaPink™ Secreted Protein Expression Kit (Invitrogen #A11151), following the manufacturer’s guidelines. Briefly, the plasmid is electroporated into competent cells of af. pastoris strain such as PischiaPink™. White colonies, expressing high levels of the cloned CP, are picked and tested for the integration of the construct. Selected colonies are grown in 100 mb of appropriate medium such as buffered complex glycerol (BMGY) medium at 28 °C in a shaking incubator at 250 rpm. When the adsorption of the culture at 600 nm reaches 2-6 absorbance units, the cells are centrifuged at l,500x g for 5 minutes and resuspended in buffered complex methanol (BMMY) medium to induce expression. Cells are grown at 15-35 °C for 1-3 days while shaking at 250 rpm. Methanol is added to the medium at a final concentration of 0%-2.0% every 24 hours to maintain the induction. To collect the secreted CP, cells are pelleted to recover the supernatant. The supernatant is concentrated via ultrafiltration (10 ED, Amicon Ultra-15, Millipore) at 4 °C at 5,000x g for 30 minutes. The expression of CP is analyzed in both pellet and supernatant fractions by SDS- PAGE on a 4-12% Bis-Tris gel in MES buffer (Thermo Scientific). To determine protein quantity, the extinction coefficient is determined using the amino acid sequence of the CP. The absorbance is measured at 280 nm and calculated based on the measurement done in triplicate.

Histidine tag cleavage of recombinant capsid proteins

[00177] In this Example, the expressed CP protein is tagged to facilitate detection and purification. Once purified, and prior to assembly into capsids, the tag is removed. For the examples shown above, the CP is cloned with a Histidine tag (His-tag) separated by the cleavage site for Tobacco Etch Virus (TEV) protease. To remove the His-tag of the purified CCMV CP, TEV cleavage is conducted in a reaction mixture comprising of about 450 pg of His-tagged CCMV CP, 30 pL of TEV protease (NEB), and 75 pL of a lOx TEV protease reaction buffer, in a total volume of 750 pL at 4 °C overnight. Subsequently, the TEV-cleaved product is purified using a HisPur Cobalt spin column (ThermoFisher). Initially, the resin is equilibrated with two resin-bed volumes of wash buffer such as 50 mM Tris pH 7.5, 500 mM NaCl, 5 mM imidazole, 2 mM TCEP, and then mixed with the TEV reaction mixture on an orbital shaker at 4 °C for 30 minutes. The flow-through containing the CCMV CP without the tag is collected, and the column is washed with 2 resin-bed volumes of wash buffer twice before being eluted with 1 resin-bed volume of elution buffer consisting of 50 mM Tris pH 7.5, 250 mM NaCl, 400 mM imidazole, 2 mM TCEP. All fractions are collected and subsequently assessed for purity and yield using previous SDS-PAGE analysis and quantitation by absorbance.

Capsid protein isolation from live variants

[00178] As an alternative to recombinant production, capsid proteins (CP) can be generated in planta and optionally purified. In certain cases, it is contemplated that in planta generation may be more efficient than recombinant generation, for example, for TMGMV and TMV.

[00179] For in planta generation, viruses are purified from infected leaf tissue using standard methods (Bruckman and Steinmetz (2014) Methods Mol Biol. 1108:173-85). Infected leaves are homogenized, filtered and the virus is purified by chloroform extraction. The aqueous phase, containing the viral particles, is precipitated with 8% polyethylene glycol (PEG)-8000, 1% (v/v) Triton-X 100 and 0.2 M NaCl, and purified by gradient separation and ultracentrifugation. Briefly, a first ultracentrifugation is performed at 160,000x g for 3 hours through a 40% sucrose cushion, followed by another ultracentrifugation of the pellet over a 10- 40% sucrose gradient at 104,000x g for 2 hours. The different bands are collected to further purify the virus by ultracentrifugation at 160,000x g for 3 hours and resuspended in endotoxin- free PBS. The virus extract is then analyzed for particle quality and quantified.

[00180] Next, the isolated particles are disassembled to obtain capsid proteins following established protocols (Lam et al. (2016) Sci Rep. (2016) 6:23803). For TMV, viral particles are incubated with 2 volumes of glacial acetic acid for 20 minutes on ice, and precipitated by centrifugation at 20,000x g for 20 minutes at 4 °C. The supernatant is transferred to a 6-8 MWCO dialysis tubing and dialyzed against water for 48 hours at 4 °C. Following dialysis, the CPs are centrifugated at 20,000x g for 20 minutes at 4 °C and the pellet is resuspended in 75 mM sodium phosphate buffer pH 7.2 overnight. For CCMV, virus particles are dialyzed using a 3.5K MWCO dialysis cassette, in a disassembly buffer consisting of 0.5 CaC12, 50 mM Tris-HCl, pH 7.5, ImM EDTA, 1 mM DTT and 0.5 mM PMSF for 4 °C for 24 hours. After dialysis, the viral RNA removed through ultracentrifugation at around 12,000x g for 30 min at 4 °C. Next, the supernatant is centrifuged at around 220,000x g for 2 hours at 4 °C, to pellet the nondisassociated virus particles. The supernatant, containing capsid protein monomers, is dialyzed again with, for instance, a 3.5K MWCO dialysis cassette in protein buffer, containing, for instance, 1 M NaCl, 20 mM Tris pH 7.2, 1 mM EDTA, 1 mM DTT and 1 mM PMSF for 24 hours and stored at 4 °C. The CP concentration is measured photometrically at 280 nm.

Example 2: Production of cargo ribonucleic acid (RNA)

[00181] MMVs are packaged with mRNA encoding one or multiple proteins of interest. This example describes the production of RNA for packaging with an MMV. In this example, mRNA encoding a reporter protein, Gaussia luciferase (Glue), is produced. This reporter protein will be used in a subsequent example to determine the ability of produced MMVs to transduce mammalian cells.

Design of RNA construct

[00182] The mRNA construct comprises a coding sequence for a gene to be expressed in human cells for either a therapeutic or a reporter purpose. mRNA is designed with two aims: 1) to interact with the MMV CP to be assembled into particles and 2) to be translated in mammalian cells. In some cases, plant virus genetic elements are introduced into the mRNA construct to modulate its interaction with the viral CP, for example, to enhance packaging and particle assembly. Other regulatory elements, such as UTRs are incorporated to modulate mRNA properties within human cells, such as stability and translation efficiency.

Cloning of reporter gene cDNA

[00183] cDNA encoding Gaussia luciferase (Glue) is synthesized for producing mRNA to be combined with CP for delivery to cells. To synthesize mRNA, the cDNA encoding Glue is cloned into an expression plasmid containing a selection marker such as Kanamycin resistance. The plasmid is transformed into a DHFR5alpha£. coli strain. A 5 mL culture is grown overnight at 37 °C in LB-Miller broth with 50 mg/mL kanamycin. The culture is harvested by centrifugation at 4,000x g for 15 minutes and the supernatant discarded. The remaining cells are lysed, and plasmid DNA is purified using the manufacturer’s instructions included with the GeneJET Plasmid Miniprep Kit (Thermo Scientific) and quantified through absorbance.

In vitro transcription of plasmid containing reporter gene cDNA

[00184] Purified plasmid DNA is linearized to serve as a template for an in vitro transcription (IVT) reaction. Specifically, the purified plasmid is linearized with Hindlll using the manufacturer’s instructions (New England Biolabs) and purified using PureLink PCR Purification Kit (Thermo Scientific) following the manufacturer’s instructions. Once linearized, the Glue DNA is run on a 1% agarose gel and further purified using the PureLink Quick Gel Extraction Kit following the manufacturer’s instructions. Finally, the linearized Glue cDNA is used as a template to generate Glue mRNA through IVT using the Takara IVTpro mRNA Synthesis System (Takara) kit following the manufacturer’s instructions.

Example 3: Assembly of RNA-containing viral capsids (MMV)

Assembly ofMMVs

[00185] The MMV CP and reporter gene mRNA are produced as described above and packaged and assembled into virus-like particles (VLPs) using the following method. MMV CP and mRNA are mixed using methods previously described (Azizgolshani et al. (2013) Virology 441 12-17; Lam and Steinmetz (2019) Biomater Sci. 7(8) : 3138-3142). The ratio between CP and mRNA is adjusted for each virus species to optimize packaging efficiency and particle formation of the desired size and shape. For example, to package mRNA in a CCMV CP, the CP and mRNA are mixed in a mass ratio of 6: 1. Specifically, 72 pg of CP and 12 pg of RNA are mixed in 400 pL of a buffer such as 50 mM Tris-HCl (pH 7.2), 1 M NaCl, 1 mM EDTA, 1 mM DTT and 1 mM PMSF. The complex is subjected to dialysis against a buffer such as 50 mM Tris-HCl (pH 7.2), 50 mM NaCl, 10 mM KC1, 5 mM MgC12 and 1 mM DTT, at 4 °C for at least 6 hours, followed by dialysis against a buffer comprising, for example, 50 mM sodium acetate, 8 mM magnesium acetate (pH 4.8) with glacial acetic acid at 4 °C overnight.

Labelling of i ’s

[00186] In some cases, the assembled particles will be modified through chemical modification to add, for instance, a fluorescent label or a functional group following similar methods used by others. This reaction is performed with methods and ratios that can be adjusted for each MMV based on their surface chemistry. For example, CCMV, which displays 300 addressable lysins, can be modified with an NHS-activated fluorescent protein such as Cyanine5, by mixing 100 molar excess of dye with CCMV in a buffer such as 0.1 M HEPES pH 7.0, 5 mM MgC12 and 10% (v/v) DMSO, overnight at room temperature (Lam and Steinmetz (2019), supra). For TMV, which has a solvent-exposed tyrosine residue in the exterior surface, this can be modified with diazonium coupling reactions (B ruckman and Steinmetz (2014), supra). Genetic engineering of CPs to introduce accessible amino acid residues that can be chemically modified is performed to modulate the number of chemical groups to be added into MMVs.

[00187] The reaction is purified through ultracentrifugation at 150,000x g for 1 hour over a 30 % (w/v) sucrose cushion.

Quality control and VLP assessment

[00188] The formation of VLPs is evaluated using dynamic light scattering (DLS) and electron microscopy.

Example 4: Transducing human cell lines with MMVs

[00189] The MMVs produced as described herein are assessed for their ability to transduce human cell lines in vitro. An array of different cell lines is tested as shown in TABLE 2. These MMVs carry an mRNA for one or multiple reporter genes such as GFP and/or luciferase, generated as described above.

TABLE 2. Cell lines used for in vitro transduction assays Cell maintenance and culture

[00190] Cells are cultured and maintained with DMEM medium (ThermoFisher) supplemented with 10% Fetal Bovine Serum (FBS) (ThermoFisher) and 1% Pen Strep (ThermoFisher) at 37 °C. Twenty -four (24) hours prior to the transduction assay, cells are seeded in 24-well plates at a density of around 1 x 10⁵ cells/mL in DMEM + 10% FBS medium without antibiotics.

Cell transduction

[00191] The day of the transduction assay, when cells are around 90% confluent, cells are treated with MMVs at a concentration of approximately 4.88 x 10¹¹ parti cles/well. As negative controls, cells are treated with an equivalent amount of naked mRNA (0.5 pg/well), with an empty MMV (containing no RNA), and with a CP monomer (without assembly). As positive controls, Lipofectamine 2000 (ThermoFisher) is used for all previously mentioned conditions. Briefly, 1 pL Lipofectamine 2000 is diluted in 50 pL OptiMEM (ThermoFisher) in a separate tube for each condition. Next, naked mRNA, CP monomer, empty MMV or full MMV, is diluted in 50 pL OptiMEM medium and mixed into its corresponding Lipofectamine 2000 tube. The solutions are incubated for 20 minutes at room temperature to allow for complex formation, before adding them into the cells. Cells are incubated with the Lipofectamine complexes for 6 hours before changing their medium to fresh DMEM + 10% FBS medium. Cells transduced with naked particles, CP or mRNA, with or without Lipofectamine 2000, are incubated for 24 hours at 37 °C to allow for mRNA transduction and translation.

Reporter assay

[00192] Twenty-four (24) hours after transduction, the reporter mRNA expression is assessed. For GFP, samples are imaged under a fluorescent microscope. In addition, fluorescence could be quantified using a plate reader. For luciferase, bioluminescence is assessed using a plate reader and the Firefly Luciferase Glow Assay kit (ThermoFisher). Briefly, cells are lysed with cell lysis buffer and plated into a 96-well white/clear bottom plate (ThermoFisher) in triplicates. Each well is treated with a working solution containing D- luciferin. After 10 minutes, the light output of each well is analyzed using a plate reader. [00193] To assess for reporter mRNA presence in the cells, RNA is extracted from the cell lysates and the reporter mRNA is amplified with RT-qPCR. The primers used for this reaction for GFP and luciferase were prepared by ThermoFisher. For this assay, a housekeeping gene such as GAPDH is used as control for normalization.

Example 5: Testing MMV biodistribution in mice

MMV sourcing and cocktail generation

[00194] To test the ability of MMVs to access mammalian tissues, mice are dosed with either 1) commercially available, 2) recombinantly produced (as described above), or 3) in-planta produced plant viruses, multiplexed in a cocktail containing between 2 and 15 different virus species. MMVs tested in this example are included in TABLE 1. The viruses are obtained as described in the examples above and each type of virus is quantified and combined into a cocktail at equal amounts.

Animal treatment and tissue collection

[00195] Female C57BL/6 mice of 7-8 weeks of age are dosed once with the virus cocktail either through oral gavage or intravenous (i.v.) injection, at doses similar to previous studies (Rae et al. (2005) Virology 343(2) 224-235; Affonso de Oliveira et al. (2022) ACS Nano.

16(11): 18315-18328; Singh et al. (2007), Vishnu Vardhan et cd. (2016) Arch Virol.

161 (10):2673-81 ; Le et al. (2019) Biomacromolecules 20(l):469-477, Wu etal. (2013) Biomacromolecules 14(11):4032-4037). Treatment groups include 7 mice treated with 100 pg and 500 pg of MMVs for i.v. and oral gavage, respectively, and 3 mice treated with PBS as negative controls. Between 24 and 48 hours after treatment, mice are sacrificed to collect different tissues to test for the presence of MMVs. Samples include blood, spleen, lungs, liver, brain, heart, kidneys, bone marrow, small intestine (duodenumjejunum, ileum), and pancreas. Tissues are collected and stored in RNAlater solution (ThermoFisher) until their processing for RNA extraction. RNA extraction and virus detection through RT-qPCR

[00196] After organ collection, total RNA is extracted using RNA extraction kits and viral genomes are measured through RT-qPCR. Briefly, qPCR primers for a viral gene that allows the identification of the different species within the cocktail used, are designed, and used for viral genome detection within each tissue’s total RNA extract. Genes used for MMV identification include the CP and/or RdRp genes. For certain experiments, multiple reactions are run simultaneously using a multiplexed qPCR protocol.

[00197] To test for recombinantly produced or reassembled MMVs, MMVs are assembled with a unique RNA sequence in each different MMV in the cocktail, which serves as a molecular barcode for their identification. In this case, total RNA extracted from each tissue is processed for RNA sequencing. Then, the barcode RNA is amplified to serve as template for a sequencing library preparation (Illumina).

Virus detection through fluorescence

[00198] Viral particles are labelled with fluorescent dyes (as described above), administered to mice, and detected in homogenized organs using fluorescence quantification.

INCORPORATION BY REFERENCE

[00199] The entire disclosure of each of the patent and scientific documents referred to herein is incorporated by reference for all purposes.

EQUIVALENTS

[00200] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein.

Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method of delivering a therapeutic agent to a specific target tissue or organ in a patient in need thereof the method comprising administering to the patient a therapeutically effective amount of a therapeutic composition comprising: a protein complex comprising a plant viral capsid protein or fragment thereof and a therapeutic agent, and a pharmaceutically acceptable excipient; thereby delivering the therapeutic agent to the patient's specific target tissue or organ.

2. A method of delivering a heterologous nucleic acid to a specific target tissue or organ in a patient in need thereof the method comprising administering to the patient a therapeutically effective amount of a composition comprising: a complex comprising a plant viral capsid protein or fragment thereof and a heterologous nucleic acid, and optionally, a pharmaceutically acceptable excipient; thereby delivering the heterologous nucleic acid to the patient’s specific target tissue or organ, optionally wherein the heterologous nucleic acid is expressed in the specific target tissue or organ.

3. A method of delivering a therapeutic agent to a specific target tissue or organ in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a therapeutic composition comprising: a complex comprising a plant viral capsid protein or fragment thereof and a therapeutic agent, and optionally, a pharmaceutically acceptable excipient; thereby delivering the therapeutic agent to the patient’s specific target tissue or organ, wherein the specific target tissue is selected from the group consisting of esophageal, thy roid, nerve, blood, muscle, breast, pituitary, lung, liver, heart, or artery -coronary tissue.

4. A method of delivering a therapeutic agent to a specific target tissue or organ in a patient in need thereof the method comprising administering to the patient a therapeutically effective amount of a therapeutic composition comprising: a complex comprising a plant viral capsid protein or fragment thereof and a therapeutic agent, and optionally, a pharmaceutically acceptable excipient; wherein the viral capsid protein or fragment thereof is a Bromoviridae, Alphaflexiviridae, Virgaviridae, Potyviridae. Solemoviridae, Closteroviridae, or Betaflexiviridae viral capsid protein or fragment thereof.

5. The method of claim 4, wherein the viral capsid protein or fragment thereof is an Alfamovirus, Anulavirus, Bromovirus, Cucumovirus, liarvirus, or Oleavirus viral capsid protein or fragment thereof.

6. The method of claim 5, wherein the viral capsid protein or fragment thereof is an alfalfa mosaic virus, a brome mosaic virus, a lilac leaf chlorosis virus, an apple necrotic mosaic virus, or a cowpea chlorotic mottle virus viral capsid protein or fragment thereof.

7. The method of claim 4, wherein the viral capsid protein or fragment thereof is an Allexivirus, Botrexvirus, Lolavirus, Platypuvirus, Poiexvirus. or Sclerodarnavirus viral capsid protein or fragment thereof.

8. The method of claim 7, wherein the viral capsid protein or fragment thereof is a clover yellow mosaic vims capsid protein or fragment thereof, a pepino mosaic virus capsid protein or fragment thereof, or a white clover mosaic virus capsid protein or fragment thereof.

9. The method of claim 4, wherein the viral capsid protein is aFurovirus, Goravirus, Hordeivirus, Pecluvirus, Pomovirus, Tohamovirus. or Tobravirus viral capsid protein or fragment thereof.

10. The method of claim 9, wherein the viral capsid protein or fragment thereof is a cucumber green mottle mosaic virus capsid protein or fragment thereof, a tobacco mild green mosaic virus capsid protein or fragment thereof, a tobacco mosaic virus capsid protein or fragment thereof, a tomato mosaic vims capsid protein or fragment thereof, or a tropical soda apple mosaic virus capsid protein or fragment thereof.

11. The method of claim 4, wherein the viral capsid protein or fragment thereof is an Arepavirus, Bevemovirus, Brambyvirus, Bymovirus. Celavirus, Ipomovirus. Macluravirus , Poacevirus. Potyvirus, Roymovirus, Rymovirus, or Tritimovirus viral capsid protein or fragment thereof.

12. The method of claim 11, wherein the viral capsid protein or fragment thereof is a pepper mild mottle virus capsid protein or fragment thereof or a potato virus Y capsid protein or fragment thereof.

13. The method of claim 4, wherein the viral capsid protein or fragment thereof is an Enamovirus, Polemovirus. Polerovirus, or Sobemovirus viral capsid protein or fragment thereof

14. The method of claim 13, wherein the viral capsid protein or fragment thereof is a poinsettia latent virus capsid protein or fragment thereof.

15. The method of claim 4, wherein the viral capsid protein or fragment thereof is a Carlavirus, Foveavirus, Robigovlrus, Caplllovlrus, Chordovirus, Cilrivirus, Divavirus. Prunevirus. Ravavirus. Tepovirus. Trichovirus, Vitivirus, or Wamavirus viral capsid protein or fragment thereof.

16. The method of claim 15, wherein the viral capsid protein or fragment thereof is an apple stem pitting virus capsid protein or fragment thereof.

17. The method of claim 4, wherein the plant viral capsid protein or fragment thereof is a Ampelovirus, Closterovirus, Crinivirus, or Velarivirus capsid protein or fragment thereof.

18. The method of claim 17, wherein the plant viral capsid protein or fragment thereof is a citrus tristeza virus capsid protein or fragment thereof.

19. The method of any one of claims 1-18, wherein the plant virus capsid protein or fragment is a modified plant virus capsid protein.

20. The method of any one of claims 1-18, wherein the plant vims capsid protein or fragment is naturally occurring.

21. The method of any one of claims 1-20. wherein an amino acid sequence of the plant viral capsid protein or fragment thereof is at least 70% identical to a naturally occurring plant viral capsid protein or fragment thereof.

22. The method of any one of claims 1-21, wherein the fragment of the plant viral capsid protein comprises at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, or at least 450 consecutive amino acids present in a corresponding full-length plant viral capsid protein.

23. The method of any one of claims 1 and 3-22, wherein the therapeutic agent is a non- naturally occurring nucleic acid.

24. The method of any one of claims 1 and 3-23, wherein the therapeutic agent is a heterologous nucleic acid.

25. The method of any one of claims 2, 23, and 24, wherein the non-naturally occurring or heterologous nucleic acid is a DNA or an RNA.

26. The method of any one of claims 2 and 23-25. w herein the non-naturally occurring or heterologous nucleic acid is expressed in the specific target tissue or organ.

27. The method of any one of claims 1, 2, and 4-26, wherein the specific target tissue is selected from the group consisting of adipose, adrenal, artery, blood, brain, breast, cervix, colon, esophagus, heart, kidney, liver, lung, muscle, nerve, ovary, pancreas, pituitary, prostate, salivary' gland, small intestine, spleen, stomach, testis, thyroid, and uterus tissue.

28. The method of any one of claims 1-27, wherein the delivery is oral or intravenous delivery.

29. A composition comprising a plant viral capsid protein or fragment thereof and a heterologous therapeutic agent, and wherein the viral capsid protein or fragment thereof is a Bromoviridae, Alphaflexiviridae. Virgciviridae, Potyviridae, Solemoviridae, Closteroviridae, or Betaflexiviridae viral capsid protein or fragment thereof.

30. The composition of claim 29, wherein the heterologous therapeutic agent is a heterologous nucleic acid.

31. The composition of claim 30, wherein the heterologous nucleic acid is a DNA or an RNA.

32. The composition of claim 31, wherein the heterologous nucleic acid is an RNA.

33. The composition of any one of claims 29-32, wherein an amino acid sequence of the plant viral capsid protein or fragment thereof is at least 90% identical to a naturally occurring plant viral capsid protein or fragment thereof.

34. The composition of any one of claims 31-33, wherein the fragment of the plant viral capsid protein comprises at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, or at least 450 consecutive amino acids present in a corresponding full-length plant viral capsid protein.

35. The composition of any one of claims 29-34 , wherein the viral capsid protein or fragment thereof is an Alfamovirus, Anulavirus, Bromovirus, Cucumovirus , liarvirus, or Oleavirus viral capsid protein or fragment thereof.

36. The composition of claim 35, wherein the viral capsid protein or fragment thereof is an alfalfa mosaic virus capsid protein or fragment thereof, a brome mosaic virus capsid protein or fragment thereof, a lilac leaf chlorosis virus capsid protein or fragment thereof, an apple necrotic mosaic virus capsid protein or fragment thereof, or a cowpea chlorotic mottle virus capsid protein or fragment thereof.

37. The composition of any one of claims 29-34, wherein the viral capsid protein or fragment thereof is an Allexivirus, Botr exvirus, Lolavirus, Platypuvirus, Potexvirus, or Sclerodarnavirus viral capsid protein or fragment thereof.

38. The composition of claim 37, wherein the viral capsid protein or fragment thereof is a clover yellow- mosaic virus capsid protein or fragment thereof, a pepino mosaic virus capsid protein or fragment thereof, or a white clover mosaic virus capsid protein or fragment thereof.

39. The composition of any one of claims 29-34, wherein the viral capsid protein or fragment thereof is a Furovirus, Goravirus, Hordeivirus, Pecluvirus, Pomovirus, Tobamovirus, or Tobravirus viral capsid protein or fragment thereof.

40. The composition of claim 39, wherein the viral capsid protein or fragment thereof is a cucumber green mottle mosaic virus capsid protein or fragment thereof, a tobacco mild green mosaic virus capsid protein or fragment thereof, a tobacco mosaic virus, a tomato mosaic virus capsid protein or fragment thereof, or a tropical soda apple mosaic virus capsid protein or fragment thereof.

41. The composition of any one of claims 29-34, wherein the viral capsid protein or fragment thereof is aArepavirus, Bevemovirus, Brambyvirus, Bymovirus, Celavirus, Ipomovirus, Macluravirus , Poacevirus, Potyvirus, Roymovirus, Rymovirus, or Tritimovirus viral capsid protein or fragment thereof.

42. The composition of claim 41, wherein the viral capsid protein or fragment thereof is a pepper mild mottle virus capsid protein or fragment thereof or a potato virus Y capsid protein or fragment thereof.

43. The composition of any one of claims 29-34, wherein the viral capsid protein or fragment thereof is a Enamovirus, Polemovirus, Polerovirus, or Sobemovirus viral capsid protein or fragment thereof.

44. The composition of claim 43, wherein the viral capsid protein or fragment thereof is a poinsettia latent virus capsid protein or fragment thereof.

45. The composition of any one of claims 29-34, wherein the viral capsid protein or fragment thereof is a Carlavirus, Foveavirus, Robigovirus, Capillovirus, Chordovirus, Citrivirus, Divavirus, Prunevirus, Ravavirus, Tepovirus, Trichovirus, Vitivirus, or Wamavirus viral capsid protein or fragment thereof.

46. The composition of claim 45, wherein the viral capsid protein or fragment thereof is an apple stem pitting virus capsid protein or fragment thereof.

47. The composition of any one of claims 29-34, wherein the viral capsid protein or fragment thereof is aAmpelovirus, Closter ovirus, Crinivirus, or Velarivirus capsid protein or fragment thereof.

48. The composition of claim 47, wherein the viral capsid protein or fragment thereof is a citrus tristeza virus capsid protein or fragment thereof.

49. A pharmaceutical composition comprising the composition of any one of claims 29-

48and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is suitable for oral delivery.

50. A pharmaceutical composition comprising the composition of any one of claims 29- 48 and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is suitable for intravenous delivery.

51. A nucleic acid or nucleic acids encoding the composition of any one of claims 29-48.

52. A host cell comprising a composition of any one of claims 29-48 or the nucleic acid or nucleic acids of claim 51.

53. The host cell of claim 52, wherein the host cell is a plant cell.

54. A method of delivering a therapeutic agent to a specific target tissue or organ in a patient, the method comprising administering to the patient a therapeutically effective amount of a therapeutic composition comprising: a complex comprising an Alfamovirus capsid protein or a fragment thereof and the therapeutic agent; and optionally, a pharmaceutically acceptable excipient; thereby delivering the therapeutic agent to the patient's specific target tissue or organ, wherein the specific target tissue or organ is selected from brain, prostate, esophagus, small intestine, breast, salivary gland, nerve, thyroid, adrenal, adipose, lung, muscle, heart, colon, artery, blood, liver, stomach, testis, pancreas, and ovary.

55. The method of claim 54, wherein the Alfamovirus capsid protein or fragment thereof is an alfalfa mosaic virus capsid protein or fragment thereof.

56. The method of claim 54 or 55, wherein the therapeutic agent is a heterologous nucleic acid.

57. The method of claim 56, wherein the heterologous nucleic acid is DNA or RNA.

58. The method of any one of claims 54-57, wherein an amino acid sequence of the Alfamovirus capsid protein or fragment thereof is at least 90% identical to a naturally occurring Alfamovirus capsid protein or fragment thereof.

59. The method of any one of claims 54-58, wherein the fragment of the Alfamovirus capsid protein comprises at least 50. at least 100, at least 150. at least 200, at least 250. at least 300, at least 350, at least 400, or at least 450 consecutive amino acids present in a corresponding full-length Alfamovirus capsid protein.

60. A method of delivering a therapeutic agent to a pituitary tissue in a patient, the method comprising administering to the patient a therapeutically effective amount of a therapeutic composition comprising: a complex comprising an Foveavirus capsid protein or a fragment thereof and the therapeutic agent; and optionally, a pharmaceutically acceptable excipient; thereby delivering the therapeutic agent to the patient’s pituitary tissue.

61. The method of claim , wherein the Foveavirus capsid protein or a fragment thereof is an apple stem pitting virus capsid protein or fragment thereof.

62. The method of claim 60 or 61, wherein the therapeutic agent is a heterologous nucleic acid.

63. The method of claim 62, wherein the heterologous nucleic acid is DNA or RNA.

64. The method of any one of claims 60-63. wherein an amino acid sequence of the Foveavirus capsid protein or fragment thereof is at least 90% identical to a naturally occurring Foveavirus capsid protein or fragment thereof.

65. The method of any one of claims 60-64. wherein the fragment of the Foveavirus capsid protein comprises at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, or at least 450 consecutive amino acids present in a corresponding full-length Foveavirus capsid protein.

66. A method of delivering a therapeutic agent to a specific tissue or organ in a patient, the method comprising administering to the patient a therapeutically effective amount of a therapeutic composition comprising: a complex comprising a Bromovirus capsid protein or a fragment thereof and the therapeutic agent; and optionally, a pharmaceutically acceptable excipient; thereby delivering the therapeutic agent to the patient's ovary⁷, prostate, brain, breast, adipose, nerve, artery, small intestine, thyroid, blood, colon, lung, liver, muscle, esophagus, adrenal, or uterus tissue.

67. The method of claim 66, wherein the Bromovirus capsid protein or a fragment thereof is a brome mosaic virus capsid protein or fragment thereof, a lilac leaf chlorosis virus capsid protein or fragment thereof, or a cowpea chlorotic mottle virus capsid protein or fragment thereof.

68. The method of claim 66 or 67. wherein the therapeutic agent is a heterologous nucleic acid.

69. The method of claim 65, wherein the heterologous nucleic acid is DNA or RNA.

70. The method of any' one of claims 66-69, wherein an amino acid sequence of the Bromovirus capsid protein or fragment thereof is at least 90% identical to a naturally occurring Bromovirus capsid protein or fragment thereof.

71. The method of any one of claims 66-70, wherein the fragment of the Bromovirus capsid protein comprises at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, or at least 450 consecutive amino acids present in a corresponding full-length Bromovirus capsid protein.

72. A method of delivering a therapeutic agent to a specific tissue or organ in a patient, the method comprising administering to the patient a therapeutically effective amount of a therapeutic composition comprising: a complex comprising a Potexvirus capsid protein or a fragment thereof and the therapeutic agent; and optionally, a pharmaceutically acceptable excipient; thereby delivering the therapeutic agent to the patient's artery, blood, brain, esophagus, adipose, stomach, breast, adrenal, thyroid, cervix, small intestine, spleen, colon, testis, ovary, nerve, muscle, heart .

73. The method of claim 72, wherein the Potexvirus capsid protein or fragment thereof is a clover yellow mosaic virus capsid protein or fragment thereof, a white clover mosaic virus capsid protein or fragment thereof, or a pepino mosaic virus capsid protein or fragment thereof.

74. The method of claim 72 or 73, wherein the therapeutic agent is a heterologous nucleic acid.

75. The method of claim 74, wherein the heterologous nucleic acid is DNA or RNA.

76. The method of any one of claims 72-75, wherein an amino acid sequence of the Potexvirus capsid protein or fragment thereof is at least 90% identical to a naturally occurring Potexvirus capsid protein or fragment thereof.

77. The method of any one of claims 72-76, wherein the fragment of the Potexvirus capsid protein comprises at least 50, at least 100, at least 150. at least 200, at least 250. at least 300, at least 350, at least 400, or at least 450 consecutive amino acids present in a corresponding full-length Potexvirus capsid protein.

78. A method of delivering a therapeutic agent to a specific tissue or organ in a patient, the method comprising administering to the patient a therapeutically effective amount of a therapeutic composition comprising: a complex comprising a Tobamovirus capsid protein or a fragment thereof and the therapeutic agent; and optionally, a pharmaceutically acceptable excipient; thereby delivering the therapeutic agent to the patient’s specific tissue or organ, wherein the specific tissue or organ is artery, brain, adipose, lung, blood, nerve, breast, esophagus, muscle, thyroid, testis, kidney, pituitary, prostate, liver, stomach, adrenal, heart, uterus, colon, small intestine, ovary, pancreas, spleen .

79. The method of claim 78, wherein the Tobamovirus capsid protein or fragment thereof is a tobacco mild green mosaic virus capsid protein or fragment thereof, a tobacco mosaic virus capsid protein or fragment thereof, a tomato mosaic virus capsid protein or fragment thereof, a tropical soda apple mosaic virus capsid protein or fragment thereof, a pepper mild mottle virus capsid protein or fragment thereof, or a cucumber green mottle mosaic virus capsid protein or fragment thereof.

80. The method of claim 78 or 79, wherein the therapeutic agent is a heterologous nucleic acid.

81. The method of claim 80, wherein the heterologous nucleic acid is DNA or RNA.

82. The method of any one of claims 78-81, wherein an amino acid sequence of the Tobamovirus capsid protein or fragment thereof is at least 90% identical to a naturally occurring Tobamovirus capsid protein or fragment thereof.

83. The method of any one of claims 78-82, wherein the fragment of the Tobamovirus capsid protein comprises at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, or at least 450 consecutive amino acids present in a corresponding full-length Tobamovirus capsid protein.

84. A method of delivering a therapeutic agent to a heart tissue in a patient, the method comprising administering to the patient a therapeutically effective amount of a therapeutic composition comprising: a complex comprising a Polemovirus capsid protein or a fragment thereof and the therapeutic agent; and optionally, a pharmaceutically acceptable excipient; thereby delivering the therapeutic agent to the patient’s heart tissue.

85. The method of claim 84, wherein the Polemovirus capsid protein or fragment thereof is a poinsettia latent virus capsid protein or fragment thereof.

86. The method of claim 84 or 85. wherein the therapeutic agent is a heterologous nucleic acid.

87. The method of claim 86, wherein the heterologous nucleic acid is DNA or RNA.

88. The method of any one of claims 84-87, wherein an amino acid sequence of the Polemovirus capsid protein or fragment thereof is at least 90% identical to a naturally occurring Polemovirus capsid protein or fragment thereof.

89. The method of any one of claims 84-88, wherein the fragment of the Polemovirus capsid protein comprises at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, or at least 450 consecutive amino acids present in a corresponding full-length Polemovirus capsid protein.

90. A method of delivering a therapeutic agent to an artery tissue in a patient, the method comprising administering to the patient a therapeutically effective amount of a therapeutic composition comprising: a complex comprising a Potyvirus capsid protein or a fragment thereof and the therapeutic agent; and optionally, a pharmaceutically acceptable excipient; thereby delivering the therapeutic agent to the patient's artery tissue.

91. The method of claim 90, wherein the Potyvirus capsid protein or fragment thereof is a potato virus Y virus capsid protein or fragment thereof.

92. The method of claim 90 or 91, wherein the therapeutic agent is a heterologous nucleic acid.

93. The method of claim 92. wherein the heterologous nucleic acid is DNA or RNA.

94. The method of any one of claims 91-93, wherein an amino acid sequence of the Potyvirus capsid protein or fragment thereof is at least 90% identical to a naturally occurring Potyvirus capsid protein or fragment thereof.

95. The method of any one of claims 91-94, wherein the fragment of the Potyvirus capsid protein comprises at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400. or at least 450 consecutive amino acids present in a corresponding full-length Potyvirus capsid protein.

96. A method of delivering a therapeutic agent to a specific organ or tissue in a patient, the method comprising administering to the patient a therapeutically effective amount of a therapeutic composition comprising: a complex comprising a Closterovirus capsid protein or a fragment thereof and the therapeutic agent; and optionally, a pharmaceutically acceptable excipient; thereby delivering the therapeutic agent to the patient's specific tissue or organ, wherein the specific tissue or organ is adipose, breast, artery, or nerve tissue.

97. The method of claim 96, wherein the Closterovirus capsid protein or fragment thereof is a citrus tristeza virus capsid protein or fragment thereof.

98. The method of claim 96 or 97, wherein the therapeutic agent is a heterologous nucleic acid.

99. The method of claim 98, wherein the heterologous nucleic acid is DNA or RNA.

100. The method of any one of claims 96-99, wherein an amino acid sequence of the Closterovirus capsid protein or fragment thereof is at least 90% identical to a naturally occurring Closterovirus capsid protein or fragment thereof.

101. The method of any one of claims 96-100, wherein the fragment of the Closterovirus capsid protein comprises at least 50. at least 100, at least 150. at least 200, at least 250. at least 300, at least 350, at least 400, or at least 450 consecutive amino acids present in a corresponding full-length Closterovirus capsid protein.

102. The method of any one of claims 54-101, wherein the therapeutic composition is orally administered to the patient.

103. The method of any one of claims 54-101, wherein the therapeutic composition is intravenously administered to the patient.