Polynucleotides containing antigenic payloads

Abstract

Providing a polynucleotide comprising an antigenic payload. [Solution] Polynucleotides, scaffolds, and cassettes are disclosed and described herein. In particular, these polynucleotides can have a formula comprising signal / leader-payload-PRM, where the signal / leader encodes a signal sequence, a leader sequence, or a sorting sequence in frame with and upstream of the payload, the payload is an antigenic payload region, a detectable agent, and a therapeutic agent, and the PRM encodes all or a portion of at least one parent receptor molecule region from one or more isoforms or proteins selected from the group consisting of CD1d, CD1e, LDLR, LDLRP, and LRP1 proteins.

Description

[Background technology]

[0001] background Stimulation of both CD8+ and CD4+ lymphocytes is desirable for effective immunotherapy with recombinant vaccines, and in recent years, vaccines based on DNA or RNA nucleic acids have become increasingly important. However, these types of vaccines have little or no stimulation of CD4+ lymphocytes, which is an important factor for the effectiveness of recombinant vaccines. Therefore, several genetic manipulations have been developed to increase the immunogenicity of vaccines, for example, by changing the primary sequence of fusions to foreign epitopes derived from bacteria or viruses, and by chimeric products consisting of antigens and immunomodulators such as cytokines or chemokines. Summary of the Invention

[0002] Abstract The present disclosure provides examples of polynucleotides, scaffolds, and cassettes. The present disclosure also provides examples of fusion molecules containing one or more polypeptide antigens, such as tumor antigens, neoantigens, patient-specific antigens, shared antigens, and infectious pathogen antigens, engineered as payloads incorporating one or more regions of a parent receptor molecule, such as a signal sequence, extracellular region, transmembrane region, and / or cytoplasmic region, for antigen presentation on the surface of a cell or in a specific cellular compartment. The present disclosure also provides examples of polynucleotides and scaffolds that can be used for many applications, including inducing immune or therapeutic responses in animals. Specifically, the polynucleotides and scaffolds of the present disclosure are based on designs that utilize CD1 and other cellular receptors.

[0003] The present disclosure also provides alternative vaccine modalities, including scaffolds and cassettes incorporating antigenic payloads for use as vaccines.

[0004] The disclosure further describes polynucleotides, e.g., DNA, RNA, or mRNA, that encode the scaffolds and cassettes, and methods of making and using them.

[0005] One embodiment of the present disclosure relates to a polynucleotide having the formula: signal / leader-payload-TMD-CYD, where signal / leader encodes a signal sequence, leader sequence, or sorting sequence in frame with and upstream of the payload; payload is selected from the group consisting of an antigenic payload region, a detectable agent, and a therapeutic agent; TMD encodes a portion of a transmembrane region from one or more proteins or isoforms selected from the group consisting of CD1d, CD1e, LDLR, LDLRP, and LRP1 proteins; and CYD encodes all or a portion of a cytoplasmic region from one or more proteins or isoforms selected from the group consisting of CD1d, CD1e, LDLR, LDLRP, and LRP1 proteins.

[0006] In some embodiments, the payload is an antigenic payload region having the formula (An1)n-Xo-(An2)p, comprising a first encoded antigenic payload (An1), where n is an integer between 1 and 10; an encoded linker region (X), where o is an integer between 0 and 10; and a second encoded antigenic payload (An2), where p is an integer between 0 and 10.

[0007] In some embodiments, the first encoded antigenic payload or the second encoded antigenic payload encodes all or a portion of a tumor antigen or an infectious agent antigen.

[0008] In some embodiments, the first encoded antigenic payload or the second encoded antigenic payload comprises the sequence SIINFEKL.

[0009] In certain embodiments, the payload is a detectable agent selected from the group consisting of a small organic molecule, an inorganic compound, a nanoparticle, an enzyme or enzyme substrate, a fluorescent material, a luminescent material, a bioluminescent material, a chemiluminescent material, a radioactive material, a contrast agent, gadolinium, iron oxide, single crystalline iron oxide nanoparticles, ultrasmall superparamagnetic iron oxide, manganese chelate, barium sulfate, an iodinated contrast agent, a microbubble, and a perfluorocarbon.

[0010] In one embodiment, the TMD and CYD are from the same isoform or protein, hi another embodiment, the TMD and CYD are from different isoforms or proteins.

[0011] In certain embodiments, the signal / leader encodes a signal sequence, leader sequence, or sorting sequence from the same isoform or protein as the TMD, CYD, or both. In one embodiment, the TMD encodes the sequence MGLIALAVLACLLFLLIVGFT. In another embodiment, the CYD encodes the sequence SRFKRQTSYQGVL. In yet another embodiment, the signal sequence encodes the sequence MGCLLFLLLWALLQAWGSA.

[0012] One aspect of the present disclosure relates to a polynucleotide having the formula: signal / leader-payload-PRM, where signal / leader encodes a signal sequence, leader sequence, or sorting sequence in frame with and upstream of the payload, payload is selected from the group consisting of an antigenic payload region, a detectable agent, and a therapeutic agent, and PRM encodes all or a portion of at least one parent receptor molecule region from one or more proteins or isoforms selected from the group consisting of CD1d, CD1e, LDLR, LDLRP, and LRP1 proteins.

[0013] In some embodiments, the parent receptor molecule is selected from the group consisting of an extracellular domain, a transmembrane domain, and a cytoplasmic domain.

[0014] One aspect of the present disclosure pertains to host cells comprising at least one of the disclosed polynucleotides.

[0015] One aspect of the present disclosure relates to a pharmaceutical composition comprising at least one of the disclosed polynucleotides or host cells. In some embodiments, the pharmaceutical composition is in the form of a vaccine. In other embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients or one or more additional active pharmaceutical ingredients. In other embodiments, the pharmaceutically acceptable excipient is selected from the group consisting of anti-adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes, emollients, emulsifiers, fillers, film-forming agents or coatings, flavorings, fragrances, glidants, lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and hydration water.

[0016] One aspect of the present disclosure relates to a therapeutic polynucleotide comprising at least one polynucleotide of the present disclosure formulated with a delivery vehicle.In one aspect, the polynucleotide is encapsulated in the delivery vehicle.In another aspect, the delivery vehicle is selected from the group consisting of amphiphilic molecules, amino lipid-added peptides, and tertiary amino lipid-added cationic peptides.

[0017] One aspect of the present disclosure relates to a therapeutic composition comprising a therapeutic polynucleotide. In another aspect, the therapeutic composition is in the form of a vaccine. In a further aspect, the therapeutic composition further comprises one or more therapeutically acceptable excipients or one or more additional therapeutically active ingredients. In some aspects, the therapeutically acceptable excipient is selected from the group consisting of anti-adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes, emollients, emulsifiers, fillers, film-forming agents or coatings, flavorings, fragrances, glidants, lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and water for hydration.

[0018] One aspect of the present disclosure includes administering at least one disclosed pharmaceutical or therapeutic composition, particularly a therapeutically effective dose, a prophylactically effective dose, or a suitable imaging dose of a pharmaceutical or therapeutic composition, to a subject in need thereof.

[0019] One aspect of the present disclosure includes a method of treating, vaccinating, or immunizing a subject in need thereof, the method comprising administering to the subject at least one disclosed polynucleotide, host cell, at least one disclosed pharmaceutical composition, or at least one disclosed therapeutic composition.

[0020] In one embodiment, the subject is a mammal, hi another embodiment, the subject is a human.

[0021] In one embodiment of the present disclosure, the polynucleotide of the present disclosure (including, but not limited to, a host cell of the present disclosure, a pharmaceutical composition of the present disclosure, a therapeutic polynucleotide of the present disclosure, a therapeutic composition of the present disclosure, or a method of the present disclosure) is for one of the following: a) enabling antigen processing and presentation; b) transporting proteins into the antigen presentation pathway; c) improving T cell activation; d) increasing clonal diversity; and e) any combination thereof.

[0022] One aspect of the present disclosure relates to a polynucleotide having the formula [signal / leader]-[(An1)n-Xo-(An2)p]-[TMD]-[CYD]], where [signal / leader] encodes any signal, leader, or sorting sequence in-frame with and upstream of the antigenic payload region; [(An1)n-Xo-(An2)p] comprises an antigenic payload region, which comprises (a) a first encoded antigenic payload (An1) that may be replicated "n" times, (b) a second encoded antigenic payload (An1) that may be replicated "n" times, and (c) a second encoded antigenic payload (An1) that may be replicated "o" times, optionally and (c) optionally, if present, a second encoded antigenic payload (An2) that can be replicated "p" times; the TMD encodes a portion of a transmembrane region from one or more proteins selected from the group consisting of CD1, LDLR, LDLRP and / or LRP1 proteins; and the CYD encodes all or a portion of a cytoplasmic region from one or more proteins selected from the group consisting of CD1, LDLR, LDLRP and / or LRP1 proteins.

[0023] In one embodiment, the TMD and CYD are derived from the CD1 isoform. In another embodiment, the TMD and CYD are derived from the CD1d isoform. In one embodiment, the TMD encodes the sequence MGLIALAVLACLLFLLIVGFT. In another embodiment, the CYD encodes the sequence SRFKRQTSYQGVL. In yet another embodiment, the signal sequence encodes the sequence MGCLLFLLLWALLQAWGSA. In another embodiment, the encoded antigenic payload comprises the sequence SIINFEKL.

[0024] Other aspects and features of the present disclosure will become apparent to those skilled in the art upon review of the following description of specific embodiments of the present disclosure in conjunction with the accompanying figures.

[0025] It should be understood that all combinations of the foregoing concepts and additional concepts discussed in more detail below (to the extent such concepts are not mutually inconsistent) are contemplated as part of the inventive subject matter disclosed herein and may be used to achieve the advantages described herein. [Brief explanation of the drawings]

[0026] [Figure 1A] FIG. 1A shows, in one example, flow cytometry results comparing antigen presentation in the JAWS dendritic cell model (naive and murine) for epitopes in different scaffold contexts.

[0027] [Figure 1B] FIG. 1B shows, in one example, flow cytometry results comparing antigen presentation in the JAWS dendritic cell model (human CD1d and human CD1b) for epitopes in different scaffold contexts.

[0028] [Figure 2A] 2A and 2B show, in one example, flow cytometry results of mRNA hCD1d MHC trafficking signals enhance CD8 T cell reactivation in vitro. [Figure 2B] 2A and 2B show, in one example, flow cytometry results of mRNA hCD1d MHC trafficking signals enhance CD8 T cell reactivation in vitro.

[0029] [Figure 3A] FIG. 3A illustrates, in one example, a comparison of the IFNg T cell responses observed using the Sec-hCD1d MHC sorting sequence against peptide, native pp65 mRNA, and pp65 mRNA Sec-MITD.

[0030] [Figure 3B]FIG. 3B shows, in one example, flow cytometry results of activated CD8 T cells in samples treated with Sec-hCD1d pp65 mRNA nanoparticles compared to native pp65 mRNA and pp65 mRNA Sec-MITD.

[0031] [Figure 4A] Figure 4A shows a comparison of CD8 T cell proliferation in cultures treated with Sec-hCD1d at 1 μg / mL of non-coding mRNA nanoparticles, 2 μg / mL of pp65 peptide, and 1 μg / mL of pp65-encoding mRNA.

[0032] [Figure 4B] FIG. 4B shows, in one example, flow cytometry results for T2 target cells.

[0033] [Figure 4C] FIG. 4C shows, in one example, flow cytometry results of naive target cells, peptide-induced CD8 T cells, and mRNA-induced CD8 T cells.

[0034] [Figure 4D] FIG. 4D shows, in one example, a comparison of %PI positive target cells for peptide-induced CD8 T cells, mRNA-induced CD8 T cells, antigen-loaded T2 target cells, and antigen-negative T2 target cells.

[0035] [Figure 5] FIG. 5 shows, in one example, clonal diversity among CD8 T cells sorted from PBMCs treated with pp65 Sec-hCD1d mRNA nanoparticles compared to those treated with pp65 peptide.

[0036] [Figures 6A-6B] Figures 6A and 6B show, in one example, HPV16 E7 protein expression in HEK293. DETAILED DESCRIPTION OF THE INVENTION

[0037] Detailed Description composition

[0038] scaffold

[0039] The scaffolds of the present disclosure are derived from one or more parent polypeptides, such as one or more regions of receptor molecules, which may include, but are not limited to, the CD1, LDLR, LDLRP, and / or LRP1 family of receptors or proteins.

[0040] In some embodiments, the parent molecule is selected from the CD1 glycoprotein family of receptors. CD1 proteins are encoded by a genetic locus on human chromosome 1. This region encodes five CD1 isoforms (CD1a-e). These proteins are expressed exclusively intracellularly, except for CD1e, which is involved in lipid processing and editing for presentation by other human CD1 isoforms and is expressed on the cell surface where it functions as an antigen-presenting molecule. CD1 isoforms traffic around the cell by association with various chaperones, such as calnexin, calreticulin, and even B2M. Newly synthesized, unoccupied CD1 isoforms exit the ER and Golgi to the plasma membrane, then internalize and enter different compartments via tyrosine-based sorting motifs that enable binding to adaptor protein complexes 2 and 3. This facilitates entry into various endosomal compartments (early endosomes, recycling endosomes, late endosomes) and lysosomes, ultimately following a transport pathway similar to that of MHC I molecules. Furthermore, CD1 isomers are transported through these endosomal compartments to load with antigen, and in many cases, CD1 and MHC I and MHC II molecules are found within the same compartment.

[0041] cassette

[0042] Constructs and scaffolds for pharmaceutical and therapeutic compositions are disclosed herein. As provided herein, the pharmaceutical or therapeutic compositions described herein may include a scaffold that carries or is capable of carrying a payload, such as an antigenic payload, a detectable agent, or a therapeutic agent. The combination of a scaffold and an antigenic payload is referred to herein as a cassette. In examples where the composition is a vaccine composition, the composition includes a scaffold that carries and delivers an antigenic payload, and this combination of a scaffold and a payload is a vaccine cassette. As used herein, a "cassette" is a polynucleotide (or its encoded polypeptide) that encodes a scaffold and an antigenic payload. In one aspect, a cassette having a scaffold and its antigenic payload can function as a vaccine. A vaccine may refer to a substance prepared from a disease-causing agent, its product, or a synthetic substitute, used to stimulate antibody production and provide immunity against one or several diseases. The cassette may be configured for direct administration or may be encoded by one or more polynucleotides for expression in cells, or encoded by DNA, RNA, or mRNA for administration.

[0043] According to the present disclosure, the cassette has the following formula: 5'UTR--signal / leader-(An1)n-Xo-(An2)p-TMD-CYD-3'UTR-polyA

[0044] In the formula, "UTR" refers to the untranslated region located at the 5' and 3' ends of the mRNA construct, and "polyA" refers to the polyadenylation site of the mRNA.

[0045] Signal / leader refers to an appropriate signal sequence, leader sequence, or sorting sequence that is in frame with and upstream of the antigenic payload region.

[0046] (An1)n-Xo-(An2)p refers to any suitable antigenic payload region, including a first antigenic payload (An1), a spacer or linker region (X), and a second antigenic payload (An2). In some examples, n is an integer greater than 1. For example, n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some examples, n can be greater than 10. In some examples, o is an integer greater than 0. For example, o can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some examples, o can be greater than 10. In some examples, p is an integer greater than 0. For example, p can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some examples, p can be greater than 10.

[0047] TMD refers to all or part of a transmembrane region from one or more CD1 isoforms, LDLR, LDLRP and / or LRP1 proteins;

[0048] CYD refers to all or part of the cytoplasmic domain from one or more CD1 isoforms, LDLR, LDLRP and / or LRP1 proteins.

[0049] In some embodiments, the cassette may comprise the following formula: 5'UTR-signal / leader-payload-PRM-3'UTR-polyA

[0050] In the formula, "UTR" refers to the untranslated region located at the 5' and 3' ends of the mRNA construct, and "polyA" refers to the polyadenylation site of the mRNA.

[0051] Signal / leader refers to an appropriate signal sequence, leader sequence, or sorting sequence that is in frame with and upstream of the antigenic payload region.

[0052] Payload refers to an antigenic payload region, a detectable agent, or a therapeutic agent;

[0053] PRM refers to all or part of at least one parent receptor molecule region from one or more proteins selected from one or more CD1 isoforms, LDLR, LDLRP, and LRP1 proteins. For example, the parent receptor molecule region can be independently selected from the extracellular region, transmembrane region, or cytoplasmic region, or any combination thereof.

[0054] In some embodiments, the scaffolds or cassettes of the present disclosure comprise a plurality of one or more signal sequences and / or cytoplasmic sorting signals of CD1 isoforms, LDLR, LDLRP and / or LRP1 isoforms that facilitate antigen routing to the endosomal and / or lysosomal compartments, ultimately enabling processing and loading of MHC class I and MHC class II molecules.

[0055] In some embodiments, the signal sequence is selected from human CD1a (MLFLLLPLLAVLPGDG); human CD1b (MLLLPFQLLAVLFPGGN); human CD1c (MLFLQFLLLALLLPGGD); human CD1d (MGCLLFLLLWALLQAWGSA); human CD1e (MLLLFLLFEGLCCPGENTA); human LDLR (MGPWGWKLRWTVALLLAAAGT); or human LRP1 (MLTPPLLLLLPLLSALVAA). According to the present disclosure, the signal sequence can be derived from any protein. Signal sequences can range from 4 to 50 amino acids and can be chimeric, tandem, repeated, or inverted. Signal sequences can include those taught herein or any signal sequence that is at least about 50%, e.g., at least about 60, about 70, about 80, about 90, about 95, about 99% or more identical to those taught herein, so long as the signaling function is substantially retained.

[0056] In some embodiments, the transmembrane domain sequence is selected from human CD1a (GFIILAVIVPLLLLIGLALWF); human CD1b (IVLAIIVPSLLLLLCLALWYM); human CD1c (NWIALVVIVPLVILIVLVLWF); human CD1d (MGLIALAVLACLLFLLIVGFT); human CD1e (SIFLILICLTVIVTLVILVVV); human LDLR (ALSIVLPIVLLVFLCLGVFLLW); or human LRP1 (HIASILIPLLLLLLLVLVAGVVFWY). According to the present disclosure, the transmembrane domain sequence can be derived from any protein. The transmembrane sequence can range from 10 to 100 amino acids and can be chimeric, tandem, repeated, or inverted. The transmembrane sequence can include those taught herein or any transmembrane sequence that is at least—e.g., at least about 60, about 70, about 80, about 90, about 95, about 99% or more—identical to those taught herein, so long as function is substantially retained.

[0057] In some embodiments, the cytoplasmic domain sequence is selected from human CD1a (RKRCFC); human CD1b (RRRSYQNIP); human CD1c (KKHCSYQDIL); human CD1d (SRFKRQTSYQGVL); human CD1e (DSRLKKQSSNKNILSPHTPSPVFLMGANTQDTKNSRHQFCLAQVSWIKNRVLKKWKTRLNQLW); human LDLR (KNWRLKNINSINFDNPVYQKTTEDEVHICHNQDGYSYPSRQMVSLEDDVA); and human LRP1 (KRRVQGAKGFQHQRMTNGAMNVEIGNPTYKMYEGGEPDDVGGLLDADFALDPDKPTNFTNPVYATLYMGGHGSRHSLASTDEKRELLGRGPEDEIGDPLA). According to the present disclosure, the cytoplasmic domain sequence can be derived from any protein. The cytoplasmic sequence can range from 10 to 100 amino acids and can be chimeric, tandem, repeat, or inverted. The cytoplasmic sequence can include those taught herein or any cytoplasmic sequence that is at least about 50%, e.g., at least about 60, about 70, about 80, about 90, about 95, about 99% or more identical to those taught herein, so long as function is substantially retained.

[0058] It should be noted that the CD1e sequence structure also contains an N-terminal propeptide sequence (APQALQSYHLAA) that is processed in the endosomal compartment and is involved in membrane association, the absence of which results in a soluble molecule.

[0059] NCBI references for each of the above parent receptor molecules are provided in Table 1. [Table 1]

[0060] Antigenic payload

[0061] The scaffold of the present disclosure is engineered so that at least one antigenic payload can be loaded or incorporated therein. When an antigenic payload is combined with the scaffold, the construct is referred to herein as a cassette. In one aspect, the scaffold is a vaccine scaffold, and therefore the construct is referred to as a vaccine cassette.

[0062] Pharmaceutical and Therapeutic Compositions

[0063] A variety of diseases, disorders, and / or conditions can be treated with the pharmaceutical composition. The pharmaceutical composition may also include one or more pharmaceutically acceptable excipients or one or more additional active pharmaceutical ingredients.

[0064] Suitable non-limiting examples of pharmaceutically acceptable excipients include anti-adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes, emollients, emulsifiers, fillers, film formers or coatings, flavors, fragrances, glidants, lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and water of hydration.

[0065] A pharmaceutically active ingredient includes any substance or mixture of substances intended to provide pharmaceutical activity or other direct effect to diagnose, cure, alleviate, treat, or prevent a disease, disorder, and / or condition.

[0066] Therapeutic compositions can be used to treat a disease, or to prevent the occurrence of a disease, or to alleviate the symptoms of such a disease.

[0067] In some embodiments, a therapeutic composition can include at least one polynucleotide of the present disclosure formulated or encapsulated in a delivery vehicle. This formulated or encapsulated polynucleotide is also referred to as a "therapeutic polynucleotide." In some examples, the delivery vehicle is an amphiphilic molecule, a peptide, an aminolipid-added peptide, or a tertiary aminolipid-added cationic peptide. The therapeutic composition can also include one or more therapeutically acceptable excipients or one or more additional therapeutically active ingredients.

[0068] Suitable non-limiting therapeutically acceptable excipients include anti-adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes, emollients, emulsifiers, fillers, film formers or coatings, flavors, fragrances, glidants, lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and water of hydration.

[0069] Therapeutically active ingredients include any substance or mixture of substances intended to provide therapeutic activity or other direct effect to diagnose, cure, alleviate, treat, or prevent a disease, disorder, and / or condition.

[0070] Such diseases include cancer or infectious diseases. When cancer is the disease to be diagnosed, cured, alleviated, treated, or prevented with the pharmaceutical or therapeutic composition of the present disclosure, the antigenic payload can encode all or part of at least one tumor antigen. The tumor antigen can be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). When an infectious disease is the disease to be diagnosed, cured, alleviated, treated, or prevented with the pharmaceutical or therapeutic composition of the present disclosure, the antigenic payload can encode all or part of at least one infectious pathogen antigen.

[0071] One example of such a pharmaceutical or therapeutic composition is a vaccine. In one example of a vaccine of the present disclosure, the vaccine cassette comprises one or more antigenic payloads derived from proteins against which an immune response is desired.

[0072] As used herein, the term "cancer" refers to any of a variety of malignant neoplasms characterized by the proliferation of undifferentiated cells that tend to invade surrounding tissues and metastasize to new body sites, and also refers to pathological conditions characterized by such malignant neoplastic growth.Cancer can be a tumor or hematological malignancy, including, but not limited to, all types of lymphoma / leukemia, carcinoma and sarcoma, such as cancers or tumors found in the anus, bladder, bile duct, bone, brain, breast, cervix, colon / rectum, endometrium, esophagus, eye, gallbladder, head and neck, liver, kidney, larynx, lung, mediastinum (chest), mouth, ovaries, pancreas, penis, prostate, skin, small intestine, stomach, spinal cord, tailbone, testicles, thyroid gland and uterus.

[0073] Types of carcinomas that can be treated with the pharmaceutical or therapeutic compositions of the present disclosure include, but are not limited to, soft tissue sarcomas (such as alveolar soft part sarcoma, angiosarcoma, dermatofibrosarcoma, desmoid tumor, desmoplastic small cell tumor, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, hemangiopericytoma, angiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma, synovial sarcoma, and Askin's tumor), Ewing's sarcoma (primitive neuroectodermal tumor), malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, and chondrosarcoma.

[0074] By way of non-limiting example, cancers that may be treated include acute granulocytic leukemia, acute lymphocytic leukemia, acute myeloid leukemia, adenocarcinoma, adenosarcoma, adrenal gland carcinoma, adrenocortical carcinoma, anal carcinoma, aplastic astrocytoma, angiosarcoma, appendix carcinoma, astrocytoma, basal cell carcinoma, B-cell lymphoma), bile duct carcinoma, bladder carcinoma, bone cancer, intestinal cancer, brain cancer, brain stem glioma, brain tumor, breast cancer, carcinoid tumor, cervical cancer, cholangiocarcinoma, chondrosarcoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colon cancer, colorectal cancer, craniopharyngioma, cutaneous lymphoma, cutaneous melanoma, diffuse astrocytoma, ductal carcinoma in situ, endometrial carcinoma, ependymoma, epithelioid sarcoma, esophageal cancer, Ewing's sarcoma, extrahepatic bile duct carcinoma, eye cancer, fallopian tube carcinoma, fibrosarcoma, gallbladder cancer, gastric cancer, cancer), gastrointestinal cancer, gastrointestinal carcinoid cancer, gastrointestinal stromal tumor, general, germ cell tumor, glioblastoma multiforme, glioma, hairy cell leukemia, head and neck cancer, hemangioendothelioma, Hodgkin's lymphoma, Hodgkin's disease, Hodgkin's lymphoma , hypopharyngeal cancer, invasive ductal carcinoma, invasive lobular carcinoma, inflammatory breast cancer, intestinal cancer, intrahepatic cholangiocarcinoma, invasive / infiltrating breast cancer, pancreatic islet cell carcinoma, jaw cancer, Kaposi's sarcoma, renal cancer, laryngeal cancer, leiomyosarcoma, leptomeningeal metastasis, leukemia , lip cancer, liposarcoma, liver cancer, lobular carcinoma in situ, low-grade astrocytoma, lung cancer, lymph node cancer, lymphoma, male breast cancer, medullary carcinoma, medulloblastoma, melanoma, meningioma, Merkel cell carcinoma, mesenchymal chondrosarcoma, mesenchymal, mesothelioma, metastatic breast cancer, metastatic melanoma, metastatic squamous cell carcinoma of the neck, mixed glioma, oral cancer, mucosal carcinoma, mucosal melanoma, multiple myeloma, nasal cancer, nasopharyngeal carcinoma, cervical cancer, neuroblastoma, neuroendocrine tumor, non-Hodgkin's lymphoma, non-small cell lung cancer, oat cell carcinoma, eye cancercancer), ocular melanoma, oligodendroglioma, oral cancer, oral cancer, oropharyngeal cancer, osteogenic sarcoma, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian primary peritoneal cancer, ovarian sex cord-stromal tumor, Paget's disease, pancreatic cancer, papillary cancer, paranasal sinus cancer, parathyroid cancer, pelvic cancer, penile cancer, peripheral nerve cancer, peritoneal cancer, pharyngeal cancer, pheochromocytoma, pilocytic astrocytoma, pineal region tumor, pineoblastoma, pituitary cancer, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma, sarcoma, bone, sarcoma, soft tissue, sarcoma, uterus, paranasal sinus cancer, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord cancer, spinal column cancer, spinal cord tumor, squamous cell carcinoma, gastric cancer cancer), synovial sarcoma, T-cell lymphoma), testicular cancer, pharyngeal cancer, thymic cancer / thymic carcinoma, thyroid cancer, tongue cancer, tonsillar cancer, transitional cell carcinoma, transitional cell carcinoma, transitional cell carcinoma, triple-negative breast cancer, fallopian tube cancer, tubular carcinoma, ureteral cancer, urethral cancer, uterine adenocarcinoma, uterine cancer, uterine sarcoma, vaginal cancer, and vulvar cancer.

[0075] Various infectious diseases can be treated with the pharmaceutical or therapeutic compositions of the present disclosure. In some examples, the cassette comprises one or more antigenic payloads derived from infectious agents or organisms. As used herein, the term "infectious disease" refers to any disorder caused by an organism such as a bacterium, virus, fungus, or parasite.Non-limiting examples of infectious diseases and / or causative agents include acute bacterial rhinosinusitis, 14-day measles, acne, acrodermatitis chronica atrophicans (ACA) - (a late-onset skin manifestation of latent Lyme disease), acute hemorrhagic conjunctivitis, acute hemorrhagic cystitis, acute rhinosinusitis, adult T-cell leukemia-lymphoma (ATLL), African sleeping sickness, AIDS (acquired immunodeficiency syndrome), alveolar hydrocephalus, amebiasis, and amebic meningoencephalitis. , Anaplasmosis, Anthrax, Arboviral or Parainfectious - (Ascariasis), Aseptic meningitis, Athlete's foot (Tinea pedis), Australian tick typhus, Avian influenza, Babesiosis, Bacterial angiomatosis, Bacterial meningitis, Bacterial vaginosis, Balanitis, Balantidiosis, Bang's disease, Bartonella forest virus infection, Bartonellosis (Peruvian wart; Carrion disease; Oroya fever), Bat lyssavirus infection, Bay sore (chiclero ulcer), raccoon infection (raccoon roundworm infection), beaver fever, beef tapeworm, Beezel (endemic syphilis), biphasic meningoencephalitis, Black Bane, Black Death, black sand fever, black water fever, blastomycosis, neonatal pyorrhea, blepharitis, boils, Bornholm disease (pleurisy), Borrelia miyamotoi disease, botulism, boutonneuse fever, Brazilian purpura, fracture fever, brill, bronchiolitis, bronchitis, brucellosis (Bang's disease), bubonic plague, bullous impetigo, Burkholderia mallei (glanders), Burkholderia pseudomallei (melioidosis), bursal ulcer (including Mycobululi ulcer), Busse-Buschke disease (cryptococcosis), California county encephalitis, campylobacteriosis, candidiasis, Canfield fever (Candicola fever; 7-day fever; Weil's disease; leptospirosis; Canfield fever), canicola fever, capillary disease, carbapenem-resistant Enterobacteriaceae (CRE), Carbuncle disease, Carrion disease, cat-scratch fever, Cave disease, Central Asian hemorrhagic fever, Central European tick disease, cervical cancer, Chagas disease, Chancroid (soft diarrhea), Chicago disease, varicella (chickenpox), chiclero ulcer, chikungunya fever, chlamydial infection, cholera, chromoblastomycosis, ciguatera, gonorrhea, Clonorchiasis (liver fluke infection), Clostridium Difficile infection, Clostridium perfringens. Perfringens (epsilon toxin), Coccidioides fungal infections (valley fever, desert rheumatism), coenurosis, Colorado tick fever, Condyloma accuminata, Condyloma accuminata (warts), Condyloma lata, Congo fever, Congo hemorrhagic fever virus, conjunctivitis, cowpox, crab, Crimean, croup, cryptococcosis, cryptosporidiosis (crypt), cutaneous larva migrans, cyclosporiasis, cystic hydatid disease, cysticercosis, cystitis, Czechoslovakian tick, D68 (EV-D68), dacryocystitis, dengue fever, Darling's disease, deer fly fever, Dengue fever (1, 2, 3 and 4), desert rheumatism, epidemic pleurisy (Devil's disease) grip), biphasic suckling fever, diphtheria, disseminated intravascular coagulation, canine tapeworm, lymphogranuloma venereum, lymphogranuloma venereum (granuloma inguinale), dracunculosis, Duke's disease, Dum-Dum disease, Durand-Nicolas-Fabre disease, dwarf tapeworm disease, Escherichia coli infection (E. coli), eastern equine encephalitis, Bola hemorrhagic fever (Ebola virus disease EVD), extra-hair fungi, ehrlichiosis (Senetz fever), encephalitis, epidemic relapsing fever, endemic syphilis, endophthalmitis, intra-hair fungi, enterobiasis (pinworm infection), enterotoxin B poisoning (staphylococcal food poisoning), enterovirus infection, epidemic keratoconjunctivitis, epidemic relapsing fever, epidemic typhus, epiglottitis, erysipelas, erysipelothrix (erysipelothrix), migrans Erythema chronica, erythema infectiosum, erythema marginale, erythema multiforme, erythema nodosum, erythema nodosum leprosum, erythrasma, espundia, fungal mycetoma, European blastomycosis, exanthema subitum (sixth disease), eyeworm, Far Eastern tick, fascioliasis, Butnews fever (Ixodes typhus), fifth disease (erythema infectiosum), Philatau-Dukes disease (scarring skin syndrome; Ritter's disease), fish tapeworm, Fitz-Hugh-Curtis syndrome, perihepatitis, Flinders Island spotted fever, influenza (influenza), folliculitis, Four Corners disease, Four Corners disease (human pulmonary syndrome (HPS)), Flambesia, Francis' disease, furuncle disease, gas gangrene, gastroenteritis, genital herpes, genital warts, German measles, Gerstmann-Straussler-Scheinker (GSS), giardiasisGilchrist's disease, gingivitis, gingivostomatitis, glanders, glandular fever (infectious mononucleosis), gnathostomiasis, gonorrhea (gonococcal infection), gonorrhea, granuloma inguinale (donovanosis), filariasis; Haemophilus influenzae disease, Hamburger's disease, Hansen's disease, Hantaan disease, Hantaan-Korean hemorrhagic fever, Hantavirus pulmonary syndrome (HPS), hard chancre, hard measles, Haverhill fever, rat bite fever, head and body lice, Heartland fever, Helicobacteriosis, hemolytic uremic syndrome (HUS), hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, herpangina, genital herpes, labial herpes, neonatal herpes, hidradenitis, histopathology Histoplasmosis, Histoplasmosis infection (Histoplasmosis), His-Werner's disease, HIV infection, Hookworm infection, Hordeola, Hordeola (hordeolum), HTLV, HTLV-associated myelopathy (HAM), Human granulocytic ehrlichiosis, Human monocytic ehrlichiosis, Human papillomavirus (HPV), Human pulmonary syndrome, Hydatid disease, Hydrophobia, Impetigo, Congenital (including German measles), Inclusion conjunctivitis, Inclusion conjunctivitis - swimming pool conjunctivitis - pannus, Infantile diarrhea, Infectious mononucleosis, Infectious myocarditis, Infectious pericarditis, Influenza, Isosporosis, Israeli spotted fever, Japanese encephalitis, Jock itch, Jorge Lobomycosis, Jungle Yellow Fever, Junin-Argentine Hemorrhagic Fever, Kala-azar, Kaposi's Sarcoma, Keloid Blastosis, Keratoconjunctivitis, Kuru, Kyasanur Forest Disease, La Crosse Encephalitis, Lassa Hemorrhagic Fever, Legionnaires' Disease, Legionnaires' Pneumonia, Lemire's Syndrome (Postanginal Septicemia), Lemming Fever, Leprosy, Leptospirosis (Sheep Fever; Weil's Disease), Listeriosis, Liver Fluke Infection, Lobomycosis, Tetanus, Loiasis, Louping Disease, Cellulitis of the Floor of the Mouth (Ludwig's Angina), Paragonimus Infection, Paragonimus Infection (Paragonimus), Lyme Disease, Lymphogranuloma Venereum Infection (LGV), Machupo Bolivian Hemorrhagic Fever, Madura del Valle pinto, malaria, malignant pustulosis, Malta fever, Marburg hemorrhagic fever, Masters' disease, maternal sepsis (puerperal fever), measles, Mediterranean spotted fever, mellioidosis (Whitemore's disease), meningitis, meningococcal disease, MERS, Milker's tubercle, molluscum contagiosum, moniliasis, monkeypox, mononucleosis, mononucleosis-like syndrome,Montezuma's Revenge, Morbilli, MRSA (Methicillin-resistant Staphylococcus aureus) infection, Mucosal mycosis-zygomycosis, Multiple Organ Dysfunction Syndrome or MODS, Multiple Organ Atrophy Syndrome (MSA), Mumps, Murine Typhus, Murray Valley Encephalitis (MVE), Mycoburuli ulcer, Mycoburuli ulcer-Buruli ulcer, Fungal vulvitis, Myositis, Mountain Fever, Necrotizing fasciitis, Necrotizing fasciitis-1, Necrotizing fasciitis-2, Negishi, New World Spotted Fever, Nocardiosis, Nongonococcal urethritis, Nonpolio (Nonpolio enteric virus), Norovirus infection, North American blastomycosis, North American tick typhus, Norwalk K virus infection, Norwegian pruritus, O'Hara disease, Omsk hemorrhagic fever, onchocerciasis, onchomycosis, opisthorchiasis, ophthalmia neonatorum, oral hairy leukoplakia, contagious impetigo, oriental cyst, oriental spotted fever, ornithosis (parrot fever, psittacosis), Oroya fever, otitis externa, otitis media, pannus, paracoccidioidomycosis, paragonimism, paralytic shellfish poisoning, paronychia (whelan), parotitis, PCP pneumonia, pediculosis, hepatic purpura, pelvic inflammatory disease, whooping cough cough), phaeochromocytosis, pharyngoconjunctival fever, sand mites (white sand mites), sand mites (black sand mites), pig bell, pink eye conjunctivitis, pinworm infection, excoriation, tinea versicolor (black catfish), plague; gland, thymus, pneumococcal disease, pneumocystiosis, pneumonia, pneumonia (plague), poliomyelitis or acute poliomyelitis, multicystic hydatid disease, Pontiac fever, suis tapeworm, Posada-Wernicke's disease, postanginal sepsis, Powassan, progressive multifocal leukoencephalopathy, progressive rubella encephalitis, prostatitis, pseudomembranous colitis, o Umbilical cord disease, puerperal fever, pustular rash (smallpox), pyelonephritis, pyelitis Q-fever, tonsillar abscess, Quintana fever (5-day fever), rabbit fever, rabies, raccoon roundworm infection, sheep roundworm infection, rat bite fever, rat tapeworm, Reiter's syndrome, relapsing fever, respiratory syncytial virus (RSV) infection, rheumatic fever, rickettsialpox, rickettsiosis, Rift Valley fever, ringworm, Litter's disease, river blindness, Rocky Mountain spotted fever, rose handler's disease (sporotrichosis), infantile roseola, roseola, Ross River fever, roundworm infection, rubella, measles, Russian spring, salmonellosis gastroenteritis,San Joaquin Valley fever, San Paulo encephalitis, San Paulo fever, SARS, Scabies infestation (Norwegian pruritus), Scalded skin syndrome, Scarlet fever (Scarlatina), Schistosomiasis, Scombroidery, Tsugamushi disease, Senec fever, Sepsis (Septic shock), Severe acute respiratory syndrome (SARS), Shiga toxin-producing Escherichia coli (STEC / VTEC), Shigella gastroenteritis (Shigella), Shinbone fever, Shingles, Shipping fever, Siberian mite typhus, Sinusitis, Sixth disease, Slapped cheek disease disease), sleeping sickness, smallpox, snail fever, chancroid, southern tick-associated rash disease, sparganosis, spelunky disease, sporadic typhus, sporotrichosis, spotted fever, spring, St. Louis encephalitis, staphylococcal food poisoning, staphylococcal infection, streptococcal pharyngitis, streptococcal disease, streptococcal toxic shock syndrome, strongyloidiasis, subacute sclerosing panencephalitis, subacute sclerosing panencephalitis (SSPE), acute respiratory syndrome, exanthema subitum, otitis externa, swimmer's itch, pool conjunctivitis, forest yellow fever, systemic inflammatory response syndrome (SIRS), tabes dorsalis (tertiary syphilis), taeniasis, taeniasis, taeniasis, taeniasis, tapeworm infection, temporal lobe encephalitis, tetanus Jaw), tetanus infection, pinworm infection, thrush, ticks, tick typhus, tinea barbae, tinea capitis, tinea corporis, tinea cruris, tinea manubriata, tinea nigra, tinea pedis, tinea unguium, tinea versicolor, torulopsosis, torulosis, toxic shock syndrome, toxoplasmosis, transmissible spongiform encephalopathy (CJD), traveler's diarrhea, trench fever, trichinosis, trichomoniasis, axillary tinea, trichinellosis, tropical spastic paraparesis (TSP), trypanosomiasis, Nucleus (TB), Tuberculosis, Tularemia, Typhoid Fever, Typhoid Fever, Soft Ulcer, Undulgent Fever, Urban Yellow Fever, Urethritis, Vaginitis, Vaginosis, Vancomycin Intermediate (VISA), Vancomycin Resistant (VRSA), Chickenpox, Venezuelan Equine Encephalitis, Peruvian Warts, Vibrio Cholera (Cholera), Vibrio Disease (Vibrio), Vincent's Disease or Oral Trench, Viral Conjunctivitis, Viral Meningitis, Viral Meningoencephalitis, Viral Exanthem, Visceral Larva Migrans, Vomito Negro, Vulvovaginitis, Warts, Waterhouse, Weil's Disease, West Nile Fever, Western Equine Encephalitis, Whipple's Disease, Whipple's Infection, White Sand Fever, Whitlow,Whitmore, Disease, winter diarrhea, Urchin fever, Uhl-Sauter's disease, yaws, yellow fever, yersiniosis, yersiniosis (Yersinia), Zahorsky's disease, Zika virus disease, shingles, zygomycosis, John Cunningham virus (JCV), human immunodeficiency virus (HIV), influenza virus, hepatitis B, hepatitis C, hepatitis D, respiratory syncytial virus (RSV), herpes simplex virus 1 and 2, human cytomegalovirus, Epstein-Barr virus, varicella-zoster virus, coronavirus, poxvirus, enterovirus 71, rubella virus, human papillomavirus, pneumococcus, streptococcal virus, Staphylococcus aureus (S. aureus), methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-intermediate Staphylococcus aureus (VISA), vancomycin-resistant Staphylococcus aureus (S. aureus) aureus) (VRSA), Staphylococcus epidermidis (S. epidermidis), Clostridium Tetani, Bordetella pertussis, Bordetella paratussis, Mycobacterium, Francisella Tularensis, Toxoplasma gondii, Candida (C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. krusei, and C. lusitaniae) and / or other infectious diseases, disorders, or syndromes.

[0076] A variety of toxins can be used as components or antigenic payloads in the vaccines or cassettes of the present disclosure. Non-limiting examples of such antigenic payloads include ricin, anthrax, shiga and shiga-like toxins, and botulinum toxins.

[0077] Various peptides or proteins derived from agents that cause tropical diseases can be used as components or antigenic payloads in the vaccines or cassettes of the present disclosure. Non-limiting examples of tropical diseases and / or causative agents of such diseases include chikungunya, dengue fever, Chagas disease, rabies, malaria, Ebola virus, Marburg virus, West Nile virus, yellow fever, Japanese encephalitis virus, and St. Louis encephalitis virus.

[0078] Various peptides or proteins derived from agents that cause foodborne illnesses can be used as components or antigenic payloads in the vaccines or cassettes of the present disclosure. Non-limiting examples of foodborne illnesses and / or causative agents of such illnesses or gastroenteritis include rotavirus, Norwalk virus (norovirus), Campylobacter jejuni, Clostridium difficile, Entamoeba histolytica, Helicobacter pylori, Staphylococcus aureus enterotoxin B, hepatitis A virus (HAV), hepatitis E, Listeria monocytogenes, Salmonella, Clostridium perfringens, and Salmonella.

[0079] A variety of peptides or proteins derived from agents that cause infections can be used as components or antigenic payloads in the vaccines or vaccine cassettes of the present disclosure. Non-limiting examples of such infectious agents include adenovirus, Anaplasma phagocytophilium, Ascaris lumbricoides, Bacillus anthracis, Bacillus cereus, Bacteriodes spp., Barmah Forest virus, Bartonella bacilliformis, Bartonella henselae, Bartonella quintana, Clostridium perfringens beta toxin, Bordetella pertussis, Bordetella parapertussis, Borrelia burgdorferi, Borrelia miyamotoi, Borrelia recurrentis, Borrelia spp., Botulinum toxin, Brucella spp., Burkholderia pseudomallei, California encephalitis virus, Campylobacter, Candida albicans, Chikungunya virus, Chlamydia psittaci, Chlamydia trachomatis, Clonorchis sinensis, Clostridium difficile bacteria, Clostridium tetani, Colorado tick fever virus, Corynebacterium diphtheriae, Corynebacterium minutissimum, Coxiella burnetii, Coxsackie A, Coxsackie B, Crimean-Congo hemorrhagic fever virus, cytomegalovirus, dengue virus, Eastern equine encephalitis virus, Ebola virus, echovirus, Ehrlichia chaffeensis, Ehrlichia equi, Ehrlichia spp., Entamoeba histolytica, Enterobacter spp., Enterococcus feacalis, Enterovirus 71, Epstein-Barr virus (EBV), Erysipelothrix rhusiopathiae, Escherichia coli (Escherichiacoli), flaviviruses, Fusobacterium necrophorum, Gardnerella vaginalis, Group B streptococcus, Haemophilus aegyptius, Haemophilus ducreyi, Haemophilus influenzae, hantavirus, Helicobacter pylori, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, herpes simplex viruses 1 and 2, human herpesvirus 6, human herpesvirus 8, human immunodeficiency viruses 1 and 2, human T-cell leukemia viruses I and II, influenza viruses (A, B, C), Jamestown Canyon virus, Japanese encephalitis antigenicity, Japanese encephalitis virus, John Cunningham virus, Junin virus, Kaposi's sarcoma-associated herpesvirus (KSHV), Klebsiella granulomatis, Klebsiella spp., Kyasanur Forest disease virus, La Crosse virus, Lassa virus, Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes, lymphocytic choriomeningitis virus, lyssavirus, Machupo virus, Marburg virus, measles virus, MERS coronavirus (MERS-CoV), Micrococcus sedentarius, Mobiluncus spp., morsipoxvirus, Moraxella catarrhalis, morbilli-measles virus, mumps virus, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma genitalium, Mycoplasma spp., Nairovirus, Neisseria gonorrhoeae, Neisseriameningitidis, Nocardia, Norwalk virus, Norovirus, Omsk hemorrhagic fever virus, Papillomavirus, Parainfluenza viruses 1-3, Parapoxvirus, Parvovirus B19, Peptostreptococccus spp., Plasmodium spp., Poliovirus types I, II, and III, Proteus spp., Pseudomonas aeruginosa, Pseudomonas pseudomallei, Pseudomonas spp., Rabies virus, Respiratory syncytial virus, Rickettsia australis, Rickettsia conori, Rickettsia honei, Rickettsia prowazekii, Ross River virus, Rotavirus, Rubella virus, St. Louis encephalitis, Salmonella Typhi, Sarcoptes scabiei, SARS-associated coronavirus (SARS-CoV), SARS-associated coronavirus (SARS-CoV-2), Serratia spp., Shiga toxin and Shiga-like toxin, Shigella spp., Sin Nombre virus, Snowshoe hare virus, Staphylococcus aureus, Staphylococcus epidermidis, Streptobacillus moniliformis, Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus A-H groups, Streptococcus pneumoniae, Streptococcus pyogenes, Treponema pallidum subsp. Pallidum, Treponema pallidum var. carateum, Treponema pallidum pallidum var. endemicum, Tropheryma whippelii, Ureaplasma urealyticum, varicella zoster virus, chickenpox virus, Vibrio cholerae, West Nile virus, yellow fever virus, Yersinia enterocolitica, Yersinia pestis, and Zika virus.

[0080] Pharmaceutical and Therapeutic Compositions: Dosing, Administration and Delivery

[0081] dosage

[0082] The present disclosure provides methods comprising administering any one or more pharmaceutical or therapeutic compositions described herein to a subject in need thereof. The pharmaceutical or therapeutic compositions can be, for example, a vaccine. They can be administered to a subject in any amount and using any route of administration effective to prevent or treat a disease, disorder, and / or symptom (e.g., a disease, disorder, and / or symptom) or to image the disease. The exact amount required will vary from subject to subject, depending on the subject's species, age, and general condition, the severity of the disease, the particular composition, its mode of administration, its mode of activity, etc.

[0083] Compositions described herein can be formulated in dosage unit form for ease of administration and dosage uniformity.However, it is understood that the total daily use amount of compositions described herein can be determined by attending physician within the scope of sound medical judgment.The specific therapeutically effective, prophylactically effective or suitable imaging dose level for any specific patient will depend on various factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound used; the specific composition used; the age, weight, general health, sex and diet of the patient; the administration time, administration route and excretion rate of the specific compound used; treatment period; the drug used in combination with or simultaneously with the specific compound used; and similar factors well known in the medical field.

[0084] Administration

[0085] The pharmaceutical or therapeutic compositions of the present disclosure may be administered by any route that achieves a therapeutically effective outcome.These include, but are not limited to, enteral (into the gut), gastrointestinal, epidural (into the dura), oral (through the mouth), transdermal, peridural, intracerebral (into the brain), intraventricular (into the ventricles of the brain), epithelial (applied to the skin), intradermal (on the skin itself), subcutaneous (under the skin), intranasal administration (through the nose), intravenous (into a vein), bolus injection, intravenous drip, intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous (into the bone marrow) injection, intrathecal (into the spinal canal), intraperitoneal (infusion or injection into the peritoneum), intravesical infusion, intravitreal (through the eye), intracavity injection (into a diseased cavity), intracavity injection (into the base of the penis), intravaginal administration, intrauterine administration, extra-amniotic administration, transdermal (diffusion through intact skin for systemic distribution), transmucosal (diffusion through mucous membranes), transvaginal, insufflation (aspiration), sublingual, sublabial, enema, ophthalmic (on the conjunctiva), eardrop, auricular (in or through the ear), buccal (directed buccal), conjunctival, skin, dental (tooth or teeth), electroosmosis, intracervical, intrasinus, intratracheal, extracorporeal, hemodialysis, infiltration, interstitial, intraperitoneal, intra-amniotic, intra-articular, intrabiliary, intra-bronchial, intrasynovial, intrachondral (into cartilage), intrasacral (in the cauda equina), intracisternal (into the cisterna magna of the cerebral medulla oblongata), intracorneal (into the cornea) intramembrane), intracoronary, intracoronary (inside the coronary arteries), corpora cavernosa (inside the expandable space of the corpus cavernosum), intradiscal (inside the disc), intraductal (inside the glandular duct), intraduodenal (inside the duodenum), intradural (inside the dura or subdural), intraepidermal (inside the epidermis), intraesophageal (inside the esophagus), intragastric (inside the stomach), intragingival (inside the gums), intraileal (inside the distal part of the small intestine), intralesional (inside a localized lesion or introduced directly), intraluminal (inside the lumen), intralymphatic (inside the lymphatic vessels), intramedullary (inside the bone marrow cavity), intrameningeal (inside the meninges), intramyocardial (inside the myocardium), intraocular (inside the eye), intraovarian (inside the ovary), intrapericardial (inside the pericardium), intrapleural (inside the pleura), intraprostatic (inside the prostate) ), intrapulmonary (inside the lungs or bronchi), intraantral (inside the nasal or periorbital sinuses), intraspinal (inside the spinal column), intrasynovial (inside the synovial cavity of a joint), intratendinous (inside the tendons), intratesticular (inside the testes), intrathecal (inside the cerebrospinal fluid at any level of the cerebrospinal axis), intrathoracic (inside the chest cavity), intratubular (inside the renal tubules of an organ), intratumoral (inside a tumor), intratympanic (inside the ear), intravascular (inside blood vessels), intraventricular (inside the heart), iontophoresis (by electric current that moves ions of soluble salts into body tissues), irrigation (by bathing or rinsing an open wound or body cavity), laryngeal (directly into the larynx), nasogastric (through the nose into the stomach), occlusive dressing method (administered by a local route.which is then covered with a dressing to occlude the area), ophthalmic (outside the eye), oropharyngeal (directly into the mouth and pharynx), parenteral, transdermal, periarticular, peridural, perineural, periodontal, rectal, respiratory (into the airways by oral or nasal inhalation for local or systemic effect), retrobulbar (behind the pons or behind the eyeball), intramyocardial (into the myocardium), soft tissue, subarachnoid, subconjunctival, submucosal, topical, transplacental (through or across the placenta), transtracheal (through the tracheal wall), transtympanic (across or through the tympanic cavity), ureteral (into the ureter), urethral (into the urethra), vaginal, caudal block, diagnostic, nerve block, biliary perfusion, cardiac perfusion, photopheresis, or spinal.

[0086] Parenteral and injectable administration

[0087] In some embodiments, pharmaceutical or therapeutic compositions of the present disclosure may be administered parenterally. Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically or therapeutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and / or elixirs. In addition to the active ingredient, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing and emulsifying agents, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, oral compositions may contain adjuvants such as wetting agents, emulsifying and suspending agents, sweeteners, flavoring agents, and / or perfuming agents. In certain embodiments for parenteral administration, the composition is mixed with a solubilizing agent such as CREMOPHOR®, alcohol, oil, modified oil, glycol, polysorbate, cyclodextrin, polymer, and / or combinations thereof. Other embodiments include a surfactant such as hydroxypropyl cellulose.

[0088] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, can be formulated according to known techniques using suitable dispersants, wetting agents, and / or suspending agents. Sterile injectable preparations can be sterile injectable solutions, suspensions, and / or emulsions in non-toxic parenterally acceptable diluents and / or solvents (e.g., 1,3-butanediol). Acceptable vehicles and solvents that can be used include water, Ringer's solution (USP), and isotonic sodium chloride solution. Sterile fixed oils are conventionally used as solvents or suspending media. For this purpose, any non-irritating fixed oil can be used, including synthetic monoglycerides or diglycerides. Fatty acids, such as oleic acid, can be used in the preparation of injectables.

[0089] The injectable preparations can be sterilized, for example, by filtration through a bacterial-retaining filter and / or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0090] To prolong the effect of an active ingredient, it is often desirable to slow the absorption of the active ingredient from subcutaneous or intramuscular injection. This can be accomplished by using a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the active ingredient depends on its dissolution rate, which in turn may depend on crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form can be achieved by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are made by forming microencapsulated matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. The rate of drug release can be controlled depending on the drug-to-polymer ratio and the nature of the particular polymer used. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[0091] Rectal and vaginal administration

[0092] In some aspects, the pharmaceutical or therapeutic compositions of the present disclosure may be administered rectally and / or vaginally. Compositions for rectal or vaginal administration are typically suppositories that can be prepared by mixing the composition with a suitable non-irritating excipient such as cocoa butter, polyethylene glycol or a suppository wax that is solid at ambient temperature but liquid at body temperature and thus melts in the rectum or vaginal cavity to release the active ingredient.

[0093] Oral administration

[0094] In some aspects, the pharmaceutical or therapeutic compositions of the present disclosure may be administered orally. Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert pharmaceutically acceptable excipient such as sodium citrate or dicalcium phosphate and / or fillers or extenders (e.g., starch, lactose, sucrose, glucose, mannitol, and silicic acid), binders (e.g., carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia), humectants (e.g., glycerol), disintegrating agents (e.g., agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate), solution retarders (e.g., paraffin), absorption accelerators (e.g., quaternary ammonium compounds), wetting agents (e.g., cetyl alcohol and glycerol monostearate), absorbents (e.g., kaolin and bentonite clay), lubricants (e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate), and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may comprise buffering agents.

[0095] Topical or transdermal administration

[0096] As described herein, the pharmaceutical or therapeutic compositions of the present disclosure can be formulated for topical administration.Because skin is easily accessible, it can be an ideal target site for delivery.Three routes are generally considered for delivering the pharmaceutical or therapeutic compositions of the present disclosure to the skin: (i) topical application (e.g., for local / regional treatment and / or cosmetic use); (ii) intradermal injection (e.g., for local / regional treatment and / or cosmetic use); and (iii) systemic delivery (e.g., for treating skin diseases that affect both the cutaneous and extracutaneous regions).The pharmaceutical compositions of the present disclosure can be delivered to the skin by several different approaches known in the art.

[0097] In some embodiments, the present disclosure provides various bandages (e.g., wound dressings) or dressings (e.g., adhesive bandages) for conveniently and / or effectively practicing the methods of the present disclosure. Typically, the bandages or dressings will contain a sufficient amount of the pharmaceutical or therapeutic compositions of the present disclosure described herein to allow the user to perform multiple treatments.

[0098] Dosage forms for topical and / or transdermal administration may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and / or patches. Generally, the active ingredient is mixed under sterile conditions with pharmaceutically acceptable excipients and / or any necessary preservatives and / or buffers. Furthermore, the present disclosure contemplates the use of transdermal patches, which often have the added advantage of providing controlled delivery of the pharmaceutical or therapeutic compositions of the present disclosure to the body. Such dosage forms may be prepared, for example, by dissolving and / or dispensing the pharmaceutical or therapeutic composition in a suitable medium. Alternatively or additionally, the rate may be controlled by providing a rate-controlling membrane and / or dispersing the pharmaceutical or therapeutic composition in a polymer matrix and / or gel.

[0099] Formulations suitable for topical administration include, but are not limited to, liquid and / or semi-liquid preparations, such as liniments, lotions, oil-in-water and / or water-in-oil emulsions, such as creams, ointments and / or pastes, and / or solutions and / or suspensions.

[0100] Topically administrable formulations may contain, for example, about 1% to about 10% (w / w) active ingredient, although the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further include one or more additional ingredients described herein.

[0101] Depot administration

[0102] As described herein, in some embodiments, the pharmaceutical or therapeutic compositions of the present disclosure are formulated into a depot for sustained release. Generally, administration is targeted to a specific organ or tissue ("target tissue").

[0103] In some embodiments of the present disclosure, the pharmaceutical or therapeutic composition of the present disclosure is spatially retained within or adjacent to a target tissue. A method of providing a pharmaceutical or therapeutic composition to a target tissue of a mammalian subject is disclosed, wherein the target tissue (including one or more target cells) is contacted with the pharmaceutical or therapeutic composition under conditions such that the target tissue is substantially retained in the target tissue, meaning that at least about 10, e.g., at least about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 85, about 90, about 95, about 96, about 97, about 98, about 99, about 99.9, about 99.99 or more of the composition is retained in the target tissue. Advantageously, retention is determined by measuring the amount of the pharmaceutical or therapeutic composition that enters one or more target cells. For example, at least about 1%, e.g., about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.9%, about 99.99%, or more than about 99.99% of the pharmaceutical or therapeutic composition administered to a subject is present intracellularly for a period following administration. For example, intramuscular injection into a mammalian subject can be performed using an aqueous composition comprising a pharmaceutical or therapeutic composition of the present disclosure and one or more transfection reagents, and retention is determined by measuring the amount of pharmaceutical or therapeutic composition present in muscle cells.

[0104] Certain aspects of the present disclosure relate to methods of providing a pharmaceutical or therapeutic composition of the present disclosure to a target tissue in a mammalian subject by contacting the target tissue (comprising one or more target cells) with the pharmaceutical or therapeutic composition under conditions such that the composition is substantially retained in such target tissue. The pharmaceutical or therapeutic composition comprises sufficient active ingredient(s) to produce a desired effect in at least one target cell. In some aspects, the pharmaceutical or therapeutic composition generally comprises one or more cell penetration agents, although "naked" formulations (e.g., without cell penetration agents or other agents) are also contemplated, with or without a pharmaceutically or therapeutically acceptable carrier.

[0105] In some embodiments, the formulation comprises a plurality of different pharmaceutical or therapeutic compositions. Optionally, the formulation can also comprise a cell-penetrating agent to aid in the intracellular delivery of the pharmaceutical or therapeutic composition. In such embodiments, the dose of the compound and / or composition required to target the target biomolecule in a substantial proportion of the cells contained in a predetermined volume of target tissue (generally not targeting the target biomolecule in adjacent or distant tissue) is determined. The determined dose is then directly introduced into the target tissue.

[0106] Pulmonary administration

[0107] In some embodiments, pharmaceutical or therapeutic compositions of the present disclosure may be prepared, packaged, and / or sold in a formulation suitable for pulmonary administration. In some embodiments, such administration occurs via the buccal cavity. In some embodiments, the formulation may comprise dry particles comprising the active ingredient. In such embodiments, the dry particles may have a diameter ranging from about 0.5 nm to about 7 nm or from about 1 nm to about 6 nm. In some embodiments, the formulation may be in the form of a dry powder for administration using a device comprising a dry powder reservoir into which a stream of propellant can be directed to disperse such powder. In some embodiments, a self-propelling solvent / powder dispensing container may be used. In such embodiments, the active ingredient may be dissolved and / or suspended in a low-boiling propellant in a sealed container. Such powders may comprise particles in which at least 98% of the particles (by weight) have a diameter greater than 0.5 nm and at least 95% of the particles (by number) have a diameter less than about 7 nm. Alternatively, at least about 95% of the particles (by weight) have a diameter greater than about 1 nm and at least about 90% of the particles (by number) have a diameter less than about 6 nm. Dry powder compositions may include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.

[0108] Low-boiling propellants generally include liquid propellants having a boiling point below 65° F. at atmospheric pressure. Generally, the propellant may comprise from about 50% to about 99.9% (w / w) of the composition, and the active ingredient may comprise from about 0.1% to about 20% (w / w) of the composition. The propellant may further include additional ingredients such as liquid nonionic and / or solid anionic surfactants and / or solid diluents (which may have a particle size on the same order as the particles containing the active ingredient).

[0109] Pharmaceutical or therapeutic compositions formulated for pulmonary delivery may provide the active ingredient in the form of droplets of a solution and / or suspension. Such formulations may be prepared, packaged, and / or sold as aqueous and / or dilute alcoholic solutions and / or suspensions (optionally sterile) containing the active ingredient, and may be conveniently administered using any nebulizer and / or atomizer device. Such formulations may further comprise one or more additional ingredients, including, but not limited to, flavoring agents such as saccharin sodium, volatile oils, buffers, surfactants, and / or preservatives such as methyl hydroxybenzoate.

[0110] Intranasal, intranasal and buccal administration

[0111] In some embodiments, the pharmaceutical or therapeutic compositions of the present disclosure may be administered nasally and / or intranasally. In some embodiments, formulations described herein as useful for pulmonary delivery may also be useful for intranasal delivery. In some embodiments, formulations for intranasal administration comprise a coarse powder containing the active ingredient and having an average particle size of about 0.2 um to about 500 um. Such formulations are administered in the manner of snuffing, i.e., by rapid inhalation through the nasal passages from a container of powder held close to the nose.

[0112] Formulations suitable for nasal administration may contain, for example, as little as about 0.1% (w / w) to as much as about 100% (w / w) of the active ingredient, and may include one or more additional ingredients described herein. Pharmaceutical or therapeutic compositions may be prepared, packaged, and / or sold in formulations suitable for buccal administration. Such formulations may be, for example, in the form of tablets and / or lozenges manufactured using conventional methods and may contain, for example, about 0.1% to about 20% (w / w) of the active ingredient, with the remainder comprising an orally soluble and / or disintegrable composition and, optionally, one or more additional ingredients described herein. Alternatively, formulations suitable for buccal administration may comprise a powder and / or aerosolized and / or atomized solution and / or suspension comprising the active ingredient. Such powdered, aerosolized, and / or aerosolized formulations, when dispersed, may have an average particle size and / or droplet size in the range of about 0.1 nm to about 200 nm and may further include any one or more additional ingredients described herein.

[0113] Ocular or otic administration

[0114] In some embodiments, the pharmaceutical or therapeutic compositions of the present disclosure may be prepared, packaged, and / or sold in a formulation suitable for ocular and / or otic administration. Such formulations may be in the form of eye drops and / or ear drops, for example, containing a 0.1 / 1.0% (w / w) solution and / or suspension of the active ingredient in an aqueous and / or oily liquid vehicle. Such drops may further contain buffering agents, salts, and / or any one or more additional ingredients described herein. Other useful ophthalmically administrable formulations include those containing the active ingredient in microcrystalline form and / or in a liposomal preparation. Subretinal inserts may also be used as dosage forms.

[0115] Delivery vehicle molecules

[0116] Delivery Mode

[0117] Pharmaceutical or therapeutic compositions can be delivered using one or more modes. These include viral vectors and particles such as lentivirus, adenovirus, adeno-associated virus, herpes simplex virus, retrovirus, etc. Other modes, such as mRNA, plasmids, and recombinant proteins, can also be used.

[0118] Lentiviral vehicle

[0119] In some embodiments, lentiviral carriers / particles can be used as a delivery modality. Lentiviruses are a subgroup of viruses in the retroviridae family, named for the requirement for reverse transcription of the viral RNA genome into DNA prior to integration into the host genome. Therefore, the most important feature of lentiviral vehicles / particles is the integration of their genetic material into the genome of target / host cells. Some examples of lentiviruses include human immunodeficiency viruses: HIV-1 and HIV-2, simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Jembrana disease virus (JDV), equine infectious anemia virus (EIAV), equine infectious anemia virus, visna-maedi, and caprine arthritis-encephalitis virus (CAEV).

[0120] The lentiviral particles that constitute the gene delivery vehicle can themselves be replication-deficient (also known as "self-inactivating"). Lentiviruses can infect both dividing and non-dividing cells by entering through an intact host nuclear envelope (Naldini L et al., Curr. Opin. Biotechnol., 1998, 9:457-463). Recombinant lentiviral vehicles / particles have been generated by multiple attenuation of HIV pathogenic genes, such as deletion of the Env, Vif, Vpr, Vpu, Nef, and Tat genes, making the vector biologically safe. Correspondingly, lentiviral vehicles derived from, for example, HIV-1 / HIV-2 can mediate efficient delivery, integration, and long-term expression of transgenes in non-dividing cells.

[0121] Lentiviral particles can be produced by co-expressing viral packaging elements and the vector genome itself in producer cells such as human HEK293T cells. These elements are usually provided in three or four separate plasmids. Producer cells are co-transfected with a plasmid (also called a packaging system) encoding lentiviral components, including the viral core (i.e., structural proteins) and enzyme components, as well as envelope proteins, and a plasmid (also called a transfer vector) encoding a genome containing an exogenous transgene to be transferred into target cells, i.e., the vehicle itself. Generally, the plasmid or vector is contained in a producer cell line. The plasmid / vector is introduced into the producer cell line via transfection, transduction, or infection. Methods for transfection, transduction, or infection are well known to those skilled in the art. By way of non-limiting example, packaging and transfer constructs can generally be introduced into a producer cell line by calcium phosphate transfection, lipofection, or electroporation along with a dominant selectable marker such as neo, DHFR, Gln synthetase, or ADA, followed by selection in the presence of the appropriate drug to isolate clones.

[0122] The producer cells produce recombinant viral particles containing a foreign gene, such as the vaccine or vaccine cassette of the present disclosure.The recombinant viral particles are recovered from the culture medium and titrated by standard methods used by those skilled in the art.The recombinant lentiviral vehicle can be used to infect target cells.

[0123] Cells that can be used to produce high-titer lentiviral particles include, but are not limited to, HEK293T cells, 293G cells, STAR cells (Relander et al., Mol. Ther., 2005, 11:452-459), and other HEK293T-based producer cell lines (e.g., Stewart et al., Hum Gene Ther. 2011, 22(3):357-369; Lee et al., Biotechnol Bioeng, 2012, 109(6):1551-1560; Throm et al., Blood. 2009, 113(21):5104-5110; the contents of each of which are incorporated herein by reference in their entirety).

[0124] In some embodiments, the envelope protein can be a heterologous envelope protein from another virus, such as the G protein of vesicular stomatitis virus (VSV G) or the baculovirus gp64 envelope protein. The VSV-G glycoprotein can be selected from among species classified in the genus Vesiculovirus, including, inter alia, Karayas virus (CJSV), Chandipura virus (CHPV), Kokar virus (COCV), Ikhfahan virus (ISFV), Maraba virus (MARAV), Piry virus (PIRYV), Vesicular stomatitis Alagoas virus (VSAV), Vesicular stomatitis Indiana virus (VSIV), and Vesicular stomatitis New Jersey virus (VSNJV), and / or strains provisionally classified in the genus Vesiculovirus, Grassroots rhabdovirus, BeAn 157575 virus (BeAn 157575), Boteke virus (BTKV), Calchaqui virus (CQIV), and Eel virus. American (EVA), Grey Lodge virus (GLOV), Yuronavirus (JURY), Klamath virus (KLAV), Quatta virus (KWAV), La Jauja virus (LJV), Malpais Spring virus (MSPV), Mount Elgon bat virus (MEBV), Perinet virus (PERV), Pike fly rhabdovirus (PFRV), Porton virus (PORV), Radi virus (RADIV), Spring viremia of carp virus (SVCV), Tubai virus (TUPV), Ulcerative disease rhabdovirus (UDRV), and Yugbogdanova virus (YBV).gp64 or other baculovirus env proteins are expressed in Autographa californica nuclear polyhedrosis virus (AcMNPV), Anagrapha falcifera nuclear polyhedrosis virus, Bombyx mori nuclear polyhedrosis virus, Choristoneura fumiferana nuclear polyhedrosis virus, Orgyia pseudotsugata single-capsid nuclear polyhedrosis virus, Epiphyas postvittana nuclear polyhedrosis virus, Hyphantria cunea nuclear polyhedrosis virus, Galleria mellonella nuclear polyhedrosis virus, Dhori virus, Thogoto virus, Antheraea pemyi nuclear polyhedrosis virus, or Batken virus.

[0125] Other elements provided in the lentiviral particle may include a retroviral LTR (long terminal repeat) at either the 5' or 3' end, a retroviral export element, optionally a lentiviral reverse response element (RRE), a promoter or active portion thereof, and a locus control region (LCR) or active portion thereof. The effector module is linked to the vector.

[0126] Methods for producing recombinant lentiviral particles are discussed in the art, e.g., U.S. Pat. Nos. 8,846,385; 7,745,179; 7,629,153; 7,575,924; 7,179,903; and 6,808,905 (the contents of each of which are incorporated herein by reference in their entirety).

[0127] Lentiviral vehicles can be plasmid-based or virus-based and are known in the art (U.S. Patent Nos. 9,260,725; 9,068,199; 9,023,646; 8,900,858; 8,748,169; 8,709,799; 8,420,104; 8,329,462; 8,076,106; 6,013,516; and 5,994,136; the contents of each of which are incorporated herein by reference in their entirety).

[0128] Adeno-associated virus particles

[0129] The delivery of any of the pharmaceutical or therapeutic compositions of the present disclosure can be achieved using recombinant adeno-associated virus (rAAV) vector.Such vector or viral particle can be designed to utilize any of known serotype capsids or serotype capsid combinations.Capsids include but are not limited to AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV5, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV10, AAV11, AAV12, AAV16.3, AAV24.1, AAV27.3, AAV42.12, AAV42-1b, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b , AAV42-8, AAV42-10, AAV42-11, AAV42-12, AAV42-13, AAV42-15, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23, A AV43-25, AAV43-5, AAV44.1, AAV44.2, AAV44.5, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAV1-7 / rh.48, AAV1-8 / rh.49, AAV2-15 / rh.62, AAV2-3 / rh.61, AAV2-4 / rh.50, AAV2-5 / rh.51, AAV3.1 / hu.6, AAV3.1 / hu.9, AAV3-9 / rh.52, AAV3- 11 / rh.53, AAV4-8 / r11.64, AAV4-9 / rh.54, AAV4-19 / rh.55, AAV5-3 / rh.57, AAV5-22 / rh.58, AAV7.3 / hu.7, AAV16.8 / hu.10, AAV16 .12 / hu.11, AAV29.3 / bb.1, AAV29.5 / bb.2, AAV106.1 / hu.37, AAV114.3 / hu.40, AAV127.2 / hu.41, AAV127.5 / hu.42, AAV128.3 / hu.44、AAV130.4 / hu.48、AAV145.1 / hu.53、AAV145.5 / hu.54、AAV145.6 / hu.55、AAV161.10 / hu.60、AAV161.6 / hu.61、AAV33.12 / hu.17、AAV33.4 / hu.15、AAV33.8 / hu.16、AAV52 / hu.19、AAV52.1 / hu.20、AAV58.2 / hu.25、AAVA3.3、AAVA3.4、AAVA3.5、AAVA3.7、AAVC1、AAVC2、AAVC5、AAV-DJ、AAV-DJ8、AAVF3、A AVF5, AAVH2, AAVH6, AAVLK03, AAVH-1 / hu.1, AAVH-5 / hu.3, AAVLG-10 / rh.40, AAVLG-4 / rh.38, AAVLG-9 / hu.39, AAVN721-8 / rh.43, AAVCh.5, AAVCh.5R1, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5R1, AAVCy.5R2, AAVCy.5R3, AAVCy.5R4, AAVcy.6, AAVhu.1, AAVhu.2, AAVhu.3, AAVhu.4, AAVhu.5, AAVh u.6、AAVhu.7、AAVhu.9、AAVhu.10、AAVhu.11、AAVhu.13、AAVhu.15、AAVhu.16、AAVhu.17、AAVhu.18、AAVhu.20、AAVhu.21、AAVhu.22、AAVhu.23、AAVhu.2 u.24、AAVhu.25、AAVhu.27、AAVhu.28、AAVhu.29、AAVhu.29R、AAVhu.31、AAVhu.32、AAVhu.34、AAVhu.35、AAVhu.37、AAVhu.39、AAVhu.40、AAVhu.41、AA Vhu.42、AAVhu.43、AAVhu.44、AAVhu.44R1、AAVhu.44R2、AAVhu.44R3、AAVhu.45、AAVhu.46、AAVhu.47、AAVhu.48、AAVhu.48R1、AAVhu.48R2、AAVhu.48R 3、AAVhu.49、AAVhu.51、AAVhu.52、AAVhu.54、AAVhu.55、AAVhu.56、AAVhu.57、AAVhu.58、AAVhu.60、AAVhu.61、AAVhu.63、AAVhu.64、AAVhu.66、AAVhu.67, AAVhu.14 / 9, AAVhu.t 19, AAVrh.2, AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh.10, AAVrh.12, AAVrh.13, AAVrh.13R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.2 0, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.3 These may include AAVrh.7R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.46, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.61, AAVrh.64, AAVrh.64R1, AAVrh.64R2, AAVrh.67, AAVrh.73, and / or AAVrh.74.

[0130] AAV vectors include not only single-stranded vectors but also self-complementary AAV vectors (scAAV). scAAV vectors contain DNA that anneal together to form a double-stranded vector genome. By skipping second-strand synthesis, scAAV allows for rapid expression in cells.

[0131] rAAV vectors can be produced in sf9 insect cells or in suspension cell cultures of human cells such as HEK293 cells by standard methods in the art, for example, by triple transfection.

[0132] Pharmaceutical or therapeutic compositions can be encoded in one or more viral genomes packaged in the AAV capsids taught herein.

[0133] Such vectors or viral genomes may also contain, in addition to at least one or two ITRs (inverted terminal repeats), certain regulatory elements required for expression from the vector or viral genome. Such regulatory elements are well known in the art and include, for example, promoters, introns, spacers, stuffer sequences, etc.

[0134] The pharmaceutical or therapeutic compositions of the present disclosure may be administered in one or more AAV particles.

[0135] In some embodiments, a pharmaceutical or therapeutic composition can be administered in one or more AAV particles, hi some embodiments, more than one pharmaceutical or therapeutic composition can be encoded in the viral genome.

[0136] Retroviral vehicles / particles (γ-retroviral vectors)

[0137] In some embodiments, retroviral vehicles / particles can be used to deliver pharmaceutical or therapeutic compositions. Retroviral vectors (RVs) allow the permanent integration of transgenes into target cells. In addition to complex HIV-1 / 2-based lentiviral vectors, simple gammaretrovirus-based retroviral vectors have been widely used to deliver therapeutic genes and have been clinically proven as one of the most efficient and powerful gene delivery systems, capable of transducing a wide range of cell types. Examples of gammaretrovirus species include murine leukemia virus (MLV) and feline leukemia virus (FeLV).

[0138] In some embodiments, gamma-retroviral vectors derived from mammalian gamma-retroviruses (e.g., murine leukemia viruses (MLV)) are recombinant. The MLV family of gammaretroviruses includes ecotropic, amphotropic, xenotropic, and polytropic subfamilies. Ecotropic viruses can infect only mouse cells using the mCAT-1 receptor. Examples of ecotropic viruses are Moloney MLV and AKV. Amphotropic viruses infect mice, humans, and other species via the Pit-2 receptor. An example of an amphotropic virus is the 4070A virus. Xenotropic and polytropic viruses utilize the same (Xpr1) receptor but differ in their species tropism. Xenotropic viruses such as NZB-9-1 can infect humans and other species but not mouse species, whereas polytropic viruses such as focus-forming virus (MCF) can infect mice, humans, and other species.

[0139] Gammaretroviral vectors can be produced in packaging cells by co-transfecting the cells with several plasmids, including one encoding the retroviral structural and enzymatic (gag-pol) polyprotein, one encoding the envelope (env) protein, and one encoding a vector mRNA containing at least one polynucleotide encoding a composition of the present disclosure that is packaged into newly formed viral particles.

[0140] In some embodiments, recombinant gamma-retroviral vectors are pseudotyped with envelope proteins from other viruses. Envelope glycoproteins are incorporated into the outer lipid layer of the viral particle and can increase / alter cell tropism. Exemplary envelope proteins include gibbon ape leukemia virus envelope protein (GALV) or vesicular stomatitis virus G protein (VSV-G), or simian endogenous retrovirus envelope proteins, or measles virus H and F proteins, or human immunodeficiency virus gp120 envelope protein, or cocalvesiculovirus envelope protein (see, e.g., U.S. Patent Application Publication No. 2012 / 164118; the contents of which are incorporated herein by reference in their entirety). In other embodiments, envelope glycoproteins can be genetically modified to incorporate targeting / binding ligands, including but not limited to peptide ligands, single-chain antibodies, and growth factors, into gamma-retroviral vectors (Waehler et al., Nat. Rev. Genet. 2007, 8(8):573-587; the contents of which are incorporated herein by reference in their entirety). These engineered glycoproteins can retarget the vectors to cells expressing their corresponding targeting moieties. In other embodiments, "molecular bridges" can be introduced to direct the vectors to specific cells. The molecular bridges have bispecificity, i.e., one end can recognize the viral glycoprotein and the other end can bind to a molecular determinant on the target cell. Such molecular bridges, such as ligand-receptor, avidin-biotin, and chemical conjugation, monoclonal antibodies, and engineered fusogenic proteins, can direct the attachment of viral vectors to target cells for transduction (Yang et al., Biotechnol. Bioeng., 2008, 101(2):357-368 and Maetzig et al., Viruses, 2011, 3, 677-713; the contents of each of which are incorporated herein by reference in their entirety).

[0141] In some embodiments, the recombinant gamma-retroviral vector is a self-inactivating (SIN) gamma-retroviral vector. The vector is replication-incompetent. SIN vectors may have a deletion in the 3'U3 region, which initially contains enhancer / promoter activity. Additionally, the 5'U3 region may be replaced with a strong promoter from cytomegalovirus or RSV (required in packaging cell lines), or a selected internal promoter and / or enhancer element. The selection of the internal promoter may be made according to the specific requirements of gene expression required for the specific purpose of the present disclosure.

[0142] In some embodiments, a polynucleotide encoding a pharmaceutical or therapeutic composition is inserted into the recombinant viral genome. Other components of the viral mRNA of a recombinant gammaretroviral vector can be modified by inserting or removing naturally occurring sequences (e.g., inserting an IRES, inserting a heterologous polynucleotide encoding a polypeptide of interest or an inhibitory nucleic acid, shuffling a more effective promoter from a different retrovirus or virus in place of the wild-type promoter, etc.). In some examples, a recombinant gammaretroviral vector can contain a modified packaging signal, and / or a primer binding site (PBS), and / or a 5'-enhancer / promoter element in the U3 region of the 5'-long terminal repeat (LTR), and / or a modified 3'-SIN element in the U3 region of the 3'-LTR. These modifications can increase titer and infectivity.

[0143] Gammaretroviral vectors suitable for the pharmaceutical or therapeutic compositions of the present disclosure may be selected from those disclosed in U.S. Pat. Nos. 8,828,718; 7,585,676; 7,351,585; U.S. Patent Application Publication No. 2007 / 048285; PCT Application Publication Nos. WO2010 / 113037; WO2014 / 121005; WO2015 / 056014; and EP1757702; EP1757703, the contents of each of which are incorporated herein by reference in their entirety.

[0144] messenger RNA (mRNA)

[0145] In some embodiments, pharmaceutical or therapeutic compositions can be designed as messenger RNA (mRNA). As used herein, the term "messenger RNA" (mRNA) refers to any polynucleotide that encodes a polypeptide of interest and can be translated in vitro, in vivo, in situ, or ex vivo to produce the encoded polypeptide. Such mRNA molecules of the present disclosure can have any of the structural components or characteristics taught in International Application No. PCT / US2013 / 030062, the contents of which are incorporated herein by reference in their entirety.

[0146] Pharmaceutical compositions, such as, for example, mRNA vaccines or mRNA vaccine cassettes of the present disclosure, may also be used in combination with other therapeutic agents, such as those disclosed in, for example, UK Patent Application No. 0316089.2 to Ribostem Limited (filed July 9, 2003, now abandoned), PCT Application No. PCT / GB2004 / 002981 (filed July 9, 2004, published as WO2005005622), U.S. Patent Application No. 10 / 563,897 (filed June 8, 2006, published as US20060247195, now abandoned), and European Patent Application National Phase Registration No. 2004743322 (filed July 9, 2004, published as EP1646714, now withdrawn); / 88060 (filed December 19, 2007, published as WO2008140615), U.S. Patent Application No. 12 / 520,072 (filed July 2, 2009, published as US20100028943) and European Patent Application No. 2007874376 (filed July 7, 2009, published as EP2104739); University of Rochester PCT Application No. PCT / US2006 / 46120 (filed December 4, 2006, published as WO2007064952), and U.S. Patent Application No. 11 / 606,995 (filed December 1, 2006, published as US20070141030); BioNTech AG's European Patent Application No. 2007024312 (filed December 14, 2007, now abandoned), PCT Application No. PCT / EP2008 / 01059 (filed December 12, 2008, published as WO2009077134), European Patent Application No. 2008861423 (filed June 2, 2010, published as EP2240572), U.S. Patent Application No. 12 / 735,060 (filed November 24, 2010, published as US20110 065103), German Patent Application No. 102005046490 (filed September 28, 2005), PCT Application No. PCT / EP2006 / 0448 (filed September 28, 2006, published as WO2007036366), European Patent No. 1934345 (published March 21, 2012) and U.S. Patent Application No. 11 / 992,638 (filed August 14, 2009, published as 20100129877); Immune Disease Institute Inc.U.S. Patent Application No. 13 / 088,009, filed April 15, 2011, published as US20120046346, and PCT Application No. PCT / US2011 / 32679, filed April 15, 2011, published as WO20110130624; U.S. Patent Application No. 12 / 957,340, filed November 20, 2010, published as US20110244026; PCT Application No. PCT / US1998 / 019492, filed September 18, 1998, published as WO1999014346, to The Scripps Research Institute's PCT Application No. PCT / US2010 / 00567 (filed February 24, 2010, published as WO2010098861) and U.S. Patent Application No. 13 / 203,229 (filed November 3, 2011, published as US20120053333); Ludwig-Maximilians-University's PCT Application No. PCT / EP2010 / 004681 (filed July 30, 2010, published as WO2011012316); Cellscript Inc., U.S. Patent No. 8,039,214 (filed June 30, 2008, granted October 18, 2011), U.S. Patent Application No. 12 / 962,498 (filed December 7, 2010, published as US20110143436), 12 / 962,468 (filed December 7, 2010, published as US20110143397), 13 / 237,451 (filed September 20, 2011, published as US20120009649), and PCT Application No. PCT / US2010 / 5930 5 (filed December 7, 2010, published as WO2011071931) and PCT / US2010 / 59317 (filed December 7, 2010, published as WO2011071936); PCT Application No. PCT / US2006 / 32372 (filed August 21, 2006, published as WO2007024708) and U.S. Patent Application No. 11 / 990,646 (filed March 27, 2009, published as US20090286852) to the Trustees of the University of Pennsylvania; and German Patent Application No. 102001027283.9 (filed June 5, 2001, published as DE10 2001 062 480) to Curevac GMBH.No. DE 20 2006 051 516 (filed December 19, 2001) and DE 20 2006 051 516 (filed October 31, 2006) (both now abandoned), European Patent No. 1392341 (granted March 30, 2005) and European Patent No. 1458410 (granted January 2, 2008), PCT application No. PCT / EP2002 / 06180 (filed June 5, 2002, published as WO2002098443), PCT / EP2002 / 14577 (filed December 19, 2002, published as WO2003051401), PCT / EP2007 / 09469 (filed December 31, 2007, published as WO No. 10 / 729,830 (filed December 5, 2003, published as US20050032730), U.S. Patent Application No. 10 / 8 ... No. 70,110 (filed June 18, 2004, published as US20050059624), U.S. Patent Application No. 11 / 914,945 (filed July 7, 2008, published as US20080267873), U.S. Patent Application No. 12 / 446,912 (filed October 27, 2009, published as US2010047261, now abandoned), U.S. Patent Application No. 12 / 522,214 (filed January 4, 2010, published as US20100189729), U.S. Patent Application No. 12 / 787,566 (filed May ... No. 12 / 787,755 (filed May 26, 2010, published as US20110077287), U.S. Patent Application No. 12 / 787,755 (filed May 26, 2010, published as US20100239608), U.S. Patent Application No. 13 / 185,119 (filed July 18, 2011, published as US20110269950), and U.S. Patent Application No. 13 / 106,548 (filed May 12, 2011, published as US20110311472), all of which are incorporated herein by reference in their entireties.

[0147] Naked Delivery

[0148] The pharmaceutical or therapeutic compositions of the present disclosure can be delivered to cells, tissues, organs, and / or organisms in naked form. As used herein, the term "naked" refers to a pharmaceutical or therapeutic composition delivered without transfection- or permeability-promoting agents or modifications. Naked pharmaceutical or therapeutic compositions can be delivered to cells, tissues, organs, and / or organisms using routes of administration known in the art and described herein. In some embodiments, naked delivery can include formulation in a simple buffer, such as saline or PBS.

[0149] Formulation Delivery

[0150] The compositions of the present disclosure may be formulated by any method known in the art.

[0151] In some aspects, pharmaceutical or therapeutic compositions of the present disclosure may be formulated using the methods described herein.

[0152] Formulations may include pharmaceutical or therapeutic compositions, which may be modified and / or unmodified.

[0153] The formulations may further include, but are not limited to, a cell penetration agent, a pharmaceutically acceptable carrier, a delivery agent, a bioerodible or biocompatible polymer, a solvent, and / or a sustained release delivery depot. The formulations of the present disclosure can be delivered to cells using routes of administration known in the art and described herein.

[0154] Pharmaceutical or therapeutic compositions may also be formulated for direct delivery to an organ or tissue in any of several ways known in the art, including, but not limited to, via a catheter, direct immersion or bath with gels, powders, ointments, creams, gels, lotions, and / or drops, using substrates such as fabrics or biodegradable materials coated or impregnated with the composition, and the like.

[0155] In one example, the compositions described herein are RNA-based (e.g., mRNA) nanoparticle pharmaceutical or therapeutic compositions. The nanoparticles may include the described polynucleotides encapsulated by delivery vehicle molecules with formulations that may be, but are not limited to, poly(lactic-co-glycolic acid) (PLGA) microspheres, lipidoids, lipoplexes, liposomes, polymers, carbohydrates (including simple sugars), cationic lipids, and combinations thereof.

[0156] In one embodiment, the delivery vehicle molecule formulation may include at least one lipid. The lipid may be selected from, but is not limited to, DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, and PEGylated lipids. In another embodiment, the lipid may be a cationic lipid, such as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, and DODMA.

[0157] In one embodiment, the delivery vehicle molecule can have a nanoparticle geometry. The delivery vehicle can be, for example, an aminolipidated peptide, which can include a tertiary aminolipidated cationic peptide, such as any of those described in PCT Application Nos. PCT / US19 / 53661, entitled "LIPID NANOPARTICLE FORMULATIONS COMPRISING LIPIDATED CATIONIC PEPTIDE COMPOUNDS FOR NUCLEIC ACID DELIVERY," filed September 27, 2019, and PCT / US19 / 53655, entitled "TERTIARY AMINO LIPIDATED CATIONIC PEPTIDES FOR NUCLEIC ACID DELIVERY," filed September 27, 2019, the contents of each of which are incorporated herein by reference in their entirety. The nanoparticle delivery vehicle can include additional lipids / components. For example, the aminolipidated peptide can include one or more phospholipids, such as MSPC or DSPC. The lipid composition can also include a quaternary amine compound, such as DOTAP.

[0158] Pharmaceutical or therapeutic compositions may be formulated using, for example, any of the delivery vehicles taught in U.S. Patent Application Publication No. 20180028688, the contents of which are incorporated herein by reference in their entirety.

[0159] Detectable Agents and Labels

[0160] The pharmaceutical or therapeutic compositions of the present disclosure may be associated with or bound to one or more radioactive or detectable agents, including various small organic molecules, inorganic compounds, nanoparticles, enzymes or enzyme substrates, fluorescent materials, luminescent materials (e.g., luminol), bioluminescent materials (e.g., luciferase, luciferin, and aequorin), chemiluminescent materials, radioactive materials (e.g., 18 F, 67 Ga, 81m Kr, 82 Rb, 111 In, 123 I,133 Xe, 201 Tl, 125 I, 35 S, 14 C. 3 H, or 99m Tc (e.g., pertechnetate (technetate(VII), TcO4 -)), and contrast agents (e.g., gold (e.g., gold nanoparticles), gadolinium (e.g., chelated Gd), iron oxides (e.g., superparamagnetic iron oxide (SPIO), single crystalline iron oxide nanoparticles (MION), and ultrasmall superparamagnetic iron oxide (USPIO)), manganese chelates (e.g., Mn-DPDP), barium sulfate, iodinated contrast agents (iohexol), microbubbles, or perfluorocarbons. Such optically detectable labels include, but are not limited to, , for example, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid; acridine and derivatives (e.g., acridine and acridine isothiocyanate); 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS); 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5-disulfonate; N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY; Brilliant Yellow; coumarin and derivatives (e.g., coumarin, 7-amino-4-methylcoumarin (AMC, coumarin 120), and 7-amino-4-trifluoromethylcoumarin (coumarin 151)); cyanine dyes; cyanosine; 4',6-diaminidino-2-phenylindole (DAPI); 5',5''-dibromopyrogallolsulfonathalene (bromopyrogallol red); 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriaminepentaacetate; 4,4 '-Diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; 5-[dimethylamino]-naphthalene-1-sulfonyl chloride (DNS, dansyl chloride); 4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); eosin and derivatives (e.g., eosin and eosin isothiocyanate); erythrosin and derivatives (e.g., erythrosin B and erythrosin isothiocyanate); ethidium;Fluorescein and derivatives (e.g., 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2',7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein, fluorescein, fluorescein isothiocyanate, X-rhodamine-5-(and -6)-isothiocyanate (QFITC ​​or XRITC), and fluorescamine); 2-[2-[3-[[1,3-dihydro-1,1-dimethyl-3-(3-sulfopropyl)-2H-benz[e]indol-2-ylidene]ethylidene]-2-[4-(ethoxycarbonyl)-1-piperazinyl]-1-cyclopenten-1-yl]ethenyl]-1,1-dimethyl-3-(3-sulfopropyl) (I)-1H-benz[e]indolium hydroxide, inner salt, compound with n,n-diethylethanamine (1:1) (IR144); 5-chloro-2-[2-[3-[(5-chloro-3-ethyl-2(3H)-benzothiazol-ylidene)ethylidene]-2-(diphenylamino)-1-cyclopenten-1-yl]ethenyl]-3-ethylbenzothiazolium perchlorate (IR140); Malachite Green isothiocyanate; 4-methylumbelliferone orthocresolphthalein; nitrotyrosine; para-aniline; phenol red; B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives (e.g., pyrene, pyrene butyrate, and succinimidyl 1-pyrene); butyrate quantum dots; Reactive Red 4 (CIBACRON™ Brilliant Red 3B-A); rhodamine and derivatives (e.g., 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride rhodanine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red), N,N,N',N' tetramethyl-6-carboxyrhodamine (TAMRA), tetramethylrhodamine, and tetramethylrhodamine isothiocyanate (TRITC)); riboflavin; rosolic acid; terbium chelate derivatives; cyanine-3 (Cy3);Examples include cyanine-5 (Cy5); cyanine-5.5 (Cy5.5), cyanine-7 (Cy7); IRD 700; IRD 800; Alexa 647; La Jolta Blue; phthalocyanines; and naphthalocyanines.

[0161] In some embodiments, the detectable agent can be an undetectable precursor that becomes detectable upon activation (e.g., a fluorogenic tetrazine-fluorophore construct such as tetrazine-BODIPY FL, tetrazine-Oregon Green 488, or tetrazine-BODIPY TMR-X) or an enzyme-activatable fluorogenic agent (e.g., PROSENSE® (VisEn Medical))). In vitro assays in which the enzyme-labeled composition can be used include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation assay, immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA), and Western blot analysis.

[0162] kit

[0163] The present disclosure includes various kits for conveniently and / or effectively carrying out the methods of the present disclosure. Typically, the kits contain components in amounts and / or numbers sufficient to allow a user to perform one or more treatments on a subject and / or to perform one or more experiments.

[0164] In one aspect, the present disclosure provides kits for inducing an immune response in a subject or patient, optionally in combination with any other suitable active agents.

[0165] The kit may further include packaging and instructions for forming the pharmaceutical composition and / or a delivery agent. The delivery agent may include, for example, saline, a buffer solution.

[0166] In a further aspect, an assay screening kit is provided. The kit includes a container for the screening assay. Instructions for use of the assay and information regarding the screening method will be included in the kit.

[0167] term

[0168] Nucleotides are referred to by their commonly accepted single-letter codes. Unless otherwise indicated, nucleic acids are written left to right in a 5' to 3' orientation. Nucleotides are referred to herein by their commonly known single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission: A is adenine, C is cytosine, G is guanine, T is thymine, and U is uracil.

[0169] Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation.

[0170] About: The term "about" as used in connection with numerical values ​​throughout this specification and claims indicates an interval of accuracy familiar and acceptable to those skilled in the art. Generally, such an interval of accuracy is + / - 10%.

[0171] Where ranges are given, the endpoints are included. Furthermore, unless otherwise indicated or otherwise apparent from the context and the understanding of one of ordinary skill in the art, values ​​expressed as ranges can assume any specific value or subrange within the ranges set forth in different aspects of this disclosure, down to one-tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0172] Administered in Combination: As used herein, the terms "administered in combination," "co-administration," "co-administration," or "combination therapy" mean that two or more agents are administered to a subject simultaneously or within an interval such that there may be an overlap in the effect of each agent on the patient. In some embodiments, they are administered within about 60, 30, 15, 10, 5, or 1 minute of each other. In some embodiments, the administration of the agents is sufficiently close to each other that a combined (e.g., synergistic) effect is achieved.

[0173] Amino acid substitution: The term "amino acid substitution" refers to the replacement of an amino acid residue present in a parent or reference sequence (e.g., a wild-type sequence) with another amino acid residue. An amino acid can be substituted in a parent or reference sequence (e.g., a wild-type polypeptide sequence) via, for example, chemical peptide synthesis or via recombinant methods known in the art. Thus, a reference to a "substitution at position X" refers to the replacement of the amino acid present at position X with an alternative amino acid residue. In some embodiments, the substitution pattern can be described according to the schema AnY, where A is the single-letter code corresponding to the amino acid naturally or originally present at position n, and Y is the replacement amino acid residue. In other embodiments, the substitution pattern can be described according to the schema An(YZ), where A is the single-letter code corresponding to the amino acid residue substituting the amino acid naturally or originally present at position X, and Y and Z are the alternative replacement amino acid residues.

[0174] In the context of the present disclosure, substitutions (even when referred to as amino acid substitutions) are made at the nucleic acid level, i.e., by substituting a codon encoding a first amino acid with a codon encoding a second amino acid, thereby substituting an amino acid residue with an alternative amino acid residue.

[0175] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to a human at any stage of development. In some embodiments, "animal" refers to a non-human animal at any stage of development. In particular embodiments, the non-human animal is a mammal (e.g., a rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and worms. In some embodiments, the animal is a transgenic animal, a genetically engineered animal, or a clone.

[0176] Antigen of interest or desired antigen: As used herein, the term "antigen of interest" or "desired antigen" or "antigen" refers to a protein and / or other biomolecule that elicits an immune response, e.g., the production of antibodies. In some embodiments, the antigen of interest may comprise any of the polypeptides or payloads or proteins described herein, or fragments or portions thereof.

[0177] Approximately: As used herein, the term "approximately," when applied to one or more values ​​of interest, refers to a value similar to a stated reference value. In certain embodiments, the term "approximately" refers to a range of values ​​that falls within 100%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater or less than) the stated reference value, unless otherwise stated or apparent from the context (except when such number exceeds 10% of possible values).

[0178] Associated: As used herein with respect to a disease, the term "associated" means that the symptom, measurement, characteristic, or condition in question is related to the diagnosis, onset, presence, or progression of the disease. Association can be, but need not be, causally related to the disease.

[0179] The terms "associated with," "conjugated," "linked with," "attached," and "tethered with," when used in reference to two or more moieties, mean that the moieties are physically associated or linked to one another, either directly or through one or more additional moieties that act as linking agents, to form a structure that is sufficiently stable so that the moieties remain physically associated under the conditions in which the structure is used, e.g., physiological conditions. The "association" need not be strictly through a direct covalent chemical bond. It may also imply that the connectivity based on ionic or hydrogen bonding or hybridization is sufficiently stable so that the "associated" entities remain physically associated.

[0180] Biocompatible: As used herein, the term "biocompatible" means compatible with living cells, tissues, organs, or systems, with little or no risk of injury, toxicity, or rejection by the immune system.

[0181] Biodegradable: As used herein, the term "biodegradable" means capable of being broken down into harmless products by the action of living organisms.

[0182] Sequence optimization: The term "sequence optimization" refers to a process or set of processes in which nucleobases in a reference nucleic acid sequence are replaced with alternative nucleobases, resulting in a nucleic acid sequence with improved properties, e.g., improved protein expression or immunogenicity.

[0183] Generally, the goal of sequence optimization is to generate synonymous nucleotide sequences that encode the same polypeptide sequence encoded by the reference nucleotide sequence, and thus there will be no amino acid substitutions (as a result of codon optimization) in the polypeptide encoded by the codon-optimized nucleotide sequence relative to the polypeptide encoded by the reference nucleotide sequence.

[0184] Codon substitution: The term "codon substitution" or "codon replacement" in the context of sequence optimization refers to the replacement of a codon present in a reference nucleic acid sequence with another codon. Codons can be replaced in a reference nucleic acid sequence, for example, through chemical peptide synthesis or through recombinant methods known in the art. Thus, reference to "substitution" or "replacement" at a specific position in a nucleic acid sequence (e.g., mRNA) or within a specific region or subsequence of a nucleic acid sequence (e.g., mRNA) refers to the replacement of a codon at such position or region with an alternative codon.

[0185] As used herein, the terms "coding region" and "coding region" and grammatical variations thereof refer to an open reading frame (ORF) in a polynucleotide that, upon expression, produces a polypeptide or protein.

[0186] Compound: As used herein, the term "compound" is meant to include all stereoisomers and isotopes of the depicted structure. As used herein, the term "stereoisomer" refers to any geometric isomer (e.g., cis- and trans-isomers), enantiomer, or diastereomer of a compound. The present disclosure encompasses any and all stereoisomers of the compounds described herein, including stereomerically pure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) and mixtures of enantiomers and stereoisomers, e.g., racemates. Mixtures of enantiomers and stereoisomers of compounds and means for resolving them into their component enantiomers or stereoisomers are well known. "Isotopes" refer to atoms having the same atomic number but different mass numbers resulting from different numbers of neutrons in the nucleus. For example, isotopes of hydrogen include tritium and deuterium. Additionally, compounds, salts, or complexes of the present disclosure can be prepared in combination with solvents or water molecules to form solvates and hydrates by routine methods.

[0187] Conservative amino acid substitution: A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, and include basic side chains (e.g., lysine, arginine, or histidine), acidic side chains (e.g., aspartic acid or glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, or cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, or histidine). Thus, if an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the amino acid substitution is considered conservative. In another embodiment, strings of amino acids can be conservatively substituted with structurally similar strings that differ in the order and / or composition of side chain family members.

[0188] Non-conservative amino acid substitutions include (i) substitutions of a residue having an electropositive side chain (e.g., Arg, His, or Lys) with or by an electronegative residue (e.g., Glu or Asp); (ii) substitutions of a hydrophilic residue (e.g., Ser or Thr) with or by a hydrophobic residue (e.g., Ala, Leu, Ile, Phe, or Val); (iii) substitutions of a cysteine ​​or proline with or by any other residue; or (iv) substitutions of a residue having a bulky hydrophobic or aromatic side chain (e.g., Val, His, Ile, or Trp) with or by a residue having a smaller side chain (e.g., Ala or Ser) or no side chain (e.g., Gly).

[0189] Other amino acid substitutions can be easily identified by those skilled in the art.For example, for amino acid alanine, substitution can be made from any one of D-alanine, glycine, β-alanine, L-cysteine ​​and D-cysteine.For lysine, substitution can be any one of D-lysine, arginine, D-arginine, homo-arginine, methionine, D-methionine, ornithine or D-ornithine. In general, substitutions in functionally important regions that can be expected to induce changes in the properties of isolated polypeptides include: (i) polar residues (e.g., serine or threonine) substituted with (or by) hydrophobic residues (e.g., leucine, isoleucine, phenylalanine, or alanine); (ii) cysteine ​​residues substituted with (or by) any other residue; (iii) residues with electropositive side chains, such as lysine, arginine, or histidine, substituted with (or by) residues with electronegative side chains, such as glutamic acid or aspartic acid; or (iv) residues with bulky side chains, such as phenylalanine, substituted with (or by) residues without such side chains, such as glycine. The likelihood that one of the aforementioned non-conservative substitutions will change the functional properties of a protein also correlates with the location of the substitution relative to the functionally important regions of the protein. Thus, some non-conservative substitutions may have little or no effect on biological properties.

[0190] Conserved: As used herein, the term "conserved" refers to nucleotide or amino acid residues of a polynucleotide or polypeptide sequence, respectively, that occur unchanged in the same position in two or more sequences being compared. Nucleotides or amino acids that are relatively conserved are those that are more conserved between related sequences than nucleotides or amino acids that appear elsewhere in the sequences.

[0191] In some embodiments, two or more sequences are said to be "completely conserved" if they are 100% identical to each other. In some embodiments, two or more sequences are said to be "highly conserved" if they are at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to each other. In some embodiments, two or more sequences are said to be "highly conserved" if they are about 70% identical, about 80% identical, about 90% identical, about 95%, about 98%, or about 99% identical to each other. In some embodiments, two or more sequences are said to be "conserved" if they are at least 30% identical, at least 40% identical, at least 50% identical, at least 60% identical, at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to each other. In some embodiments, two or more sequences are said to be "conserved" if they are about 30% identical, about 40% identical, about 50% identical, about 60% identical, about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 98% identical, or about 99% identical to one another. Sequence conservation can apply to the entire length of a polynucleotide or polypeptide, or to portions, regions, or features thereof.

[0192] Contacting: As used herein, the term "contacting" means establishing a physical connection between two or more entities. For example, contacting a mammalian cell with a composition means that the mammalian cell and the composition are caused to share a physical connection. Methods for contacting cells with external entities, both in vivo and ex vivo, are well known in the biological arts. For example, contacting a composition with a mammalian cell placed within a mammal can be performed by various routes of administration (e.g., intravenous, intramuscular, intradermal, and subcutaneous) and can include various amounts of the composition. Furthermore, two or more mammalian cells can be contacted with the composition.

[0193] Controlled Release: As used herein, the term "controlled release" refers to a pharmaceutical or therapeutic composition or compound release profile that conforms to a particular release pattern to produce a desired, e.g., therapeutic, result.

[0194] Covalent Derivatives: The term "covalent derivatives" when referring to polypeptides includes modification of the native or starting protein with organic proteinaceous or non-proteinaceous derivatizing agents and / or post-translational modifications. Covalent modifications are traditionally introduced by reacting targeted amino acid residues of the protein with organic derivatizing agents capable of reacting with selected side chains or terminal residues, or by utilizing post-translational modification mechanisms operative in selected recombinant host cells. The resulting covalent derivatives are useful in programs aimed at identifying residues important for biological activity, immunoassays, or the preparation of anti-protein antibodies for immunoaffinity purification of recombinant glycoproteins. Such modifications are within the skill of one in the art and are performed without undue experimentation.

[0195] Circular or cyclized: As used herein, the term "circular" refers to the presence of a continuous loop. A circular molecule need not be circular, merely linked to form an unbroken chain of subunits. Circular molecules, such as engineered RNA or mRNA, can be single units or multimers, or can comprise one or more components of a complex or higher-order structure.

[0196] Cytotoxic: As used herein, "cytotoxic" refers to killing or causing harmful, toxic, or fatal effects to a cell (e.g., a mammalian cell (e.g., a human cell)), bacterium, virus, fungus, protozoan, parasite, prion, or combinations thereof.

[0197] Delivery: As used herein, the term "deliver" means providing an entity to a destination. For example, delivering a polynucleotide to a subject can include administering a composition to the subject (e.g., by intravenous, intramuscular, intradermal, or subcutaneous routes). Administering a composition to a mammal or mammalian cells can include contacting one or more cells with the composition.

[0198] Delivery vehicle: As used herein, "delivery vehicle" refers to any substance that at least partially facilitates in vivo, in vitro, or ex vivo delivery of a polynucleotide to a target cell or tissue (e.g., a tumor, etc.). Referencing something as a delivery vehicle does not mean that it may not also have a therapeutic effect.

[0199] Destabilized: As used herein, the terms "unstable," "destabilize," or "destabilizing region" refer to a region or molecule that is less stable than the starting, wild-type, or native form of the same region or molecule.

[0200] Detectable Label: As used herein, "detectable label" refers to one or more markers, signals, or moieties attached to, incorporated into, or associated with another entity that are readily detected by methods known in the art, including radiography, fluorescence, chemiluminescence, enzymatic activity, absorbance, and the like. Detectable labels include radioisotopes, fluorophores, chromophores, enzymes, dyes, metal ions, ligands such as biotin, avidin, streptavidin, and haptens, quantum dots, and the like. Detectable labels can be placed at any position in the peptides or proteins disclosed herein. They can be within an amino acid, peptide, or protein, or can be located at the N-terminus or C-terminus.

[0201] Diastereomers: As used herein, the term "diastereomers" means stereoisomers that are not mirror images of one another and are not superimposable with respect to one another.

[0202] Digestion: As used herein, the term "digest" means to break down into smaller fragments or components. When referring to polypeptides or proteins, digestion results in the production of peptides.

[0203] Distal: As used herein, the term "distal" means located away from the center or from a point or area of ​​interest.

[0204] Domain: As used herein, when referring to a polypeptide, the term "domain" refers to a motif in a polypeptide having one or more identifiable structural or functional features or characteristics (e.g., binding ability that serves as a site for protein-protein interaction).

[0205] Dosage regimen: As used herein, a "medication regimen" or "dosing regimen" is a treatment, prophylaxis, or palliative care administration schedule or physician-determined regimen.

[0206] Effective amount: As used herein, the term "effective amount" of an agent is an amount sufficient to bring about a beneficial or desired result, e.g., a clinical result, and thus "effective amount" depends on the context in which it is applied. The term "effective amount" can be used interchangeably with "effective dose," "therapeutically effective amount," or "therapeutically effective dose."

[0207] Enantiomer: As used herein, the term "enantiomer" refers to an individual optically active form of a compound of the present disclosure having an optical purity or enantiomeric excess (as determined by standard methods in the art) of at least 80% (i.e., at least 90% of one enantiomer and at most 10% of the other enantiomer), at least 90%, or at least 98%.

[0208] Encapsulate: As used herein, the term "encapsulate" means to enclose, surround, or enclose.

[0209] Engineered: As used herein, an embodiment of the present disclosure is "engineered" if it is designed to have characteristics or properties, whether structural or chemical, that vary from the starting, wild-type or native molecule.

[0210] Enhanced delivery: As used herein, the term "enhanced delivery" refers to the delivery of a composition to a target tissue of interest (e.g., the liver of a mammal) at a greater (e.g., at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold) level compared to the level of delivery to the target tissue of interest by a control composition. The level of delivery can be measured by comparing the amount of protein produced in the tissue to the weight of the tissue, comparing the amount of polynucleotide in the tissue to the weight of the tissue, comparing the amount of protein produced in the tissue to the amount of total protein in the tissue, or comparing the amount of polynucleotide in the tissue to the amount of total polynucleotide in the tissue. It will be understood that enhanced delivery to a target tissue need not be determined in the subject being treated, but can be determined in a surrogate animal model (e.g., a rat model), etc.

[0211] Exosome: As used herein, an "exosome" is a vesicle secreted by mammalian cells.

[0212] Expression: As used herein, "expression" of a nucleic acid sequence refers to one or more of the following events: (1) the generation of an RNA template from a DNA sequence (e.g., by transcription); (2) the processing of the RNA transcript (e.g., by splicing, editing, 5' capping, and / or 3' end processing); (3) the translation of the RNA into a polypeptide or protein; and (4) the post-translational modification of the polypeptide or protein.

[0213] Ex vivo: As used herein, the term "ex vivo" refers to an event that occurs outside of an organism (e.g., an animal, plant, or microorganism, or cells or tissues thereof). An ex vivo event can occur in an environment that is largely unchanged from the natural (e.g., in vivo) environment.

[0214] Characteristic: As used herein, "characteristic" refers to a property, characteristic, or distinguishing element. When referring to a polypeptide, a "characteristic" is defined as a distinct amino acid sequence-based component of the molecule. Characteristics of polypeptides encoded by the polynucleotides of the present disclosure include surface expression, local conformational shape, folds, loops, half-loops, domains, half-domains, sites, termini, or any combination thereof.

[0215] Formulation: As used herein, a "formulation" comprises at least a polynucleotide or polypeptide and one or more carriers, excipients, and delivery agents or vehicles.

[0216] Forward Scatter (FSC): As used herein, forward scatter or FSC is a flow cytometry measurement that detects light scattered by cells along the path of the laser.

[0217] Fragment: As used herein, "fragment" refers to a portion. For example, a fragment of a protein can include a polypeptide obtained by digesting a full-length protein isolated from a cultured cell. In some embodiments, a fragment is a subsequence of a full-length protein (e.g., one of the subunits of IL-23) lacking an N-terminus, and / or a C-terminus, and / or an internal subsequence. In some preferred embodiments of the present disclosure, a fragment of a protein of the present disclosure is a functional fragment.

[0218] Functional: As used herein, a "functional" biomolecule is a biomolecule in a form in which it exhibits a property and / or activity by which it is characterized.

[0219] Homology: As used herein, the term "homology" refers to the overall relatedness between polymer molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules. Generally, the term "homology" refers to the evolutionary relationship between two molecules. Thus, two homologous molecules share a common evolutionary ancestry. In the context of the present disclosure, the term homology encompasses both identity and similarity.

[0220] In some embodiments, polymer molecules are considered to be "homologous" to one another if at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the monomers in the molecules are identical (exactly the same monomers) or similar (conservative substitutions). The term "homologous" necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences).

[0221] Identity: As used herein, the term "identity" refers to the overall monomer conservation between polymer molecules, for example, between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules. The percent identity of two polynucleotide sequences can be calculated, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second nucleic acid sequences for optimal alignment, and non-identical sequences can be ignored for comparison purposes). In certain embodiments, the length of the sequence aligned for comparison is 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 length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. If a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that must be introduced for optimal alignment of the two sequences. Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent.

[0222] Suitable software programs are available from various sources and for aligning both protein and nucleotide sequences. One suitable program for determining percent sequence identity is bl2seq, which is part of the BLAST suite of programs available from the U.S. government's National Center for Biotechnology Information's BLAST website (blast.ncbi.nlm.nih.gov). bl2seq performs comparisons between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs include, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Bioinformatics Institute (EBI).

[0223] Sequence alignment can be performed using methods known in the art, such as MAFFT, Clustal (ClustalW, Clustal X, or Clustal Omega), and MUSCLE.

[0224] Different regions within a single polynucleotide or polypeptide target sequence that align with a polynucleotide or polypeptide reference sequence can each have their own percent sequence identity.

[0225] In certain embodiments, the percent identity "%ID" of a first amino acid sequence (or nucleic acid sequence) to a second amino acid sequence (or nucleic acid sequence) is calculated as %ID = 100 x (Y / Z), where Y is the number of amino acid residues (or nucleic acid bases) scored as identical matches in an alignment of the first and second sequences (aligned by visual inspection or by a particular sequence alignment program), and Z is the total number of residues in the second sequence. If the length of the first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be higher than the percent identity of the second sequence to the first sequence.

[0226] Those skilled in the art will understand that the generation of sequence alignments for calculating percent sequence identity is not limited to secondary sequence-sequence comparisons driven exclusively by primary sequence data. It will also be understood that sequence alignments can be generated by integrating sequence data with data from heterogeneous sources, such as structural data (e.g., crystallographic protein structures), functional data (e.g., mutation locations), or phylogenetic data. A suitable program for integrating heterogeneous data to generate multiple sequence alignments is available at www.tcoffee.org or, for example, T-Coffee, available from EBI. It will also be understood that the final alignment used to calculate percent sequence identity can be curated automatically or manually.

[0227] Immune response: The term "immune response" refers to the action of, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules produced by the above cells, including antibodies, cytokines, and complement, that results in the selective damage, destruction, or elimination from the body of invading pathogens, pathogen-infected cells or tissues, cancerous cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues.

[0228] Inflammatory response: "Inflammatory response" refers to an immune response that includes specific and non-specific defense systems. A specific defense system response is a specific immune system reaction to an antigen. An example of a specific defense system response is an antibody response. A non-specific defense system response is an inflammatory response mediated by white blood cells that are generally incapable of immunological memory, such as macrophages, eosinophils, and neutrophils. In some embodiments, the immune response includes the secretion of inflammatory cytokines, resulting in increased levels of inflammatory cytokines.

[0229] Inflammatory cytokine: The term "inflammatory cytokine" refers to cytokines that are elevated in an inflammatory response. Examples of inflammatory cytokines include interleukin-6 (IL-6), CXCL1 (chemokine (C-X-C motif) ligand 1 (also known as GROc), interferon-γ (IFNγ), tumor necrosis factor α (TNFα), interferon-γ-inducible protein 10 (IP-10), or granulocyte colony-stimulating factor (G-CSF). The term inflammatory cytokine also includes other cytokines associated with inflammatory responses known in the art, such as interleukin-1 (IL-1), interleukin-8 (IL-8), interleukin-12 (IL-12), interleukin-13 (IL-13), interferon α (IFN-α), etc.

[0230] In vitro: As used herein, the term "in vitro" refers to events that take place not in a living organism (e.g., an animal, plant, or microorganism) but in an artificial environment, such as in a test tube or reaction vessel, in cell culture, in a petri dish, etc.

[0231] In vivo: As used herein, the term "in vivo" refers to events that take place within an organism (e.g., an animal, plant, or microorganism, or cells or tissues thereof).

[0232] Insertion and deletion variants: "Insertion variants," when referring to polypeptides, are those that have one or more amino acids inserted immediately adjacent to an amino acid at a particular position in a native or starting sequence. "Immediately adjacent to" an amino acid means linked to either the alpha-carboxy or alpha-amino functionality of the amino acid. "Deletion variants," when referring to polypeptides, are those in which one or more amino acids in the native or starting amino acid sequence have been removed. Typically, deletion variants have one or more amino acids deleted in a particular region of the molecule.

[0233] Intact: As used herein, in the context of a polypeptide, the term "intact" means retaining the amino acids corresponding to the wild-type protein, e.g., not mutating or substituting the wild-type amino acids. Conversely, in the context of a nucleic acid, the term "intact" means retaining the nucleobases corresponding to the wild-type nucleic acid, e.g., not mutating or substituting the wild-type nucleobases.

[0234] Isolated: As used herein, the term "isolated" refers to a substance or entity that has been separated from at least some of the components with which it is associated (whether in nature or in an experimental setting). Isolated substances (e.g., nucleotide sequences or protein sequences) can have various levels of purity with respect to the materials with which they are associated. Isolated substances and / or entities can be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or more of the other components with which they were initially associated. In some embodiments, isolated agents are greater than about 80%, greater than about 85%, greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, greater than about 99%, or greater than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. The term "substantially isolated" means that a compound is substantially separated from the environment in which it was formed or detected. Partial isolation can include, for example, compositions enriched for a compound of the present disclosure. Substantial isolation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% (by weight) of a compound of the present disclosure or a salt thereof.

[0235] An "isolated" polynucleotide, vector, polypeptide, cell, or any composition disclosed herein is a polynucleotide, vector, polypeptide, cell, or composition in a form not found in nature. Isolated polynucleotides, vectors, polypeptides, or compositions include those that have been purified to the extent that they are no longer in the form in which they are found in nature. In some aspects, an isolated polynucleotide, vector, polypeptide, or composition is substantially pure.

[0236] Isomer: As used herein, the term "isomer" refers to any tautomer, stereoisomer, enantiomer, or diastereomer of any compound of the present disclosure. It is recognized that the compounds of the present disclosure can have one or more chiral centers and / or double bonds and can therefore exist as stereoisomers, such as double bond isomers (i.e., geometric E / Z isomers) or diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis / trans isomers). In accordance with the present disclosure, the chemical structures depicted herein, and thus the compounds of the present disclosure, encompass all of the corresponding stereoisomers, i.e., both stereoisomerically pure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure), as well as mixtures of enantiomers and stereoisomers, e.g., racemates. Enantiomeric and stereoisomeric mixtures of the compounds of the present disclosure can typically be resolved into their component enantiomers or stereoisomers by well-known methods such as chiral-phase gas chromatography, chiral-phase high-performance liquid chromatography, crystallizing the compounds as chiral salt complexes, or crystallizing the compounds in chiral solvents, etc. Enantiomers and stereoisomers can also be obtained from stereomerically or enantiomerically pure intermediates, reagents, and catalysts by well-known asymmetric synthetic methods.

[0237] Linker: As used herein, "linker" refers to a group of atoms, e.g., 10 to 1,000 atoms, which may be composed of atoms or groups such as, but not limited to, carbon, amino, alkylamino, oxygen, sulfur, sulfoxide, sulfonyl, carbonyl, and imine. The linker can be attached at one end to a modified nucleoside or nucleotide on a nucleobase or sugar moiety and at the second end to a payload, e.g., a detectable or therapeutic agent. The linker can be of sufficient length so as not to interfere with incorporation into a nucleic acid sequence. Linkers, as described herein, can be used for any useful purpose, e.g., to form polynucleotide multimers (e.g., via linkage of two or more chimeric polynucleotide molecules or IVT polynucleotides) or polynucleotide conjugates, as well as to administer a payload.

[0238] Polypeptides, such as amino acids, or polynucleotides, such as nucleoside monomers or multipliers, can be used as linkers. For example, a short peptide can act as a linker between two proteins or polypeptides. Similarly, a series of nucleosides or nucleotides can act as a linker between two polynucleotides.

[0239] Examples of chemical groups that can be incorporated into a linker include, but are not limited to, alkyl, alkenyl, alkynyl, amide, amino, ether, thioether, ester, alkylene, heteroalkylene, aryl, or heterocyclyl, each of which may be optionally substituted as described herein. Examples of linkers include, but are not limited to, unsaturated alkanes, polyethylene glycols (e.g., ethylene or propylene glycol monomer units, e.g., diethylene glycol, dipropylene glycol, triethylene glycol, tripropylene glycol, tetraethylene glycol, or tetraethylene glycol), and dextran polymers, and derivatives thereof. Other examples include, but are not limited to, cleavable moieties within the linker, such as disulfide bonds (-SS-) or azo bonds (-N=N-), which can be cleaved using reducing agents or photolysis. Non-limiting examples of selectively cleavable bonds include amide bonds, which can be cleaved, for example, by the use of tris(2-carboxyethyl)phosphine (TCEP) or other reducing agents, and / or photolysis, as well as ester bonds, which can be cleaved, for example, by acidic or basic hydrolysis.

[0240] Method of administration: As used herein, "method of administration" can include intravenous, intramuscular, intradermal, subcutaneous, or other methods of delivering a composition to a subject. The method of administration can be selected to target delivery (e.g., for specific delivery) to a particular area or system of the body.

[0241] Modified: As used herein, "modified" refers to an altered state or structure of a molecule of the present disclosure. Molecules can be modified in many ways, including chemically, structurally, and functionally. In some embodiments, mRNA molecules of the present disclosure are modified by the introduction of non-natural nucleosides and / or nucleotides, e.g., relative to the natural ribonucleotides A, U, G, and C. Non-canonical nucleotides, such as cap structures, differ from the chemical structure of A, C, G, U ribonucleotides, but are not considered "modified."

[0242] Naturally-occurring: As used herein, "naturally-occurring" means existing in nature without artificial assistance.

[0243] Non-human vertebrate: As used herein, "non-human vertebrate" includes all vertebrates except Homo sapiens, including wild and domestic species. Examples of non-human vertebrates include, but are not limited to, mammals such as alpacas, banteng, bison, camels, cats, cows, deer, dogs, donkeys, gayal, goats, guinea pigs, horses, llamas, mules, pigs, rabbits, reindeer, sheep buffalo, and yaks.

[0244] Nucleic acid sequence: The terms "nucleic acid sequence," "nucleotide sequence," or "polynucleotide sequence" are used interchangeably and refer to a contiguous nucleic acid sequence. The sequence can be single- or double-stranded DNA or RNA, such as mRNA.

[0245] The term "nucleic acid," in its broadest sense, includes any compound and / or substance comprising a polymer of nucleotides. These polymers are often referred to as polynucleotides. Exemplary nucleic acids or polynucleotides of the present disclosure include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA including LNA having a β-D-ribo configuration, α-LNA (a diastereomer of LNA) having an α-L-ribo configuration, 2'-amino LNA having a 2'-amino functionalization, and 2'-amino-α-LNA having a 2'-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA), or hybrids, or combinations thereof.

[0246] The phrase "nucleotide sequence encoding" refers to a nucleic acid (e.g., mRNA or DNA molecule) coding sequence that encodes a polypeptide. The coding sequence can further include initiation and termination signals operably linked to regulatory elements, including a promoter and polyadenylation signal, capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered. The coding sequence can further include a sequence encoding a signal peptide.

[0247] Off-target: As used herein, "off-target" refers to any unintended effect on any one or more targets, genes, or cellular transcripts.

[0248] Open reading frame: As used herein, "open reading frame" or "ORF" refers to a sequence that does not contain a stop codon in a given reading frame.

[0249] Operably linked: As used herein, the phrase "operably linked" refers to a functional linkage between two or more molecules, constructs, transcripts, entities, moieties, etc.

[0250] Optionally substituted: As used herein, phrases of the form "optionally substituted X" (e.g., optionally substituted alkyl) are intended to be equivalent to "X, where X is optionally substituted" (e.g., "alkyl, where the alkyl is optionally substituted alkyl"). This does not imply that the feature "X" (e.g., alkyl) itself is optional.

[0251] Portion: As used herein, a "portion" or "region" of a polynucleotide is defined as any portion of the polynucleotide that is less than the full length of the polynucleotide. Similarly, a "portion" or "region" of a polypeptide is defined as any portion of the polypeptide that is less than the full length of the polynucleotide.

[0252] Patient: As used herein, "patient" refers to a subject who is seeking or in need of treatment, in need of treatment, undergoing treatment, will undergo treatment, or being cared for by a trained professional for a particular disease or condition.

[0253] Pharmaceutically acceptable: The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio.

[0254] Pharmaceutically acceptable excipient: As used herein, the phrase "pharmaceutically acceptable excipient" refers to any component other than the compounds described herein (e.g., a vehicle capable of suspending or dissolving an active compound) that has the properties of being substantially non-toxic and non-inflammatory in patients. Excipients can include, for example, anti-adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colorants), emollients, emulsifiers, fillers (diluents), film-forming agents or coatings, flavorings, fragrances, glidants (glidants), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and hydration water. Exemplary excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0255] Pharmaceutically acceptable salts: The present disclosure also includes pharmaceutically acceptable salts of the compounds described herein. As used herein, "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds, in which the parent compound is modified by converting an existing acid or base moiety into its salt form (for example, by reacting a free base group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. Representative acid addition salts include acetate, acetic acid, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanoate, and the like. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. The pharmaceutically acceptable salts of the present disclosure include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.The pharmaceutically acceptable salts of the present disclosure can be synthesized from parent compounds containing basic or acidic moieties by conventional chemical methods.Generally, such salts can be prepared by reacting the free acid or free base form of these compounds with a stoichiometric amount of an appropriate base or acid in water or an organic solvent, or a mixture of the two, and generally use non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile.A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 17. th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use, P.H. Stahl and C.G. Wermuth (eds.), Wiley-VCH, 2008, and Berge et al., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of which is incorporated herein by reference in its entirety.

[0256] Pharmaceutically acceptable solvate: As used herein, the term "pharmaceutically acceptable solvate" refers to a compound of the present disclosure in which molecules of a suitable solvent are incorporated into the crystal lattice. The suitable solvent is physiologically acceptable at the administered dosage. For example, solvates can be prepared by crystallization, recrystallization, or precipitation from a solution containing an organic solvent, water, or a mixture thereof. Examples of suitable solvents include ethanol, water (e.g., monohydrate, dihydrate, and trihydrate), N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO), N,N'-dimethylformamide (DMF), N,N'-dimethylacetamide (DMAC), 1,3-dimethyl-2-imidazolidinone (DMEU), 1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone, benzyl benzoate, and the like. When water is the solvent, the solvate is referred to as a "hydrate."

[0257] Pharmacokinetics: As used herein, "pharmacokinetics" refers to any one or more characteristics of a molecule or compound that determine the fate of a substance administered to an organism. Pharmacokinetics is divided into several areas, including the extent and rate of absorption, distribution, metabolism, and excretion. This is commonly referred to as ADME, where (A) absorption is the process of a substance entering the blood circulation; (D) distribution is the dispersion or dissemination of a substance throughout the body's fluids and tissues; (M) metabolism (or biotransformation) is the irreversible conversion of a parent compound to daughter metabolites; and (E) excretion (or elimination) refers to the removal of a substance from the body. In rare cases, some drugs irreversibly accumulate in body tissues.

[0258] Physicochemical As used herein, "physicochemical" means or relates to physical and / or chemical properties.

[0259] Polynucleotide: As used herein, the term "polynucleotide" refers to a polymer of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, their analogs, or mixtures thereof. The term refers to the primary structure of the molecule. Thus, the term includes triple-, double-, and single-stranded deoxyribonucleic acid ("DNA"), as well as triple-, double-, and single-stranded ribonucleic acid ("RNA"). It also includes modified forms of polynucleotides, for example, by alkylation and / or by capping, as well as unmodified forms. More specifically, the term "polynucleotide" includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), tRNA, rRNA, hRNA, siRNA, and mRNA (spliced ​​or not), any other type of polynucleotide that is an N- or C-glycoside of a purine or pyrimidine base, as well as other polymers containing a nornucleotide backbone, such as polyamides (e.g., peptide nucleic acids "PNAs") and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers, provided that the polymer contains nucleobases in a configuration that allows for base pairing and base stacking as found in DNA and RNA. In certain embodiments, the polynucleotide comprises mRNA. In other embodiments, the mRNA is synthetic mRNA. In some embodiments, the synthetic mRNA contains at least one unnatural nucleobase. In some embodiments, all nucleobases of a particular class are substituted with an unnatural nucleobase (e.g., all uridines in the polynucleotides disclosed herein can be replaced with an unnatural nucleobase, such as 5-methoxyuridine). In some embodiments, a polynucleotide (e.g., synthetic RNA or synthetic DNA) includes only natural nucleobases, i.e., A, C, T, and U in the case of synthetic DNA, or A, C, T, and U in the case of synthetic RNA.

[0260] Those skilled in the art will understand that while a T base in the codon maps disclosed herein occurs in DNA, the T base is replaced with a U base in the corresponding RNA. For example, a codon-nucleotide sequence disclosed herein in DNA form, such as a vector or in vitro translation (IVT) template, will have its T base transcribed as a U based on its corresponding transcribed mRNA. In this regard, both codon-optimized DNA sequences (containing T) and their corresponding RNA sequences (containing U) are considered codon-optimized nucleotide sequences of the present disclosure. Those skilled in the art will also understand that equivalent codon maps can be generated by substituting one or more bases with unnatural bases. Thus, for example, the TTC codon (DNA map) corresponds to the UUC codon (RNA map), which in turn corresponds to the "P"C codon (RNA map in which U is replaced with pseudouridine).

[0261] Standard AT and GC base pairs form under conditions that allow the formation of hydrogen bonds between the N3-H and C4-oxy of thymidine and the N1 and C6-NH2 of adenosine, respectively, and between the C2-oxy, N3 and C4-NH2 of cytidine and the C2-NH2, N'-H and C6-oxy of guanosine, respectively. Thus, for example, guanosine (2-amino-6-oxy-9-β-D-ribofuranosyl-purine) can be modified to form isoguanosine (2-oxy-6-amino-9-β-D-ribofuranosyl-purine). Such modifications result in nucleoside bases that no longer effectively form standard base pairs with cytosine. However, modification of cytosine (1-β-D-ribofuranosyl-2-oxy-4-amino-pyrimidine) to form isocytosine (1-β-D-ribofuranosyl-2-oxy-4-amino-pyrimidine) results in a modified nucleotide that does not base pair efficiently with guanosine but does base pair with isoguanosine (U.S. Patent No. 5,681,702 to Collins et al.). Isocytosine is available from Sigma Chemical Co. (St. Louis, MO); isocytidine is prepared according to the method described by Switzer et al. (1993) in Biochemistry. 32:10489-10496 and the references cited therein; 2'-deoxy-5-methyl-isocytidine can be prepared by the method of Tor et al., (1993) J. Am. Chem. Soc. 115:4461-4467, and the references cited therein; and isoguanine nucleotides can be prepared using the method described by Switzer et al., 1993, supra, and Mantsch et al., (1993) Biochem. 14:5593-5601, or by the method described in U.S. Pat. No. 5,780,610 to Collins et al. Other unnatural base pairs can be synthesized by the method described in Piccirilli et al. (1990) Nature 343:33-37 for the synthesis of 2,6-diaminopyrimidine and its complement (1-methylpyrazolo-[4,3]pyrimidine-5,7-(4H,6H)-dione). Other such modified nucleotide units that form unique base pairs are known, such as those described in Leach et al. (1992) J. Am. Chem. Soc. 114:3675-3683, and Switzer et al., supra.

[0262] Nucleic acid sequence: The terms "nucleic acid sequence," "nucleotide sequence," or "polynucleotide" are used interchangeably and refer to a contiguous nucleic acid sequence. The sequence can be single- or double-stranded DNA or RNA, such as mRNA.

[0263] The phrase "nucleotide sequence encoding" and variants thereof refer to a nucleic acid (e.g., mRNA or DNA molecule) coding sequence comprising a nucleotide sequence encoding a polypeptide described herein or a functional fragment thereof. The coding sequence can further comprise initiation and termination signals operably linked to regulatory elements, including a promoter and polyadenylation signal, capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered. The coding sequence can further comprise a sequence encoding a signal peptide.

[0264] Polypeptide: The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. A polymer can contain modified amino acids. The term also encompasses amino acid polymers that are modified naturally or by intervention (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component). For example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, e.g., homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), as well as other modifications known in the art, are also included within the definition.

[0265] As used herein, the term refers to proteins, polypeptides, and peptides of any size, structure, or function. Polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments, and other equivalents, variants, and analogs of the above. Polypeptides can be single polypeptides or multimolecular complexes such as dimers, trimers, or tetramers. They can also include single-chain or multi-chain polypeptides. Disulfide bonds are most commonly found in multi-chain polypeptides. The term polypeptide can also apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids. In some embodiments, a "peptide" can be 50 amino acids or less in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0266] Polypeptide variant: As used herein, the term "polypeptide variant" refers to a molecule whose amino acid sequence differs from that of a native or reference sequence. Amino acid sequence variants can have substitutions, deletions, and / or insertions at specific positions within the amino acid sequence compared to the native or reference sequence. Typically, variants have at least about 50% identity, at least about 60% identity, at least about 70% identity, at least about 80% identity, at least about 90% identity, at least about 95% identity, or at least about 99% identity to the native or reference sequence. In some embodiments, they are at least about 80% or at least about 90% identical to the native or reference sequence.

[0267] Inhibit: As used herein, the term "inhibit" refers to partially or completely delaying the onset of an infection, disease, disorder, and / or condition; partially or completely delaying the onset of one or more symptoms, characteristics, or clinical findings of a particular infection, disease, disorder, and / or condition; partially or completely delaying the onset of one or more symptoms, characteristics, or findings of a particular infection, disease, disorder, and / or condition; partially or completely delaying the progression from an infection, a particular disease, disorder, and / or condition; and / or reducing the risk of developing a condition associated with an infection, disease, disorder, and / or condition.

[0268] Prophylactic: As used herein, "prophylactic" refers to a therapeutic agent or course of action used to prevent the spread of disease.

[0269] Prophylaxis: As used herein, "prophylaxis" refers to measures taken to maintain health and prevent the spread of disease. "Immunoprophylaxis," e.g., a vaccine, refers to a means to generate active or passive immunity to prevent the spread of disease.

[0270] Proteolytic cleavage site: As used herein, "proteolytic cleavage site" refers to a site at which controlled cleavage of an amino acid chain can be achieved by chemical, enzymatic or photochemical means.

[0271] Protein cleavage signal: As used herein, a "protein cleavage signal" refers to at least one amino acid that labels or marks a polypeptide for cleavage.

[0272] Protein of interest: As used herein, the term "protein of interest" or "desired protein" includes those provided herein, as well as fragments, mutants, variants, and variations thereof.

[0273] Proximal: As used herein, the term "proximal" means located closer to the center or point or area of ​​interest.

[0274] Pseudouridine: As used herein, pseudouridine refers to the C-glycoside isomer of the nucleoside uridine. A "pseudouridine analog" is any modification, variant, isoform, or derivative of pseudouridine. For example, pseudouridine analogs include, but are not limited to, 1-carboxymethyl-pseudouridine, 1-propyl-pseudouridine, 1-taurominomethyl-pseudouridine, 1-taurominomethyl-4-thio-pseudouridine, 1-methylpseudouridine (m 1 ψ), 1-methyl-4-thio-pseudouridine (m 1 s 4 ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m 3ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydropseudouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp 3 ψ), and 2'-O-methyl-pseudouridine (xm).

[0275] Purified: As used herein, "purify," "purified," and "purified" mean to make substantially pure or clear from undesirable components, material contamination, admixture, or imperfections.

[0276] Reference nucleic acid sequence: The term "reference nucleic acid sequence" or "reference nucleic acid" or "reference nucleotide sequence" or "reference sequence" refers to a starting nucleic acid sequence (e.g., an RNA, e.g., an mRNA sequence) that can be sequence-optimized. In some embodiments, the reference nucleic acid sequence is a wild-type nucleic acid sequence, a fragment, or a variant thereof. In some embodiments, the reference nucleic acid sequence is a nucleic acid sequence that has previously been sequence-optimized.

[0277] Salt: In some embodiments, pharmaceutical or therapeutic compositions for intratumoral delivery disclosed herein contain salts of some of their lipid components. The term "salt" includes any anionic and cationic complexes. Non-limiting examples of anions include inorganic and organic anions, such as fluoride, chloride, bromide, iodide, oxalate (e.g., hemioxalate), phosphate, phosphonate, hydrogen phosphate, dihydrogen phosphate, oxide, carbonate, bicarbonate, nitrate, nitrite, nitride, bisulfite, sulfide, sulfite, bisulfate, sulfate, thiosulfate, hydrogen sulfate, borate, formate, acetate, benzoate, citrate, tartrate, lactate, acrylate, polyacrylate, fumarate, Included are maleate, itaconate, glycolate, gluconate, malate, mandelate, tiglate, ascorbate, salicylate, polymethacrylate, perchlorate, chlorite, hypochlorite, bromate, hypobromite, iodate, alkylsulfonate, arylsulfonate, arsenate, arsenite, chromate, dichromate, cyanide, cyanate, thiocyanate, hydroxide, peroxide, permanganate, and mixtures thereof.

[0278] Sample: As used herein, the term "sample" or "biological sample" refers to a subset of tissues, cells, or components thereof (e.g., bodily fluids including, but not limited to, blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid, and semen). Samples can also include homogenates, lysates, or extracts prepared from whole organisms or subsets of their tissues, cells, or components, or fractions or portions thereof, including, but not limited to, for example, plasma, serum, spinal fluid, lymph, external sections of skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs. Samples also refer to media such as nutrient broths or gels, which may contain cellular components such as proteins or nucleic acid molecules.

[0279] Side Scatter (SSC): Side scatter, or SSC, is a flow cytometry measurement that measures light scattered by cells at a 90 degree angle relative to the laser.

[0280] Signal sequence: As used herein, the phrases "signal sequence," "signal peptide," and "transport peptide" are used interchangeably and refer to a sequence capable of directing the transport or localization of a protein to a specific organelle, cellular compartment, or extracellular transport. The term encompasses both signal sequence polypeptides and nucleic acid sequences encoding signal sequences. Thus, reference to a signal sequence in the context of a nucleic acid actually refers to the nucleic acid sequence encoding the signal sequence polypeptide.

[0281] Similarity: As used herein, the term "similarity" refers to the overall relatedness between polymer molecules, e.g., between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules. Calculation of percent similarity of polymer molecules to each other can be done in the same manner as calculation of percent identity, except that percent similarity calculation takes into account conservative substitutions, as understood in the art.

[0282] Specific delivery: As used herein, the terms "specific delivery," "specifically deliver," or "specifically delivering" refer to the delivery of more (e.g., at least 1.5-fold more, at least 2-fold more, at least 3-fold more, at least 4-fold more, at least 5-fold more, at least 6-fold more, at least 7-fold more, at least 8-fold more, at least 9-fold more, at least 10-fold more) polynucleotide to a target tissue of interest (e.g., the liver of a mammal) compared to an off-target tissue (e.g., the spleen of a mammal). The level of delivery to a particular tissue can be measured by comparing the amount of protein produced in the tissue to the weight of the tissue, comparing the amount of polynucleotide in the tissue to the weight of the tissue, comparing the amount of protein produced in the tissue to the amount of total protein in the tissue, or comparing the amount of polynucleotide in the tissue to the amount of total polynucleotide in the tissue.

[0283] Stable: As used herein, "stable" refers to a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and, optionally, can be formulated into an efficacious therapeutic agent.

[0284] Stabilized: As used herein, the terms "stabilize," "stabilized," and "stabilized region" mean to cause to stabilize or to stabilize.

[0285] Stereoisomers: As used herein, the term "stereoisomers" refers to all possible different isomeric and conformational forms that a compound (e.g., a compound of any formula described herein) may possess, in particular all possible stereochemical and conformational isomeric forms, all diastereomers, enantiomers, and / or conformers of the basic molecular structure. Some compounds of the present disclosure may exist in different tautomeric forms, all of the latter being included within the scope of the present disclosure.

[0286] Subject: "Subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject for whom diagnosis, prognosis, or treatment is desired, particularly a mammalian subject. Mammalian subjects include, but are not limited to, humans, farm animals, livestock, zoo animals, sport animals, pet animals (such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, and dairy cows); primates (such as apes, monkeys, orangutans, and chimpanzees); canines (such as dogs and wolves); felines (such as cats, lions, and tigers); equines (such as horses, donkeys, and zebras); bears, food animals (such as cows, pigs, and sheep); ungulates (such as deer and giraffes); and rodents (such as mice, rats, hamsters, and guinea pigs). In certain embodiments, the mammal is a human subject. In other embodiments, the subject is a human patient.

[0287] Substantially: As used herein, the term "substantially" refers to the qualitative condition indicating the whole or nearly whole extent or degree of a desired characteristic or property. Those skilled in the biological arts will understand that biological and chemical phenomena rarely, if ever, proceed to completion and / or perfection or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0288] Substantially equal: As used herein in reference to the time difference between doses, this term means plus / minus 2%.

[0289] Substantially simultaneously: As used herein, and in reference to multiple doses, this term means within a few seconds (eg, 2 seconds).

[0290] Suffering from: An individual "suffering from" a disease, disorder, and / or condition has been diagnosed with the disease, disorder, and / or condition or exhibits one or more symptoms of the disease, disorder, and / or condition.

[0291] Susceptible: An individual who is "susceptible to" a disease, disorder, and / or condition may not have been diagnosed with and / or may not exhibit symptoms of the disease, disorder, and / or condition, but has a tendency to develop the disease or its symptoms. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., cancer) is characterized by one or more of the following: (1) a genetic mutation associated with the development of the disease, disorder, and / or condition; (2) a genetic polymorphism associated with the development of the disease, disorder, and / or condition; (3) an increase and / or decrease in the expression and / or activity of proteins and / or nucleic acids associated with the disease, disorder, and / or condition; (4) habits and / or lifestyle associated with the development of the disease, disorder, and / or condition; (5) a family history of the disease, disorder, and / or condition; or (6) exposure to and / or infection by a microorganism associated with the development of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition develops the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition does not develop the disease, disorder, and / or condition.

[0292] Sustained release: As used herein, the term "sustained release" refers to a pharmaceutical or therapeutic composition or compound release profile that matches the release rate over a specific period of time.

[0293] Synthetic: The term "synthetic" means produced, prepared, and / or manufactured by the hand of man. Synthesis of polynucleotides or other molecules of the disclosure can be chemical or enzymatic.

[0294] Target cell: As used herein, "target cell" refers to any one or more cells of interest. The cells may be found in vitro, in vivo, in situ, or in the tissue or organ of an organism. The organism may be an animal, preferably a mammal, more preferably a human, and most preferably a patient.

[0295] Target tissue: As used herein, "target tissue" refers to any one or more tissue types of interest in a subject in which delivery of a polynucleotide will result in a desired biological and / or pharmacological effect. Examples of target tissues of interest include specific tissues, organs, and systems or groups thereof. "Off-target tissue" refers to any one or more tissue types in which expression of the encoded protein does not result in a desired biological and / or pharmacological effect.

[0296] Targeting sequence: As used herein, the phrase "targeting sequence" refers to a sequence that is capable of directing the trafficking or localization of a protein or polypeptide.

[0297] Terminal: As used herein, the term "terminal(s)" or "terminal" when referring to a polypeptide refers to the termini of the peptide or polypeptide. Such terminals are not limited to the first or last position of the peptide or polypeptide, but can include additional amino acids in the terminal region. The polypeptide-based molecules of the present disclosure can be characterized as having both an N-terminus (terminated by an amino acid having a free amino group (NH)) and a C-terminus (terminated by an amino acid having a free carboxyl group (COOH)). Proteins of the present disclosure are sometimes composed of multiple polypeptide chains held together by disulfide bonds or non-covalent forces (multimers, oligomers). These types of proteins will have multiple N- and C-termini. Alternatively, the termini of a polypeptide can sometimes be modified to begin or end with a non-polypeptide-based moiety, such as an organic conjugate.

[0298] Therapeutic Agent: The term "therapeutic agent" refers to an agent that has a therapeutic, diagnostic, and / or prophylactic effect and / or induces a desired biological and / or pharmacological effect when administered to a subject.

[0299] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" means an amount of an agent (e.g., nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.) delivered that, when administered to a subject suffering from or susceptible to an infection, disease, disorder and / or condition, is sufficient to treat, ameliorate the symptoms of, diagnose, prevent and / or delay the onset of the infection, disease, disorder and / or condition.

[0300] Therapeutically Effective Outcome: As used herein, the term "therapeutically effective outcome" means an outcome sufficient to treat, ameliorate the symptoms of, diagnose, prevent, and / or delay the onset of an infection, disease, disorder, and / or condition in a subject suffering from or susceptible to the infection, disease, disorder, and / or condition.

[0301] Transcription: As used herein, the term "transcription" refers to a method of introducing exogenous nucleic acid into a cell. Transfection methods include, but are not limited to, chemical methods, physical treatments, and cationic lipids or mixtures.

[0302] Transfection: As used herein, "transfection" refers to the introduction of a polynucleotide into a cell, whereby a polypeptide encoded by the polynucleotide is expressed (e.g., mRNA) or the polypeptide modulates a cellular function (e.g., siRNA, miRNA). As used herein, "expression" of a nucleic acid sequence refers to the translation of a polynucleotide (e.g., mRNA) into a polypeptide or protein and / or the post-translational modification of a polypeptide or protein.

[0303] Treating, Treatment, Therapy: As used herein, the term "treating" or "treatment" or "therapy" refers to partially or completely alleviating, ameliorating, improving, reducing, delaying onset, inhibiting progression, reducing severity, and / or reducing incidence of one or more symptoms or characteristics of a hyperproliferative disease, e.g., cancer. For example, "treating" cancer can refer to inhibiting tumor survival, growth, and / or spread. Treatment can be administered to subjects who do not show signs of the disease, disorder, and / or condition and / or who show only early signs of the disease, disorder, and / or condition, for the purpose of reducing the risk of developing pathology associated with the disease, disorder, and / or condition.

[0304] Unmodified: As used herein, "unmodified" refers to any substance, compound, or molecule before it has been altered in any way. Unmodified can, but does not necessarily, refer to the wild-type or native form of a biomolecule. A molecule can undergo a series of modifications, whereby each modified molecule can serve as the "unmodified" starting molecule for subsequent modifications.

[0305] Mutant: As used in this disclosure, the term mutant refers to both naturally occurring variants (e.g., polymorphisms, isoforms, etc.) and artificial variants in which at least one amino acid residue in a native or starting sequence (e.g., a wild-type sequence) has been removed and a different amino acid inserted in its place at the same position. These variants can be described as "substitution variants." The substitutions can be single, where only one amino acid in the molecule has been substituted, or multiple, where two or more amino acids have been substituted in the same molecule. When amino acids are inserted or deleted, the resulting variant is an "insertion variant" or "deletion variant," respectively.

[0306] The details of one or more embodiments of the present disclosure are set forth in the accompanying description below. Although any materials and methods similar or equivalent to those described herein can be used in practicing or testing the present disclosure, the preferred materials and methods are described herein. Other features, objects, and advantages of the present disclosure will be apparent from the description. As used herein, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present specification controls.

[0307] The present disclosure is further illustrated by the following non-limiting examples. [Example]

[0308] Example 1. Flow cytometry analysis of SIINFEKL-MHCl presentation

[0309] Murine dendritic cells (JAWS II) were seeded at 100,000 cells / well in 24-well plates (500 μL volume) and treated with a final dose of 200 ng of mRNA (encoding a CD1 vaccine cassette containing mouse CD1d, human CD1d, and human CD1b) per formulation per well. The mRNA vaccine containing the mouse CD1d cassette has the sequence: It has. The mRNA vaccine containing the human CD1d cassette has the sequence: It has. The mRNA vaccine containing the human CD1b cassette has the sequence: It has.

[0310] Each CD1 scaffold contains a stuffer sequence (ctagc) immediately following the T7 promoter sequence.

[0311] The cells were incubated overnight at 37°C / 5% CO2. After incubation, the cells were aliquoted into 96-well plates (approximately 250 μL), blocked with Fc block (100 μL / sample), washed with FACS buffer (dPBS pH 7.5 containing 5% fetal bovine serum), and then stained with antigen-bound PE-Cy5-conjugated anti-mouse CD11c antibody and PE-conjugated anti-mouse H-2kb (SIINFEKL). Additionally, each sample was stained with Zombie near-infrared stain to distinguish live from dead cells.

[0312] These data comparing antigen presentation in the JAWS dendritic cell model for epitopes in different scaffold contexts revealed that the hCD1d scaffold performed best with 10.2% of antigen-presenting cells showing specific epitope presentation as measured by flow cytometry compared to untreated samples. See Figure 1.

[0313] Example 2. In vivo testing

[0314] In vivo study, the vaccine described herein is evaluated against commercially available materials.In this study, mRNA vaccine is evaluated for SIINFEKL presentation level on MHC-I compared with commercially available control.Reproducibility is also demonstrated (using multiple batches), and similar levels of SIINFEKL+JAWSII cells are obtained.

[0315] In this study, mRNA vaccines will be evaluated as vaccine candidates in in vivo experiments in mice. C57BL / 6 mice will be injected (IV) with either the commercial or mRNA vaccines described herein formulated in a delivery vehicle. Seven days after injection, peripheral blood will be isolated and stained with a fluorescent MHC-I tetramer specific for T cells that recognize the OVA epitope. The fraction of OVA-specific CD8+ T cells will then be quantified by flow cytometry. In this experiment, the mRNA vaccine is expected to result in an increased proportion of OVA-specific T cells in the peripheral blood compared to the commercial control, demonstrating the strength of these molecules as vaccines.

[0316] Example 3. Ex vivo stimulation in healthy donors: pp65

[0317] Example 3A

[0318] Cryopreserved human cytomegalovirus (CMV)-seropositive healthy donor peripheral blood mononuclear cells (PMNs) were thawed and resuspended in 14 mL of RPMI 1640. Cells were pelleted by centrifugation at 1200 rpm for 10 minutes. The supernatant was aspirated, and cells were resuspended in an appropriate volume of culture medium (1:1 AIM-V / RPMI 1640 + 10% filtered human AB serum + 50 μM B-mercaptoethanol (TC grade)) and counted. Cells were incubated overnight at 37°C in a CO2 incubator (5% CO2).

[0319] After incubation, cells were treated with 50 ng of mRNA encoding the native CMV pp65 protein, 50 ng of mRNA encoding pp65 with an MHC presentation-enhancing sequence, 2 μg / mL of a CMV pp65 peptide pool covering the entire pp65 molecule, or non-coding mRNA. Cells were incubated at 37°C in a CO2 incubator (5% CO2) for 24 hours.

[0320] After 24 hours, cells were harvested and washed twice with phosphate-buffered saline (PBS) pH 7.2, and then stained with Zombie Near Infrared live-dead stain (NIR) (BioLegend) in PBS for 15 minutes at room temperature (RT).

[0321] The cells were then washed and resuspended in 100 μl FACS buffer (PBS + 0.5% BSA + 0.02% sodium azide) containing fluorescent dye conjugates a-CD8, a-CD4, a-CD137, and a-CD69 (BioLegend). The cells were then incubated at room temperature for 20 minutes. After staining, the cells were washed twice with 200 μl PBS, followed by centrifugation at 1200 rpm for 10 minutes. After the final wash, the supernatant was discarded and the cells were resuspended in 200 μl PBS. The resuspended cells were then analyzed using a flow cytometer (Cytek).

[0322] Results: The percentage of activated cells is indicated by the black boxes in Figures 2A and 2B. Control groups included DMSO (negative control), CD3 (positive control), and CTR (cells treated with non-coding mRNA nanoparticles). Treatment groups included peptide (cells treated with a 2 μM CMV pp65 peptide pool covering the entire pp65 protein in overlapping sequences) and Pp65 Sec-hCD1d (cells treated with Sec-pp65-hCD1d mRNA nanoparticles).

[0323] Observation: Compared with control and peptide-treated cells, Sec-pp65-hCD1d mRNA nanoparticle-treated cells showed two-fold more activated cells. This indicates that treatment of cells with hCD1d-enhanced mRNA encoding the entire pp65 protein allows for efficient antigen processing and presentation. This enhancement leads to better and more extensive T cell activation.

[0324] Example 3B

[0325] Cryopreserved human cytomegalovirus (CMV)-seropositive healthy donor peripheral blood mononuclear cells (PMNs) were thawed and resuspended in 14 mL of RPMI 1640. Cells were pelleted by centrifugation at 1200 rpm for 10 minutes. The supernatant was aspirated, and cells were resuspended in an appropriate volume of culture medium (1:1 AIM-V / RPMI 1640 + 10% filtered human AB serum + 50 μM B-mercaptoethanol (TC grade)) and counted. Cells were incubated overnight at 37°C in a CO2 incubator (5% CO2).

[0326] After incubation, cells were treated with 50 ng of mRNA encoding the native CMV pp65 protein, 50 ng of mRNA encoding pp65 with an MHC presentation-enhancing sequence, 2 μg / mL of a CMV pp65 peptide pool covering the entire pp65 molecule, or non-coding mRNA. Cells were incubated at 37°C in a CO2 incubator (5% CO2) for 24 hours.

[0327] After 24 hours, the cells and cell culture supernatant were harvested and washed twice with phosphate-buffered saline (PBS) pH 7.2. The washed cells were then stained with Zombie Near Infrared live dead stain (NIR) (BioLegend) in PBS for 15 minutes at room temperature (RT). The cells were then washed and resuspended in 100 μl FACS buffer (PBS + 0.5% BSA + 0.02% sodium azide) containing fluorescent dye conjugates a-CD8, a-CD4, a-CD137, and a-CD69 (BioLegend). The cells were then incubated at room temperature for 20 minutes.

[0328] After staining, cells were washed twice with 200 μl of PBS, followed by centrifugation at 1200 rpm for 10 minutes. After the final wash, the supernatant was discarded and the cells were resuspended in 200 μl of PBS. The resuspended cells were then analyzed using a flow cytometer (Cytek). The supernatant was used to measure secreted interferon gamma (IFNg) using a standardized, commercially available human IFNg ELISA kit and protocol (Thermo Scientific).

[0329] Results: As shown in Figure 3A, improved IFNg T cell responses were observed using the Sec-hCD1d MHC-sorting sequence against peptides, native pp65 mRNA, and pp65 mRNA Sec-MITD. Compared to native pp65 mRNA and pp65 mRNA Sec-MITD, more activated CD8 T cells were observed in samples treated with Sec-hCD1d pp65 mRNA nanoparticles (Figure 3B). Introducing the MHC presentation-enhancing sequence improved antigen presentation and CD8 T cell activation in these PBMC samples.

[0330] Example 3C

[0331] Cryopreserved human cytomegalovirus (CMV)-seropositive healthy donor peripheral blood mononuclear cells (PMNs) were thawed and resuspended in 14 mL of RPMI 1640. Cells were pelleted by centrifugation at 1200 rpm for 10 minutes. The supernatant was aspirated, and cells were resuspended in an appropriate volume of culture medium (1:1 AIM-V / RPMI 1640 + 10% filtered human AB serum + 50 μM B-mercaptoethanol (TC grade)) and counted. Cells were incubated overnight at 37°C in a CO2 incubator (5% CO2).

[0332] After incubation, 8 × 10 6 Cells were treated with either 1 μg of mRNA encoding pp65 with the Sec-hCD1d MHC presentation-enhancing sequence, a 2 μM CMV pp65 peptide pool covering the entire pp65 molecule, or non-coding mRNA. Cells were incubated at 37°C in a CO incubator (5% CO) in culture medium without additional cytokines for 6 days to support T cell proliferation.

[0333] After 6 days, cells were harvested and CD8 T cells were isolated using a human CD8 isolation kit (STEMCELL). Viability was measured, cells were counted, and 50,000 CD8 T cells isolated from either peptide- or mRNA-treated samples were seeded in 8 replicates in 100 μl complete medium in 96-well U-bottom plates.

[0334] HLA-A2:01-expressing T2 cells (ATCC) were then labeled with a cell tracer violet dye according to the manufacturer's protocol (Thermo Scientific). After labeling, cells were washed twice with prewarmed medium and then assessed for viability and cell number.

[0335] Half of the T2 cells were pulsed, and the other half were unpulsed, and then incubated with CMV pp65 peptide for 1 hour at 37°C. After 1 hour, the cells were washed twice with prewarmed complete medium, and 10,000 pulsed or unpulsed T2 cells were added to wells containing isolated CD8 T cells. The isolated CD8 T cells and pulsed or unpulsed T2 cells were co-incubated for 4 hours at 37°C. After 4 hours, 5 μl of propidium iodide (PI) was added to each well, and CD8-mediated T2 killing was analyzed by flow cytometry (Cytek).

[0336] Results: After 6 days of passive expansion, robust CD8 T cell proliferation was observed in cultures treated with 1 μg of mRNA encoding pp65 using Sec-hCD1d. Both viability and cell numbers were superior compared to cells treated with peptide (Figure 4A). Activated and expanded CD8 T cells were able to recognize and kill T2 target cells only when they were pulsed with CMV-pp65 antigen. Significantly better killing efficacy was observed in CD8 T cells isolated from cultures treated with mRNA compared to peptide (Figures 4B-4D). This indicates that large numbers of functional (i.e., capable of killing target cells) CD8 T cells can be generated by treating the entire PBMC population with nanoparticles containing mRNA encoding the antigen and MHC trafficking signal Sec-hCD1d.

[0337] Example 3D

[0338] Cryopreserved human cytomegalovirus (CMV)-seropositive healthy donor peripheral blood mononuclear cells (PMNs) were thawed and resuspended in 14 mL of RPMI 1640. Cells were pelleted by centrifugation at 1200 rpm for 10 minutes. The supernatant was aspirated, and cells were resuspended in an appropriate volume of culture medium (1:1 AIM-V / RPMI 1640 + 10% filtered human AB serum + 50 μM B-mercaptoethanol (TC grade)) and counted. Cells were incubated overnight at 37°C in a CO2 incubator (5% CO2).

[0339] After incubation, 8 × 10 6 Cells were treated with either 1 μg of mRNA (in duplicate) encoding pp65 with the Sec-hCD1d MHC presentation-enhancing sequence, a 2 μM CMV pp65 peptide pool covering the entire pp65 molecule, or non-coding mRNA. Cells were incubated in culture medium at 37°C in a CO2 incubator (5% CO2) for 24 hours. After 24 hours, cells and cell culture supernatants were harvested, and the cells were washed twice with phosphate-buffered saline (PBS) pH 7.2. The washed cells were then stained with Zombie Near Infrared live dead stain (NIR) (BioLegend) in PBS for 15 minutes at room temperature (RT). Cells were then washed and resuspended in 100 μl FACS buffer (PBS + 0.5% BSA + 0.02% sodium azide) containing fluorescent dye conjugates a-CD8, a-CD4, a-CD137, and a-CD69 (BioLegend). The cells were then incubated at room temperature for 20 minutes.

[0340] After staining, the cells were washed twice with 200 μl of PBS, followed by centrifugation at 1200 rpm for 10 minutes. After the final wash, the supernatant was discarded, and the cells were resuspended in 200 μl of PBS. The resuspended cells were then single-cell sorted based on CD137 and CD69 expression on a flow sorter (Aria BD biosciences). CD137 and CD69 double-positive cells were sorted in Takara 10X buffer (Takara biosciences). The sorted cells were subjected to MedGenome (Medgenome) for T cell receptor sequencing.

[0341] As shown in Figure 5, higher clonal diversity was observed among CD8 T cells sorted from pp65 Sec-hCD1dmRNA nanoparticle-treated PBMCs compared with pp65 peptide treated.

[0342] Example 4. HPV16 E7 protein expression (Sec mRNA) in HEK293 402 vs. hCD1d mRNA 416)

[0343] Cultures of 50K HEK293 cells were treated overnight with 50 ng of mRNA. Supernatants and cell lysates (frozen and thawed 3 times) were collected 24 hours after transfection. HPV17 E7 protein was measured by ELISA using an HPV16 / 18 E7 ELISA kit (CellBioLabs) or by an in-house method by direct sample coating onto plates.

[0344] Results. As shown in Figures 6A and 6B, transfection of HEK293 with SEC-HPV E6-E7 (mRNA402) produced robust E7 protein compared to hCD1d mRNA-416.

[0345] Equivalence and Scope Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments according to the disclosure set forth herein. The scope of the present disclosure is not limited to the above description, but is instead set forth in the appended claims.

[0346] In the claims, articles such as "a," "an," and "the" can mean one or more than one, unless indicated to the contrary or clear from the context. A claim or description containing "or" between one or more members of a group is considered satisfied if one, more than one, or all group members are present in, used in, or otherwise relevant to a given product or process, unless indicated to the contrary or clear from the context. The present disclosure includes embodiments in which exactly one member of a group is present in, used in, or otherwise relevant to a given product or process. The present disclosure includes embodiments in which more than one, or all group members are present in, used in, or otherwise relevant to a given product or process.

[0347] It should also be noted that the term "comprising" is intended to be open, allowing for, but not requiring, the inclusion of additional elements or steps. Thus, when the term "comprising" is used herein, the term "consisting of" is also encompassed and disclosed.

[0348] Where ranges are given, the endpoints are included. Furthermore, unless otherwise indicated or otherwise apparent from the context and the understanding of one of ordinary skill in the art, it should be understood that values ​​expressed as ranges can assume any specific value or subrange within the ranges set forth in different aspects of this disclosure, down to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0349] Furthermore, it should be understood that any particular aspect of the present disclosure that falls within the prior art may be expressly excluded from any one or more claims. Such aspects may be excluded even if the exclusion is not explicitly set forth herein, because they would be deemed known to those of ordinary skill in the art. Any particular aspect of the compositions of the present disclosure (e.g., any antibiotic, therapeutic or active ingredient; any method of manufacture; any method of use, etc.) may be excluded from any one or more claims for any reason, whether related to the existence of prior art or not.

[0350] It is to be understood that the words used are words of description rather than of limitation, and changes may be made within the purview of the appended claims without departing from the true scope and spirit of the present disclosure in its broader aspects.

[0351] While the present disclosure has been described in some length and with some particularity with respect to some described embodiments, it should not be limited to such details or embodiments or to any particular embodiment, but rather should be construed with reference to the appended claims so as to provide the broadest possible interpretation of such claims in view of the prior art and thus effectively encompass the intended scope of the present disclosure.

[0352] Furthermore, as used herein, "and / or" should be interpreted as a specific disclosure of each of the two specified features or components, regardless of the presence or absence of the other. Thus, the term "and / or" used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0353] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. See, for example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide those of skill with a general dictionary of many of the terms used in this disclosure.

[0354] Whenever an embodiment is described herein with the term "comprising," other similar embodiments are also provided that are described with the terms "consisting of" and / or "consisting essentially of."

[0355] Units, prefixes, and symbols are shown in the format recognized by the International System of Units (SI). Numerical ranges are inclusive of the numbers defining the range. When a range of values ​​is described, it is understood that each intervening integer and each fractional integer between the stated upper and lower limits of that range is also specifically disclosed, along with each subrange between such values. The upper and lower limits of any range can be independently included in or excluded from the range, and ranges in which either, neither, or both limits are included are also encompassed within the disclosure. When values ​​are explicitly recited, it is understood that amounts or amounts approximately equal to the recited value are also within the scope of the disclosure. Where combinations are disclosed, each subcombination of elements of that combination is also specifically disclosed and is within the scope of the disclosure. Conversely, where different elements or groups of elements are individually disclosed, combinations thereof are also disclosed. Where any element of the disclosure is disclosed as having multiple alternatives, examples of that disclosure in which each alternative is excluded alone or in any combination with the other alternatives are hereby disclosed. More than one element of the disclosure may have such an exclusion, and all combinations of elements having such an exclusion are disclosed herein. In certain embodiments, for example, the following are provided: (Item 1) formula: Signal / Leader-Payload-TMD-CYD (In the formula, the signal / leader encodes a signal sequence, leader sequence, or sorting sequence in frame with and upstream of the payload; the payload is selected from the group consisting of an antigenic payload region, a detectable agent, and a therapeutic agent; the TMD encodes a portion of a transmembrane region from one or more proteins or isoforms selected from the group consisting of CD1d, CD1e, LDLR, LDLRP, and LRP1 proteins; the CYD encodes all or part of a cytoplasmic region from one or more proteins or isoforms selected from the group consisting of CD1d, CD1e, LDLR, LDLRP, and LRP1 proteins. A polynucleotide having the formula: (Item 2) The payload is (a) a first encoded antigenic payload (An1), where n is an integer between 1 and 10; (b) an encoded linker region (X), where o is an integer between 0 and 10; and (c) a second encoded antigenic payload (An2), where p is an integer between 0 and 10. 2. The polynucleotide of item 1, wherein the antigenic payload region has the formula (An1)n-Xo-(An2)p, comprising: (Item 3) 3. The polynucleotide of item 2, wherein the first encoded antigenic payload or the second encoded antigenic payload encodes all or part of a tumor antigen or an infectious pathogen antigen. (Item 4) 4. The polynucleotide of claim 2 or 3, wherein the first encoded antigenic payload or the second encoded antigenic payload comprises the sequence SIINFEKL. (Item 5) 2. The polynucleotide of item 1, wherein the payload is a detectable agent selected from the group consisting of small organic molecules, inorganic compounds, nanoparticles, enzymes or enzyme substrates, fluorescent materials, luminescent materials, bioluminescent materials, chemiluminescent materials, radioactive materials, contrast agents, gadolinium, iron oxide, single-crystalline iron oxide nanoparticles, ultrasmall superparamagnetic iron oxide, manganese chelates, barium sulfate, iodinated contrast agents, microbubbles, and perfluorocarbons. (Item 6) 10. The polynucleotide of any one of the preceding items, wherein the TMD and the CYD are from the same isoform or protein. (Item 7) 6. The polynucleotide of any one of items 1 to 5, wherein the TMD and the CYD are derived from different isoforms or proteins. (Item 8) The polynucleotide of any one of the preceding items, wherein the signal / leader encodes a signal sequence, leader sequence, or sorting sequence from the same isoform or protein as the TMD, the CYD, or both. (Item 9) Item 10. The polynucleotide of any one of the preceding items, wherein the TMD encodes the sequence MGLIALAVLACLLFLLIVGFT. (Item 10) Item 10. The polynucleotide of any one of the preceding items, wherein the CYD encodes the sequence SRFKRQTSYQGVL. (Item 11) Item 10. The polynucleotide of any one of the preceding items, wherein the signal sequence encodes the sequence MGCLLFLLLWALLQAWGSA. (Item 12) formula: Signal / Leader-Payload-PRM (In the formula, The signal / leader is in-frame with and upstream of the payload. , a signal sequence, a leader sequence, or a sorting sequence; the payload is selected from the group consisting of an antigenic payload region, a detectable agent, and a therapeutic agent; The PRM encodes all or part of at least one parent receptor molecule region from one or more proteins or isoforms selected from the group consisting of CD1d, CD1e, LDLR, LDLRP, and LRP1 proteins. A polynucleotide having the formula: (Item 13) 13. The polynucleotide of item 12, wherein the parent receptor molecule is selected from the group consisting of an extracellular region, a transmembrane region, and a cytoplasmic region. (Item 14) A host cell comprising a polynucleotide according to any one of the preceding items. (Item 15) A pharmaceutical composition comprising the polynucleotide according to any one of items 1 to 13 or the host cell according to item 14. (Item 16) 16. The pharmaceutical composition according to item 15, wherein the pharmaceutical composition is in the form of a vaccine. (Item 17) 17. The pharmaceutical composition according to item 15 or item 16, further comprising one or more pharmaceutically acceptable excipients or one or more additional active pharmaceutical ingredients. (Item 18) 18. The pharmaceutical composition according to item 17, wherein the pharmaceutically acceptable excipient is selected from the group consisting of anti-adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes, emollients, emulsifiers, fillers, film-formers or coatings, flavors, fragrances, glidants, lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and water of hydration. (Item 19) 14. A therapeutic polynucleotide comprising the polynucleotide of any one of items 1 to 13 formulated with a delivery vehicle. (Item 20) 20. The therapeutic polynucleotide of claim 19, wherein the polynucleotide is encapsulated in the delivery vehicle. (Item 21) 21. The therapeutic polynucleotide of item 19 or 20, wherein the delivery vehicle is selected from the group consisting of amphipathic molecules, amino lipidated peptides, and tertiary amino lipidated cationic peptides. (Item 22) A therapeutic composition comprising the therapeutic polynucleotide according to any one of items 19 to 21. (Item 23) 23. The therapeutic composition according to claim 22, wherein the therapeutic composition is in the form of a vaccine. (Item 24) 24. The therapeutic composition of claim 22 or 23, further comprising one or more therapeutically acceptable excipients or one or more additional therapeutically active ingredients. (Item 25) 25. The therapeutic composition of claim 24, wherein the therapeutically acceptable excipient is selected from the group consisting of anti-adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes, emollients, emulsifiers, fillers, film formers or coatings, flavors, fragrances, glidants, lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and water of hydration. (Item 26) A therapeutically effective dose, a prophylactically effective dose, or an appropriate image of the pharmaceutical or therapeutic composition is administered. The pharmaceutical composition according to any one of items 15 to 18 or the therapeutic composition according to any one of items 22 to 25, wherein a dosing dose is administered to a subject in need thereof. (Item 27) 26. A method for treating, vaccinating or immunizing a subject in need thereof, the method comprising administering to the subject the polynucleotide according to any one of items 1 to 13, the host cell according to item 14, the pharmaceutical composition according to any one of items 15 to 18 or the therapeutic composition according to any one of items 22 to 25. (Item 28) 28. The pharmaceutical or therapeutic composition of claim 26, or the method of claim 27, wherein the subject is a mammal. (Item 29) 28. The pharmaceutical or therapeutic composition of item 26, or the method of item 27, wherein the subject is a human. (Item 30) the polynucleotide is a) allows antigen processing and presentation; b) transporting proteins into the antigen presentation pathway; c) improve T cell activation; d) increase clonal diversity, and e) any combination thereof the polynucleotide according to any one of Items 1 to 13, the host cell according to Item 14, the pharmaceutical composition according to any one of Items 15 to 18, the therapeutic polynucleotide according to any one of Items 19 to 21, the therapeutic composition according to any one of Items 22 to 25, or the method according to any one of Items 27 to 29,

Claims

[Claim 1] The invention described in the specification.

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