Enhanced delivery of disease-targeted nanobody-sirna conjugates

Abstract

The present disclosure is directed to nanobody-siRNA conjugates and methods of producing and using same. The nanobody-siRNA conjugates include a nanobody that may direct the conjugate to a target cell. The nanobody-siRNA conjugates are delivered into the target cell via receptor-mediated transcytosis, where the siRNA is delivered to the cell cytoplasm after it exits the endosome. To treat diseases and conditions, such as cancers and autoimmune diseases, the siRNA can be efficiently passed through the BBB to modulate an immune response. The small size of nanobodies allows more efficient targeted delivery than typical antibody systems, while conjugation strategies involving click chemistry permit the multiplexed loading of siRNA to nanobodies for increased therapeutic effects.

Description

ENHANCED DELIVERY OF DISEASE-TARGETED NANOBODY-SIRNA CONJUGATESCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United States Provisional Patent Application Number 63 / 712,425 filed on October 26, 2024, the entire contents of which are hereby incorporated by reference.SEQUENCE LISTING

[0002] An electronic sequence listing (069596-00102.xml; size 32.0 KB; date of creation October 23, 2025) submitted herewith is incorporated by reference in its entirety.FIELD

[0003] The present invention relates to systems and methods of preparing and utilizing nanobody-siRNA conjugates for targeted therapeutic treatments.BACKGROUND

[0004] Over recent years, short-interfering RNA (siRNA) research has seen growth, especially resulting from the emergence of RNA-based therapeutic applications for the COVID-19 virus. In contrast to mRNA being used to enhance the expression of a gene, siRNA can regulate mRNA expression through gene knockdown. However, the delivery of naked siRNA has challenges as it is easily degradable in the bloodstream and has low cellular uptake. Further, while several siRNA drugs have been approved by the FDA, these drugs suffer from constraints, such as limited targets, large dosing, and frequent administration. Thus, challenges remain for siRNA therapies in addressing the narrow scope of present targets, siRNA instability in the body, off-target effects, and ineffective targeted delivery.SUMMARY

[0005] In an aspect of the present disclosure, there is provided a composition having a nanobody-siRNA conjugate. The conjugate includes a nanobody configured to bind a target cellreceptor and an siRNA covalently linked to the nanobody. The nanobody-siRNA conjugate is configured to enter, via receptor-mediated transcytosis, a cell displaying the target cell receptor.

[0006] In some embodiments, the siRNA is configured to knockdown target gene expression. In some instances, the target gene is selected from the group consisting of B cell lymphoma 2 (BCL-2), MYCN Proto-Oncogene (MYCN), and polo-like kinase 1 (PLK-1). In some embodiments, the target cell receptor is selected from the group consisting of EGFR, Mucl, B7H3, albumin, transferrin, CTLA-4, and PD-L1. In some embodiments, the nanobody-siRNA conjugate is configured to penetrate the blood-brain barrier.

[0007] In some embodiments, the siRNA is linked to the nanobody via strain-promoted azide-alkyne click chemistry (SPAAC), inverse electron-demand Diels- Alder cycloaddition (IEDDA), or copper-catalyzed azide-alkyne cycloaddition (CuAAC). In some instances, the siRNA is linked to the nanobody via SPAAC and a bicyclo[6.1.0]non-4-yne (BCN) group of the siRNA reacts with an azide group of the nanobody. In some instances, a BCN, azide, or tetrazine group is present on the nanobody via incorporation of one or more non-canonical amino acid (ncAA). In some cases, a reactive group present on the siRNA is conjugated to the BCN, azide, or tetrazine group of the nanobody. In some instances, an azide, BCN, DBCO, TCO, or tetrazine group is present on a C terminus of the nanobody via an engineered sortase A bioconjugation. In some cases, a reactive group present on the siRNA is conjugated to the azide, BCN, DBCO, TCO, or tetrazine group of the nanobody.

[0008] In some embodiments, the conjugate further includes a linker between the siRNA and the nanobody. In some instances, the linker includes up to 10 spacing nucleotides on the siRNA, up to 20 residues of repeating units between the nanobody and a conjugating group of the nanobody, up to 12 ethylene glycol units of a polyethylene glycol (PEG) linker, or a combination thereof. In some cases, the up to 20 residues of repeating units comprise one or more EAAAK or GGGGS repeating unit.

[0009] In another aspect of the present disclosure, there is provided a method of delivering siRNA payload to a target cell. The method includes contacting a nanobody-siRNA conjugate with a receptor of the target cell, where a nanobody of the nanobody-siRNA conjugate binds the receptor and the nanobody-siRNA conjugate enters the target cell via receptor-mediated transcytosis. The siRNA is delivered to cytoplasm of the target cell.

[0010] In some embodiments, the nanobody-siRNA conjugate penetrates the blood-brain barrier. In some embodiments, the target cell is in a brain of a subject. In some embodiments, the target cell is associated with a cancer or autoimmune disease. In some embodiments, the siRNA knockdowns expression of a target gene of the target cell.

[0011] In yet another aspect of the present disclosure, there is provided a method of treating or preventing a condition in a subject in need thereof. The method includes administering to the subject in need thereof a therapeutically effective amount of a composition containing the nanobody-siRNA conjugates of the present disclosure. In some embodiments, the condition affects central nervous system (CNS). In some instances, the condition is cancer or an autoimmune disease. In some instances, the condition is multiple sclerosis.

[0012] In yet another aspect of the present disclosure, there is provided a method of producing a nanobody- siRNA conjugate. The method includes providing a nanobody with at least one first click chemistry reactive group and contacting the nanobody with at least one siRNA having at least one second click chemistry reactive group. The nanobody is conjugated to the at least one siRNA via click chemistry at the first and second click chemistry reactive groups.

[0013] In some embodiments, the method further includes generating the at least one first click chemistry reactive group on the nanobody at its C terminus via engineered sortase A bioconjugation. In some embodiments, the method further includes generating the at least one first click chemistry reactive group on the nanobody on a surface-facing location of the nanobody by integrating a ncAA having the at least one first click chemistry reactive group at the surface-facing location. In some embodiments, the at least one first click chemistry reactive group is an azide, BCN, DBCO, TCO, or tetrazine. In some embodiments, the method further includes generating at least one cell penetrating peptide (CPP) on the nanobody before the contacting with the siRNA.BRIEF DESCRIPTION OF THE FIGURES

[0014] The present disclosure same can be better understood, by way of example only, with reference to the following drawings. The elements of the drawings are not necessarily to scale relative to each other, emphasis instead being placed upon clearly illustrating the principles of the disclosure.

[0015] FIG. 1 is a schematic representation of strategies for enhancing the stability of siRNA. Such strategies include chemical approaches (top panel) and carriers (lower panel).

[0016] FIG. 2 is a schematic representation of exemplary carriers for the delivery of RNA. Such delivery systems include nanoparticles (NPs), GalNAc, antibodies (mAbs), nanobodies, and peptides, with their respective sizes displayed.

[0017] FIG. 3 is a schematic and graphical representation comparing nanobodies with antibodies. In the left panel, a schematic representation shows the size difference between antibodies and nanobodies. In the right panel, the benefits of nanobodies are described.

[0018] FIG. 4 is a schematic representation comparing antibody-drug conjugates with nanobody-drug conjugates, where the nanobody is a single domain antibody fragment of approximately 15 kDa and can selectively bind certain antigens. The smaller size of nanobodies is desired for effective cellular uptake. Exemplary nanobodies include, but are not limited to, transferrin, ApoE40, ApoE05, and PD-L1.

[0019] FIG. 5 is a schematic representation of nanobody -siRNA conjugates of the present disclosure. The exemplary nanobody includes a bicyclo[6.1.0]non-4-yne (BCN) group, while the exemplary siRNA is modified with an azide group. The BCN group reacts with the azide, conjugating the nanobody with the siRNA to form the nanobody-siRNA conjugate.

[0020] FIG. 6 is a schematic representation of exemplary designs and applications of the nanobody-siRNA conjugates of the present disclosure. Rational protein design and engineering principles lead to the generation of a library of nanobody-siRNA conjugates (top panel) to enhance immune responses in cancer (middle panel) and autoimmune diseases (lower panel).

[0021] FIG. 7 is a schematic representation of genes of interest and changes in expression compatible with the nanobody-siRNA conjugates of the present disclosure.

[0022] FIG. 8 is a schematic representation of applications and evaluation methods for the disclosed nanobody-siRNA platform.

[0023] FIG. 9 is a schematic representation of nanobody-siRNA conjugate design for the exemplary nanobody-siRNA conjugates of the present disclosure.

[0024] FIG. 10 is a schematic representation of receptor-mediated transcytosis, by which the nanobody-siRNA conjugates of the present disclosure are delivered into cells.

[0025] FIG. 11 is a graphical representation of nPDLl-BCN as evaluated via mass spectroscopy. The sample was approximately 3 pM and was buffer exchanged into deionizedwater for about 2 hours. The concentration was determined via spectrophotometry. Between about 100-200 pL of protein and an equal amount of methanol were used, with 0.1% of trifluoroacetic acid (TFA) added to the sample, which was evaluated via mass spectroscopy.

[0026] FIG. 12 is a graphical representation of nPDLl-BCN as evaluated via mass spectroscopy, as described for FIG. 11, but with greater detail visible at the displayed range.

[0027] FIG. 13 is a schematic representation of nanobody synthesis. A plasmid encoding the gene of interest for the nanobody (1) is transformed into T7 Shuffle E.Coli. After (2) selection and (3) protein production, (4) cells are lysed to the protein may be harvested and (5) purified using (6) affinity and size exclusion chromatography. (7) SDS PAGE was used to verify the purified product.

[0028] FIG. 14 is a schematic representation of an exemplary nanobody bioconjugation using a BCN group, where exemplary PD-L1 and albumin nanobodies were expressed and conjugated to BCN via Sortase A ligation.

[0029] FIG. 15 is a gel electrophoresis image of nanobody-BCN bioconjugation products evaluated using SDS-PAGE. PD-L1 and albumin nanobodies were expressed and conjugated to BCN via Sortase A ligation, as displayed in FIG. 14.

[0030] FIG. 16 is a schematic representation and gel electrophoresis image of nanobodysiRNA conjugation. Nanobodies and siRNA were conjugated via strain-promoted azide-alkyne click chemistry (SPAAC) reaction, with products evaluated using ESI-MS and SDS-PAGE.

[0031] FIG. 17 is a schematic and graphical representation of knockdown of luciferase in vitro. Nanobody-siRNA and free siRNA were used to transfect a luciferase expressing cell line, and luciferase gene knockdown was measured with nanobody concentration. Error bars represent SEM, withN = 3.

[0032] FIG. 18A is an SDS PAGE image representation of CPP-nPD-Ll-mCherry expression. Expression is in BL21 DE3 RIL. Lane identities: LI, Arg9-nPD-Ll -mCherry clarified lysate; El, Arg9-nPD-Ll -mCherry elution; L2, TAT-nPD-Ll-mCherry clarified lysate; E2, TAT-nPD-Ll-mCherry elution; L3, GALA-nPD-L 1 -mCherry clarified lysate; E3, GALA-nPD-Ll-mCherry elution.

[0033] FIG. 18B is an SDS PAGE image representation of CPP-nPD-L 1 -mCherry expression. The gel displays purification of inclusion bodies. Lane identities: 1, TAT-nPD-Ll-mCherry; 2, Arg9-nPD-Ll -mCherry; 3, GALA-nPD-L 1 -mCherry.

[0034] FIG. 18C is an SDS PAGE image representation of CPP-nPD-Ll -mCherry expression. The gel displays clarified lysate of expression in BL21 DE3 pLysS. Lane identities: 1, TAT-nPD-Ll -mCherry; 2, Arg9-nPD-Ll -mCherry; 3, GALA-nPD-Ll -mCherry.

[0035] FIG. 18D is an SDS PAGE image representation of CPP-nPD-Ll -mCherry expression. The gel displays constructs after bdSUMO tag removal with 1 pM of bdSENPl. Lane identities: 1, TAT-nPD-Ll-mCherry; 2, Arg9-nPD-Ll -mCherry; 3, GALA-nPD-Ll-mCherry.

[0036] FIG. 19 is a schematic representation of an alternative expression strategy of CPPs using solid phase peptide synthesis.

[0037] FIG. 20 is an SDS PAGE image representation of sortase conjugation of CPPs to nPD-Ll -mCherry.

[0038] FIG. 21 A is an SDS PAGE image representation of CPPs for conjugation to nanobodies, as visualized by mCherry.

[0039] FIG. 21A is an SDS PAGE image representation of CPPs for conjugation to nanobodies, as visualized by mCherry.

[0040] FIG. 22A are image representations of cells exposed to mCherry-JTSl at 37°C.Brightfield images are shown in the left panel, while fluorescence imaging is shown in the right panel. Nuclei have a DAPI stain, lysosomes have a LysoTracker dye, and CPPs are visualized with mCherry. Scale bars represent 50 pm.

[0041] FIG. 22B are image representations of cells exposed to mCherry-E5 at 37°C.Brightfield images are shown in the left panel, while fluorescence imaging is shown in the right panel. Nuclei have a DAPI stain, lysosomes have a LysoTracker dye, and CPPs are visualized with mCherry. Scale bars represent 50 pm.

[0042] FIG. 23 A are image representations of cells exposed to mCherry-IFN7 at 37°C.Brightfield images are shown in the left panel, while fluorescence imaging is shown in the right panel. Nuclei have a DAPI stain, lysosomes have a LysoTracker dye, and CPPs are visualized with mCherry. Scale bars represent 50 pm.

[0043] FIG. 23B are image representations of cells exposed to mCherry-PHl at 37°C.Brightfield images are shown in the left panel, while fluorescence imaging is shown in the right panel. Nuclei have a DAPI stain, lysosomes have a LysoTracker dye, and CPPs are visualized with mCherry. Scale bars represent 50 pm.

[0044] FIG. 24 A is a schematic representation of nanobody-siRNA conjugate generation via SPAAC. The nanobody is ligated to include an azide handle for click chemistry, while the siRNA includes DBCO for SPAAC click chemistry.

[0045] FIG. 24B is an SDS PAGE image representation of the conjugation of an azide click chemistry handle to nPD-Ll via sortase ligation. Lane identities: L, ladder; 1, nPD-Ll; 2, eSortA; 3-5, elution of nPD-Ll -Azide.

[0046] FIG. 24C is an SDS PAGE image representation of nanobody-siRNA conjugates formed via SPAAC. An nPD-Ll nanobody with an azide click chemistry handle is conjugated with siLUC. Lane identities: L, ladder; 1-2, FPLC fractions of reaction; 3, nPD-Ll-Azide.

[0047] FIG. 25 is a graphical representation of gene knockdown of luciferase after 24 and 48 hours of transfection with 1:3 dilution of nPD-Ll-siLUC (circle markers) compared to a positive control transfected with lipofectamine (square markers). Error bars represent SEM, with N = 3.

[0048] FIG. 26 is a schematic representation of enhanced design of nanobody-siRNA conjugates by nonconical amino acids (ncAAs).

[0049] FIG. 27 is a schematic representation of mechanism of prodrug nanobody-siRNA conjugates to tumors. Activation of prodrugs via endogenous signals such as pH, ROS, or enzyme or exogenous signals such as light activation.

[0050] FIG. 28 is an image and graphical representation of biodistribution of anti-Albumin delivery. In the left panel, IVIS images of brain uptake with (right panel) quantified regions of interest at 24 hours post IV administration.

[0051] FIG. 29 is a schematic representation of immune tolerance evaluation using the present conjugates. (Top panel) Potential immunization strategy for long-lasting immunity via dosing of antigen and antagonist for APC display and T cell activation to be assessed in (lower panel) EAE proposed experimental timeline.DETAILED DESCRIPTION

[0052] Embodiments described herein can be understood more readily by reference to the following detailed description and examples. Elements and methods described herein, however, are not limited to the specific embodiments presented in the detailed description and examples. It should be recognized that these embodiments are merely illustrative of the principles of thepresent disclosure. Numerous modifications and adaptations will be readily apparent to those of skill in the art without departing from the spirit and scope of the disclosure.

[0053] In addition, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a stated range of “1.0 to 10.0” should be considered to include any and all subranges beginning with a minimum value of 1.0 or more and ending with a maximum value of 10.0 or less, e.g., 1.0 to 5.3, or 4.7 to 10.0, or 3.6 to 7.9.

[0054] All ranges disclosed herein are also to be considered to include the end points of the range, unless expressly stated otherwise. For example, a range of “between 5 and 10,” “from 5 to 10,” or “5-10” should generally be considered to include the end points 5 and 10.

[0055] Further, when the phrase “up to” is used in connection with an amount or quantity, it is to be understood that the amount is at least a detectable amount or quantity. For example, a material present in an amount “up to” a specified amount can be present from a detectable amount and up to and including the specified amount.

[0056] Additionally, in any disclosed embodiment, the terms “substantially,” “approximately,” and “about” may be substituted with “within [a percentage] of’ what is specified, where the percentage includes 0.1, 1, 5, and 10 percent.

[0057] Disjunctive language such as the phrase “at least one of X, Y, or Z,” unless specifically stated otherwise, is otherwise understood with the context as used in general to present that an item, term, etc., may be either X, Y, or Z, or any combination thereof (e.g., X, Y, and / or Z). Thus, such disjunctive language is not generally limited to, and should not, imply that certain embodiments require at least one of X, at least one of Y, or at least one of Z to each be present.

[0058] The terms “prevention”, “prevent”, “preventing”, “suppression”, “suppress” and “suppressing” as used herein refer to a course of action (such as delivery or administration of a drug, prodrug, or compound) initiated prior to the onset of a clinical manifestation of a disease state or condition so as to prevent or reduce such clinical manifestation of the disease state or condition. Such preventing and suppressing need not be absolute to be useful.

[0059] The terms “treatment”, “treat” and “treating” as used herein refers a course of action (such as delivery or administration of a drug, prodrug, or compound) initiated after the onset of a clinical manifestation of a disease state or condition so as to eliminate or reduce such clinical manifestation of the disease state or condition. Such treating need not be absolute to be useful.

[0060] In this disclosure terms such as “administering” or “administration” include acts such as prescribing, dispensing, giving, or taking a substance such that what is prescribed, dispensed, given, or taken is actually contacts the patient’s body externally or internally (or both). It is specifically contemplated that instructions or a prescription by a medical professional to a subject or patient to take or otherwise self-administer a substance is an act of administration.

[0061] The term “in need of treatment” as used herein refers to a judgment made by a caregiver that a patient requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of a caregiver's expertise, but that includes the knowledge that the patient is ill, or will be ill, as the result of a condition that is treatable by a method or compound of the present disclosure.

[0062] The term “in need of prevention” as used herein refers to a judgment made by a caregiver that a patient requires or will benefit from prevention. This judgment is made based on a variety of factors that are in the realm of a caregiver's expertise, but that includes the knowledge that the patient will be ill or may become ill, as the result of a condition that is preventable by a method or compound of the disclosure.

[0063] The term “individual”, “subject” or “patient” as used herein refers to any animal, including mammals, such as mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and humans. The term may specify male or female or both, or exclude male or female.

[0064] The term an “effective amount,” “sufficient amount” or “therapeutically effective amount” as used herein is an amount of a compound of the disclosure that is sufficient to achieve a beneficial or desired result, including clinical results. As such, the effective amount may be sufficient, for example, to treat cancer or autoimmune disease, or one or more symptoms thereof, treat a disease or condition related to a CNS target, or enhance or otherwise improve the prophylactic or therapeutic effect(s) of another therapy, such as treatment with an additional active agent. In certain embodiments, an effective amount is an amount of the compound of the disclosure that avoids or substantially attenuates undesirable side effects. In certain embodiments, an effective amount is an amount of the compound of the disclosure that stimulates immune checkpoint blockades for enhancing an antitumor immune response. In certain embodiments, an effective amount is an amount of the compound of the disclosure thatreprograms tolerance and / or increases the number of Tregs through APC activation to modulate an immune response.

[0065] In certain embodiments, the “effective amount,” “sufficient amount” or “therapeutically effective amount” in the context of the present disclosure increases an immune response in a subject suffering from cancer or an autoimmune disease or disorder by at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 99%. In some embodiments, the “effective amount,” “sufficient amount” or “therapeutically effective amount” in the context of the present disclosure increases the survival rate of a subject suffering from cancer or an autoimmune disease or disorder by at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%. In each of the foregoing, when a reduction or increase is specified, such reduction or increase may be determined with respect to a subject that has not been treated with a compound of the disclosure and that is diagnosed as suffering from cancer or an autoimmune disease or disorder.

[0066] The term “tissue” as used herein refers to an organ, part of an organ, cellular structure(s), and / or group of cells in the body of a subject. Including, but not limited to, lung, an embryo, a fetus, placenta, liver, kidney, spleen, brain, testis, or uterus.

[0067] The term “protein,” “peptide,” “polypeptides” and “oligopeptides” refers to chains of amino acids (typically L-amino acids) whose alpha carbons are linked through peptide bonds formed by a condensation reaction between the carboxyl group of the alpha carbon of one amino acid and the amino group of the alpha carbon of another amino acid. Typically, the amino acids making up a protein are numbered in order, starting at the amino terminal residue and increasing in the direction toward the carboxy terminal residue of the protein.

[0068] The term “nucleotide” as used herein refers to any such known groups, natural or synthetic. It includes conventional DNA or RNA bases (A, G, C, T, U), base analogs (e.g., inosine, 5-nitroindazole and others), imidazole-4-carboxamide, pyrimidine or purine derivatives (e.g., modified pyrimidine base 6H,8H-3,4-dihydropyrimido[4,5-c][l,2]oxazin-7-one (sometimes designated "P" base that binds A or G)) and modified purine base N6-methoxy-2,6-diaminopurine (sometimes designated "K" base that binds C or T), hypoxanthine, N-4-methyl deoxyguanosine, 4-ethyl-2'-deoxycytidine, 4,6-difluorobenzimidazole and 2,4-difluorobenzene nucleoside analogues, pyrene-functionalized LNA nucleoside analogues, deaza- or aza-modified purines and pyrimidines, pyrimidines with substituents at the 5 or 6 position and purines with substituents at the 2, 6 or 8 positions, 2-aminoadenine (nA), 2-thiouracil (sU), 2-amino-6-methylaminopurine, O-6-methylguanine, 4-thio-pyrimidines, 4-amino-pyrimidines, 4-dimethylhydrazine-pyrimidines, O-4-alkyl-pyrimidines and hydrophobic nucleobases that form duplex DNA without hydrogen bonding. Nucleobases can be joined together by a variety of linkages or conformations, including phosphodiester, phosphorothioate or methylphosphonate linkages, peptide-nucleic acid linkages.

[0069] The term “polynucleotide” as used herein refers to a multimeric compound comprising nucleotides linked together to form a polymer, including conventional RNA, DNA, LNA, BNA, copolymers of any of the foregoing, and analogs thereof.

[0070] The term “nucleic acid” as used herein refers to a single stranded polynucleotide or a duplex of two polynucleotides. Such duplexes need not be annealed at all locations and may contain gaps or overhangs.

[0071] The nanobody-siRNA conjugates of the present disclosure represent a new generation of immunotherapy to address challenges in therapeutic delivery and immune modulation within current RNA delivery technology. While siRNA delivery and vaccine design have been previously attempted, the limitations in delivery efficiency, payload versatility, and functional immune outcomes have prevented these constructs from reaching their full therapeutic potential. In contrast, the present approach introduces several innovations to address these challenges: (1) Generation of homogenous conjugates by leveraging selective conjugation (i.e. sortase ligation) for a site-specific addition of cell-penetrating peptides (CPPs) and RNA, resulting in reproducible uniform conjugates. (2) Improved selectivity via the incorporation of ncAAs in nanobodies to direct conjugation with RNA at select locations. To our knowledge, there are no reports on the use of ncAAs as click chemistry handles for multiplexed RNA loading. The disclosed design may allow for multi-drug payloads and co-delivery dynamics, offering a powerful advantage for tuning payload composition and cellular responses. (3) Incorporating CPPs to enhance RNA entry into the cytoplasm. Through the use of nanobodies for receptor specific targeting, tissue penetration and biodistribution may be improved. (4) Providing insightinto the therapeutic efficacy and immunological safety of delivering nucleic acid payloads in cancer and autoimmune disease applications to fulfill a lack of comprehensive knowledge in the field. Ultimately the disclosed platform for siRNA delivery and vaccines integrates precision engineering, modular design, and immune-specific functionality to generate a new era of targeted, programmable nucleic acid therapeutics for broad applications across immune-mediated diseases.RNA-Based Therapeutics

[0072] RNA-based therapeutics has seen much interest, particularly based on the success in developing messenger RNA (mRNA) vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Such therapies allow treatment at the genomic level for diseases and include the use of mRNA, short-interfering RNA (siRNA), microRNA (miRNA), antisense RNA (ASO), aptamers, and CRISPR Cas9 systems. Thus, the success of the SARS-CoV-2 mRNA vaccine has provided an opportunity for other types of RNA, such as siRNA, to emerge as a therapeutic tool.

[0073] mRNA-based therapies are designed to enhance a gene of interest, transcribing the single-stranded RNA into proteins. It can be used to act as a replacement for endogenous proteins or act as a vaccine to train an immune response for infectious diseases and cancer antigens. The mRNA construct can be modified to improve translation and stability through the inclusion of (1) 5’ cap, (2) 5’ untranslated region (UTR), (3) 3’ UTR, and (4) polyadenylated (poly[A]) tail. The open reading frame (ORF) encodes for the gene of interest between the two UTRs. Each of these components has been optimized to efficiently deliver mRNA, however overlying issues persist in delivering mRNA to the desired cells without off-target effects, immunogenicity, or toxicity concerns.

[0074] Through the years, 4 types of chemical modifications of the RNA have been developed to increase stability against early degradation and innate immune responses: (1) backbone, (2) base, (3) sugar, and (4) terminal modifications. Backbone modifications are typically applied to the passenger strand by changes in phosphate ester linkages within the nucleic acid to improve clinical outcomes. Sugar based modifications, commonly 2’ -modifications to the sugar ring (e g., 2’-0Me, 2’-fluoro, and 2’-O-methoxyethyl (2’-0ME)), have been shown to improve nuclease resistance, increase potency, and extend half-lives of siRNA therapeutics.

[0075] RNA therapies can theoretically regulate any gene, specifically those deemed “undruggable” using small molecules, increasing the range of therapeutic targets. In the case of rare diseases, the development of RNA therapies for a small market is more cost efficient for pharmaceutical companies once the RNA and the carrier are optimized, the therapy can be quickly generated for clinical trials.

[0076] However, delivery of RNA faces the challenges of nucleic acid stability, cellular internalization, and off-target effects. The use of siRNA is a potential method of gene silencing to target specific genes and address some or all of these limitations. siRNAs are double stranded RNA, typically 20-25 bp in length, containing complementary sequences to target mRNA. The siRNA works within the RNA interference (RNAi) pathway to downregulate the expression of target genes. Once inside the cell, siRNA forms an RNA-Induced Silencing Complex (RISC) upon interactions with various ribonuclease proteins such as the argonaute (Ago) protein. The RNA is unraveled within the RISC-complex, discarding the passenger strand, known as the sense strand, and becoming a single-strand (guide strand or antisense strand), where it can target the complementary mRNA and trigger mRNA cleavage. This ultimately prevents the translation of mRNA into proteins.

[0077] Although the U.S. Food and Drug Administration (FDA) has approved multiple siRNA-based therapeutics, many biological barriers limit their use for treating diseases. Such limitations include challenges concerning systemic or local administration, short half-life, rapid clearance rates, nonspecific binding, cell membrane penetration inability, ineffective endosomal escape, pH sensitivity, endonuclease degradation, immunological responses, and intracellular trafficking.

[0078] To overcome these barriers, various strategies have been developed to stabilize siRNA, ensuring their delivery to the target site (FIG. 1). Chemical modifications implemented with nucleotides or the phosphate backbone can reduce off-target binding and immune stimulation. For example, sugar modifications can increase stability against nuclease degradation. Such siRNA modifications include 2’ sugar modifications: 2’-0Me and 2’-Fluoro. Further, encapsulation or formulation can protect siRNA from endonuclease degradation and enhance cellular uptake while promoting endosomal escape. Additionally, various techniques such as viral vectors, aptamers, cell-penetrating peptides, liposomes, and polymers have been developed for delivering siRNA, greatly improving their bioavailability and therapeutic potential. Herein,siRNA is provided with one or more targeting modalities to allow for delivery to a target cell or tissue while minimizing off-target effects and RNA degradation.Targeting Modalities

[0079] Drug carrier systems have been used in medicine (i.e., nanocarriers, peptides, antibodies, and proteins) to improve site-specific delivery through either encapsulation or conjugation. Early advances focused on encapsulating the RNA in a lipid nanoparticle (LNP) or synthetic nanoparticles to mask the negatively charged RNA upon entry into the cell. There are several therapies using this technology on the market with FDA approval (LNP: patisiran; N-acetylgalactosamine (GalNAc): givosiran, lumasiran, vutrisiran, and inclisiran). However, despite the advances made using GalNAc and LNPs for RNAi based therapies, there is a lack of targeted delivery systems for other tissues and cell types.

[0080] Targeting modalities capable of delivering a payload to sites beyond the liver have been investigated (FIG. 2), such as lipid nanoparticles (LNPs) and monoclonal antibodies (mAbs); however, upon translation LNPs require large doses of encapsulated siRNA with frequent administration. Antibody -RNA conjugates (ARCs) are a novel class of bivalent macromolecules where the monoclonal antibody, recombinantly expressed therapeutic proteins with a molecular weight of 150 kDa, acts as the carrier for the RNA payload. ARCs can be used to target cells other than hepatocytes (liver cells) due to the interaction of the antibody and cell membrane receptors, making them a good candidate for targeting overexpressed receptors on cancer cells. For example, anti-programmed death ligand 1 (PD-L1) overexpression in cancer cells is associated with poor clinical outcomes and can accelerate tumor progression. Immunotherapies using anti-PD-Ll (e.g., Durvalumab) can inhibit PD-1 / PD-L1 binding, allowing T cells to remain active and target cancer cells. Although monoclonal antibody-drug conjugates have gained some traction and success as a targeted immunotherapy treatment in cancer and autoimmune diseases, the therapeutic potential of antibody-drug conjugates is limited due to their larger size, lowering tissue penetration of tumors and immunogenicity.

[0081] Emerging as a promising alternative to mAbs, recombinant variable domains of heavychain-only antibodies, known as nanobodies, provide similar binding capabilities with structural simplicity, smaller size, and high physiochemical stability (FIG. 3). Nanobodies are 10-15 kDa with a similar binding affinity and specificity to antigens as their antibody counterparts. As they are recombinantly expressed in microbial hosts, nanobodies are easily engineered due to a lackof glycosylation requirements, simple design, and high physicochemical stability - providing lower manufacturing costs and time for production compared to mAbs. Applications as a delivery system mimic those of mAbs (FIG. 4), where the nanobody targets cells of interest for activation or inhibition of immunomodulators and checkpoints. The present disclosure provides for the use of nanobodies to target cells and tissues of interest, where the relatively small size of nanobodies allows for effective targeting and uptake by target cells.RNA Delivery

[0082] siRNA delivery in vivo presents further challenges regarding its short half-life and cellular internalization - siRNA may be encapsulated by endosomes and must escape for successful delivery into the cell’s cytoplasm. Inefficient endosomal escape presents a limitation in the field and often lowers therapeutic effectiveness in vivo. The ionizable lipid (IL) in LNPs is primarily responsible for endosomal escape, where, though not intending to be bound by theory, it is believed that the more acidic conditions of endosomes facilitate the formation of a non-lamellar hexagonal structure. The interaction between the new structure of the LNPs and the endosomal membrane results in the rupture of the endosome, releasing the nucleic acid payloads. An alternative method to LNPs is CPPs, an emerging method to enhance cytosolic delivery. The exact mechanism of intracellular delivery remains unclear; however, though not intending to be bound by theory, it is believed that the CPPs follow endocytosis and release from the endosome through endosomal membrane fusion and a decrease in membrane stability.

[0083] Furthermore, RNA delivery into the central nervous system (CNS) and brain is hindered due to challenges, including vasculature, permeability, and penetrating the blood-brain barrier (BBB), where the tight junction of endothelial cells limits therapeutic entry. In a healthy cell, the protective barrier around the CNS protects the brain from endotoxins in the bloodstream. However, in cases such as brain cancer or autoimmune diseases such as multiple sclerosis (MS), therapeutics may be designed to target the CNS - efficiently passing through the BBB.

[0084] Transcytosis is a passive and noninvasive way to go through the BBB. This process can be classified into two types: receptor-mediated and adsorptive-mediated. Receptor-mediated transcytosis involves targeting and binding to a surface receptor in the cell that will allow the cell entry into the BBB. The therapeutic becomes encapsulated within an endosome, thus requiring endosomolytic escape. Once inside the cell, the therapeutic agent can escape the endosome releasing into the cytoplasm of the targeted cell. Whereas in adsorptive-mediated transcytosis,the therapeutic platform is adsorbed to the cell surface, where it becomes encapsulated and brought through the cell membrane.

[0085] Nanobodies may be present as a good carrier based on their small size and the ability to selectively bind protein receptors and therapeutic targets (FIG. 3). Nanobody-siRNA conjugates may successfully achieve site-specific delivery of oligo payloads, such as to knockdown AHSA1 (a house keeping gene for epidermal growth factor receptor - EGFR), vascular endothelial growth factor (VEGF), CD47, and PDL1 in cancer therapy. Nanobody-siRNA conjugates are an effective approach for targeting and regulating receptor proteins including targets in the brain due to receptor-mediated transcytosis - a noninvasive entry into the BBB and CNS. According to the present disclosure, nanobodies can be engineered to cross the BBB for site specific delivery to the brain for applications such as the treatment of brain cancer and autoimmune diseases. The generation of a modular nanobody-siRNA conjugate platform for targeting the BBB for autoimmune diseases is disclosed herein.

[0086] The immune system plays a pivotal role in maintaining homeostasis through its ability to recognize and eliminate pathogens while preserving tolerance to self-antigens. Immune tolerance refers to the prevention of the immune response to attacking self-antigens, one’s own cells and tissues, while responding effectively to foreign antigens. It can be broadly categorized as central tolerance (immune editing occurring in the thymus) or peripheral tolerance (suppression of autoreactive lymphocytes in the periphery). The immune system is generally tolerant of self-antigens, however dysregulation of this balance can result in cancer or the development of autoimmune diseases. In cancer, there are multiple mechanisms, such as alteration of antigen presentation machinery, secretion of immunosuppressive factors to induce apoptosis in lymphocytes or activate negative regulatory pathways, or expression of immune checkpoint proteins, which contribute to the ability of tumors to evade immune recognition and induce tolerance. To combat this, cancer immunotherapy strategies are designed to break tolerance by adoptive transfer of immune effectors, vaccination, or immunomodulating therapy. mAbs have been widely studied as a class of cancer therapeutics to modify tumor cell signaling cascades and stimulate immune checkpoint blockades in cancer immunology and clinical oncology. Checkpoint blockade mAbs, such as anti-CTLA-4, recognize a receptor or ligand and interfere with the interaction via binding, which can enhance the antitumor immune response. Another approach to break tolerance uses vaccination with protein antigen or peptides. Tumor-associated antigens bind directly to MHC molecules on antigen presenting cells (APCs) for increased activation of T cell signaling. Memory T cells can be developed using vaccines to break immune tolerance and stimulate an immune response upon recognition of tumor cells.

[0087] On the other hand, in autoimmune diseases there is a lack of immune tolerance. For example, multiple sclerosis (MS) is a neurodegenerative disease that affects the CNS, causing motor and vision problems ranging in severity by demyelination. In demyelination, the myelin cells are attacked which leaves the axons exposed and unprotected. Regeneration of myelin cells can occur, however with each relapse of MS, the damage to the myelin and nerves increases and the amount regenerated decreases. Current treatments focus on slowing the progression of the disease and symptom management. Knowledge of the pathogenesis of autoimmune diseases is crucial to discovering novel targets for immune modulation. For example, Thl7, a subset of proinflammatory helper T cells, mediates the adaptive immune response to inflammation and in doing so, decreases the amount of regulatory T cells. In autoimmune diseases, this results in an imbalance between Th 17 and Tregs, exacerbating the autoreactive T cells. Ultimately, a goal is to reduce the inflammation and inflammatory response at active lesions and return balance to disordered Thl / Th2 and Thl7 / Tregs expressions. Small molecules such as inhibitors offer transcription factors, such as retinoic acid-related orphan receptor yt (RORyt) and Tbet have been shown to play an important role in T17 differentiation. It has been shown that mediated knockdown of RORyt can inhibit Thl7 production, increasing the amount of Tregs. Other targets of interest for knockdown include the following: SIRT-1, a protein deacetylase enhancing transcriptional activity of RORyt, rho associated kinase 2 (ROCK2) which decreases STAT3 activation, and interleukins such as IL-6 - known to help regulate Thl7 cells.

[0088] T cells play a role in the progression of multiple sclerosis by releasing antigens and cytokines that inflict inflammation and damage in the CNS, or through activation of B cells, ultimately resulting in demyelination and neurodegeneration. Delivery of siRNA targeting CD4+ T cells conjugated with antitransferrin nanobody is possible, and may be evaluated via in vitro qPCR experiments, while validation of the nanobody structure may be undertaken with size exclusion chromatography and SDS PAGE. Embodiments (siRNA gene type, nanobody, endosomolytic peptide, and linker spacing) may be described using cellular uptake, stability, and impact of linker length on the delivery of siRNA.

[0089] The immune system is a complex network of cascading pathways that are regulated by gene expression, which in turn stimulates protein expression and cytokine production to activate an immune response. Trained immunity through APC activation, enables the development of delivery systems like vaccines that can teach the immune system to respond properly to foreign and self-antigens. To achieve the fully optimized and therapeutic translational effect in more complicated disease models, combinational therapies with synergistic long lasting immune responses are desired. Gene modulation via knockdown by siRNA can be used in addition to antigen delivery for precision targeting to enhance an immune response in both cancer and autoimmune diseases, offering dual action therapies for immunogenicity and gene silencing. According to the present disclosure, nanobodies can be engineered to deliver siRNA to enhance the immune response in cancer and autoimmune diseases.Conjugation Methods

[0090] Further, new strategies have evolved to increase control over the location of sitespecific conjugation between biologic molecules and small molecules (e.g., fluorophores and therapeutics). Enzymatic approaches such as Sortase-mediated ligation (SML) are used as a tool to assemble conjugates of proteins with peptides or small molecules. Transpeptidase Sortase A (SrtA) recognizes the motif LPXTG (where X is any amino acid) and GGG sequence to form a new amide bond between the C-terminal tagged LPXTG protein and the corresponding N-terminal oligoglycine. Despite the high specificity for LPXTG sequence, the ligation is highly limited to the terminal ends of the protein and by the relative inefficiency of the SrtA enzyme requiring large amounts of SrtA or long reaction times.

[0091] Click chemistry reactions have garnered interest within the generation of therapeutics due to their high efficiency, specificity, and mild reaction conditions. Copper-catalyzed azidealkyne cycloaddition (CuAAC), a commonly used reaction, involves reacting an alkyne with an azide to produce 1,4-regioisomers of 1,2,3-triazoles. Although CuAAC results in high yields and is compatible with a wide range of biological substrates, copper ions can harm cells, limiting the therapeutic potential of this approach. To combat this challenge, strained cyclic alkynes, such as dibenzocyclooctyne (DBCO) or bicyclononyne (BCN), have been developed to react with an azide, known as strain-promoted azide-alkyne cycloaddition (SPAAC), resulting in fast kinetics on the order of 0.1-1 M^S'1and 0.14 M^S'1respectively. Additionally, inverse electron-demand Diels-Alder cycloaddition (IEDDA) reaction of transcyclooctene (TCO) and tetrazine (Tet) is abioorthogonal alternative to SPAAC. In this case, an electron rich dienophile (e g., TCO) reacts irreversibly with an electron poor diene (e.g., 1,2,4,5-tetrazine) with high specificity. IEDDA click reactions are reported to be the most efficient bioorthogonal reaction due to exceptionally fast second order reaction rates to the order of 106NT1S'1. The present disclosure provides for the C terminal incorporation of azide, BCN, DBCO, TCO, or tetrazine into the nanobody via sortase A bioconjugation. The present disclosure also provides for the conjugation of nanobodies and siRNA through SPAAC, IEDDA, and / or CuAAC conjugation methods (FIG. 5).

[0092] Further, using non-canonical amino acids (ncAAs), which are amino acids that are not part of the twenty in the universal genetic code, orthogonal reactive residues can be incorporated to partake in highly selective click chemistry reactions at various locations within a protein. This technology allows for the generation of unique conjugates capable of multi-drug loading with minimal impact to the antigen recognition or binding of the protein. The present disclosure provides for the use of ncAAs to link siRNA to nanobodies via SPAAC, such as via a BCN group of the nanobody reacting with an azide group of the siRNA. The present disclosure also provides for the use of ncAAs in nanobodies that exhibit azide or tetrazine to link siRNA.Nanobody-siRNA Conjugates

[0093] The present disclosure provides for nanobody-siRNA conjugates to enhance the delivery and spatiotemporal release for a customizable immunotherapy across cancer types and autoimmune diseases. As described above, previous siRNA therapies have been limited by short half-life, endosomal escape, and off-target effects. The present disclosure addresses these challenges in a versatile approach (FIG. 6): (1) First, by designing and implementing a nanobody-siRNA conjugate with CPPs to increase the potency of siRNA delivery, such as for oncogenes in cancer applications. (2) Next, by incorporating site-specific locations in the nanobody sequence for bioconjugation click chemistry reactions using ncAAs to investigate spatiotemporal control and co-delivery of siRNA in metastatic cancer applications. (3) Finally, by investigating the regulation of immune tolerance in multiple sclerosis through a siRNA-based vaccine. The disclosed system will address limitations in pre-clinical delivery, enhancing the in vivo potency and efficacy for gene modulation in cancer and autoimmune diseases. The interchangeable nature of the disclosed nanobody-siRNA conjugates demonstrates the ability to easily target novel genes of interest in various applications (see FIG. 7), including within the CNS, by modifying the siRNA.

[0094] The impacts of gene knockdown may be demonstrated using qPCR, western blots, and viability in vitro (FIG. 8). Further, the nanobody-siRNA conjugate platform may be demonstrated in vivo using flow cytometry to monitor cellular uptake and off-target effects. The impact of therapeutic siRNA may be monitored in vivo to understand the efficacy of the platform and the epitope spreading. The present disclosure allows for site-specific targeting across the BBB for delivery of siRNA to enable targeted knockdown of the gene expression of interest, and for generation of novel modular nanobodies. The disclosed nanobody-siRNA conjugate platform may be used for cancer immunotherapy applications, such as targeting siRNA to upregulated genes such as B cell lymphoma 2 (BCL-2), MYCN Proto-Oncogene (MYCN), and polo-like kinase 1 (PLK-1) (see FIG. 8). It is possible to generate a library of nanobody-siRNA conjugates for selection based on desired application.Sequences

[0095] The sequences described in Table 1 are exemplary design sequences for CPPs and may be modified according to the desired application.Table 1. Design of CPPs of the present disclosureSequence Sequence SEQ Name ID NO.E5 GLFEAIAEFIEGGWEGLIEG 1 H5WYG GLFHAIAHFIHGGWHGLIHGWYG 2 IFN7 GLFEAIEGFIENGWEGMIDGWYG 3 JTS-1 GLFEAIAEF 4 pHl GLFEAIAGF 5 mCherry- TTTTGGTCTCACATGGTTTCTAAAGGCGAAGAAGATAATATG 6 E5 GCGATCATCAAGGAATTCATGCGCTTCAAAGTTCATATGGAG GGCTCCGTGAACGGTCATGAGTTTGAAATCGAGGGTGAGGG CGAAGGCCGCCCGTACGAGGGGACCCAGACGGCGAAATTAA AGGTGACCAAAGGGGGTCCATTGCCGTTCGCCTGGGACATTC TGAGCCCGCAGTTCATGTATGGCAGCAAGGCATACGTTAAG CATCCGGCGGATATTCCGGACTACCTGAAACTGAGTTTTCCT GAAGGCTTTAAGTGGGAGCGTGTGATGAATTTTGAGGATGG TGGCGTGGTAACAGTAACTCAGGATTCTAGCCTGCAGGATG GTGAGTTTATCTATAAAGTGAAGCTCCGCGGGACAAACTTTC CGTCGGATGGCCCGGTCATGCAGAAGAAAACCATGGGTTGG GAAGCCTCGTCTGAACGCATGTATCCAGAAGACGGTGCGCT CAAAGGCGAAATTAAACAGCGCCTGAAACTGAAAGATGGGGGCCACTATGACGCAGAGGTTAAAACAACCTATAAAGCCAAAAAACCGGTGCAATTACCGGGCGCATATAATGTTAATATCAA ATTAGATATCACTTCCCATAATGAGGATTATACTATTGTGGA ACAGTACGAACGTGCAGAAGGCCGCCATAGCACAGGTGGCA TGGACGAATTATATAAAGGTGGGGGTGGTTCCGGTGGTGGG GGGTCAGGCCTGTTTGAAGCCATCGCTGAATTTATTGAGGGT GGCTGGGAGGGTCTTATCGAAGGCGGCGGGGGTGGTAGCTT ACCGGAGACGGGCGGCCATCACCACCATCACCACGAACCGG AAGCGTAAGTGAGACCTTTTmCherry- TTTTGGTCTCACATGGTTTCTAAAGGCGAAGAAGATAATATG 7 H5WYG GCGATTATTAAAGAATTTATGCGGTTTAAGGTTCATATGGAA GGTTCCGTTAATGGGCACGAATTCGAAATTGAAGGCGAAGG CGAAGGGCGTCCTTACGAGGGCACCCAAACGGCAAAATTGA AGGTGACCAAAGGTGGGCCGCTCCCATTTGCGTGGGATATTC TGTCGCCGCAGTTTATGTATGGTTCCAAAGCTTATGTCAAGC ATCCGGCGGACATCCCGGATTACTTGAAATTGAGCTTCCCGG AAGGTTTTAAATGGGAGCGTGTAATGAATTTTGAAGATGGC GGTGTAGTTACCGTGACGCAGGATAGCTCGCTGCAAGACGG TGAGTTTATCTATAAAGTGAAACTGCGCGGTACTAACTTTCC GTCTGACGGTCCGGTGATGCAGAAAAAAACCATGGGGTGGG AAGCTAGCAGCGAACGCATGTATCCAGAAGACGGCGCGTTA AAGGGCGAAATTAAACAGCGCCTCAAATTGAAGGATGGCGG CCATTATGATGCCGAGGTTAAAACGACTTACAAAGCGAAAA AACCGGTGCAGCTCCCGGGCGCGTACAACGTTAACATTAAA CTCGATATTACGTCTCATAATGAGGATTATACGATCGTGGAA CAGTATGAACGCGCGGAAGGCCGCCATAGCACCGGCGGCAT GGATGAGCTGTACAAAGGCGGGGGCGGTAGTGGTGGCGGGG GTAGTGGGCTGTTTCACGCTATCGCCCATTTTATCCATGGGG GTTGGCATGGTCTGATTCATGGTTGGTATGGGGGCGGGGGTG GCGGTTCCTTGCCGGAAACTGGGGGCCACCACCATCATCATC ACGAACCAGAAGCGTAAGTGAGACCTTTTmCherry- TTTTGGTCTCACATGGTTTCTAAAGGCGAAGAAGATAATATG 8 IFN7 GCGATTATTAAAGAGTTTATGCGTTTTAAAGTGCACATGGAG GGTAGTGTAAATGGCCACGAATTTGAGATCGAGGGCGAAGG CGAGGGCCGCCCTTATGAAGGCACCCAAACCGCGAAATTGA AAGTTACCAAAGGTGGGCCGCTGCCTTTCGCTTGGGATATCT TGAGTCCTCAGTTTATGTATGGCAGTAAAGCATACGTGAAAC ATCCGGCGGACATCCCGGATTACTTGAAGCTGTCGTTCCCGG AGGGCTTTAAATGGGAGCGTGTCATGAATTTCGAGGATGGC GGGGTAGTCACGGTTACACAAGATTCTAGCCTTCAGGATGGT GAGTTCATCTACAAGGTGAAACTGCGCGGTACCAACTTCCCG AGCGATGGTCCAGTGATGCAGAAAAAAACGATGGGGTGGGA AGCATCAAGTGAACGCATGTACCCGGAGGATGGCGCACTGA AAGGGGAAATCAAGCAGCGTTTGAAATTGAAAGATGGCGGT CATTACGACGCTGAAGTGAAAACGACCTACAAAGCCAAGAA ACCTGTTCAACTGCCAGGCGCATACAATGTGAACATCAAATTGGATATTACGTCACATAATGAAGACTATACTATCGTGGAACAGTATGAACGTGCGGAAGGTCGCCACAGTACGGGTGGCATGG ATGAGCTTTATAAAGGGGGCGGTGGTAGTGGCGGTGGCGGT TCCGGGTTATTTGAAGCCATCGAAGGTTTTATTGAAAATGGT TGGGAAGGCATGATTGACGGCTGGTATGGCGGCGGTGGGGG CGGCAGTCTTCCTGAAACGGGGGGGCACCACCATCACCATC ATGAGCCGGAGGCATAAGTGAGACCTTTTmCherry- TTTTGGTCTCACATGGTTTCTAAAGGCGAAGAAGATAATATG 9 JTS-1 GCGATTATCAAAGAGTTTATGCGCTTTAAAGTCCATATGGAA GGGAGCGTCAATGGTCATGAGTTCGAAATTGAAGGCGAGGG GGAAGGCCGCCCGTATGAAGGGACGCAAACCGCGAAACTGA AAGTCACCAAAGGCGGCCCGCTCCCATTTGCATGGGATATTT TGTCTCCACAGTTTATGTACGGCAGCAAAGCATATGTGAAAC ATCCGGCGGATATTCCGGATTATCTGAAGTTAAGCTTTCCAG AGGGCTTTAAATGGGAGCGCGTGATGAACTTTGAGGATGGT GGCGTGGTGACAGTTACCCAGGATTCCTCCCTTCAGGACGGT GAGTTCATCTACAAAGTGAAACTCCGCGGCACCAACTTCCCG TCCGATGGCCCGGTGATGCAGAAGAAAACGATGGGTTGGGA AGCATCAAGCGAACGTATGTATCCAGAAGATGGCGCGCTGA AAGGGGAAATTAAACAGCGTCTGAAACTTAAAGATGGCGGT CACTATGACGCCGAGGTGAAGACCACCTATAAAGCGAAAAA ACCGGTGCAACTGCCGGGGGCTTACAACGTGAATATTAAGC TGGATATTACTAGCCATAATGAAGATTATACGATTGTCGAAC AGTATGAGCGCGCGGAGGGCCGTCATTCGACCGGCGGTATG GATGAGCTGTATAAAGGTGGGGGCGGCAGCGGGGGCGGCGG TAGTGGCCTCTTTGAAGCCATTGCAGAGTTTGGTGGTTCGGG GTTGTTCGAAGCCATTGCGGAGTTTGGCGGTGGTGGTGGTAG CCTGCCGGAAACCGGTGGTCATCACCATCACCATCACGAAC CTGAGGCGTAAGTGAGACCTTTTmCherry- TTTTGGTCTCACATGGTTTCTAAAGGCGAAGAAGATAATATG 10 pHl GCGATTATTAAGGAGTTTATGCGCTTTAAAGTGCACATGGAA GGCAGCGTGAACGGGCACGAATTTGAAATCGAAGGGGAAGG TGAAGGCCGTCCATATGAGGGTACTCAGACGGCGAAGCTGA AAGTAACGAAAGGCGGTCCGCTGCCGTTCGCGTGGGATATC TTGTCCCCTCAGTTTATGTATGGGTCGAAAGCGTATGTCAAA CACCCGGCCGATATCCCGGATTATCTGAAATTGAGTTTTCCG GAGGGTTTTAAATGGGAACGTGTCATGAATTTTGAGGATGGC GGTGTGGTGACCGTCACCCAGGATAGCAGCCTGCAAGATGG CGAATTTATCTATAAAGTAAAATTGCGCGGGACCAATTTTCC GAGTGATGGTCCGGTCATGCAAAAAAAAACGATGGGTTGGG AAGCGTCAAGCGAGCGCATGTACCCAGAAGATGGCGCACTT AAAGGCGAAATCAAACAGCGTTTGAAACTCAAAGACGGTGG TCACTATGACGCAGAAGTCAAAACTACCTATAAAGCAAAAA AACCTGTGCAGCTGCCGGGCGCATATAACGTGAATATCAAG CTGGATATCACATCCCATAATGAAGATTACACAATTGTCGAA CAGTACGAACGCGCGGAGGGTCGTCACTCGACGGGCGGGATGGACGAACTCTACAAAGGCGGTGGCGGTAGCGGTGGTGGTGGTTCGGGCCTTTTTGAAGCGATCGCGGGTTTTGGCGGGTCGG GCCTGTTTGAAGCCATCGCTGGCTTTGGTGGTGGCGGGGGCT CGTTACCGGAAACTGGTGGCCACCATCATCATCACCATGAAC CGGAGGCATAAGTGAGACCTTTTmCherry- VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYE 11 E5 AA GTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDY LKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLR GTNFP SDGP VMQKKTMGWEAS SERMYPEDGALKGEIKQRLKL KDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTI VEQYERAEGRHSTGGMDELYKGGGGSGGGGSGLFEAIAEFIEG GWEGLIEGGGGGSLPETGGHHHHHHEPEAmCherry- GTGTCGAAGGGGGAGG 12 E5 FprimermCherry- CGCTTCCGGTTCGTGG 13 E5 RprimermCherry- VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYE 14 H5WYG GTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDY AA LKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLR GTNFP SDGP VMQKKTMGWEAS SERMYPEDGALKGEIKQRLKL KDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTI VEQYERAEGRHSTGGMDELYKGGGGSGGGGSGLFHATAHFIHG GWHGLIHGWYGGGGGGSLPETGGHHHHHHEPEAmCherry- GTTTCTAAAGGCGAAGAAGATAATATGGCG 15 H5WYGF primermCherry- CGCTTCTGGTTCGTGATGATGATGG 16 H5WYGR primermCherry- VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYE 17 IFN7 AA GTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDY LKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLR GTNFP SDGP VMQKKTMGWEAS SERMYPEDGALKGEIKQRLKL KDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTI VEQYERAEGRHSTGGMDELYKGGGGSGGGGSGLFEAIEGFIEN GWEGMIDGWYGGGGGGSLPETGGHHHHHHEPEAmCherry- GTTTCTAAAGGCGAAGAAGATAATATGGCG 18 IFN7FprimermCherry- TGCCTCCGGCTCATGATGG 19 IFN7RprimermCherry- VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYE 20 JTS-1 AA GTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFP SDGP VMQKKTMGWEAS SERMYPEDGALKGEIKQRLKL KDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTI VEQYERAEGRHSTGGMDELYKGGGGSGGGGSGLFEAIAEFGGS GLFEAIAEFGGGGGSLPETGGHHHHHHEPEAmCherry- GTTTCTAAAGGCGAAGAAGATAATATGGCG 21 JTS-1 FprimermCherry- CGCCTCAGGTTCGTGATGG 22 JTS-1 RprimermCherry- VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYE 23 pHl AA GTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDY LKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLR GTNFP SDGP VMQKKTMGWEAS SERMYPEDGALKGEIKQRLKL KDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTI VEQYERAEGRHSTGGMDELYKGGGGSGGGGSGLFEAIAGFGG SGLFEAIAGFGGGGGSLPETGGHHHHHHEPEAmCherry- GTTTCTAAAGGCGAAGAAGATAATATGGCG 24 pHl FprimermCherry- TGCCTCCGGTTCATGG 25 pHl RprimerCompounds of the Disclosure

[0096] The nanobody-siRNA conjugates of the disclosure can be produced using various techniques known in the art and techniques and methods described herein. For recombinant production of polypeptides or nanobodies of the disclosure including a heterologous amino acid sequence, nucleic acid encoding polypeptides or nanobodies of the disclosure and a desired heterologous amino acid sequence can be synthesized and inserted into one or more vectors for further cloning and / or expression in host cells. Polypeptides and nanobodies of the disclosure lacking a heterologous sequence may be produced in the same manner, with the exception the cDNA does not encode the heterologous amino acid sequence. In some embodiments, polypeptides and nanobodies of the disclosure having one or more amino acid substitutions, insertions or deletions are generated by site-directed mutagenesis or other methods known in the art. Such nucleic acid may be readily isolated and sequenced using conventional procedures. Expression vectors comprising polypeptides and nanobodies of the disclosure can be transfected into the host cells or stably expressed in the host cells. Suitable host cells for cloning or expression of the polypeptides and nanobodies of the disclosure include prokaryotic oreukaryotic cells. Expression of the polypeptides and nanobodies of the disclosure can be achieved using yeast, insect, or mammalian expression systems. The expressed polypeptides and nanobodies can be purified using methods known in the art. For example, polypeptides or nanobodies of the disclosure may be purified by affinity chromatography using an appropriate monoclonal antibody or using the optional sequence tag disclosed herein. The purified polypeptides and nanobodies can be verified by using SDS PAGE or Western Blot analysis.

[0097] A skilled artisan will be able to determine suitable substitutions, insertions and deletions, including combinations thereof, of a polypeptide or nanobody as set forth in any of SEQ ID NOS: 1-25 using techniques known in the art. For identifying suitable areas of a polypeptide or nanobody that may be changed without destroying activity, one skilled in the art may target areas not believed to be important for activity. For example, when homologous polypeptides with similar activities from the same species or from other species are known, one skilled in the art may compare the amino acid sequence of a polypeptide described herein to such homologous polypeptides. With such a comparison, one can identify residues and portions of the molecules that are conserved among similar polypeptides. It will be appreciated that changes in areas of a polypeptide described herein that are not conserved relative to such homologous polypeptide would be less likely to adversely affect the biological activity and / or structure of a polypeptide described herein. One skilled in the art would also know that, even in relatively conserved regions, one may substitute chemically similar amino acids for the naturally occurring residues while retaining activity (for example, conservative amino acid substitutions). Therefore, even areas that may be important for biological activity or for structure may be subject to such amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.

[0098] The deletions, insertions, and substitutions can be selected, as would be known to one of ordinary skill in the art, to generate a desired polypeptide or nanobody variant. For example, it is not expected that deletions, insertions, and substitutions in a non-functional region of a polypeptide or nanobody would alter activity. Likewise conservative amino acid substitutions and / or substitution of amino acids with similar hydrophilic and / or hydropathic index values are expected to be tolerated in a conserved region and polypeptide activity may be conserved with such substitutions.Medicaments and Pharmaceutical Compositions

[0099] Useful compositions of the present disclosure may comprise one or more compounds of the disclosure as described above. In one embodiment, such compounds are in the form of compositions, such as but not limited to, pharmaceutical compositions and medicaments. The compositions disclosed may comprise one or more of such compounds, in combination with a pharmaceutically acceptable carrier. To form a pharmaceutically acceptable composition suitable for administration, such compositions will contain a therapeutically effective amount of a compound(s).

[0100] The compositions of the disclosure may be used in the treatment and prevention methods of the present disclosure. Such compositions are administered to a subject in amounts sufficient to deliver a therapeutically effective amount of the compound(s) so as to be effective in the treatment and prevention methods disclosed herein. The therapeutically effective amount may vary according to a variety of factors such as, but not limited to, the subject’s condition, weight, sex and age. Other factors include the mode and site of administration. The compositions may be provided to the subject in any method known in the art. Exemplary routes of administration include, but are not limited to, subcutaneous, intravenous, topical, epicutaneous, intramuscular, and pulmonary. The compositions of the present disclosure may be administered only one time to the subject or more than one time to the subject. Furthermore, when the compositions are administered to the subject more than once, a variety of regimen may be used, such as, but not limited to, one per day, once per week, once per month or once per year. The compositions may also be administered to the subject more than one time per day. The therapeutically effective amount of the nanobody-siRNA conjugates and appropriate dosing regimens may be identified by routine testing in order to obtain optimal activity, while minimizing any potential side effects. In addition, co-administration or sequential administration of other agents may be desirable.

[0101] The compositions of the present disclosure may be administered systemically, such as by intravenous administration, or locally such as by subcutaneous injection. The compositions of the present disclosure may further comprise agents which improve the solubility, half-life, absorption, etc. of the compound(s). Furthermore, the compositions of the present disclosure may further comprise agents that attenuate undesirable side effects and / or or decrease the toxicity of the compounds(s).

[0102] The compositions of the present disclosure can be administered in a wide variety of dosage forms for administration. For example, compositions can be administered in forms, such as, but not limited to, solutions, suspensions, emulsions, or solutions for intravenous administration or injection. Any of the foregoing may be modified to provide for timed release and / or sustained release formulations.

[0103] In the present disclosure, the compositions may further comprise a pharmaceutically acceptable carrier. Such carriers include, but are not limited to, vehicles, adjuvants, surfactants, suspending agents, emulsifying agents, diluents, excipients, binders, lubricants, and buffering agents. Typically, the pharmaceutically acceptable carrier is chemically inert to the active compounds and has no detrimental side effects or toxicity under the conditions of use. The pharmaceutically acceptable carriers can include polymers and polymer matrices. The nature of the pharmaceutically acceptable carrier may differ depending on the particular dosage form employed and other characteristics of the composition.

[0104] Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the patient, and aqueous and nonaqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The compound(s) may be administered in a physiologically acceptable diluent, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol such as poly(ethyleneglycol) 400, glycerol ketals, such as 2,2-dimethyl-l,3-dioxolane-4-methanol, ethers, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as, but not limited to, a soap, an oil or a detergent, suspending agent, such as, but not limited to, pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants. Suitable preservatives and buffers can be used in such formulations. Methods of Treatment and Prevention

[0105] The teachings of the present disclosure provide for the treatment and / or prevention of cancer or autoimmune disease in a subject in need of such treatment. Cancers and autoimmune disease include breast cancer, brain cancer, lung cancer, colorectal cancer, prostate cancer,cervical cancer, melanoma, kidney cancer, pancreatic cancer, thyroid cancer, bladder cancer, multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer’s disease, systemic lupus erythematosus, neuromyelitis optical, and autoimmune encephalitis.

[0106] The method of treatment and / or prevention comprises administering to the subject any of the compounds disclosed herein. The method will often further comprise identifying a subject in need of such treatment or prevention.

[0107] Said modulation is accomplished by different cells through transcytosis within the BBB and CNS. Such targeted delivery interactions are accomplished by administering a compound or pharmaceutical composition containing at least one nanobody-siRNA conjugate. Any siRNA configured to knock down a gene of interest or modulate a desired immune response may be utilized.

[0108] The following Examples are exemplary of methods and compounds described herein and should not be considered limiting unless expressly stated.WORKING EXAMPLE 1Targeted Delivery of a High-Affinity Nanobody-siRNA Conjugate

[0109] There are currently six FDA approved siRNA immunotherapies which knockdown a gene of interest. The delivery of these drugs is limited due to RNA stability in the bloodstream and ability to enter the cell. Nanobodies, proteins of about 15 kDa in size, can help direct the siRNA into the cell. Although a tenth of the size of an antibody, nanobodies have similar a binding affinity. Using click chemistry, a nanobody-siRNA conjugate is engineered (FIG. 9) for cancer immunotherapy and other targeted therapeutic applications.

[0110] Nanobody-siRNA conjugates may be delivered into the cell via receptor-mediated transcytosis (see FIG. 10). The nanobody binds to the receptor on the cell and forms an endosome. The conjugate escapes the endosome to deliver siRNA into the cytoplasm.Modulation of nanobody type, such as nPDLl (FIG. 11 and FIG. 12) to target different cells through transcytosis within the BBB and CNS is presented.

[0111] In some embodiments, the plasmid encoding a gene for the nanobody is transformed into T7 Shuffle E. Coli. The protein was harvested and purified using affinity and size exclusion chromatography. The resultant protein is verified by SDS PAGE (FIG. 13). As shown in FIG. 14, PD-L1 and Albumin nanobodies are expressed and conjugated to BCN via Sortase A ligation.Generated products are verified by ESI-MS and SDS-PAGE (FIG. 15). As shown in FIG. 16, nanobodies are expressed and conjugated to siRNA via SPAAC. Generated products are verified by ESI-MS and SDS-PAGE (FIG. 16).

[0112] The knockdown of luciferase in vitro has been demonstrated for evaluation of the structure and cellular uptake with and without an endosomolytic peptide using flow cytometry and microscopy. In FIG. 17, a luciferase expressing cell line is transfected with nanobody-siRNA and free siRNA. The nanobody increases the delivery of the siRNA into the cell, as shown in the graphical representation in FIG. 17.WORKING EXAMPLE 2Enhanced Delivery of Nanobody-siRNA Conjugates for Oncogenes

[0113] Although great strides have been made to deliver RNA using LNPs, effective delivery of siRNA into the cytoplasm remains a limitation. The disclosed design facilitates an interchangeable format, wherein each piece (CPP, nanobody, and siRNA) can be adapted for novel therapies targeting oncogenes. To address the delivery obstacle, a CPP containing nanobody-siRNA conjugate is presented for delivery to tumors. To achieve this, the design strategy relies on (1) SPAAC click chemistry to allow for direct site of conjugation of the siRNA at the N-terminal of the nanobody, (2) incorporation of CPP at the C-terminal of the nanobody and (3) using the established model of luciferase knockdown to verify enhanced delivery of siRNA.

[0114] To maximize the interchangeable design, the CPP addition and siRNA conjugation is optimized in parallel. Nanobodies are selected that can help extend half-life, human serum albumin (nAlb), or target receptors on breast cancer cells, Programmed Death-Ligand 1 (nPD-Ll). To serve as an initial representation of the platform, a small panel of previously reported CPPs were the focus: TAT, GALA, Arginine-9 (Arg9).

[0115] The present disclosure opts to include a removable N-terminal solubility enhancement domain to improve CPP-protein production. The catalytic residues 21-97 of the Brachypodium distachyon-derived small ubiquitin-related modifier (bdSUMO2i-97) protein have been reported to be efficiently cleaved by the cognate protease, bdSENPl.

[0116] The expression of CPPs can be challenging due to the repetition in the sequences. For example, the sequence of Arg9 is nine arginine residues (RRRRRRRRR) which can lead to mutations in the expression. To avoid this complication, the proteins were expressed inBL21(DE3) RJL, allowing for rare codon usage in the sequences. According to previous reports of CPP protein fusions, the proteins were expressed for 6 hours post induction at 37°C.Expressing proteins at higher temperatures and shorter induction times can lead to higher levels of protein production. To visualize expression of the construct, mCherry, a fluorescent protein, was expressed at the C terminal. If mCherry is being expressed, as confirmed by UV light, this indicates that the protein has expressed the CPP. Confirmation of these constructs is seen through sodium dodecyl sulfate gel electrophoresis (SDS-PAGE) (FIG. 18) and intact electrospray ionization mass spectrometry (ESI-MS). Expected sizes are listed as 64.8 kDa for TAT-nPD-Ll -mCherry, 52.2 kDa for Arg9-nPD-Ll -mCherry, and 52.7 kDa for GALA-nPD-Ll-mCherry.

[0117] The initial expression was optimized to increase the amount of CPP-nPDLl -mCherry expressed. The amount of time post induction and preinduction, temperature post induction, and amount of IPTG added was varied, as shown in Table 2. As demonstrated via SDS-PAGE (not shown), each CPP-nPDLl -mCherry construct demonstrated different levels of expression at each condition. It was not determined that one set of optimal conditions exists for every CPP-nanobody conjugate; however, a lower IPTG concentration and shorter time post induction at 37°C generate better expression.Table 2. Optimizing Expression ConditionsIPTG Temperature Post Time Pre Time Post Batch Concentration Induction Induction Induction (niM) (°C) (hrs) (hrs) 1 0.5 37 4.5 6 2 0.5 16 4.5 24 3 0.1 37 4.5 6 4 0.1 37 2 6 5 0.1 37 2 10

[0118] During optimization, though without wishing to be bound by theory, it was believed that the protein construct being produced was toxic, leading to a lower expression, yield or that it was trapped in an inclusion body due to misfolded or aggregate proteins. Thus, it was transformed into a new strain, BL21 DE3 pLysS, known to help express toxic proteins, in parallelwith purifying for inclusion bodies (FIGS. 18B and 18C). The initial expression conditions remained similar to the previous strain — grown for 6 hrs post induction at 37°C with an induction for a final concentration of 0.1 mM IPTG. In a typical protein purification process, any inclusion bodies would be separated from the clarified cell lysate after centrifugation at 11000 rpm for 20 minutes. It was observed that the cell pellets post sonication and centrifugation of the whole cell lysate were still purple. Without wishing to be bound by theory, it was the belief that the protein was in inclusion bodies and not all located in the clarified cell lysate. The cell pellets were washed with 5 mL of 1 M guanidine HC1. The pellets were sonicated at 18% amplitude for 1 min with 5 seconds on and 5 seconds off to make sure all cells were lysed before centrifugation at 11000 rpm for 15 minutes at 4°C. The pellets were resuspended in 6M guanidine HC1 overnight. Further, the bdSUMO tag was cleaved at the C-terminal using bdSENPl following incubation at 4°C for one hour (FIG. 18D).

[0119] As opposed to expressing the CPP with the nanobody, the CPPs may be synthesized in house using a Liberty Blue 1.0 solid phase peptide synthesizer, as demonstrated in FIG. 19. The peptide synthesizer was used at a 0.025 mmol scale with 81 mg of Fmoc-Gly-Wang Polystyrene resin, yielding around 20-25 mg of CPP. A glycine linker (GGG) was used at the N terminal of the CPP such that it can be attached to a sortase ligation tag on the nanobody. The synthesis of the peptide chains is a series of deprotections and coupling reactions, where the deprotection leaves an available amine for coupling reactions. The resin containing the synthesized peptide is collected and washed thoroughly with dichloromethane (DCM) in a 6 mL SPE polypropylene tube. To cleave the peptide from the resin, 4 mL of a 50% trifluoroacetic acid (TFA) and 50% DCM solution is added to the column and left to shake for 1 hour at room temperature. The resin is rinsed with DCM and the peptide is collected in a 100 mL round bottom flask. Methanol is added to the peptide solution to react with TFA before being placed on Heidolph rotary evaporator to remove all solvents. To validate the correct sequence was made, the CPP was tested on LC-MS (not depicted). The CPP is kept as a 10 mM stock in deionized water for use in bioconjugation reactions. FIG. 20 shows the conjugation of CPPs prepared using this method to nPD-Ll-mCherry.

[0120] Ligation of a CPP to a nanobody using a sortase ligation tag, LPET, on the CPP is possible, with examples of such CPPs visualized with mCherry shown in the images of FIG. 21 Aand FIG. 21B. Similarly, the CPPs with mCherry can be visualized after application to cells as shown in FIG. 22A and FIG. 22B, and FIG. 23 A and FIG. 23B.

[0121] The nanobodies, nPD-Ll, nAlb, and EsrtA, are purified through affinity chromatography and size exclusion chromatography via IMAC and FPLC respectively. The pure fractions are collected and concentrated for conjugation reactions. A click chemistry handle was able to be conjugated onto the protein due to the inclusion of a sortase ligation tag (LPET) at the N terminal. The reaction occurs in mild conditions (room temperature for 24 hours). Originally, a BCN was attached onto the protein, and the siRNA was modified with an azide for SPAAC, however it is more cost effective to switch the click chemistry handles (FIG. 24A). Thus, an azide click chemistry handle was conjugated onto the nanobody, nPD-Ll, as shown in FIG. 24B. The siRNA, purchased from IDT as single strands, is annealed by resuspending the siRNA in a duplex buffer (30mM HEPES, lOOmM potassium acetate) to 100 pM. The oligonucleotides are mixed at a 1:1 molar ratio in PCR tubes and heated to 95C followed by a controlled cooling. The duplexed siRNA is mixed with 1 :2 ratio of nanobody for 48 hours at room temperature. The product is purified through size exclusion chromatography via amicron spin columns and FPLC. The constructs are verified through SDS PAGE imaging using a SYPR-ruby to stain for RNA followed by a protein stain - Coomassie-blue (FIG. 24C) and ESLMS on negative mode, with the expected molecular weight of a nPD-Ll-siLUC conjugate being 25.8 kDa.

[0122] Luciferase, an enzyme that produces bioluminescence, is commonly used as a reporter assay. Common cell lines can be engineered to produce luciferase (i.e. MDA MB 231 luc) upon the addition of luciferin to the cells, for easy and cheaper validation of gene knockdown compared to qPCR. For gene knockdown of luciferase, 5k MDA MB 231 luc cells per well in 100 pL DMEM are plated in a white 96 well plate 24 hours before transfection. The next day, the cells are transfected with either nPD-Ll-siLUC or a positive control (siLUC). The positive control requires the use of lipofectamine purchased from Thermofisher, to create a lipid-siRNA conjugate. A 1:2 dilution of the materials is done using a separate 96 well plate before adding the materials to the cells. After 48 hours, a 15 mg / mL stock solution of luciferin and a working solution of luciferin in DMEM medium is prepared. 100 pL of DMEM containing luciferin is added to each well and placed into the incubator at 37°C for 5 minutes. The plate is analyzed using a plate reader for luminescence (FIG. 25).

[0123] Next, the health of cells after application of the disclosed nanobody-siRNA conjugates was evaluated by quantifying cell growth and observing cell morphology. Cells were plated and transfected with nanobody-siRNA conjugates or a positive control. Cell health was quantified in batch format using trypan blue staining and cell counter.PROPHETIC EXAMPLE 3Verification using luciferase knockdown in vivo

[0124] Cytotoxicity of the disclosed conjugates may be tested by an alive / dead cell assay. Cells are plated at 10k cells / well in 100 pL of media into a 96 well plate, leaving the cells to adhere overnight. The cells may be transfected with nPD-Ll-siLUC, nAlb-siLUC, or a positive control at a 1:2 dilution. After 2 hours, 100 pL of CellTiter-Glo Reagent, purchased from Promega, is added to 100 pL of medium containing cells for a 96-well plate. The plate is mixed for 2 minutes on an orbital shaker to induce cell lysis and then incubated at room temperature for 10 minutes to allow the luminescent signal to stabilize. The plate can then be analyzed by a plate reader to record the luminescence. The results may provide analysis of nanobody-conjugate nontoxicity.

[0125] Based on prior experiments using the CPP, the CPP best able to enhance in vitro delivery of siRNA into the cytoplasm may be determined for CPP-nAlb-siLUC or CPP-nPD-Ll-siLUC conjugates in vivo. To investigate, BALB / c mice will be inoculated with 4xl064T1 luciferase cells subcutaneously into the mammary fat pads and the tumors will be allowed to grow. Biodistribution of the nanobody conjugates can be studied using dyes (i.e. Cy7 on nanobody and Cy3 on siRNA), allowing visualization of where the conjugate is targeting within the body. The In Vivo Imaging System (IVIS) allows imaging of the mice via fluorescence and bioluminescence such that the expression of luciferase at the tumor site and biodistribution of the conjugates can be evaluated simultaneously. The mice may be split into 6 groups (CPP-nPD-Ll-siLUC, CPP-nAlb-siLUC, siLUC, nPD-Ll, nAlb, and PBS) with an n of 5. The injections may be delivered by tail vein or retroorbital. IVIS images may be collected at specific time points post injection (0 min, 5 min, 10 min, 30 min, 1 hour, 2 hour, 4 hour) to see gene knockdown over time as well as biodistribution. Rapid clearance in vivo may occur for nanobodies due to their short half-lives compared to the anti-albumin nanobody which extends half-life to 55 hours. The organs (tumor, heart, lungs, liver, spleen, and kidneys) will be harvested and blood collected viacheek bleeds for further analysis. The organs can be imaged on IVTS and then stained for flow cytometry where the immune cells present may be examined using the following panel: CD3, CD4, CD8, CD19, CD14, CD16, CD25, and CD45. The blood is analyzed using a predefined cytokine detection kit for the flow cytometer.Evaluation of targeted gene knockdown in breast cancer

[0126] Overexpression of oncogenes such as B-cell lymphoma (BCL-2), polo-like kinase 1 (PLK-1), and N-Myc proto-oncogene protein (Mycn) can lead to progression and metastasis of the tumor and are linked to progressive cases of breast cancer. Mycn and bcl-2 regulate tumor cell death; whereas plkl is a serine-threonine kinase associated with the regulation of mitosis. The conjugate system may be evaluated in triple negative breast cancer model, EMT6 and 4T1, to study these target genes. In a similar manner, the conjugates will be assembled using SPAAC to attach the siRNA onto the nanobody. The conjugates will be verified through SDS PAGE and ESI-MS. EMT6 cells and 4T1 cells will be plated in a 96 well plate prior to transfection at 5k per well in 100 pL of media to evaluate the efficiency of protein and mRNA knockdown through western blots and quantitative PCR (qPCR), respectively. Maximal mRNA knockdown may occur in 24-48 hours; however phenotypic responses can require 48-96 hours of transfection. To quantify mRNA knockdown, qPCR for each gene will be analyzed against a housekeeping gene, GAPDEI, using TaqMan. The western blot will indicate that the gene knockdown is effective not only at the genetic level, but also for protein expression and phenotypic changes. Primary antibodies for PLK1, BCL-2, and N-Myc will be able to detect any changes in protein expressions compared to a control gene, GAPDH. A secondary goat anti-rabbit antibody with HRP allows detection using a chemiluminescent substrate, SuperSignal West Femto Maximum Sensitivity Substrate. A cytotoxicity assay will also be performed in vitro to assess cell health and toxicity of the conjugate.

[0127] Further, the conjugates will be evaluated in vivo. A survival animal study will be used to understand the impact of each gene on tumor growth. The mice will be inoculated with 4xl064T1 cells subcutaneously and split into groups of n=5: CPP-nAlb-siPLK-1, CPP-nAlb-BCL-2, CPP-nAlb-Mycn, nAlb, siRNA, and PBS. Tumor volumes and growth will be monitored using a scale and calipers as the conjugates are injected intravenously via tail vein or retro-orbital, for 30 days or until euthanasia occurs if the tumor volumes are > 1500 mm3or >20% body weight loss. The organs will be harvested, and the tumor, liver, and spleen tissues will be analyzed via qPCRto validate knockdown of the target gene in comparison to a housekeeping gene, GAPDH. Blood collected from each mouse will be analyzed by a multiplex cytokine detection kit using flow cytometry.Evaluation of targeted gene modulation in breast to brain metastasis (BTBM)

[0128] Brain metastasis is a hallmark of metastatic breast cancer, with upwards of 40% of patients developing and yielding significant impacts on patient survival rates. Current treatment options are focusing on radiation or surgical options, leaving a need in the clinical field to explore immunotherapy within the brain as an alternative.

[0129] To determine genes of interest, a literature review is conducted to include bulk RNA sequencing experiments on BTBM tumors, highlighting the importance of the BTBM cascade. Genes linked to metastasis include matrix metallopeptidase 7 (MMP7), syndecan-1 (SDCP), and lipocalin-2 (LCN2), where high expression levels at tumor sites indicate poor prognosis.Commonly upregulated genes of interest in the tumor microenvironment include glial fibrillary acidic protein (GFAP), aquaporin 4 (AQP4). and interleukin enhancer-binding factor 2 (ILF2).

[0130] The conjugate system will be evaluated in a triple negative breast cancer model, EO771.LMB - a murine breast adenocarcinoma cell line, to study these target genes. In a similar manner, the conjugates will be assembled using SPAAC to attach the siRNA onto the nanobody. The conjugates will be verified through SDS PAGE and ESI-MS. EO771.LMB cells will be plated in a 96 well plate prior to transfection at 5k per well in 100 pL of media to evaluate the efficiency of protein and mRNA knockdown through western blots and quantitative PCR (qPCR), respectively. To quantify mRNA knockdown, qPCR for each gene will be analyzed against a housekeeping gene, GAPDH, using TaqMan. Phenotype changes will be assessed by western blots. A cytotoxicity assay will also be performed in vitro to assess cell health and toxicity of the conjugate. Conjugates with fluorescently labelled siRNA will be used with confocal microscopy to confirm endosomal escape and intracellular localization.

[0131] Further, the conjugates will be evaluated in vivo. A biodistribution study will be conducted using Cy5 dyed nanobody conjugates at n=3 per group. At specific timepoints (e.g., 2 hours, 4 hours, 24 hours), dependent on the nanobody used, mice will be harvested and ex-vivo IVIS will quantify uptake at each organ. A survival animal study will be used to understand the impact of each gene on tumor growth. C57BL / 6 mice will be inoculated with 4xl06EO771.LMB cells subcutaneously and split into groups of n=5: CPP-nAlb-X, CPP-nAlb- X, CPP-nAlb-X,nAlb, siRNA, and PBS where X represents the panel of siRNA. Tumor volumes and growth will be monitored using a scale and calipers as the conjugates are injected intravenously via tail vein or retroorbital, for 30 days or until euthanasia occurs if the tumor volumes are > 1500 mm3or >20% body weight loss. The organs will be harvested, and the tumor, liver, spleen, and brain tissues will be analyzed via qPCR to validate knockdown of the target gene in comparison to a house keeping gene, GAPDH. Blood collected from each mouse will be analyzed by a multiplex cytokine detection kit using flow cytometry.PROPHETIC EXAMPLE 4Engineering a dual delivery system for controlled spatiotemporal release of siRNA

[0132] Metastasis is a complex process where the cancer cells adapt to invade local tissues and blood vessels from the site of the primary tumor to form metastases throughout the body. Distant metastatic sites can dramatically impact patient survival rates. For example, the 5-year survival rate in locally invasive breast cancer is above 99% yet decreases to 30% for distant metastatic sites. Breast cancer is one of the leading causes of death worldwide. The aggressive behavior of breast tumors results from their metastasis. Notably, brain tissue is one of the common regions of metastasis, thereby reducing the overall survival of patients. Moreover, metastatic tumors demonstrate poor response or resistance to therapies. In addition, breast cancer brain metastasis provides poor prognosis of patients. Therefore, it is important to understand the mechanisms in breast cancer brain metastasis. Both cell lines and animal models have been developed for the evaluation of breast cancer brain metastasis. Moreover, different tumor microenvironment components and other factors such as lymphocytes and astrocytes can affect brain metastasis. The breast cancer cells can disrupt the blood-brain barrier (BBB) during their metastasis into brain, developing blood-tumor barrier to enhance carcinogenesis. Breast cancer brain metastasis can be increased by the dysregulation of chemokines, STAT3, Wnt, Notch and PI3K / Akt. On the other hand, effective therapeutics have been developed for brain metastasis such as introduction of nanoparticles. Moreover, the disruption of BBB by ultrasound can increase the entrance of bioactive compounds to the brain tissue. To improve specificity and selectivity, the nanoparticles for the delivery of therapeutics and crossing over BBB have been developed to suppress breast cancer brain metastasis. Brain metastasis in breast cancer is common, occurring in 15% of patients, and severely limits the survival rates with a 1-yearsurvival rate of 20%. Breast cancer to brain metastasis is treated through radiation, surgery, or chemotherapy. However, the therapeutic benefits are severely limited with these treatments, leaving a clinical need to develop immunotherapies based on the pathways initiating metastatic cascade in BCBM.

[0133] A modular delivery platform will be developed for co-delivery of oligonucleotides to cancerous tissues for gene modulation. Logic-gated approaches can be used to deliver cargo to tumor sites through the incorporation of ncAAs and CPPs into the rational protein design. This platform aims to investigate how enhanced targeted delivery of one or more oligonucleotides can be optimized to create a synergistic immunogenetic treatment in metastatic cancer. The engineered delivery platform, using ncAAs and logic gated controls, can increase the efficacy of siRNA to reduce metastasis. This strategy relies on (1) incorporating ncAAs into the protein sequence for sequential one-pot click chemistry reactions, (2) engineering the controlled release of siRNA by exogenous stimuli, and (3) efficient gene knockdown of multiple genes for a combinational therapy.

[0134] The incorporation of ncAAs has been engineered within the existing protein structure, allowing direct sites for further conjugations ultimately increasing the specificity without inhibiting the binding ability of the protein (FIG. 26). These advancements will allow the generation and cloning of plasmids containing dual ncAAs into anti-Albumin nanobodies via transformations into A. coli for separate click chemistry reactions. Computational methods can be used to choose where to include the ncAA without impacting binding ability to the receptors. Separate bioorthogonal click chemistry reactions will occur on the nanobody to account for two siRNA incorporation sites with the corresponding azide ncAA and tetrazine (tet) ncAA on the nanobody. Modified DBCO siRNA will be selective for the azide ncAA through SPAAC and unreactive to the tet ncAA leaving it to react in a sequential one-pot SPAAC reaction with BCN modified siRNA. The addition of CPPs will be through sortase-mediated ligation at the C terminal of the nanobody, where the synthesized CPPs via SPPS will contain a GGG linker at the N terminal.

[0135] Successful expression of ncAA-containing nanobodies will be purified using size exclusion chromatography and verified by SDS-PAGE and ESLMS. Fluorescence microscopy will be used to validate co-localization of oligo payloads in vitro after dual labeling of the ncAAs, evaluating the impact ncAA incorporation has on antigen recognition or binding. A classof CPP that can target the BBB, known as brain homing peptides (BHPs) will be evaluated in vitro along with CPPs (TAT, Arg9, and GALA). Peptides such as ApoE (159-167)2, ApoB (3371-3409), and HAI will be chosen for an expansion of the panel. Most of the BHPs target a receptor on the BBB, low-density lipoprotein receptor and transferrin receptor respectively for the ApoE / ApoB and HAI, whereas TAT, a CPP from the previous panel, follows adsorptive-mediated transcytosis. These peptides can be synthetically generated using a peptide synthesizer with a GGG on the N terminal of the peptide for conjugation onto the nanobody via sortase ligation.

[0136] The siRNA encoding MMP 7, SDC1, LCN2, GFAP, AQP4, &n .FF2 may be selected based on upregulation in the tumor microenvironment and association with metastasis. Top performing siRNA will be delivered with a second siRNA to enhance anti-tumor immune response, such as CD47. Gene knockdown efficiency of 4 treatment groups - PBS, nanobody with siRNA- 1, nanobody-siRNA-2, and nanobody with dual conjugation to both siRNA, with dosages (0-10 pM) for each treatment - will be evaluated in vitro using engineered breast cancer cell lines that will metastasize to the brain, such as EO771.1mb. Phenotype changes will be monitored through western blots, using proteins downstream of key pathways to verify effective gene modulation. A decrease in mRNA levels will be evaluated by qPCR. To address cytotoxicity concerns in delivering multiple siRNA, cell health will be monitored using Cell Titer Gio assay by flow cytometry and cytokine production (e.g., TNF-a, IL-10, IL-6) from the cell supernatant.

[0137] Without wishing to be bound by theory, it is believed that in delivering multiple oligo payloads using the modular conjugate platform, a synergistic immunogenetic effect can be established that allows targeting of multiple pathways in the TME to ultimately enhance antitumor immune response. To evaluate in vitro additivity or synergy, gene knockdown efficacy experiments can be used. The median-effect and combination index (CI) method will analyze the dosage-response curves to indicate synergism, summation, or antagonism effects of the codelivery through the provided user-friendly computer program. These studies will identify any potential synergistic effects and the corresponding dosages.

[0138] Further, the indicated conjugates for synergism will be tested in vivo to study pharmacokinetics, biodistribution, immunogenetic impact, and anti -tumor therapy. To monitor the delivery of the therapy, the siRNA will be dual labeled (e g., Cy7 and Cy5) and injected viaIV and IP administration to C57BL / 6 mice, inoculated with EO771 LMB cells, with PBS and NHS Fluorescein labeled nanobody as negative controls. Blood collections at predetermined timepoints (5 min, 15 min, 30 min, 1 hour, 2 hours, 4 hours, and 24 hours) will be analyzed on a plate reader for fluorescence levels to assess the kinetics. Organs will be analyzed ex vivo by IVIS at 2 hours and 24 hours indicating biodistribution to primary tumors and metastases within the brain before flow cytometry to monitor phenotype changes in immune cell populations. Tumor, liver, spleen, and brain tissues will be analyzed via qPCR to validate knockdown of the target gene in comparison to a housekeeping gene, GAPDH. The gathered data will be analyzed again via the median-effect and combinational index method to confirm in vitro results.

[0139] In targeting multiple pathways, the role of timing and controlled release in the delivery of RNA payloads may be investigated. These experiments can combine prodrug technologies, a method that stimulates the release of a drug once certain endogenous or exogenous stimuli such as pH or light are met, limiting potential off-target effects (FIG. 27). In cancer, it is well established that the tumor microenvironment has certain characteristics that are distinct from normal tissue, such as an acidic pH of 6.8 and increases in reactive oxygen species (ROS), which serve as targets for stimuli. Previous antibody-drug conjugates are designed with a cleavable pH sensitive linker consisting of chemical bonds sensitive to acid such as hydrazone bonds, imine bonds, phenylmethylimine bonds, amide bonds, and maleic acid amide (MAA) derivatives. Overall hydrazone bonds are used to induce pH cleavage and payload release. At acidic conditions, the double bond between C and N breaks to generate ketones and hydrazine functions. Alternatively, other stimuli, for example light activation with a glutathione (GSH) / visible-light tandem-responsive cleavable linker may be evaluated. After endocytosis of the conjugate, the tumor biomarker GSH cleaves the linker to enable photosensitivity, where the release of siRNA is determined by exposure to visible light.

[0140] It has been shown that the guide strand is essential for RNAi activity, such that an “AND” logic gate can be incorporated by conjugating the pH sensitive linker to the 3’ end of the guide strand to permit precise release and activation of siRNA. The hydrazone linker (e.g., methyltetrazine-PEG4-hydrazone-DBCO or DBCO-hydrazone-Mal) can be included between the 3’ end of the guide strand and the nanobody. In using the commercially available DBCO-hydrazone-Mal linker, the linker will attach to the nanobody via an azide ncAA. siRNA can be modified with a thiol for maleimide-thiol chemistry. Prodrugged conjugates may be confirmedby ESI-MS and SDS-PAGE. In vitro testing can evaluate pH dependence release through transfecting EO177.LMB cells and a control cell line with dye labelled prodrugged conjugates. After incubation at 37°C for 2h, the cells may be stained with DAPI (nuclei stain) and a lysosome stain before cell microscopy. Gene modulation efficiency may be assessed to compare the impact of pH-controlled release to the nanobody design. Further testing in vivo can study the pharmacokinetics of dual delivery by IV and IP administration over 4 h, 24h, and 48 hr.PROPHETIC EXAMPLE 5Developing a vaccine to regulate immune tolerance in multiple sclerosis and assessing antigenspecific T cell responses and immune memory

[0141] The immune response by MS is investigated since most drugs currently on the market target T and B cells to limit symptoms of the disease. T cells are lymphocytes that can help with the immune response and are classified as: CD4+ and CD8+, helper cells (Th), and regulatory cells (Treg). Helper T cells help signal the immune response and activate other immune cells such as B cells and macrophages. Regulatory T cells maintain the immune system and help reduce excessive responses. Both helper and regulatory T cells have CD4 markers. CD4+ T cells have been shown to help produce the autoimmune response in patients with MS. Recently, B cells have also been shown to play a role in the pathway of MS. B cells are lymphocytes that produce antibody molecules. The three types of B cells are memory B cells, regulatory B cells, and plasma cells. Memory B cells can provide long-term immunity by remembering a specific antigen. Regulatory B cells help suppress immune responses. B cells that produce large amounts of antibodies are plasma B cells. Overall B cells can help enhance various pathways such as the release of antibodies, secretion of proinflammatory cytokines, and antigen presentation.

[0142] In understanding these pathways, novel targets aimed to shift immune tolerance emerge for dual delivery strategies such as vaccines. A newer classification among vaccines, siRNA-vaccines, allows for precision targeting of immune pathways, capable of reaching targets other inhibitors like small molecules have trouble accessing. These targets may be incorporated to engineer a siRNA-based vaccine to reprogram tolerance, increasing the number of Tregs through APC activation and modulating immune response in an EAE model. Without wishing to be bound by theory, it is believed that a synergistic long-lasting immune response may be created through co-delivery of an antagonist (siRNA) and antigen (M0G2 peptide). The strategy toachieve this includes (1) learning and establishing in vitro and in vivo models for MS, (2) assessing synergy immunogenic impacts of antigen to antagonist dosages, and (3) investigating T cell activation on immune memory.

[0143] Due to the complex nature of MS, in vitro models are difficult to replicate. Current models can replicate parts of the disease, depending on a few factors. Myelination defects can be studied through primary oligodendrocyte precursor cells, whereas neuroinflammation is typically studied using microglia cells (such as BV-2, N9, or HMC3) or astrocytes that can be stimulated to mimic the immune environment in MS. A 2D model, especially adherent cell lines are convenient to study a specific cell type or function. Otherwise, 3D models, such as co-culture systems, are preferred to capture cell to cell interaction, which is more in line with in vivo models. A microglia and astrocyte co-culture model can be established to study the impact of targeting antigen presenting cells (APCs) - cells whose primary function is to capture, process, and present antigens (small fragments of pathogens) to lymphocytes (T-cells).

[0144] A commonly used in vivo model in clinical settings is experimental autoimmune encephalomyelitis (EAE) model, in which central nervous system inflammation occurs in the mice after immunization against a CNS-specific antigen. C57BL / 6 mice generally develop a chronic form of EAE following immunization with myelin oligodendrocyte glycoprotein (MOG 35-55) and pertussis toxin as an adjuvant. Further, SJL / J mice develop a relapse-remitting form of the disease upon injection of PLP139-151. Each mouse will be evaluated starting on day 9 after postimmunization for the presence of neurological deficits and assigned an EAE score. EAE scores are defined as the following: 0: no clinical signs, 1: limp tail, 2: moderate hindlimb weakness, 3: severe hindlimb weakness, 4: complete hindlimb paralysis, 5: quadriplegia, and 6: deceased due to EAE.

[0145] Data in FIG. 28 shows that nAlb-Cy7, injected retro-orbital to wild type FVB / N mice, has the capacity to enter the brain, verified using IVIS ex vivo. It is known that the anti-Albumin nanobody (nAlb) has a longer half-life, about 55 hours, compared to other nanobodies where the half-life is generally a few minutes due to the ability of “hitchhiking” onto human serum albumin to mimic its half-life. Prolonged biodistribution ensures that the present therapy will reach target cells before renal clearance. Due to these reasons, the present nanobody conjugates use nAlb to deliver both the antigen and antagonist.

[0146] The conjugates can be generated using click chemistry (nanobody with tet ncAA to react with BCN-modified siRNA via IEDDA click chemistry), verified by ESI-MS and SDS PAGE for expected weight shift before testing knockdown efficiency in vitro within the coculture. qPCR of the proteins and supernatant can quantify IL-17, IL-6, and IFN-y production. The immune cell types may be monitored by flow cytometry, specifically for T cell activation (CD4+ and CD8+). The cytotoxicity of the conjugates can be monitored using Cell Titer Gio Assay, evaluated on the plate reader. In parallel, the antigen may be produced via SPPS using a TCO-modified lysine residue for bioconjugation onto nAlb via IEDDA with a tet ncAA, verified by LC-MS, ESI-MS, and SDS-PAGE.

[0147] Stimulation assays with bone marrow-derived dendritic cells (BMDCs) and dendritic cells (DCs) to evaluate APC activation and antigen presentation will be conducted. The vaccine’s influence on existing auto-reactive T cell populations can be evaluated in vitro using an antigen recall assay. T cell populations may be monitored through intracellular cytokine staining (ICS) and CD69 / CD25 expression analysis by flow cytometry.

[0148] The multi-loaded nanobody conjugates (nAlb-MOG-2 and nAlb-siRNA) may be used in an EAE model to evaluate antigen specific immune responses (FIG. 29). Doses of conjugated may be inj ected at various antigen-antagonist ratios (1:1, 2:1, 3:1, 4:1) and the weights and severity of the disease may be monitored. An increase in T cell priming may be observed, as analyzed through flow cytometry and qPCR to quantify CD4+ and CD8+ T cells in the spleen, lymph nodes, and CNS (brain and spinal cords). Serum cytokine levels (e.g., IL-10, IFN-y, TNF-a) may be monitored through LegendPlex via flow cytometry to study systemic inflammatory effects. Further tests in vivo will challenge the immune system with booster antigen injections to test the memory of the immune system. The vaccine may train APCs to recognize these antigens and to stimulate an immune response towards it. Repeated antigen injections, 21 days and 40 days post immunization, will be given to study the longevity of memory immune response.EMBODIMENTS

[0149] Some additional non-limiting example Embodiments are as follows.

[0150] Embodiment 1. A composition including a nanobody-siRNA conjugate. The nanobody-siRNA conjugate includes a nanobody configured to bind a target cell receptor and ansiRNA covalently linked to the nanobody. The nanobody-siRNA conjugate is configured to enter a cell displaying the target cell receptor, with entry occurring via receptor-mediated transcytosis.

[0151] Embodiment 2. The composition according to Embodiment 1, comprising siRNA that is configured to knockdown target gene expression.

[0152] Embodiment 3. The composition of Embodiments 1 or 2, wherein the target gene is selected from the group consisting of B cell lymphoma 2 (BCL-2), MYCN Proto-Oncogene (MYCN), and polo-like kinase 1 (PLK-1).

[0153] Embodiment 4. The composition of Embodiments 1, 2, or 3, wherein the target cell receptor is selected from the group consisting of EGFR, Mucl, B7H3, albumin, transferrin, CTLA-4, and PD-L1.

[0154] Embodiment 5. The composition of Embodiments 1, 2, 3, or 4, wherein the nanobody-siRNA conjugate is configured to penetrate the blood-brain barrier.

[0155] Embodiment 6. The composition of any of Embodiments 1-5, wherein the siRNA is linked to the nanobody via strain-promoted azide-alkyne click chemistry (SPAAC), inverse electron-demand Diels- Alder cycloaddition (TEDDA), or copper-catalyzed azide-alkyne cycloaddition (CuAAC).

[0156] Embodiment 7. The composition of any of Embodiments 1-6, wherein the siRNA is linked to the nanobody via SPAAC and a bicyclo[6.1.0]non-4-yne (BCN) group of the siRNA reacts with an azide group of the nanobody.

[0157] Embodiment 8. The composition of any of Embodiment 1-6, wherein a BCN, azide, or tetrazine group is present on the nanobody via incorporation of one or more non-canonical amino acid (ncAA).

[0158] Embodiment 9. The composition of any of Embodiments 1-6 and 8, wherein a reactive group present on the siRNA is conjugated to the BCN, azide, or tetrazine group of the nanobody.

[0159] Embodiment 10. The composition of any of Embodiments 1-6, wherein an azide, BCN, DBCO, TCO, or tetrazine group is present on a C terminus of the nanobody via an engineered sortase A bioconjugation.

[0160] Embodiment 11. The composition of any of Embodiments 1-6 and 10, wherein a reactive group present on the siRNA is conjugated to the azide, BCN, DBCO, TCO, or tetrazine group of the nanobody.

[0161] Embodiment 12. The composition of any of Embodiments 1-11, further comprising a linker between the siRNA and the nanobody.

[0162] Embodiment 13. The composition of any of Embodiments 1-12, wherein the linker comprises up to 10 spacing nucleotides on the siRNA, up to 20 residues of repeating units between the nanobody and a conjugating group of the nanobody, up to 12 ethylene glycol units of a polyethylene glycol (PEG) linker, or a combination thereof.

[0163] Embodiment 14. The composition of any of Embodiments 1-13, wherein the up to 20 residues of repeating units comprise one or more EAAAK or GGGGS repeating unit.

[0164] Embodiment 15. A method of delivering siRNA payload to a target cell, the method comprising: contacting a nanobody-siRNA conjugate with a receptor of the target cell, wherein a nanobody of the nanobody-siRNA conjugate binds the receptor and the nanobody-siRNA conjugate enters the target cell via receptor-mediated transcytosis, and wherein the siRNA is delivered to cytoplasm of the target cell.

[0165] Embodiment 16. The method of Embodiment 15, wherein the nanobody-siRNA conjugate penetrates the blood-brain barrier.

[0166] Embodiment 17. The method of Embodiments 15 or 16, wherein the target cell is in a brain of a subject.

[0167] Embodiment 18. The method of Embodiments 15, 16, or 17, wherein the target cell is associated with a cancer or autoimmune disease.

[0168] Embodiment 19. The method of Embodiments 15, 16, 17, or 18, wherein the siRNA knockdowns expression of a target gene of the target cell.

[0169] Embodiment 20. A method of treating or preventing a condition in a subject in need thereof, the method comprising: administering to the subject in need thereof a therapeutically effective amount of the composition of any of Embodiments 1-14.

[0170] Embodiment 21. The method of Embodiment 20, wherein the condition affects central nervous system (CNS).

[0171] Embodiment 22. The method of Embodiments 20 or 21, wherein the condition is cancer or an autoimmune disease.

[0172] Embodiment 23. The method of Embodiments 20, 21, or 22, wherein the condition is multiple sclerosis.

[0173] Embodiment 24. A method of producing a nanobody-siRNA conjugate, comprising: providing a nanobody with at least one first click chemistry reactive group; and contacting the nanobody with at least one siRNA having at least one second click chemistry reactive group, such that the nanobody is conjugated to the at least one siRNA via click chemistry at the first and second click chemistry reactive groups.

[0174] Embodiment 25. The method of Embodiment 24, further comprising generating the at least one first click chemistry reactive group on the nanobody at its C terminus via engineered sortase A bioconjugation.

[0175] Embodiment 26. The method of Embodiment 24, further comprising generating the at least one first click chemistry reactive group on the nanobody on a surface-facing location of the nanobody by integrating a ncAA having the at least one first click chemistry reactive group at the surface-facing location.

[0176] Embodiment 27. The method of Embodiments 24, 25, or 26, wherein the at least one first click chemistry reactive group is an azide, BCN, DBCO, TCO, or tetrazine.

[0177] Embodiment 28. The method of Embodiments 24, 25, 26, or 27, further including generating at least one cell penetrating peptide (CPP) on the nanobody before the contacting with the siRNA.REFERENCES(1) Ali Zaidi, S. S.; Fatima, F.; Ali Zaidi, S. A.; Zhou, D.; Deng, W.; Liu, S. Engineering siRNA Therapeutics: Challenges and Strategies. J Nanobiotechnol 2023, 21 (1), 381. doi.org / 10.1186 / sl2951-023-02147-z.(2) Dong, Y.; Siegwart, D. J.; Anderson, D. G. Strategies, Design, and Chemistry in siRNA Delivery Systems. Advanced Drug Delivery Reviews 2019, 144, 133-147. doi.org / 10.1016 / j.addr.2019.05.004.(3) Sahin, U.; Kariko, K.; Tiireci, O. mRNA-Based Therapeutics — Developing a New Class of Drugs. Nat Rev Drug Di scov 2014, 13 (10), 759-780. doi.org / 10.1038 / nrd4278.(4) Ali, S.; Dussouillez, C.; Padilla, B.; Frisch, B.; Mason, A. J.; Kichler, A. Design of a New Cell Penetrating Peptide for DNA, siRNA and mRNA Delivery. The Journal of Gene Medicine 2022, 24 (3), e3401. doi.org / 10.1002 / jgm.3401.(5) Hu, B.; Zhong, L , Weng, Y.; Peng, L.; Huang, Y.; Zhao, Y.; Liang, X.-J. Therapeutic siRNA: State of the Art. Sig Transduct Target Ther 2020, 5 (1), 101. doi.org / 10.1038 / s41392-020-0207-x.(6) Marei, H. E.; Cenciarelli, C.; Hasan, A. Potential of Antibody-Drug Conjugates (ADCs) for Cancer Therapy. Cancer Cell Int 2022, 22 (1), 255. doi.org / 10.1186 / sl2935-022-02679-8.(7) Chiu, M. L.; Goulet, D. R.; Teplyakov, A.; Gilliland, G. L. Antibody Structure and Function: The Basis for Engineering Therapeutics. Antibodies 2019, 8 (4), 55. doi.org / 10.3390 / antib8040055.(8) Ruggiu, M.; Guerin, M. V.; Corre, B.; Bardou, M.; Alonso, R.; Russo, E.; Garcia, Z.; Feldmann, L.; Lemaitre, F.; Dusseaux, M.; Grandjean, C. L.; Bousso, P. Anti-PD-1 Therapy Triggers Tfh Cell-Dependent IL-4 Release to Boost CD8 T Cell Responses in Tumor-Draining Lymph Nodes. Journal of Experimental Medicine 2024, 227 (4), e20232104.doi.org / 10.1084 / jem.20232104.(9) Hambuchen, M. D.; Carroll, F. I.; Ruedi-Bettschen, D.; Hendrickson, H. P.;Hennings, L. J.; Blough, B. E.; Brieaddy, L. E.; Pidaparthi, R. R.; Owens, S. M. Combining Active Immunization with Monoclonal Antibody Therapy To Facilitate Early Initiation of a Long-Acting Anti -Methamphetamine Antibody Response. J. Med. Chem. 2015, 58 (11), 4665-4677. doi.org / 10.1021 / acs.jmedchem.5b00220.(10) Bannas, P.; Hambach, J.; Koch-Nolte, F. Nanobodies and Nanobody-Based Human Heavy Chain Antibodies As Antitumor Therapeutics. Front. Immunol. 2017, 8, 1603. doi.org / 10.3389 / fimmu.2017.01603.(11) Grau, M.; Wagner, E. Strategies and Mechanisms for Endosomal Escape of Therapeutic Nucleic Acids. Current Opinion in Chemical Biology 2024, 81, 102506. doi.org / 10.1016 / j.cbpa.2024.102506.(12) Narum, S.; Deal, B.; Ogasawara, H.; Mancuso, J. N.; Zhang, J.; Salaita, K. An Endosomal Escape Trojan Horse Platform to Improve Cytosolic Delivery of Nucleic Acids. ACS Nano 2024, 18 (8), 6186-6201. doi.org / 10.1021 / acsnano.3c09027.(13) Chen, L.; Cohen, J.; Song, X.; Zhao, A.; Ye, Z.; Feulner, C. J.; Doonan, P.; Somers, W.; Lin, L.; Chen, P. R. Improved Variants of SrtA for Site-Specific Conjugation on Antibodies and Proteins with High Efficiency. Sci Rep 2016, 6 (1). doi.org / 10.1038 / srep31899.(14) Ragab, S. S. Signature of Click Chemistry in Advanced Techniques for Cancer Therapeutics. RSC Adv. 2025, 75 (14), 10583-10601. doi.org / 10.1039 / d5ra01196e.(15) Pickens et al. - 2018 - Practical Considerations, Challenges, and Limitati.(16) He, X.; Li, J.; Liang, X.; Mao, W .; Deng, X.; Qin, M.; Su, H.; Wu, H. An All-in-One Tetrazine Reagent for Cysteine-Selective Labeling and Bioorthogonal Activable Prodrug Construction. Nat Comrmm 2024, 75 (1), 2831. doi.org / 10.1038 / s41467-024-47188-6.(17) Carlson, J. C. T.; Mikula, H.; Weissleder, R. Unraveling Tetrazine-Triggered Bioorthogonal Elimination Enables Chemical Tools for Ultrafast Release and Universal Cleavage. J. Am. Chem. Soc. 2018, 140 (10), 3603-3612. doi.org / 10.1021 / jacs.7bl 1217.(18) Devi, G.; Hedger, A. K.; Whitby, R. J.; Watts, J. K. Double Click: Unexpected 1:2 Stoichiometry in a Norbomene-Tetrazine Reaction. J. Org. Chem. 2023, 88 (9), 5341-5347. doi.org / 10.1021 / acs.joc.2c02861.(19) Pickens, C. J.; Johnson, S. N.; Pressnail, M. M.; Leon, M. A.; Berkland, C. J.Practical Considerations, Challenges, and Limitations of Bioconjugation via Azide-Alkyne Cycloaddition. Bioconjugate Chem. 2018, 29 (3), 686-701. doi.org / 10.1021 / acs.bioconjchem.7b00633.(20) Makkouk, A.; Weiner, G. J. Cancer Immunotherapy and Breaking Immune Tolerance: New Approaches to an Old Challenge. Cancer Research 2015, 75 (1), 5-10. doi.org / 10.1158 / 0008-5472. can-14-2538.(21) Babamohamadi, M.; Mohammadi, N.; Faryadi, E.; Haddadi, M.; Merati, A.;Ghobadinezhad, F.; Amirian, R.; Izadi, Z.; Hadjati, J. Anti-CTLA-4 Nanobody as a Promising Approach in Cancer Immunotherapy. Cell Death Dis 2024, 75 (1), 17. doi.org / 10.1038 / s41419-023-06391-x.(22) Amin, M.; Hersh, C. M. Updates and Advances in Multiple Sclerosis Neurotherapeutics. Neurodegenerative Disease Management 2023, 13 (1), 47-70.doi . org / 10.2217 / nmt-2021-0058.(23) Bierhansl, L.; Hartung, H.-P.; Aktas, O.; Ruck, T.; Roden, M.; Meuth, S. G. Thinking Outside the Box: Non-Canonical Targets in Multiple Sclerosis. Nat Rev Drug Discov 2022, 27 (8), 578-600. doi.org / 10.1038 / s41573-022-00477-5.(24) Therapeutic Potential of Targeting the Thl 7 / Treg Axis in Autoimmune Disorders. www.mdpi.com / 1420-3049 / 22 / 1 / 134 (accessed 2025-07-22).(25) siRNA -mediated knockdown of T-bet and RORyt contributes to decreased inflammation in pre-eclampsia. www.spandidos-publications.com / 10.3892 / mmr.2017.7348 (accessed 2025-07-22).(26) Straus Farber et al. - 2016 - Novel Agents for Relapsing Forms of Multiple Scler (27) Li, J.; Mao, N.; Wang, Y.; Deng, S.; Chen, K. Novel Insights into the ROCK-JAK-STAT Signaling Pathway in Upper Respiratory Tract Infections and Neurodegenerative Diseases. Molecidar Therapy 2025, 33 (1), 32-50. doi.org / 10.1016 / j.ymthe.2024.11.011.(28) Ando, Y.; Nakazawa, H.; Miura, D.; Otake, M.; Umetsu, M. Enzymatic Ligation of an Antibody and Arginine 9 Peptide for Efficient and Cell-Specific siRNA Delivery. Sci Rep 2021, 11 (1), 21882. doi.org / 10.1038 / s41598-021-01331-l.(29) Adhikari, S.; Alahmadi, T. I.; Gong, Z.; Karlsson, A. J. Expression of Cell-Penetrating Peptides Fused to Protein Cargo. Microb Physiol 2018, 28 (4), 159-168. doi.org / 10.1159 / 000494084.(30) Frey, S.; Gorlich, D. A New Set of Highly Efficient, Tag-Cleaving Proteases for Purifying Recombinant Proteins. Journal of Chromatography A 2014, 1337, 95-105.doi . org / 10.1016 / j . chroma.2014.02.029.(31) Lin, Y.; Zhao, Y .; Chen, M.; Li, Z.; Liu, Q.; Chen, J.; Ding, Y.; Ding, C.; Ding, Y.; Qi, C.; Zheng, L.; Li, J.; Zhang, R.; Zhou, J.; Wang, L.; Zhang, Q.-Q. CYD0281, a Bcl-2 BH4 Domain Antagonist, Inhibits Tumor Angiogenesis and Breast Cancer Tumor Growth. BMC Cancer 2023, 23 (1). doi.org / 10.1186 / sl2885-023-10974-4.(32) Ueda, A.; Oikawa, K.; Fujita, K.; Ishikawa, A.; Sato, E.; Ishikawa, T.; Kuroda, M.; Kanekura, K. Therapeutic Potential of PLK1 Inhibition in Triple-Negative Breast Cancer.Laboratory Investigation 2019 , 99 (9), 1275-1286. doi.org / 10.1038 / s41374-019-0247-4.(33) Zimmerli, D.; Brambillasca, C. S.; Talens, F.; Bhin, J.; Linstra, R ; Romanens, L.; Bhattacharya, A.; Joosten, S. E. P.; Da Silva, A. M.; Padrao, N.; Wellenstein, M. D.; Kersten, K.; De Boo, M.; Roorda, M.; Henneman, L.; De Bruijn, R.; Annunziato, S.; Van Der Burg, E.;Drenth, A. P.; Lutz, C ; Endres, T.; Van De Ven, M.; Eilers, M.; Wessels, L.; De Visser, K. E.; Zwart, W Fehrmann, R. S. N.; Van Vugt, M. A. T. M.; Jonkers, J. MYC Promotes Immune-Suppression in Triple-Negative Breast Cancer via Inhibition of Interferon Signaling. Nat Commun 2022, 13 (1). doi.org / 10.1038 / s41467-022-34000-6.(34) Montaudon, E.; Nikitorowicz-Buniak, J.; Sourd, L.; Morisset, L.; El Botty, R.;Huguet, L.; Dahmani, A.; Painsec, P.; Nemati, F.; Vacher, S.; Chemlali, W.; Masliah-Planchon, J.; Chateau-Joubert, S.; Rega, C.; Leal, M. F.; Simigdala, N.; Pancholi, S.; Ribas, R.; Nicolas, A.; Meseure, D.; Vincent-Salomon, A.; Reyes, C.; Rapinat, A.; Gentien, D.; Larcher, T.; Bohec, M.; Baulande, S.; Bernard, V.; Decaudin, D.; Coussy, F.; Le Romancer, M.; Dutertre, G ; Tariq, Z.; Cottu, P.; Driouch, K.; Bieche, I.; Martin, L.-A.; Marangoni, E. PLK1 Inhibition Exhibits Strong Anti-Tumoral Activity in CCND1 -Driven Breast Cancer Metastases with Acquired Palbociclib Resistance. Nat Commun 2020, 11 (1). doi.org / 10.1038 / s41467-020-17697-l.(35) Liu, L.; He, Y.; Du, H.; Tang, M.; Wang, T.; Tan, J.; Zha, L.; Yang, L.; Ashrafizadeh, M.; Tian, Y.; Zhou, H. Biological Profde of Breast Cancer Brain Metastasis, acta neuropathol commun 2025, 13 (1). doi.org / 10.1186 / s40478-025-01983-4.(36) Wu, X.; Zhang, Y.; Huang, Z.; Li, R. Differential Adaptation and Remodeling of the Tumor Microenvironment in Breast Cancer Cells During the Stages of Brain Metastasis. Cold Spring Harbor Laboratory July 15, 2023. doi.org / 10.1101 / 2023.07.13.548875.(37) Li et al. - 2025 - GRB2 Promotes Brain Metastasis in HER2 -Positive Br.(38) Zhu, L.; Liu, M.; Shang, Y.; Cheng, J.; Zhao, H.; Zhang, J.; Shen, D. Single-Cell Profiling Transcriptomic Reveals Cellular Heterogeneity and Cellular Crosstalk in Breast Cancer Lymphatic Node, Bone, and Brain Metastases. Set Rep 2025, 15 (1). doi.org / 10.1038 / s41598-025-85531-z.(39) Lan et al. - 2024 - Update on the Intriguing Roles of AQP4 Expression.(40) Zhou et al. - 2021 - Brain Penetrating Peptides and Peptide-Drug Conjug.(41) Chen, C.; Wang, R.; Chen, X.; Hou, Y.; Jiang, J. Targeting CD47 as a Novel Immunotherapy for Breast Cancer. Front. Oncol. 2022, 72, 924740.doi.org / 10.3389 / fonc.2022.924740.(42) Ma, J.; Motsinger-Reif, A. Current Methods for Quantifying Drug Synergism. 2020.(43) Chen, Y.; Clay, N.; Phan, N.; Lothrop, E.; Culkins, C.; Robinson, B.; Stubblefield, A.; Ferguson, A.; Kimmel, B. R. Molecular Matchmakers: Bioconjugation Techniques Enhance Prodrug Potency for Immunotherapy. Mol. Pharmaceutics 2025, 22 (1), 58-80. doi.org / 10.102 l / acs.molpharmaceut.4c00867.(44) Li, C.; Sun, S.; Kong, H; Xie, X.; Liang, G; Zhang, Y.; Wang, H; Li, J. A Dual-Locked Cyclopeptide-siRNA Conjugate for Tumor-Specific Gene Silencing. RSC Chem. Biol.2025, 6(1), 73-80. doi.org / 10.1039 / D4CB00247D.(45) Abd-Ellah, H. S.; Zhao, D.; Zhou, Y.; Baell, J. B. Unlocking pH-Responsive Dual Payload Release through Hydrazone Linkage Chemistry. Bioorganic & Medicinal Chemistry 2025, 723, 118172. doi.org / 10.1016 / j.bmc.2025.118172.(46) Berrino, E.; Thibaudeau, S. pH-Sensitive Prodrugs. In Advances in Prodrugs,' Elsevier, 2025; pp 23-40. doi.org / 10.1016 / B978-0-443-15635-9.00010-9.(47) Zavoiura, O.; Brunner, B.; Casteels, P.; Zimmermann, L.; Ozog, M.; Boutton, C.; Helms, M. W .; Wagenaar, T.; Adam, V.; Peterka, J.; Metz-Weidmann, C.; Deschaght, P.; Scheidler, S.; Jahn-Hofmann, K. Nanobody-siRNA Conjugates for Targeted Delivery of siRNA to Cancer Cells. Mol. Pharmaceutics 2021, 18 (3), 1048-1060. doi.org / 10.1021 / acs.molpharmaceut.0c01001.(48) Hoover, E. C.; Roy Chowdhury, C.; Ruggiero, O. M.; Day, E. S. Conjugation of Antibodies and siRNA Duplexes to Polymer Nanoparticles via Maleimide-Thiol Chemistry. ACS Omega 2024, 9 (48), 47637-47646. doi.org / 10.1021 / acsomega.4c07025.(49) Angelini, G.; Bani, A.; Constantin, G.; Rossi, B. The Interplay between T Helper Cells and Brain Barriers in the Pathogenesis of Multiple Sclerosis. Front. Cell. Neurosci. 2023, 77, 1101379. doi.org / 10.3389 / fncel.2023.1101379.(50) Lovett-Racke, A. E. Contribution of EAE to Understanding and Treating Multiple Sclerosis. Journal of Neuroimmunology 2017, 304, 40-42.doi.org / 10.1016 / j.jneuroim.2016.08.013.

Claims

CLAIMSWhat is claimed:

1. A composition comprising a nanobody-siRNA conjugate havinga nanobody configured to bind a target cell receptor; andan siRNA covalently linked to the nanobody,wherein the nanobody-siRNA conjugate is configured to enter, via receptor-mediated transcytosis, a cell displaying the target cell receptor.

2. The composition of claim 1, wherein the siRNA is configured to knockdown target gene expression.

3. The composition of claim 2, wherein the target gene is selected from the group consisting of B cell lymphoma 2 (BCL-2), MYCN Proto-Oncogene (MYCN), and polo-like kinase 1 (PLK-1).

4. The composition of claim 1, wherein the target cell receptor is selected from the group consisting of EGFR, Mucl, B7H3, albumin, transferrin, CTLA-4, and PD-L1.

5. The composition of claim 1, wherein the nanobody-siRNA conjugate is configured to penetrate the blood-brain barrier.

6. The composition of claim 1, wherein the siRNA is linked to the nanobody via strain-promoted azide-alkyne click chemistry (SPAAC), inverse electron-demand Diels- Alder cycloaddition (IEDDA), or copper-catalyzed azide-alkyne cycloaddition (CuAAC).

7. The composition of claim 6, wherein the siRNA is linked to the nanobody via SPAAC and a bicyclo[6.1 ,0]non-4-yne (BCN) group of the siRNA reacts with an azide group of the nanobody.

8. The composition of claim 6, wherein a BCN, azide, or tetrazine group is present on the nanobody via incorporation of one or more non-canonical amino acid (ncAA).

9. The composition of claim 8, wherein a reactive group present on the siRNA is conjugated to the BCN, azide, or tetrazine group of the nanobody.

10. The composition of claim 6, wherein an azide, BCN, DBCO, TCO, or tetrazine group is present on a C terminus of the nanobody via an engineered sortase A bioconjugation.

11. The composition of claim 10, wherein a reactive group present on the siRNA is conjugated to the azide, BCN, DBCO, TCO, or tetrazine group of the nanobody.

12. The composition of claim 1, further comprising a linker between the siRNA and the nanobody.

13. The composition of claim 12, wherein the linker comprises up to 10 spacing nucleotides on the siRNA, up to 20 residues of repeating units between the nanobody and a conjugating group of the nanobody, up to 12 ethylene glycol units of a polyethylene glycol (PEG) linker, or a combination thereof.

14. The composition of claim 13, wherein the up to 20 residues of repeating units comprise one or more EAAAK or GGGGS repeating unit.

15. A method of delivering siRNA payload to a target cell, the method comprising: contacting a nanobody-siRNA conjugate with a receptor of the target cell, wherein a nanobody of the nanobody-siRNA conjugate binds the receptor and the nanobody-siRNA conjugate enters the target cell via receptor-mediated transcytosis, and wherein the siRNA is delivered to cytoplasm of the target cell.

16. The method of claim 15, wherein the nanobody-siRNA conjugate penetrates the bloodbrain barrier.

17. The method of claim 15, wherein the target cell is in a brain of a subject.

18. The method of claim 15, wherein the target cell is associated with a cancer or autoimmune disease.

19. The method of claim 15, wherein the siRNA knockdowns expression of a target gene of the target cell.

20. A method of treating or preventing a condition in a subject in need thereof, the method comprising: administering to the subject in need thereof a therapeutically effective amount of the composition of claim 1.

21. The method of claim 20, wherein the condition affects central nervous system (CNS).

22. The method of claim 21, wherein the condition is cancer or an autoimmune disease.

23. The method of claim 22, wherein the condition is multiple sclerosis.

24. A method of producing a nanobody-siRNA conjugate, comprising:providing a nanobody with at least one first click chemistry reactive group; andcontacting the nanobody with at least one siRNA having at least one second click chemistry reactive group, such that the nanobody is conjugated to the at least one siRNA via click chemistry at the first and second click chemistry reactive groups.

25. The method of claim 24, further comprising generating the at least one first click chemistry reactive group on the nanobody at its C terminus via engineered sortase A bioconjugation.

26. The method of claim 24, further comprising generating the at least one first click chemistry reactive group on the nanobody on a surface-facing location of the nanobody by integrating a ncAA having the at least one first click chemistry reactive group at the surfacefacing location.

27. The method of claim 24, wherein the at least one first click chemistry reactive group is an azide, BCN, DBCO, TCO, or tetrazine.

28. The method of claim 24, further including generating at least one cell penetrating peptide (CPP) on the nanobody before the contacting with the siRNA.

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