Virus vector with engineering function and production method thereof

Abstract

Provided herein are viral vectors having engineered functionality and methods of producing the same.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 433,148, filed on December 16, 2022; the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to the fields of molecular biology and lentiviral vectors. Background Art

[0004] Lentivirus (LV) is a genus of the Retroviridae family that includes the human immunodeficiency virus (HIV). LV is an enveloped virus with a single-stranded RNA length of approximately 8 kilobases (kp). Recent studies have shown that lentiviral vectors (LVV) may be a preferred vector for gene delivery for therapeutic purposes due to their ability to express genes for a long period of time, their specificity in targeting cells, tissues, and organs, and their safety attributes. To date, several iterations of LVV modified from HIV have been used in human clinical trials as components of therapeutic agents and drugs. These include chimeric antigen receptor T cell therapy for blood cancers, and hematopoietic stem cell therapy for β-thalassemia, cerebral adrenoleukodystrophy, and X-linked severe combined immunodeficiency. Due to limitations in LVV assembly and production, it has been a technical challenge to produce LVV with novel, advanced functions, including the ability to directly target desired cells in vivo while maintaining sufficiently high delivery efficiency for therapeutic use.

[0005] Overview

[0006] The invention described herein is based, at least in part, on the discovery that the production of LVV and other enveloped vectors with desired functionality for in vivo therapy is limited by extremely low functional titers resulting from current processes for producing next-generation functional LVV. Indeed, it has been discovered that when LVV compositions add additional therapeutically relevant functionality to the surface via exogenous surface proteins, there is a trade-off between this advanced functionality and the titer of the resulting LVV that can be produced. Low titers lead to low delivery efficiency, low efficacy, and limited therapeutic applications. This is presumably due to a bottleneck in targeting the desired exogenous functional protein(s) to the site where the vector buds from the producer cell. Currently, the entire lentiviral vector field produces vectors with exogenous surface proteins by transcribing and translating plasmids or integrated gene sequences encoding those surface proteins in producer cells. However, this results in low-titer viral vector preparations of vectors with the desired exogenous targeted surface protein(s), and these low titers are insufficient for widespread therapeutic use.

[0007] Increasing the titer of lentiviral vectors is an active area of ​​research to improve therapeutic access and cost; however, the field currently focuses on bioprocess optimization, such as altering pH, dissolved oxygen levels, mixing properties, and concentration. This work stems from the assumption that optimal process conditions can lead to significant improvements in yield—an assumption that has not been proven correct to date. Here, we take a completely different approach, improving the process by fundamentally changing how lentiviral vectors are created. Currently, the industry produces these advanced functional viral vectors using a one-step production process, in which genetic instructions are delivered to producer cells to produce the viral vector with the desired exogenous functional protein on its surface. The present invention is based on the discovery that an alternative production method, in which the viral vector core is first assembled in a cell-based process and then the exogenous functional protein is added to the viral vector core using a cell-free method, should in turn lead to significant improvements in the production yield of these advanced functional viral vectors. These improvements enable an "assembly line" production method for preparing viral vectors one step at a time, offering engineered production efficiencies not available in the classic, one-step, cell-based production methods currently used in the field. The magnitude of these production improvements has facilitated the application of viral vector gene therapy in therapeutic areas that were previously inaccessible due to production limitations. The present invention is based on the discovery that chemical conjugation can be used to successfully present one or more desired exogenous surface proteins onto the viral vector core after budding from the producer cell, thereby increasing the yield of therapeutically effective viral vectors bearing one or more desired exogenous surface proteins by several orders of magnitude compared to methods of producing viral vectors by introducing gene sequences alone into producer cells.

[0008] Previously, the field was limited to producing vectors with very rudimentary targeting functionality chemically conjugated to the vector, such as targeting conferred by basic peptides. Using the methods provided by the present invention, full-length exogenous targeting proteins can be successfully added to lentiviral vectors, providing viral vectors with more precise targeting. Surprisingly, it has been discovered that the correct integration of the core viral vector production process, conjugation technology, and exogenous targeting protein and glycoprotein combination provides lentiviral vectors with improved assembly and delivery efficiency.

[0009] In light of these findings, provided herein are viral vectors with improved function and methods for producing the same.

[0010] Provided herein are viral vector cores having a first chemical handle on their surface. In some embodiments of any of the viral vector cores described herein, the first chemical handle comprises an azido sugar. In some embodiments of any of the viral vectors described herein, the first chemical handle comprises an alkyne sugar. In some embodiments of any of the viral vector cores described herein, the viral vector core contains a nucleic acid encoding one or more therapeutic proteins. In some embodiments of any of the viral vector cores described herein, the viral vector core is a lentiviral vector core.

[0011] Also provided herein are compositions comprising any of the viral vector cores described herein. Also provided herein are compositions comprising a virus-like particle core, a non-viral envelope vector core, or an extracellular vesicle core.

[0012] Also provided herein is a viral vector comprising one or more exogenous targeting proteins, one or more exogenous immune modification proteins and / or one or more polymers covalently attached to its surface by conjugation features. In some embodiments of any viral vector described herein, the conjugation features comprise a triazole bond, a thioether bond, a disulfide bond or a [4+2] cycloadduct. In some embodiments of any viral vector described herein, one or more exogenous targeting proteins or one or more exogenous immune modification proteins have a molecular weight greater than 2kDa. In some embodiments of any viral vector described herein, one or more exogenous targeting proteins comprise scFv, nano antibodies, darpin, VHH, aptamer or lectin binding domains. In some embodiments of any viral vector described herein, the polymer is polyethylene glycol (PEG). In some embodiments of any viral vector described herein, PEG has an average molecular weight of about 100 g / mol to about 20000 g / mol. In some embodiments of any viral vector described herein, the viral vector contains a nucleic acid encoding one or more therapeutic proteins. In some embodiments of any viral vector described herein, the viral vector is a lentiviral vector. In some embodiments of any of the viral vectors described herein, the viral vector has a pseudotype selected from the group consisting of VSV-G, blind VSV-G, Sindbis glycoprotein, blind Sindbis glycoprotein, murine leukemia virus glycoprotein, blind murine leukemia virus glycoprotein, Moloney murine leukemia virus glycoprotein, blind Moloney murine leukemia virus glycoprotein, cocal virus glycoprotein, blind cocal virus glycoprotein, foamy virus glycoprotein, blind foamy virus glycoprotein. In some embodiments of any of the viral vectors described herein, the viral vector is not pseudotyped with VSV-G.

[0013] Also provided herein are compositions comprising a population of any of the viral vectors described herein. In some embodiments of any of the compositions described herein, prior to concentration and conjugation of the viral vectors, the population of viral vectors has a functional titer corresponding to greater than about 1E7 TU / mL. In some embodiments of any of the compositions described herein, the ratio of functional titer to physical titer is from about 1:1000 to about 1:10. In some embodiments of any of the compositions described herein, the composition is a pharmaceutical composition.

[0014] Also provided herein is a method for preparing a viral vector, comprising: (a) contacting a viral vector core comprising a first chemical handle on its surface with an exogenous targeting protein, an exogenous immune-modifying protein, or a polymer comprising a second chemical handle, and (b) covalently conjugating the first chemical handle to the second chemical handle to produce a viral vector. In some embodiments of any of the methods described herein, the method further comprises: producing a viral vector core comprising a first chemical handle on its surface. In some embodiments of any of the methods described herein, the step of producing the viral vector core comprises a step of purifying or isolating the viral vector core by purification and / or chromatography. In some embodiments of any of the methods described herein, the step of producing the viral vector core comprises culturing production cells in a liquid culture medium comprising a small molecule that is capable of metabolically inserting the first chemical handle into a glycoprotein on the production cell membrane. In some embodiments of any of the methods described herein, the first chemical handle and the second chemical handle are azide and alkyne. In some embodiments of any of the methods described herein, the first chemical handle and the second chemical handle do not comprise BCN. In some embodiments of any of the methods described herein, step (b) is performed using a click chemistry reaction. In some embodiments of any of the methods described herein, the click chemistry reaction is performed at a temperature of about 30°C to about 40°C and a pH of about 6.5 to about 7.5.

[0015] Also provided herein are kits comprising any of the compositions described herein.

[0016] Also provided herein are methods of treating a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the compositions described herein.

[0017] The term "viral vector" refers to a modified virus that does not cause diseases associated with the original virus. The viral vector can transduce cells and deliver transgenes (e.g., one or more transgenes). One or more transgenes can, for example, encode therapeutic proteins. The viral vector can also deliver proteins. Non-limiting examples of proteins delivered by viral vectors include gene editing proteins (e.g., Cas9, Cas12a, Cas12b, adenine and cytosine base editors, primer editors, and ribonucleoproteins thereof).

[0018] The term "exogenous functional protein" refers to a peptide sequence that can alter the function of a viral vector. Non-limiting examples of altered viral vector function include targeting, altered interaction with the immune system, immune evasion, and altered biodistribution. In some embodiments, the peptide sequence is not typically present on the surface of a corresponding naturally occurring viral vector. In some embodiments, the peptide sequence is not typically present in the corresponding naturally occurring viral vector in the same amount or form as when it is introduced as an exogenous functional protein into the viral vector.

[0019] The term "exogenous targeting protein" refers to a peptide sequence that can direct a viral vector to a specific receptor or feature on the surface of a target cell through interaction between the exogenous targeting protein and the receptor or feature on the surface of the target cell. In some embodiments, the peptide sequence is not normally present in the corresponding naturally occurring viral vector. In some embodiments, the peptide sequence is not normally present on the surface of the viral vector in the same amount or form as when it is introduced as an exogenous targeting protein into the viral vector. An exogenous targeting protein is a type of exogenous functional protein.

[0020] The term "exogenous immune modifying protein" refers to a peptide sequence that can change the way a viral vector interacts with the immune system (relative to the way the viral vector interacts with the immune system in the absence of the protein). In some embodiments, the peptide sequence is not typically present in the corresponding naturally occurring viral vector. In some embodiments, the peptide sequence is not typically present on the surface of the viral vector in the same amount or form as when it is introduced into the viral vector as an exogenous targeting protein. An exogenous immune modifying protein is a type of exogenous functional protein.

[0021] The term "glycoprotein" refers to a molecule expressed on the surface of a viral vector that has one or more carbohydrate groups attached to a peptide chain.

[0022] The term "pseudotype" refers to a viral vector that has foreign viral envelope proteins not normally associated with the wild-type virus.

[0023] The term "producer cell" refers to cells that produce a viral vector or viral vector core by introducing a plasmid or other nucleic acid into the cell that enables them to produce the viral vector or a component of a viral vector. The nucleic acid can be introduced transiently or permanently, the latter of which is sometimes referred to in the industry as a stable cell line.

[0024] The term "chemical handle" refers to any chemical moiety that can be used to chemically conjugate with a second chemical moiety. Non-limiting examples of chemical handles include click chemistry handles (or click handles). Non-limiting examples of click chemistry handles are described herein.

[0025] The term "chemical conjugation" refers to the joining of two molecules to one another by covalent or non-covalent chemistry.

[0026] The term "conjugation feature" refers to a bond that remains after covalent conjugation between a first chemistry handle and a second chemistry handle. Non-limiting examples of conjugation features include a bond that remains after covalent conjugation of a first click chemistry handle to a second click chemistry handle, where such a conjugation feature can be referred to as a click feature.

[0027] The term "viral vector core" refers to a fragment of a viral vector that contains genetic material or protein delivery material within the viral capsid, but lacks a complete, functional envelope including all necessary or desired surface proteins. For example, a viral vector core may lack one or more exogenous targeting proteins and / or exogenous immune-modifying proteins.

[0028] The term "functional titer" refers to the concentration of a viral vector that has the ability to successfully deliver a transgene to a target cell type. In some embodiments, a viral vector that has the ability to successfully deliver a transgene to a target cell type may have one or more (e.g., two or more) exogenous targeting proteins on its surface that allow the transgene to be successfully delivered to the target cell type. Transgene delivery is quantified by titration in integrated units (IU) or transduction units (TU), based on the number of integrated copies and the percentage of target cells expressing the transgene at a specific titration, respectively.

[0029] The term "functional viral vector" refers to a viral vector that has the ability to transduce desired target cells and has all desired exogenous functional proteins correctly expressed in desired amounts.

[0030] The term "non-functional viral vector" refers to a viral vector that does not have the ability to transduce desired target cells and / or does not have all desired exogenous functional proteins correctly expressed in desired amounts.

[0031] The term "physical titer" refers to the total concentration of viral vectors, including functional and non-functional viral vectors. The total concentration of viral vectors is quantified by genome quantification or by the concentration of viral structural proteins.

[0032] The term "blind protein" refers to a glycoprotein or cell surface protein that normally binds to a specific target on a cell, but after amino acid modification (e.g., amino acid substitution, deletion and / or insertion) of the glycoprotein or cell surface protein, it no longer binds to the target or binds to the target with significantly reduced affinity.

[0033] Other definitions appear throughout the context of this disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and not restrictive. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, the present specification (including definitions) will prevail.

[0034] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Figure 1 is a schematic diagram of the LVV production method described herein, wherein a plasmid delivered to a production cell is capable of producing a viral vector core. These viral vector cores are then chemically conjugated to one or more exogenous functional proteins to produce a complete, functional viral vector.

[0037] Figure 2 is a detailed schematic diagram of an exemplary LVV production method described herein, in which plasmids are delivered to producer cells to produce viral vector cores. These viral vector cores are then chemically conjugated to one or more exogenous functional proteins to produce a complete, functional viral vector. The schematic depicts exemplary plasmid transfection, nuclear localization, transcription, transport to the cell membrane, assembly, and budding of a viral vector core, followed by chemical conjugation of one or more exogenous functional proteins to the surface of the viral vector core.

[0038] Figure 3 Schematic diagram of an exemplary method for producing therapeutic next-generation LVVs. An exemplary seed culture is used to grow a production cell culture, which is then transfected with a plasmid to produce a viral vector core with a therapeutic transgene. The viral vector core is then purified and chemically conjugated to exogenous functional proteins (in this figure, a CD3-targeting scFv and two exogenous immune-modifying proteins) in a cell-free process and formulated for therapeutic administration. Detailed Description of the Invention

[0040] Provided herein is a method for producing a viral vector having an exogenous functional protein on its surface. The method described herein comprises the following steps: contacting a viral vector core comprising a first chemical handle on its surface with an exogenous functional protein comprising a second chemical handle, and covalently conjugating the first chemical handle to the second chemical handle to produce a viral vector having an exogenous functional protein on its surface. Click chemistry using chemical handles has been reviewed in the following literature: for example, Devaraj et al., Chemical Reviews special issue on Click Chemistry 121(12):6697-7248, 2021; and for example, Chandrasekaran et al., Click Reactions in Organic Synthesis, Wiley 2016; and Rutjes et al., Science of Synthesis: Click Chemistry, Thieme, 2021.

[0041] In some embodiments, the exogenous functional protein comprises an exogenous targeting protein. In some embodiments, the exogenous functional protein comprises an exogenous immune evasion protein. In some embodiments, the exogenous functional protein comprises a combination of an exogenous immune evasion protein, an exogenous targeting protein and an exogenous polyethylene glycol. In some embodiments, these methods may further include the step of producing a viral vector core. For example, producing a viral vector core may include the step of culturing production cells (such as HEK293 cells, HEK293T cells and other cell types) in a culture medium that supports the growth of these cells and the production of viral vectors. Next, a plasmid complex encoding a subset of the properties of the desired viral vector composition is introduced into the culture medium to transfect the production cells, and then the transfected production cells produce the viral vector core. In some embodiments, the production cells are engineered to stably express nucleic acids encoding a subset of the properties of the desired viral vector composition that forms the viral vector core. In some embodiments, the subset of the properties of the desired viral vector composition that forms the viral vector core comprises a viral structural protein encoded by a gagpol sequence, a regulatory protein encoded by a rev sequence, a transgenic target encoded by a specific nucleic acid sequence of the protein or multiple proteins (e.g., one or more therapeutic proteins). In some embodiments, the viral vector core further comprises a glycoprotein that mediates internalization by fusion with the target cell membrane or by fusion with the endosomal membrane. In some embodiments, a first chemical handle (such as an azido sugar) is introduced into the culture medium and the production cell integrates it into the cell component. In some embodiments, these components include glycoproteins that are located on the surface of the production cell. When the viral vector core buds from the production cell, the surface of the viral vector core includes a glycoprotein that has been integrated with the first chemical handle on its surface (for example, the glycoprotein includes a first chemical handle or multiple copies of the first chemical handle, or the glycoprotein has a covalently linked first chemical handle or multiple copies of the covalently linked first chemical handle). Some embodiments of these methods also include the step of producing an exogenous functional protein comprising a second chemical handle. For example, the second chemical handle may include an alkyne moiety (DBCO, BCN, etc.). Click chemistry reactions and suitable chemical groups for click chemistry reactions that may comprise the first and second handles are well known to those skilled in the art and include, but are not limited to, terminal alkyne or alkyne groups, azide or azide groups, strained alkyne or alkyne groups, tetrazine groups including substituted forms thereof, dienes, dienophiles, alkoxyamine or alkoxyamino groups, carbonyl groups, phosphines, hydrazides, hydrazines, hydrazones, thiols, and olefin moieties. In non-limiting embodiments, the click reaction does not require a catalyst. In non-limiting embodiments, the click reaction does not require copper ions, for example, proceeding at substantially the same rate in the absence of copper ions as in the presence of copper ions.

[0042] When a chemical handle can participate in a click chemistry reaction, the chemical substance may be referred to as a click handle in this article. For example, a strained cyclic olefin, such as cyclooctene, is a click handle because it can participate in a strain-promoted cycloaddition, which is a non-limiting example of a click chemistry reaction. Typically, a click chemistry reaction requires at least two molecules (click pairs) comprising click handles that can react with each other. Within the click pair, the first click handle can provide a nucleophilic (Nu) group, and the second click handle can provide an electrophilic (E) group that can react with the Nu group of the first click handle (and vice versa). Such mutually reactive click pairs are sometimes referred to as paired click handles in this article. For example, tetrazine is a paired click handle for cyclooctene or any other olefin. In this way, a click pair can be selected and provided on separate entities (entity A and entity B) so that providing both entity A and entity B will cause the click handles of the click pair to react.

[0043] Described herein are exemplary click handles suitable for use in accordance with aspects of the present disclosure. In some embodiments, the click handles of a click pair may include (i) a terminal alkyne and an azide; (ii) a strained alkyne and an azide; (iii) a diene and a dienophile; (iv) an alkoxyamine and a carbonyl; (v) a phosphine and an azide; (vi) a hydrazide and a carbonyl; (vii) a thiol and an alkene; (viii) a cyclooctyne (e.g., a strained cyclooctyne (SCO), such as difluorooctyne (DIFO), dibenzylcyclooctyne (DIBO), biarylazacyclooctynone (BARAC), or bicyclo[6.1.0]nonyne (BCN)) and an azide; or (viii) a cyclooctene (e.g., transcyclooctene (TCO)) and a tetrazine. Additional click handles are described in Becer et al., Angew. Chem. Int. ed. 2009; 48: 4900-4908, and International Publication No. WO 2013 / 003555 and references therein, which are incorporated herein by reference for click handles and methods.

[0044] When both members of a click pair are present, the click handle will react and a click conjugate feature (or click feature) may be formed. In some embodiments, the click feature has the structure of a click feature described herein.

[0045] The click handle can further comprise a linker or linker region. Exemplary linker regions are described herein. When a linker region is present, the click feature can be present within the residual linker formed as a result of the click reaction.

[0046] In some embodiments, the click feature comprises a cyclic moiety, e.g., a heterocycle, such as a triazole, e.g., a disubstituted triazole, or a cycloadduct. In some embodiments, the click feature comprises a cyclic olefin, e.g., cyclohexene, an alkyl sulfide, a dihydropyrazine, e.g., 1,2-dihydropyrazine, a diazole, or a sulfur-containing ring, e.g., a thiopyran.

[0047] In some embodiments, the click pair comprises a first click handle and a second click handle that reacts with the first click handle. Exemplary click handles include, for example, click chemistry groups, such as one of the click pairs selected from the group consisting of: a Huisgen 1,3-dipolar cycloaddition reaction between an alkynyl group and an azide group to form a triazole-containing linker; a copper(I)-catalyzed azide-alkyne cycloaddition reaction between an alkynyl group and an azide group to form a triazole-containing linker; a strain-promoted azide-alkyne cycloaddition (SPAAC) reaction between a strained alkynyl group (e.g., present in a strained cyclooctynyl group such as DIFO, DIBO, or others described herein) and an azide group to form a triazole-containing linker; a strain-promoted alkyne-nitrone cycloaddition (SPANC) reaction between a strained alkynyl group (e.g., present in a strained cyclooctynyl group such as DIFO, DIBO, or others described herein) and a nitrone group to form an isoxazoline-containing linker; a 4-mercapto-1,4-dihydro ... Diels-Alder reactions (including reverse electron demand Diels-Alder reactions) between a diene having a π-electron system (e.g., an optionally substituted 1,3-unsaturated compound such as optionally substituted 1,3-butadiene, 1-methoxy-3-trimethylsilyloxy-1,3-butadiene, cyclopentadiene, cyclohexadiene, or furan) and a dienophile or isodienol having a 2π-electron system (e.g., an optionally substituted alkenyl or an optionally substituted alkynyl); ring-opening reactions with nucleophiles and strained heterocyclic electrophiles; splint ligation reactions with phosphorothioate and iodine groups; Staudinger reactions with phosphines and azides; and reductive amination reactions with aldehyde and amino groups.

[0048] In some embodiments, the click feature is formed or capable of forming by a cycloaddition (e.g., a 1,3-dipolar cycloaddition or a heteroDiels-Alder cycloaddition), a nucleophilic ring opening (e.g., ring opening of strained heterocyclic electrophiles such as aziridines, epoxides, cyclic sulfates, aziridinium ions, and episulfonium ions), a non-aldol carbonyl chemistry (e.g., formation of ureas, thioureas, hydrazones, oxime ethers, amides, or aromatic heterocycles), or an addition to a carbon-carbon multiple bond (e.g., epoxidation, aziridination, dihydroxylation, nitrosyl halide addition, nitrobenzoyl halide addition, or Michael addition). Examples of these types of click reactions are described in more detail in Hein et al., Pharm. Res. 2008 October; 25(10): 2216-2230, which is incorporated herein by reference in its entirety. In an embodiment, the click reaction is a metal-free [3+2] cycloaddition reaction, a Diels-Alder reaction, or a thiol-olefin free radical reaction. Examples of these types of click reactions are described in more detail in Becer et al., Angew. Chem. Int. Ed. 2009, 48, 4900-4908, which is incorporated herein by reference in its entirety.

[0049] In one embodiment, the click feature is an alkyne / azide click feature (e.g., wherein the alkyne is a cyclooctyne, an activated alkyne, or an electron-deficient alkyne), for example, the click feature comprises a triazole, such as a 1,2,3-triazole and / or a disubstituted triazole. In one embodiment, the click feature is a diene / dienophile click feature (e.g., wherein the dienophile comprises an olefin moiety), for example, the click feature comprises a cycloolefin, such as a disubstituted olefin. In an embodiment, the click feature is a tetrazine / olefin click feature, for example, the click feature comprises a dihydropyrazine, such as 1,2-dihydropyrazine. In an embodiment, the click feature is a tetrazole / olefin click feature, for example, the click feature comprises a diazole. In an embodiment, the click feature is a dithioester / diene click feature, for example, the click feature comprises a sulfur-containing ring, such as a tetrahydrothiophene, such as a disubstituted tetrahydrothiophene. In an embodiment, the click feature is a dithioester / diene feature, for example, the click feature comprises a sulfur-containing ring, such as a thiopyran. In embodiments, the click feature is a thiol / olefin click feature, eg, the click feature comprises an alkyl sulfide.

[0050] An example of a cycloaddition reaction is the Huisgen 1,3-dipolar cycloaddition of a dipolarophile and a 1,3 dipolar component, which produces a five-membered (hetero) ring. Examples of dipolarophiles are alkenes, alkynes, and molecules with related heteroatom functional groups (such as carbonyl and nitrile). Specifically, another example is the 2+3 cycloaddition of alkyl azide and acetylene. Other cycloaddition reactions include the Diels-Alder reaction of conjugated dienes and dienophiles (such as alkynes or alkenes). Examples of cycloaddition reactions are described in, for example, U.S. Patent No. 9,517,291, which is incorporated herein by reference in its entirety.

[0051] Other examples of click reactions include hydrosilylation of H-Si and simple non-activated vinyl compounds, carbamate formation from alcohols and isocyanates, Menshutkin reactions of tertiary amines with alkyl iodides or alkyl triflates, Michael additions (e.g., very efficient maleimide-thiol reactions), atom transfer radical additions between -SO2Cl and olefins (e.g., including carbon-carbon double bonds), metathesis reactions, Staudinger reactions of phosphines with alkyl azides, oxidative coupling of thiols, nucleophilic substitutions (especially of small strained rings such as epoxides and aziridine compounds), carbonyl chemistry (e.g., urea formation), and addition reactions of carbon-carbon double bonds (e.g., dihydroxylation). Thus, the attached functional group can be selected from acetylene bonds, azides, nitrile groups, acetylenes, amino groups, phosphino groups. Click chemistry reactions can result in the addition of functional groups selected from amino groups, primary amino groups, hydroxyl groups, sulfonates, benzotriazoles, bromides, chlorides, chloroformates, trimethylsilanes, phosphonium bromides, and the like.

[0052] Further click reactions, click handles, and process conditions are described elsewhere, for example, in Kolb et al., Angew. Chem. Int. Ed. 2001;40: 2004-2021; Evans, et al., Austral. J. Chem. 2007;60:384-395; Carlmark et al., Chem. Soc. Rev. 2009;38:352-362; U.S. Patent No. 8,912,323; and Lahann, Click Chemistry for Biotechnology and Materials Science, 2009, John Wiley & Sons Ltd, ISBN 978-0-470-69970-6, each of which is incorporated herein by reference in its entirety.

[0053] In some embodiments, the first and second chemical handles may comprise an azide or an alkyne. In some embodiments, the first and second chemical handles may comprise a maleimide or a thiol. In some embodiments, the thiol is the side chain of the amino acid cysteine. In some embodiments, the first and second chemical handles may comprise a diene or a dienophile. In some embodiments, the diene is a tetrazine. In some embodiments, the dienophile is an olefin. In some embodiments, the dienophile is trans-cyclooctene. In some embodiments, the first and second chemical handles may comprise one of protein A, protein G, protein AG, and an antibody. In some embodiments, the first handle and the second handle may comprise a SNAP tag and a benzylguanine derivative. In some embodiments, the first handle and the second handle may comprise a CLIP tag and a benzylcytosine derivative.

[0054] In some embodiments, the first and second chemical handles may comprise biotin, monomeric streptavidin, monomeric streptavidin 2, intein, SunTag, Isopeptag, SpyTag, SpyCatcher, SnoopTag, SnoopTagJr, SnoopCatcher, DogTag, DogCatcher, glutathione-S-transferase, CLIP, HA, FLAG, and HiBiT.

[0055] In some embodiments, the first and second chemical handles do not comprise BCN, Protein A, Protein G, Protein AG, Spytag, or Spycatcher.

[0056] In some embodiments, the first and second chemical handles do not comprise a SNAP tag, a benzylguanine, an alkyne or alkynyl group, an azide or azido group, and / or a phosphine or phosphine group.

[0057] In some embodiments, the first and second chemical handles do not comprise a cyclooctene or cyclooctenyl group, a cyclooctyne or cyclooctynyl group, a tetrazine or tetrazinyl group (e.g., 1,2,4,5-tetrazine or 1,2,4,5-tetrazinyl), a tetrazole or tetrazolyl group (e.g., 2-tetrazole or 2-tetrazolyl), a phosphine or phosphino group (e.g., triphenylphosphine or triphenylphosphino), an aldehyde, a ketone, an alkanoyl group (e.g., -C(O)R, wherein R is an organic substituent such as an alkyl or aliphatic group), a formyl group (e.g., -C(O)H), a hydrazine or hydrazinyl group (e.g., -NHNH2), and / or a hydroxylamine or hydroxylamino group (e.g., -NH2).

[0058] In some embodiments, the first chemical handle is embedded in the lipid bilayer membrane of the production cell or in the lipid bilayer membrane of the vector.

[0059] In some embodiments, the first chemical handle is not embedded in the lipid bilayer membrane of the production cell or in the lipid bilayer membrane of the vector.

[0060] In some embodiments, the first chemical handle is embedded in a glycoprotein on the surface of the production cell and the surface of the carrier membrane.

[0061] In some embodiments, the first chemical handle is not embedded in a glycoprotein on the surface of the production cell and the surface of the carrier membrane.

[0062] The produced exogenous targeting protein comprising a second chemical handle can be optionally purified and prepared in a specific stoichiometry for conjugation to the first chemical handle of the viral vector core. The conjugation reaction can be carried out, for example, under physiological conditions (e.g., at a temperature of 30°C-40°C (e.g., 37°C) or at a pH of about 6.5 to 7.5 (e.g., pH 7.3)). Unlike other methods for genetically modifying proteins on the viral envelope, this method introduces as few as 3 atoms at each conjugation site and is expected to have minimal negative physiological effects on the viral vector. Additional non-limiting aspects of these methods are described herein.

[0063] A non-limiting example of preparing an exogenous targeting molecule with a chemical handle includes first digesting the antibody with IdeS, IdeZ, or papain. Next, a molecule comprising a maleimide and an additional chemical handle is reacted with the reduced antibody fragment.

[0064] Also provided herein are viral vectors produced by these methods, populations of viral vectors produced by these methods, compositions (e.g., pharmaceutical compositions) comprising any of the viral vectors or populations of viral vectors described herein, and kits comprising any of the compositions described herein.

[0065] Also provided herein are methods of administering any of the viral vectors described herein or any of the compositions (eg, pharmaceutical compositions described herein) to a subject (eg, a human) in need thereof.

[0066] Methods for producing viral vector cores

[0067] The production of viral vector cores begins by culturing production cells in a liquid culture medium containing a small molecule that is capable of metabolically inserting a first chemical handle into a protein (e.g., a glycoprotein) in the plasma membrane of the production cell. Several production cell types for producing viral vectors are known in the art. In some embodiments, the production cell is a HEK293 cell. In some embodiments, the production cell is a HEK293T cell. Several liquid culture media for producing viral vectors are known in the art. In some embodiments, the liquid culture medium is LVMAX medium (Thermo Fisher). Several small molecules capable of metabolically inserting a first chemical handle into a protein (e.g., a glycoprotein) or a lipid bilayer membrane in the plasma membrane of the production cell are known in the art. In some embodiments, the small molecule is N-α-azidoacetylmannosamine (Ac4ManNAz), which is fully acetylated with an azido sugar.

[0068] This method can be used to produce viral vector cores derived from many viral vector types. The examples herein describe viral vector cores and viral vectors, wherein the viral vector is a lentiviral vector. In some embodiments, the viral vector can be a retroviral vector, an alphaviral vector, a filoviral vector, a rhabdoviral vector, a Moloney murine leukemia vector, or a respiratory syncytial virus. In some embodiments, the viral vector is an enveloped viral vector. In some embodiments, the viral vector is an extracellular vesicle.

[0069] Liquid culture media may optionally be supplemented with glucose or other common additives known in the art to support general cell culture.

[0070] The transfection complex can be introduced into the cell culture by a transient transfection method known in the art, and the viral vector core is produced from the cultured production cells. In this method, a plasmid is formed by combining a plasmid with a complexing agent in a specific ratio in a separate liquid medium. In some embodiments, the plasmid contained in the complex comprises a plasmid encoding the viral structural protein gagpol, a plasmid encoding the regulatory protein rev, and a plasmid encoding the target transgene (referred to in the art as a third generation lentiviral vector system). The third generation lentiviral vector system is described in, for example, Dull et al., J. Virol. 72 (11): 8463-71, 1998 and, for example, U.S. Patent No. 8,329,462 B2. In some embodiments, the plasmid encoding the target transgene contains a nucleic acid encoding one or more therapeutic proteins.

[0071] In some embodiments, the complex further comprises a plasmid encoding a glycoprotein. In this embodiment, the glycoprotein encoded by the glycoprotein plasmid can fuse the viral vector to the cell membrane or endosomal membrane, but lacks tropism for human cells, which is due to a natural lack of tropism for human cells or due to a mutation that removes the tropism of the glycoprotein that originally had tropism for human cells. In some embodiments, the glycoprotein is a vesicular stomatitis virus glycoprotein (VSV-G), a Sindbis virus glycoprotein, a murine leukemia virus glycoprotein, a Moloney murine leukemia virus glycoprotein, a Cocal virus glycoprotein, a foamy virus glycoprotein, a retrovirus family glycoprotein, a GP64 glycoprotein, a rhabdovirus family glycoprotein, a filovirus family glycoprotein, or a paramyxovirus family glycoprotein. In some embodiments, the glycoprotein is blind to its natural receptor. For example, blind VSV-G is described in US20200216502 A1, blind Sindbis virus is described in, for example, US Patent No. 7,429,481 B2, and blind Paramyxovirus is described in, for example, US20220064674 A1.

[0072] In some embodiments, polyethyleneimine (PEI) is used as a complexing agent.Additional transfection agents and transfection methods are known in the art.

[0073] When the production cells reach a target density of 3E6 (range 5E5 to 5E7) at the production volume and container, the plasmid complex is added to the cell culture. In some embodiments, 24 hours after addition (range 12h-48h), the liquid culture medium is supplemented with a transfection enhancing molecule, such as sodium butyrate. Additional transfection enhancing molecules are known in the art.

[0074] In some embodiments, viral vector cores can be produced from producer cells cultured as described above by stably introducing one or more genes encoded by the above-described plasmids into the producer cells. In some embodiments, these engineered producer cells can produce viral vector cores without the introduction of a plasmid complex. In some embodiments, these engineered producer cells can produce viral vector cores after the introduction of a plasmid complex comprising a plasmid assembly as described above for transient transfection methods.

[0075] These methods result in the production of a viral vector core that comprises a first chemical handle (eg, one or more copies of the first chemical handle) on its surface.

[0076] 48 hours after the complex is added (range 24h-96h), a step of purifying or isolating the viral vector core by centrifugation, purification, or chromatography can be performed. The purification step removes cells and cell debris. In some embodiments, a chromatography step is performed to remove protein and DNA impurities. In some embodiments, a concentration step is performed by centrifugation or filtration. In some embodiments, a buffer exchange is performed. Several concentration, filtration, and buffer exchange methods are known in the art.

[0077] At this stage of the process, the viral core has been produced, purified, and concentrated.

[0078] A viral vector core having a first chemical handle covalently attached to its surface

[0079] The viral vector core is characterized by physical titer and the presence of the first chemical handle. The physical titer assays used are ELISA for p24 lentiviral structural proteins and PCR for vector genome quantification. These assays and methods for calculating titer from the assays are known in the art.

[0080] The process of producing viral vector cores can be characterized by applying these physical titer determinations at or during several steps in the process.Before steps involving purification or isolation of the viral vector cores by cleanup and / or chromatography, the liquid culture medium is sampled and the physical titer determined.

[0081] Exogenous functional protein

[0082] Exogenous targeting proteins, exogenous immunomodulatory proteins, and polymers are prepared and covalently attached to the surface of the viral vector core by chemical conjugation. These exogenous functional proteins and polymers give the viral vector unique functional properties.

[0083] An exogenous functional protein (e.g., an exogenous targeting protein and / or an exogenous immune-modifying protein) and / or a polymer (e.g., polyethylene glycol) is synthesized with a second chemical handle that reacts with the first chemical handle present on the viral vector core. Several chemical treatments and methods for synthesizing proteins containing them are known in the art. In some embodiments, the second chemical handle is dibenzocyclooctyne (DBCO) or bicyclo[6.1.0]nonyne (BCN). In some embodiments, the second chemical handle is not BCN. A non-limiting example of preparing an exogenous targeting molecule with a chemical handle involves first digesting an antibody with IdeS, IdeZ, or papain. Next, a molecule containing a maleimide and an additional chemical handle is reacted with a reduced antibody fragment.

[0084] In some embodiments, the exogenous targeting protein comprises a second chemical handle synthesized onto an antibody, antibody fragment, single chain variable fragment (scFv), nanobody, darpin, VHH, aptamer, or lectin binding domain that binds to a surface molecule unique to the target cell (e.g., T cell or B cell). T cell targeting molecules are described, for example, in U.S. Patent No. 8,784,821 B1. Non-limiting examples of antibody fragments include F(ab')2, Fab, Fab', bispecific Fab2, bispecific antibodies, trispecific Fab3, minibodies, scFv-Fc, and diabodies.

[0085] The polymer can optionally be conjugated to the viral vector core. In some embodiments, polyethylene glycol (PEG)-DBCO is conjugated to the viral vector core. In some embodiments, PEG has an average molecular weight of about 100 g / mol to about 20,000 g / mol. Methods for chemically conjugating PEG to viral vectors are known in the art. See, for example, US 6,399,385 B1.

[0086] These exogenous functional proteins with a second chemical handle and polymers can be mixed in a solution at a defined stoichiometric ratio.

[0087] Methods for conjugating viral vector cores to functionally defined proteins and molecules

[0088] In this method for preparing a viral vector, a viral vector core comprising a first chemical handle (e.g., one or more copies of the first chemical handle) on its surface is contacted with an exogenous targeting protein, an exogenous immune-modifying protein, and / or a polymer comprising a second chemical handle. The first chemical handle is contacted with the second chemical handle, and the exogenous functional protein and / or polymer is covalently conjugated to the viral vector core to produce a viral vector.

[0089] In some embodiments, an exogenous functional protein or polymer comprising a second chemical handle is introduced into a liquid culture medium in which a viral vector core has already been produced by a producer cell. In some embodiments, prior to the introduction of the exogenous functional protein and / or polymer comprising the second chemical handle, the liquid culture medium has undergone a step comprising purification or isolation of the viral vector core by purification and / or chromatography. In some embodiments, prior to the introduction of the exogenous functional protein and / or polymer comprising the second chemical handle, the liquid culture medium has undergone a concentration and / or buffer exchange step.

[0090] The exogenous functional protein and / or polymer is introduced under physiological conditions (pH about 6.5 to about 7.5; temperature about 30° C. to about 40° C.) for 1 hour (about 30 min to about 24 hours). In some embodiments, the liquid culture medium is mixed.

[0091] After this step, a viral vector is produced having one or more exogenous targeting proteins, one or more exogenous immune modifying proteins, and / or one or more polymers covalently attached to its surface or a component on its surface via a conjugation feature. In some embodiments, the conjugation feature comprises a triazole bond. In some embodiments, the conjugation feature comprises a 1-2-3 triazole bond. In some embodiments, the conjugation feature is only a 1-2-3 triazole bond.

[0092] In some embodiments, the conjugation feature comprises a thioether bond. In some embodiments, the conjugation feature comprises a disulfide bond.

[0093] In some embodiments, the conjugation feature comprises a [4+2] cycloadduct.

[0094] In some embodiments, a next step is performed that includes purification of the viral vector by chromatography to remove unreacted substrate. Depending on the desired application, additional processing steps may be performed.

[0095] Viral vectors with exogenous functional properties produced by chemical conjugation

[0096] Viral vectors have one or more exogenous targeting proteins, one or more exogenous immune-modifying proteins, and / or one or more polymers covalently attached to their surface via conjugation features, which possess several unique functional and physical properties.

[0097] Most importantly, producing viral vectors with a single exogenous targeting protein while maintaining high functional titers has been a major challenge for the gene therapy industry. Viral vectors with other exogenous functional proteins (such as exogenous immune-modifying proteins) have shown beneficial properties; however, current industry standard production methods do not allow the production of viral vectors with multiple exogenous functional proteins. Viral vectors with multiple exogenous functional proteins at titers relevant for therapeutic use have never been described before. This limits the gene therapy industry's ability to prepare viral vectors with more advanced functions and restricts the industry's ability to expand this format to other therapeutic areas.

[0098] Notably, the methods described herein yield pharmaceutical compositions containing viral vectors harboring exogenous functional proteins and / or polymers, demonstrating significantly higher functional titers than viral vectors harboring exogenous functional proteins produced without the methods described herein. This improves delivery efficiency, potentially opening up important therapeutic areas for these therapies that are currently inaccessible to the gene therapy industry.

[0099] The physical titers between the methods of producing viral vectors described herein and previous methods of producing viral vectors are similar. Thus, the methods provided herein provide a viral vector population that is significantly enriched for functional viral vectors.

[0100] Thus, the viral vectors produced by the methods described herein represent more potent pharmaceutical compositions and pharmaceutical compositions with superior functionality than any previously described pharmaceutical compositions.

[0101] Functional titer is assessed in vitro using titration methods known in the art. In short, a solution containing the viral vector is serially diluted and the recipient cell line or primary cells are transduced. Transduction is assessed at each dilution point and IU and TU / mL are calculated. In some embodiments, Jurkat cell lines and primary T cells are used for titration. In some embodiments, HEK293 cells engineered to overexpress the target of an exogenous targeting protein are used for titration.

[0102] The ratio of functional titer to physical titer is a measure of which parts of the viral vector are functional.

[0103] Pharmaceutical compositions as described herein can be delivered intravenously to mice and non-human primates. In these animals, viral vectors transduce target cells in vivo and evade the recognition of the immune system due to exogenous immune escape proteins and exogenous PEG. After interacting with the cell types in the animal, the exogenous targeting protein mediates the binding to the specific cell receptors on the target cells. In some embodiments, after interacting with alternative cell types, alternative exogenous targeting molecules mediate the binding to other specific cell receptors. In some embodiments, the alternative exogenous functional protein triggers specific interactions or inhibits specific interactions in vivo. After the viral vector binds to the target cell, it is mediated by glycoprotein, and the viral vector fuses to the cell membrane or is internalized into the endosome and fuses with the endosomal membrane. This starts the fully characterized viral transduction process, resulting in the insertion of transgenic into the target cells in the animal. In some embodiments, transgenic encoding green fluorescent protein (GFP).

[0104] Pharmaceutical compositions and clinical applications

[0105] The use of viral vectors conjugated to exogenous targeting proteins can target specific cell types in humans and modify their genes, which will change the current practices in many medical fields. In some embodiments, viral vectors chemically conjugated to exogenous targeting proteins that target T cells in vivo and transduce them with therapeutic proteins can make it possible to produce therapeutic cancer-targeted cells directly in human patients. This will change the current medical practice of oncology and many other serious diseases (including autoimmune diseases and other diseases). Today, several cancer-targeted cell therapies have been approved, which have efficacy in patients with severe and terminal blood cancers. However, this field struggles because these therapies are manufactured in a patient-specific manner, which limits the chances of obtaining these therapies and causes many patients to die while waiting for manufacturing. In addition, the production costs of these drugs are very high, and the patients who obtain drugs outside specific regions are extremely limited.

[0106] The viral vectors described herein can create new and transformative means for treating these same cancer patients and other patients. In some embodiments, the pharmaceutical compositions described herein with high functional titers can directly generate cancer-targeted cells and other therapeutic cells in vivo and bypass the challenging manufacturing and treatment required for today's advanced cell therapies. In some embodiments, the viral vectors described herein can contain exogenous targeting proteins that target the viral vector to any cell in the body and deliver nucleic acids encoding one or more therapeutic proteins. In some embodiments, these genes are integrated genes that result in persistent expression or non-integrated genes for transient expression.

[0107] This method enables the production of viral vectors with one or more exogenous functional proteins in a population enriched for functional viral vectors. These methods and viral vectors, viral vector populations and pharmaceutical compositions may lead to the cure of patients with many prevalent diseases that cannot be treated by current state-of-the-art viral vector therapies.

[0108] Provided herein are pharmaceutical compositions comprising a population of viral vectors produced by any of the methods described herein. In some embodiments, the pharmaceutical composition comprises a functional titer of at least about 1E6 TU / mL, at least about 1E7 TU / mL, at least about 1E8 TU / mL, at least about 1E9 TU / mL, or at least about 1E10 TU / mL. In some embodiments, the pharmaceutical composition comprises a functional titer to physical titer ratio of at least about 1:10, at least about 1:100, or at least about 1:1000.

[0109] Kits for producing viral vectors

[0110] The present disclosure encompasses kits for practicing the methods provided herein. Such kits typically comprise two or more components required for producing viral vectors. Components of the kit include, but are not limited to, one or more of the following: compounds, reagents, containers, equipment, and instructions for use of the kit. Thus, the methods described herein can be performed using the prepackaged kits provided herein.

[0111] In some embodiments, a kit for producing a viral vector is provided. The kit comprises: one or more exogenous functional proteins and / or polymers comprising a second chemical handle. In some embodiments, the kit further comprises one or more culture medium components each of: a liquid culture medium, a growth supplement, a small molecule capable of metabolically inserting the first chemical handle into the producer cells, and a transduction enhancer. In some embodiments, the kit further comprises producer cells. In some embodiments, the kit further comprises all of the aforementioned components.

[0112] In some embodiments, instructions for using the kit to produce viral vectors are provided. The instructions may include one or more protocols for: liquid culture medium formulation; culture conditions, such as time, temperature, and / or gas incubation concentration; transfection protocol; harvesting protocol; and protocols for identifying viral vectors. The kit may also include materials for performing the methods of the invention, including culture plates, well plates, flasks, chromatography columns, filters, culture dishes, and the like.

[0113] Also provided herein are kits comprising a population of viral vector cores comprising a first chemical handle and one or more exogenous functional proteins comprising a second chemical handle and / or one or more PEGs comprising a second chemical handle. In some embodiments, the kit further comprises instructions for performing a chemical conjugation reaction between the first and second chemical handles.

[0114] Also provided herein are kits comprising a composition (eg, a pharmaceutical composition) comprising any of the viral vectors described herein (eg, a viral vector produced by any of the methods described herein).

[0115] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims. Example

[0116] Example 1. Production of Viral Cores with Azide Handles

[0117] In Example 1, a viral vector core is produced with a first chemical handle. In this example, the first chemical handle is an azide handle.

[0118] Viral vector core production followed the LVMAX Viral Production System manufacturer's guidelines (see LVMAX Viral Production System Manual on the ThermoFisher website), which are summarized below, with modifications noted.

[0119] HEK293 cells were seeded into 125 mL shake flasks in liquid culture medium (LVMAX medium, Thermo Fisher). Ac4ManNAz was added to the liquid culture medium at a concentration of 50 µM. Cells were counted daily using a ViCell counter. Transfection was performed when the cell density reached 3 million cells / mL.

[0120] The transfection complex was formed by combining gagpol, rev, GFP, and glycoprotein plasmids. 1.5 μg of total plasmid DNA / mL was added to OptiMem medium (Thermo Fisher) to achieve the specified ratio of the plasmids. PEIpro (Polyplus) was added to the OptiMem medium at 1.5 μg / mL. The total volume of the complexation reaction was 10% of the total culture volume. The complexation reaction took 10 minutes, after which the complexation mixture was added to the liquid culture medium.

[0121] 24 hours after transfection, 1 mM sodium butyrate (Sigma) was added to the liquid culture medium. 48 hours after transfection, the viral vector was harvested and purified through a 0.45 μm filter (Pall). The viral vector was purified using an anion exchange chromatography column (Pall). Alternatively, the vector was concentrated by ultracentrifugation and resuspended in buffer.

[0122] Samples were collected before, after, and after chromatography (or before and after ultracentrifugation). Physical and functional titers of the samples were determined. The physical titer before purification was approximately 1000 ng / mL (approximately 1.25E10 particles / mL). After purification, a loss of less than 25% in physical titer was observed. A further loss of less than 30% was observed after chromatography. The functional titer of all samples was below the limit of detection. This was expected because the viral vector core lacks the ability to transduce cells.

[0123] Host cell protein and host cell DNA were determined from these three samples using commercially available kits (Thermo Fisher). A reduction in host cell protein was observed after cleanup and chromatography.

[0124] Example 2. Production of exogenous targeting proteins with alkyne handles

[0125] In Example 2, an exogenous targeting protein with a second chemical handle is produced. In this example, the exogenous targeting protein is a F(ab') that targets a protein on the surface of T cells (e.g., CD3). In this example, the second chemical handle is an alkyne handle.

[0126] An exogenous targeting protein with an alkyne handle is prepared by first digesting a CD3 antibody with the enzyme IdeS. Next, a DBCO-PEG-maleimide molecule reacts with a thiol group on the F(ab') to form an exogenous functional protein containing a second chemical handle. Optionally, purification is performed using reverse-phase hPLC or size exclusion chromatography.

[0127] The resulting protein was assayed by SDS Page to confirm that protein bands were observed within the expected size range.

[0128] Example 3. Production of exogenous immune-modifying proteins with alkyne handles

[0129] In Example 3, an exogenous immune-modifying protein with a second chemical handle is produced. In this example, the exogenous immune-modifying protein is the extracellular domain of CD47. In this example, the second chemical handle is an alkyne handle. CD47 is an immune-modifying protein known in the art for use in viral vectors. See, for example, U.S. Patent No. 9,050,269 B2.

[0130] CD47 protein is produced using protein production methods known in the art.

[0131] DBCO-PEG5-NHS reacts with the NH2 group on the protein to form an exogenous functional protein containing a second chemical handle. Optionally, purification is performed using reverse phase hPLC or size exclusion chromatography.

[0132] The resulting proteins were assayed by SDS-PAGE to confirm that bands were observed within the expected size range.

[0133] Example 4. Production of viral vectors with exogenous immune-modifying proteins and / or exogenous targeting proteins.

[0134] In Example 4, a viral vector was produced, which contained a CD47 exogenous immune modifying protein or a CD3-targeting scFv exogenous targeting molecule, or both, and a nucleic acid encoding a GFP protein on its surface.

[0135] The exogenous functional proteins of Examples 2 and 3 were introduced individually or together into the culture medium containing the viral vector core of Example 1. The reaction was carried out in an incubator at 37° C. and a physiological pH of 7.3 for 1 hour.

[0136] As described above, the functional titer of the resulting viral vector was determined on Jurkat cells expressing CD3 and HEK293 cells engineered to overexpress CD3. Relative to viral vectors with the same exogenous functional proteins produced using traditional viral vector production methods, the functional titer of the viral vectors obtained using the methods described herein is expected to be significantly higher.

[0137] This study provides the production of T cell-targeting envelope vectors, immune-modified envelope vectors, and combinations of T cell-targeting and immune-modifying molecules on vectors while maintaining functional titers of >1E8 TU / mL. Taking into account the concentration step in producing this formulation, this is equivalent to an unconcentrated titer of >1E6 TU / mL. Previously, the functional titer of T cell-targeted lentiviral vectors was <1E6 TU / mL, as described, for example, in Bender, RR, et al (2016) Receptor-Targeted Nipah Virus Glycoproteins Improve Cell-Type Selective Gene Delivery and Reveal a Preference for Membrane-Proximal Cell Attachment." PLOS Pathogens 12(6):e1005641. Other viral vectors targeting T cells have been described, for example, in US20220064674 A1. This study provides lentiviral vector preparations with functional titers greater than 10-fold. Noting that the physical titers between this preparation method and those previously described are expected to be similar, it is expected that the viral vector population produced by this method will be enriched in functional viral vectors by approximately 10-fold. Use of this vector is expected to improve delivery efficiency relative to vectors produced without the methods described herein.

[0138] In some embodiments, the viral vector can be further conjugated to PEG, as described, for example, in US Patent No. 6,399,385 Bl. In some embodiments, the viral vectors produced in this and previous examples do not include PEG.

[0139] Example 5. Characterization of exogenous functional proteins present on viral vectors

[0140] In Example 5, the viral vector produced in Example 4 was characterized to confirm the presence of the exogenous functional protein on the viral vector.

[0141] The viral vector of Example 4 was stained with antibodies against the CD3 targeting molecule, CD47, and viral glycoprotein. Next, the fraction of viral vectors expressing the two exogenous surface proteins was counted using flow cytometry. This method was repeated for viral vectors with these exogenous surface proteins produced using conventional methods. As an alternative to flow cytometry, the viral vectors were run on a reducing SDS-page gel and stained with a secondary antibody against the exogenous surface protein to confirm the presence of the expected band size.

[0142] It is expected that a substantial portion of viral vectors produced using the methods herein will express both exogenous surface proteins, whereas double-positive expression of viral vectors produced using traditional production methods employed in the industry will be below the limit of detection.

[0143] Example 6. Viral vector transduction of primary T cells

[0144] In Example 6, the viral vectors produced in Example 4 were characterized for their ability to transduce primary human T cells.

[0145] Primary human T cells (Stem Cell Technologies) were thawed and cultured in 96-well plates according to the manufacturer's protocol. The viral vector of Example 4 was serially diluted and introduced into each well of the 96-well plate in a titration assay. Primary T cells were assayed by flow cytometry for the GFP transgene and integration of the transgene into the T cell genome by qPCR. It is expected that the viral vector will be able to transduce T cells within a certain dilution range as determined by integration unit and transduction unit assays.

[0146] Example 7. Transduction of T cells by viral vectors in mixed cultures

[0147] In Example 7, the viral vectors produced in Example 4 were characterized for their ability to transduce Jurkat T cells when mixed with other cell lines in culture.

[0148] Seven wells of a 96-well plate were seeded in quadruplicate with 10,000 cells of various combinations of Jurkat and HEK293 cells: 100% HEK293, 1:1000 Jurkat:HEK293, 1:200 Jurkat:HEK293, 1:100 Jurkat:HEK293, 1:20 Jurkat:HEK293, 1:10 Jurkat:HEK293, 100% Jurkat.

[0149] Each well was transduced with different dilutions of viral vector. After 3 days, the cells were analyzed for GFP expression by flow cytometry. It was expected that the frequency of GFP+ cells would correspond to the frequency of Jurkat cells in the culture.

[0150] In addition, the T cell transduction ability of the vector produced in Example 4 in peripheral blood mononuclear cell (PBMC) colonies containing both T cells and non-T cells was characterized. Human PBMCs were thawed and cultured in 6-well plates. The vector was added to these cultures and incubated for 12 to 72 hours. The cells were then stained for markers of different cell types and analyzed by flow cytometry. Only the T cell colony was GFP positive, indicating that the vector only transduced T cells.

[0151] Example 8. Viral vectors do not transduce cells that do not express the CD3 surface marker

[0152] In Example 8, off-target transduction of the viral vector produced in Example 4 was characterized.

[0153] A panel of cell lines was cultured according to the supplier's instructions, representing various cell types found in vivo. This panel included the following cell lines: HEK293, CD3-overexpressing HEK293, HeLa, Jurkat, HepG2, and AC16. These cells were stained with an anti-CD3 antibody and analyzed by flow cytometry to confirm CD3 expression or lack thereof. Only the CD3-overexpressing HEK293 and Jurkat lines were confirmed to express CD3.

[0154] Next, the panel of cell lines was transduced with dilutions of the viral vector of Example 4 found to be within the linear range in the titration assay, including one dilution above and one below the linear range. All cells were analyzed by flow cytometry. It was expected that only cells expressing CD3 would be GFP-positive, indicating that the viral vector only transduced cells expressing CD3.

[0155] Example 9. Alternative Methods for Chemically Adding Exogenous Targeting Molecules to Vectors

[0156] Two other methods are used to conjugate exogenous targeting molecules to vectors (cell type-specific delivery by modular envelope design, Strebinger et al. Nature Communications, 2023). Viral vector cores containing VSVg glycoprotein or GP64 glycoprotein are produced. The first handle is introduced into the vector core. In this example, the handle is an engineered protein comprising the protein AG coding sequence between the secretory domain and the transmembrane domain of VSV-G; alternatively, the handle is a SNAP tag. These vectors are conjugated with antibodies (using interactions with the Fc domain of protein AG) or benzylguanine-modified antibodies, respectively. These two vector conjugation methods were prepared with anti-CD3, anti-CD5, and anti-CD46 antibodies. The transduction ability of the resulting vectors to Jurkat cells was determined, and efficient gene delivery to Jurkat cells was observed. These methods were also used to produce CD117-targeted vectors, and transduction of Kasumi-1 cells was observed.

[0157] In this example, several additional vectors were produced using the Protein AG conjugation system with different glycoproteins. These glycoproteins included: blind Cocal virus glycoprotein, blind Rhabdovirus glycoprotein, and GP64 glycoprotein.

[0158] In this example, a vector was also produced whose core was a Moloney murine leukemia virus vector (rather than a lentiviral vector) with a SNAP tag conjugated to anti-CD5 and anti-CD3 antibodies. These vectors efficiently transduced Jurkat cells (Figure 5 and Supplementary Figure 5 in Strebinger et al. 2023).

[0159] Using vectors with a blinded VSVG glycoprotein and a SNAP-tag handle conjugated to anti-CD3, anti-CD28, or anti-CD4 antibodies, these vectors efficiently targeted and transduced T cells in mixed peripheral blood mononuclear cell cultures with GFP and did not transduce non-T cells (Figure 6 of Strebinger et al. 2023).

[0160] A vector assembled with blinded VSVG glycoprotein and CD5 antibody conjugated to a SNAP tag was then produced and injected into mice. Five days after injection, different cell subsets in the spleen were analyzed (Strebinger et al. 2023).

[0161] Example 10. In vivo transduction

[0162] Wild-type mice were injected intravenously with either a viral vector formulation or saline. The viral vector was engineered with an exogenous targeting protein that targets T cells and encodes green fluorescent protein. One and two weeks after administration, blood was drawn from the mice and analyzed by flow cytometry for green fluorescent protein expression in T cells. The animals were also weighed and examined for any adverse health effects of the viral vector. Two weeks after administration, the animals were sacrificed and tissue samples from each organ were harvested and analyzed for off-target GFP expression in these tissues.

[0163] Mice were also injected with an engineered viral vector formulation containing an exogenous targeting protein that targets T cells and encodes the alkaline phosphatase protein. One and two weeks after administration, blood was drawn and analyzed by flow cytometry. Two weeks later, the animals were sacrificed and tissue samples from each organ were harvested and assayed for off-target alkaline phosphatase expression.

[0164] These data indicate that this method can be used to express any relevant exogenous functional protein.

[0165] Other embodiments

[0166] It should be understood that although the invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate rather than limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

1. A viral vector core having a first chemical handle on its surface.

2. The viral vector core of claim 1, wherein the first chemical handle comprises an azido sugar.

3. The viral vector core of claim 1, wherein the first chemical handle comprises an alkyne sugar.

4. The viral vector core of any one of claims 1-3, wherein the viral vector core contains a nucleic acid encoding one or more therapeutic proteins.

5. The viral vector core of any one of claims 1-4, wherein the viral vector core is a lentiviral vector core.

6. A composition comprising the viral vector core according to any one of claims 1 to 5.

7. A viral vector comprising one or more exogenous targeting proteins, one or more exogenous immune modifying proteins and / or one or more polymers covalently attached to its surface via a conjugation feature.

8. The viral vector of claim 7, wherein the conjugation feature comprises a triazole bond, a thioether bond, a disulfide bond, or a [4+2] cycloadduct.

9. The viral vector of claim 7 or 8, wherein the one or more exogenous targeting proteins or the one or more exogenous immune modifying proteins have a molecular weight greater than 2 kDa.

10. The viral vector of claim 7 or 8, wherein the one or more exogenous targeting proteins comprise a scFv, a nanobody, a darpin, a VHH, an aptamer, or a lectin binding domain.

11. The viral vector of claim 7 or 8, wherein the polymer is polyethylene glycol (PEG).

12. The viral vector of claim 11, wherein the PEG has an average molecular weight of about 100 g / mol to about 20,000 g / mol.

13. The viral vector of any one of claims 7-12, wherein the viral vector contains a nucleic acid encoding one or more therapeutic proteins.

14. The viral vector of any one of claims 7-13, wherein the viral vector is a lentiviral vector.

15. The viral vector of any one of claims 7-14, wherein the viral vector has a pseudotype selected from the group consisting of: VSV-G, blind VSV-G, Sindbis glycoprotein, blind Sindbis glycoprotein, murine leukemia virus glycoprotein, blind murine leukemia virus glycoprotein, Moloney murine leukemia virus glycoprotein, blind Moloney murine leukemia virus glycoprotein, Cocal virus glycoprotein, blind Cocal virus glycoprotein, foamy virus glycoprotein, blind foamy virus glycoprotein, and GP64 glycoprotein.

16. The viral vector of any one of claims 7-14, wherein the viral vector does not have a VSV-G pseudotype.

17. A composition comprising a population of the viral vector of any one of claims 7-16.

18. The composition of claim 17, wherein the population of viral vectors has a functional titer corresponding to greater than about 1E7 TU / mL prior to concentrating and conjugating the viral vectors.

19. The composition of claim 17 or 18, wherein the ratio of functional titer to physical titer is from about 1:1000 to about 1:

10.

20. The composition of any one of claims 17-19, wherein the composition is a pharmaceutical composition.

21. A method for preparing a viral vector, wherein the method comprises: (a) contacting a viral vector core comprising a first chemical handle on its surface with an exogenous targeting protein, an exogenous immune-modifying protein, or a polymer comprising a second chemical handle, and (b) covalently conjugating the first chemical handle to the second chemical handle to produce the viral vector.

22. The method of claim 21, wherein the method further comprises: A viral vector core is produced that comprises the first chemical handle on its surface.

23. The method of claim 22, wherein the step of producing the viral vector core comprises the step of purifying or isolating the viral vector core by cleanup and / or chromatography.

24. The method of claim 22, wherein the step of producing the viral vector core comprises culturing producer cells in a liquid culture medium comprising a small molecule that is capable of metabolically inserting the first chemical handle into a glycoprotein on the cell membrane of the producer cells.

25. The method of any one of claims 22-24, wherein the first chemical handle and the second chemical handle are an azide and an alkyne.

26. The method of any one of claims 22-24, wherein the first chemical handle or the second chemical handle comprises DBCO.

27. The method of any one of claims 22-26, wherein step (b) is performed using a click chemistry reaction.

28. The method of claim 27, wherein the click chemistry reaction is performed at a temperature of about 30°C to about 40°C and a pH of about 6.5 to about 7.

5.

29. A kit comprising the composition of any one of claims 17-20.

30. A method of treating a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the composition of any one of claims 17-20.

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