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  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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  • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
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  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
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  • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
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  • C12N2760/20211—Vesiculovirus, e.g. vesicular stomatitis Indiana virus
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  • C12N2810/00—Vectors comprising a targeting moiety
  • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
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  • C12N2810/00—Vectors comprising a targeting moiety
  • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
  • C12N2810/80—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates
  • C12N2810/85—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian
  • C12N2810/859—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian from immunoglobulins

Definitions

• Retroviruses akin to the human immunodeficiency virus (HIV) have been adapted to accomplish the delivery and integration of genetic payloads associated with cell and gene therapies.
• Cancer cell lines can be leveraged as hosts to make these retroviruses by transfecting these host cell lines with multiple plasmids or vectors.
• a transfer vector encodes the genome of the retrovirus to be produced by the host cell.
• the transfer vector thus includes the genetic payload, e.g., therapeutic genes or gene circuits, that is to be integrated into the genome of a cell targeted by the retrovirus.
• a packaging plasmid encodes the viral Gag-Pol proteins that enable both production of the retrovirus within the host cell, and integration of the genetic payload into the host cell genome.
• An envelope plasmid encodes a binding protein that enables the retrovirus to target host cells and begin the viral entry process.
• the envelope proteins direct viruses toward a surface receptor on the host cell, binding the two together and beginning the viral entry process. Once bound, proteins encoded on the transfer plasmid fuse the cell and virus together, releasing the contents of the virus into the cell’s cytoplasm.
• the viral genome is reverse transcribed from RNA to DNA and is brought into the nucleus, where a viral integrase semi-randomly integrates the viral DNA into the genome.
• the introduced payload can then divide with the cell and is subject to all other genomic processes, creating a long-term change that enables both cell and gene therapy.
• retroviruses massively reducing their utility in more advanced cell and gene therapies. These therapies increasingly require selective transduction to only desired cell types, and achieving such selectivity remains extremely difficult for multiple reasons.
• viral envelope proteins often do not target specific cells of interest.
• the most ubiquitous envelope protein used with retroviruses is VSV-G, which binds LDL-receptor proteins. This family of surface proteins, though, are not present or adequately expressed on many therapeutically important cell types.
• Natural Killer (NK) cells are a type of immune cells having extremely exciting potentials as cures for cancers, autoimmune disorders, and diseases of aging.
• Natural Killer cells do not express LDL and are therefore difficult to adapt into an engineered cell therapy.
• Use of other envelope proteins such as RD114 (that binds to RDR surface proteins) or BaEV (that binds to ASCT surface proteins) can at least somewhat overcome this issue, but often at the cost of less efficient viral manufacturing or more limited knowledge of the envelope protein binding partner.
• viral envelope proteins lack selectivity. The binding partners of these proteins are not restricted to being expressed by a specific cell type. As a result, retroviruses expressing a particular envelope protein generally cannot target a specific tissue when delivered in situ.
• Retroviruses also typically cannot target a specific cell type in a mixture when the viruses are administered ex vivo.
• currently available technology does not allow the engineering of only the T cells in a peripheral blood mononuclear cell (PBMC) culture by using a retrovirus as an engineering tool.
• PBMC peripheral blood mononuclear cell
• the disclosure provides a retroviral vector system including an envelope plasmid, a packaging plasmid, and a transfer plasmid.
• the envelope plasmid encodes a viral membrane fusion protein of a retrovirus.
• the packaging plasmid encodes Gag-Pol proteins of the retrovirus.
• the transfer plasmid includes one or more genes of interest for transfer from the retrovirus to a target cell.
• One or both of the envelope plasmid and the packaging plasmid further encodes a binding moiety.
• the binding moiety directly or indirectly binds a surface feature of the target cell.
• the disclosure provides a retrovirus-packaging cell.
• the retrovirus-packaging cell includes any of the retroviral packing systems disclosed herein.
• the disclosure provides a retrovirus including a viral membrane fusion protein, a viral genome, and a binding moiety.
• the viral genome includes one or more genes of interest for transfer from the retrovirus to a target cell.
• the binding moiety binds a ligand that includes an antibody, an antibody mimetic, a single-chain variable fragment (scFv), or a derivative or fragment thereof.
• the ligand binds a surface feature of the target cell.
• the disclosure provides a virus-like particle including a binding moiety.
• the binding moiety binds a ligand that includes an antibody, an antibody mimetic, a single-chain variable fragment (scFv), or a derivative or fragment thereof.
• the ligand binds a surface feature of the target cell.
• the disclosure provides a method for producing a retrovirus.
• FIG. 12 is a graph plotting the transduction of primary T cells by a provided retrovirus having a FITC single-chain variable fragment (scFv) binding moiety, where the T cells have been engineered with antibodies against subtype marker CD4 or subtype marker CD8.
• FIG. 13 is a graph plotting the specific transduction of CD4 + T cells, and not CD8 + T cells, by a provided retrovirus having a FITC binding moiety conjugated to a CD4 antibody.
• FIG. 14 is a graph plotting the specific transduction of CD8 + T cells, and not CD4 + T cells, by a provided retrovirus having a FITC binding moiety conjugated to a CD8 antibody DETAILED DESCRIPTION I.
• the present disclosure generally provides materials and methods related to retroviruses and virus-like particles engineered to possess modular binding moieties on their surfaces.
• the particular binding moieties described herein decorate the retroviruses and virus- like particles, and allow them either to bind directly to cells of interest, or to bind an antibody, antibody fragment, or other ligand that binds to the cells.
• the moieties can advantageously bind to readily available off-the-shelf antibodies, to easily customized antibodies, or to surface features already present on the cells being targeted.
• the modular plug-and-play manner in which the binding moieties can be designed or selected offers several significant advantages over existing retrovirus methodologies.
• the transduction space available to the retroviruses and virus-like particles can be increased to include additional cell types that cannot be transduced by traditional lentivirus approaches.
• these additional cell types include many, such as Natural Killer (NK) cells, that are prime candidates for targeting in developing cellular and genetic therapies.
• NK Natural Killer
• the binding moieties also can enable the universal pseudotyping of a retrovirus or virus-like particle, such that one retrovirus or virus-like particle design can be conjugated with any antibody to target any cell.
• the targeting can also advantageously be more specific than can be achieved with other retrovirus approaches.
• binding moieties for the provided retroviruses and virus-like particles are encoded on the packaging and/or envelope plasmids of the viruses or particles. As a result, the genes encoding the binding moieties beneficially do not become integrated into host cells producing the viruses or particles or targeted for transduction.
• the systems for generating the provided retroviruses and virus-like particles can include a minimal number of plasmids, simplifying processes and increasing efficiency.
• a plasmid system for generating a provided retrovirus can include as few as three plasmids: the packaging and envelope plasmids, at least one of which will encode the binding moiety, and a transfer plasmid that carries the genetic material to be transferred to the target cell.
• the production of the provided retroviruses and virus-like particles is also advantageously facile and can easily be incorporated into standard viral production procedures.
• viral producing cells such as HEK293T cells
• a transfer plasmid encoding a viral genome
• an envelope plasmid encoding the viral envelope
• one or more packaging plasmids encoding the viral Gag-Pol proteins.
• the envelope and/or packaging plasmid in such a system can contain the binding moiety.
• Viral producing cells containing these plasmids then generate functional viruses that present the binding moieties on their surface.
• antibodies can be incubated with purified retrovirus to conjugate the antibody to the virus. The antibody then can direct the retrovirus to the target cells.
• Universal retroviruses can be created by engineering the packaging plasmid or, as shown in panel (A) of the figure, the envelope plasmid, with a binding moiety.
• the binding moiety can thus include a binding domain directly fused to a transmembrane domain.
• the binding moiety can be encoded directly upstream or downstream of the envelope or Gag-Pol proteins, and can be separated from the protein post-translationally by a 2A tag.
• the cognate envelope protein such as VSV-G, can further be mutated to no longer bind to its cognate ligand.
• the binding moiety can contain either a cell binding domain (CBD, top) or an antibody/antibody-fragment/ligand binding domain (ABD, bottom).
• CBD cell binding domain
• ABD antibody/antibody-fragment/ligand binding domain
• the binding moiety directs the virus toward a specific surface protein of the target cell, enabling the virus to transduce this particular cell type.
• the binding moiety contains an ABD
• the binding moiety binds a secondary binder such as an antibody, and the combined virus and antibody together transduce specific targeted cell types.
• Retrovirus refers to a member of the Retroviridae virus family.
• a retrovirus has a single-stranded, diploid, positive-sense RNA genome that is reverse-transcribed into a DNA intermediate that can then be incorporated into a host cell genome.
• Retroviridae-derived viruses are generally enveloped particles with a diameter of 80-120 nm.
• Retroviral vectors or plasmids can be replication-deficient viral particles derived from the Retroviridae virus family.
• the plasmids can contain group-specific antigen (Gag) and Pol proteins, a single-stranded RNA genome, and envelope proteins. Retroviral plasmids can also include psi elements and long terminal repeats (LTRs) that can be required for efficient packing and reverse transcription in DNA. Retroviruses include alpharetroviruses, gammaretroviruses, and lentiviruses. Representative species of lentiviruses include the human immunodeficiency virus (HIV). Representative species of gammaretroviruses include murine leukemia virus and the feline leukemia virus. [0039] Integrase-deficient retroviruses and retroviral vectors cannot integrate the retroviral vector genome in the host cell genome.
• Integrase-deficient retroviral vectors or plasmids can be derived from conventional retroviral vectors, and lack, or contain a mutated form of, the retroviral integrase.
• the retroviral vector genome of an integrase-deficient retrovirus is reverse-transcribed in the cytoplasm, and delivered into the nucleus, but not stably integrated into the host cell genome.
• the term “transduction” refers to the processes of a virus entering a host cell and delivering an RNA genome, and the expression of a thus-delivered gene of interest by the host cell.
• virus-like particle and “VLP” refer to particles that resemble viruses, but are not infecting or transducing because they contain no viral genetic material encoding the proteins of the virus-like particle.
• the expression of viral structural proteins, such as envelope or capsid proteins, can result in the assembly of virus-like particles.
• Virus-like particles can be used to deliver proteins and/or nucleic acids to the cytoplasm of target cells.
• plasmid refers to a circular, double-stranded DNA containing one or more sequences of interest, for example, sequences encoding one or more particular proteins.
• a plasmid can further include regulatory sequences or other genetic elements that are operatively linked to a sequence encoding a particular protein.
• the terms “viral membrane fusion protein, “membrane fusion protein,” “fusion protein,” and “fusogen” refer to a polypeptide that causes or enhances fusion of biological membranes, e.g., a viral envelope and a cell wall.
• a viral membrane fusion protein can be a transmembrane protein or a functional fragment or derivative thereof.
• the term “cell” generally refers to a biological cell. A cell can be the basic structural, functional and/or biological unit of a living organism.
• a cell can originate from any organism having one or more cells.
• Some non-limiting examples include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant (e.g., cells from plant crops, fruits, vegetables, grains, soy bean, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, ferns, clubmosses, hornworts, liverworts, mosses), an algal cell, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, and the like), seaweeds (e.g., kelp), a fun
• a cell does not originate from a natural organism (e.g., a cell can be a synthetically made, sometimes termed an artificial cell).
• the term “variant,” as used in the context of polypeptides described herein, refers to polypeptides having a high degree of structural similarity to one another, with structural differences resulting from differences in polynucleotides encoding the polypeptide variants.
• Polypeptide variants may have amino acid sequences that are at least 80% similar to one another (% identity) , e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similar to one another (% identity).
• Polypeptide variants may have the same biological functions as one another. For example, where polypeptide variants are enzymes, the polypeptide variants may each catalyze the same reaction.
• an antibody refers to a polypeptide of the immunoglobulin family or a polypeptide comprising fragments of an immunoglobulin that is capable of noncovalently, reversibly, and in a specific manner binding to an epitope of a corresponding antigen.
• the term includes, but is not limited to, polyclonal or monoclonal antibodies of the isotype classes IgA, IgD, IgE, IgG, and IgM, derived from human or other mammalian cells, including natural or genetically modified forms such as humanized, human, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitro generated antibodies.
• conjugates including but not limited to proteins containing an immunoglobulin moiety (e.g., chimeric or bispecific antibodies or single chain Fv’s (scFv’s)), and fragments, such as Fab, F(ab')2, Fv, scFv, Fd, dAb and other compositions.
• scFv single chain Fv
• fragments such as Fab, F(ab')2, Fv, scFv, Fd, dAb and other compositions.
• single-chain variable fragment As used herein, the terms “single-chain variable fragment,” “single chain Fv,” and “scFv” refer to an antibody in which the variable domains of the heavy chain and of the light chain of a traditional two chain antibody have been joined to form one chain. Typically, a linker peptide is inserted between the two chains to allow for proper folding and creation of an active binding site.
• the terms “nanobody” or “single-domain antibody” refer to an antibody fragment comprised of a single monomeric variable antibody domain, having a molecular weight of less than 20 kDa, and able to bind selectively to a specific antigen.
• the term “epitope” refers to the localized site on the antigen that is recognized and bound by the antibody. Protein epitopes can include a few amino acids or portions of a few amino acids, e.g., 5 or 6 or more, or 20 or more amino acids or portions of those amino acids.
• Epitopes can also include non-protein components, e.g., nucleic acid (e.g., RNA or DNA), carbohydrate, lipid, or a combination thereof.
• the epitope can be a three- dimensional moiety.
• the epitope can include consecutive amino acids, or amino acids from different parts of the protein that are brought into proximity by protein folding (e.g., a discontinuous epitope).
• a discontinuous epitope e.g., a discontinuous epitope.
• target molecules such as DNA and chromatin, which form three- dimensional structures.
• detectable binding agents that are proteins
• specific binding is determinative of the presence of the protein in a heterogeneous population of proteins and other biologics.
• the specified antibodies bind to a particular protein sequence, thereby identifying its presence.
• Specific binding to an antibody under such conditions requires an antibody that is selected for its specificity for a particular protein.
• antibodies raised against a particular protein polymorphic variants, alleles, orthologs, and conservatively modified variants, or splice variants, or portions thereof, can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with the protein of interest and not with other proteins. This selection may be achieved by subtracting out antibodies that cross-react with other molecules.
• a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
• solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).
• Methods for determining whether two molecules specifically interact are disclosed herein, and methods of determining binding affinity and specificity are well known in the art (see, for example, Harlow and Lane, Antibodies: A laboratory manual (Cold Spring Harbor Laboratory Press, 1988); Friefelder, "Physical Biochemistry: Applications to biochemistry and molecular biology” (W.H. Freeman and Co.1976)).
• switch receptor and “chimeric switch receptor” refer to a molecule designed to switch a negative signal transduction signal into a positive signal.
• the switch receptor can be a chimeric protein comprising a first protein or fragment thereof associated with a negative signal, and a second protein or fragment thereof associated with a positive signal.
• proteins associated with a negative signal include, without limitation, CTLA-4, PD-1, BTLA, TIM-3 and the like.
• proteins associated with a positive signal include, without limitation, CD28, ICOS, 4-1BB, TGF ⁇ R and the like
• selectable marker refers to a gene that encodes a protein that allows the cell expressing the gene to be identified and/or isolated from other cells in a population. Selectable markers include, but are not limited to, genes that encode drug resistance, fluorescence, and essential genes for growth in limiting conditions.
• subject refers to a vertebrate, and preferably to a mammal. Mammalian subjects for which the provided composition is suitable include, but are not limited to, mice, rats, simians, humans, farm animals, sport animals, and pets.
• the subject is human. In some embodiments, the subject is male. In some embodiments, the subject is female. In some embodiments, the subject is an adult. In some embodiments, the subject is an adolescent. In some embodiments, the subject is a child. In some embodiments, the subject is above 10 years of age, e.g., above 20 years of age, above 30 years of age, above 40 years of age, above 50 years of age, above 60 years of age, above 70 years of age, or above 80 years of age.
• the subject is less than 80 years of age, e.g., less than 70 years of age, less than 60 years of age, less than 50 years of age, less than 40 years of age, less than 30 years of age, less than 20 years of age, or less than 10 years of age.
• administering refers to oral administration, administration as a suppository, topical contact, parenteral, intravenous, intraperitoneal, intramuscular, intralesional, intranasal or subcutaneous administration, intrathecal administration, or the implantation of a slow-release device e.g., a mini-osmotic pump, to the subject.
• the terms “treat”, “treating” and “treatment” refer to a procedure resulting in any indicia of success in the elimination or amelioration of an injury, pathology, condition, or symptom (e.g., pain), including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the symptom, injury, pathology or condition more tolerable to the patient; decreasing the frequency or duration of the symptom or condition; or, in some situations, preventing the onset of the symptom.
• the treatment or amelioration of symptoms can be based on any objective or subjective parameter; including, e.g., the result of a physical examination.
• the terms “pharmaceutically acceptable excipient” and “pharmaceutically acceptable carrier” refer to a substance that aids the administration of an active agent to and absorption by a subject and may be included in the compositions of the present disclosure without causing a significant adverse toxicological effect on the subject.
• pharmaceutically acceptable excipients and carriers include water, NaCl, normal saline solutions, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, and the like.
• pharmaceutically acceptable excipients and carriers include water, NaCl, normal saline solutions, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, and the like.
• the term “therapeutically effective amount” refers to an amount or dose of a compound, composition, or formulation that produces therapeutic effects for which it is administered.
• the exact amount or dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).
• the term “vaccine” refers to a composition comprising at least one antigen or immunogen, or comprising a nucleic acid molecule encoding at least one antigen or immunogen, in a pharmaceutically acceptable carrier, that is useful for inducing an immune response against the antigen or immunogen in a subject, for the purpose of improving immunity against a disease and/or infection in the subject.
• vaccine refers to a composition comprising at least one antigen or immunogen, or comprising a nucleic acid molecule encoding at least one antigen or immunogen, in a pharmaceutically acceptable carrier, that is useful for inducing an immune response against the antigen or immunogen in a subject, for the purpose of improving immunity against a disease and/or infection in the subject.
• the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise.
• reference to “a polymer” optionally includes a combination of two or more polymers, and the like.
• the term “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).
• the terms “including,” “comprising,” “having,” “containing,” and variations thereof, are inclusive and open-ended and do not exclude additional, unrecited elements or method steps beyond those explicitly recited.
• the phrase “consisting of” is closed and excludes any element, step, or ingredient not explicitly specified.
• the phrase “consisting essentially of” limits the scope of the described feature to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the disclosed feature. III.
• a retroviral vector system includes plasmids that can be used to transform a retrovirus-packaging cell, enabling the cell to produce retroviruses according to the contents of the vector system.
• the disclosed retroviral vector system can therefore provide many of the surprising advantages discussed herein, particularly when used in therapeutic applications such as gene or cell therapies.
• the retroviral vector system can be used as a tool for producing retroviruses engineered to specifically and/or selectively transduce one or more particular cell types or populations.
• the engineered retroviruses produced using the retroviral vector system can also advantageously have a cell specificity that is easily switchable.
• the provided retroviral vector systems include one or more envelope plasmids, one or more packaging plasmids, and one or more transfer plasmids.
• An advantage of the provided vector systems is that a small number of plasmids can be effective in transfecting a virus-packing cell to enable the cell to produce a functional retrovirus having the binding moiety disclosed herein.
• the retroviral vector system includes only one envelope plasmid.
• the retroviral vector system includes only one packaging plasmid. In some embodiments, the retroviral vector system includes only one transfer plasmid. In some embodiments, the retroviral vector system includes only one envelope plasmid and only one packaging plasmid. In some embodiments, the retroviral packaging system includes only one envelope plasmid and only one transfer plasmid. In some embodiments, the retroviral vector system includes only one packaging plasmid and only one transfer plasmid. In some embodiments, the retroviral vector system includes only one envelope plasmid, only one packaging plasmid, and only one transfer plasmid.
• the envelope plasmid of the provided retroviral vector system generally encodes a viral membrane fusion protein of the retrovirus to be produced by a virus-packaging cell transfected with the vector system.
• the packaging plasmid of the retroviral vector system generally encodes Gag-Pol proteins of the retrovirus.
• the transfer plasmid of the retroviral vector system generally encodes one or more genes of interest for transfer from the retrovirus to a target cell.
• At least one plasmid of the provided retroviral vector system encodes a binding moiety that directly or indirectly binds a surface feature of a target cell.
• the binding moiety of the provided retroviral vector system generally includes an extramembrane domain, where the extramembrane domain is a binding domain that recognizes a surface feature of a target cell, or that recognizes a secondary binding partner that recognizes a surface feature of the target cell.
• the binding moiety includes a transmembrane domain that positions the binding moiety in the envelope of the retrovirus. In some embodiments, the extramembrane domain of the binding moiety is fused to the transmembrane domain of another viral envelope protein.
• the binding moiety can be encoded directly upstream of an envelope protein on the envelope plasmid of the provided retroviral vector system.
• the binding moiety can be encoded directly downstream of an envelope protein on the envelope plasmid (FIG. 2).
• the binding moiety can be encoded directly upstream of a Gag-Pol protein on the packaging plasmid of the retroviral vector system.
• the binding moiety can be encoded directly downstream of a Gag-Pol protein on the packaging plasmid.
• the binding moiety is post-translationally separated from the adjacent protein of the plasmid, e.g., with a 2A tag.
• the binding moiety of the provided retroviral vector system is designed or selected to bind with a ligand, where the ligand is designed or selected to bind with a target cell, e.g., to a surface feature of the target cell (FIG. 1).
• the ligand bound by the binding moiety includes an antibody, an antibody mimetic, a single- chain variable fragment (scFv), or a derivate or fragment thereof.
• the binding moiety can be described as including an antibody binding domain (ABD).
• the ligand is a conjugated derivative of an antibody, an antibody mimetic, or an scFv, where the ligand is conjugated to a small molecule recognized by the binding moiety (FIG. 5).
• a retroviral vector system can be engineered such that it can be used to produce universal pseudotyped retroviruses.
• These universal pseudotyped retroviruses each include a binding moiety that binds to a particular small molecule, and any conjugate between a ligand and the small molecule can easily be created so that the ligand is compatible with the binding moiety having an affinity for the particular small molecule of the conjugate.
• a single universal design of a provided retrovirus e.g.
• the binding moiety includes a fluorescein isothiocyanate (FITC)-binding domain, and the ligand is FITC-conjugated.
• FITC fluorescein isothiocyanate
• the binding moiety can include an anti-fluorescein scFv or fluorescein-binding anticalin.
• the ligand recognized by this binding moiety can be, for example, a FITC-conjugated antibody.
• the binding moiety includes a biotin-binding domain, and the ligand is biotin-conjugated.
• the binding moiety can include an anti-biotin scFv, biotin-binding anticalin, or an avidin-family protein, e.g., avidin or streptavidin.
• the ligand recognized by this binding moiety can be, for example, a biotin-conjugated antibody.
• a single design of a universal pseudotyped retrovirus with a biotin- binding moiety can thus specifically transduce a variety of cell types.
• the ligands recognized by an antibody binding domain of a provided binding moiety can themselves bind to a wide variety of surface features of target cells.
• Many off-the- shelf antibodies suitable for use with the provided materials and methods are available, where the available antibodies have different specificity and selectivity for various antigens and epitopes.
• Still other antibodies and antibody conjugates can be developed for binding to further target cell surface features.
• the ligands can be used to indirectly bind the provided binding moiety to one or more of CD3, CD4, CD7, CD8, CD19, CD20, CD56, CD71, or CTLA4.
• the binding moiety of the provided retroviral vector system is designed or selected to bind directly with a target cell, e.g., to a surface feature of the target cell (FIG. 1).
• the binding moiety can be described as a including a cell binding domain (CBD).
• the cell binding domain includes an antibody, an antibody mimetic, an scFv, a nanobody, another ligand, or a derivative or fragment thereof.
• binding moieties including cell binding domains can also bind to a wide variety of surface features of target cells.
• the cell binding domain can be designed or selected to bind the provided binding moiety to one or more of CD3, CD4, CD7, CD8, CD19, CD20, CD56, CD71, or CTLA4.
• a wide variety of cell types can be targeted by the provided retroviral vector systems.
• the target cell is an immune cell, including any cell that is involved in an immune response.
• the targeting of immune cells such as optionally allogenic natural NK cells, iPSC-derived NK cells, and/or macrophage cells can greatly facilitate treatment of solid tumor while avoiding side effects.
• the target cell includes granulocytes such as basophils, eosinophils, and neutrophils; mast cells; monocytes which can develop into macrophages; antigen-presenting cells such as dendritic cells; and lymphocytes such as natural killer cells (NK cells), B cells, and T cells.
• the target cell is an immune effector cell.
• An immune effector cell is an immune cell that can perform a specific function in response to a stimulus.
• the target cell is an immune effector cell which can induce cell death.
• the target cell is a lymphocyte.
• the lymphocyte is an NK cell.
• the lymphocyte is a T cell.
• the T cell is an activated T cell.
• T cells include both naive and memory cells (e.g., central memory or TCM, effector memory or TEM and effector memory RA or TEMRA), effector cells (e.g., cytotoxic T cells or CTLs or Tc cells), helper cells (e.g., Thl, Th2, Th3, Th9, Th7, TFH), regulatory cells (e.g., Treg, and Trl cells), natural killer T cells (NKT cells), tumor infiltrating lymphocytes (TILs), lymphocyte-activated killer cells (LAKs), ⁇ ⁇ cells, ⁇ ⁇ cells, and similar unique classes of the T cell lineage.
• naive and memory cells e.g., central memory or TCM, effector memory or TEM and effector memory RA or TEMRA
• effector cells e.g.
• T cells can be divided into two broad categories: CD8+ T cells and CD4+ T cells, based on which protein is present on the cell's surface. T cells can carry out multiple functions, including killing infected cells and activating or recruiting other immune cells. CD8+ T cells are referred to as cytotoxic T cells or cytotoxic T lymphocytes (CTLs). CD4+ T cells can be subdivided into four sub-sets – Th1, Th2, Th17, and Treg, with “Th” referring to “T helper cell,” although additional sub-sets may exist. Th1 cells can coordinate immune responses against intracellular microbes, especially bacteria. They can produce and secrete molecules that alert and activate other immune cells, like bacteria-ingesting macrophages.
• CTLs cytotoxic T lymphocytes
• the engineered retroviruses produced using the retrovirus-packaging cell can also advantageously have a target cell specificity that is easily switchable.
• This switchable specificity can be realized by, for example, using facile procedures to alter the binding moieties of the retroviruses or to alter a secondary binder, e.g., an antibody, to which the retrovirus binding moieties conjugate.
• a population of retrovirus-packaging cells is provided.
• each host cell of the population independently includes a retroviral vector system as disclosed herein.
• a cell culture comprising a population of cells as described herein is also provided. Methods for the culture and production of many cells, including cells of bacterial (for example E.
• the genes of interest and the target cell can by any of those disclosed herein regarding the retroviral vector system.
• the provided retrovirus further includes a binding moiety that can be any of those disclosed herein regarding the retroviral vector system.
• the binding moiety includes an antibody binding domain (ABD) of any of the types disclosed herein.
• the binding moiety of the provided retrovirus can include a ligand that is an antibody, an antibody mimetic, a single-chain variable fragment (scFv), or a derivate or fragment thereof, where the ligand binds a surface feature of the target cell.
• Disorders suitable for treating with the provided method include, but are not limited to, X- linked severe combined immune deficiency, sickle cell anemia, thalassemia, hemophilia, neoplasia, cancer, age-related macular degeneration, schizophrenia, trinucleotide repeat disorders, fragile X syndrome, prion-related disorders, amyotrophic lateral sclerosis, drug addiction, autism, Alzheimer’s disease, Parkinson’s disease, cystic fibrosis, blood and coagulation disease or disorders, inflammation, facioscapulohumeral muscular dystrophy, retinitis pigmentosa, Leber congenital amaurosis, glaucoma, immune-related diseases or disorders, metabolic diseases and disorders, liver diseases and disorders, kidney diseases and disorders, muscular/skeletal diseases and disorders, neurological and neuronal diseases and disorders, cardiovascular diseases and disorders, pulmonary diseases and disorders, and ocular diseases and disorders.
• a tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years.
• the size of a tumor or the number of tumor cells is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more.
• the tumor is completely eliminated, or reduced below a level of detection.
• a subject remains tumor free (e.g. in remission) for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks following treatment.
• a subject remains tumor free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months following treatment. In some embodiments, a subject remains tumor free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years after treatment.
• Suitable anti-cancer agents for combination therapy include, without limitation, cytotoxins and agents such as antimetabolites, alkylating agents, anthracyclines, antibiotics, antimitotic agents, procarbazine, hydroxyurea, asparaginase, corticosteroids, interferons, radiopharmaceuticals, peptides with anti-tumor activity such as TNF- ⁇ , pharmaceutically acceptable salts thereof; derivatives thereof, prodrugs thereof, and combinations thereof.
• a pharmaceutical composition comprising the provided retroviral vector systems, retrovirus-packaging cells, retroviruses, and/or virus-like particles can be administered to a patient before, during, or after administration of an anti-cancer agent or combination of anti- cancer agents either before, during, or after chemotherapy.
• treatment with the provided retroviral vector systems, retrovirus-packaging cells, retroviruses, and/or virus-like particles results in stable disease, partial remission or complete remission in the subject (e.g., the methods described herein comprise administering to the subject a dose of the provided retroviral vector systems, retrovirus-packaging cells, retroviruses, and/or virus-like particles that kills or otherwise slows the growth or progression of cancer cells and leads to stable disease or to partial or complete remission of the cancer in the subject).
• treatment with the provided retroviral vector systems, retrovirus-packaging cells, retroviruses, and/or virus-like particles results in a reduction in metastases of the cancer in the subject (e.g., the methods described herein comprise administering to the subject a dose of the provided retroviral vector systems, retrovirus-packaging cells, retroviruses, and/or virus-like particles that reduces metastases of the cancer in the subject).
• treatment with the provided retroviral vector systems, retrovirus-packaging cells, retroviruses, and/or virus-like particles results in a reduction in volume, size, or growth of a tumor in the subject (e.g., the methods described herein comprise administering to the subject a dose of the provided retroviral vector systems, retrovirus-packaging cells, retroviruses, and/or virus-like particles that reduces the volume, size, or growth of a tumor in the subject).
• treatment with the provided retroviral vector systems, retrovirus-packaging cells, retroviruses, and/or virus-like particles results in an increased responsiveness of the cancer to a subsequently administered anti-cancer agent (e.g., the methods described herein comprise administering to the subject a dose of the provided retroviral vector systems, retrovirus- packaging cells, retroviruses, and/or virus-like particles that increases responsiveness of the cancer to a subsequently administered anti-cancer agent).
• the prevented or treated disease is an infectious disease.
• the infectious disease can be, for example, a viral infectious disease.
• the infectious disease can be, for example, a bacterial infectious disease.
• the innate immune system In the case of bacterial infections, the innate immune system must recognize specific markers of the microbes in order to clear the pathogen. These pathogen associated molecular patterns are recognized by various receptors, most notably the TLRs that are common to all immune cells and work to activate immune pathways that turn on their bactericidal capacities. In the case of macrophages, recognition of microbial pathogens through a TLR activates their unique ability to engulf the bacteria within themselves and destroy the pathogen by acidification. However, bacteria have mechanisms to evade macrophages by hiding the molecules that cause this activation.
• retroviral vector systems can “rewire” these TLRs to recognize the constituents of the biofilm itself as opposed to the bacteria, such that macrophages are activated by the evasion mechanisms, destroying the infection and allowing these devices to be more safely implanted.
• the provided retroviral vector systems, retrovirus-packaging cells, retroviruses, and/or virus-like particles are administered to a subject once, twice, three times, four times, or five times over a course of treatment. Subsequent administration of the provided retroviral vector systems, retrovirus-packaging cells, retroviruses, and/or virus-like particles can occur at defined intervals of time, separated by days, weeks, or months.
• the provided retroviral vector systems, retrovirus-packaging cells, retroviruses, and/or virus-like particles are administered at a subsequent time if a tumor or cancerous cells reappear, continue to grow, or otherwise are not fully treated after the first administration of the provided retroviral vector systems, retrovirus-packaging cells, retroviruses, and/or virus- like particles.
• the provided retroviral vector systems, retrovirus-packaging cells, retroviruses, and/or virus-like particles are administered again at a subsequent time if the subject does not have a complete response to the first treatment, experiences a partial response, a stable response or progressive disease.
• the provided method further includes obtaining a test sample from the subject.
• the test sample can include, for example, a blood sample, a tissue sample, a urine sample, a saliva sample, a cerebrospinal fluid sample, or a combination thereof.
• the provided method further includes determining the level of one or more biomarkers in the obtained test sample. Determining the presence or level of biomarkers(s) can be used to, as non-limiting examples, determine response to treatment or to select an appropriate composition for the prevention or treatment of the disease. [0104] In some embodiments, the provided method further includes comparing the determined level of the one of more biomarkers in the obtained test sample to the level of the one or more biomarkers in a reference sample.
• the reference sample can be obtained, for example, from the subject, with the reference sample being obtained prior to the obtaining of the test sample, e.g., prior to the administering to the subject of the therapeutically effective amount of the provided materials.
• the reference sample can provide information about baseline levels of the biomarkers in the sample before the treatment
• the test sample can provide information about levels of the biomarkers after the treatment.
• the reference sample can be obtained, for example, from a different subject, e.g., a subject in which the treatment is not provided according to the provided methods. In this way, the reference sample can provide information about baseline levels of the biomarkers without treatment, and the test sample can provide information about levels of the biomarkers with treatment.
• the reference sample can also be obtained, for example, from a population of subjects, e.g., subjects in which the treatment is not provided according to the provided method. In this way, the reference sample can provide population-averaged information about baseline levels of the biomarkers without treatment, and the test sample can provide information about levels of the biomarkers with treatment.
• the reference sample can also be obtained from an individual or a population of individuals after treatment is provided according to the provided methods, and can serve as, for example, a positive control sample. In some embodiments, the reference sample is obtained from normal tissue. In some embodiments, the reference sample is obtained from abnormal tissue.
• an increase or a decrease relative to a normal control or reference sample can be indicative of the presence of a disease, or response to treatment for a disease.
• an increased level of a biomarker in a test sample, and hence the presence of a disease, e.g., an infectious disease or cancer, increased risk of the disease, or response to treatment is determined when the biomarker levels are at least, 1.1- fold, e.g., at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 3-fold, at least 4- fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10- fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least
• a decreased level of a biomarker in the test sample, and hence the presence of the disease, increased risk of the disease, or response to treatment is determined when the biomarker levels are at least 1.1-fold, e.g., at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7- fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5- fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11- fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, or at least 20-fold lower in comparison to a negative control.
• the biomarker levels can be detected using any method known in the art, including the use of antibodies specific for the biomarkers.
• Exemplary methods include, without limitation, polymerase chain reaction (PCR), Western Blot, dot blot, ELISA, radioimmunoassay (RIA), immunoprecipitation, immunofluorescence, FACS analysis, electrochemiluminescence, and multiplex bead assays, e.g., using Luminex or fluorescent microbeads.
• PCR polymerase chain reaction
• Western Blot Western Blot
• dot blot ELISA
• radioimmunoassay RIA
• immunoprecipitation immunofluorescence
• FACS analysis e.g., electrochemiluminescence
• electrochemiluminescence e.g., electrochemiluminescence
• multiplex bead assays e.g., using Luminex or fluorescent microbeads.
• nucleic acid sequencing is employed.
• the presence of decreased or increased levels of one or more biomarkers is indicated by a detectable signal, e.g., a blot, fluorescence, chemiluminescence, color, or radioactivity, in an immunoassay or PCR reaction, e.g., quantitative PCR.
• a detectable signal e.g., a blot, fluorescence, chemiluminescence, color, or radioactivity
• This detectable signal can be compared to the signal from a reference sample or to a threshold value.
• the results of the biomarker level determinations are recorded in a tangible medium.
• the results of diagnostic assays e.g., the observation of the presence or decreased or increased presence of one or more biomarkers, and the diagnosis of whether or not there is an increased risk or the presence of a disease, e.g., an infectious disease or cancer, or whether or not a subject is responding to treatment can be recorded, for example, on paper or on electronic media, e.g., audio tape, a computer disk, a CD-ROM, or a flash drive.
• the provided method further includes the step of providing to the subject a diagnosis and/or the results of treatment. VIII. Methods for Inducing an Immune Response
• the present disclosure provides various method for inducing an immune response in a subject.
• the methods generally include administering to the subject any of the retroviral vector systems, retrovirus-packaging cells, retroviruses, or virus-like particles disclosed herein, e.g., in Sections III, IV, V, and VI.
• an immunogenic composition can be formed including any of the provided retroviral vector systems, retrovirus-packaging cells, retroviruses, or virus-like particles.
• the immunogenic composition can be a vaccine, and the administration of the immunogenic composition can include vaccinating the subject with the vaccine.
• the disclosed immunogenic compositions can be administered using the provided method as a single dose or as multiple doses, for example, two doses administered at an interval of about one week, two weeks, three weeks, one month, about two months, about three months, about six months, or about 12 months.
• Other suitable dosage schedules can be determined by a medical practitioner.
• additional compounds or medications can be co-administered to the subject.
• Such compounds or medications can be co-administered to, for example, alleviate signs or symptoms of the disease being treated, or to reduce side effects caused by induction of the immune response.
• IX. Pharmaceutical compositions [0114] In another aspect, a pharmaceutical composition is provided.
• the provided pharmaceutical composition includes one or more, e.g., two or more, of any of the retroviral vector systems, retrovirus-packaging cells, retroviruses, or virus-like particles disclosed herein, e.g., in Sections III, IV, V, and VI.
• the provided pharmaceutical compositions can, for example, better allow the retroviruses or virus-like particles disclosed herein to deliver genetic payloads in situ to a subject in need thereof.
• the pharmaceutical composition includes a therapeutically effective amount of a pharmaceutically acceptable excipient.
• the pharmaceutical composition includes one or more of a diluent, adjuvant, or carrier in a formulation suitable for administration, e.g., administration to a mammal.
• the pharmaceutical compositions can also include additional substances to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, wetting agents, and detergents.
• additional substances such as pH adjusting and buffering agents, toxicity adjusting agents, wetting agents, and detergents.
• auxiliary, thickening, lubricating, and coloring agents can alternatively or additionally be used.
• Pharmaceutical compositions can be formulated into preparations in solid, semisolid, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols.
• the provided pharmaceutical composition can also include any of a variety of stabilizing agents, such as an antioxidant for example.
• the polypeptide can be complexed with various well-known compounds that enhance the in vivo stability of the polypeptide, or otherwise enhance its pharmacological properties (e.g., increase the half-life of the polypeptide, reduce its toxicity, and/or enhance solubility or uptake).
• modifications or complexing agents include sulfate, gluconate, citrate, and phosphate.
• the nucleic acids or polypeptides of a composition can also be complexed with molecules that enhance their in vivo attributes. Such molecules include, for example, carbohydrates, polyamines, amino acids, other peptides, ions (e.g., sodium, potassium, calcium, magnesium, manganese), and lipids.
• Embodiment 1 A retroviral vector system comprising: an envelope plasmid encoding a viral membrane fusion protein of a retrovirus; a packaging plasmid encoding Gag- Pol proteins of the retrovirus; and a transfer plasmid comprising one or more genes of interest for transfer from the retrovirus to a target cell; wherein one or both of the envelope plasmid and the packaging plasmid further encodes a binding moiety that directly or indirectly binds a surface feature of the target cell.
• Embodiment 27 A retrovirus comprising: a viral membrane fusion protein; a viral genome comprising one or more genes of interest for transfer from the retrovirus to a target cell; and a binding moiety that binds a ligand comprising an antibody, an antibody mimetic, a single-chain variable fragment (scFv), or a derivate or fragment thereof, wherein the ligand binds a surface feature of the target cell.
• Embodiment 28 An embodiment of embodiment 27, wherein the viral membrane fusion protein is an engineered variant of a wild-type viral membrane fusion protein.

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