WO2024059569A2 - Composition et méthode de rétrovirus pseudotypés universels - Google Patents

Composition et méthode de rétrovirus pseudotypés universels Download PDF

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WO2024059569A2
WO2024059569A2 PCT/US2023/073983 US2023073983W WO2024059569A2 WO 2024059569 A2 WO2024059569 A2 WO 2024059569A2 US 2023073983 W US2023073983 W US 2023073983W WO 2024059569 A2 WO2024059569 A2 WO 2024059569A2
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retrovirus
cell
retroviral vector
binding
vector system
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WO2024059569A3 (fr
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Lei S. QI
Michael Chavez
Paul B. FINN
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The Board Of Trustees Of The Leland Stanford Junior University
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16045—Special targeting system for viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20211—Vesiculovirus, e.g. vesicular stomatitis Indiana virus
    • C12N2760/20222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
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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

  • Gene therapies may be used to correct genetic disorders by editing the genome, e.g., through the use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) gene editing sequences. Gene therapies also may address genetic disorders by introducing new corrective genetic information, e.g. through the use of clinical-grade viruses.
  • Cell therapies may be applied to reprogram cells, directing the human body’s natural, complex functionality toward new targets. This opens an enormous space of possible therapeutic modalities that were previously unavailable using old paradigms. Both cell and gene therapies rely on successful delivery of therapeutic genetic payloads to precisely targeted cells or tissues. Many of these therapies also need the delivered genetic information to be directly integrated into the genome, enabling long-term, heritable, and stable expression of the gene.
  • 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.
  • the method includes transfecting a host cell with any of the retroviral vector systems disclosed herein.
  • the disclosure provides a method for preventing or treating a disease in a subject.
  • the method includes administering to the subject any of the retroviral vector systems, retrovirus-packaging cells, retroviruses, or virus-like particles disclosed herein.
  • FIG.1 is an illustration of a retroviral vector system, retrovirus-packaging cells, and retroviruses in accordance with provided embodiments.
  • FIG. 2 is a schematic illustration of an envelope plasmid encoding a binding moiety in accordance with a provided embodiment.
  • FIG. 1 is an illustration of a retroviral vector system, retrovirus-packaging cells, and retroviruses in accordance with provided embodiments.
  • FIG. 2 is a schematic illustration of an envelope plasmid encoding a binding moiety in accordance with a provided embodiment.
  • FIG. 1 is an illustration of a retroviral vector system, retrovirus-packaging cells, and retroviruse
  • FIG. 3 presents results from an example demonstrating lymphocyte transduction by a provided retrovirus having a CD7-targeting nanobody as a binding moiety.
  • FIG. 4 is a graph plotting the transduction of Natural Killer (NK) cells by a provided retrovirus or by a comparative lentivirus.
  • FIG. 5 is an illustration of a universal pseudotyped retrovirus in accordance with a provided embodiment.
  • FIG. 6 is a graph plotting the transduction of Jurkat cells at different concentrations of (1) a provided retrovirus having monomeric streptavidin (mSA) as a binding moiety, and (2) a CD71 antibody conjugated to biotin.
  • mSA monomeric streptavidin
  • FIG. 7 is a graph plotting the transduction of Jurkat cells at different concentrations of (1) a provided retrovirus having an anti-fluorescein isothiocyanate ( ⁇ FITC) binding moiety, and (2) a CD71 antibody conjugated to FITC.
  • FIG. 8 is a graph plotting the magnitude of retrovirus-delivered mCherry expression in the Jurkat cells of FIG.6.
  • FIG. 9 is a graph plotting the magnitude of retrovirus-delivered mCherry expression in the Jurkat cells of FIG.7. [0025] FIG.
  • FIG. 10 is a graph plotting the transduction of Jurkat cells by a provided retrovirus having a FITC single-chain variable fragment (scFv) binding moiety, where the Jurkat cells have been engineered with antibodies against lymphocyte markers (CD7) or T cell markers (CD3).
  • FIG. 11 is a graph plotting the transduction of Raji cells by a provided retrovirus having a FITC single-chain variable fragment (scFv) binding moiety, where the Raji cells have been engineered with antibodies against B cell marker CD19 or B cell marker CD20.
  • FIG. 10 is a graph plotting the transduction of Jurkat cells by a provided retrovirus having a FITC single-chain variable fragment (scFv) binding moiety, where the Raji cells have been engineered with antibodies against B cell marker CD19 or B cell marker CD20.
  • 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.
  • Encoding the binding moiety with one of the plasmids of the vector system advantageously ensures that all retroviruses produced using the system will include the binding moiety.
  • only one plasmid of the retroviral vector system encodes the binding moiety.
  • two or more different plasmids of the retroviral vector system encode the binding moiety.
  • each different plasmid of the retroviral vector system encodes the binding moiety.
  • 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
  • Th2 cells are involved in coordinating immune responses against extracellular pathogens, like helminths (parasitic worms), by alerting B cells, granulocytes, and mast cells.
  • Th17 cells can produce interleukin 17 (IL-17), a signaling molecule that activates immune and non-immune cells.
  • IL-17 interleukin 17
  • Th17 cells are important for recruiting neutrophils.
  • the retroviral vector system can be engineered to target cell types that mediate cell differentiation and programming. This can allow site-specific differentiation of cells and tissues to repair or regenerate a damaged or aged body.
  • the target cell is a stem cell.
  • the target cell can be, for example, an induced pluripotent stem cell (iPSC), an embryonic stem cell (ESC), an adult stem cell, or a mesenchymal stem cell (MSC).
  • the target cell is a progenitor cell.
  • the target cell can be, for example, a neural progenitor cell, a skeletal progenitor cell, a muscle progenitor cell, a fat progenitor cell, a heart progenitor cell, a chondrocyte, or a pancreatic progenitor cell.
  • the provided retroviral vector system is designed to produce retroviruses that target cells of one or more specific tissues.
  • the target cells include muscle cells.
  • the target cells include neural cells.
  • the target cells include pancreatic cells, e.g., islets.
  • the provided retroviral vector system is designed to produce retroviruses that target two or more different cell types with a different payload delivered to each of the different cell types.
  • a retroviral vector system can include a FITC- binding domain recognizing a FITC-conjugated antibody, where the FITC-conjugated antibody targets a first antigen for delivery of a first payload.
  • the same retroviral vector system can further include a biotin-binding domain recognizing a biotin-conjugated antibody, where the biotin-conjugated antibody targets a second antigen for delivery of a second payload.
  • the provided retroviral vector systems can be used to deliver any genetically encoded material.
  • the systems, and the retroviruses they create can be used to deliver, for example, human genes, chimeric and engineered genes, gene editors, and genes from other species.
  • vector system transfer plasmid genes encode receptors including but not limited to chimeric antigen receptors (CARs) (including first, second, third, or fourth generation receptors), switch receptors, MIMIC receptors, or modified versions thereof.
  • CARs chimeric antigen receptors
  • the transfer vector genes include one or more nucleases such as CRISPR-associated (Cas) proteins or Cas nucleases including type I CRISPR-associated (Cas) polypeptides, type II CRISPR-associated (Cas) polypeptides, type III CRISPR- associated (Cas) polypeptides, type IV CRISPR-associated (Cas) polypeptides, type V CRISPR-associated (Cas) polypeptides, and type VI CRISPR-associated (Cas) polypeptides; zinc finger nucleases (ZFN); transcription activator-like effector nucleases (TALEN); meganucleases; RNA-binding proteins (RBP); CRISPR-associated RNA binding proteins; recombinases; flippases; transposases; Argonaute (Ago) proteins (e.g., prokaryotic Argonaute (pAgo), archaeal Argonaute (aAgo), eukary
  • the transfer vector genes include one or more reporter genes or selectable markers such as a fluorescent protein, an antibiotic resistance gene, or a derivative or fragment thereof.
  • the provided transfer plasmid includes more than one gene in one or more polycistronic element.
  • the retroviral vector system can be used to produce retroviruses that transduce host cells to express more than one gene of a polycistronic element using, for example, IRES or 2A tags.
  • the transfer plasmid includes one or more regulatory elements such as promoters, introns, enhancers, post-transcriptional elements, or post-translational elements, that can regulate transduced gene expression.
  • the viral membrane fusion protein of the provided retroviral vector system envelope plasmid is the vesicular stomatitis virus G (VSV-G) protein, or is an engineered variant thereof.
  • the viral membrane fusion protein is a membrane fusion protein from feline endogenous virus RD114, or is an engineered variant thereof.
  • the viral membrane fusion protein is a membrane fusion protein from baboon endogenous virus BaEV, or is an engineered variant thereof.
  • the viral membrane fusion protein is an engineered variant that does not bind to a cognate binding partner of the corresponding wild-type viral membrane fusion protein.
  • the engineered variant includes one or more, e.g., two or more, point mutations to the sequence of the wild-type viral membrane fusion protein, such that the protein loses its cognate binding ability.
  • the engineered variant is a truncated mutant of the wild-type viral membrane fusion protein, such that the protein loses its cognate binding ability.
  • an integrase enzyme of the provided retroviral vector system is mutated, such that the vector system can be used to produce integrase deficient retroviruses.
  • Retrovirus-Packaging Cells In another aspect of the disclosure, a retrovirus-packaging cell is provided.
  • the retrovirus-packaging cell is a cell that has been transformed with any of the provided retroviral vector systems, e.g., any of those described in Section III.
  • the retrovirus-packaging cell can therefore provide many of the surprising advantages discussed herein, particularly when used in therapeutic applications such as gene or cell therapies.
  • the retrovirus-packaging cell 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 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.
  • a retrovirus in another aspect of the disclosure, can be produced using any of the provided retroviral vector systems and/or retrovirus-packaging systems. Accordingly, the retroviruses can provide the surprising advantages discussed herein, including broad applicability, high specificity and selectivity, switchable targeting, and universal pseudotyping.
  • the provided retrovirus includes a viral membrane fusion protein.
  • the viral membrane fusion protein can be any of those disclosed herein regarding the retroviral vector system.
  • the provided retrovirus further includes a viral genome including one or more genes of interest for transfer from the retrovirus to a target cell.
  • 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.
  • the virus-like particle includes many of the same advantages of the provided retroviruses, including broad applicability, high specificity and selectivity, switchable targeting, and universal pseudotyping. Unlike the provided retroviruses, the virus-like particle does not include a viral genome, and therefore cannot be used to integrate delivered genetic material into the genome of a target cell. The virus-like particles therefore offer an attractive alternative to retroviruses where such integration is not desired.
  • the provided virus-like particles include a binding moiety that can be any of those disclosed herein regarding the retroviral vector system. In some embodiments, the binding moiety includes an antibody binding domain (ABD) of any of the types disclosed herein.
  • ABS antibody binding domain
  • 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.
  • a method for preventing or treating a disease includes administering to a 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.
  • compositions containing retroviral vector systems, retrovirus-packaging cells, retroviruses, or virus-like particles described herein, e.g., in Section IX can be administered for prophylactic and/or therapeutic treatments.
  • these compositions can be administered to a subject already suffering from a disease or condition, in an amount sufficient to cure or at least partially arrest the symptoms of the disease or condition, or to cure, heal, improve, or ameliorate the condition.
  • Amounts effective for this use can vary based on the severity and course of the disease or condition, previous therapy, the subject’s health status, weight, and response to the drugs, and the judgment of the treating physician.
  • Retroviral vector systems, retrovirus-packaging cells, retroviruses, or virus-like particles described herein can be administered before, during, or after the occurrence of a disease or condition, and the timing of administering the composition can vary.
  • a pharmaceutical compositions can be used as a prophylactic and can be administered continuously to subjects with a propensity to conditions or diseases in order to prevent the occurrence of the disease or condition.
  • the pharmaceutical compositions can be administered to a subject during or as soon as possible after the onset of the symptoms. The administration can be initiated within the first 48 hours of the onset of the symptoms, within the first 24 hours of the onset of the symptoms, within the first 6 hours of the onset of the symptoms, or within 3 hours of the onset of the symptoms.
  • the initial administration can be via any route practical, such as by any route described herein using any formulation described herein.
  • a composition can be administered as soon as is practicable after the onset of a disease or condition is detected or suspected, and for a length of time necessary for the treatment of the disease, such as, for example, from about 1 month to about 3 months.
  • the length of treatment can vary for each subject.
  • a wide variety of diseases can be prevented or treated using the provided methods.
  • the methods are suitable for broad cell or gene therapy applications to treat, for example, blood or solid cancer, viral infections, bacterial infections, genetic diseases, wound healing, autoimmunity, regenerative medicine, CNS diseases, and anti-aging.
  • the prevented of treated disease is a genetic disorder.
  • 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.
  • the prevented or treated disease is a cancer.
  • the prevented or treated disease is a cancerous tumor.
  • the cancerous tumor can be a solid cancerous tumor or a liquid cancerous tumor.
  • the liquid cancerous tumor can be, for example, a lymphoma or a leukemia.
  • a tumor treated with the methods disclosed herein can result in stabilized tumor growth (e.g., one or more tumors do not increase more than 1%, 5%, 10%, 15%, or 20% in size, and/or do not metastasize).
  • a tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks.
  • a tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months.
  • 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.
  • Suitable diluents, adjuvants, or carriers can include, for example, lipids, e.g., liposomes, e.g., liposome dendrimers; liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like; gum acacia; gelatin; starch paste; talc; keratin; colloidal silica; urea; and the like.
  • suitable diluents include distilled water, buffered water, physiological saline, PBS, Ringer's solution, dextrose solution, and Hank's solution.
  • 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 2 An embodiment of embodiment 1, wherein the viral membrane fusion protein is an engineered variant of a wild-type viral membrane fusion protein.
  • Embodiment 3 An embodiment of embodiment 2, wherein the engineered variant does not bind to a cognate binding partner of the wild-type viral membrane fusion protein.
  • Embodiment 4 An embodiment of embodiment 2 or 3, wherein the engineered variant is a truncated mutant of the wild-type viral membrane fusion protein.
  • Embodiment 5 An embodiment of any one of embodiments 1-4, wherein the binding moiety comprises an extramembrane domain and a transmembrane domain.
  • Embodiment 6 An embodiment of any one of embodiments 1-5, wherein the envelope plasmid encodes the binding moiety.
  • Embodiment 7 An embodiment of any one of embodiments 1-5, wherein the packaging plasmid encodes the binding moiety.
  • Embodiment 8 An embodiment of any one of embodiments 1-7, wherein the binding moiety binds a ligand comprising an antibody, an antibody mimetic, a single-chain variable fragment (scFv), or a derivate or fragment thereof, and wherein the ligand binds the surface feature of the target cell.
  • scFv single-chain variable fragment
  • Embodiment 9 An embodiment of embodiment 8, wherein the binding moiety comprises a fluorescein isothiocyanate (FITC)-binding domain, and wherein the ligand is FITC-conjugated.
  • Embodiment 10 An embodiment of embodiment 9, wherein the binding moiety comprises an anti-fluorescein scFv or fluorescein-binding anticalin.
  • Embodiment 11 An embodiment of embodiment 9 or 10, wherein the ligand is an FITC-conjugated antibody.
  • Embodiment 12 An embodiment of embodiment 8, wherein the binding moiety comprises a biotin-binding domain, and wherein the ligand is biotinylated.
  • Embodiment 13 An embodiment of embodiment 12, wherein the binding moiety comprises an anti-biotin scFv, a polyclonal anti-biotin antibody, a monoclonal anti-biotin antibody, biotin-binding anticalin, or an avidin-family protein.
  • Embodiment 14 An embodiment of embodiment 12 or 13, wherein the ligand is a biotinylated antibody.
  • Embodiment 15 An embodiment of any one of embodiments 8-14, wherein the surface feature of the target cell comprises CD3, CD4, CD7, CD8, CD19, CD20, CD56, CD71, or CTLA4.
  • Embodiment 16 An embodiment of any one of embodiments 1-7, wherein the binding moiety directly binds the surface feature of the target cell.
  • Embodiment 17 An embodiment of embodiment 16, wherein the binding moiety comprises an scFv or a nanobody.
  • Embodiment 18 An embodiment of embodiment 16 or 17, wherein the surface feature of the target cell comprises CD19 or CD7.
  • Embodiment 19 An embodiment of any one of embodiments 1-18, wherein the target cell is a T cell, a B cell, a natural killer (NK) cell, an astrocyte, a dendritic cell (DC), or a monocyte.
  • NK natural killer
  • DC dendritic cell
  • Embodiment 20 An embodiment of any one of embodiments 1-19, wherein the retrovirus is an alpharetrovirus, a gammaretrovirus, or a lentivirus.
  • Embodiment 21 An embodiment of any one of embodiments 1-20, wherein the viral membrane fusion protein is the vesicular stomatitis virus G (VSV-G) protein, is from feline endogenous virus RD114, or is from baboon endogenous virus BaEV.
  • Embodiment 22 An embodiment of any one of embodiments 1-21, wherein the retrovirus is an integrase-deficient retrovirus.
  • Embodiment 23 An embodiment of any one of embodiments 1-22, wherein the one or more genes of interest encode a chimeric antigen receptor (CAR), a switch receptor, or a derivative or fragment thereof.
  • Embodiment 24 An embodiment of any one of embodiments 1-23, wherein the one or more genes of interest encode a CRISPR-associated (Cas) protein, a zinc finger nuclease (ZFN).
  • Cas CRISPR-associated protein
  • ZFN zinc finger nuclease
  • TALEN transcription activator-like effector nuclease
  • RBP RNA- binding protein
  • a recombinase a flippase
  • base editor a prime editor
  • nuclease- impaired Cas nuclease-dead Cas
  • CRISPRa/i transcriptional modifier
  • transposase an Argonaute (Ago) protein, an adenosine deaminase acting on RNA (ADAR), a Pumilio RNA-binding family (PUF) protein, a homing endonuclease, or a derivative or fragment thereof.
  • ADAR adenosine deaminase acting on RNA
  • PEF Pumilio RNA-binding family
  • Embodiment 25 An embodiment of any one of embodiments 1-24, wherein the one or more genes of interest encode a fluorescent protein, an antibiotic resistance gene, or a derivative or fragment thereof.
  • Embodiment 26 A retrovirus-packaging cell comprising the retroviral vector system of any one of embodiments 1-25.
  • 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.
  • Embodiment 29 An embodiment of embodiment 28, wherein the engineered variant does not bind to a cognate binding partner of the wild-type viral membrane fusion protein.
  • Embodiment 30 An embodiment of embodiment 28 or 29, wherein the engineered variant is a truncated mutant of the wild-type viral membrane fusion protein.
  • Embodiment 31 An embodiment of any one of embodiments 27-30, wherein the binding moiety comprises an extramembrane domain and a transmembrane domain.
  • Embodiment 32 An embodiment of embodiment 29 or 30, wherein the engineered variant of the wild-type viral membrane fusion protein comprises the binding moiety.
  • Embodiment 33 An embodiment of any one of embodiments 27-32, wherein the binding moiety comprises a fluorescein isothiocyanate (FITC)-binding domain, and wherein the ligand is FITC-conjugated.
  • Embodiment 34 An embodiment of embodiment 33, wherein the binding moiety comprises an anti-fluorescein scFv or fluorescein-binding anticalin.
  • Embodiment 35 An embodiment of embodiment 33 or 34, wherein the ligand is an FITC-conjugated antibody.
  • Embodiment 36 An embodiment of any one of embodiments 27-32, wherein the binding moiety comprises a biotin-binding domain, and wherein the ligand is biotinylated.
  • Embodiment 37 An embodiment of embodiment 36, wherein the binding moiety comprises an anti-biotin scFv, biotin-binding anticalin, or an avidin-family protein.
  • Embodiment 38 An embodiment of embodiment 36 or 37, wherein the ligand is a biotinylated antibody.
  • Embodiment 39 An embodiment of any one of embodiments 27-38, wherein the surface feature of the target cell comprises CD3, CD4, CD7, CD8, CD19, CD20, CD56, CD71, or CTLA4.
  • Embodiment 40 An embodiment of any one of embodiments 27-39, wherein the retrovirus is an alpharetrovirus, a gammaretrovirus, or a lentivirus.
  • Embodiment 41 An embodiment of any one of embodiments 27-40, wherein the viral membrane fusion protein is the vesicular stomatitis virus G (VSV-G) protein, is from feline endogenous virus RD114, or is from baboon endogenous virus BaEV.
  • VSV-G vesicular stomatitis virus G
  • Embodiment 42 An embodiment of any one of embodiments 27-41, wherein the retrovirus is an integrase-deficient retrovirus.
  • Embodiment 43 An embodiment of any one of embodiments 27-42, wherein the one or more genes of interest encode a chimeric antigen receptor (CAR), a switch receptor, or a derivative or fragment thereof.
  • Embodiment 44 An embodiment of any one of embodiments 27-43, wherein the one or more genes of interest encode a CRISPR-associated (Cas) protein, a zinc finger nuclease (ZFN).
  • Cas CRISPR-associated protein
  • ZFN zinc finger nuclease
  • TALEN transcription activator-like effector nuclease
  • RBP RNA-binding protein
  • TALEN transcription activator-like effector nuclease
  • RBP RNA-binding protein
  • a recombinase a flippase
  • base editor a prime editor
  • nuclease-impaired Cas nuclease-dead Cas
  • CRISPRa/i transcriptional modifier
  • transposase a transcription activator-like effector nuclease
  • ADAR adenosine deaminase acting on RNA
  • PEF Pumilio RNA-binding family
  • Embodiment 45 A virus-like particle comprising 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 a target cell.
  • Embodiment 46 An embodiment of embodiment 45, wherein the binding moiety comprises a fluorescein isothiocyanate (FITC)-binding domain, and wherein the ligand is FITC-conjugated.
  • Embodiment 47 An embodiment of embodiment 46, wherein the binding moiety comprises an anti-fluorescein scFv or fluorescein-binding anticalin.
  • Embodiment 48 An embodiment of embodiment 46 or 47, wherein the ligand is an FITC-conjugated antibody.
  • Embodiment 49 An embodiment of embodiment 45, wherein the binding moiety comprises a biotin-binding domain, and wherein the ligand is biotinylated.
  • Embodiment 50 An embodiment of embodiment 49, wherein the binding moiety comprises an anti-biotin scFv, biotin-binding anticalin, or an avidin-family protein.
  • Embodiment 51 An embodiment of embodiment 49 or 50, wherein the ligand is a biotinylated antibody.
  • Embodiment 52 An embodiment of any one of embodiments 45-51, wherein the surface feature of the target cell comprises CD3, CD4, CD7, CD8, CD19, CD20, CD56, CD71, or CTLA4.
  • Embodiment 53 An embodiment of any one of embodiments 45-52, comprising a CRISPR-associated (Cas) protein, a zinc finger nuclease (ZFN).
  • Cas CRISPR-associated protein
  • ZFN zinc finger nuclease
  • TALEN transcription activator-like effector nuclease
  • RBP RNA-binding protein
  • TALEN transcription activator-like effector nuclease
  • RBP RNA-binding protein
  • a recombinase a flippase
  • base editor a prime editor
  • nuclease-impaired Cas nuclease-dead Cas
  • CRISPRa/i transcriptional modifier
  • transposase a transcription activator-like effector nuclease
  • ADAR adenosine deaminase acting on RNA
  • PEF Pumilio RNA-binding family
  • Embodiment 54 A method for producing the retroviral vector system of any one of embodiments 1-25, the method comprising transfecting a host cell with the retroviral vector system of any one of embodiments 1-25.
  • Embodiment 55 A method for preventing or treating a disease in a subject, the method comprising administering to the subject the retroviral vector system of any one of embodiments 1-25, the retrovirus-packaging cell of embodiment 26, the retrovirus of any one of embodiments 27-44, or the virus-like particle of any one of embodiments 45-53.
  • Embodiment 56 An embodiment of embodiment 55, wherein the disease comprises a genetic disorder.
  • Embodiment 57 An embodiment of embodiment 56, wherein the disease comprises a cancer.
  • Embodiment 58 An embodiment of embodiment 57, wherein the cancer comprises a solid tumor.
  • Embodiment 59 An embodiment of embodiment 55, wherein the disease comprises an infection.
  • Embodiment 60 An embodiment of embodiment 59, wherein the infection is associated with a biomedical device implant of the subject.
  • Embodiment 61 A method for repairing or regenerating a damaged or aged tissue of a subject, the method comprising administering to the subject the retroviral vector system of any one of embodiments 1-25, the retrovirus-packaging cell of embodiment 26, the retrovirus of any one of embodiments 27-44, or the virus-like particle of any one of embodiments 45-53, wherein the target cell comprises an induced pluripotent stem cell, an embryonic stem cell, an adult stem cell, a mesenchymal stem cell, or a progenitor cell.
  • Embodiment 62 An embodiment of embodiment 61, wherein the damaged or aged tissue comprises muscle cells, neural cells, or pancreatic cells.
  • Example 1 Transduction of lymphocytes by a retrovirus with a cell binding domain
  • a retrovirus in accordance with a provided embodiment was created having a CD7- targeting nanobody as a cell binding domain. Wild-type Jurkat cells that do not natively express GFP (FIG. 3, top row) were transduced with a traditional lentivirus having VSV-G envelope protein and encoding an expression cassette for GFP. The cells transduced with the traditional lentivirus exhibited GFP activity (FIG. 3, second row).
  • the VSV-G of the lentivirus was then mutated to ablate its natural binding ability.
  • the mutated lentivirus was unable to effectively transduce the Jurkat cells (FIG. 3, third row).
  • a feline endogenous virus pseudotyped retrovirus (RD114) was also unable to transduce the cells (FIG. 3, fourth row).
  • the provided retrovirus with the CD7 cell binding domain however, produced transduction results similar to that seen with the traditional lentivirus (FIG. 3, bottom row).
  • NK cells Transduction of NK cells by a retrovirus with a cell binding domain
  • Natural killer (NK) cells from two donors were engineering ex vivo with a chimeric receptor protein based on Toll Like Receptor 5 using two different retroviruses.
  • the first retrovirus a traditional lentivirus having VSV-G envelope protein, exhibited a transduction efficiency of less than 10% for each of the two donor populations of NK cells (FIG. 4).
  • the second retrovirus a retrovirus having a CD7-binding cell binding domain in accordance with a provided embodiment, exhibited 2-fold to 8-fold higher transduction efficiency than seen with the traditional lentivirus (FIG. 4).
  • Example 4 Targeting of different cell markers and epitopes with a universal pseudotyped retrovirus [0184] A universal pseudotyped retrovirus was created in accordance with a provided embodiment, where the retrovirus included a FITC-binding scFv and an mCherry expression cassette. The retrovirus was used to transduce Jurkat cells (FIG.
  • a universal pseudotyped retrovirus was produced in accordance with a provided embodiment, where the retrovirus included a FITC-binding scFv and an mCherry expression cassette.
  • a transfer plasmid containing the genetic payload of interest, a packaging plasmid, and modified envelope plasmid, as described in Section III are transfected into HEK293T cells.
  • the modified envelope plasmid is mutated to ablate the binding properties of VSV-G and engineered to encode a binding moiety.
  • the binding moiety can be one configured or selected to bind a cell marker directly, or can be one configured or selected to bind a moiety contained on a secondary binder such as a biotin- or FITC- conjugated antibody.
  • the virus is isolated and purified.
  • the purified virus and secondary binder are incubated together for a suitable period of time prior to administration.
  • the payload of interest is GFP
  • the binding moiety is either a CD7 nanobody (nbCD7) or a monomeric streptavidin (mSA).
  • nbCD7 CD7 nanobody
  • mSA monomeric streptavidin
  • the virus is incubated with biotin conjugated CD7 antibody for 30 min.
  • the virus is injected into mice intravenously. About one week later, the spleens and peripheral blood of the mice are taken and processed to isolate immune cells (splenocytes or peripheral blood mononuclear cells respectively).

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Abstract

L'invention concerne des systèmes de vecteurs rétroviraux utiles pour produire des rétrovirus pseudotypés universels pour des thérapies cellulaires et géniques. Les systèmes vectoriels comprennent un plasmide d'enveloppe et un plasmide d'encapsidation, au moins l'un de ces plasmides codant pour une fraction de liaison qui se lie directement à une cellule cible par l'intermédiaire d'un domaine de liaison cellulaire, ou qui se lie indirectement à une cellule cible par l'intermédiaire d'un domaine de liaison d'anticorps. L'invention concerne également des cellules d'encapsidation de rétrovirus associées, des rétrovirus, des particules de type viral, ainsi que des procédés pour leur production et des méthodes pour leur utilisation dans la prévention ou le traitement d'une maladie.
PCT/US2023/073983 2022-09-12 2023-09-12 Composition et méthode de rétrovirus pseudotypés universels WO2024059569A2 (fr)

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