Materials and methods for viral manipulation

Abstract

The present invention provides materials and methods for viral manipulation, including, for example, the production of vectors and viral particles useful for gene therapy.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority to U.S. Provisional Patent Application No. 63 / 511,977, filed on 5 July 2023, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Retroviruses are RNA viral molecules that infect host cells, reverse transcribe their RNA molecules into DNA, and encapsulate their genome in a viral protein envelope that stably integrates it into the host cell's genome. Systems for packaging retroviral vectors into viral particles to form virions have been developed to facilitate the transfer of exogenous genes into target cells for gene therapy purposes. Retroviral vectors are modified DNA or RNA that serve as vectors for recombinant DNA. Retroviral vectors can transfer genes into a wide range of cell types from many different species. [Overview of the Initiative]

[0003] The present invention relates, in part, to a method for manipulating a virus to include two or more envelope glycoproteins.

[0004] In some embodiments, the present invention relates to a method for producing a multipseudotype virus, comprising: providing a mammalian cell line; adding a cell medium to the mammalian cell line; contacting the mammalian cell line with a composition comprising at least one packaging plasmid encoding at least one glycoprotein; incubating the mammalian cell line with the composition; and recovering the multipseudotype virus 48 hours and 72 hours after contacting the mammalian cell line with the composition, wherein the recovery of the multipseudotype virus yields a virus with a higher titer compared to a method for producing a virus containing a single pseudotype.

[0005] In one embodiment, culturing is carried out at a temperature of approximately 37°C in a humidified incubator with approximately 5% to 10% CO2 for approximately 24 to 36 hours. In one embodiment, a composition comprising at least one packaging plasmid encoding at least one glycoprotein further comprises a transfer vector comprising a gene encoding the transgene. In one embodiment, the at least one glycoprotein comprises a first glycoprotein selected from the group consisting of ecotropic virus envelope glycoproteins and amphotropic envelope glycoproteins. In one embodiment, the at least one glycoprotein comprises a first glycoprotein selected from the group consisting of RD114, VSV-G, 4070A, gap70, 10A1, and GALV. In one embodiment, the at least one glycoprotein is two glycoproteins, three glycoproteins, or four glycoproteins. In one embodiment, the at least one glycoprotein comprises a second glycoprotein selected from the group consisting of ecotropic virus envelope glycoproteins and amphotropic envelope glycoproteins, and is different from the first glycoprotein. In one embodiment, at least one glycoprotein comprises a second glycoprotein selected from the group consisting of RD114, VSV-G, 4070A, gap70, 10A1, and GALV, and is different from the first glycoprotein. In one embodiment, the two glycoproteins are RD114 and VSV-G. In one embodiment, the two glycoproteins are GALV and VSV-G. In one embodiment, the two glycoproteins are GALV and 10A1. In one embodiment, at least one packaging plasmid encoding at least one glycoprotein is in a ratio of about 1 / 5 to 1 / 10 compared to the total DNA.

[0006] In some embodiments, the present invention relates to a method for producing a multipseudotype virus, comprising: providing a mammalian cell line for transfection and virus production; culturing the mammalian cell line, wherein the culturing is performed in a humidified incubator at a temperature of about 37°C and a CO2 content of about 5% to about 10%; treating the mammalian cell line with trypsin; adding a cell medium to the mammalian cell line; contacting the mammalian cell line with a composition comprising i) a transfer vector containing a transgene, and ii) at least one packaging plasmid encoding at least one glycoprotein; incubating the mammalian cell line with the composition; and recovering the multipseudotype virus 48 hours and 72 hours after contacting the mammalian cell line with the composition, wherein the recovery of the multipseudotype virus yields a virus with a higher titer compared to a method for producing a virus containing a single pseudotype.

[0007] In some embodiments, the method further comprises filtering the multipseudotype virus, which is carried out using a low-protein-binding filter. In one embodiment, the filtration is carried out using a filter containing cellulose acetate or polysulfonate. In some embodiments, the method further comprises concentrating the virus. In some embodiments, the method produces multipseudotype viruses with titers about 20, 30, 40, 50, 60, 70, 80, or 90 times higher than methods for producing viruses containing a single envelope glycoprotein, including RD114, VSV-G, GALV, or 10A1.

[0008] In some embodiments, the virus includes retroviruses.

[0009] In some embodiments, the present invention relates to an engineered multipsydetype virus comprising at least two envelope glycoproteins. In one embodiment, the engineered multipsydetype virus is produced by the method of the present invention.

[0010] In some embodiments, the present invention relates to a method for increasing the transduction efficiency in γδT cells, the method comprising providing engineered viral particles, wherein the viral particles comprise a protein-encoding nucleic acid and a multipseudotype virus; providing negatively isolated γδT cells for transduction; and transduction of the engineered viral particles into γδT cells. In some embodiments, the protein comprises a chimeric antigen receptor.

[0011] In some embodiments, the present invention relates to a system for carrying out a method for generating γδT cells containing a transgene, the system comprising one or more components capable of carrying out a method comprising the steps of: providing γδT cells; activating the γδT cells for about 24 to 48 hours using about 1000 U of IL2 or OKT3 antibody; transducing the γδT cells with an engineered multipseudotype virus containing a sequence encoding the transgene; and analyzing the transduction efficiency, wherein the transduction efficiency is increased by the multipseudotype virus compared to transduction by a singlepseudotype virus. In some embodiments, the virus includes a γ retrovirus.

[0012] In some embodiments, the present invention is a method for increasing virus titer, comprising providing a mammalian cell line, adding a cell culture medium to the mammalian cell line, providing a composition containing a transfer vector comprising a gene encoding a transgene to the mammalian cell line, providing a first packaging plasmid encoding at least one glycoprotein to the mammalian cell line, contacting the mammalian cell line with a composition comprising at least a first packaging plasmid encoding at least one glycoprotein to the mammalian cell line, incubating the mammalian cell line with the composition, and recovering a multi-pseudotype virus 48 hours and 72 hours after contacting the mammalian cell line with the composition, wherein recovering the multi-pseudotype virus results in a higher titer of virus compared to a method of producing a virus comprising a single pseudotype.

[0013] In some embodiments, the multi-pseudotype virus comprises two or more envelope glycoproteins, is produced at an enhanced titer, and includes the properties of the envelope glycoproteins. In some embodiments, one or more envelope glycoproteins are modified to provide different binding properties to the virus. In some embodiments, the modification is cleavage, masking, or redirection. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The following detailed description of preferred embodiments of the present invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the present invention, the drawings show currently preferred embodiments. However, it should be understood that the present invention is not limited to the precise arrangements and instrumentalities of the illustrated embodiments. [Figure 1]A representative bar graph is drawn showing that RD114+VSV-G dual pseudotype gamma-retrovirus produced a higher titer of virus supernatant when compared with RD114 single pseudotype gamma-retrovirus. The dual pseudotype virus has a titer approximately 79-fold higher than the single pseudotype virus. [Figure 2] A representative bar graph is drawn showing that the RD114+VSV-G dual pseudotype gamma-retrovirus (central bar graph) exhibited transduction efficiency comparable to that of RD114 single pseudotype gamma-retrovirus-mediated transduction (left bar graph). The VSV-G single pseudotype gamma-retrovirus did not sufficiently transduce primary gamma delta T cells (right bar graph). (1 (Donor 1), 2 (Donor 2), 3 (Donor 3), 4 (Donor 4), 5 (alpha beta T cell donor). [Figure 3] A representative line graph is drawn showing that, using primary GD T cell samples from four healthy donors, RD114+VSV-G dual pseudotype gRV-transduced CAR T showed tumor-associated antigen (TAA)-dependent killing similar to that of RD114 single pseudotype gamma-retrovirus-transduced CAR T (Light gray circles: tumor only, black squares: GD donor 1 dual pseudotype, black triangles: GD donor 1 RE114 pseudotype, gray inverted triangles: GD donor 1 non-transduced, black diamonds: GD donor 2 dual pseudotype, white circles: GD donor 2 RD pseudotype, gray squares: GD donor 2 non-transduced, white triangles: donor 3 dual pseudotype, white inverted triangles: GD donor 3 RD pseudotype, gray diamonds: GD donor 3 non-transduced, black circles: GD donor 4 dual pseudotype, black stars: GD donor 4 RD pseudotype, and gray stars: GD donor 4 non-transduced). The left panel is antigen-positive and the right panel is antigen-negative. [Figure 4]Using primary GD T cell samples from four healthy donors, we create a representative line graph showing that RD114+VSV-G dual pseudotype gRV-transduced CAR T cells exhibited similar TAA-dependent death to RD114 single pseudotype gamma retrovirus-transduced CAR T cells. (Light gray circles: tumors only, black squares: GD donor 1 dual pseudotype, black triangles: GD donor 1 RE114 pseudotype, gray inverted triangles: GD donor 1 untransduced, black diamonds: GD donor 2 dual pseudotype, white circles: GD donor 2 RD114 pseudotype, gray squares: GD donor 2 untransduced, white triangles: donor 3 dual pseudotype, white inverted triangles: GD donor 3 RD114 pseudotype, gray diamonds: GD donor 3 untransduced, black circles: GD donor 4 dual pseudotype, black stars: GD donor 4 RD114 pseudotype, and gray stars: GD donor 4 untransduced). The left panel is antigen-positive, and the right panel is antigen-negative. [Figure 5] Using primary GD T cell samples from four healthy donors, we create representative line graphs demonstrating that RD114+VSV-G dual pseudotype gRV-transduced CAR T cells exhibited TAA-dependent cytokine secretion similar to that of RD114 single pseudotype gamma retrovirus-transduced CAR T cells. The experiment was conducted under antigen-positive conditions. (1: Donor 1, 2: Donor 2, 3: Donor 3, 4: Donor 4, 5: Donor 5, AB T cell donor, 6: Tumor only). [Figure 6] Using primary GD T cell samples from four healthy donors, we create representative line graphs demonstrating that RD114+VSV-G dual pseudotype gRV-transduced CAR T cells exhibited TAA-dependent cytokine secretion similar to that of RD114 single pseudotype gamma retrovirus-transduced CAR T cells. The experiment was conducted under antigen-negative conditions. (1: Donor 1, 2: Donor 2, 3: Donor 3, 4: Donor 4, 5: Donor 5, AB T cell donor, 6: Tumor only). [Modes for carrying out the invention]

[0015] The inventors of the present invention recognized, based on the successful transduction of target cells for gene therapy in humans, that viral packaging cell lines should (i) produce a large amount of retroviral particle supernatant, and (ii) produce viral particles that efficiently transduce target cells. The inventors addressed the need for such materials and methods, including cell lines and methods relating thereto, thereby satisfying the need in the art for improved methods of gene therapy, particularly for the treatment or prevention of disorders. The present invention provides solutions to these problems and other problems plaguing the art.

[0016] The present invention relates in part to the development of improved methods for recombinant retroviral transduction. In one embodiment, the present invention includes retroviruses pseudotyped with one or more envelope glycoproteins, as well as methods for producing and using the same. In one embodiment, the retrovirus is a gamma retrovirus. In one embodiment, the retrovirus is a lentivirus. In one embodiment, the retrovirus is pseudotyped with one or more envelope glycoproteins, variants thereof, or fragments thereof. In one embodiment, the retrovirus is pseudotyped with two envelope glycoproteins, three envelope glycoproteins, four envelope glycoproteins, five envelope glycoproteins, six envelope glycoproteins, seven envelope glycoproteins, eight envelope glycoproteins, nine envelope glycoproteins, or ten or more envelope glycoproteins. In one embodiment, the envelope glycoproteins are selected from the group consisting of ecotropic virus envelope glycoproteins and amphotropic envelope glycoproteins. In one embodiment, the envelope glycoprotein is selected from the group consisting of RD114, VSV-G, 4070A, gap70, and 10A1. In one embodiment, more than one selected envelope glycoprotein is different. In one embodiment, the retrovirus is pseudotyped with RD114 envelope glycoprotein and VSV-G envelope glycoprotein. In one embodiment, the retrovirus is pseudotyped with GALV envelope glycoprotein and VSV-G envelope glycoprotein. In one embodiment, the retrovirus is pseudotyped with GALV envelope glycoprotein and 10A1 envelope glycoprotein. In one embodiment, the envelope glycoprotein is further modified.

[0017] The present invention further relates to a method for producing a high-titer retrovirus. In one embodiment, the retrovirus comprises one or more envelope glycoproteins. In one embodiment, the method comprises the use of nucleic acids encoding the envelope glycoproteins. In one embodiment, the nucleic acids encoding the envelope glycoproteins are located in a plasmid. In one embodiment, one or more envelope glycoproteins are encoded. In one embodiment, the one or more envelope glycoproteins are not identical. In one embodiment, the one or more envelope glycoproteins are encoded by the use of one or more nucleic acids. In one embodiment, each of the one or more nucleic acids encodes an envelope glycoprotein. In one embodiment, the one or more envelope glycoproteins are encoded by the use of nucleic acids encoding one or more envelope glycoproteins. In one embodiment, the titer of the retrovirus can be further enriched.

[0018] In one embodiment, the present invention relates to a method for producing a high-titer retrovirus, the method comprising transfecting a host cell with one or more nucleic acid molecules encoding one or more envelope glycoproteins.

[0019] The present invention further relates to an improved method for transducing cells with a polynucleotide of interest. In one embodiment, the method for transducing cells involves the use of a retrovirus pseudotyped with one or more envelope glycoproteins. In one embodiment, the cells to be transduced are immune cells. In one embodiment, the method for transducing cells involves contacting the cells of interest with a retrovirus pseudotyped with one or more envelope glycoproteins. In one embodiment, the immune cells are gamma delta T cells (γδ T cells). In one embodiment, the polynucleotide of interest encodes a chimeric antigen receptor.

[0020] The present invention further relates to the use of transduced cells for cell therapy. In one embodiment, cells are transduced using a retrovirus pseudotyped with one or more envelope glycoproteins. In one embodiment, the transduced cells are immune cells. In one embodiment, the immune cells are T cells. In one embodiment, the immune cells are transduced with a chimeric antigen receptor. In one embodiment, the immune cells transduced with a chimeric antigen receptor kill tumor cells. In one embodiment, the transduced immune cells are introduced into a target.

[0021] The present invention also relates to the use of transduced cells for any type of cell therapy. In one embodiment, transduced cells can be used to deliver any desired cargo. For example, the cargo may include, but is not limited to, proteins, CRISPR-CAS systems, gene editing systems, CARs, TCRs, etc. Therefore, the present invention is not limited to any type of cargo.

[0022] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the invention pertains. Terms used herein in describing the invention are used solely for the purpose of describing specific embodiments and are not intended to limit the invention.

[0023] As used herein, each of the following terms shall have the meaning associated with it in this section.

[0024] The articles "a" and "an" are used herein to refer to one or more than one (i.e., at least one) object articles. For example, "an element" means one or more elements.

[0025] Furthermore, unless otherwise specified, the use of "or" means "and / or". Similarly, "comprise", "comprises", "comprising", "include", "includes", and "including" are interchangeable and not intended to be limiting.

[0026] As used herein, the terms “about” or “approximately” refer to a quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length that varies by 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% relative to the reference quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length. In one embodiment, the terms “about” or “approximately” refer to a range of quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% relative to the reference quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length.

[0027] As used herein, the term “virus” is intended to mean a physical virus or a retroviral particle. A “retrovirus” is a virus that has an RNA genome.

[0028] "Lentivirus" refers to a genus of retroviruses that can infect both dividing and non-dividing cells. "Dividing" cells are those that undergo active mitosis or meiosis. "Non-dividing" cells refer to those that do not undergo mitosis. Some examples of lentiviruses include HIV (human immunodeficiency virus, including HIV types 1 and HIV 2), the pathogen of acquired immunodeficiency syndrome (AIDS); Visna-Maedi, which causes encephalitis (visna) or pneumonia (maedi) in sheep; Caprin arthritis-encephalitis virus, which causes immunodeficiency, arthritis, and encephalopathy in goats; Equine infectious anemia virus, which causes autoimmune hemolytic anemia and encephalopathy in horses; feline immunodeficiency virus (FIV), which causes immunodeficiency in cats; bovine immunodeficiency virus (BIV), which causes lymphadenopathy, lymphocytosis, and sometimes central nervous system infection in cattle; and simian immunodeficiency virus (SIV), which causes immunodeficiency and encephalopathy in great apes.

[0029] "Gamma retrovirus" refers to a genus of the family Retroviridae. Examples of gamma retroviruses include, but are not limited to, mouse stem cell virus, mouse leukemia virus, feline leukemia virus, feline sarcoma virus, and reticuloendotheliosis virus.

[0030] As used herein, “hybrid virus” refers to a virus having components from one or more other viral vectors, including an element from a non-retroviral vector, such as an adenovirus-retrovirus hybrid. As used herein, a hybrid vector having a retroviral component is considered to fall within the scope of a retrovirus.

[0031] As used herein, the viral "envelope" protein, or "Env" protein, refers to any polypeptide sequence present on the surface lipid bilayer of a retroviral virion, whose function is to mediate its adsorption to and penetration into infection-susceptible host cells. The retroviral envelope is formed by a cell-derived lipid bilayer into which proteins encoded by the env region of the viral genome are inserted. Envelope proteins are typically glycoproteins and usually contain transmembrane (TM) and surface (SU) components linked together by disulfide bonds. The viral structure is described in detail, for example, Coffin, et al., Retroviruses, 1997, Cold Spring Harbor Laboratory Press.

[0032] As used herein, the viral "capsid" refers to the major structural protein of the virion core, derived from the central region of the Gag polyprotein. In mature viral particles, the capsid protein forms a shell surrounding the ribonucleoprotein complex containing the genomic nucleic acid. This shell, containing additional proteins, is also referred to as the capsid. The capsid shell can exist as a component of the virion without surrounding the genomic nucleic acid.

[0033] In this specification, “virion,” “viral particle,” and “retroviral particle” are used to refer to a single virus comprising an RNA genome, a pol gene-derived protein, a gag gene-derived protein, and a lipid bilayer presenting an envelope (glyco) protein. The RNA genome is typically a recombinant RNA genome and may therefore contain RNA sequences that are exogenous with respect to the native viral genome. The RNA genome may also contain deficient endogenous viral sequences.

[0034] As used herein, the terms “pseudotyped” or “pseudotyping” refer to a virus in which the viral envelope protein is replaced with the envelope protein of another virus having preferred characteristics. A “pseudotyped” retrovirus is a retroviral particle having an envelope protein derived from a virus other than the RNA genome from which the retroviral genome originates. The envelope protein may be derived from, for example, different retroviral or non-retroviral origins, but is not limited to these. The envelope protein may be a native envelope protein or an envelope protein that has been modified, mutated, or manipulated as described herein. Thus, a pseudotyping element as used herein may include a “binding polypeptide” comprising one or more polypeptides (typically glycoproteins) that identify and bind to a target host cell, and one or more “fusion polypeptides” that mediate the fusion of the retroviral membrane with the target host cell membrane, thereby enabling the retroviral genome to enter the target host cell.

[0035] As used herein, the terms “packaging signal” or “packaging sequence” refer to a sequence located within the retroviral genome required for the insertion of viral RNA into a viral capsid or particle; see, for example, Clever et al., 1995. J. of Virology, Vol. 69, No. 4; pp. 2101-2109. Some retroviral vectors use the minimal packaging signal (psi[Ψ] or [Ψ] required for capsid formation of the viral genome. + The ] sequence (also referred to as the ] sequence is used. Therefore, as used herein, the terms “packaging sequence,” “packaging signal,” “psi,” and the symbol “Ψ” are used in reference to non-coding sequences required for capsid formation of the retroviral RNA strand during viral particle formation.

[0036] The terms “vector,” “vector construct,” and “expression vector” refer to a vehicle capable of introducing nucleic acid molecules, such as DNA or RNA sequences (e.g., foreign genes), into host cells in order to transform the host and promote the expression (e.g., transcription and translation) of the introduced sequence. Vectors typically contain DNA of a transmissible material into which foreign DNA encoding a protein has been inserted by restriction enzyme technique. A common type of vector is a “plasmid,” which is generally a self-contained molecule of double-stranded DNA that can readily accept additional (foreign) DNA and be readily introduced into a suitable host cell.

[0037] The term "viral vector" can refer to a virus or viral particle capable of transferring nucleic acids into a cell, or the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and / or functional genetic elements primarily derived from viruses. As will be apparent to those skilled in the art, the term "viral vector" is broadly used to refer to either a nucleic acid molecule (e.g., a transfer plasmid) containing a virus-derived nucleic acid element that facilitates the transfer or integration of nucleic acid molecules into a cell's genome, or a viral particle that mediates nucleic acid transfer. Viral particles typically contain various viral components, and sometimes host cell components, in addition to nucleic acids.

[0038] As used herein, the term “packaging vector” refers to an expression vector or viral vector that lacks a packaging signal and contains polynucleotides encoding one, two, three, four, or more viral structural genes and / or accessory genes. Typically, a packaging vector is contained in a packaging cell and introduced into the cell via transfection, transduction, or infection. Methods for transfection, transduction, or infection are well known to those skilled in the art.

[0039] As used herein, the term “cell line” refers to cultured cells that can be passaged (divided) more than once.

[0040] As used herein, the term “packaging cell line” refers to a cell line that does not contain a packaging signal but stably or transiently expresses viral structural proteins and replication enzymes (e.g., gag, pol, and env) necessary for the precise packaging of viral particles.

[0041] As used herein, the term "producer cell line" refers to a cell line capable of producing recombinant retroviral particles, including a packaging cell line and a transfer vector construct containing a packaging signal.

[0042] As defined herein, “transformation” describes the process by which exogenous DNA enters a target cell. Transformation may depend on any known method for inserting an exogenous nucleic acid sequence into a prokaryotic or eukaryotic host cell and may include, but is not limited to, viral infection, electroporation, heat shock, lipofection, and particle impact. “Transformed” cells include stably transformed cells in which the inserted nucleic acid is capable of replicating either as an autonomously replicating plasmid or as part of a host chromosome. Cells that transiently express the gene of interest are also included.

[0043] As used herein, the terms “transfect” or “transfection” are intended to mean the transfer of at least one extrinsic nucleic acid into a cell. The nucleic acid may be RNA, DNA, or a combination of both. An extrinsic nucleic acid refers to a nucleic acid not found as a result of host cell division or proliferation.

[0044] As used herein, the terms “transient expression” and “transient expression” are intended to mean the transient expression period of the genetic material and / or that the genetic material is not permanently and stably integrated into the host cell’s genome and therefore does not have the same expressive capacity over time as the host cell’s innate genetic material.

[0045] The delivery of genes or other polynucleotide sequences using retroviral or lentiviral vectors via viral infection rather than transfection is called "transduction." A target cell is "transduced" if it contains genes or other polynucleotide sequences delivered to the cell by infection using a virus or retroviral vector.

[0046] As used herein, the terms “stable expression” and “stable expression” are intended to mean that the genetic material is stably expressed and / or permanently and stably integrated into the host cell’s genome, and therefore has the same expression capacity over time as the host cell’s innate genetic material.

[0047] "Host cells" include cells that have been transfected, infected, or transduced in vivo, ex vivo, or in vitro with the recombinant vector or polynucleotide of the present invention. Host cells may include packaging cells, producer cells, and cells infected with the viral vector. The term "target cells" is used interchangeably with "host cells" and refers to transfected, infected, or transduced cells of a desired cell type.

[0048] As used herein, the terms nucleic acid, polynucleotide and nucleotide are interchangeable and refer to any nucleic acid, whether or not it consists of phosphodiester bonds or modified bonds (e.g., phosphotriesters, phosphoramidates, siloxanes, carbonates, carboxymethyl esters, acetamidates, carbamates, thioethers, cross-linked phosphoramidates, cross-linked methylenephosphonates, cross-linked phosphoramidates, cross-linked phosphoramidates, cross-linked methylenephosphonates, phosphorothioates, methylphosphonates, phosphorodithioates, cross-linked phosphorothioates or sultone bonds, and combinations of such bonds).

[0049] The terms nucleic acids, polynucleotides, and nucleotides also specifically include nucleic acids composed of bases other than the five biologically present bases (adenine, guanine, thymine, cytosine, and uracil).

[0050] As used herein, a nucleic acid molecule is said to be “isolated” if it is substantially separated from contaminating nucleic acid molecules that encode other polypeptides.

[0051] "Transgene" means any nucleotide sequence, particularly a DNA sequence, that is incorporated into one or more chromosomes of a host cell by human intervention, for example, by the method of the present invention. The transgene preferably includes the "target gene".

[0052] The “gene of interest” can be any nucleic acid, without limitation, that is desired to be delivered to, incorporated into, transcribed, translated, and / or expressed in a target cell, by any means whatsoever. The gene of interest may encode a functional product, such as a protein or an RNA molecule. The gene of interest is generally operably ligated to other sequences useful for obtaining the desired expression of the gene of interest, such as transcriptional regulatory sequences. The term “polynucleotide of interest” refers to one or more polynucleotides, e.g., polynucleotides encoding a polypeptide (i.e., the polypeptide of interest), inserted into an expression vector that is desired to be expressed.

[0053] The term “operatably coupled” refers to a juxtaposition of components that are in a relationship that allows them to function in their intended manner. “Functional relationship” and “operatably coupled” mean that, with respect to the gene of interest, the gene’s expression is affected when the gene is in the correct position and orientation with respect to the promoter and / or enhancer, and the promoter and / or enhancer is in contact with the appropriate molecule.

[0054] The terms “expression” and “expression” mean enabling or revealing information about a gene or DNA sequence, for example, producing a protein by activating cellular functions involved in the transcription and translation of the corresponding gene or DNA sequence. A DNA sequence is expressed within or by a cell to form “expression products,” such as proteins. The expression product itself, for example, the resulting protein, may also be said to have been “expressed” by a cell. Polynucleotides or polypeptides are recombinantly expressed, for example, when they are expressed or produced in a foreign host cell under the control of a foreign or native promoter, or in a native host cell under the control of a foreign promoter.

[0055] The term "regulate" refers to suppressing gene expression when a gene is overexpressed, or increasing gene expression when a gene is underexpressed.

[0056] The terms “regulatory element” and “expression regulatory element” are used interchangeably and refer to nucleic acid molecules that can influence the transcription and / or translation of a coding sequence operably linked in a particular environment. These terms are widely used and encompass all elements that promote or regulate transcription, including promoters, core elements necessary for the fundamental interaction between RNA polymerase and transcription factors, upstream elements, enhancers, and response elements (see, for example, Lewin, “Genes V” (Oxford University Press, Oxford), pages 847–873). Exemplary regulatory elements in prokaryotes include promoters, operator sequences, and ribosome binding sites. Regulatory elements used in eukaryotic cells may include, but are not limited to, promoters, enhancers, splicing signals, and polyadenylation signals.

[0057] The term "nucleic acid regulatory domain" collectively refers to promoter sequences (e.g., pol II promoter sequences), polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, enhancers, etc., which collectively provide for the replication, transcription, and translation of coding sequences in recipient cells. Not all of these regulatory sequences are always necessary, as long as the selected coding sequence can be replicated, transcribed, and translated in appropriate host cells.

[0058] The term “promoter region” is used herein in its usual sense to refer to a nucleotide region containing a DNA regulatory sequence, which originates from a gene capable of binding to RNA polymerase and initiating transcription of a downstream (3' direction) coding sequence. The regulatory sequence may be homogeneous or heterogeneous to the desired gene sequence.

[0059] The term "enhancer" refers to a segment of DNA that contains sequences capable of providing transcriptional enhancement and, in some examples, can function independently of their orientation relative to other regulatory sequences. Enhancers can function cooperatively or additionally with promoters and / or other enhancer elements. The term "promoter / enhancer" refers to a segment of DNA that contains sequences capable of providing both promoter and enhancer functions.

[0060] An internal ribosome entry site (IRES) typically refers to a segment of nucleic acid that facilitates ribosome entry or retention during translation of the coding sequence at the 3' end of the IRES. In some embodiments, the IRES may include a splice acceptor / donor site, but a preferred IRES lacks a splice acceptor / donor site.

[0061] "Endogenous" regulatory sequences are those naturally linked to a given gene in the genome. "Exogenous" regulatory sequences are sequences that are placed in parallel with a gene by genetic engineering (i.e., molecular biological techniques), so that the transcription of that gene is directed by the linked enhancer / promoter. "Heterogeneous" regulatory sequences are exogenous sequences derived from a different species than the cell being genetically engineered. "Synthetic" regulatory sequences may include elements of another endogenous and / or exogenous sequence, and / or sequences determined in vitro or in silico to provide optimal promoter and / or enhancer activity for a particular gene therapy.

[0062] As used herein, the term “constitutive regulatory sequence” refers to a promoter, enhancer, or promoter / enhancer that enables continuous or continuous transcription of an operablely linked sequence. A constitutive regulatory sequence may be a “ubiquitous” promoter, enhancer, or promoter / enhancer that enables expression in a wide range of cell and tissue types, or a “cell-specific,” “cell type-specific,” “cell lineage-specific,” or “tissue-specific” promoter, enhancer, or promoter / enhancer that enables expression in a limited range of cell and tissue types, respectively.

[0063] As used herein, “conditional expression” can mean any type of conditional expression, including, but is not limited to, inducible expression; repressible expression; or expression in cells or tissues having a particular physiological, biological, or disease state. This definition is not intended to exclude cell type or tissue-specific expression.

[0064] Conditional expression can also be achieved by using site-specific DNA recombinases. As used herein, the terms “recombinase” or “site-specific recombinase” include excised or incorporated proteins, enzymes, cofactors or related proteins involved in a recombination reaction that includes one or more recombination sites (e.g., 2, 3, 4, 5, 7, 10, 12, 15, 20, 30, 50, etc.), which may be wild-type proteins (see Landy, (1993) Current Opinion in Biotechnology 3:699-707), or their variants, derivatives (e.g., fusion proteins containing recombinant protein sequences or fragments thereof), fragments, and variants. Exemplary examples of recombinases suitable for use in specific embodiments of the present invention include, but are not limited to, Cre, Int, IHF, Xis, Flop, Fis, Hin, Gin, ΦC31, Cin, Tn3 resolverase, TndX, XerC, XerD, TnpX, Hjc, Gin, SpCCE1, and ParA.

[0065] As used herein, the terms “recombinant sequence,” “recombinant site,” or “site-specific recombinant site” refer to a specific nucleic acid sequence that a recombinase recognizes and binds to.

[0066] As used herein, the terms “heterogeneic” nucleic acid sequence or transgene mean (i) a sequence not normally present in wild-type retroviruses, (ii) a sequence derived from an alien species, or (iii) a sequence that, if derived from the same species, can be substantially modified from its original form. Alternatively, an unaltered nucleic acid sequence that is not normally expressed in a cell is a heterogeneic nucleic acid sequence.

[0067] Antibodies (Ab) and immunoglobulins (Ig) are glycoproteins with the same structural characteristics. As used herein, the term "antibody" refers to a protein containing at least one immunoglobulin variable domain or immunoglobulin variable domain sequence. For example, an antibody may contain a heavy (H) chain variable region (abbreviated herein as VH) and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody may contain two heavy (H) chain variable regions and two light (L) chain variable regions. Antibodies exhibit binding specificity to a particular antigen, while immunoglobulins include both antibodies and other antibody-like molecules that lack antigen specificity. The latter type of polypeptide is produced at low levels by the lymphoid system, for example, and at high levels by myeloma.

[0068] The term "antibody" is used in its broadest sense, specifically encompassing human, non-human (e.g., mouse), chimeric, and humanized monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, and antibody fragments insofar as they exhibit the desired biological activity. Therefore, the term "antibody" includes antigen-binding fragments of antibodies (e.g., single-chain antibodies, Fab fragments, F(ab').sub.2, Fd fragments, Fv fragments, and dAb fragments) as well as complete antibodies. Typically, fragments compete with the intact antibody from which they originate for specific binding to the antigen.

[0069] The term "monospecific antibody" refers to an antibody that exhibits a single binding specificity and affinity for a specific target, such as an epitope. This term includes, for example, "monoclonal antibody," which refers to an antibody produced as a single molecular species from a homogeneous population of isolated cells. A "monoclonal antibody composition" refers to a preparation of an antibody or a fragment thereof in a composition containing a single molecular species of antibody.

[0070] The term "epitope" also refers to the site of an antigen to which B cells and / or T cells respond. B cell epitopes can be formed from both juxtaposed continuous amino acids by tertiary folding of proteins, or from discontinuous amino acids. Epitopes formed from continuous amino acids are typically retained when exposed to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically contain at least three, more commonly at least five, or eight to ten amino acids in their unique spatial structure. Methods for determining the spatial structure of epitopes include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996). Antibodies that recognize the same epitope can be identified in a simple immunoassay demonstrating the ability of one antibody to block the binding of another antibody to its target antigen. T cells recognize a sequence of epitopes consisting of approximately 9 amino acids for CD8 cells, or approximately 13-15 amino acids for CD4 cells. T cells that recognize an epitope are primed by T cells that respond to the epitope. 3 As determined by H-thymidine incorporation, it can be identified by an in vitro assay measuring antigen-dependent proliferation (see Burke, above; Tigges, above).

[0071] The term “immunological” or “immune” response refers to the occurrence of a beneficial humoral (antibody-mediated) and / or cellular (antigen-specific T cell-mediated or their secretion-mediated) response to amyloid peptides in a recipient patient. Such responses can be active responses induced by the administration of immunogens, or passive responses induced by the administration of antibodies or primed T cells. Cellular immune responses are triggered by the presentation of polypeptide epitopes associated with class I or class II MHC molecules and antigen-specific CD4 + T helper cells and / or CD8 +It activates cytotoxic T cells. The response may also involve the activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia, eosinophils, or other components of innate immunity. The presence of a cell-mediated immunological response is confirmed in proliferation assays (CD4 + This can be determined by T cell (T cell) or CTL (cytotoxic T lymphocyte) assay (Burke et al., J.Inf.Dis.170, 1110-19 (1994)), antigen-dependent cell death (cytotoxic T lymphocyte assay, Tigges et al., J.Immunol.156, 3901-3910), or cytokine secretion. The relative contributions of humoral and cellular responses to the protective or therapeutic effect of an immunogen can be distinguished by separately isolating IgG and T cells from immunized syngenes and measuring their protective or therapeutic effect in a second target.

[0072] "Immunogenic agents" or "immunogens," when administered to patients in combination with an adjuvant (optional), can induce an immunological response to themselves.

[0073] The term "adjuvant" refers to a compound that, when administered with an antigen, increases, enhances, and / or boosts the immune response to the antigen, but, when administered alone, does not generate an immune response to the antigen. Adjuvants can be administered as a single composition with the recombinant virus of the present invention, or before, simultaneously with, or after the administration of the recombinant virus of the present invention. Adjuvants can enhance the immune response by several mechanisms, including, for example, lymphocyte recruitment, stimulation of B cells and / or T cells, and stimulation of macrophages.

[0074] As used herein, “Subject” includes any animal exhibiting symptoms of a monogenic disease, disorder, or condition that can be treated with gene therapy vectors, cell-based therapeutic agents, and methods disclosed elsewhere herein.

[0075] Suitable subjects (e.g., patients) include laboratory animals (such as mice, rats, rabbits, or guinea pigs), livestock, and domestic animals or pets (such as cats or dogs). Non-human primates, preferably human patients, are also included. Typical subjects include animals exhibiting abnormal levels of one or more physiological activities (lower or higher than those in "normal" or "healthy" subjects) that can be modulated by gene therapy.

[0076] As used herein, “treatment” or “to treat” includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, and may even include a minimal reduction of one or more measurable markers of the disease or condition being treated. Treatment may optionally involve either a reduction or improvement of the symptoms of the disease or condition, or a delay in the progression of the disease or condition. “Treatment” does not necessarily mean the complete eradication or cure of the disease or condition, or its associated symptoms.

[0077] As used herein, “prevent” and similar words such as “prevented” and “preventing” refer to an approach to prevent, inhibit, or reduce the likelihood of the onset or recurrence of a disease or condition. It also means delaying the onset or recurrence of the disease or condition, or delaying the onset or recurrence of the symptoms of a disease or condition. As used herein, “prevention” and similar words also include reducing the intensity, effect, symptoms and / or burden of a disease or condition before the onset or recurrence of the disease or condition.

[0078] The term "therapeutic" is used in a general sense and includes therapeutic agents, prophylactic agents, and supplements. The term "therapeutic" can refer to an effect that prevents, reverses, or delays the natural course of a disease or its symptoms. A therapeutic effect can be prophylactic, curative, or merely mitigating, and does not mean that an affected human or animal patient will not die from the disease.

[0079] As used herein, the term “amount” means an “effective amount” or “effective dose” of the virus or transduction therapy cells to achieve a beneficial or desired prophylactic or therapeutic outcome, including clinical results.

[0080] The “prophylactically effective dose” refers to the amount of virus or transduced therapeutic cells that is effective in achieving the desired prophylactic outcome. Typically, but not always, the prophylactically effective dose is less than the therapeutically effective dose, because prophylactic doses are used in subjects before or in the early stages of disease.

[0081] The “therapeutically effective amount” of a virus or transduced therapeutic cell may vary depending on factors such as the individual disease state, age, sex, and weight, and the ability of the stem cells and progenitor cells to elicit the desired response in each individual. The therapeutically effective amount is also the amount in which the therapeutically beneficial effects of the virus or transduced therapeutic cell outweigh any toxic or adverse effects. The term “therapeutically effective amount” includes an amount effective in “treating” a subject (e.g., a patient).

[0082] The term "pharmaceutically acceptable" refers to molecular entities and compositions that do not cause allergic reactions or similar adverse reactions when administered to humans. The preparation of aqueous compositions containing proteins as active ingredients is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions, and solid forms suitable for dissolution or suspension in liquid before injection may also be prepared. Preparations may also be further emulsified.

[0083] As used herein, “carrier” includes any and all solvents, dispersions, vehicles, coatings, diluents, antimicrobial and antifungal agents, isotonic and absorption retardants, buffers, carrier solutions, suspensions, colloids, etc. The use of such media and substances with pharmaceutically active substances is well known in the art. Any conventional medium or substance is intended for use in therapeutic compositions unless it is incompatible with the active ingredient. Auxiliary active ingredients may also be incorporated into the composition.

[0084] As used herein, “pharmaceutically acceptable carrier” includes any and all of physiologically compatible solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic and absorption retardants, and pharmaceutically acceptable cell culture media.

[0085] The following description provides certain specific details in order to give a complete understanding of the various embodiments of the present invention. However, those skilled in the art will understand that the present invention can be carried out without these details.

[0086] Throughout this specification, any reference to “one embodiment” or “a certain embodiment” means that a particular feature, structure, or characteristic described in relation to an embodiment is included in at least one embodiment of the present invention. Therefore, the appearance of the phrase “in one embodiment” or “in a certain embodiment” in various places throughout this specification does not necessarily refer to the same embodiment. Furthermore, a particular feature, structure, or characteristic can be combined in any preferred manner in one or more embodiments.

[0087] All publications referenced herein are incorporated herein in their entirety by reference for the purpose of describing and disclosing methodologies described herein that may be used in connection with the description herein. The publications discussed herein are provided only for disclosures prior to the filing date of this application. Nothing included herein shall be construed as an acknowledgment by the inventors that they have no prior rights to such disclosures on the grounds of prior invention.

[0088] explanation The present invention relates to compositions and methods for use in improving the efficiency of virus production and viral transduction into target cells. In one embodiment, the present invention includes compositions and methods for improved recombinant virus production and viral transduction. In one embodiment, the present invention includes pseudotyped retroviruses with one or more envelope glycoproteins and methods thereof.

[0089] In one embodiment, the present invention provides compositions and methods for producing high-titer retroviruses. In one embodiment, the present invention provides an improved method for transducing cells using a polynucleotide of interest. Thus, the present invention provides compositions and methods applicable to any type of gene therapy. For example, the present invention is applicable to transduction of exogenous genes into any target cells and is therefore applicable to any type of gene therapy setting.

[0090] Virus production using packaging cells The present invention provides compositions and methods for improving the efficiency of viral transduction into target cells. In one embodiment, the present invention provides compositions and methods for producing retroviral packaging cells comprising a suitable mammalian cell having (i) a first recombinant nucleic acid comprising a first envelope gene and (ii) a second recombinant nucleic acid comprising a second envelope gene. In one embodiment, the first envelope gene and the second envelope gene are different from each other. In one embodiment, the suitable mammalian cell is HEK293 cell. As used herein, “packaging cell” is a mammalian cell capable of expressing retroviral genes and assembling retroviral particles.

[0091] In one embodiment, the present invention provides a composition and method for producing retroviral packaging cells comprising (i) a suitable mammalian cell having therein at least one recombinant nucleic acid comprising at least one envelope gene. In some embodiments, the mammalian cell comprises a first recombinant nucleic acid comprising a first envelope gene and a second envelope gene.

[0092] The advantage of producing retroviral packaging cells, including suitable mammalian cells having (i) a first recombinant nucleic acid containing a first envelope gene and (ii) a second recombinant nucleic acid containing a second envelope gene, is the generation of a multipseudotype virus. In one embodiment, the multipseudotype virus of the present invention enables the production of retroviral supernatant with a higher titer. In one embodiment, the method further includes the step of collecting and concentrating the retroviral supernatant.

[0093] Multipsydetype retrovirus The present invention is intended to provide an improved method for recombinant retroviral transduction. In one embodiment, the present invention includes a retrovirus pseudotyped with two or more envelope glycoproteins and a method thereof.

[0094] In some embodiments, the present invention provides methods and compositions for generating a multipseudotype virus comprising at least two different envelope glycoproteins. In some embodiments, the virus is a retrovirus. In some embodiments, the retrovirus is a gammaretrovirus. In some embodiments, the virus is a lentivirus.

[0095] Retroviruses can originate from members of the family Retroviridae. The classification of this family has changed several times over the past 10-15 years. Currently, the family Retroviridae consists of two subfamilies: the Spumaretrovirinae, which has a single genus of spumaviruses (or foamy viruses) such as human and simian foamy virus (HFV); and the Orthoretrovirinae, which has six genera—beta-retroviruses (e.g., MMTV), gamma-retroviruses (e.g., MLV), alpha-retroviruses (e.g., ALV), delta-retroviruses (e.g., BLV and HTLV-1), lentiviruses (e.g., HIV-1), and epsilon-retroviruses (e.g., ocular dermatosarcoma virus). These classifications are based on common molecular features, such as the relative reading frames of gag, pol, and env, polyprotein processing, individual tRNAs used to prime reverse transcription, and the properties of the LTR structure. The first method of classifying retroviruses classified them into groups A, B, C, and D based on particle morphology observed under an electron microscope during viral maturation. Type A particles represent immature particles of type B and D viruses found in the cytoplasm of infected cells. These particles are not infectious. Type B particles bud from the plasma membrane as mature virions by encapsulating type A particles within the cytoplasm. Within the membrane, they have a 75 nm toroidal core from which long glycoprotein spikes protrude. After budding, type B particles contain an eccentrically located, electron-dense core. Beta-retroviruses, such as mouse mammary tumor virus (MMTV), have a type B morphology, although beta-retroviruses can also have a type D structure. Type D particles are similar to type B particles in that they exhibit a ring-like structure budding from the cell surface in the cytoplasm of infected cells, but the virions incorporate short surface glycoprotein spikes. The electron-dense core is also eccentrically located within the particle. The Mason Pfizer monkey virus (MPMV) is also a beta-retrovirus and a prototype D-type virus.In cells infected with type C viruses, cytoplasmic particles are not observed. Instead, mature particles bud directly from the cell surface via crescent-shaped "C" condensation, then close themselves and are surrounded by the plasma membrane. Envelope glycoprotein spikes can be seen along with a core of uniform electron density. Budding can occur from the surface plasma membrane or directly into the intracellular vacuole. Alpha-retroviruses, gamma-retroviruses, delta-retroviruses, and epsilon-retroviruses all have the appearance of a type C structure.

[0096] In some embodiments, recombinant retroviral particles or envelope glycoproteins may be derived from the genera alpha-retrovirus, beta-retrovirus, gamma-retrovirus, delta-retrovirus, epsilon-retrovirus, lentivirus, or spumavirus. Many retroviruses exist that are suitable for use in the methods disclosed herein. For example, murine leukemia virus (MLV), human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), mouse mammary cancer virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), and Avian myelocytomatosis virus-29. Virus-29 (MC29) and Avian erythroblastosis virus (AEV) can be used. A detailed list of retroviruses can be found in Coffin et al. ("Retroviruses" 1997 Cold Spring Harbor Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 758-763). Details on the genome structure of some retroviruses can be found in the relevant field. For example, details on HIV can be found in the NCBI Genbank (i.e., genome accession number AF033819).

[0097] In some embodiments, retroviruses are derived from the lentivirus genus. Exemplary lentiviruses include, but are not limited to, HIV (human immunodeficiency virus, including HIV types 1 and HIV 2); visna-maedi virus (VMV) virus; caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV).

[0098] In some embodiments, the retroviruses are derived from the genus Gammaretrovirus. Exemplary Gammaretroviruses include, but are not limited to, reticuloendotheliosis virus, gibbon leukemia virus, koala retrovirus, Moloney's mouse leukemia virus, Ebelson's mouse leukemia virus, heterotropic mouse leukemia-associated virus, feline leukemia virus, and Friend virus.

[0099] Retroviruses are defined by the way they replicate their genetic material. During replication, RNA is converted to DNA. After infecting a cell, a double-stranded DNA molecule is produced from two RNA molecules contained within the viral particle by a molecular process known as reverse transcription. The DNA morphology is covalently integrated into the host cell genome as a provirus, from which viral RNA is expressed with the help of cellular and / or viral factors. The expressed viral RNA is packaged within a particle and released as an infectious virion.

[0100] Retroviral particles consist of two identical RNA molecules. Each wild-type genome has a positive-sense single-stranded RNA molecule capped at the 5' end and polyadenylated at the 3' tail. Diploid viral particles contain two RNA strands that form a complex with the gag protein, viral enzyme (pol gene product), and host tRNA molecule within a "core" structure of the gag protein. Surrounding and protecting this capsid is a lipid bilayer derived from the host cell membrane, containing the viral envelope (env) protein. The env protein binds to the viral cell receptor, and the particle typically enters the host cell via receptor-mediated endocytosis and / or membrane fusion.

[0101] The viral envelope protein (env) determines the range of host cells that can ultimately be infected and transformed by recombinant retroviruses generated from a cell line. In the case of lentiviruses such as HIV-1, HIV-2, SIV, FIV, and EIV, the env proteins include gp41 and gp120. Preferably, the viral env proteins expressed by the packaging cells of the present invention are encoded on a vector separate from the viral gag and pol genes, as described later.

[0102] Examples of retrovirus-derived env genes that can be used in the present invention include, but are not limited to, the MLV envelope, 10A1 envelope, BAEV, FeLV-B, RD114, SSAV, Ebola, Sendai virus, FPV (avian plague virus), and influenza virus envelope. Similarly, genes encoding envelopes from RNA viruses (e.g., RNA virus families of Picornaviridae, Calciviridae, Astroviridae, Togaviridae, Flaviviridae, Coronaviridae, Paramyxoviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae, Bunyaviridae, Arenaviridae, Reoviridae, Birnaviridae, and Retroviridae) and genes encoding envelopes from DNA viruses (families of Hepadnaviridae, Circoviridae, Parvoviridae, Papovaviridae, Adenoviridae, Herpesviridae, Poxviridae, and Iridoviridae) may be used. Typical examples include FeLV, VEE, HFVW, WDSV, SFV, rabies, ALV, BIV, BLV, EBV, CAEV, SNV, ChTLV, STLV, MPMV, SMRV, RAV, FuSV, MH2, AEV, AMV, CT10, EIAV, VSV-G, 4070A, gap70, and GALV. In some embodiments, the retrovirus-derived env gene that can be used in the present invention includes any ecotropic virus envelope glycoprotein and amphotropic envelope glycoprotein.

[0103] As used herein, the terms “pseudotype” or “pseudotyping” refer to a virus in which the viral envelope protein is replaced with the envelope protein of another virus having desirable characteristics. For example, HIV can be pseudotyped with the vesicular stomatitis virus G-protein (VSV-G) envelope protein, which allows HIV to infect a wider range of cells, as the HIV envelope protein (encoded by the env gene) typically targets the virus to CD4+ presenting cells.

[0104] In some embodiments, the multipsydetype virus contains at least one envelope glycoprotein. In some embodiments, the multipsydetype virus contains at least two envelope glycoproteins. In some embodiments, the multipsydetype virus contains at least three envelope glycoproteins. In some embodiments, the multipsydetype virus contains at least four envelope glycoproteins. In some embodiments, the multipsydetype virus contains at least five envelope glycoproteins. In some embodiments, the multipsydetype virus contains at least six envelope glycoproteins. In some embodiments, the multipsydetype virus contains at least seven envelope glycoproteins. In some embodiments, the multipsydetype virus contains at least eight envelope glycoproteins. In some embodiments, the multipsydetype virus contains at least nine envelope glycoproteins. In some embodiments, the multipsydetype virus contains at least ten envelope glycoproteins.

[0105] In some embodiments, the first envelope glycoprotein for pseudotyping the viruses of the present invention includes any viruses of the following types: feline endogenous virus (RD114), influenza A, influenza B, influenza C viruses such as H1N1, H1N2, H3N2 and H5N1 (avian influenza), hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, rotavirus, Norwalk virus group, lyssaviruses such as enteric adenovirus, parvovirus, dengue fever virus, empox, mononegavirus, rabies virus, Lagos bat virus, mocola virus, dubengee virus, European bat virus 1 and 2, as well as Australian bat virus, ephemerovirus, becyclovirus, vesicular stomatitis virus (VSV), herpesviruses such as herpes simplex virus 1 and 2, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus. EBV, human herpesviruses (HHV), human herpesvirus types 6 and 8, human immunodeficiency virus (HIV), papillomavirus, mouse gamma herpesvirus, Argentine hemorrhagic fever virus, Bolivian hemorrhagic fever virus, Sabia-associated hemorrhagic fever virus, Venezuelan hemorrhagic fever virus, Lassa fever virus, Machupo virus, lymphocytic choriomeningitis virusThe first envelope glycoprotein is selected from the group consisting of arenaviruses such as virus (LCMV), Bunyaviridae such as Crimean-Congo hemorrhagic fever virus, Hantavirus, hemorrhagic fevers including viruses that cause hemorrhagic fever with renal syndrome, Rift Valley fever virus, Filoviridae (filoviruses) including Ebola hemorrhagic fever and Marburg hemorrhagic fever, Flaviviridae including Kasanur forest disease virus, Omsk hemorrhagic fever virus, Paramyxoviridae such as tick-borne encephalitis viruses and Hendra virus and Nipah virus, smallpox major and minor, Venezuelan horse encephalitis virus, Eastern horse encephalitis virus, Western horse encephalitis virus, SARS-related coronavirus (SARS-CoV), West Nile virus, any encephalitis-causing virus, 4070A, gap70, 10A1, and GALV. In some embodiments, the first envelope glycoprotein is selected from the group consisting of ecotropic virus envelope glycoproteins and amphotropic virus envelope glycoproteins.

[0106] In some embodiments, the multipseudotype virus contains two envelope glycoproteins.In some embodiments, the second envelope glycoprotein for pseudotyping viruses of the present invention differs from the first envelope glycoprotein in that it can pseudotype viruses such as feline endogenous virus (RD114), influenza A, influenza B, influenza C viruses such as H1N1, H1N2, H3N2 and H5N1 (avian influenza), hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, rotavirus, any virus of the Norwalk virus group, enteric adenovirus, parbow virus. Lyssaviruses such as Virus, dengue fever virus, empox, mononegavirus, and rabies virus; Lagos bat virus, mocola virus, Dubengee virus, European bat virus 1 and 2, as well as Australian bat virus, ephemerovirus, becyclovirus, vesicular stomatitis virus (VSV), herpesviruses such as herpes simplex virus type 1 and 2, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus (EBV), human herpesvirus (HHV), and human herpesvirus Arenaviruses such as 147 virus types 6 and 8, human immunodeficiency virus (HIV), papillomavirus, mouse gamma herpesvirus, Argentine hemorrhagic fever virus, Bolivian hemorrhagic fever virus, Sabia-associated hemorrhagic fever virus, Venezuelan hemorrhagic fever virus, Lassa fever virus, Machupo virus, lymphocytic choriomeningitis virus (LCMV), Bunyaviridae such as Crimean-Congo hemorrhagic fever virus, hantavirus, hemorrhagic fevers caused by viruses that cause hemorrhagic fever with renal syndrome, Rift Valley fever virus, Ebola hemorrhagic fever, and Marburg hemorrhagic fever The group is selected from the family Filoviridae (filoviruses), including Kasanul Forest disease virus, Flaviviridae, Omsk hemorrhagic fever virus, tick-borne encephalitis viruses and Paramyxoviridae such as Hendra virus and Nipah virus, smallpox major and minor, Venezuelan horse encephalitis virus, Eastern equine encephalitis virus, Western equine encephalitis virus, SARS-related coronavirus (SARS-CoV), West Nile virus, any encephalitis-causing virus, 4070A, gap70, 10A1, and GALV.In some embodiments, the second envelope glycoprotein is selected from the group consisting of ecotropic virus envelope glycoproteins and amphotropic virus envelope glycoproteins.

[0107] In some embodiments, the multipseudotype virus contains three envelope glycoproteins.In some embodiments, the third envelope glycoprotein for pseudotyping viruses of the present invention differs from the first and second envelope glycoproteins in that it can pseudotype any virus from the group of viruses including feline endogenous virus (RD114), influenza A, influenza B, influenza C viruses such as H1N1, H1N2, H3N2 and H5N1 (avian influenza), hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, rotavirus, and Norwalk virus. Lyssaviruses such as enteric adenovirus, parvovirus, dengue virus, empox, mononegavirus, and rabies virus; Lagos bat virus, mocola virus, Dubengee virus, European bat virus 1 and 2, as well as Australian bat virus, ephemerovirus, becyclovirus, vesicular stomatitis virus (VSV), herpesviruses such as herpes simplex virus type 1 and 2; varicella-zoster virus, cytomegalovirus, Epstein-Barr virus (EBV), and human herpesvirus (H) HV), human herpesvirus types 6 and 8, human immunodeficiency virus (HIV), papillomavirus, mouse gamma herpesvirus, Argentine hemorrhagic fever virus, Bolivian hemorrhagic fever virus, Sabia-associated hemorrhagic fever virus, Venezuelan hemorrhagic fever virus, Lassa fever virus, Machupo virus, arenaviruses such as lymphocytic choriomeningitis virus (LCMV), Bunyaviridae such as Crimean-Congo hemorrhagic fever virus, hantavirus, hemorrhagic fever with viruses causing hemorrhagic fever with renal syndrome, Rift Valley fever virus, Ebola hemorrhagic fever and Marlborough hemorrhagic fever The group is selected from the family Filoviridae (filoviruses), including Lug hemorrhagic fever; Flaviviridae, including Kasanur Forest disease virus; Omsk hemorrhagic fever virus; Paramyxoviridae, including tick-borne encephalitis viruses and Hendra virus and Nipah virus; smallpox major and minor; Venezuelan horse encephalitis virus; Eastern equine encephalitis virus; Western equine encephalitis virus; SARS-related coronavirus (SARS-CoV); West Nile virus; any encephalitis-causing virus; 4070A, gap70, 10A1, and GALV.In some embodiments, the third envelope glycoprotein is selected from the group consisting of ecotropic virus envelope glycoproteins and amphotropic virus envelope glycoproteins.

[0108] In some embodiments, the multipseudotype virus contains four envelope glycoproteins.In some embodiments, the fourth envelope glycoprotein for pseudotyping viruses of the present invention differs from the first, second, and third envelope glycoproteins in that it is used to pseudotype viruses such as feline endogenous virus (RD114), influenza A, influenza B, influenza C viruses such as H1N1, H1N2, H3N2 and H5N1 (avian influenza), hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, rotavirus, Norwalk virus, and others. Any virus from the Virus group, enteric adenovirus, parvovirus, dengue virus, empox, mononegavirus, rabies virus and other lyssaviruses, Lagos bat virus, mocola virus, Dubengee virus, European bat virus 1 and 2, as well as Australian bat virus, ephemerovirus, becyclovirus, vesicular stomatitis virus (VSV), herpesviruses such as herpes simplex virus type 1 and 2, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus (EBV), human herpesvirus Hemorrhagic fevers include: pesvirus (HHV), human herpesvirus types 6 and 8, human immunodeficiency virus (HIV), papillomavirus, mouse gamma herpesvirus, Argentine hemorrhagic fever virus, Bolivian hemorrhagic fever virus, Sabia-associated hemorrhagic fever virus, Venezuelan hemorrhagic fever virus, Lassa fever virus, Machupo virus, arenaviruses such as lymphocytic choriomeningitis virus (LCMV), Bunyaviridae such as Crimean-Congo hemorrhagic fever virus, hantavirus, hemorrhagic fevers caused by viruses that cause hemorrhagic fever with renal syndrome, Rift Valley fever virus, Ebola hemorrhagic fever, and The group is selected from the family Filoviridae (filoviruses), including Marburg hemorrhagic fever; Flaviviridae, including Kasanur Forest disease virus; Omsk hemorrhagic fever virus; Paramyxoviridae, including tick-borne encephalitis viruses and Hendra virus and Nipah virus; smallpox major and minor; Venezuelan horse encephalitis virus; Eastern equine encephalitis virus; Western equine encephalitis virus; SARS-related coronavirus (SARS-CoV); West Nile virus; any encephalitis-causing virus; 4070A, gap70, 10A1, and GALV.In some embodiments, the fourth envelope glycoprotein is selected from the group consisting of ecotropic virus envelope glycoproteins and amphotropic virus envelope glycoproteins.

[0109] In some embodiments, the multipseudotyped virus comprises five envelope glycoproteins. In some embodiments, the fifth envelope glycoprotein for pseudotyping the virus of the present invention differs from the first, second, third, and fourth envelope glycoproteins in relation to influenza A, influenza B, influenza C viruses, hepatitis A virus, hepatitis B virus, and hepatitis C virus, such as feline endogenous virus (RD114), H1N1, H1N2, H3N2, and H5N1 (avian influenza). , hepatitis D virus, hepatitis E virus, rotavirus, any virus from the Norwalk virus group, lyssaviruses such as enteric adenovirus, parvovirus, dengue fever virus, empox, mononegavirus, and rabies virus, Lagos bat virus, mocola virus, dubengee virus, European bat virus 1 and 2, as well as Australian bat virus, ephemerovirus, becyclovirus, vesicular stomatitis virus (VSV), herpes simplex virus type 1 and 2, and other herpes viruses. Arenaviruses such as varicella-zoster virus, cytomegalovirus, Epstein-Barr virus (EBV), human herpesvirus (HHV), human herpesvirus types 6 and 8, human immunodeficiency virus (HIV), papillomavirus, mouse gamma herpesvirus, Argentine hemorrhagic fever virus, Bolivian hemorrhagic fever virus, Sabia-associated hemorrhagic fever virus, Venezuelan hemorrhagic fever virus, Lassa fever virus, Machupo virus, lymphocytic choriomeningitis virus (LCMV), and Crimean-Congo hemorrhagic fever virus. Bunyaviridae viruses such as Viruses, Hantaviruses, Hemorrhagic fevers including viruses that cause hemorrhagic fever with renal syndrome, Rift Valley fever virus, Filoviridae (Filoviruses) including Ebola hemorrhagic fever and Marburg hemorrhagic fever, Flaviviridae including Kasanur Forest disease virus, Omsk hemorrhagic fever virus, Tick-borne encephalitis viruses and Paramyxoviridae such as Hendra virus and Nipah virus, Smallpox major and minor, Venezuelan horse encephalitis virus, Eastern horse encephalitis virus, Western horse encephalitis virus,The fifth envelope glycoprotein is selected from the group consisting of SARS-related coronavirus (SARS-CoV), West Nile virus, any encephalitis-causing virus, 4070A, gap70, 10A1, and GALV. In some embodiments, the fifth envelope glycoprotein is selected from the group consisting of ecotropic virus envelope glycoproteins and amphotropic virus envelope glycoproteins.

[0110] In some embodiments, 10 or more envelope glycoproteins are used to pseudotype the viruses of the present invention, and all envelope glycoproteins are different, including feline endogenous virus (RD114), influenza A, influenza B, influenza C virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, rotavirus, any virus of the Norwalk virus group, and enteric adenovirus. Viruses, including parvovirus, dengue fever virus, empox, mononegavirus, rabies virus, lyssaviruses, Lagos bat virus, mocola virus, dubengee virus, European bat virus 1 and 2, as well as Australian bat virus, ephemerovirus, becyclovirus, vesicular stomatitis virus (VSV), herpesviruses such as herpes simplex virus type 1 and 2, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus (EBV), and human herpesvirus (HHV). Human herpesviruses types 6 and 8, human immunodeficiency virus (HIV), papillomavirus, mouse gamma herpesvirus, Argentine hemorrhagic fever virus, Bolivian hemorrhagic fever virus, Sabia-associated hemorrhagic fever virus, Venezuelan hemorrhagic fever virus, Lassa fever virus, Machupo virus, arenaviruses such as lymphocytic choriomeningitis virus (LCMV), Bunyaviridae such as Crimean-Congo hemorrhagic fever virus, hantavirus, hemorrhagic fevers with viruses causing hemorrhagic fever with renal syndrome, Rift Valley fever virus, Ebola hemorrhagic fever and Marburg hemorrhagic fever The group is selected from the family Filoviridae (filoviruses), including hemorrhagic fever; Flaviviridae, including Kasanur Forest disease virus; Omsk hemorrhagic fever virus; Paramyxoviridae, including tick-borne encephalitis viruses and Hendra virus and Nipah virus; smallpox major and minor; Venezuelan horse encephalitis virus; Eastern equine encephalitis virus; Western equine encephalitis virus; SARS-related coronavirus (SARS-CoV); West Nile virus; any encephalitis-causing virus; 4070A, gap70, 10A1, and GALV.In some embodiments, 10 or more envelope glycoproteins are selected from the group consisting of ecotropic virus envelope glycoproteins and amphotropic virus envelope glycoproteins.

[0111] In further embodiments, the retrovirus is pseudotyped with any combination of envelope glycoproteins selected from the group consisting of RD114, VSV-G, 4070A, gap70, 10A1, and GALV. In one embodiment, at least one envelope glycoprotein is selected from the group consisting of RD114, VSV-G, 4070A, gap70, 10A1, and GALV. In one embodiment, the first envelope glycoprotein is selected from the group consisting of RD114, VSV-G, 4070A, gap70, 10A1, and GALV. In one embodiment, the second envelope glycoprotein is selected from the group consisting of RD114, VSV-G, 4070A, gap70, 10A1, and GALV. In one embodiment, the third envelope glycoprotein is selected from the group consisting of RD114, VSV-G, 4070A, gap70, 10A1, and GALV. In one embodiment, the fourth envelope glycoprotein is selected from the group consisting of RD114, VSV-G, 4070A, gap70, 10A1, and GALV. In one embodiment, the fifth envelope glycoprotein is selected from the group consisting of RD114, VSV-G, 4070A, gap70, 10A1, and GALV.

[0112] In another embodiment, the retrovirus is pseudotyped with any combination of envelope glycoproteins selected from the group consisting of ecotropic virus envelope glycoproteins and amphotropic virus envelope glycoproteins. In one embodiment, at least one envelope glycoprotein is selected from the group consisting of ecotropic virus envelope glycoproteins and amphotropic virus envelope glycoproteins. In one embodiment, the first envelope glycoprotein is selected from the group consisting of ecotropic virus envelope glycoproteins and amphotropic virus envelope glycoproteins. In one embodiment, the second envelope glycoprotein is selected from the group consisting of ecotropic virus envelope glycoproteins and amphotropic virus envelope glycoproteins. In one embodiment, the third envelope glycoprotein is selected from the group consisting of ecotropic virus envelope glycoproteins and amphotropic virus envelope glycoproteins. In one embodiment, the fourth envelope glycoprotein is selected from the group consisting of ecotropic virus envelope glycoproteins and amphotropic virus envelope glycoproteins. In one embodiment, the fifth envelope glycoprotein is selected from the group consisting of ecotropic virus envelope glycoproteins and amphotropic virus envelope glycoproteins.

[0113] In one embodiment, the combination of envelope glycoproteins is RD114 and VSV-G. In another embodiment, the combination of envelope glycoproteins is GALV and VSV-G. In yet another embodiment, the combination of envelope glycoproteins is GALV and 10A1.

[0114] In some embodiments, the envelope glycoprotein may be modified or altered. In some embodiments, the envelope glycoprotein may be derived from the above-described envelope glycoprotein. In one embodiment, the modification or alteration is cleavage, masking, redirection, or a combination thereof. In some embodiments, the modification or alteration confers different binding properties to the virus. In an exemplary example, the envelope glycoprotein may be modified or altered so that an antibody-binding domain is bound to it. In an exemplary example, the CD3 antibody-binding domain is bound to a modified or altered VSV-G envelope glycoprotein that directs the virus to CD3+ cells. In one embodiment, the modification or alteration is cleavage. In one embodiment, the modification or alteration is masking. In one embodiment, the modification or alteration is redirection.

[0115] In another exemplary example, the GALV envelope glycoprotein is modified on the cytoplasmic surface as described by Tomas et al. (Tomas HA, et al., 2019, Mol Ther Methods Clin Dev. 15:1-8). In this exemplary example, the GALV envelope glycoprotein further comprises a synthetic amino acid sequence or an HIV-1 matrix capsid amino acid sequence. In some embodiments, this modification improves the transduction efficiency of the virus.

[0116] In various embodiments, the multipseudotype viruses described herein, comprising the described envelope glycoprotein, can infect host cells. After the outer envelope is shed, the viral RNA is copied to DNA by reverse transcription. This is catalyzed by a reverse transcriptase encoded by a pol region, using host cell tRNA packaged in the virion as a primer for DNA synthesis. In this way, the RNA genome is converted to a more complex DNA genome.

[0117] The double-stranded linear DNA produced by reverse transcription may or may not be circularized in the nucleus. Proviruses now have two identical repeats, known as long terminal repeats (LTRs), at either end. The ends of the two LTR sequences create a site recognized by the pol product (integrase protein) that catalyzes integration, resulting in the provirus always joining to host DNA by two base pairs (bp) from the ends of the LTRs. Cellular sequence duplication is observed at the ends of both LTRs, reminiscent of the integration patterns of transferable genetic elements. Integration is thought to occur essentially randomly within the target cell genome. However, it is possible to control the integration of retroviral genomes by modifying the long terminal repeats.

[0118] The transcription, RNA splicing, and translation of incorporated viral DNA are mediated by host cell proteins. Various spliced ​​transcripts are produced. In the case of human retroviruses, HIV-1 / 2 and HTLV-I / II viral proteins are also used to regulate gene expression. The interaction between cellular and viral factors is a factor in controlling viral latency and the temporal order in which viral genes are expressed.

[0119] Retroviruses can be transmitted horizontally and vertically. Efficient infectious transmission of retroviruses requires the expression of receptors on target cells that specifically recognize the viral envelope protein, although viruses can use receptor-independent, nonspecific entry pathways with lower efficiency. Furthermore, the target cell type must be able to support all stages of the replication cycle after the virus has bound and entered the cell. Vertical transmission occurs when the viral genome is incorporated into the host germline. Proviruses are then passaged from generation to generation, as if they were cellular genes. Thus, endogenous proviruses are established that are often latent but can be activated when the host is exposed to appropriate triggers.

[0120] As described above, the incorporated DNA intermediate is referred to as a provirus. Previous gene therapy or gene delivery systems have used methods and retroviruses that require the transcription of a provirus and assembly into an infectious virus in a cell line containing a suitable sequence that enables capsid formation in the presence of a suitable helper virus or without the co-production of a contaminating helper virus. Helper viruses are not required for the production of the recombinant retrovirus of this disclosure, as the sequence for capsid formation is provided in the genome, and therefore a replicable retroviral vector for gene delivery or gene therapy is provided, as described below.

[0121] The retroviral genomes and proviral DNAs of this disclosure have at least three genes: gag, pol, and env, which may have one or two long-terminal repeats (LTRs) adjacent to each other, or, in the case of proviruses, two long-terminal repeats (LTRs) adjacent to a sequence containing a cis-acting sequence such as psi. The gag gene encodes internal structure (matrix, capsid, and nucleocapsid) proteins; the pol gene encodes RNA-dependent DNA polymerase (reverse transcriptase), proteases, and integrases; and the env gene encodes viral envelope glycoproteins. The 5' and / or 3' LTRs function to facilitate transcription and polyadenylation of virion RNA. The LTRs contain all other cis-acting sequences necessary for viral replication. Lentiviruses have additional genes, including vif, vpr, tat, rev, vpu, nef, and vpx (in HIV-1, HIV-2, and / or SIV).

[0122] Adjacent to the 5'LTR are the sequences necessary for reverse transcription of the genome (tRNA primer binding sites) and the sequences necessary for efficient capsid formation of viral RNA into particles (Psi sites). If the sequences necessary for capsid formation (or packaging of retroviral RNA into infectious virions) are lost from the viral genome, the result is a cis-deficient that prevents capsid formation of genomic viral RNA. This type of modified vector is typically used in earlier gene delivery systems (i.e., systems lacking elements necessary for virion capsid formation).

[0123] Methods of production and collection In some embodiments, the present invention provides methods and compositions for generating multipseudotype viruses.

[0124] Some aspects of this disclosure include, or are, cells used as packaging cells for producing recombinant retroviruses or recombinant retroviral particles, including, in exemplary cases, mammalian cells. According to the present invention, any of a wide variety of cells can be selected for the in vitro production of viruses or viral particles, e.g., redirected recombinant retroviral particles. The term “packaging cell line” is used in reference to cell lines that do not contain packaging signals but stably or transiently express viral structural proteins and replication enzymes (e.g., gag, pol, and env) necessary for the precise packaging of viral particles. Packaging cells of the present invention can be prepared using any suitable cell line. Cell lines for packaging viruses are well known in the art. Eukaryotic cells are typically used. Generally, the cells are mammalian cells. In certain embodiments, the cells used to produce the packaging cell line are human cells. Suitable cell lines that can be used include, for example, CHO cells, BHK cells, MDCK cells, C3H 10T1 / 2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRCS cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211A cells.

[0125] In one embodiment, the packaging cells include 293 cells, HEK 293, 293T cells, GP2-293 cells, phoenix GP, 293-vec RD114, RetroPack PT67, Plat A, etc.

[0126] As used herein, the term “producer cell line” refers to a cell line capable of producing recombinant retroviral particles, comprising a packaging cell line and a transfer vector construct containing a packaging signal. The production of infectious viral particles and viral stock solutions can be carried out using conventional techniques. Methods for preparing viral stock solutions are known in the art and are exemplified, for example, by Y. Soneoka et al. (1995 Nucl. Acids Res. 23:628-633) and NRLandau et al. (1992). J. Virol. 66:5110-5113.

[0127] In certain embodiments, viral particles can be generated by co-expressing a virion packaging element and a transfer vector in producer cells. These cells can be transiently transfected with a number of plasmids. Typically, three to four plasmids are used, but the number may be greater, depending on the extent to which the retroviral components are broken down into separate units. For example, one plasmid may encode the core and enzymatic components of an HIV-1 virion. This plasmid is called a packaging plasmid. Another plasmid typically encodes an envelope protein, most commonly the G protein of vesicular stomatitis virus (VSV G) due to its high stability and broad tropism. In one embodiment, the plasmid encodes an envelope glycoprotein selected from the group of ecotropic virus envelope glycoproteins, amphotropic virus envelope glycoproteins, and any combination thereof. In one embodiment, the plasmid encodes an envelope glycoprotein selected from the group consisting of RD114, VSV-G, 4070A, gap70, 10A1, GALV, and any combination thereof. This plasmid may be called an envelope expression plasmid. In one embodiment, one or more envelope glycoproteins are used to generate viral particles. In one embodiment, each envelope expression plasmid expresses one type of envelope glycoprotein. In one embodiment, the envelope expression plasmid is manipulated to express one or more envelope glycoproteins. For example, in the generation of a multipseudotype virus, one or more envelope expression plasmids expressing envelope glycoproteins selected from the group consisting of RD114, VSV-G, 4070A, gap70, 10A1, and GALV are used, and each envelope expression plasmid expresses a different envelope glycoprotein.In another example, one or more envelope glycoproteins selected from the group consisting of RD114, VSV-G, 4070A, gap70, 10A1, and GALV are expressed from one or more envelope expression plasmids, and one expression plasmid expresses multiple envelope glycoproteins.

[0128] In another example, one or more envelope expression plasmids are used that express envelope glycoproteins selected from the group consisting of ecotropic virus envelope glycoproteins and amphotropic virus envelope glycoproteins, with each envelope expression plasmid expressing a different envelope glycoprotein. In yet another example, one or more envelope glycoproteins selected from the group consisting of ecotropic virus envelope glycoproteins and amphotropic virus envelope glycoproteins are expressed from one or more envelope expression plasmids, with one expression plasmid expressing multiple envelope glycoproteins.

[0129] For example, in the generation of a multipsydetype virus, an envelope expression plasmid expressing VSV-G and another envelope expression plasmid expressing RD114 are used. In another example, VSV-G and RD114 are expressed from a single envelope expression plasmid. In yet another example, in the generation of a multipsydetype virus, an envelope expression plasmid expressing VSV-G and another envelope expression plasmid expressing GALV are used. In yet another example, VSV-G and GALV are expressed from a single envelope expression plasmid. In yet another example, in the generation of a multipsydetype virus, an envelope expression plasmid expressing 10A1 and another envelope expression plasmid expressing GALV are used. In yet another example, 10A1 and GALV are expressed from a single envelope expression plasmid.

[0130] However, any combination of desired envelopes can be used with any desired number of expression plasmids.

[0131] Another plasmid encodes the genome, or vector itself, that is transferred to the target cell, and is called a transfer vector. Packaging plasmids can be introduced into human cell lines by known techniques, including calcium phosphate transfection, lipofection, or electroporation. Recombinant viruses with titers of several million transduction units per milliliter (TU / ml) can be generated by this technique and its variants.

[0132] In some embodiments, at least one packaging plasmid encoding at least one glycoprotein is in a ratio of about 1 / 10 to 1 / 50 compared to the total DNA. In some embodiments, at least one packaging plasmid encoding at least one glycoprotein is in a ratio of about 1 / 50 to 1 / 100 compared to the total DNA. In some embodiments, at least one packaging plasmid encoding at least one glycoprotein is in a ratio of about 1 / 100 to 1 / 1000 compared to the total DNA. In some embodiments, at least one packaging plasmid encoding at least one glycoprotein is in a ratio of about 1 / 1000 to 1 / 100,000 compared to the total DNA. In some embodiments, at least one packaging plasmid encoding at least one glycoprotein is in a ratio of about 1 / 100,000 to 1 / 100,000,000 compared to the total DNA.

[0133] In some embodiments, the at least one packaging plasmid encoding at least one glycoprotein is in a ratio of about 1 / 5 to 1 / 10 of the total DNA. In some embodiments, the at least one packaging plasmid encoding at least one glycoprotein is in a ratio of about 1 / 4 to 1 / 5 of the total DNA. In some embodiments, the at least one packaging plasmid encoding at least one glycoprotein is in a ratio of about 1 / 3 to 1 / 4 of the total DNA. In some embodiments, the at least one packaging plasmid encoding at least one glycoprotein is in a ratio of about 1 / 2 to 1 / 3 of the total DNA. In some embodiments, the at least one packaging plasmid encoding at least one glycoprotein is the only DNA.

[0134] Infectious viral particles can be collected from packaging cells using conventional techniques. For example, infectious particles can be collected by cell lysis or collection of the supernatant of cell culture, as is known in the art. If desired, the collected viral particles may be purified. Suitable purification techniques are well known to those skilled in the art. Viral particles may be concentrated. Methods for concentrating viruses are well known to those skilled in the art.

[0135] Viral particles can be collected by centrifugation or ultracentrifugation. After ultracentrifugation, a concentrated stock can be obtained. The virus can be used to infect cells in vivo, ex vivo, or in vitro using techniques well known in the art. For example, if cells are transduced ex vivo, the vector particles can generally be incubated with the cells using a desired dose.

[0136] Genetic composition of introduced genes Depending on the intended use of the retroviral vector of this disclosure, any number of heterogeneous polynucleotides or nucleic acid sequences may be inserted into the retroviral vector.

[0137] The polynucleotides of the present invention, regardless of the length of the coding sequence itself, can be combined with other DNA sequences, such as promoters and / or enhancers, untranslated regions (UTRs), Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosomal entry sites (IRESs), recombinase recognition sites (e.g., LoxP, FRT, and Att sites), stop codons, transcription termination signals, and polynucleotides encoding self-cleaving polypeptides, epitope tags, as disclosed elsewhere in this specification or as known in the art, so that their total length can vary considerably. Therefore, polynucleotide fragments of almost any length may be used, and the total length is preferably intended to be limited by the ease of preparation and use in the intended recombinant DNA protocol.

[0138] The term "expression regulatory sequence" refers to a polynucleotide sequence comprising one or more promoters, enhancers, or other transcriptional regulatory elements, or combinations thereof, that can operably direct, increase, regulate, or control the transcription or expression of a polynucleotide.

[0139] In certain embodiments, the vector of the present invention includes one or more expression regulatory sequences specific to a particular cell, cell type, or cell lineage, for example, target cells. That is, the expression of a polynucleotide operably linked to an expression regulatory sequence specific to a particular cell, cell type, or cell lineage is expressed in target cells but not in other non-target cells. Each of the one or more expression regulatory sequences in the cell-specific vector may be expressed in the same or different cell types depending on the desired treatment.

[0140] In certain embodiments, the vectors of the present invention include extrinsic, endogenous, or heterologous regulatory sequences such as promoters and / or enhancers. “Endogenous” regulatory sequences are those naturally ligated to a given gene in the genome. “Exogenous” regulatory sequences are sequences positioned parallel to a gene by genetic engineering (i.e., molecular biological techniques), resulting in the transcription of that gene being directed by the ligated enhancer / promoter. “Heterologous” regulatory sequences are extrinsic sequences derived from a different species than the genetically engineered cell. “Synthetic” regulatory sequences may include elements of another endogenous and / or extrinsic sequence, and / or sequences determined in vitro or in silico to provide optimal promoter and / or enhancer activity for a particular gene therapy.

[0141] In one embodiment, a nucleic acid expression control sequence (such as a promoter and / or enhancer, or other expression control sequence) and a second polynucleotide sequence, for example, a polynucleotide of interest, are operably linked, and the expression control sequence directs the transcription of the nucleic acid corresponding to the second sequence.

[0142] A constitutive regulatory sequence is a promoter, enhancer, or promoter / enhancer that enables continuous or sequential transcription of a operably linked sequence. A constitutive regulatory sequence may be a “ubiquitous” promoter, enhancer, or promoter / enhancer that enables expression in a wide range of cell and tissue types, or a “cell-specific,” “cell type-specific,” “cell lineage-specific,” or “tissue-specific” promoter, enhancer, or promoter / enhancer that enables expression in a limited range of cell and tissue types, respectively.Exemplary ubiquitous regulatory sequences include the cytomegalovirus (CMV) very early promoter, Simian virus 40 (SV40) (e.g., early or late), Moloney's mouse leukemia virus (MoMLV) LTR promoter, Roussarcoma virus (RSV) LTR, herpes simplex virus (HSV) (thymidine kinase) promoter, vaccinia virus-derived H5, P7.5, and P11 promoters, elongation factor 1-alpha (EF1a) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and eukaryotic translation initiation factor 4A1. Examples of such promoters include, but are not limited to, 4A1 (EIF4A1), heat shock 70kDa protein 5 (HSPA5), heat shock protein 90kDa beta, member 1 (HSP90B1), heat shock protein 70kDa (HSP70), β-kinesin (β-KIN), human ROSA 26 locus (Irions et al., (2007) Nature Biotechnology 25, 1477-1482), ubiquitin C promoter (UBC), phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer / chicken β-actin (CAG) promoter, and β-actin promoter.

[0143] In certain embodiments, it may be desirable to use cell, cell type, cell lineage, or tissue-specific expression regulatory sequences to achieve cell type-specific, lineage-specific, or tissue-specific expression of a desired polynucleotide sequence (for example, expressing a specific nucleic acid encoding a polypeptide in only a subset of cell types, cell lineages, or tissues, or during a specific stage of development).

[0144] Exemplary examples of tissue-specific promoters include the B29 promoter (B cell expression), the lanto transcription factor (CBFa2) promoter (stem cell-specific expression), the CD14 promoter (monocyte expression), the CD43 promoter (leukocyte and platelet expression), the CD45 promoter (hematopoietic cell expression), the CD68 promoter (macrophage expression), the CYP450 3A4 promoter (hepatocyte expression), the desmin promoter (muscle expression), the elastase 1 promoter (pancreatic acinar cell expression), the endoglin promoter (endothelial cell expression), the fibroblast-specific protein 1 (FSP1) promoter (fibroblast expression), the fibronectin promoter (fibroblast expression), the fins-related tyrosine kinase 1 (FLT1) promoter (endothelial cell expression), and the glial fibrillary acidic protein promoter. protein, GFAP promoter (astrocytic cell expression), insulin promoter (pancreatic beta cell expression), integrin, alpha 2b (integrin,Alpha 2b (ITGA2B) promoter (megakaryocytes), intracellular adhesion molecule 2 (ICAM-2) promoter (endothelial cells), interferon beta (IFN-β) promoter (hematopoietic cells), keratin 5 promoter (keratinocyte expression), myoglobin (MB) promoter (muscle expression), myogenic differentiation 1 (MYOD1) promoter (muscle expression), nephrin promoter (podocyte expression), bone gamma-carboxyglutamate protein 2 (OG-2) promoter (osteoblast expression), 3-oxoacid CoA transferase 2B (Oxct2B) promoter (haploid spermatogonial expression), surfactant protein B (SP-B) promoter (lung expression), synapsin promoter (neuron expression), Wiskott-Aldrich syndrome protein Examples include, but are not limited to, protein and WASP promoters (hematopoietic cell expression).

[0145] In certain embodiments of the present invention, the conditional expression of a polynucleotide of interest is provided, for example, by subjecting cells, tissues, organisms, etc., to a process or condition that causes them to express the polynucleotide or to increase or decrease the expression of the polynucleotide encoded by the polynucleotide of interest.

[0146] Examples of inductive promoters / systems include, but are not limited to, steroid-inducible promoters (e.g., promoters of genes encoding glucocorticoids or estrogen receptors (inducible by treatment with the corresponding hormones)), metallothione promoters (inducible by treatment with various heavy metals), MX-1 promoters (inducible by interferon), the "GeneSwitch" mifepristone-regulated system (Sirin et al., (2003) Gene, 323:67), the kmet-inducible gene switch (International Publication No. 2002 / 088346), and tetracycline-dependent regulatory systems.

[0147] Conditional expression can also be achieved by using site-specific DNA recombinases. According to certain embodiments of the present invention, the vector contains at least one (typically two) sites for recombination mediated by site-specific recombinases. As used herein, the terms “recombinase” or “site-specific recombinase” include excised or incorporated proteins, enzymes, cofactors or related proteins involved in a recombination reaction involving one or more recombination sites (e.g., 2, 3, 4, 5, 7, 10, 12, 15, 20, 30, 50, etc.), which may be wild-type proteins (see Landy, (1993) Current Opinion in Biotechnology 3:699-707), or their variants, derivatives (e.g., fusion proteins containing recombinant protein sequences or fragments thereof), fragments, and variants. Exemplary examples of recombinases suitable for use in specific embodiments of the present invention include, but are not limited to, Cre, Int, IHF, Xis, Flop, Fis, Hin, Gin, ΦC31, Cin, Tn3 resolverase, TndX, XerC, XerD, TnpX, Hjc, Gin, SpCCE1, and ParA.

[0148] A vector may contain one or more recombination sites for any of the wide variety of site-specific recombinases. It should be understood that the target site of the site-specific recombinase is added to any site required for integration into the vector, such as a retroviral vector or lentiviral vector. As used herein, the terms “recombinant sequence,” “recombinant site,” or “site-specific recombinant site” refer to the specific nucleic acid sequence that a recombinase recognizes and binds to.

[0149] For example, one recombination site of Cre recombinase is loxP, a 34-base pair sequence containing two 13-base pair reverse repeats (which function as recombinase binding sites) adjacent to an 8-base pair core sequence (see Figure 1 in Sauer, B., (1994) Current Opinion in Biotechnology 5:521-527). Other exemplary loxP sites include, but are not limited to, lox511 (Hoess et al., 1996, Bethke and Sauer, 1997), lox5171 (Lee and Saito, 1998), lox2272 (Lee and Saito, 1998), m2 (Langer et al., 2002), lox71 (Albert et al., 1995), and lox66 (Albert et al., 1995).

[0150] Suitable recognition sites for FLP recombinase include, but are not limited to, FRT (McLeod, et al., 1996), F1, F2, F3 (Schlake and Bode, 1994), F4, F5 (Schlake and Bode, 1994), FRT(LE) (Senecoff et al., 1988), and FRT(RE) (Senecoff et al., 1988).

[0151] Other examples of recognition sequences are the attB, attP, attL, and attR sequences recognized by the recombinase enzyme λ-integrase, e.g., phi-c31. The φC31 SSR mediates recombination only between the heterotype sites attB (length 34 bp) and attP (length 39 bp) (Groth et al., 2000). Named after the attachment sites of phage integrases on bacterial and phage genomes, respectively, attB and attP both contain incomplete reverse repeats that are likely to be bound by the φC31 homodimer (Groth et al., 2000). The product sites, attL and attR, are effectively inactive to further φC31-mediated recombination (Belteki et al., 2003), making the reaction irreversible. To catalyze insertion, DNA containing attB has been found to be more readily inserted into genomic attP sites than attP sites into genomic attB sites (Thyagarajan et al., 2001; Belteki et al., 2003). Therefore, a typical strategy is to position a “docking site” containing attP at a defined locus by homologous recombination, which is then paired with an incoming sequence containing attB for insertion.

[0152] Certain embodiments of the present invention also include polypeptide "variants." The term polypeptide "variant" refers to a polypeptide that is distinguished from a reference polypeptide by the addition, deletion, cleavage, and / or substitution of at least one amino acid residue and that retains biological activity. In certain embodiments, the polypeptide variant is distinguished from the reference polypeptide by one or more substitutions, which may be conserved or non-conservative, as known in the art.

[0153] In certain embodiments, the variant polypeptide comprises an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity or similarity to the corresponding sequence of the reference polypeptide. In certain embodiments, the addition or deletion of amino acids occurs at the C-terminus and / or N-terminus of the reference polypeptide.

[0154] As described above, the polypeptides of the present invention can be modified in various ways, including amino acid substitution, deletion, cleavage, and insertion. Methods for such operations are generally known in the art. For example, amino acid sequence variants of a reference polypeptide can be prepared by mutation in DNA. Methods for mutagenesis and nucleotide sequence modification are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA. 82:488-492, Kunkel et al., (1987) Methods in Enzymol, 154:367-382, U.S. Patent No. 4,873,192, Watson, J. Det. al., (1987) Molecular Biology of the Gene, Fourth Edition, Benjamin / Cummings, Menlo Park, Calif., and the references cited therein. Guidance on appropriate amino acid substitutions that do not affect the biological activity of the target protein can be found in the model in Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC).

[0155] Those skilled in the art will readily understand that there are no limitations on the possible heterogeneous polynucleotides or nucleic acid sequences that can be inserted into the viral vector introduced into host cells by the present invention.

[0156] A vector typically contains a transmissible DNA into which a protein-coding foreign DNA has been inserted by restriction enzyme technique. A common type of vector is a "plasmid," which is generally a self-contained double-stranded DNA molecule that can readily accept additional (foreign) DNA and be readily introduced into a suitable host cell. Numerous vectors, including plasmids and fungal vectors, have been described for replication and / or expression in various eukaryotic and prokaryotic hosts. Non-limiting examples include pKK plasmid (Clonetech), pUC plasmid, pET plasmid (Novagen, Inc., Madison, Wis.), pRSET or pREP plasmid (Invitrogen, San Diego, Calif.), or pMAL plasmid (New England Biolabs, Beverly, Mass.), and many suitable host cells, using methods disclosed or cited herein, or otherwise known to those skilled in the art in the relevant art. Recombinant cloning vectors often include one or more replication systems for cloning or expression, one or more markers for selection in the host (e.g., antibiotic resistance), and one or more expression cassettes.

[0157] In some embodiments, the transfer vector includes a nucleic acid sequence. In some embodiments, the transfer vector includes a gene encoding a transgene. In some embodiments, the transgene is one or more selected from the group consisting of siRNA, miRNA, mRNA, antibody, protein, and peptide. In some embodiments, the transgene is one or more CARs having secreted / fixed proteins.

[0158] Chimeric Antigen Receptor (CAR) In one embodiment, the transgene encodes a CAR. In one embodiment, the CAR includes an antigen-binding domain specific to at least one marker of at least one cancer cell or at least one pathogen. In some embodiments, upon binding to at least one cancer cell or at least one pathogen, at least one modified target cell promotes the destruction of at least one cancer cell or at least one pathogen (e.g., by phagocytosis, T cell-mediated cytotoxicity, etc.), thereby treating or preventing a disease or disorder in the subject (e.g., cancer).

[0159] As used herein, the terms “chimeric antigen receptor” or “CAR” refer to an artificial cell receptor expressed on immune effector cells such as NK cells, macrophages, B cells, or dendritic cells, which is engineered to specifically bind to an antigen on at least one cancer cell or at least one pathogen. CARs may be used as therapeutic agents in conjunction with adoptive cell transfer. Generally, the immune cells of interest are removed from a patient and modified to express a receptor specific to a particular form of antigen. In some embodiments, the CAR has specificity for at least one cancer cell or at least one pathogen. The CAR may also include an intracellular activation domain, a transmembrane domain, and an extracellular domain containing an antigen-binding region that specifically binds to at least one cancer cell or at least one pathogen.

[0160] In various embodiments, a CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding element, also called an antigen-binding domain. In some embodiments, the extracellular domain also includes a hinge domain. In certain embodiments, the intracellular domain, or otherwise the cytoplasmic domain, includes a costimulatory signaling region and a zeta chain portion. The costimulatory signaling region refers to the portion of the CAR that includes the intracellular domain of the costimulatory molecule. The costimulatory molecule is a cell surface molecule other than the antigen receptor or its ligand necessary for the efficient response of lymphocytes to an antigen.

[0161] A spacer domain may be incorporated between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR. As used herein, the term “spacer domain” generally means any oligo or polypeptide that functions to link the transmembrane domain to either the extracellular domain or the cytoplasmic domain in the polypeptide chain. The spacer domain may contain up to 5 amino acids, or 10 amino acids, or 20 amino acids, or 30 amino acids, or 40 amino acids, or 50 amino acids, or 60 amino acids, or 70 amino acids, or 80 amino acids, or 90 amino acids, or 100 amino acids, or 110 amino acids, or 120 amino acids, or 130 amino acids, or 140 amino acids, or 150 amino acids, or 160 amino acids, or 170 amino acids, or 180 amino acids, or 190 amino acids, or 200 amino acids, or 210 amino acids, or 220 amino acids, or 230 amino acids, or 240 amino acids, or 250 amino acids, or 260 amino acids, or 270 amino acids, or 280 amino acids, or 290 amino acids, or 300 amino acids.

[0162] The extracellular domain, transmembrane domain, and intracellular domain can be derived from any desired source of such domains.

[0163] CAR antigen-binding domain The antigen-binding domain may be obtained from any of a wide variety of extracellular domains or secreted proteins involved in ligand binding and / or signal transduction. In one embodiment, the antigen-binding domain may consist of an Ig heavy chain that can be covalently bound to an Ig light chain by the presence of CH1 and hinge regions, respectively, or it may be covalently bound to another Ig heavy / light chain complex by the presence of hinge, CH2, and CH3 domains. In the latter case, the heavy / light chain complex conjugated to the chimeric construct may constitute an antibody having specificity different from that of the chimeric construct. Depending on the antibody function, desired structure, and signal transduction, the entire chain may be used, or cleaved chains may be used, or all or part of the CH1, CH2, or CH3 domains may be removed, or all or part of the hinge region may be removed.

[0164] In various embodiments, the CAR antigen-binding domain may be humanized or may contain a complete human sequence.

[0165] CAR transmembrane domain With respect to the transmembrane domain, the CARs of this disclosure can be designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain naturally associated with one of the domains within the CAR is used. In some examples, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domain to the transmembrane domains of the same or different surface membrane proteins, thereby minimizing interaction with other members of the receptor complex.

[0166] The transmembrane domain may originate from either a natural or synthetic source. If the source is natural, the domain may originate from any membrane-bound or transmembrane protein. Particularly useful transmembrane regions in the present invention may originate from the alpha, beta, or zeta chains of T cell receptors, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154 (i.e., may include at least their transmembrane regions). Alternatively, the transmembrane domain may be synthetic, in which case it mainly consists of hydrophobic residues such as leucine and valine. In one embodiment, a triplet of phenylalanine, tryptophan, and valine may be found at each end of the synthetic transmembrane domain. Optionally, short oligo or polypeptide linkers, for example, but not limited to those 2 to 10 amino acids long, may form a link between the transmembrane domain of the CAR and the cytoplasmic signaling domain. In another embodiment, the linker comprises a glycine-serine doublet.

[0167] CAR intracellular domain In various embodiments, the cytoplasmic domain of a CAR, or otherwise an intracellular domain, may be responsible for activating at least one of the normal effector functions of the immune cell on which the CAR is expressed. The term “effector function” refers to a specific function of the cell. The effector function of a T cell may be, for example, cytolytic activity or helper activity, including cytokine secretion. The term “intracellular signaling domain” refers to the portion of the protein that transmits the effector function signal and instructs the cell to perform the specific function. Usually, the entire intracellular domain can be used, but often it is not necessary to use the entire chain. To the extent that a cleavage portion of the intracellular domain is used, such a cleavage portion can be used in place of the intact chain, as long as it transmits the effector function signal. Thus, the term intracellular domain means that it includes any cleavage portion of the intracellular domain sufficient to transmit the effector function signal.

[0168] Examples of intracellular domains for use in the CARs of this disclosure include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that act in coordination to initiate signal transduction after antigen receptor ligation, as well as any derivatives or variants of these sequences and any synthetic sequences having the same functional capabilities.

[0169] It is known that signals generated via the TCR alone are insufficient for complete T cell activation, and that secondary or co-stimulatory signals are also required. Therefore, T cell activation can be said to be mediated by two classes of intracellular signaling sequences: sequences that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences), and sequences that act in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences).

[0170] Primary intracellular signaling sequences regulate the primary activation of the TCR complex either stimulatingly or inhibitorily. Stimulating primary intracellular signaling sequences may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs.

[0171] Examples of ITAMs containing primary intracellular signaling sequences particularly useful in the present invention include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In one embodiment, the intracellular signaling molecule in the CAR of the present invention includes an intracellular signaling sequence derived from CD3 zeta.

[0172] In another embodiment, the intracellular domain of the CAR may include the CD3-zeta signaling domain itself or be designed to be combined with any other desired cytoplasmic domain useful in the context of the CAR of the present invention. For example, the intracellular domain of the CAR may include the CD3 zeta chain portion and the costimulatory signaling region. The costimulatory signaling region refers to a portion of the CAR that includes the intracellular domain of a costimulatory molecule. The costimulatory molecule is a cell surface molecule other than the antigen receptor or its ligand necessary for the efficient response of lymphocytes to an antigen. Examples of such molecules include ligands that specifically bind to CD2, CD27, CD28, 4-1BB (CD137), Ox40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.

[0173] The intracellular signaling sequences within the intracellular domain of the CAR of the present invention can be linked to each other in a random or specific order. Optionally, short oligo or polypeptide linkers, e.g., 2 to 10 amino acid lengths, can form the linkage. Glycine-serine doublets provide suitable linkers in some embodiments.

[0174] In one embodiment, the intracellular domain is designed to include a CD3 zeta signaling domain and a CD28 signaling domain. In yet another embodiment, the intracellular domain is designed to include a CD3 zeta signaling domain and a 4-IBB signaling domain.

[0175] In various embodiments, the CAR can be a "first-generation," "second-generation," "third-generation," "fourth-generation," or "fifth-generation" CAR (e.g., Sadelain et al., Cancer Discov.3(4):388-398(2013), Jensen et al., Immunol.Rev.257:127-133(2014), Sharpe et al., Dis.Model Mech.8(4):337-350(2015), Brentjens et al., Clin.Cancer Res.13:5426-5435(2007), Gade et al., Cancer Res.65:9080-9088(2005), Maher et al., Nat.Biotechnol.20:70-75(2002), Kershaw et al. See al., J.Immunol. 173:2143-2150 (2004), Sadelain et al., Curr.Opin.Immunol. (2009), and Hollyman et al., J.Immunother. 32:169-180 (2009). Each of these is incorporated in its entirety by reference.

[0176] The “first-generation” CARs for use in this invention comprise a single-chain variable fragment (scFv) fused to a transmembrane domain, which is fused to an antigen-binding domain, e.g., the cytoplasmic / intracellular domain of a T cell receptor chain. “First-generation” CARs typically have an intracellular domain derived from the CD3ζ chain, which is the primary signaling molecule from the endogenous T cell receptor (TCR). “First-generation” CARs can provide de novo antigen recognition in a single fusion molecule, independently of HLA-mediated antigen presentation, via their CD3ζ chain signaling domains, and induce activation of both CD4+ and CD8+ T cells.

[0177] The “second-generation” CAR for use in this invention comprises an antigen-binding domain, e.g., a single-chain variable fragment (scFv), fused to an intracellular signaling domain capable of activating T cells, and a costimulatory domain designed to enhance the potency and persistence of T cells (Sadelain et al., Cancer Discov. 3:388-398 (2013)). Thus, the CAR design can combine antigen recognition with signaling, and the two functions are physiologically supported by two distinct complexes, the TCR heterodimer and the CD3 complex. The “second-generation” CAR contains intracellular domains from various costimulatory molecules, e.g., CD28, 4-1BB, ICOS, OX40, etc., in the cytoplasmic tail of the CAR to provide additional signaling to the cell.

[0178] "Second-generation" CARs provide both, for example, co-stimulation via the CD28 or 4-1BB domain, and activation via, for example, the CD3ζ signaling domain. Preclinical studies have shown that "second-generation" CARs can improve the antitumor activity of T cells. For example, the potent efficacy of "second-generation" CAR-modified T cells was demonstrated in clinical trials targeting the CD19 molecule in patients with chronic lymphoblastic leukemia (CLL) and acute lymphoblastic leukemia (ALL) (Davila et al., Oncoimmunol. 1(9):1577-1583 (2012)).

[0179] "Third-generation" CARs provide multiple co-stimuli, for example, by including both the CD28 and 4-1BB domains, and provide activation, for example, by including the CD3ζ activation domain.

[0180] "Fourth-generation" CARs provide, for example, co-stimulation by the CD28 or 4-1BB domain, and activation by the CD3ζ signaling domain in addition to, for example, constitutive or inducible chemokine components.

[0181] "Fifth-generation" CARs provide, for example, co-stimulation by CD28 or 4-1BB domains, as well as activation by, for example, CD3ζ signaling domains, constitutive or inducible chemokine components, and cytokine receptors, such as the intracellular domain of IL-2Rβ.

[0182] In various embodiments, CARs may be included in a multivalent CAR system, such as a DualCAR or "TandemCAR" system. A multivalent CAR system includes a system or cell comprising multiple CARs, and a system or cell comprising a bivalent / bispecific CAR that targets more than one antigen.

[0183] In embodiments disclosed herein, the CAR generally comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain, as described above. In certain non-limiting embodiments, the antigen-binding domain is scFv.

[0184] In one embodiment, the antigen-binding domain of the CAR molecule is a targeting domain, which directs cells expressing the CAR to at least one cancer cell or at least one pathogen. For example, in one embodiment, the targeting domain includes an antibody, antibody fragment, or peptide that specifically binds to an antigen (e.g., an autoantigen or an exogenous antigen), thereby directing cells expressing the CAR to at least one cancer cell or at least one pathogen, where the at least one cancer cell or at least one pathogen expresses the antigen.

[0185] In one embodiment, the antigen-binding domain of the CAR molecule of the present invention can be generated to be reactive to any desired antigen or fragment thereof of interest, including but not limited to tumor antigens, exogenous antigens (e.g., bacterial antigens, viral antigens, etc.), or autoantigens on the surface of at least one cancer cell or at least one pathogen. In some embodiments, the antigen on the surface of at least one cancer cell is a tumor antigen.

[0186] Tumor antigens are proteins produced by tumor cells that trigger an immune response. The selection of the antigen-binding domain in the VM domain-containing fusion molecule of the present invention depends on the specific type of cancer being treated. Tumor antigens are well known in the art, and include, for example, glioma-associated antigens, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostain, PSMA, Her2 / neu, survivorbin and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, and insulin growth factor. Examples of tumor antigens include factor, IGF-I, IGF-II, IGF-I receptor, and mesothelin. Another exemplary tumor antigen is chondroitin sulfate proteoglycan 4 (CSPG4) (also known as melanoma-associated chondroitin sulfate proteoglycan (MCSP), high-molecular-weight melanoma-associated antigen (HMW-MAA), or neuron-glial antigen 2 (NG2)).

[0187] In one embodiment, a tumor antigen comprises one or more antigenic cancer epitopes associated with a malignant tumor. Malignant tumors express numerous proteins that can function as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase, and GP 100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens are oncoemetic antigens such as carcinoembryonic antigens (CEAs). In B-cell lymphoma, tumor-specific idiotype immunoglobulins constitute truly tumor-specific immunoglobulin antigens that are unique to individual tumors. B-cell differentiation antigens such as CD19, CD20, and CD37 are other candidate target antigens in B-cell lymphoma. Some of these antigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targets for passive immunotherapy using monoclonal antibodies, but success has been limited.

[0188] The types of tumor antigens referred to in this invention may be tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs). TSAs are specific to tumor cells and are not present in other cells in the body. TAA-associated antigens are not specific to tumor cells and instead are expressed on normal cells under conditions that do not induce a state of immune tolerance to the antigen. Antigen expression on tumors can occur under conditions that allow the immune system to respond to the antigen. TAAs may be antigens expressed on normal cells during fetal development when the immune system is immature and unable to respond, or they may be antigens that are normally present at very low levels on normal cells but are expressed at much higher levels on tumor cells.

[0189] Non-limiting examples of TSA or TAA antigens include: differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multisystem antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, and p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, and HER-2 / neu; unique tumor antigens arising from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, and MYL-RAR; and viral antigens, such as Epstein-Barr virus antigen (EBVA) and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3, CA 27.29, BCAA, CA 195, CA Examples include 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophyllin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0190] In some embodiments, the antigen on the surface of at least one pathogen comprises an exogenous antigen, which comprises a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen or a fragment thereof, or a variant thereof.

[0191] Transduction of cells The present invention aims to provide compositions and methods for transducing host cells with high titer and high efficiency in vitro, ex vivo, and in vivo.

[0192] Host cells can be transfected, infected, or transduced in vivo, ex vivo, or in vitro using the recombinant vector or polynucleotide of the present invention. Host cells may include packaging cells, producer cells, and cells infected with the viral vector. In certain embodiments, host cells infected with the viral vector of the present invention are administered to a subject requiring treatment. In certain embodiments, the term “target cells” is used interchangeably with “host cells” and refers to transfected, infected, or transduced cells of a desired cell type. In some embodiments, target cells are dividing or non-dividing cells. In some embodiments, target cells are mammalian cells. In some embodiments, target cells are human cells. In some embodiments, cells are immune cells. In some embodiments, the cells are one or more selected from the group consisting of alpha-beta T cells (αβT cells), gamma-delta T cells (γδT cells), natural killer (NK) cells, dendritic cells, macrophages, natural killer T (NKT) cells, B cells, neutrophils, mast cells, hematopoietic stem cells (HSCs), induced pluripotent stem cells (IPSCs), IPSC-derived αβT cells, IPSC-derived NK cells, IPSC-derived γδT cells, CD34+ cells, umbilical cord blood-derived stem cells, lymphoid tissue inducer (LTi) cells, group 1 innate lymphoid cells (ILC-1), group 2 innate lymphoid cells (ILC-2), and group 3 innate lymphoid cells (ILC-3). In some embodiments, the cells are gamma-delta T cells (γδT cells).

[0193] In some embodiments, host cells are activated before, during, or after transfection, infection, or transduction. In some embodiments, host cells are activated before, during, and after transfection, infection, or transduction. In some embodiments, host cells are activated before transduction. In some embodiments, host cells are activated during transfection, infection, or transduction. In some embodiments, host cells are activated after transfection, infection, or transduction.

[0194] Methods for activating host cells are known in the art. In some embodiments, cells are activated by a TCR agonist. In some embodiments, the TCR agonist is one or more selected from the group consisting of anti-αβTCR antibodies and anti-γδTCR antibodies. In some embodiments, cells are activated by one or more selected from the group consisting of anti-NKG2D antibodies, anti-CD2 antibodies, anti-NkP46 antibodies, anti-NkP30 antibodies, anti-NkP44 antibodies, anti-CD16 antibodies, IL2 and / or anti-CD3 (e.g., OKT3) antibodies. In some embodiments, cells are activated by IL2 and / or anti-CD3 (e.g., OKT3) antibodies. In some embodiments, cells are activated by IL2. In some embodiments, cells are activated by anti-CD3 antibodies. In some embodiments, the cells to be activated are immune cells. In some embodiments, the cells to be activated are T cells.

[0195] In some embodiments, cells are activated with 0.1U to 0.1U of one or more antibodies selected from the group consisting of anti-NKG2D antibody, anti-CD2 antibody, anti-NkP46 antibody, anti-NkP30 antibody, anti-Nkp44 antibody, anti-CD16 antibody, IL2 and / or anti-CD3 (e.g., OKT3) antibody. In some embodiments, cells are activated with 1 U to 10 U of one or more antibodies selected from the group consisting of anti-NKG2D antibody, anti-CD2 antibody, anti-NkP46 antibody, anti-NkP30 antibody, anti-Nkp44 antibody, anti-CD16 antibody, IL2, and / or anti-CD3 (e.g., OKT3) antibodies. In some embodiments, cells are activated with 10 U to 100 U of one or more antibodies selected from the group consisting of anti-NKG2D antibody, anti-CD2 antibody, anti-NkP46 antibody, anti-NkP30 antibody, anti-Nkp44 antibody, anti-CD16 antibody, IL2, and / or anti-CD3 (e.g., OKT3) antibodies. In some embodiments, cells are activated with 100 U to 1000 U of one or more antibodies selected from the group consisting of anti-NKG2D antibody, anti-CD2 antibody, anti-NkP46 antibody, anti-NkP30 antibody, anti-Nkp44 antibody, anti-CD16 antibody, IL2, and / or anti-CD3 (e.g., OKT3) antibodies. In some embodiments, cells are activated with 1000 U to 1,000,000 U of one or more antibodies selected from the group consisting of anti-NKG2D antibody, anti-CD2 antibody, anti-NkP46 antibody, anti-NkP30 antibody, anti-Nkp44 antibody, anti-CD16 antibody, IL2, and / or anti-CD3 (e.g., OKT3) antibodies.

[0196] In some embodiments, cells are activated with 0.0000001U to 0.1U of IL2. In some embodiments, cells are activated with 0.1U to 1U of IL2. In some embodiments, cells are activated with 1U to 10U of IL2. In some embodiments, cells are activated with 10U to 100U of IL2. In some embodiments, cells are activated with 100U to 1000U of IL2. In some embodiments, cells are activated with 1000U to 1,000,000U of IL2.

[0197] In some embodiments, cells are activated with 0.0000001U to 0.1U of anti-CD3 antibody. In some embodiments, cells are activated with 0.1U to 1U of anti-CD3 antibody. In some embodiments, cells are activated with 1U to 10U of anti-CD3 antibody. In some embodiments, cells are activated with 10U to 100U of anti-CD3 antibody. In some embodiments, cells are activated with 100U to 1000U of anti-CD3 antibody. In some embodiments, cells are activated with 1000U to 1,000,000U of anti-CD3 antibody.

[0198] In some embodiments, cells are activated for 0 to 1 hour. In some embodiments, cells are activated for 1 to 6 hours. In some embodiments, cells are activated for 6 to 12 hours. In some embodiments, cells are activated for 12 to 24 hours. In some embodiments, cells are activated for 24 to 48 hours. In some embodiments, cells are activated for 48 to 72 hours. In some embodiments, cells are activated for 3 days to 1 week. In some embodiments, cells are activated for 1 week to 1 month. In some embodiments, cells are activated for 1 month to 1 year. In some embodiments, cells are activated for 1 year to 10 years.

[0199] In some embodiments, the recombinant retroviral vector contains the polynucleotide / gene of interest.

[0200] Method of Use in Treatment The virus can be delivered according to the virus titer (TU / mL), which can be measured, for example, by using a commercially available p24 titer assay, which is an ELISA for the p24 virus coat protein. The following formula can be used to calculate pg / mL of p24: There are approximately 2000 molecules of p24 per physical particle (PP) of lentivirus: (2×10 3 )×(24×10 3 Da of p24 per PP), 48×10 6 / Avogadro = PP per (48×10 6 ) / (6×10 23 ) = 8×10 -17 g of p24, approximately 1 PP per 1×10 -16 g of p24, 1×10 4 PP per 1 pg of p24. A reasonably well-packaged recombinant retroviral vector has an infectivity index in the range of 1 TU per 1000 physical particles (PP) to 1 TU per 100 PP (or less). Thus, the range is approximately 10 - 100 TU / pg of p24. Through this conversion, TU / mL is obtained.

[0201] The virus can be delivered to a subject in vivo by direct injection into the cells, tissues, or organs that require treatment. As a non-limiting exemplary example, the virus can be delivered for regenerative medicine. As a further non-limiting exemplary example, the virus can be used in the fields of oncology, immunology, cardiovascular, autoimmune, aging, musculoskeletal, ophthalmic treatment, and dermatology. Direct injection requires a multiplicity of infection (MOI) of about 1 - 50, which also corresponds to 1×10 5 to 50×10 5 transducing units of the viral vector per cell.

[0202] ​​Based on previous experience, the amount of recombinant retrovirus directly injected is determined by the total TU and can vary based on both the volume that can be feasibly injected into the site and the type of tissue to be injected. For example, the brain injection site may only be able to inject a very small amount of virus, and therefore a high-titer preparation is preferred, with approximately 1 × 10⁶ per injection. 6 ~1 × 10 7 , about 1×10 6 ~1 × 10 8 , 1 x 10 6 ~1 × 10 9 , about 1×10 7 ~1 × 10 10 , 1 x 10 8 ~1 × 10 11 , about 1×10 8 ~1 × 10 12 , or approximately 1 x 10 10 ~1 × 10 12 The above TUs can be used. However, whole-body delivery can accommodate much larger TUs, 1 × 10 8 , 1 x 10 9 , 1 x 10 10 , 1 x 10 11 , 1 x 10 12 , 1 x 10 13 , 1 x 10 14 , or 1 × 10 15 It can deliver the load.

[0203] Embodiment The present invention includes at least the following numbered embodiments. 1. A method for generating a multipseudotype virus, the method comprising: providing a mammalian cell line; adding a cell medium to the mammalian cell line; contacting the mammalian cell line with a composition comprising at least one packaging plasmid encoding at least one glycoprotein; incubating the mammalian cell line with the composition; and recovering the multipseudotype virus 48 hours and 72 hours after contacting the mammalian cell line with the composition, wherein the recovery of the multipseudotype virus yields a virus with a higher titer compared to a method for producing a virus containing a single pseudotype. 2. The method according to Embodiment 1, wherein the culture is carried out at a temperature of approximately 37°C in a humidified incubator with approximately 5% to 10% CO2 for approximately 24 to 36 hours. 3. The method according to Embodiment 1 or Embodiment 2, wherein the composition comprising at least one packaging plasmid encoding at least one glycoprotein further comprises a transfer vector containing a gene encoding an introduced gene. 4. The method according to any one of Embodiments 1 to 3, wherein at least one glycoprotein comprises a first glycoprotein selected from the group consisting of ecotropic virus envelope glycoproteins and amphotropic envelope glycoproteins. 5. The method according to any one of Embodiments 1 to 3, wherein at least one glycoprotein comprises a first glycoprotein selected from the group consisting of RD114, VSV-G, 4070A, gap70, 10A1, and GALV. 6. The method according to any one of Embodiments 1 to 5, wherein at least one glycoprotein is two glycoproteins, three glycoproteins, or four glycoproteins. 7. The method according to any one of Embodiments 1 to 6, wherein at least one glycoprotein comprises a second glycoprotein selected from the group consisting of ecotropic virus envelope glycoproteins and amphotropic envelope glycoproteins, and is different from the first glycoprotein. 8. The method according to any one of Embodiments 1 to 6, wherein at least one glycoprotein comprises a second glycoprotein selected from the group consisting of RD114, VSV-G, 4070A, gap70, 10A1, and GALV, and is different from the first glycoprotein. 9. The method according to Embodiment 8, wherein the two glycoproteins are RD114 and VSV-G. 10. The method according to Embodiment 8, wherein the two glycoproteins are GALV and VSV-G. 11. The method according to Embodiment 8, wherein the two glycoproteins are GALV and 10A1. 12. The method according to any one of Embodiments 1 to 11, wherein at least one packaging plasmid encoding at least one glycoprotein is in a ratio of about 1 / 5 to 1 / 10 of the total DNA. 13. A method for producing a multipseudotype virus, the method comprising: providing a mammalian cell line for transfection and virus production; culturing the mammalian cell line, wherein the culturing is performed at a temperature of approximately 37°C in a humidified incubator with approximately 5% to 10% CO2; treating the mammalian cell line with trypsin; adding cell medium to the mammalian cell line; contacting the mammalian cell line with a composition comprising i) a transfer vector containing a transgene, and ii) at least one packaging plasmid encoding at least one glycoprotein; incubating the mammalian cell line with the composition; and recovering the multipseudotype virus 48 hours and 72 hours after contacting the mammalian cell line with the composition, wherein the recovery of the multipseudotype virus yields a virus with a higher titer compared to a method for producing a virus containing a single pseudotype. 14. The method according to Embodiment 13, wherein at least one glycoprotein comprises a first glycoprotein selected from the group consisting of ecotropic virus envelope glycoproteins and amphotropic envelope glycoproteins. 15. The method according to Embodiment 13, wherein the first envelope glycoprotein is selected from the group consisting of RD114, VSV-G, 4070A, gap70, 10A1, and GALV. 16. The method according to any one of Embodiments 13 to 15, wherein at least one glycoprotein is two glycoproteins, three glycoproteins, or four glycoproteins. 17. The method according to any one of Embodiments 13 to 16, wherein the second envelope glycoprotein is selected from the group consisting of ecotropic virus envelope glycoproteins and amphotropic envelope glycoproteins, and the second envelope glycoprotein is different from the first envelope glycoprotein. 18. The method according to any one of Embodiments 13 to 16, wherein the second envelope glycoprotein is selected from the group consisting of RD114, VSV-G, 4070A, gap70, 10A1, and GALV, and the second envelope glycoprotein is different from the first envelope glycoprotein. 19. The method according to any one of Embodiments 13 to 18, wherein the two glycoproteins are RD114 and VSV-G. 20. The method according to any one of Embodiments 13 to 18, wherein the two glycoproteins are GALV and VSV-G. 21. The method according to any one of Embodiments 13 to 18, wherein the two glycoproteins are GALV and 10A1. 22. The method according to any one of Embodiments 1 to 21, further comprising filtering for multipseudotype viruses, wherein the filtering is carried out using a low protein binding filter. 23. The method according to Embodiment 22, wherein filtration is carried out using a filter containing cellulose acetate or polysulfonate. 24. The method according to any one of Embodiments 1 to 23, further comprising concentrating the virus. 25. The method according to any one of Embodiments 1 to 24, wherein the method produces a multipseudotype virus with a titer about 20, 30, 40, 50, 60, 70, 80, or 90 times higher than a method for producing a virus containing a single envelope glycoprotein including RD114, VSV-G, GALV, or 10A1. 26. The method according to any one of Embodiments 1 to 25, wherein the virus includes a retrovirus. 27. An engineered multipseudotype virus containing at least two envelope glycoproteins. 28. An engineered multipseudotype virus produced by the method described in any one of Embodiments 1 to 26. 29. The manipulated multipseudotype virus described in Embodiment 27 or Embodiment 28, wherein the virus includes a retrovirus. 30. A method for increasing the transduction efficiency in γδT cells, the method comprising: providing engineered viral particles, wherein the viral particles comprise a protein-coding nucleic acid and a multipseudotype virus; providing negatively isolated γδT cells for transduction; and transducing the engineered viral particles into γδT cells. 31. The method according to Embodiment 30, wherein the protein comprises a chimeric antigen receptor. 32. A system for carrying out a method for generating γδT cells containing a transgene, the system comprising one or more components capable of carrying out a method comprising: providing γδT cells; activating the γδT cells for about 24 to 48 hours using about 1000 U of IL2 or OKT3 antibody; transducing the γδT cells with an engineered multipseudotype virus containing a sequence encoding the transgene; and analyzing the transduction efficiency, wherein the transduction efficiency is increased by the multipseudotype virus compared to transduction by a singlepseudotype virus. 33. The system according to Embodiment 32, wherein the virus comprises a gamma retrovirus. 34. A method for increasing viral titer, the method comprising: providing a mammalian cell line; adding a cell medium to the mammalian cell line; providing the mammalian cell line with a composition comprising a transfer vector comprising a gene encoding a transgene; providing the mammalian cell line with a first packaging plasmid encoding at least one glycoprotein; contacting the mammalian cell line with the composition comprising at least one first packaging plasmid encoding at least one glycoprotein to the mammalian cell line; incubating the mammalian cell line with the composition; and recovering multipseudotype viruses 48 hours and 72 hours after contacting the mammalian cell line with the composition, wherein the recovery of multipseudotype viruses yields a virus with a higher titer compared to a method for producing a virus including a single pseudotype. 35. The method according to Embodiment 34, wherein at least one glycoprotein comprises a first glycoprotein selected from the group consisting of ecotropic virus envelope glycoproteins and amphotropic envelope glycoproteins. 36. The method according to Embodiment 34, wherein at least one glycoprotein comprises a first glycoprotein selected from the group consisting of RD114, VSV-G, 4070A, gap70, 10A1, and GALV. 37. The method according to any one of embodiments 34 to 36, wherein at least one glycoprotein is two glycoproteins, three glycoproteins, or four glycoproteins. 38. The method according to any one of embodiments 34 to 37, wherein at least one glycoprotein comprises a second glycoprotein selected from the group consisting of ecotropic virus envelope glycoproteins and amphotropic envelope glycoproteins, and is different from the first glycoprotein. 39. The method according to any one of embodiments 34 to 37, wherein at least one glycoprotein comprises a second glycoprotein selected from the group consisting of RD114, VSV-G, 4070A, gap70, 10A1, and GALV, and is different from the first glycoprotein. 40. The method according to any one of embodiments 34 to 39, wherein the two glycoproteins are RD114 and VSV-G. 41. The method according to any one of embodiments 34 to 39, wherein the two glycoproteins are GALV and VSV-G. 42. The method according to any one of embodiments 34 to 39, wherein the two glycoproteins are GALV and 10A1. 43. The method according to any one of Embodiments 1 to 26 or Embodiments 34 to 42, wherein a multipseudotype virus comprises two or more envelope glycoproteins, is produced with an enhanced titer, and possesses the properties of an envelope glycoprotein. 44. The method according to any one of Embodiments 1 to 26 or Embodiments 34 to 43, wherein one or more envelope glycoproteins are modified to provide different binding properties to the virus. 45. The method according to Embodiment 44, wherein the modification is cutting, masking, or redirection.

[0204] Experimental Examples The present invention will be described in more detail with reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to limit the invention unless otherwise specified. Therefore, the present invention should not be construed as being limited in any way to the following examples, but rather as encompassing all possible modifications that become apparent as a result of the teachings provided herein.

[0205] Without further explanation, it is expected that those skilled in the art can carry out and use the present invention and implement the claimed method by utilizing the above description and the following exemplary embodiments. Therefore, the following practical embodiments should not be construed as limiting any other part of the present disclosure.

[0206] Example 1: Dual Pseudotyping The results discussed herein are based on experiments designed to address the current need in the art for obtaining high-titer gamma retroviruses (gRVs) in large-scale virus production scenarios. For example, experiments were designed to evaluate whether dual-pseudotyping gRVs enable high-titer retroviral supernatant production. Dual-pseudotyping viruses are viruses containing two envelope glycoproteins. Experiments were designed to evaluate whether VSV-G and RD114 dual-pseudotyping produce enhanced titers compared to RD114 single-pseudotyping.

[0207] These studies demonstrated that dual pseudotyping can efficiently combine parental envelope characteristics such as infectivity and titer to produce viruses that exhibit the characteristics of both viral envelopes and are inherently functional. For example, using a representative model, experiments were conducted using GP2 cells constitutively expressing gag / pol as a retrovirus-producing cell line. These cells were co-transfected with a transfer plasmid encoding the target gene along with an envelope plasmid. The envelope plasmids tested were either a) RD114 alone, b) VSV-G alone, or c) and d) RD114+VSV-G in two different ratios. Surprisingly, we observed that RD114+VSV-G dual pseudotyped viruses produced titers 40–80 times higher than RD114 single pseudotyped viruses (Table 1 and Figure 1). The results from this experiment showed that dual pseudotyping using RD114 and VSV-G unexpectedly enables the production of high-titer viral supernatant that can be used in downstream experiments without further enrichment, which is not the case with RD114 single pseudotype retrovirus.

[0208] [Table 1] * The value is the CAR+ cell percentage.

[0209] Example 2 Further experiments were designed to evaluate whether the RD114+VSV-G dual pseudotyped gRV efficiently transduced primary gamma delta T cells in a similar manner to the RD114 single pseudotyped gRV. Briefly, using a representative model, primary gamma delta T cells from four healthy donors were transduced using both single RD114 pseudotyped gRV and dual RD114+VSV-G pseudotyped gRV. Transduction efficiency was measured 4–9 days post-transduction using CAR detection reagents and flow cytometry. It was observed that the RD114+VSV-G dual pseudotyped gRV efficiently transduced primary gamma delta T cells from four healthy donors, resulting in a transduction efficiency of 30–50% (Figure 2). The results from this experiment showed that the dual pseudotyped virus not only produced a high-titer virus but also a functional virus that could be successfully used to transduce primary cells such as alpha-beta T cells or gamma delta T cells.

[0210] Example 3 To evaluate whether RD114+VSV-G dual pseudotyped gRV transducers (CAR Ts) exhibit similar TAA-dependent cytotoxicity to RD114 pseudotyped gamma retrovirus transducers (CAR Ts), we designed an experiment. Briefly, primary gamma delta T cells from four healthy donors were transduced using both single RD114 pseudotyped gRVs and dual RD114+VSV-G pseudotyped gRVs. These CAR Ts were used in an Incucyte-based 3D spheroid assay to determine tumor cell line death over a 10-day period. The data showed tumor cell proliferation over this period, measured using nuclear markers present in the tumor cell lines (Figure 3). It was observed that RD114+VSV-G dual pseudotyped gRV transducers (CAR Ts) exhibited similar TAA-dependent death to RD114 single pseudotyped gamma retrovirus transducers (CAR Ts) using primary gamma delta T cell samples from four healthy donors. The results from this experiment showed that dual pseudotype viruses not only produced high-titer viruses, but also functional viruses that could be successfully used to transduce primary cells such as alpha-beta T cells or gamma-delta T cells, and that the resulting CAR T cells could successfully eliminate tumor cells in an antigen-dependent manner.

[0211] Example 4 Furthermore, we designed an experiment to evaluate whether RD114+VSV-G dual pseudotyped gRV transducers (CAR Ts) exhibit similar TAA-dependent cytotoxicity to RD114 pseudotyped gamma retrovirus transducers (CAR Ts). Briefly, primary gamma delta T cells from four healthy donors were transduced using both single RD114 pseudotyped gRVs and dual RD114+VSV-G pseudotyped gRVs. These CAR Ts were used in an Incucyte-based 3D spheroid cytotoxicity assay to determine tumor cell line death over a 10-day period. The data showed tumor cell proliferation over this period, measured using nuclear markers present in the tumor cell lines (Figure 4). It was observed that RD114+VSV-G dual pseudotyped gRV transducers (CAR Ts) exhibited similar TAA-dependent death to RD114 single pseudotyped gamma retrovirus transducers (CAR Ts) in this study, using primary gamma delta T cell samples from four healthy donors. The results from this experiment showed that dual pseudotype viruses not only produce high-titer viruses, but also functional viruses that can be successfully used to transduce primary cells such as alpha-beta T cells or gamma-delta T cells, and that the resulting CAR T cells can successfully eliminate tumor cells in an antigen-dependent manner.

[0212] Example 5 To evaluate whether RD114+VSV-G dual pseudotyped gRV transducers (CAR Ts) exhibit similar TAA-dependent cytokine secretion and pluripotency to RD114 pseudotyped gamma retrovirus transducers (CAR Ts), we designed experiments using representative models. Briefly, both single RD114 pseudotyped gRVs and dual RD114+VSV-G pseudotyped gRVs were used to transduce primary gamma delta T cells from four healthy donors. These CAR Ts were used in co-culture assays to determine cytokine secretion in response to tumor cell lines over 24 hours (Figures 5 and 6). Using primary gamma delta T cell samples from four healthy donors, RD114+VSV-G dual pseudotyped gRV transducers (CAR Ts) were observed to exhibit similar TAA-dependent cytokine secretion to RD114 single pseudotyped gamma retrovirus transducers (CAR Ts) in this study. The surprising results from this experiment showed that the dual pseudotype virus not only produced high-titer viruses, but also functional viruses that could be successfully transduced into primary cells such as alpha-beta T cells or gamma-delta T cells, and that the resulting CAR T cells could successfully secrete multiple cytokines in response to antigens.

[0213] Example 6: Exemplary protocol for transfection and RV generation using GP2 cells The materials and methods are described here. However, it should be understood that the present invention is not limited to the strict protocols disclosed herein. Those skilled in the art will understand that modifications may be made to the exemplary protocols mentioned herein based on the extensive teachings in this application.

[0214] material:

[0215] [Table 2]

[0216] Example procedure: 1. Complete DMEM medium: Prepare DMEM containing 10% thermally inactivated FBS and free of antibiotics for transfection experiments. 2. Thaw the cell vial rapidly in a 37°C water bath with gentle agitation. Immediately after thawing, wipe the outside of the vial with 70% ethanol. All subsequent operations should be performed under strict sterile conditions in a laminar flow tissue culture hood. Gently twist off the top of the vial and, using a pipette, transfer the contents of the vial to a 15 mL conical centrifuge tube containing 1 mL of pre-warmed culture medium. Mix gently. 3. Slowly add an additional 4 mL of fresh, pre-warmed culture medium to the tube and mix gently. 4. Add an additional 5 mL of pre-warmed culture medium to the tube and mix gently. Centrifuge at 100 × g for 5 minutes, carefully aspirate the supernatant, and gently resuspend the cells in complete culture medium. 5. Thoroughly mix the cell suspension and add it to a suitable culture vessel. Gently shake or swirl the dish / flask to distribute the cells evenly across the growth surface, and incubate in a humidified incubator at 37°C (5-10% CO2 if necessary) for 24 hours. 6. The following day, examine the cells under a microscope. Once the cells have sufficiently adhered and become confluent, they can be subcultured for use. If most of the cells have not sufficiently adhered, continue culturing for another 24 hours. Complete adhesion of newly thawed cultures of cell lines based on HEK 293 may require up to 48 hours. 7. Once the culture is initiated and the cells are growing normally, they can be used for transfection and virus seeding.

[0217] For cell lines based on HEK 293, collagen-coated plates or flasks are used for efficient culture of frozen stocks. Containers coated with compounds other than collagen may also provide suitable growth substrates (e.g., poly-L-lysine). After harvesting, cells may be cultured directly on tissue culture plastic. However, if adhesion is insufficient, it is recommended to use only collagen-coated blood vessels.

[0218] - Day 1: Seed the cells 1. Remove the culture medium from the cultured GP2 cells and wash the flask with 10 mL of DPBS (do not apply the DPBS directly to the cells, but add it to the side of the flask). 2. Remove the DPBS, add 3 mL of 0.05% trypsin / EDTA to the T75 flask, and incubate in a 37°C incubator. 3. After 2 minutes, add 5 mL of complete medium to the T75 flask containing the GP2 cells. 4. Transfer the culture medium containing the cells to a 15 mL centrifuge tube and count the cells on the Moxicyte. 5,100mm 2 5x10 6 GP2 cells of strain 293T were seeded in a total of 10 mL of complete medium in round-bottom culture dishes coated with Corning® BioCoat® Collagen I and incubated overnight at 37°C. In some cases, 150 mm plates were used instead of 100 mm plates (Corning, catalog no. 354551). The same volume of all reagents listed above was maintained without modification for the 150 mm plates.

[0219] Day 0: Transfection 1. After plate inspection, the cells should be 70-90% confluent. Prepare the transfection mixture as follows: 2. For each plate to be transfected, warm approximately 5 ml of complete DMEM in a centrifuge tube in a 37°C bath in preparation for subsequent medium exchange. 3. For each flask to be transfected, add 1.5 mL of OptiMEM to a 15 mL centrifuge tube. 4. Gently tap the tube to mix. 5. Carefully add 15 μL of lipofectamine 2000 to the center of the culture medium, being careful not to touch the walls of the tube. 6. Gently tap the tube to mix, then maintain at room temperature for 15 minutes. 7. While the lipofectamine and OptiMEM are being mixed, prepare the master mix for the following plasmids. 8. Resuspend the following plasmid in 1500 μL of OptiMEM.

[0220] The following conditions apply to the RD114 single pseudo-type.

[0221] [Table 3]

[0222] The following conditions apply to the VSV-G single pseudo-type.

[0223] [Table 4]

[0224] The following conditions apply to the RD114+VSV-G pseudotype (one-fifth of the total DNA).

[0225] [Table 5]

[0226] The following conditions apply to the RD114+VSV-G pseudotype (one-tenth of the total DNA).

[0227] [Table 6] 9. Combine the lipofectamine and plasmid mixes from step 5 (1.5 mL) and step 8 (1.5 mL) to obtain a total volume of 3 mL. 10. Incubate at room temperature for 20-25 minutes. 11. Meanwhile, remove the used culture medium from the GP2 cell plate and replace it with 5 mL of fresh culture medium. 12. Add the transfection mixture dropwise to the GP2 cells and gently swirl the plate to mix. 13. Return the plate to the incubator. After 14.6 hours, remove the medium and carefully replace it with 10 mL of pre-warmed complete medium, then return the plate to the incubator. 15. Collect the retrovirus 48 hours and 72 hours after transfection.

[0228] Day 2: Collection in 48 hours 1. Warm approximately 7 mL of culture medium per plate 30 minutes before collection. 2. Carefully tilt the plate to collect the culture medium from the flask and transfer it to a 15 mL centrifuge tube. Add 3.7 mL of warmed culture medium to the flask and return it to the incubator. 4. Rotate the conical container containing the collected supernatant at 1500 rpm for 5 minutes to collect only the viruses, and discard any cell pellets that have formed. 5. Filter the virus through a 0.45 μm filter to remove cell debris. 6. Prepare 100 μL aliquots of each virus for titration using the standard titer protocol (used by Lenti Core). Use Jurkat cells instead of SUPT1 cells for titration.

[0229] The filter used is cellulose acetate or polysulfonate (low protein binding) instead of nitrocellulose or its equivalent. Nitrocellulose binds to proteins present in the retrovirus membrane, destroying the virus.

[0230] Day 3: Addition of Retro-X concentrate for virus concentration. 1.1 volumes of Retro-X concentrate are combined with 3 volumes of clarified supernatant. 2. Incubate the mixture overnight at 4°C. 3. Centrifuge the sample at 1500xg for 45 minutes at 4°C. After centrifugation, an off-white pellet will be visible. 4. Carefully remove the supernatant, taking care not to disturb the pellet (using a pipette). 5. Gently resuspend the pellet in R10 medium to 1 / 10 to 1 / 100 of its original volume. 6. Titrate the same solution immediately, or store in disposable aliquots at -80°C.

[0231] Lenti X concentrate can also be used to concentrate retroviruses according to the manufacturer's instructions.

[0232] Protocol for titration: Titer assay (or seeding can be done using Jurkat instead of Sup T1) 1. Seed 20,000 SupT1 cells per well in 100 μL of culture medium in a U-bottom 96-well plate. 2. Dilute the thawed lentivirus three-fold in the culture medium (first well), then dilute it three-fold again in the culture medium for a dilution series, leaving the final well untreated. 3. This assay used an 11-dilution series. (Alternatively, a 7-dilution series using 8 wells without viral conditions can be used.) 4. Virus dilution: Mix six times with 1 / 3 of the well volume, and replace the tip after each dilution series. 5. Add 50 μL of the virus dilution plate to 100 μL of cells. (Alternatively, an additional rotation step of 5 minutes at 1500 rpm can be applied). 6. The cells were incubated with the virus at 37°C for 4 days. 7. Cells were stained for CAR expression using 50 μL of chromosome product. 8. The staining reagent was used in multiplex in this assay. 9. CAR staining and viability dye (staining reagent per tested transgene). 10. Incubate at 4°C for 60 minutes. (Alternatively, 30 minutes at room temperature). 11. Wash three times with 200 μL of FACS buffer (spin at 400 g / 4°C / 4 minutes). (Alternatively, wash once for 5 minutes at 1500 rpm in 200 μL of FACS buffer). 12. Read on a flow cytometer.

[0233] Example 7: Transfection Using GP2 Cells for the Generation of GALV and VSV-G Pseudotyped Viruses and Protocol for RV Generation Here, the materials and methods are described.

[0234] Materials:

[0235] [Table 7]

[0236] The first step for the generation of GALV and VSV-G pseudotyped viruses is the same as the procedure steps and seeding steps on day -1 described in Example 2 and is referred to. To test the increase in the titer of GALV and VSV-G dual pseudotyped viruses, controls of GALV pseudotyped virus and VSV-G pseudotyped virus are generated. The VSV-G pseudotyped virus is generated as described in Example 2. The transfection of cells for the generation of GALV pseudotyped virus and GALV+VSV-G dual pseudotyped virus is described in the following steps.

[0237] [[ID=3 4]]Day 0: Transfection 1. After inspection of the plate, the cells should be 70 - 90% confluent. Prepare the transfection mixture as follows. 2. For each plate to be transfected, warm approximately 5 ml of complete DMEM in a centrifuge tube in a 37°C bath in preparation for subsequent medium exchange. 3. For each flask to be transfected, add 1.5 mL of OptiMEM to a 15 mL centrifuge tube. 4. Gently tap the tube to mix. 5. Carefully add 15 μl of lipofectamine 2000 to the center of the culture medium, being careful not to touch the walls of the tube. 6. Gently tap the tube to mix, then maintain at room temperature for 15 minutes. 7. While the lipofectamine and OptiMEM are being mixed, prepare the master mix for the following plasmids. 8. Resuspend the following plasmid in 1500 μl of OptiMEM.

[0238] The following conditions apply to GALV single pseudotype.

[0239] [Table 8]

[0240] The following conditions apply to the GALV+VSV-G (one-fifth of the total DNA) pseudotype.

[0241] [Table 9]

[0242] The following conditions apply to the GALV+VSV-G pseudotype (one-tenth of the total DNA).

[0243] [Table 10] 9. Combine the lipofectamine and plasmid mixes from step 5 (1.5 mL) and step 8 (1.5 mL) to obtain a total volume of 3 mL. 10. Incubate at room temperature for 20-25 minutes. 11. During that time, remove the used medium from the GP2 cell plate and replace it with 5 mL of fresh medium. 12. Drop the transfection mixture onto the GP2 cells and gently rotate the plate to mix. 13. Return the plate to the incubator. 14. After 6 hours, remove the medium and carefully replace it with 10 mL of pre-warmed complete medium, then return the plate to the incubator. 15. Recover the retrovirus 48 hours and 72 hours after transfection.

[0244] The virus collection process and concentration process are described in Example 2. When measuring the titers of all viruses (single pseudotype and dual pseudotype), the titer of the dual pseudotype virus increases compared to the method of producing a virus containing only a single pseudotype of either VSV-G or GALV.

[0245] The dual pseudotype of GALV+VSV-G increases the transduction efficiency in γδ T cells compared to a virus containing only a single pseudotype of either VSV-G or GALV.

[0246] Using any one combination of RD114, VSV-G, eco (ecotropic), ampho (amphotropic), 10A1, or GALV for the dual pseudotype virus results in a virus with a higher titer at the time of recovery compared to the method of producing a virus containing only any one of the aforementioned single pseudotypes.

[0247] Example 8: Transfection and RV production using GP2 cells for the generation of GALV and 10A1 pseudotype viruses Here, the materials and methods are described.

[0248] Materials:

[0249] [Table 11]

[0250] The initial steps for generating GALV and 10A1 pseudotype viruses are the same as the procedure and seeding steps on day 1 described in Example 2. To test the increase in titers of GALV and 10A1 dual pseudotype viruses, controls of GALV pseudotype viruses and controls of 10A1 pseudotype viruses are also generated. GALV pseudotype viruses are generated as described in Example 3. Cell transfection for generating 10A1 pseudotype viruses and GALV+10A1 dual pseudotypes is described in the following steps.

[0251] Day 0: Transfection 1. After plate inspection, the cells are 70-90% confluent. Prepare the transfection mixture as follows: 2. For each plate to be transfected, warm approximately 5 ml of complete DMEM in a centrifuge tube in a 37°C bath in preparation for subsequent medium exchange. 3. For each flask to be transfected, add 1.5 mL of OptiMEM to a 15 mL centrifuge tube. 4. Gently tap the tube to mix. 5. Carefully add 15 μL of lipofectamine 2000 to the center of the culture medium, being careful not to touch the walls of the tube. 6. Gently tap the tube to mix, then maintain at room temperature for 15 minutes. 7. While the lipofectamine and OptiMEM are being mixed, prepare the master mix for the following plasmids. 8. Resuspend the following plasmid in 1500 μl of OptiMEM.

[0252] The following conditions apply to the 10A1 single pseudo-type.

[0253] [Table 12]

[0254] The following conditions apply to the GALV+10A1 (one-fifth of the total DNA) pseudotype.

[0255] [Table 13]

[0256] The following conditions apply to the GALV+10A1 (one-tenth of the total DNA) pseudotype.

[0257] [Table 14] 9. Combine the lipofectamine and plasmid mixes from step 5 (1.5 mL) and step 8 (1.5 mL) to obtain a total volume of 3 mL. 10. Incubate at room temperature for 20-25 minutes. 11. Meanwhile, remove the used culture medium from the GP2 cell plate and replace it with 5 mL of fresh culture medium. 12. Add the transfection mixture to the GP2 cells and gently rotate the plate to mix. 13. Return the plate to the incubator. After 14.6 hours, remove the medium and carefully replace it with 10 mL of pre-warmed complete medium, then return the plate to the incubator. 15. Collect the retrovirus 48 hours and 72 hours after transfection.

[0258] The virus collection and concentration steps are described in Example 2. When the titers of all viruses are measured, the titers of dual pseudotype viruses are increased compared to the method that produces viruses containing only single pseudotypes, either 10A1 or GALV.

[0259] The dual pseudotype of GALV+10A1 increases the transduction efficiency in γδ T cells compared to viruses containing only a single pseudotype, either VSV-G or 10A1.

[0260] Having a combination of any one of RD114, VSV-G, eco(ecotropic), ampho(amphotropic), 10A1, or GALV for dual pseudotype viruses results in a higher titer virus upon recovery compared to a method that produces a virus containing only one of the aforementioned single pseudotypes.

[0261] Example 9: Multipseudotype virus Multipsydetype viruses are generated as described in Examples 2-4. The concentration of the plasmid encoding the envelope sequence is optimized to achieve multipsydetype viruses with enhanced titer and transduction efficiency. The first step for generating singlepsydetype viruses is the same as the procedure and day 1 seeding step described in the previous examples.

[0262] Cell transfection is as described above. However, to generate a multipseudotype virus having two or three or more envelope glycoproteins containing three or four or more of any combination of RD114, VSV-G, eco(ecotropic), ampho(amphotropic), 10A1, or GALV, the plasmid concentration is optimized for transfection.

[0263] The materials and methods will now be described.

[0264] The following conditions apply to the triple pseudo-type.

[0265] [Table 15]

[0266] The following conditions apply to the quadruple pseudo type.

[0267] [Table 16]

[0268] The transfection may be optimized by adjusting the concentration of any one of the plasmids encoding the envelope glycoproteins as appropriate. The limit on the number of pseudotypes is adjustable, and it is understood that adding more than two envelope glycoproteins may enhance the titer of the produced virus and increase the transduction efficiency.

[0269] Example 10: A multipseudotype virus containing modified glycoproteins. Improved glycoproteins have been previously described (Tomas HA, et al., 2019, Mol Ther Methods Clin Dev. 15:1-8). These include dual-pseudotypes or multi-pseudotypes that use combinations of different glycoproteins having modified envelope glycoproteins that may be cleaved or have additional modifications.

[0270] To increase viral titer production and enhance the transduction level, dual pseudotyped viruses are produced using modified GALV glycoproteins with modifications to the cytoplasmic tail, as described, for example, by Tomas et al. (Tomas HA, et al., 2019, Mol Ther Methods Clin Dev. 15:1-8). As described, GALV further contains either a synthetic amino acid sequence or an HIV-1 matrix capsid amino acid sequence (Tomas HA, et al., 2019, Mol Ther Methods Clin Dev. 15:1-8). The combination of modified GALV glycoproteins with a second pseudotype is observed to increase the infection stage and improve transduction efficiency.

[0271] Any patents, patent applications, and publications disclosed herein by reference are incorporated herein by reference in their entirety. While the present invention is disclosed with reference to specific embodiments, it will be apparent that other embodiments and modifications of the invention can be devised by those skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed as encompassing all such embodiments and equivalent modifications.

Claims

1. A method for generating a multipseudotype virus, wherein the method is a. To provide mammalian cell lines, b. Adding the cell culture medium to the mammalian cell line, c. Contacting the mammalian cell line with a composition comprising at least one packaging plasmid encoding at least one glycoprotein, d. Incubating the mammalian cell line with the composition, e. A method comprising recovering the multipseudotype virus 48 hours and 72 hours after contacting the mammalian cell line with the composition, wherein the recovery of the multipseudotype virus yields a virus with a higher titer compared to a method for producing a virus including a single-pseudotype.

2. Culturing can be done at a temperature of approximately 37°C, CO ２ The method according to claim 1, wherein the process is carried out for approximately 24 to 36 hours in a humidified incubator with a humidity of approximately 5% to approximately 10%.

3. The method according to claim 1 or 2, wherein the composition comprising at least one packaging plasmid encoding at least one glycoprotein further comprises a transfer vector comprising a gene encoding a transgene.

4. The method according to any one of claims 1 to 3, wherein the at least one glycoprotein comprises a first glycoprotein selected from the group consisting of ecotropic virus envelope glycoproteins and amphotropic envelope glycoproteins.

5. The method according to any one of claims 1 to 3, wherein the at least one glycoprotein comprises a first glycoprotein selected from the group consisting of RD114, VSV-G, 4070A, gap70, 10A1, and GALV.

6. The method according to any one of claims 1 to 5, wherein the at least one glycoprotein is two glycoproteins, three glycoproteins, or four glycoproteins.

7. The method according to any one of claims 1 to 6, wherein the at least one glycoprotein comprises a second glycoprotein selected from the group consisting of ecotropic virus envelope glycoproteins and amphotropic envelope glycoproteins, and is different from the first glycoprotein.

8. The method according to any one of claims 1 to 6, wherein the at least one glycoprotein comprises a second glycoprotein selected from the group consisting of RD114, VSV-G, 4070A, gap70, 10A1, and GALV, and is different from the first glycoprotein.

9. The method according to claim 8, wherein the two glycoproteins are RD114 and VSV-G.

10. The method according to claim 8, wherein the two glycoproteins are GALV and VSV-G.

11. The method according to claim 8, wherein the two glycoproteins are GALV and 10A1.

12. The method according to any one of claims 1 to 11, wherein the at least one packaging plasmid encoding the at least one glycoprotein is in a ratio of about 1 / 5 to 1 / 10 of the total DNA.

13. A method for generating a multipseudotype virus, wherein the method is a. To provide mammalian cell lines for transfection and virus production, b. Culturing the mammalian cell line, wherein the culturing is performed at a temperature of approximately 37°C, CO ２ This process is carried out in a humidified incubator with approximately 5% to 10% humidity. c. Treating the mammalian cell line with trypsin, d. Adding the cell culture medium to the mammalian cell line, e. The mammalian cell line, i. A transfer vector containing the introduced gene, and ii. Contacting with a composition comprising at least one packaging plasmid encoding at least one glycoprotein, f. Incubating the mammalian cell line with the composition, g. A method comprising recovering the multipseudotype virus 48 hours and 72 hours after contacting the mammalian cell line with the composition, wherein the recovery of the multipseudotype virus yields a virus with a higher titer compared to a method for producing a virus including a single-pseudotype.

14. The method according to claim 13, wherein the at least one glycoprotein comprises a first glycoprotein selected from the group consisting of ecotropic virus envelope glycoproteins and amphotropic envelope glycoproteins.

15. The method according to claim 13, wherein the first envelope glycoprotein is selected from the group consisting of RD114, VSV-G, 4070A, gap70, 10A1, and GALV.

16. The method according to any one of claims 13 to 15, wherein the at least one glycoprotein is two glycoproteins, three glycoproteins, or four glycoproteins.

17. The method according to any one of claims 13 to 16, wherein the second envelope glycoprotein is selected from the group consisting of ecotropic virus envelope glycoproteins and amphotropic envelope glycoproteins, and the second envelope glycoprotein is different from the first envelope glycoprotein.

18. The method according to any one of claims 13 to 16, wherein the second envelope glycoprotein is selected from the group consisting of RD114, VSV-G, 4070A, gap70, 10A1, and GALV, and the second envelope glycoprotein is different from the first envelope glycoprotein.

19. The method according to any one of claims 13 to 18, wherein the two glycoproteins are RD114 and VSV-G.

20. The method according to any one of claims 13 to 18, wherein the two glycoproteins are GALV and VSV-G.

21. The method according to any one of claims 13 to 18, wherein the two glycoproteins are GALV and 10A1.

22. The method according to any one of claims 1 to 21, further comprising filtering the multipseudotype virus, wherein the filtering is carried out using a low protein binding filter.

23. The method according to claim 22, wherein the filtration is carried out using a filter containing cellulose acetate or polysulfonate.

24. The method according to any one of claims 1 to 23, further comprising concentrating the virus.

25. The method according to any one of claims 1 to 24, wherein the method produces a multipseudotype virus with a titer about 20, 30, 40, 50, 60, 70, 80, or 90 times higher than a method for producing a virus comprising a single envelope glycoprotein including RD114, VSV-G, GALV, or 10A1.

26. The method according to any one of claims 1 to 25, wherein the virus includes a retrovirus.

27. A modified multipseudotype virus containing at least two envelope glycoproteins.

28. An engineered multipseudotype virus produced by the method described in any one of claims 1 to 26.

29. The manipulated multipseudotype virus according to claim 27 or 28, wherein the virus includes a retrovirus.

30. A method for increasing the transduction efficiency in γδ T cells, wherein the method is a. To provide manipulated viral particles, wherein the viral particles include a protein-encoding nucleic acid and a multipseudotype virus, b. To provide negatively isolated γδ T cells for transduction, c. A method comprising transducing the manipulated virus particles into the γδ T cells.

31. The method according to claim 30, wherein the protein comprises a chimeric antigen receptor.

32. A system for carrying out a method for generating γδT cells containing a transgene, wherein the system a. A step of providing γδ T cells, b. A step of activating the γδ T cells for about 24 to 48 hours using approximately 1000 U of IL2 or OKT3 antibody, c. A step of transduction into the γδ T cells with a modified multipseudotype virus containing a sequence encoding the transgene, d. A system comprising one or more components capable of carrying out a method including a step of analyzing the transduction efficiency, wherein the transduction efficiency is increased by the multipseudotype virus compared to transduction by a singlepseudotype virus.

33. The system according to claim 32, wherein the virus includes a gamma retrovirus.

34. A method for increasing viral titer, wherein the method is a. To provide mammalian cell lines, b. Adding the cell culture medium to the mammalian cell line, c. Providing the mammalian cell line with a composition comprising a transfer vector containing a gene encoding the introduced gene, d. To provide the mammalian cell line with a first packaging plasmid encoding at least one glycoprotein, e. Contacting the mammalian cell line with a composition comprising at least the first packaging plasmid encoding at least one glycoprotein to the mammalian cell line, f. Incubating the mammalian cell line with the composition, g. A method comprising recovering a multipseudotype virus 48 hours and 72 hours after contacting the mammalian cell line with the composition, wherein the recovery of the multipseudotype virus yields a virus with a higher titer compared to a method for producing a virus including a single-pseudotype.

35. The method according to claim 34, wherein the at least one glycoprotein comprises a first glycoprotein selected from the group consisting of ecotropic virus envelope glycoproteins and amphotropic envelope glycoproteins.

36. The method according to claim 34, wherein the at least one glycoprotein comprises a first glycoprotein selected from the group consisting of RD114, VSV-G, 4070A, gap70, 10A1, and GALV.

37. The method according to any one of claims 34 to 36, wherein the at least one glycoprotein is two glycoproteins, three glycoproteins, or four glycoproteins.

38. The method according to any one of claims 34 to 37, wherein the at least one glycoprotein comprises a second glycoprotein selected from the group consisting of ecotropic virus envelope glycoproteins and amphotropic envelope glycoproteins, and is different from the first glycoprotein.

39. The method according to any one of claims 34 to 37, wherein the at least one glycoprotein comprises a second glycoprotein selected from the group consisting of RD114, VSV-G, 4070A, gap70, 10A1, and GALV, and is different from the first glycoprotein.

40. The method according to any one of claims 34 to 39, wherein the two glycoproteins are RD114 and VSV-G.

41. The method according to any one of claims 34 to 39, wherein the two glycoproteins are GALV and VSV-G.

42. The method according to any one of claims 34 to 39, wherein the two glycoproteins are GALV and 10A1.

43. The method according to any one of claims 1 to 26 or 34 to 42, wherein the multipsydetype virus comprises two or more envelope glycoproteins, is produced with an enhanced titer, and possesses the properties of the envelope glycoproteins.

44. The method according to any one of claims 1 to 26 or 34 to 43, wherein one or more envelope glycoproteins are modified to provide different binding properties to the virus.

45. The method according to claim 44, wherein the modification is cutting, masking, or redirection.

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