Materials and methods for viral engineering

Abstract

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

Description

Materials and Methods for Viral EngineeringCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No.63 / 511,977, filed July 5, 2023, which is incorporated by reference herein in its entirety.BACKGROUND

[0002] A retrovirus is an RNA viral molecule encapsulated in a viral protein envelope which infects a host cell, reverse transcribes its RNA molecule into DNA, and integrates its genome stably 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 the purpose of gene therapy. A retroviral vector is DNA or RNA that has been modified to serve as a vector for recombinant DNA.Retroviral vectors can transfer genes into a wide variety of cell types from many different species.SUMMARY

[0003] The present invention relates, in part, to methods of engineering viruses to comprise two or more envelope glycoproteins.

[0004] In some embodiments, the invention relates to methods for generating multi pseudotyped viruses, the method comprising providing a mammalian cell line, adding 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 harvesting the multi pseudotyped virus 48 hours and 72 hours after contacting the mammalian cell line with the composition, wherein the harvesting of the multi pseudotyped virus yields a higher titer of virus as compared to a method of producing a virus comprising a single pseudotype.

[0005] In one embodiment, the culturing is performed at a temperature of about 37°C and in a humidified incubator with about 5% to about 10% CO2 for about 24 to 36 hours. In one embodiment, the composition comprising at least one packaging plasmidencoding at least one glycoprotein further comprises a transfer vector comprising a gene encoding a transgene. In one embodiment, the at least one glycoprotein comprises a first glycoprotein selected from the group consisting of an ecotropic virus envelope glycoprotein and an amphotropic envelope glycoprotein. 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 consisting of an ecotropic virus envelope glycoprotein and an amphotropic envelope glycoprotein; and is different from the first glycoprotein. In one embodiment, 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. 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, the at least one packaging plasmid encoding at least one glycoprotein is at a ratio of about 1 / 5 to 1 / 10 compared to total DNA.

[0006] In some embodiments, the invention relates to a method for generating a multi pseudotyped virus, the method comprising, providing a mammalian cell line for transfection and viral production, culturing the mammalian cell line; wherein the culturing is performed at a temperature of about 37 °C and in a humidified incubator with about 5% to about 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 comprising a transgene and ii) at least one packaging plasmid encoding at least one glycoprotein, incubating the mammalian cell line with the composition, and harvesting the multi pseudotyped virus 48 hours and 72 hours after contacting the mammalian cell line with the composition, wherein the harvesting of the multi pseudotyped virus yields a higher titer of virus as compared to a method of producing a virus comprising a single pseudotype.

[0007] In some embodiments, the method further comprises filtering the multi pseudotyped virus, wherein the filtering is performed with low protein binding filters. Inone embodiment, the filtering is performed with filters comprising cellulose acetate or polysulfonate. In some embodiments, the method further comprises concentrating the virus. In some embodiments, the method produces about 20, 30, 40, 50, 60, 70, 80 or 90 times more titres of the multi pseudotyped virus than a method of producing a virus comprising a single envelope glycoprotein, wherein the single envelope glycoprotein comprises RD114, VSV-G, GALV, or 10A1.

[0008] In some embodiments, the virus comprises a retrovirus.

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

[0010] In some embodiments, the invention relates to a method of increasing transduction efficiency in y8 T cells, the method comprising providing an engineered viral particle, wherein the viral particle comprises a nucleic acid encoding a protein and a multi pseudotyped virus, providing a negatively isolated y8 T cells for transduction, and transducing the y8 T cells with the engineered viral particle. In some embodiments, the protein comprises a chimeric antigen receptor.

[0011] In some embodiments, the invention relates to a system for performing a method of generating y8 T cells comprising a transgene, wherein the system comprises one or more components capable of performing a method comprising the following steps: providing y8 T cells, activating the y8 T cells with about 1000U IL2 or OKT3 antibody for about 24 to 48 hours, transducing the y8 T cells with an engineered multi pseudotyped virus comprising a sequence encoding a transgene, and analyzing transduction efficiency, wherein the multi pseudotyped virus increases transduction efficiency as compared to transduction with a single pseudotyped virus. In some embodiments, the virus comprises a y-retrovirus.

[0012] In some embodiments, the invention relates to a method of increasing viral titers, the method comprising providing a mammalian cell line, adding cell medium to the mammalian cell line, providing a composition comprising 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 themammalian cell line with a composition comprising at least the first packaging plasmid encoding at least one glycoprotein to the mammalian cell line, incubating the mammalian cell line with the composition, and harvesting a multi pseudotyped virus 48 hours and 72 hours after contacting the mammalian cell line with the composition, wherein the harvesting of the multi pseudotyped virus yields a higher titer of virus as compared to a method of producing a virus comprising a single pseudotype.

[0013] In some embodiments, the multi-pseudotyped virus comprises two or more envelope glycoproteins, is produced with enhanced titer, and comprises the properties of the envelope glycoproteins. In some embodiments, one or more envelope glycoprotein is altered to provide the virus different binding properties. In some embodiments, the alteration is a truncation, masking, or redirecting.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0015] Figure 1 depicts a representative bar graph demonstrating thatRD114+VSV-G dual pseudotyped gamma retroviruses produced higher titers of viral supernatant when compared to RD114 single pseudotyped gamma retrovirus. The dual pseudotyped virus has about 79 times more titers than the single pseudotyped virus.

[0016] Figure 2 depicts a representative bar graph demonstrating that RD 114+VSV-G dual pseudotyped gamma retroviruses (middle bar graph) exhibited comparable transduction efficiency to RD114 single pseudo typed gamma retrovirus mediated transduction (left bar graph). VSV-G single pseudotyped gamma retrovirus did not transduce primary gamma delta T cells well (right bar graph). (1 (Donor 1 ), 2 (Donor 2), 3, (Donor 3), 4 (Donor 4), 5 (Alpha beta T cell donor).

[0017] Figure 3 depicts a representative line graph demonstrating that RD 114+VSV-G dual pseudotyped gRV-transduced CAR T displayed tumor associatedantigen (TAA)-dependent killing similar to RD114 single pseudotyped gamma retrovirus-transduced CAR T using 4 healthy donor-derived primary GD T cell samples, (light grey circle: tumor only, black square: GD Donor 1 dual pseudotyped, black triangle: GD donor 1 RE114 pseudotyped, upside down grey triangle: GD Donor 1 Untransduced, black diamond: GD donor 2 dual pseudotyped, open circle: GD donor 2 RD114 pseudotyped, grey square GD donor 2 untransduced, open triangle: donor 3 dual pseudotyped, upside down open triangle GD donor 3 RD114 pseudotyped, grey diamond: GD donor 3 untransduced, black circle: GD donor 4 dual pseudotyped, black star: GD donor 4 RD114 pseudotyped and grey star GD donor 4 un transduced). Left panel is antigen positive, right panel is antigen negative.

[0018] Figure 4 depicts a representative line graph demonstrating thatRD114+VSV-G dual pseudotyped gRV-transduced CAR T displayed TAA-dependent killing similar to RD114 single pseudotyped gamma retrovirus-transduced CAR T using 4 healthy donor-derived primary GD T cell samples, (light grey circle: tumor only, black square: GD Donor 1 dual pseudotyped, black triangle: GD donor 1 RE 114 pseudotyped, upside down grey triangle: GD Donor 1 Un-transduced, black diamond: GD donor 2 dual pseudotyped, open circle: GD donor 2 RD114 pseudotyped, grey square GD donor 2 untransduced, open triangle: donor 3 dual pseudotyped, upside down open triangle GD donor 3 RD114 pseudotyped, grey diamond: GD donor 3 untransduced, black circle: GD donor 4 dual pseudo typed, black star: GD donor 4 RD114 pseudo typed and grey star GD donor 4 untransduced). Left panel is antigen positive, right panel is antigen negative.

[0019] Figure 5 depicts a representative bar graph demonstrating thatRD 114+VSV-G dual pseudotyped gRV-transduced CAR T displayed TAA-dependent cytokine secretion similar to RD114 single pseudotyped gamma retrovirus -transduced CAR T using 4 healthy donor-derived primary GD T cell samples. Experiments were performed in 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).

[0020] Figure 6 depicts a representative bar graph demonstrating thatRD 114+VSV-G dual pseudotyped gRV-transduced CAR T displayed TAA-dependent cytokine secretion similar to RD114 single pseudotyped gamma retrovirus -transduced CAR T using 4 healthy donor-derived primary GD T cell samples. Experiments wereperformed in 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).DETAILED DESCRIPTION

[0021] It was appreciated by the inventors of the present invention that successful transduction a target cell for gene therapy in humans, a viral packaging cell line should (i) produce large quantities of retroviral particle supernatants; (ii) produce viral particles that efficiently transduce the target cell. The present inventors addressed the need for such materials and methods, including cell lines and methods related thereto, and accordingly, met needs in the art for improved methods of gene therapy and, in particular, for the treatment or prevention of disorders. The present invention offers solutions to these and other problems that plague the art.

[0022] This invention partly relates to, inter alia, the development of an improved method of recombinant retroviral transduction. In one embodiment, the invention includes retroviruses pseudotyped with more than one envelope glycoprotein and methods of making and methods of using thereof. 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 more than one envelope glycoprotein, a variant thereof, or a fragment thereof. In one embodiment, the retrovirus is pseudotyped with two envelope glycoproteins, with three envelope glycoproteins, with four envelope glycoproteins, with five envelope glycoproteins, with six envelope glycoproteins, with seven envelope glycoproteins, with eight envelope glycoproteins, with nine envelope glycoproteins, or with ten or more envelope glycoproteins. In one embodiment, the envelope glycoproteins are selected from the group consisting of an ecotropic virus envelope glycoprotein and an amphotropic envelope glycoprotein. In one embodiment, the envelope glycoproteins are selected from the group consisting of RD114, VSV-G, 4070A, gap70, and 10A1. In one embodiment, the more than one envelope glycoproteins selected are different. In one embodiment, the retrovirus is pseudotyped with the RD114 and VSV-G envelope glycoproteins. In one embodiment, the retrovirus is pseudotyped with the GALV and VSV-G envelope glycoproteins. In one embodiment, the retrovirus ispseudotyped with the GALV and 10A1 envelope glycoproteins. In one embodiment, the envelope glycoproteins are further modified.

[0023] The invention further relates to a method of producing a high titer of a retrovirus. In one embodiment, the retrovirus comprises more than one envelope glycoprotein. In one embodiment, the method comprises the use of nucleic acids encoding envelope glycoproteins. In one embodiment, the nucleic acid encoding the envelope glycoproteins is in a plasmid. In one embodiment, more than one envelope glycoprotein is encoded. In one embodiment, the more than one envelope glycoproteins are not the same. In one embodiment, the more than one envelope glycoproteins is encoded by the use of more than one nucleic acid. In one embodiment, the more than one nucleic acid each encode an envelope glycoprotein. In one embodiment, the more than one envelope glycoprotein is encoded by the use of a nucleic acid encoding more than one envelope glycoprotein. In one embodiment, the titer of retrovirus can be further concentrated.

[0024] In one embodiment, the invention relates to a method of producing a high titer of retrovirus comprising transfecting a host cell with one or more nucleic acid molecules encoding more than one envelope glycoprotein.

[0025] The invention further relates to an improved method of transducing cells with a polynucleotide of interest. In one embodiment, the method of transducing cells comprises the use of a retrovirus pseudotyped with more than one envelope glycoprotein. In one embodiment, the cell being transduced is an immune cell. In one embodiment, the method of transducing cells comprises contacting a retrovirus pseudotyped with more than one envelope glycoprotein to a cell of interest. In one embodiment, the immune cell is a gamma delta T cell (y8 T cell). In one embodiment, the polynucleotide of interest encodes a chimeric antigen receptor.

[0026] The invention further relates to the use of the transduced cells for cell therapy. In one embodiment, the cells were transduced using a retrovirus pseudotyped with more than one envelope glycoprotein. In one embodiment, the transduced cell is an immune cell. In one embodiment, the immune cell is a T cell. In one embodiment, the immune cell is transduced with a chimeric antigen receptor. In one embodiment, theimmune cell transduced with a chimeric antigen receptor kills tumor cells. In one embodiment, the transduced immune cell is introduced into a subject.

[0027] The invention also relates to the use of the transduced cells for any type of cell therapy. In one embodiment, transduced cells can be used for the delivery of any desired cargo. For example, the cargo can include but is not limited to a protein, CRISPR CAS system, gene editing system, CARs, TCRs, and the likes. Accordingly, the invention is not limited to any type of cargo.Definitions

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0029] As used herein, each of the following terms has the meaning associated with it in this section.

[0030] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0031] Also, the use of “or” means “and / or” unless stated otherwise. Similarly, “comprise,” “comprises,” “comprising” “include,” “includes,” and “including” are interchangeable and not intended to be limiting.

[0032] As used herein, the term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In one embodiment, the term “about” or “approximately” refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% about a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0033] The term “virus” as used herein is intended to mean the physical virus or retrovirus particle. “Retroviruses” are viruses having an RNA genome.

[0034] “Lentivirus” refers to a genus of retroviruses that are capable of infecting dividing and non-dividing cells. By “dividing” cell is meant a cell that undergoes active mitosis, or meiosis. The phrase “non-dividing” cell refers to a cell that does not go through mitosis. Several examples of lentiviruses include, but are not limited to, HIV (human immunodeficiency virus: including HIV type 1, and HIV type 2), the etiologic agent of the human acquired immunodeficiency syndrome (AIDS); visna-maedi, which causes encephalitis (visna) or pneumonia (maedi) in sheep, the caprine arthritisencephalitis virus, which causes immune deficiency, arthritis, and encephalopathy in goats; equine infectious anemia virus, which causes autoimmune hemolytic anemia, and encephalopathy in horses; feline immunodeficiency virus (FIV), which causes immune deficiency in cats; bovine immune deficiency virus (BIV), which causes lymphadenopathy, lymphocytosis, and possibly central nervous system infection in cattle; and simian immunodeficiency virus (SIV), which cause immune deficiency and encephalopathy in sub-human primates.

[0035] “Gammaretrovirus” refers to a genus of the retroviridae family. Exemplary gammaretroviruses include, but are not limited to, mouse stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis viruses.

[0036] A “hybrid virus” as used herein refers to a virus having components from one or more other viral vectors, including element from non-retroviral vectors, for example, adenoviral-retroviral hybrids. As used herein hybrid vectors having a retroviral component are to be considered within the scope of the retroviruses.

[0037] A viral “envelope” protein, or “Env” protein, as used herein, refers to any polypeptide sequence that resides on the surface lipid bilayer of a retroviral virion whose function is to mediate the adsorption to and the penetration of host cells susceptible to infection. A 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 comprise a transmembrane (TM) and a surface(SU) component linked together by disulfide bonds. Virus structure is described in detail in, for example, Coffin, et al., Retroviruses, 1997, Cold Spring Harbor Laboratory Press.

[0038] A viral “capsid,” as used herein, refers to the principal structural protein of the virion core derived from the central region of the Gag polyprotein. The capsid protein in a mature viral particle forms a shell surrounding the ribonucleoprotein complex that contains the genomic nucleic acid. This shell, which includes additional proteins, is also referred to as a capsid. A capsid shell can exist as a component of a virion without surrounding a genomic nucleic acid.

[0039] “Virion,” “viral particle” and “retroviral particle” are used herein to refer to a single virus comprising an RNA genome, pol gene derived proteins, gag gene derived proteins and a lipid bilayer displaying an envelope (glyco )protein. The RNA genome is usually a recombinant RNA genome and thus may contain an RNA sequence that is exogenous to the native viral genome. The RNA genome may also comprise a defective endogenous viral sequence.

[0040] The terms “pseudotype” or “pseudotyping” as used herein, refer to a virus whose viral envelope proteins have been substituted with those of another virus possessing preferable characteristics. A “pseudotyped” retrovirus is a retroviral particle having an envelope protein that is from a virus other than the virus from which the RNA genome is derived. The envelope protein can be, for example and without limitation, from a different retrovirus or from a non-retroviral origin. The envelope protein can be a native envelope protein or an envelope protein that is modified, mutated or engineered as described herein. A pseudotyping element as used herein can thus include a “binding polypeptide” that includes one or more polypeptides, typically glycoproteins, that identify and bind the target host cell, and one or more “fusogenic polypeptides” that mediate fusion of the retroviral and target host cell membranes, thereby allowing a retroviral genome to enter the target host cell.

[0041] As used herein, the term “packaging signal” or “packaging sequence” refers to sequences located within the retroviral genome which are required for insertion of the viral RNA into the viral capsid or particle, see e.g., Clever et al., 1995. J. of Virology, Vol. 69, No. 4; pp. 2101-2109. Several retroviral vectors use the minimal packaging signal (also referred to as the psi [ ] or [+] sequence) needed forencapsidation of the viral genome. Thus, as used herein, the terms “packaging sequence,” “packaging signal,” “psi” and the symbolare used in reference to the non-coding sequence required for encapsidation of retroviral RNA strands during viral particle formation.

[0042] The terms “vector”, “vector construct” and “expression vector” mean the vehicle by which a nucleic acid molecule such as a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence. Vectors typically comprise the DNA of a transmissible agent, into which foreign DNA encoding a protein is inserted by restriction enzyme technology. A common type of vector is a “plasmid”, which generally is a self-contained molecule of double-stranded DNA that can readily accept additional (foreign) DNA and which can readily introduced into a suitable host cell.

[0043] The term viral vector may refer either to a virus or viral particle capable of transferring a nucleic acid into a cell or to the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and / or functional genetic elements that are primarily derived from a virus. As will be evident to one of skill in the art, the term “viral vector” is widely used to refer either to a nucleic acid molecule (e.g., a transfer plasmid) that includes virus-derived nucleic acid elements that typically facilitate transfer of the nucleic acid molecule or integration into the genome of a cell or to a viral particle that mediates nucleic acid transfer. Viral particles will typically include various viral components and sometimes also host cell components in addition to nucleic acid(s).

[0044] As used herein, the term “packaging vector” refers to an expression vector or viral vector that lacks a packaging signal and comprises a polynucleotide encoding one, two, three, four or more viral structural and / or accessory genes. Typically, the packaging vectors are included in a packaging cell, and are introduced into the cell via transfection, transduction or infection. Methods for transfection, transduction or infection are well known by those of skill in the art.

[0045] The term “cell line” as used herein refers to cultured cells that can be passed (divided) more than once.

[0046] As used herein, the term “packaging cell lines” is used in reference to cell lines that do not contain a packaging signal, but do stably or transiently express viral structural proteins and replication enzymes (e.g., gag, pol and env) which are necessary for the correct packaging of viral particles.

[0047] As used herein, the term “producer cell line” refers to a cell line which is capable of producing recombinant retroviral particles, comprising a packaging cell line and a transfer vector construct comprising a packaging signal.

[0048] “Transformation,” as defined herein, describes a process by which exogenous DNA enters a target cell. Transformation may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell and may include, but is not limited to, viral infection, electroporation, heat shock, lipofection, and particle bombardment. “Transformed” cells include stably transformed cells in which the inserted nucleic acid is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome. Also included are cells that transiently express a gene of interest.

[0049] The terms “transfecting” or “transfection” as used herein are intended to mean the transfer of at least one exogenous nucleic acid into a cell. The nucleic acid may be RNA, DNA or a combination of both. The exogenous nucleic acid refers to nucleic that is not found as a result of host cell division or host cell multiplication.

[0050] The expressions “transient expression” and “transiently expressing” as used herein are intended to mean that the genetic material temporal expression period and / or is not integrated permanently and stably in the genome of the host cell, and thus does not have the same expression potential over time as the native genetic material of the host cell.

[0051] The delivery of a gene(s) or other polynucleotide sequences using a retroviral or lentiviral vector by means of viral infection rather than by transfection is referred to as “transduction.” A target cell, is “transduced” if it comprises a gene or other polynucleotide sequence delivered to the cell by infection using a viral or retroviral vector.

[0052] The expressions “stable expression” and “stably expressing” as used herein are intended to mean that the genetic material that is being stably expressed and / oris integrated permanently and stably in the genome of the host cell, and thus has the same expression potential over time as the native genetic material of the host cell.

[0053] A “host cell” includes cells transfected, infected, or transduced in vivo, ex vivo, or in vitro with a recombinant vector or a polynucleotide of the invention. Host cells may include packaging cells, producer cells, and cells infected with viral vectors. The term “target cell” is used interchangeably with host cell and refers to transfected, infected, or transduced cells of a desired cell type.

[0054] As used herein, the terms nucleic acid, polynucleotide and nucleotide are interchangeable and refer to any nucleic acid, whether composed of phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphoramidate, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sultone linkages, and combinations of such linkages.

[0055] The terms nucleic acid, polynucleotide and nucleotide also specifically include nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil).

[0056] As used herein, a nucleic acid molecule is said to be “isolated” when the nucleic acid molecule is substantially separated from contaminant nucleic acid molecules encoding other polypeptides.

[0057] By “transgene” is meant any nucleotide sequence, particularly a DNA sequence, that is integrated into one or more chromosomes of a host cell by human intervention, such as by the methods of the present invention. The transgene preferably comprises a “gene of interest.”

[0058] A “gene of interest” is not limited in any way and may be any nucleic acid, without limitation, that is desired to be delivered to, integrated, transcribed, translated, and / or expressed in a target cell. The gene of interest may encode a functional product, such as a protein or an RNA molecule. The gene of interest is generally operatively linked to other sequences that are useful for obtaining the desired expression of the gene of interest, such as transcriptional regulatory sequences. The term“polynucleotide(s)-of-interest” refers to one or more polynucleotides, e.g., a polynucleotide encoding a polypeptide (i.e., a polypeptide-of-interest), inserted into an expression vector that is desired to be expressed.

[0059] The term “operably linked”, refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A “functional relationship” and “operably linked” mean, with respect to the gene of interest, that the gene is in the correct location and orientation with respect to the promoter and / or enhancer that expression of the gene will be affected when the promoter and / or enhancer is contacted with the appropriate molecules.

[0060] The terms “express” and “expression” mean allowing or causing the information in a gene or DNA sequence to become manifest, for example producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence. A DNA sequence is expressed in or by a cell to form an “expression product” such as a protein. The expression product itself, e.g. the resulting protein, may also be said to be “expressed” by the cell. A polynucleotide or polypeptide is expressed recombinantly, for example, when it is 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.

[0061] The term “modulate” envisions the suppression of expression of a gene when it is over-expressed, or augmentation of expression when it is under-expressed.

[0062] The term “regulatory element” and “expression control element” are used interchangeably and refer to nucleic acid molecules that can influence the transcription and / or translation of an operably linked coding sequence in a particular environment. These terms are used broadly and cover all elements that promote or regulate transcription, including promoters, core elements required for basic interaction of RNA polymerase and transcription factors, upstream elements, enhancers, and response elements (see, e.g., Lewin, “Genes V” (Oxford University Press, Oxford) pages 847-873). Exemplary regulatory elements in prokaryotes include promoters, operator sequences and a ribosome binding sites. Regulatory elements that are used in eukaryotic cells may include, without limitation, promoters, enhancers, splicing signals and polyadenylation signals.

[0063] The term “nucleic acid regulatory domain” refers collectively to promoter sequences (e.g., pol II promoter sequences), polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, enhancers and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control sequences need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell.

[0064] The term “promoter region” is used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3 '-direction) coding sequence. The regulatory sequence may be homologous or heterologous to the desired gene sequence.

[0065] The term “enhancer” refers to a segment of DNA which contains sequences capable of providing enhanced transcription and in some instances can function independent of their orientation relative to another control sequence. An enhancer can function cooperatively or additively with promoters and / or other enhancer elements. The term “promoter / enhancer” refers to a segment of DNA which contains sequences capable of providing both promoter and enhancer functions.

[0066] An internal ribosome entry sites (“IRES”) refers to a segment of nucleic acid that promotes the entry or retention of a ribosome during translation of a coding sequence usually 3' to the IRES. In some embodiments the IRES may comprise a splice acceptor / donor site, however, preferred IRESs lack a splice acceptor / donor site.

[0067] An “endogenous” control sequence is one which is naturally linked to a given gene in the genome. An “exogenous” control sequence is one which is placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques) such that transcription of that gene is directed by the linked enhancer / promoter. A “heterologous” control sequence is an exogenous sequence that is from a different species than the cell being genetically manipulated. A “synthetic” control sequence may comprise elements of one more endogenous and / or exogenous sequences, and / or sequences determined in vitro or in silico that provide optimal promoter and / or enhancer activity for the particular gene therapy.

[0068] As used herein, the term “constitutive expression control sequence” refers to a promoter, enhancer, or promoter / enhancer that continually or continuously allows for transcription of an operably linked sequence. A constitutive expression control sequence may be a “ubiquitous” promoter, enhancer, or promoter / enhancer that allows expression in a wide variety of cell and tissue types or a “cell specific,” “cell type specific,” “cell lineage specific,” or “tissue specific” promoter, enhancer, or promoter / enhancer that allows expression in a restricted variety of cell and tissue types, respectively.

[0069] As used herein, “conditional expression” may refer to any type of conditional expression including, but not limited to, inducible expression; repressible expression; expression in cells or tissues having a particular physiological, biological, or disease state, etc. This definition is not intended to exclude cell type or tissue specific expression.

[0070] Conditional expression can also be achieved by using a site specific DNA recombinase. As used herein, the terms “recombinase” or “site specific recombinase” include excisive or integrative proteins, enzymes, co-factors or associated proteins that are involved in recombination reactions involving one or more recombination sites (e.g., two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.), which may be wild-type proteins (see Landy, (1993) Current Opinion in Biotechnology 3:699-707), or mutants, derivatives (e.g., fusion proteins containing the recombination protein sequences or fragments thereof), fragments, and variants thereof. Illustrative examples of recombinases suitable for use in particular embodiments of the present invention include, but are not limited to: Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, C31 , Cin, Tn3 resolvase, TndX, XerC, XerD, TnpX, Hjc, Gin, SpCCEl, and ParA.

[0071] As used herein, the terms “recombination sequence,” “recombination site,” or “site specific recombination site” refer to a particular nucleic acid sequence to which a recombinase recognizes and binds.

[0072] As used herein, the term “heterologous” nucleic acid sequence or transgene refers to (i) a sequence that does not normally exist in a wild-type retrovirus, (ii) a sequence that originates from a foreign species, or (iii) if from the same species, it may be substantially modified from its original form. Alternatively, an unchanged nucleicacid sequence that is not normally expressed in a cell is a heterologous nucleic acid sequence.

[0073] “Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins having the same structural characteristics. As used herein, the term “antibody” refers to a protein that includes at least one immunoglobulin variable domain or immunoglobulin variable domain sequence. For example, an antibody can include 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 includes two heavy (H) chain variable regions and two light (L) chain variable regions. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules that lack antigen specificity. Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas.

[0074] The term “antibody” is used in the broadest sense and specifically covers human, non-human (e.g. murine), chimeric, and humanized monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multi- specific antibodies (e.g., bispecific antibodies), single-chain antibodies, and antibody fragments so long as they exhibit the desired biological activity. The term “antibody” thus encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab fragments, F(ab').sub.2, a Fd fragment, a Fv fragments, and dAb fragments) as well as complete antibodies. Typically, fragments compete with the intact antibody from which they were derived for specific binding to an antigen.

[0075] The term “monospecific antibody” refers to an antibody that displays a single binding specificity and affinity for a particular target, e.g., epitope. This term includes a “monoclonal antibody” which refers to an antibody that is produced as a single molecular species, e.g., from a population of homogenous isolated cells. A “monoclonal antibody composition” refers to a preparation of antibodies or fragments thereof of in a composition that includes a single molecular species of antibody.

[0076] The term “epitope” or “antigenic determinant” refers to a site on an antigen to which B and / or T cells respond. B-cell epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of aprotein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., 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 showing the ability of one antibody to block the binding of another antibody to a target antigen. T- cells recognize continuous epitopes of about nine amino acids for CD8 cells or about 13- 15 amino acids for CD4 cells. T cells that recognize the epitope can be identified by in vitro assays that measure antigen-dependent proliferation, as determined by3H-thymidine incorporation by primed T cells in response to an epitope (see Burke, supra; Tigges, supra).

[0077] The term “immunological” or “immune” response is the development of a beneficial humoral (antibody mediated) and / or a cellular (mediated by antigen- specific T cells or their secretion products) response directed against an amyloid peptide in a recipient patient. Such a response can be an active response induced by administration of immunogen or a passive response induced by administration of antibody or primed T- cells. A cellular immune response is elicited by the presentation of polypeptide epitopes in association with Class I or Class II MHC molecules to activate antigen-specific CD4+T helper cells and / or CD8+cytotoxic T cells. The response may also involve activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia cells, eosinophils or other components of innate immunity. The presence of a cell-mediated immunological response can be determined by proliferation assays (CD4+T cells) or CTL (cytotoxic T lymphocyte) assays (Burke et al., J. Inf. Dis. 170, 1110-19 (1994)), by antigen-dependent killing (cytotoxic T lymphocyte assay, Tigges et al., J. Immunol. 156, 3901-3910) or by 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 an immunized syngeneic animal and measuring protective or therapeutic effect in a second subject.

[0078] An “immunogenic agent” or “immunogen” is capable of inducing an immunological response against itself on administration to a patient, optionally in conjunction with an adjuvant.

[0079] The term “adjuvant” refers to a compound that when administered in conjunction with an antigen augments, enhances, and / or boosts the immune response to the antigen, but when administered alone does not generate an immune response to the antigen. An adjuvant can be administered with the recombinant virus of the invention as a single composition, or can be administered before, concurrent with or after administration of the recombinant virus of the invention. Adjuvants can enhance an immune response by several mechanisms including lymphocyte recruitment, stimulation of B and / or T cells, and stimulation of macrophages.

[0080] A “subject,” as used herein, includes any animal that exhibits a symptom of a monogenic disease, disorder, or condition that can be treated with the gene therapy vectors, cell-based therapeutics, and methods disclosed elsewhere herein.

[0081] Suitable subjects (e.g., patients) include laboratory animals (such as mouse, rat, rabbit, or guinea pig), farm animals, and domestic animals or pets (such as a cat or dog). Non-human primates and, preferably, human patients, are included. Typical subjects include animals that exhibit aberrant amounts (lower or higher amounts than a “normal” or “healthy” subject) of one or more physiological activities that can be modulated by gene therapy.

[0082] As used herein “treatment” or “treating,” includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, and may include even minimal reductions in one or more measurable markers of the disease or condition being treated. Treatment can involve optionally either the reduction or amelioration of symptoms of the disease or condition, or the delaying of the progression of the disease or condition. “Treatment” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof.

[0083] As used herein, “prevent,” and similar words such as “prevented,” “preventing” etc., indicate an approach for preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of, a disease or condition. It also refers to delaying the onset or recurrence of a disease or condition or delaying the occurrence orrecurrence of the symptoms of a disease or condition. As used herein, “prevention” and similar words also includes reducing the intensity, effect, symptoms and / or burden of a disease or condition prior to onset or recurrence of the disease or condition.

[0084] The term “therapeutic” is used in a generic sense and includes treating agents, prophylactic agents, and replacement agents. The term “therapeutic” can to an action that prevents, reverses, or slows the natural course of a disease, or its symptoms. A therapeutic action can be preventive, curative or merely palliative, and does not mean that the affected human or animal patient will not die from the disease.

[0085] As used herein, the term “amount” refers to “an amount effective” or “an effective amount” of a virus or transduced therapeutic cell to achieve a beneficial or desired prophylactic or therapeutic result, including clinical results.

[0086] A “prophylactically effective amount” refers to an amount of a virus or transduced therapeutic cell effective to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount is less than the therapeutically effective amount.

[0087] A “therapeutically effective amount” of a virus or transduced therapeutic cell may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the stem and progenitor cells to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the virus or transduced therapeutic cells are outweighed by the therapeutically beneficial effects. The term “therapeutically effective amount” includes an amount that is effective to “treat” a subject (e.g., a patient).

[0088] The phrase “pharmaceutically-acceptable” refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human. The preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified.

[0089] As used herein, “carrier” includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0090] As used herein “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible, including pharmaceutically acceptable cell culture media.

[0091] In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments of the invention. However, one skilled in the art will understand that the invention may be practiced without these details.

[0092] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0093] All publications mentioned herein are incorporated herein by reference in full for the purpose of describing and disclosing the methodologies, which are described in the publications, which might be used in connection with the description herein. The publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure.Description

[0094] The present invention relates to compositions and methods for use in improving virus production and transduction efficiency of viruses into target cells. In one embodiment, the invention includes compositions and methods for improved recombinant viral production and viral transduction. In one embodiment, the invention includes retroviruses pseudotyped with more than one envelope glycoprotein and methods thereof.

[0095] In one embodiment, the invention provides compositions and methods for producing a high titer of a retrovirus. In one embodiment, the invention provides an improved method of transducing cells with a polynucleotide of interest. Accordingly, the invention provides compositions and methods that are applicable to any type of gene therapy. For example, the invention is applicable to transduction of an exogenous gene into any cell of interest and therefore is applicable to any type of gene therapy setting.Virus production using packaging cells

[0096] This invention provides compositions and methods for improving transduction efficiency of viruses into target cells. In one embodiment, the invention provides compositions and methods to produce a retroviral packaging cell comprising a suitable mammalian cell having therein (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 and second envelope genes are different from one another. In one embodiment, the suitable mammalian cell is a HEK 293 cell. As used herein, a “packaging cell” is a mammalian cell capable of expressing retroviral genes and assembling retroviral particles.

[0097] In one embodiment, the invention provides compositions and methods to produce a retroviral packaging cell comprising a suitable mammalian cell having therein (i) 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.

[0098] A benefit of producing a retroviral packaging cell comprising a suitable mammalian cell having therein (i) a first recombinant nucleic acid comprising a first envelope gene, and (ii) a second recombinant nucleic acid comprising a second envelope gene is generation of a multi pseudotyped virus. In one embodiment, the multipseudotyped virus of the invention allows for the production of higher-titered retroviral supernatant. In one embodiment, the method further comprises the steps of collecting and concentrating the retroviral supernatant.Multi-Pseudotyped Retrovirus

[0099] This invention contemplates an improved method of recombinant retroviral transduction. In one embodiment, the invention includes retroviruses pseudotyped with more than one envelope glycoprotein and methods thereof.

[0100] In some embodiments, the invention provides methods and compositions for generating multi pseudotyped viruses comprising at least two different envelope glycoproteins. In some embodiments, the virus is a retrovirus. In some embodiments, the retrovirus is a gamma retrovirus. In some embodiments, the virus is a lentivirus.

[0101] Retroviruses can be derived from members of the retroviridae family. The classification of this family has changed several times over the last ten to fifteen years. Currently the Retroviridae family consists of two sub-families: the Spumaretrovirinae- which has a single genus, the spumavirus (or foamy viruses) such as the human and simian foamy virus (HFV) and the Orthoretroviriniae sub-family which has 6 genus — betaretrovirus (e.g. MMTV), gammaretrovirus (e.g MLV), alpharetrovirus (e.g. ALV) deltaretrovirus (e.g. BLV and HTLV-1) lentivirus (e.g. HIV 1) and epsilon retrovirus (e.g. wall eye dermal sarcoma virus). These classifications are made on the basis of common molecular features such as the relative reading frames for gag, pol and env, the processing of the polyproteins, the individual tRNAS used for priming reverse transcription, and the nature of the LTR structures. The original method of classification of retroviruses was into groups A, B, C and D on the basis of particle morphology, as seen under the electron microscope during viral maturation. A-type particles represent the immature particles of the B- and D-type viruses seen in the cytoplasm of infected cells. These particles are not infectious. B-type particles bud as mature virion from the plasma membrane by the enveloping of intracytoplasmic A-type particles. At the membrane they possess a toroidal core of 75 nm, from which long glycoprotein spikes project. After budding, B-type particles contain an eccentrically located, electron-dense core. The betaretrovirus, Mouse mammary tumor virus (MMTV) has a B-type morphology, butbetaretroviruses can also have a D-type structure. D-type particles resemble B-type particles in that they show as ring-like structures in the infected cell cytoplasm, which bud from the cell surface, but the virion incorporate short surface glycoprotein spikes. The electron-dense cores are also eccentrically located within the particles. Mason Pfizer monkey virus (MPMV), also a betaretrovirus, is the prototype D-type virus. No intracytoplasmic particles can be observed in cells infected by C-type viruses. Instead, mature particles bud directly from the cell surface via a crescent ‘C’ -shaped condensation which then closes on itself and is enclosed by the plasma membrane. Envelope glycoprotein spikes may be visible, along with a uniformly electron-dense core. Budding may occur from the surface plasma membrane or directly into intracellular vacuoles. Alpha retroviruses, gamma retroviruses, delta retroviruses and epsilon retroviruses all have the C-type structural appearance.

[0102] In some embodiments, the recombinant retroviral particles or the envelope glycoproteins can be derived from the Alpha retrovirus genus, the Beta retrovirus genus, the Gamma retrovirus genus, the Delta retrovirus genus, the Epsilon retrovirus genus, the Lentivirus genus, or the Spumavirus genus. There are many retroviruses suitable for use in the methods disclosed herein. For example, murine leukemia virus (MLV), human immunodeficiency virus (HIV), equine infectious anaemia virus (EIAV), mouse mammary tumor 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), Avian myelocytomatosis virus-29 (MC29), and Avian erythroblastosis virus (AEV) can be used. A detailed list of retroviruses may be found in Coffin et al (“Retroviruses” 1997 Cold Spring Harbor Laboratory Press Eds: J M Coffin, S M Hughes, H E Varmus pp 758-763). Details on the genomic structure of some retroviruses may be found in the art. By way of example, details on HIV may be found from the NCBI Genbank (i.e. Genome Accession No. AF033819).

[0103] In some embodiments, the retrovirus is derived from the Lentivirus genus. Illustrative lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2); visna-maedi virus (VMV) virus; the caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV);feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV).

[0104] In some embodiments, the retrovirus is derived from the Gammaretrovirus genus. Illustrative gamma-retroviruses include but are not limited to: reticuloendotheliosis virus, gibbon ape leukemia virus, koala retrovirus, Moloney murine leukemia virus, Abelson murine leukemia virus, xenotropic murine leukemia-related virus, feline leukemia virus, friend virus

[0105] Retroviruses are defined by the way in which they replicate their genetic material. During replication the RNA is converted into DNA. Following infection of the cell a double-stranded molecule of DNA is generated from the two molecules of RNA which are carried in the viral particle by the molecular process known as reverse transcription. The DNA form becomes covalently integrated in the host cell genome as a provirus, from which viral RNAs are expressed with the aid of cellular and / or viral factors. The expressed viral RNAs are packaged into particles and released as infectious virion.

[0106] The retrovirus particle is composed of two identical RNA molecules. Each wild-type genome has a positive sense, single-stranded RNA molecule, which is capped at the 5' end and polyadenylated at the 3' tail. The diploid virus particle contains the two RNA strands complexed with gag proteins, viral enzymes (pol gene products) and host tRNA molecules within a ‘core’ structure of gag proteins. Surrounding and protecting this capsid is a lipid bilayer, derived from host cell membranes and containing viral envelope (env) proteins. The env proteins bind to a cellular receptor for the virus and the particle typically enters the host cell via receptor-mediated endocytosis and / or membrane fusion.

[0107] Viral envelope proteins (env) determine the range of host cells which can ultimately be infected and transformed by recombinant retroviruses generated from the cell lines. In the case of lentiviruses, such as HIV-1, HIV-2, SIV, FIV and EIV, the env proteins include gp41 and gpl20. Preferably, the viral env proteins expressed by packaging cells of the invention are encoded on a separate vector from the viral gag and pol genes as described later.

[0108] Illustrative examples of retroviral-derived env genes which can be employed in the invention include, but are not limited to: MLV envelopes, 10A1 envelope, BAEV, FeLV-B, RD114, SSAV, Ebola, Sendai, FPV (Fowl plague virus), and influenza virus envelopes. 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, Retroviridae) as well as from the DNA viruses (families of Hepadnaviridae, Circoviridae, Parvoviridae, Papovaviridae, Adenoviridae, Herpes viridae, Poxyiridae, and Iridoviridae) may be utilized. Representative examples include, FeEV, VEE, HFVW, WDSV, SFV, Rabies, AEV, BIV, BEV, EBV, CAEV, SNV, ChTLV, STLV, MPMV, SMRV, RAV, FuSV, MH2, AEV, AMV, CT 10, EIAV, VSV-G, 4070A, gap70, and GALV. In some embodiments, the retroviral-derived env genes which can be employed in the invention include any ecotropic virus envelope glycoproteins and amphotropic envelope glycoproteins

[0109] The terms “pseudotype” or “pseudotyping” as used herein, refer to a virus whose viral envelope proteins have been substituted with those of another virus possessing preferable characteristics. For example, HIV can be pseudotyped with vesicular stomatitis virus G-protein (VSV-G) envelope proteins, which allows HIV to infect a wider range of cells because HIV envelope proteins (encoded by the env gene) normally target the virus to CD4+ presenting cells.

[0110] In some embodiments, the multi pseudotyped virus comprises at least one envelope glycoprotein. In some embodiments, the multi pseudotyped virus comprises at least two envelope glycoproteins. In some embodiments, the multi pseudotyped virus comprises at least three envelope glycoproteins. In some embodiments, the multi pseudotyped virus comprises at least four envelope glycoproteins. In some embodiments, the multi pseudotyped virus comprises at least five envelope glycoproteins. In some embodiments, the multi pseudotyped virus comprises at least six envelope glycoproteins. In some embodiments, the multi pseudotyped virus comprises at least seven envelope glycoproteins. In some embodiments, the multi pseudotyped virus comprises at least eight envelope glycoproteins. In some embodiments, the multi pseudotyped viruscomprises at least nine envelope glycoproteins. In some embodiments, the multi pseudotyped virus comprises at least ten envelope glycoproteins.

[0111] In some embodiments, the first envelope glycoprotein for pseudotyping a virus of present invention is selected from the group consisting of: Feline endogenous virus (RD114), Influenza A such as H1N1, H1N2, H3N2 and H5N1 (bird flu), 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, enteric adenoviruses, parvovirus, Dengue fever virus, Monkey pox, Mononegavirales, Lyssavirus such as rabies virus, Lagos bat virus, Mokola virus, Duvenhage virus, European bat virus 1 & 2 and Australian bat virus, Ephemero virus, Vesiculovirus, Vesicular Stomatitis Virus (VSV), Herpesviruses such as Herpes simplex virus types 1 and 2, varicella zoster, cytomegalovirus, Epstein-Bar virus (EBV), human herpesviruses (HHV), human herpesvirus type 6 and 8, Human immunodeficiency virus (HIV), papilloma virus, murine gammaherpesvirus, Arenaviruses such as 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), Bunyaviridiae such as Crimean-Congo hemorrhagic fever virus, Hantavirus, hemorrhagic fever with renal syndrome causing virus, Rift Valley fever virus, Filoviridae (filovirus) including Ebola hemorrhagic fever and Marburg hemorrhagic fever, Flaviviridae including Kaysanur Forest disease virus, Omsk hemorrhagic fever virus, Tick-borne encephalitis causing virus and Paramyxoviridae such as Hendra virus and Nipah virus, variola major and variola minor (smallpox), alphaviruses such as Venezuelan equine encephalitis virus, eastern equine encephalitis virus, western equine encephalitis virus, SARS-associated coronavirus (SARS-CoV), West Nile virus, any encephaliltis 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.

[0112] In some embodiments, the multi pseudotyped virus comprises two envelope glycoproteins. In some embodiments, the second envelope glycoprotein for pseudotyping a virus of present invention is different from the first envelope glycoprotein and is selected from the group consisting of: Feline endogenous virus (RD114), InfluenzaA such as H1N1, H1N2, H3N2 and H5N1 (bird flu), 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, enteric adenoviruses, parvovirus, Dengue fever virus, Monkey pox, Mononegavirales, Lyssavirus such as rabies virus, Lagos bat virus, Mokola virus, Duvenhage virus, European bat virus 1 & 2 and Australian bat virus, Ephemero virus, Vesiculovirus, Vesicular Stomatitis Virus (VSV), Herpesviruses such as Herpes simplex virus types 1 and 2, varicella zoster, cytomegalovirus, Epstein-Bar virus (EBV), human herpesviruses (HHV), human herpesvirus type 6 and 8, Human immunodeficiency virus (HIV), papilloma virus, murine gammaherpesvirus, Arenaviruses such as 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), Bunyaviridiae such as Crimean-Congo hemorrhagic fever virus, Hantavirus, hemorrhagic fever with renal syndrome causing virus, Rift Valley fever virus, Filoviridae (filovirus) including Ebola hemorrhagic fever and Marburg hemorrhagic fever, Flaviviridae including Kaysanur Forest disease virus, Omsk hemorrhagic fever virus, Tick-borne encephalitis causing virus and Paramyxoviridae such as Hendra virus and Nipah virus, variola major and variola minor (smallpox), alphaviruses such as Venezuelan equine encephalitis virus, eastern equine encephalitis virus, western equine encephalitis virus, SARS-associated coronavirus (SARS-CoV), West Nile virus, any encephaliltis 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.

[0113] In some embodiments, the multi pseudotyped virus comprises three envelope glycoproteins. In some embodiments, the third envelope glycoprotein for pseudotyping a virus of present invention is different from the first envelope glycoprotein and the second envelope glycoprotein and is selected from the group consisting of: Feline endogenous virus (RD114), Influenza A such as H1N1, H1N2, H3N2 and H5N1 (bird flu), 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 virusgroup, enteric adenoviruses, parvovirus, Dengue fever virus, Monkey pox, Mononegavirales, Lyssavirus such as rabies virus, Lagos bat virus, Mokola virus, Duvenhage virus, European bat virus 1 & 2 and Australian bat virus, Ephemerovirus, Vesiculovirus, Vesicular Stomatitis Virus (VSV), Herpesviruses such as Herpes simplex virus types 1 and 2, varicella zoster, cytomegalovirus, Epstein-Bar virus (EBV), human herpesviruses (HHV), human herpesvirus type 6 and 8, Human immunodeficiency virus (HIV), papilloma virus, murine gammaherpesvirus, Arenaviruses such as 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), Bunyaviridiae such as Crimean-Congo hemorrhagic fever virus, Hantavirus, hemorrhagic fever with renal syndrome causing virus, Rift Valley fever virus, Filoviridae (filovirus) including Ebola hemorrhagic fever and Marburg hemorrhagic fever, Flaviviridae including Kaysanur Forest disease virus, Omsk hemorrhagic fever virus, Tick-borne encephalitis causing virus and Paramyxoviridae such as Hendra virus and Nipah virus, variola major and variola minor (smallpox), alphaviruses such as Venezuelan equine encephalitis virus, eastern equine encephalitis virus, western equine encephalitis virus, SARS-associated coronavirus (SARS-CoV), West Nile virus, any encephaliltis 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.

[0114] In some embodiments, the multi pseudotyped virus comprises four envelope glycoproteins. In some embodiments, the fourth envelope glycoprotein for pseudotyping a virus of present invention is different from the first envelope glycoprotein, the second envelope glycoprotein, and the third envelope glycoprotein and is selected from the group consisting of: Feline endogenous virus (RD114), Influenza A such as H1N1, H1N2, H3N2 and H5N1 (bird flu), 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, enteric adenoviruses, parvovirus, Dengue fever virus, Monkey pox, Mononegavirales, Lyssavirus such as rabies virus, Lagos bat virus, Mokola virus, Duvenhage virus, European bat virus 1 & 2 andAustralian bat virus, Ephemero virus, Vesiculovirus, Vesicular Stomatitis Virus (VSV), Herpesviruses such as Herpes simplex virus types 1 and 2, varicella zoster, cytomegalovirus, Epstein-Bar virus (EBV), human herpesviruses (HHV), human herpesvirus type 6 and 8, Human immunodeficiency virus (HIV), papilloma virus, murine gammaherpesvirus, Arenaviruses such as 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), Bunyaviridiae such as Crimean-Congo hemorrhagic fever virus, Hantavirus, hemorrhagic fever with renal syndrome causing virus, Rift Valley fever virus, Filoviridae (filovirus) including Ebola hemorrhagic fever and Marburg hemorrhagic fever, Flaviviridae including Kaysanur Forest disease virus, Omsk hemorrhagic fever virus, Tick-borne encephalitis causing virus and Paramyxoviridae such as Hendra virus and Nipah virus, variola major and variola minor (smallpox), alphaviruses such as Venezuelan equine encephalitis virus, eastern equine encephalitis virus, western equine encephalitis virus, SARS-associated coronavirus (SARS-CoV), West Nile virus, any encephaliltis 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.

[0115] In some embodiments, the multi pseudotyped virus comprises five envelope glycoproteins. In some embodiments, the fifth envelope glycoprotein for pseudotyping a virus of present invention is different from the first envelope glycoprotein, the second envelope glycoprotein, the third envelope glycoprotein, and the fourth envelope glycoprotein and is selected from the group consisting of: Feline endogenous virus (RD114), Influenza A such as H1N1, H1N2, H3N2 and H5N1 (bird flu), 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, enteric adenoviruses, parvovirus, Dengue fever virus, Monkey pox, Mononegavirales, Lyssavirus such as rabies virus, Lagos bat virus, Mokola virus, Duvenhage virus, European bat virus 1 & 2 and Australian bat virus, Ephemerovirus, Vesiculovirus, Vesicular Stomatitis Virus (VSV), Herpesviruses such as Herpes simplexvirus types 1 and 2, varicella zoster, cytomegalovirus, Epstein-Bar virus (EBV), human herpesviruses (HHV), human herpesvirus type 6 and 8, Human immunodeficiency virus (HIV), papilloma virus, murine gammaherpesvirus, Arenaviruses such as 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), Bunyaviridiae such as Crimean-Congo hemorrhagic fever virus, Hantavirus, hemorrhagic fever with renal syndrome causing virus, Rift Valley fever virus, Filoviridae (filovirus) including Ebola hemorrhagic fever and Marburg hemorrhagic fever, Flaviviridae including Kaysanur Forest disease virus, Omsk hemorrhagic fever virus, Tick-borne encephalitis causing virus and Paramyxoviridae such as Hendra virus and Nipah virus, variola major and variola minor (smallpox), alphaviruses such as Venezuelan equine encephalitis virus, eastern equine encephalitis virus, western equine encephalitis virus, SARS-associated coronavirus (SARS-CoV), West Nile virus, any encephaliltis 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.

[0116] In some embodiments, the ten or more envelope glycoproteins are used for pseudotyping a virus of present invention wherein all envelope glycoproteins are different and are selected from the group consisting of: Feline endogenous virus (RD114), Influenza A such as H1N1, H1N2, H3N2 and H5N1 (bird flu), 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, enteric adenoviruses, parvovirus, Dengue fever virus, Monkey pox, Mononegavirales, Lyssavirus such as rabies virus, Lagos bat virus, Mokola virus, Duvenhage virus, European bat virus 1 & 2 and Australian bat virus, Ephemero virus, Vesiculovirus, Vesicular Stomatitis Virus (VSV), Herpesviruses such as Herpes simplex virus types 1 and 2, varicella zoster, cytomegalovirus, Epstein-Bar virus (EBV), human herpesviruses (HHV), human herpesvirus type 6 and 8, Human immunodeficiency virus (HIV), papilloma virus, murine gammaherpesvirus, Arenaviruses such as 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), Bunyaviridiae such as Crimean-Congo hemorrhagic fever virus, Hantavirus, hemorrhagic fever with renal syndrome causing virus, Rift Valley fever virus, Filoviridae (filovirus) including Ebola hemorrhagic fever and Marburg hemorrhagic fever, Flaviviridae including Kaysanur Forest disease virus, Omsk hemorrhagic fever virus, Tick-borne encephalitis causing virus and Paramyxoviridae such as Hendra virus and Nipah virus, variola major and variola minor (smallpox), alphaviruses such as Venezuelan equine encephalitis virus, eastern equine encephalitis virus, western equine encephalitis virus, SARS-associated coronavirus (SARS-CoV), West Nile virus, any encephaliltis causing virus, 4070A, gap70, 10A1, and GAEV. In some embodiments, the ten or more envelope glycoprotein is selected from the group consisting of ecotropic virus envelope glycoproteins and amphotropic virus envelope glycoproteins.

[0117] In a further embodiment, the retrovirus is pseudotyped with any combination of envelope glycoproteins selected from the group consisting of RD114, VSV-G, 4070A, gap70, 10A1, and GAEV. 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.

[0118] 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. Inone 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.

[0119] In one embodiment, the combination of envelope glycoproteins is RD114 and VSV-G. In one embodiment, the combination of envelope glycoproteins is GALV and VSV-G. In one embodiment, the combination of envelope glycoproteins is GALV and 10A1.

[0120] In some embodiments, the envelope glycoprotein may be altered or modified. In some embodiments, the envelope glycoprotein may be derived from the above envelope glycoproteins. In one embodiment, the alteration or modification is a truncation, masking, redirecting, or a combination thereof. In some embodiments, the alteration or modification confers a different binding property to the virus. In an illustrative example, an envelope glycoprotein may be altered or modified such that an antibody binding region is attached. In an illustrative example, a CD3 antibody binding domain is attached to an altered or modified VSV-G envelope glycoprotein which directs the virus towards CD3+ cells. In one embodiment, the alteration or modification is a truncation. In one embodiment, the alteration or modification is a masking. In one embodiment, the alteration or modification is a redirecting.

[0121] In another illustrative example, a GALV envelope glycoprotein is modified on the cytoplasmic table as described in Tomas et al. (Tomas HA, et al., 2019, Mol Ther Methods Clin Dev.15: 1-8). In an illustrative example, the GALV envelope glycoprotein further comprises a synthetic amino acid sequence or HIV-1 matrix capsidamino acid sequence. In some embodiments, this modification improves the transduction efficiency of the virus.

[0122] In various embodiments, the multi psuedotyped virus, described herein, comprising the described envelope glycoproteins are capable of infecting a host cell. After the outer envelope is shed, the viral RNA is copied into DNA by reverse transcription. This is catalyzed by the reverse transcriptase enzyme encoded by the pol region and uses the host cell tRNA packaged into the virion as a primer for DNA synthesis. In this way the RNA genome is converted into the more complex DNA genome.

[0123] The double-stranded linear DNA produced by reverse transcription may, or may not, have to be circularized in the nucleus. The provirus now has two identical repeats at either end, known as the long terminal repeats (LTR). The termini of the two LTR sequences produces the site recognized by a pol product — the integrase protein — which catalyzes integration, such that the pro virus is always joined to host DNA two base pairs (bp) from the ends of the LTRs. A duplication of cellular sequences is seen at the ends of both LTRs, reminiscent of the integration pattern of transposable genetic elements. Integration is thought to occur essentially at random within the target cell genome. However, by modifying the long-terminal repeats it is possible to control the integration of a retroviral genome.

[0124] Transcription, RNA splicing and translation of the integrated viral DNA is mediated by host cell proteins. Variously spliced transcripts are generated. In the case of the human retroviruses HIV- 1 / 2 and HTLV-I / II viral proteins are also used to regulate gene expression. The interplay between cellular and viral factors is a factor in the control of virus latency and the temporal sequence in which viral genes are expressed.

[0125] Retroviruses can be transmitted horizontally and vertically. Efficient infectious transmission of retroviruses requires the expression on the target cell of receptors which specifically recognize the viral envelope proteins, although viruses may use receptor-independent, nonspecific routes of entry at low efficiency. In addition, the target cell type must be able to support all stages of the replication cycle after virus has bound and penetrated. Vertical transmission occurs when the viral genome becomes integrated in the germ line of the host. The provirus will then be passed from generationto generation as though it were a cellular gene. Hence endogenous proviruses become established which frequently lie latent, but which can become activated when the host is exposed to appropriate agents.

[0126] As mentioned above, the integrated DNA intermediate is referred to as a provirus. Prior gene therapy or gene delivery systems use methods and retroviruses that require transcription of the provirus and assembly into infectious virus while in the presence of an appropriate helper virus or in a cell line containing appropriate sequences enabling encapsidation without coincident production of a contaminating helper virus. As described below, a helper virus is not required for the production of the recombinant retrovirus of the disclosure, since the sequences for encapsidation are provided in the genome thus providing a replication competent retroviral vector for gene delivery or therapy.

[0127] The retroviral genome and the proviral DNA of the disclosure have at least three genes: the gag, the pol, and the env, these genes may be flanked by one or two long terminal (LTR) repeat, or in the provirus are flanked by two long terminal repeat (LTR) and sequences containing cis-acting sequences such as psi. The gag gene encodes the internal structural (matrix, capsid, and nucleocapsid) proteins; the pol gene encodes the RNA-directed DNA polymerase (reverse transcriptase), protease and integrase; and the env gene encodes viral envelope glycoproteins. The 5' and / or 3' LTRs serve to promote transcription and polyadenylation of the virion RNAs. The LTR contains 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).

[0128] Adjacent to the 5' LTR are sequences necessary for reverse transcription of the genome (the tRNA primer binding site) and for efficient encapsidation of viral RNA into particles (the Psi site). If the sequences necessary for encapsidation (or packaging of retroviral RNA into infectious virion) are missing from the viral genome, the result is a cis defect which prevents encapsidation of genomic viral RNA. This type of modified vector is what has typically been used in prior gene delivery systems (i.e., systems lacking elements which are required for encapsidation of the virion).Methods of Production and Collection

[0129] In some embodiments, the invention provides methods and compositions for generating multi pseudotyped viruses.

[0130] Some aspects of the present disclosure include or are cells, in illustrative examples, mammalian cells, that are used as packaging cells to make recombinant retroviruses or recombinant retroviral particles. Any of a wide variety of cells can be selected for in vitro production of a virus or virus particle, such as a redirected recombinant retroviral particle, according to the invention. The term “packaging cell lines” is used in reference to cell lines that do not contain a packaging signal, but do stably or transiently express viral structural proteins and replication enzymes (e.g., gag, pol and env) which are necessary for the correct packaging of viral particles. Any suitable cell line can be employed to prepare packaging cells of the invention. Cell lines that package viruses are well known in the art. Eukaryotic cells are typically used. Generally, the cells are mammalian cells. In a particular embodiment, the cells used to produce the packaging cell line are human cells. Suitable cell lines which 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.

[0131] In one embodiment, the packaging cells are 293 cells, HEK 293, 293T cells, GP2-293 cells, phoenix GP, 293-vec RD114, RetroPack PT67, Plat A, and the likes.

[0132] As used herein, the term “producer cell line” refers to a cell line which is capable of producing recombinant retroviral particles, comprising a packaging cell line and a transfer vector construct comprising a packaging signal. The production of infectious viral particles and viral stock solutions may be carried out using conventional techniques. Methods of preparing viral stock solutions are known in the art and are illustrated by, e.g., Y. Soneoka et al. (1995) Nucl. Acids Res. 23:628-633, and N. R. Landau et al. (1992). J. Virol. 66:5110-5113.

[0133] In particular embodiments, viral particles may be generated by coexpressing the virion packaging elements and the transfer vector in a producer cell. Thesecells may be transiently transfected with a number of plasmids. Typically from three to four plasmids are employed, but the number may be greater depending upon the degree to which the retroviral components are broken up into separate units. For example, one plasmid may encode the core and enzymatic components of the virion, derived from HIV-1. This plasmid is termed the packaging plasmid. Another plasmid typically encodes the envelope protein(s), most commonly the G protein of vesicular stomatitis virus (VSV G) because of its high stability and broad tropism. In one embodiment, the plasmid encodes the envelope glycoproteins selected from the group of ecotropic virus envelope glycoproteins, amphotropic virus envelope glycoproteins, and any combination thereof. In one embodiment, the plasmid encodes the envelope glycoproteins selected from the group of RD114, VSV-G, 4070A, gap70, 10A1, GALV, and any combination thereof. This plasmid may be termed the envelope expression plasmid. In one embodiment, one or more envelope glycoprotein is used for the generation of viral particles. In one embodiment, each envelope expression plasmid expresses a type of envelope glycoprotein. In one embodiment, an envelope expression plasmid is engineered to express one or more types of envelope glycoprotein. For example, in the generation of a multi-pseudotyped virus, one or more envelope expression plasmids expressing an envelope glycoprotein selected from the group consisting of RD114, VSV-G, 4070A, gap70, 10A1, and GALV is used, wherein each envelope expression plasmid expresses a different envelope glycoprotein. In another example, one or more envelope glycoprotein selected from the group consisting of RD114, VSV-G, 4070A, gap70, 10A1, and GALV is expressed from one or more envelope expression plasmid, wherein one expression plasmid expresses multiple envelope glycoprotein.

[0134] In another example, one or more envelope expression plasmids expressing an envelope glycoprotein selected from the group consisting of ecotropic virus envelope glycoproteins and amphotropic virus envelope glycoproteins is used, wherein each envelope expression plasmid expresses a different envelope glycoprotein. In another example, one or more envelope glycoprotein selected from the group consisting of ecotropic virus envelope glycoproteins and amphotropic virus envelope glycoproteins is expressed from one or more envelope expression plasmid, wherein one expression plasmid expresses multiple envelope glycoprotein.

[0135] For example, in the generation of a multi-pseudotyped virus, an envelope expression plasmid expressing VSV-G and another envelope expression plasmid expressing RD114 is used. In another example, VSV-G and RD114 are expressed from one envelope expression plasmid. In another example, in the generation of a multipseudotyped virus, an envelope expression plasmid expressing VSV-G and another envelope expression plasmid expressing GALV is used. In another example, VSV-G and GALV are expressed from one envelope expression plasmid. In another example, in the generation of a multi-pseudotyped virus, an envelope expression plasmid expressing 10A1 and another envelope expression plasmid expressing GALV is used. In another example, 10A1 and GALV are expressed from one envelope expression plasmid.

[0136] However, any combination of desired envelopes can be used with any desired number of expression plasmids.

[0137] Yet another plasmid encodes the genome to be transferred to the target cell, that is, the vector itself, and is called the transfer vector. The 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 millions of transducing units per milliliter (TU / ml) can be generated by this technique and variants thereof.

[0138] In some embodiments, the at least one packaging plasmid encoding at least one glycoprotein is at a ratio of about 1 / 10 to 1 / 50 compared to total DNA. In some embodiments, the at least one packaging plasmid encoding at least one glycoprotein is at a ratio of about 1 / 50 to 1 / 100 compared to total DNA. In some embodiments, the at least one packaging plasmid encoding at least one glycoprotein is at a ratio of about 1 / 100 to 1 / 1000 compared to total DNA. In some embodiments, the at least one packaging plasmid encoding at least one glycoprotein is at a ratio of about 1 / 1000 to 1 / 100,000 compared to total DNA. In some embodiments, the at least one packaging plasmid encoding at least one glycoprotein is at a ratio of about 1 / 100,000 to 1 / 100,000,000 compared to total DNA.

[0139] In some embodiments, the at least one packaging plasmid encoding at least one glycoprotein is at a ratio of about 1 / 5 to 1 / 10 compared to total DNA. In some embodiments, the at least one packaging plasmid encoding at least one glycoprotein is ata ratio of about 1 / 4 to 1 / 5 compared to total DNA. In some embodiments, the at least one packaging plasmid encoding at least one glycoprotein is at a ratio of about 1 / 3 to 1 / 4 compared to total DNA. In some embodiments, the at least one packaging plasmid encoding at least one glycoprotein is at a ratio of about 1 / 2 to 1 / 3 compared to total DNA. In some embodiments, the at least one packaging plasmid encoding at least one glycoprotein is the only DNA.

[0140] Infectious virus particles may be collected from the packaging cells using conventional techniques. For example, the infectious particles can be collected by cell lysis, or collection of the supernatant of the cell culture, as is known in the art.Optionally, the collected virus particles may be purified if desired. Suitable purification techniques are well known to those skilled in the art. The viral particles may be concentrated. Methods of concentrating viruses are well known to those skilled in the art.

[0141] Viral particles may be collected by centrifugation or ultracentrifugation. After ultracentrifugation concentrated stocks can be obtained. Viruses may be used to infect cells in vivo, ex vivo, or in vitro using techniques well known in the art. For example, when cells are transduced ex vivo, the vector particles may be incubated with the cells using a desired dose generally.Genetic Composition of Transgene

[0142] Depending upon the intended use of the retroviral vector of the disclosure any number of heterologous polynucleotide or nucleic acid sequences may be inserted into the retroviral vector.

[0143] The polynucleotides of the present invention, regardless of the length of the coding sequence itself, may 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 (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Att sites), termination codons, transcriptional termination signals, and polynucleotides encoding self-cleaving polypeptides, epitope tags, as disclosed elsewhere herein or as known in the art, such that their overall length may vary considerably. It is therefore contemplated that a polynucleotide fragment of almost any length may beemployed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.

[0144] The term “expression control sequence” refers to a polynucleotide sequence that comprises one or more promoters, enhancers, or other transcriptional control elements or combinations thereof that are capable of directing, increasing, regulating, or controlling the transcription or expression of an operatively linked polynucleotide.

[0145] In particular embodiments, vectors of the invention comprise one or more expression control sequences that are specific to particular cells, cell types, or cell lineages e.g., target cells; that is, expression of polynucleotides operatively linked to an expression control sequence specific to particular cells, cell types, or cell lineages is expressed in target cells and not in other non-target cells. Each one of the one or more expression control sequences in a vector that are cell specific may express in the same or different cell types depending on the therapy desired.

[0146] In particular embodiments, a vector of the invention comprises exogenous, endogenous, or heterologous control sequences such as promoters and / or enhancers. An “endogenous” control sequence is one which is naturally linked to a given gene in the genome. An “exogenous” control sequence is one which is placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques) such that transcription of that gene is directed by the linked enhancer / promoter. A “heterologous” control sequence is an exogenous sequence that is from a different species than the cell being genetically manipulated. A “synthetic” control sequence may comprise elements of one more endogenous and / or exogenous sequences, and / or sequences determined in vitro or in silico that provide optimal promoter and / or enhancer activity for the particular gene therapy.

[0147] 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, e.g., a polynucleotide-of-interest are operably linked, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0148] A constitutive expression control sequence is a promoter, enhancer, or promoter / enhancer that continually or continuously allows for transcription of an operably linked sequence. A constitutive expression control sequence may be a “ubiquitous” promoter, enhancer, or promoter / enhancer that allows expression in a wide variety of cell and tissue types or a “cell specific,” “cell type specific,” “cell lineage specific,” or “tissue specific” promoter, enhancer, or promoter / enhancer that allows expression in a restricted variety of cell and tissue types, respectively. Illustrative ubiquitous expression control sequences include, but are not limited to, a cytomegalovirus (CMV) immediate early promoter, a viral simian virus 40 (SV40) (e.g., early or late), a Moloney murine leukemia virus (MoMLV) LTR promoter, a Rous sarcoma virus (RSV) LTR, a herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, and Pl l promoters from vaccinia virus, an elongation factor 1-alpha (EFla) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70 kDa protein 5 (HSPA5), heat shock protein 90 kDa beta, member 1 (HSP90B1), heat shock protein 70 kDa (HSP70), P-kinesin (P-KIN), the human ROSA 26 locus (Irions et al., (2007) Nature Biotechnology 25, 1477-1482), a Ubiquitin C promoter (UBC), a phosphoglycerate kinase- 1 (PGK) promoter, a cytomegalovirus enhancer / chicken P-actin (CAG) promoter, and a P-actin promoter.

[0149] In a particular embodiment, it may be desirable to use a cell, cell type, cell lineage or tissue specific expression control sequence to achieve cell type specific, lineage specific, or tissue specific expression of a desired polynucleotide sequence (e.g., to express a particular nucleic acid encoding a polypeptide in only a subset of cell types, cell lineages, or tissues or during specific stages of development).

[0150] Illustrative examples of tissue specific promoters include, but are not limited to: an B29 promoter (B cell expression), a runt transcription factor (CBFa2) promoter (stem cell specific expression), an CD 14 promoter (monocytic cell expression), an CD43 promoter (leukocyte and platelet expression), an CD45 promoter (hematopoietic cell expression), an CD68 promoter (macrophage expression), a CYP450 3A4 promoter (hepatocyte expression), an desmin promoter (muscle expression), an elastase 1 promoter (pancreatic acinar cell expression, an endoglin promoter (endothelial cell expression), afibroblast specific protein 1 promoter (FSP1) promoter (fibroblast cell expression), a fibronectin promoter (fibroblast cell expression), a fins-related tyrosine kinase 1 (FLT1) promoter (endothelial cell expression), a glial fibrillary acidic protein (GFAP) promoter (astrocyte expression), an insulin promoter (pancreatic beta cell expression), an integrin, alpha 2b (ITGA2B) promoter (megakaryocytes), an intracellular adhesion molecule 2 (ICAM-2) promoter (endothelial cells), an interferon beta (IFN-P) promoter (hematopoietic cells), a keratin 5 promoter (keratinocyte expression), a myoglobin (MB) promoter (muscle expression), a myogenic differentiation 1 (MYODI) promoter (muscle expression), a nephrin promoter (podocyte expression), a bone gamma-carboxyglutamate protein 2 (OG-2) promoter (osteoblast expression), an 3-oxoacid CoA transferase 2B (Oxct2B) promoter, (haploid-spermatid expression), a surfactant protein B (SP-B) promoter (lung expression), a synapsin promoter (neuron expression), a Wiskott-Aldrich syndrome protein (WASP) promoter (hematopoietic cell expression).

[0151] Certain embodiments of the invention provide conditional expression of a polynucleotide-of-interest, e.g., expression is controlled by subjecting a cell, tissue, organism, etc., to a treatment or condition that causes the polynucleotide to be expressed or that causes an increase or decrease in expression of the polynucleotide encoded by the polynucleotide-of-interest.

[0152] Illustrative examples of inducible promoters / systems include, but are not limited to, steroid-inducible promoters such as promoters for genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormone), metallothionine promoter (inducible by treatment with various heavy metals), MX-1 promoter (inducible by interferon), the “GeneSwitch” mifepristone -regulatable system (Sirin et al., (2003) Gene, 323:67), the cumate inducible gene switch (WO 2002 / 088346), tetracycline-dependent regulatory systems, etc.

[0153] Conditional expression can also be achieved by using a site specific DNA recombinase. According to certain embodiments of the invention the vector comprises at least one (typically two) site(s) for recombination mediated by a site specific recombinase. As used herein, the terms “recombinase” or “site specific recombinase” include excisive or integrative proteins, enzymes, co-factors or associated proteins that are involved in recombination reactions involving one or more recombination sites (e.g.,two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.), which may be wild-type proteins (see Landy, (1993) Current Opinion in Biotechnology 3:699-707), or mutants, derivatives (e.g., fusion proteins containing the recombination protein sequences or fragments thereof), fragments, and variants thereof. Illustrative examples of recombinases suitable for use in particular embodiments of the present invention include, but are not limited to: Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, C31 , Cin, Tn3 resolvase, TndX, XerC, XerD, TnpX, Hjc, Gin, SpCCEl, and ParA.

[0154] The vectors may comprise one or more recombination sites for any of a wide variety of site specific recombinases. It is to be understood that the target site for a site specific recombinase is in addition to any site(s) required for integration of a vector, e.g., a retroviral vector or lentiviral vector. As used herein, the terms “recombination sequence,” “recombination site,” or “site specific recombination site” refer to a particular nucleic acid sequence to which a recombinase recognizes and binds.

[0155] For example, one recombination site for Cre recombinase is loxP which is a 34 base pair sequence comprising two 13 base pair inverted repeats (serving as the recombinase binding sites) flanking an 8 base pair core sequence (see FIG. 1 of 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).

[0156] Suitable recognition sites for the FLP recombinase include, but are not limited to: FRT (McLeod, et al., 1996), Fl, F2, F3 (Schlake and Bode, 1994), F4, F5 (Schlake and Bode, 1994), FRT(LE) (Senecoff et al., 1988), FRT(RE) (Senecoff et al., 1988).

[0157] Other examples of recognition sequences are the attB, attP, attL, and attR sequences, which are recognized by the recombinase enzyme X Integrase, e.g., phi-c31. The cpC31 SSR mediates recombination only between the heterotypic sites attB (34 bp in length) and attP (39 bp in length) (Groth et al., 2000). attB and attP, named for the attachment sites for the phage integrase on the bacterial and phage genomes, respectively, both contain imperfect inverted repeats that are likely bound by cpC31 homodimers (Groth et al., 2000). The product sites, attL and attR, are effectively inert to further cpC31-mediated recombination (Belteki et al., 2003), making the reaction irreversible. For catalyzing insertions, it has been found that attB-bearing DNA inserts into a genomic attP site more readily than an attP site into a genomic attB site (Thyagarajan et al., 2001; Belteki et al., 2003). Thus, typical strategies position by homologous recombination an attP-bearing “docking site” into a defined locus, which is then partnered with an attB- bearing incoming sequence for insertion.

[0158] Particular embodiments of the invention also include polypeptide “variants.” The recitation polypeptide “variant” refers to polypeptides that are distinguished from a reference polypeptide by the addition, deletion, truncations, and / or substitution of at least one amino acid residue, and that retain a biological activity. In certain embodiments, a polypeptide variant is distinguished from a reference polypeptide by one or more substitutions, which may be conservative or non-conservative, as known in the art.

[0159] In certain embodiments, a variant polypeptide includes 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 a corresponding sequence of a reference polypeptide. In certain embodiments, amino acid additions or deletions occur at the C-terminal end and / or the N-terminal end of the reference polypeptide.

[0160] As noted above, polypeptides of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a reference polypeptide can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations 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. Pat. No. 4,873,192, Watson, J. D. et al., (1987) Molecular Biology of the Gene, Fourth Edition, Benjamin / Cummings, Menlo Park, Calif., and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl.Biomed. Res. Found., Washington, D.C.).

[0161] One skilled in the art will readily appreciate that there is no limit to the possible heterologous polynucleotide or nucleic acid sequences that may be inserted into the viral vector to be introduced to a host cell by way of this invention.

[0162] Vectors typically comprise the DNA of a transmissible agent, into which foreign DNA encoding a protein is inserted by restriction enzyme technology. A common type of vector is a “plasmid”, which generally is a self-contained molecule of doublestranded DNA that can readily accept additional (foreign) DNA and which can readily introduced into a suitable host cell. A large number of vectors, including plasmid and fungal vectors, have been described for replication and / or expression in a variety of eukaryotic and prokaryotic hosts. Non-limiting examples include pKK plasmids (Clonetech), pUC plasmids, pET plasmids (Novagen, Inc., Madison, Wis.), pRSET or pREP plasmids (Invitrogen, San Diego, Calif.), or pMAL plasmids (New England Biolabs, Beverly, Mass.), and many appropriate host cells, using methods disclosed or cited herein or otherwise known to those skilled in the relevant art. Recombinant cloning vectors will 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.

[0163] In some embodiments, the transfer vector comprises a nucleic acid sequence. In some embodiments, the transfer vector comprises a gene encoding a transgene. In some embodiments, the transgene is one or more selected from the group consisting of: siRNA, miRNA, mRNA, antibodies, proteins, and peptides. In some embodiments, the transgene is one or more CARs with secre ted / anchored proteins.Chimeric Antigen Receptors (CARs)

[0164] In one embodiment, the transgene encodes a CAR. In one embodiment, the CAR comprises an antigen binding domain which is specific for at least one marker of at least one cancer cell or at least one pathogen. In some embodiments, once bound to the at least one cancer cell or at least one pathogen, the at least one modified target cell facilitates the destruction of the 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 (e.g., cancer, etc.) in the subject.

[0165] The term “chimeric antigen receptor” or “CAR,” as used herein, refers to an artificial cell receptor that is engineered to be expressed on an immune effector cell, such as an NK cell, a macrophage, a B cell, or a dendritic cell, and specifically bind an antigen on at least one cancer cell or at least one pathogen. CARs may be used as a therapy with adoptive cell transfer. Generally, immune cells of interest, are removed from a patient and modified so that they express the receptors specific to a particular form of antigen. In some embodiments, the CARs have specificity to at least one cancer cell or at least one pathogen. CARs may also comprise an intracellular activation domain, a transmembrane domain and an extracellular domain comprising an antigen binding region that specifically binds to at least one cancer cell or at least one pathogen.

[0166] In various embodiments, the CARs contemplated herein comprise an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain comprises a target-specific binding element otherwise referred to as an antigen binding domain. In some embodiments, the extracellular domain also comprises a hinge domain. In certain embodiments, the intracellular domain or otherwise the cytoplasmic domain comprises, a costimulatory signaling region and a zeta chain portion. The costimulatory signaling region refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigens receptors or their ligands that are required for an efficient response of lymphocytes to antigen.

[0167] Between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR, there may be incorporated a spacer domain. 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. A spacer domain may comprise 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 240amino 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.

[0168] The extracellular domain, transmembrane domain, and intracellular domain can be derived from any desired source of such domains.CAR antigen binding domain

[0169] The antigen binding domain may be obtained from any of the wide variety of extracellular domains or secreted proteins associated with ligand binding and / or signal transduction. In one embodiment, the antigen binding domain may consist of an Ig heavy chain which may in turn be covalently associated with Ig light chain by virtue of the presence of CHI and hinge regions, or may become covalently associated with other Ig heavy / light chain complexes by virtue of the presence of hinge, CH2 and CH3 domains. In the latter case, the heavy / light chain complex that becomes joined to the chimeric construct may constitute an antibody with a specificity distinct from the antibody specificity of the chimeric construct. Depending on the function of the antibody, the desired structure and the signal transduction, the entire chain may be used or a truncated chain may be used, where all or a part of the CHI, CH2, or CH3 domains may be removed or all or part of the hinge region may be removed.

[0170] In various embodiments, the CAR antigen binding domain may be humanized or comprise a fully human sequence.CAR transmembrane domain

[0171] With respect to the transmembrane domain, a CAR of the disclosure can be designed to comprise a transmembrane domain that is fused to the extracellular domain of the CAR. In one embodiment, the transmembrane domain that naturally is associated with one of the domains in the CAR is used. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0172] The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from anymembrane-bound or transmembrane protein. Transmembrane regions of particular use in this invention may be derived from (i.e., comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD 154. Alternatively, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In one embodiment, a triplet of phenylalanine, tryptophan and valine can be found at each end of a synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, for example, but not limited to between 2 and 10 amino acids in length, may form the linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. In another embodiment, the linker comprises a glycine-serine doublet.CAR intracellular domain

[0173] In various embodiments, the cytoplasmic domain or otherwise the intracellular domain of a CAR may be responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR is expressed. The term "effector function" refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity, including the secretion of cytokines. The term "intracellular signaling domain" refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The term intracellular domain is thus meant to include any truncated portion of the intracellular domain sufficient to transduce the effector function signal.

[0174] Examples of intracellular domains for use in the CARs of the disclosure include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability.

[0175] It is known that signals generated through the TCR alone are insufficient for full activation of the T cell and that a secondary or co-stimulatory signal is also required. Thus, T cell activation can be said to be mediated by two classes of intracellular signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences) and those that act in an antigenindependent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences).

[0176] Primary intracellular signaling sequences regulate primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary intracellular signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or IT AMs.

[0177] Examples of IT AMs containing primary intracellular signaling sequences that are of particular use in the 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 invention comprises an intracellular signaling sequence derived from CD3 zeta.

[0178] In another embodiment, the intracellular domain of the CAR can be designed to comprise the CD3-zeta signaling domain by itself or combined with any other desired cytoplasmic domain(s) useful in the context of the CAR of the invention. For example, the intracellular domain of the CAR can comprise a CD3 zeta chain portion and a costimulatory signaling region. The costimulatory signaling region refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or their ligands that is required for an efficient response of lymphocytes to an antigen. Examples of such molecules include CD2, CD27, CD28, 4- IBB (CD 137), 0x40, CD30, CD40, PD- 1, ICOS, lymphocyte function-associated antigen-1 (EFA-1), CD2, CD7, EIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, and the like.

[0179] The intracellular signaling sequences within the intracellular domain of the CAR of the invention may be linked to each other in a random or specified order. Optionally, a short oligo- or polypeptide linker, for example, between 2 and 10 aminoacids in length may form the linkage. A glycine-serine doublet provides a suitable linker in some embodiments.

[0180] In one embodiment, the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD28. In yet another embodiment, the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of 4- IBB.

[0181] In various embodiments, the CAR can be a “first generation,” “second generation,” “third generation,” “fourth generation” or “fifth generation” CAR (see, for example, Sadelain et al., Cancer Discov. 3(4):388-398 (2013); Jensen et al., Immunol. Rev. 257: 127-133 (2014); Sharpe et al., Dis. Model Meeh. 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., J. Immunol. 173:2143-2150 (2004); Sadelain et al., Curr. Opin. Immunol. (2009);Hollyman et al., J. Immunother. 32: 169-180 (2009)), each of which are incorporated by reference in its entirety).

[0182] “First generation” CARs for use in the invention comprise an antigen binding domain, for example, a single-chain variable fragment (scFv), fused to a transmembrane domain, which is fused to a cytoplasmic / intracellular domain of the T cell receptor chain. “First generation” CARs typically have the intracellular domain from the CD3^-chain, which is the primary transmitter of signals from endogenous T cell receptors (TCRs). “First generation” CARs can provide de novo antigen recognition and cause activation of both CD4+ and CD8+ T cells through their CD3^ chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation.

[0183] “Second-generation” CARs for use in the invention comprise an antigen binding domain, for example, a single-chain variable fragment (scFv), fused to an intracellular signaling domain capable of activating T cells and a co-stimulatory domain designed to augment T cell potency and persistence (Sadelain et al., Cancer Discov. 3:388-398 (2013)). CAR design can therefore combine antigen recognition with signal transduction, two functions that are physiologically borne by two separate complexes, the TCR heterodimer and the CD3 complex. “Second generation” CARs include an intracellular domain from various co-stimulatory molecules, for example, CD28, 4-1BB,ICOS, 0X40, and the like, in the cytoplasmic tail of the CAR to provide additional signals to the cell.

[0184] “Second generation” CARs provide both co-stimulation, for example, by CD28 or 4- IBB domains, and activation, for example, by a CD3^ signaling domain. Preclinical studies have indicated that “Second Generation” CARs can improve the antitumor activity of T cells. For example, robust efficacy of “Second Generation” CAR modified T cells was demonstrated in clinical trials targeting the CD 19 molecule in patients with chronic lymphoblastic leukemia (CLL) and acute lymphoblastic leukemia (ALL) (Davila et al., Oncoimmunol. 1(9): 1577-1583 (2012)).

[0185] “Third generation” CARs provide multiple co-stimulation, for example, by comprising both CD28 and 4- IBB domains, and activation, for example, by comprising a CD3^ activation domain.

[0186] “Fourth generation” CARs provide co-stimulation, for example, by CD28 or 4- IBB domains, and activation, for example, by a CD3^ signaling domain in addition to a constitutive or inducible chemokine component.

[0187] “Fifth generation” CARs provide co-stimulation, for example, by CD28 or 4- IBB domains, and activation, for example, by a CD3^ signaling domain, a constitutive or inducible chemokine component, and an intracellular domain of a cytokine receptor, for example, IL-2RP.

[0188] In various embodiments, the CAR can be included in a multivalent CAR system, for example, a DualCAR or “TandemCAR” system. Multivalent CAR systems include systems or cells comprising multiple CARs and systems or cells comprising bivalent / bispecific CARs targeting more than one antigen.

[0189] In the embodiments disclosed herein, the CARs generally comprise an antigen binding domain, a transmembrane domain and an intracellular domain, as described above. In a particular non-limiting embodiment, the antigen-binding domain is an scFv.

[0190] In one embodiment, the antigen binding domain of the CAR molecule is a targeting domain, wherein the targeting domain directs the cell expressing the CAR to at least one cancer cell or at least one pathogen For example, in one embodiment, the targeting domain comprises an antibody, antibody fragment, or peptide that specificallybinds to an antigen (e.g., a self-antigen or a foreign antigen) thereby directing the cell expressing the CAR to at least one cancer cell or at least one pathogen, wherein the at least one cancer cell or at least one pathogen expresses the antigen.

[0191] In one embodiment, the antigen binding domain of the CAR molecule of the invention can be generated to be reactive to any desirable antigen of interest, or fragment thereof, including, but not limited to a tumor antigen, a foreign antigen (e.g, a bacterial antigen, a viral antigen, etc.) or a self-antigen, on the surface of the at least one cancer cell or at least one pathogen. In some embodiments, the antigen on the surface of the at least one cancer cell is a tumor antigen.

[0192] Tumor antigens are proteins that are produced by tumor cells that elicit an immune response. The selection of the antigen binding domain of the VM -domain containing fusion molecule of the invention will depend on the particular type of cancer to be treated. Tumor antigens are well known in the art and include, for example, a glioma-associated antigen, carcinoembryonic antigen (CEA), P-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN- CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO- 1, LAGE- la, p53, prostein, PSMA, Her2 / neu, survivin and telomerase, prostatecarcinoma tumor antigen- 1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin. Another exemplary tumor antigen is chondroitin sulfate proteoglycan 4 (CSPG4) (also referred to as melanoma-associated chondroitin sulfate proteoglycan (MCSP), high-molecular- weight melanoma-associated antigen (HMW-MAA), or neuron-glial antigen 2 (NG2)).

[0193] In one embodiment, the tumor antigen comprises one or more antigenic cancer epitopes associated with a malignant tumor. Malignant tumors express a number of proteins that can serve as target antigens for an 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 onco-fetal antigens such as carcinoembryonic antigen (CEA). In B celllymphoma the tumor-specific idiotype immunoglobulin constitutes a truly tumor-specific immunoglobulin antigen that is unique to the individual tumor. B cell differentiation antigens such as CD 19, CD20 and CD37 are other candidates for target antigens in B cell lymphoma. Some of these antigens (CEA, HER-2, CD 19, CD20, idiotype) have been used as targets for passive immunotherapy with monoclonal antibodies with limited success.

[0194] The type of tumor antigen referred to in the invention may also be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). A TSA is unique to tumor cells and does not occur on other cells in the body. A TAA associated antigen is not unique to a tumor cell and instead is also expressed on a normal cell under conditions that fail to induce a state of immunologic tolerance to the antigen. The expression of the antigen on the tumor may occur under conditions that enable the immune system to respond to the antigen. TAAs may be antigens that are 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 extremely low levels on normal cells but which are expressed at much higher levels on tumor cells.

[0195] Non-limiting examples of TSA or TAA antigens include the following: differentiation antigens such as MART-l / MelanA (MART-I), gplOO (Pmel 17), tyrosinase, TRP- 1 , TRP-2 and tumor-specific multilineage antigens such as MAGE- 1 , MAGE-3, BAGE, GAGE-1, GAGE-2, pl 5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP- 180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, 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 15-3XCA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68XP1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, M0V18, NB / 70K, NY-CO-1,RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, and TPS.

[0196] In some embodiments, the antigen on the surface of the at least one pathogen comprises a foreign antigen wherein the foreign antigen comprises a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen or fragment thereof, or variant thereof.Transduction of Cells

[0197] The present invention contemplates compositions and methods that provide high titers and high efficiency transduction of host cells in vitro, ex vivo, and in vivo.

[0198] Host cells can be transfected, infected, or transduced in vivo, ex vivo, or in vitro with a recombinant vector or a polynucleotide of the invention. Host cells may include packaging cells, producer cells, and cells infected with viral vectors. In particular embodiments, host cells infected with viral vector of the invention are administered to a subject in need of therapy. In certain embodiments, the term “target cell” is used interchangeably with host cell and refers to transfected, infected, or transduced cells of a desired cell type. In some embodiments, the target cell is a dividing cell or a non-dividing cell. In some embodiments, the target cell is a mammalian cell. In some embodiments, the target cell is a human cell. In some embodiments, the cell is an immune cell. In some embodiments, the cell is one or more selected from the group consisting of: an alpha beta T cell (aP T cell), a gamma delta T cell (y8 T cell), a natural killer (NK) cell, a dendritic cell, a macrophage, a natural killer T (NKT) cell, a B cell, a neutrophil, a mast cell, a hematopoietic stem cells (HSC), an induced pluripotent stem cells (IPSC), an IPSC- derived aP T cell, an IPSC-derived NK cell, an IPSC-derived y8 T cell, a CD34+ cell, a cord blood-derived stem cell, a lymphoid tissue inducer (LTi) cell, a group 1 innate lymphoid cell (ILC-1), a group 2 innate lymphoid cell (ILC-2), and a group 3 innate lymphoid cell (ILC-3). In some embodiments, the cell is a gamma delta T cell (y8 T cell).

[0199] In some embodiments, the host cell is activated before, during, or after the transfection, infection, or transduction. In some embodiments, the host cell is activated before, during, and after the transfection, infection, or transduction. In someembodiments, the host cell is activated before the transduction. In some embodiments, the host cell is activated during the transfection, infection, or transduction. In some embodiments, the host cell is activated after the transfection, infection, or transduction.

[0200] Methods of activating host cells are known in the art. In some embodiments, the cell is activated by a TCR agonist. In some embodiments, the TCR agonist is one or more selected from the group consisting of an anti-aP TCR antibody and an anti-yb TCR antibody. In some embodiments, the cell is activated by one or more 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, the cell is activated by IL2 and or anti-CD3 (e.g. OKT3) antibody. In some embodiments, the cell is activated by IL2. In some embodiments, the cell is activated by anti-CD3 antibody. In some embodiments, the cell to be activated is an immune cell. In some embodiments, the cell to be activated is a T cell.

[0201] In some embodiments, the cell is activated with 0000001U to 0.1U of one or more 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, the cell is activated with 0.1U to 1U of one or more 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, the cell is activated with 1U to 10U of one or more 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, the cell is activated with 10U to 100U of one or more 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, the cell is activated with 100U to 1000U of one or more 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 someembodiments, the cell is activated with 1000U to l,000,000U of one or more 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. 0KT3) antibody.

[0202] In some embodiments, the cell is activated with 0.0000001U to 0.1U IL2. In some embodiments, the cell is activated with 0.1U to 1U IL2. In some embodiments, the cell is activated with 1U to 10U IL2. In some embodiments, the cell is activated with 10U to 100U IL2. In some embodiments, the cell is activated with 100U to 1000U IL2. In some embodiments, the cell is activated with 1000U to l,000,000U IL2.

[0203] In some embodiments, the cell is activated with 0.0000001U to 0.1U anti- CD3 antibody. In some embodiments, the cell is activated with 0.1U to 1U anti-CD3 antibody. In some embodiments, the cell is activated with 1U to 10U anti-CD3 antibody. In some embodiments, the cell is activated with 10U to 100U anti-CD3 antibody. In some embodiments, the cell is activated with 100U to 1000U anti-CD3 antibody. In some embodiments, the cell is activated with 1000U to l,000,000U anti-CD3 antibody.

[0204] In some embodiments, the cell is activated for 0 to 1 hour. In some embodiments, the cell is activated for 1 to 6 hours. In some embodiments, the cell is activated for 6 to 12 hours. In some embodiments, the cell is activated 12 to 24 hours. In some embodiments, the cell is activated 24 to 48 hours. In some embodiments, the cell is activated 48 to 72 hours. In some embodiments, the cell is activated 3 days to a week. In some embodiments, the cell is activated a week to 1 month. In some embodiments, the cell is activated a month to a year. In some embodiments, the cell is activated 1 year to 10 years.

[0205] In some embodiments, the recombinant retroviral vector comprises a polynucleotide / gene of interest.Methods of Use in Therapy

[0206] Viruses may be delivered according to viral titer (TU / mL), which can be measured, for example, by using a commercially available p24 titer assay, which is an ELISA against the p24 viral coat protein. The following formula can be used to calculate the pg / mL of p24: there are approximately 2000 molecules of p24 per physical particle(PP) of lentivirus: (2xl03)x(24xl03Da of p24 per PP), 48xl06 / Avogadro=(48xl06) / (6xl023)=8xl017g of p24 per PP, approximately 1 PP per IxlO16g of p24, IxlO4PP per pg of p24. A reasonably well packaged recombinant retrovirus vector will have 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 to 100 TU / pg of p24. It is through this conversion that TU / mL is obtained.

[0207] Viruses may be delivered to a subject in vivo, by direct injection to the cell, tissue, or organ in need of therapy. As non-limiting illustrative examples, viruses may be delivered for regenerative medicine. As additional non-limiting illustrative examples, viruses may be used in areas such as oncology, immunology, cardiovascular, auto immunity, aging, musculoskeletal, ocular therapy and dermatology. Direct injection requires on the order of between 1 to 50 multiplicities of infection (MOI) which also corresponds to IxlO5to 50xl05transducing units of the viral vector per 105cells.

[0208] Based on previous experience, the amount of recombinant retrovirus directly injected is determined by total TU and can vary based on both the volume that could be feasibly injected to the site and the type of tissue to be injected. For example, a brain injection site may only allow for a very small volume of virus to be injected, so a high titer prep would be preferred, a TU of about IxlO6to IxlO7, about IxlO6to IxlO8, IxlO6to IxlO9, about IxlO7to IxlO10, IxlO8to IxlO11, about IxlO8to IxlO12, or about IxlO10to IxlO12or more per injection could be used. However, a systemic delivery could accommodate a much larger TU, a load of IxlO8, IxlO9, IxlO10, IxlO11, IxlO12, IxlO13, IxlO14, or IxlO15, could be delivered.EmbodimentsThe invention includes at least the following numbered embodiments:1. A method for generating a multi pseudotyped virus, the method comprising providing a mammalian cell line, adding 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 harvesting the multi pseudotyped virus 48hours and 72 hours after contacting the mammalian cell line with the composition, wherein the harvesting of the multi pseudotyped virus yields a higher titer of virus as compared to a method of producing a virus comprising a single pseudotype. The method of embodiment 1, wherein the culturing is performed at a temperature of about 37°C and in a humidified incubator with about 5% to about 10% CO2 for about 24 to 36 hours. The method of embodiment 1 or embodiment 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. The method of any one of embodiments 1-3, wherein the at least one glycoprotein comprises a first glycoprotein selected from the group consisting of an ecotropic virus envelope glycoprotein and an amphotropic envelope glycoprotein. The method of any one of embodiments 1-3, wherein the at least one glycoprotein comprises a first glycoprotein selected from the group consisting of RD114, VSV- G, 4070A, gap70, 10A1, and GALV. The method of any one of embodiments 1-5, wherein the at least one glycoprotein is two glycoproteins, three glycoproteins, or four glycoproteins. The method of any one of embodiments 1-6, wherein the at least one glycoprotein comprises a second glycoprotein selected from the group consisting of consisting of an ecotropic virus envelope glycoprotein and an amphotropic envelope glycoprotein; and is different from the first glycoprotein. The method of any one of embodiments 1-6, 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.The method of embodiment 8, wherein the two glycoproteins are RD114 and VSV-G. The method of embodiment 8, wherein the two glycoproteins are GALV and VSV-G. The method of embodiment 8, wherein the two glycoproteins are GALV and 10A1. The method of any one of embodiments 1-11, wherein the at least one packaging plasmid encoding at least one glycoprotein is at a ratio of about 1 / 5 to 1 / 10 compared to total DNA. A method for generating a multi pseudotyped virus, the method comprising providing a mammalian cell line for transfection and viral production, culturing the mammalian cell line; wherein the culturing is performed at a temperature of about 37°C and in a humidified incubator with about 5% to about 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 comprising a transgene and ii) at least one packaging plasmid encoding at least one glycoprotein, incubating the mammalian cell line with the composition, and harvesting the multi pseudotyped virus 48 hours and 72 hours after contacting the mammalian cell line with the composition, wherein the harvesting of the multi pseudotyped virus yields a higher titer of virus as compared to a method of producing a virus comprising a single pseudotype. The method of embodiment 13, wherein the at least one glycoprotein comprises a first glycoprotein selected from the group consisting of an ecotropic virus envelope glycoprotein and an amphotropic envelope glycoprotein.The method of embodiment 13, wherein the first envelope glycoprotein is selected from the group consisting of RD114, VSV-G, 4070A, gap70, 10A1, and GALV. The method of any one of embodiments 13-15, wherein the at least one glycoprotein is two glycoproteins, three glycoproteins, or four glycoproteins. The method of any one of embodiments 13-16, wherein the second envelope glycoprotein is selected from the group consisting of an ecotropic virus envelope glycoprotein and an amphotropic envelope glycoprotein; and wherein the second envelope glycoprotein is different from the first envelope glycoprotein. The method of any one of embodiments 13-16, wherein the second envelope glycoprotein is selected from the group consisting of RD114, VSV-G, 4070A, gap70, 10A1, and GALV; and wherein the second envelope glycoprotein is different from the first envelope glycoprotein The method of any one of embodiments 13-18, wherein the two glycoproteins are RD 114 and VSV-G. The method of any one of embodiments 13-18, wherein the two glycoproteins are GALV and VSV-G. The method of any one of embodiments 13-18, wherein the two glycoproteins are GALV and 10A1. The method of any one of embodiments 1-21, further comprising filtering the multi pseudotyped virus, wherein the filtering is performed with low protein binding filters. The method of embodiment 22, wherein the filtering is performed with filters comprising cellulose acetate or polysulfonate.The method of any one of embodiments 1-23, the method further comprising concentrating the virus. The method of any one of embodiments 1-24, wherein the method produces about 20, 30, 40, 50, 60, 70, 80 or 90 times more titres of the multi pseudotyped virus than a method of producing a virus comprising a single envelope glycoprotein, wherein the single envelope glycoprotein comprises RD 114, VSV-G, GALV, or 10A1. The method of any one of embodiments 1-25, wherein the virus comprises a retrovirus. An engineered multi pseudotyped virus comprising at least two envelope glycoprotein. The engineered multi pseudotyped virus manufactured by the method of any one of embodiments 1-26. The engineered multi pseudotyped virus of embodiment 27 or embodiment 28, wherein the virus comprises a retrovirus. A method of increasing transduction efficiency in y8 T cells, the method comprising providing an engineered viral particle, wherein the viral particle comprises a nucleic acid encoding a protein and a multi pseudotyped virus, providing a negatively isolated y8 T cells for transduction, and transducing the y8 T cells with the engineered viral particle. The method of embodiment 30, wherein the protein comprises a chimeric antigen receptor. A system for performing a method of generating y8 T cells comprising a transgene, wherein the system comprises one or more components capable ofperforming a method comprising the following steps providing y8 T cells, activating the y8 T cells with about 1000U IL2 or 0KT3 antibody for about 24 to 48 hours, transducing the y8 T cells with an engineered multi pseudotyped virus comprising a sequence encoding a transgene, and analyzing transduction efficiency, wherein the multi pseudotyped virus increases transduction efficiency as compared to transduction with a single pseudotyped virus. The system of embodiment 32, wherein the virus comprises a y-retrovirus. A method of increasing viral titers, the method comprising providing a mammalian cell line, adding cell medium to the mammalian cell line, providing a composition comprising 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 the first packaging plasmid encoding at least one glycoprotein to the mammalian cell line, incubating the mammalian cell line with the composition, and harvesting a multi pseudotyped virus 48 hours and 72 hours after contacting the mammalian cell line with the composition, wherein the harvesting of the multi pseudotyped virus yields a higher titer of virus as compared to a method of producing a virus comprising a single pseudotype. The method of embodiment 34, wherein the at least one glycoprotein comprises a first glycoprotein selected from the group consisting of an ecotropic virus envelope glycoprotein and an amphotropic envelope glycoprotein. The method of embodiment 34, wherein the at least one glycoprotein comprises a first glycoprotein selected from the group consisting of RD114, VSV-G, 407 OA, gap70, 10A1, and GALV. The method of any one of embodiments 34-36, wherein the at least one glycoprotein is two glycoproteins, three glycoproteins, or four glycoproteins.The method of any one of embodiments 34-37, wherein the at least one glycoprotein comprises a second glycoprotein selected from the group consisting of an ecotropic virus envelope glycoprotein and an amphotropic envelope glycoprotein; and is different from the first glycoprotein. The method of any one of embodiments 34-37, 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. The method of any one of embodiments 34-39, wherein the two glycoproteins are RD 114 and VSV-G. The method of any one of embodiments 34-39, wherein the two glycoproteins are GALV and VSV-G. The method of any one of embodiments 34-39, wherein the two glycoproteins are GALV and 10A1. The method of any one of embodiments 1-26 or embodiments 34-42, wherein the multi-pseudotyped virus comprises two or more envelope glycoproteins, is produced with enhanced titer, and comprises the properties of the envelope glycoproteins. The method of any one of embodiments 1-26 or embodiments 34-43, wherein one or more envelope glycoprotein is altered to provide the virus different binding properties. The method of embodiment 44, wherein the alteration is a truncation, masking, or redirecting.EXPERIMENTAL EXAMPLES

[0209] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0210] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore are not to be construed as limiting in any way the remainder of the disclosure.Example 1: Dual Psuedotyping

[0211] The results discussed herein were based on experiments designed to address the current need in the art for obtaining high titers of gamma retroviruses (gRVs) in a large-scale virus production scenarios. For example, experiments were designed to assess whether dual pseudotyping gRV would allow high titer retroviral supernatant production. A dual pseudotyped virus is a virus comprising two envelope glycoproteins. Experiments were designed to assess whether VSV-G and RD114 dual pseudotyping would produce enhanced titers compared to RD114 single pseudotype.

[0212] The dual pseudotyping generated by these studies showed that properties of parental envelopes such as infectivity and titer can be efficiently combined to produce viruses that display properties of both viral envelopes and are functional in nature. For example, using representative models, experiments were conducted where GP2 cells constitutively expressing gag / pol were used as the retroviral producer cell line. To these cells, the transfer plasmid encoding the gene of interest was co-transfected along with the envelope plasmid(s). The envelope plasmids tested were either a) RD114 alone, b)VSV- G alone, or c) and d) RD114+VSV-G at two different ratios. It was surprisingly observed that RD 114+VSV-G dual pseudotyped viruses gave higher titers by 40-80 fold compared to RD114 single pseudotyped virus (Table 1 and Figure 1). The results from this experiment demonstrated that dual pseudotyping with RD114 and VSV-G unexpectedlyallows production of high titer viral supernatant which can be used in downstream experiments without further concentration which is not the case for RD114 single pseudotyped retrovirus.Table 1: Percent CAR transgene expression from RD114 alone, VSV-G alone, and dual RD114+ VSV-G pseudotyped gRV* Values are percent CAR+ cellsExample 2

[0213] Experiments were further designed to assess whether RD114+VSV-G dual pseudotyped gRV efficiently transduces primary gamma delta T cells in a manner similar to RD114 single pseudotyped gRV. Briefly, using a representative model, both single RD114 pseudotyped gRV and dual RD114+VSV-G pseudotyped gRV were used to transduce primary gamma delta T cells from four healthy donors. The transduction efficiency was measured days 4-9 post transduction using CAR detection reagent and flow cytometry. It was observed that RD 114+VSV-G dual pseudotyped gRV efficiently transduced primary gamma delta T cells from four healthy donors and resulted in transduction efficiency of 30-50% (Figure 2). The results from this experimentdemonstrated that dual pseudotyped virus not only resulted in high titers of virus but also resulted in functional virus which can be successfully used to transduce primary cells such as alpha beta or gamma delta T cells.Example 3

[0214] Experiments were designed to assess whether RD 114+VSV-G dual pseudotyped gRV-transduced CAR T would display TAA dependent cytotoxicity similar to RD114 pseudotyped gamma retrovirus-transduced CAR T. Briefly, both single RD114 pseudotyped gRV and dual RD 114+VSV-G pseudotyped gRV were used to transduce primary gamma delta T cells from four healthy donors. These CAR T were used in an Incucyte based 3D spheroid assay to determine killing of tumor cell lines over a period of 10 days. The data showed growth of tumor cells over this time period as measured using an intra-nuclear marker present in the tumor cell line (Figure 3). It was observed that RD 114+VSV-G dual pseudotyped gRV-transduced CAR T showed TAA-dependent killing similar to RD114 single pseudotyped gamma retrovirus transduced-CAR T using 4 healthy donor derived primary gamma delta T cell samples. The results from this experiment demonstrated that dual pseudotyped virus not only resulted in high titers of virus but also resulted in functional virus which can be successfully used to transduce primary cells such as alpha beta or gamma delta T cells and the resultant CAR T cells generated can successfully clear tumor cells in an antigen-dependent fashion.Example 4

[0215] Further, experiments were designed to assess whether RD 114+VSV-G dual pseudotyped gRV-transduced CAR T would display TAA dependent cytotoxicity similar to RD114 pseudotyped gamma retrovirus-transduced CAR T. Briefly, both single RD114 pseudotyped gRV and dual RD 114+VSV-G pseudotyped gRV were used to transduce primary gamma delta T cells from four healthy donors. These CAR T were used in an Incucyte based 3D spheroid cytotoxicity assay to determine killing of tumor cell lines over a period of 10 days. Data showed growth of tumor cells over this time period as measured using an intra- nuclear marker present in the tumor cell line (Figure 4). It was observed that RD114+VSV-G dual pseudotyped gRV-transduced CAR T showed TAA-dependent killing similar to RD114 single pseudotyped gamma retrovirus-transduced CAR T in this study using 4 healthy donor-derived primary gamma delta T cell samples. The results from this experiment demonstrated that dual pseudotyped virus not only result in high titers of virus but also result in functional virus which can be successfully used to transduce primary cells such as alpha beta or gamma delta T cells and the resultant CAR T cells generated can successfully clear tumor cells in an antigen dependent fashion.Example 5

[0216] Experiments were designed using representative models to assess whether RD114+VSV-G dual pseudotyped gRV transduced CAR T would display TAA dependent cytokine secretion and polyfunctionality similar to RD114 pseudotyped gamma retrovirus transduced CAR T. Briefly, both single RD114 pseudotyped gRV and dual RD 114+VSV-G pseudotyped gRV were used to transduce primary gamma delta T cells from four healthy donors. These CAR T were used in a co-culture assay to determine secretion of cytokines in response to tumor cell lines over 24 hours (Figure 5 and Figure 6). It was observed that RD114+VSV-G dual pseudotyped gRV-transduced CAR T displayed TAA-dependent cytokine secretion similar to RD114 single pseudotyped gamma retrovirus transduced-CAR T in this study using 4 healthy donor derived primary gamma delta T cell samples. The surprising results from this experiment demonstrated that dual pseudotyped virus not only resulted in high titers of virus but also resulted in functional virus which can be successfully used to transduced primary cells such as alpha beta or gamma delta T cells and the resultant CAR T cells generated can successfully secrete multiple cytokines in response to antigen.Example 6: Exemplary protocol for transfection and RV generation using GP2 cellsThe materials and methods are described herein. It is to be understood, however, that the present invention is not restricted to the precise protocol described herein. A person having ordinary skill in the art will understand, based on the extensive teachings in the present application, that adjustments may be made to the exemplary protocols referred to herein.MATERIALS:Table 2: Reagents for producing RD114 and VSV-G gRVsEXEMPLARY PROCEDURE(S):1. Prepare complete DMEM medium: DMEM with 10% heat inactivated FBS, without antibiotics for transfection experiments.2. Thaw the vial of cells rapidly in a 37°C water bath with gentle agitation. Immediately upon thawing, wipe the outside of the vial with 70% ethanol. All the operations from this point on should be carried out in a laminar flow tissue culture hood under strict aseptic conditions. Unscrew the top of the vial slowly and, using a pipet, transfer the contents of the vial to a 15 mL conical centrifuge tube containing 1 mL of prewarmed medium. Mix gently.3. Slowly add an additional 4 mL of fresh, prewarmed medium to the tube and mix gently.4. Add an additional 5 mL of prewarmed medium to the tube, mix gently. Centrifuge at 100 x g for 5 min, carefully aspirate the supernatant, and gently resuspend the cells in complete medium.5. Mix the cell suspension thoroughly and add to a suitable culture vessel. Gently rock or swirl the dish / flask to distribute the cells evenly over the growth surface and place it in a 37°C humidified incubator (5-10% CO2 as appropriate) for 24 hr.6. The next day, examine the cells under a microscope. If the cells are well-attached and confluent, they can be passaged for use. If most cells are not well-attached, continue culturing for another 24 hr. Complete attachment of newly thawed cultures of HEK 293-based cell lines, may require up to 48 hr.7. Once the culture has been started and the cells are growing normally, cells may be used to seed for transfection and virus production.

[0217] For HEK 293-based cell lines, collagen-coated plates or flasks for efficient culturing of frozen stocks are used. Vessels coated with compounds other than collagen may also provide suitable growth substrates (e.g. poly-L-lysine). Once recovered, the cells may be cultured directly on tissue culture plastic. However, if adherence is poor, it is recommended to use only collagen-coated vessels.Day -1: Seeding cells1. Remove medium from cultured GP2 cells, wash flask using 10 mL of DPBS (Don’t apply DPBS to cells directly, add to sides of flask).2. Remove DPBS, add 3 mL of 0.05% Trypsin / EDTA to T75 flask and incubate in 37°C incubator.3. After 2 minutes add 5 mL of complete medium to T75 flask containing GP2 cells4. Transfer cell-containing medium to 15 mL centrifuge tube and count cells on Moxicyte.5. Seed 5xl06293T based GP2 cells per 100 mm2round-bottomed Corning™ BioCoat™ Collagen I coated culture dish in a total of 10 mL of complete mediaand incubate at 37°C overnight. In some situations, 150 mm plates were used (Corning, catalog# 354551) as a substitute for 100 mm plates. Same volumes of all reagents as mentioned above were maintained for 150 mm plates with no changes.Dav 0: Transfection1. After examination of plates, the cells should be 70-90% confluent. Prepare the transfection mix as follows.2. For each plate to be transfected, warm ~5ml of complete DMEM in centrifuge tubes in the 37°C bath for subsequent media change.3. For each flask to be transfected, add 1.5 mL of OptiMEM to 15 mL centrifuge tubes.4. Gently flick the tube to mix.5. Carefully add 15 pL of Lipof ectamine 2000 to the center of the media, not touching the walls of the tube.6. Flick the tube gently to mix and keep at room temperature for 15 minutes.7. While the Lipofectamine and OptiMEM is mixing, make a master mix of the plasmids below.8. Re-suspend below plasmids in 1500 pL of OptiMEM.This condition below is for the RD114 alone pseudotype:Table 3: Reagents for producing RD114 pseudotyped gRVThis condition below is for the VSV-G alone pseudotype:Table 4: Reagents for producing VSV-G pseudotyped gRVThis condition below is for the RD114+VSV-G (175thtotal DNA) pseudotype:Table 5: Reagents for producing RD114+VSV-G pseudotyped gRVThis condition below is for the RD 114+VSV-G (1710thtotal DNA) pseudotype:Table 6: Reagents for producing RD114+VSV-G pseudotyped gRV9. Combine lipofectamine and plasmid mixes from steps 5 (1.5 mL) and 8 (1.5 mL) to get a total of 3 ml volume.10. Incubate at room temperature for 20 to 25 minutes.11. In the meantime, remove spent media from GP2 cell plates and replace with 5 mL of fresh media.12. Add the transfection mixture to GP2 cells dropwise and gently swirl the plate to mix.13. Place the plates back in the incubator.14. After 6 hours, remove media and carefully replace with 10 mL pre-warmed complete media and put the plate back in the incubator.15. Harvest retrovirus 48- and 72-hours post-transfection.Day 2: 48 Hour Collection1. Half hour prior to collection, warm ~7 mL of media per plate.2. Carefully tilt the plate, collect the media from the flask, and put it into the 15 mL centrifuge tube.3. Add 7 mL of warmed media to the flask and put back into the incubator.4. Spin the conical with the collected supernatant for 5 minutes at 1500 rpm and collect only virus while discarding any cell pellet formed.5. Filter the virus through a 0.45 pm filter to remove cellular debris.6. Set aside a 100 pL aliquot of each virus for titer calculations using the standard titer protocol (used by Lenti Core). Use Jurkat cells for titration instead of SUPT1 cells.

[0218] The filter used is cellulose acetate, or polysulfonate (low protein binding), instead of nitrocellulose or an equivalent thereof. Nitrocellulose binds proteins present in the membrane of retrovirus and destroys the virus.Day 3: Addition of Retro-X Concentrator for concentration of virus1. Combine 1 volume of Retro-X Concentrator with 3 volumes of clarified supernatant.2. Incubate the mixture overnight at 4°C.3. Centrifuge sample at 1500 x g for 45 minutes at 4°C. After centrifugation an off- white pellet will visible.4. Carefully remove the supernatant, taking care not to disturb the pellet (use pipette).5. Gently resuspend the pellet in 1 / 10thto 17100thof the original volume using RIO media.6. Immediately titrate the same or store at -80°C in single use aliquots.

[0219] Lenti X concentrator can also be used to concentrate retrovirus as per manufacturer instructions.Protocol for titration:Titer Assay (Alternatively, Jurkats instead of Sup T1 can be used to seed)1. Seed 20,000 SupTl cells per well in 100 pL media in U bottom 96 well plates.2. Thawed lentivirus is diluted 3-fold in media (1st point) then diluted 3-fold in media for the dilution series, leaving final well as untreated.3. This assay used an 11-point dilution series. (Alternatively, a 7-point dilution series with the eight well having no virus condition can be used).4. Virus Dilution: mixed 6X at 1 / 3 well volume & tips changed after each dilution series.5. 50 pL from viral dilution plate added on top of 100 pL cells. (Alternatively, an added spin step at 1500 rpm for 5 mins can be applied).6. Cells incubated with virus at 37°C for 4 days7. Cells stained for CAR expression in 50 pL staining volume:8. Staining Reagents used in multiplex for this assay:9. CAR stain & Viability Dye:(Staining reagent as per transgene tested.10. 60-minute incubation at 4°C. (Alternatively, 30 mins at room temp).11. Wash 3-times with 200 pL of FACS buffer (spin 400g / 4°C / 4 minutes). (Alternatively, one wash in 200 pL FACS buffer 1500rpm for 5 mins).12. Read on flow cytometer.Example 7: Protocol for transfection and RV generation using GP2 cells for the generation of GALV and VSV-G pseudotyped virusThe materials and methods are described herein.MATERIALS:Table 7: Reagents for producing GALV and VSV-G gRVs

[0220] The initial steps for the generation of the GALV and VSV-G pseudotyped virus are similar to the Procedure steps and Day - 1 seeding steps as described in Example 2 and are referred to. In order to test for increased titers of the GALV and VSV-G dual pseudotyped virus, controls of the GALV pseudotyped virus and VSV-G pseudotyped virus are generated. The VSV-G pseudotyped virus is generated as described in Example 2. Transfection of the cells to generate the GALV pseudotyped virus and the GALV + VSV-G dual pseudotype is described in the steps below.Dav 0: Transfection1. After examination of plates, the cells should be 70-90 percent confluent. Prepare the transfection mix as follows.2. For each plate to be transfected, warm ~5ml of complete DMEM in centrifuge tubes in the 37°C bath for subsequent media change.3. For each flask to be transfected, add 1.5 mL of OptiMEM to 15 mL centrifuge tubes.4. Gently flick the tube to mix.5. Carefully add 15 pl of Lipofectamine 2000 to the center of the media, not touching the walls of the tube6. Flick the tube gently to mix and keep at room temperature for 15 minutes.7. While the Lipofectamine and OptiMEM is mixing, make a master mix of the plasmids below.8. Re-suspend below plasmids in 1500 pl of OptiMEMThis condition below is for the GALV alone pseudotype:Table 8: Reagents for producing GALV pseudotyped gRVThis condition below is for the GALV+VSV-G (175thtotal DNA) pseudotype:Table 9: Reagents for producing GALV+VSV-G pseudotyped gRVThis condition below is for the GALV+VSV-G (1710thtotal DNA) pseudotype:Table 10: Reagents for producing GALV+VSV-G pseudotyped gRV9. Combine lipofectamine and plasmid mixes from steps 5 (1.5 ml) and 8 (1.5 ml) to get a total of 3 ml volume.10. Incubate at room temperature for 20 to 25 minutes.11. In the meantime, remove spent media from GP2 cell plates and replace with 5 ml of fresh media.12. Add the transfection mixture to GP2 cells dropwise and gently swirl the plate to mix.13. Place the plates back in the incubator.14. After 6 hours, remove media and carefully replace with 10 mL pre-warmed complete media and put the plate back in the incubator.15. Harvest retrovirus 48- and 72-hours post-transfection.

[0221] Collection and concentration steps of the virus are described in Example 2. The titers of all viruses (single and dual pseudotyped) are measured and the titers for the dual pseudotyped virus are increased as compared to a method of producing a virus comprising the single pseudotype of either VSV-G or GALV alone.

[0222] The dual pseudotype of GALV+VSV-G increases transduction efficiency in y8 T cells as compared to a virus comprising the single pseudotype of either VSV-G or GALV alone.

[0223] A combination of any one of RD114, VSV-G, eco (ecotropic), ampho (amphotropic), 10A1 or GALV for a dual pseudotyped virus is used and upon harvesting, yields a higher titer of virus as compared to a method of producing a virus comprising any one of the aforementioned single psuedotype alone.Example 8: Transfection and RV generation using GP2 cells for the generation of GALV and 10A1 pseudotyped virusThe materials and methods are described herein.MATERIALS:Table 11: Reagents for producing GALV and VSV-G gRVsLipofectamine™ 2000 Transfection ThermoFisherReagent Scientific

[0224] The initial steps for the generation of the GALV and 10A1 pseudotyped virus are similar to the Procedure steps and day 1 seeding steps as described in Example2. In order to test for increased titers of the GALV and 10A1 dual pseudotyped virus, controls of the GALV pseudotyped virus and 10A1 pseudotyped virus are also generated. The GALV pseudotyped virus is generated as described in Example 3. Transfection of the cells to generate the 10A1 pseudotyped virus and the GALV + 10A1 dual pseudotype is described in the steps below.Day 0: Transfection1. After examination of plates, the cells are 70-90% confluent. Prepare the transfection mix as follows.2. Lor each plate to be transfected, warm ~5ml of complete DMEM in centrifuge tubes in the 37°C bath for subsequent media change.3. Lor each flask to be transfected, add 1.5 mL of OptiMEM to 15 mL centrifuge tubes.4. Gently flick the tube to mix.5. Carefully add 15 pl of Lipofectamine 2000 to the center of the media, not touching the walls of the tube.6. Elick the tube gently to mix and keep at room temperature for 15 minutes.7. While the Lipofectamine and OptiMEM is mixing, make a master mix of the plasmids below.8. Re-suspend below plasmids in 1500 pl of OptiMEM.This condition below is for the 10A1 alone pseudotype:Table 12. Reagents for producing 10A1 pseudotyped gRVThis condition below is for the GALV+10A1 (l / 5thtotal DNA) pseudotype:Table 13. Reagents for producing GALV+10A1 pseudotyped gRVThis condition below is for the GALV+10A1 (1710thtotal DNA) pseudotype:Table 14. Reagents for producing GALV+10A1 gRV9. Combine lipofectamine and plasmid mixes from steps 5 (1.5 ml) and 8 (1.5 ml) to get a total of 3 ml volume.10. Incubate at room temperature for 20 to 25 minutes.11. In the meantime, remove spent media from GP2 cell plates and replace with 5 ml of fresh media.12. Add the transfection mixture to GP2 cells dropwise and gently swirl the plate to mix.13. Place the plates back in the incubator.14. After 6 hours, remove media and carefully replace with 10 mL pre-warmed complete media and put the plate back in the incubator.15. Harvest retrovirus 48- and 72-hours post-transfection.

[0225] Collection and concentration steps of the virus are described in Example 2. The titers of all viruses are measured and the titers for the dual pseudotyped virus are increased as compared to a method of producing a virus comprising the single pseudotype of either 10A1 or GALV alone.

[0226] Dual pseudotype of GALV+10A1 would increase transduction efficiency in y8 T cells as compared to a virus comprising the single pseudotype of either VSV-G or 10A1 alone.

[0227] One has a combination of any one of RD114, VSV-G, eco (ecotropic), ampho (amphotropic), 10A1 or GALV for a dual pseudotyped virus and upon harvesting, yielding a higher titer of virus as compared to a method of producing a virus comprising any one of the aforementioned single psuedotype alone.Example 9: Multi-Pseudotyped viruses

[0228] Multi-pseudotyped viruses are generated as described Examples 2-4. Concentrations of the plasmids encoding the sequence of the envelope are optimized in order to achieve multi pseudotyped viruses with enhanced titers and transduction efficiencies. The initial steps for the generation of the single pseudotyped virus is similar to the Procedure steps and day 1 seeding steps as described in the previous examples.

[0229] Transfection of the cells is as previously described. However, for the generation of the multi-psuedotyped viruses which has two or more of any one of the envelope glycoproteins comprising three or more of any one of the combinations ofRD114, VSV-G, eco (ecotropic), ampho (amphotropic), 10A1 or GALV, the plasmid concentrations will be optimized for the transfection.The materials and methods are described herein.This condition below is for a triple pseudotype:Table 15. Reagents for producing triple pseudotyped gRVThis condition below is for a quadruple pseudotype:Table 16. Reagents for producing quadruple pseudotyped gRV

[0230] Accordingly, the concentrations of any one of the plasmids encoding the envelope glycoprotein may be adjusted to optimize the transfection. It should beunderstood that the limit of the number of pseudotypes may be adjustable and that addition of more than two envelope glycoproteins may enhance the titers of the virus produced and may increase the transduction efficiency.Example 10: Multi pseudotyped viruses comprising modified glycoproteins.

[0231] Improved glycoproteins have been previously described (Tomas HA, et al., 2019, Mol Ther Methods Clin Dev.l5:l-8) Accordingly, dual pseudotypes or multipseudotypes using a combination of different glycoproteins having altered envelope glycoproteins that may be truncated or have additional modifications are included.

[0232] In order to increase the production of the viral titers and to increase transduction levels, a dual pseudotype virus is produced using an improved GALV glycoprotein, wherein the GALV glycoprotein comprises modifications on the cytoplasmic tail as described in Tomas et al (Tomas HA, et al., 2019, Mol Ther Methods Clin Dev.15: 1-8), for example. As described, the GALV further comprises a synthetic amino acid sequence or a HIV-1 Matrix capsid amino acid sequence (Tomas HA, et al., 2019, Mol Ther Methods Clin Dev.15: 1-8). The combination of a modified GALV glycoprotein in combination with a second pseudotype is observed to increase infectious tiers and improve transduction efficiency.

[0233] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMSWhat is claimed is:

1. A method for generating a multi pseudotyped virus, the method comprising: a. providing a mammalian cell line; b. adding cell 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; and e. harvesting the multi pseudotyped virus 48 hours and 72 hours after contacting the mammalian cell line with the composition, wherein the harvesting of the multi pseudotyped virus yields a higher titer of virus as compared to a method of producing a virus comprising a single pseudotype.

2. The method of claim 1, wherein the culturing is performed at a temperature of about 37°C and in a humidified incubator with about 5% to about 10% CO2 for about 24 to 36 hours.

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

5. The method of any one of claims 1-3, 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 of any one of claims 1-5, wherein the at least one glycoprotein is two glycoproteins, three glycoproteins, or four glycoproteins.

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

8. The method of any one of claims 1-6, 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 of claim 8, wherein the two glycoproteins are RD114 and VSV-G.

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

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

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

13. A method for generating a multi pseudotyped virus, the method comprising: a. providing a mammalian cell line for transfection and viral production; b. culturing the mammalian cell line; wherein the culturing is performed at a temperature of about 37 °C and in a humidified incubator with about 5% to about 10% CO2; c. treating the mammalian cell line with Trypsin; d. adding cell medium to the mammalian cell line; e. contacting the mammalian cell line with a composition comprising i. a transfer vector comprising a transgene; and ii. at least one packaging plasmid encoding at least one glycoprotein;f. incubating the mammalian cell line with the composition; and g. harvesting the multi pseudotyped virus 48 hours and 72 hours after contacting the mammalian cell line with the composition, wherein the harvesting of the multi pseudotyped virus yields a higher titer of virus as compared to a method of producing a virus comprising a single pseudotype.

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

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

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

18. The method of any one of claims 13-16, wherein the second envelope glycoprotein is selected from the group consisting of RD114, VSV-G, 4070A, gap70, 10A1, and GALV; and wherein the second envelope glycoprotein is different from the first envelope glycoprotein19. The method of any one of claims 13-18, wherein the two glycoproteins are RD 114 and VSV-G.

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

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

22. The method of any one of claims 1-21, further comprising filtering the multi pseudotyped virus, wherein the filtering is performed with low protein binding filters.

23. The method of claim 22, wherein the filtering is performed with filters comprising cellulose acetate or polysulfonate.

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

25. The method of any one of claims 1-24, wherein the method produces about 20, 30, 40, 50, 60, 70, 80 or 90 times more titres of the multi pseudotyped virus than a method of producing a virus comprising a single envelope glycoprotein, wherein the single envelope glycoprotein comprises RD 114, VSV-G, GALV, or 10A1.

26. The method of any one of claims 1-25, wherein the virus comprises a retrovirus.

27. An engineered multi pseudotyped virus comprising at least two envelope glycoprotein.

28. The engineered multi pseudotyped virus manufactured by the method of any one of claims 1-26.

29. The engineered multi pseudotyped virus of claim 27 or 28, wherein the virus comprises a retrovirus.

30. A method of increasing transduction efficiency in y8 T cells, the method comprising:a. providing an engineered viral particle, wherein the viral particle comprises a nucleic acid encoding a protein and a multi pseudotyped virus; b. providing a negatively isolated y8 T cells for transduction; and c. transducing the y8 T cells with the engineered viral particle.

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

32. A system for performing a method of generating y8 T cells comprising a transgene, wherein the system comprises one or more components capable of performing a method comprising the following steps: a. providing y8 T cells; b. activating the y8 T cells with about 1000U IL2 or OKT3 antibody for about 24 to 48 hours; c. transducing the y8 T cells with an engineered multi pseudotyped virus comprising a sequence encoding a transgene; and d. analyzing transduction efficiency, wherein the multi pseudotyped virus increases transduction efficiency as compared to transduction with a single pseudo typed virus.

33. The system of claim 32, wherein the virus comprises a y-retrovirus.

34. A method of increasing viral titers, the method comprising: a. providing a mammalian cell line; b. adding cell medium to the mammalian cell line; c. providing a composition comprising a transfer vector comprising a gene encoding a transgene to the mammalian cell line; d. providing a first packaging plasmid encoding at least one glycoprotein to the mammalian cell line;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; and g. harvesting a multi pseudotyped virus 48 hours and 72 hours after contacting the mammalian cell line with the composition, wherein the harvesting of the multi pseudotyped virus yields a higher titer of virus as compared to a method of producing a virus comprising a single pseudotype.

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

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

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

39. The method of any one of claims 34-37, 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 of any one of claims 34-39, wherein the two glycoproteins are RD 114 and VSV-G.

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

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

43. The method of any one of claims 1-26 or 34-42, wherein the multi -pseudotyped virus comprises two or more envelope glycoproteins, is produced with enhanced titer, and comprises the properties of the envelope glycoproteins.

44. The method of any one of claims 1-26 or 34-43, wherein one or more envelope glycoprotein is altered to provide the virus different binding properties.

45. The method of claim 44, wherein the alteration is a truncation, masking, or redirecting.

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