Polymer-encapsulated viral vectors for gene therapy

Abstract

The present invention relates to a nanoparticle comprising a viral vector coated with a polymer or a mixture of polymers, wherein the viral vector comprises a transgene, and wherein the nanoparticle functions as a vehicle for delivering the transgene to eukaryotic cells, wherein the viral vector lacks an envelope protein.

Description

Polymer-encapsulated viral vectors for gene therapy Cross-reference to related applications This application claims priority over U.S. Provisional Application No. 62 / 618,156, filed January 17, 2018, entitled "Viral Vector Nanoparticles for In Vitro and In Vivo Cell Targeting," U.S. Provisional Application No. 62 / 661,990, filed April 24, 2018, entitled "Polymer-Encapsulated Viral Vectors Lacking Envelope Protein," and U.S. Provisional Application No. 62 / 715,007, filed August 6, 2018, entitled "Polymer-Encapsulated Viral Vectors Lacking Envelope Protein." Each of these provisional applications is incorporated herein by reference in its entirety. Background of the invention Gene therapy delivers exogenous genetic material to target cells to correct genetic abnormalities or treat disease by altering cellular function. Viral and non-viral gene delivery methods have been used for this purpose, but both approaches still have significant limitations. Several viral vector applications have already been translated to the clinic, either for gene therapy or vaccination protocols. However, the / UU1 viral vectors currently used have certain drawbacks. For example, targeting specific cells using a viral vector is challenging. In some gene therapy protocols, cell targeting is achieved by purifying the target cells and transducing them ex vivo, then expanding the transduced cells in vitro before reimplanting them into the patient. In vaccination protocols, a direct injection of the vector is performed, and nonspecific cell transduction is often used to elicit an immune response against the gene product encoded by the vector. Cell targeting may be necessary to increase the efficacy and safety of the treatment. There is a need for improved viral vector-based systems for targeted delivery of genetic material. Summary of the invention The technology described herein provides synthetic packaged viral vector nanoparticle compositions and methods for using them to deliver an enhanced supply of genetic material for gene therapy and vaccine applications. The vector compositions and methods can be employed in any situation where transduction of cells with one or more transgenes is useful. For example, they can be used to treat cancer, infectious diseases, metabolic diseases, neurological diseases, or inflammatory conditions, or to correct genetic defects. The viral vector nanoparticle compositions of the present technology include an outer coating containing a polymer or polymer mixture that encapsulates the vector. In certain embodiments, the nanoparticle polymer coating contains one or more oligopeptide-derived poly(beta-aminoester) species having the general formula Pep-S S-Pep wherein Pep is a peptide, such as an oligopeptide, and R is OH, CH3, or a cholesterol group, and wherein m ranges from 1 to 20, n ranges from 1 to 100, and i ranges from 1 to 10. In preferred embodiments, the peptide includes at least two, or at least three, amino acids selected from the group consisting of arginine (R), lysine (K), histidine (H), glutamic acid (E), aspartic acid (D), and cysteine ​​(C). See also WO2014 / 136100 and WO2016 / 116887 for a more detailed description of oligopeptide-derived PBAEs that may be used in the present technology. Cysteine ​​may be included to provide a covalent attachment site for the peptide to the polymer. Although it has a slight negative charge at pH 7, it does not significantly alter the charge when used with positively charged amino acids. Illustrative peptides are CRRR (SEQ ID NO: 1), CHHH (SEQ ID NO: 2), CKKK (SEQ ID NO: 3), CEEE (SEQ ID NO: 4), and CDDD (SEQ ID NO: 5).In other formulations, any naturally occurring amino acid can be included in the Pep portions. The peptide sequence is selected to promote cellular uptake and targeting of the polymer-coated vector. In formulations that utilize and lack viral envelope proteins, the oligopeptide sequences and net charge are selected to promote cellular uptake, endosomal uptake, and / or endosomal leakage of polymer-encapsulated viral vectors into the desired target cells. The peptides at each of the two ends of the polymer are generally the same in a given polymer molecule, but they can be different. The polymer molecules in a mixture used to coat a vector can be the same or different; if different, they may differ in the polymer backbone or in the terminal peptides.In preferred embodiments, the peptide at both ends of the polymer has the amino acid sequence CRRR (SEQ ID NO: 1), or CHHH (SEQ ID NO: 2), or CKKK (SEQ ID NO: 3), or CEEE (SEQ ID NO: 4), or CDDD (SEQ ID NO: 5). The polymer or polymer mixture may have a net positive or negative charge, or may be uncharged. Oligopeptides containing two or more residues of a single amino acid type, such as R, K, H, D, or E, may be used. The polymer may comprise a sugar or sugar alcohol grafted onto the side chain attached to the polymer backbone. One or more of the polymer molecules encapsulating each nanoparticle vector may optionally comprise a targeting portion linked to the polymer at any desired position on the polymer molecule, such as at R. The polymer molecules may be crosslinked or non-crosslinked.Polymer molecules can be attached to the surface of the viral vector non-covalently or covalently, such as by covalent bonding to a lipid molecule (e.g., cholesterol, a phospholipid, or a fatty acid) that is cleaved in the vector's lipid bilayer, or to a protein embedded in the lipid bilayer. In some embodiments, the polymer simply coats the vector but is attached non-specifically; that is, it is not attached to the vector by specific covalent or non-covalent interactions, but only by non-specific interactions such as net charge, hydrophobic character, or van der Waals interactions. In certain embodiments, the ratio of the polymer or polymer mixture to the retroviral vector is approximately 1:100 to approximately 5:1 by weight, or the number of polymer molecules per vector particle is approximately 10⁶ to approximately 10¹². Viral vector nanoparticles comprise a viral vector. The viral vector can be selected based on desired characteristics (e.g., immunogenicity, ability to accommodate constructs of different sizes, integrative and replicative properties, expression level, duration of expression, tissue tropism or targeting characteristics, ability to infect dividing and / or quiescent cells, etc.). The viral vector can be a retroviral vector such as a lentiviral vector. In preferred embodiments, the viral vector is a retroviral vector. However, the invention can also be implemented with other types of viruses and / or viral vectors, including those derived from DNA or RNA viruses. The nanoparticles may include one or more targeting portions exposed on the surface of the encapsulated vector nanoparticles, either through covalent or non-covalent association with the polymer coating. One or more different targeting portions may be present on each viral vector nanoparticle. Targeting portions can be, for example, antibodies, antibody fragments (including Fab), scFvs, antibody-like protein scaffolds, oligopeptides, aptamers, L-RNA aptamers, or ligands for cell surface receptors. In one embodiment, the targeting portion is an anti-CD3 antibody or aptamer for targeting the nanoparticles to T cells. In another embodiment, the transgene encodes a chimeric antigen receptor.The nanoparticle is capable of acting as a gene delivery vehicle by transducing cells in vivo in a subject to whom the vehicle is administered, or by transducing cells in vitro. In certain embodiments, the nanoparticle is capable of transducing a specific cell type or class, generally through the action of the targeting portion and / or through the action of the polymer or polymer mixture. In some embodiments, the polymer of the polymer-coated vector particles can promote cell transduction not only by binding to target cell surfaces, but also through endosomal uptake, such as by endocytosis, micropinocytosis, phagocytosis, or other mechanisms, and endosomal leakage (through membrane fusion after a reduction in pH within the endosomal vesicle).In preferred modalities, the amino acid sequence of polymer-associated oligopeptides can specifically promote cellular transduction through the endosomal pathway. While in some embodiments of the current technology the viral vectors are pseudotyped, in others the vectors lack certain viral envelope proteins or are pseudotyped. In pseudotyped vectors, envelope proteins, such as the HIV gpl20gp41 complex or the vesicular stomatitis virus glycoprotein (VSV-G), form spike-like structures on the outer surface of the viral envelope, facilitating the binding of vector particles to host cells and viral entry into host cells. Pseudotype proteins such as VSV-G can also be used to protect or bind the vector during purification (e.g., protection during ultracentrifugation, binding to an affinity column or other affinity matrix, or gel purification).However, in the context of viral vector polymer packaging, these structures can interfere with a tight association between the vector particle envelope and the polymers. Viral envelope proteins also have a pH-dependent charge, which can limit or interfere with polymer association, particularly with charged polymers. Furthermore, packaged viral vectors harboring pseudotyping proteins can undergo destabilization and destruction of the envelope in vitro and in vivo, thereby releasing the viral vector capable of nonspecifically transducing cells, which can lead to a reduced safety profile. Vectors lacking envelope proteins can improve viral vector packaging using polymers.Coated vectors lacking envelope protein exhibit an improved safety profile compared to vectors with envelope protein because, after destabilization or destruction of the coating, they cannot transduce cells in vitro or in vivo. Nanoparticles of the present technology, in which the viral vector lacks viral envelope protein, have a built-in safety mechanism for use in gene therapy or immunotherapy. This is because the nanoparticles are only capable of transducing a mammalian cell above a certain threshold number of polymer molecules per vector. Below this threshold, the amount of polymer is insufficient to fully coat the vector, which can cause the nanoparticles to become structurally unstable or subject to dissociation of the polymer molecules from the nanoparticle.Once these vectors lacking an envelope protein lose an effective polymer coating, they become unable to transduce mammalian cells, including human cells. Such nanoparticles have an improved safety profile for in vivo use compared to a vector or polymer-encapsulated vector lacking the threshold characteristic. In some embodiments, certain membrane proteins, such as proteins that are not viral fusion or envelope proteins and that do not otherwise form spike-like structures on the outer surface of the membrane, such as proteins from vector-producing cells that are not used for pseudotyping, may be present in the lipid membrane of the viral vector particle. In certain embodiments of the present technology, viral envelope proteins are excluded from viral vector particles, such as those having a significant mass protruding from the outer surface of the envelope lipid bilayer, such as at least 20%, at least 30%, at least 40%, or at least 50% of their mass protruding from the outer surface of the envelope. Nanoparticle compositions may optionally contain additional components, such as lipid molecules, surfactants, nucleic acids, protein molecules, or small-molecule drugs. The present technology also encompasses pharmaceutical formulations or compositions containing the nanoparticles along with one or more excipients, vehicles, pH regulators, salts, or liquids, making the delivery vehicle suitable for oral, intranasal, or parenteral administration, such as intravenous, intramuscular, subcutaneous, or peritumoral injection. ML / a / ZUZ 1 / UU I4ZJ intratumoral, or for in vitro administration to cells in an ex vivo gene transfer protocol. Such compositions and formulations may also be lyophilized for stabilization during storage. The viral vector nanoparticle of current technology can serve as a gene delivery vehicle. The nanoparticle includes a viral vector coated on its outer surface with a layer containing a polymer or a mixture of polymers. In some embodiments, the viral vector is pseudotyped and possesses an envelope protein that promotes fusion and comprises a transgene. In other embodiments, the viral vector lacks any native or recombinant envelope protein and comprises a transgene. In certain embodiments, the retroviral vector specifically lacks the viral envelope protein that would typically be included in the envelope of similar retroviral vectors.In certain embodiments, the viral vector specifically lacks any envelope protein from the natural or modified viral vector, such as wild-type or modified VSV-G, HIV gpl20, HIV gp41, MMTV gp52, MMTV qp36, MLV qp71, syncytin, wild-type or modified Sindbis virus envelope protein, measles virus hemagglutinin (H) and fusion glycoproteins (F), and HEMO. In other embodiments, the vector contains one or more of these envelope proteins. Another aspect of the technology is a method for manufacturing the nanoparticle / gene delivery vehicle described above (viral vector nanoparticles). The method includes the steps of: (a) providing a viral vector that possesses or lacks an envelope protein and contains a transgene; (b) providing a polymer or polymer mixture; and (c) bringing the viral vector and the polymer or polymer mixture into contact, whereby the viral vector and the polymer / polymer mixture combine to form the nanoparticle, which contains the viral vector coated with the polymer or polymer mixture. An additional aspect of the technology is an in vivo method for treating a disease using the viral vector nanoparticles (gene delivery vehicles) described above. This method involves parenterally administering a composition containing the nanoparticles to a patient in need, whereupon the cells within the patient are transduced by the viral vector and the transgene is expressed in the transduced cells. In one modality, the disease to be treated is cancer. For in vivo administration, modalities in which viral envelope proteins such as VSV-G are absent are preferred, as they provide an improved safety profile because they lack the ability to transduce untargeted cells in the host and because of the more specific targeting provided by the use of polymer encapsulation to restore the cellular uptake and / or endosomal uptake and / or endosomal leakage provided by the envelope protein. Another aspect of the technology is an in vitro cell transduction method. This method involves bringing isolated live cells into contact with the viral vector nanoparticles described above, so that at least some of the cells are transduced by the retroviral vector and the therapeutic protein or marker protein is expressed in the transduced cells. In another aspect of the technology, a method for performing gene therapy is provided that requires bringing cells in an in vitro culture or cells within a living organism into contact with the nanoparticle / gene delivery vehicle described above, through which the transgene is expressed in the cells. The transgene can replace or modify a defective gene or replace a missing gene. Current technology can be further summarized with the following list of modalities. A nanoparticle comprising a viral vector coated with a polymer or a mixture of polymers, wherein the viral vector comprises a transgene, and wherein the nanoparticle functions as a vehicle for delivering the transgene to eukaryotic cells. 2. The nanoparticle in accordance with the modality 1, where the viral vector lacks an envelope protein. 3. The nanoparticle according to modality or modality 2, wherein the polymer or polymer mixture comprises a poly(beta-amino ester) having the formula where each Pep is an oligopeptide; where R is OH, CH3, or cholesterol; and where m ranges from 1 to 20, n ranges from 1 to 100, and yo ranges from 1 to 10. 4. The nanoparticle in accordance with the modality 3, wherein each oligopeptide comprises at least three amino acid residues selected from the group consisting of arginine (R), glutamic acid (E), lysine (K), aspartic acid (D), histidine (H) and cysteine ​​(C). 5. The nanoparticle in accordance with modality or modality 4, wherein the amino acid sequence of at least one of the oligopeptides is CRRR (SEQ ID NO: 1), or CHHH (SEQ ID NO: 2), or CKKK (SEQ ID NO: 3), or CEEE (SEQ ID NO: 4), or CDDD (SEQ ID NO: 5). 6. The nanoparticle in accordance with any of the above modalities, wherein the polymer or polymer mixture has a net positive charge or a net negative charge at pH 7. 7. The nanoparticle in accordance with any of the above modalities, wherein the nanoparticle contains from 108 to 1012 polymer molecules per vector. 8. The nanoparticle in accordance with modality 7, wherein the nanoparticle contains approximately 109 to approximately 1010 polymer molecules per vector. 9. The nanoparticle in accordance with any of the above modalities, wherein the viral vector is a retroviral vector. 10. The nanoparticle in accordance with modality 9, wherein the retroviral vector is a lentiviral vector. 11. The nanoparticle in accordance with any of the above embodiments, further comprising one or more targeting portions attached to one or more polymer molecules. 12. The nanoparticle in accordance with modality 11, wherein the targeted portion is selected from the group consisting of antibodies, antibody fragments, scFv, antibody-like protein scaffolds, oligopeptides, aptamers, L-RNA aptamers, and ligands for cell surface receptors. 13. The nanoparticle in accordance with modality 12, wherein the targeting portion is an anti-CD3 antibody or an anti-CD3 aptamer. 14. The nanoparticle in accordance with any of the above modalities, wherein the transgene encodes a chimeric antigen receptor. 15. The nanoparticle in accordance with any of the above modalities having a zeta potential of approximately -15 mV to approximately +15 mV. 16. The nanoparticle in accordance with any of the above modalities that lacks VSV-G envelope protein. 17. The nanoparticle conforming to modality 16, which has an improved safety profile for in vivo use compared to a similar nanoparticle comprising the VSV-G envelope protein. 18. The nanoparticle in accordance with any of the above modalities, wherein the viral vector lacks viral envelope protein, and wherein the nanoparticle is only capable of transducing a mammalian cell above a threshold number of polymer molecules per vector. 19. The nanoparticle in accordance with modality 18, wherein below said threshold number of polymer molecules per vector, the amount of polymer is insufficient to completely coat the vector. 20. The nanoparticle in accordance with modality 18 or modality 19, wherein below such threshold number of polymer molecules per vector, the nanoparticle is structurally unstable or is subject to dissociation of the polymer molecules from the nanoparticle. 21. The nanoparticle conforming to any of modalities 18-20 that has an improved safety profile for in vivo use compared to a vector or polymer-encapsulated vector that lacks such a threshold. 22. A method for manufacturing the nanoparticle in accordance with any of the above embodiments, the method comprising the steps of: (a) provide a viral vector comprising a transgene; (b) providing a polymer or polymer mixture, and optionally one or more targeting portions; and (c) bringing the viral vector and the polymer or polymer mixture, and optionally one or more targeting portions, into contact, whereby the viral vector and the polymer or polymer mixture combine to form such nanoparticle. 23. The method in accordance with modality 22, wherein the viral vector lacks envelope protein. 24. The method of compliance with modality 22 or ML / a / ZUZ 1 / UU 14ZJ modality 23, where step (o) is performed at a pH of approximately 5.0 to approximately 6.5. 25. The method in accordance with modality 24, wherein step (c) is performed at a pH of approximately 5.5 to 6.0. 26. The method in accordance with any of embodiments 22-25, wherein the polymer or polymer mixture comprises a poly(beta-amino ester) having the formula where each Pep is an oligopeptide; where R is OH, CH3, or cholesterol; and where m ranges from 1 to 20, n ranges from 1 to 100 and i ranges from 1 to 10. 27. The method in accordance with modality 26, wherein each oligopeptide comprises at least three amino acid residues selected from the group consisting of R, E, K, D, H and C. 28. The method in accordance with modality 26, wherein the amino acid sequence of at least one of the oligopeptides is CRRR (SEQ ID NO: 1), or CHHH (SEQ ID NO: 2), or CKKK (SEQ ID NO: 3), or CEEE (SEQ ID NO: 4) or CDDD (SEQ ID NO: 5). 29. The method in accordance with any of the modalities 22-28, wherein the polymer or polymer mixture has a net positive charge or a net negative charge at pH 7. 30. The method in accordance with any of the modalities 22-29, wherein the nanoparticle contains from 108 to 1012 polymer molecules. 31. The method in accordance with modality 30, wherein the nanoparticle contains from approximately 109 to approximately 1010 polymer molecules. 32. The method in accordance with any of the modalities 22-31, wherein the retroviral vector is a retroviral vector. 33. The method in accordance with modality 32, wherein the retroviral vector is a lentiviral vector. 34. The method in accordance with any of the modes 22-33, further comprising contacting the vector with one or more addressing portions, optionally wherein the addressing portions are attached to the polymer or particle mixture. 35. The method in accordance with modality 34, wherein the targeting portion is selected from the group consisting of antibodies, antibody fragments, scFv, antibody-like protein scaffolds, oligopeptides, aptamers, L-RNA aptamers, and ligands for cell surface receptors. 36. The method in accordance with modality 35, wherein the targeting portion is an anti-CD3 antibody or an anti-CD3 aptamer. 37. The method in accordance with any of the modalities 22-36, wherein the transgene encodes a chimeric antigen receptor. 38. The method in accordance with any of the modalities 22-37, wherein the viral vector lacks VSV-G envelope protein. 39. The method in accordance with any of modalities 22-38, wherein step (c) is performed at a pH at which the polymer or polymer mixture has a net positive charge and the retroviral vector has a negative surface potential. 40. A method for treating a disease, the method comprising administering a composition comprising a plurality of nanoparticles in accordance with any of modalities 1-21 to a subject in need thereof, whereby the subject's cells are transduced by the retroviral vector and the transgene is expressed in the transduced cells. 41. The method in accordance with modality 40, where the disease is cancer. 42. The method of conformity with modality 40 or modality 41, where the subject is human. 43. A method for transducing cells in vitro, the method comprising contacting cultured cells with a plurality of nanoparticles in accordance with any of modalities 1-21, whereby at least some of the cultured cells are transduced by the retroviral vector and the transgene is specifically expressed in the transduced cells. 44. The method in accordance with modality 43, wherein the nanoparticles are targeted to CD3-positive cells and the transgene is specifically expressed in CD3-positive cells. 45. The method in accordance with modality 43 or modality 44, wherein the transgene encodes a chimeric antigen receptor. 46. ​​A method for performing gene therapy, the method comprising contacting cells in vitro or within a living organism with a plurality of nanoparticles in accordance with any of modalities 1-21, whereby the transgene is expressed in the cells. 47. A nanoparticle comprising a viral vector coated with a polymer or a mixture of polymers, wherein the viral vector comprises a reduced level of envelope protein and comprises a transgene, wherein the nanoparticle functions as a vehicle for delivering the transgene to eukaryotic cells, and wherein the reduced level of envelope protein is insufficient to promote cell entry of the vector and cell transduction by the vector. 48. The nanoparticle in accordance with modality 47, wherein the reduced level of envelope protein is 5% or less of an amount of envelope protein in a functional virus from which the vector is derived. Brief description of the drawings Figure 1A shows a schematic diagram of one embodiment of a gene delivery nanoparticle. The nanoparticle contains a polymer-encapsulated viral vector particle lacking an envelope protein. Figure 1B shows a schematic diagram of another embodiment of a gene delivery nanoparticle, in which the viral vector has envelope proteins. Figure 2 shows a schematic diagram of one embodiment of a gene delivery nanoparticle, in which the viral vector is encapsulated in multiple polymer layers. Although not shown, the viral vector envelope proteins may be present in some embodiments. Figure 3 shows a schematic diagram of one modality of a gene delivery nanoparticle, wherein the nanoparticle includes a targeting portion attached to the polymer layer (or outer polymer layer if a polymer multilayer is used). Figures 4A-4C are schematic representations of a cell transduction process with a lentiviral vector containing a GFP transgene (Figure 4A), inhibiting transduction using an anti-VSV-G antibody (Figure 4B) and avoiding antibody-mediated inhibition by polymeric encapsulation of the vector particles (Figure 4C). Figures 5A-5C are schematic representations of a process similar to that depicted in Figures 4A-4C, but using a lentiviral vector that lacks VSV-G protein. Figures 6A-6F show results of zeta potential measurements in bald lentiviral vectors encapsulated in the indicated PBAE polymers. Figure 7 shows the results of HEK293T cell transduction with a lentiviral vector containing a GFP transgene as a function of incubation time in pH regulator formulations that vary in composition and pH. Figures 8A–8H show the physical stability of a lentiviral vector lacking VSV-G protein as a function of free p24 released after increasing incubation times (hours) in the indicated formulation pH regulators, which vary in composition and pH. Figures 8A–8D show p24 associated with LV, and Figures 8E–8H show free p24 released from LV. Figure 9 shows the results of HEK293T cell transduction with a lentiviral vector lacking VSV-G protein and containing a GFP transgene as a function of polymer / vector R particle ratio, formulation buffer composition, and pH. Figure 10 shows the results of an experiment similar to that shown in Figure 9 but using a mixture of R / H polymers in which both polymers were either conjugated or not with cholesterol. Figure 11 shows the results of an experiment similar to that shown in Figure 9 but using a pseudotyped lentiviral vector containing a GFP transgene. Figure 12 shows the results of GFP expression and cell viability after transduction using encapsulated VSV-G+ lentivectors with the indicated PBAE polymers and polymer / cell molecule ratios, with and without VSV-G antibody. Data are normalized, with transduction in the absence of the antibody equal to 100%. Figures 13A-13D show the results of mCherry expression and cell viability after transduction using lentivectors VSV-G(bald) encapsulated using the indicated PBAE polymers and the polymer molecule / cell ratios. Figures 14A to 14F show the transduction results of non-encapsulated G+ VSV lentiviral vectors of human lymphocytes (14A), non-encapsulated bald LV (14B), PBAE-encapsulated bald LV (14C), and PBAE-encapsulated bald LV with a Fab PGA-anti-CD3 model targeting portion (14D), all analyzed by fluorescence-activated cell sorting (comparing CD3 antibody labeling and GFP expression). In Figure 14E, lymphocytes were also transduced with a CD19-specific CAR, and CD3-expressing cells are plotted against CD19-expressing CARs. In Figure 14F, the same lymphocytes that were used for the experiment depicted in Figure 9E were brought into contact with human CD19+ cancer cells (Ramos cells) and cytotoxicity against the cancer cells was assessed by the loss of CD19+ cells after 4 hours. Figure 15 shows the results of transduction (GFP expression) in different mouse PBMC populations after treatment with each of the indicated vectors and titers. Detailed description Packaged viral vector nanoparticle compositions are provided that enable enhanced delivery of genetic material for gene therapy and vaccine applications. These compositions include a viral vector encapsulated by a polymer or a mixture of polymers. The polymer coating provides protection for the viral vector particle in the bloodstream and tissues after parenteral administration to a subject and, in some formulations, can aid in delivering the vector to its target cell. Figures 1A and 1B show two embodiments of viral vector nanoparticles, also referred to herein as gene delivery vehicles, according to the present technology. The basic structure of the vehicle is that of nanoparticle 10, which contains a retroviral vector encapsulated by the polymer shell 11. The retroviral vector includes the membrane envelope 12, the protein capsid 13 arranged within the envelope, and one or more nucleic acid molecules 14 arranged within the capsid. The viral envelope is a lipid bilayer-based structure that, in the embodiment shown in Figure 1B, contains one or more types of viral envelope or fusion proteins 20 embedded within or attached to the bilayer membrane; such envelope or fusion proteins are absent in the embodiment shown in Figure 1A.The viral vector envelope, with or without envelope proteins, is encapsulated, both functionally and essentially completely, by the polymer matrix 11. The vector particle may be encapsulated by more than one polymer coat (see, for example, Figure 2), i.e., it may be encapsulated by a multilayer polymer coat. Figure 3 shows one modality in which the targeting portion 40 is associated with the polymer coat. In some embodiments, this technology provides a new generation of synthetic viral vectors, particularly lentiviral vectors, in which the viral vector particles lack a viral envelope protein. This new platform technology offers several advantages compared to using viral vector particles containing an envelope protein, including cheaper production of viral vector particles by transient transfection. 1) Because the plasmid encoding the viral envelope protein is removed and the vector does not transduce the producing cells during production, the elimination of the envelope protein increases the useful production time and, therefore, the yields of the vectors produced.2) Vectors lacking envelope protein are safer for use in in vivo gene therapy because uncoated vectors, which lack a viral envelope protein, cannot transduce cells and have reduced immunogenicity. 3) Vectors lacking envelope protein provide greater precision and efficiency of transduction due to the use of polymer-mediated targeting. 4) Polymer-encapsulated viral vectors, particularly lentiviral vectors, are more stable at low pH than normal (uncoated) viral vectors, improving their stability and effectiveness, especially when endosomal uptake mechanisms are involved.The removal of viral envelope protein from viral vector particles is particularly compatible with the use of polymer-coated viral vector particles, as it essentially eliminates the ability of uncoated vector particles to transduce cells, and the polymer coating can provide a specific target; therefore, the risk of transducing untargeted cells is drastically reduced. Furthermore, the removal of viral envelope protein promotes the close association of the polymer molecules with the vector envelope surface, providing better stability and more reliable targeting compared to polymer-coated viral vector particles containing viral envelope proteins. To produce the nanoparticulate form of the vector, the polymer molecules must be firmly attached to the vector particles. Loose association of the polymer molecules with the vector can lead to delamination and loss of the polymer coating from the surface of the viral vector particles, resulting in a loss of activity. Retrovirus-based viral vectors generally possess a core structure, or capsid, containing a transgene and other necessary nucleic acid sequences. This core structure is surrounded by a membrane envelope. The envelope typically contains a viral fusion protein that facilitates the attachment and fusion of the viral envelope to the host cell membrane, enabling the viral vector to enter the cell.However, in the embodiment of the present technology, the role of cell targeting, binding, and entry is assumed (and restored in the case of bald LV) by the polymer coat of the nanoparticulate vector particles, which can act at the cell uptake and / or endosomal level to transduce cells, making the viral fusion protein unnecessary. Furthermore, the presence of a viral fusion protein in the vector's envelope interferes with the firm and stable binding of the polymer molecules to the envelope. Therefore, in certain embodiments of the present technology, the viral vector nanoparticles have been genetically engineered to lack envelope proteins, particularly viral fusion or spike proteins.The result is a more robust nanoparticle structure and a more stable vector that exhibits better activity and a longer half-life when administered to a patient, such as by intravenous administration. Poly(beta-amino ester) nanoparticles In current technology, for a viral vector to lack envelope protein means that the vector's envelope, specifically its lipid bilayer, lacks any proteins typically found on the surface of enveloped viruses, whether in their natural or modified form, such as the Env protein complex or viral envelope glycoproteins. Examples include the HIV gpl20-gp41 complex, vesicular stomatitis virus (VSV-G) glycoprotein, influenza virus hemagglutinin, measles virus H and F proteins, and wild-type or modified Sindbis virus envelope protein. Such proteins, which are absent in the vector nanoparticles of the present technology, would project outward from the vector's envelope lipid bilayer and interfere with the required stable contact between the outer polymer layer and the lipid bilayer.In different embodiments, viral vector nanoparticles of the present technology may i) lack envelope proteins entirely, ii) essentially lack envelope proteins (i.e., contain only a trace amount such as 0.1% or less of typical viral vector particles), or iii) have a small amount of envelope protein, such as less than 10%, less than 5%, less than 3%, less than 2%, or less than 1% of the typical amount. A typical amount of envelope protein is an amount present in a viral vector particle that relies on the envelope protein for cell entry and transduction, or an amount found in the envelope of an intact, functional virus particle.The viral vector envelope can be engineered to be entirely devoid of envelope protein, for example, by removing any genes encoding an envelope protein during expression in the cells from which the vector is produced. However, membrane-embedded or membrane-bound proteins that do not form spike-like structures on the outer surface of the envelope may be present in the lipid bilayer of viral vector nanoparticles in certain modalities. In one embodiment, the polymers used to form the viral vector nanoparticle include one or more poly(beta-amino ester) (PBAE) species having the formula OO where Pep is an oligopeptide and R is OH, CH3 or cholesterol, a phospholipid molecule or a fatty acid or an acyl chain, where m preferably ranges from 1 to 20, n preferably ranges from 1 to 100, and i ranges from 1 to 10. Other polymers and mixtures thereof with each other and with PBAE may also be used to encapsulate viral vector particles. Additional coating mechanisms using lipids may also be used, such as in the form of unilamellar or multilamellar lipid liposomes or micelle-like structures that include surfactants and / or lipid molecules. Such coatings may be particularly useful for encapsulating viral vector particles that lack envelope proteins (bald vectors). Polybutylated amino acids (PBAEs) have been described as non-viral polynucleotide delivery vectors capable of condensing both DNA and RNA into discrete nanoparticles (Green, JJ et al. Acc. Chem. Res. 41, 749-759 (2008)), although they exhibit low transduction efficiency and high toxicity. PBAEs can be formed from the reaction of di(acrylate ester) monomers with amine monomers, resulting in polymers with terminal acrylate groups that can be functionalized with end modifications. End groups are functionalities or constitutional units located at the end of a macromolecule or oligomer. End modification of PBAEs allows for the enhancement of their biological properties. Chemical modification of PBAEs' terminals with primary amines, for example, results in greater transfection efficiency than acrylate-terminated polymers. Alternatively, PBAEs can be modified at the ends with oligopeptides, which can target the vector to particular cell types, depending on the specific oligopeptide sequence. An oligopeptide, as defined here, comprises a chain of at least two, or in some embodiments at least three, amino acids linked together by peptide bonds. Examples of suitably modified PBAEs are described in documents WO2014 / 136100 and WO2016 / 116887, which are incorporated herein by reference in full. In some embodiments, the oligopeptide comprises one, two, three, four, five, or more amino acids selected from the group consisting of arginine (R), glutamic acid (E), lysine (K), aspartic acid (D), histidine (H), and cisternae (C). In some embodiments, the oligopeptide contains one, two, three, or more amino acids that are positively charged (such as R, K, or H), or at least partially positively charged, under physiological conditions. In other embodiments, the oligopeptide contains one or more negatively charged amino acids (such as D or E), either in the absence of positively charged amino acids or mixed with positively charged amino acids. The data shown in the examples reveal that the inclusion of negatively charged amino acids improves the performance of bald lentiviral vectors, likely by enhancing endosomal leakage.The oligopeptides at each end of the polymer or polymers comprising the polymer mixture are preferably identical. The PBAE nanoparticle ensures that the vector remains intact during transit through the patient's circulation. The nanoparticle can help protect the viral vector from the host's immune system, for example, by ensuring that the claimed nanoparticles can maintain high transduction efficiency even in the presence of neutralizing antibodies against the viral envelope. Furthermore, the particle can ensure that the vector transfects only the desired immune cells: nanoparticle modifications and / or the presence of targeting portions can direct the nanoparticle specifically to the tissues of interest. Preferably, the PBAEs employed in the present technology have a molecular weight of 1,000 to 100,000 g / mol, very preferably 2,000 to 50,000 g / mol, and very preferably still 5,000 to 40,000 g / mol. The polymer layer or coating that covers the viral vector envelope may form a single homogeneous polymer layer, or it may form two or more overlapping layers of the same or different polymers, or polymers that have different net charge or degree of crosslinking. Other polymers that are not PBAE may be mixed with PBAE or added above or below a PBAE layer to form the polymer coating. The surface charge (or surface potential or zeta potential) of nanoparticles is important for tissue targeting and cell transduction and can be finely tuned through polymer modifications. Preferably, the zeta potential of nanoparticles under physiological conditions of use is in the range of approximately -30 mV to approximately +30 mV, or very preferably in the range of approximately -15 mV to approximately +15 mV. The polymeric nanoparticle may include one or more types of polymer. In some embodiments, the polymer coating forms a single-layer coating, a double-layer coating, or a multi-layer coating. Figure 2 shows one embodiment of the vector nanoparticle that includes the first layer 30, the second layer 31, and the third layer 32 of different polymer coatings encapsulating the retroviral vector.In some embodiments, the multilayer coating includes an anionic and a cationic polymer. In some embodiments, the cationic polymer includes one or more PBAEs. In some embodiments, the anionic polymer is polyglutamic acid (PGA), chondroitin sulfate, and / or hyaluronic acid. In certain embodiments, the viral vector is encapsulated within two or more polymer layers, such as alternating layers with alternating net electrical charges; that is, a positively charged layer is followed by a negatively charged layer, and then by a positively charged layer. In some embodiments, the net charge of the nanoparticle is positive at pH 7. In some embodiments, the net charge of the nanoparticle is negative at pH 7. In some embodiments, the net charge of the nanoparticle is tissue-dependent. In some embodiments, the particle is negatively charged, but in the tumor microenvironment, it becomes positively charged.In some modalities, the particle responds to the weakly acidic tumor microenvironment and the endo / lysosome through the disintegration or cleavage of certain chemical bonds, whereby the PBAE layer underneath is exposed and exerts a proton sponge effect. As used herein, the term nanoparticles refers to a solid particle with a diameter of approximately 1 to approximately 1000 nm. The average diameter of the nanoparticles of the present technology can be determined by methods known in the art, preferably by dynamic light scattering. In particular, the technology relates to nanoparticles that are solid particles with a diameter of approximately 1 to approximately 1000 nm when analyzed by dynamic light scattering at a scattering angle of 90° and a temperature of 25°C, using a sample suitably diluted with filtered PBS and a suitable instrument such as one of the ZETASIZER instruments from Malvern Instruments (UK).When a particle is said to have a diameter of x nm, there will generally be a distribution of particles around this mean, but at least 50% in number (e.g., > 60%, > 70%, > 80%, > 90%, or more) of the particles will have a diameter within the range x + 20%. The nanoparticle diameter can be from approximately 5 nm to approximately 999 nm, and preferably from approximately 50 nm to approximately 400 nm, most preferably from approximately 100 nm to approximately 300 nm. Alternatively, the nanoparticle diameter can be from approximately 10 nm to approximately 100 nm. In one embodiment, the nanoparticles exhibit a degree of agglomeration of less than 10%, preferably less than 5%, and preferably less than 1%. Preferably, the nanoparticles are substantially non-agglomerated, as determined by transmission electron microscopy or another method. Retroviral vectors Retroviruses are RNA viruses that can stably integrate into the host genome. In preferred embodiments, the retroviral vector in the gene delivery vehicle is a lentiviral vector. Lentiviral vectors are derived from lentiviruses, which are a genus of slow-moving viruses in the family Retroviridae. Lentiviruses include human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), equine infectious encephalitis virus (EIAV), caprine arthritis encephalitis virus (CAEV), bovine immunodeficiency virus (BIV), and feline immunodeficiency virus (FIV). Lentiviruses can integrate into the genome of both dividing and non-dividing cells. Furthermore, they can persist indefinitely in their hosts and replicate continuously. In some embodiments described here, the lentiviral vector has a capsid but lacks an envelope protein.Preferably, the vector is non-replicative in cells of the intended host or treatment subject. Preferably, the vector does not transduce or alter gene expression in non-target cells. Preferably, the vector lacks genes that cause virulence, particularly virulence manifested as inducing unwanted cell death in non-target cells. Lentiviral vectors offer several advantages as tools for immunotherapy. They are less toxic compared to other vectors and are capable of transducing non-dividing cells, including dendritic cells (He et al. 2007, Expert Rev Vaccines, 6(6):913-24). However, lentiviruses are more complex than other retroviruses due to the presence of accessory genes (e.g., vif, vpr, vpu, nef, tat, and rev), which are required for in vivo replication. The bioproduction of integrative but replication-defective lentiviral vectors relies on the separation of cis- and trans-action sequences of lentiviruses. Transduction in non-dividing cells requires the presence of two cis-action sequences in the lentiviral genome: the central polypurine tract (cPPT) and the central termination sequence (CTS). This leads to the formation of a triple-stranded DNA flap, which maximizes the efficiency of gene import into the nuclei of non-dividing cells, including dendritic cells (DCs) (Zennou et al., 2000, Cell, 101(2)): 173-85; Arhel et al., 2007, EMBO J, 26(12): 3025-2537). Furthermore, the removal of the LTR U3 sequence has resulted in self-inactivating vectors that are completely devoid of viral promoter and enhancer sequences and are safer. Lentiviral particles, which contain lentiviral vectors, can be produced by transient transfection of HEK 293 cells (with or without the T antigen, adherent or grown in suspension) using a combination of DNA plasmids, for example: (i) a packaging plasmid expressing Gag, Rol, Rev, Tat, and optionally structural proteins or enzymes required for packaging the transfer construct; (ii) a proviral transfer plasmid containing an expression cassette and cis-HIV action factors required for packaging, reverse transcription, and integration; and (iii) a plasmid encoding an envelope protein, such as vesicular stomatitis virus glycoprotein (VSV-G), a protein that allows the formation of mixed particles (pseudotypes) that can transduce a wide variety of cells, especially antigen-presenting cells (ARGs).including dendritic cells (DCs) and macrophages. In the present technology, the plasmid encoding an envelope protein is completely omitted in certain modalities, while in others it is included but its expression is maintained at a low level. Lentiviral particle vectors can also be continuously produced by stably inserting the packaging genes and the proviral coding DNA into the cellular genome. A combination of integrated plasmids and transient transfection can also be used. However, it should be noted that, in certain modalities, the lentiviral vectors suitable for use in the gene delivery vehicle described herein lack an envelope protein. Non-integrative lentiviral vectors lacking the envelope protein can also be used in gene transfer vehicles. Examples of non-integrative lentiviral vectors can be found in Coutant et al., PLOS ONE 7 (11): e48644 (2102); Karwacz et al., J. Virol. 83 (7): 3094-3103 (2009); Negri et al., Molecular Therapy 15 (9): 1716-1723 (2007); and Hu et al., Vaccine 28: 6675-6683 (2010). Because the 5'LTR of the proviral sequence lacks U3, transgene expression is activated by an internal promoter. Viral promoters, such as the CMV promoter, are preferably avoided due to the presence of strong enhancers. Ubiquitous promoters, such as those for human genes encoding ubiquitin, PGK, β-actin, GAPDH, β-kinesin, and similar genes, can be used. Alternatively, cell-active promoters can be used. IVIA / a / ZUZ I / UU I T and APC, such as the promoter for human MHC class I genes. In all cases, the promoter preferably does not contain an enhancer. The promoter is preferably selected to achieve a therapeutic level of transgene expression in the target cells. In some modalities, the promoter is specific to the target cells, i.e., it allows a therapeutic level of transgene expression in the target cells, and preferably allows a higher level of transgene expression in the target cells than in non-target cells. In certain modalities, the promoter allows little or no transgene expression in non-target cells. Encapsulation of viral vectors in polymer Previous technologies using polymer-encapsulated vehicles for cell transfection have involved plasmids or other nucleic acid molecules directly coated with polymers such as PBAE. Some of these technologies have achieved transfection using nucleic acid molecules (US2016 / 0145348A1, Mangraviti et al. 2015, Anderson et al. 2004, WO2016 / 116887). Poor stability of PBAE polymers at pH 7 has been reported (Lynn et al. 2000). Such preparations can cause cellular toxicity, resulting in cell death upon exposure to polymer-coated plasmids or other nucleic acid molecules. Therefore, the dose of the PBAE-encapsulated vector administered to cells in vivo or in vitro should be kept to the minimum necessary for useful transduction to avoid toxic effects. However, viral vectors encapsulated in PBAE advantageously resist degradation at low pH. To the inventors' knowledge, the encapsulation of viral vectors such as LV in polymers has not been previously achieved. The inventors have developed methods for encapsulating LV and other viral vectors in polymers that result in stable polymer-encapsulated LV particles with low toxicity and high cell transduction effectiveness. Important factors in the production of viable encapsulated vectors include the type of polymer used (e.g., its charge and charge distribution), the ratio of polymer molecules to vector particles, and the pH used for encapsulation. The effect of these parameters was investigated and is described in Examples 1 and 2 below. Using PBAE and LV polymers, successful encapsulation, resulting in particles capable of efficient transduction of target cells, requires approximately 108 to 1011 polymer molecules per vector particle; higher ratios may produce toxicity. Furthermore, the optimum pH for successful encapsulation differs depending on whether the LV contains VSV-G+ or not (VSV-G” or bald LV). For VSV-G+LV, optimum PBAE encapsulation occurs at approximately pH 5, as indicated by the high level of antibody-resistant VSV-G transduction at pH 5, while little or no encapsulation occurred at higher pH values. In contrast, for VSV-G” LV, optimum PBAE encapsulation efficiency occurs over a broader and lower pH range, such as approximately 5.0, 5.5, 6.0, or 6.5, or 5.0–6.5, or 5.0–6.0, or 5.5–6.0, or 5.5–6.5, whereas for VSV-G-LV, optimum PBAE encapsulation efficiency occurs over a lower pH range, such as approximately 5.0, 5.5, 6.0 or 6.5, or 5.0-6.5, or 5.0-6.0, or 5.5-6.0, or 5.5-6.5. See Example 2. Finally, the optimum pH for successful encapsulation that does not affect the viability of the transduced cells is the same whether the LV contains VSV-G+ or not, such as 5.0 or 5.5. Nanoparticles can be formed by mixing a solution comprising the required materials and then incubating the solution. For example, nanoparticles can be formed by mixing a solution comprising PBAE with a solution comprising the viral vector, and then incubating the solution. In some embodiments, the solution contains a sugar, such as glucose. In some embodiments, the solution contains 5% glucose. In some embodiments, the solution contains a pH regulator. In some embodiments, the pH regulator is sodium acetate, and the final concentration of sodium acetate pH regulator is 2 to 50 mM, 10 to 30 mM, or approximately 25 mM. In some embodiments, the pH regulator is Tris-HCl pH regulator, and the final concentration of Tris-HCl pH regulator is 2 to 40 mM, 5 to 30 mM, or 15 to 25 mM. In some embodiments, the solution is incubated for at least 5 minutes.In some formulations, the solution is incubated for at least 30 minutes. In some formulations, the solution is incubated overnight. The solution can be incubated between 4 and 40 °C, or between 17 and 25 °C; preferably, the solution is incubated at 4 °C. In some embodiments, nanoparticles are formed by layering ionic polymers onto retrovirus particles, thus forming multi-layered coated viral vectors. In other embodiments, a solution containing ionic polymers is contacted with a solution containing viral vector particles. The polymer-to-viral vector particle ratio can range from approximately 1:100 to approximately 5:1 by weight. This ratio can be varied to ensure that the nanoparticles contain, on average, one viral vector particle per polymer-coated nanoparticle. For example, the weight ratio of all polymers to viral vector can range from approximately 1:100 to approximately 1:1, or from approximately 1:50 to about 1:1, or from about 1:20 to about 1:1, or from about 1:10 to about 2:1, or from about 1:1 to approximately 5:1. In some formulations, one or more polymers are added to a suspension of retrovirus particles in glucose or another sugar compatible with lyophilization and direct injection after reconstitution in a human or other subject. In some formulations, negatively charged and positively charged polymers are added sequentially to form polymer layers with alternating charges. In some formulations, the polymers are added at 30-minute intervals. In some formulations, there are five polymer layers. In some formulations, multi-layered coated viral vectors have a net negative charge at pH 7. In some formulations, multi-layered coated viral vectors have a net positive charge at pH 7. The number and polarity of the layers can be chosen to optimize transduction efficiency for particular cell types of interest. Preferably, the nanoparticles are formed at a pH at which the surface charge of the viral vector particle, the surface potential, or the zeta potential is opposite to that of one or more polymers forming the polymer coat around the viral vector particle. In some embodiments, the pH is above the isoelectric point of one or more retroviral envelope proteins, or the envelope as a whole. For example, the pH may be equal to or greater than approximately 4.0, 4.5, 5.0, 5.4, 5.5, 5.6, 5.8, 6.0, 6.2, 6.4, 6.5, 6.8, or 7.0. In some embodiments, the nanoparticles are formed in a solution having a pH of approximately 5. Addressing portions The nanoparticle vector delivery vehicle can target specific cells or tissues requiring therapy, or where protein expression by the vector is desired. Another targeting modality is shown in Figure 3, where the delivery vehicle contains targeting portions covalently or non-covalently attached to the polymer surface. The targeting portions can be directed to specific cells or cell classes in vivo or in vitro. Examples of targeting portions include proteins (e.g., antibodies, including human, mouse, camelid, and shark antibodies, single-chain antibodies, nanobodies, diabodies, and antigen-binding antibody fragments), peptides, nucleic acids such as aptamers or oligonucleotides, or ligands that bind to cell-surface receptors or extracellular matrix components. In one modality, the delivery vehicle specifically targets T cells for the expression of one or more therapeutic proteins, such as chimeric antigen receptor (CAR) proteins, to enhance the destruction of cells (e.g., tumor cells or pathogens) to which the chimeric receptor binds. In a preferred modality, the target cells for the vector are T cells (such as CD3+ T cells) or NK cells. Targeting can be achieved using one or more DNA aptamers or antibodies that have specificity for a protein on the surface of the target cells. The aptamer or antibody serves as a targeting portion when integrated into the nanoparticle and exposed to the nanoparticle surface. For example, the targeting portion can specifically bind to CD3 or CD8, or a combination of CD3 and CD8.Alternatively, or in addition to this, the targeting portion can specifically bind to a surface protein found only on intact T cells, such as CD45RA, CCR7, CD62L, CD27, CD28, IL7-Ra, or DC-SIGN. Combinations of these targeting portions can also be used. However, targeting is not limited to CAR T-cell technology or hematologic cells. Any cell type can be targeted. For example, in certain gene therapy applications, a specific tissue or cell type (e.g., hematopoietic cells, myocytes, islet cells, etc.) can be targeted to repair or replace a defective function. In other applications, dormant cells can be directed to dividing cells to reduce the risk of recombination events. In still other applications, dividing cells, such as cancer cells, can be targeted, for example, in a cancer vaccine. Transgenes and their expression products The viral vector in the gene delivery vehicle described herein contains one or more transgenes that, after entering a cell, are expressed within the cell and produce expression products. The viral vector may contain one, two, three, or more transgenes. Transgenes are nucleic acid molecules that encode any natural or artificial (i.e., recombinant) protein, polypeptide, or peptide, including a therapeutic protein or a marker protein. Such proteins expressed in vectors preferably have therapeutic value in the subject in which they are expressed and are therefore therapeutic proteins. One type of therapeutic protein is a chimeric antigen receptor (CAR).The CARs used in the technology described herein are recombinant antigen receptors, such as T-cell receptors, with binding specificity for cell-surface antigens that do not require peptide processing or HLA expression to bind. The antigen-binding portion can be an antibody-derived scFv, a Fab selected from a library, or a natural ligand for a cell-surface receptor. It can also be a nanobody (VHH). The remainder of the CAR molecule couples the antigen-binding domain to intracellular signal transduction domains. Specifically, a CAR of the technology includes one or more of each of the following domains: an extracellular antigen-binding domain, a hinge and transmembrane domain, and intracellular signaling domains (CD3zeta and / or CD28 or 41-BB / OX40).Additional details of CARs suitable for use in the delivery vehicle for genes and sequences to encode them in a lentiviral vector can be found in document WO2016 / 012623A1, which is incorporated herein by reference in its entirety. Other genes that can be delivered using the gene delivery vehicles of this technology include interleukins and cytokines, such as interleukin 1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, GM-CSF, and G-CSF. Genes encoding antigens, such as viral, bacterial, fungal, or parasitic antigens, can also be delivered. Viruses whose antigens can be expressed include picornaviruses, coronaviruses, togaviruses, flaviviruses, rhabdoviruses, paramyxoviruses, orthomyxoviruses, bunyaviruses, arenviruses, reoviruses, retroviruses, papovaviruses, parvoviruses, herpesviruses, poxviruses, hepadnaviruses, and spongiform viruses. Preferred viral targets include influenza viruses, herpes simplex viruses 1 and 2, measles viruses, smallpox viruses, polio viruses, and HIV. Pathogens include trypanosomes, tapeworms, roundworms, and helminths.Tumor antigens, such as fetal antigen or prostate-specific antigen, can also be targeted in this manner. Administration of a vector using current technology for vaccination purposes can be carried out according to any known vaccination strategy. Vaccination of an individual can be performed according to any desired schedule; preferably, vaccination is only required infrequently, such as annually or biennially, and provides long-term immunological protection against the infectious agent. A viral vector transgene can also encode an anti-checkpoint protein. Examples of anti-checkpoint proteins that can be encoded by the transgene include inhibitors of CTLA-4, PD1, PDL1, LAG-3, TIM 3, B7-H3, ICOS, IDO, 4-1BB, CD47, and combinations thereof. Uses of gene delivery vehicles The gene delivery vehicles of this technology can be used in any application requiring the efficient transduction of genetic material into cells of interest, such as mammalian or human cells, using a retroviral vector, such as a lentiviral vector. The delivery vehicles transduce cells with high efficiency and exhibit low toxicity and low immunogenicity. They can permanently integrate genetic material into the host cell genome, making the system particularly suitable for the permanent labeling of cells and their progeny.Given their high specificity, low toxicity, and high transduction capacity even in vivo, these vehicles are particularly well-suited for gene transfer and gene therapy applications, both in humans and animals, as well as in animal models. This allows for the investigation of the molecular mechanisms underlying different biological functions and their translation into therapy. However, some preparations of polymer-encapsulated viral vector nanoparticles may be limited to transducing certain cell types, such as preferentially transducing dividing cells and not transducing non-dividing cells. The delivery vehicles of this technology can be used to genetically manipulate cells for biotechnological applications, such as the manufacture of biological products and improvements in livestock and plants. Precise and stable insertion of the gene or genes of interest is essential for these activities and can be achieved consistently with the technology's delivery vehicle. The vehicles can be used to insert genes that were not originally present in the cells used for manufacturing purposes, such as transformed cells (Chinese hamster ovary (CHO) cells, HEK293 cells) or primary cells. Alternatively, the vehicles can be used to inactivate the expression of certain genes present in these cells, such as genes involved in protein glycosylation, a common cause of immunogenicity.Vehicles can be used, for example, to prevent, treat or cure diseases that affect livestock, or to improve their economic value by accelerating growth and maturation. The gene delivery vehicle can be used to deliver genetic material as part of gene therapy in vitro or in vivo. In gene therapy, the gene delivery vehicle can be used to replace a disease-causing mutated version of a gene with a healthy copy, to inactivate or suppress the expression of a non-functioning gene, and / or to introduce a new gene into a cell, such as to combat a disease. The use of retroviral vectors, in general, allows for the permanent insertion of genetic material into a cell of interest. The use of lentiviral vectors, in particular, allows for the permanent insertion of genetic material even into non-dividing cells. Gene delivery vehicles are capable of transducing cells in vivo, leading to functional transgene expression, and can therefore be used to perform gene therapy. The vector delivery vehicle can be administered parenterally, such as intravenously, intramuscularly, or intratumorally. Alternatively, the vector delivery vehicle can be administered externally, such as orally or intranasally. The gene delivery vehicle can be administered by any other suitable route, including intracerebral and pulmonary. The gene delivery vehicles of this technology are also capable of transducing cells in vitro, leading to functional transgene expression, and can therefore be used for both in vitro and ex vivo gene therapy. Patient cells can be obtained (e.g., via a blood or bone marrow sample, or a biopsy) and introduced into one of the described vector delivery vehicles in a laboratory setting. The transduced cells can then be returned to the patient to help treat a disease. Diseases that can be treated with gene therapy using gene delivery vehicles include inherited disorders such as severe combined immunodeficiency (SCID), chronic granulomatous disease (CGD), Wiskott-Aldrich syndrome (WAS), relapsing acute lymphoblastic leukemia (ALL), adrenoleukodystrophy (ALD), hemophilia B, adenosine deaminase (ADA) deficiency, cystic fibrosis (CF), beta-thalassemia, sickle cell disease, Duchenne muscular dystrophy, familial hypercholesterolemia, and hereditary blindness. Treatable diseases also include neurodegenerative disorders such as Parkinson's disease, Alzheimer's disease, and Huntington's disease. Additionally, they include acquired diseases such as heart disease, diabetes, musculoskeletal disorders, and infectious diseases (e.g., influenza, HIV, hepatitis, among others).Other treatable diseases include cancer, such as prostate, pancreatic, brain, skin, liver, colon, breast, and kidney cancer. Example 1. Production of polymer-coated lentiviral vectors Polymer-coated lentiviral vectors for use in cell transduction studies were made using the following materials and methods. Materials The transfer vector plasmid was paraCMV-GFP or paraCMV-mCherry or para or para-hUBC-CDl9. A kanamycin-resistant plasmid encoded for the provirus (a non-pathogenic, non-replicating recombinant proviral DNA derived from HIV-1, strain NL4-3) was cloned into which an expression cassette was inserted. The insert contained the transgene, the promoter for transgene expression, and added sequences to enhance transgene expression and enable the lentiviral vector to transduce all cell types, including non-mitotic cells. The coding sequences corresponded to the gene encoding either green fluorescent protein (GFP), mCherry (red fluorescent protein), or CD19. The promoter was either the human ubiquitin promoter or the CMV promoter. It was devoid of any enhancer sequences and promoted gene expression at a high level in a ubiquitous manner.The non-coding sequences and expression signals corresponded to Long Terminal Repeat (LTR) sequences with the complete cis-active elements for the 5' LTR (U3-R-U5) and those removed for the 3' LTR, thus lacking the promoter region (AU3-R-U5). For transcription and integration experiments, encapsidation sequences (SD and 5'Gag), the Central Polypurine Tract / Central Termination Site for nuclear translocation of the vectors, and the BGH polyadenylation site were added. The packaging plasmid was para-Pack. The kanamycin-resistant plasmid encoded the structural lentiviral proteins (GAG, ROL, TAT, and REV) used in trans for the encapsidation of the lentiviral provirus. The coding sequences corresponded to a polycistronic gene gag-pol-tat-rev, which encoded the structures (Matrix MA, Capsid CA, and Nucleocapsid NC), enzymatic proteins (Protease PR, Integrase IN, and Reverse Transcriptase RT), and regulatory proteins (TAT and REV). The non-coding sequences and expression signals corresponded to a minimal CMV promoter for transcription initiation, a polyadenylation signal from the insulin gene for transcription termination, and an HIV-1 Rev-Sensitive Element (RRE) involved in the nuclear export of the packaging RNA. The envelope plasmid, when used, was pENVl. This kanamycin-resistant plasmid encoded the glycoprotein G of the Indiana strain of Stomatitis Virus. Vesicular (VSV-G), used for pseudotyping some lentiviral vectors. The VSV-G genes were codon-optimized for expression in human cells, and the gene was cloned into the pVAXl plasmid (Invitrogen). The coding sequences corresponded to the codon-optimized VSV-G gene, and the non-coding sequences and expression signals corresponded to a minimal CMV promoter for transcription initiation and the BGH polyadenylation site for RNA stabilization. Production of Lentiviral Vector Particles (bald) VSV-G LV293 cells were seeded at 5 x 10⁵ cells / ml into two 3000 ml Erlenmeyer flasks (Corning, 431252) containing 1000 ml of LVmax Production Medium (Gibco, A3583401). Both Erlenmeyer flasks were incubated at 37°C, 65 rpm under 8% humidified CO₂. The day after seeding, transient transfection was performed. PEIPro transectant reagent (PolyPlus, 115-010) was mixed with the transfer vector plasmid (pARA-CMV-GFP or pARA-CMV-mCherry or pARA or pArahUBC-CD19) and the packaging plasmid (pARA-Pack). After incubation at room temperature, the PEIPro / plasmid mixture was added dropwise to the cell line and incubated at 37°C, 65 rpm under 8% humidified CO2. On day 3, lentivector production was stimulated by sodium butyrate at a final concentration of 5 mM. The bulk mixture was incubated at 37°C, 65 rpm under 8% humidified CO2 for 24 hours. After clarification by deep filtration to 5 and 0.At 5 pm (Pall, 609-122U), the clarified bulk mixture was incubated for 1 hour at room temperature for treatment with DNase. The lentivector was purified by chromatography (Mustang Q membrane) and eluted by NaCl gradient. Tangential flow filtration was performed on a 100 kDa HYDROSORT membrane (Sartorius, 3M81446802E-SW), allowing for volume reduction and formulation in a specific pH buffer at pH 7, ensuring at least two years of stability. After sterile filtration to 0.22 µm (Millipore, SLGVM33RS), the bulk drug product was filled into 2 mL glass vials with aliquots of less than 1 mL, then labeled, frozen, and stored at <-70°C. The number of bald LVs was assessed by physical titer quantification. The assay was performed by detection and quantification of the lentivirus-associated HIV-1 core protein p24 only (Cell Biolabs P24 ELISA Lentivirus Titer QUICK TITER Kit). Production, purification and quantification of polymer 5 PBAE Poly(p-amino ester) polymers were synthesized following a two-step procedure similar to that described by Dosta et al. 5-Amino-l-pentanol (Sigma-Aldrich 3.9 g, 38 mmol) and 1-hexylamine (Sigma-Aldrich 3.9 g, 38 mmol) were mixed in a round-bottom flask and combined with 1,4-butanediol diacrylate (Aldrich 18 g, 82 mmol) in a 2.1:1 molar ratio of diacrylate to amine monomers. The mixture was heated to 90°C with stirring for 20 hr. It was then cooled to room temperature to form a light yellow viscous solid oil and stored at -20°C until further use. The synthesized structures were confirmed by 1H-NMR spectroscopy. NMR spectra were recorded on a 400 MHz Varian NMR instrument (Varian NMR Instruments, Clarendon Hills, IL) using d4 methanol as the solvent. Molecular weight determination was performed on an Elite LaChrom HPLC system (VWR-Hitachi) equipped with a SHODEX KF-603 GPC column (approximately 6.0 x 150 mm) and THF as the mobile phase. Molecular weight was calculated by comparing it to the retention times of polystyrene standards. The weight-average molecular weight (Mw) was 4400, and the number-average molecular weight (Mn) was 2900. Oligopeptide-modified PBAEs were obtained by final modification of the acrylate-terminated C6 polymer with thiol-terminated oligopeptide at a molar ratio of 1:2.5 in dimethyl sulfoxide (DMSO). The following synthetic procedure for obtaining the tri-arginine-modified PBAE polymer, C6-CR3, is shown as an example: A solution of C6 (113 mg, 0.054 mmol) dissolved in DMSO (1.1 mL) and a solution of H-Cys-Arg-ArgArg-NHz hydrochloride (CR3-95% purity - acquired from Ontores) (99 mg, 0.13 mmol) in DMSO (1 mL) were mixed in a Teflon-coated screw-cap vial and shaken in a water bath at a controlled temperature of 20°C for 20 hr. The capped polymer was precipitated with 5 volumes of diethyl ether. After centrifugation (10 minutes at 3000 g), the polymer was washed with 2 volumes of fresh mixture of diethyl ether and acetone (7:3 v / v), then the residue was vacuum dried before forming a 100 mg / ml supply in DMSO, which was stored at -20°C. In another example, the tri-lysine-end-modified PBAE polymer, C6-CK3, was obtained by mixing a solution of C6 (100 mg, 0.048 mmol) dissolved in DMSO (1.1 mL) and a solution of H-Cys-Lys-Lys-Lys-NH2 (CK3) hydrochloride (78 mg, 0.12 mmol) in DMSO (1.8 mL) and processing the coupling reaction as previously described for C6-CR3. For the trihistidine-end-modified PBAE polymer, C6-CH3, a solution of C6 (65 mg, 0.031 mmol) dissolved in DMSO (1.1 mL) was mixed with a solution of H-Cys-His-His-His-NH2 (CH3) hydrochloride (53 mg, 0.078 mmol) in DMSO (1.2 mL). The PBAE polymer modified at the ends with tri-aspartate, C6-CD3, the C6 solution (96 mg, 0.046 mmol) dissolved in DMSO (1.1 mL) that was mixed with an H-Cys-Asp-Asp-Asp-NHz (CD3) hydrochloride solution (96 mg, 0.046 mmol) in DMSO (1.7 mL). Finally, the tri-glutamate-modified PBAE polymer, C6-CE3, the C6 solution (280 mg, 0.13 mmol) dissolved in DMSO (1.1 mL) was mixed with an H-Cys-Glu-GluGlu-NH2(CE3) hydrochloride solution (184 mg, 0.34 mmol) in DMSO (4.6 mL). The chemical structure of the oligopeptide-modified PBAEs was confirmed by 1H-NMR spectroscopy by the disappearance of acrylate signals and the presence of signals generally associated with amino acid segments. The residual free peptide content was quantified by UV detection (wavelength 220 nm) after separation using the ACQUITY UPLC system (Waters) equipped with a BEH C18 column (130 Å, 1.7 pm, 2.1 x 50 mm, temperature 35°C) using an acetonitrile gradient. Fab-PGA Production Polyglutamic acid (PGA) of 15 kDa (Alamanda Polymers) was dissolved in MES pH 6.0 pH buffer at 20 mg / mL and then sonicated for 10 minutes in a sonicator bath. 1.8 mL of PGA solution was added to 0.9 mL of ethyl-N'-(3-dimethylaminopropyl)carbodiimide-HCl (4 mg / mL, 16 equiv.) and 0.9 mL of sulfo-NHS (2.75 w / w EDC) in both the MES pH 6.0 pH buffer and the solution at room temperature for 15 minutes. The resulting activated PGA was added to a mouse anti-CD3e F(ab')2 fragment solution (InVivoMAb) in phosphate-buffered saline (PBS) at a 4:1 molar ratio and mixed at room temperature for 2 hours. Excess reagents and unbound PGA were removed by filtration (30,000 MWCO Vivaspin column), and the pH buffer was exchanged 7 times against PBS. Antibody concentration was determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific). Stability kinetics of VSV-G+LV as a function of pH 10 μA of drug pseudotyping lentivectors or bald lentivectors were diluted in 10 μA of different pH regulators (pH 5 to 7) for different times (0, 15, 30, 60, 90 and 120 minutes) at +4°C. The titration of the pseudotyping lentivectors was performed by flow cytometry as follows. HEK293T cells (8 × 10⁵ cells / well) were seeded into 24-well plates and incubated at 37°C in a humidified atmosphere with 5% CO₂. After 4 hours, the culture medium was removed and the cells were added to each well. 300 μA of fresh DMEM containing 10% FBS, 1% PS, and 10 μA of each condition. After 2 hr, each well was supplemented with 0.5 ml of fresh culture medium. Three days post-infection, HEK293T cells in each well were trypsinized, fixed (BD CellFIX solution # 340181), and the number of fluorescently positive cells was determined using flow cytometry (AttuneNXT; Invitrogen, Inc.). The following formula was used to convert the percentage of cells expressing GFP for a specific dilution in TU: TU / ml = (% of cells expressing eGFP / 100) χ total number of HEK293T cells at the time of infection / volume of aggregated virus supply (mi). The titration of bald lentivectors was performed by physical titer quantification. The assay was conducted by detection and quantification of the lentivirus-associated HIV-1 core protein p24 (using the Cell Biolabs QUICK TITER P24 ELISA lentivirus titration kit). Pretreatment samples allowed for the differentiation of free p24 from destroyed lentivectors. Encapsulation of VSV-G+LV and bald LV with oligopeptide-modified PBAE Lentiviral vector encapsulation was performed as follows. The lentiviral vectors were diluted in 25 mM citrate pH buffer, pH 5.0 (or in an appropriate pH buffer system at a different defined pH) to prepare a final volume of 75 pL per replicate. PBAE polymers were diluted in the same pH buffer as the lentiviral vectors (75 μL per replicate) and vortexed for 2 s to ensure homogenization. The diluted polymers were added to the diluted vectors in a 1:1 (v / v) ratio, the mixtures were gently swirled for 10 seconds, and incubated for 30 minutes at ±4°C. Finally, an equal volume of culture medium (150 pL) was added to the encapsulation reactions before transferring them to the cells. VSB-G+LV coating encapsulated in PBAE and bald LV with PGA-Fab The encapsulation method described above was modified as follows. Lentiviral vectors were diluted in 25 mM citrate pH 5.0 buffer (or in an appropriate pH buffer system to a defined pH) to prepare a final volume of 75 pL per replicate. PBAE were diluted in the same pH buffer as for the lentiviral vectors (75 pL per replicate) and vortexed for 2 s to ensure homogenization. The diluted polymers were added to the diluted vectors in a 1:1 (v / v) ratio. The mixtures were gently swirled for 10 s and incubated for 30 min at 4°C. PGA-Fab polymer diluted in 25 mM citrate pH 5.0 buffer (or in an appropriate pH buffer system to a defined pH) was added to the encapsulation mixture and gently swirled for 10 s to ensure homogenization. The coating reaction mixture was incubated for 30 minutes at +4°C and the volume was made up to 300 pL with culture medium before being transferred to the cells. Example 2. Potential Zeta measurements of lentiviral vectors encapsulated in PBAE. Surface charge measurements of LV / PBAE / PGA-Fab complex particles were determined by dynamic light scattering (DLS) at room temperature using a Zetasizer Nano ZS (Malvern Instruments Ltd, UK, 4 mW laser) at a wavelength of 633 nm. Measurements were performed by mixing 200 pL of complexes with 800 pL of D-PBS Ix pH buffer solution (pH 7.1). The results were plotted as mean and standard deviation of three intensity-analyzed measurements and are shown in Figures 6A–6F. The results showed potential surface modification through encapsulation of the vector particles with the polymer according to the polymer charge, indicating successful encapsulation with the polymer. Example 3. Transduction of HEK293T cells by polymer-coated lentiviral vectors. To test the effect of polymer coating on the transduction capacity of lentiviral vectors, peptide-conjugated poly(beta-amino ester) polymers were prepared and used to coat lentiviral vectors and test HEK293T cell transduction. The lentiviral vectors carried a green fluorescent protein (GFP) transgene, and transduction was quantified by GFP expression in the transduced host cells (see Figure 4A). Peptides with different charged amino acid groups were conjugated to the polymer backbone to test the effect of charge. Vector particles with and without VSV-G protein in their envelopes were coated with the polymer.The effectiveness of polymer encapsulation was assessed by determining the transduction capacity of the vectors in the presence of antibodies against VSV-G, which can block transduction dependent on exposed VSV-G proteins (see Figure 4B), but does not block transduction by the vector that is effectively encapsulated in the polymer and does not rely on exposed VSV-G for transduction (see Figure 4C). Similar experiments were performed with VSV-G-deficient LVs; this strategy is depicted in Figures 5A–5C. These bald LVs were unable to transduce due to the lack of VSV-G protein, but became transducible after encapsulation with PBAE polymers. The polymer-enabled transfection was not expected to be blocked by the anti-VSV-G antibody. The encapsulated vectors were prepared as follows. Vector dilutions were prepared in a first set of 1.5 mL microtubes to a final volume of 150 μL per replicate, and polymer dilutions were prepared in a second set of 1.5 mL microtubes to the same final volumes. The diluted polymer was then added to the diluted vector, and the mixture was homogenized by gentle pipetting to avoid bubbles or foam. The encapsulation mixtures were incubated for 30 minutes at ±4°C. For transduction quantification, cells were seeded in 24-well plates at a density of 8 x 10⁴ cells per well in complete medium containing 10% FBS and incubated for 4 hr to adhere. To test for neutralization of transduction by the anti-VSV-G antibody, 300 µA of encapsulated vector was mixed with 300 µA of complete cell culture medium or with 300 µA of polyclonal rabbit anti-VSV-g serum diluted 100-fold in complete cell culture medium, and incubated for 30 min at 37°C, 5% CO₂. The cells were then transduced by replacing the medium with 300 μA of vector in antibody neutralization medium or culture medium lacking antibody, followed by incubation at 37°C, 5% CO2 for 2 hr. After adsorption, 1 ml of complete medium was added to each well.At 72 hr after transduction, the cells were trypsinized and resuspended in 200 pL of Cellfix IX, and the percentage of cells expressing GFP was determined using an Attune NxT flow cytometer using the BL1 channel. The polymers used in the experiments of MA / a / ZUZ 1 / UU I4ZJ encapsulation polymers were poly(beta-amino ester) (PBAE) polymers as shown in the following formula, conjugated with charged peptides (Pep) and optionally cholesterol (R). Polymer R refers to PBAE conjugated with peptide CRRR (same peptide at both ends). Polymer H refers to PBAE conjugated with peptide CHHH. Polymer E refers to PBAE conjugated with peptide CEEE. Polymer D refers to PBAE conjugated with peptide CDDD. Mixtures of the peptides were tested in different ratios indicated in the following tables. R / H refers to a 60 / 40 mixture of polymers R and H. Polymers indicated with col were also conjugated with cholesterol as the R group indicated in the structure below. The m value was 3 and the o value was 1. The following tables show the polymer blends and the ratio of polymer molecules per LV particle used for encapsulation and tested for transduction capability. Table 1. Polymer coatings used to test the effect of loading Influence of load on LV coating (HEK293T) Ia. Series: Test of negatively charged polymers (with pilot batch LV-GFP) Polymer R mixed with polymer E: 100 / 0 - 66 / 33 - 33 / 66 - 0 / 100 Polymer R-col mixed with polymer E-col: 100 / 0 - 66 / 33 - 33 / 66 - 0 / 100 Polymer R mixed with polymer E-col: 100 / 0 - 66 / 33 - 33 / 66 - 0 / 100 Polymer R mixed with polymer E: 100 / 0 - 66 / 33 - 33 / 66 - 0 / 100 Test on 3 polymer / LV ratios: IO9, 1010 and 10--, + / - antibody Table 2. Other polymer coatings used to test the effect of loading Improvement of LV coating with D and K polymers (HEK293T) 2a. Series: If negatively charged, polymers improve encapsulation (with batch LV- Polymer Polymer Polymer Polymer Polymer Polymer Polymer Polymer Pilot GFP Test) R mixed with polymer E: R mixed with polymer D: R-col mixed with polymer E-col: R-col mixed with polymer D-col: K mixed with polymer E: K mixed with polymer D: K-col mixed with polymer E-col: K-col mixed with polymer D-col: On the best polymer / LV ratio: + / - 100 / 0 - 66 / 33 100 / 0 66 / 33 100 / 0 - 66 / 33 100 / 0 - 66 / 33 100 / 0 - 66 / 33 100 / 0 - 66 / 33 100 / 0 - 66 / 33 100 / 0 - 66 / 33 antibody - 33 / 66 - 0 / 100 33 / 66 0 / 100 - 33 / 66 - 0 / 100 - 33 / 66 - 0 / 100 - 33 / 66 - 0 / 100 - 33 / 66 - 0 / 100 - 33 / 66 - 0 / 100 Table 3. Polymer coatings used with LV without VSV-G. Evaluation of LV coating without VSV-G (HEK293T) 2nd Series: If negatively charged, the polymers do not improve the encapsulation test of LV without VSV-G (with pilot LV-GFP batch and UC batches of LV GFP + / - VSV G) R / H polymer mixed with polymer E: 100 / 0 - 66 / 33 - 33 / 66 - 0 / 100 R / H polymer mixed with polymer D: 100 / 0 - 66 / 33 - 33 / 66 - 0 / 100 R / H-col polymer mixed with polymer E-col: 100 / 0 - 66 / 33 - 33 / 66 - 0 / 100 R / H-col polymer mixed with polymer D-col: 100 / 0 - 66 / 33 - 33 / 66 - 0 / 100 Test on 3 polymer / LV ratios: 10% 10'iCy 10:% ​​+ / - antibody In order to test the effect of polymer coating on the transduction capacity of polymer-coated lentiviral vectors lacking viral envelope protein, peptides having amino acids with different charges were conjugated to the polymer backbone, and the resulting polymers were used to coat lentiviral vectors and test HEK293T cell transduction. GFP expression data were obtained from flow cytometry results after transduction with vectors having the indicated polymer-to-lentiviral vector (LV) particle ratios. In each condition, the addition of polyclonal anti-G-syndrome rabbit serum reduced the transduction rate by a ratio of 10⁹, but the antibody failed to reduce transduction by ratios of 10¹⁰ and 10¹¹, indicating that at least approximately 10¹⁰ polymers per LV particle were required for effective encapsulation. The data also confirmed that polymer-encapsulated LV was able to perform transduction effectively, and that the positively charged polymer was more effective, with transduction efficiency decreasing with increasing negative charge in the polymer. A similar experiment was conducted using cholesterol-conjugated PBAE polymer. The results were similar to those obtained for the non-cholesterol-conjugated polymer, for both polymer R and polymer E. A similar experiment was conducted using LV lacking the VSV-G envelope protein (bald LV). The results indicated that uncoated bald LV failed to induce transduction. However, polymer-encapsulated bald LV was able to effectively transduce cells at a polymer / LV molecule ratio of 10⁹, 10¹⁰, or 10¹¹. As expected, the addition of anti-VSV-G antibodies had no effect. Positively charged polymers were much more effective than neutral or negatively charged polymers. Similar results were obtained using cholesterol-conjugated polymer, and also for polymer mixtures containing R + H and D peptides, with or without cholesterol. Example 4. Effect of pH during polymer coating of lentiviral vectors ± VSV-G. PBAE-coated LVs lacking or expressing VSV-G were prepared, and their transduction into Jurkat cells was performed as described in Example 3, except that the cells were cultured in suspension and the pH of the coating solution was varied. Acetate pH buffer was used at pH 4.5–6, citrate pH buffer at pH 4.5–6, histidine pH buffer at pH 6–7, and Tris pH buffer at pH 7–8. At each pH condition, the number of polymer molecules per LV particle was varied from 10⁶ to 10¹⁰. The stability of VSV-G + LV is shown in Figure 7. The vectors were stable in the pH range of 4.5–8.0 and could be maintained in these pH ranges for up to 120 minutes. Figures 8A–8H show the results of an experiment to test the stability of bald LV particles in different pH buffers without polymer encapsulation. For VSV-G+ LV, integrity was assessed by measuring its ability to transduce HEK293T cells and induce GFP expression (detected by FACS). For VSVG- LV (bald), integrity was assessed by measuring the release of the p24 protein (an indicator of vector particle lysis; p24 was measured in the supernatants by ELISA), since transduction was not possible using uncoated bald LV. Physical integrity was determined after 15, 30, 60, 90, and 120 minutes in the different pH buffers. The results indicated that there was no loss of integrity for any of the LV types up to 120 min, although higher pH conditions were slightly less favorable for LV viability than lower pH conditions.The raw data for the retention and release of LV calvo p24 are shown in Figures 8A-8H. Figure 9 shows the results for the encapsulation of bald LV at different pH values, followed by HEK293T cell transduction and GFP expression detected by FACS. As expected from other experiments described in Example 1, complete polymer coating of bald LV, resulting in transducible LV, required at least approximately 10⁹ molecules of PBAE per LV particle to obtain viable particles. A clear pH dependence was observed for the coating process. A higher pH (pH 7) yielded virtually no infectious LV, while coating at pH in the range of approximately 5.0 to approximately 6.5 gave good transduction results. The R / H derivative PBAE (PBAE conjugated with CRRR peptides mixed with PBAE-CHHH in a 60 / 40 vol / vol ratio, see Example 1) was used for coating in this experiment.Significantly, Figure 9 also shows that partial polymer encapsulation results in a lack of transduction capability, demonstrating that using viral vectors lacking envelope protein will improve the safety profile. Without this feature, vector particles whose polymer coating is partially or completely lost or partially degraded, or vector particles that have been incompletely coated during production, could pose a safety problem due to the potential transduction of non-target cells. Figure 10 shows the results of an experiment similar to that depicted in Figure 9, but using a (60 / 40) mixture of the polymers R-cholesterol-PBAE and H-cholesterol-PBAE. The addition of cholesterol reduced the polymer particle / LV ratio required to completely encapsulate the bald LV at low pH. Figure 11 shows an experiment testing the coating of VSV-G+ LV at different pH values. The polymer R-PBAE was used for encapsulation, and sensitivity to anti-VSV-G+ antibody was also tested. The results indicate low pH sensitivity to overall transduction rates, but sensitivity to antibodies (indicative of incomplete polymer coating) was higher at pH 6.5 than at lower pH values. Further evidence of more competitive coating at lower pH is seen in the higher basal transduction in the presence of anti-VSV-G+ antibody at pH 5. ML / a / ZUZ 1 / UU 14ZJ Example 4. Lymphocyte transduction using encapsulated lentiviral vectors ± VSV-G. Transduction of Jurkat cells and human lymphocytes For transduction quantification, Jurkat cells were seeded in 24-well plates at a density of 8 x 10⁴ cells per well in complete medium containing 10% FBS and 1% penicillin / streptomycin. 300 pL of encapsulated vector were added to the cells. After 2 h of incubation at 37°C, 5% CO₂, 500 pL of fresh complete medium was added to each well. The percentage of cells expressing GFP or mCherry transgenes was determined 72 h post-transduction using an Attune NxT flow cytometer (Thermo Fisher) with the BL1 or YL1 channel, respectively. The phenotype of transduced cells expressing the GFP transgene was determined by flow cytometry staining with different specific antibodies for the following cell types following the manufacturer's instructions (BD Biosciences): CD3 (T lymphocytes) and CD19 (B lymphocytes). Human lymphocyte transduction was performed using the same method, except that 105 cells / well were seeded. Cytotoxicity Human lymphocytes were seeded in 24-well plates at a density of 5 x 10⁵ cells per well in complete medium containing 10% FBS and 1% penicillin / streptomycin. 300 pL of encapsulated CAR CD19 expression vector was added to the cells. After 2 h of incubation, 500 µA of fresh complete medium was added to each well. After 48 h of incubation at 37°C, 5% CO₂, CD19-positive Ramos humoral cells were added at a lymphocyte-to-tumor cell ratio of 50:1 or 1:1. After 4 hr of incubation at 37°C, 5% CO2, CAR CD19-expressing cells were stained with protein L and detected using an ATTUNE NXT flow cytometer (Thermo Fisher) with the YL1 channel. CD19-positive cells and lymphocytes were quantified by flow cytometry staining with specific CD19 or CD3 antibodies (BD Biosciences) according to the manufacturer's instructions. Mouse PBMC preparation and transduction Freshly prepared mouse peripheral blood mononuclear cells (PBMCs) were isolated from heparinized whole blood using LEUCOSEP tubes (Greiner bio-one) filled with FICOLL-PAQUE PREMIUM 1.084 (GE Healthcare). After blood dilution with DPBS, the PBMCs were separated on a FICOLL density gradient, washed twice with DPBS, and resuspended to the desired cell density in culture medium. Cells were seeded in 24-well plates at a density of 2 x 10⁵ cells per well in RPMI medium containing 10% FBS and 1% penicillin / streptomycin. 300 pL of encapsulated vector were added to the cells. After 2 h of incubation at 37°C, 5% CO₂, 500 pL of fresh complete medium was added to each well. The percentage of cells expressing GFP was determined 48 h post-transduction using an Attune NxT flow cytometer with channel BL1. The phenotype of transduced cells expressing the GFP transgene was determined by flow cytometry staining with different specific antibodies for the following cell types following the manufacturer's instructions (BD Biosciences): CD4 (helper T lymphocytes), CD8 (cytotoxic lymphocytes), CD19 (B lymphocytes), Ly-6G (granulocytes), CDllb (macrophages), CDllc (dendritic cells) and F4 / 80 (monocytes). Figure 12 shows GFP expression and cell viability of Jurkat cells transduced with the LV encapsulated in the indicated PBAE in the presence and absence of anti-VSV-G antibody. Results are normalized to antibody-negative conditions as 100%. A wide variety of encapsulation polymers and polymer / vector ratios failed to block antibody inhibition, indicating that the VSV-G protein protruded through the polymer coating under all tested and mediated transduction conditions. In Figures 13A-13D, mCherry expression is shown as a measure of Jurkat cell transduction by bald LV encapsulated by the indicated PBAE polymers, with and without cholesterol conjugation, and in a variety of different polymer / vector ratios and net polymer charge. The results of human lymphocyte transduction using different vectors are shown in Figures 14A–14D. The number of vector particles per lymphocyte increases from left to right in each figure. Figure 14A shows the results obtained for typical VSV-G+LV; little transduction was observed, as was also the case for the unencapsulated VSVG→ ...The experiment whose results are shown in Figure 14E demonstrates that additional transduction of human lymphocytes with a CD19-specific CAR was compatible with strong transduction of the LV transgene. Furthermore, the results shown in Figure 14F reveal that human lymphocytes transduced with CD19-specific CARs were capable of cytotoxic action against CD19-positive cancer cells. Figure 15 shows the results of experiments in which mouse PBMCs were transjected with non-encapsulated LV containing VSV-G+ (two bars to the left) or bald LV encapsulated in PBAE (two bars to the right). The encapsulated bald LV showed much stronger transduction than the VSV-G+ vectors for each cell type present in the PBMC preparation. References Nayerossadat, Maedeh, T, Ali, PA. Viral and nonviral delivery systems for gene delivery, Adv Biomed Res, 2102, 1:27. Green, JJ Langer, R, y Anderson, DG, A. Combinatoria! Polymer Library Approach Yields Insight into Nonviral Gene Delivery, Acc. Chem. Res, 2008, 41 (6), pp 749759 . He Y, Munn D, Falo LD Jr. Recombinant lentivector as a genetic immunization vehicle for antitumor immunity, Expert Rev Vaccines, Dic de 2007; 6(6):913-24. Zennou V, Petit C, Guetard D, Nerhbass U, Montagnier L, Charneau P. HIV-1 genome nuclear import is mediated by a central DNA flap, Cell. 14 de Abril de 2000; 101 (2) :173-85. Arhel NJ, Souquere-Besse S, Munier S, Souque P, Guadagnini S, Rutherford S, Prévost MC, Alien TD, Charneau P. HIV-1 DNA Flap formation promotes uncoating of the preintegration complex at the nuclear pore, EMBO J. 20 de Junio de 2007 26(12) : 3025-37 . Coutant F, Sánchez David RY, Félix T, Boulay A, Caleechurn L, Souque P, Thouvenot C, Bourgouin 0, Beignon AS, Charneau P. A nonintegrative lentiviral vector-based vaccine provides long-term sterile protection against malaria, PLoS One. 2012; 7 (11) . Karwacz K, Mukherjee S, Apolonia L, Blundell MP, Bouma G, Escors D, Collins MK, Thrasher AJ. Nonintegrating lentivector vaccines stimulate prolonged T-cell and antibody responses and are effective in tumor therapy, J Virol. April 2009; 83(7):3094-103. Anderson, DJ, Proc. Nati. Acad. Sci. USA 2004, 101:16028-33 . Lynn DM, Langer, R, J. Am Chem Soc 2000, 122:1076110768. Mangraviti, et al., ACS Nano 2015, 9:1236-1249. Negri DR, Michelini Z, Baroncelli S, Spada M, Vendetti S, Buffa V, Bona R, Leone P, Klotman ME, Cara A. Successful immunization with a single injection of non-integrating lentiviral vector, Mol Ther. September 2007; 15(9) : 1716-23. Hu B, Dai B, Wang P. Vaccines delivered by integration-deficient lentiviral vectors targeting dendritic cells induces strong antigen-specific immunity, Vaccine. September 24, 2010; 28(41):6675-83. Montenarh M. Biochemical properties of the growth suppressor / oncoprotein p53. Oncogene. September 1992; 7 (9):1673-80. As used herein, "essentially" permits the inclusion of materials or steps that do not materially affect the basic and novel features of the claim. Any mention herein of the term "comprising," particularly in a description of components of a composition or in a description of elements of a device, may be replaced by "essentially" or "consisting of." Although the present technology has been described together with certain preferred modalities, a person skilled in the art, after reading the above specification, may make various changes, substitutions of equivalents, and other alterations to the compositions and methods set forth in this document.

Claims

1. A nanoparticle comprising a viral vector coated with a polymer or a mixture of polymers, wherein the viral vector comprises a transgene, and wherein the nanoparticle functions as a vehicle for delivering the transgene to eukaryotic cells.

2. The nanoparticle according to claim 1, wherein the viral vector lacks envelope protein.

3. The nanoparticle according to claim 1 or claim 2, wherein the polymer or polymer mixture comprises a poly(beta-amino ester) having the formula wherein each Pep is an oligopeptide; wherein R is OH, CH3, or cholesterol; and wherein m ranges from 1 to 20, n ranges from 1 to 100, and yo ranges from 1 to 10.

4. The nanoparticle according to claim 3, wherein each oligopeptide comprises at least three amino acid residues selected from the group consisting of arginine (R), glutamic acid (E), lysine (K), aspartic acid (D), histidine (H), and cysteine ​​(C).

5. The nanoparticle according to claim 3 or claim 4, wherein the amino acid sequence of at least one of the oligopeptides is CRRR (SEQ ID NO: 1), or CHHH (SEQ ID NO: 2), or CKKK (SEQ ID NO: 3), or CEEE (SEQ ID NO: 4), or CDDD (SEQ ID NO: 5).

6. The nanoparticle according to any of the preceding claims, wherein the polymer or polymer mixture has a net positive charge or a net negative charge at pH 7.

7. The nanoparticle according to any of the preceding claims, wherein the nanoparticle contains from 108 to 1012 polymer molecules per vector.

8. The nanoparticle according to claim 7, wherein the nanoparticle contains from approximately 109 to approximately 1010 polymer molecules per vector.

9. The nanoparticle according to any of the preceding claims, wherein the viral vector is a retroviral vector.

10. The nanoparticle according to claim 9, wherein the retroviral vector is a lentiviral vector.

11. The nanoparticle according to any of the preceding claims, further comprising one or more targeting portions attached to one or more polymer molecules.

12. The nanoparticle according to claim 11, wherein the targeted portion is selected from the group consisting of antibodies, antibody fragments, scFv, antibody-like protein scaffolds, oligopeptides, aptamers, L-RNA aptamers, and ligands for cell surface receptors.

13. The nanoparticle according to claim 12, wherein the targeted portion is an anti-CD3 antibody or an anti-CD3 aptamer.

14. The nanoparticle according to any of the preceding claims, wherein the transgene encodes a chimeric antigen receptor.

15. The nanoparticle according to any of the preceding claims having a zeta potential of approximately -15 mV to approximately +15 mV.

16. The nanoparticle according to any of the preceding claims, lacking VSV-G envelope protein.

17. The nanoparticle according to claim 16, having an improved safety profile for in vivo use compared to a similar nanoparticle comprising the VSV-G envelope protein.

18. The nanoparticle according to any of the preceding claims, wherein the viral vector lacks viral envelope protein, and wherein the nanoparticle is only capable of transducing a mammalian cell above a threshold number of polymer molecules per vector.

19. The nanoparticle according to claim 18, wherein below said threshold number of polymer molecules per vector, the amount of polymer is insufficient to completely coat the vector.

20. The nanoparticle according to claim 18 or claim 19, wherein below said threshold number of polymer molecules per vector, the nanoparticle is structurally unstable or subject to dissociation of the polymer molecules from the nanoparticle.

21. The nanoparticle according to any of claims 18 to 20 having an improved safety profile for in vivo use compared to a vector or polymer-encapsulated vector lacking such a threshold.

22. A method for manufacturing the nanoparticle according to any of the preceding claims, comprising the steps of: (a) providing a viral vector comprising a transgene; (b) providing a polymer or polymer mixture, and optionally one or more targeting portions; and (c) contacting the viral vector and the polymer or polymer mixture, and optionally one or more targeting portions, whereby the viral vector and the polymer or polymer mixture combine to form said nanoparticle.

23. The method according to claim 22, wherein the viral vector lacks envelope protein.

24. The method according to claim 22 or claim 23, wherein step (c) is performed at a pH of approximately 5.0 to approximately 6.

5.

25. The method according to claim 24, wherein step (c) is performed at a pH of approximately 5.5 to 6.

0.

26. The method according to any of claims 22 to 25, wherein the polymer or polymer mixture comprises a poly(beta-amino ester) having the formula O S-Pep wherein each Pep is an oligopeptide; wherein R is OH, CH3, or cholesterol; and wherein m ranges from 1 to 20, n ranges from 1 to 100, and i ranges from 1 to 10.

27. The method according to claim 26, wherein each oligopeptide comprises at least three amino acid residues selected from the group consisting of R, E, K, D, H and C.

28. The method according to claim 26, wherein the amino acid sequence of at least one of the oligopeptides is CRRR (SEQ ID NO: 1), or CHHH (SEQ ID NO: 2), or CKKK (SEQ ID NO: 3), or CEEE (SEQ ID NO: 4) or CDDD (SEQ ID NO: 5).

29. The method according to any of claims 22 to 28, wherein the polymer or polymer mixture has a net positive charge or a net negative charge at pH 7.

30. The method according to any of claims 22 to 29, wherein the nanoparticle contains from 108 to 1017 polymer molecules.

31. The method according to claim 30, wherein the nanoparticle contains from approximately 109 to approximately 1010 polymer molecules.

32. The method according to any of claims 22 to 31, wherein the retroviral vector is a retroviral vector.

33. The method according to claim 32, wherein the retroviral vector is a lentiviral vector.

34. The method according to any of claims 22 to 33, further comprising contacting the vector with one or more addressing portions, optionally wherein the addressing portions are attached to the polymer or particle mixture.

35. The method according to claim 34, wherein the targeted portion is selected from the group consisting of antibodies, antibody fragments, scFv, antibody-like protein scaffolds, oligopeptides, aptamers, L-RNA aptamers, and ligands for cell surface receptors.

36. The method according to claim 35, wherein the targeted portion is an anti-CD3 antibody or an anti-CD3 aptamer.

37. The method according to any of claims 22 to 36, wherein the transgene encodes a chimeric antigen receptor.

38. The method according to any of claims 22 to 37, wherein the viral vector lacks VSV-G envelope protein.

39. The method according to any of claims 22 to 38, wherein step (c) is performed at a pH where the polymer or polymer mixture has a net positive charge and the retroviral vector has a negative surface potential.

40. A method for treating a disease, the method comprising administering a composition comprising a plurality of nanoparticles according to any of claims 1 to 21 to a subject in need thereof, wherein the subject's cells are transduced by the retroviral vector and the transgene is expressed in the transduced cells.

41. The method according to claim 40, wherein the disease is cancer.

42. The method according to claim 40 or claim 41, wherein the subject is a human.

43. A method for transducing cells in vitro, the method comprising contacting cultured cells with a plurality of nanoparticles according to any of claims 1 to 21, whereby at least some of the cultured cells are transduced by the retroviral vector and the transgene is specifically expressed in the transduced cells.

44. The method according to claim 43, wherein the nanoparticles are directed to CD3 positive cells and the transgene is specifically expressed in CD3 positive cells.

45. The method according to claim 43 or claim 44, wherein the transgene encodes a chimeric antigen receptor.

46. ​​A method for performing gene therapy, the method comprising contacting cells in vitro or within a living organism with a plurality of nanoparticles according to any of claims 1 to 21, whereby the transgene is expressed in the cells.

47. A nanoparticle comprising a viral vector coated with a polymer or a mixture of polymers, wherein the viral vector comprises a reduced level of envelope protein and comprises a transgene, wherein the nanoparticle functions as a vehicle for delivering the transgene to eukaryotic cells, and wherein the reduced level of envelope protein is insufficient to promote cell entry of the vector and cell transduction by the vector.

48. The nanoparticle according to claim 47, wherein the reduced level of envelope protein is 5% or less of an amount of envelope protein in a functional virus from which the vector is derived.