Compositions and methods for enhancing transduction efficiency of viral vectors with heparin and analogues thereof

Abstract

The present invention provides methods of enhancing the efficiency of viral transduction in cells comprising administering to cells an effective amount of heparin or an analogue thereof and an effective amount of a viral vector.

Description

[0001] COMPOSITIONS AND METHODS FOR ENHANCING TRANSDUCTION EFFICIENCY OF VIRAL VECTORS WITH HEPARIN AND ANALOGUES THEREOF

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Application No.: 63 / 556,040, filed February 21, 2024, the contents of which are hereby incorporated by reference in their entirety.

[0004] INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0005] Incorporated by reference in its entirety herein is a computer-readable sequence listing submitted concurrently herewith and identified as follows: One 3,785 Byte xml file named “Sequence_listing.xml,” created on February 20, 2025.

[0006] FIELD OF THE INVENTION

[0007] The field of the invention relates to viral vectors and gene therapy, in particular to compositions and methods for improving transduction efficiency of viral vectors.

[0008] BACKGROUND

[0009] Viral vectors are commonly used in molecular biology and gene therapy, facilitating the transfer of genetic material into specific cells. These vectors are derived from viruses that have been genetically modified to eliminate their pathogenic features, while preserving their ability to infect cells and transfer genetic material. Viral vectors are extensively used in research and therapeutic applications due to their effective and adaptable gene delivery capacities. Common examples include retroviral vectors, lentiviral vectors, adenoviral (AV) vectors and adeno-associated viral (AAV) vectors (J. T. Bulcha et al., Target. Ther., (2021), 6:doi: 10.1038 / s41392-021-00487-6). Viral vector-based drugs and therapies have received approval or authorization for medical use in various applications.

[0010] In general, viruses can be classified into two different types: enveloped and nonenveloped viruses. Enveloped viruses are characterized by a lipid envelope surrounding the viral capsid containing the viral genome. Prominent examples of such viruses are influenza virus, Human Immunodeficiency Virus (HIV), herpes simplex virus (HSV), and SARS-CoV-2 virus, which is responsible for causing COVID-19 (Zeng et al., Environ. Eleal., 1:15-31, (2023), doi: 10.1021 / cnvhcalth.3c00005). In contrast, non-cnvclopcd viruses are characterized by their lack of an outer lipid envelope. These viruses, such as AAV, norovirus, rotavirus, and human papillomavirus (HPV), comprise protein capsid, which protects their genetic material (Liang et al., Nat. Rev. Microbiol., 19:514-527, (2021), doi: 10.1038 / s41579-021-00536-5).

[0011] Viruses utilize different mechanisms for cell entry, such as receptor- mediated endocytosis, membrane fusion, direct penetration, phagocytosis, and micropinocytosis (Caveolin et al., Multifaceted Functions of Host Cell, (2020)). One of the most common mechanisms is interaction with heparan sulfate proteoglycan (HSPG), essential for SARS- CoV2 uptake (Somiya et al., N ano therano sties, (2017), 1:415-429, doi: 10.7150 / ntno.21723.). HSPG is a crucial viral binding receptor used by many types of viruses (both enveloped and non-enveloped). HSPG chains on the cell surface interact with regions of the viral envelope proteins. The first interaction between viral attachment proteins and heparan sulfate enables the initial contact between the virus and the host cell. The interaction is an essential prerequisite for following steps in viral entry. These glycosaminoglycans (GAGs) have similar structural features, including negatively charged sulfate groups, which viruses can interact with during the initial stages of infection (Stroh et al., Ann . Rev. Virol., (2014), 1:285-306, doi: 10.1146 / annurev-virology-031413- 085417; De Pasquale et al., Int. J. Mol. Sei., (2021), 22:doi: 10.3390 / ijms22126574).

[0012] Heparin is a polysaccharide structurally similar to heparan sulfate but is highly sulfated, and it possesses well-characterized stability and pharmacokinetic properties. It has primarily been used as an anticoagulant (Piran et al., Blood, (2019), 133:425-435, doi: 10.1182 / blood-2018-06-820746). Heparin and its analogues have also been shown to mitigate pulmonary thrombosis in individuals infected with the SARS-CoV-2 virus, which predominantly targets the respiratory system (Tang et al., J. Thromb. Haemost., (2020), 18:1094-1099, doi: 10.1111 / jth.14817). Several biochemical investigations have furthermore shown the inhibitory effects of heparin and its analogues on the binding and utilization of the angiotensin-converting enzyme 2 (ACE2) receptor and heparan sulfate co-receptors by the virus. (Zhang et al., Intensive Care Med., (2020), 46:586-590, doi: 10.1007 / s00134-020-05985-9; Tandon et al., J. Virol., (2021), 95:1-12, 2021, doi: 10.1128 / jvi.Ol 987-20; Tanaka et al., J. Virol., (2017), 91 :doi: 10.1128 / jvi.00432- 17; Clausen et al., Cell, (2020), 183:1043-1057.cl5, 2020, doi: 10.1016 / j.ccll.2020.09.033). It has also been shown to inhibit the infectivity of other respiratory viruses, such as respiratory syncytial virus (RSV), by binding to viral glycoproteins (Bourgeois J. Virol., (1998), 72:7221-7227, doi: 10.1128 / jvi.72.9.7221-7227.1998; Donalisio et al.,Antimicrob. Agents Chemother., (2012), 56:5278-5288, doi: 10.1128 / AAC.00771-12).

[0013] Heparin is naturally produced by mast cells and can be found abundantly in the gastrointestinal tract, liver, and respiratory system of both vertebrates and invertebrates (Li Fu et al., J. Pharm. Sci., (2013), 102:1447-1457 doi: https: / / doi.org / 10.1002 / jps.23501). It is a polysaccharide characterized by a highly sulfated chain composed of disaccharide subunits. The predominant constituents of this chain are 2-O- sulphated iduronic acid and 6-O-sulphated glucosamine (Zhang et al., Carbohydr. Polym., (2018), 203:87-94, doi: 10.1016 / j.carbpol.2018.08.108). In addition, it is worth noting that heparin exhibits a significant degree of negative charge density, with a value of 3.3 negative charges per disaccharide unit (Weiss, et al., Org. Biomol. Chem., (2017), 15:5656-5668, doi: 10.1039 / c7ob01058c). Consequently, a negative charge on heparin facilitates a strong affinity for binding to various proteins, such as serine protease inhibitor antithrombin III (Esko et al., J. Clin. Invest., (2001), 108:169-173, doi: 10.1172 / JCI200113530). Furthermore, heparin can be categorized into two main types: unfractionated heparin (UFH), which has a molecular weight range of 3-30 kDa, and low molecular weight heparin (LMWH) analogues which have a molecular weight range of 4-7 kDa. The low molecular weight analogues are derived from the UFH by chemical, physical, and enzymatic digestion methods. Heparin, through electrostatic interactions with positively charged proteins, competes with HSPG. This competition reduces inflammation by inhibiting the binding of chemokines and selectins to heparan sulfate on endothelial cells. Consequently, this inhibition hinders the migration of leukocytes (Farrugia et al., J. Histochem. Cytochem., (2018), 66:321-336, doi: 10.1369 / 0022155417740881).

[0014] What is needed are new and improved compositions and methods for enhancing viral delivery to cells, for in vitro, in vivo and ex vivo applications. This background information is provided for informational purposes only. No admission is necessarily intended, nor should it be construed that any of the preceding information constitutes prior art against the present invention.

[0015] SUMMARY OF THE INVENTION

[0016] It is to be understood that both the foregoing general description of the embodiments and the following detailed description are exemplary and thus do not restrict the scope of the embodiments.

[0017] Viral vectors have been extensively used in gene therapy applications. However, viral transduction efficiency in certain cell types is often insufficient. Many polymers, lipids, peptides and polycationic compounds have been employed as additives to improve the transduction efficacy of viral vectors. Polycationic additives such as protamine sulfate and polybrene enhance the transduction efficiency, while anionic molecules such as pyran and heparin typically inhibit the transduction efficiency. In this disclosure, the present inventors have surprisingly observed a significant improvement in the transduction efficiency of Respiratory syncytial virus (RSV), and lentiviruses upon using low concentrations of heparin and analogues thereof.

[0018] Additionally, it is described herein that lactoferrin, a specific heparan-sulfate proteoglycans (HSPGs) inhibitor, prevented heparin-induced transduction, supporting, and without being bound by theory, that at low concentrations, heparin and its analogues enhance transduction by bridging the interaction between the virus and HSPGs. In contrast, they compete with viral binding to HSPGs at high concentrations. The results provided herein indicate that the effect of polyanionic molecules on viral uptake is concentrationdependent and that these molecules enhance viral transduction at low concentrations. Thus, they can be utilized in various applications, including gene therapy.

[0019] In one aspect, the invention provides a method of enhancing the efficiency of viral transduction in cells, comprising administering to the cells an effective amount of heparin or an analogue thereof and an effective amount of a viral vector, wherein the effective amount of the heparin or analogue thereof enhances the efficiency of transduction of the viral vector in the cells.

[0020] In another aspect, the invention provides a method of treating or preventing a condition or disease in a subject, comprising administering to the subject an effective amount of heparin or an analogue thereof and an effective amount of a viral vector, wherein the effective amount of heparin or analogue thereof enhances the efficiency of transduction of the viral vector in one or more cells of the subject.

[0021] In another aspect, the invention provides a composition, comprising an effective amount of a viral vector and an effective amount of heparin or an analogue thereof, wherein the effective amount of heparin or analogue thereof enhances the efficiency of transduction of the viral vector in one or more cells when the composition is administered to the subject.

[0022] In another aspect, the invention provides a method for identifying heparin or an analogue thereof that enhances efficiency of transduction of a viral vector in cells, comprising i) administering an amount of heparin or an analogue to the cells; ii) administering a viral vector to the cells; iii) assaying for transduction efficiency of the viral vector following administration of the heparin or analogue thereof; iv) assaying for transduction efficiency of the viral vector in the absence of administering the heparin or analogue thereof; and v) comparing transduction efficiency of the viral vector in parts iii) and iv) whereby when the transduction efficiency of part iii) is greater than the transduction efficiency of part iv), a heparin or analogue thereof that enhances transduction efficiency is identified.

[0023] In another aspect, the invention provides a pharmaceutical composition comprising transduced cells that have been prepared in accordance with the methods herein.

[0024] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0025] BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way. FIG. 1 . A549 cells infected with RSV. The transduction efficiency of respiratory syncytial virus (RSV) in A549 cells cultured in VP scrum-frcc medium was assessed using flow cytometry after a 48-hour incubation period. The A549 cells were treated with various concentrations (1 mg / mL, 100 pg / mL, 10 pg / mL, 1 pg / mL, 0.5 pg / mL, 0.25 pg / mL, and 0.125 pg / mL) of A) heparin B) enoxaparin C) dalteparin, and D) tinzaparin. The values are the average of three independent experiments with triplicate measurements (mean ± SEM). The p-values were calculated using the one-way analysis of variance (ANOVA) with Dunnell's multiple comparison test. The levels of significance were classified as nonsignificant (ns), significant at p < 0.05 (*), highly significant at p < 0.01 (**), and extremely significant at p < 0.0001 (****).

[0027] FIG. 2. Enhancing Transduction Efficiency of Foamy Envelope Lentivirus in a T- cell-line with Low Concentrations of Heparin and Its Analogues. The transduction efficiency of GFP-encoding lentivirus pseudotyped with foamy envelope in Jurkat cells cultured in serum-free RPMI medium was assessed after 48 hours using flow cytometry. The transduction efficiency was then compared to Jurkat cells transduced with the same viral titer and treated with four different poly anions; A) heparin, B) enoxaparin, C) dalteparin, and D) tinzaparin, at various concentrations ranging from 1 mg / mL to 0.125 pg / mL. The values are the average of three independent experiments with triplicate measurements (mean ± SEM). The p-values were calculated using the one-way analysis of variance (ANOVA) with Dunnett's multiple comparison test. The levels of significance were classified as non-significant (ns), significant at p < 0.05 (*), highly significant at p < 0.01 (**), and extremely significant at p < 0.0001 (****).

[0028] FIG. 3. Enhanced Transduction Efficiency of Foamy Envelope Lentivirus in Macrophage-Like Cells with Low Concentrations of Heparin and Its Analogues. The transduction efficiency of GFP-encoding lentivirus pseudotyped with foamy envelope in RAW 264.7 cells cultured in serum-free DMEM medium. After a 48-hour incubation period, the cells were analyzed using flow cytometry. The transduction efficiency of the lentivirus was then compared with the RAW 264.7 cells transduced with the viral titer and subjected to different treatments: A) heparin, B) enoxaparin, C) dalteparin, and D) tinzaparin. These treatments were provided at various doses ranging from 1 mg / mL to 0.125 pg / mL. The values are the average of three independent experiments with triplicate measurements (mean ± SEM). The p-values were calculated using the one-way analysis of variance (ANOVA) with Dunnctt's multiple comparison test. The levels of significance were classified as non-significant (ns), significant at p < 0.05 (*), highly significant at p < 0.01 (**), and extremely significant at p < 0.0001 (****).

[0029] FIG. 4. Visualizing the Improved Transduction Efficiency of Foamy Envelope Lentivirus in Macrophage-Like Cells. Raw 264.7 cells were subjected to infection with GFP encoding foamy envelope lentivirus compared to the cells that were transduced with the same virus and treated with heparin, enoxaparin, dalteparin, and tinzaparin at concentrations of 10 pg / mL, 1 pg / mL, and 0.25 pg / mL. These cells were cultured in DMEM serum-free media at 37°C with 5% CO2 for 48 hours. Afterward, transduced cells were fixed using 4% paraformaldehyde (PFA) and images were taken using an Olympus 1X81 fluorescent microscope. (20x magnification; scale bar = 100 pm).

[0030] FIG. 5. Enhancing Transduction Efficiency of VSV-G Envelope Lentivirus in T Cells with Low Concentrations of Heparin and Its Analogues. The transduction efficiency of GFP-encoding lentivirus pseudotyped with VSV-G envelope in Jurkat cells in serumcontaining RPMI medium was evaluated after 48 h by flow cytometry and then compared to the Jurkat cells transduced with the same virus and treated with A) heparin, B) enoxaparin at 1 mg / ml, 100 pg / mL. The values are the average of three independent experiments with triplicate measurements (mean ± SEM). The p-values were calculated using the independent t-test. The levels of significance were classified as non-significant (ns), significant at p < 0.05 (*), highly significant at p < 0.01 (**), and extremely significant at p < 0.0001 (****).

[0031] FIG. 6. Probing cellular uptake of GFP-labelled lentiviruses. VSV-G envelope lentivirus labelled with GFP was added to Jurkat cells cultured in RPMI media supplemented with serum for 8 hours. Subsequently, the cells were washed with PBS and fixed before being analyzed using flow cytometry. The values are the average of three independent experiments with triplicate measurements (mean ± SEM). The p-values were calculated using the one-way analysis of variance (ANOVA) with Dunnett's multiple comparison test. The levels of significance were classified as non-significant (ns), significant at p < 0.05 (*), highly significant at p < 0.01 (**), and extremely significant at p < 0.0001 (****). FIG. 7. Lactoferrin Inhibits the Entry of Foamy Envelope Lentivirus in T Cells with Low Concentrations of Heparin and Its Analogues. Jurkat cells were treated with bovine milk-derived Lactoferrin and subjected to incubation at 37°C with a CO2 concentration of 5% for one hour. Subsequently, heparin, enoxaparin, and dalteparin were used at concentrations of 100 pg / mL, 10 pg / mL, and 1 pg / mL, respectively, before initiating viral infection. Following a 48-hour incubation period, the cells were subjected to fixation and then processed using flow cytometry. The p-values were calculated using the independent t-test. The levels of significance were classified as non-significant (ns), significant at p < 0.05 (*), highly significant at p < 0.01 (**), and extremely significant at p < 0.0001 (****).

[0032] FIG. 8. Illustrative mechanism of enveloped lenti viral bridging with the cells.

[0033] FIG. 9. The MFI of jurkat cells transduced with foamy envelope lentivirus in serum free media. After a 48-hour incubation period, the flow cytometry study was performed on jurkat cells seeded in RPMI media without serum, which were treated with GFP- encoding lentivirus pseudotyped with foamy envelope. The main focus of the analysis was to determine the mean fluorescence intensity. The obtained data was afterwards compared to those of jurkat cells transduced with a similar virus but subjected to different doses of heparin (A), enoxaparin (B), dalteparin (C), and tinzaparin (D) at concentrations ranging from 1 mg / ml to 0.125 pg / mL. The data represents the average values obtained from three separate experiments, with each study including three repeated measurements. These values are presented as the mean ± standard error of the mean (SEM). The p-values were calculated using the one-way analysis of variance (ANOVA) method, using Dunnett's multiple comparison test. 0.001 (***). 0.0001 (****).

[0034] FIG. 10. The MFI of jurkat cells transduced with foamy envelope lentivirus in serum containing media. After a 48-hour incubation period, the flow cytometry study was performed on jurkat cells seeded in RPMI media with serum, which were treated with GFP- encoding lentivirus pseudotyped with foamy envelope. The main focus of the analysis was to determine the mean fluorescence intensity. The obtained data was afterwards compared to those of jurkat cells transduced with a similar virus but subjected to different doses of heparin (A), enoxaparin (B), dalteparin (C), and tinzaparin (D) at concentrations ranging from 1 mg / ml to 0.125 pg / mL. The data represents the average values obtained from three separate experiments, with each study including three repeated measurements. These values are presented as the mean ± standard error of the mean (SEM). The p-values were calculated using the one-way analysis of variance (ANOVA) method, using Dunnctt's multiple comparison test. 0.001 (***). 0.0001 (****).

[0035] FIG. 11. The GFP expression in jurkat cells transduced with foamy envelope lentivirus in serum containing media. The fold increase over GFP positive jurkat cells transduced with foamy envelope lentivirus in serum-containing RPMI medium were evaluated after 48 hrs by flow cytometry then compared to the jurkat cells transduced with the same virus and treated with A) heparin, B) enoxaparin C) dalteparin and, D) tinzaparin at 1 mg / ml, 100 pg / mL, 10 pg / mL, 1 pg / mL, 0.5 pg / mL, 0.25 pg / mL, 0.125 pg / mL doses. The values are the average of three independent experiments with triplicate measurements (mean ± SEM). The p-values were calculated using the one-way analysis of variance (ANOVA) with Dunnett's multiple comparison test. The levels of significance were classified as non-significant (ns), significant at p < 0.05 (*), highly significant at p < 0.01 (**), and extremely significant at p < 0.0001 (****).

[0036] FIG. 12. The MFI of RAW 264.7 cells transduced with foamy envelope lentivirus in serum free media. The mean fluorescent intensity of GFP-encoding lentivirus pseudotyped with foamy envelope in Raw 246.7 cells in serum-free DMEM medium were evaluated after 48 hrs by flow cytometry then compared to the RAW 264.7 cells transduced with the same virus and treated with A) heparin, B) enoxaparin C) dalteparin and, D) tinzaparin at 1 mg / ml, 100 pg / mL, 10 pg / mL, 1 pg / mL, 0.5 pg / mL, 0.25 pg / mL, 0.125 pg / mL doses. The values are the average of three independent experiments with triplicate measurements (mean ± SEM). The p-values were calculated using the one-way analysis of variance (ANOVA) with Dunnett's multiple comparison test. The levels of significance were classified as non-significant (ns), significant at p < 0.05 (*), highly significant at p < 0.01 (**), and extremely significant at p < 0.0001 (****).

[0037] FIG. 13. The GFP expression in RAW 264.7 cells transduced with foamy envelope lentivirus in serum containing media. The transduction efficiency of GFP-encoding lentivirus pseudotyped with foamy envelope in RAW 264.7 cells in serum-containing DMEM medium were evaluated after 48 hrs by flow cytometry then compared to the RAW 264.7 cells transduced with the same virus and treated with A) heparin, B) enoxaparin C) dalteparin and, D) tinzaparin at 1 mg / ml, 100 pg / mL, 10 pg / mL, 1 pg / mL, 0.5 pg / mL, 0.25 pg / mL, 0.125 pg / mL doses. The values are the average of three independent experiments with triplicate measurements (mean ± SEM). The p-valucs were calculated using the oneway analysis of variance (ANOVA) with Dunnett's multiple comparison test. The levels of significance were classified as non-significant (ns), significant at p < 0.05 (*), highly significant at p < 0.01 (**), and extremely significant at p < 0.0001 (****).

[0038] FIG. 14. The MFI of RAW 264.7 cells transduced with foamy envelope lentivirus in serum containing media. The mean fluorescent intensity of GFP-encoding lentivirus pseudotyped with foamy envelope in RAW 264.7 cells in serum-containing DMEM medium were evaluated after 48 hrs by flow cytometry then compared to the Raw 264.7 cells transduced with the same virus and treated with A) heparin, B) enoxaparin C) dalteparin and, D) tinzaparin at 1 mg / ml, 100 pg / mL, 10 pg / mL, 1 pg / mL, 0.5 pg / mL, 0.25 pg / mL, 0.125 pg / mL doses. The values are the average of three independent experiments with triplicate measurements (mean ± SEM). The p-values were calculated using the oneway analysis of variance (ANOVA) with Dunnett's multiple comparison test. The levels of significance were classified as non-significant (ns), significant at p < 0.05 (*), highly significant at p < 0.01 (**), and extremely significant at p < 0.0001 (****).

[0039] FIG. 15. The flowcytometry analysis of jurkat cells in serum free media. The percentage of GFP expression in jurkat cells transduced with lentivirus pseudotyped with foamy envelope in serum-free RPMI medium were evaluated after 48 hrs by flow cytometry then compared to the jurkat cells transduced with the same virus and treated with A) Heparin, B) enoxaparin C) dalteparin and, D) tinzaparin 1 mg / ml, 100 pg / mL, 10 pg / mL, 1 pg / mL, 0.5 pg / mL, 0.25 pg / mL, 0.125 pg / mL doses.

[0040] FIG. 16. The flowcytometry analysis of jurkat cells in serum containing media. The percentage of GFP expression in jurkat cells transduced with lentivirus pseudotyped with foamy envelope in serum-containing RPMI medium were evaluated after 48 hrs by flow cytometry then compared to the jurkat cells transduced with the same virus and treated with A) heparin, B) enoxaparin C) dalteparin and, D) tinzaparin at 1 mg / ml, 100 pg / mL, 10 pg / mL, 1 pg / mL, 0.5 pg / mL, 0.25 pg / mL, 0.125 pg / mL ml doses.

[0041] FIG. 17. Lentivirus with foamy envelope in serum free media at different MOI after 48 hrs. The percentage of GFP expression in jurkat cells transduced with lentivirus pseudotyped with foamy envelope at different MOI in serum-containing RPMI medium were evaluated after 48 hrs by flow cytometry then compared to the jurkat cells transduced with the same virus and treated with 10 pg / mL cnoxaparin.

[0042] FIG. 18. The flowcytometry analysis of RAW 264.7 cells in serum containing media. The percentage of GFP expression in RAW 264.7 cells transduced with lentivirus pseudotyped with foamy envelope in serum-containing DMEM medium were evaluated after 48 hrs by flow cytometry then compared to the jurkat cells transduced with the same virus and treated with A) heparin, B) enoxaparin C) dalteparin and, D) tinzaparin at 1 mg / ml, 100 pg / mL, 10 pg / mL, 1 pg / mL, 0.5 pg / mL, 0.25 pg / mL, 0.125 pg / mL doses.

[0043] FIG. 19. Live image scanning in different time points. GFP expression in Jurkat cells transduced with lentivirus pseudotyped with foamy envelope in serum containing RPMI medium were evaluated in different time points 24 hrs, 48 hrs, 72 hrs, 96 hrs and 120 hrs then compared to the jurkat cells transduced with the same virus and treated with 1 pg / mL of heparin, 10 pg / mL of enoxaparin and 10 pg / mL of dalteparin.

[0044] FIG. 20. The MFI of jurkat cells transduced with VSV-G envelope lentivirus in serum free media. The mean fluorescent intensity of GFP-encoding lentivirus pseudotyped with VSV-G envelope in jurkat cells in serum-containing RPMI medium were evaluated after 48 hrs by flow cytometry then compared to the jurkat cells transduced with the same virus and treated with A) heparin, B) enoxaparin at 1 mg / mL doses. The values are the average of three independent experiments with triplicate measurements (mean ± SEM). The p-values were calculated using the one-way analysis of variance (ANOVA) with Dunnett's multiple comparison test. The levels of significance were classified as nonsignificant (ns), significant at p < 0.05 (*), highly significant at p < 0.01 (**), and extremely significant at p < 0.0001 (****).

[0045] FIG. 21. VSV-G uptake in jurkat cells. The percentage of GFP expression in jurkat cells transduced with lentivirus pseudotyped with VSV-G envelope in serum-containing RPMI medium were evaluated after 8 hrs by flow cytometry then compared to the jurkat cells transduced with the same virus and treated with A) heparin, B) enoxaparin C) dalteparin and, D) tinzaparin at 1 mg / ml, 100 pg / mL, 10 pg / mL, 1 pg / mL, 0.5 pg / mL, 0.25 pg / mL, 0.125 pg / mL doses. DETAILED DESCRIPTION OF THE INVENTION

[0046] The present inventors have surprisingly found that administering low concentrations of heparin or heparin analogues enhances transduction efficiency of viral vectors in eukaryotic cells.

[0047] Described in more detail herein, the present inventors investigated the effects of heparin and its analogues on the uptake and transduction efficacy of several viruses known to enter cells via the heparan sulfate pathway, including RSV and Lentivirus (Cagno et al., Viruses, (2019), 11:1-24, doi: 10.3390 / v 11070596). As expected, and in line with previous literature, high heparin doses blocked viral transduction (Seffer et al., Heparin 2, (2021), 28-34, doi: 10.1159 / 000508647). Interestingly, however, it was discovered that low concentrations of heparin and its analogues increased viral transduction in several cell lines. The results herein indicate that low-dose heparin can be utilized as a transduction enhancer for cell line development and also in ex vivo and in vivo viral gene therapy.

[0048] Reference will now be made in detail to the presently preferred embodiments of the invention which, together with the drawings and the following examples, serve to explain the principles of the invention. These embodiments describe in sufficient detail to enable those skilled in the art to practice the invention, and it is understood that other embodiments may be utilized, and that structural, biological, and chemical changes may be made without departing from the spirit and scope of the present invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0049] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al., 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Harries & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. I. Higgins eds. 1984); Culture Of Animal Cells (R. T. Freshney, Alan R. Liss, Tnc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Pcrbal, A Practical Guide To Molecular Cloning (1984); the scries, Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds. -in-chief, Academic Press, Inc., New York), specifically, Vols.154 and 155 (Wu et al. eds) and Vol. 185, "Gene Expression Technology" (D. Goeddel, ed); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

[0050] Unless specifically defined herein, all technical and scientific terms used have the same meaning as commonly understood by a skilled artisan in the fields of gene therapy, biochemistry, genetics, immunology, cancer and molecular biology. Definitions of common terms in molecular biology may be found, for example, in Benjamin Lewin, Genes VII, published by Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al. (eds); The Encyclopedia of Molecular Biology, published by Blackwell Publishers,

[0051] 1994 (ISBN 0632021829); and Robert A. Meyers (ed), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Wiley, John & Sons, Inc.,

[0052] 1995 (ISBN 0471186341).

[0053] For the purpose of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with the usage of that word in any other document, including any document incorporated herein by reference, the definition set forth below shall always control for purposes of interpreting this specification and its associated claims unless a contrary meaning is clearly intended (for example in the document where the term is originally used). The use of "or" means "and / or" unless stated otherwise. As used in the specification and claims, the singular' form "a," "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof. The use of “comprise,” “comprises,” “comprising,” “include,” “includes,” and “including” are interchangeable and not intended to be limiting. Furthermore, where the description of one or more embodiments uses the term “comprising,” those skilled in the art would understand that, in some specific instances, the embodiment or embodiments can be alternatively described using the language “consisting essentially of’ and / or “consisting of.”

[0054] As used herein, the term "about" means plus or minus 10% of the numerical value of the number with which it is being used.

[0055] Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art.

[0056] As used herein, "recombinant" includes reference to a cell or vector, that has been modified by the introduction of a heterologous nucleic acid sequence or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found in identical form within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all as a result of deliberate human intervention.

[0057] As used herein, a "recipient" is a patient that receives a transplant, such as a transplant containing a population of engineered cells, such as T-cells. The transplanted cells administered to a recipient may be, e.g., autologous, syngeneic, or allogeneic cells.

[0058] As used herein, a "donor" is a human or animal from which one or more cells are isolated prior to administration of the cells, or progeny thereof, into a recipient. The one or more cells may be, e.g., a population of immune cells or hematopoietic stem cells to be engineered, expanded, enriched, or maintained according to the methods of the invention prior to administration of the cells or the progeny thereof into a recipient.

[0059] "Expansion" in the context of cells refers to an increase in the number of a characteristic cell type, or cell types, from an initial cell population of cells, which may or may not be identical. The initial cells used for expansion may not be the same as the cells generated from expansion. For example, in some embodiments, cells transduced in vitro, or ex vivo can be expanded prior to administration to a subject for therapy. As used herein, the term "pharmaceutical composition" refers to the active agent in combination with a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0060] As used herein, the term "administering," refers to the placement of a compound, viral vector, cell, or population of cells as disclosed herein into a subject or to cells by a method or route which results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions comprising the compounds, vectors or cells disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject.

[0061] As used herein, "nucleic acid" or "polynucleotides" refers to nucleotides and / or polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action. Nucleic acid molecules can be composed of monomers that are naturally occurring nucleotides (such as DNA and RNA), or analogues of naturally occurring nucleotides (e.g., enantiomeric forms of naturally occurring nucleotides), or a combination of both. Modified nucleotides can have alterations in sugar moieties and / or in pyrimidine or purine base moieties. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars can be functionalized as ethers or esters. Moreover, the entire sugar moiety can be replaced with sterically and electronically similar structures, such as aza-sugars and carbocyclic sugar analogues. Examples of modifications in a base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes. Nucleic acid monomers can be linked by phosphodiester bonds or analogues of such linkages. Nucleic acids can be either single stranded or double stranded.

[0062] The terms "polypeptide," "peptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues. The term also applies to amino acid polymers in which one or more amino acids are chemical analogues or modified derivatives of corresponding naturally occurring amino acids.

[0063] The term "identity" relates to an exact nucleotide-to-nucleotide or amino acid-to- amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) can be compared by determining their percent identity. Calculations of homology or sequence identity between two sequences (the terms are used interchangeably herein) are performed as follows. The sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The optimal alignment is determined as the best score using the GAP program in the GCG software package with a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frame shift gap penalty of 5. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percentage identity between the two sequences is a function of the number of identical positions shared by the sequences.

[0064] As used herein, the terms "treat," "treatment," "treating," and the like, refer to obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. "Treatment," as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, e.g., causing regression of the disease, e.g., to completely or partially remove symptoms of the disease.

[0065] The term "subject" or "patient" as used herein includes all members of the animal kingdom including non-human primates and humans. An "effective amount" or "therapeutically effective amount" refers to that amount of a composition described herein which, when administered to a subject or to cells (e.g., human), is sufficient to aid in treating a disease or condition or achieve some desired effect, e.g., increasing the transduction efficiency of a viral vector or infection of cells by a viral vector. The amount of a composition that constitutes a "therapeutically effective amount" will vary depending on the cell or viral vector preparations, the condition and its severity, the manner of administration, and the age of the subject to be treated, but can be determined routinely by one of ordinary skill in the art having regard to his own knowledge and to this disclosure. When referring to an individual active ingredient or composition, administered alone, a therapeutically effective dose refers to that ingredient or composition alone. When referring to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients, compositions or both that result in the therapeutic effect, whether administered serially, concurrently or simultaneously.

[0066] By "vector" is meant a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. A "vector" in the present invention preferably comprises a viral vector. Large numbers of suitable vectors are known to those of skill in the art and commercially available. Viral vectors include retrovirus, adenovirus, parvovirus (e.g., adeno-associated viruses (AAV), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., measles and Sendai), positive strand RNA viruses such as picomavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox and canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of retroviruses include avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996). In one embodiment, the invention provides a method of enhancing the efficiency of viral transduction in cells comprising administering to the cells an effective amount of heparin or an analogue thereof and an effective amount of a viral vector.

[0067] In another embodiment, the invention provides a method of treating or preventing a condition or disease in a subject, comprising administering to the subject an effective amount of cells (or their progeny) that have been transduced in accordance with the methods herein.

[0068] In another embodiment, the invention provides a method of treating or preventing a condition or disease in a subject, comprising administering to the subject an effective amount of heparin or an analogue thereof and an effective amount of a viral vector, wherein the effective amount of heparin or analogue thereof enhances the efficiency of transduction of the viral vector in one or more cells of the subject.

[0069] In another embodiment, the invention provides a composition comprising an effective amount of a viral vector and an effective amount of heparin or an analogue thereof, wherein the effective amount of heparin or analogue thereof enhances the efficiency of transduction of the viral vector in one or more cells when the composition is administered to the subject.

[0070] In another aspect, the invention provides a method for identifying a heparin or an analogue thereof that enhances efficiency of transduction of a viral vector in cells, comprising i) administering an amount of heparin or an analogue to the cells; ii) administering a viral vector to the cells; iii) assaying for transduction efficiency of the viral vector following administration of the heparin or analogue thereof; iv) assaying for transduction efficiency of the viral vector in the absence of administering the heparin or analogue thereof; and v) comparing transduction efficiency of the viral vector in parts iii) and iv) whereby when the transduction efficiency of part iii) is greater than the transduction efficiency of part iv), a heparin or analogue thereof that enhances transduction efficiency is identified. The term “transducing” in the context of cell modulation using viral vectors is well known in the art and has no other meaning herein. Briefly, the term refers to the process of introducing genetic material into a cell and, optionally, its subsequent integration into the genome of said cell via a viral vector. In some embodiments, the genetic material comprises or consists of viral nucleic acid combined with one or more target nucleic acid sequences (hereinafter referred to as target or heterologous sequences) comprised in said vector intended for integration into the genome of a target cell.

[0071] The term “administering” or “contacting” as used in the context of the transduction methods of the invention refers to bringing into contact a target cell with a viral vector and a heparin or analogue thereof so that the efficiency of the transduction event is enhanced. Conditions for contacting that allow the transduction event to occur can depend to a certain extent on the target cells and the viral vector chosen. For example, some target cells are harder to transfect than other cells and may need to be transitioned into a specific culture medium before transduction with a viral vector can be achieved. Corresponding methods and conditions are described for example in Jacome et al. (Lentiviral-mediated Genetic Correction of Hematopoietic and Mesenchymal Progenitor Cells From Fanconi Anemia Patients. Mol Ther. 2009 June; 17(6): 1083-1092), Chu et al. (Efficient and Stable Gene Expression into Human Osteoclasts Using an HIV-l-Based Lentiviral Vector. DNA Cell Biol. 2008 June; 27(6): 315-320), or Poczobutt et al. (Benign mammary epithelial cells enhance the transformed phenotype of human breast cancer cells. BMC Cancer. 2010; 10: 373). Exemplary conditions are described in the example section. The viral vector and the heparin or analogue thereof can be added simultaneously, e.g. as a mixture, to the target cells or in sequential mode, as long as both compounds are simultaneously in contact with the target cell to allow an enhanced efficiency of transduction.

[0072] In some embodiments, the target cell, viral vector and heparin or analogue thereof are contacted for at least 1 hour, such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, and at least 12 hours. Also envisaged are longer contacting times such as at least 13, at least 14, at least 15, at least 16, or at least 24 hours. In some embodiments, the viral vector and heparin or analogue thereof are added simultaneously to the cells or subject. By "enhance" or "promote," or "increase" or "expand" refers generally to the ability of the compositions and / or methods contemplated herein to elicit, cause, or produce higher numbers of transduced cells compared to the number of cells transduced by either vehicle or a control molecule / composition. For example, a hematopoietic stem or progenitor cell transduced with compositions and methods contemplated herein may comprise an increase in the number of transduced cells compared to existing transduction compositions and methods, or in the absence of effective amounts of heparin or analogues thereof. Increases in cell transduction, can be ascertained using methods known in the art, such as reporter assays, RT-PCR, and cell surface protein expression, among others. An "increased" or "enhanced" amount of transduction is typically a "statistically significant" amount, and may include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the number of cells transduced by vehicle, a control composition, or other transduction method.

[0073] In some embodiments, transduction efficiency is significantly increased by contacting cells, in vitro, ex vivo, or in vivo, with effective amounts of a viral vector and heparin or an analogue thereof.

[0074] In some embodiments, transduction efficiency is significantly increased by contacting cells, in vitro, ex vivo, or in vivo, with a viral vector and heparin or an analogue in combination with an effective amount of one or more other substances, such as, for example, polycationic additives such as protamine sulfate and polybrene, one or more poloxamers (e.g., having a molecular weight of 12.8 kDa to about 15 kDa), or (5Z)-7- Oxozeaenol. See, e.g., U.S. Patent No. 10,815,498 B2 and U.S. Pat. Appl. Pub. No. 2019 / 0262309 Al, which are incorporated by reference in their entireties herein. In some embodiments, the combination is additive or synergistic.

[0075] In some embodiments, the transduction efficiency in combination with an effective amount of one or more other substances is enhanced by at least about 10%, at least about 15%, at least about 25%, at least about 50%, at least about 75%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 225%, at least about 250%, at least about 275%, or at least about 300%, relative to the transduction efficiency with heparin or an analogue thereof alone. In some embodiments, the one or more other substances is (5Z)-7-Oxozcacnol.

[0076] The heparin or analogue that can be used in the present invention is not particularly limiting. In some embodiments, the heparin is unfractionated heparin (UFH), which has a molecular weight range of about 3-30 kDa. In some embodiments, the heparin analogue is a low molecular weight heparin (LMWH) analogue which has a molecular weight range of about 4-7 kDa. In some embodiments, low molecular weight analogues of heparin can be derived from the UFH by chemical, physical, and enzymatic digestion methods. In some embodiments, the low molecular weight heparin analogue is selected from enoxaparin, dalteparin, tinzaparin, and combinations thereof.

[0077] As provided herein, an effective amount of heparin or an analogue thereof is an amount that is capable of enhancing the efficiency of transduction of viral vectors in cells, in vitro, ex vivo, or in vivo. In some embodiments, an effective amount of heparin includes a concentration that ranges from about 0.05 pg / ml to about 5 pg / l upon administration in vitro or ex vivo. In some embodiments, the effective amount of heparin includes a concentration that ranges from about 0.1 pg / ml to about 2 pg / ml upon administration in vitro or ex vivo. In some embodiments, the effective amount of heparin includes a concentration that ranges from about 0.125 pg / ml to about 1 pg / ml upon administration in vitro or ex vivo.

[0078] In some embodiments, a heparin analogue is administered. The analogue of heparin is not limiting, provided it enhances efficiency of transduction of the viral vector. In some embodiments, the analogue is selected from the group consisting of enoxaparin, dalteparin, tinzaparin, bemiparin, certoparin, nadroparin, parnaparin, reviparin and ardaparin and combinations thereof. In some embodiments, the effective amount of analogue includes a concentration that ranges from about 0.125 pg / ml to about 250 pg / ml upon administration in vitro or ex vivo. In some embodiments, the effective amount of analogic includes a concentration that ranges from about 0.250 pg / ml to about 100 pg / ml upon administration in vitro or ex vivo. In some embodiments, the effective amount of analogue includes a concentration that ranges from about 0.5 pg / ml to about 10 pg / ml upon administration in vitro or ex vivo. In some embodiments, an effective amount of enoxaparin includes a concentration that ranges from about 0.125 pg / ml to about 250 pg / l upon administration in vitro or ex vivo. In some embodiments, the effective amount of enoxaparin includes a concentration that ranges from about 0.250 pg / ml to about 100 pg / ml upon administration in vitro or ex vivo. In some embodiments, the effective amount of enoxaparin includes a concentration that ranges from about 0.5 pg / ml to about 10 pg / ml upon administration in vitro or ex vivo.

[0079] In some embodiments, an effective amount of dalteparin includes a concentration that ranges from about 0.125 pg / ml to about 50 pg / ml upon administration in vitro or ex vivo. In some embodiments, the effective amount of dalteparin includes a concentration that ranges from about 0.250 pg / ml to about 25 pg / ml upon administration in vitro or ex vivo. In some embodiments, the effective amount of dalteparin includes a concentration that ranges from about 0.5 pg / ml to about 10 pg / ml upon administration in vitro or ex vivo.

[0080] In some embodiments, an effective amount of tinzaparin includes a concentration that ranges from about 0.125 pg / ml to about 50 pg / ml upon administration in vitro or ex vivo. In some embodiments, the effective amount of tinzaparin includes a concentration that ranges from about 0.250 pg / ml to about 25 pg / ml upon administration in vitro or ex vivo. In some embodiments, the effective amount of tinzaparin includes a concentration that ranges from about 0.25 pg / ml to about 10 pg / ml upon administration in vitro or ex vivo.

[0081] In some embodiments, heparin or an analogue thereof is administered in vivo in amounts effective to achieve concentrations that enhance transduction efficiency in cells. In some embodiments, the heparin or analogue is administered locally or topically to a cell or tissue to be transduced. In some embodiments, the heparin or analogue thereof is administered systemically. The concentrations of the heparin or analogue can be appropriately tailored taking into account any dilution upon administration and in some embodiments, taking into account the cell density of the cells to be transduced.

[0082] In some embodiments, for local administration, the same concentration can be administered as what is disclosed for in vitro or ex vivo administration, but can also include amounts that are 10, 20, 50, 100, 200, 500 and 1000-fold in excess to the amounts administered in vitro or ex vivo. In some embodiments, the compositions described herein may be administered to a patient locally or topically, e.g., subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intrathecally, intramuscularly, ocularly, intraccrcbrovcntricularly (ICV), and to the car or inner or outer car.

[0083] The viral vector to be used in accordance with the present disclosure is not particularly limiting. As used herein, “viral” or “viruses” relates to natural occurring viruses as well as artificial viruses. In the context of the present invention, a viral vector can be from an enveloped or non-enveloped virus. Parvoviruses such as adeno-associated viruses (or AAVs) are illustrative non-enveloped viruses. In some embodiments, the viral vectors are from enveloped viruses. In some embodiments, the viral vectors are retroviruses and in particular lentiviruses.

[0084] For example, in some embodiments the viral vector is made from paramyxovirus (such as respiratory syncytial virus, measle virus), orthomyxovirus (such as influenza virus), flavivirus (such as hepatitis C virus), hepadnavirus (such as hepatitis B virus), rhabdovirus (such as rabies, VSV), coronavirus (such as SARS), togavirus (such as Sindbis virus, Chikungunya virus), filovirus (such as ebola virus), arenavirus, poxvirus, herpesvirus, bunyavirus, bomavirus, arterivirus, baculovirus, and parvovirus such as adeno-associated virus. According to a particular embodiment, the viruses are artificial viruses, which may for instance comprise a heterologous sequence of interest (i.e., cargo sequence), such as a sequence useful for a therapeutic, diagnostic or any other purpose (e.g. useful for conducting functional studies within a target cell). Illustrative cargos include nucleic acids such as a DNA or RNA sequence encoding a product, in particular a gene therapy product (for example a protein or RNA, such as an antisense RNA, shRNA, piRNA, miRNA or siRNA), a system for editing or modifying the genomic or mitochondrial DNA, or a diagnostic product.

[0085] In some embodiments, the viral vector is made from any enveloped virus.

[0086] In some embodiments, the viral vector is selected from the group consisting of herpes simplex virus, cytomegalovirus, human herpes virus, foot and mouth disease virus, John Cunningham polyomavirus, respiratory syncytial virus, dengue virus, pseudorabies virus, human papilomavirus, Venezuelan equine encephalitis virus, enterovirus, parainfluenza virus, echovirus, Merkel cell polyomavirus, hepatitis C virus, sindbis virus, human metapneumovirus, echovirus, hepatitis B virus / hepatitis, delta virus, adeno- associated virus, Semliki forest virus, zika virus, North American eastern equine encephalitis virus, vaccinia vims, human immunodeficiency vims, rhinovirus, Adenovims, filovims, coronavirus, norovirus, Akabanc vims, Schmallenberg virus, Rift valley fever vims, coxsackie vims, rabies vims, yellow fever vims, swine vesicular disease virus, Japanese encephalitis virus, Theiler murine encephalomyelitis vims, west nile virus, human parechovims, Hendra and Nipah vimses, tick-bome encephalitis virus, porcine reproductive and respiratory syndrome vims, human T cell leukemia virus, porcine circovirus, hepatitis E vims, Chikungunya vims, and Murray Valley encephalitis virus.

[0087] "Adeno-associated viruses," from the parvovirus family, are small vimses with a genome of single stranded DNA. These viruses can insert genetic material at a specific site on chromosome 19 and can be used in some embodiments because they are not associated with pathogenic disease in humans. The adeno-associated viral vector that can be used is not particularly limiting.

[0088] AAV vectors do not typically include viral genes associated with pathogenesis. Such vectors typically have one or more of the wild type AAV genes deleted in whole or in part, for example, rep and / or cap genes, but retain at least one functional flanking ITR sequence, as necessary for the rescue, replication, and packaging of the recombinant vector into an AAV vector particle. For example, in some embodiments, only the essential parts of vector e.g., the ITR and LTR elements, respectively, arc included. An AAV vector genome would therefore include sequences required in cis for replication and packaging (e.g., functional ITR sequences).

[0089] Recombinant AAV vectors, as well as methods and uses thereof, can include any viral strain or serotype. As a non-limiting example, a recombinant AAV vector can be based upon any AAV genome, such as AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, or AAV-2i8, for example. Such vectors can be based on the same strain or serotype (or subgroup or variant) or be different from each other. As a non-limiting example, a recombinant AAV vector based upon one serotype genome can be identical to one or more of the capsid proteins that package the vector. In addition, a recombinant AAV vector genome can be based upon an AAV (e.g., AAV2) serotype genome distinct from one or more of the AAV capsid proteins that package the vector. For example, the AAV vector genome can be based upon AAV2, whereas at least one of the three capsid proteins could be a AAV1 , AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12, or AAV-2i8 or variant thereof, for example. AAV variants include variants and chimeras of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 and AAV-2i8 capsids.

[0090] In some embodiments, adeno-associated virus (AAV) vectors include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV-2i8, as well as variants (e.g., capsid variants, such as amino acid insertions, additions, substitutions and deletions) thereof, for example, as set forth in WO 2013 / 158879 (International Application PCT / US2013 / 037170), WO 2015 / 013313 (International Application PCT / US 2014 / 047670) and US 2013 / 0059732 (US Application No. 13 / 594,773, discloses LK01, LK02, LK03, etc).

[0091] AAV and AAV variants (e.g., capsid variants) serotypes (e.g., VP1, VP2, and / or VP3 sequences) may or may not be distinct from other AAV serotypes, including, for example, AAV1-AAV12 (e.g., distinct from VP1, VP2, and / or VP3 sequences of any of AAV1-AAV12 serotypes).

[0092] As used herein, the term "serotype" is a distinction used to refer to an AAV having a capsid that is serologically distinct from other AAV serotypes. Serologic distinctiveness is determined on the basis of the lack of cross -reactivity between antibodies to one AAV as compared to another AAV. Such cross-reactivity differences are usually due to differences in capsid protein sequences / antigenic determinants (e.g., due to VP1, VP2, and / or VP3 sequence differences of AAV serotypes). Despite the possibility that AAV variants including capsid variants may not be serologically distinct from a reference AAV or other AAV serotype, they differ by at least one nucleotide or amino acid residue compared to the reference or other AAV serotype.

[0093] Under the traditional definition, a serotype means that the virus of interest has been tested against serum specific for all existing and characterized serotypes for neutralizing activity and no antibodies have been found that neutralize the virus of interest. As more naturally occurring virus isolates are discovered and / or capsid mutants are generated, there may or may not be serological differences with any of the currently existing serotypes. Thus, in cases where the new virus (e.g., AAV) has no serological difference, this new virus (e.g., AAV) would be a subgroup or variant of the corresponding serotype. In many cases, serology testing for neutralizing activity has yet to be performed on mutant viruses with capsid sequence modifications to determine if they are of another serotype according to the traditional definition of serotype. Accordingly, for the sake of convenience and to avoid repetition, the term "serotype" broadly refers to both serologically distinct viruses (e.g., AAV) as well as viruses (e.g., AAV) that are not serologically distinct that may be within a subgroup or a variant of a given serotype.

[0094] In various exemplary embodiments, an AAV vector related to a reference serotype has a polynucleotide, polypeptide or subsequence thereof that includes or consists of a sequence at least 80% or more (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, etc.) identical to one or more AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV-2i8 (e.g., such as an ITR, or a VP1, VP2, and / or VP3 sequences).

[0095] In some embodiments, compositions, methods and use of the invention include AAV sequences (polypeptides and nucleotides), and subsequences thereof that exhibit less than 100% sequence identity to a reference AAV serotype such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV-2i8, but are distinct from and not identical to known AAV genes or proteins, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12, or AAV-2i8, genes or proteins, etc. In one embodiment, an AAV polypeptide or subsequence thereof includes or consists of a sequence at least 75% or more identical, e.g., 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical to any reference AAV sequence or subsequence thereof, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV-2i8 (e.g., VP1, VP2 and / or VP3 capsid or ITR). In particular aspects, an AAV variant has 1, 2, 3, 4, 5, 5-10, 10-15, 15-20 or more amino acid substitutions.

[0096] Recombinant AAV vectors, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AA112, or AAV-2i8 and variant, related, hybrid and chimeric sequences, can be constructed using recombinant techniques that are known to the skilled artisan, to include one or more nucleic acid sequences (transgenes) flanked with one or more functional AAV ITR sequences.

[0097] In some embodiments the viral vector comprises a heterologous nucleic acid. A “heterologous” nucleic acid merely refers to a polynucleotide inserted into a viral vector for purposes of viral vector mediated transfer / delivery of the polynucleotide into a cell. Heterologous polynucleotides are typically distinct from the viral nucleic acid, i.e., are “non-native” with respect to the particular virus used for delivery. Once transferred / delivered into the cell, a heterologous polynucleotide, contained within the viral vector can be expressed (e.g., transcribed, and translated if appropriate). Alternatively, a transferred / delivered heterologous polynucleotide in a cell, contained within the viral vector, need not be expressed. Although the term “heterologous” is not always used herein in reference to polynucleotides, reference to a polynucleotide even in the absence of the modifier “heterologous” includes heterologous polynucleotides in spite of the omission.

[0098] In some embodiments, the heterologous nucleic acid encodes a protein. Such proteins can be wild-type or a variant, modified or chimeric protein. A "variant protein" can mean a modified protein such that the modified protein has an amino acid alteration compared to wild-type protein. A "human protein" for use in the vectors of the invention is preferably a highly conserved protein which would not be recognized as a foreign or non-self antigen by the human immune system. Proteins encoded by a nucleic acid include therapeutic proteins.

[0099] The “polypeptides,” “proteins” and “peptides” encoded by nucleic acid sequences include full-length native sequences, as with naturally occurring proteins, as well as functional subsequences, modified forms or sequence variants. In methods and uses of the invention, such polypeptides, proteins and peptides encoded by the polynucleotide sequences can be but are not required to be identical to an endogenous protein that is defective, or whose expression is insufficient, or deficient in the treated mammal.

[0100] A heterologous nucleic acid can also refer to a sequence which produces a transcript when transcribed. Such transcripts can be RNA, such as inhibitory RNA (RNAi, e.g., small or short hairpin (sh)RNA, microRNA (miRNA), piRNA, small or short interfering (si)RNA, trans-splicing RNA, or antisense RNA). In some embodiments, the nucleic acid comprises a sequence encoding an RNA molecule that modulates expression of a gene in the cell. In some embodiments, the expression of the gene is inhibited by the RNA molecule.

[0101] In some embodiments, the nucleic acid comprises a sequence for use in editing the genome of the transduced cell. In some embodiments, the nucleic acid comprises a sequence encoding a nuclease. The particular nuclease is not limiting. In some embodiments, the nuclease is selected from the group consisting of a CRISPR associated protein (Cas proteins, e.g., Cas9), Zinc finger nuclease (ZFN), Transcription Activator- Like Effector Nuclease (TALEN), and meganuclease.

[0102] In some embodiments, the heterologous nucleic acid comprises a sequence encoding a CRISPR / Cas system comprising: (a) a gRNA molecule comprising a targeting domain which is complementary with a target domain sequence of a gene in the cell and (b) a Cas9 molecule, wherein the CRISPR / Cas system is capable of modifying the gene. In some embodiments, the nucleic acid further comprises a template nucleic acid providing a corrected sequence used to edit the genome in the cell. In some embodiments, more than one viral vector or nucleic acid delivery vehicles (e.g., nanoparticles) are used to supply the components necessary for editing a genome.

[0103] In some embodiments, the nucleic acid can comprise an "expression operon" possessing operably linked transcriptional and translational control sequences, such as promoters, enhancers, translational start signals (e.g., ATG or AUG codons), polyadenylation signals, terminators, and the like, and which facilitate the expression of a polypeptide coding sequence in a cell or organism. The term "operably linked" means that the regulatory sequences necessary for expression of the coding sequence are placed in the DNA molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of transcription units and other transcription control elements (e.g. enhancers) in an expression vector.

[0104] In some embodiments, the nucleic acid comprises a promoter, and a polyadenylation sequence. The term "promoters" or "promoter" as used herein can refer to a DNA sequence that is located adjacent to a DNA sequence that encodes a recombinant product. A promoter is preferably linked operatively to an adjacent DNA sequence. A promoter typically increases an amount of recombinant product expressed from a DNA sequence as compared to an amount of the expressed recombinant product when no promoter exists. A promoter from one organism can be utilized to enhance recombinant product expression from a DNA sequence that originates from another organism. For example, a vertebrate promoter may be used for the expression of jellyfish GFP in vertebrates. In addition, one promoter element can increase an amount of recombinant products expressed for multiple DNA sequences attached in tandem. Hence, one promoter element can enhance the expression of one or more recombinant products. Multiple promoter elements are well-known to persons of ordinary skill in the art.

[0105] In some embodiments, high-level constitutive expression will be desired. Examples of such promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter / enhancer, the cytomegalovirus (CMV) immediate early promoter / enhancer (see, e.g., Boshart et al., Cell, 41:521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the cytoplasmic [3-actin promoter and the phosphoglycerol kinase (PGK) promoter.

[0106] In another embodiment, inducible promoters may be desired. Inducible promoters are those which are regulated by exogenously supplied compounds, either in cis or in trans, including without limitation, the zinc-inducible sheep metallothionine (MT) promoter; the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter; the T7 polymerase promoter system (WO 98 / 10088); the tetracycline-repressible system (Gossen etal., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)); the tetracycline-inducible system (Gossen et al., Science, 268:1766-1769 (1995); see also Harvey et al., Curr. Opin. Chem. Biol., 2:512-518 (1998)); the RU486-inducible system (Wang etal., Nat. Biotech., 15:239- 243 (1997) and Wang et al., Gene Ther., 4:432-441 (1997)]; and the rapamycin-inducible system (Magari et al., J. Clin. Invest., 100:2865-2872 (1997); Rivera et al., Nat. Medicine. 2:1028-1032 (1996)). Other types of inducible promoters which may be useful in this context are those which are regulated by a specific physiological state, e.g., temperature, acute phase, or in replicating cells only.

[0107] In another embodiment, the native promoter for a transgene or nucleic acid sequence of interest will be used. The native promoter may be preferred when it is desired that expression of the transgene or the nucleic acid sequence should mimic the native expression. The native promoter may be used when expression of the transgene or other nucleic acid sequence must be regulated temporally or developmentally, or in a tissuespecific manner, or in response to specific transcriptional stimuli. In a further embodiment, other native expression control elements, such as enhancer elements, polyadenylation sites or Kozak consensus sequences may also be used to mimic the native expression.

[0108] In one embodiment, the viral vector, e.g., is a recombinant AAV vector comprising a transgene operably linked to a tissue-specific promoter. Examples of promoters that are tissue-specific are known for liver albumin, Miyatake et al., J. Virol., 71:5124-32 (1997); hepatitis B vims core promoter, Sandig et al., Gene Ther. 3:1002-9 (1996); alphafetoprotein (AFP), Arbuthnot et al., Hum. Gene Ther., 7:1503- 14 (1996)], bone (osteocalcin, Stein etal., Mol. Biol. Rep., 24:185-96 (1997); bone sialoprotein, Chen et al., J. Bone Miner. Res. 11:654-64 (1996)), lymphocytes (CD2, Hansal et al., J. Immunol., 161:1063-8 (1998); immunoglobulin heavy chain; T cell receptor a chain), neuronal (neuron- specific enolase (NSE) promoter, Andersen et al. Cell. Mol. Neurobiol., 13:503- 15 (1993); neurofilament light-chain gene, Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991); the neuron- specific vgf gene, Piccioli etal., Neuron, 15:373-84 (1995)]; among others.

[0109] The term "enhancers" or "enhancer" as used herein can refer to a nucleic acid sequence that is located adjacent to the DNA sequence that encodes a recombinant product. Enhancer elements are typically located upstream of a promoter element or can be located downstream of or within a coding DNA sequence (e.g., a DNA sequence transcribed or translated into a recombinant product or products). Hence, an enhancer element can be located 100 base pairs, 200 base pairs, or 300 or more base pairs upstream or downstream of a DNA sequence that encodes recombinant product. Enhancer elements can increase an amount of recombinant product expressed from a DNA sequence above increased expression afforded by a promoter element. Multiple enhancer elements are readily available to persons of ordinary skill in the art.

[0110] In one embodiment, the heterologous nucleic acid encodes a "therapeutic molecule." In one embodiment, the therapeutic molecule is a peptide or protein that may alleviate or reduce symptoms that result from an absence or defect in a protein in a cell or subject. In some embodiments, a "therapeutic" peptide or protein encoded by a transgene is one that confers a benefit to a subject, c.g., to correct a genetic defect, to correct a gene (expression or functional) deficiency, or an anti-cancer effect.

[0111] Therapeutic peptides and proteins include, but are not limited to, cystic fibrosis transmembrane regulator protein (CFTR), dystrophin, utrophin, blood coagulation (clotting) factor (e.g., Factor XIII, Factor IX, Factor X, Factor VIII, Factor Vila, protein C, Factor VII, B domain-deleted Factor VIII, or a high-activity or longer half-life variant of coagulation factor, or an active or inactive form of a coagulation factor), a monoclonal antibody, retinal pigment epithelium- specific 65 kDa protein (RPE65), erythropoietin, LDL receptor, lipoprotein lipase, ornithine transcarbamylase, P-globin, a-globin, spectrin, a-antitrypsin, adenosine deaminase (ADA), a metal transporter (ATP7A or ATP7), sulfamidase, an enzyme involved in lysosomal storage disease (ARSA), hypoxanthine guanine phosphoribosyl transferase, P-25 glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase, branched-chain keto acid dehydrogenase, a hormone, a growth factor, insulin-like growth factor 1 or 2, platelet derived growth factor, epidermal growth factor, nerve growth factor, neurotrophic factor -3 and -4, brain-derived neurotrophic factor, glial derived growth factor, transforming growth factor a and p, a cytokine, a- interferon, P-interferon, interferon-y, interleukin-2, interleukin-4, interleukin- 12, granulocyte-macrophage colony stimulating factor, lymphotoxin, a suicide gene product, herpes simplex virus thymidine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine kinase, tumor necrosis factor, a drug resistance protein, a tumor suppressor protein (e.g., p53, Rb, Wt-1, NF1, Von Hippel-Lindau (VHL), SERCA2a, adenomatous polyposis coli (APC)), VEGF, microdystrophin, lysosomal acid lipase, arylsulfatase A and B, ATP7A and B, a peptide with immunomodulatory properties, a tolerogenic or immunogenic peptide or protein Tregitope or hCDRl, insulin, glucokinase, guanylate cyclase 2D (LCA-GUCY2D), Rab escort protein 1 (Choroideremia), LCA 5 (LCA-Lebercilin), ornithine ketoacid aminotransferase (Gyrate Atrophy), Retinoschisin 1 (X-linked Retinoschisis), USH1C (Usher’s Syndrome 1C), X-linked retinitis pigmentosa GTPase (XLRP), MERTK (AR forms of RP: retinitis pigmentosa), DFNB1 (Connexin 26 deafness), ACHM 2, 3 and 4 (Achromatopsia), PKD-1 or PKD-2 (Polycystic kidney disease), TPP1, CLN2, a gene product implicated in lysosomal storage diseases (e.g., sulfatases, N-acetylglucosamine-l-phosphate transferase, cathepsin A, GM2-AP, NPC1 , VPC2, a sphingolipid activator protein, or one or more zinc finger nucleases for genome editing, or donor sequences used as repair templates for genome editing, and any other peptide or protein that has a therapeutic effect in a subject in need thereof.

[0112] Further exemplary therapeutic peptides or proteins encoded by the heterologous nucleic acid include those that may be used in the treatment of a disease or disorder including, but not limited to, cystic fibrosis (and other diseases of the lung), hemophilia A, hemophilia B, thalassemia, anemia and other blood disorders, AIDS, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, epilepsy, and other neurological disorders, cancer, diabetes mellitus, muscular dystrophies (e.g., Duchenne, Becker), Gaucher's disease, Hurler's disease, adenosine deaminase deficiency, glycogen storage diseases and other metabolic defects, retinal degenerative diseases (and other diseases of the eye), and diseases of solid organs (e.g., brain, liver, kidney, heart).

[0113] As set forth herein, heterologous nucleic acid sequences can comprise inhibitory and antisense nucleic acid sequences. Inhibitory, antisense, siRNA, piRNA, miRNA, shRNA, RNAi and antisense oligonucleotides can modulate expression of a target gene. Such molecules include those able to inhibit expression of a target gene involved in mediation of a disease process, thereby reducing, inhibiting or alleviating one or more symptoms of a disease.

[0114] A “siRNA” refers to a therapeutic molecule involved in the RNA interference process for a sequence- specific post-transcriptional gene silencing or gene knockdown. siRNAs have homology with the sequence of the cognate mRNA of the targeted gene. In some embodiments, specific siRNA sequences for inhibiting mRNA of a target gene can be between 15-50 nucleotides in length, and more typically about 20-30 nucleotides in length. Such nucleic acid molecules can be incorporated into the viral vectors disclosed herein.

[0115] In some embodiments, the nucleic acid encodes an antigen that is present or overexpressed in a disease or condition. The disease can be, for instance, a cancer or infection by a pathogen such as a virus. An antigen associated with a disease state, can be an antigen present in cancer cells or tumors. In some embodiments, a tumor antigen refers to mutated forms of a protein, which only appears in that form in tumors, while the non-mutated form is observed in non-tumoral tissues.

[0116] In some embodiments, the antigen is from an infectious disease-causing pathogen. In some embodiments, the pathogen is a bacterial or viral pathogen. In some embodiments, the pathogen is selected from the group consisting of Streptococcus pneumonia, Neisseria meningitidis, Haemophilus influenza, Klebsiella spp., Pseudomonas spp., Salmonella spp., Shigella spp., and Group B streptococci, Bacillus anthracis adenoviruses; Bordetella pertussus; Botulism; bovine rhinotracheitis; Brucella spp.; Branhamella catarrhalis; canine hepatitis; canine distemper; Chlamydiae; Cholera; coccidiomycosis; cowpox; tularemia; filoviruses; arenaviruses; bunyaviruses; cytomegalovirus; cytomegalovirus; Dengue fever; dengue toxoplasmosis; Diphtheria; encephalitis; Enterotoxigenic Escherichia coli; Epstein Barr virus; equine encephalitis; equine infectious anemia; equine influenza; equine pneumonia; equine rhinovirus; feline leukemia; flavivirus; Burkholderia mallei; Globulin; Haemophilus influenza type b; Haemophilus influenzae; Haemophilus pertussis; Helicobacter pylori; Hemophilus spp.; hepatitis; hepatitis A; hepatitis B; Hepatitis C; herpes viruses; HIV; HIV-1 viruses; HIV-2 viruses; HTLV; Influenza; Japanese encephalitis; Klebsiellae spp. Legionella pneumophila; leishmania; leprosy; lyme disease; malaria immunogen; measles; meningitis; meningococcal; Meningococcal Polysaccharide Group A, Meningococcal Polysaccharide Group C; mumps; Mumps Virus; mycobacteria; Mycobacterium tuberculosis; Neisseria spp; Neisseria gonorrhoeae; ovine blue tongue; ovine encephalitis; papilloma; SARS and associated coronaviruses; Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV- 2) (COVID-19); parainfluenza; paramyxovirus; paramyxoviruses; Pertussis; Plague; Coxiella bumetti; Pneumococcus spp.; Pneumocystis carinii; Pneumonia; Poliovirus; Proteus species; Pseudomonas aeruginosa; rabies; respiratory syncytial virus; rotavirus; Rubella; Salmonellae; schistosomiasis; Shigellae; simian immunodeficiency virus; Smallpox; Staphylococcus aureus; Staphylococcus spp.; Streptococcus pyogenes; Streptococcus spp.; swine influenza; tetanus; Treponema pallidum; Typhoid; Vaccinia; varicella-zoster virus; and Vibrio cholera and combinations thereof.

[0117] Particular- non-limiting examples of genes (e.g., genomic DNA) or transcript of a pathogenic gene (e.g., RNA or mRNA) that can be targeted with an inhibitory heterologous nucleic acid in a viral vector include, but are not limited to: pathogenic genes associated with polynucleotide repeat diseases such as huntingtin (HTT) gene, a gene associated with dentatorubropallidolusyan atropy (e.g., atrophin 1, ATN1); androgen receptor on the X chromosome in spinobulbar muscular atrophy, human Ataxin-1, -2, -3, and -7, Cav2.1 P / Q voltage-dependent calcium channel is encoded by the (CACNA1A), TATA-binding protein, Ataxin 8 opposite strand, also known as ATXN80S, Serine / threonine-protein phosphatase 2A 55 kDa regulatory subunit B beta isoform in spinocerebellar ataxia (type 1, 2, 3, 6, 7, 8, 12 17), FMRI (fragile X mental retardation 1) in fragile X syndrome, FMRI (fragile X mental retardation 1) in fragile X-associated tremor / ataxia syndrome, FMRI (fragile X mental retardation 2) or AF4 / FMR2 family member 2 in fragile XE mental retardation; Myotonin-protein kinase (MT-PK) in myotonic dystrophy; Frataxin in Friedreich’s ataxia; a mutant of superoxide dismutase 1 (SOD1) gene in amyotrophic lateral sclerosis ; a gene involved in pathogenesis of Parkinson’ s disease and / or Alzheimer’ s disease; apolipoprotein B (APOB) and proprotein convertase subtilisin / kexin type 9 (PCSK9), hypercoloesterolemia; HIV Tat, human immunodeficiency virus transactivator of transcription gene, in HIV infection; HIV TAR, HIV TAR, human immunodeficiency virus transactivator response element gene, in HIV infection; C-C chemokine receptor (CCR5) in HIV infection; Rous sarcoma virus (RSV) nucleocapsid protein in RSV infection, liver-specific microRNA (miR-122) in hepatitis C virus infection; p53, acute kidney injury or delayed graft function kidney transplant or kidney injury acute renal failure; protein kinase N3 (PKN3) in advance recurrent or metastatic solid malignancies; LMP2, LMP2 also known as proteasome subunit beta-type 9 (PSMB 9), metastatic melanoma; LMP7, also known as proteasome subunit beta-type 8 (PSMB 8), metastatic melanoma; MECL1 also known as proteasome subunit beta-type 10 (PSMB 10), metastatic melanoma; vascular endothelial growth factor (VEGF) in solid tumors; kinesin spindle protein in solid tumors, apoptosis suppressor B-cell CLL / lymphoma (BCL-2) in chronic myeloid leukemia; ribonucleotide reductase M2 (RRM2) in solid tumors; Furin in solid tumors; polo-like kinase 1 (PLK1) in liver tumors, diacylglycerol acyltransferase 1 (DGAT1) in hepatitis C infection, beta-catenin in familial adenomatous polyposis; beta2 adrenergic receptor, glaucoma; RTP801 / Reddl also known as DAN damage-inducible transcript 4 protein, in diabetic macular edema (DME) or age-related macular degeneration; vascular endothelial growth factor receptor I (VEGFR1) in age-related macular degeneration or choroidal neovascularization, caspase 2 in non-artcritic ischaemic optic neuropathy; Keratin 6A N17K mutant protein in pachyonychia congenital; influenza A virus genome / gene sequences in influenza infection; severe acute respiratory syndrome (SARS) coronavirus genome / gene sequences in SARS infection; respiratory syncytial vims genome / gene sequences in respiratory syncytial vims infection; Ebola filovims genome / gene sequence in Ebola infection; hepatitis B and C virus genome / gene sequences in hepatitis B and C infection; herpes simplex virus (HSV) genome / gene sequences in HSV infection, coxsackievims B3 genome / gene sequences in coxsackievims B3 infection; silencing of a pathogenic allele of a gene (allele-specific silencing) like torsin A (T0R1A) in primary dystonia, pan-class I and HLA-allele specific in transplant; or mutant rhodopsin gene (RHO) in autosomal dominantly inherited retinitis pigmentosa (adRP).

[0118] The cells to be transduced are not limiting. The “cell” or “target cell” to be transduced according to the invention can be any cell that is targeted for transduction with the viral vector. The term “cell” as used in connection with the present invention can refer to a single and / or isolated cell or to a cell that is part of a multicellular entity such as a tissue, an organism or a cell culture. In other words, the method can be performed in vivo, ex vivo or in vitro. The cell is a eukaryotic cell. A eukaryotic cell as used herein, refers to any cell of a multi-cellular eukaryotic organism, including cells from animals like vertebrates. In some embodiments, the cell is a mammalian cell. The term “mammalian cell” as used herein, is well known in the art and refers to any cell belonging to or derived from an animal that is grouped into the class of mammalia. Furthermore, within a species one may choose a cell to be used in the method of the invention based on the tissue type and / or capacity to differentiate equally depending on the goal to be achieved by modifying the genome via transducing a target cell according to the method of the invention.

[0119] Three basic categories of cells, which in principle can be transduced with the method of the invention, make up the mammalian body: germ cells, somatic cells and stem cells. A germ cell is a cell that gives rise to gametes and thus is continuous through the generations. Stem cells can divide and differentiate into diverse specialized cell types as well as self-renew to produce more stem cells. In mammals there are two main types of stem cells: embryonic stem cells and adult stem cells. Somatic cells include all cells that are not a gametes, gametocytes or undifferentiated stem cells. The cells of a mammal can also be grouped by their ability to differentiate. A totipotent (also known as omnipotent) cell is a cell that is able to differentiate into all cell types of an adult organism including placental tissue such as a zygote (fertilized oocyte) and subsequent blastomeres, whereas pluripotent cells, such as embryonic stem cells, cannot contribute to extraembryonic tissue such as the placenta, but have the potential to differentiate into any of the three germ layers endoderm, mesoderm and ectoderm. Multipotent progenitor cells have the potential to give rise to cells from multiple, but limited number of cell lineages. Further, there are oligopotent cells that can develop into only a few cell types and unipotent cells (also sometimes termed a precursor cell) that can develop into only one cell type. There are four basic types of tissues: muscle tissue, nervous tissue, connective tissue and epithelial tissue that a cell to be used in the method of the invention can be derived from, such as, for example hematopoietic stem cells or neuronal stem cells. To the extent human cells are envisaged for use in the method of the invention, it is preferred that such human cells are not obtained from a human embryo, in particular not via methods entailing destruction of a human embryo. On the other hand, human embryonic stem cells are at the skilled person's disposal such as being taken from existent embryonic stem cell lines commercially available. Accordingly, the present invention may be worked with human embryonic stem cells without any need to use or destroy a human embryo. Alternatively, or instead of human embryonic stem cells, pluripotent cells that resemble embryonic stem cells such induced pluripotent stem (iPS) cells may be used, the generation of which is state of the art (Hargus G etal., 2010, Proc Natl Acad Sei USA, 107:15921-15926; Jaenisch R. and Young R„ 2008, Cell 132:567-582; Saha K, and Jaenisch R., 2009, Cell Stem Cell 5:584-595).

[0120] In some embodiments, the cells can be transduced ex vivo (e.g., in cells removed from a donor or removed from a subject to be treated). In some embodiments, the cells are transduced in vivo, by administering the heparin or analogues and viral vector to a subject. The target cells can be any kind of eukaryotic cells such as mammalian cells, in particular human, mouse, rat, monkey, dog or hamster cells.

[0121] Cells that may be transduced include cells of any tissue or organ type, of any origin (e.g., mesoderm, ectoderm or endoderm). Non-limiting examples of cells include liver (e.g., hepatocytes, sinusoidal endothelial cells), pancreas (e.g., beta islet cells), lung, central or peripheral nervous system, such as brain (e.g., neural, glial or ependymal cells) or spine, kidney, eye (e.g., retinal, cell components), spleen, skin, thymus, testes, lung, diaphragm, heart (cardiac), muscle or psoas, or gut (e.g., endocrine), adipose tissue (white, brown or beige), muscle (e.g., fibroblasts), synoviocytes, chondrocytes, osteoclasts, epithelial cells, endothelial cells, salivary gland cells, inner ear nervous cells or hematopoietic (e.g., blood or lymph) cells. Additional examples include stem cells, such as pluripotent or multipotent progenitor cells that develop or differentiate into liver (e.g., hepatocytes, sinusoidal endothelial cells), pancreas (e.g., beta islet cells), lung, central or peripheral nervous system, such as brain (e.g., neural, glial or ependymal cells) or spine, kidney, eye (retinal, cell components), spleen, skin, thymus, testes, lung, diaphragm, heart (cardiac), muscle or psoas, or gut (e.g., endocrine), adipose tissue (white, brown or beige), muscle (e.g., fibroblasts), synoviocytes, chondrocytes, osteoclasts, epithelial cells, endothelial cells, salivary gland cells, inner ear nervous cells or hematopoietic (e.g., blood or lymph) cells.

[0122] In some embodiments, the target cell to be transduced is a stem cell or multipotent cell. In some embodiments, the cell comprises hematopoietic cells. Hematopoietic stem cells (HSCs) give rise to committed hematopoietic progenitor cells (HPCs) that are capable of generating the entire repertoire of mature blood cells over the lifetime of an organism. The term "hematopoietic stem cell" or "HSC" refers to multipotent stem cells that give rise to the all the blood cell types of an organism, including myeloid (e.g., monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells), and lymphoid lineages (e.g., T-cells, B-cells, NK-cells), and others known in the ail (See Fei, R., et al., U.S. Patent No. 5,635,387 ; McGlave, et al., U.S. Patent No. 5,460,964 ; Simmons, P., et al., U.S. Patent No. 5,677,136 ; Tsukamoto, et al., U.S. Patent No. 5,750,397 ; Schwartz, et al., U.S. Patent No. 5,759,793 ; DiGuisto, et al., U.S. Patent No. 5,681,599 ; Tsukamoto, et al., U.S. Patent No. 5,716,827). When transplanted into irradiated animals or humans, hematopoietic stem and progenitor cells can repopulate the erythroid, neutrophil-macrophage, megakaryocyte and lymphoid hematopoietic cell pool.

[0123] As used herein, the term "progenitor" or "progenitor cells" refers to cells that have the capacity to self-renew and to differentiate into more mature cells. Many progenitor cells differentiate along a single lineage but may have quite extensive proliferative capacity. In some embodiments the cells are CD34+ cells, in particular a CD34+ cell collected from a subject in need of a gene therapy of his / her hematopoietic lineage.

[0124] In some embodiments, the cells are immune cells, such as lymphocytes. In some embodiments, the immune cells are selected from B lymphocytes, T lymphocytes, dendritic cells, macrophages, natural killer cells, and NK92 cells. In some embodiments, the cells are T-cells that are engineered according to the methods herein to express on their cell surface a chimeric antigen receptor (CAR) directed against one or more antigens. By “chimeric antigen receptor” or “CAR” is generally meant a synthetic receptor comprising a targeting moiety that is associated with one or more signaling domains in a single fusion molecule. As defined herein, the term “chimeric antigen receptor” covers single chain CARs as well as multi-chain CARs. In some embodiments, the binding moiety of a CAR comprises an antigen-binding domain of a single-chain antibody (scFv), comprising light chain and heavy chain variable fragments of a monoclonal antibody joined by a flexible linker. Binding moieties based on receptor or ligand domains have also been used successfully. The signaling domains for first generation CARs are derived from the cytoplasmic region of the CD3zeta or the Fc receptor gamma chains. First generation CARs have been shown to successfully redirect T cell cytotoxicity. However, they failed to provide prolonged expansion and anti-tumor activity in vivo. Signaling domains from costimulatory molecules including CD28, OX-40 (CD134), and 4-1BB (CD137) have been added alone (second generation) or in combination (third generation) to enhance survival and increase proliferation of CAR modified T cells. CARs are not necessarily only single chain polypeptides, as multi-chain CARs are also possible. According to the multi-chain CAR architecture, for instance as described in WO 2014 / 039523, the signalling domains and co- stimulatory domains are located on different polypeptide chains. Such multi-chain CARs can be derived from FcsRI, by replacing the high affinity IgE binding domain of FcsRI alpha chain by an extracellular ligand-binding domain such as scFv, whereas the N- and / or C-termini tails of FcsRI beta and / or gamma chains are fused to signal transducing domains and co- stimulatory domains respectively. The extracellular ligand binding domain has the role of redirecting T-cell specificity towards cell targets, while the signal transducing domains activate the immune cell response. In some embodiments, the cells to be transduced are primary cells. The term "primary cell" as used herein is known in the art to refer to a cell that has been isolated from a tissue and has been established for growth in vitro or ex vivo. Corresponding cells have undergone very few, if any, population doublings and are therefore more representative of the main functional component of the tissue from which they are derived in comparison to continuous cell lines, thus representing a more representative model to the in vivo state. Methods to obtain samples from various tissues and methods to establish primary cell lines are well-known in the art (see, e.g., Jones and Wise, Methods Mol Biol. 1997). Primary cells may be derived from, e.g., blood, lymphoma and epithelial tumors.

[0125] The cells and methods herein can be part of an autologous or part of an allogenic treatment. By autologous, it is meant that cells used for treating patients originate from said patient. By allogeneic is meant that the cells or population of cells used for treating patients are not originating from said patient but from a donor.

[0126] The administration of the cells, viral vector and heparin or analogues thereof to a subject can be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous or intralymphatic injection, or intraperitoneally. In one embodiment, the cell compositions are administered by intravenous injections, where there are capable of migrating to their desired site action.

[0127] An effective amount means an amount which provides a therapeutic or prophylactic benefit. The dosage administrated will be dependent upon the age, health and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment and the nature of the desired effect. In some embodiments, the administration of the cells or population of cells comprises administration of about 104- 109cells per kg body weight. In some embodiments, about 105to 106cells / kg body weight are administered. All integer values of cell numbers within those ranges are contemplated. In some embodiments, about IxlO7to about 5xlOnviral particles are administered to the subject.

[0128] The cells, viral vector, and heparin or analogue thereof, can be administrated in one or more doses. In another embodiment, an effective amount of cells, viral vector, or heparin or analogue thereof are administrated as a single dose. In another embodiment, an effective amount of cells, viral vector, and heparin or analogue thereof arc administrated as more than one dose over a period of time. Timing of administration is within the judgment of managing physician and depends on the clinical condition of the patient.

[0129] In certain embodiments, the transduced cells are administered to the subject as combination therapy comprising immunosuppressive agents. Exemplary immunosuppressive agents include sirolimus, tacrolimus, cyclosporine, mycophenolate, anti-thymocyte globulin, corticosteroids, calcineurin inhibitor, anti-metabolite, such as methotrexate, post-transplant cyclophosphamide or any combination thereof. In some embodiments, the subject is pretreated with only sirolimus or tacrolimus as prophylaxis against GVHD. In some embodiments, the cells are administered to the subject before an immunosuppressive agent. In some embodiments, the cells are administered to the subject after an immunosuppressive agent. In some embodiments, the cells are administered to the subject concurrently with an immunosuppressive agent. In some embodiments, the cells are administered to the subject without an immunosuppressive agent. In some embodiments, the patient receiving genetically modified cells receives an immunosuppressive agent for less than 6 months, 5 months, 4 months, 3 months, 2 months, 1 month, 3 weeks, 2 weeks, or 1 week.

[0130] In some embodiments, large scale viral particle production is often necessary to achieve a reasonable viral titer. Viral particles are produced by transfecting a transfer vector into a packaging cell line that comprises viral structural and / or accessory genes, e.g., gag, pol, env, tat, rev, vif, vpr, vpu, vpx, or nef genes or other retroviral genes.

[0131] As used herein, the term "packaging vector" refers to an expression vector or viral vector that lacks a packaging signal and comprises a polynucleotide encoding one, two, three, four or more viral structural and / or accessory genes. Typically, the packaging vectors are included in a packaging cell, and are introduced into the cell via transfection, transduction or infection. Methods for transfection, transduction or infection are well known by those of skill in the art. A lentiviral transfer vector contemplated may be introduced into a packaging cell line, via transfection, transduction or infection, to generate a producer cell or cell line. The packaging vectors can be introduced into human cells or cell lines by standard methods including, e.g., calcium phosphate transfection, lipofection or electroporation. The packaging vectors may be introduced into the cells together with a dominant selectable marker, such as neomycin, hygromycin, puromycin, blastocidin, zeocin, thymidine kinase, DHFR, Gin synthetase or ADA, followed by selection in the presence of the appropriate drug and isolation of clones. A selectable marker gene can be linked physically to genes encoding by the packaging vector, e.g., by IRES or self-cleaving viral peptides.

[0132] Viral envelope proteins (env) determine the range of host cells which can ultimately be infected and transformed by recombinant retroviruses generated from the cell lines. In the case of lentiviruses, such as HIV-1, HIV-2, SIV, FIV and EIV, the env proteins include gp41 and gpl20. Preferably, the viral env proteins expressed by packaging cells are encoded on a separate vector from the viral gag and pol genes, as has been previously described.

[0133] Illustrative examples of retroviral-derived env genes which can be employed include, but are not limited to: MLV envelopes, 10A1 envelope, BAEV, FeLV-B, RD114, SSAV, Ebola, Sendai, FPV (Fowl plague virus), and influenza virus envelopes. Similarly, genes encoding envelopes from RNA viruses (e.g., RNA virus families of Picomaviridae, Calciviridae, Astroviridae, Togaviridae, Flaviviridae, Coronaviridae, Paramyxoviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae, Bunyaviridae, Arenaviridae, Reoviridae, Bimaviridae, Retroviridae) as well as from the DNA viruses (families of Hepadnaviridae, Circoviridae, Parvoviridae, Papovaviridae, Adenoviridae, Herpesviridae, Poxyiridae, and Iridoviridae) may be utilized. Representative examples include, FeLV, VEE, HFVW, WDSV, SFV, Rabies, ALV, BIV, BLV, EBV, CAEV, SNV, ChTLV, STLV, MPMV, SMRV, RAV, FuSV, MH2, AEV, AMV, CT10, and EIAV.

[0134] Envelope proteins for pseudotyping a virus may include, but may not be limited to any of the following virus: Influenza A such as H INI, H1N2, H3N2 and H5N1 (bird flu), Influenza B, Influenza C virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rotavirus, any virus of the Norwalk virus group, enteric adenoviruses, parvovirus, Dengue fever virus, Monkey pox, Mononegavirales, Lyssavirus such as rabies virus, Lagos bat virus, Mokola virus, Duvenhage virus, European bat virus 1 & 2 and Australian bat vims, Ephemerovirus, Vesiculovirus, Vesicular Stomatitis Vims (VSV), Herpesvimses such as Herpes simplex vims types 1 and 2, varicella zoster, cytomegalovirus, Epstein-Bar virus (EBV), human herpesviruses (HHV), human herpesvirus type 6 and 8, Human immunodeficiency virus (HIV), papilloma virus, murine gammaherpesvirus, Arenaviruses such as Argentine hemorrhagic fever virus, Bolivian hemorrhagic fever virus, Sabia-associated hemorrhagic fever virus, Venezuelan hemorrhagic fever virus, Lassa fever virus, Machupo virus, Lymphocytic choriomeningitis virus (LCMV), Bunyaviridiae such as Crimean-Congo hemorrhagic fever virus, Hantavirus, hemorrhagic fever with renal syndrome causing virus, Rift Valley fever virus, Filoviridae (filovirus) including Ebola hemorrhagic fever and Marburg hemorrhagic fever, Flaviviridae including Kaysanur Forest disease virus, Omsk hemorrhagic fever virus, Tick- borne encephalitis causing virus and Paramyxoviridae such as Hendra virus and Nipah virus, variola major and variola minor (smallpox), alphaviruses such as Venezuelan equine encephalitis virus, eastern equine encephalitis virus, western equine encephalitis virus, SARS-associated coronavirus (SARS-CoV), West Nile virus, any encephaliltis causing virus.

[0135] Packaging cells which produce recombinant retrovirus, e.g., lentivirus, pseudotyped with the VSV-G glycoprotein may be contemplated.

[0136] The terms "pseudotype" or "pseudotyping" as used herein, refer to a virus whose viral envelope proteins have been substituted with those of another virus possessing preferable characteristics. For example, HIV can be pseudotyped with vesicular stomatitis virus G-protein (VSV-G) envelope proteins, which allows HIV to infect a wider range of cells because HIV envelope proteins (encoded by the env gene) normally target the virus to CD4+ presenting cells. Lentiviral envelope proteins may be pseudotyped with VSV-G. Packaging cells may produce recombinant retrovirus, e.g., lentivirus, pseudotyped with the VSV-G envelope glycoprotein.

[0137] As used herein, the term "packaging cell lines" is used in reference to cell lines that do not contain a packaging signal but do stably or transiently express viral structural proteins and replication enzymes (e.g., gag, pol and env) which are necessary for the correct packaging of viral particles. Suitable cell lines may be employed to prepare packaging cells. Generally, the cells are mammalian cells. The cells used to produce the packaging cell line may be human cells. Suitable cell lines which can be used include, for example, CHO cells, BHK cells, MDCK cells, C3H 10T1 / 2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B- 50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211A cells. The packaging cells may be 293 cells, 293T cells, 293F cells, or A549 cells.

[0138] As used herein, the term "producer cell line" refers to a cell line which is capable of producing recombinant retroviral particles, comprising a packaging cell line and a transfer vector construct comprising a packaging signal. The production of infectious viral particles and viral stock solutions may be carried out using conventional techniques. Methods of preparing viral stock solutions are known in the art and are illustrated by, e.g., Y. Soneoka et al., (1995) Nucl. Acids Res. 23:628-633, and N. R. Landau et al., (1992) J. Virol. 66:5110-5113. Infectious virus particles may be collected from the packaging cells using conventional techniques. For example, infectious particles can be collected by cell lysis, or collection of the supernatant of the cell culture, as is known in the art. Optionally, the collected virus particles may be purified if desired. Suitable purification techniques are well known to those skilled in the art, e.g., Kutner et al., BMC Biotechnol. 2009;9:10. doi: 10.1186 / 1472-6750-9-10; Kutner et al., Nat. Protoc. 2009;4(4):495-505. doi: 10.1038 / nprot.2009.22.

[0139] The pharmaceutical compositions can be formulated according to known methods for preparing pharmaceutically acceptable useful compositions and may include a pharmaceutically acceptable carrier. The carrier may be liquid, solid, or semi-solid for example. Formulations are described in a number of sources which are well known to those of skill in the art. The physical and / or chemical characteristics of compositions of the inventions may be modified or optimized according to skill in the art, depending on the mode of administration. The compositions may be in any suitable form, depending on the desired method of administration.

[0140] The pharmaceutical composition may be adapted for administration by any appropriate route, for example by the oral, rectal, nasal, topical, vaginal or parenteral routes.

[0141] Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation substantially isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. Excipients which may be used for injectable solutions include water, alcohols, polyols, glycerine and vegetable oils, for example. The compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in freeze-dried conditions requiring only the addition of a sterile liquid immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. The pharmaceutical compositions may contain preserving agents, solubilizing agents, stabilizing agents, wetting agents, emulsifiers, salts, buffers, antioxidants, etc.

[0142] Application of the teachings of the present invention to a specific problem is within the capabilities of one having ordinary skill in the art in light of the teaching contained herein. Examples of the compositions and methods of the invention appear in the following non-limiting Examples.

[0143] EXAMPLES

[0144] Example 1. Heparin and Its Low Molecular Analogues Improve Transduction Efficiency of Enveloped Viruses

[0145] This example demonstrates that the transduction efficiency of lentivirus with foamy envelope in Jurkat and Raw 264.7 cells, both in serum-free and serum-containing media, can be improved by low concentrations of heparin and its low molecular weight analogues. Materials and Methods

[0146] Cell Lines and culture conditions:

[0147] A549 (human lung carcinoma), RAW 264.7 (tumor-derived macrophage cell lineage from BALB / c mice), Jurkat (immortalized T lymphocyte cell line), HEK293T (derived from a human embryonic kidney) and Hep2 (a malignant tumor known as an epidermoid carcinoma, derived explicitly from the larynx) cell lines were cultured and maintained in high glucose Dulbecco’ s modified Eagle’ s medium DMEM (Gibco, USA) or RPMI 1640 (Gibco, USA), supplemented with 10% fetal bovine serum (FBS) (Invitrogen) except that in Hep2 cells we used 5% FBS at 37 °C, in a humidified incubator with 5% CO2. All culture media were supplemented with 1% Penicillin lOOU / mL and Streptomycin 100 pg / mL. Cloning and viral particle production

[0148] For the production of RS V, we followed the protocol previously described (Ezzat et al., Nat. Commun., (2019), 10:1-16, doi: 10.1038 / s41467-019-10192). Briefly, Hep2 cells were infected with RSV when the cell confluency reached 70% to 80%. The multiplicity of infection (MOI) used was 4, and the cells were incubated for 6 days in VP-SFM medium (Thermo Fisher Scientific) supplemented with 50 pg / mL of gentamycin. Subsequently, the cells were gently scraped, vigorously mixed using a vortex mixer and subjected to sonication for 10 minutes. Following this, the sample was centrifuged at 1000 g for 5 minutes. The supernatant was collected and used for the heparin experiment. The infectious virus titers were assessed as the median tissue culture infectious dose (TCID50). MOI applied for infectivity assays were based on genome copies / mL determined by qPCR with the following probe and primers RSV-A probe 5'- CACCATCCAACGGAGCACAGGAGAT-3' (SEQ ID NO:1) (5' labeled with 6-FAM and three tagged with BHQ1), RSV-A forward primer 5'- AGATCAACTTCTGTCATCCAGCAA-3' (SEQ ID NOG), and RSV-A reverse 5'- TTCTGCACATCATAATTAGGAG-3' (SEQ ID NOG).

[0149] In the process of lentivirus production, 20 million HEK293T cells were cotransfected with 22.5 pg of a lentiviral vector. This vector was designed with a cytomegalovirus promoter and encoded inside a p2CL9IPw5 plasmid. The plasmid itself contains a fusion of CD63 and green fluorescent protein (GFP). 3.5 pg of a human foamy virus envelope pcoPE or vesicular stomatitis vims glycoprotein (VSV-G) envelope and 22.5 pg of packaging plasmid pCD / NL-BH was performed in 3 mL of Opti-MEM media. Additionally, 3 mL of Opti-MEM medium mixed with a polyethylenimine (PEI) ratio of 3:1 was used for transfection. The plasmids were then added to PEI, and the whole mixture was left to incubate for 20 minutes at room temperature. Subsequently, 20 mL of a DMEM medium supplemented with 10% FBS was mixed with the transfection reagent, and the mixture was subsequently added to the HEK293T cells and incubated at 37° C. At 24 h, 10 uM sodium butyrate (Sigma- Aldrich) was added to the cells to induce the gene expression, after which cells were cultured for another 6-8 hours before having their media replaced with new DMEM media containing serum and incubated for another 24 hrs. Viral particles were harvested first by removing the debris at low- speed centrifugation of 500g for 5 minutes and filtration through a 0.45 pm filter. Second, the viruses were concentrated by high-speed centrifugation at 25k g for 1.5 hours using Amicon-20 columns. Then, the concentrated virus was stored at -80° C in serum-free DMEM or RPMI media. We quantified the infectious lentivirus vectors in HEK293T cells to determine MOI by infecting the cells with a serially diluted viral stock. After 72 hrs, we evaluated the transduced cells via flow cytometry.

[0150] Cell transduction

[0151] Cells from three different lines (A549, Jurkat, and RAW 264.7) were each seeded in a 96- well plate (10,000 cells / well) and grown in medium with or without serum. The next day, the cells were treated with heparin sodium salt from porcine intestinal mucosa (Sigma H3393), enoxaparin sodium (EQ 180000), dalteparin sodium (Sigma D0070000), and tinzaparin (Sigma T 1490000) at concentrations of Img / mL, 100 pg / mL, 10 pg / mL, 0.5 pg / mL, 0.25 pg / mL, and 0.125 pg / mL and then infected with the viruses for 48 hours at 37° C and 5% CO2.

[0152] Cell fixation and screening by flow cytometry

[0153] After 2 days of cellular infection, the cells were washed twice with PBS and stained with a LIVE / DEAD flexible far red cell stain kit (Invitrogen) and incubated for 30 minutes at 4°C. Cells were then washed with PBS and trypsinized for 5 minutes at 37°C, after which cells were resuspended with DMEM serum-containing media. After that, cells were centrifuged at 1000 g, and the media was aspirated gently. 100 pl of 4% paraformaldehyde (PFA) 4% (VWR Chemicals) was added to the cells and incubated for 10 minutes at room temperature. Afterward, cells were centrifuged at 1000 g for 5 minutes, and PFA was removed. Cells were subsequently resuspended with PBS containing 0.5% FBS. After fixation, the transduced cells were analyzed by flow cytometry (MACSquant, Milteny) using FlowJo 10.8.1 software (LLC, Ashland, OR, USA) to detect GFP signals corresponding to transduction efficacy.

[0154] Fluorescence microscopy

[0155] Cell imaging was performed using a fluorescence microscope (Olympus 1X81, Olympus America Inc. Center Valley, PA, USA) to visualize the GFP protein in RAW 264.7 transduced with foamy envelope GFP lentivirus with or without heparin / heparin analogues. Live cell imaging assays

[0156] Live cell imaging was performed as described in (Rasul et al., Cure. Issues Mol. Biol., (2022), 44:3859-3871, doi: 10.3390 / cimb44090265). The IncuCyte machine was placed in a humidified incubator at 37 °C and 5% CO2. 10,000 Jurkat cells were seeded per well in a 96-well plate. Before analysis, the foamy envelope GFP lentivirus and different concentrations of heparin, enoxaparin and dalteparin were added to the cells. The number of green cells was monitored (phase + green, four images / well) every four hours over 120 hours using an IncuCyte® S3 Live Cell Analysis System (Sartorius).

[0157] Heparan Sulfate Proteoglycan Inhibition

[0158] Lactoferrin from bovine milk (Sigma L9507) was added to the cells and incubated at 37°C with 5% CO2 for one hour. After that, heparin, enoxaparin and dalteparin at 100 pg / mL, 10 pg / mL and 1 pg / mL were added, followed by virus infection. After 48 hours, the cells were fixed and run with the FACS machine.

[0159] Statistical Analysis

[0160] Statistical analysis was assessed by performing at least two independent experiments by applying the standard error of the mean ± SEM. Significant. Dunnetf s multiple comparison test and one-way ANOVA were applied to evaluate statistical significance. Moreover, Bonferroni's multiple comparison test or unpaired t-test was applied using GraphPad Prism 9 software developed by GraphPad Software (San Diego, CA, USA). Significant differences were determined for all cases with a significance level of p < 0.05 (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

[0161] Results

[0162] GFP expression of RSV enhanced in A549 cells

[0163] During the COVID-19 pandemic, we started screening heparin and some analogues; heparin (~ 15k Da), enoxaparin (-4500 Da), dalteparin (-6500 Da), and tinzaparin (-6500 Da), to test their potential use as inhibitors for respiratory viral infections. RSV virus served as a good model system for such an initial screen. In addition to sharing respiratory symptoms, RSV and COVID-19 viruses contain similar viral envelopes and spike proteins necessary for cell entry

[0031] . We infected A549 cells with GFP-expressing RSV at a MOI of 5 for two days in the presence of heparin, enoxaparin, dalteparin, or tinzaparin. While inhibition of the transduction efficiency was evident at high dosages of 1 mg / mL and 100 pg / mL for enoxaparin, dalteparin, and tinzaparin, a notable enhancement in transduction efficiency was observed at lower concentrations of 1 pg / mL, 0.5 pg / mL, 0.25 pg / mL, and 0.125 pg / mL. Notably, this enhancement was only apparent at a heparin concentration of 0.125 g / mL.

[0164] Heparin and its low molecular weight analogues enhance the transduction efficiency of foamy-enveloped lentivirus in Jurkat and RAW 264.7 cells

[0165] Immune cells are known to be notoriously refractory to transfection with nonviral delivery reagents. Therefore, viral vectors such as lentiviruses have been extensively explored (Swainson et al., Methods Mol. Biol., (2008), 415:301-320, doi: 10.1007 / 978-1- 59745-570- 1_18). However, high viral titers are frequently needed. Therefore, finding ways to enhance viral transduction would greatly benefit its clinical use. To this end, and in light of the observed transduction enhancement of RSV at low heparin doses, we next studied how heparin and its analogues would impact lentiviral transduction (Turner et al., CD46-Mediated Transduction of a Species D Adenovirus Vaccine Improves Mucosal Vaccine Efficacy, (2014), 374:364-374, doi: 10.1089 / hum.2013.215). To demonstrate the effect of high and low heparin dosages on viral transduction, we examined the transduction efficiency of lentivirus with a foamy envelope at MOI 80 in Jurkat and MOI 400 in RAW 264.7 cells. At low dosages (0.125-10 pg / mL) in serum-free medium, the transduction efficiency was significantly enhanced in Jurkat and RAW 264.7 cells (Figures 2, 3, and 15). The transduction efficiency of Jurkat cells treated with heparin and its analogues were 2.5-fold higher than that of transduced cells with no additives (Figure 2 and 15).

[0166] Additionally, additive-treated RAW 264.7 cells had 5.5-fold higher transduction efficiency than untreated RAW 264.7 cells (Figure 3). Moreover, a similar enhancement pattern was observed when we analyzed the mean fluorescence intensity of Jurkat and RAW 264.7 cells in serum-free media (Figures 9 and 12). As expected, transduction efficiency was dramatically reduced with high doses of heparin and its analogues.

[0167] In serum-containing media, the highest transduction efficiency of Jurkat cells treated with enoxaparin and dalteparin was at two doses: 100 pg / mE and 10 pg / mE. However, the observed enhancement effect was at or below a 100 pg / mL dosage for both heparin and tinzaparin (Figures 11 and 16). Furthermore, transduction efficiency in RAW 264.7 cells was enhanced using low molecular weight analogues at 100 pg / mL and 10 pg / mL concentrations. Tn addition, unfractionated heparin showed significant enhancement at doses below 10 pg / mL (Figures 10, 13, 14, 18). Again, high doses of these polyanions blocked viral infectivity, in keeping with results from the literature.

[0168] The transduction efficiency of foamy envelope lentivirus was dramatically increased in Jurkat cells treated with 10 pg / mL enoxaparin at various MOIs, as shown in Figure 17, when the same experiment was applied in a serum-containing medium. The transduction efficiency of enoxaparin-treated Jurkat cells was almost 80% at a MOI of 500, whereas that of untreated Jurkat cells was 10% irrelevant to the MOI of the virus. Furthermore, the Jurkat cells treated with enoxaparin showed steady transduction enhancement at higher MOIs than untreated cells, whose transduction progressively increased over time.

[0169] Next, our findings were further confirmed using fluorescence microscopy. RAW 264 cells were infected with the foamy envelope lentivirus and then treated with heparin and its analogues at 100 pg / mL, 10 pg / mL, or 1 pg / mL concentrations before being imaged. We found that the expression of GFP was significantly enhanced in cells treated with heparin and its low molecular weight analogues, as compared to the non-heparin treated cells (Figure 4).

[0170] Additionally, the results of the Jurkat cells’ transduction at 24, 48, 72, 96 and 120 hrs, monitored by incuCyte, showed increased GFP expression over time. Furthermore, the transduction of the Jurkat cells exposed to the foamy-enveloped virus in the presence of 1 pg / mL of dalteparin exhibited a more rapid expression rate (within 24 hours) than the other treatment conditions (Figure 19).

[0171] Heparin and enoxaparin enhance the transduction efficiency of VSV-G enveloped lentivirus in Jurkat cells.

[0172] To investigate the potential efficacy of transduction between heparin and its analogues when used with another viral glycoprotein, we tested a VSV-G enveloped GFP encoding lentivirus. Specifically, we used two treatments, heparin and enoxaparin at Img / mL concentration, to evaluate their respective impacts on transduction efficiency. We observed that the expression of GFP was increased after two days at the concentration of Img / mL in Jurkat cells cultured in RPMI serum-containing media (Figure 5). In addition, the mean fluorescence intensity increased significantly (Figure 20). Heparin and analogues enhance viral transduction efficiency by enhancing viral uptake.

[0173] To discern whether the enhancements in transduction efficacy were a result of enhanced viral uptake into cells, GFP-labelled VSV-G envelope lentivirus was added to Jurkat cells at MOI 53 cultured in RPMI serum-containing medium for 8 hrs, followed by subsequent analysis by flow cytometry. Figures 6 and 21 show that the results revealed a significant uptake enhancement when exposed to heparin and its analogues at various concentrations. This effect was most prominent at 1 mg / mL compared to Jurkat cells treated with the virus without additives (Figure 6).

[0174] The HSPG-binding protein lactoferrin reverses the transduction- enhancing effects of heparin and analogues.

[0175] We hypothesized that the enhancement in transduction results from heparin binding to both the virus and HSPGs on the cell surface, thereby acting as a bridge for uptake.

[0176] To test this hypothesis, we used Lactoferrin, a selective inhibitor of HSPGs, in Jurkat cells treated with foamy-enveloped lentivirus. The results clearly showed that Lactoferrin significantly inhibited heparin-induced enhancement of the transduction (Figure 7).

[0177] Discussion

[0178] Viral transduction is widely used to deliver genetic material into cells for various research and medical applications. However, it has some limitations. Targeting specific tissues or organs might be difficult if the vector has low transduction efficiency. Additionally, some viral vectors have a limited capacity to carry genetic material (J. T. Bulcha et al., Target. Ther., (2021), 6:doi: 10.1038 / s41392-021-00487-6). Finally, certain viral vectors, such as retroviruses, can integrate the transgene into the host cell's genome by a non-specific process that randomly disrupts crucial genes or regulatory elements (Butt et al., Genes (Basel)., (2022), 13:doi: 10.3390 / genes 13081370).

[0179] Several techniques have been developed to enhance the transduction efficiency of viral vectors. These techniques include genetically modifying viruses, physical methods, and chemical methods. The latter contains chemical additives that are either covalently or noncovalently bound to the virus's outer surface. Non-covalent strategies are more common, but excessive toxicity and non-biodegradability of chemical additives is still a limitation (Kamimura et al., Pharmaceut. Med., (2011 ), 25:293-306:doi: 10.2165 / 11594020-000000000-00000).

[0180] It is reported that heparin treatment has an inhibitory effect on most viruses that use HSPGs as co-receptors for cellular entry, especially at high concentrations of 100 pg / mL or higher (Esko et al., J. Clin. Invest., (2001), 108:169-173, doi: 10.1172 / JCI200113530; Cagno et al., Viruses, (2019), 11:1-24, doi: 10.3390 / vl 1070596). Our study shows that heparin and its analogues increase the transduction efficiency at low doses via bridging the viral envelope to cellular receptors. This discovery has significant implications for understanding heparin's involvement in viral infection.

[0181] Heparin is a polysaccharide that binds with the binding site of heparan sulfate proteoglycans on the viral envelope to inhibit the entry of the virus into the cells (Mycroft- West et al., Thromb. Haemost., (2020), 120:1700-1715, doi: 10.1055 / s-0040-1721319). Surprisingly, we found that RS V infectivity was enhanced at low doses of heparin and its analogues. The transduction inhibition was noticeable at Img / mL for all treatments. These results agree with the data obtained by Krusat et al. (Krusat et al., Arch. Virol., (1997), 142:1247-1254, doi: 10.1007 / s007050050156), who demonstrated that the inhibition of RSV started at 0.32 pg / mL and higher doses. However, our study observed the enhancement at 0.125 pg / mL for heparin and 1 pg / mL to 0.125 pg / mL for low molecular weight analogues (Figure 1).

[0182] Following our observation with RSV, we sought to broaden our study to other viruses with potential medical applications, such as lentiviruses used in gene therapy to transport therapeutic genes into target cells to address genetic disorders. The lentiviral vectors can integrate the therapeutic gene into the host cell's genome, ensuring the longterm expression of the desired protein. Lentiviral vectors have been used to engineer immune cells, such as T cells, to express chimeric antigen receptors (CARs) for use in cancer immunotherapy. CAR-T cell therapy, for example, has shown remarkable success in developing drugs for certain types of blood cancers, particularly B cell lymphoma, such as Kymriah and Yescarta (U.S. Food and Drug Administration, BLA Approv., (2017), Available:

[0183] U.S. Food and Drug Administration, BLA Approv., (2017), Available: http: / / www.fda.gov / Drugs / GuidanceComplianceRegulatoryInformation / Surveillance / %0 Ahttps: / / www.fda.gov / downloads / BiologicsBloodVaccincs / CcllularGcncThcrapyProduct s / Appro vedProductsZUCM581259.pdf). Our work investigated enhancing the transduction efficiency in lymphocytes such as Jurkat cells and macrophages such as RAW 264.7 cells that can be genetically modified.

[0184] Here, we demonstrate that the transduction efficiency of lentivirus with foamy envelope in Jurkat and Raw 264.7 cells, both in serum-free and serum-containing media, can be improved by low concentrations of heparin and its low molecular weight analogues (Figure 2 and Figure 3). Additionally, we used VSV-G as a pseudotyping envelope protein, a prevalent technique to generate viral vectors intended for gene delivery. The VSV-G protein is recognized for its extensive cellular tropism by binding to the LDL-R abundant in many cell types (Sun et al., Virology, (2005), 338:53-60, doi:

[0185] 10.1016 / j.virol.2005.05.006). In this context, we noted a substantial increase in viral binding to cells and viral transduction when heparin and enoxaparin were introduced to Jurkat cells at a concentration of 1 mg / mL. In contrast what was reported before with the inhibitory impact became noticeable at 50 ug / mL concentration in HT1080 cells (Guibinga et al., Mol. Ther., (2002), 5:538-546, doi: 10.1006 / mthe.2002.0578).

[0186] Consequently, the varying responses seen in differently coated viruses suggest distinct uptake mechanisms, necessitating tailored adjustments in heparin dosage for each virus type. We hypothesized that heparin and its low molecular equivalents bridged with HSPGs, significantly increasing viral entry. In a manner consistent with other work with viral bridging, Gas6 has been shown to promote Ebola virus entrance by binding tyrosineprotein kinase receptor 3 (TYRO3)-AXL on target cells to the phosphatidylserine expressed on the viral envelope (Morizono et al., Cell Host Microbe, 9:286-298, (2011), doi: 10.1016 / j.chom.2011.03.012). In addition, these results are in line with other work demonstrating that the presence of blood coagulation factor X (FX) can facilitate viral entry by acting as a bridge between the adenovirus 5 (Ad5) capsid and heparan sulfate proteoglycans HSPGs in the liver of mice with a deleted Ext I gene (Zaiss et al. , J. Virol. , (2016), 90:412-420, doi: 10.1128 / jvi.01939-15).

[0187] To further elucidate whether HSPGs arc responsible for the improved transduction efficiency, Jurkat cells were treated with Lactoferrin before treatment with heparin, enoxaparin, and dalteparin. Lactoferrin binds to HSPGs, which the virus uses to enter cells, thereby preventing the entry mechanism (Hara et al., Hepatol. Res., (2002), 24:228-235, doi: 10.1016 / S 1386-6346(02)00088-8; Kell et al., Front. Immunol., (2020), 11:1-15, doi: 10.3389 / fimmu.2020.01221). In line with the previous experiments, Lactoferrin treatment led to a significantly decrease in virus transduction (Figure 7). These findings suggest that Lactoferrin competes with heparin and its equivalents for cell binding, preventing the HSPGs from bridging the viral envelope.

[0188] To summarize, we discovered that heparin and its analogues form a bridge between the cells that use the HSPGs as a co-receptor and the enveloped viruses and that this bridge is not based on electrostatic interaction but on lipid-lipid interaction, hence facilitating viral entrance (Figure 8).

[0189] Example 2. Transduction of NK92 Cells with Heparin, Enoxaparin with or without (5Z)- 7-Oxozeaenol.

[0190] This example aims to evaluate the effect of heparin, enoxaparin, with and without (5Z)-7-Oxozeaenolon the lentiviral transduction efficiency of NK92 cells. Using MOI 50, transduction efficiency will be assessed at 72 hours post-infection, incorporating spinfection for enhanced viral entry and delayed IL-2 addition for optimal NK92 cell survival.

[0191] Materials & Reagents

[0192] Cell Culture & Medium

[0193] • NK92 cells (ATCC CRL-2407)

[0194] • SCGM medium (Stem Cell Growth Medium) with 20% FBS (no IL-2 at transduction)

[0195] • Proleukin (IL-2, 500 U / mL, added after 8 hours post-infection) Lentiviral Constructs & Transduction

[0196] • Lentiviral vector (e.g., GFP-encoding lentivirus)

[0197] • Multiplicity of infection (MOI): 50

[0198] • PolyBrene (optional, Sigma TR-1003-G)

[0199] Heparin & (5Z)-7-OxozeaenolTreatment

[0200] • Heparin sodium salt (Sigma H3393)

[0201] • Enoxaparin sodium (E0180000) • (5Z)-7-Oxozeaenol(Stock: 6 mM, Final: 7 pM)

[0202] Additional Reagents

[0203] • LIVE / DEAD™ Far Red Cell Stain Kit (Invitrogen)

[0204] • Dulbecco's Modified Eagle Medium (DMEM) with serum

[0205] • 4% Paraformaldehyde (PFA) (VWR Chemicals)

[0206] • PBS containing 0.5% FBS

[0207] Equipment

[0208] • 96- well V-bottom plate

[0209] • Centrifuge (for spinfection and media change)

[0210] • 37 °C incubator with 5% CO?

[0211] • Flow Cytometer (MACSquant, Miltenyi)

[0212] • FlowJo 10.8.1 software (LLC, Ashland, OR, USA)

[0213] Experimental Procedure

[0214] 1. Cell Seeding and Treatment (Day 0 - Same Day as Transduction)

[0215] A. Seed 20,000 NK92 cells per well in a 96-well V-bottom plate using SCGM medium with 20% FBS (without IL-2).

[0216] B. Treat cells immediately with either heparin or enoxaparin at different concentrations (1 mg / mL, 100 pg / mL, 10 pg / mL, 0.5 pg / mL, 0.25 pg / mL, 0.125 pg / mL).

[0217] C. In designated wells, add (5Z)-7-Oxozeaenol to achieve a final concentration of 7 pM.

[0218] 2. Lentiviral Transduction

[0219] A. Prepare the lentiviral particles at MOI 50.

[0220] B. Add the lentivirus directly to each well containing treated NK92 cells.

[0221] C. Centrifuge the plate at 1000 x g for 1 hour at 32°C to facilitate viral entry.

[0222] D. Following centrifugation, incubate the cells at 37 °C with 5% CO2 for 8 hours.

[0223] 3. Media Change & IL-2 Addition (After 8 Hours Post-Transduction)

[0224] A. Centrifuge the plate at 400 x g for 5 minutes to pellet the cells.

[0225] B. Carefully aspirate the supernatant without disturbing the cell pellet. C. Replace with fresh SCGM medium containing 20% FBS and Proleukin (IL-2, 500 U / mL).

[0226] D. Return the plate to the incubator at 37 °C with 5% CO2 and continue culturing the cells for 72 hours.

[0227] 4. Cell Fixation and Screening by Flow Cytometry (After 72 Hours Post-Transduction)

[0228] A. Wash the cells twice with PBS to remove excess debris.

[0229] B. Stain the cells with LIVE / DEAD™ Far Red Cell Stain Kit (Invitrogen) and incubate at 4°C for 30 minutes.

[0230] C. Wash the stained cells with PBS to remove excess dye.

[0231] D. Centrifuge the cells at 1000 x g, aspirate the media gently, and resuspend in 100 pL of 4% paraformaldehyde (PFA).

[0232] E. Incubate for 10 minutes at room temperature to fix the cells.

[0233] F. Centrifuge the fixed cells at 1000 x g for 5 minutes, aspirate the PFA, and resuspend the pellet in PBS containing 0.5% FBS.

[0234] G. Analyze transduced cells by flow cytometry (MACSquant, Miltenyi) using FlowJo 10.8.1 software to detect GFP expression, which corresponds to transduction efficiency.

[0235] Transduction efficiency with heparin or enoxaparin in combination (5Z)-7- Oxozeaenol is enhanced by at least about 10%, at least about 15%, at least about 25%, at least about 50%, at least about 75%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 225%, at least about 250%, at least about 275%, or at least about 300%, relative to the transduction efficiency with heparin or enoxaparin alone.

[0236] While the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the ait.

[0237] Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this invention pertains.

Claims

CLAIMSWc claim:

1. A method of enhancing the efficiency of viral transduction in cells comprising administering to cells an effective amount of heparin or an analogue thereof and an effective amount of a viral vector.

2. The method of claim 1, wherein the viral vector comprises a heterologous nucleic acid.

3. The method of claim 1 or 2, wherein the nucleic acid comprises a sequence encoding a protein.

4. The method of any of claims 1-3, wherein the nucleic acid comprises a sequence for use in editing the genome of the cell.

5. The method of any of claims 1-4, wherein the sequence encodes a nuclease.

6. The method of claim 5, wherein the nuclease is selected from the group consisting of a CRISPR associated protein (Cas proteins, e.g., Cas9), Zinc finger nuclease (ZFN), Transcription Activator-Like Effector Nuclease (TALEN), and meganuclease.

7. The method of any of claims 1-6, wherein the nucleic acid comprises a sequence encoding a CRISPR / Cas system comprising: (a) a gRNA molecule comprising a targeting domain which is complementary with a target domain sequence of a gene in the cell and (b) a Cas9 molecule, wherein the CRISPR / Cas system is capable of modifying the gene.

8. The method of any of claims 1-2, wherein the nucleic acid comprises a sequence encoding an RNA molecule that modulates expression of a gene in the cell.

9. The method of claim 8, wherein the RNA is selected from the group consisting of siRNA, piRNA, miRNA and combinations thereof.

10. The method of claim 3, wherein the protein is an antigen.

11. The method of claim 10, wherein the antigen comprises an antigen from cancer cells.

12. The method of claim 1, wherein the viral vector is an envelope virus.

13. The method of claim 1, wherein the viral vector is a non-envelope vims.

14. The method of claim 1, wherein the viral vector is a retrovirus.

15. The method of claim 1 , wherein the viral vector is a lentiviral vector.

16. The method of claim 1, wherein the viral vector is selected from the group consisting of herpes simplex virus, cytomegalovirus, human herpes virus, foot and mouth disease virus, John Cunningham polyomavirus, respiratory syncytial virus, dengue virus, pseudorabies virus, human papilomavirus, Venezuelan equine encephalitis virus, enterovirus, parainfluenza virus, echovirus, Merkel cell polyomavirus, hepatitis C virus, sindbis virus, human metapneumovirus, echovirus, hepatitis B virus / hepatitis, delta virus, adeno-associated virus, Semliki forest virus, zika virus, North American eastern equine encephalitis virus, vaccinia virus, human immunodeficiency virus, rhinovirus, Adenovirus, filovirus, coronavirus, norovirus, Akabane virus, Schmallenberg virus, Rift valley fever virus, coxsackie virus, rabies virus, yellow fever virus, swine vesicular disease virus, Japanese encephalitis virus, Theiler murine encephalomyelitis virus, west nile virus, human parechovirus, Hendra and Nipah viruses, tick-borne encephalitis virus, porcine reproductive and respiratory syndrome virus, human T cell leukemia virus, porcine circovirus, hepatitis E virus, Chikungunya virus, and Murray Valley encephalitis virus.

17. The method of claim 1, wherein the viral vector is an adeno-associated virus.

18. The method of claim 1, wherein the viral vector is a respiratory syncytial virus.

19. The method of any of claims 1-18, wherein the efficiency of viral transduction is dependent on the presence of heparan sulfate proteoglycans on the surface of the cells.

20. The method of any of claims 1-19, wherein the heparin or analogue thereof and viral vector are administered ex vivo or in vitro to the cells.

21. The method of claim 20, wherein the method further comprises treating a disease or condition in a subject, comprising administering to the subject an effective amount of the transduced cells.

22. The method of claim 21, wherein the cells are autologous.

23. The method of claim 21, wherein the cells are allogenic from a donor and the subject is a recipient.

24. The method of any of claims 1-23, wherein the cells are selected from liver (e.g., hepatocytes, sinusoidal endothelial cells), pancreas (e.g., beta islet cells), lung,central or peripheral nervous system, brain (e.g., neural, glial or ependymal cells), spine, kidney, eye (e.g., retinal, cell components), spleen, skin, thymus, testes, lung, diaphragm, heart (cardiac), gut (e.g., endocrine), adipose tissue (white, brown or beige), muscle, synoviocytes, chondrocytes, osteoclasts, epithelial cells, endothelial cells, salivary gland cells, inner ear nervous cells, hematopoietic (e.g., blood or lymph) cells, stem cells, pluripotent or multipotent progenitor cells, and hematopoietic (e.g., blood or lymph) cells.

25. The method of claim 23, wherein the cells comprise T lymphocytes, natural killer cells or NK92 cells.

26. The method of any of claims 1-25, wherein the heparin or analogue thereof and viral vector are administered in separate compositions.

27. The method of any of claims 1-25, wherein the heparin or analogue thereof and viral vector are administered in the same composition.

28. The method of any of claims 1-27, further comprising administering an effective amount of one or more agents that enhance transduction efficiency of the viral vector in cells.

29. The method of claim 28, wherein the one or more agents is selected from (5Z)-7- Oxozeaenol and a polaxamer compound.

30. The method of claim 28, wherein the (5Z)-7-Oxozeaenol is administered in an amount sufficient to achieve a concentration from about 0.04 pM to about 10 pM.

31. The method of claim 28, wherein the effective amount of (5Z)-7-Oxozeaenol is from 0.4 pM to about 1.5 pM.

32. The method of claim 28, wherein the effective amount of (5Z)-7-Oxozeaenol is from about 0.5 pM to about 6 pM.

33. The method of any of claims 21-32, wherein the disease or condition is a genetic disorder.

34. The method of claim 33, wherein the genetic disorder is selected from the group consisting of 21 -hydroxylase deficiency, achondroplasia, acute intermittent porphyria, adenylosuccinate lyase deficiency, Adrenoleukodystrophy, Alagille syndrome, Alexander disease, Alstrom syndrome, Amelogenesis imperfecta, biotinidase deficiency, CGD Chronic granulomatous disorder, DiGeorge'ssyndrome, fanconi anemia, G6PD deficiency, lipoprotein lipase deficiency, Muscular dystrophy (Duchenne type), Sidcrius X-linkcd mental retardation syndrome caused by mutations in the PHF8 gene, X-linked severe combined immunodeficiency (X-SCID), or X-linked sideroblastic anemia (XLSA).

35. The method of any of claims 21-32, wherein the disease or condition is cancer.

36. The method of claim 35 wherein the cancer is selected from: carcinomas, sarcomas, lymphomas, leukemias, and blastomas: acute lymphoblastic leukemia (all), acute myeloid leukemia, adrenocortical carcinoma, aids-related cancers, anal cancer, astrocytoma, basal-cell carcinoma, extrahepatic bile duct cancer (cholangiocarcinoma), bladder cancer, bone tumor (osteosarcoma / malignant fibrous histiocytoma), brainstem glioma, brain cancer, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma, breast cancer, bronchial adenomas / carcinoids, burkitt's lymphoma, central nervous system lymphoma, cervical cancer, chondrosarcoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous t-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (gist), extracranial, extragonadal, or ovarian germ cell tumor, gestational trophoblastic tumor, glioma of the brain stem, childhood cerebral astrocytoma glioma, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, hodgkin lymphoma, intraocular melanoma, islet cell carcinoma (endocrine pancreas), kaposi sarcoma, kidney cancer (renal cell cancer), acute lymphoblastic leukaemia (also called acute lymphocytic leukemia), acute myeloid leukemia (also called acute myelogenous leukemia), chronic lymphocytic leukemia, chronic myelogenous leukemia (also called chronic myeloid leukemia), hairy cell leukemia, lip and oral cavity cancer, liposarcoma, non-small cell lung cancer, small cell lung cancer, macroglobulinemia, Waldenstrom, male breast cancer, malignant fibrous histiocytoma of bone / osteo sarcoma, medulloblastoma, melanoma, intraocular(eye)melanoma, Merkel cell cancer, mesothelioma, metastatic squamous neck cancer with occult primary, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndromes, myelodysplastic / myeloproliferative diseases, chronic myelogenous leukemia, acute myeloid leukemia, myeloid leukemia, multiple myeloma (cancer of the bone-marrow), myeloproliferative disorders, myxoma, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-small cell lung cancer, oligodendroglioma, oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer (surface epithelial-stromal tumor), ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, pancreatic cancer, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary adenoma, plasma cell neoplasia / multiple myeloma, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), renal pelvis and ureter transitional cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, soft tissue sarcoma, uterine sarcoma, Sezary syndrome, melanoma and non-melanoma skin cancer, merkel cell skin carcinoma, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer with occult primary, stomach cancer, supratentorial primitive neuroectodermal tumor, t- cell lymphoma (mycosis fungoides and sezary syndrome), testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, trophoblastic tumor, ureter and renal pelvis transitional cell cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma, vulvar cancer, Waldenstrom macroglobulinemia, Wilms tumor (kidney cancer).

37. A method of treating or preventing a condition or disease in a subject, comprising administering to the subject an effective amount of heparin or an analogue thereof and an effective amount of a viral vector, wherein the effective amount of heparinor analogue thereof enhances the efficiency of transduction of the viral vector in one or more cells of the subject.

38. The method of claim 37, wherein the viral vector comprises a heterologous nucleic acid.

39. The method of claim 37 or 38, wherein the nucleic acid comprises a sequence encoding a protein.

40. The method of any of claims 37-39, wherein the nucleic acid comprises a sequence for use in editing the genome of the cell.

41. The method of any of claims 37-40, wherein the sequence encodes a nuclease.

42. The method of claim 41, wherein the nuclease is selected from the group consisting of a CRISPR associated protein (Cas proteins, e.g., Cas9), Zinc finger nuclease (ZFN), Transcription Activator-Like Effector Nuclease (TALEN), and meganuclease.

43. The method of any of claims 37-42, wherein the nucleic acid comprises a sequence encoding a CRISPR / Cas system comprising: (a) a gRNA molecule comprising a targeting domain which is complementary with a target domain sequence of a gene in the cell and (b) a Cas9 molecule, wherein the CRISPR / Cas system is capable of modifying the gene.

44. The method of any of claims 37 or 38, wherein the nucleic acid comprises a sequence encoding an RNA molecule that modulates expression of a gene in the cell.

45. The method of claim 44, wherein the RNA is selected from the group consisting of siRNA, piRNA, miRNA and combinations thereof.

46. The method of claim 39, wherein the protein is an antigen.

47. The method of claim 46, wherein the antigen comprises an antigen from a cancer cell.

48. The method of claim 37, wherein the viral vector is an envelope virus.

49. The method of claim 37, wherein the viral vector is a non-envelope virus.

50. The method of claim 37, wherein the viral vector is a retrovirus.

51. The method of claim 37, wherein the viral vector is a lentiviral vector.

52. The method of claim 37, wherein the viral vector is selected from the group consisting of herpes simplex virus, cytomegalovirus, human herpes virus, foot and mouth disease virus, John Cunningham polyomavirus, respiratory syncytial virus, dengue virus, pseudorabies virus, human papilomavirus, Venezuelan equine encephalitis virus, enterovirus, parainfluenza virus, echovirus, Merkel cell polyomavirus, hepatitis C virus, sindbis virus, human metapneumovirus, echovirus, hepatitis B virus / hepatitis, delta virus, adeno-associated virus, Semliki forest virus, zika virus, North American eastern equine encephalitis virus, vaccinia virus, human immunodeficiency virus, rhinovirus, Adenovirus, filovirus, coronavirus, norovirus, Akabane virus, Schmallenberg virus, Rift valley fever virus, coxsackie virus, rabies virus, yellow fever virus, swine vesicular disease virus, Japanese encephalitis virus, Theiler murine encephalomyelitis virus, west nile virus, human parechovirus, Hendra and Nipah viruses, tick-borne encephalitis virus, porcine reproductive and respiratory syndrome virus, human T cell leukemia virus, porcine circovirus, hepatitis E virus, Chikungunya virus, and Murray Valley encephalitis virus.

53. The method of claim 37, wherein the viral vector is an adeno-associated virus.

54. The method of claim 37, wherein the viral vector is a respiratory syncytial virus.

55. The method of any of claims 37-54, wherein the efficiency of viral transduction is dependent on the presence of heparan sulfate proteoglycans on the surface of the cells.

56. The method of any of claims 37-55, wherein the cells are selected from liver (e.g., hepatocytes, sinusoidal endothelial cells), pancreas (e.g., beta islet cells), lung, central or peripheral nervous system, brain (e.g., neural, glial or ependymal cells), spine, kidney, eye (e.g., retinal, cell components), spleen, skin, thymus, testes, lung, diaphragm, heart (cardiac), gut (e.g., endocrine), adipose tissue (white, brown or beige), muscle, synoviocytes, chondrocytes, osteoclasts, epithelial cells, endothelial cells, salivary gland cells, inner ear nervous cells, hematopoietic (e.g., blood or lymph) cells, stem cells, pluripotent or multipotent progenitor cells, and hematopoietic (e.g., blood or lymph) cells.

57. The method of claims 37-55, wherein the cells comprise hematopoietic cells, immune cells, dendritic cells, or stem cells.

58. The method of claim 57, wherein the cells comprise T lymphocytes, natural killer cells or NK92 cells.

59. The method of any of claims 37-58, wherein the heparin or analogue thereof and viral vector are administered in separate compositions.

60. The method of any of claims 37-58, wherein the heparin or analogue thereof and viral vector are administered in the same composition.

61. The method of any of claims 37-60, further comprising administering an effective amount of one or more agents that enhance transduction efficiency of the viral vector in cells.

62. The method of claim 61, wherein the one or more agents is selected from (5Z)-7- Oxozeaenol and a polaxamer compound.

63. The method of claim 62, wherein the (5Z)-7-Oxozeaenol is administered in an amount sufficient to achieve a concentration from about 0.04 pM to about 10 pM.

64. The method of claim 62, wherein the effective amount of (5Z)-7-Oxozeaenol is from 0.4 pM to about 1.5 pM.

65. The method of claim 62, wherein the effective amount of (5Z)-7-Oxozeaenol is from about 0.5 pM to about 6 pM.

66. The method of any of claims 37-65, wherein the disease or condition is a genetic disorder.

67. The method of claim 66, wherein the genetic disorder is selected from the group consisting of 21 -hydroxylase deficiency, achondroplasia, acute intermittent porphyria, adenylosuccinate lyase deficiency, Adrenoleukodystrophy, Alagille syndrome, Alexander disease, Alstrom syndrome, Amelogenesis imperfecta, biotinidase deficiency, CGD Chronic granulomatous disorder, DiGeorge's syndrome, fanconi anemia, G6PD deficiency, lipoprotein lipase deficiency, Muscular dystrophy (Duchenne type), Siderius X-linked mental retardation syndrome caused by mutations in the PHF8 gene, X-linked severe combined immunodeficiency (X-SCID), or X-linked sideroblastic anemia (XLSA).

68. The method of any of claims 37-65, wherein the disease or condition is cancer.

69. The method of claim 65, wherein the cancer is selected from: carcinomas, sarcomas, lymphomas, leukemias, and blastomas: acute lymphoblastic leukemia(all), acute myeloid leukemia, adrenocortical carcinoma, aids-related cancers, anal cancer, astrocytoma, basal-cell carcinoma, extrahepatic bile duct cancer (cholangiocarcinoma), bladder cancer, bone tumor (osteosarcoma / malignant fibrous histiocytoma), brainstem glioma, brain cancer, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma, breast cancer, bronchial adenomas / carcinoids, burkitt's lymphoma, central nervous system lymphoma, cervical cancer, chondrosarcoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous t-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (gist), extracranial, extragonadal, or ovarian germ cell tumor, gestational trophoblastic tumor, glioma of the brain stem, childhood cerebral astrocytoma glioma, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, hodgkin lymphoma, intraocular melanoma, islet cell carcinoma (endocrine pancreas), kaposi sarcoma, kidney cancer (renal cell cancer), acute lymphoblastic leukaemia (also called acute lymphocytic leukemia), acute myeloid leukemia (also called acute myelogenous leukemia), chronic lymphocytic leukemia, chronic myelogenous leukemia (also called chronic myeloid leukemia), hairy cell leukemia, lip and oral cavity cancer, liposarcoma, non-small cell lung cancer, small cell lung cancer, macroglobulinemia, Waldenstrom, male breast cancer, malignant fibrous histiocytoma of bone / osteosarcoma, medulloblastoma, melanoma, intraocular (eye)melanoma, Merkel cell cancer, mesothelioma, metastatic squamous neck cancer with occult primary, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndromes, myelodysplastic / myeloproliferative diseases, chronic myelogenous leukemia, acute myeloid leukemia, myeloid leukemia, multiple myeloma (cancer of the bone-marrow), myeloproliferative disorders, myxoma, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-small celllung cancer, oligodendroglioma, oral cancer, oropharyngeal cancer, ostcosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer (surface epithelial-stromal tumor), ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, pancreatic cancer, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary adenoma, plasma cell neoplasia / multiple myeloma, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), renal pelvis and ureter transitional cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, soft tissue sarcoma, uterine sarcoma, Sezary syndrome, melanoma and non-melanoma skin cancer, merkel cell skin carcinoma, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer with occult primary, stomach cancer, supratentorial primitive neuroectodermal tumor, t- cell lymphoma (mycosis fungoides and sezary syndrome), testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, trophoblastic tumor, ureter and renal pelvis transitional cell cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma, vulvar cancer, Waldenstrom macroglobulinemia, Wilms tumor (kidney cancer).

70. A pharmaceutical composition, comprising an effective amount of a viral vector and an effective amount of heparin or an analogue thereof, wherein the effective amount of heparin or analogue thereof enhances the efficiency of transduction of the viral vector in one or more cells when the composition is administered to the subject.

71. A method for identifying a heparin analogue that enhances efficiency of transduction of a viral vector in cells, comprising i) administering a heparin analogue to the cells; ii) administering a viral vector to the cells;iii) assaying for transduction efficiency of the viral vector following administration of the heparin analogue; iv) assaying for transduction efficiency of the viral vector in the absence of administering the heparin analogue; and v) comparing transduction efficiency of the viral vector in parts iii) and iv) whereby when the transduction efficiency of part iii) is greater than pail iv), a heparin analogue that enhances transduction efficiency is identified.

72. The method of any of claims 28-36 or 61-71, wherein the transduction efficiency is enhanced by at least about 10%, at least about 15%, at least about 25%, at least about 50%, at least about 75%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 225%, at least about 250%, at least about 275%, or at least about 300%, relative to the transduction efficiency with heparin or an analogue thereof alone.

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