Pdgfr and EGFR inhibitors as viral production enhancersand methods and uses thereof

Abstract

The present application relates to viral sensitizers. More specifically, the present application relates to inhibitors of EGFR and / or PDGFR, as well as methods of using such compounds as viral sensitizers, specifically for increasing permissiveness of a cell to the production of virus from reverse genetics systems, replicating viruses and increasing transduction.

Description

PDGFR and EGFR INHIBITORS AS VIRAL PRODUCTION ENHANCERS AND METHODS AND USES THEREOFCROSS REFERENCE TO RELATED APPLICATION

[0001] The present application claims the benefit of priority of co-pending U.S. Provisional Patent Application No. 63 / 683,000 which was filed August 14, 2024, the contents of which are incorporated herein by reference in their entirety.FIELD

[0002] The present application relates to the field of molecular biology, virology, and genetic engineering and viral sensitizers. Specifically, it focuses on methods and compositions for enhancing or increasing viral production in or from cells, for example through transfection, and for the production of various viral vectors, including, but not limited to, lentivirus and adeno- associated virus (AAV). The application contemplates Platelet Derived Growth Factor Receptor (PDGFR) and / or Epidermal Growth Factor Receptor (EGFR) inhibitors and methods of using such inhibitors to enhance the yield of replicating viruses or reverse genetics systems geared toward the production of non-replicating or replicating viruses, or their encoded genetic material.BACKGROUND

[0003] Gene and cellular therapy products have the potential to transform the therapeutic landscape for a wide range of diseases encompassing genetic disease, cancer, and auto immunity. The vast majority of commercial and clinical pipeline gene and cell therapeutics rely on the use of recombinant viruses either as the therapeutic, or a component of the manufacturing process.

[0004] The production of viruses for use in the development of therapeutics including cell and gene therapy and vaccine vectors, is complex resulting in clinical and commercial success being hindered by low yields and poor quality. There are multiple strategies to address production efficiencies, including bioreactor design, media formulations, plasmid DNA design, and cell line development. A complementary strategy is to realize gains in productivity via small molecule additives to biomanufacturing processes. This has been exemplified in the literature as well as several patents demonstrating the use of small molecules to increase production, replication and spread of a wide variety of viruses. Virus sensitizing compounds have been described in PCT / CA2010 / 001057, PCT / CA2014 / 050564, PCT / CA2016 / 050061 , PCT / CA2016 / 050062, PCT / CA2017 / 051176, PCT / CA2018 / 051492, andPCT / CA2022 / 050713, through targeting of cellular innate antiviral pathways, culminating in the suppression of type 1 interferon. This approach was further exemplified inW02024003718A1 , which describes the use of type 1 interferon inhibitors to increase production of recombinant AAV and Lentiviral vectors. Additionally, WO2024081927A1 describes the use of HDAC inhibitors to increase production of AAV vectors. Despite these advances there is a continued need to develop additional small molecule enhancers targeting alternative pathways.SUMMARY OF THE INVENTION

[0005] Several compounds which specifically inhibit Platelet Derived Growth Factor Receptor (PDGFR) and / or Epidermal Growth Factor Receptor (EGFR) that significantly enhanced AAV production have been identified using plasmid transfection in suspension HEK293 cells. Given plasmid-based reverse genetics systems have been developed and described for many viruses including, but not limited to, influenza A virus (IAV), Ebola virus (EBOV), respiratory syncytial virus (RSV), vesicular stomatitis virus (VSV) and Newcastle disease virus (NDV), the present application also relates to improving the production of both replicating and nonreplicating viruses, as well as increasing the spread of replicating viruses, viral transduction, as well as the production of transgenes encoded by such reverse genetics systems.

[0006] It has been shown that specific inhibitors of PDGFR and / or EGFR are effective to increase permissiveness of a cell to the production of virus from a reverse genetics system and thus exhibit viral sensitizing activity, with high potency and versatility.

[0007] Accordingly, the present application includes a method of increasing permissiveness of a cell to one or more nucleic acids, comprising contacting the cell with an effective amount of a PDGFR inhibitor.

[0008] In one embodiment, the PDGFR inhibitor is CP673451 , Crenolanib (CP-868596), Sunitinib, Sorfafenib, Pazopanib, Motesanib, Cediranib, Axitininb, Linifanib, or an analogue or a derivative thereof, or a pharmaceutically acceptable salt, where applicable, solvate and / or prodrug thereof.

[0009] The present application also includes a method of increasing permissiveness of a cell to one or more nucleic acids, comprising contacting the cell with an effective amount of an EGFR inhibitor.

[0010] In one embodiment, the EGFR inhibitor is Ibrutinib and Rociletinib, or an analogue or a derivative thereof, or a pharmaceutically acceptable salt, where applicable, solvate and / or prodrug thereof.

[0011] In another embodiment, the one or more nucleic acids encode one or more components of a virus.

[0012] In another embodiment, the one or more nucleic acids comprise one or more plasmids.

[0013] In another embodiment, the virus is an adeno-associated virus (AAV), optionally wherein the AAV is selected from the group consisting of AAV serotypes 1-9 and recombinant serotypes thereof.

[0014] In another embodiment, the virus is a retrovirus, optionally a lentivirus, optionally where the lentivirus is selected from the group consisting of HIV-1 , HIV-2, SIV, and recombinant lentiviruses.

[0015] In another embodiment, the cell is contacted with the inhibitor before, after, or concurrently with the one or more nucleic acids.

[0016] In another embodiment, the cell is a mammalian cell.

[0017] In another embodiment, production of the virus by the cell is increased by at least 1.1 fold compared to a control cell. In another embodiment, production of the virus by the cell is increased by at least 1 .5 fold compared to a control cell.

[0018] The present application also includes a method of increasing production of a virus in a cell, comprising contacting the cell with an effective amount of a PDGFR and / or EGFR inhibitor, in combination with one or more nucleic acids encoding components of the virus.

[0019] In one embodiment, the one or more nucleic acids comprise one or more plasmids.

[0020] In another embodiment, the virus is an adeno-associated virus (AAV), optionally wherein the AAV is selected from the group consisting of AAV serotypes 1-9 and recombinant serotypes thereof.

[0021] In another embodiment, the virus is a retrovirus, optionally a lentivirus, optionally wherein the lentivirus is selected from the group consisting of HIV-1 , HIV-2, SIV, and recombinant lentiviruses.

[0022] In another embodiment, the cell is contacted with the compound before, after, or concurrently with the one or more nucleic acids.

[0023] In another embodiment, the cell is a mammalian cell.

[0024] In another embodiment, production of the virus by the cell is increased by at least 1.1 fold compared to a control cell. In another embodiment, production of the virus by the cell is increased by at least 1 .5 fold compared to a control cell.

[0025] The present application further includes a method of increasing transgene expression in a cell comprising contacting the cell with an effective amount of a PDGFR and / or EGFR inhibitor, in combination with one or more nucleic acids, wherein at least one of the nucleic acids encodes the transgene.

[0026] In one embodiment, the transgene is a virally-encoded transgene and the one or more nucleic acids further comprise one or more components of a virus.

[0027] In another embodiment, the transgene is a therapeutic gene.

[0028] In another embodiment, the virus is an adeno-associated virus (AAV), optionally wherein the AAV is selected from the group consisting of AAV serotypes 1-9 and recombinant serotypes thereof.

[0029] In another embodiment, the virus is a retrovirus, optionally a lentivirus, optionally wherein the lentivirus is selected from the group consisting of HIV-1 , HIV-2, SIV, and recombinant lentiviruses.

[0030] The present application also includes a method of increasing transduction of a virus into a cell, comprising contacting the cell with an effective amount of a PDGFR inhibitor.

[0031] The present application further includes a method of increasing transduction of a virus into a cell, comprising contacting the cell with an effective amount of an EGFR inhibitor.

[0032] The present application also includes a method of increasing production of an adeno- associated virus (AAV), comprising contacting the cell with an effective amount of a PDGFR and / or EGFR inhibitor, in combination with one or more nucleic acids encoding components of the AAV.

[0033] In one embodiment, the cell is contacted with the compound before, after, or concurrently with the one or more nucleic acids.

[0034] In another embodiment, production of the virus by the cell is increased by at least 1.1 fold compared to a control cell. In another embodiment, production of AAV is increased by at least 1 .5 fold compared to a control cell.

[0035] In another embodiment, the AAV is selected from the group consisting of AAV serotypes 1-9 and recombinant serotypes thereof.

[0036] The present application further includes a method of increasing transduction of a virus into a cell, comprising contacting the cell with an effective amount of a PDGFR and / or EGFR inhibitor.

[0037] The present application also includes a composition comprising a PDGFR and / or EGFR inhibitor, and a one or more nucleic acids encoding components of a virus.

[0038] The present application also includes use of a composition of the present application for increasing production of a virus in a cell.

[0039] Other features and advantages of the present application will become apparent from the following detailed description. It should be understood, however, that the detaileddescription and the specific examples, while indicating embodiments of the application, are given by way of illustration only and the scope of the claims should not be limited by these embodiments, but should be given the broadest interpretation consistent with the description as a whole.BRIEF DESCRIPTION OF DRAWINGS

[0040] The embodiments of the application will now be described in greater detail with reference to the attached drawings in which:

[0041] FIG.1A, FIG.1B, FIG.1C and FIG.1D show a comparative analysis for enhancement of AAV2 production for exemplary PDGFR and EGFR inhibitors. FIG.1A is a bar graph illustrating the observed fold enhancement above library vehicle control (1 .05% DMSO) in relative light units (RLU), generated through virus-mediated luciferase activity for CP673451 at 0.1 , 1 , and 10 pM. The values represent the average of three technical replicates (n=3). An asterisk (*) indicates statistical significance with p-values <0.01 calculated using unpaired, two-tailed t-test. FIG.1B shows fold enhancement above library vehicle control for Crenolanib at 0.1 , 1 , and 10 pM. The values represent the average of three technical replicates (n=3) and an asterisk (*) indicates statistical significance with p-values <0.01 calculated using unpaired, two-tailed t-test. FIG.1C shows fold enhancement above library vehicle control for Ibrutinib at 0.1 , 1 , and 10 pM. FIG.1D shows fold enhancement above library vehicle control for Rociletinib at 0.1 , 1 , and 10 pM. The values represent the average of three technical replicates (n=3) and an asterisk (*) indicates statistical significance with p-values <0.01 calculated using unpaired, two-tailed t-test.

[0042] FIG. 2A and FIG. 2B shows fold increase in AAV2 genomic titer of CP-673451 and Crenolanib (exemplary PDGFR inhibitors) treated VPC2.0 HEK293 cells at various concentrations. This was measured by the AAV qPCR assay harvested from 125ml_ shake flasks. CP-673451 and Crenolanib both enhanced viral genomic titer at 5, 10, 15 and 20 pM relative to the untreated control. The values represent the average of two technical replicates (n=2). An asterisk (*) indicates statistical significance with p-values <0.01 calculated using unpaired, two-tailed t-test.

[0043] FIG. 3A and FIG. 3B shows fold increase in AAV8 viral genomic titer of CP-673451 and Crenolanib (exemplary PDGFR inhibitors) treated VPC2.0 HEK293 cells as measured by qPCR harvested from 125ml_ shake flasks. Enhancement of AAV8 viral genomic titer was observed in response to 1 , 5, 10 and 15 pM of CP-673451 , and 10 pM of Crenolanib. The values represent the average of two technical replicates (n=2). An asterisk (*) indicates statistical significance with p-values <0.01 calculated using unpaired, two-tailed t-test.

[0044] FIG. 4 shows the fold increase in RLU equivalent to AAV2 functional titer of CP-673451 (exemplary PDGFR inhibitor) treated VPC2.0 HEK293 cells across a wide range of concentrations relative to control as measured by the luciferase reporter assay. Enhancement of functional viral titer was observed in VPC2.0 HEK293 cells transfected with FectoVir-AAV reagent or PEIpro reagent. 5 pM and 10 pM of CP-673451 led to enhanced functional titer in VPC 2.0 HEK 293 cells with FectoVir-AAV and 10 pM of CP-673451 increased functional titer in VPC 2.0 HEK 293 cells with PEIpro. The values represent the average of two technical replicates (n=2). An asterisk (*) indicates statistical significance with p-values <0.01 calculated using unpaired, two-tailed t-test.

[0045] FIG. 5A and FIG. 5B shows fold increase in replication-competent VSVA51 virus functional titer of CP-673451 and Crenolanib (exemplary PDGFR inhibitors) treated 786-0 cells, respectively, as measured by luciferase reporter assay. CP-673451 increases VSVA51 viral functional titer from 4 to 18 pM (FIG. 5A), and Crenolanib increases VSVA51 viral functional titer at 5, 10 and 15 pM relative to control (FIG. 5B). The values represent the average of three technical replicates (n=3). An asterisk (*) indicates statistical significance with p-values <0.01 calculated using unpaired, two-tailed t-test.

[0046] FIG. 6A and FIG. 6B shows fold enhancement of Adenovirus 5-encoded GFP expression CP-673451 and Crenolanib (exemplary PDGFR inhibitors) treated HEK293 cells, respectively, as measured by quantification of GFP fluorescence. FIG. 6A shows CP-673451 at 1 and 5 pM increased Ad5- encoded GFP expression relative to control. FIG. 6B shows Crenolanib at 1 pM increased Ad5- encoded GFP expression relative to control. The values represent the average of three technical replicates (n=3). An asterisk (*) indicates statistical significance with p-values <0.01 calculated using unpaired, two-tailed t-test.

[0047] FIG. 7A and FIG. 7B shows fold increase % GFP-positive cells equivalent to lentiviral transduction of human primary pan T-cells (CD3+) treated with Ibrutinib (exemplary EGFR inhibitor) and Crenolanib (exemplary PDGFR inhibitor) as measured by flow cytometry. FIG. 7A shows fold enhancement of T-cell transduction with Ibrutinib at 10 and 15 pM relative to the control. FIG. 7B shows enhancement of T-cell transduction with Crenolanib at 10 pM relative to the control. The values represent the average fold change enhancement of % transduction by based on two technical replicates (n=2). An asterisk (*) indicates statistical significance with p-values <0.01 calculated using unpaired, two-tailed t-test.

[0048] FIG. 8A and FIG. 8B shows fold increase of VSVA51 - encoded GFP expression in 786-0 cells treated with CP-673451 and Crenolanib (exemplary PDGFR inhibitors) at 10, 20 and 40 pM relative to control as determined by quantification of GFP fluorescence. The valuesrepresent the average of three technical replicates (n=3). An asterisk (*) indicates statistical significance with p-values <0.01 calculated using unpaired, two-tailed t-test.

[0049] FIG. 9 shows fold enhancement of Adenovirus 5 functional titer of CP-673451 (exemplary PDGFR inhibitor) treated HEK293 cells. CP-673451 at 5 pM increased Ad5 functional titer by 1 .5-fold relative to control. The values represent the average of three technical replicates (n=3).DETAILED DESCRIPTIONI. Definitions

[0050] Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the present application herein described for which they are suitable as would be understood by a person skilled in the art.

[0051] As used in this application and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "include" and "includes") or "containing" (and any form of containing, such as "contain" and "contains"), are inclusive or open-ended and do not exclude additional, unrecited elements or process steps.

[0052] The term “consisting” and its derivatives as used herein are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, and also exclude the presence of other unstated features, elements, components, groups, integers and / or steps.

[0053] The term “consisting essentially of’, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and / or steps as well as those that do not materially affect the basic and novel characteristic(s) of these features, elements, components, groups, integers, and / or steps.

[0054] The terms "about", “substantially” and “approximately” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies or unless the context suggests otherwise to a person skilled in the art.

[0055] As used in the present application, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise. For example, an embodiment including “a compound” should be understood to present certain aspects with one compound, or two or more additional compounds.

[0056] In embodiments comprising an “additional” or “second” component, such as an additional or second compound, the second component as used herein is chemically different from the other components or first component. A “third” component is different from the other, first, and second components, and further enumerated or “additional” components are similarly different.

[0057] The term “and / or” as used herein means that the listed items are present, or used, individually or in combination. In effect, this term means that “at least one of’ or “one or more” of the listed items is used or present. The term “and / or” with respect to salts, solvates and / or prodrugs thereof means that the compounds of the application exist as individual salts, solvates and prodrugs, as well as a combination of, for example, a salt of a solvate of a compound of the application.

[0058] The term “compound of the application” or “compound of the present application” and the like as used herein refers a compound which has shown PDGFR and / or EGFR inhibition activity, or a pharmaceutically acceptable salt (where applicable), solvate and / or prodrug thereof, including those disclosed herein.

[0059] The term “composition of the application” or “composition of the present application” and the like as used herein refers to a composition comprising one or more compounds of the application and optionally one or more viruses and / or one or more nucleic acids encoding components of a virus.

[0060] The term “suitable” as used herein means that the selection of the particular composition or conditions would depend on the specific steps to be performed, the identity of the components to be transformed and / or the specific use for the compositions, but the selection would be well within the skill of a person trained in the art.

[0061] The present description refers to a number of chemical terms and abbreviations used by those skilled in the art. Nevertheless, definitions of selected terms are provided for clarity and consistency.

[0062] The term “cell” as used herein refers to a single cell or a plurality of cells and includes a cell in a cell culture or a cell in a subject.

[0063] The term “subject” as used herein includes all members of the animal kingdom including mammals, and suitably refers to humans. Thus, the methods and uses of the present application are applicable to both human therapy and veterinary applications.

[0064] The term “pharmaceutically acceptable” means compatible with the treatment of subjects, for example humans.

[0065] The term “pharmaceutically acceptable carrier” means a non-toxic solvent, dispersant, excipient, adjuvant or other material which is mixed with the active ingredient in order to permit the formation of a pharmaceutical composition, i.e., a dosage form capable of administration to a subject.

[0066] The term “pharmaceutically acceptable salt” means either an acid addition salt or a base addition salt which is suitable for, or compatible with the treatment of subjects.

[0067] The term “solvate” as used herein means a compound, or a salt and / or prodrug of a compound, wherein molecules of a suitable solvent are incorporated in the crystal lattice. A suitable solvent is physiologically tolerable at the dosage administered.

[0068] The term “prodrug” as used herein means a compound, or salt and / or solvate of a compound, that, after administration, is converted into an active drug.

[0069] The term “treating” or “treatment” as used herein and as is well understood in the art, means an approach for obtaining beneficial or desired results, including clinical results.

[0070] The term “administered” as used herein means administration of a therapeutically effective amount of a compound, or one or more compounds, or a composition of the application to a cell either in cell culture or in a subject. As used herein, “administration” is an example of “contacting” a cell either in cell culture or in a subject compound with one or more compounds, or a composition of the application.

[0071] As used herein, the term “effective amount” or “therapeutically effective amount” means an amount of a compound, or one or more compounds, of the application that is effective, at dosages and for periods of time necessary to achieve a desired result.

[0072] The term “cancer” as used herein refers to cellular-proliferative disease states.

[0073] The terms “sensitization” or “sensitizing” as used herein, in the context of a cell, refers to a decreased or altered cellular response to an outside agent such that the outside agent has an increased or altered effect on the cell. Where the outside agent is a virus, the terms “sensitization” or “sensitizing” (also referred to as “viral sensitization” or “viral sensitizing”) refers to a decreased or altered cellular response to the virus, thereby increasing the ability of the virus to infect and / or replicate in the cell. Where the outside agent is genetic material encoding a virus, the terms “sensitization” or “sensitizing” (also referred to as “viral sensitization” or “viral sensitizing”) refers to a decreased or altered cellular response to the genetic material, thereby increasing ability of the genetic material to transfect and / or expressed in the cell.

[0074] The term “permissiveness” as used herein, in the context of a cell, refers to an increase in the uptake of an outside agent by the cell and / or an increase in the activity of the outsideagent in the cell and / or a reduction in cellular defenses that would otherwise inhibit the expression, replication or stability of the outside agent in the cell. When the outside agent is a virus, “permissiveness” refers to the ability of a virus to infect and / or transduce a cell. When the outside agent is genetic material, “permissiveness” refers to the ability of genetic material; to transfect a cell and / or get processed by a cell into viral particle(s).

[0075] The terms “reverse genetics” and “reverse genetic systems”, as used herein, refer to a method or platform for studying gene function and producing viruses and proteins by first identifying or manipulating a known nucleotide sequence and subsequently observing the phenotypic effects of these manipulations. As used herein, “reverse genetic systems” include any method of providing a nucleic acid to a cell, including transfection, lipofection, electroporation, viral transduction and microinjection. “Reverse genetics” systems are contrasted with “forward genetics” approaches where a mutant cell or organism is identified, and the genetic changes associated with the phenotype are determined. Reverse genetics can include directed gene deletions and point mutations, for example.

[0076] The term “plasmid-based reverse genetics systems”, as used herein, refers to a method or platform as described above for “reverse genetics systems” but involves the generation of a gene product, including but not limited to recombinant virus particles, using one or more plasmids comprising at least one nucleic acid of interest.

[0077] The terms “transduction” or “transducing” as used herein, refer to the introduction of a virus containing an exogenous nucleic acid into a cell leading to expression of the nucleic acid. The nucleic acid is optionally a gene (also referred to as a transgene). The gene is optionally a therapeutic gene.

[0078] The term “genetic material encoding components of a virus” or “one or more nucleic acids” encoding components of a virus refers to a nucleic acid, and chemically modified variants thereof, that carry viral-like sequences of nucleotides. The sequences encode viral- like proteins and / or functional sequences for targeting, integration, promotion, etc.

[0079] The term “increase” or “increasing” as used herein refers to any detectable increase or enhancement in a function or characteristic in the presence of one or more test variables, compared to otherwise the same conditions except in the absence of the one or more test variables.

[0080] The term “decrease” or “decreasing” as used herein refers to any detectable decrease or reduction in a function or characteristic in the presence of one or more test variables, compared to otherwise the same conditions except in the absence of the one or more test variables.

[0081] The term “EGFR” refers to epidermal growth factor receptor. EGFR is a transmembrane protein that is a receptor for members of the epidermal growth factor family (EGF family) of extracellular protein ligands.

[0082] The term “epidermal growth factor receptor inhibitor” or “EGFR inhibitor” refers to an agent that decreases the function or activity of EGFR. Inhibitors of EGFR include, but are not limited to, small molecule inhibitors of EGFR, antibodies to EGFR, such as cetuximab, panitumumab, nimotuzumab, and necitumumab, protein or peptide inhibitors of EGFR and nucleic acid inhibitors of EGFR such as inhibitory RNA (for example, siRNA).

[0083] The term “PDGFR” refers to platelet-derived growth factor receptor. PDGFRs are cell surface tyrosine kinase receptors for members of the platelet-derived growth factor (PDGF) family. “PDGFR” includes both the alpha and beta forms of PDGFR.

[0084] The term “platelet-derived growth factor receptor” or “PDGFR inhibitor” refers to an agent that decreases the function or activity of PDGFR. Inhibitors of PDGFR include, but are not limited to, small molecule inhibitors of PDGFR, antibodies to PDGFR, protein or peptide inhibitors of PDGFR and nucleic acid inhibitors of PDGFR such as inhibitory RNA (for example, siRNA).II. Compounds and Compositions of the Application

[0085] The PDGFR and EGFR signaling cascades are related and have been implicated as a key driver of many cancers. Within the context of viral replication, there are several papers which implicate either PDGFR and / or EGFR inhibitors at antivirals. For example, Meineke et al. demonstrated known EGFR / PDGFR kinase inhibitors showed antiviral activity against pandemic and seasonal influenza A. Additionally Tani et al., exemplified the use of targeted PDGFR inhibitors as antivirals for severe fever with thrombocytopenia syndrome virus (SFTSV).

[0086] Given the demonstration of PDGFR and / or EGFR inhibitors as anti-virals, it was surprisingly shown herein that compounds which are known to inhibit PDGFR and / or EGFR of the present application (compounds of the application) are effective to increase permissiveness of a cell to a reverse genetics system suitable for production of a virus, and thus exhibit viral sensitizing activity, with high potency and versatility.

[0087] Accordingly, in the methods and uses of the present application, the compound of the application comprises a compound which inhibits EFGR and / or PDGFR, or a pharmaceutically acceptable salt (where applicable), solvate and / or prodrug thereof, optionally with a pharmaceutically acceptable carrier for use in increasing permissiveness of a cell to one or more nucleic acids.

[0088] In some embodiments, the PDGFR inhibitor (or compound of the application) is a compound selected from CP673451 , Crenolanib (CP-868596), Sunitinib, Sorfafenib, Pazopanib, Motesanib, Cediranib, Axitininb, Linifanib, or an analogue, pharmaceutically acceptable salt (where applicable), solvate and / or prodrug thereof.

[0089] In some embodiments, the EGFR inhibitor (or compound of the application) is a compound selected from Ibrutinib and Rociletinib, or an analogue, salt, solvate and / or prodrug thereof.

[0090] In some embodiments, the PDGFR and / or EGFR inhibitor is selected from:or a pharmaceutically acceptable salt (where applicable), solvate and / or prodrug thereof.

[0091] In some embodiments, the salt is an acid addition salt or a base addition salt. In some embodiments, for pharmaceutical methods and uses on human or animal subjects, the salt is a pharmaceutically acceptable salt. The selection of a suitable salt may be made by a person skilled in the art. Suitable salts include acid addition salts that may, for example, be formed bymixing a solution of a compound with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, acetic acid, trifluoroacetic acid, or benzoic acid. Additionally, acids that are generally considered suitable for the formation of pharmaceutically useful salts from basic pharmaceutical compounds are discussed, for example, by P. Stahl et al, Camille G. (eds.) and Handbook of Pharmaceutical Salts. Properties, Selection and Use. (2002) Zurich: Wiley VCH; S. Berge et al, Journal of Pharmaceutical Sciences 1977 66(1) 1-19; P. Gould, International J. of Pharmaceutics (1986) 33 201-217; Anderson et al, The Practice of Medicinal Chemistry (1996), Academic Press, New York; and in The Orange Book (Food & Drug Administration, Washington, D.C. on their website).

[0092] An acid addition salt suitable for, or compatible with, the treatment of subjects is any non-toxic organic or inorganic acid addition salt of any basic compound. Basic compounds that form an acid addition salt include, for example, compounds comprising an amine group. Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulfuric, nitric and phosphoric acids, as well as acidic metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids which form suitable salts include mono-, di- and tricarboxylic acids. Illustrative of such organic acids are, for example, acetic, trifluoroacetic, propionic, glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, hydroxymaleic, benzoic, hydroxybenzoic, phenylacetic, cinnamic, mandelic, salicylic, 2-phenoxybenzoic, p- toluenesulfonic acid and other sulfonic acids such as methanesulfonic acid, ethanesulfonic acid and 2-hydroxyethanesulfonic acid. In some embodiments, exemplary acid addition salts also include acetates, ascorbates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, fumarates, hydrochlorides, hydrobromides, hydroiodides, lactates, maleates, methanesulfonates (“mesylates”), naphthalenesulfonates, nitrates, oxalates, phosphates, propionates, salicylates, succinates, sulfates, tartarates, thiocyanates, toluenesulfonates (also known as tosylates) and the like. In some embodiments, the mono- or di-acid salts are formed and such salts exist in either a hydrated, solvated or substantially anhydrous form. In general, acid addition salts are more soluble in water and various hydrophilic organic solvents and generally demonstrate higher melting points in comparison to their free base forms. The selection criteria for the appropriate salt will be known to one skilled in the art. Other non-pharmaceutically acceptable salts such as but not limited to oxalates may be used, for example in the isolation of compounds of the application for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt.

[0093] A base addition salt suitable for, or compatible with, the treatment of subjects is any non-toxic organic or inorganic base addition salt of any acidic compound. Acidic compoundsthat form a basic addition salt include, for example, compounds comprising a carboxylic acid group. Illustrative inorganic bases which form suitable salts include lithium, sodium, potassium, calcium, magnesium or barium hydroxide as well as ammonia. Illustrative organic bases which form suitable salts include aliphatic, alicyclic or aromatic organic amines such as isopropylamine, methylamine, trimethylamine, picoline, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Exemplary organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine. The selection of the appropriate salt may be useful, for example, so that an ester functionality, if any, elsewhere in a compound is not hydrolyzed. The selection criteria for the appropriate salt will be known to one skilled in the art. In some embodiments, exemplary basic salts also include ammonium salts, alkali metal salts such as sodium, lithium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases (for example, organic amines) such as dicyclohexylamine, Abutyl amine, choline and salts with amino acids such as arginine, lysine and the like. Basic nitrogen containing groups may be quarternized with agents such as lower alkyl halides (e.g., methyl, ethyl and butyl chlorides, bromides and iodides), dialkyl sulfates (e.g., dimethyl, diethyl and dibutyl sulfates), long chain halides (e.g., decyl, lauryl and stearyl chlorides, bromides and iodides), aralkyl halides (e.g., benzyl and phenethyl bromides) and others. Compounds carrying an acidic moiety can be mixed with suitable pharmaceutically acceptable salts to provide, for example, alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal salts (e.g., calcium or magnesium salts) and salts formed with suitable organic ligands such as quaternary ammonium salts. Also, in the case of an acid (-COOH) or alcohol group being present, pharmaceutically acceptable esters can be employed to modify the solubility or hydrolysis characteristics of the compound.

[0094] All such acid salts and base salts are intended to be pharmaceutically acceptable salts within the scope of the application and all acid and base salts are considered equivalent to the free forms of the corresponding compounds for purposes of the application. In addition, when a compound of the application contains both a basic moiety, such as, but not limited to an aliphatic primary, secondary, tertiary or cyclic amine, an aromatic or heteroaryl amine, pyridine or imidazole and an acidic moiety, such as, but not limited to tetrazole or carboxylic acid, zwitterions (“inner salts”) may be formed and are included within the terms “salt(s)” as used herein. It is understood that certain compounds of the application may exist in zwitterionic form, having both anionic and cationic centers within the same compound and a net neutralcharge. Such zwitterions are included within the application. It is also understood by a person skilled in the art that not all compounds of the application contain a basic and / or acidic moiety to allow the formation of pharmaceutically acceptable salts, therefore no salt forms of such compounds are intended to be included within the present application. Such compounds include, for example, Sorafenib, and Pazopanib.

[0095] Solvates of compounds of the application include, for example, those made with solvents that are pharmaceutically acceptable. Examples of such solvents include water (resulting solvate is called a hydrate) and ethanol and the like. Suitable solvents are physiologically tolerable at the dosage administered.

[0096] The compounds of the present application may further exist in varying amorphous and polymorphic forms and it is contemplated that any amorphous forms, polymorphs, or mixtures thereof, which form are included within the scope of the present application.

[0097] The compounds of the present application may further be radiolabeled and accordingly all radiolabeled versions of the compounds of the application are included within the scope of the present application. The compounds of the application also include those in which one or more radioactive atoms are incorporated within their structure.

[0098] In some embodiments, the compounds of the present application are suitably formulated in a conventional manner into compositions using one or more carriers, optionally in combination with one or more reverse genetics systems encoding viral and non-viral components. Accordingly, the present application also includes a composition comprising one or more compounds of the application and a carrier. The present application also includes a composition comprising one or more compounds of the application, one or more reverse genetics systems and a carrier.

[0099] In some embodiments a compound of the application is coupled with soluble polymers as targetable drug carriers. Such polymers include, for example, polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamide-phenol, polyhydroxy-ethylaspartamide-phenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues. Furthermore, in some embodiments, a compound of the application is coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and crosslinked or amphipathic block copolymers of hydrogels.

[0100] The compositions of the application are particularly amenable to administration with the aid of nano-carrier systems, such as liposomes, micelles, nanoparticles, nano-emulsions, lipidic nano-systems and the like (see for example, Bhat, M. et al. Chem. and Phys, of Lipids,2021 , 236, 105053). Accordingly, the present application includes a composition comprising one or more compounds of the application, optionally one or more reverse genetics systems suitable to the production of viruses and one or more components of a nano-carrier system.

[0101] Also included is a kit comprising the compound of the application and a) a reverse genetics system suitable for the production of a virus, suitably an attenuated or genetically modified virus, a gene therapy vector, b) one or more production cells; c) a pharmaceutically acceptable carrier, diluent or excipient; d) cell culture media; f) a transfection reagent g) a cell culture plate, multi-well dish or large-scale cell culture system h) an apparatus to deliver the viral sensitizing compound to a cell, medium or to a subject; i) instructions for using the viral sensitizing agent; orj) a carrier diluent or excipient, or any combination of a)-j).

[0102] In some embodiments, which are not meant to be limiting in any manner, included is a kit comprising a compound of the application and a medium for growing, culturing or transfecting cells with reverse genetics system suitable for producing a virus and optionally, one or more cells which are capable of producing virus from reverse genetics systems.

[0103] In some embodiments, the kit comprises instructions for using any component or combination of components and / or practicing any method as described herein.

[0104] In the above, the term "a compound" also includes embodiments wherein one or more compounds are referenced.III. Methods and Uses of the Application

[0105] The application also provides uses and methods relating to the compounds and compositions of the application described herein.

[0106] The compounds and compositions of the application have been shown to increase production of a virus by a cell. Thus, the compounds and compositions exhibit viral sensitizing activity, with high potency and versatility. Accordingly, the compounds and compositions of the application are useful for increasing permissiveness of a cell to one or more nucleic acids suitable for producing a virus and encoded transgene(s) useful fortreating diseases, disorders or conditions. The compounds and compositions of the application are also useful for increasing permissiveness of a cell to genetic material encoding components of a virus, increasing virus production, increasing transduction, increasing reverse genetics system- encoded transgene expression, increasing virally-encoded transgene expression and increasing virus growth and / or spread.Methods and uses of increasing permissiveness of a cell to one or more nucleic acids

[0107] Accordingly, the present application includes a method, optionally an in vitro method, of increasing permissiveness of a cell to one or more nucleic acids, comprising administeringan effective amount of a compound inhibiting PDGFR, or a salt, solvate and / or prodrug thereof, (i.e. a compound of the application) to the cell.

[0108] The present application also includes a method, optionally an in vitro method, of increasing permissiveness of a cell to one or more nucleic acids, comprising administering an effective amount of a compound inhibiting EGFR, or a salt, solvate and / or prodrug thereof, (i.e. a compound of the application) to the cell.

[0109] Also provided is use of a compound of the application to increase permissiveness of a cell to one or more nucleic acids. In another embodiment, a compound of the application is used in the manufacture of a therapeutic to increase permissiveness of a cell to one or more nucleic acids. In yet another embodiment, a compound of the application is for use in increasing permissiveness of a cell to one or more nucleic acids.

[0110] The cell is optionally in vivo, ex vivo or in vitro. In some embodiments, the cell is a cell in vitro, for example a cell line or a cell culture. In some embodiments, the cell is an in vivo cell in a subject.

[0111] As used herein, the expression “contacting” refers to any means for exposing a cell or a subject to a compound or administering a compound to a cell or a subject. For example, a cell in cell culture may be contacted with a compound by adding the compound to the culture medium.

[0112] In some embodiments, the compound of the application is contacted with the cell or subject prior, concurrent with, or after provision of the one or more nucleic acids to the cell. Methods of providing nucleic acids for uptake by a cell can include viral infection and / or via a non-viral process, generally known as transfection, and include any a carrier or method commonly known to a person skilled in the art. In no way limiting, this includes transfection using PEI or lipid-based reagents, electroporation, and nanoparticles, as is generally known in the art.

[0113] In some embodiments, the one of more nucleic acids encode components of a virus. The term “components of a virus” refers to nucleic acids, and chemically modified variants thereof, that comprise or consist of viral and / or viral-like sequences. The sequences can encode viral proteins, viral-like proteins and / or functional sequences for targeting, integration, promotion, etc.

[0114] In some embodiments, the nucleic acids are comprised in one or more plasmids, for example DNA plasmids. In some embodiments, the one or more nucleic acids encoding components of a virus are present on a plasmid.

[0115] Delivery of nucleic acids to a cell is generally described as transfection. Transfection efficiency refers to the degree to which a supplied source of genetic material is taken up and functional for its intended purpose in a cell.

[0116] Such nucleic acids may be delivered to the cell directly, or they may be provided in a carrier to enhance delivery and uptake by the cell. Examples of carriers include diverse polymers known in the art, which may be designed in a variety of nanoparticle formats, either loosely organized or more precision designed.

[0117] In one example, lentivirus, gamma-Retrovirus, or AAV are produced following transfection of plasmids encoding lentivirus, gamma-Retrovirus, or AAV viral or viral-like sequences into a cell.

[0118] In some embodiments, the one or more nucleic acids are DNA. In some embodiments, the one or more nucleic acids comprise one or more DNA plasmids. In some embodiments, one or more nucleic acids comprise alternatives to DNA plasmids for example but not limited to Doggy bone DNA, Bacterial artificial chromosomes (BACs), Yeast artificial chromosomes (YACs), P1 -derived artificial chromosomes (PACs), Phagemids, or Synthetic chromosomes.

[0119] In some embodiments, the method is used for production of a virus that encodes a non-viral gene product.

[0120] In some embodiments, the method is used for the production of a therapeutic.

[0121] In some embodiments, the virus is an interferon (IFN)-sensitive virus.

[0122] In some embodiments, the virus is a retrovirus (for example a lentivirus (LV), an attenuated virus, a genetically modified virus, a non-replicating virus, a replicating virus or an oncolytic virus.

[0123] In some embodiments, virus is a non-replicating viral vector, optionally an adenovirus (Ad), an adeno-associated virus (AAV) or lentivirus (LV).

[0124] In some embodiments, the LV is pseudo-typed with alternative glycoproteins including but not limited to VSV glycoprotein, Baboon Endogenous virus glycoprotein, Murine Lukemia Virus glycoprotein, Sindbis virus glycoprotein, Ebola virus glycoproetien, Rabies virus glycoprotein, Measles virus glycoproteins, LCMV virus glycoprotein, Junin virus glycoprotein.

[0125] In some, embodiments, of the virus is a double-stranded DNA virus, including but not limited to herpes simplex virus (HSV), vaccinia virus, or viral vectors derived therefrom.

[0126] In some embodiments, the virus is a gene therapy vector. As used herein, the term “gene therapy vector” is refers to a viral vector designed to deliver therapeutic genetic material to a cell or subject. Examples of gene therapy vectors include, but are not limited to humanAd5, Ad3, Ad11 , Ad35, canine Ad2, chimp Ad26, chimp AdOx1 , or recombinant serotypes thereof, AAV serotypes 1-9 or recombinant serotypes thereof, Lentivirus, gamma-retrovirus, Annellovirus, or Baculovirus.

[0127] In some embodiments, the virus is a component of a vaccine such as, but not limited to, a live attenuated vaccine such as, measles, mumps, rubella, rotavirus, chickenpox, yellow fever or a viral vector vaccine encoding a vaccine antigen transgene such as rVSVAG- ZEBOV-GP (Ervebo) or ChadOx1-S (Vaxzevria).

[0128] In some embodiments, the virus is a rhabdovirus, a togavirus, or an orthomyxovirus.

[0129] In some embodiments, rhabdovirus is vesicular stomatitis virus (VSV), engineered mutants of SV (VSVA51), an oncolytic non-VSV rhabdovirus, or a recombinant oncolytic non- VSV rhabdovirus encoding one or more of rhabdoviral N, P, M, G and / or L protein, or variant thereof including chimeras and fusion proteins thereof, having an amino acid identity of at least or at most 20, 30, 40, 50, 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, 98, 99, 100%, including all ranges and percentages there between, to the N, P, M, G and / or L protein of Arajas virus, Chandipura virus, Cocal virus, Isfahan virus, Maraba virus, Piry virus, Vesicular stomatitis Alagoas virus, BeAn 157575 virus, Boteke virus, Calchaqui virus, Eel virus American, Gray Lodge virus, Jurona virus, Klamath virus, Kwatta virus, La Joya virus, Malpais Spring virus, Mount Elgon bat virus, Perinet virus, Tupaia virus, Farmington, Bahia Grande virus, Muir Springs virus, Reed Ranch virus, Hart Park virus, Flanders virus, Kamese virus, Mosqueiro virus, Mossuril virus, Barur virus, Fukuoka virus, Kern Canyon virus, Nkolbisson virus, Le Dantec virus, Keuraliba virus, Connecticut virus, New Minto virus, Sawgrass virus, Chaco virus, Sena Madureira virus, Timbo virus, Almpiwar virus, Aruac virus, Bangoran virus, Bimbo virus, Bivens Arm virus, Blue crab virus, Charleville virus, Coastal Plains virus, DakArK 7292 virus, Entamoeba virus, Garba virus, Gossas virus, Humpty Doo virus, Joinjakaka virus, Kannamangalam virus, Kolongo virus, Koolpinyah virus, Kotonkon virus, Landjia virus, Manitoba virus, Marco virus, Nasoule virus, Navarro virus, Ngaingan virus, Oak-Vale virus, Obodhiang virus, Oita virus, Quango virus, Parry Creek virus, Rio Grande cichlid virus, Sandjimba virus, Sigma virus, Sripur virus, Sweetwater Branch virus, Tibrogargan virus, Xiburema virus, Yata virus, Rhode Island, Adelaide River virus, Berrimah virus, Kimberley virus, or Bovine ephemeral fever virus.

[0130] In some embodiments, the togavirus is sindbis, semliki forest virus or M1 virus.

[0131] In some embodiments, the orthomyxovirus is influenza A, influenza B, influenza C, influenza D, isavirus, thogotovirus or quanranjavirus.

[0132] In some embodiments, the cell is a eukaryotic cell, for example a human or other mammalian cell. In some embodiments, the cell is a prokaryotic cell.

[0133] In some embodiments, the cell is one or more types of immortalized cells in vitro or in vivo from any cell, cell line, tissue or organism, not limited to, human, rat, mouse, cat, dog, pig, primate, horse and the like, for example, without limitation: Vero, HEK-293 cells or derivatives, HEK293T cells, VPC 1.0, VPC 2.0, Expi293F, EB-66 cells, EbX cells, PER. C6 cells, AGE1.CR, Agel.O S, Agel.HN, Agel.RO, Q0R2 / 2E11 , UMNSAH-DF1 , CHO, hybridoma cells, sf9 cells, HT 1080 or R4 cells.

[0134] In some embodiments, permissiveness of the cell to the one or more nucleic acids is increased 1.1 fold or more, 1.2 fold or more, 1.5 fold or more, 2 fold or more, 2.5 fold or more, 3 fold or more, 5 fold or more, or 10 fold or more, e.g., compared to permissiveness of the cell, or a comparable cell (also referred to herein as a control cell) prior to, or in absence of, the cell being contacted with a compound or composition of the application. In some cases, the method includes measuring the permissiveness to the cell to the one or more nucleic acids or measuring the production of viruses by the cell.Methods and uses of increasing production of viruses

[0135] The present application also includes a method, optionally an in vitro method, of increasing production of a virus by a cell comprising administering a compound of the application to the cell. Also included is a use of a compound of the application for increasing production of a virus by a cell, as well as a compound of the application for use in increasing production of a virus by a cell.

[0136] In some embodiments, the method comprises growing the virus in an appropriate medium in the presence of a compound of the application.

[0137] In some embodiments, the compound of the application is contacted with the cell or subject prior, concurrent with, or after provision of the one or more nucleic acids to the cell. Methods of providing nucleic acids for uptake by a cell can include viral infection and / or via a non-viral process, generally known as transfection, and include any a carrier or method commonly known to a person skilled in the art. In no way limiting, this includes transfection using PEI or lipid-based reagents, electroporation, and nanoparticles, as is generally known in the art.

[0138] In some embodiments, the one or more nucleic acids encode components of a virus. The term “components of a virus” refers to nucleic acids, and chemically modified variants thereof, that comprise or consist of viral and / or viral-like sequences. The sequences can encode viral proteins, viral-like proteins and / or functional sequences for targeting, integration, promotion, etc.

[0139] In some embodiments, the nucleic acids are comprised in one or more plasmids, for example DNA plasmids. In some embodiments, the one or more nucleic acids encoding components of a virus are present on a plasmid.

[0140] Delivery of nucleic acids to a cell is generally described as transfection. Transfection efficiency refers to the degree to which a supplied source of genetic material is taken up and functional for its intended purpose in a cell.

[0141] Such nucleic acids may be delivered to the cell directly, or they may be provided in a carrier to enhance delivery and uptake by the cell. Examples of carriers include diverse polymers known in the art, which may be designed in a variety of nanoparticle formats, either loosely organized or more precision designed.

[0142] In one example, lentivirus, gamma-Retrovirus, or AAV are produced following transfection of plasmids encoding lentivirus, gamma-Retrovirus, or AAV viral or viral-like sequences into a cell.

[0143] In some embodiments, the one or more nucleic acids are DNA. In some embodiments, the one or more nucleic acids comprise one or more DNA plasmids. In some embodiments, one or more nucleic acids comprise alternatives to DNA plasmids for example but not limited to Doggy bone DNA, Bacterial artificial chromosomes (BACs), Yeast artificial chromosomes (YACs), P1 -derived artificial chromosomes (PACs), Phagemids, or Synthetic chromosomes.

[0144] In some embodiments, the method is used for production of a virus that encodes a non-viral gene product.

[0145] In some embodiments, the method is used for the production of a therapeutic.

[0146] In some embodiments, the virus is an interferon (IFN)-sensitive virus.

[0147] In some embodiments, the virus is among those disclosed in Methods and uses of increasing permissiveness of a cell to one or more nucleic acids.

[0148] In some embodiments, the cells are those disclosed in Methods and uses of increasing permissiveness of a cell to one or more nucleic acids.

[0149] In some embodiments, the virus produced by the cell is an oncolytic virus, gene therapy vector or a vaccine.

[0150] In some embodiments, production of the virus increased 1.1 fold or more, 1.2 fold or more, 1.5 fold or more, 2 fold or more, 2.5 fold or more, 3 fold or more, 5 fold or more, or 10 fold or more, e.g., compared to production of the virus by the cell, or a comparable cell, prior to the method or in the absence of the method. In some cases, the method includes measuring the increase in production of the virus, for example by determining the level of the virus in thecell and / or in the cell culture medium. Methods for determining virus production are known in the art and include, but are not limited to plaque assays, TCID50, PCR, ddPCR, ELISA, SRID and HPLC.

[0151] In some embodiments, the quality of the produced virus is improved by 1.1 fold or more, e.g., compared to the quality of the virus produced by the cell, or a comparable cell, prior to the method or in the absence of the method. In some cases, the method includes measuring the quality of virus, for example by determining the purity, potency, integrity or infectivity of the virus. Methods for determining virus quality are known in the art and include, but are not limited to, measurements of viral titer, genome integrity, infectivity assays, potency assays, electron microscopy, capillary electrophoresis and high-performance liquid chromatography (HPLC).Methods and uses of increasing transduction

[0152] The present application also includes a method, optionally an in vitro method, of increasing transduction of a virus into a cell comprising administering a compound of the application and the virus to the cell. Also included is a use of a compound of the application for increasing transduction of a virus into a cell, as well as a compound of the application for use in increasing transduction of a virus into a cell.

[0153] “Transduction” or “transducing” as used herein, refers to the introduction of a virus containing an exogenous gene into a cell leading to expression of the gene, e.g., the transgene in the cell. The gene is optionally a therapeutic gene.

[0154] In some embodiments, the compound of the application is contacted with the cell or subject prior, concurrent with, or after provision of a virus containing an exogenous gene.

[0155] In some embodiments, the method is used for the production of a therapeutic.

[0156] In some embodiments, the virus is an interferon (IFN)-sensitive virus.

[0157] In some embodiments, the virus is among those disclosed in Methods and uses of increasing permissiveness of a cell to one or more nucleic acids.

[0158] In some embodiments, the cells are those disclosed in Methods and uses of increasing permissiveness of a cell to one or more nucleic acids.

[0159] In some embodiments, the cell is a primary cell, namely a cell that is used for cell therapy. Examples of cell therapy cells include, but are not limited to, T-cells, NK-cells, TILs, Macrophages, mesenchymal stem cells, hematopoietic stem cells, induced-pluripotent stem cells, embryonic stem cells and adult stem cells.

[0160] As used herein, the expression “increasing transduction” includes increasing transduction efficiency.

[0161] In some embodiments, transduction of the virus is increased 1.1 fold or more, 1.2 fold or more, 1.5 fold or more, 2 fold or more, 2.5 fold or more, 3 fold or more, 5 fold or more, or 10 fold or more, e.g., compared to transduction of the virus by the cell, or a comparable cell, prior to the method or in the absence of the method. In some cases, the method includes measuring the increase transduction of the virus, for example by determining the level of the virus in the cell. Methods of measuring transduction efficiency are known in the art and include, but are not limited to Fluorescence imaging, in vitro and in vivo luminometry, immunohistochemistry, PCR, ddPCR and flow cytometry.Methods and uses of increasing transgene expression

[0162] The present application also includes a method, optionally an in vitro method, of increasing transgene expression comprising contacting the cell with an effective amount of a compound of the application, in combination with one or more nucleic acids, wherein at least one of the nucleic acids encodes the transgene.

[0163] Also included is a use of a compound of the application for increasing transgene expression, as well as a compound of the application for use in increasing transgene expression. Optionally, the transgene is a therapeutic gene.

[0164] In some embodiments, the compound of the application is contacted with the cell or subject prior, concurrent with, or after provision of the one or more nucleic acids or virus a containing an exogenous gene to the cell. Methods of providing nucleic acids for transgene expression by a cell can include viral infection and / or via a non-viral process, generally known as transfection, and include any a carrier or method commonly known to a person skilled in the art. In no way limiting, this includes transfection using PEI or lipid-based reagents, electroporation, and nanoparticles, as is generally known in the art.

[0165] In some embodiments, the one or more nucleic acids encode components of a virus. The term “components of a virus” refers to nucleic acids, and chemically modified variants thereof, that comprise or consist of viral and / or viral-like sequences. The sequences can encode viral proteins, viral-like proteins and / or functional sequences for targeting, integration, promotion, etc.

[0166] In some embodiments, the nucleic acids are comprised in one or more plasmids, for example DNA plasmids. In some embodiments, the one or more nucleic acids encoding components of a virus are present on a plasmid.

[0167] Delivery of nucleic acids to a cell is generally described as transfection. Transfection efficiency refers to the degree to which a supplied source of genetic material is taken up and functional for its intended purpose in a cell.

[0168] Such nucleic acids may be delivered to the cell directly, or they may be provided in a carrier to enhance delivery and uptake by the cell. Examples of carriers include diverse polymers known in the art, which may be designed in a variety of nanoparticle formats, either loosely organized or more precision designed.

[0169] In one example, lentivirus, gamma-Retrovirus, or AAV are produced following transfection of plasmids encoding lentivirus, gamma-Retrovirus, or AAV viral or viral-like sequences into a cell.

[0170] In some embodiments, the one or more nucleic acids are DNA. In some embodiments, the one or more nucleic acids comprise one or more DNA plasmids. In some embodiments, one or more nucleic acids comprise alternatives to DNA plasmids for example but not limited to Doggy bone DNA, Bacterial artificial chromosomes (BACs), Yeast artificial chromosomes (YACs), P1 -derived artificial chromosomes (PACs), Phagemids, or Synthetic chromosomes.

[0171] In some embodiments, the method is used for production of a virus that encodes a non-viral gene product.

[0172] In some embodiments, the method is used for the production of a therapeutic.

[0173] In some embodiments, the virus is an interferon (IFN)-sensitive virus.

[0174] In some embodiments, the virus is among those disclosed in Methods and uses of increasing permissiveness of a cell to one or more nucleic acids.

[0175] In some embodiments, the cells are those disclosed in Methods and uses of increasing permissiveness of a cell to one or more nucleic acids.

[0176] In some embodiments, the transgene is a virally-encoded transgene and at least one of the nucleic acids encodes one or more components of the virus.

[0177] In some of the embodiments, the transgene is a non-viral gene, and may include a chimeric antigen receptor, therapeutic protein, antibody, enzyme, or versions thereof.

[0178] In some embodiments, the compound of the application is administered to the cell before, after and / or concurrently with the nucleic acid encoding the transgene.

[0179] In some embodiments, expression of the transgene is increased 1.1 fold or more, 1.2 fold or more, 1 .5 fold or more, 2 fold or more, 2.5 fold or more, 3 fold or more, 5 fold or more, or 10 fold or more, e.g., compared to expression of the transgene, prior to the method or in the absence of the method. In some cases, the method includes measuring the expression ofthe transgene. Measuring expression levels of a transgene can be done by any method known in the art, including but not limited to measuring levels of nucleic acid expression or expression levels of protein encoded by the transgene.Methods and uses of increasing virus growth and / or virus spread

[0180] The present application also includes a method, optionally an in vitro method, of increasing virus growth and / or virus spread in cells comprising administering a compound of the application to the cells prior to, after or concurrently with the virus. Also included is a use of a compound of the application for increasing virus growth and / or virus spread, as well as a compound of the application for use in increasing virally-encoded transgene expression for increasing virus growth and / or virus spread.

[0181] In some embodiments, the compound of the application is contacted with the cell or subject prior, concurrent with, or after provision of the one or more nucleic acids or virus to the cell. Methods of providing nucleic acids for uptake by a cell can include viral infection and / or via a non-viral process, generally known as transfection, and include any a carrier or method commonly known to a person skilled in the art. In no way limiting, this includes transfection using PEI or lipid-based reagents, electroporation, and nanoparticles, as is generally known in the art.

[0182] In some embodiments, the one or more nucleic acids encode components of a virus. The term “components of a virus” refers to nucleic acids, and chemically modified variants thereof, that comprise or consist of viral and / or viral-like sequences. The sequences can encode viral proteins, viral-like proteins and / or functional sequences for targeting, integration, promotion, etc.

[0183] In some embodiments, the nucleic acids are comprised in one or more plasmids, for example DNA plasmids. In some embodiments, the one or more nucleic acids encoding components of a virus are present on a plasmid.

[0184] Delivery of nucleic acids to a cell is generally described as transfection. Transfection efficiency refers to the degree to which a supplied source of genetic material is taken up and functional for its intended purpose in a cell.

[0185] Such nucleic acids may be delivered to the cell directly, or they may be provided in a carrier to enhance delivery and uptake by the cell. Examples of carriers include diverse polymers known in the art, which may be designed in a variety of nanoparticle formats, either loosely organized or more precision designed.

[0186] In one example, lentivirus, gamma-Retrovirus, or AAV are produced following transfection of plasmids encoding lentivirus, gamma-Retrovirus, or AAV viral or viral-like sequences into a cell.

[0187] In some embodiments, the one or more nucleic acids are DNA. In some embodiments, the one or more nucleic acids comprise one or more DNA plasmids. In some embodiments, one or more nucleic acids comprise alternatives to DNA plasmids for example but not limited to Doggy bone DNA, Bacterial artificial chromosomes (BACs), Yeast artificial chromosomes (YACs), P1 -derived artificial chromosomes (PACs), Phagemids, or Synthetic chromosomes.

[0188] In some embodiments, the method is used for production of a virus that encodes a non-viral gene product.

[0189] In some embodiments, the method is used for the production of a therapeutic.

[0190] In some embodiments, the virus is an interferon (IFN)-sensitive virus.

[0191] In some embodiments, the virus is among of those disclosed in Methods and uses of increasing permissiveness of a cell to one or more nucleic acids.

[0192] In some embodiments, the cells are those disclosed in Methods and uses of increasing permissiveness of a cell to one or more nucleic acids.

[0193] In some embodiments, virus growth and / or virus spread is increased 1.1 fold or more, 1.2 fold or more, 1.5 fold or more, 2 fold or more, 2.5 fold or more, 3 fold or more, 5 fold or more, or 10 fold or more, e.g., compared to virus growth and / or virus spread, prior to the method or in the absence of the method. In some cases, the method includes measuring virus growth and / or virus spread.Methods and uses for improving manufacturing of therapeutic viral-based products

[0194] Compounds and compositions of the application are useful for improving the manufacturing processes of viral vectors or gene therapies by enhancing the viral production or transduction of cells used in manufacturing. Therefore, the compounds and compositions of the present application are useful in bioprocessing and the application also includes a compound or composition for use in combination with viral vectors or gene therapies.

[0195] Accordingly, the present application includes a method for improving manufacturing of viral vectors or gene therapies by increasing the viral production or transduction of cells. This method comprises administering a therapeutically effective amount of a compound of the application to cells used in the manufacturing process, thereby enhancing the yield and quality of the viral vectors or gene therapies produced.

[0196] In some embodiments, the compound of the application is contacted with the cell or subject prior, concurrent with, or after provision of the one or more nucleic acids, or virus containing an exogenous gene to the cell. Methods of providing nucleic acids for uptake by a cell can include viral infection and / or via a non-viral process, generally known as transfection, and include any a carrier or method commonly known to a person skilled in the art. In no way limiting, this includes transfection using PEI or lipid-based reagents, electroporation, and nanoparticles, as is generally known in the art.

[0197] In some embodiments, the one or more nucleic acids encode components of a virus. The term “components of a virus” refers to nucleic acids, and chemically modified variants thereof, that comprise or consist of viral and / or viral-like sequences. The sequences can encode viral proteins, viral-like proteins and / or functional sequences for targeting, integration, promotion, etc.

[0198] In some embodiments, the nucleic acids are comprised in one or more plasmids, for example DNA plasmids. In some embodiments, the one or more nucleic acids encoding components of a virus are present on a plasmid.

[0199] Delivery of nucleic acids to a cell is generally described as transfection. Transfection efficiency refers to the degree to which a supplied source of genetic material is taken up and functional for its intended purpose in a cell.

[0200] Such nucleic acids may be delivered to the cell directly, or they may be provided in a carrier to enhance delivery and uptake by the cell. Examples of carriers include diverse polymers known in the art, which may be designed in a variety of nanoparticle formats, either loosely organized or more precision designed.

[0201] In one example, lentivirus, gamma-Retrovirus, or AAV are produced following transfection of plasmids encoding lentivirus, gamma-Retrovirus, or AAV viral or viral-like sequences into a cell.

[0202] In some embodiments, the one or more nucleic acids are DNA. In some embodiments, the one or more nucleic acids comprise one or more DNA plasmids. In some embodiments, one or more nucleic acids comprise alternatives to DNA plasmids for example but not limited to Doggy bone DNA, Bacterial artificial chromosomes (BACs), Yeast artificial chromosomes (YACs), P1 -derived artificial chromosomes (PACs), Phagemids, or Synthetic chromosomes.

[0203] In some embodiments, the method is used for production of a virus that encodes a non-viral gene product.

[0204] In some embodiments, the method is used for the production of a therapeutic.

[0205] In some embodiments, the virus is an interferon (IFN)-sensitive virus.

[0206] In some embodiments, the virus is those disclosed in Methods and uses of increasing permissiveness of a cell to one or more nucleic acids.

[0207] In some embodiments, the cells are those disclosed in Methods and uses of increasing permissiveness of a cell to one or more nucleic acids.

[0208] In some embodiments, the cell is a primary cell, namely a cell that is used for cell therapy. Examples of cell therapy cells include, but are not limited to, T-cells, NK-cells, TILs, Macrophages, mesenchymal stem cells, hematopoietic stem cells, induced-pluripotent stem cells, embryonic stem cells and adult stem cells.

[0209] In some embodiments, the compound of the application is administered to the cells before, during, and / or after the introduction of genetic material encoding viral components. In further embodiments, the compound of the application is used to pre-treat the cells, enhancing their capacity to support viral production and transgene expression, thus improving the overall efficiency of the manufacturing process.

[0210] In some embodiments, the compound of the application is administered to the cell during manufacturing as a batch feed, pulse feed, continuous feed, gradient feed, or controlled release.

[0211] In some embodiments, the compound of application is administered to the cell through closed or open systems.

[0212] In some embodiments, the compound of the application allows for a reduction in the amount of upstream manufacturing components like genetic material or cells, thereby lowering production costs and increasing the scalability of the manufacturing process.

[0213] In some embodiments, the compound of application can be integrated within existing production processes in a simple way.

[0214] In some embodiments, the compound of application can be removed or significantly reduced from the final product using typical viral vector purification procedures resulting in residual traces.

[0215] In some embodiments, the compound can enhance the replication and packaging efficiency of the viral vectors within the manufacturing cells.

[0216] In some embodiments, the compound of the application can be used to improve the production of viral vectors or gene therapies for a variety of conditions, including genetic disorders, and infectious diseases. For example, the compound can enhance the production of viral vectors designed for use in gene therapy for inherited diseases.

[0217] In some embodiments, the compound can be applied to enhancing viral production or transduction of cells within large-scale culture systems. In some embodiments, large-scale culture systems include and are not limited to stirred-take bioreactors, wave bioreactors, airlift bioreactors, hollow fiber bioreactors, fixed bed bioreactors and single-use bioreactors.

[0218] In some embodiments, the compound can be applied to enhancing viral production or transduction of cells within small-scale culture systems. In some embodiments, small-scale culture systems include and are not limited to spinner flasks, T-flasks, roller bottles, cell stacks, hyper stacks, shake flasks, perfusion systems and wave-mixed bag systems.

[0219] To be clear, in the above methods and uses, the term “a compound of the application” also includes embodiments wherein one or more compounds of the application are referenced or formulated in a composition as described herein.IV. Methods of Preparing the Compounds and Compositions of the Application

[0220] Compounds of the present application can be prepared by various synthetic processes. The choice of particular structural features and / or substituents may influence the selection of one process over another. The selection of a particular process to prepare a given compound of the application is within the purview of the person of skill in the art. Some starting materials for preparing compounds of the present application are available from commercial chemical sources or may be extracted from cells, plants, animals or fungi. Other starting materials, for example as described below, are readily prepared from available precursors using straightforward transformations that are well known in the art.

[0221] Salts of the compounds of the application are generally formed by dissolving the neutral compound in an inert organic solvent and adding either the desired acid or base and isolating the resulting salt by either filtration or other known means.

[0222] The formation of solvates of the compounds of the application will vary depending on the compound and the solvate. In general, solvates are formed by dissolving the compound in the appropriate solvent and isolating the solvate by cooling or using an antisolvent. The solvate is typically dried or azeotroped under ambient conditions. The selection of suitable conditions to form a particular solvate can be made by a person skilled in the art. Examples of suitable solvents are ethanol, water and the like. When water is the solvent, the molecule is referred to as a “hydrate”.

[0223] Prodrugs of the compounds of the present application may be, for example, conventional esters formed with available hydroxy, thiol, amino or carboxyl groups. For example, available hydroxy or amino groups may be acylated using an activated acid in the presence of a base, and optionally, in inert solvent (e.g. an acid chloride in pyridine). Somecommon esters which have been utilized as prodrugs are phenyl esters, aliphatic (C1-C24) esters, acyloxymethyl esters, carbamates and amino acid esters.

[0224] Throughout the processes, it is to be understood that, where appropriate, suitable protecting groups will be added to, and subsequently removed from, the various reactants and intermediates in a manner that will be readily understood by one skilled in the art. Conventional procedures for using such protecting groups as well as examples of suitable protecting groups are described, for example, in “Protective Groups in Organic Synthesis", T.W. Green, P.G.M. Wuts, Wiley-lnterscience, New York, (1999). It is also to be understood that a transformation of a group or substituent into another group or substituent by chemical manipulation can be conducted on any intermediate or final product on the synthetic path toward the final product, in which the possible type of transformation is limited only by inherent incompatibility of other functionalities carried by the molecule at that stage to the conditions or reagents employed in the transformation. Such inherent incompatibilities, and ways to circumvent them by carrying out appropriate transformations and synthetic steps in a suitable order, will be readily understood to one skilled in the art. Examples of transformations are given herein, and it is to be understood that the described transformations are not limited only to the generic groups or substituents for which the transformations are exemplified. References and descriptions of other suitable transformations are given in “Comprehensive Organic Transformations - A Guide to Functional Group Preparations” R.C. Larock, VHC Publishers, Inc. (1989). References and descriptions of other suitable reactions are described in textbooks of organic chemistry, for example, “Advanced Organic Chemistry", March, 4th ed. McGraw Hill (1992) or, “Organic Synthesis", Smith, McGraw Hill, (1994). Techniques for purification of intermediates and final products include, for example, straight and reversed phase chromatography on column or rotating plate, recrystallisation, distillation and liquid-liquid or solid-liquid extraction, which will be readily understood by one skilled in the art.EXAMPLESExample 1Materials and MethodsCell lines

[0225] Cell lines: HEK293T (human embryonic kidney epithelium), 786-0 (human renal carcinoma), and Vero (monkey kidney) cells were obtained from the American Type Culture Collection and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum and buffered with 30mM Hepes. HEK293 cells were obtained from the American Type Culture Collection and maintained in DMEM Medium supplemented with 2% FBS. VPC 2.0 HEK 293 (human embryonic kidney epithelium) were obtained fromThermoFisher and maintained in Viral Production Media. HEK293T, 786-0, Vero, and HEK293 cells were incubated at 37°C with 5% C02and the VPC 2.0 HEK293 cells were incubated at 37°C with 8% C02.

[0226] Primary human pan (CD3+) T-cells were obtained from STEMCELL Technologies, maintained in 4-Cell Nutri-T Media and IL-2. T-cells were activated using T-Cell TransACT obtained from Miltenyi.Viruses

[0227] VSVA51 is a recombinant variant of the Indiana serotype of VSV harbouring a deletion of the 51stmethionine in the M protein. VS A51 expressing firefly luciferase (FLuc) and VSVA51 expressing green fluorescent protein (GFP) were a recombinant derivative of VS A51. All virus stocks were propagated in Vero cells, purified on Optiprep gradient and tittered on Vero cells as described in (Diallo et al. Methods Mol Bio. 2012;797:127:40).

[0228] Lentiviral vector encoding EmGFP (>1e8 TU / mL) was obtained from Genscript.

[0229] Ad5 expressing GFP was purchased from Vector Biolabs (Malvern, PA, USA), amplified on HEK293 cells, purified by CsCI gradient, and quantified using the Adeno-X Rapid Titer Kit from Takara (Palo Alto, CA, USA).Compound testing in 786-0 cells for enhanced production of VSVA51

[0230] 786-0 cells were seeded at 1.05e4 cells / well in 96well format (100pL / well) in order to reach 100% confluency, the following day. Cells were then treated with compounds at multiple concentrations ranging from 1 pM to 40pM for 4 hours. After compound treatment, cells were infected with VSVA51 at MOI of 0.05. Infected cells were incubated at 37°C with 5% C02for 48 hours.

[0231] One day following infection, Vero cells were plated in 96well format at 1 ,2e4 cells / well to reach 100% confluency the following day. These cells are susceptible to VSVA51 infection and allow for high throughput virus quantification. At 48 hours following infection of 786-0 cells, VSVA51 in the supernatant was transferred on to Vero cells for quantification of VSVA51- fLuc expression. A standard curve was also added to Vero cells in a similar manner as described herein. Infected Vero cells were incubated at 37°C with 5% CO2for two hours.

[0232] After five hours, Vero cells were read for luminescent activity in a similar manner as described herein. Luminescent data was obtained and analyzed as described herein.Compound testing in HEK293 cells for enhanced production of Adenovirus 5

[0233] HEK293 cells were seeded at 3e4 cells / well in 96well plate format (100 pL / well) in order to reach 80% confluency the following day. Cells were treated with CP-673451 and Crenolanib at 1 , 5, 10, 20 and 40 pM. After treatment with an exemplary compound of the application, cells were infected with Ad5-GFP at MOI of 5 and infected cells were incubated at 37°C with 5% CO2for 48 hours.

[0234] One day prior to transduction, HEK293 cells were plated in 96well format at 1e4 cells / well to reach 50% confluency the following day. These cells are susceptible to Ad5 infection and allow for high throughput virus quantification. At 48 hours following infection of HEK293 cells, Ad5-containing supernatant (after 3 freeze-thaw cycles) was transferred on to HEK293 reporter cells for quantification of Ad5-GFP expression. A standard curve was also added to HEK293 cells in a similar manner as described herein. Infected HEK293 cells were incubated at 37°C with 5% CO2for 24 hours.

[0235] After 24 hours, the Agilent Cytation was used to quantify GFP fluorescence, equivalent to functional titer.Compound testing for enhanced Adenovirus 5 encoded GFP expression

[0236] HEK293 cells were seeded at 3e4 cells / well in 96well plate format (100 pL / well) in order to reach 80% confluency the following day. Cells were then treated with CP-673451 and Crenolanib at 1 , 5, 10, 20 and 40 pM. After compound treatment, cells were infected with Ad5- GFP at MOI of 5 and infected cells were incubated at 37°C with 5% C02for 40 hours.

[0237] Ad5 encoded GFP expression was measured by quantifying GFP fluorescence using the Agilent Cytation.Compound testing for enhanced VSVA51 encoded GFP expression

[0238] 786-0 cells were seeded at 1.05e4 cells / well in 96well plate format (100 pL / well) in order to reach 100 % confluency the following day. Cells were treated with CP-673451 and Crenolanib at 10, 20 and 40 pM. After compound treatment for 4 hours, cells were infected with VSVA51-GFP at MOI of 0.05. Infected cells were incubated at 37°C with 5% C02for 24 hours. VSVA51 -encoded GFP expression was measured by quantifying GFP fluorescence using Thermo Scientific Celli nsight CX5.AAV vectors

[0239] A three-plasmid system was used for the production of AAV2 consisting of pAAV-RC2, pHelper and pAAV-transgene, engineered to express a firefly luciferase (fLuc) transgene and manufactured using transient transfection of VPC2.0 HEK293 suspension cells.

[0240] A three-plasmid system was used for the production of AAV8 consisting of pAAV-RC8, pHelper and pAAV-transgene, engineered to express a firefly luciferase (fLuc) transgene and manufactured using transient transfection of VPC 2.0 HEK293 suspension cells.Compound testing in VPC2.0 HEK293 for enhanced AAV2 production

[0241] Compounds were tested in VPC 2.0 HEK293 in 96 well format or 125mL shake flask format following a batch (reverse) transfection method. VPC 2.0 HEK293 cells were suspended at 3e6 cells / mL in Viral Production Media prior to transfection. A transfection mixture was prepared containing the three AAV2 plasmids, and a corresponding amount of Fectovir™ transfection reagent. The plasmid and Fectovir mixtures were allowed to complex for 15 minutes at room temperature prior to addition to the VPC 2.0 HEK293 cell mixture.

[0242] In the 96 well format, batch transfected VPC2.0 HEK293 cells were then plated in 96 well format at 5.3e4 cells / well, in triplicate, using a high-throughput liquid dispenser. Immediately following plating, VPC 2.0 HEK293 cells were treated with exemplary compounds of the application at a concentration of 0.1 .M, 1 pM and 10 .M, or a corresponding amount of library vehicle control (Dimethyl sulfoxide (DMSO)) using a high-throughput liquid handler. Treated cells were incubated at 37°C with 8% C02for 72 hours.

[0243] In the 125mL shake flask format, 30mL of batch transfected VPC2.0 HEK293 cells are aliquoted per flask, in duplicate. The VPC2.0 HEK293 cells were then treated with exemplary compounds of the application at concentrations ranging from 5 pM to 40 .M.

[0244] Two days following transfection, HEK293T reporter cells were plated in 96well format at 6.26e4 cells / well, in order to reach 65% confluency the following day. These cells acted as reporter cells that were easily transduced by AAV-containing lysate from producer cells and allowed for high throughput virus quantification. At 72 hours following transient transfection of VPC2.0 HEK 293 production cultures, AAV2 from harvested cell lysate was transferred on to the HEK293T reporter cell line for transduction analysis and quantification of AAV2-driven fLuc expression. A standard curve was also added to the HEK293T reporter plates in a similar manner as described herein. Transduced HEK293T cells were incubated at 37°C with 5% CO2.

[0245] At 72 hours following the transduction, HEK293T reporter cells were read for luminescent activity in a similar manner as described herein. Luminescent data was obtained and analyzed as described herein.Compound testing in VPC2.0 HEK293 for enhanced AAV8 production

[0246] Compounds were tested in VPC 2.0 HEK293 cells in 125mL Shake flask format following a batch (reverse) transfection method. VPC 2.0 HEK293 cells were suspended at 3e6 cells / mL in Viral Production Media prior to transfection with either the Fectovir-AAV or PEIpro transfection reagent.

[0247] To prepare the Fectovir-AAV transfection mixture, three AAV8 plasmids were added to a corresponding amount of Fectovir-AAV transfection reagent. The plasmids and transfection reagent mixtures were allowed to complex for 30 minutes at room temperature prior to addition to the VPC 2.0 HEK293 cell mixture. Following complexation, the mixture was added to the VPC 2.0 HEK293 cells to transfect cells.

[0248] For the PEIpro transfection system, three AAV8 plasmids were added to the corresponding amount of PEIpro transfection reagent and allowed to complex for 15 minutes at room temperature. Following complexation, the mixture was added to the VPC 2.0 HEK293 cells to transfect cells.

[0249] 30mL of batch transfected VPC2.0 HEK293 cells were aliquoted in each 125mL shake flask, in duplicate. Transfected VPC 2.0 HEK293 cells were immediately treated with test compounds at relevant concentrations or a corresponding amount of library vehicle control (Dimethyl sulfoxide (DMSO)). Treated cells were incubated at 37°C with 8% C02for 72 hours. Three days following transfection, AAV crude lysate was harvested using chemical lysis.

[0250] Genomic titer was quantified using qPCR (ABM materials, cat. #G931.) per the manufacturer’s protocol.Compound testing for enhanced lentiviral transduction of T-cells

[0251] Primary human pan (CD3+) T-cells were thawed and re-suspended in media. Following centrifugation at 800xg for 5 minutes, the cell pellet was resuspended in 4Cell Nutri- T Advanced Media supplemented with IL-2. Thawed cells were then activated using T-cell TransAct and incubated with agitation at 37°C and 5% CO2for 24 hours.

[0252] Activated T-cells were seeded in 384-well plates (at 25pL / well) and treated with test compounds at a concentration of 10 and 15 mM for 30 minutes. GFP-encoding lentivirus vector was added to the T-cells to achieve a final MOI of 4. Spinoculation was performed by centrifugation at 800xg for 1 hour at 32°C. Plates were then incubated at 37°C and 5% CO2for 72 hours. To quantify transduction efficiency, the percentage of GFP-expressing cells was quantified by flow cytometry using the MACSQuant Analyzer 16.EXPERIMENTAL RESULTSExample 2

[0253] The ability of compounds of the application to enhance plasmid-based viral vector production in the VPC 2.0 HEK293 suspension cell line was tested, since this cell line, its related parent line (HEK293) and subclonal derivatives are commonly used to produce viral vectors including but not limited to lentivirus and adeno-associated virus. The reverse genetic system employed consisted of transient transfection of three circular plasmids appropriate to produce AAV encoding a luciferase transgene in small-scale format. The compounds were tested at a 0.1 p.M, 5p.M and 10p.M concentration using the firefly luciferase (fLuc)-encoding AAV production system using a high-throughput batch transfection assay described herein, measuring AAV2 output from HEKVPC 2.0 cells from a secondary transduction assay carried out in an adherent ATCC HEK293T reporter cell line.

[0254] Impact on AAV2 production was inferred by measuring the luciferase expression resulting from transfer of AAV-containing supernatants (after 3 freeze-thaw cycles) from compound-treated I AAV plasmid-transfected VPC 2.0 HEK293 cells. In this assay, the luciferase signal (Relative Light Units or RLU) ensued from the transduction of adherent HEK293T reporter cells, which correlated with quantity of functional virus as determined from a standard curve tested of AAV2 in parallel.

[0255] Several PDGFR inhibitors were identified, most notably Crenolanib and CP673451 , as well as EGFR inhibitors Ibrutinib and Rociletinib as positive drivers of AAV2 production at several doses (FIG.1). All compounds provided at least 2-fold statistically significant enhancements in AAV production.Example 3

[0256] To evaluate the ability of PDGFR inhibitors to enhance viral vector production across AAV serotypes, CP-673451 and Crenolanib were evaluated for their ability to enhance AAV8 genomic titer in HEK 293 VPC 2.0 cells transfected with Fectovir-AAV reagent. Fold enhancement in genomic titer was calculated relative to transfection reagent-matched untreated control conditions. Consistent with prior observations of AAV2 production, CP- 673451 enhanced AAV8 production at 1 , 5, 10 and 15 pM relative to the untreated control as shown in FIG. 3A. Crenolanib statistically enhanced AAV8 production at 10 pM as shown in FIG. 3B. These results extend the application of PDGFR inhibitors across multiple AAV serotypes, cell lines, and transfection reagents.Example 4

[0257] PDGFR inhibitor CP-673451 was tested to assess the ability of enhancing AAV2 viral production across different transfection reagents in 125ml_ shake flasks. HEK293 VPC2.0 cells were transfected with either Feet oVir- AAV or PEIpro reagents. CP-673451 was added at 5, 10, 20 and 40 pM. Fold change of RLU equivalent to functional AAV2 titer output was measured from a secondary transduction assay described herein. Results are summarized in FIG. 4. CP-673451 at 5 and 10 pM enhanced functional titer of AAV2 in HEK293 VPC2.0 transfected with either FectoVir-AAV or PEIpro reagents. These results extend the application of PDGFR inhibitors across multiple transfection reagents.Example 5

[0258] To test the applicability of these compounds with enhancing replicating viruses including but not limited to rhabdoviruses, the PDGFR inhibitors CP-673451 and Crenolanib were investigated for their ability to enhance replication of a recombinant variant of vesicular stomatitis virus (VSV). CP-673451 was tested at various concentrations ranging from 4pM to 40 pM in 786-0 cells using the methods described herein.

[0259] As shown in FIG. 5A and Fig. 5B, fold enhancement in replication-competent VS A51 functional titers over untreated control was assessed by measuring VSV-mediated reporter signal in target Vero cells following exposure to cell-free viral supernatant from treated and infected 786-0 cells. The assay quantified functional viral output of VS A51 in response to PDGFR inhibitors CP-673451 and Crenolanib.

[0260] Crenolanib most notably increased VSV replication at 5, W and 15 pM over the control.

[0261] Statistically significant enhancement of VSV replication was observed at 4, 5 and 8 pM of CP-673451 . These findings demonstrate that PDGFR inhibitors CP-673451 and Crenolanib not only enhances non-replicating AAV vector production but also augments the output of actively replicating rhabdovirus, supporting broader applicability of PDGFR inhibitors in viral production systems.Example 6

[0262] The ability of PDGFR inhibitors to enhance virally-encoded transgenes was evaluated by testing their effect on Ad5-encoded and VSV A51 -encoded GFP expression in HEK293 cells and 786-0 cells, respectively. As shown in FIG. 6A and FIG. 6B, CP-673451 and Crenolanib were tested at 1 , 5, 10, 20 and 40 pM for their impact on Adenovirus - encoded GFP expression. CP-673451 significantly enhanced Ad5-encoded GFP expression at 1 and 5 pM, and Crenolanib significantly increased Ad5-encoded GFP expression at 1 pM. As shown in FIG. 8A and FIG. 8B, CP-673451 and Crenolanib both significantly enhanced VSVA51-encoded GFP expression in 786-0 cells at 1 pM. This demonstrates that PDGFR inhibitors enhance virally-encoded transgenes in replicating and non-replicating viruses.Example 7

[0263] T o investigate the ability of PDGFR and EGFR inhibitors to enhance virus transduction, Ibrutinib and Crenolanib were evaluated for their impact on primary T-cell transduction using a GFP-encoding lentiviral vector. Human CD3+T cells were activated and subsequently transduced in the presence of 10 and 15 pM of Ibrutinib or Crenolanib. Transduction efficiency was measured by flow cytometry as the percentage of GFP-positive cells 72 hours posttransduction, and fold enhancement was calculated relative to untreated control.

[0264] As shown in FIG. 7A, 15 pM of Ibrutinib enhanced lentiviral transduction in primary T cells by 1.6-fold. As shown in FIG. 7B, statistically significant enhancement of lentiviral transduction in primary T-cells was observed with 10 pM of Crenolanib. Elevated transduction levels indicate that PDGFR and EGFR inhibitors are effective at also increasing virus transduction.Example 8

[0265] Adenovirus viral vector production was evaluated in adherent HEK293 cells. HEK293 cells were infected Ad5-GFP virus at MOI of 5 and treated with CP-673451 (exemplary PDGFR inhibitor) at 1 , 5, 10, 20 and 40 pM. 48 hours later, Ad5-containing samples were transferred on HEK293 reporter cells and incubated for 24 hours. GFP fluorescence was quantified and indicative of Adenovirus 5 functional. As shown in FIG. 9, CP-673451 enhanced Adenovirus 5 production at 5 pM by 1.5-fold relative to the control, demonstrating the ability of PDGFR inhibitors enhance the production of various viral vectors.

[0266] While the applicant's teachings described herein are in conjunction with various embodiments for illustrative purposes, it is not intended that the applicant's teachings be limited to such embodiments as the embodiments described herein are intended to be examples. On the contrary, the applicant's teachings described and illustrated herein encompass various alternatives, modifications, and equivalents, without departing from the embodiments described herein, the general scope of which is defined in the appended claims.References:

[0267] Robert Meineke, Sonja Stelz, Maximilian Busch, Christopher Werlein, Mark Kuhnel, Danny Jonigk, Guus F. Rimmelzwaan, Husni Elbahesh, FDA-approved Abl / EGFR / PDGFR kinase inhibitors show potent efficacy against pandemic and seasonal influenza A virus infections of human lung explants, iScience, Volume 26, Issue 4, 2023, 106309, ISSN 2589- 0042, https: / / doi.orq / 10.1016Zi.isci.2023.106309.

[0268] Hideki Tani, Miyuki Kimura, Hiroshi Yamada, Hikaru Fujii, Satoshi Taniguchi, Masayuki Shimojima, Shuetsu Fukushi, Shigeru Morikawa, Masayuki Saijo, Activation of platelet-derived growth factor receptor p in the severe fever with thrombocytopenia syndrome virus infection, Antiviral Research, Volume 182, 2020, 104926, ISSN 0166-3542, https: / / doi.Org / 10.1016 / j. antiviral.2020.104926.

Claims

Claims1. A method of increasing permissiveness of a cell to one or more nucleic acids, comprising contacting the cell with an effective amount of a PDGFR inhibitor.

2. The method of claim 1 , wherein the PDGFR inhibitor is CP673451 , Crenolanib (CP- 868596), Sunitinib, Sorfafenib, Pazopanib, Motesanib, Cediranib, Axitininb, Linifanib or an analogue or a derivative thereof, or a pharmaceutically acceptable salt, where applicable, solvate and / or prodrug thereof.

3. A method of increasing permissiveness of a cell to one or more nucleic acids, comprising contacting the cell with an effective amount of an EGFR inhibitor.

4. The method of claim 3, wherein the EGFR inhibitor is Ibrutinib and Rociletinib or an analogue or a derivative thereof, or a pharmaceutically acceptable salt, solvate and / or prodrug thereof.

5. The method of any one of claims 1 to 4, wherein the one or more nucleic acids encode one or more components of a virus.

6. The method of any one of claims 1 to 5, wherein the one or more nucleic acids comprise one or more plasmids.

7. The method of claim 5, wherein the virus is an adeno-associated virus (AAV), optionally wherein the AAV is selected from the group consisting of AAV serotypes 1-9 and recombinant serotypes thereof.

8. The method of claim 5, wherein the virus is a retrovirus.

9. The method of claim 8, wherein the retrovirus is a lentivirus, optionally wherein the lentivirus is selected from the group consisting of HIV-1 , HIV-2, SIV, and recombinant lentiviruses.

10. The method of any one of claims 1 to 9, wherein the cell is contacted with the inhibitor before, after, or concurrently with the one or more nucleic acids.11 . The method of any one of claims 1 to 10, wherein the cell is a mammalian cell.

12. The method of claim 5, wherein production of the virus by the cell is increased by at least 1.1 fold compared to a control cell.

13. The method of claim 5, wherein production of the virus by the cell is increased by at least 1 .5 fold compared to a control cell.

14. A method of increasing production of a virus in a cell, comprising contacting the cell with an effective amount of a PDGFR and / or EGFR inhibitor, in combination with one or more nucleic acids encoding components of the virus.

15. The method of claim 14, wherein the one or more nucleic acids comprise one or more plasmids.

16. The method of claim 14 or 15, wherein the virus is an adeno-associated virus (AAV), optionally wherein the AAV is selected from the group consisting of AAV serotypes 1-9 and recombinant serotypes thereof.

17. The method of claim 14 or 15, wherein the virus is a retrovirus.

18. The method of claim 17, wherein the retrovirus is a lentivirus, optionally wherein the lentivirus is selected from the group consisting of HIV-1 , HIV-2, SIV, and recombinant lentiviruses.

19. The method of claim 14, wherein the one or more nucleic acids are introduced by viral infection.

20. The method of claim 19, wherein the virus is an RNA virus.21 . The method of claim 19, wherein the virus is a DNA virus.

22. The method of claim 19, wherein the virus is a Rhabdovirus, recombinant or derivative thereof23. The method of claim 19, wherein the virus is an Adenovirus, serotype, recombinant or derivative thereof24. The method of any one of claims 11 to 23, wherein the cell is contacted with the compound before, after, or concurrently with the one or more nucleic acids.

25. The method of any one of claims 11 to 24, wherein the cell is a mammalian cell.

26. The method of any one of claims 11 to 25, wherein production of the virus by the cell is increased by at least 1.1 fold compared to a control cell.

27. The method of any one of claims 11 to 25, wherein production of the virus by the cell is increased by at least 1.5 fold compared to a control cell.

28. A method of increasing transgene expression in a cell comprising contacting the cell with an effective amount of a PDGFR and / or EGFR inhibitor, in combination with one or more nucleic acids, wherein at least one of the nucleic acids encodes the transgene.

29. The method of claim 28, wherein the transgene is a virally-encoded transgene and the one or more nucleic acids further comprise one or more components of a virus.

30. The method of claim 28 or 29, wherein the transgene is a therapeutic gene.

31. The method of any one of claims 28 to 30, wherein the virus is an adeno-associated virus (AAV), optionally wherein the AAV is selected from the group consisting of AAV serotypes 1-9 and recombinant serotypes thereof.

32. The method of any one of claims 28 to 30, wherein the virus is a retrovirus.

33. The method of claim 32, wherein the retrovirus is a lentivirus, optionally wherein the lentivirus is selected from the group consisting of HIV-1 , HIV-2, SIV, and recombinant lentiviruses.

34. A method of increasing transduction of a virus into a cell, comprising contacting the cell with an effective amount of a PDGFR inhibitor.

35. The method of claim 34, wherein the PDGFR inhibitor is CP673451 , Crenolanib (CP-868596), Sunitinib, Sorfafenib, Pazopanib, Motesanib, Cediranib, Axitininb, Linifanib or an analogue or a derivative thereof, or a pharmaceutically acceptable salt, where applicable, solvate and / or prodrug thereof.

36. A method of increasing transduction of a virus into a cell, comprising contacting the cell with an effective amount of an EGFR inhibitor.

37. The method of claim 36, wherein the EGFR inhibitor is Ibrutinib and Rociletinib or an analogue or a derivative thereof, or a pharmaceutically acceptable salt, solvate and / or prodrug thereof.

38. The method of any one of claims 34 to 37, wherein the virus is a retrovirus.

39. The method of claim 38, wherein the retrovirus is a lentivirus, optionally where the lentivirus is selected from the group consisting of HIV-1 , HIV-2, SIV, and recombinant lentiviruses.

40. A method of increasing production of an adeno-associated virus (AAV), comprising contacting the cell with an effective amount of a PDGFR and / or EGFR inhibitor, in combination with one or more nucleic acids encoding components of the AAV.

41. The method of claim 40, wherein the cell is contacted with the compound before, after, or concurrently with the one or more nucleic acids.

42. The method of claim 40 or 41 , wherein the production of AAV is increased by at least 1.1 fold compared to a control cell.

43. The method of claim 40 or 41 , wherein the production of AAV is increased by at least 1 .5 fold compared to a control cell.

44. The method of any one of claims 40 to 43, wherein the AAV is selected from the group consisting of AAV serotypes 1-9 and recombinant serotypes thereof.

45. A composition comprising a PDGFR and / or EGFR inhibitor, and a one or more nucleic acids encoding components of a virus.

46. Use of the composition of claim 45 for increasing production of a virus in a cell.

47. Use of the composition of claim 45 for increasing transduction of a virus in a cell.

48. A method of increasing transduction of a virus into a cell, comprising contacting the cell with an effective amount of PDGFR and / or EGFR inhibitor.

49. The method of claim 48, wherein the virus is a retrovirus.

50. The method of claim 49, wherein the retrovirus is a lentivirus, optionally wherein the lentivirus is selected from HIV-1 , HIV-2, SIV, and recombinant lentiviruses.

51. The method of claim 48, wherein the virus is an Adeno Associated Virus or a serotype, recombinant, or derivative thereof.

52. The method of claim 48, wherein the virus is a Rhabdovirus, recombinant, or derived thereof.

53. The method of claim 48, wherein the virus is an Adenovirus, serotype, recombinant, or derivative thereof.

54. The method of any one of claims 47 to 52, wherein the cell is an immune cell or stem cell.

55. The method of any one of claims 47 to 52, wherein the cell is a T-cell.

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