Viral production enhancers, methods and uses thereof

Abstract

The present application relates to viral sensitizers More specifically, the present application relates to compounds of Formula (I), 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

[0005] These compounds have also been successfully applied commercially to improve the production of viral-based products. Small molecules present a number of advantages over other methods to improve virus yields because they are simple additives that can be introduced during the transfection or infection stages of viral production. They integrate seamlessly into existing workflows without requiring significant changes to established protocols, making them user-friendly for current manufacturing processes. Further, small molecule compounds are easily eliminated using typical viral vector purification procedures and do not generally leave residual traces after purification, ensuring that the viral vectors remain pure and suitable for therapeutic use. This characteristic simplifies the downstream purification processes, reducing the need for additional steps to remove contaminants and ensuring regulatory compliance. From this perspective, there is a need to use compounds that have low mutagenic, carcinogenic, and teratogenic potential.

[0006] While different viral sensitizing compounds can be effective in a broad range of applications; their efficacy can fluctuate depending on the context owing to differences in their physicochemical properties as well as their compound-specific off-target effects.

[0007] As such, there is need to provide improved viral sensitizing compounds, for example, for viral-based gene therapies and viral vector production.SUMMARY

[0008] Several compounds that significantly enhance lentivirus production were identified. Compounds also demonstrated activity in enhancing AAV production from HEK293 cells, showing broader applicability across plasmid-based reverse genetics production systems. Given plasmid-based reverse genetics systems have been developed and previously 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 applies to improving the production of both replicating and non-replicating 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.

[0009] It has been shown that compounds of Formula (I) of the present application 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.

[0010] Accordingly, the present application includes a method of increasing permissiveness of a cell to a reverse genetics system, comprising administering an effective amount of a compound of Formula (I), or a salt, solvate and / or prodrug thereof, to the cell.

[0011] Accordingly, the present application includes a compound of Formula (I), or a salt, solvate and / or prodrug thereof (compond of the application).210490331

[0012] Further included is a method of increasing permissiveness of a cell to a virus, comprising administering an effective amount of a compound of the present application to the cell.

[0013] The present application also includes a method of increasing permissiveness of a cell to genetic material encoding components of a virus, comprising administering an effective amount of a compound of the present application to the cell in combination with provision of the genetic material encoding components of a virus to the cell.

[0014] Also included is a method of increasing production of a virus by a cell comprising administering a compound of the present application to the cell.

[0015] The present application further includes a method of increasing virus propagation in a cell comprising administering a compound of the present application and the virus to the cell.

[0016] Also included is a method of increasing expression of a transgene, including but in no way limited to viral genes, comprising administering a compound of the present application and reverse genetics system to a cell.

[0017] Also included is a method of increasing virally-encoded transgene expression comprising administering a compound of the present application and the virus to a cell.

[0018] Further included is a method of increasing virus growth in cells comprising administering a compound of the present application to the cells in combination with provision of the virus to the cells.

[0019] 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 compound of the application, in combination with the virus.

[0020] Other features and advantages of the present application will become apparent from the following detailed description. It should be understood, however, that the detailed description 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 THE DRAWINGS

[0021] The embodiments of the application will now be described in greater detail with reference to the attached drawings in which:

[0022]

[0250] FIG.1A, FIG.1B and FIG.1C show a comparative analysis for enhancement of lentivirus production and producer cell viability for exemplary compounds of the application. For all panels, the values represent the average of three technical replicates (n=3). An asterisk (*) indicates statistical significance with p-values <0.05 calculated using unpaired, two-tailed310490331t-test. FIG.1A (from left to right, compounds compounds 1-1 to I-4, I-6, I-5, I-7 to I-33, I-44, I- 54, I-55, I-62, I-64 to I-66, I-79 to 1-88) 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. FIG.1B (from left to right, compounds compounds 1-1 to I-5, I-7, I-6, I-8 to I-33, I-44, I-54, I-55, I-62, I-64 to I-66, I-79 to 1-88) shows fold enhancement above library vehicle control in viral expression units. FIG.1C (from left to right, compounds compounds 1-1 to I-5, I-7, I-6, I-8 to I-33, I-44, I-54, I-55, I-62, I-64 to I-66, I- 79 to 1-88) shows the relative cell viability in producer HEK293T cells for each of the compounds included in FIG.1A and FIG.1B. Average cell viability was measured using a resazurin-based cell metabolic activity assay in HEK293T producer cells, 48 hours posttreatment and post-transfection.

[0023] FIG.2 shows lentivirus fold enhancement data in two adherent HEK293T subclones, for a subset of the exemplary compounds of the application highlighted in FIG.1 , and Table 1. A subset of these compounds were selected for additional testing. This further testing was conducted using the same transient batch transfection methods used for all other studies, at three concentrations (5 .M, 10 .M and 15 .M). FIG.2 (from left to righ, compounds I-7, I-9, I- 12, 1-13, I-22) is a bar graph showing the fold enhancement of lentivirus production based on virus-mediated luminescent signal over library vehicle control in both HEK293T (solid bars) and HEK293T / 17 (dotted bars) producer cells. The values represent the average of three technical replicates (n=3), with asterisks (*) denoting statistically significant enhancements with p-values <0.05 calculated through unpaired, two-tailed t-test.

[0024] FIG.3 presents a bar graph illustrating greater than 1.1 -fold enhancement of AAV2 production in adherent HEK293 cells as determined by virus-driven luminescent signal for a selected subset of the exemplary compounds of the application (from left to right, compounds 1-1 , I-4, I-7, 1-13, 1-16, 1-15, 1-17 to 1-19, 1-21 to I-23, I-26, I-28, 1-31 , I-33 to 1-78). These compounds were tested at a concentration of 10 .M, and the values represent the average fold change in viral production compared to the library vehicle control (1.05% DMSO). This data is averaged from three technical replicates (n=3), and statistical significance is indicated by asterisk (*), with p-values <0.05 (calculated by unpaired, two-tailed t-test). This figure indicates that the identified class of molecules are also effective in enhancing the production of AAV2, suggesting their applicability to a range of plasmid-based reverse genetic systems.

[0025] FIG. 4A, FIG. 4B, FIG. 4C and FIG. 4D shows greater than 1.1-fold enhancement of lentivirus production in suspension adherent and suspension HEK293 derived cells with multiple transfection reagents in 96 well format for a subset of exemplary compounds of the application (from left to right, compounds I-7, I-9, 1-12, 1-13 and I-35) at differing peak concentrations. All panels show fold change of RLU equivalent to lentivirus functional titer and410490331values represent the average of three technical replicates (n=3). More specifically, in FIG. 4A, these exemplary compounds were tested at concentrations varying from 0.25 pM to 80 pM with adherent HEK293T / 17 cells with PEIPro™ transfection reagent. In FIG. 4B, these exemplary compounds were tested from 0.25 pM to 80 pM in suspension VPC1.0 HEK293 cells with PEIPro™ transfection reagent. FIG. 4C shows fold enhancement of RLU equivalent to lentivirus functional titer in VPC2.0 HEK293 cells transfected with FectoVir-LV reagent and treated with exemplary compounds at 5 pM to 40 .M. In FIG. 4D, these exemplary compounds were tested at varying concentrations for lentivirus functional titer in VPC1.0 HEK293 cells with LV-MAX™ transfection reagent, LV-MAX™ supplement and enhancer.

[0026] FIG. 5A, FIG. 5B and FIG. 5C shows greater than 1.1 -fold enhancement of AAV2 production in adherent and suspension HEK293 based cells with two transfection reagents in 96 well format for a subset of exemplary compounds of the application (from left to right, compounds I-7, 1-13, I-23 and I-35) at differing peak concentrations. All panels show fold change of RLU equivalent to functional titer and values represent the average of three technical replicates (n=3). More specifically, in FIG. 5A, these exemplary compounds were tested at concentrations varying from 0.25 pM to 80 pM in Expi293F™ suspension cells transfected with FectoVir-AAV™ reagent. In FIG. 5B, these exemplary compounds were tested at concentrations ranging from 0.25 pM to 80 pM in HEK293 cells transfected with PEIPro™ reagent. FIG. 5C shows fold enhancement of AAV2 functional in VPC2.0 HEK293 suspension cells transfected with FectoVir-AAV™ reagent and treated with exemplary compounds at concentrations ranging from 0.25 pM to 80 pM.

[0027] FIG. 6 shows fold increase of AAV2 functional titer (RLU equivalent) and genomic titer (VG / ml) of I-7 and I-35 (exemplary compounds of the application) treated VPC2.0 HEK293 cells harvested from 125mL shake flasks, in comparison to control. Compounds I-7 and I-35 were tested at 5, 10, 15 and 20 pM and production (RLU) was measured by the luciferase reporter assay to evaluate fold change of functional titer. Enhancement of genomic titer was measured by qPCR. I-7 and I-35 (exemplary compounds of the application) enhanced both functional and capsid titer by 1.1 -fold or higher at varying peak concentrations and generally increased functional titer to a greater extent than genomic titer. The values represent the average of two technical replicates (n=2).

[0028] FIG. 7 shows greater than 1.1 -fold enhancement of AAV8 production in suspension VPC2.0 HEK293 cells transfected with either FectoVir-AAV reagent or AAV-MAX kit in 96 well format for a subset of exemplary compounds of the application (from left to right, compounds I-4, I-7, I-9, 1-13, I-23 and I-35). All panels show fold change of RLU equivalent to functional titer and values represent the average of three technical replicates (n=3). Compounds I-4, I-5104903317, 1-9, 1-13, I-23 and I-35 led to enhanced AAV8 production at varying peak concentrations in VPC2.0 HEK293 cells across both transfection reagents.

[0029] In FIG. 8, I-35 (exemplary compound of the application) was tested at 10, 20 and 30 pM in 125ml_ shake flasks for changes in LV functional titer and LV capsid titer in VPC2.0 HEK293 cells transfected with FectoVir-LV, with and without sodium butyrate. The values represent the average of two technical replicates (n=2). I-35 (exemplary compound of the application) led to greater than 1 .1 -fold enhancement of RLU equivalent to functional titer and capsid titer indicative of physical titer, in the presence and absence of sodium butyrate. I-35 (exemplary compound of the application) generally increased functional titer to a greater extent than capsid titer.

[0030] FIG. 9 shows the fold- change of functional and capsid titer of lentivirus encoding therapeutic transgene (LV-CD19CAR-GFP) following transfection of VPC2.0 HEK293 cells and treatment with 10 pM of I-35 (exemplary compound of the application) in 125ml_ shake flasks. I-35 enhanced the percentage of GFP-positive cells, equivalent to functional titer, and capsid titer (VP / mL) by greater than 1.1 -fold relative to control. The values represent the average of two technical replicates (n=2).

[0031] FIG. 10 shows greater than 2-fold increase in % GFP-positive cells equivalent to lentiviral transduction of human primary pan T-cells (CD3+) treated with 15 pM of (from left to right) compounds I-36, I-53, I-55 and I-60 (exemplary compounds of the application) as measured by flow cytometry. The values represent the average fold change enhancement of % transduction by GFP expression based on two technical replicates (n=2).

[0032] FIG. 11 show greater than 1.1-fold enhancement in replication-competent VSVA51 virus functional titer of (from left to right) compounds I-34, I-36, I-47, I-50, I-54, I-55, I-59, I-60 and 1-61 (exemplary compounds of the application) treated 786-0 cells at 15 pM relative to control, as measured by the luciferase reporter assay. The values represent the average of three technical replicates (n=3).

[0033] FIG. 12 shows greater than 1.1-fold enhancement of lentivirus-encoded GFP expression in human primary pan T-cells (CD3+) treated with 15 pM of (from left to right) compounds I-4, I-36, I-40, I-43, I-44, I-47, I-50, 1-51 , I-52, I-53, I-54, I-55, I-58, I-59, I-60 and 1-61 (exemplary compounds of the application) relative to control. GFP expression was determined by quantification of mean fluorescence intensity of GFP following transduction of lentivirus - encoding GFP in T-cells. The values represent the average based on two technical replicates (n=2).

[0034] FIG. 13 shows fold increase of VSVA51 - encoded GFP expression in 786-0 cells treated with I-35 (exemplary compound of the application) at 10, 20 and 40 pM relative to610490331control as determined by quantification of GFP fluorescence. The values represent the average of three technical replicates (n=3).DETAILED DESCRIPTIONI. Definitions

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.710490331

[0042] 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.

[0043] The term “compound of the application” or “compound of the present application” and the like as used herein refers to any Compound of Formula (I), including those disclosed herein as well as salts, solvates and / or prodrugs thereof.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] The term “alkyl” as used herein, whether it is used alone or as part of another group, means straight or branched chain, saturated alkyl groups. The number of carbon atoms that are possible in the referenced alkyl group are indicated by the prefix “Cni-n2”, wherein n1 represents the lowest number of carbon atoms and n2 the highest number of carbon atoms in the group. For example, the term Ci- alkyl means an alkyl group having 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.

[0048] The term “alkylene”, whether it is used alone or as part of another group, means straight or branched chain, saturated alkylene group, that is, a saturated carbon chain that contains substituents on two of its ends. The number of carbon atoms that are possible in the referenced alkylene group are indicated by the prefix “Cni-n2”, wherein n1 represents the lowest number of carbon atoms and n2 the highest number of carbon atoms in the group. For example, the term C2-6alkylene means an alkylene group having 2, 3, 4, 5 or 6 carbon atoms.

[0049] The term “alkenyl” as used herein, whether it is used alone or as part of another group, means straight or branched chain, unsaturated alkyl groups containing at least one double bond. The number of carbon atoms that are possible in the referenced alkylene group are indicated by the prefix “Cni-n2”, wherein n1 represents the lowest number of carbon atoms and810490331n2 the highest number of carbon atoms in the group. For example, the term C2-6alkenyl means an alkenyl group having 2, 3, 4, 5 or 6 carbon atoms and at least one double bond.

[0050] The term “alkynyl” as used herein, whether it is used alone or as part of another group, means straight or branched chain, unsaturated alkynyl groups containing at least one triple bond. The number of carbon atoms that are possible in the referenced alkyl group are indicated by the prefix “Cni-n2”, wherein n1 represents the lowest number of carbon atoms and n2 the highest number of carbon atoms in the group. For example, the term C2-6alkynyl means an alkynyl group having 2, 3, 4, 5 or 6 carbon atoms.

[0051] The term “cycloalkyl,” as used herein, whether it is used alone or as part of another group, means a saturated carbocyclic group containing from 3 to 10 carbon atoms and one or more rings. The number of carbon atoms that are possible in the referenced cycloalkyl group are indicated by the numerical prefix “Cni-n2”, wherein n1 represents the lowest number of carbon atoms and n2 the highest number of carbon atoms in the group. For example, the term C3.iocycloalkyl means a cycloalkyl group having 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.

[0052] The term “aryl” as used herein, whether it is used alone or as part of another group, refers to carbocyclic groups containing at least one aromatic ring and contains either 6 to 10 carbon atoms.

[0053] The term “heterocyclyl” as used herein, whether it is used alone or as part of another group, refers to cyclic groups containing at least one non-aromatic ring containing from 3 to 10 atoms in which one or more of the atoms are a heteroatom selected from O, S and N and the remaining atoms are C. Heterocyclyl groups are either saturated or unsaturated (i.e. contain one or more double bonds). When a heterocycloalkyl group contains the prefix Cni-n2this prefix indicates the number of carbon atoms in the corresponding carbocyclic group, wherein n1 represents the lowest number of carbon atoms and n2 the highest number of carbon atoms in the group, in which one or more, suitably 1 to 5, of the ring atoms is replaced with a heteroatom as selected from O, S and N and the remaining atoms are C. Heterocyclyl groups are optionally benzofused.

[0054] The term “heteroaryl” as used herein, whether it is used alone or as part of another group, refers to cyclic groups containing at least one heteroaromatic ring containing 5-10 atoms in which one or more of the atoms are a heteroatom selected from O, S and N and the remaining atoms are C. When a heteroaryl group contains the prefix Cni-n2this prefix indicates the number of carbon atoms in the corresponding carbocyclic group, wherein n1 represents the lowest number of carbon atoms and n2 the highest number of carbon atoms in the group, in which one or more, suitably 1 to 5, of the ring atoms is replaced with a heteroatom as defined above. Heteroaryl groups are optionally benzofused.910490331

[0055] All cyclic groups, including aryl, heteroaryl, heterocyclyl and cycloalkyl groups, contain one or more than one ring (i.e. are polycyclic). When a cyclic group contains more than one ring, the rings may be fused, bridged, spirofused or linked by a bond.

[0056] The term “fluoroalkyl” refers to the substitution of one or more, including all, available hydrogens in an alkyl group with fluoro.

[0057] The terms “halo” or “halogen” as used herein, whether it is used alone or as part of another group, refers to a halogen atom and includes fluoro, chloro, bromo and iodo.

[0058] 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.

[0059] 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.

[0060] The term “pharmaceutically acceptable” means compatible with the treatment of subjects, for example humans.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] The term “cancer” as used herein refers to cellular-proliferative disease states.1010490331

[0069] 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.

[0070] 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 outside agent 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).

[0071] 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.

[0072] In the context of virus and protein production, reverse genetics systems enable the construction and recovery of virus particles from cloned DNA or RNA. This allows for the generation of recombinant viruses with specific mutations or engineered traits, facilitating the study of viral pathogenesis, vaccine development, cell and gene therapy development and antiviral drug testing. Additionally, reverse genetics systems are used to produce proteins by manipulating gene sequences to express desired proteins in various host cells, aiding in the development of therapeutic proteins, enzymes, and biopharmaceuticals.

[0073] 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 the1110490331generation 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.II. Compounds and Compositions of the Application

[0078] It has been shown herein that compounds of Formula (I) of the present application (compounds of the application) are effective to increase permissiveness of a cell to nucleic acids suitable for production of a virus, and thus exhibit viral sensitizing activity, with high potency and versatility.

[0079] Accordingly, the present application includes a composition comprising a compound of Formula (I), or a pharmaceutically acceptable salt, solvate and / or prodrug thereof (i.e. a compound of the application) and a virus or genetic material encoding components of the virus, optionally with a pharmaceutically acceptable carrier or excipient.

[0080] In some embodiments, the compound of Formula (I) or a salt, solvate and / or prodrug thereof (or compound of the application) is:1210490331wherein:Z is NR6, S, O, SO2or CHR7;— - is a single or a double bond;Y is N or CR8when — - is a double bond; or Y is NR8or CHR8when — - is a single bond;R1is selected from H, halo, Ci.6alkyl, OCi.6alkyl, C(O)NH2and X1Cy1; or R1is =0 and — - is a single bond;R2is selected from H, halo, Ci.6alkyl, OCi.6alkyl and X2Cy2;R3is selected from H, halo, Ci.6alkyl, OCi.6alkyl and X3Cy3;R4is selected from H, halo, Ci.6alkyl, OCi.6alkyl, NH(CO)Ci.6alkyl and X4Cy4;R5is selected from H, halo, Ci.6alkyl, OCi.6alkyl and X5Cy5;R6is selected from H, Ci.6alkyl, Ci.6alkyleneOCi.6alkyl, (CO)NHCi.6alkyl and X6Cy6;R76s selected from H, halo, Ci.6alkyl, OCi.6alkyl and X7Cy7;R8is selected from H, halo, Ci.6alkyl, OCi.6alkyl, NHC(O)Ci-6alkyleneOCi-6alkyl, and X8Cy8;Cy1, Cy2, Cy3, Cy4, Cy5, Cy6, Cy7and Cy8are each independently a cyclic group selected from 6- to 16-membered aryl, 5- to 16-membered heterocyclyl, 5- to 16- membered heteroaryl and 3- to 16-membered cycloalkyl, wherein when Cy1, Cy2, Cy3, Cy4, Cy5, Cy6, Cy7and Cy8comprises 9- to 16-members, the cyclic group is either a bicyclic or tricyclic, fused and / or spiro cyclic group, and each of Cy1, Cy2, Cy3, Cy4, Cy5, Cy6, Cy7and Cy8is, independently, unsubstituted or substituted with one to four substituents independently selected from OH, halo, Ci_6alkyl, OC1-13104903316alkyl, CN, =0, C(O)Ci.6alkyl and C(O)N(Ci-6alkyl)2and / or substituted with one substituent selected from X9phenyl, X10C3-6cycloalkyl, X10C3-6heterocycloalkyl and X10C5-6heteroaryl, wherein each phenyl, C3-6cycloalkyl, C3-6heterocycloalkyl and C5. eheteroaryl is unsubstituted or substituted with one or more substituents independently selected from OH, halo, Ci_6alkyl, OCi.6alkyl, CN, C(O)Ci.6alkyl, SCi.6alkyl, S(O)Ci.6alkyl, SO2Ci-6alkyl and NHCi.6alkyl; or R1and R6or R7, R1and R8, R2and R6or R3and R4are linked to form, together with the atoms therebetween, a 5- to 16-membered monocyclic, bicyclic, and / or spirocyclic, saturated and / or unsaturated cyclic group, optionally comprising one or more heteromoieties independently selected from O, S, N and NR9and unsubstituted or substituted with one to four substituents independently selected from halo, Ci_6alkyl, OCi.6alkyl, CN and C(O)Ci.6alkyl, and / or substituted with one substituent selected from X11phenyl and X12C5-6heteroaryl, wherein each phenyl and C5-6heteroaryl is, unsubstituted or substituted with one or more substituents independently selected from OH, halo, Ci_6alkyl, OCi.6alkyl, CN, C(O)Ci.6alkyl, SCi.6alkyl, S(O)Ci.6alkyl, SO2Ci.6alkyl and NHCi.6alkyl; each X1, X2, X3, X4, X5, X7, X8, X9, X10, X11and X12is independently selected from a direct bond, Ci.6alkylene, C(O), O, NH, N(Ci.4alkyl), S, S(O), SO2, SCi.6alkylene, Ci.6alkyleneS, S(O)Ci.6alkylene, Ci.6alkyleneS(O), SO2Ci.6alkylene, Ci. 6alkyleneSO2, NHCi.6alkylene, Ci.6alkyleneNH, N(Ci.4alkyl)Ci.6alkylene, Ci- 6alkyleneN(Ci.4alkyl), OCi.6alkylene, Ci.6alkyleneO, C(O)NH, NHC(O), C(O)N(Ci. 4alkyl), SO2NH, NHSO2, NHC(O)NH, SO2N(Ci-4alkyl), N(Ci-4alkyl)SO2, C(O)Ci- 6alkylene, Ci.6alkyleneC(O), NHC(O)Ci.6alkylene, NHC(O)Ci.66lkyleneC(O), C(O)Ci.6alkyleneNHC(O), NHC(O)NHCi.6alkyleneC(O), C(O)Ci.6alkyleneNHC(O)NH, Ci.6alkyleneC(O)NH, NHC(O)Ci.6alkyleneO, OCi. 6alkyleneC(O)NH, NHC(O)Ci.6alkyleneS, SCi.6alkyleneC(O)NH, NHC(O)Ci. 6alkyleneS(O), S(O)Ci.6alkyleneC(O)NH, NHC(O)Ci.6alkyleneSO2and SO2Ci. 6alkyleneC(O)NH;X6is selected from a direct bond, Ci.6alkylene, C(O), Ci.6alkyleneS, S(O)Ci. 6alkylene, Ci-ealkyleneS(O), Ci-ealkyleneSO2, Ci-ealkyleneNH, Ci-ealkyleneN(Ci. 4alkyl), Ci.6alkyleneO, C(O)NH, C(O)N(Ci.4alkyl), SO2NH, SO2N(Ci.4alkyl), C(O)Ci. 6alkylene, Ci.6alkyleneC(O) and Ci.6alkyleneC(O)NH;R9is selected from H, C(O)Ci-ealkyl, CO2Ci-ealkyl, SO2Ci-ealkyl and Ci-ealkyl; at least 4 of R1, R2, R3, R4, R5, R6, R7and R8are selected from H and Ci_6alkyl; and1410490331all available hydrogen atoms are optionally and independently replaced with a halogen and all available atoms are optionally and independently replaced with an alternate isotope thereof.

[0081] In some embodiments, R1is selected from H, Ci-4alkyl, C(O)NH2 and X1Cy1. In some embodiments, R1is selected from H, CH3, CH2CH3, C(O)NH2and X1Cy1.

[0082] In some embodiments, X1is selected from a direct bond, S, Ci.4alkylene, SO.4alkylene, Ci.4alkyleneS, NHCi.4alkylene, Ci.4alkyleneNH, N(Ci-4alkyl)Ci-6alkylene, Ci-6alkyleneN(Ci.4alkyl), OCi.6alkylene, Ci.6alkyleneO, C(O)NH, C(O), NHC(O), NH, C(O)NHCi.4alkylene and C(O)N(Ci.4alkyl). In some embodiments, X1is selected from a direct bond, S, CH2S, SCH2, CH2CH2S CH2O, NHCH2. CH2NH, C(O)NH, C(O)NCH3, C(O), NHC(O), NH and CH2CH2CH2.

[0083] In some embodiments, Cy1is selected from 6- to 8-membered aryl, 6-, 9-, 10- or 13- membered heterocyclyl, and 5-, 6- or 9-membered heteroaryl. In some embodiments, Cy1is selected from phenyl, pyridinyl, piperidinyl, benzimidazolyl, imidazolyl, thiazolyl, and benzodioxinyl, each of which are unsubstituted or substituted with one to four substituents independently selected from F, Cl, CN, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, C(CH3)3, C(O)CH3, C(O)CH2CH3, OCH3, and OCH2CH3. In some embodiments, Cy1is selected from phenyl, pyridinyl, piperidinyl, benzimidazolyl, imidazolyl, thiazolyl, and benzodioxinyl, each of which are unsubstituted or substituted with one to four substituents independently selected from NHC(O)phenyl, NHC(O)Ci.4alkylene- Ophenyl, NHC(O)pyrimidinyl and NHC(0)Ci.4alkylenepyrazolyl wherein each phenyl, pyrimidinyl and pyrazolyl group is unsubstituted or substituted with one to four substituents independently selected from F, Cl, SO2CH3, SO2CH2CH3, and NHCi.6alkyl.

[0084] In some embodiments, R1is selected from H, =0, CH3, C(O)NH2, phenyl,1510490331wherein I indicates the point of attachment to the remainder of the compound.

[0085] In some embodiments, R1and R6or R7, R1and R8, R2and R6or R3and R4are linked, together with the atoms therebetween to form a 6- membered monocyclic, a 10-membered bicyclic or a 15-membered spirocyclic, optionally comprising one to three NH and unsubstituted or substituted with one to four substituents independently selected from X11phenyl, wherein each phenyl is unsubstituted or substituted with one to four substituents independently selected from CH3, CH2CH3, OCH3, and OCH2CH3. In some embodiments, R11710490331and R6or R7, R1and R8, R2and R6or R3and R4are linked, together with the atoms therebetween to form:wherein i indicates the point of attachment to the remainder of the compound.

[0086] In some embodiments, R2is selected from H, CH3, CH2CH3and X2Cy2.

[0087] In some embodiments, X2is NHC(O) or C(O)NH.

[0088] In some embodiments, Cy2is phenyl unsubstituted or substituted with one to four substituents independently selected from CN, F, Cl, CH3or CH2CH3.

[0089] In some embodiments, R2is selected from H and10490331

[0091] In some embodiments, R4is selected from H, Cl, F, OCH3, OCH2CH3, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2and X4Cy4.

[0092] In some embodiments, X4is selected from NHC(O), C(O)NH, C(O)NHCI.4alkylene, NHCi.4alkylene, NHC(O)Ci-4alkyleneO, NHC(O)CI.4alkylene, NHC(O)NH, and C(O).

[0093] In some embodiments, Cy4is phenyl, thiophenyl, tetrazolyl, cyclopentyl, triazolopyridinyl, pyridinyl or azepanyl, each of which is unsubstituted or substituted with one to four substituents independently selected from OCH3, OCH2CH3, CH3, or CH2CH3.wherein1indicates the point of attachment to the remainder of the compound.

[0095] In some embodiments, R3and R4are linked, together with the atoms therebetween to form:

[0096] In some embodiments, R5is selected from H, F, Cl, OCH3, OCH2CH3, CH3, CH2CH3,CH2CH2CH3, and CH(CH3)2andsome embodiments, R5is selected from H, F, Cl and CH3.1910490331

[0097] In some embodiments, R6is selected from H, CH3, CH2CH3, (CH2)2OCH3and X6Cy6.

[0098] In some embodiments, X6is selected from a direct bond, Ci.4alkyleneO, C1-4 alkylene, C(O), C(O)NH, Ci.4alkyleneC(O) and SO2.

[0099] In some embodiments, Cy6is phenyl, 10- to 13-membered heterocyclyl, morpholinyl, each of which is unsubstituted or substituted with one to four substituents independently selected from F, Cl, CH3, or CH2CH3.

[0100] In some embodiments, R6is selected from H, CH3, CH2CH3, (CH2)2OCH3, C(O)NHCH3,wherein1indicates the point of attachment to the remainder of the compound.

[0101] In some embodiments, R8is selected from H, OCH3, OCH2CH3, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, Cl and X8Cy8.

[0102] In some embodiments, X8is selected from a direct bond, C(O)NH, NHC(O)NH, Ci.4alkyleneO, Ci.4alkylene, C(O), Ci.4alkyleneC(O), Ci.4alkyleneC(O)NH, C(O)Ci.4alkyleneO, C(O)Ci.6alkyleneNHC(O)NH and SO2.

[0103] In some embodiments, Cy8is phenyl, 10- to 13-membered heterocyclyl, 5- to 9-membered heteroaryl, pyridinyl, or morpholinyl, each of which is unsubstituted or substituted with one to four substituents independently selected from F, Cl, CH3, or CH2CH3.

[0104] In some embodiments, R8is selected from H, CH3, CH2CH3, Cl,2010490331

[0105] wherein i indicates the point of attachment to the remainder of the compound. In some embodiments, the compound of the application is selected from a compound in Table 1 , or a pharmaceutically acceptable salt, solvate and / or prodrug thereof.Table 1 : Compounds of Formula (I)1049033123or a pharmaceutically acceptable salt (where applicable), solvate and / or prodrug thereof.

[0106] ln 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 (where applicable). 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 by mixing a solution of a compound with a solution of a pharmaceutically3810490331acceptable 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 197766(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).

[0107] 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.

[0108] 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 3910490331compounds that 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.

[0109] 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 and4010490331a net neutral charge. 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.

[0110] 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.

[0111] It is understood and appreciated that in some embodiments, compounds of the present application may have at least one chiral center and therefore can exist as enantiomers and / or diastereomers. It is to be understood that all such isomers and mixtures thereof in any proportion are encompassed within the scope of the present application. It is to be further understood that while the stereochemistry of the compounds may be as shown in any given compound listed herein, such compounds may also contain certain amounts (for example, less than 20%, suitably less than 10%, more suitably less than 5%) of compounds of the present application having an alternate stereochemistry. It is intended that any optical isomers, as separated, pure or partially purified optical isomers or racemic mixtures thereof are included within the scope of the present application.

[0112] In some embodiments, the compounds of the present application can also include tautomeric forms, such as keto-enol tautomers and the like. Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution. It is intended that any tautomeric forms which the compounds form, as well as mixtures thereof, are included within the scope of the present application.

[0113] 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.

[0114] 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.

[0115] 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-viral4110490331components. 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.

[0116] 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.

[0117] The compositions of the application are particularly amenable to administration with the aid of nano-carrier systems, such as liposomes, micelles, nanoparticles, nanoemulsions, 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.

[0118] In some embodiments, the compounds of the application, when administered to a subject or cell in vivo, they are formulated in pharmaceutical compositions in a variety of forms depending on the selected route of administration, as will be understood by those skilled in the art. For example, a compound of the application is administered by oral, inhalation, parenteral, buccal, sublingual, insufflation, epidurally, nasal, rectal, vaginal, patch, pump, minipump, topical or transdermal administration and the pharmaceutical compositions formulated accordingly. Conventional procedures and ingredients for the selection and preparation of suitable compositions are described, for example, in Remington's Pharmaceutical Sciences (2000 - 20th edition) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999.

[0119] 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 transfection4210490331reagent 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; or j) a carrier diluent or excipient, or any combination of a)-j).

[0120] 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.

[0121] In some embodiments, the kit comprises instructions for using any component or combination of components and / or practicing any method as described herein.

[0122] In the above, the term "a compound" also includes embodiments wherein one or more compounds are referenced.III. Methods and Uses of the Application

[0123] The application also provides uses and methods relating to the compounds and compositions described herein.

[0124] 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 for treating 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

[0125] 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 administering an effective amount of a compound of Formula (I), or a salt, solvate and / or prodrug thereof, (i.e. a compound of the application) to the cell.

[0126] 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.4310490331

[0127] 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.

[0128] 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.

[0129] 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 virual infection and / or a non viral process, is 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.

[0130] 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.

[0131] 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.

[0132] 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.

[0133] 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. Common carriers of genetic material are described, for example, in Cullis and Hope (2017) and in Mitchell et al. (2021).

[0134] 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.4410490331

[0135] 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.

[0136] In some embodiments, the method is used for production of a virus that encodes a non-viral gene product.

[0137] In some embodiments, the method is used for the production of a therapeutic.

[0138] In some embodiments, the virus is an interferon (IFN)-sensitive virus.

[0139] 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.

[0140] In some embodiments, virus is a non-replicating viral vector, optionally an adenovirus (Ad), an adeno-associated virus (AAV) or lentivirus (LV).

[0141] 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.

[0142] In some, embodiments, the virus is a double-stranded DNA virus, including but not limited to herpes simplex virus (HSV), vaccinia virus, or viral vectors derived therefrom.

[0143] In some embodiments, the virus is a gene therapy vector. As used herein, the term “gene therapy vector” 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 human Ad5, Ad3, Ad11 , Ad35, canine Ad2, chimp Ad26, chimp AdOx1 , or recombinant serotypes thereof, AAV serotypes 1-9 or recombinant serotypes thereof, Lentivirus, gammaretrovirus, Annellovirus, or Baculovirus.

[0144] 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).

[0145] In some embodiments, the virus is a rhabdovirus, a togavirus, or an orthomyxovirus.4510490331

[0146] In some embodiments, rhabdovirus is vesicular stomatitis virus (VSV), engineered mutants of VSV (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.

[0147] In some embodiments, the togavirus is sindbis, semliki forest virus or M1 virus.

[0148] In some embodiments, the orthomyxovirus is influenza A, influenza B, influenzaC, influenza D, isavirus, thogotovirus or quanranjavirus.

[0149] 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.

[0150] 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 cells, 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, HT1080 cells or R4 cells.

[0151] 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 4610490331or 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

[0152] 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.

[0153] In some embodiments, the method comprises growing the virus in an appropriate medium in the presence of a compound of the application.

[0154] 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.

[0155] 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.

[0156] 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.

[0157] 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.

[0158] 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 include4710490331diverse polymers known in the art, which may be designed in a variety of nanoparticle formats, either loosely organized or more precision designed.

[0159] 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.

[0160] 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.

[0161] In some embodiments, the method is used for production of a virus that encodes a non-viral gene product.

[0162] In some embodiments, the method is used for the production of a therapeutic.

[0163] In some embodiments, the virus is an interferon (IFN)-sensitive virus.

[0164] In some embodiments, the virus produced by the cell is an oncolytic virus, gene therapy vector or a vaccine.

[0165] 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.

[0166] In some embodiments, the cells are those disclosed in Methods and uses of increasing permissiveness of a cell to one or more nucleic acids.

[0167] 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 the cell 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.

[0168] 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 or4810490331infectivity 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

[0169] 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.

[0170] “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.

[0171] As used herein, the expression “increasing transduction” includes increasing transduction efficiency.

[0172] In some embodiments, the compound of the application is administered to the cell before, after and / or concurrently with a virus containing an exogenous gene.

[0173] In some embodiments, the method is used for the production of a therapeutic.

[0174] In some embodiments, the virus is an interferon (IFN)-sensitive virus.

[0175] 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.

[0176] In some embodiments, the cells are those disclosed in Methods and uses of increasing permissiveness of a cell to one or more nucleic acids.

[0177] 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.

[0178] 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 in4910490331the 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

[0179] 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.

[0180] 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.

[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 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.

[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.5010490331

[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.

[0188] In some embodiments, the one or more nucleic acids comprise one or more DNA plasmids.

[0189] 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.

[0190] In some embodiments, the method is used for production of a virus that encodes a non-viral gene product.

[0191] In some embodiments, the method is used for the production of a therapeutic.

[0192] In some embodiments, the virus is an interferon (IFN)-sensitive virus.

[0193] 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.

[0194] In some embodiments, the cells are those disclosed in Methods and uses of increasing permissiveness of a cell to one or more nucleic acids.

[0195] 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.

[0196] 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.

[0197] 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.

[0198] 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 of the 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.5110490331Methods and uses of increasing virus growth and / or virus spread

[0199] 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.

[0200] 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.

[0201] 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.

[0202] 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.

[0203] 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.

[0204] 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.

[0205] 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.

[0206] 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 5210490331embodiments, 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.

[0207] In some embodiments, the method is used for production of a virus that encodes a non-viral gene product.

[0208] In some embodiments, the method is used for the production of a therapeutic.

[0209] In some embodiments, the virus is an interferon (IFN)-sensitive virus.

[0210] 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.

[0211] In some embodiments, the cells are those disclosed in Methods and uses of increasing permissiveness of a cell to one or more nucleic acids.

[0212] 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.

[0213] 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.Methods and uses for improving manufacturing of therapeutic viral-based products

[0214] 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.

[0215] Accordingly, the present application includes a method for improving the upstream 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.

[0216] 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 5310490331virus 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.

[0217] 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.

[0218] 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.

[0219] 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.

[0220] 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.

[0221] 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.

[0222] 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.

[0223] In some embodiments, the method is used for production of a virus that encodes a non-viral gene product.

[0224] In some embodiments, the method is used for the production of a therapeutic.

[0225] In some embodiments, the virus is an interferon (IFN)-sensitive virus.5410490331

[0226] 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.

[0227] In some embodiments, the cells are those disclosed in Methods and uses of increasing permissiveness of a cell to one or more nucleic acids.

[0228] 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.

[0229] 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, virus transduction and transgene expression, thus improving the overall efficiency of the manufacturing process.

[0230] 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.

[0231] In some embodiments, the compound of application is administered to the cell through closed or open systems.

[0232] 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.

[0233] In some embodiments, the compound of application can be integrated within existing production processes in a simple way.

[0234] 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.

[0235] In some embodiments, the compound can enhance the replication and packaging efficiency of the viral vectors within the manufacturing cells.

[0236] 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.5510490331

[0237] 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.

[0238] 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.

[0239] 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 of the Application

[0240] 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.

[0241] 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.

[0242] 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”.

[0243] Prodrugs of the compounds of the present application may be, for example, conventional esters formed with available hydroxy, thiol, amino or carboxyl groups. For5610490331example, 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). Some common esters which have been utilized as prodrugs are phenyl esters, aliphatic (C1-C24) esters, acyloxymethyl esters, carbamates and amino acid esters.

[0244] 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 liquidliquid or solid-liquid extraction, which will be readily understood by one skilled in the art.EXAMPLESExample 1 Materials and MethodsCell lines

[0245] Cell lines: HEK293T (human embryonic kidney epithelium), HEK293, and HT1080 (human connective fibrosarcoma), 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. HEK 293T / 17 (human embryonic kidney 5710490331epithelium) was clonally derived from HEK293T. Expi293FTM(human embryonic kidney epithelium) cells were obtained from ThermoFisher and maintained in Expi293 Expression Media. VPC 1.0 HEK293, VPC 2.0 HEK 293 (human embryonic kidney epithelium) were obtained from ThermoFisher and maintained in Viral Production Media or LV-MAX Production Media. HEK293T, HEK293, HT1080, 786-0 and Vero cell lines were incubated at 37°C with 5% CO2. VPC1 .0, VPC 2.0 HEK293 and Expi293F cells were incubated at 37°C with 8% CO2.

[0246] 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.Lentiviral vectors

[0247] The lentivirus construct for this study is a third-generation self-inactivating vector based on the HIV-1 backbone pseudotyped with the VSV-G glycoprotein, engineered to express a firefly luciferase (fLuc) transgene and manufactured using transient transfection of culture-adapted adherent HEK293T, suspension VPC1.0 HEK293 or VPC2.0 HEK293 cells with a four-plasmid system.

[0248] A pLenti6.3 lentiviral vector backbone was engineered to express a CD19CAR-P2A-GFP transgene (CD19CAR-GFP) by Genscript and was manufactured using transient transfection of VPC2.0 HEK293 cells.Compound Testing of HEK293T cells for enhanced LV production

[0249] Compounds of high chemical diversity and low predicted mutagenic, carcinogenic and teratogenic properties were tested in HEK293T in 96 well format following a batch (reverse) transfection method. HEK293T cells were suspended at 4.5e5 cells / mL in complete DMEM prior to transfection. A transfection mixture was prepared containing the 4 lentiviral plasmids and a corresponding amount of PEIpro transfection reagent in OptiMEM reduced serum media. The plasmid and PEIpro mixtures were allowed to complex for 30 minutes at room temperature prior to addition to the HEK293T cell mixture. Batch transfected HEK293T cells were immediately plated in 96well format at 4.5e4 cells / well, in triplicate, using a high-through liquid dispenser. Immediately following plating, HEK 293T cells were treated with either compounds at a concentration of 10 .M, or a corresponding amount of vehicle control (1.05% dimethyl sulfoxide (DMSO)) using a high-throughput liquid handler. Treated cells were incubated at 37°C with 5% C02for 48 hours.

[0250] The next day following transfection, HT 1080 reporter cells were plated in 96well format at 3e3 cells / well, in order to reach 50% confluence the following day. These cells act as reporter cells that are easily transduced by lentivirus-containing supernatant from producer5810490331cells and allow for high throughput virus quantification. 48 hours following transient transfection, a sample of lentivirus containing supernatant from HEK 293T production cultures was transferred on to the HT 1080 reporter cell line for transduction analysis and quantification of lentivirus driven fLuc expression. A standard curve was also added to the HT1080 reporter plates at the time of supernatant transfer from the HEK 293T producer cells. The standard curve was prepared by adding known amounts of fLuc-encoding lentivirus (in transducing units / mL) to the HT1080 cells at the same time as the transfer of supernatant. Using this standard curve, it is possible to convert the observed luminescence from transduced HT1080 cells into viral expression units (VEU) by, for example, plotting input virus level versus observed luminescence (relative light units) and performing a four-parameter non-linear regression analysis. During our studies we observed that increase in raw luminescence corresponded linearly with increases in calculated VEUs. After the supernatant transfer sample had been removed from the HEK 293T producer plates, cell viability was assessed using resazurin assay to determine relative metabolic activity relative to transfected cells treated only with vehicle control (1.05% DMSO). This allows for interrogation of the relative drop in cell viability caused only by the compounds and not remnant cytotoxicity from the DMSO vehicle. Transduced HT1080 cells were incubated at 37°C with 5% CO2.

[0251] 72 hours following the transduction of with lentivirus-virus containing supernatant from the HEK 293T producer cells, HT1080 reporter cells were read for luminescent activity. Since each well of the reporter cells were transduced with the same volume of supernatant containing luciferase-encoding lentivirus, increases in luminescent output would correspond to increased levels of virus produced following compound treatment and transfection of the producer cell line. The HT1080 reporter cells were treated with a 2mg / ml_ solution of luciferin salt in phosphate buffered saline, were shaken for 5 seconds and allowed to incubate at room temperature in the dark for 30 seconds to allow the luminescent reaction to occur. Luminescent signal, measured as relative light units per second (RLU / second), was recorded using a high-throughput plate reader.

[0252] Luminescent data obtained from the HT1080 reporter cells was analyzed by calculating the average luminescent value for each compound (in RLU / second) for each of the triplicate wells, and then determining the relative fold change in signal above the average of vehicle controls (1.05% DMSO) from wells on the same plates. This is reported as the Average Fold Change Above Vehicle. Significance was assessed using an unpaired, two-tailed t-test to produce a p-value. Az-score was also calculated for each compound compared to all other compounds in the same experiment.5910490331

[0253] The criteria used to classify a compound as active was based on average fold change above vehicle greater than or equal to 1 .1 fold, p-value of less than 0.05 and z-score greater than 1.0.Compound testing in HEK293T clones for enhanced LV production

[0254] Experiments were carried out using the same methods described in above, with the exception being that two different HEK 293T clones (HEK 293T and HEK 293T / 17) were used as production cells and three concentrations (5 .M, 10pM, and 15 .M) of drug were used to assess their impact on lentivirus production. Each condition was run in triplicate and supernatant from both producer HEK 293 clones were individually transferred onto HT1080 reporter cells as previously described. Analysis of luminescent signal and calculation of fold changes above vehicle and p-values for significance was performed as previously described in the above sections.

[0255] The average fold change above vehicle and p-values for each concentration of compound was calculated for both cell lines. The criteria used to classify a compound as active for this experiment was an average fold change above vehicle greater than 1.1 -fold and a p- value less than 0.05.Compound testing in HEK293T / 17, VPC1.0 HEK293 and VPC2.0 HEK293 cells for enhanced LV production

[0256] Compounds were tested in 96 well format in adherent HEK293T / 17, suspension VPC1.0 HEK293 and VPC2.0 HEK293 cells following a batch (reverse) transfection using one of three transfection reagents (PEIPro, FectoVir-LV or LV-MAX).

[0257] For adherent HEK293T / 17 cells transfected with PEIPro transfection reagent in 96 well plate format, experiments were carried out using the same methods described in above, except that nine concentrations of the exemplary compounds, ranging from 0.25 pM to 80 pM, were tested.

[0258] For the suspension cell lines, VPC1 .0 HEK293 and VPC2.0 HEK293 cells were suspended at 4e6 cells / mL in LV-MAX Production Media prior to transfection, and exemplary compounds of the application were added at 9 concentrations ranging from 0.25 to 80 .M at the time of transfection. VPC1.0 HEK293 cells were transfected with either the LV-MAX transfection kit or PEIPro transfection reagent and VPC2.0 HEK293 cells were transfected with FectoVir-LV transfection reagent in 96 well format.

[0259] For the LV-MAX transfection kit, the LV-MAX supplement was added to VPC1.0 HEK293 cells and incubated at 37°C with 8% CO2 for 30 to 60 minutes. While the supplement and cell mixture incubated, the transfection mixture was prepared. Four LV plasmids were 6010490331added to the LV-MAX transfection reagent and allowed to complex for 10 minutes at room temperature. Following complexation, the mixture was added to supplement treated VPC1.0 HEK293 cells for transfection. Batch transfected cells were plated at 4e5 cells / well, in triplicate, using a high-throughput liquid dispenser. Immediately following plating, transfected cells were treated with exemplary compounds of the application at concentrations ranging from 0.25 pM to 80 pM or a corresponding amount of library vehicle control (Dimethyl sulfoxide (DMSO)). Treated cells were incubated at 37°C with 8% CO2. 14 to 18 hours post transfection, LV-MAX enhancer was added to the transfected and treated cells.

[0260] For VPC1 .0 HEK293 cells to be transfected with PEIPro transfection reagent, four lentivirus plasmids were added to the PEIPro transfection reagent and allowed to complex for 15 to 20 minutes at room temperature. Following complexation, the mixture was added to 4e6 cells / mL of VPC1.0 HEK293 cells in LV-MAX production media. Batch transfected cells were plated at 4e5 cells / well, in triplicate, using a high-throughput liquid dispenser. Immediately following plating, transfected cells were treated with exemplary compounds of the application at concentrations ranging from 0.25 pM to 80 pM or a corresponding amount of library vehicle control (Dimethyl sulfoxide (DMSO)).

[0261] VPC2.0 HEK293 cells were suspended at 4e6cells / mL in LV-MAX Production media prior to transfection with FectoVir-LV transfection reagent. Four LV plasmids were added to the FectoVir-LV transfection reagent and allowed to complex for 15 minutes at room temperature. Following complexation, the mixture was added to VPC2.0 HEK293 cells to transfect. Batch transfected cells were plated at 4e5 cells / well, in triplicate, using a high- throughput liquid dispenser. Immediately following plating, transfected cells were treated with exemplary compounds of the application at concentration ranging from 0.25 pM to 80 pM or a corresponding amount of library vehicle control (Dimethyl sulfoxide (DMSO)).

[0262] Treated cells were incubated at 37°C with 8% CO2. 48 hours following transient transfection, a sample of lentivirus-containing supernatant from production cultures was transferred on to the HT1080 reporter cell line for transduction analysis and quantification of lentivirus driven fLuc expression, as described herein.Compound testing in VPC2.0 HEK293 cells for enhanced LV functional and capsid titer

[0263] Exemplary compound of the application was tested in 125mL shake flask format for enhanced production of LV-fLuc and LV-CD19CAR-GFP in suspension VPC2.0 HEK293 cells following a batch (reverse) transfection with FectoVir-LV transfection reagent.

[0264] VPC2.0 HEK293 cells were suspended at 4e6cells / mL in LV-MAX Production media prior to transfection with FectoVir-LV transfection reagent. Four LV plasmids were added to the FectoVir-LV transfection reagent and allowed to complex for 15 minutes at room 6110490331temperature. Following complexation, the mixture was added to VPC2.0 HEK293 cells to transfect. 30ml_ of batch transfected cells were aliquoted per 125ml_ shake flask, in duplicate. Transfected cells were immediately treated with exemplary compound of the application ranging from application at concentrations of 10, 20 and 30 pM or a corresponding amount of library vehicle control (Dimethyl sulfoxide (DMSO)) and incubated at 37°C with 8% CO2. 18 hours post transfection, sodium butyrate was added to the transfected and treated cells.

[0265] 48 to 55 hours later, LV-fLuc sample of lentivirus containing supernatant was transferred onto HT1080 reporter cells to quantify functional titer using the luciferase reporter assay (described herein). Viral particles by capsid titer was quantified using an Enzyme-Linked Immunosorbent Assay (ELISA) assay (Cell Biolabs, cat. #VPK-107).

[0266] 48 to 55 hours later, LV-CD19CAR-GFP sample of lentivirus containing supernatant was transferred onto HT1080 reporter cells to quantify the percentage of GFP- positive cells by flow cytometry using the MACSQuant™ Analyzer 16. Viral particles by capsid titer was quantified using an Enzyme-Linked Immunosorbent Assay (ELISA) assay (Cell Biolabs, cat. #VPK-107).AAV vectors

[0267] 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 adherent HEK293 cells, suspension VPC1.0 HEK293 cells, VPC2.0 HEK293 cells and Expi293F cells.

[0268] 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 for enhanced AAV2 production in HEK293 cells

[0269] Compounds were tested in HEK293 cells in 96 well format following a batch (reverse) transfection method. HEK293 cells were suspended at 5.3e5 cells / mL in complete DMEM prior to transfection. A transfection mixture was prepared containing the three AAV2 plasmids, and a corresponding amount of PEIpro transfection reagent in serum-free DMEM. The plasmid and PEIpro mixtures were allowed to complex for 15 minutes at room temperature prior to addition to the HEK293 cell mixture. Batch transfected 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, HEK293 cells were treated with either exemplary compounds of the application at a concentration ranging from 0.25 pM to 80 pM, or a corresponding6210490331amount of vehicle control (Dimethyl sulfoxide (DMSO) using a high-throughput liquid handler. Treated cells were incubated at 37°C with 5% C02for 72 hours.

[0270] 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 act as reporter cells that are easily transduced by AAV-containing lysate (after 3 freeze-thaw cycles) from producer cells and allow for high throughput virus quantification. At 72 hours following transient transfection of 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.

[0271] 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 in herein.Compound testing for enhanced AAV2 production in Expi293F and VPC2.0 HEK293 cells

[0272] Compounds were tested in Expi293FTMor VPC2.0 HEK293 cells in 96 well format following a batch (reverse) transfection using FectoVir-AAV transfection reagent. Compounds were also tested in 125mL shake flask format in VPC2.0 HEK293 cells with FectoVir-AAV transfection reagent.

[0273] Expi293FTMcells were suspended at 1.8e6 cells / mL in Expi293 Expression Media prior to transfection with FectoVir-AAV transfection reagent. Three AAV plasmids were added to the FectoVir-AAV transfection reagent and allowed to complex for 30 minutes at room temperature. Following complexation, the mixture was added to Expi293FTMcells to transfect. In the 96 well format, batch transfected Expi293FTMcells were plated at 1.8e5 cells / well, in triplicate, using a high-throughput liquid dispenser. Immediately following plating, transfected cells were treated with exemplary compounds of the application at concentrations ranging from 0.25 to 80 pM or a corresponding amount of library vehicle control (Dimethyl sulfoxide (DMSO)).

[0274] VPC2.0 HEK293 cells were suspended at 3e6 cells / mL in Viral Production Media prior to transfection with FectoVir-AAV transfection reagent. Three AAV plasmids were added to the FectoVir-AAV transfection reagent and allowed to complex for 30 minutes at room temperature. Following complexation, the mixture was added to VPC2.0 HEK293 cells to transfect. In the 96 well format, batch transfected cells were plated at 3e5 cells / well, in triplicate, using a high-throughput liquid dispenser. Immediately following plating, transfected cells were treated with exemplary compounds of the application at concentrations ranging6310490331from 1 to 100 pM or a corresponding amount of library vehicle control (Dimethyl sulfoxide (DMSO)). In the 125ml_ shake flask format, 30ml_ of batch transfected VPC2.0 HEK293 cells were aliquoted per flask, in duplicate. Cells were immediately treated with exemplary compounds of the application at concentrations ranging from 5, 10, 15 and 20 pM or a corresponding amount of library vehicle control (Dimethyl sulfoxide (DMSO)).

[0275] 72 hours following transient transfection, a sample of AAV-containing lysate from producer cells from the 96-well format and the 125ml_ shake flask format was freeze- thawed and transferred on to the HEK293T reporter cell line for transduction analysis and quantification of AAV-driven fLuc expression, as described herein.

[0276] 72 hours following transient transient transfection of VPC2.0 HEK293 cells in125ml_ shake flasks, AAV crude lysate was harvested using chemical lysis. A sample of AAV crude lysate was tested to quantify genomic titer using qPCR.Compound testing for enhanced AAV8 production in VPC2.0 HEK293 cells

[0277] Exemplary compounds of the application were tested in VPC2.0 HEK293 cells in 96 well format following a batch (reverse) transfection method. VPC2.0 HEK293 cells were suspended at 3e6 cells / mL in Viral Production Media prior to transfection with either FectoVir- AAV transfection reagent or AAV-MAX transfection kit.

[0278] 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.

[0279] For the AAV-MAX transfection system, the AAV-MAX enhancer was added to the VPC 2.0 HEK293 cells and incubated at 37° C with 8% CO2for 30 minutes. While the enhancer and cell mixture incubated, the transfection mixture was prepared. The three AAV8 plasmids were added to the corresponding AAV-MAX transfection reagent and booster and allowed to complex for 10 minutes at room temperature. Following complexation, the mixture was added to the VPC 2.0 HEK293 cells treated with enhancer, to transfect.

[0280] 100 pL of batch transfected VPC2.0 HEK293 cells were aliquoted in each well, in triplicate. Transfected cells were immediately treated with 25 pL exemplary compounds of the application at a range of 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, AAV8 from harvested cell lysate was transferred on to the HEK293T reporter cell line and incubated at 37°C with 5% CO2for 72 hours prior to6410490331transduction analysis and quantification of AAV8-driven fLuc expression in a similar manner as described herein.Viruses

[0281] VSVA51 is a recombinant variant of the Indiana serotype of VSV harbouring a deletion of the 51stmethionine in the M protein. VSVA51 expressing firefly luciferase (FLuc) and VSVA51 expressing green fluorescent protein (GFP) were recombinant derivatives of VSVA51. 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).

[0282] Lentiviral vector encoding EmGFP (>1 e8 TU / mL) was obtained from Genscript.Compound testing in 786-0 cells for enhanced output of VSVA51

[0283] 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 exemplary compounds at a concentration of 15pM for 4 hours. After compound treatment, cells were infected with VS A51 at MOI of 0.05. Infected cells were incubated at 37°C with 5% C02for 48 hours.

[0284] 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-fl_uc 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.

[0285] 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 for enhanced VSVA51 encoded GFP expression

[0286] 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 exemplary compounds 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% CO2for 24 hours. VSV A51 -encoded GFP expression was measured by quantifying GFP fluorescence using Thermo Scientific Celli nsight CX5.Compound testing for enhanced lentiviral transduction and virally-encoded transgene expression of T-cells6510490331

[0287] Primary human Pan T-cells (CD3+) 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% CO2 for 24 hours.

[0288] Activated T-cells were seeded in 384-well plates (at 25pL / well) and treated with exemplary compounds of the application at a concentration of 15 pM 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.

[0289] To quantify transduction efficiency, the percentage of GFP-expressing cells from the live cell population was quantified by flow cytometry using the MACSQuant™ Analyzer 16.

[0290] Lentivirus-encoded GFP transgene expression was measured by quantifying the mean fluorescent intensity of GFP in transduced cells. This was done flow cytometry using the MACSQuant™ Analyzer 16.EXPERIMENTAL RESULTS

[0291] The ability of compounds from the present application to enhance plasmidbased viral vector production in the HEK293T cell line was tested, since this cell line and 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 we employed consisted of transient transfection of four circular plasmids appropriate to produce lentivirus encoding a luciferase transgene in small-scale format. The compounds of high chemical diversity and low predicted mutagenic, carcinogenic and teratogenic properties were tested at a 10 .M concentration using the firefly luciferase (fLuc)-encoding lentivirus production system using a high-throughput batch transfection assay described herein, measuring lentivirus output from HEK293T cells from a secondary transduction assay carried out in an HT1080 reporter cell line.

[0292] Impact on lentivirus production was inferred by measuring the luciferase expression resulting from transfer of lentivirus-containing supernatants from compound- treated I LV plasmid-transfected HEK293T cells. In this assay, the luciferase signal (Relative Light Units or RLU) ensues from the transduction of HT1080 reporter cells, which correlates with quantity of functional virus as determined from a standard curve tested of LV in parallel. Many actives compounds increasing lentivirus production by at least a 1.1-fold as measured by luciferase transgene over the vehicle control (DMSO) were identified..6610490331

[0293] Compounds of the application can be seen in Table 1 and a detailed overview of their activity in terms of enhancing luminescent signal, correlated viral expression units (VEU), and cytotoxicity profile can be seen in FIG.1A, FIG.1B, and FIG.1C, respectively. The structurally similar active molecules were found to decrease producer cell viability by no more than 30% as seen in FIG.1C. Results for these fifty molecules can be seen in Table 2.Table 2. Lentivirus primary data of Compounds of Formula (I)Compound Fold Fold p-value p-valueI.D. change change (RLU) (VEU)Above AboveVehicle Vehicle(RLU) (VEU)1-1 1 -6 1.6 3.70E-03 3.08E-03I-2 1.7 1.7 6.08E-04 1.36E-03I-3 1.6 1.6 5.63E-07 2.88E-08I-4 1.9 1.9 5.37E-12 3.74E-13I-5 1.7 1.7 4.21 E-06 8.45E-08I-6 2.3 2.3 4.28E-12 5.29E-15I-7 2.4 2.4 5.53E-06 1.69E-09I-8 1.5 1.5 6.45E-03 9.14E-04I-9 2.5 2.4 3.55E-06 8.01 E-121-10 1.7 1.7 3.51 E-05 3.79E-031-11 2.0 2.0 4.42E-06 1.11 E-041-12 2.1 2.1 4.63E-06 1.76E-101-13 3.1 3.1 2.54E-11 1.58E-191-14 1.5 1.5 6.52E-02 1.13E-021-15 2.2 2.2 1.37E-07 5.69E-121-16 1.7 1.7 4.77E-04 3.86E-051-17 2.0 1.9 1.15E-07 2.10E-091-18 2.3 2.2 1.76E-11 2.56E-131-19 1 -5 1.5 7.83E-06 9.03E-07I-20 1.7 1.7 1.87E-05 8.23E-081-21 1.7 1.7 8.39E-06 3.59E-09I-22 1.7 1.7 1.60E-03 1.03E-04I-23 2.1 2.1 5.01 E-05 1.07E-07I-24 1.6 1.6 4.82E-07 6.45E-05I-25 1.6 1.6 9.98E-10 7.57E-08I-26 1.9 1.9 2.79E-12 1.06E-10I-27 1.7 1.7 3.12E-04 4.17E-09I-28 1.8 1.8 4.04E-06 6.96E-10I-29 1.6 1.5 9.16E-05 2.46E-066710490331I-30 2.2 2.2 2.11 E-11 6.22E-15 1-31 1.6 1 6 1.51 E-04 2.70E-08 I-32 1.7 1.7 3.36E-03 9.26E-04 I-33 2.0 2.0 5.64E-05 3.42E-05I-44 1.5 1.5 3.13E-03 1.89E-02 I-54 1.9 1.9 8.79E-07 5.67E-10 I-55 2.4 2.4 5.36E-08 6.99E-12 I-62 1.9 1.9 1.57E-08 1.90E-11I-64 1.9 1.9 1.45E-11 5.77E-13 I-65 1.8 1.8 3.21 E-04 1.84E-05 I-79 2.4 2.4 1.65E-06 1.49E-08 I-80 2.3 2.3 5.48E-06 1.73E-091-81 2.1 2.1 8.99E-09 8.94E-10 I-82 1.9 1 9 5.41 E-08 2.08E-08 I-83 1.8 1.8 5.00E-09 5.31 E-08 I-84 1.7 1.7 1.81 E-05 1.32E-08I-85 1.7 1 7 1.48E-02 1.61 E-02 I-86 1.6 1.6 2.78E-03 7.31 E-05 I-87 1.6 1 6 2.14E-03 3.78E-03 I-88 1.5 1.5 4.51 E-06 1.21 E-04

[0294] A subset of compounds with high and low activity were selected for subsequent testing in HEK293T and an alternative HEK293T subclone, HEK293T / 17. In particular, I-7, I- 9, 1-12, 1-13 and I-22 were tested at 3 concentrations (5 .M, 10 .M, 15 .M). These experiments were carried out using the same method used in the primary testing for assessing relative changes in LV-luciferase production. Data for this subset can be found in FIG.2, demonstrating similar activity across the two HEK293T subclones.Example 3

[0295] To further confirm the applicability of these molecules for additional plasmidbased reverse genetic systems, a subset of actives were investigated for their ability to enhance the production of adeno-associated virus-2 (AAV2) in HEK293 production cells. 1-1 , I-4, I-7, 1-13, 1-15 to 1-19, 1-21 to I-23, I-26, I-28, 1-31 , I-33 to 1-78 were tested at 10pM in HEK293 cells. These experiments were carried out using plasmids for the production of AAV2 and the methods as described herein. Data for this experiment can be found in FIG.3 and Table 3. All compounds tested showed statistically significant enhancement of AAV2 production and indicates that the identified class of molecules of Formula (I) and listed in Table 1 are also effective in enhancing the production of AAV2, suggesting their applicability to a6810490331range of plasmid-based reverse genetics systems, for example but not limited to the production of LV or AAV.Table 3. AAV production data of Viral Sensitizing Compounds / Compounds of Formula (I)6910490331Example 4

[0296] To confirm the applicability of these compounds for LV production in various cell lines and with various transfection reagents, 1-7, 1-9, 1-12, 1-13 and I-35 (exemplary compounds of the application) were used to treat adherent HEK293T / 17 cells with PEIPro, suspension VPC1.0 HEK293 cells transfected with PEIPro and suspension VPC2.0 HEK293 cells transfected with FectoVir-LV transfection reagent. As shown in FIG. 4A, FIG. 4B and FIG. 4C, all five compounds at varying peak concentrations led to greater than 1.1 fold enhancement in RLU, equivalent to functional lentivirus titer relative to vehicle control. In FIG. 4D, I-7, I-9, 1-13 and I-35 (exemplary compounds of the application) enhanced functional lentivirus titer by greater than 1.1 fold at varying peak concentrations in suspension VPC1.0 HEK293 cells transfected with LV-MAX transfection reagent including LV-MAX supplement and enhancer.7010490331

[0297] Enhanced lentivirus production in the prescence of two other enhancers in the LV-MAX transfection kit indicate the compatibility of these exemplary compounds of the application with other enhancers of virus production. This data of LV enhancement in adherent and suspension cell lines transfected with various transfection reagents suggests that exemplary compounds of the application are broadly-acting lentivirus production enhancers.Example 5

[0298] To further confirm the applicability of these compounds with enhancing AAV2 production in various cell lines and multiple transfection reagents, 1-7, 1-13, I-23 and I-35 (exemplary compounds of the application) were evaluated for fold change in RLU, indicative of functional titer. As show in FIG. 5A, all compounds enhanced AAV2 production in Expi293FTMcells with FectoVIR-AAV™ transfection reagent by greater than 1 .1 -fold at varying peak concentrations. I-7, 1-13, I-23 and I-35 also enhanced AAV2 production in adherent HEK293 cells transfected with PEIPro (FIG. 5B) and in VPC2.0 HEK 293 cells transfected with FectoVir-AAV™ transfection reagent (FIG. 5C). This data confirms that exemplary compounds of the application are broadly-activing AAV2 production enhancers based on increased activity in adherent and suspension cell lines transfected with various transfection reagents.Example 6

[0299] To test these exemplary compounds further, I-7 and I-35 (exemplary compounds of the application) were assessed for increased viral production in multiple plasmid-based reverse genetic systems at a larger scale.

[0300] I-7 and I-35 (exemplary compounds of the application) were treated in VPC2.0HEK293 cells with FectoVir-AAV in 125ml_ shake flasks and assessed for their impact on AAV2 functional titer by luciferase reporter assay and genomic titer by qPCR. I-7 and I-35 led to greater than 1.1-fold enhancement of genomic titer at 5pM and greater than 2-fold enhancement of functional titer at 5, 10 and 20pM compared to the untreated control (FIG. 6). Generally, increases in functional titer were greater than increases in genomic titers at peak concentrations. Since functional titer measures the infectious particles capable of transduction and genomic titer measures total number of genome-containing particles, this data indicates an ability of the compounds of the present application to increase the ratio of functional particles to physical particles (e.g., by increase of full:empty particle ratios).

[0301] I-35 was treated in VPC2.0 HEK293 cells with FectoVir-LV with and without sodium butyrate in 125ml_ shake flasks and assessed fortheir impact on LV functional titer by luciferase reporter assay and capsid titer by ELISA. As shown in FIG. 8, I-35 led to greater7110490331than 1.1 fold enhancement of RLU equivalent to functional titer and capsid titer (VP / mL) at varying peak concentrations.

[0302] I-35 also enhanced functional and capsid titer in the presence of sodium butyrate (an inhibitor of histone deacetylases (HDAC)) by greater than 1.1 -fold, indicating the compatibility of exemplary compounds of this application with other enhancers of virus production.

[0303] Generally, increases in functional titer were greater than increases in capsid titers at peak concentrations. Since functional titer measures the infectious particles capable of transduction and capsid titer measures total number of particles, this data indicates an ability of the compounds of the present application to increase the ratio of functional particles to physical particles (e.g., by increase of full:empty particle ratios).Example 7

[0304] To test the ability of exemplary compounds of the application to enhance viral vector production across AAV serotypes, I-4, I-7, I-9, 1-13, I-23 and I-35 were tested for their impact on AAV8 functional titer in VPC2.0 HEK293 cells using two different plasmid delivery reagents. Transfections of VPC2.0 HEK 293 cells were performed using either AAV-MAX transfection kit (including the kit-supplied enhancer and booster) or FectoVir-AAV transfection reagent and compounds were evaluated from 5 to 30 pM. Fold enhancement in functional titer was calculated relative to transfection reagent-matched untreated control conditions and results are summarized in FIG. 7.

[0305] Enhanced AAV production in the presence of the enhancer and booster in the AAV-MAX transfection kit indicate the compatibility of these exemplary compounds of the application with other enhancers of virus production. Consistent with prior observations using AAV2 vector, exemplary compounds of the application enhanced RLU equivalent to AAV8 functional titer by greater than 1.1 -fold at varying peak concentrations across transfection methods. These results extend the application of these exemplary compounds of the application across multiple AAV serotypes, cell lines, and transfection reagents.Example 8

[0306] To test the ability of exemplary compounds of the application enhance the production of therapeutic transgene-encoded lentivirus, 10pM of I-35 (exemplary compound of the application) was used to treat VPC2.0 HEK293 cells transfected with FectoVir-LV reagent and plasmids encoding CD19CAR-GFP. As shown in FIG. 9., I-35 enhanced both RLU equivalent to functional lentivirus titer and capsid titer (VP / mL) by greater than 1.1 fold. This7210490331data demonstrates the ability of exemplary compounds of this application to enhance the viral production of therapeutic transgenes.Example 9

[0307] To investigate the ability of compounds in this application to enhance virus transduction, various compounds were evaluated for its impact on primary T-cell transduction using a GFP-encoding lentiviral vector. Human CD3+T cells were activated and subsequently transduced with lentivirus in the presence of 15 pM of each compound. T ransduction 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.

[0308] As shown in FIG. 10, 15 pM of I-36, I-53, I-55 and I-60 enhanced lentiviral transduction in primary T cells by greater than 2-fold. Elevated transduction levels indicate that compounds of this application are also effective at increasing the number of virus- transduced cells and consequent expression of virally encoded transgenes in the overall cell population.Example 10

[0309] To test the applicability of these compounds with enhancing replicating viruses including but not limited to rhabdoviruses, compounds were investigated for its ability to enhance replication of a recombinant variant of vesicular stomatitis virus (VSV). Compounds were tested at a concentration of 15 pM in 786-0 cells using the methods described herein.

[0310] As shown in FIG. 11 , fold enhancement in functional VSVA51 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 quantifies functional viral output of VSVA51 in response to compounds.

[0311] Enhancement of VSV replication was observed with I-34, I-36, I-47, I-50, I-54, I-55, I-59, I-60 and 1-61 at 15 pM. These findings demonstrate that compounds of this application not only enhance plasmid-based reverse genetic systems like AAV and LV production, but also augment the output of actively replicating rhabdovirus, supporting broader applicability of these compounds in viral production systems.Example 11

[0312] The ability of exemplary compounds to enhance virally-encoded transgenes was evaluated by testing their effect on lentivirus-encoded GFP expression in human primary (CD34+) T-cells and VSV A51 -encoded GFP expression 786-0 cells. As shown in FIG. 12, I-73104903314, 1-36, 1-40, 1-43, 1-44, 1-47, 1-50, 1-51 , 1-52, 1-53, 1-54, 1-55, 1-58, 1-59, 1-60 and 1-61 (exemplary compounds of the application) at 15 pM enhanced lentivirus-encoded GFP expression by more than 1.1-fold in primary T-cells. In FIG. 13, 1-35 (exemplary compound of the application) enhanced VSVA51 -encoded GFP expression in 786-0 cells in a dose-dependent manner from 10 to 40 pM. This demonstrates that exemplary compounds of this application enhance virally- encoded transgenes in transduced cells.

[0313] 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.7410490331

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 compound of formula (I), or a pharmaceutically acceptable salt, solvate or prodrug thereof:wherein:Z is NR6, S, O, SO2or CHR7;— - is a single or a double bond;Y is N or CR8when — - is a double bond; or Y is NR8or CHR8when — - is a single bond;R1is selected from H, halo, Ci-ealkyl, OCi-ealkyl, C(O)NH2 and X1Cy1; or R1is =0 and — - is a single bond;R2is selected from H, halo, Ci.6alkyl, OCi.6alkyl and X2Cy2;R3is selected from H, halo, Ci-ealkyl, OCi-ealkyl and X3Cy3;R4is selected from H, halo, Ci-ealkyl, OCi-ealkyl, NH(CO)Ci.6alkyl and X4Cy4;R5is selected from H, halo, Ci-ealkyl, OCi.6alkyl and X5Cy5;R6is selected from H, Ci-ealkyl, Ci.ealkyleneOCi.ealkyl, (CO)NHCi-ealkyl and X6Cy6;R7is selected from H, halo, Ci-ealkyl, OCi.6alkyl and X7Cy7;R8is selected from H, halo, Ci-ealkyl, OCi-ealkyl, NHC(O)Ci.ealkyleneOCi.ealkyl, and X8Cy8;Cy1, Cy2, Cy3, Cy4, Cy5, Cy6, Cy7and Cy8are each independently a cyclic group selected from 6- to 16-membered aryl, 5- to 16-membered heterocyclyl, 5- to 16- membered heteroaryl and 3- to 16-membered cycloalkyl, wherein when Cy1, Cy2,10490331Cy3, Cy4, Cy5, Cy6, Cy7and Cy8comprises 9- to 16-members, the cyclic group is either a bicyclic or tricyclic, fused and / or spiro cyclic group, and each of Cy1, Cy2, Cy3, Cy4, Cy5, Cy6, Cy7and Cy8is, independently, unsubstituted or substituted with one to four substituents independently selected from OH, halo, Ci-salkyl, OC1.6alkyl, CN, =0, NH2, SCi.6alkyl, C(O)Ci.6alkyl and C(O)N(Ci-6alkyl)2and / or substituted with one substituent selected from X9phenyl, X10C3-6cycloalkyl, X10C3.6heterocycloalkyl and X10C5-6heteroaryl, wherein each phenyl, C3.6cycloalkyl, C3.6heterocycloalkyl and C5-6heteroaryl is unsubstituted or substituted with one or more substituents independently selected from OH, halo, Ci.6alkyl, OCi.6alkyl, CN, C(O)Ci-ealkyl, SCi-ealkyl, S(O)Ci-ealkyl, SO2Ci-ealkyl and NHCi-ealkyl; or R1and R6or R7, R1and R8, R2and R6or R3and R4are linked to form, together with the atoms therebetween, a 5- to 16-membered monocyclic, bicyclic, and / or spirocyclic, saturated and / or unsaturated cyclic group, optionally comprising one or more heteromoieties independently selected from O, S, N and NR9and unsubstituted or substituted with one to four substituents independently selected from halo, Ci-salkyl, OCi-ealkyl, CN and C(O)Ci-ealkyl, and / or substituted with one substituent selected from X11phenyl and X12C5-6heteroaryl, wherein each phenyl and Cs-eheteroaryl is, unsubstituted or substituted with one or more substituents independently selected from OH, halo, Ci_6alkyl, OCi.6alkyl, CN, C(O)Ci.6alkyl, SCi.6alkyl, S(O)Ci.6alkyl, SO2Ci.6alkyl and NHCi-ealkyl; each X1, X2, X3, X4, X5, X7, X8, X9, X10, X11and X12is independently selected from a direct bond, Ci-ealkylene, C(O), O, NH, N(Ci-4alkyl), S, S(O), SO2, SCi-ealkylene, Ci-6alkyleneS, S(O)Ci.6alkylene, Ci.6alkyleneS(O), SO2Ci.6alkylene, Ci.6alkyleneSO2, NHCi.6alkylene, Ci.6alkyleneNH, N(Ci-4alkyl)Ci-6alkylene, Ci- 6alkyleneN(Ci-4alkyl), OCi.6alkylene, Ci.6alkyleneO, C(O)NH, NHC(O), C(O)N(Ci.4alkyl), SO2NH, NHSO2, NHC(O)NH, SO2N(Ci.4alkyl), N(Ci.4alkyl)SO2, C(O)Ci.6alkylene, Ci.6alkyleneC(O), NHC(O)Ci.6alkylene, NHC(O)Ci.6alkyleneC(O), C(O)Ci-6alkyleneNHC(O), NHC(O)NHCi-6alkyleneC(O), C(O)Ci-6alkyleneNHC(O)NH, Ci.6alkyleneNHC(O)NH, Ci.6alkyleneC(O)NH, Ci.6alkyleneNHC(O), NHC(O)Ci-6alkyleneO, OCi-6alkyleneC(O)NH, NHC(O)Ci-6alkyleneS, SCi.6alkyleneC(O)NH, NHC(O)Ci.6alkyleneS(O), S(O)Ci.6alkyleneC(O)NH, NHC(O)Ci-6alkyleneSO2and SO2Ci-6alkyleneC(O)NH;X6is selected from a direct bond, Ci.6alkylene, C(O), SO2, Ci.6alkyleneS, S(O)Ci.6alkylene, Ci.6alkyleneS(O), Ci.6alkyleneSO2, Ci.6alkyleneNH, Ci-6alkyleneN(Ci-4alkyl), Ci.6alkyleneO, C(O)NH, C(O)N(Ci.4alkyl), SO2NH, SO2N(Ci.4alkyl), C(O)Ci.766alkylene, Ci.6alkyleneC(O), C(O)Ci.6alkyleneO, Ci.6alkyleneNHC(O), and Ci. 6alkyleneC(O)NH;R9is selected from H, C(O)Ci.6alkyl, CO2Ci-6alkyl, SO2Ci-6alkyl and Ci.6alkyl; at least 4 of R1, R2, R3, R4, R5, R6, R7and R8are selected from H and Ci_6alkyl; and all available hydrogen atoms are optionally and independently replaced with a halogen and all available atoms are optionally and independently replaced with an alternate isotope thereof.

2. The method of claim 1 , wherein the one or more nucleic acids encode components of a virus.

3. The method of claim 2, wherein the method comprises contacting the cell with an effective amount of the compound of formula (I), or a pharmaceutically acceptable salt, solvate, or prodrug thereof, in combination with the one or more nucleic acids, and the method is for increasing production of the virus by the cell.

4. The method of any one of claims 1 to 3, wherein the one or more nucleic acids comprise one or more plasmids.

5. The method of claim 2 or 3, wherein the virus is an adeno-associated virus (AAV), optionally wherein the AAV is selected from AAV serotypes 1 -9 and recombinant serotypes thereof.

6. The method of claim 2 or 3, wherein the virus is a retrovirus.

7. The method of claim 6, wherein the retrovirus is a lentivirus, optionally where the lentivirus is selected from HIV-1 , HIV-2, SIV, and recombinant lentiviruses.

8. The method of any one of claims 1 to 7, wherein the cell is a mammalian cell.

9. The method of claim 2 or 3, wherein production of the virus by the cell is increased by at least 1.1 fold compared to a control cell.

10. The method of claim 3, wherein the one or more nucleic acids are introduced by viral infection of the cell by a virus.1 1 . The method of claim 10, wherein the virus is an RNA virus.

12. The method of claim 10, wherein the virus is a DNA virus.

13. The method of claim 10, wherein the virus is a Rhabdovirus.

14. The method of claim 10, wherein the virus is an Adenovirus.771049033115. The method of claim 1 , wherein at least one of the one or more nucleic acids encodes a transgene, the method comprises contacting the cell with an effective amount of the compound of formula (I), or a salt, solvate or prodrug thereof, in combination with the one or more nucleic acids, and the method is for increasing expression of the transgene in the cell.

16. The method of claim 15, wherein the transgene is a virally-encoded transgene and wherein at least one of the nucleic acids encodes one or more components of the virus.

17. The method of claim 15 or 16, wherein the transgene is a therapeutic gene.

18. The method of any one of claims 15 to 17, wherein the virus is an adeno-associated virus (AAV), optionally wherein the AAV is selected from AAV serotypes 1-9 and recombinant serotypes thereof.

19. The method of any one of claims 15 to 18, wherein the virus is a retrovirus.

20. The method of claim 19, wherein the retrovirus is a lentivirus, optionally where the lentivirus is selected from HIV-1 , HIV-2, SIV, and recombinant lentiviruses.21 . Th2 method of claims 15 to 17, wherein the virus is a Rhabdovirus.

22. The method of claim 1 , wherein the one or more nucleic acids are encapsulated in a virus, the method comprises contacting the cell with an effective amount of the compound of formula (I), or a pharmaceutically acceptable salt, solvate, or prodrug thereof, in combination with the virus, and the method is for increasing transduction of the virus into the cell.

23. The method of claim 22, wherein the virus is an adeno-associated virus (AAV), optionally wherein the AAV is selected from AAV serotypes 1-9 and recombinant serotypes thereof.

24. The method of claim 23, wherein the virus is a retrovirus.

25. The method of claim 24, wherein the retrovirus is a lentivirus, optionally where the lentivirus is selected from HIV-1 , HIV-2, SIV, and recombinant lentiviruses.

26. The method of claim 22, wherein the virus is a Rhabdovirus.

27. The method of claim 22, wherein the virus is an Adenovirus.

28. The method of any one of claims 22 to 27, wherein the cell is an immune cell or a T-cell.781049033129. The method of claim 1 , wherein the one or more nucleic acids encode components of a lentivirus, the method comprises contacting the cell with an effective amount of the compound of formula (I), or a pharmaceutically acceptable salt, solvate, or prodrug thereof, in combination with the one or more nucleic acids, and the method is for increasing production of the lentivirus in the cell.

30. The method of any one of claims 1 to 29, wherein the cell is contacted with the compound before, after, or concurrently with the one or more nucleic acids.31 . The method of claim 29 or 30, wherein the compound is used in combination with a booster or an enhancer.

32. The method of claim 31 , wherein the booster or enhancer is an histone deacetylase (HDAC) inhibitor.

33. The method of claim 32, wherein the HDAC inhibitor is sodium butyrate.

34. The method of claim 31 , wherein the enhancer is the LV-MAX supplement and LV- MAX enhancer.

35. The method of any one of claims 29 to 34, wherein production of the lentivirus is increased by at least 1.1 fold compared to a control cell.

36. The method of any one of claims 29 to 35, wherein the lentivirus is selected from HIV-1 , HIV-2, SIV, and recombinant lentiviruses.

37. The method of claim 1 , wherein the one or more nucleic acids encode components of an adeno-associated virus (AAV), the method comprises contacting the cell with an effective amount of the compound of the formula (I), or a pharmaceutically acceptable salt, solvate, or prodrug thereof, in combination with the one or more nucleic acids, and the method is for increasing production of the AAV in the cell.

38. The method of claim 37, wherein the cell is contacted with the compound before, after, or concurrently with the one or more nucleic acids.

39. The method of claim 37 or 38, wherein the compound is used in combination with a booster or an enhancer.

40. The method of claim 39, wherein the booster or enhancer is the AAV-MAX booster or the AAV-MAX enhancer.

41. The method of any one of claims 37 to 40, wherein the production of AAV is increased by at least 1.1 fold compared to a control cell.791049033142. The method of any one of claims 37 to 41 , wherein the AAV is selected from AAV serotypes 1-9 and recombinant serotypes thereof.

43. The method of any one of claims any one of claims 3 to 14 and 29 to 42, wherein the ratio of functional titer to physical titer of the virus is increased by contacting the compound of formula (I) compared to a control virus44. The method of any one of claims 1 to 43, wherein R1is selected from H, Ci. 4alkyl, C(O)NH2and X1Cy1.

45. The method of any one of claims 1 to 43, wherein R1is selected from H, CH3, CH2CH3, C(O)NH2and X1Cy1.

46. The method of any one of claims 1 to 45, wherein X1is selected from a direct bond, N(Ci-4 C(O)N47. The method of any one of claims 1 to 45, wherein X1is selected from a direct bond, S, CH2S, SCH2, CH2CH2S CH2O, NHCH2, CH2NH, C(O)NH, C(O)NCH3, C(O), NHC(O), NH and CH2CH2CH2.

48. The method of any one of claims 1 to 45, wherein Cy1is selected from 6- to 8- membered aryl, 6-, 9-, 10- or 13-membered heterocyclyl, and 5-, 6- or 9-membered heteroaryl.

49. The method of any one of claims 1 to 48, wherein Cy1is selected from phenyl, pyridinyl, piperidinyl, benzimidazolyl, imidazolyl, thiazolyl, and benzodioxinyl, each of which are unsubstituted or substituted with one to four substituents independently selected from F, Cl, CN, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, C(CH3)3, C(O)CH3, C(O)CH2CH3, OCH3, and OCH2CH3.

50. The method of any one of claims 1 to 48, wherein Cy1is selected from phenyl, pyridinyl, piperidinyl, benzimidazolyl, imidazolyl, thiazolyl, and benzodioxinyl, each of which are unsubstituted or substituted with one to four substituents independently selected from NHC(O)phenyl, NHC(O)Ci-4alkylene-Ophenyl, NHC(O)pyrimidinyl and NHC(0)Ci-4alkylenepyrazolyl wherein each phenyl, pyrimidinyl and pyrazolyl group is unsubstituted or substituted with one to four substituents independently selected from F, Cl, SO2CH3, SO2CH2CH3, and NHCi.6alkyl.51 . The method of any one of claims 1 to 43, wherein R1is selected from H, =0, CH3, C(O)NH2, phenyl,8010490331wherein1indicates the point of attachment to the remainder of the compound.

52. The method of any one of claims 1 to 43, wherein R1and R6or R7, R1and R8, R2and R6or R3and R4are linked, together with the atoms therebetween to form a 6- membered monocyclic, a 10-membered bicyclic or a 15-membered spirocyclic, optionally comprising one to three NH and unsubstituted or substituted with one to four substituents independently selected from X11phenyl, wherein each phenyl is10490331unsubstituted or substituted with one to four substituents independently selected from CH3, CH2CH3, OCH3, and OCH2CH3.

53. The method of any one of claims 1 to 43, wherein R1and R6or R7, R1and R8, R2and R6or R3and R4are linked, together with the atoms therebetween to form:wherein i indicates the point of attachment to the remainder of the compound.

54. The method of any one of claims 1 to 51 , wherein R2is selected from H, CH3, CH2CH3 and X2Cy2.

55. The method of any one of claims 1 to 54, wherein X2is NHC(O) or C(O)NH.

56. The method of any one of claims 1 to 55, wherein Cy2is phenyl unsubstituted or substituted with one to four substituents independently selected from CN, F, Cl, CH3or CH2CH3.

57. The method of any one of claims 1 to 51 , wherein R2is selected from H and1049033158. The method of any one of claims 1 to 57, wherein R3is selected from H, Cl, OCH3, OCH2CH3, OCH2CH2CH3, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, and X3Cy3.

59. The method of any one of claims 1 to 57, wherein R3is selected from H, Cl,60. The method of any one of claims 1 to 59, wherein R4is selected from H, Cl, F, OCH3, OCH2CH3, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2and X4Cy4.61 . The method of any one of claims 1 to 60, wherein X4is selected from NHC(O), C(O)NH, C(O)NHCI.4alkylene, NHC1.4 alkylene, NHC(O)CI.4alkyleneO, , NHC(O)CI.4alkylene, NHC(O)NH and C(O).

62. The method of any one of claims 1 to 61 , wherein Cy4is phenyl, thiophenyl, tetrazolyl, cyclopentyl, triazolopyridinyl, pyridinyl or azepanyl, each of which is unsubstituted or substituted with one to four substituents independently selected from OCH3, OCH2CH3, CH3, or CH2CH3.

63. The method of any one of claims 1 to 62, wherein R4is selected from H, Cl, F, OCH3, CH3,8410490331JW I\A wherein i indicates the point of attachment to the remainder of the compound.

64. The method of any one of claims 1 to 43, wherein R3and R4are linked, together c ) with the atoms therebetween to form: .

65. The method of any one of claims 1 to 64, wherein R5is selected from H, F, Cl, OCH3, OCH2CH3, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2.

66. The method of any one of claims 1 to 64, wherein R5is selected from H, F, Cl, and CH3.

67. The method of any one of claims 1 to 66, wherein R6is selected from H, CH3, CH2CH3, (CH2)2OCH3and X6Cy6.

68. The compound of any one of claims 1 to 67, wherein X6is selected from a direct bond, Ci.4alkyleneO, C1-4 alkylene, C(O), C(O)NH, Ci.4alkyleneC(O) and SO2.

69. The method of any one of claims 1 to 68, wherein Cy6is phenyl, 10- to 13- membered heterocyclyl, morpholinyl, each of which is unsubstituted or substituted with one to four substituents independently selected from F, Cl, CH3, or CH2CH3.851049033170. The method of any one of claims 1 to 69, wherein R6is selected from H, CH3, CH2CH3, (CH2)2OCH3, C(O)NHCH3,« / vw I \ wherein1indicates the point of attachment to the remainder of the compound.

71. The method of any one of claims 1 to 70, wherein R8is selected from H, OCH3, OCH2CH3, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, Cl and X8Cy8.

72. The method of any one of claims 1 to 71 wherein X8is selected from a direct bond, , C(O)NH, NHC(O)NH, Ci-4alkyleneO, Ci-4alkylene, C(O), Ci.4alkyleneC(O), Cv 4alkyleneC(O)NH, C(O)Ci.4alkyleneO, C(O)Ci.6alkyleneNHC(O)NH and SO2.

73. The method of any one of claims 1 to 72, wherein Cy8is phenyl, 10- to 13- membered heterocyclyl, 5- to 9-membered heteroaryl, pyridinyl, or morpholinyl, each of which is unsubstituted or substituted with one to four substituents independently selected from F, Cl, CH3, or CH2CH3.

74. The method of any one of claims 1 to 73, wherein R8is selected from H, CH3, CH2CH3, Cl,8610490331wherein I indicates the point of attachment to the remainder of the compound.

75. The method of any one of claims 1 , 3, 15, 22, 29 and 37, wherein the compound of formula (I) is selected from selected from Table 1.

76. The method of any one of claims 1 to 74, wherein the compound of formula (I) is compound of Formula (l-A), or a pharmaceutically acceptable salt, solvate or prodrug thereof:wherein R1, R2, R3, R4, R5and R6are as defined in any one of claims 1 to 74.

77. The method of any one of claims 1 to 74, wherein the compound of formula (I) is compound of Formula (l-B), or a pharmaceutically acceptable salt, solvate or prodrug thereof:10490331wherein R1, R2, R3, R4, R5, R6and R8are as defined in any one of claims 1 to 74.

78. The method of any one of claims 1 to 74, wherein the compound of formula (I) is compound of Formula (l-C) or a pharmaceutically acceptable salt, solvate or prodrug thereof:wherein R1, R2, R3, R4, and R5are as defined in any one of claims 1 to 74.

79. The method of any one of claims 1 to 74, wherein the compound of formula (I) is compound of Formula (l-D) or a pharmaceutically acceptable salt, solvate or prodrug thereof:wherein R1, R2, R3, R4, and R5are as defined in any one of claims 1 to 74, wherein — z is a single bond or a double bond and R8 is absent is when — - is a double bond and as defined in any one of claims 1 to 74 when — - is a single bond.

80. A composition comprising a compound of the formula (I) as defined in any one of claims 1 to 79, or a pharmaceutically acceptable salt, solvate, or prodrug thereof,10490331and a one or more nucleic acids encoding one or more components of a virus or encapsulated in a virus.81 . Use of the composition of claim 80 for increasing production of a virus in a cell.

82. Use of the composition of claim 80 for increasing transduction of a virus in a cell.

83. Use of the composition of claim 80 for increasing transgene expression in a cell.8910490331

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